RANBP1 Regulates NOTCH3-Mediated Autophagy in High Glucose-Induced Vascular Smooth Muscle Cells.

Abstract

Background: Vascular smooth muscle cells(VSMCs) phenotypic switching under hyperglycemic conditions accelerates atherosclerotic progression. Notch receptor 3(NOTCH3), a critical stabilizer of VSMC homeostasis implicated in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) pathogenesis, ensures vascular integrity; however, its interplay with RAN Binding Protein 1(RANBP1) during pathological hyperglycemia remains uncharacterized. We hypothesize that hyperglycemia-induced autophagic dysregulation is mechanistically governed by theNotch receptor 3 (NOTCH3)/RANBP1 axis, proliferative capacity, and apoptotic signaling in high glucose (HG)-stimulated VSMCs. The aim of this study was to elucidate the regulatory mechanisms of autophagy in VSMCs under HG conditions, with a focus on the NOTCH3/RANBP1 axis and its implications for vascular health.

Methods: Bioinformatics analysis was performed on NOTCH3 sequencing data, including weighted gene co-expression network analysis (WGCNA), screening of differentially expressed genes (DEGs), and construction of a protein-protein interaction (PPI) network, to identify the key gene, RANBP1. In vitro experiments, including cell counting kit-8 (CCK-8) assays, quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting (WB), and flow cytometry, were conducted to examine the effects of NOTCH3 knockdown combined with RANBP1 overexpression on glucose-induced autophagy marker expression and cell viability in VSMCs.

Results: NOTCH3 knockdown suppressed VSMC proliferation and induced apoptosis, and the cell cycle was stopped at the S phase. Analysis of VSMC sequencing data revealed 38 overlapping genes between the turquoise module and DEGs, 11 (HPF1, RANBP1, CRNKL1, LGALS3, RDX, ECM1, CXCL5, PA2G4, CENPS, ZNF830, and HIST1H4L) of which were significantly underexpressed in VSMC samples with si-NOTCH3. In a dose-dependent manner, HG therapy altered the expression of autophagy-related markers, upregulated NOTCH3, and downregulated phosphorylated mammalian target of rapamycin (p-mTOR). Downregulation of NOTCH3 aggravated the effects of HG on cell viability and autophagy, whereas overexpression of RANBP1 reversed these effects, suggesting an offsetting effect on HG-induced autophagy.

Conclusion: On the basis of sequencing technology, bioinformatics analysis and cell experiments, we conclude that the RANBP1/NOTCH3 axis is essential for the control of autophagy and survival of VSMCs under hyperglycemic stress and could provide new insight for the clinical treatment of VSMC-related diseases.