PDF(Pubmed)
Abstract：
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: Our findings indicate the contribution of the USF1/USP14/NLRC5 axis to atherosclerosis development via promoting EndMT, which provide effective therapeutic targets.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: Our findings indicate the contribution of the USF1/USP14/NLRC5 axis to atherosclerosis development via promoting EndMT, which provide effective therapeutic targets.
摘要：
背景：内皮-间充质转化（EndMT）在动脉粥样硬化的病理进展过程中在内皮功能障碍中起关键作用；然而，其详细机制尚不清楚。在这里，我们探讨了EndMT中上游刺激因子1(USF1)在动脉粥样硬化过程中的生物学功能和机制.
方法：在高脂饮食喂养的ApoE-/-小鼠和ox-LDL暴露的人脐静脉内皮细胞（HUVECs）中建立体内和体外动脉粥样硬化模型。斑块的形成,胶原蛋白和脂质沉积，并通过苏木精和伊红（HE）评估主动脉组织的形态学变化，Masson,油红O和Verhoeff-VanGieson（EVG）染色，分别。通过EndMT相关蛋白的表达水平测定EndMT。通过RT-qPCR和Western印迹检测靶分子表达。通过ELISA测量促炎细胞因子的释放。通过transwell和划痕测定检测HUVEC的迁移。通过双荧光素酶报告实验研究了分子机制，ChIP,和Co-IP测定。
结果：USF1在动脉粥样硬化患者中表达上调。USF1敲低通过上调CD31和VE-Cadherin抑制EndMT，同时下调α-SMA和波形蛋白，从而抑制炎症，和在暴露于ox-LDL的HUVECs中的迁移。此外,USF1转录激活泛素特异性蛋白酶14（USP14），这促进了NLR家族CARD结构域含5（NLRC5）的去泛素化和上调，以及随后的Smad2/3途径激活。sh-USF1或sh-USP14对EndMT的抑制作用被USP14或NLRC5过表达部分逆转。最后,USF1敲低通过抑制小鼠EndMT延迟动脉粥样硬化进展。
结论：我们的发现表明USF1/USP14/NLRC5轴通过促进EndMT对动脉粥样硬化发展的贡献，提供有效的治疗靶点。
方法：在高脂饮食喂养的ApoE-/-小鼠和ox-LDL暴露的人脐静脉内皮细胞（HUVECs）中建立体内和体外动脉粥样硬化模型。斑块的形成,胶原蛋白和脂质沉积，并通过苏木精和伊红（HE）评估主动脉组织的形态学变化，Masson,油红O和Verhoeff-VanGieson（EVG）染色，分别。通过EndMT相关蛋白的表达水平测定EndMT。通过RT-qPCR和Western印迹检测靶分子表达。通过ELISA测量促炎细胞因子的释放。通过transwell和划痕测定检测HUVEC的迁移。通过双荧光素酶报告实验研究了分子机制，ChIP,和Co-IP测定。
结果：USF1在动脉粥样硬化患者中表达上调。USF1敲低通过上调CD31和VE-Cadherin抑制EndMT，同时下调α-SMA和波形蛋白，从而抑制炎症，和在暴露于ox-LDL的HUVECs中的迁移。此外,USF1转录激活泛素特异性蛋白酶14（USP14），这促进了NLR家族CARD结构域含5（NLRC5）的去泛素化和上调，以及随后的Smad2/3途径激活。sh-USF1或sh-USP14对EndMT的抑制作用被USP14或NLRC5过表达部分逆转。最后,USF1敲低通过抑制小鼠EndMT延迟动脉粥样硬化进展。
结论：我们的发现表明USF1/USP14/NLRC5轴通过促进EndMT对动脉粥样硬化发展的贡献，提供有效的治疗靶点。
