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Departments of
* Oncology,
Bone Marrow Transplantation,
Pharmacology, and
§ Ophthalmology, Hadassah-Hebrew University Hospital, Jerusalem 91120; and
¶ Institute of Animal Science, the Volcany Center, Bet Dagan, Israel
1Correspondence: Department of Oncology, Hadassah Hospital, POB 12000, Jerusalem, 91120, Israel. E-mail: vlodavsk{at}cc.huji.ac.il
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1(I) and matrix metalloproteinase 2 (MMP-2) gene expression.
Halofuginone also effectively suppresses tumor progression and
metastasis in mice. These results together with the well-documented
role of extracellular matrix (ECM) components and matrix degrading
enzymes in formation of new blood vessels led us to investigate the
effect of halofuginone on the angiogenic process. In a variety of
experimental system, representing sequential events in the angiogenic
cascade, halofuginone treatment resulted in profound inhibitory effect.
Among these are the abrogation of endothelial cell MMP-2 expression and
basement membrane invasion, capillary tube formation, and vascular
sprouting, as well as deposition of subendothelial ECM. The most
conclusive anti-angiogenic activity of halofuginone was demonstrated
in vivo (mouse corneal micropocket assay) by showing a
marked inhibition of basic fibroblast growth factor (bFGF) -induced
neovascularization in response to systemic administration of
halofuginone, either i.p. or in the diet. The ability of halofuginone
to interfere with key events in neovascularization, together with its
oral bioavailability and safe use as an anti-parasitic agent, make it a
promising drug for further evaluation in the treatment of a wide range
of diseases associated with pathological angiogenesis.—Elkin, M.,
Miao, H.-Q., Nagler, A., Aingorn, E., Reich, R., Hemo, I., Dou, H.-L.,
Pines, M., Vlodavsky, I. Halofuginone: a potent inhibitor of critical
steps in angiogenesis progression,
Key Words: neovascularization • type I collagen • halofuginone • matrix metalloproteinase-2 • extracellular matrix
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
.
Moreover, angiogenesis is clearly implicated in tumor growth and
metastasis (4
, 5)
. Expansion of microscopic solid tumor
beyond 
1–2 mm in size requires continuous recruitment of new blood
vessels. These vessels also serve as a gateway for cancer cells to
enter the blood circulation and spread to distant organs. Developing
new approaches to interfere with molecular and cellular events in the
angiogenic process is therefore regarded as a highly effective means in
the treatment of cancer and a wide range of human disorders, including
rheumatoid arthritis, psoriasis, and diabetic retinopathy (3
, 4
, 6
7
8)
.
Regardless of the initiating event, the angiogenic stimulus results in
local proliferation of endothelial cells, followed by degradation of
the subendothelial capillary basement membrane (BM) and formation of
vascular sprouts (2)
. One of the rate-limiting steps in
this process is the activity of matrix metalloproteinases (MMPs) in
degrading structural ECM proteins. Endothelial cells were shown to
produce a variety of MMPs (9)
; of these, MMP-2 is the most
studied for its role in angiogenesis (2
, 10
, 11)
. MMP-2
(72 kDa collagenase type IV) cleaves primarily type IV collagen
(12)
, the protein backbone of the endothelial basement
membrane (13)
. The advancing front of the migrating column
of endothelial cells (vascular sprout) uses MMP-2 enzymatic activity to
migrate through the basement membrane and ECM.
Different ECM components are thought to be implicated in vascular tube
formation. It was proposed that type I collagen is involved in
directing the migration and assembly of endothelial cells into the
newly formed blood vessels (14
, 15)
. This hypothesis was
supported by the observation that exogenous type I collagen promoted
rapid tube formation by cultured confluent human dermal microvascular
endothelial cells (16)
. The tubes contained collagen
fibrils in the lumen space, suggesting that the endothelial cells use
the fibrils to fold and align into tube structures (16)
.
After a vascular tube has been formed, a new basement membrane is
eventually deposited by the endothelial cells, a lumen is developed;
finally, blood flow is established in the newly vascularized region
(1
, 2
, 17)
. The tight association of endothelial cells
with the underlying BM plays an important role in the maintenance of
their quiescence and differentiated phenotype (18
19
20)
.
In previous studies, we have demonstrated that halofuginone, a
low molecular weight quinazolinone alkaloid (495 Da, structure
presented in the Merck Index) isolated from the plant Dichroa Febrifuga
and used as a coccidiostat in chickens and turkeys (21)
,
suppresses MMP-2 (22)
and collagen type 1 (I)
(23
24
25)
gene expression. A strong inhibitory effect of
halofuginone on MMP-2 gene transcription activity was observed in human
and murine bladder carcinoma cell lines (22)
. Specific
inhibition of collagen type I expression was demonstrated in a broad
range of cell types of chicken, mouse, rat, and human origin, both
in vitro and in experimental animals (21
, 23
24
25
26)
. Moreover, exposure to halofuginone inhibited deposition
of ECM by vascular smooth muscle (25)
and kidney mesangial
(27)
cells.
In the present study, we investigated the effect of halofuginone on angiogenesis in different in vitro and in vivo experimental models. Halofuginone was found to exert a moderate anti-proliferative effect on vascular endothelial cells. A more pronounced inhibitory effect was noted on MMP-2 activity, resulting in a marked decrease in reconstituted BM (Matrigel) invasion by endothelial cells, capillary tube formation on ECM substrata, and aortic sprouting. In addition, halofuginone was found to inhibit deposition of BM-like ECM by cultured endothelial cells. Taken together, halofuginone appears to inhibit several essential stages of the angiogenic process, namely, endothelial cell proliferation, MMP-2 expression, and BM invasion as well as capillary tube formation and ECM deposition by the newly formed vessels. The most conclusive anti-angiogenic activity of halofuginone was demonstrated in vivo by showing a marked inhibition of bFGF-induced neovascularization by systemic treatment with halofuginone, administered either intraperitoneally (i.p.) or in the diet, using the mouse corneal micropocket model.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells
Clonal populations of bovine aortic endothelial cells (BAEC)
were established and cultured in DMEM (1 g glucose/l) supplemented with
10% calf serum, as described (28)
. Cells were cultured at
37°C in a 10% CO2 humidified incubator and the
experiments were performed with early (5
6
7
8
9
10)
cell
passages. Recombinant human bFGF (1 ng/ml) was added every other day
during the phase of active cell growth.
Cell proliferation
Cell proliferation was evaluated by measuring
[3H]-thymidine incorporation. BAEC were plated
(4x104 cells/16 mm well/ml) in DMEM (1 g
glucose/l) supplemented with 10% CS. Four days after plating, the
subconfluent cells were exposed to increasing concentrations of
halofuginone (25–400 ng/ml) in the presence of 1 ng/ml bFGF.
3H-thymidine (1 µCi/well) was then added and
DNA synthesis was assayed 48 h later by measuring the
radioactivity incorporated into trichloroacetic acid (TCA) insoluble
material (29)
.
MMP-2 gelatinolytic activity
Subconfluent BAEC cell cultures were incubated for 24 h in
the absence or presence of increasing concentrations (10–100 ng/ml) of
halofuginone in serum-free DMEM, and aliquots of the resultant
conditioned medium were analyzed for collagenolytic activity.
Collagenase activity was determined on gelatin impregnated (1 mg/ml,
Difco, Detroit, Mich.) sodium dodecyl sulfate (SDS) 8% polyacrylamide
gels, as described previously (10
, 22)
. Briefly, samples
of the culture media, normalized for equal cell protein, were separated
on the substrate-impregnated gels under nonreducing conditions,
followed by 30 min incubation in 2.5% Triton X-100 (BDH, England). The
gels were then incubated for 16 h at 37°C in 50 mM Tris, 0.2 M
NaCl, 5 mM CaCl2, 0.02% Brij 35 (w/v) at pH 7.6.
At the end of the incubation period, the gels were stained with 0.5%
Coomassie G 250 (Bio-Rad, Richmond, Calif.) in methanol/acetic
acid/H2O (30:10:60).
Matrigel invasion assay
Blind well chemotaxis chambers with filters 13 mm in diameter
were used. Polyvinyl pyrrolidine-free polycarbonate filters with pores
8 mm in size (Costar Scientific, Cambridge, Mass.) were coated with
basement membrane Matrigel (25 µg/filter) as described
(30)
. Cells (2x105) suspended in
DMEM containing 0.1% bovine serum albumin were added to the upper
chamber in the absence or presence of increasing concentrations (1–50
ng/ml) of halofuginone. Medium conditioned by 3T3 fibroblasts was
applied as a chemoattractant and placed in the lower compartment of the
Boyden chamber (30)
. Cells were assayed at 37°C in 5%
CO2 for 6 h. More than 90% of the cells
attached to the filter after a 2 h incubation period. At the end
of the incubation, the cells on the upper surface of the filter were
removed by wiping with a cotton swab and cells on the lower surface of
the filter were stained with Diff-Quick kit. Cells in different areas
of the lower surface were counted and each assay was performed in
triplicate. For chemotaxis studies, filters were coated with collagen
type IV alone (5 µg/filter) to promote cell adhesion. This amount of
collagen does not form a barrier to the migrating cells but rather an
attachment substratum (30)
.
Organization of BAEC on a basement membrane substratum in
vitro
Twenty-four-well plates were coated with 320 µl of
reconstituted basement membrane Matrigel (10 mg/ml) per well, which was
allowed to polymerize for 30 min at 37°C. DMEM (500 µl) was added
into each culture well. BAEC suspended in 500 µl of culture medium
were then added to each well to a final concentration of 4 x
104 cells per 1 ml. Halofuginone was added to
some of the wells at various concentrations ranging from 10 to 200
ng/ml, and the plates were incubated for 18 h at 37°C. After
removal of the medium by aspiration, the culture was fixed and
capillary-like structures were visualized by phase microscopy and
photographed (10)
.
In vitro angiogenesis: rat aortic ring assay
SIS gel (31
32
33)
was prepared according to the
manufacturer’s instructions. Briefly, 4 ml of chilled SIS gel solution
were mixed with 0.6 ml 10x phosphate buffered saline; 0.3 ml of
ice-cold 0.5 N NaOH was then added. This solution was brought to pH 7.4
with 0.04 N HCl, aliquoted into 24-well plates (0.2 ml/well), and
gelled at 37°C for 30 min. Thoracic aortas were obtained from
2-month-old Sprague-Dawley rats. The fibroadipose tissue was carefully
removed under a dissecting microscope; aortic rings were sectioned (1
mm long) and placed on top of 0.2 ml SIS gel in 16 mm culture wells.
SIS gel mixture (0.4 ml) was carefully poured on top of the ring. After
the gel was formed, 0.4 ml of serum-free endothelial growth medium
(Gibco BRL, Grand Island, N.Y.) was added and replaced every other day
by fresh medium containing the increasing concentrations of
halofuginone (25–500 ng/ml). Microvessel outgrowth was visualized by
phase microscopy and photographed.
ECM deposition by cultured endothelial cells
For preparation of sulfate-labeled ECM, BAEC were seeded into
24-well plates at a confluent density (2.5x105
cells/well), within 4–6 h forming a growth-arrested cell layer. Under
these conditions, the cells remained viable and retained their normal
monolayer configuration and morphological appearance up to a
concentration of 2 µg/ml halofuginone.
Na235SO4
(40 µCi/ml) and increasing concentrations of halofuginone (10–200
ng/ml) were added 1 and 3 days after seeding and the cultures were
incubated without a change of medium. Nine days after seeding the ECM
was exposed by dissolving (5 min, room temperature) the cell layer with
phosphate-buffered saline (PBS) containing 0.5% Triton X-100 and 20 mM
NH4OH, followed by four washes with PBS
(25
, 27)
. The ECM was then digested with collagenase (3
U/ml, 18 h, 37°C), followed by trypsin (25 µg/ml, 24 h,
37°C), and the solubilized material was counted in a ß-counter.
In vivo angiogenesis: mouse corneal micropocket
assay
A corneal micropocket was created in C57BL/6 mice (7–9 wk of
age). A sucralfate and Hydron pellet containing bFGF was implanted into
each pocket. Briefly, pellets were made of the slow-release polymer
Hydron (polyhydroxyethylmethacrylate [polyHEMA] (Interferon Sciences,
New Brunswick, N.J.) (10 µl of 12% Hydron in ethanol), containing
sucralfate (10 mg) and/or bFGF (10 µg), as described
(34)
. The suspension was then embedded onto a sterilized
nylon mesh (TETKO, 3–300/50, approximate pore size 0.4x0.4 mm) and
allowed to dry for 30 min. Subsequently, the fibers of the mesh were
pulled apart and 30–40 uniformly sized pellets containing 80–100
ng bFGF per pellet were collected. C57Bl/6 mice were anesthetized, the
eye globe was proposed, and a lamellar micropocket was dissected toward
the temporal limbus as described (34)
. A single pellet was
then introduced into the pocket. Halofuginone was administered either
in the food (5 mg/kg) or by i.p. injections (2 µg/mouse, once a day).
On postoperative days 3 and 7, corneas were examined by slit lamp
biomicroscopy and photographed to determine the neovascular response.
The area (mm2) of angiogenesis was calculated,
using the formula of an ellipse, by measuring the maximal vessel length
and the contiguous circumferential zone of neovascularization.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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80% inhibition was
obtained at 400 ng/ml (Fig. 1
).
|
Effect of halofuginone on MMP-2 gelatinolytic activity expressed by
vascular endothelial cells
We investigated the effect of halofuginone on MMP-2 activity
expressed by BAEC. For this purpose, subconfluent cultures were treated
(24 h) with increasing concentrations (10–100 ng/ml) of
halofuginone in serum-free medium. Aliquots of the conditioned medium
were applied onto gelatin embedded SDS-polyacrylamide gel. The zymogram
shown in Fig. 2a
demonstrates that the gelatinolytic activity of the 72 kDa
MMP-2 enzyme was markedly reduced by halofuginone. A noticeable
decrease in MMP-2 activity was already revealed in cells treated with
10 ng/ml halofuginone and 80-90% inhibition of MMP-2 activity was
exerted by 100 ng/ml halofuginone (Fig. 2a
). Activation of
the proenzyme yielding a 68 kDa processed enzyme was even more readily
affected by halofuginone (Fig. 2a
). Addition of halofuginone
(100–200 ng/ml) to the zymogram reaction buffer had no effect on MMP-2
activity (not shown), indicating that the drug does not exert a direct
inhibitory effect on the MMP-2 enzyme itself.
|
Effect of halofuginone on BAEC invasion through Matrigel-coated
filters
A necessary step in blood vessel formation is degradation of the
subendothelial basement membrane by proteases. MMP-2 has previously
been shown to play a central role in basement membrane degradation and
endothelial cell invasion during the angiogenic process (reviewed in
ref 2
). We therefore tested the effect of halofuginone on
BAEC invasion using the Matrigel invasion assay (30)
. The
presence of as little as 50 ng/ml halofuginone during the chemoinvasion
assay resulted in a 60% inhibition of the ability of BAEC to invade
through Matrigel-coated filters (Fig. 2b
, filled bars). The
BAEC readily traversed through filters coated with 5 µg collagen type
IV to a similar extent in the absence or presence of 50 ng/ml
halofuginone (Fig. 2b
, empty bars), indicating no effect of
halofuginone on cell viability and motility.
Inhibition of capillary-like tube formation by halofuginone
Vascular endothelial cells incubated on a Matrigel substratum
align and form tubular structures (Fig. 3a
), mimicking certain steps in angiogenesis such as invasion,
migration, and differentiation. Tube formation by endothelial cells was
shown to be dependent on MMP-2 activity (10)
. We therefore
investigated the effect of halofuginone on the ability of BAEC to
organize into capillary-like tubes on Matrigel. As shown in Fig. 3
,
halofuginone inhibited the formation of tubular networks on Matrigel in
a dose-dependent manner. A remarkable inhibition of tube formation was
observed in the presence of 25 ng/ml halofuginone (Fig. 3b
)
and complete inhibition was obtained at 50 ng/ml halofuginone (Fig. 3c
). At the latter concentration, most of the cells appeared
as unorganized cell aggregates (Fig. 3c
). Similar studies
performed with human umbilical vein endothelial cells yielded the same
pattern of inhibition (not shown).
|
Halofuginone inhibits endothelial cell sprouting in rat aortic
rings
We tested the ability of halofuginone to inhibit microvessel
formation in vitro, using rat aortic rings embedded in small
intestinal submucosa gel (31
32
33)
. SIS gel represents a
physiological ECM that has been derived from porcine intestine while
preserving its natural composition and architecture (31)
.
Due to these features, SIS gel enables several cell types, including
endothelial cells, to maintain in culture their in vivo
phenotype and 3-dimensional organization (32
, 33)
. In our
experiments, branching microvessels forming a capillary network of
tubes and loops were developed, starting on day 7–9, at the periphery
of untreated aortic rings embedded in SIS gel, reaching a maximal
degree of sprouting on days 12–14 (Fig. 4a
). In contrast, microvessel formation was almost completely
inhibited in the presence of 60 ng/ml halofuginone. Under those
conditions, single cells migrated out of the aortic ring, but failed to
align into microvessel tubes (Fig. 4b
). Similar results were
obtained with aortic explants embedded in collagen type I gel, commonly
used in previous studies (35)
. A quantitative analysis of
the number of out-growing microvessels revealed a 50% inhibition of
vascular tube formation at 25 ng/ml halofuginone and complete
inhibition at 100 ng/ml halofuginone.
|
Effect of halofuginone on ECM deposition by cultured vascular
endothelial cells
The last stage of angiogenesis is characterized by
deposition of new basement membrane (17
, 20)
. Deposition
of basement membrane-like ECM by cultured vascular and corneal
endothelial cells has previously been demonstrated (36)
.
It was also found that the level of incorporated sulfate is a reliable
measure of the amount of ECM deposited by these cells (25
, 27)
. To assess the effect of halofuginone on BM deposition,
labeled sulfate
(Na235SO4)
and increasing concentrations of halofuginone were added to confluent
BAEC 24 and 96 h after seeding. On day 9, the cell layer was
dissolved in order to expose the underlying ECM. The ECM was then
digested with collagenase and the remaining material was trypsinized.
The solubilized radioactivity was counted in a ß-scintillation
counter to evaluate the effect of halofuginone on the amount of
collagenase digestible and nondigestible ECM constituents. As shown in
Fig. 5
, deposition of ECM was inhibited by 80–85% in the presence of 50
ng/ml halofuginone. This observation was supported by a microscopic
examination of the denuded culture dishes, revealing a very thin or no
ECM produced in the presence of halofuginone.
|
Anti-angiogenic activity of halofuginone in vivo
A murine corneal angiogenesis model was applied to evaluate the
inhibitory effect of halofuginone in vivo. The angiogenic
stimulant bFGF was applied into a corneal micropocket in a slow release
polymer, as described in Materials and Methods, and halofuginone was
administered either in the food (5 mg/kg) or i.p. (2 µg/mouse/day)
for 7 consecutive days. Control mice received a regular diet. On
postoperative days 3 and 7, corneas were examined by slit lamp
microscopy to determine the neovascular response and photographed. As
demonstrated in Fig. 6
, neovascularization from the corneal limbus to the pellet occurred in
the eyes of control mice (Fig. 6a
, left). A remarkable
inhibition of the neovascular response to the pellet was observed in
mice receiving oral halofuginone (Fig. 6a
, middle and
right). On postoperative day 7, the average area of neovascularization
in the control group was 1.75 ± 0.27 mm2,
as compared to 0.65 ± 0.23 and 0.63 ± 0.19
mm2 in mice that received halofuginone in the
food or by i.p. injections, respectively (Fig. 6b
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Independent of
this effect, halofuginone was found to be a specific inhibitor of
collagen type 1(I) gene expression. Halofuginone was shown to
inhibit collagen synthesis and ECM deposition in a variety of cell
types (21
, 23
24
25
26
27)
, resulting in amelioration of a number
of fibrotic disorders (24
25
26)
. Halofuginone also
effectively inhibits MMP-2 gene expression, and in combination with its
other activities, exerts profound antitumor and anti-metastatic effects
in various experimental models (22
, 37
, 38)
. Decrease in
vascular density and neovascularization in primary tumor tissue was
reported after a systemic administration of halofuginone, indicating a
possible anti-angiogenic activity of the drug (37
, 38)
.
In the present study, we demonstrate that halofuginone is a potent
anti-angiogenic agent, affecting the main molecular and cellular events
associated with the multistep angiogenic process. First, we tested the
effects of halofuginone on the initial stages of angiogenesis,
involving local proliferation of endothelial cells, degradation of the
underlying basement membrane, and endothelial cell invasion into the
underlying substratum. A 40–60% decrease in endothelial cell
proliferation was obtained in the presence of 100–200 ng/ml
halofuginone. The inhibitory effect of halofuginone on MMP-2 expression
by endothelial cells, their invasion through reconstituted BM
(Matrigel) and capillary-like tube formation was more pronounced,
reaching almost complete inhibition at 50–100 ng/ml. These findings
are consistent with an inhibitory effect of halofuginone on MMP-2
promoter activity (22)
. It has previously been shown that
sprouting angiogenesis is clearly associated with enzymatic degradation
of the subendothelial BM by MMP-2 secreted by endothelial cells
(reviewed in ref 2
). Treatment of endothelial cells with
exogenous MMP-2 was reported to induce a dose-dependent morphological
change consistent with an angiogenic response (i.e., capillary-like
tube formation) (10)
. Inhibition of enzymatic degradation
of collagen type IV resulted in suppression of experimental
tumor-associated angiogenesis (39
, 40)
. The significance
of MMP-2 in angiogenesis was further emphasized by showing a marked
inhibition of tumor-induced angiogenesis and tumor progression in MMP-2
deficient mice (11)
. It is conceivable, therefore, that
the observed suppression by halofuginone of both the invasive ability
of endothelial cells and their differentiation into tubular structures
on Matrigel is primarily due to the inhibition of MMP-2 expression.
Furthermore, inhibition of collagen type 1(I) gene expression by
halofuginone may also play an important role in the anti-angiogenic
activity of the compound. The role of collagen type I in angiogenesis
was emphasized in numerous studies. Type I collagen is believed to aid
endothelial cell migration during sprouting angiogenesis and their
proper assembly into newly formed capillaries (14
, 15)
.
In vitro studies suggest that expression of type I collagen
is initiated when endothelial cells undergo angiogenic sprouting
(14)
. Experimentally induced angiogenesis on the chicken
chorioallantoic membrane (CAM) is associated with the deposition of
large amounts of collagen (41)
. Moreover, transcriptional
activation of the collagen 1(I) gene was observed in endothelial
cells undergoing angiogenesis in vitro (15)
. It
was also reported that when bovine aortic endothelial cells were
exposed to angiogenic stimuli, collagen type I synthesis was
up-regulated 4.5-fold (41)
. Type I collagen fibrils were
found to induce formation of capillary-like tubes by endothelial cells
in vitro (16)
. Electron microscopy revealed
that the lumen of the tubes consisted of collagen fibrils, with the
surrounding cells having typical endothelial junctional complexes
(16)
. Histological examination of normal, tumor, and
experimentally induced angiogenesis demonstrated that collagen type I
is localized within endothelial cords and tubes (42)
. On
the other hand, metabolic inhibition of collagen type I synthesis
inhibits capillary formation on the CAM (43)
. Endothelial
cell migration from aortic explants embedded within collagen gels is
inhibited when maintained in the presence of the proline analog
cis-hydroxyproline, once again indicating the need for
normal collagen synthesis (44)
. In light of the above
data, it seems likely that collagen 1(I) suppression by halofuginone
is responsible, at least in part, for abrogation of capillary tube
formation on Matrigel and in the rat aortic ring assay, as reported in
this study.
Basement membrane deposition by endothelial cells during the final
phase of angiogenesis is the hallmark of blood vessel maturation
(17
, 20)
. We demonstrated here that low concentrations
(50 ng/ml) of halofuginone almost completely inhibited the
deposition of BM-like ECM by vascular endothelial cells. It should be
noted that the inhibitory effect of halofuginone on MMP-2 expression,
as well as invasiveness, tube formation, and ECM deposition by
endothelial cells, was not due to its antiproliferative activity.
Although endothelial cell proliferation was inhibited (60–70%) by
halofuginone at concentrations as high as 200–400 ng/ml halofuginone,
all the other anti-angiogenic effects investigated in this study were
exerted at 25–50 ng/ml of the compound. Thus, the potent
anti-angiogenic activity of halofuginone both in vitro
(Matrigel tube formation and aortic ring assays) and in vivo
(corneal micropocket model) should be attributed primarily to the
combined action of the compound on MMP-2 and collagen 1(I) gene
expression and ECM deposition rather than to a direct inhibition of
endothelial cell proliferation.
The diverse nature of angiogenesis and the range of its molecular and
cellular mechanisms make it unlikely that inhibition of any single
target in this process will abrogate neovascularization. Most of the
proposed or clinically tested anti-angiogenic drugs are directed
against a particular stage of the angiogenic cascade (i.e., factors
promoting degradation of the subendothelial BM, endothelial cell
proliferation, migration, interaction with ECM components, cell
survival) (reviewed in ref 8
). Our present results
indicate that, in fact, halofuginone interferes with several sequential
events during new blood vessel formation. The anti-angiogenic activity
of halofuginone was best demonstrated in the mouse corneal micropocket
model in which neovascularization was induced by bFGF and halofuginone
was administered systemically, either in the diet or by i.p.
injections. The oral bioavailability, lack of toxic effects, and
long-term safe use in farm animals make halofuginone a promising
candidate for further evaluation in cancer therapy and treatment of
other angiogenesis related pathologies such as rheumatoid arthritis,
psoriasis, and diabetic retinopathy.
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ACKNOWLEDGMENTS |
|---|
Received for publication May 22, 2000. Accepted for publication June 5, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) or by i.p. injections (2
µg/mouse, once a day) (•). Control mice received regular diet
without halofuginone and i.p. injections of saline (
). The area
(mm2) of vascularized zone was calculated as described in
Materials and Methods. The results are the mean ± SD,
n = 6.