Participation And Regulation Effect Of Autophagy In Calcified Aortic Valve Disease

Part I Expression of autophagy-related proteins Beclin1and LC3in patients with calcified aortic valvesObjective:To study the expression and significance of autophagy-related proteins in the aortic valve of patients with hyperlipidemia complicated with calcifed aortic valve disease (CAVD).Methods:Twenty patients (aged>55years) with hyperlipidemia complicated with CAVD (serum total cholesterol>5.7mmol/L) and having lesions in aortic valve (experimental group) were compared with10patients (aged<40years) with normal blood lipids (control group). The morphological structure of aortic valve was observed by hematoxylin and eosin (HE), Masson, and anti-smooth muscle antibodies (aSMA) immunofluorescence staining methods. Deposition of calcium salts was observed using Von Kossa staining. The expression and distribution of calcification-related proteins and autophagy-related proteins in the aortic valve were observed by immunohistochemical staining.Results:HE, Masson, and aSMA immunofluorescence staining methods showed that the aortic valves of patients in experimental group were thickened obviously with hyperplasia of muscle fibers, and the collagen fiber and valvular interstitial cell phenotype were transformed to myofibroblast. Von Kossa staining showed calcium salt deposition in the aortic valves. The immunohistochemical staining showed high expression of calcification-related proteins (osteocalcin, Wnt3a, P-catenin) in the aortic valves. The expression level of autophagy-related proteins Beclinl and LC3was also higher. However, all these staining methods did not show similar observations in the aortic valves of patients in the control group (P<0.01or0.05).Conclusion:Calcification in the aortic valves was accompanied by increased expression of autophagy-related Beclinl and LC3proteins in patients with hyperlipidemia complicated with CAVD, suggesting the role of autophagy in valvular calcification. Part II Expression of autophagy-related proteins Beclinl and LC3in a mice model with calcified aortic valveObjective:To study the expression and significance of autophagy-related proteins Beclinl and LC3in mice model with calcified aortic valve disease (CAVD).Methods:Six-to eight-week-old C57BL/6j male mice with apoE-/-were divided into two groups randomly:control group (n=10) with normal feeding and experimental group with Western diet feeding (n=20). After six months of feeding in specific pathogen free condition, the following tests were performed and the results were compared between the groups:endocardiograhy, blood glucose and lipid levels, hematoxylin and eosin (HE) staining, Von Kossa staining, and immunohistochemical staining.Results:Endocardiograhy showed that the orifice area of aortic valve was much smaller in the experimental group than the control group (P<0.05). Blood lipid level in the experimental group was significantly higher than that in the control group (P<0.01), while the blood glucose level had no obvious statistical difference. The HE staining showed that the valves were obviously thicker than control; the Von Kossa staining was positive in the experimental group was positive, while it was negative in the control group (P<0.01); and the immunohistochemical staining showed that calcification-related proteins (osteocalcin, Wnt3a, β-catenin) and autophagy marker proteins (Beclinl, LC3) were expressed only in the experiment group, but not in the control group (P<0.05).Conclusion:Calcification in the aortic valves was accompanied by increased expression of autophagy-related Beclinl and LC3proteins in mice model with CAVD, suggesting the role of autophagy in valvular calcification by affecting the calcificationg-related signaling pathways. Part III Effect of regulating Beclinl expression on the process of excessive autophagy and calcification in human valvular interstitial cells induced by oxidized low-density lipoproteinObjective:To observe the effect of regulating expression of proteins related with autophagic death and calcification in human valvular interstitial cells (huVICs) induced by oxidized low-density lipoprotein (ox-LDL).Methods:Primary huVICs were isolated from the normal valve of heart transplantation patients, underwent Dil-ox-LDL uptake test, and were observed through immunofluorescence. The level of reactive oxygen species (ROS) was detected after intervention25,50, and100μg/mL of ox-LDL. After adding the antibody of lectin-like ox-LDL receptor1(LOX-1) to block the receptors, the level of LOX-1in huVICs was detected by Dil-ox-LDL uptake test and Western Blot. After transfection the adenovirus with Beclinl overexpression and silent gene, the transfection efficiency was detected by real time polymerase chain reaction and Western Blot method. After completing intervention, the proteins related with autophagy, apoptosis, Wnt signaling pathway and calcification were detected by Western Blot method.Results:The immunofluorescence staining showed positive expression of a-SMA and vimentin. The fluorescence microscopy confirmed that huVICs could take in ox-LDL. After intervention of huVICs with different concentrations of ox-LDL for48h, the flow cytometry found that ROS levels increased with the increase in ox-LDL concentration (P<0.01). The immunoblot technique test showed increase in the expression level of autophagy-related Beclinl and LC3proteins; autophagic death-related hMOF protein; acetyl-histone H4(Lys16); and promoting apoptosis factors Caspase3, Bax, Wnt3a, and P-Catenin; but decrease in the expression of inhibiting apoptosis factor Bcl-2and Bcl-xL protein decreased (P<0.01). Conclusion:Study results confirmed the involvement of Beclinl-dependent pathway in regulation of autophagic death in huVICs, by affecting the activation of Wnt signaling pathways.