BMPR2 Research Articles

Abstract 8895: Clinical Outcomes of Mutation Carriers in Pulmonary Arterial Hypertension in Children

Background: In heritable pulmonary arterial hypertension (HPAH) and idiopathic pulmonary arterial hypertension (IPAH), mutations have previously been described in genes for the bone morphogenetic protein receptor type 2 ( BMPR2 ) and the activin receptor-like kinase 1 ( ALK1 ). It is, however, not known, especially in childhood, how these mutations relate to the clinical characteristics. Methods: Sixty four childhood patients whose onset of IPAH/HPAH was under 16 years old were selected. Mutations were monitored by direct sequencing and multiplex ligation-dependent probe amplification in BMPR2, ALK1, endoglin, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, SMAD7, SMAD8 , BMPR1A and BMPR1B genes. One SMAD8 mutation carrier and 2 BMPR1B mutation carriers were excluded from the present analysis because of minority of cases. Functional characteristics, hemodynamic parameters, and clinical outcomes were compared among BMPR2 mutation group, ALK1 mutation group, and noncarrier group. Results: Among these 61 childhood IPAH/HPAH patients, 18 had BMPR2 mutation and 7 had ALK1 mutation. At the time of diagnosis, age, WHO functional class, mean pulmonary artery pressure, pulmonary resistance, serum brain natriuretic peptide level, and 6-minute walking distance were similar among 3 groups. Medications including bosentan and sildenafil were also similar among 3 groups. Among 61 patients, 5-year and 10-year survival were 77% and 74%, respectively. Despite targeted therapy, 5-year survival in the BMPR2 mutation group (55%) was lowest among these 3 groups. The 5-year survival in the ALK1 mutation group (64%) tended to be lower than that in the noncarrier group (91%). Intravenous epoprostenol therapy strongly ameliorated the WHO functional class in all groups. Especially in the ALK1 mutation group, pulmonary vasodilator therapies were not effective except for intravenous epoprostenol. Conclusions: Prognosis of IPAH/HPAH in children with BMPR2 and ALK1 mutation is poor. Aggressive treatment including intravenous epoprostenol may be warranted in those children.

Gene expression analysis of a murine model with pulmonary vascular remodeling compared to end-stage IPAH lungs

BackgroundIdiopathic pulmonary arterial hypertension (IPAH) continues to be one of the most serious intractable diseases that might start with activation of several triggers representing the genetic susceptibility of a patient. To elucidate what essentially contributes to the onset and progression of IPAH, we investigated factors playing an important role in IPAH by searching discrepant or controversial expression patterns between our murine model and those previously published for human IPAH. We employed the mouse model, which induced muscularization of pulmonary artery leading to hypertension by repeated intratracheal injection of Stachybotrys chartarum, a member of nonpathogenic and ubiquitous fungus in our envelopment.MethodsMicroarray assays with ontology and pathway analyses were performed with the lungs of mice. A comparison was made of the expression patterns of biological pathways between our model and those published for IPAH.ResultsSome pathways in our model showed the same expression patterns in IPAH, which included bone morphogenetic protein (BMP) signaling with down-regulation of BMP receptor type 2, activin-like kinase type 1, and endoglin. On the other hand, both Wnt/planar cell polarity (PCP) signaling and its downstream Rho/ROCK signaling were found alone to be activated in IPAH and not in our model.ConclusionsActivation of Wnt/PCP signaling, in upstream positions of the pathway, found alone in lungs from end stage IPAH may play essential roles in the pathogenesis of the disease.

Altered expression and signal transduction of endothelin‐1 receptors in heritable and idiopathic pulmonary arterial hypertension

Human pulmonary arterial smooth muscle cells (PASMC) were isolated from elastic pulmonary arteries dissected from lungs of individuals with and without pulmonary arterial hypertension (PAH). Reflecting increased smooth muscle constriction in cells from PAH subject, Ca(2+) influx in response to endothelin-1 (ET-1) increased in all the PAH PASMC populations relative to the normal donor control cells. The ETA receptor mRNA levels remained unchanged, whereas the ETB receptor mRNA levels decreased in both heritable and idiopathic PAH-derived PASMC. All the PASMC populations expressed considerably higher ETA compared to ETB receptor number. Both ETA and ETB receptor numbers were reduced in bone morphogenetic protein receptor type II (BMPR2) mutation PAH. ETB receptors showed a particular reduction in number. Phospho-antibody array analysis of normal and BMPR2 deletion PASMC illustrated ERK and Akt activation to be the most prominent and to be taking place principally through ETB receptors in normal PASMC, but primarily through ETA receptors in PASMC from BMPR2 PAH subjects. Additionally in the PAH cells the total relative ET-1 signal response was markedly reduced. Western analysis from the BMPR2 PASMC duplicated the array results, whereas PASMC from iPAH subjects showed variability with most samples continuing to signal through ETB. In sum, these results indicate that generally both receptors are reduced in PAH particularly ETB, and that ETB signaling through protein kinases becomes markedly reduced in BMPR2 PASMC, while it continues in IPAH. Importantly, the data suggest that caution must be taken when applying ET-1 receptor antagonist therapy to PAH patients.

5′UTR Repeat Polymorphisms of the BMPR2 gene in Children with Pulmonary Hypertension associated with Congenital Heart Disease

The mutations of bone morphogenetic protein receptor type 2 (BMPR2) in patients with idiopathic pulmonary hypertension has been well defined. We investigated the occurrence of BMPR2 mutation and genetic polymorphisms in children with pulmonary hypertension associated with congenital heart disease (aPH/CHD) and correlated with the pulmonary haemodynamic and vasoreactivity. BMPR2 mutation/polymorphisms were determined in 30 aPH/CHD children. All children underwent cardiac catheterisation to obtain baseline haemodynamic data. The 5'UTR containing promoter region and all the exons [1-13] of BMPR2 gene were genotyped for possible genetic variants that may be related to the aPH/CHD. None of our 30 patients (median-age 90 months) with aPH/CHD (mean PAP 48±17mmHg, PVR 6.7±4.2WUm(2)) has had any BMPR2 mutation. Fifteen of them had single nucleotide polymorphism, rs1061157 and/or 5'UTR-polymorphism, specifically GGC repeat variant in seven patients; AGC repeat variant in one patient; and nine base pairs duplication (CTTCTTCGG) in one patient. The GGC repeat ≥13 was found in three out of six of children with aPH/CHD with normal PVR vs. two out of 24 children with aPH/CHD with high PVR. The odd ratio between these two subgroups of aPH/CHD is 0.09 (95% CI 0.02-0.34). In our cohort, there was no BMPR2 mutation in children with aPH/CHD while nine out of 30 of them have 5'UTR repeat polymorphisms. Our data suggests the occurrence of GGC repeat ≥13 at the 5'UTR region may have some protective effect towards pulmonary vasculopathy in children who have been exposed to high pulmonary blood flow due to CHD.

SMAD versus Non-SMAD Signaling Is Determined by Lateral Mobility of Bone Morphogenetic Protein (BMP) Receptors

Bone (or body) morphogenetic proteins (BMPs) belong to the TGFβ superfamily and are crucial for embryonic patterning and organogenesis as well as for adult tissue homeostasis and repair. Activation of BMP receptors by their ligands leads to induction of several signaling cascades. Using fluorescence recovery after photobleaching, FRET, and single particle tracking microscopy, we demonstrate that BMP receptor type I and II (BMPRI and BMPRII) have distinct lateral mobility properties within the plasma membrane, which is mandatory for their involvement in different signaling pathways. Before ligand binding, BMPRI and a subpopulation of BMPRII exhibit confined motion, reflecting preassembled heteromeric receptor complexes. A second free diffusing BMPRII population only becomes restricted after ligand addition. This paper visualizes time-resolved BMP receptor complex formation and demonstrates that the lateral mobility of BMPRI has a major impact in stabilizing heteromeric BMPRI-BMPRII receptor complexes to differentially stimulate SMAD versus non-SMAD signaling.

Longitudinal Analysis Casts Doubt on the Presence of Genetic Anticipation in Heritable Pulmonary Arterial Hypertension

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. 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. 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). 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. Genetic anticipation is likely an artifact of incomplete time of observation of kindreds with HPAH due to BMPR2 mutations.

The Estrogen Puzzle in Pulmonary Arterial Hypertension

> New Scientist: What do you think most about during the day? > > Stephen Hawking: Women. They are a complete mystery. > > —Stephen Hawking at 70 – exclusive interview at the New Scientist, Jan 4, 2012 The biology of pulmonary arterial hypertension (PAH) is full of mysteries, and one of its longer-standing ones has also intrigued and inspired both scientists and artists throughout history: the female sex. Although affecting patients of all ages and both sexes, PAH preferentially affects young women, suggesting that the female sex is a risk factor for PAH. Even in heritable PAH associated with autosomal dominant mutations in the gene encoding the bone morphogenetic protein receptor type 2 (BMPR2), women after puberty are ≈2.5 times more likely to develop PAH than males.1 In idiopathic PAH the female/male ratio ranges from 1.7:1 or 1.9:1 to 4.1:1 in 3 published PAH registries (the National Institutes of Health registry in the 80s,2 the French registry,3 and the Registry to Evaluate Early and Long-term PAH Disease Management (REVEAL),4 respectively). On the other hand, the disease can be more severe in men. Female animals with PAH tend to have lower pulmonary artery pressures and better outcomes compared with males,5 and similarly, male PAH patients have higher mortality than females.6 The basis for this apparent paradox remains unknown. Article see p 1087 Estrogens exert their effects through both genomic effects, by activating their cytosolic or membrane-bound receptors, and nongenomic effects, by activating membrane G protein–coupled estrogen receptors. For example, via the nongenomic effects, estrogens promote the release of both prostacyclin and nitric oxide while, via the genomic effects, they decrease the expression of endothelin 1 under hypoxia, all promoting vasoconstriction in the pulmonary circulation.5 Yet, the pathology of PAH is driven not by vasoconstriction, but rather by a proproliferative and antiapoptotic diathesis throughout the pulmonary arterial wall. The main circulating estrogen in …

Chasing Pulmonary Hypertension: 1980–2012

After years of effort, investigators at Vanderbilt and Columbia found the gene for heritable pulmonary hypertension (PH) nearly simultaneously in the year 2000. Mutations in the so-called bone morphogenetic protein receptor type 2 (BMPR2) are now known to be responsible for about 75% of cases of heritable pulmonary arterial hypertension (PAH). In the other 25% of families, either the mutation remains unknown, or it is in the ALK-1 or endoglin genes, SMAD 8 or caveolin 1. Undoubtedly, other rarer mutations that cause disease will be found. In most registries, known heritable PAH accounts for about 6% of the population enrolled, so 6 out of every 100 cases. However, surveys of mutation status in idiopathic PAH have revealed a prevalence of about 10%20% of BMPR2 mutation carriers. Thus, as shown in Figure 1, the largest number of patients with the gene raising the risk of PH is in the idiopathic group, and these actually outnumber the known family cases. One of the scientists vital to the discovery of BMPR2 mutations, William Nichols, PhD, at the University of Cincinnati, now has National Institutes of Health (NIH) funding to obtain DNA and RNA from a large number of patients with idiopathic PAH from multiple centers, to discover the actual prevalence of BMPR2 mutations in a USA cohort, and to look for other genetic associations with PH. The next phase in our journey with heritable PAH was to look for genetic/ genomic associations that might help explain the incomplete penetrance of BMPR2 mutations. For many years, based on discussions at the Evian World Health Organization (WHO) Pulmonary Hypertension Conference, the penetrance of the BMPR2 gene has been estimated to be about 20%. This means that having the mutation is not enough, and that environmental, genetic, or biological differences must have a role. Newer data from the Vanderbilt PAH Registry suggests that the penetrance is about 27%, with a 3:1 female to male ratio, giving females about a 42% penetrance and males 14%. Nonetheless, the reduced penetrance gave rise to several years of difficult experiments designed to discover a major second gene that might explain penetrance. Although multiple genes were discovered to have a minor role, no major gene has been found. Genes that have been found to possibly have influence on the likelihood of disease include serotonin transporter, Cyp, angiotensin converting enzyme, and nitric oxide synthase. Undoubtedly, a large number of interacting gene products will have major influence on the development of PAH. Part of the problem is that PAH is not one disease and therefore does not have one etiology. This is a very complex problem to address. It also must include epigenetic and other somatic gene modifications that are not discoverable by germline approaches. The problem is summarized in Figure 2. Although basic important work is progressing on mechanisms that regulate BMPR2 expression and function, the modifier gene approach must await newer modalities such as whole exome and genome sequencing to discover rare variants that may influence development of pulmonary vascular disease. This approach will also discover rare variants that have a Mendelian effect on the development of PH in families that remain with undiscovered mutations. The current thrust of our work is along 4 lines. The first is the study of the gender difference and sex hormones in PH. The greatest risk for development of either idiopathic or heritable PH is female gender, 3:1 over males. Eric Austin in our laboratory is studying estrogen metabolites and the role of cytochrome enzymes in determining circulating levels of estrogen metabolites and their impact on PH risk. Others are following similar experimental approaches. These studies will certainly yield insights into development of PH and perhaps lead to preventive or management therapies. Key Words—BMPR2, genetics, heritable pulmonary hypertension, idiopathic pulmonary hypertension Correspondence: john.newman@vanderbilt.edu

Rescuing the BMPR2 Pathway: How and Where Can We Intervene?

Rescuing the BMPR2 Pathway: How and Where Can We Intervene?

The Hedgehog Signal Induced Modulation of Bone Morphogenetic Protein Signaling: An Essential Signaling Relay for Urinary Tract Morphogenesis

BackgroundCongenital diseases of the urinary tract are frequently observed in infants. Such diseases present a number of developmental anomalies such as hydroureter and hydronephrosis. Although some genetically-modified mouse models of growth factor signaling genes reproduce urinary phenotypes, the pathogenic mechanisms remain obscure. Previous studies suggest that a portion of the cells in the external genitalia and bladder are derived from peri-cloacal mesenchymal cells that receive Hedgehog (Hh) signaling in the early developmental stages. We hypothesized that defects in such progenitor cells, which give rise to urinary tract tissues, may be a cause of such diseases.Methodology/Principal FindingsTo elucidate the pathogenic mechanisms of upper urinary tract malformations, we analyzed a series of Sonic hedgehog (Shh) deficient mice. Shh−/− displayed hydroureter and hydronephrosis phenotypes and reduced expression of several developmental markers. In addition, we suggested that Shh modulation at an early embryonic stage is responsible for such phenotypes by analyzing the Shh conditional mutants. Tissue contribution assays of Hh-responsive cells revealed that peri-cloacal mesenchymal cells, which received Hh signal secreted from cloacal epithelium, could contribute to the ureteral mesenchyme. Gain- and loss-of-functional mutants for Hh signaling revealed a correlation between Hh signaling and Bone morphogenetic protein (Bmp) signaling. Finally, a conditional ablation of Bmp receptor type IA (BmprIA) gene was examined in Hh-responsive cell lineages. This system thus made it possible to analyze the primary functions of the growth factor signaling relay. The defective Hh-to-Bmp signaling relay resulted in severe urinary tract phenotypes with a decrease in the number of Hh-responsive cells.Conclusions/SignificanceThis study identified the essential embryonic stages for the pathogenesis of urinary tract phenotypes. These results suggested that Hh-responsive mesenchymal Bmp signaling maintains the population of peri-cloacal mesenchyme cells, which is essential for the development of the ureter and the upper urinary tract.

Bone morphogenetic protein 2 (BMP2); BMP4; bone morphogenetic protein receptor type IA (BMPR1A)

Bone morphogenetic protein 2 (BMP2); BMP4; bone morphogenetic protein receptor type IA (BMPR1A)

Inhibition of endogenous antagonists with an engineered BMP-2 variant increases BMP-2 efficacy in rat femoral defect healing

Bone morphogenetic proteins (BMP) have been used successfully by orthopedic clinicians to augment bone healing. However, these osteoinductive proteins must be applied at high concentrations to induce bone formation. The limited therapeutic efficacy may be due to the local expression of BMP antagonists such as Noggin that neutralize exogenous and endogenous BMPs. If so, inhibiting BMP antagonists may provide an attractive option to augment BMP induced bone formation. The engineered BMP-2 variant L51P is deficient in BMP receptor type I binding, but maintains its affinity for BMP receptor type II and BMP antagonists including Noggin, Chordin and Gremlin. This modification makes L51P a BMP receptor-inactive inhibitor of BMP antagonists. We implanted β-tricalcium phosphate ceramics loaded with BMP-2 and/or L51P into a critical size defect model in the rat femur to investigate whether the inhibition of BMP antagonist with L51P enhances the therapeutic efficacy of exogenous BMP-2. Our study reveals that L51P reduces the demand of exogenous BMP-2 to induce bone healing markedly, without promoting bone formation directly when applied alone.

MicroRNA-100 regulates osteogenic differentiation of human adipose-derived mesenchymal stem cells by targeting BMPR2

Elucidation of the molecular mechanisms governing human adipose-derived mesenchymal stem cells (hASCs) osteogenic differentiation is of great importance for improving the treatment of bone-related diseases. In this study, we examined the role of microRNA (miR)-100 on the osteogenesis of hASCs. Overexpression of miR-100 inhibited osteogenic differentiation of hASCs in vitro, whereas downregulation of miR-100 enhanced the process. Target prediction analysis and dual luciferase report assay confirmed that bone morphogenetic protein receptor type II (BMPR2) was a direct target of miR-100. Furthermore, knockdown of BMPR2 by RNA interference inhibited osteogenic differentiation of hASCs, similar as the effect of upregulation miR-100. Taken together, our findings imply that miR-100 plays a negative role in osteogenic differentiation and might act through targeting BMPR2.

Outcomes of Childhood Pulmonary Arterial Hypertension in BMPR2 and ALK1 Mutation Carriers

Mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene and the activin receptor-like kinase 1 (ALK1) gene have been reported in heritable pulmonary arterial hypertension (HPAH) and idiopathic pulmonary arterial hypertension (IPAH). However, the relation between clinical characteristics and each gene mutation in IPAH and HPAH is still unclear, especially in childhood. The aim of this study was to determine, in a retrospective study, the influence and clinical outcomes of gene mutations in childhood IPAH and HPAH. Fifty-four patients with IPAH or HPAH whose onset of disease was at <16 years of age were included. Functional characteristics, hemodynamic parameters, and clinical outcomes were compared in BMPR2 and ALK1 mutation carriers and noncarriers. Overall 5-year survival for all patients was 76%. Eighteen BMPR2 mutation carriers and 7 ALK1 mutation carriers were detected in the 54 patients with childhood IPAH or HPAH. Five-year survival was lower in BMPR2 mutation carriers than mutation noncarriers (55% vs 90%, hazard ratio 12.54, p = 0.0003). ALK1 mutation carriers also had a tendency to have worse outcome than mutation noncarriers (5-year survival rate 64%, hazard ratio 5.14, p = 0.1205). In conclusion, patients with childhood IPAH or HPAH with BMPR2 mutation have the poorest clinical outcomes. ALK1 mutation carriers tended to have worse outcomes than mutation noncarriers. It is important to consider aggressive treatment for BMPR2 or ALK1 mutation carriers.

Parathyroid hormone induces differentiation of mesenchymal stromal/stem cells by enhancing bone morphogenetic protein signaling

Parathyroid hormone (PTH) stimulates bone remodeling and induces differentiation of bone marrow mesenchymal stromal/stem cells (MSCs) by orchestrating activities of local factors such as bone morphogenetic proteins (BMPs). The activity and specificity of different BMP ligands are controlled by various extracellular antagonists that prevent binding of BMPs to their receptors. Low-density lipoprotein receptor-related protein 6 (LRP6) has been shown to interact with both the PTH and BMP extracellular signaling pathways by forming a complex with parathyroid hormone 1 receptor (PTH1R) and sharing common antagonists with BMPs. We hypothesized that PTH-enhanced differentiation of MSCs into the osteoblast lineage through enhancement of BMP signaling occurs by modifying the extracellular antagonist network via LRP6. In vitro studies using multiple cell lines, including Sca-1(+) CD45(-) CD11b(-) MSCs, showed that a single injection of PTH enhanced phosphorylation of Smad1 and could also antagonize the inhibitory effect of noggin. PTH treatment induced endocytosis of a PTH1R/LRP6 complex and resulted in enhancement of phosphorylation of Smad1 that was abrogated by deletion of PTH1R, β-arrestin, or chlorpromazine. Deletion of LRP6 alone led to enhancement of pSmad1 levels that could not be further increased with PTH treatment. Finally, knockdown of LRP6 increased the exposure of endogenous cell-surface BMP receptor type II (BMPRII) significantly in C2C12 cells, and PTH treatment significantly enhanced cell-surface binding of (125) I-BMP2 in a dose- and time-dependent manner, implying that LRP6 organizes an extracellular network of BMP antagonists that prevent access of BMPs to BMP receptors. In vivo studies in C57BL/6J mice and of transplanted green fluorescent protein (GFP)-labeled Sca-1(+) CD45(-) CD11b(-) MSCs into the bone marrow cavity of Rag2(-/-) immunodeficient mice showed that PTH enhanced phosphorylation of Smad1 and increased commitment of MSCs to osteoblast lineage, respectively. These data demonstrate that PTH enhancement of MSC differentiation to the osteoblast lineage occurs through a PTH- and LRP6-dependent pathway by endocytosis of the PTH1R/LRp6 complex, allowing enhancement of BMP signaling.

Expression of BMP-receptor type 1A correlates with progress of osteoarthritis in human knee joints with focal cartilage lesions

Bone morphogenetic protein-2 (BMP-2) and its receptor type 1A (BMPR-1A) play significant roles in cartilage metabolism. The aim of this study was to evaluate a possible correlation between intra-articular expression of these proteins and the degree of osteoarthritis (OA) in human knees. Biopsies of synovia and debrided cartilage were taken in 15 patients undergoing autologous chondrocyte implantation. Expression of BMP-2 and BMPR-1A was evaluated semi-quantitatively by immunohistologic staining. These data were complemented by grading of cartilage lesions according to International Cartilage Repair Society (ICRS), defect size, duration of complaints, knee osteoarthritis scoring system (KOSS) and Henderson and Kellgren-Lawrence scores. General histologic stainings were used to determine Mankin, Pritzker and Krenn scores. The expression of BMPR-1A but not of BMP-2 was significantly higher in cartilage biopsies taken in type 3 lesions with intact subchondral layer compared with type 4 defects (P < 0.05). In cartilage areas of bordering sectors, the intensity of immunohistologic staining of BMPR-1A was statistically significantly higher in mature cartilage compared with repair zones (P < 0.05). Expression of BMP-2 and its receptor 1A correlated in the cartilage biopsies (P < 0.02) but not in the synovia. The degree of OA was scored in all biopsies according to Mankin and Pritzker, and these scores correlated statistically significantly with BMPR-1A expression in the synovia (P < 0.05). In patients with an osteochondritis dissecans, the degree of OA was higher compared with other causes of chondromalacia, as evaluated by defect size, ICRS score, duration of complaints, Pritzker score and expression of BMPR-1A in cartilage (P < 0.05). These data support the role of BMPR-1A as an indicator of OA progression in human knees with circumscribed cartilage lesions.

BuMPy road of delayed graft function

BuMPy road of delayed graft function

Plumbagin reverses proliferation and resistance to apoptosis in experimental PAH

Like cancer, pulmonary arterial hypertension (PAH) is characterised by a pro-proliferative and anti-apoptotic phenotype. In PAH, pulmonary artery smooth muscle cell (PASMC) proliferation is enhanced and apoptosis suppressed. The sustainability of this phenotype requires the activation of pro-survival transcription factors, such as signal transducer and activator of transcription (STAT)3 and nuclear factor of activated T-cells (NFAT). There are no drugs currently available that are able to efficiently and safely inhibit this axis. We hypothesised that plumbagin (PLB), a natural organic compound known to block STAT3 in cancer cells, would reverse experimental pulmonary hypertension. Using human PAH-PASMC, we demonstrated in vitro that PLB inhibits the activation of the STAT3/NFAT axis, increasing the voltage-gated K(+) current bone morphogenetic protein receptor type II (BMPR2), and decreasing intracellular Ca(2+) concentration ([Ca(2+)](i)), rho-associated coiled-coil containing protein kinase (ROCK)1 and interleukin (IL)-6, contributing to the inhibition of PAH-PASMC proliferation and resistance to apoptosis (proliferating cell nuclear antigen (PCNA), TUNEL, Ki67 and anexine V). In vivo, PLB oral administration decreases distal pulmonary artery remodelling, mean pulmonary artery pressure and right ventricular hypertrophy without affecting systemic circulation in both monocrotaline- and suden/chronic hypoxia-induced PAH in rats. This study demonstrates that the STAT3/NFAT axis can be therapeutically targeted by PLB in human PAH-PASMC and experimental PAH rat models. Thus, PLB could be considered a specific and attractive future therapeutic strategy for PAH.

Whole exome sequencing to identify a novel gene (caveolin-1) associated with human pulmonary arterial hypertension.

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. 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. 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.

Metabolomic Analysis of Bone Morphogenetic Protein Receptor Type 2 Mutations in Human Pulmonary Endothelium Reveals Widespread Metabolic Reprogramming

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease of the lung vasculature for which the molecular etiologies are unclear. Specific metabolic alterations have been identified in animal models and in PAH patients, though existing data focus mainly on abnormalities of glucose homeostasis. We hypothesized that analysis of the entire metabolome in PAH would reveal multiple other metabolic changes relevant to disease pathogenesis and possible treatment. Layered transcriptomic and metabolomic analyses of human pulmonary microvascular endothelial cells (hPMVEC) expressing two different disease-causing mutations in the bone morphogenetic protein receptor type 2 (BMPR2) confirmed previously described increases in aerobic glycolysis but also uncovered significant upregulation of the pentose phosphate pathway, increases in nucleotide salvage and polyamine biosynthesis pathways, decreases in carnitine and fatty acid oxidation pathways, and major impairment of the tricarboxylic acid (TCA) cycle and failure of anaplerosis. As a proof of principle, we focused on the TCA cycle, predicting that isocitrate dehydrogenase (IDH) activity would be altered in PAH, and then demonstrating increased IDH activity not only in cultured hPMVEC expressing mutant BMPR2 but also in the serum of PAH patients. These results suggest that widespread metabolic changes are an important part of PAH pathogenesis, and that simultaneous identification and targeting of the multiple involved pathways may be a more fruitful therapeutic approach than targeting of any one individual pathway.