Vascular Smooth Muscle Cells (VSMCs) Derived From The Spontaneously Hypertensive Rat (SHR) Are Prone To Senescence And Calcification In Vitro And Its Mechanism

Objectives:To study the role of fibronectin(FN)and its receptorα5-integrin in the senescence and calcification of thoracic aortic smooth muscle cells of Spontaneously hypertensive rat(SHR)in vitro,and to explore the mechanism of senescence and calcification of vascular smooth muscle cells(VSMCs)derived from hypertensive rat in vitro.Methods:VSMCs isolated from SHR and WKY(Wistar-Kyoto rat)of 12 weeks were cultured in vitro.The cells of passage three were used for all experiments.Based on VSMCs were coated with or without exogenous FN or added with or without GRGDSP which is the inhibitor of integrin,the experimental group were set up as follows:(1)the group of SHR+FN(group S-F),(2)the group of SHR+GRGDSP(group S-G),(3)the group of WKY(group W),(4)the group of SHR(group S).(1)and(2)were the experimental groups;(3)and(4)were the control groups.The levels of senescence,calcification and expressions of FN、EDA-FN、α5-integrin of VSMCs for 3d、6d、9d、12d were detected respectively by Senescenceβ-Galactosidase Staining,von kossa Staining and immunocytochemistry dynamically.The expression of ALP,which was a marker of phenotypic transformation,was analyzed by Western blot between group W and group S in all time points,and in all groups which were cultured for 12d,and in Group S which was coated with different concentrations of FN respectively that was cultured for 12d.Results:(1)Senescence of VSMCs:The VSMCs senescent level in every group became more and more serious as time went on(3d〈6d〈9d〈12d).At the time of 3d,the changes had no significant difference between group W and group S(P>0.05),but at the time of6d、9d、12d,group S was higher than group W(P<0.05).At the same time point,group S-F was higher than group S(P<0.05),and group S-G was lower than group S(P<0.05).(2)Calcification of VSMCs:The calcification of VSMCs in every group became more and more serious as time went on(3d〈6d〈9d〈12d).At the time of 3d,the changes had no significant difference between group W and group S(P>0.05),but at the time of6d、9d、12d,group S was higher than group W(P<0.05).At the same time point,group S-F was higher than group S(P<0.05),and group S-G was lower than group S(P<0.05).(3)The expressions of FN、EDA-FN、α5-integrin in VSMCs:The expressions of the three protein in every group increased as time went on(3d〈6d〈9d〈12d).(1)The expressions of FN、EDA-FN:at the time of 3d,the changes had no significant difference between group W and group S(P>0.05),but at the time of 6d、9d、12d,group S was higher than group W(P<0.05).At the same time point,group S-F was higher than group S(P<0.05),there was no significant difference between group S-G and group S(P>0.05).(2)The expression ofα5-integrin:At the same time point,group S was higher than group W(P<0.05),group S-F was higher than group S(P<0.05),and there was no significant difference between group S-G and group S(P>0.05).(4)The expressions of ALP:(1)between group W and group S:the expression of ALP increased gradually as time went on.At the time of 3d,the changes had no significant difference(P>0.05),but at the time of 6d、9d and 12d,group S was higher than group W(P<0.05).(2)At the time of 12d,group S was higher than group W(P<0.05),group S-F was higher than group S(P<0.05),and group S-G was lower than group S(P<0.05).(3)12 days after different concentrations of FN(5ug/cm~2、10ug/cm~2、15ug/cm~2)respectively coated to VSMCs of group S,the level of ALP became higher and higher with the concentration of FN increased(P<0.05).Conclusions:VSMCs derived from SHR are prone to senescence and calcification than that of WKY in vitro.Its mechanism may be that when hypertension happens,the expressions of FN including EDA-FN and integrin receptor on VSMCs upregulate,therefore the binding of FN to integrin receptor increases and then induces VSMCs tranform to osteoblast-like phenotype.