Ma Nina, Wu Fangfang, Liu Jiayu, Wu Ziru, Wang Lu, Li Bochuan, Liu Yuming, Dong Xue, Hu Junhao, Fang Xi, Zhang Heng, Ai Ding, Zhou Jing, Wang Xiaohong
Department of Pharmacology, Tianjin Key Laboratory of Inflammation Biology, State Key Laboratory of Experimental Hematology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics (N.M., F.W., Z.W., L.W., Y.L., X.D., X.W.), School of Basic Medical Sciences, Tianjin Medical University, China.
Department of Ophthalmology, Laboratory of Molecular Ophthalmology and Tianjin Key Laboratory of Ocular Trauma, Ministry of Education International Joint Laboratory of Ocular Diseases, Tianjin Medical University General Hospital, China (N.M., F.W., Z.W., L.W., Y.L., X.D., X.W.).
Circ Res. 2024 Dec 6;135(12):1141-1160. doi: 10.1161/CIRCRESAHA.124.324773. Epub 2024 Nov 4.
Atheroprotective shear stress preserves endothelial barrier function, while atheroprone shear stress enhances endothelial permeability. Yet, the underlying mechanisms through which distinct flow patterns regulate EC integrity remain to be clarified. This study aimed to investigate the involvement of Kindlin-2, a key component of focal adhesion and endothelial adherens junctions crucial for regulating endothelial cell (EC) integrity and vascular stability.
Mouse models of atherosclerosis in EC-specific knockout mice () were used to study the role of Kindlin-2 in atherogenesis. Pulsatile shear (12±4 dynes/cm) or oscillatory shear (0.5±4 dynes/cm) were applied to culture ECs. Live-cell imaging, fluorescence recovery after photobleaching assay, and OptoDroplet assay were used to study the liquid-liquid phase separation (LLPS) of Kindlin-2. Co-immunoprecipitation, mutagenesis, proximity ligation assay, and transendothelial electrical resistance assay were used to explore the underlying mechanism of flow-regulated Kindlin-2 function.
We found that Kindlin-2 localization is altered under different flow patterns. mice showed heightened vascular permeability. were bred onto mice to generate ; mice, which displayed a significant increase in atherosclerosis lesions. In vitro data showed that in ECs, Kindlin-2 underwent LLPS, a critical process for proper focal adhesion assembly, maturation, and junction formation. Mass spectrometry analysis revealed that oscillatory shear increased arginine methylation of Kindlin-2, catalyzed by PRMT5 (protein arginine methyltransferase 5). Functionally, arginine hypermethylation inhibits Kindlin-2 LLPS, impairing focal adhesion assembly and junction maturation. Notably, we identified R290 of Kindlin-2 as a crucial residue for LLPS and a key site for arginine methylation. Finally, pharmacologically inhibiting arginine methylation reduces EC activation and plaque formation.
Collectively, our study elucidates that mechanical force induces arginine methylation of Kindlin-2, thereby regulating vascular stability through its impact on Kindlin-2 LLPS. Targeting Kindlin-2 arginine methylation emerges as a promising hemodynamic-based strategy for treating vascular disorders and atherosclerosis.
具有抗动脉粥样硬化作用的剪切应力可维持内皮屏障功能，而易于引发动脉粥样硬化的剪切应力则会增强内皮通透性。然而，不同血流模式调节内皮细胞（EC）完整性的潜在机制仍有待阐明。本研究旨在探究黏着斑蛋白2（Kindlin-2）的作用，它是黏着斑和内皮紧密连接的关键组成部分，对调节内皮细胞完整性和血管稳定性至关重要。
利用内皮细胞特异性敲除小鼠（）的动脉粥样硬化小鼠模型，研究Kindlin-2在动脉粥样硬化发生过程中的作用。对培养的内皮细胞施加脉动剪切力（12±4达因/平方厘米）或振荡剪切力（0.5±4达因/平方厘米）。采用活细胞成像、光漂白后荧光恢复测定法和光控液滴测定法研究Kindlin-2的液-液相分离（LLPS）。运用免疫共沉淀、诱变、邻近连接测定法和跨内皮电阻测定法，探究血流调节Kindlin-2功能的潜在机制。
我们发现，在不同血流模式下，Kindlin-2的定位会发生改变。小鼠表现出血管通透性增加。将小鼠与小鼠杂交培育出小鼠，其动脉粥样硬化病变显著增加。体外实验数据表明，在内皮细胞中，Kindlin-2会发生液-液相分离，这是黏着斑正确组装、成熟和连接形成的关键过程。质谱分析显示，振荡剪切力会增加由蛋白精氨酸甲基转移酶5（PRMT5）催化的Kindlin-2的精氨酸甲基化。在功能上，精氨酸高度甲基化会抑制Kindlin-2的液-液相分离，损害黏着斑组装和连接成熟。值得注意的是，我们确定Kindlin-2的R290是液-液相分离的关键残基和精氨酸甲基化的关键位点。最后，通过药物抑制精氨酸甲基化可减少内皮细胞活化和斑块形成。
总的来说，我们的研究阐明了机械力诱导Kindlin-2的精氨酸甲基化，从而通过其对Kindlin-2液-液相分离的影响来调节血管稳定性。靶向Kindlin-2精氨酸甲基化成为一种有前景的基于血流动力学的治疗血管疾病和动脉粥样硬化的策略。
