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.