title: Homocysteine-induced biochemical stress predisposes to cytoskeletal remodeling in stretched endothelial cells.
abstract: Cellular cytoskeletal remodeling reflects alterations in local biochemical and mechanical changes in terms of stress that manifests relocation of signaling molecules within and across the cell. Although stretching due to load and chemical changes by high homocysteine (HHcy) causes cytoskeletal re-arrangement, the synergism between stretch and HHcy is unclear. We investigated the contribution of HHcy in cyclic stretch-induced focal adhesion (FA) protein redistribution leading to cytoskeletal re-arrangement in mouse aortic endothelial cells (MAEC). MAEC were subjected to cyclic stretch (CS) and HHcy alone or in combination. The redistribution of FA protein, and small GTPases were determined by Confocal microscopy and Western blot techniques in membrane and cytosolic compartments. We found that each treatment induces focal adhesion kinase (FAK) phosphorylation and cytoskeletal actin polymerization. In addition, CS activates and membrane translocates small GTPases RhoA with minimal effect on Rac1, whereas HHcy alone is ineffective in both GTPases translocation. However, the combined effect of CS and HHcy activates and membrane translocates both GTPases. Free radical scavenger NAC (N-Acetyl-Cysteine) inhibits CS and HHcy-mediated FAK phosphorylation and actin stress fiber formation. Interestingly, CS also activates and membrane translocates another FA protein, paxillin in HHcy condition. Cytochalasin D, an actin polymerization blocker and PI3-kinase inhibitor Wortmannin inhibited FAK phosphorylation and membrane translocation of paxillin suggesting the involvement of PI3K pathway. Together our results suggest that CS- and HHcy-induced oxidative stress synergistically contribute to small GTPase membrane translocation and focal adhesion protein redistribution leading to endothelial remodeling.
authors.affiliation: Department of Physiology & Biophysics, HSC, University of Louisville School of Medicine, A-1215, 500 South Preston Street, Louisville, KY 40202, USA.
authors.forename: Utpal
authors.initials: U
authors.lastname: Sen
authors.forename: Karni S
authors.initials: KS
authors.lastname: Moshal
authors.forename: Mahavir
authors.initials: M
authors.lastname: Singh
authors.forename: Neetu
authors.initials: N
authors.lastname: Tyagi
authors.forename: Suresh C
authors.initials: SC
authors.lastname: Tyagi
mesh_terms: D000199:Actins / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001696:Biomechanical Phenomena; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects* / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D006710:Homocysteine / Q000494:pharmacology*; D020138:Hyperhomocysteinemia / Q000201:enzymology; D051379:Mice; D018384:Oxidative Stress / Q000187:drug effects*; D051419:Paxillin / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D000077191:Wortmannin; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D000199:Actins; D000730:Androstadienes; D051419:Paxillin; D006710:Homocysteine; D015638:Cytochalasin D; D019869:Phosphatidylinositol 3-Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D020744:rac GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D000077191:Wortmannin