The Effects Of Angiotensin-(1-7) On Vascular Calcification In Human Umbilical Vein Smooth Muscle Cell And The Underlying Mechanism

Objectives:In the present study, we establish the human umbilical vein smooth muscle cell (HU- -SMCs) calcification model which induced byβ-Glycerophosphate, and observed the effect of Angiotensin-(1-7)[Ang-(1-7)] in vascular calcification, investigating the possible mech- -anisms which was Ang-(1-7) delay or inhibit the HUSMCs calcium deposition.Method:1. Reproduction of vascular calcific model1.1. Usingβ-glycerolphosphate (β-GP) stimulate the human umbilical vein cells which cultured in vitro. The cells were randomly divided into 5 groups (n = 6): the control group (CON), 10mmol/L, 12mmol/L, 14mmol/L, 16mmol/Lβ-GP (I.e:10mmol/L group, 12mmol/L group, 14mmol/L group,16 mmol/L group). Calcium deposition in HUSMCs were detected by Von Kossa staining; and calculated Von Kossa staining by micro-image analysis system;the activities of alkaline phosphatase (ALP) in HUSMCs were verified by pheny1 diphosphate-2-sodium;the total calcium contents were calculated by ultraviolet spectrophotometry.2. Angiotensin-(1-7) effects on HUSMCs calcification2.1. Intervented angiotensin-(1-7) in calcified nutrient solution., and experimental group were randomly divided into 6 groups (n = 6):the control group (CON), calcification group (CAL), pure angiotensin-(1-7) group [Ang-(1-7) group], angiotensin-(1-7) with high-dose in calcified nutrient solution [Ang-(1-7)H group], angiotensin-(1-7) with moderate dose in the calcified nutrient solution [Ang-(1-7)M Group], angiotensin-(1-7) with low-dose in calcified nutrient solution group [Ang-(1-7)L Group].2.2. AnaLysis:①Calcium deposition in HUSMCs were detected by Von Kossa staining.②Von Kossa staining calculated by micro-image analysis system.③The activities of alkaline phosphatase (ALP) in HUSMCs were verified by pheny1 diphosphate-2-sodium; the total calcium contents in HUSMCs were calculated by ultraviolet spectrophotometry.④Activity of TGFβ1 in the nutrient solution were analysised by enzyme-linked immunosorbent assay, determineing the change of the level of TGFβ1 in HUSMCs.⑤The expression of core binding factorα1 (cbfα1) in HUSMCs were analyzed by Western-blot.⑥Osteocalcin (OC) contents in the nutrient solution were determined by radio immunoassay.Results:1.HUSMCs calcification were reproduced successfully: usingβ-glycerolphosphate-induced calcification in vitro HUSMCs. After Von Kossa staining, a large number of daisy-shaped HUSMCs were significantly detected in group 12mmol/L. There were brown nodules among smooth muscle cell. Compared with CON group, Micro-image analysis of quantitative calculations show that the percentage of calcified cells in 12mmol/L group were significantly increased (P<0.05), 10mmol/L group, 14mmol/L group, 16mmol/L group were also increased (respectively P<0.01); and cell calcium content and ALP levels were increased respectively (P<0.001).But 12mmol/L group was significantly increased. 2. Angiotensin-(1-7) effects on HUSMCs calcification①Ang-(1-7) can significantly reduce the calcification of the HUSMCs. Compared with CAL group, Von Kossa staining of Ang-(1-7)H group, Ang-(1-7)M group, Ang-(1-7)L group showed brown nodules were significantly reduced (P<0.001); Compared with CAL group, the percentage of positive cells in calcification, the calcium content and ALP levels of Ang-(1-7)H group, Ang-(1-7)M group, Ang-(1-7)L group was significantly reduced respectively(P<0.001);②Compared with the CAL group, TGFβ1 levels of Ang-(1-7)H group, Ang-(1-7)M group, Ang-(1-7)L group were reduced respectively (P<0.001), expression of cbfα1 in HUSMCs and osteocalcin content in culture medium were decreased.( respectively P<0.01).Conclusion:1.12mmol/Lβ-glycerolphosphate-stimulated HUSMCs can replicate calcium deposition model in vitro.2.Micro-image analysis software can be more objective and accurate interpretated Von Kossa staining of vascular calcification, and reduce the impact of subjective factors.3.Ang-(1-7) could inhibit the HUSMCs calcification.4.The role of Ang-(1-7) inhibited HUSMCs calcification through influenced TGFβ1-Smads- -cbfα1 signaling, weakening the key signaling molecule TGFβ1, cbfα1 expression and reducing OC contents.