Aberrant TGFβ/BMP signalling in connective tissue disease associated pulmonary hypertension

Up to 10 percent of systemic sclerosis (SSc) patients develop pulmonary arterial hypertension (PAH). This risk persists throughout the disease and is time dependent, suggesting that SSc operates as a susceptibility factor. Outcomes for SSc-PAH patients remain poor compared with heritable (HPAH) or idiopathic (HPAH) forms, despite clinical and pathological similarities. Whereas susceptibility in HPAH and HPAH is strongly associated with gene mutations that lead to reduced expression of functional bone morphogenetic protein type II receptor (BMPRII), these mutations have not been observed in SSc-PAH. My initial aim was to investigate BMPRII expression and downstream signalling pathways in whole lung tissue and explant cultured fibroblasts derived from a murine model of SSc (TβRIIΔk-fib) that is susceptible to developing PAH Complementary studies examined SSc or control lung tissue and fibroblasts. My results suggest reduced BMPRII levels, impaired signalling and altered receptor turnover could be due to increased TGFβ activity in a 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 TGFβ activity. Proteasomal inhibition restored BMPRII expression and cellular responses. Collectively suggesting that impaired TGFβ/BMP signalling leading to increased receptor degradation, may promote PAH susceptibility in SSc and provide a unifying mechanism across different forms of PAH. Since more than one cell type contribute to the development of PAH and the pathophysiology of the disease BMPRII expression and TGFβ responses in pulmonary arterial smooth muscle cells (PASMCs) were also investigated. Initial studies generated a synthetic “disease” like PASMC that also displayed a reduction in BMPRII expression and increased response to TGFβ, which was similar to IPAH cells Finally, the role of epigenetic inhibition in the TβRIIΔk-fib model was investigated. The epigenetic inhibitor JQ1 was able to attenuate the spontaneous development of PAH in the TβRIIΔk-fib model of PAH. Taken together, work described in this thesis strongly suggests that a reduction in BMPRII is a susceptibility factor to the development of PAH in a pre-clinical model of SSc and in SSc patients

Bromodomain Inhibitor JQ1 Modulates Collagen Processing and Ameliorates Bleomycin Induced Dermal Fibrosis in Mice

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 (n≥3) 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 (n≥6) or 0.1U/ml bleomycin (n≥6) for 14 days and treated with 12mg/kg/day JQ1 (n≥6) or vehicle (n≥6). 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<0.05) inhibited in a dose dependent manner by JQ1. Consistent with this JQ1 attenuated SSc collagen deposition and processing (P˂0.05). Assessment of JQ1 in a pre-clinical model of dermal fibrosis demonstrated a markedly attenuation of dermal thickening in vivo(P˂0.05). Consistent with this we observed significant reduction in fibrogenic markers including αSMA, and collagen expression in the skin (P˂0.05). Furthermore secretion of the inflammatory marker, IL-6 was significantly attenuated (P˂0.05). 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 the excessive deposition and processing of collagen type I by SSc fibroblasts. In keeping with Brd proteins playing a pivotal role in the development and progression of dermal fibrosis, JQ1 significantly inhibited ECM deposition in vivo. Our data suggests a key role for Brd proteins in the persistence of the SSc dermal fibroblast phenotype

Bromodomain Inhibitor JQ1 Modulates Collagen Processing and Ameliorates Bleomycin Induced Dermal Fibrosis in Mice

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 (n≥3) 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 (n≥6) or 0.1U/ml bleomycin (n≥6) for 14 days and treated with 12mg/kg/day JQ1 (n≥6) or vehicle (n≥6). 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<0.05) inhibited in a dose dependent manner by JQ1. Consistent with this JQ1 attenuated SSc collagen deposition and processing (P˂0.05). Assessment of JQ1 in a pre-clinical model of dermal fibrosis demonstrated a markedly attenuation of dermal thickening in vivo(P˂0.05). Consistent with this we observed significant reduction in fibrogenic markers including αSMA, and collagen expression in the skin (P˂0.05). Furthermore secretion of the inflammatory marker, IL-6 was significantly attenuated (P˂0.05). 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 the excessive deposition and processing of collagen type I by SSc fibroblasts. In keeping with Brd proteins playing a pivotal role in the development and progression of dermal fibrosis, JQ1 significantly inhibited ECM deposition in vivo. Our data suggests a key role for Brd proteins in the persistence of the SSc dermal fibroblast phenotype

Endothelial to Mesenchymal Transition (EndoMT) leads to a loss of normal endothelial cell function and may contribute to the development of pulmonary arterial hypertension

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 αSMA. EndoMT was induced in human PAECs (n=3) in vitro by TNFα [5ng/ml], IL-1β [0.1ng/m;] and TGFβ [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, αSMA, 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 αSMA was observed in ≤5% of pulmonary arteries from SSc-PAH patients and hypoxia/SU5416 mice. PAECs treated with TNFα, IL-1β and TGFβ 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 PAECs alone. Consistent with this, EndoMT cells co-cultured with PAECs in a ratio of 1:10 led to 2.5-fold significant (P>0.05) increase in permeability. Conclusion The co-localisation of vWF and αSMA present in the pulmonary arteries of SSc-PAH patients and pre-clinical models of PAH, is indicative of EndoMT. We demonstrate EndoMT leads to a loss of normal PAEC morphology and an enhanced secretion of pro-inflammatory chemokines. Furthermore EndoMT cells failed to form integral biological barriers and contributed to enhanced permeability of PAEC barriers. Collectively our data suggests that EndoMT may contribute to the loss of normal endothelium function and the development of SSc-PAH

Prevention of SU5416-Induced Pulmonary Hypertension in a TGF beta Dependent Genetic Mouse Model of Scleroderma Using the Endothelin Receptor Antagonist Macitentan.

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 (TβRIIΔk-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 TβRIIΔk-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<0.05). The increase in RV systolic pressure in TG animals treated with SU5416 was also abrogated by macitentan (figure 1) without any significant change in systemic arterial blood pressure in any group. Explanted TG lung fibroblasts showed an increase in proliferation and migration with upregulation of VEGF and TGFbeta signalling and downregulation of endothelin receptor A compared with WT littermates. 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. 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 experimental therapeutic studies as well as providing insight into pathogenic mechanisms of disease

Endothelin Receptor Blockade Prevents Development of Pulmonary Hypertension in a Mouse Model of Scleroderma

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 < 0.05). The increase in RV systolic pressure in TG animals treated with SU5416 was also abrogated by macitentan (TG vehicle 28.8 mmHg 0.72, TG macitentan 22.0 1.62, P < 0.001) without 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. Conclusion: Macitentan prevents the development of histological and haemodynamic PH in this mouse model of SSc. These findings suggest a pivotal role for perturbed endothelin activity in the development of PH associated with altered TGFb and VEGF signalling. Our results also validate this model as a platform for experimental therapeutic studies and provide further insight into pathogenic mechanisms of PAH in SSc

Impaired bone morphogenetic protein receptor II signaling in a transforming growth factor-β-dependent mouse model of pulmonary hypertension and in systemic sclerosis.

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 (TβRIIΔk-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 TβRIIΔk-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)-β-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-β 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-β-dependent increased receptor degradation, may promote PAH susceptibility in SSc and provide a unifying mechanism across different forms of PAH

Impaired bone morphogenetic protein receptor II signaling in a transforming growth factor-β-dependent mouse model of pulmonary hypertension and in systemic sclerosis.

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 (TβRIIΔk-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 TβRIIΔk-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)-β-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-β 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-β-dependent increased receptor degradation, may promote PAH susceptibility in SSc and provide a unifying mechanism across different forms of PAH