Expression analysis of MMP2 and TIMP2 genes in patients with aortic dissection

Thoracic aortic dissection (TAD) is an extremely lethal disease that occurs inside the aorta. It is characterized by the separation of the layers from each other by a tear starting in the inner layer of the aorta, the largest artery in the human body, extending along the vessel. Between the separated layers, false lumen formation is observed that is parallel to the true lumen. The media layer, which is the middle layer of the aorta, contains the extracellular matrix (ECM) which provides flexibility and strength to the aorta. Elastic fibers and peptidoglycan structures are the components of ECM. In the TAD cases, the extracellular matrix in the aorta is fragmented. In the TAD pathogenesis, the changes in the structure and amount of fiber that provide the flexibility to the aorta, an imbalance between the enzymes and regulators that break down the ECM proteins, and an increase in cell fragmentation activities are observed accordingly. Matrix metalloproteinases (MMP) are a large endopeptidase family having the Zn2+ in their active site. They can be synthesized by the cell types such as endothelial cells, smooth muscle cells, vascular smooth muscle cells, fibroblast, inflammatory cells, and the wall of aorta. MMPs have functions such as the degradation of extracellular matrix proteins, the maintenance and control of ECM, and the regulation of signalling. MMPs are found in the tissue with their natural inhibitors. Tissue inhibitors of metalloproteinases (TIMPs) are actively involved in both activation and inhibition of MMPs. Our first aim in this thesis study is to determine the mRNA level expression differences of the MMP2, MMP9, TIMP2 and TIMP3 genes, which are thought to have an important role in the pathogenesis of TAD, in aortic vascular tissue obtained from 10 TAD patients and 10 control individuals. The second aim of the study is to compare the gene expression levels of MMP2, MMP9, TIMP2 and TIMP3 genes in peripheral blood-derived monocyte cells obtained from the study groups with the gene expression in aortic vascular tissue. Our third aim is to determine the methylation patterns in the promoter regions of target genes by using methylation sensitive high-resolution melting (MS-HRM) method, and to investigate the relation between the target gene expression levels and the methylation pattern in the aortic vascular tissue. The gene expression analysis of the MMP2, MMP9, TIMP2 and TIMP3 genes are conducted by using the digital droplet polymerase chain reaction (ddPCR) method. The statistical significance levels of the changes in the obtained gene expression results between TAD patients and control groups were evaluated with the non-parametric "Mann Whitey U" test. "Receiver Operating Characteristics" (ROC) curve analysis was used to control the sensitivity and specificity between the patient and control group. As a result of the analyses performed, it is observed that the gene expression of MMP2 (p=0.85) has no change in the aortic tissue of TAD patients compared to the control group, and that MMP9 (p=0.043), TIMP2 (p=0.0089) and TIMP3 (p=0.028) was found to be significantly reduced. In the gene expression analysis performed in monocyte cells, it was found that MMP2 (p=0.15), MMP9 (p=0.11), TIMP2 (p=0.25) and TIMP3 (p=0.37) did not show a significant change in TAD patients compared to the control group. In addition, MMP/TIMP ratios in aortic vascular tissue and monocyte cells were also investigated. MMP2/TIMP2 ratio (p=0.063) increased in aortic vascular tissue, but it was not statistically significant. While the ratio of MMP2/TIMP3 (p=0.012) was found to be significantly different in TAD samples compared to controls, no significant increase was observed in the ratios of MMP9/TIMP2 (p=0.25) and MMP9/TIMP3 (p=0.80). In monocyte cells, MMP2/TIMP2 (p=0.45), MMP2/TIMP3 (p=0.68), MMP9/TIMP2 (p=0.80) and MMP9/TIMP3 (p=0.28) were found. As a result of the MS-HRM method used to investigate the methylation pattern in the promoter regions of target genes, the percentage of methylation in the targeted region of the MMP9 gene was determined as 0% in TAD patients and in the control group. In the TIMP3 gene, the mean methylation level of the promoter region was found to be 10% in TAD patients, while it was 12.5% in the control group. Additionally, it was found that there was no significant difference (p=0.91) between the TAD patient and the control group regarding the methylation patterns of target genes. In the conducted thesis, in TAD patients compared to control tissues the statistically significant decrease in the expression of MMP9, TIMP2 and TIMP3 genes, the statistically significant increase in the ratio of MMP2/TIMP3, and an increase in MMP2/TIMP2 ratio that is very close to the statistical significance level show that selected target genes may have an important role in the pathogenesis of TAD. In addition, decreased MMP9 and TIMP3 gene expression in monocyte cells and increased MMP9/TIMP2, and MMP9/TIMP3 gene expression rates in aortic vascular tissue are also compatible with the TAD scenario although the results could not reach statistical significance. Furthermore, as a result of the methylation analysis, it can be said that DNA methylation has no contribution on the regulation of gene expression of MMP9 and TIMP3 genes in TAD cases.