Influence of oxidatively modified LDL on monocyte-macrophage differentiation |
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Authors: | Achuthan Radhika Shiney S Jacob Perumana R Sudhakaran |
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Institution: | (1) Department of Physiology & Biophysics, HSC, University of Louisville School of Medicine, A-1215, 500 South Preston Street, Louisville, KY 40202, USA;(2) Biotechnology and Cell Biology, Potentia Pharmaceuticals Inc., Louisville, KY 40202, USA |
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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. |
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Keywords: | Focal adhesion Cytoskeletal signaling GTPase translocation Oxidative stress |
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