4144e082a4c70169c5e03e7956dc7de3 337151.pdf 10924d92c9315e63eabbd853745c0bdeb688f312 337151.pdf ecbf1d5e37f28a7e375f9d058defabb714f3e26cc4bd99b8165c21e002035f44 337151.pdf Title: Gene Expression Pattern Associated with Cytoskeletal Remodeling in Lipid-Loaded Human Vascular Smooth Muscle Cells: Crosstalk Between C3 Complement and the Focal Adhesion Protein Paxillin Subject: 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 M2 (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. Keywords: cell migration; cell adhesion; cytoskeleton; actin organization; gene expression Author: Maisa Garcia-Arguinzonis, Rafael Escate, Roberta Lugano, Esther Peña, Maria Borrell-Pages, Lina Badimon and Teresa Padro Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Tue Aug 12 16:09:34 2025 CEST ModDate: Tue Aug 12 16:32:10 2025 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2291645 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- JBWCSP+URWPalladioL-Ital Type 1 Custom yes yes yes 132 0 XVRKKP+URWPalladioL-Bold Type 1 Custom yes yes yes 133 0 YHZIWR+URWPalladioL-Roma Type 1 Custom yes yes yes 134 0 VVSYYB+EURM10 Type 1 Builtin yes yes yes 135 0 QMDDXF+CMSY10 Type 1 Builtin yes yes yes 173 0 MPUDFL+PazoMath Type 1 Builtin yes yes yes 215 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2026-10-31 04:14:15 CET RepresentationInformation: 337151.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2026-10-30 10:54:38 CET Size: 2291645 Format: PDF Version: 1.5 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 417 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Materials and Methods Children: Item: Title: hVSMC Culture and LDL Preparation Item: Title: Scratch-Wound Assay Item: Title: Cell Adhesion Assay Item: Title: RNA Extraction and Real-Time PCR Analysis Item: Title: In Silico Analysis Item: Title: Western Blot Analysis Item: Title: Confocal Microscopy Item: Title: Statistical Analysis Item: Title: Results Children: Item: Title: Gene Profiling Related to Cell Migration of hVSMCs Item: Title: Gene Ontology (GO) Terms for the Differential Gene Pattern in Migrating hVSMCs Item: Title: Differential Pattern of Genes Related to Actin Cytoskeleton in Migrating hVSMCs Item: Title: Signaling Pathway Linked to Cell Migration of hVSMCs Item: Title: Effect of Atherogenic LDL on PXN Expression and Intracellular Localization Item: Title: PXN as a Target of iC3b-Integrin Signaling: In Silico Predictions and Confocal Analysis Item: Title: iC3b Modulates the Cytoskeleton Remodeling of PXN in Lipid-Loaded Adherent hVSMCs Item: Title: Discussion Item: Title: Conclusions Item: Title: References Info: Title: Gene Expression Pattern Associated with Cytoskeletal Remodeling in Lipid-Loaded Human Vascular Smooth Muscle Cells: Crosstalk Between C3 Complement and the Focal Adhesion Protein Paxillin Author: Maisa Garcia-Arguinzonis, Rafael Escate, Roberta Lugano, Esther Peña, Maria Borrell-Pages, Lina Badimon and Teresa Padro Subject: 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 M2 (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. 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