Effect Of Calcitriol On Osteogenic Differentiation Of Arotic Valve Interstitial Cells Induced By Lipopolysaccharide

Backgroud:During the process of calcific aortic valve disease,osteogenic differentiation of valve interstitial cells could promote calcification of valves.It was found that human vitamin D₃ deficiency is closely related to the occurrence and development of cardiovascular diseases.Themostimportantactivemetaboliteofvitamin D₃-calcitriol[1α,25-dihydroxycholecalciferol,1,25（OH）₂D₃]playsan important biological role in regulating the proliferation and differentiation of cells,but it is not clear whether it can regulate the osteogenic differentiation of aortic valve interstitial cells.Objective:Toinvestigatetheeffectof calcitriol[1α,25-dihydroxycholecalciferol,1,25（OH）₂D₃]onosteogenic differentiation of porcine aortic valve interstitial cells induced by lipopolysaccharide（LPS）,and to provide a theoretical basis for the prevention of calcific aortic valve disease.Methods:Six healthy male domestic pigs（8¹0 months old,body weight 100¹20 kg）were sacrificed and the aortic valve leaflets were aseptically removed immediately.The aortic valve interstitial cells were isolatedbycontinuouscollagenasedigestion,itsmorphological characteristics were observed and the phenotypes were identified by immunofluorescence staining.The conditioned medium induced osteogenic differentiation of VICs for the establishment of calcification model of aortic valve interstitial cells in vitro,then treated with different concentrations of calcitriol for 72 h,and also established blank control group and vehicle control group.LPS induced osteogenic differentiation of VICs,then selected the optimal LPS effect concentration;Experimental groups as:blank control group;calcitriol group;LPS group;calcitriol+LPS group;VICs were treated with 1 nmol/L calcitriol and 100 ng/mL LPS for48 h.The expression of mRNA and protein levels of bone morphogenetic protein 2（BMP-2）and osteocalcin（OC）were detected by real-time fluorescence quantitative PCR and Western blot,respectively;and alizarin red staining was used to evaluate the formation of late calcium nodules.Results:The The primary porcine aortic valve interstitial cells were successfully isolated and the staining ofα-smooth muscle actin（α-SMA）and vimentin were positive,the staining of von Willebrand factor（vWF）was negative.Calcitriol inhibited osteogenic differentiation of VICs induced by conditioned medium,the expression of BMP-2 and OC were significantly lower than that of blank control group and vehicle control group（P<0.05）.The treatment of LPS promoted osteogenic differentiation of VICs,Compared with other concentrations of LPS group,the expression of BMP-2 and OC of 100 ng/mL LPS group was the most（P<0.05）,Pretreatment with calcitriol can reduce the expression of the markers of osteogenic differentiation induced by LPS,the expression of BMP-2 and OC of calcitriol+LPS group were lower than of LPS group（P<0.05）,and the formation of late calcium nodules decreased.Conclusions:Calcitriol can inhibit the osteogenic differentiation of aortic valve interstitial cells induced by conditioned medium and LPS,which may have a protective effect on aortic valve calcification.