FSTL1 Was Associated With Calcific Aortic Valve Disease:FSTL1 Regulating On Calcification Of Valvular Interstitial Cells

[Objective]With the development of economy and the aging of population,the incidence of calcific aortic valve disease(CAVD)has a up-trend,and CAVD has been the kind of cardiovascular disease second only to the coronary heart disease(CAD)and hypertension.CAVD is characterized by mineralized nodules on the valve cusps and typically results in progressive aortic valve stenosis.During disease,valvular interstitial cells(VICs)from mineralized valves activate molecular pathways associated with valve development and bone differentiation.The expression of osteogenic gene markers,such as BMP2(bone morphogenetic protein-2),Runx2(runt-related transcription factor 2)and Msx2(Muscle Segment Homeobox,Homolog of 2),could participate in the progression of aortic valve calcification.FSTL1(Follistatin-like protein 1)is a kind of secreted glycoprotein which is produced mainly by cells of mesenchymal origin.FSTL1 has mainly been regarded as a cardioprotective factor secreted by the heart in the research field of cardiovascular diseases.In addition,there have been many experiments on animals reported that FSTL1 could protect the function of heart in acute myocardial infarction and heart failure.However,there has not been any studies on the effect of FSTL1 on aortic valve calcification.To investigate the mechanism of the effect of FSTL1 on calcification aortic valve disease,the study mainly has three parts including the collection of clinical data,in vivo experiments and the experiments on animals.In this study,the serum and aortic valves of patients were used to detect the change of expression of FSTL1;secondly,the data of serum concentration of FSTL1 was used to annlyze the association of FSTL1 and CAVD;thirdly,the primary VICs were used to observe the effects of FSTL1 on the deposition of calcium salt;fourth,the primary VICs were used to detect the effects of FSTL1 on the expression of osteogenic gene markers including BMP2,Runx2 and Msx2;finally,the effects ofFSTL1 on the deposition of calcium salt and cardiac functions were observed by the aortic valves of mice and echocardiography;[Methods]1.Serum FSTL1 levels were determined in CAVD patients and non-CAVD controls by ELISA(enzyme-linked immunosorbent assay).Human calcific and non-calcific aortic valves were used for histological and immunochemical analysis and FSTL1 in the valves was detected by IHC.2.Primary VICs from C57/BL6 mice were stimulated with different concentrations(0?25?50?100ng/mL)for 21 days.Then the calcification of VICs were detected by Alizarin Red S and Alkaline Phosphatase Diethanolamine(ALP)activity.3.Primary VICs from C57/BL6 mice were stimulated with 100 ng/mL rhFSTL1 for 7 days,14 days and 21 days,then detected the calcification of VICs.4.100 ng/mL rhFSTL1 was used to stimulate the primary VICs for 21 days,and then the Western Blot,Real time-Polymerase Chain Reaction(RT-PCR)and Immunofluorescence(IF)were used to detect the expression of osteogenic gene markers including BMP2,Runx2 and Msx2 in VICs.5.The ApoE knockout mice were fed on high-fat diet for 6 months.At the beginning of the high-fat diet,the mice were injected negative control and FSTL1 overexpression lentivirus through vena caudalis.Alizarin Red S and ALP activity were used to detect the calcification of the aortic valves of mice.Visitech BP-2000 was used to record the blood pressure of mice.Cardiac echocardiography was used to assess mice's cardiac function.[Results]1.The FSTL1 concentration in serum and the calcific aortic valves were both significantly lower than that in the control non-calcified aortic valves.2.The serum FSTL1 level was negatively related to the prevalence rate of CAVD,which indicated that FSTL1 could be a protective factor associated withCAVD.In addition,the increase of serum FSTL1 concentration may dcrease the hazard of CAVD.3.When the serum FSTL1 concentration was at low level,the protective effect of FSTL1 on CAVD was dose-dependent.However,when serum FSTL1 reached certain concentration range,the protective effect of FSTL1 on CAVD reached saturation.4.The stimulation of rhFSTL1 could reduce the calcium deposits and expression of osteogenic gene markers in VICs.5.Compared with the negative control mice,the group of mice injected with FSTL1 overexpression lentivirus showed less aortic valve calcification and lower systolic blood pressure.However,there was no significant differences in the facet of diastolic blood pressure.The cardiac echocardiography results showed that the EF(ejection fraction)and FS(fractional shortening)were higher in the group of mice injected with FSTL1 overexpression lentivirus,but had no significant differences.[Conclusions]A reduction in FSTL1 was associated with aortic valve calcification,and FSTL1 participated in the mineralization of VICs by regulating specific osteogenic genes and the activity of ALP.