J Pickworth

Inhibition of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) reverses experimental pulmonary hypertension.

Genetic deletion of TRAIL or antibody blockade prevents the development of pulmonary arterial hypertension and can reverse vascular remodeling in established disease.

MicroRNA-140-5p and SMURF1 regulate pulmonary arterial hypertension

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 signaling. Evaluation of human tissue revealed that SMURF1 is increased in patients with PAH. miR-140-5p mimic or SMURF1 knockdown in PASMCs altered BMP signaling, further supporting these factors as regulators of BMP signaling. Finally, Smurf1 deletion protected mice from PAH, demonstrating a critical role in disease development. Together, these studies identify both miR-140-5p and SMURF1 as key regulators of disease pathology and as potential therapeutic targets for the treatment of PAH.

Serum osteoprotegerin is increased and predicts survival in idiopathic pulmonary arterial hypertension

We previously reported that osteoprotegerin (OPG) is regulated by pathways associated with pulmonary arterial hypertension (PAH), and is present at elevated levels within pulmonary vascular lesions and sera from patients with idiopathic PAH (IPAH). Since OPG is a naturally secreted protein, we investigated the relationship between serum OPG and disease severity and outcome in patients with IPAH and animal models. OPG mRNA expression was measured in pulmonary artery smooth muscle cells (PASMC) from pulmonary arteries of patients with and without IPAH. Serum concentrations of OPG were measured in a retrospective and prospective group of patients. OPG levels were compared with phenotypic data and other putative PAH biomarkers. Prognostic significance was assessed and levels compared with healthy controls. Correlation of OPG and pulmonary vascular remodeling was also performed in rodent models of PAH. OPG mRNA was significantly increased 2-fold in PASMC isolated from explanted PAH lungs compared with control. Serum OPG concentrations were markedly elevated in IPAH compared with controls. In Cohort 1 OPG levels significantly correlated with mean right atrial pressure and cardiac index, while in Cohort 2 significant correlations existed between age-adjusted OPG levels and gas transfer. In both cohorts an OPG concentration above a ROC-derived threshold of 4728 pg/ml predicted poorer survival. In two rodent models, OPG correlated with the degree of pulmonary vascular remodeling. OPG levels are significantly elevated in patients with idiopathic PAH and are of prognostic significance. The role of OPG as a potential biomarker and therapeutic target merits further investigation.

T5 MicroRNA-140–5p Regulates Disease Phenotype in Experimental Pulmonary Arterial Hypertension via SMURF1

Introduction and objectives Clinical therapies for the treatment of pulmonary arterial hypertension (PAH) target vasoconstriction. However, the proliferative pulmonary vascular remodelling that drives disease persists contributing to significant patient morbidity and mortality. MicroRNA (miR) are short non-coding RNA that mediate post-transcriptional regulation of mRNA targets. We hypothesise that dysregulation of miR leads to de-repression of cellular targets central to disease pathogenesis. We sought to identify dysregulated circulating miR in patients with PAH, determine their phenotypic effect using in vitro and in vivo models and identify key mechanistic regulators that may represent novel therapeutic targets. Methods Two patient cohorts were used to identify and validate differential expression of miR in whole blood by microarray and single assay qPCR. Binding site and network analysis was used to identify key miR targets. Effect of miR on identified targets and disease phenotype was determined in pulmonary artery smooth muscle cells (PASMC) and in the monocrotaline (MCT) and Sugen5416 plus Hypoxia (SuHx) models of PAH. Results Expression of miR-140–5p was reduced in whole blood samples from patients with PAH and experimental models of PAH. Network and pathway analysis identified key regulators of TGFß and PDGF signalling as miR-140–5p targets. Transfection with miR-140–5p inhibitor resulted in increased proliferation and migration of PASMC and de-repression of key targets. Nebulised delivery of miR-140–5p mimic prevented the development of PAH in the MCT rat model and attenuated progression of established PAH in MCT and SuHx rat models. In experimental models levels of SMURF1 protein correlated inversely with miR-140–5p. Direct regulation of SMURF1 by miR-140–5p was demonstrated in vitro by 3’UTR luciferase activity. Both miR-140–5p mimic and SMURF1 siRNA increased BMP response element activity identifying SMURF1 as a key negative regulator of BMP signalling in PASMC. Genetic ablation of SMURF1 in C57BL6 mice conferred allele dependent protection from SuHx induced PAH. Finally, whole blood mRNA and pulmonary vascular immunoreactivity of SMURF1 was increased in patients with PAH. Conclusions These studies suggest that miR-140–5p and SMURF1 are key regulators of BMP signalling and disease pathology in PAH and highlight SMURF1 as a potential novel therapeutic target.

Eplerenone attenuates pathological pulmonary vascular rather than right ventricular remodeling in pulmonary arterial hypertension

BackgroundAldosterone 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.MethodsThe 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.ResultsPreventive 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 gene expression in the right ventricle without direct effects on either RV structure (Cardiomyocyte hypertrophy, Fibrosis) or function (assessed by non-invasive echocardiography along with intra-cardiac pressure volume measurements), but significantly lowered systemic blood pressure.ConclusionsOur data indicate that aldosterone antagonism with Eplerenone attenuates pulmonary vascular rather than RV remodeling in PAH.

E microRNA-140-5p and SMURF1 Regulate Pulmonary Arterial Hypertension

Background Clinical therapies for the treatment of pulmonary arterial hypertension (PAH) target vasoconstriction. However, the proliferative pulmonary vascular remodelling that drives disease persists contributing to significant patient morbidity and mortality. MicroRNA (miR) are short non-coding RNA that mediate post-transcriptional regulation of mRNA targets. We hypothesise that dysregulation of miR leads to de-repression of cellular targets central to disease pathogenesis. Objective To identify dysregulated circulating miR in patients with PAH, determine their phenotypic effect using in vitro and in vivo models and identify key mechanistic regulators that may represent novel therapeutic targets. Methods and results Expression of miR-140-5p was reduced in whole blood samples from patients with PAH. Reduced expression of miR-140-5p at the time of diagnosis identified patients with adverse clinical characteristics. Transfection with miR-140-5p inhibitor resulted in increased proliferation PASMC and de-repression of key targets. Nebulised delivery of miR-140-5p mimic attenuated progression of established PAH in the SuHx rat model. Network and pathway analysis identified SMAD Specific E3 Ubiquitin Protein Ligase 1 (SMURF1) as a key miR-140-5p target. Whole blood mRNA and pulmonary vascular immunoreactivity of SMURF1 was increased in patients with PAH implicating SMURF1 in the pathology of human disease. Direct regulation of SMURF1 by miR-140-5p was demonstrated in-vitro by 3’UTR luciferase activity. Both miR-140-5p mimic and SMURF1 siRNA increased BMP response element activity identifying SMURF1 as a key negative regulator of BMP signalling in PASMC. Genetic ablation of SMURF1 in C57BL6 mice conferred allele dependent protection from SuHx induced PAH. Conclusions These studies suggest that miR-140 and SMURF1 are key regulators of BMP signalling and disease pathology in PAH and highlight SMURF1 as a potential novel therapeutic target.

T6 TRAIL is a potential novel therapeutic target in pulmonary arterial hypertension

Introduction and Objectives Pulmonary Arterial Hypertension (PAH) is a life threatening disease characterised by the progressive narrowing and occlusion of small pulmonary arteries, driven by the dysregulated growth of vascular cells. Current therapies are unable to reverse PAH and so identifying key pathways in disease pathogenesis should permit the development of more targeted therapeutics. The cytokine, Tumour Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) induces EC apoptosis and SMC proliferation in the systemic circulation, but hitherto has not been studied in PAH. We recently determined that TRAIL is a mitogen for human PA-SMCs in-vitro and was associated with pulmonary vascular lesions in humans and rodent models. We thus hypothesised that TRAIL is an important mediator in the pathogenesis of PAH and now describe the potential therapeutic benefits of targeting TRAIL in-vivo using two animal models. Methods To test whether blocking TRAIL could prevent the development of PAH, an anti-TRAIL antibody was delivered via osmotic mini-pump coincident with the induction of PAH in the MCT rat model. We also tested whether genetic deletion of TRAIL (ApoE−/−/TRAIL−/− mice) would confer protection to diet-induced PAH. To test whether inhibiting TRAIL could reverse established disease we again treated both models with an anti-TRAIL antibody starting from day 21 in the rat and 8 weeks in the ApoE−/− mouse. Phenotyping included echocardiography, closed chest cardiac catheterisation followed by immuno-histological and biochemical analyses of the lung. Results Antibody blockade (MCT) and genetic deletion (ApoE−/−) of TRAIL prevented the development of PAH in both models. Interestingly a PAH disease phenotype was restored in ApoE−/−/TRAIL−/− mice by the administration of recombinant TRAIL. In rodents with established PAH, an anti-TRAIL antibody, significantly increased survival and reduced pulmonary vascular remodelling in the fatal rat MCT model (p<0.05 cf control). In the murine model, an anti-TRAIL antibody treatment reversed both haemodynamics (RVSP 27 mm Hg vs 88 mm Hg, p<0.001) and pulmonary vascular remodelling. Conclusions Our preclinical investigations are the first to determine the importance of TRAIL to disease pathogenesis and highlight its potential as a novel and rational target to direct future translational therapies for PAH.

Differential IL-1 signaling induced by BMPR2 deficiency drives pulmonary vascular remodeling.

Bone morphogenetic protein receptor type 2 (BMPR2) mutations are present in patients with heritable and idiopathic pulmonary arterial hypertension (PAH). Circulating levels of interleukin-1 (IL-1) are raised in patients and animal models. Whether interplay between BMP and IL-1 signaling can explain the local manifestation of PAH in the lung remains unclear. Cell culture, siRNA, and mRNA microarray analysis of RNA isolated from human pulmonary artery (PASMC) and aortic (AoSMC) smooth muscle cells were used. R899X+/– BMPR2 transgenic mice fed a Western diet for six weeks were given daily injections of IL-1ß prior to assessment for PAH and tissue collection. PASMC have reduced inflammatory activation in response to IL-1ß compared with AoSMCs; however, PASMC with reduced BMPR2 demonstrated an exaggerated response. Mice treated with IL-1ß had higher white blood cell counts and significantly raised serum protein levels of IL-6 and osteoprotegerin (OPG) plasma levels recapitulating in vitro data. Phenotypically, IL-1ß treated mice demonstrated increased pulmonary vascular remodeling. IL-1ß induces an exaggerated pulmonary artery specific transcriptomic inflammatory response when BMPR2 signaling is reduced.

DEFICIENCY OF TOLL-LIKE RECEPTOR 3 (TLR3) EXACERBATES PULMONARY HYPERTENSION IN MICE

Introduction The mechanisms regulating aberrant vascular remodelling in pulmonary arterial hypertension (PAH) are poorly understood and treatments targeted at halting or reversing this process are lacking. Toll-like receptor 3 (TLR3) is a viral sensor and more recently has been established as a sensor of endogenous damage signals, responding to mRNA released by damaged cells. TLR3 signalling induces pro- and anti-inflammatory cytokine production and regulates inflammation-associated apoptosis and tyrosine kinase signalling. In a model of systemic arterial injury, TLR3 signalling was shown to modulate neointimal remodelling in a protective manner. TLR3 is also expressed in pulmonary artery smooth muscle (PASMCs) and endothelial cells (PAECs). We therefore hypothesised that TLR3 would play roles in pulmonary vascular remodelling. Methods TLR3-deficient (TLR3−/−) or wild-type C57BL/6 (WT) mice were exposed to hypoxia (10% Oxygen) and given Sugen 5416 (weekly 20 mg/kg subcutaneous injections) or maintained in normoxic conditions for 3 weeks. Haemodynamic (cardiac catheterisation and echocardiography) and histological assessments were performed after 3 weeks. Human PASMCs were serum-starved before stimulation with PDGF or poly(I:C) and proliferation was assessed after 72 hours. Results TLR3−/− mice developed a markedly exaggerated phenotype of PAH in response to Sugen/Hypoxia with increased right ventricular systolic pressures (WT 51.6 mmHg ± 4.6 vs. TLR3−/− 73.0 mmHg ± 6.8; p < 0.05, mean ± SEM, n = 6), increased muscularisation of small pulmonary arteries and reduced right ventricular cardiac output (WT 424.2 RVUmin-1 ± 84.2 vs. TLR3−/− 283.3 RVUmin-1 ± 18.4, mean ± SEM, min n = 6) after 3 weeks. Poly(I:C) suppressed PDGF-induced PASMC proliferation in a dose-dependent manner. Conclusions We have shown that mice deficient in TLR3 develop a markedly exaggerated haemodynamic pulmonary hypertension phenotype and human PASMC proliferation is suppressed by the TLR3 ligand, poly(I:C). Together these data imply that TLR3 signalling in disease mediates a protective phenotype in keeping with that observed in systemic vascular remodelling, and identify a protective pathway potentially amenable to therapeutic targeting.

S85 Reduction of CD68 macrophages causes gender specific spontaneous pulmonary arterial hypertension in mice

Introduction Macrophages are proposed to play an important regulatory role in the pathogenesis of pulmonary arterial hypertension (PAH) as excessive infiltration detected around vascular lesions in patients and animal models. The exact ‘causal’ role for macrophages, and whether their presence or absence is required for the vascular remodelling seen in PAH remains unclear. Objectives Using a novel inducible macrophage depletion model (MacLow mouse) we aimed to determine the role of macrophages in pulmonary arterial remodelling associated with PAH. Methods Macrophage depletion was induced in MacLow mice by administration of doxycycline, where macrophage-specific induction of the cytotoxic diphtheria toxin A chain (DTA) is driven by the CD68 promoter. Mice were phenotyped for PAH by echocardiography, closed chest cardiac catheterization and immunohistochemistry (IHC) after 6 weeks. To investigate the origin of the effector cells, male chimeric mice were generated, and the disease stimulated by inducing macrophage ablation with doxycycline. Furthermore, to study gender-specificity of the disease phenotype, MacLow mixed gender chimeric mice were produced, and macrophage ablation induced as previous. Results Interestingly male but not female MacLow mice developed a PAH phenotype compared to controls (RVSP of 66.1 mmHg vs 24.5 mmHg, p < 0.0001, n = 5–8), associated with increased right ventricular Hypertrophy (RVH 0.264 vs 0.226, p < 0.001, n = 8) and pulmonary vascular remodelling. IHC analysis of diseased lungs demonstrated increased iNOS- |CD206+ |F4/80+ macrophages suggesting a M2-like macrophage population drive the PAH phenotype in these mice. The bone marrow transplant studies shows that bone marrow (BM) derived cells contribute in the development of the disease phenotype as wild type BM cells attenuate disease progression. Moreover, female BM transplanted into male mice alleviate but does not protect them from developing PAH. Conclusion Development of PAH in male MacLow mice suggests that macrophages play a causal role in pulmonary vascular remodelling. Results suggest that the phenotype is driven by lung resident M2- like macrophages with a contribution from bone marrow derived cells. A study to examine the probable protective effect of Oestrogen is now underway to further investigate the implication of gender difference in the incidence of PAH in this model.