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Molecular and Cellular Biochemistry

, Volume 302, Issue 1–2, pp 133–143 | Cite as

Homocysteine-induced biochemical stress predisposes to cytoskeletal remodeling in stretched endothelial cells

  • Utpal Sen
  • Karni S. Moshal
  • Mahavir Singh
  • Neetu Tyagi
  • Suresh C. Tyagi
Article

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.

Keywords

Focal adhesion Cytoskeletal signaling GTPase translocation Oxidative stress 

Abbreviations

β-MHC

β-myosin heavy chain

CS

Cyclic stretch

FA(K)

Focal adhesion (kinase)

HHcy

Hyperhomocysteinemia

MAEC

Mouse aortic endothelial cell

NAC

N-Acetyl-Cysteine

PVDF

Polyvinylidene difluoride

ROK

Rho kinase

ROS

Reactive oxygen species

Notes

Acknowledgments

This research was supported in part by American Heart Association Post-Doctoral training grant (award # 0625579B) (to Karni S. Moshal) and NIH grants HL-71010 and HL-74185 (to Suresh C. Tyagi). Mahavir Singh sincerely appreciates the Indian Council of Agricultural Research (ICAR), New Delhi, Indian Veterinary Research Institute (IVRI), Izatnagar and the Department of Biotechnology (DBT), New Delhi, India for promoting the Advanced Biotechnology Research Initiatives.

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Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Utpal Sen
    • 1
  • Karni S. Moshal
    • 1
  • Mahavir Singh
    • 1
    • 2
  • Neetu Tyagi
    • 1
  • Suresh C. Tyagi
    • 1
  1. 1.Department of Physiology & Biophysics, HSCUniversity of Louisville School of MedicineLouisvilleUSA
  2. 2.Biotechnology and Cell BiologyPotentia Pharmaceuticals Inc.LouisvilleUSA

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