我們實驗室先前的研究顯示，在迷你豬的腹主動脈末端進行窄化12週後，會誘導腹主動脈瘤(abdominal aortic aneurysm, AAA)的形成。血管窄化所誘導的腹主動脈瘤(coarctation-induced AAA)呈現血管壁的彈性蛋白斷裂且分解、膠原蛋白重塑和平滑肌細胞排列雜亂的現象，這些現象與人類退化性腹主動脈瘤的觀察結果一致。明膠酶譜(gelatin zymography)分析的結果顯示腹主動脈瘤形成的過程中，間質金屬蛋白酶9 (matrix metalloproteinase,(MMP)-9)和 MMP-2 的活性明顯上升。即時聚合酶鏈式反應(real-time PCR)分析顯示MMP-3(4週)、MMP-9(4週)、MMP-13 (4週與12週)和MMP-19(4週) 訊息核醣核酸(mRNA)的表現量上升。本研究的目標為探討血管窄化所誘導的腹主動脈瘤之中表現上升的MMPs潛在的角色，並且探討彈性蛋白降解胜肽(elastin-derived peptides, EDPs)是否刺激血管平滑肌細胞MMP的表現、分泌和活化。免疫組織染色的結果顯示，MMP-3在血管窄化後4週和12週的血管壁中層平滑肌細胞表現增加，MMP-13的表現則沒有明顯變化，MMP-19在血管窄化後4週的血管壁中層與內層表現增加。使用酪蛋白酶譜(casein zymography)偵測血管窄化後，血管組織中MMP-3的活性，結果沒有顯著差異。我們進一步使用慢病毒感染人類主動脈平滑肌細胞以送入MMP-3基因，使平滑肌細胞持續表現MMP-3，以探討MMP-3過度表現對於平滑肌細胞移行的活性和肌動蛋白絲(actin filament)細胞骨架組織的影響。相對於控制組細胞，過度表現MMP-3的平滑肌細胞受由血小板衍生的生長因子(platelet-derived growth factor, (PDGF)-BB)所刺激的細胞侵入活性有增加的趨勢。初步結果顯示，相較於控制組細胞，過度表現MMP-3的平滑肌細胞在植入由纖連蛋白塗覆的聚丙烯酰胺凝膠(fibronectin-coated polyacrylamide gels, 表面硬度大約20 kPa，接近主動脈壁硬度)後，有較好的黏附與擴展性，並且表現較粗而明顯的actin filaments。我們進一步探討EDPs對血管平滑肌細胞功能的影響，發現經由EDPs處理24小時會刺激血管平滑肌細胞分泌pro-MMP-2、 pro-MMP-3和pro-MMP-9，並且具有劑量依賴性。酶譜分析發現血管平滑肌細胞分泌的pro-MMP-9的活性受到EDP的刺激，但pro-MMP-2與pro-MMP-3 的活化則不受影響。相對地，pro-MMP-13與pro-MMP-19在血管平滑肌細胞的表現與分泌量都很低且EDPs的處理亦無影響。此外，EDPs的處理對血管平滑肌細胞的細胞增生(細胞計數法)和細胞移行(傷口癒合試驗) 皆無影響。上述結果顯示，血管窄化所誘導的腹主動脈瘤形成的過程中，MMP-3的表現與(或)分泌量的增加，可能促進血管平滑肌細胞actin filament細胞骨架組織和細胞移行的活性，因而影響血管重塑。此外，由彈性蛋白碎裂所產生的EDPs可能透過刺激血管平滑肌細胞分泌與(或)活化pro-MMP-2、 pro-MMP-3和 pro-MMP-9，促進血管重塑。

Previous studies from our laboratory showed that prolonged coarctation of a terminal segment of the abdominal aorta for 12 weeks induced the formation of abdominal aortic aneurysm (AAA) in mini pigs. The coarctation-induced AAA exhibited elastic lamella fragmentation and degradation, collagen remodeling, and smooth muscle disarray, consistent with degenerative AAA observed in human. Gelatin zymography showed that activation of both matrix metalloproteinase (MMP)-9 and MMP-2 increased during AAA formation. Real-time polymerase chain reaction (PCR) analysis further showed that mRNA levels of MMP-3 (4w), MMP-9 (4w), MMP-13 (4w and 12w), and MMP-19 (4w) were elevated in the AAA segment. This study was aimed to investigate the potential roles of upregulated MMPs in the coarctation-induced AAA formation and whether elastin-derived peptides (EDPs) induce MMP expression, secretion, and activation in vascular smooth muscle cells (VSMCs). Immunohistochemical staining showed that MMP-3 levels increased in the medial VSMCs at 4w and 12w post-coarctation, MMP-13 levels did not change markedly, whereas MMP-19 increased in the media and intima at 4w post-coarctation. In tissue homogenates of the distal AA, MMP-3 activity detected with casein zymography did not change significantly. The effect of MMP-3 overexpression on VSMC migration activity and actin cytoskeleton organization was further examined in cultured human aortic VSMCs overexpressing MMP-3 encoded by lentivirus. VSMCs overexpressing mCherry-MMP-3 exhibited apparently higher invasion activity stimulated with platelet-derived growth factor (PDGF)-BB than cells expressing mCherry vector alone. On fibronectin-coated polyacrylamide gels with stiffness close to the aortic wall (~20 kPa), preliminary results showed that MMP-3-expressing VSMCs exhibited better adhesion and spreading with more prominent actin filaments compared to vector-expressing VSMCs. In human aortic VSMCs, treatment of EDPs for 24 hours increased the secretion of pro-MMP-2, pro-MMP-3, and pro-MMP-9 in a dose-dependent manner. Zymography analysis showed that activation of secreted pro-MMP-9, but not pro-MMP-2 or pro-MMP-3, was stimulated by EDPs treatment. In contrast, the expression and secretion levels of pro-MMP-13 and pro-MMP-19 in VSMCs were low and EDPs treatment had no effect. In addition, EDPs treatment did not affect VSMCs proliferation rate and migration activities assessed with cell counting and wound-healing assay. These results suggested that increased MMP-3 expression and/or secretion during coarctation-induced AAA formation may promote actin filament reorganization and migration activity of VSMCs and hence vascular remodeling. In addition, EDPs derived from elastic lamella fragmentation may contribute to vascular remodeling through stimulating the secretion and/or activation of pro-MMP-2, pro-MMP-3, and pro-MMP-9.

誌謝 1
中文摘要 2
Abstract 4
Introduction 6
Objectives of this study 9
Materials and Methods 10
Results 18
Discussion 22
References 26
Tables 32
Figures 35
Appendix 57

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