Nina Ma, Fangfang Wu, Jiayu Liu, Ziru Wu, Lu Wang, Bochuan Li, Yuming Liu, Xue Dong, Junhao Hu, Xi Fang, Heng Zhang, Ding Ai, Jing Zhou, Xiaohong Wang
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Pulsatile shear (2±4 dynes/cm<sup>2</sup>) or oscillatory shear (0.5±4 dynes/cm<sup>2</sup>) 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.</p><p><strong>Results: </strong>We found that Kindlin-2 localization is altered under different flow patterns. <i>Kindlin-2</i><sup><i>iΔEC</i></sup> mice showed heightened vascular permeability. <i>Kindlin-2</i><sup><i>iΔEC</i></sup> were bred onto <i>ApoE</i><sup><i>-/-</i></sup> mice to generate <i>Kindlin-2</i><sup><i>iΔEC</i></sup>; <i>ApoE</i><sup><i>-</i></sup><sup><i>/-</i></sup> 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.</p><p><strong>Conclusions: </strong>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.</p><p><strong>Registration: </strong>URL: https://www.clinicaltrials.gov; Unique identifier: NCT02783300.</p>","PeriodicalId":10147,"journal":{"name":"Circulation research","volume":" ","pages":""},"PeriodicalIF":16.5000,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Kindlin-2 Phase Separation in Response to Flow Controls Vascular Stability.\",\"authors\":\"Nina Ma, Fangfang Wu, Jiayu Liu, Ziru Wu, Lu Wang, Bochuan Li, Yuming Liu, Xue Dong, Junhao Hu, Xi Fang, Heng Zhang, Ding Ai, Jing Zhou, Xiaohong Wang\",\"doi\":\"10.1161/CIRCRESAHA.124.324773\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>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.</p><p><strong>Methods: </strong>Mouse models of atherosclerosis in EC-specific <i>Kindlin-2</i> knockout mice (<i>Kindlin-2</i><sup><i>iΔEC</i></sup>) were used to study the role of Kindlin-2 in atherogenesis. Pulsatile shear (2±4 dynes/cm<sup>2</sup>) or oscillatory shear (0.5±4 dynes/cm<sup>2</sup>) 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.</p><p><strong>Results: </strong>We found that Kindlin-2 localization is altered under different flow patterns. <i>Kindlin-2</i><sup><i>iΔEC</i></sup> mice showed heightened vascular permeability. <i>Kindlin-2</i><sup><i>iΔEC</i></sup> were bred onto <i>ApoE</i><sup><i>-/-</i></sup> mice to generate <i>Kindlin-2</i><sup><i>iΔEC</i></sup>; <i>ApoE</i><sup><i>-</i></sup><sup><i>/-</i></sup> 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.</p><p><strong>Conclusions: </strong>Collectively, our study elucidates that mechanical force induces arginine methylation of Kindlin-2, thereby regulating vascular stability through its impact on Kindlin-2 LLPS. 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Kindlin-2 Phase Separation in Response to Flow Controls Vascular Stability.
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 (2±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.
期刊介绍：
Circulation Research is a peer-reviewed journal that serves as a forum for the highest quality research in basic cardiovascular biology. The journal publishes studies that utilize state-of-the-art approaches to investigate mechanisms of human disease, as well as translational and clinical research that provide fundamental insights into the basis of disease and the mechanism of therapies.
Circulation Research has a broad audience that includes clinical and academic cardiologists, basic cardiovascular scientists, physiologists, cellular and molecular biologists, and cardiovascular pharmacologists. The journal aims to advance the understanding of cardiovascular biology and disease by disseminating cutting-edge research to these diverse communities.
In terms of indexing, Circulation Research is included in several prominent scientific databases, including BIOSIS, CAB Abstracts, Chemical Abstracts, Current Contents, EMBASE, and MEDLINE. This ensures that the journal's articles are easily discoverable and accessible to researchers in the field.
Overall, Circulation Research is a reputable publication that attracts high-quality research and provides a platform for the dissemination of important findings in basic cardiovascular biology and its translational and clinical applications.
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