Effects Of Angiotensin ? Type 2 Receptor And Angiotensin-(1-7) On Vascular Calcification

Vascular calcification is one of the common vascular lesions and major complications of atherosclerosis,chronic kidney disease,hypertension and diabetes.When vascular calcification occurs,it indicates worse clinical outcomes and adverse cardiovascular events,but the mechanism leading to vascular calcification remains unclear.Vascular calcification is associated with disorders of mineral metabolism in chronic kidney disease.Among them,serum phosphorus has become an important regulator of vascular calcification in patients with chronic kidney disease.In a mouse model of chronic kidney disease,a high phosphorus diet induces calcification of vascular smooth muscle cells(VSMC)in the middle layer of the aorta.In vitro experiments also showed that the same level of phosphorus as it in hyperphosphatism individuals can directly promote the calcification of VSMC,resulting in VSMC from contractile phenotype to osteochondral phenotype.Renin angiotensin system(RAS)is an endocrine system that regulates body fluid,electrolyte balance and blood pressure.Chronic kidney disease is associated with the persistent activation of RAS,and angiotensin ?(Ang?)is also associated with the process of arterial calcification in chronic kidney disease.Ang ? binds to 2 receptor subtypes,type 1(AT1)and type 2(AT2)receptors.Most effects of Ang ? is mediated by AT1 receptor,while the beneficial effect mediated by AT2 receptor is opposite to the effect of AT1 receptor.AT2 receptor has low homology with AT1 receptor,but Ang ?shows similar affinity to both AT1 and AT2 receptors.AT2 receptor is highly expressed in fetal organs,but very low in most tissues of adult organisms.However,AT2 receptor can be re-expressed in pathological conditions such as congestive heart failure,renal failure,or in events such as skin injury,vascular injury myocardial infarction,cerebral ischemia,or peripheral neurotomy.For many years,it is difficult to reveal the physiological or potential pathophysiological functions of AT2 receptor.Because AT2 receptors are hardly detectable in many tissues,including cardiovascular related tissues under normal physiological conditions.Although it is difficult to assess its physiological role in healthy tissues or in fetal development,AT2 receptor mediated beneficial effects have been described in recent years in cardiovascular and renal diseases.However,there is no report about the role of AT2 receptor in the formation of vascular calcification.Angiotensin-(1-7)(Ang-(1-7))is a new bioactive component in the RAS system,which acts by binding to specific Mas receptor.Ang-(1-7)has been found to counterbalance the role of Ang 11 in the heart,blood vessels,lungs,kidneys,brain and liver.The effect of Ang-(1-7)on blood pressure is mainly caused by its direct action in blood vessels,kidneys and brain.In adult type ? diabetic mice,administration of Ang-(1-7)for 6 weeks prevented the increase of systolic blood pressure,while the Mas receptor inhibitor A-779 could eliminate the increase of systolic blood pressure.Ang-(1-7)can also improve cardiac parameters associated with hypertension,such as reducing cardiac hypertrophy,total collagen deposition,type ? collagen and fibronectin gene expression.Studies have shown that Ang-(1-7)reduces the diameter of myocardial cells in hypertensive animals.More and more evidences show that Ang-(1-7)has cardiovascular protective effects.Ang-(1-7)has been shown to inhibit vascular calcification induced by vitamin D3 and nicotine in rats.However,the effect of Ang-(1-7)on vascular calcification is still not very clear and needs further investigation.Therefore,this study is to investigate the role of AT2 receptor and Ang-(1-7)in vascular calcification and its mechanisms.In vivo,5/6 nephrectomized mice were used to establish the in vivo vascular calcification model.In vitro,vascular smooth muscle cells were cultured in calcified medium as the in vitro vascular calcification model.We investigate the effects of AT2 receptor and Ang-(1-7)on vascular calcification and try to find potential targets for the therapy of vascular calcification.MethodsIn vivo experiments,ApoE and ApoE/AT2KO mice were used to prepare 5/6 nephrectomy model for chronic kidney disease.Two operations were performed under anesthesia.For the first time,1/3 parts of the upper and lower poles of the left kidney were resected respectively.After two weeks,second operations were performed and all the right kidneys were ligated and resected.Some mice were treated with osmotic pump by intraperitoneal administration of Ang-(1-7)at 0.5mg/kg/days for 6 weeks.Serum samples were taken before operation and 2 weeks after operation for biochemical analysis.The contents of urea nitrogen(BUN),creatinine(Cre),inorganic phosphate(IP)and calcium(Ca)were determined.Bone morphogenetic protein-2(BMP-2),osteoprotegerin(OPG),osteocalcin(OCN)populationand osteogenic transcription factor(RUNX2)in aorta of mice were detected by real-time quantitative PCR 6 weeks after operation.Primary cultured thoracic aortic vascular smooth muscle cells(VSMC)were obtained from WT and SMAT2 mice.The cells were cultured in a calcified medium containing 5%FBS,0.25 mM ascorbic acid and 10 mM ?-glycerophosphate for 14 days.Some was added Ang-(1-7)at 100nM for 14 days.The deposition of calcium in VSMC was identified by Alizarin Red S staining.The absorption of Alizarin Red eluent was determined by spectrophotometer,which was used for quantitative analysis.The expressions of BMP-2,OPG,OCN,RUNX2,osteopontin(OPN),AT1 receptor and Mas receptor in VSMC cultured in calcified medium for 14 days were detected by real-time quantitative PCR.Results1.Compared with ApoE mice,in ApoE/AT2KO mice the levels of BUN and IP in serum were increased 2 weeks after operation,and the expressions of BMP-2,OPG and RUNX2 were significantly changed,suggesting that the aortic calcification caused by chronic kidney disease in AT2 receptor knockout mice was more serious.2.Compared with WT+IP group,in SmAT2+IP group the absorbance of Alizarin Red S eluent was lower,and the expressions of BMP-2,OPG,OCN,RUNX 2 and OPN were significantly changed,which proved that the high expression of AT2 receptor could prevent the formation of VSMC calcification.3.Compared with the model group of ApoE mice,in Ang-(1-7)group the levels of BUN,Cre and IP in serum were decreased,and the expressions of BMP-2 and RUNX2 were decreased,suggesting that Ang-(1-7)could improve the formation of aortic calcification induced by chronic kidney disease in mice.4.Compared with IP group,in IP+Ang-(1-7)group the absorbance of Alizarin Red S eluent was lower,and the expressions of BMP-2,OPG,OCN,RUNX 2 and OPN were significantly changed,which proved that Ang-(1-7)could prevent the formation of VSMC calcification.5.Compared with the model group in ApoE/AT2KO mice,in Ang-(1-7)group the expressions of BMP-2,OPG,OCN and RUNX2 did not change,indicating that Ang-(1-7)could not improve the formation of aortic calcification induced by chronic renal failure in AT2 receptor knockout mice.So the effect of Ang-(1-7)on improving aortic calcification induced by chronic kidney disease was related to the expression of AT2.ConclusionThis study demonstrated that both the expression of AT2 receptor and the administration of Ang-(l-7)could prevent the formation of vascular calcification by regulating the expressions of osteocyte phenotype-related proteins.Moreover,the protective effect of Ang-(1-7)on vascular calcification is closely related to the expression of AT2 receptor.