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Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111
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ABSTRACT |
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 TOP
 ABSTRACT
 NORMAL ANGIOGENESIS
TUMOR-INDUCED ANGIOGENESIS
SUMMARY
REFERENCES
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Often those diseases most evasive to therapeutic intervention usurp the human body's own cellular machinery or deregulate normal physiological processes for propagation. Tumor-induced angiogenesis is a pathological condition that results from aberrant deployment of normal angiogenesis, an essential process in which the vascular tree is remodeled by the growth of new capillaries from preexisting vessels. Normal angiogenesis ensures that developing or healing tissues receive an adequate supply of nutrients. Within the confines of a tumor, the availability of nutrients is limited by competition among actively proliferating cells, and diffusion of metabolites is impeded by high interstitial pressure (Jain RK. Cancer Res 47: 3039-3051, 1987). As a result, tumor cells induce the formation of a new blood supply from the preexisting vasculature, and this affords tumor cells the ability to survive and propagate in a hostile environment. Because both normal and tumor-induced neovascularization fulfill the essential role of satisfying the metabolic demands of a tissue, the mechanisms by which cancer cells stimulate pathological neovascularization mimic those utilized by normal cells to foster physiological angiogenesis. This review investigates mechanisms of tumor-induced angiogenesis. The strategies used by cancer cells to develop their own blood supply are discussed in relation to those employed by normal cells during physiological angiogenesis. With an understanding of blood vessel growth in both normal and abnormal settings, we are better suited to design effective therapeutics for cancer.
blood vessel; growth; cancer; endothelium; pericyte
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NORMAL ANGIOGENESIS |
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TOP
ABSTRACT
NORMAL ANGIOGENESIS
TUMOR-INDUCED ANGIOGENESIS
SUMMARY
REFERENCES
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The adult vasculature is derived from a network of blood vessels that is initially created in the embryo by vasculogenesis, a process whereby vessels are formed de novo from endothelial cell precursors termed angioblasts (202). During vasculogenesis, angioblasts proliferate and coalesce into a primitive network of vessels known as the primary capillary plexus. The endothelial cell lattice created by vasculogenesis then serves as a scaffold for angiogenesis.
After the primary capillary plexus is formed, it is remodeled by the sprouting and branching of new vessels from preexisting ones in the process of angiogenesis. Most normal angiogenesis occurs in the embryo, where it establishes the primary vascular tree as well as an adequate vasculature for growing and developing organs (73). Angiogenesis occurs in the adult during the ovarian cycle and in physiological repair processes such as wound healing (123). However, very little turnover of endothelial cells occurs in the adult vasculature (48).
Maturation and remodeling of newly formed microvessels is accomplished
by the coordination of several diverse processes in the
microvasculature (124) that are summarized in Fig.
1. For new blood vessel sprouts to form,
mural cells (pericytes) must first be removed from the branching
vessel. Endothelial cell basement membrane and extracellular matrix are
then degraded and remodeled by specific proteases such as matrix
metalloproteinases (161), and new matrix synthesized by
stromal cells is then laid down. This new matrix, coupled with soluble
growth factors, fosters the migration and proliferation of endothelial
cells. After sufficient endothelial cell division has occurred,
endothelial cells arrest in a monolayer and form a tubelike structure.
Mural cells (pericytes in the microvasculature, smooth muscle cells in
larger vessels) are recruited to the abluminal surface of the
endothelium, and vessels uncovered by pericytes regress. Blood flow is
then established in the new vessel.
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Under normal circumstances, angiogenesis is a highly ordered process
under tight regulation, because it requires inducing quiescent
endothelial cells in a monolayer to divide and spread the vascular
network only to the extent demanded by the demands of growing tissues.
Many positively and negatively acting factors influence angiogenesis;
among these are soluble polypeptides, cell-cell and cell-matrix
interactions, and hemodynamic effects. The soluble growth factors,
membrane-bound molecules, and mechanical forces that mediate these
signals are summarized in Table
1 and are discussed
below in terms of their contribution to the mechanism of normal
angiogenesis.
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Soluble Factors
Vascular endothelial growth factor. Perhaps the most well-characterized angiogenic factor is vascular endothelial growth factor (VEGF). Alternative splicing of a single gene generates six isoforms of VEGF composed of 121, 145, 165, 183, 189, and 206 amino acids, although VEGF165 is the most commonly expressed isoform (59, 205). Interestingly, although all isoforms demonstrate identical biological activities, VEGF121 and VEGF165 are secreted into the extracellular environment whereas VEGF189 and VEGF206, and to some extent VEGF165, remain cell- or matrix-associated via their affinity for heparan sulfates (108). VEGF is a highly conserved disulfide-bonded dimeric glycoprotein, with a molecular mass of 34-45 kDa, that loses biological activity in the presence of reducing agents (40).
A wide variety of human and animal tissues express low levels of VEGF, but high levels are produced where angiogenesis is required such as in fetal tissue, the placenta, and the corpus luteum as well as in a vast majority of human tumors (59). Many mesenchymal and stromal cells produce VEGF (70). VEGF binds to at least three known tyrosine kinase receptors: Flt-1 (VEGFR-1) (47), KDR/Flk-1 (VEGFR-2) (235), and Flt-4 (VEGFR-3) (174). Functional VEGF receptors were originally characterized as endothelial cell specific (174), but they have recently been found on other normal cell types including vascular smooth muscle cells and monocytes/macrophages (113, 192). Therefore, VEGF mediates several actions derived from varied sources and may utilize endothelial as well as other cell types as effectors. VEGF receptors belong to the "7-Ig," or flt, gene family characterized by seven extracellular immunoglobulin-like domains, one membrane-spanning segment, and a conserved intracellular tyrosine kinase domain (192, 210). VEGFR-1 has the highest affinity for VEGF (Kd = 10-30 pM) (15), and it is expressed in the endothelium of adult and embryonic mice as well as in healing skin wounds (185). VEGFR-1 also is expressed on vascular smooth muscle cells (113) and monocytes (111). Interestingly, no direct migratory, proliferative, or cytoskeletal effects appear to be mediated by VEGFR-1 (255). Nonetheless, VEGFR-2, a tyrosine kinase with lower affinity (Kd
75-760 pM) for VEGF than VEGFR-1, mediates endothelial cell mitogenesis, chemotaxis, and shape changes (255).
VEGFR-2 is expressed on endothelial and hematopoietic precursors
(61) as well as on proliferating endothelial cells in the
embryo (156), but in quiescent endothelium of the adult
vasculature, VEGFR-2 RNA is dramatically reduced
(156). A third receptor tyrosine kinase, VEGFR-3, is
mainly expressed adult lymphatic endothelium and may be involved in
lymphangiogenesis (114). VEGFR-3 does not bind VEGF
but, rather, complexes VEGF-related proteins VEGF-C and VEGF-D
(see below).
A VEGF receptor distinct from flt family members is
neuropilin. Neuropilin-1 is a neuronal receptor for members of the
collapsing/semaphoring family (95, 129). However,
neuropilin-1 also is expressed on normal endothelial cells
(221). Neuropilin-1 binds VEGF165, but not VEGF121, fosters its own binding to VEGFR-2, and
enhances its own chemotactic effects (221).
Neuropilin-1 may be involved in angiogenesis, because in transgenic
mice, neuropilin-1-overexpressing mice have a high density of dilated
blood vessels and die at embryonic day 17.5 (122).
Furthermore, neuropilin-1-deficient mice exhibit disrupted blood
vessels and insufficient development of vascular networks
(115).
VEGF exerts several effects on vascular endothelial cells. It was
initially isolated from tumor cell-conditioned medium as a protein that
increased the permeability of small blood vessels to circulating
metabolites (212). VEGF may increase endothelial cell
permeability by enhancing the activity of vesicular-vacuolar organelles, clustered vesicles in endothelial cells lining small vessels that facilitate transport of metabolites between luminal and
abluminal plasma membranes (128). Alternatively, VEGF may enhance permeability by loosening adherens junctions between
endothelial cells in a monolayer via rearrangement of cadherin/catenin
complexes (64, 117). Increased vascular permeability may
allow for the extravasation of plasma proteins and formation of
extracellular matrix favorable to endothelial and stromal cell
migration (58). In addition, VEGF stimulates endothelial
cell the production of plasminogen activators (uPA and tPA)
(182), plasminogen activator inhibitor-1 (PAI-1)
(182), and interstitial collagenase (247). Therefore, VEGF induces a balanced system of proteolysis that can
remodel extracellular matrix components necessary for angiogenesis.
Many laboratories have described the ability of VEGF to stimulate
endothelial cell proliferation in vitro (41, 67, 91). This
effect is specific to vascular endothelial cells, because VEGF does not
induce proliferation of other cell types such as smooth muscle cells,
corneal endothelial cells, lens epithelial cells, fibroblasts, and
adrenal cortex cells (67, 91). VEGF also enhances
endothelial cell migration in vitro (52), an initial step
in the branching of endothelium from the preexisting vasculature. Interestingly, VEGF inhibits endothelial cell apoptosis
(87) and thus acts as a survival factor. Thus VEGF
demonstrates many effects on endothelial cells.
Many experiments also implicate VEGF in angiogenesis in vivo. In the
cornea and healing bone grafts, VEGF induces growth of capillary
sprouts from preexisting blood vessels (41). Mice deficient in the gene for VEGF and the VEGF receptor Flk-1 are virtually devoid of vascular structures and are thus defective in the
very early events in blood vessel formation that characterize vasculogenesis (68, 216). However, Flt-1 (VEGFR-1)-null
mice form vascular structures but are impaired in the assembly of
vessels (79). Therefore, Flt-1 appears to have a role in
vascular remodeling in angiogenesis rather than creation of blood
vessels de novo in vasculogenesis. Interestingly, these results suggest
that Flk-1 and Flt-1 effect different signaling cascades upon VEGF
binding. The molecular nature of these differences, however, remains unclear.
VEGF production is regulated by local oxygen concentration
(219). Hypoxia stimulates VEGF production through the
binding of hypoxia-inducible factor (HIF) to cis elements in
the VEGF promoter, and HIF increases VEGF gene transcription and mRNA
stability (120). Therefore, not only does VEGF stimulate
the entire angiogenic process through its various effects on
endothelial cells, but it also acts to stimulate angiogenesis upon
demand of low oxygen concentration. Although this mechanism ensures
that developing tissues and hypoxic environments become vascularized
and oxygenated, as described below, it also underlies pathologies
associated with angiogenesis.
Interestingly, VEGF is one member of a family of growth factors that
share significant amino acid homology. Platelet-derived growth factor
(PDGF) shares between 18 and 24% total amino acid homology (116,
242) and eight conserved cysteine residues (242) with VEGF. Though these conserved cysteine residues suggest a common
mode of intra- and interchain disulfide bond formation (254), PDGF and VEGF bind distinct receptors.
Other VEGF-related molecules bind VEGF receptors. For example, placenta
growth factor (PlGF), which shares 53% amino acid identity with the
PDGF-like region in VEGF (144), binds VEGFR-1 (177) and neuropilin-1 (154). Under normal
conditions, PlGF is preferentially expressed in placenta
(145). Interestingly, embryonic angiogenesis is unaffected
in PlGF-deficient mice (33). However, PlGF may synergize
with VEGF by forming PlGF/VEGF heterodimers (27, 53) or by
potentiating VEGF signaling (177).
VEGF-B is a growth factor that shares ~43% amino acid sequence
identity with VEGF164 and 30% identity with PlGF
(169). It is expressed predominantly in embryonic and
adult muscle tissues and to a lesser extent in many other tissues such
as brain, lung, and kidney (169). VEGF-B binds and
activates VEGFR-1 as well as neuropilin-1 (148). Because
mice deficient in VEGF-B are overtly normal and exhibit only minor
cardiac defects (2, 17), VEGF-B does not appear necessary
for angiogenesis. Nonetheless, VEGF-B is mitogenic for endothelial
cells (169) and, similarly to PlGF, may cooperate with
VEGF through its ability to form heterodimers with VEGF (2,
170).
Other VEGF-related molecules share less homology with VEGF. VEGF-C and
VEGF-D form a subfamily of their own based on their structural
similarity (205). VEGF-C (136) and VEGF-D
(3), respectively, share 32 and 31% identity with
VEGF121 and VEGF165. Interestingly, both bind
and activate VEGFR-2 and VEGFR-3 and are mitogenic for endothelial
cells in vitro (3, 136). This mitogenic activity, however,
is significantly less potent (5- to 100-fold) than that induced by VEGF
(3, 136). Both VEGF-C (29, 184) and VEGF-D
(150) stimulate angiogenesis in vitro and in vivo.
Nonetheless, localization of VEGF-C and its preferred receptor,
VEGFR-3, suggest that VEGF-C may play a paracrine role in angiogenesis
of lymphatic vessels during development (131) and
maintenance of differentiated lymphatic endothelium in the adult
(114). VEGF-D is induced by c-fos
(171), and its high expression in embryonic lung suggests
a role in lung development (65). Physiological roles of
VEGF-C and VEGF-D are, however, still largely undefined.
Finally, VEGF-E refers to a group of VEGF-related proteins encoded by
the orf virus, a parapoxvirus that infects sheep, goats, and
occasionally humans, that share between 16 and 27% amino acid identity
to mammalian VEGF (143). Interestingly, these viral proteins have retained VEGF function, because they signal through VEGFR-2 and stimulate angiogenesis in vitro and in vivo
(153). Furthermore, orf virus lesions exhibit
dermal vascular endothelial proliferation and dilation
(143). VEGF-E may be a product of genetic drift from a
VEGF gene acquired by the orf virus from a mammalian host
(143). Use of both VEGF and the actions of a VEGF-related
protein for propagation and growth in organisms as distantly related as
mammals and viruses underlies the functional importance and versatility
of VEGF in fostering processes inherent for survival.
Angiopoietins and tie receptors. The angiopoietins belong to a family of secreted proteins, four of which have been identified to date, that bind Tie family receptors. Angiopoietins and Tie receptors have been reported to play a major role in angiogenesis. Tie receptors were discovered before angiopoietins in attempts to characterize novel tyrosine kinases in endothelium and heart tissue (56, 179).
TIE RECEPTORS. Expression patterns of the two Tie receptors identified so far, Tie1 and Tie2 (Tek), mimic those of VEGF receptors and appear to be specific for vascular endothelium (261), although cells in the hematopoietic cell lineage such as the tumor cell line K562 (178) also express Tie receptors. Tie1 mRNA is robustly expressed in embryonic angioblasts (endothelial cell precursors), vascular endothelium, and endocardium, whereas in adult tissues Tie1 mRNA is expressed weakly in endocardium but strongly in lung capillaries (130). Tie2 mRNA shows a similar embryonic localization but is detected earlier (day 7.5) than Tie1 (day 8.5), and it is expressed weakly in adult endocardium and vasculature endothelium (56). Therefore, expression patterns of both Tie receptors suggest a role in developmental angiogenesis. Genetic studies also implicate the Tie receptors in angiogenesis. Tie1-deficient mice develop extensive edema and hemorrhage and die either perinatally (209) or at embryonic day 14.5 (190). Although blood vessels are established in these mice, vascular integrity is severely compromised, suggesting that Tie1 is not necessary for endothelial cell differentiation in vasculogenesis but, rather, for integrity and survival of endothelial cells during angiogenesis (190). Tie2-deficient mice die embryonically and exhibit a reduction in the number of endothelial cells in blood vessels compared with wild-type littermates, underdeveloped hearts, vasodilation, and abnormal vascular network formation including lack of sprouting and branching vessels (57, 209). Therefore, whereas these studies suggest that both Tie1 and Tie2 are important for vascular integrity, they imply that Tie2 is crucial for sprouting and branching of vessels characteristic of angiogenesis. ANGIOPOIETINS. The angiopoietins are ~70-kDa secreted ligands for Tie2. A ligand for Tie1 has not yet been identified. mRNA for angiopoietin-1 (Ang1), the most extensively characterized member of this family, is found at embryonic days 9-11 in heart myocardium surrounding the endocardium and later in mesenchyme surrounding blood vessels (46). Although human neuroepithelioma and mouse myoblast cell lines are sources of Ang1, in situ localization studies suggest that mesenchymal cells closely associated with endothelium produce Ang1 (124). Interestingly, Ang1 does not induce endothelial cell proliferation or tube formation in vitro (46), but it does stimulate sprout formation from confluent endothelial cells cultured on microcarrier beads and embedded in three-dimensional fibrin gels (125). Accordingly, mice deficient in Ang1 exhibit many defects similar to those seen in Tie2-deficient mice: a grossly normal primary vasculature develops, but the mice die at embryonic day 12.5 because of incomplete vascular remodeling (228). The most severe defects are in the heart, where endocardial and trabecular development is notably impaired. Furthermore, branching of the vascular network and organization into large and small vessels is defective, blood vessels are dilated, and periendothelial cells are absent from underdeveloped tissue folds that are thought to be involved in normal vessel branching. When overexpressed in transgenic mice, Ang1 induces blood vessels that are more numerous, more highly branched, and larger in diameter than those in wild-type mice (229). In addition, overexpression of Ang1 causes blood vessels to be resistant to leakage induced by inflammatory agents or coexpressed VEGF (241). Hence, the vessel branching and remodeling stimulated by Ang1 signaling appears to be mechanistically related to its ability to increase the girth and stability of endothelium in newly formed angiogenic sprouts. Another angiopoietin family member, angiopoietin-2 (Ang2), was first characterized as a structural homolog of Ang1 that bound Tie2 and antagonized Ang1 (147). However, even though Ang2 blocks Ang1-mediated Tie2 autophosphorylation in endothelial cells, which express endogenous Tie2, Ang2 stimulates Tie2 autophosphorylation in NIH/3T3 cells ectopically expressing Tie2 (147). Thus Ang2 may be an Ang1 antagonist only in the context of the vasculature. Ang2 mRNA is expressed embryonically in the dorsal aorta and in punctate regions of the vasculature, and in adult tissues it is expressed in the placenta, ovary, and uterus, primary sites for angiogenesis in the adult (147). Furthermore, overexpression of Ang2 in vascular structures results in embryonic lethality with similar, yet more severe, defects (such as endothelial discontinuities) as those observed in mice lacking Ang1 or Tie2 (147). Therefore, Ang2 antagonizes Ang1 in the vasculature in vivo and may act as a check on Ang1/Tie2-mediated angiogenesis to prevent excessive branching and sprouting of blood vessels by promoting destabilization of blood vessels. In addition, vessel destabilization induced by Ang2 may allow angiogenic sprouts to be plastic and sensitive to remodeling factors. The angiogenic mechanism established by stimulation from VEGF and Ang1 and inhibition from Ang2 thus plays a major role in the regulation of normal blood vessel remodeling.Fibroblast growth factor. Basic [isoelectric point (pI) = 9.6] and acidic (pI = 5) fibroblast growth factors (bFGF and aFGF, respectively) are ubiquitously expressed 18- to 25-kDa polypeptides that are members of a large family of structurally related growth regulators (238) and have been thought to play a role in normal angiogenesis. In fact, aFGF was the first growth factor to be associated with angiogenesis. Like VEGF, both bFGF and aFGF induce processes in endothelial cells in vitro that are critical to angiogenesis. FGFs stimulate endothelial cell proliferation (90) and migration (236) as well as endothelial cell production of plasminogen activator and collagenase (158). In addition, bFGF causes endothelial cells to form tubelike structures in three-dimensional collagen matrices (160). Thus FGFs appear to induce many processes involved in angiogenesis. Nevertheless, unlike VEGF, which is mitogenic primarily for endothelial cells, FGF stimulates proliferation of most, if not all, cells derived from embryonic mesoderm and neuroectoderm, including pericytes, fibroblasts, myoblasts, chondrocytes, and osteoblasts (238).
Perhaps the most convincing evidence for a role of FGFs in angiogenesis is the fact that, like VEGF, FGFs induce sprouting of preexisting blood vessels toward an implanted bolus in vivo in the cornea and chick chorioallantoic membrane (CAM) (77, 140). Nevertheless, FGFs do not appear to play a major role in angiogenesis in vivo because vascular development is normal in mice deficient in both aFGF and bFGF (159). A clue to the way in which FGFs are delivered and signal to cells in vivo comes from the observations that aFGF and bFGF lack a signal sequence and therefore are not secreted proteins. Most FGF remains cytoplasmic or is bound to the extracellular matrix (76, 96) because of an intrinsic affinity for heparin. Thus FGF may be released upon cell disruption by an injury and might have a role in local reparative angiogenesis following tissue injury where it is deposited in the extracellular matrix. Indeed, mice deficient in FGFs display mild defects in wound healing (159). Therefore, bFGF does not appear to play a general role in all angiogenic responses but, rather, may be necessary for blood vessel remodeling associated with tissue repair.Platelet-derived growth factor.
As its name suggests, PDGF was originally purified from platelets;
however, it has since been found in many other cell types including
fibroblasts, keratinocytes, myoblasts, astrocytes, epithelial cells,
and macrophages (for review see Ref. 97). PDGFs exist as
45-kDa homodimers (PDGF-AA or -BB) or heterodimers (PDGF-AB) composed
of PDGF chains A and B. Most cells express both PDGF A and B, although
a few express only one isoform. PDGF receptors are also dimeric in
nature; they are made up of complexes between 
- and 
-subtypes
(97); receptor can bind both PDGF A and B chains,
whereas receptor can bind only PDGF B. Therefore, of the three
types of receptors (, , and ) only one () can
bind all three PDGF isoforms. PDGF receptor expression follows a
pattern similar to that of PDGF; however, a majority of cell types
express only one isoform ( or ).
receptor and are stimulated by PDGF-BB not only
to increase DNA synthesis (11, 14) but also to form
angiogenic chords and sprouts in vitro (14, 164). Although endothelial cells produce PDGF-BB, an autocrine feedback loop for PDGF
in endothelial cells is unlikely because little convincing evidence
exists regarding coexpression of PDGF-BB and PDGF- receptor in
endothelial cells (14, 15). PDGF also stimulates the
proliferation of cultured smooth muscle cells and pericytes (60), both of which have been shown to express PDGF-
receptor (97). In addition, PDGF may contribute indirectly
to cardiac angiogenesis, because PDGF-AB induces von Willebrand factor
as well as VEGF and VEGF-R2 in cardiac microvascular endothelial cells
in vitro (60).
PDGF also has been shown to be important for angiogenesis in vivo.
Although mice deficient in PDGF-B or PDGF- receptor develop blood
vessels that appear normal by gross inspection, they die perinatally
from hemorrhage and edema and lack mesangial cells, the counterparts of
pericytes in the kidney (138, 223). Closer inspection
indicated that a lack of pericytes (PDGF- receptor-positive mural
cells) in the microvasculature of these mutant mice is responsible for
capillary dilation and leakiness (139). Interestingly,
PDGF- receptor-positive mural cells are found around arteries in
these mutant mice. Further study indicated that pericytes are initially recruited to microvessels independent of PDGF but that proliferation and migration of pericytes along angiogenic sprouts is mediated by PDGF
(98). However, because pericytes were localized indirectly by PDGF receptor (139) as well as desmin and smooth muscle
actin (98) staining in these studies, accurate delineation
of microvascular pericytes in control and mutant mice remains
questionable. Nevertheless, electron microscopic analysis of
PDGF-B-deficient mouse brain capillaries (139) clearly
shows the absence of pericytes and dilated vessel lumen. Therefore,
PDGF may play a role in recruitment of pericytes to preformed
capillaries or in inducing the proliferation of pericytes previously
recruited by a PDGF-independent mechanism, and it thus helps to
maintain capillary wall stability.
Transforming growth factor-.
The transforming growth factor-s (TGF-) represent a family of
highly conserved 25-kDa disulfide-linked homodimeric cytokines typified
by TGF-1 (151). Before secretion from the cell,
cleavage by a furin peptidase generates a COOH-terminal 112-amino acid peptide that noncovalently associates with the NH2-terminal
pro region (called latency-associated peptide, or LAP) and dimerizes to
form mature TGF- (187). Secreted TGF- cannot bind
TGF- receptors and is biologically inactive; the latent complex is activated by proteases such as plasmin and cathepsin D, low pH, chaotropic agents such as urea, and heat (134, 142).
Exposure to low pH or protease cleavage most likely activates latent
TGF- in vivo.
is expressed by a wide variety of normal and transformed cells,
and TGF- receptors are broadly expressed in virtually all mammalian
and avian cells (50, 151). Like bFGF, TGF- is found in
extracellular matrix of many tissues (240). In the
microvasculature, both endothelial cells and pericytes produce TGF-
(7, 208) and possess TGF- receptors. Therefore, TGF-
exerts its effects in many cell types, including those comprising the vasculature.
TGF- was originally characterized by its ability to support
anchorage-independent growth of fibroblasts (203) and has
since been associated with a variety of functions in several different cell types. It can stimulate or inhibit cell proliferation; control cell adhesion by regulating production of extracellular matrix, protease inhibitors, and integrins; and induce cellular differentiation (151). Much evidence points to an important role for
TGF- in the vasculature.
Several in vitro studies have demonstrated the importance of TGF- in
vascular cells. TGF- significantly inhibits the proliferation and
migration of endothelial cells (183). However, one study claims that it stimulates growth at low doses and inhibits at high
doses (163). TGF- also regulates endothelial cell
migration and formation of tubelike structures in a collagen gel,
features characteristic of in vitro angiogenesis. Interestingly,
similar to its effects on endothelial cell proliferation, TGF- may
either stimulate (152) or inhibit (162, 172)
in vitro endothelial tube formation. At a lower doses (
0.5 ng/ml),
TGF-1 stimulates tube formation (152), but at higher
doses (1-5 ng/ml), it inhibits this angiogenic activity
(152, 162). This effect of TGF- is also isoform
specific; unlike TGF-1, TGF-2 has no effect on in vitro vessel
formation at low concentrations, but at higher doses it stimulates
endothelial tube formation (152).
The effects of TGF- on endothelial tube formation may be mediated by
its effects on proteolytic activity. TGF- can produce a net
antiproteolytic activity in these cultures by modulating uPA
(urokinase-like plasminogen activator), and PAI levels
(181). Furthermore, TGF- can inhibit the production of
proteases, such as transin, and stimulate the production of protease
inhibitors, such as tissue inhibitor of metalloproteinase
(183). These effects prevent matrix remodeling and inhibit angiogenesis.
Other studies show that TGF- promotes angiogenesis by another
mechanism. When endothelial cells are cocultured with either pericytes
or vascular smooth muscle cells, latent TGF- is cleaved, most likely
by plasmin, to generate active TGF- (7, 208) that
affects both endothelial cells and pericytes. Active TGF- mediates
the inhibition in endothelial cell growth observed upon endothelial
cell-mural cell contact (172). In addition, active TGF-
binds to pericytes and induces expression of vascular smooth muscle
actin and myogenic determination (unpublished observations). Together, these studies suggest that TGF- may function to establish the structural integrity of newly formed capillary sprouts during angiogenesis. By inhibiting endothelial cell proliferation and promoting mural cell differentiation, TGF- helps to form and strengthen the vessel wall, and its matrix-modulating effects stimulate
tube assembly.
TGF- also has been shown to be involved in angiogenesis in vivo,
although results vary depending on the experimental conditions. TGF-
will stimulate robust angiogenesis if administered subcutaneously into
mice (204), applied to the chick embryo CAM
(262), or implanted into the rabbit cornea
(186) and rabbit ear dermal ulcers (188). However, in most cases, new blood vessels were accompanied by inflammation. Because TGF- is chemotactic for a wide range of cells,
including monocytes (253) and fibroblasts
(189), angiogenesis in these studies is likely indirectly
mediated by the effect of TGF- on recruiting these cells and
directly mediated by angiogenic factors produced by them. Moreover,
when overexpressed in the vessel wall and a variety of other tissues,
TGF- does not induce angiogenesis or an inflammatory response
(183). Thus TGF- is not angiogenic in vivo in the
absence of inflammatory mediators. Another possibility, however, is
that TGF- may not have been activated from its latent form in the
instances when angiogenesis was not observed in vivo. Activation of
TGF- requires certain conditions that have been mimicked in vitro,
including direct contact between two specific cell types (7,
208), but may not be present those systems studied in vivo.
Genetic studies also suggest a role for TGF- in angiogenesis. In
embryos of mice lacking TGF-1, differentiation of mesodermal precursors into endothelial cells appears normal, but embryonic lethality results because of defects in the yolk sac vasculature and
hematopoietic system (51). In these embryos, blood vessel walls are frail because of disrupted endothelial cell contacts. Similarly, TGF- receptor I-deficient mice show a similar phenotype: blood vessels are formed but are dilated and exhibit disrupted cell
contacts (173). Thus TGF- does appear to play a role in establishing vessel wall integrity.
Together, the in vitro and in vivo studies demonstrate important roles
for TGF- in angiogenesis. Through modulation of the synthesis of
extracellular matrix components, proteases, and protease inhibitors,
TGF- establishes a scaffold favorable to formation of vessel tubes.
This cytokine also acts to establish and strengthen the vessel wall
through regulation of endothelial cell quiescence, stability of
cell-cell contacts, and differentiation of mural cells. Furthermore,
TGF- indirectly stimulates angiogenesis by the recruitment of
inflammatory mediators that secrete angiogenic factors. Therefore,
TGF- stimulates vascular remodeling through its pleiotropic effects
on different cell types.
Other soluble factors.
Many other soluble factors have been proposed to function in
angiogenesis, but their effects on the vasculature are not as widespread as the above-described growth factors. For example, other
growth factors such as tumor necrosis factor- (TNF-), epidermal
growth factor (EGF), transforming growth factor- (TGF-), and the
colony-stimulating factors (CSFs) exhibit angiogenic properties. TNF- is secreted mainly by activated macrophages and some tumor cells, and although it is primarily involved in inflammation and immunity (19), it shares many properties with TGF-.
Both stimulate angiogenesis in vivo (in the CAM and cornea for TNF-)
(81), promote endothelial cell tube formation in vitro
(137), and inhibit endothelial cell growth (81,
123). EGF and TGF-, both of which bind the EGF receptor
(243), are 5- to 6-kDa proteins that are mitogenic for
endothelial cells in vitro and induce angiogenesis in vivo in the
hamster cheek pouch (211). In addition, granulocyte colony
stimulating factor (G-CSF) and granulocyte/macrophage
colony-stimulating factor (GM-CSF), proteins required for growth and
differentiation of hematopoietic precursors (38), induce
migration and proliferation of endothelial cells to a limited extent
(26).
inhibits capillary formation and endothelial cell proliferation in vitro (83, 99, 100, 246). In addition, cortisone (74),
thrombospondin (194), platelet factor IV
(146), protamine (233), and the more recently
discovered angiostatin (166) and endostatin
(167) inhibit angiogenesis in vivo in the CAM or corneal
pocket assays. Although thrombospondin (194) inhibits
endothelial cell migration and platelet factor IV (146)
inhibits the proliferation of endothelial cells in vitro, the
mechanisms whereby most of these substances inhibit angiogenesis in
vivo are not clear. Whether these substances inhibit physiological angiogenesis is unclear, but they are potent inhibitors of tumor angiogenesis (see below).
The soluble factors mentioned above are, in many cases, derived from
several different sources, but in all cases their effects are felt
directly or indirectly at the level of the endothelial and mural cells.
The fact that such a large number of discovered (and probably yet
undiscovered) soluble factors contribute to angiogenesis attests to its
complex nature and highlights its multiple modes of positive and
negative regulation in vivo.
Membrane-Bound Factors
In addition to factors that are secreted from cells and act at a distance from their sites of synthesis, several membrane-bound proteins play prominent roles in angiogenesis. These molecules require close cell-cell or cell-matrix contact for their effects to be felt. Integrins, cadherins, and ephrins are endothelial membrane proteins that mediate many functions involved in blood vessel assembly. In particular,v3-integrin, VE-cadherin, and ephrin-2B have been reported to play important roles in normal angiogenesis.
v3-Integrin.
Integrins are heterodimeric complexes composed of - and -subunits
that are receptors for extracellular matrix proteins and membrane-bound
polypeptides on other cells. More than 16 - and 8 -subunits can
combine to form a diverse array of >20 different integrins
(63). Extracellular matrix substrates for integrins include polysaccharide glycosaminoglycans as well as fibrous proteins such as fibronectin, vitronectin, collagen, laminin, and elastin (237). Some integrins bind short peptide sequences, such
as the RGD (Arg-Gly-Asp) sequence found in fibronectin and vitronectin, but others recognize three-dimensional conformations (8).
v3,
which binds von Willebrand factor, vitronectin, fibronectin, and fibrin (36), is highly expressed in vitro on endothelial cells
exposed to growth factors such as bFGF (230) and VEGF
(214). Integrin v3 also
mediates in vitro endothelial cell attachment, spreading, and migration
(135), and it is transiently localized to endothelial cells at the tips of capillary sprouts during wound repair
(37). v3 is thus important
for endothelial cell functions in neovascularization. In addition,
v3 is abundantly expressed on angiogenic
blood vessels in granulation tissue but not on vessels from normal
skin. Newly formed blood vessels induced by bFGF in the CAM assay also show robust v3 expression that is not
seen in untreated CAMs (22).
A requisite role for v3-integrin in
angiogenesis is suggested by the observation that neutralizing
antibodies to v3 inhibit bFGF-induced
vessel sprouting in the CAM, whereas the antibody has no effect on
preexisting vessels (22). In addition,
v3 is highly expressed on angioblasts
before and during vasculogenesis in the quail embryo, and injection of
an antibody to v3 results in abnormal
patterning of blood vessels characterized by discontinuous lumens and
incomplete vascular networks (55). Therefore,
v3 appears to be crucial for aspects of
vasculogenesis as well as angiogenesis in the developing embryo. Thus
v3 mediates endothelial cell functions in
vitro and plays an important role in angiogenesis in vivo.
The role of v3 in blood vessel growth has
been examined in embryonic vascular development.
v-Integrin knockout mice exhibit vessel abnormalities
and hemorrhaging in brain and intestinal vasculature (10),
and mice deficient in 3-integrins exhibit extensive
bleeding but demonstrate grossly normal vasculature (104).
Surprisingly, in both knockout experiments, extensive vasculogenesis
and angiogenesis proceeded normally. Nevertheless, these studies are
difficult to interpret because the functional redundancy of many
integrins (63) raises the possibility of compensatory
mechanisms in the absence of v- and
3-integrins.
The role of v3 in mediating angiogenesis
is not limited to binding of extracellular matrix components.
v3 also binds matrix metalloproteinase-2
and localizes the active form of the enzyme at the tips of angiogenic
blood vessels (24). Therefore,
v3 may regulate localized degradation of
the extracellular matrix and then mediate endothelial cell migration by
adhering to the modulated matrix. v3
ligation also induces mitogen-activated protein kinase activation
(62) and suppresses apoptosis (227) in endothelial cells. Thus v3 may mediate
endothelial cell survival by activating intracellular pathways that
promote proliferation and activation. By several mechanisms, then,
v3 mediates angiogenesis.
Other integrins have been implicated in regulating angiogenesis. For
example, in both the CAM and rabbit corneal pocket assays, anti-v3 inhibits bFGF-induced
angiogenesis, whereas anti-v5 suppresses
VEGF-stimulated angiogenesis (82). In addition, the collagen receptor integrins 11 and
21 are induced by VEGF, and antibodies to
them drastically inhibit VEGF-driven angiogenesis (212).
Antibodies to 51 inhibit angiogenesis
induced by several growth factors but not that induced by VEGF
(119). These studies suggest that different growth factors
may induce the expression of similar, yet distinct, integrins that
mediate the growth of new blood vessels. These different integrins may
regulate adhesion of endothelial cells to distinct substrates and
facilitate migration through several extracellular matrices.
Furthermore, 5-integrins, in general, appear to be
involved in angiogenesis, because although 5-null mouse
embryos develop a vascular system, their blood vessels are dilated and
leaky (263). Given the diverse functions of integrins in
angiogenesis, including adhesion to extracellular matrix, localization of proteases to capillary sprouts, and enhancement of endothelial cell
survival, endothelial cell expression of a variety of integrins may
stimulate distinct intracellular pathways that all contribute to the
progression of angiogenesis.
VE-cadherin.
Cadherins comprise a large family of Ca2+-binding
transmembrane molecules that promote homotypic cell-cell interactions
(94). These proteins serve diverse purposes in many cells.
The intracellular domain of cadherins mediates a linkage to the
cytoskeleton by binding to -catenin and plakoglobulin, two proteins
that are anchored to cortical actin by -catenin (94).
Cadherins also mediate intracellular signaling by controlling
cytoplasmic levels of b-catenins and plakoglobulin, which, when
released from cadherins, can translocate to the nucleus and regulate
gene transcription (16).
Eph-B4/ephrin-B2. A unique class of receptor/ligand pair, eph receptors and ephrin ligands play a prominent role in blood vessel development. Eph receptors belong to the largest known family of receptor tyrosine kinases consisting of at least 14 membrane-bound proteins, and 8 transmembrane ligands (ephrins) for them have been identified (260). Interestingly, not only does an ephrin expressed on the surface of one cell bind and activate its cognate eph receptor on another cell, but through a reciprocal signaling mechanism the ephrin is also activated upon receptor engagement (107). These molecules have been well characterized in the nervous system, where they appear to assist axon guidance through repulsive signals and establish borders between neuronal compartments (72). They also are found at compartment boundaries in several other embryonic tissues, including early somites and limb precursors (85). The requirement of cell-cell contact for their engagement and activation has suggested that they are involved generally in the formation of spatial boundaries that establish the developing body plan during embryogenesis (85).
One member of the ephrin family, ephrin-B2, is expressed on arterial endothelial cells of the developing embryo, and its receptor, eph-B4, is exclusively localized to venous endothelial cells; ephrin-B2 colocalizes with eph-4B at arterial/venous interfaces after vasculogenesis has established the primary capillary plexus but before angiogenesis has remodeled it (258). Indeed, the importance of ephrin-B2 and its interaction with eph-4B during angiogenesis is highlighted by mice with a null mutation in ephrin-B2 that exhibit normal vasculogenesis but demonstrate defects in angiogenesis of the head and yolk sac vasculature and in myocardial trabeculation (258). Interestingly, ephrin-2B also is expressed in a variety of nonvascular tissues, including caudal somites (257). However, eph-4B is exclusively localized on vascular endothelial and endocardial cells, and mice with a targeted mutation in eph-4B exhibit phenotypes similar to those seen in the ephrin-2B-null mice (88). These results suggest that establishment of contact and signaling between arterial and venous compartments mediated by ephrin-B2 and eph-4B is necessary for remodeling of the established primary capillary plexus. Other ephrin/eph family members appear to play a role in angiogenesis as well. Ephrin-A1 is required for angiogenesis stimulated by TNF-,
but not bFGF, in the rat cornea (175). Interestingly, a
soluble Ig chimera of ephrin-A1 is chemotactic for endothelial cells in
vitro (175). Furthermore, transfection of human umbilical vein endothelial cells (HUVECs) with a dominant negative form of
eph-2A, the receptor for ephrin-2A, results in an impaired ability to
form capillary tubes in vitro (168). Thus ephrin family members other than ephrin-2B are important for endothelial cell events
involved in angiogenesis.
Interestingly, ephrins (particularly ephrin-2A) exhibit growth factor
specificity in angiogenesis (175) in a manner similar to
that reported for integrins (82, 119). These results
reinforce the concept that diverse members of transmembrane signaling
molecule families are induced by distinct stimuli during angiogenesis
and that each has an important role in mediating the varied angiogenic processes in vivo. Thus mechanisms of angiogenesis cannot be modeled merely as summations of growth factor signals through singular pathways. Rather, angiogenesis results from a complex coordination of
positive and negative regulators on many different cellular systems.
Biomechanical Forces
In addition to the soluble and membrane-bound molecules described, mechanical forces acting on vascular endothelium also contribute to the pruning and remodeling processes characteristic of normal angiogenesis. The mechanical forces mediated by blood flow have profound effects on vessel growth. Vessels that are not perfused with blood eventually regress (202). This phenomenon is most apparent in the regulated cycles of angiogenesis occurring in the female reproductive system where periodic growth and regression of blood vessels cyclically remodel the ovarian, uterine, and placental tissues (199). For example, some of the highest rates of blood flow on a weight basis are observed in these tissues and are associated with extensive proliferation of vascular endothelial cells (199). On the contrary, it has been suggested that a reduction in ovarian blood flow leads to luteal regression (198), a process associated with extensive capillary bed degeneration. Furthermore, in skeletal muscle, increased blood flow induced by electrical or chemical stimulation results in capillary angiogenesis and arterial growth (5), and decreased blood flow causes a reduction in size and number of arterioles (256).Careful in vitro and in vivo characterization of the effects of blood
flow on capillary cells has revealed a mechanism by which it affects
vessel growth. In vitro, fluid shear stress induces a dramatic increase
in endothelial cell stress fiber expression (if flow is laminar)
(80), promotes endothelial cells to divide (if flow is
turbulent) (45), and stimulates the transcription of genes
for PDGF and TGF- (197), which promote angiogenesis as
described above. In vivo, increased shear stress in rabbit ear vessels
associated with enhanced blood flow correlates with an increase in
microvascular area (109). Therefore, shear stress induced
by blood flow modulates blood vessel morphogenesis. Laminar flow
stabilizes and protects the vessel wall by increasing stress fiber
expression in endothelial cells, and turbulent flow leads to further
blood vessel growth. This is an efficient mechanism of remodeling the
primary vascular plexus, because vasculogenesis results in the
overproduction of blood vessels. Nonperfused capillaries regress,
probably by endothelial cell apoptosis (201),
whereas those in which blood flow is established persist and become a stable part of the vasculature.
Thus microvascular blood vessels are remodeled in angiogenesis through several diverse mechanisms. Growth factors secreted from distant cells, transmembrane proteins binding to extracellular matrix components or to receptors on other cells, and hemodynamic forces all act in concert to regulate normal angiogenesis. In a physiological setting, these factors exert both positive and negative influences on blood vessel growth to ensure that angiogenesis is confined to metabolic demands of growing and healing tissues. However, certain pathological conditions usurp these mechanisms to enhance the spread of disease. One of the most characterized of these is tumor angiogenesis, which is discussed below in terms of its relation to normal angiogenesis.
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TUMOR-INDUCED ANGIOGENESIS |
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TOP
ABSTRACT
NORMAL ANGIOGENESIS
TUMOR-INDUCED ANGIOGENESIS
SUMMARY
REFERENCES
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Tumors are populations of host-derived cells that have lost the ability to regulate growth and therefore proliferate aberrantly. Though several features distinguish them from their nontransformed counterparts, many aspects of tumor cells are similar to those of normal ones. One major similarity is the requirement for an adequate supply of oxygen and nutrients and an effective means to remove wastes in order for metabolic processes to occur and survival to be maintained. Proximity to a vascular supply fulfills these requirements for mammalian cells. Normal cells and tissues rely on physiological vasculogenesis and angiogenesis (described in detail above) to provide them with a vasculature that fulfills their metabolic demands. Tumor cells, on the other hand, can induce their own blood supply from the preexisting vasculature in a process that mimics normal angiogenesis.
The Tumor Vasculature
Tumors can establish their own blood supply by several means. Figure 2 is a schematic of tumor-induced neovascularization. In a process very similar to normal angiogenesis, a tumor may elicit the formation of blood vessels from preexisting capillaries. In addition, tumor cells are able to grow around an existing vessel and hence, at least initially, do not need to induce angiogenesis for adequate vascularization (105). Furthermore, circulating endothelial precursors, angioblast-like cells derived from bone marrow but reported to be present in the adult circulation, have recently been suggested to contribute to tumor-derived blood vessels (193). Though tumor-induced vessels form a conduit for the delivery of metabolites, ultrastructurally they are abnormal. Many lack functional pericytes (18), they are dilated and convoluted, and they are exceptionally permeant due to the presence of fenestrae and transcellular holes and lack of a complete basement membrane (32) (see Fig. 2). Furthermore, tumor vessel walls may be made up of both endothelial cells and tumor cells (35). These structural abnormalities in tumor vessels reflect the pathological nature of their induction, yet their ability to support cell growth also underlies the use of physiological mechanisms of angiogenesis that tumors commandeer for their propagation.
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Factors Involved in Tumor Angiogenesis
The induction of new blood vessel growth by a tumor is mediated through the action of many molecules, some of which are involved in normal angiogenesis. Those substances that are well characterized in tumor neovascularization are summarized in Table 2 and are described below.
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Vascular endothelial growth factor. As in normal angiogenesis, tumor angiogenesis appears to rely heavily on VEGF. Many tumor cell lines secrete VEGF in vitro (215), and by in situ hybridization VEGF mRNA is highly upregulated in most human cancers including lung, breast, gastrointestinal tract, kidney, bladder, ovary, and endometrial carcinomas, intracranial tumors, glioblastomas, and capillary hemangioblastomas (68). Both VEGF and its receptor (Flk-1) are highly expressed in metastatic human colon carcinomas and their associated endothelial cells, respectively, and production of these two proteins correlates directly with the degree of tumor vascularization (231). Furthermore, increased VEGF expression is closely associated with increased intratumoral microvessel density and poor prognosis in breast cancer patients (244). In addition to producing VEGF themselves, tumors may induce the production of VEGF in their surrounding stromal tissue (84). In this study, green fluorescent protein driven by the VEGF promoter was robustly expressed for weeks in fibroblasts surrounding both implanted and spontaneous tumors. Therefore, high levels of VEGF production in a wide variety of tumor and tumor-associated cells and robust expression of its receptor in tumor-associated blood vessels suggest that VEGF plays an important role in tumor angiogenesis.
A causative role for VEGF in tumor angiogenesis is suggested by inhibition studies. Intraperitoneal administration of anti-VEGF antibody in nude mice harboring tumors derived from injected sarcoma and glioblastoma cells significantly decreases tumor vessel density and suppresses tumor cell growth (118). By intravital examination of blood vessels stimulated by tumor spheroids administered to mice, anti-VEGF was shown to almost completely inhibit tumor neovascularization (21). These observations indicate that a general inhibition of VEGF activity in vivo results in reduced tumor angiogenesis and tumor growth. A more direct role of tumor cell-derived VEGF in stimulating angiogenesis in vivo was suggested by the dramatically impaired ability of embryonic stem (ES) cells with a targeted inactivation of the VEGF gene to form teratocarcinomas in nude mice compared with control ES cells (69). Interestingly, blood vessels induced by the VEGF
/ ES cells were lower
in number and less branched than those induced by control ES cells.
Inhibition of VEGF receptor signaling also suppresses tumor growth in
vivo. Retrovirus-mediated expression of a dominant negative VEGF
receptor (Flk-1) dramatically inhibits the growth of a variety of
tumors, including mammary, ovarian, and lung carcinomas
(156) as well as C6 glioblastomas (157) in
nude mice. Histological examination of these inhibited neoplasms demonstrates that the growth of blood vessels in the tumors is also
severely reduced. These studies have far-reaching clinical implications
because, aside from the reduction in tumor vascularization and growth,
host animals were largely unaffected by inhibition in VEGF or its receptor.
The mechanism of VEGF-mediated tumor angiogenesis most likely relies on
regulation by oxygen tension. The fact that solid tumors contain a
central region of necrotic tissue resulting form poor delivery of
oxygen has been known for some time (239). In fact,
hypoxia associated with poorly vascularized areas of tumors selects for
cells that are resistant to damaging effects of hypoxia or that can
induce oxygenation. In the former case, hypoxia is able to select for
apoptosis-defective cells lacking p53 and may be partially
responsible for the observation that p53 is one of the most commonly
mutated genes in human cancer (93). The latter case is
indicative of the ability of the tumor environment to stimulate VEGF
production and angiogenesis by cancer cells. In situ hybridization has
identified VEGF mRNA in hypoxic regions of glioblastoma immediately
adjacent to necrotic areas, and capillary bundles have been found next
to the VEGF-producing cells (219). These observations
suggest that induction of VEGF mRNA in tumor cells by exposure to
hypoxia in vitro (120) is also relevant in vivo.
Together, these results strongly implicate VEGF as having a prominent
role in inducing tumor angiogenesis. Mechanistically, the mediation of
blood vessel growth by VEGF in tumors is similar to that of
physiological angiogenesis, i.e., low oxygen tension induces
neovascularization to satisfy metabolic demands (4). Furthermore, perhaps the most clinically relevant aspect of a tumor is
its ability to metastasize to distant sites. In addition to stimulating
blood vessel growth, VEGF increases vascular permeability (64,
117, 128, 213). In such a manner VEGF can induce the formation
of leaky blood vessels with fragmented membranes that can easily be
penetrated by neoplastic cells (206) to disseminate a
primary tumor. Thus VEGF may have multiple roles in tumor angiogenesis. Nevertheless, although VEGF is important for these processes, other
polypeptides complement its actions in inducing blood vessel growth in tumors.
Fibroblast growth factor. The first tumor-derived factor known to stimulate endothelial cell proliferation and induce neovascularization in vivo was isolated by Folkman et al. (78) in the early 1970s. However, purification of this factor was difficult because of a lack of suitable bioassays, and the angiogenic activity was simply known as "tumor angiogenesis factor" (78). The advent of heparin-affinity chromatography facilitated the identification of FGF as the first known tumor-derived angiogenic factor (75, 217, 218).
A direct role for FGF in tumor angiogenesis has been suggested in an inhibition study using a soluble form of the bFGF receptor. Administration of a soluble form of the FGF receptor to mice injected with pancreatic-cell tumors induced by SV40 T antigen expression driven by the rat insulin promoter dramatically suppresses tumor growth and decreases tumor vessel density (39). In
addition, if biopsied pancreatic -cell tumors are incubated with
HUVECs in a three-dimensional collagen matrix, the HUVECs
proliferate and migrate toward the tumor. However, HUVECs induced to
secrete soluble FGF receptor demonstrate a very limited angiogenic
response (39). Therefore, these results suggest that FGF
production by neoplastic cells induces angiogenesis that stimulates
tumor survival and growth in vivo.
Because of the observation that soluble FGF receptor impedes tumor
growth at a later time point after tumor induction than does soluble
VEGF receptor, it has been speculated that whereas VEGF acts to
initiate tumor angiogenesis, FGF is important for its maintenance
(39). Indeed, bFGF has been reported to cooperate with
VEGF in stimulating angiogenesis. VEGF and bFGF synergized in vitro to
increase the rate of proliferation and formation of cordlike structures
by bovine capillary endothelial cells in a collagen gel
(92) and in vivo to induce collateral vessel development following hindlimb ischemia in rabbits (9). FGF
also may help to augment the production of VEGF. Exogenous expression
of FGF4, an FGF family member that is secreted from cells, in normal
mouse mammary cells renders them tumorigenic in nude mice and
angiogenic for HUVECs cultured in a collagen gel (49). The
angiogenic effect is mediated by stimulation of VEGF mRNA and protein
production by FGF4 expression (49). In addition, bFGF
induces an increase of VEGF mRNA in vascular smooth muscle cells
(224) and an increase in VEGF receptor in microvascular
endothelial cells (149). It is very likely, then, that FGF
can stimulate tumor angiogenesis in vivo via several mechanisms
including activation of, and synergism with, VEGF.
Heparanase also can be considered a separate inducer of tumor
angiogenesis, but its mechanism of action appears to be mediated by
bFGF. Heparanase promotes angiogenesis directly by stimulating invasion
of endothelial cells and vascular sprouting as well as indirectly by
releasing heparan sulfate-bound bFGF from its sites of deposition in
the extracellular matrix (250). Heparanase mRNA and
protein are enriched in metastatic cell lines as well as specimens of
human melanomas and carcinomas vs. normal tissues, and transfection of
nonmetastatic T lymphoma and melanoma cell lines with the heparanase gene renders them highly metastatic in vivo (250).
Furthermore, in vivo angiogenic activity of heparanase is evidenced by
the significant increase in neovascularization induced by T lymphoma cells in the Matrigel plug assay when these cells are transfected with
heparanase (250). Thus heparanase may be necessary to
evoke the blood vessel growth by tumor cells.
FGF may therefore both directly and indirectly stimulate tumor
angiogenesis. Inhibition studies indicate that FGF is in part necessary, but not sufficient, to induce blood vessel growth by tumors.
FGF and VEGF are two of the many factors that cooperatively mediate
neovascularization in the tumor microenvironment.
Ang2. Recent evidence strongly implicates Ang2 in tumor angiogenesis. As mentioned above, the angiopoietins play prominent roles in normal angiogenesis. Ang1 signaling through the Tie2 receptor remodels newly formed capillary tubes and stabilizes them through interactions between endothelial cells and surrounding support cells (228, 229, 241). Ang2 is an antagonist of Ang1 and destabilizes blood vessels (147). In the absence of VEGF production, Ang2 mediates blood vessel regression; however, in the presence of VEGF, Ang2-induced destabilization of vessels renders them plastic and more responsive to VEGF-mediated growth (147).
Based on Ang2 and VEGF functions in normal angiogenesis, an interesting model has been proposed for angiogenesis induced by several tumors. Contrary to initial reports that most tumors, especially metastases, originate in an environment devoid of blood vessels, many tumors start growing around existing vessels and initially do not need to induce angiogenesis to survive (261). As the tumor grows larger, however, mural cells progressively disengage from the endothelium of these co-opted vessels, and the blood vessels regress (106) by endothelial cell apoptosis. Interestingly, Ang2 is induced in the endothelium of these vessels even before they regress (105). Furthermore, robust expression of VEGF in the growing tumor cells then results in angiogenesis, and the newly formed vessels also express high levels of Ang2 mRNA (105). Therefore, Ang2 plays a dual role in tumor angiogenesis. In the early stages of tumor cell growth around an existing blood vessel, tumor cells do not produce VEGF. Instead, they induce Ang2 expression in the blood vessel, which results in vessel destabilization and regression. As the tumor grows and its metabolic demands become greater, VEGF production by the tumor induces neovascularization, and Ang2 induction by tumor cells in endothelial cells of newly formed vessels facilitates this process by rendering endothelium unstable and plastic. Indeed, by in situ hybridization, Ang2 mRNA is expressed in endothelial cells of tumor vessels but not in normal blood vessels, and it is one of the earliest markers of tumor-induced neovascularization (265). Block of the stabilizing effect of Ang1 on newly formed blood vessels by Ang2 is probably a major contribution to leakiness and fragility of tumor vessels (32). Therefore, Ang2 contributes significantly to tumor angiogenesis. Like FGF, it cooperates with VEGF to induce blood vessel growth.Interleukin-8 and matrix metalloproteinase-2. A growth factor that is not well characterized in normal angiogenesis but has attracted attention in tumor neovascularization is interleukin-8 (IL-8). An angiogenic role for IL-8 in angiogenesis was first suggested by the observation that macrophages produce IL-8 and mediate angiogenesis in chronic inflammatory diseases such as psoriasis and rheumatoid arthritis (126, 127). Subsequently, it was shown that not only is IL-8 mitogenic and chemotactic for HUVECs in vitro, but it also stimulates angiogenesis in the rat cornea (127).
A role for IL-8 in tumor angiogenesis is suggested by the findings that IL-8 mRNA is upregulated in neoplastic tissues, such as non-small cell lung cancer (264) and melanoma (141), vs. normal ones in vivo and that its expression correlates with the extent of neovascularization. In addition, overexpression of IL-8 in nonmetastatic, IL-8-negative melanoma cells not only increases their ability to invade Matrigel-coated filters but also renders them highly tumorigenic and metastatic in nude mice (12). Also, stable transfection of gastric carcinoma cells that produce low amounts of endogenous IL-8 with the IL-8 gene allows them to produce rapidly growing, highly vascular neoplasms that are not seen with control-transfected cells (121). Furthermore, conditioned medium from the IL-8-transfected cells stimulates HUVEC proliferation (121). Although these results suggest a role for IL-8 in the induction of endothelial cell proliferation in the tumor vasculature, another mechanism may mediate the role of IL-8 in tumor angiogenesis. An important observation made in the studies using IL-8-transfected melanoma cells was that the cells exhibit an increase in MMP-2 mRNA and activity and an increase in MMP-2 promoter-driven reporter gene activity (141). Therefore, angiogenesis induced by IL-8 may have been mediated in part by its ability to stimulate production of MMP-2, which degrades basement membranes and remodels the extracellular matrix for cell invasion and migration. One of the first steps in angiogenesis, normal or pathogenic, is degradation of the extracellular matrix (124). Indeed, MMP-2 has been shown to directly modulate melanoma cell adhesion and spreading on extracellular
