Effects Of A Novel Butyrolactone Derivative On Angiogenesis And The Corresponding Mechanisms

Angiogenesis plays essential role in physiological processes such as embryonic development and in pathologic conditions(eg,tumor growth and atherosclerosis).The process of angiogenesis consists of several signal transductions,which include proliferation,survival,migration,extracellular matrix degradation,differentiation and morphogenesis.Inhibition of VEC apoptosis providing VEC survival is thought to be an essential issue during angiogenesis.In our previous study,it was found that a novel butyrolactone derivative, 3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one(3BDO), could effectively suppress human umbilical vascular endothelial cell(HUVEC) apoptosis induced by deprivation of fibroblast growth factor-2(FGF-2)and serum. From the chemical genetics perspective,we investigate the role of 3BDO and its molecular mechanism in regulation of angiogenesis.METHODS:1.HUVECs and VSMCs were gained as described previously[Jaffe et al,1973;Leik et al,2004].2.Rat aorta ring was cultured as described previously[Nicosia & Ottinetti,1990]3.Angiogenesis assay in vitro:capillary-like tube formation on Matrigel as described previously[Kureishi Y et al,2000]4.Cell migration assay in vitro:monolayer cell wound healing assay as described previously[Bürk,1973;Vasvari et al,2007]5.The morphological changes were observed under phase contrast microscope.6.Analysis of nuclear fragmentation by acridine orange staining combined with laser scan confocol microscope.7.The cell proliferation was measured by MTT assay.8.Na~+K~+-ATPase activity assay was performed by the Na~+K~+-ATPase detection kit.9.Mitochondrial membrane potential(MMP)assay:fluorescent probe(TMRM) combined with laser scan confocol microscope as described previously[Falchi et al, 2005] RESULTS:1.120μM 3BDO had selective effects on VSMCs and VECs in angiogenesis of rat aortic rings.2.Capillary-like tube formation assay on Matrigel in vitro showed that in the absent of serum and FGF-2,after exposed to 120μM 3BDO for 48 h,HUVECs formed capillary-like structures on matrigel.3.Monolayer cell wound healing assay in vitro showed that after exposed to 120μM 3BDO for 24 h,3BDO maintained the ability of migration in HUVECs.4.The nuclei remained complete as that in control group at 24 h after the treatment of 3BDO,after exposed to 120μM 3BDO for 24 h,3BDO inhibited the growth and migration of VSMCs.5.After exposed to 120μM 3BDO for24 h,3BDO inhibited the activity of Na, K-ATPase of HUVECs,but in VSMCs treated with 120μM 3BDO,the activity of Na,K-ATPase was not changed.6.120μM 3BDO depressed mitochondria membrane potential obviously,but in VECs treated with 120μM 3BDO,the activity of mitochondria membrane potential was not changed.CONCLUSION:1.This study is the first to investigate the effect of 3BDO on angiogenesis.3BDO has a selective effect on VECs and VSMCs in angiogenesis.2.3BDO could block the migration and proliferation of VSMCs,inhibit VEC apoptosis and maintain their ability to form capillary-like tubes on Matrigel.The distinct role of 3BDO on VECs and VSMCs may be beneficial for the controls of endothelium functional integrity and atherosclerosis development.3.3BDO might perform its role through the distinct effects on the activity of Na, K-ATPase and the mitochondria membrane potential in VECs and VSMCs.