Background—: Heritable and idiopathic pulmonary arterial hypertension (PAH) are phenotypically identical and associated with mutations in several genes related to transforming growth factor (TGF) beta signaling, including bone morphogenetic protein receptor type 2, activin receptor-like kinase 1, endoglin, and mothers against decapentaplegic 9. Approximately 25% of heritable cases lack identifiable mutations in any of these genes. Methods and Results—: We used whole exome sequencing to study a 3-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.

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 (1.4±1.3% triglyceride versus 0.22±0.11% triglyceride, P =0.02). Ceramide, a mediator of lipotoxicity, was increased in PAH RVs versus controls. Using an animal model of heritable PAH, we demonstrated reduced FA oxidation via failure of palmitoylcarnitine to stimulate oxygen consumption in the PAH RV. Conclusions—: Abnormalities in FA metabolism can be detected in the blood and myocardium in human PAH and are associated with in vivo cardiac steatosis and lipotoxicity. Murine data suggest that lipotoxicity may arise from reduction in FA oxidation.

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 ^delx4+ mice). When the mutation was activated after birth, mice developed increased pulmonary artery pressure, RV/LV+S ratio, and pulmonary arterial muscularization with no increase in systemic arterial pressure. Studies with SM22-tet-BMPRII ^delx4+ mice support the hypothesis that loss of BMPRII signaling in smooth muscle is sufficient to produce the pulmonary hypertensive phenotype.

Rationale: Analysis of the age of onset in heritable pulmonary arterial hypertension (HPAH) has led to the hypothesis that genetic anticipation causes younger age of onset and death in subsequent generations. With accrual of pedigree data over multiple decades, we retested this hypothesis using analyses that eliminate the truncation of data that exists with shorter duration of follow-up. Objectives: To analyze the pedigrees of families with mutations in bone morphogenetic protein receptor type 2 (BMPR2), afflicted in two or more generations with HPAH, eliminating time truncation bias by including families for whom we have at least 57 years of data. Methods: We analyzed 355 individuals with BMPR2 mutations from 53 families in the Vanderbilt Pulmonary Hypertension Registry. We compared age at diagnosis or death in affected individuals (n = 249) by generation within families with multigenerational disease. We performed linear mixed effects models and we limited time-truncation bias by restricting date of birth to before 1955. This allowed for 57 years of follow-up (1955–2012) for mutation carriers to develop disease. We also conducted Kaplan-Meier analysis to include currently unaffected mutation carriers (n = 106). Measurements and Main Results: Differences in age at diagnosis by generation were found in a biased analysis that included all birth years to the present, but this finding was eliminated when the 57-year observation limit was imposed. By Kaplan-Meier analysis, inclusion of currently unaffected mutation carriers strengthens the observation that bias of ascertainment exists when recent generations are included. Conclusions: Genetic anticipation is likely an artifact of incomplete time of observation of kindreds with HPAH due to BMPR2 mutations.

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 aBMPR2mutation.Genotypes forCYP1B1 Asn453Ser(N453S) were determined for 140BMPR2mutation 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 carriersversussix 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-OHE₁ratio 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.

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⁻¹intravenously).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.