Abstract
Purpose : To investigate the effect of withaferin A (WFA) on the proliferation and migration of brain endothelial cells. Methods : BALB-5023 mouse microvascular cells were treated with a range of withaferin A (WFA) concentrations from 10 to 100 ng/mL. Dojindo’s CCK-8 cell proliferation kit was used for the analysis of cell proliferation. Transwell cell culture inserts were used to determine the migration potential of WFAtreated endothelial cells. Absorbance was measured at 450 nm on an enzyme-linked immunosorbent (ELISA) reader. Results : The results revealed a significant increase in the proliferation and migration of endothelial cells following treatment with a low concentration (30 ng/mL) of WFA compared with the higher concentration (> 10 ng/mL). The effect was further enhanced when WFA was used in combination with soluble Fas ligand (sFasL). Autocrine signaling of vascular endothelial growth factor (VEGF) by endothelial cells was significantly increased following treatment with WFA or in combination with sFasL. WFA increased the expression of Fas on endothelial cells, suggesting the involvement of sFasL in the proliferation and migration of brain endothelial cells. Conclusion : Thus, WFA promotes the proliferation and migration of endothelial cells through increase in the expression of Fas and secretion of VEGF. Keywords : Endothelial cells, Vascular endothelial growth factor, Microvascular, Vascular disease, Withaferin A
Highlights
At present the leading cause of morbidity and mortality throughout the globe is cardiovascular diseases and stroke
The cells treated with withaferin A (WFA) alone or combined with soluble FasL for 72 h were washed twice in PBS followed by addition of Lysis buffer (50 mM Tris-HCl pH 7.4, 137 mM NaCl, 10 % glycerol, 100 mM sodium vanadate, 1 mM PMSF, 10 mg/ml aprotinin, 10 mg/mL leupeptin, 1 % NP-40, and 5 mM cocktail)
The results revealed that WFA stimulated the proliferation of the endothelial cells significantly at a low-dosage, ranging from 0.025 to 0.25 ng/mL, compared to
Summary
At present the leading cause of morbidity and mortality throughout the globe is cardiovascular diseases and stroke. After 24 h, different concentrations of WFA (Chinese National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) or soluble FasL (Sigma, St. Louis, MO, USA) were added to each well containing endothelial cells for 48 h. In 6-well plates, endothelial cells were treated for 72 h with either WFA or WFA and soluble FasL. To each well 100 μl assay diluents, followed by 100 μl standard, control or cell culture supernatant was added. The cells treated with WFA alone or combined with soluble FasL for 72 h were washed twice in PBS followed by addition of Lysis buffer (50 mM Tris-HCl pH 7.4, 137 mM NaCl, 10 % glycerol, 100 mM sodium vanadate, 1 mM PMSF, 10 mg/ml aprotinin, 10 mg/mL leupeptin, 1 % NP-40, and 5 mM cocktail).
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