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«BY VASCULAR ENDOTHELIAL GROWTH INHIBITOR (VEGI) by Paulina H. Liang Bachelor of Science, University of Pittsburgh 2003 Submitted to the Graduate ...»

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MODULATION OF BONE MARROW-DERIVED ENDOTHELIAL PROGENITOR

CELLS BY VASCULAR ENDOTHELIAL GROWTH INHIBITOR (VEGI)

by

Paulina H. Liang

Bachelor of Science, University of Pittsburgh 2003

Submitted to the Graduate Faculty of

The School of Medicine in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy

University of Pittsburgh

2010

i

UNIVERSITY OF PITTSBURGH

SCHOOL OF MEDICINE

This dissertation was presented by Paulina H. Liang It was defended on December 2, 2010 and approved by Shi-Yuan Cheng, PhD, Department of Pathology Tao Cheng, MD, Department of Radiation Oncology Beth R. Pflug, PhD, Department of Urology Committee Chair: Donna B. Stolz, PhD, Department of Pathology Dissertation Advisor: Lu-Yuan Li, PhD, Department of Pathology ii

MODULATION OF BONE MARROW-DERIVED ENDOTHELIAL PROGENITOR

CELLS BY VASUCLAR ENDOTHELIAL GROWTH INHIBITOR (VEGI)

Paulina H. Liang, PhD University of Pittsburgh, 2010 Bone marrow (BM)-derived endothelial progenitor cells (EPCs) have a critical role in tumor vasculogenesis, mobilizing to tumors and supporting de novo formation of blood vessels essential for tumor growth and metastasis. Vascular endothelial growth inhibitor (VEGI; TL1A) is a member of the tumor necrosis superfamily (TNFSF15) and is produced predominantly by endothelial cells (ECs). VEGI has been shown to act in an autocrine manner by specifically targeting ECs to inhibit their proliferation and induce apoptosis, resulting in elimination of ECs in established tumor vasculature and inhibition of angiogenesis. However, it remains unclear whether VEGI exerts its function solely on fully differentiated ECs or if it is able to modulate BM-derived EPCs as well. Here, the effect of recombinant VEGI on BM-derived EPC function is evaluated in an effort to establish the potential therapeutic value of VEGI. We found that VEGI inhibits the differentiation of EPCs from murine BM under EC stimulating culture conditions. Consistently, VEGI treatment decreases the capability of the cells to adhere, migrate and form capillary-like structures necessary for vascular formation. Additionally, differentiated BM-derived EPCs in cultures underwent apoptosis in response to VEGI treatment. To investigate the impact of VEGI on BM-derived EPC-supported tumor vasculogenesis, mice bearing Lewis lung carcinoma (LLC) tumors were treated with intraperitoneal injection of recombinant VEGI. VEGI treatment significantly decreased the population of BM-derived EPCs found in the tumors while increasing their population in the bone marrow. Furthermore, an

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treatment. Our results indicate VEGI prevents incorporation of BM-derived EPCs into LLC tumors, resulting in the inhibition of EPC-supported tumor vasculogenesis and tumor growth.

Together, these findings suggest that VEGI takes part in the modulation of tumor vasculogenesis by inhibiting BM-derived EPC differentiation and mobilization as well as inducing apoptosis.

These studies yield important insights into the function of VEGI in postnatal vasculogenesis, helping to facilitate the development of therapeutic uses of VEGI in cancer.

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TABLE OF CONTENTS

LIST OF FIGURES

PREFACE

1.0 INTRODUCTION

1.1 NEOVASCULARIZATION

1.1.1 Angiogenesis

1.1.2 Vasculogenesis

1.1.3 Tumor Neovascularization

1.2 ENDOTHELIAL PROGENITOR CELLS

1.2.1 Discovery

1.2.2 Location

1.2.3 Isolation and Characterization

1.2.4 Mobilization

1.3 ROLE OF EPC IN TUMOR VASCULOGENESIS

1.4 VASCULAR ENDOTHELIAL GROWTH INHIBITOR

1.4.1 Identification

1.4.2 Structure and Isoforms

1.4.3 Expression

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1.4.5 Anticancer Activity

1.5 PURPOSE

2.0 VEGI INHIBITS BM-DERIVED EPC DIFFERENTIATION AND FUNCTION IN

VIVO

2.1 ABSTRACT

2.2 INTRODUCTION

2.3 MATERIALS AND METHODS

2.3.1 Antibodies and reagents

2.3.2 Mice

2.3.3 Cell Preparation

2.3.4 Flow cytometry

2.3.5 Cell adhesion assay

2.3.6 Cell migration assay

2.3.7 Tube formation assay

2.3.8 Cellomics Array Scan

2.3.9 Cell proliferation and viability assay

2.3.10 Cell apoptosis assay

2.3.11 Caspase-3 activity assay

2.3.12 Immunofluorescence staining

2.3.13 Western blot analysis

2.3.14 Statistical analysis

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2.4.1 Characterization of mouse BM-derived EPC

2.4.2 VEGI inhibits EC marker expression on BM-derived EPC

2.4.3 VEGI inhibits EPC migration and formation of capillary-like structures..

2.4.4 VEGI inhibits EPC from adhering to fibronectin and vitronectin........... 49 2.4.5 Effect of VEGI on EPC viability and apoptosis

2.4.6 VEGI activates caspase-3 in late stage EPC

2.4.7 VEGI inhibits Akt, activates Erk and p38 in early stage EPC.................. 59 2.5 CONCLUSIONS





3.0 VEGI INHIBITS BM-DERIVED EPC SUPPORTED TUMOR VASCULOGENESIS

3.1 ABSTRACT

3.2 INTRODUCTION

3.3 MATERIALS AND METHODS

3.3.1 Antibodies and reagents

3.3.2 Cells

3.3.3 Mice

3.3.4 Bone marrow transplantation and engraftment analysis

3.3.5 Tumor inoculation and VEGI administration

3.3.6 Flow cytometry

3.3.7 Immunohistochemistry

3.3.8 Detection of apoptotic cells

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3.4 RESULTS

3.4.1 VEGI inhibits tumor growth and vascularization

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3.4.3 VEGI inhibits BM-derived EPC incorporation into LLC tumors............ 76 3.4.4 VEGI induces apoptosis of BM-derived cells

3.5 CONCLUSIONS

4.0 DISCUSSION

4.1 MODULATION OF BM-DERIVED EPC IN VITRO

4.1.1 VEGI inhibits differentiation of BM-derived EPC

4.1.2 VEGI inhibits BM-derived EPC functionality

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4.3 MODULATION OF BM-DERIVED EPC IN VIVO

4.3.1 BM-derived EPC-supported vasculogenesis

4.3.2 VEGI inhibits BM-derived EPC in circulation

4.3.3 VEGI inhibits BM-derived EPC in tumors

4.4 CONCLUDING REMARKS

BIBLIOGRAPHY

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Figure 1. Schematic representation of angiogenesis and vasculogenesis.

Figure 2. The progression of a tumor to a malignant phenotype.

Figure 3. EPCs are involved in blood vessel repair and tumor angiogenesis.

Figure 4. Gene structure of human VEGI and proposed generation of isoforms.

Figure 5. Morphology changes of BM-derived EPCs in culture.

Figure 6. Expression pattern of BM-derived EPC surface markers

Figure 7. VEGI inhibits differentiation of BM-derived EPC in culture.

Figure 8. VEGI treatment decreases expression of EC markers.

Figure 9. Analysis of fluorescence intensity of EPC markers.

Figure 10. VEGI inhibits BM-derived EPC migration.

Figure 11. VEGI inhibits capillary-like tube formation

Figure 12. EPC adhesion to extracellular matrix proteins.

Figure 13. VEGI decreases cell adhesion signaling in EPCs

Figure 14. VEGI inhibits integrin expression on BM-derived EPCs

Figure 15. VEGI inhibits BM-derived EPC adhesion in culture.

Figure 16. Relationship between EPC differentiation and apoptosis.

Figure 17. VEGI induces apoptosis of adhered cells.

Figure 18. VEGI downregulates expression of E-selectin in adhered cells.

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Figure 20. BM-derived EPCs express DR3.

Figure 21. VEGI-induced apoptosis mediated by DR3.

Figure 22. Cell signaling changes induced by VEGI treatment in early EPCs

Figure 23. VEGI inhibits the growth of LLC tumors.

Figure 24. VEGI treatment inhibits tumor vascularization.

Figure 25. Effect of VEGI treatment on peripheral blood EPCs.

Figure 26. Effect of VEGI treatment on bone marrow HSCs.

Figure 27. VEGI inhibits EPC incorporation into LLC tumors.

Figure 28. Induction of apoptosis of BM-derived cells by VEGI-treatment.

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First and foremost, I would like to give an enormous thanks to my dissertation advisor, Dr. LuYuan Li, for years of guidance and for helping me develop my potential as a scientist. He has been an outstanding mentor and I am extremely lucky to have had the opportunity to work with him. I also want to thank my committee for their assistance in the development of my research and for collectively mentoring me thought my graduate career. Thank you to my committee chair, Dr. Donna Stolz for sharing her expertise in immunostaining and confocal imaging. I am grateful to Dr. Shi-Yuan Cheng for advising me on experimental procedures and for allowing me to use the cryostat. I would like to thank Dr. Beth Pflug, for her insight into my research and going the extra distance to be a part of my committee. I also want to express my gratitude to Dr.

Tao Cheng, for seeing me as more than a lab technician and persuading me to apply to the graduate program at Pitt.

I want to thank the Li lab, a group of amazing scientists who have not only been extremely helpful collaborators in research, but have been supportive as friends in life.

Particular thanks goes out to Dr. Fang Tian, who helped me every day along the way and was instrumental in the planning and execution of many of the in vitro experiments. I would also like to thank Dr. Yi Lu for her assistance with the in vivo experiments and sparing me from having to pick up squirming mice.

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have struggled and wondered if the end was ever going to be within reach, they are the ones who have never doubted my abilities and potential. I am forever grateful to all the people in my life who have never wavered in their support and encouragement throughout this journey. Thank you to my wonderful husband, Dan, who has traveled this bumpy road with me and never got sick. He has been my rock and the one person who could always make me smile. I cannot begin to describe how much I owe to my biggest supporter, my mom, Lynn. She has believed in me from day one and has sacrificed so much to get me to where I am today. I know I can prevail over any obstacle in life because of her continuous example of courage and strength.

This work was supported in part by grants to L.Y.L. from the National Institutes of Health (Washington, DC; CA113875), Pennsylvania Department of Health (Harrisburg, PA), the Hillman Foundation (Pittsburgh, PA), and the Chinese Ministry of Science and Technology (Beijing, China; 2009CB918900). One chapter of this dissertation contains data from a published manuscript: Tian F, Liang PH, Li LY. “Inhibition of endothelial progenitor cell differentiation by VEGI.” Blood. 2009 May 21;113(21):5352-60. In recognition of their scientific and intellectual contributions, special thanks go out to: Dr. Brain Nolan, Adam Farkas, Richard Demarco, Dr. Hui Yu, Dr. Richard XuFeng, and Mark Ross for their skillful technical assistance.

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Anti-angiogenesis is an important avenue and approach to cancer therapy. The development of anti-angiogenic drugs has focused on endogenous angiogenesis inhibitors that innately modulate endothelial cell (EC) growth. Increasing evidence points to tumor vasculogenesis as a principle mechanism for the support and growth of tumors. Therefore, the discovery of endogenous inhibitors which are able to not only suppress tumor angiogenesis, but tumor vasculogenesis as well, is of high therapeutic value.

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1.1.1 Angiogenesis The vascular endothelium is an essential component of the cardiovascular system and provides a dynamic barrier between circulating blood and surrounding tissues in the body. The endothelium monolayer plays a vital role in regulating vascular homeostasis, coagulation and inflammation, and as such, restoration of the monolayer is essential following damage or cell death [1, 2].

Under normal conditions, the endothelium undergoes a low turnover rate, retaining ECs in a quiescent state in order to maintain vascular homeostasis [3]. However, the number of

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neovascularization, such that occurs following acute stress or injury of the vascular endothelium.

It has been commonly believed that postnatal neovascularization, the vascular growth and remodeling in newborns and adults, is mainly attributed to angiogenesis, a process of capillary sprouting from pre-existing capillary vessels (Figure 1) [4]. The process of angiogenesis can occur during normal physiological conditions such as embryonic development and the female reproductive cycle. Angiogenesis can also be engaged under pathological conditions such as tumor growth, macular degeneration, ischemia and rheumatoid arthritis [1, 5].



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