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tumor necrosis factor related apoptosis-inducing ligand axis in the pathogenesis of pulmonary arterial hypertension
Vascular Pharmacology 63 (2014) 114–117
Contents lists available at ScienceDirect
Vascular Pharmacology journal homepage: www.elsevier.com/locate/vph
Review
The role of the osteoprotegerin/tumor necrosis factor related apoptosis-inducing ligand axis in the pathogenesis of pulmonary arterial hypertension☆ Allan Lawrie ⁎ Department of Cardiovascular Science, Faculty of Medicine, Dentistry & Health, University of Sheffield, Sheffield S10 2RX, United Kingdom
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Article history: Received 10 August 2014 Received in revised form 25 September 2014 Accepted 4 October 2014 Available online 18 October 2014 Keywords: Pulmonary arterial hypertension Osteoprotegerin TRAIL
a b s t r a c t Pulmonary arterial hypertension (PAH) is a fatal condition driven by a progressive remodelling of the small pulmonary arteries through sustained vasoconstriction, and vascular cell proliferation. This process causes a substantial reduction in luminal area increasing pulmonary vascular resistance and blood pressure leading to right heart failure. Current medical therapies can alleviate some symptoms and reduce the vasoconstrictive aspects of disease but new treatments are required that target the vascular cell proliferation if we are to develop new therapies. Expression of the tumour necrosis factor related apoptosis-inducing ligand (TRAIL) and osteoprotegerin (OPG) proteins are increased in IPAH. Specifically OPG is increased within the serum of patients with idiopathic pulmonary arterial hypertension (IPAH) and has prognostic utility, and both OPG and TRAIL are increased within pulmonary vascular lesions of patients with IPAH, and are mitogens for pulmonary artery smooth muscle cells in vitro. We have demonstrated that genetic deletion, or antibody blockade of TRAIL prevents, and critically reverses the development of PAH in multiple rodent models. The role OPG plays in this process both through interacting with TRAIL, and indirectly through other mechanisms is currently unclear these but data highlight the critical importance of this pathway in PAH pathogenesis, and its potential for future therapies. © 2014 Elsevier Inc. All rights reserved.
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . 2. Tumor necrosis factor (TNF) related apoptosis inducing-ligand 3. Osteoprotegerin. . . . . . . . . . . . . . . . . . . . . . 4. Summary and future directions . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Abbreviations: BM, bone marrow; BMDC, bone marrow derived cells; BMT, bone marrow transplant; MCT, monocrotaline; SuHx, sugen 5416 & hypoxia; OPG, osteoprotegerin; PAH, pulmonary arterial hypertension; PASMC, pulmonary artery smooth muscle cell; PCNA, proliferating cell nuclear antigen; PH, pulmonary hypertension; RVSP, right ventricular systolic pressure; SMC, smooth muscle cell; TRAIL, tumor necrosis factor related apoptosis-inducing ligand; TUNEL, TdT-mediated dUTP nick-end labelling. ☆ This article is one of a series based on presentations at the 8th International Workshop on Cardiovascular Biology & Medicine, London, September 2013. ⁎ Department of Cardiovascular Science, University of Sheffield, The Medical School, Beech Hill Road, Sheffield S10 2RX, United Kingdom. Tel.: +44 114 271 3176; fax: +44 114 271 1863. E-mail address: a.lawrie@sheffield.ac.uk.
http://dx.doi.org/10.1016/j.vph.2014.10.002 1537-1891/© 2014 Elsevier Inc. All rights reserved.
Pulmonary hypertension (PH) describes a group of progressive conditions each with a different origin but sharing a common haemodynamic diagnosis of mean pulmonary artery pressure greater than or equal to 25 mm Hg at rest [1]. The World Health Organisation (WHO) classification segregates PH into six groups based on aetiology and pathology that are — 1. Pulmonary arterial hypertension; 1′. Pulmonary veno-occlusive disease and/or pulmonary capillary haemangiomatosis; 3. PH due to left heart disease; 4. PH due to lung diseases and/or hypoxia; 4. Chronic thromboembolic pulmonary hypertension; and 5. PH with unclear and/or multifactorial mechanisms [2]. The work
A. Lawrie / Vascular Pharmacology 63 (2014) 114–117
highlighted in this article is primarily focused on addressing the pathogenesis of group 1 pulmonary arterial hypertension (PAH) where the haemodynamic alterations are driven by progressive pulmonary vascular remodelling. PAH is a rare disease with approximately 3000 prevalent cases in the UK [3] that often affects the young, with a 1:2.3 female gender bias. The disease significantly limits physical capacity and confers a life expectancy of 2.8 years without treatment [4]. Pathologically, PAH is characterised by sustained vasoconstriction and a progressive obliteration of small resistance pulmonary arteries and arterioles through a process of medial thickening, intimal fibrosis and the formation of angioproliferative (plexiform) lesions [5]. Endothelial dysfunction and pulmonary artery endothelial cell (PA-EC) apoptosis/dysfunction are thought to play an important early role in disease pathogenesis. Subsequent proliferation and migration of medial cells including smooth muscle cells (PA-SMC), fibroblasts and PA-EC [5] drive the pulmonary vascular remodelling. Current treatments target the sustained pulmonary vasoconstriction via either the prostacyclin, endothelin or nitric oxide pathway [6] in isolation or combination, but do little to address the underlying proliferative vascular disease. Subsequently, there is still no curative treatment for PAH other than transplantation, and the 3 and 5 year survival for PAH in its idiopathic form remains low at 38% and 17% respectively [7]. The last 10–15 years have seen some major breakthroughs in our understanding of the pathobiology of PAH. There are now wellestablished mechanistic insights into disease pathogenesis, for example, bone morphogenetic protein receptor type II (BMP-RII) mutations, the involvement of serotonin pathway, inflammation, mitochondria metabolism and many others [8,9]. These have manifested in many preclinical and small clinical studies evaluating the ‘therapeutic potential’ of manipulating new pathways implicated in vasoconstriction and/or the progressive remodelling of the pulmonary vasculature, such as vasoactive intestinal peptide (VIP) [10], Rho kinases [11], serotonin [12], apelin [13], the innate and acquired immunity system [14], epidermal growth factor [15,16], and peroxisome proliferator-activated receptor (PPAR)γ/ β-catenin complex [17,18], and fatty acid omega three [19]. Despite these important insights, the precise cell and molecular mechanisms leading to disease manifestation, and driving pathogenesis remain poorly understood. Dissecting the molecular mechanisms underlying
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PAH is therefore crucial if effective treatments for a condition that has a worse prognosis than many malignancies are to be developed. 2. Tumor necrosis factor (TNF) related apoptosis inducing-ligand Tumor necrosis factor (TNF) related apoptosis inducing-ligand (TRAIL, Apo2L), is a type II transmembrane protein that is widely expressed and detected in a variety of human tissues, most predominantly in spleen, lung, and prostate [20]. TRAIL can be alternatively spliced to produce a number of different isoforms [21] although little is known about their function. In humans there are four transmembrane TRAIL receptors, death receptor 4 (DR4, TRAIL-R1) [22], DR5 (TRAIL-R2) [22–25], decoy receptor 1 (DcR1, TRAIL-R3) [26–28], DcR2 (TRAIL-R4) [29–31] and the soluble protein osteoprotegerin (OPG) [32]. Both TRAIL-R1 and TRAIL-R2 contain a conserved death domain (DD) motif and mediate the extrinsic apoptosis pathway by TRAIL [33], TRAIL-R3 lacks an intracellular domain and TRAIL-R4 has a truncated DD, both are therefore considered decoy receptors to antagonize TRAIL-induced apoptosis by competing for ligand binding along with OPG [28,33,34] (Fig. 1). TRAIL was initially heralded as an anti-cancer therapy [35] due to its apparent selective ability to induce apoptosis in a variety of transformed or tumour cells while leaving normal, untransformed cells unaffected [20,36]. Unfortunately many cancer cells have subsequently been found to be resistant to TRAIL-induced apoptosis [35] via a variety of mechanism thought to include the regulation TRAIL receptor expression by genetic [37] and epigenetic mechanisms [38], as well as modulation of OPG expression [39,40]. TRAIL has also been shown to be important in the early resolution of inflammation through regulating inflammatory cell clearance by apoptosis [41–43] and to have immunosuppressive and immunoregulatory functions that are important for lymphocyte homeostasis and the transition between innate-to-adaptive immunity [44]. Interactions between inflammation and vascular cells are a key aspect of vascular injury/ repair and are considered to have an important role in cardiovascular disease. TRAIL mRNA and protein expression has previously been described in normal human pulmonary arteries [45]. We have demonstrated that TRAIL protein is associated with concentric and plexiform pulmonary vascular lesions from patients with IPAH [46] and from a
Fig. 1. Multiple signalling pathway implicated in PAH pathogenesis can feed into, and cause an increase in recruitment/expression of OPG and/or TRAIL. TRAIL binds to TRAIL-R3 on the cell surface of pulmonary artery smooth muscle cells (PA-SMC) and activates the phosphorylation of ERK 1/2 driving a pro-proliferative phenotype. OPG, either by interaction with TRAIL, or another un-associated cell surface receptor similarly mediates a pro-survival phenotype by a yet to be defined mechanism.
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A. Lawrie / Vascular Pharmacology 63 (2014) 114–117
murine model of severe PAH [47]. Other investigators have reported pro-proliferative effects of TRAIL on systemic VSMC [48,49], and propose a role in neointimal formation in mice following a vascular injury [50], in atherosclerosis [51] and coagulation [52]. Specifically in the context of pulmonary vascular disease my group has recently shown that PASMCs isolated from patients with IPAH expressed increased levels of TRAIL, and it's receptors, and that TRAIL is a mitogen and pro-migratory stimulus to human PASMCs in vitro via the phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) downstream of TRAIL-R3 [53] (Fig. 1). By utilising a range of pre-clinical models of pulmonary hypertension we demonstrated that TRAIL is required for the development of PAH in rodent models through either genetic deletion of TRAIL in the hypoxia, sugen/hypoxia [54] or the high fat diet (HFD)-induced model [47] when crossed with ApoE−/− mice. Furthermore prophylactic treatment with an anti-TRAIL antibody prevented disease in the monocrotaline rat model [53]. Interestingly in the ApoE−/−/TRAIL−/− mice fed a HFD treatment with exogenous, recombinant TRAIL to the mice resulted in the re-establishment of PAH [53] providing further evidence of the key role TRAIL plays in disease pathogenesis. Data from TRAIL chimeric mice suggest that TRAIL expression by cells within tissue, rather than bone marrow derived is the main source. TRAIL is a membrane anchored protein that can be enzymatically cleaved by cysteine proteases to generate a soluble form of TRAIL [55]. Cysteine proteases, including caspases, calpain and papain are abundant within the vessel wall [56]. Calpain in particular has recently been shown to be important in pulmonary vascular remodelling in animal models of PAH [57]. Cleavage of TRAIL by cysteine proteinases, such as calpain may play an important role in allowing paracrine effects of cleaved soluble TRAIL on multiple cell types. This may also explain why the systemic delivery of recombinant soluble form of TRAIL, without direct presentation by another cell, resulted in the development of a PAH phenotype suggesting that soluble/cleaved levels within the vessel wall are critically important. In therapeutic studies on models with established disease, TRAIL blockade significantly reduced pulmonary vascular remodelling and increased survival in a rat monocrotaline (MCT) model, and in a murine model of severe PAH resulted in full regression of pulmonary vascular remodelling with virtual normalisation of right ventricular pressures [53]. These therapeutic effects were associated with a reduction in proliferating cells and an increase in apoptotic cells within the distal pulmonary arteries and arterioles suggesting that TRAIL is a key regulator of the proliferative phenotype of pulmonary arteriole smooth muscle cells in these models of PAH (Fig. 1). 3. Osteoprotegerin Current dogma suggests that the predominant function of OPG is to regulate osteoclastogenesis via direct binding to RANKL, with data from mice demonstrating that reduced OPG expression results in osteoporosis [58] and over-expression of OPG causes osteopetrosis [59]. However, as described above OPG has also been shown to bind to TRAIL to inhibit TRAIL-induced apoptosis in TRAIL sensitive cells [32]. We have demonstrated that both TRAIL and OPG can be regulated by a number of pathways associated with PAH including bone morphogenetic proteins, 5-HT and inflammatory cytokines [46,53] (Fig. 1). As described above, PA-SMC and endothelial cell migration and proliferation are key processes in the pathogenesis of pulmonary arterial hypertension (PAH). The molecular and cellular mechanisms involved are complex and involve cross-talk between several signalling pathways including the transforming growth factor beta (TFG-β)/bone morphogenetic protein (BMP), growth factors e.g. PDGF, and vasoactive proteins e.g. endothelin-1 (ET-1) [9]. The secreted glycoprotein osteoprotegerin (OPG, TNFRSF11B) is emerging as an important regulatory molecule in vascular biology and we have shown that it's expression and release from PASMCs are modulated by BMPs, 5-HT, and interleukin-1 [46]. Recombinant OPG added to human PA-SMC in culture stimulates
proliferation and migration in a dose dependent manner [46] indicating that OPG is capable of acting as an autocrine factor stimulating pulmonary vascular remodelling (Fig. 1). OPG mRNA expression is greater than 2-fold higher in PASMCs isolated from patients with IPAH compared to control [60], and immunoreactivity to OPG protein was markedly increased in concentric and plexiform lesions from patients with IPAH compared to control sections [46]. Since OPG is a naturally secreted protein, and its expression is increased within remodelled pulmonary arteries in patients with IPAH we have also investigated the relationship between serum OPG and disease severity and outcome in patients with IPAH and animal models. We have measured serum concentrations of OPG in both a retrospective (on PH therapies, collected at Papworth, UK) and prospective group of treatment naïve patients collected in Sheffield. Statistical analysis was performed to compare OPG levels with clinical phenotypic data and other putative biomarkers to assess the prognostic significance serum levels of OPG. In both cohorts serum OPG concentrations were demonstrated to be markedly elevated in IPAH compared with controls [60]. In the retrospective cohort OPG levels significantly correlated with mean right atrial pressure and cardiac index, while in prospective cohort significant correlations existed between age-adjusted OPG levels and gas transfer, the difference is possibly due to their relative stage of disease progression and the potential influence of current PAHtreatments on disease pathogenesis. However, in both cohorts, a ROCderived serum concentration for OPG of greater than 4728 pg/ml predicted poorer survival. Furthermore, studies in two rodent models of PAH, namely the monocrotaline rat, HFD-ApoE−/− mouse, and HFDApoE−/−/IL-1R1−/− mouse, demonstrate that OPG serum levels correlate strongly with the degree of pulmonary vascular remodelling in both a time course, and treatment study [60]. More recently, Jaisewicz et al. have similarly reported that OPG levels are elevated in PAH and are associated with indicators of disease severity and prognosis. Interestingly they also reported that sRANKL was a better discriminant between PAH and left ventricular associated heart failure than OPG [61]. These data demonstrate that OPG can be regulated by multiple pathways associated with PAH, has increased expression in PAH, and that it can regulate PASMC proliferation and migration (Fig. 1). OPG may provide a common link between the different pathways associated with the disease, potentially playing an important role in the pathogenesis of PAH. As a secreted molecule, OPG therefore shows potential as a biomarker and therapeutic target in PAH. 4. Summary and future directions Taken together, these data highlight the OPG/TRAIL axis as a new important pathway in the pathogenesis of PAH, and therefor highlight it as a therapeutic target. Further work is clearly required to further characterise the direct effect of the OPG–TRAIL interaction in PAH, and to determine whether the OPG-driven PASMC phenotype is mediated via TRAIL, or another novel signalling mechanism. These critical studies are currently underway, and we hope the data will provide strong evidence for the development of a new therapeutic agent targeting the key molecular processes in this pathway. Acknowledgments Funding for this study has been provided by a British Heart Foundation Senior Basic Science Research Fellow (FS/13/48/3045), Medical Research Council Career Development Award (G0800318); British Heart Foundation Clinical Research Training Fellowship (FS/08/061/ 25740); National Institute for Health Research Sheffield Cardiovascular Biomedical Research Unit; and Cambridge National Institute of Health Research Biomedical Research Centre; and used services provided by the Sheffield National Institute for Health Research Clinical Research Facility.
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Contents lists available at ScienceDirect
Vascular Pharmacology journal homepage: www.elsevier.com/locate/vph
Review
The role of the osteoprotegerin/tumor necrosis factor related apoptosis-inducing ligand axis in the pathogenesis of pulmonary arterial hypertension☆ Allan Lawrie ⁎ Department of Cardiovascular Science, Faculty of Medicine, Dentistry & Health, University of Sheffield, Sheffield S10 2RX, United Kingdom
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Article history: Received 10 August 2014 Received in revised form 25 September 2014 Accepted 4 October 2014 Available online 18 October 2014 Keywords: Pulmonary arterial hypertension Osteoprotegerin TRAIL
a b s t r a c t Pulmonary arterial hypertension (PAH) is a fatal condition driven by a progressive remodelling of the small pulmonary arteries through sustained vasoconstriction, and vascular cell proliferation. This process causes a substantial reduction in luminal area increasing pulmonary vascular resistance and blood pressure leading to right heart failure. Current medical therapies can alleviate some symptoms and reduce the vasoconstrictive aspects of disease but new treatments are required that target the vascular cell proliferation if we are to develop new therapies. Expression of the tumour necrosis factor related apoptosis-inducing ligand (TRAIL) and osteoprotegerin (OPG) proteins are increased in IPAH. Specifically OPG is increased within the serum of patients with idiopathic pulmonary arterial hypertension (IPAH) and has prognostic utility, and both OPG and TRAIL are increased within pulmonary vascular lesions of patients with IPAH, and are mitogens for pulmonary artery smooth muscle cells in vitro. We have demonstrated that genetic deletion, or antibody blockade of TRAIL prevents, and critically reverses the development of PAH in multiple rodent models. The role OPG plays in this process both through interacting with TRAIL, and indirectly through other mechanisms is currently unclear these but data highlight the critical importance of this pathway in PAH pathogenesis, and its potential for future therapies. © 2014 Elsevier Inc. All rights reserved.
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . 2. Tumor necrosis factor (TNF) related apoptosis inducing-ligand 3. Osteoprotegerin. . . . . . . . . . . . . . . . . . . . . . 4. Summary and future directions . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Abbreviations: BM, bone marrow; BMDC, bone marrow derived cells; BMT, bone marrow transplant; MCT, monocrotaline; SuHx, sugen 5416 & hypoxia; OPG, osteoprotegerin; PAH, pulmonary arterial hypertension; PASMC, pulmonary artery smooth muscle cell; PCNA, proliferating cell nuclear antigen; PH, pulmonary hypertension; RVSP, right ventricular systolic pressure; SMC, smooth muscle cell; TRAIL, tumor necrosis factor related apoptosis-inducing ligand; TUNEL, TdT-mediated dUTP nick-end labelling. ☆ This article is one of a series based on presentations at the 8th International Workshop on Cardiovascular Biology & Medicine, London, September 2013. ⁎ Department of Cardiovascular Science, University of Sheffield, The Medical School, Beech Hill Road, Sheffield S10 2RX, United Kingdom. Tel.: +44 114 271 3176; fax: +44 114 271 1863. E-mail address: a.lawrie@sheffield.ac.uk.
http://dx.doi.org/10.1016/j.vph.2014.10.002 1537-1891/© 2014 Elsevier Inc. All rights reserved.
Pulmonary hypertension (PH) describes a group of progressive conditions each with a different origin but sharing a common haemodynamic diagnosis of mean pulmonary artery pressure greater than or equal to 25 mm Hg at rest [1]. The World Health Organisation (WHO) classification segregates PH into six groups based on aetiology and pathology that are — 1. Pulmonary arterial hypertension; 1′. Pulmonary veno-occlusive disease and/or pulmonary capillary haemangiomatosis; 3. PH due to left heart disease; 4. PH due to lung diseases and/or hypoxia; 4. Chronic thromboembolic pulmonary hypertension; and 5. PH with unclear and/or multifactorial mechanisms [2]. The work
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highlighted in this article is primarily focused on addressing the pathogenesis of group 1 pulmonary arterial hypertension (PAH) where the haemodynamic alterations are driven by progressive pulmonary vascular remodelling. PAH is a rare disease with approximately 3000 prevalent cases in the UK [3] that often affects the young, with a 1:2.3 female gender bias. The disease significantly limits physical capacity and confers a life expectancy of 2.8 years without treatment [4]. Pathologically, PAH is characterised by sustained vasoconstriction and a progressive obliteration of small resistance pulmonary arteries and arterioles through a process of medial thickening, intimal fibrosis and the formation of angioproliferative (plexiform) lesions [5]. Endothelial dysfunction and pulmonary artery endothelial cell (PA-EC) apoptosis/dysfunction are thought to play an important early role in disease pathogenesis. Subsequent proliferation and migration of medial cells including smooth muscle cells (PA-SMC), fibroblasts and PA-EC [5] drive the pulmonary vascular remodelling. Current treatments target the sustained pulmonary vasoconstriction via either the prostacyclin, endothelin or nitric oxide pathway [6] in isolation or combination, but do little to address the underlying proliferative vascular disease. Subsequently, there is still no curative treatment for PAH other than transplantation, and the 3 and 5 year survival for PAH in its idiopathic form remains low at 38% and 17% respectively [7]. The last 10–15 years have seen some major breakthroughs in our understanding of the pathobiology of PAH. There are now wellestablished mechanistic insights into disease pathogenesis, for example, bone morphogenetic protein receptor type II (BMP-RII) mutations, the involvement of serotonin pathway, inflammation, mitochondria metabolism and many others [8,9]. These have manifested in many preclinical and small clinical studies evaluating the ‘therapeutic potential’ of manipulating new pathways implicated in vasoconstriction and/or the progressive remodelling of the pulmonary vasculature, such as vasoactive intestinal peptide (VIP) [10], Rho kinases [11], serotonin [12], apelin [13], the innate and acquired immunity system [14], epidermal growth factor [15,16], and peroxisome proliferator-activated receptor (PPAR)γ/ β-catenin complex [17,18], and fatty acid omega three [19]. Despite these important insights, the precise cell and molecular mechanisms leading to disease manifestation, and driving pathogenesis remain poorly understood. Dissecting the molecular mechanisms underlying
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PAH is therefore crucial if effective treatments for a condition that has a worse prognosis than many malignancies are to be developed. 2. Tumor necrosis factor (TNF) related apoptosis inducing-ligand Tumor necrosis factor (TNF) related apoptosis inducing-ligand (TRAIL, Apo2L), is a type II transmembrane protein that is widely expressed and detected in a variety of human tissues, most predominantly in spleen, lung, and prostate [20]. TRAIL can be alternatively spliced to produce a number of different isoforms [21] although little is known about their function. In humans there are four transmembrane TRAIL receptors, death receptor 4 (DR4, TRAIL-R1) [22], DR5 (TRAIL-R2) [22–25], decoy receptor 1 (DcR1, TRAIL-R3) [26–28], DcR2 (TRAIL-R4) [29–31] and the soluble protein osteoprotegerin (OPG) [32]. Both TRAIL-R1 and TRAIL-R2 contain a conserved death domain (DD) motif and mediate the extrinsic apoptosis pathway by TRAIL [33], TRAIL-R3 lacks an intracellular domain and TRAIL-R4 has a truncated DD, both are therefore considered decoy receptors to antagonize TRAIL-induced apoptosis by competing for ligand binding along with OPG [28,33,34] (Fig. 1). TRAIL was initially heralded as an anti-cancer therapy [35] due to its apparent selective ability to induce apoptosis in a variety of transformed or tumour cells while leaving normal, untransformed cells unaffected [20,36]. Unfortunately many cancer cells have subsequently been found to be resistant to TRAIL-induced apoptosis [35] via a variety of mechanism thought to include the regulation TRAIL receptor expression by genetic [37] and epigenetic mechanisms [38], as well as modulation of OPG expression [39,40]. TRAIL has also been shown to be important in the early resolution of inflammation through regulating inflammatory cell clearance by apoptosis [41–43] and to have immunosuppressive and immunoregulatory functions that are important for lymphocyte homeostasis and the transition between innate-to-adaptive immunity [44]. Interactions between inflammation and vascular cells are a key aspect of vascular injury/ repair and are considered to have an important role in cardiovascular disease. TRAIL mRNA and protein expression has previously been described in normal human pulmonary arteries [45]. We have demonstrated that TRAIL protein is associated with concentric and plexiform pulmonary vascular lesions from patients with IPAH [46] and from a
Fig. 1. Multiple signalling pathway implicated in PAH pathogenesis can feed into, and cause an increase in recruitment/expression of OPG and/or TRAIL. TRAIL binds to TRAIL-R3 on the cell surface of pulmonary artery smooth muscle cells (PA-SMC) and activates the phosphorylation of ERK 1/2 driving a pro-proliferative phenotype. OPG, either by interaction with TRAIL, or another un-associated cell surface receptor similarly mediates a pro-survival phenotype by a yet to be defined mechanism.
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murine model of severe PAH [47]. Other investigators have reported pro-proliferative effects of TRAIL on systemic VSMC [48,49], and propose a role in neointimal formation in mice following a vascular injury [50], in atherosclerosis [51] and coagulation [52]. Specifically in the context of pulmonary vascular disease my group has recently shown that PASMCs isolated from patients with IPAH expressed increased levels of TRAIL, and it's receptors, and that TRAIL is a mitogen and pro-migratory stimulus to human PASMCs in vitro via the phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) downstream of TRAIL-R3 [53] (Fig. 1). By utilising a range of pre-clinical models of pulmonary hypertension we demonstrated that TRAIL is required for the development of PAH in rodent models through either genetic deletion of TRAIL in the hypoxia, sugen/hypoxia [54] or the high fat diet (HFD)-induced model [47] when crossed with ApoE−/− mice. Furthermore prophylactic treatment with an anti-TRAIL antibody prevented disease in the monocrotaline rat model [53]. Interestingly in the ApoE−/−/TRAIL−/− mice fed a HFD treatment with exogenous, recombinant TRAIL to the mice resulted in the re-establishment of PAH [53] providing further evidence of the key role TRAIL plays in disease pathogenesis. Data from TRAIL chimeric mice suggest that TRAIL expression by cells within tissue, rather than bone marrow derived is the main source. TRAIL is a membrane anchored protein that can be enzymatically cleaved by cysteine proteases to generate a soluble form of TRAIL [55]. Cysteine proteases, including caspases, calpain and papain are abundant within the vessel wall [56]. Calpain in particular has recently been shown to be important in pulmonary vascular remodelling in animal models of PAH [57]. Cleavage of TRAIL by cysteine proteinases, such as calpain may play an important role in allowing paracrine effects of cleaved soluble TRAIL on multiple cell types. This may also explain why the systemic delivery of recombinant soluble form of TRAIL, without direct presentation by another cell, resulted in the development of a PAH phenotype suggesting that soluble/cleaved levels within the vessel wall are critically important. In therapeutic studies on models with established disease, TRAIL blockade significantly reduced pulmonary vascular remodelling and increased survival in a rat monocrotaline (MCT) model, and in a murine model of severe PAH resulted in full regression of pulmonary vascular remodelling with virtual normalisation of right ventricular pressures [53]. These therapeutic effects were associated with a reduction in proliferating cells and an increase in apoptotic cells within the distal pulmonary arteries and arterioles suggesting that TRAIL is a key regulator of the proliferative phenotype of pulmonary arteriole smooth muscle cells in these models of PAH (Fig. 1). 3. Osteoprotegerin Current dogma suggests that the predominant function of OPG is to regulate osteoclastogenesis via direct binding to RANKL, with data from mice demonstrating that reduced OPG expression results in osteoporosis [58] and over-expression of OPG causes osteopetrosis [59]. However, as described above OPG has also been shown to bind to TRAIL to inhibit TRAIL-induced apoptosis in TRAIL sensitive cells [32]. We have demonstrated that both TRAIL and OPG can be regulated by a number of pathways associated with PAH including bone morphogenetic proteins, 5-HT and inflammatory cytokines [46,53] (Fig. 1). As described above, PA-SMC and endothelial cell migration and proliferation are key processes in the pathogenesis of pulmonary arterial hypertension (PAH). The molecular and cellular mechanisms involved are complex and involve cross-talk between several signalling pathways including the transforming growth factor beta (TFG-β)/bone morphogenetic protein (BMP), growth factors e.g. PDGF, and vasoactive proteins e.g. endothelin-1 (ET-1) [9]. The secreted glycoprotein osteoprotegerin (OPG, TNFRSF11B) is emerging as an important regulatory molecule in vascular biology and we have shown that it's expression and release from PASMCs are modulated by BMPs, 5-HT, and interleukin-1 [46]. Recombinant OPG added to human PA-SMC in culture stimulates
proliferation and migration in a dose dependent manner [46] indicating that OPG is capable of acting as an autocrine factor stimulating pulmonary vascular remodelling (Fig. 1). OPG mRNA expression is greater than 2-fold higher in PASMCs isolated from patients with IPAH compared to control [60], and immunoreactivity to OPG protein was markedly increased in concentric and plexiform lesions from patients with IPAH compared to control sections [46]. Since OPG is a naturally secreted protein, and its expression is increased within remodelled pulmonary arteries in patients with IPAH we have also investigated the relationship between serum OPG and disease severity and outcome in patients with IPAH and animal models. We have measured serum concentrations of OPG in both a retrospective (on PH therapies, collected at Papworth, UK) and prospective group of treatment naïve patients collected in Sheffield. Statistical analysis was performed to compare OPG levels with clinical phenotypic data and other putative biomarkers to assess the prognostic significance serum levels of OPG. In both cohorts serum OPG concentrations were demonstrated to be markedly elevated in IPAH compared with controls [60]. In the retrospective cohort OPG levels significantly correlated with mean right atrial pressure and cardiac index, while in prospective cohort significant correlations existed between age-adjusted OPG levels and gas transfer, the difference is possibly due to their relative stage of disease progression and the potential influence of current PAHtreatments on disease pathogenesis. However, in both cohorts, a ROCderived serum concentration for OPG of greater than 4728 pg/ml predicted poorer survival. Furthermore, studies in two rodent models of PAH, namely the monocrotaline rat, HFD-ApoE−/− mouse, and HFDApoE−/−/IL-1R1−/− mouse, demonstrate that OPG serum levels correlate strongly with the degree of pulmonary vascular remodelling in both a time course, and treatment study [60]. More recently, Jaisewicz et al. have similarly reported that OPG levels are elevated in PAH and are associated with indicators of disease severity and prognosis. Interestingly they also reported that sRANKL was a better discriminant between PAH and left ventricular associated heart failure than OPG [61]. These data demonstrate that OPG can be regulated by multiple pathways associated with PAH, has increased expression in PAH, and that it can regulate PASMC proliferation and migration (Fig. 1). OPG may provide a common link between the different pathways associated with the disease, potentially playing an important role in the pathogenesis of PAH. As a secreted molecule, OPG therefore shows potential as a biomarker and therapeutic target in PAH. 4. Summary and future directions Taken together, these data highlight the OPG/TRAIL axis as a new important pathway in the pathogenesis of PAH, and therefor highlight it as a therapeutic target. Further work is clearly required to further characterise the direct effect of the OPG–TRAIL interaction in PAH, and to determine whether the OPG-driven PASMC phenotype is mediated via TRAIL, or another novel signalling mechanism. These critical studies are currently underway, and we hope the data will provide strong evidence for the development of a new therapeutic agent targeting the key molecular processes in this pathway. Acknowledgments Funding for this study has been provided by a British Heart Foundation Senior Basic Science Research Fellow (FS/13/48/3045), Medical Research Council Career Development Award (G0800318); British Heart Foundation Clinical Research Training Fellowship (FS/08/061/ 25740); National Institute for Health Research Sheffield Cardiovascular Biomedical Research Unit; and Cambridge National Institute of Health Research Biomedical Research Centre; and used services provided by the Sheffield National Institute for Health Research Clinical Research Facility.
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