Analysis Of Angiotensin Ⅱ-induced Dysfunctions Of Cardiac Microvascular Endothelial Cells

Cardiac remodeling,which typically results from chronic hypertension or following an acute myocardial infarction,is a major risk factor for the development of heart failure and, ultimately,death.The renin-angiotensin aldosterone system(RAAS)plays a key role in the regulation of blood pressure,acting via the effects of the hormone angiotensin(Ang)Ⅱ.AngⅡincreases blood pressure and can exert growth-promoting effects leading to end-organ damage.Excess RAAS activity has been shown to be a major underlying cause of hypertension,heart failure,and related cardiovascular disorders.The renin-angiotensin system(RAS) has previously been established to play an important role in the progression of cardiac remodeling,and inhibition of a hyperactive RAS provides protection from cardiac remodeling and subsequent heart failure.And,angiogenesis plays an important role incardiac remodeling,AngⅡcan induce apoptosis and remodeling of myocardial cell.However,the relationgship between AngⅡand microvascular endothelial cell(MMVEC) in the heart remains unexplored.It has been recently reported that dysfunctions of angiogenesis in myocardium are critical to the progress of heart failure.Therefore our studies try to investigate whether angiotensinⅡmight induce dysfunctions of cultured cardiac microvascular endothelial cells and the possible mechanism involved in the effect of angiotensinⅡ.This paper is composed of three parts as follows.PartⅠ.Wistar rats(male,8-9 weeks of age) were selected for primary cell culture by method of planting myocardial tissue.And the molecular marker-vWF was observed and identified by immunocytochemistry to determin the purity of microvascular endothelial cells.To study the function of MMVEC,the capillary tube-like formation was used.The results showed that purity of microvascular endothelial cells characterized by staining with vWF was about 95%,and the cells showed a high ability to form the capillary tube-like structures after grown in matrigel for 18 hours.MMVEC cultured by this method was able to be used.PartⅡ.ERK phosphorylation and p53 expression were determined by methods of Western blot and PT-PCR,respectively,after treatment with angiotensinⅡ(10~-7-10~-6 M) in microvascular endothelial cells at different times.The results showed that phosphorylation of ERKs after addition of angiotensinⅡto the microvascular endothelial cells for 5min or 10 min was significantly increased than those without angiotensinⅡaddition(p＜0.05). And the gene expression of p53 was slightly enhanced by stimulation with angiotensinⅡfor 30 min,1 hour,2 hours,6 hours,12 hours or 24 hours as compared to those without the stimulation.So,we presume that persistent treatment with angiotensinⅡfor a long time might impaired cardiac microvascular endothelial cells,which might associate with the upregulation of p53 expression;Pretreatment for a short time with angiotensinⅡmight protect the cells,which might be attributed to a transient increase in the activities of ERKs,and the protection of the cells by ERKs might be through inhibiting the expression of p53,but the results showed that the expression of p53 has no degression.So,the activities of ERKs and the expression of p53 in MMVEC after treated with angi otensinⅡhave no significant relationship.PartⅢ.The capillary tube-like formation was also observed under a microscope after incubation of cardiac microvascular endothelial cells with angiotensinⅡ.The results showed that the formation of capillary-like tubes by microvascular endothelial cells was decreased after incubation with angiotensinⅡ(10~-7-10~-6 M ) for 18 hours as compared to those without angitensinⅡtreatment(p＜0.05 or p＜0.01);However,it was significantly improved when the cells were pretreated with angiotensin for 5 min or 10 min before a long time(18 hours) incubation comparing to those with angiotensinⅡtreatment for 18 hour alone(p＜0.05).Consider that:angiotensinⅡimpaired the function of MMVEC,persistent treatment with angiotensinⅡfor a long time might impaired cardiac microvascular endothelial cells,and pretreatment for a short time with angiotensinⅡmight protect the cells.In summary,the present study demonstrated that persistent treatment with angiotensinⅡfor a long time significantly impaired the ability of cardiac microvascular endothelial cells to form the capillary tube-like structures,which might associate with the upregulation of p53 expression;Pretreatment for a short time before the long term treatment with angiotensinⅡsignificantly improved the dysfunctions of microvascular endothelial cells induced by the treatment with angiotensinⅡ,which might be attributed to a transient increase in the activities of ERKs and the protection of the cells by ERKs migtht not be through inhibiting the expression of p53.