Homocysteine-induced myofibroblast differentiation in mouse aortic endothelial cells

✍ Scribed by Utpal Sen; Karni S. Moshal; Neetu Tyagi; Ganesh K. Kartha; Suresh C. Tyagi

• Publisher: John Wiley and Sons
• Year: 2006
• Tongue: English
• Weight: 350 KB
• Volume: 209
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/jcp.20752

✦ Synopsis

Abstract

Differentiation of myofibroblast, as evidenced by α‐smooth muscle actin (α‐SMA) expression, is largely mediated by transforming growth factor‐β1 (TGF‐β1). This mechanism often follows inflammatory events such as endothelial damage due to oxidative stress, which can further leads to vascular thickening, stiffness, and fibrosis. We hypothesized that hyperhomocysteinemia (HHcy)‐induced oxidative stress lead to vascular stiffness, in part due to endothelial–myofibroblast differentiation and alteration of collagen homeostasis in the extracellular matrix (ECM). We tested our hypothesis in vitro using mouse aortic endothelial cells (MAEC). Our result shows that Hcy induces α‐SMA and collagen type‐1 expression in MAEC as evidenced by immunoblot and confocal imaging. RT‐PCR shows robust increase of α‐SMA and collagen type‐1 mRNA level in Hcy‐induced condition. We demonstrated that Hcy induces autophosphorylation of focal adhesion kinase (FAK) (a member of the protein tyrosine kinase (PTK) family) at Tyr‐397. PP2 (general PTK inhibitor) as well as FAK siRNA abrogates Hcy‐mediated α‐SMA formation. In addition to that, Hcy‐mediated TGF‐β1 induction was inhibited by TGF‐β R1 kinase inhibitor II (ALK5 inhibitor II) and attenuated FAK phosphorylation and α‐SMA expression. Furthermore, we showed that Hcy activates ERK‐44/42 (extracellular signal‐regulated kinase) pathway and augments collagen type‐1 deposition. Studies with pharmacological ERK blocker, PD98059 and ERK siRNA attenuated ERK‐44/42 phosphorylation and collagen type‐1 synthesis. Taken together our results demonstrate that Hcy‐mediated TGF‐β1 upregulation triggers endothelial–myofibroblast differentiation secondary to FAK phosphorylation and that Hcy‐induced ERK activation is involved in ECM remodeling by altering collagen type‐1 homeostasis. J. Cell. Physiol. 209: 767–774, 2006. © 2006 Wiley‐Liss, Inc.