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

Bone fractures and pathologic conditions like chronic wounds significantly reduce the quality of life for the patients, which is especially dramatic in an elderly population with considerable multi-morbidity and lead to substantial socio-economic costs. To improve the wound healing capacity of these patients, new strategies for the design of novel multi-functional biomaterials are required: they should be able to decrease extensive pathologic tissue degradation and specifically control angiogenesis in damaged vascularized tissues like bone and skin. Glycosaminoglycans (GAGs) like hyaluronan (HA) and chondroitin sulfate (CS) as important extracellular matrix (ECM) components are involved in several biological processes such as matrix remodeling and growth factor signaling, either by directly influencing the cellular response or by interacting with mediator proteins. This could be useful in functionalizing biomaterials, but native sulfated GAGs (sGAGs) show a high batch-to-batch variability and are limited in their availability. Chemically modified HA and CS derivatives with much more defined characteristics regarding their carbohydrate backbone, sulfate group distribution and sulfation degree are favorable to study the structure-function relationship of GAGs in their interaction with mediator proteins and/or cells and this might be used to precisely modulate activity profiles to stimulate wound healing. By combining collagen type I as the main structural protein of the bone and skin ECM with these GAG derivatives, 2.5-dimensional (2.5D) and 3D artificial ECM (aECM) coatings and hydrogels were developed. These biomaterials as well as the respective GAG derivatives alone were compared to native GAGs and used to analyze how the sulfation degree, pattern and carbohydrate backbone of GAGs influence: i) the activity of tissue inhibitor of metalloproteinase-3 (TIMP-3) and vascular endothelial growth factor-A (VEGF-A) as main regulators of ECM remodeling and angiogenesis, ii) the composition and characteristics of the developed 2.5D and 3D aECMs, iii) the enzymatic degradation of collagen-based aECMs and HA/collagen-based hydrogels, iv) the proliferation and functional morphology of endothelial cells. Surface plasmon resonance (SPR) and enzyme linked immunosorbent assay (ELISA) binding studies revealed that sulfated HA (sHA) derivatives interact with TIMP-3 and VEGF-A in a sulfation-dependent manner. sHA showed an enhanced interplay with these proteins compared to native GAGs like heparin (HEP) or CS, suggesting a further impact of the carbohydrate backbone and sulfation pattern. sGAGs alone were weak modulators of the matrix metalloproteinase-1 and -2 (MMP-1 and -2) activity and did not interfere with the inhibitory potential of TIMP-3 against these proteinases during enzyme kinetic analyses. However, the formation of TIMP 3/GAG complexes reduced the binding of TIMP-3 to cluster II and IV of its endocytic receptor low-density lipoprotein receptor-related protein-1 (LRP-1, mediates the up-take and degradation of TIMP-3 from the extracellular environment) in a sulfation- and GAG type-dependent manner. It is of note that the determined complex stabilities of TIMP-3 with cluster II and IV were almost identical indicating for the first time that both clusters contribute to the TIMP-3 binding. Competitive SPR experiments demonstrated that GAG polysaccharides interfere stronger with the TIMP 3/LRP-1 interplay than GAG oligosaccharides. The importance of the position of sulfation is highlighted by the finding that a sHA tetrasaccharide exclusively sulfated at the C6 position of the N-acetylglucosamine residues significantly blocked the receptor binding, while CS and HEP hexasaccharides had no detectable effects. Thus, sHA derivatives as part of biomaterials could be used to sequester and accumulate TIMP 3 in aECMs in a defined manner where sHA-bound TIMP-3 could decrease the matrix breakdown by potentially restoring the MMP/TIMP balance. GAG binding might extend the beneficial presence of TIMP-3 into wounds characterized by excessive pathologic tissue degradation (e.g. chronic wounds, osteoarthritis). Mediator protein interaction studies with sHA coated surfaces showed the simultaneous binding of TIMP-3 and VEGF-A, even though the sHA/VEGF-A interplay was preferred. Moreover, kinetic analysis revealed almost comparable affinities of both proteins for VEGF receptor-2 (VEGFR-2), explaining their competition that mainly regulates the activation of endothelial cells. Additional SPR measurements demonstrated that the binding of sGAGs to TIMP-3 or VEGF-A decreases the binding of the respective mediator protein to VEGFR-2. Likewise, a sulfation-dependent reduction of the binding signal was observed after pre-incubation of a mixture of TIMP-3 and VEGF-A with sGAG poly- and oligosaccharides. The biological consequences of GAGs interfering with VEGF-A/VEGFR-2 and TIMP-3/VEGFR 2 were assessed in vitro using porcine aortic endothelial cells stably transfected with VEGFR 2 (PAE/KDR cells). The presence of sHA both decreased VEGF-A activity and the activity of TIMP-3 to inhibit the VEGF-A-induced VEGFR-2 phosphorylation. The same decreased activities could be observed for the migration of endothelial cells. However, if sHA, TIMP-3 and VEGF-A were present simultaneously, sHA partially restored the TIMP-3-mediated blocking of VEGF-A activity. These findings provide novel insights into the regulatory potential of sHA during endothelial cell activation as an important aspect of angiogenesis, which could be translated into the design of biomaterials to treat abnormal angiogenesis. These sHA-containing materials might control the angiogenic response by modulating the activity of TIMP 3 and VEGF-A. The in vitro fibrillogenesis of collagen type I in the presence of sHA derivatives led to 2.5D collagen-based aECM coatings with stable collagen contents and GAG contents that resemble the organic part of the bone ECM. A burst release of GAGs was observed during the first hour of incubation in buffer with the GAG content remaining almost constant afterwards, implying that the number of GAG-binding sites of collagen restricts the amounts of associated GAGs. Moreover, two differently sulfated HA derivatives could for the first time be incorporated into one multi-GAG aECM as verified via agarose gel electrophoresis and fluorescence measurements. This illustrates the multiple options to modify the aECM composition and thereby potentially their functionality. Atomic force microscopy showed that the presence of sHA derivatives during fibrillogenesis significantly reduced the resulting fibril diameter in a concentration- and sulfation-dependent manner, indicating an interference of the GAGs with the self-assembly of collagen monomers. In line with enzyme kinetic results, none of the GAGs as part of aECMs altered the enzymatic collagen degradation via a bacterial collagenase. Thus aECMs were proven to be biodegradable independent from their composition, which is favorable concerning a potential biomedical usage of the aECMs e.g. as implant coatings. HA/collagen-based hydrogels containing fibrillar collagen embedded into a network of crosslinked HA and sGAGs were developed as 3D aECMs. Scanning electron microscopy demonstrated a porous structure of the gels after lyophilization, which could favor the cultivation of cells. The presence of collagen markedly enhanced the stability of the gels against the enzymatic degradation via hyaluronidase, something beneficial to clinical use as this is often limited by the generally fast breakdown of HA. Binding and release experiments with lysozyme, as positively charged model protein for e.g. pro-inflammatory cytokines, and VEGF A revealed that the sulfation of GAGs increased the protein binding capacity for pure GAG coatings and retarded the protein release from hydrogels compared to hydrogels without sGAGs. Moreover, the additional acrylation of sHA was shown to strongly reduce the interaction with both proteins when the primary hydroxyl groups were targets of acrylation. This stresses the influence of the substitution pattern on the protein binding properties of the GAG derivatives. However, hydrogel characteristics like the elastic modulus remained unaffected. The different interaction profiles of lysozyme and VEGF-A with GAGs demonstrated a protein-specific preference of different monosaccharide compositions, suggesting that the mediator protein binding could be simultaneously adjusted for several proteins by combining different GAG derivatives. This might allow the scavenging of pro-inflammatory cytokines and at the same time a binding and release of wound healing stimulating growth factors. Since there is a growing demand for biomaterials to regenerate injured vascularized tissues like bone and skin, endothelial cells were used to examine the direct effects of solute GAGs and hydrogels containing these GAGs in vitro. In both cases, sHA strongly enhanced the proliferation of PAE/KDR cells. A VEGFR-2-mediated effect of GAGs on endothelial cells as underlying mechanism is unlikely since GAGs alone did not bind to VEGFR-2 and had no influence on VEGFR-2 phosphorylation. Other factors like GAG-induced alterations of cell-matrix interactions and cell signaling could be responsible. In accordance with SPR results, a decreased endothelial cell proliferation stimulating activity of VEGF-A was observed in the presence of solute GAGs or after binding to hydrogels compared to the respective treatment without VEGF-A. However, tube formation could be observed in the presence of solute VEGF A and GAGs and within hydrogels with sGAGs that released sufficient VEGF-A amounts over time. Overall the presence of GAGs and VEGF-A strongly promoted the endothelial cell proliferation compared to the treatment with GAGs or VEGF-A alone. Thus, HA/collagen-based hydrogels functionalized with sHA derivatives offer a promising option for the design of “intelligent” biomaterials that direct and regulate the cellular behavior instead of simply acting as inert filling material. They could be used for the controlled delivery and/or scavenging of multiple mediator proteins, thus enhancing the local availability or reducing the activity of these GAG-interacting mediator proteins, or by directly influencing the cellular response. This might be applied to a range of pathological conditions by tuning the biomaterial compositions to patient-specific needs. However, extensive in vivo validation is required to show whether these in vitro findings could be used to control the biological activity of for instance TIMP-3 and VEGF-A, especially under the pathological conditions of extended matrix degradation and dysregulated angiogenesis.

Bone fractures and pathologic conditions like chronic wounds significantly reduce the quality of life for the patients, which is especially dramatic in an elderly population with considerable multi-morbidity and lead to substantial socio-economic costs. To improve the wound healing capacity of these patients, new strategies for the design of novel multi-functional biomaterials are required: they should be able to decrease extensive pathologic tissue degradation and specifically control angiogenesis in damaged vascularized tissues like bone and skin. Glycosaminoglycans (GAGs) like hyaluronan (HA) and chondroitin sulfate (CS) as important extracellular matrix (ECM) components are involved in several biological processes such as matrix remodeling and growth factor signaling, either by directly influencing the cellular response or by interacting with mediator proteins. This could be useful in functionalizing biomaterials, but native sulfated GAGs (sGAGs) show a high batch-to-batch variability and are limited in their availability. Chemically modified HA and CS derivatives with much more defined characteristics regarding their carbohydrate backbone, sulfate group distribution and sulfation degree are favorable to study the structure-function relationship of GAGs in their interaction with mediator proteins and/or cells and this might be used to precisely modulate activity profiles to stimulate wound healing. By combining collagen type I as the main structural protein of the bone and skin ECM with these GAG derivatives, 2.5-dimensional (2.5D) and 3D artificial ECM (aECM) coatings and hydrogels were developed. These biomaterials as well as the respective GAG derivatives alone were compared to native GAGs and used to analyze how the sulfation degree, pattern and carbohydrate backbone of GAGs influence: i) the activity of tissue inhibitor of metalloproteinase-3 (TIMP-3) and vascular endothelial growth factor-A (VEGF-A) as main regulators of ECM remodeling and angiogenesis, ii) the composition and characteristics of the developed 2.5D and 3D aECMs, iii) the enzymatic degradation of collagen-based aECMs and HA/collagen-based hydrogels, iv) the proliferation and functional morphology of endothelial cells. Surface plasmon resonance (SPR) and enzyme linked immunosorbent assay (ELISA) binding studies revealed that sulfated HA (sHA) derivatives interact with TIMP-3 and VEGF-A in a sulfation-dependent manner. sHA showed an enhanced interplay with these proteins compared to native GAGs like heparin (HEP) or CS, suggesting a further impact of the carbohydrate backbone and sulfation pattern. sGAGs alone were weak modulators of the matrix metalloproteinase-1 and -2 (MMP-1 and -2) activity and did not interfere with the inhibitory potential of TIMP-3 against these proteinases during enzyme kinetic analyses. However, the formation of TIMP 3/GAG complexes reduced the binding of TIMP-3 to cluster II and IV of its endocytic receptor low-density lipoprotein receptor-related protein-1 (LRP-1, mediates the up-take and degradation of TIMP-3 from the extracellular environment) in a sulfation- and GAG type-dependent manner. It is of note that the determined complex stabilities of TIMP-3 with cluster II and IV were almost identical indicating for the first time that both clusters contribute to the TIMP-3 binding. Competitive SPR experiments demonstrated that GAG polysaccharides interfere stronger with the TIMP 3/LRP-1 interplay than GAG oligosaccharides. The importance of the position of sulfation is highlighted by the finding that a sHA tetrasaccharide exclusively sulfated at the C6 position of the N-acetylglucosamine residues significantly blocked the receptor binding, while CS and HEP hexasaccharides had no detectable effects. Thus, sHA derivatives as part of biomaterials could be used to sequester and accumulate TIMP 3 in aECMs in a defined manner where sHA-bound TIMP-3 could decrease the matrix breakdown by potentially restoring the MMP/TIMP balance. GAG binding might extend the beneficial presence of TIMP-3 into wounds characterized by excessive pathologic tissue degradation (e.g. chronic wounds, osteoarthritis). Mediator protein interaction studies with sHA coated surfaces showed the simultaneous binding of TIMP-3 and VEGF-A, even though the sHA/VEGF-A interplay was preferred. Moreover, kinetic analysis revealed almost comparable affinities of both proteins for VEGF receptor-2 (VEGFR-2), explaining their competition that mainly regulates the activation of endothelial cells. Additional SPR measurements demonstrated that the binding of sGAGs to TIMP-3 or VEGF-A decreases the binding of the respective mediator protein to VEGFR-2. Likewise, a sulfation-dependent reduction of the binding signal was observed after pre-incubation of a mixture of TIMP-3 and VEGF-A with sGAG poly- and oligosaccharides. The biological consequences of GAGs interfering with VEGF-A/VEGFR-2 and TIMP-3/VEGFR 2 were assessed in vitro using porcine aortic endothelial cells stably transfected with VEGFR 2 (PAE/KDR cells). The presence of sHA both decreased VEGF-A activity and the activity of TIMP-3 to inhibit the VEGF-A-induced VEGFR-2 phosphorylation. The same decreased activities could be observed for the migration of endothelial cells. However, if sHA, TIMP-3 and VEGF-A were present simultaneously, sHA partially restored the TIMP-3-mediated blocking of VEGF-A activity. These findings provide novel insights into the regulatory potential of sHA during endothelial cell activation as an important aspect of angiogenesis, which could be translated into the design of biomaterials to treat abnormal angiogenesis. These sHA-containing materials might control the angiogenic response by modulating the activity of TIMP 3 and VEGF-A. The in vitro fibrillogenesis of collagen type I in the presence of sHA derivatives led to 2.5D collagen-based aECM coatings with stable collagen contents and GAG contents that resemble the organic part of the bone ECM. A burst release of GAGs was observed during the first hour of incubation in buffer with the GAG content remaining almost constant afterwards, implying that the number of GAG-binding sites of collagen restricts the amounts of associated GAGs. Moreover, two differently sulfated HA derivatives could for the first time be incorporated into one multi-GAG aECM as verified via agarose gel electrophoresis and fluorescence measurements. This illustrates the multiple options to modify the aECM composition and thereby potentially their functionality. Atomic force microscopy showed that the presence of sHA derivatives during fibrillogenesis significantly reduced the resulting fibril diameter in a concentration- and sulfation-dependent manner, indicating an interference of the GAGs with the self-assembly of collagen monomers. In line with enzyme kinetic results, none of the GAGs as part of aECMs altered the enzymatic collagen degradation via a bacterial collagenase. Thus aECMs were proven to be biodegradable independent from their composition, which is favorable concerning a potential biomedical usage of the aECMs e.g. as implant coatings. HA/collagen-based hydrogels containing fibrillar collagen embedded into a network of crosslinked HA and sGAGs were developed as 3D aECMs. Scanning electron microscopy demonstrated a porous structure of the gels after lyophilization, which could favor the cultivation of cells. The presence of collagen markedly enhanced the stability of the gels against the enzymatic degradation via hyaluronidase, something beneficial to clinical use as this is often limited by the generally fast breakdown of HA. Binding and release experiments with lysozyme, as positively charged model protein for e.g. pro-inflammatory cytokines, and VEGF A revealed that the sulfation of GAGs increased the protein binding capacity for pure GAG coatings and retarded the protein release from hydrogels compared to hydrogels without sGAGs. Moreover, the additional acrylation of sHA was shown to strongly reduce the interaction with both proteins when the primary hydroxyl groups were targets of acrylation. This stresses the influence of the substitution pattern on the protein binding properties of the GAG derivatives. However, hydrogel characteristics like the elastic modulus remained unaffected. The different interaction profiles of lysozyme and VEGF-A with GAGs demonstrated a protein-specific preference of different monosaccharide compositions, suggesting that the mediator protein binding could be simultaneously adjusted for several proteins by combining different GAG derivatives. This might allow the scavenging of pro-inflammatory cytokines and at the same time a binding and release of wound healing stimulating growth factors. Since there is a growing demand for biomaterials to regenerate injured vascularized tissues like bone and skin, endothelial cells were used to examine the direct effects of solute GAGs and hydrogels containing these GAGs in vitro. In both cases, sHA strongly enhanced the proliferation of PAE/KDR cells. A VEGFR-2-mediated effect of GAGs on endothelial cells as underlying mechanism is unlikely since GAGs alone did not bind to VEGFR-2 and had no influence on VEGFR-2 phosphorylation. Other factors like GAG-induced alterations of cell-matrix interactions and cell signaling could be responsible. In accordance with SPR results, a decreased endothelial cell proliferation stimulating activity of VEGF-A was observed in the presence of solute GAGs or after binding to hydrogels compared to the respective treatment without VEGF-A. However, tube formation could be observed in the presence of solute VEGF A and GAGs and within hydrogels with sGAGs that released sufficient VEGF-A amounts over time. Overall the presence of GAGs and VEGF-A strongly promoted the endothelial cell proliferation compared to the treatment with GAGs or VEGF-A alone. Thus, HA/collagen-based hydrogels functionalized with sHA derivatives offer a promising option for the design of “intelligent” biomaterials that direct and regulate the cellular behavior instead of simply acting as inert filling material. They could be used for the controlled delivery and/or scavenging of multiple mediator proteins, thus enhancing the local availability or reducing the activity of these GAG-interacting mediator proteins, or by directly influencing the cellular response. This might be applied to a range of pathological conditions by tuning the biomaterial compositions to patient-specific needs. However, extensive in vivo validation is required to show whether these in vitro findings could be used to control the biological activity of for instance TIMP-3 and VEGF-A, especially under the pathological conditions of extended matrix degradation and dysregulated angiogenesis.