| Summary: | 碩士 === 國立成功大學 === 細胞生物及解剖學研究所 === 100 === Abdominal aortic aneurysm (AAA) is a common dilating disorder of the aorta and a major cause of death upon rupture. The key processes leading to AAA formation includes degradation of extracellular matrix (ECM), inflammation and smooth muscle cell (SMC) death. Our laboratory developed a coarctation-induced AAA model in Taiwanese Lanyu mini pigs characterized by high induction rate and elastic fiber degradation. Coarctation was performed in the infrarenal abdominal aorta (AA) for 4 weeks (4w), 8 weeks (8w), or 12 weeks (12w). AAA was detected in the AA segment distal to the coarctation at 12w post-coarctation. This study examined the role of matrix metalloproteinase-2 (MMP-2) and MMP-9 in the coarctation-induced AAA. The expression and activity of MMP-2 and MMP-9 were examined in three AA segments, i.e. suprarenal AA, proximal AA and distal AA. Suprarenal AA is located proximal to the renal arteries and is less likely to be affected by coarctation. Proximal AA is located proximal to the coarctation, which has laminar flow but is exposed to lower shear stress after coarctation. Messenger RNA levels, protein expression and activity levels of MMP-2 and MMP-9 were detected at 4w, 8w, and 12w post-coarctation with reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting, and gelatinase zymography, respectively. In the distal AA segment, MMP-2 and MMP-9 mRNA levels were higher at 8w post-coarctation. Protein expression and activity levels of MMP-9 were higher at 4w post-coarctation while those of MMP-2 didn’t vary. In the proximal AA segment, MMP-2 mRNA expression was up-regulated throughout the experimental period but no change in protein expression and activity was detected after coarctation. MMP-9 activity was markedly higher at 8w post-coarctation. In the suprarenal AA segment, no difference in MMP-2 mRNA, protein or activity levels was detected between experimental and sham control groups whereas MMP-9 expression and activity were barely detectable. Comparison among the three AA segments revealed that both MMP-9 protein expression and activity in the distal AA were higher at 4w post-coarctation. Because MMP activities in vivo are regulated by tissue inhibitors of MMPs (TIMPs), the activities of TIMPs were examined by reverse zymography. The activities of TIMP-1, -2, -3, and -4 in all three AA segments did not change compared with the sham control. Interestingly, the proximal AA segment showed higher TIMP activity compared with other AA segments. TIMPs inhibit MMPs by forming 1:1 enzyme-inhibitor complex. Therefore, we assessed MMP/TIMP ratios to determine effective activity levels of MMP-2 and -9. In the distal AA segment, MMP-2/TIMP-2 and MMP-9/TIMP-3/4 ratios were elevated at 4w post-coarctation compared with sham control. Furthermore, MMPs/TIMPs ratios in the distal AA segment were higher than those of the other AA segments. Interestingly, in sham group, the distal AA also exhibited higher MMP-2/TIMP-2 and MMP-2/TIMP-3/4 ratios compared with other two AA segments. Plasmin activity is essential for MMPs activation and plasmin activator uPA plays important role in AAA formation, thus, we examined mRNA expression of uPA and tPA. In the distal AA segment, uPA mRNA levels increased at 4w post-coarctation and were higher than those of other AA segments. In contrast, tPA mRNA expression did not differ among AA segments and treatment groups. These results suggest that MMP-2 and -9 in combinations with TIMPs play important roles in the coarctation-induced AAA formation.
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