Department of Cardiology, Zhengzhou University People's Hospital, Henan Provincial People's Hospital, Zhengzhou, Henan, 450000, China.
Department of Cardiology, Central China Fuwai Hospital, Zhengzhou, Henan, 450000, China.
Mol Med. 2024 Feb 29;30(1):32. doi: 10.1186/s10020-024-00798-8.
Endothelial-to-Mesenchymal Transformation (EndMT) plays key roles in endothelial dysfunction during the pathological progression of atherosclerosis; however, its detailed mechanism remains unclear. Herein, we explored the biological function and mechanisms of upstream stimulating factor 1 (USF1) in EndMT during atherosclerosis.
The in vivo and in vitro atherosclerotic models were established in high fat diet-fed ApoE mice and ox-LDL-exposed human umbilical vein endothelial cells (HUVECs). The plaque formation, collagen and lipid deposition, and morphological changes in the aortic tissues were evaluated by hematoxylin and eosin (HE), Masson, Oil red O and Verhoeff-Van Gieson (EVG) staining, respectively. EndMT was determined by expression levels of EndMT-related proteins. Target molecule expression was detected by RT-qPCR and Western blotting. The release of pro-inflammatory cytokines was measured by ELISA. Migration of HUVECs was detected by transwell and scratch assays. Molecular mechanism was investigated by dual-luciferase reporter assay, ChIP, and Co-IP assays.
USF1 was up-regulated in atherosclerosis patients. USF1 knockdown inhibited EndMT by up-regulating CD31 and VE-Cadherin, while down-regulating α-SMA and vimentin, thereby repressing inflammation, and migration in ox-LDL-exposed HUVECs. In addition, USF1 transcriptionally activated ubiquitin-specific protease 14 (USP14), which promoted de-ubiquitination and up-regulation of NLR Family CARD Domain Containing 5 (NLRC5) and subsequent Smad2/3 pathway activation. The inhibitory effect of sh-USF1 or sh-USP14 on EndMT was partly reversed by USP14 or NLRC5 overexpression. Finally, USF1 knockdown delayed atherosclerosis progression via inhibiting EndMT in mice.
Our findings indicate the contribution of the USF1/USP14/NLRC5 axis to atherosclerosis development via promoting EndMT, which provide effective therapeutic targets.
内皮-间充质转化（EndMT）在动脉粥样硬化的病理进程中对内皮功能障碍起着关键作用，但其中的详细机制尚不清楚。本研究旨在探索上游刺激因子 1（USF1）在动脉粥样硬化中的 EndMT 中的生物学功能和机制。
通过高脂饮食喂养 ApoE 敲除小鼠和氧化型低密度脂蛋白（ox-LDL）诱导的人脐静脉内皮细胞（HUVEC）建立体内和体外动脉粥样硬化模型。采用苏木精和伊红（HE）、Masson、油红 O 和 Van Gieson（EVG）染色分别评估主动脉组织中的斑块形成、胶原和脂质沉积以及形态学变化。通过 EndMT 相关蛋白的表达水平来确定 EndMT 的发生情况。通过 RT-qPCR 和 Western blot 检测靶分子的表达水平。通过 ELISA 法检测促炎细胞因子的释放。通过 Transwell 和划痕实验检测 HUVEC 的迁移。通过双荧光素酶报告基因检测、染色质免疫沉淀（ChIP）和免疫共沉淀（Co-IP）实验来探究分子机制。
USF1 在动脉粥样硬化患者中呈上调表达。USF1 敲低通过上调 CD31 和 VE-Cadherin 、下调 α-SMA 和 vimentin 抑制 ox-LDL 诱导的 HUVEC 中的 EndMT，从而抑制炎症和迁移。此外，USF1 转录激活泛素特异性蛋白酶 14（USP14），促进其去泛素化并上调 NLR 家族含 CARD 结构域蛋白 5（NLRC5），进而激活 Smad2/3 通路。用 USP14 或 NLRC5 过表达部分逆转 sh-USF1 或 sh-USP14 对 EndMT 的抑制作用。最后，USF1 敲低通过抑制 EndMT 而在小鼠中延缓动脉粥样硬化的进展。
本研究结果表明，USF1/USP14/NLRC5 轴通过促进 EndMT 参与动脉粥样硬化的发展，为其提供了有效的治疗靶点。
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