Garcia-Arguinzonis Maisa, Escate Rafael, Lugano Roberta, Peña Esther, Borrell-Pages Maria, Badimon Lina, Padro Teresa
Institut Recerca Sant Pau (IR-Sant Pau), 08041 Barcelona, Spain.
Centro de Investigación Biomédica en Red Cardiovascular (CIBERCV), Instituto de Salud Carlos III, 28029 Madrid, Spain.
Cells. 2025 Aug 12;14(16):1245. doi: 10.3390/cells14161245.
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.
血管壁中的机械力和收缩力调节平滑肌细胞迁移。我们之前在人类主动脉的动脉粥样硬化病变中发现了C3补体产物，并表明C3衍生片段可促进脂质负载的人类血管平滑肌细胞（hVSMC）中的关键细胞过程，如肌动蛋白细胞骨架组织和细胞迁移。在本研究中，我们旨在研究迁移的hVSMC中与细胞骨架重塑和细胞粘附相关的基因表达谱，特别关注C3补体途径对这些过程的调节作用。我们使用实时PCR和计算机网络分析来分析迁移和未迁移的hVSMC中的基因表达。此外，我们通过蛋白质印迹和共聚焦显微镜研究细胞骨架重塑。PCR分析显示，与未迁移的对照细胞相比，迁移的hVSMC中有30个基因的表达发生了显著变化。计算机分析确定其中6个基因——PXN、AKT1、RHOA、VCL、CTNNB1和FN1——与细胞骨架重塑和粘着斑相关，PXN在相互作用网络中占据中心位置。在迁移的脂质负载hVSMC中，PXN的转录本和蛋白质水平均降低，且亚细胞定位发生改变。使用STRING进行的蛋白质-蛋白质相互作用分析预测PXN与整合素复合物αMβ2（由ITGAM（CD11b）和ITGB2（CD18）组成）之间存在关联，该复合物作为iC3b补体片段的受体发挥作用。细胞粘附结构的共聚焦成像显示，与脂质负载的对照细胞相比，用iC3b刺激的脂质负载hVSMC显示出更分散的PXN分布，并且在活跃的细胞质区域中PXN-F-肌动蛋白共定位显著增加。PXN-F-肌动蛋白共定位从(1.26%)增加到(19.68%)°亚细胞分级分离进一步证实了膜组分中PXN富集增强，而胞质或细胞骨架组分中未观察到显著变化。总之，脂质负载的hVSMC中iC3b介导的分子信号改变了PXN分布并增强了细胞骨架重塑，揭示了血管重塑和动脉粥样硬化病变进展中的新型分子相互作用。
