Vascular smooth muscle cells (VSMCs) show a remarkable phenotypic plasticity reflecting their ability to acquire contractile or synthetic states. Numerous signaling pathways and conditions are known, which cause loss of contractility and attainment of a synthetic state but knowledge about signaling pathways promoting the contractile state is limited and in vivo evidence is critically missing. Here, we investigated the role of BMP9, BMP10, activin receptor-like kinase 1 (ALK1) and Smad7 for controlling formation of contractile VSMCs. Effects of Bmp9 and Bmp10 inactivation, conditional deletion of Alk1 in SMCs, overexpression of Bmp10 in endothelial cells (ECs) and overexpression of Smad7 in SMCs were evaluated in respect to coverage of vessels by VSMC, their contractile state and blood pressure. The molecular mechanisms underlying effects of BMP9/BMP10 were studied by treating dedifferentiated SMCs with BMP9/BMP10 in vitro. Concomitant genetic inactivation of BMP9/BMP10 in mice led to a dramatic diminution of VSMC layer, loss of contractile VSMCs and decreased systemic blood pressure. Consistently, overexpression of Bmp10 in ECs of adult mice dramatically enhanced VSMC contractility and increased formation of contractile VSMCs, indicating that BMP9/BMP10 play fundamental roles for differentiation and contractility of VSMCs. Importantly, treatment of BMP9/BMP10 induced an ALK1-dependent phenotypic switch from synthetic to contractile state in de-differentiated VSMCs. SMC-specific overexpression of inhibitory Smad7 completely suppressed differentiation and proliferation of VSMCs during embryonic development and reiterated the defects observed in BMP9/BMP10 double mutants in adults. Likewise, deletion of Alk1 in SMCs recapitulated attenuated VSMCs coverage and loss of contractile VSMCs seen in BMP9/BMP10 mutants but only in pulmonary and not in aortic or coronary arteries. Bulk RNA-seq and single molecule RNA-fluorescent in situ hybridization (RNA-FISH) uncovered heterogeneous expression of BMP type I receptors in distinct vessel beds, explaining phenotypic differences between Alk1 and Bmp9/Bmp10 inactivation. Furthermore, analysis of BMP9/BMP10 stimulated VSMCs by ATAC-seq combined with RNA-seq revealed that YAP/TEAD and BMP9/BMP10/Smad-signaling converge at promoters of several VSMC genes. Gain-and loss-of-function studies of the YAP-TEAD axis demonstrated that YAP-TEAD axis antagonized BMP9/BMP10 induced VSMCs differentiation and formation of contractile VSMCs.In conclusion, the study indicated that BMP9 and BMP10 are released into the blood stream by the liver and the right atrium, respectively and bypass the endothelial barrier to directly induce and maintain contractile VSMCs. Importantly, the effects of BMP9/BMP10 in VSMCs are modulated by different combination of BMP type one receptors in a vessel bed specific manner.
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