Transactivation Domain Of Krüppel-like Factor 15 Negatively Regulates Angiotensin Ⅱ-induced Vascular Remodeling

Background:Hypertensive-induced vascular remodeling is an important pathological basis for human cardiovascular and cerebrovascular events,but its pathogenesis has not yet been clarified.Ours and others’previous studies have proved that adventitia inflammation and fibrosis play an important role in vascular remodeling.Adventitia fibroblasts are first activated during vascular injury,which participates in vascular remodeling with the increased secretion of cytokines and inflammatory mediators.It has been reported that Krueppel-like factor 15（KLF15）inhibits target organ fibrosis,but whether it participates in angiotensin Ⅱ（Ang Ⅱ）-induced vascular remodeling and its mechanism remains unclear.Objective:Make it clear that whether KLF15 participates in angiotensin Ⅱ-induced vascular inflammation,fibrosis and explore its potential mechanisms.Method and Results:1.The effect of Ang Ⅱ on the expression of KLF15:1)Mice were infused with Ang Ⅱ（1000ng/kg/min）for 14 days.Ang Ⅱ down-regulated the expression of KLF15 in the thoracic aorta of mice.2)The primary rat adventitial fibroblasts（AFs）were extracted and stimulated with Ang Ⅱ at different concentrations and time.Ang Ⅱ down-regulated the expression of KLF15 in AFs in a time-and dose-dependent manner.2.Effects of AdKLF15on Ang Ⅱ-induced blood pressure and vascular remodeling:1)AdCTL or AdKLF15 or AdKLF15-ΔTAD was respectively injected intravenously on day 0 or day 7 during Ang Ⅱ infusion.In vivo administration of AdKLF15 but not AdKLF15-ΔTAD improved Ang Ⅱ-induced blood pressure and heart rate,thoracic aortic wall thickness and wall area,inflammation and fibrosis.2)The common carotid artery was perivascularly infected with AdCTL,AdKLF15,AdKLF15-ΔTAD,and then subcutaneously infused with Ang Ⅱ for 14 days.Local delivery of AdKLF15,AdKLF15-ΔTAD around the common carotid artery had no effect on the increase of blood pressure and heart rate induced by Ang Ⅱ.AdKLF15 but not AdKLF15-ΔTAD attenuated the vascular remodeling,inflammation and fibrosis of the common carotid artery wall.3.Effect of KLF15 on adventitial fibroblasts:AFs were transfected with KLF15 small interfering RNA（siRNA）,AdKLF15 and AdKLF15-ΔTAD,respectively,and stimulated with Ang Ⅱ(10^-7 M).KLF15 negatively regulates Ang Ⅱ-induced increase of proliferation,migration and differentiation of AFs by TAD.In additition,KLF15 negatively regulates Ang Ⅱ-induced inflammation and fibrosis through TAD.4.Mechanism research:1)HEK293T cells were infected with AdKLF15 and AdKLF15-ΔTAD,and stimulated by Ang Ⅱ(10^-7 M),dual luciferase reporter system demonstrated that KLF15 down-regulated CCL2 promoter activity through TAD.Co-immunoprecipitation and Chromatin immunoprecipitation assay demonstrated that KLF15 directly regulates the promoter activity of CCL2 by binding to Smad2/3.2)AFs were inhibited with AT1receptor,AT2 receptor,PLCγ1,Src,PI3 kinase and MEK1/2,followed by Ang Ⅱ(10^-7 M)stimulation.Ang Ⅱ down-regulates the expression of KLF15 via the AT1receptor/PLCγ1/ERK1/2 signaling pathway.Conclusion:1.Ang Ⅱ down-regulates the expression of KLF15 by AT1 receptor/PLCγ1/ERK1/2 signalling.2.KLF15 inhibits Ang Ⅱ-induced vascular fibrosis and inflammation by negatively regulating Smad2/3 mediated CCL2 expression by TAD.