FREE ELECTRONIC LIBRARY - Dissertations, online materials

Pages:   || 2 | 3 | 4 | 5 |   ...   | 12 |

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

-- [ Page 1 ] --




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





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



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

–  –  –

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.

–  –  –






1.1.1 Angiogenesis

1.1.2 Vasculogenesis

1.1.3 Tumor Neovascularization


1.2.1 Discovery

1.2.2 Location

1.2.3 Isolation and Characterization

1.2.4 Mobilization



1.4.1 Identification

1.4.2 Structure and Isoforms

1.4.3 Expression

–  –  –

1.4.5 Anticancer Activity







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

–  –  –

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





3.3.1 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

–  –  –


3.4.1 VEGI inhibits tumor growth and vascularization

–  –  –

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




4.1.1 VEGI inhibits differentiation of BM-derived EPC

4.1.2 VEGI inhibits BM-derived EPC functionality

–  –  –


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



–  –  –

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.

–  –  –

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.

–  –  –

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.

–  –  –

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.

–  –  –

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.

–  –  –

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

–  –  –

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].

Pages:   || 2 | 3 | 4 | 5 |   ...   | 12 |

Similar works:

«SEXUAL OFFENDER TREATMENT: A PARADIGM ANALYSIS OF ACADEMIC JOURNALS by VIJAY F. CHILLAR A thesis submitted in partial fulfillment of the requirements for the Honors in the Major Program in Criminal Justice in the College of Health and Public Affairs and in The Burnett Honors College at the University of Central Florida Orlando, Florida Spring Term 2014 Thesis Chair: Dr. Roberto Potter ABSTRACT Many criminologists and psychologists have theorized the possible causes behind an individual who...»

«ACT Government Gazette Gazetted Notices for the week beginning 19 February 2015 Published by Shared Services | 26 February 2015 | © Australian Capital Territory, Canberra, 2015 ACT Government Gazette | 26 February 2015 EXECUTIVE NOTICES Capital Metro Engagement Stephen Anderson – Director, Planning and Design (E745) Section 72 of the Public Sector Management Act 1994 Melanie Taylor – Director, Communications and Stakeholder Engagement (E733) Section 72 of the Public Sector Management Act...»

«9. Infections and sports Authors Göran Friman, MD, PhD, Professor, Department of Infectious Diseases, Uppsala University Hospital, Uppsala, Sweden Lars Wesslén, MD, Department of Infectious Diseases, Gävle Hospital, Gävle, Sweden Ola Rønsen, MD, Chief Medical Officer for Norway’s Olympic Team, Olympiatoppen, Oslo, Norway Summary In general, physical activity in association with infections is accompanied by certain medical risks, in part for the infected individual, in part for other...»

«Guideline for Managing Occupational Health and Safety in an Influenza Pandemic The aim of the guideline is to provide guidance and information from an Occupational Health and Safety perspective to the University community in the event of a pandemic. This Guideline links to the UQ Crisis Management Plan which aims to protect and support students, staff and the general public in crisis situations http://www.uq.edu.au/ohs/oh-s-policies-procedures-guidelines. and to the Managing OHS in an Influenza...»

«The Determinants of Health Expenditure: A Country-Level Panel Data Analysis by Xu Kea, Priyanka Saksenaa and Alberto Hollyb,c a. World Health Organization, Geneva, Switzerland b. University of Lausanne, Geneva, Switzerland c. Nova University, Lisbon, Portugal December 2011 Abstract The rapid growth of health expenditure has become a great concern for both households and governments. There is extensive literature on the determinants of health expenditure in OECD countries, but the same is not...»

«Handling Frozen/ Thawed Meat and Prey United States Department of Agriculture Items Fed to Captive Agricultural Research Exotic Animals Service National Agricultural A Manual of Standard Library Animal and Operating Procedures Plant Health Inspection Service May 2001 United States Handling Frozen/ Department of Agriculture Thawed Meat and Prey Agricultural Research Service Items Fed to Captive National Agricultural Exotic Animals Library Animal and Plant Health A Manual of Standard Inspection...»

«Barking and Dagenham, Havering and Redbridge System Resilience Group Barking and Dagenham CCG | Havering CCG | Redbridge CCG | Barking, Havering and Redbridge University Hospitals NHS Trust | NELFT | London Borough of Barking and Dagenham | London Borough of Havering | London Borough of Redbridge | Together First | Havering Health | Healthbridge Direct | Partnership of East London Cooperatives | London Ambulance Service | NHS England area team | Healthwatch | Local Pharmaceutical Committee...»

«JPET Fast Forward. Published on March 24, 2015 as DOI: 10.1124/jpet.114.222414 This article has not been copyedited and formatted. The final version may differ from this version. JPET #222414 The Journal of Pharmacology and Experimental Therapeutics Donepezil reverses intermittent stress-induced generalized chronic pain syndrome in mice Takehiro Mukae, Hitoshi Uchida, Hiroshi Ueda Downloaded from jpet.aspetjournals.org at ASPET Journals on November 15, 2016 Department of Pharmacology and...»

«Solo System ® User Manual Form 94006 May 29, 2002 / ISO Reference 7.2.2 DEVICE Congratulations! You are now the owner of a Sunlighten sauna! Are you ready to experience ultimate relaxation and rejuvenation? Just follow our assembly and usage guidelines and you will soon be in Sunlighten heaven!DISCLAIMER: The Solo System® is a Personal Sauna. Any information presented within this manual is for educational or reference purposes only. The content of this manual is not intended for diagnosis or...»

«Health, Safety and Reclamation Code for Mines in British Columbia Ministry of Energy, Mines and Petroleum Resources Mining and Minerals Division Victoria, British Columbia 2008 Canadian Cataloguing in Publication Data Main entry under title: Select Standing Health, safety and reclamation code for mines in British Columbia 2008 reprinting of 2003 ed. Includes Index. ISBN 978-0-7726-6011-4 1. Mine safety — Law and legislation — British Columbia. 2. Mineral industries — Health aspects —...»

«Michigan Ear Institute Head Noise or Tinnitus www.michiganear.com Jack M. Kartush, MD DOCTORS Dennis I. Bojrab, MD Michael J. LaRouere, MD John J. Zappia, MD, FACS Eric W. Sargent, MD, FACS Seilesh C. Babu, MD Eleanor Y. Chan, MD LOCATIONS Providence Medical Building 30055 Northwestern Highway Suite 101 Farmington Hills, MI 48334 Beaumont Medical Building 3535 W. Thirteen Mile Road Suite 444 Royal Oak, MI 48073 Oakwood Medical Building 18181 Oakwood Blvd. Suite 202 Dearborn, MI 48126 Providence...»

«Preventing and Minimising Gambling Harm Six-year strategic plan 2010/11–2015/16 i Preventing and Minimising Gambling Harm: Six-year strategic plan 2010/11–2015/16 Citation: Ministry of Health. 2010. Preventing and Minimising Gambling Harm: Six-year strategic plan 2010/11–2015/16. Wellington: Ministry of Health. Published in May 2010 by the Ministry of Health PO Box 5013, Wellington 6145, New Zealand ISBN: 978-0-478-35963-3 (Print) ISBN: 978-0-478-35964-0 (Online) HP 5108 This document is...»

<<  HOME   |    CONTACTS
2016 www.dissertation.xlibx.info - Dissertations, online materials

Materials of this site are available for review, all rights belong to their respective owners.
If you do not agree with the fact that your material is placed on this site, please, email us, we will within 1-2 business days delete him.