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.

Cystathionine-β-synthase gene transfer and 3-deazaadenosine ameliorate inflammatory response in endothelial cells

“…Mouse aortic endothelial cells (MAEC) were a generous gift from Kathleen Bove Stratton, VA Medical Center, Albany, NY. Cells were maintained in DMEM/F-12 50/50 (Cellgro, Mediatech, Herndon, VA) as described earlier (29) in a humid chamber at 37°C in an atmosphere of 5% CO 2 and 95% air. Confluent T-25 flasks were trypsinized (0.25% trypsin-2.21 mM EDTA in Hanks' balanced salt solution without sodium bicarbonate, calcium, and magnesium) (Cellgro, Mediatech, Herndon, VA) to detach cells, resuspended in fresh complete culture medium, plated onto a 12-well cell culture plate, and allowed to grow about 80% confluence before experiments.…”

The Protective Effects of Bevacizumab in Bleomycin-Induced Experimental Scleroderma

“…If so, migrated local and bone marrow derived endothelial cells may transform into the different cells for instance fibroblastic cells and thus may abrogate the fibrotic process instead of neovascularization. Endothelial cells handled by oxidative stress [12] and cytokines [11] have been reported to transform into fibroblastic cells.…”

“…Endothelial cells cultured with fibroblast growth factor [10], and exposed to tumor necrosis factor alpha (TNF-α) or IL-1β [11] are observed to transform into myofibroblastic cells. Endothelial-myofibroblastic cell transformation is also reported by the application of homocysteine, a potent oxidant [12].…”

Genistein protects dermal fibrosis in bleomycin-induced experimental scleroderma

“…Endothelial cells cultured with fibroblast growth factor (8) and exposed to tumor necrosis factor alpha (TNF-α) or IL-1β (9) have been observed to transform into myofibroblastic cells in in vitro experimental studies. Endothelial-myofibroblastic cell transformation by the application of homocysteine, a potent oxidant, has also been reported (10).…”