Applying side effects of anti fibrotic compounds to promote neovascularization for tissue engineering 1

Acknowledgements I would like to express my heartfelt thanks to the people who have contributed to and supported my graduate studies in various ways, in particular: My supervisor, Associate Professor Michael Raghunath, and co-supervisor, Associate Professor Ge Ruowen, for their guidance and teaching during the course of my graduate studies; Dr Muhammad Farooq, for teaching me all about zebrafish; Ms Lee Yunqin and Ms Teo En Wei, our research attachment students, for their assistance in immunohistochemistry, VEGF ELISA and quantification of co-culture angiogenesis; Laboratory members of Tissue Modulation Laboratory, Division of Bioengineering, for your friendship, help and suggestions; Fellow classmates from the Graduate Programme in Bioengineering (GPBE), particularly those from the class of 2003; DSO National Laboratories, for the opportunity and funding for my graduate studies; My dear family members and friends, for your love; And last, but certainly not the least, God, for His love, grace, strength and inspiration that enables me to all things. i TABLE OF CONTENTS Page No Acknowledgements i Table of Contents ii Summary (Abstract) vi List of Figures ix List of Tables xi List of Abbreviations xii 1. LITERATURE REVIEW 1.1 Neovascularization in Tissue Engineering 1.2 HIF-1α: An Alternative Approach to Neovascularization 1.3 Regulation of HIF-1 1.4 HIF-1 Target Genes 1.5 HIF-1α Promotes Angiogenesis 1.6 HIF Prolyl 4-Hydroxylases 1.7 Prolyl 4-Hydroxylase Inhibitors (PHi) Promote Angiogenesis by Stabilizing HIF-1α 1.8 In Vitro Angiogenesis Assays 12 1.9 In Vivo Angiogenesis Models 14 1.10 Fibrosis in Tissue Engineering – A Result of Foreign Body Reaction and Wound Healing 19 1.11 Collagen Biosynthesis 21 1.12 Inhibition of Collagen Biosynthesis Using Prolyl Hydroxylase Inhibitors 22 1.13 Selection of Prolyl 4-Hydroxylase Inhibitors 23 ii 2. MATERIALS AND METHODS 24 2.1 Cell Culture 24 2.2 Chemicals and Reagents 25 Hypoxia Treatment Preparation of Prolyl Hydroxylase Inhibitorsand Recombinant Human VEGF 25 25 Sequential Co-cultures 26 Admixed Co-Cultures 26 Immunohistochemistry 26 Quantification of Angiogenesis by Image J and Metamorph 28 VEGF Production 29 Gel Electrophoresis and Western Blotting 30 Growth Factor Reduced Matrigel Assay 31 Co-cultures in PLLA Scaffolds 32 Collagen Biosynthesis in Cell Culture 33 Zebrafish Embryo Collection and Drug Treatment 34 Screening Ectopic SIV in Zebrafish Embryos 35 Collagen Analysis of Zebrafish Embryos by Peptic Digest and SDSPAGE Cytotoxicity Assay 36 37 3. RESULTS 3.1 35 In Vitro Angiogenesis 37 3.1.1 37 CD31 and von Willebrand Factor: Markers for Endothelial Cells iii 3.2 3.1.2 Development of Sequential Co-culture for Proangiogenesis 40 3.1.3 PHi Induced Angiogenesis in Sequential Co-cultures 49 3.1.4 Quantification of Co-culture Angiogenesis 52 HIF-1α 54 3.2.1 Stabilization of HIF-1α by Hypoxia 54 3.2.2 PHi Induced HIF-1α Stabilization 57 3.2.3 PHi Upregulated VEGF, a HIF-1α Target and Angiogenic Growth Factor 62 3.3 HDZ, CPX and PDCA Did Not Augment Tube Formation in Matrigel Assay 65 3.4 In Vivo Angiogenesis: Zebrafish Embryo 67 3.4.1 Optimization of Zebrafish Embryo Angiogenesis Assay 67 3.4.2 Characterization of PHi Effects on Zebrafish Embryo Angiogenesis 68 3.4.3 PHi Dose Dependently Induced Ectopic SIVs in Zebrafish Embryos 72 3.5 Cytotoxicity of PHi 74 3.6 Inhibition of Collagen Biosynthesis 76 3.6.1 PHi Inhibited Collagen Biosynthesis in vitro 76 3.6.2 PHi Inhibited Collagen Biosynthesis in Zebrafish Embryos (in vivo) 80 3.7 Optimizing Admixed Co-cultures in Two-Dimensional Cultures 83 3.8 Applications in Tissue Engineering – Prevascularization of Scaffolds 85 iv 4. DISCUSSION 89 4.1 Sequential Co-culture Assay 90 4.2 Cell Density Affects HUVEC Morphology 91 4.3 Quantification of Sequential Co-culture Angiogenesis 92 4.4 Fibroblasts Are Required For HUVEC Formation of Capillary Like Structures 93 4.5 PHi Induced HIF-1α and VEGF 95 4.6 Zebrafish Angiogenesis 96 4.7 PHi Inhibited Collagen Biosynthesis in vitro and in vivo 99 4.8 Applications in Tissue Engineering: Pre-vascularizing PLLA Scaffolds 101 4.9 Advantages of Using PHi in Tissue Engineering 105 5. CONCLUSIONS 108 6. REFERENCES 111 v SUMMARY (ABSTRACT) Neovascularization and fibrosis are two challenges in regenerative medicine and tissue engineering which can limit the survival, viability and function of implanted tissue engineered constructs and biomaterials. Slow neovascularization can compromise delivery of oxygen and nutrients to cells in the interior of a tissue construct; while fibrosis may occur as a result of a foreign body reaction to encapsulate the tissue construct with fibrous collagenous tissue. Prolyl 4-hydroxylase is an enzyme involved in both collagen biosynthesis and hypoxia inducible factor-1α (HIF-1α) degradation. While prolyl 4-hydroxylase inhibitors (PHi) have been shown to inhibit collagen biosynthesis, limited literature is available on the potential of these substances to promote angiogenesis by stabilization of the transcription factor HIF-1α and subsequent upregulation of relevant gene targets. The use of this approach to improve vascularization of tissue engineered constructs is also novel. Coupled with anti-fibrotic properties, PHi may be a potent aid for tissue engineering, by tackling both the problem of fibrosis and vascularization requirements. Therefore, the aim of this research study was to determine if selected PHi, namely hydralazine (HDZ), pyridine-2,4-dicarboxylate (PDCA) and ciclopirox olamine (CPX), would inhibit collagen biosynthesis as well as promote angiogenesis via stabilization of HIF-1α, to assess their potential use in tissue engineering applications. vi A sequential co-culture using fibroblasts and endothelial cells was modified and optimized for assessment of in vitro pro-angiogenesis. In this inducible system, elliptical clusters were formed by HUVECs in untreated controls, a distinctly different morphology from capillary-like structures (CLS) formed when induced with VEGF or PHi. This occurred in a dose-dependent manner, quantified by measuring the length of CLS formed. In dissecting the respective roles of HUVECs and fibroblasts, differential responses of these cell types towards PHi treatment were discovered. PHi induced stronger HIF-1α nuclear accumulation in fibroblasts than in HUVECs in co-cultures, confirmed by Western Blot comparisons of similarly treated monocultures. Using VEGF secreted into culture medium as a measure of HIF-1α activation, VEGF production by fibroblasts similarly surpassed that of HUVECs. These findings highlighted the importance fibroblasts or other non-endothelial cells in PHi induced in vitro angiogenesis. Zebrafish embryos were used to investigate in vivo angiogenesis. Ectopic subintestinal vessels (SIV) were observed either as vessel outgrowths induced by HDZ and VEGF, or as an enlarged SIV basket with more than arcades induced by PDCA, evidence of accelerated angiogenesis. To examine their effects on collagen biosynthesis, peptic treatment of conditioned medium from PHi treated fibroblast cultures or zebrafish embryos was used to analyze collagen content. PHi decreased collagen production in vitro and lowered collagen content in zebrafish embryos at concentrations that exhibited angiogenic effects. vii Finally, we investigated if angiogenesis could be similarly induced in three-dimensional scaffolds. CPX induced endothelial cells to form more interconnected CLS than untreated controls, in a similar manner to VEGF, confirming that it was able to accelerate formation of vascular analogues by endothelial cells within three dimensional scaffolds. This demonstrated a proof of concept that PHi could improve neovascularization for tissue engineering applications by pre-vascularization of three dimensional scaffolds. viii LIST OF FIGURES Figure 1. Schematic representation of HIF-1α protein domain structures. Figure 2. Regulation of the HIF-1 transcription factor. Figure 3. Some HIF-1 target gene products. Figure 4. Collagen synthesis, processing and assembly. Figure 5. CD31 and von Willebrand Factor as markers of HUVECs. Figure 6. Formation of elliptical clusters in normal sequential co-cultures. Figure 7. Schematic diagram indicating the distribution of HUVEC within a 24 well with different cell seeding densities Figure 8. Sequential co-cultures on Day to Day 4. Formation of elliptical clusters and capillary like structures. Figure 9. Capillary like structures (CLS) formed by HUVEC in sequential co-cultures at 4x, 10x and 20x magnification. Figure 10. Increasing the HUVEC density in sequential co-cultures causes an increase in the size of elliptical clusters formed. Figure 11. Increasing fibroblast cell density caused formation of capillary like structures in untreated sequential co-cultures. Figure 12. Fibroblasts and cell to cell contact with HUVECs were required for formation of capillary like structures. Figure 13. Effects of various concentrations of HDZ, CPX, PDCA and 10ng/ml VEGF on optimized sequential co-cultures of fibroblasts and HUVEC. Figure 14. Suramin inhibits angiogenesis in sequential co-cultures and attenuates angiogenic effect of CPX. Figure 15. Quantification of co-culture angiogenesis. Figure 16. Hypoxia (Immunofluorescence). induced HIF-1α stabilization in nuclei of fibroblasts Figure 17. Hypoxia induced HIF-1α stabilization in nuclei of fibroblasts (HRP based immunohistochemistry). ix Figure 18. PHi induced nuclear stabilization of HIF-1α in fibroblasts, particularly CPX and PDCA. Figure 19. 100µM HDZ induced HIF-1α (red) stabilization in fibroblasts. Figure 20. Western blot of HIF-1α in fibroblast lysates induced by various concentrations of HDZ, CPX and PDCA. Figure 21. Differential (immunofluorescence). HIF-1α expression in fibroblasts and HUVEC Figure 22. HIF-1α Western blot of fibroblasts and HUVEC monoculture lysates show that HUVEC expression of HIF-1α was much weaker than fibroblasts with CPX treatment. Figure 23. VEGF in conditioned medium from PHi sequential co-cultures analyzed by ELISA. Figure 24. ELISA analysis of VEGF in conditioned medium from separately cultured HUVEC and fibroblast monocultures treated with PHi. Figure 25. Effects of PHi in Matrigel tube formation assay. Figure 26. Illustrations of zebrafish embryo at 50% epiboly and shield stage. Figure 27. TG(fli1:EGFP) zebrafish embryos at 72 hpf. Figure 28. Development of ectopic subintestinal vessels (SIV) in HDZ, PDCA and VEGF treated zebrafish embryos at 72 hpf. 10x magnification. Figure 29. 72hpf TG(Fli-1:EGFP) zebrafish embryos treated with HDZ, PDCA and VEGF under bright field and fluorescence microscopy; 2.5x magnification. Figure 30. Percentage of zebrafish embryos exhibiting ectopic SIV following HDZ, PDCA and VEGF treatment. Figure 31. Relative cytotoxicity of HDZ, CPX and PDCA in monocultures or mixed cultures of IMR90 fibroblasts and HUVECs. Figure 32. PHi inhibition of fibroblast collagen production in vitro. SDS-PAGE and desitometric analysis. Figure 33. PHi inhibition of fibroblast collagen production in vitro. Immunofluorescence staining of collagen I and protein disulphide isomerase. x Figure 34. 400µM HDZ inhibited fibroblast collagen production. SDS-PAGE and densitometric analysis. Figure 35. PDCA and HDZ inhibited collagen biosynthesis in zebrafish embryos. SDSPAGE and densitometric analysis. Figure 36. Admixed co-cultures of fibroblasts and HUVEC with or without ascorbate. Figure 37. Confocal microscopy (z-stack) of CPX and VEGF treated admixed co-culture of fibroblasts and HUVEC in PLLA scaffolds. 20x magnification. Figure 38. Confocal microscopy (z-stack) of µM CPX treated admixed co-cultures in PLLA scaffolds. 10x magnification. Formation of CLS not follow scaffold fiber orientation. LIST OF TABLES Table 1. Summary of the in vitro and in vivo angiogenic and anti-fibrotic effects of PDCA, CPX, HDZ and VEGF. xi LIST OF ABBREVIATIONS Ascorbate AF bFGF BSA CAM CLS CO2 Col CPX C-TAD DA DAB DAPI DFO DLAV DMEM DMOG DTT EBM-2 EGM-2MV EGFP ELISA EPO FBS FIH G6PD Gly HDZ HIF-1 HRP HUVEC HVSMC IGF-1 ISV MW N2 N-TAD ODD PBS PDCA PDGF PHi PLLA PS Asc Alexa Fluor (fluorescent dyes) Basic fibroblast growth factor bovine serum albumin Chorioallantoic membrane Capillary like structures Carbon dioxide Collagen Ciclopirox olamine C-terminal transactivation domain Dorsal aorta Diaminobenzidine 4’6-diamidino-2-phenylindole, dihydrochloride desferrioxamine or deferioxamine dorsal longitudinal anastomotic vessel Dulbecco's Modified Eagle's Medium dimethyloxalyglycine dithiothreitol Endothelial basal medium Endothelial growth medium Enhanced green fluorescent protein Enzyme linked immunosorbent assay Erythropoietin Fetal bovine serum Factor inhibiting HIF-1 glucose –phosphate dehydrogenase Glycine Hydralazine Hypoxia inducible factor-1 Horse radish peroxidase Human umbilical vein endothelial cell human vascular smooth muscle cells Insulin-like growth factor-1 Intersegmental vessel Molecular weight Nitrogen N-terminal transactivation domain Oxygen dependent domain Phosphate buffered saline Pyridine-2,4-dicarboxylic acid or pyridine-2,4-dicarboxylate Platelet derived growth factor Prolyl 4-hydroxylase inhibitors Poly-L-lactide Penicillin Streptomycin xii SIV TBS TBST TGF-β VEGF VEGF-R vWF Subintestinal vessels Tris buffered saline Tris buffered saline with tween-20 Transforming growth factor- β Vascular endothelial growth factor Vascular endothelial growth factor receptor von Willebrand Factor xiii [...]... scaffolds 10 x magnification Formation of CLS do not follow scaffold fiber orientation LIST OF TABLES Table 1 Summary of the in vitro and in vivo angiogenic and anti- fibrotic effects of PDCA, CPX, HDZ and VEGF xi LIST OF ABBREVIATIONS Ascorbate AF bFGF BSA CAM CLS CO2 Col CPX C-TAD DA DAB DAPI DFO DLAV DMEM DMOG DTT EBM-2 EGM-2MV EGFP ELISA EPO FBS FIH G6PD Gly HDZ HIF -1 HRP HUVEC HVSMC IGF -1 ISV MW... pyridine-2,4-dicarboxylate Platelet derived growth factor Prolyl 4-hydroxylase inhibitors Poly-L-lactide Penicillin Streptomycin xii SIV TBS TBST TGF-β VEGF VEGF-R vWF Subintestinal vessels Tris buffered saline Tris buffered saline with tween-20 Transforming growth factor- β Vascular endothelial growth factor Vascular endothelial growth factor receptor von Willebrand Factor xiii ... fluorescent protein Enzyme linked immunosorbent assay Erythropoietin Fetal bovine serum Factor inhibiting HIF -1 glucose –phosphate dehydrogenase Glycine Hydralazine Hypoxia inducible factor -1 Horse radish peroxidase Human umbilical vein endothelial cell human vascular smooth muscle cells Insulin-like growth factor -1 Intersegmental vessel Molecular weight Nitrogen N-terminal transactivation domain Oxygen... densitometric analysis Figure 35 PDCA and HDZ inhibited collagen biosynthesis in zebrafish embryos SDSPAGE and densitometric analysis Figure 36 Admixed co-cultures of fibroblasts and HUVEC with or without ascorbate Figure 37 Confocal microscopy (z-stack) of CPX and VEGF treated admixed co-culture of fibroblasts and HUVEC in PLLA scaffolds 20x magnification Figure 38 Confocal microscopy (z-stack) of 8... fibroblast growth factor bovine serum albumin Chorioallantoic membrane Capillary like structures Carbon dioxide Collagen Ciclopirox olamine C-terminal transactivation domain Dorsal aorta Diaminobenzidine 4’6-diamidino-2-phenylindole, dihydrochloride desferrioxamine or deferioxamine dorsal longitudinal anastomotic vessel Dulbecco's Modified Eagle's Medium dimethyloxalyglycine dithiothreitol Endothelial basal . vi List of Figures ix List of Tables xi List of Abbreviations xii 1. LITERATURE REVIEW 1 1. 1 Neovascularization in Tissue Engineering 1 1. 2 HIF -1 : An Alternative Approach to Neovascularization. Stabilizing HIF -1 9 1. 8 In Vitro Angiogenesis Assays 12 1. 9 In Vivo Angiogenesis Models 14 1. 10 Fibrosis in Tissue Engineering – A Result of Foreign Body Reaction and Wound Healing 19 1. 11 Collagen. in Tissue Engineering: Pre-vascularizing PLLA Scaffolds 10 1 4.9 Advantages of Using PHi in Tissue Engineering 10 5 5. CONCLUSIONS 10 8 6. REFERENCES 11 1 vi SUMMARY (ABSTRACT) Neovascularization