USF1 transcriptionally activates USP14 to drive atherosclerosis by promoting EndMT through NLRC5/Smad2/3 axis

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. Conclusion Our findings indicate the contribution of the USF1/USP14/NLRC5 axis to atherosclerosis development via promoting EndMT, which provide effective therapeutic targets.

Hydroxysafflor yellow A, a natural food pigment, ameliorates atherosclerosis in ApoE−/− mice by inhibiting the SphK1/S1P/S1PR3 pathway

“…The inflammatory response of vascular endothelial cells mediated by ox‐LDL is the early behavior and main signal of atherosclerosis (Mehta & Malik, 2006 ). Therefore, we used ox‐LDL (100 μg/mL, Yiyuan Biotechnology, Shanghai, China) (Zhang et al., 2024 ) to stimulate HUVECs for 24 h as an in vitro model of vascular endothelial hyperpermeability in AS. To investigate the effect of HSYA on SphK/S1P/S1PR‐mediated vascular endothelial permeability, cells were stimulated with the SphK1 agonist K6PC‐5 (10 μM, Monmouth Junction, NJ, USA) (Shao et al., 2015 ), the S1P agonist FTY‐720 (50 ng/mL, Monmouth Junction, NJ, USA) (Milford et al., 2022 ), or the S1PR3 agonist CYM5541 (100 nM, Monmouth Junction, NJ, USA) (Ziegler et al., 2024 ).…”

The role of deubiquitinases in cardiovascular diseases: mechanisms and therapeutic implications

“…Further experiments revealed that overexpression of USP14 alleviates ox-LDL-induced endothelial inflammation, primarily by deubiquitinating NLRC5, thereby inhibiting NF- κ B activation and reducing the release of pro-inflammatory cytokines ( 20 ). Interestingly, another study by Zhang et al reported that USP14 expression is upregulated in the serum of atherosclerosis patients, and overexpression of USP14 promotes the activation of the Smad2/3 signaling pathway via NLRC5 deubiquitination ( 21 ), leading to increased secretion of pro-inflammatory cytokines. These seemingly contradictory findings, however, can be reasonably explained.…”