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 12 CAS plaques obtained following carotid endarterectomy (CEA) and carotid arteries from mice were isolated by fluorescence-activated cell sorting and analyzed using ScRNA-seq

颈动脉粥样硬化（Carotid atherosclerosis, CAS）是缺血性脑卒中的主要致病因素，血管钙化可推动疾病进展。然而，CAS中驱动血管钙化的分子机制仍未阐明。既往研究已证实骨形态发生蛋白（Bone Morphogenetic Proteins, BMPs）在钙化过程中发挥关键作用，但BMP6信号通路在血管钙化中的调控机制尚不明确。本研究旨在探究BMP6在CAS血管钙化中的作用及其潜在机制。研究人员通过对人CAS斑块样本进行单细胞RNA测序，鉴定出一群高表达BMP6的内皮细胞（Endothelial Cells, ECs），该类细胞可通过BMP信号通路与特定的血管平滑肌细胞（Vascular Smooth Muscle Cells, VSMCs）发生相互作用。体外实验证实，BMP6可诱导VSMCs发生成骨分化。进一步研究发现，BMP6可通过结合ACVR1-BMPR2受体复合物激活p-SMAD1/5/8信号通路，而该通路的药物抑制可减弱成骨分化过程。内皮特异性BMP6敲除（BMP6ECKOApoE-/-）及过表达（AAV-Cdh5-BMP6）小鼠的实验结果证实，BMP6可加剧血管钙化，而敲低BMP6则可减轻钙化病变。此外，紊乱血流可通过抑制Krüppel样因子4（Krüppel-like factor 4, KLF4）上调BMP6的表达，从而将血流动力与BMP6介导的钙化过程联系起来。上述研究结果表明，BMP6是CAS中血管钙化的关键调控因子，其作用由EC-VSMC相互作用及血流动力应激所驱动。本研究共收集12例经颈动脉内膜切除术（Carotid Endarterectomy, CEA）获取的CAS斑块样本，并分离小鼠颈动脉组织，通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）纯化目标细胞后，利用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）开展分析。