Altered Growth Responses of Pulmonary Artery Smooth Muscle Cells From Patients With Primary Pulmonary Hypertension to Transforming Growth Factor-β ₁ and Bone Morphogenetic Proteins

Abstract

Background-Mutations in the type II receptor for bone morphogenetic protein (BMPR-II), a receptor member of the transforming growth factor-␤ (TGF-␤) superfamily, underlie many cases of familial and sporadic primary pulmonary hypertension (PPH). We postulated that pulmonary artery smooth muscle cells (PASMCs) from patients with PPH might demonstrate abnormal growth responses to TGF-␤ superfamily members. Methods and Results-For studies of 3 H-thymidine incorporation or cell proliferation, PASMCs (passages 4 to 8) were derived from main pulmonary arteries. In control cells, 24-hour incubation with TGF-␤ 1 (10 ng/mL) or bone morphogenetic protein (BMP)-2, -4, and -7 (100 ng/mL) inhibited basal and serum-stimulated 3 H-thymidine incorporation, and TGF-␤ 1 and BMPs inhibited the proliferation of serum-stimulated PASMCs. In contrast, TGF-␤ 1 stimulated 3 H-thymidine incorporation (200%; PϽ0.001) and cell proliferation in PASMCs from PPH but not from patients with secondary pulmonary hypertension. In addition, BMPs failed to suppress DNA synthesis and proliferation in PASMCs from PPH patients. Reverse transcription-polymerase chain reaction of PASMC mRNA detected transcripts for type I (TGF-␤RI, Alk-1, ActRI, and BMPRIB) and type II (TGF-␤RII, BMPR-II, ActRII, ActRIIB) receptors. Receptor binding and cross-linking studies with 125 I-TGF-␤ 1 confirmed that the abnormal responses in PPH cells were not due to differences in TGF-␤ receptor binding. Mutation analysis of PASMC DNA failed to detect mutations in TGF-␤RII and Alk-1 but confirmed the presence of a mutation in BMPR-II in 1 of 5 PPH isolates. Conclusions-We

Involvement of the bone morphogenetic protein system in endothelin- and aldosterone-induced cell proliferation of pulmonary arterial smooth muscle cells isolated from human patients with pulmonary arterial hypertension

“…In contrast, BMP-4 and BMP-6 had no effect on PASMC mitogenicity. PDGF-BB (100 ng ml À1 ), which is known to be a potent PASMC growth factor, 8 increased DNA synthesis by both PASMC types by up to Endothelin, aldosterone and BMP in PAH pathogenesis R Yamanaka et al approximately twofold (Figure 3). We further examined the functional interrelationship between the BMP system and ET1 or aldosterone in PASMC proliferation.…”

“…3 Subsequent functional studies have provided compelling evidence that PAH cells that harbor the BMPR2 mutations exhibit aberrant BMPRII function and disrupted BMP signaling. 8,9 However, given the fact that almost half of all PAH patients do not have the BMPR2 mutation, the entire mechanism of pulmonary artery smooth muscle cell (PASMC) mitogenesis in PAH lungs is still unclear. BMP ligands that are involved in the aberrant signaling caused by the BMPRII mutations in PAH patients have also not been identified.…”

Pathology of Pulmonary Hypertension

“…The identification of mutations in BMPR2 led to several in vitro studies aimed at relating smooth muscle cell proliferation to abnormal BMPR2 signaling. IPAH smooth muscle cells isolated from proximal arteries (i.e., elastic vessels, >500 μm in diameter) exhibit decreased inhibitory effect on cell proliferation mediated by TGF-β1 or BMP-4 when compared with smooth muscle cells isolated from normal human pulmonary arteries [49]. Of note, these altered responses are not due to abnormal expression of smad signaling or ligand binding to its receptors in IPAH smooth muscle cells.…”

Pathogenesis of Pulmonary Arterial Hypertension

“…Furthermore, Smad proteins have been reported to interact with calmodulin, a Ca 2ϩ -sensitive protein in the cytosol. Overexpression of calmodulin inhibits Smad activation and attenuates the response of BMP signal transduction, suggesting that a rise in [Ca 2ϩ ] cyt may exert an inhibitory effect on the BMP signaling pathway by activating calmodulin and attenuating BMP-mediated antiproliferative or proapoptotic effects on human PASMCs.In cells isolated from normal subjects, BMPs inhibit proliferation in PASMCs, 19 induce apoptosis in PASMCs, 20 and enhance survival in PAECs and endothelial progenitor cells. 21 The BMP-mediated apoptosis in normal PASMCs appears to be at least partially due to downregulation of Bcl-2, an antiapoptotic protein.…”