Department of Vascular Surgery, Institute of Vascular Surgery, Zhongshan Hospital, Fudan University, Shanghai, China.
Department of Orthodontics, School & Hospital of Stomatology, Tongji University, Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai, 200072, China.
Atherosclerosis. 2018 Aug;275:58-67. doi: 10.1016/j.atherosclerosis.2018.05.046. Epub 2018 May 24.
Carotid atherosclerotic plaque is one of the main sources of ischemic stroke, and endothelial-to-mesenchymal transition (EndMT) is a major feature of atherosclerosis. Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) activation, stimulated by high glucose, plays an important role in EndMT, and circadian locomotor output cycles protein kaput (Clock) deficiency leads to hyperglycemia and enhanced atherosclerosis in Clockapolipoprotein E (ApoE) mice. These findings point to a mechanism whereby CLOCK exerts a protective effect against EndMT and atherosclerotic plaque accumulation.
Cultured human umbilical vein endothelial cells (HUVECs) were stimulated with 66 mM glucose for 120 h to induce EndMT. The expression of CLOCK and ROCK1 was assayed, as were their effects on EndMT. We also conducted molecular and morphometric examination of carotid artery plaques from patients with carotid artery stenosis to assess the clinical relevance of these findings.
Upon EndMT, HUVECs exhibited decreased CLOCK expression and increased ROCK1 expression. Notably, CLOCK silencing increased high glucose-induced EndMT, migration ability, and ROCK1 activation, while overexpressing CLOCK attenuated these characteristics. Moreover, inhibition of ROCK1 largely blocked EndMT induced by high-glucose or transforming growth factor (TGF)-β1 but failed to rescue the reduced CLOCK expression. The vulnerability of human carotid artery plaque was strongly correlated with loss of CLOCK expression, activation of TGF-β/ROCK1 signaling, and the extent of EndMT.
The data indicate that loss of protective endothelial CLOCK expression aggravates TGF-β/ROCK1-modulated EndMT progression, which contributes to the vulnerability of human carotid plaque.
颈动脉粥样硬化斑块是缺血性脑卒中的主要来源之一，而内皮到间充质转化（EndMT）是动脉粥样硬化的主要特征之一。高血糖刺激下 Rho 相关卷曲螺旋蛋白激酶 1（ROCK1）的激活在 EndMT 中起重要作用，而昼夜节律运动输出周期蛋白 kaput（Clock）缺失会导致 Clock 载脂蛋白 E（ApoE）小鼠的高血糖和增强的动脉粥样硬化。这些发现表明了 CLOCK 通过发挥对 EndMT 和动脉粥样硬化斑块积累的保护作用的机制。
用 66mmol/L 葡萄糖刺激培养的人脐静脉内皮细胞（HUVECs）120 小时诱导 EndMT。检测 CLOCK 和 ROCK1 的表达，并观察它们对 EndMT 的影响。我们还对颈动脉狭窄患者的颈动脉斑块进行了分子和形态学检查，以评估这些发现的临床相关性。
在 EndMT 过程中，HUVECs 中 CLOCK 的表达降低，ROCK1 的表达增加。值得注意的是，沉默 CLOCK 增加了高糖诱导的 EndMT、迁移能力和 ROCK1 激活，而过表达 CLOCK 则减弱了这些特征。此外，ROCK1 的抑制在很大程度上阻断了高糖或转化生长因子（TGF）-β1 诱导的 EndMT，但未能挽救 CLOCK 表达的降低。人类颈动脉斑块的脆弱性与 CLOCK 表达的丧失、TGF-β/ROCK1 信号的激活以及 EndMT 的程度密切相关。
数据表明，保护性内皮 CLOCK 表达的丧失加剧了 TGF-β/ROCK1 调节的 EndMT 进展，这导致了人类颈动脉斑块的脆弱性。
