Dysfunctional Smad Signaling Contributes to Abnormal Smooth Muscle Cell Proliferation in Familial Pulmonary Arterial Hypertension

Abstract

Abstract-Mutations in the bone morphogenetic protein type II receptor gene (BMPR2) are the major genetic cause of familial pulmonary arterial hypertension (FPAH). Although smooth muscle cell proliferation contributes to the vascular remodeling observed in PAH, the role of BMPs in this process and the impact of BMPR2 mutation remains unclear. Studies involving normal human pulmonary artery smooth muscle cells (PASMCs) suggest site-specific responses to BMPs. Thus, BMP-4 inhibited proliferation of PASMCs isolated from proximal pulmonary arteries, but stimulated proliferation of PASMCs from peripheral arteries, and conferred protection from apoptosis. These differences were not caused by differential activation of BMP signaling pathways because exogenous BMP-4 led to phosphorylation of Smad1, p38 MAPK , and ERK1/2 in both cell types. However, the proproliferative effect of BMP-4 on peripheral PASMCs was found to be p38 MAPK /ERK-dependent. Conversely, overexpression of dominant-negative Smad1 converted the response to BMP-4 in proximal PASMCs from inhibitory to proliferative. Furthermore, we confirmed that proximal PASMCs harboring kinase domain mutations in BMPR2 are deficient in Smad signaling and are unresponsive to the growth suppressive effect of BMP-4. Moreover, we show that the pulmonary vasculature of patients with familial and idiopathic PAH are deficient in the activated form of Smad1. We conclude that defective Smad signaling and unopposed p38 MAPK /ERK signaling, as a consequence of mutation in BMPR2, underlie the abnormal vascular cell proliferation observed in familial PAH. Key Words: vascular remodeling Ⅲ pulmonary hypertension Ⅲ transforming growth factor-␤ Ⅲ smooth muscle cells Ⅲ cell signaling P rimary, or idiopathic, pulmonary arterial hypertension (IPAH) is a rare disorder that is progressive and often fatal, leading to death within a median of 3 years from right ventricular failure without treatment. 1 The disease is characterized by vascular cell proliferation and obliteration of small pulmonary arteries by smooth muscle cells and myofibroblasts. 2 In addition, plexiform lesions comprising endothelial cells and myofibroblasts are found in Ϸ50% of cases. 3 Genetic studies have revealed heterozygous mutations in the BMPR2 gene encoding the type II bone morphogenetic protein receptor (BMPR-II), a member of the TGF-␤ superfamily of receptors, 4,5 underlying the familial form of the disease (FPAH). Subsequently, BMPR2 mutations were found in Ϸ25% of apparently sporadic cases of IPAH, many of which are examples of familial transmission with low disease gene penetrance. 6,7 Although these genetic studies point toward a critical role for the TGF-␤ superfamily in the regulation of pulmonary vascular cell growth and differentiation, the precise molecular mechanisms leading to disease pathogenesis remain to be elucidated.Signaling by BMP receptors involves heterodimerization of 2 transmembrane serine/threonine kinases: the constitutively active type II receptor, BMPR-II, and a corresponding type I re...

Bone morphogenetic protein signalling in heritable versus idiopathic pulmonary hypertension

“…Moreover, noncysteine substitutions localise to the cell surface but also exhibit defects in BMP signalling activity [31]. In contrast, mutations in the cytoplasmic C-terminal domain only moderately inhibit Smad signalling [31,32]. Therefore, in the present study, all reported mutations would be susceptible to be deleterious by changing the protein sequence at important functional sites of the receptor and the associated protein functions.…”

“…We selected a dose of BMP4 of 100 ng?mL -1 on the basis of available literature [18,19,32] and preliminary testing showing maximum efficacy of 100 ng?mL -1 compared with 10 ng?mL -1 and 1 ng?mL -1 in discriminating PASMCs with and without BMPR-2 mutations. However, a recent study showed maximum efficacy at a lower dose of 10 ng?mL -1 of BMP4 [33].…”

Pathology of Pulmonary Hypertension

“…Similar studies were extended to more peripheral smooth muscle cells (i.e., from arteries less than 2 mm in diameter-(that are still elastic arteries) obtained from normal pulmonary arteries, which undergo cell proliferation and protection against apoptosis when exposed to BMP-4, a ligand for BMPR2. Therefore, loss of function mutations in MPR2 would be predicted to cause smooth muscle cell arrest and increased cell death [50], i.e., paradoxically opposite to that predicted to occur in familial IPAH in which mutations would facilitate smooth muscle cell growth and remodeling. Given these somewhat discrepant results [50], the evidence of reduced expression of BMPR2 found in IPAH alveolar septa [12], and the finding of decreased levels of the activated form of smad 1 (the signaling smad for BMPR2) in smooth muscle cells of muscular pulmonary arteries in IPAH, it remains unclear how BMPR2 mutations either can cause or trigger the disease.…”

“…Therefore, loss of function mutations in MPR2 would be predicted to cause smooth muscle cell arrest and increased cell death [50], i.e., paradoxically opposite to that predicted to occur in familial IPAH in which mutations would facilitate smooth muscle cell growth and remodeling. Given these somewhat discrepant results [50], the evidence of reduced expression of BMPR2 found in IPAH alveolar septa [12], and the finding of decreased levels of the activated form of smad 1 (the signaling smad for BMPR2) in smooth muscle cells of muscular pulmonary arteries in IPAH, it remains unclear how BMPR2 mutations either can cause or trigger the disease. Indeed, when compared with wild type mice, heterozygous mice lacking a single copy of BMPR2 have no pulmonary vascular phenotype at baseline or under chronic hypoxia [51], but show moderately increased pulmonary artery pressures and minimal remodeling when stressed with intratracheal delivery of a 5-lipoxygenase expressing vector [51].…”

“…These findings are supported by the report that mice deficient in 5HTT or 5-HT-2B are protected against PH caused by hypoxia alone [52] or hypoxia combined with the anorexigen dexfenfluramine [54], respectively. Of interest is the observation that increased serotonin levels affect signaling pathways downstream of mutated BMPR2, as serotonin infusion enhances normoxic and hypoxic pulmonary pressures and vascular remodeling in BMPR2 heterozygous mice as compared with wild type mice [50].…”