Mechanism Of Nuclear Receptor NR3C1 In Aortic Valve Calcification

BackgroundCalcific aortic valve disease(CAVD)is one of the common heart valve diseases in cardiovascular surgery.The main cause of aortic valve stenosis is valve calcification,which can lead to angina,syncope,heart failure,and even sudden cardiac death.Untreated CAVD patients with symptoms have poor prognosis and significantly increased mortality,which puts a burden on the healthcare system.With the accelerated aging of the population,the incidence and prevalence of CAVD are increasing year by year,and there are currently no effective drugs to prevent or slow the progression of the disease.The traditional concept regards the pathogenesis of CAVD as a passive calcium deposition process,but more and more studies have shown that aortic valve calcification is an active and multifactorial pathological change,and its progression is similar to that of atherosclerotic cardiovascular disease.The valve is mainly composed of valvular interstitial cells(VIC s),valvular endothelial cells,and various extracellular matrices.VICs account for about 80%of valve cells,and together with extracellular matrices,they maintain the normal structure and function of the valve.VICs have the potential to differentiate into various phenotypes,and their differentiation into osteoblast-like cells is an important pathological feature of aortic valve calcification and a research focus of CAVD pathogenesis.、Glucocorticoids(GC)are the most widely used anti-inflammatory drugs and immunosuppressants in clinical practice,and their side effects in the cardiovascular system mainly manifest as inducing vascular calcification.The glucocorticoid receptor NR3C1(Nuclear Receptor Subfamily 3 Group C Member 1)can act as a transcription factor,bind to glucocorticoid response elements in the promoter of glucocorticoid-responsive genes,and activate their transcription,or as a regulatory factor for other transcription factors.Studies have shown that NR3C1 is a key regulatory factor in osteogenic differentiation,and its regulatory effects are complex and diverse,but there have been no reports on its role in the pathogenesis of valve calcification.Objectives1.Determining the expression profile of NR3C1 in aortic valve calcification and osteogenic differentiation of VICs2.Elucidating the regulation of NR3C1 on VICs osteogenic differentiation and aortic valve calcification3.Clarifing the molecular mechanism of NR3C1 promoting VICs osteogenic differentiation and aortic valve calcificationvMethods1.Collection and expression identification of aortic valve specimensThe aortic valve of patients who were diagnosed with calcific aortic valve stenosis and underwent aortic valve replacement surgery was collected,while excluding patients with rheumatic heart valve disease,autoimmune diseases,congenital aortic valve disease,genetic diseases,and infective endocarditis.The aortic valve of the recipient who underwent orthotopic heart transplantation surgery for dilated cardiomyopathy during the same period was collected as a negative control.The basic clinical data of patients were statistically analyzed using SPSS 25.0 software.The aortic valve tissue was embedded in paraffin and cut into slices,which were then stained with hematoxylin and eosin to identify tissue and cell structures.Alizarin red and silver nitrate staining were used to detect calcium deposition.Western blot and immunohistochemistry experiments were used to detect the expression levels of osteogenic markers(OPN and Runx2)and nuclear receptor NR3C1.2.Isolation and identification of VICs and construction of calcification model in vitroPrimary human VICs were isolated using an improved type Ⅱ collagenase digestion method,and the cell purity was identified by immunofluorescence staining for Vimentin and CD31.VICs at passages 4-6 with stable cell status were selected for subsequent cell experiments.An in vitro calcification model was constructed using osteogenic induction medium(OIM medium:DMEM medium containing 50 μg/mL ascorbic acid,10-7mol/L insulin,2 mmol/L sodium phosphate,and 5%fetal bovine serum).After 7 days of induction culture,VIC osteogenic differentiation and calcification levels were identified by alkaline phosphatase activity detection,Alizarin Red S staining,and intracellular calcium ion concentration measurement.The expression changes of osteogenic markers(OPN and Runx2)and NR3C1 were detected by Western Blot,and the expression localization of NR3C1 was determined by immunofluorescence staining.3.Construction and Identification of CAVD Rat ModelA CAVD rat model was constructed using intraperitoneal injection of vitamin D and a high-fat diet.The control group of rats received a regular diet and intraperitoneal injection of an equal amount of saline,and both groups were continuously fed for four months.The rat aortic valve calcification and stenosis degree were evaluated by echocardiography to measure the transvalvular pressure gradient and regurgitation volume.Aortic valve tissue was collected and frozen sections were stained with HE to assess valve thickening and structure.Alizarin red staining was used to detect valve calcification,and immunohistochemistry and immunofluorescence were used to detect the expression and localization of osteogenic markers(OPN and Runx2)and NR3C1.4.Regulatory effect of NR3C1 expression on osteogenic differentiation of VICsNR3C1 was overexpressed in human VICs by lentivirus(pLV-NR3C1)transfection,and NR3C1 expression in human VICs was inhibited by siRNA(siNR3C1).48 hours after transfection,qRT-PCR experiments were performed to detect the expression level of NR3C1,and to verify the overexpression and inhibition efficiency.Transfect VICs with pLV-NR3C1 or siNR3C1,and after OIM-induced culture,detect the calcium salt deposition,calcium ion concentration,alkaline phosphatase activity and the expression of osteogenic markers in VICs to determine the effect of NR3C1 on the osteogenic differentiation of VICs.5.Effects of NR3C1 activity on osteogenic differentiation of VICs and calcification of rat aortic valveUnder the induction of OIM,adding dexamethasone(1 μmol/L)or mifepristone(1μmol/L)activated or inhibited the activity of NR3C1,respectively.The calcium salt deposition,calcium ion concentration,alkaline phosphatase activity and osteogenic markers of VICs were detected to determine the effect of NR3C1 activity on the osteogenic differentiation of VICs.On the basis of the CAVD rat model,intraperitoneal injection of dexamethasone(1 mg/kg/day)or subcutaneous injection of mifepristone(10 mg/kg/day)was performed to activate or inhibit the activity of NR3C1 in vivo.After 4 months of modeling,echocardiography was used to detect the transvalvular pressure difference,regurgitation flow and calcification degree of the rat aorta;histological staining of frozen sections was used to detect the valve structure and calcium salt deposition.The expression of osteogenic markers was detected by immunohistochemical method,and the effect of NR3C1 activity intervention on rat aortic valve calcification was determined.6.The molecular mechanism of NR3C1 promoting osteogenic differentiation of VICThe differentially expressed genes that NR3C1 promotes the osteogenic differentiation of VICs were screened by transcriptome sequencing technology,and bioinformatics analysis was performed,and the biological processes,cell composition,molecular functions and signaling pathways involved in these differentially expressed genes were enriched by GO analysis and KEGG analysis,and screened the key pathways that NR3C1 regulates the calcification process of VICs.In VICs overexpressing NR3C1,qRT-PCR and Western Blot were used to determine the impact of NR3C1 on potential signaling pathways,and cell rescue experiments were performed using signaling pathway inhibitors to clarify the molecular mechanism of NR3C1 promoting VICs calcification.Results1.Expression changes of NR3C1 in human calcified aortic valve tissueAccording to the inclusion and exclusion criteria,21 cases of calcified aortic valve and 12 cases of normal aortic valve were included.Statistical analysis of the basic clinical data of the patients showed that the CAVD group was matched with the control group in terms of gender and age,and there was no significant difference in baseline data(except for history of hypertension and LVEF)(P>0.05).Compared with the normal aortic valve,the tissue of the calcified aortic valve was thickened and the cell arrangement was disordered,there were obvious calcium nodules in the valve tissue,and the expressions of calcification-related proteins(OPN and Runx2)were significantly increased.The expression of NR3C1 was also significantly up-regulated,and it was mainly localized in Vimentin-positive VICs.2.The expression changes of NR3C1 in the process of rat aortic valve calcificationCompared with the rats in the control group,the aortic valve transvalvular pressure gradient(0.644±0.068 mmHg vs.1.964±0.059 mmHg,P<0.05)and the maximum aortic valve velocity(0.419±0.016 m/s vs.0.801±0.004 m/s,P<0.05)in the CAVD group were larger,and the aortic valve was significantly narrowed.The results of histological staining showed that after 4 months of modeling,the rat aortic valve had obvious calcified nodules,and the expression of calcification-related proteins(OPN and Runx2)increased significantly.Immunofluorescence results showed that the expression of NR3C1 began to increase after 3 months of modeling and maintained a high expression level.3.Regulatory effect of NR3C1 expression on osteogenic differentiation of VICsAfter OIM induction for 3 days,the calcium ion content in VICs increased significantly(0.5±0.577 μmol/mg vs.4.250±1.258 μmol/mg,P<0.01),the activity of ALP increased significantly(0.004±0.002 unit/mg vs.0.021 ±0.001 unit/mg,P<0.01),and the expression of calcification-related proteins(OPN and Runx2)increased significantly.After continuous induction for 7 days,calcified nodules of VICs gradually formed.The expression of NR3C1 increased significantly after 3 days of OIM induction,and it accumulated into the nucleus.After transfection with lentivirus pLV-NR3C1,the expression of NR3C1 in VICs was significantly increased,and the number of calcium nodules,calcium ion content,ALP activity and expression of calcification-related proteins were significantly higher than those in the empty virus control group.After transfection with siNR3C1,the expression of NR3C1 was significantly reduced,and the degree of calcification of VICs was significantly alleviated,which was manifested by the reduction of the number of calcium nodules and calcium ion content,the reduction of ALP activity and the expression of calcification-related proteins.4.Effect of NR3C1 activity on osteogenic differentiation of VICs and aortic valve calcificationAfter treatment with OIM containing dexamethasone,the number of calcium nodules and calcium ion content in VICs increased significantly,and the activity of ALP and the expression of calcification-related proteins increased significantly.After treatment with OIM containing mifepristone,the degree of calcification of VICs was significantly relieved,manifested as a reduction in the number of calcium nodules and calcium ion content,as well as the reduction of ALP activity and expression of calcification-related proteins.In the CAVD rat model,compared with rats in the calcified group,the aortic valve transvalvular pressure difference(2.482±0.479 mmHg vs.1.846±0.067 mmHg,P<0.05)and maximum aortic valve flow velocity were significantly higher in the dexamethasone-treated group(0.889±0.062 m/s vs.0.741±0.024 m/s,P<0.05)and calcium salt deposition was more severe in aortic valve tissue sections;whereas the transvalvular pressure difference(1.446±0.424 mmHg vs.1.964±0.059 mmHg,P<0.05)and maximum aortic flow velocity(0.669±0.076 m/s vs.0.801 ±0.004 m/s,P<0.05)were attenuated.5.The molecular mechanism of NR3C1 regulating the osteogenic differentiation of VICsTranscriptome sequencing results showed that in OIM-induced VICs,overexpression of NR3C1 caused significant abnormal expression of 43 genes,of which 19 genes were upregulated and 24 genes were down-regulated.The results of GO enrichment analysis showed that these differentially expressed genes were mainly enriched in biological processes such as signal transduction,biological regulation,growth and metabolism;they were mainly located in cell components such as cell envelope and organelle components.Differential genes are mainly involved in molecular functions such as molecular adhesion,catalytic activity,and enzyme activity regulation.KEGG enrichment analysis results showed that differentially expressed genes were mainly enriched in signaling pathways such as Wnt pathway and apoptosis pathway.In OIM-induced cultured VICs,overexpression of NR3C1 significantly up-regulated Wnt pathway-related proteins(β-catenin,Wnt5,Wnt11).At the same time,after adding the Wnt pathway inhibitor nitazoxanide to inhibit the Wnt pathway,the effect of NR3C1 on promoting the osteogenic differentiation of VICs was significantly alleviated.ConclusionsThe increase of NR3C1 expression may be closely related to the process of aortic valve calcification.NR3C1 promotes the osteogenic differentiation of VICs and exacerbates aortic valve calcification by activating the Wnt pathway.NR3C1 may be a potential target for prevention and treatment of CAVD.