PCSK9通过诱导血管平滑肌细胞表型转换促进动脉粥样硬化

PCSK9 promotes atherosclerosis by inducing phenotypic transformation of vascular smooth muscle cells

摘要:

目的]探讨前蛋白转化酶枯草溶菌素9(PCSK9)在动脉粥样硬化(As)发生发展中的作用及分子机制。 [方法]采用血小板源性生长因子-BB(PDGF-BB)对小鼠原代血管平滑肌细胞(VSMC)进行不同时间的处理,通过划痕实验评估细胞迁移能力,并利用Western blot检测表型转换标志物及PCSK9的表达。使用Ad-PCSK9腺病毒感染小鼠原代VSMC,检测收缩型与合成型标志蛋白的表达情况,以及细胞增殖与迁移能力。通过腺病毒过表达或小干扰RNA(siRNA)抑制PCSK9表达,检测核因子κB(NF-κB)、Krüppel样因子4(KLF4)和心肌素(MYOCD)的表达水平。采用免疫组织化学、免疫荧光、油红O染色和苏木精-伊红(HE)染色,分析VSMC特异性过表达PCSK9(PCSK9^sm OE)小鼠与对照组(PCSK9^flox/flox)小鼠主动脉斑块中相关分子的表达、脂质沉积及斑块面积。 [结果]PCSK9与血管中膜及As斑块内的VSMC共定位；与中膜收缩型VSMC相比,其在斑块合成型VSMC中表达显著升高。PDGF-BB以时间依赖方式上调PCSK9及合成表型标志蛋白骨桥蛋白(OPN)、表皮调节素(EREG)表达,下调收缩表型标志蛋白平滑肌肌球蛋白重链(SMMHC)、α-平滑肌肌动蛋白(α-SMA)、钙调理蛋白、平滑肌22α(SM22α)表达,并增强细胞迁移能力。Ad-PCSK9可促进VSMC由收缩表型向合成表型转换,显著增强DNA复制活性与细胞迁移能力,同时,下调MYOCD表达,上调KLF4与NF-κB p65表达；而siRNA抑制PCSK9则呈现相反效应。组织学分析表明,VSMC过表达PCSK9可促进As小鼠主动脉斑块形成并增加斑块不稳定性。 [结论]PCSK9可诱导VSMC由收缩表型向合成表型转换,加剧小鼠As病变,该机制可能与NF-κB、KLF4及MYOCD之间的信号串扰有关。

Abstract:

Aim To investigate the role and molecular mechanism of proprotein convertase subtilisin/kexin type 9 (PCSK9) in the occurrence and development of atherosclerosis (As). Methods Mouse primary vascular smooth muscle cells (VSMCs) were treated with platelet-derived growth factor-BB (PDGF-BB) for different durations. Cell migration ability was evaluated by scratch assay, and the expression of phenotypic transition markers and PCSK9 was detected by Western blot. Mouse primary VSMCs were infected with Ad-PCSK9 adenovirus to examine the expression of contractile and synthetic marker proteins, as well as cell proliferation and migration capabilities. PCSK9 expression was overexpressed via adenovirus or inhibited by small interfering RNA (siRNA), and the expression levels of nuclear factor-κB (NF-κB), Krüppel-like factor 4 (KLF4), and myocardin (MYOCD) were detected. Immunohistochemistry, immunofluorescence, Oil Red O staining, and hematoxylin-eosin (HE) staining were used to analyze the expression of related molecules, lipid deposition, and plaque area in aortic plaques of VSMC-specific PCSK9 overexpression (PCSK9^sm OE) mice and PCSK9 conditional knockout (PCSK9^flox/flox) mice. Results PCSK9 was colocalized with VSMCs in the medial layer and atherosclerotic plaques. Compared with contractile VSMCs in the medial layer, PCSK9 expression was significantly elevated in synthetic VSMCs within the plaques. PDGF-BB upregulated PCSK9 in a time-dependent manner, along with the synthetic phenotypic marker proteins osteopontin (OPN) and epiregulin (EREG), while downregulating the contractile marker proteins smooth muscle myosin heavy chain (SMMHC), α-smooth muscle actin (α-SMA), calponin, and smooth muscle 22α (SM22α), and enhanced cell migration capacity. Ad-PCSK9 promoted the phenotypic transformation of VSMCs from contractile to synthetic phenotype, significantly enhanced DNA replication activity and cell migration ability, downregulated MYOCD expression, and upregulated KLF4 and NF-κB p65 expression. Conversely, siRNA-mediated PCSK9 inhibition showed opposite effects. Histological analysis revealed that VSMC-specific overexpression of PCSK9 promoted aortic plaque formation and increased plaque instability in atherosclerotic mice. Conclusions PCSK9 can induce VSMC phenotypic transformation from the contractile to synthetic phenotype, exacerbating the atherosclerotic lesions in mice. This mechanism may involve the signaling crosstalk among NF-κB, KLF4, and MYOCD.