Senescence of vascular smooth muscle cells (VSMCs) is a key contributor to plaque vulnerability in atherosclerosis (AS), which is affected by endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production. However, the crosstalk between ER stress and ROS production in the pathogenesis of VSMC senescence remains to be elucidated. ER-associated degradation (ERAD) is a complex process that clears unfolded or misfolded proteins to maintain ER homeostasis. HRD1 is the major E3 ligase in mammalian ERAD machineries that catalyzes ubiquitin conjugation to the unfolded or misfolded proteins for degradation. Our results showed that HRD1 protein levels were reduced in human AS plaques and aortic roots from ApoE^−/− mice fed with high-fat diet (HFD), along with the increased ER stress response. Exposure to cholesterol in VSMCs activated inflammatory signaling and induced senescence, while reduced HRD1 protein expression. CRISPR Cas9-mediated HRD1 knockout (KO) exacerbated cholesterol- and thapsigargin-induced cell senescence. Inhibiting ER stress with 4-PBA (4-Phenylbutyric acid) partially reversed the ROS production and cell senescence induced by HRD1 deficiency in VSMCs, suggesting that ER stress alone could be sufficient to induce ROS production and senescence in VSMCs. Besides, HRD1 deficiency led to mitochondrial dysfunction, and reducing ROS production from impaired mitochondria partly reversed HRD1 deficiency-induced cell senescence. Finally, we showed that the overexpression of HDR1 reversed cholesterol-induced ER stress, ROS production, and cellular senescence in VSMCs. Our findings indicate that HRD1 protects against senescence by maintaining ER homeostasis and mitochondrial functionality. Thus, targeting HRD1 function may help to mitigate VSMC senescence and prevent vascular aging related diseases.

Trial registration

A real-world study based on the discussion of primary and secondary prevention strategies for coronary heart disease, URL:https://www.clinicaltrials.gov, the trial registration number is [2022]-02–121-01.

中文翻译：

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胆固醇诱导的 HRD1 减少通过刺激内质网应激诱导的活性氧加速血管平滑肌细胞衰老

血管平滑肌细胞（VSMC）的衰老是动脉粥样硬化（AS）斑块脆弱性的关键因素，其受到内质网（ER）应激和活性氧（ROS）产生的影响。然而， VSMC衰老发病机制中ER应激和ROS产生之间的相互影响仍有待阐明。ER 相关降解 (ERAD) 是一个复杂的过程，可清除未折叠或错误折叠的蛋白质以维持 ER 稳态。HRD1 是哺乳动物 ERAD 机器中的主要 E3 连接酶，可催化泛素与未折叠或错误折叠的蛋白质缀合以进行降解。我们的结果表明，人类 AS 斑块和喂食高脂饮食 (HFD)^{的 ApoE −/−}VSMC 中暴露于胆固醇会激活炎症信号并诱导衰老，同时降低 HRD1 蛋白表达。CRISPR Cas9 介导的 HRD1 敲除 (KO) 加剧了胆固醇和毒胡萝卜素诱导的细胞衰老。用 4-PBA（4-苯基丁酸）抑制 ER 应激可部分逆转 VSMC 中 HRD1 缺陷诱导的 ROS 产生和细胞衰老，表明仅 ER 应激就足以诱导 VSMC 中 ROS 产生和衰老。此外，HRD1 缺陷导致线粒体功能障碍，而受损线粒体产生的 ROS 减少部分逆转了 HRD1 缺陷引起的细胞衰老。最后，我们发现 HDR1 的过度表达可逆转胆固醇诱导的 ER 应激、ROS 产生和 VSMC 细胞衰老。我们的研究结果表明，HRD1 通过维持内质网稳态和线粒体功能来防止衰老。因此，靶向HRD1功能可能有助于减轻VSMC衰老并预防血管老化相关疾病。

试用注册

一项基于冠心病一级和二级预防策略讨论的真实世界研究，网址： https: //www.clinicaltrials.gov，试验注册号为[2022]-02–121-01。