Kopych Vadym, Da Costa Avelino Dos Santos, Park Kwideok
Center for Biomaterials, Korea Institute of Science and Technology (KIST), Seoul, 02792, Republic of Korea.
Division of Bio-Medical Science and Technology, KIST School, University of Science and Technology (UST), Seoul, 02792, Republic of Korea.
Tissue Eng Regen Med. 2025 Jul 11. doi: 10.1007/s13770-025-00733-w.
Endothelial cells (ECs) are key regulators of vascular function, adapting to mechanical forces, such as shear stress to maintain vascular homeostasis. Disruption of this adaptation, particularly in the regions of disturbed flow, contributes to endothelial dysfunction and the development of atherosclerosis later on.
We prepared a custom-designed PDMS-based flow chamber to apply controlled shear stress (2 or 7 dynes/cm) to human umbilical vein endothelial cells. ECs were cultured on gelatin-coated coverslips and exposed to different shear flows for up to 12 h. Cell alignment was confirmed by angle measurements using ImageJ. Gene expression of SIRT4, PIEZO1, NOTCH1, and LOX-1 was determined via qPCR, and protein levels were assessed by western blot. Specific gene knockdown was also conducted using siRNAs, targeting either PIEZO1 or SIRT4. Oxidized LDL uptake was evaluated using DiI-labeled Ox-LDL and quantified by fluorescence imaging. Immunofluorescence staining of ECs was performed to visualize VE-cadherin, F-actin, and nuclei. All quantitative data were subjected to statistical analysis.
We demonstrated that the mechanosensitive ion channel PIEZO1, regulates SIRT4 expression in response to shear stress. Under atheroprotective shear stress (7 dyne/cm), PIEZO1-mediated upregulation of SIRT4 was observed, while atheroprone shear stress (2 dyne/cm) led to reduced expression. Functional assays showed that SIRT4 protects endothelial cells from Ox-LDL uptake, a key factor in atherosclerosis. SIRT4 silencing increased Ox-LDL accumulation even under protective flow. This effect, and its link to LOX-1, was dependent on PIEZO1 signaling.
Current findings suggest that the PIEZO1-SIRT4 axis may modulate endothelial responses to shear stress, offering a protective mechanism against Ox-LDL-induced dysfunction and pathology. Our study underscores the potential of SIRT4 as a therapeutic target to mitigate vascular disorders associated with oxidative stress and disturbed blood flow.
内皮细胞（ECs）是血管功能的关键调节因子，可适应机械力，如剪切应力，以维持血管稳态。这种适应性的破坏，特别是在血流紊乱的区域，会导致内皮功能障碍，并在以后促进动脉粥样硬化的发展。
我们制备了一个定制设计的基于聚二甲基硅氧烷（PDMS）的流动腔，以对人脐静脉内皮细胞施加可控的剪切应力（2或7达因/平方厘米）。将内皮细胞培养在明胶包被的盖玻片上，并暴露于不同的剪切流中长达12小时。使用ImageJ通过角度测量来确认细胞排列。通过定量聚合酶链反应（qPCR）测定SIRT4、PIEZO1、NOTCH1和凝集素样氧化低密度脂蛋白受体1（LOX-1）的基因表达，并通过蛋白质印迹法评估蛋白质水平。还使用小干扰RNA（siRNAs）进行特异性基因敲低，靶向PIEZO1或SIRT4。使用碘化油（DiI）标记的氧化低密度脂蛋白（Ox-LDL）评估氧化低密度脂蛋白摄取，并通过荧光成像进行定量。对内皮细胞进行免疫荧光染色以可视化血管内皮钙黏蛋白（VE-钙黏蛋白）、F-肌动蛋白和细胞核。所有定量数据均进行统计分析。
我们证明了机械敏感离子通道PIEZO1可响应剪切应力调节SIRT4表达。在抗动脉粥样硬化的剪切应力（7达因/平方厘米）下，观察到PIEZO1介导的SIRT4上调，而促动脉粥样硬化的剪切应力（2达因/平方厘米）导致表达降低。功能试验表明，SIRT4可保护内皮细胞免受氧化低密度脂蛋白摄取，氧化低密度脂蛋白摄取是动脉粥样硬化的一个关键因素。即使在保护性血流条件下，SIRT4沉默也会增加氧化低密度脂蛋白的积累。这种效应及其与LOX-1的联系依赖于PIEZO1信号传导。
目前的研究结果表明，PIEZO1-SIRT4轴可能调节内皮细胞对剪切应力的反应，提供一种针对氧化低密度脂蛋白诱导的功能障碍和病理的保护机制。我们的研究强调了SIRT4作为减轻与氧化应激和血流紊乱相关的血管疾病的治疗靶点的潜力。
