Integrated transcriptomic and metabolomic analysis reveals that cathepsin D regulates the phenotypic transition of vascular smooth muscle cells by inhibiting glycolysis

This study investigates the role of Cathepsin D (CTSD) in diabetic vascular complications, particularly its impact on the phenotypic transformation of vascular smooth muscle cells (VSMCs) induced by advanced glycation end-products (AGEs), and explores its potential molecular mechanisms. CTSD was overexpressed in VSMCs using lentiviral vectors. Various methods, including CCK-8, immunofluorescence, SA-ß-Gal staining, EdU assay, scratch assay, cell cycle analysis, and Western blotting, were employed to assess VSMC viability, proliferation, migration, senescence, and apoptosis. Additionally, transcriptomic and metabolomic analyses were conducted to investigate the molecular mechanisms underlying CTSD overexpression in VSMCs. AGEs treatment significantly inhibited CTSD expression in VSMCs, leading to reduced cell viability, enhanced proliferation and migration, increased senescence, and apoptosis. In contrast, overexpression of CTSD effectively inhibited AGEs-induced VSMC proliferation, migration, senescence, and apoptosis. Combined transcriptomic and metabolomic analyses suggested that CTSD may affect VSMC phenotypic transformation by inhibiting the glycolysis pathway. This study highlights the critical role of CTSD in the phenotypic transformation of VSMCs induced by AGEs and provides a new perspective for cardiovascular and cerebrovascular disease treatment. CTSD may emerge as a novel therapeutic target, though its specific molecular mechanisms and clinical application prospects in VSMC phenotypic transformation require further investigation. Overall design: To investigate the effects of AGEs on rat thoracic aortic VSMCs, the cells were incubated with 200 µg/mL for 24 h.To examine the impact of CTSD over-expression on AGEs-induced VSMCs, five cell groups were established: normal VSMCs treated with BSA (C-B group), empty vector-transfected VSMCs treated with BSA (N-B group), normal VSMCs treated with AGEs (C-A group), empty vector-transfected VSMCs treated with AGEs (N-A group), and overexpression-transfected VSMCs treated with AGEs (T-A group).We then performed gene expression profiling using data obtained from RNA-seq of cells from three different treatment groups (N-B, N-A, T-A).Comparative analysis of gene expression profiles of RNA-seq data of VSMCs cells from T-A and N-B groups, respectively, with those from N-A group, reveals the function of CTSD