BACKGROUND:Vascular calcification, which is characterized by calcium deposition in arterial walls and the osteochondrogenic differentiation of vascular smooth muscle cells, is an actively regulated process that involves complex mechanisms. Vascular calcification is associated with increased cardiovascular adverse events. The role of 4-hydroxynonenal (4-HNE), which is the most abundant stable product of lipid peroxidation, in vascular calcification has been poorly investigated.METHODS:Serum was collected from patients with chronic kidney disease and controls, and the levels of 4-HNE and 8-iso-prostaglandin F2α were measured. Sections of coronary atherosclerotic plaques from donors were immunostained to analyze calcium deposition and 4-HNE. A total of 658 patients with coronary artery disease who received coronary computed tomography angiography were recruited to analyze the relationship between coronary calcification and the rs671 mutation in aldehyde dehydrogenase 2 (ALDH2). ALDH2 knockout (ALDH2^-/-) mice, smooth muscle cell–specific ALDH2 knockout mice, ALDH2 transgenic mice, and their controls were used to establish vascular calcification models. Primary mouse aortic smooth muscle cells and human aortic smooth muscle cells were exposed to medium containing β-glycerophosphate and CaCl₂ to investigate cell calcification and the underlying molecular mechanisms.RESULTS:Elevated 4-HNE levels were observed in the serum of patients with chronic kidney disease and model mice and were detected in calcified artery sections by immunostaining. ALDH2 knockout or smooth muscle cell–specific ALDH2 knockout accelerated the development of vascular calcification in model mice, whereas overexpression or activation prevented mouse vascular calcification and the osteochondrogenic differentiation of vascular smooth muscle cells. In patients with coronary artery disease, patients with ALDH2 rs671 gene mutation developed more severe coronary calcification. 4-HNE promoted calcification of both mouse aortic smooth muscle cells and human aortic smooth muscle cells and their osteochondrogenic differentiation in vitro. 4-HNE increased the level of Runx2 (runt-related transcription factor-2), and the effect of 4-HNE on promoting vascular smooth muscle cell calcification was ablated when Runx2 was knocked down. Mutation of Runx2 at lysine 176 reduced its carbonylation and eliminated the 4-HNE–induced upregulation of Runx2.CONCLUSIONS:Our results suggest that 4-HNE increases Runx2 stabilization by directly carbonylating its K176 site and promotes vascular calcification. ALDH2 might be a potential target for the treatment of vascular calcification.

中文翻译：

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Runx2 在 K176 处被 4-Hydroxynonenal 羰基化可加速血管钙化

背景：血管钙化的特征是动脉壁钙沉积和血管平滑肌细胞的骨软骨分化，是一个主动调节的过程，涉及复杂的机制。血管钙化与心血管不良事件的增加有关。4-羟基壬烯醛 (4-HNE) 是最丰富的脂质过氧化稳定产物，其在血管钙化中的作用尚未得到充分研究。 方法：收集慢性肾病患者和对照者的血清，并检测 4-羟基壬烯醛 (4-HNE) 的水平。测量了-HNE和8-异前列腺素F2α。对供体的冠状动脉粥样硬化斑块切片进行免疫染色，以分析钙沉积和 4-HNE。共招募658例接受冠状动脉CT血管造影的冠心病患者，分析冠状动脉钙化与乙醛脱氢酶2（ALDH2）rs671突变之间的关系。采用ALDH2基因敲除(ALDH2 ^-/- )小鼠、平滑肌细胞特异性ALDH2基因敲除小鼠、ALDH2转基因小鼠及其对照小鼠建立血管钙化模型。将原代小鼠主动脉平滑肌细胞和人主动脉平滑肌细胞暴露于含有β-甘油磷酸和CaCl ₂的培养基中，以研究细胞钙化及其潜在分子机制。结果：慢性肾病患者血清中4-HNE水平升高疾病和模型小鼠，并通过免疫染色在钙化动脉切片中检测到。ALDH2 敲除或平滑肌细胞特异性 ALDH2 敲除加速了模型小鼠血管钙化的发展，而过度表达或激活则阻止小鼠血管钙化和血管平滑肌细胞的骨软骨分化。在冠状动脉疾病患者中，ALDH2 rs671基因突变的患者出现更严重的冠状动脉钙化。4-HNE 促进小鼠主动脉平滑肌细胞和人主动脉平滑肌细胞的钙化及其体外骨软骨分化。4-HNE增加Runx2（runt相关转录因子2）的水平，当Runx2被敲低时，4-HNE促进血管平滑肌细胞钙化的作用被消除。Runx2 在赖氨酸 176 处的突变减少了其羰基化，并消除了 4-HNE 诱导的 Runx2 上调。结论：我们的结果表明，4-HNE 通过直接羰基化其 K176 位点来增加 Runx2 的稳定性，并促进血管钙化。ALDH2可能是治疗血管钙化的潜在靶点。