BACKGROUND 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. METHODS Mouse models of atherosclerosis in EC-specific Kindlin-2 knockout mice (Kindlin-2iΔEC) were used to study the role of Kindlin-2 in atherogenesis. Pulsatile shear (12±4 dynes/cm2) or oscillatory shear (0.5±4 dynes/cm2) 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. RESULTS We found that Kindlin-2 localization is altered under different flow patterns. Kindlin-2iΔEC mice showed heightened vascular permeability. Kindlin-2iΔEC were bred onto ApoE-/- mice to generate Kindlin-2iΔEC; ApoE-/- 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. CONCLUSIONS 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.

中文翻译：

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响应血流的 Kindlin-2 相分离控制血管稳定性。


背景 动脉粥样硬化保护性剪切应力保持内皮屏障功能，而动脉粥样硬化诱导剪切应力增强内皮通透性。然而，不同流型调节 EC 完整性的潜在机制仍有待阐明。本研究旨在探讨 Kindlin-2 的参与，Kindlin-2 是黏着斑的关键成分，内皮粘附连接对调节内皮细胞 （EC） 完整性和血管稳定性至关重要。方法 使用 EC 特异性 Kindlin-2 敲除小鼠 （Kindlin-2iΔEC） 动脉粥样硬化小鼠模型研究 Kindlin-2 在动脉粥样硬化生成中的作用。脉冲剪切 （12±4 dynes/cm2） 或振荡剪切 （0.5±4 dynes/cm2） 应用于培养 EC。采用活细胞成像、光漂白后荧光恢复和 OptoDroplet 法研究 Kindlin-2 的液-液相分离 （LLPS）。采用免疫共沉淀、诱变、邻位连接试验和跨内皮电阻测定探讨血流调节 Kindlin-2 功能的潜在机制。结果 我们发现 Kindlin-2 定位在不同的流型下发生改变。Kindlin-2iΔEC 小鼠表现出更高的血管通透性。将 Kindlin-2iΔEC 培育到 ApoE-/- 小鼠上以生成 Kindlin-2iΔEC;ApoE-/- 小鼠，其动脉粥样硬化病变显着增加。体外数据显示，在 EC 中，Kindlin-2 经历了 LLPS，这是正确粘着斑组装、成熟和连接形成的关键过程。质谱分析显示，在 PRMT5 （蛋白精氨酸甲基转移酶 5） 的催化下，振荡剪切增加了 Kindlin-2 的精氨酸甲基化。 在功能上，精氨酸高甲基化抑制 Kindlin-2 LLPS，损害粘着斑组装和连接成熟。值得注意的是，我们发现 Kindlin-2 的 R290 是 LLPS 的关键残基和精氨酸甲基化的关键位点。最后，药理学抑制精氨酸甲基化可减少 EC 活化和斑块形成。结论 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 LLPS 的影响来调节血管稳定性。靶向 Kindlin-2 精氨酸甲基化成为治疗血管疾病和动脉粥样硬化的一种有前途的基于血流动力学的策略。