Endothelial BMP6 Drives Hemodynamic-Dependent VSMCs Calcification in Carotid Atherosclerosis

Carotid atherosclerosis (CAS) is a major contributor to ischemic stroke, with vascular calcification driving disease progression. However, the molecular mechanisms driving vascular calcification in CAS remain unelucidated. Previous studies have confirmed that bone morphogenetic proteins (BMPs) play essential roles in calcification; however, the regulatory mechanisms of BMP6 signaling in vascular calcification remain unclear. This study aimed to investigate the role of BMP6 in vascular calcification in CAS and the underlying mechanisms. A subset of endothelial cells (ECs) with high BMP6 expression, which interacted with specific vascular smooth muscle cells (VSMCs) via the BMP signaling pathway, was identified using single-cell RNA sequencing of human CAS plaque samples. In vitro experiments demonstrated BMP6-induced osteogenic differentiation of VSMCs. Moreover, BMP6 activated the p-SMAD1/5/8 signaling pathway by binding to the ACVR1–BMPR2 receptor complex, and pharmacological inhibition of this pathway attenuated osteogenic differentiation. Experimental results from endothelium-specific BMP6 knockout (BMP6ECKOApoE-/-) and overexpression (AAV-Cdh5-BMP6) mice confirmed that BMP6 exacerbated vascular calcification, whereas its knockdown reduced calcific lesions. Additionally, disturbed flow conditions upregulated BMP6 expression by suppressing Krüppel-like factor 4 and linking hemodynamic forces to BMP6-mediated calcification. These findings suggest that BMP6 is a key regulator of vascular calcification in CAS, driven by EC–VSMC interactions and hemodynamic stress