Sarah Trinder

Dr Sarah L Trinder


Teaching Fellow in Pharmacology
PhD, MPharmacol (Hons), G.Cert L&T, FHEA

Academic and research departments

School of Biosciences and Medicine.

Biography

University roles and responsibilities

  • Programme Director BSc Biochemistry
  • Programme Director MSc Pharmaceutical Medicine/Clinical Pharmacology

Affiliations and memberships

Fellow of the Higher Education Academy

Courses I teach on

Undergraduate

Postgraduate taught

My publications

Publications

Trinder S, Tarriela M, Gilbane A, Good A, Shi-Wen X, Abraham D, Holmes A (2014) Bromodomain Inhibitor JQ1 Modulates Collagen Processing and Ameliorates Bleomycin Induced Dermal Fibrosis in Mice, ARTHRITIS & RHEUMATOLOGY 66 pp. S760-S760 Wiley-Blackwell
Background/Purpose: Systemic sclerosis (SSc) is a complex pro-inflammatory scarring disease, characterised by elevated deposition of extracellular matrix (ECM) proteins, in particular collagen type I. The disease is heterogeneous affecting both the skin and visceral organs including kidney, lung and heart. The SSc fibroblast is a key cell which promotes a pro-inflammatory and fibrotic microenvironment that can lead to the loss of normal tissue architecture and organ function. The mechanisms that contribute to the formation and persistence of the SSc dermal fibroblast remain unclear. We have previously shown the epigenetic bromodomain and extra-terminal domain-containing proteins (Brd) which bind to acetylated histone residues, play a significant role in pulmonary fibrosis. Here we seek to explore the contribution of Brd proteins in the development of dermal fibrosis using a specific inhibitor of Brd proteins (Brd 2, 3, 4 and T), JQ1.

Methods: We investigated the dose-response of JQ1 on SSc and healthy control (HC) donor (ne3) dermal fibroblasts. We assessed the effects on collagen deposition and processing using the Scar-in-a-Jar in vitrofibrosis assay, by western blot and immuno-florescence microscopy for collagen type I (n=4). To determine the effect of JQ1 in a pre-clinical model of skin fibrosis, female C57BL/6 mice were given three weekly subcutaneous injections of 100µl sterile saline (ne6) or 0.1U/ml bleomycin (ne6) for 14 days and treated with 12mg/kg/day JQ1 (ne6) or vehicle (ne6). After 14 days histological analysis for fibrogenic proteins and ECM was performed on skin, and pro-inflammatory chemokines in sera assessed by ELISA.

Results: IL-6 and MCP-1 secretion by SSc and HC donor fibroblasts was significantly (P

Conclusion: We have assessed the functional effects of the Brd inhibitor, JQ1, on SSc dermal fibroblasts and the development of dermal fibrosis in a pre-clinical model of dermal fibrosis. We demonstrate that JQ1 markedly attenuated

Good R, Trinder S, Abdi S, Yu R, Denton C, Abraham D, Holmes A (2014) Characterisation of late-outgrowth endothelial progenitor cells from systemic sclerosis patients, Clinical and Experimental Rheumatology 32 (2) pp. S32-S33 CLINICAL & EXPER RHEUMATOLOGY, VIA SANTA MARIA 31, 56126 PISA, ITALY
Introduction. Vascular complications associated with systemic sclerosis (SSc)
including pulmonary arterial hypertension (PAH-SSc), result from endothelial
damage and loss of barrier function. The causes of endothelial dysfunction are
unclear, but the integrity of the endothelium is likely to be significantly diminished
in SSc. Endothelial progenitor cells (EPCs) derived from peripheral blood
mononuclear cells (PBMCs) express endothelial and haematopoietic markers. It
is thought they home to sites of vascular injury and differentiate into endothelial
cells and restore the barrier. In SSc patients circulating levels of EPCs are
reduced. This study aimed to: (i) develop a robust method to isolate and grow
healthy control (HC) and SSc EPCs from PBMCs. (ii) Compare the cellular functions
of EPCs to mature endothelial cells.
Methods. Peripheral blood was taken from HC (n=10) and SSc donors (n=10).
EPCs were cultured from PBMCs, and EPC colonies grown to passage 4. EPCs
and human pulmonary artery endothelial cells (hPAECs) were seeded into transwell
inserts and grown to confluence. Cells were incubated with TNF-± (10ng/
S-33
3rd Systemic Sclerosis World Congress Poster Tours - Clinical
ml), and their capacity to form biological barriers and support immune cell influx
was assessed using FITC-albumin (0.5mg/ml) and neutrophil transmigration.
We further assessed the responses of EPCs to TNF-± stimulation by ELISA to
quantify pro-inflammatory cytokine release.
Results. We demonstrate that EPCs form biological barriers with similar capabilities
as mature hPAECs in vitro. TNF-± significantly enhanced permeability
of EPCs (p similar cellular activities as mature endothelial cells, TNFa stimulated
neutrophil transmigration in monolayers of EPCs (p and enhanced the secretion of IL-8 in both EPCs (p We sought to determine the frequency of EPC colony formation from PBMCs
and found no significant difference in the capacity to form EPC colonies in HC
and SSc patient PBMCs.
Discussion. We have developed a robust method for isolating EPCs from PBMCs.
We have demonstrated that endothelial progenitors can maintain an endothelial
barrier consistent with that observed by mature hPAECs in vitro. We
have established that EPCs respond to TNF-± in a similar manner to mature
PAECs, secreting pro-inflammatory cytokines such as IL-8 and supporting neutrophil
transmigr
Trinder S, Shi-wen X, Ahmed-Abdi B, Good R, Gilbane A, Denton C, Budd D, Abraham D, Holmes A (2014) The role of MCSF and endothelin 1 in fibrocyte differentiation, Clinical and Experimental Rheumatology 32 (2) pp. S59-S60 CLINICAL & EXPER RHEUMATOLOGY, VIA SANTA MARIA 31, 56126 PISA, ITALY
Good RB, Gilbane AJ, Trinder SL, Denton CP, Coghlan G, Abraham DJ, Holmes AM (2015) Endothelial to Mesenchymal Transition Contributes to Endothelial Dysfunction in Pulmonary Arterial Hypertension, American Journal of Pathology 185 (7) pp. 1850-1858
© 2015 American Society for Investigative Pathology.Pulmonary arterial hypertension (PAH) is a progressive disease characterized by lung endothelial cell dysfunction and vascular remodeling. Normally, the endothelium forms an integral cellular barrier to regulate vascular homeostasis. During embryogenesis endothelial cells exhibit substantial plasticity that contribute to cardiac development by undergoing endothelial-to-mesenchymal transition (EndoMT). We determined the presence of EndoMT in the pulmonary vasculature in vivo and the functional effects on pulmonary artery endothelial cells (PAECs) undergoing EndoMT in vitro. Histologic assessment of patients with systemic sclerosis-associated PAH and the hypoxia/SU5416 mouse model identified the presence von Willebrand factor/±-smooth muscle actin-positive endothelial cells in up to 5% of pulmonary vessels. Induced EndoMT in PAECs by inflammatory cytokines IL-1², tumor necrosis factor ±, and transforming growth factor ² led to actin cytoskeleton reorganization and the development of a mesenchymal morphology. Induced EndoMT cells exhibited up-regulation of mesenchymal markers, including collagen type I and ±-smooth muscle actin, and a reduction in endothelial cell and junctional proteins, including von Willebrand factor, CD31, occludin, and vascular endothelial-cadherin. Induced EndoMT monolayers failed to form viable biological barriers and induced enhanced leak in co-culture with PAECs. Induced EndoMT cells secreted significantly elevated proinflammatory cytokines, including IL-6, IL-8, and tumor necrosis factor ±, and supported higher immune transendothelial migration compared with PAECs. These findings suggest that EndoMT may contribute to the development of PAH.
Hobbs A, Bubb K, Trinder S, Baliga R, MacAllister R (2014) Phosphodiesterase 2 is a novel therapeutic target in pulmonary hypertension, Basic & Clinical Pharmacology & Toxicology 115 (40-41) Wiley-Blackwell
Trinder S, Shi-wen X, Ahned-Abdi B, Denton C, Budd D, Abraham D, Holmes A (2014) Modulating the fibrotic effects of fibrocytes from scleroderma patients, Rheumatology 53 pp. 173-173 Oxford University Press
Background: SSc is a complex autoimmune fibrotic disease,
characterized by elevated deposition of extracellular matrix (ECM)
proteins, in particular collagen type I. The disease is heterogeneous;
organs commonly affected by fibrosis are the skin, kidney, lung and
heart. Vascular complications include pulmonary arterial hypertension
(PAH), which occurs in 12?40% of patients. CD14þmonocytes are afunctionally heterogeneous cell type. They have been noted to
differentiate into a number of cell phenotypes including macrophages
and collagen-producing fibrocytes. In culture fibrocytes adopt a
spindle shape, co-express haematopoietic CD45RO and 25F9, along
with mesenchymal markers including aSMA and collagen type I.
Fibrocytes amplify the inflammatory/immune response through distinct
mechanisms, including antigen presentation, cytokine and chemokine
secretion, and the production of MMPs. We and others have shown
fibrocyte differentiation is enhanced by fibrogenic cytokines including
PDGF. Here we seek to understand the mechanism by which SSc
fibrocytes influence the local microenvironment of the tissue.
Methods: CD14þ peripheral blood mononuclear cells (PBMCs) were
isolated from SSc patient and healthy control blood. PBMCs were
cultured in the presence of macrophage colony stimulating factor
(MCSF; n > 10) and/or endothelin-1 (ET-1; n > 10); after 14 days of
culture number of fibrocytes was assessed. The effect of pharmacological
inhibitors including ETRA and ETRB antagonism on fibrocyte
differentiation (n ¼ 6 SSc and control) was investigated. Secreted
factors in culture media from SSc and control fibrocytes were
assessed by ELISA (n ¼ 6), and the effects of conditioned media
explored in 3D-collagen gel.
Results: Both MCSF and ET-1 significantly induced fibrocyte
differentiation, in combination differentiation was significantly augmented
(P more readily differentiated from CD14þ PBMCs than healthy control
donors in response to MCSF (P MCSF with ET-1 in combination (P BQ123 and BQ788 (respectively), and Bosentan (a dual ETR
antagonist) inhibited MCSF induced fibrocyte differentiation in a
concentration dependant manner. Furthermore SSc fibrocytes
secreted significantly more connective tissue growth factor (CTGF)
than control fibrocytes (P fibrocytes acting in a paracrine manner, conditioned me
Derrett-Smith E, Dooley A, Trinder S, Holmes A, Abraham D, Denton C (2014) Pulmonary endothelial injury in the context of perturbed transforming growth factor beta signalling as a unique model of pulmonary hypertension in scleroderma, Lancet 383 (1) pp. S11-S11 Elsevier
Background

The development of pulmonary arterial hypertension in scleroderma remains an important contributor to mortality in this condition, despite substantial improvements in outcomes due to modern therapeutic strategies. No animal models of scleroderma develop this important complication. We describe the constitutive vascular phenotype of a mouse model of scleroderma and show that pulmonary endothelial injury replicates the pathological changes of pulmonary arterial hypertension seen in human disease.

Methods

The T²RII”k-fib mouse strain expresses a kinase-deficient type II transforming growth factor ² (TGF²) receptor driven by a fibroblast-specific promoter leading to ligand-dependent upregulation of TGF² signalling; this mouse strain replicates key fibrotic features of scleroderma. We did structural, biochemical, and functional assessments of pulmonary and systemic vessels, including in-vivo haemodynamic studies, before and after vascular endothelial growth factor receptor (VEGFR) inhibition with SU5416, which induced pulmonary endothelial cell apoptosis. These assessments included biochemical analysis of the TGF², endothelin, and VEGF signalling axes in vivo; tissue sections; and explanted pulmonary arterial smooth muscle cells.

Findings

In the T²RII”k-fib mouse strain, a constitutive pulmonary vasculopathy with medial thickening, a perivascular proliferating chronic inflammatory cell infiltrate, and mildly raised pulmonary artery pressures resemble the well-described chronic hypoxia model of pulmonary hypertension. After administration of SU5416, the pulmonary vascular phenotype was more florid, with pulmonary arteriolar luminal obliteration by apoptosis-resistant proliferating endothelial cells; the result was right ventricular hypertrophy confirming haemodynamically significant pulmonary arterial hypertension. Altered TGF², endothelin, and ligand and receptor expression of VEGF were consistent with a scleroderma phenotype.

Interpretation This study replicates key features of scleroderma-associated pulmonary arterial hypertension in a mouse model. Our results suggest that pulmonary endothelial cell injury in a genetically susceptible mouse strain triggers this complication and support functional interplay between TGF², endothelin, and VEGF that provides insight into pathogenesis.

Baliga R, Scotton C, Trinder S, Chambers R, MacAllister R, Hobbs A (2011) Protective Role Of Natriuretic Peptides In Pulmonary Fibrosis: A Novel Therapeutic Target?, American Journal of Respiratory and Critical Care Medicine 183 American Thoracic Society
Trinder S (2013) Altered BMP Signalling In a TGF beta Dependent Murine Model Of Scleroderma May Contribute To Development Of Pulmonary Arterial Hypertension, Arthritis and Rheumatism 65 (SI) Wiley-Blackwell
Baliga RS, Scotton CJ, Trinder SL, Chambers RC, MacAllister RJ, Hobbs AJ (2014) Intrinsic defence capacity and therapeutic potential of natriuretic peptides in pulmonary hypertension associated with lung fibrosis, British Journal of Pharmacology 171 (14) pp. 3463-3475
Background and Purpose Idiopathic pulmonary fibrosis (IPF) is a progressive fibro-proliferative disorder refractory to current therapy commonly complicated by the development of pulmonary hypertension (PH); the associated morbidity and mortality are substantial. Natriuretic peptides possess vasodilator and anti-fibrotic actions, and pharmacological augmentation of their bioactivity ameliorates renal and myocardial fibrosis. Here, we investigated whether natriuretic peptides possess an intrinsic cytoprotective function preventing the development of pulmonary fibrosis and associated PH, and whether therapeutics targeting natriuretic peptide signalling demonstrate efficacy in this life-threatening disorder. Experimental Approach Pulmonary haemodynamics, right ventricular function and markers of lung fibrosis were determined in wild-type (WT) and natriuretic peptide receptor (NPR)-A knockout (KO) mice exposed to bleomycin (1 mg·kg-1). Human myofibroblast differentiation was studied in vitro. Key Results Exacerbated cardiac, vascular and fibrotic pathology was observed in NPR-A KO animals, compared with WT mice, exposed to bleomycin. Treatment with a drug combination that raised circulating natriuretic peptide levels (ecadotril) and potentiated natriuretic peptide-dependent signalling (sildenafil) reduced indices of disease progression, whether administered prophylactically or to animals with established lung disease. This positive pharmacodynamic effect was diminished in NPR-A KO mice. Atrial natriuretic peptide and sildenafil synergistically reduced TGF²-induced human myofibroblast differentiation, a key driver of remodelling in IPF patients. Conclusions and Implications These data highlight an endogenous host-defence capacity of natriuretic peptides in lung fibrosis and PH. A combination of ecadotril and sildenafil reversed the pulmonary haemodynamic aberrations and remodelling that characterize the disease, advocating therapeutic manipulation of natriuretic peptide bioactivity in patients with IPF. © 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.
Baliga RS, Milsom AB, Ghosh SM, Trinder SL, MacAllister RJ, Ahluwalia A, Hobbs AJ (2012) Dietary nitrate ameliorates pulmonary hypertension: Cytoprotective role for endothelial nitric oxide synthase and xanthine oxidoreductase, Circulation 125 (23) pp. 2922-2932
Background-Pulmonary hypertension (PH) is a multifactorial disease characterized by increased pulmonary vascular resistance and right ventricular failure; morbidity and mortality remain unacceptably high. Loss of nitric oxide (NO) bioactivity is thought to contribute to the pathogenesis of PH, and agents that augment pulmonary NO signaling are clinically effective in the disease. Inorganic nitrate (NO 3-) and nitrite (NO 2-) elicit a reduction in systemic blood pressure in healthy individuals; this effect is underpinned by endogenous and sequential reduction to NO. Herein, we determined whether dietary nitrate and nitrite might be preferentially reduced to NO by the hypoxia associated with PH, and thereby offer a convenient, inexpensive method of supplementing NO functionality to reduce disease severity. Methods and Results-Dietary nitrate reduced the right ventricular pressure and hypertrophy, and pulmonary vascular remodeling in wild-type mice exposed to 3 weeks of hypoxia; this beneficial activity was mirrored largely by dietary nitrite. The cytoprotective effects of dietary nitrate were associated with increased plasma and lung concentrations of nitrite and cGMP. The beneficial effects of dietary nitrate and nitrite were reduced in mice lacking endothelial NO synthase or treated with the xanthine oxidoreductase inhibitor allopurinol. Conclusions-These data demonstrate that dietary nitrate, and to a lesser extent dietary nitrite, elicit pulmonary dilatation, prevent pulmonary vascular remodeling, and reduce the right ventricular hypertrophy characteristic of PH. This favorable pharmacodynamic profile depends on endothelial NO synthase and xanthine oxidoreductase-catalyzed reduction of nitrite to NO. Exploitation of this mechanism (ie, dietary nitrate/nitrite supplementation) represents a viable, orally active therapy for PH. © 2012 American Heart Association, Inc.
Good R, Trinder S, Abdi B, Yu R, Denton C, Abraham D, Holmes A (2013) Endothelial progenitor cells form biological exclusion barriers similar to that of mature endothelial cells- a therapeutic potential in systemic sclerosis?, Thorax 68 pp. A147-A147 BMJ Publishing group
Abstract
Abstract Vascular complications associated with systemic sclerosis (SSc) including pulmonary arterial hypertension (PAH-SSc), result from endothelial damage and loss of barrier function. Endothelial progenitor cells (EPCs) express endothelial (VEGFR2+, CD31+ ) and haematopoietic (CD133+ ) markers. They home to sites of vascular injury and differentiate into endothelial cells restoring the endothelium. In SSc patients circulating levels of EPCs are reduced. This study aimed to develop a robust method to grow EPCs from peripheral blood mononuclear cells (PBMCs) and to compare cellular functions to mature endothelial cells.

Methods EPCs and human pulmonary artery endothelial cells (hPAECs) were seeded into transwell inserts and grown to confluence. Cells were incubated with TNFa (50ng/ml), and their capacity to form biological barriers assessed using FITC-albumin (5mg/ml). FITC-albumin ?leak? was quantified by fluorescent absorbance over time. We further assessed the responses of EPCs to TNFa stimulation by ELISA to quantify pro-inflammatory cytokine release.

Results EPCs form a biological exclusion barrier with similar capabilities as mature hPAECs. TNFa significantly enhanced the permeability of EPCs (P

Discussion We developed a robust method for isolating EPCs from PBMCs. We have demonstrated that EPCs can maintain an endothelial barrier consistent with that observed by mature hPAECs in vitro. We have established that EPCs respond to TNFa in a similar manner to mature PAECs. We have shown no significant difference in the capacity of PBMCs from SSc patients to form EPC colonies compared to HCs.

Derrett-Smith E, Sobanski V, Trinder S, Gilbane A, Iglarz M, Abraham D, Holmes A, Denston C (2015) Endothelin Receptor Blockade Prevents Development of Pulmonary Hypertension in a Mouse Model of Scleroderma, Rheumatology 2015 Abstracts 54 (Suppl 1) pp. i159-i160 Oxford University Press
Background: Pulmonary arterial hypertension (PAH) is an important
complication of SSc that occurs in around 10% of cases. We have
previously shown that a TGFb dependent transgenic mouse strain
(TbRIIk-fib) is susceptible to organ based pathology relevant to SSc
including development of pulmonary hypertension (PH) after pharmacological pulmonary endothelial injury by SU5416, a VEGF receptor inhibitor. In this study, we have prevented the development of PH in this mouse strain using macitentan, a novel dual ETA/B receptor
antagonist recently licensed to treat PAH in CTD based upon a
significant effect on morbidity and mortality in PAH.
Methods: SU5416, a VEGF receptor inhibitor, was administered to all TbRIIk-fib transgenic (TG) mice and wild-type (WT) littermate
controls to induce endothelial injury with subsequent endothelial
proliferation and PH in transgenic mice only. Mice were treated with
either 50 mg/kg macitentan daily by oral gavage or vehicle alone (n
¼ 8 for each group) either before SU5416 injection or on day 8 following
injection. The development of PH in each group was assessed by
histology and immunohistochemistry of vessel architecture, in vivo
haemodynamic studies and RV mass index measurements.
Biochemical responses to TGFb, endothelin and VEGF stimulation
before and after macitentan were examined in cultured T
bRII k-fib lung fibroblasts.
Results: Compared with WT littermates, after SU5416, all TG mice
developed a prominent perivascular chronic inflammatory infiltrate and
smooth muscle layer hypertrophy, as previously described. RV mass
index was elevated in TG animals receiving vehicle compared with
other groups (TG vehicle 0.29 0.007, TG macitentan 0.24
0.007, P 28.8 mmHg 0.72, TG macitentan 22.0 1.62, P any significant change in systemic arterial blood pressure in any
group. Treatment with macitentan after day 8 was sufficient to
normalize haemodynamic consequences of SU5416 administration.
There was obliterative pulmonary arteriolar occlusion in 21% of
vessels in TG mice treated with vehicle. In contrast, no vessels in
WT mice or TG mice treated with macitentan developed this
histological change. Explanted TG lung fibroblasts showed an increase
in proliferation and migration with upregulation of VEGF and TGFb
signalling and downregulation of endothelin receptor A compared with
WT littermates
Gilbane AJ, Derrett-Smith E, Trinder SL, Good RB, Pearce A, Denton CP, Holmes AM (2015) Impaired bone morphogenetic protein receptor II signaling in a transforming growth factor-²-dependent mouse model of pulmonary hypertension and in systemic sclerosis, American Journal of Respiratory and Critical Care Medicine 191 (6) pp. 665-677
Copyright © 2015 by the American Thoracic Society.Rationale: Up to 10% of patients with systemic sclerosis (SSc) develop pulmonary arterial hypertension (PAH). This risk persists throughout the disease and is time dependent, suggesting that SSc is a susceptibility factor. Outcome for SSc-PAH is poor compared with heritable or idiopathic forms, despite clinical and pathological similarities. Although susceptibility in heritable PAH and idiopathic PAH is strongly associated with gene mutations leading to reduced expression of bone morphogenetic protein receptor (BMPR) II, these mutations have not been observed in SSc-PAH. Objectives: To explore BMPRII expression and function in a mouse model of SSc (T²RII”k-fib) that is susceptible to developing pulmonary hypertension and in SSc lung. Methods: BMPRII and downstream signaling pathways were profiled in lung tissue and fibroblasts from the T²RII”k-fib model, which develops pulmonary vasculopathy with pulmonary hypertension that is exacerbated by SU5416. Complementary studies examined SSc or control lung tissue and fibroblasts. Measurements and Main Results: Our study shows reduced BMPRII, impaired signaling, and altered receptor turnover activity in a transforming growth factor (TGF)-²-dependent mouse model of SSc-PAH. Similarly, a significant reduction in BMPRII expression is observed in SSc lung tissue and fibroblasts. Increased proteasomal degradation of BMPRII appears to underlie this and may result from heightened TGF-² activity. Conclusions: We found reduced BMPRII protein in patients with SSc-PAH and a relevant mouse model associated with increased proteasomal degradation of BMPRII. Collectively, these results suggest that impaired BMP signaling, resulting from TGF-²-dependent increased receptor degradation, may promote PAH susceptibility in SSc and provide a unifying mechanism across different forms of PAH.
Derrett-Smith E, Sobanski V, Gilbane A, Trinder S, Bauer Y, Renault B, Iglarz M, Abraham D, Holmes A, Denton C (2015) Macitentan Responsiveness Supports the Validity of a Murine Model of Pulmonary Hypertension in Scleroderma Associated with Altered Tgfbeta/BMPR2 Signalling, ARTHRITIS & RHEUMATOLOGY 67 (10) Wiley Blackwell
Background/Purpose: Pulmonary arterial hypertension (PAH) is an important complication
of systemic sclerosis (SSc) that occurs in around 10% of cases. We have previously shown that an imbalance
between TGFbeta and BMPRII signalling
in the transgenic mouse strain T²RII”k-fib
contributes to the development of PH following pulmonary endothelial injury. In
this study, we have both prevented and treated PH in this mouse strain using macitentan,
an endothelin receptor antagonist licensed to treat PAH in connective tissue
disease.

Methods: SU5416 was administered to all T²RII”k-fib transgenic (TG) mice and littermate wildtype (WT) animals to induce endothelial injury with
subsequent endoluminal proliferation and PH in
transgenic mice only. Mice were treated daily with either macitentan or vehicle
alone (n=8 each group) from either 2 days before or 8 days following SU5416
administration to assess prevention or treatment respectively. The development
of PH in each group was assessed by histology and immunohistochemistry of vessel
architecture, in vivo haemodynamic studies and RV mass index measurements
following 3 weeks of treatment. Microarray analysis of right ventricular tissue
was performed.

Results:
: All TG mice developed a perivascular inflammatory
infiltrate and smooth muscle layer hypertrophy after SU5416 administration. RV mass index was elevated in TG
animals receiving vehicle compared to other groups. In particular
co-administration of macitentan to TG animals treated with SU5416 resulted in
normal RV mass (TG vehicle 0.29±0.007, TG macitentan at day -2 0.24±0.007,
p SU5416 was abrogated by macitentan (TG vehicle 28.8±3.2, TG+macitentan
at day -2 22.0±2.9, TG+macitentan at day +8, 24.4±1.8,
p Explanted TG lung fibroblasts showed an increase in TGFbeta signalling and downregulation of BMPRII compared with
WT littermates following macitentan treatment. Pulmonary
arteriolar occlusion occurred in 21% of vessels in TG mice treated with vehicle
with no occlusion in any other vessels. Gene expression analysis of whole right
ventricle showed alterations in key genes known to be associated with cardiac
muscle remodelling and failure. Figure 1 shows the
cluster analysis of TG mice treated with SU5416 compared with those also
treated with macitentan.

Conclusion:
Macitentan prevents and treats the development of
his

Feeney M, Syed F, Khan K, Shiwen X, Sully K, Trinder S, Wilson P, Abraham D, Holmes A, Denton C (2015) Oncostatin M As a Potential Molecular Target in Systemic Sclerosis, ARTHRITIS & RHEUMATOLOGY 67 (10) Wiley-Blackwell
Background/Purpose:
Oncostatin M (OSM) is a pleiotropic member of the gp130/ IL-6 cytokine family,
produced by a variety of immune cells, including macrophages, neutrophils and
activated T cells. OSM signals through two receptors; gp130/ LIF low affinity
receptor (LIFR) and gp130/ OSM high affinity receptor (OSMR); activating
JAK/STAT, ERK1/2 and p38 MAPK pathways. The rationale for the role of OSM in
Systemic Sclerosis (SSc) lies in that key disease components, i.e.
inflammation, vascular dysregulation and fibrosis, complement the biological
activities of the target.

Methods: Serum OSM levels
were measured by custom OSM ELISA in 63 diffuse cutaneous (Dc) SSc and 18
age-matched healthy donors (HD). Transcriptomics data derived from published
DcSSc skin biopsies were mined for OSM and known OSM-regulated genes. The presence of OSM, OSMR, and phospho(p)STAT3 in skin
biopsies from HD (n=12), involved limited cutaneous (Lc)SSc (n=12), and
involved and uninvolved DcSSc (n=13) patients was assessed by
immunohistochemistry (IHC). Primary dermal fibroblasts isolated from HD or
DcSSc skin were incubated with OSM at 2, 20 or 200ng/ml and cell associated
collagen type I (Col-1), and connective tissue growth factor (CTGF) assessed by
Western blot analysis at 24hrs and 72hrs.

Results: Serum OSM levels
were significantly (P=0.004) increased in DcSSc patients compared with HDs (Table
1). OSM and OSM-regulated genes, including S100A9 and VCAM-1, were upregulated
in the DcSSc skin. IHC analysis confirmed OSM, OSMR and pSTAT3 expression in
skin biopsies from SSc patients and HDs, with a similar expression pattern in
both. SSc skin biopsies exhibited significantly higher inflammatory cell
infiltrates compared to HDs, and were strongly positive for OSM, OSMR and
pSTAT3. Uninvolved DcSSc skin had significantly more OSM positive immune cell
infiltrates and increased pSTAT3 expression than HDs, but not compared to
involved DcSSc skin. Involved DcSSc skin exhibited significantly more pSTAT3
positive fibroblasts compared to HDs (p positive immune cell infiltrates was further significantly enhanced in involved
compared to uninvolved tissue. Increased numbers of positive OSM and pSTAT3
fibroblasts were present in involved versus uninvolved skin. OSM induced CTGF
after 24hrs in 4/5 DcSSc dermal fibroblast cell lines tested. No induction
above baseline was observed in the two HD dermal fibroblast cell lines.
Following 72hrs stimulation,

Trinder S, Gilbane A, Pontikos M, Donovan J, Denton C, Abraham D, Holmes A (2012) A Putative Role for the TGF beta Accessory Receptors Betaglycan and Endoglin in pulmonary Complications of Scleroderma., Arthritis and Rheumatism 64 (10) pp. S974-S974 Wiley-Blackwell
Trinder S, Shi-Wen X, Abdi B, Denton C, Budd D, Abraham D, Holmes A (2013) The role of endothelin receptors (etra/b) in fibrocyte differentiation, Thorax 68 pp. A72-A72 BMJ Publishing Group
Introduction Scleroderma (SSc) is an autoimmune connective tissue disease of unknown aetiology. Pulmonary involvement including the development of pulmonary arterial hypertension (PAH) is characterised by vascular remodelling, collagen deposition and expression of connective tissue growth factor (CTGF). CD14+ monocytes can differentiate into spindle shaped cells termed ?fibrocytes?. Fibrocytes express haematopoietic and mesenchymal markers including collagen, and amplify inflammatory/immune responses via antigen presentation and chemokine secretion. Fibrocyte differentiation is enhanced by fibrogenic cytokines including PDGF. The role fibrocytes play in promoting PAH in SSc is unknown.

Methods CD14+ PBMCs were isolated from SSc and healthy donor blood. Fibrocyte differentiation in the presence of MCSF and/or ET-1 was assessed after 14 days. The effect of endothelin receptor (ETR) antagonists (selective/dual) on fibrocyte differentiation (n = 6) was investigated. SSc and control fibrocyte secretomes were assessed by ELISA (n = 6), and the effects on fibroblast-mediated gel contraction determined.

Results MCSF and ET-1 alone and in combination induced fibrocyte differentiation (P

Discussion CD14+ SSc PBMCs readily differentiate into fibrocytes in response to ET-1 and MCSF via ETRA and ETRB. Our data suggests fibrocytes contribute to the development of PAH in SSc via a paracrine mechanism modulating the functional activities of resident tissue fibroblasts.

Good R, Gilbane A, Trinder S, Denton C, Coghlan G, Abraham D, Holmes A (2014) Endothelial to Mesenchymal Transition (EndoMT) leads to a loss of normal endothelial cell function and may contribute to the development of pulmonary arterial hypertension, Molecular Biology of the Cell 25 American Society of Cell Biology
Background/Purpose

Vascular complications in Scleroderma (SSc) patients are associated with high mortality, particularly in patients who develop pulmonary arterial hypertension (SSc-PAH). Vascular complications, thought to arise from initial activation and dysfunction of the endothelium can lead to: elevated vascular leak, inflammation, mesenchymal hypertrophy by activation of resident smooth muscle cells and fibroblasts, and neointima formation. Recent studies suggest that as well as resident mesenchymal cells, endothelial cells can undergo endothelial-mesenchymal transition (EndoMT), and acquire a mesenchymal phenotype which may contribute to the expansion of the mesenchymal cell population. Here we sought to determine the prevalence of EndoMT in SSc-PAH patients and pre-clinical models of PAH, and assess the cellular effects on pulmonary artery endothelial cells (PAECs) functions.

Methods

Using lung tissue from SSc-PAH patients (n=3), healthy control (HC) donors (n=3), and from the hypoxia/SU5416 pre-clinical murine model of PAH (n=5), EndoMT was determined by immunofluorescence based on co-expression of vWF and ±SMA. EndoMT was induced in human PAECs (n=3) in vitro by TNF± [5ng/ml], IL-1² [0.1ng/m;] and TGF² [5ng/ml] in combination. Morphological changes were assessed by light microscopy and phalloidin staining. Western blotting and immunofluorescence was used to quantify: CD31, vWF, occludin, VE-cadherin, ±SMA, calponin and collagen type 1 expression. Conditioned media was collected from PAECs, PAECs following treatment to initiate EndoMT and SSc-PAH and HC fibroblasts; levels of inflammatory secretion was quantified by MSD arrays. The capacity of homogenous EndoMT monolayers (n=6) and mixed cultures of 1:10 EndoMT:PAECs (n=6) cells to form exclusion barriers was assessed using trans-well permeability FITC-albumin assays.

Results

Co-localisation of vWF and ±SMA was observed in d5% of pulmonary arteries from SSc-PAH patients and hypoxia/SU5416 mice. PAECs treated with TNF±, IL-1² and TGF² exhibited significant changes in morphology, loss of endothelial markers and elevated expression of mesenchymal markers by day 6. There was a significant (P0.01) 5-fold increase in permeability compared to PA

Good R, Trinder S, Denton C, Abraham D, Holmes A (2015) Blood Outgrowth Endothelial Cells Isolated from Systemic Sclerosis Patients Exhibit a Pro-Inflammatory Phenotype, ARTHRITIS & RHEUMATOLOGY 67 (10) Wiley/Blackwell
Background/Purpose:

Vascular complications are a key pathological feature of systemic sclerosis (SSc) affecting the microcirculation and arterioles. Under normal circumstances the endothelium acts as a biological barrier supporting controlled permeability, immune surveillance and cellular trafficking. In SSc endothelial damage, contributes to barrier dysfunction and elevated immune cell infiltration and inflammation. The cause(s) of the initial endothelial dysfunction in SSc is unclear. Blood outgrowth endothelial cells (BOECs) are thought to home to sites of vascular injury and differentiate into endothelial cells, aiding the repair and restoration of normal endothelial functions. Circulating levels of BOECs have been shown to be reduced in SSc patients, and recent studies suggest that BOECs may be dysfunctional in vascular diseases. Here we sought to assess SSc-BOECs and their contribution to vascular dysfunction in SSc.

Methods:

BOECs were established from peripheral blood PBMCs from healthy donors (HD) and SSc patients. BOECs from HD (n=5) and SSc patients (n=6) were profiled using Illumina HT12 gene arrays and secreted inflammatory cytokines profiled by meso discovery scale (MSD) arrays. The capacity of SSc-BOECs (n=4) and HD-BOECs (n=4) to: 1) Establish biological barriers alone or in co-culture with mature endothelial cells, was assessed using electric cell-substrate impedance sensing (ECIS); 2) Support PBMC (n=4) trans-endothelial migration, by forming monolayers in 24-well transwell inserts stimulated with or without TNF± (10ng/ml).

Results: Using gene set enrichment analysis, it was determined that SSc-BOECs exhibit a significantly altered gene expression profile including inflammatory chemokines and cytokines. In addition SSc-BOECs exhibited significantly elevated pro-inflammatory cytokine secretion compared to HD-BOECs, including IL-6 (P

Conclusion:

We have demonstrated that BOECs isolated from SSc patients exhibit a significantly altered gene profile compared to that

Bubb KJ, Trinder SL, Baliga RS, Patel J, Clapp LH, MacAllister RJ, Hobbs AJ (2014) Inhibition of phosphodiesterase 2 augments cGMP and cAMP signaling to ameliorate pulmonary hypertension, Circulation 130 (6) pp. 496-507
Background: Pulmonary hypertension (PH) is a life-threatening disorder characterized by increased pulmonary artery pressure, remodeling of the pulmonary vasculature, and right ventricular failure. Loss of endothelium-derived nitric oxide (NO) and prostacyclin contributes to PH pathogenesis, and current therapies are targeted to restore these pathways. Phosphodiesterases (PDEs) are a family of enzymes that break down cGMP and cAMP, which underpin the bioactivity of NO and prostacyclin. PDE5 inhibitors (eg, sildenafil) are licensed for PH, but a role for PDE2 in lung physiology and disease has yet to be established. Herein, we investigated whether PDE2 inhibition modulates pulmonary cyclic nucleotide signaling and ameliorates experimental PH. Methods and Results: The selective PDE2 inhibitor BAY 60-7550 augmented atrial natriuretic peptide- and treprostinilevoked pulmonary vascular relaxation in isolated arteries from chronically hypoxic rats. BAY 60-7550 prevented the onset of both hypoxia- and bleomycin-induced PH and produced a significantly greater reduction in disease severity when given in combination with a neutral endopeptidase inhibitor (enhances endogenous natriuretic peptides), trepostinil, inorganic nitrate (NO donor), or a PDE5 inhibitor. Proliferation of pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension was reduced by BAY 60-7550, an effect further enhanced in the presence of atrial natriuretic peptide, NO, and treprostinil. Conclusions: PDE2 inhibition elicits pulmonary dilation, prevents pulmonary vascular remodeling, and reduces the right ventricular hypertrophy characteristic of PH. This favorable pharmacodynamic profile is dependent on natriuretic peptide bioactivity and is additive with prostacyclin analogues, PDE5 inhibitor, and NO. PDE2 inhibition represents a viable, orally active therapy for PH. © 2014 American Heart Association, Inc.
Derret-Smith E, Sobanski V, Trinder S, Gilbane A, Iglarz M, Abraham D, Holmes A, Denton C (2014) Prevention of SU5416-Induced Pulmonary Hypertension in a TGF beta Dependent Genetic Mouse Model of Scleroderma Using the Endothelin Receptor Antagonist Macitentan., ARTHRITIS & RHEUMATOLOGY 66 (10) pp. S751-S752 Wiley-Blackwell
Background/Purpose: Pulmonary arterial hypertension (PAH) is an important complication of systemic sclerosis (SSc) that occurs in around 10% of cases. We have previously shown that a TGFbeta dependent transgenic mouse strain (T²RII”k-fib) is susceptible to organ based pathology relevant to SSc and that pulmonary endothelial injury is associated with development of PH with perturbed VEGF, BMP and endothelin signalling. In this study, we have prevented the development of PH in this mouse strain using macitentan, a potent endothelin receptor antagonist recently licensed to treat PAH in connective tissue disease based upon a significant effect on morbidity and mortality in PAH.

Methods: SU5416, a VEGF receptor inhibitor, was administered to all T²RII”k-fib transgenic (TG) mice and littermate wildtype (WT) animals to induce endothelial injury with subsequent endoluminal proliferation and PH in transgenic mice only. Mice were treated with either 50mg/kg macitentan daily by oral gavage or vehicle alone (n=8 each group). The development of PH in each group was assessed by histology and immunohistochemistry of vessel architecture, in vivo haemodynamic studies and RV mass index measurements.

Results: Compared with WT littermates, after SU5416, all TG mice developed a prominent perivascular chronic inflammatory infiltrate and smooth muscle layer hypertrophy, as previously described. RV mass index was elevated in TG animals receiving vehicle compared to other groups (TG vehicle 0.29±0.007, TG macitentan 0.24±0.007, p

Conclusion: Macitentan prevents the development of histological and haemodynamic PH in this mouse model of SSc. These findings support a pivotal role for perturbed endothelin activity in a model that is induced by altered TGFbeta signalling and triggered by experimental VEGF inhibition. It underpins the value of this model as a platform for expe

Derrett-Smith EC, Dooley A, Gilbane AJ, Trinder SL, Khan K, Baliga R, Holmes AM, Hobbs AJ, Abraham D, Denton CP (2013) Endothelial injury in a transforming growth factor ²-dependent mouse model of scleroderma induces pulmonary arterial hypertension, Arthritis and Rheumatism 65 (11) pp. 2928-2939
Objective To delineate the constitutive pulmonary vascular phenotype of the T²RII”k-fib mouse model of scleroderma, and to selectively induce pulmonary endothelial cell injury using vascular endothelial growth factor (VEGF) inhibition to develop a model with features characteristic of pulmonary arterial hypertension (PAH). Methods The T²RII”k-fib mouse strain expresses a kinase-deficient transforming growth factor ² (TGF²) receptor type II driven by a fibroblast-specific promoter, leading to ligand-dependent up-regulation of TGF² signaling, and replicates key fibrotic features of scleroderma. Structural, biochemical, and functional assessments of pulmonary vessels, including in vivo hemodynamic studies, were performed before and following VEGF inhibition, which induced pulmonary endothelial cell apoptosis. These assessments included biochemical analysis of the TGF² and VEGF signaling axes in tissue sections and explanted smooth muscle cells. Results In the T²RII”k-fib mouse strain, a constitutive pulmonary vasculopathy with medial thickening, a perivascular proliferating chronic inflammatory cell infiltrate, and mildly elevated pulmonary artery pressure resembled the well-described chronic hypoxia model of pulmonary hypertension. Following administration of SU5416, the pulmonary vascular phenotype was more florid, with pulmonary arteriolar luminal obliteration by apoptosis-resistant proliferating endothelial cells. These changes resulted in right ventricular hypertrophy, confirming hemodynamically significant PAH. Altered expression of TGF² and VEGF ligand and receptor was consistent with a scleroderma phenotype. Conclusion In this study, we replicated key features of systemic sclerosis-related PAH in a mouse model. Our results suggest that pulmonary endothelial cell injury in a genetically susceptible mouse strain triggers this complication and support the underlying role of functional interplay between TGF² and VEGF, which provides insight into the pathogenesis of this disease. Copyright © 2013 by the American College of Rheumatology.