Nw Morrell

Fibrinogen Aα Thr312Ala polymorphism is associated with chronic thromboembolic pulmonary hypertension

Although chronic thromboembolic pulmonary hypertension (CTEPH) is characterised by the persistence of organised thrombus, few pro-thrombotic risk factors have been identified in subjects with the disease. The aim of the present study was to compare the prevalence of eight functionally relevant haemostatic polymorphisms between CTEPH subjects and healthy controls. Genomic DNA was isolated from 214 CTEPH subjects and 200 healthy controls, and analysed for Factor V Leiden, prothrombin guanine (G) to adenine (A) substitution at nucleotide 20210 (20210G>A), plasminogen activator inhibitor-1 4G/5G, tissue plasminogen activator 7351 cytosine (C)>thymidine (T), Factor XIII 100G>T, fibrinogen Aα substitution of threonine with alanine at position 312 (Thr312Ala), fibrinogen Bβ substitution of arginine with lysine at position 448 (Arg448Lys) and fibrinogen Bβ 455G>A polymorphisms. A significant difference was demonstrated in fibrinogen Aα Thr312Ala genotype and allele frequencies between CTEPH subjects and controls. The presence of the alanine allele significantly increased the risk of CTEPH. The fibrinogen Aα alanine 312 allele alters fibrinogen α–α chain cross-linkage and has previously been associated with both increased risk of embolisation and increased resistance to thrombolysis. An association between this polymorphism and chronic thromboembolic pulmonary hypertension, therefore, supports an embolic aetiology for this disease, and may provide a mechanism by which thrombus persists following an acute event.

Current differences in referral patterns for pulmonary endarterectomy in the UK

Pulmonary endarterectomy (PEA) surgery is the treatment of choice in surgically accessible chronic thromboembolic pulmonary hypertension and is potentially curative. The UK is served by seven specialist pulmonary hypertension centres and, consequently, there are regions which do not have a specialist unit. Since 2000, Papworth Hospital (Papworth Everard, UK) has been the sole PEA provider for the UK, offering the opportunity to study the national incidence of operable disease and give potential insight into factors that might affect geographical distribution within the UK. All 262 UK residents who underwent PEA surgery between April 2000 and May 2006 were included in the present study. The age-adjusted cumulative referral rates were compared between regions to test for uniformity. Overall, observed rates differed significantly from expected, with evidence of significant nonuniformity across the UK. The highest rates were observed in proximity to the nationally designated specialist centres and in particular in East Anglia and the West Midlands, nearest Papworth. These two regions differed by >2×sd from the national mean rate. The present study demonstrates wide geographical variation in the number of patients referred for pulmonary endarterectomy surgery. This suggests that there may be patients who are not presently being offered this potentially curative option.

Inhibition of Hyaluronan Synthesis Attenuates Pulmonary Hypertension Associated with Lung Fibrosis

Group III pulmonary hypertension (PH) is a highly lethal and widespread lung disorder that is a common complication in idiopathic pulmonary fibrosis (IPF) where it is considered to be the single most significant predictor of mortality. While increased levels of hyaluronan have been observed in IPF patients, hyaluronan‐mediated vascular remodelling and the hyaluronan‐mediated mechanisms promoting PH associated with IPF are not fully understood.

S84 Identification of MIR-124a as a major regulator of enhanced endothelial cell glycolysis in pulmonary arterial hypertension

Introduction Pulmonary arterial hypertension (PAH) is a rare desease characterised by profound vascular abnormalities in the peripheral arteries of the lung, leading to a progressive increase in pulmonary vascular resistance, right heart failure and death. The disease exists in several forms including a heritable form (HPAH) caused primarily by mutations in bone morphogenetic protein receptor type 2 (BMPR2) and an idiopathic form (IPAH). Endothelial cell (EC) dysfunction is considered a critical initiating factor in the pathobiology of PAH, manifested by increased susceptibility to apoptosis, heightened permeability and enhanced endothelial proliferation. Substantial changes in bioenergetics of ECs, including higher rates of glycolysis, have been reported in PAH patients. However, the mechanisms underlying alterations in energy production have not been identified. Methods We measured glycolysis in blood outgrowth endothelial cells (BOECs) from HPAH patients carrying mutations in BMPR2 and IPAH patients to confirm the metabolic abnormalities previously. We also employed an unbiased genome-wide microarray and proteomic screening approach to detect miRNAs and proteins dysregulated in the same groups to determine the mechanisms underlying abnormal endothelial glycolysis. Results HPAH and IPAH BOECs recapitulated the metabolic phenotype previously observed in PAECs. These alterations were found to be associated with the downregulation of miR-124 and the upregulation of its known target, splicing factor polypyrimidine-tract-binding protein (PTBP1). We also demonstrated that increased PTBP1 promotes the switching in expression of two forms of pyruvate kinase, PKM1 and PKM2, resulting in an increase of aerobic glycolysis, consequently increasing cell proliferation (mechanism schematized in Figure 1). Overexpression of miR-124, or siRNA silencing of PTPB1, restoring normal expression levels of PKM2, also restored normal proliferation and glycolysis in HPAH BOECs. Finally, we observed reduced miR-124 and increased PTPB1 and PKM2 expression in a well-established rat model of PAH, characterised by endothelial proliferation, supporting the presence of this mechanism in vivo. Conclusions Loss of function of BMPR2 results in the downregulation of miR-124 and consequently in the glycolytic abnormalities reported in PAH ECs. Therefore, the manipulation of this miRNA, or its targets, could represent a novel and effective strategy to achieve clinical benefits in the treatment of PAH. Abstract S84 Figure 1

S67 Characterisation of the endothelial outgrowth cell

The existence of cells circulating in the peripheral blood with the capacity of an endothelial progenitor cell (EPC) remains controversial. This is best exemplified by the original cell posited by Asahara and colleagues to be an EPC in 1997,1 which has now been clearly phenotyped as a monocyte with dendritic features.2 There remains a viable candidate known as the endothelial colony-forming cell (ECFC) or endothelial outgrowth cell (OEC), named because it cannot be seen in ex vivo culture for at least 10u2005days. These cells proliferate in culture, form a cobblestone monocellular layer, and form networks in ex vivo assays. Given the lack of understanding of previous candidate cells we sought to better characterise the EOC. Here we present data from electron microscopy studies, immunohistochemistry, ligand stimulation studies, and mRNA microarrays that demonstrate the EOC is a true endothelial cell. Furthermore we demonstrate that these cells can potentially be used as endothelial surrogates in patients with pulmonary hypertension due to a mutation in the bone morphogenetic protein type II receptor (BMPRII). Cells taken from the peripheral blood of patients have a deficiency in intracellular signalling in the BMPRII pathway on stimulation with BMP9. This can be demonstrated by reduced phosphoSmad 1/5 protein on western blotting, and downstream with reduced Id1 gene induction by qPCR. Therefore regardless of its origin and biologically intended function, the EOC is an endothelial cell, and has the capacity to act as an easily derivable endothelial surrogate from patients in whom vascular tissue is not normally obtainable.

S99 The anti-malarial drug and lysosomal inhibitor, chloroquine, increases cell surface expression of BMPR-II

Bone morphogenetic protein receptor type II (BMPR-II) is a member of the transforming growth factor ß (TGFß) receptor superfamily. Mutations in BMPR2 are the major cause of familial pulmonary arterial hypertension (PAH). Reduced BMPR-II expression is significantly reduced in both familial and idiopathic PAH patients. We have shown previously that BMPR-II expression is regulated via a lysosomal degradative pathway. The anti-malarial drug, chloroquine, blocks lysosomal degradation by raising lysosomal pH and impairs autophagic protein degradation. Using an experimental rat model of PAH we observed that chloroquine administration prevented an increase in right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and vascular remodelling following monocrotaline (MCT) treatment. BMPR-II expression was significantly increased in lungs from chloroquine treated rats. Furthermore, in cellular localisation studies chloroquine increased BMPR-II cell surface expression. Methods Male Sprague-Dawley rats received a single subcutaneous injection of MCT to induce PAH. To assess prevention or inhibition of PAH progression, animals received chloroquine, or vehicle, by daily intraperitoneal injection from day 1 to 21 or day 21 to 31 post MCT injections, respectively. Rats were anaesthetised for haemodynamic assessment and lung tissue collected for immunohistochemistry and protein isolation. Muscularisation of small pulmonary arteries was assessed in lung tissue sections by staining with anti-smooth muscle actin. BMPR-II protein expression was determined in frozen lung tissue using western blotting. Cellular localisation of BMPR-II expression in a lung fibroblast cell line that stably expresses green fluorescent protein (GFP) tagged BMPR-II was determined using immunofluorescence and biotinylation of cell surface BMPR-II using NHS-Biotin-SS labelling and precipitation with avidin agarose beads. BMPR-II cell surface expression was then determined by GFP immunoblotting. Results Chloroquine prevented experimental PAH by significantly decreasing RVSP, RVH and muscularisation in MCT-treated rats. Treatment with chloroquine dramatically increased BMPR-II protein levels in the lung. Furthermore, chloroquine treatment inhibited MCT-induced PAH. This was associated with an increase in cell surface BMPR-II expression in a cell line that stably expresses the GFP-tagged receptor. Conclusion This study demonstrates the potential use of chloroquine as a therapeutic agent in the treatment of PAH by potentially increasing levels of BMPR-II at the cell surface.

S97 BMPR2 R899X knock-in mice developed age-related pulmonary hypertension

Background Heterozygous germline mutations in the gene encoding the bone morphogenetic protein type II receptor (BMPR2) underlie the majority (>70%) of cases of heritable pulmonary arterial hypertension (hPAH) and a variable proportion of idiopathic PAH (15%–40%). There are also reports of PAH in patients with mutations in the downstream Smad signalling proteins. However, to date there is no mouse model that mimics the genetic mutations in human disease. Methods We developed a knock-in mouse harbouring a heterozygous (±) human disease causing mutation in BMPR-II: a nonsense mutation in the cytoplasmic tail (R899X) to determine the in vivo physiologic consequences of this BMPR2 mutation. In addition, we crossed this animal with Smad1± knockout mice to determine the effect of additional loss of signalling via this pathway. Haemodynamic, and morphometric data were collected at 3u2005months and 6u2005months of age. Results At 3u2005months of age pulmonary haemodynamics and vascular morphometry of R899X± and Smad1± mice were similar to wild-type littermate controls. In contrast, at 6u2005months of age R899X± and Smad1± mice developed mild pulmonary hypertension with pulmonary vascular remodelling compared with wild-types. Pulmonary artery smooth muscle cells from R899X± mice were hyperproliferative in serum and exhibited defects in Smad signalling in response to BMPs. When R899X± mice were crossed with Smad1± animals, double heterozygous mice had significantly higher right ventricular systolic pressures than single heterozygous mice. Conclusion These findings demonstrate that knockin of a human disease causing BMPR-II mutation causes age-related pulmonary hypertension in mice. In addition, we show that the accumulation of defects in the BMP/Smad signalling pathway increases the susceptibility to pulmonary hypertension, highlighting the central role of this pathway in disease.Abstract S97 Figure 1

S122 A role for the bone morphogenetic protein type 2 receptor (bmpr2) in differentiation of the common myeloid progenitor lineage in mice and humans

Rationale There is increasing evidence of a link between abnormalities in the myeloid cell lineage and pulmonary arterial hypertension (PAH). Heterozygous mutations in the gene encoding the bone morphogenetic protein type 2 receptor (BMPR2) are the most common genetic cause of PAH. We sought to characterise the impact of the genetic loss/reduction of BMPR2 function in the myeloid lineage in mice and humans, and whether this altered susceptibility to PAH. Methods Mx1-cre mice were crossed with bmpr2flox/flox mice. At approximately 8 weeks of age cre-recombinase was induced with polyinosinic-polycytidylic acid (Poly I:C). Control mice (bmpr2flox/flox mice with no cre) were also induced with Poly I:C. At approximately 16 weeks post-induction mice underwent right-heart catheterisation, exsanguination and tissue was removed for analysis. The spleens were weighed and histology was performed on the femurs. Mouse data are presented as mean ±SEM. In a large cohort of PAH patients with (n=160) and without (n=831) BMPR2 mutations blood count indices were analysed. Data presented as median [IQR]. Results 16 weeks after induction of cre-recombinase in Mx1-cre/bmpr2flox/flox mice we observed significant increases (p<0.05) in red blood cells (x106/mm3) (12.7±0.9 compared with 12.1±0.2), haematocrit (%) (64.8±0.7 compared with 62.6±1) and haemoglobin (g/dl) (16±0.9 compared with 15.4±0.2) compared with bmpr2flox/flox mice alone. A significant increase in circulating monocytes (x103/mm3) was also observed (p<0.05) (0.4±0.05 compared with 0.3±0.05). In addition, we identified a significant increase (p<0.05) in megakaryocytes in the femurs (80±10 compared with 17±5) and a significant increase (p<0.01) in the ratio of spleen weight/body weight (0.003±0.0001 compared with 0.002±0.0001) in Mx1-cre/bmpr2flox/flox mice. During right heart catheterisation right ventricular systolic pressures were similar in both groups. In PAH patients significant differences (p<0.05) were seen in haemoglobin (BMPR2 mutation: 162u2009g/L [151.75–173]) vs. no mutation: 150u2009g/L [135 – 163]), haematocrit (0.48 [0.45–0.52] vs. 0.44 [0.41–0.48]) and white blood cells (8.8 [7.3–10.4] vs. 8.11 [6.77–9.61]). Conclusions we have identified a role for bmpr2 in the differentiation of the mouse myeloid lineage, which was also confirmed in PAH patients with BMPR2 mutations. BMPR2 appears particularly important in the differentiation of megakaryocyte-erythrocyte lineage.

S110 Hif2a deletion in the pulmonary endothelium prevents hypoxia-induced pulmonary hypertension

Pulmonary arterial hypertension is a progressive and irreversible disease that leads eventually to right heart failure and death. The pathogenesis of this condition involves proliferation of endothelial and smooth muscle cells resulting in vascular remodelling of the pulmonary arterioles. Several factors are implicated in the remodelling process driven by hypoxia including stabilisation of hypoxia-inducible transcription factors (HIFs), HIF1α and HIF2α. Previous studies have shown that heterozygous deletions of HIF1α or HIF2α partially attenuate many of the remodelling process associated with the development of PAH. Consistent with these observations we have found that pulmonary endothelial specific deletion of HIF2α, achieved using murine cre-loxp technologies (L1 or alk1-cre), offers protection against hypoxia-induced PAH. The rise in pulmonary artery pressure (PAP) normally observed following chronic hypoxic challenge was absent in mice with pulmonary endothelial HIF2α deletion. The right ventricular systolic pressure of L1cre- HIF2α mice post hypoxic challenge (26.17±1.67u2009mmHg, n=7) was not significantly different from untreated WT mice (22.48±1.19u2009mmHg, n=9) and much lower than the hypertensive values seen in WT littermate controls (41.91±1.88u2009mmHg, n=12, p<0.0001) and L1cre-HIF1α mice (36.25±2.37u2009mmHg, n=7, p<0.005). Only minimal remodelling was observed in lung sections from L1cre-HIF2α mice reflecting the normal physiological PAPs following chronic hypoxia. We next questioned whether deletion of lung endothelial HIF2α would be sufficient to reduce downstream arginase-1 and −2 gene expression and in turn influence plasma nitrite/nitrate (NO(X)) concentrations, which would be indicative of changes in nitric oxide homeostasis. The expression of both arginase-1 and −2 were significantly reduced in hypoxia-conditioned whole lung samples from L1cre-HIF2α mice relative to WT littermate controls. Plasma NO(X) concentrations were also significantly elevated in the HIF2α mutant mice when compared to plasma from WT control mice. These observations fit a model whereby reduced arginase-1/2 expression leads to increased availability of l-arginine, and in turn increased NO synthesis via NO synthases. These data offer new insights into the role of pulmonary endothelial HIF2α in causing PAH, and offer new therapeutic opportunities for the treatment of this condition.

S108 Genome-wide association study in chronic thromboembolic pulmonary hypertension reveals new insights into aetiology

Introduction Chronic thromboembolic pulmonary hypertension (CTEPH) is an infrequent but important complication of acute pulmonary embolism (PE). Thrombophilias and non-O blood groups are genetic risk factors for venous thromboembolism (VTE), however they are not independently associated with CTEPH. Identifying genetic risk factors for CTEPH would provide important insights into pathobiology and might allow risk-stratification following PE. We undertook a genome-wide association study (GWAS) in CTEPH to identify novel disease loci. Methods To date, 1457 Caucasian CTEPH patients were enrolled from 10 European and US Centres and compared to 1536 healthy Caucasian controls from the Wellcome Trust Case Control Consortium. Genotyping was performed using the HumanOmniExpressExome-8 array. Quality control criteria and statistical analysis are summarised in figure 1. Results 1250 CTEPH cases, 1492 controls and 7u2009million single-nucleotide polymorphisms (SNPs) were included after quality control exclusions. Two loci, in chromosomes 4 and 9 were significantly associated with CTEPH (figure 1). The lead SNP in chr9 (rs532436, OR=2.38, p=4.6x10-32) is highly correlated with the tagging SNP for the A1 blood group (rs507666, R2=0.99). Reconstructing genetic ABO groups confirmed an over-representation of the A1A1 group in CTEPH compared to controls (7% vs. 2.9%, OR 4.5). Additionally, there were 11 significant SNPs in the chr9 ADAMTS13 gene locus that is moderately correlated with ABO (R2=0.33). The lead SNP in chr4 (rs13130318, OR=1.4, p=5.6x10-8) is highly correlated with a missense variant in FGG (rs6050, R2=0.89) associated with decreased fibrinogen protein and increased resistance to fibrinolysis in CTEPH. There were no associations at the F5 locus, which is highly significant in VTE. Conclusions We report the first GWAS in CTEPH, identifying at least 2 genetic loci associated with the disease. The ABO association is driven by the A1 blood group and represents the largest population attributable genetic risk factor for CTEPH, which is higher than previously reported for VTE. The potential ADAMTS13 association is a plausible biological candidate, and further work will establish whether it is independent from ABO. The lack of associations with other loci found in VTE suggests that ABO might have a pathobiological role in CTEPH in addition to its contribution to VTE. Abstract S108 Figure 1 Manhattan plot of significant loci in chromosome 4 and 9 associated with CTEPH. Quality control exclusion thresholds and chromosome 9 regional association plot shown within figure. Dotted line represents a Genome-wide significance threshold of p=5x10-8 (Bonferroni). Imputaton was performed from the HapIotype Reference Consortium (Sanger imputation service). An additive model of association was applied using logistic regression with gender and 1 principal component as covariates. HWE (Hardy-Weinberg equilibrium), IBD (iderity by descent), MAF (minor allele frequency), PCA (principal component analysis), SNP (single nucleotide polymorphism).