From the Renal Division, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
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Endostatin, a carboxyl-terminal fragment of
collagen XVIII, has been shown to regress tumors in mice. In this
study, we have analyzed the mechanism of endostatin action on
endothelial cells and nonendothelial cells. Endostatin treatment of cow
pulmonary artery endothelial cells caused apoptosis, as demonstrated by three methods, annexin V-fluorescein isothiocyanate staining, caspase
3, and terminal deoxynucleotidyl transferase-mediated dUTP
nick-end-labeling assay. Moreover, addition of endostatin led to a
marked reduction of the Bcl-2 and Bcl-XL
anti-apoptotic protein, whereas Bax protein levels were unaffected.
These effects were not seen in several nonendothelial cells.
Collectively, these findings provide important mechanistic insight into
endostatin action.
Angiogenesis is required for tumors to grow beyond a few
millimeters in size (1). Advantages of anti-angiogenic therapy include
ease of access of drugs to the endothelial cell compartment, targeting
of a genetically stable cell population (endothelial versus
tumor cells) thereby lessening the chance of drug resistance and
"amplification" achieved, because one endothelial cell supports the
growth of 50-100 tumor cells (2-7).
Fragments of proteins have recently been described with anti-angiogenic
action. Endostatin, a 20-kDa C-terminal fragment of collagen XVIII, is
a specific inhibitor of endothelial cell proliferation (8), migration
(9), and angiogenesis (8). Endostatin regresses established syngeneic
Lewis lung carcinomas, T241 fibrosarcoma, and B16 melanoma tumors in
xenograft models (8). Recently, tumor dormancy was induced following
repeated cycles of endostatin treatment (10). Administration of
recombinant mouse endostatin to tumor-bearing animals increases the
apoptotic index of the tumor cells without affecting their
proliferation index (8), but no data on endothelial cells has been described.
The mechanism of endostatin action is unknown. In vitro
results from this laboratory and others (8, 9) show that endostatin inhibits the proliferation of endothelial cells but has no effect on
tumor cells or nonendothelial cell types, including fibroblasts and
smooth muscle cells. Thus, the action of endostatin seems to
be endothelial cell specific. Because the presence of endostatin in
endothelial cell culture affects proliferation (and migration), we
asked whether the endothelial cells are undergoing programmed cell death.
Our studies show that soluble yeast-derived mouse endostatin altered
the morphology of endothelial cells leading to the characteristic features of cells undergoing apoptosis. Endostatin-treated cells shifted in annexin
V-FITC1-labeling by FACS, and
after roughly 12 h, externalization of phosphatidylserine on the
cell surface ensued. In addition, endostatin treatment increased the
activity of the intracellular protease caspase 3, resulting in DNA
degradation in the nucleus, as independently confirmed via the terminal
deoxynucleotidyl transferase-mediated dUTP nick-end-labeling) (TUNEL)
assay. Western blot analysis showed reduction of the Bcl-2 and
Bcl-XL anti-apoptotic proteins without any effect on
the level of the Bax pro-apoptotic protein. In nonendothelial cells,
viz., NIH3T3 and A10 smooth muscle cells, endostatin did not
show any effects in any of the above assays, indicating specificity of
endostatin toward endothelial cells.
Reagents
The mouse endostatin protein was produced and purified from
yeast, as described elsewhere (9). Some batches contained DNA, which
had no effect on apoptosis. Anti-Fade reagent was purchased from
Molecular Probes, Inc. Tumor necrosis factor(TNF)- Cell Lines
Cow pulmonary artery endothelial (C-PAE) cells, obtained from
ATCC (Manassas, VA), were maintained in DMEM supplemented with 10%
fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. The cells were incubated
in a humidified environment at 37 °C in the presence of 5%
CO2. Bovine aortic endothelial cells, a gift from C. Ferran, Beth Israel Deaconess Medical Center, Boston, were maintained
in DMEM containing 10% fetal calf serum. Bovine adrenal cortex
capillary endothelial cells were a gift from Dr. J. Folkman,
Children's Hospital, Boston. HUVE and HMVE-L cells were obtained from
Clonetics Corp. (San Diego, CA). IMR-90, lung fibroblast, NIH3T3
fibroblast, A10 smooth muscle cell, H9c2 (2-1)-myoblast cell line, and
786-0 renal clear cell carcinoma cells were all purchased from ATCC.
Annexin V-FITC Assay
Annexin V, a calcium-dependent phospholipid-binding
protein with a high affinity for phosphatidylserine (PS) was used to
detect early stage apoptosis (11). Briefly, 200,000 cells were plated onto a fibronectin-coated 6-well plate in DMEM containing 2% FCS and 3 ng/ml bFGF. Different concentrations of recombinant mouse endostatin
were added to each well, and cells were harvested and processed
18 h after treatment. For the time course study, 10 µg/ml
endostatin was added and cells were processed after 3, 4, 6, 12, and
18 h. Human recombinant TNF- Caspase 3 Assay
This assay was performed in a 75-cm2 tissue culture
flask or on fibronectin-coated 6-well plates. For the 6-well plates,
0.5-1 × 106 cells (75-cm2 flask, 2 × 106 cells) were seeded and maintained overnight in DMEM
with 10% FCS. The following day, the old medium was replaced with
fresh medium (2% FCS), and the cells were incubated overnight at
37 °C. Following starvation, the cells were stimulated with bFGF (3 ng/ml) in DMEM (2% FCS). Along with bFGF, yeast endostatin (10 µg/ml, final concentration) was added, and the cells were grown for
24 h. For the control plate, only the PBS buffer was added. As a
positive control, TNF- Microscopic Detection of TUNEL Staining
For Adherent Cells--
C-PAE cells were seeded at a density of
5,000 cells/well on fibronectin-coated (10 µg/ml) Lab-Tek chamber
slides and grown in 0.4 ml of DMEM with 10% FCS. After 2 days, the old
medium was aspirated and fresh DMEM with 2% FCS was added, and the
cells were starved overnight. The following day, 0.36 ml of new medium (with 2% FCS) containing 3 ng/ml bFGF was added along with yeast endostatin (10 µg/ml) or TNF-
The TUNEL assay was performed as described in the ApoAlert DNA
fragmentation assay kit user manual (CLONTECH),
except that the final concentration of propidium iodide (Sigma) used
was 1 µg/ml. After the assay, a drop of anti-fade solution was added, and the treated portion of the slide was covered with a glass coverslip
with the edges sealed with clear nail polish. Slides were viewed
immediately under a fluorescent microscope using a dual filter set for
green (520 nm) and red fluorescence (>620 nm). The images were
captured using a digital microscope (Nikon Microphot-SA) and processed
using SPOT software version 1.1.02. For all samples except the positive
control (TNF- For Cells in Suspension--
Floating cells were collected by
centrifugation at 300 × g for 10 min at 4 °C. The
old medium was aspirated, and the cells were resuspended in 500 µl of
PBS (pH 7.4). Cells were centrifuged again, the PBS removed, and the
pellet was resuspended in 75 µl of fresh PBS. Resuspended cells were
spread on a poly-L-lysine-coated slide (Labscientific,
Inc.) using a clean slide. The cells were fixed by immersing the
slides in fresh 4% formaldehyde/PBS at 4 °C for 25 min. The rest of
the protocol was carried out as explained above.
Western Blot Analysis
C-PAE cells (1 × 106) were seeded in 10-cm
Petri dishes precoated with fibronectin (10 µg/ml) in the presence of
2% FCS containing 3 ng/ml bFGF. Endostatin was added at 10 µg/ml,
and cells were harvested at 12, 24, and 28 h after treatment.
Cells were washed three times in PBS buffer, pH 7.4, and the cells were
resuspended in 1 ml of 1× EBC buffer (50 mM Tris-HCl, pH
8.0, 120 mM NaCl, 1% Nonidet P-40) containing freshly
added complete protease inhibitor tablet (Boehringer Mannheim), 100 µg/ml Pefabloc, 1 µg/ml pepstatin. The protein concentration in
whole cell lysate was measured by the bicinchoninic acid method. 30 µg of whole cell extract was loaded onto a 4-15% gradient
polyacrylamide gel. Transfer was performed using a semi-dry transblot
apparatus (Bio-Rad). The membrane was blocked in wash buffer (1×
Tris-buffered saline) with 5% nonfat dry milk and incubated at
37 °C for 1 h. Goat antibody directed against human Bcl-2
(N-19) {sc-492-G} was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Affinity purified mouse polyclonal antibody against
Bax (B-9) {sc7490} and Bcl-XS/L {sc1690} were
purchased from the same manufacturer. Polyclonal anti-actin antibody
(Sigma) was used to normalize for protein loading. Secondary antibodies
were anti-goat, mouse and rabbit immunoglobulin conjugated to
horseradish peroxidase (Amersham Pharmacia Biotech). The
immunoreactivity was detected with an enhanced chemiluminescence
reagent (Pierce). Images were scanned using a flat bed scanner
(Scan-Jet 4C) and quantitated by the NIH image 1.61 software.
Normalization was done by dividing the Bcl-2 signal by that of actin
within each experiment.
Endostatin Changes the Morphology of Endothelial Cells--
When
proliferating, C-PAE cells were treated with mouse endostatin at a
concentration of 10 µg/ml, the cells rounded (Fig. 1A) and detached from the
fibronectin-coated plate. In the controls, the cells showed intact
endothelial cell morphology, whereas endostatin-treated cells appeared
rounded and showed membrane blebbing characteristic of apoptotic cells.
We have also observed similar effects with endostatin-treated bovine
adrenal cortex capillary endothelial and bovine aorta endothelial cells
(data not shown), but C-PAE cells showed the most dramatic effect.
Interestingly, these effects of endostatin were most pronounced when
bFGF was added to subconfluent monolayers (2 × 104/well in a 24-well plate and 0.1 × 106/well in a 6-well plate) when cells were in exponential
growth. This, perhaps, mimics the in vivo situation,
i.e. endostatin appears to be selective for tumor
vasculature where there is endothelial cell division. Importantly,
endostatin at 10 µg/ml on nonendothelial cells, viz. A10
and H9c2 (2-1)-myoblast, did not show any effect as was seen in
endothelial cells (Fig. 1, B and C). Moreover, because cells plated efficiently on fibronectin-coated plates even in
the presence of endostatin, endostatin's effect was not simply via
altering cell-matrix interactions. Next, we checked for other hallmarks
of apoptosis, phosphatidylserine externalization, intracellular
protease activation, and nuclear condensation with DNA
fragmentation.
Endostatin Translocates Phosphatidylserine from the Inner Face of
the Plasma Membrane to the Cell Surface--
After initiation of
apoptosis, most cell types translocate the membrane phospholipid PS
from the inner surface of the plasma membrane to the outside (11-13).
PS can be detected by staining with an FITC conjugate of annexin V,
38-kDa protein that binds naturally to PS. During programmed cell
death, PS externalization typically precedes membrane bleb formation
and DNA fragmentation (12).
Fig. 2A shows annexin V-FITC
staining in conjunction with PI staining in C-PAE cells treated for
18 h with different concentrations of endostatin. Endostatin at 10 µg/ml showed a distinct shift in annexin fluorescence intensity. The
mean fluorescence intensity difference between control and
endostatin-treated cell was significant (p = 0.01) at 5 and 10 µg/ml. The shift in fluorescence intensity was similar for
endostatin at 10 µg/ml and the positive control TNF-
Morphological examination of FACS-analyzed samples with fluorescence
microscopy (Nikon) showed annexin V staining localized to the cell
membrane at 12 h with no staining in the cytoplasm (data not
shown). During this period, the majority of the cells were negative for
PI, implicating the early stage of apoptosis. With increased exposure
time (24-36 h), in addition to membrane staining with annexin V some
of the cells turned positive for PI (data not shown) (12, 14),
consistent with a more advanced stage of apoptosis.
Similar levels of annexin V staining were observed in two other
endothelial cell lines studied, namely, bovine aorta endothelial and
bovine adrenal cortex capillary endothelial (data not shown). We have
also tested the effect of human endostatin on these three bovine
endothelial cell lines. We failed to detect annexin V staining in the
presence of human endostatin added to these cells, whereas when human
endothelial cell lines were used (HUVE and HMVE-L), it resulted in a
marked shift in annexin V
fluorescence.2 These data
indicate that apoptosis, as assessed by annexin V staining, occurs in
diverse endothelial cells in response to mouse and human endostatin.
With regard to nonendothelial cells, 786-0 and NIH3T3 cells, we failed
to see any distinct annexin positivity (Fig. 2, C and D). In addition, we screened other nonendothelial cells
(IMR-90, A10, and H9c2 (2-1)-myoblast) and found no effect of
endostatin (data not shown). Based on these results, endostatin's
action appears to be selective for endothelial cells.
Endostatin Increases the Intracellular Activity of Caspase
3--
Caspase 3 (CPP32) is an intracellular protease activated early
during apoptosis of mammalian cells and initiates cellular breakdown by
degrading specific structural, regulatory, and DNA repair proteins (12,
14-16). This protease activity can be measured spectrophotometrically
by detection of the chromophore (p-nitroanilide) after
cleavage from the labeled substrate (DEVD-pNA). First, we performed a
time course experiment with 10 µg/ml of endostatin and checked for
increase in caspase 3 activity. There was no difference in caspase 3 activity between the treated and the control samples at 2, 4, 8, and
14 h (data not shown). However, caspase 3 activity was elevated
24 h after treatment with endostatin over controls. The caspase
activity of the endostatin and TNF- Endostatin Induces DNA Fragmentation in the Nucleus of Endothelial
Cells--
Fragmentation of nuclear DNA is one of the distinct
morphological changes occurring in the nucleus of an apoptotic cell. A TUNEL assay was performed on endostatin-treated, TNF-
We counted the number of apoptotic cells in several fields, and the
percent of apoptotic cells (green divided by the number of red cells
per field) is plotted in Fig. 4B. The apoptosis rate in the
control cells was 1.24%. In the endostatin-treated cells, a 30-fold
increase in the apoptosis rate was observed in suspension cells
(38.3%), whereas a 15-fold increase was observed in the attached cells
(19.4%). With TNF- Bcl-2 and Bax Expression by Western Blot
Analysis--
Anti-apoptotic members such as Bcl-2 and
Bcl-XL prevent programmed cell death in response to
numerous stimuli (18, 19). Conversely, pro-apoptotic proteins such as
Bax and Bak can accelerate cell death, and in certain cases, they are
sufficient to cause apoptosis independent of additional signals (19).
We tested whole cell extract of endostatin-treated and control C-PAE
cells for Bcl-2 and Bax expression levels. In growth arrested C-PAE cells, Bcl-2 expression was high. It was relatively constant up to
28 h; in contrast, endostatin-treated cells showed a marked decrease in Bcl-2 (Fig. 5A).
Densitometry revealed that the levels of Bcl-2 compared with control
was 1.2-, 1.5-, and 3-fold less at 12, 24, and 28 h, respectively,
after treatment, with actin levels used as normalization controls. In
contrast, Bax expression was similar between control and treated
cultures (Fig. 5B).
Bcl-2 protein was not detected in NIH3T3 and IMR-90 cells. Bax
expression levels were not affected by endostatin treatment in these
cell lines (Fig. 6, A and
B). In C-PAE cells, at the early time point (12 h),
Bcl-XL level was reduced by 2-fold, whereas its expression
was unchanged in NIH3T3 cell (Fig. 6, C and D). Interestingly, we have detected only the larger pro-apoptotic form of
Bcl-X in C-PAE, whereas both smaller and larger forms were detected in
NIH3T3.
These findings suggest that endostatin exerts its regulatory activity
by altering Bcl-2 expression. Interestingly, vascular endothelial
growth factor has been shown to augment Bcl-2 levels in endothelial
cells (20). Because endostatin antagonizes VEGF's proliferative
effects (data not shown), Bcl-2 appears to be one of potentially many
down-regulated proteins. Recent studies indicate that the Bcl-2 protein
binds to other proteins, such as Bax, Bcl XS, Bik, and Bad,
which ultimately enhance cell survival (21, 22). The function of
another Bcl-2 homologue, Bax, remains enigmatic (23). Bcl-2 and
Bcl-XL function through heterodimerization with Bax (24,
25) and overexpression of Bax accelerates apoptosis (23). Recently, it
was shown that FGF-2 inhibited endothelial cell apoptosis by
Bcl-2-dependent and -independent mechanisms (26). In our
study, we did see differences in Bcl-2 (and Bcl-XL) expression in endostatin-treated cultures but no difference in Bax
levels. It is possible that Bcl-2 may act independently of Bax, as has
been shown for T cells (26). Studies are in progress to delineate the
importance of other anti-apoptotic and pro-apoptotic proteins and their
phosphorylation status in response to endostatin treatment.
In summary, we have demonstrated that endostatin causes apoptosis of
endothelial cells in vitro, but not nonendothelial cells. This finding sheds the first mechanistic insight into the action of
endostatin. The intracellular events that trigger the apoptotic response are under current investigation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
was obtained from
R & D Systems, Inc. 16% methanol-free formaldehyde, used for the
fixing solutions, was purchased from Polysciences, Inc. The annexin
V-FITC-labeling kit was purchased from
CLONTECH.
(40 ng/ml) was used as a positive
control. The cells were washed in PBS and resuspended in binding buffer
(10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Annexin V-FITC was added to a final
concentration of 100 ng/ml, and the cells were incubated in the dark
for 10 min, then washed again in PBS and resuspended in 300 µl of
binding buffer. 10 µl of propidium iodide (PI) was added to each
sample before flow cytometric analysis. The cells were analyzed using a
Becton Dickinson FACStar plus flow cytometer. Electronic compensation was used to eliminate bleed-through fluorescence. In each sample, a
minimum of 10,000 cells was counted and stored in listmode. Data
analysis was performed with standard Cell Quest software (Becton-Dickinson). The quadrant settings were set so that the negative
control allowed less than 1% positivity. Endostatin was added to
nonendothelial cells (NIH3T3 and 786-0) at 10 µg/ml, and the cells
were processed and analyzed as described above.
was used at a final concentration of 20 ng/ml. After 24 h, the supernatant cells were centrifuged and
collected. The wells (flasks) were trypsinized to collect the attached
cells and combined with the supernatant cells. The cells were counted
and resuspended in cell lysis buffer (CLONTECH) at
a concentration of 4 × 107 cells/ml. The rest of the
protocol followed the manufacturer's instruction
(CLONTECH). A specific inhibitor of caspase 3, DEVD-fmk, was used to confirm the specificity as suggested by the
manufacturer. The absorbance was measured in a microplate reader
(Bio-Rad) at 405 nm. Fold-increase in protease activity (caspase 3) was
determined by comparing the results of the induced sample (yeast
endostatin or TNF-
) with the uninduced control. Similarly
nonendothelial cells (NIH3T3 and H9c2 (2-1)-myoblast) were used and
analyzed as described above.
(20 ng/ml). For control samples, fresh medium (2% FCS) containing bFGF (3 ng/ml) was added. Following induction (24 h), the slides were washed twice with PBS, and
subsequently fixed in fresh 4% formaldehyde/PBS at 4 °C for 25 min.
The slides were washed in PBS and the cells permeabilized in 0.2%
Triton X-100/PBS for 5 min on ice, then washed with fresh PBS twice for 5 min each at room temperature, and the TUNEL assay was performed as
described below.
: 5 fields), 15 random fields were chosen, and the
number of green and red cells per field were counted.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
A, C-PAE cell morphology is altered
following endostatin treatment as monitored by phase contrast
microscopy. Confluent C-PAE cells were trypsinized, plated at
approximately 50% confluency in 2% FCS containing 3 ng/ml bFGF with
simultaneous addition of endostatin at 10 µg/ml. Pictures were taken
24 h after treatment (400× magnification). The typical morphology
of apoptotic cells, membrane blebbing, cytoplasmic shrinkage, and
chromatin condensation, was seen in endostatin-treated cells.
B-D, morphology of nonendothelial cells treated
with endostatin; A10 smooth muscle cells (B), 786-0 renal
carcinoma cells (C), and IMR-90 fibroblasts (200×
magnification) (D).
(40 ng/ml).
Concentrations of endostatin below 0.1 µg/ml did not show any
significant annexin V positivity (data not shown). To investigate the
earliest time point at which endostatin caused externalization of PS,
we conducted a time course experiment (Fig. 2B). The effect
of endostatin was significant (p = 0.01) at 12 h
after treatment. Time points before 6 h did not show a difference between control and treated samples.
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Fig. 2.
A, annexin V-FITC staining was performed
on C-PAE cells treated with increasing concentrations of endostatin
(0.1-10 µg/ml). FACS was done to quantify the percentage of cells
undergoing apoptosis. Cells were stained with PI just before analysis.
Gating was performed to analyze only annexin V positive and PI negative
cells. TNF- (40 ng/ml) was used as a positive control. Control
(black); endostatin, 10 µg/ml (red); 5 µg/ml
(yellow); 1 µg/ml (green); TNF-
, 40 ng/ml
(blue). FL1-height represents the annexin
fluorescence intensity as a log scale. B, flow cytometric
analysis of apoptosis induced by endostatin treatment. Endostatin was
added at 10 µg/ml and cells were harvested at 3, 4, 6, 12, and
18 h after treatment. Processing of cells was performed as
described earlier. The cell populations were gated to analyze only
annexin V positive/PI negative cells. Control (black);
3 h (red); 6 h (green); 12 h
(blue); and 18 h (orange).
FL1-height represents the annexin fluorescence intensity as
a log scale. C and D, flow cytometric analysis of
apoptosis induced by endostatin on nonendothelial cells. 786-0, renal
clear cell carcinoma cells (C) and NIH3T3 fibroblasts
(D). FL1-height represents the annexin
fluorescence intensity as a log scale. Control (black);
endostatin, 10 µg/ml (green); 5 µg/ml (red);
2.5 µg/ml (dark blue); 1 µg/ml (orange); and
0.5 µg/ml (light blue).
(positive control) treated
samples is shown in Fig. 3A.
When compared with controls, endostatin-treated cells showed a 1.8-fold
increase in caspase 3 activity after 24 h, whereas TNF-
gave a
comparable (1.75-fold) increase. When a specific inhibitor of caspase 3 (DEVD-fmk) was included in the same samples, the protease activity was
at baseline (comparison of the dark box with the corresponding white
box), indicating that the increase in the measured activity was
specific for caspase 3. For NIH3T3 cells only, a marginal increase in
caspase 3 was seen, whereas in myoblast cells there was no difference in caspase 3 levels between treated and control cultures (Fig. 3B).
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Fig. 3.
A, endostatin treatment increases
caspase 3 activity. Samples from control and treated cells were
processed, and caspase 3 activity was detected by reading the samples
in a microplate reader at 405 nm. The A405
reading was plotted in the presence (black box) and absence
(open box) of the inhibitor (DEVD-fmk). The error bars
represent mean ± S.E. B, caspase 3 activity in
nonendothelial cells. NIH3T3 and H9c2 (2-1)-myoblast cells were
treated with endostatin at 10 µg/ml. The cells were harvested and
processed as described for C-PAE cells.
-treated, and
control cells. Fig. 4A shows
the immunofluorescent pictures of adherent cells on slides. In the
presence of the enzyme terminal deoxynucleotidyltransferase, both
endostatin- and TNF-
-treated slides showed numerous positive cells
under green fluorescence (panels f and h),
whereas no positive cells were seen in the control (panel
b). Without the enzyme, the endostatin-treated slide showed background cell fluorescence (panel d). Panels a,
c, e, and g represent the same field
of cells stained with propidium iodide.
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Fig. 4.
A, detection of fragmented DNA in
adherent C-PAE cells by the TUNEL assay. Adherent cells were fixed with
fresh 4% formaldehyde/PBS at 4 °C for 25 min. The cells were
permeabilized with prechilled 0.2% Triton X-100/PBS for 5 min on ice.
After permeabilization, the TUNEL assay was performed along with
propidium iodide dye staining. On staining, pictures were taken
immediately using a fluorescent microscope attached to a digital camera
and analyzed using the SPOT software. Panels a,
c, e, and g are fields of cells
stained with propidium iodide (1 µg/ml). Panels b,
d, f, and h are the same fields under
green fluorescent light. Control cells were incubated in DMEM with 2%
FCS. Endostatin concentration in the terminal
deoxynucleotidyltransferase+ and terminal
deoxynucleotidyltransferase samples was 10 µg/ml.
TNF-
was added at a final concentration of 20 ng/ml. B,
quantitative determination of apoptosis. After TUNEL and propidium
iodide staining, the cells were counted in 15 fields for control and
endostatin-treated attached cells. For TNF-
-treated cells, five
fields were counted. The percentage of green divided by the number of
red cells in a given field was determined and an average (mean ± S.E.) of the different fields is presented.
, the apoptosis rate was 6.4%. In contrast, the
percent of TUNEL-positive in the angiostatin-treated bovine adrenal
cortex capillary endothelial cells was 2% when compared with the
control cells (1.2%), a 1.6-fold increase (17), suggesting that
endostatin is a stronger apoptotic agent than angiostatin.
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Fig. 5.
A, Western blot analysis of C-PAE cell
lysate for Bcl-2 protein levels. C-PAE cells were treated with
endostatin (10 µg/ml) for the indicated period of time. Total cell
lysate from control ( ) and treated (+) cultures was analyzed as
described earlier. 30 µg of total protein was subjected to
SDS-polyacrylamide gel electrophoresis analysis followed by Western
blot transfer. Immunoblot analysis was performed with antibodies
directed against Bcl-2. Reprobing of the same blot with actin antibody
was performed to normalize for protein loading. B,
immunoblot detection of total cell lysate for Bax expression levels. 30 µg of total cell lysate was separated on 4-15% gradient gel.
SDS-polyacrylamide gel electrophoresis analysis was followed by
membrane transfer and probing with Bax antibody. Actin probing is
shown, as well.
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Fig. 6.
Western blot analysis of nonendothelial cell
lysate for Bax protein levels. 30 µg of total protein was
subjected SDS-polyacrylamide gel electrophoresis analysis followed by
Western blot transfer. Immunoblot analysis was performed with
antibodies directed against Bax (2 µg/ml). Reprobing of the same blot
with actin antibody was performed to normalize for protein loading.
( ) and (+) refer to control and endostatin-treated (10 µg/ml)
samples. A, NIH3T3 cell lysate; B, IMR-90 cell
lysate. Immunoblot analysis was performed with antibodies directed
against Bcl-XS/L (2 µg/ml). C, C-PAE cell
lysate; D, NIH3T3 cell lysate.
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ACKNOWLEDGEMENTS |
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We thank Dr. A. Saluja for use of the digital microscopy instrumentation facility, and Dr. M. Werner for excellent support in FACS cell sorting.
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FOOTNOTES |
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* This work was supported by seed funds from Beth Israel Deaconess Medical Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipients of salary support from a National Institutes of Health basic science training grant.
These authors made equal contributions to this work.
¶ To whom correspondence should be addressed: Beth Israel Deaconess Medical Center, Dana 517, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-2105; Fax: 617-667-7843; E-mail: vsukhatm{at}bidmc.harvard.edu.
2 M. Dhanabal, R. Volk, R. Ramchandran, M. Simons, and V. P. Sukhatme, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorting; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling; TNF, tumor necrosis factor; DMEM, Dulbecco's modified Eagle's medium; C-PAE, cow pulmonary artery endothelial; PBS, phosphate-buffered saline; PI, propidium iodide; FGF, fibroblast growth factor; PS, phosphatidylserine; FCS, fetal calf serum.
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