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Vol. 52, Issue 2, 237-268, June 2000
Tumor Angiogenesis Laboratory (A.W.G.), Department of Internal Medicine, University Hospital Maastricht, Maastricht; Groningen University Institute for Drug Exploration (G.M.), Department of Pathology and Laboratory Medicine, Tumor Immunology Laboratory, and Department of Pharmacokinetics and Drug Delivery, Groningen, The Netherlands
Abstract
I. General Aspects of Angiogenesis
A. Introduction
B. Function of Endothelial Cells in Normal Physiology
C. Molecular Control of Angiogenesis
1. Initiation of the Angiogenic Response.
2. Endothelial Cell Migration and Proliferation.
3. Maturation of the Neovasculature.
4. Other Mechanisms Implicated in Angiogenesis Control.
II. Angiogenesis Stimulation
A. Target Diseases for Angiogenesis Stimulation
B. Proangiogenic Compounds
1. Vascular Endothelial Growth Factor.
2. Fibroblast Growth Factors.
3. Angiopoietin-1.
C. Effects of Angiogenesis Stimulation in Preclinical Studies
D. First Clinical Studies on Angiogenesis Stimulation
III. Angiogenesis Inhibition
A. Angiogenesis and Cancer
B. In Vitro and in Vivo Models to Study Angiogenesis
C. Ways to Interfere with Angiogenesis
1. Intervention with Endothelial Cell Growth.
2. Intervention with Endothelial Cell Adhesion and Migration.
3. Intervention with Metalloproteinases.
D. Preclinical Use of Angiogenesis Inhibitors in Cancer
E. Clinical Trials with Inhibitors of Angiogenesis for Cancer Treatment
F. Novel Approaches to Interfere with Tumor Blood Flow
1. Targeted Strategies to Induce Tumor Blood Coagulation.
2. Targeted Strategies to Kill Tumor Endothelial Cells.
3. The Quest for New Targets on Tumor Endothelium.
IV. The Interplay between Angiogenesis and Cells of the Immune System
A. Angiogenesis Regulates Leukocyte Recruitment
B. The Role of Angiogenesis in Chronic Inflammation
C. Inhibition of Angiogenesis in Chronic Inflammation
D. Clinical Trials with Inhibitors of Angiogenesis for Noncancerous Diseases
V. Back to the Drawing Board
A. Angiogenesis Stimulation
B. Antiangiogenic Strategies in Cancer Therapy
C. Antiangiogenic Strategies in Chronic Inflammation
VI. Concluding Remarks
Acknowledgments
References
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Angiogenesis, or the formation of new blood vessels out of pre-existing capillaries, is a sequence of events that is fundamental to many physiologic and pathologic processes such as cancer, ischemic diseases, and chronic inflammation. With the identification of several proangiogenic molecules such as the vascular endothelial cell growth factor, the fibroblast growth factors (like in FGFs), and the angiopoietins, and the recent description of specific inhibitors of angiogenesis such as platelet factor-4, angiostatin, endostatin, and vasostatin, it is recognized that therapeutic interference with vasculature formation offers a tool for clinical applications in various pathologies. Whereas inhibition of angiogenesis can prevent diseases with excessive vessel growth such as cancer, diabetes retinopathy, and arthritis, stimulation of angiogenesis would be beneficial in the treatment of diseases such as coronary artery disease and critical limb ischemia in diabetes. In this review we highlight the current knowledge on angiogenesis regulation and report on the recent findings in angiogenesis research and clinical studies. We also discuss the potentials, limitations, and challenges within this field of research, in light of the development of new therapeutic strategies for diseases in which angiogenesis plays an important role.
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I. General Aspects of Angiogenesis |
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A. Introduction
The formation of new blood vessels out of
pre-existing capillaries, or angiogenesis, is a sequence of events that
is of key importance in a broad array of physiologic and pathologic
processes. Normal tissue growth, such as in embryonic development,
wound healing, and the menstrual cycle, is characterized by dependence on new vessel formation for the supply of oxygen and nutrients as well
as removal of waste products. Also, a large number of different and
nonrelated diseases is associated with formation of new vasculature.
Among these pathologies are diseases, such as tissue damage after
reperfusion of ischemic tissue or cardiac failure, where angiogenesis
is low and should be enhanced to improve disease conditions (Carmeliet
et al., 1999
; Ferrara and Alitalo, 1999). In several diseases,
excessive angiogenesis is part of the pathology. These diseases include
cancer (both solid and hematologic tumors), cardiovascular diseases
(atherosclerosis), chronic inflammation (rheumatoid arthritis, Crohn's
disease), diabetes (diabetic retinopathy), psoriasis, endometriosis,
and adiposity. These diseases may benefit from therapeutic inhibition
of angiogenesis (Folkman, 1995; Hanahan and Folkman, 1996).
The initial recognition of angiogenesis being a therapeutically
interesting process began in the area of oncology in the early 1970s,
when Drs. Folkman and Denekamp put forward the idea that tumors are
highly vascularized and thereby vulnerable at the level of their blood
supply. In those early years, it was already hypothesized that the
process of angiogenesis might be a target for therapy. It was only
after the discovery of the first compounds with specific angiostatic
effects in the early 1990s (Ingber et al., 1990; O'Reilly et al.,
1994) that the research field of angiogenesis rapidly expanded and
provided an increasing body of evidence that inhibition of angiogenesis
could attenuate tumor growth. More recently, novel angiogenesis
inhibitors have shown great potential in the treatment of cancer in
preclinical studies. Several of those compounds are currently being
tested in clinical trials (Molema and Griffioen, 1998). With increasing
insight into the role of angiogenesis in other diseases as well,
modulation of vascular outgrowth is now also regarded as a therapeutic
target in these diseases.
To date, antiangiogenesis therapy is considered, worldwide, a promising
approach, supposedly leading to the desperately needed breakthrough in
cancer therapy and other proangiogenic diseases. Nevertheless, many
questions remain unanswered and many concepts unverified at present.
For example, it has to be established whether the exciting effects seen
in preclinical investigations using xenogeneic and syngeneic tumor
transplant models and transgenic systems also prevail in the human
situation. Furthermore, it has been shown that the angiogenesis
inhibitors angiostatin and endostatin (O'Reilly et al., 1994, 1997) do
not elicit drug-induced resistance on prolonged treatment in
tumor-bearing animals, although being highly effective in tumor growth
reduction. This observation is of extreme importance, because it opens
possibilities for long-term treatment or the development of treatment
modalities for the prevention of disease in high-risk populations prone
to develop tumors. It remains to be seen whether this scenario can be
extended to other angiogenesis inhibitors as well as to other
proangiogenic diseases of interest. Another important issue is the
concept of cancer treatment with angiogenesis inhibitors as a
single-compound strategy. Is this a feasible treatment strategy or
should antiangiogenic therapy be used in combination with other
treatment modalities such as immuno- or chemotherapy? Also, although
antiangiogenesis therapy is considered to have low toxicity, there is
as yet little information on the safety of therapeutic angiostatic
strategies; there is little or no information to what extent inhibition
of angiogenesis as tumor treatment will affect normal physiological processes in embryonic development or in wound healing and what the
long-term side effects will be.
Although current interest in angiogenesis comes mainly from oncology researchers, also nononcological research fields have now recognized that modulation of angiogenesis may provide a tool for clinical interventions. This demonstrates that angiogenesis is a multidisciplinary theme from a pharmacologic target point of view. In addition, many disciplines of biomedical origin are contributing to basic angiogenesis research, because the processes involved are so complex. In this review, the molecular players of vessel growth, methodology of angiogenesis research, and preclinical and clinical use of angiogenesis as a target for therapy will be discussed.
B. Function of Endothelial Cells in Normal Physiology
The blood vessels in the body have long been considered to merely function as a transport compartment of the blood. Nowadays, it is appreciated that the vasculature is one of the main organs in the body, extending more than 900 m2 and playing a major role in maintaining the body's integrity in various ways.
Blood vessels consist of endothelial cells that are in direct contact
with the blood, and subendothelially located pericytes, smooth muscle
cells, fibroblasts, basement membrane
(BM),1 and
extracellular matrix (ECM). Depending on the location in the body, the
organ microenvironment, the cellular constituents, BM, and ECM of the
vasculature differ in phenotype, composition, and function (Rajotte et
al., 1998).
The endothelial cells form a monolayer in every single blood vessel in
the circulation and are actively involved in several regulatory
processes in the body (Fig. 1). Besides
being metabolically active and selectively permeable for small solutes
and peptides/proteins, they regulate blood coagulation. When
their integrity is maintained, endothelial cells exert
anticoagulative properties via the synthesis of thrombomodulin, tissue
factor (TF) pathway inhibitor and tissue-type plasminogen activator
(t-PA). On activation or damage, endothelial cells quickly release
proteins like multimeric von Willebrand factor (vWF), which promotes
platelet adhesion and aggregation, and plasminogen activator
inhibitor-1, a member of the serpin family. In addition, TF expression
by endothelium leads to initiation of the extrinsic blood coagulation
pathway (Verstraete, 1995). Another important feature of endothelial
cells is their ability to direct cells of the immune system to specific
sites in the body. Constitutively expressed or cytokine-inducible
cellular adhesion molecules [e.g., E-selectin and intercellular
adhesion molecule-1 (ICAM-1)] and soluble factors such as
chemoattractants, cytokines, and chemokines act in concert to recruit
the immune cells to lymphoid organs or inflammatory sites (Carlos and
Harlan, 1994). Last, endothelial cells are actively involved in
vascular remodeling during, for example, ovulation, wound healing,
tumor growth, and diabetic retinopathy. Although complex in regulation and sometimes difficult to functionally analyze in vitro, as well as
during disease progression, data have become available that link (parts
of) these endothelial cell functions to various steps in the angiogenic
cascade.
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C. Molecular Control of Angiogenesis
In vasculogenesis during embryonic development, new endothelial
cells differentiate from stem cells. In contrast, in angiogenesis new
blood vessels mainly emerge from pre-existing ones (Risau, 1997). In
adult life, physiologic stimuli during wound healing and the
reproductive cycle in women lead to angiogenesis, whereas vasculogenesis is absent. Pathologic conditions such as tumor growth,
rheumatoid arthritis, and diabetic retinopathy are characterized by
abundant angiogenesis. The active vascular remodeling phase in tumors,
e.g., is reflected by the fact that tumor endothelial cells proliferate
20 to 2000 times faster than normal tissue endothelium in the adult
(Denekamp, 1984). In the last decade, several molecular players have
been identified that significantly contribute to the molecular
processes leading to new blood vessel formation. In the following
sections, recent advances in this area of research are discussed.
1. Initiation of the Angiogenic Response.
Angiogenesis is
rapidly initiated in response to hypoxic or ischemic conditions.
Vascular relaxation, for example, mediated by nitric oxide (NO) is a
prerequisite for endothelial cells to enter the angiogenic cascade.
Likely, morphologic changes of the endothelial cells lead to a decrease
in confluency status to make them susceptible to mitogens (Folkman,
1997). In all types of angiogenesis, either under physiologic or
pathologic conditions, endothelial cell activation is the first process
to take place. Cytokines from various sources are released in response
to hypoxia or ischemia. It is suggested that vascular endothelial
growth factor (VEGF) is a major player in angiogenesis initiation based on its ability to induce vasodilation via endothelial NO production and
its endothelial cell permeability increasing effect (Ziche et al.,
1997). This allows plasma proteins to enter the tissue to form a
fibrin-rich provisional network (Dvorak, 1986). The observation that
VEGF production is under control of hypoxia inducible factor (HIF)
strengthens the suggestion of an early involvement of VEGF in the
angiogenic response. Moreover, VEGF receptor (VEGFR) expression is
up-regulated under hypoxic or ischemic conditions as well (Forsythe et
al., 1996).
). Besides
affecting vasodilation and vascular permeability, VEGF can induce the
expression of proteases and receptors important in cellular invasion
and tissue remodeling and is able to prevent endothelial cell apoptosis
(Ferrara and Keyt, 1997; Gupta et al., 1999). That angiogenesis is not
completely dependent on VEGF production was recently shown by
Hansen-Algenstaedt et al. (1999); the consequences of which will be
discussed in more detail in Section V. For a more detailed
overview on the role of VEGF in the regulation of angiogenesis, the
reader is referred to a recently published review by Ferrara (1999).
After proper activation of the endothelial cells, endothelial
penetration into new areas of the body is achieved by degradation of
the BM by matrix metalloproteinases (MMPs). These extracellular endopeptidases are secreted as zymogens that become activated in the
ECM compartment and subsequently selectively degrade components of the
ECM (Stetler Stevenson, 1999). They are produced by a variety of cells,
including epithelial cells, fibroblasts, inflammatory cells, and
endothelial cells. MMP activity and, hence, angiogenesis is
counteracted by the family of tissue inhibitors of metalloproteinase (TIMPs) (Gomez et al., 1997; Valente et al., 1998).
2. Endothelial Cell Migration and Proliferation. Plasminogen activators u-PA and t-PA convert the ubiquitous plasma protein plasminogen to plasmin. Plasmin has a broad trypsin-like specificity and degrades, e.g., fibronectin, laminin, and the protein core of proteoglycans. In addition, plasmin activates certain metalloproteinases. Plasmin is believed to be the most important protease for the mobilization of fibroblast growth factor-2 (FGF-2 or basic FGF) from the ECM pool.
FGF members are directly acting proangiogenic molecules. FGF-2 consists of, in two modifications, an 18-kDa low-molecular weight form and a 22- to 24-kDa high-molecular weight form. During angiogenesis, low-molecular weight FGF-2 binding to endothelium induces FGF receptor (FGF-R) down-regulation, increased motility, proliferation and proteinase activity, and modulates integrin levels. High-molecular weight FGF-2 may act on endothelial cell proliferation after nuclear translocation in the endothelial cells (Gleizes et al., 1995; Klein et
al., 1997). Recently, it was shown that a secreted FGF-2-binding
protein could bind FGF-2 that is normally inactive due to strong
adherence to heparan sulfate proteoglycans in the ECM. The displaced
FGF-2 molecules were thus released to mediate biological function. Of
note is the observation in this and other studies that angiogenesis
seems exquisitely sensitive to small changes in factors such as VEGF
and FGF-2 that drive the angiogenic process. This may have important
therapeutic implications in treating angiogenesis-driven disorders
(Czubayko et al., 1997). Besides its effect on angiogenesis initiation,
VEGF also affects endothelial cell proliferation. This effect can be
(partly) attributed to NO and cGMP-mediated activation of the
mitogen-activated protein kinase (MAPK) family (see Section
II.B. for a more detailed description on VEGF and FGF-2-mediated
signal transduction and cell activation pathways).
Integrins are transmembrane proteins composed of an 
and 
subunit
in over 20 different heterodimeric combinations. They bind to ECM
proteins or cell surface ligands through short peptide sequences.
Combinations of different integrins on cell surfaces allow cells to
recognize and respond to a variety of different ECM proteins (Varner,
1997). They are able to transduce signals from within the cells to the
outside as well as from the outside into the cell (Aplin et al., 1998).
Integrin-mediated cell adhesion impacts two key aspects of growth
regulation. First, it can influence the activity of the basal cell
cycle machinery consisting of cyclin-dependent kinase complexes.
Second, integrins play a pivotal role in anchorage-dependent cell death
or anoikis (Frisch and Ruoslahti, 1997; Howe et al., 1998). Integrin
v3 mediates cellular
adhesion to vitronectin, fibrinogen, laminin, collagen, vWF, or
osteopontin through their exposed tripeptide Arg-Gly-Asp (RGD) moiety
(Cheresh, 1993). v3 is minimally expressed on normal resting endothelium, but significantly up-regulated on activated endothelium and is believed to play a
critical role in angiogenesis. Both peptide and antibody inhibitors of
v3 induced
endothelial cell apoptosis, suggesting a role for this integrin in
endothelial cell survival during angiogenesis (Brooks et al., 1994a).
Another v integrin associated with
angiogenesis is v5.
Whereas FGF-2 or tumor necrosis factor- (TNF-) induced v3-dependent
angiogenesis in vivo, VEGF or transforming growth factor- (TGF-)
initiated an angiogenesis pathway merely dependent on
v5 (Friedlander et
al., 1995).
Components of the ECM also contribute to the regulation of endothelial
cell morphology and function. Thrombospondin, for example, inhibits
endothelial cell proliferation when added in soluble form. When
endothelial cells on the other hand are plated on matrix bound
thrombospondin, they become more permissive for proliferative signals.
Furthermore, through binding to and activation of TGF- and affecting
protease activity, thrombospondin may be able to influence cell growth,
migration, and differentation as well (DiPietro, 1997). In patients
with invasive bladder cancer, low-thrombospondin expression in the
tumor was associated with increased recurrence rates, decreased overall
survival, and high-microvessel density counts. These data are
suggestive of an antiangiogenic role for this ECM constituent under
physiologic conditions (Grossfeld et al., 1997). Laminin is another ECM
protein with functions in endothelial cell attachment, growth
promotion, protease secretion, and interactions with other ECM
components. Laminin can bind to cell surface-binding proteins including
integrins, which leads to integrin signaling (Grant and Kleinman,
1997). SPARC (secreted protein
acidic and rich in cysteine), also
known as BM40 or osteonectin, is a protein of which the expression is
elevated under stress conditions such as endotoxin stimulation, heat
shock, and sparse cell density. SPARC overexpression has been observed
in tumors such as human esophageal carcinoma and cutaneous malignant
melanoma (Porte et al., 1998; Massi et al., 1999). Furthermore,
transient expression of SPARC during endothelial cell injury and
cellular activation indicated a role in tissue repair, remodeling and
angiogenesis (Jendraschak and Sage, 1996).
3. Maturation of the Neovasculature.
Endothelial cell
interaction with ECM and mesenchymal cells is a prerequisite to form a
stable vasculature. Therefore, after endothelial cell proliferation and
maturation, and the formation of endothelial tube structures,
surrounding vessel layers composed of mural cells (pericytes in small
vessels and smooth muscle cells in large vessels), need to be
recruited. Endothelial cells may accomplish this via the synthesis and
secretion of platelet-derived growth factor (PDGF), a mitogen and
chemoattractant for a variety of mesenchymal cells. Subsequent
differentiation of the mural precursor cells into pericytes and smooth
muscle cells is believed to be a cell-cell contact-dependent process.
On endothelial cell-mural cell contact, a latent form of TGF-,
produced by both endothelium and mural cells, is activated in a
plasmin-mediated process. Activated TGF- can induce changes in
myofibroblasts and pericytes, which may contribute to the formation of
a quiescent vessel, ECM production, and maintenance of growth control.
The coinciding investment of growing capillaries by pericytes with the
deposition of BM and cessation of vessel growth during wound healing
also indicates vessel growth regulation by pericytes (Hirschi and
D'Amore, 1997). FGF-1 is also implicated in endothelial cell
differentiation leading to vascular tube formation. Besides inducing
plasminogen activator and endothelial cell proliferation and migration,
FGF-1 receptor signaling resulted in endothelial tube formation in
collagen (Kanda et al., 1996).
). Tie1 function is related to endothelial cell
differentiation and the establishment of blood vessel integrity. Tie2,
on the other hand, is particularly important for vascular network
formation (Dumont et al., 1994; Puri et al., 1995; Sato et al., 1995).
Tie2 expression is restricted to the endothelial cells. Surprisingly,
Tie2 was present on quiescent as well as angiogenic endothelium in the
adult rat. Moreover, the receptor tyrosine kinase was constitutively
phosphorylated in both types of vasculature. These data suggest that
Tie2 has a dual function involving both angiogenesis and vascular
maintenance (Wong et al., 1997). Angiopoietin-1 (Ang-1) and
Angiopoietin-2 (Ang-2) are Tie2-specific ligands that activate or
antagonize Tie2 signaling in endothelium, respectively. In postnatal
neovascularization, Ang-1 is likely to promote vascular network
maturation (see Section II.B. for a more detailed
description of Ang-1 as a proangiogenic protein for therapeutic
purposes). In contrast, Ang-2 rendered endothelium sensitive to
angiogenic factors via induction of smooth muscle cell/pericyte loss
and hence destabilized the neovasculature (Maisonpierre et al., 1997;
Asahara et al., 1998). The observation that Ang-2 was able to
phosphorylate Tie2 when expressed by fibroblasts, indicates that in
endothelial cells other regulatory mechanism(s) prevail leading to
antagonistic activity. Whereas Ang-1 is widely expressed, Ang-2 is only
found at sites of vascular remodeling. Here it may block vessel
stabilization, maturation, or survival signals from Tie2 (Maisonpierre
et al., 1997; Korpelainen and Alitalo, 1998). In human glioblastomas, a
cell-specific up-regulation of Tie2, Ang-1, and Ang-2 during tumor
progression was demonstrated in a pattern compatible with a role in
tumor-induced angiogenesis (Stratmann et al., 1998). Using homology
based cloning, two new members of the angiopoietins, Ang-3
(mouse-specific) and Ang-4 (human-specific) were identified. They are
distributed differently in the respective species, where Ang-3 acts as
an antagonist and Ang-4 as an agonist of receptor tyrosine kinase
signaling. Their respective roles in vascular maintenance have not been
established yet (Valenzuela et al., 1999).
4. Other Mechanisms Implicated in Angiogenesis Control.
Although the roles of several factors during angiogenesis
have been discussed here separately, it is important to note that the
activity of an angiogenesis-regulating cytokine depends on the presence
and concentration of other factors or cytokines in the environment of
the responding endothelium (Pepper et al., 1998). For example,
exogenous factors such as hormones can affect conditions leading to
angiogenesis (Schiffenbauer et al., 1997). Isoforms of VEGF that bind
to ECM-associated heparan sulfate proteoglycans can release ECM-stored
FGF-2 in a bioactive form (Jonca et al., 1997), and angiopoietins
potentiate the effects of VEGF (Asahara et al., 1998).
; Blair et
al., 1997). T lymphocytes were able to activate endothelial expression
of various metalloproteinases via CD40/CD40-ligand interactions. As a
consequence, increased tube formation in a three-dimensional gel was
observed (Mach et al., 1999). Based on the effect of cells of the
immune system on angiogenic parameters and the overt neovascularization
in chronic inflammatory diseases, antiangiogenic strategies were put
forward as treatment modalities for these diseases as well (see
Section IV).
Recently, Keshet and coworkers identified the importance of the
presence of periendothelial cells in the microenvironment as a control
mechanism of angiogenesis. Loss of VEGF by androgen ablation therapy
led to selective apoptosis of endothelial cells in vessels devoid of
periendothelial cells. Based on this observation, it is now
hypothesized that VEGF is required to maintain cell anchorage to a
provisional ECM until periendothelial cells facilitate a more permanent
mode of adhesion (Benjamin et al., 1999).
Besides the already mentioned proangiogenic factors, VEGF, FGF-1, and
FGF-2, many others have now been identified in various settings of
physiologic and pathologic angiogenesis. Among them are TGF- and
TGF-, granulocyte macrophage-colony-stimulating factor, epidermal
growth factor, interleukin-1 (IL-1), scatter factor,
platelet-activating factor, IL-8, and substance P (Bouck et al., 1996;
Yoshida et al., 1997). Their effects can be either directly or
indirectly on the endothelium via activation of surrounding cells to
produce other factors with proangiogenic activity or modulation of
receptors/receptor activities (Yoshida et al., 1997; Giraudo et al.,
1998).
Hypoxia is an important environmental factor that leads to
neovascularization. In the case of tumor growth, however,
cancer-causing genetic changes, possibly in conjunction with
environmental influences, are able to induce angiogenesis as well (Rak
et al., 1995; Okada et al., 1998). Many oncogenes, among which
c-myb, sis, and src, were shown to stimulate the
expression of a wide variety of molecules that induce angiogenesis.
Furthermore, mutant ras oncogenes strongly up-regulated the
proangiogenic factors TGF-, TGF-, and VEGF. Activated oncogenes
can also indirectly contribute to the angiogenic phenotype by affecting
the production and activation of BM and ECM-degrading enzymes (Bouck et
al., 1996; Okada et al., 1998). Tumor suppressor genes have now also
been identified to play a role in angiogenic activities of cells.
Inactivation of p53, for example, down-regulated the antiangiogenic ECM
component thrombospondin (Dameron et al., 1994; Grossfeld et al.,
1997). In nonsmall cell lung cancer, loss of p53 was associated with a
high-vascular maturation index (Kakolyris et al., 1999). Besides the
involvement of tumor cell-associated changes in p53, this tumor
suppressor gene also plays a role in endothelial cell-mediated control
of angiogenesis. Adenovirus-mediated overexpression of endothelial p53
inhibited human umbilical vein endothelial cells (HUVEC) proliferation
and capillary network formation in vitro (Riccioni et al., 1998). In
endothelial cells existing in atherosclerotic and normal human aorta,
variations in p53 expression levels could be detected. Moreover, the
multinucleated variant endothelial cells expressed a mutant p53 type,
which may be indicative for loss of endothelial cell growth control
(Satoh et al., 1998). As with tumor cells, it is most likely that in
vivo a combination of mutations in various tumor suppressor genes and
oncogenes leads to a proliferative proangiogenic character of the
endothelial cells.
Most of the experimental data on angiogenesis control published so far
deal with angiogenesis in cancer. The advances made in this area are of
prime importance for understanding molecular players involved in the
regulatory pathways. It should be realized, however, that the process
of angiogenesis may be differentially regulated in the various disease
settings. Therefore, care should be taken in extrapolating data on,
e.g., regulatory pathways and their activators and inhibitors from
these tumor growth-related experiments to other diseases.
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II. Angiogenesis Stimulation |
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Much attention has been payed to therapeutic strategies that are
able to stop the angiogenic cascade in tumor growth (see Section
III) and more recently, in chronic inflammatory situations such as
rheumatoid arthritis (see Section IV). There are, however, various diseases affecting millions of people every year that would
benefit from the induction of angiogenesis, so-called therapeutic angiogenesis (Takeshita et al., 1994). Although the number of studies
reported in this area of research are not nearly as high as the number
of studies on antiangiogenic therapies, the approach appeared to
be quite successful in a preclinical setting as well as in the recently
performed first clinical trials.
A. Target Diseases for Angiogenesis Stimulation
The disease conditions that may benefit from therapeutic
angiogenesis encompass ischemic diseases such as ischemic coronary artery disease, critical limb ischemia with various etiologies, and
decubitus. In these diseases, functional blood flow is partially lost
in an organ or limb. For coronary artery disease, the leading cause of
morbidity and mortality in Western countries, the therapeutic options
(reducing the risk factors, restoration of the blood flow by
angioplasty, or coronary bypass grafting), are insufficient. In
critical limb ischemia, estimated to develop in 500 to 1000 individuals
per million per year, the anatomic extent and the distribution of the
arterial occlusions render the patients unsuitable for operative or
percutaneous revascularization. At present, no pharmacologic treatment
could favorably affect the ischemia. Often, loss of the limb by
amputation is the recommended treatment for these patients (Baumgartner
et al., 1998). A specific form of vascular occlusive disease that leads
to critical limb ischemia, is thromboangiitis obliterans, or Buerger's
disease. The disease afflicts arteries of young smokers and is
characterized by the onset of distal extremity ischemic symptoms,
leading to ulceration and gangrene (Isner et al., 1998). Gastroduodenal
ulcers, also caused by local insufficient perfusion, have been subject
of angiogenesis stimulation therapies (Wolfe et al., 1995). It has
recently been suggested that for congestive heart failure, possibly a
result of myocardial ischemia, stimulation of angiogenesis may also
become a therapeutic option (Carmeliet et al., 1999; Isner and Losordo, 1999).
The treatment of arterial occlusions by balloon angioplasty is
frequently associated with delinquent re-endothelialization and smooth
muscle cell proliferation. One therapeutic option to reduce subsequent
intimal thickening is the induction of apoptosis in infiltrating immune
cells (Sata et al., 1998). Therapeutic angiogenesis to facilitate
endothelial cell regeneration in this specific pathology has been
proposed as well (Callow et al., 1994; Asahara et al., 1996).
In the case of organ transplantation, surgical procedures dictate loss
of vessel integrity and function of the transplanted organ (Taub et
al., 1998). Transplantation of encapsulated pancreatic islets as a
treatment modality for type I diabetes, for example, may be more
successful when prevascularized solid supports are used or solid
supports are pretreated with proangiogenic factors (de Vos et al.,
1997).
B. Proangiogenic Compounds
Ischemic diseases from different etiologies may improve when treated with agents that induce neovascularization. Although a vast number of proangiogenic factors are available (see Section I.C), to date mostly VEGF and FGF-2 have been explored for this purpose. More recently, the proangiogenic protein angiopoietin-1 (Ang-1), ligand for the Tie2 receptor on endothelium, has been applied in therapeutic angiogenesis strategies as well.
1. Vascular Endothelial Growth Factor.
Angiogenesis is driven
by numerous mediators produced by numerous cells under a variety of
conditions. These mediators are either soluble, ECM or membrane bound
growth factors, or components of the ECM itself. Of the soluble
factors, one of the best studied and the most potent proangiogenic
factor is VEGF, discovered in the early eighties by Dvorak and
colleagues (Senger et al., 1983). VEGF (also known as VEGF-A) isoforms
VEGF-121, -145, -165, -183, -189, and -205 are a result of alternative
splicing from a single VEGF gene located on chromosome 6 (Mattei et
al., 1996). Together with VEGF-B, -C, and -D, they belong to the
VEGF/PDGF super family. Recently, a viral VEGF family member,
designated VEGF-E, was described (Meyer et al., 1999). The two
VEGF-specific tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2
(KDR/Flk-1), are expressed on vascular endothelium, and to a lesser
extent on monocytes/macrophages and certain tumor cell types. VEGFR-3
(Flt-4), which binds VEGF-C and VEGF-D, is mainly expressed on
lymphatic endothelium (Kaipainen et al., 1995). Interaction of VEGF
with VEGFR-2 is a critical requirement to induce the full spectrum of
VEGF biologic responses. Intracellular signal transduction pathways in
endothelial cells through VEGFR-2 dimerization lead to permeability
enhancement, cellular proliferation, and migration, as schematically
shown in Fig. 2 (Abedi and Zachary, 1997;
Kroll and Waltenberger, 1997; Wheeler Jones et al., 1997; Ziche et al.,
1997; Gerber et al., 1998; Hood and Granger, 1998; Wellner et al.,
1999; Doanes et al., 1999; Shen et al., 1999; Yu and Sato, 1999). All
these studies were performed in vitro, exploiting either HUVEC or
vascular endothelium from bovine or porcine origin. Evidence for at
least partly similar VEGFR-mediated signal transduction pathways in
vivo was recently provided using intact microvessels of mouse mesentery
(Mukhopadhyay et al., 1998).
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). VEGF-mediated signaling was
also able to confer a proliferation inhibitory signal in endothelium through regulating cell cycle progression by p38 MAPK activation (Yu
and Sato, 1999) to induce endothelial expression of ECM-degrading enzymes and to recruit pericytes (Puri et al., 1995; Lamoreaux et al.,
1998). VEGFR-2 and endothelial NO synthase colocalized with caveolin in
plasma membrane caveolae, suggestive of VEGF signaling events within
the caveolar compartment of endothelium (Feng et al., 1999).
JNK kinases participate in cellular processes via the modulation of
transcriptional activation factors such as AP-1. Other transcriptional
activation factors downstream of the VEGF signal transduction pathways
shown to be functional in endothelial cells are nuclear factor of
activated T cells (NFAT) and nuclear transcription factor ETS
(Chen et al., 1997). Whereas AP-1 and NFAT switch on genes regulating
tissue factor expression upon VEGF activation, ETS regulates the
expression of u-PA, MMPs, and integrin 3 (Iwasaka et al., 1996; Oda et
al., 1999).
The pleiotropic effects of VEGF in vitro on endothelial cell growth,
proliferation and migration, among others, have now been extensively
documented by many groups. However, plasma leakage and subsequent
fibrin formation is one of the hallmarks of angiogenesis initiation in
solid tumor growth and initiation of wound healing. The fibrin provides
a functional matrix for endothelial cells to become activated, to
proliferate, and to migrate (Dvorak, 1986). Therefore, it may well be
that in vivo the permeability inducing capacity of VEGF is its most
important function in regulating physiologic and pathologic angiogenesis.
2. Fibroblast Growth Factors.
Members of the FGF family are
also potent inducers of angiogenesis. Cellular responses mediated by
FGFs include cell migration, proliferation, and differentiation (Kanda
et al., 1997). The FGF family consists of nine structurally related
polypeptides, of which FGF-1 (acidic FGF) and FGF-2 (basic FGF) are the
most extensively studied. Both FGF-1 and FGF-2 are devoid of a signal
sequence for secretion. Export from cells without compromising cell
integrity or requiring cell death possibly follows a nonclassical,
synaptotagmin-1-dependent exocytotic pathway (LaVallee et al., 1998).
). In
addition, low-affinity FGF receptors exist, that consist of polysaccharide components of heparan sulfate proteoglycans on cell
surfaces and in ECM. Binding to the latter receptors has been proposed
as a mechanism to stabilize and protect FGF from inactivation. Heparan
sulfate on cell surfaces, on the other hand, plays a more active role
in displacing ECM-bound FGF-2 and subsequent presentation to the
high-affinity signal transducing receptors (Miao et al., 1996).
Receptor dimerization by FGF is facilitated by heparin. It results in
protein kinase activity and receptor autophosphorylation. As with VEGFR
signaling, this autophosphorylation enables adaptor proteins such as
Grb2, Shc, and Nck to bind and subsequently activate the Ras/Raf-MAPK
pathway of endothelial cell proliferation activation (Klein et al.,
1997). p42 MAPK activation was also implicated in endothelial
cell motility regulatory responses to FGF. p42 MAPK driven
phosphorylation of cytoplasmic phospholipase-A2
enabled arachidonic acid release upon FGF-2 activation of bovine aortic cells (Sa et al., 1995). Besides activating MAPK-mediated cell proliferation, FGF-2 induced murine brain endothelial cell
proliferation via a serine/threonine kinase that phosphorylates
ribosomal protein S6 (p70S6K). This proliferation
activation route was restricted to endothelial cells cultured on
fibronectin. When allowed to differentiate to form tube-like structures
in collagen gels, p70S6K was not activated (Kanda
et al., 1997).
In addition to initiating receptor signaling, FGF-1 can be endocytosed
and transported to the cell nucleus. This transport affects the cell
cycle in the late G1 stage, promoting transition to the S stage (Imamura et al., 1994). Entrance of FGF-2 into the
nucleus correlated with phosphorylation of nucleolin and subsequent increases of rDNA transcription, likely to be mediated by the protein
kinase CKII (Bouche et al., 1994). FGF-2 uptake by endothelial cell was
furthermore shown to be a route of growth factor degradation and can
therefore act to regulate FGF-2 activity (Gleizes et al., 1995).
3. Angiopoietin-1.
Ang-1 is an endogenously secreted
glycoprotein of approximately 75 kDa. Its receptor, Tie2, is generally
restricted to the endothelium and of importance in angiogenesis during
development, tumor growth, and wound healing (Sato et al., 1995; Lin et
al., 1997; Wong et al., 1997; Stratmann et al., 1998). In vitro, Ang-1 stimulated tyrosine phosphorylation of Tie2 in endothelial cells, inhibited serum starvation-induced endothelial apoptosis, induced sprouting angiogenesis, and stabilized HUVEC network organization (Koblizek et al., 1998; Korpelainen and Alitalo, 1998; Kwak et al.,
1999). When combined with other angiogenic factors such as VEGF or
growth factor supplements containing FGF-1, the survival of both
endothelial cells and vascular networks increased even more (Kwak et
al., 1999; Papapetropoulos et al., 1999). Although being chemotactic
for endothelial cells and Tie2-transfected fibroblasts, no mitogenic
responses of endothelial cells to Ang-1 could be observed (Koblizek et
al., 1998; Witzenbichler et al., 1998).
C. Effects of Angiogenesis Stimulation in Preclinical Studies
In various animal models, the effects of either plasmid DNA
encoding angiogenic factors or their respective protein have been studied. In pigs, gradual narrowing of the coronary artery leading to
complete blood vessel occlusion was induced by use of an ameroid constructor. In this model, the continuous perivascular administration of VEGF via an osmotic pump, or FGF-2 containing heparin-alginate microspheres, led to improved resting and stress-induced collateral blood flow values. Although the number of (vWF positive) blood vessels
in nonischemic heart tissue was unchanged, the vessel density
significantly increased in the ischemic areas (Lopez and Simons, 1996).
In a mouse model of hindlimb ischemia, created by femoral artery
ligation, the question of whether diabetes leads to impaired neovascularization was addressed, because diabetes is a major risk
factor for artery diseases. It was shown, that nonobese diabetic mice
exerted a lower angiogenic response in ischemic tissue compared with
normal mice. This impaired response could be reduced by intramuscular (i.m.) gene therapy with recombinant adenovirus expressing murine VEGF
cDNA (Rivard et al., 1999). Local administration of a plasmid encoding
the 165-amino acid isoform of human VEGF165
(phVEGF165) during balloon cathetherization of
the femoral arteries of rabbits resulted in an increased rate of
re-endothelialization. This effect was also observed in the
contralateral femoral artery that underwent simultaneous balloon injury
but was not transfected. As a result of the increased
re-endothelialization, the intimal thickening was diminished in both
limbs, thrombotic occlusions were less frequent, and recovery of the
endothelial cell-dependent vasomotor reactivity was accelerated
(Asahara et al., 1996).
For reconstructive surgery and organ transplantation procedures,
hypoxia or ischemia of the organs will negatively influence organ
viability and function. The local administration of VEGF cDNA or FGF-2
protein into ischemic experimental skin flaps in rats and rabbits,
respectively, significantly enhanced survival time of isolated skin
flaps after 1 week (Hickey et al., 1998; Taub et al., 1998). In the
case of a solid support system exploited for the grafting of, e.g.,
pancreatic islets as a means of bioartificial organ development,
incorporation of FGF-1 led to ingrowth of blood vessels 4 weeks after
implantation under the liver. Engrafting of pancreatic islets into
these FGF-1 prevascularized solid support systems resulted in a better
survival of the graft compared with islets engrafted without a solid
support, although islet function was somewhat less than in normal rats
(de Vos et al., 1997). This study indicates that therapeutic
angiogenesis may also have a potential in organ transplantation and
bioartificial organ development. It should be realized, however, that
in the case of allotransplantation, immune cell activation will occur.
Increased neovascularization may facilitate immune cell infiltration by
virtue of the fact that more blood vessels allow better access to the
graft. On the other hand, endothelium under the influence of
proangiogenic factors may exhibit impaired leukocyte recruitment
functions (see Section IV.A).
After i.m. administration of a plasmid encoding the human Ang-1 gene in
a rabbit ischemic hindlimb model, human Ang-1 mRNA could be detected 3 to 14 days after gene transfer. No mRNA was found in sites distant from
the ischemic hindlimb. Both the angiographic score and the capillary
density were increased in the hindlimb 40 days after Ang-1 encoding
plasmid administration (Shyu et al., 1998).
The increase in re-endothelialization of balloon injured vessels
in the femoral artery that was not treated with the
phVEGF165 cDNA (Asahara et al., 1996), poses the
question whether angiogenic therapy for a specific purpose may affect
other sites in the body as well. For example, the question comes to
mind whether therapeutic angiogenesis in a patient with myocardial
ischemia is able to induce angiogenesis in an otherwise dormant,
undetected, tumor nodule. Until now, however, laboratory studies did
not demonstrate that stimulation of angiogenesis alone was sufficient
for malignant growth (Isner et al., 1996).
D. First Clinical Studies on Angiogenesis Stimulation
So far, results are available from several pilot studies on the
clinical application of therapeutic angiogenesis. In all three studies
described, naked plasmid DNA encoding human
phVEGF165 under the cytomegalovirus
promoter/enhancer was administered. In the case of ischemic limbs in
patients with critical limb ischemia or thromboangiitis obliterans, the
DNA was i.m. injected in the ischemic limb (Baumgartner et al., 1998;
Isner et al., 1998). The DNA was administered directly in the
myocardium of patients suffering from myocardial ischemia. In patients
suffering from myocardial ischemia, the DNA was administered directly
in the myocardium (Losordo et al., 1998).
Using contrast angiography, newly formed collateral blood vessels could be visualized in critical limb ischemia and thromboangiitis obliterans patients treated with phVEGF165. Ischemic ulcers markedly improved or healed, resulting in successful limb salvage in several patients. Documented adverse effects were transient ankle or calf edema in some limbs. Patients suffering from myocardial ischemia had significant reduction in angina and reduced ischemia after phVEGF165 treatment.
VEGF was also administered as a protein to patients with angina.
Although the 120-day follow-up showed promising results, the 60-day
follow-up showed no difference in exercise time or improvement of
angina compared with placebo. An unexpected improvement in the placebo
group may be the reason for this result. Furthermore, a clinical study
on the applicability of FGF-2 in a similar patient group has started.
Results are expected to be presented early 2000 (anonymous, 1999a).
It was concluded from these preliminary studies that therapeutic
angiogenesis was able to induce neovascularization, and if instituted
at the proper time, it may improve ischemic disease conditions in
humans. The finding that endothelial progenitors can be isolated from
human peripheral blood opens another possibility to augment collateral
vessel growth to ischemic tissue (Asahara et al., 1997). The homing
potential of these progenitors to foci of angiogenesis may be exploited
for their application as autologous vectors for gene therapy with,
e.g., cDNA encoding VEGF after angiogenesis induction with nontargeted
plasmid DNA.
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III. Angiogenesis Inhibition |
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A. Angiogenesis and Cancer
Virchow was among the first to demonstrate the high
vascularization in tumors in his publication Die Krankhaften
Geschwülste published in 1863. He suggested that this was
associated with the disorganized nature of tumor cells. The origin of
the observed blood vessels was uncertain by then, it either developed
from the transformed tumor cells or, alternatively, from normal cells that had been derived from the neighboring benign tissues. In a later
period it was suggested that the trigger for enhanced blood vessel
growth in tumors emanated from the invading malignant cells. It was
proposed that the ability to attract new vasculature from the host was
a characteristic feature of tumor cells. Recently, two paradigms were
added to the vascular processes thought to prevail in tumor-induced
neovascularization. During vessel co-option, tumors will initially
exploit the host vasculature for survival, which coincides with host
vasculature regression. Ongoing tumor cell growth will subsequently
lead to initiation of angiogenesis (Holash et al., 1999). Furthermore,
circulating endothelial progenitor cells can form an additional source
for postnatal vasculogenesis in tumor growth (Asahara et al., 1999).
Since then many researchers have studied angiogenesis in a variety of
test systems. Only after it was recognized that new vessels at the
tumor site were absolutely required for solid tumor expansion beyond
the size of approximately 1 to 2 mm in diameter, it was suggested that
the process of angiogenesis might be a target for therapy. Recent
studies have demonstrated that lymphoproliferative diseases, such as
leukemia and lymphoma, are dependent on angiogenesis as well. Elevated
expression of FGF and VEGF has been observed in acute myeloid leukemia,
acute lymphoblastoid leukemia, and lymphomas (Fiedler et al., 1997;
Foss et al., 1997; PerezAtayde et al., 1997). These studies indicate,
therefore, that angiogenesis might also be a therapeutical target for
hematologic tumors.
The first molecule identified as an angiogenic factor was described in
1984 (Shing et al., 1984) after which a large number of angiogenic
factors followed. These factors can be produced by the tumor cells
themselves, cells present in the tumor stroma such as fibroblasts,
smooth muscle cells, or by infiltrating immune cells. To complicate
matters, these cells are all able to produce angiogenesis inhibitors as
well. More recent attention has been paid to the isolation and
characterization of these angiogenesis inhibitors because they may have
potential as therapeutic agents. Most of them have been studied for
their applicability in cancer therapy but may also be suitable for the
therapy of chronic inflammatory conditions (see Section
IV.C).
B. In Vitro and in Vivo Models to Study Angiogenesis
Angiogenesis can be qualitatively and quantitatively measured in a
large variety of in vitro and in vivo model systems. As discussed in
Section I.C, the angiogenic cascade can be dissected in
different sequential steps so that can each be studied separately in
vitro. Research has mainly focused on proliferation and migration of
endothelial cells. For this research, different endothelial cell
sources can be applied. For human research most laboratories make use
of HUVECs. This is the best available source of human endothelium, but
the major drawback of these cells is their macrovascular origin, which
makes them less suitable for studies on angiogenesis, a microvascular
process. Although more laborious, human microvascular endothelial cells
can be isolated from other organs such as foreskin or adipose tissue.
For all primary isolates, the number of in vitro passages (4-5, and in
the presence of growth factors, approximately 10) is, however, limited,
which poses a major problem for their application. The required regular
isolations furthermore introduce significant donor variation. To
circumvent these drawbacks, one can use immortalized endothelial cell
lines, such as HMEC-1 (Ades et al., 1992), EA.hy926 (Edgell et al.,
1983), or ECL4n (Griffioen et al., 1996b). ECV304, a spontaneous
immortalized endothelial cell line, has been applied for in vitro
angiogenesis studies, although recently it has become apparent that
this cell line contains a nonendothelial background as well.
Endothelial cells from other species are also available, e.g., bovine
capillary endothelial cells or cell lines from mouse and rat origin. It
should be kept in mind that the effects of angiogenesis-inducing or
-inhibiting factors can be different in the different species.
Assays to study proliferation of endothelial cells are based on cell counting or radiolabeled thymidine incorporation, or on colorimetric systems for measurement of mitochondrial activity [cell counting kit-8 (CCK-8) or dimethylthiazol diphenyl tetrazolium bromide (MTT)]. Alternatively, proliferation of endothelial cells can be analyzed by DNA profiling or determination of cell cycle-dependent expression of molecules such as proliferating cell nuclear antigen or Ki-67. Also, detection of cell death is a commonly used approach to average cell growth; e.g., apoptosis induction can be studied by detection of subdiploid cells or analysis of DNA degradation profiles, cell morphology or nick-end labeling by terminal dUTP nick-end labeling analysis.
For analysis of migration of endothelial cells, Boyden chambers are primarily used. An easier method is the wound assay. This assay system is based on wounding of a confluent monolayer of endothelial cells and measurement of the wound width in time.
Although the advantage of these in vitro assays is clearly the control
over the few parameters present, the angiogenic cascade consists of
multiple steps. This as a more extended process, can be studied in
vitro, too. Most of these assays studying more complex processes of
angiogenesis are based on tube formation of long-term cultured
endothelial cells in a 3-dimensional seminatural matrix microenvironment. The most commonly used assay system to measure tube
formation is the growth factor-induced sprouting of capillary-like structures from a confluent monolayer of endothelial cells grown on a
thick gel. These gels can either be composed of a seminatural matrix
with or without growth factors (e.g., Matrigel), or be based on
collagen (Barendsz-Janson et al., 1998) or fibrin (Koolwijk et al.,
1996). The demonstration of lumina in these endothelial cell sprouts is
regarded as a criterion for vessel growth, in contrast to just
migration of endothelial cells in the matrix or just rearrangement of
endothelial cells on the gel. An elegant method to measure
capillary formation has been described where endothelial cells grown on
gelatin-coated cytodex-3 microcarrier beads were cultured in a fibrin
gel (Nehls and Drenckhahn, 1995; Trochon et al., 1998). Quantification
of sprouting can subsequently be performed by either measurement of
maximal sprouting distance or by computer-based determination of total
vessel length. Assays based on the sprouting of capillaries out of
fresh tissue embedded in matrix gels more closely reflect the in vivo
situation. This has been described for rat aortic rings (Nicosia and
Ottinetti, 1990; Malinda et al., 1999) and human placenta tissue (Brown
et al., 1996). This procedure is not applicable for all tissues because it has been described that, e.g., tumor biopsies often produce too much
proteases digesting the matrix and thereby prevent endothelial cell
sprouting (Barendsz-Janson et al., 1998). Figure
3 shows some examples of in vitro
angiogenesis assays. A recently published novel way of measurement of
angiogenesis in vitro, which is even more close to the in vivo
situation, is the use of embryoid bodies (Wartenberg et al., 1998). In
vitro cultured mouse blastocyst cells (Evans and Kaufman, 1981)
recapitulate several steps of murine embryogenesis, including
vasculogenesis and angiogenesis (Risau et al., 1988). There is a
complete blood vessel development in these embryoid bodies (Vittet et
al., 1996) making this system suitable for the study of angiogenesis
modulators.
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Besides the advantages that in vitro angiogenesis assays clearly have,
the major drawback of all these assays is that they require the
endothelial cells to be removed from their natural microenvironment,
which may alter their physiologic properties. To study angiogenesis in
vivo, the most frequently used assay systems are the chicken
chorioallantoic membrane assay (Nguyen et al., 1994), the corneal
pocket (Conrad et al., 1994), transparent chamber preparations such as
the dorsal skin-fold chamber (Algire, 1943; Lichtenbeld et al., 1998),
the cheek pouch window (Shubik et al., 1976), and the polymer matrix
implants (Mahadevan et al., 1989; Plunkett and Hailey, 1990). However,
in vivo assays also have several disadvantages: the pharmacokinetic
properties of the compounds tested, necessary for proper interpretation
of results, are often not known and the host will respond
nonspecifically to the implantation. In this review, these assays will
not be discussed in more detail because recently an elegant review on this issue appeared, discussing the pro's and con's of in vivo quantitative angiogenesis assays (Jain et al., 1997).
C. Ways to Interfere with Angiogenesis
A broad spectrum of strategies for modulation of angiogenesis has been described. As discussed in Section I.C, angiogenesis mainly depends on proper activation, proliferation, adhesion, migration, and maturation of endothelial cells. Most approaches to modulate angiogenesis are therefore focused on these endothelial functions during blood vessel formation.
1. Intervention with Endothelial Cell Growth.
The most
successful approach to modulate angiogenesis, to date, is the use of
agents that specifically inhibit the growth of the endothelial cells.
One of the first compounds identified to exhibit inhibitory effects on
cell growth with specificity for endothelial cells was
O-chloroacetylcarbamoyl fumagillol or AGM-1470/TNP-470, an
analog of the fungus-derived antibiotic fumagillin (Ingber et al.,
1990; Kusaka et al., 1991). The mechanism of action of this compound
was found to be prevention of endothelial cells to enter
G1 phase of the cell cycle, resulting in a
decrease in proliferation (reviewed in Castronovo and Belotti, 1996).
In later years, several endogenous molecules with angiostatic activity were described. Among these molecules are thrombospondin-1 (Rastinejad et al., 1989; Good et al., 1990; Grossfeld et al., 1997), platelet factor-4 (Gupta et al., 1995; Kolber et al., 1995), and
interferon-inducible protein-10 (Luster et al., 1995). Two other
members of this class of endogenously produced antiangiogenic proteins
are angiostatin (O'Reilly et al., 1994) and endostatin (O'Reilly et
al., 1997). Angiostatin is an internal fragment of plasminogen with
multiple antiangiogenic activities in vitro and in vivo. Endostatin is a proteolytic fragment of collagen XVIII that affects endothelial cell
survival via the induction of an imbalance between the antiapoptotic proteins Bcl-2 and Bcl-XL and the proapoptotic
protein Bax (Dhanabal et al., 1999). Both induced tumor regression, not
only growth inhibition, in tumor-bearing mice, an effect that was most
pronounced with endostatin and demonstrates the potential of these
proteins. Direct inhibition of endothelial cell growth was also
obtained with two other recently described endogenously produced
angiostatic proteins, namely vasostatin (Pike et al., 1998) and restin
(Ramchandran et al., 1999). Detailed mechanisms of action have not been
described yet for these angiogenesis inhibitors.
). This can be performed by treatment with humanized
blocking antibodies to these factors, antibodies to their receptors,
with soluble receptors functioning as antagonists, dominant negative
growth factor variants, or VEGF antisense constructs. Functional
interference with growth factor signaling can also be performed by
specific inhibitors of growth factor receptor signaling, as has been
described for SU5416, a specific inhibitor of VEGFR-2 phosphorylation
(Fong et al., 1999).
Recently a new nonendothelial cell-specific inhibitor of angiogenesis
was described. Carboxyamidotriazole (CAI) is an inhibitor of tumor cell
motility; the mechanism of action is the inhibition of transmembrane
calcium influx. The inhibition of calcium influx prevents the
activation of the focal adhesion kinase and RhoA pathways. CAI
inhibited invasion by its ability to decrease the production of MMPs,
blocked migration of cells, and caused cytostasis in tumor cells and
endothelial cells. By interference in the biochemical pathways involved
in endothelial spreading on extracellular matrix, the integrity of the
vascular tube as well as stabilization of newly formed vessels were
affected. Local administration of CAI inhibited capillary expansion in
the chick chorioallantoic membrane assay. In vivo studies confirmed the
antiangiogenic and anticancer effect of CAI for, e.g., ovarian cancer
(Kohn and Liotta, 1995; Kohn et al., 1995).
2. Intervention with Endothelial Cell Adhesion and
Migration.
Because the process of angiogenesis also depends on
endothelial cell adhesion events to, and migration of cells through,
the extracellular matrix, effort is put in the search for modulators of
these interactions. The first identified member of this group of
compounds is the endogenously produced cytokine interferon. Antiendothelial activity was recognized by the observation that interferon could inhibit the migration of capillary endothelial cells
(Brouty and Zetter, 1980). Subsequently, both interferons and were shown to have in vivo antiangiogenic activity (Sidky and Borden,
1987). Although interferons are probably not sufficiently active for
treatment of all tumors, benign tumors predominantly comprised of
endothelial cells are particularly sensitive to treatment with
interferon (Ezekowitz et al., 1992). When it was found that activated
endothelial cells up-regulate receptors for extracellular matrix
components (Re et al., 1994; Frisch et al., 1996; Griffioen et al.,
1997), interaction of endothelial cells with the matrix was chosen as a
target for inhibition of angiogenesis. This proved to be a relevant
approach by the demonstration that
v3 integrin molecules, the biological function of which is binding of vitronectin and other RGD-containing matrix components, are overexpressed in
angiogenically stimulated blood vessels. Ligation of these receptors
with an antibody called LM609 interferes with endothelial cell growth
leading to inhibition of subsequent tumor growth (Brooks et al.,
1994a). In addition, the exposure of endothelial cells to
anti-v3 antibodies
resulted in the induction of apoptosis in these cells via loss of cell
anchorage to the extracellular matrix (Brooks et al., 1994b). This is
most likely the mechanism by which proliferation of endothelial cells
and angiogenesis in vivo is blocked by
v3-directed antibodies.
3. Intervention with Metalloproteinases.
Another mechanism of
angiogenesis inhibition, related to the inhibition of endothelial cell
adhesion and migration, is the use of specific inhibitors of
proteinases that dissolve the connective tissue, thereby facilitating
endothelial cell migration and subsequent vessel formation. Matrix
metalloproteinases are a homologous family of enzymes that are involved
in tissue remodeling and morphogenesis. Collectively, these enzymes are
capable of degrading all components of the extracellular matrix
(Rasmussen and McCann, 1997). Increased activity of these enzymes has
been observed in tumor formation, and therefore inhibitors of MMPs
represent an attractive approach to treat cancer. MMP inhibitors can be
divided in synthetic protease inhibitors and naturally occurring MMP
inhibitors, the tissue inhibitors of metalloproteinase. Belonging to
the former group, batimastat, marimastat, and prinomastat/AG3340 are
potent broad-spectrum inhibitors of the major MMPs and can prevent or
reduce spread and growth of several different malignant tumors in
numerous animal models (Brown and Giavazzi, 1995; Shalinsky et al.,
1999). Cell adhesion and proteolytic mechanisms are functionally
associated, as recently demonstrated by the observation that the
collagenase MMP-2 can bind to integrin
v3 on angiogenic
blood vessels. Most interestingly, it was found that a naturally
occurring MMP-2 breakdown product, called PEX, can inhibit
cell-associated collagenolytic activity. It is suggested that this
breakdown product is an important regulator of protease activity during
angiogenesis and vasculogenesis. A recombinant form of PEX was useful
in blocking angiogenesis and tumor growth in vivo, providing a novel
therapeutic approach for angiogenesis inhibition at this level (Brooks
et al., 1998).
D. Preclinical Use of Angiogenesis Inhibitors in Cancer
The pivotal role of angiogenesis in tumor progression and
metastasis has urged researchers to test newly developed angiogenesis inhibitors in a broad variety of animal tumor growth models. Studies with one of the first angiogenesis inhibitors, AGM-1470/TNP-470, were
performed in the early 1990s. Although in vitro the sensitivity for
TNP-470 was not completely restricted to endothelial cells, doses of
the compound had to be 10 to 100 times higher for comparable inhibition
of tumor cell lines. Treatment of tumor-bearing mice resulted in a
significantly increased survival time of 260% over untreated control
animals. Daily treatment was not necessary; optimal treatment regimens
were s.c. or i.v. administration once every three days. Oral
administration had weaker effects on tumor growth, likely a result of
lower bioavailability. The fact that sensitivity of tumor cells in
vitro and effect on tumor growth in vivo did not correlate is seen as
evidence for antitumor effects through the tumor vasculature (Ingber et
al., 1990; Yamaoka et al., 1993). Angiogenesis inhibitors have also
been expected to be efficient metastasis inhibitors based on the
concept that tumors require new vasculature for spreading and outgrowth
at a secondary site. TNP-470 reduced both the number and size of
metastases of the B16BL6 melanoma and the M5076 reticulum cell sarcoma
cell line in mice by 80 to 90% (Yamaoka et al., 1993). Also in rats, inhibition of tumor growth and metastasis was observed (Futami et al.,
1996).
The next breakthrough in the search for novel antiangiogenic compounds
occurred when the hypothesis that a primary tumor, whereas capable of
stimulating angiogenesis for its own blood supply, can produce
angiogenesis inhibitors that suppress the outgrowth of distant
metastases, was proven to hold true. This hypothesis came from the
observation that the removal of primary tumors could lead to the
accelerated growth of metastases (Sugarbaker et al., 1977). To test
this hypothesis, the Lewis lung carcinoma mouse model was used in which
the primary tumor completely suppresses the growth of its metastases.
From the urine of these mice a cleavage fragment of plasminogen called
angiostatin was purified, which completely replaced the inhibitory
activity of the primary tumor (O'Reilly et al., 1994). Treatment of
tumor-bearing mice with angiostatin almost completely prevented
metastasis formation in the lung. Using a similar strategy, endostatin
was discovered (O'Reilly et al., 1997). Treatment of mice carrying
different syngeneic malignant tumors with endostatin led to a rapid
regression of tumors. As with angiostatin, there was no sign of
toxicity, and continued endostatin therapy maintained the tumors in a
state of dormancy. Discontinuation of treatment led to renewed growth at the primary site, which would eventually lead to death of the animals. However, when therapy was restarted tumors regressed again for
nearly 100%. Subsequent intermittent cycles of treatment were able to
maintain the tumors in a state of dormancy and showed no sign of
drug-induced resistance (Boehm et al., 1997). When angiostatin and
endostatin therapy were combined for 25 days, both at relatively high
doses (20 mg/kg/day), tumors regressed completely yielding tumor-free
survival of up to 11 months after start of therapy.
The nature of this dormancy is still obscure since these tumors have
the capacity of renewed outgrowth when transplanted to the other flank
or to another animal. This makes the involvement of the immune system,
i.e., the development of a specific antitumor immune response in these
mice, unlikely. It has been suggested that the sequential accumulation
of endostatin during these cycles of growth and regression can locally
lead to a high concentration, and hence keep the tumor dormant (Black
and Agner, 1998). The data on endostatin suggest that the endothelial
cell compartment is directly involved in the dormancy of the tumor
cells, demonstrating the powerful control exerted by the vascular
endothelial cell population over the tumor cell population.
The discovery of overexpression of
v3 integrin in tumor
vessels and the dependence of angiogenesis on this matrix receptor (Brooks et al., 1994a) introduced the first strategy for angiogenesis inhibition by targeting specific tumor endothelial cell determinants. Antibody and specific RGD motif containing peptide ligands both inhibited human tumor growth in animal models (Brooks et al., 1994b;
Friedlander et al., 1995). The prevention of the formation of a
suitable matrix to migrate through by the inhibition of MMPs is related
to this inhibition of cell adhesion to suppress angiogenesis. Treatment
of mice carrying the highly metastatic tumor B16BL6 with batimastat
resulted in reduction of lung colony number. It was found that not cell
arrest in the lungs but actually extravasation of the cells was
inhibited (Chirivi et al., 1994). Batimastat also inhibited primary
tumor cell outgrowth. A 58% inhibition was observed when treatment was
started from the day of tumor cell inoculation. In numerous other
models, such as ovarian cancer and an orthotopic colon cancer model,
similar results were obtained (Davies et al., 1993; Wang et al., 1994).
Several other low-molecular weight synthetic MMP inhibitors is
currently under development. One of them is prinomastat/AG3340, which
has one of the lowest Ki values
(Ki is the concentration that inhibits
enzyme activity by 50%) for gelatinase A (MMP-2) and gelatinase B
(MMP-9). In a chemoresistant human nonsmall cell lung carcinoma mouse
model, AG3340 was well tolerated at 400 mg/kg/day (administered twice daily, 7 days a week) and exhibited significant inhibition of angiogenesis (up to 77%) and tumor growth (up to 65%). A suboptimal dose of AG3340 in combination with carboplatin or paclitaxel resulted in greater inhibition than was observed with either agent alone. In
colon and prostatic tumors, angiogenesis was inhibited by approximately 50% (Shalinsky et al., 1998).
The potential of the use of inhibitors of specific signal transduction
pathways is nicely exemplified by the use of CAI. This inhibitor of
calcium mobilization, arachidonic acid release, and generation of
inositol phosphates, inhibits growth of tumor cell lines. In the same
way that it inhibits tumor cells, endothelial cells are affected,
resulting in inhibition of angiogenesis. Oral administration of CAI to
mice carrying human xenografts resulted in a >80% inhibition of tumor
outgrowth (Kohn et al., 1992). CAI was not toxic as determined by
macroscopic and histological evaluation. These studies are highly
interesting because one might expect that pharmacologic interference
with intracellular signaling pathways, which are so fundamental to
cellular function in both pathologic and normal tissues, will lead to
massive toxicity. Because this is not the case, the conclusion seems
justified that tumors display an enhanced dependence and sensitivity to
these second messenger pathways or are exposed to higher drug
concentrations by increased uptake or accumulation at the tumor site.
E. Clinical Trials with Inhibitors of Angiogenesis for Cancer Treatment
Currently some 30 antiangiogenesis compounds are being tested in human clinical anticancer trials. Most of them are in phase I or II testing, to determine optimal dosage, nature and severeness of side effects, safety and preliminary effectivity. Only a few compounds, as discussed in this chapter, are further down the clinical road and currently being tested in phase III trials. Usually the angiogenesis targeted studies are designed in a way that patients receive either standard (chemo- or immuno-) therapy and a placebo, or standard therapy combined with a new antiangiogenic drug. The National Cancer Institute, which is involved in some of these clinical trials, has classified these drugs in different categories. Without trying to be complete, a few examples of clinical studies with these compounds will be discussed here.
i. Drugs that inhibit endothelial cell proliferation directly. As a member of the "first generation" of angiogenesis inhibitors, TNP-470 was used in the first formal clinical trial of antiangiogenic therapy. In cooperation with the National Cancer Institute, currently three clinical studies are operative: 1) a phase I study of TNP-470 in patients with recurrent or refractory pediatric solid tumors, lymphomas, and acute leukemias; 2) a phase II study for advanced or recurrent squamous cell carcinoma of the cervix; and 3) a phase III randomized study of TNP-470 versus synchronous radiotherapy and chemotherapy for locally advanced nonresectable nonmetastatic pancreatic cancer. Recently, one phase II multi-institutional clinical study in metastatic renal cell carcinoma patients was reported. In the first 20 patients with an adequate follow-up, there was one partial response and one minor (40% shrinkage) response. Five of these 20 patients remained progression-free for 16 weeks or more. The mild toxicity reported and the high incidence of apparent prolonged progression-free survival in this heavily pretreated cohort is encouraging (Stadler et al., 1998).
ii. Drugs that block matrix breakdown.
Among
the clinical trials with angiogenesis inhibitors, the metalloproteinase
inhibitors have probably been the most extensively studied in clinical
trials to date. The first trial in patients with a MMP inhibitor,
batimastat, started in 1991 in patients with ovarian carcinoma. Because
the drug showed low bioavailability, other administration routes were
used. A phase I study using an i.p. formulation yielded high and
sustained plasma concentrations of the drug, low toxicity, and early
signs of efficacy. A randomized controlled trial of marimastat in 400 patients with advanced pancreatic cancer has now been completed. The
study was designed to compare the effect of orally and twice daily
administered marimastat with the conventional treatment with
gemcitabine. The study did not meet its primary endpoint, designated as
a 16% or greater reduction in mortality versus gemcitabine treatment,
i.e., there was no significant difference between the survival curves
for gemcitabine and the highest marimastat dose. Safety data revealed
no difference between the treatment groups other than the expected
musculoskeletal events (stiffness and pain) caused by the MMP inhibitor
that have been reported in most studies. Although this study has not
shown any benefit of treatment with marimastat over the conventional treatment, there is evidence that marimastat is as effective as the
"drug of first choice" treatment. This holds promise for the use of
marimastat in future combination regimens, studies which are currently
being enrolled. Oral administration of prinomastat/AG3340 was tested in
a single dose phase I study in healthy volunteers. Favorable absoption,
pharmacokinetics, and toxicity profiles were observed. In a phase I
safety and tolerability study of AG3340 administered orally and twice
daily in patients with advanced cancer, including lung, prostate,
kidney, and colorectal cancers as well as sarcoma and melanoma, disease
was stabilized in more than 25% of 47 evaluable patients who were
treated for periods of 16 to 40 weeks. Three patients (one each with
nonsmall cell lung cancer, renal carcinoma, and melanoma) were found to
have minor reductions in tumor volume. AG3340 was found to be generally well tolerated.
A phase III clinical trial of AG3340 has been initiated in combination
with chemotherapy in patients with advanced nonsmall cell lung cancer.
This trial is designed to evaluate the safety and efficacy of AG3340 as
part of first-line therapy in combination with chemotherapy. In a study
being conducted at sites in North America, Europe, and Australia,
patients with advanced nonsmall cell lung cancer will be randomized to
receive either AG3340 (tablet form) in combination with gemcitabine and
cisplatin or placebo in combination with gemcitabine and cisplatin. The
primary objective of this study is to compare time of overall survival
between patients receiving AG3340 or placebo in combination with
gemcitabine and cisplatin.
iii. Drugs that inhibit endothelial-specific
integrin/survival signaling.
The results of a completed phase I
clinical trial with Vitaxin, the humanized
v3-directed LM609
monoclonal antibody have been presented (Gutheil et al., 1998). This
trial of 14 patients revealed no significant toxicities, and in
addition, showed that two-thirds of the patients experienced an
objective clinical response to the Vitaxin treatment. Most recently, a
phase I/II study was initiated to determine the optimal dosage, whereas
a phase II clinical trial to determine efficacy is planned to start
early 2000.
iv. Drugs with nonendothelial cell-specific
mechanism(s) of action.
Phase I clinical trials with CAI, the
inhibitor of transmembrane calcium influx, have been completed,
demonstrating that oral treatment with a single dose daily was
relatively well tolerated (Kohn et al., 1997). Cytostasis in the form
of disease stabilization was observed in almost half the solid tumor
patients treated. Several refractory ovarian cancer patients were found
to have disease stabilization lasting up to 10 months. This led to the initiation of a phase II clinical trial for women with relapsed epithelial ovarian cancer and subjected to no more than three prior
therapeutic regimens. This single agent trial is aimed at an outcome of
6 or more months of disease stabilization.
v. Drugs that block activators of
angiogenesis.
Angiogenesis seems exquisitely sensitive to small
changes in factors such as VEGF and FGF-2 that drive the angiogenic
process. This has important therapeutic implications in treating
angiogenesis-driven disorders (Rak and Kerbel, 1997). Next to anti-VEGF
antibodies used to neutralize the cytokine in serum/extracellular
fluids, another approach is the blockade of VEGF receptor signaling.
SU5416 is a specific inhibitor of the signaling pathway of the VEGFR-2 found on endothelial cells. This compound entered phase I clinical testing in the fall of 1997 and has shown good safety profiles in
approximately 100 patients with solid tumors. The phase III clinical
study in colorectal and lung carcinoma patients will compare the
efficacy of standard chemotherapy regimens with the efficacy when
combined with SU5416. SU6668 is a broad-spectrum inhibitor of
angiogenesis and tumor growth, acting by interference with VEGF, FGF,
and PDGF receptors. This drug is currently entering phase I studies in
both i.v. and oral formulations.
A listing of angiogenesis inhibitors that are currently in clinical
trials, can be found on an internet website of the National Cancer
Institute (http://cancertrials.nci.nih.gov). Moreover, most of the data
available so far can only be retrieved from the internet. Inhibition of
angiogenesis is a new strategy of cancer therapy, in which the concept
of treatment is fundamentally different from conventional therapies. In
standard chemotherapy the desired endpoint is shrinkage of the tumor
and complete remission, whereas in antiangiogenic therapy the endpoint
aimed at is achievement of stable disease and prolonged
progression-free periods leading to a relatively long survival. This
underscores the novelty of antiangiogenesis therapy because until now,
to our knowledge, there has never been a drug for oncological use that
has been approved by the FDA on the basis of stabilization of the
disease. As a consequence, the surrogate endpoints, such as
stabilization or decrease of circulating tumor markers [e.g., prostate
specific antigen (PSA) in prostate cancer, carcino-embryonic antigen
(CEA) in gastrointestinal cancer, and CA15 in breast cancer], do not necessarily need to be the same. An important issue in antiangiogenesis treatment that concerns most clinicians, is the question whether there
are surrogate endpoints for angiostatic treatment. The most commonly
used parameter for ongoing angiogenesis is the circulating (plasma)
level of VEGF. However, this is an indirect method and in some
instances, e.g., during treatment with VEGF-blocking antibody, this
parameter cannot be used. More useful surrogate endpoints have not been
described yet, although circulating levels of E-selectin and vascular
cell adhesion molecule-1 (VCAM-1) have been put forward for this
purpose (Ferrara, 1995; Koch et al., 1995). Considering the fact that
their counter ligands are abundantly present in the serum, it is
however likely that these soluble adhesion molecules are rapidly
cleared from the circulation, and thereby mask ongoing angiogenesis.
Although stabilization of tumor size in antiangiogenesis therapy may be
an endpoint in the clinic, in preclinical animal studies it is obvious
that the most promising angiogenesis inhibitors do not stabilize the
tumor. Instead, they induce partial or sometimes even complete
regression. This may partly be explained by the rapid turnover of cells
in animal tumors, even in a stabilized tumor, in which case inhibition
of angiogenesis would lead to a disturbed balance between cell growth
and cell death leading to an overall enhanced cell death.
Animal experiments also may provide a clue to whether benefit from
antiangiogenesis therapy should be expected in patients with large
tumor burdens. Some unpublished studies indicate that the largest
benefit from angiogenesis inhibition is expected when the therapies are
started as early as possible, with the largest effects seen on starting
the treatment even before the angiogenic switch occurred. Because this
does not meet the situation in the clinic, where cancer is normally
diagnosed at a much later stage, combination therapies are most likely
to give the best results.
F. Novel Approaches to Interfere with Tumor Blood Flow
Angiogenesis inhibitors are compounds that specifically interfere with regulatory processes of the various steps of angiogenesis, as described above. Tumor vasculature targeting aims at specifically delivering pharmacologic agents to the site of the blood vessels of a tumor. The pharmacologically active moieties can exert a variety of activities, but in general all strategies aim to block tumor blood flow and hence tumor growth. Moreover, as a consequence, site-specific immunologic responses may be brought about and potentiate anti-tumor effects. Besides the choice of the effector modality (e.g., coagulation factors or toxins), the choice of the target epitope is of great importance for feasibility and effectiveness of these approaches, as will be discussed below.
1. Targeted Strategies to Induce Tumor Blood Coagulation.
At
present, two studies on targeting blood coagulation-inducing activity
to tumor endothelium as a therapy for cancer have been reported. In the
first study, an animal model with artificially induced target epitopes
on the tumor vasculature was exploited. In this model, transfected
tumor cells s.c. inoculated locally produced high amounts of interferon
(IFN), leading to tumor endothelium-specific expression of major
histocompatibility complex (MHC) class II. Bispecific antibodies (BsAb)
against MHC class II and a truncated form of the activator of the
extrinsic coagulation pathway, tTF, were subsequently developed.
Intravenous administration of a mixture consisting of BsAb and tTF
(BsAb·tTF), the so called "coaguligand" formulation, to mice with
clinically relevant tumor burden resulted in dramatic tumor reduction
without concurrent toxicity in other organs. Site-specific blood
coagulation in the tumor blood vessels caused an almost instantaneous
and persisting blockade of tumor blood flow. Treatment of mice bearing
s.c. tumors twice with BsAb·tTF coaguligand lead to 38% complete
tumor regressions and 24% partial responses (Huang et al., 1997). The
attractiveness of the coaguligand approach is the use of a truncated
form of TF that is completely devoid of coagulation-inducing activity
as long as it is kept from complexation with the lipophilic factor X at
cell membranes. On cross-linking of the hydrophilic tTF with the target
cell membranes by the BsAb, tTF becomes complexed with factor X in the
presence of factor VII/VIIa, leading to the induction of blood
coagulation (Fig. 4). It is thought that
a threshold in the number of tTF cross-linked to factor X-containing
cell membranes exists, above which the coagulation cascade is
initiated. This allows the use of target epitopes that are highly but
not exclusively expressed on tumor endothelium.
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). The antitumor effect was not
as dramatic as that seen in the MHC class II model. Possibly, the
number of tTF molecules delivered at the site of the tumor endothelium
was not sufficient to create a rapid and more or less generalized blood
coagulation throughout the tumor vasculature. Only if the blood in the
majority of vessels becomes coagulated by the coaguligand, the number
of tumor cells that will be deprived of nutrients will be sufficient
enough to result in strong antitumor effects. Furthermore,
anticoagulative activities may be strong enough to counteract the
effects when the kinetics of coagulation induction by the coaguligand
are insufficient to perturb local pro- and anticoagulative activities.
The coagulation induction potency of coaguligand formulations are
mainly determined by the following factors: i) the number of target
epitopes on the tumor endothelium that allow BsAb-mediated interaction
between tTF and factor X on the target cell membrane; ii) locally
present counteracting anti-coagulative activity; and iii) kinetics of
cross-linking of the BsAb and the target epitopes in relation to the
kinetics of coagulation induction capacity. No detailed information is
available on species differences in coagulation induction activity of
the tTF coaguligand formulation, although we are aware of differences
in pro- and anticoagulative properties between species. Furthermore,
the number of MHC class II and VCAM-1 molecules expressed on the tumor
vasculature of the animal models discussed, were high, as were the
antibody affinities. This enabled a significant number of tTF to be
rapidly cross-linked to the target cell membrane. For clinically
relevant target epitopes and targeting devices, it needs to be
established whether these parameters are as important with respect to
therapy efficacy.
The research in the area of coaguligand therapy for cancer is still in
its infancy. Important for its future is to define appropriate target
epitopes on human tumor endothelium (Section III.F.3),
qualitatively as well as quantitatively. Furthermore, the issue of the
occurrence of distant embolism following local thrombus formation in
the tumor vasculature, needs to be addressed in the appropriate
settings. By obtaining more insight in these and other above-discussed
processes in the coming years, we will be able to better understand the
critical players in this type of therapy, and hence the value of its
potential for future clinical application.
2. Targeted Strategies to Kill Tumor Endothelial
Cells.
In the development of drug-targeting approaches for
therapy of cancer, toxins, chemotherapeutics, and ionizing radiation
have been extensively studied as effector molecules (Meijer and Molema, 1995). The majority of approaches aim at targeting the effector molecules to the tumor cells, thereby increasing therapeutic efficacy and decreasing toxicity elsewhere in the body. Treatment of solid tumors, however, has not been successful, which is believed to be due
to poor penetration of the drug-targeting conjugates in the solid tumor
mass (Jain, 1996; Molema et al., 1997). In this respect, tumor
endothelial cells are now considered a better target candidate for
cancer therapy, because they are easily accessible for blood-borne
therapeutics. Furthermore, hundreds of tumor cells rely on the
functionality of only one blood vessel formed by relatively few
endothelial cells. The number of target cells to be destroyed is
therefore significantly less than when tumor cells themselves are the
target for killing.
). Treatment of tumor-bearing mice with the immunotoxin
caused complete occlusion of the tumor vasculature and strong
regression of large solid tumors. Another tumor endothelial cell-targeted immunotoxin recently reported consisted of deglycosylated ricin A chain conjugated with antibody against endoglin, a TGF-binding protein specifically expressed on proliferating cells (Matsuno et al.,
1999). In addition, "angiotoxins" consisting of diphtheria toxin
domains fused or chemically conjugated to VEGF isoforms, VEGF-121 and
-165, have been investigated for this purpose. These approaches were
potent in inhibiting angiogenesis and tumor outgrowth in in vitro and
in vivo angiogenesis models (Ramakrishnan et al., 1996; Olson et al.,
1997; Arora et al., 1999).
Targeted radioimmunotherapy of pulmonary micrometastases was feasible
in mice with an antibody directed against thrombomodulin, expressed
selectively and in large amounts on the luminal surfaces of capillaries
and small blood vessels in the lungs. The short-lived (t1/2 = 45 min) -particle emitter
213Bi conjugated to the antibody was delivered to
healthy lung and tumor capillaries, resulting in significant tumor
growth reduction and extended life span of animals treated at low
doses. At higher doses, tumors almost completely regressed, but animals
died of lung fibrosis induced as a result of concurrent damage to
healthy tissue (Kennel et al., 1999).
By covalently conjugating the chemotherapeutic agent doxorubicin to a
peptide containing an RGD motif, Arap et al. (1998) constructed
drug-targeting conjugates specific for integrins
v3 and
v5 on endothelial
cell of tumor vasculature. Treatment of human breast carcinoma-bearing
mice with the conjugate at a doxorubicin equivalent 10 to 40 times
lower than that of free doxorubicin, caused vascular damage in the
tumors and a strong antitumor effect, whereas liver and heart toxicity
was less compared with free doxorubicin (Arap et al., 1998). Whether
this effect was caused by the selective delivery of the
chemotherapeutic drug to the tumor endothelial cells and/or tumor
cells, direct caspase-3 activation (Buckley et al., 1999), or a
combination has not been studied yet. More recently, the applicability
of this RGD peptide-mediated tumor endothelium targeting was extended
to dipeptides consisting of the RGD-targeting peptide and an
apoptosis-inducing peptide. The dipeptides were selectively toxic to
angiogenic endothelium and showed anticancer activity in mice (Ellerby
et al., 1999).
Some major steps forward have been made in the development of novel
drug-targeting approaches aimed at selectively killing tumor
endothelial cells. The extensive "from the bench to the bed"
experience with tumor cell targeted immunotoxins (Frankel et al., 1996)
will be used for the further development of these tumor endothelial
cell-targeted strategies.
3. The Quest for New Targets on Tumor Endothelium.
The
angiogenic phenotype of the tumor vasculature provides the most
prominent markers for differentiation between tumor and normal
endothelial cells. One of the first studies on the development of
molecules specifically recognizing tumor endothelium was published by
Hagemeier et al. (1986). They developed an antibody recognizing a 30.5-kDa antigen present at the tip of budding capillaries in proliferating tissue (placenta, umbilical vein, intestine) and in acute
inflammatory reactions and tumors. This study was followed by several
others, all describing molecular markers more or less selectively
expressed on endothelial cells in tumor tissue (Table 1).
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v3 integrin and
VEGFR. Not only was the
v3 targeted doxorubicin a potent inhibitor of tumor endothelial cell proliferation in mice (Arap et al., 1998),
v3 was also a
suitable target for the detection of tumor angiogenesis by magnetic
resonance imaging in rabbits (Sipkins et al., 1998). The conjugation of
diptheria toxin to VEGF resulted in an angiotoxin with endothelial cell killing activity in vivo (see Section III.F.2). One may
consider modifying these small peptide conjugates, because clearance
from the systemic circulation can be rapid and unwanted. Modifications can be brought about by increasing molecular weight, for example, an
approach also followed in the development of antibody fragments with
unfavorable pharmacokinetics. A longer circulation time of the
conjugates allows longer exposure of the target endothelium to the
conjugates, although unwanted toxicity elsewhere may simultaneously increase.
Induction of target epitopes by irradiation of the tumor or cytokine
administration may be considered an option. Using X-ray radiation,
P-selectin was rapidly translocated to the vascular lumen of tumors in
rats and mice (Hallahan et al., 1998). It has been known for quite some
time, that TNF, combined with IFN, exerts selective cytotoxic effect on
tumor macro- and microvasculature. In vitro studies demonstrated a
decrease in v3
integrin expression on exposure to these cytokines (Ruegg et al.,
1998). One can hypothesize that this combination of cytokines is likely
to affect the differential expression of other molecules as well.
Up-regulated molecules may then be identified as potential targets.
Especially in the context of multiple metastases in an organ, which can
be subjected to isolated organ perfusion (e.g., limb, liver, kidney),
cytokine-induced target epitopes may be worth considering.
CM101 is a polysaccharide exotoxin of group B streptococcus. In
tumor-bearing mice, i.v. injection of CM101 led to a rapid binding of
the compound to tumor neovasculature. As a result, complement was
activated and leukocytes started to infiltrate, followed by the
production of proinflammatory cytokines (Yan et al., 1998). Besides
having a quite potent effect on tumor growth itself, CM101 could be
exploited as a carrier to deliver pharmacologically active compounds at
the site of the tumor vasculature. By incorporating two entities with a
different mechanism of action, an approach called "dual targeting"
(i.e., the carrier itself and the attached drug both exert an effect),
synergistic effects can occur (Meijer et al., 1996). Similarly
endostatin and TNF may be considered carrier molecules for dual
targeting purposes, because tumor endothelium is more prone to
endostatin- and TNF-induced cytotoxicity. The recent demonstration that
the putative binding sites for endostatin are present on endothelial
cells in normal tissue as well (Chang et al., 1999) challenges the use
of endostatin for this purpose. As a result of its high toxicity,
application of TNF for such an approach seems only feasible in isolated
organ perfusion systems (Eggermont et al., 1996).
The observation in the MHC class II directed coaguligand studies that
tumor endothelium of those animals not responding to treatment were
devoid of the target epitope is important in light of intra- and
intertumoral heterogeneity in human. In human tumors, the vasculature
exists in different stages of activation and angiogenesis. In some
areas the endothelium is quiescent, whereas at the same time in other
areas neovessel maturation takes place. Therefore, targeting effector
molecules to one target epitope only will not be successful in blocking
the majority of the tumor blood flow. Conjugates targeted to a
combination of epitopes should be applied for effective solid tumor
debulking. In this respect, one can think of targeting to VEGFR in the
early angiogenic cascade,
v3 integrin expressed
in later stages and Tie2 during the maturation phase.
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IV. The Interplay between Angiogenesis and Cells of the Immune System |
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A. Angiogenesis Regulates Leukocyte Recruitment
It is well established that the immune system plays an important
role in the regulation of angiogenesis. Multiple studies indicate that
leukocytes can induce vascular proliferation (Sidky and Auerbach, 1975;
Polverini et al., 1977; Camussi et al., 1997), and specific
leukocyte-derived cytokines have been identified to induce angiogenesis
(Koch et al., 1992; Hashimoto et al., 1994; Richardson et al., 1994;
Vanhee et al., 1994; Freeman et al., 1995). Next to the regulation of
angiogenesis by leukocytes, angiogenic processes can have a major
impact on cells of the immune system and on the development of an
immune response as well. Normal endothelial cells contribute to the
recruitment of immune cells to the site of inflammation by the
expression of adhesion molecules (depicted in Fig. 1). Different
families of adhesion molecules play a role in this process. The most
important families identified at present are 1) the Ig superfamily of
Ig related molecules, such as ICAM-1, VCAM-1, and CD31; 2) the
selectins, molecules that initiate the adhesion cascade by mediating
leukocyte rolling through recognition of carbohydrate epitopes; and 3)
a group consisting of among others, CD34 an L-selectin binding
glycoprotein that is expressed on hematopoietic progenitor cells and on
the luminal side of vascular endothelial cells (Kuzu et al., 1992) and
CD44, the lymphocyte homing receptor that is expressed on activated
endothelial cells (Griffioen et al., 1997). Expression of endothelial
adhesion molecules is controlled by cytokines such as TNF, IL-1, and
IFN. These cytokines facilitate leukocyte adhesion to endothelial cells
and extravasation into tissues by inducing an enhanced expression of
ICAM-1, VCAM-1, and E-selectin among others (Carlos and Harlan, 1994).
In angiogenically stimulated tissues angiogenesis mediates the
formation of new blood vessels (Hanahan and Folkman, 1996). The
exposure of endothelial cells to angiogenic factors down-regulates adhesion molecule expression (Kitayama et al., 1994; Griffioen et al.,
1996b, 1999b). Also, the induction of adhesion molecule expression by
the proinflammatory cytokines TNF, IL-1, or IFN, is severely hampered
(Fig. 5), a phenomenon called endothelial cell anergy (Griffioen et al., 1996a). These observations are in line
with and provided a mechanistic background for earlier observations of
reduced leukocyte-vessel wall interactions in tumors (Wu et al., 1994;
Dellian et al., 1995; Fukumura et al., 1995; Borgstrom et al., 1997).
In one study (Melder et al., 1996), it was found that during
angiogenesis FGF-2 and VEGF have opposite functions with regard to
natural killer cell adhesion to endothelium. Whereas FGF-2 inhibited
adhesion both in vivo and in vitro, the effect of VEGF, which was only
studied in vitro, consisted of stimulation of adhesion. The above
results may partly explain the findings that some adhesion molecules
that are absent on resting endothelial cells become overexpressed on
tumor endothelial cells (Kraling et al., 1996).
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A link between leukocyte-endothelium adhesion and angiogenesis seems to
be present as suggested by the observation that endothelial markers
originally identified to play a role in leukocyte recruitment appear to
be involved in neovascularization as well (Ferrara, 1995). E-selectin
has a function in the formation of capillaries (Nguyen et al., 1993),
whereas platelet endothelial cell adhesion molecule-1 or CD31 has been
reported to play a role in tube formation of endothelial cells (Berger
et al., 1993; Fox et al., 1995; Delisser et al., 1997). These
observations were extended for VCAM-1 being expressed in medullo- and
neuroblastoma, lung and renal cell carcinoma (Kuzu et al., 1993; Patey
et al., 1996). The description of soluble VCAM-1 and E-selectin as
activators and attractors of endothelial cells (Koch et al., 1995),
added to this leukocyte endothelial cell adhesion-angiogenesis link.
At the molecular level, expression of endothelial cell adhesion molecules required to facilitate leukocyte recruitment is differentially regulated from the expression of adhesion molecules in angiogenesis. Although inflammatory signals increase expression, angiogenic stimuli often down-regulate adhesion molecules involved in leukocyte-endothelial cell interactions. The net adhesion molecule receptor make-up on the endothelium is hence determined by both signals.
Recently, additional studies addressing the role of VEGF and FGF in
leukocyte vessel wall interactions showed that in general a strong
induction of angiogenesis inhibits leukocyte adhesion (Booth et al.,
1999). These studies are especially important in the light of tumor
biology because these phenomena contribute to the escape of tumors from
immune surveillance. It has been suggested that this "aberrant"
responsiveness in adhesion molecule expression is not specific for
tumors but has its origin in embryonic development where growing tissue
should not be infiltrated by excessive numbers of leukocytes.
Inhibition of angiogenesis may counteract the down-regulation of
adhesion molecules and endothelial cell anergy in tumors, and thereby
restore the inflammatory response of endothelial cells and enhance the
infiltration of leukocytes (Griffioen et al., 1999a) (Fig. 5). This
would, next to the direct inhibition of tumor growth by prevention of
blood vessel formation, be beneficial in the treatment of cancer and
could be enhanced even further by combination of the therapy with,
e.g., bispecific antibodies (Kroesen et al., 1998).
B. The Role of Angiogenesis in Chronic Inflammation
The relation between angiogenesis and leukocyte infiltration in cardiovascular diseases and chronic inflammation has attracted a lot of attention in the last years. In these diseases, the leukocyte involvement, e.g., macrophage infiltration in atherosclerotic plaques and the immune response against cartilage in rheumatoid arthritis, is expected to mediate the pathology.
In the first acute phase of inflammation, functional changes in the
vasculature such as dilatation, increase in permeability, and
endothelial activation occur. In the second subacute phase, capillaries
and venules remodel with extensive endothelial mitotic activity (Majno,
1998). Upon chronic stimulation, both increases in capillary density
and vascular dilatation can be observed, although these responses can
differ significantly between strains of mice and possibly between
species (Thurston et al., 1998). In many chronic inflammatory diseases
in human, neovascularization can be identified in the inflamed lesions.
In rheumatoid arthritis, neovascularization, which is one of the
earliest histopathological findings, is thought to be required for
pannus development. Rheumatoid synovial endothelium is constantly
subjected to remodeling. Besides nurturing the pannus, the blood
vessels also play an active role in the inflammation by being a source
of cytokines, chemokines, and proteases (Storgard et al., 1999).
Synoviocytes in the rheumatoid lesions exhibit characteristics of tumor
cells, including somatic mutations in regulatory genes such as
Ha-ras and p53. Therefore, the rheumatoid synovium can be
envisioned as a tumor-like mass that invades and destroys its local
environment and is enriched in angiogenesis-promoting cytokines, such
as FGF-2, VEGF, and IL-8, and soluble adhesion molecules VCAM-1 and
E-selectin (Koch, 1998; Firestein, 1999). Also in psoriasis, expansion
of the dermal microvasculature is a prominent feature (Creamer et al.,
1997). Capillary leakiness and vascular anomalies develop in psoriatic skin after the occurrence of epidermal alterations. TGF, overexpressed in psoriatic epidermis, induced VEGF production by keratinocytes in
vitro. Furthermore, extensive VEGF production in psoriatic skin lesions
coincided with VEGFR-2 overexpression in lesional endothelium (Detmar
et al., 1994). Interleukin-8 may play a key role in psoriasis because
it is expressed in the stratum granulosum, attracts polymorphonuclear
cells, and stimulates angiogenesis and keratinocyte mitogenesis
(Konstantinova et al., 1996).
As is demonstrated for psoriasis, obesity is also dependent on
angiogenesis (Crandall et al., 1997). Rats that are exposed to cold
perform adaptive hyperplasia of adipose tissue. This coincides with
abundant overexpression of VEGF in this tissue. This reaction was
regulated by -adrenergic stimulation of the adipose tissue and was
mediated by VEGF120, the short heparin binding
site lacking isoform of VEGF (Asano et al., 1997).
Interestingly, the endothelial adhesion molecule E-selectin, which was
identified many years ago as a prime mediator of leukocyte tethering
from the blood stream, was more recently identified as a marker of
angiogenic endothelium (Luscinskas et al., 1989; Nguyen et al., 1993).
It may well be that specific isoforms of E-selectin prevail during the
angiogenic process, although at present, the mechanism(s) leading to
this and the functional consequences of such molecular modulations have
not been extensively investigated (Verkarre et al., 1999).
In vitro, activated T cells were able to induce endothelial expression
of MMPs and vascular tube formation in a three-dimensional gel assay by
CD40-CD40 ligand interactions. In sites of chronic inflammation such as
atherosclerotic plaques, vascular expression of CD40 and its ligand has
been reported. These data suggest that ligation of CD40 on endothelium
can mediate several aspects of vascular remodeling and
neovascularization during atherogenesis and other chronic inflammatory
diseases (Mach et al., 1999). In addition, VEGF was shown to be
moderately to strongly expressed in atherosclerotic human arteries.
Smooth muscle cells as well as macrophage-derived foam cells and ECM
near macrophages contained extensive VEGF levels. In regions rich in
macrophages, the prominent T cell infiltrate was a major source of VEGF
(Couffinhal et al., 1997; Inoue et al., 1998; Chen et al., 1999a). The
more the atherosclerotic lesions advanced, the more often the lesions
contained intimal blood vessels. VEGFR-1 and -2 were both distinctly
upregulated in macrophages and endothelial cells in the lesions (Inoue
et al., 1998).
The increased serum levels of VEGF that correlated with disease
activity in patients with inflammatory bowel diseases, Crohn's disease
and ulcerative colitis, indicate a role for this cytokine in promoting
inflammation in these chronic inflammatory diseases as well. The
mechanism of action may be through increasing the vascular permeability
and/or wound healing via its proangiogenic effects (Bousvaros et al.,
1999).
In endometriosis, excessive endometrial angiogenesis is proposed as a
mechanism in the pathogenesis of this disease. The endometrium of women
with endometriosis has an increased capacity to proliferate, implant,
and grow in the peritoneal cavity. There is enhanced endothelial cell
proliferation in the endometrium of women with this disease, and these
vessels express the cell adhesion molecule v3 integrin. This
demonstrates that endometriosis is a disease with a proangiogenic
character, which suggests that novel new medical treatments for
endometriosis can be aimed at the inhibition of angiogenesis (Rogers
and Gargett, 1999).
Intraocular neovascularization causes a functional disorder of the eye
and contributes to loss of vision. It is associated with diseases such
as diabetic retinopathy, retinal vein occlusion, and age-related
macular degeneration. In diabetic retinopathy and other retinal
disorders, vitreous levels of soluble E-selectin and ICAM-1 were
significantly elevated, indicating a strong inflammatory component in
these diseases (Esser et al., 1995; Limb et al., 1999). Furthermore,
several types and layers of retinal cells can produce VEGF, the major
mediator of intraocular neovascularization and permeability (Brown et
al., 1997). Proangiogenic factors such as placenta growth factor and
TGF-1 may also be involved in deposition of other proangiogenic
proteins in the blood vessel walls leading to neovascularization
(Spirin et al., 1999). Recently, a hierarchical relationship between
insulin-like growth factor-1 and VEGFR signaling in retinopathy was
identified. This can (partially) explain the observation in diabetic
patients who are treated with insulin that a rise in insulin-like
growth factor-1 levels is followed by VEGF-induced retinopathy (Smith
et al., 1999).
In various tumor types, inflammatory cells are present in the stromal
areas. These cells may account for the production of cytokines that
attract additional inflammatory cells but can have a proangiogenic
activity themselves as well (Chen et al., 1999b; Goede et al., 1999).
In wound healing, inflammation is followed by angiogenesis before
resolution of the wound occurs (Witte and Barbul, 1997). Similarly, in
foreign body reactions against biomaterial implants, neovascularization
and giant cell formation take place in concert. Recently, a
macrophage-derived peptide, PR39, was shown to inhibit
proteasome-mediated degradation of HIF-1. This resulted in accelerated
vasculature formation in mice (Li et al., 2000). All these observations
indicate that in a variety of conditions, angiogenesis and inflammation
exist at the same time. The differences in the expression of molecules
such as CD40 and in the composition of cellular infiltrates between,
e.g., rheumatoid arthritis lesions and solid tumors are highly
suggestive of pathology-related variations in the angiogenic and
inflammatory processes. Possibly, mechanisms driving the angiogenic
cascade are differentially regulated depending on the disease
pathology. Future studies will provide clues on whether regulatory fine
tuning of angiogenic processes under the various pathologic conditions
can be exploited therapeutically. Such an approach seems justified when
undesired side effects in physiologic angiogenesis occur during therapy
directed against pathologic angiogenesis.
C. Inhibition of Angiogenesis in Chronic Inflammation
In theory, several advantages of antiangiogenic therapy for chronic inflammatory diseases can be envisioned, similar to those of tumor growth-directed antiangiogenic strategies. First of all, suppression of blood vessel growth leads to diminished nutrient supply to the metabolically active cells present in inflamed tissue. Second, by preventing blood vessel formation, the entry route of inflammatory cells into the tissue becomes blocked. A third potential advantage of inhibiting endothelial cell activation, proliferation and vascular remodeling in chronically inflamed lesions is the inhibition of the production of endothelial cell-derived soluble factors such as MMPs and cytokines.
One of the first studies on the pharmacologic effects of angiogenesis
inhibition in chronic inflammatory disease was reported by Peacock et
al. (1992). They showed that the antiangiogenic fumagillin analog
AGM-1470 (TNP-470) could prevent and even reverse established arthritis
in rats. In combination with taxol, a microtubule inhibitor interfering
with cell mitosis, migration, chemotaxis, and intracellular transport
functions, an even greater reduction of arthritis was observed (Oliver
et al., 1994).
Recent research on atherosclerosis investigated the role of
angiogenesis (Carmeliet and Collen, 1998). In apolipoprotein
E-deficient mice, neovascularization similar to that observed in human
atherosclerotic lesions occurs. Treatment with either endostatin or
AGM-1470 for 16 weeks inhibited plaque growth by 85 and 70%,
respectively, whereas intimal smooth muscle cell content did not change
during the treatment period (Moulton et al., 1999). This study clearly demonstrated that neovascularization is involved in the promotion of
plaque formation and that antiangiogenic therapy in this disease may be
an effective strategy.
Thalidomide is a drug that has now been tried in humans for various
diseases, among which are rheumatoid arthritis and gastrointestinal ulcerations. It is believed to act primarily as an inhibitor of TNF
expression by destabilization of TNF mRNA. It may, however, also impair
angiogenesis, possibly by down-regulating endothelial integrin
expression (Marriott et al., 1999; Sands and Podolsky, 1999). In an
arthritis rat model, on the other hand, inhibited collagen induced
arthritis by mechanisms other than TNF or VEGF down-regulation (Oliver
et al., 1998). Also, thrombospondin-1 has been studied in arthritis
models. Surprisingly, this angiogenesis inhibitor augmented the
severity of the disease (Koch et al., 1998). These findings may be
explained by interference in the numerous angiogenesis-independent
functions of thrombospondin-1. Alternatively, it may reflect
disadvantageous features of antiangiogenic treatment options for
arthritic disease.
The observation that synovial blood vessels from rheumatoid arthritis
patients have an increased expression of integrin
v3 prompted Cheresh
and collaborators (Storgard et al., 1999) to study the effects of
inhibition of this integrin on arthritic disease in rabbits.
Intra-articular administration of a cyclic peptide antagonist of
v3 induced vascular
apoptosis and inhibition of synovial angiogenesis. This was paralleled
by a reduction of joint swelling, synovial infiltrate, and pannus
formation in both early and well established arthritis. Moreover, the
antagonist was able to protect against cartilage erosions.
Antiangiogenic strategies have enormous potential for clinical
application in the treatment of arthritis and other chronic inflammatory diseases accompanied by overt neovascularization (Firestein, 1999). Furthermore, application of antiangiogenic strategies will allow us to study in more detail the functional role of
angiogenesis during chronic inflammation. Initiation of the angiogenic
cascade during inflammation is most likely caused by activated and
proliferating cells that are in continuous demand of nutrients and
oxygen. It may well be that during a prolonged inflammatory response,
cells involved in the angiogenic cascade start to function
autonomously. Continuous production of cytokines involved in
angiogenesis as well as leukocyte recruitment and activation may be the
consequence. These issues need to be studied to better understand the
role of angiogenesis in the pathology of chronic inflammation and to
design tailor made drugs to interfere at specific levels of regulation
of the disease.
D. Clinical Trials with Inhibitors of Angiogenesis for Noncancerous Diseases
At present, data on the effects of drugs in patients suffering from chronic inflammatory diseases with respect to their antiangiogenic activity are limited. One study on interference with macular degeneration was announced to be initiated soon. The neovascular form of age-related macular degeneration is the leading cause of blindness and vision impairment in people aged 60 years and older. A phase II clinical trial will assess the safety of the MMP inhibitor AG3340 in approximately 100 patients aged 50 years and older affected by this disease and determine the optimal dose and regimen to use in subsequent phase III trials.
In rheumatoid arthritis patients treated with TNF--blocking
antibodies, a decrease in synovial vascularity was observed. This
effect is likely to be mediated by VEGF, because treatment with
anti-TNF- antibodies significantly decreased serum VEGF levels.
Studies with human rheumatoid arthritis synovial membrane cultures
furthermore demonstrated that a combined neutralization of TNF- and
IL-1 elicited a more pronounced reduction of VEGF production than
separate inhibition of either cytokine. Whether IL-1 also participates
in in vivo effects exerted by anti-TNF- antibodies is not clear yet
(Paleolog et al., 1998).
Thalidomide was shown to be efficacious in some patients with
refractory Crohn's disease. In an open label trial, 9 of 22 patients
with either luminal disease or fistulas achieved clinical remissions
(Ehrenpreis et al., 1999). Although at present the mechanism through
which thalidomide induced these remissions remains to be elucidated, it
is believed to act via a combination of direct inhibition of TNF
production and angiogenesis.
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V. Back to the Drawing Board |
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An enormous research effort in the last decade has resulted in important advances in our understanding of the role of angiogenesis in health and disease. Still, several issues will have to be addressed in the coming years before we can fully appreciate the options and limitations of modulation of angiogenesis as a therapeutic tool.
A. Angiogenesis Stimulation
The first results of the angiogenesis stimulation trials in
patients with ischemic disease are encouraging. However, the company that developed VEGF protein for this purpose recently announced that
they would not pursue further clinical trials with this protein. This
decision followed disappointing results with a phase II trial in 178 patients. The 60-day results of this trial showed no difference in
clinical improvement compared with placebo, and although the 120-day
follow-up was more promising, the trial was halted. Because the protein
was very effective in animal models, it has now taken back into the
laboratory to improve its effectiveness An enormous research effort in
the last decade has resulted in important advances in our understanding
of the role of angiogenesis in health and disease. Still, several
issues will have to be addressed in the coming years before we can
fully appreciate the options and limitations of modulation of
angiogenesis as a therapeutic tool. An important obstacle in this
respect is the fact that the responses of patients suffering from
ischemia and those of healthy animals with a relatively short-term
ischemic insult are likely to be incomparable (Ferrara and Alitalo,
1999).
As with angiogenesis inhibition, it may well be that the effects of angiogenesis induction also takes months to become evident. Although the clinical studies on the naked plasmid VEGF administration in myocardial ischemia patients challenges this, it is important to note that these were not placebo-controlled studies. The development of a catheter that can inject the plasmid without the need for opening the chest now allows placebo-controlled studies and proper evaluation of the effects. If indeed prolonged levels of proangiogenic factors will be required for treatment success, the effects of prolonged treatment on tumor dormancy will be an issue to reconsider. Even though it is believed that the angiogenic factors alone do not affect a tumor's angiogenic switch, this belief has been based on relatively short-term studies in animals. Markers other than VEGF or FGF-2 plasma levels used to measure the occurrence of tumor-specific angiogenesis would be enormously useful in this respect.
At present, all studies on angiogenesis induction made use of a single
proangiogenic factor. Given the complexity of the angiogenic cascade,
this may result in incompletely functioning or unstable endothelial
channels with defects in differentiation (Ferrara and Alitalo, 1999).
B. Antiangiogenic Strategies in Cancer Therapy
One of the main messages during the angiogenesis symposia at the
1999 Annual Meeting of the American Association of Cancer Research was
that "we need to go back to the drawing board". Although many
antiangiogenic therapies for treating cancer were highly active in
animal models, clinical results so far are disappointing. This may
either be a result of the most promising antiangiogenic compounds
having not been tested in the clinic yet or that the read-out systems
available for measuring clinical efficacy of antitumor drugs are not
suitable for measuring antiangiogenic effects. For a better
understanding of the pharmacologic effects of antiangiogenic therapies
in cancer patients, methods need to be developed that are able to
determine the response(s) of the body to the therapy. In the clinical
trials recently performed on angiogenesis stimulation with
phVEGF165, markers of blood flow, imaging
techniques, and simply increased limb viability nicely demonstrated
clinical improvement. For antiangiogenic therapy for tumor growth or
chronic inflammatory diseases, the read-out system is less clear. In
addition to this, biological agents need a longer period of time to
induce a response, in contrast to, e.g., cytotoxic drugs. An example of
this has been described by Folkman and colleagues (Ezekowitz et al.,
1992), who treated a child with hemangioma with IFN- for
almost a year before disease improvement was visible. Furthermore,
biological agents are likely to exert their effects in a
concentration-dependent way. At higher concentrations, antagonistic
effects or loss of effects may occur, as has been described for
TNF- and granulocyte macrophage-colony-stimulating factor, for example. A complete inventory on cytokine levels in sera/plasma of patients before and after treatment with antiangiogenic drugs would be ideal to see whether it is possible to define a set of
(surrogate) markers for antiangiogenic effects. In several tumor types,
VEGF plasma levels, for example, can be of value in predicting tumor
vascularity and disease outcome, and hence serve as a parameter of
treatment effectiveness. These levels are, however, not applicable to
all tumor types and disease stages (Salven et al., 1998; Gadducci et
al., 1999). It is likely, therefore, that tumor type-specific sets of
markers need to be defined. In this respect, it would be of enormous
help, but extremely difficult to achieve, to define markers related to
endothelial cell death and decreased endothelial cell proliferation
rate, because these are the common denominators of
antiangiogenic activity.
Of great importance is the observation by Kennel et al. (1999) that a
better antitumor response of targeted 213Bi was
observed in immunocompetent mice. Many antitumor studies, either
exploiting "conventional" chemotherapeutic drugs or drugs with a
pronounced antiangiogenic effect, are performed in immunodeficient mice. They accept human tumor grafts but lack additional antitumor mechanisms brought about by cells of the immune system. It is very
possible, that upon tumor endothelial cell inhibition/killing or blood
coagulation induction, an inflammatory response is initiated when an
intact immune system is available. In contrast to cell killing
via apoptosis, a mechanism aimed at eliminating the dying cells without
eliciting an inflammatory reaction, target cell killing via necrosis is
expected to be more effective in initiating such an inflammatory
response. These effects are underestimated in immunodeficient animal
models. In cancer patients, defects in the immune defense may exist,
and additional immunologic responses on antiangiogenic therapies may or
may not be present. To date, we are not capable of fully interpreting
the consequences of the lack of certain immunologic specificities on
the effects of antiangiogenic therapies due to lack of knowledge.
Another major drawback of the use of xenograft mouse models is the
xenogeneic interplay between the mouse vessels and the human tumor. It
is unknown to what extent mouse vessels in a human microenvironment are
more fragile than vessels in an entirely syngeneic mouse model.
An important issue, which has not been addressed to the full extent, is
the potential occurrence of side effects during antiangiogenic treatment. Recently, Ferrara and colleagues (Gerber et al., 1999) demonstrated that VEGF-driven angiogenesis is an important feature of
bone formation in the growth plate. This may have important implications for antiangiogenic therapies in the elderly when the
growth plate undergoes active remodeling. From the clinical studies
with MMP inhibitors, it was observed that at generally well tolerated
doses, in most cases musculoskeletal- and joint-related events occurred
approximately 4 weeks after the start of treatment. These events
included joint stiffness and swelling, and, limited to a few patients,
the mobility of certain joints. The side effects typically begin in a
dose- and time-dependent manner and may be explained by unintentional
inhibition of proteolytic processes of macrophages that are involved in
bone and cartilage remodeling in the joints. The side effects are
reversible and can be managed by treatment rests and subsequent dose
reduction (Wilding et al., 1998). The recent withdrawal of two MMP
inhibitors from clinical trials indicates the occurrence of toxicities
that were not obvious in animal models (Anonymous, 1999b).
Because angiogenesis is a process that occurs during many normal physiologic processes, great care should be taken to address the issue of side effects of the tumor vasculature-directed antiangiogenic therapy in patients as well.
One of the advantages of antiangiogenic therapy is believed to be the
lack of induction of resistance to therapy (Boehm et al., 1997). This
idea is based on the fact that processes exerted by endothelial cells
in angiogenesis are not a result of endothelial cell genetic
alterations in the oncogene/tumor suppressor gene activity. In other
words, the absence of drug resistance to angiogenesis inhibitors is
most likely explained by the fact that endothelial cells are
genetically stable cells that are considered not to mutate into
drug-resistant variants. Although this is a highly promising feature of
this kind of treatment, absence of resistance strongly depends on the
type of angiostatic therapy applied. If the therapy is aimed
interference at the level of tumor cell produced (growth) signals, it
can be expected that the therapy eventually will run into drug-induced
resistance due to adaptation or mutation of the tumor cells. For
example, when the approach consists of intervention at VEGF signaling,
regardless of which approach (blocking antibodies, soluble receptors,
or inhibition of signal transduction), the tumor will either switch to
dependence on other growth factors, or selection of other growth
factor-dependent cells will occur. The recent observation by Jain and
coworkers (Hansen-Algenstaedt et al., 1999), that upon long-term
blocking of VEGF activity a second wave of VEGF-independent
angiogenesis occurred, shows the importance of this concept. Next to
this phenomenon, the success of the use of angiogenesis inhibitors that
are not specific for endothelial cells, such as MMP inhibitors and the
calcium influx inhibitor CAI, may also very well be dependent on
effects on the tumor cells. In that case, mutation of tumor cells will
reduce the efficacy of the compound. Also, it may well be that the dual activity of these compounds dictates their efficacy and loss of one
effector function reduces therapeutic outcome. A therapeutic consequence of these considerations is that treatment of excessive angiogenesis with only one angiogenesis inhibitor is not an option. Moreover, one should realize that in animal models of either tumor growth or inflammatory angiogenesis, the majority of the vasculature is
in a proangiogenic state. In contrast, in human tumors the percentage
of proangiogenic vessels is variable, often quite low, and hence
antiangiogenic therapy may only affect the minority of vessels.
Furthermore, fine-tuned strategies to target specific stages of the
disease progression and hence angiogenesis were recently proposed
(Bergers et al., 1999). Such strategies require an enormous preclinical
research effort on the most potent formulations, dosing regimens, and
so on, and therefore, clinical applications of these optimized
strategies are not expected to be started soon.
The heterogeneity in angiogenic stages in human tumor vasculature makes
that antiangiogenic therapy alone is believed to be never sufficiently
effective on its own. Combining antiangiogenic therapy with
conventional, tumor cell-directed therapies seem contradictory;
blockade of the formation of new blood vessels will hamper the
availability of the chemotherapeutics in the tumor tissue.
Whereas antiangiogenic therapeutics act on vessels in hypoxic tumor
sites, chemotherapeutics would become available in sites where the
tumor vasculature is at rest. One can ask the question whether the
tumor cells in such a site are nondividing cells, requiring few
nutrients and being unresponsive to chemotherapeutics acting
on cell cycle regulatory processes. On the other hand, by lowering the
tumor mass with antiangiogenic drugs, intratumoral pressure may
concurrently drop. As a result, the availability of chemotherapeutics
can increase. Furthermore, radiotherapy had an additional antitumor
effect when combined with angiostatin in animals and hence may be
considered for combination therapies as well (Mauceri et al., 1998).
Still, one has to keep in mind that these therapies have all been
tested in animal models with a homogeneous proangiogenic tumor
vasculature make-up. Lack of proper knowledge on the mechanistic
backgrounds of this potentiating effect prohibits any prediction on
usefulness in cancer patients at this moment.
C. Antiangiogenic Strategies in Chronic Inflammation
Many of the above considerations on tumor-directed antiangiogenic
therapies seem to be applicable to chronic inflammatory diseases. The
codependence of angiogenesis and chronic inflammation (Jackson et al.,
1997) seems to justify the development of inhibitors of chronic
inflammation for specific tumor types and inhibitors of angiogenesis
for the treatment of chronic inflammation. Still, the data obtained so
far do not allow a definite conclusion about whether
inflammation-induced angiogenesis and tumor growth-induced angiogenesis
are analogous processes. For example, in animal models, thrombospondin-1 inhibited tumor-induced angiogenesis but worsened the
disease parameters in adjuvant-induced arthritis (Koch et al., 1998).
Therefore, care should be taken in just extrapolating the knowledge on
tumor angiogenesis to the situation of chronic inflammation. And
similar to anticancer therapy, knowledge on the stages of angiogenesis
during chronic inflammation in human and animal models is a
prerequisite for designing optimal treatment strategies.
Maintenance of the vascular wall integrity is a general function of
endothelial cells. If the integrity of endothelium is lost, the
integrity of the whole body will be negatively affected. Therefore,
regulatory processes in endothelial cells are such that they acquire a
phenotype that protects them from activation-induced damage, e.g.,
cytokine-induced apoptosis. With this function, the endothelium
distinguishes itself from almost all other cell types in the body
(Badrichani et al., 1999). This characteristic is believed to play a
pivotal role in endothelial cell function and possibly dysfunction
especially under proinflammatory conditions. This subject of protective
gene expression has up to now only been extensively addressed in
transplantation-related research. One can easily envision that under
chronic inflammatory conditions, both proangiogenic and proinflammatory
factors affect these specific functions of the endothelium. Moreover,
they may significantly contribute to the outcome of therapeutic
interventions aimed at proangiogenic processes in chronic inflammation.
Addressing these issues in full detail and comparing the status of the
endothelium in patient lesions and lesions in animal models with
respect to these specific characteristics will provide insight based on
which drugs can be developed.
Taking into account that inhibitors of angiogenesis may be quite toxic to normal tissues, it is worthwhile to consider the selective delivery of drugs to endothelial cells to block their proangiogenic behavior. But, in contrast to endothelial cell killing as an effector modality in tumor endothelial cell targeting, a more sophisticated approach will be required to obtain anti-inflammatory effects.
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VI. Concluding Remarks |
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Angiogenesis is an important process during normal physiology and pathologic conditions such as ischemic diseases, chronic inflammatory diseases, and tumor growth. Important advances have been made in unraveling the regulatory pathways involved in the various steps that take place during angiogenesis. The cellular players, soluble factors, and environmental conditions that are able to affect one or more of the angiogenic steps have been identified in studies referred to in this review and in many others. In the coming years, it will be a great challenge to unravel the causal versus the consequential relation between the various disease pathologies and the spatiotemporal disbalance in pro- and antiangiogenic entities in human disease in light of the development of better therapies. In this respect, the advent of novel molecular biological techniques such as the isolation of single cells from patient biopsies and subsequent profiling of gene expression with DNA array technology will be a helpful tool.
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Acknowledgments |
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This work was supported by research grants from the University Hospital Maastricht (to A.W.G.), and The Royal Netherlands Academy of Arts and Sciences KNAW (G.M.). We thank Drs. H.F.P. Hillen (Maastricht, The Netherlands), H. Kleinman (Bethesda, MD), W. Leenders (Nijmegen, The Netherlands), D.W.J. van der Schaft (Maastricht) for valuable input.
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Footnotes |
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1 Address for correspondence: Dr. A.W. Griffioen, Tumor Angiogenesis Laboratory, Dept. of Internal Medicine, University Hospital Maastricht, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands. E-mail: a.griffioen{at}intmed.unimaas.nl
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Abbreviations |
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BM, basement membrane; Ang-1, angiopoietin-1; BsAb, bispecific antibody; CAI, carboxyamidotriazole; ECM, extracellular matrix; HIF, hypoxia-inducible factor; HUVEC, human umbilical vein endothelial cells; FGF, fibroblast growth factor; FGF-R, FGF receptor; ICAM-1, intercellular adhesion molecule-1; IFN, interferon; IL, interleukin; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; MMP, matrix metalloproteinase; NFAT, nuclear factor of activated T cells; NO, nitric oxide; PDGF, platelet-derived growth factor; t-PA/u-PA, tissue type/urokinase plasminogen activator; SPARC, secreted protein acidic and rich in cysteine; TGF, transforming growth factor; TNF, tumor necrosis factor; tTF, truncated tissue factor; VCAM-1, vascular cell adhesion molecule-1; VEGF, vascular endothelial growth factor; VEGF-R, VEGF receptor; phVEGF165, plasmid-encoding human VEGF165 isoform; vWF, von Willebrand factor/factor VIII-related antigen.
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W. J. M. Mulder, D. W. J. van der Schaft, P. A. I. Hautvast, G. J. Strijkers, G. A. Koning, G. Storm, K. H. Mayo, A. W. Griffioen, and K. Nicolay Early in vivo assessment of angiostatic therapy efficacy by molecular MRI FASEB J, February 1, 2007; 21(2): 378 - 383. [Abstract] [Full Text] [PDF]
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A. Rusk, E. McKeegan, F. Haviv, S. Majest, J. Henkin, and C. Khanna Preclinical Evaluation of Antiangiogenic Thrombospondin-1 Peptide Mimetics, ABT-526 and ABT-510, in Companion Dogs with Naturally Occurring Cancers Clin. Cancer Res., December 15, 2006; 12(24): 7444 - 7455. [Abstract] [Full Text] [PDF]
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A. Rusk, E. Cozzi, M. Stebbins, D. Vail, J. Graham, V. Valli, J. Henkin, R. Sharpee, and C. Khanna Cooperative Activity of Cytotoxic Chemotherapy with Antiangiogenic Thrombospondin-I Peptides, ABT-526 in Pet Dogs with Relapsed Lymphoma Clin. Cancer Res., December 15, 2006; 12(24): 7456 - 7464. [Abstract] [Full Text] [PDF]
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D. M. Brantley-Sieders, W. B. Fang, Y. Hwang, D. Hicks, and J. Chen Ephrin-A1 Facilitates Mammary Tumor Metastasis through an Angiogenesis-Dependent Mechanism Mediated by EphA Receptor and Vascular Endothelial Growth Factor in Mice Cancer Res., November 1, 2006; 66(21): 10315 - 10324. [Abstract] [Full Text] [PDF]
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P. Sanz-Cameno, S. Martin-Vilchez, E. Lara-Pezzi, M. J. Borque, J. Salmeron, P. Munoz de Rueda, J. A. Solis, M. Lopez-Cabrera, and R. Moreno-Otero Hepatitis B Virus Promotes Angiopoietin-2 Expression in Liver Tissue: Role of HBV X Protein Am. J. Pathol., October 1, 2006; 169(4): 1215 - 1222. [Abstract] [Full Text] [PDF]
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E.-O. Lee, H.-J. Lee, H.-S. Hwang, K.-S. Ahn, C. Chae, K.-S. Kang, J. Lu, and S.-H. Kim Potent inhibition of Lewis lung cancer growth by heyneanol A from the roots of Vitis amurensis through apoptotic and anti-angiogenic activities Carcinogenesis, October 1, 2006; 27(10): 2059 - 2069. [Abstract] [Full Text] [PDF]
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J. R. van Beijnum, R. P. Dings, E. van der Linden, B. M. M. Zwaans, F. C. S. Ramaekers, K. H. Mayo, and A. W. Griffioen Gene expression of tumor angiogenesis dissected: specific targeting of colon cancer angiogenic vasculature Blood, October 1, 2006; 108(7): 2339 - 2348. [Abstract] [Full Text] [PDF]
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S. I. Zittermann and A. C. Issekutz Endothelial growth factors VEGF and bFGF differentially enhance monocyte and neutrophil recruitment to inflammation J. Leukoc. Biol., August 1, 2006; 80(2): 247 - 257. [Abstract] [Full Text] [PDF]
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A. E. M. Dirkx, M. G. A. oude Egbrink, K. Castermans, D. W. J. van der Schaft, V. L. J. L. Thijssen, R. P. M. Dings, L. Kwee, K. H. Mayo, J. Wagstaff, J. C. A. B. ter Steege, et al. Anti-angiogenesis therapy can overcome endothelial cell anergy and promote leukocyte-endothelium interactions and infiltration in tumors FASEB J, April 1, 2006; 20(6): 621 - 630. [Abstract] [Full Text] [PDF]
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S. I. Zittermann and A. C. Issekutz Basic Fibroblast Growth Factor (bFGF, FGF-2) Potentiates Leukocyte Recruitment to Inflammation by Enhancing Endothelial Adhesion Molecule Expression Am. J. Pathol., March 1, 2006; 168(3): 835 - 846. [Abstract] [Full Text] [PDF]
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L.-H. Zhang, L. Wu, H. K. Raymon, R. S. Chen, L. Corral, M. A. Shirley, R. Krishna Narla, J. Gamez, G. W. Muller, D. I. Stirling, et al. The Synthetic Compound CC-5079 Is a Potent Inhibitor of Tubulin Polymerization and Tumor Necrosis Factor-{alpha} Production with Antitumor Activity Cancer Res., January 15, 2006; 66(2): 951 - 959. [Abstract] [Full Text] [PDF]
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E.-H. Park, J. M. Lee, J. D. Blais, J. C. Bell, and J. Pelletier Internal Translation Initiation Mediated by the Angiogenic Factor Tie2 J. Biol. Chem., June 3, 2005; 280(22): 20945 - 20953. [Abstract] [Full Text] [PDF]
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S. H. Lee, D. Lopes de Menezes, J. Vora, A. Harris, H. Ye, L. Nordahl, E. Garrett, E. Samara, S. L. Aukerman, A. B. Gelb, et al. In vivo Target Modulation and Biological Activity of CHIR-258, a Multitargeted Growth Factor Receptor Kinase Inhibitor, in Colon Cancer Models Clin. Cancer Res., May 15, 2005; 11(10): 3633 - 3641. [Abstract] [Full Text] [PDF]
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H. Jiang, A. S. Weyrich, G. A. Zimmerman, and T. M. McIntyre Endothelial Cell Confluence Regulates Cyclooxygenase-2 and Prostaglandin E2 Production That Modulate Motility J. Biol. Chem., December 31, 2004; 279(53): 55905 - 55913. [Abstract] [Full Text] [PDF]
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I. Vucenik, A. Passaniti, M. I. Vitolo, K. Tantivejkul, P. Eggleton, and A. M. Shamsuddin Anti-angiogenic activity of inositol hexaphosphate (IP6) Carcinogenesis, November 1, 2004; 25(11): 2115 - 2123. [Abstract] [Full Text] [PDF]
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R. Grau, M. A. Iniguez, and M. Fresno Inhibition of Activator Protein 1 Activation, Vascular Endothelial Growth Factor, and Cyclooxygenase-2 Expression by 15-Deoxy-{Delta}12,14-Prostaglandin J2 in Colon Carcinoma Cells: Evidence for a Redox-Sensitive Peroxisome Proliferator-Activated Receptor-{gamma}-Independent Mechanism Cancer Res., August 1, 2004; 64(15): 5162 - 5171. [Abstract] [Full Text] [PDF]
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Y. Ikeda, I. Hayashi, E. Kamoshita, A. Yamazaki, H. Endo, K. Ishihara, S. Yamashina, Y. Tsutsumi, H. Matsubara, and M. Majima Host Stromal Bradykinin B2 Receptor Signaling Facilitates Tumor-Associated Angiogenesis and Tumor Growth Cancer Res., August 1, 2004; 64(15): 5178 - 5185. [Abstract] [Full Text] [PDF]
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A Scott, K M Khan, C R Roberts, J L Cook, and V Duronio What do we mean by the term "inflammation"? A contemporary basic science update for sports medicine Br. J. Sports Med., June 1, 2004; 38(3): 372 - 380. [Abstract] [Full Text] [PDF]
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J. Dixelius, L. Jakobsson, E. Genersch, S. Bohman, P. Ekblom, and L. Claesson-Welsh Laminin-1 Promotes Angiogenesis in Synergy with Fibroblast Growth Factor by Distinct Regulation of the Gene and Protein Expression Profile in Endothelial Cells J. Biol. Chem., May 28, 2004; 279(22): 23766 - 23772. [Abstract] [Full Text] [PDF]
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J Varayoud, J G Ramos, V L Bosquiazzo, M Munoz-de-Toro, and E H Luque Mast cells degranulation affects angiogenesis in the rat uterine cervix during pregnancy Reproduction, March 1, 2004; 127(3): 379 - 387. [Abstract] [Full Text] [PDF]
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C.-R. Yang, S.-L. Hsieh, C.-M. Teng, F.-M. Ho, W.-L. Su, and W.-W. Lin Soluble Decoy Receptor 3 Induces Angiogenesis by Neutralization of TL1A, a Cytokine Belonging to Tumor Necrosis Factor Superfamily and Exhibiting Angiostatic Action Cancer Res., February 1, 2004; 64(3): 1122 - 1129. [Abstract] [Full Text] [PDF]
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S. Sengupta, L. A. Sellers, T. Cindrova, J. Skepper, E. Gherardi, R. Sasisekharan, and T.-P. D. Fan Cyclooxygenase-2-selective Nonsteroidal Anti-Inflammatory Drugs Inhibit Hepatocyte Growth Factor/Scatter Factor-induced Angiogenesis Cancer Res., December 1, 2003; 63(23): 8351 - 8359. [Abstract] [Full Text] [PDF]
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Q. G. de Lussanet, W. H. Backes, A. W. Griffioen, J. M. A. van Engelshoven, and R. G. H. Beets-Tan Gadopentetate Dimeglumine versus Ultrasmall Superparamagnetic Iron Oxide for Dynamic Contrast-enhanced MR Imaging of Tumor Angiogenesis in Human Colon Carcinoma in Mice Radiology, November 1, 2003; 229(2): 429 - 438. [Abstract] [Full Text] [PDF]
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P. M. Winter, S. D. Caruthers, A. Kassner, T. D. Harris, L. K. Chinen, J. S. Allen, E. K. Lacy, H. Zhang, J. D. Robertson, S. A. Wickline, et al. Molecular Imaging of Angiogenesis in Nascent Vx-2 Rabbit Tumors Using a Novel {alpha}{nu}{beta}3-targeted Nanoparticle and 1.5 Tesla Magnetic Resonance Imaging Cancer Res., September 15, 2003; 63(18): 5838 - 5843. [Abstract] [Full Text] [PDF]
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B. Ruggeri, J. Singh, D. Gingrich, T. Angeles, M. Albom, H. Chang, C. Robinson, K. Hunter, P. Dobrzanski, S. Jones-Bolin, et al. CEP-7055: A Novel, Orally Active Pan Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases with Potent Antiangiogenic Activity and Antitumor Efficacy in Preclinical Models Cancer Res., September 15, 2003; 63(18): 5978 - 5991. [Abstract] [Full Text] [PDF]
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T. Ishida, R. K. Kundu, E. Yang, K.-i. Hirata, Y.-D. Ho, and T. Quertermous Targeted Disruption of Endothelial Cell-selective Adhesion Molecule Inhibits Angiogenic Processes in Vitro and in Vivo J. Biol. Chem., September 5, 2003; 278(36): 34598 - 34604. [Abstract] [Full Text] [PDF]
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I. Pollet, C. J. Opina, C. Zimmerman, K. G. Leong, F. Wong, and A. Karsan Bacterial lipopolysaccharide directly induces angiogenesis through TRAF6-mediated activation of NF-{kappa}B and c-Jun N-terminal kinase Blood, September 1, 2003; 102(5): 1740 - 1742. [Abstract] [Full Text] [PDF]
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L. Favot, S. Martin, T. Keravis, R. Andriantsitohaina, and C. Lugnier Involvement of cyclin-dependent pathway in the inhibitory effect of delphinidin on angiogenesis Cardiovasc Res, August 1, 2003; 59(2): 479 - 487. [Abstract] [Full Text] [PDF]
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A. E. M. Dirkx, M. G. A. oude Egbrink, M. J. E. Kuijpers, S. T. van der Niet, V. V. T. Heijnen, J. C. A. B.-t. Steege, J. Wagstaff, and A. W. Griffioen Tumor Angiogenesis Modulates Leukocyte-Vessel Wall Interactions in Vivo by Reducing Endothelial Adhesion Molecule Expression Cancer Res., May 1, 2003; 63(9): 2322 - 2329. [Abstract] [Full Text] [PDF]
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Y. Qian, J. Luo, S. S. Leonard, G. K. Harris, L. Millecchia, D. C. Flynn, and X. Shi Hydrogen Peroxide Formation and Actin Filament Reorganization by Cdc42 Are Essential for Ethanol-induced in Vitro Angiogenesis J. Biol. Chem., April 25, 2003; 278(18): 16189 - 16197. [Abstract] [Full Text] [PDF]
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S. Sengupta, E. Gherardi, L. A. Sellers, J. M. Wood, R. Sasisekharan, and T.-P. D. Fan Hepatocyte Growth Factor/Scatter Factor Can Induce Angiogenesis Independently of Vascular Endothelial Growth Factor Arterioscler. Thromb. Vasc. Biol., January 1, 2003; 23(1): 69 - 75. [Abstract] [Full Text] [PDF]
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T. Tarui, M. Majumdar, L. A. Miles, W. Ruf, and Y. Takada Plasmin-induced Migration of Endothelial Cells. A POTENTIAL TARGET FOR THE ANTI-ANGIOGENIC ACTION OF ANGIOSTATIN J. Biol. Chem., September 6, 2002; 277(37): 33564 - 33570. [Abstract] [Full Text] [PDF]
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M. Trikha, Z. Zhou, J. Timar, E. Raso, M. Kennel, E. Emmell, and M. T. Nakada Multiple Roles for Platelet GPIIb/IIIa and {alpha}v{beta}3 Integrins in Tumor Growth, Angiogenesis, and Metastasis Cancer Res., May 1, 2002; 62(10): 2824 - 2833. [Abstract] [Full Text] [PDF]
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B. A. Ruggeri, C. Robinson, T. Angeles, J. Wilkinson IV, and M. L. Clapper The Chemopreventive Agent Oltipraz Possesses Potent Antiangiogenic Activity in Vitro, ex Vivo, and in Vivo and Inhibits Tumor Xenograft Growth Clin. Cancer Res., January 1, 2002; 8(1): 267 - 274. [Abstract] [Full Text] [PDF]
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A. Otani, B. M. Slike, M. I. Dorrell, J. Hood, K. Kinder, K. L. Ewalt, D. Cheresh, P. Schimmel, and M. Friedlander A fragment of human TrpRS as a potent antagonist of ocular angiogenesis PNAS, January 1, 2002; (2002) 12601899. [Abstract] [Full Text] [PDF]
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D. GOUKASSIAN, A. DIEZ-JUAN, T. ASAHARA, P. SCHRATZBERGER, M. SILVER, T. MURAYAMA, J. M. ISNER, and V. ANDRES Overexpression of p27Kip1 by doxycycline-regulated adenoviral vectors inhibits endothelial cell proliferation and migration and impairs angiogenesis FASEB J, September 1, 2001; 15(11): 1877 - 1885. [Abstract] [Full Text] [PDF]
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E. Pillebout, M. Burtin, H. T. Yuan, P. Briand, A. S. Woolf, G. Friedlander, and F. Terzi Proliferation and Remodeling of the Peritubular Microcirculation after Nephron Reduction : Association with the Progression of Renal Lesions Am. J. Pathol., August 1, 2001; 159(2): 547 - 560. [Abstract] [Full Text]
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M. S. Pepper Role of the Matrix Metalloproteinase and Plasminogen Activator-Plasmin Systems in Angiogenesis Arterioscler. Thromb. Vasc. Biol., July 1, 2001; 21(7): 1104 - 1117. [Abstract] [Full Text] [PDF]
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C.-H. Yeh, H.-C. Peng, R.-S. Yang, and T.-F. Huang Rhodostomin, A Snake Venom Disintegrin, Inhibits Angiogenesis Elicited by Basic Fibroblast Growth Factor and Suppresses Tumor Growth by A Selective alpha vbeta 3 Blockade of Endothelial Cells Mol. Pharmacol., April 16, 2001; 59(5): 1333 - 1342. [Abstract] [Full Text]
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C. D. Kim, H. H. Kim, Y. K. Kim, Y. K. Kwak, S.-O. Kim, S.-E. Yoo, and K. W. Hong Antiangiogenic Effect of KR31372 in Rat Sponge Implant Model J. Pharmacol. Exp. Ther., March 1, 2001; 296(3): 1085 - 1090. [Abstract] [Full Text]
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E. Middleton Jr., C. Kandaswami, and T. C. Theoharides The Effects of Plant Flavonoids on Mammalian Cells:Implications for Inflammation, Heart Disease, and Cancer Pharmacol. Rev., December 1, 2000; 52(4): 673 - 751. [Abstract] [Full Text] [PDF]
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R. C. Savani, G. Cao, P. M. Pooler, A. Zaman, Z. Zhou, and H. M. DeLisser Differential Involvement of the Hyaluronan (HA) Receptors CD44 and Receptor for HA-mediated Motility in Endothelial Cell Function and Angiogenesis J. Biol. Chem., September 21, 2001; 276(39): 36770 - 36778. [Abstract] [Full Text] [PDF]
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L. C. Boujaoude, C. Bradshaw-Wilder, C. Mao, J. Cohn, B. Ogretmen, Y. A. Hannun, and L. M. Obeid Cystic Fibrosis Transmembrane Regulator Regulates Uptake of Sphingoid Base Phosphates and Lysophosphatidic Acid. MODULATION OF CELLULAR ACTIVITY OF SPHINGOSINE 1-PHOSPHATE J. Biol. Chem., September 14, 2001; 276(38): 35258 - 35264. [Abstract] [Full Text] [PDF]
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A. Otani, B. M. Slike, M. I. Dorrell, J. Hood, K. Kinder, K. L. Ewalt, D. Cheresh, P. Schimmel, and M. Friedlander A fragment of human TrpRS as a potent antagonist of ocular angiogenesis PNAS, January 8, 2002; 99(1): 178 - 183. [Abstract] [Full Text] [PDF]
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, phospholipase C-
