Department of Infection, Immunity, and Cardiovascular Disease, INSIGNEO Institute for In Silico Medicine, and the Bateson Centre, University of Sheffield, United Kingdom (L.C., S.S., M.R.D., E.W., B.T.A., S.F., T.J.A.C., P.C.E., J.S.-C.).
National Heart and Lung Institute, Imperial College London, United Kingdom (D.P.).
Arterioscler Thromb Vasc Biol. 2023 Apr;43(4):547-561. doi: 10.1161/ATVBAHA.122.318110. Epub 2023 Feb 16.
Hemodynamic wall shear stress (WSS) exerted on the endothelium by flowing blood determines the spatial distribution of atherosclerotic lesions. Disturbed flow (DF) with a low WSS magnitude and reversing direction promotes atherosclerosis by regulating endothelial cell (EC) viability and function, whereas un-DF which is unidirectional and of high WSS magnitude is atheroprotective. Here, we study the role of EVA1A (eva-1 homolog A), a lysosome and endoplasmic reticulum-associated protein linked to autophagy and apoptosis, in WSS-regulated EC dysfunction.
The effect of WSS on EVA1A expression was studied using porcine and mouse aortas and cultured human ECs exposed to flow. EVA1A was silenced in vitro in human ECs and in vivo in zebrafish using siRNA (small interfering RNA) and morpholinos, respectively.
EVA1A was induced by proatherogenic DF at both mRNA and protein levels. silencing resulted in decreased EC apoptosis, permeability, and expression of inflammatory markers under DF. Assessment of autophagic flux using the autolysosome inhibitor, bafilomycin coupled to the autophagy markers LC3-II (microtubule-associated protein 1 light chain 3-II) and p62, revealed that knockdown promotes autophagy when ECs are exposed to DF, but not un-DF . Blocking autophagic flux led to increased EC apoptosis in -knockdown cells exposed to DF, suggesting that autophagy mediates the effects of DF on EC dysfunction. Mechanistically, expression was regulated by flow direction via TWIST1 (twist basic helix-loop-helix transcription factor 1). In vivo, knockdown of orthologue in zebrafish resulted in reduced EC apoptosis, confirming the proapoptotic role of EVA1A in the endothelium.
We identified EVA1A as a novel flow-sensitive gene that mediates the effects of proatherogenic DF on EC dysfunction by regulating autophagy.
血流作用于血管内皮的血流切应力（WSS）决定了动脉粥样硬化病变的空间分布。低切应力和方向逆转的紊乱流（DF）通过调节内皮细胞（EC）的活力和功能促进动脉粥样硬化，而单向高切应力的未紊乱流（un-DF）则具有抗动脉粥样硬化作用。在这里，我们研究了 EVA1A（ev-1 同源物 A）在 WSS 调节的 EC 功能障碍中的作用，EVA1A 是一种与自噬和细胞凋亡相关的溶酶体和内质网相关蛋白。
使用猪和鼠主动脉以及暴露于流动的培养的人 EC 研究 WSS 对 EVA1A 表达的影响。使用 siRNA（小干扰 RNA）和形态发生素来分别在体外沉默人 EC 中的 EVA1A 和体内沉默斑马鱼中的 EVA1A。
EVA1A 在 mRNA 和蛋白水平上均由促动脉粥样硬化的 DF 诱导。在 DF 下，沉默会导致 EC 凋亡、通透性和炎症标志物表达减少。使用自噬体抑制剂巴弗洛霉素与自噬标志物 LC3-II（微管相关蛋白 1 轻链 3-II）和 p62 结合评估自噬通量，结果表明在 DF 下，EC 暴露于 DF 时， 敲低会促进自噬，但在非 DF 下不会。在 DF 下，阻断自噬通量会导致 敲低的细胞凋亡增加，这表明自噬介导了 DF 对 EC 功能障碍的影响。在机制上，通过 TWIST1（扭转型基因 1），EVA1A 的表达受流动方向调节。在体内，斑马鱼中 EVA1A 同源物的敲低导致 EC 凋亡减少，证实了 EVA1A 在血管内皮中的促凋亡作用。
我们鉴定出 EVA1A 是一种新的血流敏感基因，通过调节自噬，介导促动脉粥样硬化 DF 对 EC 功能障碍的影响。
