Oroxylin A Inhibits Glycolysis-dependent Vascular Smooth Muscle Cell Phenotypic Switching And Vascular Remodeling

BackgroundOcclusive artery diseases such as atherosclerosis,pulmonary arterial hypertension,and restenosis after angioplasty are main causes of death in patients with cardiovascular disease.Effective control of the occurrence and progression of these diseases is a key unmet need today.Vascular smooth muscle cells(VSMCs)are highly differentiated cells located in the tunica media of arteries,and undergo significant dedifferentiation under the stimulation of injury or cytokines such as platelet derived growth factor(PDGF),which manifests a phenotypic switching from contractile phenotype to proliferative phenotype and causes luminal stenosis as well as decreased blood vessel elasticity.Metabolic reprogramming is always involved in cell proliferation,aerobic glycolysis refers to a condition in which tumor cells produce energy by a high rate of glycolysis in the presence of oxygen,which enhances greatly energy supply and promotes cell growth and proliferation.This condition is also referred to as the Warburg effect.A growing number of studies have demonstrated that this condition also exists in non-tumor cells,especially in endothelial cells,however,the role of aerobic glycolysis in VSMCs remains unclear.Oroxylin A is a flavonoid compound that has anti-inflammatory,neuroprotective,and vasodilator effects,and has been shown to exert anti-tumor effects via regulating glycolysis recently.AimsThis study aims to dissect the role of glycolysis in VSMC phenotype switching,and the mechanism of Oroxylin A in regulating VSMC phenotype switching and vascular remodeling.Methods1.Post 7 days,western blot analysis was used to detect the changes of VSMC phenotypic markers and other proteins in the tunica media of mice with carotid artery ligation.Neointimal formation model was induced after the carotid artery ligation or carotid artery wire injury for 28 days in mice.HE staining and immunofluorescence were used to detect the effects of glycolysis inhibitors and Oroxylin A on arterial remodeling and phenotypic conversion of VSMCs or glycolytic enzyme expression in vivo,respectively.Oroxylin A(once two days)or3PO(once a day)were dissolved in DMSO(dimethyl sulfoxide)to 40 mg/ml final concentration for animal experiments.All vehicle group was injected with DMSO(1ml/kg body weight)as same frequence.2.Rat aortic vascular SMCs were isolated from male Sprague-Dawley rats(6-8w)which were euthanized by CO2 and the aorta was separated to remove adhering peri-adventitial tissue and the endothelium denuded with a catheter.All VSMCs cultures used in this study were less than 8 passages.3.Brd U staining,cell count,and wst-1 proliferation assay were used to detect the effects of PDGF,glycolytic agonists and inhibitors,and Oroxylin A on VSMC proliferation in vitro.4.The XFe-96 Glycolysis Stress Test and Cell Mito Stress Test Kit from Seahorse Biosciences was used to examine the effects of PDGF,glycolysis agonists and inhibitors,and Oroxylin A on the levels of glycolysis and oxidative phosphorylation of VSMCs in vitro.5.The effects of PDGF,glycolytic agonists and inhibitors,and Oroxylin A on phenotypic switching of VSMCs and intracellular kinase signaling pathway in vitro were detected using Western blot,while q PCR was used to investigate the effects of PDGF and Oroxylin A on the m RNA of VSMC phenotypic switching markers and glycolytic key enzymes.Methods and Results1.PFKFB3 was highly expressed in the tunica intima of hyperplasia in human coronary artery atherosclerotic plaques.Vascular injury induced VSMC phenotypic switching was correlated with significantly increased expression of glycolytic key enzymes,P<0.01.2.PDGF induced the VSMCs from contractive type to proliferative type in vitro,and promoted the proliferation and migration of VSMC,associated with the increase of glycolysis level,P<0.01.3.Enhanced glycolysis level promoted VSMC from contractive type to proliferative type,while blocking glycolysis could partially reverse the effect of PDGF on VSMC phenotypic switching in vitro.4.Glycolytic enzyme PFKFB3 specific inhibitor 3PO inhibited VSMC phenotypic switching in vivo,and 3PO significantly inhibited vascular remodeling,P<0.01.5.The flavonoid Oroxylin A reverses the PDGF-induced VSMC phenotypic switching via regulating glycolysis rather than oxidative phosphorylation.Further more,Oroxylin A inhibited cell proliferation induced by glucose or PDGF,but the over-expression of HIF-1α could partially inhibited the effect of Oroxylin A.6.Oroxylin A alleviates glycolysis through acting as a specific inhibitor of TSC2 phosphorylation on Ser939,thereby blocking the TSC2-m TOC1-HIF-1α axis.7.Oroxylin A prevents vascular injury-induced phenotypic switching and glycolysis elevation in VSMCs as well as neointima formation in vivo,P<0.01.ConclusionGlycolysis promotes VSMC phenotype switching,and glycolysis inhibitors inhibit VSMC phenotype switching and vascular remodeling.Oroxylin A inhibits glycolysisdependent VSMC phenotype switching and neointima formation by regulating the TSC2-m TORC1-HIF-1α axis.