Prevention of vein graft failure: mechanisms involved and therapeutic strategies.

• Other Authors: Hu, Jia
• Format: Others
• Language: English; Chinese
• Published: 2012
• Subjects: Thrombophlebitis > Prevention; Metalloproteinases; Thrombophlebitis > prevention & control; Tissue Inhibitor of Metalloproteinase-3
• Online Access: http://library.cuhk.edu.hk/record=b5549582; http://repository.lib.cuhk.edu.hk/en/item/cuhk-328258

冠狀動脈旁路移植術是治療合併左主幹及多隻冠狀動脈狹窄性病變患者的理想方法。然而靜脈橋失效極大地限制了冠脈搭橋手術的遠期療效。基於對靜脈橋失效潛在機制的研究，近年來開發出了多種針對性的防治手段。但是除了積極的降脂治療，目前尚未有其它療法獲得臨床證實可以有效改善靜脈橋遠期通暢率。所以，本研究旨在探索與防治靜脈橋再狹窄相關的新型生物標靶和防治策略。 === 我們應用豬大隱靜脈植入頸內動脈模型，觀察骨橋蛋白是否參與靜脈橋動脈化進程以及其與基質金屬蛋白酶功能活動的關係。我們發現骨橋蛋白表達在靜脈橋動脈化過程中顯著增加，並且與基質金屬蛋白酶2/9和增殖細胞數量的變化同步。此外，骨橋蛋白富集區域在靜脈橋內的再分佈與血管壁重構進程相關。這些結果表明, 骨橋蛋白積極參與了靜脈橋壁重構，而抑制骨橋蛋白表達作為防治靜脈橋失效的治療策略值得深入研究。 === 我們運用體外培養的方法研究了在高糖環境中骨成形蛋白4與靜脈內皮細胞舒張功能障礙的關係。我們發現，骨成形蛋白4在糖尿病患者的大隱靜脈與高糖培養的人臍靜脈內皮細胞中顯著增加；而骨成形蛋白4的高表達與靜脈血管內皮細胞依賴性舒張功能受損有關。本研究結果為解釋糖尿病患者有著較高的冠脈搭橋術後靜脈橋失效率提供了新證據，同時也為改善此類患者靜脈橋通暢率提出了潛在的治療靶點。 === 通過轉染金屬蛋白酶-3抑制物 (TIMP-3)基因來針對性地抑制血管中層平滑肌細胞的遷移和增殖，可以有效地減少靜脈橋新生內膜增生。基於前期研究，我們觀察了在豬模型中運用重組腺病毒轉載TIMP-3(RAdTIMP-3) 防治靜脈橋狹窄的遠期效果(3個月)。結果發現，即使在腺病毒載體已被清除的情況下，RAdTIMP-3對靜脈橋的良性保護作用仍持續存在。此外，我們通過比較術後7天與3個月獲取的橋血管中炎性標記物表達的差異，發現腺病毒轉染並未對靜脈橋造成長期的炎性損害。因此，我們認為RAd-TIMP3基因能夠安全有效地防治靜脈橋遠期狹窄。本研究結果為TIMP-3基因治療轉化至臨床實踐提供了可靠的前期證據。 === Coronary artery bypass grafting (CABG) remains the “gold standard“ for treating high-risk patients with unprotected left-main or multi-vessel coronary lesions. However, the long-term success of CABG is largely limited by an inadequate patency of saphenous vein grafts. To date, various therapeutic strategies targeting at the underlying mechanisms involved in the pathogenesis of vein graft failure (VGF) have been proposed and tested. However, apart from lipid-lowering therapy, no other intervention appears to have sustained benefits on improving vein graft patency in the clinical setting. Therefore, the aim of this study is to explore novel sets of molecular targets and effective therapeutic strategies to prevent VGF. === Novel molecules involved in the pathogenesis of vein graft failure === Using a porcine model, we assessed the involvement of osteopontin (OPN) in the venous arterialization and its relationship with the matrix metalloproteinases (MMPs). We found that the expression of OPN was significantly increased over the 3-month study period. Moreover, the expression of OPN at different time points well correlated with the fluctuating activities of MMP-2/9 and the number of proliferative cells. We also observed a time-dependent redistribution of OPN protein accumulating in different layers of the venous wall. These findings suggest a contributory role of OPN protein involved in the process of vein graft wall remodeling. === We used pig and human saphenous veins (SVs), as well as human umbilical endothelial cells (HUVECs), to investigate the changes of bone morphogenic protein-4 (BMP4) expression and its effects on endothelium-dependent relaxations (EDRs) under hyperglycemic conditions. Our results demonstrated a marked increase of BMP4 expression in SVs from diabetic patients and in HUVECs cultured with hyperglycemic medium. Moreover, such an increase of BMP4 contributes significantly to the impaired EDRs in venous conduits. Our findings add novel evidence that helps explain the high prevalence of VGF in diabetic patients undergoing CABG, and also suggest BMP4 as a potential therapeutic target to improve vein graft patency in this population. === Novel Therapeutic Strategy -- Gene Therapy === Aiming at blocking the development of neointima formation caused by vascular smooth muscle cells migration and proliferation, genetic transfection of tissue inhibitor of metalloproteinases-3 (TIMP-3) to vein grafts has shown promising results. Based on our previous study, we used recombinant adenoviruses that carry TIMP-3 (RAdTIMP-3) as a therapeutic gene to evaluate its long-term (3 months) effects on the pathological vein graft wall thickening in vivo. We found that the RAdTIMP-3-treated vein grafts had significantly reduced intimal and medial thickness compared with grafts from the control groups at 3 months, even after adenoviruses had already been cleared from transduced tissue. Furthermore, by assessing the amount of macrophages and the level of three inflammatory biomarkers within grafts harvested at 7 days and 3 months after implantation, we did not observe any detrimental effects of adenoviral transfection on the inflammatory status within the vein grafts. We therefore concluded that overexpression of TIMP-3 could effectively inhibit vein graft wall over-thickening in the longer-term. Our findings suggested the ex vivo RAdTIMP-3 gene therapy an attractive candidate for future clinical translation. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Hu, Jia. === Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. === Includes bibliographical references (leaves 109-143). === Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. === Abstract also in Chinese. === Abstract --- p.i === Declaration --- p.vii === Acknowledgement --- p.viii === Table of Contents --- p.x === List of Abbreviations --- p.xvi === List of Figures/Tables --- p.xviii === Chapter Chapter I --- INTRODUCTION --- p.1 === Chapter 1.1 --- SAPHENOUS VEIN GRAFTS IN CORONARY REVASCULARIZATION --- p.3 === Chapter 1.1.1 --- The use of venous conduits in CABG --- p.3 === Chapter 1.1.2 --- The long-term patency of saphenous vein grafts --- p.4 === Chapter 1.1.3 --- PCI for vein graft diseases --- p.6 === Chapter 1.1.4 --- Vein graft failure and adverse clinical outcomes --- p.7 === Chapter 1.2 --- MORPHOLOGY AND PHYSIOLOGY OF A NORMAL SAPHENOUS VEIN --- p.8 === Chapter 1.3 --- THE PATHOPHYSIOLOGY OF VEIN GRAFT FAILURE --- p.10 === Chapter 1.3.1 --- The quality of vein grafts prior to grafting --- p.10 === Chapter 1.3.1.1 --- Pre-existing endothelial dysfunction --- p.10 === Chapter 1.3.1.2 --- Surgical injuries --- p.11 === Chapter 1.3.2 --- Mechanisms of the pathological vein graft wall thickening --- p.12 === Chapter 1.3.2.1 --- Platelet activation and coagulant cascade --- p.13 === Chapter 1.3.2.2 --- Leukocytes recruitment and inflammation --- p.13 === Chapter 1.3.2.3 --- Hemodynamic forces --- p.14 === Chapter 1.3.2.4 --- Growth factors and VSMCs activation --- p.15 === Chapter 1.3.2.5 --- Contribution of adventitial and graft-extrinsic cells --- p.16 === Chapter 1.3.2.6 --- Oxidative stress --- p.17 === Chapter 1.3.2.7 --- Concomitant risk factors and vein graft atherosclerosis --- p.17 === Chapter 1.4 --- STRATEGIES FOR THE PREVENTION OF VEIN GRAFT FAILUR --- p.18 === Chapter 1.4.1 --- Minimizing surgical injuries --- p.18 === Chapter 1.4.2 --- Pharmacologic interventions --- p.19 === Chapter 1.4.3 --- External supports --- p.21 === Chapter 1.4.4 --- Genetic engineering of the vein graft --- p.23 === Chapter 1.4.4.1 --- Delivery systems --- p.23 === Chapter 1.4.4.2 --- Therapeutic strategies of the genetic modulation --- p.25 === Chapter 1.4.4.2.1 --- Antithrombotic and anticoagulant strategies --- p.25 === Chapter 1.4.4.2.2 --- Therapies for endothelial protection and regeneration --- p.27 === Chapter 1.4.4.2.3 --- Reducing inflammation and atherosclerosis --- p.28 === Chapter 1.4.4.2.4 --- Antioxidative therapy --- p.29 === Chapter 1.4.4.2.5 --- Therapies targeting at the cellular proliferation --- p.29 === Chapter 1.4.4.2.6 --- Inhibiting extracellular matrix reorganization --- p.31 === Chapter 1.5 --- CONCLUSIONS --- p.32 === Chapter Chapter II --- MATERIALS AND METHODS --- p.34 === Chapter 2.1 --- MATERIALS --- p.35 === Chapter 2.1.1 --- Reagents and equipment --- p.35 === Chapter 2.1.1.1 --- General materials and equipment for animal model --- p.35 === Chapter 2.1.1.2 --- General reagents and equipment for western blot --- p.35 === Chapter 2.1.1.3 --- General reagents and equipment for immunohistochemistry --- p.36 === Chapter 2.1.1.4 --- General reagents and equipment for venous ECs functional studies --- p.37 === Chapter 2.1.2 --- Buffers --- p.37 === Chapter 2.1.2.1 --- Buffers for human and animal samples --- p.37 === Chapter 2.1.2.2 --- Buffers for western blot --- p.38 === Chapter 2.1.2.3 --- Immunohistochemistry buffers --- p.39 === Chapter 2.1.2 --- Antibodies and adenoviral vectors --- p.41 === Chapter 2.2 --- METHODS --- p.41 === Chapter 2.2.1 --- Animal model --- p.41 === Chapter 2.2.2 --- Functional studies --- p.44 === Chapter 2.2.3 --- Human endothelial cells culture --- p.44 === Chapter 2.2.4 --- Western blot analysis --- p.45 === Chapter 2.2.5 --- Immunochemistry and immunofluorescence --- p.46 === Chapter Chapter III --- ROLE OF BMP4 IN VENOUS ENDOTHELIAL DYSFUNCTION --- p.47 === Chapter 3.1 --- INTRODUCTION --- p.48 === Chapter 3.2 --- MATERIALS AND METHODS --- p.49 === Chapter 3.2.1 --- Patient characteristics --- p.49 === Chapter 3.2.2 --- Preparation of human vein segments --- p.51 === Chapter 3.2.3 --- Porcine saphenous veins culture --- p.51 === Chapter 3.2.4 --- Functional studies of vein segments --- p.52 === Chapter 3.2.5 --- Cell culture --- p.53 === Chapter 3.3.6 --- Western blot analysis of BMP4 --- p.53 === Chapter 3.3.7 --- ROS measurement by dihydroethidium fluorescence imaging --- p.54 === Chapter 3.2.8 --- Statistical analysis --- p.54 === Chapter 3.3 --- RESULTS --- p.54 === Chapter 3.3.1 --- ACh-induced EDRs are impaired in diabetic veins --- p.54 === Chapter 3.3.2 --- The expression of BMP4 is upregulated under hyperglycemic condition --- p.55 === Chapter 3.3.3 --- BMP4 induces venous endothelial dysfunction in diabetes --- p.56 === Chapter 3.3.4 --- BMP4 impairs EDRs in cultured porcine saphenous veins --- p.58 === Chapter 3.4 --- DISCUSSION --- p.59 === Chapter 3.5 --- CONCLUSIONS --- p.62 === Chapter Chapter IV --- ROLE OF OSTEOPONTIN IN VEIN GRAFT REMODELING --- p.63 === Chapter 4.1 --- INTRODUCTION --- p.64 === Chapter 4.2 --- MATERIALS AND METHODS --- p.66 === Chapter 4.2.1 --- Surgical procedures --- p.66 === Chapter 4.2.2 --- Immunohistochemistry --- p.67 === Chapter 4.2.3 --- Western blot --- p.68 === Chapter 4.2.4 --- Gelatin zymography --- p.69 === Chapter 4.2.5 --- Cell proliferation --- p.69 === Chapter 4.2.6 --- Statistical analysis --- p.69 === Chapter 4.3 --- RESULTS --- p.70 === Chapter 4.3.1 --- Expression and redistribution of OPN protein within the venous wall --- p.70 === Chapter 4.3.2 --- The fluctuating expression of the matrix metalloproteinases --- p.72 === Chapter 4.3.3 --- Vascular smooth muscle cells proliferation --- p.74 === Chapter 4.4 --- DISCUSSION --- p.75 === Chapter 4.5 --- CONCLUIONS --- p.79 === Chapter Chapter V --- TIMP-3 GENE THERAPY FOR NEOINTIMA FORMATION --- p.81 === Chapter 5.1 --- INTRODUCTION --- p.82 === Chapter 5.2 --- MATERIALS AND METHODS --- p.84 === Chapter 5.2.1 --- Materials --- p.84 === Chapter 5.2.2 --- Grafting of pig saphenous veins and adenoviral transfection --- p.84 === Chapter 5.2.3 --- Histologic and morphometric analysis of the vein graft --- p.87 === Chapter 5.2.4 --- Immunocytochemistry --- p.87 === Chapter 5.2.5 --- Data analysis and statistics --- p.88 === Chapter 5.3 --- RESULTS --- p.89 === Chapter 5.3.1 --- Histologic and morphometric analysis of the vein graft --- p.89 === Chapter 5.3.2 --- Overexpression of TIMP-3 in porcine interposition grafts --- p.91 === Chapter 5.3.3 --- Endothelial cell coverage and VSMCs content --- p.92 === Chapter 5.3.4 --- Inflammation in vein grafts --- p.92 === Chapter 5.4 --- DISCUSSION --- p.97 === Chapter Chapter VI --- SUMMARY AND DISCUSSION OF MAJOR FINDINGS --- p.103 === Chapter 6.1 --- SUMMARY AND DISCUSSION --- p.104 === Chapter 6.1.1 --- The role of BMP4 in the pathogenesis of venous endothelial dysfunction --- p.104 === Chapter 6.1.2 --- The involvement of osteopontin in the process of vein graft remodeling --- p.105 === Chapter 6.1.3 --- Sustained benefits of adenoviruses-mediated TIMP-3 gene transfer in reducing vein graft neointima formation --- p.106 === Chapter 6.1.4 --- The inflammatory responses induced by adenoviral transfection --- p.106 === Chapter 6.1.5 --- Perspectives: novel therapeutic targets and clinical translation --- p.107 === Chapter 6.2 --- CONCLUSIONS --- p.108 === REFERENCES --- p.109 === PUBLICATION LIST --- p.144