IN VITRO ASSAYS OF ANGIOGENIC AND ANTIANGIOGENIC FACTORS

The search for proangiogenic and antiangiogenic molecules has
been made possible by methods that enable the measurement of
angiogenesis activity. Unlike cell proliferation or death, which
can be easily evaluated in convenient in vitro assays, angiogenesis
involves complex interactions between endothelial cells
and their surroundings. Because of this, angiogenesis measurements
are subject to substantial assay-dependent biases. Several
angiogenesis bioassays that represent various in vivo phases of
angiogenesis have been developed. The assays used most often
are those of endothelial cell proliferation and migration, two
biologic events that are critical to the formation of new blood
vessels. Proliferation is evaluated by measuring cell number
changes, cell cycle alterations, or DNA incorporation, whereas
migration assays usually involve scoring cells that migrate
across a porous membrane toward a putative chemoattractant
(Boyden chamber assay).
Additional assays include the tube formation assay, aortic
ring assay, and chick-embryo chorioallantoic-membrane bioassay.
The tube formation assay involves visualizing tubelike
structures that form when endothelial cells are cultured on an
artificial ECM, and the aortic ring assay evaluates similar structures
that grow from a slice of rat aorta in culture. The chickembryo
chorioallantoic-membrane bioassay uses the vascular
structures in a fertilized chick egg, either in uvo or in vitro. 96
The rabbit cornea implant assay involves placing a pellet of
sustained-release polymers in a normally avascular cornea and
examining the evolving blood vessels. 97 The matrigel-plug assay
is a simpler version of the cornea implant assay. It involves the
subcutaneous injection of a matrigel plug into a mouse and
evaluation of its vascularization. None of these assays is flawless;
thus, caution must be exercised when interpreting results
from a single type of angiogenesis assay.
MAJOR MOLECULAR PLAYERS
VEGF Signal Transduction Pathway
VEGF Family Members VEGF, also called VEGF-A, increases
endothelial permeability 98 ; therefore, it was initially
named vascular permeability factor. This protein was found to
function as a mitogenic and survival factor, specific to endothelial
cells. 99 VEGF is a heparin-binding glycoprotein with at
least five family members, VEGF-A to -D and placental growth
factor (PlGF). VEGF binds mostly Flt-1(fms-like tyrosine
kinase-1)/VEGFR-1 and Flk-1(fetal liver kinase-1)/VEGFR-2/
KDR (kinase domain region), parts of a family of tyrosine
kinase receptors. VEGFR-1 and -2 are mainly expressed by
vascular endothelial cells 100 but are also expressed by monocytes,
101 hematopoietic stem cells, 102 and some cancer cells. 103
VEGFR-2 is a major positive regulator of the vascular system.
Its ligands are VEGF, VEGF-D, and VEGF-C. It activates the
proliferation and migration of endothelial cells and acts as a
survival factor for these cells. The role of VEGFR-1 in angiogenesis
is less clear, demonstrating a higher binding affinity of
VEGF than for VEGFR-2 but lower kinase activity and no mitogenic
response. Mice manipulated not to express VEGFR-1
have an abnormal vascular system because of an increased
number of hemangioblasts. 104 Interestingly, mice expressing a
VEGFR-1 that lacks the kinase domain developed normally,
suggesting that this protein functions as a negative regulator
of the VEGF pathway by trapping ligand molecules. 105 The
ligands of VEGFR-1 include VEGF, VEGF-B, and PlGF.
The differences between VEGFR-1 and VEGFR-2 lie mostly
in the intra-cellular C-terminal domain of these receptors. 106
VEGF-C and VEGF-D mainly regulate lymph vessel formation
through activation of VEGFR-3, 107 although VEGFR-3
activity seems to be required also for blood vessel angiogenesis.
108 Interestingly, LKB-1, a serine/threonine kinase that is
mutated in almost a third of lung cancers, was found to repress
VEGF-C, among other targets 109 (see Fig. 8.2 for a scheme of
the VEGF pathway).
The induction of VEGF production can result from
various signals. Physiologically, it is controlled by the HIF-1
pathway in response to hypoxia. Mitogenic signals, including
phosphatidylinositol 3-kinase (PI3K) and Ras, also increase
VEGF mRNA levels. 110,111 Various isoforms of protein kinase
C (PKC) have also been shown to induce VEGF. 112 EGFR
inhibition reduced VEGF levels in a HIF-1–dependent and
in a HIF-1–independent manner. 113 In addition, activated
oncogenes and deregulated tumor suppressor genes contribute
to VEGF’s activation in tumors. Src kinase was found to be
induced by hypoxia and to activate the VEGF promoter. 114,115
Wild-type p53 suppressed VEGF promoter activity, whereas
mutant p53 had no effect or activated it. 115,116 HIF-1 degradation
induced by p53 was also demonstrated, secondary to
p53- dependent activation of Mdm2. 117
VEGF receptors are activated by receptor dimerization
induced by binding of the ligand dimer. This dimerization
allows cross-phosphorylation of specific tyrosine residues of
the receptors, creating docking sites for adapter molecules.
The phosphorylation map and site-specific adapter proteins
of VEGFR-2 have been delineated. 118 Various signaling pathways
are activated downstream of VEGF receptors. VEGFR-2
is a survival factor for endothelial cells through PI3K and
AKT. 119 VEGFR-2 also activates phospholyphase C (PLC ),
which is not activated by VEGFR-1, EGF, or FGF. Signaling
through VEGFR-2 independently activated phospholyphase
C , PKC , and PI3K. 120 VEGFR-2 activates mitogen- activated
protein kinase (MAPK) through mitogen-activated protein kinase
kinase (MEK) and phospholyphase C . Blockage of PKC
reduced DNA replication induced by VEGF but had no effect
on DNA replication induced by FGF or EGF. 120 Therefore,
the signaling pathways used by VEGFR-2 are different from
those used by other mitogens, although the end points of these
cascades overlap. The MEK-ERK, PKC, and PI3K control
DNA replication, cell proliferation, and survival signals in response
to VEGF.
Some modulation of the VEGF pathway occurs through
regulation of the receptor. For example, vascular endothelial
(VE)-cadherin, a cell–cell adhesion molecule that is specifically
expressed by endothelial cells, modulates signaling
relayed by VEGFR-2. By blocking the internalization of this
receptor, VE-cadherin inhibits its downstream signaling in
confluent endothelial cells. 121 Downregulation of VEGFR-2
may occur in a PKC-regulated manner. 122 Another important
modulator of VEGF receptor activity is neuropilin-1, a coreceptor
of VEGF, originally recognized as a semophorin coreceptor
that participates in neuronal guidance. Neuropilin-1
enhances the activation of VEGFR-2 by VEGF and the
mitogenic and chemotactic response it evokes. It functions
in an isoform-specific manner, binding VEGF165 but not
VEGF121 (see below). 123 VEGFR-1 can also bind neuropilin-
1, possibly as a negative regulator of its activity. 124
Neuropilin-1 is expressed by endothelial cells and cancer cells
and contributes to their migration and in vivo progression.
Neuropilin-1 mRNA levels were measured in the tumors of
60 NSCLC patients and were found to be an independent
negative prognostic factor. 125
New blood vessel formation requires endothelial cell migration,
a biologic phenomena that involves complex modifications
of cell–cell and cell–matrix adhesions. VEGF activates
actin cytoskeleton remodeling and motility. These phenomena
are mediated through VEGFR-2 and various adapter proteins,
including src family kinase proteins such as Fyn and the small
GTPases Cdc42, RhoA, and Rac-1. SAPK2/p38 activation
modulates actin fiber polymerization, and focal adhesion kinase
controls the formation of focal adhesion. 126,127 These
molecular events are critical for the motility and migration of
endothelial cells in response to VEGF signaling.
VEGF signaling apparently affects not only the cells
that surround the tumor but also distant tissues. Activation
of VEGFR-1 in the lungs of mice, in a premetastatic phase,
was required for the induction of MMP9. This induction was
evident only in the lungs of tumor-bearing mice; thus, it was
the result of a tumor-originated influence. Lung endothelial
cells and lung macrophages had elevated MMP9 levels in
tumor-bearing mice, and this increase was essential for the formation
of lung metastasis. 128 VEGFR-1 bearing hematopoietic
progenitor cells localized to a premetastatic niche and were
followed by metastasis formation in another mouse model. 129
Five isoforms of VEGF are expressed by human cells, all
of which are produced from the same VEGF gene through
alternative splicing. These isoforms are named VEGF121,
VEGF145, VEGF165, VEGF189, and VEGF206, according
to the number of amino acids of the produced protein. 130
VEGF121 is the only isoform that does not bind heparin and
is thus freely diffusible. The other four isoforms bind heparin;
the two shortest, VEGF145 and VEGF165, are also secreted.
VEGF145 binds ECM in a heparin-independent manner. 131
VEGF189 and VEGF206 are basic large proteins that are associated
with the cell surface through their high affinity to
proteoglycans or bound to the ECM, where they can activate
VEGF signaling and be released to a soluble form. 132
Interestingly, VEGF189 was found to be prognostically important
in NSCLC, 133 whereas VEGF165 had prognostic
implications in osteosarcoma patients. 134 Thus, the various
isoforms of VEGF exert different biologic activities in different
tissues.
The RIP-Tag mice model offers insight into the activation
of the VEGF pathway during the angiogenic switch.
Targeted knockout of VEGF or inhibition of VEGFR-2
by small molecule inhibitors in the RIP-Tag mice, reduced
tumor initiation and progression, demonstrating the critical
role of this molecule. 8 Progression of carcinomas in this model
was accompanied by an increased release of VEGF from the
tumor mass and increased binding of the ligand to VEGFR-2.
Interestingly, no increase in the expression of this protein or
its receptors was found. MMP9 expression was found to be
increased in stromal cells in progressing tumors. It was shown
to increase the release of ECM-bound VEGF and thus its
ability to activate the receptor. 9 Therefore, nontumorigenic
cells in the tumor stroma can control the angiogenic switch by
producing MMP and modulating the release of VEGF from
the ECM.
Angiopoietins and Tie Receptors Angiopoietins are a
family of angiogenesis modulators composed of four ligands
that bind the Tie-2 tyrosine kinase receptor. Ang-1 and
Ang-4 function mostly as positive regulators, whereas Ang-2
and Ang-3 are mostly antiangiogenic. However, these roles
are context dependent. Knockout Ang-1 mice do not form a
normal vascular network in utero, at least partly because of
reduced pericyte coverage. Ang-2 overexpression has a similar
phenotype to that of Ang-1 knockout, indicating that Ang-2
has an antagonistic role in Tie-2 activity. Ang-2 antagonizes
the Ang-1-dependent recruitment of pericytes to new blood
vessels, thus preventing their stabilization. However, Ang-2
knockout mice have defects in adult vascular sprouting. This
finding suggests that destabilization of the vessel structure is
needed for angiogenesis to progress. Ang-2 seems to have a
proangiogenic role when VEGF is abundant but an antiangiogenic
role when VEGF levels are low. 135 The Tie-2 receptor
is expressed by endothelial cells and by pericytes and smooth
muscle cells. Its expression by blood monocytes might be important
for their recruitment to tumor tissues. 136 Importantly,
Ang-2 expression has negative prognostic implications in lung
cancer patients, especially when VEGF expression is high 137
(see succeeding discussion). Ang-2 and Tie-2 apparently regulate
the survival of vessels co-opted by cancer 138 (see previous
discussion). Interestingly, Ang-1 has a negative role in tumor
angiogenesis, probably secondary to enhanced pericyte vessel
coverage and reduced vessel permeability. 139 The resultant vessels
do not allow extravasation of plasma proteins and a less
proangiogenic environment is formed.
Unexpected Outcomes of VEGF Inhibition In studies in
which the VEGF pathway was effectively attenuated, rebound
angiogenesis was observed despite persistent inhibition of the
VEGF pathway. Alternative angiogenic mechanisms were upregulated
when the VEGF pathway was suppressed, including
induction of FGF family members, angiopoietins, and
vessel co-option. 10 The activation of additional proangiogenic
pathways may be triggered by hypoxia in tumors subjected to
VEGF inhibition. However, increased levels of some of these
factors persist when no hypoxia is apparent. Therefore, pathologic
angiogenesis is controlled by various signaling pathways
that may be important therapeutic targets. 97
The other important finding in models of VEGFdependent
tumor growth treated with VEGF inhibitors is
increased invasiveness of tumors that thrive in these conditions.
9,10 This phenomenon could be secondary to hypoxia-induced
activation of the HGF-Met pathway, 140 urokinase-type
plasminogen activator, 141 or other survival pathways. Hypoxia
may select for tumor cells with increased aggressiveness, such
as through loss of p53 142,143 or other genetic events. 144 As
VEGF pathway inhibition is assayed as a cancer therapeutic
strategy, further understanding about cancer escape mechanisms
is needed.
HIF HIF-1 is the major transcription factor that regulates
the response of tissues to oxygen deprivation. 145 It is expressed
ubiquitously in humans, where it upregulates erythropoiesis
and blood vessel formation and controls metabolic pathways.
HIF-1 subunits are tightly regulated by hypoxia through control
of their degradation, in addition to hypoxia-independent
regulation by growth factor signaling. 146 Under normoxia,
they are marked for degradation via the 26S proteosome by its
E3-ubiquitin ligase von Hippel-Lindau (VHL), a tumor suppressor
gene. 147 Oxygen levels are sensed by prolyl hydoxylase,
an enzyme that hydroxylates specific prolyl residues of HIF-1
when oxygen is available. This modified domain of HIF-1 is
the binding site of VHL, which activates its rapid degradation.
Lack of oxygen thus prevents VHL-dependent degradation of
HIF-1 , causing an accumulation of this protein. 148,149
HIF-1 is expressed constitutively, forming a heterodimer
with a HIF subunit, thus activating more than 70 genes
whose products carry out the complex regulation mastered by
HIF-1. HIF-1 and HIF-2 are the products of two different
genes, regulated similarly by hypoxia, both of which can
heterodimerize with HIF-1 and activate each a distinct set of
genes. 150 Both subunits are produced by various cell types,
including endothelial cells. 151,152 The relative contributions of
HIF-1 or -2 differ under different conditions. 153 HIF-1 expression
by tumor cells was found to contribute to tumor progression.
154 HIF-2 is highly expressed in tumor-infiltrating
macrophages, and this expression was correlated with tumor
angiogenesis in a series of breast carcinomas. 155 Thus, HIF-
2 may participate in the contribution of the tumor microenvironment
to tumor vascularity. 152 In addition, the role of
HIF-3 has yet to be defined. A splice variant of HIF-3 was
found to antagonize the transactivation of hypoxia-inducible
genes by HIF. 156
Endogenous Antiangiogenic Factors Some antiangiogenic
factors are produced by cleavage of larger proteins. For
example, angiostatin, a potent angiogenesis inhibitor, is the
cleavage product of plasminogen, a component of the coagulation
control mechanism. 157 Macrophage-derived methalloelastase
is thought to be responsible for the in vivo conversion
of plasminogen to angiostatin. 158 Thus, tumor-infiltrating
macrophages may determine the production of antiangiogenic
factors. In addition, cancer cells may secrete enzymes that can
produce angiostatin. 159 The conversion of plasminogen to
the proangiogenic plasmin is essential for the production of
angiostatin. 159 Angiostatin’s mechanism of action might be
through inhibition of plasmin production. 160 This cross-talk
between an angiogenic molecule and an antiangiogenic factor
is consistent with the continuous balancing mechanisms
between these effects.
Endostatin is an antiangiogenic factor that was shown to
be generated by tumor cell lines. A biochemical analysis indicated
that it is a fragment of collagen XVIII. 161 Recently,
(II) collagen prolyl-4-hydroxylase [ (II)PH] was found to
catalyze a rate-limiting step in collagen synthesis that is apparently
required in endostatin production. Importantly,
(II)PH was found to be a direct transcriptional target of
p53, a well- recognized tumor suppressor gene, in tumor
cells. p53 expression was found to increase the expression of
(II)PH, leading to enhanced endostatin production. By
an apparently similar mechanism, p53 led to the production
of tumstatin from collagen IV. 143 Thrombospondin-1
is another antiangiogenic molecule induced by p53, through
transcriptional activation. 33 Thrombospondin-1 secretion decreases
with malignant progression, 162 possibly a reflection of
the common loss of p53 transcriptional activity in tumor cells.
Endostatin and tumstatin function through binding of integrins
and modulating various signaling pathways, 163,164 whereas
the mechanism of action of thrombosponsdin-1 involves inhibition
of MMP9 activation 165 and activating endothelial cell
apoptosis through CD36. 166
Additional potent antiangiogenic factors are produced by
the cleavage of common proteins, 167 suggesting a paradigm.
C-terminal fragments of various collagens are cryptic antiangiogenic
agents, becoming active once released from the
parent collagen. 168 The control of angiogenesis seems to require
reserves of antiangiogenic factors available for rapid
mobilization.
PDGF Platelet-derived growth factor beta polypeptide (PDGFBB)
and PDGF- receptor are the major molecules involved in
regulating the pericytes perivascular envelope. 169,170 PDGF-B
is secreted by tumor endothelial cells, and tumor cells in some
cases, and forms the active dimer of PDGF-BB. PDGF- receptor
is expressed mainly by pericytes, although it has also been
found on tumor endothelial cells. The activation of the PDGF-
receptor results in the recruitment of pericytes to the developing
tumor vessel, as in physiologic angiogenesis. 171 Pericytes
have a supportive and modulating role in the evolving vessel.
Abnormal pericyte coverage in PDGF-B null mice resulted in
vessel dilatation, leakage, aneurism formation, and hemorrhage
in late gestation. 172 Interestingly, the retention of PDGF-B on
the surface of the secreting cell is important for correct pericyte
deposition, at least during embryonic development. Local retention
of growth factors is a mechanism whereby extracellular
matrix and proteases affect biologic phenomena. The retention
of PDGF-B is mediated by its C-terminal motif of positively
charged amino acids, which bind negatively charged heparan
sulphate proteoglycans. 173 Blood vessels of tumors inoculated
into mice that expressed an ECM retention-defective mutant
PDGF-B had defects in pericyte coverage. PDGF-B expression
by tumor cells only partially compensated for this defect, suggesting
that PDGF-B must be expressed by endothelial cells 174
to facilitate the correct homing of pericytes to blood vessels.
bFGF bFGF is a potent angiogenic factor that stimulates the
proliferation and migration of endothelial cells as well as the
production of MMPs. However, unlike VEGF, bFGF affects
various cell types, including endothelial cells, smooth muscle
cells, fibroblasts, and epithelial cells. 175,176 bFGF can be found
in high levels in some low-proliferation tissues, indicating tight
control of its activity after its production. bFGF is at least partly
regulated by its extracellular export. 177,178 In addition, binding
of the FGF receptor by bFGF requires the presence of an
ECM/basement membrane proteoglycan such as perlecan. 179
Capillary endothelial cells express and secrete bFGF and thus
induce their own proliferation and migration. 180 bFGF signaling
cross-talks with additional angiogenic pathways. Hypoxic
induction of HIF-1 in endothelial cells is bFGF dependent. 181
bFGF signaling has been shown to activate transcription of the
PDGF receptor, whereas PDGF-BB amplified bFGF receptor
expression. 182 bFGF has a major role in angiogenesis, but its
involvement in many other pathologic and normal processes
makes it a problematic therapeutic target.
The overexpression of FGF-10 in respiratory epithelial
lung cells in mice induced multifocal pulmonary adenomas
within 1 to 4 weeks. Interestingly, all these tumors regressed
shortly after withdrawal of the transgene-activating agent,
indicating that no irreversible carcinogenic change occurred
and that the tumors were dependent on FGF-10 expression.
Angiogenesis measures were not reported for this model, but
given the importance of the FGF pathway in tumor angiogenesis,
an angiogenic switch was plausibly involved. 183
Chemokines Chemokines are small (8–10 kDa) proteins that
regulate mainly leukocyte trafficking. Various chemokines also influence
angiogenesis, both negatively and positively. The C-X-C
chemokine family can be divided to two subfamilies. Those that
contain a Glu-Leu-Arg motif at the NH3- terminus (ELR ), activate
the CXCR2 receptor, and are mostly angiogenic. CXC chemokines
that lack this motif (ELR ), act through CXCR3 and
are angiostatic. Platelet factor-4 (PF-4, also called CXCL4) is an
example of an ELR chemokine, which is a strong inhibitor of
angiogenesis. A variant of PF-4/CXCL4 exists, which is a stronger
inhibitor of angiogenesis than PF-4/CXCL4. 184 IL-8/CXCL8 is
an ELR chemokine and is indeed a potent angiogenic factor. 82
SDF-1/CXCL12 is an exception to the aforementioned rule,
being an ELR chemokine, but activating the CXCR4 receptor,
and thought to have an angiogenic effect. 185 In a mouse model of
lung cancer, inhibition of SDF1/CXCL12 abrogated metastasis
formation but did not affect angiogenesis, 35 suggesting it might
not have a major angiogenic role in lung cancer. The C-X-C chemokines
act through the activation of G-protein–coupled serpentine
(seven-transmembrane spanning) receptors.
Cell–Cell Adhesion Molecules The various cellular events
that occur during the formation of new blood vessels involve
interactions among endothelial cells, pericytes, smooth muscle
cells, inflammatory cells, and epithelial cells that are governed
mainly by cell–cell adhesion molecules. Several of these molecules
have a role in modulating angiogenesis.
Platelet endothelial cell–cell adhesion molecule-1
(PECAM-1 or CD31) is commonly used as a marker of endothelial
cells in immunohistochemical studies. 186 Binding
of this molecule by an inhibitory antibody suppresses angiogenic
activity. 187 PECAM-1 is expressed also by platelets
and inflammatory cells. It plays a role in leukocyte migration,
and is involved in several signaling pathways. 186 PECAM-1
might contribute to endothelial cells function. It can also be
speculated that the contribution of this molecule to angiogenesis
might be through recruitment of inflammatory cells, or
through the anchoring of platelets in angiogenic sites. Platelets
seem to have an important role in storing and delivering angiogenic
and antiangiogenic factors. 97,188
Intercellular adhesion molecule 2 (ICAM-2), a member of
the immunoglobulin superfamily, is a transmembrane protein
that is involved in binding various integrins and other molecules.
In addition, through homophilic interactions (binding
the same protein expressed on another cell), ICAM-2 is involved
in endothelial cell survival and migration. It is commonly
expressed in endothelial cell–cell junctions 189 and was
found in a mouse model to be involved in angiogenesis. Mice
that did not express this protein were defective in angiogenesis
in in vivo assays, and endothelial cells from these mice demonstrated
defective migration and increased apoptosis. 190
Importantly, plasma ICAM levels were found to be prognostic
in a clinical study of lung cancer patients treated with an anti-
VEGF antibody (see later). 191
Cadherins are transmembrane proteins that populate adherence
junctions. The extracellular domains of cadherins form
calcium-dependent, homophilic transdimers when they bind
similar proteins on neighboring cells, mediating cell–cell adhesions.
The cytoplasmic tails of cadherins bind to several potential
signaling proteins, most notably -catenin, a transcriptional
cofactor in Wnt signaling. -catenin bound to a cadherin intracellular
domain participates in cell–cell interactions and does
not function as a transcription factor. When cadherin membranous
localization is disrupted, -catenin is released from the
cytoskeleton and can enter the nucleus, where it functions as
a transcription factor with oncogenic features. Membranous
E-cadherin expression in NSCLC tumor specimens was found
to be prognostic. 192–194 Tumors that expressed low levels of
membranous E-cadherin were more likely to metastasize to regional
lymph nodes, accompanied by reduced survival. It can
be speculated that low membranous localization of E-cadherin
allows enhanced oncogenic transcriptional activity of -catenin,
and an angiogenic switch (see succeeding discussion).
TGF- and EGFR Pathway Transforming growth factor
(TGF)- is a pleiotropic factor that can have different effects
on different cells. It is required for endothelial differentiation
195 and induces angiogenesis in vivo 196 ; however, in a
different experimental setup, disruption of TGF- signaling
induced angiogenesis and tumor formation. 197 TGF- was
shown to cause endothelial cell apoptosis, which appears to be
essential for angiogenesis. 198
This apparent correlation between endothelial cell apoptosis
and enhanced angiogenesis underscores the complexity
of angiogenesis and the potential biases of studies that use endothelial
cell proliferation or survival as a surrogate for the in
vivo end point. Importantly, TGF- levels in adenocarcinoma
NSCLC tumor samples were correlated positively with MVD
and negatively with prognosis. 199 Tumor endothelial cell survival
involves also the EGFR pathway, demonstrated to depend
on EGF secretion by tumor cells. 200 ERK activation downstream
of EGFR was found to activate a crosstalk between cancer
cells and endothelial cells that promotes angiogenesis. 201
Some prostaglandins (a large group of signal mediators) also
induce angiogenesis; the most notable of these is prostaglandin
E2. The angiogenic effect of prostaglandin E2 is dependent on
TGF- and MMPs. 202
Nitric Oxide Nitric oxide is a small signaling molecule
that is involved in many biologic phenomena. It affects angiogenesis
both negatively and positively, depending on its
concentration. 203 For example, nitric oxide regulates thrombospondin
1 levels in a triphasic manner. 204 It participates in
the biologic response to both angiogenic and antiangiogenic
factors, possibly through their effects on nitric oxide synthase.
The nitric oxide synthase 2 isoform was found to be correlated
with VEGF levels and MVD in 106 NSCLC tissue
specimens. 205 Treatment with nitric oxide donors combined
with chemotherapy given to NSCLC patients, improved their
outcome in a large randomized phase II trial. 206 The complex
and dosage-dependent effects of nitric oxide on various aspects
of cancer biology makes it difficult to predict the possible effect
it might have had on angiogenesis in those lung cancers.