PCSK9促进线粒体功能障碍加速腹主动脉瘤的进程

PCSK9 promotes mitochondrial dysfunction to accelerate the process of abdominal aortic aneurysm

摘要:

目的]探讨前蛋白转化酶枯草溶菌素9(PCSK9)影响腹主动脉瘤(AAA)的进展及机制。 [方法]选取6～8周龄ApoE－/－小鼠构建小鼠AAA模型:通过皮下植入微型渗透泵,持续输注血管紧张素Ⅱ(AngⅡ),高脂喂养28天后处死。收集人和小鼠正常腹主动脉血管和AAA标本,用免疫组织化学和免疫荧光检测PCSK9在腹主动脉平滑肌细胞中的表达。不同浓度的AngⅡ处理原代培养的C57BL/6小鼠血管平滑肌细胞(mVSMC)24 h后,检测PCSK9 mRNA和蛋白表达。建立PCSK9过表达和敲低细胞模型,检测线粒体活性氧(mtROS)、线粒体膜电位(MMP)、线粒体通透性转换孔(MPTP)开放及Z-DNA结合蛋白1(ZBP1)蛋白表达。通过生物信息学对多个单细胞测序的数据集进行差异表达分析来获取关键差异表达基因,并探讨其在AAA中的表达及作用。 [结果]免疫组织化学和免疫荧光结果显示,在人和小鼠的AAA组织中PCSK9的表达增加(P＜0.01),并与平滑肌存在共定位。AngⅡ呈浓度依赖性促进mVSMC中PCSK9的表达,2.0 μmol/L AngⅡ组PCSK9 mRNA和蛋白的表达分别增加2.9倍和1.1倍(P＜0.01),这一效应最为显著。成功构建PCSK9过表达和PCSK9干扰mVSMC模型后,PCSK9过表达导致细胞内mtROS增加,MMP下降,MPTP开放增加,细胞活性下降(P＜0.01)；PCSK9敲低可以减少AngⅡ引起的mtROS增加、MMP下降和MPTP开放；与siNC＋AngⅡ组相比,siPCSK9＋AngⅡ组mtROS减少,MMP和MPTP的荧光亮度增加(P＜0.05)。生物信息学分析发现,ZBP1是AAA中核心差异表达基因。免疫组织化学和免疫荧光结果显示,在人和小鼠的AAA组织中ZBP1的表达增高,并与平滑肌存在共定位；Western blot结果显示,过表达PCSK9或2.0 μmol/L AngⅡ处理均可增加ZBP1蛋白表达(P＜0.01),敲低PCSK9可减轻AngⅡ引起的ZBP1表达增高(P＜0.05)。 [结论]PCSK9可能通过诱导平滑肌细胞中的线粒体损伤,激活下游分子ZBP1导致细胞损伤,从而促进AAA的发展。

Abstract:

Aim To explore the impact and mechanism of proprotein convertase subtilisin kexin 9 (PCSK9) on the progression of abdominal aortic aneurysm (AAA). Methods 6～8 week old ApoE－/－ mice were selected to establish the AAA model. AngiotensinⅡ (AngⅡ) was continuously infused through subcutaneous implantation of a micro-osmotic pump. The mice were fed with high-fat diet and killed after 28 days. The expression of PCSK9 in abdominal aortic smooth muscle cells was detected by immunohistochemistry and immunofluorescence in normal abdominal aortic blood vessels and AAA samples in human and mice. Primary cultured murine vascular smooth muscle cells (mVSMC) of C57BL/6 mice were treated with different concentrations of AngⅡ for 24 h, and the expression of PCSK9 mRNA and protein was detected. PCSK9 overexpression and knockdown cell models were established, and mitochondrial reactive oxygen species (mtROS), mitochondrial membrane potential (MMP), mitochondrial permeability transition pore (MPTP) opening, and Z-DNA binding protein 1 (ZBP1) protein expression were detected. Bioinformatics was used to analyze the differential expression of multiple single-cell sequencing datasets to obtain the key differentially expressed genes, and to study their expression and role in AAA. Results Immunohistochemistry and immunofluorescence results showed that PCSK9 expression in human and mouse AAA increased (P＜0.01), and co-localized with smooth muscle. AngⅡpromoted PCSK9 expression in mVSMC in a concentration-dependent manner, the 2.0 μmol/L AngⅡ group showed a 2.9-fold and 1.1-fold increase in the expression of PCSK9 mRNA and protein, respectively (P＜0.01), with the most significant effect observed. After successfully constructing PCSK9 overexpression and PCSK9 interference mVSMC models, PCSK9 overexpression led to an increase in intracellular mtROS, a decrease in MMP, an increase in MPTP opening, and a decrease in cellular activity (P＜0.01); PCSK9 knockdown could reduce AngⅡinduced increase in mtROS, decrease in MMP and MPTP opening; compared with the siNC＋AngⅡgroup, the siPCSK9＋AngⅡ group showed a decrease in mtROS and an increase in the fluorescence brightness of MMP and MPTP (P＜0.05). Bioinformatics analysis revealed that ZBP1 was a core differentially expressed gene in AAA. Immunohistochemistry and immunofluorescence results showed that ZBP1 expression in human and mouse AAA tissues increased, and co-localized with smooth muscle. Western blot results showed that PCSK9 overexpression or treatment with 2.0 μmol/L AngⅡ could increase ZBP1 protein expression (P＜0.01), while PCSK9 knockdown could alleviate the increased ZBP1 expression caused by AngⅡ (P＜0.05). Conclusion PCSK9 may induce mitochondrial damage in smooth muscle cells, activate downstream molecule ZBP1 to cause cell damage, and promote the development of AAA.