Professor Ali Mobasheri

Professor of Musculoskeletal Physiology & Head of Department of Pre-clinical Animal Science & Veterinary Pre-Clinical Sciences

Qualifications: BSc ARCS (Hons), MSc, DPhil (Oxon)

Email:
Phone: Work: 01483 68 9398
Room no: 05 DK 05

Office hours

Please contact Catherine Coats for an appointment on 01483 689015 or by e-mail: c.coats@surrey.ac.uk

Further information

Biography

Ali Mobasheri graduated in Biochemistry from Imperial College London in 1990. He was awarded an Open Fellowship by the University of Toronto and completed a Master’s degree in 1993. He returned to the UK to undertake a PhD sponsored by the Arthritis and Rheumatism Council (now known as Arthritis Research UK) at Wolfson College, University of Oxford. After completing his PhD in 1997 he worked as a lecturer in London (1997-2000) before moving to the University of Liverpool, where he was lecturer from 2000-2005 and senior lecturer from 2005-2006. In 2006 he moved to the University of Nottingham as Associate Professor and Reader in Comparative Physiology. He currently holds the post of Professor of Musculoskeletal Physiology at the University of Surrey.

Research Interests

Ali’s primary research focuses on cartilage biology. He is the Scientific Co-ordinator of the EU Framework 7 funded D-BOARD consortium, which brings together leading European academic institutions and SMEs to focus on the identification, validation and qualification of new biomarkers for degenerative and inflammatory diseases of joints.


http://www.d-board.eu/dboard/index.aspx
http://cordis.europa.eu/projects/rcn/105314_en.html
http://ec.europa.eu/research/health/medical-research/severe-chronic-diseases/projects/d-board_en.html


Ali’s track record of professional achievement is backed up by more than 167 primary peer-reviewed publications. He has contributed to more than twenty book chapters and has ongoing collaborative research with industry and leading academic investigators in established centres of excellence in Europe and the Middle East. Ali is a member of the Board of Directors of the Osteoarthritis Research Society International (OARSI) and chairs OARSI's Strategic Alliances Committee. He currently serves on the BBSRC’s Committee A, the BBSRC Industrial CASE Studentship Committee and is a member of the BBSRC Pool of Experts. Ali also serves as a Faculty Member in the Musculoskeletal Repair & Regeneration/Rheumatology & Clinical Immunology sections of Faculty of 1000 Ltd.


Goals: To contribute to the development of high quality research and teaching as well as professional consultancy services for healthcare, veterinary, diagnostics and pet food and agri-food industries.

Publications

Journal articles

  • Musumeci G, Mobasheri A, Trovato FM, Szychlinska MA, Graziano AC, Lo Furno D, Avola R, Mangano S, Giuffrida R, Cardile V. (2014) 'Biosynthesis of collagen I, II, RUNX2 and lubricin at different time points of chondrogenic differentiation in a 3D in vitro model of human mesenchymal stem cells derived from adipose tissue.'. Acta Histochem,

    Abstract

    The first aim of the study was to identify the most appropriate time for differentiation of adipose tissue derived mesenchymal stem cells (MSCs) to chondrocytes, through the self-assembly process. For this purpose, the expression of some chondrocyte markers, such as collagen type I, collagen type II, RUNX2 and lubricin was investigated at different times (7, 14, 21 and 28 days) of chondrogenic differentiation of MSCs, by using immunohistochemistry and Western blot analysis. The second aim of the study was to demonstrate that the expression of lubricin, such as the expression of collagen type II, could be a possible biomarker for the detection of chondrocytes well-being and viability in the natural self-assembling constructs, called 'cell pellets'. Histology (hematoxylin and eosin) and histochemistry (alcian blue staining) methods were used to assess the chondrogenic differentiation of MSCs. The results showed that after 21 days the differentiated chondrocytes, when compared with MSCs cultured without chondrogenic medium (CD44, CD90 and CD105 positive; CD45, CD14 and CD34 negative), were able to produce significant quantities of collagen type I, collagen type II, and lubricin, suggesting hyaline cartilage formation. During the differentiation phase, the cells showed a reduced expression of RUNX2, a protein expressed by osteoblasts. Our studies demonstrated that 21 days is the optimum time for the implantation of chondrocytes differentiated from adipose tissue-derived MSCs. This information could be useful for the future development of cell-based repair therapies for degenerative diseases of articular cartilage.

  • Musumeci G, Szychlinska MA, Mobasheri A. (2014) 'Age-related degeneration of articular cartilage in the pathogenesis of osteoarthritis: molecular markers of senescent chondrocytes.'. Histol Histopathol,

    Abstract

    Aging is a natural process by which every single living organism approaches its twilight of existence in a natural way. However, aging is also linked to the pathogenesis of a number of complex diseases. This is the case for osteoarthritis (OA), where age is considered to be a major risk factor of this important and increasingly common joint disorder. Half of the world's population, aged 65 and older, suffers from OA. Although the relationship between the development of OA and aging has not yet been completely understood, it is thought that age-related changes correlate with other risk factors. The most prominent hypothesis linking aging and OA is that chondrocytes undergo premature aging due to several factors, such as excessive mechanical load or oxidative stress, which induce the so called "stress-induced senescent state", which is ultimately responsible for the onset of OA. This review focuses on molecular markers and mechanisms implicated in chondrocyte aging and the pathogenesis of OA. We discuss the most important age-related morphological and biological changes that affect articular cartilage and chondrocytes. We also identify the main senescence markers that may be used to recognize molecular alterations in the extracellular matrix of cartilage as related to senescence. Since the aging process is strongly associated with the onset of osteoarthritis, we believe that strategies aimed at preventing chondrocyte senescence, as well as the identification of new increasingly sensitive senescent markers, could have a positive impact on the development of new therapies for this severe disease.

  • Musumeci G, Castrogiovanni P, Trovato FM, Imbesi R, Giunta S, Szychlinska MA, Loreto C, Castorina S, Mobasheri A. (2014) 'Physical activity ameliorates cartilage degeneration in a rat model of aging: A study on lubricin expression.'. Scand J Med Sci Sports,

    Abstract

    Osteoarthritis (OA) is a common musculoskeletal disorder characterized by slow progression and joint tissue degeneration. Aging is one of the most prominent risk factors for the development and progression of OA. OA is not, however, an inevitable consequence of aging and age-related changes in the joint can be distinguished from those that are the result of joint injury or inflammatory disease. The question that remains is whether OA can be prevented by undertaking regular physical activity. Would moderate physical activity in the elderly cartilage (and lubricin expression) comparable to a sedentary healthy adult? In this study we used physical exercise in healthy young, adult, and aged rats to evaluate the expression of lubricin as a novel biomarker of chondrocyte senescence. Immunohistochemistry and western blotting were used to evaluate the expression of lubricin in articular cartilage, while enzyme-linked immunosorbent assay was used to quantify lubricin in synovial fluid. Morphological evaluation was done by histology to monitor possible tissue alterations. Our data suggest that moderate physical activity and normal mechanical joint loading in elderly rats improve tribology and lubricative properties of articular cartilage, promoting lubricin synthesis and its elevation in synovial fluid, thus preventing cartilage degradation compared with unexercised adult rats.

  • Matta C, Mobasheri A, Gergely P, Zákány R. (2014) 'Ser/Thr-phosphoprotein phosphatases in chondrogenesis: neglected components of a two-player game.'. Cell Signal, England: 26 (10), pp. 2175-2185.

    Abstract

    Protein phosphorylation plays a determining role in the regulation of chondrogenesis in vitro. While signalling pathways governed by protein kinases including PKA, PKC, and mitogen-activated protein kinases (MAPK) have been mapped in great details, published data relating to the specific role of phosphoprotein phosphatases (PPs) in differentiating chondroprogenitor cells or in mature chondrocytes is relatively sparse. This review discusses the known functions of Ser/Thr-specific PPs in the molecular signalling pathways of chondrogenesis. PPs are clearly equally important as protein kinases to counterbalance the effect of reversible protein phosphorylation. Of the main Ser/Thr PPs, some of the functions of PP1, PP2A and PP2B have been characterised in the context of chondrogenesis. While PP1 and PP2A appear to negatively regulate chondrogenic differentiation and maintenance of chondrocyte phenotype, calcineurin is an important stimulatory mediator during chondrogenesis but becomes inhibitory in mature chondrocytes. Furthermore, PPs are implicated to be mediators during the pathogenesis of osteoarthritis that makes them potential therapeutic targets to be exploited in the close future. Among the many yet unexplored targets of PPs, modulation of plasma membrane ion channel function and participation in mechanotransduction pathways are emerging novel aspects of signalling during chondrogenesis that should be further elucidated. Besides the regulation of cellular ion homeostasis, other potentially significant novel roles for PPs during the regulation of in vitro chondrogenesis are discussed.

  • Henrotin Y, Marty M, Mobasheri A. (2014) 'What is the current status of chondroitin sulfate and glucosamine for the treatment of knee osteoarthritis?'. Maturitas, 78 (3), pp. 184-187.

    Abstract

    Chondroitin sulfate and glucosamine sulfate exert beneficial effects on the metabolism of in vitro models of cells derived from synovial joints: chondrocytes, synoviocytes and cells from subchondral bone, all of which are involved in osteoarthritis (OA). They increase type II collagen and proteoglycan synthesis in human articular chondrocytes and are able to reduce the production of some pro-inflammatory mediators and proteases, to reduce the cellular death process, and improve the anabolic/catabolic balance of the extracellular cartilage matrix (ECM). Clinical trials have reported a beneficial effect of chondroitin sulfate and glucosamine sulfate on pain and function. The structure-modifying effects of these compounds have been reported and analyzed in recent meta-analyses. The results for knee OA demonstrate a small but significant reduction in the rate of joint space narrowing. Chondroitin sulfate and glucosamine sulphate are recommended by several guidelines from international societies for the management of knee and hip OA, while others do not recommend these products or recommend only under condition. This comprehensive review clarifies the role of these compounds in the therapeutic arsenal for patients with knee OA. © 2014 The Authors.

  • Mobasheri A, Batt ME, Kalamegam G, Musumeci G. (2014) 'Chondrocyte and mesenchymal stem cell-based therapies for cartilage repair in osteoarthritis and related orthopaedic conditions'. Maturitas, 78 (3), pp. 188-198.

    Abstract

    Osteoarthritis (OA) represents a final and common pathway for all major traumatic insults to synovial joints. OA is the most common form of degenerative joint disease and a major cause of pain and disability. Despite the global increase in the incidence of OA, there are no effective pharmacotherapies capable of restoring the original structure and function of damaged articular cartilage. Consequently cell-based and biological therapies for osteoarthritis (OA) and related orthopaedic disorders have become thriving areas of research and development. Autologous chondrocyte implantation (ACI) has been used for treatment of osteoarticular lesions for over two decades. Although chondrocyte-based therapy has the capacity to slow down the progression of OA and delay partial or total joint replacement surgery, currently used procedures are associated with the risk of serious adverse events. Complications of ACI include hypertrophy, disturbed fusion, delamination, and graft failure. Therefore there is significant interest in improving the success rate of ACI by improving surgical techniques and preserving the phenotype of the primary chondrocytes used in the procedure. Future tissue-engineering approaches for cartilage repair will also benefit from advances in chondrocyte-based repair strategies. This review article focuses on the structure and function of articular cartilage and the pathogenesis of OA in the context of the rising global burden of musculoskeletal disease. We explore the challenges associated with cartilage repair and regeneration using cell-based therapies that use chondrocytes and mesenchymal stem cells (MSCs). This paper also explores common misconceptions associated with cell-based therapy and highlights a few areas for future investigation. © 2014 The Authors.

  • Musumeci G, Trovato FM, Loreto C, Leonardi R, Szychlinska MA, Castorina S, Mobasheri A. (2014) 'Lubricin expression in human osteoarthritic knee meniscus and synovial fluid: a morphological, immunohistochemical and biochemical study.'. Acta Histochem, Germany: 116 (5), pp. 965-972.

    Abstract

    The purpose of this study was to investigate the expression of lubricin, the product of the human PRG4 (proteoglycan 4) gene, in menisci and synovial fluid from normal donors and patients with osteoarthritis (OA), using a combination of histology, immunohistochemistry, ELISA and Western blotting analysis, to provide further insight on the role of this protein in the progression of OA and pathological processes in the meniscus. Lubricin expression was studied in samples from 40 patients and in 9 normal donors after arthroscopic partial meniscectomy. Histological analysis confirmed normal microanatomy and the absence of structural changes in control samples. Menisci derived from OA patients showed evidence of structural alterations, fibrillations and clefts. Immunohistochemical analysis revealed very strong lubricin immunostaining in normal menisci in contrast to weak/moderate staining seen in osteoarthritic menisci. Quantitative ELISA and Western blot analysis confirmed the above results. The findings of this study support the notion that changes in lubricin expression and boundary-lubricating ability of cartilage is followed by the development of OA. This study could provide the biological foundation for the development of novel therapeutic treatments, to be applied before the surgery, for the prevention of post-traumatic cartilage damage.

  • Musumeci G, Loreto C, Szychlinska MA, Castorina S, Trovato FM, Leonardi R, Mobasheri A. (2014) 'Lubricin expression in human osteoarthritic knee meniscus and synovial fluid: A morphological, immunohistochemical and biochemical study'. Acta Histochemica, 116 (5), pp. 965-972.

    Abstract

    The purpose of this study was to investigate the expression of lubricin, the product of the human PRG4 (proteoglycan 4) gene, in menisci and synovial fluid from normal donors and patients with osteoarthritis (OA), using a combination of histology, immunohistochemistry, ELISA and Western blotting analysis, to provide further insight on the role of this protein in the progression of OA and pathological processes in the meniscus. Lubricin expression was studied in samples from 40 patients and in 9 normal donors after arthroscopic partial meniscectomy. Histological analysis confirmed normal microanatomy and the absence of structural changes in control samples. Menisci derived from OA patients showed evidence of structural alterations, fibrillations and clefts. Immunohistochemical analysis revealed very strong lubricin immunostaining in normal menisci in contrast to weak/moderate staining seen in osteoarthritic menisci. Quantitative ELISA and Western blot analysis confirmed the above results. The findings of this study support the notion that changes in lubricin expression and boundary-lubricating ability of cartilage is followed by the development of OA. This study could provide the biological foundation for the development of novel therapeutic treatments, to be applied before the surgery, for the prevention of post-traumatic cartilage damage. © 2014 Elsevier GmbH.

  • Mobasheri A, Kalamegam G, Musumeci G, Batt ME. (2014) 'Chondrocyte and mesenchymal stem cell-based therapies for cartilage repair in osteoarthritis and related orthopaedic conditions.'. Maturitas, Ireland: 78 (3), pp. 188-198.

    Abstract

    Osteoarthritis (OA) represents a final and common pathway for all major traumatic insults to synovial joints. OA is the most common form of degenerative joint disease and a major cause of pain and disability. Despite the global increase in the incidence of OA, there are no effective pharmacotherapies capable of restoring the original structure and function of damaged articular cartilage. Consequently cell-based and biological therapies for osteoarthritis (OA) and related orthopaedic disorders have become thriving areas of research and development. Autologous chondrocyte implantation (ACI) has been used for treatment of osteoarticular lesions for over two decades. Although chondrocyte-based therapy has the capacity to slow down the progression of OA and delay partial or total joint replacement surgery, currently used procedures are associated with the risk of serious adverse events. Complications of ACI include hypertrophy, disturbed fusion, delamination, and graft failure. Therefore there is significant interest in improving the success rate of ACI by improving surgical techniques and preserving the phenotype of the primary chondrocytes used in the procedure. Future tissue-engineering approaches for cartilage repair will also benefit from advances in chondrocyte-based repair strategies. This review article focuses on the structure and function of articular cartilage and the pathogenesis of OA in the context of the rising global burden of musculoskeletal disease. We explore the challenges associated with cartilage repair and regeneration using cell-based therapies that use chondrocytes and mesenchymal stem cells (MSCs). This paper also explores common misconceptions associated with cell-based therapy and highlights a few areas for future investigation.

  • Matta C, Mobasheri A. (2014) 'Regulation of chondrogenesis by protein kinase C: Emerging new roles in calcium signalling.'. Cell Signal, England: 26 (5), pp. 979-1000.

    Abstract

    During chondrogenesis, complex intracellular signalling pathways regulate an intricate series of events including condensation of chondroprogenitor cells and nodule formation followed by chondrogenic differentiation. Reversible phosphorylation of key target proteins is of particular importance during this process. Among protein kinases known to be involved in these pathways, protein kinase C (PKC) subtypes play pivotal roles. However, the precise function of PKC isoenzymes during chondrogenesis and in mature articular chondrocytes is still largely unclear. In this review, we provide a historical overview of how the concept of PKC-mediated chondrogenesis has evolved, starting from the first discoveries of PKC isoform expression and activity. Signalling components upstream and downstream of PKC, leading to the stimulation of chondrogenic differentiation, are also discussed. Although it is evident that we are only at the beginning to understand what roles are assigned to PKC subtypes during chondrogenesis and how they are regulated, there are many yet unexplored aspects in this area. There is evidence that calcium signalling is a central regulator in differentiating chondroprogenitors; still, clear links between intracellular calcium signalling and prototypical calcium-dependent PKC subtypes such as PKCalpha have not been established. Exploiting putative connections and shedding more light on how exactly PKC signalling pathways influence cartilage formation should open new perspectives for a better understanding of healthy as well as pathological differentiation processes of chondrocytes, and may also lead to the development of novel therapeutic approaches.

  • Henrotin Y, Marty M, Mobasheri A. (2014) 'What is the current status of chondroitin sulfate and glucosamine for the treatment of knee osteoarthritis?'. Maturitas, Ireland: 78 (3), pp. 184-187.

    Abstract

    Chondroitin sulfate and glucosamine sulfate exert beneficial effects on the metabolism of in vitro models of cells derived from synovial joints: chondrocytes, synoviocytes and cells from subchondral bone, all of which are involved in osteoarthritis (OA). They increase type II collagen and proteoglycan synthesis in human articular chondrocytes and are able to reduce the production of some pro-inflammatory mediators and proteases, to reduce the cellular death process, and improve the anabolic/catabolic balance of the extracellular cartilage matrix (ECM). Clinical trials have reported a beneficial effect of chondroitin sulfate and glucosamine sulfate on pain and function. The structure-modifying effects of these compounds have been reported and analyzed in recent meta-analyses. The results for knee OA demonstrate a small but significant reduction in the rate of joint space narrowing. Chondroitin sulfate and glucosamine sulphate are recommended by several guidelines from international societies for the management of knee and hip OA, while others do not recommend these products or recommend only under condition. This comprehensive review clarifies the role of these compounds in the therapeutic arsenal for patients with knee OA.

  • Bullock CM, Wookey P, Bennett A, Mobasheri A, Dickerson I, Kelly S. (2014) 'Peripheral calcitonin gene-related peptide receptor activation and mechanical sensitization of the joint in rat models of osteoarthritis pain.'. Arthritis Rheumatol, United States: 66 (8), pp. 2188-2200.

    Abstract

    To investigate the role of the sensory neuropeptide calcitonin gene-related peptide (CGRP) in peripheral sensitization in experimental models of osteoarthritis (OA) pain.

  • Starowicz A, Grzesiak M, Szoltys M, Mobasheri A. (2014) 'Immunolocalization of aquaporin 5 during rat ovarian follicle development and expansion of the preovulatory cumulus oophorus'. Acta Histochemica, 116 (3), pp. 457-465.

    Abstract

    Immunofluorescent localization of aquaporin 5 (AQP5) was investigated in rat ovarian follicles during development and preovulatory cumulus oophorus expansion. Ampullary cumuli oophori complexes (COCs) were examined. Analysis revealed that AQP5 immunostaining appeared in preantral follicles and formed a characteristic ring encircling and touching the oolemma. The staining represented most likely AQP5 functioning at the ends of corona radiata cell projections, anchoring on the oocyte surface. However, several hours after the presumptive preovulatory LH surge, when the process of expansion of COCs started, the AQP5 staining appeared also on the cumulus granulosa cells and in the extracellular matrix. In the postovulatory ampullary COCs the fluorescent ring was not observed, which may be the result of the well-established preovulatory withdrawal of projections from the zona pellucida. At that time-point immunofluorescent staining of AQP5 appeared in most oocytes and was also present in the apical membrane of epithelial cells of the oviduct ampulla. The latter observation suggests that after ovulation AQP5 is involved in the transcellular movement of water in the oviduct ampulla and oocytes in rats. © 2013 Elsevier GmbH.

  • Hdud IM, Mobasheri A, Loughna PT. (2014) 'Effect of osmotic stress on the expression of TRPV4 and BKCa channels and possible interaction with ERK1/2 and p38 in cultured equine chondrocytes.'. Am J Physiol Cell Physiol, United States: 306 (11), pp. C1050-C1057.

    Abstract

    The metabolic activity of articular chondrocytes is influenced by osmotic alterations that occur in articular cartilage secondary to mechanical load. The mechanisms that sense and transduce mechanical signals from cell swelling and initiate volume regulation are poorly understood. The purpose of this study was to investigate how the expression of two putative osmolyte channels [transient receptor potential vanilloid 4 (TRPV4) and large-conductance Ca(2+)-activated K(+) (BKCa)] in chondrocytes is modulated in different osmotic conditions and to examine a potential role for MAPKs in this process. Isolated equine articular chondrocytes were subjected to anisosmotic conditions, and TRPV4 and BKCa channel expression and ERK1/2 and p38 MAPK protein phosphorylation were investigated using Western blotting. Results indicate that the TRPV4 channel contributes to the early stages of hypo-osmotic stress, while the BKCa channel is involved in responding to elevated intracellular Ca(2+) and mediating regulatory volume decrease. ERK1/2 is phosphorylated by hypo-osmotic stress (P < 0.001), and p38 MAPK is phosphorylated by hyperosmotic stress (P < 0.001). In addition, this study demonstrates the importance of endogenous ERK1/2 phosphorylation in TRPV4 channel expression, where blocking ERK1/2 by a specific inhibitor (PD98059) prevented increased levels of the TRPV4 channel in cells exposed to hypo-osmotic stress and decreased TRPV4 channel expression to below control levels in iso-osmotic conditions (P < 0.001).

  • Mobasheri A, Barrett-Jolley R. (2014) 'Aquaporin water channels in the mammary gland: from physiology to pathophysiology and neoplasia.'. J Mammary Gland Biol Neoplasia, United States: 19 (1), pp. 91-102.

    Abstract

    Aquaporins are membrane proteins that play fundamental roles in water and small solute transport across epithelial and endothelial barriers. Recent studies suggest that several aquaporin proteins are present in the mammary gland. Immunohistochemical techniques have confirmed the presence of aquaporin 1 (AQP1) and AQP3 water channels in rat, mouse, bovine and human mammary glands. Studies suggest that in addition to AQP1 and AQP3 AQP4, AQP5 and AQP7 proteins are expressed in different locations in the mammary gland. Aquaporins play key roles in tumor biology and are involved in cell growth, migration and formation of ascites via increased water permeability of micro-vessels. Emerging evidence suggests that expression of these proteins is altered in mammary tumors and in breast cancer cell lines although it is not yet clear whether this is a cause or a consequence of neoplastic development. This review analyzes the expression and potential functional roles of aquaporin water channels in the mammary gland. The physiological mechanisms involved in the transport of water and small solutes across mammary endothelial and epithelial barriers are discussed in the context of milk production and lactation. This paper also reviews papers from the recent cancer literature that implicate aquaporins in mammary neoplasia.

  • Williams S, Horner J, Orton E, Green M, McMullen S, Mobasheri A, Freeman SL. (2014) 'Water intake, faecal output and intestinal motility in horses moved from pasture to a stabled management regime with controlled exercise.'. Equine Vet J,

    Abstract

    A change in management from pasture to stabling is a risk factor for equine colic.

  • Hdud IM, Mobasheri A, Loughna PT. (2014) 'Effects of cyclic equibiaxial mechanical stretch on α-BK and TRPV4 expression in equine chondrocytes.'. Springerplus, Switzerland: 3

    Abstract

    Chondrocytes are regularly exposed to load-induced stimuli and have the capability to sense and respond to applied mechanical stress. However, the mechanisms involved in chondrocyte mechanotransduction are not clearly understood. The purpose of this study was to explore the effects of cyclic equibiaxial mechanical stretch on the expression of α-BK and TRPV4 channels.

  • Henrotin Y, Marty M, Mobasheri A. (2014) 'Corrigendum to: What is the current status of chondroitin sulfate and glucosamine for the treatment of knee osteoarthritis? Maturitas 78 (2014) 184-187 (DOI:10.1016/j.maturitas.2014.04.015)'. Maturitas,
  • Swan AL, Mobasheri A, Allaway D, Liddell S, Bacardit J. (2013) 'Application of machine learning to proteomics data: classification and biomarker identification in postgenomics biology.'. OMICS, United States: 17 (12), pp. 595-610.

    Abstract

    Mass spectrometry is an analytical technique for the characterization of biological samples and is increasingly used in omics studies because of its targeted, nontargeted, and high throughput abilities. However, due to the large datasets generated, it requires informatics approaches such as machine learning techniques to analyze and interpret relevant data. Machine learning can be applied to MS-derived proteomics data in two ways. First, directly to mass spectral peaks and second, to proteins identified by sequence database searching, although relative protein quantification is required for the latter. Machine learning has been applied to mass spectrometry data from different biological disciplines, particularly for various cancers. The aims of such investigations have been to identify biomarkers and to aid in diagnosis, prognosis, and treatment of specific diseases. This review describes how machine learning has been applied to proteomics tandem mass spectrometry data. This includes how it can be used to identify proteins suitable for use as biomarkers of disease and for classification of samples into disease or treatment groups, which may be applicable for diagnostics. It also includes the challenges faced by such investigations, such as prediction of proteins present, protein quantification, planning for the use of machine learning, and small sample sizes.

  • Mobasheri A. (2013) 'The future of osteoarthritis therapeutics: emerging biological therapy.'. Curr Rheumatol Rep, United States: 15 (12)

    Abstract

    Biological therapy is a thriving area of research and development, and is well established for chronic forms of rheumatoid arthritis (RA) and ankylosing spondylitis (AS). However, there is no clinically validated biological therapy for osteoarthritis (OA). Chronic forms of OA are increasingly viewed as an inflammatory disease. OA was largely regarded as a "wear and tear disease". However, the disease is now believed to involve "low grade" inflammation and the growth of blood vessels and nerves from the subchondral bone into articular cartilage. This realization has focused research effort on the development and evaluation of biological therapy that targets proinflammatory mediators, angiogenic factors and cytokines in articular cartilage, subchondral bone and synovium in chronic forms of OA. This review article provides an overview of emerging biological therapy for OA, and discusses recent molecular targets implicated in angiogenesis and neurogenesis and progress with antibody-based therapy, calcitonin, and kartogenin, the small molecule stimulator of chondrogenesis.

  • Henrotin Y, Priem F, Mobasheri A. (2013) 'Curcumin: a new paradigm and therapeutic opportunity for the treatment of osteoarthritis: curcumin for osteoarthritis management.'. Springerplus, 2 (1)

    Abstract

    The management of osteoarthritis represents a real challenge. This complex and multi-factorial disease evolves over decades and requires not only the alleviation of symptoms, i.e. pain and joint function but also the preservation of articular structure without side effects. Nutraceuticals are good candidates for the management of OA due to their safety profile and potential efficacy. However, they are not part of the treatment guidelines and published recommendations. Curcumin is the yellow pigment isolated from the rhizomes of Curcuma longa, commonly known as turmeric. Curcumin is a highly pleiotropic molecule with an excellent safety profile. Strong molecular evidence has been published for its potency to target multiple inflammatory diseases. However, naturally occurring curcumin cannot achieve its optimum therapeutic outcomes due to its low solubility and poor bioavailability. Nevertheless, curcumin presents great potential for treating OA and has been categorized as having preclinical evidence of efficacy. This review aimed at gathering most of the available information to document the potential efficacy of curcumin based on the results obtained in in vitro models of cartilage and osteoarthritis and in other diseases.

  • Starowicz A, Grzesiak M, Mobasheri A, Szoltys M. (2013) 'Immunolocalization of aquaporin 5 during rat ovarian follicle development and expansion of the preovulatory cumulus oophorus.'. Acta Histochem, Germany: 116 (3), pp. 457-465.

    Abstract

    Immunofluorescent localization of aquaporin 5 (AQP5) was investigated in rat ovarian follicles during development and preovulatory cumulus oophorus expansion. Ampullary cumuli oophori complexes (COCs) were examined. Analysis revealed that AQP5 immunostaining appeared in preantral follicles and formed a characteristic ring encircling and touching the oolemma. The staining represented most likely AQP5 functioning at the ends of corona radiata cell projections, anchoring on the oocyte surface. However, several hours after the presumptive preovulatory LH surge, when the process of expansion of COCs started, the AQP5 staining appeared also on the cumulus granulosa cells and in the extracellular matrix. In the postovulatory ampullary COCs the fluorescent ring was not observed, which may be the result of the well-established preovulatory withdrawal of projections from the zona pellucida. At that time-point immunofluorescent staining of AQP5 appeared in most oocytes and was also present in the apical membrane of epithelial cells of the oviduct ampulla. The latter observation suggests that after ovulation AQP5 is involved in the transcellular movement of water in the oviduct ampulla and oocytes in rats.

  • Lewis R, May H, Mobasheri A, Barrett-Jolley R. (2013) 'Chondrocyte channel transcriptomics: do microarray data fit with expression and functional data?'. Channels (Austin), United States: 7 (6), pp. 459-467.

    Abstract

    To date, a range of ion channels have been identified in chondrocytes using a number of different techniques, predominantly electrophysiological and/or biomolecular; each of these has its advantages and disadvantages. Here we aim to compare and contrast the data available from biophysical and microarray experiments. This letter analyses recent transcriptomics datasets from chondrocytes, accessible from the European Bioinformatics Institute (EBI). We discuss whether such bioinformatic analysis of microarray datasets can potentially accelerate identification and discovery of ion channels in chondrocytes. The ion channels which appear most frequently across these microarray datasets are discussed, along with their possible functions. We discuss whether functional or protein data exist which support the microarray data. A microarray experiment comparing gene expression in osteoarthritis and healthy cartilage is also discussed and we verify the differential expression of 2 of these genes, namely the genes encoding large calcium-activated potassium (BK) and aquaporin channels.

  • Mobasheri A. (2013) 'The future of osteoarthritis therapeutics: targeted pharmacological therapy.'. Curr Rheumatol Rep, United States: 15 (10)

    Abstract

    Osteoarthritis (OA) is one of the most common forms of degenerative joint disease and a major cause of pain and disability affecting the aging population. It is estimated that more than 20 million Americans and 35 to 40 million Europeans suffer from OA. Analgesics and non-steroidal anti-inflammatory drugs (NSAIDs) are the only therapeutic treatment options for OA. Effective pharmacotherapy for OA, capable of restoring the original structure and function of damaged cartilage and other synovial tissue, is urgently needed, and research into such disease-modifying osteoarthritis drugs (DMOADs) is in progress. This is the first of three reviews focusing on OA therapeutics. This paper provides an overview of current research into potential structure-modifying drugs and more appropriately targeted pharmacological therapy. The challenges and opportunities in this area of research and development are reviewed, covering the most up-to-date initiatives, trends, and topics.

  • Mobasheri A, Shakibaei M. (2013) 'Is tendinitis an inflammatory disease initiated and driven by pro-inflammatory cytokines such as interleukin 1β?'. Histol Histopathol, Spain: 28 (8), pp. 955-964.

    Abstract

    Tendonitis and tendinitis are terms used to describe an inflamed and painful tendon. Tendinopathy, is a descriptive term for describing clinical conditions arising from tendon injury and overuse both within and around tendons. The aim of this mini-review is to explore the role of pro-inflammatory cytokines, particularly interleukin-1β (IL-1β) in tendon disorders. A number of investigators including our group have proposed that pro-inflammatory cytokines such as IL-1β are initiators of tendinopathies, stimulating inflammation, apoptosis and extracellular matrix (ECM) degradation. This is one of the reasons why IL-1β is frequently used in culture models of tendon inflammation to study the inflammatory and catabolic responses of tenocytes. However, some researchers oppose this view and suggest that although IL-1β may play a role in rheumatoid arthritis (RA) and osteoarthritis (OA), the involvement of IL-1β in the development of tendinopathy is questionable. This mini-review discusses the relevant papers published in this area and summarises the evidence for and against the involvement of pro-inflammatory cytokines such as IL-1β in tendonitis. Reaching a consensus will be important for the development and refinement of biomimetic models of tendon inflammation and the formulation of new therapeutic strategies for the treatment of tendon injuries.

  • Rufino AT, Rosa SC, Judas F, Mobasheri A, Lopes MC, Mendes AF. (2013) 'Expression and function of K(ATP) channels in normal and osteoarthritic human chondrocytes: possible role in glucose sensing.'. J Cell Biochem, United States: 114 (8), pp. 1879-1889.

    Abstract

    ATP-sensitive potassium [K(ATP)] channels sense intracellular ATP/ADP levels, being essential components of a glucose-sensing apparatus in various cells that couples glucose metabolism, intracellular ATP/ADP levels and membrane potential. These channels are present in human chondrocytes, but their subunit composition and functions are unknown. This study aimed at elucidating the subunit composition of K(ATP) channels expressed in human chondrocytes and determining whether they play a role in regulating the abundance of major glucose transporters, GLUT-1 and GLUT-3, and glucose transport capacity. The results obtained show that human chondrocytes express the pore forming subunits, Kir6.1 and Kir6.2, at the mRNA and protein levels and the regulatory sulfonylurea receptor (SUR) subunits, SUR2A and SUR2B, but not SUR1. The expression of these subunits was no affected by culture under hyperglycemia-like conditions. Functional impairment of the channel activity, using a SUR blocker (glibenclamide 10 or 20 nM), reduced the protein levels of GLUT-1 and GLUT-3 by approximately 30% in normal chondrocytes, while in cells from cartilage with increasing osteoarthritic (OA) grade no changes were observed. Glucose transport capacity, however, was not affected in normal or OA chondrocytes. These results show that K(ATP) channel activity regulates the abundance of GLUT-1 and GLUT-3, although other mechanisms are involved in regulating the overall glucose transport capacity of human chondrocytes. Therefore, K(ATP) channels are potential components of a broad glucose sensing apparatus that modulates glucose transporters and allows human chondrocytes to adjust to varying extracellular glucose concentrations. This function of K(ATP) channels seems to be impaired in OA chondrocytes.

  • Arrighi S, Aralla M, Fracassetti P, Mobasheri A, Cremonesi F. (2013) 'Aquaporin water channels in the canine gubernaculum testis.'. Acta Histochem, Germany: 115 (6), pp. 541-548.

    Abstract

    The jelly-like gubernaculum testis (GT) is a hydrated structure consisting of a concentric sheath of dense connective tissue around a loose mesenchymal core, with two cords of skeletal muscle cells asymmetrically placed alongside. Expansion of the GT occurs during the transabdominal phase of testicular descent, linked to cell proliferation together with modifications of the hydric content of the organ. The aim of this study was to detect immunohistochemically the presence of aquaporins (AQPs), integral membrane proteins permitting passive transcellular water movement, in the canine GTs. Samples (n=15) were obtained from pregnancies of 9 medium sized bitches and dissected from healthy fetuses. Five fetuses were aged 35-45 days of gestation, 10 fetuses from 46 days of gestation to delivery, thus offering us the opportunity to study the progressive maturation of the gubernacula. The presence of AQP3, 4, 7, 8 and -9 was assessed in the muscular components of the GT, some of them (AQP3, AQP4, AQP7) with increasing intensity through the second half of pregnancy up to term. AQP1 was localized in the capillary and venous endothelia in the younger fetuses, also in the artery adventitia and in the nerve perineurium in progressively older fetuses. These data demonstrate the potential importance and contribution of AQP-mediated water flux in hydration and volume modification of the growing GT in a canine model.

  • Mobasheri A, Shakibaei M. (2013) 'Osteogenic effects of resveratrol in vitro: potential for the prevention and treatment of osteoporosis.'. Ann N Y Acad Sci, United States: 1290, pp. 59-66.

    Abstract

    There are a number of pharmacological agents for the treatment of bone mineral loss and osteoporosis. Hormone replacement therapy (HRT) with estrogen is an established treatment, but it has several adverse side effects and can increase the risk of cancer, heart disease, and stroke. There is increasing interest in nutritional factors and naturally occurring phytochemical compounds with the potential for preventing age-related and postmenopausal bone loss. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a polyphenolic phytoestrogen with osteogenic and osteoinductive properties. It can modify the metabolism of bone cells and has the capacity to modulate bone turnover. This paper provides an overview of current research on resveratrol and its effects on bone cells in vitro, highlighting the challenges and opportunities facing this area of research, especially in the context of providing nutritional support for postmenopausal women who may not benefit from HRT and older patients with various forms of arthritis, metabolic bone disease, and osteoporosis.

  • Mobasheri A. (2013) 'Exploring the serum proteome in dogs: setting the scene for the discovery of new biomarkers in canine lymphoma.'. Vet J, England: 196 (3), pp. 286-287.
  • Lewis R, Feetham CH, Gentles L, Penny J, Tregilgas L, Tohami W, Mobasheri A, Barrett-Jolley R. (2013) 'Benzamil sensitive ion channels contribute to volume regulation in canine chondrocytes.'. Br J Pharmacol, England: 168 (7), pp. 1584-1596.

    Abstract

    Chondrocytes exist within cartilage and serve to maintain the extracellular matrix. It has been postulated that osteoarthritic (OA) chondrocytes lose the ability to regulate their volume, affecting extracellular matrix production. In previous studies, we identified expression of epithelial sodium channels (ENaC) in human chondrocytes, but their function remained unknown. Although ENaC typically has Na(+) transport roles, it is also involved in the cell volume regulation of rat hepatocytes. ENaC is a member of the degenerin (Deg) family, and ENaC/Deg-like channels have a low conductance and high sensitivity to benzamil. In this study, we investigated whether canine chondrocytes express functional ENaC/Deg-like ion channels and, if so, what their function may be.

  • Henrotin Y, Chevalier X, Herrero-Beaumont G, McAlindon T, Mobasheri A, Pavelka K, Schön C, Weinans H, Biesalski H, Participants at the Hohenheim Consensus Conference in August 29th 2011 . (2013) 'Physiological effects of oral glucosamine on joint health: current status and consensus on future research priorities.'. BMC Res Notes, England: 6

    Abstract

    The aim of this paper was to provide an overview of the current knowledge and understanding of the potential beneficial physiological effects of glucosamine (GlcN) on joint health. The objective was to reach a consensus on four critical questions and to provide recommendations for future research priorities. To this end, nine scientists from Europe and the United States were selected according to their expertise in this particular field and were invited to participate in the Hohenheim conference held in August 2011. Each expert was asked to address a question that had previously been posed by the chairman of the conference. Based on a systematic review of the literature and the collection of recent data, the experts documented the effects of GlcN on cartilage ageing, metabolic/kinetic and maintenance of joint health as well as reduction of risk of OA development. After extensive debate and discussion the expert panel addressed each question and a general consensus statement was developed, agreeing on the current state-of-the-art and future areas for basic and clinical studies. This paper summarizes the available evidence for beneficial effects of GlcN on joint health and proposes new insight into the design of future clinical trials aimed at identifying beneficial physiological effect of GlcN on joint tissues.

  • Mobasheri A, Mendes AF. (2013) 'Physiology and pathophysiology of musculoskeletal aging: current research trends and future priorities.'. Front Physiol, Switzerland: 4
  • Swan AL, Hillier KL, Smith JR, Allaway D, Liddell S, Bacardit J, Mobasheri A. (2013) 'Analysis of mass spectrometry data from the secretome of an explant model of articular cartilage exposed to pro-inflammatory and anti-inflammatory stimuli using machine learning.'. BMC Musculoskelet Disord, England: 14

    Abstract

    Osteoarthritis (OA) is an inflammatory disease of synovial joints involving the loss and degeneration of articular cartilage. The gold standard for evaluating cartilage loss in OA is the measurement of joint space width on standard radiographs. However, in most cases the diagnosis is made well after the onset of the disease, when the symptoms are well established. Identification of early biomarkers of OA can facilitate earlier diagnosis, improve disease monitoring and predict responses to therapeutic interventions.

  • Shakibaei M, Mobasheri A, Lueders C, Busch F, Shayan P, Goel A. (2013) 'Curcumin enhances the effect of chemotherapy against colorectal cancer cells by inhibition of NF-κB and Src protein kinase signaling pathways.'. PLoS One, United States: 8 (2)

    Abstract

    Development of treatment resistance and adverse toxicity associated with classical chemotherapeutic agents highlights the need for safer and effective therapeutic approaches. Herein, we examined the effectiveness of a combination treatment regimen of 5-fluorouracil (5-FU) and curcumin in colorectal cancer (CRC) cells.

  • Williams A, Smith JR, Allaway D, Harris P, Liddell S, Mobasheri A. (2013) 'Carprofen inhibits the release of matrix metalloproteinases 1, 3, and 13 in the secretome of an explant model of articular cartilage stimulated with interleukin 1β.'. Arthritis Res Ther, England: 15 (6)

    Abstract

    Arthritic diseases are characterized by the degradation of collagenous and noncollagenous extracellular matrix (ECM) components in articular cartilage. The increased expression and activity of matrix metalloproteinases (MMPs) is partly responsible for cartilage degradation. This study used proteomics to identify inflammatory proteins and catabolic enzymes released in a serum-free explant model of articular cartilage stimulated with the pro-inflammatory cytokine interleukin 1β (IL-1β). Western blotting was used to quantify the release of selected proteins in the presence or absence of the cyclooxygenase-2 specific nonsteroidal pro-inflammatory drug carprofen.

  • Clutterbuck AL, Allaway D, Harris P, Mobasheri A. (2013) 'Curcumin reduces prostaglandin E2, matrix metalloproteinase-3 and proteoglycan release in the secretome of interleukin 1β-treated articular cartilage.'. F1000Res, England: 2

    Abstract

    Curcumin (diferuloylmethane) is a phytochemical with potent anti-inflammatory and anti-oxidant properties, and has therapeutic potential for the treatment of a range of inflammatory diseases, including osteoarthritis (OA). The aim of this study was to determine whether non-toxic concentrations of curcumin can reduce interleukin-1beta (IL-1β)-stimulated inflammation and catabolism in an explant model of cartilage inflammation.

  • Lorenz W, Buhrmann C, Mobasheri A, Lueders C, Shakibaei M. (2013) 'Bacterial lipopolysaccharides form procollagen-endotoxin complexes that trigger cartilage inflammation and degeneration: implications for the development of rheumatoid arthritis.'. Arthritis Res Ther, England: 15 (5)

    Abstract

    We have previously reported that bacterial toxins, especially endotoxins such as lipopolysaccharides (LPS), might be important causative agents in the pathogenesis of rheumatoid arthritis (RA) in an in vitro model that simulates the potential effects of residing in damp buildings. Since numerous inflammatory processes are linked with the nuclear factor-κB (NF-κB), we investigated in detail the effects of LPS on the NF-κB pathway and the postulated formation of procollagen-endotoxin complexes.

  • Mobasheri A. (2012) 'Osteoarthritis year 2012 in review: biomarkers.'. Osteoarthritis Cartilage, England: 20 (12), pp. 1451-1464.

    Abstract

    Biomarkers provide useful diagnostic information by detecting cartilage degradation in osteoarthritis (OA), reflecting disease-relevant biological activity and predicting the course of disease progression. They also serve as surrogate endpoints in the drug discovery process. The aim of this narrative review was to focus on OA biomarker-related papers published between the osteoarthritis research society international (OARSI) 2011 meeting in San Diego and the OARSI 2012 meeting in Barcelona.

  • Mobasheri A. (2012) 'Intersection of inflammation and herbal medicine in the treatment of osteoarthritis.'. Curr Rheumatol Rep, United States: 14 (6), pp. 604-616.

    Abstract

    Herbal remedies and dietary supplements have become an important area of research and clinical practice in orthopaedics and rheumatology. Understanding the risks and benefits of using herbal medicines in the treatment of arthritis, rheumatic diseases, and musculoskeletal complaints is a key priority of physicians and their patients. This review discusses the latest advances in the use of herbal medicines for treating osteoarthritis (OA) by focusing on the most significant trends and developments. This paper sets the scene by providing a brief introduction to ethnopharmacology, Ayurvedic medicine, and nutrigenomics before discussing the scientific and mechanistic rationale for targeting inflammatory signalling pathways in OA by use of herbal medicines. Special attention is drawn to the conceptual and practical difficulties associated with translating data from in-vitro experiments to in-vivo studies. Issues relating to the low bioavailability of active ingredients in herbal medicines are discussed, as also is the need for large-scale, randomized clinical trials.

  • Busch F, Mobasheri A, Shayan P, Lueders C, Stahlmann R, Shakibaei M. (2012) 'Resveratrol modulates interleukin-1β-induced phosphatidylinositol 3-kinase and nuclear factor κB signaling pathways in human tenocytes.'. J Biol Chem, United States: 287 (45), pp. 38050-38063.

    Abstract

    Resveratrol, an activator of histone deacetylase Sirt-1, has been proposed to have beneficial health effects due to its antioxidant and anti-inflammatory properties. However, the mechanisms underlying the anti-inflammatory effects of resveratrol and the intracellular signaling pathways involved are poorly understood. An in vitro model of human tenocytes was used to examine the mechanism of resveratrol action on IL-1β-mediated inflammatory signaling. Resveratrol suppressed IL-1β-induced activation of NF-κB and PI3K in a dose- and time-dependent manner. Treatment with resveratrol enhanced the production of matrix components collagen types I and III, tenomodulin, and tenogenic transcription factor scleraxis, whereas it inhibited gene products involved in inflammation and apoptosis. IL-1β-induced NF-κB and PI3K activation was inhibited by resveratrol or the inhibitors of PI3K (wortmannin), c-Src (PP1), and Akt (SH-5) through inhibition of IκB kinase, IκBα phosphorylation, and inhibition of nuclear translocation of NF-κB, suggesting that PI3K signaling pathway may be one of the signaling pathways inhibited by resveratrol to abrogate NF-κB activation. Inhibition of PI3K by wortmannin attenuated IL-1β-induced Akt and p65 acetylation, suggesting that p65 is a downstream component of PI3K/Akt in these responses. The modulatory effects of resveratrol on IL-1β-induced activation of NF-κB and PI3K were found to be mediated at least in part by the association between Sirt-1 and scleraxis and deacetylation of NF-κB and PI3K. Overall, these results demonstrate that activated Sirt-1 plays an essential role in the anti-inflammatory effects of resveratrol and this may be mediated at least in part through inhibition/deacetylation of PI3K and NF-κB.

  • Mobasheri A, Lewis R, Ferreira-Mendes A, Rufino A, Dart C, Barrett-Jolley R. (2012) 'Potassium channels in articular chondrocytes.'. Channels (Austin), United States: 6 (6), pp. 416-425.

    Abstract

    Chondrocytes are the resident cells of cartilage, which synthesize and maintain the extracellular matrix. The range of known potassium channels expressed by these unique cells is continually increasing. Since chondrocytes are non-excitable, and do not need to be repolarized following action potentials, the function of potassium channels in these cells has, until recently, remained completely unknown. However, recent advances in both traditional physiology and "omic" technologies have enhanced our knowledge and understanding of the chondrocyte channelome. A large number of potassium channels have been identified and a number of putative, but credible, functions have been proposed. Members of each of the potassium channel sub-families (calcium activated, inward rectifier, voltage-gated and tandem pore) have all been identified. Mechanotransduction, cell volume regulation, apoptosis and chondrogenesis all appear to involve potassium channels. Since evidence suggests that potassium channel gene transcription is altered in osteoarthritis, future studies are needed that investigate potassium channels as potential cellular biomarkers and therapeutic targets for treatment of degenerative joint conditions.

  • Busch F, Mobasheri A, Shayan P, Stahlmann R, Shakibaei M. (2012) 'Sirt-1 is required for the inhibition of apoptosis and inflammatory responses in human tenocytes.'. J Biol Chem, United States: 287 (31), pp. 25770-25781.

    Abstract

    Tendon overuse injuries and tendinitis are accompanied by catabolic processes and apoptosis of tenocytes. However, the precise molecular mechanisms of the destructive processes in tendon are not fully understood. Sirt-1, a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase, has been linked to transcriptional silencing and appears to play a key role in inflammation. The purpose of this study was to examine whether down-regulation of Sirt-1 using antisense oligonucleotides (ASO) affects inflammatory and apoptotic signaling in tenocytes. Transient transfection of tenocytes with ASO against Sirt-1 induced expression of Bax and other proteins involved in apoptosis (cleaved caspase-3 and poly(ADP-ribose)polymerase), acetylation of tumor suppressor p53, and mitochondrial degradation. Interestingly, Sirt-1 was found to interact directly with p53. In contrast, Sirt-1 activator resveratrol inhibited interleukin-1β (IL-1β)- and nicotinamide-induced NF-κB activation and p65 acetylation and suppressed the activation of IκB-α kinase. Resveratrol also reversed the IL-1β- or nicotinamide-induced up-regulation of various gene products that mediate inflammation (cyclooxygenase-2) and matrix degradation (matrix metalloproteinase-9) that are known to be regulated by NF-κB. Knockdown of Sirt-1 by using ASO abolished the inhibitory effects of resveratrol on inflammatory and apoptotic signaling including Akt activation and SCAX suppression. Down-regulation of histone deacetylase Sirt-1 by mRNA interference abrogated the effect of resveratrol on NF-κB suppression, thus highlighting the crucial homeostatic role of this enzyme. Overall, these results suggest for the first time that Sirt-1 can regulate p53 and NF-κB signaling via deacetylation, demonstrating a novel role for resveratrol in the treatment of tendinitis/tendinopathy.

  • Mobasheri A. (2012) 'Identification and phenotypic characterisation of chondroprogenitor cells for the repair of equine articular cartilage.'. Vet J, England: 192 (3), pp. 260-261.
  • Aralla M, Mobasheri A, Groppetti D, Cremonesi F, Arrighi S. (2012) 'Expression of aquaporin water channels in canine fetal adnexa in respect to the regulation of amniotic fluid production and absorption.'. Placenta, England: 33 (6), pp. 502-510.

    Abstract

    Amniotic fluid (AF) is created by the flow of fluid from the fetal lung and bladder and reabsorbed in part by fetal swallowing and partly by the transfer across the amnion to the fetal circulation. Placental water flux is an important factor in determining AF volume and fetal hydration. In addition the fetal membranes might be involved in the regulation of fluid composition. To understand the mechanisms responsible for maintaining a correct balance of AF volume we evaluated the expression of aquaporins (AQPs) in canine fetal adnexa. AQPs are a family of integral membrane proteins permitting passive but physiologically rapid transcellular water movement. The presence of AQP1, 3, 5, 8 and -9 was immunohistochemically assessed in canine fetal adnexa, collected in early, middle and late-gestation during ovario-hysterectomies performed with fully informed owners' consent. Changes in AF volume and biochemical composition were also evaluated throughout pregnancy. Our results show distinct aquaporin expression patterns in maternal and extraembryonic tissues in relation to pregnancy period. AQP1 was localized in placental endothelia, allantochorion, amnion, allantois and yolk sac. AQP3 was present in the placental labyrinth, amnion, allantois and yolk sac. AQP8 was especially evident on the epithelia lining the glandular chambers, the amniotic and allantois sacs. AQP9, a channel highly permeable to water and urea, was observed in epithelia of amnion, allantois and yolk sac. In summary, AQP1, 3, 5, 8 and -9 have distinct expression patterns in canine fetal membranes and placenta in relation to pregnancy period, suggesting an involvement in mediating the AF changes during gestation.

  • Mobasheri A, Kelly S, Buhrmann C, Shakibaei M, Clutterbuck AL. (2012) 'Three-Dimensional, High-Density and Tissue Engineered Culture Models of Articular Cartilage'. Replacing Animal Models: A Practical Guide to Creating and Using Culture-Based Biomimetic Alternatives, , pp. 167-192.
  • Mobasheri A, Moskaluk CA, Marplesc D, Shakibaeid M. (2012) 'The role of aquaporins in human synovitis'. Arthritis: Types, Treatment and Prevention, , pp. 65-93.

    Abstract

    Rheumatoid arthritis (RA) is an autoimmune disorder characterized by synovial proliferation (synovitis), articular cartilage and subchondral bone degradation and synovial inflammation. Joint swelling and oedema often accompany pannus formation and joint chronic inflammation in RA. Clinical evidence suggests that joint swelling and oedema frequently accompany the chronic inflammation observed in synovial joints of RA patients. Although joint swelling is understood to be a major problem in synovitis, very little is known about the molecular mechanisms responsible for the oedema fluid formation that is associated with joint inflammation. Recent studies from our laboratory have shown that articular chondrocytes and synoviocytes express aquaporin 1 (AQP1) water channels. Aquaporins are a family of small integral membrane proteins related to the major intrinsic protein (MIP or AQP0). In recent studies we have used immunohistochemistry to investigate whether the expression of the AQP1 water channel is altered in synovitis. Our data suggests that this membrane protein is upregulated in the synovium derived from RA and psoriatic arthritis patients. In this chapter these observations are discussed in the context of RA and psoriatic arthritis. AQP1 and other aquaporins may play an important role in joint swelling and the vasogenic oedema fluid formation and hydrarthrosis associated with synovial inflammation. © 2012 by Nova Science Publishers, Inc. All rights reserved.

  • Mobasheri A. (2012) 'Mammary glands, aquaporins and milk production'. Milk Production, , pp. 1-12.

    Abstract

    The mammary gland is a specialized, enlarged sudoriferous or sweat (apocrine) gland that produces and secretes milk during lactation. Milk consists of simple sugars, lipids, proteins, vitamins and minerals dissolved in water, which accounts for up to 88% per unit volume of milk. The water content of milk will vary depending on the animal species under investigation and the physiological state of the lactating animal. Current knowledge suggests that water is secreted across the mammary epithelium in a transcellular manner, in response to an osmotic gradient produced largely by the lactose content of milk. Milk yield and quality are important criteria for the dairy industry. Despite the economic importance of milk yield little is known about the physiological mechanisms responsible for water transport in the bovine mammary gland. Recent studies suggest that several aquaporin proteins are present in the rodent, bovine and human mammary gland. Aquaporins play fundamental roles in water and small solute transport across epithelial and endothelial barriers. Immunohistochemical techniques have confirmed the presence of AQP1 and AQP3 water channels in rat, mouse, bovine and human mammary glands. Studies from our laboratory suggest that in addition to AQP1 and AQP3 the AQP4, AQP5 and AQP7 proteins are expressed in different epithelial and endothelial locations in the mammary gland. This chapter discusses the potential functional role of aquaporin water channels in the transport of water and small solutes across endothelial and epithelial barriers in the mammary gland and explores how these membrane proteins may be involved in milk production. © 2012 Nova Science Publishers, Inc. All rights reserved.

  • Mobasheri A, Henrotin Y, Biesalski HK, Shakibaei M. (2012) 'Scientific evidence and rationale for the development of curcumin and resveratrol as nutraceutricals for joint health.'. Int J Mol Sci, Switzerland: 13 (4), pp. 4202-4232.

    Abstract

    Interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) are key cytokines that drive the production of inflammatory mediators and matrix-degrading enzymes in osteoarthritis (OA). These proinflammatory cytokines bind to their respective cell surface receptors and activate inflammatory signaling pathways culminating with the activation of nuclear factor κB (NF-κB), a transcription factor that can be triggered by a host of stress-related stimuli including, excessive mechanical stress and ECM degradation products. Once activated, NF-κB regulates the expression of many cytokines, chemokines, adhesion molecules, inflammatory mediators, and several matrix-degrading enzymes. Therefore, proinflammatory cytokines, their cell surface receptors, NF-κB and downstream signaling pathways are therapeutic targets in OA. This paper critically reviews the recent literature and outlines the potential prophylactic properties of plant-derived phytochemicals such as curcumin and resveratrol for targeting NF-κB signaling and inflammation in OA to determine whether these phytochemicals can be used as functional foods.

  • Penny J, Harris P, Shakesheff KM, Mobasheri A. (2012) 'The biology of equine mesenchymal stem cells: phenotypic characterization, cell surface markers and multilineage differentiation.'. Front Biosci (Landmark Ed), United States: 17, pp. 892-908.

    Abstract

    Mesenchymal stem cells (MSCs) are multipotent stem cells that can give rise to a range of connective tissue cells including osteoblasts, chondrocytes and adipocytes. MSCs have been isolated from humans and a variety of animal species including rodents, dogs, horses and rabbits. There is currently no consensus on how these cells are identified and characterized. This is partly due to the lack of standardized specific cell surface markers for MSCs. The aim of this review is to examine the literature on equine MSCs and establish whether there is a well-defined phenotype for these cells. Equine MSCs have been obtained from four main sources, bone marrow, adipose tissue, umbilical cord (blood and matrix) and peripheral blood. MSCs from these tissue sources have been shown to undergo chondrogenic, adipogenic and osteogenic differentiation. However the markers used to identify these cells vary significantly in the literature. Despite this, CD90 and CD34 seem to be reliable positive and negative markers respectively. Our understanding of the biology of equine MSCs will benefit from better reagents for their phenotypic characterization. The antibodies and molecular probes needed for the reliable identification of equine MSCs are not standardized and this is a high priority for future research.

  • Henrotin Y, Mobasheri A, Marty M. (2012) 'Is there any scientific evidence for the use of glucosamine in the management of human osteoarthritis?'. Arthritis Res Ther, England: 14 (1)

    Abstract

    Glucosamine in its acetylated form is a natural constituent of some glycosaminoglycans (for example, hyaluronic acid and keratan sulfate) in the proteoglycans found in articular cartilage, intervertebral disc and synovial fluid. Glucosamine can be extracted and stabilized by chemical modification and used as a drug or a nutraceutical. It has been approved for the treatment of osteoarthritis (OA) in Europe to promote cartilage and joint health and is sold over the counter as a dietary supplement in the United States. Various formulations of glucosamine have been tested, including glucosamine sulfate and glucosamine hydrochloride. In vitro and in vivo studies have uncovered glucosamine's mechanisms of action on articular tissues (cartilage, synovial membrane and subchondral bone) and justified its efficacy by demonstrating structure-modifying and anti-inflammatory effects at high concentrations. However, results from clinical trials have raised many concerns. Pharmacokinetic studies have shown that glucosamine is easily absorbed, but the current treatment doses (for example, 1,500 mg/day) barely reach the required therapeutic concentration in plasma and tissue. The symptomatic effect size of glucosamine varies greatly depending on the formulation used and the quality of clinical trials. Importantly, the effect size reduces when evidence is accumulated chronologically and evidence for the structure-modifying effects of glucosamine are sparse. Hence, glucosamine was at first recommended by EULAR and OARSI for the management of knee pain and structure improvement in OA patients, but not in the most recent NICE guidelines. Consequently, the published recommendations for the management of OA require revision. Glucosamine is generally safe and although there are concerns about potential allergic and salt-related side effects of some formulations, no major adverse events have been reported so far. This paper examines all the in vitro and in vivo evidence for the mechanism of action of glucosamine as well as reviews the results of clinical trials. The pharmacokinetics, side effects and differences observed with different formulations of glucosamine and combination therapies are also considered. Finally, the importance of study design and criteria of evaluation are highlighted as new compounds represent new interesting options for the management of OA.

  • Gabay O, Mobasheri A. (2012) 'Epigenetics in arthritis'. Current Rheumatology Reviews, 8 (2), pp. 77-79.
  • Mobasheri A, Shakibaei M. (2012) 'The role of proinflammatory cytokines in osteoarthritis: New therapeutic approaches using anti-cytokine therapy and NF-κB inhibitors'. Cytokines: Mechanisms, Functions and Abnormalities, , pp. 147-169.

    Abstract

    Osteoarthritis (OA) is the most common form of joint disease. It is the most common cause of chronic disability in older adults. OA has enormous socioeconomic consequences for healthcare systems in North America, Europe and throughout the rest of the developed world. OA is associated with articular cartilage destruction, subchondral bone remodeling and synovial thickening. Although OA was traditionally viewed as a "wear and tear" degenerative condition of synovial joints, recent studies have demonstrated a significant inflammatory component to the disease, which includes increased expression and activity of several secreted proinflammatory cytokines in the joint tissues that drive production of matrix-degrading enzymes. Interleukin 1ß (IL-1ß) and tumor necrosis factor a (TNF-a) are key cytokines involved in degeneration of articular cartilage matrix, which makes them and their downstream signaling pathways prime targets for novel therapeutic strategies. Apart from IL-1ß and TNF-a, several other cytokines and chemokines including IL-6, IL-8 and IL-17 are implicated in OA. These proinflammatory cytokines bind to their respective cell surface receptors and activate inflammatory signaling pathways culminating with the activation of nuclear factor κB (NF-κB) a transcription factor that can be triggered by stress-related stimuli, including excessive mechanical stress and extracellular matrix (ECM) degradation products. Once activated, NF-κB regulates the expression of many cytokines, chemokines, adhesion molecules, inflammatory mediators, and several matrix-degrading enzymes. Therefore, proinflammatory cytokines and their cell surface receptors, as well as NF-κB and associated signaling pathways can be targeted in OA. This chapter reviews the current state of play regarding the role of proinflammatory cytokines in the pathophysiology of OA, and addresses the potential of anti-cytokine and anti-NF-κB therapy in the treatment of this disease. We also discuss the potential for complementary therapies using natural products such as curcumin and resveratrol. © 2012 by Nova Science Publishers, Inc. All rights reserved.

  • Henrotin Y, Mobasheri A. (2012) 'Soluble biomarkers in osteoarthritis - current status and future prospects?'. European Musculoskeletal Review, 7 (4), pp. 217-220.

    Abstract

    There is an acute need for reliable soluble biomarkers that can facilitate earlier diagnosis of osteoarthritis (OA), inform the prognosis and monitor and predict the responses of patients to therapeutic interventions. The number of biomarkers that have been validated and qualified is relatively small. This article summarises the current state of play in the field of OA soluble biomarkers. It discusses the barriers that prevent biomarkers from achieving their full potential and provides some perspectives for basic researchers and clinicians. © Touch Medical Media 2012.

  • Mobasheri A. (2012) 'Glucose: an energy currency and structural precursor in articular cartilage and bone with emerging roles as an extracellular signaling molecule and metabolic regulator.'. Front Endocrinol (Lausanne), Switzerland: 3

    Abstract

    In the skeletal system glucose serves as an essential source of energy for the development, growth, and maintenance of bone and articular cartilage. It is particularly needed for skeletal morphogenesis during embryonic growth and fetal development. Glucose is vital for osteogenesis and chondrogenesis, and is used as a precursor for the synthesis of glycosaminoglycans, glycoproteins, and glycolipids. Glucose sensors are present in tissues and organs that carry out bulk glucose fluxes (i.e., intestine, kidney, and liver). The beta cells of the pancreatic islets of Langerhans respond to changes in blood glucose concentration by varying the rate of insulin synthesis and secretion. Neuronal cells in the hypothalamus are also capable of sensing extracellular glucose. Glucosensing neurons use glucose as a signaling molecule to alter their action potential frequency in response to variations in ambient glucose levels. Skeletal muscle and adipose tissue can respond to changes in circulating glucose but much less is known about glucosensing in bone and cartilage. Recent research suggests that bone cells can influence (and be influenced by) systemic glucose metabolism. This focused review article discusses what we know about glucose transport and metabolism in bone and cartilage and highlights recent studies that have linked glucose metabolism, insulin signaling, and osteocalcin activity in bone. These new findings in bone cells raise important questions about nutrient sensing, uptake, storage and processing mechanisms and how they might contribute to overall energy homeostasis in health and disease. The role of glucose in modulating anabolic and catabolic gene expression in normal and osteoarthritic chondrocytes is also discussed. In summary, cartilage and bone cells are sensitive to extracellular glucose and adjust their gene expression and metabolism in response to varying extracellular glucose concentrations.

  • Mobasheri A, Asplin K, Clutterbuck A, Shakibaei M. (2012) 'Nutraceuticals: From research to legal and regulatory affairs'. Nonpharmacological Therapies in the Management of Osteoarthritis, , pp. 115-126.

    Abstract

    In this chapter we define the term "nutraceutical" and its relevance to arthritic diseases, and summarize recent research on nutraceuticals for joint disorders, particularly osteoarthritis (OA). The nutraceutical industry is not regulated. Consequently there are concerns about the purity, labeling and advertising of nutraceuticals. Manufacturers of nutraceuticals have a duty to communicate the benefits of their products with supporting scientific and clinical evidence. Nutraceutical products should be clearly labelled and advertising campaigns should be truthful and well balanced. The public and the scientific community require greater transparency and uniformity of commercially produced nutraceuticals. Consumers need to be able to make an informed choice about nutraceuticals based on evidence. We advocate greater regulation and regular independent testing of these products in order to ensure uniformity and greater reliability. The intended audience for this article include clinicians, basic researchers, producers of nutraceuticals and functional foods and advertising and marketing companies worldwide, particularly multi-national companies requiring information on issues relating to nutraceutical regulation. © 2012 Bentham Science Publishers. All rights reserved.

  • Shakibaei M, Shayan P, Busch F, Aldinger C, Buhrmann C, Lueders C, Mobasheri A. (2012) 'Resveratrol mediated modulation of Sirt-1/Runx2 promotes osteogenic differentiation of mesenchymal stem cells: potential role of Runx2 deacetylation.'. PLoS One, United States: 7 (4)

    Abstract

    Osteogenic repair in response to bone injury is characterized by activation and differentiation of mesenchymal stem cells (MSCs) to osteoblasts. This study determined whether activation of Sirt-1 (a NAD(+)-dependent histone deacetylase) by the phytoestrogen resveratrol affects osteogenic differentiation.

  • Hdud IM, El-Shafei AA, Loughna P, Barrett-Jolley R, Mobasheri A. (2012) 'Expression of Transient Receptor Potential Vanilloid (TRPV) Channels in Different Passages of Articular Chondrocytes.'. Int J Mol Sci, Switzerland: 13 (4), pp. 4433-4445.

    Abstract

    Ion channels play important roles in chondrocyte mechanotransduction. The transient receptor potential vanilloid (TRPV) subfamily of ion channels consists of six members. TRPV1-4 are temperature sensitive calcium-permeable, relatively non-selective cation channels whereas TRPV5 and TRPV6 show high selectivity for calcium over other cations. In this study we investigated the effect of time in culture and passage number on the expression of TRPV4, TRPV5 and TRPV6 in articular chondrocytes isolated from equine metacarpophalangeal joints. Polyclonal antibodies raised against TRPV4, TRPV5 and TRPV6 were used to compare the expression of these channels in lysates from first expansion chondrocytes (P0) and cells from passages 1-3 (P1, P2 and P3) by western blotting. TRPV4, TRPV5 and TRPV6 were expressed in all passages examined. Immunohistochemistry and immunofluorescence confirmed the presence of these channels in sections of formalin fixed articular cartilage and monolayer cultures of methanol fixed P2 chondrocytes. TRPV5 and TRPV6 were upregulated with time and passage in culture suggesting that a shift in the phenotype of the cells in monolayer culture alters the expression of these channels. In conclusion, several TRPV channels are likely to be involved in calcium signaling and homeostasis in chondrocytes.

  • Shakibaei M, Allaway D, Nebrich S, Mobasheri A. (2012) 'Botanical Extracts from Rosehip (Rosa canina), Willow Bark (Salix alba), and Nettle Leaf (Urtica dioica) Suppress IL-1β-Induced NF-κB Activation in Canine Articular Chondrocytes.'. Evid Based Complement Alternat Med, United States: 2012

    Abstract

    The aim of this study was to characterize the anti-inflammatory mode of action of botanical extracts from rosehip (Rosa canina), willow bark (Salix alba), and nettle leaf (Urtica dioica) in an in vitro model of primary canine articular chondrocytes. Methods. The biological effects of the botanical extracts were studied in chondrocytes treated with IL-1β for up to 72 h. Expression of collagen type II, cartilage-specific proteoglycan (CSPG), β1-integrin, SOX-9, COX-2, and MMP-9 and MMP-13 was examined by western blotting. Results. The botanical extracts suppressed IL-1β-induced NF-κB activation by inhibition of IκBα phosphorylation, IκBα degradation, p65 phosphorylation, and p65 nuclear translocation. These events correlated with downregulation of NF-κB targets including COX-2 and MMPs. The extracts also reversed the IL-1β-induced downregulation of collagen type II, CSPG, β1-integrin, and cartilage-specific transcription factor SOX-9 protein expression. In high-density cultures botanical extracts stimulated new cartilage formation even in the presence of IL-1β. Conclusions. Botanical extracts exerted anti-inflammatory and anabolic effects on chondrocytes. The observed reduction of IL-1β-induced NF-κB activation suggests that further studies are warranted to demonstrate the effectiveness of plant extracts in the treatment of OA and other conditions in which NF-κB plays pathophysiological roles.

  • Mobasheri A, Trujillo E, Arteaga MF, Martín-Vasallo P. (2012) 'Na(+), K(+)-ATPase Subunit Composition in a Human Chondrocyte Cell Line; Evidence for the Presence of α1, α3, β1, β2 and β3 Isoforms.'. Int J Mol Sci, Switzerland: 13 (4), pp. 5019-5034.

    Abstract

    Membrane transport systems participate in fundamental activities such as cell cycle control, proliferation, survival, volume regulation, pH maintenance and regulation of extracellular matrix synthesis. Multiple isoforms of Na(+), K(+)-ATPase are expressed in primary chondrocytes. Some of these isoforms have previously been reported to be expressed exclusively in electrically excitable cells (i.e., cardiomyocytes and neurons). Studying the distribution of Na(+), K(+)-ATPase isoforms in chondrocytes makes it possible to document the diversity of isozyme pairing and to clarify issues concerning Na(+), K(+)-ATPase isoform abundance and the physiological relevance of their expression. In this study, we investigated the expression of Na(+), K(+)-ATPase in a human chondrocyte cell line (C-20/A4) using a combination of immunological and biochemical techniques. A panel of well-characterized antibodies revealed abundant expression of the α1, β1 and β2 isoforms. Western blot analysis of plasma membranes confirmed the above findings. Na(+), K(+)-ATPase consists of multiple isozyme variants that endow chondrocytes with additional homeostatic control capabilities. In terms of Na(+), K(+)-ATPase expression, the C-20/A4 cell line is phenotypically similar to primary and in situ chondrocytes. However, unlike freshly isolated chondrocytes, C-20/A4 cells are an easily accessible and convenient in vitro model for the study of Na(+), K(+)-ATPase expression and regulation in chondrocytes.

  • Lewis R, Asplin KE, Bruce G, Dart C, Mobasheri A, Barrett-Jolley R. (2011) 'The role of the membrane potential in chondrocyte volume regulation.'. J Cell Physiol, United States: 226 (11), pp. 2979-2986.

    Abstract

    Many cell types have significant negative resting membrane potentials (RMPs) resulting from the activity of potassium-selective and chloride-selective ion channels. In excitable cells, such as neurones, rapid changes in membrane permeability underlie the generation of action potentials. Chondrocytes have less negative RMPs and the role of the RMP is not clear. Here we examine the basis of the chondrocyte RMP and possible physiological benefits. We demonstrate that maintenance of the chondrocyte RMP involves gadolinium-sensitive cation channels. Pharmacological inhibition of these channels causes the RMP to become more negative (100 µM gadolinium: ΔV(m)  = -30 ± 4 mV). Analysis of the gadolinium-sensitive conductance reveals a high permeability to calcium ions (PCa/PNa ≈80) with little selectivity between monovalent ions; similar to that reported elsewhere for TRPV5. Detection of TRPV5 by PCR and immunohistochemistry and the sensitivity of the RMP to the TRPV5 inhibitor econazole (ΔV(m)  = -18 ± 3 mV) suggests that the RMP may be, in part, controlled by TRPV5. We investigated the physiological advantage of the relatively positive RMP using a mathematical model in which membrane stretch activates potassium channels allowing potassium efflux to oppose osmotic water uptake. At very negative RMP potassium efflux is negligible, but at more positive RMP it is sufficient to limit volume increase. In support of our model, cells clamped at -80 mV and challenged with a reduced osmotic potential swelled approximately twice as much as cells at +10 mV. The positive RMP may be a protective adaptation that allows chondrocytes to respond to the dramatic osmotic changes, with minimal changes in cell volume.

  • Medina-Torres CE, Mason SL, Floyd RV, Harris PA, Mobasheri A. (2011) 'Hypoxia and a hypoxia mimetic up-regulate matrix metalloproteinase 2 and 9 in equine laminar keratinocytes.'. Vet J, England: 190 (2), pp. e54-e59.

    Abstract

    The aim of this study was to determine if hypoxia and the hypoxia mimetic cobalt chloride regulate the activity of matrix metalloproteinase (MMP)-2 and -9 in cultures of equine hoof keratinocytes. These effects were assessed in primary cultures of laminar keratinocytes using gelatin zymography. Incubation of keratinocytes with cobalt chloride significantly increased the levels of active MMP-2 compared to untreated controls. Hypoxia significantly increased the expression of active MMP-2 and -9 in keratinocyte cultures. This up-regulation was observed after 6h and peaked at 24h. The study findings provide novel evidence of a potential link between hypoxia within the hoof and up-regulation of MMPs which may in turn result in damage to the lamellar basement membrane.

  • Mobasheri A. (2011) 'Identification and validation of early biomarkers of osteoarthritis in companion animals: are we ready for the challenge?'. Vet J, England: 190 (1), pp. 1-4.
  • Shakibaei M, Buhrmann C, Mobasheri A. (2011) 'Anti-inflammatory and anti-catabolic effects of TENDOACTIVE® on human tenocytes in vitro.'. Histol Histopathol, Spain: 26 (9), pp. 1173-1185.

    Abstract

    Tendons have a limited capacity for self-repair due to the low density and mitotic activity of tenocytes. Pro-inflammatory cytokines such as interleukin-1β (IL-1β) have been identified as the main initiators of tendinopathies, stimulating inflammation, apoptosis and extracellular matrix (ECM) degradation. The aim of this study was to evaluate the potential of Tendoactive®, a newly developed proprietary nutraceutical formulation that includes mucopolysaccharides, collagen and vitamin C, in an in vitro model of tendon inflammation. The effects of Tendoactive® were studied in primary cultures of human tenocytes treated with IL-1β for up to 72 h. Expression of collagen type I, integrin β1, cyclo-oxygenase-2 (COX-2), caspase-3 and matrix metalloproteinase-1 (MMP-1) was monitored by western blotting. The effects of Tendoactive® on the expression, phosphorylation and nuclear translocation of protein components of the NF-κB system were studied by western blotting and immunofluorescence respectively. Treatment of tenocytes with Tendoactive® suppressed IL-1β-induced NF-κB activation and p65 nuclear translocation. These events correlated with down-regulation of NF-κB targets including COX-2, MMP-1 and activated caspase-3. Tendoactive® also reversed the IL-1β-induced down-regulation of collagen type I and β1-integrin receptor expression. These results indicate that Tendoactive® has nutraceutical potential as an anti-inflammatory agent for treating tendinopathy through suppression of NF-κB mediated IL-1β catabolic signalling pathways in tenocytes.

  • Schneider PR, Buhrmann C, Mobasheri A, Matis U, Shakibaei M. (2011) 'Three-dimensional high-density co-culture with primary tenocytes induces tenogenic differentiation in mesenchymal stem cells.'. J Orthop Res, United States: 29 (9), pp. 1351-1360.

    Abstract

    Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and tissue engineering and may represent an attractive option for tendon repair and regeneration. Thus far the ability of MSCs to differentiate into tenocytes in vitro has not been investigated. Experiments were performed with and without growth factors (IGF-1, TGF-β1, IGF-1/TGF-β1, PDGF-BB, and BMP-12), in co-cultures of tenocytes and MSCs mixed in different ratios and by culturing MSCs with spent media obtained from primary tenocytes. Tenogenesis was induced in MSCs through a combination of treatment with IGF-1 and TGF-β1, in high-density co-cultures and through cultivation with the spent media from primary tenocytes. Electron microscopy and immunoblotting were used to demonstrate up-regulation of collagen I/III, decorin, tenomodulin, β1-Integrin, MAPKinase pathway (Shc, Erk1/2), and scleraxis in the co-cultures and provide simultaneous evidence for the inhibition of apoptosis. In monolayer co-cultures extensive intercellular contacts between MSCs and tenocytes were observed. Cells actively exchanged vesicles, which were labeled by using immunofluorescence and immunogold techniques, suggesting the uptake and interchange of soluble factors produced by the MSCs and/or tenocytes. We conclude that MSCs possess tenogenic differentiation potential when provided with relevant stimuli and a suitable microenvironment. This approach may prove to be of practical benefit in future tissue engineering and tendon regenerative medicine research.

  • Buhrmann C, Mobasheri A, Busch F, Aldinger C, Stahlmann R, Montaseri A, Shakibaei M. (2011) 'Curcumin modulates nuclear factor kappaB (NF-kappaB)-mediated inflammation in human tenocytes in vitro: role of the phosphatidylinositol 3-kinase/Akt pathway.'. J Biol Chem, United States: 286 (32), pp. 28556-28566.

    Abstract

    Inflammatory processes play essential roles in the pathogenesis of tendinitis and tendinopathy. These events are accompanied by catabolic processes initiated by pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Pharmacological treatments for tendinitis are restricted to the use of non-steroidal anti-inflammatory drugs. Recent studies in various cell models have demonstrated that curcumin targets the NF-κB signaling pathway. However, its potential for the treatment of tendinitis has not been explored. Herein, we used an in vitro model of human tenocytes to study the mechanism of curcumin action on IL-1β-mediated inflammatory signaling. Curcumin at concentrations of 5-20 μm inhibited IL-1β-induced inflammation and apoptosis in cultures of human tenocytes. The anti-inflammatory effects of curcumin included down-regulation of gene products that mediate matrix degradation (matrix metalloproteinase-1, -9, and -13), prostanoid production (cyclooxygenase-2), apoptosis (Bax and activated caspase-3), and stimulation of cell survival (Bcl-2), all known to be regulated by NF-κB. Furthermore, curcumin suppressed IL-1β-induced NF-κB activation via inhibition of phosphorylation and degradation of inhibitor of κBα, inhibition of inhibitor of κB-kinase activity, and inhibition of nuclear translocation of NF-κB. Furthermore, the effects of IL-1β were abrogated by wortmannin, suggesting a role for the phosphatidylinositol 3-kinase (PI-3K) pathway in IL-1β signaling. Curcumin suppressed IL-1β-induced PI-3K p85/Akt activation and its association with IKK. These results demonstrate, for the first time, a potential role for curcumin in treating tendon inflammation through modulation of NF-κB signaling, which involves PI-3K/Akt and the tendon-specific transcription factor scleraxis in tenocytes.

  • Shakibaei M, Mobasheri A, Buhrmann C. (2011) 'Curcumin synergizes with resveratrol to stimulate the MAPK signaling pathway in human articular chondrocytes in vitro.'. Genes Nutr, Germany: 6 (2), pp. 171-179.

    Abstract

    The mitogen-activated protein kinase (MAPK) pathway is stimulated in differentiated chondrocytes and is an important signaling cascade for chondrocyte differentiation and survival. Pro-inflammatory cytokines such as interleukin 1β (IL-1β) play important roles in the pathogenesis of osteoarthritis (OA) and rheumatoid arthritis (RA). In this study, we investigated whether curcumin and resveratrol can synergistically inhibit the catabolic effects of IL-1β, specifically the inhibition of the MAPK and subsequent apoptosis in human articular chondrocytes. Chondrocytes were either left untreated or treated with 10 ng/ml IL-1β or 1 μM U0126, a specific inhibitor of MAPK pathway alone for the indicated time periods or pre-treated with 10 μM curcumin, 10 μM resveratrol or 10 μM resveratrol and 10 μM curcumin for 4 h followed by co-treatment with 10 ng/ml IL-1β or 1 μM U0126 and 10 μM resveratrol, 10 μM curcumin or 10 μM resveratrol and 10 μM curcumin for the indicated time periods. Cultures were evaluated by immunoblotting and transmission electron microscopy. Incubation of chondrocytes with IL-1β resulted in induction of apoptosis, downregulation of β1-integrins and the extracellular signal-regulated kinase 1/2 (Erk1/2). Interestingly, U0126 induced apoptosis and blocked the above-mentioned proteins in a similar way to IL-1β. Furthermore, curcumin and resveratrol inhibited IL-1β- or U0126-induced apoptosis and downregulation of β1-integrins and Erk1/2 in human articular chondrocytes. These results suggest that combining these two natural compounds activates MEK/Erk signaling, a pathway that is involved in the maintenance of chondrocyte differentiation and survival.

  • Clutterbuck AL, Smith JR, Allaway D, Harris P, Liddell S, Mobasheri A. (2011) 'High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation.'. J Proteomics, Netherlands: 74 (5), pp. 704-715.

    Abstract

    This study employed a targeted high-throughput proteomic approach to identify the major proteins present in the secretome of articular cartilage. Explants from equine metacarpophalangeal joints were incubated alone or with interleukin-1beta (IL-1β, 10ng/ml), with or without carprofen, a non-steroidal anti-inflammatory drug, for six days. After tryptic digestion of culture medium supernatants, resulting peptides were separated by HPLC and detected in a Bruker amaZon ion trap instrument. The five most abundant peptides in each MS scan were fragmented and the fragmentation patterns compared to mammalian entries in the Swiss-Prot database, using the Mascot search engine. Tryptic peptides originating from aggrecan core protein, cartilage oligomeric matrix protein (COMP), fibronectin, fibromodulin, thrombospondin-1 (TSP-1), clusterin (CLU), cartilage intermediate layer protein-1 (CILP-1), chondroadherin (CHAD) and matrix metalloproteinases MMP-1 and MMP-3 were detected. Quantitative western blotting confirmed the presence of CILP-1, CLU, MMP-1, MMP-3 and TSP-1. Treatment with IL-1β increased MMP-1, MMP-3 and TSP-1 and decreased the CLU precursor but did not affect CILP-1 and CLU levels. Many of the proteins identified have well-established extracellular matrix functions and are involved in early repair/stress responses in cartilage. This high throughput approach may be used to study the changes that occur in the early stages of osteoarthritis.

  • Shakibaei M, Buhrmann C, Mobasheri A. (2011) 'Resveratrol-mediated SIRT-1 interactions with p300 modulate receptor activator of NF-kappaB ligand (RANKL) activation of NF-kappaB signaling and inhibit osteoclastogenesis in bone-derived cells.'. J Biol Chem, United States: 286 (13), pp. 11492-11505.

    Abstract

    Resveratrol is a polyphenolic phytoestrogen that has been shown to exhibit potent anti-oxidant, anti-inflammatory, and anti-catabolic properties. Increased osteoclastic and decreased osteoblastic activities result in bone resorption and loss of bone mass. These changes have been implicated in pathological processes in rheumatoid arthritis and osteoporosis. Receptor activator of NF-κB ligand (RANKL), a member of the TNF superfamily, is a major mediator of bone loss. In this study, we investigated the effects of resveratrol on RANKL during bone morphogenesis in high density bone cultures in vitro. Untreated bone-derived cell cultures produced well organized bone-like structures with a bone-specific matrix. Treatment with RANKL induced formation of tartrate-resistant acid phosphatase-positive multinucleated cells that exhibited morphological features of osteoclasts. RANKL induced NF-κB activation, whereas pretreatment with resveratrol completely inhibited this activation and suppressed the activation of IκBα kinase and IκBα phosphorylation and degradation. RANKL up-regulated p300 (a histone acetyltransferase) expression, which, in turn, promoted acetylation of NF-κB. Resveratrol inhibited RANKL-induced acetylation and nuclear translocation of NF-κB in a time- and concentration-dependent manner. In addition, activation of Sirt-1 (a histone deacetylase) by resveratrol induced Sirt-1-p300 association in bone-derived and preosteoblastic cells, leading to deacetylation of RANKL-induced NF-κB, inhibition of NF-κB transcriptional activation, and osteoclastogenesis. Co-treatment with resveratrol activated the bone transcription factors Cbfa-1 and Sirt-1 and induced the formation of Sirt-1-Cbfa-1 complexes. Overall, these results demonstrate that resveratrol-activated Sirt-1 plays pivotal roles in regulating the balance between the osteoclastic versus osteoblastic activity result in bone formation in vitro thereby highlighting its therapeutic potential for treating osteoporosis and rheumatoid arthritis-related bone loss.

  • Mobasheri A, Kendall BH, Maxwell JE, Sawran AV, German AJ, Marples D, Luck MR, Royal MD. (2011) 'Cellular localization of aquaporins along the secretory pathway of the lactating bovine mammary gland: an immunohistochemical study.'. Acta Histochem, Germany: 113 (2), pp. 137-149.

    Abstract

    In this study we examined the cellular localization of aquaporins (AQPs) along the secretory pathway of actively lactating bovine mammary glands using immunohistochemistry. Mammary tissues examined included secretory ducts and acini, gland cisterns, teats, stromal and adipose tissues. Aquaporin 1 (AQP1) was localized in capillary endothelia throughout the mammary gland in addition to myoepithelial cells underlying teat duct epithelia. AQP2 and AQP6 were not detected and AQP9 was found only in leukocytes. AQP3 and AQP4 were observed in selected epithelial cells in the teat, cistern and secretory tubuloalveoli. AQP5 immunopositivity was prominent in the cistern. AQP3 and AQP7 were found in smooth muscle bundles in the teat, secretory epithelial cells and duct epithelial cells. These immunohistochemical findings support a functional role for aquaporins in the transport of water and small solutes across endothelial and epithelial barriers in the mammary gland and in the production and secretion of milk.

  • Mobasheri A, Barrett-Jolley R. (2011) 'Transient receptor potential channels: emerging roles in health and disease.'. Vet J, England: 187 (2), pp. 145-146.
  • Ghuman SP, Morris R, Scherzer J, Routly JE, Mobasheri A, Smith RF, Dobson H. (2011) 'Neuronal responses in the brainstem and hypothalamic nuclei following insulin treatment during the late follicular phase in the ewe.'. Reprod Domest Anim, Germany: 46 (1), pp. 121-129.

    Abstract

    The aim of this study was to determine the neuronal responses following insulin administration during the late follicular phase. Intact ewes were given either saline or insulin (5 IU/kg, i.v.) at 35 h after progesterone withdrawal and killed 3 h later. There was a marked increase in the number of Fos-positive noradrenergic neurones in the caudal brainstem of insulin-treated ewes. In the hypothalamic paraventricular nucleus, insulin treatment increased the presence of Fos-positive corticotrophin-releasing hormone neurones (from 2% to 98%) and Fos-positive arginine vasopressin parvocellular neurones (from 2% to 46%). Interestingly, after insulin treatment, despite a general increase in Fos-positive neurones in the arcuate nucleus (ARC), there was a marked reduction (from 47% to 1%) in Fos-positive β-endorphin neurones. Similarly, colocalized Fos and oestradiol receptor (ER) α-positive neurones decreased in the ARC after insulin (from 7% to 3%). Conversely, in the ventromedial nucleus, ERα-positive neurones with Fos increased (from 7% to 22%) alongside a general increase in Fos-positive neurones. Overall, a complex system of neurones in brainstem and hypothalamus is activated following insulin administration during the late follicular phase.

  • Mobasheri A. (2011) 'Applications of proteomics to osteoarthritis, a musculoskeletal disease characterized by aging.'. Front Physiol, Switzerland: 2

    Abstract

    The incidence of age-related musculoskeletal impairment is steadily rising throughout the world. Musculoskeletal conditions are closely linked with aging and inflammation. They are leading causes of morbidity and disability in man and beast. Aging is a major contributor to musculoskeletal degeneration and the development of osteoarthritis (OA). OA is a degenerative disease that involves structural changes to joint tissues including synovial inflammation, catabolic destruction of articular cartilage and alterations in subchondral bone. Cartilage degradation and structural changes in subchondral bone result in the production of fragments of extracellular matrix molecules. Some of these biochemical markers or "biomarkers" can be detected in blood, serum, synovial fluid, and urine and may be useful markers of disease progression. The ability to detect biomarkers of cartilage degradation in body fluids may enable clinicians to diagnose sub-clinical OA as well as determining the course of disease progression. New biomarkers that indicate early responses of the joint cartilage to degeneration will be useful in detecting early, pre-radiographic changes. Systems biology is increasingly applied in basic cartilage biology and OA research. Proteomic techniques have the potential to improve our understanding of OA physiopathology and its underlying mechanisms. Proteomics can also facilitate the discovery of disease-specific biomarkers and help identify new therapeutic targets. Proteomic studies of cartilage and other joint tissues may be particularly relevant in diagnostic orthopedics and therapeutic research. This perspective article discusses the relevance and potential of proteomics for studying age-related musculoskeletal diseases such as OA and reviews the contributions of key investigators in the field.

  • Mobasheri A, Clutterbuck AL, Henrotin Y, Mathy-Hartert M, Allaway D, Lodwig EM, Harris P, Shakibaei M. (2011) 'Nutritional biochemistry of curcumin (diferuloylmethane) and a review of its biological actions on articular chondrocytes'. Handbook of Nutritional Biochemistry: Genomics, Metabolomics, and Food Supply, , pp. 289-315.

    Abstract

    Curcumin (diferuloylmethane) is the principle biochemical component of the spice turmeric and has been shown to possess potent anti-catabolic, anti-inflammatory and anti-oxidant, properties. In this chapter we provide a brief review of osteoarthritis (OA) and discuss the anti-inflammatory properties of curcumin in the context of its actions on articular chondrocytes. We highlight its potential benefits and drawbacks for treating OA and related osteoarticular conditions. Recent work has shown that curcumin protects human chondrocytes from the catabolic actions of interleukin (IL)-1 beta (IL-1) including matrix metalloproteinase-3 (MMP-3) upregulation, inhibition of collagen type II and down-regulation of β1-integrin expression. Curcumin blocks IL-1-induced proteoglycan degradation, Nuclear Factor Kappa B (NF-B) signaling, chondrocyte apoptosis and activation of caspase-3. The available data from in vitro and in vivo studies suggest that curcumin may be a beneficial complementary treatment for OA in humans and companion animals. Nevertheless, before initiating extensive clinical trials, more basic research is required to improve its absorption and bioavailability and gain additional information about its safety and efficacy in different species. Once these obstacles have been overcome, curcumin and structurally related biochemicals could become suitable nutritional and nutraceutical alternatives to the non-steroidal anti-inflammatory drugs that are currently used for the treatment of OA. © 2010 Nova Science Publishers, Inc. All rights reserved.

  • Acosta E, Avila J, Mobasheri A, Martin-Vasallo P. (2011) 'Na+, K+-ATPase genes are down-regulated during adipose stem cell differentiation.'. Front Biosci (Elite Ed), United States: 3, pp. 1229-1240.

    Abstract

    The expression of Na+, K+-ATPase alpha and beta subunits isoforms, FXYD2 and FXYD7 were studied in rat adipose stem cell (ASC) by qRT-PCR and immunofluorescence. ASCs were able to differentiate to chondrocytes or adipocytes. All studied genes were expressed in freshly isolated ASCs and in all passages checked. Immunostaining for alpha1 isoform was found in plasma membrane and nuclear envelope, alpha2 signal was lower and alpha3 staining was variable among cells. Beta isoforms signal was abundant and displayed an isoform-specific picture. Staining for FXYD7 was homogeneous in plasma membrane and cytosol. Chondrocytes differenciated from ASC showed identical Na+, K+-ATPase subunits isoforms expression patterns to chondrocytes in cartilage. The expression pattern of Na+, K+-ATPase genes in ASCs exhibits a unique phenotypic signature that implies functional differences in Na+ and K+ transport rates. Furthermore, this phenotypic signature may also be used as a complementary marker for studies of mesenchymal stem cell differentiation. We propose a possible 'moonlighting' role of Na+, K+-ATPase beta isoforms that could be essential for the study of mesenchymal stem cell function and differentiation.

  • Mobasheri A. (2011) 'Proteomic profiling and its applications to muscle aging and sarcopenia.'. Front Physiol, Switzerland: 2
  • Williams A, Smith JR, Allaway D, Harris P, Liddell S, Mobasheri A. (2011) 'Applications of proteomics in cartilage biology and osteoarthritis research.'. Front Biosci (Landmark Ed), United States: 16, pp. 2622-2644.

    Abstract

    In osteoarthritis (OA) the turnover of extracellular matrix (ECM) macromolecules is disrupted by catabolic changes that lead to the production of a range of inflammatory mediators and the loss and fragmentation of proteoglycans, fibrillar and non-fibrillar collagens. These events result in the degradation and release of ECM fragments, which are potential biomarkers that can be detected in synovial fluid, blood and urine. Proteomics is increasingly applied in cartilage research and has the potential to advance our understanding of the biology of this tissue. It can also provide mechanistic insight into disease pathogenesis and progression and facilitate biomarker discovery. Here we review the area of cartilage and chondrocyte proteomics and published studies relevant to arthritis and OA biomarkers, highlighting areas of current and future research and development. Markers of tissue turnover in joints have the capacity to reflect disease-relevant biological activity potentially enabling a more rational approach to healthcare management. Therefore proteomic studies of cartilage, chondrocytes and their subcellular fractions and other joint cells and tissues may be particularly relevant in diagnostic orthopedics and therapeutic research.

  • Montaseri A, Busch F, Mobasheri A, Buhrmann C, Aldinger C, Rad JS, Shakibaei M. (2011) 'IGF-1 and PDGF-bb suppress IL-1β-induced cartilage degradation through down-regulation of NF-κB signaling: involvement of Src/PI-3K/AKT pathway.'. PLoS One, United States: 6 (12)

    Abstract

    Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that plays a key role in the pathogenesis of osteoarthritis (OA). Growth factors (GFs) capable of antagonizing the catabolic actions of cytokines may have therapeutic potential in the treatment of OA. Herein, we investigated the potential synergistic effects of insulin-like growth factor (IGF-1) and platelet-derived growth factor (PDGF-bb) on different mechanisms participating in IL-1β-induced activation of nuclear transcription factor-κB (NF-κB) and apoptosis in chondrocytes.

  • Mobasheri A, Shakibaei M. (2011) 'Applications of mesenchymal stem cells in cartilage tissue engineering-part 1'. Recent Patents on Regenerative Medicine, 1 (1), pp. 30-41.

    Abstract

    Arthritic diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA) cause considerable pain, reduced mobility and significant disability among affected patients and present a major challenge to clinicians and basic scientists due to the limited inherent repair capacity of articular cartilage. The poor capacity of articular cartilage for self-repair is largely due to its avascular nature and has resulted in the development of a variety of surgical treatments including Autologous Chondrocyte Implantation (ACI) or Autologous Chondrocyte Transplantation (ACT), microfracture and mosaicplasty. Mesenchymal stem cells (MSCs) are multipotent progenitor cells with significant potential for chondrogenesis and new cartilage formation. Novel approaches using MSCs derived from bone marrow and adipose tissue have been proposed as alternatives to patient derived chondrocytes. In this paper we provide a scientific background to the biology of articular cartilage biology and its degeneration in arthritis. We also summarize some of the recent patents on applications of MSCs in articular cartilage tissue engineering and regenerative medicine for OA, RA and other joint diseases that involve cartilage degradation. © 2011 Bentham Science Publishers Ltd.

  • Graham NS, Clutterbuck AL, James N, Lea RG, Mobasheri A, Broadley MR, May ST. (2010) 'Equine transcriptome quantification using human GeneChip arrays can be improved using genomic DNA hybridisation and probe selection.'. Vet J, England: 186 (3), pp. 323-327.

    Abstract

    Affymetrix GeneChip arrays are a powerful tool for transcriptome profiling and have been applied to a wide range of species. A genomic DNA (gDNA)-based probe selection method has been developed which broadens the range of species to which GeneChips may be successfully applied. This study demonstrated that gDNA-based probe selection on the Affymetrix U133+2 GeneChip array can be used to study the equine transcriptome which, to date, has received only limited attention. More than 29,000 transcripts can be detected in equine brain and liver and in primary cultures of equine articular chondrocytes. Gene ontology analysis of differentially expressed genes revealed the presence of expected categories within each tissue. The level of gene expression could also be correlated with the phenotypes and specialised functions of each tissue. The results demonstrated that probe selection on a human chip can be successfully used to study the equine transcriptome.

  • Mobasheri A, Henrotin Y. (2010) 'Identification, validation and qualification of biomarkers for osteoarthritis in humans and companion animals: mission for the next decade.'. Vet J, England: 185 (2), pp. 95-97.
  • Mobasheri A, Cassidy JP. (2010) 'Biomarkers in veterinary medicine: Towards targeted, individualised therapies for companion animals.'. Vet J, England: 185 (1), pp. 1-3.
  • Freeman A, Hetzel U, Cripps P, Mobasheri A. (2010) 'Expression of the plasma membrane markers aquaporin 1 (AQP1), glucose transporter 1 (GLUT1) and Na, K-ATPase in canine mammary glands and mammary tumours.'. Vet J, England: 185 (1), pp. 90-93.

    Abstract

    This study investigated the expression of the plasma membrane markers aquaporin 1 (AQP1), glucose transporter 1 (GLUT1) and the alpha1 subunit of Na, K-ATPase in normal canine mammary glands and in benign and malignant mammary tumours, using immunohistochemistry and semi-quantitative histomorphometry. AQP1 immunoreactivity was absent from the majority of specimens studied. GLUT1 immunoreactivity was observed in normal mammary tissue and particularly in the epithelial and mesenchymal cells of benign, and in the epithelial cells of malignant tumours, respectively. Na, K-ATPase immunoreactivity was present in normal and neoplastic mammary epithelium and was significantly increased in the epithelium of both benign and malignant tumours. These results suggest that GLUT1 is more highly expressed in neoplastic epithelium and mesenchyme and that Na, K-ATPase is more highly expressed in neoplastic mammary epithelium. In consequence, these membrane proteins may have potential as diagnostic and prognostic biomarkers of canine mammary neoplasia.

  • Airley R, Evans A, Mobasheri A, Hewitt SM. (2010) 'Glucose transporter Glut-1 is detectable in peri-necrotic regions in many human tumor types but not normal tissues: Study using tissue microarrays.'. Ann Anat, Germany: 192 (3), pp. 133-138.

    Abstract

    The hypoxic tumor microenvironment is associated with malignant progression and poor treatment response. The glucose transporter Glut-1 is a prognostic factor and putative hypoxia marker. So far, studies of Glut-1 in cancer have utilized conventional immunohistochemical analysis in a series of individual biopsy or surgical specimens. Tissue microarrays, however, provide a rapid, inexpensive means of profiling biomarker expression. To evaluate hypoxia markers, tissue cores must show the architectural features of hypoxia; i.e. viable tissue surrounding necrotic regions. Glut-1 may be a useful biomarker to validate tissue microarrays for use in studies of hypoxia-regulated genes in cancer. In this study, we carried out immunohistochemical detection of Glut-1 protein in many tumor and normal tissue types in a range of tissue microarrays. Glut-1 was frequently found in peri-necrotic regions, occurring in 9/34 lymphomas, 6/12 melanomas, and 5/16 glioblastomas; and in 43/54 lung, 22/84 colon, and 23/60 ovarian tumors. Expression was rare in breast (6/40) and prostate (1/57) tumors, and in normal tissue, was restricted to spleen, tongue, and CNS endothelium. In conclusion, tissue microarrays enable the observation of Glut-1 expression in peri-necrotic regions, which may be linked to hypoxia, and reflect previous studies showing differential Glut-1 expression across tumor types and non-malignant tissue.

  • Mobasheri A, Lewis R, Maxwell JE, Hill C, Womack M, Barrett-Jolley R. (2010) 'Characterization of a stretch-activated potassium channel in chondrocytes.'. J Cell Physiol, United States: 223 (2), pp. 511-518.

    Abstract

    Chondrocytes possess the capacity to transduce load-induced mechanical stimuli into electrochemical signals. The aim of this study was to functionally characterize an ion channel activated in response to membrane stretch in isolated primary equine chondrocytes. We used patch-clamp electrophysiology to functionally characterize this channel and immunohistochemistry to examine its distribution in articular cartilage. In cell-attached patch experiments, the application of negative pressures to the patch pipette (in the range of 20-200 mmHg) activated ion channel currents in six of seven patches. The mean activated current was 45.9 +/- 1.1 pA (n = 4) at a membrane potential of 33 mV (cell surface area approximately 240 microm(2)). The mean slope conductance of the principal single channels resolved within the total stretch-activated current was 118 +/- 19 pS (n = 6), and reversed near the theoretical potassium equilibrium potential, E(K+), suggesting it was a high-conductance potassium channel. Activation of these high-conductance potassium channels was inhibited by extracellular TEA (K(d) approx. 900 microM) and iberiotoxin (K(d) approx. 40 nM). This suggests that the current was largely carried by BK-like potassium (MaxiK) channels. To further characterize these BK-like channels, we used inside-out patches of chondrocyte membrane: we found these channels to be activated by elevation in bath calcium concentration. Immunohistochemical staining of equine cartilage samples with polyclonal antibodies to the alpha1- and beta1-subunits of the BK channel revealed positive immunoreactivity for both subunits in superficial zone chondrocytes. These experiments support the hypothesis that functional BK channels are present in chondrocytes and may be involved in mechanotransduction and chemotransduction.

  • Mobasheri A, Moskaluk CA, Marples D, Shakibaei M. (2010) 'Expression of aquaporin 1 (AQP1) in human synovitis.'. Ann Anat, Germany: 192 (2), pp. 116-121.

    Abstract

    Rheumatoid arthritis (RA) is an autoimmune disorder characterized by synovial proliferation (synovitis), articular cartilage and subchondral bone degradation as well as joint swelling. Joint swelling and edema often accompany pannus formation and chronic joint inflammation in RA. We have recently shown that human chondrocytes and synoviocytes express aquaporin 1 (AQP1) water channels and that AQP1 is upregulated in RA cartilage. Clinical evidence suggests that joint swelling and edema accompany the chronic inflammation observed in synovial joints of RA patients. Therefore we hypothesized that AQP1 water channels may be involved in joint swelling and synovial edema formation. To test this hypothesis, we performed immunostaining of normal and human synovitis tissue microarrays (TMAs) to investigate whether the expression of AQP1 water channels is altered in the synovium in synovitis. Immunohistochemistry revealed that AQP1 is expressed in synovial micro-vessels and synoviocytes from normal joints (n=20 normal subjects). Semi-quantitative histomorphometric analysis of AQP1 expression in the TMAs revealed upregulation of the membrane protein in the synovium derived from RA (n=10) and psoriatic arthritis (n=8) patients. These results indicate a potential role for synovial AQP1 and other aquaporins in joint swelling and the vasogenic edema fluid formation and hydrarthrosis associated with synovial inflammation. Future experiments will need to determine whether the expression of other aquaporins is altered in synovitis.

  • Floyd RV, Wray S, Quenby S, Martín-Vasallo P, Mobasheri A. (2010) 'Expression and distribution of Na, K-ATPase isoforms in the human uterus.'. Reprod Sci, United States: 17 (4), pp. 366-376.

    Abstract

    Na, K-ATPase activity relies on the composition of its catalytic alpha, beta, and FXYD constituents, all of which are expressed as multiple isoforms (4alpha, 4beta, and 7 FXYD). We used reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry to study Na, K-ATPase expression in uterine samples from nonlaboring elective and laboring emergency caesarean sections (CSs). Transcripts of alpha1 to 3, beta1 to 3, and FXYD1 isoforms were detected in all samples, but FXYD2 was only present in hysterectomy samples. Abundant immunoreactivity of alpha1 and moderate alpha2 was localized in myometrial smooth muscle and secretory glands of all groups. Smooth muscle and gland epithelia showed diffuse cytoplasmic alpha3 immunoreactivity. beta isoforms were detected in all groups but beta3 showed much denser immunoreactivity in myometrial samples taken from women in labor. In pregnancy, there was a switch in isoform expression, resulting in increased beta3 and decreased FXYD2 at the protein and messenger RNA (mRNA) levels. Na, K-ATPase isoform alterations may modulate uterine contractility during labor.

  • Rauch C, Pluen A, Foster N, Loughna P, Mobasheri A, Lagadic-Gossmann D, Counillon L. (2010) 'On some aspects of the thermodynamic of membrane recycling mediated by fluid phase endocytosis: evaluation of published data and perspectives.'. Cell Biochem Biophys, United States: 56 (2-3), pp. 73-90.

    Abstract

    The theoretical and experimental description of fluid phase endocytosis (FPE) requires an asymmetry in phospholipid number between the two leaflets of the cell membrane, which provides the biomechanical torque needed to generate membrane budding. Although the motor force behind FPE is defined, its kinetic has yet to be determined. Based on a body of evidences suggesting that the mean surface tension is unlikely to be involved in endocytosis we decided to determine whether the cytosolic hydrostatic pressure could be involved, by considering a constant energy exchanged between the cytosol and the cell membrane. The theory is compared to existing experimental data obtained from FPE kinetic studies in living cells where altered phospholipid asymmetry or changes in the extracellular osmotic pressure have been investigated. The model demonstrates that FPE is dependent on the influx and efflux of vesicular volumes (i.e. vesicular volumes recycling) rather than the membrane tension of cells. We conclude that: (i) a relationship exists between membrane lipid number asymmetry and resting cytosolic pressure and (ii) the validity of Laplace's law is limited to cells incubated in a definite hypotonic regime. Finally, we discuss how the model could help clarifying elusive observations obtained from different fields and including: (a) the non-canonical shuttling of aquaporin in cells, (b) the relationship between high blood pressure and inflammation and (c) the mechanosensitivity of the sodium/proton exchanger.

  • Floyd RV, Wray S, Martín-Vasallo P, Mobasheri A. (2010) 'Differential cellular expression of FXYD1 (phospholemman) and FXYD2 (gamma subunit of Na, K-ATPase) in normal human tissues: a study using high density human tissue microarrays.'. Ann Anat, Germany: 192 (1), pp. 7-16.

    Abstract

    FXYD proteins have been proposed to function as regulators of Na, K-ATPase function by lowering affinities of the system for potassium and sodium. However, their distribution in normal human tissues has not been studied. We have therefore used immunohistochemistry and semi-quantitative histomorphometric analysis to determine the relative expression at the protein level and distribution of FXYD1 (phospholemman) and FXYD2 (gamma subunit of Na, K-ATPase) in human Tissue MicroArrays (TMAs). Expression of FXYD1 was abundant in heart, kidney, placenta, skeletal muscle, gastric and anal mucosa, small intestine and colon. Lower FXYD1 expression was detected in uterine, intestinal and bladder smooth muscle, choroid plexus, liver, gallbladder, spleen, breast, prostate and epididymis. The tissue distribution of FXYD2 was less extensive compared to that of FXYD1. There was an abundant expression in kidney and choroid plexus and moderate expression in placenta, amniotic membranes, breast epithelium, salivary glands, pancreas and uterine endometrium. Weaker FXYD2 expression was detected in the adrenal medulla, liver, gallbladder, bladder and pancreas. The common denominator in the distribution of FXYD1 and FXYD2 was expression in highly active transport epithelia of the kidney, choroid plexus, placenta and salivary glands. This study reveals, in human tissues, the specific expression of FXYD proteins, which may associate with Na, K-ATPase in selected cell types and modulate its catalytic properties.

  • Henrotin Y, Clutterbuck AL, Allaway D, Lodwig EM, Harris P, Mathy-Hartert M, Shakibaei M, Mobasheri A. (2010) 'Biological actions of curcumin on articular chondrocytes.'. Osteoarthritis Cartilage, England: 18 (2), pp. 141-149.

    Abstract

    Curcumin (diferuloylmethane) is the principal biochemical component of the spice turmeric and has been shown to possess potent anti-catabolic, anti-inflammatory and antioxidant, properties. This article aims to provide a summary of the actions of curcumin on articular chondrocytes from the available literature with the use of a text-mining tool. We highlight both the potential benefits and drawbacks of using this chemopreventive agent for treating osteoarthritis (OA). We also explore the recent literature on the molecular mechanisms of curcumin mediated alterations in gene expression mediated via activator protein 1 (AP-1)/nuclear factor-kappa B (NF-kappaB) signalling in chondrocytes, osteoblasts and synovial fibroblasts.

  • Boone M, Mobasheri A, Fenton RA, van Balkom BW, Wismans R, van der Zee CE, Deen PM. (2010) 'The lysosomal trafficking regulator interacting protein-5 localizes mainly in epithelial cells.'. J Mol Histol, Netherlands: 41 (1), pp. 61-74.

    Abstract

    Endocytosis, subsequent protein sorting into multivesicular bodies (MVBs), and eventual degradation in lysosomes compose an important mechanism for controlling protein expression on the plasma membrane. The lysosomal trafficking regulator interacting protein-5 (LIP5) is part of the complex protein machinery involved in MVB biosynthesis. LIP5 interacts with other players of the ESCRT machinery as well as with two known cargo proteins, AQP2 and EGFR, whose degradation is affected upon reduction of LIP5 expression. To investigate the expression and localization pattern of LIP5, we studied LIP5 protein expression in a mouse tissue panel and subjected various rodent and human tissues to immunohistochemistry. Immunoblotting revealed that, except for jejunum, LIP5 is expressed as a 42 kDa protein in all mouse tissues tested. Alternatively-spliced gene products could not be detected. Immunohistochemical studies revealed that in tissues positive for LIP5, LIP5 is detected in virtually all epithelial cells of the examined rodent and human tissues. The observed LIP5 expression in epithelial tissues suggests that LIP5 is of particular importance in the MVB sorting and degradation of proteins expressed in polarized cells.

  • Noble K, Floyd R, Shmygol A, Shmygol A, Mobasheri A, Wray S. (2010) 'Distribution, expression and functional effects of small conductance Ca-activated potassium (SK) channels in rat myometrium.'. Cell Calcium, Netherlands: 47 (1), pp. 47-54.

    Abstract

    Calcium-activated potassium channels are important in a variety of smooth muscles, contributing to excitability and contractility. In the myometrium previous work has focussed on the large conductance channels (BK), and the role of small conductance channels (SK) has received scant attention, despite the finding that over-expression of an SK channel isoform (SK3) results in uterine dysfunction and delayed parturition. This study therefore characterises the expression of the three SK channel isoforms (SK1-3) in rat myometrium throughout pregnancy and investigates their effect on cytosolic [Ca] and force and compares this with that of BK channels. Consistent expression of all SK isoform transcripts and clear immunostaining of SK1-3 was found. Inhibition of SK1-3 channels (apamin, scyllatoxin) significantly inhibited outward current, caused membrane depolarisation and elicited action potentials in previously quiescent cells. Apamin or scyllatoxin increased the amplitude of [Ca] and force in spontaneously contracting myometrial strips throughout gestation. The functional effect of SK inhibition was larger than that of BK channel inhibition. Thus we show for the first time that SK1-3 channels are expressed and translated throughout pregnancy and contribute to outward current, regulate membrane potential and hence Ca signals in pregnant rat myometrium. They contribute more to quiescence that BK channels.

  • Cornock R, Langley-Evans SC, Mobasheri A, McMullen S. (2010) 'The impact of maternal protein restriction during rat pregnancy upon renal expression of angiotensin receptors and vasopressin-related aquaporins.'. Reprod Biol Endocrinol, England: 8

    Abstract

    Maternal protein restriction during rat pregnancy is known to impact upon fetal development, growth and risk of disease in later life. It is of interest to understand how protein undernutrition influences the normal maternal adaptation to pregnancy. Here we investigated the mechanisms regulating renal haemodynamics and plasma volume during pregnancy, in the context of both normal and reduced plasma volume expansion. The study focused on expression of renal angiotensin receptors (ATR) and vasopressin-related aquaporins (AQP), hypothesising that an alteration in the balance of these proteins would be associated with pregnancy per se and with compromised plasma volume expansion in rats fed a low-protein diet.

  • Summerfield N, Peters ME, Hercock CA, Young IS, Mobasheri A. (2010) 'Immunohistochemical evidence for expression of fast-twitch type sarco(endo)plasmic reticulum Ca ATPase (SERCA1) in German shepherd dogs with dilated cardiomyopathy myocardium'. Journal of Veterinary Cardiology, 12 (1), pp. 17-23.

    Abstract

    Objectives: Dilated cardiomyopathy (DCM) is one of the most common acquired canine heart diseases. It is particularly common in large and giant breed dogs. Although a great deal is known about the clinical progression and manifestations of the disease, the underlying cellular and molecular mechanisms remain poorly understood. One widely held belief is that calcium-handling abnormalities are critically involved in the disease process. This study investigates the changes in expression of the sarco(endo)plasmic reticulum calcium ATPase (SERCA) isoforms in DCM myocardium from German shepherd dogs. Animals, materials and methods: Affected tissue samples were obtained from German shepherd dogs with DCM, euthanized for intractable congestive heart failure while normal myocardial tissue samples were obtained from German shepherd dogs, euthanized for non-cardiovascular reasons. Tissue microarrays containing normal and DCM myocardium samples were prepared, immunostained with SERCA1 and SERCA2 antibodies and analyzed. Results: We were able to demonstrate, for the first time, that while there is little change in the expression of the cardiac isoform (SERCA2), there is clear expression of the fast-twitch skeletal muscle isoform SERCA1 in the myocardium of dogs diagnosed with DCM. Conclusion: We propose that SERCA1 expression is evidence of a natural adaptive response to the impaired Ca handling thought to occur in German shepherd dogs with DCM and heart failure. © 2010 Elsevier B.V. All rights reserved.

  • Summerfield N, Peters ME, Hercock CA, Mobasheri A, Young IS. (2010) 'Immunohistochemical evidence for expression of fast-twitch type sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1) in German shepherd dogs with dilated cardiomyopathy myocardium'. Journal of Veterinary Cardiology,
  • Richardson SM, Hoyland JA, Mobasheri R, Csaki C, Shakibaei M, Mobasheri A. (2010) 'Mesenchymal stem cells in regenerative medicine: opportunities and challenges for articular cartilage and intervertebral disc tissue engineering.'. J Cell Physiol, United States: 222 (1), pp. 23-32.

    Abstract

    Defects of load-bearing connective tissues such as articular cartilage and intervertebral disc (IVD) can result from trauma, degenerative, endocrine, or age-related disease. Current surgical and pharmacological options for the treatment of arthritic rheumatic conditions in the joints and spine are ineffective. Cell-based surgical therapies such as autologous chondrocyte transplantation (ACT) have been in clinical use for cartilage repair for over a decade but this approach has shown mixed results. This review focuses on the potential of mesenchymal stem cells (MSCs) as an alternative to cells derived from patient tissues in autologous transplantation and tissue engineering. Here we discuss the prospects of using MSCs in regenerative medicine and summarize the advantages and disadvantages of these cells in articular cartilage and IVD tissue engineering. We discuss the conceptual and practical difficulties associated with differentiating and pre-conditioning MSCs for subsequent survival in a physiologically harsh extracellular matrix, an environment that will be highly hypoxic, acidic, and nutrient deprived. Implanted MSCs will be exposed to traumatic physical loads and high levels of locally produced inflammatory mediators and catabolic cytokines. We also explore the potential of culture models of MSCs, fully differentiated cells and co-cultures as "proof of principle" ethically acceptable "3Rs" models for engineering articular cartilage and IVD in vitro for the purpose of replacing the use of animals in arthritis research.

  • Buhrmann C, Mobasheri A, Matis U, Shakibaei M. (2010) 'Curcumin mediated suppression of nuclear factor-κB promotes chondrogenic differentiation of mesenchymal stem cells in a high-density co-culture microenvironment.'. Arthritis Res Ther, England: 12 (4)

    Abstract

    Osteoarthritis (OA) and rheumatoid arthritis (RA) are characterised by joint inflammation and cartilage degradation. Although mesenchymal stem cell (MSC)-like progenitors are resident in the superficial zone of articular cartilage, damaged tissue does not possess the capacity for regeneration. The high levels of pro-inflammatory cytokines present in OA/RA joints may impede the chondrogenic differentiation of these progenitors. Interleukin (IL)-1β activates the transcription factor nuclear factor-κB (NF-κB), which in turn activates proteins involved in matrix degradation, inflammation and apoptosis. Curcumin is a phytochemical capable of inhibiting IL-1β-induced activation of NF-κB and expression of apoptotic and pro-inflammatory genes in chondrocytes. Therefore, the aim of the present study was to evaluate the influence of curcumin on IL-1β-induced NF-κB signalling pathway in MSCs during chondrogenic differentiation.

  • Clutterbuck AL, Harris P, Allaway D, Mobasheri A. (2010) 'Matrix metalloproteinases in inflammatory pathologies of the horse.'. Vet J, England: 183 (1), pp. 27-38.

    Abstract

    The extracellular matrix (ECM) of connective tissue is constantly being remodelled to allow for growth and regeneration. Normal tissue maintenance requires the ECM components to be degraded and re-synthesised in relatively equal proportions. This degradation is facilitated by matrix metalloproteinases (MMPs) and their proteolytic action is controlled primarily by the tissue inhibitors of metalloproteinases (TIMPs). Both MMPs and TIMPs exist in a state of dynamic equilibrium, with a slight excess of one or the other depending on the need for either ECM breakdown or synthesis. Long-term disruption to this balance between MMPs and TIMPs will have pathological consequences. Matrix metalloproteinases are involved in a number of diseases in mammals, including the horse. Excess MMP activity can cause ECM destruction, as seen in the lamellar basement membrane in laminitis and the articular cartilage in osteoarthritis. Matrix metalloproteinase under-activity can potentially impede healing by preventing fibrinolysis in fibrotic conditions and the removal of scar tissue in wounds. Matrix metalloproteinases also degrade non-ECM proteins and regulate cell behaviour via the release of growth factors from the substrates they cleave, increasing the scope of their effects. This review looks at the involvement of MMPs in equine health and pathologies, whilst exploring the potential consequences of therapeutic intervention.

  • Barrett-Jolley R, Lewis R, Fallman R, Mobasheri A. (2010) 'The emerging chondrocyte channelome.'. Front Physiol, Switzerland: 1

    Abstract

    Chondrocytes are the resident cells of articular cartilage and are responsible for synthesizing a range of collagenous and non-collagenous extracellular matrix macromolecules. Whilst chondrocytes exist at low densities in the tissue (1-10% of the total tissue volume in mature cartilage) they are extremely active cells and are capable of responding to a range of mechanical and biochemical stimuli. These responses are necessary for the maintenance of viable cartilage and may be compromised in inflammatory diseases such as arthritis. Although chondrocytes are non-excitable cells their plasma membrane contains a rich complement of ion channels. This diverse channelome appears to be as complex as one might expect to find in excitable cells although, in the case of chondrocytes, their functions are far less well understood. The ion channels so far identified in chondrocytes include potassium channels (K(ATP), BK, K(v), and SK), sodium channels (epithelial sodium channels, voltage activated sodium channels), transient receptor potential calcium or non-selective cation channels and chloride channels. In this review we describe this emerging channelome and discuss the possible functions of a range of chondrocyte ion channels.

  • Mobasheri A, Cathro HP, German A, Marples D, Martín-vasallo P, Canessa CM. (2009) 'Applications of Tissue Microarrays in Renal Physiology and Pathology'. Renal and Urinary Proteomics: Methods and Protocols, , pp. 97-118.
  • Clutterbuck AL, Asplin KE, Harris P, Allaway D, Mobasheri A. (2009) 'Targeting matrix metalloproteinases in inflammatory conditions.'. Curr Drug Targets, Netherlands: 10 (12), pp. 1245-1254.

    Abstract

    The matrix metalloproteinases (MMPs) and their endogenous regulators, the tissue inhibitors of MMPs (TIMPs) are responsible for the physiological remodelling of the extracellular matrix (ECM) in healthy connective tissues. MMPs are also involved in the regulation of cell behaviour via the release of growth factors and cytokines from the substrates they cleave, increasing the magnitude of their effects. Excess MMP activity is associated with ECM destruction in various inflammatory conditions, such as osteoarthritis (OA), while MMP under-activity potentially impairs healing by promoting fibrosis and preventing the effective removal of scar tissue. Both direct (TIMPs, small molecule MMP inhibitor drugs, blocking antibodies and anti-sense technologies) and indirect (glucocorticoids and non-steroidal anti-inflammatory drugs, statins, anti-sense technologies and various phytochemicals) strategies for MMP inhibition have been proposed and investigated. The strategy of MMP inhibition for degenerative and neoplastic diseases has been relatively unsuccessful due to undesired sequelae, often caused by non-selectivity of the MMP inhibition method. Therapeutic strategies for MMP-related conditions ideally should regulate MMP activity in order to maintain the optimum balance between MMPs and TIMPs. By avoiding complete inhibition it may be possible to prevent the complications of MMP over- and under-activity. Furthermore, MMP sub-type specificity is critical for minimising detrimental off-target effects that have been observed with broad-spectrum MMP inhibitors. Any potential MMP inhibitor or modulator must be subjected to rigorous pharmacokinetic, toxicity and safety studies and data obtained using in vitro models must be verified in clinically relevant animal models before therapeutic use is considered.

  • Eckersall PD, Slater K, Mobasheri A. (2009) 'Biomarkers in veterinary medicine: establishing a new international forum for veterinary biomarker research.'. Biomarkers, England: 14 (8), pp. 637-641.
  • Mobasheri A, Davidson M. (2009) 'Exciting future predicted for animal health'. European Life Science Journal, (2), pp. 20-21.
  • Clutterbuck AL, Mobasheri A, Shakibaei M, Allaway D, Harris P. (2009) 'Interleukin-1beta-induced extracellular matrix degradation and glycosaminoglycan release is inhibited by curcumin in an explant model of cartilage inflammation.'. Ann N Y Acad Sci, United States: 1171, pp. 428-435.

    Abstract

    Osteoarthritis (OA) is a degenerative and inflammatory disease of synovial joints that is characterized by the loss of articular cartilage, for which there is increasing interest in natural remedies. Curcumin (diferuloylmethane) is the main polyphenol in the spice turmeric, derived from rhizomes of the plant Curcuma longa. Curcumin has potent chemopreventive properties and has been shown to inhibit nuclear factor kappaB-mediated inflammatory signaling in many cell types, including chondrocytes. In this study, normal articular cartilage was harvested from metacarpophalangeal and metatarsophalangeal joints of eight horses, euthanized for reasons other than research purposes, to establish an explant model mimicking the inflammatory events that occur in OA. Initially, cartilage explants (N= 8) were stimulated with increasing concentrations of the proinflammatory cytokine IL-1beta to select effective doses for inducing cartilage degeneration in the explant model. Separate cartilage explants were then cotreated with IL-1beta at either 10 ng/mL (n= 3) or 25 ng/mL (n= 3) and curcumin (0.1 micromol/L, 0.5 micromol/L, 1 micromol/L, 10 micromol/L, and 100 micromol/L). After 5 days, the percentage of glycosaminoglycan (GAG) release from the explants was assessed using a dimethylmethylene blue colorimetric assay. Curcumin (100 micromol/L) significantly reduced IL-1beta-stimulated GAG release in the explants by an average of 20% at 10 ng/mL and 27% at 25 ng/mL back to unstimulated control levels (P < 0.001). Our results suggest that this explant model effectively simulates the proinflammatory cytokine-mediated release of articular cartilage components seen in OA. Furthermore, the evidence suggests that the inflammatory cartilage explant model is useful for studying the effects of curcumin on inflammatory pathways and gene expression in IL-1beta-stimulated chondrocytes.

  • Ratcliffe L, Mian S, Slater K, King H, Napolitano M, Aucoin D, Mobasheri A. (2009) 'Proteomic identification and profiling of canine lymphoma patients.'. Vet Comp Oncol, England: 7 (2), pp. 92-105.

    Abstract

    This study employed proteomic and bioinformatic approaches to identify serum biomarkers in canine lymphoma patients. Chilled serum samples derived from non-lymphoma (n = 92) and lymphoma (n = 87) patients were shipped from first opinion veterinary practices, subjected to ion exchange chromatography and analysed by surface-enhanced laser desorption ionization mass spectrometry. Nineteen serum protein peaks were identified between the two groups as being significantly different (P < 0.05) based upon their normalized ion intensities. Two biomarkers were identified that were capable of differentiating lymphoma and non-lymphoma patients. Analysis of the test data provided a positive predictive value (PPV) of 82%. A clinical follow-up study was carried out on 96 canine patients suspected of having lymphoma. Evaluation of this data gave a specificity value of 91%, sensitivity of 75%, PPV of 80% and negative predictive value of 88%. In conclusion, the expression pattern of two serum biomarkers has enabled serum samples to be classified into either lymphoma or non-lymphoma categories.

  • Peansukmanee S, Vaughan-Thomas A, Carter SD, Clegg PD, Taylor S, Redmond C, Mobasheri A. (2009) 'Effects of hypoxia on glucose transport in primary equine chondrocytes in vitro and evidence of reduced GLUT1 gene expression in pathologic cartilage in vivo.'. J Orthop Res, United States: 27 (4), pp. 529-535.

    Abstract

    Articular chondrocytes exist in an environment lacking in oxygen and nutrients due to the avascular nature of cartilage. The main source of metabolic energy is glucose, which is taken up by glucose transporters (GLUTs). In diseased joints, oxygen tensions and glucose availability alter as a result of inflammation and changes in vascularisation. Accordingly, in this study we examined the effects of hypoxia and the hypoxia mimetic cobalt chloride (CoCl(2)) on glucose transport in equine chondrocytes and compared expression of the hypoxia responsive GLUT1 gene in normal and diseased cartilage. Monolayers of equine chondrocytes were exposed to 20% O(2), 1% O(2), CoCl(2) (75 microM), or a combination of 1% O(2) and CoCl(2). Glucose uptake was measured using 2-deoxy-D-[2,6-(3)H] glucose. GLUT1 protein and mRNA expression were determined by FACS analysis and qPCR, respectively. GLUT1 mRNA expression in normal and diseased cartilage was analyzed using explants derived from normal, OA, and OCD cartilage. Chondrocytes under hypoxic conditions exhibited a significantly increased glucose uptake as well as upregulated GLUT1 protein expression. GLUT1 mRNA expression significantly increased in combined hypoxia-CoCl(2) treatment. Analysis of clinical samples indicated a significant reduction in GLUT1 mRNA in OA samples. In OCD samples GLUT1 expression also decreased but did not reach statistical significance. The increase in glucose uptake and GLUT1 expression under hypoxic conditions confirms that hypoxia alters the metabolic requirements of chondrocytes. The altered GLUT1 mRNA expression in diseased cartilage with significance in OA suggests that reduced GLUT1 may contribute to the failure of OA cartilage repair.

  • Mobasheri A, Csaki C, Clutterbuck AL, Rahmanzadeh M, Shakibaei M. (2009) 'Mesenchymal stem cells in connective tissue engineering and regenerative medicine: applications in cartilage repair and osteoarthritis therapy.'. Histol Histopathol, Spain: 24 (3), pp. 347-366.

    Abstract

    Defects of load-bearing connective tissues such as articular cartilage, often result from trauma, degenerative or age-related disease. Osteoarthritis (OA) presents a major clinical challenge to clinicians due to the limited inherent repair capacity of articular cartilage. Articular cartilage defects are increasingly common among the elderly population causing pain, reduced joint function and significant disability among affected patients. The poor capacity for self-repair of chondral defects has resulted in the development of a large variety of treatment approaches including Autologous Chondrocyte Transplantation (ACT), microfracture and mosaicplasty methods. In ACT, a cartilage biopsy is taken from the patient and articular chondrocytes are isolated. The cells are then expanded after several passages in vitro and used to fill the cartilage defect. Since its introduction, ACT has become a widely applied surgical method with good to excellent clinical outcomes. More recently, classical ACT has been combined with tissue engineering and implantable scaffolds for improved results. However, there are still major problems associated with the ACT technique which relate mainly to chondrocyte de-differentiation during the expansion phase in monolayer culture and the poor integration of the implants into the surrounding cartilage tissue. Novel approaches using mesenchymal stem cells (MSCs) as an alternative cell source to patient derived chondrocytes are currently on trial. MSCs have shown significant potential for chondrogenesis in animal models. This review article discusses the potential of MSCs in tissue engineering and regenerative medicine and highlights their potential for cartilage repair and cell-based therapies for osteoarthritis and a range of related osteoarticular disorders.

  • Csaki C, Matis U, Mobasheri A, Shakibaei M. (2009) 'Co-culture of canine mesenchymal stem cells with primary bone-derived osteoblasts promotes osteogenic differentiation.'. Histochem Cell Biol, Germany: 131 (2), pp. 251-266.

    Abstract

    Tissue engineering of bone grafts with osteogenic progenitor cells such as adult mesenchymal stem cells (MSC) represents a promising strategy for the treatment of large bone defects. The aim of this experimental study was to evaluate the osteogenic potential of primary osteoblasts on MSCs in co-culture at different ratios. The co-cultures were treated with or without a specific osteogenic induction medium in monolayer and high density cultures. In monolayer co-cultures, MSCs and osteoblasts actively searched for cell-cell contact leading to cell proliferation and only in treated monolayer co-cultures osteogenesis was observed. Ultrastructural evaluation of high density co-cultures using electron microscopy demonstrated osteogenesis with no significant difference between treated or untreated co-cultures. Immunoblotting confirmed expression of collagen type I, beta1-Integrin, the osteogenic-specific transcription factor Cbfa-1 and induction of the MAPKinase pathway (Shc, Erk1/2) in both treated and untreated co-cultures. Although treatment with the induction medium enhanced osteogenesis in the co-cultures compared to untreated co-cultures, the quality of osteogenesis was proportional to the quantity of osteoblasts in the co-cultures. Fifty percent osteoblasts in the co-cultures markedly increased osteogenesis; even the presence of ten percent osteoblasts in the co-culture strongly promoted osteogenesis. This data leads us to conclude that co-culture of MSCs with osteoblasts combined with the three-dimensional environment of the high density culture strongly promotes osteogenesis and stabilizes the osteogenic potential of MSCs. This approach may prove to be of practical benefit in future tissue engineering and regenerative medicine research.

  • Clutterbuck A, Harris P, Mobasheri A. (2009) 'Comment on: comparison between chondroprotective effects of glucosamine, curcumin and diacerein in IL-1beta-stimulated C-28/I2 chondrocytes.'. Osteoarthritis Cartilage, England: 17 (1), pp. 135-136.
  • Sutton S, Clutterbuck A, Harris P, Gent T, Freeman S, Foster N, Barrett-Jolley R, Mobasheri A. (2009) 'The contribution of the synovium, synovial derived inflammatory cytokines and neuropeptides to the pathogenesis of osteoarthritis.'. Vet J, England: 179 (1), pp. 10-24.

    Abstract

    Osteoarthritis (OA) is one of the most common and disabling chronic joint disorders affecting horses, dogs and humans. Synovial inflammation or synovitis is a frequently observed phenomenon in osteoarthritic joints and contributes to the pathogenesis of OA through formation of various catabolic and pro-inflammatory mediators altering the balance of cartilage matrix degradation and repair. Catabolic mediators produced by the inflamed synovium include pro-inflammatory cytokines, nitric oxide, prostaglandin E(2) and several neuropeptides, which further contribute to the pathogenesis of OA by increasing cartilage degradation. Recent studies suggest that substance P, corticotropin-releasing factor, urocortin and vasoactive intestinal peptide may also be involved in OA development, but the precise role of these neuropeptides in the pathogenesis of OA is not known. Since increased production of matrix metalloproteinases by the synovium is stimulated by pro-inflammatory cytokines, future anti-inflammatory therapies should focus on the synovium as a means of controlling subsequent inflammatory damage.

  • Rosa SC, Gonçalves J, Judas F, Mobasheri A, Lopes C, Mendes AF. (2009) 'Impaired glucose transporter-1 degradation and increased glucose transport and oxidative stress in response to high glucose in chondrocytes from osteoarthritic versus normal human cartilage.'. Arthritis Res Ther, England: 11 (3)

    Abstract

    Disorders that affect glucose metabolism, namely diabetes mellitus (DM), may favor the development and/or progression of osteoarthritis (OA). Thus far, little is known regarding the ability of chondrocytes to adjust to variations in the extracellular glucose concentration, resulting from hypoglycemia and hyperglycemia episodes, and so, to avoid deleterious effects resulting from deprivation or intracellular accumulation of glucose. The aim of this study was to compare the ability of normal and OA chondrocytes to regulate their glucose transport capacity in conditions of insufficient or excessive extracellular glucose and to identify the mechanisms involved and eventual deleterious consequences, namely the production of reactive oxygen species (ROS).

  • Csaki C, Mobasheri A, Shakibaei M. (2009) 'Synergistic chondroprotective effects of curcumin and resveratrol in human articular chondrocytes: inhibition of IL-1beta-induced NF-kappaB-mediated inflammation and apoptosis.'. Arthritis Res Ther, England: 11 (6)

    Abstract

    Currently available treatments for osteoarthritis (OA) are restricted to nonsteroidal anti-inflammatory drugs, which exhibit numerous side effects and are only temporarily effective. Thus novel, safe and more efficacious anti-inflammatory agents are needed for OA. Naturally occurring polyphenolic compounds, such as curcumin and resveratrol, are potent agents for modulating inflammation. Both compounds mediate their effects by targeting the NF-kappaB signalling pathway.

  • Shakibaei M, Csaki C, Nebrich S, Mobasheri A. (2008) 'Resveratrol suppresses interleukin-1beta-induced inflammatory signaling and apoptosis in human articular chondrocytes: potential for use as a novel nutraceutical for the treatment of osteoarthritis.'. Biochem Pharmacol, England: 76 (11), pp. 1426-1439.

    Abstract

    Osteoarthritis is an inflammatory disease of load-bearing synovial joints that is currently treated with drugs that exhibit numerous side effects and are only temporarily effective on pain, the main symptom of the disease. Consequently, there is an acute need for novel, safe and more effective chemotherapeutic agents for the treatment of osteoarthritis and related arthritic diseases. Resveratrol is a phytoalexin stilbene produced naturally by plants including red grapes, peanuts and various berries. Recent research in various cell models has demonstrated that resveratrol is safe and has potent anti-inflammatory properties. However, its potential for treating arthritic conditions has not been explored. In this study we provide experimental evidence that resveratrol inhibits the expression of VEGF, MMP-3, MMP-9 and COX-2 in human articular chondrocytes stimulated with the pro-inflammatory cytokine IL-1beta. Since these gene products are regulated by the transcription factor NF-kappaB, we investigated the effects of resveratrol on IL-1beta-induced NF-kappaB signaling pathway. Resveratrol, like N-Ac-Leu-Leu-norleucinal (ALLN) suppressed IL-1beta-induced proteasome function and the degradation of IkappaBalpha (an inhibitor of NF-kappaB) without affecting IkappaBalpha kinase activation, IkappaBalpha-phosphorylation or IkappaBalpha-ubiquitination which suppressed nuclear translocation of the p65 subunit of NF-kappaB and its phosphorylation. Furthermore, we observed that resveratrol as well as ALLN inhibited IL-1beta-induced apoptosis, caspase-3 activation and PARP cleavage in human articular chondrocytes. In summary, our results suggest that resveratrol suppresses apoptosis and inflammatory signaling through its actions on the NF-kappaB pathway in human chondrocytes. We propose that resveratrol should be explored further for the prophylactic treatment of osteoarthritis in humans and companion animals.

  • Clutterbuck AL, Mobasheri A, Allaway D, Harris P. (2008) 'Toxic effects of curcumin (Diferuloylmethane) on equine articular chondrocytes and synoviocytes In Vitro'. European Cells and Materials, 16 (SUPPL. 3), pp. 43-43.
  • Richardson SM, Knowles R, Marples D, Hoyland JA, Mobasheri A. (2008) 'Aquaporin expression in the human intervertebral disc.'. J Mol Histol, Netherlands: 39 (3), pp. 303-309.

    Abstract

    The nucleus pulposus (NP) of the human intervertebral disc (IVD) is a hyperosmotic tissue that is subjected to daily dynamic compressive loads. In order to survive within this environment the resident chondrocyte-like cells must be able to control their cell volume, whilst also controlling the anabolism and catabolism of their extra-cellular matrix. Recent studies have demonstrated expression of a range of bi-directional, transmembrane water and solute transporters, named aquaporins (AQPs), within chondrocytes of articular cartilage. The aim of this study was to use immunohistochemsitry to investigate the expression of aquaporins 1, 2 and 3 within the human IVD. Results demonstrated expression of both AQP-1 and -3 by cells within the NP and inner annulus fibrosus (AF), while outer AF cells lacked expression of AQP-1 and showed very low numbers of AQP-3 immunopositive cells. Cells from all regions were negative for AQP-2. Therefore this study demonstrates similarities in the phenotype of NP cells and articular chondrocytes, which may be due to similarities in tissue osmolarity and mechanobiology. The decrease in expression of AQPs from the NP to the outer AF may signify changes in cellular phenotype in response to differences in mechanbiology, osmolarity and hydration between the gelatinous NP and the fibrous AF.

  • Richardson SM, Knowles R, Tyler J, Mobasheri A, Hoyland JA. (2008) 'Expression of glucose transporters GLUT-1, GLUT-3, GLUT-9 and HIF-1alpha in normal and degenerate human intervertebral disc.'. Histochem Cell Biol, Germany: 129 (4), pp. 503-511.

    Abstract

    The glucose transporters GLUT-1 and GLUT-3 are targets of the hypoxia-inducible transcription factor HIF-1alpha and it has been shown that nucleus pulposus (NP) cells in rat intervertebral discs (IVD) express both HIF-1alpha and GLUT-1. However, there is limited data on the expression of HIF-1alpha and GLUTs in human IVD. The aim here was to (1) determine whether, like articular chondrocytes, human IVD cells express GLUT-1, 3 and 9 and whether there was any co-expression with HIF-1alpha; and (2) to localise expression of the GLUT isoforms in the disc and identify any changes during degeneration. Real-time PCR was used to identify expression of GLUT1, 3, 9 and HIF-1alpha mRNAs and immunohistochemistry was used to analyse protein expression and localisation of GLUTs in normal and degenerate IVD biopsies. Results confirmed HIF-1alpha, GLUT1, 3 and 9 mRNA expression in NP and AF and co-expression of each GLUT isoform with HIF-1alpha in the NP, but not the AF. Immunohistochemistry demonstrated regional differences in GLUT expression, with the highest expression being in the NP. GLUT expression also changed as degeneration progressed. This study demonstrates that NP and AF cells have different GLUT expression profiles that suggest regional differences in the metabolic nature of the human IVD and that this environment changes during degeneration.

  • Mobasheri A, Bondy CA, Moley K, Mendes AF, Rosa SC, Richardson SM, Hoyland JA, Barrett-Jolley R, Shakibaei M. (2008) 'Glucose transporter expression and regulation in embryonic and mesenchymal stem cells'. Advances in Anatomy Embryology and Cell Biology, 200, pp. 57-66.

    Abstract

    Stem cells have the remarkable potential to develop into many different cell types in the body. Serving as a sort of repair system for the body, they can theoretically divide without limit to replenish other cells as long as the person or animal is still alive. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell. © 2008 Springer-Verlag Berlin Heidelberg.

  • Mobasheri A, Bondy CA, Moley K, Mendes AF, Rosa SC, Richardson SM, Hoyland JA, Barrett-Jolley R, Shakibaei M. (2008) 'Mammalian sugar transporter families: GLUT and SGLT'. Advances in Anatomy Embryology and Cell Biology, 200, pp. 22-31.

    Abstract

    Sugar transport across the plasma membrane of mammalian cells is mediated by members of the GLUT/SLC2A family of facilitative sugar transporters (Joost and Thorens 2001; Mueckler 1994; Seatter and Gould 1999; Uldry and Thorens 2004) and the SGLT/SLC5A family of Na+-dependent sugar transporters (Wood and Trayhurn 2003). These proteins belong to a larger superfamily of proteins known as the major facilitator superfamily (MFS) or uniporter-symporter-anti-porter family (Saier et al. 1999a) The MFS family is one of the two largest families of membrane transporters in nature and accounts for nearly half of the solute transporters encoded within the genomes of microorganisms (bacteria, yeasts) and higher organisms such as plants and animals. The MFS was originally thought to function primarily in the uptake of sugars, but more detailed studies of members of this family have revealed that drug efflux systems, Krebs cycle metabolites, organophosphate: phosphate exchangers, oligosaccharide: H1 symport permeases, and bacterial aromatic acid permeases are also members of the MFS superfamily. These observations led to the probability that the MFS is far more widespread in nature and far more diverse in function than had been thought previously (Pao et al. 1998; Saier et al. 1999a, 1999b, 1998). Thus far 17 subgroups of the MFS have been identified. The human genome project has identified 14 members of the GLUT/SLC2A family which have been cloned in humans (Wood and Trayhurn 2003; Wu and Freeze 2002) (Fig. 6, Table 1). GLUT proteins are characterized by the presence of 12 membrane spanning helices and several conserved sequence motifs (Joost and Thorens 2001). The GLUT/SLC2A proteins are expressed in a tissue- and cell-specific manner and exhibit distinct kinetic and regulatory properties that reflect their functional and tissue-specific roles. The full definitions and unique functional characteristics for each of the GLUT protein isoforms are outlined in Table 1. The sequence similarities of the GLUT/SLC2A family members are now well defined and the family is now divided into three subclasses (Fig. 6). Five of the mammalian facilitated glucose carriers (GLUTs 1-5) have been very well characterized, but significantly less is known about the remaining nine glucose carriers (GLUTs 6-14) since their discovery in late 2001 (Joost and Thorens 2001) and much remains to be learned about their expression, tissue distribution, and transport functions (Uldry and Thorens 2004). Of

  • Mobasheri A, Bondy CA, Moley K, Mendes AF, Rosa SC, Richardson SM, Hoyland JAJA, Barrett-Jolley R, Shakibaei M. (2008) 'Molecular diversity of facilitative glucose transporters in articular chondrocytes'. Advances in Anatomy Embryology and Cell Biology, 200, pp. 31-50.

    Abstract

    In order to appreciate the physiological basis for the molecular diversity of facilitative glucose transporters in chondrocytes we need to re-examine the importance of glucose as a nutrient and structural precursor. Glucose is an important nutrient in fully developed articular cartilage due to the poor vascularization and highly glycolytic nature of the tissue, a situation that is further exacerbated by low oxygen tensions and ongoing anaerobic glycolysis by chondrocytes (Mobasheri et al. 2002c; Otte 1991; Rajpurohit et al. 2002). Therefore, even modest changes in glucose concentrations in the extracellular microenvironment of chondrocytes could impair anabolic and catabolic activities (Mobasheri et al. 2002c; Shikhman et al. 2001a). Fully developed adult chondrocytes express mRNA for multiple isoforms of the GLUT/SLC2A family of glucose transporters including GLUT1, GLUT3, GLUT5, GLUT6, GLUT8, GLUT9, GLUT10, GLUT11, and GLUT12 (Mobasheri et al. 2002b, 2002c; Richardson et al. 2003; Shikhman et al. 2001a) (Fig. 8). The reason for such GLUT isoform diversity in chondrocytes has not yet been satisfactorily explained but several hypotheses have been put forward: GLUT isoform diversity in chondrocytes suggests that the transmembrane uptake of glucose, fructose, and other related hexose sugars is highly specialized and requires several proteins with the capacity to transport structurally different sugars. The observed diversity of GLUT proteins in chondrocytes may possibly reflect a cartilage-specific requirement for 'fast' (i.e., GLUT3) and baseline (GLUT1) glucose transporters that operate more efficiently at low substrate concentrations under physiological conditions (Mobasheri et al. 2002c; Richardson et al. 2003). The presence of GLUT1 in chondrocytes has also been linked to the acute requirement of these cells for glycolytic energy metabolism under the low oxygen tension conditions that are prevalent in avascular load-bearing articular cartilage and intervertebral disc (Pfander et al. 2003; Rajpurohit et al. 2002; Schipani et al. 2001). GLUT1 has also been shown to be a cytokine inducible glucose transporter in cartilage since it is induced by catabolic, proinflammatory cytokines (Phillips et al. 2005a; Richardson et al. 2003; Shikhman et al. 2004, 2001a) (Fig. 9). © 2008 Springer-Verlag Berlin Heidelberg.

  • Mobasheri A, Bondy CA, Moley K, Mendes AF, Rosa SC, Richardson SM, Hoyland JA, Barrett-Jolley R, Shakibaei M. (2008) 'Facilitative glucose transporters in articular chondrocytes. Expression, distribution and functional regulation of GLUT isoforms by hypoxia, hypoxia mimetics, growth factors and pro-inflammatory cytokines.'. Adv Anat Embryol Cell Biol, Germany: 200, pp. 1 p following vi-84.

    Abstract

    Articular cartilage is a unique and highly specialized avascular connective tissue in which the availability of oxygen and glucose is significantly lower than synovial fluid and plasma. Glucose is an essential source of energy during embryonic growth and fetal development and is vital for mesenchymal cell differentiation, chondrogenesis, and skeletal morphogenesis. Glucose is an important metabolic fuel for differentiated chondrocytes during postnatal development and in adult articular cartilage and is a common structural precursor for the synthesis of extracellular matrix glycosaminoglycans. Glucose metabolism is critical for growth plate chondrocytes which participate in long bone growth. Glucose concentrations in articular cartilage can fluctuate depending on age, physical activity, and endocrine status. Chondrocytes are glycolytic cells and must be able to sense the concentration of oxygen and glucose in the extracellular matrix and respond appropriately by adjusting cellular metabolism. Consequently chondrocytes must have the capacity to survive in an extracellular matrix with limited nutrients and low oxygen tensions. Published data from our laboratories suggest that chondrocytes express multiple isoforms of the GLUT/SLC2A family of glucose/polyol transporters. In other tissues GLUT proteins are expressed in a cell-specific manner, exhibit distinct kinetic properties, and are developmentally regulated. Several GLUTs expressed in chondrocytes are regulated by hypoxia, hypoxia mimetics, metabolic hormones, and proinflammatory cytokines. In this multidisciplinary text we review the molecular and morphological aspects of GLUT expression and function in chondrocytes and their mesenchymal and embryonic stem cell precursors and propose key roles for these proteins in glucose sensing and metabolic regulation in cartilage.

  • Mobasheri A, Bondy CA, Moley K, Mendes AF, Rosa SC, Richardson SM, Hoyland JA, Barrett-Jolley R, Shakibaei M. (2008) 'Glucose: An essential metabolite and structural precursor for articular cartilage'. Advances in Anatomy Embryology and Cell Biology, 200, pp. 19-22.

    Abstract

    Glucose is an important metabolite for all living cells and with other simple sugars and related molecules derived from these sugars it provides sources of readily available energy for cells. Sugars also provide basic carbon skeletons for the biosynthesis of other macromolecules. These include proteins, lipids, nucleic acids, and complex storage polysaccharides (glycogen). Furthermore, hexose sugars are building blocks of glycoproteins such as cartilage-specific proteoglycans. In addition to their role as structural components of the cartilage ECM (Fig. 4), proteoglycans also fulfil adhesive and informational functions. © 2008 Springer-Verlag Berlin Heidelberg.

  • Mobasheri A, Bondy CA, Moley K, Mendes AF, Rosa SC, Richardson SM, Hoyland JA, Barrett-Jolley R, Shakibaei M. (2008) 'Articular cartilage: Structure, function, and pathophysiology'. Advances in Anatomy Embryology and Cell Biology, 200, pp. 5-13.

    Abstract

    In the following two subsections we discuss the structure and function of articular cartilage and give a broad background to the pathophysiology of arthritis before discussing the links between nutrition and arthritis and how diet affects skeletal development. We also discuss how nutrition might predispose humans and animals to arthritis or protect us from it. We then discuss the links between nutrition and joint disease in the context of our studies on glucose transporters in chondrocytes. © 2008 Springer-Verlag Berlin Heidelberg.

  • Shakibaei M, Csaki C, Mobasheri A. (2008) 'Concluding remarks'. Advances in Anatomy Embryology and Cell Biology, 197, pp. 47-49.

    Abstract

    Although this review has covered the diverse roles of integrin receptors in articular cartilage, we suspect that the research carried out to date has merely revealed the tip of the iceberg. Here, we summarise the main points concerning the involvement of integrins in cartilage function and pathophysiology: During growth and development, the skeletal system, particularly the cartilage lining the articulating surfaces of bones, optimises its extracellular matrix architecture by subtle adaptations to the prevailing mechanical loads. The mechanisms for adaptation involve a multi-step process of cellular mechanotransduction including mechanocoupling, conversion of mechanical forces into local mechanical signals, (i.e. shear stresses, that initiate a response by chondrocytes), biochemical coupling, transduction of a mechanical signal to a biochemical response involving pathways within the cell membrane and cytoskeleton; and finally modulation of gene expression and cell behaviour. © 2008 Springer-Verlag Berlin Heidelberg.

  • Mobasheri A, Bondy CA, Moley K, Mendes AF, Rosa SC, Richardson SM, Hoyland JA, Barrett-Jolley R, Shakibaei M. (2008) 'Glucose transporters in the intervertebral disc'. Advances in Anatomy Embryology and Cell Biology, 200, pp. 53-57.

    Abstract

    The intervertebral disc is a cartilaginous structure that resembles articular cartilage in its biochemistry and cell biology, but morphologically it is clearly different (Roberts 2002; Urban and Roberts 2003). The disc shows degenerative and ageing changes earlier than any other connective tissue in the human body (Urban and Roberts 2003). The maintenance of the ECM in the NP of the adult human disc is dependent on the functional integrity of the cartilage end plate cells (Pritzker 1977). Cartilage end plate senescence is followed by compensatory cartilaginous metaplasia of annulus fibrosus cells. It has been proposed that intervertebral disc narrowing and collapse are related to metabolic failure of matrix production by end plate and annulus fibrosus cells (Pritzker 1977). © 2008 Springer-Verlag Berlin Heidelberg.

  • Shakibaei M, Csaki C, Mobasheri A. (2008) 'Diverse roles of integrin receptors in articular cartilage.'. Adv Anat Embryol Cell Biol, Germany: 197, pp. 1-60.

    Abstract

    Integrins are heterodimeric integral membrane proteins made up of alpha and beta subunits. At least eighteen alpha and eight beta subunit genes have been described in mammals. Integrin family members are plasma membrane receptors involved in cell adhesion and active as intra- and extracellular signalling molecules in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response and metastatic spread of tumour cells. Integrin beta 1 (beta1-integrin), the protein encoded by the ITGB1 gene (also known as CD29 and VLAB), is a multi-functional protein involved in cell-matrix adhesion, cell signalling, cellular defense, cell adhesion, protein binding, protein heterodimerisation and receptor-mediated activity. It is highly expressed in the human body (17.4 times higher than the average gene in the last updated revision of the human genome). The extracellular matrix (ECM) of articular cartilage is a unique environment. Interactions between chondrocytes and the ECM regulate many biological processes important to homeostasis and repair of articular cartilage, including cell attachment, growth, differentiation and survival. The beta1-integrin family of cell surface receptors appears to play a major role in mediating cell-matrix interactions that are important in regulating these fundamental processes. Chondrocyte mechanoreceptors have been proposed to incorporate beta1-integrins and mechanosensitive ion channels which link with key ECM, cytoskeletal and signalling proteins to maintain the chondrocyte phenotype, prevent chondrocyte apoptosis and regulate chondrocyte-specific gene expression. This review focuses on the expression and function of beta1-integrins in articular chondrocytes, its role in the unique biology of these cells and its distribution in cartilage.

  • Mobasheri A, Bondy CA, Moley K, Mendes AF, Rosa SC, Richardson SM, Hoyland JA, Barrett-jolley R, Shakibaei M. (2008) 'Does arthritis have a nutritional etiology?'. Advances in Anatomy Embryology and Cell Biology, 200, pp. 13-17.

    Abstract

    Despite the recognition that degenerative cartilage disorders like OA and OCD may have nutritional abnormalities at the root of their pathogenesis, the role of nutrition in the etiology of these disorders is poorly studied (Mobasheri et al. 2002c). A huge amount of research effort and funding is focused on nutraceuticals, nutritional supplements, and naturally occurring bioactive components of foods (Goggs et al. 2005; Mobasheri et al. 2002c; Shakibaei et al. 2007a, 2007b, 2005). It is clear that balanced dietary supplementation programs have played a secondary role in the management of joint diseases. Nutritional factors such as glucose and glucose-derived sugars (i.e., glucosamine sulfate and vitamin C) are important for the development, maintenance, repair, and remodeling of cartilage, bone, and other load-bearing connective tissues. In the following sections we review the links between nutrition and joint disease in order to justify the physiological and pathophysiological relevance of our studies on glucose transporters in chondrocytes. © 2008 Springer-Verlag Berlin Heidelberg.

  • Shakibaei M, Csaki C, Mobasheri A. (2008) 'Integrins in articular cartilage'. Advances in Anatomy Embryology and Cell Biology, 197, pp. 10-47.

    Abstract

    In the following sections, we primarily review our own work on the expression, tissue distribution and function of integrins in selected in vitro models of articular chondrocytes and limb bud mesenchymal cells. The expression pattern of α1-, α3-, αv- and α5β1-integrins and their specific ligand binding were investigated in monolayer cultures of chondrocytes from 17-day-old mouse embryos using morphological and immunomorphological methods. After a 3-h culture period of chondrocytes from 17-day-old mouse embryos, numerous cells had already adhered in the form of a monolayer. After a 1-day culture period αv-, α3- and α5β1-integrins were observed on the chondrocytes. During the first 4 days, the number of the cells had increased, a matrix became perceptible. After a 5-day culture period, the flat fibroblast-like cells, often of bipolar shape, increased in number at the expense of the chondrocytes. © 2008 Springer-Verlag Berlin Heidelberg.

  • Mobasheri A, Bondy CA, Moley K, Mendes AF, Rosa SC, Richardson SM, Hoyland JA, Barrett-Jolley R, Shakibaei M. (2008) 'Regulation of glucose transport by nonsteroidal anti-inflammatory drugs'. Advances in Anatomy Embryology and Cell Biology, 200, pp. 50-53.

    Abstract

    Cartilage destruction in arthritis and OA is linked to aberrant proinflammatory cytokine and growth factor expression in the joint (Chikanza and Fernandes 2000; Malemud et al. 2003). The proinflammatory cytokines TNF-α and IL-β have been found in significantly elevated levels in the synovial fluid of OA joints (Goldring 1999, 2000a; van den Berg 1999). Catabolic pathways are activated by TNF-α and IL-β, which are both upregulated in OA (Malemud et al. 2003). These proinflammatory mediators cause an increase in cartilage matrix degradation through increased MMP and aggrecanase activity in the joint. In addition, TNF-α and IL-β downregulate ECM protein biosynthesis while concomitantly up-regulating matrix MMP gene and protein expression. When MMPs are activated, cartilage ECM degradation ensues apparently because levels of endogenous cartilage MMP inhibitors cannot regulate MMP activity (Malemud et al. 2003). © 2008 Springer-Verlag Berlin Heidelberg.

  • Mobasheri A, Bondy CA, Moley K, Mendes AF, Rosa SC, Richardson SM, Hoyland JA, Barrett-Jolley R, Shakibaei M. (2008) 'Metabolic dysfunction in arthritis'. Advances in Anatomy Embryology and Cell Biology, 200, pp. 17-19.

    Abstract

    Healthy bones and joints depend on a normally functioning endocrine system. It is a fact of clinical significance that excess IGF-I and growth hormone (GH) causes major joint pathology (Stavrou and Kleinberg 2001). Endocrine disorders not only affect soft connective tissues but also implicate load-bearing musculoskeletal structures including bone, cartilage, synovium, tendon, and ligament. Damage to soft connective tissue and associated innervation is a hallmark of acromegaly, hypothyroidism,and diabetes mellitus (Liote and Orcel 2000). Acromegaly normally presents with quite severe arthritis involving degeneration of the spine and articular cartilage in peripheral joints (Stavrou and Kleinberg 2001). Severe diabetes mellitus increases the risk of neuroarthropathy as a direct result of infection, neuropathy, and vasculopathy. Pituitary tumors can have manifestations similar to rheumatological disease and may cause connective tissue disorders as a result of overproduction or deficiencies of pituitary hormones (Stavrou and Kleinberg 2001). Excessive GH production by the pituitary gland causes cartilage destruction. GH deficiency on the other hand increases the risk of bone fractures. © 2008 Springer-Verlag Berlin Heidelberg.

  • Csaki C, Matis U, Mobasheri A, Ye H, Shakibaei M. (2007) 'Chondrogenesis, osteogenesis and adipogenesis of canine mesenchymal stem cells: a biochemical, morphological and ultrastructural study.'. Histochem Cell Biol, Germany: 128 (6), pp. 507-520.

    Abstract

    Musculoskeletal diseases with osteochondrotic articular cartilage defects, such as osteoarthritis, are an increasing problem for humans and companion animals which necessitates the development of novel and improved therapeutic strategies. Canine mesenchymal stem cells (cMSCs) offer significant promise as a multipotent source for cell-based therapies and could form the basis for the differentiation and cultivation of tissue grafts to replace damaged tissue. However, no comprehensive analysis has been undertaken to characterize the ultrastructure of in vitro differentiated cMSCs. The main goal of this paper was to focus on cMSCs and to analyse their differentiation capacity. To achieve this aim, bone marrow cMSCs from three canine patients were isolated, expanded in monolayer culture and characterized with respect to their ability for osteogenic, adipogenic and chondrogenic differentiation capacities. cMSCs showed proliferative potential and were capable of osteogenic, adipogenic and chondrogenic differentiation. cMSCs treated with the osteogenic induction medium differentiated into osteoblasts, produced typical bone matrix components, beta1-integrins and upregulated the osteogenic specific transcription factor Cbfa-1. cMSCs treated with the adipogenic induction medium showed typical adipocyte morphology, produced adiponectin, collagen type I and beta1-integrins, and upregulated the adipogenic specific transcription factor PPAR-gamma. cMSCs treated with the chondrogenic induction medium exhibited a round to oval shape, produced a cartilage-specific extracellular matrix, beta1-integrins and upregulated the chondrogenic specific transcription factor Sox9. These results demonstrate, at the biochemical, morphological and ultrastructural levels, the multipotency of cMSCs and thus highlight their potential therapeutic value for cell-based tissue engineering.

  • Richardson SM, Mobasheri A, Freemont AJ, Hoyland JA. (2007) 'Intervertebral disc biology, degeneration and novel tissue engineering and regenerative medicine therapies.'. Histol Histopathol, Spain: 22 (9), pp. 1033-1041.

    Abstract

    Degeneration of the intervertebral disc (IVD) is a major cause of low back pain affecting a large percentage of the population at some point in their lives. Consequently IVD degeneration and its associated low back pain has a huge socio-economic impact and places a burden on health services world-wide. Current treatments remove the symptoms without treating the underlying problem and can result in reoccurrence in the same or adjacent discs. Tissue engineering offers hope that new therapies can be developed which can regenerate the IVD. Combined with this, development of novel biomaterials and an increased understanding of mesenchymal stem cell and IVD cell biology mean that tissue engineering of the IVD may soon become a reality. However for any regenerative medicine approach to be successful there must first be an understanding of the biology of the tissue and the pathophysiology of the disease process. This review covers these key areas and gives an overview of the recent developments in the fields of biomaterials, cell biology and tissue engineering of the IVD.

  • Richardson SM, Freemont AJ, Hoyland JA, Mobasheri A. (2007) 'Intervertebral disc biology, degeneration and novel tissue engineering and regenerative medicine therapies'. Histology and Histopathology, 22 (7-9), pp. 1033-1041.

    Abstract

    Degeneration of the intervertebral disc (IVD) is a major cause of low back pain affecting a large percentage of the population at some point in their lives. Consequently IVD degeneration and its associated low back pain has a huge socio-economic impact and places a burden on health services world-wide. Current treatments remove the symptoms without treating the underlying problem and can result in reoccurrence in the same or adjacent discs. Tissue engineering offers hope that new therapies can be developed which can regenerate the IVD. Combined with this, development of novel biomaterials and an increased understanding of mesenchymal stem cell and IVD cell biology mean that tissue engineering of the IVD may soon become a reality. However for any regenerative medicine approach to be successful there must first be an understanding of the biology of the tissue and the pathophysiology of the disease process. This review covers these key areas and gives an overview of the recent developments in the fields of biomaterials, cell biology and tissue engineering of the IVD.

  • Mobasheri A. (2007) 'Colony-stimulating factors in veterinary medicine: an unexplored potential.'. Vet J, England: 174 (1), pp. 8-9.
  • Floyd RV, Mason SL, Proudman CJ, German AJ, Marples D, Mobasheri A. (2007) 'Expression and nephron segment-specific distribution of major renal aquaporins (AQP1-4) in Equus caballus, the domestic horse.'. Am J Physiol Regul Integr Comp Physiol, United States: 293 (1), pp. R492-R503.

    Abstract

    Aquaporins (AQPs) play fundamental roles in water and osmolyte homeostasis by facilitating water and small solute movement across plasma membranes of epithelial, endothelial, and other tissues. AQP proteins are abundantly expressed in the mammalian kidney, where they have been shown to play essential roles in fluid balance and urine concentration. Thus far, the majority of studies on renal AQPs have been carried out in laboratory rodents and sheep; no data have been published on the expression of AQPs in kidneys of equines or other large mammals. The aim of this comparative study was to determine the expression and nephron segment localization of AQP1-4 in Equus caballus by immunoblotting and immunohistochemistry with custom-designed rabbit polyclonal antisera. AQP1 was found in apical and basolateral membranes of the proximal convoluted tubules and thin descending limbs of the loop of Henle. AQP2 expression was specifically detected in apical membranes of cortical, medullary, and papillary collecting ducts. AQP3 was expressed in basolateral membranes of cortical, medullary, and papillary collecting ducts. Immunohistochemistry also confirmed AQP4 expression in basolateral membranes of cells lining the distal convoluted and connecting tubules. Western blots revealed high expression of AQP1-4 in the equine kidney. These observations confirm that AQPs are expressed in the equine kidney and are found in similar nephron locations to mouse, rat, and human kidney. Equine renal AQP proteins are likely to be involved in acute and chronic regulation of body fluid composition and may be implicated in water balance disorders brought about by colic and endotoxemia.

  • Shakibaei M, John T, Schulze-Tanzil G, Lehmann I, Mobasheri A. (2007) 'Suppression of NF-kappaB activation by curcumin leads to inhibition of expression of cyclo-oxygenase-2 and matrix metalloproteinase-9 in human articular chondrocytes: Implications for the treatment of osteoarthritis.'. Biochem Pharmacol, England: 73 (9), pp. 1434-1445.

    Abstract

    Pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) play a key role in the pathogenesis of osteoarthritis (OA). Anti-inflammatory agents capable of suppressing the production and catabolic actions of these cytokines may have therapeutic potential in the treatment of OA and a range of other osteoarticular disorders. The purpose of this study was to examine the effects of curcumin (diferuloylmethane), a pharmacologically safe phytochemical agent with potent anti-inflammatory properties on IL-1beta and TNF-alpha signalling pathways in human articular chondrocytes maintained in vitro. The effects of curcumin were studied in cultures of human articular chondrocytes treated with IL-1beta and TNF-alpha for up to 72h. Expression of collagen type II, integrin beta1, cyclo-oxygenase-2 (COX-2) and matrix metalloproteinase-9 (MMP-9) was monitored by western blotting. The effects of curcumin on the expression, phosphorylation and nuclear translocation of protein components of the NF-kappaB system were studied by western blotting and immunofluorescence, respectively. Treatment of chondrocytes with curcumin suppressed IL-1beta-induced NF-kappaB activation via inhibition of IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation and p65 nuclear translocation. Curcumin inhibited the IL-1beta-induced stimulation of up-stream protein kinase B Akt. These events correlated with down-regulation of NF-kappaB targets including COX-2 and MMP-9. Similar results were obtained in chondrocytes stimulated with TNF-alpha. Curcumin also reversed the IL-1beta-induced down-regulation of collagen type II and beta1-integrin receptor expression. These results indicate that curcumin has nutritional potential as a naturally occurring anti-inflammatory agent for treating OA through suppression of NF-kappaB mediated IL-1beta/TNF-alpha catabolic signalling pathways in chondrocytes.

  • John T, Kohl B, Mobasheri A, Ertel W, Shakibaei M. (2007) 'Interleukin-18 induces apoptosis in human articular chondrocytes.'. Histol Histopathol, Spain: 22 (5), pp. 469-482.

    Abstract

    Elevated levels of the pro-inflammatory cytokine, interleukin-18 (IL-18) have recently been demonstrated in osteoarthritic cartilage. However, the effects of IL-18 on chondrocyte signalling and matrix biosynthesis are poorly understood. Therefore, the present study was undertaken to further characterize the impact of IL-18 on human articular chondrocyte in vitro. Human articular chondrocytes were stimulated with various concentrations of recombinant human IL-18 (1, 10, 100 ng/ml) for 0, 4, 8, 12, 24, 48, 72 h in vitro. The effects of IL-18 on the cartilage-specific matrix protein collagen type II, the cytoskeletal protein vinculin, the cell matrix signal transduction receptor beta-integrin, key signalling proteins of the MAPKinase pathway (such as SHC (Sarc Homology Collagen) and activated MAPKinase [ERK-1/-2]), the pro-inflammatory enzyme cyclo-oxygenase-2 (COX-2) and the apoptosis marker activated caspase-3 were evaluated by Western blot analysis and immunofluorescence labelling. Morphological features of IL-18 stimulated chondrocytes were estimated by transmission electron microscopy. IL-18 lead to inhibition of collagen type II-deposition, decreased beta-integrin receptor and vinculin synthesis, SHC and MAPKinase activation, increased COX-2 synthesis and activation of caspase-3 in chondrocytes in a time- and dose-dependent manner. Furthermore, chondrocytes treated with IL-18 exhibited typical morphological features of apoptosis as revealed by transmission electron microscopy. Taken together, the results of the present study underline key catabolic events mediated by IL-18 signalling in chondrocytes such as loss of cartilage-specific matrix and apoptosis. Inhibition of MAPKinase signalling is hypothesized to contribute to these features. Future therapeutics targeting IL-18 signalling pathways may be beneficial in rheumatoid arthritis and osteoarthritis therapy.

  • John T, Kohl B, Ertel W, Mobasheri A, Shakibaei M. (2007) 'Interleukin-18 induces apoptosis in human articular chondrocytes'. Histology and Histopathology, 22 (4-6), pp. 469-482.

    Abstract

    Elevated levels of the pro-inflammatory cytokine, interleukin- 18 (IL-18) have recently been demonstrated in osteoarthritic cartilage. However, the effects of IL-18 on chondrocyte signalling and matrix biosynthesis are poorly understood. Therefore, the present study was undertaken to further characterize the impact of IL- 18 on human articular chondrocyte in vitro. Human articular chondrocytes were stimulated with various concentrations of recombinant human IL-18 (1, 10, 100 ng/ml) for 0, 4, 8, 12, 24, 48, 72 h in vitro. The effects of IL-18 on the cartilage-specific matrix protein collagen type II, the cytoskeletal protein vinculin, the cell matrix signal transduction receptor β-integrin, key signalling proteins of the MAPKinase pathway (such as SHC (Sarc Homology Collagen) and activated MAPKinase [ERK-1/-2]), the pro-inflammatory enzyme cyclo-oxygenase-2 (COX-2) and the apoptosis marker activated caspase-3 were evaluated by Western blot analysis and immunofluorescence labelling. Morphological features of IL-18 stimulated chondrocytes were estimated by transmission electron microscopy. IL-18 lead to inhibition of collagen type II- deposition, decreased β-integrin receptor and vinculin synthesis, SHC and MAPKinase activation, increased COX-2 synthesis and activation of caspase-3 in chondrocytes in a time- and dose-dependent manner. Furthermore, chondrocytes treated with IL-18 exhibited typical morphological features of apoptosis as revealed by transmission electron microscopy. Taken together, the results of the present study underline key catabolic events mediated by IL-18 signalling in chondrocytes such as loss of cartilage- specific matrix and apoptosis. Inhibition of MAPKinase signalling is hypothesized to contribute to these features. Future therapeutics targeting IL-18 signalling pathways may be beneficial in rheumatoid arthritis and osteoarthritis therapy.

  • Mobasheri A, Gent TC, Nash AI, Womack MD, Moskaluk CA, Barrett-Jolley R. (2007) 'Evidence for functional ATP-sensitive (K(ATP)) potassium channels in human and equine articular chondrocytes.'. Osteoarthritis Cartilage, England: 15 (1), pp. 1-8.

    Abstract

    Chondrocytes are highly sensitive to variations in extracellular glucose and oxygen levels in the extracellular matrix. As such, they must possess a number of mechanisms to detect and respond to alterations in the metabolic state of cartilage. In other organs such as the pancreas, heart and brain, such detection is partly mediated by a family of potassium channels known as K(ATP) (adenosine 5'-triphosphate-sensitive potassium) channels. Here we investigate whether chondrocytes too express functional K(ATP) channels, which might, potentially, serve to couple metabolic state with cell activity.

  • Shakibaei M, John T, Seifarth C, Mobasheri A. (2007) 'Resveratrol inhibits IL-1 beta-induced stimulation of caspase-3 and cleavage of PARP in human articular chondrocytes in vitro.'. Ann N Y Acad Sci, United States: 1095, pp. 554-563.

    Abstract

    Resveratrol is a polyphenolic phytoalexin that is present in various fruits, in the skin of red grapes and peanuts. Recent studies have shown that resveratrol exhibits potent antioxidant properties and is able to exert anti-inflammatory and anti-catabolic properties in several cell types. The pro-inflammatory cytokine interleukin-1beta (IL-1beta) plays a pivotal role in the pathogenesis of osteoarthritis (OA) in humans and animals. In this article we investigated whether resveratrol is able to block the effects of IL-1beta, specifically the activation of caspase-3 and subsequent cleavage of poly (ADP-ribose) polymerase (PARP) in human articular chondrocytes. Cultures of human chondrocytes were prestimulated with 10 ng/mL IL-1beta for 1, 12, and 24 h before being co-treated with IL-1beta and 100 microM resveratrol or 50 microM of the caspase inhibitor Z-DEVD-FMK for 1, 12, and 24 h, respectively in vitro. Resveratrol significantly reduced the IL-1beta-induced inhibition of expression of cartilage-specific collagen type II and signal transduction receptor beta1-integrin in a time-dependent manner. Incubation of chondrocytes with IL-1beta resulted in the activation of caspase-3 and PARP cleavage. These effects were abolished through co-treatment with resveratrol. Furthermore, co-treatment of IL-1beta-stimulated cells with the caspase inhibitor Z-DEVD-FMK blocked activation of caspase-3 and PARP cleavage, suggesting that this process is a caspase-dependent pathway. In summary, our results confirm that resveratrol is an effective inhibitor of chondrocyte apoptosis in vitro. These findings suggest that this dietary polyphenolic compound may have future applications in the nutraceutical-based therapy of human and animal OA.

  • Mobasheri A, Marples D, Young IS, Floyd RV, Moskaluk CA, Frigeri A. (2007) 'Distribution of the AQP4 water channel in normal human tissues: protein and tissue microarrays reveal expression in several new anatomical locations, including the prostate gland and seminal vesicles.'. Channels (Austin), United States: 1 (1), pp. 29-38.

    Abstract

    Aquaporins facilitate osmotically driven water movement across cell membranes. Aquaporin 4 (AQP4) is a major water channel in the central nervous system where it participates in cerebral water balance. AQP4 is also present in basolateral membranes of lower respiratory tract airway and renal collecting duct epithelial cells, gastric parietal cells and skeletal muscle cells. However, the distribution of AQP4 in many other tissues is still unknown. The aim of this study was to determine the expression and relative abundance of AQP4 in human Tissue MicroArrays (TMAs) and human protein microarrays by immunohistochemistry and chemiluminescence. In the central nervous system AQP4 was abundantly expressed in the cerebral cortex, cerebellar cortex (purkinje/granular layer), ependymal cell layer, hippocampus and spinal cord. Lower levels were detected in choroid plexus, white matter and meninges. In the musculoskeletal system AQP4 was highly expressed in the sarcolemma of skeletal muscle from the chest and neck. In the male genital system AQP4 was moderately expressed in seminiferous tubules, seminal vesicles, prostate and epidiymis. In the respiratory system AQP4 was moderately expressed in lung and bronchus. AQP expression was abundant in the kidney. In the gastrointestinal system AQP4 was moderately present in basolateral membranes of parietal cells at the base of gastric glands. AQP4 was also detected in salivary glands, adrenals, anterior pituitary, prostate and seminal vesicles. Human protein microarrays verified the TMA data. Our findings suggest that AQP4 is expressed more widely than previously thought in human organs and may be involved in prostatic and seminal fluid formation.

  • Csaki C, Matis U, Mobasheri A, Putz R, Ye H, Shakibaei M. (2007) 'Chondrogenesis in co-culture: An intensive interaction between mesenchymal stem cells and primary chondrocytes.'. J Stem Cells Regen Med, India: 2 (1), pp. 117-118.
  • Airley RE, Mobasheri A. (2007) 'Hypoxic regulation of glucose transport, anaerobic metabolism and angiogenesis in cancer: novel pathways and targets for anticancer therapeutics.'. Chemotherapy, Switzerland: 53 (4), pp. 233-256.

    Abstract

    Cancer cells require a steady source of metabolic energy in order to continue their uncontrolled growth and proliferation. Accelerated glycolysis is one of the biochemical characteristics of cancer cells. Recent work indicates that glucose transport and metabolism are essential for the posttreatment survival of tumor cells, leading to poor prognosis. Glycolytic breakdown of glucose is preceded by the transport of glucose across the cell membrane, a rate-limiting process mediated by facilitative glucose transporter proteins belonging to the facilitative glucose transporter/solute carrier GLUT/SLC2A family. Tumors frequently show overexpression of GLUTs, especially the hypoxia-responsive GLUT1 and GLUT3 proteins. There are also studies that have reported associations between GLUT expression and proliferative indices, whilst others suggest that GLUT expression may be of prognostic significance. In this article we revisit Warburg's original hypothesis and review the recent clinical and basic research on the expression of GLUT family members in human cancers and in cell lines derived from human tumors. We also explore the links between hypoxia-induced genes, glucose transporters and angiogenic factors. Hypoxic tumors are significantly more malignant, metastatic, radio- and chemoresistant and have a poor prognosis. With the discovery the oxygen-sensitive transcription factor hypoxia-inducible factor (HIF-1) has come a new understanding of the molecular link between hypoxia and deregulated glucose metabolism. HIF-1 induces a number of genes integral to angiogenesis, e.g. vascular endothelial growth factor (VEGF), a process intimately involved with metastatic spread. This knowledge may enhance existing chemotherapeutic strategies so that treatment can be more rationally applied and personalized for cancer patients.

  • Mobasheri A, Platt N, Thorpe C, Shakibaei M. (2006) 'Regulation of 2-deoxy-D-glucose transport, lactate metabolism, and MMP-2 secretion by the hypoxia mimetic cobalt chloride in articular chondrocytes.'. Ann N Y Acad Sci, United States: 1091, pp. 83-93.

    Abstract

    Articular cartilage is an avascular tissue with significantly reduced levels of oxygen and nutrients compared to plasma and synovial fluid. Therefore, chondrocyte survival and cartilage homeostasis require effective mechanisms for oxygen and nutrient signaling. To gain a better understanding of the mechanisms responsible for oxygen and nutrient sensing in chondrocytes, we investigated the effects of hypoxic stimulation induced by cobalt chloride treatment (a hypoxia-mimetic) on glucose uptake and lactate production in chondrocytes. We also studied the effects of cobalt chloride and glucose deprivation on the expression and secretion of active MMP-2. Primary cultures of articular chondrocytes were either maintained in 20% O(2) (normoxia) or exposed to the hypoxia-mimetic cobalt chloride for up to 24 h at the following concentrations: 15 microM, 37.5 microM, and 75 microM. Glucose transport was determined by measuring the net uptake of nonmetabolizable 2-deoxy-D-[2, 6-(3)H] glucose into chondrocytes. Active MMP-2 secretion was assayed by gelatin zymography. Lactic acid production was assayed using a lactate kit. Exposure to cobalt chloride significantly increased the uptake of 2-deoxy-D-[2, 6-(3)H] glucose and the production of lactate. Glucose deprivation and cobalt chloride treatment increased levels of active MMP-2 in the culture medium. Our results suggest that these metabolic alterations are important events during adaptation to hypoxia. Upregulation of MMP-2 and the build-up of lactic acid will have detrimental effects on the extracellular matrix and may contribute to the pathogenesis and progression of osteoarthritis (OA).

  • Shakibaei M, Seifarth C, John T, Rahmanzadeh M, Mobasheri A. (2006) 'Igf-I extends the chondrogenic potential of human articular chondrocytes in vitro: molecular association between Sox9 and Erk1/2.'. Biochem Pharmacol, England: 72 (11), pp. 1382-1395.

    Abstract

    Expansion of articular chondrocytes in monolayer culture leads to loss of the unique chondrocyte phenotype and the cells' redifferentiation capacity. Dedifferentiation of chondrocytes in monolayer culture is a challenging problem for autologous chondrocyte transplantation (ACT). It is well established that Igf-I exerts positive anabolic effects on chondrocytes in vivo and in vitro. Accordingly, in this study, we examined whether the anabolic insulin-like growth factor-I (Igf-I) is capable of extending the chondrogenic potential of dedifferentiated chondrocytes in vitro. Chondrocyte monolayers were cultured up to 10 passages. At each passage chondrocytes were stimulated with Igf-I (10ng/ml) and introduced to high-density cultures for up to 7 days. Expression of collagen type II, cartilage-specific proteoglycans, activated caspase-3, integrin beta1, extracellular signal-regulated kinase (Erk) and Sox9 was examined by Western blotting, immunoprecipitation and immunomorphological techniques. Monolayer chondrocytes rapidly lost their differentiated phenotype. When introduced to high-density cultures, only chondrocytes from P1-P4 redifferentiated. In contrast, Igf-I treated cells from P1 up to P7 redifferentiated and formed cartilage-like tissue in high-density culture. P8-P10 cells exhibited apoptotic alterations and produced significantly less matrix. Igf-I markedly increased expression of integrin beta1, Erk and Sox9. Immunoprecipitation revealed that phosphorylated Erk1/2 physically interacts with Sox9 in chondrocyte nuclei, suggesting a previously unreported functional association which was markedly enhanced by Igf-I. Treatment of chondrocyte cultures with Igf-I stabilizes chondrogenic potential, stimulates Sox9 and promotes molecular interactions between Erk and Sox9. These effects appear to be regulated by the integrin/MAPK signaling pathways.

  • Mobasheri A. (2006) 'Comment on: cloning and characterization of porcine aquaporin 1 water channel expressed extensively in the gastrointestinal system.'. World J Gastroenterol, China: 12 (27), pp. 4437-4439.
  • Mobasheri A. (2006) 'A new low temperature method for high yield isolation of chondrocytes from elderly human subjects and its applicability to mature articular cartilage from older animals.'. Osteoarthritis Cartilage, England: 14 (7), pp. 720-721.
  • Lorenz W, Sigrist G, Shakibaei M, Mobasheri A, Trautmann C. (2006) 'A hypothesis for the origin and pathogenesis of rheumatoid diseases.'. Rheumatol Int, Germany: 26 (7), pp. 641-654.

    Abstract

    It is well established that a correlation exists between rheumatoid arthritis (RA) and microbial damage. Material analyses have suggested that bacteria may be causative agents. This study was undertaken to further characterize the microbial agent responsible for pathogenesis of RA. In order to investigate whether substances in moist building materials can affect human cartilage, extracts from moist building materials were analysed for microbial components. Exposure of chondrocyte cultures to extracts in vitro showed that they were damaging the cultures. A direct correlation between strength of damage and concentration of MMP3 demonstrated that the effect was dose-dependent. High quantities of LPS were detected in the extracts. Experiments after deactivation with Polymyxin B showed that LPS are the causative agents. The present study leads to the hypothesis that LPS may bind to procollagen, as they bind to scavenger receptors. This procollagen endotoxin complex may block tropocollagen synthesis.

  • Haisch A, Marzahn U, Mobasheri A, Schulze-Tanzil G, Shakibaei M. (2006) 'Development and phenotypic characterization of a high density in vitro model of auricular chondrocytes with applications in reconstructive plastic surgery.'. Histol Histopathol, Spain: 21 (5), pp. 467-476.

    Abstract

    Cultivation of phenotypically stable auricular chondrocytes will have applications in autologous chondrocyte transplantation and reconstructive surgery of cartilage. Chondrocytes grown in monolayer culture rapidly dedifferentiate assuming a fibroblast-like morphology and lose their cartilage-specific pattern of gene expression. Three-dimensional high-density culture models mimic more closely the in vivo conditions of cartilage. Therefore, this study was undertaken to test whether the high-density cultures might serve as a suitable model system to acquire phenotypically and functionally differentiated auricular chondrocytes from porcine cartilage. Freshly isolated porcine auricular chondrocytes were cultured for 7 passages in monolayer culture. From each passage (passage 0 and 1-7) cells were introduced to high-density cultures and examined by transmission electron microscopy. Western blotting was used to analyse the expression of cartilage-specific markers, such as collagen type II and cartilage specific proteoglycan, fibronectin, cell adhesion and signal transduction receptor beta1-integrin, matrix metalloproteinases (MMP-9, MMP-13), cyclo-oxygenase (COX)-2 and the apoptosis commitment marker, activated caspase-3. When dedifferentiated auricular chondrocytes from monolayer passages 0-4 were cultured in high-density culture, they recovered their chondrocytic phenotype and formed cartilage nodules surrounded by fibroblast-like cells and synthesised collagen type II, proteoglycans, fibronectin and beta1-integrins. However, chondrocytes from monolayer passages 5-7 did not redifferentiate to chondrocytes even when transferred to high-density culture, and did not synthesize a chondrocyte-specific extracellular matrix. Instead, they produced increasing amounts of MMP-9, MMP-13, COX-2, activated caspase-3 and underwent apoptosis. Three-dimensional high-density cultures may therefore be used to obtain sufficient quantities of fully differentiated auricular chondrocytes for autologous chondrocyte transplantation and reconstructive plastic surgery.

  • Mobasheri A, Shakibaei M, Mobasheri R, Richardson SM, Hoyland JA. (2006) 'Glucose sensing in chondrocytes via GLUT1 and GLUT3: Implications for articular cartilage and intervertebral disc metabolism'. Current Rheumatology Reviews, 2 (2), pp. 109-121.

    Abstract

    Glucose is an essential source of energy for embryonic growth and fetal development. Glucose is vital for chondroblasts during chondrogenesis and for articular and growth plate chondrocytes during post-natal development. Glucose also plays a key role in extracellular matrix synthesis as a precursor for glycosaminoglycans. The quantity of glucose available to cells in avascular connective tissues such as articular cartilage and intervertebral disc is normally significantly lower than plasma and synovial fluid. Glucose concentrations in cartilage can fluctuate depending on age, physical activity and endocrine status. Chondrocytes are glycolytic cells and must be able to sense the amount of glucose available to them in the extracellular matrix and respond appropriately by adjusting cellular metabolism. Studies are currently in progress to identify glucose sensors in tissues that carry out bulk glucose fluxes (intestine, kidney and liver) where the sodium dependent glucose transporter (SGLT1) has been proposed to be involved. GLUT2 and hexokinase have been identified as important components of the glucose sensing apparatus in the pancreas. Chondrocytes do not appear to express GLUT2 or SGLT1 and glucose sensing in these cells is likely to involve other facilitative glucose transporters. In this article we review selected literature on glucose sensing in the yeast, and in pancreas, kidney, brain and gut in an attempt to understand how different cells sense and respond to alterations in extracellular glucose. We present a new hypothesis that implicates the hypoxia responsive GLUT1 and GLUT3 glucose transporters and the hypoxia-inducible transcription factor (HIF-1α) in glucose sensing in chondrocytes. © 2006 Bentham Science Publishers Ltd.

  • Haisch A, Marzahn U, Mobasheri A, Schulze-Tanzil G, Shakibaei M. (2006) 'Development and phenotypic characterization of a high density in vitro model of auricular chondrocytes with applications in reconstructive plastic surgery'. Histology and Histopathology, 21 (4-6), pp. 467-476.

    Abstract

    Cultivation of phenotypically stable auricular chondrocytes will have applications in autologous chondrocyte transplantation and reconstructive surgery of cartilage. Chondrocytes grown in monolayer culture rapidly dedifferentiate assuming a fibroblast-like morphology and lose their cartilage-specific pattern of gene expression. Three-dimensional high-density culture models mimic more closely the in vivo conditions of cartilage. Therefore, this study was undertaken to test whether the high-density cultures might serve as a suitable model system to acquire phenotypically and functionally differentiated auricular chondrocytes from porcine cartilage. Freshly isolated porcine auricular chondrocytes were cultured for 7 passages in monolayer culture. From each passage (passage 0 and 1-7) cells were introduced to high-density cultures and examined by transmission electron microscopy. Western blotting was used to analyse the expression of cartilage-specific markers, such as collagen type II and cartilage specific proteoglycan, fibronectin, cell adhesion and signal transduction receptor β1-integrin, matrix metalloproteinases (MMP-9, MMP-13), cyclo-oxygenase (COX)-2 and the apoptosis commitment marker, activated caspase-3. When dedifferentiated auricular chondrocytes from monolayer passages 0-4 were cultured in high-density culture, they recovered their chondrocytic phenotype and formed cartilage nodules surrounded by fibroblast-like cells and synthesised collagen type II, proteoglycans, fibronectin and β1-integrins. However, chondrocytes from monolayer passages 5-7 did not redifferentiate to chondrocytes even when transferred to high-density culture, and did not synthesize a chondrocyte-specific extracellular matrix. Instead, they produced increasing amounts of MMP-9, MMP-13, COX-2, activated caspase-3 and underwent apoptosis. Three-dimensional high-density cultures may therefore be used to obtain sufficient quantities of fully differentiated auricular chondrocytes for autologous chondrocyte transplantation and reconstructive plastic surgery.

  • Simpson DM, Mobasheri A, Haywood S, Beynon RJ. (2006) 'A proteomics study of the response of North Ronaldsay sheep to copper challenge.'. BMC Vet Res, England: 2

    Abstract

    The objective of this proteomics study was to identify proteins that changed expression as a result of copper challenge in the uniquely copper sensitive North Ronaldsay sheep and further, to compare those changes in expression with the more copper tolerant Cambridge breed. Such data gives us a proteome-centered perspective of the pathogenesis of copper-induced oxidative stress in this breed.

  • Shakibaei M, Schulze-Tanzil G, John T, Mobasheri A. (2005) 'Curcumin protects human chondrocytes from IL-l1beta-induced inhibition of collagen type II and beta1-integrin expression and activation of caspase-3: an immunomorphological study.'. Ann Anat, Germany: 187 (5-6), pp. 487-497.

    Abstract

    Interleukin 1beta (IL-1beta) is a pleiotropic pro-inflammatory cytokine that plays a key role in mediating cartilage degradation in osteoarticular disorders such as osteoarthritis (OA) and rheumatoid arthritis (RA). At the cellular level, IL-1beta activates matrix degrading enzymes, down-regulates expression of matrix components and induces chondrocyte apoptosis. Curcumin (diferuloylmethane) is an anti-inflammatory phytochemical agent that has recently been shown to antagonize the pro-inflammatory effects of cytokines in chondrocytes and other cells. To test the hypothesis that curcumin also protects chondrocytes from morphological alterations induced by IL-1beta, we investigated its in vitro effects on apoptotic signalling proteins and key cartilage-specific matrix components in IL-1beta-stimulated chondrocytes. Human articular chondrocytes were pre-treated with 10 ng/mI IL-1beta alone for 30 min before being co-treated with IL-1beta and 50 microM curcumin for 5, 15 or 30 min, respectively. The ultrastructural morphology of chondrocytes was investigated by transmission electron microscopy. The production of collagen type II, the adhesion and signal transduction receptor beta1-integrin, the apoptosis marker activated caspase-3 was analysed by immunohistochemistry, immunoelectron microscopy and Western blotting. Transmission electron microscopy of chondrocytes stimulated with IL-1beta revealed early degenerative changes which were relieved by curcumin co-treatment. The suppression of collagen type II and beta1-integrin synthesis by IL-1beta was inhibited by curcumin. Additionally, curcumin antagonized IL-1beta-induced caspase-3 activation in a time-dependent manner. This study clearly demonstrates that curcumin exerts anti-apoptotic and anti-catabolic effects on IL-1beta-stimulated articular chondrocytes. Therefore curcumin may have novel therapeutic potential as an adjunct nutraceutical chondroprotective agent for treating OA and related osteoarticular disorders.

  • Mobasheri A, Richardson S, Mobasheri R, Shakibaei M, Hoyland JA. (2005) 'Hypoxia inducible factor-1 and facilitative glucose transporters GLUT1 and GLUT3: putative molecular components of the oxygen and glucose sensing apparatus in articular chondrocytes.'. Histol Histopathol, Spain: 20 (4), pp. 1327-1338.

    Abstract

    Articular cartilage is an avascular connective tissue in which the availability of oxygen and glucose is significantly lower than synovial fluid and plasma. Glucose is an important metabolic fuel and structural precursor that plays a key role in the synthesis of extracellular matrix macromolecules in articular cartilage. However, glucose concentrations in cartilage can fluctuate depending on age, physical activity and endocrine status. Chondrocytes are glycolytic cells and must be able to sense the quantities of oxygen and glucose available to them in the extracellular matrix and respond appropriately by adjusting cellular metabolism. Consequently chondrocytes must have the capacity to survive in an extracellular matrix with limited nutrients and low oxygen tensions. The molecular mechanisms responsible for allowing chondrocytes to adapt to these harsh environmental conditions are poorly understood. In this article we present a novel "dual" model of oxygen and glucose sensing in chondrocytes based on recent experimental data. This model incorporates the hypoxia-inducible factor alpha (HIF-1alpha) as an oxygen sensor and the hypoxia responsive facilitative glucose transporters, GLUT1 and GLUT3 as putative components of the glucose sensing apparatus in chondrocytes. Recent studies have shown that GLUT1 and GLUT3 are both expressed in chondrocytes and their HIF-1alpha-mediated transcription may be dually stimulated in response to hypoxia and low glucose conditions which in turn promote anaerobic glycolysis in favor of oxidative phosphorylation. This working model provides, for the first time, a unifying hypothesis to explain how chondrocytes might sense and respond to low oxygen tensions and alterations in extracellular glucose.

  • Mobasheri A, Gent TC, Womack MD, Carter SD, Clegg PD, Barrett-Jolley R. (2005) 'Quantitative analysis of voltage-gated potassium currents from primary equine (Equus caballus) and elephant (Loxodonta africana) articular chondrocytes.'. Am J Physiol Regul Integr Comp Physiol, United States: 289 (1), pp. R172-R180.

    Abstract

    In this comparative study, we have established in vitro models of equine and elephant articular chondrocytes, examined their basic morphology, and characterized the biophysical properties of their primary voltage-gated potassium channel (Kv) currents. Using whole cell patch-clamp electrophysiological recording from first-expansion and first-passage cells, we measured a maximum Kv conductance of 0.15 +/- 0.04 pS/pF (n = 10) in equine chondrocytes, whereas that in elephant chondrocytes was significantly larger (0.8 +/- 0.4 pS/pF, n = 4, P

  • Mobasheri A, Airley R, Hewitt SM, Marples D. (2005) 'Heterogeneous expression of the aquaporin 1 (AQP1) water channel in tumors of the prostate, breast, ovary, colon and lung: a study using high density multiple human tumor tissue microarrays.'. Int J Oncol, Greece: 26 (5), pp. 1149-1158.

    Abstract

    Aquaporin 1 (AQP1) water channels are membrane proteins that control the permeability of endothelial and epithelial barriers by facilitating water movement across cell membranes. Recent studies suggest that AQP1 may be responsible for the high vascular permeability and interstitial fluid pressure in tumors of the brain, colon, breast and pancreas. AQP1 may also play a role in tumor angiogenesis and may be involved in development of effusions or edema fluid. The aim of the present study was to use immunohistochemistry and semi-quantitative histomorphometric analysis to compare the distribution and relative abundance of AQP1 on NCI TARP human multiple tumor tissue microarrays (TMAs) with normal tissues represented on the CHTN TMAs. Immunohistochemistry and semi-quantitative histomorphometric analysis were used to compare the distribution of AQP1 in tumors of the prostate, colon, lung, breast and ovary represented on TARP TMAs with their normal counterparts on CHTN TMAs. AQP1 was expressed in capillary endothelia of all normal tissues. In most tumors AQP1 was confined to endothelial barriers. AQP1 expression was marginally higher in microvascular structures in prostate and ovarian tumors and was higher in advanced mammary and colorectal carcinomas where AQP1 immunoreactivity was also seen in some neoplastic tumor cells. In conclusion, the AQP1 water channel is an excellent marker of microvasculature but it is heterogeneously expressed in different human tumors and not necessarily expressed in all neoplastic cells. Increased AQP1 expression in some human adenocarcinomas may be a consequence of angiogenesis and important for the formation or clearance of tumor edema.

  • Mobasheri A, Dobson H, Mason SL, Cullingham F, Shakibaei M, Moley JF, Moley KH. (2005) 'Expression of the GLUT1 and GLUT9 facilitative glucose transporters in embryonic chondroblasts and mature chondrocytes in ovine articular cartilage.'. Cell Biol Int, England: 29 (4), pp. 249-260.

    Abstract

    Glucose transport across the chondrocyte membrane is essential for chondrogenesis and the development of the skeletal system. We have previously used RT-PCR to show that fully developed human articular chondrocytes express transcripts for the GLUT1 and GLUT9 glucose transporters. In this study we report on the expression and immunohistochemical localization of the GLUT1 and GLUT9 proteins in embryonic and mature ovine cartilage. We also provide Western blot evidence for GLUT1 and GLUT9 expression in mature ovine chondrocytes. Ovine embryos (developmental stages E32 to E36 and E42 to E45) were obtained from pregnant ewes humanely killed by injection with sodium pentobarbitone. Embryos were fixed and processed for immunohistochemistry. Polyclonal antibodies to GLUT1 and GLUT9 revealed that both transporters are expressed in developing chondrocytes in ovine embryos and in the superficial, middle and deep layers of ovine cartilage from mature animals. GLUT1 expression was observed in erythrocytes and organs including heart, liver, and kidney. GLUT9 was also found in heart, kidney and liver. Western blotting confirmed the presence of the GLUT1 protein which migrated between the 50 and 64 kDa markers and two specific GLUT9 bands migrating under the 50 and 60 kDa markers, respectively. The presence of GLUT1 and GLUT9 in developing joints of ovine embryos suggests that these proteins may be important in glucose delivery to developing chondroblasts. Expression of these GLUT isoforms may be an important bioenergetic adaptation for chondrocytes in the extracellular matrix of developing cartilage.

  • Phillips T, Ferraz I, Bell S, Clegg PD, Carter SD, Mobasheri A. (2005) 'Differential regulation of the GLUT1 and GLUT3 glucose transporters by growth factors and pro-inflammatory cytokines in equine articular chondrocytes.'. Vet J, England: 169 (2), pp. 216-222.

    Abstract

    Glucose serves as the major energy substrate for articular chondrocytes and as the main precursor for the synthesis of extracellular matrix glycosaminoglycans in cartilage. Chondrocytes have been shown to express several glucose transporter (GLUT) isoforms including GLUT1 and GLUT3. The aim of this investigation was to determine the effects of endocrine and cytokine factors on the capacity of equine articular chondrocytes for transporting 2-deoxy-d-[2,6-3H] glucose and on the expression levels of GLUT1 and GLUT3. Chondrocytes maintained in monolayer culture were stimulated for 24 h with TNF-alpha (100 ng mL(-1)), IL-1beta (100 ng mL(-1)), IGF-I (20 ng mL(-1)), TGF-beta (20 ng mL(-1)) and insulin (12.5 microg mL(-1)) before measuring uptake of non-metabolizable 2-deoxyglucose in the presence and absence of the glucose transport inhibitor cytochalasin B. Polyclonal antibodies to GLUT1 and GLUT were used to compare GLUT1 and GLUT3 expression in stimulated and un-stimulated alginate encapsulated chondrocytes by Western blotting. Results indicated that 2-deoxyglucose uptake was inhibited by up to 95% in the presence of cytochalasin B suggesting that glucose uptake into equine chondrocytes is GLUT-mediated. Insulin had no effect on glucose uptake, but treatment with IGF-I, TGF-beta, IL-1beta and TNF-alpha resulted in a significant increase (>65%) in 2-deoxyglucose uptake compared to control values. GLUT1 was found to be increased in chondrocytes stimulated with all the growth factors and cytokines but GLUT 3 was only upregulated by IGF-I. The data presented support a critical role for glucose in the responses of equine articular chondrocytes to pro-inflammatory cytokines and anabolic endocrine factors.

  • Duquette RA, Shmygol A, Vaillant C, Mobasheri A, Pope M, Burdyga T, Wray S. (2005) 'Vimentin-positive, c-kit-negative interstitial cells in human and rat uterus: a role in pacemaking?'. Biol Reprod, United States: 72 (2), pp. 276-283.

    Abstract

    The mechanism underlying spontaneous pacemaker potential in the uterus is not clearly understood. Several spontaneously active smooth muscles have interstitial cells of Cajal (ICCs) or ICC-like cells. We therefore examined cells from freshly dispersed uterine muscle strips (from pregnant human and rat myometrium) and in situ uterine preparations to determine the cell types present. Both preparations revealed numerous ICC-like cells; they were multipolar, with spider-like projections and enlarged central regions. These cells were readily distinguished from uterine myocytes by their morphology and ultrastructure, i.e., no myofilaments, numerous mitochondria, caveolae, and filaments. In addition, the ICC-like cells were noncontractile. These cells were negative to c-kit, a classic marker for ICCs. They stained positive for the intermediate filament, vimentin, a marker for cells of mesenchymal origin but not differentiated myocytes. The ICC-like cells had a more or less stable resting membrane potential of -58+/-7 mV compared with smooth-muscle cells, -65+/-13 mV, and produced outward current in response to voltage clamp pulses. However, in contrast with uterine myocytes, inward currents were not observed. This is the first description of ICC-like cells in myometrium and their role in the uterus is discussed, as possible inhibitors of intrinsic smooth-muscle activity.

  • Mobasheri A, Wray S, Marples D. (2005) 'Distribution of AQP2 and AQP3 water channels in human tissue microarrays.'. J Mol Histol, Netherlands: 36 (1-2), pp. 1-14.

    Abstract

    The objective of this investigation was to use semi-quantitative immunohistochemistry to determine the distribution and expression levels of AQP2 and AQP3 proteins in normal human Tissue MicroArrays. Expression of the vasopressin regulated AQP2 was observed in a limited number of tissues. AQP2 was prominent in the apical and subapical plasma membranes of cortical and medullary renal collecting ducts. Surprisingly, weak AQP2 immunoreactivity was also noted in pancreatic islets, fallopian tubes and peripheral nerves. AQP2 was also localized to selected parts of the central nervous system (ependymal cell layer, subcortical white matter, hippocampus, spinal cord) and selected cells in the gastrointestinal system (antral and oxyntic gastric mucosa, small intestine and colon). These findings corroborate the restricted tissue distribution of AQP2. AQP3 was strongly expressed in many of the human tissues examined particularly in basolateral membranes of the distal nephron (medullary collecting ducts), distal colon, upper airway epithelia, transitional epithelium of the urinary bladder, tracheal, bronchial and nasopharyngeal epithelium, stratified squamous epithelial cells of the esophagus, and anus. AQP3 was moderately expressed in basolateral membranes of prostatic tubuloalveolar epithelium, pancreatic ducts, uterine endometrium, choroid plexus, articular chondrocytes, subchondral osteoblasts and synovium. Low AQP3 levels were also detected in skeletal muscle, cardiac muscle, gastric pits, seminiferous tubules, lymphoid vessels, salivary and endocrine glands, amniotic membranes, placenta and ovary. The abundance of basolateral AQP3 in epithelial tissues and its expression in many non-epithelial cells suggests that this aquaglyceroporin is a major participant in barrier hydration and water and osmolyte homeostasis in the human body.

  • Goggs R, Vaughan-Thomas A, Clegg PD, Carter SD, Innes JF, Mobasheri A, Shakibaei M, Schwab W, Bondy CA. (2005) 'Nutraceutical therapies for degenerative joint diseases: a critical review.'. Crit Rev Food Sci Nutr, United States: 45 (3), pp. 145-164.

    Abstract

    There is growing recognition of the importance of nutritional factors in the maintenance of bone and joint health, and that nutritional imbalance combined with endocrine abnormalities may be involved in the pathogenesis of osteoarthritis (OA) and osteochondritis dissecans (OCD). Despite this, dietary programs have played a secondary role in the management of these connective tissue disorders. Articular cartilage is critically dependent upon the regular provision of nutrients (glucose and amino acids), vitamins (particularly vitamin C), and essential trace elements (zinc, magnesium, and copper). Therefore, dietary supplementation programs and nutraceuticals used in conjunction with non-steroidal, anti-inflammatory drugs (NSAIDs) may offer significant benefits to patients with joint disorders, such as OA and OCD. This article examines the available clinical evidence for the efficacy of nutraceuticals, antioxidant vitamin C, polyphenols, essential fatty acids, and mineral cofactors in the treatment of OA and related joint disorders in humans and veterinary species. This article also attempts to clarify the current state of knowledge. It also highlights the need for additional targeted research to elucidate the changes in nutritional status and potential alterations to the expression of plasma membrane transport systems in synovial structures in pathophysiological states, so that current therapy and future treatments may be better focused.

  • Schulze-Tanzil G, Mobasheri A, Sendzik J, John T, Shakibaei M. (2004) 'Effects of curcumin (diferuloylmethane) on nuclear factor kappaB signaling in interleukin-1beta-stimulated chondrocytes.'. Ann N Y Acad Sci, United States: 1030, pp. 578-586.

    Abstract

    Curcumin (diferuloylmethane) is a nontoxic dietary pigment in tumeric and curry and a potent inhibitor of the common transcription factor Nuclear Factor kappaB (NF-kappaB) in several cell types. It is well established that some of the catabolic effects of the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha in osteoarthritis are regulated by the activation of NF-kappaB. Therefore, the aim of this study was to determine whether curcumin modifies the catabolic response of chondrocytes to IL-1beta. Human articular chondrocytes were prestimulated with 10 ng/mL IL-1beta for 0, 4, 8, 12, or 24 h and then cotreated with 50 microM curcumin for 0, 12, 24, 36, or 48 h. Synthesis of the cartilage-specific collagen type II and matrix-degrading enzyme matrix metalloproteinase-3 (MMP-3) was investigated in chondrocytes by Western blot analysis. Activation and nuclear translocation of NF-kappaB were observed by immunofluorescence microscopy. IL-1beta induced a decrease in collagen type II and upregulation of MMP-3 in a time-dependent manner. Upregulation of MMP-3 was inhibited by curcumin in a time-dependent manner. In addition, IL-1beta-induced a decrease in type II collagen, which was relieved by curcumin treatment. In response to IL-1beta, NF-kappaB translocated to the nucleus, but translocation was inhibited by curcumin, as revealed by immunofluorescence microscopy. Taken together, these results confirmed an IL-1beta-mediated upregulation of proinflammatory MMP-3 in chondrocytes via an NF-kappaB activation mechanism. Curcumin protected chondrocytes from the catabolic effects of IL-1beta, such as MMP-3 upregulation, and interestingly also relieved cytokine-induced suppression of matrix protein synthesis. Therefore, curcumin antagonizes crucial catabolic effects of IL-1beta signaling that are known to contribute to the pathogenesis of osteoarthritis.

  • Mobasheri A, Trujillo E, Bell S, Carter SD, Clegg PD, Martín-Vasallo P, Marples D. (2004) 'Aquaporin water channels AQP1 and AQP3, are expressed in equine articular chondrocytes.'. Vet J, England: 168 (2), pp. 143-150.

    Abstract

    Chondrocytes exist in an unusual and highly variable ionic and osmotic environment in the extracellular matrix of articular cartilage. Alterations to the ionic and osmotic environment of chondrocytes influence the volume and ionic content of the cells, which, in turn, modifies the rate at which extracellular matrix macromolecules are synthesized and degraded. Thus, regulation of the water and solute content of chondrocytes will profoundly affect their anabolic and catabolic functions. The water content of cells is effectively influenced by the abundance of aquaporin (AQP) water channels. Recent studies have shown that several AQP water channel isoforms are expressed in chondrocytes from Meckel's cartilage, developing teeth and other orofacial tissues. The aim of the present investigation was to determine if chondrocytes from equine articular cartilage express AQP water channels. Polyclonal antibodies to AQP1, AQP2 and AQP3 were used in conjunction with immunohistochemistry, immunoblotting and quantitative flow cytometry to determine if AQP1, AQP2 and AQP3 are expressed in equine articular chondrocytes. Our studies show that AQP1 and AQP3 are expressed by chondrocytes in articular cartilage in situ and in isolated chondrocytes. We found no evidence for expression of AQP2, the vasopressin-regulated water channel in chondrocytes. AQP1 and AQP3 may be involved in the transport of water and small solutes and osmotically active metabolites across the chondrocyte plasma membrane during volume regulatory behaviour. AQP1 may be involved in transporting metabolic water. AQP3 may participate in the transport of glycerol and structurally related molecules.

  • Schulze-Tanzil G, Mobasheri A, Clegg PD, Sendzik J, John T, Shakibaei M. (2004) 'Cultivation of human tenocytes in high-density culture.'. Histochem Cell Biol, Germany: 122 (3), pp. 219-228.

    Abstract

    Limited supplies of tendon tissue for use in reconstructive surgery require development of phenotypically stable tenocytes cultivated in vitro. Tenocytes in monolayer culture display an unstable phenotype and tend to dedifferentiate, but those in three-dimensional culture may remain phenotypically and functionally differentiated. In this study we established a three-dimensional high-density culture system for cultivation of human tenocytes for tissue engineering. Human tenocytes were expanded in monolayer culture before transfer to high-density culture. The synthesis of major extracellular matrix proteins and the ultrastructural morphology of the three-dimensional cultures were investigated for up to 2 weeks by electron microscopy, immunohistochemistry, immunoblotting and quantitative, real-time PCR. Differentiated tenocytes were able to survive over a period of 14 days in high-density culture. During the culture period tenocytes exhibited a typical tenocyte morphology embedded in an extensive extracellular matrix containing cross-striated collagen type I fibrils and proteoglycans. Moreover, expression of the tendon-specific marker scleraxis underlined the tenocytic identity of these cells. Taken together, we conclude that the three-dimensional high-density cultures may be useful as a new approach for obtaining differentiated tenocytes for autologous tenocyte transplantation to support tendon and ligament healing and to investigate the effect of tendon-affecting agents on tendon in vitro.

  • Schulze-Tanzil G, Mobasheri A, de Souza P, John T, Shakibaei M. (2004) 'Loss of chondrogenic potential in dedifferentiated chondrocytes correlates with deficient Shc-Erk interaction and apoptosis.'. Osteoarthritis Cartilage, England: 12 (6), pp. 448-458.

    Abstract

    If dedifferentiated chondrocytes could be induced to redifferentiate in vitro, then we might thereby be furnished with a population of phenotypically stable cells for autologous implantation in reconstructive surgery. We therefore investigated the redifferentiation capabilities of chondrocytes which, having migrated from alginate beads to form a monolayer, were subsequently passaged. We also characterized the molecular traits of irreversibly dedifferentiated cells.

  • Mobasheri A, Shakibaei M, Marples D. (2004) 'Immunohistochemical localization of aquaporin 10 in the apical membranes of the human ileum: a potential pathway for luminal water and small solute absorption.'. Histochem Cell Biol, Germany: 121 (6), pp. 463-471.

    Abstract

    A new member of the aquaporin family (AQP10) has recently been identified in the human small intestine by molecular cloning and in situ hybridization. Ribonuclease protection assay and northern blotting have demonstrated that AQP10 is expressed in the human duodenum and jejunum. However, the subcellular distribution of the AQP10 protein and its plasma membrane polarization have not yet been established. The objective of this study was to determine the distribution of the AQP10 protein in the human ileum by immunohistochemistry and western blotting using a polyclonal antibody raised against a unique 17-amino acid peptide derived from the human AQP10 sequence. The distribution of the AQP1 and AQP3 proteins was also studied by immunohistochemical staining using affinity-purified polyclonal antibodies. Results revealed that the AQP10 protein is preferentially targeted to the apical membrane domain of absorptive intestinal epithelial cells, whereas AQP3 is located in the basolateral membrane of the cells and AQP1 expression is restricted to the mucosal microvascular endothelia. The presence of AQP10 in the apical membrane of intestinal villi suggests that this protein may represent an entry pathway for water and small solutes from the lumen across to the mucosal side.

  • Trujillo E, González T, Marín R, Martín-Vasallo P, Marples D, Mobasheri A. (2004) 'Human articular chondrocytes, synoviocytes and synovial microvessels express aquaporin water channels; upregulation of AQP1 in rheumatoid arthritis.'. Histol Histopathol, Spain: 19 (2), pp. 435-444.

    Abstract

    Recent studies have shown that aquaporin water channels are expressed in human Meckel's cartilage. The aim of the present investigation was to determine if human articular chondrocytes and synoviocytes express aquaporin 1 (AQP1) water channels and to establish if there are any alterations in AQP1 expression in osteoarticular disorders such as osteoarthritis (OA) and rheumatoid arthritis (RA). Immunohistochemistry was employed semi-quantitatively to compare the expression of AQP1 in human chondrocytes derived from normal, OA and RA joints. PCR, cloning and sequencing confirmed the presence of AQP1 transcripts in chondrocytes. Normal human tissue microarrays including samples of kidney, choroid plexus and pancreas were used as positive controls for AQP1 expression. In most tissues AQP1 was expressed along endothelial barriers. In the kidney AQP1 was present in the glomerular capillary endothelium, proximal tubule and descending thin limbs. AQP1 was also localized to pancreatic ducts and acini and the apical membrane domain of the choroid plexus. Immunohistochemistry showed that AQP1 is expressed in synovial micro-vessels, synoviocytes and predominantly in chondrocytes located in the deep zone of articular cartilage. Image analysis of normal, OA and RA cartilage suggested that AQP1 may be upregulated in RA. This is the first report of AQP1 mRNA and protein expression in articular chondrocytes and synoviocytes. These findings suggest a potential role for AQP1 and possibly other members of the AQP gene family in the movement of extracellular matrix and metabolic water across the membranes of chondrocytes and synoviocytes for the purposes of chondrocyte volume regulation and synovial homeostasis.

  • Mobasheri A, Critchlow K, Clegg PD, Carter SD, Canessa CM. (2004) 'Chronic equine laminitis is characterised by loss of GLUT1, GLUT4 and ENaC positive laminar keratinocytes.'. Equine Vet J, England: 36 (3), pp. 248-254.

    Abstract

    Equine laminitis is a multifactorial connective tissue disorder with major implications for the welfare of horses. There are few published studies on phenotypic markers for identification of equine laminar keratinocytes using immunohistochemical techniques.

  • Mobasheri A, Marples D. (2004) 'Expression of the AQP-1 water channel in normal human tissues: a semiquantitative study using tissue microarray technology.'. Am J Physiol Cell Physiol, United States: 286 (3), pp. C529-C537.

    Abstract

    Aquaporin water channels are a family of membrane proteins that facilitate water movement across biological membranes. Aquaporin-1 (AQP-1) has been found to be important in osmotic water movement across cell membranes of epithelial and endothelial barriers. However, the distribution of AQP-1 in many normal human tissues is still unknown. The aim of this study was to use immunohistochemistry and semiquantitative histomorphometric analysis to determine the tissue distribution and relative expression of AQP-1 in normal human tissues using tissue microarray (TMA) technology. The normal human TMAs employed in this study included cardiovascular, respiratory, gastrointestinal, hepatic and pancreatobiliary, oral, salivary, nasal, mammary, fetal, endocrine, genital tract, central and peripheral nervous systems, urinary tract, skin, cartilage, and other soft connective tissues. Immunohistochemistry and semiquantitative histomorphometric analysis confirmed the presence of AQP-1 in endothelial barriers of almost all tissues and in many epithelial barriers. AQP-1 was highly expressed in the renal cortex, choroid plexus, and pancreatic ducts. AQP-1 expression levels were surprisingly high in the anus, gallbladder, and liver; moderate expression was also detected in the hippocampus and ependymal cells of the central nervous system. This is the first report of AQP-1 protein distribution in normal human TMAs. These findings confirm the presence of AQP-1 in human endothelia and selected water-transporting epithelia and several new locations, including mammary epithelium, articular chondrocytes, synoviocytes, and synovial microvessels where AQP-1 may be involved in milk production, chondrocyte volume regulation, synovial fluid secretion, and homeostasis, respectively.

  • Mobasheri A, Marples D. (2004) 'Expression of the AQP-1 water channel in normal human tissues: A semiquantitative study using tissue microarray technology'. American Journal of Physiology - Cell Physiology, 286 (3 55-3)

    Abstract

    Aquaporin water channels are a family of membrane proteins that facilitate water movement across biological membranes. Aquaporin-1 (AQP-1) has been found to be important in osmotic water movement across cell membranes of epithelial and endothelial barriers. However, the distribution of AQP-1 in many normal human tissues is still unknown. The aim of this study was to use immunohistochemistry and semiquantitative histomorphometric analysis to determine the tissue distribution and relative expression of AQP-1 in normal human tissues using tissue microarray (TMA) technology. The normal human TMAs employed in this study included cardiovascular, respiratory, gastrointestinal, hepatic and pancreatobiliary, oral, salivary, nasal, mammary, fetal, endocrine, genital tract, central and peripheral nervous systems, urinary tract, skin, cartilage, and other soft connective tissues. Immunohistochemistry and semiquantitative histomorphometric analysis confirmed the presence of AQP-1 in endothelial barriers of almost all tissues and in many epithelial barriers. AQP-1 was highly expressed in the renal cortex, choroid plexus, and pancreatic ducts. AQP-1 expression levels were surprisingly high in the anus, gallbladder, and liver; moderate expression was also detected in the hippocampus and ependymal cells of the central nervous system. This is the first report of AQP-1 protein distribution in normal human TMAs. These findings confirm the presence of AQP-1 in human endothelia and selected water-transporting epithelia and several new locations, including mammary epithelium, articular chondrocytes, synoviocytes, and synovial microvessels where AQP-1 may be involved in milk production, chondrocyte volume regulation, synovial fluid secretion, and homeostasis, respectively.

  • Mobasheri A, Airley R, Foster CS, Schulze-Tanzil G, Shakibaei M. (2004) 'Post-genomic applications of tissue microarrays: basic research, prognostic oncology, clinical genomics and drug discovery.'. Histol Histopathol, Spain: 19 (1), pp. 325-335.

    Abstract

    Tissue microarrays (TMAs) are an ordered array of tissue cores on a glass slide. They permit immunohistochemical analysis of numerous tissue sections under identical experimental conditions. The arrays can contain samples of every organ in the human body, or a wide variety of common tumors and obscure clinical cases alongside normal controls. The arrays can also contain pellets of cultured tumor cell lines. These arrays may be used like any histological section for immunohistochemistry and in situ hybridization to detect protein and gene expression. This new technology will allow investigators to analyze numerous biomarkers over essentially identical samples, develop novel prognostic markers and validate potential drug targets. The ability to combine TMA technology with DNA microarrays and proteomics makes it a very attractive tool for analysis of gene expression in clinically stratified tumor specimens and relate expression of each particular protein with clinical outcome. Public domain software allows researchers to examine digital images of individual histological specimens from TMAs, evaluate and score them and store the quantitative data in a relational database. TMA technology may be specifically applied to the profiling of proteins of interest in other pathophysiological conditions such as congestive heart failure, renal disease, hypertension, diabetes, cystic fibrosis and neurodegenerative disorders. This review is intended to summarize the strengths and weaknesses of TMA technology which will have an increasingly important role in the laboratories of the post-genomic era.

  • Arteaga MF, Gutiérrez R, Avila J, Mobasheri A, Díaz-Flores L, Martín-Vasallo P. (2004) 'Regeneration influences expression of the Na+, K+-atpase subunit isoforms in the rat peripheral nervous system.'. Neuroscience, United States: 129 (3), pp. 691-702.

    Abstract

    Neural injury triggers changes in the expression of a large number of gene families. Particularly interesting are those encoding proteins involved in the generation, propagation or restoration of electric potentials. The expression of the Na+, K+-ATPase subunit isoforms (alpha, beta and gamma) was studied in dorsal root ganglion (DRG) and sciatic nerve of the rat in normal conditions, after axotomy and during regeneration. In normal DRG, alpha1 and alpha2 are expressed in the plasma membrane of all cell types, while there is no detectable signal for alpha3 in most DRG cells. After axotomy, alpha1 and alpha2 expression decreases evenly in all cells, while there is a remarkable onset in alpha3 expression, with a peak about day 3, which gradually disappears throughout regeneration (day 7). beta1 Is restricted to the nuclear envelope and plasma membrane of neurons and satellite cells. Immediately after injury, beta1 shows a homogeneous distribution in the soma of neurons. No beta2 expression was found. Beta3 Specific immunofluorescence appears in all neurons, although it is brightest in the smallest, diminishing progressively after injury until day 3 and, thereafter, increasing in intensity, until it reaches normal levels. FXYD7 is expressed weakly in a few DRG neurons (less than 2%) and Schwann cells. It increases intensely in satellite cells immediately after axotomy, and in all cell types at day 3. Transient switching of members of the Na+, K+-ATPase isoform family elicited by axotomy suggests variations in the sodium pump isozymes with different affinities for Na+, K+ and ATP from those in intact nerve. This adaptation may be important for regeneration.

  • Schwab W, Schulze-Tanzil G, Mobasheri A, Dressler J, Kotzsch M, Shakibaei M. (2004) 'Interleukin-1beta-induced expression of the urokinase-type plasminogen activator receptor and its co-localization with MMPs in human articular chondrocytes.'. Histol Histopathol, Spain: 19 (1), pp. 105-112.

    Abstract

    The urokinase-type plasminogen activator receptor (uPAR) plays a critical role in cartilage degradation during osteoarthritis as it regulates pericellular proteolysis mediated by serine proteinases. Another important family of proteinases responsible for ECM destruction in arthritis are the matrix metalloproteinases (MMPs). MMPs are regulated by IL-1beta, a cytokine that plays a pivotal role in pathogenesis of osteoarthritis. This study was undertaken to address two questions: 1. Is uPAR-expression regulated by proinflammatory cytokines such as IL-1beta? 2. Does a functional co-localization exist between uPAR and MMPs? By immunohistochemical analysis we observed enhanced expression of uPAR on chondrocytes derived from osteoarthritic human cartilage compared to non-osteoarthritic controls. We found an IL-1beta-mediated expression of uPAR by immunoelectron microscopy. Western blot analysis demonstrated that IL-1beta-stimulated expression of uPAR on chondrocytes in vitro increased in a dose-dependent manner. Furthermore, we found a functional co-localization between uPAR and MMP-9 on IL-1beta-stimulated chondrocytes by means of a co-immunoprecipitation assay. Expression of uPAR in osteoarthritic cartilage but not in healthy cartilage suggests that uPAR plays a role in cartilage breakdown. We propose that uPAR-mediated effects e.g. pericellular proteolysis are one of other cytokine (IL-1beta)-mediated events that contribute to the pathogenesis of osteoarthritis. Furthermore, we found that MMPs and uPAR were part of the same cell surface complexes in chondrocytes. This finding underlines a functional interaction between MMPs and the serine proteinase system in the fine regulation of pericellular proteolysis. Interfering with uPAR signaling may present a novel target in arthritis therapy to prevent excessive proteolytic degradation.

  • Mobasheri A, Pestov NB, Papanicolaou S, Kajee R, Cózar-Castellano I, Avila J, Martín-Vasallo P, Foster CS, Modyanov NN, Djamgoz MB. (2003) 'Expression and cellular localization of Na,K-ATPase isoforms in the rat ventral prostate.'. BJU Int, England: 92 (7), pp. 793-802.

    Abstract

    To determine the expression and plasma membrane domain location of isoforms of Na,K-ATPase in the rat ventral prostate.

  • Goggs R, Carter SD, Schulze-Tanzil G, Shakibaei M, Mobasheri A. (2003) 'Apoptosis and the loss of chondrocyte survival signals contribute to articular cartilage degradation in osteoarthritis.'. Vet J, England: 166 (2), pp. 140-158.

    Abstract

    Apoptotic death of articular chondrocytes has been implicated in the pathogenesis of osteoarthritis (OA). Apoptotic pathways in chondrocytes are multi-faceted, although some cascades appear to play a greater in vivo role than others. Various catabolic processes are linked to apoptosis in OA cartilage, contributing to the reduction in cartilage integrity. Recent studies suggest that beta1-integrin mediated cell-matrix interactions provide survival signals for chondrocytes. The loss of such interactions and the inability to respond to IGF-1 stimulation may be partly responsible for the hypocellularity and matrix degradation that characterises OA. Here we have reviewed the literature in this area of cartilage cell biology in an effort to consolidate the existing information into a plausible hypothesis regarding the involvement of apoptosis in the pathogenesis of OA. Understanding of the interactions that promote chondrocyte apoptosis and cartilage hypocellularity is essential for developing appropriately targeted therapies for inhibition of chondrocyte apoptosis and the treatment of OA.

  • Shakibaei M, Schulze-Tanzil G, Mobasheri A, Beichler T, Dressler J, Schwab W. (2003) 'Expression of the VEGF receptor-3 in osteoarthritic chondrocytes: stimulation by interleukin-1 beta and association with beta 1-integrins.'. Histochem Cell Biol, Germany: 120 (3), pp. 235-241.

    Abstract

    Recent studies have demonstrated enhanced expression of vascular endothelial growth factor and vascular endothelial growth factor receptor (VEGFR)-1 and -2 in chondrocytes of rheumatoid and osteoarthritic cartilage. Since expression of VEGFR-3 ( Flt-4) in chondrocytes has not yet been investigated, we studied the distribution of VEGFR-3 in osteoarthritic cartilage samples by immunohistochemistry and immunoelectron microscopy. Furthermore, we looked for functional colocalization of VEGFR-3 with the signal transduction receptor beta(1)-integrin. Superficial osteoarthritic chondrocytes exhibited VEGFR-3 expression in the cytoplasm and on the cell membrane. Using western blotting we could demonstrate that interleukin-1 beta (IL-1 beta) stimulates the expression of VEGFR-3 in chondrocytes in vitro in a dose-dependent manner. By coimmunoprecipitation assay we found a functional complex between the beta(1)-integrin and VEGFR-3 in IL-1 beta-stimulated chondrocytes indicating that activated VEGFR-3 may interact with beta(1)-integrin and associated subcellular pathways in osteoarthritic chondrocytes. Taken together with results of previous studies showing that beta(1)-integrins were also associated with other surface receptors and proteins in chondrocytes that cause cartilage destruction in arthritis (for example, urokinase-type plasminogen activator receptor and matrix metalloproteinases), we can hypothesize that signal transduction by these receptor complexes via beta(1)-integrins may play a crucial role in pathogenesis of osteoarticular disorders. Further work needs to be done to elucidate downstream signaling events activated by these receptors.

  • Clegg PD, Mobasheri A. (2003) 'Chondrocyte apoptosis, inflammatory mediators and equine osteoarthritis.'. Vet J, England: 166 (1), pp. 3-4.
  • Mobasheri A, Fox R, Evans I, Cullingham F, Martín-Vasallo P, Foster CS. (2003) 'Epithelial Na, K-ATPase expression is down-regulated in canine prostate cancer; a possible consequence of metabolic transformation in the process of prostate malignancy.'. Cancer Cell Int, 3 (1)

    Abstract

    BACKGROUND: An important physiological function of the normal prostate gland is the synthesis and secretion of a citrate rich prostatic fluid. In prostate cancer, citrate production levels are reduced as a result of altered cellular metabolism and bioenergetics. Na, K-ATPase is essential for citrate production since the inward Na+ gradients it generates are utilized for the Na+ dependent uptake of aspartate, a major substrate for citrate synthesis. The objective of this study was to compare the expression of previously identified Na, K-ATPase isoforms in normal canine prostate, benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma (PCa) using immunohistochemistry in order to determine whether reduced citrate levels in PCa are also accompanied by changes in Na, K-ATPase expression. RESULTS: Expression of Na, K-ATPase alpha1 and beta1 isoforms was observed in the lateral and basolateral plasma membrane domains of prostatic epithelial cells in normal and BPH prostates. Canine kidney was used as positive control for expression of Na, K-ATPase alpha1 and gamma isoforms. The alpha1 isoform was detected in abundance in prostatic epithelial cells but there was no evidence of alpha2, alpha3 or gamma subunit expression. In advanced PCa, Na, K-ATPase alpha1 isoform expression was significantly lower compared to normal and BPH glands. The abundant basolateral immunostaining observed in normal and BPH tissue was significantly attenuated in PCa. CONCLUSION: The loss of epithelial structure and function and the transformation of normal epithelial cells to malignant cells in the canine prostate have important implications for cellular metabolism and are accompanied by a down regulation of Na, K-ATPase.

  • Shakibaei M, Mobasheri A. (2003) 'β1-integrins co-localize with Na, K-ATPase, epithelial sodium channels (ENaC) and voltage activated calcium channels (VACC) in mechanoreceptor complexes of mouse limb-bud chondrocytes'. Histology and Histopathology, 18 (2), pp. 343-351.

    Abstract

    Interactions between chondrocytes and their extracellular matrix are partly mediated by β1-integrin receptors. Recent studies have shown that β1-integrins co-localize with a variety of cytoskeletal complexes, signaling proteins and growth factor receptors. Since mechanosensitive ion channels and integrins have been proposed to participate in skeletal mechanotransduction, in this study, we investigated the possible co-localization of β1-integrins with two ion channels and a P-type ATPase in mouse limb-bud chondrocytes. The β1 subunits of Na, K-ATPase, the epithelial sodium channel (ENaC) and the voltage activated calcium channel (VACC) were immunostained in organoid cultures derived from limb-buds of 12-day-old mice using well-characterized antibodies . Indirect immunofluorescence revealed abundant expression of β1-integrins and each of the selected systems in limb-bud chondrocytes. Two-fluorochrome immunostaining demonstrated that β1-integrin, Na, K-ATPase, ENaC and VACC co-localize in chondrocytes. Co-imunoprecipitation experiments revealed co-localization and association of integrins with ENaC, VACC and Na, K-ATPase. Cellular responses and signaling cascades initiated by the influx of calcium or sodium through putative mechanosensitive channels may be regulated more effectively if such channels were organized around integrins with receptors, kinases and cytoskeletal complexes clustered about them. The close proximity of ATPase ion pumps such as Na, K-ATPase to chondrocyte mechanoreceptor complexes could facilitate rapid homeostatic responses to the ionic perturbations brought about by activation of mechanically gated cation channels and efficiently regulate the intracellular milieu of chondrocytes.

  • Shakibaei M, Mobasheri A. (2003) 'Beta1-integrins co-localize with Na, K-ATPase, epithelial sodium channels (ENaC) and voltage activated calcium channels (VACC) in mechanoreceptor complexes of mouse limb-bud chondrocytes.'. Histol Histopathol, Spain: 18 (2), pp. 343-351.

    Abstract

    Interactions between chondrocytes and their extracellular matrix are partly mediated by beta1-integrin receptors. Recent studies have shown that beta1-integrins co-localize with a variety of cytoskeletal complexes, signaling proteins and growth factor receptors. Since mechanosensitive ion channels and integrins have been proposed to participate in skeletal mechanotransduction, in this study, we investigated the possible co-localization of beta1-integrins with two ion channels and a P-type ATPase in mouse limb-bud chondrocytes. The alpha subunits of Na, K-ATPase, the epithelial sodium channel (ENaC) and the voltage activated calcium channel (VACC) were immunostained in organoid cultures derived from limb-buds of 12-day-old mice using well-characterized antibodies. Indirect immunofluorescence revealed abundant expression of beta1-integrins and each of the selected systems in limb-bud chondrocytes. Two-fluorochrome immunostaining demonstrated that beta1-integrin, Na, K-ATPase, ENaC and VACC co-localize in chondrocytes. Co-imunoprecipitation experiments revealed co-localization and association of integrins with ENaC, VACC and Na, K-ATPase. Cellular responses and signaling cascades initiated by the influx of calcium or sodium through putative mechanosensitive channels may be regulated more effectively if such channels were organized around integrins with receptors, kinases and cytoskeletal complexes clustered about them. The close proximity of ATPase ion pumps such as Na, K-ATPase to chondrocyte mechanoreceptor complexes could facilitate rapid homeostatic responses to the ionic perturbations brought about by activation of mechanically gated cation channels and efficiently regulate the intracellular milieu of chondrocytes.

  • Mobasheri A, Evans I, Martín-Vasallo P, Foster CS. (2003) 'Expression and cellular localization of Na,K-ATPase isoforms in dog prostate in health and disease.'. Ann N Y Acad Sci, United States: 986, pp. 708-710.
  • Floyd R, Mobasheri A, Martín-Vasallo P, Wray S. (2003) 'Na,K-ATPase isoforms in pregnant and nonpregnant rat uterus.'. Ann N Y Acad Sci, United States: 986, pp. 614-616.
  • Richardson S, Neama G, Phillips T, Bell S, Carter SD, Moley KH, Moley JF, Vannucci SJ, Mobasheri A. (2003) 'Molecular characterization and partial cDNA cloning of facilitative glucose transporters expressed in human articular chondrocytes; stimulation of 2-deoxyglucose uptake by IGF-I and elevated MMP-2 secretion by glucose deprivation.'. Osteoarthritis Cartilage, England: 11 (2), pp. 92-101.

    Abstract

    Recent evidence suggests that human chondrocytes express several facilitative glucose transporter (GLUT) isoforms and also that 2-deoxyglucose transport is accelerated by cytokine stimulation. The aim of the present investigation was to determine if human articular chondrocytes express any of the recently identified members of the GLUT/SLC2A gene family and to examine the effects of endocrine factors, such as insulin and IGF-I on the capacity of human chondrocytes for transporting 2-deoxyglucose.

  • Mobasheri A, Vannucci SJ, Bondy CA, Carter SD, Innes JF, Arteaga MF, Trujillo E, Ferraz I, Shakibaei M, Martín-Vasallo P. (2002) 'Glucose transport and metabolism in chondrocytes: a key to understanding chondrogenesis, skeletal development and cartilage degradation in osteoarthritis.'. Histol Histopathol, Spain: 17 (4), pp. 1239-1267.

    Abstract

    Despite the recognition that degenerative cartilage disorders like osteoarthritis (OA) and osteochondritis dissecans (OCD) may have nutritional abnormalities at the root of their pathogenesis, balanced dietary supplementation programs have played a secondary role in their management. This review emphasizes the importance and role of nutritional factors such as glucose and glucose-derived sugars (i.e. glucosamine sulfate and vitamin C) in the development, maintenance, repair, and remodeling of cartilage. Chondrocytes, the cells of cartilage, consume glucose as a primary substrate for ATP production in glycolysis and utilize glucosamine sulfate and other sulfated sugars as structural components for extracellular matrix synthesis and are dependent on hexose uptake and delivery to metabolic and biosynthetic pools. Data from several laboratories suggests that chondrocytes express multiple isoforms of the GLUT/SLC2A family of glucose/polyol transporters. These facilitative glucose transporter proteins are expressed in a tissue and cell-specific manner, exhibit distinct kinetic properties, and are developmentally regulated. They may also be regulated by endocrine factors like insulin and insulin-like growth factor I (IGF-I) and cytokines such as interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF-alpha). Recent studies suggest that degeneration of cartilage may be triggered by metabolic disorders of glucose balance and that OA occurs coincident with metabolic disease, endocrine dysfunction and diabetes mellitus. Based on these metabolic, endocrine and developmental considerations we present a novel hypothesis regarding the role of glucose transport and metabolism in cartilage physiology and pathophysiology and speculate that supplementation with sugar-derived vitamins and nutraceuticals may benefit patients with degenerative joint disorders.

  • Francis MJ, Lees RL, Trujillo E, Martín-Vasallo P, Heersche JN, Mobasheri A. (2002) 'ATPase pumps in osteoclasts and osteoblasts.'. Int J Biochem Cell Biol, England: 34 (5), pp. 459-476.

    Abstract

    Osteoblasts, osteocytes and osteoclasts are specialised cells of bone that play crucial roles in the formation, maintenance and resorption of bone matrix. Bone formation and resorption critically depend on optimal intracellular calcium and phosphate homeostasis and on the expression and activity of plasma membrane transport systems in all three cell types. Osteotropic agents, mechanical stimulation and intracellular pH are important parameters that determine the fate of bone matrix and influence the activity, expression, regulation and cell surface abundance of plasma membrane transport systems. In this paper the role of ATPase pumps is reviewed in the context of their expression in bone cells, their contribution to ion homeostasis and their relation to other transport systems regulating bone turnover.

  • Mobasheri A. (2002) 'Role of chondrocyte death and hypocellularity in ageing human articular cartilage and the pathogenesis of osteoarthritis.'. Med Hypotheses, Scotland: 58 (3), pp. 193-197.

    Abstract

    Apoptotic death of articular chondrocytes has been implicated in the pathogenesis of human osteoarthritis. Although nitric oxide and Fas ligand have been shown to be inducers of chondrocyte apoptosis in vitro and in vivo, the contribution of other triggers such as hypoxia, matrix acidosis, abnormal shear stress and catabolic cytokines like interleukin-1beta and tumour necrosis factor alpha has not been examined. It is also not known if growth factors such as insulin like growth factor 1 or anabolic cytokines prevent apoptosis. The intracellular mechanism of effecting apoptotic death depend on whether damage to the mitochondrion or receptor ligation is the primary apoptotic stimulus, since these activate different initiator caspases which then deliver the apoptotic signal to common downstream effector caspases and other proteases. The hypothesis proposed here suggests that by using chondrocytes derived from control and osteoarthritis joints and established human chondrocyte cell lines, it is possible to investigate the relative contributions of major cell death inducing mechanisms and correspondingly which initiating caspase is activated. This understanding is essential for developing appropriately targeted anti-protease therapies for the inhibition of chondrocyte apoptosis in the rational treatment of osteoarthritis.

  • Mobasheri A, Neama G, Bell S, Richardson S, Carter SD. (2002) 'Human articular chondrocytes express three facilitative glucose transporter isoforms: GLUT1, GLUT3 and GLUT9.'. Cell Biol Int, England: 26 (3), pp. 297-300.

    Abstract

    Glucose is an important metabolite and a structural precursor for articular cartilage and its transport has significant consequences for cartilage development and functional integrity. In this study the expression of facilitative glucose transporters (GLUTs) in human chondrocytes was investigated. Results showed that at least three GLUT isoforms (GLUT1, GLUT3 and GLUT9) are expressed by normal chondrocytes. Given the central role of glucose in chondrocyte physiology and metabolism, its regular provision via GLUTs will influence the metabolic activity and survival of chondrocytes in cartilage matrices.

  • Mobasheri A, Carter SD, Martín-Vasallo P, Shakibaei M. (2002) 'Integrins and stretch activated ion channels; putative components of functional cell surface mechanoreceptors in articular chondrocytes.'. Cell Biol Int, England: 26 (1), pp. 1-18.

    Abstract

    Perception of mechanical signals and the biological responses to such stimuli are fundamental properties of load bearing articular cartilage in diarthrodial joints. Chondrocytes utilize mechanical signals to synthesize an extracellular matrix capable of withstanding high loads and shear stresses. Recent studies have shown that chondrocytes undergo changes in shape and volume in a coordinated manner with load induced deformation of the matrix. These matrix changes, together with alterations in hydrostatic pressure, ionic and osmotic composition, interstitial fluid and streaming potentials are, in turn, perceived by chondrocytes. Chondrocyte responses to these stimuli are specific and well coordinated to bring about changes in gene expression, protein synthesis, matrix composition and ultimately biomechanical competence. In this hypothesis paper we propose a chondrocyte mechanoreceptor model incorporating key extracellular matrix macromolecules, integrins, mechanosensitive ion channels, the cytoskeleton and subcellular signal transduction pathways that maintain the chondrocyte phenotype, prevent chondrocyte apoptosis and regulate chondrocyte-specific gene expression.

  • Mobasheri A, Neama G, Bell S, Carter SD. (2001) 'Molecular evidence for two glucose transporters (GLUT1 and GLUT3) in adult human articular cartilage'. International Journal of Experimental Pathology, 82 (6)
  • Shakibaei M, Mobasheri A. (2001) 'Sodium potassium pumps, epithelial sodium channels and voltage gated calcium channels colocalize with β1-integrins in mouse limb-bud chondrocytes in organoid culture'. International Journal of Experimental Pathology, 82 (6)
  • Mobasheri A, Oukrif D, Dawodu SP, Sinha M, Greenwell P, Stewart D, Djamgoz MB, Foster CS, Martín-Vasallo P, Mobasheri R. (2001) 'Isoforms of Na+, K+-ATPase in human prostate; specificity of expression and apical membrane polarization.'. Histol Histopathol, Spain: 16 (1), pp. 141-154.

    Abstract

    The cellular distribution of Na+, K+-ATPase subunit isoforms was mapped in the secretory epithelium of the human prostate gland by immunostaining with antibodies to the alpha and beta subunit isoforms of the enzyme. Immunolabeling of the alpha1, beta1 and beta2 isoforms was observed in the apical and lateral plasma membrane domains of prostatic epithelial cells in contrast to human kidney where the alpha1 and beta1 isoforms of Na+, K+-ATPase were localized in the basolateral membrane of both proximal and distal convoluted tubules. Using immunohistochemistry and PCR we found no evidence of Na+, K+-ATPase alpha2 and alpha3 isoform expression suggesting that prostatic Na+, K+-ATPase consists of alpha1/beta1 and alpha1/beta2 isozymes. Our immunohistochemical findings are consistent with previously proposed models placing prostatic Na+, K+-ATPase in the apical plasma membrane domain. Abundant expression of Na+, K+-ATPase in epithelial cells lining tubulo-alveoli in the human prostate gland confirms previous conclusions drawn from biochemical, pharmacological and physiological data and provides further evidence for the critical role of this enzyme in prostatic cell physiology and ion homeostasis. Na+, K+-ATPase most likely maintains an inwardly directed Na+ gradient essential for nutrient uptake and active citrate secretion by prostatic epithelial cells. Na+, K+-ATPase may also regulate lumenal Na+ and K+, major counter-ions for citrate.

  • Mobasheri A, Avila J, Cózar-Castellano I, Brownleader MD, Trevan M, Francis MJ, Lamb JF, Martín-Vasallo P. (2000) 'Na+, K+-ATPase isozyme diversity; comparative biochemistry and physiological implications of novel functional interactions.'. Biosci Rep, UNITED STATES: 20 (2), pp. 51-91.

    Abstract

    Na+, K+-ATPase is ubiquitously expressed in the plasma membrane of all animal cells where it serves as the principal regulator of intracellular ion homeostasis. Na+, K+-ATPase is responsible for generating and maintaining transmembrane ionic gradients that are of vital importance for cellular function and subservient activities such as volume regulation, pH maintenance, and generation of action potentials and secondary active transport. The diversity of Na+, K+-ATPase subunit isoforms and their complex spatial and temporal patterns of cellular expression suggest that Na+, K+-ATPase isozymes perform specialized physiological functions. Recent studies have shown that the alpha subunit isoforms possess considerably different kinetic properties and modes of regulation and the beta subunit isoforms modulate the activity, expression and plasma membrane targeting of Na+, K+-ATPase isozymes. This review focuses on recent developments in Na+, K+-ATPase research, and in particular reports of expression of isoforms in various tissues and experiments aimed at elucidating the intrinsic structural features of isoforms important for Na+, K+-ATPase function.

  • Brownleader MD, Hopkins J, Mobasheri A, Dey PM, Jackson P, Trevan M. (2000) 'Role of extensin peroxidase in tomato (Lycopersicon esculentum Mill.) seedling growth'. PLANTA, 210 (4), pp. 668-676.
  • Mobasheri A, Trujillo E, Golding S, Ellory JC, Pagakis SN, Pocock AE, Martin-Vasallo P, Francis MJO. (2000) 'Isoforms of Na,K-ATPase in primary human bone derived osteoblasts'. Journal of Biochemistry, Molecular Biology and Biophysics, 4 (5), pp. 343-357.

    Abstract

    Osteoblasts play a critical role in bone formation and mineralization, a process that depends on optimal calcium and phosphate homeostasis. Transcellular transport of free calcium [Ca], uptake of inorganic phosphate (P(i)) and numerous other transport systems in osteoblasts depend on a low intracellular Na:K ratio furnished by (Na+K)-stimulated adenosine triphosphatase (Na,K-ATPase), an enzyme embedded in the plasma membrane. In this study, we have examined, for the first time, the expression of the catalytic α and regulatory β subunit isoforms of Na,K-ATPase in primary human bone derived osteoblasts using isoform specific monoclonal and polyclonal antibodies. Immunofluorescence was used to detect the α1, β1 and β2 isoforms of Na,K-ATPase in dispersed osteoblasts. Laser scanning confocal microscopy also revealed an abundance of Na,K-ATPase isoforms in subcellular compartments. The existence of α1, β1 and β2 suggests that at least two major isozyme combinations of Na,K-ATPase are present in human bone (α1β1,α1β2).

  • Trujillo E, Alvarez de la Rosa D, Mobasheri A, Avila J, González T, Martín-Vasallo P. (1999) 'Sodium transport systems in human chondrocytes. I. Morphological and functional expression of the Na+,K(+)-ATPase alpha and beta subunit isoforms in healthy and arthritic chondrocytes.'. Histol Histopathol, SPAIN: 14 (4), pp. 1011-1022.

    Abstract

    The chondrocyte is the cell responsible for the maintenance of the articular cartilage matrix. The negative charges of proteoglycans of the matrix draw cations, principally Na+, into the matrix to balance the negative charge distribution. The Na+,K(+)-ATPase is the plasma membrane enzyme that maintains the intracellular Na+ and K+ concentrations. The enzyme is composed of an alpha and a beta subunit, so far, 4 alpha and 3 beta isoforms have been identified in mammals. Chondrocytes are sensitive to their ionic and osmotic environment and are capable of adaptive responses to ionic environmental perturbations particularly changes to extracellular [Na+]. In this article we show that human fetal and adult chondrocytes express three alpha (alpha 1, alpha 2 and the neural form of alpha 3) and the three beta isoforms (beta 1, beta 2 and beta 3) of the Na+,K(+)-ATPase. The presence of multiple Na+,K(+)-ATPase isoforms in the plasma membrane of chondrocytes suggests a variety of kinetic properties that reflects a cartilage specific and very fine specialization in order to maintain the Na+/K+ gradients. Changes in the ionic and osmotic environment of chondrocytes occur in osteoarthritis and rheumatoid arthritis as result of tissue hydration and proteoglycan loss leading to a fall in tissue Na+ and K+ content. Although the expression levels and cellular distribution of the proteins tested do not vary, we detect changes in p-nitrophenylphosphatase activity "in situ" between control and pathological samples. This change in the sodium pump enzymatic activity suggests that the chondrocyte responds to these cationic environmental changes with a variation of the active isozyme types present in the plasma membrane.

  • Trujillo E, Alvarez de la Rosa D, Mobasheri A, González T, Canessa CM, Martín-Vasallo P. (1999) 'Sodium transport systems in human chondrocytes. II. Expression of ENaC, Na+/K+/2Cl- cotransporter and Na+/H+ exchangers in healthy and arthritic chondrocytes.'. Histol Histopathol, SPAIN: 14 (4), pp. 1023-1031.

    Abstract

    In this article, the second of two, we continue our studies of sodium-dependent transport systems in human cartilage from healthy individuals and with osteoarthritis (OA) and rheumatoid arthritis (RA). We demonstrate the presence of the epithelial sodium channel (ENaC), previously undescribed in chondrocytes. This system is composed of three subunits, alpha, beta and gamma. We have shown that the human chondrocytes express at least the alpha and the beta subunit of ENaC. The expression of these subunits is altered in arthritic chondrocytes. In RA samples the quantity of alpha and beta is significantly higher than in control samples. On the other hand, ENaC alpha and beta subunits are absent in the chondrocytes of OA cartilage. Human chondrocytes also possess three isoforms of the Na+/H+ exchanger (NHE), NHE1, NHE2 and NHE3. The NHE system is composed of a single protein and is believed to participate in intracellular pH regulation. Furthermore, our studies indicate that at least one isoform of the electroneutral Na+/K+/2Cl- cotransporter (NKCC) is present in human chondrocytes. There are no obvious variations in the relative expression of NHE isoforms or NKCC between healthy and arthritic cartilage. Our data suggests that chondrocytes from arthritic cartilage may adapt to changes in their environmental sodium concentration through variations in ENaC protein levels. ENaC is also likely to serve as a major sodium entry mechanism, a process that, along with cytoskeletal proteins, may be part of mechanotransduction in cartilage.

  • Mobasheri A. (1999) 'Brefeldin A influences the cell surface abundance and intracellular pools of low and high ouabain affinity Na+, K(+)-ATPase alpha subunit isoforms in articular chondrocytes.'. Histol Histopathol, SPAIN: 14 (2), pp. 427-438.

    Abstract

    The catalytic alpha isoforms of the Na+, K(+)-ATPase and stimuli controlling the plasma membrane abundance and intracellular distribution of the enzyme were studied in isolated bovine articular chondrocytes which have previously been shown to express low and high ouabain affinity alpha isoforms (alpha 1 and alpha 3 respectively; alpha 1 > alpha 3). The Na+, K(+)-ATPase density of isolated chondrocyte preparations was quantified by specific 3H-ouabain binding. Long-term elevation of extracellular medium [Na+] resulted in a significant (31%; p < 0.05) upregulation of Na+, K(+)-ATPase density and treatment with various pharmacological inhibitors (Brefeldin A, monensin and cycloheximide) significantly (p < 0.001) blocked the upregulation. The subcellular distribution of the Na+, K(+)-ATPase alpha isoforms was examined by immunofluorescence confocal laser scanning microscopy which revealed predominantly plasma membrane immunostaining of alpha subunits in control chondrocytes. In Brefeldin A treated chondrocytes exposed to high [Na+], Na+, K(+)-ATPase alpha isoforms accumulated in juxta-nuclear pools and plasma membrane Na+, K(+)-ATPase density monitored by 3H-ouabain binding was significantly down-regulated due to Brefeldin A mediated disruption of vesicular transport. There was a marked increase in intracellular alpha 1 and alpha 3 staining suggesting that these isoforms are preferentially upregulated following long-term exposure to high extracellular [Na+]. The results demonstrate that Na+, K(+)-ATPase density in chondrocytes is elevated in response to increased extracellular [Na+] through de novo protein synthesis of new pumps containing alpha 1 and alpha 3 isoforms, delivery via the endoplasmic reticulum-Golgi complex constitutive secretory pathway and insertion into the plasma membrane.

  • Brownleader MD, Jackson P, Mobasheri A, Pantelides AT, Sumar S, Trevan M, Dey PM. (1999) 'Molecular aspects of cell wall modifications during fruit ripening'. CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION, 39 (2), pp. 149-164.
  • Mobasheri A, Martín-Vasallo P. (1999) 'Epithelial sodium channels in skeletal cells; a role in mechanotransduction?'. Cell Biol Int, ENGLAND: 23 (4), pp. 237-240.
  • Mobasheri A. (1999) 'Regulation of Na+, K+-ATPase density by the extracellular ionic and osmotic environment in bovine articular chondrocytes.'. Physiol Res, CZECH REPUBLIC: 48 (6), pp. 509-512.

    Abstract

    The abundance of Na+, K+-ATPase in cartilage is controlled by the ionic composition of the extracellular environment of chondrocytes, and specifically depends on the local concentration of polyanionic matrix proteoglycans. In this study, it was found that the plasma membrane density of Na+, K+-ATPase in isolated chondrocytes is sensitive to both ionic and osmotic changes in the extracellular environment. The upregulation observed experimentally was similar in magnitude as measured by 3H-ouabain binding, which indicates that chondrocytes respond adaptively to both ionic and osmotic stimuli. The precise mechanism for this novel mode of Na+, K+-ATPase regulation has yet to be elucidated. Physiological perturbation of the ionic and osmotic environment of chondrocytes may alter intracellular Na+ concentration and this may be one of a number of stimuli responsible for alterations to the expression and plasma membrane abundance of Na+, K+-ATPase in the cells.

  • Mobasheri A, Mobasheri R, Francis MJ, Trujillo E, Alvarez de la Rosa D, Martín-Vasallo P. (1998) 'Ion transport in chondrocytes: membrane transporters involved in intracellular ion homeostasis and the regulation of cell volume, free [Ca2+] and pH.'. Histol Histopathol, SPAIN: 13 (3), pp. 893-910.

    Abstract

    Chondrocytes exist in an unusual and variable ionic and osmotic environment in the extracellular matrix of cartilage and are responsible for maintaining the delicate equilibrium between extracellular matrix synthesis and degradation. The mechanical performance of cartilage relies on the biochemical properties of the matrix. Alterations to the ionic and osmotic extracellular environment of chondrocytes have been shown to influence the volume, intracellular pH and ionic content of the cells, which in turn modify the synthesis and degradation of extracellular matrix macromolecules. Physiological ion homeostasis is fundamental to the routine functioning of cartilage and the factors that control the integrity of this highly evolved and specialized tissue. Ion transport in cartilage is relatively unexplored and the biochemical properties and molecular identity of membrane transport mechanisms employed by chondrocytes in the control of intracellular ion concentrations and pH is not fully defined and this review focuses on these processes. Chondrocytes have been shown to express voltage and stretch activated ion channels, passive exchangers and ATP dependent ion pumps. In addition, recent studies of transport systems in chondrocytes have demonstrated the presence of isozyme diversity that includes Na+/H+ exchange (NHE1, NHE3), Na+, K(+)-ATPase (several isoforms) and others each of which possess considerably different kinetic properties and modes of regulation. This multitude of isozyme diversity indicates the highly specialized handling of ions and protons in order to accomplish a fine regulation of their transmembrane fluxes. The complexities of these transport systems and their patterns of isoform expression underscore the subtlety of ion homeostasis and pH regulation in normal cartilage. Perturbations in these mechanisms may affect the physiological turnover of cartilage and thus increase the susceptibility to degenerative joint disease.

  • Basavappa S, Mobasheri A, Errington R, Huang CC, Al-Adawi S, Ellory JC. (1998) 'Inhibition of Na+, K+-ATPase activates swelling-induced taurine efflux in a human neuroblastoma cell line.'. J Cell Physiol, UNITED STATES: 174 (2), pp. 145-153.

    Abstract

    The Na+ pump (Na+, K+-ATPase) has been implicated in the regulation of many cellular functions, including cell volume regulation. The effects of inhibiting Na+ pump activity on cell volume and taurine efflux were evaluated in the human neuroblastoma cell line CHP-100. Cell volume changes monitored with the Coulter Multisizer technique and confocal microscopy showed that neuroblastoma cells exposed to ouabain swelled by 22 +/- 4% (n = 5). The rapid cell swelling was followed by regulatory volume decrease (RVD). In cells treated with ouabain, 14C-taurine efflux increased by 183 +/- 11% compared with controls. However, cells exposed simultaneously to ouabain and hypoosmotic solution resulted in a 14C-taurine efflux of 207 +/- 18%. Western blot and immunofluorescence microscopy with specific monoclonal antibodies for the catalytic alpha isoforms of Na+, K+-ATPase demonstrated high levels of the ubiquitously expressed alpha1 and the neuronal-specific alpha3. Ouabain-binding data showed that CHP-100 cells express approximately 3 x 10(5) pump units/cell. The present data indicate that efflux of taurine may be involved during volume recovery subsequent to blockade of Na+, K+-ATPase in CHP-100 cells.

  • Mobasheri A. (1998) 'Correlation between [Na+], [glycosaminoglycan] and Na+/K+ pump density in the extracellular matrix of bovine articular cartilage.'. Physiol Res, CZECH REPUBLIC: 47 (1), pp. 47-52.

    Abstract

    The plasma membrane abundance of the Na+/K+ pump or Na+,K+-ATPase depends on the intracellular concentration of Na+ in almost all animal cells. In cartilage, chondrocytes are surrounded by an extracellular matrix which consists of collagen and proteoglycan, a ground substance composed of glycosaminoglycan (GAG) side chains with a high fixed negative charge density. The polyanionic nature of the GAGs tends to draw monovalent cations into the matrix resulting in high [Na+] which may exceed 250 mM. As the concentration of GAGs in the tissue increases, so does [Na+]. In this study, it was found that the density of the Na+/K+ pump, measured by 3H-ouabain binding, correlates with the concentration of GAGs in the tissue. This indicates that chondrocytes are sensitive to their ionic environment and respond to local [Na+] variations by regulating the abundance of the Na+/K+ pump.

  • Mobasheri A, Golding S, Pagakis SN, Corkey K, Pocock AE, Fermor B, O'BRIEN MJ, Wilkins RJ, Ellory JC, Francis MJ. (1998) 'Expression of cation exchanger NHE and anion exchanger AE isoforms in primary human bone-derived osteoblasts.'. Cell Biol Int, ENGLAND: 22 (7-8), pp. 551-562.

    Abstract

    The authors used isoform-specific antibodies against cation (NHE) and anion (AE) exchange isoforms in order to establish their specific expression and localization in dispersed human bone-derived cells. Immunocytochemical preparations of permeabilized osteoblasts probed with polyclonal antibodies were optically analysed by conventional immunofluorescence and con-focal laser scanning microscopy. These techniques demonstrated the abundant presence of epitopes of the cation exchangers NHE1 and NHE3 and the anion exchanger AE2 in these cells. The NHE1 and NHE3 isoform proteins were predominantly located in subplasmalemmal and nucleoplasmic vesicles. The AE2 isoform was densely localized to a subcellular location characteristic of the Golgi complex. The molecular identity of the AE and NHE isoforms was investigated by RT-PCR that confirmed the presence of NHE1 and NHE3 transcripts in addition to NHE4. RT-PCR and diagnostic restriction analysis of amplified AE cDNA established preferential AE2 expression. Since AE2 has been shown to act as a sulfate transporter at low pH, it is possible that it performs this function in the osteoblast Golgi complex where sulfation reactions occur post-translationally on numerous extracellular matrix macromolecules prior to secretion and mineralization. The Na(+)/H(+)exchanger proteins are regulated by mitogenic and non-mitogenic stimuli in the osseus environment and are involved in the large fluxes of ions and protons that necessarily occur during bone formation and resorption and thus play an important role in intracellular ion homeostasis in osteoblasts.

  • Mobasheri A, Errington RJ, Golding S, Hall AC, Urban JP. (1997) 'Characterization of the Na+, K(+)-ATPase in isolated bovine articular chondrocytes; molecular evidence for multiple alpha and beta isoforms.'. Cell Biol Int, ENGLAND: 21 (4), pp. 201-212.

    Abstract

    We have used isoform-specific antibodies against the Na+, K(+)-ATPase alpha (alpha 1, alpha 2 and alpha 3) and beta (beta 1 and beta 2) subunit isoforms in order to establish their specific localization in isolated bovine articular chondrocytes. Immunoblotting confirmed the presence of the alpha 1 and alpha 3 isoforms, although alpha 1 expression was significantly greater than alpha 3 as assessed by immunofluorescence confocal laser scanning microscopy and PCR. A similar approach revealed the presence of the beta 1 and beta 2 isoforms in chondrocytes, although beta 2 immunostaining on the plasma membrane was more punctate than beta 1 which in contrast predominated in a subcellular compartment. The plasma membrane abundance of the Na+, K(+)-ATPase was found to be sensitive to the extracellular ionic concentration and long-term elevation of extracellular Na+ concentration significantly upregulated Na+, K(+)-ATPase density as measured by specific 3H-ouabain binding. Our observations suggest that the expression of alpha 3 and beta 2 is not restricted to excitable tissues as previously reported. The physiological relevance of alpha 3 expression in chondrocytes may be related to its low affinity for intracellular Na+ in an extracellular environment where Na+ concentration is unusually high (260-350 mM) compared to other cell types (140 mM). Glycoproteins and their branched carbohydrates have been implicated in cell recognition events, thus the beta 2 subunit glycoprotein may allow the chondrocyte to detect changes in its extracellular environment by physically interacting with components of the cellular cytoskeleton and matrix macromolecules.

  • Mobasheri A, Hall AC, Urban JP, France SJ, Smith AL. (1997) 'Immunologic and autoradiographic localisation of the Na+, K(+)-ATPase in articular cartilage: upregulation in response to changes in extracellular Na+ concentration.'. Int J Biochem Cell Biol, ENGLAND: 29 (4), pp. 649-657.

    Abstract

    The maintenance of a relatively low intracellular Na+:K+ ratio is essential for the functioning of a wide range of cellular processes, and is achieved principally by the activity of the membrane-bound Na+, K(+)-ATPase. Chondrocytes, the cells of articular cartilage, exist in an ionic environment where the free extracellular [Na+] is higher (250-400 mM) than that of most other tissues (approximately 140 mM) owing to the fixed negative charges on glycosaminoglycans in the extracellular matrix. This can increase further during static joint loading when fluid expression occurs. To determine aspects of how chondrocytes regulate their ionic composition, in this study, the in situ distribution, pattern of isoform expression and density of the Na+, K(+)-ATPase within cartilage has been investigated. The density of the Na+, K(+)-ATPase was found to be high in the mid-zone, but lower in the surface and deep zones. Immunofluorescence microscopy using monoclonal antibodies to the catalytic alpha subunits of the Na+, K(+)-ATPase revealed the expression of isoforms alpha 1 and alpha 3. Alterations to the extracellular [Na+] (from 80-220 mM, or 120-220 mM) significantly elevated Na+, K(+)-ATPase density of in situ chondrocytes. The results indicate that the Na+, K(+)-ATPase is abundantly expressed in articular chondrocytes and its density is sensitive to the extracellular [Na+]. The expression of the alpha 3 isoform is surprising for a non-neuronal cell, and may indicate a physiological adaptation to the unusually high extracellular [Na+] to which chondrocytes are exposed in the extracellular matrix of cartilage.

  • Wilkins RJ, Mobasheri A, Hall AC. (1996) 'Characterisation of Na × H exchange in isolated bovine articular chondrocytes'. FASEB Journal, 10 (3)

    Abstract

    We have previously shown that intracellular pH (pH,) in the articular chondrocyte is regulated by a single mechanism, Na Hexchange. HCO3-dependent mechanisms play no role, possibly because of the low numbers of anions in the extracellular matrix, and the uptake of SO,2' and Zn2 by modified anion exchangers. Since pH, can modify matrix metabolism, regulation of Nax Hexchange will have important effects on cartilage integrity. We have therefore performed further experiments to characterise the properties of the chondrocyte Nax Hexchange. Recovery from ammonium-induced intracellular acidosis is 'smart', showing allosteric modification with increasing levels of intracellular acidity, sensitive to amiloride (K = SμM) and is stimulated by cell shrinkage, exposure to serum, and by application of hydrostatic pressure, seemingly by changes to Nax Hexchange phosphorylation state. These properties are consistent with the NHE-1 isoform of Nax Hexchange. However, using antibodies directed against Nax Hproteins we find that both NHE-1 and NHE-3 are present in these cells. Funded by The Wellcome Trust, UK.

Conference papers

  • Csaki C, Mobasheri A, Matis U, Putz R, Shakibaei M. (2007) 'Chondrogenic induction of canine mesenchymal stem cells in high density co-culture with chondrocytes: Potential applications in regenerative medicine'. MARY ANN LIEBERT INC TISSUE ENGINEERING, London, ENGLAND: Conference of the Tissue-Engineering-and-Regenerative-Medicine-International-Society (TERMIS-EU) 13 (7), pp. 1656-1656.

Book chapters

  • Bachmann R, Zaheer A. (2006) 'Introduction'. in Bachmann R, Zaheer A (eds.) Handbook of Trust Research Cheltenham : Edward Elgar , pp. 1-12.

Teaching

Ali has been involved in teaching cell biology, renal, endocrine and musculoskeletal physiology.

More

Section Editor (Pathophysiology of Musculoskeletal Disorders), BMC Musculoskeletal Disorders
http://www.biomedcentral.com/bmcmusculoskeletdisord/about/edboard

Section Editor (Pain Aspects of Arthritis), Current Pain and Headache Reports
http://www.springer.com/medicine/journal/11916?detailsPage=editorialBoard

Hirsch (h) index: 33

ResearcherID: http://www.researcherid.com/
http://www.researcherid.com/rid/D-5770-2011

ORCID ID: 0000-0001-6261-1286
http://orcid.org/0000-0001-6261-1286

ResearchGate
https://www.researchgate.net/profile/Ali_Mobasheri/

Scopus Author ID: 7003311894
http://www.scopus.com/authid/detail.url?authorId=7003311894

LinkedIn Profile:
http://uk.linkedin.com/pub/ali-mobasheri/12/97/680

Page Owner: am0083
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