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The role of tumor-associated and normal human brain endothelial cells in angiogenesis

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The role of tumor-associated and normal human brain endothelial cells in angiogenesis

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Content THE ROLE OF TUMOR-ASSOCIATED AND NORMAL HUMAN BRAIN ENDOTHELIAL CELLS IN ANGIOGENESIS by Christiana Charalambous A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (MOLECULAR MICROBIOLOGY AND IMMUNOLOGY) December 2005 Copyright 2005 Christiana Charalambous R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. UMI Number: 3220092 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 3220092 Copyright 2006 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. DEDICATION I would like to dedicate this work to my beloved parents, Athos and Anthi Charalambous. They were my constant source of inspiration and encouragement from elementary school until graduate school. It would have been impossible for me to accomplish any of my academic achievements without their love, encouragement and continuous support throughout my studies. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. ACKNOWLEDGEMENTS First of all, I would like to thank and express my deep appreciation to my mentors, Dr Florence Hofman and Dr Thomas Chen, for their constant help and guidance and tremendous support through my whole studies, in finding and developing my project, designing experiments, interpreting results and writing my manuscripts and my dissertation. I also wish to thank my committee members, Dr Axel Schonthal and Dr Joseph Landolph for sacrificing their valuable time to give me valuable suggestions and help for this project by reviewing my manuscripts and this dissertation. In addition, I would like to thank Dr Raphael Zidovetzki for his help in data analysis and manuscript editing. In addition, I would like to thank Dr Dixon Gray and Mr. Hal Soucier for their precious help in flow cytometry analysis. I would also like to thank Adel Kardosh and Lubna Qazi-Abdullah for assisting me with several experiments of my project. Furthermore, I would like to express my great appreciation to Ligaya Pen and Susan Su, for isolating the normal human brain endothelial cells and glioblastoma-associated endothelial cells used in this project, respectively. I would also like to thank Dr Schonthal for providing us with most of the cyclin antibodies used in this study. Special thanks also go to Dezheng Dong for his help with the P-galactosidase assay and Dr Randall Widelitz and the staff in Doheny Core Facility for their help with photography. Finally, I wish to thank the rest of the students and staff in Dr Hofman’s, Dr Chen’s and Dr Schonthal’s laboratories, Mark Jabbour, Vinay Gupta, Wei-Jun Wang, Jennilyn Virrey, Peter Pyrko, Nathaniel Soriano, Christian Samuelson and Encouse Golden. iii R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. TABLE OF CONTENTS Dedication ii Acknowledgements iii List of Tables vi List of Figures vii Abbreviations ix Abstract x Chapter I: Introduction 1 1.1. Preface 2 1.2. Glioblastoma multiforme 2 1.3. Angiogenesis 6 1.4. Regulation of angiogenesis 9 1.5. Comparison of normal and tumor endothelium 20 1.6. Purpose of study 26 Chapter II: Functional and phenotypic differences between tumor -associated and normal human brain endothelial cells 28 2.1. Introduction 29 2.2. Materials and Methods 31 2.3. Results 37 2.4. Discussion 51 Chapter III: Interleukin-8 (IL-8) differentially regulates migration of tumor-associated and normal human endothelial cells 56 3.1. Introduction 57 3.2. Materials and Methods 60 3.3. Results 68 3.4. Discussion 83 Chapter IV: Senescent phenotype of tumor-associated endothelial cells 88 4.1. Introduction 89 4.2. Materials and Methods 92 4.3. Results 97 4.4. Discussion 110 iv R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Chapter V: Conclusions and Prospects 117 5.1. Conclusions 118 5.2. Prospects and Future directions 123 References 127 v R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. LIST OF TABLES Table 1-1: CNS vascular and perivascular cell markers Table 2-1: Summary of endothelial cell markers expression on BEC and TuBEC as determined by immunohistochemistry Table 4-1: Cell cycle analysis of BEC and TuBEC R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. LIST OF FIGURES Figure 1-1: Astrocytoma progression to glioblastoma multiforme 3 Figure 1-2: The angiogenesis cascade 8 Figure 1-3: Schematic overview of tumor cell -blood vessel interactions and signaling pathways involved in growth factor induced stimulation of angiogenesis 15 Figure 1-4: Blood vessel structure 22 Figure 1-5: Differentiation cascade of endothelial cells 24 Figure 2-1: Comparison of BEC and TuBEC morphology and detection of Factor VIII, GFAP and CD1 lb expression in TuBEC 38 Figure 2-2: Differential expression of specific markers in TuBEC compared to BEC 40 Figure 2-3: Comparison of BEC and TuBEC proliferation 41 Figure 2-4: Comparison of BEC and TuBEC apoptosis 44 Figure 2-5: Comparison of BEC and TuBEC migration rate 48 Figure 2-6: Comparison of Et-1 and VEGF production by BEC and TuBEC 49 Figure 3-1: IL-8 effect on BEC and TuBEC migration 69 Figure 3-2: IL-8 receptor expression in BEC and TuBEC 72 Figure 3-3: Comparison of IL-8 production by BEC and TuBEC 75 Figure 3-4: Effect of TGF-pl and VEGF on IL-8 production by TuBEC 78 Figure 3-5: Effect of anti-CXCRl, anti-CXCR2 and anti-IL-8 neutralizing antibodies on BEC and TuBEC migration 81 Figure 4-1: Cell cycle analysis of TuBEC and BEC and cell cycle protein expression 98 Figure 4-2: Effects of growth factors on TuBEC proliferation 100 v ii R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 4-3: Immunocytochemical detection of P-galactosidase 102 Figure 4-4: PDGF-A and PDGF-B expression in TuBEC and BEC 103 Figure 4-5: Cytotoxic effects of CPT-11 and temozolomide on TuBEC and BEC 105 Figure 4-6: Effect of glioma supernatant on endothelial cell morphology and P-galactosidase expression 108 Figure 5-1: Proposed hypothetical model of tumor-endothelial cells interactions 123 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. ABBREVIATIONS BEC Brain Endothelial Cells BrdU Bromodeoxy-Uridine EC Endothelial Cells ECGS Endothelial Cells Growth Supplement ELISA Enzyme Linked Immunosorbent Assay Et-1 Endothelin-1 FACS Fluorescence Activated Cell Sorter FCS Fetal Calf Serum FITC Fluorescein Isothiocyanate GBM Glioblastoma Multiforme GFAP Glial Fibrillary Acidic Protein ICAM Intercellular Adhesion Molecule Ig Immunoglobulin IL-8 Interleukin-8 MTT Monotetrazolium PBS Phosphate Buffered Saline SEM Standard Error of Mean SMA Smooth Muscle Actin TuBEC Tumor-associated Brain Endothelial cells TUNEL Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling VEGF Vascular Endothelial Growth Factor vWF von Willebrand Factor R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. ABSTRACT Glioblastoma multiforme are highly vascular brain tumors characterized by abnormal vascular structures in vivo. This suggests that tumor-associated brain endothelial cells (TuBEC) may have intrinsically different properties than normal human brain endothelial cells (BEC). Therefore, the aim of this investigation was to identify the functional and phenotypic characteristics of these glioblastoma-associated endothelial cells in order to use these cells as a potential target for anti-angiogenic therapy. Our data demonstrate that TuBEC differ phenotypically and functionally from BEC. TuBEC display a large, flat, and veil-like appearance, in contrast to BEC, which are small and plump cells. In addition, TuBEC proliferate slower and undergo less apoptosis than BEC. However, TuBEC migrate faster than BEC and do not respond to growth factor stimulation. TuBEC constitutively produce higher levels of growth factors such as Et-1, IL-8, PDGF and VEGF, compared to BEC, which may act in an autocrine manner to induce their activation, independent of any exogenous stimulation. Our data demonstrate that production of IL-8 by the TuBEC is the result of VEGF and TGF-pi stimulation. Further examination of TuBEC demonstrated that these cells have properties of senescent cells. TuBEC cultures exhibit senescence-associated p-galactosidase staining, cell cycle arrest at Go/Gi, and increased levels of the cell cycle inhibitors p21 and p27, compared to BEC. TuBEC also demonstrate increased expression of the tumor suppressor protein p53 and resistance to cytotoxic drugs. Since TuBEC are resistant to x R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. conventional chemotherapy, our results are important for the development of novel anti- angiogenic agents specifically targeting tumor-associated endothelial cells. This work is especially significant because it helps us understand the physiological properties of TuBEC, and some of the basic mechanisms responsible for their altered functions. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. CHAPTER I INTRODUCTION R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 1.1. PREFACE The work presented in this study focuses on the differences between normal and glioblastoma-associated endothelial cells and their role in angiogenesis. Glioblastoma multiforme is a hypervascular tumor characterized by extensive angiogenesis. The current treatments for glioblastoma have poor results, therefore, anti-angiogenic therapy, that targets endothelial cells, is considered as an alternative therapeutic approach. Since endothelial cells participate in angiogenesis, the formation of new blood vessels, it is essential to characterize and understand the differences of tumor- associated endothelial cells in comparison to normal endothelial cells. Angiogenesis is tightly regulated by several growth factors that act on endothelial cells, such as IL-8, Et- 1, and VEGF. Therefore, the production of these growth factors by endothelial cells and their effects on both normal and tumor-associated endothelial cells, are of great interest. 1.2. GLIOBLASTOMA MULTIFORME 1.2.1. Astrocytomas Gliomas are tumors of glial cells such as astrocytes and oligodendrocytes. However, the most common gliomas are astrocytomas (Mentlein and Held-Feindt, 2000). Astrocytomas are divided by WHO (World Health Organization) into four grades of malignancy, as shown in Figure 1-1. Glioblastoma multiforme (GBM) is the most malignant, poorly differentiated, and highly vascularized brain tumor. It is highly angiogenic, and consists of areas of active proliferation and areas of necrosis in the less angiogenic regions. Because of their highly malignant phenotype, glioblastomas cause 2 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. patients to have a poor prognosis with an average survival time of 5-12 months after diagnosis. Glioblastomas serve as a model for studying tumor angiogenesis and anti- angiogenic therapies due to their highly vascularized nature. P rogfisssion Biology Censtfe events Angiogenic events Normal Brain ' ‘~d r ?si&£k j i s M j S M U-XlSt Infiltrating Astrocytoma (WHO grade II) Infiltration pS l tats V IG F T Anaplasttc Astrocytoma (WHO grade H I) Proliferation/expansion Glioblastoma Mtslliforme (WHO grade IV) Hypoxia/necrosis p1«, p14A«F lots EGFR amplification PDGFR amplification chromosome 10 losses (PTBN) PDGFR T Angiogenesis VEGF f t Microvascular hyperplasia Figure 1-1. Astrocytoma progression to glioblastoma multiforme. In grade II (infiltrating astrocytoma) single tumor cells infiltrate the CNS parenchyma. At grade III (anaplastic astrocytoma) more tumor cells are present characterized by mitosis and atypia. At grade IV (glioblastoma multiforme) tumor cells aggregate surrounding a necrotic area (black arrows). Microvascular proliferation is present (white arrow). The events that take place during progression to glioblastoma are shown below the figure (Figure adapted from Brat et al., 2003). 3 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 1.2.2. Glioblastoma Treatment: Conventional Vs Anti-Angiogenic Therapy Currently, surgery, radiotherapy, and chemotherapy, which are used as the standard therapies for anaplastic astrocytoma and glioblastoma multiforme, have limited efficacy (Mentlein and Held-Feindt, 2003; Hofer and Herman, 2000). A common problem associated with surgery is the infiltrative nature of glioblastoma, which causes a high degree of recurrence. Chemotherapy is often used in combination with radiotherapy. However, chemotherapy also presents significant problems, such as limited drug delivery through the blood brain barrier and chemoresistance of the tumor. Current chemotherapies used for glioblastomas include temozolomide, procarbazine and BCNU (bis-chloroethyl-nitrosourea) as the first line of treatment and irinotecan (CPT-11) and etoposide as secondary agents (Engelhard, 2000; Hofer and Herman, 2000). Other drugs occasionally used to treat glioblastoma are cytostatic agents such as tamoxifen, paclitaxel (taxol), celecoxib and thalidomide (Parney and Chang, 2003). Most of the drugs used in glioma chemotherapy, such as temozolomide and BCNU, are cytotoxic alkylating agents that inhibit DNA repair in the tumor cells and therefore they also interfere with DNA replication causing cell cycle arrest and cell death. More specifically, temozolomide and CPT-11 block DNA replication by inhibiting FEN-1 (flap endonuclease) and topoisomerase I respectively, important enzymes involved in DNA repair (Alimonti et al., 2004). Taxol, on the other hand, inhibits microtubules, causing cytoskeletal dysfunction and inhibition of the mitotic spindle in cells (Wilson and Jordan, 2004). All these drugs result in inhibition of DNA replication and therefore cell death. 4 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Since gliomas are highly vascular tumors, anti-angiogenic therapy is an alternative promising approach. Anti-angiogenic therapy targets endothelial cells thus indirectly inhibiting the blood and nutrient supply to the tumor. Therefore, the characterization of the glioblastoma-associated endothelial cells is extremely important for targeting the tumor-associated blood vessels. Thalidomide is a cytostatic anti-angiogenic agent that has been shown to inhibit tumor growth both in vitro and in vivo and has shown promising results in combination with chemotherapy and radiation (Short et al., 2001; Fine et al., 2000). Thalidomide exerts its anti-angiogenic effects by inhibiting growth factor mediated endothelial cell proliferation and therefore neovascularization. However, the exact mechanism of action has not been determined (Short et al., 2001). Anti-angiogenic therapy has several advantages in comparison to conventional chemotherapy. First, there are differences in the proliferation capacity of normal versus tumor-associated endothelial cells, which can be taken advantage of in therapy. Second, endothelial cells are more genetically stable than tumor cells, and therefore more homogeneous. Thus, endothelial cells are easier to target. Furthermore, tumor endothelium expresses specific proteins and antigens that are absent from the normal endothelium. These antigens may serve as targets for specific drugs. Third, endothelial cells are more accessible than tumors to drugs, via the systemic circulation. Fourth, anti-angiogenic agents have reduced toxicity in comparison to cytotoxic agents because they do not affect cell division (Short et al., 2001). Finally, recent data demonstrated that thalidomide combination with dexamethasone or BCNU had synergistic results in multiple myeloma and glioblastoma patients respectively (Weber, 2003; Fine et al., 5 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 2003). Consequently, since tumor cells are depended on endothelial cells for their growth, combination of anti-angiogenic drugs with cytotoxic agents may have synergistic effects because these agents specifically target two distinct cell populations, endothelial cells and tumor cells respectively (Longo et al., 2002). 1.3. ANGIOGENESIS Angiogenesis is the development of new capillaries from pre-existing blood vessels in contrast to vasculogenesis, the development of new blood vessels from endothelial precursor cells. Angiogenesis is a multistep process that involves proliferation, migration and invasion of endothelial cells through basement membranes, and extracellular matrix remodeling (Folkman et al., 1992). Angiogenesis normally occurs during wound healing and embryonic development (Mentlein and Held-Feindt, 2003; Longo et al., 2002). Tumors require oxygen and nutrients to grow; therefore, they cannot grow bigger than 1 mm without efficient blood supply (Folkman, 1990; Cameliet and Jain, 2000). In order to grow, tumors recruit blood vessels via angiogenesis, which occurs in the early stages of tumorigenesis (Folkman and Shing, 1992; Flanahan and Folkman, 1996). Tumor formation results in local hypoxia, which in turn results in the production of several pro-angiogenic factors, such as bFGF, VEGF, IL-8 and Et-1. These factors trigger the angiogenic cascade (Dawas et al., 1999; Bagnato and Spinella, 2003). They growth factors are also produced in response to other conditions such as local stress, low glucose levels and inflammation. However, hypoxia is the most common condition 6 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. in tumors. Under hypoxic conditions, HIF-1 (hypoxia inducible factor-1) is activated and binds to the HRE (hypoxia response element). This results in transcriptional activation of several angiogenic factors, such as VEGF and endothelin-1 (Et-1). Consequently, the angiogenesis cascade is triggered. 1.3.1. The Angiogenesis Cascade Angiogenesis can be divided into four major consecutive phases: the initiation phase, which involves vasodilation and ECM degradation, the extension phases, which involve the endothelial cell proliferation and migration phases, and maturation phase which involves recruitment of perivascular cells to the vessel wall. At phase I (initiation), VEGF, produced due to hypoxia, induces NOS (nitric oxide synthase) activation. This leads to cGMP activation and smooth muscle cell relaxation, thus inducing vasodilation. Angiopoietin-2 (Ang-2), which is upregulated by hypoxia and VEGF, and several proteases such as plasminogen activator, chymases, and matrix metalloproteinases induce the dissociation of smooth muscle cells and pericytes from the blood vessels (Distler et al., 2003). Phase II includes endothelial cell proliferation which is mediated by several growth factors including VEGF, FGF, EGF, IL-8. Phase III involves migration of endothelial cells and invasion through the basement membrane and extracellular matrix. Finally, phase IV (maturation) includes the recruitment of mesenchymal cells into the blood vessels, which in turn differentiate under the influence of angiogenic factors such as PDGF, to mural cells. Mural cells are perivascular cells located at the abluminal side of the blood vessels, such as pericytes 7 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. and smooth muscle cells, that provide structural support to the blood vessel wall. A diagram summarizing the major angiogenesis phases is shown in figure 1-2 (Distler et al., 2003). Figure 1-2. The angiogenesis cascade. For more information refer to the text above (Figure adaptedfrom Distler et al., 2003). 1.3.2. The Migration Cascade Since endothelial cell migration is critical for neovascularization in tumors, this study specifically focused on the regulation brain endothelial cells migration. Cell migration is basically divided in 4 consecutive steps: (a) lamellipodia formation, (b) attachment of lamellipodia to the extracellular matrix, (c) cell contraction and (d) recycling of adhesion and signaling molecules (Rousseau et al., 2000). Several signaling pathways are involved in the initiation of endothelial cell migration. Chemokines usually act via their receptors to induce signaling through the MAPK/p38 pathway (Rousseau et al., 1997) or through focal adhesion kinase (FAK) (Rousseau et R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Vasodilatation, permeability t Vessel TGF-fl PDGF Augl/Tie2 Pr oliferation and differentiation ofM C intoPC and SMC Angiogenesis Detachment of SMC, ECM degradation Ang2/Tk2 TGFfl MMPs VEGFs PGFs EC migration, tune lorraation EC proliferation 8 al, 2000), which can in turn activate Akt, a protein kinase (also known as protein kinase B and Rac), leading to actin polymerization. FAK participates in the assembly of focal adhesions which are required for the formation of stress fibers (Aplin et al., 1998). Other molecules that participate are Rho GTPases such as RhoA which also induces the formation of stress fibers and cdc42/Rac, which induce the formation of lamellipodia and membrane ruffles (Chrzanowska-Wondnicka and Burridge, 1996). More specifically, activation of RhoA leads to myosin light chain phosphorylation, which in turn generates tension in the actin filaments, thus inducing the formation of stress fibers (Chrzanowska-Wondnicka and Burridge, 1996). Actin polymerization, in turn, results in the formation of lamellipodiae and stress fibers, which are required for migration (Lavoie et al., 1993; Rousseau et al., 1997). 1.4. REGULATION OF ANGIOGENESIS Angiogenesis is regulated by the balance between pro-angiogenic and anti- angiogenic growth factors. The major angiogenic factors are VEGF, IL-8, Et-1, bFGF, PDGF, EGF, TNF-a and TGF-P, whereas the major anti-angiogenic factors present in tumors are angiostatin, endostatin, thrombospondin and somatostatin (O’ Reilly et al., 1994). 1.4.1. IL-8 Interleukin -8 (IL-8) is a member of the CXC family of chemokines which have a CXC motif comprised of two highly conserved cysteine residues separated by a non- 9 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. conserved amino acid residue (Belperio et al., 2000). IL-8 exists in two isoforms, the 77 amino acid isoform, which is derived from endothelial cells and fibroblasts, and the 72 amino acid isoform, which is derived from macrophages and monocyte. IL-8 is produced during hypoxia in cancer by macrophages and endothelial cells (Van Meir et al., 1992). The production of IL-8 is induced by several stimuli, such as LPS, IL-1, TNF-a, Et-1, and stress conditions, such as hypoxia. On the other hand, factors such as IL-4, IL-10 and TGF-P downregulate IL-8 production in human umbilical vein endothelial cells (HUVEC) (Pirtskhalaishvili and Nelson, 2000). In contrast, previous data from our laboratory have demonstrated that in human brain endothelial cells, although TGF-p 1 is involved in downregulation of IL-8 production, IL-10 has no effect (Hofman et al., 1998). IL-8 has been shown to promote endothelial cell migration, proliferation, survival, tubule formation (endothelial structures similar to the internal wall of capillaries) and matrix metalloproteinases (MMP) production (Koch et al., 1992; Heidemman et al, 2003; Li et al, 2002) as well as tumor associated angiogenesis (Belperio et al., 2000). IL-8 also upregulates beta-2 integrin expression on leukocytes, thus enhancing their trans- endothelial migration (Huber et al., 1991). However, there is little information known on the role of IL-8 in the regulation of endothelial cells integrins. IL-8 appears to be overexpressed in many tumors such as melanoma and prostate cancer (Pirtskhalaishvili and Nelson, 2000). Glioblastoma cells, have actually been shown to produce IL-8 themselves (Tada et al, 1993; Van Meir et al, 1992) which may be acting in both an autocrine and paracrine manner to stimulate their growth. The 10 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. reason for the increased expression of IL-8 in tumors is usually the local hypoxia in the tumor microenvironment (Takeda, 2002). However, other conditions, such as shear stress, can also induce IL-8 production. IL-8 expression is associated with increased metastatic potential of tumors. In addition to IL-8, the IL-8 receptors CXCR1 and CXCR2 are also upregulated in tumors such as colon carcinoma (Takeda, 2002). IL-8 is a critical factor in glioma angiogenesis. It has been shown that in gliomas two major pathways control angiogenesis, one mediated by VEGF/FGF and one mediated by IL-8 (Wakabayashi et al., 1995). VEGF and FGF act via tyrosine kinase receptors activating the MAPK cascade whereas IL-8 acts via G-protein coupled receptors activating the Rho kinase and protein kinase B (PKB) pathways. In addition, it has been previously shown that two other major angiogenic factors, endothelin-1 (Et-1) and VEGF, stimulate production of IL-8 in human brain endothelial cells (Hofman et al., 1998; Lee et al., 2002). However, there is little information known regarding the production and role of IL-8 in regulation of tumor endothelial cells. Therefore, since IL-8 is a major factor in glioma angiogenesis, our studies focused on the production and regulation of tumor-associated and normal endothelial cells by IL-8. IL-8 acts on endothelial cells via two types of receptors, CXCR1 (IL8-RA) and CXCR2 (IL8-RB), causing cytoskeletal rearrangement resulting in chemotaxis (Schraufstatter et al., 2001). However, not all the types of endothelial cells express both types of IL-8 receptors. For example, human intestinal microvascular endothelial cells express only CXCR2, whereas human lung and dermal microvascular endothelial cells and HUVEC express both receptors. Both receptors belong in the family of the 7- 11 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. transmembrane domain G-coupled receptors. CXCR1 is specific for IL-8 whereas CXCR2 is promiscuous, binding to several other peptides such as GRO, NAP-2 and GCP-2 (Schraufstatter et al., 2001). Peptide binding and activation of CXCR1 leads to activation of Rho/Rho kinase pathway, which in turn leads to PLD (phospholipase D) activation, whereas activation of CXCR2 leads to Rac activation which in turn activates PAK (Schraufstatter et al., 2001; Feniger-Barish, 2003). The activation of the Rac and Rho pathways activation results in NF-kB activation in the nucleus and transcriptional activation of several genes including the IL-8 gene (Schraufstatter et al., 2001), which contains a binding site for NF-kB (Brat et al., 2005). CXCR1 rapidly responds to high IL-8 concentrations, upon cell activation, whereas CXCR2 responds to the circulating low IL-8 concentration in neutrophils (Hauser et al., 1999). The receptors autoregulate their expression, with CXCR1 stimulation leading to a decrease in CXCR2 expression, whereas CXCR2 stimulation leads to increased expression of CXCR1 (Hauser et al., 1999). Engagement of CXCR1 and CXCR2 also leads to FAK (focal adhesion kinase) activation and thus initiation of the migration cascade (Feniger-Barish et al., 2003). 1.4.2. Endothelin-1 Endothelin-1 (Et-1) is a 21 amino acid peptide primarily produced by endothelial and vascular smooth muscle cells (Levin, 1996). Et-1 acts on the micro vessel endothelial cells via two types of G protein-coupled, seven transmembrane domain receptors, the EtA and Et b receptors (Rubanji and Polokoff, 1994). Binding of Et-1 to these receptors results in stimulation of the proliferation and migration of several types 12 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. of endothelial cells, such as HUVEC. One mechanism by which Et-1 promotes angiogenesis is indirectly via the induction of the expression other angiogenic factors, such as VEGF and IL-8 (Pedram et al., 1997; Hofman et al., 1998). Elevated levels of Et-1 are found in several types of cancers, such as ovarian, colorectal, lung, prostate, and breast carcinoma (Rosano et al., 2001; Salani et al., 2000; Dawas et al., 1999). In addition, increased levels of Et-1 receptors are found in several cancers. The EtA receptor has been shown to increase in several cancer types, such as in ovarian carcinoma cells, meningiomas, and the microvessels associated with glioblastomas (Rosano et al., 2001; Egidy et al., 2000). Human glioblastomas express the EtB receptor, whereas the glioblastoma-associated blood vessels express high levels of EtA receptor (Edigy et al., 2000). Therefore, since Et-1 is an important factor in glioma angiogenesis, the production and regulation of tumor and normal endothelial cells by Et-1 was investigated in our studies. 1.4.3. VEGF Another factor produced by tumors during hypoxia is VEGF (Vascular Endothelial Growth factor). VEGF is produced by several cell types, such as macrophages, T-cells, smooth muscle cells, tumor cells, keratinocytes, osteoclasts and kidney cells (Klagsbrun and D’Amore, 1996). To date, six isoforms of VEGF have been shown to be produced by alternative splicing of VEGF mRNA. VEGF121 and VEGF165 are the most abundant isoforms of VEGF (Klagsbrun and D’Amore, 1996). In addition to size, the difference between them is that VEGF121 is a totally secreted form, whereas VEGF165 exists in 13 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. both a secreted and a cell- associated form (Park et al, 1993). VEGF induces endothelial cell proliferation, migration, and survival (Chavakis and Dimmeler, 2002; Neufeld et al., 1999). VEGF165 is the most mitogenic form of VEGF for endothelial cells (Keyt et al., 1996). VEGF exerts its cellular functions by interacting mainly with VEGF-R1 (Fit -1) and VEGF-R2 (Flk-l/KDR) receptors expressed on endothelial cells and enabling tyrosine phosphorylation of several cytosolic proteins, which ultimately signal to the nucleus (Pedram et al., 1997). VEGF acts through the PI3K/Akt and MAPK pathways to mediate its effects on endothelial cells. Both VEGF-R1 and VEGF- R2 are up-regulated in certain primary human tumors (Guo et al., 2001). VEGF expression is induced by hypoxia in tumors (Bagnato and Spinella, 2002) and therefore is upregulated in several tumor types, including glioblastomas (Plate et al., 1992). VEGF is thought to act at the initiation of angiogenesis in immature blood vessels. This is in contrast to PDGF, which is thought to act by promoting vessel maturation by recruitment of pericytes (Jain and Booth, 2003). VEGF, together with bFGF and IL-8 , exerts an important role in glioma angiogenesis (Wakabayashi et al., 1995). However, there is little information known regarding the production and role of VEGF in regulation of tumor endothelial cells. Therefore, the production and regulation of normal and tumor endothelial cells by VEGF were investigated in this study. A comprehensive model of tumor angiogenesis, including the roles of Et-1 and VEGF, is shown in Figure 1-3. 14 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. ET*R antagonist o ABT627 • ET -1 * VEGF Tumor colt Endothelial cell* JSS ffi§'FSX KS SysSwE Ssm Figure 1-3. Schematic overview of tumor cell -blood vessel interactions and signaling pathways involved in growth factor induced stimulation of angiogenesis. Endothelin-1 and VEGF are used as examples of growth factors. Other cytokines can act in a similar manner. Stimulation of HIF-1 leads to the induction of VEGF production by tumor cells. For more details refer to text above (Figure adapted from Spinella and Bagnato, 2003). 1.4.4. TGF-P Besides VEGF, Et-land IL-8, glioma cells also produced several other angiogenic factors, such as transforming growth factor-p (TGF-p). TGF-P is a pleiotropic factor involved in the regulation of cell growth, immunosuppression, and differentiation. Three TGF-P isoforms are known to date (TGF-P 1, TGF-p2, TGF-P3). TGF-p is secreted as a latent molecule which is proteolytically cleaved from the extracellular matrix and released as an active factor (Rifkin et al., 1999). TGF-P induces migration in endothelial cells (Merzak et al., 1994) and has tumor-promoting immunosuppressive activity by inhibiting the activity of NK cells and tumor-infiltrating lymphocytes, thus enabling tumor growth and spread (Wojtowicz-Praga, 1997). Many of the angiogenic 15 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. effects of TGF-P include induction of VEGF, bFGF and PDGF expression, induction of PDGFR-P and EGFR expression, and induction of collagen and fibronectin secretion (Pepper, 1997; Kavanaugh et al., 1988). TGF-P acts on three types of receptors named TGFR- I, TGFR-II and TGFR-III. TGF receptors I and II belong to the family of serine/threonine receptor kinases whereas TGFRIII (endoglin; CD 105) is an internal co receptor molecule that regulates TGF-P interaction with its receptor (Massague, 1998). Activation of the TGF-p receptors leads to phosphorylation and activation of different SMAD proteins (Lebrin et al., 2005). SMADs are a family of transcription factors involved in transduction of TGF-p signaling to the nucleus. Glioblastoma multiforme is known to produce high levels of TGF-P2 (Letterio and Roberts, 1998). In addition, TGF-p receptors are also overexpressed in glioblastomas (Yamada et al., 1995). TGF-P exerts differential effects depending on which receptor it binds to and which intracellular signal cascade pathway it activates (Lebrin et al., 2005). Therefore, TGF-p can have both pro-angiogenic and anti-angiogenic activities depending on the experimental conditions, such as the type of assay and the concentration in which it is used (Pepper et al., 1997; Goumans et al., 2003; Lebrin et al., 2005). Most in vivo studies suggest that TGF-p is pro-angiogenic, whereas most in vitro assays suggest that it is anti-angiogenic because it inhibits endothelial cells proliferation (Dunn et al., 2000). Previous studies from our laboratory have demonstrated that TGF-pi inhibits Et-1 induced IL-8 production in BEC cells. 16 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. However, the result of TGF-(31 stimulation in TuBEC is not known. Therefore, our study investigated the role of TGF-p 1 in the regulation of IL-8 production by TuBEC. 1.4.5. PDGF Platelet derived growth factor (PDGF) is a heparin binding growth factor, produced by several cell types, such as platelets, endothelial cells and vascular smooth muscle cells (Kaplan et al., 1979; Pirtshalaishvili and Nelson, 2000). PDGF has several roles in wound healing and embryonic development as well as a role in angiogenesis, by participating in maturation of blood vessels and by inducing the recruitment of pericytes to the newly formed blood vessels (Jain and Booth, 2003). PDGF is a 30 kDa protein consisting of two polypeptide chains, A and B, which form either a homodimer or a heterodimer, dimerizing as PDGF-AA, PDGF-AB or PDGF-BB. PDGF acts via a tyrosine kinase receptor to activate the MAPK cascade. Two receptor subunits exist; PDGFR-a and PDGFR-P, which dimerize upon PDGF binding, resulting in autophosphorylation and signal transduction pathways activation (Dunn et al., 2000). However, PDGF-A binds only to PDGFR-a, whereas PDGF-B can bind to both PDGFR-a and PDGFR-p, although it has higher affinity for PDGFR-P (Dunn et al., 2000). Consequently. PDGF-BB is the most mitogenic of the PDGF dimers, because it can bind any receptor dimer combination. Gliomas express high levels of PDGF which correlates with the tumor grade (Hermansson et al., 1988). In addition, PDGF-B and PDGFR-P are overexpressed in tumor endothelial cells (Hermansson et al., 1988). This is consistent with the fact 17 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. that PDGF stimulates migration and proliferation of endothelial cells (Risau et al., 1992). In addition to its direct role as a chemotactic factor for endothelial cells, together with bFGF and EGF, PDGF has an indirect angiogenic role by inducing VEGF expression in tumors (Tsai et al., 1995). Since it is not known whether tumor endothelial cells produce PDGF, this was also investigated in this study. 1.4.6. TNF- a TNF-a is a pro-inflammatory cytokine that has been shown to induce endothelial cell proliferation, migration, tubule formation and in vivo angiogenesis (Distler et al., 2003). In addition, TNF-a induces the expression of endothelial adhesion molecules, thus mediating migration and angiogenesis (Kofler et al., 2005). Therefore, TNF-a was mostly used in our experiments as a positive control, to induce endothelial cell proliferation and growth factor production. 1.4.7. Pleiotropic Angiogenic Factors Besides the growth factors described above, several other factors are involved in the induction of angiogenesis, such as fibroblast growth factors (FGF), epidermal growth factors (EGF), angiopoietins Ang-1 and Ang-2, granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF), insulin-like growth factor (IGF), angiogenin, angiotropin, tissue factor, and erythropoietin (Distler et al., 2003; Dunn et al., 2000; Pirtskhalaishvili and Nelson, 2000; Lopes, 2003). 18 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 1.4.8. Angiostatic Factors Besides angiogenic factor, several angiostatic factors are involved in the inhibition of angiogenesis such as endostatin, angiostatin, thrombospondins, somatostatins, and interferons, such as IFN-y (Mentlein and Held -Feindt, 2003; Distler et al., 2003). 1.4.9. Endothelial Adhesion Molecules Several endothelial adhesion molecules have been ascribed a role in angiogenesis and regulation of tumor development. The most important of these are ICAM-1, CD31 (PECAM-1), and VE-cadherin (CD 144). Intercellular adhesion molecules ICAM-1 and ICAM-2 participate in the adhesion and transmigration of leukocytes by interaction with the ICAM receptor, which is known as LFA-1 (leukocyte-function-associated antigen) (Williams and Barclay, 1988). ICAM-1 is usually expressed both in normal and tumor endothelium. However, conflicting reports exist regarding ICAM-1 expression on tumor endothelium. The role of ICAM-1 is really important, since it participates in the immune response against tumor progression by mediating lymphocyte trans-migration at the tumor site (Renkoven et al., 1992). Therefore, our studies examined the expression of ICAM-1 on tumor endothelial cells in comparison to normal endothelial cells. Platelet endothelial cell adhesion molecule (PECAM-1) or CD31 is also a member of the Ig-superfamily, similarly to ICAM-1. It is one of the most reliable endothelial cell markers as it is expressed on both normal and tumor endothelium. CD31 exerts an important role in leukocyte trans-migration to the inflammation site or tumor site and 19 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. angiogenesis as it is a prerequisite for tubule formation by endothelial cells (Cao et al., 2002). Furthermore, it was demonstrated that blocking of CD31 inhibits angiogenesis in vivo (Cao et al., 2002; De Lisser et al., 1997). Although CD31 was reported to be expressed both on normal and tumor endothelium, redistribution of CD31 to the intercellular junction has been observed in several tumors, such as melanoma (Berger et al., 1993). Therefore, our study examines and compares the expression of CD31 in normal and tumor endothelial cells. VE-cadherin belongs to the family of calcium-dependent transmembrane proteins called cadherins that are involved in the assembly of intercellular junctions (Melder et al., 1996). VE-cadherin exerts an important role in the migration of endothelial cells through the extracellular matrix (Griffioen, 1997). In addition, VE-cadherin regulates endothelial cell proliferation by destabilization of intercellular junctions (Caveda et al., 1996). Since there is little information known regarding the expression of VE-cadherin on tumor endothelial cells, this was also examined in our study. 1.5. COMPARISON OF NORMAL AND TUMOR ENDOTHELIUM 1.5.1. Vascular Endothelium Blood vessels are comprised of the endothelial cells, the mural cells (pericytes and smooth muscle cells), and the basement membrane (Kalluri, 2003; Figure 1-4). Endothelial cells can be identified from other perivascular cells, such as mural cells, by their immunopositivity for vWF (von Willebrand’s factor), CD31 (PECAM-1) and CD 105 (Table 1-1). 20 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. The normal endothelium is a normally quiescent but metabolically active cell layer involved in several physiologic functions such as homeostasis control, vasomotor tone control, cell and nutrient exchange, and angiogenesis (Aird, 2004). The endothelium serves as an input-output device which senses changes in the extracellular environment (e.g., hypoxia, growth factors, temperature, pH) and transforms them into phenotypic changes (e.g., migration, proliferation). Consequently, endothelial cells have different phenotypes depending on the site or organ that they are derived from (Aird, 2003). Endothelial cell heterogeneity is observed in terms of morphology, filaments, vesicles, and tight junctions. In addition, heterogeneity is observed in cell surface glycoprotein expression and lectin binding patterns, protein levels, mRNA expression, and signaling pathways. Finally, endothelial cells are unique in their functional properties such as homeostasis, barrier functions, leukocyte traffickling, survival, migration, proliferation, and antigen presentation properties (Aird, 2003). For example, brain endothelial cells have tight junctions which regulate traffickling via the blood-brain barrier, whereas aortic endothelial cells do not (Craig et al., 1998). Another typical example of endothelial cell heterogeneity is the differences in the expression of the Et-1 receptors Et-A and Et-B between brain microvascular endothelial cells and human umbilical vein endothelial cells (HUVEC). Brain endothelial cells express both receptors, whereas HUVEC only express Et-B (Salani et al., 2000; Hofman et al., 1998). In addition, the expression of the IL-8 receptors, CXCR1 and CXCR2 also depends on the organ origin of endothelial cells. Human intestinal microvascular endothelial cells express only CXCR2 (Heidemann et al., 2003) whereas HUVEC, human dermal cells and human 21 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. lung microvascular endothelial cells express both receptors (Murdoch et al.,1999; Salcedo et al., 2000; Schraufstatter et al., 2001). Most differences between HUVEC and microvascular endothelial cells are due to the fact that HUVEC are macrovascular endothelial cells. Several differences have been observed in the structural and phenotypic properties between the macrovascular and microvascular endothelial cells (Craig et al., 1998). Endothelial celts Btoodi vessefcapilary Vascular basement membrane Pericyte Figure 1-4. Blood vessel structure. {Figure adapted from Kalluri, 2003). Table 1-1. CNS vascular and perivascular cell markers* Maiker/cell a-SM A De&min Vimentiii vWF GSA CD 11b ED-2 CD45 MHC class II GFAP PC + — + — — + — — - SMC + + - - - - - - - - EC - - + + + - - - - - PvM - - + - + + + + -■ AS — — — — — — — — — •F *ot-SMA = a-Smooth Muscle Actin; GSA = Griffonia Simplidfolia Agglutinin; GFAP = glial fibrillary acidic protein; vWF = von Willebrand Factor, PC = pericyte; SMC = smooth muscle cell; EC = endothelial cell; PvM = perivascular macrophage; AS = astrocyte. 1.5.2. Endothelial Cell Differentiation Angiogenesis mainly occurs by the migration and proliferation of pre-existing endothelial cells. However, because of their limited proliferation capacity, since they are terminally differentiated cells, differentiation of endothelial precursor cells into mature endothelial cells is sometimes also required (Hristov and Weber, 2004). 22 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Recent studies have actually demonstrated that, in human tumors, vasculogenesis is much less prelevant than angiogenesis (Peters et al., 2005). Nevertheless, vasculogenesis may still be an important process in tumorigenesis in addition to angiogenesis. There is actually a current debate on whether or not tumor-associated endothelial cells are derived from a common endothelial progenitor cell as normal endothelial cells or they just differentiated into tumor endothelial cells under the influence of growth factors. Mature endothelial cells present in blood vessels are derived from endothelial progenitor cells, which are present in the bone marrow and in the peripheral blood (Hristov and Weber, 2004; Rumpold et al., 2004). Once endothelial cells are required for angiogenesis, the endothelial precursor cells differentiate into mature endothelial cells which express Factor VIII, CD31 and CD105 (Hristov and Weber, 2004; Figure 1- 5). There is a current debate on whether or not tumor-associated endothelial cells are derived from a common endothelial progenitor cell as normal endothelial cells or they just differentiated into tumor endothelial cells under the influence of growth factors. 2 3 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. IM S T u W strwi.j! I tils hom otcpcofc p ro .;u '": L '.'i o iroi'i-m- hemonjiobiost «d lymphoid <«ltf m yeloid cells .■ter V E O F F .* a.*ivdt>a< by p trip jic iv l signals i.-j v c & n lone ri.jrrcti •Jf.yictlos! C0133* C O M * VE0FP.-2* early EPC aMtlnC CD 133* vWF' CD34* VE-cadhtrinr V E O rK -J* E : H « ! u i CD31* «NO& CE-I5.; vW P COW*1 VE-ca<thertn* VEC-FR-2* E-*d«tta* CD31* eNOS* Figure 1-5. Differentiation cascade of endothelial cells. For more information refer to text above (Adaptedfrom Hristov and Weber, 2004). 1.5.3. Tumor Endothelial Cells Tumor blood vessels are usually structurally and functionally different from normal vessels. Tumor vasculature is disorganized and tumor blood vessels are tortuous, dilated, leaky, and hemorrhagic, often displaying dead end structures. These observations suggest that the tumor endothelial cells have different phenotypic and functional properties, compared to normal endothelial cells. There are currently three hypotheses on the development and origin of tumor endothelial cells. The first hypothesis is that they develop from normal endothelial cells recruited into the tumor site, and under the influence of angiogenic growth factors, differentiate in tumor endothelial cells. The second hypothesis is that tumor endothelial cells develop from a common endothelial progenitor cell as the normal endothelial 2 4 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. cells. A third, less popular hypothesis, is that TuBEC develop from de-differentiated tumor cells and hence retain several properties of tumor cells (Bussolati et al., 2003). There are several reports suggesting that tumor associated endothelial cells are phenotypically and functionally different from normal endothelial cells. Tumor endothelial cell lines derived from renal cell carcinomas, ovarian carcinomas, and brain tumors express von Willebrand Factor (vWF), CD 105, CD31 and VE-cadherin, similar to control endothelial cells (Bussolati et al., 2003; Alessandri et al., 1999). In addition, these studies demonstrate that tumor endothelial cells express high levels of VEGF, IL- 8 ICAM-1, E-selectin, CD44 and the VEGF receptors (Bussolati et al., 2003; Alessandri et al., 1999; Griffioen, 1997). Furthermore, tumor endothelial cells express increased survival factors, such as bcl-2 and higher integrin expression (Bussolati et al., 2003). Another characteristic of tumor microvessels is that they lose the expression of tight junction proteins. In fact, it has been demonstrated that in glioblastomas, tumor microvessels lose the expression of claudin-1 and claudin-5 and occludin (Liebner et al., 2000; Papadopoulos et al., 2001). Consequently, this leads to opening of the microvessels junctions and leaking of fluid into the brain (Davies, 2002). Tumor endothelium is considered in general to express lower amounts of leukocyte adhesion molecules, resulting in a diminished leukocyte interaction. However, conflicting data exist on the expression of several adhesion molecules by tumor endothelial cells. For example, ICAM-1 expression on lymphoma derived endothelial cells was shown to be upregulated whereas downregulation of ICAM-1 was reported on endothelial cells derived from colon, ovarian and renal cell carcinomas (Griffioen, 25 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 1997). Similarly, contradictory reports exist for VCAM-1 expression (Kuzu et al., 1993; Piali et al., 1995; Nelson et al., 1994). E-selectin, on the other hand, an activation marker for endothelial cells, was reported to be absent from normal endothelium and present on the tumor vascular endothelium (Ye et al., 1995). Interestingly, it was demonstrated that renal carcinoma and glioblastoma tumor-derived human endothelial cells have high levels of ICAM-1, VCAM-1, and E-selectin (Alessandri et al., 1999). The expression of CD34, a marker for endothelial progenitor cells, was reported to be elevated on the tumor blood vessels, whereas the expression of CD 105 (endoglin) was downregulated (Griffioen et al., 1996). These differences between the normal and the tumor endothelial cells may actually allow tumor endothelial cells to evade anti- angiogenic therapy. Since anti-angiogenic agents often target molecules expressed on normal endothelial cells that may be absent from tumor endothelial cells, the differences between them can form the basis for the development of anti-angiogenic agents that would target both tumor endothelial cells as well as normal endothelial cells. 1.6. PURPOSE OF STUDY Previous research demonstrates that tumor blood vessels have some differences from normal blood vessels (Griffioen, 1997; Bussolati et al., 2003; Alessandri et al., 1999). However, there are several conflicting reports regarding the endothelial markers, adhesion molecules and growth factors that tumor endothelial cells express. In addition, there is little information known on their functional properties such as migration and proliferation. Furthermore, similarly to normal endothelial cells, there is evidence that 26 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. the functional and phenotypic properties of tumor endothelial cells vary depending on their origin (Griffioen, 1997; Alessandri et al., 1999). Since little information is available on CNS tumor endothelial cells (Alessandri et al., 1999), and since brain endothelial cells have some specialized properties as a part of the blood brain barrier, it would be highly significant to characterize brain tumor endothelial cells. Consequently, the purpose of this study is to extensively characterize tumor-associated brain endothelial cells (TuBEC) and compare their functional and phenotypic properties with those of normal human brain endothelial cells (BEC). More specifically, the specific aims of this study are: (a) to characterize and compare the phenotypic and functional properties of glioblastoma-associated endothelial cells with normal brain endothelial cells, (b) to investigate and compare the role of IL-8 in the regulation of TuBEC and BEC migration, and (c) to determine whether TuBEC have phenotypic and functional characteristics of senescent cells. This work is highly significant because it helps us understand the physiological properties of TuBEC, and some of the basic mechanisms responsible for their altered phenotype and functions. This is very important because tumor endothelial cells may have different characteristics from normal endothelial cells and therefore conventional anti-angiogenic therapy may not be able to target them. Consequently, since little information is available on CNS tumor endothelial cells, characterization of TuBEC as a potential target for anti-angiogenic therapy is important as it could lead to the development of novel anti-angiogenic therapies specifically targeting tumor endothelial cells. 2 7 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. CHAPTER II FUNCTIONAL AND PHENOTYPIC DIFFERENCES BETWEEN GLIOBLASTOMA-DERIVED ENDOTHELIAL CELLS AND NORMAL HUMAN BRAIN ENDOTHELIAL CELLS R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 2.1. INTRODUCTION In the specific type of brain tumor called glioblastoma multiforme (GBM), tumor progression has been correlated with extensive blood vessel growth. A histologic examination of GBM tissue reveals that these blood vessels are a chaotic network of irregularly shaped, dilated and tortuous vessels (Balabanov and Dore-Duffy, 1998). Moreover, these vessels are leaky and hemorrhagic, often displaying dead end structures (Davies, 2002). These observations suggest that the endothelial cells, which compose the glioblastoma-derived vasculature (TuBEC) could have different phenotypic and functional properties, compared to normal brain-derived endothelial cells (BEC). Comparisons of tumor-derived and normal endothelial cells have been made for a variety of systemic tumors (Bussolati et al., 2003; Griffioen, 1997). Tumor-associated endothelial cell lines derived from renal cell carcinomas express von Willebrand Factor (vWF), and CD105, similar to control endothelial cells (Bussolati et al., 2003). These studies also demonstrated that tumor cell lines constitutively express VEGF receptors 1, 2, 3, and the angiopoietin receptor Tie-2 (Bussolati et al., 2003). Other reports showed that tumor endothelium has the phenotype of activated endothelial cells as reflected in the high expression of the adhesion molecules: ICAM-1, E-selectin, and CD44 in the tumor endothelial cells (Griffioen, 1997). While these studies compare tumor vasculature to normal endothelial cells in systemic tumors, there is little information regarding endothelial cells derived from central nervous system (CNS) tumors. Liebner and coworkers did show that the junctional proteins claudin-1 and claudin-5 are altered 29 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. in blood vessels derived from human GBM, compared to normal brain (Liebner et al., 2000). These studies also suggest intrinsic morphologic differences between tumor- derived blood vessels and normal blood vessels. Brain endothelial cells, comprising the blood-brain-barrier, have highly specialized properties and functions, including tight-junctions, selective transport, and altered pinocytotic activity (Kniesel and Wolburg, 2000). Therefore, studies were initiated to identify the morphology, phenotypic characteristics, and functions of brain endothelial cells derived from gliomas. The results demonstrate that endothelial cells from the glioma vasculature proliferate less than normal BEC. However, these TuBEC are highly active in migration and secretion of angiogenic factors. These data emphasize that the tumor microenvironment significantly alters the characteristics of the normal vasculature, making these cells a potential target for anti-angiogenic therapy. 3 0 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 2.2. MATERIALS AND METHODS 2.2.1. Cell Culture and Reagents BEC and TuBEC were characterized using the following specific markers: von- Willebrand factor (vWF) (DAKO, Carpinteria, CA) and CD 105 (Ancell corporation, MN) for endothelial cells, glial fibrillary acidic protein (GFAP) (DAKO) for astrocytes and glioma cells, C D llb (Immunotech, Villepinte, France) for macrophages/microglia, and SMA (smooth muscle actin) (DAKO) for smooth muscle cells. Cell viability was determined to be greater than 95% as assessed by trypan blue exclusion. BEC and TuBEC were cultured in RPMI 1640 medium (GIBCO Laboratories, Grand Island, NY) supplemented with 100 ng/ml endothelial cell growth supplement (ECGS, Upstate Biotechnologies, NY), 2 mmol/L L-glutamine (GIBCO), 10 mmol/L HEPES (GIBCO), 24 mmol/L sodium bicarbonate (GIBCO), 300 U heparin USP (Sigma-Aldrich, St.Louis, MO), 1% penicillin/streptomycin (GIBCO), and 10% fetal calf serum (FCS) (Omega Scientific, Tarzana, CA). VEGF was purchased from R&D systems (Minneapolis, MN) and Et-1 was purchased from Bachem (Torrance, CA). The ABC and AEC kits used for immunohistochemistry were purchased from Vector Laboratories (Burlingame, CA). 2.2.2. Endothelial Cell Isolation and Cultivation Normal endothelial cells and glioblastoma- derived endothelial cells were isolated from either normal human brain or human glioblastoma tissue. Institutional Review Board (IRB) approval was obtained for discarded brain tissue from trauma lobectomies 31 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. or glioblastoma specimens. Glioblastoma specimens were obtained from patients with newly diagnosed or recurrent glioblastoma. No identification of tissue to patient was made. The tissue was washed three times with RPMI 1640 medium containing penicillin/streptomycin. The tissue was then cut into small pieces in medium containing 2% FCS. Subsequently, fresh medium was added, and the mixture was transferred to a centrifuge tube. An equal volume of a 30% dextran solution was added, bringing the mixture to a final concentration of 15%. The resulting mixture was then centrifuged for 10 min at 10000 rpm to isolate the brain microvessels. The microvessel pellet was resuspended in 1 mg/ml collagenase/dispase in RPMI 1640 containing 2% FCS, and incubated in a 37 °C water bath with a shaker for 1 hour. Subsequently, 10 ml of RPMI 2% FCS was added to the cells, and the suspension was centrifuged at 1200 rpm for 5 min. The pellet was resuspended in 20 ml of the RPMI 2% FCS medium and centrifuged again. The final pellet was resuspended in RPMI 1640 supplemented with 10% FCS, 100 ng/ml ECGS and 10% Nu-serum culture supplement (BD Biocoat, Bedford, MA). Microvessels were plated on precoated gelatin flasks for 3-4 days. Medium was changed every 3 or 4 days until cultures became 80% confluent. Endothelial cells were then purified from the cellular mixture by FACS cell sorting based on expression of dil-acetylated-LDL. Following the sort, the purity of the endothelial cell population was confirmed by immunostaining with vWF, GFAP, CD1 lb and SMA. Only cells from passage 3-5 were used for the experiments reported here. 3 2 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 2.2.3. Endothelial Cell Migration Assay Migration assays were performed using modified transwell chambers as described previously (Rosano et al., 2001). Briefly, 6.5 mm diameter polyethylene filters (BD Biocoat, Bedford, MA) with 8 pm pore-size were coated on top and bottom by immersion into 0.5% gelatin for 2 hours and air-dried overnight. Subconfluent cultures of BEC were harvested using trypsin/EDTA (0.05%) solution (GIBCO); 5xl04 cells were then resuspended in RPMI medium supplemented with 1 % FCS and placed on top of the filter in the upper chamber (100 pl/well). Chemo-attractants were placed in the lower compartment (600 pl/well). Transwell plates were then incubated at 37 °C in 5% CO2 for 6 hours. At the termination of experiments, the medium was removed from the inserts and remaining cells on the upper side of the chamber were removed with a wet Q-tip. The filters were stained with the Diff-Quick staining solution (Dade Behring Inc., Newark, DE). Cells that migrated through to the underside of the membrane were counted under high-power magnification (HPM; 40X); 10 fields per filter were counted. All experimental groups were plated in triplicate. 2.2.4. MTT Cell Growth Assay Cells (2.5 xl03 /well) were seeded in 96-well plates (100 pl/well) in quadruplicate. Medium was changed to RPMI 1% FCS or 10% FCS immediately after attachment of the cells. At daily intervals, for 5 consecutive days, the MTT reagent was added (1:10 dilution) to the cultures for 4 hours, according to the manufacturer’s instructions (Sigma-Aldrich, St Louis, MO). Medium was then removed, dimethylsulfoxide 33 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. (DMSO) was added (150 |il/well) and the color intensity, which is proportional to the cell number, was measured at 570 nm in a Dynatech Laboratories MRX spectrophotometer. 2.2.5. BrdU Cell Proliferation Assay Cells were seeded in quadruplicate in 96-well plates at 2.5 xlO3 cells/ well. The BrdU reagent was added to cells for 18 hrs and proliferation was then evaluated using the BrdU labeling and detection kit III (Roche Diagnostics, Indianapolis, IN). The number of proliferating cells was proportional to absorbance at 405 nm, which was proportional to BrdU incorporation. 2.2.6. Immunocytochemistry Cells were immunostained as previously described (Li et al., 2003). Briefly, cytocentrifuge preparations of cells were fixed in acetone, blocked with 5% normal horse or goat serum in PBS (phosphate buffered saline) for 20 min and incubated for 2 hours with rabbit anti-Et-1 antibody (Bachem, San Carlos, CA), or overnight with anti- VEGF (Chemicon, Temecula, CA), Ki-67 (DAKO), VE-cadherin (BD Pharmingen, San Diego, CA), CD31 (BD Pharmingen), CD105, C D llb, GFAP or a-SMA antibodies. Slides were then incubated with biotinylated goat anti-rabbit IgG or biotinylated horse anti-mouse IgG for 45 min (Vector Laboratories, CA). Subsequently, slides were treated with ABC elite (avidin biotin complex) (Vector Laboratories) for 30 min followed by AEC (aminoethyl carbazol) substrate kit (Vector Laboratories) according to 34 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. the manufacturer’s instructions. A red precipitate identified positive staining; slides were lightly (1 min) counterstained with hematoxylin, for nuclear definition. 2.2.7. ELISA Cells (105 cells/well) were seeded in 6-well plates. After attachment of the cells (6 hours), the media was changed to RPMI 1% FCS. Supernatants from cells cultured for 48 hrs in media containing 1% FCS were collected and analyzed for VEGF and Et-1 concentrations. Quantities of secreted VEGF and Et-1 were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN) as per the manufacturer's instructions. 2.2.8. Flow Cytometry The percent of apoptotic cells was determined by annexin-V-FITC staining. Briefly, subconfluent cell cultures were harvested by trypsinization, washed twice with PBS and incubated with annexin-V-FITC (BD Pharmingen, San Diego, CA) for 15 min. Stained cells were then analyzed using the FACScan machine with appropriate software (Becton Dickinson, Bedford, MA). Data, expressed as percent positive cells per total number of cells, represented the apoptotic cell population. 2.2.9. TUNEL Assay To determine apoptosis, the commercially available ApopTag In Situ detection kit (TUNEL assay) (Chemicon International, Temecula, CA) was used as per 35 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. manufacturers protocol. Briefly, cytocentrifuge preparations were made, and cells fixed with 2% paraformaldehyde. The cells were post-fixed with ethanol: acetic acid (2:1 dilution v/v) and incubated with the TdT enzyme (terminal deoxynucleotide transferase), followed by an incubation with anti-digoxigenin peroxidase conjugate (Chemicon). Finally, the cells were incubated with the peroxidase substrate and counterstained with haematoxylin. The red nuclei represented apoptotic nuclei. 2.2.10. Cell Death Detection •a BEC and TuBEC were plated in 96-well plates in quadruplicates at 7.5x10 cells/well (100 pl/well). Cells were lysed after 24 or 48 hr, depending upon experimental protocol, and lysates were analysed using the Cell death detection ELISA Plus kit (Roche diagnostics) for the presence of nucleosomes. Absorbance was measured at 405 nm. 2.2.11. Statistical Analysis Values are presented as the mean ± SEM. Statistical significance was evaluated using Student's t-test for paired comparison. P<0.05 was considered statistically significant. 3 6 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 2.3. RESULTS 2.3.1. Morphology TuBEC, isolated from glioma tissue, were characterized and utilized in these studies. A total of six different TuBEC cultures were collected and studied. As described in the Methods section, these cells were identified as pure endothelial cell cultures, based on staining with vWF (Figure 2-1 A) and CD 105 (data not shown). At no time was there any evidence of glioma cells, glia, or macrophages in these cultures, as judged by negative staining for GFAP (Figure 2-1B) and CDllb (Figure 2-1C) and positive staining for endothelial cell markers. TuBEC cultures were clearly distinguishable from control BEC morphologically. Subconfluent cultures of TuBEC were large, flat, adherent cells with small, distinct nuclei and abundant cytoplasm (Figure 2-ID). In contrast, subconfluent cultures of control BEC exhibited plump cells, with large nuclei and limited cytoplasm (Figure 2-IE). BEC grown to confluence demonstrated typical cobblestone-like appearance (Figure 2-1G) whereas confluent TuBEC did not have this appearance (Figure 2-IF). TuBEC were not vacuolated and were greater than 97% viable, as determined by the trypan blue dye exclusion technique. Approximately 90% of the cells had this morphology. The remaining cells had the appearance of normal BEC. The morphologic characteristics of TuBEC were consistent among the six different TuBEC cultures tested. 3 7 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 2-1. Comparison of BEC and TuBEC morphology and detection of factor VIII, GFAP and C D llb expression in TuBEC. A-C. Immunostaining of tumor- derived human brain endothelial cells (TuBEC) with Factor VIII, GFAP and C D llb. TuBEC stained with anti-vWF exhibit greater than 98% intense positive staining (A). TuBEC stained with anti-GFAP (B) or CDllb (C) were negative (Mag. =10X). D-G. Comparison of human tumor-derived brain endothelial cell (TuBEC) and normal brain endothelial cell (BEC) morphology. Subconfluent TuBEC are adherent with abundant cytoplasm, multiple processes, and flat appearance (D). Subconfluent BEC demonstrate adherent, plump, and angular cells (E). Confluent TuBEC do not demonstrate the typical endothelial cobblestone appearance (F). Confluent BEC display the typical cobblestone appearance (G). (Mag. = 20X). 38 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 2.3.2. Surface and Cytoplasm Marker Expression in TuBEC Based on the appearance of distinct phenotypic differences, expression of specific surface and cellular markers was analyzed using immunocytochemistry. The results in Figures 2-2A and B demonstrate a decreased expression of VE-cadherin in TuBEC compared to BEC; BEC (>95%) were positive for VE-cadherin (Figure 2-2B) whereas TuBEC were faint (Figure 2-2A). In addition, although the number of CD31 positive cells in TuBEC and BEC cultures was similar, the distribution of staining was different. CD31 was localized to the cell membrane in BEC (Figure 2-2D), and to the cytoplasm in TuBEC (Figure 2-2C). Antibody to a- smooth muscle actin (SMA) stained approximately 50% of TuBEC (Figure 2-2E), and was virtually negative in BEC (Figure 2-2F). These data are summarized in Table 2-1. Table 2-1: Summary of endothelial cell markers expression on BEC and TuBEC as determined by immunohistochemistry. BEC TuBEC Factor VIII ++++ ++++ GFAP - - C D llb - - CD105 ++++ ++++ SMA* - ++ (50% positive) CD31 ** +++ +++ VE-cadherin +++ + GFAP, glial fibrillary acidic protein; SMA, a-smooth muscle actin. Legend of staining intensity: - = negative;+ = faint; ++ = low;+++ = moderate;++++ = high.* 50% of TuBEC show intense positivity for SMA. ** CD31 distribution was different in TuBEC compared to BEC. 3 9 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 2-2. Differential expression of specific markers in TuBEC compared to BEC. TuBEC and BEC were stained with a series of antibodies, which resulted in different expression patterns. Staining with anti-VE-cadherin demonstrated decreased expression in TuBEC (A) compared to BEC (B). Staining with anti-CD31 showed different localization in TuBEC (C) compared to BEC (D). TuBEC exhibited significant staining with a-smooth muscle actin (SMA) (E) compared to BEC, which showed negligible reactivity (F) (Mag.= 20X). 40 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 2.3.3. Proliferation Based on evidence that the microvessel density in gliomas is greater than in normal brain tissue, the proliferation rates of TuBEC and BEC were examined. Equal numbers (2.5 xlO3 cells/well) of TuBEC and BEC were cultured in media containing either 1% FCS, 10% FCS, or ECGS for 24, 48, 72, 96 hrs, then analyzed for cell number using the MTT assay. The results show (Figures 2-3A and B) that in these environments, the number of cells increased to a greater extent in BEC cultures compared to TuBEC. To confirm the actual difference in cell replication, TuBEC and BEC cultures were analyzed for proliferation using BrdU incorporation. Data show that TuBEC proliferate more slowly than BEC (Figure 2-3C). To further confirm these results, TuBEC and BEC were stained with Ki-67, an antibody detecting proliferating cells. The results show significantly greater proliferation in BEC compared to TuBEC (P=0.003; Figure 2-3D). These data were confirmed using 3 different TuBEC and 3 different BEC samples. TuBEC and BEC growth 140 - i 120 - Q ° 1 0 0 - 6 24 48 72 96 — *— BEC - - m - - TuBEC Hours 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2-3: Continued B Growth of TuBEC and BEC cultured in different media 250 g 200 ® 150 < 0 (0 £ 100 U * 50 0 ECGS 10% FCS 1% FCS ■ BEC ■ TuBEC TuBEC and BEC proliferation at 72 hrs under different culture conditions 2.00 'g 1.50 c o 1.00 o 0.50 0.00 I BEC ITuBEC ECGS 10% FCS 1% FCS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. D TuBEC and BEC proliferation using KI-67 expression = 7 0 ] 8 6 0 - 8 5 0 BEC TuBEC Figure 2-3. Comparison of BEC and TuBEC proliferation. A. TuBEC and BEC, cultured in media with 10% FCS, were monitored over a 96 hr period using the MTT assay. Data are expressed as percent increase in optical density (OD) relative to OD immediately after cell attachment. OD was measured at 570 nm. Bars correspond to standard error. TuBEC and BEC, cultured under different conditions, were analyzed after 72 hr using the MTT (B), and BrdU assays (C). Data is recorded as OD measured at 570 nm and 405 nm respectively. D. TuBEC and BEC cultures were stained with anti-Ki-67 to determine number of dividing cells. The data are expressed as average percent proliferating cells per total population; 10 high power (40X) fields were counted. Bars correspond to standard error. (ECGS: endothelial cell growth supplement). 2.3.4. Apoptosis To determine whether differences in rate of growth of BEC and TuBEC were related to differences in apoptosis, several apoptosis assays were performed. The TUNEL assay demonstrated negligible apoptosis (< 1% of the cells) in both BEC and TuBEC cultured in media containing 1 0 % FCS, for 24, 48 and 72 hrs (data not shown). However, incubation for 24 or 48 hrs in serum-free media caused significant apoptosis in the BEC, with little apoptosis in the TuBEC population ( P 4 8 h = 0 . 0 0 0 4 ) (Figure 2-4A). Cell death ELISA assays were also performed using lysates from cultures of BEC and 4 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TuBEC grown in serum-free media for 24 and 48 hrs. The data showed that cell death in BEC cultures was significantly higher than in TuBEC (P48h=0.01) (Figure 2-4B). Using media supplemented with 1% FCS, flow cytometric analysis of annexin-V stained BEC and TuBEC revealed an increase in apoptotic cells in the BEC population as compared to TuBEC (Figure 2-4C). A Apoptosis in TuBEC and BEC using the TUNEL ■ BEC ■ TuBEC 24 h 48 h Hours 4 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2-4: Continued B Comparison of TuBEC and BEC death usi ng the nucleosomal release cell death assay - 0.6 c 0.5 IO §, 0.4 o 0.3 | 0.2 £ 0.1 .a < n I BEC I TuBEC 24 h 48 h Hours TuBEC and BEC apoptosis using annexin-V- C FITC staining 70.0 ^ 60.0 o 50.0 ■| 40.0 -S . 30.0 % 20.0 55 10.0 0.0 BEC TuBEC Figure 2-4. Comparison of BEC and TuBEC apoptosis. A. TuBEC and BEC were cultured in serum-free media for 24 and 48 hr, then analyzed using the TUNEL assay. The data are expressed as percent apoptotic cells per total population, by counting 10 high power fields. B. TuBEC and BEC were cultured in serum-free media for 24 and 48 hr, and assayed using the nucleosomal release cell death assay. The data are expressed as OD at 405 nm, which is proportional to the number of dead cells per well. Each experiment was performed in quadruplicates. C. TuBEC and BEC were cultured in 1% FCS media for 72 hr, then stained for annexin -V. The data are analyzed using flow cytometry, and expressed as percent annexin-V-FITC positive cells per total population. 4 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.3.5. Function Since the tumor vasculature is actively participating in angiogenesis in gliomas, the question was raised whether these TuBEC function differently in specific angiogenic processes, compared to normal BEC. Endothelial cell migration is critical to angiogenesis. Therefore, migration was tested using a modified Boyden chamber technique. TuBEC and BEC (5x104 cells) were placed on top of a porous filter, and after 6 hrs, the numbers of cells migrating through the pores were counted. The results n show greater TuBEC migration compared to BEC migration (P=8 xlO' )(Figures 2-5A and B). These results were consistent using 3 different TuBEC and 3 different BEC cultures. Thus TuBEC were intrinsically more active in the absence of a chemotaxis agent. When chemotactic factors such as Et-1 and VEGF were added to the lower chamber, BEC migration rate increased, whereas the TuBEC were unresponsive to these factors (Figures 2-5 A and B). The concentrations of VEGF and Et-1 used are the optimal for BEC migration as determined by testing different concentrations (Et-1, 1- 1000 nM; VEGF, 1-30 ng/ml). TuBEC did not respond at any of the concentrations tested (data not shown). To determine whether TuBEC were active in the production of growth factors, BEC and TuBEC were cultured in 1% FCS for 48 hrs and supernatants were tested for the angiogenic factors, VEGF and Et-1. The data demonstrated that unstimulated TuBEC constitutively produced more VEGF-1 and Et-1 compared to unstimulated BEC (PEt- 1=0.008; PV egf=0.02) (Figures 2-6A and B). Immunostaining with anti- Et-1 antibody showed that over 95% of TuBEC and BEC were positive for Et-1, but TuBEC exhibited 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. significantly more intense staining compared to BEC (Figures 2-6C and D). In addition, in BEC, the staining pattern demonstrated that Et-1 was associated with plasma membrane, whereas in TuBEC staining appeared diffusely distributed throughout the cytoplasm (Figures 2-6C and D). To determine the percent population producing VEGF, immunocytochemistry was performed on TuBEC and BEC. The results demonstrated that over 95% of the TuBEC and BEC were producing VEGF (Figures 2-6E and F), but the intensity of staining was significantly greater in TuBEC (Fig 2-6E). 4 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TuBEC and BEC migration in response to Et-1 30 i ■ control ■ Et-1 (100 nM) BEC TuBEC B TuBEC and BEC migration in resp o n se to VEGF 45 n ■ control ■ VEGF (1 ng/ml) BEC TuBEC Figure 2-5. Comparison of BEC and TuBEC migration rate. TuBEC and BEC were tested in the modified Boyden chamber for rate of migration. After 6 hours incubation, TuBEC migrated faster compared to BEC; but TuBEC were unresponsive to stimulation with Et-1 (A) or VEGF (B). The data are expressed as the mean of the number of migrated cells in 10 fields (40X); bars represent standard error. (HPF: high power fields). 4 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Et-1 production by TuBEC and BEC VEGF production by TuBEC and BEC Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2-6: Continued Figure 2-6. Comparison of Et-1 and VEGF production by BEC and TuBEC. Supernatants from TuBEC and BEC cultures, incubated for 48 hr in 1% FCS, were tested for Et-1 (A) and VEGF (B) using the ELISA technique. In parallel, cytocentrifuge preparation of TuBEC and BEC were stained with anti-Et-1 (C and D) or anti-VEGF (E and F). Bars represent standard error. 5 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.4. DISCUSSION Angiogenesis is a critical requirement for solid tumor growth (Lopes, 2003). These infiltrating blood vessels may be derived from pre-existing normal, neighboring, benign tissue vasculature (Holash et al., 1999), or from circulating endothelial progenitor cells (Asahara et al., 1999), or a combination of both sources (Bussolati et al., 2003). What is becoming apparent is that extensive exposure of normal endothelium to the tumor microenvironment, results in an altered phenotype and aberrant functions of endothelial cells within the tumor, particularly glioma-derived brain endothelial cells. The in vitro studies presented here were performed on endothelial cells derived from human glioblastoma tissue. These cells were determined to be pure endothelial cells, as documented by immunostaining. In contrast to normal BEC, TuBEC exhibited alpha- smooth muscle actin expression, in addition to the common endothelial cell markers (vWF, CD 105, CD31). SMA is reportedly expressed on smooth muscle cells, and pericytes (Balabanov and Dore-Duffy, 1998), and not a typical endothelial cell marker. Since TuBEC were consistently vWF and CD 105 positive, these data suggest that TuBEC have the unique property of expressing both endothelial and pericyte markers. The TuBEC tested in the present studies were derived from six different surgical glioblastoma multiforme specimens. We believe that this tissue heterogeneity makes our data even more compelling, as the data that we report was seen in all the specimens reported. Moreover, as there is significant heterogeneity based on genetic diversity as well as previous adjunctive therapy of the human tissue samples, all observations and experiments were based on data from at least three different tumor specimens. Thus, 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the in vitro model used here to study TuBEC is likely to have general characteristics of glioblastomas, and should provide direct information regarding the properties of the glioma vasculature. A common feature of all TuBEC cultures examined is their unique morphology. This is consistent with evidence that these cells function differently than normal BEC cells. The data show that TuBEC migrate faster than normal BEC. This migration is not based on chemotaxis, but more on random movement (chemokinetic), suggesting that TuBEC are active in the absence of exogenous factors. Other evidence that TuBEC are constitutively activated cells, are derived from data showing that TuBEC actively secrete the growth factors VEGF and Et-1, demonstrated by immunostaining and ELISA assays. These growth factors likely activate TuBEC in an autocrine fashion, resulting in greater migration and stimulation of growth factor production. VEGF and Et-1 were reported to stimulate endothelial cell migration (Jensen, 1998; Li et al., 2003; Rosano et al., 2001). Both TuBEC and BEC express receptors for these angiogenic factors, as identified by flow cytometry and immunocytochemistry (data not shown). The reason for this lack of response in TuBEC to VEGF or Et-1 is not clear. However, the constant presence of these growth factors may saturate the receptors and thus cause non-responsiveness. The mechanisms responsible for the increased chemotactic activity of TuBEC are not clear. However, the autocrine effect of these factors on the endothelial cells may, at least in part, be responsible for this enhanced activity. A surprising finding presented here is that TuBEC proliferated less than normal BEC. These data were confirmed using the MTT assay, BrdU incorporation, and Ki-67 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. expression. This decreased proliferation for TuBEC was observed in media containing either 10% or 1% fetal calf serum. These results are in contrast to a previous observation that tumor endothelial cells have a high rate of proliferation compared to normal endothelium (Denekamp, 1984). This discrepancy may be due to the fact that the previously published study examined tumor tissue explants in vitro, while our studies tested purified, characterized glioblastoma-derived endothelial cells. In addition, our results are consistent with the observations that there may be a temporal dichotomy between migration and proliferation, as glioma cells with decreased motility demonstrate increased proliferation (Giese et al., 1996). The decreased number of TuBEC in culture is not a reflection of increased apoptosis. Our studies show that levels of apoptosis for both BEC and TuBEC were relatively low under optimal growth conditions; however, in suboptimal conditions (i.e. low or serum-free media) there was less apoptosis in the TuBEC population. This is consistent with published data (Bussolati et al., 2003) showing that renal carcinoma-derived endothelial cells have increased survival and increased expression of the anti-apoptotic protein Bcl-2. Indeed, cDNA microarray studies have shown that migrating cells have a global down- regulation of pro-apoptotic genes and up-regulation of anti-apoptotic genes (Mariani et al., 2001a; Mariani et al., 2001b). This is also consistent with our data that TuBEC constitutively produce the survival factor, VEGF (Bussolati et al., 2003). Thus, TuBEC exhibit enhanced survival and are functionally active in the tumor microenvironment (i.e., poor nutrient conditions). 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TuBEC were shown here to have altered expression of CD31 (also known as platelet endothelial cell adhesion molecule-1, PE-CAM-1), and CD 144 (VE-cadherin), as demonstrated by immunostaining. In TuBEC, CD31 was present in the cytoplasm, whereas in BEC, CD31 was predominantly expressed on the cell surface. CD31 is a receptor present on both endothelial cells and leukocytes, and is involved in leukocyte transmigration, angiogenesis, and apoptosis (Jackson, 2003). It has been shown in the past (Griffioen, 1997) that there are low numbers of infiltrating, cytotoxic T cells in gliomas. This may be due to a diminished leukocyte-endothelium interaction, and subsequent reduced transmigration. Based on these studies, the altered distribution and expression of CD31 on TuBEC membrane may be an excellent topic for further investigation. CD 144 is known to be critical for angiogenesis; blocking CD 144 leads to abnormal capillary junctions, and aberrant tubule formation (Griffioen, 1997; Wright et al., 2002). Our studies demonstrated that VE-cadherin is significantly reduced in TuBEC. This lack of junctional proteins may contribute to tumor blood vessel leakiness and abnormal structure (Davies, 2002). There are several reports of reduced CD 105 expression on tumor microvessels (Griffioen et al., 1996) and contradictory reports regarding CD34 expression (Hellwig et al., 1997; Traweek et al., 1991). In our studies, we have not detected any differences in CD34 and CD 105 expression between normal BEC and TuBEC (data not shown). Our data demonstrated phenotypic and functional differences between BEC and TuBEC. It is unclear whether genotypic differences are also present in these two distinct populations. Whether these genotypic differences are permanent, or transient 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. secondary to the tumor microenvironment, will need to be determined. Recently, Croix and coworkers have demonstrated that tumor and normal endothelium are distinct at a genetic level (Croix et al., 2000). Comparing gene expression levels of endothelial cells derived from blood vessels of normal and malignant colorectal tissues, they found differential expression and selective upregulation of endothelial derived genes in tumor versus normal endothelium (Croix et al., 2000). This differential gene expression of endothelial cells may be secondary to distinct angiogenic states (Glienke et al., 2000). In conclusion, we have demonstrated that TuBEC have different proliferation, migration, adhesion, and growth factor production properties from normal BEC. These different properties may be key to understanding the mechanism by which normal BEC gain the characteristics of TuBEC. This knowledge will provide information leading to the treatment of gliomas through selective anti-angiogenic chemotherapy. 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER III INTERLEUKIN-8 (IL-8) DIFFERENTIALLY REGULATES MIGRATION OF TUMOR-ASSOCIATED AND NORMAL HUMAN BRAIN ENDOTHELIAL CELLS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.1. INTRODUCTION Angiogenesis is the multistep process which generates new blood vessels from pre existing ones. This process occurs during tumor growth, as well as during wound healing (Rosano et al., 2001; Longo et al., 2002). Angiogenesis involves activation of endothelial cells, which includes proliferation, migration, extracellular matrix remodeling, and tubule formation (Longo et al., 2002; Li et al., 2003; Heidemann et al., 2003). Interleukin-8 (IL-8), a member of the ELR (glutamic acid-leucine-arginine) CXC chemokine family, is reported to be a potent angiogenic factor, specifically enhancing endothelial cell migration (Li et al., 2003; Heidemann et al., 2003). IL-8 is produced by activated endothelial cells and by a variety of tumors, including gliomas (Wakabayashi et al., 1995). In fact, it was shown that in human gliomas, two pathways control angiogenesis in a paracrine manner, one mediated by VEGF and/or FGF, and the other through IL-8 (Wakabayashi et al., 1995). In addition, since both VEGF and Et-1, angiogenic factors produced in gliomas, stimulate expression of IL-8 in endothelial cells (Lee et al., 2002; Hofman et al., 1998), the role of IL-8 in migration of normal and tumor-derived endothelial cells is critical. Consequently, IL-8 is a highly significant mediator of angiogenesis, and its role in tumor development is of interest (Wakabayashi et al., 1995; Brat et al., 2005; Belperio et al., 2000). IL-8 binds to two related 7 transmembrane, domain G -protein coupled receptors on target cells (Belperio et al., 2005; Schraufstatter et al., 2001). CXCR1 binds specifically to IL-8, while CXCR2 binds promiscuously to several CXC chemokines, 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. including IL-8, GRO-a,p,y (growth regulated oncogenes), NAP-2 (neutrophil activating peptide-2) and ENA-78 (epithelial neutrophil-activating peptide-78) (Schraufstatter et al., 2001). Ligand binding to the two receptors leads to sequential activation of multiple signaling pathways which leads to activation of enzymes such as PLD (phospholipase D), PAK (p-21 activated kinase), FAK (focal adhesion kinase) and the MAPK (mitogen activated protein kinase) cascade (Schraufstatter et al., 2001; Feniger-Barish et al., 2003; Jones et al., 1995). Microvessels commonly express CXCR1 and CXCR2 mRNA and protein constitutively (Li et al., 2003; Salcedo et al., 2000). Both of these receptors are involved in endothelial cell migration (Salcedo et al., 2000). Most reports examining the role of IL-8 in tumor angiogenesis have been performed on normal endothelial cells (Li et al., 2003; Heidemann et al., 2003; Wakabayashi et al., 1995). Few studies have investigated primary cultures of endothelial cells derived from tumor tissue (Bussolati et al., 2003). Therefore, it is not clear whether tumor-associated brain endothelial cells (TuBEC) respond the same or differently to angiogenic factors compared to normal brain endothelial cells (BEC). Since the ultimate goal of this study is to determine the mechanism by which brain tumors regulate neovascularization, in these studies we used human brain endothelial cells obtained from tumor tissue. In the normal human brain, the cerebral endothelial cells, which comprise the blood- brain-barrier (BBB), differ from systemic endothelial cells by having decreased pinocytotic activity, increased expression of tight junction proteins, and the presence of unique transport proteins (Kniesel et al., 2000). In glioblastoma multiforme, the most malignant form of glioma, tumor-associated blood vessels are characterized by 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. microvessel hypertrophy and proliferation in both the intratumoral, peri-necrotic regions of the glioblastoma and in the peritumoral brain tissue (Burger et al., 1991). Microvessels of brain tumors undergo active angiogenesis, resulting in “leaky” vessels, increased expression of av05 integrins and a breakdown of the blood brain barrier (Bello et al., 2001; Davies, 2002). Cerebral endothelial cells within tumors show increased pinocytosis, decreased tight junction proteins, such as claudin-1 and occludin, and increased permeability (Davies, 2002). Thus, it is evident from our studies and others, in human and animal brain tumor models, that cerebral blood vessels in tumors function differently from normal vessels (Charalambous et al., 2005). In our studies, we analyzed the response and function of isolated, purified, and characterized human BEC and TuBEC with respect to regulation of endothelial cell migration and chemokine production. The experiments demonstrate that TuBEC constitutively secrete IL-8, which acts in an autocrine manner to induce endothelial cell migration. This autocrine stimulation is not down-regulated by TGF-pl, as in normal endothelial cells. Furthermore, these data suggest that TuBEC contribute growth factors to the pro-angiogenic microenvironment produced by tumors, and emphasize the functional differences between TuBEC and normal brain endothelial cells. 5 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.2. MATERIALS AND METHODS 3.2.1. Cell Culture and Reagents Human brain endothelial cells, both BEC and TuBEC, were isolated from normal and glioblastoma brain tissue respectively as previously described (Charalambous et al., 2005). TuBEC were derived from untreated patients diagnosed with stage IV glioblastoma multiforme; BEC were derived from brain tissue obtained following trauma. Institutional Review Board (IRB) approval was obtained for brain tissue that was discarded following trauma surgeries and glioblastoma specimens. BEC and TuBEC were characterized as 99% pure using the following specific markers: von Willebrand factor (vWF) (DAKO, Carpinteria, CA) for endothelial cells, glial fibrillary acidic protein (GFAP) (DAKO) for astrocytes and glioma cells, CD lib (Immunotech, Villepinte, France) for macrophages and microglia, and a-SMA (smooth muscle actin) (DAKO). Cell viability was determined to be greater than 99% as assessed by trypan blue exclusion. BEC and TuBEC were cultured in RPMI 1640 medium (GIBCO Laboratories, Grand Island, NY) supplemented with 100 ng/ml endothelial cell growth factor (Endogro, Upstate biotechnologies, NY), 2 mmol/L L-glutamine (GIBCO), 10 mmol/L HEPES (GIBCO), 24 mmol/L sodium bicarbonate (GIBCO), 300 U heparin USP (Sigma-Aldrich, St.Louis, MO), 1% penicillin/streptomycin (GIBCO) and 10% fetal calf serum (FCS) (Omega Scientific, Tarzana, CA). Cells were used in passage 4 or 5 only, unless otherwise noted. The experiments presented in this study were performed using 6 different cases of normal BEC and 4 different cases of TuBEC. 6 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Recombinant human IL-8 (R&D Systems, Minneapolis, MN), tumor necrosis factor- alpha (TNF-a) (Roche Diagnostics, Indianapolis, IN), vascular endothelial growth factor (VEGF)(Upstate Biotechnologies), anti-VEGF antibody (Chemicon, Temecula, CA), transforming growth factor beta (TGF-pi) (R&D systems, Minneapolis, MN) and endothelin-1 (Et-1) (Bachem, Torrance, CA) were purchased from commercial suppliers. IL-8 receptor CXCR1 and CXCR2 neutralizing antibodies, and mouse isotype IgG control sera were purchased (BD Pharmingen, San Diego, CA). The anti- IL-8 neutralizing antibody was purchased from R&D systems (Minneapolis, MN).The ABC (avidin biotin complex) and AEC (aminoethyl carbazol) kits used for immunohistochemistry were purchased (Vector Laboratories, Burlingame, CA). Saponin used for permeabilization studies was purchased (Sigma-Aldrich). 3.2.2. Endothelial Cell Migration Assay Migration assays were performed using transwell chambers as described previously (Rosano et al., 2001). Briefly, 8 pm pore, 6.5 mm diameter polyethylene terephthalate filters (BD Biocoat, Bedford, MA) were coated on top and bottom by immersion in 0.5 % gelatin for at least 2 hrs, and air-dried overnight. Subconfluent cultures of BEC were harvested using trypsin/EDTA (0.05%) solution (GIBCO); 5xl04 cells were then resuspended in RPMI medium supplemented with 1% FCS and placed on the filter membrane in the top chamber (100 pl/well). Chemoattractants were placed in the lower compartment (600 pl/well). For receptor blocking studies, IL-8 receptor anti-CXCRl and anti-CXCR2 antibodies (4 pg/ml each) were added to the lower chamber 30 min 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. before the addition of IL-8 (1 ng/ml) or VEGF (1 ng/ml). Mouse isotype IgG served as the negative control for the CXCR1 and CXCR2 antibodies (BD Pharmingen). The isotype control IgG was also added to the cells 30 min before the addition of IL-8 at the same concentration as the IL-8 receptor neutralizing antibodies. Transwell plates were then incubated at 37°C in 5% CO2 for 6 hrs. At the termination of the experiment, media was removed from inserts, and the remaining cells on the upper side of the chamber were removed with a wet Q-tip. The filters were then stained with Diff-Quick staining solution (Dade Behring Inc., Newark, DE). Cells that migrated through to the underside of the filter membrane were counted under high-power magnification; 10 fields per filter were counted using the 40X objective. Groups were plated in triplicate; experiments; were repeated at least twice. 3.2.3. MTT Proliferation Assay BEC and TuBEC were seeded into 96-well plates (2.5 x l0 3 /well; 100 pi of cell suspension/well) in quadruplicate. Media was changed to RPMI 1% FCS 24 hrs before treatment with reagents. Cells were treated with IL-8 (1 ng/ml) and TNF-a (10 ng/ml) for 6, 24, 48 or 72 hours. At the end of treatment, the MTT (dimethyl thiazol diphenyl tetrazolium bromide) reagent was added (1:10 dilution) to the cultures for 4 hrs according to the manufacturer’s instructions (Sigma-Aldrich). The media was then removed, and dimethylsulfoxide (DMSO) was added (150 pl/well). The color intensity, which is proportional to the number of living cells, was quantified using the 570 nm filter wavelength. 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.2.4. Immunocytochemistry Cells were immunostained using a previously described method (Hofman et al., 1994). Briefly, cells (2.5xl04 cells/well) were plated on glass coverslips (Fisherbrand Microscope Cover Glass, Fisher Scientific, Tustin, CA) and coated with 1% gelatin. The cells were fixed in acetone, blocked with 5% normal horse serum in PBS, and incubated overnight with primary anti-IL-8 antibody (5 pg/ml) (R&D systems) or anti- CXCR1 and anti-CXCR2 antibodies (Chemicon). Samples were then incubated with biotinylated horse anti-goat IgG (1:100 dilution; Vector Laboratories) or biotinylated horse anti- mouse IgG (1:200 dilution; 0.01 mg/ml; Vector Laboratories, CA) for 45 min. Subsequently, slides were treated with the ABC (avidin biotin complex) elite (Vector Laboratories) for 30 min followed by aminoethyl carbazol (AEC) substrate kit (Vector Laboratories) according to the manufacturer’s instructions. Slides were counterstained with hematoxylin. A red precipitate identified positive staining. Pictures were taken using an Olympus BH2 microscope (Fluor lOx objective; numerical aperture 0.25) and a Nikon Coolpix 990 digital camera. 3.2.5. Flow Cytometry Subconfluent cell cultures were prepared as single cell preparations. Total IL-8 receptor expression in cells was determined by fixing cells in 2% paraformaldehyde for 15 min followed by permeabilization with 0.1% saponin for 15 min. For cell surface receptor expression detection, cells were harvested using Hanks-based dissociation buffer (GIBCO), and were not fixed or permeabilized. Cells were then incubated first 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. with primary mouse anti-human monoclonal antibody to either CXCR1 or CXCR2 for 30 min, followed by a 30 min incubation with secondary goat anti-mouse FITC conjugated antibody. Negative controls included the use of secondary FITC-labeled antibody only. Cells were then analyzed using the FAC Scan machine with appropriate software (BD). Data are expressed as percent positive cells. 3.2.6. IL-8 ELISA In order to determine whether there were differences in the production of IL-8 by BEC and TuBEC, endothelial cells were plated in triplicate in equal numbers in 6-well plates. Cells were cultured in RPM I1% FCS media for 48 hrs. Supernatants from cells were then collected and analyzed for IL-8 levels using the commercially available enzyme-linked immunosorbent assay (ELISA) kit as per manufacturer’s instructions (R&D systems). BEC cultured in media containing 10% FCS exhibited similar baseline levels of IL-8 in the ELISA assay with cells cultured in media containing 1% FCS (Hofman FM, unpublished observations). To determine the potential effect of VEGF on IL-8 production, TuBEC were plated in triplicate in equal numbers in 6-well plates. Media was changed to RPMI 1% FCS overnight, and cells were then incubated with rabbit anti-human VEGF antibody (10 pg/ml). Cells treated with rabbit serum (Vector Laboratories) served as an isotype control for the rabbit-anti human VEGF antibody. Supernatants from cells were collected after 48 hrs, and analyzed for IL-8 levels using the commercially available IL- 8 ELISA kit. 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. To determine the potential effects of TGF-pl on IL-8 production, BEC and TuBEC were plated in triplicate in equal numbers in 6-well plates. Media was changed to RPMI 1% FCS overnight and cells were treated with TGF-pi (10 ng/ml) or Et-1 (100 nM). Supernatants from cells cultured for 72 hrs in media containing 1% FCS were collected and analyzed for IL-8 levels. The concentrations of secreted IL-8 were determined using the commercially available IL-8 ELISA kit as per manufacturer's instructions. 3.2.7. RT-PCR IL-8 mRNA was detected in the following manner: Equal numbers of BEC and TuBEC (5 xlO4 cells) were lysed in cold cell lysis buffer (100 pi), and treated with DNAse I, according to the manufacturer’s instructions (Cells to cDNA II kit) (Ambion, Austin, TX). The lysate RNA was reverse-transcribed directly into cDNA according to the manufacturer's instructions (Cells to cDNA II kit) (Ambion). PCR was preformed using specific IL-8 gene primers according to the manufacturer’s instructions (IL-8 Gene Specific Relative RT-PCR kit; Ambion) using a PTC-100 thermocycler with an initial denaturation step at 94 °C (2 min), then 35 cycles at 94 °C (30 sec), followed by 57 0 C (30 sec), and 72 0 C (30 sec). Final extension of cDNA was perfomed at 72 °C (5 min). For negative control, cDNA was replaced with nuclease-free water in the PCR; and for positive control IL-8 DNA was amplified. The PCR mixture was heated to 94 °C (2 min), then 35 cycles at 94 °C (30 sec), followed by 57 ° C (30 sec), and 72 ° C (30 sec). Further extension of cDNA was performed at 72 °C for 5 min. The cDNA 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. samples were analyzed using gel electrophoresis in 2% agarose gel (Cambrex, Rockland, ME) containing 0.05 pg/ml ethidium bromide (Sigma-Aldrich). Bands were visualized under UV light and photographed using a BioRad Fluor-S Multi-imager (Hercules, CA). Equal loading was confirmed by amplification of 18S rRNA. IL-8 receptor mRNA was detected using the following protocol: Neutrophils were isolated by Ficoll-Hypaque (Amersham Pharmacia Biotech, Arlington Heights, IL) density centrifugation from peripheral blood. Total RNA was isolated from subconfluent BEC, TuBEC and neutrophils by a single step guanidium thiocyanate/phenol-chloroform extraction using TRIZOL reagent (Invitrogen, Carlsbad, CA). Genomic DNA was removed by DNase I treatment (Ambion). For reverse transcription, cDNA was generated using 2.5 pg of RNA in a total volume of 20 pi according to the manufacturer's protocol (Gene Specific Relative RT-PCR kit) (Ambion). PCR was performed using primers synthesized by the USC Microchemical Core facility of the USC/Norris Comprehensive Cancer Center (Unicersity of Southern California, Los Angeles, CA). The primer sequences used were: CXCR1 sense 5'- GGG GCC ACA CCA ACC TTC-3' and antisense 5'-AGT GCC TGC CTC AAT GTC TCC-3' (product size 363 bp); CXCR2 sense 5'-GGG CAA CAA TAC AGC AAA CT- 3' and antisense 5'-GCA CTT AGG CAG GAG GTC TT-3' (499 bp); p-actin sense 5'- CCA GAG CAA GAG AGG CAT CC-3' and antisense 5’ - CTG TGG TGG TGA AGC TGT AG-3' (436 bp). For each reaction, 5 pi of cDNA generated by RT reaction was amplified using specific primers (5 pM) in a final reaction volume of 50 pi. For CXCR1, PCR was performed for 40 cycles of 94 °C (45 sec), 57 °C (30 sec) and 72 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. °C (60 sec) followed by a final 72 °C step (6 min). For CXCR2 and (3-actin, PCR samples were subjected to 40 cycles of 94 °C (60 sec), 57 °C (150 sec) and 72 °C (60 sec) followed by a final 72 °C step (6 min). PCR products were separated on a 2% agarose gel and visualized by ethidium bromide staining (0.05 pg/ml). cDNA-minus PCR was used as a negative control, while cDNA from neutrophils served as a positive control. Equal loading of cDNA was confirmed by amplification of p-actin. 3.2.8. Statistical Analysis Values are presented as the mean ± SEM. Statistical significance was evaluated using Student's t-test for paired comparison. P<0.05 was considered statistically significant. 6 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.3. RESULTS 3.3.1. TuBEC Are Unresponsive to IL-8 Induced Migration Studies were performed to determine whether normal BEC or TuBEC responded to IL-8 treatment by migration. Cells were plated in the upper chamber of transwell plates, and IL-8 (1 ng/ml) was added to the lower chamber. Cells were then allowed to migrate for 6 hrs. All experiments were performed in triplicate cultures. The results (Figure 3-1 A) show that in untreated control cultures, TuBEC migration is greater than control BEC (P=0.02). Furthermore, upon activation with IL-8, normal BEC migration increases (P=0.003), while TuBEC do not respond to IL-8 (P=0.5). To determine whether this difference in response to IL-8 is dose-dependent, BEC and TuBEC were treated with a range of IL-8 concentrations (0.001-10 ng/ml). The results (Figure 3-1B) show that the migration of BEC cells is dependent on the amount of IL-8 added. The optimal dose of IL-8 for BEC is 1 ng/ml. However, there is no significant response of TuBEC cells to IL-8 within this concentration range. Based on these results, we used 1 ng/ml IL-8 for all the experiments presented in this study. Since previous studies by others (Heidemann et al., 2003) have shown that IL-8 induces endothelial cell proliferation, we asked whether increased migration of IL-8-treated BEC and TuBEC was a result of increased proliferation rather than migration. To test this, TuBEC and BEC, either stimulated with IL-8 or untreated, were analyzed using the MTT assay. MTT was added for the 4 hours after the completion of IL-8 treatment (6 hrs or 24 hrs), and cell cultures were then evaluated for proliferation. The results show that IL-8 does not induce proliferation of either BEC or TuBEC within 6 or 24 hrs of treatment; 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. additional experiments demonstrate that IL-8 does not induce proliferation after 48 and 72 hrs either (data not shown). TNF-a stimulated endothelial cell proliferation within 24 hrs, serving as a positive control. Thus, the constitutively high migration rate of TuBEC, and the increased BEC migration in response to IL-8 are not a result of an increase in cell number. These data demonstrate that TuBEC and BEC respond differently to IL-8 in migration. Effect of IL-8 on BEC and TuBEC migration ■ control ■ IL-8 TUBEC 6 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-1: Continued B Dose response effect of IL-8 on BEC and TuBEC migration 160 » « ® 140 o o ?■§ 120 a 9 100 o i M ■ o o k. ( 0 Q . E o o 0.001 0.01 0.1 1 10 0 ■BEC -TuBEC IL-8 concentration (ng/ml) Figure 3-1. IL-8 effect on BEC and TuBEC migration. A. Comparison of migration response to IL-8 in normal and tumor-associated BEC. Equal numbers of BEC or TuBEC (5x105 cells/ml) were plated in the upper chamber of transwell plates. After 6 hrs, cells that migrated through the filter membrane were counted. Data are expressed as the number of cells in 10 high power fields (HPF) under a 40X objective, and are the means of results from each experimental group, performed in triplicate. Bars correspond to standard error. Student t-test values correspond to: P(bec+ il8/bec) =0.003, P(T u B ec/bec)~0.02 and P(tu bec+ il8/ T u B E C ) = 0 . 5 . This experiment is representative of 10 experiments. B. Titration of IL-8 activity in BEC and TuBEC migration assay. Equal numbers of BEC and TuBEC were plated in the upper chamber of transwell plates. After 6 hrs, cells that migrated through the filter membrane were counted. Data are expressed as the percent increase in migration in response to different IL-8 concentrations. Each point represents triplicate values. Bars correspond to standard error. The data are representative of two experiments. 3.3.2. BEC and TuBEC Express Both CXCR1 and CXCR2 Receptors There is considerable controversy over whether both or only one IL-8 receptor is expressed in human microvessel endothelial cells (Heidemann et al., 2003). To address 7 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. this question, flow cytometry experiments were performed on BEC and TuBEC using specific monoclonal antibodies to the IL-8 receptors, CXCR1 and CXCR2. In the first set of experiments, endothelial cells were not permeabilized, so that only the surface receptors were examined. The results demonstrate that both receptors are expressed on BEC and TuBEC to similar extents (Figure 3-2A). We next permeabilized the cells, which resulted in staining of surface and cytoplasmic receptor determinants. These data exhibit higher receptor expression (Figure 3-2B); however the proportion of cells expressing both receptors was similar in both TuBEC and BEC. Flow cytometric analysis of IL-8 receptors expression was performed on TuBEC and BEC at an earlier passage (p-3). The results were similar with those obtained with passage 5 cells (data not shown). To confirm flow cytometry studies, TuBEC and BEC were immunostained on coverglass slides, using anti-CXCRl and anti-CXCR2 antibodies (Figures 3-2C). The results demonstrate that differences in the responses of BEC and TuBEC to IL-8 are not likely due to the absence or reduced expression of IL-8 receptors in TuBEC. To confirm these findings at the mRNA level, CXCR1 and CXCR2 mRNA expression was measured using semi-quantitative RT-PCR. The data show that both BEC and TuBEC have similar levels of CXCR1 and CXCR2 mRNA (Figure 3-2D). Polymorphonuclear granulocytes (PMNs) served as the positive control; experimental bands were evaluated based on p-actin levels. 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cell surface expression of IL-8 receptors in BEC and TuBEC 18 16 » a > 14 0 ® 12 1 10 o 9 - 8 c c O 6 X 4 ICXCR1 ICXCR2 BEC TUBEC B Total cellular expression of IL-8 receptors in BEC and TuBEC 60.0 50.0 g 40.0 > ( 0 o 30.0 0 £ O X o 20.0 10.0 0.0 ■ CXCR1 ■ C X C R 2 BEC TuBEC Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-2: Continued C CXCR1 BEC TuBEC J < \ CXCR2 * ' S ’ ' X - ' V - X V . X ’ f \ ■ 7 c , x h , > ' 1 x'. A ■ l r f i f : s SL A sA a i l s ? ■■£:'I* s~ 4 X - v- * X ♦ V ' \ X X * •**> 4 V "• '' ' < \ ' ■ # % s ! f c s ; L: W- t t % . < \ s * i S P ^ ' i M * * A ' 4 k l i l t - , 1 ■ 1 V " X • • Jk « « s - # . ; ^ Isotype control y g B B lliMi 1 ^ ( V i . ■ I B B S Figure 3-2. IL-8 receptor expression in BEC and TuBEC. A. Cell surface expression of IL-8 receptors on normal and tumor derived brain endothelial cells. Cells were labeled with anti-CXCRl or anti-CXCR2 antibody and analyzed using flow cytometry. 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Data are expressed as percent of receptor positive cells and are the means of results from each experimental group, performed in triplicate. These data are representative of 4 experiments. Bars correspond to standard error. B. Intracellular expression of IL-8 receptors in BEC and TuBEC. Cells were fixed, permeabilized, labeled with anti- CXCR1 or anti-CXCR2 antibody and analyzed using flow cytometry. Data are expressed as percent of receptor positive cells and are the means of results from each experimental group, performed in triplicate. These data are representative of 4 experiments. Bars correspond to standard error. C. CXCR1 and CXCR2 immunostaining in BEC and TuBEC. BEC and TuBEC were grown on glass coverlips and immunostained with anti-CXCRl and anti-CXCR2 antibodies. Positive cells were identified by the presence of a red precipitate (100 X magnification) .D. CXCR1 and CXCR2 mRNA detection in BEC and TuBEC using RT-PCR. Polymorphonuclear granulocytes (PMNs) served as the positive control for the expression of the receptor mRNA. P-actin served as the internal control. 3.3.3. TuBEC Constitutively Produce IL-8 Experiments were performed to determine whether the lack of responsiveness of TuBEC to IL-8 was a result of endogenous IL-8 production. To determine this, unstimulated TuBEC were cultured for 48 hrs. Subsequently, culture supernatants were collected and analyzed for the presence of secreted IL-8 using the ELISA technique. Results show (Figure 3-3A) that TuBEC constitutively produce approximately 3-fold higher amounts of IL-8 compared to BEC (P=0.004). In three different experiments, TuBEC consistently secrete 2 to 6 times higher levels of IL-8, compared to BEC. To determine whether the increased production of IL-8 was due to a few cells secreting large quantities of IL-8, or the general population of TuBEC producing higher levels of IL-8, endothelial cells were cultured on coverslips and directly immunostained for IL-8. The results demonstrate that TuBEC are significantly more positive for IL-8 than BEC (Figure 3-3B). The intensity of IL-8 staining is greater in TuBEC compared to BEC 7 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Figure 3-3B) and relatively homogeneous. Based on the number of positive cells and intensity of immunostaining, TuBEC exhibit higher IL-8 production compared to BEC. To determine whether increased production of IL-8 in TuBEC is a result of upregulation of mRNA expression, semi-quantitative RT-PCR for IL-8 mRNA was performed on untreated TuBEC and BEC. IL-8 bands were normalized to 18S rRNA bands. The data (Figure 3-3C) demonstrate a 4- fold increase in IL-8 mRNA in TuBEC, compared to BEC. To determine whether TuBEC could be stimulated to further secrete IL-8, these cells were treated with TNF-a and Et-1, reagents known to induce IL-8 production. Both factors stimulate IL-8 production in TuBEC, as well as BEC (data not shown). Thus, TuBEC constitutively produced IL-8, but can be further stimulated with appropriate mediators. IL-8 concentration in BEC and TuBEC supernatants 1800 ^ 1600 = 1400 o 1200 o 1000 800 600 400 ” 200 0 P=0.004 o > a. o o BEC TuBEC 7 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-3: Continued B BEC ■ i l B IllM t H mm ■ I ■ ill mmm TuBEC * 5 3 f < 8 | y ' i c * • ^ ~ > * V \ - K - ' - y * , % . ' v * ■ *• ‘ ' ■t... _ , . - , BEC TuBEC IL-8 mRNA 18s rRNA Figure 3-3. Comparison of IL-8 production by BEC and TuBEC. A. IL-8 secretion by TuBEC and BEC. Equal numbers of normal and tumor-associated endothelial cells (5xl04 /well) were plated in 6-well plates. After 48 hrs, IL-8 concentration was measured in culture supernatants; triplicate samples were assayed. Data are expressed 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. as pg of IL-8/105 cells. Bars correspond to the SEM. Student t-test value comparing untreated BEC and TuBEC corresponds to P ( t ub e c / b e c ) = 0.004. B. IL-8 immunostaining of BEC and TuBEC. Untreated cells were stained with primary mouse anti-human IL-8 antibody (5 pg/ml). Positive cells were identified by the red precipitate (100 X magnification). C. Detection of IL-8 mRNA expression in BEC and TuBEC using RT-PCR. mRNA in cell lysates from equal numbers of BEC and TuBEC (5xl04 cells) was converted directly into cDNA, which was then amplified using IL-8 primers (0.4 pM). 18S rRNA cDNA was also amplified as the internal control. D. IL-8 mRNA expression in BEC and TuBEC.The graph represents the relative ratio of the IL-8 bands intensity in comparison to the 18S internal control. The data are expressed as percentage intensity of IL-8 bands in BEC and TuBEC in comparison to their corresponding 18S rRNA bands. 3.3.4. Role of TGF-pi and VEGF in the Constitutive Production of IL-8 by TuBEC We hypothesized that a potential mechanism causing the constitutive production of IL-8 in TuBEC may be the lack of negative feedback regulation by inhibitory cytokines. One such suppressive growth factor, TGF-pi, has previously been shown to inhibit Et- 1-induced IL-8 production in BEC (Hofman et al., 1998). To test this hypothesis, we treated TuBEC and BEC with TGF-pl (10 ng/ml) for 72 hrs, harvested the supernatants and quantified the IL-8 content using an IL-8 ELISA. Optimal concentrations of Et-1 and TGF-pl were determined in previous studies carried out in this laboratory (Hofman et al., 1998). The results in Figure 3-4A demonstrated, as expected (Hofman et al., 1998), that TGF-pi inhibits Et-1-induced IL-8 production (P=0.004). By contrast, TGF-pi did not inhibit the production of IL-8 in TuBEC (Figure 3-4B). In fact, TGF- pi stimulated IL-8 production in TuBEC (P = 0.0006). We previously showed that TuBEC constitutively secrete VEGF (Charalambous et al., 2005) and this growth factor induces IL-8 production in brain endothelial cells (Lee et al., 2002). Thus another potential mechanism for constitutive IL-8 production in 77 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TuBEC may be the secretion and autocrine activation of VEGF. To test this, TuBEC were treated with anti-VEGF neutralizing antibody (10 |ag/ml) for 48 hrs. Supernatants were then harvested and IL-8 content was quantified as described. The data show that anti-VEGF treated TuBEC exhibit a significant decrease in IL-8 production (P=0.00002; Figure 3-4C). Thus endogenous VEGF produced by TuBEC, can induce IL-8 production in these cells. Therefore both TGF-pi and VEGF may be responsible, at least in part, for the observed constitutive production of IL-8 in TuBEC. A IL-8 production in TGF-|J 1 treated BEC 1000 900 _ 800 I 700 J* 600 ° 500 O . « . a. 400 3 300 200 100 0 control Et-1 (100nM) TGF-IJ1 (10 Et-1+TGF-G1 ng/m I) B IL-8 production in TGF-p1 treated TuBEC 3500 £ 3000 g 2500 “ o 2000 i) 1500 f 1000 d 500 0 7 8 ■ ■ I I control Et-1 (100nM) TGF-IS1 (10 Et-1+TGF-G1 ng/ml) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-4: Continued Effect of VEGF on IL -B production in TuBEC I S 50 1050 ■ 1455 - if 1255 - j “ o 1055 - 2 850 • I 655 • 450 ■ 200 ■ 0 ■ control 808-VEGF A b (10 fig/ml) isotype control a arum Figure 3-4. Effect of TGF-pl and VEGF on IL-8 production by TuBEC. A. TGF- pi inhibits Et-1 induced IL-8 production in BEC. Equal numbers of BEC (5xl04 /well) were plated in 6-well plates. The media was changed to RPMI containing 1% FCS, 24 hrs before initiation of the experiments. Cells were then treated with TGF- pl (10 ng/ml) and endothelin (Et)-1 (100 nM) for 72 hrs. All groups were plated in triplicate. After treatment, supernatant was collected from triplicate cultures and measured for IL-8 concentration levels. Data are expressed as IL-8 pg/ 105 cells. Student t-test values in comparison to the untreated control group correspond to: P(E t- i)=0.002; P(tgf-pi)=0.1; P(tgf-pi+ em)=0.33. Bars correspond to the SEM. B. TGF-pl does not inhibit IL-8 production by TuBEC. Equal numbers of TuBEC (5xl04 /well) were plated in 6-well plates. The media was changed to RPMI containing 1% FCS, 24 hrs before initiation of the experiments. Cells were then treated with TGF-pi (10 ng/ml) and Et-1 (100 nM) for 72 hrs. After treatment, supernatant was harvested from triplicate cultures and measured for IL-8 concentration. Data are expressed as IL-8 pg/10 cells. Student t-test values in comparison to the untreated control group correspond to: P (E t- i)=0.04; P(tgf-pi)=0.0006; P(tgf-pi+ em) =0.001.Bars correspond to the SEM. C. Anti- VEGF antibody treatment inhibits IL-8 production in TuBEC. Equal numbers of TuBEC (5xl04 /well) were plated in 6-well plates. The media was changed to RPMI containing 1% FCS, 24 hrs before initiation of experiments. Cells were then treated with anti-VEGF neutralizing antibody (10 pg/ml) for 48 hrs. As an isotype control, cells were treated with polyclonal rabbit serum (1:1000 dilution). After treatment, supernatants were harvested from the triplicate cultures and measured for IL-8 concentration values. Data are expressed IL-8 pg/105 cells. Bars correspond to SEM. 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Student t-test values in comparison to the untreated control group correspond to: P ( V E G F )-10 . 00002 ; P ( i s o t y p e c o n t r o l s e r u m ) - 0 - 8 . 3.3.5. Both CXCR1 and CXCR2 Are Responsible for IL-8 Induced Migration TuBEC and BEC were analyzed for potential functional differences in IL-8 receptor expression with respect to cell migration. To identify the IL-8 receptor subtype responsible for IL-8-induced BEC migration, BEC were pretreated with neutralizing anti-CXCRl (4 pg/ml) or anti-CXCR2 antibody (4 pg/ml), or both antibodies in transwell plates. The results show that after 6 hrs, either anti-CXCRl or anti-CXCR2 antibodies completely block IL-8-induced migration (P=0.00006 and 0.0003 respectively; Figure 3-5A). To confirm the specificity of these neutralizing antibodies, BEC were stimulated with VEGF and treated with both IL-8 receptor antibodies; these neutralizing antibodies do not block VEGF-induced migration (data not shown). As an additional control, isotype matched IgG does not block IL-8-induced migration (P=0.3; Figure 3-5A). To determine which IL-8 receptor is responsible for TuBEC migration, TuBEC were treated with anti-CXCRl or anti-CXCR2 or both reagents, as described above. The results (Figure 3-5B) demonstrate that, in contrast to BEC, each antibody partially blocks TuBEC migration while both antibodies have an additive effect in blocking TuBEC migration (P<0.05). Experiments were performed to determine whether IL-8 neutralizing antibody is able to block TuBEC migration. The results demonstrate that anti-IL-8 antibody significantly reduces TuBEC migration (P=0.02; Figure 3-5C). These data suggest that both TuBEC and BEC respond to IL-8 through 8 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. both IL-8 receptors, however the mechanism of this response may differ. In addition, the data suggest that IL-8 acts in an autocrine manner to induce TuBEC migration. A Inhibition of IL-8 induced m igration in BEC by anti- CXCRl and anti-CXCR2 antibodies 40 £ 35 5 30 ( 0 75 25 'R 20 o S 15 c 10 iilt I i 1 1 1 ^ S > ^ 9 ^ 9 8 > 8 > 8 > S > V kA j y ^ ^ r< ^ c<P o ® /v f ? ” 4 C iO .(j- ® ^ CA CA rT - A 0 . p r . p r CA < $ ' N x A sir B Inhibition of TuBEC m igration by anti-CXCR1 and anti-CXCR2 a n tib o d ies 40.0 35.0 H £ 30.0 1 25.0 © £ 20.0 0 < 5 15.0 1 io.o c 5.0 0.0 P=0.5 P= 0.00002 ■ H T p=o.oooi I I I ! control IL-8 anti- anti- a- CXCR1 Ab CXCR2 Ab CXCR1+a- CXCR2Ab 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-5 continued C Inhibition of TuBEC migration in resp o n se to anti- IL-8 neutralizing antibody control anti-IL-8 Ab anti-IL-8 Ab control serum (500 ng/ml) (1500 ng/ml) Figure 3-5. Effect of anti-CXCRl, anti-CXCR2, and anti-IL-8 neutralizing antibodies on BEC and TuBEC migration. A. Both CXCR1 and CXCR2 are responsible for IL-8 induced BEC migration. Equal numbers of cells (5x104 cells/well) were plated in the upper chamber of transwell plates and the migration assays were preformed as previously described. Neutralizing antibodies to the IL-8 receptors (4 pg/ml each) or isotype control IgG (4 pg/ml) were added to cells 30 min before addition of IL-8 (1 ng/ml). After 6 hrs, cells were counted and evaluated as described previously. Data are expressed as the mean of cells in 10 high power fields (HPF) with a 40X objective. All groups were set up in triplicate. The bars correspond to standard error. Student t-test values in comparison to the IL-8 treated BEC correspond to: P(a-cxcRi A b ) = 0.00006; P(a-cxcR2 A b ) =0.0003; P(a-cxcRi + a -cxcR2 A b ) = 0.0001. B. Anti- CXCRl and CXCR2 neutralizing antibodies (4 pg/ml) block TuBEC migration. Cells were plated in transwell chambers and migration was evaluated as described previously. After 6 hrs, cells that migrated through the filter membrane were counted. The bars correspond to standard error. Student t-test values (P values) shown on the graph correspond to the experimental groups compared to the untreated control. For groups treated with both anti-CXCRl and anti-CXCR2 antibodies, the P value in comparison to groups treated with a single blocking antibody was significant at 0.003. C.Anti-IL-8 neutralizing antibody blocks TuBEC migration. Cells were plated in transwell chambers, treated with anti-IL-8 antibody (0.5 and 1.5 pg/ml) and migration was evaluated as described previously. After 6 hrs, cells that migrated through the filter membrane were counted. The bars correspond to standard error. 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.4. DISCUSSION Glioblastomas are angiogenesis-dependent tumors, exhibiting a significant increase in blood vessel density during progression from low to high grade tumor (Brat and Mapstone, 2003). To provide an increasing blood supply as the tumor grows, these cancer cells secrete a number of pro-angiogenic growth factors such as IL-8, VEGF, basic fibroblast growth factor (bFGF), and transforming growth factor-alpha (TGF-a) (Koch et al., 2002; Plate and Risau, 1995; Jensen, 1998). IL-8 has been reported to regulate angiogenesis and metastasis in several different tumor types, as well as differentially affect endothelial cell migration and proliferation (Li et al., 2003; Heidemann et al., 2003). Data presented here demonstrate that untreated TuBEC have a higher baseline migration rate than BEC. This is likely the result of the constitutive production of angiogenic factors. Our studies show that TuBEC constitutively produce IL-8. These results are consistent with previous studies showing that endothelial cells derived from ovarian and kidney tumors constitutively produce high levels of IL-8 (Alessandri et al., 1999). To eliminate the possibility that contaminating glioma cells are responsible for this IL-8 production (Wakabayashi et al., 1995; Tada et al., 1993; Van Meir et al., 1997), extensive analyses were performed to confirm that TuBEC were exclusively endothelial cells (Charalambous et al., 2005). Recent studies have shown that normal human brain microvascular endothelial cells produce IL-8 when stimulated with VEGF (Lee et al., 2002). Tumor cells, as well as renal tumor-derived endothelial cells, express high levels of endogenous VEGF, compared to negligible levels of VEGF in their normal endothelial cell counterparts 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Bussolati et al, 2003). Similarly, our previously published work showed that TuBEC derived from glioma tissue, secreted high amounts of VEGF (Charalambous et al., 2005). Therefore, we examined the role of VEGF as a potential trigger for the observed constitutive IL-8 production by TuBEC. Our results demonstrate that treatment of TuBEC with anti-VEGF neutralizing antibody decreases IL-8 production, suggesting that VEGF plays a vital role in the ability of TuBEC to produce IL-8. These studies imply that TuBEC-derived VEGF is, at least in part, responsible for the production of IL-8 in these cells. The potential action of IL-8 on VEGF production is not clear. There is little evidence in the literature referring to the effects of IL-8 on VEGF production in brain endothelial cells. Our studies show that IL-8 does not affect VEGF receptor expression in brain microvessels (data not shown). Thus the reciprocal effect of IL-8 on VEGF production seems unlikely. Differences between TuBEC and BEC responsiveness to IL-8 are not likely due to variations in IL-8 receptor expression, as demonstrated by flow cytometry and receptor neutralization data. In the case of IL-8, high levels of this chemokine are associated with receptor desensitization, due to receptor internalization. Once the ligand is removed, the receptor is recycled to the cell surface (Murphy, 1997; Matityahu et al., 2002). Since TuBEC do not appear to be responsive to IL-8, the possibility exists that there may be aberrant recycling of the IL-8 receptor. However, both ligand bound and unbound receptors are recycling from the cell membrane (Salcedo et al., 1999; Salcedo et al., 2000). Therefore, it is not surprising that intracellular expression of IL-8 receptors is similar in TuBEC and BEC. The data in figures 3-2A and 3-2B indicate 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. that the differences in function between TuBEC and BEC were not likely due to differences in numbers of receptors, either on the cell surface, or those internalized within the cell. These studies, however, do not determine whether the receptor affinities of these cells are similar. Expression levels of IL-8 receptors on all endothelial cells are not homogeneous, but organ and tissue dependent. Human intestinal microvascular endothelial cells only express CXCR2 (Heidemann et al., 2003), while both CXCR1 and CXCR2 were detected on human dermal microvascular endothelial cells and human umbilical vein endothelial cells (HUYEC) (Li et al., 2003). The results here demonstrate that quantitatively, IL-8 receptor expression on endothelial cells derived from the normal brain and from tumor microenvironment is similar. However, our inhibition studies suggest that IL-8 receptor function in TuBEC may differ from BEC. In TuBEC, inhibition of migration is achieved by blocking both receptors. In normal BEC, either receptor antibody will block IL-8 induced migration. These differences in responses between BEC and TuBEC may represent potential differences in IL-8 signaling pathways. Inhibition of TuBEC migration by IL-8 receptors antibodies and IL-8 antibody suggests that endogenous IL-8 produced by TuBEC acts in an autocrine manner to induce their migration. Normal BEC secrete IL-8 upon activation, and respond to TGF-pl by downregulating IL-8 (Hofman et al., 1998). Extensive studies were performed by this and other laboratories showing that TGF-pi acts as an inhibitory growth factor on normal endothelial cells, smooth muscle cells, and fibroblasts (Hofman et al., 1998; Ma et al., 2000; Debacq-Chainiaux et al., 2005). However, in different pathologic 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. conditions, such as rheumatoid arthritis and atherosclerosis, cells become resistant to the suppressive activity of TGF- pi (Cheon et al., 2002; Me Caffrey et al, 2000). These studies correlate resistance to TGF-P 1, to a decrease in type II TGF-P 1 receptor expression (Me Caffrey et al., 2000). Our data show that TuBEC do not respond to TGF-pi as BEC. In fact, TGF-pi appears to have a stimulatory effect on IL-8 production in TuBEC. In various normal cell types, IL-8 expression was shown to be differentially downregulated by TGF-pi, IL-4 or IL-10 (Chen and Manning, 1996). However, several reports demonstrate that TGF-pl can also have a stimulatory effect on various cell types. TGF-pi has been shown to stimulate IL-8 production in smooth muscle cells (Fong et al., 2000). TGF-pi also induces SDF-1 signaling in human macrophages by increasing CXCR-4 (Stromal Derived Factor-1 receptor) expression (Chen et al., 2005). In addition, TGF-pl induces VEGF production from conjuctival fibroblasts (Asano-Kato, 2005). Gliomas have been shown to produce high levels of both TGF-pl and TGF-P2, but not IL-10 and IL-4 (Pamey et al., 2000; Sakaki et al., 1995), while normal human astrocytes do not produce either IL-4 or IL-10, and only produce TGF-P in its latent form (Hori et al., 1999). Therefore in TuBEC, TGF-pi may function in a stimulatory manner on endothelial cells, rather than inhibitory. This growth factor has indeed been shown to be pro-angiogenic in specific environments (Benckert et al., 2003; Duff et al., 2003; Buschmann et al., 2003; Nagakawa et al., 2004). Therefore, the stimulation of IL-8 production by TGF-P 1 in TuBEC may contribute to the observed constitutive production of IL-8 by these cells. 8 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In summary, these studies show that primary cultures of human tumor-associated brain endothelial cells constitutively produce IL-8, which acts in an autocrine manner to induce TuBEC migration. The production of this chemokine is stimulated by VEGF and TGF-P 1. Understanding the functions and regulatory processes of tumor-associated endothelial cells is critical for developing appropriate anti-angiogenic therapies. 8 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER IV SENESCENT PHENOTYPE OF TUMOR-ASSOCIATED ENDOTHELIAL CELLS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.1. INTRODUCTION A major concept in cancer biology is that tumor growth is dependent on the blood supply provided to the tumor (Hanahan and Folkman, 1996). One proposed source of these vessels is the expansion of normal preexisting microvessels surrounding the tumor, a process known as angiogenesis (Cameliet, 2003). Circulating endothelial stem cells may also lodge in the tumor and become sites of blood vessel growth, a process known as vasculogenesis (Jain, 2003). Both angiogenesis and vasculogenesis are normal events occurring during wound healing and development (Hanahan and Folkman, 1996). However, blood vessels within the tumor, particularly in gliomas, are structurally and functionally very different from normal vessels (Bergers and Benjamin, 2003; Charalambous et al., 2005). Tumor-associated blood vessels are leaky and hemorrhagic, displaying tortuous and disorganized structures, while normal vessels form orderly networks with tight junctions (Bergers and Benjamin, 2003). The specific stimulus for abnormal blood vessel development in cancer is not known. However, the tumor microenvironment is thought to play an important role (Vakjoczy et al., 2002; Cameliet and Jain, 2000; Ferrara, 2001). Due to the highly proliferating nature of neoplastic cells, the tumor environment is often hypoxic and depleted of essential nutrients (Yancopoulos et al., 2000). Tumor cells also secrete an array of growth factors, many of which regulate and stimulate endothelial cell proliferation and survival (Cameliet and Jain, 2000; Ferrara, 2001). These growth factors, particularly vascular endothelial growth factor (VEGF), have been shown to induce endothelial cell proliferation, and migration (Yancopoulos et al., 2000). Growth factors involved in 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. normal wound healing are secreted locally and have a limited half-life, remaining active for the specific period of infection or tissue remodeling (Bergers and Benjamin, 2003). By contrast, in the neoplastic process, tumor-associated brain endothelial cells (TuBEC) are exposed to a constant environment of pro-angiogenic factors for months or years. In this study we show that chronic long-term exposure of TuBEC to this highly mitogenic and stressful environment results in profound changes consistent with the senescent phenotype. In an earlier study, we demonstrated that TuBEC have unique morphologic and functional features compared to primary cultures of normal brain-derived control endothelial cells (BEC) (Charalambous et al., 2005). TuBEC are large, irregularly shaped, and demonstrate a veil-like appearance, unlike normal endothelial cells (Charalambous et al., 2005). Importantly, TuBEC cultures exhibit markedly reduced proliferative activity, and have a decreased rate of apoptosis compared to BEC (Charalambous et al., 2005). These unique features of TuBEC are also characteristics of a variety of cell types, including fibroblasts, lymphocytes, and endothelial cells, that have reached end stage senescence in cell culture after extensive proliferation or as a consequence of oxidative stress (Krtolinca et al., 2001; Effros, 2005; Foreman and Tang, 2003). If TuBEC are indeed senescent endothelial cells, their angiogenic characteristics, and resistance to cell death in response to cytotoxic drugs might be fundamentally different from normal non-senescent cells. To investigate the cellular characteristics of tumor-associated endothelial cells, primary cultures of these cells 9 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were isolated from human glioblastoma tumor tissue, analyzed for purity, and evaluated by a variety of functional criteria. The results reported here show that TuBEC, both in culture and in situ within the tumor, stain positively for senescence-associated P-galactosidase (SA-P-gal). Furthermore, TuBEC are arrested at Gi phase of the cell cycle, are more resistant to cytotoxic drugs, and produce more growth factors compared to normal BEC. These data support the notion that tumor-associated endothelial cells show characteristics of typical senescent cells. A thorough understanding of the physiological properties of tumor-associated endothelial cells is essential for the development of effective and selective anti-angiogenic therapies. 91 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.2. METHODS 4.2.1. Cell Culture TuBEC and BEC were isolated from glioblastoma multiforme and normal human brain tissue respectively as previously described (Charalambous et al., 2005). All tissues were obtained within the guidelines of the Institutional Review Board of the Keck School of Medicine, University of Southern California. Endothelial cells were cultured in RPMI 1640 medium (GIBCO Laboratories, Grand Island, NY) supplemented with 100 ng/ml endothelial cell growth supplement (ECGS) (Upstate biotechnologies, Rochester, NY), 2 mM L-glutamine (GIBCO), 10 mM HEPES (GIBCO), 24 mM sodium bicarbonate (GIBCO), 300 U heparin USP (Sigma-Aldrich, St.Louis, MO), 1% penicillin/streptomycin (GIBCO) and 10% fetal calf serum (FCS) (Omega Scientific, Tarzana, CA). A 172 and LN229 glioblastoma cell lines were cultured in RPMI 1640 and DMEM (GIBCO) media respectively supplemented with 10% FCS. BEC and TuBEC were used up to passage 5 only. Cell viability was determined to be greater than 99% as assessed by trypan blue exclusion. 4.2.2. Reagents For cell characterization, the following reagents were used: von-Willebrand factor (vWF) (DAKO, Carpinteria, CA), CD31 (Santa Cruz Biotechnology, Santa Cruz, CA), and CD 105 (Santa Cruz Biotechnology) for endothelial cells, glial fibrillary acidic protein (GFAP) (DAKO) for astrocytes and glioma cells, CDllb (Immunotech, Villepinte, France) for macrophages/microglia, and SMA (smooth muscle actin) 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (DAKO). Tumor supernatants were derived from the LN229 glioblastoma cell line; supernatants were harvested every 48 hr, centrifuged and filtered using a micropore filter (0.2 pm pore). 4.2.3. Cell Cycle Analysis Cells were stained with propidium iodide (PI), and their DNA content was analyzed using flow cytometry. Briefly, cells were fixed in 70% ethanol, incubated with the PI staining solution (containing 10 pg/ml PI and 100 pg/ml RNase) for 15 min at 37 °C, and analyzed for DNA content using flow cytometry. 4.2.4. Immunohistochemistry Cells were immunostained using a previously described method (Hofman et al., 1998). Briefly, cytocentrifuge cell preparations were fixed in acetone, blocked with 5% normal goat serum in PBS, and incubated overnight with primary anti-PDGF-A or anti- PDGF-B polyclonal antibodies (1:100) (Santa Cruz Biotechnology). Samples were then incubated with biotinylated goat anti-rabbit antibody (1:400 dilution; Vector Laboratories, Burlingame, CA) for 45 min. Subsequently, slides were treated with the ABC (avidin biotin complex) (Vector Laboratories) for 30 min followed by AEC- aminoethyl carbazol substrate kit (Vector Laboratories) according to the manufacturer’s instructions. The red precipitate identifies positive staining; slides were counterstained with blue hematoxylin. 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.2.5. MTT Cell Growth Assay Cells were seeded in 96-well plates (2.5 xlO /well; 100 pl/well) in quadruplicate in RPMI 10% FCS. After attachment, cells were treated with a range of concentrations of CPT-11 (0 - 200 pM) (Pharmacia, New York, NY) for 96 hr. The MTT reagent was then added (1:10 dilution) to cultures for 4 hr, according to the manufacturer’s instructions (Sigma-Aldrich, St Louis, MO). Medium was removed, dimethylsulfoxide (DMSO) was added (150 pl/well), and color intensity, proportional to the viable number of cells, was measured at 570 nm. 4.2.6. Western Blot Analysis Cell extracts from endothelial cells cultured in RPMI supplemented with 10% FCS were quantitated using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Equal amounts of proteins (50 pg/lane) were separated on SDS-PAGE and electrotransferred to 0.45 pm nitrocellulose membranes. The membranes were then blocked with 5% non-fat dry milk in TBST and probed with anti-p21 (1:500), anti-p27 (1:500), anti-cyclin A (1:500), anti-cyclin E (1:500), anti-cyclin D1 (1:500), anti-cyclin B (1:500), anti-PDGF-A (1:500) and anti-PDGF-B (1:500) polyclonal antibodies (Santa Cruz) or anti-p53 (1:500) and anti-GAPDH (1:5000) monoclonal antibodies followed by HRP-conjugated anti-rabbit or anti-mouse IgG respectively. Protein bands were detected by chemiluminescence using the SuperSignal™ substrate (Pierce) according to the manufacturer’s protocol and analyzed using a Phosphorlmager (Hope Micro-max, Freedom Imaging, Anaheim, CA). 94 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.2.7. Cell Death ELISA Endothelial cells were plated in 96-well plates in quadruplicates at 7.5xl03 /cells (100 pl/well). Cells were lysed after 96 hr; lysates were analyzed for the presence of nucleosomes using the Cell death detection ELISA Plus kit (Roche Diagnostics, Indianapolis, IN). Absorbance was measured at 405 nm. 4.2.8. Senescence-Associated P-Galactosidase Assay Cytochemical detection of senescence associated (3-galactosidase (SA-p-gal) was performed at pH 6, as previously described (Dimri et al., 1995; van der Loo et al., 1998). Briefly, cells and tissue were fixed in 2% parafomaldehyde for 5 min or 1% neutral-buffered formalin for 1 min respectively, washed with PBS, and transferred overnight at 37 °C to a solution containing 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM MgCl2, 0.02% NP-40, 0.01% deoxycholate, 40 mM sodium citrate, 150 mM NaCl and 1 mg/ml X-gal (X-galactosidase; Calbiochem, San Diego, CA). The solution was titrated to pH 6 with NaH2P0 4 . Stained cultures were viewed under a light microscope and assessed for P-gal content. 4.2.9. BrdU cell Proliferation Assay Cells were seeded in quadruplicate in 96-well plates at 2.5x103 cells/well. Medium was changed to RPMI with 1% FCS 24 hr before treatment. Cells were treated with Et- 1 or VEGF for 48 hr. The BrdU reagent was added to cells for the last 18 hr of treatment and proliferation evaluated using the BrdU labeling and detection kit III 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Roche Diagnostics, Indianapolis, IN). The number of proliferating cells was proportional to absorbance at 405 nm, which was proportional to BrdU incorporation. 4.2.10. Statistical Analysis Values are presented as the mean + SEM. Statistical significance was evaluated using Student's two-tailed t-test. P<0.05 was considered statistically significant. 9 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.3. RESULTS 4.3.1. TuBEC Demonstrate an Arrest in Go/Gi Phase of Cell Cycle Our previous study demonstrated that the rate of replication and doubling time of TuBEC cultures were slower than those of BEC (Charalambous et al., 2005). To determine whe ther there was an arrest in cell cycle, subconfluent cultures of TuBEC and BEC, at equivalent passage, were cultured in RPMI containing 10% FCS, and analyzed using propidium iodide (PI) staining. The results in Figure 4-1A show that 81.6 ± 1.4% of the TuBEC population are in the Go/Gi phase of cell cycle, a 26.4 ± 1.07% increase compared to BEC (P=0.002). There was a correspondingly substantial decrease in the proportion of S phase (P=0.03) as well as a decrease in the G2/M phase cells in the TuBEC cultures (P=0.01). Three different primary TuBEC and three different BEC cultures were tested, all showing similar data. To identify the specific phase of cell cycle arrest in TuBEC, cell cycle proteins and inhibitors were measured using western blot analysis. The results show that TuBEC have a decreased expression of cyclin A and increased expression of cyclin Di compared to BEC (Figure 4-1B). The cell cycle inhibitors p21 and p27 were also examined and shown to be increased (Figure 4-IB). The levels of cyclins B and E remained relatively constant. Since accumulation of p53 is one of the major characteristics of cells reaching senescence in vitro (Donehower, 2002), TuBEC were tested for expression of p53 using western blot analysis. The results in Figure 4-IB demonstrate that p53 is highly expressed in TuBEC compared to BEC. To eliminate the possibility that the observed decreased proliferation rate in TuBEC was due to the absence of tumor growth factors usually 97 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. present in tumor microenvironment, TuBEC were treated every 48 hours with 50% glioma supernatant for 15 consecutive days. Cell cycle analysis demonstrated that under these conditions, the percent of TuBEC in Go/Gi phase did not change compared to the untreated TuBEC (data not shown). Cell cycle analysis of BEC and TuBEC B BEC TuBEC Cyclin A Cyclin B Cyclin Di Cyclin E p27 p21 p53 GAPDH 98 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4-1: Continued Cell cycle relative protein expression in BEC and TuBEC o w w £ Q . S < 1 ) > < u 0 £ I BEC I TuBEC Cyc A Cyc B Cyc D1 Cyc E p21 p27 p53 Figure 4-1. Cell cycle analysis of BEC and TuBEC and cell cycle protein expression. A. Cell cycle analysis of TuBEC and BEC. Untreated TuBEC and BEC were stained with the propidium iodide (PI) solution for 15 min and analyzed for cell cycle phase distribution using FACS. The data are expressed as a percentage of positive cells in the respective cell cycle phase. Bars correspond to the standard errors of the mean. B. Cell cycle proteins and p53 expression. Cyclin enzymes and inhibitors, as well as p53 expression in untreated TuBEC and BEC were examined using western blot analysis. Cellular proteins were visualized using antibodies to cyclin A, cyclin B, cyclin Di, cyclin E, p21, p27, p53 (1:500 dilution) and GAPDH (1:5000 dilution). These data are representative of 3 different TuBEC and 3 different BEC samples. C. Comparison of relative cell cycle proteins expression in TuBEC and BEC. Data are expressed as ratio of western blot band intensity: loading control (GAPDH) intensity. Table 4-1. Cell cycle analysis of BEC and TuBEC. Cell cycle phase BEC (%) TuBEC (%) G0 /G, 63.2 81.6 S 16.1 6.9 g 2 /m 21.2 11.7 9 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.3.2. TuBEC Do Not Proliferate in Response to Cytokines and Growth factors To determine whether TuBEC can be induced to proliferate, cells were treated for 48 hr with endothelin-1 (Et-1) (100 nM) or YEGF (10 ng/ml). Proliferation was assayed using BrdU incorporation. Both Et-1 and YEGF induce BEC proliferation (Pem=0.01, Pvegf=0.001), with little effect on TuBEC proliferation (Pem=0.3, PV egf=0.2; Figure 4- 2). Thus TuBEC can not be stimulated to proliferate by typical pro-angiogenic factors. Effect of Et-1 and VEGF on BEC and TuBEC 180 -| 160 - 140 - O O 120- c < d 100- < o C D _ _ 2 80 4 o ■ E 60 - £ 40 - 20 0 I BEC ITuBEC Et-1 (100 nM) VEGF (10 ng/ml) Figure 4-2. Effect of growth factors on TuBEC proliferation. TuBEC and BEC were incubated with VEGF (10 ng/ml) or Et-1 (100 nM) for 48 hr. Proliferation was then quantitated using BrdU incorporation. The data are expressed as percentage increase in OD of treated versus untreated cells. Each experiment was performed in quadruplicates. The bars correspond to standard error. 100 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.3.3. TuBEC Express Senescence-Associated P-Galactosidase (SA-P-gal) The signature characteristic for a variety of cell types that reach senescence in vitro is positive staining for P-galactosidase (SA (3-gal) at pH 6. This marker has also been validated on cell samples isolated from donors of different ages (Dimri et al., 1995). To confirm that the TuBEC population contains senescent cells, subconfluent cultures of TuBEC were stained for SA p-gal at pH 6 (Dimri et al., 1995; van den Loo et al., 1998). Results show that typically, over 80% of the TuBEC were positive for SA p-gal (Figure 4-3A), while control BEC exhibited negligible SA P-gal staining (Figure 4-3B); cells at the same passage were used. SA P-gal was consistently observed in the five TuBEC cases tested. To determine whether this senescent characteristic is acquired in vitro or is a true reflection of the in vivo status of the endothelial cells within the tumor, glioma tissue and normal brain samples were stained for SA P-gal. The results demonstrate that blood vessels within the glioma tissue are positive for SA p-gal (Figure 4-3C); normal brain tissue does not stain (Figure 4-3D). These results demonstrate that endothelial cells within tumors, in situ and in culture, express SA p-gal, a defining characteristic of senescence. 101 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4-3. Immunocytochemical detection of P-galactosidase. Cytocentrifuge preparations of TuBEC (A) and BEC (B) were fixed and stained with X-gal at pH 6.0. The intense blue color in the cytoplasm signifies positive staining. (400X magnification.) Glioblastoma multiforme tissue (C) and normal brain tissue (D) were fixed and stained with X-gal solution as described above. The detection of blue vascular structures in the tissue demonstrates positive staining (100X magnification). 4.3.4. TuBEC Are Functionally Active Cells Replicative senescent cells, although non-proliferating, are metabolically active and produce a variety of soluble factors (Krtolica et al., 2001; Effros et al., 1995; Smith and Pereira-Smith, 1996). To determine whether TuBEC have retained the capacity to produce growth factors, we compared the levels of intracellular PDGF in TuBEC and BEC. Using immunohistochemistry, we show that TuBEC (Figure 4-4A) have higher cytoplasmic expression of PDGF-A compared to BEC (Figure 4-4B); similar results 102 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were observed with PDGF-B (data not shown). To further verify the differences in growth factor expression, western blot analysis was performed (Figure 4-4C). These data show that PDGF-A and B are constitutively upregulated in TuBEC. Therefore, these data demonstrate that TuBEC are metabolically active and able to produce high levels of growth factors. B C BEC TuBEC A172 : I m J V J I E 9 GAPDH Figure 4-4. PDGF-A and PDGF-B expression in TuBEC and BEC. To determine whether TuBEC are constitutively producing growth factors, untreated cells were analyzed for PDGF-A and PDGF-B protein expression. Cytocentrifuge preparations of TuBEC (A) and BEC (B) were immunostained with anti-PDGF-A. The red precipitate within the cytoplasm denoted positive staining (200X magnification). C. Western blot analysis was performed on untreated TuBEC and BEC using anti-PDGF-A and -B antibodies. The glioma cell line A172 served as the positive control. 103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.3.5. TuBEC Are More Resistant to Chemotherapeutic Drugs than BEC Senescence in many cell types is associated with resistance to cell death. Our own previous studies have shown that TuBEC, cultured in serum-free media, undergo less spontaneous apoptosis than BEC (Charalambous et al., 2005). To determine whether TuBEC are more resistant to cancer chemotherapeutic drugs compared to BEC, endothelial cultures were treated with the chemotherapeutic agents irinotecan (CPT-11) and temozolomide, and tested for cell death using the MTT assay and cell death ELISA. TuBEC and BEC were incubated with CPT-11 at doses ranging from 0-200 pM for 96 hr. The results demonstrate that TuBEC are consistently more resistant to CPT-11 as compared to BEC. After 96 hr of CPT-11 treatment at 50 pM (Figure 4-5 A), TuBEC are approximately 3.1-fold more resistant to the drug compared to BEC (P=0.03). The glioma cell line, A172, served as the positive control for drug-induced cell death. To confirm that the observed differences in the MTT assay were due to cell death, the cell death ELISA was performed. Figure 4-5B demonstrates that treatment of endothelial cells with CPT-11 (50 pM) induces significantly greater cell death in BEC compared to TuBEC (P=0.001). In addition, BEC and TuBEC were treated with different concentrations of temozolomide for 7 days. Similar to CPT-11, the results also demonstrate that TuBEC are resistant to temozolomide-induced cell death. These data demonstrated that TuBEC are relatively resistant to drug-induced cell death, similar to other types of senescent cells, and different from control BEC. 104 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B Effect of CPT-11 on BEC and TuBEC cell death E c i n o Q O 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 BBEC ■TuBEC 0 50 CPT-11 concentration (^M) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4-5: Continued Effect of tem o zo lo m id e long- term tre a tm e n t on BEC a n d TuBEC E c IO © Q O 0.400 0.350 0.300 0.250 0.200 0.150 0.100 0.050 0.000 ■ BEC ■ TuBEC 0 300 Temozolomide concentration (pM) Figure 4-5. Cytotoxic effects of CPT-11 and temozolomide on TuBEC and BEC. Endothelial cells were treated for 96 hr with a range of CPT-11 concentrations (0-200 pM). At the termination of the experiments, cell viability was determined using the MTT assay (A), and cell death measured using the Cell death ELISAplus kit (B). The MTT data, measured as OD at 570 nm, are expressed as percentage of viable cells per well as compared to the untreated control cells. The glioma cell line, A 172 served as the positive control. The cell death ELISA data, analyzed at an OD of 405 nm, are proportional to the number of nucleosomes present and therefore number of dead cells per well. Each experiment was performed in quadruplicates. The bars correspond to the standard error. C. Endothelial cells were treated for 7 days with temozolomide (300 pM). At the termination of the experiment, cell death was determined using the Cell death ELISAplus kit. Each experiment was performed in quadruplicates. The bars correspond to the standard error 4.3.6. Tumor-Derived Supernatants Induce Morphologic Characteristics of Senescent Cells in BEC To determine whether the tumor microenvironment may be responsible for the phenotypic changes observed in TuBEC, BEC were exposed to supernatants derived 106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. from LN229 glioblastoma tumor cells or normal endothelial cells, over the course of 20 population doublings (PDs). The results demonstrate that continuous exposure to fresh tumor-derived supernatants induced changes in BEC morphology. Figure 4-6A demonstrates that BEC cultures treated for 20 PDs in untransformed-cell culture supernatants have the appearance of normal endothelial cells, while BEC treated with tumor cell-derived supernatants become large, flattened, and veil-like cells. This unique morphology was similar to that observed for TuBEC. In addition, in order to determine whether long-term culture of BEC with glioma supernatant would induce the expression of senescent cells, the cultures were stained and evaluated for p-gal expression. The results (Figure 4-6B and C) demonstrate that the percentage of P-gal positive cells is higher in the cells cultured with glioma supernatant in comparison to the cells cultures with regular culture media. Consequently, these data demonstrate that a consequence of chronic exposure of BEC to soluble pro-angiogenic factors derived from tumors can be the induction of TuBEC phenotypic characteristics. 107 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. BEC (p15) + control media V * ■ r * — ( ■ « »* TuBEC (p5) r f i • V * m i — * r ; . \ * ; I i ni O ^ x « — i r f * h* V * ■ I , , M * //* * * * A & _ T r f l BEC (p15) + LN229 supernatant % V XK 5 — « f-i ► t V 4U ^**21 B BEC control BEC + LN229 supernatant m i 108 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4-6 continued C E x p ressio n of s e n e s c e n t cells in BEC tre a te d with gliom a s u p e rn a ta n t 18 control LN229 sup Figure 4-6. Effect of glioma supernatant on endothelial cell morphology and (3 - galactosidase expression. BEC were cultured for 20 population doublings with supernatants derived from normal endothelial cells or supernatants from the glioblastoma cell line, LN229. (A) Morphology of BEC treated with normal media or LN229 supernatant after 20 population doublings as compared to morphology of untreated TuBEC at passage 5. (100X magnification). (B) BEC treated with normal media or LN229 supernatant after 20 population doublings were stained for P-gal. (C) Comparison of P-gal expression in BEC treated with normal media or LN229 supernatant after 20 population doublings. 109 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.4. DISCUSSION The results of our study provide the first demonstration that glioblastoma-associated TuBEC have characteristics of senescence. The data show that a significant proportion of TuBEC, approximately 50-80%, have many of the unique and identifiable traits that have been attributed to senescent cells. In normal tissue, endothelial cells are relatively quiescent, undergoing short periods of proliferation when stimulated (Foreman and Tang, 2003). In contrast, tumors provide a microenvironment composed of a variety of powerful growth factors, particularly VEGF (Yancopoulos et al., 2000). In addition to stimulating replication, these growth factors can also accelerate the accumulation of senescent cells (Kurz et al., 2003). The relationship between replication and senescence was established over 40 years ago; Hayflick and others showed that normal, untransformed cells replicate for approximately 50 cycles before undergoing irreversible cell cycle arrest and replicative senescence (Hayflick and Moonhead, 1961). Although senescent cells do not proliferate, these cells are functionally and metabolically active (Krtolica et al., 2001; Roninson et al., 2001). Senescence appears to be the result of two simultaneous events: strong mitogen stimulation and a block in cyclin-dependent kinases activity (Blagosklonny, 2003). The data presented here demonstrate that TuBEC have become senescent. The trigger for the induction of this process is not clear. TuBEC senescence may be the result of constant mitogenic stimulation by the tumor environment, resulting in a combination of a high rate of replication and increased stress to the cells. Recent evidence has shown that there is a common sub-fraction of genes activated in both 110 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. replicative and stress-induced senescence, while other genes and proteins are different (Dierick et al., 2002). Nevertheless, the terms replicative or stress-induced senescence are often used interchangeably; and we will continue to use the generic term senescence in the present study (Shay and Roninson, 2004). Data from a variety of studies have validated SA P-gal staining at pH 6 as an essential hallmark of senescent cells (Dimri et al., 1995; van der Loo, 1998; Kurz et al., 2000). We show here that TuBEC stain positively for SA p-gal; furthermore, this staining correlates with typical morphologic features of senescence, such as large, flat, veil-like cells with a decreased growth rate (van der Loo, 1998; Kurz et al., 2000). We have previously described the morphologic characteristics of TuBEC without addressing the association to senescence (Charalambous et al., 2005). In all five TuBEC samples tested, SA p-gal was positive in 50-80% of cells. Since senescence can be a result of cell survival following drug exposure (Roninson et al., 2001), it is important to emphasize that SA P-gal was positive in TuBEC derived from both treated and untreated glioblastoma patients. The percent positive cells varied from case to case, and reflects the heterogeneous endothelial cell population in TuBEC cultures. However, unlike the normal BEC cultures, which can be passaged up to passage 20, TuBEC cultures become static at passage 6 and do not proliferate further, while remaining viable. Thus TuBEC cultures contain senescent cells, and a subpopulation of pre-senescent endothelial cells. In addition to these in vitro results, SA P-gal staining was also detected in the typically tortuous blood vessels within tumor tissue, while normal brain specimens 111 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were negative. SA p-gal staining in tumor tissue appeared to be regional, suggesting that not all TuBEC in the tumor tissue are senescent. The formation of senescent vessels in the tumor is likely to be dynamic process, with regions of actively proliferating cells at the periphery of the tumor, and senescent TuBEC in areas closer toward the center of the tumor (Me Lendon et al., 1998). Pathological analysis of gliomas emphasizes that microvascular proliferation typically occurs most conspicuously near the periphery of tumors (Me Lendon et al., 1998). This variance in endothelial cell proliferation may be dependent on the extent of exposure of microvessels to pro-angiogenic factors, with vessels further into the tumor exposed to higher concentrations of secreted products derived from tumor cells. Replicative senescence is associated with p53 accumulation, G1 arrest, and induction of p27 (Yoon et al., 2002; Campisi, 2005). An upregulation and accumulation of p53 in the cell is a response to DNA damage, hypoxia, or other stress- inducing environments, and is directly associated with senescent cells and SA P-gal staining of endothelial cells (Oren, 2003; Goodwin and Di Maio,2001; Fang et al.,1999). Our studies demonstrate that p53 and p21 expression is upregulated in TuBEC. When activation of p53 leads to cell-cycle arrest, p 2 lW A F 1/clpl cyclin- dependent kinase inhibitor, a transcriptional target of p53, is upregulated (Fang et al., 1999; Brown et al., 1997). Prolonged irreversible cell cycle arrest leading to cellular senescence has been shown to directly increase p21 expression (Bringold and Serrano, 2000). Another cell-cycle inhibitor in the Cip/Kip family is p27K lpl, also upregulated in TuBEC. Upregulation of p27 expression induces cell-cycle arrest and accelerated 112 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. aging (Wagner et al., 2001; Lafuente et al., 1999). Both p21 and p27 have been shown to inhibit the formation of cyclin-dependent kinase cdk-2/cyclin A complex; an excess of these proteins results in cell-cycle arrest (Jain and Booth, 2003). Consistent with the results of the present study, it was reported that replicative senescent HUVEC arrested in Gj phase of cell cycle, express increased levels of p21 and p27 proteins, decreased cyclin A and increased cyclin Di (Wagner et al, 2001). Cyclin A is involved in the G2/M transition. Therefore its levels are significantly decreased in cells that have undergone Gi arrest (Wagner et al., 2001). Similarly, cyclins Di and E are involved in the Gj/S transition and therefore they accumulate in cells that are arrested in Gi (Wagner et al., 2001). Senescent cells are metabolically active, producing significant quantities of growth factors (Krtolica et al., 2001; Roninson et al., 2001). Indeed, we have previously demonstrated that TuBEC produce higher amounts of VEGF and endothelin-1 (Et-1) compared to untreated BEC (Charalambous et al., 2005). In addition, we demonstrate here that TuBEC produce higher basal levels of PDGF-A and PDGF-B than BEC. PDGF, present in glioma tissue (Lafuente et al., 1999), is responsible for the abnormal distribution of pericytes on vessels, leading to blood vessel leakiness (Jain and Booth, 2003). PDGF is also produced by activated endothelial cells (Totani et al., 1998). Our results show that compared to BEC, TuBEC constitutively produce higher levels of PDGF, as well as increased amounts of other pro-angiogenic factors, such as VEGF and Et-1 (Charalambous et al., 2005). The increased production of these proliferation- 113 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. inducing cytokines and growth factors illustrates that TuBEC have a high metabolic activity, and is likely to contribute to tumor growth in a positive feedback manner. There is considerable controversy over the effects of replicative senescence on apoptosis. Several reports using fibroblasts or CD8 T cells demonstrated that replicative senescent cells are resistant to apoptosis and cell death induced by cytotoxic drugs and radiation (Donehower, 2002; Roninson et al., 2001; Marcotte, 2004; Posnett et al., 1999; Yeo et al., 2000). We have previously shown that TuBEC undergo less spontaneous apoptosis in culture than BEC (Charalambous et al., 2005). Other studies indicate that senescent CD4 T and HUVEC have an increased tendency toward apoptosis (Wagner et al., 2001; Hyland et al., 2001; Unterluggauer et al., 2003). This variation in apoptosis may be related to significant differences between HUVEC and microvessel endothelial cell structure and functions (Maher, 1992; Utgaard et al., 1998; King et al., 2004). The present data show that TuBEC are significantly more resistant to the cytotoxic drugs CPT-11 and temozolomide (Figure 4-5). Temozolomide is a FEN-1 (flap endonuclease) inhibitor whereas CPT-11 is a semisynthetic derivative of camptothecin that interacts with topoisomerase I (Alimonti et al., 2004; Mehra et al., 2005). Since the cytotoxic actions of these drugs are mediated during DNA repair, cells that are not proliferating would be predicted to be less susceptible to these agents (Mathijssen et al., 2001). Recent studies using murine tumor-derived endothelial cells have also observed that these cells are more drug-resistant compared to their normal counterpart (Arbiser et al., 1999). It should be noted that extrapolations between the two species are not necessarily warranted, because cells of rodent and human origin 114 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. are fundamentally different with respect to properties of senescence (Shay and Wright, 2001; Wright and Shay, 2001). A newly appreciated feature of senescent cells is the acquisition of novel functional properties. Studies show that senescent T cells acquire suppressor functions, and exhibit altered cytokine production profiles (Effros et al., 2005). Similarly, senescent fibroblasts enhance the growth of pre-malignant cells in vitro and in vivo, thus producing a microenvironment that promotes mutagenesis leading to tumor formation (Krtolica et al., 2001). The data presented here demonstrate that senescent brain endothelial cells within the tumor may play a key role in inducing or perpetuating tumors by the increased production of key growth factors, in addition to providing a site for nutrient-waste exchange required for tumor growth. Furthermore, the data presented in Figure 4-6, which demonstrate that normal BEC can transition to TuBEC-like cells in the presence of glioma supernatants, imply that the mechanism of BEC-TuBEC transition involves tumor-secreted factors. Thus, this research provides a novel framework for understanding the previously documented aberrant characteristics of TuBEC. Revision of our conceptual paradigm regarding the nature of endothelial cells comprising the tumor-associated vasculature, will enable the development of new strategies for anti-angiogenic therapies. Perhaps the approach of using multiple drugs focusing on the different growth patterns of the tumor vasculature would be more therapeutically effective, with apoptosis agents designed to specifically target senescent endothelial cells, as well as anti-angiogenic agents blocking fast-growing, newly 115 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. forming vessels within the tumor. Understanding the nature of human tumor-derived endothelial cells is critical to identifying effective anti-vascular therapies. 116 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER V CONCLUSIONS AND PROSPECTS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.1. CONCLUSIONS This research is significant for many reasons. In general, it provided us with important information regarding the unique functional properties of tumor- associated endothelial cells that can lead to the development of more efficient anti-angiogenic therapies that would target the tumor vasculature. More specifically, one of the first and most important things we learned about primary culture glioblastoma-associated endothelial cells is that they have a lower proliferation rate than normal brain endothelial cells (Charalambous et al., 2005), in contrast to what has been previously shown with tumor-associated endothelial cell lines (Bussolati et al., 2003; Alessandri et al., 1999). This evidence is particularly important in terms of anti-tumor therapy, as most anti-angiogenic drugs currently used for tumor therapy are based on the fact that they target rapidly proliferating cells. Consequently, conventional anti-angiogenic drugs will be less effective in targeting the tumor blood vessels. This fact is also supported by further studies showing that tumor endothelial cells are resistant to both spontaneous and drug-induced apoptosis (Charalambous et al., 2005). Our studies demonstrated that TuBEC are resistant to drugs used in glioma therapy such as CPT-11 and temozolomide. These results are not surprising as previous studies by others have shown that tumor endothelial cells were resistant to apoptosis and express high amounts of anti-apoptotic proteins such as bcl-2 (Bussolati et al., 2003). In addition, this study demonstrates that TuBEC constitutively produce several growth factors such as IL-8, Et-1, VEGF and PDGF, which possibly induce angiogenesis independent of any exogenous stimulus. Our data also demonstrate that 118 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. constitutive production of IL-8 by TuBEC is a result of autocrine stimulation by VEGF and paracrine stimulation by TGF-f31 produced by gliomas. Production of growth factors by TuBEC is extremely important as not only they are not depended on external growth factors for their growth but also they promote tumor angiogenesis, Our studies have shown that TuBEC do not proliferate as fast as BEC, and they actually have a high percentage of terminally differentiated senescent cells, which are unable to proliferate or undergo apoptosis. Cellular senescence is defined as an irreversible cell cycle arrest associated with G1 arrest in the cell cycle and increase in tumor suppressor proteins. There are two types of senescence: replicative senescence which occurs in aged cells and stress- induced senescence which occurs in cells exposed to stress conditions such as oxidative damage, DNA damage, or mitogens (Campisi, 2005). Senescence stops the accumulation of damaged cells such as cells with mutations. Cellular senescence is therefore one of the most potent anticancer mechanisms. However, senescent cells, such as TuBEC, are abnormal, and they may instead promote the development of cancer as they accumulate by supplying the tumor with nutrients (Campisi, 2005). Even though they are senescent cells, TuBEC are metabolically active, producing growth factors which facilitate tumor angiogenesis. Therefore, they need to be eliminated in order to inhibit tumorigenesis. However, their senescence phenotype likely renders them unresponsive to conventional drug therapy. Our findings may suggest a hypothetic model of tumor-endothelial cell interactions, in which tumors induce the transition of endothelial cells into senescent endothelial cells, which in turn, facilitate tumor angiogenesis by producing their own growth 119 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. factors. Although TuBEC are slow-proliferating cells and may not participate to the active angiogenesis occurring in the periphery of the tumor, they may still be part of the blood vessels and maintaining the internal part of the tumor with valuable nutrients. Consequently, even if tumor cells and the actively proliferating endothelial cells are eliminated by chemotherapy, tumor endothelial cells will remain intact, producing growth factors to promote angiogenesis and tumor recovery. In addition, based on our data that tumor supernatant induces the appearance of senescent cell characteristics in BEC, this research supports one of the possible hypothesis that have been previously proposed regarding the origin of TuBEC, which is that they are derived from normal endothelial cells that under the influence of growth factors become tumor endothelial cells (Bussolati et al., 2003). The other two popular hypotheses were that tumor endothelial cells are derived either from endothelial progenitor cells, or tumor cells that de-differentiate into tumor endothelial cells (Bussolati et al., 2003). Our results suggest that TuBEC are possibly derived from normal endothelial cells that were over- stimulated by tumor- derived growth factors to proliferate, and therefore they have reached their maximum population doublings and hence replicative senescence in vivo, in the human brain. Consequently, tumor-associated endothelial cells express characteristics of senescent cells. Since tumor endothelial cells facilitate angiogenesis in the tumor microenvironment, new approaches have to be developed which eliminate TuBEC by targeting the specific characteristics of TuBEC that are different from normal BEC. Targeting the tumor vasculature with drugs that also target TuBEC would be more effective. The currently 120 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. used anti-angiogenic approaches are focusing on molecules and proteins are differentially expressed on tumor and normal vasculature. Some of the approaches used involve targeting VEGF receptors, endoglin, and vascular integrins, such as avP3, which is overexpressed on tumor endothelium (Neri and Bicknell, 2005). More specifically, vitaxin, an agent that is currently under clinical trials, consists of fully humanized anti-avp3 integrin antibodies. Similarly, avastin, another agent that is currently used for cancer therapy, consists of anti-VEGF antibodies (Neri and Bicknell, 2005). Other approaches focus on the development of anti-tumor vaccines. For example, immunization with an anti-VEGF vaccine generated anti-Flk-1 specific antibodies. Finally, targeted gene delivery has been used in order to selectively deliver therapeutic genes to the tumor vessels by utilizing coupling to-avP3 integrin ligand. However, most of these approaches present problems such as partial patient response to them and toxicity. Our studies have verified and expanded the knowledge of phenotypic and functional characteristics of primary tumor endothelial cells. Most approaches target rapidly proliferating cells such as tumor cells and proliferating endothelial cells. However, our studies have demonstrated that tumor endothelial cells are not rapidly proliferating as previous studies have shown and therefore future therapies must be focused on the low proliferative potential of these cells. Possibly the approach of using multiple drugs focusing on the different growth pattern and different marker expression of the tumor vasculature would be therapeutically more effective. Combination therapies are currently used in many cancers, including gliomas. For example, combination of a 121 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. topoisomerase inhibitor such as CPT-11 with DNA alkylating agents such as procarbazine and BCNU or temozolomide and the microtubule targeting agent vincristine generated synergistic effects in clinical trials for glioma therapy (Reardon et al., 2003). Another effective approach would be to develop and use drugs that inhibit survival proteins or growth factors that they overexpress. In general, this research provides novel information required for understanding the aberrant characteristics of the tumor vasculature. Therefore, our results are important for the development of novel anti-angiogenic therapies that specifically target senescent tumor associated endothelial cells. Anti-angiogenic therapy needs to use such targets as proteins or growth factors such as IL-8 and VEGF that are expressed on tumor vasculature but not on normal vessels. Such approaches will increase drug specificity and selectivity and reduce drug toxicity, because it will target tumor blood vessels directly, without causing toxicity to other non-malignant cells. Therefore, understanding the characteristics of tumor endothelial cells and their interactions with normal endothelial cells and tumor cells is essential for the development of more selective anti-angiogenic therapies with better discrimination between normal and tumor endothelial cells. 122 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Normal vessels TuBEC vessels Activatt vessels Tumor periphery Tumor Necrotic center Normal brain Figure 5-1. Proposed hypothetical model of tumor-endothelial cells interactions. Active proliferation of endothelial cells and angiogenesis occurs mostly in the periphery of the tumor. The center of the tumor is usually necrotic whereas the area between the periphery and the necrotic center of the tumor is the area that probably has most of the senescent tumor endothelial cells. Consequently, most of the BEC are located in the vessels of the tumor periphery, and most of the TuBEC in the vessels of the senescence area. 5.2. PROSPECTS AND FUTURE DIRECTIONS Several future directions of research and projects can be derived from this work. First of all, an important aspect of angiogenesis is the regulation of cell survival by growth factors. This research has emphasized the important role of IL-8, Et-1 and VEGF in the regulation of endothelial angiogenesis. One aspect affecting angiogenesis is the role of these growth factors in the regulation of survival of BEC and TuBEC. This remains to be investigated as the endogenous growth factors produced by TuBEC may be responsible for inducing the expression of survival factors in TuBEC such as bcl-2 and survivin and downregulating apoptosis factors such as bax. Bcl-2 is an anti- apoptotic protein which binds to bax, an apoptotic protein, and inhibits it from translocating to the mitochondria and inducing cytochrome-c release and caspase 9 123 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. activation (Tsujimoto, 1998). Our preliminary data demonstrate that TuBEC have increased expression of the anti-apoptotic protein survivin, which inhibits the caspase cascade by inhibiting caspases 3,7 and 9 activation (Schimmer, 2004). IL-8 and VEGF have already been demonstrated to be important survival factors for endothelial cells by inducing bcl-2 expression (Li et al., 2003). Consequently, the hypothesis that the increased survival of TuBEC may be due to the endogenous expression of these growth factors needs to be investigated. Another interesting project would be to investigate the mechanisms of increased drug resistance of TuBEC. One mechanism that may be involved in their drug resistance is overexpression of the glucose regulated protein GRP-78 (Dong et al., 2005). GRP-78 is an endoplasmic reticulum chaperone that is involved in survival of cells and is found to be overexpressed in many tumors (Dong et al., 2005). Therefore, the potential regulation of GRP-78 by IL-8, VEGF and Et-1 in BEC and TuBEC needs to be investigated. Furthermore, another important aspect of angiogenesis that remains to be investigated is the role and regulation of matrix metalloproteinases (MMPs) in BEC and TuBEC. MMP production is highly significant for tumors, as these enzymes mediate extracellular matrix degradation and hence tumor cell invasion, angiogenesis and metastasis (Bjorklund and Koivunen, 2005). Our preliminary data suggest that TuBEC have increased expression of MMP-2 in comparison to BEC. Upregulated expression of the gelatinases MMP-2 and MMP-9 may be responsible for the higher migration rate of TuBEC in contrast to BEC. More specifically, IL-8 has already been shown to induce 124 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MMP-2 expression in endothelial cells (Li et al., 2003). Therefore, the expression and regulation of MMPs in BEC and TuBEC should be examined. Other interesting projects that could be investigated are the signaling pathways that are involved in BEC and TuBEC migration. Understanding the molecular pathways involved in angiogenesis is very important for the development of drugs targeting the appropriate signaling enzymes. Previous work from our laboratory has investigated the molecular pathways involved in Et-1 and IL-8 induced BEC migration (Milan et al., Charalambous et al., unpublished observations). Similar work investigating the signaling pathways involved in TuBEC migration would help to identify specific drugs to block the signaling pathways and therefore TuBEC functions. Finally, in terms of TuBEC characterization, it would be interesting to investigate the expression of tight junction proteins. Preliminary data demonstrate that the expression of some tight junction proteins such as VE-cadherin and claudin-5 are downregulated in tumor endothelial cells (Charalambous et al., 2005; Liebner et al., 2000). VE-cadherin regulates angiogenesis by inducing endothelial cell migration and proliferation by destabilization of intercellular junctions (Caveda et al., 1996; Griffioen, 1997). Since tight junction proteins exert an important role in angiogenesis (Griffioen, 1997; Distler et al., 2003), it is important to investigate their expression and regulation by various growth factors. 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Creator Charalambous, Christiana (author)

Core Title The role of tumor-associated and normal human brain endothelial cells in angiogenesis

Degree Doctor of Philosophy

Degree Program Molecular Microbiology and Immunology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

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