Gene Expression Pattern Associated with Cytoskeletal Remodeling in Lipid-Loaded Human Vascular Smooth Muscle Cells:...

Mechanical and contractile forces in the vascular wall regulate smooth muscle cell migration. We previously demonstrated the presence of C3 complement products in atherosclerotic lesions of human aortas and showed that that C3-derived fragments promote key cellular processes, such as actin cytoskeleton organization and cell migration, in lipid-loaded human vascular smooth muscle cells (hVSMCs). In the present study, we aimed to investigate gene expression profiles related to cytoskeletal remodeling and cell adhesion in migrating hVSMCs with a particular focus on modulatory effect of the C3 complement pathway on these processes. We analyzed gene expression in migrating and non-migrating hVSMCs using real-time PCR and in silico network analysis. Additionally, we investigated cytoskeletal remodeling through Western blotting and confocal microscopy. PCR profiling revealed 30 genes with significantly altered expression in migrating hVSMCs compared to non-migrating control cells. In silico analysis identified six of these genes-PXN, AKT1, RHOA, VCL, CTNNB1, and FN1-as being associated with cytoskeletal remodeling and focal adhesion, with PXN occupying a central position in the interaction network. PXN expression was reduced at both the transcript and protein levels and showed altered subcellular localization in migrating lipid-loaded hVSMCs. Protein-protein interaction analysis using STRING predicted an association between PXN and the integrin complex αMβ2 (comprising ITGAM (CD11b) and ITGB2 (CD18)), which functions as receptors for the iC3b complement fragment. Confocal imaging of cell adhesion structures revealed that lipid-loaded hVSMCs stimulated with iC3b displayed a more diffuse PXN distribution and significantly increased PXN-F-actin colocalization in active cytoplasmic regions compared to lipid-loaded control cells. PXN-F-actin colocalization increased from 1.26% to 19.68%. Subcellular fractionation further confirmed enhanced PXN enrichment in the membrane fraction, with no significant changes observed in the cytosolic or cytoskeletal compartments. In conclusion, iC3b-mediated molecular signaling in lipid-loaded hVSMCs alters PXN distribution and enhances cytoskeletal remodeling, revealing novel molecular interactions in vascular remodeling and the progression of atherosclerotic lesions.