| Background:Coronary heart disease(CHD)has a high incidence and poor prognosis,which is an urgent medical and public health problem.However,the current understanding of its pathogenesis is still quite insufficient,and new biological mechanisms are required to explore therapeutic targets,which is beneficial for reducing a large number of unknown cardiovascular risks.Aging is a major risk factor for atherosclerosis cardiovascular diseases and has an impact on the onset and outcome of CHD.Therefore,anti-aging therapy is very important for cardiovascular research and clinical practice.Klotho is an endogenous pleiotropic protein observed in both tissues and blood.Circulating Klotho decreases with aging and negatively correlates with renal function.Tissue Klotho is regard as membrane receptors and mainly involved in the regulation of calcium/phosphorus metabolism.Circulating Klotho,as a hormone-like factor is considered to play an important biological role of anti-oxidation and anti-aging.Klotho knockout mice show vascular aging phenotypes including vascular stiffness,vascular calcification,and atherosclerosis,while administration of klotho can significantly attenuate severe vascular disease.The results indicate that Klotho is a protective factor regulating vascular aging,therefore it is possible to participate in the atherosclerosis process.Cellular senescence includes replicative senescence and stress-induced aging,which is characterized by decreased proliferation,morphological changes,cycle arrest,and dysfunction.Autophagy is a cellular self-stabilizing mechanism in response to stress,self-renewing by degrading its own components.Senescence is closely related with autophagy,both senescence and autophagy are the adaptive mechanisms involved in stress damage.It was reported that knocking out autophagy-related genes resulted in malformation and death in mice,and the aging process was accompanied by inhibition of autophagy,while activating autophagy can delay the progression of aging,suggesting that autophagy may help maintain stability and health.In several cardiovascular studies,activation of autophagy helps reduce stress-induced damage to vascular cells and plays an important role in vascular remodeling and plaque stabilization.Among the vascular components,endothelial cells are the direct performer of physiological functions,alleviation of endothelial aging and restoration of vascular homeostasis may be an important therapy strategy for anti-atherosclerosis.Therefore,our study aims to investigate the role of Klotho in metabolic stress-induced endothelial senescence.In addition,in view of the important biological effect of autophagy,our study will also explore the relationship between Klotho,autophagy and aging,in order to extend understanding of Klotho's anti-aging mechanism.Inflammation and metabolic disorders are both important risk factors for atherosclerosis and interact with each other.On the one hand,metabolic disorders can upregulate systemic inflammation,promote vascular lipid deposition,and activate vascular inflammation phenotyped characterized by monocyte/macrophage,thereby promoting atherosclerosis;on the other hand,inflammation also mediates insulin resistance,thus exacerbates metabolic disorders and significantly increases the risk of cardiovascular diseases.Chronic inflammation and metabolic disorders are also associated with aging,but this correlation is mainly based on estimation of age.So far there is still a lack of aging markers quantifying cardiovascular risk.Therefore,our study aims to investigate the correlation of anti-aging protein Klotho with inflammation and metabolic disorder,and to further evaluate the association between Klotho and coronary artery disease.Methods:1.Role of Klotho in metabolic stress-induced endothelial senescence and its underlying mechanism(1)Cultured human coronary artery endothelial cells(HCAECs)were treated with Ox-LDL at different concentrations(0 ?g/mL,25 ?g/mL,50 ?g/mL,75 ?g/mL)and different durations(0 h,24 h,48 h,72 h),or intervention of Klotho(400pM).Cell viability by CCK-8 assay,senescent cells detected by SA-?-GAL staining,intracellular expression of P53 and P16 by immunofluorescence,and quantitative analysis of P53 and P16 by western blot.(2)Cultured HCAECs were treated with Ox-LDL at different concentrations(0 ?g/mL,25 ?g/mL,50 ?g/mL,75 ?g/mL)and different durations(0 h,24 h,48 h,72 h).Autophagy-related proteins LC3 and P62 expression were detected by western blot.(3)Cultured HCAECs were treated with Klotho(400 pM),rapamycin(5?M),and chloroquine(2?M)respectively under Ox-LDL exposure(75 ?g/mL).LC3 and P62 expression were detected by western blot.Adenovirus GFP-LC3 was transfected into endothelial cells,and intracellular green fluorescent spots were counted.(4)Cultured HCAECs were treated with Klotho(400 pM),rapamycin(5?M),and chloroquine(2?M)respectively under Ox-LDL exposure(75 ?g/mL).Cell viability by CCK-8 assay,senescent cells detected by SA-?-GAL staining,and quantitative analysis of P53 and P16 by western blot.2.Association of Klotho with inflammation and metabolic disordersThis was a cross-sectional study involved in patients with cardiovascular diseases who were admitted between December 2017 and March 2018 in Xinqiao Hospital.A total of 308 patients were enrolled.According to coronary angiography,the subjects were divided into coronary artery disease(CAD)group and non-CAD group.The clinical data of all patients were collected,and serum Klotho was detected by ELISA assay.The steps are as follows:(1)comparison between CAD group and non-CAD group in general information;(2)correlation between Klotho and inflammation markers and glucose and lipid metabolism indicators by Spearman rank correlation analysis;(3)Klotho's diagnostic power for dyslipidemia,glucose metabolism disorder,and metabolic syndrome by ROC curve analyses;(4)evaluation of the association between Klotho and metabolic syndrome by logistic regression model.3.Association between Klotho and coronary artery disease severityFollow the methods above,coronary angiography data were obtained from all patients,and serum Klotho was detected by ELISA assay.The steps are as follows:(1)the data of coronary artery disease were shown;(2)Klotho's diagnostic power for coronary artery disease by ROC curve analysis,and correlation between Klotho and SYNTAX score in patients with CAD by Spearman rank correlation analysis;(3)comparison of Klotho levels between the patients with complicated coronary lesions and without complicated coronary lesions,and Klotho's diagnostic power for complicated coronary lesions by ROC curve analyses;(4)evaluation of the association between Klotho and severe coronary artery disease by logistic regression model.Results:1.Role of Klotho in metabolic stress-induced endothelial senescence and its underlying mechanism(1)Ox-LDL induced HCAECs senescence in a time-dependent or dose-dependent manner to some extent.(2)Klotho alleviated Ox-LDL-induced inhibition of HCAECs viability,the proportion of senescent cells,and the expression of aging-related proteins P53 and P16.(3)Ox-LDL inhibited autophagy of HCAECs in a time-dependent or dose-dependent manner to some extent.(4)Klotho activated autophagy impaired by Ox-LDL and alleviated autophagic flux inhibited by chloroquine in HCAECs.(5)Klotho attenuated Ox-LDL-induced inhibition of HCAECs viability,the proportion of senescent cells,and the expression of P53 and P16 via activation of autophagy.2.Association of Klotho with inflammation and metabolic disorders(1)The patients in CAD group were older than those in non-CAD group(P = 0.003).The CAD group had more males(P = 0.013)and smokers(P = 0.001)than non-CAD group;combined with elevated inflammation(P < 0.001),worse renal function(P = 0.005),dyslipidemia(P < 0.001),glucose metabolism disorder(P < 0.001),and metabolic syndrome(P = 0.026).Serum Klotho also decreased significantly in CAD group(P < 0.001).(2)Klotho was significantly negatively correlated with TG,HDL,FBG,HbA1 c,TG/HDL-C ratio,and TyG index(P < 0.001).Klotho was significantly negatively correlated with MHR(P < 0.001);MHR was significantly positively correlated with CRP(P < 0.001);patients with metabolic syndrome had higher MHR and CRP levels(P < 0.001).(3)Klotho levels were lower in patients with dyslipidemia,glucose metabolism disorder,and metabolic syndrome(P < 0.001);and areas under the ROC curve were 0.731,0.728,and 0.713,respectively(P < 0.001).(4)It was shown that Klotho was independent associated with metabolic syndrome and might be a protective factor for metabolism disorders in multivariate logistic regression model(OR = 0.991,P < 0.001).3.Association between Klotho and coronary artery disease severity(1)SYNTAX scores were negatively correlated with Klotho levels in CAD group(R =-0.527,P < 0.001);Klotho also showed a strong diagnostic performance for coronary artery disease(AUC = 0.964,P < 0.001).(2)Klotho levels were lower in patients with three-vessel lesions,calcified lesions,and SYNTAX ? 23(P < 0.001);and areas under the ROC curve were 0.794,0.796,and 0.803,respectively(P < 0.001).(3)Multivariate logistic regression showed that Klotho was independently associated with severe coronary artery disease and might be a protective factor for severe coronary lesions(OR = 0.974,P < 0.001).Conclusion:1.The anti-atherosclerosis effect of Klotho may be attributed to its underlying mechanism involved in anti-endothelial aging;Klotho can ameliorate metabolic stress-induced endothelial cell senescence via activation of autophagy.2.Klotho is significantly associated with inflammation and metabolic disorders;low circulating Klotho may be an independent risk factor for metabolic syndrome.3.Klotho is significantly associated with coronary artery disease;low circulating Klotho may be an independent risk factor for severe coronary artery disease. |
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