| Background: With the aging of China’s population,the incidence of vascular calcification(VC)has been found to be increasing year by year;according to the epidemiological survey,more than 90% of males and 65% of females aged 70 years and above suffer from vascular calcification.Vascular calcification is a pathological manifestation of ectopic deposition of hydroxyapatite in the vascular wall,which is a common feature of aging and a variety of internal environmental disorders,and is an independent risk factor for cardiovascular events in diabetes mellitus and other diseases.The currently recognized pathological mechanism is the dedifferentiation of the contractile phenotype of vascular smooth muscle cells into an osteogenic phenotype,which is reflected in the altered expression of various phenotypic markers such as α-smooth muscle actin(α-SMA)and bone morphogenetic protein-2(BMP2).Reports have shown that estrogen has significant cardiovascular protective effects,but estrogen replacement therapy can also increase the risk of associated tumor diseases.It has been found that estrogen,in the form of a ligand,activates the transcriptional activity of the transcription factor estrogen receptor alpha(ERα),thereby inhibiting the development of vascular calcification.In addition,estrogen receptor is also dependent on co-regulatory factors to regulate the activity and stability of ERα,which together affect the transcription and expression of target genes.Studies have shown that epigenetic regulation influences the progression of vascular calcification in many ways,and that cofactors play an important role in the pathological process of the disease,so it is crucial to explore the cofactors that influence vascular calcification.It is crucial to explore the co-modulatory factors affecting vascular calcification.In-depth study of the regulation of estrogen receptor by co-modulatory factors and promotion of the protective mechanism of estrogen receptor in vascular calcification are of great clinical significance to reduce the adverse cardiovascular events.Objective: To investigate the role of estrogen receptor in vascular calcification and to explore the mechanism of action of the epigenetic regulator histone acetyltransferase KAT8 affecting the transcriptional regulation of estrogen receptor smad7.Methods:1.In an in vivo experiment,aortic calcification was induced in naturally menopausal aged mice by subcutaneous injection of cholecalciferol/vitamin D3.Estradiol levels were determined by vaginal secretion smear,ovarian index,hematoxylin-eosin staining of ovarian frozen sections,and enzyme-linked immunosorbent assay to determine the entry of old mice into menopause;aortic stiffness was assessed by pulse tail-collar method noninvasive blood pressure measurement system during the modeling period;calcification was analyzed using whole segment vascular alizarin red staining,micro-computed tomography(micro-CT),and protein immunoblotting The expression of relevant factors was analyzed using micro-computed tomography(micro-CT)and protein immunoblotting,and mineral deposition was observed to judge the success of calcification modeling.2.The aortic tissues of aged control and aged calcified mice were subjected to transcriptome library sequencing,quality control overview of sequencing data,reference genome comparison,quantitative gene expression analysis,and RNA-seq correlation analysis before screening differentially expressed genes(DEGs)using DESeq2 software,performing functional enrichment analysis(GO)and KEGG pathway annotation on DEGs,using STRING database and Cytoscape software to construct protein interaction networks(PPI)and identify pivotal genes with high module scores.Screening of epigenetically relevant differentially expressed genes;enrichment of TGF-β pathway regulating osteogenic differentiation in KEGG;quantitative reverse transcription q PCR to verify the expression of differentially expressed genes in each group of tissues.3.Eight-month-old female BALB/C mice were selected,and bilateral ovaries were removed via a dorsal incision under gas anesthesia with anesthesia ventilator,the fallopian tubes were ligated,and denudation was given;one month later,subcutaneous injection of cholecalciferol/vitamin D3 induced aortic calcification in ovary-removed mice;mineral deposition was observed using alizarin red staining of vascular paraffin sections and silver nitrate staining;protein immunoblotting analysis of calcification-related factors Observation of phenotypic transformation and differences in expression of co-regulatory factors KAT8 and Smad7,a member of the TGF-β family;immunohistochemical staining to assess the expression of KAT8 and Smad7;real-time fluorescence quantitative polymerase chain reaction assay to view changes in osteogenic versus contractile phenotypic markers and changes in expression of KAT8 and Smad7.4.A type II diabetic female rat model was established to observe aortic calcification in rats.The rats were fed a high-fat and high-sugar diet for 8 weeks,and after intraperitoneal injection of streptozotocin(STZ),body weight,coat color,bedding and feces were recorded,random blood glucose was measured,and glucose tolerance test(OGTT)was performed to determine whether the type II diabetic rat model was successfully established.Osteogenic differentiation and changes in KAT8 and Smad7 expression in female rats with diabetes mellitus compared with control rats were detected by protein immunoblotting.5.In vitro experiments,calcification was induced in human aortic smooth muscle cells(HASMCs)using β-glycerophosphate disodium salt hydrate(β-GP);calcium deposition was detected by alizarin red staining,formic acid quantification,and calcium quantification assays;western blot and q PCR measured the expression of calcificationrelated markers to assess osteogenic differentiation,and the expression of the co-regulatory factor KAT8 alteration.KAT8-3×HA overexpression plasmid was constructed,si KAT8 knockdown gene was designed,and changes in calcium deposition were observed using alizarin red staining,formic acid quantification,and calcium quantification assays;western blot and q PCR assays were performed to investigate the effects of KAT8 overexpression and knockdown on osteogenic differentiation and calcification in vascular smooth muscle cells.Protein immunoprecipitation assay was performed to investigate the interaction between estrogen receptor ERα and KAT8 at the protein level;dual luciferase reporter assay was performed to verify that KAT8 affects the transcriptional activity of estrogen receptor ERα in vascular smooth muscle cells and to investigate the binding of KAT8 to the structural domain of ERα and thus the transcriptional activity of ERα;WB and q PCR were used to investigate the effect of overexpression of KAT8 on vascular smooth muscle cells under the influence of estrogen.The effect of KAT8 on the expression of estrogen receptor α(ERα)and the downstream target gene Smad7 in vascular smooth muscle cells was investigated by WB and q PCR.The acetyltransferase active site mutant plasmid KAT8-K274 R with mutation of lysine to arginine at position 274 was constructed based on the KAT8 wild-type plasmid,and dual luciferase reporter assays were performed to verify whether the effect of KAT8 on the transcriptional activity of estrogen receptor ERαin vascular smooth muscle cells was dependent on acetylase activity;vascular smooth muscle cells were transfected under calcification induction with wild-type as well as mutant Vascular smooth muscle cells were transfected with wild-type and mutant KAT8 plasmids under calcification induction,given estrogen stimulation or blank reagent according to the grouping,and WB experiments were performed to observe the changes in cellular phenotypic markers and the altered expression of Smad7,a downstream target gene of ERα.Small interfering RNA knockdown gene of Smad7 was designed,and WB experiments were performed to observe the effect of si Smad7 on cell phenotype under calcification induction.Vascular smooth muscle cells were transfected with wild-type KAT8 plasmid under calcification induction for 24 h,then transfected with si Smad7 for gene knockdown,and WB experiments were performed to observe the changes of cell phenotype markers.Results:1.In female mice,at about 18 to 19 months of age,vaginal discharge was stagnant in the interestrus(predominant)or preestrus(minority)phase;ovarian index decreased significantly(0.23±0.02 mg/g versus 0.81±0.07 mg/g,P<0.05),ovarian sections showed a significant decrease in the number of follicles and a relative increase in white bodies as well as interstitium,as an aging change;estradiol levels decreased(19.58±4.55 比 44.32±8.7pg/ml,P<0.001),suggesting that the mice entered menopause.The systolic blood pressure gradually increased during the modeling period until the middle of the modeling period;micro-CT showed significant calcification foci in the aortic vessel wall,and the thoracic and ventral segments of the full-length aorta were stained with alizarin red,and protein immunoblotting suggested elevated expression of calcification-related factors,indicating the successful establishment of the calcification model in aged mice.2.Transcriptome sequencing was set as the cut-off value of |log2(Fold Change)| >= 1,p<=0.05 for quantitative gene expression analysis,and a total of 2741 differential genes were screened,of which 1135 were up-regulated and 1606 were down-regulated.Differential gene function enrichment analysis(GO)was performed,and the term associated with histones was screened in GO biological process(BP),and the top ten were screened by-log10(p.adj)sorting,and the second positively regulated histone modification pathway,Rich factor was higher than other pathways(13/1473),and the protein interaction network of 13 differential genes enriched by this pathway was selected The data files were imported into cytoscape software to screen Hub genes,and the top three scores were DNMT3 B,KMT2A and KAT8;the pathways associated with histones were screened in GO molecular function(MF)and sorted by-log10(p.adj),suggesting that the top 4 rankings were all related to histone acetylation modification;the screening analysis of the BP and MF methods was integrated,and it was concluded that KAT8 histone acetyltransferase may be more important in epigenetics for vascular calcification effects.The TGF-β pathway,which regulates osteogenic differentiation,was enriched in the KEGG pathway with significant differences(P=0.0002);Smad7,a member of the TGF-β family that plays a negative regulatory role,is a downstream target gene of ERα and is transcriptionally regulated by the transcription factor ERα,so the role of Smad7 in calcification was analyzed.q PCR verified that the differentially expressed genes KAT8,Smad7 in significantly down-regulated in the aged calcification group,consistent with the sequencing trend.3.In young BALB/C mice,calcification was induced by vitamin D3 one month after ovarian excision to establish a calcification model.Immunohistochemical staining indicated that the expression of KAT8 and Smad7 decreased significantly in the calcification group.q PCR assay showed that the expression of osteogenic phenotypic markers BMP2 and RUNX2 in aortic tissue was significantly increased in the estrogendeficient calcification model group,and the expression of contractile phenotypic markersα-SMA and calcium inhibitory factor OPN was decreased.m RNA quantification showed that both old and decompensated groups showed osteogenic phenotypic transformation after calcification induction,and the expression of KAT8 decreased,and the degree of transformation in old The m RNA quantification analysis showed that both old and depressed groups exhibited osteogenic phenotypic transformation and reduced KAT8 expression,and the degree of transformation was more obvious in old mice than in depressed group,which may be related to aging.4.The female diabetic rat model generally exhibited the typical "three more and one less" as the duration of streptozotocin administration increased,with gradual weight loss;random blood glucose was greater than 16.7 mmol/L(18.5±0.73 mmol/L vs.7.82±0.30mmol/L,P<0.001);blood glucose Peak was delayed,blood glucose could not be recovered2 hours after meal,and glucose tolerance was impaired(AUC area 926±27.6 minmmol/L/h versus 2395±48.07 min-mmol/L/h,P<0.001),suggesting that the type II diabetes mellitus rat model was successfully established.In the aortic tissue,the expression of osteogenic phenotypic markers BMP2 and RUNX2 was significantly increased in the diabetic group,and the expression of contractile phenotypic markers α-SMA and calcium inhibitory factor OPN was decreased,and the content of KAT8 and Smad7 was relatively reduced in the diabetic group.5.After calcification-induced culture of vascular smooth muscle cells in β-GP-containing medium,alizarin red staining,formic acid quantification,and calcium quantification assays suggested mineral deposition in the cells.western blot and q PCR suggested osteogenic phenotype transformation,and KAT8 expression gradually decreased with the osteogenic differentiation process of vascular smooth muscle cells.Overexpression of KAT8 inhibited β-GP-induced calcification in vascular smooth muscle cells,and silencing KAT8 promoted calcification.Immunoprecipitation suggested that KAT8 and ERα could bind to each other at the protein level with or without estrogen stimulation;dual luciferase reporter assay suggested that overexpression of KAT8 could upregulate ERαtranscriptional activity,and estrogen stimulation could increase the upregulation activity of KAT8 on ERα;dual reporter assay showed that KAT8 had no regulatory effect on the ligand non-dependent AF1 structural domain,while the addition of estrogen stimulation KAT8 can significantly promote the ligand-dependent AF2 structural domain as well as ERα full-length.Overexpression of KAT8 stabilized the protein level of ERα and promoted the expression of Smad7,a downstream target gene of ERα.Estrogen stimulation increased the inhibitory effect of KAT8 on β-GP-induced vascular smooth muscle calcification,which could further promote the upregulation of Smad7 by KAT8;transfection of acetylase active site mutant plasmid KAT8-K274 R in HASMCs failed to recapitulate the upregulation of ERα transcriptional activity by wild-type KAT8,and could not play the calcium suppressive role of wild-type KAT8,and had no upregulation of smad7 expression was not upregulated,and administration of estrogen stimulation only mildly promoted the upregulation of smad7.Knockdown of Smad7 gene promoted β-GPinduced calcification in HASMCs.Overexpression of KAT8 partially attenuated calcification-induced osteogenic transformation of vascular smooth muscle cells,while knockdown of Smad7 on top of overexpression of KAT8 increased calcification again.Conclusion:KAT8 is lowly expressed in vascular calcification and is a protective factor;by binding to ERα,it upregulates ERα-mediated gene transcription and promotes the transcriptional expression of smad7,a downstream target gene of ERα,thereby inhibiting the BMP/Smad signaling pathway that promotes osteogenic differentiation and plays an inhibitory role in vascular calcification;estrogen can further promote the calciumsuppressive effect of KAT8. |
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