BMPR2 haploinsufficiency as the inherited molecular mechanism for primary pulmonary hypertension

Primary pulmonary hypertension (PPH) is a potentially lethal disorder, because the elevation of the pulmonary arterial pressure may result in right-heart failure. Histologically, the disorder is characterized by proliferation of pulmonary-artery smooth muscle and endothelial cells, by intimal hyperplasia, and by in situ thrombus formation. Heterozygous mutations within the bone morphogenetic protein type II receptor (BMPR-II) gene (BMPR2), of the transforming growth factor beta (TGF-beta) cell-signaling superfamily, have been identified in familial and sporadic cases of PPH. We report the molecular spectrum of BMPR2 mutations in 47 additional families with PPH and in three patients with sporadic PPH. Among the cohort of patients, we have identified 22 novel mutations, including 4 partial deletions, distributed throughout the BMPR2 gene. The majority (58%) of mutations are predicted to lead to a premature termination codon. We have also investigated the functional impact and genotype-phenotype relationships, to elucidate the mechanisms contributing to pathogenesis of this important vascular disease. In vitro expression analysis demonstrated loss of BMPR-II function for a number of the identified mutations. These data support the suggestion that haploinsufficiency represents the common molecular mechanism in PPH. Marked variability of the age at onset of disease was observed both within and between families. Taken together, these studies illustrate the considerable heterogeneity of BMPR2 mutations that cause PPH, and they strongly suggest that additional factors, genetic and/or environmental, may be required for the development of the clinical phenotype.     

Truncating and missense BMPR2 mutations differentially affect the severity of heritable pulmonary arterial hypertension

Background

Autosomal dominant inheritance of germline mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene are a major risk factor for pulmonary arterial hypertension (PAH). While previous studies demonstrated a difference in severity between BMPR2 mutation carriers and noncarriers, it is likely disease severity is not equal among BMPR2 mutations. We hypothesized that patients with missense BMPR2 mutations have more severe disease than those with truncating mutations.

Methods

Testing for BMPR2 mutations was performed in 169 patients with PAH (125 with a family history of PAH and 44 with sporadic disease). Of the 106 patients with a detectable BMPR2 mutation, lymphocytes were available in 96 to functionally assess the nonsense-mediated decay pathway of RNA surveillance. Phenotypic characteristics were compared between BMPR2 mutation carriers and noncarriers, as well as between those carriers with a missense versus truncating mutation.

Results

While there was a statistically significant difference in age at diagnosis between carriers and noncarriers, subgroup analysis revealed this to be the case only for females. Among carriers, there was no difference in age at diagnosis, death, or survival according to exonic location of the BMPR2 mutation. However, patients with missense mutations had statistically significant younger ages at diagnosis and death, as well as shorter survival from diagnosis to death or lung transplantation than those with truncating mutations. Consistent with this data, the majority of missense mutations were penetrant prior to age 36 years, while the majority of truncating mutations were penetrant after age 36 years.

Conclusion

In this cohort, BMPR2 mutation carriers have more severe PAH disease than noncarriers, but this is only the case for females. Among carriers, patients with missense mutations that escape nonsense-mediated decay have more severe disease than those with truncating mutations. These findings suggest that treatment and prevention strategies directed specifically at BMPR2 pathway defects may need to vary according to the type of mutation.     

Retinoic acid down-regulates VPAC(1) receptors and TGF-beta 3 but up-regulates TGF-beta 2 in lung cancer cells

The effects of retinoic acid (RA) on lung cancer cells were investigated. Both all-trans (t-RA) and 13-cis RA (c-RA) decreased specific ¹²⁵I-VIP binding to NCI-H1299 cells in a time- and concentration-dependent manner. After 20 hr, 30 μM t-RA decreased specific ¹²⁵I-VIP binding by 60%. By Scatchard analysis, the density of VIP binding sites but not the affinity was reduced by 42%. NCI-H1299 VPAC₁ receptor mRNA was reduced by 48%. VIP caused a 3-fold elevation in the NCI-H1299 cAMP, and the increase in cAMP caused by VIP was reduced by 38% if the NCI-H1299 cells were treated with t-RA. Using the MTT assay, 3 μM t-RA and 3 μM c-RA inhibited NCI-H1299 proliferation by 60 and 23% respectively. Also, transforming growth factor (TGF)-β2 increased after treatment of NCI-H1299 cells with t-RA whereas TGF-β1 mRNA was unaffected and TGF-β3 mRNA was decreased. These results suggest that RA may inhibit lung cancer growth by down-regulating VPAC₁ receptor and TGF-β3 mRNA but up-regulating TGF-β2 mRNA.     

Inflammation, endothelial injury, and persistent pulmonary hypertension in heterozygous BMPR2-mutant mice

Heterozygous bone morphogenetic protein receptor-II-knockout (BMPR2(+/-)) mice have a similar genetic trait like that in some idiopathic pulmonary arterial hypertension patients. To examine the effect of pulmonary endothelial injury in BMPR2(+/-) mice, we challenged the mice with two injections of monocrotaline combined with intratracheal instillation of replication-deficient adenovirus expressing 5-lipoxygenase (MCT+Ad5LO). After the challenge (1 wk), BMPR2(+/-) mice exhibited a doubling of right ventricular systolic pressure that was greater than that of wild-type mice and remained elevated for 3 wk before heart failure developed. Muscularization and thickening of small pulmonary arterioles was evident in the BMPR2(+/-) lungs at 2 wk after the challenge and became severe at 3 wk. Marked perivascular infiltration of T cells, B cells, and macrophages was associated with the remodeled vessels. Real-time PCR analysis showed that the expression of six endothelial cell markers in lung tissue was decreased to 20-40% of original levels at 1 wk after the challenge in both BMPR2(+/-) and wild-type mice and largely recovered in wild-type (50-80%) but not BMPR2(+/-) lungs (30-50%) at 3 wk after the challenge. Macrophage inflammatory protein-1alpha and fractalkine receptor expression doubled in BMPR2(+/-) compared with wild-type lungs. Expression of type I and type II BMP receptors, but not transforming growth factor-beta receptors, in the challenged BMPR2(+/-) and wild-type lungs showed a similar pattern of expression as that of endothelial markers. Apoptotic responses at 1 wk after MCT and Ad5LO challenge were also significantly greater in the BMPR2(+/-) lungs than the wild-type lungs. These data show that BMPR2(+/-) mice are more sensitive to MCT+Ad5LO-induced pulmonary hypertension than wild-type mice. Greater endothelial injury and an enhanced inflammatory response could be the underlying causes of the sensitivity and may work in concert with BMPR2 heterozygosity to promote the development of persistent pulmonary hypertension.     

Alterations in TGF-beta1 expression in lambs with increased pulmonary blood flow and pulmonary hypertension

The mechanisms responsible for pulmonary vascular remodeling in congenital heart disease with increased pulmonary blood flow remain unclear. We developed a lamb model of congenital heart disease and increased pulmonary blood flow utilizing an in utero placed aortopulmonary vascular graft (shunted lambs). Morphometric analysis of barium-injected pulmonary arteries indicated that by 4 wk of age, shunts had twice the pulmonary arterial density of controls (P < 0.05), and their pulmonary vessels showed increased muscularization and medial thickness at both 4 and 8 wk of age (P < 0.05). To determine the potential role of TGF-beta1 in this vascular remodeling, we investigated vascular changes in expression and localization of TGF-beta1 and its receptors TbetaRI, ALK-1, and TbetaRII in lungs of shunted and control lambs at 1 day and 1, 4, and 8 wk of life. Western blots demonstrated that TGF-beta1 and ALK-1 expression was elevated in shunts compared with control at 1 and 4 wk of age (P < 0.05). In contrast, the antiangiogenic signaling receptor TbetaRI was decreased at 4 wk of age (P < 0.05). Immunohistochemistry demonstrated shunts had increased TGF-beta1 and TbetaRI expression in smooth muscle layer and increased TGF-beta1 and ALK-1 in endothelium of small pulmonary arteries at 1 and 4 wk of age. Moreover, TbetaRI expression was significantly reduced in endothelium of pulmonary arteries in the shunt at 1 and 4 wk. Our data suggest that increased pulmonary blood flow dysregulates TGF-beta1 signaling, producing imbalance between pro- and antiangiogenic signaling that may be important in vascular remodeling in shunted lambs.     

Chronic allergic inflammation causes vascular remodeling and pulmonary hypertension in BMPR2 hypomorph and wild-type mice

Loss-of-function mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene have been identified in patients with heritable pulmonary arterial hypertension (PAH); however, disease penetrance is low, suggesting additional factors play a role. Inflammation is associated with PAH and vascular remodeling, but whether allergic inflammation triggers vascular remodeling in individuals with BMPR2 mutations is unknown. Our goal was to determine if chronic allergic inflammation would induce more severe vascular remodeling and PAH in mice with reduced BMPR-II signaling. Groups of Bmpr2 hypomorph and wild-type (WT) Balb/c/Byj mice were exposed to house dust mite (HDM) allergen, intranasally for 7 or 20 weeks to generate a model of chronic inflammation. HDM exposure induced similar inflammatory cell counts in all groups compared to controls. Muscularization of pulmonary arterioles and arterial wall thickness were increased after 7 weeks HDM, more severe at 20 weeks, but similar in both groups. Right ventricular systolic pressure (RVSP) was measured by direct cardiac catheterization to assess PAH. RVSP was similarly increased in both HDM exposed groups after 20 weeks compared to controls, but not after 7 weeks. Airway hyperreactivity (AHR) to methacholine was also assessed and interestingly, at 20 weeks, was more severe in HDM exposed Bmpr2 hypomorph mice versus WT. We conclude that chronic allergic inflammation caused PAH and while the severity was mild and similar between WT and Bmpr2 hypomorph mice, AHR was enhanced with reduced BMPR-II signaling. These data suggest that vascular remodeling and PAH resulting from chronic allergic inflammation occurs independently of BMPR-II pathway alterations.     

Cytoskeletal defects in Bmpr2-associated pulmonary arterial hypertension

The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.     

ANG-1 TIE-2 and BMPR signalling defects are not seen in the nitrofen model of pulmonary hypertension and congenital diaphragmatic hernia

Background

Pulmonary hypertension (PH) is a lethal disease that is associated with characteristic histological abnormalities of the lung vasculature and defects of angiopoetin-1 (ANG-1), TIE-2 and bone morphogenetic protein receptor (BMPR)-related signalling. We hypothesized that if these signalling defects cause PH generically, they will be readily identifiable perinatally in congenital diaphragmatic hernia (CDH), where the typical pulmonary vascular changes are present before birth and are accompanied by PH after birth.

Methods

CDH (predominantly left-sided, LCDH) was created in Sprague-Dawley rat pups by e9.5 maternal nitrofen administration. Left lungs from normal and LCDH pups were compared at fetal and postnatal time points for ANG-1, TIE-2, phosphorylated-TIE-2, phosphorylated-SMAD1/5/8 and phosphorylated-ERK1/2 by immunoprecipitation and Western blotting of lung protein extracts and by immunohistochemistry on lung sections.

Results

In normal lung, pulmonary ANG-1 protein levels fall between fetal and postnatal life, while TIE-2 levels increase. Over the corresponding time period, LCDH lung retained normal expression of ANG-1, TIE-2, phosphorylated-TIE-2 and, downstream of BMPR, phosphorylated-SMAD1/5/8 and phosphorylated-p44/42.

Conclusion

In PH and CDH defects of ANG-1/TIE-2/BMPR-related signalling are not essential for the lethal vasculopathy.     

Right ventricular size and function under riociguat in pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension (the RIVER study)

Background

Riociguat is a soluble guanylate cyclase stimulator approved for pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTPEH). The objective of this study was to evaluate right heart size and function assessed by echocardiography during long term treatment with riociguat.

Methods

Patients who started riociguat treatment (1.0–2.5?mg tid) within the trials phase II, PATENT, PATENTplus, EAS, CHEST and continued treatment for 3–12?months were included in this study. Echocardiography was analysed off-line at baseline, after 3, 6 and 12?months by investigators who were blinded to clinical data. Last and baseline observation carried forward method (LOCF, BOCF) were performed as sensitivity analysis.

Results

Seventy-one patients (45% PAH, 55% CTEPH; 53.5% female; 60?±?13?years, mean pulmonary arterial pressure 46?±?10?mmHg, mean PVR 700?±?282dynes·sec·cm-5) were included. After 6?months, RA and RV area, RV thickness tricuspid regurgitation velocity showed a significant reduction. After 12?months, patients receiving riociguat therapy showed a significant reduction in right atrial (??2.6?±?4.4?cm2, 95% CI -3.84, ??1.33; p?<?0.001, n?=?49) and right ventricular (RV) area (??3.5?±?5.2?cm2, 95% CI -5.1, ??1.9; p?<?0.001; n?=?44), RV thickness (??0.76?±?2.2?mm, 95% CI -1.55, 0.03; n?=?32), and a significant increase in TAPSE (2.95?±?4.78?mm, 95% CI 1.52, 4.39; n?=?45) and RV fractional area change (8.12?±?8.87?mm, 95% CI 4.61, 11.62; n?=?27).Both LOCF and BOCF showed similar results but lower effect sizes.

Conclusion

Patients under long-term treatment with riociguat show significantly reduced right heart size and improved RV function in PAH and CTEPH. Further controlled prospective studies are needed to confirm these results.

     

Nitric oxide and pulmonary arterial pressures in pulmonary hypertension

Decreased production of vasodilator substances such as nitric oxide (NO) has been proposed as important in development of pulmonary arterial hypertension (PAH). We hypothesize that NO measured over time serves as a non invasive marker of severity of PAH and response to therapy. We prospectively and serially measured exhaled NO and carbon monoxide (CO), a vasodilator and anti-inflammatory product of heme oxygenases, in 17 PAH patients in conjunction with hemodynamic parameters over 2 years. Although pulmonary artery pressures and NO were similar in all patients at entry to the study, NO increased in the 12 individuals who survived to complete the study, and correlated with change in pulmonary artery pressures. In contrast, CO did not change or correlate with hemodynamic parameters. Investigation of NO-oxidant reaction products in PAH in comparison to controls suggests that NO synthesis is impaired in the lung and that reactive oxygen species may be involved in the pathophysiology of pulmonary hypertension. Endogenous NO is inversely related to pulmonary artery pressure in PAH, with successful therapy of PAH associated with increase in NO.     

t-plasminogen activator inhibitor-1 polymorphism in idiopathic pulmonary arterial hypertension

AIM:

The aim of the present study was to identify the possible genotypic association of 3’UTR Hind III polymorphism of Plasminogen activator Inhibitor-1 (PAI-1) gene with idiopathic pulmonary arterial hypertension (IPAH).

BACKGROUND:

IPAH is a disorder with abnormally raised mean pulmonary arterial pressure and increase in the resistance to blood flow in pulmonary artery. One of the pathological features seen is development of intraluminal thrombin deposition leading to thrombosis. Plasminogen activator inhibitor-1 is an important inhibitor of the fibrinolytic system; its up-regulation may suppress fibrinolysis and result in an increased risk of thrombosis.

METHOD:

Blood samples from 54 IPAH patients and 100 healthy voluntary donors were analyzed by PCR-RFLP method for 3’UTR Hind III polymorphism.

RESULTS AND DISSCUSSION:

A significant association of Hd2 allele with the disease was observed. Raised mean level of right ventricular systolic pressure was observed in the Hd2/Hd2 genotypic patients, strengthening the role of Hd2 allele in the disease progression. Our data suggests an association of Hd2/Hd2 genotype, which may lead to the up-regulation of PAI-1 gene leading to increased levels of PAI-1, which is seen in IPAH. PAI-1 competes with plasminogen activators and hinders the normal mechanism of plasminogen activation system and leads to thrombosis and formation of plexiform lesions in the lung tissue, further strengthening its role in tissue remodeling and disease progression.     

Assignment of the rat genes coding for DOPA decarboxylase (DDC) and glutamic acid decarboxylases (GAD1 and GAD2)

By use of rat cDNA probes and a panel of cell hybrids segregating rat chromosomes, the genes encoding three pyridoxal 5-phosphate (PLP)-dependent decarboxylases—namely, DOPA-decarboxylase (Ddc), glutamic acid decarboxylase 1 and 2 (Gad1 and Gad2)—were assigned to rat Chromosomes (Chrs) 14, 3, and 17, respectively. If one takes into account chromosome localizations in the human and the mouse, the present results (i) show that a synteny group is retained on rat Chr 14, human Chr 7, and mouse Chr 11 (Ddc); (ii) strengthen the homology relation known between rat Chr 3 and human and mouse Chrs 2 (Gad1); (iii) suggest that rat Chr 17 has no extensive homology to any human chromosome; and (iv) suggest the order (Prl, Fdp)-Tpl2-Gad2 on the rat Chr 17.     

TRPC6介导肺动脉高压大鼠肺动脉张力和肺动脉平滑肌细胞Ca~(2+)浓度的升高

经典瞬时感受器电位通道6(transient receptor potential channel6,TRPC6)蛋白是受体操纵性Ca2+通道(ROCC)的分子基础。本文旨在研究TRPC6/ROCC在野百合碱(monocrotaline,MCT)诱发的肺动脉高压大鼠模型中的作用。Sprague-Dawley大鼠随机分为正常对照组(CON组)和MCT组,CON组正常饲养三周,而MCT组按60mg/kg剂量一次性腹腔注射2%MCT,建立MCT诱导的慢性肺动脉高压大鼠模型。通过测定右心室收缩压(RVSP)和右心室重量指数(RVMI)、HE染色观察肺动脉血管形态,分析肺动脉结构重建。半定量RT-PCR和Western blot检测大鼠肺动脉TRPC6 mRNA和蛋白表达水平。血管张力实验中用可特异性激活ROCC、可透膜的DAG拟似物1-oleoyl-2-acetyl-sn-glycerol(OAG)检测大鼠离体肺动脉环的收缩效应。用荧光探针Fluo3-AM测定OAG诱导大鼠肺动脉平滑肌细胞(PASMCs)胞浆游离Ca2+浓度([Ca2+]i)。结果显示,与CON组相比,MCT组的RVSP、RVMI均明显增高(P0.01);形态学观察可见肺小动脉平滑肌层明显增厚,管腔减小;TRPC6的mRNA和蛋白质表达无明显变化。在CON组,OAG几乎不引起肺动脉环收缩,而在MCT组,肺动脉环的收缩反应显著增强,差别有显著性意义(P0.01)。相比较于CON组,MCT也可使OAG触发的PASMCs[Ca2+]i增量值显著升高(P0.05)。上述结果提示,MCT预处理对肺动脉TRPC6mRNA和蛋白质水平的表达无显著增强效应,但可促进TRPC6/ROCC介导的PASMCsCa2+内流和肺动脉张力升高,诱导大鼠产生肺动脉高压,并进一步诱发肺血管及右心室重构。     

Mouse smad8 phosphorylation downstream of BMP receptors ALK-2, ALK-3, and ALK-6 induces its association with Smad4 and transcriptional activity

Smads are intracellular signaling mediators for TGF-beta superfamily. Smad1 and Smad5 are activated by BMP receptors. Here, we have cloned mouse Smad8 and functionally characterized its ability to transduce signals from BMP receptors. Constitutively active BMP type I receptors, ALK-3 and ALK-6, as well as ALK-2, were phosphorylated Smad8 and induced Smad8 interaction with Smad4. Nuclear translocation of Smad8 was stimulated by constitutively active BMP type I receptors. In contrast, constitutively active TGF-beta type I receptor, ALK-5, did not exhibit any action on Smad8. Smad8 and Smad4 cooperatively induced the promoter of Xvent2, a homeobox gene that responds specifically to BMP signaling. Dominant-negative Smad8 was shown to inhibit the increase of alkaline phosphatase activity induced by BMP-2 on pluripotent mesenchymal C3H10T1/2 and myoblastic C2C12 cell lines. The presence of Smad8 mRNA in mouse calvaria cells and osteoblasts suggests a role of Smad8 in the osteoblast differentiation and maturation.     

Trapping of BMP receptors in distinct membrane domains inhibits their function in pulmonary arterial hypertension

Bone morphogenetic proteins (BMPs) are pleiotrophic growth factors that influence diverse processes such as skeletal development, hematopoiesis, and neurogenesis. They play crucial roles in diseases such as pulmonary arterial hypertension (PAH). In PAH, mutants of the BMP type II receptors (BMPR2) were detected, and their functions were impaired during BMP signaling. It is thought that expression levels of these receptors determine the fate of BMP signaling, with low levels of expression leading to decreased Smad activation in PAH. However, our studies demonstrate, for the first time, that the localization of receptors on the plasma membrane, in this case BMPR2, was misdirected. Three BMPR2 mutants, D485G, N519K, and R899X, which are known to be involved in PAH, were chosen as our model system. Our results show that all three BMPR2 mutants decreased BMP-dependent Smad phosphorylation and Smad signaling. Although the three mutants reached the cell membrane and their expression was lower than that of BMPR2, they formed smaller clusters and associated differently with membrane domains, such as caveolae and clathrin-coated pits. The disruption of these domains restored the Smad signaling of D485G and N519K to the level of wild-type BMPR2, showing that these mutants were trapped in the domains, rather than just expressed at a lower level on the surface. Therefore, new treatment options for PAH should also target receptor localization, rather than just expression level.     

Mediators and modulators of pulmonary arterial hypertension

Pulmonary hypertension (PH), defined as a mean pulmonary arterial (PA) pressure of >25 mmHg at rest or >30 mmHg during exercise, is characterized by a progressive and sustained increase in pulmonary vascular resistance that eventually leads to right ventricular failure. Clinically, PH may result from a variety of underlying diseases (Table 1 and Refs. 50, 113, 124). Pulmonary arterial hypertension (PAH) may be familial (FPAH) or sporadic (idiopathic, IPAH), formerly known as primary pulmonary hypertension, i.e., for which there is no demonstrable cause. More often, PAH is due to a variety of identifiable diseases including scleroderma and other collagen disorders, liver disease, human immunodeficiency virus, and the intake of appetite-suppressant drugs such as phentermine and fenfluramine (72). Other, more common, causes of PAH include left ventricular failure (perhaps the most common cause), valvular lesions, chronic pulmonary diseases, sleep-disordered breathing, and prolonged residence at high altitude. This classification, now widely accepted, was first proposed at a meeting in Evian, France, in 1998, and modified in Venice, Italy, in 2003 (124).