Bone morphogenic protein-2 regulates the myogenic differentiation of PMVECs in CBDL rat serum-induced pulmonary microvascular remodeling.

Hepatopulmonary syndrome (HPS) is characterized by an arterial oxygenation defect induced by intrapulmonary vasodilation (IPVD) that increases morbidity and mortality. In our previous study, it was determined that both the proliferation and the myogenic differentiation of pulmonary microvascular endothelial cells (PMVECs) play a key role in the development of IPVD. However, the molecular mechanism underlying the relationship between IPVD and the myogenic differentiation of PMVECs remains unknown. Additionally, it has been shown that bone morphogenic protein-2 (BMP2), via the control of protein expression, may regulate cell differentiation including cardiomyocyte differentiation, neuronal differentiation and odontoblastic differentiation. In this study, we observed that common bile duct ligation (CBDL)-rat serum induced the upregulation of the expression of several myogenic proteins (SM-α-actin, calponin, SM-MHC) and enhanced the expression levels of BMP2 mRNA and protein in PMVECs. We also observed that both the expression levels of Smad1/5 and the activation of phosphorylated Smad1/5 were significantly elevated in PMVECs following exposure to CBDL-rat serum, which was accompanied by the down-regulation of Smurf1. The blockage of the BMP2/Smad signaling pathway with Noggin inhibited the myogenic differentiation of PMVECs, a process that was associated with relatively low expression levels of both SM-α-actin and calponin in the setting of CBDL-rat serum exposure, although SM-MHC expression was not affected. These findings suggested that the BMP2/Smad signaling pathway is involved in the myogenic differentiation of the PMVECs. In conclusion, our data highlight the pivotal role of BMP2 in the CBDL-rat serum-induced myogenic differentiation of PMVECs via the activation of both Smad1 and Smad5 and the down-regulation of Smurf1, which may represent a potential therapy for HPS-induced pulmonary vascular remodeling.

MiR-206 controls the phenotypic modulation of pulmonary arterial smooth muscle cells induced by serum from rats with hepatopulmonary syndrome by regulating the target gene, annexin A2.

BACKGROUND: Hepatopulmonary syndrome (HPS) is a serious complication of advanced liver disease that is characterised by intrapulmonary vascular dilatation (IPVD) and arterial hypoxemia. Pulmonary vascular remodelling (PVR) is an important pathological feature of HPS, but the potential mechanisms underlying PVR remain undefined. Recent findings have established the essential role of changes in Annexin A2 (ANXA2) in controlling the phenotypic modulation of pulmonary artery smooth muscle cells (PASMCs) in PVR associated with HPS. However, the mechanism by which upstream signalling regulates ANXA2 is unclear. METHODS: In the present study, computational analysis was used to predict which miRNA might target the 3´-untranslated region (3´-UTR) of the ANXA2 mRNA. Real-time PCR and western blotting were performed to study the level of correlation between ANXA2 and the differentiation marker with the predicted miRNAs in PASMCs stimulated with serum from normal rats or those with HPS. Functional analysis of the miRNA and a luciferase reporter assay were performed to demonstrate that the predicted miRNA suppressed ANXA2 expression by directly targeting the predicted 3´-UTR site of the ANXA2 mRNA. RESULTS: Computational analysis predicted that miR-206 would target the 3´-UTR of ANXA2 mRNA. In HPS rat serum-stimulated PASMCs, the expression of miR-206 displayed an inverse correlation with ANXA2, while a positive correlation was observed with the phenotypic marker smooth muscle α-actin (SM α-actin). The miRNA functional analysis and luciferase reporter assay demonstrated that miR-206 effectively downregulated the expression of ANXA2 by binding to the 3´-UTR of the ANXA2 mRNA. Consistently, miR-206 effectively inhibited the HPS rat serum-induced phenotypic modulation and proliferation, while these effects were reversed in ANXA2-overexpressing PASMCs. CONCLUSION: This study demonstrates that miR-206 inhibits the HPS rat serum-induced phenotypic modulation and proliferation in PASMCs by down-regulating ANXA2 gene expression.

Over-expression of PKGIα inhibits hypoxia-induced proliferation, Akt activation, and phenotype modulation of human PASMCs: the role of phenotype modulation of PASMCs in pulmonary vascular remodeling.

The proliferation of pulmonary artery smooth muscle cells (PASMCs) plays a role in pulmonary vascular remodeling (PVR). Recently, it was shown that vascular smooth muscular cell phenotype modulation is important for their proliferation in other diseases. However, little is known about the role of human PASMC phenotype modulation in the proliferation induced by hypoxia and its molecular mechanism during PVR. In this study, we found using primary cultured human PASMCs that hypoxia suppressed the expression of endogenous PKGIα, which was reversed by transfection with a recombinant adenovirus containing the full-length cDNA of PKGIα (Ad-PKGIα). Ad-PKGIα transfection significantly attenuated the hypoxia-induced downregulation of the expression of smooth muscle α-actin (SM-α-actin), myosin heavy chain (MHC) and calponin in PASMCs, indicating that hypoxia-induced phenotype modulation was blocked. Furthermore, flow cytometry and (3)H-TdR incorporation demonstrated that hypoxia-induced PASMC proliferation was suppressed by upregulation of PKGIα. These results suggest that enhanced PKGIα expression inhibited hypoxia-induced PASMC phenotype modulation and that it could reverse the proliferation of PASMCs significantly. Moreover, our previous work has demonstrated that Akt protein is activated in the process of hypoxia-induced proliferation of human PASMCs. Interestingly, we found that Akt was not activated by hypoxia when PASMC phenotype modulation was blocked by Ad-PKGIα. This result suggests that blocking phenotype modulation might be a key up-stream regulatory target.