The Antiangiogenic Factor 16K PRL Induces Programmed Cell Death in Endothelial Cells by Caspase Activation
Jean-François Martini,
Christophe Piot1,
Laurent M. Humeau2,
Ingrid Struman3,
Joseph A. Martial3 and
Richard I. Weiner
Center for Reproductive Sciences Department of Obstetrics,
Gynecology and Reproductive Sciences University of California
School of Medicine San Francisco, California 94143
 |
ABSTRACT
|
---|
We asked whether the antiangiogenic action
of 16K human PRL (hPRL), in addition to blocking mitogen-induced
vascular endothelial cell proliferation, involved activation of
programmed cell death. Treatment with recombinant 16K hPRL increased
DNA fragmentation in cultured bovine brain capillary endothelial (BBE)
and human umbilical vein endothelial (HUVE) cells in a time- and
dose-dependent fashion, independent of the serum concentration. The
activation of apoptosis by 16K hPRL was specific for endothelial cells,
and the activity of the peptide could be inhibited by heat
denaturation, trypsin digestion, and immunoneutralization, but not by
treatment with the endotoxin blocker, polymyxin-B. 16K hPRL-induced
apoptosis was correlated with the rapid activation of caspases 1 and 3
and was blocked by pharmacological inhibition of caspase activity.
Caspase activation was followed by inactivation of two caspase
substrates, poly(ADP-ribose) polymerase (PARP) and the inhibitor of
caspase-activated deoxyribonuclease (DNase) (ICAD). Furthermore, 16K
hPRL increased the conversion of Bcl-X to its proapoptotic form,
suggesting that the Bcl-2 protein family may also be involved in 16K
hPRL-induced apoptosis. These findings support the hypothesis that the
antiangiogenic action of 16K hPRL includes the activation of programmed
cell death of vascular endothelial cells.
 |
INTRODUCTION
|
---|
Angiogenesis, the formation of new blood vessels, is a necessary
component of physiological processes (1) as well as pathological
conditions such as tumor growth and metastasis (2, 3). During
angiogenesis, capillary endothelial cells proliferate, migrate into
tissues, and organize into vessels. Migration and organization of
endothelial cells into vessels requires concomitant activation of
proteases necessary for tissue remodeling. This cascade of events is
under the control of a balance of angiogenic and antiangiogenic
factors. Factors known to stimulate the formation of new capillaries
in vivo (1) include members of the fibroblast growth factors
family (FGF) e.g. basic FGF (4) and vascular endothelial
growth factor (VEGF) (5), angiogenin (6), and angiopoietin-1 (7).
Factors with antiangiogenic activity include platelet factor 4 (8),
thrombospondin (9), the 16-kDa N-terminal fragment of PRL (16K PRL)
(10, 11), angiostatin (12), and endostatin (13). More recently, Struman
et al. (14) demonstrated that recombinant 16-kDa N-terminal
fragments of related PRL family members including human GH, GH variant,
and placental lactogen also have antiangiogenic activity (14).
The antiangiogenic action of 16K PRL appears to affect the abilities of
capillary endothelial cells to proliferate, migrate, and organize into
vessels. A high-affinity, saturable, 16K PRL binding site that was
independent from the PRL receptor was characterized on capillary
endothelial cells (15). We showed that 16K PRL inhibited the
proliferative effects of both basic FGF (bFGF) and VEGF on bovine brain
capillary endothelial (BBE) cells (10, 11). Considerable evidence
supports the hypothesis that VEGF stimulates endothelial cell
proliferation via activation of the mitogen-activated protein kinase
(MAPK) signaling cascade. Binding of VEGF to its receptor (Flk-1/KDR)
results in autophosphorylation of the receptor, recruitment of the
Shc/Grb2/Sos coupling proteins, and activation of Ras and downstream
kinases leading to MAPK activation. We demonstrated that 16K human PRL
(hPRL) inhibited VEGF-induced activation of MAPK (16). The blockade
occurred distal to the activation of the Flk-1 and its association with
coupling proteins (17). However, VEGF-induced Ras activation was
inhibited by 16K hPRL, which is consistent with the inhibition of the
downstream kinases, Raf-1 and MAPK.
16K hPRL was shown to block the organization of BBE cells into
polarized capillaries when cultured in type 1 collagen gels (11).
In vivo tissue remodeling, which accompanies the formation
of new vessels, is dependent upon the activation of urokinase (uPA)
(18). We showed that 16K hPRL inhibited uPA activity by activation of
plasminogen activator inhibitor-type 1 (PAI-1) expression (19).
In addition to the inhibition of endothelial cell proliferation by
antiangiogenic factors, there is recent evidence that angiostatin (20)
and thrombospondin (21) also activate programmed cell death of
endothelial cells. Extensive studies in past few years have led to the
identification of many genes regulating programmed cell death during
various physiological and pathological processes. Cells undergoing
apoptosis are characterized by cytoplasmic shrinkage, membrane
blebbing, chromatin condensation, DNA cleavage, and finally, cell
fragmentation into membrane-bound apoptotic bodies.
Caspase-mediated proteolysis of specific proteins results in this
irreversible commitment to cell death (22). At least 13 caspases
function as initiators or effectors of the apoptotic signaling pathway
(23). Caspases exist as inactive proenzymes, which are activated by
cleavage at specific aspartate residues, followed by assembly
into heterotetramers. Activation of a caspase can result in stimulation
of additional caspases, e.g. autocatalytic activation of
caspase 1 by self-aggregation in turn activates caspase 3 (24). Another
regulatory component of the apoptotic pathway is the Bcl-2
protein family. These proteins are believed to act at the level of, or
upstream to, the caspases to inhibit or facilitate the apoptotic
cascade. Antiapoptotic factors include Bcl-2 and
Bcl-XL, while proapoptotic factors include Bax,
Bak, Bad, and Bcl-XS (25).
In the current study, we asked whether 16K hPRL could activate
programmed cell death as an additional mechanism for inhibiting
angiogenesis. We show that 16K hPRL, but not native 23-kDa PRL,
activates DNA fragmentation, and that apoptosis requires activation of
the caspase cascade. We demonstrate that the apoptosis-inducing action
of the 16K hPRL preparations is dependent on the activity of the
peptide and not on endotoxin contamination. Unlike the inhibition of
mitogen-induced cell proliferation by 16K hPRL, treatment with 16K hPRL
directly activates apoptosis. These findings provide the first detailed
and specific analysis of the mechanisms regulating the activation of
apoptosis by an antiangiogenic factor.
 |
RESULTS
|
---|
Stimulation of DNA Fragmentation by 16K hPRL in Vascular
Endothelial Cells
The level of DNA fragmentation associated with apoptosis was
estimated by measuring the levels of cytosolic mono- and
oligonucleosomes with an enzyme-linked immunosorbent assay (ELISA)
(25). Experiments were performed in BBE cells deprived of serum [0.5%
calf serum (CS)] for 2 days, a treatment that caused a 5-fold increase
in nucleosome formation compared with cells cultured in 10% CS (Fig. 1A
). Treatment with
2 or 10 nM 16K hPRL for the second day of serum deprivation
increased DNA fragmentation 2.3- and 4.4-fold compared with cells
cultured in 0.5% CS, respectively. Interestingly, bFGF (0.5
nM) partially reversed the effect of 10 nM 16K
hPRL by 50%. To a lesser degree, treatment with VEGF also reduced the
effect of 10 nM 16K hPRL by 32%.

View larger version (39K):
[in this window]
[in a new window]
|
Figure 1. 16K hPRL Induces DNA Fragmentation in Bovine
Endothelial Cells
A, BBE cells were cultured in 0.5% CS or 10% CS for 24 h. For a
second 24 h cells were cultured in 0.5% or 10% CS, or were
treated with 0.5 nM bFGF, 2 nM VEGF, 2 or 10
nM 16K hPRL, or 16K hPRL in combination with the mitogens
in 0.5% CS. If not mentioned in the figure legend, this design was
used throughout the studies. Endotoxin-1 (0.5 EU/ml) was added as a
control for endotoxin contamination in the 16K hPRL preparation. DNA
fragmentation was measured using an ELISA assay and is expressed as an enrichment factor (ratio of the absorbance of
the sample to that measured for 10% CS). Each bar
represents the mean ± SD, n = 6. B, Similar
experiments were performed in bovine aortic endothelial (BAE) cells.
The caspase inhibitor, z-VAD-fmk (20 µM), was added to
determine whether the ability of 16K hPRL to stimulate DNA
fragmentation in BAE cells depended on caspase activation. Each
bar represents the mean ± SD, n =
4. C, Dose-dependent stimulation of DNA fragmentation by increasing
concentrations of 16K hPRL in BBE cells. Each bar
represents the mean ± SD, n = 3. The
asterisk denotes a P value of <
0.05 vs. 0.5% CS control. D, Kinetics of 16K
hPRL-induced BBE cell DNA fragmentation. Cells were cultured in 0.5%
CS for 24 h and subsequently treated or not (open
square and circle) with 2 nM 16K
hPRL for the indicated time (filled square and
circle). DNA fragmentation was measured in cells
attached to the plastic dish (square) or in cells
floating in the conditioned medium (circle). Each
point represents the mean ± SD, n
= 3. (E and F) To test whether the stimulation of DNA fragmentation by
16K hPRL was dependent on serum concentration, BBE cells were cultured
in 0.5, 1, 5, or 10% CS for 48 h, while HUVE cells were cultured
in 0% or 10% serum. E, Cells were treated with nothing or 2, or 10
nM 16K hPRL for the last 24 h. F, Cells were treated
with nothing, 1 or 10 nM 16K hPRL, 5 ng/ml TNF , 10 EU/ml
endotoxin-1, or 5 ng/ml endotoxin-2. Each bar represents
the mean ± SD, n = 5. The
asterisk denotes a P value of <
0.05 vs. the nonstimulated control.
|
|
The effect of 16K hPRL was similar in other bovine vascular endothelial
cells including aortic endothelial (BAE) and the adrenal capillary
endothelial (BAC) cells (Fig. 1B
and data not shown). Serum deprivation
of BAE cells increased DNA fragmentation 2-fold, while 2
nM 16K hPRL caused a further 5-fold increase. Treatment
with 0.5 nM bFGF had no effect on the serum
deprivation-induced effect; however, it reduced the increase by 16K
hPRL by 25% (Fig. 1B
).
The action of 16K hPRL on DNA fragmentation was dose dependent.
Increasing concentrations of 16K hPRL from 0.1 to 25 nM
caused an exponential increase in DNA fragmentation in BBE cells (Fig. 1C
). The highest concentration of 16K hPRL caused a 14-fold increase in
DNA fragmentation as compared with 0.5% CS. The first significant
increase in DNA fragmentation (2-fold) after treatment with 2
nM 16K hPRL was detectable at 1 h in floating cells
and at 2 h in attached cells (Fig. 1D
). 16K hPRL-induced DNA
fragmentation continued to increase for up to 24 h. The time
course of the response in floating and attached cells was parallel for
8 h, but was then divergent, as it increased linearly in attached
cells and plateaued in floating cells.
To distinguish the effect of 16K hPRL on apoptosis from that of serum
deprivation, we studied the effect of 16K hPRL on DNA fragmentation in
different concentrations of serum in BBE cells. Increasing serum
concentration decreased the amplitude of 16K hPRL-induced DNA
fragmentation (Fig. 1E
). As previously observed in BBE cells cultured
in 0.5% CS, 2 and 10 nM 16K hPRL caused a 2- and 4-fold
increase in DNA fragmentation, respectively. In 1% and 5% CS,
treatment with 10 nM 16K hPRL induced a 4- and 10-fold
increase in DNA fragmentation compared with the untreated control,
respectively. Finally, in 10% CS, 2 and 10 nM 16K hPRL
caused a 3- and 8-fold increase in DNA fragmentation, respectively
(Fig. 1E
). These results show that the stimulation of DNA fragmentation
by 16K hPRL is not dependent on serum concentration. In human umbilical
vein endothelial (HUVE) cells, similar results were obtained. In 10%
FCS, 1 and 10 nM 16K hPRL caused a 3.2- and 6.5-fold
increase in DNA fragmentation, respectively (Fig. 1F
). Interestingly,
tumor necrosis factor-
(TNF
) (5 ng/ml) had no significant effect
on nucleosome formation in 10% FCS, while it caused a 7.1-fold
increase under serum-starved conditions. Finally, endotoxin standards-1
[E-Toxate kit, Sigma, St. Louis, MO; 10 endotoxin units
(EU)/ml] and -2 (Re 595, Sigma, 5 ng/ml) induced a
3.5 ± 0.2 and 4.4 ± 0.3 fold increase DNA fragmentation,
respectively, which were significantly less than the 6.5 ± 0.3
fold increase due to 10 nM 16K hPRL (n = 5,
P < 0.05).
Specificity of 16K hPRL Preparation
Since the 16K hPRL was produced in Escherichia coli it
was important to demonstrate that the activity of the preparation used
was not caused by a contaminant, e.g. endotoxin. A well
defined property of endotoxin [or lipopolysaccharide (LPS)] in many
cultured cells is the induction of programmed cell death (26, 27). In
BBE cells, while 0.5 EU/ml endotoxin-1 had no effect (Fig. 1A
), the
first detectable increase in DNA fragmentation was observed at a
concentration of 1 EU/ml of endotoxin-1, which caused a significant
2-fold increase vs. 0.5% CS control (P <
0.05) (data not shown). Treatment with 10 nM 16K
hPRL increased DNA fragmentation 5.7-fold, while treatment with 10
EU/ml endotoxin-1 caused a similar 6.1-fold increase compared with
culture in 0.5% CS (Fig. 2
). This is
1300 times the amount of endotoxin present in 320 ng of 16K hPRL
preparation used throughout the study (amount present in 2 ml of a 10
nM solution).

View larger version (43K):
[in this window]
[in a new window]
|
Figure 2. Stimulation of DNA Fragmentation by 16K hPRL Is Not
Dependent on Endotoxin Contamination
BBE cells were cultured in 0.5% or 10% CS and treated for 24 h
with 10 nM 16K hPRL or 10 EU/ml endotoxin-1. 16K hPRL and
endotoxin samples were also pretreated with Polymyxin-B, boiling for 2
min, and trypsin digestion before being added to the cells. Each
bar represents the mean ± SD, n =
3. The asterisk denotes a P value of
< 0.05 vs. the corresponding 0.5% CS control.
|
|
Heat denaturation of 16K hPRL by boiling the sample for 2 min before
adding it to the cells completely inhibited the 16K hPRL stimulation of
DNA fragmentation. Similarly, the complete digestion of 16K hPRL by
treatment with trypsin abolished its activity (Fig. 2
). Neither
treatment had any effect on the action of endotoxin-1. Furthermore,
pretreatment with 10 µg/ml (final concentration) of the endotoxin
blocker, polymyxin-B, completely abolished the endotoxin effect, but
had no effect on the action of 16K hPRL.
To further dissociate the action of 16K hPRL from endotoxin, we showed
that 16K hPRL caused a 4.8-fold increase in DNA fragmentation over
serum deprivation in HUVE cells, a response similar to that seen in BBE
cells. In comparison 5 ng/ml of TNF
caused a similar 4.8-fold
increase in DNA fragmentation, while 10 EU/ml of endotoxin-1 caused a
3.2-fold increase. However, in primary endometrial stromal cells and in
endometrial epithelial (Ishikawa) cells, while 10 nM 16K
hPRL had no effect on DNA fragmentation, 5 ng/ml of TNF
and 10 EU/ml
of endotoxin-1 stimulated DNA fragmentation in both cell types (Fig. 3
, B and C).

View larger version (32K):
[in this window]
[in a new window]
|
Figure 3. 16K hPRL Induces DNA Fragmentation Specifically in
Endothelial Cells
HUVE (A), human primary stromal endometrial (B), and human epithelial
endometrial (C) cells were cultures in 10% serum or serum starved and
treated for 24 h with either 10 nM 16K hPRL, 5 ng/ml
TNF , or 10 EU/ml endotoxin-1. Each bar represents the
mean ± SD, n = 4. The asterisk
denotes a P value of < 0.05 vs. the
serum-starved control.
|
|
Immunoneutralization of 16K hPRL was able to block its ability to
induce DNA fragmentation in BBE cells (Fig. 4A
). Both a 1:500 and 1:1000 dilution of
monoclonal antibodies 4G7 (16K hPRL specific) or 6E4 (recognizes both
16K hPRL and intact 23-kDa hPRL) inhibited 16K hPRL activity. However,
preincubation of 16K hPRL with a 1:500 dilution of an unrelated
monoclonal antibody (Ctrl) had no effect on the stimulation of DNA
fragmentation. Monoclonal antibodies 6E4 and 4G7 (1:500 dilution) also
abolished 16K hPRL activity in HUVE cells, but had no effect on TNF
and endotoxin-2 activity (Fig. 4B
). Finally, a neutralizing antibody
against human TNF
was able to block the TNF
effect, but neither
the 16K hPRL nor the endotoxin-2 effects. Preincubation with the
unrelated antibody had no effect on any response.

View larger version (39K):
[in this window]
[in a new window]
|
Figure 4. Immunoneutralization of 16K hPRL Effect
A, BBE cells were cultured in 0.5% or 10% CS and treated for 24
h. Samples containing 10 nM 16K hPRL were preincubated with
either a monoclonal antibody that recognizes both native 23 kDa hPRL
and 16K hPRL (6E4), a monoclonal antibody that is specific for 16K hPRL
(4G7), or a control monoclonal antibody that is unrelated to 16K hPRL
(Ctrl). B, HUVE cells were cultured in 10% serum or serum starved and
treated for 24 h with 10 nM 16K hPRL, 5 ng/ml TNF ,
or 5 ng/ml endotoxin-2. Indicated samples were preincubated with the
monoclonal antibodies 6E4 or 4G7, or an immunoneutralizing
antibody specific for human TNF , or the control monoclonal antibody
(control mAb). Each bar represents the mean ±
SD, n = 3. The asterisk denotes a
P value of < 0.05 vs. the
corresponding serum-starved control.
|
|
These results demonstrate that the ability of the 16K hPRL preparations
to stimulate DNA fragmentation was dependent on the activity of the
peptide, and not on endotoxin or contaminating proteins in the
preparation. The results also show that the action of 16K hPRL is
specific for vascular endothelial cells. Furthermore, the experiments
in HUVE cells show that the action of 16K hPRL is not mediated by the
stimulation of the release of TNF
.
Action of 16K hPRL Is Not Mediated via PRL or GH Receptors
To demonstrate that 16K hPRL is not interacting with the GH or PRL
receptor family to transduce its signal, we tested the effect of hGH
and hPRL alone or in combination with 16K hPRL (Fig. 5
). Addition of 10 and 50 nM
of either recombinant hGH or hPRL alone after serum deprivation of BBE
cells had no significant effects on DNA fragmentation. Once again these
results argue against the possibility that the action of 16K hPRL is
due to endotoxin contamination, since both recombinant hPRL and hGH had
no effect on DNA fragmentation even though the concentration used had
equivalent or higher endotoxin content than the 16K hPRL preparation.
Furthermore, addition of 25 times more hGH or hPRL (50 nM)
had no antagonistic effect on the ability of 2 nM 16K hPRL
to stimulate DNA fragmentation (Fig. 5
). The results suggested that, as
for inhibition of mitogen-induced cell proliferation (11) or activation
of PAI-1 expression (19), the 16K hPRL effects are not mediated by the
PRL or GH receptors, but rather by a yet unknown type of receptor.

View larger version (45K):
[in this window]
[in a new window]
|
Figure 5. Effect of 16K hPRL Is Not Mediated by the GH or PRL
Receptors
BBE cells were cultured in normal 10% CS conditions, and then serum
deprived or not for 24 h, and finally treated for another 24
h with increasing concentrations of hGH or hPRL alone, with 2
nM 16K hPRL alone, or in combination with 50 nM
hGH or hPRL to test the agonistic or antagonistic effects of 16K hPRL
to hPRL and hGH. Each bar represents the mean ±
SD, n = 3. The asterisk denotes a
P value of < 0.05 vs. 0.5% CS
control.
|
|
Effect of 16K hPRL on DNA Cell Content
To directly investigate the effect of 16K hPRL on the number of
cells entering programmed cell death, we analyzed the DNA content of
16K hPRL-treated BBE cells using flow cytometry of propidium
iodide-stained cells. After 2 days of serum deprivation, BBE cells were
relatively well synchronized in
G0/G1 phase (Fig. 6A
, top panel), with 17% of
the cells having reduced DNA content (undergoing programmed cell
death). After 24 h of 16K hPRL treatment (2
nM), more than 50% of the cells were undergoing
apoptosis. Treatment with 16K hPRL drastically diminished the
population of cells in G2/M phase, as well as the
population of cells in
G0/G1 phase (Fig. 6A
, bottom panel). Kinetically, we observed the same pattern of
changes in DNA content after 16K hPRL treatment as had been observed
for DNA fragmentation (Fig. 6B
). The dying cell population doubled
after 1 h of treatment and was still increasing at 24 h.
These results indicate that 16K hPRL not only increased the DNA
fragmentation in cells already undergoing apoptosis after serum
deprivation, but also recruited new cells into this pathway.

View larger version (44K):
[in this window]
[in a new window]
|
Figure 6. Stimulation of Apoptosis by 16K hPRL as Determined
by DNA Content Analysis by Flow Cytometry
BBE cells were cultured in 0.5% CS (A) or 10% CS (C) for 24 h
and then treated with nothing (Control) or 2 nM 16K hPRL
for 24 h. Events falling under the 100 (0 to 100) FL2 channel
scale are considered as apoptotic events resulting from DNA
fragmentation. Percents represent the proportion of gated cells
undergoing apoptosis. Position of the G0/G1, S,
G2/M, and apoptosis phase is indicated on top of the
right panel (A). B and D, Percentage of cells undergoing
apoptosis after treatment with 2 nM 16K hPRL with time.
Each point represents the mean value of two separate experiments.
|
|
Similar effects on DNA content were observed when experiments were
performed in 10% CS. Cells cultured in 10% CS showed three distinct
populations, 52% of the cell being in
G0/G1 phase, 38% being in
S or G2/M phases, and only 10% of the cells
undergoing programmed cell death (Fig. 6C
, top panel). After
24 h treatment with 2 nM 16K hPRL, the
population of cells undergoing mitosis was dramatically diminished, to
7%, as was the population of cells in
G0/G1 phase (24%). A
similar percentage of cells (68%) were undergoing apoptosis in 10% CS
as seen with 0.5% CS (50%) (Fig. 6C
, bottom panel).
Kinetically, we observed the same dynamics of the changes in DNA
content after 16K hPRL treatment as seen for cells cultured in 0.5%
CS. The apoptotic cell population doubled after 1 h and was still
growing between 8 and 24 h (Fig. 6D
).
16K hPRL Activates the Caspase Cascade
We then asked whether the stimulation of apoptosis by 16 K hPRL
was associated with activation of the caspase cascade. By Western
blotting the 32-kDa proform of caspase 3 was expressed in BBE cells
before and after serum deprivation (Fig. 7A
). The 17-kDa active subunit of caspase
3 was not present in cells cultured in 10% CS, while after culture in
0.5% CS for 24 h a faint 17-kDa band corresponding to the active
subunit of caspase 3 began to appear. Treatment with 16K hPRL alone or
in combination with bFGF induced a large increase in the formation of
the active 17-kDa form, while the amount of the 32-kDa proform was
decreased (treatment with 16K hPRL in combination with bFGF) or
unchanged (treatment with 16K hPRL alone).

View larger version (66K):
[in this window]
[in a new window]
|
Figure 7. Activation of Caspases 1 and 3 by 16K hPRL
A, Western blot analysis of caspase 3 in lysates of BBE cells treated
for 24 h with 0.5 nM bFGF, 2 nM 16K hPRL,
or both in 0.5% CS. The molecular masses of the inactive proform (32
kDa), partially processed inactive form (28 kDa), and active subunit
(17 kDa) of caspase 3 are indicated on the left side. B,
upper panel: Western blot analysis using an anti-PARP
antibody (PARP). The active 116 kDa form of PARP was converted to the
inactive 85 kDa form after activation of caspase 3. B, bottom
panel: The mean ± SD (n = 4) relative
intensity of the 116 and 85 kDa bands determined by ImageQuant Software
(Molecular Dynamics, Inc.). The asterisk
denotes a P value of < 0.05 vs.
0.5% CS control. C, Western blot analysis of the inactive 45 kDa
proform, partially processed inactive 30 kDa form, and 20 kDa active
form of caspase 1, after treatment with 2 or 10 nM 16K
hPRL, 0.5 nM bFGF, and zVAD, alone or in combination. The
effects of treatment with 23K hPRL or endotoxin-1 were also tested. D,
Effect of CS concentration (1%, 5%, 10%, or 0.5%) on the activation
of caspase 1 by 16K hPRL. E, Time course of the activation of caspases
1 and 3 and inactivation of caspase 3 substrates, PARP and ICAD, after
treatment with 2 nM 16K hPRL in 0.5% or 10% CS. Sizes of
the expected proteins are indicated in between the two
panels.
|
|
To confirm that the increase in the 17-kDa band was associated with an
increase in caspase 3 activity, we analyzed one of the known substrates
of caspase 3, the poly(ADP-ribose) polymerase (PARP). This 116-kDa DNA
repair enzyme is cleaved to an inactive 85-kDa fragment by caspase 3
(Fig. 7B
, upper panel). By densitometric analysis, treatment
with 16K hPRL resulted in the conversion of 65% of PARP into the
inactive 85-kDa fragment (Fig. 7B
, lower panel). Serum
deprivation only resulted in the conversion of 15% of the PARP, while
at the basal level, in 10% CS, the conversion level was 5%. Treatment
with bFGF was not able to significantly reverse the effect of 16K
hPRL.
We then asked whether treatment with 16K hPRL activated caspase 1, a
potential upstream step in the caspase cascade. Caspase 1 has been
shown to activate caspase 3 in response to TNF
(28), as well as
during photooxidative stress (29). By Western blotting, caspase 1 was
present in BBE cells as the inactive 45-kDa proenzyme (Fig. 7C
).
Treatment with 16K hPRL, at two different doses (2 and 10
nM), or in combination with bFGF, stimulated the formation
of the 20-kDa fragment of caspase 1, known to be part of the active
protease complex. This band was absent before serum deprivation and in
most of the experiments after serum deprivation, or after treatment
with bFGF or native hPRL. The effect of 10 nM 16K hPRL on
caspase 1 activation was independent of serum concentration, since it
was observed in 1, 5, and 10% CS (Fig. 7D
). Treatment with 1
nM 16K hPRL was capable of inducing the formation of the
20-kDa fragment in 10% CS, while serum deprivation resulted in the
appearance of only a faint band (Fig. 7D
). Endotoxin-1 (1 EU/ml) was
not able to induce the processing of caspase 1 to its active form (Fig. 7C
). The effect of 16K hPRL was inhibited by treatment with the general
caspase inhibitor, z-VAD (30). A similar effect of z-VAD treatment was
observed in BAE cells. The increase in DNA fragmentation induced by 2
nM 16K hPRL in 0.5% CS was abolished by pretreatment of
the cells with 20 µM z-VAD (Fig. 1B
). These results were
consistent with the idea that 16K hPRL specifically activated an
apoptotic cascade involving the activation of caspase 1.
The kinetics of caspases 1 and 3 processing were similar
under 0.5 and 10% CS culture conditions (Fig. 7E
, 0.5% CS and
10% CS). The active forms of the proteases were observed as early as
15 min after treatment with 2 nM 16K hPRL. By 30 min,
activation of caspase 3 resulted in the cleavage of PARP (116 kDa),
resulting in the appearance of the inactive 85-kDa form. The 85-kDa
cleaved form was observed throughout the 24-h treatment, while the
intact PARP could no longer be observed after 3 h in 10% CS and
was almost undetectable in 0.5% CS. The inhibitor of the
caspase-activated DNase (ICAD) (31, 32), another potential substrate of
caspase 3, was cleaved to its inactive 34-kDa form in a similar pattern
by treatment with 16K hPRL. This suggests that 16K hPRL specifically
activated an apoptotic cascade involving the activation of caspases,
like caspase 1 or 3, and subsequently the inactivation of downstream
substrates like PARP or ICAD.
Effects of 16K hPRL on Expression of the Bcl-2 Family
Among the large Bcl-2 protein family, we analyzed the expression
of one antiapoptotic protein, Bcl-2, and two proapoptotic proteins, Bax
and Bak, by Western blotting (33). After serum deprivation, the amount
of Bcl-2 was markedly increased (Fig. 8A
). Treatment of the BBE cells with 2 or
10 nM 16K hPRL did not significantly modify this increased
expression. The levels of Bax and Bak were unchanged after culture
in 0.5% CS or treatment with 2 or 10 nM 16K hPRL.
Densitometric analysis and expression of the results as the
Bcl-2/Bax ratio also showed no changes (Fig. 8B
).

View larger version (43K):
[in this window]
[in a new window]
|
Figure 8. Effect of Treatment with 16K hPRL on Levels of
Bcl-2, Bax, Bak, and Bcl-X
A, Western blot analysis of Bcl-2, Bax, and Bak after treatment with 2
or 10 nM 16K hPRL in 0.5% CS as compared with culture in
0.5 or 10% CS. Size of the corresponding proteins was indicated on the
right side. B, The relative intensity of 26 and 23 kDa
bands, corresponding to Bcl-2 and Bax, respectively, and the mean
(n = 2) Bcl-2/Bax ratio calculated from the intensity of the bands
determined by the ImageQuant Software (Molecular Dynamics, Inc.). C, Time course of the effect of treatment with 2
nM 16K hPRL on the level of Bcl-2, Bcl-X, Bax, and Bak as
analyzed by Western blot in 0.5 and 10% CS. The 29 kDa antiapoptotic
form of Bcl-X (Bcl-XL) was converted to the 21 kDa
proapoptotic form (Bcl-XS) by treatment with 16K hPRL.
Sizes of the expected proteins are indicated in between the two
panels.
|
|
Similarly, in kinetic studies, treatment with 2 nM 16K hPRL
had little effect on the level of Bcl-2, Bax, or Bak over a 24-h period
(Fig. 8C
, 0.5% CS and 10% CS). However, treatment with 16K hPRL
stimulated the conversion of Bcl-X from its antiapoptotic 29-kDa form
(Bcl-XL) to its proapoptotic 21-kDa form
(Bcl-XS) (34). Clear increases in
Bcl-XS were observed 1 h after treatment
with 16K hPRL, and at 3 h, 50 and 20% of
Bcl-XL was converted to
Bcl-XS in 0.5% CS and 10% CS, respectively.
These results suggest that 16K hPRL had no major effect on Bcl-2, Bax,
and Bak levels. However, stimulation of the conversion of
Bcl-XL to inactive Bcl-XS
by 16K hPRL is consistent with the activation of apoptosis. This
observation may help to explain why the increase in Bcl-2 expression
observed after culture in 0.5% CS was not sufficient to prevent the
apoptotic effect of 16K hPRL.
 |
DISCUSSION
|
---|
These findings demonstrate that 16K hPRL specifically stimulates
programmed cell death of vascular endothelial cells by multiple
criteria: DNA fragmentation; fluorescence-activated cell sorting
(FACS) analysis of BBE cell DNA content; activation of the caspase
cascade; and regulation of the Bcl-2 family members. In addition,
treatment with 16K hPRL inhibited entry of BBE cells into the cell
cycle at the G0/G1
transition. After 24 h of 16K hPRL treatment, more than 50% of
the cells were undergoing apoptosis, while the reminder of cells were
mainly in the G0/G1 state.
These data support the earlier observations that 16K hPRL inhibits
FGF-induced BBE cell proliferation, which was followed by rounding up
of the cells (11); they show in addition that 16K hPRL stimulates
apoptosis. This induction of programmed cell death was dependent on the
caspase cascade activation, as well as the inactivation of Bcl-X.
Bacterial endotoxin (LPS) has been shown to increase PAI-1 expression,
stimulate apoptosis, and inhibit cell proliferation of endothelial
cells (35, 36). The observation regarding the similarity of the
signaling pathways activated by LPS and 16K hPRL made it imperative to
demonstrate that the antiangiogenic actions of the recombinant 16K hPRL
preparations was not due to contamination with endotoxin. First the
endotoxin level of the 16K hPRL preparation used in this study was more
than a 1,000-fold less than necessary to observe any biological effect.
The ability of the 16K hPRL preparations to stimulate DNA fragmentation
could be completely blocked by digestion of the protein with trypsin,
or denaturation of the protein by boiling. The action of LPS was
unaffected by boiling but was blocked by the addition of polymyxin-B.
However, polymyxin-B treatment had no effect on the action of 16K hPRL.
That the activity of the 16K hPRL could depend on complexing with LPS
in the preparations appears unlikely, since for every 3,000 16K hPRL
molecules, there is approximately 1 LPS molecule. Human endothelial
cells, especially HUVE cells, have been described as 1,000 times less
sensitive to endotoxin than bovine endothelial cells (37). However, we
found that HUVE cells responded to 16K PRL (10 nM) and
TNF
(5 ng/ml) in a similar manner, and to a lesser degree to
endotoxin-1 (10 EU/ml). We also demonstrated that the response to 16K
hPRL was specific for endothelial cells, while endotoxin and TNF
activated DNA fragmentation in endothelial cells as well as stromal and
epithelial cells. Most importantly, the ability of 16K hPRL to activate
DNA fragmentation in both bovine and human endothelial cells was
abolished by immunoneutralization using specific monoclonal antibodies.
The unlikely possibility that the activity of the recombinant
preparations is due to some unknown heat-sensitive contaminant that
coprecipitates with 16K hPRL cannot be ruled out.
Within 15 min 16K hPRL treatment resulted in conversion of the inactive
proforms of caspase 1 and 3 to their active fragments. Activation of
caspase 3 was directly confirmed by the cleavage and inactivation of
the substrates, PARP and ICAD. Cleavage of both PARP (38) and ICAD (31, 39) by caspase 3 have been shown to be important steps in the apoptotic
pathway. Inactivation of ICAD allows caspase-activated DNase (CAD) to
enter the nucleus and degrade genomic DNA, while inactivation of PARP
inhibits its DNA repair activity. The combination of these two events
facilitates cellular disassembly and ensures the completion and
commitment of the cell to the apoptotic pathway. Activation of the
caspase cascade was essential for the apoptotic action of 16K hPRL. The
processing of caspase 1 and the stimulation of DNA fragmentation by 16K
hPRL were totally blocked by treatment with the caspase inhibitor,
z-VAD.
Although the levels of Bcl-2, Bax, and Bak were unaffected by treatment
with 16K hPRL, the conversion of Bcl-X was stimulated from its
antiapoptotic form, Bcl-XL, to its proapoptotic
form, Bcl-XS. Increased expression of
Bcl-XL appears to play an important role in the
ability of nuclear factor (NF)-
B to inhibit apoptosis (40). The
inhibition of the antiapoptotic action of
Bcl-XL by 16K hPRL treatment is consistent with
its stimulation of apoptosis.
The signaling mechanisms mediating the multiple antiangiogenic actions
of 16K hPRL on vascular endothelial cells are still poorly understood.
Although a specific, high-affinity, saturable binding site for 16K hPRL
has been described on capillary endothelial cells (15), the identity of
the receptor molecule is unknown. The effects of 16K hPRL are not
mediated via an action on the PRL receptor as an agonist or antagonist.
As with the effects of 16K hPRL on cell proliferation (11) and
stimulation of PAI-1 expression (19), intact 23-kDa hPRL has no effect
on stimulating apoptosis or inhibiting the ability of 16K hPRL to
stimulate apoptosis. The question of how binding of 16K hPRL to its
receptor activates the caspase cascade and apoptosis, inhibits
activation of Ras and presumably cell proliferation, and increases the
expression of PAI-1 remains unclear. However, activation of multiple
signaling pathways appears to be the rule rather than the exception
with ligands acting on endothelial cells. For example, another
antiangiogenic factor thrombospondin inhibits endothelial cell
proliferation and stimulates apoptosis (21, 41, 42). Several other
cytokines have also been shown to affect multiple signaling pathways in
endothelial cells, e.g. TNF
and transforming growth
factor-ß (TGFß) (43, 44, 45).
Tumor growth and progression have been shown to be dependent on
development of a new microvasculature (2). Inhibition of the action of
the angiogenic factor VEGF has been shown to result in tumor
endothelial cell apoptosis and tumor necrosis (46). As previously seen,
withdrawal of bFGF and/or serum induces endothelial cell apoptosis
in vitro (47). These observations support the importance of
the regulation of apoptosis as a control mechanism for tumor
angiogenesis. The ability of 16K hPRL to inhibit the stimulation of
endothelial cell proliferation by the angiogenic factors VEGF and bFGF
and to directly stimulate apoptosis strongly supports the potential of
16K hPRL as an antitumor drug.
In conclusion, we have clearly demonstrated that 16K hPRL protein in
our recombinant preparation is responsible for promoting programmed
cell death of cultured endothelial cells. Furthermore, we have
presented detailed functional evidence that the caspase cascade is
fundamental for 16K hPRL-induced apoptosis in a dose- and
time-dependent manner. These findings reveal a novel and important
mechanism for 16K hPRL to regulate angiogenesis and emphasize its
potential antitumor properties.
 |
MATERIALS AND METHODS
|
---|
Production of Recombinant Proteins
For the production of recombinant 16K hPRL the cDNA encoding
hPRL minus the corresponding signal peptide was inserted into the pT7L
expression vector (48). An ATG was genetically engineered 5' to the
first codon of the cDNA. The codon for Cys 58 (TGC) was mutated to a
Ser codon (TCC) to prevent the formation of incorrect disulfide bounds
during refolding of the 16K hPRL (11). To get high levels of expression
of the recombinant protein in E. coli the full-length hPRL
molecule was expressed, purified, and then cleaved (14). To accomplish
this the nucleotide sequence coding for amino acids 139144
(Pro-Glu-Thr-Lys-Glu-Asn: CCT-GAA-ACC-AAA-GAA-AAT) in hPRL was replaced
with the nucleotide sequence coding for the cleavage site of IgA
protease (Pro-Arg-Pro-Pro-Thr-Pro: CCT-AGA-CCC-CCA-ACA-CCT).
Cleavage occurred between Pro 142 and Thr 143.
In brief, intact protein expression was induced in E. coli
BL21 DE3 with isopropyl ß-D-thiogalactopyranoside and the
inclusion bodies were isolated. After washing, the inclusion bodies
were solubilized in denaturation buffer (8 M
urea, 20 mM ethanolamine-HCl, pH 10, 1%
2-mercaptoethanol, 0.5 mM PMSF) for 10 min at 55
C and overnight at room temperature. After renaturation by dialysis
against 20 mM ethanolamine-HCl, pH 9, the
proteins were purified as previously described (48). The mutated hPRL
was enzymatically cleaved with IgA protease (0.05%, 25 C, overnight;
Roche Molecular Chemicals, Indianapolis, IN). The 16K hPRL
was purified by ion-exchange chromatography (HiTrap Q, Pharmacia Biotech, Piscataway, NJ).
The purity of each recombinant protein exceeded 95% as estimated by
silver staining. The Limulus amoebocyte lysate assay
(E-Toxate kit, Sigma) was used to detect and quantify
endotoxin levels. The endotoxin level of the 16K hPRL preparation used
in the studies was 47 x 10-6 EU/ng
protein.
Cell Culture
BBE and BAE cells were isolated as previously described (Refs.
49, 50), respectively. The cells were grown and serially passaged in
low glucose DMEM supplemented with 10% calf serum (CS), 2
mM L-glutamine, and antibiotics (100 U of
penicillin/streptomycin per ml and 2.5 mg of fungizone per ml).
Recombinant human basic FGF (bFGF, Promega Corp., Madison,
WI) was added (1 ng/ml) to the cultures every other day. Experiments
were initiated with confluent cells between passages 612. Primary
human umbilical vein endothelial (HUVE) cells were obtained from
Clonetics Corp. (San Diego, CA), grown according to specification, and
retained for up to six passages. Primary human endometrial stromal
cells and human endometrial epithelial cell line (Ishikawa) were kindly
provided by Drs. R. N. Taylor and D. Lebovic (University of
California, San Francisco). Primary stromal cells were prepared and
cultured from endometrial biopsies as described (51). Ishikawa cells
were routinely grown in DMEM/F-12 (1:1) supplemented with 10% FBS,
penicillin-streptomycin, and sodium pyruvate.
Cell Stimulation and Preparation of Cell Extracts
Confluent cell cultures were dispersed and plated at the density
of 1520 x 103
cells/cm2 culture plate (one plate per condition)
in appropriate media. Thirty six hours after plating, cells were either
serum starved or left in medium containing 10% serum for another
24 h. Cells were left untreated or treated with 2
nM recombinant human VEGF165 (VEGF,
Genentech, Inc., South San Francisco, CA), 2
nM VEGF plus 2 nM 16K hPRL, 0.5 nM
bFGF (Promega Corp.), 0.5 nM bFGF plus 2
nM 16K hPRL, various concentrations of 16K hPRL,
recombinant human TNF
(Pepro Tech, Rocky Hill, NJ), endotoxin-1
(Limulus amoebocyte lysate, E-Toxate kit,
Sigma) or endotoxin-2 (Salmonella
minnesota Re 595, a minimal naturally occurring endotoxic
structure of LPS, Sigma) for 24 h.
Incubations were terminated by aspiration of the medium, two washes
with ice-cold PBS, and addition of 200 µl of lysis buffer (1% Triton
X-100 lysis buffer containing 20 mM Tris-HCl, pH 8.0, 137
mM NaCl, 10% (vol/vol) glycerol, 2 mM EDTA,
with (for detection of nuclear proteins) or without 3 M
urea, 1 mM pefabloc, 0.14 U aprotinin, 20 µM
leupeptin, and 1 mM sodium orthovanadate) at 4 C as
previously described (16).
The peptidyl fluoromethyl ketone (fmk) caspase inhibitor z-VAD-fmk (20
µM, Enzyme Systems Products, Livermore, CA)
was added at the time cells were serum deprived and/or treatment
added.
Endotoxin Controls
To study the effect of the addition of endotoxin (LPS), several
approaches were used to demonstrate that the apoptotic activity of 16K
hPRL preparations was not due to endotoxin contamination. Polymyxin-B
(Sigma), an antibiotic that binds and inactivates LPS, was
preincubated with samples for 10 min at 37 C in a final concentration
of 10 µg/ml. Alternatively, 16K hPRL activity was destroyed in the
sample to be added to the culture cells by boiling for 2 min, or
proteolytic digestion with trypsin (Sigma) for 16 h
at 37 C according to the manufacturers protocol. Efficiency of the
digestion was tested by Coomassie blue staining (Bio-Rad Laboratories, Inc.) of SDS-PAGE gels (16% acrylamide). Finally
16K hPRL activity was immunoneutralized by preincubation for 45 min at
4 C in serum free medium containing either a specific monoclonal
antibody for 16K hPRL (4G7, Dr. Martial), a monoclonal antibody that
recognizes both the 23 kDa native hPRL and 16K hPRL with equal potency
(6E4, Dr. Martial) and, as controls, an unrelated monoclonal antibody
(pp54) or a neutralizing monoclonal antibody antihuman TNF
(Upstate Biotechnology, Inc., Lake Placid, NY; 1:750
dilution).
DNA Fragmentation ELISA Assay
A hallmark of programmed cell death by apoptosis is the
formation of multinucleosomal sized genomic DNA fragments. DNA
fragments are multiples of 180 bp subunits associated with core
histones. Accumulation of mono- and oligonucleosomes into the cytoplasm
of apoptotic cells is due to the fact that DNA fragmentation occurs
before the breakdown of the plasma membrane, which is not the case with
cell death by necrosis. The levels of mono- and oligonucleosomal DNA
released in the cytosol of apoptotic cells were measured using the Cell
Death Detection ELISA kit (Roche Molecular Biochemicals,
Indianapolis, IN). This is a quantitative sandwich-enzyme-immunoassay
using antibodies against DNA and histones. Levels of DNA fragmentation
were expressed as an enrichment factor, calculated by dividing the
absorbance of a given sample by the absorbance of the corresponding
10% CS control.
DNA Content Analysis
BBE cells were cultured and treated as described above. The
cells were harvested by trypsinization, washed in cold PBS, and fixed
in 80% ethanol in PBS at 4 C for 30 min. After centrifugation, the
cell pellets were suspended into PBS and passed through a 70 µm nylon
cell strainer (Falcon). The cells were stained with 10 µg/ml
propidium iodide and treated with 200 µg/ml RNase A at 37 C for 30
min. The fluorescence of individual cells was measured with a
FACScalibur cytofluorometer equipped with the CellQuest software
(Becton Dickinson and Co., Franklin Lakes, NJ).
Western Blotting
To detect processing of apoptotic proteases (caspases 1 and 3),
the cleavage of poly(ADP-ribose) polymerase (PARP), and to survey the
protein expression of ICAD, Bax, Bak, Bcl2, Bcl-X, equal quantities of
lysates from BBE cells were resolved by SDS/PAGE (8%, 12%, or 15%)
and transferred to polyvinylidene fluoride membranes
(Millipore Corp., Bedford, MA). Separate Western blots
were performed using a variety of antibodies including an antihuman
caspase 1 (ICE) rabbit polyclonal antibody (Upstate Biotechnology, Inc. 1:1000 dilution) that recognizes the
proenzyme of 45 kDa, the partially cleaved inactive form of 30 kDa, and
the p20 subunit of active caspase 1; an antihuman caspase 3
(YAMA/Apopain/CPP32) rabbit polyclonal antibody (Upstate Biotechnology, Inc. 1:1000 dilution; Santa Cruz Biotechnology, Inc., 1:500 dilution) that recognizes the caspase
3 precursor (32 kDa), the partially processed inactive form of 28 kDa,
and the p17 subunit of the active caspase 3; an antihuman PARP rabbit
polyclonal antibody (Upstate Biotechnology, Inc. 1:750
dilution) that recognizes both the 116 kDa form of active PARP and its
85 kDa proteolytic fragment; an antihuman DFF-45/ICAD rabbit polyclonal
antibody (Upstate Biotechnology, Inc. 1:1000 dilution)
that recognizes the N-terminal part of both the full-length molecule
(45 kDa) and the caspase 3 generated form of approximately 34 kDa; an
antihuman Bcl-2 oncoprotein mouse monoclonal antibody
(Upstate Biotechnology, Inc. 1:500; Santa Cruz Biotechnology, Inc., 1:500 dilution) that recognizes a protein
of 26 kDa; an antihuman Bak rabbit polyclonal antibody (Upstate Biotechnology, Inc. 1:1000 dilution) that recognizes a protein
of 29 kDa; an antihuman Bax rabbit polyclonal antibody (Upstate Biotechnology, Inc. 1:500 dilution) that recognizes a protein of
23 kDa; and an antichicken
Bcl-XL/XS rabbit polyclonal
antibody (Upstate Biotechnology, Inc. 1:1000 dilution)
that recognizes both proteins of 21 (Bcl-XS) and
29 kDa (Bcl-XL). Nuclear extract of human Hela,
human A431 cells stimulated with or without EGF, and mouse 3T3 cell
lysates were used appropriately as positive controls for all
antibodies. Western blots were incubated with the appropriate antibody
and then washed in Tris-buffered saline containing 0.1% Tween 20.
Antigen-antibody complexes were detected with horseradish
peroxidase-coupled secondary antibodies and the enhanced
chemiluminescence system (Renaissance, NEN Life Science Products, Boston, MA). Finally, the blots were developed on
reflection NEF film (NEN Life Science Products).
Statistical Analysis
All values are expressed as mean ± SD.
Comparisons between treatment conditions were assessed by one-way ANOVA
with the post hoc analysis with the StudentNewman-Keuls test.
Statistical significance was defined as a value of P <
0.05.
 |
ACKNOWLEDGMENTS
|
---|
The authors are grateful to Drs. R.N. Taylor, D. Lebovic, F.
Bentzien, and N. Lomri for reagents, useful advice, and discussion.
Technical assistance was provided by A. Choi.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Richard I. Weiner, Ph.D., Department of Obstetrics/Gynecology/Reproductive Science, Box 0556, 513 Parnassus Avenue, University of California, San Francisco, California 94143-0556. e-mail: weinerr@obgyn.ucsf.edu.
This work was supported by University of California/Chiron Corp. bioSTAR Grant.
1 Current address: Centre Hospitalier Regional et Universitaire,
Service de Cardiologie B, 34295 Montpellier Cedex 5, France. 
2 Current address: Gastrointestinal Division, University of California
School of Medicine, San Francisco, California 94143. 
3 Current address: Laboratoire de Biologie Moléculaire et de
Génie Génétique, Université de Liège,
Allée du 6 Août, B6, B-4000 Sart Tilman, Belgium. 
Received for publication April 14, 2000.
Revision received July 13, 2000.
Accepted for publication July 17, 2000.
 |
REFERENCES
|
---|
-
Hanahan D, Folkman J 1996 Patterns and emerging
mechanisms of the angiogenic switch during tumorigenesis. Cell 86:353364[Medline]
-
Folkman J 1995 Angiogenesis inhibitors generated by tumors.
Mol Med 1:1202[Medline]
-
Pepper MS 1997 Manipulating angiogenesis. From basic science
to the bedside. Arterioscler Thromb Vasc Biol 17:605619[Abstract/Free Full Text]
-
Esch F, Baird A, Ling N, Ueno N, Hill F, Denoroy L, Klepper
R, Gospodarowicz D, Bohlen P, Guillemin R 1985 Primary structure of
bovine pituitary basic fibroblast growth factor (FGF) and comparison
with the amino-terminal sequence of bovine brain acidic FGF. Proc Natl
Acad Sci USA 82:65076511[Abstract]
-
Ferrara N, Henzel WJ 1989 Pituitary follicular cells secrete
a novel heparin-binding growth factor specific for vascular endothelial
cells. Biochem Biophys Res Commun 161:851858[Medline]
-
Fett JW, Strydom DJ, Lobb RR, Alderman EM, Bethune JL,
Riordan JF, Vallee BL 1985 Isolation and characterization of
angiogenin, an angiogenic protein from human carcinoma cells.
Biochemistry 24:54805486[Medline]
-
Davis S, Aldrich TH, Jones PF, Acheson A, Compton DL, Jain V,
Ryan TE, Bruno J, Radziejewski C, Maisonpierre PC, Yancopoulos GD 1996 Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by
secretion-trap expression cloning. Cell 87:11611169[Medline]
-
Maione TE, Gray GS, Petro J, Hunt AJ, Donner AL, Bauer SI,
Carson HF, Sharpe RJ 1990 Inhibition of angiogenesis by recombinant
human platelet factor-4 and related peptides. Science 247:7779[Medline]
-
Good DJ, Polverini PJ, Rastinejad F, Le Beau MM, Lemons RS,
Frazier WA, Bouck NP 1990 A tumor suppressor-dependent inhibitor of
angiogenesis is immunologically and functionally indistinguishable from
a fragment of thrombospondin. Proc Natl Acad Sci USA 87:66246628[Abstract]
-
Ferrara N, Clapp C, Weiner R 1991 The 16K fragment of
prolactin specifically inhibits basal or fibroblast growth factor
stimulated growth of capillary endothelial cells. Endocrinology 129:896900[Abstract]
-
Clapp C, Martial JA, Guzman RC, Rentier-Delure F, Weiner RI 1993 The 16-kilodalton N-terminal fragment of human prolactin is a
potent inhibitor of angiogenesis. Endocrinology 133:12921299[Abstract]
-
OReilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses
M, Lane WS, Cao Y, Sage EH, Folkman J 1994 Angiostatin: a novel
angiogenesis inhibitor that mediates the suppression of metastases by a
Lewis lung carcinoma. Cell 79:31528[Medline]
-
OReilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS,
Flynn E, Birkhead JR, Olsen BR, Folkman J 1997 Endostatin: an
endogenous inhibitor of angiogenesis and tumor growth. Cell 88:277285[Medline]
-
Struman I, Bentzien F, Lee H, Mainfroid V, DAngelo G, Goffin
V, Weiner RI, Martial JA 1999 Opposing actions of intact and N-terminal
fragments of the human prolactin/growth hormone family members on
angiogenesis: an efficient mechanism for the regulation of
angiogenesis. Proc Natl Acad Sci USA 96:12461251[Abstract/Free Full Text]
-
Clapp C, Weiner RI 1992 A specific, high affinity, saturable
binding site for the 16-kilodalton fragment of prolactin on capillary
endothelial cells. Endocrinology 130:13801386[Abstract]
-
DAngelo G, Struman I, Martial J, Weiner RI 1995 Activation
of mitogen-activated protein kinases by vascular endothelial growth
factor and basic fibroblast growth factor in capillary endothelial
cells is inhibited by the antiangiogenic factor 16-kDa N-terminal
fragment of prolactin. Proc Natl Acad Sci USA 92:63746378[Abstract]
-
DAngelo G, Martini JF, Iiri T, Fantl WJ, Martial J, Weiner
RI 1999 16K human prolactin inhibits vascular endothelial growth
factor-induced activation of Ras in capillary endothelial cells. Mol
Endocrinol 13:692704[Abstract/Free Full Text]
-
Pepper MS, Vassalli JD, Montesano R, Orci L 1987 Urokinase-type plasminogen activator is induced in migrating capillary
endothelial cells. J Cell Biol 105:25352541[Abstract]
-
Lee H, Struman I, Clapp C, Martial J, Weiner RI 1998 Inhibition of urokinase activity by the antiangiogenic factor 16K
prolactin: activation of plasminogen activator inhibitor 1 expression.
Endocrinology 139:36963703[Abstract/Free Full Text]
-
Claesson-Welsh L, Welsh M, Ito N, Anand-Apte B, Soker S,
Zetter B, OReilly M, Folkman J 1998 Angiostatin induces endothelial
cell apoptosis and activation of focal adhesion kinase independently of
the integrin-binding motif RGD. Proc Natl Acad Sci USA 95:55795583[Abstract/Free Full Text]
-
Guo N, Krutzsch HC, Inman JK, Roberts DD 1997 Thrombospondin 1 and type I repeat peptides of thrombospondin 1
specifically induce apoptosis of endothelial cells. Cancer Res 57:17351742[Abstract]
-
Nicholson DW, Thornberry NA 1997 Caspases: killer proteases.
Trends Biochem Sci 22:299306[CrossRef][Medline]
-
Cryns V, Yuan J 1998 Proteases to die for. Genes Dev 12:15511570[Free Full Text]
-
Gagliardini V, Fernandez PA, Lee RK, Drexler HC, Rotello RJ,
Fishman MC, Yuan J 1994 Prevention of vertebrate neuronal death by the
crmA gene. Science 263:826828[Medline]
-
Au JL, Panchal N, Li D, Gan Y 1997 Apoptosis: a new
pharmacodynamic endpoint. Pharmacol Res 14:16591671[CrossRef]
-
Bannerman DD, Sathyamoorthy M, Goldblum SE 1998 Bacterial
lipopolysaccharide disrupts endothelial monolayer integrity and
survival signaling events through caspase cleavage of adherens junction
proteins. J Biol Chem 273:3537135380[Abstract/Free Full Text]
-
Lawson JA, Fisher MA, Simmons CA, Farhood A, Jaeschke H 1998 Parenchymal cell apoptosis as a signal for sinusoidal sequestration and
transendothelial migration of neutrophils in murine models of endotoxin
and Fas-antibody-induced liver injury. Hepatology 28:761767[Medline]
-
Thome M, Hofmann K, Burns K, Martinon F, Bodmer JL, Mattmann
C, Tschopp J 1998 Identification of CARDIAK, a RIP-like kinase that
associates with caspase-1. Curr Biol 8:885888[Medline]
-
Krishnamoorthy RR, Crawford MJ, Chaturvedi MM, Jain SK,
Aggarwal BB, Al-Ubaidi MR, Agarwal N 1999 Photo-oxidative stress
down-modulates the activity of nuclear factor-kappaB via involvement of
caspase-1, leading to apoptosis of photoreceptor cells. J Biol
Chem 274:37343743[Abstract/Free Full Text]
-
Slee EA, Zhu H, Chow SC, MacFarlane M, Nicholson DW, Cohen GM 1996 Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK)
inhibits apoptosis by blocking the processing of CPP32. Biochem J 315:2124[Medline]
-
Liu X, Zou H, Slaughter C, Wang X 1997 DFF, a
heterodimeric protein that functions downstream of caspase-3 to
trigger DNA fragmentation during apoptosis. Cell 89:175184[Medline]
-
Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S 1998 A caspase-activated DNase that degrades DNA during apoptosis, and
its inhibitor ICAD. Nature 391:4350[CrossRef][Medline]
-
Adams JM, Cory S 1998 The Bcl-2 protein family: arbiters of
cell survival. Science 281:13221326[Abstract/Free Full Text]
-
Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T,
Turka LA, Mao X, Nunez G, Thompson CB 1993 bcl-x, a bcl-2-related gene
that functions as a dominant regulator of apoptotic cell death. Cell 74:597608[Medline]
-
Gallicchio M, Hufnagl P, Wojta J, Tipping P 1996 IFN-gamma
inhibits thrombin- and endotoxin-induced plasminogen activator
inhibitor type 1 in human endothelial cells. J Immunol 157:26102617[Abstract]
-
Messmer UK, Briner VA, Pfeilschifter J 1999 Tumor necrosis
factor-alpha and lipopolysaccharide induce apoptotic cell death in
bovine glomerular endothelial cells. Kidney Int 55:23222337[CrossRef][Medline]
-
Harlan JM, Harker LA, Striker GE, Weaver LJ 1983 Effects of
lipopolysaccharide on human endothelial cells in culture. Thromb Res 29:1526[Medline]
-
Oliver FJ, de la Rubia G, Rolli V, Ruiz-Ruiz MC, de Murcia G,
Murcia JM 1998 Importance of poly(ADP-ribose) polymerase and its
cleavage in apoptosis. Lesson from an uncleavable mutant. J Biol
Chem 273:3353333539[Abstract/Free Full Text]
-
Tang D, Kidd VJ 1998 Cleavage of DFF-45/ICAD by multiple
caspases is essential for its function during apoptosis. J Biol
Chem 273:2854928552[Abstract/Free Full Text]
-
Lee HH, Dadgostar H, Cheng Q, Shu J, Cheng G 1999 NF-
B-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for
CD40 survival signaling in B lymphocytes. Proc Natl Acad Sci USA 96:9136141[Abstract/Free Full Text]
-
Panetti TS, Chen H, Misenheimer TM, Getzler SB, Mosher
DF 1997 Endothelial cell mitogenesis induced by LPA: inhibition by
thrombospondin-1 and thrombospondin-2. J Lab Clin Med 129:208216[Medline]
-
Hugo CP, Pichler RP, Schulze-Lohoff E, Prols F, Adler S,
Krutsch HC, Murphy-Ullrich JE, Couser WG, Roberts DD, Johnson RJ 1999 Thrombospondin peptides are potent inhibitors of mesangial and
glomerular endothelial cell proliferation in vitro and
in vivo. Kidney Int 55:22362249[CrossRef][Medline]
-
Hudson SJ, Cai JP, Thomas V, Chin YH 1996 Intracellular
signaling of tumor necrosis factor-
in brain microvascular
endothelial cells is mediated by a protein tyrosine kinase and protein
kinase C-dependent pathway. J Neuroimmunol 70:199206[CrossRef][Medline]
-
Modur V, Zimmerman GA, Prescott SM, McIntyre TM 1996 Endothelial cell inflammatory responses to tumor necrosis factor
.
Ceramide-dependent and -independent mitogen-activated protein kinase
cascades. J Biol Chem 271:1309413102[Abstract/Free Full Text]
-
Zhang Y, Derynck R 1999 Regulation of Smad signalling by
protein associations and signalling crosstalk. Trends Cell Biol 9:274279[CrossRef][Medline]
-
Benjamin LE, Keshet E 1997 Conditional switching of vascular
endothelial growth factor (VEGF) expression in tumors: induction of
endothelial cell shedding and regression of hemangioblastoma-like
vessels by VEGF withdrawal. Proc Natl Acad Sci USA 94:87618766[Abstract/Free Full Text]
-
Araki S, Shimada Y, Kaji K, Hayashi H 1990 Apoptosis of
vascular endothelial cells by fibroblast growth factor deprivation.
Biochem Biophys Res Commun 168:11941200[Medline]
-
Paris N, Rentier-Delrue F, Defontaine A, Goffin V, Lebrun JJ,
Mercier L, Martial JA 1990 Bacterial production and purification of
recombinant human prolactin. Biotechnol Appl Biochem 12:436449[Medline]
-
Gospodarowicz D, Cheng J, Lirette M 1983 Bovine brain
and pituitary fibroblast growth factors: comparison of their abilities
to support the proliferation of human and bovine vascular endothelial
cells. J Cell Biol 97:16771685[Abstract]
-
Pepper MS, Montesano R, el Aoumari A, Gros D, Orci L, Meda P 1992 Coupling and connexin 43 expression in microvascular and large
vessel endothelial cells. Am J Physiol 262:C12461257
-
Ryan IP, Schriock ED, Taylor RN 1994 Isolation,
characterization, and comparison of human endometrial and endometriosis
cells in vitro. J Clin Endocrinol Metab 78:642649[Abstract]