Pregnancy-induced Hypertension-related Regulation Of Metalloproteinase Expression And Methylation

Pregnancy-induced hypertension syndrome(PIH) is a multisystem disease classically defined on the basis of hypertension and proteinuria after 20 weeks gestation.The etiology of the disease remains poorly understood but it is known that altered expression of some metalloproteinases resulted extracellular matrix degrading disorder may be responsible for the abnormal trophoblast invasion.Objective:To perform a systematic expression analysis of MMP-9 and ADAMTS family (ADAAMTS-1,-4,-5) genes in normal and pathological placentas and to pinpoint epigenetic alterations inside promoter regions of genes.Methods:Placentas were obtained from 20 patients with PIH and 18 normal pregnancies in the third trimester.Immunohistochemistry of paraffin wax-embedded slices and Quantitative Real-Time PCR methods were used to detect the protein and mRNA expression level of different genes.The methylation status of the promoter regions was analyzed with methylation-sensitive restriction enzymes,followed by PCR amplification in order to study the association between demethylation of specific CpG sitesand abnormal expression.Results:1) Immunohistochemistry:MMP-9 was highly expressed in trophoblast and Villous stromal cells in PIH samples,while much lower in normal controls;ADAMTS-4 was highly expressed syncytiotrophoblast cells and lower in cytotrophoblast cells,while there was almost no diction in normal controls;ADAMTS-5 expressed highly in trophoblast cells,apparently higher than normal controls.There was no suitable antibody for ADAMTS-1.2) Quantitative Real-Time PCR results showed MMP-9,ADAMTS-1,-4,-5 were expressed higher in PIH samples than normal controls.3) The methylation status of the promoter regions:There was no significant higher level of MMP-9 demethylation between PIH samples and normal controls.But the percentage of unmethylated-712 site was higher in preeclampsia patients(90%) comparing with controls 44%(p＜0.05);ADAMTS-1 was significant higher demethylation in PIH samples than normal controls,the percentage was 48.8%vs. 30.6%(p＜0.05),with the specific site-944 which the unmethylation percentage was 55%vs.22.2%(p＜0.05);ADAMTS-4 was significant higher demethylation in PIH samples than normal controls,the percentage was 20%vs.4.4%(p＜0.05),with the specific site -323 which the unmethylation percentage was 25%vs.0(p＜0.05); ADAMTS-5 was significant higher demethylation in PIH samples than normal controls,the percentage was 40%vs.20.8%(p＜0.05).with the specific site +72 which the unmethylation percentage was 33%vs.0(p＜0.05).Conclusion:1) Demonstrated the association between the altered synthesis of ADAMTS-1,-4,-5 and PIH etiology for the first time;2) For the first time to study the expression of MMP-9,ADAMTS-1,-4,-5 may result from epigenetic changes of the methylation status of CpG sites in the promoter region and was responsible for PIH happens.3) Screened out specific sites of promoter regions may play key roles in gene regulation. This study provided new biomarkers for early diagnose and drug investigations.