Association Of TLR4 And MMP9 Gene Polymorphisms And Its Differential Expression With The Risk Of Aortic Aneurysm, And Its Molecular Mechanisms

Objective:Aortic aneurysm?AA?is a local or diffuse abnormal aortic expansion and can be divided into abdominal aortic aneurysm?AAA?and thoracic aortic aneurysm?TAA?according to the anatomy.AA is the result of polygenic and multifactor interaction.According to the latest guidelines for diagnosis and treatment of aortic diseases,the occurrence and development of AA is a complex process,and its main pathological changes include extracellular matrix degradation and infiltration of immune inflammatory factors.TLR4 is a pattern recognition receptor of the natural immune system.Previous studies showed that the immune inflammation pathway mediated by TLR4 was associated with a variety of cardiovascular diseases.In addition,TLR4 plays an important role in maintaining physiological aortic homeostasis and inducing pathological aortic phenotype transformation.MMP9 is the most important member of the MMP family,which has the role of degrading IV collagen and elastin in the matrix of vascular wall,playing an important role in the reconstruction of aortic wall and the development of AA.Related studies have shown that MMP9 could be highly expressed in serum and aortic wall,leading to the degradation of the extracellular matrix of aorta and the degeneration of middle layer of the artery,which resulted in AA formation.Li et al reported that TLR4 activation could induce human aorta smooth muscle cells to secrete MMP9.Qin et al proved that TLR4 antagonist or TLR4 gene knockout would reduce the MMP2 and MMP9 activation and pro-inflammatory factors secretion in the rat model of AAA formation induced by AngII,and verified that the expression level of TLR4 in human AA tissues was significantly higher than that in normal tissues.Therefore,TLR4plays an important role in the pathological change of AA and the connection with other AA-related signal pathways.However,above evidence was generally based on animal experiments,and genetic association studies were rare.Single nucleotide polymorphism?SNP?is the most common form of genetic variation,which leads to different functions of the same gene coding products,different susceptibility of individuals to diseases,diversity of clinical manifestations,different disease outcomes and prognosis.Although the relationship of MMP9 polymorphism and its expression with AA has been widely concerned,the research results are not uniform.However,studies on TLR4polymorphism and its expression linked to AA risk were rarely reported,which needed to be further clarified.To sum up,this subject aimed to explore the relationship of TLR4and MMP9 gene polymorphisms and their differential expression with the risk of aortic aneurysmal disease and its molecular mechanism,so as to provide theoretical basis for diagnosis and individualized treatment of AA.Methods:1.Genetic polymorphism was detected using KASP genotype method.We used multivariate logistic regression to analyze the relationship of polymorphism with disease risk.The SNP-SNP interactions and the interaction between SNP and hypertension,diabetes and dyslipidemia were performed using the whole factor model and likelihood ratio method of logistic regression.2.Serum TLR4 and MMP9 levels were detected by ELISA.Spearman rank correlation was used to examine the correlation between TLR4 and MMP9 expression levels and their correlation with the serum AA-related biomarkers and the maximum diameter of AA.ROC curve was used to evaluate the diagnostic efficacy of serum TLR4 and MMP9for AA and its subtypes.3.The protein contents of TLR4 and MMP9 in AA tissues and normal aortic tissues were detected by immunohistochemistry,and the expression levels of TLR4 and MMP9 in situ were compared.4.To compare serum and in situ tissue expression levels among different genotypes of TLR4 and MMP9 tagSNP.5.Luciferase activity of TLR4rs1927914 polymorphic allele reporter plasmids was detected using luciferase reporter system,and differences in promoter activity of different alleles were compared.Results:1.Association analysis of TLR4 and MMP9 polymorphisms with the risk of AAMMP9rs17576 AA genotype carriers had an increased risk of AA and AAA?OR=1.897,95%CI=1.132-3.177;OR=2.291,95%CI=1.253-4.190?.In the recessive model,rs17576 AA carriers significantly increased the risk of AA and AAA.TLR4rs1927914 TC genotype could increase the risk of AA in male populations?OR=1.435,95%CI=1.029-2.002,P=0.033?.MMP9rs17576 AA genotype and recessive model?AA vs.GA+GG?were associated with increased risk of AA and its subtypes in male populations,and also significantly increased risk of AA and AAA in people aged?60 years.2.Association analysis of the polymorphic interactions of TLR4 and MMP9 in the risk of AAThere were interactions between the dominant model of TLR4rs1927914 and the recessive model of MMP9rs17576,which could significantly increase the risk of TAA(P_interaction=0.038,OR=6.186).The rs1927914 dominant model with rs17576 AA genotype and rs17576 recessive model with rs1927914 TC+CC genotype could significantly increase the risk of TAA.With the rs1927914 TC+CC genotype,the rs17576 recessive model could significantly increase the risk of AA and AAA?OR=2.333and 2.519,respectively?.In addition,as the number of risk genotypes increased,the risk of AA and AAA can be significantly elevated(P_trend=0.032 and 0.031,respectively).3.The interaction effects of TLR4 and MMP9 polymorphisms with cardiovascular risk factors in the risk of AATLR4rs11536889 CC genotype had an interaction with dyslipidemia,which significantly increased the risk of TAA(P_interaction=0.001).TLR4rs1927914 CC genotype has interaction with hypertension,which could significantly increase the risk of overall AA and AAA(P_interaction=0.018 and 0.039,respectively).Diabetes and TLR4rs1927914CC genotype have interaction on increasing the risk of AA and TAA(P_interaction=0.032 and0.018,respectively),and there was an interaction between diabetes and TC+CC genotype in increasing the risk of AAA(P_interaction=0.040).4.Association analysis of TLR4,MMP9 serum and in situ protein expression with the risk of AAIn total comparison,TLR4 and MMP9 levels of AAA and TAA were significantly higher than those in control group.The stratification results showed that serum TLR4expressions in AAA or TAA group were obviously higher compared with control group?P<0.05?in any stratified analysis,except that there was no significant difference in diabetic stratification between TAA and control group.In the subgroups of normal blood lipid,normal blood glucose and aged<65 years,serum MMP9 levels were gradually increased from control group to AAA group to TAA group?P<0.05?.Compared with control group,serum MMP9 levels of patients with AAA and TAA were significantly increased in both male and female subgroups,while the MMP9 levels were also significantly elevated in hypertensive subgroup?P<0.05?.In addition,TLR4 and MMP9showed different levels of in situ expression in AA tissues,while negative expression was found in normal aortic tissues.There were significant differences in the ratio and expression level of TLR4 and MMP9 proteins between the two groups?P<0.001?.Serum TLR4 and MMP9 levels were significantly positively correlated?r=0.412,P<0.001?,and TLR4 and MMP9 in situ expression levels were also significantly positively correlated?r=0.718,P<0.001?.5.The relationship of serum TLR4 and MMP9 levels with circulating AA-related biomarkers and the maximum diameter of AAThere was a significant association between serum TLR4 and circulating AA-related biomarkers including CRP,D-dimer,Fg,Hcy and Cys-c?P<0.05?.Serum MMP9 was positively correlated with CRP,D-dimer and Hcy?P<0.05?.However,serum TLR4 and MMP9 levels were not related to the maximum diameter of AA?P>0.05?.6.The diagnostic and predictive value of serum TLR4 and MMP9 for aortic aneurysmal diseaseThe optimal cut-off values of TLR4 for diagnosis of AAA and TAA were 13.86ng/ml and 11.31ng/m,and the corresponding area under the curve?AUC?was 0.662 and 0.689with the sensitivity was 45.9%and 57.7%,and the specificity was 84.7%and 72.1%,respectively.The optimal cut-off values of serum MMP9 for AAA and TAA were 385.32ng/ml and 393.00 ng/m,and the corresponding AUC was 0.686 and 0.834 with the sensitivity of 49.5%and 69.6%,and the specificity of 87.5%and 91.1%,respectively.7.Effects of TLR4 and MMP9 polymorphisms on TLR4 and MMP9 expressionTLR4rs1927914 polymorphism was correlated with serum TLR4 expression.In the general population and patients with AA,serum TLR4 protein expression was significantly increased in heterozygous?TC vs.TT?and dominant model?TC+CC vs.TT?carriers?P<0.05?,while TLR4rs11536889 and MMP9rs17576 polymorphisms did not affect serum TLR4 and MMP9 expression?P>0.05?.There was no significant relationship of TLR4 and MMP9 polymorphisms with their expression in situ in AA group.8.The effect of TLR4rs1927914 on promoter activity by functional experiment in vitroThe results of the luciferase reporter gene experiment showed that the promoter activity of TLR4rs1927914 C allele was significantly higher than that of the T allele?0.589±0.004 vs.0.340±0.014,P<0.001?.Conclusion:1.The results of single polymorphism study suggested that MMP9rs17576gene polymorphism was associated with increased risk of AA and AAA.TLR4rs1927914was correlated with an increased risk of AA in male population.MMP9rs17576 was associated with an increased risk of AA and its subtype in male population,as well as an elevated risk of AA and AAA in subjects aged?60 years.2.The results of SNP-SNP interaction showed that TLR4rs1927914 and MMP9rs17576had interaction effects in susceptibility to TAA.TLR4rs1927914 and MMP9rs17576 also had epistatic and cumulative effects in the increased risk of AA and AAA.3.The results of the interaction between SNP and cardiovascular risk factors indicated that there was an interaction effect between TLR4rs11536889 and dyslipidemia in susceptibility to TAA,TLR4rs1927914 and hypertension had interaction effects in the pathogenesis of AA and AAA,while TLR4rs1927914 and diabetes had interaction effects in the risk of AA and its subtypes.4.High TLR4 and MMP9 expression in either serum or tissue in situ was associated with the risk of aortic aneurysmal disease,and serum TLR4 and MMP9 were of important diagnostic and predictive value for aortic aneurysmal disease.5.TLR4rs1927914 polymorphism was correlated with serum TLR4 expression,and serum TLR4 levels were higher in the heterozygous and dominant model carriers than those in the wild homozygous carriers.6.The rs1927914 polymorphism in the TLR4 gene 5'-UTR region could change the promoter activity,further clarifying the possible mechanism by which rs1927914increased the risk of aortic aneurysmal disease.