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Regular Articles |
From the Departments of Pathology*
and
Pharmacology,
Boyer Center for Molecular
Medicine, Yale University School of Medicine, New Haven, Connecticut;
and ISIS Pharmaceuticals,
Carlsbad, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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- or ceramide-induced cell
death, enhanced caspase-3 activity, promoted the
generation of a 
17-kd active caspase-3 subunit, and
increased cleavage of the caspase substrate, polyADP ribose
polymerase. In contrast, the survivin antisense oligonucleotide
had no effect on EC viability in the absence of VEGF. Antisense
oligonucleotides to platelet-endothelial cell adhesion molecule-1
(PECAM-1, CD31), lymphocyte function-associated
molecule-3 (LFA-3, CD58), or intercellular adhesion
molecule-1 (ICAM-1, CD54) did not reduce the anti-apoptotic
function of VEGF in endothelium. When tested on other angiogenic
activities mediated by VEGF, survivin antisense treatment
induced rapid regression of three-dimensional vascular capillary
networks, but did not affect EC migration/chemotaxis. These
data suggest that the anti-apoptotic properties of VEGF during
angiogenesis are primarily mediated by the induced expression of
survivin in ECs. Manipulation of this pathway may increase EC viability
in compensatory angiogenesis or facilitate EC apoptosis and promote
vascular regression during tumor angiogenesis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B signaling.12
Specifically, stimulation of ECs
by VEGF or Ang-1 resulted in up-regulation of anti-apoptotic bcl-2 and
A1 molecules,13,14
and expression of inhibitor of
apoptosis (IAP) proteins,15
survivin, and
XIAP.16-18 In this study, we used an antisense targeting strategy to dissect the relative contribution of survivin to the anti-apoptotic activities of VEGF in endothelium.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human umbilical vein ECs were maintained in M199 medium containing 20% fetal calf serum (FCS), 50 µg/ml EC growth supplement, 100 µg/ml heparin, 100 µg/ml penicillin, and 100 µg/ml streptomycin (all from Life Technologies, Grand Island, NY) in 5% CO2 at 37°C as described previously.16 Subconfluent ECs were rendered quiescent by an 18-hour culture in M199 plus 0.1% FCS. Cells were detached with 0.05% trypsin/0.02% ethylenediaminetetraacetic acid (EDTA), seeded in C6-well plates (Costar Corp., New Bedford, MA), grown to 70% confluency, and used between passages 2 and 3.
Gene Targeting by Antisense
Quiescent EC monolayers were incubated with 50 ng/ml of recombinant VEGF (Collaborative Biomedical Products, Bedford, MA) for 24 hours at 37°C in M199 plus 0.1% FCS. At the end of the incubation, ECs were washed, harvested by trypsin/EDTA, and lysed in 0.5% Triton X-100, 0.5% Nonidet P-40, 0.05 mol/L Tris-HCl, 0.15 mol/L NaCl plus protease inhibitors. Protein-normalized aliquots of cell extracts were electrophoresed on sodium dodecyl sulfate-polyacrylamide gradient gels, transferred to nylon membranes (Millipore Corp., Bedford, MA) for 1 hour at 1 A, and immunoblotted with 2 µg/ml of a rabbit antibody to survivin19 or a mouse monoclonal antibody to bcl-2 (Transduction Laboratories, San Diego, CA), followed by chemiluminescence (Amersham, Arlington Heights, IL) and autoradiography. Samples were sequentially analyzed by Western blotting with a mouse antibody to ß-actin to confirm equal protein loading. To determine the contribution of survivin to EC protection mediated by VEGF, 2'-O-methoxyethyl chimeric phosphorothioate oligonucleotides were used. A survivin antisense oligonucleotide with the sequence 5'-TGTGCTATTCTGTGAATT-3' was characterized previously for its ability to suppress endogenous survivin mRNA expression in T24 bladder and HeLa epithelial carcinoma cells.20 A scrambled oligonucleotide with the sequence 5'-TAAGCTGTTCTATGTGTT-3' was used as a control, and also characterized in previous cell culture assays.20 Oligonucleotides were synthesized with uniform phosphorothioate linkages, and underlined nucleosides correspond to 2'-O-methoxyethyl nucleosides. Antisense oligonucleotides to platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31), lymphocyte function-associated molecule-3 (LFA-3, CD58), and intercellular adhesion molecule-1 (ICAM-1, CD54) were synthesized as described above and characterized in previous studies.21 For transfection experiments, increasing concentrations of control or the various antisense oligonucleotides (50 to 500 nmol/L) were mixed with 1 ml of OPTI-MEM and 6 µl of Lipofectin according to the manufacturer’s instructions (Life Technologies), and incubated with serum-starved ECs for 8 hours. The transfection medium was replaced with M199 plus 0.1% FCS for an additional 18 hours followed by VEGF stimulation for 24 hours. Transfection efficiency monitored by fluorescence microscopy using fluorescein isothiocyanate-conjugated oligonucleotides was always >85%. To determine the effect of antisense treatment on survivin mRNA expression, proliferating ECs were transfected with control or the survivin antisense oligonucleotide, harvested after a 24-hour culture at 37°C, and total RNA was extracted using the Qiagen RNeasy reagent, according to the manufacturer’s specifications. Samples were separated on 1% agarose-formaldehyde gels, transferred to Hybond nylon membranes and hybridized with a 32P-random-primed labeled survivin cDNA22 with visualization of radioactive bands by autoradiography. Northern blots were reprobed with random primed 32P-labeled human G3PDH cDNA to confirm equal loading.
Determination of EC Apoptosis
ECs were transfected with increasing concentrations of control or
the various antisense oligonucleotides, stimulated with 50 ng/ml VEGF
for 16 hours at 37°C, and incubated in the presence of 25 µmol/L of
C-6 ceramide or the combination of tumor necrosis factor (TNF)- (10
ng/ml; Endogen, Woburn, MA) plus cycloheximide (10 µg/ml, Sigma
Chemical Co., St. Louis, MO) for an additional 12 hours at 37°C. At
the end of the incubation, ECs (floaters plus attached cells) were
harvested, fixed in 70% ethanol, stained with 10 µg/ml propidium
iodide plus 100 µg/ml RNase A and 0.05% Triton X-100 in
phosphate-buffered saline (PBS), pH 7.4, and analyzed for DNA content
by flow cytometry as described previously.16
In other
experiments, transfected ECs stimulated with VEGF and incubated with
C-6 ceramide for 12 hours at 37°C were harvested, washed in PBS, pH
7.4, and fixed in 4% paraformaldehyde containing 0.25% Triton X-100
for 10 minutes at 22°C. Cell nuclei were stained with 6.5 µg/ml
4,6-diamidino-2-phenylindole (DAPI, Sigma), 16% polyvinyl alcohol (Air
Products and Chemicals, Allentown, PA), and 40% glycerol. The
percentage of apoptotic cells was calculated by counting the average
cells with nuclear morphology of apoptosis (chromatin condensation, DNA
fragmentation) in three independent high-power fields (x400), each
containing 150 cells using a Zeiss fluorescent microscope.
Caspase Activation
ECs transfected with the various oligonucleotides were stimulated with 50 ng/ml VEGF, incubated with C-6 ceramide as described above, and detergent-solubilized cell extracts were assayed for caspase-3-dependent hydrolysis of the fluorogenic substrate Ac-DEVD-AMC (N-acetyl-Asp-Glu-Val-Asp-aldehyde; Pharmingen, San Diego, CA). Fluorescence emissions were quantitated on a spectrofluorometer with excitation wavelength of 360 nm and emission of 460 nm. For biochemical markers of caspase activation, transfected ECs treated with VEGF plus ceramide were lysed in 0.25% Triton X-100, 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 20 mmol/L HEPES, plus protease inhibitors. Protein-normalized aliquots of the various cell extracts were separated by sodium dodecyl sulfate-gel electrophoresis, transferred to nylon membranes (Millipore Corp.), and immunoblotted with a 1:5000 dilution of a rabbit antibody to caspase 3 (Transduction Laboratories), or a 1:1000 dilution of a mouse antibody to PolyADP ribose polymerase (Pharmingen, San Diego, CA), followed by chemiluminescence and autoradiography.
EC Migration
Migration assays were performed using a Boyden chamber (Neuroprobe) as described.23 Briefly, quiescent ECs were transfected with control or the survivin antisense oligonucleotide, stimulated with VEGF, and detached using 0.05% trypsin and 0.53 mmol/L EDTA. Twenty thousand cells were suspended in M199 medium containing 0.1% bovine serum albumin and added to the lower chamber. Polycarbonate filters (8-µm diameter) were coated with 100 µg/ml type I collagen. The top half of the chamber was attached and the chamber was incubated in an inverted position for 2 hours at 37°C. Increasing concentrations (1 to 500 ng/ml) of VEGF or D-erythro-sphyngosine-1-phosphate (SPP-1; Calbiochem, La Jolla, CA) were separately added to the upper chamber and incubated for an additional 5 hours at 37°C. At the end of the incubation, cells were fixed in 70% ethanol and nonmigrating ECs on the upper surface of the filter were removed. Migrated cells were stained with Giemsa and counted at x400 magnification in three random fields per well.23 Each experiment was performed in triplicate and migration was expressed as the total number of cells counted per well.
Three-Dimensional Capillary Formation
Monolayers of quiescent ECs (80% confluency) in C6-well plates were transfected with 500 nmol/L of control or the survivin antisense oligonucleotide. After an 8-hour incubation, the transfection medium was replaced with M199 medium containing 0.1% FCS for an additional 18 hours at 37°C. Rat-tail type I collagen (3 mg/ml; Becton Dickinson, Bedford, MA) in 1/10 volume of 10x Dulbecco’s modified Eagle’s medium was neutralized with sterile 1 mol/L NaOH and kept on ice. Suspended ECs were added to the collagen suspension to a final concentration of 1 x 106 cells/ml of collagen. Ten drops (0.1 ml each) of the EC-collagen mixture were added to a 35-mm plate. Plates were placed in a humidified incubator at 37°C, and the EC-collagen mixtures were allowed to gel for 10 minutes, after which 3 ml of M199 medium containing 20% FCS, 50 µg/ml EC growth supplement, 100 µg/ml heparin, 100 µg/ml penicillin, and 100 µg/ml streptomycin were added to each plate. ECs were allowed to form capillary-like vascular tubes throughout a 24-hour incubation in the presence of 16 nmol/L phorbol myristate acetate (PMA, Sigma). Addition of PMA in this experimental protocol results in a potent morphogenic effect promoting the formation of endothelial tube-like structures, which closely mimics capillary formation in vivo via a PKC-, MAPK-, and PI3-kinase-dependent pathway.24 As determined in previous studies, PMA withdrawal under these conditions is associated with rapid regression of capillary structures and EC apoptosis, in vitro.24 After an additional 24-hour incubation, ECs were washed three times in PBS, pH 7.4, and supplemented with fresh M199 growth medium in the presence or in the absence of 50 ng/ml VEGF. The cultures were examined by phase-contrast microscopy for the presence of capillary-like vascular tubes during an additional 48-hour incubation at 37°C as described.25
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies demonstrated that a survivin
2'-O-methoxyethyl chimeric phosphorothioate antisense
oligonucleotide (5'-TGTGCTATTCTGTGAATT-3')
inhibited survivin mRNA and protein expression in HeLa and T24 cancer
cell lines.20
Consistent with these observations,
increasing concentrations of the survivin antisense oligonucleotide
suppressed survivin mRNA expression in proliferating ECs in a
dose-dependent manner, by Northern blotting (Figure 1A)
. In contrast, comparable
concentrations of a control scrambled oligonucleotide
(5'-TAAGCTGTTCTATGTGTT-3'), did not decrease
survivin mRNA levels in ECs (Figure 1A)
. In parallel experiments, VEGF
stimulation resulted in an approximate fourfold increased survivin
expression in quiescent endothelium (Figure 1B)
, and in agreement with
previous observations.16-18
Pretreatment of ECs with the
survivin antisense, but not control oligonucleotide, suppressed VEGF
induction of survivin in a concentration-dependent manner by Western
blotting (Figure 1B)
. In contrast, transfection with control or the
survivin antisense oligonucleotide did not reduce antiapoptotic bcl-2
expression in endothelium by Western blotting13,14
(Figure 1C)
.
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Exposure of quiescent ECs to C-6 ceramide resulted in induction of
apoptosis as determined by chromatin condensation and DNA
fragmentation, by DAPI nuclear staining (Figure 2, A and B)
. Addition of VEGF attenuated
ceramide-induced EC apoptosis and restored normal nuclear morphology
(Figure 2, A and B)
. Under these experimental conditions, transfection
of ECs with the survivin antisense oligonucleotide completely reversed
the anti-apoptotic function of VEGF against ceramide-induced apoptosis,
whereas the control oligonucleotide was without effect (Figure 2, A and B)
. Similarly, treatment with ceramide or the combination of TNF-
plus cycloheximide resulted in an approximate sevenfold increase in EC
apoptosis, as determined by the appearance of a cell fraction with
hypodiploid, ie, sub-G1, DNA content, by propidium iodide staining
and flow cytometry (Figure 2, C and D)
. Addition of VEGF counteracted
both ceramide- and TNF--induced apoptosis in ECs by 45% (Figure 2, C and D)
. However, and consistent with the data presented above, EC
transfection with the survivin antisense, but not control
oligonucleotide, suppressed VEGF cytoprotection against both cell
death-inducing stimuli, and restored EC apoptosis to levels observed in
the absence of VEGF (Figure 2, C and D)
.
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. This was associated with proteolytic
cleavage of 32-kd proform caspase-3 and generation of active
subunits of 19 and 17 kd (Figure 3B)
,26
and cleavage
of the 115-kd caspase substrate polyADP ribose polymerase (PARP) to
an 85-kd apoptotic fragment (Figure 3C)
. Consistent with its
survival properties in EC, addition of VEGF reduced ceramide-induced
caspase-3 activity, nearly completely inhibited the generation of
17-kd active caspase-3 subunit, and of 85-kd PARP fragment
(Figure 3; A, B, and C
). Under these experimental conditions, EC
transfection with the survivin antisense, but not control
oligonucleotide, restored caspase-3 catalytic activity, which was
associated with proteolytic generation of a 17-kd active caspase-3
subunit and apoptotic fragmentation of PolyADP ribose polymerase
(Figure 3, A, B, and C)
.
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Next, we wished to determine whether antisense treatment affected
a constitutive versus VEGF-inducible population of survivin
molecules in ECs. Incubation of ECs in 0% serum for 24 hours resulted
in increased caspase-3 activity by DEVD hydrolysis as compared with
continuously growing cultures (Figure 4A)
, and appearance of an apoptotic cell
fraction with hypodiploid DNA content by propidium iodide staining and
flow cytometry (Figure 4B)
. Addition of ceramide to these cells further
increased caspase-3 activity and generation of ECs with hypodiploid DNA
content (Figure 4, A and B)
. However, in the absence of VEGF, and at
variance with the data presented above, transfection of ECs with
control or the survivin antisense oligonucleotide did not further
enhance caspase-3 activity, or the generation of apoptotic cells in the
presence or absence of ceramide (Figure 4, A and B)
. These data suggest
that antisense targeting of survivin affects EC viability only in the
presence of VEGF stimulation.
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, thus confirming the
specificity of the survivin antisense approach. In contrast, EC
transfection with the survivin antisense, but not control
oligonucleotide, completely blocked the cytoprotective effect of VEGF
in ceramide-treated cultures (Figure 5)
, in agreement with the data
presented above.
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The potential role of survivin targeting on other angiogenic
responses induced by VEGF3
was next investigated. First,
stimulation with VEGF or SPP-1 resulted in EC chemotaxis and migration
in a specific and concentration-dependent manner (Figure 6)
, in agreement with previous
observations.23
However, transfection of VEGF-stimulated
ECs with inhibitory concentrations of control or the survivin antisense
oligonucleotide failed to decrease EC migration in response to VEGF or
SPP-1 (Figure 6)
.
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7).24
In contrast, no
viable capillaries were formed in the absence of PMA (not shown), and
withdrawal of VEGF resulted in rapid involution of three-dimensional
vascular networks (Figure 7)
.24
Under these experimental
conditions, transfection of ECs with control oligonucleotide did not
affect the ability of VEGF to maintain EC viability and stabilize
vascular capillaries after PMA withdrawal in vitro (Figure 7)
. In contrast, transfection of EC with the survivin antisense
oligonucleotide completely suppressed the anti-apoptotic function
of VEGF in preserving EC viability, and resulted in complete involution
of three-dimensional vascular networks throughout a 3-day culture
(Figure 7)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced apoptosis, enhanced caspase-3 activity, generation of
17-kd active caspase-3 subunit, and involution of three-dimensional
capillary-like networks in vitro. In contrast, other
angiogenic functions of VEGF, including EC migration and chemotaxis
were not affected by the survivin antisense, and, survivin targeting
had no effect on cell viability of quiescent endothelium in the absence
of VEGF. One of the cornerstone requirements for new blood vessel formation, ie, angiogenesis, is the continuous preservation of endothelium viability, a multifaceted process that involves activation of pro-survival signaling pathways,9,10 optimal cell adhesion mechanisms,7,8 and de novo expression of specialized anti-apoptotic genes.11 In this context, previous studies identified survivin as one of the target genes induced by VEGF in endothelium, which was associated with prominent up-regulation of survivin in newly formed blood vessels during angiogenesis in vivo.16 Using selective antisense targeting, the data presented here suggest that most of the anti-apoptotic properties of VEGF are mediated via the de novo induction of survivin in endothelium. Several lines of evidence corroborate the specificity of the antisense approach used here. First, transfection with the survivin antisense had no effect on EC viability in the absence of VEGF, in agreement with the undetectable levels of survivin in quiescent endothelium in vitro and in vivo.16 Secondly, a panel of antisense oligonucleotides targeting critical adhesion molecules expressed on ECs and including ICAM-1 (CD54), PECAM-1 (CD31), or LFA-3 (CD58) did not decrease the anti-apoptotic function of VEGF in endothelium. Thirdly, survivin targeting did not modify the levels of anti-apoptotic bcl-2, which has also been potentially implicated in VEGF cytoprotection.13,14 Moreover, a similar antisense targeting strategy has been recently used to further dissect cell death pathways in endothelium and identify differential roles of bcl-XL or A1 in mediating EC cytoprotection.27
Differently from other members of the IAP gene family,15 survivin has recently attracted attention for its cell cycle-regulated expression in G2/M and localization to the mitotic apparatus, which suggested a potential role at the interface between apoptosis and cell division control.28 Although an evolutionary conserved role of survivin in the regulation of mitosis and cytokinesis has been recognized,20,29,30 the mechanisms by which survivin may control apoptosis are far from elucidated. However, physical proximity/association between survivin and initiator/effector caspases has been reported by several groups in vitro and in vivo.31-33 Moreover, interference with expression/function of survivin by antisense (see below) or dominant-negative mutant was sufficient to cause increased caspase-3 activity and apoptosis at the G2/M transition.20,28 This suggests that survivin, alone or in combination with as yet unidentified regulatory molecules, may influence the assembly/function of the cell death machinery and affect downstream caspase processing/topography2 in a cell cycle-dependent manner. Consistent with this view, interference with mitotic phosphorylation of survivin on Thr34 by p34cdc2-cyclin B1 resulted in mislocalization of initiator caspase-9 and apoptosis of cells traversing mitosis.33 Although this pathway is clearly operative in proliferating cells and thus consistent with the mitogenic effect of VEGF on endothelium, the data presented here suggest that survivin may also mediate EC protection independently of cell cycle progression. In this context, antisense suppression of survivin abrogated the anti-apoptotic function of VEGF in a morphogenic, nonproliferative model of three-dimensional capillary formation in vitro.24 Although the molecular requirements of survivin-dependent cytoprotection in nonproliferating endothelium remain to be elucidated, previous studies demonstrated that EC stimulation with Ang-1, which provides a nonproliferative, stabilizing signal in angiogenesis also resulted in increased survivin expression and apoptosis inhibition via an Akt-dependent pathway.18
Differently from other apoptosis regulators of the bcl-234 or IAP15 gene families, survivin is undetectable in normal adult tissues but becomes abundantly expressed in most human cancers in vivo.22 Identified in genome-wide searches as the top fourth "transcriptome" found in common neoplasms,35 expression of survivin has been associated with abbreviated survival rates, unfavorable prognosis, and accelerated recurrences.36 This is consistent with the general view that dysregulation of apoptosis constitutes a critical pathogenic factor in cancer, aberrantly extending cell viability and facilitating the insurgence of transforming mutations and resistance to therapy.37 Using an antisense approach similar to that described here, targeting survivin in cancer cell lines was sufficient, alone, to induce a profound dysregulation of mitosis,20 associated with spontaneous apoptosis and sensitization of cancer cells to chemotherapy-induced cell death.38,39 The data presented here extend these observations and suggest that manipulation of the survivin pathway may be also beneficial to promote EC apoptosis during tumor angiogenesis, thus potentially accelerating regression of newly formed blood vessels and reducing the incidence of metastatic disease.40 The selected expression of survivin in tumor cells and angiogenically stimulated ECs may provide a high degree of specificity for potential survivin antagonists to enhance both anti-angiogenic and anti-neoplastic therapeutic strategies.
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Footnotes |
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Supported by National Institutes of Health grants HL54131 and CA78810 (to D. C. A.), HL61371 and HL64793 (to W. C. S.), HL51014 (to J. S. P.), and HL10112 (to M. M.).
Accepted for publication February 5, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor -induced apoptosis. J Exp Med 1998, 188:211-216This article has been cited by other articles:
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  C. G. Leung, Y. Xu, B. Mularski, H. Liu, S. Gurbuxani, and J. D. Crispino Requirements for survivin in terminal differentiation of erythroid cells and maintenance of hematopoietic stem and progenitor cells J. Exp. Med., July 9, 2007; 204(7): 1603 - 1611. [Abstract] [Full Text] [PDF]
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