James Iremonger

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.

Epigenetic Dysregulation of the Drp1 Binding Partners MiD49 and MiD51 Increases Mitotic Mitochondrial Fission and Promotes Pulmonary Arterial Hypertension: Mechanistic and Therapeutic Implications

Background: Mitotic fission is increased in pulmonary arterial hypertension (PAH), a hyperproliferative, apoptosis-resistant disease. The fission mediator dynamin-related protein 1 (Drp1) must complex with adaptor proteins to cause fission. Drp1-induced fission has been therapeutically targeted in experimental PAH. Here, we examine the role of 2 recently discovered, poorly understood Drp1 adapter proteins, mitochondrial dynamics protein of 49 and 51 kDa (MiD49 and MiD51), in normal vascular cells and explore their dysregulation in PAH. Methods: Immunoblots of pulmonary artery smooth muscle cells (control, n=6; PAH, n=8) and immunohistochemistry of lung sections (control, n=6; PAH, n=6) were used to assess the expression of MiD49 and MiD51. The effects of manipulating MiDs on cell proliferation, cell cycle, and apoptosis were assessed in human and rodent PAH pulmonary artery smooth muscle cells with flow cytometry. Mitochondrial fission was studied by confocal imaging. A microRNA (miR) involved in the regulation of MiD expression was identified using microarray techniques and in silico analyses. The expression of circulatory miR was assessed with quantitative reverse transcription–polymerase chain reaction in healthy volunteers (HVs) versus patients with PAH from Sheffield, UK (plasma: HV, n=29, PAH, n=27; whole blood: HV, n=11, PAH, n=14) and then confirmed in a cohort from Beijing, China (plasma: HV, n=19, PAH, n=36; whole blood: HV, n=20, PAH, n=39). This work was replicated in monocrotaline and Sugen 5416-hypoxia, preclinical PAH models. Small interfering RNAs targeting MiDs or an miR mimic were nebulized to rats with monocrotaline-induced PAH (n=4–10). Results: MiD expression is increased in PAH pulmonary artery smooth muscle cells, which accelerates Drp1-mediated mitotic fission, increases cell proliferation, and decreases apoptosis. Silencing MiDs (but not other Drp1 binding partners, fission 1 or mitochondrial fission factor) promotes mitochondrial fusion and causes G1-phase cell cycle arrest through extracellular signal-regulated kinases 1/2– and cyclin-dependent kinase 4–dependent mechanisms. Augmenting MiDs in normal cells causes fission and recapitulates the PAH phenotype. MiD upregulation results from decreased miR-34a-3p expression. Circulatory miR-34a-3p expression is decreased in both patients with PAH and preclinical models of PAH. Silencing MiDs or augmenting miR-34a-3p regresses experimental PAH. Conclusions: In health, MiDs regulate Drp1-mediated fission, whereas in disease, epigenetic upregulation of MiDs increases mitotic fission, which drives pathological proliferation and apoptosis resistance. The miR-34a-3p-MiD pathway offers new therapeutic targets for PAH.

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.

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.

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.

P245 Whole blood levels of microrna-34a predict survival and regulate genes associated with pulmonary arterial hypertension

Introduction Despite advanced therapies for pulmonary arterial hypertension (PAH), the hyperproliferative pulmonary vasculopathy persists. Circulatory microRNAs (miR) offer considerable promise as both a prognostic biomarker, and to identify molecular mechanisms underlying PAH. Previous study from our lab identified whole blood miR-34a as downregulated in patients with PAH. Objectives To validate changes in whole blood miR-34a levels in patients with PAH and relate them to disease severity and survival, and determine the phenotypic effect on pulmonary artery smooth muscle cells (PASMC). Methods Whole blood RNA was isolated from 27 treatment-naive patients with PAH, 12 age-matched healthy volunteers (HV) and experimental models of PAH (Monocrotaline-MCT, Sugen5416/hypoxia-SuHx and controls, n = 5/group). Whole blood miR-34a-5p and −3p levels were measured by qPCR. The phenotypic effect of miR-34a-5p and −3p levels was assessed on PASMC in-vitro. Differences between groups were determined by Student’s t-test or ANOVA-Tukey. Results Whole blood miR-34a-5p was reduced in patients with PAH (p < 0.0001) and experimental models of PAH (MCT p < 0.05, SuHx p < 0.001). Receiver operating characteristic curve identified that miR-34a-5p levels discriminates patients with PAH from HV (AUC = 0.86, p = 0.001). MiR-34a-5p levels were significantly lower in patients with severe PAH, as defined by a cardiac index of <2 vs >2.5 l/min/m2 (p < 0.05) and NT-proBNP > 300 vs <300 ng/l (p < 0.001) and predict survival at 5 years. MiR-34a-5p levels were negatively correlated with pulmonary vascular resistance (r = −0.4, p < 0.05) and pulmonary arterial wedge pressure (r = −0.4, p < 0.05). Preliminary data showed that whole blood miR-34a-3p was reduced in patients with PAH (p = 0.0267) and experimental models of PAH (MCT p < 0.01, SuHx p < 0.01); and delineates patients with PAH from HV (AUC = 0.925, P = 0.01). Transfection of PDGF-stimulated PASMC with miR-34a-5p or −3p inhibitor promote PASMC proliferation (p < 0.001). In contrast, miR-34a-5p and −3p mimic suppress PASMC proliferation (p < 0.05 and p < 0.001 respectively). Additionally, transfection with miR-34a-3p increases caspase-3/7 activities in PASMC (p < 0.0001). Conclusions Reduced miR-34a-5p levels associate with increased disease severity and poor prognosis in PAH. MiR-34a-5p and −3p levels regulate PASMC proliferative-phenotype in response to PDGF. This research identifies miR-34a-5p and −3p as potential biomarkers, subsequent network analysis may identify novel disease mechanisms. Further experiments in preclinical models are currently underway.

S3 Reduced BMPR2 expression potentiates a pulmonary artery smooth muscle cell specific IL-1ß response

Introduction and objectives Bone morphogenetic protein receptor type 2 (BMPR2) mutations are found in heritable and idiopathic pulmonary arterial hypertension however penetrance is incomplete implying necessity for a ‘second hit’. IL-1ß and IL-6 are increased in PAH patients and animal models and are thought to have a role in disease. We aimed to determine pulmonary specific interplay between BMPR2 and IL-1ß signalling through assessing IL-1ß responsiveness of pulmonary artery and aortic smooth muscle cells (Ao/PA SMC) and determine the effects of reduced BMPR2. Methods Microarray analysis of PASMC and AoSMC mRNA was performed using microarray on mRNA isolated from cells cultured in SMGM-2 (Lonza) +/- functional BMPR2 (by use of siRNA) and stimulation with 10 ng/ml IL-1ß for 6 h. Subsequent bioinformatics was performed using R. Findings were validated using quantitative PCR and western blotting. Furthermore R899X+/- BMPR2 transgenic mice were fed western diet for six weeks and injected daily with IL-1ß then assessed for inflammatory activation and PAH phenotype (catheter/echo). mRNA and protein changes were measured by TaqMan PCR, western blotting and serum ELISA. Immuno-staining of paraffin embedded lung sections assessed pulmonary vascular remodelling. Results Array data shows reduced inflammatory activation in response to IL-1ß in PASMC compared with AoSMCs, analysis of cells lacking functional BMPR2 identified an exaggerated inflammatory response to IL-1ß in PASMC lacking BMPR2 (siRNA). Significant up-regulation of IL-6, IL-1α and adhesion molecules (>2-fold) shown by array analysis was validated by qPCR. In the absence of BMPR2 a 1.5 fold increase in proliferation was observed in response to IL-1ß compared to PASMC with functional BMPR2. Mice treated with IL-1ß show higher white blood cell counts (1.7-fold), and protein levels of OPG and IL-6 (serum) matching in vitro data. Conclusion IL-1ß induces a pulmonary specific transcriptome altered by suppression of BMPR2 signalling indicating cross-talk between the pathways. In the presence of BMPR2, PASMCs show limited response to IL-1ß however reducing BMPR2 exacerbated this response increasing the likelihood of a PAH phenotype in PASMCs. This highlights a mechanism that increased IL-1ß may provide “second hit” to reduced BMPR2 to stimulate development of PAH.

183 Novel OPG Protein Interactions Regulate Survival, Proliferation And Pah-associated Gene Expression in Pulmonary Arterial Smooth Muscle Cells

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.