Mark Crabtree

Dr Mark Crabtree

Senior Lecturer
BSc, PhD

Academic and research departments

Section of Cardiovascular Sciences, School of Biosciences.



Research interests

Research projects

Research collaborations


Postgraduate research supervision



Mark Crabtree, Gang Hao, Steven S Gross (2003)Detection of cysteine S-nitrosylation and tyrosine 3-nitration in kidney proteins, In: Methods in molecular medicine86pp. 373-384
Matthew S Alkaitis, Mark J Crabtree (2012)Recoupling the cardiac nitric oxide synthases: tetrahydrobiopterin synthesis and recycling, In: Current heart failure reports9(3)pp. 200-210

Nitric oxide (NO), a key regulator of cardiovascular function, is synthesized from L-arginine and oxygen by the enzyme nitric oxide synthase (NOS). This reaction requires tetrahydrobiopterin (BH4) as a cofactor. BH4 is synthesized from guanosine triphosphate (GTP) by GTP cyclohydrolase I (GTPCH) and recycled from 7,8-dihydrobiopterin (BH2) by dihydrofolate reductase. Under conditions of low BH4 bioavailability relative to NOS or BH2, oxygen activation is "uncoupled" from L-arginine oxidation, and NOS produces superoxide (O (2) (-) ) instead of NO. NOS-derived superoxide reacts with NO to produce peroxynitrite (ONOO(-)), a highly reactive anion that rapidly oxidizes BH4 and propagates NOS uncoupling. BH4 depletion and NOS uncoupling contribute to overload-induced heart failure, hypertension, ischemia/reperfusion injury, and atrial fibrillation. L-arginine depletion, methylarginine accumulation, and S-glutathionylation of NOS also promote uncoupling. Recoupling NOS is a promising approach to treating myocardial and vascular dysfunction associated with heart failure.

LM Fan, G Douglas, JK Bendall, E McNeill, Mark J Crabtree, AB Hale, A Mai, JM Li, MA McAteer, JE Schneider, RP Choudhury, KM Channon (2014)Endothelial cell-specific reactive oxygen species production increases susceptibility to aortic dissection., In: Circulation129(25)pp. 2661-2672

BACKGROUND: Increased production of reactive oxygen species (ROS) throughout the vascular wall is a feature of cardiovascular disease states, but therapeutic strategies remain limited by our incomplete understanding of the role and contribution of specific vascular cell ROS to disease pathogenesis. To investigate the specific role of endothelial cell (EC) ROS in the development of structural vascular disease, we generated a mouse model of endothelium-specific Nox2 overexpression and tested the susceptibility to aortic dissection after angiotensin II (Ang II) infusion. METHODS AND RESULTS: A specific increase in endothelial ROS production in Nox2 transgenic mice was sufficient to cause Ang II-mediated aortic dissection, which was never observed in wild-type mice. Nox2 transgenic aortas had increased endothelial ROS production, endothelial vascular cell adhesion molecule-1 expression, matrix metalloproteinase activity, and CD45(+) inflammatory cell infiltration. Conditioned media from Nox2 transgenic ECs induced greater Erk1/2 phosphorylation in vascular smooth muscle cells compared with wild-type controls through secreted cyclophilin A (CypA). Nox2 transgenic ECs (but not vascular smooth muscle cells) and aortas had greater secretion of CypA both at baseline and in response to Ang II stimulation. Knockdown of CypA in ECs abolished the increase in vascular smooth muscle cell Erk1/2 phosphorylation conferred by EC conditioned media, and preincubation with CypA augmented Ang II-induced vascular smooth muscle cell ROS production. CONCLUSIONS: These findings demonstrate a pivotal role for EC-derived ROS in the determination of the susceptibility of the aortic wall to Ang II-mediated aortic dissection. ROS-dependent CypA secretion by ECs is an important signaling mechanism through which EC ROS regulate susceptibility of structural components of the aortic wall to aortic dissection.

Marina Diotallevi, Thomas Nicol, Faseeha Ayaz, Jade Bailey, Andrew Shaw, Eileen McNeill, Ben Davies, Keith M. Channon, Mark J. Crabtree (2022)Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology, In: Journal of Visualized Experiments2022(183) MyJove Corporation

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Paul M. Heerdt, Paul Lane, Brian Y. Pan, Ulrich Schaefer, Mark Crabtree, Roger Hong, Andrew A. M. Singer, Roberto Levi, Bernard J. Park (2007)Nitrosative Stress and Myocardial Sarcoplasmic Endoreticular Calcium Adenosine Triphosphatase Subtype 2a Activity after Lung Resection in Swine, In: Anesthesiology (Philadelphia)107(6)pp. 954-962

Background Chronic, disease-associated oxidative stress induces myocardial peroxynitrite formation that may lead to nitrosative inhibition of the calcium cycling protein sarcoplasmic endoreticular calcium adenosine triphosphatase subtype 2a (SERCA2a). The current study was designed to test the hypothesis that the acute oxidative stress associated with lung resection also induces myocardial nitrosative stress and alters SERCA2a activity. Methods Ventricular myocardium from 16 swine was studied; 11 animals had undergone left upper lobectomy (n = 7) or sham thoracotomy (n = 4) 3 days before harvest, and 5 were nonoperated controls. Tissue peroxynitrite was assessed by measurement of 3-nitrotyrosine incorporation into proteins. SERCA2a activity was determined from indo-1 uptake by isolated sarcoplasmic reticular membranes. Expression of SERCA2a and its regulatory protein phospholamban were determined by Western blotting, as was the phospholamban phosphorylation state (when dephosporylated, phospholamban inhibits SERCA2a). Mechanical significance of changes in SERCA2a activity was assessed from the force-frequency relation of isometric myocardial trabeculae. Results Relative to both the control and sham groups, lobectomy animals exhibited a greater than twofold higher myocardial 3-nitrotyrosine incorporation and an approximately 50% lower SERCA2a activity, but no difference in SERCA2a or phospholamban expression or phospholamban phosphorylation. Concomitantly, whereas the trabecular force-frequency relation of control animals was positive, that of lobectomy animals was negative, consistent with impaired calcium cycling. Conclusions These data indicate that oxidative/nitrosative stress associated with lung resection influences SERCA2a activity independent of any influence on protein expression or phospholamban phosphorylation. The findings link an acute event with a subcellular process primarily described for chronic illness and suggest a biochemical basis for perioperative changes in myocardial mechanical reserve.

Mark A. Richards, Jillian N. Simon, Ruichong Ma, Aminah A. Loonat, Mark J. Crabtree, David J. Paterson, Richard P. Fahlman, Barbara Casadei, Larry Fliegel, Pawel Swietach (2020)Nitric oxide modulates cardiomyocyte pH control through a biphasic effect on sodium/hydrogen exchanger-1, In: Cardiovascular research116(12)pp. 1958-1971 Oxford Univ Press

Aim When activated, Na+/H+ exchanger-1 (NHE1) produces some of the largest ionic fluxes in the heart. NHE1-dependent H+ extrusion and Na+ entry strongly modulate cardiac physiology through the direct effects of pH on proteins and by influencing intracellular Ca2+ handling. To attain an appropriate level of activation, cardiac NHE1 must respond to myocyte-derived cues. Among physiologically important cues is nitric oxide (NO), which regulates a myriad of cardiac functions, but its actions on NHE1 are unclear. Methods and results NHE1 activity was measured using pH-sensitive cSNARF1 fluorescence after acid-loading adult ventricular myocytes by an ammonium preputse solution manoeuvre. NO signalling was manipulated by knockout of its major constitutive synthase nNOS, adenoviral nNOS gene delivery, nNOS inhibition, and application of NO-donors. NHE1 flux was found to be activated by tow [NO], but inhibited at high [NO]. These responses involved cGMP-dependent signalling, rather than S-nitros(yDation. Stronger cGMP signals, that can inhibit phosphodiesterase enzymes, allowed [cAMP] to rise, as demonstrated by a FRET-based sensor. Inferring from the actions of membrane-permeant analogues, cGMP was determined to activate NHE1, whereas cAMP was inhibitory, which explains the biphasic regulation by NO. Activation of NHE1-dependent Na+ influx by low [NO] also increased the frequency of spontaneous Ca2+ waves, whereas high [NO] suppressed these aberrant forms of Ca2+ signalling. Conclusions Physiological levels of NO stimulation increase NHE1 activity, which boosts pH control during acid-disturbances and results in Natdriven cellular Ca2+ loading. These responses are positively inotropic but also increase the tiketihood of aberrant Ca2+ signals, and hence arrhythmia. Stronger NO signals inhibit NHE1, leading to a reversal of the aforementioned effects, ostensibly as a potential cardioprotective intervention to curtail NHE1 overdrive. [GRAPHICS] .

Jade D. Bailey, Andrew Shaw, Eileen McNeill, Thomas Nicol, Marina Diotallevi, Surawee Chuaiphichai, Jyoti Patel, Ashley Hale, Keith M. Channon, Mark J. Crabtree (2020)Isolation and culture of murine bone marrow-derived macrophages for nitric oxide and redox biology, In: Nitric oxide100pp. 17-29 Elsevier

Macrophages are mononuclear phagocytes derived from haematopoietic progenitors that are widely distributed throughout the body. These cells participate in both innate and adaptive immune responses and lie central to the processes of inflammation, development, and homeostasis. Macrophage physiology varies depending on the environment in which they reside and they exhibit rapid functional adaption in response to external stimuli. To study macrophages in vitro, cells are typically cultured ex vivo from the peritoneum or alveoli, or differentiated from myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). BMDMs represent an efficient and cog-effective means of studying macrophage biology. However, the inherent sensitivity of macrophages to biochemical stimuli (such as cytokines, metabolic intermediates, and RNS/ROS) makes it imperative to control experimental conditions rigorously. Therefore, the aim of this study was to establish an optimised and standardised method for the isolation and culture of BMDMs. We used classically activated macrophages isolated from WT and nitric oxide (NO)-deficient mice to develop a standardised culture method, whereby the constituents of the culture media are defined. We then methodically compared our standardised protocol to the most commonly used method of BMDM culture to establish an optimal protocol for the study of nitric oxide (NO)-redox biology and immunometabolism in vitro.

Surawee Chuaiphichai, Grace Z. Yu, Cheryl M.J. Tan, Christopher Whiteman, Gillian Douglas, Yasmin Dickinson, Edward N. Drydale, Mahesh Appari, Wei Zhang, Mark J. Crabtree, Eileen McNeill, Ashley B. Hale, Adam J. Lewandowski, Nicholas J. Alp, Manu Vatish, Paul Leeson, Keith M. Channon (2021)Endothelial GTPCH (GTP Cyclohydrolase 1) and Tetrahydrobiopterin Regulate Gestational Blood Pressure, Uteroplacental Remodeling, and Fetal Growth, In: Hypertension (Dallas, Tex. 1979)78(6)pp. 1871-1884 Lippincott Williams & Wilkins

Supplemental Digital Content is available in the text. Abnormal uteroplacental remodeling leads to placental hypoperfusion, causing fetal growth restriction and pregnancy-related hypertension, which are associated with endothelial dysfunction and markers of reduced vascular NO bioavailability and oxidative stress. Tetrahydrobiopterin (BH4) is a redox cofactor for eNOS (endothelial NO synthase) with a required role in NO generation. Using mice models and human samples, we investigated the physiological requirement for endothelial cell BH4 in uteroplacental vascular adaptation and blood pressure regulation to pregnancy. In pregnant mice, selective maternal endothelial BH4 deficiency resulting from targeted deletion of Gch1 caused progressive hypertension during pregnancy and fetal growth restriction. Maternal endothelial cell Gch1 deletion caused defective functional and structural remodeling in uterine arteries and in spiral arteries, leading to placental insufficiency. Using primary endothelial cells isolated from either normal or hypertensive pregnancies, we found that hypertensive pregnancies are associated with reduced endothelial cell BH4 levels, impaired eNOS activity, and reduced endothelial cell proliferation, mediated by reduced GTPCH (GTP cyclohydrolase 1) protein. In rescue experiments, high blood pressure and fetal growth restriction in pregnant endothelial cell Gch1 deficient mice was not rescued by oral BH4 supplementation, due to systemic oxidation of BH4 to dihydrobiopterin. However, the fully reduced folate, 5-methyltetrahydrofolate prevented BH4 oxidation, reduced blood pressure to normal levels, and normalized fetal growth. We identify a critical requirement for maternal endothelial cell BH4 biosynthesis in uteroplacental vascular remodeling in pregnancy. Restoration of endothelial cell BH4 with reduced folates identifies a novel therapeutic target for the prevention and treatment of pregnancy-related hypertension such as preeclampsia.

Laurienne Edgar, Naveed Akbar, Adam T Braithwaite, Thomas Krausgruber, Héctor Gallart-Ayala, Jade Bailey, Alastair L Corbin, Tariq E Khoyratty, Joshua T Chai, Mohammad Alkhalil, André F Rendeiro, Klemen Ziberna, Ritu Arya, Thomas J Cahill, Christoph Bock, Jurga Laurencikiene, Mark J Crabtree, Madeleine E Lemieux, Niels P Riksen, Mihai G Netea, Craig E Wheelock, Keith M Channon, Mikael Rydén, Irina A Udalova, Ricardo Carnicer, Robin P Choudhury (2021)Hyperglycemia Induces Trained Immunity in Macrophages and Their Precursors and Promotes Atherosclerosis, In: Circulation (New York, N.Y.)144(12)pp. 961-982

Cardiovascular risk in diabetes remains elevated despite glucose-lowering therapies. We hypothesized that hyperglycemia induces trained immunity in macrophages, promoting persistent proatherogenic characteristics. Bone marrow-derived macrophages from control mice and mice with diabetes were grown in physiological glucose (5 mmol/L) and subjected to RNA sequencing (n=6), assay for transposase accessible chromatin sequencing (n=6), and chromatin immunoprecipitation sequencing (n=6) for determination of hyperglycemia-induced trained immunity. Bone marrow transplantation from mice with (n=9) or without (n=6) diabetes into (normoglycemic) mice was used to assess its functional significance in vivo. Evidence of hyperglycemia-induced trained immunity was sought in human peripheral blood mononuclear cells from patients with diabetes (n=8) compared with control subjects (n=16) and in human atherosclerotic plaque macrophages excised by laser capture microdissection. In macrophages, high extracellular glucose promoted proinflammatory gene expression and proatherogenic functional characteristics through glycolysis-dependent mechanisms. Bone marrow-derived macrophages from diabetic mice retained these characteristics, even when cultured in physiological glucose, indicating hyperglycemia-induced trained immunity. Bone marrow transplantation from diabetic mice into (normoglycemic) mice increased aortic root atherosclerosis, confirming a disease-relevant and persistent form of trained innate immunity. Integrated assay for transposase accessible chromatin, chromatin immunoprecipitation, and RNA sequencing analyses of hematopoietic stem cells and bone marrow-derived macrophages revealed a proinflammatory priming effect in diabetes. The pattern of open chromatin implicated transcription factor Runt-related transcription factor 1 (Runx1). Similarly, transcriptomes of atherosclerotic plaque macrophages and peripheral leukocytes in patients with type 2 diabetes were enriched for Runx1 targets, consistent with a potential role in human disease. Pharmacological inhibition of Runx1 in vitro inhibited the trained phenotype. Hyperglycemia-induced trained immunity may explain why targeting elevated glucose is ineffective in reducing macrovascular risk in diabetes and suggests new targets for disease prevention and therapy.

Jade D. Bailey, Marina Diotallevi, Thomas Nicol, Eileen McNeill, Andrew Shaw, Surawee Chuaiphichai, Ashley Hale, Anna Starr, Manasi Nandi, Elena Stylianou, Helen McShane, Simon Davis, Roman Fischer, Benedikt M. Kessler, James McCullagh, Keith M. Channon, Mark J. Crabtree (2019)Nitric Oxide Modulates Metabolic Remodeling in Inflammatory Macrophages through TCA Cycle Regulation and Itaconate Accumulation, In: Cell reports (Cambridge)28(1)pp. 218-230.e7 Elsevier Inc

Classical activation of macrophages (M(LPS+IFNγ)) elicits the expression of inducible nitric oxide synthase (iNOS), generating large amounts of NO and inhibiting mitochondrial respiration. Upregulation of glycolysis and a disrupted tricarboxylic acid (TCA) cycle underpin this switch to a pro-inflammatory phenotype. We show that the NOS cofactor tetrahydrobiopterin (BH4) modulates IL-1β production and key aspects of metabolic remodeling in activated murine macrophages via NO production. Using two complementary genetic models, we reveal that NO modulates levels of the essential TCA cycle metabolites citrate and succinate, as well as the inflammatory mediator itaconate. Furthermore, NO regulates macrophage respiratory function via changes in the abundance of critical N-module subunits in Complex I. However, NO-deficient cells can still upregulate glycolysis despite changes in the abundance of glycolytic intermediates and proteins involved in glucose metabolism. Our findings reveal a fundamental role for iNOS-derived NO in regulating metabolic remodeling and cytokine production in the pro-inflammatory macrophage. [Display omitted] •NO orchestrates metabolic remodeling in macrophages responding to LPS+IFNγ•NO regulates itaconate metabolism in two models of infection and inflammation•NO determines Complex I subunit abundance in inflammatory macrophages•Glycolysis is increased in activated NO-deficient cells despite metabolic changes Metabolic remodeling underpins inflammatory macrophage activation, but the modulatory mechanisms are still being elucidated. Bailey et al. show that NO regulates specific changes in the abundance of TCA cycle metabolites, itaconate, and catalytic subunits of Complex I in the respiratory chain in inflammatory murine macrophages both in vitro and in vivo.

Hasan El-Hassan, Khurshid Anwar, Patricia Macanas-Pirard, Mark Crabtree, Sek C Chow, Victoria L Johnson, Pauline C Lee, Richard H Hinton, Shirley C Price, George E.N Kass (2003)Involvement of mitochondria in acetaminophen-induced apoptosis and hepatic injury: Roles of cytochrome c, Bax, Bid, and caspases, In: Toxicology and applied pharmacology191(2)pp. 118-129 Elsevier Inc

The role of apoptosis in acetaminophen (AAP)-induced hepatic injury was investigated. Six hours after AAP administration to BALB/c mice, a significant loss of hepatic mitochondrial cytochrome c was observed that was similar in extent to the loss observed after in vivo activation of CD95 by antibody treatment. AAP-induced loss of mitochondrial cytochrome c coincided with the appearance in the cytosol of a fragment corresponding to truncated Bid (tBid). At the same time, tBid became detectable in the mitochondrial fraction, and concomitantly, Bax was found translocated to mitochondria. However, AAP failed to activate the execution caspases 3 and 7 as evidenced by a lack of procaspase processing and the absence of an increase in caspase-3-like activity. In contrast, the administration of the pan-inhibitor of caspases, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (but not its analogue benzyloxycarbonyl-Phe-Ala-fluoromethylketone) prevented the development of liver injury by AAP and the appearance of apoptotic parenchymal cells. This correlated with the inhibition of the processing of Bid to tBid. The caspase inhibitor failed to prevent both the redistribution of Bax to the mitochondria and the loss of cytochrome c. In conclusion, apoptosis is an important causal event in the initiation of the hepatic injury inflicted by AAP. However, as suggested by the lack of activation of the main execution caspases, apoptosis is not properly executed and degenerates into necrosis.

Mark J. Crabtree, Amy L. Tatham, Yasir Al-Wakeel, Nicholas Warrick, Ashley B. Hale, Shijie Cai, Keith M. Channon, Nicholas J. Alp (2009)Quantitative Regulation of Intracellular Endothelial Nitric-oxide Synthase (eNOS) Coupling by Both Tetrahydrobiopterin-eNOS Stoichiometry and Biopterin Redox Status INSIGHTS FROM CELLS WITH TET-REGULATED GTP CYCLOHYDROLASE I EXPRESSION, In: The Journal of biological chemistry284(2)pp. 1136-1144 Amer Soc Biochemistry Molecular Biology Inc

Tetrahydrobiopterin (BH4) is a critical determinant of endothelial nitric-oxide synthase (eNOS) activity. In the absence of BH4, eNOS becomes "uncoupled" and generates superoxide rather than NO. However, the stoichiometry of intracellular BH4/eNOS interactions is not well defined, and it is unclear whether intracellular BH4 deficiency alone is sufficient to induce eNOS uncoupling. To address these questions, we developed novel cell lines with tet-regulated expression of human GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in BH4 synthesis, to selectively induce intracellular BH4 deficiency by incubation with doxycycline. These cells were stably co-transfected to express a human eNOS-green fluorescent protein fusion protein, selecting clones expressing either low (GCH/eNOS-LOW) or high (GCH/eNOS-HIGH) levels. Doxycycline abolished GTPCH mRNA expression and GTPCH protein, leading to markedly diminished total biopterin levels and a decreased ratio of BH4 to oxidized biopterins in cells expressing eNOS. Intracellular BH4 deficiency induced superoxide generation from eNOS, as assessed by N-nitro-L-arginine methyl ester inhibitable 2-hydroxyethidium generation, and attenuated NO production. Quantitative analysis of cellular BH4 versus superoxide production between GCH/eNOS-LOW and GCH/eNOS-HIGH cells revealed a striking linear relationship between eNOS protein and cellular BH4 stoichiometry, with eNOS uncoupling at eNOS: BH4 molar ratio >1. Furthermore, increasing the intracellular BH2 concentration in the presence of a constant eNOS: BH4 ratio was sufficient to induce eNOS-dependent superoxide production. This specific, reductionist approach in a cell-based system reveals that eNOS: BH4 reaction stoichiometry together with the intracellular BH4:BH2 ratio, rather than absolute concentrations of BH4, are the key determinants of eNOS uncoupling, even in the absence of exogenous oxidative stress.

Germaine Korner, Tanja Scherer, Dea Adamsen, Alexander Rebuffat, Mark Crabtree, Anahita Rassi, Rossana Scavelli, Daigo Homma, Birgit Ledermann, Daniel Konrad, Hiroshi Ichinose, Christian Wolfrum, Marion Horsch, Birgit Rathkolb, Martin Klingenspor, Johannes Beckers, Eckhard Wolf, Valérie Gailus-Durner, Helmut Fuchs, Martin Hrabě de Angelis, Nenad Blau, Jan Rozman, Beat Thöny (2016)Mildly compromised tetrahydrobiopterin cofactor biosynthesis due to Pts variants leads to unusual body fat distribution and abdominal obesity in mice, In: Journal of inherited metabolic disease39(2)pp. 309-319

Tetrahydrobiopterin (BH4) is an essential cofactor for the aromatic amino acid hydroxylases, alkylglycerol monooxygenase, and nitric oxide synthases (NOS). Inborn errors of BH4 metabolism lead to severe insufficiency of brain monoamine neurotransmitters while augmentation of BH4 by supplementation or stimulation of its biosynthesis is thought to ameliorate endothelial NOS (eNOS) dysfunction, to protect from (cardio-) vascular disease and/or prevent obesity and development of the metabolic syndrome. We have previously reported that homozygous knock-out mice for the 6-pyruvolytetrahydropterin synthase (PTPS; Pts-ko/ko) mice with no BH4 biosynthesis die after birth. Here we generated a Pts-knock-in (Pts-ki) allele expressing the murine PTPS-p.Arg15Cys with low residual activity (15% of wild-type in vitro) and investigated homozygous (Pts-ki/ki) and compound heterozygous (Pts-ki/ko) mutants. All mice showed normal viability and depending on the severity of the Pts alleles exhibited up to 90% reduction of PTPS activity concomitant with neopterin elevation and mild reduction of total biopterin while blood L-phenylalanine and brain monoamine neurotransmitters were unaffected. Yet, adult mutant mice with compromised PTPS activity (i.e., Pts-ki/ko, Pts-ki/ki or Pts-ko/wt) had increased body weight and elevated intra-abdominal fat. Comprehensive phenotyping of Pts-ki/ki mice revealed alterations in energy metabolism with proportionally higher fat content but lower lean mass, and increased blood glucose and cholesterol. Transcriptome analysis indicated changes in glucose and lipid metabolism. Furthermore, differentially expressed genes associated with obesity, weight loss, hepatic steatosis, and insulin sensitivity were consistent with the observed phenotypic alterations. We conclude that reduced PTPS activity concomitant with mildly compromised BH4-biosynthesis leads to abnormal body fat distribution and abdominal obesity at least in mice. This study associates a novel single gene mutation with monogenic forms of obesity.

Charalambos Antoniades, Cheerag Shirodaria, Mark Crabtree, Ruth Rinze, Nicholas Alp, Colin Cunnington, Jonathan Diesch, Dimitris Tousoulis, Christodoulos Stefanadis, Paul Leeson, Chandi Ratnatunga, Ravi Pillai, Keith M. Channon (2007)Altered plasma versus vascular biopterins in human atherosclerosis reveal relationships between endothelial nitric oxide synthase coupling, endothelial function, and inflammation, In: Circulation (New York, N.Y.)116(24)pp. 2851-2859 Lippincott Williams & Wilkins

Background-Tetrahydrobiopterin ( BH4) is a key regulator of endothelial nitric oxide synthase (eNOS) activity and coupling. However, the extent to which vascular and/or systemic BH4 levels are altered in human atherosclerosis and the importance of BH4 bioavailability in determining endothelial function and oxidative stress remain unclear. We sought to define the relationships between plasma and vascular biopterin levels in patients with coronary artery disease and to determine how BH4 levels affect endothelial function, eNOS coupling, and vascular superoxide production. Methods and Results-Samples of saphenous veins and internal mammary arteries were collected from 219 patients with coronary artery disease undergoing coronary artery bypass grafting. We determined plasma and vascular levels of biopterins, vasomotor responses to acetylcholine, and vascular superoxide production in the presence and absence of the eNOS inhibitor N-G-nitro-L-arginine methyl ester. High vascular BH4 was associated with greater vasorelaxations to acetylcholine (P < 0.05), whereas high plasma BH4 was associated with lower vasorelaxations in response to acetylcholine (P < 0.05). Furthermore, an inverse association was observed between plasma and vascular biopterins (P < 0.05 for both saphenous veins and internal mammary arteries). High vascular (but not plasma) BH4 was associated with reduced total and NG-nitro-L-arginine methyl ester-inhibitable superoxide, suggesting improved eNOS coupling. Finally, plasma but not vascular biopterin levels were correlated with plasma C-reactive protein levels (P < 0.001). Conclusions-An inverse association exists between plasma and vascular biopterins in patients with coronary artery disease. Vascular but not plasma BH4 is an important determinant of eNOS coupling, endothelium-dependent vasodilation, and superoxide production in human vessels, whereas plasma biopterins are a marker of systemic inflammation.

Valerio Azzimato, Ping Chen, Emelie Barreby, Cecilia Morgantini, Laura Levi, Ana Vankova, Jennifer Jager, Andre Sulen, Marina Diotallevi, Joanne X. Shen, Anne Miller, Ewa Ellis, Mikael Ryden, Erik Naslund, Anders Thorell, Volker M. Lauschke, Keith M. Channon, Mark J. Crabtree, Arvand Haschemi, Siobhan M. Craige, Mattia Mori, Francesco Spallotta, Myriam Aouadi (2021)Hepatic miR-144 Drives Fumarase Activity Preventing NRF2 Activation During Obesity, In: Gastroenterology (New York, N.Y. 1943)161(6) Elsevier

BACKGROUND AND AIMS: Oxidative stress plays a key role in the development of metabolic complications associated with obesity, including insulin resistance and the most common chronic liver disease worldwide, nonalcoholic fatty liver disease. We have recently discovered that the microRNA miR-144 regulates protein levels of the master mediator of the antioxidant response, nuclear factor erythroid 2-related factor 2 (NRF2). On miR-144 silencing, the expression of NRF2 target genes was significantly upregulated, suggesting that miR-144 controls NRF2 at the level of both protein expression and activity. Here we explored a mechanism whereby hepatic miR144 inhibited NRF2 activity upon obesity via the regulation of the tricarboxylic acid (TCA) metabolite, fumarate, a potent activator of NRF2. METHODS: We performed transcriptomic analysis in liver macrophages (LMs) of obese mice and identified the immuno-responsive gene 1 (Irg1) as a target of miR-144. IRG1 catalyzes the production of a TCA derivative, itaconate, an inhibitor of succinate dehydrogenase (SDH). TCA enzyme activities and kinetics were analyzed after miR-144 silencing in obese mice and human liver organoids using single-cell activity assays in situ and molecular dynamic simulations. RESULTS: Increased levels of miR-144 in obesity were associated with reduced expression of Irg1, which was restored on miR-144 silencing in vitro and in vivo. Furthermore, miR144 overexpression reduces Irg1 expression and the production of itaconate in vitro. In alignment with the reduction in IRG1 levels and itaconate production, we observed an upregulation of SDH activity during obesity. Surprisingly, however, fumarate hydratase (FH) activity was also upregulated in obese livers, leading to the depletion of its substrate fumarate. miR-144 silencing selectively reduced the activities of both SDH and FH resulting in the accumulation of their related substrates succinate and fumarate. Moreover, molecular dynamics analyses revealed the potential role of itaconate as a competitive inhibitor of not only SDH but also FH. Combined, these results demonstrate that silencing of miR-144 inhibits the activity of NRF2 through decreased fumarate production in obesity. CONCLUSIONS: Herein we unravel a novel mechanism whereby miR-144 inhibits NRF2 activity through the consumption of fumarate by activation of FH. Our study demonstrates that hepatic miR-144 triggers a hyperactive FH in the TCA cycle leading to an impaired antioxidant response in obesity.

Anna Starr, Claire A Sand, Lamia Heikal, Peter D Kelly, Domenico Spina, Mark Crabtree, Keith M Channon, James M Leiper, Manasi Nandi (2014)Overexpression of GTP cyclohydrolase 1 feedback regulatory protein is protective in a murine model of septic shock, In: Shock (Augusta, Ga.)42(5)pp. 432-439

Overproduction of nitric oxide (NO) by inducible NO synthase contributes toward refractory hypotension, impaired microvascular perfusion, and end-organ damage in septic shock patients. Tetrahydrobiopterin (BH4) is an essential NOS cofactor. GTP cyclohydrolase 1 (GCH1) is the rate-limiting enzyme for BH4 biosynthesis. Under inflammatory conditions, GCH1 activity and hence BH4 levels are increased, supporting pathological NOS activity. GCH1 activity can be controlled through allosteric interactions with GCH1 feedback regulatory protein (GFRP). We investigated whether overexpression of GFRP can regulate BH4 and NO production and attenuate cardiovascular dysfunction in sepsis. Sepsis was induced in mice conditionally overexpressing GFRP and wild-type littermates by cecal ligation and puncture. Blood pressure was monitored by radiotelemetry, and mesenteric blood flow was quantified by laser speckle contrast imaging. Blood biochemistry data were obtained using an iSTAT analyzer, and BH4 levels were measured in plasma and tissues by high-performance liquid chromatography. Increased BH4 and NO production and hypotension were observed in all mice, but the extents of these pathophysiological changes were attenuated in GFRP OE mice. Perturbations in blood biochemistry were similarly attenuated in GFRP OE compared with wild-type controls. These results suggest that GFRP overexpression regulates GCH1 activity during septic shock, which in turn limits BH4 bioavailability for iNOS. We conclude that the GCH1-GFRP axis is a critical regulator of BH4 and NO production and the cardiovascular derangements that occur in septic shock.

Chieh-Ju Lu, Guoliang Hao, Natalia Nikiforova, Hege E Larsen, Kun Liu, Mark J Crabtree, Dan Li, Neil Herring, David J Paterson (2015)CAPON modulates neuronal calcium handling and cardiac sympathetic neurotransmission during dysautonomia in hypertension, In: Hypertension (Dallas, Tex. 1979)65(6)pp. 1288-1297

Genome-wide association studies implicate a variant in the neuronal nitric oxide synthase adaptor protein (CAPON) in electrocardiographic QT variation and sudden cardiac death. Interestingly, nitric oxide generated by neuronal NO synthase-1 reduces norepinephrine release; however, this pathway is downregulated in animal models of cardiovascular disease. Because sympathetic hyperactivity can trigger arrhythmia, is this neural phenotype linked to CAPON dysregulation? We hypothesized that CAPON resides in cardiac sympathetic neurons and is a part of the prediseased neuronal phenotype that modulates calcium handling and neurotransmission in dysautonomia. CAPON expression was significantly reduced in the stellate ganglia of spontaneously hypertensive rats before the development of hypertension compared with age-matched Wistar-Kyoto rats. The neuronal calcium current (ICa; n=8) and intracellular calcium transient ([Ca(2+)]i; n=16) were significantly larger in the spontaneously hypertensive rat than in Wistar-Kyoto rat (P

Mark J. Crabtree, Caroline L. Smith, George Lam, Michael S. Goligorsky, Steven S. Gross (2008)Ratio of 5,6,7,8-tetrahydrobiopterin to 7,8-dihydrobiopterin in endothelial cells determines glucose-elicited changes in NO vs. superoxide production by eNOS, In: American journal of physiology. Heart and circulatory physiology294(4)

5,6,7,8-Tetrahydrobiopterin (BH 4 ) is an essential cofactor of nitric oxide synthases (NOSs). Oxidation of BH 4 , in the setting of diabetes and other chronic vasoinflammatory conditions, can cause cofactor insufficiency and uncoupling of endothelial NOS (eNOS), manifest by a switch from nitric oxide (NO) to superoxide production. Here we tested the hypothesis that eNOS uncoupling is not simply a consequence of BH 4 insufficiency, but rather results from a diminished ratio of BH 4 vs. its catalytically incompetent oxidation product, 7,8-dihydrobiopterin (BH 2 ). In support of this hypothesis, [ 3 H]BH 4 binding studies revealed that BH 4 and BH 2 bind eNOS with equal affinity ( K d ≈ 80 nM) and BH 2 can rapidly and efficiently replace BH 4 in preformed eNOS-BH 4 complexes. Whereas the total biopterin pool of murine endothelial cells (ECs) was unaffected by 48-h exposure to diabetic glucose levels (30 mM), BH 2 levels increased from undetectable to 40% of total biopterin. This BH 2 accumulation was associated with diminished calcium ionophore-evoked NO activity and accelerated superoxide production. Since superoxide production was suppressed by NOS inhibitor treatment, eNOS was implicated as a principal superoxide source. Importantly, BH 4 supplementation of ECs (in low and high glucose-containing media) revealed that calcium ionophore-evoked NO bioactivity correlates with intracellular BH 4 : BH 2 and not absolute intracellular levels of BH 4 . Reciprocally, superoxide production was found to negatively correlate with intracellular BH 4 :BH 2 . Hyperglycemia-associated BH 4 oxidation and NO insufficiency was recapitulated in vivo, in the Zucker diabetic fatty rat model of type 2 diabetes. Together, these findings implicate diminished intracellular BH 4 :BH 2 , rather than BH 4 depletion per se, as the molecular trigger for NO insufficiency in diabetes.

Eileen McNeill, Mark J. Crabtree, Natasha Sahgal, Jyoti Patel, Surawee Chuaiphichai, Asif J. Iqbal, Ashley B. Hale, David R. Greaves, Keith M. Channon (2015)Regulation of iNOS function and cellular redox state by macrophage Gch1 reveals specific requirements for tetrahydrobiopterin in NRF2 activation, In: Free radical biology & medicine79pp. 206-216 Elsevier Inc

Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele (Gch1fl/fl) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes (Gch1fl/flTie2cre). Macrophages from Gch1fl/flTie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1fl/flTie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1fl/flTie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1fl/flTie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1fl/flTie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes (gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1fl/flTie2cre macrophages. These findings identify BH4-dependent iNOS regulation and NO generation as specific requirements for NRF2-dependent responses in macrophage inflammatory activation. [Display omitted] •Gch1−/− macrophages lack GTPCH protein expression, rendering cells BH4 deficient.•Gch1−/− cells express normal iNOS protein levels, but lack nitric oxide production.•BH4-deficient cells exhibit higher cellular ROS production.•Alterations in NO and ROS production can be reversed by pharmacological BH4 rescue.•Stimulated BH4-deficient cells show defective NRF2-dependent gene expression.

Jade Bailey, Andrew Shaw, Roman Fischer, Brent J. Ryan, Benedikt M. Kessler, James McCullagh, Richard Wade-Martins, Keith M. Channon, Mark J. Crabtree (2017)A novel role for endothelial tetrahydrobiopterin in mitochondrial redox balance, In: Free radical biology & medicine104pp. 214-225 Elsevier

The redox co-factor tetrahydrobiopterin (BH4) regulates nitric oxide (NO) and reactive oxygen species (ROS) production by endothelial NOS (eNOS) and is an important redox-dependent signalling molecule in the endothelium. Loss of endothelial BH4 is observed in cardiovascular disease (CVD) states and results in decreased NO and increased superoxide (O-2(-)) generation via eNOS uncoupling. Genetic mouse models of augmented endothelial BH4 synthesis have shown proof of concept that endothelial BH4 can alter CVD pathogenesis. However, clinical trials of BH4 therapy in vascular disease have been limited by systemic oxidation, highlighting the need to explore the wider roles of BH4 to find novel therapeutic targets. In this study, we aimed to elucidate the effects of BH4 deficiency on mitochondrial function and bioenergetics using targeted knockdown of the BH4 synthetic enzyme, GTP Cyclohydrolase I (GTPCH). Knockdown of GTPCH by > 90% led to marked loss of cellular BH4 and a striking induction of O-2(-) generation in the mitochondria of murine endothelial cells. This effect was likewise observed in BH4-depleted fibroblasts devoid of NOS, indicating a novel NOS-independent role for BH4 in mitochondrial redox signalling. Moreover, this BH4-dependent, mitochondria-derived ROS further oxidised mitochondrial BH4, concomitant with changes in the thioredoxin and glutathione antioxidant pathways. These changes were accompanied by a modest increase in mitochondrial size, mildly attenuated basal respiratory function, and marked changes in the mitochondrial proteome and cellular metabolome, including the accumulation of the TCA intermediate succinate. Taken together, these data reveal a novel NOS independent role for BH4 in the regulation of mitochondrial redox signalling and bioenergetic metabolism.

R. Cerrato, C. Cunnington, M. J. Crabtree, C. Antoniades, J. Pernow, K. M. Channon, F. Bohm (2012)Endothelin-1 increases superoxide production in human coronary artery bypass grafts, In: Life sciences (1973)91(13-14)pp. 723-728 Elsevier

Aims: Endothelin-1 (ET-1) has been shown to increase endothelial superoxide (O-2(-)) production in experimental animal models. It is unclear whether ET-1 increases O-2(-) production in humans. We sought to elucidate whether ET-1 increases O-2(-) production in human vessels and to identify the mechanism behind this effect. Main methods: Segments of internal mammary artery (IMA) and human saphenous vein (HSV) were harvested from 90 patients undergoing elective coronary artery bypass graft surgery. Paired vessel rings were incubated in the presence and absence of ET-1 (10(-10) M), the ETA receptor antagonist BQ123 alone, or in combination with the ETB receptor antagonist BQ788 (dual BQ) and known inhibitors of sources of O-2(-) and further analysed for O-2(-) production using lucigenin-enhanced chemiluminescence and DHE fluorescence. Key findings: ET-1 increased O-2(-) production in both IMA (2.6 +/- 1.5 vs. 1.4 +/- 0.8 relative light units/s/mg tissue (RLU); n = 33; p

Paul M. Heerdt, Paul B. Lane, Mark J. Crabtree, Bernard J. Park (2006)Systemic oxidative stress associated with lung resection during single lung ventilation, In: European journal of cardio-thoracic surgery30(3)pp. 568-569 Elsevier Science B.V
Amy L Tatham, Mark J Crabtree, Nicholas Warrick, Shijie Cai, Nicholas J Alp, Keith M Channon (2009)GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression, In: The Journal of biological chemistry284(20)pp. 13660-13668

GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression.

Ricardo Carnicer, Mark J. Crabtree, Vidhya Sivakumaran, Barbara Casadei, David A. Kass (2013)Nitric Oxide Synthases in Heart Failure, In: Antioxidants & redox signaling18(9)pp. 1078-1099 Mary Ann Liebert, Inc

Significance: The regulation of myocardial function by constitutive nitric oxide synthases (NOS) is important for the maintenance of myocardial Ca 2+ homeostasis, relaxation and distensibility, and protection from arrhythmia and abnormal stress stimuli. However, sustained insults such as diabetes, hypertension, hemodynamic overload, and atrial fibrillation lead to dysfunctional NOS activity with superoxide produced instead of NO and worse pathophysiology. Recent Advances: Major strides in understanding the role of normal and abnormal constitutive NOS in the heart have revealed molecular targets by which NO modulates myocyte function and morphology, the role and nature of post-translational modifications of NOS, and factors controlling nitroso-redox balance. Localized and differential signaling from NOS1 (neuronal) versus NOS3 (endothelial) isoforms are being identified, as are methods to restore NOS function in heart disease. Critical Issues: Abnormal NOS signaling plays a key role in many cardiac disorders, while targeted modulation may potentially reverse this pathogenic source of oxidative stress. Future Directions: Improvements in the clinical translation of potent modulators of NOS function/dysfunction may ultimately provide a powerful new treatment for many hearts diseases that are fueled by nitroso-redox imbalance. Antioxid. Redox Signal . 18, 1078–1099.

Jade Bailey, Simon Davis, Andrew Shaw, Marina Diotallevi, Roman Fischer, Matthew A Benson, Hanneng Zhu, James Brown, Shoumo Bhattacharya, Benedikt M Kessler, Keith M Channon, Mark J Crabtree (2018)Tetrahydrobiopterin modulates ubiquitin conjugation to UBC13/UBE2N and proteasome activity by S-nitrosation, In: Scientific reports8(1)

Nitric Oxide (NO) is an intracellular signalling mediator, which affects many biological processes via the posttranslational modification of proteins through S-nitrosation. The availability of NO and NOS-derived reactive oxygen species (ROS) from enzymatic uncoupling are determined by the NO synthase cofactor Tetrahydrobiopterin (BH4). Here, using a global proteomics "biotin-switch" approach, we identified components of the ubiquitin-proteasome system to be altered via BH4-dependent NO signalling by protein S-nitrosation. We show S-nitrosation of ubiquitin conjugating E2 enzymes, in particular the catalytic residue C87 of UBC13/UBE2N, leading to impaired polyubiquitylation by interfering with the formation of UBC13~Ub thioester intermediates. In addition, proteasome cleavage activity in cells also seems to be altered by S-nitrosation, correlating with the modification of cysteine residues within the 19S regulatory particle and catalytic subunits of the 20S complex. Our results highlight the widespread impact of BH4 on downstream cellular signalling as evidenced by the effect of a perturbed BH4-dependent NO-Redox balance on critical processes within the ubiquitin-proteasome system (UPS). These studies thereby uncover a novel aspect of NO associated modulation of cellular homeostasis.

Tim Van-Assche, Veronique Huygelen, Mark J Crabtree, Charalambos Antoniades (2013)Gene delivery strategies targeting stable atheromatous plaque, In: Current pharmaceutical design19(9)pp. 1626-1637

Conventional therapeutic options to treat chronic angina pectoris are pharmacological interventions, coronary bypass surgery (CABG) and percutaneous coronary intervention (PCI). In animal models, it was shown that gene delivery strategies harbour an exciting potential to support and maybe even replace conventional anti-angina treatments, but the translation of the basic science to clinical practise appears problematic. Gene therapy targeting key elements of neointima formation (e.g. cell cycle regulators, metalloproteinases, inflammation and oxidative stress) reduces vein graft and stent failure in experimental models. Additionally, systemic gene delivery of genes targeting NO production, oxidative stress, inflammation and foam cell formation has been shown to prevent atherosclerosis in different animal models. During CABG the vein graft can be transfected ex vivo and during PCI, a stent carrying transfection vectors can be deployed. Both strategies result in the induction of local transgene expression at the site of interest. This limits unwarranted transgene expression and the toxicity seen with systemic gene delivery. However, with the development of new transfection vectors, able to induce local transgene expression without detrimental side effects, systemic anti-inflammatory and anti-oxidative, gene delivery could be a powerful tool in secondary prevention.

Marina Diotallevi, Faseeha Ayaz, Thomas Nicol, Mark J Crabtree (2021)Itaconate as an inflammatory mediator and therapeutic target in cardiovascular medicine, In: Biochemical Society transactions49(5)pp. 2189-2198

Inflammation is a critical component of cardiovascular disease (CVD), encompassing coronary artery disease (CAD), cerebrovascular events and heart failure and is the leading cause of mortality worldwide. In recent years, metabolism has been placed centrally in the governance of the immune response. Termed immunometabolism, immune cells adapt cellular metabolic pathways to meet demands of activation and thus function. This rewiring influences not only the bioenergetics of the cell but altered metabolites act as signalling molecules to regulate cellular response. In this review, we focus on the TCA cycle derivative, itaconate, as one such metabolite with promising immunomodulatory and therapeutic potential in inflammatory cardiovascular disease.

Achini K Vidanapathirana, Jarrad M Goyne, Anna E Williamson, Benjamin J Pullen, Pich Chhay, Lauren Sandeman, Julien Bensalem, Timothy J Sargeant, Randall Grose, Mark J Crabtree, Run Zhang, Stephen J Nicholls, Peter J Psaltis, Christina A Bursill (2022)Biological Sensing of Nitric Oxide in Macrophages and Atherosclerosis Using a Ruthenium-Based Sensor, In: Biomedicines10(8)

Macrophage-derived nitric oxide (NO) plays a critical role in atherosclerosis and presents as a potential biomarker. We assessed the uptake, distribution, and NO detection capacity of an irreversible, ruthenium-based, fluorescent NO sensor (Ru-NO) in macrophages, plasma, and atherosclerotic plaques. In vitro, incubation of Ru-NO with human THP1 monocytes and THP1-PMA macrophages caused robust uptake, detected by Ru-NO fluorescence using mass-cytometry, confocal microscopy, and flow cytometry. THP1-PMA macrophages had higher Ru-NO uptake (+13%, p < 0.05) than THP1 monocytes with increased Ru-NO fluorescence following lipopolysaccharide stimulation (+14%, p < 0.05). In mice, intraperitoneal infusion of Ru-NO found Ru-NO uptake was greater in peritoneal CD11b+F4/80+ macrophages (+61%, p < 0.01) than CD11b+F4/80− monocytes. Infusion of Ru-NO into Apoe−/− mice fed high-cholesterol diet (HCD) revealed Ru-NO fluorescence co-localised with atherosclerotic plaque macrophages. When Ru-NO was added ex vivo to aortic cell suspensions from Apoe−/− mice, macrophage-specific uptake of Ru-NO was demonstrated. Ru-NO was added ex vivo to tail-vein blood samples collected monthly from Apoe−/− mice on HCD or chow. The plasma Ru-NO fluorescence signal was higher in HCD than chow-fed mice after 12 weeks (37.9%, p < 0.05). Finally, Ru-NO was added to plasma from patients (N = 50) following clinically-indicated angiograms. There was lower Ru-NO fluorescence from plasma from patients with myocardial infarction (−30.7%, p < 0.01) than those with stable coronary atherosclerosis. In conclusion, Ru-NO is internalised by macrophages in vitro, ex vivo, and in vivo, can be detected in atherosclerotic plaques, and generates measurable changes in fluorescence in murine and human plasma. Ru-NO displays promising utility as a sensor of atherosclerosis.

Mark J. Crabtree, Amy L. Tatham, Ashley B. Hale, Nicholas J. Alp, Keith M. Channon (2009)Critical Role for Tetrahydrobiopterin Recycling by Dihydrofolate Reductase in Regulation of Endothelial Nitric-oxide Synthase Coupling RELATIVE IMPORTANCE OF THE DENOVO BIOPTERIN SYNTHESIS VERSUS SALVAGE PATHWAYS, In: The Journal of biological chemistry284(41)pp. 28128-28136 Amer Soc Biochemistry Molecular Biology Inc

Tetrahyrobiopterin (BH4) is a required cofactor for the synthesis of nitric oxide by endothelial nitric-oxide synthase (eNOS), and BH4 bioavailability within the endothelium is a critical factor in regulating the balance between NO and superoxide production by eNOS (eNOS coupling). BH4 levels are determined by the activity of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in de novo BH4 biosynthesis. However, BH4 levels may also be influenced by oxidation, forming 7,8-dihydrobiopterin (BH2), which promotes eNOS uncoupling. Conversely, dihydrofolate reductase (DHFR) can regenerate BH4 from BH2, but the functional importance of DHFR in maintaining eNOS coupling remains unclear. We investigated the role of DHFR in regulating BH4 versus BH2 levels in endothelial cells and in cell lines expressing eNOS combined with tet-regulated GTPCH expression in order to compare the effects of low or high levels of de novo BH4 biosynthesis. Pharmacological inhibition of DHFR activity by methotrexate or genetic knockdown of DHFR protein by RNA interference reduced intracellular BH4 and increased BH2 levels resulting in enzymatic uncoupling of eNOS, as indicated by increased eNOS-dependent superoxide but reduced NO production. In contrast to the decreased BH4:BH2 ratio induced by DHFR knockdown, GTPCH knockdown greatly reduced total biopterin levels but with no change in BH4:BH2 ratio. In cells expressing eNOS with low biopterin levels, DHFR inhibition or knockdown further diminished the BH4:BH2 ratio and exacerbated eNOS uncoupling. Taken together, these data reveal a key role for DHFR in eNOS coupling by maintaining the BH4:BH2 ratio, particularly in conditions of low total biopterin availability.

Sandy M. Chu, Lisa C. Heather, Surawee Chuaiphichai, Thomas Nicol, Benjamin Wright, Matthieu Miossec, Jennifer K. Bendall, Gillian Douglas, Mark J. Crabtree, Keith M. Channon (2023)Cardiomyocyte tetrahydrobiopterin synthesis regulates fatty acid metabolism and susceptibility to ischaemia–reperfusion injury, In: Experimental physiology108(6)pp. 874-890
Surawee Chuaiphichai, Mark J Crabtree, Eileen Mcneill, Ashley B Hale, Lucy Trelfa, Keith M Channon, Gillian Douglas (2017)A key role for tetrahydrobiopterin-dependent endothelial NOS regulation in resistance arteries: studies in endothelial cell tetrahydrobiopterin-deficient mice, In: British journal of pharmacology174(8)pp. 657-671

The cofactor tetrahydrobiopterin (BH4) is a critical regulator of endothelial NOS (eNOS) function, eNOS-derived NO and ROS signalling in vascular physiology. To determine the physiological requirement for de novo endothelial cell BH4 synthesis for the vasomotor function of resistance arteries, we have generated a mouse model with endothelial cell-specific deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme for BH4 biosynthesis, and evaluated BH4-dependent eNOS regulation, eNOS-derived NO and ROS generation. The reactivity of mouse second-order mesenteric arteries was assessed by wire myography. High performance liquid chromatography was used to determine BH4, BH2 and biopterin. Western blotting was used for expression analysis. Gch1 Tie2cre mice demonstrated reduced GTPCH protein and BH4 levels in mesenteric arteries. Deficiency in endothelial cell BH4 leads to eNOS uncoupling, increased ROS production and loss of NO generation in mesenteric arteries of Gch1 Tie2cre mice. Gch1 Tie2cre mesenteric arteries had enhanced vasoconstriction to U46619 and phenylephrine, which was abolished by L-NAME. Endothelium-dependent vasodilatations to ACh and SLIGRL were impaired in mesenteric arteries from Gch1 Tie2cre mice, compared with those from wild-type littermates. Loss of eNOS-derived NO-mediated vasodilatation was associated with increased eNOS-derived H O and cyclooxygenase-derived vasodilator in Gch1 Tie2cre mesenteric arteries. Endothelial cell Gch1 and BH4-dependent eNOS regulation play pivotal roles in maintaining vascular homeostasis in resistance arteries. Therefore, targeting vascular Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of microvascular dysfunction in patients with cardiovascular disease.

Sergey V Brodsky, Olga Gealekman, Jun Chen, Fan Zhang, Nobuhiko Togashi, Mark Crabtree, Steven S Gross, Alberto Nasjletti, Michael S Goligorsky (2004)Prevention and reversal of premature endothelial cell senescence and vasculopathy in obesity-induced diabetes by ebselen, In: Circulation research94(3)pp. 377-384

Although the accelerated atherosclerosis and premature aging of the cardiovascular system in patients with metabolic syndrome have been appreciated, the mechanisms of their development and potential therapeutic interventions remain unresolved. Our previous studies implicated advanced glycosylation end products in development of premature senescence preventable with a peroxynitrite scavenger, ebselen. Therefore, the effect of ebselen on endothelial senescence and vasculopathy in a model of metabolic syndrome--Zucker diabetic rats (ZDF)--was investigated. Ebselen decreased the abundance of 3-nitrotyrosine-modified proteins in ZDF rats. A 6-fold increase in the number of senescent endothelial cells in 22-week-old ZDF was prevented by ebselen. Development of vasculopathy, as collectively judged by the acetylcholine-induced vasorelaxation, NO production, angiogenic competence, and number of circulating microparticles, was almost completely prevented when ebselen was administered from 8 to 22 weeks and partially reversed when the treatment interval was 13 to 22 weeks. In conclusion, premature senescence of endothelial cells is progressively rampant in ZDF rats and is associated with the signs of severe vasculopathy. In addition, prevention of premature senescence of vascular endothelium through controlled decrease in nitrotyrosine formation was chronologically associated with the amelioration of vasculopathy, lending support to the idea of the pathogenetic role of premature senescence of endothelial cells in diabetic macrovasculopathy.

Gillian Douglas, Jennifer K. Bendall, Mark J. Crabtree, Amy L. Tatham, Emma E. Carter, Ashley B. Hale, Keith M. Channon (2012)Endothelial-specific Nox2 overexpression increases vascular superoxide and macrophage recruitment in ApoE−/− mice, In: Cardiovascular research94(1)pp. 20-29 Oxford University Press
Mark J. Crabtree, Rachel Brixey, Helen Batchelor, Ashley B. Hale, Keith M. Channon (2013)Integrated Redox Sensor and Effector Functions for Tetrahydrobiopterin- and Glutathionylation-dependent Endothelial Nitric-oxide Synthase Uncoupling, In: The Journal of biological chemistry288(1)pp. 561-569 American Society for Biochemistry and Molecular Biology

Background: Both tetrahydrobiopterin (BH4) and S -glutathionylation are important regulators of eNOS activity and coupling. Results: S -Glutathionylation and BH4 deficiency induce eNOS uncoupling through distinct mechanisms but are mutually regulated by changes in BH4 oxidation and cellular GSH:GSSG ratio. Conclusion: BH4-dependent and S -glutathionylation-induced eNOS uncoupling are mechanistically independent but functionally linked. Significance: BH4 and S -glutathionylation exemplify eNOS as an integrated redox signaling “hub.” Endothelial nitric-oxide synthase (eNOS) is a critical regulator of vascular homeostasis by generation of NO that is dependent on the cofactor tetrahydrobiopterin (BH4). When BH4 availability is limiting, eNOS becomes “uncoupled,” resulting in superoxide production in place of NO. Recent evidence suggests that eNOS uncoupling can also be induced by S -glutathionylation, although the functional relationships between BH4 and S -glutathionylation remain unknown. To address a possible role for BH4 in S -glutathionylation-induced eNOS uncoupling, we expressed either WT or mutant eNOS rendered resistant to S -glutathionylation in cells with Tet-regulated expression of human GTP cyclohydrolase I to regulate intracellular BH4 availability. We reveal that S -glutathionylation of eNOS, by exposure to either 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or glutathione reductase-specific siRNA, results in diminished NO production and elevated eNOS-derived superoxide production, along with a concomitant reduction in BH4 levels and BH4:7,8-dihydrobiopterin ratio. In eNOS uncoupling induced by BH4 deficiency, BCNU exposure further exacerbates superoxide production, BH4 oxidation, and eNOS activity. Following mutation of C908S, BCNU-induced eNOS uncoupling and BH4 oxidation are abolished, whereas uncoupling induced by BH4 deficiency was preserved. Furthermore, BH4 deficiency alone is alone sufficient to reduce intracellular GSH:GSSG ratio and cause eNOS S -glutathionylation. These data provide the first evidence that BH4 deficiency- and S -glutathionylation-induced mechanisms of eNOS uncoupling, although mechanistically distinct, are functionally related. We propose that uncoupling of eNOS by S -glutathionylation- or by BH4-dependent mechanisms exemplifies eNOS as an integrated redox “hub” linking upstream redox-sensitive effects of BH4 and glutathione with redox-dependent targets and pathways that lie downstream of eNOS.

Mark J. Crabtree, Keith M. Channon (2009)Dihydrofolate reductase and biopterin recycling in cardiovascular disease, In: Journal of molecular and cellular cardiology47(6)pp. 749-751 Elsevier
Winifred O Idigo, Svetlana Reilly, Mei Hua Zhang, Yin Hua Zhang, Raja Jayaram, Ricardo Carnicer, Mark J Crabtree, Jean-Luc Balligand, Barbara Casadei (2012)Regulation of endothelial nitric-oxide synthase (NOS) S-glutathionylation by neuronal NOS: evidence of a functional interaction between myocardial constitutive NOS isoforms, In: The Journal of biological chemistry287(52)pp. 43665-43673

Myocardial constitutive No production depends on the activity of both endothelial and neuronal NOS (eNOS and nNOS, respectively). Stimulation of myocardial β(3)-adrenergic receptor (β(3)-AR) produces a negative inotropic effect that is dependent on eNOS. We evaluated whether nNOS also plays a role in β(3)-AR signaling and found that the β(3)-AR-mediated reduction in cell shortening and [Ca(2+)](i) transient amplitude was abolished both in eNOS(-/-) and nNOS(-/-) left ventricular (LV) myocytes and in wild type LV myocytes after nNOS inhibition with S-methyl-L-thiocitrulline. LV superoxide (O(2)(·-)) production was increased in nNOS(-/-) mice and reduced by L-N(ω)-nitroarginine methyl ester (L-NAME), indicating uncoupling of eNOS activity. eNOS S-glutathionylation and Ser-1177 phosphorylation were significantly increased in nNOS(-/-) myocytes, whereas myocardial tetrahydrobiopterin, eNOS Thr-495 phosphorylation, and arginase activity did not differ between genotypes. Although inhibitors of xanthine oxidoreductase (XOR) or NOX2 NADPH oxidase caused a similar reduction in myocardial O(2)(·-), only XOR inhibition reduced eNOS S-glutathionylation and Ser-1177 phosphorylation and restored both eNOS coupled activity and the negative inotropic and [Ca(2+)](i) transient response to β(3)-AR stimulation in nNOS(-/-) mice. In summary, our data show that increased O(2)(·-) production by XOR selectively uncouples eNOS activity and abolishes the negative inotropic effect of β(3)-AR stimulation in nNOS(-/-) myocytes. These findings provide unequivocal evidence of a functional interaction between the myocardial constitutive NOS isoforms and indicate that aspects of the myocardial phenotype of nNOS(-/-) mice result from disruption of eNOS signaling.

Brent J Ryan, Mark J Crabtree, Keith M Channon, Richard Wade-Martins (2015)Parkinson's disease in GTP cyclohydrolase 1 mutation carriers, In: Brain (London, England : 1878)138(Pt 5)pp. e348-U10
Rita Upmacis, Mark Crabtree, Ruba Deeb, Hao Sheen, Paul Lane, Lea Benguigui, Nobuyo Maeda, David Hajjar, Steven Gross (2007)Profound biopterin oxidation and protein tyrosine nitration in tissues of ApoE-null mice on an atherogenic diet: contribution of inducible nitric oxide synthase, In: American journal of physiology. Heart and circulatory physiology293(5)pp. H2878-2887 American Physiological Society

Diminished nitric oxide (NO) bioactivity and enhanced peroxynitrite formation have been implicated as major contributors to atherosclerotic vascular dysfunctions. Hallmark reactions of peroxynitrite include the accumulation of 3-nitrotyrosine (3-NT) in proteins and oxidation of the NO synthase (NOS) cofactor, tetrahydrobiopterin (BH^sub 4^). The present study sought to 1) quantify the extent to which 3-NT accumulates and BH^sub 4^ becomes oxidized in organs of apolipoprotein E-deficient (ApoE...) atherosclerotic mice and 2) determine the specific contribution of inducible NOS (iNOS) to these processes. Whereas protein 3-NT and oxidized BH^sub 4^ were undetected or near the detection limit in heart, lung, and kidney of 3-wk-old ApoE... mice or ApoE... mice fed a regular chow diet for 24 wk, robust accumulation was evident after 24 wk on a Western (atherogenic) diet. Since 3-NT accumulation was diminished 3- to 20-fold in heart, lung, and liver in ApoE... mice missing iNOS, iNOS-derived species are involved in this reaction. In contrast, iNOS-derived species did not contribute to elevated protein 3-NT formation in kidney or brain. iNOS deletion also afforded marked protection against BH^sub 4^ oxidation in heart, lung, and kidney of atherogenic ApoE... mice but not in brain or liver. These findings demonstrate that iNOS-derived species are increased during atherogenesis in ApoE... mice and that these species differentially contribute to protein 3-NT accumulation and BH^sub 4^ oxidation in a tissue-selective manner. Since BH^sub 4^ oxidation can switch the predominant NOS product from NO to superoxide, we predict that progressive NOS uncoupling is likely to drive atherogenic vascular dysfunctions. (ProQuest: ... denotes formulae/symbols omitted.)

Tim S. Schmidt, Eileen McNeill, Gillian Douglas, Mark J. Crabtree, Ashley B. Hale, Jeffrey Khoo, Charles A. O'Neill, Alphonsus Cheng, Keith M. Channon, Nicholas J. Alp (2010)Tetrahydrobiopterin supplementation reduces atherosclerosis and vascular inflammation in apolipoprotein E-knockout mice, In: Clinical science (1979)119(3-4)pp. 131-142 Portland Press Ltd

BH4 (tetrahydrobiopterin) supplementation improves endothelial function in models of vascular disease by maintaining eNOS (endothelial nitric oxide synthase) coupling and NO (nitric oxide) bioavailability. However, the cellular mechanisms through which enhanced endothelial function leads to reduced atherosclerosis remain unclear. We have used a pharmaceutical BH4 formulation to investigate the effects of BH4 supplementation on atherosclerosis progression in ApoE-KO (apolipoprotein E-knockout) mice. Single oral dose pharmacokinetic studies revealed rapid BH4 uptake into plasma and organs. Plasma BH4 levels returned to baseline by 8 h after oral dosing, but remained markedly increased in aorta at 24 h. Daily oral BH4 supplementation in ApoE-KO mice from 8 weeks of age, for a period of 8 or 12 weeks, had no effect on plasma lipids or haemodynamic parameters, but significantly reduced aortic root atherosclerosis compared with placebo-treated animals. BH4 supplementation significantly reduced VCAM-I (vascular cell adhesion molecule I) mRNA levels in aortic endothelial cells, markedly reduced the infiltration of T-cells, macrophages and monocytes into plaques, and reduced T-cell infiltration in the adjacent adventitia, but importantly had no effect on circulating leucocytes. GCH (GTP cyclohydrolase I)-transgenic mice, with a specific increase in endothelial BH4 levels, exhibited a similar reduction in vascular immune cell infiltration compared with BH4-deficient controls, suggesting that BH4 reduces vascular inflammation via endothelial cell signalling. In conclusion, BH4 supplementation reduces vascular immune cell infiltration in atherosclerosis and may therefore be a rational therapeutic approach to reduce the progression of atherosclerosis.

Surawee Chuaiphichai, Sandy M. Chu, Ricardo Carnicer, Matthew Kelly, Jenifer K. Bendall, Jillian N. Simon, Gillian Douglas, Mark J. Crabtree, Barbara Casadei, Keith M. Channon (2023)Endothelial cell-specific roles for tetrahydrobiopterin in myocardial function, cardiac hypertrophy, and response to myocardial ischemia-reperfusion injury, In: American journal of physiology. Heart and circulatory physiology324(4)pp. H430-H442 Amer Physiological Soc

The cofactor tetrahydrobiopterin (BH4) is a critical regulator of nitric oxide synthase (NOS) function and redox signaling, with reduced BH4 implicated in multiple cardiovascular disease states. In the myocardium, augmentation of BH4 levels can impact on cardiomyocyte function, preventing hypertrophy and heart failure. However, the specific role of endothelial cell BH4 biosynthesis in the coronary circulation and its role in cardiac function and the response to ischemia has yet to be elucidated. Endothelial cell-specific Gch1 knockout mice were generated by crossing Gch1fl/fl with Tie2cre mice, generating Gch1fl/flTie2cre mice and littermate controls. GTP cyclohydrolase protein and BH4 levels were reduced in heart tissues from Gch1fl/flTie2cre mice, localized to endothelial cells, with normal cardiomyocyte BH4. Deficiency in coronary endothelial cell BH4 led to NOS uncoupling, decreased NO bioactivity, and increased superoxide and hydrogen peroxide productions in the hearts of Gch1fl/flTie2cre mice. Under physiological conditions, loss of endothelial cell-specific BH4 led to mild cardiac hypertrophy in Gch1fl/flTie2cre hearts. Endothelial cell BH4 loss was also associated with increased neuronal NOS protein, loss of endothelial NOS protein, and increased phospholamban phosphorylation at serine-17 in cardiomyocytes. Loss of cardiac endothelial cell BH4 led to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia-reperfusion injury. Taken together, these studies reveal a specific role for endothelial cell Gch1/BH4 biosynthesis in cardiac function and the response to cardiac ischemia-reperfusion injury. Targeting endothelial cell Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction and ischemia-reperfusion injury. NEW & NOTEWORTHY We demonstrate a critical role for endothelial cell Gch1/BH4 biosynthesis in coronary vascular function and cardiac function. Loss of cardiac endothelial cell BH4 leads to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia/reperfusion injury. Targeting endothelial cell Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction, ischemia injury, and heart failure.

Surawee Chuaiphichai, Eileen McNeill, Gillian Douglas, Mark J Crabtree, Jennifer K Bendall, Ashley B Hale, Nicholas J Alp, Keith M Channon (2014)Cell-autonomous role of endothelial GTP cyclohydrolase 1 and tetrahydrobiopterin in blood pressure regulation, In: Hypertension (Dallas, Tex. 1979)64(3)pp. 530-540

Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) function and NO generation. Augmentation of BH4 levels can prevent eNOS uncoupling and can improve endothelial dysfunction in vascular disease states. However, the physiological requirement for de novo endothelial cell BH4 biosynthesis in eNOS function remains unclear. We generated a novel mouse model with endothelial cell-specific deletion of GCH1, encoding GTP cyclohydrolase 1, an essential enzyme for BH4 biosynthesis, to test the cell-autonomous requirement for endothelial BH4 biosynthesis in vivo. Mice with a floxed GCH1 allele (GCH1(fl/fl)) were crossed with Tie2cre mice to delete GCH1 in endothelial cells. GCH1(fl/fl)Tie2cre mice demonstrated virtually absent endothelial NO bioactivity and significantly greater O2 (•-) production. GCH1(fl/fl)Tie2cre aortas and mesenteric arteries had enhanced vasoconstriction to phenylephrine and impaired endothelium-dependent vasodilatations to acetylcholine and SLIGRL. Endothelium-dependent vasodilatations in GCH1(fl/fl)Tie2cre aortas were, in part, mediated by eNOS-derived hydrogen peroxide (H2O2), which mediated vasodilatation through soluble guanylate cyclase. Ex vivo supplementation of aortic rings with the BH4 analogue sepiapterin restored normal endothelial function and abolished eNOS-derived H2O2 production in GCH1(fl/fl)Tie2cre aortas. GCH1(fl/fl)Tie2cre mice had higher systemic blood pressure than wild-type littermates, which was normalized by NOS inhibitor, NG-nitro-L-arginine methyl ester. Taken together, these studies reveal an endothelial cell-autonomous requirement for GCH1 and BH4 in regulation of vascular tone and blood pressure and identify endothelial cell BH4 as a pivotal regulator of NO versus H2O2 as alternative eNOS-derived endothelial-derived relaxing factors.

Gillian Douglas, Ashley B. Hale, Mark J Crabtree, Brent J. Ryan, Alex Hansler, Katrin Watschinger, Steven S Gross, Craig A. Lygate, Nicholas J. Alp, Keith M. Channon (2015)A requirement for Gch1 and tetrahydrobiopterin in embryonic development, In: Developmental biology399(1)pp. 129-138 Elsevier Inc

GTP cyclohydrolase I (GTPCH) catalyses the first and rate-limiting reaction in the synthesis of the enzymatic cofactor, tetrahydrobiopterin (BH4). Loss of function mutations in the GCH1 gene lead to congenital neurological diseases such as DOPA-responsive dystonia and hyperphenylalaninemia. However, little is known about how GTPCH and BH4 affects embryonic development in utero, and in particular whether metabolic replacement or supplementation in pregnancy is sufficient to rescue genetic GTPCH deficiency in the developing embryo. Gch1 deficient mice were generated by the insertion of loxP sites flanking exons 2–3 of the Gch1 gene. Gch1fl/fl mice were bred with Sox2cre mice to generate mice with global Gch1 deficiency. Genetic ablation of Gch1 caused embryonic lethality by E13.5. Despite loss of Gch1 mRNA and GTPCH enzymatic activity, whole embryo BH4 levels were maintained until E11.5, indicating sufficient maternal transfer of BH4 to reach this stage of development. After E11.5, Gch1−/− embryos were deficient in BH4, but an unbiased metabolomic screen indicated that the lethality was not due to a gross disturbance in metabolic profile. Embryonic lethality in Gch1−/− embryos was not caused by structural abnormalities, but was associated with significant bradycardia at E11.5. Embryonic lethality was not rescued by maternal supplementation of BH4, but was partially rescued, up to E15.5, by maternal supplementation of BH4 and l-DOPA. These findings demonstrate a requirement for Gch1 in embryonic development and have important implications for the understanding of pathogenesis and treatment of genetic BH4 deficiencies, as well as the identification of new potential roles for BH4. •Generation and developmental analysis of Gch1 knockout mice.•Global deficiency in Gch1 is embryonically lethal between E11.5 and E13.5.•Gch1−/− embryos have no gross structural abnormalities but are bradycardic at E11.5.•Embryo lethality cannot be rescued by maternal supplementation.•There is a requirement for Gch1 and BH4 in embryo development.

Jennifer K. Bendall, Gillian Douglas, Eileen McNeill, Keith M. Channon, Mark J. Crabtree (2014)Tetrahydrobiopterin in Cardiovascular Health and Disease, In: Antioxidants & redox signaling20(18)pp. 3040-3077 Mary Ann Liebert, Inc

Tetrahydrobiopterin (BH4) functions as a cofactor for several important enzyme systems, and considerable evidence implicates BH4 as a key regulator of endothelial nitric oxide synthase ( eNOS) in the setting of cardiovascular health and disease. BH4 bioavailability is determined by a balance of enzymatic de novo synthesis and recycling, versus degradation in the setting of oxidative stress. Augmenting vascular BH4 levels by pharmacological supplementation has been shown in experimental studies to enhance NO bioavailability. However, it has become more apparent that the role of BH4 in other enzymatic pathways, including other NOS isoforms and the aromatic amino acid hydroxylases, may have a bearing on important aspects of vascular homeostasis, inflammation, and cardiac function. This article reviews the role of BH4 in cardiovascular development and homeostasis, as well as in pathophysiological processes such as endothelial and vascular dysfunction, atherosclerosis, inflammation, and cardiac hypertrophy. We discuss the therapeutic potential of BH4 in cardiovascular disease states and attempt to address how this modulator of intracellular NO-redox balance may ultimately provide a powerful new treatment for many cardiovascular diseases.

Matthew A. Benson, Helen Batchelor, Surawee Chuaiphichai, Jade Bailey, Hanneng Zhu, Dennis J. Stuehr, Shoumo Bhattacharya, Keith M. Channon, Mark J. Crabtree (2013)A Pivotal Role for Tryptophan 447 in Enzymatic Coupling of Human Endothelial Nitric Oxide Synthase (eNOS), In: The Journal of biological chemistry288(41)pp. 29836-29845 American Society for Biochemistry and Molecular Biology

Background: Interaction of tetrahydrobiopterin (BH4) with a key tryptophan residue in the NOS active site is critical for activity. Results: Mutation of tryptophan 447 causes eNOS uncoupling and monomerization. Conclusion: Tryptophan 447 determines enzymatic coupling of human eNOS. Significance: The development of BH4-based strategies to restore NOS function must consider the structural effects of BH4 binding and their role in NOS coupling. Tetrahydrobiopterin (BH4) is a required cofactor for the synthesis of NO by NOS. Bioavailability of BH4 is a critical factor in regulating the balance between NO and superoxide production by endothelial NOS (eNOS coupling). Crystal structures of the mouse inducible NOS oxygenase domain reveal a homologous BH4-binding site located in the dimer interface and a conserved tryptophan residue that engages in hydrogen bonding or aromatic stacking interactions with the BH4 ring. The role of this residue in eNOS coupling remains unexplored. We overexpressed human eNOS W447A and W447F mutants in novel cell lines with tetracycline-regulated expression of human GTP cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis, to determine the importance of BH4 and Trp-447 in eNOS uncoupling. NO production was abolished in eNOS-W447A cells and diminished in cells expressing W447F, despite high BH4 levels. eNOS-derived superoxide production was significantly elevated in W447A and W447F versus wild-type eNOS, and this was sufficient to oxidize BH4 to 7,8-dihydrobiopterin. In uncoupled, BH4-deficient cells, the deleterious effects of W447A mutation were greatly exacerbated, resulting in further attenuation of NO and greatly increased superoxide production. eNOS dimerization was attenuated in W447A eNOS cells and further reduced in BH4-deficient cells, as demonstrated using a novel split Renilla luciferase biosensor. Reduction of cellular BH4 levels resulted in a switch from an eNOS dimer to an eNOS monomer. These data reveal a key role for Trp-447 in determining NO versus superoxide production by eNOS, by effects on BH4-dependent catalysis, and by modulating eNOS dimer formation.

Gillian Douglas, Ashley B Hale, Jyoti Patel, Surawee Chuaiphichai, Ayman Al Haj Zen, Victoria S Rashbrook, Lucy Trelfa, Mark J Crabtree, Eileen McNeill, Keith M Channon (2018)Roles for endothelial cell and macrophage Gch1 and tetrahydrobiopterin in atherosclerosis progression, In: Cardiovascular research114(10)pp. 1385-1399

GTP cyclohydrolase I catalyses the first and rate-limiting reaction in the synthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases (NOS). Both eNOS and iNOS have been implicated in the progression of atherosclerosis, with opposing effects in eNOS and iNOS knockout mice. However, the pathophysiologic requirement for BH4 in regulating both eNOS and iNOS function, and the effects of loss of BH4 on the progression of atherosclerosis remains unknown. Hyperlipidemic mice deficient in Gch1 in endothelial cells and leucocytes were generated by crossing Gch1fl/flTie2cre mice with ApoE-/- mice. Deficiency of Gch1 and BH4 in endothelial cells and myeloid cells was associated with mildly increased blood pressure. High fat feeding for 6 weeks in Gch1fl/flTie2CreApoE-/- mice resulted in significantly decreased circulating BH4 levels, increased atherosclerosis burden and increased plaque macrophage content. Gch1fl/flTie2CreApoE-/- mice showed hallmarks of endothelial cell dysfunction, with increased aortic VCAM-1 expression and decreased endothelial cell dependent vasodilation. Furthermore, loss of BH4 from pro-inflammatory macrophages resulted in increased foam cell formation and altered cellular redox signalling, with decreased expression of antioxidant genes and increased reactive oxygen species. Bone marrow chimeras revealed that loss of Gch1 in both endothelial cells and leucocytes is required to accelerate atherosclerosis. Both endothelial cell and macrophage BH4 play important roles in the regulation of NOS function and cellular redox signalling in atherosclerosis.

Eileen McNeill, Elena Stylianou, Mark J Crabtree, Rachel Harrington-Kandt, Anna-Lena Kolb, Marina Diotallevi, Ashley B Hale, Paulo Bettencourt, Rachel Tanner, Matthew K O'Shea, Magali Matsumiya, Helen Lockstone, Julius Müller, Helen A Fletcher, David R Greaves, Helen McShane, Keith M Channon (2018)Regulation of mycobacterial infection by macrophage Gch1 and tetrahydrobiopterin, In: Nature communications9(1)5409

Inducible nitric oxide synthase (iNOS) plays a crucial role in controlling growth of Mycobacterium tuberculosis (M.tb), presumably via nitric oxide (NO) mediated killing. Here we show that leukocyte-specific deficiency of NO production, through targeted loss of the iNOS cofactor tetrahydrobiopterin (BH4), results in enhanced control of M.tb infection; by contrast, loss of iNOS renders mice susceptible to M.tb. By comparing two complementary NO-deficient models, Nos2 mice and BH4 deficient Gch1 Tie2cre mice, we uncover NO-independent mechanisms of anti-mycobacterial immunity. In both murine and human leukocytes, decreased Gch1 expression correlates with enhanced cell-intrinsic control of mycobacterial infection in vitro. Gene expression analysis reveals that Gch1 deficient macrophages have altered inflammatory response, lysosomal function, cell survival and cellular metabolism, thereby enhancing the control of bacterial infection. Our data thus highlight the importance of the NO-independent functions of Nos2 and Gch1 in mycobacterial control.

Tim Van-Assche, Veronique Huygelen, Mark J Crabtree, Charalambos Antoniades (2011)Gene therapy targeting inflammation in atherosclerosis, In: Current pharmaceutical design17(37)pp. 4210-4223

The extensive cross-talk between the immune system and vasculature leading to the infiltration of immune cells into the vascular wall is a major step in atherogenesis. In this process, reactive oxygen species play a crucial role, by inducing the oxidation of LDL and the formation of foam cells, and by activating a number of redox-sensitive transcriptional factors such as nuclear factor kappa B (NFkappa B) or activating protein 1 (AP1), that regulate the expression of multiple pro/anti inflammatory genes involved in atherogenesis. Delivery of genes encoding antioxidant defense enzymes (e.g. superoxide dismutase, catalase, glutathione peroxidase or heme oxygenase- 1) or endothelial nitric oxide synthase (eNOS), suppress atherogenesis in animal models. Similarly, delivery of genes encoding regulators of redox sensitive transcriptional factors (e.g. NF-kappa B, AP-1, Nrf2 etc) or reactive oxygen species scavengers have been successfully used in experimental studies. Despite the promising results from basic science, the clinical applicability of these strategies has proven to be particularly challenging. Issues regarding the vectors used to deliver the genes (and the development of immune responses or other side effects) and the inability of sufficient and sustained local expression of these genes at the target-tissue are some of the main reasons preventing optimism regarding the use of these strategies at a clinical level. Therefore, although premature to discuss about effective "gene therapy" in atherosclerosis at a clinical level, gene delivery techniques opened new horizons in cardiovascular research, and the development of new vectors may allow their extensive use in clinical trials in the future.

Mark J Crabtree, Ashley B Hale, Keith M Channon (2011)Dihydrofolate reductase protects endothelial nitric oxide synthase from uncoupling in tetrahydrobiopterin deficiency, In: Free radical biology & medicine50(11)pp. 1639-1646

Tetrahydrobiopterin (BH4) is a required cofactor for the synthesis of NO by endothelial nitric oxide synthase (eNOS), and endothelial BH4 bioavailability is a critical factor in regulating the balance between NO and superoxide production (eNOS coupling). Biosynthesis of BH4 is determined by the activity of GTP-cyclohydrolase I (GTPCH). However, BH4 levels may also be influenced by oxidation, forming 7,8-dihydrobiopterin (BH2), which promotes eNOS uncoupling. Conversely, dihydrofolate reductase (DHFR) can regenerate BH4 from BH2, but whether DHFR is functionally important in maintaining eNOS coupling remains unclear. To investigate the mechanism by which DHFR might regulate eNOS coupling in vivo, we treated wild-type, BH4-deficient (hph-1), and GTPCH-overexpressing (GCH-Tg) mice with methotrexate (MTX), to inhibit BH4 recycling by DHFR. MTX treatment resulted in a striking elevation in BH2 and a decreased BH4:BH2 ratio in the aortas of wild-type mice. These effects were magnified in hph-1 but diminished in GCH-Tg mice. Attenuated eNOS activity was observed in MTX-treated hph-1 but not wild-type or GCH-Tg mouse lung, suggesting that inhibition of DHFR in BH4-deficient states leads to eNOS uncoupling. Taken together, these data reveal a key role for DHFR in regulating the BH4 vs BH2 ratio and eNOS coupling under conditions of low total biopterin availability in vivo.

Ricardo Carnicer, Ashley B. Hale, Silvia Suffredini, Xing Liu, Svetlana Reilly, Mei Hua Zhang, Nicoletta C. Surdo, Jennifer K. Bendall, Mark J. Crabtree, Gregory B. S. Lim, Nicholas J. Alp, Keith M. Channon, Barbara Casadei (2012)Cardiomyocyte GTP Cyclohydrolase 1 and Tetrahydrobiopterin Increase NOS1 Activity and Accelerate Myocardial Relaxation, In: Circulation research111(6)pp. 718-727 Lippincott Williams & Wilkins

Rationale: Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthases (NOS). Oral BH4 supplementation preserves cardiac function in animal models of cardiac disease; however, the mechanisms underlying these findings are not completely understood. Objective: To study the effect of myocardial transgenic overexpression of the rate-limiting enzyme in BH4 biosynthesis, GTP cyclohydrolase 1 (GCH1), on NOS activity, myocardial function, and Ca2+ handling. Methods and Results: GCH1overexpression significantly increased the biopterins level in left ventricular (LV) myocytes but not in the nonmyocyte component of the LV myocardium or in plasma. The ratio between BH4 and its oxidized products was lower in mGCH1-Tg, indicating that a large proportion of the myocardial biopterin pool was oxidized; nevertheless, myocardial NOS1 activity was increased in mGCH1-Tg, and superoxide release was significantly reduced. Isolated hearts and field-stimulated LV myocytes (3 Hz, 35 degrees C) overexpressing GCH1 showed a faster relaxation and a PKA-mediated increase in the PLB Ser(16) phosphorylated fraction and in the rate of decay of the [Ca2+](i) transient. RyR2 S-nitrosylation and diastolic Ca2+ leak were larger in mGCH1-Tg and I-Ca density was lower; nevertheless the amplitude of the [Ca2+](i) transient and contraction did not differ between genotypes, because of an increase in the SR fractional release of Ca2+ in mGCH1-Tg myocytes. Xanthine oxidoreductase inhibition abolished the difference in superoxide production but did not affect myocardial function in either group. By contrast, NOS1 inhibition abolished the differences in I-Ca density, Ser(16) PLB phosphorylation, [Ca2+](i) decay, and myocardial relaxation between genotypes. Conclusions: Myocardial GCH1 activity and intracellular BH4 are a limiting factor for constitutive NOS1 and SERCA2A activity in the healthy myocardium. Our findings suggest that GCH1 may be a valuable target for the treatment of LV diastolic dysfunction. (Circ Res. 2012;111:718-727.)

Mark J. Crabtree, Keith M. Channon (2011)Synthesis and recycling of tetrahydrobiopterin in endothelial function and vascular disease, In: Nitric oxide25(2)pp. 81-88 Elsevier

Nitric oxide, generated by the nitric oxide synthase (NOS) enzymes, plays pivotal roles in cardiovascular homeostasis and in the pathogenesis of cardiovascular disease. The NOS cofactor, tetrahydrobiopterin (BH4), is an important regulator of NOS function, since BH4 is required to maintain enzymatic coupling of L-arginine oxidation, to produce NO. Loss or oxidation of BH4 to 7,8-dihydrobiopterin (BH2) is associated with NOS uncoupling, resulting in the production of superoxide rather than NO. In addition to key roles in folate metabolism, dihydrofolate reductase (DHFR) can 'recycle' BH2, and thus regenerate BH4 [1,2]. It is therefore likely that net BH4 cellular bioavailability reflects the balance between de nova BH4 synthesis, loss of BH4 by oxidation to BH2, and the regeneration of BH4 by DHFR. Recent studies have implicated BH4 recycling in the direct regulation of eNOS uncoupling, showing that inhibition of BH4 recycling using DHFR-specific siRNA and methotrexate treatment leads to eNOS uncoupling in endothelial cells and the hph-1 mouse model of BH4 deficiency, even in the absence of oxidative stress. These studies indicate that not only BH4 level, but the recycling pathways regulating BH4 bioavailability represent potential therapeutic targets and will be discussed in this review. (C) 2011 Elsevier Inc. All rights reserved.

Ruha Cerrato, Mark Crabtree, Charalambos Antoniades, Karolina Kublickiene, Ernesto L Schiffrin, Keith M Channon, Felix Böhm (2018)Effects Of Endothelin-1 On Intracellular Tetrahydrobiopterin Levels In Vascular Tissue, In: Scandinavian cardiovascular journal : SCJ52(3)pp. 163-169

Tetrahydrobiopterin (BH4) is the essential cofactor of endothelial nitric oxide synthase (eNOS) and intracellular levels of BH4 is regulated by oxidative stress. The aim of this paper was to describe the influence of exogenous endothelin-1 on intracellular BH4 and its oxidation products dihydrobiopterin (BH2) and biopterin (B) in a wide range of vascular tissue. Segments of internal mammary artery (IMA) and human saphenous vein (SV) from 41 patients undergoing elective surgery were incubated in ET-1 (0.1 μM). Aorta and lung from transgenic mice overexpressing ET-1 in the endothelium (ET-TG) were analysed with regards to intracellular biopterin levels. Human umbilical vein endothelial cells (HUVEC) were incubated in ET-1 (0.1 μM) and intracellular biopterin levels were analysed. From 6 healthy women undergoing caesarean section, subcutaneous fat was harvested and the resistance arteries in these biopsies were tested for ET-mediated endothelial dysfunction. In HUVEC, exogenous ET-1 (0.1 μM) did not significantly change intracellular BH4, 1.54 ± 1.7 vs 1.68 ± 1.8 pmol/mg protein; p = .8. In IMA and SV, exogenous ET-1(0.1 μM) did not change intracellular BH4 n = 10, p = .4. In aorta from wild type vs ET-TG mice there was no significant difference in intracellular BH4 between the groups: 1.3 ± 0.49 vs 1.23 ± 0.3 pmol/mg protein; p = .6. In resistance arteries (n = 6) BH4 together with DTE (an antioxidant) was not able to prevent ET-mediated endothelial dysfunction. ET-1 did not significantly alter intracellular tetrahydrobiopterin levels in IMA, SV, HUVEC or aorta from ET-TG mice. These findings are important for future research in ET-1 mediated superoxide production and endothelial dysfunction.

Manish Kalla, Minesh Chotalia, Charles Coughlan, Guoliang Hao, Mark J. Crabtree, Jakub Tomek, Gil Bub, David J. Paterson, Neil Herring (2016)Protection against ventricular fibrillation via cholinergic receptor stimulation and the generation of nitric oxide, In: The Journal of physiology594(14)pp. 3981-3992 Wiley

Implantable cardiac vagal nerve stimulators are a promising treatment for ventricular arrhythmia in patients with heart failure. Animal studies suggest the anti-fibrillatory effect may be nitric oxide (NO) dependent, although the exact site of action is controversial. We investigated whether a stable analogue of acetylcholine could raise ventricular fibrillation threshold (VFT), and whether this was dependent on NO generation and/or muscarinic/nicotinic receptor stimulation. VFT was determined in Langendorff perfused rat hearts by burst pacing until sustained VF was induced. Carbamylcholine (CCh, 200 nmol l(-1), n = 9) significantly (P < 0.05) reduced heart rate from 292 +/- 8 to 224 +/- 6 b.p.m. Independent of this heart rate change, CCh caused a significant increase in VFT (control 1.5 +/- 0.3 mA, CCh 2.4 +/- 0.4 mA, wash 1.1 +/- 0.2 mA) and flattened the restitution curve (n = 6) derived from optically mapped action potentials. The effect of CCh on VFT was abolished by a muscarinic (atropine, 0.1 mu mol l(-1), n = 6) or a nicotinic receptor antagonist (mecamylamine, 10 mu mol l(-1), n = 6). CCh significantly increased NOx content in coronary effluent (n = 8), but not in the presence of mecamylamine (n = 8). The neuronal nitric oxide synthase inhibitor AAAN (N-(4S)-4-amino-5-[aminoethyl]aminopentyl-N'-nitroguanidine; 10 mu mol l(-1), n=6) or soluble guanylate cyclase (sGC) inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 10 mu mol l(-1), n = 6) prevented the rise in VFT with CCh. The NO donor sodium nitrprusside (10 mu mol l(-1), n = 8) mimicked the action of CCh on VFT, an effect that was also blocked by atropine (n = 10). These data demonstrate a protective effect of CCh on VFT that depends upon both muscarinic and nicotinic receptor stimulation, where the generation of NO is likely to be via a neuronal nNOS/sGC-dependent pathway.

Praveen N Chander, Olga Gealekman, Sergey V Brodsky, Saban Elitok, Akihiro Tojo, Mark Crabtree, Steven S Gross, Michael S Goligorsky (2004)Nephropathy in Zucker diabetic fat rat is associated with oxidative and nitrosative stress: Prevention by chronic therapy with a peroxynitrite scavenger ebselen, In: Journal of the American Society of Nephrology15(9)pp. 2391-2403 Lippincott Williams & Wilkins
Raja Jayaram, Mark J Crabtree, Michael Jones, Svetlana Reilly, Nikhil Pal, Nicola Goodfellow, Keshav Nahar, Jillian Simon, Ricardo Carnicer, Ravi DeSilva, Chandana Ratnatunga, Mario Petrou, Rana Sayeed, Andrea Roalfe, Keith M Channon, Yaver Bashir, Timothy Betts, Michael Hill, Barbara Casadei (2022)Atrial nitroso-redox balance and refractoriness following on-pump cardiac surgery: a randomized trial of atorvastatin, In: Cardiovascular research118(1)pp. 184-195

Systemic inflammation and increased activity of atrial NOX2-containing NADPH oxidases have been associated with the new onset of atrial fibrillation (AF) after cardiac surgery. In addition to lowering LDL-cholesterol, statins exert rapid anti-inflammatory and antioxidant effects, the clinical significance of which remains controversial. We first assessed the impact of cardiac surgery and cardiopulmonary bypass (CPB) on atrial nitroso-redox balance by measuring NO synthase (NOS) and GTP cyclohydrolase-1 (GCH-1) activity, biopterin content, and superoxide production in paired samples of the right atrial appendage obtained before (PRE) and after CPB and reperfusion (POST) in 116 patients. The effect of perioperative treatment with atorvastatin (80 mg once daily) on these parameters, blood biomarkers, and the post-operative atrial effective refractory period (AERP) was then evaluated in a randomized, double-blind, placebo-controlled study in 80 patients undergoing cardiac surgery on CPB. CPB and reperfusion led to a significant increase in atrial superoxide production (74% CI 71-76%, n = 46 paired samples, P 

Daria M. Svistunova, Jillian N. Simon, Elzbieta Rembeza, Mark Crabtree, Wyatt W. Yue, Peter L. Oliver, Mattéa J. Finelli (2019)Oxidation resistance 1 regulates post-translational modifications of peroxiredoxin 2 in the cerebellum, In: Free radical biology & medicine130pp. 151-162 Elsevier Inc

Protein aggregation, oxidative and nitrosative stress are etiological factors common to all major neurodegenerative disorders. Therefore, identifying proteins that function at the crossroads of these essential pathways may provide novel targets for therapy. Oxidation resistance 1 (Oxr1) is a protein proven to be neuroprotective against oxidative stress, although the molecular mechanisms involved remain unclear. Here, we demonstrate that Oxr1 interacts with the multifunctional protein, peroxiredoxin 2 (Prdx2), a potent antioxidant enzyme highly expressed in the brain that can also act as a molecular chaperone. Using a combination of in vitro assays and two animal models, we discovered that expression levels of Oxr1 regulate the degree of oligomerization of Prdx2 and also its post-translational modifications (PTMs), specifically suggesting that Oxr1 acts as a functional switch between the antioxidant and chaperone functions of Prdx2. Furthermore, we showed in the Oxr1 knockout mouse that Prdx2 is aberrantly modified by overoxidation and S-nitrosylation in the cerebellum at the presymptomatic stage; this in-turn affected the oligomerization of Prdx2, potentially impeding its normal functions and contributing to the specific cerebellar neurodegeneration in this mouse model. [Display omitted] •Oxr1 interacts with the multifunctional antioxidant enzyme, Prdx2.•Oxr1 modulates the oligomerization and post-translational modifications of Prdx2.•Oxr1 acts as a functional switch between Prdx2 antioxidant and chaperone functions.•Loss of Oxr1 leads to aberrantly modified Prdx2 that contributes to neurodegeneration.

Brent J Ryan, Lara L Lourenço-Venda, Mark J Crabtree, Ashley B Hale, Keith M Channon, Richard Wade-Martins (2014)α-Synuclein and mitochondrial bioenergetics regulate tetrahydrobiopterin levels in a human dopaminergic model of Parkinson disease, In: Free radical biology & medicine67pp. 58-68

Parkinson disease (PD) is a multifactorial disease resulting in preferential death of the dopaminergic neurons in the substantia nigra. Studies of PD-linked genes and toxin-induced models of PD have implicated mitochondrial dysfunction, oxidative stress, and the misfolding and aggregation of α-synuclein (α-syn) as key factors in disease initiation and progression. Many of these features of PD may be modeled in cells or animal models using the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)). Reducing oxidative stress and nitric oxide synthase (NOS) activity has been shown to be protective in cell or animal models of MPP(+) toxicity. We have previously demonstrated that siRNA-mediated knockdown of α-syn lowers the activity of both dopamine transporter and NOS activity and protects dopaminergic neuron-like cells from MPP(+) toxicity. Here, we demonstrate that α-syn knockdown and modulators of oxidative stress/NOS activation protect cells from MPP(+)-induced toxicity via postmitochondrial mechanisms rather than by a rescue of the decrease in mitochondrial oxidative phosphorylation caused by MPP(+) exposure. We demonstrate that MPP(+) significantly decreases the synthesis of the antioxidant and obligate cofactor of NOS and TH tetrahydrobiopterin (BH4) through decreased cellular GTP/ATP levels. Furthermore, we demonstrate that RNAi knockdown of α-syn results in a nearly twofold increase in GTP cyclohydrolase I activity and a concomitant increase in basal BH4 levels. Together, these results demonstrate that both mitochondrial activity and α-syn play roles in modulating cellular BH4 levels.

Gillian Douglas, Erik Van Kampen, Ashley B. Hale, Eileen McNeill, Jyoti Patel, Mark J. Crabtree, Ziad Ali, Robert A. Hoerr, Nicholas J. Alp, Keith M. Channon (2013)Endothelial cell repopulation after stenting determines in-stent neointima formation: effects of bare-metal vs. drug-eluting stents and genetic endothelial cell modification, In: European heart journal34(43)pp. 3378-3388 Oxford Univ Press

Understanding endothelial cell repopulation post-stenting and how this modulates in-stent restenosis is critical to improving arterial healing post-stenting. We used a novel murine stent model to investigate endothelial cell repopulation post-stenting, comparing the response of drug-eluting stents with a primary genetic modification to improve endothelial cell function. Endothelial cell repopulation was assessed en face in stented arteries in ApoE(/) mice with endothelial-specific LacZ expression. Stent deployment resulted in near-complete denudation of endothelium, but was followed by endothelial cell repopulation, by cells originating from both bone marrow-derived endothelial progenitor cells and from the adjacent vasculature. Paclitaxel-eluting stents reduced neointima formation (0.423 0.065 vs. 0.240 0.040 mm(2), P 0.038), but decreased endothelial cell repopulation (238 17 vs. 154 22 nuclei/mm(2), P 0.018), despite complete strut coverage. To test the effects of selectively improving endothelial cell function, we used transgenic mice with endothelial-specific overexpression of GTP-cyclohydrolase 1 (GCH-Tg) as a model of enhanced endothelial cell function and increased NO production. GCH-Tg ApoE(/) mice had less neointima formation compared with ApoE(/) littermates (0.52 0.08 vs. 0.26 0.09 mm(2), P 0.039). In contrast to paclitaxel-eluting stents, reduced neointima formation in GCH-Tg mice was accompanied by increased endothelial cell coverage (156 17 vs. 209 23 nuclei/mm(2), P 0.043). Drug-eluting stents reduce not only neointima formation but also endothelial cell repopulation, independent of strut coverage. In contrast, selective targeting of endothelial cell function is sufficient to improve endothelial cell repopulation and reduce neointima formation. Targeting endothelial cell function is a rational therapeutic strategy to improve vascular healing and decrease neointima formation after stenting.

Colin Cunnington, Tim Van Assche, Cheerag Shirodaria, Ilias Kylintireas, Alistair C. Lindsay, Justin M. Lee, Charalambos Antoniades, Marios Margaritis, Regent Lee, Ruha Cerrato, Mark J. Crabtree, Jane M. Francis, Rana Sayeed, Chandi Ratnatunga, Ravi Pillai, Robin P. Choudhury, Stefan Neubauer, Keith M. Channon (2012)Systemic and Vascular Oxidation Limits the Efficacy of Oral Tetrahydrobiopterin Treatment in Patients With Coronary Artery Disease, In: Circulation (New York, N.Y.)125(11)pp. 1356-1366 Lippincott Williams & Wilkins

Background-The endothelial nitric oxide synthase cofactor tetrahydrobiopterin (BH4) plays a pivotal role in maintaining endothelial function in experimental vascular disease models and in humans. Augmentation of endogenous BH4 levels by oral BH4 treatment has been proposed as a potential therapeutic strategy in vascular disease states. We sought to determine the mechanisms relating exogenous BH4 to human vascular function and to determine oral BH4 pharmacokinetics in both plasma and vascular tissue in patients with coronary artery disease. Methods and Results-Forty-nine patients with coronary artery disease were randomized to receive low-dose (400 mg/d) or high-dose (700 mg/d) BH4 or placebo for 2 to 6 weeks before coronary artery bypass surgery. Vascular function was quantified by magnetic resonance imaging before and after treatment, along with plasma BH4 levels. Vascular superoxide, endothelial function, and BH4 levels were determined in segments of saphenous vein and internal mammary artery. Oral BH4 treatment significantly augmented BH4 levels in plasma and in saphenous vein (but not internal mammary artery) but also increased levels of the oxidation product dihydrobiopterin (BH2), which lacks endothelial nitric oxide synthase cofactor activity. There was no effect of BH4 treatment on vascular function or superoxide production. Supplementation of human vessels and blood with BH4 ex vivo revealed rapid oxidation of BH4 to BH2 with predominant BH2 uptake by vascular tissue. Conclusions-Oral BH4 treatment augments total biopterin levels in patients with established coronary artery disease but has no net effect on vascular redox state or endothelial function owing to systemic and vascular oxidation of BH4. Alternative strategies are required to target BH4-dependent endothelial function in established vascular disease states.