title: Homocysteine-induced myofibroblast differentiation in mouse aortic endothelial cells.
abstract: Differentiation of myofibroblast, as evidenced by alpha-smooth muscle actin (alpha-SMA) expression, is largely mediated by transforming growth factor-beta1 (TGF-beta1). 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 alpha-SMA and collagen type-1 expression in MAEC as evidenced by immunoblot and confocal imaging. RT-PCR shows robust increase of alpha-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 alpha-SMA formation. In addition to that, Hcy-mediated TGF-beta1 induction was inhibited by TGF-beta R1 kinase inhibitor II (ALK5 inhibitor II) and attenuated FAK phosphorylation and alpha-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-beta1 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.
authors.affiliation: Department of Physiology & Biophysics, HSC, University of Louisville School of Medicine, 500 South Preston St., 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: Neetu
authors.initials: N
authors.lastname: Tyagi
authors.forename: Ganesh K
authors.initials: GK
authors.lastname: Kartha
authors.forename: Suresh C
authors.initials: SC
authors.lastname: Tyagi
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000166:cytology*; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D006710:Homocysteine / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
chemical_list: D000199:Actins; D024042:Collagen Type I; D004791:Enzyme Inhibitors; D034741:RNA, Small Interfering; D053773:Transforming Growth Factor beta1; D006710:Homocysteine; D051417:Focal Adhesion Kinase 1; C496051:Ptk2 protein, mouse; D048049:Extracellular Signal-Regulated MAP Kinases