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Regular Articles |
From the Department of Oral Medicine, Pathology, and
Oncology*
and the Department of Biologic and
Material Sciences,
University of Michigan
School of Dentistry, Ann Arbor, Michigan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The angiogenic property of VEGF has been attributed to several distinct
functions associated with this cytokine. VEGF is an endothelial cell
mitogen and permeability-enhancing factor that influences the egress of
plasma proteins and cells that both directly and indirectly stimulate
angiogenesis.11-13
Recent studies suggest that VEGF may
also function as a survival factor for endothelial
cells.14-16
Spyridopoulos reported that VEGF is able to
support the survival of endothelial cells exposed to the
apoptosis-inducing cytokine tumor necrosis factor
(TNF)-
.15
Watanabe and Dvorak16
have shown
that endothelial cells exposed to VEGF used vitronectin and 5ß5 to
sustain their survival when grown on a nonsupportive (hydrophobic
polystyrene) surface. In both instances, VEGF was able to enhance the
survival of endothelial cells by inducing endothelial cells to produce
a scaffold of matrix molecules that maximized cell adherence and
proliferation.
Apoptosis is a genetically controlled, morphologically unique form of cell death that plays a central role in reinforcing appropriate cellular patterns and in regulating cell number by eliminating cells that are harmful or no longer needed. Conversely, disruption of this program has been shown to contribute to the pathogenesis of several developmental, inflammatory and degenerative diseases, including cancer.17,18 The Bcl-2 gene family consists of a group of homologous proteins that function to either promote or suppress cell death.17,19-21 A role for Bcl-2 in endothelial cell survival has recently been proposed by Kondo et al.22 They showed that aortic endothelial cells overexpressing Bcl-2 were protected from the apoptogenic effects of basic fibroblast growth factor withdrawal. More recently, Gerber et al23 have shown that VEGF is able to protect serum-starved human umbilical vein endothelial cells from apoptosis in association with a significant up-regulation in the anti-apoptotic proteins Bcl-2 and A1.
To further define the mechanisms, by which VEGF promotes angiogenesis and the significance of enhanced endothelial cell survival in this process, we examined the effects of VEGF on the expression of several Bcl-2 family members during the development of angiogenic responses in vitro and in vivo. We report here that growth-factor-enriched and serum-deprived cultures of human dermal microvascular endothelial cells (HDMECs) grown on collagen type I gels in the presence of VEGF exhibited a significant reduction in apoptosis. Enhanced HDMEC survival was associated with a dose-dependent increase in Bcl-2 expression and a decrease in the expression of the processed forms of the cysteine protease caspase-3. HDMECs transduced with and overexpressing Bcl-2 exhibited enhanced protection from apoptosis induced by growth-factor deprivation and showed an increased responsiveness to growth stimuli and an enhanced ability to form spontaneous sprout-like structures in culture. Furthermore, we show that HDMECs overexpressing Bcl-2 exhibit a sustained increase in the number of functioning microvessels that developed at 7 and 14 days after implantation into SCID mice. Our results indicate that angiogenic responses triggered by VEGF may be due in part to its ability to enhance the survival of endothelial cells by up-regulating expression of the anti-apoptotic protein Bcl-2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of VEGF to induce endothelial cells to proliferate and organize into capillary-like sprouts was examined using HDMECs grown on type I collagen gels as previously described.24 Briefly, 3.0 x 105 HDMECs (Cell Systems Corp., Kirkland, WA) were seeded in 60-mm tissue culture dishes (Corning Costar Corp., Cambridge, MA) that were coated with a 1.5 ml of gelled solution of bovine dermal type I collagen (Vitrogen 100, Collagen Biomaterials, Palo Alto, CA). Gelation was achieved by exposing the collagen solution to ammonia vapors for 15 minutes. Cells were suspended in MCDB 131 media supplemented with epidermal growth factor (10 ng/ml), hydrocortisone (1 µg/ml), bovine brain extract containing 10 µg/ml heparin, fetal bovine serum (FBS; 5%), 50 µg/ml gentamicin, and 50 ng/ml amphotericin-B (Endothelial Cell Growth Medium, EGM, Clonetics Corp., San Diego, CA), allowed to attach for 24 hours, and washed to remove nonadherent cells. Beginning on day 1 and at 2-day intervals thereafter, cells were fed with either 50 ng/ml recombinant human VEGF165 (Intergen Co., Purchase, NY) or 50 ng/ml recombinant human interleukin (IL)-8 (R&D Systems, Minneapolis, MN). At daily intervals, the number of cells in 10 random high-power fields (x200) as well as the number of capillary-like sprouts (x100) were counted as previously described.25 The data were obtained from triplicate dishes per condition at each time point.
TUNEL Assay and Flow Cytometry
HDMECs were seeded in collagen or on the surface of plastic culture dishes and exposed for 3 days to either 50 ng/ml VEGF165 or 50 ng/ml IL-8 added to either EGM or MCDB 131 medium devoid of growth factors (Endothelial Cell Basal Medium, EBM, Clonetics Corp.) supplemented only with 1% FBS. HDMECs were then retrieved from collagen gels with a solution of 2.5 mg/ml collagenase (Sigma Chemical Co., St. Louis, MO) and from the surface culture dishes with a solution of 0.5% trypsin/5.3 mmol/L EDTA (Gibco BRL, Gaithersburg, MD), fixed in 1% paraformaldehyde for 15 minutes, and stored in 70% ethanol at -20°C. The percentage of apoptotic cells was evaluated using the APO-BRDU terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) assay26 according to the manufacturer's instructions (Phoenix Flow Systems, Phoenix, AZ). Apoptotic cells were quantitated by flow cytometry using an argon laser excited at 488 nm (Epics XL, Coulter Corp., Hialeah, FL). A total of 10,000 cells were examined for each condition. The data shown are representative of three independent experiments.
DNA Ladder Analysis
The ability of VEGF to protect endothelial cells from DNA fragmentation was evaluated using a DNA ladder assay.27 Briefly, HDMEC cultures grown in the presence of either 50 ng/ml VEGF165 or 50 ng/ml IL-8 in EBM (Clonetics) with 1% FBS were retrieved from gels as previously described. Cells were fixed in 70% ethanol at -20°C overnight, centrifuged, and resuspended in 40 µl of phosphate-citrate buffer at room temperature for 30 minutes. Supernatants were concentrated in a SpeedVac (Savant Instruments, Farmingdale, NY) and resuspended in 3 µl of 0.25% Nonidet P-40 (Sigma), followed by 3 µl of 1 mg/ml RNAse (Sigma). After 30 minutes of incubation at 37°C, 3 µl of 1 mg/ml proteinase K (Sigma) was added to the extract and incubated for an additional 30 minutes at 37°C. The DNA was resolved in a 1.5% agarose gel and visualized under ultraviolet light after staining with ethidium bromide. Positive controls consisted of HDMECs cultured in suspension in a solution of 1.68% methylcellulose (Sigma) for 72 hours.28
Northern and Western Analyses
Total HDMEC RNA was extracted with TRIzol (Gibco BRL), subjected
to electrophoresis in 1% agarose gel, and transferred to a Hybond
nylon membrane (Amersham Corp., Arlington Heights, IL).
32P-radiolabeled cDNA probes were prepared using the random
primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN). After
hybridization, blots were washed and exposed to X-Omat AR film (Eastman
Kodak Corp., Rochester, NY). The membranes were rehybridized with a
ß-actin probe to control for equal loading. For Western analysis,
whole-cell lysates were prepared as described29
and run in
a 12% Tris-glycine gel at 125 V, and the protein was transferred to a
nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). Blots
were blocked for 2 hours in PBS/0.1% Tween containing 5% nonfat dried
milk and then probed overnight at 4°C with the appropriate primary
antibody. The primary antibodies and the concentrations used were as
follows: monoclonal hamster anti-Bcl-2, 0.1 µg/ml (15131A,
Pharmingen, San Diego, CA); monoclonal mouse anti-flag, 10 µg/ml (M2,
Eastman Kodak); monoclonal mouse anti-caspase-3, 1 µg/ml (AM20,
Oncogene Research Products, Cambridge, MA); polyclonal rabbit anti-Bax
antibody, 1/1000, and rabbit polyclonal anti-Bcl-xL/S
1/1000 (a gift from G. Nuñez). Blots were exposed for 2 hours to
the appropriate peroxidase-coupled secondary antibodies (Amersham) and
washed, and the bound antibody was detected with the ECL system
(Amersham). The membranes were reprobed with a monoclonal mouse anti
-tubulin antibody (CP06, Calbiochem-Novabiochem Corp., San Diego,
CA) to control for equal gel loading. The relative band densities were
calculated with NIH Image 1.61b7 software.
Retroviral Vector Construction and HDMEC Transduction
A 753-bp cassette containing human bcl-2 from the expression plasmid pcDNA3-hu bcl-2-flag (a gift from G. Nuñez) was inserted in the EcoRI cloning site of a retroviral vector (LXSN, gift from D. Miller).30 The Bcl-2 construct or the vector alone was transfected into PA317 amphotropic packaging cells with Superfect transfection reagent (Qiagen, San Clarita, CA). Viral supernatants from confluent cultures of vector-producing cells were collected after 24 hours, centrifuged, filtered, and stored at -70°C. HDMECs were transfected with either Bcl-2 or vector alone by incubating 1 x 106 HDMECs overnight with a 1/10 dilution of the viral supernatant in the presence of 4 µg/ml Polybrene (Sigma). The viral supernatant was aspirated, cells were washed with Hanks' balanced salt solution (Gibco BRL), and EGM-MV medium containing 250 µg/ml G418 (Gibco BRL) was used to select for resistant clones. Bcl-2 expression was confirmed by examining total RNA and protein by Northern and Western blot analysis, respectively.
Sponge Implants in SCID Mice
Porous poly (L-lactic acid) (PLA) sponges were
fabricated as previously described.31
Briefly, PLA (Aldrich
Chemical Co., Milwaukee, WI) was dissolved in chloroform to yield a
solution of 10% polymer (w:v), and 0.12 ml of this solution was loaded
into Teflon cylinders packed with 0.4 g of sodium chloride
particles. The solvent was allowed to evaporate, and then the sponges
were immersed for 16 hours in an aqueous solution containing 10 mg/ml
polyvinyl alcohol (Aldrich Chemical) in PBS. The sponges (measuring
approximately 6 mm x 6 mm x 1 mm) with an average pore
diameter of 180 µm were dried, lyophilized, and sterilized by
exposure to 
-radiation. The sponges were then soaked in 100%
ethanol for 2 hours, washed 1 hour in PBS, and then left overnight in
fresh PBS. Just before implantation, 1 x 106
Bcl-2-transduced HDMECs (HDMEC-Bcl-2), vector control (HDMEC-LXSN),
or parental HDMEC (untransduced) were resuspended in a 1:1 mixture of
EBM-MV/Matrigel (Collaborative Biomedical Products, Cambridge, MA) and
allowed to adsorb into the sponges. Male SCID mice (CB.17.SCID,
Taconic, Germantown, NY), 3 to 4 weeks old, were anesthetized with
ketamine and xylazine, and two sponges were implanted subcutaneously in
the dorsal region of each mouse. At 7 and 14 days after
transplantation, mice were sacrificed, and the sponges were retrieved,
fixed overnight in 10% buffered formalin, dehydrated through graded
ethanol, embedded in paraffin, and mounted on Superfrost (Fisher
Scientific, Pittsburgh, PA) glass slides for histological examination.
Three sponges from four to five mice were evaluated for cell type at
each time point.
Immunolocalization of CD34 Antigen and in Situ TUNEL Assay
Deparaffinized tissue sections were prepared as described earlier, and antigen retrieval was achieved by microwaving tissue sections for 14 minutes in citrate buffer (2.1 g/L citric acid, pH 6.0). Tissue sections were incubated with 2 µg/ml monoclonal mouse anti-human CD34 (Serotec, Raleigh, NC) for 1 hour at 37°C. The sections were processed using a Vectastain Elite ABC kit, (Vector Laboratories, Burlingame, CA), and a solution of 0.014 g of 3-amino-9-ethyl carbazole (Sigma) in 2.5 ml of N,N-dimethylformamide (Sigma) was used to visualize the bound antibody. An in situ TUNEL assay (ApopTag peroxidase In situ apoptosis detection kit, Oncor, Gaithersburg, MD) was used according to manufacturer's instructions to determine the percentage of apoptotic cells in the sponge implants. The number of CD34+ blood vessels and TUNEL-positive cells was counted blindly in 10 random fields per sponge using an optical microscope (x400).
Statistical Analyses
The statistical analyses were performed with SigmaStat (Sigma). A descriptive analysis was performed initially, followed by z-test or one-way ANOVA, according to experimental design.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the relationship between endothelial survival and the
development of the angiogenic phenotype we examined the ability of VEGF
and IL-8, two well described pro-angiogenic mediators, to induce HDMECs
grown on collagen gels to proliferate and form sprout-like
structures.32
In this model system, endothelial cells
when exposed to angiogenic factors proliferate, migrate, and
organize into sprout-like structures that mimic stages in the
development of microvessels in vivo.25
For these
experiments HDMECs were grown in the presence of 50 ng/ml of either
VEGF or IL-8, and the total number of endothelial cells as well as the
number of capillary-like sprouts that developed were counted each day
for 7 days. After 7 days exposure to VEGF, HDMECs exhibited a twofold
increase in cell number (P 
0.01) as compared
with untreated HDMECs (Figure 1A)
. We
then compared the mitogenic potency of VEGF to the pro-angiogenic
chemokine IL-8.32
Cultures of HDMECs treated with IL-8
exhibited a rapid increase in cell number that gradually declined to
levels that were at or below levels encountered in untreated HDMEC
cultures (Figure 1A)
. In addition to their mitogenic effect, both VEGF
and IL-8 were able to induce HDMECs to organize and differentiate into
capillary-like structures. On the addition of VEGF to HDMEC
cultures, a significant increase in the number of sprout-like
structures was observed (Figure 1B)
. By day 7, the number of HDMECs
exposed to IL-8 developed far fewer sprouts when compared with
VEGF-treated HDMECs (P 0.01). Cultures of
untreated HDMECs showed little or no capacity to form spontaneous
sprout-like structures.
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DNA ladder analysis and TUNEL staining were performed to evaluate
the ability of VEGF and IL-8 to protect HDMECs from apoptosis when
grown in nutrient-rich endothelial growth medium and under conditions
of growth factor deprivation. In untreated HDMEC cultures, DNA ladder
analysis showed a typical pattern of DNA fragmentation (Figure 2A)
. Flow analysis of TUNEL-stained cells
revealed a baseline level of apoptosis of 19.8%. Cultures treated with
IL-8 showed a DNA ladder profile similar to untreated HDMEC cultures
with a comparable percentage (20.4%) of TUNEL-positive cells. However,
when VEGF was added to HDMEC cultures, DNA fragmentation was barely
detectable. Under these conditions, less than 5%
(P 0.01) of the cells were apoptotic (Figure 2B)
. A further test of the protective effect of VEGF on HDMEC apoptosis
was undertaken by growing HDMECs in basal medium devoid of
exogenous growth factors and supplemented only with 1% FBS. DNA ladder
analysis revealed that VEGF was able to confer substantial protection
on HDMECs from apoptosis in this experimental setting (Figure 2A)
. This
result was confirmed by flow cytometry which demonstrated that when
HDMECs were grown in presence of VEGF, less than 50% of endothelial
cells were apoptotic (P 0.01), as compared
with 78.1% in the untreated group and 70.5% in the IL-8-treated group
(Figure 2B)
. Microscopically, the proportion of cells that rounded up
and detached from the collagen was higher in the untreated (Figure 2C)
and IL-8-treated groups (Figure 2E)
, as compared with the VEGF-treated
HDMECs (Figure 2D)
. VEGF also protected HDMECs from undergoing
apoptosis when grown in nutrient-rich endothelial growth medium. Under
these conditions, 19.8% and 20.4% of the cells were apoptotic in
untreated and IL-8-treated cultures, respectively. However, when VEGF
was added, less than 5% (P 0.01) of the
cells were apoptotic (Figure 2B)
. Flow cytometry also revealed that
neither VEGF nor IL-8 was able to rescue HDMECs grown in surface
culture from apoptotic stimuli. We observed (data not shown) that 8.5%
of HDMECs were apoptotic in cultures fed for 3 days with EGM-MV
supplemented with 50 ng/ml VEGF and 10.2% in the group supplemented
with IL-8, as compared with 9.8% for untreated controls. Taken
together, these data indicate that VEGF, but not IL-8, protected HDMECs
cultured in type 1 collagen gels from baseline levels of apoptosis as
well as growth-factor-deficiency-induced apoptosis.
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Western blots were used to evaluate the effect of VEGF on the
expression of several members of the Bcl-2 family of proteins. VEGF
when added to cultures of HDMECs grown in complete medium induced an
11.2-fold increase (measured by relative band density in Figure 3A
) in the level of expression of Bcl-2,
which was dose dependent for concentration up to 100 ng/ml (Figure 3C)
.
However, VEGF had no detectable effect on the expression of Bax,
Bcl-xL, and Bcl-xS (Figure 3A)
. IL-8 had no
detectable effect on the expression of any of the proteins examined
(Figure 3A)
. Interestingly, induction of Bcl-2 expression by VEGF was
observed only when the cells were seeded in collagen (Figure 3B)
,
indicating an apparent substrate requirement for expression of
Bcl-2.15
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Western blots were also used to evaluate the relationship between
VEGF-induced expression of Bcl-2 and cleavage of the interleukin-1
ß-converting enzyme-like aspartate-specific protease caspase 3.
Activation of this caspase results in downstream activation of
endonucleases and DNA fragmentation.33-35
Cultures of
HDMECs exposed to VEGF showed an approximately threefold reduction in
the activated forms of the protease at all time periods examined
(Figure 3D)
. To further demonstrate the ability of Bcl-2 to protect
endothelial cells from apoptosis, HDMECs were stably transduced with
the bcl-2 gene in a retrovirus vector. Northern (Figure 4A)
and Western (Figure 4B)
analyses
confirmed that transduced HDMECs exhibit a marked increase in the
expression of Bcl-2 as compared with HDMECs transduced with the vector
alone or untransduced HDMECs. This was also confirmed using anti-flag
antibody, which showed a positive band only for HDMEC-Bcl-2. Western
blot analysis confirmed the stability of transduction with Bcl-2 for at
least eight passages (data not shown). Flow cytometry was then
performed to evaluate the effect of overexpression of Bcl-2 on survival
of HDMECs. When HDMECs were grown in complete EGM-MV medium, only 2.7%
of the HDMEC-Bcl-2 cells were apoptotic after 3 days as compared with
19.1% of the HDMEC-LXSN and 13.2% of the parental HDMECs (Figure 4C)
.
The same protective effect of Bcl-2 was observed for cells grown in
medium deprived of growth factors. In this setting, only 33.9% of
HDMEC-Bcl-2 were apoptotic compared with 81.6% of the HDMEC-LXSN or
78.1% of the parental HDMECs (Figure 4C)
.
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The effect of overexpression of Bcl-2 on the ability of HDMECs to
form sprouts in vitro and microvessels in vivo
was also evaluated. After 7 days, the number of cells (Figure 5A)
and the number of sprout-like
structures (Figure 5B)
that developed in cultures of HDMEC-Bcl-2 were
significantly higher (P 0.01) as compared
with HDMEC-LXSN and parental HDMECs. This tendency for enhanced
growth and spontaneous sprout formation by HDMEC-Bcl-2 persisted
throughout the duration of the assay (7 days). To see whether
overexpression of Bcl-2 influenced capillary growth in vivo,
we examined the ability of HDMEC-Bcl-2 to organize into functional
microvessels after implantation into SCID mice. CD34, a marker
that exhibits high specificity for endothelial cells, was used
to identify blood vessels populated by human endothelial
cells.36
When sponges were examined 7 and 14 days after
implantation, the number of CD34+ blood vessels in the SCID
mice was significantly higher (P = 0.01) in
sponges populated with HDMEC-Bcl-2 than in sponges containing either
HDMEC-LXSN or parental HDMECs (Figure 6, A, C, and D)
. The increased
vascularization encountered in sponges populated by HDMEC-Bcl-2 was
associated with a decrease in the number of cells undergoing apoptosis
(P 0.01) at both 7 and 14 days after
implantation (Figure 7)
. Histological
analysis revealed that only blood vessels in the interior of the
sponges were stained with the anti-human CD34 antibody and not the
mouse blood vessels in the connective tissue surrounding the sponge
implant (Figure 6B)
. Immunostaining with anti-human CD31 antibody and
anti-flag antibody confirmed that the blood vessels in the interior of
the sponges were of human origin (data not shown). These findings
demonstrate that transplanted HDMECs were able to organize into
capillary structures, connect with mouse microvessels, and become
functional blood vessels. Moreover, our results indicate that HDMECs
overexpressing Bcl-2 are able to participate in a vigorous and
sustained angiogenic response in vivo (Figure 6D)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When cultured endothelial cells were deprived of growth factors and essential nutrients, the anti-apoptotic effects of VEGF were also pronounced. Under these conditions, large numbers of endothelial cells were dead or dying after 3 days in culture. The addition of VEGF to the medium increased the survival of cultured HDMECs by 30%, as measured by flow cytometry. These results were confirmed by DNA ladder analysis where a marked reduction in DNA fragmentation was observed. VEGF consistently induced expression of the Bcl-2 in HDMECs seeded in collagen; however, this phenomenon was not observed when cells were grown directly on the surface of tissue culture dishes. This suggests that conditions that lead to enhanced survival of endothelial cells require that these cells interact with a suitable substrate. Recently, Gerber et al reported that Bcl-2 and A1 mediated the survival of human umbilical vein endothelial cells (HUVECs) in vitro.23 In contrast to the survival-enhancing effects of VEGF, IL-8 did not potentiate endothelial cell survival nor did it up-regulate Bcl-2. IL-8 is a potent mediator of angiogenesis that is produced by many different cell types, including endothelial cells and macrophages.32,37 Although both VEGF and IL-8 have been implicated in angiogenic responses, the mechanism by which IL-8 induces angiogenesis appears different from VEGF. This suggests that inducers of angiogenesis may employ a different mechanism to induce a similar phenotypic response in endothelial cells. Overexpression of Bcl-2 is sufficient to block apoptosis by retaining cytochrome c in the mitochondria, thereby inhibiting caspase-3 activation and subsequent downstream apoptotic events.33-35 We investigated the ability of VEGF to limit the proteolytic cleavage of caspase-3 into its smaller derivatives in endothelial cells. We found that endothelial cells exposed to VEGF demonstrated a threefold reduction in cleaved forms of caspase-3. These data further corroborate our finding that VEGF when added to the culture medium protects endothelial cells from apoptosis.
To investigate the role of Bcl-2 in angiogenesis, we generated a clonal population of HDMECs that stably overexpressed Bcl-2. The HDMEC-Bcl-2 proliferated at a rate comparable to parental cells exposed to VEGF and demonstrated the ability to align and differentiate into sprouts in vitro without the addition of exogenous VEGF. These cells were also remarkably resistant to apoptosis when grown in either complete EGM-MV medium and in growth-factor-deficient medium. Perhaps the most informative analysis of the role of Bcl-2 in angiogenesis was obtained from the implantation of HDMECs overexpressing Bcl-2 (HDMEC-Bcl-2) in SCID mice. The number of human microvessels that developed in sponges populated with HDMEC-Bcl-2 was significantly higher than in sponges containing parental HDMECs or HDMECs transfected with vector alone. In addition, the number of apoptotic cells in sponges containing HDMEC-Bcl-2 was significantly lower as compared with the controls. This suggests that overexpression of Bcl-2 in endothelial cells has at least one of the following functions in vivo, neither of which is mutually exclusive. First, Bcl-2 may protect endothelial cells from apoptosis induced by inhibitors of angiogenesis resulting in a net gain in new blood vessels. Indeed, HDMECs overexpressing Bcl-2 are refractory to the apoptotic and angiosuppressive effects of the angiogenesis inhibitor thrombospondin 1 (J. E. Nör, R. J. Mitra, M. M. Sutorik, D. J. Mooney, V. P. Castle, and P. J. Polverini, in preparation). Second, Bcl-2 might potentiate the ability of endothelial cells to differentiate into functional blood vessels. Our SCID mouse model of angiogenesis would suggest that HDMECs overexpressing Bcl-2 were able to participate in a vigorous and sustained angiogenic response through at least 14 days.
In conclusion, we have demonstrated that the angiogenic activity of VEGF is mediated in part by its ability to induce expression of Bcl-2 in endothelial cells. In addition, VEGF was able to enhance the endothelial cell survival when grown in a growth-factor-deficient environment. Several studies have demonstrated increased expression of VEGF adjacent to sites of necrosis in solid tumors. This is an environment that under normal circumstances imposes severe constraints on endothelial cell growth and survival. Our findings suggest a mechanism whereby VEGF-induced expression of Bcl-2 may function to enhance the survival of endothelial cells in the toxic, oxygen-deficient environment of tumors and ensure the continuous, uninterrupted flow of nutrients to the tumor.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grants HL39926 and CA64416 (P.J. Polverini) and CAPES grant 2889/92-3 (J.E. Nör).
Accepted for publication November 5, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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: balance between growth and death signals. J Mol Cell Cardiol 1997, 29:1321-1330[Medline]
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B. D. Zeitlin, E. Joo, Z. Dong, K. Warner, G. Wang, Z. Nikolovska-Coleska, S. Wang, and J. E. Nor Antiangiogenic Effect of TW37, a Small-Molecule Inhibitor of Bcl-2. Cancer Res., September 1, 2006; 66(17): 8698 - 8706. [Abstract] [Full Text] [PDF]
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 M.S. Pinsky, W. Song, Z. Dong, K. Warner, B. Zeitlin, E. Karl, D.E. Hall, and J.E. Nor Activation of iCaspase-9 in Neovessels Inhibits Oral Tumor Progression. J. Dent. Res., May 1, 2006; 85(5): 436 - 441. [Abstract] [Full Text] [PDF]
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 M. M. de Resende, S. L. Amaral, D. H. Munzenmaier, and A. S. Greene Role of endothelial cell apoptosis in regulation of skeletal muscle angiogenesis during high and low salt intake Physiol Genomics, April 13, 2006; 25(2): 325 - 335. [Abstract] [Full Text] [PDF]
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L. Wang, D. P. Dittmer, C. C. Tomlinson, F. D. Fakhari, and B. Damania Immortalization of Primary Endothelial Cells by the K1 Protein of Kaposi's Sarcoma-Associated Herpesvirus. Cancer Res., April 1, 2006; 66(7): 3658 - 3666. [Abstract] [Full Text] [PDF]
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I. A. Siddiqui, N. Zaman, M. H. Aziz, S. R. Reagan-Shaw, S. Sarfaraz, V. M. Adhami, N. Ahmad, S. Raisuddin, and H. Mukhtar Inhibition of CWR22R{nu}1 tumor growth and PSA secretion in athymic nude mice by green and black teas Carcinogenesis, April 1, 2006; 27(4): 833 - 839. [Abstract] [Full Text] [PDF]
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G. K. Hong, P. Kumar, L. Wang, B. Damania, M. L. Gulley, H.-J. Delecluse, P. J. Polverini, and S. C. Kenney Epstein-Barr Virus Lytic Infection Is Required for Efficient Production of the Angiogenesis Factor Vascular Endothelial Growth Factor in Lymphoblastoid Cell Lines J. Virol., November 15, 2005; 79(22): 13984 - 13992. [Abstract] [Full Text] [PDF]
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 C. E. Cameron, N. L. Brouwer, L. M. Tisch, and J. M. Y. Kuroiwa Defining the Interaction of the Treponema pallidum Adhesin Tp0751 with Laminin Infect. Immun., November 1, 2005; 73(11): 7485 - 7494. [Abstract] [Full Text] [PDF]
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) or 50 ng/ml IL-8
(
) or in the absence
of additional cytokines
(
). C and
D: Representative microscopic field
(x200) of HDMECs seeded
in collagen for 5 days in the absence of additional cytokines
(C) or fed with EGM-MV supplemented
with 50 ng/ml VEGF (D). At daily
intervals, the number of cells and sprouts was counted in 10 random
fields from three independent experiments.
