Background Heritable and idiopathic pulmonary arterial hypertension (PAH) are phenotypically identical and associated with mutations in several genes related to TGF beta signaling, including bone morphogenetic protein receptor type 2 (BMPR2), activin receptor-like kinase 1 (ALK1), endoglin (ENG), and mothers against decapentaplegic 9 (SMAD9). Approximately 25% of heritable cases lack identifiable mutations in any of these genes. Methods and Results We used whole exome sequencing to study a three generation family with multiple affected family members with PAH but no identifiable TGF beta mutation. We identified a frameshift mutation in Caveolin-1 (CAV1), which encodes a membrane protein of caveolae abundant in the endothelium and other cells of the lung. An independent de novo frameshift mutation was identified in a child with idiopathic PAH. Western blot analysis demonstrated a reduction in caveolin-1 protein, while lung tissue immunostaining studies demonstrated a reduction in normal caveolin-1 density within the endothelial cell layer of small arteries. Conclusions Our study represents successful elucidation of a dominant Mendelian disorder using whole exome sequencing. Mutations in CAV1 are associated in rare cases with PAH. This may have important implications for pulmonary vascular biology as well as PAH-directed therapeutic development.

Mutations in bone morphogenetic protein receptor type 2 (BMPR2) cause familial pulmonary arterial hypertension (FPAH), but the penetrance is reduced and females are significantly overrepresented. In addition, gene expression data implicating the oestrogen-metabolising enzyme CYP1B1 suggests a detrimental role of oestrogens or oestrogen metabolites. We examined genetic and metabolic markers of altered oestrogen metabolism in subjects with a BMPR2 mutation. Genotypes for CYP1B1 Asn453Ser (N453S) were determined for 140 BMPR2 mutation carriers (86 females and 54 males). Nested from those subjects, a case–control study of urinary oestrogen metabolite levels (2-hydroxyoestrogen (2-OHE) and 16α-hydroxyoestrone (16α-OHE1)) was conducted in females (five affected mutation carriers versus six unaffected mutation carriers). Among females, there was four-fold higher penetrance among subjects homozygous for the wild-type genotype (N/N) than those with N/S or S/S genotypes (p=0.005). Consistent with this finding, the 2-OHE/16α-OHE1 ratio was 2.3-fold lower in affected mutation carriers compared to unaffected mutation carriers (p=0.006). Our findings suggest that variations in oestrogens and oestrogen metabolism modify FPAH risk. Further investigation of the role of oestrogens in this disease with profound sex bias may yield new insights and, perhaps, therapeutic interventions.

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Pulmonary arterial hypertension (PAH) is a deadly disease with no cure. Alternate conversion of angiotensin II (AngII) to angiotensin-(1-7) (Ang-(1-7)) by angiotensin-converting enzyme 2 (ACE2) resulting in Mas receptor (Mas1) activation improves rodent models of PAH. Effects of recombinant human (rh) ACE2 in human PAH are unknown. Our objective was to determine the effects of rhACE2 in PAH.We defined the molecular effects of Mas1 activation using porcine pulmonary arteries, measured AngII/Ang-(1-7) levels in human PAH and conducted a phase IIa, open-label pilot study of a single infusion of rhACE2 (GSK2586881, 0.2 or 0.4 mg·kg intravenously).Superoxide dismutase 2 (SOD2) and inflammatory gene expression were identified as markers of Mas1 activation. After confirming reduced plasma ACE2 activity in human PAH, five patients were enrolled in the trial. GSK2586881 was well tolerated with significant improvement in cardiac output and pulmonary vascular resistance. GSK2586881 infusion was associated with reduced plasma markers of inflammation within 2-4 h and increased SOD2 plasma protein at 2 weeks.PAH is characterised by reduced ACE2 activity. Augmentation of ACE2 in a pilot study was well tolerated, associated with improved pulmonary haemodynamics and reduced markers of oxidant and inflammatory mediators. Targeting this pathway may be beneficial in human PAH.

Abstract-Bone morphogenetic peptides (BMPs), a family of cytokines critical to normal development, were recently implicated in the pathogenesis of familial pulmonary arterial hypertension. The type-II receptor (BMPRII) is required for recognition of all BMPs, and targeted deletion of BMPRII in mice results in fetal lethality before gastrulation. To overcome this limitation and study the role of BMP signaling in postnatal vascular disease, we constructed a smooth muscle-specific transgenic mouse expressing a dominant-negative BMPRII under control of the tetracycline gene switch (SM22-tet-BMPRII delx4ϩ mice). When the mutation was activated after birth, mice developed increased pulmonary artery pressure, RV/LVϩS ratio, and pulmonary arterial muscularization with no increase in systemic arterial pressure. Studies with SM22-tet-BMPRII delx4ϩ mice support the hypothesis that loss of BMPRII signaling in smooth muscle is sufficient to produce the pulmonary hypertensive phenotype. Key Words: artery Ⅲ bone morphogenetic peptide Ⅲ hypertension Ⅲ smooth muscle Ⅲ vascular A ssociation between abnormal bone morphogenetic peptide (BMP) signaling and pulmonary vascular disease was suggested by genetic studies of familial pulmonary arterial hypertension (PAH), a disorder characterized by the pathological development of increased pressure and structural remodeling in the pulmonary circulation later in life. 1,2 Fifteen to 25% of cases of PAH occur in families, with autosomal-dominant inheritance, and in the year 2000, two groups independently identified the presence of mutations in the BMRII gene in familial PAH. 3,4 Subsequently, approximately 25% of sporadic cases of PAH were also found to be associated with mutations in BMPRII. 5 Dissecting the function of BMPII using transgenic mice has identified a critical role for BMP signaling in development. 6 Although there is redundancy at the level of ligands, type I receptors, intra-and extracellular inhibitors and downstream signaling via SMADs, functional BMPRII is an absolute requirement for BMP signaling. Consequently, BM-PRII Ϫ/Ϫ mice die early in development, before gastrulation, whereas BMPRII ϩ/Ϫ mice develop normally and have no apparent phenotype. 7 To overcome the developmental lethality identified by traditional transgenic approaches, we constructed a conditional, tissue-specific BMPRII transgenic mouse, using a smooth muscle cell-specific promotor and a dominant-negative BMPRII identified in a family with PAH. 8 Using these mice, we asked two questions: (1) is expression of a strong loss-of-function mutation in BMPRII sufficient to produce pulmonary hypertension, and (2) is loss of BMPRII function in smooth muscle alone sufficient to produce pulmonary hypertension? Materials and Methods Construction of SM22-tet-BMPRII delx4؉ Transgenic MiceThe mice were generated at the University of Cincinnati Transgenic Mouse Science from plasmids we provided. The murine SM22 promotor (488 bp starting 340 bp before the start site and extending 148 bp into the 5Ј UTR) was used to drive...

R ight ventricular (RV) failure is the predominant cause of death in pulmonary arterial hypertension (PAH), but no RV-specific therapies exist because the underlying mechanisms are poorly understood. Abnormalities of glucose homeostasis and insulin resistance are well described in PAH, 1-4 but less is known about lipid metabolism despite the interrelated nature of glucose and lipid homeostasis. Abnormalities in fatty acid metabolism have been described in experimental models of PAH, 5,6 but systemic and myocardial fatty acid metabolism have not been studied in human PAH. Clinical Perspective on p 1944Given the heart's preference for fatty acids (FAs) as an energy source, 7 understanding FA metabolism may be particularly relevant to understanding RV adaptation to elevated afterload in PAH. We recently showed that RV failure is associated with myocardial steatosis and accumulation of the lipotoxic and proapoptotic mediator ceramide in human heritable PAH because of mutation in bone morphogenetic protein receptor type II (BMPR2). 8 Others and we have also shown indirect evidence of abnormal fatty acid oxidation (FAO) in experimental models of PAH. [9][10][11] The generalizability of these abnormalities in FA metabolism to idiopathic PAH and whether they are a systemic feature in human PAH are unknown.We hypothesized that reduced FA metabolism is ubiquitous in PAH and associated with lipotoxic cardiac steatosis in the RV. We tested this hypothesis by studying blood, RV Background-The mechanisms of right ventricular (RV) failure in pulmonary arterial hypertension (PAH) are poorly understood. Abnormalities in fatty acid (FA) metabolism have been described in experimental models of PAH, but systemic and myocardial FA metabolism has not been studied in human PAH. Methods and Results-We used human blood, RV tissue, and noninvasive imaging to characterize multiple steps in the FA metabolic pathway in PAH subjects and controls. Circulating free FAs and long-chain acylcarnitines were elevated in PAH patients versus controls. Human RV long-chain FAs were increased and long-chain acylcarnitines were markedly reduced in PAH versus controls. With the use of proton magnetic resonance spectroscopy, in vivo myocardial triglyceride content was elevated in human PAH versus controls ( Sample Collection and AnalysisFasting peripheral blood samples were obtained at the time of clinic visits or at the Vanderbilt General Clinical Research Center. Plasma samples were collected into ethylenediaminetetraacetic acid plasma tubes. Ethylenediaminetetraacetic acid tubes were centrifuged within 45 minutes at 4000 rpm and the plasma fraction immediately aliquoted as 20-µL aliquots and stored at -80ºC. Plasma acylcarnitine samples were analyzed as described previously. 13 The Hormone Assay Core of the Mouse Metabolic Phenotypic Center at Vanderbilt University quantified plasma-free fatty acids by using standard enzymatic reactions. RV Gene Expression ArrayRNA isolation and Microarray techniques have been described previously. 8 All array results...

Rationale: Shorter survival in heritable pulmonary arterial hypertension (HPAH), often due to BMPR2 mutation, has been described in association with impaired right ventricle (RV) compensation. HPAH animal models are insulin resistant, and cells with BMPR2 mutation have impaired fatty acid oxidation, but whether these findings affect the RV in HPAH is unknown.Objectives: To test the hypothesis that BMPR2 mutation impairs RV hypertrophic responses in association with lipid deposition.Methods: RV hypertrophy was assessed in two models of mutant Bmpr2 expression, smooth muscle-specific ( Sm22 R899X ) and universal expression (Rosa26 R899X ). Littermate control mice underwent the same stress using pulmonary artery banding (Low-PAB). Lipid content was assessed in rodent and human HPAH RVs and in Rosa26 R899X mice after metformin administration. RV microarrays were performed using human HPAH and control subjects. Conclusions: These data demonstrate that Bmpr2 mutation affects RV stress responses in a transgenic rodent model. Impaired RV hypertrophy and triglyceride and ceramide deposition are present as a function of RV mutant Bmpr2 in mice; fatty acid oxidation impairment in human HPAH RVs may underlie this finding. Further study of how BMPR2 mediates RV lipotoxicity is warranted.

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Here we define a population of resident lung mesenchymal stem cells (luMSC) that function to regulate the severity of bleomycin injury via modulation of the T-cell response. Bleomycin induced loss of these endogenous luMSC and elicited fibrosis (PF), inflammation and pulmonary arterial hypertension (PAH). Replacement of resident stem cells by administration of isolated luMSC attenuated the bleomycin-associated pathology and mitigated the development of PAH. In addition, luMSC modulated a decrease in numbers of lymphocytes and granulocytes in bronchoalveolar fluid and demonstrated an inhibition of effector T cell proliferation in vitro. Global gene expression analysis indicated that the luMSC are a unique stromal population differing from lung fibroblasts in terms of proinflammatory mediators and pro-fibrotic pathways. Our results demonstrate that luMSCs function to protect lung integrity following injury however when endogenous MSC are lost this function is compromised illustrating the importance of this novel population during lung injury. The definition of this population in vivo in both murine and human pulmonary tissue facilitates the development of a therapeutic strategy directed at the rescue of endogenous cells to facilitate lung repair during injury.