| 摘要: | 血管新生是一種重要的生理現象，其發生是經由原先存在於血管中之內皮細胞再新生而形成血管分支的過程；其在維持血腦屏障之完整性及早期發育過程中扮演著重要的角色。因內皮細胞的遷移作用為血管新生過程之所需，芳香烴受體抑制內皮細胞的遷移作用可能造成血管新生的阻礙。近來有研究指出中樞神經系統中的血管內皮層具有被戴奧辛神經毒物標記之潛力。阮淑慧 博士先前的研究已顯示芳香烴受體之促效劑三甲基膽蒽會降低人類臍靜脈內皮細胞的增生作用及血管新生作用。這些結果透露出活化態的芳香烴受體可能會進一步抑制內皮細胞的遷移作用並降低細胞與細胞及細胞與基質間的交互作用，因而損害血管新生及血腦屏障的完整性。然而，以上調控之分子機轉仍未釐清，因此本篇論文主要研究動機為探討活化態的芳香烴受體在血管內皮細胞中血管新生作用與血腦屏障的完整性之分子調控機制。於論文第一部分，其研究動機為探討透過芳香烴受體而改變小G蛋白RhoA/黏著斑激酶的表現量，其降低人類臍靜脈內皮細胞的運動性之分子機制。在人類臍靜脈內皮細胞中，芳香烴受體透過其促效劑三甲基膽蒽來降低黏著斑激酶的表現量及增加小G蛋白RhoA的表現情形。同樣地，利用電泳遷移率變動分析法及染色質免疫沉澱法的分析，三甲基膽蒽的投予，顯現人類臍靜脈內皮細胞中芳香烴受體結合在小G蛋白RhoA及黏著斑激酶之基因促進子上「生物性毒素反應單元結合位－5’-GCGTG-3’」的表現量有明顯改變情形。 此外，三甲基膽蒽透過抑制黏著斑激酶及p190Rho水解酶活化蛋白對小G蛋白RhoA的負回饋機制來增加其活性。隨後進一步地觀察到：三甲基膽蒽所活化的小G蛋白RhoA增加人類臍靜脈內皮細胞中肌動蛋白張力絲及黏著斑複合體的形成，而小G蛋白RhoA的抑制劑及芳香烴受體的結抗劑可降低其造成的細胞骨架之動態變化。利用細胞移動能力試驗觀察到三甲基膽蒽的投予明顯地降低人類臍靜脈內皮細胞遷移能力。以上研究可進一步透過動物實驗中的基底膜基質形成試驗分析來驗證。總結以上，芳香烴受體之促效劑三甲基膽蒽改變小G蛋白RhoA及黏著斑激酶的基因表現進而促使小G蛋白RhoA活化，而抑制人類臍靜脈內皮細胞中的遷移作用及血管新生作用。於論文第二部分，是更進一步以小鼠腦微血管內皮細胞來研究及探討：透過芳香烴受體之促效劑三甲基膽蒽所活化的小G蛋白RhoA在血腦屏障的瓦解作用之分子機制。由芳香烴受體之促效劑三甲基膽蒽所調控的「磷酸肌醇磷酸酶－蛋白激脢Cδ－肝醣合成酶激酶-3β－β-鏈蛋白之分子傳遞鏈」可能牽涉血腦屏障完整性的改變，將進一步來實驗求證。芳香烴受體之促效劑三甲基膽蒽所活化的小G蛋白RhoA透過增加磷酸肌醇磷酸酶的磷酸化表現，來增加由「蛋白激脢Cδ－肝醣合成酶激酶-3β傳遞鏈」所調控的β-鏈蛋白之磷酸化表現，使β-鏈蛋白走向蛋白酶體之降解作用。β-鏈蛋白表現量下降造成了內皮細胞的細胞外基質「纖連蛋白」及其受體α5β1之表現量下降。除此之外，黏著斑蛋白群包括：黏著斑激酶、血管內皮細型-鈣黏蛋白、紐蛋白及β-肌動蛋白之間的交互作用也減弱而促使黏附連結區穩定度下降。利用泳遷移率變動分析法及染色質免疫沉澱法的分析，三甲基膽蒽的投予，顯現小鼠腦微血管內皮細胞中β-鏈蛋白結合在纖連蛋白及其受體α5β1之基因促進子上「T細胞因子/淋巴增強因子結合位」的表現量下降。此外，辛伐斯汀及他伐斯汀的投予可透過抑制小G蛋白RhoA的活性來降低三甲基膽蒽在小鼠腦內皮細胞中之調控與表現，並利用小鼠為模型來分析其在血腦障壁通透性的影響。因此，在動物實驗中，辛伐斯汀可緩解由芳香烴受體之促效劑三甲基膽蒽活化的小G蛋白RhoA透過β-鏈蛋白之降解作用所造成內皮細胞屏障的機能障礙。以上結果顯示，由三甲基膽蒽活化的小G蛋白RhoA透過調控「磷酸肌醇磷酸酶－蛋白激脢Cδ－肝醣合成酶激酶-3β－β-鏈蛋白之分子傳遞鏈」來降低血管內皮細胞所表現的細胞外基質「纖連蛋白」及其受體α5β1，造成血腦屏障的瓦解作用，以上調控機制可由辛伐斯汀修復。總括來說，由本論文的實驗中發現：透過芳香烴受體之促效劑三甲基膽蒽所活化的小G蛋白RhoA及其下游調節蛋白群增加了血管新生的抑制作用及瓦解內皮細胞屏障的完整性。
Angiogenesis is the physiological process involving the growth of new blood vessels from pre-existing vessels, and it plays an important role in formation and integrity of blood brain barrier in early developmental processes. Since endothelial cell migration is one of the major events essential for angiogenesis, the finding of the anti-migration effect of aryl-hydrocarbon receptor (AhR) activation in endothelial cells suggests to that activating AhR might also exert an anti-angiogenic activity. Recent studies suggested that the vascular endothelium in central nervous system (CNS) might be a potential target of dioxin neurotoxicity. Dr. Juan’s lab previously demonstrated the anti-proliferative and anti-angiogenic effects of 3-methylcholanthrene (3MC), an AhR agonist, in human umbilical vascular endothelial cells (HUVECs). These findings showed that activated AhR might induce anti-migration, disruption of cell-cell and cell-matrix interactions, which led to increases in impediment of angiogenesis and BBB integrity, but the underlying mechanism is unclear. The aim of this study was to investigate the molecular mechanisms underlying ligand-bound AhR on angiogenesis and on the maintenance of endothelial barrier integrity. In the first part of this study, the research was aimed to investigate the molecular mechanisms underlying AhR-mediated inhibition of motility in HUVEC through alterations of RhoA/FAK expressions. FAK was downregulated whereas RhoA was up-regulated upon AhR agonist 3MC treatment. Consequently, novel functional XRE binding sites containing the core sequence 5’-GCGTG-3’in the promoter regions of RhoA and FAK were identified by electrophoretic mobility shift and chromatin immunoprecipitation assays. These results suggested the alterations of the binding activities of AhR in HUVECs treated with 3MC. Moreover, 3MC increased membrane-bound RhoA levels via suppression of a negative feedback pathway of FAK/p190RhoGAP. Subsequently, the formations of actin stress fiber and focal adhesion complex were observed with an increase in activated form of RhoA in 3MC-treated cells, and these dynamic changes of cytoskeleton were prevented by a RhoA inhibitor and AhR antagonists. Apparently, in vitro by a transwell approach showed that these compounds significantly prevented 3MC-induced inhibition of migration in HUVECs. The in vitro findings were further confirmed using an animal model of Matrigel formation in Balb/c mice. Collectively, AhR-mediated changes in genomic regulation of FAK/RhoA, triggering RhoA activation, cause the anti-migratiory and anti-angiogenic effects in HUVECs by 3MC administration. In the second part of this study, the signaling cascade of AhR-mediated up-regulation of RhoA expression was further studied to investigate the molecular mechanisms of the disruption of BBB upon 3MC treatment in MCVECs. The AhR/RhoA-mediated molecular mechanisms of PTEN/PKCδ/pGSK3β/β-catenin signaling pathway, which might implicate in alterations to blood-brain barrier integrity, were verified. PTEN phosphorylation, which was increased by 3MC-mediated AhR/RhoA activation, facilitated the proteasomal degradation of β-catenin through PKCδ/pGSK3β-mediated β-catenin phosphorylation. The decrease in β-catenin causes down-regulation of fibronectin and α5β1 integrin. In addition, the interactions of focal adhesion proteins and junctional proteins among FAK, VE-cadherin, vinculin, and β-actin were simultaneously decreased, suggesting adherens junction instability. Novel functional TCF/LEF1 binding sites in the promoter regions of fibronectin and α5/β1 integrins were identified by electrophoretic mobility shift and chromatin immunoprecipitation assays, resulting in the decreases in the binding activities of β-catenin in 3-MC-treated MCVECs. Furthermore, 3MC-mediated alterations in MCVECs were prevented by simvastatin and pravastatin treatment through inhibiting RhoA activation, and the in vitro findings were substantiated by an in vivo blood-brain barrier assay. Therefore, endothelial barrier dysfunction due to 3MC occurs through AhR/RhoA-mediated β-catenin down-regulation, which is prevented by simvastatin treatment in vivo. These results suggest that 3MC-induced up-regulation of RhoA contributed to the expressions of PTEN/PKCδ/GSK3β/β-catenin signaling pathway, which in turn decreased vascular endothelial cell’s ECM “fibronection” and its integrins “α5β1”, and the 3-MC-mediated disruption of endothelial barrier was prevented by RhoA inhibitors intervention. Taken together, the findings from the present study suggest that 3-MC/AhR-mediated alterations in vascular endothelial cells increase inhibition of angiogenesis and disruption of endothelial barrier integrity through regulating activation of RhoA and its downstream effectors. |
