| Part 1:Expression of Matrix metalloproteinase-12 in the peripheral blood of patients with rheumatic heart diseaseObjective:The pathogenesis of rheumatic heart disease(RHD)is characterized by inflammation.Rheumatic mitral stenosis(RMVS),is the most common in patients with RHD,often accompanied by varying degrees of pulmonary hypertension(PAH).Matrix metalloproteinase(MMPs)is involved in the pathogenesis of PAH.Matrix metalloproteinase-12(MMP-12),which belongs to the family of the MMPs,isimplicated in various cardiovascular diseases.MMP-12 is highly expressed on pulmonary arterioles in certain pathological conditions.Accordingly,the aim of this study was to investigate the relationship between the expression of MMP-12 and RMVS in peripheral blood in Han population of Yunnan province.Methods:The case group was from 50 patients with RMVS before valve replacementsurgery.Then 50 healthy volunteers were chosen as control group.The case group was subdivided into mild to moderate mitral stenosis(MS)group and severe MS group according to the degree of MS.The venous blood samples were taken for determination of serum MMP-12 activity and concentration.The activity of MMP-12 in peripheral blood was detected by fluorescence resonance energy transfer(FRET)activity assay.The expression level of MMP-12 in peripheral blood was detected by ELISA.The relationship between the expression of MMP-12 in peripheral blood and RMVS was elucidated by statistical analysis.Results:In the case group and control group,the results of FRET assay indicated that the relative fluorescence unit(RFU)of MMP-12 in the case group was significantly higher than that in the control group(1108.57±45.37 vs.1054.98±41.47,P=0.006).The results of ELISA detection showed that serum MMP-12concentration in the case group was significantly higher than that in the control group(2.55+1.61 ng/ml vs.1.75+0)..93,P=0.003).Logistic regression analysis showed that MMP-12 enzyme activity was an independent predictor of RMVS(P=0.010,OR=1.003,95%CI:1.001-1.006).Subgroup analysis in the case group,the results of fluorescence enzymology test showed that there was no statistical difference between the mild to moderate MS group and the severe MS group(1326.06+ 329.84 vs.1313.82+283.57,P=0.890).ELISA test showed that the serum MMP-12 concentration in severe RMVS group was significantly higher than that in mild to moderate RMVS group(2.09+1.35ng/mlvs.3.08+1/75 ng/ml,P=0.03).Spearman correlation analysis showed that serum MMP-12 concentration was positively correlated with the pulmonary arterial pressure(r=0.365,P=0.009)and negatively correlated with the lymphocyte count(LYM)in the case group(r=-0.340,P=0.016).Conclusions:The activity and expression level of MMP-12 in peripheral blood of patients with RMVS are increased.MMP-12 proteolytic activity exists within the blood samples can be used as a predictor of RMVS.Pulmonary arterial hypertension(PASP)is associated with serum MMP-12 concentration in patients with RMVS.Detection of serum MMP-12 by FRET assay and ELISA can be used as a diagnostic method for RMVS.Part 2:Expression of Matrix metalloproteinase-12 in mitral valve tissue of patients with rheumatic heart diseaseObjective:calcification is a common degenerative disorder in rheumatic valvular disease.Matrix metalloproteinase-12(matrix metalloproteinase-12,MMP-12)is closely related to cardiac valve calcification.Therefore,the aim of this study was to investigate the implication of MMP-12 in rheumatic mitral valve and association between MMP-12 and the severity of rheumatic mitral valve stenosis(RMVS)in Han population of Yunnan province.Methods:24 patients with rheumatic mitral stenosis(RMVS)were selected as the case group and 3 patients with non rheumatic mitral valve as control group.The pathological changes were observed with microscopy after hematoxylin and cosin(HE)staining.The expression of MMP-12 in mitral valve tissue was observed by immunohistochemical staining.The expression of MMP-12mRNA in mitral valve tissue was studied by RT-PCR,and the quantitative expression of MMP-12 protein in mitral valve tissue was studied by Western blottting.Results:HE staining showed that the normal mitral valve tissue was replaced by fibrin proliferation,capillary proliferation and inflammatory cell infiltration.Some degenerative disorders were found in the mitral valve tissue,such as calcification,myxoid degeneration and collagen fiber hyaline degeneration.Immunohistochemical staining showed that MMP-12 was expressed in the cytoplasm of valvular vascular endothelial cells(VEC)in mitral valve tissue from patients with RHD and non-RHD.Through RT-PCR,we demonstrated that MMP-12mRNA was expressed in rheumatic and non rheumatic mitral valves.Using Western blotting,we further confirmed the expression of MMP-12 in rheumatic and non rheumatic mitral valve tissues.By quantitative analysis,the expression of MMP-12 protein in rheumatic mitral valve tissue is higher than that of non rheumatic mitral valve tissue(P<0.001).and the expression of MMP-12 protein in mitral valve tissue with severe stenosis was significantly higher than that in mitral valve tissue with moderate and mild stenosis(P<0.001).Conclusions:This study demonstrated that MMP-12 proteolytic activity existed in rheumatic and non rheumatic mitral valve tissues.The expression level of MMP-12 is associated with severity of patients with RMVS.MMP-12 may be involved in the remodeling of rheumatic mitral valve.Part 3:Association of matrix metalloprotease 1,3,and 12 promoter polymorphisms with rheumatic heart diseaseObjective:Genetic susceptibility is associated with the pathogenesis of rheumatic heart disease(RHD).MMP-1,-3,and-12 are localized on the same chromosome and these 3 loci are considered to act in cooperation with each other.Studies have shown that MMP-1,-3 and-12 gene promoter polymorphisms are involved in the pathogenesis of various of cardiovascular disease.The aim of this study was to investigate the association between MMP-1,-3 and-12 promoter polymorphisms and RHD in Han population of Yunnan province.Methods:DNA samples were obtained from 90 adult patients with RHD and 90 control subjects.DNA was extracted from the peripheral blood samples and polymerase chain reaction(PCR)was performed to amplify the gene promoter sequence of MMP-1,-3 and-12.Polymorphisms in MMP-1-1607,MMP-3-1612 and MMP-12-82 were genotyped by direct sequencing.The gene type of heterozygote was verified by cloning sequencing.Differences in genotype and allele frequencies of these polymorphisms were compared between the cases and the controls using Unconditional logistic regression models and Chi-squared test.Results:The frequency distribution of MMP-1-1607 genotype was not significantly different between the case group and the control group(P=0.072).Compared with the genotype frequency distribution of 1G/1G,the genotype frequency distribution of 2G/2G was significantly higher in the case group than in the control group(45.6%vs.34.4%;p=0.03;OR=3.227,95%CI:1.118-9.31),There was no significant difference in genotype frequency distribution of 1G/2G between the case group and the control group(47.8%vs,48.9%;p=0.09;OR=2.455,95%CI:0.87-6.926).The frequency distribution of 2G allele in the case group was significantly higher than that of the control group(69.4%vs.58.9%;p=0.048;OR=0.644,95%CI:0.416-0.).996).There was no significant difference in the frequency distribution f of MMP-3-1612 genotype between the case group and the control group(P=0.509).Compared with the genotype frequency distribution of 6A/6A,there was no the no statistical difference in the frequency distribution of 5A/6A genotype between the case group and the control group(23.3%vs.24.4%;P=0.973;OR=0.993,95%CI:0.498-1.981),there was no statistical difference in the frequency distribution of 5A/5A genotype between the case group and the control group(4.4%vs.1.1%;P=0.230;OR=3.908,95% CI:0.421-36.242).Compared with the 6A allele frequency distribution,5A allele frequency distribution was not statistically different between the case group and the control group(16.1%vs.13.3%;P=0.473;OR=1.24,95%CI:0.689-2.231).There was no significant difference in the frequency distribution of MMP-12-82 genotype between the case group and the control group(P=0.767).Compared with the genotype frequency distribution of A/A,the genotype frequency distribution of A/G had no statistical difference between the case group and the control group(5.6%vs.7.8%;P=0.59;OR=0.708,95%CI:0.215-2.324);Compared with A allele frequency distribution,there was no statistically significant difference in G allele frequency distribution between the case group and the control group(2.8%vs.3.9%;P=0.576;OR=0.717,95%CI:0.223-2.307).In addition,A cytosine(C)to thymidine(T)missense mutation was identified within the MMP-12 gene signal peptide(SP)sequence(rs191825816).and the C/T genotype of MMP-12(rs191825816)was found in 2 RHD patients that was excluded in 90 healthy controls.There was no significant difference in the overall genotype frequency of MMP-12 gene(rs191825816)between the case group and the control group(P=0.497).Compared with the genotype frequency of C/C,the genotype frequency of C/T had no statistical difference between the case group and the control group(P=0.497;OR=0.978,95%CI:0.948-1.009).compared with C allele frequency,T allele frequency distribution was no difference between the case group and the control group(P=0.499;OR=0.989,95%CI:0.974-1.004).Conclusions:Our results suggest that MMP-1-1607 1G/2G polymorphism is associated with RHD.Individuals carrying 2G allele are likely more susceptible to RHD.A single nucleotide polymorphism(SNP)in SP sequence of MMP-12 does appear to exist in RHD group.Cloning sequencing is an effective way to verify heterozygous genotype. |
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