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Targeting vessel maturation: an anti-angiogenesis based cancer therapy
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Content
TARGETING VESSEL MATURATION: AN ANTI-ANGIOGENESIS BASED
CANCER THERAPY
by
Jeffrey S. Scehnet
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PATHOBIOLOGY)
December 2010
Copyright 2010 Jeffrey S. Scehnet
ii
DEDICATION
This dissertation is dedicated to my family and friends.
What a long, strange trip it’s been…
iii
ACKNOWLEDGMENTS
First, I would like to thank my advisor and mentor, Parkash Gill, for his patience,
guidance, and continued commitment to my growth and development. He has gifted me
his scientific, professional, and personal wisdom, my best interests always at heart. I will
always be indebted, thank you.
Second, I would like to express my sincere appreciation to the other members of my
committee – David Hinton, Alan Epstein, Randy Widelitz, and Gage Crump – each of
whom provided their time, expertise, and kindness.
Third, I would like to thank Valery Krasnoperov for his constant support, guidance,
intellectual stimulation, and an authentic wool hat that keeps my head warm throughout
the cold winter months.
Fourth, I would like to thank Eric Ley and Theo Sadler for their support, guidance,
friendship, and conversations while shuttling me to the airport every Friday or
overindulging in libations at The Biltmore Gallery Bar and Cognac Room. Cheers!
Lastly, I would like to thank all the members of the Gill laboratory that contributed to my
success. Thank you for your friendship and support.
iv
TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGMENTS iii
LIST OF FIGURES v
ABSTRACT vi
CHAPTER 1: INTRODUCTION
Chapter 1: Angiogenesis 1
Chapter 1: Major Players in Angiogenesis (VEGF) 2
Chapter 1: Major Players in Angiogenesis (Delta-Notch) 3
Chapter 1: Major Players in Angiogenesis (Eph/Ephrin) 4
Chapter 1: Anti-Angiogenic Therapy 5
Chapter 1: References 7
CHAPTER 2: INHIBITION OF DLL4-MEDIATED SIGNALING INDUCES
PROLIFERATION OF IMMATURE VESSELS AND RESULTS IN POOR
TISSUE PERFUSION.
Chapter 2: Abstract 11
Chapter 2: Introduction 12
Chapter 2: Materials and Methods 14
Chapter 2: Results 21
Chapter 2: Discussion 33
Chapter 2: References 37
CHAPTER 3: THE ROLE OF EPHS, EPHRINS AND GROWTH FACTORS IN
KAPOSI SARCOMA AND IMPLICATIONS OF EPHRINB2 BLOCKADE.
Chapter 3: Abstract 40
Chapter 3: Introduction 41
Chapter 3: Materials and Methods 43
Chapter 3: Results 51
Chapter 3: Discussion 63
Chapter 3: References 67
CHAPTER 4: CONCLUSIONS
Chapter 4: Summary and Future Directions 70
Chapter 4: Concluding Remarks 74
Chapter 4: References 75
BIBLIOGRAPHY 77
v
LIST OF FIGURES
Figure 2.1 Dll4+/- mutant mice show defective increase in vascular proliferation 23
Figure 2.2 Biochemical properties of sDll4 25
Figure 2.3 sDll4 induces tubule formation in vitro 26
Figure 2.4 sDll4 induces sprouting in endothelial cell spheroids in vitro 27
Figure 2.5 sDll4 induces vessel response but lacks perfusion in murine
Matrigel assay 29
Figure 2.6 sDll4 inhibits the tumor growth in a murine tumor xenograft model 32
Figure 3.1 The Eph and Ephrin expression in KS cell lines KS-SLK and
KS-IMM, lymphatic endothelial cells transformed with
HHV-8 (LEC/HHV-8), and KS tumor biopsy 52
Figure 3.2 Expression and characterization of sEphB4 and sEphB4-HAS 54
Figure 3.3 sEphB4-HSA activity in KS migration, invasion, and survival 56
Figure 3.4 sEphB4-HSA inhibits KS tumor growth in a murine tumor
xenograft model 59
Figure 3.5 Analysis of vascular perfusion, vessel density, tumor cell
proliferation, apoptosis and hypoxia 62
vi
ABSTRACT
Angiogenesis is an extremely complex process regulated by several different
signaling pathways. The identification of novel molecular targets and understanding of
their interaction with angiogenesis pathways are required to design rational anti-
angiogenesis therapies. Notch-Dll4 and EphB4-EphrinB2 receptor-ligand pairs are key
players in these pathways. The following studies provide evidence for two potential anti-
angiogenesis therapies, soluble Dll4 (sDll4) and soluble EphB4 (sEphB4). First, the role
of Dll4 in vascular
remodeling at sites of angiogenesis, including tumor vasculature was
investigated.
Results showed that loss of Dll4 function promotes endothelial cell
migration, excessive vascular network formation, and reduction
in pericyte recruitment,
both in embryos and adult mice. Soluble
forms of Dll4 interrupted Dll4-Notch signaling
and recapitulated
the vascular alterations seen in the gene knock-out mice, including
increased vascular network formation, decreased or absent vascular
lumen, and reduced
recruitment of pericytes resulting in tissue
hypoxia and decreased tumor growth. Second,
a comprehensive analysis of the Eph receptor tyrosine kinases was conducted to
determine which members are expressed and contribute to Kaposi Sarcoma (KS)
pathogenesis. In addition, the biological effects of blocking EphrinB2 in vitro and in vivo
using a soluble form of EphB4 (sEphB4) was studied. sEphB4 blocks EphB4-EphrinB2
interaction, and inhibits KS tumor growth with marked reduction in vessel density, vessel
maturity and vessel perfusion. Its anti-tumor activity was comparable or better than
VEGF moAb in KS treatment.
vii
Combination of soluble EphB4 and VEGF moAb resulted in additive anti-tumor effect.
Thus, more effective anti-angiogenic therapeutics may arise from a better understanding
of the molecular and cellular mechanisms involved in tumor angiogenesis and the
response to anti-angiogenic therapies in a wide range of cancers.
1
CHAPTER 1: INTRODUCTION
Chapter 1: Angiogenesis
Almost all tissues in the body develop a vascular network that provides cells with
oxygen and nutrients, and enables the elimination of metabolic wastes. Once formed, the
vascular network is stable and regenerates slowly. In physiological conditions,
angiogenesis occurs primarily during embryonic development, during wound healing,
and in response to ovulation. However, the rapid formation of an imperfect vascular
network during pathological angiogenesis is implicated in over 20 diseases, including
cancer, psoriasis, and age-related macular degeneration.
1-3
Pathological angiogenesis
performs a critical role in the development of cancer. Tumor cells, in response to
hypoxia, release pro-angiogenic molecules, such as VEGF, that attract endothelial cells
and promote their proliferation. Endothelial cells differentiate and secrete matrix
metalloproteases (MMPs), which digest the blood-vessel walls, allowing migration of
endothelial cells toward the site of the angiogenic stimuli. During angiogenic growth,
certain endothelial cells, known as tip cells, lead the growing sprout. Notch receptors and
their Delta-like-4 (DLL4) ligand are essential for sprouting. Dll4 expression is induced in
the tip cell, whereas the activation of Notch signaling in neighboring endothelial cells is
thought to suppress sprouting of these cells.
4,5
The mechanism by which Notch imposes
differential behavior on endothelial cells that are exposed to similar pro-angiogenic
stimuli appears to be directly connected to VEGFA signaling. Dll4 expression is induced
in response to VEGFA, whereas the activation of Notch by DLL4, suppresses the
expression of VEGFR2, and in zebrafish, VEGFR3 (flt4).
6
2
Therefore, Dll4 expression may correspond to an upregulated VEGF-mediated signal
transduction in certain endothelial cells, whereas Notch activation by neighboring cells
inhibits a pro-angiogenic response. Growing vascular sprouts generate another
concentration gradient, namely for platelet-derived growth factor B (PDGFB). High
levels of PDGFB in tip cells promote the recruitment of pericytes that express the PDGF
receptor β (PDGFRβ). This process ensures that the endothelium of growing vessels is
sufficiently stabilized by supporting mural cells, such as pericytes and vascular smooth
muscle cells. Pericytes establish direct cell-cell contact with ECs and cover capillaries
and immature blood vessels, whereas vSMCs cover mature and larger diameter vessels,
such as arteries and veins, resulting in a continuous blood flow. Therefore, facilitating
cancer progression and providing a route for distant metastases.
Chapter 1: Major Players in Angiogenesis (VEGF)
The VEGF pathway is essential to vascular and haematopoietic systems, and is
conserved from zebrafish to mammals. In mammals, there are five secreted ligands
(VEGF-A, VEGF-B, VEGF-C, VEGF-D and PlGF) and three receptors (VEGF-R1,
VEGF-R2, VEGF-R3). In general, the ligands are broadly expressed and display
multiple splice isoforms. Expression of VEGF-A is induced by hypoxia, which is a
common feature of rapidly growing solid tumors, and constitutes a key signal from a
tissue calling for more blood vessels and/or increased vascular function. VEGF-R1 and
VEGF-R2 are primarily expressed on vascular endothelial cells, whereas, VEGF-R3 is
predominately expressed on lymphatic endothelial cells.
3
VEGF is the primary growth factor involved in the creation of the primary vascular
network and drives secondary angiogenesis during vascular development.
7, 8
VEGF is
essential for the specification, morphogenesis, and homeostasis of blood vessels. VEGF
signaling influences endothelial cell proliferation, migration, and survival. In addition,
VEGFs regulate genes that provide arterial or venous identity to endothelial cells, such as
induction of EphrinB2 that phenotypically defines arterial endothelial cells and pericytes,
and represses EphB4 that defines venous endothelial cells.
9-12
Chapter 1: Major Players in Angiogenesis (Delta-Notch)
The Notch pathway consists of four single pass transmembrane receptors
(Notch1-Notch4) and five ligands (Jagged1, Jagged2, Dll1, Dll3, and Dll4). The Notch
pathway functions through cell-cell interaction such that the extracellular domain of cell
membrane bound ligand interacts with the extracellular domain of the receptor on an
adjacent cell. Binding of the ligand promotes two proteolytic processing events. The first,
an ADAM-family metalloprotease called TACE (Tumor Necrosis Factor Alpha
Converting Enzyme) cleaves the Notch protein just outside the membrane.
13
This releases
the extracellular portion of Notch. The ligand and the Notch extracellular domain is
endocytosed by the ligand-expressing cell. Next, an enzyme called γ-secretase cleaves the
remaining Notch protein just inside the cell membrane. This releases the Notch
intracellular domain (NICD), allows translocation of this fragment into the nucleus,
followed by activation of target genes.
14
4
The Notch pathway is important for cell-cell communication, neuronal function and
development, and stabilization of arterial endothelial fate and angiogenesis.
15-17
Early
studies in zebrafish suggests that VEGF signaling acts upstream of Notch during arterial-
venous differentiation.
18
In contrast, Notch signaling downstream of VEGF/VEGF-R2
signaling results in a negative feedback loop. Thus, VEGF signaling is reduced.
19-21
Also,
downregulation of VEGF-R2 has been shown in cultured endothelial cells (Taylor et al.
2002).
21
Inversely, increased levels of VEGF-R2 were observed in vessels of
heterozygous mice and as a result of Dll4 blockade.
5
Chapter 1: Major Players in Angiogenesis (Eph/Ephrin)
The Eph family of receptors is the largest family of receptor tyrosine kinases. It is
comprised of fourteen different structurally related receptors which selectively bind eight
membrane-associated ligands called Ephrins. Both Eph receptors and Ephrin ligands are
subdivided into sub-families named EphAs and EphBs, EphrinAs and EphrinBs based on
their preferential binding properties. EphA receptors mostly bind to EphrinAs, proteins
linked to the cell membrane via GPI-linkages, and EphBs preferably bind to EphrinBs -
proteins with a single transmembrane domain. Within EphB family, EphB4 receptor is
specific for EphrinB2.
22
It has been shown that EphB4/EphrinB2 interaction plays a
critical role in vessel maturation as knockout of either protein is embryonically lethal in
mice due to vascular arrest at the primitive capillary plexus stage.
11, 23
Furthermore,
EphB4 has generated considerable enthusiasm for their role in tumor biology.
5
Several recent papers have showed an important role for EphB4 expression in the
progression of epithelial cancers. EphB4 is specifically expressed in tumor cells at higher
levels than adjacent normal tissue.
24-29
The level of expression of EphB4 correlates with
tumor stage, grade and aggressiveness.
25, 29
EphB4 transcripts are more abundant in the
invasive front of the tumor.
24, 25, 27-29
In addition, EphB4 on tumor cells can engage
EphrinB2 on tumor endothelial cells augmenting tumor angiogenesis.
30
Chapter 1: Anti-Angiogenic Therapy
In 1971, Dr. Judah Folkman proposed that tumor growth and metastasis are
angiogenesis-dependent.
31
Therefore, blocking angiogenesis could prevent tumor
progression and metastasis. The tumor cells would be starved of nutrients and oxygen,
and unable to eliminate metabolic wastes. This idea paved the way for a new type of
therapeutic. Anti-angiogenesis therapy has several advantages compared to conventional
therapy. First, endothelial cells are genetically stable and represent a more uniform target
as compared to the tumor cells.
32, 33
Second, endothelial cells can be easily reached by
systemic administration. Third, certain anti-angiogenic agents may have a synergistic
effect when combined with traditional cytotoxic drugs or radiation therapy.
34
The first
drug developed as an inhibitor of angiogenesis, Bevacizumab, was approved by the Food
and Drug Administration (FDA) in February 2004 for use in combination with i.v. 5-
fluorouracil
(5-FU)-based chemotherapy for the first-line treatment of patients
with
metastatic carcinoma of the colon and rectum (CRC).
35, 36
Bevacizumab, a humanized
monoclonal antibody, is an inhibitor of the VEGF pathway.
6
It has been shown to be effective at inhibiting tumor angiogenesis and growth. However,
certain tumors exhibit varied sensitivity to VEGF blockade. Furthermore, WEHI3 and
MV-522 xenograft tumors that are initially sensitive to a block in VEGF signaling may
eventually progress while still on treatment.
19
This resistance is believed to result from
the recruitment of additional angiogenic signals beyond VEGF. Therefore, it is important
to develop alternative anti-angiogenic therapeutics targeting distinct signaling pathways
to synergize with VEGF blockade. Thus, more effective anti-angiogenic therapeutics may
arise from a better understanding of the molecular and cellular mechanisms involved in
tumor angiogenesis and the response to anti-angiogenic therapies.
7
Chapter 1: References
1. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med
1995, 1:27-31.
2. Folkman J. Angiogenesis inhibitors: a new class of drugs. Cancer Biol Ther. 2003 Jul-
Aug;2(4 Suppl 1):S127-33.
3. Folkman J. Fundamental concepts of the angiogenic process. Curr Mol Med.
2003;3:643-651.
4. Hellstrom M, Phng LK, Hofmann JJ, et al. Dll4 signalling through Notch1 regulates
formation of tip cells during angiogenesis. Nature. 2007;445:776-780.
5. Suchting S, Freitas C, le Noble F, et al. The Notch ligand Delta-like 4 negatively
regulates endothelial tip cell formation and vessel branching. Proc Natl Acad Sci U S A.
2007;104:3225-3230.
6. Adams R and Alitalo K. Molecular regulation of angiogenesis and lymphangiogenesis.
Nature 2007, 8: 464-478.
7. Ferrara N, Carver-Moore K, Chen H, et al. Heterozygous embryonic lethality induced
by targeted inactivation of the VEGF gene. Nature. 1996;380:439-442.
8. Ferrara N. The role of VEGF in the regulation of physiological and pathological
angiogenesis. EXS 2005;(94):209-31.
9. Masood R, Cai J, Zheng T, Smith DL, Naidu Y, Gill PS. Vascular endothelial growth
factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi
sarcoma. Proc Natl Acad Sci U S A. 1997;94:979-984.
10. Wang HU, Chen ZF, Anderson DJ. Molecular distinction and angiogenic interaction
between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4.
Cell. 1998;93:741-753.
8
11. Gerety SS, Wang HU, Chen ZF, Anderson DJ. Symmetrical mutant phenotypes of the
receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular
development. Mol Cell. 1999;4:403-414.
12. Sivakumar R, Sharma-Walia N, Raghu H, et al. Kaposi’s sarcoma-associated
herpesvirus induces sustained levels of vascular endothelial growth factors A and C early
during in vitro infection of human microvascular dermal endothelial cells: biological
implications. J Virol. 2008;82: 1759-1776.
13. Brou C, Logeat F, Gupta N, Bessia C, LeBail O, Doedens JR, Cumano A, Roux P,
Black RA, Israel A. A novel proteolytic cleavage involved in Notch signaling: the role of
the disintegrin-metalloprotease TACE. Mol Cell. 2000 (2):207-16.
14. Lai EC. Notch signaling: control of cell communication and cell fate. Development.
2004:131:965-73.
15. Gaiano N, Fishell G. The role of notch in promoting glial and neural stem cell fates.
Annu. Rev. Neurosci. 2002:25:471-90.
16. Bolos V, Grego-Bessa J, de la Pompa JL. Notch signaling in development and cancer.
Endocr. Rev. 2007:28(3):339-63.
17. Liu ZJ, Shirakawa T, Li Y, et al. Regulation of Notch1 and Dll4 by vascular
endothelial growth factor in arterial endothelial cells: implications for modulating
arteriogenesis and angiogenesis. Mol Cell Biol. 2003;23:14-25.
18. Lawson ND, Vogel AM, Weinstein BM. Sonic hedgehog and vascular endothelial
growth factor act upstream of the Notch pathway during arterial endothelial
differentiation. Dev Cell. 2002;3:127-136.
19. Ridgway J, Zhang G, Wu Y, et al. Inhibition of Dll4 signalling inhibits tumour
growth by deregulating angiogenesis. Nature. 2006;444:1083-1087.
9
20. Williams CK, Li JL, Murga M, Harris AL, Tosato G. Up-regulation of the Notch
ligand Delta-like 4 inhibits VEGF-induced endothelial cell function. Blood.
2006;107:931-939.
21. Taylor KL, Henderson AM, Hughes CC. Notch activation during endothelial cell
network formation in vitro targets the basic HLH transcription factor HESR-1 and down-
regulates VEGFR-2/KDR expression. Microvasc Res 2002;64:372-83.
22. Sakano S, Serizawa R, Inada T, Iwama A, Itoh A, Kato C, Shimizu Y, Shinkai F,
Shimizu R, Kondo S, Ohno M, Suda T. Characterization of a ligand for receptor protein-
tyrosine kinase HTK expressed in immature hematopoietic cells. Oncogene 1996;
13:813-22.
23. Adams RH, Wilkinson GA, Weiss C, et al. Roles of ephrinB ligands and EphB
receptors in cardiovascular development: demarcation of arterial/venous domains,
vascular morphogenesis, and sprouting angiogenesis. Genes Dev. 1999;13: 295-306.
24. Berclaz G, Karamitopoulou E, Mazzucchelli L, Rohrbach V, Dreher E, Ziemiecki A,
Andres AC. Activation of the receptor protein tyrosine kinase EphB4 in endometrial
hyperplasia and endometrial carcinoma. Ann Oncol. 2003;14:220-6.
25. Wu Q, Suo Z, Risberg B, Karlsson MG, Villman K, Nesland JM. Expression of
Ephb2 and Ephb4 in breast carcinoma. Pathol Oncol Res. 2004;10:26-33.
26. Lee YC, Perren JR, Douglas EL, Raynor MP, Bartley MA, Bardy PG, Stephenson
SA. Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate
carcinoma. BMC Cancer. 2005;20;5:119.
27. Kumar SR, Singh J, Xia G, et al. Receptor tyrosine kinase EphB4 is a survival factor
in breast cancer. Am J Pathol. 2006;169:279-293.
28. Kumar SR, Masood R, Spannuth WA, et al. The receptor tyrosine kinase EphB4 is
overexpressed in ovarian cancer, provides survival signals and predicts poor outcome. Br
J Cancer. 2007;96:1083-1091.
10
29. Kumar SR, Scehnet JS, Ley EJ, Singh J, Krasnoperov V, Liu R, Manchanda PK,
Ladner RD, Hawes D, Weaver FA, Beart RW, Singh G, Nguyen C, Kahn M, Gill PS.
Preferential induction of EphB4 over EphB2 and its implication in colorectal cancer
progression. Cancer Res. 2009 May 1;69(9):3736-45.
30. Noren NK, Lu M, Freeman AL, Koolpe M, Pasquale EB. Interplay between EphB4
on tumor cells and vascular ephrin-B2 regulates tumor growth. Proc Natl Acad Sci USA
2004;101:5583-8.
31. Folkman J. Tumor Angiogenesis: Therapeutic implications. N Engl J Med. 1971 Nov
18; 285(21):1182-6.
32. Kerbel RS. Inhibition of tumor angiogenesis as a strategy to circumvent acquired
resistance to anti-cancer therapeutic agents. Bioessays 1991;13: 31-6.
33. Rak J, Kerbel RS. Treating cancer by inhibiting angiogenesis: New hopes and
potential pitfalls. Cancer Metastasis Rev 1996; 15:231-6.
34. Kakeji Y, Teicher BA. Preclinical studies of the combination of angiogenic inhibitors
with cytotoxic agents. Invest New Drugs 1997; 15: 39-48.
35. Cohen MH, Gootenberg J, Keegan P, Pazdur R. FDA Drug Approval Summary:
Bevacizumab (Avastin®) Plus Carboplatin and Paclitaxel as First-Line Treatment of
Advanced/Metastatic Recurrent Nonsquamous Non-Small Cell Lung Cancer Oncologist,
Jun 2007; 12: 713 - 718.
36. Kerbel RS. Tumor angiogenesis. N Engl J Med. 2008 May 8;358(19):2039-49.
11
CHAPTER 2: INHIBITION OF DLL4-MEDIATED SIGNALING INDUCES
PROLIFERATION OF IMMATURE VESSELS AND RESULTS IN POOR TISSUE
PERFUSION.
Scehnet JS, Jiang W, Kumar SR, Krasnoperov V, Trindade A, Benedito R, Djokovic D,
Borges C, Ley EJ, Duarte A, Gill PS. Inhibition of Dll4-mediated signaling induces
proliferation of immature vessels and results in poor tissue perfusion. Blood. 2007 Jun
1;109(11):4753-60.
Chapter 2: Abstract
Vascular development is dependent on various growth factors
and certain
modifiers critical for providing arterial or venous
identity, interaction with the
surrounding stroma and tissues,
hierarchic network formation, and recruitment of
pericytes.
Notch receptors and ligands (Jagged and Delta-like) play a critical
role in this
process in addition to VEGF. Dll4 is one of the
Notch ligands that regulates arterial
specification and maturation
events. In the current study, we have shown that loss of
function
by either targeted allele deletion or use of a soluble form
of Dll4 extracellular
domain leads to inhibition of Notch signaling,
resulting in increased vascular proliferation
but defective
maturation. Newly forming vessels have thin caliber, a markedly
reduced
vessel lumen, markedly reduced pericyte recruitment,
and deficient vascular perfusion.
sDll4 similarly induced defective
vascular response in tumor implants leading to reduced
tumor
growth. Interference with Dll4-Notch signaling may be particularly
desirable in
tumors that have highly induced Dll4-Notch pathway.
12
Chapter 2: Introduction
Primary vasculogenesis serves as the template from which a higher
order of
branching network is generated by the process defined
as angiogenesis.
1–3
During
angiogenesis, branching of
arterial and venous components is orchestrated such that the
capillaries from these 2 compartments fuse in symmetry, anchored
in place by interaction
with matrix proteins.
4
Vascular endothelial
growth factor (VEGF) is indispensable for the
formation of primary
vascular network and secondary angiogenesis.
5
VEGF however
requires
the presence of precise quantities of several other constituents
within well-
defined temporal and spatial constraints to construct
and remodel the vascular system.
Specifically the Notch signaling
pathway is necessary to provide signals for phenotypic
determination
of arteries and veins, and regulated vessel migration and branching
leading
to the vascular morphogenesis and remodeling (Bray
6
and W.J. and P.S.G., unpublished
data, December 2006).
In mammals, the Notch family of proteins is composed of 4 single-pass
transmembrane receptors (notch1-4) and 5 membrane-bound ligands
(jagged1, 2 and
Dll1, 3, and 4). Mutations of Notch receptors
and ligands in mice and humans lead to
abnormalities in the
vascular system.
7
The Notch pathway functions through cell-cell
interaction such that the extracellular domain of cell membrane–bound
ligand interacts
with the extracellular domain of the receptor
on an adjacent cell. Notch receptor
activation requires cleavage
of Notch intracellular domain (NICD) and translocation to
the
nucleus, and activation of target genes.
8
13
Differentiation of vascular cells to arterial or venous compartments
was
previously thought to depend on physical factors such as
blood pressure and oxygen
concentration. Over the past few years,
however, the differential and restricted expression
of a number
of genes in arterial or venous endothelial cells prior to the
onset of circulation
suggested the potential for genetic determination
of the arterial and venous fate of
primary endothelial cells.
Among these genes are NOTCH1,
9
NOTCH4,
10
DLL4,
11
and the
Dll4-Notch–regulated
genes EPHB4 and EFNB2 specifically expressed in venous
12
and
arterial endothelial cells, respectively.
13,14
Vascular expression of Dll4 and its cognate receptors Notch1
and Notch4 is
restricted to arterial endothelium. Dll4 is one
of the earliest genes expressed in arterial
endothelial cells,
is induced by VEGF-VEGFR signaling, and is essential for
establishment
of the arterial endothelial cell fate.
14–16
Haploinsufficiency
of Dll4, like that
of VEGF, leads to embryonic lethality due
to defects in vascular development.
14,17
The
observed defects
include loss of expression of arterial markers, reduced arterial
phenotype
and augmented venous phenotypes, reduced arterial
lumen, and premature fusion among
the arterial and venous compartment
leading to short circuiting of the vascular network.
14
14
In this study, we investigated the role of Dll4 in vascular
remodeling at sites of
angiogenesis, including tumor vasculature.
We show that loss of Dll4 function promotes
endothelial cell
migration, excessive vascular network formation, and reduction
in
pericyte recruitment, both in embryos and adult mice. Soluble
forms of Dll4 interrupt
Dll4-Notch signaling and recapitulate
the vascular alterations seen in the gene knock-out
mice, including
increased vascular network formation, decreased or absent vascular
lumen, and reduced recruitment of pericytes resulting in tissue
hypoxia and decreased
tumor growth.
Chapter 2: Materials and Methods
Analysis of Dll4 germ-line mutant mice in embryos and adults
Dll4 knock-out mice were generated in CD1 background and described
previously.
14
Dll4
–/–
and most Dll4
+/–
mouse
embryos have a lethal phenotype. The
vasculature of Dll4
+/–
embryos was visualized with platelet endothelial cell adhesion
molecule (PECAM) and alpha smooth muscle actin ( -SMA) staining.
Dll4
+/–
mice that
survived to adulthood were studied for
alterations in the vasculature. Dll4
+/–
CD1 male
mice
and wild-type mice (6-8 weeks old) received a transplant of
5 x 10
6
tumor cells
(S180 mouse sarcoma cell line). Tumors were
harvested after 2 weeks for analysis. Dll4
expression was studied
in Dll4
+/–
mice in the tumor tissue and adjacent normal
tissue by
the use of a lacZ reporter included in the targeting
vector. Whole-mount embryo
immunohistochemistry (PECAM antibody
was from Pharmingen, San Diego, CA) and
lacZ staining were carried
out by standard techniques.
18
15
Reverse-transcription–polymerase chain reaction (RT-PCR) analysis
First-strand cDNA was synthesized from total RNA using a SuperScript
Preamplification System kit (GIBCOBRL, Grand Island, NY) and
used (0.1 µg) for PCR
with specific primers for Dll4,
HEY1, HEY2, HES1, and HES2
(Dll4-For-taaggcaggagcctacctg, Dll4-Rev- gatgccagacagacacccaa,
HEY1-For-tgcagatgaccgtggatcac, HEY1-Rev-tgcattgggagacagtaagtg,
HEY2-For-tgaagatgcttcaggcaacag, HEY2-Rev-gaggcacttctgaagttgtgg,
HES1-For-atagctcgcggcattccaagc, HES1-Rev-cggaggtgccgctgttgctg,
HES2-For-tgtgtgcaagagctagctgc, HES2-Rev-ctggcattacagacgtgagc).
PCR products were
visualized by ethidium bromide staining.
Antibodies and other reagents
Anti-PECAM (M20) was purchased from Santa Cruz Biotechnology
(Santa Cruz,
CA); anti– -SMA (Dako, Carpinteria, CA), IgG-Fc
fragment, and anti–human Fc, from
Jackson Laboratories
(Bar Harbor, ME); Notch1-Fc and Notch3-Fc, from R&D Systems
(Minneapolis, MN); hypoxyprobe-1, from Chemicon International
(Temecula, CA);
rhodamine-labeled ricinus communis agglutinin
I (RCA), from Vector Laboratories
(Burlingame, CA); and alkaline
phosphatase substrate PNPP, from Sigma Chemicals (St
Louis,
MO).
16
Cell culture
Normal human umbilical vein endothelial cells (HUVECs) and human
umbilical
arterial endothelial cells (HUAECs) were obtained
from Cambrex (Walkersville, MD)
and maintained in EGM2-supplemented
medium (Invitrogen, Carlsbad, CA). For all
experiments, HUVECs
and HUAECs were used at passages 4 or below and collected
from
a confluent dish. ChoK cell line was obtained from American
Type Culture
Collection (ATCC, Manassas, VA) and cultured under
recommended conditions.
Dll4 constructs
Full-length human Dll4 gene was cloned by PCR amplification
from human
cDNA (Clontech, Mountain View, CA) made from fetal
lung tissues. Both full-length
(amino acid residues 1-685) and
C-terminally His-tagged extracellular domain (amino
acid residues
1-486) proteins were expressed from pcDNA3.1 expression vector
(Invitrogen). Fc fusion protein was expressed from pCXFc vector
(Invitrogen). AP fusion
protein was expressed from pAPtag-2
vector (GeneHunter, Nashville, TN). All proteins
were transiently
expressed in ChoK cells (ATCC) using Lipofectamine 2000 (Invitrogen).
His-tagged and Fc-fusion Dll4 proteins were purified through
nickel-NTA column and
protein A–Sepharose column.
19
17
Notch receptor binding and activation pathway
Notch1-Fc and Notch3-Fc (5 µg/mL each) were coated overnight
at 4°C in PBS
on 96-well plates. Dll4-AP was diluted in
PBS and 0.1% Tween-20 (PBST); 50 µL of
each dilution was
incubated with Notch-Fc and blocked with 5% milk in PBS for
one
hour. Wells were washed 3 times with PBST, developed with
PNPP, and read at OD405.
Typically, HUVECs were grown in 100-mm
dishes until 80% confluence and were
cocultured with ChoK cells
transiently expressing full-length Dll4 (1:1 ratio) or ChoK
cells transfected with vector alone. Cocultures were treated
with either rDll4-His or rDll4-
Fc for a period of 24 hours,
cells were harvested, and total RNA was isolated for further
analysis.
20
Cell sorting
For sorting transfected cells, the MACSelect 4.1 transfected
cell selection kit
(Miltenyi Biotech, Auburn, CA) was used as
per manufacturer's instructions. In brief,
cells were cotransfected
with expression vector containing the plasmid of interest and
pMACS 4.1 plasmid. After 36 hours, cells were harvested with
5 mM EDTA and
incubated with MACSelect 4 Microbeads for 15 minutes
at 4°C. The cell suspension was
then passed via an MS+ column
in a magnetic field. After 3 washes, the column was
removed
from the field and selected cells were eluted in culture medium.
Selection
efficiency was confirmed by fluorescence-activated
cell sorter (FACS) analysis of sorted
cells with fluorescent
Dll4 monoclonal antibody (data not shown).
18
Endothelial cell (EC) tube formation assay
Matrigel (250 µL; BD Biosciences, Palo Alto, CA) was placed
in each well of an
ice-cold 24-well plate. The plate was allowed
to sit at room temperature for 15 minutes,
and at 37°C for
30 minutes for Matrigel to polymerize. HUVECs in EGM2 medium
were
plated at a concentration of 1 x 10
4
cells/well with test
material at various concentrations
in triplicates. After 6-hour
and 24-hour incubations, pictures were taken for each
concentration
using a Bioquant Image Analysis system (Bioquant, Nashville,
TN). Length
of cords formed and number of junctions were compared
among various groups using
ImageJ software (NIH, Bethesda, MD).
Experiments were repeated twice.
19
Vessel sprouting
Endothelial cell spheroids were generated by suspending equal
number of
endothelial cells (1000 cells/well) in culture medium
containing 0.25% (wt/vol)
carboxymethylcellulose and seeded
in nonadherent round-bottom 96-well plates.
Endothelial cells
were suspended to form a single spheroid per well. Spheroids
were
embedded into collagen gels and cultured for at least 24
hours. Sprouting was recorded
digitally (ocular grid at 100x
magnification) using the digital imaging DP-Soft (Olympus,
Center
Valley, PA) analyzing at least 10 spheroids per experimental
group and
experiment. Sprouting was also quantitated by measuring
the length of the sprouts by
ImageJ.
19
19
Murine Matrigel plug angiogenesis assay
In vivo angiogenesis was assayed using the Matrigel plug assay.
Matrigel rapidly
forms a solid gel at body temperature, trapping
the factors to allow slow release and
prolonged exposure to
surrounding tissues. Matrigel (8.13 mg/mL, 0.5 mL) in liquid
form
at 4°C was mixed with vehicle alone (PBS containing
0.25% BSA) or VEGF, or sDll4, or
VEGF and sDll4 together. Matrigel
(0.5 mL) was injected into the abdominal
subcutaneous tissue
of female Balb/C nu/nu mice (6 weeks old, 5 mice per group)
along
the peritoneal midline. On day 6, mice were humanely killed
and plugs were recovered,
weighed, and divided for hemoglobin
measurement and immunohistochemical analysis.
Vascular identity
of the infiltrating cells was established with PECAM immunostaining.
The experiment was repeated 3 times. The vascularized area in
each section was
calculated using ImageJ. Hemoglobin in one
half of the Matrigel plug was measured
using the Drabkin method
(Drabkin reagent kit 525; Sigma, St Louis, MO) using the
manufacturer's
recommended protocol.
Immunohistochemistry and immunofluorescence
Sections (5 µm) of formalin-fixed paraffin-embedded tissues
were processed
using standard methods.
14,19
Sections were incubated
with primary antibody overnight at
4°C and appropriate secondary
antibody for 1 hour at room temperature. Antibody
binding was
localized with ABC staining kit from Vector Laboratories according
to the
manufacturer's instructions and peroxidase activity detected
using DAB substrate solution
(Vector Laboratories).
20
Routine
negative controls were exclusion of primary and secondary antibody
and
substitution of normal IgG isotope for primary antibody.
The positive staining area was
estimated using ImageJ and analyzed
by Student t test.
Fluorescent immunostaining was
performed in a similar fashion
to detect the expression level of EC-specific markers
including
PECAM. Appropriate fluorescein-conjugated secondary antibodies
(Sigma-
Aldrich, St Louis, MO) were used and nuclei were counterstained
with 4', 6-diamidino-2-
phenylindole dihydrochloride hydrate
(DAPI). Slides were mounted with Vectashield
antifade mounting
solution (Vector Laboratories) and images obtained using an
Olympus
AX70 fluorescence microscope and Spot v2.2.2 (Diagnostic
Instruments, Sterling
Heights, MI) digital imaging system.
Murine tumor xenografts
Tumor cells (1.5 x 10
6
) HT29 (human colon cancer cell line)
or KS-IMM (human
Kaposi sarcoma cancer cell line) were implanted
subcutaneously in flanks of male
athymic BalbC nu/nu mice (6-8
weeks old, 6 mice/group and repeated twice). For
assessing local
effects of sDll4, tumor cells were mixed with Matrigel (1:1
vol/vol; BD
Biosciences) with or without 5 µg/mL sDll4.
Tumor volume was measured on day 14
estimated as 0.52 x a x
b
2
, where a and b are the largest and smallest lengths of the
palpable tumor, respectively. The Student t test was used to
compare tumor volumes, with
P < .05 being considered significant.
Animals were humanely killed, and tumor and
adjacent normal
tissues were harvested. Harvested tissues were divided and either
fixed in
formalin or frozen in OCT for analysis.
21
Distribution
and intensity of hypoxia were studied using hypoxyprobe-1 (HP1-100;
Chemicon International) infused intraperitoneally at a dose
of 60 mg/kg one hour prior to
the tumor harvest and localized
using recommended protocol. Vessel perfusion was
studied using
rhodamine-labeled ricinus communis agglutinin 1 (Vector Laboratories)
infused 10 to 15 minutes prior to the tumor harvest and analyzed
using the manufacturer's
recommended protocol. All procedures
were approved by our Institutional Animal Care
and Use Committees
and performed in accordance with the Animal Welfare Act
regulations.
Chapter 2: Results
Vascular proliferation in embryonic and adult Dll4
+/–
mutant mice
Dll4
–/–
and most of Dll4
+/–
mice die in utero
due to defective vascular
development.
17
Close examination of
the Dll4
+/–
embryos showed normal vasculogenesis
until
E8.75, when the first vascular defect became apparent. There
was increased vascular
proliferation appearing like honeycomb
and lacking hierarchic arterial branching and
maturation (Figure 2.1A).
We next studied vascular response and remodeling in adult
Dll4
+/–
mutant and wild-type mice. Mice (6 weeks old) received implants
of S180 tumor
cells. Tumor and adjacent tissue harvested after
10 days was examined for vascular
response by PECAM, and -SMA
immunolocalization. Wild-type mice showed
increased vascular
response in the tumor (Figure 2.1B) and the vessels had organized
network. In comparison, Dll4
+/–
mice showed an even greater
increase in the vascular
response (1.5-fold increase, P <
.05).
22
Furthermore, the vessels showed lack of architecture and
loss of hierarchy. Thus vascular
response was increased but
maturation was lacking. Maturation of newly forming vessels
accompanies the recruitment of pericytes. We hypothesized that
newly forming vessels in
Dll4
+/–
mice may be defective
in pericyte recruitment. Thus localization of pericytes with
-SMA antibodies showed abundant signal in tumor vessels in wild-type
mice, whereas
tumor vessels in Dll4
+/–
mice showed a profound
deficiency in pericyte coverage.
Reduced recruitment of pericytes
may contribute to the lack of vascular hierarchy in
Dll4
+/–
mice tumor vessels. Furthermore, these findings reveal a novel
function of Dll4 in
the recruitment of pericytes to newly forming
vessels. We next wished to determine if
defective vascular response
in adult mice leads to alteration in gene expression, in
particular
Dll4. To this end, we used the LacZ reporter included in the
targeting vector
used to generate mutant mice to observe Dll4
promoter activity. Dll4
+/–
mutant mice
showed highly structured
LacZ-expressing vessels in the normal tissue adjacent to the
tumor (Figure 2.1C), whereas LacZ activity was markedly increased
in vessels within the
tumor vessels (Figure 2.1C), indicative
of Dll4 activation in the tumor vasculature.
PECAM localization
in serial sections of the tumor vessels was done to determine
the
extent of Dll4 activation in tumor vasculature. Dll4 is
expressed in the majority but not in
all tumor vessels.
23
Figure 2.1 Dll4 +/- mutant mice show
defective increase in vascular
proliferation.
(A) The vasculature of wild-type and
Dll4+/- embryos were examined using
PECAM whole-mount immunostaining.
Dorsal aorta and cardinal vein are labeled
as a and v, respectively. Absence of large
vessels and an increase in vessel
branching and density was seen in
Dll4+/- embryos at E10.5 compared to
wild type. (B) Vascular response in
Dll4+/- adult mice was examined as in
panel A after tumor implantation. Wild-
type mice showed organized vascular
proliferation in the tumor (left half),
while mutant mice showed markedly
increased vascular response that lacks
organization and vascular hierarchy. (C)
Expression of Dll4 in tumor and normal
regions in Dll4+/- mutant mice was
examined by b-gal staining. Dll4
expression was observed (arrows) in a
few discrete vessels in the normal tissue,
while the tumor region showed many b-
gal–positive vessels of similar appearance
indicative of Dll4 induction in tumor
vessels. (D) Pericyte coverage around
newly forming vessels was examined by
a-SMA localization. In wild-type mice,
the vessels showed colocalization of
PECAM and a-SMA (left panel). In
Dll4+/- mice tumor vessels, however, the
number of a-SMA–positive cells lining
the endothelial cells was profoundly
reduced (right panel). Images in panel A
were viewed under an Olympus SZX12
stereomicroscope (Tokyo, Japan) with
Leica PL Fluotar 0, 5, 20_/0.5 NA dry
objective (Wetzlar, Germany), captured
with an Olympus C4040 camera, and
processed with Olympus
DP-Soft 3.2. Images in panels B-D were
viewed under a Leica DMRA2
fluorescence microscope with Leica HC
PL Fluotar 0, 5, 20_/0.5 NA dry
objective, captured using Photometrics
CoolSNAP HQ, (Photometrics,
Friedland, Denmark), and processed with
Metamorph 4.6-5 (Molecular Devices,
Sunnyvale, CA).
24
Soluble Dll4 inhibits Dll4-Notch signaling
We next wished to determine if soluble Dll4 could antagonize
Notch activation.
Extracellular domain of human Dll4 fused either
to AP, Fc, or His tag were expressed in
mammalian cells, purified,
and determined to bind Notch1-Fc (Figure 2.2A) and Notch4-
Fc (data
not shown) but not Notch3-Fc (Figure 2.2A) or Fc alone (data not
shown). Notch
activation is dependent on the expression of Dll4
in the cellular context. To test that the
soluble forms of Dll4
do not induce Notch activation, we introduced various Dll4
constructs
in endothelial cells and examined the induction of downstream
Notch-
responsive genes (Hey1, Hey2, Hes1, and Hes2) by RT-PCR.
Representative data for the
absence of Notch activation are
shown by the lack of Hes2 induction by Dll4-Fc or Dll4-
His (Figure 2.2B).
Hes2 is downstream of Notch and induced by full-length Dll4
when
presented in the cellular context (Figure 2.2C). We next
determined if sDll4 can inhibit
the activity of cellular Dll4
in inducing Notch signaling. To this end, full-length Dll4
(Dll4-FL)
was introduced into ChoK cells and cocultured with HUVECs expressing
target
Notch1 and Notch 4. Dll4-FL induces Notch-regulated genes,
Hey1, Hey2, Hes1, and
Hes2 (Figure 2.2C), in human endothelial
cells using human gene-specific primer pairs.
In identical experiments,
addition of human sDll4-Fc and sDll4-His blocked Dll4-FL–
induced
activation of Hey1, Hey2, Hes1, and Hes2 (Figure 2.2C). Thus soluble
Dll4
functions as an antagonist of Dll4-Notch signaling. Quantitation
of gene expression
showed that Dll4-Fc inhibited Hey1, Hey2,
Hes1, and Hes2 to 69%, 26%, 29%, and 46%
of control, respectively,
while sDll4-His reduced their expression to 48%, 3%, 10%, and
28%, respectively.
25
Figure 2.2 Biochemical properties of sDll4. (A) Notch-Fc fusion protein was coated directly on enzyme-linked
immunosorbent assay (ELISA) plates. sDll4-AP was allowed to bind Notch-Fc, and the bound Dll4 was quantitated by
the addition of AP substrate. sDll4-AP bound efficiently to Notch1 and not Notch3 (left panel). Binding of sDll-4Fc
and sDll4-His to Notch1 was examined. (B) HUVECs were transfected with expression vectors for sDll4-Fc, sDll4-His,
or vector alone. Notch-responsive Hes-2 gene expression was not induced by sDll4 proteins. (C) Notch activation
measured by the induction in Hes-1, Hey-1, and Hes-2 when HUVECs were cocultivated with ChoK expressing Dll4-
FL (full length). Addition of recombinant sDll4-Fc and sDll4-His reduced the induction of Notch responsive genes.
Two independent experiments produced similar results.
Soluble Dll4 induces sprouting and tube formation
We intended to determine if soluble Dll4 could mimic the dll4
loss-of-function
phenotype. sDll4-Fc and sDll4-His were tested
in tube formation assays using endothelial
cells placed on polymerized
Matrigel to promote the formation of tubelike structures.
Minimal
amounts of tubes were formed in the absence of growth factors,
and abundant
tube formation was observed with the addition of
VEGF. Dll4-His showed a dose-
dependent induction of tube formation
in the absence of additional growth factors (Figure
2.3). Quantitative
measurement of junction formation and length of tubes increased
in a
dose-dependent manner (Figure 2.3). Similar results were
seen with Dll4-Fc (data not
shown).
26
Figure 2.3 sDll4 induces tubule formation in vitro. (A) HUVECs were cultured on standard Matrigel in growth
factor–deficient conditions in triplicates in 2 independent experiments with either sDll4 or VEGF for 24 hours. Shown
are representative pictures from triplicate wells repeated twice. (B) Quantitative analysis for tube length and the
number of junctions in sDll4-treated HUVECs (Bioquant Image Analysis; mean SEM from triplicate wells in 2
repetition experiments). Similar results were seen with human arterial endothelial cell assay (data not shown). *P < .05
compared to no growth factor. Photomicrographs in panel A were taken with a Nikon Plan Fluor , 0.17, 4X/0.12 NA
objective and 10X eyepiece and processed with Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MA).
We next extended these studies in an assay in which endothelial
cell spheroids
placed in collagen show proliferation and outward
migration of vessel-like structures.
VEGF profoundly increases
sprouting, while no sprouts were observed in growth factor–
deficient
conditions. sDll4-Fc and sDll4-His when examined alone induced
sprouting in a
dose-dependent manner. Representative data with
sDll4-His are shown in Figure 2.4.
27
Similar results were seen with
sDll4-Fc (data not shown). These data indicate that
inhibition
of endogenous Notch signaling leads to increased endothelial
cell migration
and organization to make tubes and promotes vascular
sprouting that is analogous to the
observed murine Dll4
+/–
phenotype.
Figure 2.4 sDll4 induces sprouting in endothelial
cell spheroids in vitro. (A) HUVEC spheroids were
cultured on Matrigel in growth factor–deficient
conditions in triplicates with either sDll4 or VEGF
for 24 to 72 hours. Shown are representative pictures
using sDll4-His from triplicate wells repeated twice.
(B) Quantitative analysis for vascular area is shown
(Bioquant Image Analysis; mean SEM from triplicate
wells in 2 repetition experiments). Similar results
were seen with sDll4-Fc (data not shown).
Photomicrographs were taken using a Nikon Coolpix
5000 camera and a Carl Zeiss Invertoskop
microscope with a Nikon Plan Fluor , 0.17, 4X/0.12
NA objective and 10X eyepiece and processed with
Image-Pro Plus 6.0. Experiments were repeated with
similar results. (B) *P < .05 compared to no growth
factor.
28
sDll4 induces vascularization of Matrigel plugs in vivo
To further demonstrate that sDll4 can directly inhibit angiogenesis
in vivo, we
performed a murine Matrigel plug experiment. Matrigel
was supplemented with VEGF,
sDll4-Fc, sDll4-His, or various
combinations, and injected into the ventral abdominal
subcutaneous
tissue of Balb/C nu/nu mice. Matrigel plugs without growth factors
had
virtually no vascularization after 6 days (Figure 2.5A), while
VEGF recruited endothelial
cells and formed various stages of
vascular structures including those with open lumen
containing
red blood cells throughout the plug. sDll4 in the context of
VEGF showed a
marked increase in vascular structures (Figure 2.5),
which appeared like thin strings, and
mostly lacked lumen. Similarly,
sDll4 alone, in the absence of VEGF, was also capable of
inducing
angiogenesis. Immunochemical examination with PECAM further
demonstrates
the contrast between VEGF-induced large vessel
filled with red blood cells and sDll4-
induced vessels that express
PECAM but lack lumen and lack perfusion defined by the
presence
of red blood cells (Figure 2.5B). Hemoglobin was also quantitated
specifically
by the Drabkin method using a Drabkin reagent kit
and fold change was compared with
control measurement represented
as 1. Median hemoglobin levels thus were 1, 9.7, 2.5,
and 3
g/L/g plug in control, VEGF, Dll4-Fc, and Dll4-His groups, respectively.
There was
a near 4-fold decrease in hemoglobin concentration
in sDll4-containing Matrigel plugs,
compared to VEGF alone.
29
Figure 2.5 sDll4 induces vessel response but lacks perfusion in murine Matrigel assay. (A) Matrigel was injected
subcutaneously into Balb/C nu/nu mice. After 6 days, plugs were removed and processed in paraffin. Individual
sections were stained with H&E, and representative photographs at X20 magnification from triplicate plugs in 2
independent experiments are shown. (B) Matrigel plugs were stained for PECAM. Photomicrographs were taken with
an Olympus BX51 microscope with an Olympus UPlan FL , 0.17 20X/0.5 NA dry objective mounted with a Retiga
2000R camera (QImaging, Burnaby, BC, Canada) and processed with Image-Pro Plus 6.0. Quantitation of vascularized
area averaged (SEM) from all plugs (Scion Image software; Scion, Frederick, MD) in bar graph. *P < .05 compared to
no growth factor.
sDll4 inhibits the growth of human tumors in athymic mice
Tumor vessels have distinctive gene expression profile over
resting vessels. Dll4
is one of the genes induced in tumor vessels
in certain human and murine tumors (Figure
2.1C). Dll4 induction
may be a generalized feature of tumor vessels, one that could
be
beneficial for tumor growth. Dll4 expression is seen predominantly
in the tumor
vasculature. To determine the effect of sDll4 on
tumor cells, tumor cell viability in vitro
with various concentrations
of sDll4 was tested (HT29, MCF-7, SCC-15, B16, PC3, and
KS-SLK
cell lines), and no effect was observed (data not shown).
30
Given
the ability of Dll4 to profoundly affect angiogenesis in vivo,
and the observed
sDll4 alteration of the vascular response,
we speculated that sDll4 may modulate tumor
growth in vivo.
We therefore examined the activity of sDll4 in vivo in tumor
xenograft
models. HT29 (human colon carcinoma cell line) and
KS-SLK (human Kaposi sarcoma
cell line) cells were premixed
with Matrigel-containing vehicle or sDll4 and implanted
subcutaneously.
Compared to control tumors, xenografts supplemented with sDll4
exhibited a significantly reduced tumor growth over 2 weeks
(Figure 2.6A). Similar
results were obtained in KS-SLK. Median
tumor volume of control tumors at 2 weeks
was 585 mm
3
, while
that of tumors in Matrigel containing sDll4-His was 267 mm
3
.
We next studied the effect of sDll4 when produced by tumor cells.
HT29 and KS-
SLK cells were transfected with expression vectors
to produce Dll4-FL, sDll4-Fc, and
sDll4-His, and expression
of each protein was confirmed in Western blot assays (data not
shown). Coexpression of truncated CD4 allowed sorting of transfected
cells to more than
90% purity. Equal numbers of cells (1 x 10
6
per injection site) were implanted in athymic
mice (6-8 tumors
per group), and tumor volume was measured for 2 weeks. Tumor
volume was similar in vector alone and Dll4-FL, while sDll4-Fc
and sDll4-His had
markedly reduced tumor volume (more than 70%
reduction with sDll4-His) (Figure
2.6B). Tumors harvested at the
time were examined for vascular density using PECAM
immunostaining.
Tumors expressing Dll4-FL or vector alone showed highly structured
vessels (Figure 2.6C). In contrast, sDll4-expressing tumors showed
marked changes in
the vessel architecture. There were many more
branching points in sDll4-expressing
tumor vasculature compared
to vector alone or Dll4-FL (Figure 2.6).
31
Similar results were
obtained in KS-SLK tumor xenografts. Remarkably, the vessels
appeared thin and often lacking apparent lumen. These characteristics
were reminiscent
of blood vessel branching in Dll4
+/–
mice, and in Matrigel plugs impregnated with sDll4.
Consistent
with poorly forming thin vessel lacking lumen, we examined the
areas of
hypoxia. Analysis of hypoxia focused on viable tumor
regions only. There were large and
wide areas of hypoxia in
sDll4-Fc and sDll4-His. Quantitation of these areas showed
marked
increase in hypoxic regions of sDll4-expressing tumors compared
to both Dll4-
FL– and vector-expressing tumors (Figure 2.6D).
Tumor perfusion was also measured
using fluorescent-labeled
lectin, which binds to the luminal surface of the blood vessels.
There was very limited perfusion in the sDll4-expressing tumors
compared to Dll4-FL–
and vector-transfected cells (Figure 2.6E).
In addition, we determined the presence of
pericytes on newly
forming tumor vessels by localizing -SMA expression. Tumor
vessels
in wild-type mice showed normal pericyte coverage, whereas tumor
vessels in
Dll4
+/–
mice present a dramatic reduction of
pericyte coverage as determined by the
number of -SMA–positive
cells lining the endothelial cells. sDll4 similarly reduced
the
number of pericytes in tumor vessels in athymic mice bearing
human tumors. Taken
together, these data provide strong evidence
for the role of Dll4 in pericyte recruitment to
newly forming
vessels.
32
Figure 2.6 sDll4 inhibits the tumor growth in a murine tumor xenograft model. (A) Mice (n = 6/group) were
implanted with 1 X 10
6
HT29 cells in a Matrigel preparation with PBS or sDll4-Fc or sDll4-His (5 ug/mL) and tumor
volumes (mean SEM) were measured after 2 weeks; tumors were then harvested and analyzed. Tumor volumes were
significantly smaller in the sDll4 arm. *P < .05 compared to control. The experiment was repeated twice. (B) In
assessing the effect of endogenous expression of sDll4, HT29 cells were transfected with expression vector with Dll4-
FL, sDll4-Fc, sDll4-His, or vector alone. Coexpression of truncated CD4 was done to allow sorting of the transfected
cells. Equal numbers of the transfected cells were implanted in mice (n = 6/group). Tumor volumes (mean SEM) were
assessed. Tumor volumes were significantly smaller in the sDll4 groups. *P < .05 compared to vehicle. (C)
Microvasculature was assessed by PECAM immunostaining, and the blood vessel volume was quantitated as described
in “Materials and methods.” Mean SEM. *P < .05 compared to vehicle. (D) Hypoxy probe was infused prior to tumor
harvest; tumor sections were then probed with MAb and fluorescent-labeled secondary antibody as described in
“Materials and methods.” Hypoxic areas were quantitated (mean SEM) using ImageJ as described in “Materials and
methods.” All values are expressed as mean SEM. *P < .01. Photomicrographs were taken with a Nikon Eclipse 80
microscope with a Nikon Plan Fluor , 0.17 10X/0.3 NA dry objective mounted with a Photometrics CoolSNAP camera
and processed with Metamorph V 6.3r2. (E) Vascular perfusion was determined by injecting fluorescent-labeled lectin
10 to 15 minutes prior to killing mice and harvesting tumors. Lectin was localized to perfused areas, while blood
vessels were delineated with PECAM staining. Lectin and PECAM colocalized in control group, while sDll4 group
showed marked deficiency of perfusion. (F) Localization of a-SMA in tumor vessel. Control group showed
colocalization of a-SMA and PECAM, while sDll4 group had paucity of a-SMA–positive cells in the microvessels.
33
Chapter 2: Discussion
Dll4, Notch1, and Notch4 are expressed in endothelial cells.
Notch1/Notch4 double-
mutant embryos show severe vascular remodeling
defects,
9,14
and putative downstream
targets of Notch pathway
including Hey1/Hey2 double mutants develop vascular defects
with lesser severity.
14,16
Dll4 thus profoundly effects vascular
development and vessel
maturation. Notably, Dll4
–/–
and most ( 70% on CD1, 100% on C57BL6 and 129Sv/J
genetic backgrounds)
Dll4
+/–
mutant embryos die at midgestation due to defective
vascular development. Lethal haploinsufficiency of Dll4 is analogous
to that of VEGF,
5,21
which lies upstream of Notch signaling
in arterial development.
15
We thus focused on Dll4 in vascular development. Analysis of
vasculature in the
heterozygote mice has been revealing in that
the vasculature develops normally until E8.5
when the defects
become apparent and manifest by increased vascular network combined
with less mature large vessels that eventually branch and fuse
with adjacent vessels and
result in short circuit and failing
circulatory system. We wished to determine if Dll4
deficiency
was manifest in a small fraction of mice that survives to adult
life. Use of
tumor cell implants in Dll4
+/–
mice was compared
to age-matched wild-type mice for
localized vascular response.
Vascular response in adult Dll4
+/–
mice was similar to
the
embryonic defects including enhanced vascular density, nearly
2-fold higher than in wild-
type mice. Secondly, unlike the wild-type
mice, the vessels in the mutants have narrow
caliber and lack
hierarchic branching pattern (Figure 2.1A-B). Dll4 may thus be
required
for functions in branching and maturation of newly
forming vessels.
34
Furthermore, recruitment of pericytes to the
newly forming vessels was markedly reduced
in Dll4
+/–
mice tumor vessels. Similar defect was observed in tumor vessels
in response to
sDll4. It is thus possible that some of the observed
defects in Dll4-deficient signaling are
due to the paucity of
pericytes and smooth muscle.
VEGF and Dll4 have also been shown to be up-regulated by hypoxia,
22,23
which is
one of the environmental factors that regulates vascular
patterning and growth. We thus
wished to determine if Dll4 expression
was changed in tumor vessels compared to those
in the adjacent
normal tissue. Dll4
+/–
mice tumor implants were examined
for Dll4
expression. LacZ insertion in the Dll4 targeting vector
allows its expression under the
Dll4 promoter. Thus β-gal
staining highlighted a few well-organized vessels in the
normal
subcutaneous tissue. In the tumor tissue, however, we observed
a marked increase
in vascular β-gal staining and in the
density of vascular structures. This role of Dll4 in
modifying
vascular response and its overexpression in tumor vessels may
provide a
therapeutic opportunity to alter tumor response.
Dll4 induces Notch signaling when presented in the cellular
context, by activating
Notch processing and release of the intracellular
domain, which is subsequently
translocated to the nucleus to
modify expression of target genes including Hey and Hes.
24
Various
Dll4 variants containing only the extracellular domain indeed
block Dll4-induced
Hey and Hes expression. sDll4 thus allowed
us to assess the consequences of blocking
Dll4-Notch signaling
in various distinct functions. sDll4 treatment recapitulates
several
features seen in Dll4
+/–
mutant mice including
increased endothelial cell tube formation
and vascular sprouting.
35
Using Matrigel plug assays, we noticed that sDll4 markedly increased
endothelial cell
migration. Endothelial cells created stringlike
vascular structures, which do not reveal a
prominent lumen and
display a notable paucity of red blood cells, unlike those observed
upon VEGF treatment, which produces large vascular structures
with open lumen filled
with red blood cells. sDll4 thus modifies
newly forming vessels that are highly deficient
in perfusion.
Overexpression of Dll4 in tumor vessels and profound alteration
of newly
forming vessels by disruption of Dll4-Notch signaling
provides an opportunity to
influence tumor growth. Use of sDll4
indeed showed a marked reduction in tumor
growth. Inhibition
of tumor growth was also reproduced by engineering tumor cells
to
produce soluble Dll4. Both sDll4-Fc and sDll4-His markedly
reduced the tumor growth
combined with changes in tumor vessels
similar to those seen in Dll4
+/–
mutant mice and
to the
effect of sDll4 in the Matrigel assays. Perfusion deficiency
was confirmed by
examining the areas of hypoxia in the tumor,
which showed a marked increase in sDll4-
expressing tumor cells.
In summary, Dll4 has profound effect in vascular biology. It
appears to be essential for regulating migration of vessels
in response to VEGF, and
necessary for maturation of newly forming
vessels (including regulation of lumen
formation) and vascular
perfusion. Inhibition of Dll4 function thus has a paradoxical
effect in inducing an excessive density of newly forming vessels,
but defective vessel
maturation, lumen formation, recruitment
of pericytes, and perfusion.
36
It raises many questions as to
how Dll4 regulates endothelial cell migration, vessel
branching,
lumen size, and endothelial interaction with pericytes and matrix
proteins.
Inhibition of Dll4 with soluble protein or neutralizing
antibodies or knock down of gene
expression with short interference
RNA may provide opportunities to treat cancer and
vascular proliferative
diseases.
37
Chapter 2: References
1. Risau W. Mechanisms of angiogenesis. Nature. 1997;386:671-674.
2. Rossant J, Hirashima M. Vascular development and patterning: making the right
choices. Curr Opin Genet Dev. 2003;13:408-412.
3. Folkman J. Fundamental concepts of the angiogenic process. Curr Mol Med.
2003;3:643-651.
4. Carmeliet P. Angiogenesis in life, disease and medicine. Nature. 2005;438:932-936.
5. Ferrara N, Carver-Moore K, Chen H, et al. Heterozygous embryonic lethality induced
by targeted inactivation of the VEGF gene. Nature. 1996;380:439-442.
6. Bray SJ. Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell
Biol. 2006;7:678-689.
7. Iso T, Hamamori Y, Kedes L. Notch signaling in vascular development. Arterioscler
Thromb Vasc Biol. 2003;23:543-553.
8. Hainaud P, Contreres JO, Villemain A, et al. The role of the vascular endothelial
growth factor delta-like 4 ligand/Notch4-Ephrin B2 cascade in tumor vessel remodeling
and endothelial cell functions. Cancer Res. 2006;66:8501-8510.
9. Krebs LT, Xue Y, Norton CR, et al. Notch signaling is essential for vascular
morphogenesis in mice. Genes Dev. 2000;14:1343-1352.
10. Uyttendaele H, Marazzi G, Wu G, Yan Q, Sassoon D, Kitajewski J. Notch4/int-3, a
mammary proto-oncogene, is an endothelial cell-specific mammalian Notch gene.
Development. 1996; 122:2251-2259.
38
11. Shutter JR, Scully S, Fan W, et al. Dll4, a novel Notch ligand expressed in arterial
endothelium. Genes Dev. 2000;14:1313-1318.
12. Wang HU, Chen ZF, Anderson DJ. Molecular distinction and angiogenic interaction
between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4.
Cell. 1998;93:741-753.
13. Gerety SS, Wang HU, Chen ZF, Anderson DJ. Symmetrical mutant phenotypes of the
receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular
development. Mol Cell. 1999;4:403-414.
14. Duarte A, Hirashima M, Benedito R, et al. Dosage-sensitive requirement for mouse
Dll4 in artery development. Genes Dev. 2004;18:2474-2478.
15. Lawson ND, Vogel AM, Weinstein BM. sonic hedgehog and vascular endothelial
growth factor act upstream of the Notch pathway during arterial endothelial
differentiation. Dev Cell. 2002;3:127-136.
16. Fischer A, Schumacher N, Maier M, Sendtner M, Gessler M. The Notch target genes
Hey1 and Hey2 are required for embryonic vascular development. Genes Dev.
2004;18:901-911.
17. Krebs LT, Shutter JR, Tanigaki K, Honjo T, Stark KL, Gridley T. Haploinsufficient
lethality and formation of arteriovenous malformations in Notch pathway mutants. Genes
Dev. 2004;18:2469-2473.
18. Hogan B, Beddington R, Constantini F, Lacy E. Manipulating the mouse embryo.
Cold Spring Harbor, NY: Laboratory Press; 1994.
19. Kertesz N, Krasnoperov V, Reddy R, et al. The soluble extracellular domain of
EphB4 (sEphB4) antagonizes EphB4-EphrinB2 interaction, modulates angiogenesis, and
inhibits tumor growth. Blood. 2006;107:2330-2338.
39
20. Xia G, Kumar SR, Hawes D, et al. Expression and significance of vascular
endothelial growth factor receptor 2 in bladder cancer. J Urol. 2006; 175:1245-1252.
21. Carmeliet P, Ferreira V, Breier G, et al. Abnormal blood vessel development and
lethality in embryos lacking a single VEGF allele. Nature. 1996; 380:435-439.
22. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by
hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 1992;359:843-845.
23. Mailhos C, Modlich U, Lewis J, Harris A, Bicknell R, Ish-Horowicz D. Delta4, an
endothelial specific notch ligand expressed at sites of physiological and tumor
angiogenesis. Differentiation. 2001;69:135-144.
24. Diez H, Fischer A, Winkler A, et al. Hypoxia-mediated activation of Dll4-Notch-
Hey2 signaling in endothelial progenitor cells and adoption of arterial cell fate. Exp Cell
Res. 2007;313:1-9.
40
CHAPTER 3: THE ROLE OF EPHS, EPHRINS AND GROWTH FACTORS IN
KAPOSI SARCOMA AND IMPLICATIONS OF EPHRINB2 BLOCKADE.
Scehnet JS, Ley EJ, Krasnoperov V, Liu R, Manchanda PK, Sjoberg E, Kostecke AP,
Gupta S, Kumar SR, Gill PS. The role of Ephs, Ephrins, and growth factors in Kaposi
sarcoma and implications of EphrinB2 blockade. Blood. 2009 Jan 1;113(1):254-63.
Chapter 3: Abstract
Kaposi sarcoma (KS) is associated with human herpesvirus (HHV)-8 and
dependent on induction of vascular endothelial growth factors including VEGF, VEGF-C
and their cognate receptors VEGR1,-2,-3. VEGF regulates genes that provide arterial or
venous identity to endothelial cells, such as induction of EphrinB2 that phenotypically
defines arterial endothelial cells and pericytes, and represses EphB4 that defines venous
endothelial cells. We conducted a comprehensive analysis of the Eph receptor tyrosine
kinases to determine which members are expressed and therefore contribute to KS
pathogenesis. We demonstrated limited Eph/Ephrin expression, notably, the only ligand
highly expressed is EphrinB2. We next studied the biological effects of blocking
EphrinB2 using the extracellular domain of EphB4 fused with human serum albumin
(sEphB4-HSA). sEphB4-HSA inhibited migration and invasion of the KS cells in vitro in
response to various growth factors. Lastly, we determined the biological effects of
combining sEphB4-HSA and an antibody to VEGF. sEphB4-HSA was more active than
the VEGF antibody and combination of the two had at least additive activity.
41
sEphB4-HSA reduced blood vessel density, pericyte recruitment, vessel perfusion, and
increased hypoxia with an associated increase in VEGF and DLL4 expression.
Combination of sEphB4-HSA and VEGF antibody is a rational treatment combination for
further investigation.
Chapter 3: Introduction
Kaposi sarcoma (KS) is a highly vascularized tumor that is associated with human
herpesvirus (HHV)-8. KS manifests as an angioproliferative disease most frequently in
the skin; in advanced cases KS involves visceral organs such as the liver, lungs or GI
tract which can be fatal. KS lesions exhibit an extensive vascular network of slit-like
spaces with abnormal spindle-shaped endothelial cells lining the tumor vessels, which
lack basement membranes. Defective vasculature results in an accumulation of the blood
components including albumin and red and mononuclear cells in the lesions.
1
Vascular endothelial growth factors including VEGF, VEGF-C and their cognate
receptors VEGFR1,-2,-3 are highly expressed in KS cells and induced by HHV-82.
VEGFs function as autocrine growth factors.
2
Furthermore, VEGFs induce tumor vessels
in a paracrine manner, and regulate endothelial cell proliferation and migration. VEGFs
regulate genes that provide arterial or venous identity to endothelial cells, such as
induction of EphrinB2 that phenotypically defines arterial endothelial cells and pericytes,
and represses EphB4 that defines venous endothelial cells.
2-4
The EphB4/EphrinB2 interaction plays a critical role in vessel maturation as
knockout of either protein is embryonically lethal in mice due to vascular arrest at the
42
primitive capillary plexus stage.
5, 6
We previously noted the disorganized KS vasculature
was due to unbalanced expression of EphB4 and its ligand EphrinB2 and that the HHV-8
virus associated with KS regulates EphB4 and EphrinB2. Infection of venous endothelial
cells with HHV-8 results in a switch from expression of EphB4 to EphrinB2, similar to
that seen with vascular endothelial growth factor (VEGF).
2
We also observed EphrinB2
expression is required for KS cell viability by knock down with siRNA.
2
EphrinB2 may also regulate biological functions of cell migration, adhesion and
invasion in KS. EphrinB2 can interact with other members of Eph receptors including
EphB1, EphB2, EphB3, EphB4, and EphA4.
7-9
Secondly EphrinB2 can modulate
vascular response by binding to EphB4 in adjacent tumor vessels.
3, 10-12
In the current
work we conducted a comprehensive analysis of the Eph receptor tyrosine kinases to
determine which of the other members may be expressed and contribute to KS
pathogenesis. Next we studied the biological effects of blockingEphrinB2 in vitro and in
vivo using the soluble form of EphB4 (sEphB4) consisting of the extracellular domains
of the receptor.
13
Lastly, we determined the biological effects of combining sEphB4 fused
to human serum albumin (sEphB4-HSA) and an antibody to VEGF.
43
Chapter 3: Materials and Methods
Antibodies and other reagents
Antibodies to Eph receptors and ligands were obtained from R&D systems
(Minneapolis, MN), EphB2, EphB4 and EphrinB2 were generated at Vasgene
Therapeutics Inc., anti- D31 (M20) from Santa Cruz Biotechnology (Santa Cruz, CA),
anti-Ki-67 from Dako (Carpenteria, CA), anti-SMA (Dako, Carpinteria, CA), IgG-Fc
fragment, and anti–human Fc, from Jackson Laboratories (Bar Harbor, ME);
hypoxyprobe-1, from Chemicon International (Temecula, CA); rhodamine-labeled
ricinus communis agglutinin I (RCA), from Vector Laboratories (Burlingame, CA); and
alkaline phosphatase substrate para-Nitrophenylphosphate (pNPP), from Sigma
Chemicals (St Louis, MO).
Expression and purification of sEphB4 fused to human serum albumin:
cDNA encoding amino acids 1-539 of sEphB4 representing the entire
extracellular domain was cloned upstream of the mature human serum albumin
pCRscript/sEphB4-HSA and placed into the mammalian expression vector under control
of the CMV promoter stably expressed in the CHO cell line. Supernatants were
concentrated by tangential flow filtration and diafiltered into 20mM Tris pH 8.0, 50mM
NaCl. This was applied to a Q-FF column equilibrated in the same buffer. The sEphB4-
HSA fusion protein was eluted with 25mM NaCl steps in 20mM Tris pH 8.0.
44
The elution was followed by NuPAGE Gel. Fractions containing sEphB4-HSA were
diafiltered into10mM sodium phosphate buffer pH 7.0, 50mM NaCl and applied to a
hydroxyapatite column equilibrated in the same buffer. The sEphB4-HSA was eluted
from the column using 10mM sodium phosphate steps at pH 7.0 in 50mM NaCl. The
elution was followed by NuPAGE Gel.
Characterization of sEphB4-HSA by Saturation Binding
To characterize the functionality of sEphB4-HSA and sEphB4, a solid phase
ELISA was developed in which sEphB4-HSA is trapped on 96 well ELISA plates (Nunc,
Maxisorb) using EphB4 monoclonal antibody. Samples were diluted serially in PBS and
run in triplicate. After incubation at 40C and washing with PBS, sEphrinB2-alkaline
phosphatase, generated at VasGene Therapeutics, Inc., was added to the wells and
incubated at 40C for 1 hour. After washing with PBS, 100μl of pNPP substrate
(Calbiochem) in 50mM carbonate buffer pH 9.0 was added to each well. The colorimetric
reaction was developed at 37
0
C for 4 hours and read on a Victor Wallac 1420 96 well
multi-label plate reader. Absorbance at 405nm was read, data transferred into GraphPad
Prism and the dissociation constant was calculated using non-linear regression.
Intraperitoneal Pharmacokinetic Analysis
Mice (n = 6) were given a single I.P. bolus injection of 10mg/kg dose of either
sEphB4 (545pmole) or sEphB4-HSA (250pmole). Plasma samples were obtained at 0.25,
1, 2, 4, 8, 16, 24, 48 and 72 hours.
45
ELISAs were performed on serially diluted plasma samples and concentrations were
obtained from linear regression of standard curves on each ELISA plate. Area under the
time-concentration curve was calculated using GraphPad Prism.
According to first order kinetics, the elimination half life was determined from 0.693/Ke.
The elimination constants were determined using the linear slope of the Log
concentration –time curves during the elimination phase. All animal studies were
performed under the approval of the USC-Keck School of Medicine. Phosphorylation
analysis of EphB4: Endothelial cells were grown in 60mm dishes until 100% confluence,
serum-starved, and were treated with recombinant mouse EphrinB2/Fc fusion protein or
Fc alone at increasing concentrations for 10 min as previously described.
14
Lysates were
prepared with buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% (v/v)
Triton-X100, 1 mM EDTA, 1 mM PMSF, 1 mM sodium vanadate, and centrifuged at
50,000g for 60 min at 4°C. The immunoprecipitated complexes were probed with anti-p-
yr-specific antibody 4G10. EphB4 precipitation efficiency was tested by probing with
EphB4 antibody.
Cell culture
KS-SLK, KS-IMM were cultured as previously described.
2
Normal HUVECs and
human umbilical arterial ECs (HUAECs) were obtained from Cambrex (Walkersville,
MD) and maintained in EGM2-supplemented medium (Invitrogen).
46
For all experiments, HUVECs were used at passages 4 or below and collected from a
confluent dish. HT29 colon cancer cell lines were obtained from American Type Culture
Collection (Manassas, VA) and cultured under recommended conditions.
Reverse-transcription-polymerase chain reaction (RT-PCR) analysis
First-strand cDNA was synthesized from total RNA using ImProm-IITM reverse
transcription system (Promega, Madison, WI) and used for PCR with specific primers.
15
Equal amounts of cDNA from each cell line were used in each reaction (normalized to
actin). Gene-specific amplification consisted of 35 cycles of denaturation at 94°C for 30
sec, annealing at 60°C for 45 sec, and extension at 72°C for 1 min. PCR products were
separated on 1.5% agarose gels and stained with ethidium bromide. HT-29 cell line was
used as a control. Q-PCR analysis was performed as previously described.
14
Western Blot
Cell lysates were prepared using cell lysis buffer (GeneHunter, Basgvukke, TN)
supplemented with protease inhibitor cocktail (Pierce, Rockford, IL), total protein was
determined using the DC reagent system (Bio-Rad, Hercules, CA). Typically, 20μg
whole cell lysate was run on 4% to 20% Tris-glycine gradient gel, as previously
described.
14
47
Migration assay
SLK cells were seeded onto six-well plates and cultured in RPMI 1640 until
confluent. At time zero the monolayer of cells was scraped using a sterile pipette tip. The
migration process was examined dynamically after treatment with various concentration
of sEphB4 and recorded with a Nikon Coolpix 5000 digital camera with microscope
adapter, as previously described.
16, 17
Filled area was quantitated in 5 independent fields
using ImageJ software from NIH (Bethesda, MD).
Invasion assay
SLK cells (5 x 10
4
) were seeded into 8 μm Matrigel-precoated inserts (BD
Bioscience, Palo Alto, CA). The inserts were placed in companion wells containing
RPMI supplemented with 5% FBS and VEGF, EGF, PDFG, or FGF as chemoattractants.
Following 22 hours of incubation, the inserts were removed and the noninvading cells on
the upper surface were removed with a cotton swab. The cells on the lower surface of the
membrane were fixed in 100% methanol for 15 minutes, air-dried, and stained with Diff-
Quik stain for 2 minutes14. The cells were counted in five individual high-powered fields
for each membrane under a light microscope.
2
Assays were done in triplicate for each
treatment group.
48
Cell Viability Assay
KS cells were seeded in 48-well plates at a density of 1 × 10
4
cells/well in a total
volume of 500 μl. Medium was changed after cells were attached, and triplicate samples
were treated with sEphB4-HSA. Cell viability was assessed by using 3-(4,5-
dimethylthiazol-2- l)-2,5-diphenyltetrazolium bromide (MTT) as described previously.
14
Cell apoptosis was also studied by plating cells as described.
14
Cells were serum starved
overnight and apoptosis was quantified using Annexin-V FITC.
Immunohistochemistry and immunofluorescence
Fresh frozen tissue embedded in OCT were sectioned at 5 μm and fixed in
phosphate-buffered 4% paraformaldehyde and washed in PBS. For studies on formalin-
fixed paraffin-embedded tissues, 5μm sections were deparafinized and hydrated, and
antigen epitope retrieval was performed by boiling slides in 10 mM sodium citrate buffer
(pH 8.5) at 80°C for 20 min. Endogenous peroxidase activity was blocked by incubation
in 3% H2O2, followed by blocking of nonspecific sites with SuperBlock blocking buffer
(Peirce, IL). Sections were then incubated with primary antibody overnight at 4°C. After
washing in PBS, antibody binding was localized with appropriate secondary antibodies.
Nuclei were counterstained with 6-diamidino-2-phenylindole dihydrochloride hydrate
(DAPI). The number of cells staining positive was counted by a blinded observer in 5
random ×40 fields and compared by Student t test. Images were obtained using an
Olympus AX70 fluorescence microscope and Spot v2.2.2 (Diagnostic Instruments,
Sterling Heights, MI) digital imaging system.
49
Apoptosis was detected in deparaffinized sections of tumor samples by TdT-mediated
dUTP nick-end labeling (TUNEL) assay using the in situ cell death detection kit (Roche,
Piscataway, NJ). Distribution and intensity of hypoxia were studied using the recommend
protocol for hypoxyprobe-1 (HP1-100; Chemicon International). Murine tumor xenograft
and metastatic models: KS-SLK and KS-IMM cells were propagated, collected by trypsin
digestion, and re- suspended in serum-free medium. 2x10
6
cells were injected in the flank
of 10- to 12-week-old male Balb/C athymic mice. Tumor growth was measured two
times a week and volume estimated as 0.52 x a x b2, where a and b are the largest and
smallest lengths of the palpable tumor. On day 4 after cell implantation, tumor volumes
were calculated to ensure uniformity in size and animals were divided randomly into four
groups (n=4 mice per group, repeated twice). Each group was administered three times
per week by intraperitoneal injection of 200μl PBS: PBS alone, 10mg/kg VEGF
monoclonal antibody (VEGF moAb; moAb A.4.6.1), 20 mg/kg sEphB4-HSA, or
combination of VEGF moAb and sEphB4-HSA at the same dose levels. Animals were
killed and tumors and normal organs harvested after 5 weeks or when tumor volume was
considered inhumane. Distribution and intensity of hypoxia were studied using
hypoxyprobe-1 (HP1-100; Chemicon International) infused intraperitoneally at a dose of
60 mg/kg one hour prior to the tumor harvest (Chemicon International). Vessel perfusion
was studied using rhodamine-labeled ricinus communis agglutinin 1 (Vector
Laboratories) infused 10 to 15 minutes prior to the tumor harvest and analyzed using the
manufacturer's recommended protocol.
50
For the murine metastatic model, after appropriate anesthesia and sterile alcohol
preparation, a left lateral flank incision was made with a 15 blade knife in the 8- to 10-
week-old male Balb/C athymic mouse. The spleen was exposed through the incision and
1 x 10
6
SLK cells were injected. After 5 minutes, the splenic hilum was ligated,
splenectomy was performed, the incision was closed with 4-0 silk sutures, and the mouse
was recovered. Starting on post operative day 3, each group was administered three times
per week by intraperitoneal injection of 200μl PBS: PBS alone (11 mice), 10mg/kg
VEGF monoclonal antibody (5 mice), 20 mg/kg sEphB4-HSA (5 mice), or combination
of VEGF moAb and sEphB4-HSA at the same dose levels (5 mice). After five weeks of
treatment, mice were sacrificed and livers evaluated for gross metastatic deposits. All
procedures were approved by our Institutional Animal Care and Use Committees and
performed in accordance with the Animal Welfare Act regulations.
Statistical Analysis
Data are presented as mean plus or minus SE. Differences in tumor volume in vivo and
number of cells staining positively were analyzed by Student’s t-test, and significance
was set at P < 0.05.
51
Chapter 3: Results
Expression of the Eph receptors and Ephrin ligands in Kaposi Sarcoma
The Eph and Ephrin expression profiles of the Kaposi Sarcoma cell lines KS-SLK
and KS-IMM, HHV-8 transformed lymphatic endothelial cells (LEC/HHV8), and
primary KS tissue were determined by RT-PCR (Figure 3.1A, B). HT29, a colon cancer
cell line, expresses many members of Eph/Ephrin family and thus was included as a
control. Of the 13 Eph receptors studied, the KS-SLK and KS-IMM cell lines expressed
only EphA2, EphA3, EphA4, EphA5 and EphB2. Only two Ephrin ligands were
expressed in the KS cell lines, EphrinB2 and EphrinA3. LEC/HHV-8 and primary KS
tissue showed similar expression pattern. In addition, EphA1 expression was also noted
in LEC/HHV-8 and KS tumor biopsy. EphrinB2 was the only consistently expressed
Ephrin ligand in KS cell lines, LEC/HHV-8, and KS biopsy. Furthermore in protein
analysis, EphrinB2 was highly expressed in both KS cell lines and KS tissue, while there
was no appreciable level of EphrinA3. Thus of the 8 different ligands only EphrinB2 is
expressed on western blot. Protein analysis for Eph receptors shows expression of EphB2
and EphA4, both of which are known receptors for EphrinB2. EphA2 was not observed at
the protein level. Thus EphrinB2 and its receptors are the pertinent Eph/Ephrin members
of importance in KS.
HUVEC expression analysis demonstrated abundant expression of EphB4 by RT-
PCR and immunoblotting. Additionally EphB2, EphA2 and low levels of EphrinB2 were
expressed in endothelial cells, with very little expression of other Eph/Ephrin members
(Figure 3.1A).
52
EphrinB2 from KS cells or adjacent endothelial cells can activate Eph receptors in KS.
Furthermore, EphrinB2 from endothelial cells may activate EphB4 on adjacent vascular
endothelial cells and regulate a vascular response.
13
Inhibition of the EphrinB2
interaction with its cognate receptors using the soluble form of EphB4 with improved
pharmacokinetics was thus studied further. sEphB4-HSA was chosen for its specificity
for EphrinB2 and lack of binding to any other Ephrin ligand.
13
Figure 3.1 The Eph and Ephrin expression in KS cell lines KS-SLK and KS-IMM, lymphatic endothelial cells
transformed with HHV-8 (LEC/HHV-8), and KS tumor biopsy. (A) The mRNA expression of 13 Eph receptors and
8 Ephrin ligands was determined with RT-PCR. HT29 colon cancer cell line and HUVEC were included as controls.
(B) Western blot analysis to for protein expression of selected expressed Eph-Ephrins. b-actin expression was done to
show comparable protein loading.
53
Expression and purification of the soluble form of EphB4 fused to human serum albumin
In order to develop a long acting form of sEphB4, we choose to fuse the coding
region of the extracellular domain of EphB4 and human serum albumin. This fusion
protein (sEphB4-HSA) has the potential to provide long retention time in the circulation
while retaining the antagonistic function of monomeric sEphB4. The fusion protein was
thus expressed in CHO cells and purified from the supernatant to purity over 95% (Figure
3.2A). When the sEphB4-HSA binding kinetics were compared directly to sEphB4 in
saturation binding studies, the affinity of the fusion protein to sEphrinB2-AP was not
adversely affected as indicated by the comparable dissociation constants (Figure 3.2B).
The sEphB4-HSA fusion protein also retained activity to inhibit EphB4 phosphorylation
upon treatment with dimeric EphrinB2. Recombinant sEphB4-HSA was tested for its
ability to block EphrinB2-induced EphB4 phosphorylation in HUVECs
13
and MCF7 cells
(Figure 3.2B). Pharmacokinetic studies of the purified protein were conducted in mice
and compared to sEphB4 lacking albumin. Circulation half life for sEphB4-HSA was 25
hours compared to 9 hours for sEphB4; sEphB4-HSA area under the curve was 3.8 fold
higher than sEphB4 (Figure 3.2C, D). From here on all studies were completed using the
albumin fusion derivative of sEphB4.
54
Figure 3.2 Expression and characterization of sEphB4 and sEphB4-HSA. (A) sEphB4-HSA was expressed in CHO
cells and purified to near homogeneity and separated on SDS-PAGE (Coomassie staining) under reducing and
nonreducing conditions. (B) Saturation binding kinetics of sEphB4 and sEphB4-HSA in a solid-phase ELISA.
Interaction of increasing concentrations of sEphB4 or sEphB4-HSA with sEphrinB2-AP was determined in a solid-
phase ELISA. Each point was determined in triplicate. Dissociation constants were calculated with the use of nonlinear
regression and Graphpad Prism. (C) Systemic pharmacokinetics of sEphB4 and sEphB4-HSA administered
intraperitoneally. Mice were injected with a 10 mg/kg dose of either sEphB4 or sEphB4-HSA administered
intraperitoneally. Each point represents the average of 2 separate experiments. Error bars represent the SEM. (D)
Pharmacokinetic and saturation binding constants of sEphB4 and sEphB4-HSA. (E) Tyrosine phosphorylation of
EphB4 receptor in MCF7 cells in response to stimulation with EphrinB2-Fc (15 minutes) in the absence or presence of
EphB4-derived soluble proteins.
55
Effects of sEphB4-HSA on KS cell migration and invasion
Given that EphrinB2 is the only major Eph ligand expressed in KS cells, we
studied the effect of blocking EphrinB2 binding to various receptors on adhesion,
migration and invasion. Specifically we determined whether sEphB4-HSA affects KS
cell migration, invasion, and proliferation. KS cells grown to confluence were wounded
and treated with sEphB4-HSA or diluents, and the occupancy of the cell free zone was
examined over time with photography. sEphB4-HSA markedly inhibited the migration of
KS cells to occupy the cell free zone decreasing from 100% confluence at 24 hours with
no treatment, to 17% confluence with 10nm sEphB4-HSA, to 2% confluence with 100nm
sEphB4-HSA (Figure 3.3A).
We then performed an in vitro invasion assay to measure KS cell ability of to
degrade basement membrane and migrate toward a growth factor stimulus. Various
growth factors including FGF, VEGF, EGF and PDGF were used as chemoattractants.
sEphB4-HSA markedly inhibited the invasion of KS cells in response to each of the
growth factors; at 100nm sEphB4-HSA a 5 to 10- fold reduction was demonstrated. Thus
sEphB4-HSA is active regardless of the growth factor used (Figure 3.3B). Growth factors
increased the attachment of KS cells; however sEphB4-HSA treated cells markedly
reduced binding (data not shown). Lastly, sEphB4-HSA was found to have no
appreciable cytotoxic effect on KS cells as assessed by MTT (Figure 3.3C).
56
Figure 3.3 sEphB4-HSA activity in KS migration, invasion, and survival. (A) KS-SLK cells were grown to
confluence, scraped, and treated with varied concentrations of sEphB4-HSA. Cell migration in the clear zone was
documented by photographs at various time points at 20X fields. (B) KS cell invasion in response to growth factors.
Modified Boyden chamber assay was used to determine KS cell invasion across Matrigel-precoated inserts. Data are
presented as number of invading cells plus or minus SE from duplicate wells in 2 experiments. Photomicrographs in
panels A and B were taken with a Nikon Coolpix 5000 camera (Nikon, Tokyo, Japan) and a Carl Zeiss Invertoskop
microscope (Zeiss, Goettingen, Germany) with a 4_/0.12 NA objective and 10X eyepiece.
57
Figure 3.3 sEphB4-HSA activity in KS migration, invasion, and survival. (C) Cell viability assay. KS cells were
grown in triplicate in the presence of increasing concentrations of sEphB4-HSA for 72 hours. Cell viability was
assessed by MTT assay. The experiment was repeated twice with similar results.
sEphB4-HSA inhibits KS tumor growth in a murine tumor xenograft model
sEphB4-HSA inhibits the EphrinB2 interaction with Eph receptors on both other
KS tumor cells, as well as the Eph receptors expressed on newly formed vessels in vivo.
We next studied the in vivo effect of sEphB4-HSA on KS cells as well as the new host
vessels growing into the tumor using a KS tumor xenograft model (Figure 3.4A).
KS-SLK and KS-IMM cells (2 x 10
6
cells) were injected into the flank of 8- to
10-week old, male Balb/C athymic mice. On day 4 after cell implantation, mice were
randomly placed into four groups. Each group was then randomly chosen for systemic
treatment three times a week with PBS (negative control), VEGF moAb (positive
control), sEphB4-HSA or combined sEphB4-HSA and VEGF moAb. Relative to PBS,
sEphB-HSA inhibited tumor growth in both SLK and IMM tumors with tumor volume
reduced to 15.9% and 37.2% of control. sEphB4-HSA was more active in SLK tumors
compared to VEGF moAb (p value < 0.01) and it was equally as active as VEGF moAb
in IMM tumors (p value > 0.05).
58
The combination of sEphB4-HSA and VEGF moAb was superior to each alone in both
tumor types with tumor volumes of 12.1% for KS-SLK (P value < 0.001) and 12.6% for
KS-IMM (P value < 0.001).
KS is known to involve the GI tract, liver and lungs. We thus studied metastasis
of KS to the liver as a representative metastatic model. 1X10
6
SLK cells were injected
into the spleens of 8- to 10- week old, male Balb/C athymic mice exposed by a flank
incision. Splenectomy was performed after 5 minutes and mice were recovered. sEphB4-
HSA was administered three times a week for a period of five weeks and liver metastasis
were quantified by visual examination. Metastatic deposits were noted in the livers of 8
of 11 mice treated with PBS, 2 of 5 mice treated with VEGF moAb (p = NS), 2 of 5 mice
treated with sEphB4-HSA (p = NS), and 0 of 5 mice treated with sEphB4-HSA/VEGF
moAb (P = 0.004, Figure 3.4B). Liver metastases were smaller in size in the mice
receiving sEphB4-HSA or VEGF moAb individually. These results demonstrate the
activity of each agent alone and the additive activity from combination therapy.
59
Figure 3.4 sEphB4-HSA inhibits KS tumor growth in a murine tumor xenograft model. (A) Mice (n = 8/group)
were implanted with 2 X 106 KS-SLK or KS-IMM cells and treated with sEphB4-HSA, VEGF moAb, or combination
sEphB4-HSA/VEGF moAb; tumor volumes were measured 2 times a week; and the data are presented as tumor
volume. After 5 weeks, tumor volumes were as follows; sEphB4-HSA (KS-SLK = 15.9% of control, P < .001; KS-
IMM = 37.2% of control, P < .001) or combination sEphB4-HSA plus VEGF moAb (KS-SLK = 12.1% of control, P <
.001; KS-IMM = 12.6% reduction, P = .001). (B) Mice spleens were injected with 106 SLK cells and treated with
sEphB4-HSA, VEGF moAb, or combination sEphB4 plus VEGF moAb. After 5 weeks, livers were harvested and
examined for the number of tumor metastases (tumors/livers) and are as follows; control (8/11), VEGF moAb (2/5),
sEphB4-HSA (2/5), and combination of sEphB4-HSA and VEGF moAb (0/5).
60
sEphB4-HSA affects tumor vessel density and perfusion
Tumors were harvested and examined for tumor vessel perfusion and tumor
vessel growth. Vascular perfusion was determined by injecting fluorescent-labeled lectin
10 to 15 minutes prior to sacrificing the mice and harvesting KS tumors and lectin was
localized to the perfused areas. Blood vessel density was delineated with PECAM
staining. Lectin and PECAM colocalized in the control group, while the treatment groups
showed marked deficiency of perfusion and vessel density (Figure 3.5A). Adjacent
normal tissue vessel density and perfusion were unaffected by sEphB4-HSA. Deeper
tumor tissue shows progressive decrease in vessel density and even greater decrease in
vessel perfusion (Figure 3.5B). Vessel density decreased with sEphB4-HSA (38.6% of
control), VEGF moAb (62.5% of control) and even more with combination treatment
(16.9% of control). Perfusion also decreased with sEphB4-HSA (20.6% of control) and
VEGF moAb (61.7% of control) (Figure 3.5A). Notably, after VEGF moAb treatment the
remaining vessels perfused well. In sharp contrast among the sEphB4-HSA treatment
group, only a small fraction of the vessels showed perfusion indicating lack of maturation
or collapse of the poorly formed vessels. In both instances, combination treatment with
sEphB4-HSA and VEGF moAb decreased both vessel perfusion and vessel density more
than sEphB4-HSA or VEGF moAb alone.
61
sEphB4-HSA decreases proliferation and increases apoptosis and hypoxia in KS
murine tumor xenograft model
Hypoxia in situ (hypoxyprobe-1) analysis showed that sEphB4-HSA treatment
resulted in increased areas of tumor hypoxia. Both sEphB4-HSA and VEGF moAb
decreased tumor cell proliferation as evaluated by Ki-67 staining (a 2- to 3- fold
reduction in proliferative cells). Apoptosis was measured by TUNEL assay and
combination therapy demonstrated a marked increase in TUNEL-positive cells consistent
with induction of apoptosis.
sEphB4-HSA treatment affects regulators of blood vessel formation
We next examined the differential regulation of the VEGF family of receptors as
well as the VEGF signaling proteins in response to sEphB4-HSA treatment. The levels of
the VEGF, VEGFR1 and VEGFR2 levels in KS tumors were assessed by RT-PCR after
treatment with VEGF moAb, sEphB4-HSA and combination sEphB4-HSA/VEGF moAb.
Treatment with VEGF moAb minimally decreased the levels of VEGF, while treatment
with sEphB4-HSA increased VEGF levels, most likely from the marked increase in
hypoxia. However combination treatment with sEphB4-HSA and VEGF moAb still had
high VEGF levels (Figure 3.5C). Treatment with VEGF MoAb sEphB4-HSA alone or in
combination did not affect VEGFR1 and VEGFR2 levels. EphB4-HSA also showed
increase in murine Dll4 (mDll4) which is induced by VEGF (Figure 3.5C). While VEGF
MoAB had no effect, sEphB4-HSA also showed marked increase in PDGFR-s levels
which was sustained even in the combination group (Figure 3.5C).
62
PDGFR-β is expressed on pericytes and regulates pericyte recruitment to endothelial
cells. sEphB4-HSA treated tumor have poorly formed blood vessels. Increase in PDGFR-
β may be due to feedback from defective vessel maturation.
Figure 3.5 Analysis of vascular perfusion, vessel density, tumor cell proliferation, apoptosis and hypoxia.
(A) Tumors were harvested at completion of the study and examined by hematoxylin and eosin staining. Just before
harvest, mice were infused with RCA-Lectin and hypoxia probe. Nuclei were counterstained with DAPI. RCA-Lectin
localized the perfused vessels, CD31 localized microvascular endothelial cells, and the merged picture shows perfusion
of total vessels in the field. Quantitation was performed with the use of Bioquant Image Analysis (Bioquant, Nashville,
TN). Proliferating cells within the tumor were assessed by immunohistochemical detection of Ki-67 protein and
quantified as described. All values are expressed as mean plus or minus SEM. *P < .01 by Student t test. Ki-67 pictures
were taken with Carl Zeiss Invertoskop microscope with a 4X/0.12 NAobjective and 10X eyepiece. Photomicrographs
were taken using a Nikon Coolpix 5000 camera and a Nikon Eclipse E400 microscope with a 10X eyepiece.
Magnification was as 40X/0.75 NA objectives.
63
Figure 3.5 Analysis of vascular perfusion, vessel density, tumor cell proliferation, apoptosis and hypoxia.
(B) sEphB4-HSA–treated tumor and adjacent normal tissue vessel density and perfusion. Dotted line demarcates the
skin showing autofluorescence. Vessels in the subcutaneous tissue (subcutis) and the margin of the tumor show
perfusion. Deeper tumor tissue shows progressive decrease in the vessel density and even greater decrease in vessel
perfusion. (C) Gene expression analysis of tumor tissues was performed by quantitative PCR for VEGF, VEGFR1,
VEGFR2, PDGF-b, and Dll4. Gene expression levels were corrected for b-actin levels.
Chapter 3: Discussion
Eph is the largest family of receptor tyrosine kinases with 14 members and 8 ligands.
The Eph-Ephrin interaction plays important functions in morphogenesis, affecting
nervous system, pancreas, gastro-intestinal and genito-urinary tract development.
6, 18-20
In
vasculogenesis and angiogenesis, EphB4 and EphrinB2 play critical roles and notably
lack redundancy in their important functions. A comprehensive review of the Eph-Ephrin
family in KS demonstrated expression of limited members.
64
Notably, only EphrinB2 is expressed at substantial levels in all KS cell lines, HHV-8
transformed lymphatic endothelial cells (LEC/HHV8), and KS tissue. In addition,
EphrinB2 was previously reported in lymphatic endothelial cells, and this does not
contradict the lineage studies in KS.
21
EphrinB2 is also induced by HHV-8 and a number
of growth factors for KS including VEGF, VEGF-C, and IL-8.
1, 2, 4
EphrinB2 can bind
several Eph receptors of which EphB2 and EphA4 are expressed on KS cells while
EphB4 is expressed in the vessels. These findings suggested that EphrinB2 plays an
important role in KS biology. To address this question we employed the soluble
extracellular domain of EphB4 as an antagonist. sEphB4 only binds EphrinB2 and thus
its function is highly predictable. However, this binding characteristic prevents EphrinB2
from binding to any other receptor. We have shown previously that sEphB4 is a highly
active antagonist of the EphB4/EphrinB2 interaction.
13
sEphB4 has suboptimal
pharmacokinetics in mice. We thus developed an optimal version of sEphB4 by fusing it
with the human albumin coding region placed on its C-terminus. This protein retains
binding and functions as an antagonist to EphrinB2. Furthermore the kinetics in the
mouse are markedly improved such that sEphB4-HSA can be considered suitable for
human clinical development.
To test the consequences of blocking EphrinB2 function in KS, we first conducted
studies in vitro. sEphB4-HSA demonstrated a dramatic effect on the migration of KS
cells in vitro. Furthermore, sEphB4-HSA inhibited the invasion of KS cells across matrix
proteins especially when stimulated with growth factors that promote KS growth and
migration including VEGF, bFGF, EGF and PDGF-B.
65
We have shown previously that sEphB4 inhibits angiogenesis in response to various
growth factors. Given that KS cells express EphrinB2, which is rare among cancers, its
interaction with EphB4 will further enhance angiogenesis. Thus, in vivo studies of
sEphB4-HSA were expected to be highly effective. This indeed was the case; sEphB4-
HSA is very active in vivo.
Given that VEGF inhibitors are active in many cancers and that KS is among the
highest VEGF producers, we compared a VEGF antibody to sEphB4-HSA in vivo.
sEphB4-HSA was substantially more active than VEGF moAb in KS-SLK and equally
active in KS-IMM. Combination of the two has at least additive activity. This is not
surprising, since sEphB4-HSA induced severe hypoxia in the tumor tissue and increased
VEGF expression. Combination of VEGF inhibitor and sEphB4-HSA is thus a potent
combination. sEphB4-HSA also increased murine Dll4 (mDll4) levels consistent with the
induction of VEGF and hypoxia both which induce Dll4. mDll4 expression in tumor
vessels may inhibit VEGFR expression and thus inhibit VEGF activity and promote
vessel maturation. Combination of sEphB4-HSA with VEGF antibody as expected
reduced the mDll4 levels.
KS is lethal when spread to vital organs including the lungs, GI tract and liver; as
sEphB4-HSA had profound effects on KS cell migration and invasion. We envisioned
sEphB4-HSA would reduce the ability of KS to spread to distant sites. To test this
function, we utilized a model of tumor cell migration, invasion and growth by injecting
KS-SLK cells into the spleen and monitoring for metastasis in the liver.
66
KS metastasis indeed grew in the liver and sEphB4-HSA and VEGF moAb markedly
reduced these metastasis to the liver. The Combination of sEphB4-HSA and VEGF
moAb was remarkable for complete blockade of liver metastasis.
In summary, the monomeric form of sEphB4-HSA ectodomain displays the
following: (1) functions as an EphrinB2 antagonist (2) has markedly improved
pharmacokinetics compared to sEphB4, (3) inhibits KS adhesion, migration and invasion;
(3) inhibits the activity of several growth factors including VEGF, bFGF, EGF and
PDGF; and (4) inhibits KS tumor growth with marked reduction in vessel density, vessel
maturity and vessel perfusion; (5) comparable or better than VEGF moAb in KS
treatment with additive effect in combination. sEphB4-HSA is a candidate for
investigation on how it prevents vessel maturation, and consideration for clinical
investigation in KS and possibly other cancers.
67
Chapter 3: References
1. Regezi JA, Macphail LA, Daniels TE, De Souza YG, Greenspan JS, Greenspan D.
Human immunodeficiency virus-associated oral Kaposi’s sarcoma: a heterogeneous cell
population dominated by spindle-shaped endothelial cells. Am J Pathol. 1993;143:240-
249.
2. Masood R, Cai J, Zheng T, Smith DL, Naidu Y, Gill PS. Vascular endothelial growth
factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi
sarcoma. Proc Natl Acad Sci U S A. 1997;94:979-984.
3. Wang HU, Chen ZF, Anderson DJ. Molecular distinction and angiogenic interaction
between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4.
Cell. 1998;93:741-753.
4. Sivakumar R, Sharma-Walia N, Raghu H, et al. Kaposi’s sarcoma-associated
herpesvirus induces sustained levels of vascular endothelial growth factors A and C early
during in vitro infection of human microvascular dermal endothelial cells: biological
implications. J Virol. 2008;82: 1759-1776.
5. Gerety SS, Wang HU, Chen ZF, Anderson DJ. Symmetrical mutant phenotypes of the
receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular
development. Mol Cell. 1999;4:403-414.
6. Adams RH, Wilkinson GA, Weiss C, et al. Roles of ephrinB ligands and EphB
receptors in cardiovascular development: demarcation of arterial/venous domains,
vascular morphogenesis, and sprouting angiogenesis. Genes Dev. 1999;13: 295-306.
7. Kullander K, Klein R. Mechanisms and functions of Eph and ephrin signalling. Nat
Rev Mol Cell Biol. 2002;3:475-486.
8. Mellitzer G, Xu Q, Wilkinson DG. Eph receptors and ephrins restrict cell
intermingling and communication. Nature. 1999;400:77-81.
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9. Poliakov A, Cotrina M, Wilkinson DG. Diverse roles of eph receptors and ephrins in
the regulation of cell migration and tissue assembly. Dev Cell. 2004;7:465-480.
10. Augustin HG, Reiss Y. EphB receptors and ephrinB ligands: regulators of vascular
assembly and homeostasis. Cell Tissue Res. 2003;314:25-31.
11. Gale NW, Baluk P, Pan L, et al. Ephrin-B2 selectively marks arterial vessels and
neovascularization sites in the adult, with expression in both endothelial and smooth-
muscle cells. Dev Biol. 2001;230:151-160.
12. Hayashi S, Asahara T, Masuda H, Isner JM, Losordo DW. Functional ephrin-B2
expression for promotive interaction between arterial and venous vessels in postnatal
neovascularization. Circulation. 2005;111:2210-2218.
13. Kertesz N, Krasnoperov V, Reddy R, et al. The soluble extracellular domain of
EphB4 (sEphB4) antagonizes EphB4-EphrinB2 interaction, modulates angiogenesis, and
inhibits tumor growth. Blood. 2006;107:2330-2338.
14. Kumar SR, Singh J, Xia G, et al. Receptor tyrosine kinase EphB4 is a survival factor
in breast cancer. Am J Pathol. 2006;169:279-293.
15. Fox BP, Kandpal RP. Invasiveness of breast carcinoma cells and transcript profile:
Eph receptors and ephrin ligands as molecular markers of potential diagnostic and
prognostic application. Biochem Biophys Res Commun. 2004;318:882-892.
16. Kumar SR, Masood R, Spannuth WA, et al. The receptor tyrosine kinase EphB4 is
overexpressed in ovarian cancer, provides survival signals and predicts poor outcome. Br
J Cancer. 2007;96:1083-1091.
17. Xia G, Kumar SR, Masood R, et al. EphB4 expression and biological significance in
prostate cancer. Cancer Res. 2005;65:4623-4632.
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18. Klagsbrun M, Eichmann A. A role for axon guidance receptors and ligands in blood
vessel development and tumor angiogenesis. Cytokine Growth Factor Rev. 2005;16:535-
548.
19. van Eyll JM, Passante L, Pierreux CE, Lemaigre FP, Vanderhaeghen P, Rousseau
GG. Eph receptors and their ephrin ligands are expressed in developing mouse pancreas.
Gene Expr Patterns. 2006;6:353-359.
20. Flanagan JG, Vanderhaeghen P. The ephrins and Eph receptors in neural
development. Annu Rev Neurosci. 1998;21:309-345.
21. Makinen T, Adams RH, Bailey J, et al. PDZ interaction site in ephrinB2 is required
for the remodeling of lymphatic vasculature. Genes Dev. 2005;19:397-410.
70
CHAPTER 4: CONCLUSIONS
Chapter 4: Summary and Future Directions
Almost all tissues in the body develop a vascular network that provides cells with
oxygen and nutrients, and enables the elimination of metabolic wastes. Once formed, the
vascular network is stable and regenerates slowly. In physiological conditions,
angiogenesis occurs primarily during embryonic development, during wound healing,
and in response to ovulation. However, the rapid formation of an imperfect vascular
network during pathological angiogenesis is implicated in over 20 diseases, including
cancer, psoriasis, and age-related macular degeneration.
1-3
Pathological angiogenesis
performs a critical role in the development of cancer. In 1971, Dr. Judah Folkman
proposed that tumor growth and metastasis are angiogenesis-dependent.
4
Therefore;
blocking angiogenesis could prevent tumor progression and metastasis. The tumor cells
would be starved of nutrients and oxygen, and unable to eliminate metabolic wastes. This
idea paved the way for a new type of therapeutic. Angiogenesis, from the recruitment of
endothelial cells to vessel maturation, is an extremely complex process regulated by
several different signaling pathways. The identification of novel molecular targets and
understanding of their interaction with angiogenesis pathways are required to design
effective anti-angiogenesis therapies for cancer.
71
The previous studies provided initial evidence for two potential therapeutic
molecules, soluble Dll4 (sDll4) and soluble EphB4 (sEphB4). sDll4 inhibits Notch
signaling,
resulting in increased vascular proliferation but defective
maturation.
The newly forming vessels have thin caliber, reduced vessel lumen, reduced pericyte
recruitment,
and deficient vascular perfusion leading to reduced tumor
growth.
5
sEphB4
blocks EphB4-EphrinB2 interaction, and inhibits Kaposi Sarcoma (KS) tumor growth
with marked reduction in vessel density, vessel maturity and vessel perfusion. Its anti-
tumor activity is comparable or better than VEGF moAb in KS treatment. Combination
of soluble EphB4 and VEGF moAb results in additive anti-tumor effect.
6
Since
angiogenesis is required for progression and metastasis of solid tumors, sDll4 and
sEphB4 have the potential to treat a wide range of cancers. However, answers to several
key questions remain: 1) What downstream signals are modulated by blockage of ligand-
receptor interaction by these two molecules, and do these downstream molecules have
any therapeutic potential? 2) What are the side effects on non-vascular organs and cells
known to express Dll4 and EphrinB2? 3) What happens to the non-functional vasculature
when the treatment is stopped? 4) What is the kinetics of the response to these treatments,
and what would be the best treatment cycles for maximal inhibition of the tumor growth?
5) What are the therapeutic potentials on other pathologies associated with deregulated
angiogenesis? All these questions should be addressed in future studies.
72
More recently, in vivo and in vitro studies on the role of DLL4–Notch in
angiogenesis further strengthen a model in which DLL4 helps to regulate the cellular
actions of VEGF. Specifically, VEGF-mediated signaling in the sprouting endothelial tip
cells induces the expression of DLL4, which then provides signals to adjacent
downstream Notch receptor-bearing endothelial stalk cells to downregulate VEGFR2 and
VEGFR3, thereby suppressing the tip-cell phenotype, and tip-cell phenotype suppression
cell-autonomously promotes the stalk-cell phenotype.
7
Together, this balances tip-cell
and stalk-cell selection. Thus, it limits the number of sprouting vessels. Genetic or
pharmacological disruption of Dll4-Notch signaling leads to excessive tip-cell formation
and vessel sprouting in cultured cells, zebrafish, mouse embryos, and during tumor
angiogenesis.
7-14
The increased vascular density in response to DLL4 inhibition in tumors
is due in part to a failure to downregulate this sprouting and branching. Although the
blockade of DLL4–Notch signaling in tumors results in an increased density of vascular
sprouts and branches, they are poorly functional.
5
. Under normal circumstances, the
combined actions of VEGF and DLL4–Notch help to generate new vessels and promote a
coordinated vascular response to VEGF by downregulation of sprouting and branching in
the downstream stalk cells. This is accompanied by the formation of a vessel lumen and
interactions with pericytes.
15
This may also be regulated by Notch signaling and may be
lacking in vessels in which DLL4 has been blocked. This role for DLL4–Notch is
consistent with its association with cell fate decisions.
73
Interestingly, many of the highly branched and sprouted vascular structures in the tumors
in which DLL4 has been blocked are not perfused due to either a lack of a vessel lumen
15
or the structures are too disorganized to support adequate perfusion.
5
In most normal adult tissues, where VEGF signaling is low and the vessels are
quiescent and specialized, blockade of DLL4 has few consequences. However, in tumors,
where VEGF signaling is high and the angiogenic process is already active, DLL4
blockade can exaggerate the pathological features of the tumor vessels, by allowing
overactive VEGF signaling. Although DLL4–Notch signaling is clearly induced by
VEGF and helps to regulate VEGF-mediated vascular growth, there are indications that it
also has functions in angiogenesis that are independent of VEGF. Recent studies have
shown that blockade of DLL4 can have potent vascular effects on tumors that are
resistant to VEGF inhibition.
11
Furthermore, simultaneously blocking both VEGF and
DLL4 has more potent anti-tumor effects than the blockade of either pathway alone,
which would not be expected if the VEGF and DLL4 signals were in a simple linear
pathway. Unfortunately, a current study raises concern that chronic DLL4 blockade has
the potential to disrupt normal organ homeostasis and produce significant pathology in
multiple organs. Refined strategies might be necessary to harness this pathway safely as a
powerful tool to disrupt tumor angiogenesis without causing prohibitive side effects.
16
74
Chapter 4: Concluding Remarks
Angiogenesis research has grown drastically since Dr. Folkman proposed that
tumor growth and metastasis are angiogenesis-dependent, therefore, blocking
angiogenesis could prevent tumor progression and metastasis.
4
Consequently, we have
seen several treatments advance from the bench to the bedside. However, this progression
must be maintained through new avenues of research and a better understanding of the
molecular and cellular mechanisms involved in tumor growth and metastasis. Currently,
the primary strategy of anti-angiogenic therapy is to inhibit the effect of only one or a few
pro-angiogenic factors. The redundancy of pro-angiogenic signals secreted by tumor cells
or indirectly via tumor stroma may limit the therapeutic efficacy of drugs that block the
effects of a single pro-angiogenic protein.
17
Therefore, characterization of a tumor’s
angiogenic profile and responses to anti-angiogenic therapy are critical steps towards
designing multi-targeted anti-angiogenic drug combinations. If the compensatory
mechanisms against anti-angiogenic therapy are known, it might be possible to use this
strategy to force tumors to become dependent on specific angiogenic factors.
17
Subsequent treatment could then be directed against the selected angiogenic factors to
exploit the full therapeutic potential of the anti-angiogenic therapy. It is also likely that
combination of anti-angiogenic agents with conventional chemo and/or radio therapy will
yield better clinical responses compared to either treatment alone. The challenge remains
as to how to best combine these treatments and how to measure the responses seen with
these treatments in order to better manage the disease.
75
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Notch ligand Deltalike4 restricts angiogenesis. Development 2007, 134:839-844.
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11. Ridgway J, Zhang G, Wu Y, et al. Inhibition of Dll4 signalling inhibits tumour
growth by deregulating angiogenesis. Nature 2006, 444:1083-1087.
12. Sainson RC, Aoto J, Nakatsu MN, et al. Cell-autonomous notch signaling regulates
endothelial cell branching and proliferation during vascular tubulogenesis. FASEB J
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13. Siekmann AF, Lawson ND. Notch signalling limits angiogenic cell behaviour in
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14. Suchting S, Freitas C, le Noble F, et al. The Notch ligand Delta-like 4 negatively
regulates
endothelial tip cell formation and vessel branching. Proc Natl Acad Sci 2007, 104:3225-
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utilizing endothelial tip cell filopodia. J. Cell Biol. 2003;161, 1163–1177.
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neoplasms. Nature. 2010;463:E6-7.
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Abstract (if available)
Abstract
Angiogenesis is an extremely complex process regulated by several different signaling pathways. The identification of novel molecular targets and understanding of their interaction with angiogenesis pathways are required to design rational anti-angiogenesis therapies. Notch-Dll4 and EphB4-EphrinB2 receptor-ligand pairs are key players in these pathways. The following studies provide evidence for two potential anti-angiogenesis therapies, soluble Dll4 (sDll4) and soluble EphB4 (sEphB4). First, the role of Dll4 in vascular remodeling at sites of angiogenesis, including tumor vasculature was investigated. Results showed that loss of Dll4 function promotes endothelial cell migration, excessive vascular network formation, and reduction in pericyte recruitment, both in embryos and adult mice. Soluble forms of Dll4 interrupted Dll4-Notch signaling and recapitulated the vascular alterations seen in the gene knock-out mice, including increased vascular network formation, decreased or absent vascular lumen, and reduced recruitment of pericytes resulting in tissue hypoxia and decreased tumor growth. Second, a comprehensive analysis of the Eph receptor tyrosine kinases was conducted to determine which members are expressed and contribute to Kaposi Sarcoma (KS) pathogenesis. In addition, the biological effects of blocking EphrinB2 in vitro and in vivo using a soluble form of EphB4 (sEphB4) was studied. sEphB4 blocks EphB4-EphrinB2 interaction, and inhibits KS tumor growth with marked reduction in vessel density, vessel maturity and vessel perfusion. Its anti-tumor activity was comparable or better than VEGF moAb in KS treatment. Combination of soluble EphB4 and VEGF moAb resulted in additive anti-tumor effect. Thus, more effective anti-angiogenic therapeutics may arise from a better understanding of the molecular and cellular mechanisms involved in tumor angiogenesis and the response to anti-angiogenic therapies in a wide range of cancers.
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Scehnet, Jeffrey S.
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Targeting vessel maturation: an anti-angiogenesis based cancer therapy
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2010-12
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