|
Angiogenesis Research: Guidelines for Translation
to Clinical Application |
Judah Folkman,
Timothy Browder, Jan Palmblad1
Children’s Hospital and Harvard Medical
School, Boston, Massachusetts, USA and
1Huddinge University Hospital
and the Karolinska Institute, Stockholm,
Sweden
Correspondence to: Judah Folkman, M.D.,
Children’s Hospital, Hunne-well 103, 300
Longwood Avenue, Boston, Massachusetts
02115, USA — Tel.: 1.617-355-7661; Fax:
1.617-355-7662; E-mail: judah.folkman@tch.Harvard.edu |
Key words
Angiogenesis, leukemia, antiangiogenic
therapy, angiogenesis inhibitor |
Summary
Angiogenesis research is being translated
to the clinic. Certain guidelines may
facilitate this effort. Recruitment of
endothelial cells by a tumor is an early
event in angiogenesis, a process regulated
at genetic and epigenetic levels. The
microvascular endothelial cell has become
an important second target in cancer therapy.
Angiogenesis inhibitors are either "direct"
or "indirect" and their optimal dosing
depends on a different logic than conventional
chemotherapy. Conversely, antiangiogenic
scheduling of chemotherapy can by-pass
drug resistance. Like all solid tumors,
hematologic malignancies are angiogenesis-dependent.
Further, angiogenesis is modulated by
proteins and cells from the hematopoietic
and hemostatic systems. Clinical testing
of angiogenesis inhibitors has accentuated
the need for surrogate markers of tumor
angiogenesis activity. Microvessel density,
so valuable as a prognostic indicator
of metastatic risk, cannot determine efficacy
of an angiogenesis inhibitor. In the future,
angiogenesis inhibitors may be added to
chemotherapy or to radiotherapy, or to
other modalities. Also, combinations of
angiogenesis inhibitors may be administered
together.
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Introduction
These laboratory studies are currently
being translated into clinical trials
for patients with neoplastic and non-neoplastic
diseases. Because angiogenic and antiangiogenic
therapies differ in many respects from
conventional therapies, it may be useful
to examine how an understanding of the
angiogenic process per se can facilitate
its clinical application. |
| Microvascular
Endothelium — A Second Target in Cancer |
The cancer cell
has been virtually the sole target of
various anticancer therapies over the
past half-century. The cancer genome however,
"appears to be far more unstable than
previously thought" (12). "The time-dependent
nature of the cancer genome complicates
matters further. Aberrations continually
accrue which alter the character of both
the primary tumor and its metastases."
The resulting high mutation rate of the
cancer cell permits it to eventually escape
most chemotherapies or immunotherapies
directed to it.
In contrast, the microvascular endothelial
cell has a stable genome and an extremely
low or non-detectable mutation rate. Microscopic
or barely visible in situ tumors
cannot usually expand to a larger tumor
until after they have recruited
new capillary blood vessels. The endothelial
cells which line these microvessels are
now considered to be a powerful control
point in tumor growth. While it is has
long been recognized that the microvessels
generated by endothelial cells provide
oxygen and nutrients and remove catabolites
and carbon dioxide, the paracrine role
of endothelial cells in a tumor has only
recently been appreciated. Endothelial
cells are known to supply at least 20
different growth factors and antiapoptotic
factors to tumor cells (13). Each endothelial
cell controls the growth of approximately
50 to 100 tumor cells (7) (Fig. 1). As
a result, the microvascular endothelial
cell, recruited by a tumor, has become
an important second target in cancer
therapy. |
| |
Cross-section
of human breast cancer (MCa-IV) in a mouse
showing the microcylinders of tumor cells
that surround each vessel. Large and small
thin-walled microvessels in breast tumor
labeled by vascular perfusion of green
(FITC) fluorescent lectin staining. The
perivascular cuff of tumor tissue is 100
microns thick, which is within the range
of the oxygen diffusion limit. Two endothelial
cells (red cytoplasm with white nuclei)
have been drawn in the lumen to approximate
scale. Yellow tumor cells with brown-red
nuclei occupy the perivascular cuff of
tumor tissue. Each microvessel is surrounded
by a cuff of tumor cells. At this high
power different types of cancer would
look similar except for small differences
in intercapillary distances. Vessels were
preserved in the open state by vascular
perfusion of fixative. (Courtesy of Donald
M. McDonald, University of California,
San Francisco), (with permission from
[101]). [Drawings of superimposed cells
by J. Folkman and Kristin Gullage]
Angiogenesis inhibitors work directly
or indirectly (see below) to selectively
or specifically inhibit proliferating
and migrating endothelial cells. These
inhibitors have emerged as a new class
of drugs. Endothelial cells, because of
their stable genome, do not usually acquire
resistance to an inhibitor which targets
them directly (14, 15). Antiangiogenic
therapy and cytotoxic chemotherapy are
not mutually exclusive. It has been proposed
that treating both the cancer cell and
the microvascular endothelial cell in
a tumor, may be more effective than treating
the cancer cell alone (7, 8). Clinical
trials with combinations of an angiogenesis
inhibitor plus cytotoxic chemotherapy,
as well as trials in which an angiogenesis
inhibitor is added to radiotherapy are
now in progress. Furthermore, at least
20 novel angiogenesis inhibitors themselves
are in clinical trials in more than 100
medical centers in the USA, as well as
in other centers in the U.K. and in Europe
(16) (Table 1). |
| |
Table1
At least 22 angiogenesis inhibitors are
in clinical trials in more than 100 medical
centers throughout the USA. Of these 7
have reached Phase III. Others are being
developed and tested in Europe and the
UK
At least seven angiogenesis inhibitors
have reached Phase III. Eventually physicians
should be able to choose from a group
of angiogenesis inhibitors. These inhibitors
may be administered in combinations with
each other, or with conventional therapies,
or with new modalities. The immediate
goal of introducing first generation angiogenesis
inhibitors into the clinic is to reduce
the harsh side-effects of cytotoxic chemotherapy
and to decrease the risk of drug resistance. |
Direct
vs. Indirect Angiogenesis Inhibitors
Angiogenesis inhibitors which block
the endothelial cell locomotion or proliferation
induced by angiogenic factors such as
vascular endothelial growth factor (VEGF)
or basic fibroblast growth factor (bFGF)
(among others), can be considered as
direct angiogenesis inhibitors
(Fig. 2).
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Fig.2
Diagram of the endothelial cell compartment
and the tumor-cell compartment in a tumor.
Each cell population stimulates growth
of the other by releasing mitogens and
survival factors. This figure also illustrates
the critical difference between "direct"
and "indirect" antiangiogenic therapy
in terms of the risk of relapse. "Direct"
antiangiogenic therapy, which specifically
targets endothelial cells, has a low risk
of relapse. In contrast, "indirect" antiangiogenic
therapy which depends on blockade of tumor-derived
angiogenic protein(s), has a higher risk
of relapse because tumor cells may eventually
appear that release a different angiogenic
protein. For example, a tumor that is
producing mainly VEGF may eventually generate
a subpopulation of tumor cells that produces
bFGF. Conventional chemotherapy, which
targets mainly tumor cells, commonly induces
drug resistance. From (102), with permission
For example, the angiogenesis inhibitor,
pigment epithelium derived factor (PEDF)
inhibited endothelial cell migration in
the presence of a wide range of positive
regulators of angiogenesis including,
aFGF, VEGF, PDGF, IL-8, and lysophosphatidic
acid. PEDF also inhibited endothelial
proliferation induced by bFGF (17). Angiostatin,
endostatin, TNP-470 (18) and thrombospondin
are other examples. Direct angiogenesis
inhibitors induce little or no drug resistance
in their endothelial cell targets (14,
15) and therefore can be administered
for prolonged periods of time without
relapse. The very low mutation rate of
endothelial cells and of bone marrow cells
may account for their lack of "resistance"
to antiangiogenic therapy and to cytotoxic
chemotherapy respectively (19), in contrast
to the high mutation rate of most cancer
cells. An indirect angiogenesis inhibitor
inhibits a tumor cell’s production of
an angiogenic factor, neutralizes the
angiogenic factor itself, or blocks its
receptor on endothelial cells. Examples
of FDA approved indirect angiogenesis
inhibitors are interferon-alpha (20),
which inhibits production of bFGF by tumor
cells (21) and Herceptin which inhibits
VEGF production by tumor cells (98). Because
an indirect angiogenesis inhibitor blocks
a tumor cell product, i.e., a specific
angiogenic protein, the emergence of a
mutant tumor cell which produces a different
angiogenic protein may nullify the effectiveness
of the angiogenesis inhibitor. This hypothesis
has not yet been formally demonstrated
in animals, but it serves to distinguish
the "relapse" which may occur after prolonged
administration of an indirect angiogenesis
inhibitor, from the classical drug "resistance"
which emerges after prolonged administration
of a cytotoxic agent.
If this distinction holds up, it suggests
that in the case of "relapse" following
prolonged administration of an indirect
angiogenesis inhibitor, that the inhibitor
should not be discontinued. Instead, a
different angiogenesis inhibitor could
be added which blocks the new angiogenic
factor from the mutant clone of tumor
cells. For example, the cause of a relapse
on Herceptin could be the emergence of
mutant tumor cells producing bFGF or IL-8
(interleukin-8). Because discontinuation
of Herceptin would leave VEGF production
unopposed, it might be prudent to add
an angiogenesis inhibitor which blocked
bFGF or IL-8. Some angiogenesis inhibitors
such as 2-methoxyestradiol (22), may target
both endothelial cells and tumor cells
in a tumor.
Titration
of Antiangiogenic Therapy versus
Maximum Tolerated Dosing for Conventional
Chemotherapy
Experimental and clinical studies reveal
that administration of antiangiogenic
therapy for cancer requires a different
logic than conventional cytotoxic therapy.
Maximum tolerated dosing is the hallmark
of most chemotherapy. The dose is set
by what the host can tolerate; the choice
of chemotherapeutic agent is determined
by tumor type.
In contrast, the dose of an angiogenesis
inhibitor is best titrated against the
total angiogenic output of a tumor. Angiogenic
output can be defined operationally as
the sum total of angiogenic activity from
positive regulators of angiogenesis released
by a tumor (e.g., bFGF, VEGF, IL-8, etc.)
minus the antiangiogenic activity due
to negative regulators of angiogenesis
generated by a tumor (e.g., thrombospondin,
angiostatin, endostatin, etc.) (23-26).
The angiogenic output of a primary tumor
may differ from that of its metastases
and may also differ among tumors of the
same type, e.g., breast cancers. Toxicity
to the host is usually not limiting, at
least for direct angiogenesis inhibitors
(i.e., angiostatin or endostatin) (Table
2). |
| |
Table2
Differences between cytotoxic chemotherapy
and direct and indirect antiangiogenic
therapy. The major difference is that
for cytotoxic chemotherapy, dose is the
maximum tolerated by the host and choice
of agent isdictated by tumor type. In
contrast, for a direct angiogenesis inhibitor,
dose is titrated to the angiogenic output
of the tumor; a maximum tolerated dose
has not yet been found for some direct
inhibitors. Choice of a direct angiogenesis
inhibitor is not dictated as stringently
by tumor type as is choice of chemotherapy
However, an urgent challenge for future
angiogenesis research is to develop an
assay based on a simple blood or urine
test that could quantify the total angiogenic
output of a patient’s tumor or tumor burden.
At the least, a test is needed to detect
the presence or disappearance of angiogenic
activity in the body. It would be unnecessary
to locate the angiogenic site. Angiogenesis
is such a rare event in the body and physiological
angiogenesis is so brief, that detection
of persistent or abnormally intense angiogenic
activity should suffice. In current clinical
trials the determination of an optimum
dose for an angiogenesis inhibitor relies
on stabilization or regression of tumor
growth. Microvessel density of tumor biopsies,
while useful as a prognostic indicator
of future risk of metastasis or mortality
(7), may not be helpful as a measurement
of the angiogenic activity of a tumor
(see below for the basis of this argument).
Plasma or urinary levels of angiogenic
factors such as VEGF or bFGF have not
yet been demonstrated to reliably correlate
with total angiogenic output, presumably
because it would be necessary to know
the whole range of positive and negative
regulators released by a tumor. In the
absence of a method to quantify angiogenic
output, titration of dosing for an angiogenesis
inhibitor is difficult, not unlike administering
insulin without a blood glucose test or
coumadin without a prothrombin test. Because
E-selectin is highly up-regulated on a
focus of proliferating or activated endothelium
(27), the measurement of soluble circulating
E-selectin is one possible candidate for
a surrogate marker of angiogenic activity
in a tumor (28).
Certain Cytotoxic Chemotherapeutic
Agents Also Inhibit Angiogenesis
Conventional chemotherapy must traverse
vascular endothelium to reach tumor cells.
Therefore, cytotoxic agents should in
principle, inhibit angiogenesis. But,
if certain cytotoxic agents have antiangiogenic
activity, why does cytotoxic chemotherapy
induce drug resistance? Timothy Browder
in Folkman’s laboratory (19) proposed
that the traditional dose-schedule regimen
for chemotherapeutic agents fails to provide
the sustained blockade of angiogenesis
achieved by an angiogenesis inhibitor.
Conventional chemotherapy is usually administered
at a maximum tolerated dose up-front,
followed by an extended treatment-free
interval to allow recovery of bone marrow
and gastrointestinal tract (7). During
the period of an average treatment-free
interval of 2 -3 weeks, microvascular
endothelial cells in the tumor bed may
also recover and resume their proliferation.
The resulting new microvessels could support
regrowth of tumor cells and increase the
risk of emergence of drug-resistant tumor
cells. By more frequent administration
of a standard cytotoxic agent — cyclophosphamide,
without a treatment-free interval and
at an overall lower dose, Browder achieved
a more sustained apoptosis of endothelial
cells in the vascular bed of a murine
tumor (19). This "antiangiogenic schedule"
of chemotherapy more effectively controlled
tumor growth in mice, regardless of whether
the tumor cells were drug resistant. The
antiangiogenic schedule of cyclophosphamide
dramatically reduced side-effects and
eliminated bone marrow suppression. This
improvement in chemotherapy resulted from
the application of new logic to an old
drug.
These results in mice may help to explain
why some patients who are receiving long-term
maintenance or even palliative chemotherapy
continue to have stable disease beyond
the time that the tumor cells would have
been expected to develop drug resistance.
Furthermore antiangiogenic scheduling
may explain the improved outcome of empiric
treatment of slower growing human cancers
employing continuous infusion 5-fluorouracil
(29-31), weekly paclitaxel (32-34) or
daily oral etoposide (35-37). Robert Kerbel
also reported that frequent low dosing
of vinblastine was antiangiogenic and
that it improved control of tumor growth
(38). Antiangiogenic chemotherapy has
also been called"metronomic therapy" (39).
These data further suggest that other
chemotherapeutic agents may have antiangiogenic
activity in addition to their cytotoxic
activity (40). Moreover, vascular targeting
of very low doses of cytotoxic agents
may increase antiangiogenic activity.
For example, extremely low concentrations
of doxorubicin conjugated to vascular
integrin-binding peptides were targeted
to the angiogenic vessels in a tumor and
led to significant tumor suppression without
side-effects on host tissues (41).
Leukemia
and Other Hematologic Malignancies Are
Angiogenic
It has long been thought that leukemias
and other hematologic malignancies differ
from solid neoplasms by neither stimulating
nor requiring angiogenesis. However, in
the past 8 years this view has changed
radically. In 1993, in an address before
the American Society of Hematology, Folkman
presented a hypothesis that the leukemias
could be angiogenesis-dependent (42).
This idea was based on his finding that
the angiogenic protein, bFGF was abnormally
elevated in the urine of newly diagnosed
leukemic patients to higher levels than
in any other malignancy (43), taken together
with the finding by Brunner et al. (44)
that bone marrow cells expressed bFGF.
A year later, Vacca and colleagues showed
that bone marrow was angiogenic in patients
with multiple myeloma (45). They employed
a method previously described to quantify
angiogenesis in breast cancer by microvessel
density (46) to determine that angiogenesis
in the bone marrow of patients with multiple
myeloma was 5-times higher than normal.
This finding was confirmed by subsequent
reports of increased angiogenesis in multiple
myeloma (47, 48). In 1995 came the first
report that bone marrow was highly neovascularized
in a B-cell leukemia (49) and two years
later bone marrow biopsies from children
with newly diagnosed untreated acute lymphoblastic
leukemia revealed a 6 to 7-fold increase
in microvessel density in contrast to
control bone marrows from children undergoing
staging evaluations at the time of diagnosis
of solid tumor (50). This study also demonstrated
by confocal microscopy the three-dimensional
packing of new microvessels in leukemic
bone marrow (Fig. 3). |
| |
Fig.3
Confocal microscopic sections of bone
marrow biopsies. Microvessels stained
with antibody to von Willebrand factor,
from children (a.) without leukemia and
(b.) with newly diagnosed untreated acute
lymphoblastic leukemia. Intense neovascularization
is observed in the leukemic bone marrow
(b). From (7) with permission
The close configuration of neoplastic
cells and vascular endothelial cells was
similar to solid tumors. Urinary levels
of the angiogenic protein, bFGF were approximately
7-fold higher in the leukemic children
than in controls. By 1999, cellular VEGF
was shown to be a predictor of outcome
in patients with multiple myeloma (51).
A year later it was clear that angiogenesis
per se was a prognostic indicator in multiple
myeloma (52) and further that in myelofibrosis
and in acute myeloid leukemia that blood
vessels were "highly malformed with numerous
branches, pathological caliber changes
and other features" (53). At the American
Society of Hematology meeting in 2000
there were more than 100 reports of increased
bone marrow angiogenesis in a wide variety
of hematological malignancies (54) and
it is now clear that malignant cells in
these diseases may recruit new microvessels
by similar molecular signals as tumor
cells in solid tumors. In fact, the myeloproliferative
diseases polycythemia vera, chronic myelocytic
leukemia and myelofibrosis, all reveal
significantly increased neovascularity
(53, 55) (for review see [56]).
However, the induction of angiogenesis
per se does not prove angiogenesis-dependence.
While the hypothesis first proposed by
Folkman in 1971 that solid tumors are
angiogenesis-dependent (1) has been confirmed
by molecular and genetic methods (for
review see [7]), preliminary evidence
that a hematologic malignancy may be angiogenesis-dependent
has just been reported this year (2001)
from experiments in which gene therapy
with the angiogenesis inhibitors angiostatin
(24) and endostatin (25), inhibited experimental
leukemia in mice (57). Also, Browder in
the Folkman laboratory has shown that
mice inoculated with B-cell, T-cell or
myelogenous leukemias and treated only
with endostatin, live significantly longer
(and without toxicity) than if treated
with any other conventional chemotherapy
(personal communication, unpublished data).
In 1994, D’Amato and Folkman reported
that thalidomide is an angiogenesis inhibitor
(58). Corneal neovascularization in rabbits
induced by bFGF or VEGF was blocked by
orally administered thalidomide. Histologic
sections of the pre-treated neovascularized
corneas were virtually free of inflammatory
cells. Thalidomide also inhibited corneal
neovascularization in mice, but higher
doses were required because mice do not
metabolize thalidomide effectively. Thalidomide
is a very weak inhibitor of tumor-necrosis
factor-alpha (TNF-alpha), but experimental
evidence suggests that the antiangiogenic
activity of thalidomide is not related
to TNF-alpha suppression. Potent inhibitors
of TNF-alpha do not inhibit corneal angiogenesis
(Table 3). |
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Table3
Compounds which inhibit activity of tumor
necrosis factor-alpha (TNF-alpha) more
potently thanthalidomide, do not inhibit
angiogenesis in the mousecornea, but thalidomide
does inhibit angiogenesis (despite the
fact that it is the weakest inhibitor
of TNF-alpha in this series)
Thus, pentoxifylline has approximately
the same anti-TNF-alpha activity as thalidomide,
but thalidomide inhibits cornea angiogenesis
and pentoxifylline does not. Furthermore,
dexamethasone is a more potent inhibitor
of TNF-alpha production by stimulated
blood monocytes than thalidomide (47%
vs. 31% respectively), but dexamethasone
does not inhibit corneal neovascularization
or is much weaker than thalidomide. Finally,
ibuprofen increases TNF-alpha in the serum
of mice by 2-fold, but it inhibits angiogenesis!
The first report of thalidomide therapy
for multiple myeloma was in 1999 (59).
In patients with disease refractory to
all other therapy, there was a 32% response
as assessed by reduction of myeloma protein
in serum, or Bence Jones protein in urine
that lasted for at least 6 weeks. Two
patients out of 76 achieved complete remission.
These results have been confirmed by numerous
groups (60-65) (for review see [56]).
As our understanding of the role of angiogenesis
in hematological malignancies continues
to expand, we are provided with a conceptual
basis for the future use of angiogenesis
inhibitors in these diseases. Angiogenesis
inhibitors are becoming available both
from clinical trials and also from drugs
found to inhibit angiogenesis after they
had been approved for another use. These
include: interferon alpha (20); celecoxib
(66); rosiglitazone (67); arsenic trioxide
(68); and thalidomide (58). Therefore,
hematologists and oncologists can now
choose from an enlarging family of FDA
approved angiogenesis inhibitors to design
clinical trials for patients with hematological
malignancies refractory to conventional
therapies.
Increased microvessel density in bone
marrow is associated with refractory multiple
myeloma or in previously untreated multiple
myeloma in virtually all reports to date.
However, some authors report that increased
angiogenesis persists even after complete
response (59). Also, increased bone marrow
angiogenesis has been reported to persist
in multiple myeloma after complete response
to stem cell transplantation (69). The
failure of microvessel density to decrease
coincident with remission of disease has
been interpreted to mean that thalidomide
is operating by some mechanism other than
inhibition of angiogenesis (70). We believe
this to be an erroneous interpretation
based on the misperception that measurements
of microvessel density made during therapy
may be used to evaluate the efficacy of
antiangiogenic agents.
In the early 1990s Weidner et al., demonstrated
the validity of tumor microvessel density
as an independent prognostic indicator
of risk of metastasis and/or mortality
in breast (46, 71) and prostate cancer
(72). Since then, the use of microvessel
density as an independent prognostic indicator
has been confirmed in many other tumor
types in more than 100 reports (for review
see [7] and [73]). Although some studies
indicate no correlation, the majority
support the predictive value of microvessel
density for the future outcome of a patient.
However, a tumor’s microvessel density
is not a measure of ist degree of angiogenic
dependence (for review see [74]). Microvessel
density is a measure of vessel count per
high power field which itself is governed
by intercapillary distance. Intercapillary
distance is determined by cuff size of
tumor cells packed around each capillary
vessel. Cuff size is inversely related
to tumor metabolic demand. Tumors which
have high oxygen/nutrient demands have
a small average cuffsize. While a minimal
vessel density is usually determined by
tumor cell metabolic demand, vessel density
can greatly exceed the metabolic demands
of a tumor. Angiogenic factor expression
can become uncoupled from normal regulatory
controls and angiogenic factors can be
constituitively expressed at high levels.
The measurement of microvessel density
is not predictive for tumor response to
antiangiogenic therapy. Nor is microvessel
density a measure of the total angiogenic
output of a tumor. While a decrease in
microvessel density during antiangiogenic
therapy implies that the agent is working,
the absence of a decrease in microvessel
density does not mean that the antiangiogenic
agent is not working. This is because
under antiangiogenic therapy, endothelial
apoptosis and/or capillary dropout precedes
tumor cell dropout (19). In certain animal
tumors, a large tumor may regress to a
microscopic size with no change in microvessel
density. The small residual tumor will
have the same microvessel density as the
original untreated tumor, because the
remaining tumor cells have accumulated
around the few remaining microvessels
without a change in cuff size. Conversely,
in tumors in which neovascularization
has exceeded metabolic demand, inhibition
of an overexpressed angiogenic factor
will be followed by rapid reduction in
microvessel density until the minimum
required vessel density is reached. In
fact, in a very over-vascularized tumor,
significant capillary dropout and a decrease
in microvessel density could occur without
a slowing of tumor growth — until a minimum
required microvessel density had been
reached.
When these results are understood, clinical
investigators should be able to avoid
the trap of using microvessel density
to determine efficacy of an angiogenesis
inhibitor during the course of treatment.
Thus, the determination of microvessel
density by repeated biopsies during the
course of antiangiogenic therapy is unlikely
to be of value.
The Hemostatic
System as a Regulator of Angiogenesis
Angiogenesis and blood coagulation share
several characteristics. Both processes
remain quiescent for prolonged periods
of time, but can be rapidly activated
during wound healing. Both processes share
certain proteins. "Proteins generated
by the hemostatic system coordinate the
spatial localization and temporal sequence
of clot/endothelial cell stabilization
followed by endothelial cell growth and
repair of a damaged blood vessel" (75).
Platelets contain at least 14 positive
regulators of angiogenesis including VEGF-A,
VEGF-C, bFGF, hepatocyte growth factor
(HGF), angiopoietin-1, insulin-like growth
factor-1, epidermal growth factor (EGF),
platelet-derived growth factor (PDGF),
and sphingosine 1-phosphate (Table 4).
Serum levels of VEGF are significantly
higher than plasma levels. In fact, serum
levels of VEGF highly correlate with platelet
counts during chemotherapy (76) and may
not necessarily reflect tumor burden.
For this reason serum VEGF may not be
a useful surrogate marker for efficacy
of cancer therapy. We have observed that
large hemangiomas or hemangioendotheliomas
which trap platelets (Kassabach-Merritt
syndrome) can rapidly develop edema (within
an hour) after a platelet transfusion
administered to treat thrombocytopenia
(personal observation, J.F.). This edema
may be due to vascular leakage caused
by VEGF (VPF) released from trapped platelets.
For this reason, we refrain from administering
platelets in children with large hemangiomas,
especially around the airway or in the
mediastinum, unless thrombocytopenia is
life-threatening. Platelets have been
reported to contribute to metastasis formation
and tumorgrowth (see [76] for review).
Angiogenesis in these lesions may be stimulated
by the VEGF transported by platelets (76).
"Inhibition of formation and growth of
metastases in animals by thrombopenia,
antiplatelet therapy, or administration
of anticoagulants," may operate in part
by prevention of platelet trapping or
release of VEGF in tumors (76).
Platelets also contain inhibitors of angiogenesis,
including platelet factor 4, thrombospondin-1,
TGF-beta1, plasminogen activator inhibitor
type-1 (PAI-1), alpha2-antiplasmin and
alpha2-macroglobulin (Table 4).
Among the approximately 40 proteins which
regulate the hemostatic system are found
at least 6 proteins which contain cryptic
angiogenic regulators (Fig. 4 and Table
4). |
| |
Fig.4
Cryptic antiangiogenic fragments within
the hemostatic system. Cryptic fragments
derived from coagulation and fibrinolytic
proteins which suppressangiogenesis are
indicated in bold red. Coagulation
factors are depicted by Roman numerals
and activation is indicated by a small
a. Coagulation cascade andfibrinolytic
pathway inhibitors are indicated by a
dashed arrow. C1-INH, complement
factor-1 esterase inhibitor; PL,
anionic phospholipids; TF, tissue
factor; TFPI, tissue factor pathway
inhibitor; tPA, tissue-type plasminogen
activator. From (75) with permission |
| |
Table4
Positive
and negative regulators of angiogenesis
carried by platelets
These can be released by enzymatic cleavage
(75). We speculate that "during the first
days of wound healing as nascent clot
bridges and stabilizes the vessel defect,
any initiation of angiogenesis directed
by platelet-derived angiogenic stimulators,
or by thrombin and fibrin, must be counteracted",
temporarily in order to prevent re-bleeding
(75). Thus, early endothelial mobilization
into a clot may be prevented by negative
regulators of angiogenesis in platelets
as well as by cryptic fragments released
from clotting proteins. Subsequently,
endothelial mobilization into the clot
is induced so that vessel repair can proceed.
Very little is known about the coordination
and regulation of this intricate mechanism
of early suppression and later stimulation
of angiogenesis in a clot, but it is a
fruitful area for future research.
Role of Bone Marrow-Derived Endothelial
Precursors in Angiogenesis at Other Sites
In the past 4 years it has been discovered
that circulating cells from bone marrow
can be identified as precursor endothelial
cells by specific markers for endothelial
cells (87-94). These cells have been found
to incorporate themselves into experimental
neovascular foci of VEGF in subcutaneous
Matrigel pellets or into the vascular
bed of tumors. However, most investigators
have found that in a tumor vascular bed,
not more than 1-2% of endothelial cells
are bone marrow-derived and even these
do not appear to be proliferating (Thomas
Quertermous, personal communication).
In fact, in our own experience (Shay Soker,
personal communication to J.F.), many
of the bone marrow derived endothelial
cells which reach a tumor vascular bed,
come to rest in the peri-vascular tissue
among pericytes. Therefore, at this writing
it is not clear what role bone marrow-derived
endothelial precursor cells play in tumor
angiogenesis. However, Lyden et al. (5)
reported that tumor cell inoculations
will not induce neovascularization in
mice carrying deletions of one allele
of Id1 and 2 alleles of Id3. The tumors
remain dormant and avascular. If these
mice then receive a bone marrow transplant
from wild-type Id1/Id3 mice, circulating
endothelial cells from this bone marrow
arrive at the tumor in sufficient numbers
to produce a neovascular bed which permits
rapid growth of the tumor, metastases,
and death of the host (Robert BenEzra,
personal communication).
Recently, Kocher et al., reported that
adult human bone marrow contains endothelial
precursors with phenotypic and functional
characteristics of angioblasts (95). When
these endothelial precursors were injected
intravenously into athymic nude rats with
acute myocardialinfarction, the injected
cells arrived in the infarct-bed where
they induced neovascularization. The neoangiogenesis
prevented apoptosis of cardiac myocytes,
an interesting result which may be analogous
to the endothelial-derived paracrine factors
released in tumor angiogenesis which are
mitogenic and anti-apoptotic for tumor
cells (13).
To those investigators working in the
field of angiogenesis research, the recently
discovered role of bone marrow endothelial
precursors asa source of angiogenesis
in tumors or in ischemic tissue, is an
exciting new direction.
Tumor Angiogenesis
is Regulated by Oncogenes and Tumor Suppressor
Genes
The switch to the angiogenic phenotype
has been documented in animal and human
tumors as the point at which an in
situ non-angio-genic tumor that is
dormant at a microscopic size or a size
of less than 1-2 mm, begins to recruit
new capillary blood vessels (96). Recent
evidence (reviewed by Kerbel et al. [97]
and by Rak et al. [98], indicates that
activated or over-expressed oncogenes
which are responsible for increased mitogenesis
and resistance to apoptosis in neoplastic
cells, also encode protein products which
drive tumor angiogenesis. Some oncogenes
also concomitantly down-regulate proteins
which normally inhibit angiogenesis. Certain
tumor suppressor genes are now known to
normally code for proteins which inhibit
angiogenesis. These findings indicate
that angiogenesis is regulated at a genetic
level in addition to its epigenetic regulation
by changes in oxygen or pH. These results
also have important implications for development
of novel angiogenesis inhibitors, and
also for understanding how to optimally
design clinical trials for such therapeutic
agents, and how to best use them in cancer
patients.
Oncogenes which are pro-angiogenic are
listed in Table 5 (from 98). For example,
K-ras and H-ras upregulate
VEGF a positive regulator of angiogenesis
and downregulate thrombospondin-1, an
inhibitor of angiogenesis. v-src acts
similarly. c-myb downregulates
thrombospondin2. HER-2 upregulates
VEGF expression, and EGFR upregulatesthree
angiogenic proteins, VEGF, bFGF and IL-8.
In contrast, p53 is a prime example of
a tumor suppressor gene which negatively
regulates angiogenesis by activating expression
of thrombospondin (17). |
| |
Table5
Examples
of oncogenes which are pro-angiogenic
either because they upregulate expression
of an angiogenic protein, or because they
downregulate expression of an angiogenesis
inhibitor, or because they do both. (Table
assembled from data in [13])
When an oncogene is used as a target to
screen for small molecular weight anticancer
drugs, it is not unusual to find that
the novel drug turns out to be an "indirect"
angiogenesis inhibitor in addition to
ist antimitogenic effect or apoptotic
effect on tumor cells. Some examples are
diagrammed in Figure 5 (assembled from
[13, 97, 98]). |
| |
Fig.5
Examples
of oncogenes or potential oncogenes which
are pro-angiogenic and the small molecules
or monoclonal antibodies which have been
developed to block the oncogenes. ras,
HER-2/neu, and EGF adapted
from (98) and Bcr-Abl adapted from
(99)
Thus, farnesyltransferase inhibitors against
ras block production of VEGF by
tumor cells. Herceptin blocks production
of VEGF by breast cancer cells which over
express HER-2/neu (erbB2). The C225 monoclonal
antibody to the EGF receptor (Iressa),
blocks VEGF, bFGF and IL-8. STI571 (Glivec)
against Bcr-Abl (98) may also block the
receptor for PDGF, (PDGF-R). In fact,
Uehara et al. in IJ Fidler’s laboratory
reported that STI571 inhibits angiogenesis
and tumor growth of human prostate cancer
in the bones of nude mice, by blockade
of PDGF-R signalling in endothelial cells
(99). Therefore, STI571, originally developed
to treat chronic myelogenous leukemia
by inhibiting the Bcr-Abl tyrosine kinase
in these cells, may also have antiangiogenic
activity against those selected tumors
which secrete or release PDGF to upregulate
the PDGF receptor on vascular endothelium
in the tumor bed.
Summary and
Future Directions
Three unifying concepts which have recently
emerged in the field of angiogenesis research
are discussed here: (i) Hematologic malignancies
are angiogenesis-dependent; (ii) Proteins
and cells from the hematopoietic and hemostatic
systems modulate angiogenesis; and (iii)
Oncogenes and tumor suppressor genes regulate
angiogenesis at a geneticlevel.
For hematologists and oncologists an important
lesson from angiogenesis research is to
think about a tumor as containing two
cell populations which stimulate each
other: the endothelial cell compartment
and the tumor cell compartment. Anticancer
therapy may be more effective if both
cell populations are targeted. The
rate of mutation in the tumor cell population
is high, but low in the endothelial cell
population. This is why it may be possible
to administer direct angiogenesis inhibitors
for the long term either alone, or together
with conventional chemotherapy, or with
radiotherapy, or with other modalities
(7).
Acknowledgements
We thank Wendy Foss and Emmy Chen for
help with references. Supported by U.S.P.H.S.
Grant #2 R01 CA64481-06 from the National
Cancer Institute to J.F., and a grant
to Children’s Hospital, Boston, Mass.
from EntreMed, Inc., Rockville, Maryland.
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