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

TABLE OF CONTENTS Page No 1.2 HIF-1α: An Alternative Approach to Neovascularization 3 1.7 Prolyl 4-Hydroxylase Inhibitors PHi Promote Angiogenesis by Stabilizing HIF-1α 9 1.10 Fibrosis i

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 do all things

TABLE OF CONTENTS Page No

1.2 HIF-1α: An Alternative Approach to Neovascularization 3

1.7 Prolyl 4-Hydroxylase Inhibitors (PHi) Promote Angiogenesis by

Stabilizing HIF-1α

9

1.10 Fibrosis in Tissue Engineering – A Result of Foreign Body

Reaction and Wound Healing

19

1.12 Inhibition of Collagen Biosynthesis Using Prolyl Hydroxylase

Inhibitors

22

1.13 Selection of Prolyl 4-Hydroxylase Inhibitors 23

2 MATERIALS AND METHODS 24

Preparation of Prolyl Hydroxylase Inhibitorsand Recombinant

Human VEGF

25

Quantification of Angiogenesis by Image J and Metamorph 28

Zebrafish Embryo Collection and Drug Treatment 34

Collagen Analysis of Zebrafish Embryos by Peptic Digest and

SDS-PAGE

35

3.1.1 CD31 and von Willebrand Factor: Markers for Endothelial

Cells

37

3.1.2 Development of Sequential Co-culture for

Pro-angiogenesis

40

3.1.3 PHi Induced Angiogenesis in Sequential Co-cultures 49 3.1.4 Quantification of Co-culture Angiogenesis 52

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

3.4.1 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

3.6.1 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

4 DISCUSSION 89

4.3 Quantification of Sequential Co-culture Angiogenesis 92

4.4 Fibroblasts Are Required For HUVEC Formation of Capillary

Like Structures

93

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

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

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 7 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

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

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 1 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 induced HIF-1α stabilization in nuclei of fibroblasts (Immunofluorescence)

Figure 17 Hypoxia induced HIF-1α stabilization in nuclei of fibroblasts (HRP based immunohistochemistry)

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 HIF-1α expression in fibroblasts and HUVEC (immunofluorescence)

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

Figure 34 400µM HDZ inhibited fibroblast collagen production SDS-PAGE and densitometric analysis

Figure 35 PDCA and HDZ inhibited collagen biosynthesis in zebrafish embryos SDS-PAGE 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 µM CPX treated admixed co-cultures in PLLA scaffolds 10x 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

LIST OF ABBREVIATIONS

Ascorbate Asc

AF Alexa Fluor (fluorescent dyes)

bFGF Basic fibroblast growth factor

BSA bovine serum albumin

CAM Chorioallantoic membrane

CLS Capillary like structures

CO2 Carbon dioxide

CPX Ciclopirox olamine

C-TAD C-terminal transactivation domain

DAB Diaminobenzidine

DAPI 4’6-diamidino-2-phenylindole, dihydrochloride

DFO desferrioxamine or deferioxamine

DLAV dorsal longitudinal anastomotic vessel

DMEM Dulbecco's Modified Eagle's Medium

DMOG dimethyloxalyglycine

DTT dithiothreitol

EBM-2 Endothelial basal medium

EGM-2MV Endothelial growth medium

EGFP Enhanced green fluorescent protein

ELISA Enzyme linked immunosorbent assay

EPO Erythropoietin

FBS Fetal bovine serum

FIH Factor inhibiting HIF-1

G6PD glucose –phosphate dehydrogenase

HDZ Hydralazine

HIF-1 Hypoxia inducible factor-1

HRP Horse radish peroxidase

HUVEC Human umbilical vein endothelial cell

HVSMC human vascular smooth muscle cells

IGF-1 Insulin-like growth factor-1

ISV Intersegmental vessel

MW Molecular weight

N-TAD N-terminal transactivation domain

ODD Oxygen dependent domain

PBS Phosphate buffered saline

PDCA Pyridine-2,4-dicarboxylic acid or pyridine-2,4-dicarboxylate

PDGF Platelet derived growth factor

PHi Prolyl 4-hydroxylase inhibitors

PLLA Poly-L-lactide

SIV Subintestinal vessels

TBS Tris buffered saline

TBST Tris buffered saline with tween-20

TGF-β Transforming growth factor- β

VEGF Vascular endothelial growth factor

VEGF-R Vascular endothelial growth factor receptor

vWF von Willebrand Factor