Bone Morphogenetic Protein Receptor 2 Signaling Research Articles

STING Contributes to Pulmonary Hypertension by Targeting IFN and BMPR2 Signaling through Regulating of F2RL3.

Pulmonary hypertension (PH) is an incurable disease characterized by pulmonary vascular remodeling. Endothelial injury and inflammation are the key triggers of disease initiation. Recent findings suggest that STING (stimulator of IFN genes) activation plays a critical role in endothelial dysfunction and IFN signaling. Here, we investigated the involvement of STING in the pathogenesis of PH. Patients with PH and rodent PH model samples, a Sugen 5416/hypoxia PH model, and pulmonary artery endothelial cells (PAECs) were used to evaluate the hypothesis. We found that the cyclic guanosine monophosphate-AMP synthase-STING signaling pathway was activated in lung tissues from rodent PH models and patients with PH and in TNF-α-induced PAECs invitro. Specifically, STING expression was significantly elevated in the endothelial cells in PH disease settings. In the Sugen 5416/hypoxia mouse model, genetic knockout or pharmacological inhibition of STING prevented the progression of PH. Functionally, knockdown of STING reduced the proliferation and migration of PAECs. Mechanistically, STING transcriptionally regulates its binding partner F2RL3 (F2R-like thrombin or trypsin receptor 3) through the STING-NF-κB axis, which activated IFN signaling and repressed BMPR2 (bone morphogenetic protein receptor 2) signaling both invitro and invivo. Further analysis revealed that F2RL3 expression was increased in PH settings and identified negative feedback regulation of F2RL3/BMPR2 signaling. Accordingly, a positive correlation of expression amounts between STING and F2RL3/IFN-stimulated genes was observed invivo. Our findings suggest that STING activation in PAECs plays a critical role in the pathobiology of PH. Targeting STING may be a promising therapeutic strategy for preventing the development of PH.

Heritable pulmonary arterial hypertension

Heritable pulmonary arterial hypertension (PAH) can be triggered by at least 18genes. The most frequently altered gene is the bone morphogenetic protein receptor2 (BMPR2). Further genes from the same pathway are also well known PAH-causing genes. Genetic testing can aid to confirm differential diagnoses such as apulmonary veno-occlusive disease. It also enables the testing of healthy family members. In addition to the PAH patient population particularly served by genetic testing, this article touches on the mode of inheritance and provides insights into the first treatments soon on the market that rebalance the BMPR2 signaling pathway.

BMPR2 Mutation and Metabolic Reprogramming in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension forms the first and most severe of the 5 categories of pulmonary hypertension. Disease pathogenesis is driven by progressive remodeling of peripheral pulmonary arteries, caused by the excessive proliferation of vascular wall cells, including endothelial cells, smooth muscle cells and fibroblasts, and perivascular inflammation. Compelling evidence from animal models suggests endothelial cell dysfunction is a key initial trigger of pulmonary vascular remodeling, which is characterised by hyperproliferation and early apoptosis followed by enrichment of apoptosis-resistant populations. Dysfunctional pulmonary arterial endothelial cells lose their ability to produce vasodilatory mediators, together leading to augmented pulmonary arterial smooth muscle cell responses, increased pulmonary vascular pressures and right ventricular afterload, and progressive right ventricular hypertrophy and heart failure. It is recognized that a range of abnormal cellular molecular signatures underpin the pathophysiology of pulmonary arterial hypertension and are enhanced by loss-of-function mutations in the BMPR2 gene, the most common genetic cause of pulmonary arterial hypertension and associated with worse disease prognosis. Widespread metabolic abnormalities are observed in the heart, pulmonary vasculature, and systemic tissues, and may underpin heterogeneity in responsivity to treatment. Metabolic abnormalities include hyperglycolytic reprogramming, mitochondrial dysfunction, aberrant polyamine and sphingosine metabolism, reduced insulin sensitivity, and defective iron handling. This review critically discusses published mechanisms linking metabolic abnormalities with dysfunctional BMPR2 (bone morphogenetic protein receptor 2) signaling; hypothesized mechanistic links requiring further validation; and their relevance to pulmonary arterial hypertension pathogenesis and the development of potential therapeutic strategies.

Novel Pyrazolo[3,4-b] Pyridine Derivative (HLQ2g) Attenuates Hypoxic Pulmonary Hypertension via Restoring cGKI Expression and BMP Signaling Pathway.

Pulmonary hypertension (PH) is an extremely serious cardiopulmonary disease, finally leading to progressive right ventricular failure and death. Our previous studies have nominated HLQ2g, a pyrazolo[3,4-b] pyridine derivative stimulating soluble guanylate cyclase (sGC), as a new candidate for the treatment of PH, but the specific mechanism is still not clear. The PH model induced by hypoxia was established in rats. Right ventricular systolic pressure (RVSP) was assessed by jugular vein catheterization. RV weight was the index to evaluate RV hypertrophy. The protein levels of cGMP-dependent protein kinase type I (cGKI), bone morphogenetic protein receptor 2 (BMPR2), phosphorylated Smad1/5/8 (p-Smad1/5/8), and inhibitor of differention 1 (Id1) in pulmonary artery and human pulmonary artery smooth muscle cells (HPASMCs) were determined by western blotting. Cell proliferation and migration were evaluated. In the whole experiment, the first clinically available sGC stimulator Riociguat was used as the reference. In hypoxic PH rat model, elevated RVSP and RV hypertrophy were significantly reduced by HLQ2g treatment. Both Riociguat and HLQ2g attenuated vascular remodeling accompanied with up-regulated cGKI expression and BMP signaling pathway, which was characterized by elevated expression of BMPR2, p-Smad1/5/8, and Id1 in HPH rats. In addition, HLQ2g inhibited proliferation and migration of HPASMCs induced by hypoxia and platelet-derived growth factor (PDGF), restored BMPR2 signaling, which was recalled by Rp-8-Br-PET-cGMPS, the inhibitor of cGKI. In summary, the novel pyrazolo[3,4-b] pyridine derivative HLQ2g can alleviate HPH progression by up-regulating cGKI protein and BMP signaling pathway.

Bone morphogenetic protein receptor 2 inhibition destabilizes microtubules promoting the activation of lysosomes and cell death of lung cancer cells

BackgroundRecent studies have shown that bone morphogenetic protein receptor 2 (BMPR2) regulates cell survival signaling events in cancer cells independent of the BMP type 1 receptor (BMPR1) or the Smad-1/5 transcription factor. Mutations in BMPR2 trafficking proteins leads to overactive BMP signaling, which leads to neurological diseases caused by BMPR2 stabilization of the microtubules. It is not known whether BMPR2 regulates the microtubules in cancer cells and what effect this has on cell survival. It is also not known whether alterations in BMPR2 trafficking effects activity and response to BMPR2 inhibitors.MethodsWe utilized BMPR2 siRNA and the BMP receptor inhibitors JL5 and Ym155, which decrease BMPR2 signaling and cause its mislocalization to the cytoplasm. Using the JL5 resistant MDA-MD-468 cell line and sensitive lung cancer cell lines, we examined the effects of BMPR2 inhibition on BMPR2 mislocalization to the cytoplasm, microtubule destabilization, lysosome activation and cell survival.ResultsWe show that the inhibition of BMPR2 destabilizes the microtubules. Destabilization of the microtubules leads to the activation of the lysosomes. Activated lysosomes further decreases BMPR2 signaling by causing it to mislocalizated to the cytoplasm and/or lysosome for degradation. Inhibition of the lysosomes with chloroquine attenuates BMPR2 trafficking to the lysosome and cell death induced by BMPR2 inhibitors. Furthermore, in MDA-MD-468 cells that are resistant to JL5 induced cell death, BMPR2 was predominately located in the cytoplasm. BMPR2 failed to localize to the cytoplasm and/or lysosome following treatment with JL5 and did not destabilize the microtubules or activate the lysosomes.ConclusionsThese studies reveal that the inhibition of BMPR2 destabilizes the microtubules promoting cell death of cancer cells that involves the activation of the lysosomes. Resistance to small molecules targeting BMPR2 may occur if the BMPR2 is localized predominantly to the cytoplasm and/or fails to localize to the lysosome for degradation.9X69BgY5xE-34q9NhE79bcVideo

Significance of BMPR2 mutations in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a debilitating disease that results from progressive remodeling and inflammation of pulmonary arteries. PAH develops gradually, is difficult to diagnose, and has a high mortality rate. Although mutation in the bone morphogenetic protein receptor 2 (BMPR2) gene has been identified as the main genetic cause of PAH, the underlying pathways involving the pathophysiology of PAH are complex and still not fully understood. Endothelial dysfunction has been observed in PAH development that results in a multitude of disturbances in the cellular processes in pulmonary vessels. Changes in the pulmonary vasculature caused by the disruption of BMPR2 signaling are observed in three main vascular components; endothelial cells, smooth muscle cells, and fibroblasts. BMPR2 also has a prominent role in maintenance of the immune system. The disruption of BMPR2 signaling pathway causes an increased degree of inflammation and decreases the ability of the immune system to resolve it. Inflammatory processes and changes in pulmonary vasculature interact with one another, resulting in the progression of chronic PAH. In this review, we highlight the various components of vascular remodeling and immune response that are caused by disruption of BMPR2 signaling, including the clinical evidence and the prospects of these components as a potential target for PAH therapy. Indeed, development of drugs to target the pathogenic pathways involved in PAH may complement existing treatment regimens and improve patient prognosis.

Novel Advances in Modifying BMPR2 Signaling in PAH.

Pulmonary Arterial Hypertension (PAH) is a disease of the pulmonary arteries, that is characterized by progressive narrowing of the pulmonary arterial lumen and increased pulmonary vascular resistance, ultimately leading to right ventricular dysfunction, heart failure and premature death. Current treatments mainly target pulmonary vasodilation and leave the progressive vascular remodeling unchecked resulting in persistent high morbidity and mortality in PAH even with treatment. Therefore, novel therapeutic strategies are urgently needed. Loss of function mutations of the Bone Morphogenetic Protein Receptor 2 (BMPR2) are the most common genetic factor in hereditary forms of PAH, suggesting that the BMPR2 pathway is fundamentally important in the pathogenesis. Dysfunctional BMPR2 signaling recapitulates the cellular abnormalities in PAH as well as the pathobiology in experimental pulmonary hypertension (PH). Approaches to restore BMPR2 signaling by increasing the expression of BMPR2 or its downstream signaling targets are currently actively explored as novel ways to prevent and improve experimental PH as well as PAH in patients. Here, we summarize existing as well as novel potential treatment strategies for PAH that activate the BMPR2 receptor pharmaceutically or genetically, increase the receptor availability at the cell surface, or reconstitute downstream BMPR2 signaling.

Targeting CCL5 for Treatment of BMP-Mediated Pulmonary Hypertension

Purpose Pulmonary arterial hypertension (PAH), a progressive disease characterized by progressive obliteration of small pulmonary arteries (PAs) that leads to elevated pulmonary arterial pressure and right heart failure. Most patients with severe PAH demonstrate persistent structural alterations in small PAs, including marked proliferation of pulmonary artery endothelial cells (PAECs), smooth muscle cells (PASMCs) and fibroblasts. Altered BMPR2 signaling is an essential piece in the pathobiologic puzzle of PAH. Reduced bone morphogenetic protein receptor 2 (BMPR2) expression in patients with PAH can impair pulmonary arterial endothelial cells (PAECs) function. This can adversely affect PAEC survival and promote PASMCs proliferation. We hypothesized that interventions to normalize the expression of genes that are targets of the BMPR2 signaling could restore PAECs function and prevent or reverse PAH. Methods Here have characterized for the first time, in human PAECs, chemokine (C-C motif) ligand 5 (CCL5/RANTES) deficiency restore bone morphogenetic protein (BMP)-mediated PAECs function. In cell culture experiments, we showed that CCL5 deficiency increased apoptosis and tube formation of PAECs, whereas it suppressed proliferation and migration of PASMCs. Results Silence the CCL5 expression in PAH PAECs restored BMP signaling responses, promoted phosphorylation of SMADs and transcription of ID genes. Moreover, CCL5 deficiency inhibited angiogenesis by increasing pSMAD-dependent and-independent BMPR2 signaling. This was linked mechanistically to enhanced interaction of BMPR2 with caveolin-1 via CCL5 deficiency-mediated stabilization of endothelial surface caveolin-1. Consistent with these functions, deletion of CCL5 significantly attenuated development of Sugen5416/hypoxia-induced PAH by restoring BMPR2 signaling in mice. Conclusion Taken together, our findings suggest that CCL5 deficiency could reverses obliterative changes in pulmonary arteries via caveolin-1-dependent amplification of BMPR2 signaling, shed a new light on our understanding of the disease pathobiology, and provides a possible novel therapeutic target for PAH. ADDIN EN.REFLIST.

HMGB1/TLR4 promotes hypoxic pulmonary hypertension via suppressing BMPR2 signaling

High mobility group box 1 (HMGB1), a critical nonclassical inflammatory cytokine, has been found up-regulated in patients with idiopathic pulmonary arterial hypertension (IPAH), but its role in vascular remodeling of pulmonary hypertension (PH) is still unknown. In present study, we demonstrated that the plasma level of inflammatory cytokine including HMGB1, interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) were elevated in hypoxia-induced pulmonary hypertension rats model. Moreover, expressions of HMGB1 and Toll like receptor-4 (TLR4) in pulmonary arteries were obviously up-regulated accompanied with down-regulation of bone morphogenetic protein receptor 2 (BMPR2) signaling, characterized by decline of phosphorylated Smad1/5/8 (p-Smsd1/5/8) and inhibitor of differention 1 (Id1) expression. In cultured primary pulmonary arterial smooth muscle cells (PASMCs), we found that HMGB1 incubation significantly promoted proliferation and migration of PASMCs, down-regulated p-Smsd1/5/8 and Id1 expression, which can be abrogated by HMGB1 inhibitors saquinavir, glycyrrhizn and TLR4 inhibitors TAK-242. Furthermore, saquinavir, glycyrrhizn and TAK-242 treatment significantly attenuated the development of PH in rats by recovering homodynamic parameters, pulmonary vascular remodeling and BMPR2 signaling pathway. In summary, our results suggest that HMGB1/TLR4 signaling promotes hypoxia-induced pulmonary hypertension via suppressing BMPR2 signaling.

A pro-con debate: current controversies in PAH pathogenesis at the American Thoracic Society International Conference in 2017.

The following review summarizes the pro-con debate about current controversies regarding the pathogenesis of pulmonary arterial hypertension (PAH) that took place at the American Thoracic Society Conference in May 2017. Leaders in the field of PAH research discussed the importance of the immune system, the role of hemodynamic stress and endothelial apoptosis, as well as bone morphogenetic protein receptor-2 signaling in PAH pathogenesis. Whereas this summary does not intend to resolve obvious conflicts in opinion, we hope that the presented arguments entice further discussions and draw a new generation of enthusiastic researchers into this vibrant field of science to bridge existing gaps for a better understanding and therapy of this fatal disease.

CCL5 deficiency rescues pulmonary vascular dysfunction, and reverses pulmonary hypertension via caveolin-1-dependent BMPR2 activation

Pulmonary arterial hypertension (PAH) is a devastating cardiopulmonary disorder characterized by pulmonary arterial remodeling mainly due to excess cellular proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Reduced bone morphogenetic protein receptor 2 (BMPR2) expression in patients with PAH impairs pulmonary arterial endothelial cells (PAECs) function. This can adversely affect PAEC survival and promote PASMCs proliferation. We hypothesized that interventions to normalize the expression of genes that are targets of the BMPR2 signaling could restore PAECs function and prevent or reverse PAH. Here we characterized for the first time, in human PAECs, chemokine (C-C motif) ligand 5 (CCL5/RANTES) deficiency restore BMP-mediated PAECs function.In the cell culture experiments, we found that CCL5 deficiency increased apoptosis and tube formation of PAECs, but suppressed proliferation and migration of PASMCs. Silencing CCL5 expression in PAH PAECs restored bone morphogenetic protein (BMP) signaling responses and promoted phosphorylation of SMADs and transcription of ID genes. Moreover, CCL5 deficiency inhibited angiogenesis by increasing pSMAD-dependent and-independent BMPR2 signaling. This was linked mechanistically to enhanced interaction of BMPR2 with caveolin-1 via CCL5 deficiency-mediated stabilization of endothelial surface caveolin-1. Consistent with these functions, deletion of CCL5 significantly attenuated development of Sugen5416/hypoxia-induced PAH by restoring BMPR2 signaling in mice. Taken together, our findings suggest that CCL5 deficiency could reverse obliterative changes in pulmonary arteries via caveolin-1-dependent amplification of BMPR2 signaling. Our results shed light on better understanding of the disease pathobiology and provide a possible novel target for the treatment of PAH.

In Pulmonary Arterial Hypertension, Reduced BMPR2 Promotes Endothelial-to-Mesenchymal Transition via HMGA1 and Its Target Slug

We previously reported high-throughput RNA sequencing analyses that identified heightened expression of the chromatin architectural factor High Mobility Group AT-hook 1 (HMGA1) in pulmonary arterial endothelial cells (PAECs) from patients who had idiopathic pulmonary arterial hypertension (PAH) in comparison with controls. Because HMGA1 promotes epithelial-to-mesenchymal transition in cancer, we hypothesized that increased HMGA1 could induce transition of PAECs to a smooth muscle (SM)-like mesenchymal phenotype (endothelial-to-mesenchymal transition), explaining both dysregulation of PAEC function and possible cellular contribution to the occlusive remodeling that characterizes advanced idiopathic PAH. We documented increased HMGA1 in PAECs cultured from idiopathic PAH versus donor control lungs. Confocal microscopy of lung explants localized the increase in HMGA1 consistently to pulmonary arterial endothelium, and identified many cells double-positive for HMGA1 and SM22α in occlusive and plexogenic lesions. Because decreased expression and function of bone morphogenetic protein receptor 2 (BMPR2) is observed in PAH, we reduced BMPR2 by small interfering RNA in control PAECs and documented an increase in HMGA1 protein. Consistent with transition of PAECs by HMGA1, we detected reduced platelet endothelial cell adhesion molecule 1 (CD31) and increased endothelial-to-mesenchymal transition markers, αSM actin, SM22α, calponin, phospho-vimentin, and Slug. The transition was associated with spindle SM-like morphology, and the increase in αSM actin was largely reversed by joint knockdown of BMPR2 and HMGA1 or Slug. Pulmonary endothelial cells from mice with endothelial cell-specific loss of Bmpr2 showed similar gene and protein changes. Increased HMGA1 in PAECs resulting from dysfunctional BMPR2 signaling can transition endothelium to SM-like cells associated with PAH.

Elafin Reverses Pulmonary Hypertension via Caveolin-1-Dependent Bone Morphogenetic Protein Signaling.

Pulmonary arterial hypertension is characterized by endothelial dysfunction, impaired bone morphogenetic protein receptor 2 (BMPR2) signaling, and increased elastase activity. Synthetic elastase inhibitors reverse experimental pulmonary hypertension but cause hepatotoxicity in clinical studies. The endogenous elastase inhibitor elafin attenuates hypoxic pulmonary hypertension in mice, but its potential to improve endothelial function and BMPR2 signaling, and to reverse severe experimental pulmonary hypertension or vascular pathology in the human disease was unknown. To assess elafin-mediated regression of pulmonary vascular pathology in rats and in lung explants from patients with pulmonary hypertension. To determine if elafin amplifies BMPR2 signaling in pulmonary artery endothelial cells and to elucidate the underlying mechanism. Rats with pulmonary hypertension induced by vascular endothelial growth factor receptor blockade and hypoxia (Sugen/hypoxia) as well as lung organ cultures from patients with pulmonary hypertension were used to assess elafin-mediated reversibility of pulmonary vascular disease. Pulmonary arterial endothelial cells from patients and control subjects were used to determine the efficacy and mechanism of elafin-mediated BMPR2 signaling. In Sugen/hypoxia rats, elafin reduced elastase activity and reversed pulmonary hypertension, judged by regression of right ventricular systolic pressure and hypertrophy and pulmonary artery occlusive changes. Elafin improved endothelial function by increasing apelin, a BMPR2 target. Elafin induced apoptosis in human pulmonary arterial smooth muscle cells and decreased neointimal lesions in lung organ culture. In normal and patient pulmonary artery endothelial cells, elafin promoted angiogenesis by increasing pSMAD-dependent and -independent BMPR2 signaling. This was linked mechanistically to augmented interaction of BMPR2 with caveolin-1 via elafin-mediated stabilization of endothelial surface caveolin-1. Elafin reverses obliterative changes in pulmonary arteries via elastase inhibition and caveolin-1-dependent amplification of BMPR2 signaling.

Loss of Bone Morphogenetic Protein Receptor 2 Is Associated with Abnormal DNA Repair in Pulmonary Arterial Hypertension

Occlusive vasculopathy with intimal hyperplasia and plexogenic arteriopathy are severe histopathological changes characteristic of pulmonary arterial hypertension (PAH). Although a phenotypic switch in pulmonary endothelial cells (ECs) has been suggested to play a critical role in the formation of occlusive lesions, the pathobiology of this process is poorly understood. The goal of this study was to identify novel molecular mechanisms associated with EC dysfunction and PAH-associated bone morphogenetic protein receptor 2 (BMPR2) deficiency during PAH pathogenesis. A bioinfomatics approach, patient samples, and in vitro experiments were used. By combining a metaanalysis of human idiopathic PAH (iPAH)-associated gene-expression microarrays and a unique gene expression-profiling technique in rat endothelium, our bioinformatics approach revealed a PAH-associated dysregulation of genes involving chromatin organization, DNA metabolism, and repair. Our hypothesis that altered DNA repair and loss of genomic stability play a role in PAH was supported by in vitro assays where pulmonary ECs from patients with iPAH and BMPR2-deficient ECs were highly susceptible to DNA damage. Furthermore, we showed that BMPR2 expression is tightly linked to DNA damage control because excessive DNA damage leads to rapid down-regulation of BMPR2 expression. Moreover, we identified breast cancer 1 (BRCA1) as a novel target for BMPR2 signaling and a novel modulator of pulmonary EC homeostasis. We show here that BMPR2 signaling plays a critical role in the regulation of genomic integrity in pulmonary ECs via genes such as BRCA1. We propose that iPAH-associated EC dysfunction and genomic instability are mediated through BMPR2 deficiency-associated loss of DNA damage control.

Repurposing FK506 to Increase BMPR2 Signaling and Improve Pulmonary Arterial Hypertension: A Fast Track From Cells to People

To improve long-term survival in pulmonary arterial hypertension (PAH), new treatment approaches that target occlusive vasculopathy instead of vasoconstriction of pulmonary arteries are necessary. Identifying genes or pathways that unify different pathologies and etiologies in PAH is a crucial first step for drug development. The bone morphogenetic protein receptor 2 (BMPR2) pathway, originally described as the cause of familial PAH, has gained significant interest over the past few years as a potential master switch in PAH, and therefore presents a promising treatment target in PAH. FK506 (tacrolimus) was found in a high-throughput screen of US Food and Drug Administration (FDA)-approved drugs to significantly increase BMPR2 signaling. Low-dose FK506 was able to restore normal BMPR2 signaling and function in patient pulmonary artery (PA) endothelial cells, inhibit proliferation and induce apoptosis in PA smooth muscle cells, and prevent and reverse pulmonary hypertension (PH) in 3 experimental rodent models of PH. As an FDA-approved drug, it was possible to relatively quickly (in approximately 2 years) translate the basic science discoveries from the bench to the clinic. A Phase 2 proof-of-concept safety and tolerability trial is underway (ClinicalTrials.gov Identifier: NCT01647945) to evaluate the use of low-dose FK506 in PAH and to identify patients who might respond best to FK506 depending on their impairment in BMPR2 signaling.

FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension

Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.

Disruption of the Bone Morphogenetic Protein Receptor 2 Pathway in Nitrofen‐Induced Congenital Diaphragmatic Hernia

Congenital diaphragmatic hernia (CDH) remains a major therapeutic challenge despite advances in neonatal resuscitation and intensive care. The high mortality and morbidity in CDH has been attributed to pulmonary hypoplasia and persistent pulmonary hypertension (PH). Bone morphogenetic protein receptor 2 (BMPR2) plays a key role in pulmonary vasculogenesis during the late stages of fetal lung development. BMPR2 is essential for control of endothelial and smooth muscle cell proliferation. Dysfunction of BMPR2 and downstream signaling have been shown to disturb the crucial balance of proliferation of smooth muscle cells contributing to the pathogenesis of human and experimental PH. We designed this study to investigate the hypothesis that BMPR2 signaling is disrupted in nitrofen-induced CDH. Pregnant rats were treated with nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and divided into CDH and control. Quantitative real-time polymerase chain reaction, Western blotting, and confocal-immunofluorescence were performed to determine pulmonary gene expression levels and protein expression of BMPR2 and related proteins. Pulmonary Bmpr2 gene expression levels were significantly decreased in nitrofen-induced CDH compared to controls. Western blotting and confocal microscopy revealed decreased pulmonary BMPR2 protein expression and increased activation of p38(MAPK) in CDH compared to controls. The observed disruption of the BMPR2 signaling pathway may lead to extensive vascular remodeling and contribute to PH in the nitrofen-induced CDH model. BMPR2 may therefore represent a potential target for the treatment of PH in CDH.

Disruption of PPARγ/β-catenin–mediated regulation of apelin impairs BMP-induced mouse and human pulmonary arterial EC survival

Reduced bone morphogenetic protein receptor 2 (BMPR2) expression in patients with pulmonary arterial hypertension (PAH) can impair pulmonary arterial EC (PAEC) function. This can adversely affect EC survival and promote SMC proliferation. We hypothesized that interventions to normalize expression of genes that are targets of BMPR2 signaling could restore PAEC function and prevent or reverse PAH. Here we have characterized, in human PAECs, a BMPR2-mediated transcriptional complex between PPARγ and β-catenin and shown that disruption of this complex impaired BMP-mediated PAEC survival. Using whole genome-wide ChIP-Chip promoter analysis and gene expression microarrays, we delineated PPARγ/β-catenin-dependent transcription of target genes including APLN, which encodes apelin. We documented reduced PAEC expression of apelin in PAH patients versus controls. In cell culture experiments, we showed that apelin-deficient PAECs were prone to apoptosis and promoted pulmonary arterial SMC (PASMC) proliferation. Conversely, we established that apelin, like BMPR2 ligands, suppressed proliferation and induced apoptosis of PASMCs. Consistent with these functions, administration of apelin reversed PAH in mice with reduced production of apelin resulting from deletion of PPARγ in ECs. Taken together, our findings suggest that apelin could be effective in treating PAH by rescuing BMPR2 and PAEC dysfunction.

Activation of AKT and endothelial nitric oxide synthase by BMP2 and BMP4 in bovine pulmonary artery endothelial cells

Mutations in the bone morphogenetic protein (BMP) receptor‐2 (BMPR2) are causally linked to familial pulmonary arterial hypertension (PAH). The mechanistic link between loss of BMPR2 signaling and development of PAH, however, remains unclear. Recently it was demonstrated that BMPR2 signaling promotes survival in pulmonary artery endothelial cells (ECs). Activation of the protein kinase AKT and its downstream target endothelial nitric oxide synthase (eNOS) are known to promote EC survival. We hypothesized that treatment of ECs with the BMPR2 ligands, BMP2 or BMP4, would result in activation of AKT and eNOS and result in nitric oxide (NO) release from ECs. Serum starved ECs were treated with vascular endothelial growth factor (VEGF; 50 ng/ml), BMP2 (30 ng/ml) or BMP4 (30 ng/ml) for 0, 5, 15, or 30 min. Western blot analysis revealed that both AKT and eNOS were phosphorylated in response to BMP2 and BMP4 similar to the AKT/eNOS activator VEGF. Likewise BMP2 and BMP4 treatment resulted in increased NO release from ECs as determined by nitrite accumulation in the cell culture medium. These data provide the first evidence that BMP signaling leads activation of this important survival pathway in ECs and represent a possible mechanism by which loss of BMPR2 signaling could lead to the pathogenesis of PAH. This work was supported by seed funds provided by the Department of Surgery, University of Pittsburgh School of Medicine.

Bone Morphogenetic Protein Receptor-2 Signaling Promotes Pulmonary Arterial Endothelial Cell Survival

Mutations in the bone morphogenetic protein (BMP) receptor-2 (BMPR2) have been found in patients with idiopathic pulmonary arterial hypertension (IPAH); however, the mechanistic link between loss of BMPR2 signaling and the development of pulmonary arterial hypertension is unclear. We hypothesized that, contrary to smooth muscle cells, this pathway promotes survival in pulmonary artery endothelial cells (ECs) and loss of BMPR2 signaling will predispose to EC apoptosis. ECs were treated with BMP-2 or BMP-7 (200 ng/mL) for 24 hours in regular or serum-free (SF) medium, with and without addition of tumor necrosis factor alpha, and apoptosis was assessed by flow cytometry (Annexin V), TUNEL, or caspase-3 activity. Treatment for 24 hours in SF medium increased apoptosis, and both BMP-2 and BMP-7 significantly reduced apoptosis in response to serum deprivation to levels not different from serum controls. Transfection with 5 microg of small interfering RNAs for BMPR2 produced specific gene silencing assessed by RT-PCR and Western blot analysis. BMPR2 gene silencing increased apoptosis almost 3-fold (P=0.0027), even in the presence of serum. Circulating endothelial progenitor cells (EPCs) isolated from normal subjects or patients with IPAH were differentiated in culture for 7 days and apoptosis was determined in the presence and absence of BMPs. BMP-2 reduced apoptosis induced by serum withdrawal in EPCs from normal subjects but not in EPCs isolated from patients with IPAH. These results support the hypothesis that loss-of-function mutations in BMPR2 could lead to increased pulmonary EC apoptosis, representing a possible initiating mechanism in the pathogenesis of pulmonary arterial hypertension.