Introduction Pulmonary arterial hypertension (PAH) is a devastating disease with a high mortality... more Introduction Pulmonary arterial hypertension (PAH) is a devastating disease with a high mortality and prognosis worse than many cancers. Pathologically, PAH is characterised by progressive arteriole remodelling driven by pulmonary artery smooth muscle cell (PASMC) proliferation and migration. Although current treatments alleviate symptoms, they do not reverse the underlying progressive pulmonary vascular proliferation. We have previously shown that the secreted glycoprotein osteoprotegerin (OPG, TNFRSF11B) is increased within pulmonary vascular lesions and serum from patients with idiopathic PAH, and promotes the proliferation and migration of PASMCs in vitro . Recent experiments have shown that administration of an anti-OPG antibody can prevent and reverse PAH in rodent models of disease. However, how OPG signals to mediate PASMC phenotype remains unknown. We hypothesise that OPG mediates these effects through a previously undescribed cell surface receptor. We aim to identify this receptor on PASMC and characterise the OPG signalling cascade leading to the proliferative phenotype. Methods Quiesced PASMCs (Lonza, Basel, Switzerland) were stimulated with 0.2% FCS (negative), and OPG (50 ng/ml) for 10 and 60 min. Phosphorylation targets were identified from protein lysates by Kinex antibody microarray (Kinexus, Canada). Selected targets were verified by western blotting. Transcriptomic analysis of PASMCs stimulated with OPG for 6 h was performed using an RNA expression microarray (Agilent) and confirmed by TaqMan RT-PCR. Novel OPG binding proteins were identified following reverse transfection of HEK293 cells with 2054 human membrane proteins (Retrogenix, Sheffield, UK) and confirmed in PASMC by co-immunoprecipitation. To assess the effect of Fas blockade, proliferation was assessed in PASMCs pre-incubated with Fas neutralising antibody (500 ng/ml) 30 min before 72h stimulation with OPG. Results OPG stimulation of PASMC resulted in significant activation of CDK4 and 5, HSP27 and ERK1/2, and significant decrease in phospho-mTOR. OPG increased TRAIL, PDGFRA, TNC, Cav-1 and reduced VIP receptor gene expression. Four novel OPG interactions with IL1RAP, Fas, TMPRSS11D and GAP43 were identified by the Retrogenix cell microarray. We have confirmed OPG interaction with IL1RAP and Fas in PASMC by co-immunoprecipitation. Fas protein expression is elevated in the pulmonary artery and right ventricle of IPAH patients. Fas RNA expression is increased in PASMCs from IPAH patient lungs. Furthermore, blocking Fas with a neutralising antibody reduces OPG-induced proliferation, PDGFRA and TNC RNA expression. Conclusions These data highlight novel binding partners for OPG. In particular the OPG-FAS interaction regulates a diverse and important intracellular signalling cascade by which OPG regulates proliferation, apoptosis and autophagy proteins, and PAH associated genes in PASMC. These data further highlight therapeutic potential of targeting OPG in PAH.
Catheterization and Cardiovascular Interventions, 2007
While several endovascular techniques have been developed for treating arterial bifurcation lesio... more While several endovascular techniques have been developed for treating arterial bifurcation lesions, there is, as yet, no single, widely accepted technique for treating left main stem (LMS) bifurcation lesions with stents. The simultaneous kissing stent (SCS) technique seems particularly suited for such lesions. The authors describe a consecutive cohort of patients with LMS bifurcation stenosis treated with this technique and present mechanistic insights from a porcine model. Thirty consecutive patients with LMS bifurcation stenosis +/- multivessel disease were treated with SCS technique using paclitaxel-eluting stents. The technique involves simultaneous implantation of two stents (LMS-LAD and LMS-Cx) so that the proximal end of both stents lie at the same level in the body of LMS. Symptom status was recorded and follow-up angiography was performed. In addition, four pigs underwent SCS implantation of the LMS and were studied by direct examination, histology, and scanning electron microscopy. The patients' mean age was 63, 26 were elective and 19 were male. Paclitaxel-eluting stents (Taxus, Boston Scientific) were used. There was one in-hospital death in a highly unstable patient. At 6-month follow-up angiography, two patients required target lesion revascularization. At 13 +/- 3 month follow-up, the remaining patients remain well, with no other major adverse cardiac events. The animals all survived with patent stents. SEM demonstrated full re-endothelialization and histology showed a thin but complete neointima covering the stent struts. The SCS technique for treating LMS bifurcation disease with paclitaxel-eluting stents is safe and feasible. Medium term clinical and angiographic results are good. An endothelium and a thin neointima cover the metal struts.
Objective: Restenosis following angioplasty involves processes that may be influenced by local pr... more Objective: Restenosis following angioplasty involves processes that may be influenced by local production of cytokines. We investigated the expression of active and total transforming growth factor b (TGFb) following porcine coronary angioplasty (PTCA), and have correlated this with the expression of potential in vivo activators of TGFb: mannose-6-phosphate / insulin-like growth factor-II (M6P/ IGF-II) receptor and thrombospondin-1. Methods: Oversized porcine PTCA was performed and the arteries excised after selected intervals. Levels of in situ active and total (active plus latent) TGFb were determined using a modified plasminogen activator-inhibitor / luciferase bioassay. Results: Levels of active TGFb significantly increased 2 h to 7 days after angioplasty, compared to non-injured controls. Levels returned to baseline by 28 days. Active TGFb in tissues adjacent to the injured artery did not change. Total TGFb was significantly higher than controls 2-6 h after injury. M6P/ IGF-II receptor mRNA was upregulated between 6 h and 3 days after injury, with protein detectable at 3-28 days. Thrombospondin-1 was detected between 1 h and 14 days. Conclusions: We conclude that balloon injury causes an early rapid increase in levels of active TGFb, that correlates with the expression of TGFb activators. Thus, TGFb is a good potential target for anti-restenotic therapies.
Objective: To determine the influence of IL-1 on the arterial response to experimental injury in ... more Objective: To determine the influence of IL-1 on the arterial response to experimental injury in porcine models of percutaneous coronary intervention (PCI). Methods: An intravenous (i.v.) bolus of 0.5 mg/kg followed by a subcutaneous (s.c.) infusion of 2 mg/kg/24 h of human IL-1 receptor antagonist (IL-1ra) inhibited neutrophil recruitment in response to intradermal IL-1. Using this dose regimen, five groups of pigs were studied: Group 1, oversized balloon angioplasty of 2 coronary vessels (14-day infusion, 28th day sacrifice and analysis); Groups 2, 3, 4, and 5, oversized stenting of 2 coronary vessels (Group 2: 14-day infusion, 28th day analysis; Group 3: 14-day infusion, 14th day analysis; Group 4: 28-day infusion, 28th day analysis; Group 5: 28-day infusion, 90th day analysis). Neointimal area was quantified by standard means. Results: In Group 1, IL-1ra resulted in a 23% decrease in neointimal area (p = 0.04); in Group 2, a 34% increase (p = 0.001); in Group 3, a 38% decrease (p < 0.0001); in Group 4, a 34% decrease (p = 0.0004); and in Group 5, a 41% decrease (p = 0.00001). Conclusions: IL-1ra was associated with a sustained, significant reduction in neointima after vessel wall injury as long as it is given for the duration of the stimulation of the IL-1 system, in this case at least 28 days. This suggests that therapies based on the antagonism of IL-1 may modulate the coronary artery response to injury.
Inflammatory mechanisms are proposed to play a significant role in the pathogenesis of pulmonary ... more Inflammatory mechanisms are proposed to play a significant role in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have described PAH in fat-fed apolipoprotein E knockout (ApoE ؊/؊) mice. We have reported that signaling in interleukin-1-receptor-knockout (IL-1R1 ؊/؊) mice leads to a reduction in diet-induced systemic atherosclerosis. We subsequently hypothesized that doublenull (ApoE ؊/؊ /IL-1R1 ؊/؊) mice would show a reduced PAH phenotype compared with that of ApoE ؊/؊ mice. Male IL-1R1 ؊/؊ , ApoE ؊/؊ , and ApoE ؊/؊ /IL-1R1 ؊/؊ mice were fed regular chow or a high-fat diet (Paigen diet) for 8 weeks before phenotyping for PAH. No abnormal phenotype was observed in the IL-1R1 ؊/؊ mice. Fat-fed ApoE ؊/؊ mice developed significantly increased right ventricular systolic pressure and substantial pulmonary vascular remodeling. Surprisingly, ApoE ؊/؊ /IL-1R1 ؊/؊ mice showed an even more severe PAH phenotype. Further molecular investigation revealed the expression of a putative, alternatively primed IL-1R1 transcript expressed within the lungs but not aorta of ApoE ؊/؊ /IL-1R1 ؊/؊ mice. Treatment of ApoE ؊/؊ and ApoE ؊/؊ /IL-1R1 ؊/؊ mice with IL-1-receptor antagonist prevented progression of the PAH phenotype in both strains. Blocking IL-1 signaling may have beneficial effects in treating PAH, and alternative IL-1-receptor signaling in the lung may be important in driving PAH pathogenesis.
Development of the atherosclerotic plaque involves a complex interplay between a number of cell t... more Development of the atherosclerotic plaque involves a complex interplay between a number of cell types and an extensive inter-cellular communication via cell bound as well as soluble mediators. The family of tribbles proteins has recently been identified as novel controllers of pro-inflammatory signal transduction. The objective of this study was to address the expression pattern of all three tribbles proteins in atherosclerotic plaques from a mouse model of atherosclerosis. Each tribbles were expressed in vascular smooth muscle cells, endothelial cells as well as in resident macrophages of mouse atherosclerotic plaques. The role of IL-1 mediated inflammatory events in controlling tribbles expression was also addressed by inducing experimental atherosclerosis in ApoE í/í IL1R1 í/í (double knockout) mice. Immunohistochemical analysis of these mice showed a selective decrease in the percentage of trb-1 expressing macrophages, compared to the ApoE í/í cohort (14.7% r 1.55 vs. 26.3% r 1.19). The biological significance of this finding was verified in vitro where overexpression of trb-1 in macrophages led to a significant attenuation (~70%) of IL-6 production as well as a suppressed IL-12 expression induced by a proinflammatory stimulus. In this in vitro setting, expression of truncated trb-1 mutants suggests that the kinase domain of this protein is sufficient to exert this inhibitory action.
The Journal of clinical investigation, Jul 23, 2016
Loss of the growth-suppressive effects of bone morphogenetic protein (BMP) signaling has been dem... more Loss of the growth-suppressive effects of bone morphogenetic protein (BMP) signaling has been demonstrated to promote pulmonary arterial endothelial cell dysfunction and induce pulmonary arterial smooth muscle cell (PASMC) proliferation, leading to the development of pulmonary arterial hypertension (PAH). MicroRNAs (miRs) mediate higher order regulation of cellular function through coordinated modulation of mRNA targets; however, miR expression is altered by disease development and drug therapy. Here, we examined treatment-naive patients and experimental models of PAH and identified a reduction in the levels of miR-140-5p. Inhibition of miR-140-5p promoted PASMC proliferation and migration in vitro. In rat models of PAH, nebulized delivery of miR-140-5p mimic prevented the development of PAH and attenuated the progression of established PAH. Network and pathway analysis identified SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) as a key miR-140-5p target and regulator of BMP si...
Pulmonary arterial hypertension (PAH) is a progressive lung disease diagnosed by an increase in p... more Pulmonary arterial hypertension (PAH) is a progressive lung disease diagnosed by an increase in pulmonary arterial blood pressure that is driven by a progressive vascular remodelling of small pulmonary arterioles. We have previously reported that tumor necrosis factor apoptosis-inducing ligand (TRAIL) protein expression is increased in pulmonary vascular lesions and pulmonary artery smooth muscle cells (PASMC) of patients with idiopathic PAH. The addition of recombinant TRAIL induces the proliferation and migration of PASMCs in vitro. TRAIL is required for hypoxia-induced pulmonary hypertension in mice, and blockade of TRAIL prevents and reduces disease development in other rodent models of PAH. Due to the availability of knockout and transgenic mice, murine models of disease are key to further advances in understanding the complex and heterogeneous pathogenesis of PAH. However, murine models vary in their disease severity, and are often criticized for lacking the proliferative pulmonary vascular lesions characteristic of PAH. The murine Sugen-hypoxic (SuHx) mouse model has recently been reported to have a more severe PAH phenotype consisting advanced pulmonary vascular remodelling. We therefore aimed to determine whether TRAIL was also required for the development of PAH in this model. C57BL/6 and TRAIL −/− mice were exposed to normoxia, Sugen5416 alone, hypoxia or both Sugen5416 and hypoxia (SuHx). We report here that SuHx treated C57BL/6 mice developed more severe PAH than OPEN ACCESS Diseases 2014, 2 261 hypoxia alone, and that TRAIL −/− mice were protected from disease development. These data further emphasise the importance of this pathway and support the use of the SuHx mouse model for investigating the importance of potential mediators in PAH pathogenesis.
Arteriosclerosis, Thrombosis, and Vascular Biology
Objective: To determine whether global reduction of CD68 macrophages impacts the development of e... more Objective: To determine whether global reduction of CD68 macrophages impacts the development of experimental pulmonary arterial hypertension (PAH) and whether this reduction affects the balance of pro- and anti-inflammatory macrophages within the lung. Additionally, to determine whether there is evidence of an altered macrophage polarization in patients with PAH. Approach and Results: Macrophage reduction was induced in mice via doxycycline-induced CD68-driven cytotoxic diphtheria toxin A chain expression (macrophage low [MacLow] mice). Chimeric mice were generated using bone marrow transplant. Mice were phenotyped for PAH by echocardiography and closed chest cardiac catheterization. Murine macrophage phenotyping was performed on lungs, bone marrow–derived macrophages, and alveolar macrophages using immunohistochemical and flow cytometry. Monocyte-derived macrophages were isolated from PAH patients and healthy volunteers and polarization capacity assessed morphologically and by flow...
AimsPulmonary hypertension (PH) is associated with significant morbidity and mortality and leads ... more AimsPulmonary hypertension (PH) is associated with significant morbidity and mortality and leads to progressive right heart failure. In patients with PAH, haemodynamic parameters measured at catheterisation relate to clinical worsening events, in patients with heart failure proactive pulmonary artery pressure based therapeutic intervention reduces hospitalisation. We therefore investigated use of a novel implanted pulmonary artery (PA) pressure monitor to detect clinically relevant changes in pressure in large animal models of pulmonary hypertension (PH).Methods and ResultsPrototype pulmonary artery pressure sensors (Endotronix) were implanted using standard interventional techniques. Acute PH was induced by infusion of thromboxane A2 in domestic swine. Over a physiological range pressure monitors remained concordant to reference catheter (bias −0.43, 95%CI-5.3-4.4). Chronic PH was induced by i.p. injection of monocrotaline. Implanted pressure sensors demonstrated a gradual rise in ...
Pulmonary arterial hypertension (PAH) is a rare but fatal disease. Current treatments increase li... more Pulmonary arterial hypertension (PAH) is a rare but fatal disease. Current treatments increase life expectancy but have limited impact on the progressive pulmonary vascular remodelling that drives PAH. Osteoprotegerin (OPG) is increased within serum and lesions of patients with idiopathic PAH and is a mitogen and migratory stimulus for pulmonary artery smooth muscle cells (PASMCs). Here, we report that the pro-proliferative and migratory phenotype in PASMCs stimulated with OPG is mediated via the Fas receptor and that treatment with a human antibody targeting OPG can attenuate pulmonary vascular remodelling associated with PAH in multiple rodent models of early and late treatment. We also demonstrate that the therapeutic efficacy of the anti-OPG antibody approach in the presence of standard of care vasodilator therapy is mediated by a reduction in pulmonary vascular remodelling. Targeting OPG with a therapeutic antibody is a potential treatment strategy in PAH.
Aldosterone is a mineralocorticoid hormone critically involved in arterial blood pressure regulat... more Aldosterone is a mineralocorticoid hormone critically involved in arterial blood pressure regulation. Although pharmacological aldosterone antagonism reduces mortality and morbidity among patients with severe left-sided heart failure, the contribution of aldosterone to the pathobiology of pulmonary arterial hypertension (PAH) and right ventricular (RV) heart failure is not fully understood. The effects of Eplerenone (0.1% Inspra® mixed in chow) on pulmonary vascular and RV remodeling were evaluated in mice with pulmonary hypertension (PH) caused by Sugen5416 injection with concomitant chronic hypoxia (SuHx) and in a second animal model with established RV dysfunction independent from lung remodeling through surgical pulmonary artery banding. Preventive Eplerenone administration attenuated the development of PH and pathological remodeling of pulmonary arterioles. Therapeutic aldosterone antagonism - starting when RV dysfunction was established - normalized mineralocorticoid receptor ...
Idiopathic pulmonary arterial hypertension (IPAH) is increasingly diagnosed in elderly patients w... more Idiopathic pulmonary arterial hypertension (IPAH) is increasingly diagnosed in elderly patients who also have an increased risk of co-morbid atherosclerosis. Apolipoprotein E-deficient (ApoE-/-) mice develop atherosclerosis with severe PAH when fed a high-fat diet (HFD) and have increased levels of endothelin (ET)-1. ET-1 receptor antagonists (ERAs) are used for the treatment of PAH but less is known about whether ERAs are beneficial in atherosclerosis. We therefore examined whether treatment of HFD-ApoE-/- mice with macitentan, a dual ETA/ETB receptor antagonist, would have any effect on both atherosclerosis and PAH. ApoE-/- mice were fed chow or HFD for eight weeks. After four weeks of HFD, mice were randomized to a four-week treatment of macitentan by food (30 mg/kg/day dual ETA/ETB antagonist), or placebo groups. Echocardiography and closed-chest right heart catheterization were used to determine PAH phenotype and serum samples were collected for cytokine analysis. Thoracic aort...
American journal of respiratory and critical care medicine, Jan 13, 2018
Pulmonary arterial hypertension (PAH) is characterized by vascular cell proliferation and endothe... more Pulmonary arterial hypertension (PAH) is characterized by vascular cell proliferation and endothelial cell apoptosis. Toll-like receptor 3 (TLR3) is a receptor for double-stranded RNA and has been recently implicated in vascular protection. The goal was to study the expression and role of TLR3 in PAH and to determine whether a TLR3 agonist reduces Pulmonary Hypertension in preclinical models. Lung tissue and endothelial cells from PAH patients were investigated by polymerase chain reaction, immunofluorescence and apoptosis assays. TLR3 and TLR3 mice were exposed to chronic hypoxia and SU5416. Chronic hypoxia or chronic hypoxia/SU5416 rats were treated with the TLR3 agonist polyinosinic:polycytidylic acid [Poly(I:C)]. TLR3 expression was reduced in PAH patient lung tissue and endothelial cells, and TLR3 mice exhibited more severe Pulmonary Hypertension following exposure to chronic hypoxia/SU5416. TLR3 knockdown promoted double-stranded RNA signaling via other intracellular RNA recep...
Pulmonary arterial hypertension is a devastating disease with high morbidity and mortality and li... more Pulmonary arterial hypertension is a devastating disease with high morbidity and mortality and limited treatment options. Recent studies have shown that pulmonary artery denervation improves pulmonary hemodynamics in an experimental model and in an early clinical trial. We aimed to evaluate the nerve distribution around the pulmonary artery, to determine the effect of radiofrequency pulmonary artery denervation on acute pulmonary hypertension induced by vasoconstriction, and to demonstrate denervation of the pulmonary artery at a histological level. Histological evaluation identified a circumferential distribution of nerves around the proximal pulmonary arteries. Nerves were smaller in diameter, greater in number, and located in closer proximity to the luminal aspect of the pulmonary arterial wall beyond the pulmonary artery bifurcation. To determine the effect of pulmonary arterial denervation acute pulmonary hypertension was induced in 8 pigs by intravenous infusion of thromboxane...
Local drug delivery from polymer-coated coronary stents may reduce the incidence of in-stent rest... more Local drug delivery from polymer-coated coronary stents may reduce the incidence of in-stent restenosis. Angiopeptin, an inhibitor of smooth muscle cell proliferation, may reduce the clinical impact of restenosis. The objectives of this study were to characterize the release kinetics and distribution of angiopeptin-loaded phosphorylcholine (PC)-coated drug delivery (DD) BiodivYsio stents and assess their safety and efficacy at reducing neointima formation. I125-angiopeptin-loaded DD-PC-coated stents were implanted into human saphenous vein segments ex vivo, and I125 angiopeptin was detected in the medial layer at 1 hour. When implanted in pig coronary arteries, I125 angiopeptin was found adjacent to the stent at intervals up to 28 days. No significant amounts were found elsewhere. To assess efficacy, twelve angiopeptin-loaded DD-PC-coated stents, twelve non-loaded DD-PC stents, ten standard PC-coated stents and 8 uncoated stents were implanted into normal porcine coronary arteries. Stents were harvested at 28 days and neointima formation was assessed by computerized morphometry. No adverse tissue reactions were seen with any of the PC-coated stents. No significant differences were seen in neointimal or luminal cross-sectional areas between study groups. Local delivery of I125 angiopeptin into the vascular wall can be achieved using a PC-coated stent. Delivery of angiopeptin from drug delivery PC-coated stents is safe, but does not lead to a significant reduction in neointimal growth at 28 days within the parameters of this study.
To investigate the relative importance of stent induced arterial stretch and deep injury to the d... more To investigate the relative importance of stent induced arterial stretch and deep injury to the development of in-stent neointima. Normal porcine coronary arteries 30 BiodivYsio stents (Biocompatibles) were deployed at a stent to artery ratio of 1.25:1 (a moderate injury) and harvested at 28 days. Multiple serial cross sections were analysed morphometrically and the neointimal areas were correlated with the type and degree of injury. Arterial stretch occurred in 78% of struts (77% of sections) and produced moderate neointimal growth (neointimal area 1.93 (0.13) mm2). Deep injury (rupture of the internal elastic lamina) occurred in 20% of struts (23% of sections) and produced a 1.7-fold increase in neointimal area (3.33 (0.41) mm2) compared with stretch only (p = 0.0002). With even deeper injury (rupture of the external elastic lamina), there was a 2.6-fold increase in neointimal area (5.01 (0.48) mm2) compared with stretch only (p = 0.02). A new injury score, incorporating both stretch and deep injury, correlated with neointimal area (r = 0.60, p &amp;amp;lt; 0.001). Stretch of the coronary artery in a stent is common, and a major contributor to neointima formation, even in the absence of deep injury. Deep injury is, however, a more potent stimulus to neointima formation than stretch. Greater degrees of stretch are associated with thicker neointima. Where neither deep injury nor stretch are seen, the stent has no effect upon the development of neointima.
Pulmonary Arterial Hypertension (PAH) is a life threatening disease characterised by the progress... more Pulmonary Arterial Hypertension (PAH) is a life threatening disease characterised by the progressive narrowing and occlusion of small pulmonary arteries. Current therapies fail to fully reverse this vascular remodelling. Identifying key pathways in disease pathogenesis is therefore required for the development of new-targeted therapeutics. We have previously reported Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) immunoreactivity within pulmonary vascular lesions from patients with idiopathic PAH and animal models. Since TRAIL can induce both endothelial cell apoptosis and smooth muscle cell proliferation in the systemic circulation, we hypothesised that TRAIL is an important mediator in the pathogenesis of PAH. We demonstrate here for the first time that TRAIL is a potent stimulus for pulmonary vascular remodelling in human cells, and rodent models. Furthermore antibody blockade or genetic deletion of TRAIL prevents the development of PAH in three independent...
Introduction Pulmonary arterial hypertension (PAH) is a devastating disease with a high mortality... more Introduction Pulmonary arterial hypertension (PAH) is a devastating disease with a high mortality and prognosis worse than many cancers. Pathologically, PAH is characterised by progressive arteriole remodelling driven by pulmonary artery smooth muscle cell (PASMC) proliferation and migration. Although current treatments alleviate symptoms, they do not reverse the underlying progressive pulmonary vascular proliferation. We have previously shown that the secreted glycoprotein osteoprotegerin (OPG, TNFRSF11B) is increased within pulmonary vascular lesions and serum from patients with idiopathic PAH, and promotes the proliferation and migration of PASMCs in vitro . Recent experiments have shown that administration of an anti-OPG antibody can prevent and reverse PAH in rodent models of disease. However, how OPG signals to mediate PASMC phenotype remains unknown. We hypothesise that OPG mediates these effects through a previously undescribed cell surface receptor. We aim to identify this receptor on PASMC and characterise the OPG signalling cascade leading to the proliferative phenotype. Methods Quiesced PASMCs (Lonza, Basel, Switzerland) were stimulated with 0.2% FCS (negative), and OPG (50 ng/ml) for 10 and 60 min. Phosphorylation targets were identified from protein lysates by Kinex antibody microarray (Kinexus, Canada). Selected targets were verified by western blotting. Transcriptomic analysis of PASMCs stimulated with OPG for 6 h was performed using an RNA expression microarray (Agilent) and confirmed by TaqMan RT-PCR. Novel OPG binding proteins were identified following reverse transfection of HEK293 cells with 2054 human membrane proteins (Retrogenix, Sheffield, UK) and confirmed in PASMC by co-immunoprecipitation. To assess the effect of Fas blockade, proliferation was assessed in PASMCs pre-incubated with Fas neutralising antibody (500 ng/ml) 30 min before 72h stimulation with OPG. Results OPG stimulation of PASMC resulted in significant activation of CDK4 and 5, HSP27 and ERK1/2, and significant decrease in phospho-mTOR. OPG increased TRAIL, PDGFRA, TNC, Cav-1 and reduced VIP receptor gene expression. Four novel OPG interactions with IL1RAP, Fas, TMPRSS11D and GAP43 were identified by the Retrogenix cell microarray. We have confirmed OPG interaction with IL1RAP and Fas in PASMC by co-immunoprecipitation. Fas protein expression is elevated in the pulmonary artery and right ventricle of IPAH patients. Fas RNA expression is increased in PASMCs from IPAH patient lungs. Furthermore, blocking Fas with a neutralising antibody reduces OPG-induced proliferation, PDGFRA and TNC RNA expression. Conclusions These data highlight novel binding partners for OPG. In particular the OPG-FAS interaction regulates a diverse and important intracellular signalling cascade by which OPG regulates proliferation, apoptosis and autophagy proteins, and PAH associated genes in PASMC. These data further highlight therapeutic potential of targeting OPG in PAH.
Catheterization and Cardiovascular Interventions, 2007
While several endovascular techniques have been developed for treating arterial bifurcation lesio... more While several endovascular techniques have been developed for treating arterial bifurcation lesions, there is, as yet, no single, widely accepted technique for treating left main stem (LMS) bifurcation lesions with stents. The simultaneous kissing stent (SCS) technique seems particularly suited for such lesions. The authors describe a consecutive cohort of patients with LMS bifurcation stenosis treated with this technique and present mechanistic insights from a porcine model. Thirty consecutive patients with LMS bifurcation stenosis +/- multivessel disease were treated with SCS technique using paclitaxel-eluting stents. The technique involves simultaneous implantation of two stents (LMS-LAD and LMS-Cx) so that the proximal end of both stents lie at the same level in the body of LMS. Symptom status was recorded and follow-up angiography was performed. In addition, four pigs underwent SCS implantation of the LMS and were studied by direct examination, histology, and scanning electron microscopy. The patients&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; mean age was 63, 26 were elective and 19 were male. Paclitaxel-eluting stents (Taxus, Boston Scientific) were used. There was one in-hospital death in a highly unstable patient. At 6-month follow-up angiography, two patients required target lesion revascularization. At 13 +/- 3 month follow-up, the remaining patients remain well, with no other major adverse cardiac events. The animals all survived with patent stents. SEM demonstrated full re-endothelialization and histology showed a thin but complete neointima covering the stent struts. The SCS technique for treating LMS bifurcation disease with paclitaxel-eluting stents is safe and feasible. Medium term clinical and angiographic results are good. An endothelium and a thin neointima cover the metal struts.
Objective: Restenosis following angioplasty involves processes that may be influenced by local pr... more Objective: Restenosis following angioplasty involves processes that may be influenced by local production of cytokines. We investigated the expression of active and total transforming growth factor b (TGFb) following porcine coronary angioplasty (PTCA), and have correlated this with the expression of potential in vivo activators of TGFb: mannose-6-phosphate / insulin-like growth factor-II (M6P/ IGF-II) receptor and thrombospondin-1. Methods: Oversized porcine PTCA was performed and the arteries excised after selected intervals. Levels of in situ active and total (active plus latent) TGFb were determined using a modified plasminogen activator-inhibitor / luciferase bioassay. Results: Levels of active TGFb significantly increased 2 h to 7 days after angioplasty, compared to non-injured controls. Levels returned to baseline by 28 days. Active TGFb in tissues adjacent to the injured artery did not change. Total TGFb was significantly higher than controls 2-6 h after injury. M6P/ IGF-II receptor mRNA was upregulated between 6 h and 3 days after injury, with protein detectable at 3-28 days. Thrombospondin-1 was detected between 1 h and 14 days. Conclusions: We conclude that balloon injury causes an early rapid increase in levels of active TGFb, that correlates with the expression of TGFb activators. Thus, TGFb is a good potential target for anti-restenotic therapies.
Objective: To determine the influence of IL-1 on the arterial response to experimental injury in ... more Objective: To determine the influence of IL-1 on the arterial response to experimental injury in porcine models of percutaneous coronary intervention (PCI). Methods: An intravenous (i.v.) bolus of 0.5 mg/kg followed by a subcutaneous (s.c.) infusion of 2 mg/kg/24 h of human IL-1 receptor antagonist (IL-1ra) inhibited neutrophil recruitment in response to intradermal IL-1. Using this dose regimen, five groups of pigs were studied: Group 1, oversized balloon angioplasty of 2 coronary vessels (14-day infusion, 28th day sacrifice and analysis); Groups 2, 3, 4, and 5, oversized stenting of 2 coronary vessels (Group 2: 14-day infusion, 28th day analysis; Group 3: 14-day infusion, 14th day analysis; Group 4: 28-day infusion, 28th day analysis; Group 5: 28-day infusion, 90th day analysis). Neointimal area was quantified by standard means. Results: In Group 1, IL-1ra resulted in a 23% decrease in neointimal area (p = 0.04); in Group 2, a 34% increase (p = 0.001); in Group 3, a 38% decrease (p < 0.0001); in Group 4, a 34% decrease (p = 0.0004); and in Group 5, a 41% decrease (p = 0.00001). Conclusions: IL-1ra was associated with a sustained, significant reduction in neointima after vessel wall injury as long as it is given for the duration of the stimulation of the IL-1 system, in this case at least 28 days. This suggests that therapies based on the antagonism of IL-1 may modulate the coronary artery response to injury.
Inflammatory mechanisms are proposed to play a significant role in the pathogenesis of pulmonary ... more Inflammatory mechanisms are proposed to play a significant role in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have described PAH in fat-fed apolipoprotein E knockout (ApoE ؊/؊) mice. We have reported that signaling in interleukin-1-receptor-knockout (IL-1R1 ؊/؊) mice leads to a reduction in diet-induced systemic atherosclerosis. We subsequently hypothesized that doublenull (ApoE ؊/؊ /IL-1R1 ؊/؊) mice would show a reduced PAH phenotype compared with that of ApoE ؊/؊ mice. Male IL-1R1 ؊/؊ , ApoE ؊/؊ , and ApoE ؊/؊ /IL-1R1 ؊/؊ mice were fed regular chow or a high-fat diet (Paigen diet) for 8 weeks before phenotyping for PAH. No abnormal phenotype was observed in the IL-1R1 ؊/؊ mice. Fat-fed ApoE ؊/؊ mice developed significantly increased right ventricular systolic pressure and substantial pulmonary vascular remodeling. Surprisingly, ApoE ؊/؊ /IL-1R1 ؊/؊ mice showed an even more severe PAH phenotype. Further molecular investigation revealed the expression of a putative, alternatively primed IL-1R1 transcript expressed within the lungs but not aorta of ApoE ؊/؊ /IL-1R1 ؊/؊ mice. Treatment of ApoE ؊/؊ and ApoE ؊/؊ /IL-1R1 ؊/؊ mice with IL-1-receptor antagonist prevented progression of the PAH phenotype in both strains. Blocking IL-1 signaling may have beneficial effects in treating PAH, and alternative IL-1-receptor signaling in the lung may be important in driving PAH pathogenesis.
Development of the atherosclerotic plaque involves a complex interplay between a number of cell t... more Development of the atherosclerotic plaque involves a complex interplay between a number of cell types and an extensive inter-cellular communication via cell bound as well as soluble mediators. The family of tribbles proteins has recently been identified as novel controllers of pro-inflammatory signal transduction. The objective of this study was to address the expression pattern of all three tribbles proteins in atherosclerotic plaques from a mouse model of atherosclerosis. Each tribbles were expressed in vascular smooth muscle cells, endothelial cells as well as in resident macrophages of mouse atherosclerotic plaques. The role of IL-1 mediated inflammatory events in controlling tribbles expression was also addressed by inducing experimental atherosclerosis in ApoE í/í IL1R1 í/í (double knockout) mice. Immunohistochemical analysis of these mice showed a selective decrease in the percentage of trb-1 expressing macrophages, compared to the ApoE í/í cohort (14.7% r 1.55 vs. 26.3% r 1.19). The biological significance of this finding was verified in vitro where overexpression of trb-1 in macrophages led to a significant attenuation (~70%) of IL-6 production as well as a suppressed IL-12 expression induced by a proinflammatory stimulus. In this in vitro setting, expression of truncated trb-1 mutants suggests that the kinase domain of this protein is sufficient to exert this inhibitory action.
The Journal of clinical investigation, Jul 23, 2016
Loss of the growth-suppressive effects of bone morphogenetic protein (BMP) signaling has been dem... more Loss of the growth-suppressive effects of bone morphogenetic protein (BMP) signaling has been demonstrated to promote pulmonary arterial endothelial cell dysfunction and induce pulmonary arterial smooth muscle cell (PASMC) proliferation, leading to the development of pulmonary arterial hypertension (PAH). MicroRNAs (miRs) mediate higher order regulation of cellular function through coordinated modulation of mRNA targets; however, miR expression is altered by disease development and drug therapy. Here, we examined treatment-naive patients and experimental models of PAH and identified a reduction in the levels of miR-140-5p. Inhibition of miR-140-5p promoted PASMC proliferation and migration in vitro. In rat models of PAH, nebulized delivery of miR-140-5p mimic prevented the development of PAH and attenuated the progression of established PAH. Network and pathway analysis identified SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) as a key miR-140-5p target and regulator of BMP si...
Pulmonary arterial hypertension (PAH) is a progressive lung disease diagnosed by an increase in p... more Pulmonary arterial hypertension (PAH) is a progressive lung disease diagnosed by an increase in pulmonary arterial blood pressure that is driven by a progressive vascular remodelling of small pulmonary arterioles. We have previously reported that tumor necrosis factor apoptosis-inducing ligand (TRAIL) protein expression is increased in pulmonary vascular lesions and pulmonary artery smooth muscle cells (PASMC) of patients with idiopathic PAH. The addition of recombinant TRAIL induces the proliferation and migration of PASMCs in vitro. TRAIL is required for hypoxia-induced pulmonary hypertension in mice, and blockade of TRAIL prevents and reduces disease development in other rodent models of PAH. Due to the availability of knockout and transgenic mice, murine models of disease are key to further advances in understanding the complex and heterogeneous pathogenesis of PAH. However, murine models vary in their disease severity, and are often criticized for lacking the proliferative pulmonary vascular lesions characteristic of PAH. The murine Sugen-hypoxic (SuHx) mouse model has recently been reported to have a more severe PAH phenotype consisting advanced pulmonary vascular remodelling. We therefore aimed to determine whether TRAIL was also required for the development of PAH in this model. C57BL/6 and TRAIL −/− mice were exposed to normoxia, Sugen5416 alone, hypoxia or both Sugen5416 and hypoxia (SuHx). We report here that SuHx treated C57BL/6 mice developed more severe PAH than OPEN ACCESS Diseases 2014, 2 261 hypoxia alone, and that TRAIL −/− mice were protected from disease development. These data further emphasise the importance of this pathway and support the use of the SuHx mouse model for investigating the importance of potential mediators in PAH pathogenesis.
Arteriosclerosis, Thrombosis, and Vascular Biology
Objective: To determine whether global reduction of CD68 macrophages impacts the development of e... more Objective: To determine whether global reduction of CD68 macrophages impacts the development of experimental pulmonary arterial hypertension (PAH) and whether this reduction affects the balance of pro- and anti-inflammatory macrophages within the lung. Additionally, to determine whether there is evidence of an altered macrophage polarization in patients with PAH. Approach and Results: Macrophage reduction was induced in mice via doxycycline-induced CD68-driven cytotoxic diphtheria toxin A chain expression (macrophage low [MacLow] mice). Chimeric mice were generated using bone marrow transplant. Mice were phenotyped for PAH by echocardiography and closed chest cardiac catheterization. Murine macrophage phenotyping was performed on lungs, bone marrow–derived macrophages, and alveolar macrophages using immunohistochemical and flow cytometry. Monocyte-derived macrophages were isolated from PAH patients and healthy volunteers and polarization capacity assessed morphologically and by flow...
AimsPulmonary hypertension (PH) is associated with significant morbidity and mortality and leads ... more AimsPulmonary hypertension (PH) is associated with significant morbidity and mortality and leads to progressive right heart failure. In patients with PAH, haemodynamic parameters measured at catheterisation relate to clinical worsening events, in patients with heart failure proactive pulmonary artery pressure based therapeutic intervention reduces hospitalisation. We therefore investigated use of a novel implanted pulmonary artery (PA) pressure monitor to detect clinically relevant changes in pressure in large animal models of pulmonary hypertension (PH).Methods and ResultsPrototype pulmonary artery pressure sensors (Endotronix) were implanted using standard interventional techniques. Acute PH was induced by infusion of thromboxane A2 in domestic swine. Over a physiological range pressure monitors remained concordant to reference catheter (bias −0.43, 95%CI-5.3-4.4). Chronic PH was induced by i.p. injection of monocrotaline. Implanted pressure sensors demonstrated a gradual rise in ...
Pulmonary arterial hypertension (PAH) is a rare but fatal disease. Current treatments increase li... more Pulmonary arterial hypertension (PAH) is a rare but fatal disease. Current treatments increase life expectancy but have limited impact on the progressive pulmonary vascular remodelling that drives PAH. Osteoprotegerin (OPG) is increased within serum and lesions of patients with idiopathic PAH and is a mitogen and migratory stimulus for pulmonary artery smooth muscle cells (PASMCs). Here, we report that the pro-proliferative and migratory phenotype in PASMCs stimulated with OPG is mediated via the Fas receptor and that treatment with a human antibody targeting OPG can attenuate pulmonary vascular remodelling associated with PAH in multiple rodent models of early and late treatment. We also demonstrate that the therapeutic efficacy of the anti-OPG antibody approach in the presence of standard of care vasodilator therapy is mediated by a reduction in pulmonary vascular remodelling. Targeting OPG with a therapeutic antibody is a potential treatment strategy in PAH.
Aldosterone is a mineralocorticoid hormone critically involved in arterial blood pressure regulat... more Aldosterone is a mineralocorticoid hormone critically involved in arterial blood pressure regulation. Although pharmacological aldosterone antagonism reduces mortality and morbidity among patients with severe left-sided heart failure, the contribution of aldosterone to the pathobiology of pulmonary arterial hypertension (PAH) and right ventricular (RV) heart failure is not fully understood. The effects of Eplerenone (0.1% Inspra® mixed in chow) on pulmonary vascular and RV remodeling were evaluated in mice with pulmonary hypertension (PH) caused by Sugen5416 injection with concomitant chronic hypoxia (SuHx) and in a second animal model with established RV dysfunction independent from lung remodeling through surgical pulmonary artery banding. Preventive Eplerenone administration attenuated the development of PH and pathological remodeling of pulmonary arterioles. Therapeutic aldosterone antagonism - starting when RV dysfunction was established - normalized mineralocorticoid receptor ...
Idiopathic pulmonary arterial hypertension (IPAH) is increasingly diagnosed in elderly patients w... more Idiopathic pulmonary arterial hypertension (IPAH) is increasingly diagnosed in elderly patients who also have an increased risk of co-morbid atherosclerosis. Apolipoprotein E-deficient (ApoE-/-) mice develop atherosclerosis with severe PAH when fed a high-fat diet (HFD) and have increased levels of endothelin (ET)-1. ET-1 receptor antagonists (ERAs) are used for the treatment of PAH but less is known about whether ERAs are beneficial in atherosclerosis. We therefore examined whether treatment of HFD-ApoE-/- mice with macitentan, a dual ETA/ETB receptor antagonist, would have any effect on both atherosclerosis and PAH. ApoE-/- mice were fed chow or HFD for eight weeks. After four weeks of HFD, mice were randomized to a four-week treatment of macitentan by food (30 mg/kg/day dual ETA/ETB antagonist), or placebo groups. Echocardiography and closed-chest right heart catheterization were used to determine PAH phenotype and serum samples were collected for cytokine analysis. Thoracic aort...
American journal of respiratory and critical care medicine, Jan 13, 2018
Pulmonary arterial hypertension (PAH) is characterized by vascular cell proliferation and endothe... more Pulmonary arterial hypertension (PAH) is characterized by vascular cell proliferation and endothelial cell apoptosis. Toll-like receptor 3 (TLR3) is a receptor for double-stranded RNA and has been recently implicated in vascular protection. The goal was to study the expression and role of TLR3 in PAH and to determine whether a TLR3 agonist reduces Pulmonary Hypertension in preclinical models. Lung tissue and endothelial cells from PAH patients were investigated by polymerase chain reaction, immunofluorescence and apoptosis assays. TLR3 and TLR3 mice were exposed to chronic hypoxia and SU5416. Chronic hypoxia or chronic hypoxia/SU5416 rats were treated with the TLR3 agonist polyinosinic:polycytidylic acid [Poly(I:C)]. TLR3 expression was reduced in PAH patient lung tissue and endothelial cells, and TLR3 mice exhibited more severe Pulmonary Hypertension following exposure to chronic hypoxia/SU5416. TLR3 knockdown promoted double-stranded RNA signaling via other intracellular RNA recep...
Pulmonary arterial hypertension is a devastating disease with high morbidity and mortality and li... more Pulmonary arterial hypertension is a devastating disease with high morbidity and mortality and limited treatment options. Recent studies have shown that pulmonary artery denervation improves pulmonary hemodynamics in an experimental model and in an early clinical trial. We aimed to evaluate the nerve distribution around the pulmonary artery, to determine the effect of radiofrequency pulmonary artery denervation on acute pulmonary hypertension induced by vasoconstriction, and to demonstrate denervation of the pulmonary artery at a histological level. Histological evaluation identified a circumferential distribution of nerves around the proximal pulmonary arteries. Nerves were smaller in diameter, greater in number, and located in closer proximity to the luminal aspect of the pulmonary arterial wall beyond the pulmonary artery bifurcation. To determine the effect of pulmonary arterial denervation acute pulmonary hypertension was induced in 8 pigs by intravenous infusion of thromboxane...
Local drug delivery from polymer-coated coronary stents may reduce the incidence of in-stent rest... more Local drug delivery from polymer-coated coronary stents may reduce the incidence of in-stent restenosis. Angiopeptin, an inhibitor of smooth muscle cell proliferation, may reduce the clinical impact of restenosis. The objectives of this study were to characterize the release kinetics and distribution of angiopeptin-loaded phosphorylcholine (PC)-coated drug delivery (DD) BiodivYsio stents and assess their safety and efficacy at reducing neointima formation. I125-angiopeptin-loaded DD-PC-coated stents were implanted into human saphenous vein segments ex vivo, and I125 angiopeptin was detected in the medial layer at 1 hour. When implanted in pig coronary arteries, I125 angiopeptin was found adjacent to the stent at intervals up to 28 days. No significant amounts were found elsewhere. To assess efficacy, twelve angiopeptin-loaded DD-PC-coated stents, twelve non-loaded DD-PC stents, ten standard PC-coated stents and 8 uncoated stents were implanted into normal porcine coronary arteries. Stents were harvested at 28 days and neointima formation was assessed by computerized morphometry. No adverse tissue reactions were seen with any of the PC-coated stents. No significant differences were seen in neointimal or luminal cross-sectional areas between study groups. Local delivery of I125 angiopeptin into the vascular wall can be achieved using a PC-coated stent. Delivery of angiopeptin from drug delivery PC-coated stents is safe, but does not lead to a significant reduction in neointimal growth at 28 days within the parameters of this study.
To investigate the relative importance of stent induced arterial stretch and deep injury to the d... more To investigate the relative importance of stent induced arterial stretch and deep injury to the development of in-stent neointima. Normal porcine coronary arteries 30 BiodivYsio stents (Biocompatibles) were deployed at a stent to artery ratio of 1.25:1 (a moderate injury) and harvested at 28 days. Multiple serial cross sections were analysed morphometrically and the neointimal areas were correlated with the type and degree of injury. Arterial stretch occurred in 78% of struts (77% of sections) and produced moderate neointimal growth (neointimal area 1.93 (0.13) mm2). Deep injury (rupture of the internal elastic lamina) occurred in 20% of struts (23% of sections) and produced a 1.7-fold increase in neointimal area (3.33 (0.41) mm2) compared with stretch only (p = 0.0002). With even deeper injury (rupture of the external elastic lamina), there was a 2.6-fold increase in neointimal area (5.01 (0.48) mm2) compared with stretch only (p = 0.02). A new injury score, incorporating both stretch and deep injury, correlated with neointimal area (r = 0.60, p &amp;amp;lt; 0.001). Stretch of the coronary artery in a stent is common, and a major contributor to neointima formation, even in the absence of deep injury. Deep injury is, however, a more potent stimulus to neointima formation than stretch. Greater degrees of stretch are associated with thicker neointima. Where neither deep injury nor stretch are seen, the stent has no effect upon the development of neointima.
Pulmonary Arterial Hypertension (PAH) is a life threatening disease characterised by the progress... more Pulmonary Arterial Hypertension (PAH) is a life threatening disease characterised by the progressive narrowing and occlusion of small pulmonary arteries. Current therapies fail to fully reverse this vascular remodelling. Identifying key pathways in disease pathogenesis is therefore required for the development of new-targeted therapeutics. We have previously reported Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) immunoreactivity within pulmonary vascular lesions from patients with idiopathic PAH and animal models. Since TRAIL can induce both endothelial cell apoptosis and smooth muscle cell proliferation in the systemic circulation, we hypothesised that TRAIL is an important mediator in the pathogenesis of PAH. We demonstrate here for the first time that TRAIL is a potent stimulus for pulmonary vascular remodelling in human cells, and rodent models. Furthermore antibody blockade or genetic deletion of TRAIL prevents the development of PAH in three independent...
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