Note: Descriptions are shown in the official language in which they were submitted.
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SURVIVIN PROMOTION OF ANGIOGENESIS
INVENTORS: Dario C. Altieri and William C. Sessa
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No.
60/172,991, filed December 21, 1999, which is herein incorporated by reference
in its
entirety. This application is related to U.S. application 08/975,080, filed
November 20,
1997, which is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
' The invention relates generally to the modulation of survivin to induce or
inhibit
angiogenesis.
BACKGROUND OF THE INVENTION
The genetic control of cell death/viability (apoptosis) preserves tissue and
organ
homeostasis by eliminating senescent or damaged cells (Vaux et al., 1999).
This process
involves different gene families of inhibitors and stimulators of cell death,
and culminates
with activation of intracellular cysteine proteases lmown as caspases
(Salvesen et al.,
1997). Aberrations of the apoptosis moieties are known to contribute to human
diseases,
including cancer (Thompson, 1995) and vascular disorders (Rudin et al., 1997).
Specifically, aberrantly increased cell death has been shown to influence
atherosclerotic
plaque instability (Bjorkerud et al., 1996), congestive heart failure
(Olivetti et al., 1997),
coronary disease (Olivetti et al., 1996), and ischemic neuronal loss (Chen et
al., 1998).
The endothelium is one of the most critical sites for the control of apoptosis
in
vascular injury and vascular remodeling (Karsan et al., 1996). In
inflammation, a
heterogeneous group of "protective"genes activated by nuclear factor KB,
opposes cell
death and proinflammatory changes in endothelial cells (EC) induced by
cytokines, i. e.
tumor necrosis factor a (TNFa) (Bath et al., 1997). Inhibition of apoptosis
may also be
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2
obligatorily required during vascular remodeling and new blood vessel
formation (Risau,
1997). In this context, EC specific mitogens, including vascular endothelial
cell growth
factor (VEGF) or basic fibroblast .growth factor (bFGF), transduce survival
signals
critically maintaining EC viability, ih vivo (Benjamin et al., 1997; Alon et
al., 1995;
Yuan et al., 1996). However, the downstream effector genes coupling mitogen-
dependent survival to the anti-apoptotic machinery in EC have not been
completely
elucidated.
It has been shown that angiopoietin-1 (Ang-1) is an endothelium-specific
ligand
essential for embryonic vascular stabilization, branching morphogenesis, and
post-natal
angiogenesis. During angiogenesis, EC receive cues from growth factors to
initiate
mitosis, migration and organization of endothelial cells into primitive
angiotubes and
patent vascular networks (Risau 1997; Hanahan 1997). These processes
critically depend
on preservation of endothelial.cell viability. Disruption of endothelial cell-
matrix
contacts or interference with extracellular survival signals is sufficient to
initiate caspase-
dependent apoptosis in endothelium, culminating with rapid involution of
vascular
structures (Brooks et al., 1994; O'Reilly et al., 1996). Unlike most
angiogenic regulators,
including fibroblast growth factor (bFGF) or vascular endothelial growth
factor (VEGF),
Ang-I does not stimulate endothelial cell growth, but rather promotes
stabilization of
vascular networks and branching morphogenesis, ih vivo and i~ vitro (Davis et
al., 1996;
Koblizelc et al., 1998; Papapetropoulos et al., 1999; Witzenbichler et al.,
1998). Little is
known about the signaling requirements of these responses, and it is unclear
if the role of
Ang-1 in angiogenesis includes protection of EC from apoptosis
(Papapetropoulos et al.,
1999; Kontos et al., 1998).
Numerous inhibitors of apoptosis (IAP) characterized with anti-apoptotic
functions have been identified. These molecules are highly conserved
evolutionarily;
they share a similar architecture organized in two or three approximately 70
amino acid
amino terminus Cys/His baculovirus IAP repeats (BIR) and by a carboxy terminus
zinc-
binding domain, designated RING finger (Duckett et al., 1996; Hay et al.,
1995; Liston et
a1.,1996; Rothe, M. et a1.,1995; Roy et al., 1995). Recombinant expression of
IAP
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proteins blocks apoptosis induced by various stimuli ih vitro (Duckett et
a1.,1996; Liston
et a1.,1996), and promotes abnormally prolonged cell survival in the
developmentally-
regulated model of the Drosophila eye, in vivo (Hay et a1.,1995).
Survivin has recently been identified as a novel member of the IAP family.
Survivin is a 16.5 kDa cytoplasmic protein containing a single partially
conserved BIR
domain, and a highly charged carboxyl-terminus coiled-coil region instead of a
RING
finger, which inhibits apoptosis induced by growth factor (IL-3) withdrawal
when
transferred in B cell precursors (Ambrosini et al., 1997). Based on overall
sequence
conservation, the absence of a carboxy terminus RING f nger and the presence
of a
single, partially conserved, BIR domain, survivin is the most distantly
related member of
the IAP family, shaxing the highest degree of similarity with NAIP (Roy et
a1.,1995).
Additionally, unlike other IAP proteins, survivin is undetectable in adult
tissues, but
becomes prominently expressed in all the most common human cancers of lung,
colon,
breast, pancreas, and prostate, and in ~50% of high-grade non-Hodgkin's
lymphomas, ih
vivo. Moreover, survivin does not bind caspases in a cell-free system (Roy et
al., 1997).
Although survivin has been characterized as a cell cycle regulated apoptosis
inhibitor, the
role of survivin on EC viability and angiogenesis has not been previously
discovered.
Angiogenesis, i. e. the formation of new blood vessels from existing ones
(Risau,
W., 1997) is an indispensable process for organ and tissue development, and
genetic
dysregulation of these mechanisms has been associated with embryonic lethality
(Hanahan, D. 1997). In the adult organism, angiogenesis provides for
beneficial
compensatory mechanisms that increase blood supply in response to hypoxia or
during
tissue repair and/or remodeling (Carmeliet, P., 2000). The same process,
however, .may
have disastrous consequences in cancer, where increased vascularization due to
de hovo
vessel formation contributes to tumor progression and metastatic dissemination
(Hanahan
et al., 1996). Targeted inhibition of angiogenesis by either disrupting cell-
cell
(Stromblad et al., 1996) and cell-matrix interactions (Brooks et al., 1998) or
by
interfering with receptor-initiated intracellular signals (Lin et al., 1998;
Claeson-Welsh et
al., 1998), results in rapid involution of newly formed blood vessels ih
vitr°o and in vivo,
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and the appearance of classical morphologic features of apoptosis in the
targeted
endothelium (Alon et al., 1995; Yuan et al., 1996; Lin et al., 1998).
Accordingly, the
continuous suppression of EC apoptosis (Bach et al., 1997) may constitute one
of the
critical requirements of angiogenesis, consistent with the up regulation of
protective
genes of the bcl-2 (Gerber et al., 1998a; Nor et al.,1999) or IAP (O'Connor et
al., 2000a;
Tran et al., 1999; Papapetropoulos et al., 2000) gene families in endothelium
stimulated
by VEGF or Ang-1.
SUMMARY OF THE INVENTION
The present invention is based on the discovery that angiogenesis stimulation
strongly induces survivin expression in endothelium during vascular remodeling
and
angiogenesis, ih vits°o and i~c vivo. Survivin expression and function
affect endothelial
cell viability during the proliferative and the stabilizing phase of
angiogenesis.
Therapeutic manipulation of survivin expression/function in endothelium may
influence
compensatory or pathologic (tumor) angiogenesis.
The present invention is also based on the discovery that Ang-1 prevents
endothelial cell apoptosis by activating a critical survival messenger, Ald,
and by up-
regulating survivin. The activation of anti-apoptotic pathways mediated by Akt
and
survivin in endothelial. cells may.contribute to Ang-1 stabilization of
vascular structures
during angiogenesis, ih vivo. Accordingly, targeted manipulation of Ang-
1/Alct/Survivin
may be exploited to improve endothelial cell viability and favor therapeutic
angiogenesis,
in vivo.
Based on these observations, the present invention provides methods of
promoting or inhibiting angiogenesis and methods of treating conditions by
inducing
compensatory angiogenesis. In one embodiment, the disclosed methods are useful
for
treating ischemic diseases caused by myocardial infarction, peripheral
vascular occlusion,
brain ischemia, or stroke. In another embodiment, the disclosed methods of
inhibiting
angiogenesis are useful for treating vasculoproliferative disease such as
cancer,
restenosis, vascular bypass graft occlusion, or transplant coronary
vasculopathy.
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The disclosed methods comprise providing to a cell or tissue an apoptosis
modulating agent, wherein the. agent is selected from the group consisting of
a survivin
polypeptide, a survivin transgene, a survivin antisense molecule, a survivin
peptidomimetic, or an agent that modulates the expression or activity of
survivin in a cell
or tissue, such as Ang-1 or Akt. In one embodiment, the agent is provided in
an implant.
The implant maybe a stmt and the implant maybe coated or impregnated with a
survivin
transgene that is operatively linked to an expression control element in a
vector. In
another embodiment the survivin transgene or antisense molecule is contained
within a
transfection facilitating composition, such as a transfection facilitating
lipid or a
transfection facilitating particle.
In one embodiment, the present invention includes a method of inhibiting
angiogenesis comprising administering an agent that inhibits survivin.
Examples of
agents that inlubit survivin include, but are not limited to, survivin
antibodies, survivin
antisense molecules, inhibitors of Akt phosphorylation, and other inhibitors
of survivin
function or expression. In another embodiment, the method inhibits the
angiogenesis of
tumors or inhibits the metastasis of cancerous cells and the agent is a
survivin antisense
molecule. Preferably, the method inhibits VEGF induced functions such as
ceramide or
TNFcc induced apoptosis and capillary formation and maintenance. Most
preferably, the
method inhibits VEGF induced capillary formation and maintenance.
As shown in the Examples below, survivin zs involved not only in the
proliferative phase of angiogenesis but also the remodeling and stabilizing
phase of
angiogenesis. Survivin antisense molecule is sufficient to induce endothelial
cell
apoptosis and regression of the capillary-like structures.
In one aspect, the present invention contemplates the use of inlubitors of
survivin
expression and function as agents that inhibits angiogenesis, such as an
antagonist. In
another aspect, the present invention contemplates the use of survivin and
inducers of
survivin expression and fiulction as agents that promotes angiogenesis, such
as an
agonist.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figures lA-C. Modulation of survivin expression in EC.
A. Quiescent EC were incubated with medium or serum (10% FCS), VEGF (100
ng/ml), bFGF (5 ng/ml), TNFa(10 ng/ml) or IL-1 (2 ng/ml) for I6 h at
37°C. Cells were
harvested, SDS-extracted and analyzed for expression of survivin or (3-actin,
by
immunoblotting. B. Control or EC were stimulated with the indicated increasing
concentrations of VEGF for 16 h at 37°C and analyzed for expression of
survivin or (3-
actin by immunoblotting. C. Total RNA was extracted fiom EC stimulated with
100
ng/ml VEGF at the indicated time intervals, separated on agarose-formaldehyde
denaturing gels and hybridized with probes to survivin or control ~3-actin.
Figures 2A-D. Expression of survivin in three-dimensional EC culture.
EC were grown in three-dimensional fibronectin-collagen gels, paraffm
embedded and analyzed for survivin expression by immunohistochemistry. A.
Survivin
expression in control, two-dimensional EC culture. B. Survivin expression in
three-
dimensional EC culture. C. Control staining of three-dimensional EC culture
with
preimmune antibody. D. Two (2-D)- or three (3-D)-dimensional EC cultures were
harvested, homogenized in a tissue grinder, and analyzed for survivin
expression by
immunoblotting.
Figures 3A-F. Expression of survivin in proliferating and non-proliferating
skin
capillaries.
Five-~,m sections of formalin-fixed, paraffin-embedded skin biopsies
containing
granulation tissue and normal skin were analyzed for survivin expression by
immunohistochemistry after antigen retrieval by pressure-coolcing. A. Strong
cytoplasmic expression of survivin in EC of dermal capillaries in granulation
tissue.
Inset, detail representation of dermal capillaries stained for survivin
expression. C.
Expression of survivin in endothelium of large vessel in granulation tissue at
the
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dermis/hypodermis junction. B, D. Control staining for panels A and C,
respectively, in
the absence of primary antibody. E. Expression of survivin in non-
proliferating
capillaries of non-inflamed normal skin. F. Control incubation for panel E, in
the
presence of preimmune antibody. Magnification x200 (A, E, F) and x400 (B, C,
D).
Figures 4A-B. Anti-apoptotic function of survivin in EC.
A. Sub-confluent bovine aortic EC were transfected with GFP-vector or GFP-
survivin by lipofectin, cultivated for 35.h at 37°C and treated with 5
ng/ml TNFa/5 ~,g/ml
cycloheximide for additional 8-h at 37°C. GFP-expressing cells were
analyzed for DNA
content by propidium iodide staining and flow cytometry. The percentage of
cells with
hypodiploid DNA content (sub-G1-fraction) is indicated in parenthesis for each
condition
tested. B. Untreated or EC transfected with GFP-vector or GFP-survivin were
incubated
with the indicated concentrations of TNFa/10 ~,g/ml cycloheximide, harvested,
and
analyzed for caspase-3 activity by hydrolysis of the fluorogenic substrate
DEVD-AMC in
the presence or in the absence of the caspase-3 inhibitor DEVD-CHO. Data are
the
mean~ SD of replicates of a representative experiment.
Figures 5A-D. Ang-1 stimulates Akt phosphorylation and lcinase activity.
A. MVEC were incubated with~Ang-1 (250 ng/ml) for 15 min and analyzed
for Alct phosphorylation (serine 473, upper panel) or total Akt expression
(lower
panel) by Western blotting. Cells were treated as described, and cell lysates
prepared
for Akt kinase assays (see B). 20 ~,g of protein was separated on SDS-
polyacrylamide
gel electrophoresis gel (SDS-PAGE) and transferred onto a polyvinylidene
difluoride
membrane (Millipore). After blocking with T-PBS (PBS containing 0.2% Tween 20)
containing 5% milk for 1 h, the membrane was incubated with anti-Akt antibody
(Santa Cruz), phospho-specific Akt antibody (New England Biolabs). ECL
(Amersham) was used for detection. B. Akt was immunoprecipitated from MVEC
and analyzed for lcinase activity using histone 2B as a substrate. (Cells were
washed
twice with PBS and lysed with cell lysis buffer (1% Nonidet P-40, 10%
glycerol, 137
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mM NaCI, 20 mM Tris-HCI, pH 7.4, 20 mM NaF, 2 ~,g/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride). Lysates were precleared with protein G-agarose
for
30 min at 4°C, and immunoprecipitated for 2 h with anti-Akt antibodies
in the
presence of 2 mg/ml bovine serum albumin with or without 16 ~,g/ml competitor
peptides (Santa Cruz). Immunoprecipitates were washed twice with cell lysis
buffer,
once with water, and once with l~inase buffer (20 mM HEPES, pH 7.2, 10 mM
MgCl2,
mM IVInCI2). Immunoprecipitated proteins were incubated in 50 ~,1 of kinase
buffer
containing 2 ~,g of histone H2B (Roche Molecular Biochemicals) and [32P-]ATP
(5
~,M, 10 ~,Ci) for 30 min at room temperature. Kinase reactions were stopped by
the
addition of SDS sample buffer and samples subjected to Cerekenov counting and
SDS-PAGE followed by autoradiography. Parallel samples were processed to
confirm equal amounts of immunoprecipitated Akt. C. Time-dependent activation
of
Akt by Ang-1. MVEC were incubated with Ang-1 for increasing amount of time and
Alct activation determined as above. D. Ang-1 induced Akt phosphorylation is
blocked by soluble Tie-2 and Ang-2, but not by soluble Tie 1. MVEC were
incubated
with vehicle (TBS plus CHAPS), or the various indicated combinations of Ang-1
(250
ng/ml), Ang-2 (alone (2.5 ~,g/ml), soluble Tie 1 or Tie 2 receptors (2.5
~.g/ml) for 15
min before determination of Akt phosphorylation or total Akt expression by
Western
blotting. Ang-1-induced Alct phosphorylation is blocked by soluble Tie-2, but
not by
Tie 1 or Ang-2. For all panels, data are representative of at least 3
experiments.
Figures 6A-C. Ang-1 inhibits endothelial cell apoptosis via a PI-3 kinase/Alct
pathway.
A. MVEC were plated onto bacteriological dishes in serum-free media for 18h
in the absence or presence of Ang-1 (250 ng/ml) or wortmannin (WM, 200 nM),
before determination of apoptosis by propidium iodide staining and flow
cytometry.
(MVEC were plated on bacteriological dishes in serum free medium in the
presence or
either vehicle (TBS containing CHAPS) or Ang-1 (250ng/ml). Cells were
incubated
for 18hr and both floating and adherent cells were collected. To determine the
number
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of subdiploid cells, MVEC were fixed for 1 hour in 70% ethanol and stained
with a
solution containing 500 ~,glml RNAase H and 50 ~,g/ml propidium iodide and
analyzed by using a fluorescence activated cell sorter (FACS). At Ieast 5000
events
were analyzed, and the percentage of cells in the sub-G1 population
calculated.) B.
The experimental conditions are the same as in A, except that MVEC were
infected
with adenovirus encoding ~i-galactosidase or dominant-negative AA-Akt for 24
h,
followed by placement into suspension and treatment with Ang-1 for 18 h. For
each
panel, the percentage of MVEC with hypodiploid (apoptotic) DNA content is
indicated. C. Ang-1 activates Akt in an integrin-independent manner. MVEC
plated
on bacteriological dishes in serum free medium were treated with vehicle or
Ang-1
(250 ng/ml) in the absence or in the presence of WM (200 nM) for 15 min, and
immunoblotted fox phosphorylated Akt (upper panel) or total Akt (lower panel).
For
all panels, data are representative of 3 independent experiments.
Figures 7A-D. Ang-1 induces survivin expression via a PI-3 kinase/Akt
pathway.
A. Time dependent expression of survivin RNA. Serum starved MVEC were
treated with Ang-1 for the indicated time intervals and survivin, GAPDH and
bcl-2
RNA expression were examined by Northern hybridization. B. Soluble Tie 2
prevents Ang-1 induction of survivin RNA. The experimental conditions axe the
same
as in A, except that Ang-1 was preincubated in the absence or in the presence
of a
soluble Tie 2 receptor before addition to MVEC for 24 h and determination of
survivin, GAPDH or bcl-2 RNA expression. C. Ang-1 stimulates survivin promoter
activity. MVEC were co-transfected with plasmids encoding a promoterless
luciferase
cassette (pLUC-42), or a 1.2 kb survivin promoter fragment (pLUC-cycl.2) with
(3-
galactosidase and relative luciferase activity was determined. D. VEGF and Ang-
1
increase survivin protein expression. ~HUVEC were treated with VEGF (50 ng/ml)
or
Ang-1 (250 ng/ml) under the various conditions tested, were harvested after 18
h and
analyzed for survivin, actin and bcl-2 protein expression by Western blotting.
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Numbers below the survivin panel indicate relative levels based on
densitometry. For
all panels, data are representative of 2-4 experiments.
Figures 8A-B. Survivin mediates the anti-apoptotic effect of Ang-1.
A. TNFa/cycloheximide. B. Anoikis. MVEC were transfected with GFP
vector, GFP-survivin (survivin) or GFP-C84A survivin (C84A survivin), followed
by
treatment with TNFa (5 ng/ml) plus cycloheximide (5 g/ml) (A), or by plating
in serum-
free medium on bacteriological dishes (B), in the absence or in the presence
of Ang-1
(250 ng/ml). Apoptosis under the various conditions tested was determined by
propidium iodide staining and flow cytometry. The percentage of cells with
hypodiploid
DNA content quantified in the GFP-expressing population is shown in each
histogram.
Data are representative of 2 experiments in duplicate.
Figures 9A-C Antisense Inhibition of Survivin Expression in EC.
A. EC were transfected with the indicated increasing concentrations of control
scrambled (Control) or the survivin antisense oligonucleotide (Smvivin AS)
followed by
hybridization with cDNAs for survivin or GAPDH. Densitometric quantitation of
hybridizing bands under the various conditions tested is shown in the bottom
panel. B.
EC were serum-starved for 18 h in the presence of 0.1 % FCS, stimulated with
VEGF (50
ng/ml) in the presence of the indicated oligonucleotide concentrations.
Protein-
normalized aliquots of detergent-solubilized EC extracts were immunoblotted
with an
antibody to survivin or ~3-actin followed by chemiluminescence. C. The
experimental
conditions are the same as in B, except that EC extracts treated with control
or the
survivin antisense oligonucleotide were analyzed with an antibody to bcl-2 by
Western
blotting. For panels B and C, molecular weight marl~ers in l~Da are indicated
on the left.
WB, Western blot.
Figures l0A-D. Inhibition of VEGF cytoprotection by survivin targeting.
A. EC were transfected with control or the survivin antisense oligonucleotide,
treated with VEGF (50 ng/rnl) and exposed to C-6 ceramide (25 ~,g/ml). Cell
nuclei were
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stained with 6.5 ~,g/ml 4,6- diamidino-2-phenylindole (DAPI, Sigma), 16%
polyvinyl
alcohol and 40% glycerol and scored morphologically for apoptosis (chromatin
condensation, DNA fragmentation) using a Zeiss fluorescent microscope.
Photographs
of phase contrast or DAPI staining of each field are from a representative
experiment out
of at least three independent determinations. B. The experimental conditions
are the
same as in A. Data are expressed as percent of apoptosis as determined by
nuclear
morphology by DAPI staining, and represent the mean~ SEM of three independent
experiments. C and D. EC transfected with control or the survivin antisense
oligonucleotide were incubated with C-6 cera~nide (25 ~,g/ml, C) or the
combination of
TNFa (10 ng/ml) plus cycloheximide (10 ~,g/ml, D) for 12 h at 37°C.
Cells were
analyzed for DNA content by propidium iodide staining and flow cytometry. The
percentage of apoptotic cells with hypodiploid, i. e., sub-G1, DNA content is
indicated per
each condition tested. Data are representative of an experiment of at least
two
independent determinations. Comparable transfection efficiencies were
demonstrated by
fluorescence microscopy of EC transfected with FITC-conjugated
oligonucleotides.
Figures 11A-C. Modulation of Caspase Activity by Survivin Targeting.
A. The conditions are as described in Figure 2. Ceramide-treated EC stimulated
with VEGF and transfected with control or the survivin antisense
oligonucleotide were
analyzed for caspase-3 activity by hydrolysis of the fluorogenic substrate
DEVD-AMC in
the presence or absence of the caspase inhibitor DEVD-CHO. Data are the mean~
SD of
two independent determinations. B. EC extracts under the various indicated
conditions
were analyzed for caspase-3 proteolytic cleavage with an antibody to caspase-3
by
Western blotting. The.positions of ~32.1cDa proform caspase-3, of the
intermediate
product of ~24 lcDa, and of active subunits of ~17 and ~19 lcDa are indicated.
C. EC
extracts were analyzed for proteolytic cleavage of the caspase-3 substrate,
PARP by
Western blotting. Densitometric quantitation under the various conditions
tested was
carried out on the ~17 kDa active caspase-3 subunit (E) or the apoptotic ~85
kDa PARP
fragment (C).
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Figures 12A and B. Effect of survivin targeting on quiescent EC.
Starved EC were transfected with the indicated oligonucleotides and incubated
in
the absence (-Ceramide) or presence (+Ceramide) of ceramide. Cell extracts
were
analyzed for caspase-3 activity by hydrolysis of the fluorogenic substrate
DEVD-AMC,
in the presence or in the absence of DEVD-CHO (A), or by DNA content by
propiditun
iodide staining and flow cytometry (B). Data are expressed as the mean~SD of
three
independent experiments. In B, the percentage of apoptotic cells with
hypodiploid, sub-
Gl, DNA content is indicated.
Figure 13. Effect of Antisense Oligonucleotides to EC Adhesion Molecules on
VEGF Cytoprotection.
The experimental conditions are as in Figure 10, except that quiescent EC were
transfected with the indicated ant'isense oligonucleotides, stimulated with
VEGF and
exposed to C-6 ceramide. Cells were analyzed for nuclear morphology by DAPI
staining
after a 12 h culture at 37°C. Data are the mean ~SEM of three
independent transfection
experiments.
Figure 14. Effect of survivin targeting on VEGF-induced EC
migration/chemotaxis.
EC were transfected with control or the survivin antisense oligonucleotide,
stimulated with VEGF and exposed to the indicated increasing concentrations of
VEGF
or control SPP-1 in a Boyden chamber. After a 5 h incubation at 37°C,
migrated cells
were counted microscopically by Giemsa. Data are the mea~SEM of triplicates of
a
representative experiment out of three independent determinations.
Figure 15. Effect of survivin targeting on capillary formation.
EC transfected with the control or the survivin antisense oligonucleotide were
cultured in collagen gels in the presence of PMA, and stabilized in the
absence (None) or
presence of VEGF. Three-dimensional capillary networks were analyzed by phase
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contrast microscopy during a 3-d culture at 37°C. Pictures are
representative of one
experiment out of at Ieast three independent determinations. Magnification
x100.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
I. General Description
T.he present invention is based in part on the discovery that survivin is a
growth
factor-inducible protective gene expressed by endothelial cells during
angiogenesis.
Stimulation of quiescent endothelial cells with mitogens, including vascular
endothelial
growth factor or basic fibroblast growth factor, induced up to ~16-fold up-
regulation of
survivin. Mitogen stimulation rapidly increased survivin RNA expression in
endothelial
cells, which peaked after 6-10 h culture and decreased by 24-h. Inflammatory
cytokines,
tumor necrosis factox a or interleukin-1 did not induce survivin expression in
endothelial
cells. Formation of three-dimensional vascular tubes, i~ vitro, was associated
with strong
induction of survivin in endothelial cells, as compared with two-dimensional
cultures.
By immunohistochemistry, survivin was minimally expressed in endothelium of
non-
proliferating capillaries of normal skin, whereas it became massively
upxegulated in
newly formed blood vessels of granulation tissue, ~in vivo. Recombinant
expression of
Green Fluorescent Protein-survivin in endothelial cells reduced caspase-3
activity and
counteracted apoptosis induced by tumor necrosis factor a, /cycloheximide.
These
findings identify survivin as a novel growth factor-inducible protective gene
expressed by
endothelial cells during angiogenesis.
The present invention is also based on the discovery that Ang-1 acting via the
Tie-2 receptor induces phosphorylation of the survival serine/threonine
l~inase Akt (or
protein kinase B). This is associated with up-regulation of survivin in
endothelial cells
and protection of endothelium from death-inducing stimuli. Moreover, a
dominant
negative survivin mutant negates the ability of Ang-1 to protect cells from
undergoing
apoptosis. The activation of anti-apoptotic pathways mediated by Akt and
survivin in
endothelial cells may contribute to Ang-1 stabilization of vascular structures
during
angiogenesis, i~c vivo.
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14
Further, the present invention is based on the finding that an antisense
oligonucleotide to the apoptosis inhibitor survivin suppressed survivin
expression in
endothelial cells induced by vascular endothelial cell growth factor (VEGF).
In contrast,
the survivin antisense oligonucleotide did not affect anti-apoptotic bcl-2
levels in
endothelium. When assessed in cell death assays, antisense targeting of
survivin
abolished the anti-apoptotic function of VEGF against TNFa - or ceramide-
induced cell
death, enhanced caspase-3 activity, promoted the generation of a ~17 kDa
active caspase-
3 subunit, and increased cleavage of the caspase substrate, poly-ADP ribose
polymerase.
In contrast, the survivin antisense oligonucleotide had no effect on
endothelial cell
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-l, CD54) did not
reduce the
anti apoptotic function of VEGF in endothelium. When tested on other
angiogenic
functions of VEGF, antisense survivin targeting induced rapid regression of
three-
dimensional capillary networks, but did not affect endothelial cell
migratiouchemotaxis.
These data suggest that the anti-apoptotic functions of VEGF during
angiogenesis are
largely mediated by the induced expression of survivin in endothelial cells.
Accordingly,
the present invention provides methods of modulating this pathway to increase
endothelial cell viability in compensatory angiogenesis or to facilitate
endothelial cell
apoptosis and promote vascular regression during tumor angiogenesis.
II. Specific Embodiments
A. Survivin Molecules
1. Survivin Polypeptides
The present invention employs survivin protein, as well as allelic variants of
the
survivin protein, and conservative amino acid substitutions of the survivin
protein. As
used herein, the term "survivin protein" or~ "surviviri" refers in part to a
protein that has
the amino acid sequence of human survivin. The term also includes naturally
occurring
allelic variants of survivin, which include naturally occurring proteins that
have a slightly
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different amino acid sequence than that specifically recited above. Allelic
variants,
though possessing a slightly different amino acid sequence than those recited
above, will
still have the requisite ability to inhibit cellular apoptosis. As used
herein, the survivin
family of proteins also refers to surviviii proteins that have been isolated
from organisms
in addition to humans.
As disclosed above, the survivin proteins of the present invention further
include
conservative variants of the survivin proteins herein described. As used
herein, a
conservative variant refers to,alterations in the amino acid sequence that do
not adversely
affect the ability of the survivin protein to bind to a survivin binding or
signaling partner
and/or to inhibit cellular apoptosis. A substitution, insertion or deletion
is.said to
adversely affect the suyvivin protein when the altered sequence prevents the
survivin
protein from associating with a sm-vivin binding or signaling partner and/or
prevents the
survivin protein from inhibiting cellular apoptosis. For example, the overall
charge,
structure or hydrophobic/hydrophilic properties of survivin can be altered
without
adversely affecting the activity of survivin. Accordingly, the amino acid
sequence of
survivin can be altered, for example to render the peptide more hydrophobic or
hydrophilic, without adversely affecting the activity of survivin.
The allelic variants, the conservative substitution variants and the members
of the
survivin family of proteins, will have the ability to inhibit cellular
apoptosis. Such
proteins will ordinarily have an amino acid sequence having at least about 75%
amino
acid sequence identity with the human survivin sequence, more preferably at
least about
80%, even more preferably at least about 90%, and most preferably at least
about 95%.
Identity or homology with respect to such sequences is defined herein as the
percentage
of amino acid residues in the candidate sequence that are identical with the
known
peptides, after aligning the seqixences,and introducing gaps, if necessary, to
achieve the
maximum percent homology, and including any conservative substitutions as
being
homologous. N-terminal, C-terminal or internal extensions, deletions, ~or
insertions into
the peptide sequence shall not be construed as affecting homology.
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16
Thus, the survivin proteins of the present invention include molecules having
full
length amino acid sequence of naturally occurring proteins; fragments thereof
or peptides
having a consecutive sequence of at least about 3, 5, 10, 15, or more amino
acid residues
of the survivin protein; amino acid sequence variants of such sequence wherein
an amino
acid residue has been inserted N- or C-terminal to, or within, the sequence of
a naturally
occurring survivin protein; amino acid sequence variants of the disclosed
survivin
sequence, or their fragments as defined above,.that have been substituted by
another
residue. Contemplated variants further include those containing predeternlined
mutations
by, e.g., homologous recombination, site-directed or PCR mutagenesis, and the
corresponding survivin proteins of other animal species, including but not
limited to
rabbit, rat, marine, porcine, bovine, ovine, equine and non-human primate
species, and
the alleles or other naturally occmTing variants of the survivin family of
proteins; and
derivatives wherein the survivin protein has been covalently modified by
substitution,
chemical, enzymatic, or other appropriate means with a moiety other than a
naturally
occurring amino acid (for example a detectable moiety such as an enzyme or
radioisotope.
The present invention also includes or employs survivin peptidomimetics.
Survivin peptidomimetics are compounds that mimic the activity of survivin
peptides.
They are structurally similar to survivin peptides but have a chemically
modified peptide
backbone. Peptidomimetics may have significant advantages over polypeptide
embodiments, including, for example: more economical production; greater
chemical
stability; enhanced pharmacological properties (half life, absorption,
potency, efficacy,
etc.); altered specificity (e.g., a broad-spectrum of biological activities);
reduced
antigenicity; and others.
2. Survivin Nucleic acids
The present invention also employs nucleic acid molecules or transgenes that
encode survivin, and the related survivin proteins. As used herein, "nucleic
acid" is
defined as RNA or DNA that encodes a peptide as defined above, or is
complementary to
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a nucleic acid sequence encoding such peptides, or hybridizes to such a
nucleic acid and
remains stably bound to it under stringent conditions, or encodes a
polypeptide sharing at
least 75% sequence identity, preferably at least 80%, and more preferably at
least 85%,
with the peptide sequences. Specifically contemplated are genomic DNA, cDNA,
mRNA
and antisense molecules, as well as nucleic acids based on an alternative
backbone or
including alternative bases whether derived from natural sources or
synthesized.
As used herein, "stringent conditions" are conditions in which hybridization
yields a clear and detectable sequence. Stringent conditions are those that
(1) employ
low ionic strength and high temperature for washing, for example, O.O15M
NaCI/O.OO15M sodium titratel0.l% SDS at 50°C., or (2) employ during
hybridization a
denaturing agent such as formamide, for example, 50% (vol/vol) formamide with
0.1
bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyTOlidone/50 mM sodium
phosphate
buffer at pH 6.5 with 750 mM NaCI, 75 mM sodium citrate at 42°C.
Another example is
use of 50% formamide, 5 x SSC (0.75M NaCI, 0075 M sodium citrate), 50 mM
sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution,
sonicated
salmon sperm DNA (50 (g/ml), 0.1% SDS, and 10% dextran sulfate at
42°C., with
washes at 42°C. in 0.2 x SSC and 0.1% SDS. A skilled artisan can
readily determine and
vary the stringency conditions appropriately to obtain a clear and detectable
hybridization signal.
The present invention further employs fragments of the survivin encoding
nucleic
acid molecule. As used herein, a fragment of a survivin encoding nucleic acid
molecule
refers to a small portion of the entire protein encoding sequence. The size of
the
fragment will be determined by the intended use. For example, if the fragment
is chosen
so as to encode an active portion of the survivin protein, such as the C-
terminal coils or
the IAP motif, the fragment will need to be large enough to encode the
functional
regions) of the Survivin protein.
Modifications to the primary structure itself by deletion, addition, or
alteration of
the amino acids incorporated into the protein sequence during translation can
be made
without destroying the activity of the protein. Such substitutions or other
alterations
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result in proteins having an amino acid sequence encoded by DNA falling within
the
contemplated scope of the present invention.
B. Survivin Antisense Molecules
An antisense survivin molecule is complementary to and capable of hybridizing
or annealing with the RNA encoded by a survivin gene (the "sense gene"). An
antisense
survivin molecule is used to inhibit the expression of the survivin gene
thereby inhibiting
angiogenesis and preventing and,treating diseases associated with
angiogenesis.
Antisense nucleic acids are preferably constructed by inverting the coding
region
of the sense gene relative to its normal presentation for transcription to
allow for
transcription of its complement, hence the complementariness of the respective
RNAs
encoded by these DNA's. In order to block the production of mRNA produced by
the
sense gene, the antisense DNA should preferably be expressed at approximately
the same
time as the sense gene if the antisense nucleic acid is to be expressed in the
cell. The
timing must be approximate in the sense that the antisense RNA must be present
within
the cell to block the function of the RNA encoded by the sense gene. To
accomplish this
result, the coding region of the antisense DNA is often placed under the
control of the
same promoter as found in the sense gene thereby causing both to be
transcribed at the
same time.
For reviews of the design considerations and use of antisense
oligonucleotides,
see Uhlmann et al. (1990) and Milligan et al. (1993), the disclosures of which
are hereby
incorporated by reference.
While in principle, antisense nucleic acids having a sequence complementary to
any region of the survivin gene may be useful in the angiogenesis inhibiting
methods of
the present invention, nucleic acid molecules complementary to a portion of
the survivin
mRNA transcript including the translation initiation codon are particularly
preferred.
Also preferred are nucleic acid molecules complementary to a portion of the
survivin
mRNA transcript lying within about 40 nucleotides upstream (the 5' direction)
or about
40 nucleotides downstream (the 3' direction) from the translation initiation
codon.
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In another embodiment, antisense oligonucleotides which hybridize or anneal to
at least a portion of the survivin mRNA in a cell may be used in the methods
of the
invention. Such oligonucleotides are typically short in length and fairly
easily diffusible
into a cell. Such antisense oligonucleotides include, but are not limited to,
polydeoxynucleotides containing 2'-deoxy-D-ribose, polyribonucleotides
containing
D-ribose, any other type of polynucleotide which is an N-glycoside of a purine
or
pyrimidine base, or other polymers containing nonnucleotide baclcbones (e.g.,
protein
nucleic acids and synthetic sequence specific nucleic acid polymers
commercially
available) or nonstandard linkages, providing that the polymers contain
nucleotides in a
configuration which allows for base pairing and base stacking such as is found
in DNA
aazd RNA. They may include double- and single-stranded DNA, as well as double-
and
single-stranded RNA and DNA:RNA hybrids, and also include, as well as
unmodified
forms of the polynucleotide or oligonucleotide, known types of modifications,
for
example, labels which are known to those skilled in the art, "caps",
methylation,
substitution of one or more of the naturally occurring nucleotides with
analogue,
internucleotide modifications such as, for example, those with uncharged
linkages (e.g.,
methyl phosphonates, phosphorotriesters, phosphoramidates, caxbamates, etc.)
and with
charged linkages or sulfur-containing linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), those containing pendant moieties, such as, for
example,
proteins (including nucleases, nuclease inhibitors, toxins, antibodies, signal
peptides,
poly-L-lysine, etc.) and sacchaxides (e.g., monosacchaxides, etc.), those with
intercalators
(e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals,
radioactive metals,
boron, oxidative metals, etc.), those containing allcylating agents, and those
with
modified linlcages (e.g., alpha anomeric nucleic acids, etc.).
The terms "nucleoside", "nucleotide" and "nucleic acid" as used concerning
survivin antisense nucleic acid molecules, include those moieties which
contain not only
the known purine and pyrimidine bases, but also other heterocyclic bases which
have
been modified. Such rilodifications include methylated purines and
pyrimidines, acylated
purines and pyrimidines, or other heterocycles. Modified nucleosides or
nucleotides will
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also include modifications on the sugar moiety, e.g., wherein one or more of
the
hydroxyl groups are replaced yvith halogen, aliphatic groups, or are
functionalized as
ethers, amines, or the like.
C. Angiogenesis
Angiogenesis is the process by which newblood vessels are formed (Folkman et
al. 1992). Thus, angiogenesis is essential in reproduction, development, and
wound
repair. However, inappropriate angiogenesis can have severe consequences. For
example, it is only after many solid tumors are vascularized as a result of
angiogenesis
that the tumors begin to grow rapidly and metastasize. Because angiogenesis is
so
critical to these functions, it must be carefully regulated in order to
maintain health. The
angiogenesis process is believed to begin with the degradation of the basement
membrane by proteases secreted from EC activated by mitogens such as vascular
endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF).
The cells
migrate and proliferate, leading to the formation of solid endothelial cell
sprouts into~the
stromal space, then, vascular loops are formed and capillary tubes develop
with formation
of tight junctions and deposition of new basement membrane.
The rate of angiogenesis involves a change in the local equilibrium between
positive and negative regulators of the growth of microvessels. Abnormal
angiogenesis
occurs when the body loses its control of angiogenesis, resulting in either
excessive or
insufficient blood vessel growth. For instance, conditions such as myocardial
infarction,
peripheral vascular occlusion, brain ischemia, or stroke may result from the
absence of
angiogenesis normally required for natural healing. On the contrary, excessive
blood
vessel proliferation may favor tumor growth and spreading, blindness,
psoriasis and
rheumatoid arthritis.
Recently, the feasibility of gene therapy for modulating angiogenesis has been
demonstrated. For example, promoting angiogenesis in the treatment of ischemia
was
demonstrated in a rabbit model and in human cliucal trials with VEGF using a
Hydrogel-coated angioplasty balloon as the gene delivery system. Successful
transfer
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21
and sustained expression of the VEGF gene in the vessel wall subsequently
augmented
neovascularization in the ischemic limb (Takeshita et al., (1996); Isner et
al., 1996).
Alternative methods for regulating angiogenesis are still desirable for a
number of
reasons. For example, it is believed that native endothelial cell (EC) number
andlor
viability decreases over time. Thus, in certain patient populations, e.g., the
elderly, the
resident population of ECs that is competent to respond to administered
angiogenic
cytokines may be limited. Moreover, while agents promoting or inhibiting
angiogenesis
may be useful at one location, they may be undesirable at another location.
Thus, means
to more precisely regulate angiogenesis at a given location are desirable.
The present invention provides a method of regulating angiogenesis by
providing
survivin or a modulator of survivin expression to a cell or tissue. A
modulator of
survivin expression is a molecule. that alters the expression of survivin in a
cell.
1. Methods of Inducing Angiogenesis
In one embodiment, the present invention provides methods of inducing
angiogenesis. The present invention is based on the findings that survivin, an
apoptosis
inhibitor, is expressed by EC during angiogenesis and vascular remodeling,
that Ang-1
prevents EC apoptosis by phosphorylation of Akt, and by up-regulating
survivin, and that
phosphorylation of Akt is required for survivin expression. Since inhibition
of apoptosis
may be required during vascular remodeling and angiogenesis, survivin and
agents that
increase the expression of survivin or the activity of survivin are useful for
inducing
angiogenesis. As used herein, the term "survivin activity" refers to the
activities
associated with survivin, for example, inhibition of apoptosis by survivin.
Accordingly,
survivin, Ang-l, Akt, or any other agent that increases the expression of
survivin or
promotes the functional activity of survivin can be used to induce
angiogenesis.
Administering the molecules in an amount that is effective to inhibit
apoptosis may be
sufficient to induce angiogenesis. Likewise, an apoptosis inhibiting amount of
survivin,
Ang-1, Akt, and or other agent that increases the expression of survivin would
be
effective in treating diseases and conditions that require inducing
compensatory
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22
angiogenesis. Examples of diseases and conditions that can be treated by these
molecules include myocardial infarction, peripheral vascular occlusion, brain
ischemia,
and stroke.
2. Methods of Preventing Angiogenesis
In another embodiment, the present invention provides methods of inhibiting
angiogenesis using agents that inhibit the expression of survivin. An example
of an
inhibitor of survivin expression is an antisense molecule. Alternatively, a
modulator that
inhibits the expression of survivin or a survivin dominant negative mutant can
be used
such as the C84A mutant (see Li et al., (1999), which is herein incorporated
by reference
in its entirety). An antisense survivin molecule or an agent that iWibits the
expression of
survivin is useful to prevent diseases or conditions such as restenosis,
vascular bypass
graft occlusion, transplant coronary vasculopathy, rheumatoid arthritis,
psoriasis, ocular
neovascularization, diabetic retinopathy, neovascular glaucoma, angiogenesis
dependent
tumors, and tumor metastasis.
Agents that inhibit the functions of survivin are also useful in inhibiting
angiogenesis, especially in cancerous cells to prevent metastases. Examples of
such
agents include, but are not limited to, survivin antibodies, inhibitors of Akt
phosphorylation, and inhibitors of survivin function.
D. Methods of Delivering a Survivin Transgene or Antisense Molecule
Gene therapy is a method for delivering functionally active therapeutic or
other
forms of genes into targeted cells. Initial efforts of gene transfer into
somatic tissues
have relied on indirect means called ex vivo gene therapy, wherein target
cells are
removed from the body, transfected or infected with vectors carrying
recombinant genes,
and re-implanted into the body. Techniques currently used to transfer DNA in
vitro into
cells include calcium phosphate-DNA precipitation, DEAE-Dextran transfection,
electroporation, liposome mediated DNA transfer or transduction with
recombinant viral
vectors. These transfection protocols have been used to transfer DNA into
different cell
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23
types including epithelial cells (U.S. Pat. No. 4,868,116; Morgan et al.,
1987),
endothelial cells (W089/05345), hepatocytes (Ledley et al., 1987; Wilson et
al., 1990)
fibroblasts (Rosenberg et al., 1988; U.S. Pat. No. 4,963,489), lymphocytes
(U.S. Pat. No.
5,399,346; Blaese et al., 1995) and hematopoietic stem cells (Lim et al.,
1989; U.S. Pat.
No. 5,399,346).
Direct i~ vivo gene transfer has been carried out with formulations of DNA
trapped in liposomes (Ledley et al., 1987), or in proteoliposomes that contain
viral
envelope receptor proteins (Nicolau et al., 1983), and with DNA coupled to a
polylysine-glycoprotein carrier complex. In addition, "gene guns" have been
used for
gene delivery into cells (Australian Patent No. 9068389). Lastly, naked DNA,
or DNA
associated with liposomes, can be formulated in liquid carrier solutions for
injection into
interstitial spaces for transfer of DNA into cells (W090/11092).
Viral vectors are often the most efficient gene therapy system, and
recombinant
replication-defective viral vectors have been used to transduce (i.e., infect)
cells both ex
vivo and i~ vivo. Such vectors include retroviral, adenovirus and adeno-
associated and
herpes viral vectors. Accordingly, in one embodiment the survivin transgene or
survivin
antisense molecule can be subcloned into an appropriate vector and transferred
into a cell
or tissue by gene transfer techniques discussed above.
In another embodiment, the survivin transgene or the survivin antisense
molecule
can be provided to the cell or tissue using a transfection facilitating
composition, such as
cationic liposomes containing desired polynucleotide. The desired
polynucleotide is the
survivin transgene or the survivin antisense molecule.
E. Methods of Delivering Survivin Expressing Cells
The present invention provides a method of delivering endothelial cells
engineered to express an apoptosis inhibiting amount of survivin to a patient.
Genetically
engineered endothelial cells, preferably autologous, may be implanted directly
into the
patient, where they produce and deliver survivin. In one preferred embodiment,
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autologous endothelial cells are engineered to express an apoptosis inhibiting
amount of
survivin.
Delivery of vertebrate cells genetically engineered to express and secrete
high
levels of a desired protein to a patient is well known. Typically, autologous
cells derived
from the patient are stably transfected with the nucleic acid encoding the
desired protein.
They are harvested from tissue culture dishes, placed in an implantation
device, and
implanted at a variety of sites including subcutaneous, intraperitoneal,
intrasplenic,
intraomental, inguinal, intrathecal, intraventricular, and intramuscular
sites, as well as
within lymph nodes or within adipose tissue.
Methods for obtaining cells stably transfected with the nucleic acid encoding
survivin are discussed above.
F. Implantable Devices
As described above, the localized induction of angiogenesis using a
Hydrogel-coated angioplasty balloon as the gene delivery system has been
successfully
demonstrated (Takeshita et al., 1996; Isner et al., 1996). These trials
demonstrated the
sustained expression of the VEGF gene in the vessel wall which subsequently
augmented
neovascularization in the ischemic limb. Accordingly, such delivery systems
may be
used to provide a survivin encoding nucleic acid molecule or transgene to a
patient in
need thereof. Such a system may also be used to locally deliver an agent which
modulates survivin expression iri contacted cells. In one preferred
embodiment, both a
survivin encoding nucleic acid molecule and the VEGF gene may be delivered in
combination or sequentially to induce localized angiogenesis.
In another embodiment, survivin or an agent that modulates survivin expression
can be provided to the tissue as an implant. The preparation of implants
requires such
supports suitable for being placed in contact with cells and various factors
promoting the
adhesion of these cells to the support if necessary, under conditions such
that the different
constituents present conserve their principal natural structural and
functional properties.
Examples of suitable implant materials are fibrous collagen and type II
collagen. The
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implant material is coated or impregnated with the agent that is to be
provided to the cell
or tissue. Other implantation device consists of a solid, unitary piece of
collagen gel (a
"collagen matrix") in which the cells.are embedded (LT.S. Pat. No. 4,485,096).
Other
substances, such as polytetrafluoro-ethylene (PTFE) fibers (Moullier et al.,
1993; WO
94/24298), may be included in the collagen implant to impart strength or other
desirable
characteristics to the collagen gel. Matrices may be implanted in a variety of
sites. A
surgical incision at the appropriate site is made, the matrix inserted, and
the incision
closed.
In a further embodiment, the implant can be provided to the cell or tissue as
a
stmt. Implantable stems are small tubes of a few millimeters in diameter and a
few
centimers in length used to deliver therapeutic agents directly to the target
site. Stents are
suitable for local delivery of viral vectors encoding a survivin nucleic acid
molecule or
survivin inducing molecule to target sites and are often designed to be
capable of
degradation into products that are nontoxic to the cells of the vessel wall
where they are
implanted (Raiasubramanian et al., 1994). Examples of biodegradable polymeric
material for making sterits are mixtures of poly-L-lactic acid (PLLA) and Poly-
E-
caprolactone (PLC) (Raiasubramanian et al., 1994) and poly-beta-
hydroxybutanoic acid
(LTS Patent No. 5,935,506). The stents can be impregnated with a survivin
nucleic acid
molecule, a survivin antisense molecule, or an agent that induces survivin
expression to
be delivered and surgically implanted at the target site.
G. Pharmaceutical Composition and Methods of Delivery of Agents
The agents that promotes or inhibits angiogenesis can be administered via
parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal,
transdermal, or
buccal routes. Alternatively, or concurrently, administration may be by the
oral route.
The dosage administered will be dependent upon the age, health, and weight of
the
recipient, kind of concurrent treatment, if any, frequency of treatment, and
the nature of
the effect desired. For example, as a means of blocking angiogenesis in tumor
cells, a
survivin inhibiting agent such as a survivin antibody, an inhibitor of
survivin expression
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Z6
or function, or an inhibitor of Akt phosphorylation, is administered
systemically or
locally to the individual being treated. Alternatively, as a means of
promoting
angiogenesis, an agent such as survivin itself, Ang-1, Akt, or any other agent
that
increases the expression of survivin or induces the function of survivin is
administered
systemically or locally to the individual. As described below, there are many
methods
that can readily be adapted to administer such agents.
The present invention contemplates compositions containing one or more agents
that promotes or inhibits angiogenesis. ° While individual needs vary,
a determination of
optimal ranges of effective amounts of each component in the composition is
within the
skill of the art. Typical dosages comprise 0.1 to 100 mglkg body wt. The
preferred
dosages comprise 0.1 to 10 mg/~g body wt. The most preferred dosages comprise
0.1 to
1 mg/kg body wt.
In addition to the pharmacologically active agent, the compositions of the
present
invention may contain suitable pharmaceutically acceptable carriers comprising
excipients and auxiliaries which facilitate processing of the active compounds
into
preparations which can be used pharmaceutically for delivery to the site of
action.
Suitable formulations for parenteral administration include aqueous solutions
of
the active compounds in water-soluble form, for example, water-soluble salts.
In
addition, suspensions of the active compounds as appropriate oily injection
suspensions
may be administered. Suitable lipophilic solvents or vehicles include fatty
oils, for
example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate
or
triglycerides. Aqueous injection suspensions may contain substances which
increase the
viscosity of the suspension include, for example, sodium carboxymethyl
cellulose,
sorbitol, and/or dextran. Optionally, the suspension may also contain
stabilizers.
Liposomes can also be used to encapsulate the agent for delivery into the
cell.
As described above, the pharmaceutical formulations for systemic
administration
according to the invention may be formulated for enteral, paxenteral or
topical
administration. Indeed, all three, types of formulations may be used
simultaneously to
achieve systemic administration of the active ingredient.
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Suitable formulations for oral administration include hard or soft gelatin
capsules,
pills, tablets, including coated tablets, elixirs, suspensions, syrups or
inhalations and
controlled release forms thereof.
In practicing the methods of this invention, the agents that induce or inhibit
angiogenesis may be used alone or in combination, or in combination with other
therapeutic or diagnostic agents: In certain preferred embodiments, the agents
may be
coadministered along with other compounds typically prescribed for these
conditions
according to generally accepted medical practice, such as chemotherapeutic
agents.
In one aspect, the present invention contemplates the use of agents that
modulate
survivin expression or function in combination with an immunomodulatory agent
in an
immunosuppressive therapy, such as those used to treat patients who have
received a
transplant or graft. Transplants of healthy organs or cells into a patient
suffering from a
disease are often rejected by the body due to an immune response initiated in
response to
the foreign tissue or cells. At present, the only method to inhibit this
immune response is
to administer chronic nonspecific immunosuppression agents. Agents that
modulate
survivin expression or function can inhibit. or induce angiogenesis depending
on the
condition, and immunomodulatory agents can suppress undesirable immune
response.
The combination would facilitate the recovery of patients who have undergone
transplantation.
Without further description, it is believed that one of ordinary slcill in the
art
can, using the preceding description and the following illustrative examples,
make and
utilize the compounds of the present invention and practice the claimed
methods. The
.,.
following working examples therefore, specifically point out preferred
embodiments of
the present invention, and are not to be construed as limiting in any way the
remainder of
the disclosure.
EXAMPLES
Example 1
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Materials and Methods
Cells and Cell Culture.
Human umbilical vein EC were maintained in M199 medium supplemented with
20% fetal calf serum (FCS), 50 ~,g/ml Endothelial Cell Growth Supplement
(ECGS), 100
~g/ml heparin, 100 ~g/ml penicillin, 100 ~,g/ml streptomycin (all from Life
Technologies, Grand Island, NY), in 5% COZ at 37°C. Bovine aortic EC
were isolated
and maintained in culture as described by De Luca et al. (1994). Subconfluent
EC were
rendered quiescent by a 24 h-culture in M199 plus 0.1% FCS. Cells were
detached with
0.05% trypsin/0.02% EDTA, seeded in C6-well plates (Costar Corp., New Bedford,
MA), growxn to 70% confluency and used between passage 2 and 3.
Modulation of Survivin Expression in EC.
Quiescent sub-confluent EC were incubated with VEGF (Collaborative
Biomedical Products, Bedford, MA; 10-100 ng/ml), basic fibroblast growth
factor (bFGF
Calbiochem Corp., La Jolla, CA; 5 ng/ml), 10% FCS, or recombinant IL-1 (R&D,
Minneapolis, MN; 2 ng/ml, 200 U/ml) or TNFa (lOng/ml, Endogen, Woburn, MA) for
14 h at 37°C in M199 plus 0.1% FCS. Cells were washed, harvested by
trypsin/EDTA
and extracted in 4% SDS plus protease inhibitors. Protein normalized aliquots
of cell
extracts were electrophoresed on a 13.5% SDS polyacrylamide gel, transferred
to nylon
membranes (Millipore, Corp.) for ~l h at 1 A,~ and immunoblotted with 1 ~glml
of a rabbit
antibody to survivin followed by,chemiluminescence (Amersham, Arlington
Heights,
IL). 18 Samples were analyzed for equal protein loading by immunoblotting with
a
mouse antibody to (3-actin. For Northern hybridization, serum-deprived EC were
stimulated with 100 ng/ml VEGF and harvested at increasing time intervals
between 1.5-
24 h culture at 37°C. Total RNA was extracted using the TRI Reagent (10
6 cells/0.2 ml,
Molecular Research Center, Cincinnati, OH), and further processed for Northern
hybridization with a 32 Pa-dCTP-random-primed labeled survivin cDNA or control
(3-
actin probe, as described (Ambrosini et..al. 1997).
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Three-Dimensional EC Culture.
EC were suspended at a density of 3x106 /ml in a liquefied matrix of rat-tail
type I
collagen (1.5 mg/ml) and human plasma-derived fbronectin (0.15 mg/ml) in M199,
pH
7.5. One ml of the EC suspension was transferred into each well of rat-tail
type I-coated
C6 wells, and warmed to 37°C to allow polymerization of the matrix.
After a 24-h
incubation at 37°C in 1VI199 plus 20% FCS, 50 ~,g/ml ECGS, 100 ~,g/ml
heparin, 100
~,g/ml penicillin and 100 ~g/ml streptomycin, the three dimensional culture
was placed in
OCT and paraffin-embedded for immunohistochemical analysis. Alternatively, two-
or
three-dimensional EC cultures were homogenized in a tissue grinder and
immunoblotted
for survivin expression. During the incubation period, EC throughout the gel
were
observed to elongate and form multicellular tubular structures, as described
(Sierra-
Honigman et al., 1998).
Immunohistochemis
Four skin biopsies, containing granulation tissue or normal, non-inflamed skin
by
hematoxylin-eosin staining were collected=from the archives of Yale-New Haven
Hospital. Five ~,m sections were prepared from paraffin-embedded tissues,
deparaffinized in xylene, and rehydrated in graded alcohol with quenching of
endogenous
peroxidase in 2% HZOz in methanol. Immunolocalization of survivin was carried
out as
described previously (Ambrosini et al., 1997) after antigen retrieval by
pressure cooking
for 5 min in 0.01 M citrate buffer pH 6Ø Binding of the primary antibody was
revealed
by addition of 3,3'-diaminobenzidine,.or, alternatively, 3-amino-9-
ethylcaxbazol (AEC,
Vector), as a substrate. Control experiments were carried out in the absence
of primary
antibody, or in the presence of preimmune rabbit IgG.
EC Protection by Survivin.
The cDNA of wild type survivin (Ambrosini et al., 1997) was inserted in frame
in
the EcoRI site of Green Fluorescence Protein (GFP.)-encoding plasmid, pEGFPcl
(Clontech, San Francisco). The correct orientation and reading frame of
pEGFPcl fusion
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plasmid were confirmed by DNA sequencing. Bovine aortic EC were seeded in C6-
well
plates at 40-50% confluency and transfected with GFP-vector or GFP-survivin by
lipofectin for 6 h at 37°C. After removal of the DNA-lipid mixture, the
EC monolayer
was placed in complete growth medium for 35 h at 37°C, and incubated
with 5 ng/ml
TNFa plus 5 ~,g/ml cycloheximide for additional 8h at 37°C. Cells
(floaters plus attached
cells) were fixed in 70% ethanol, stained with 10 ~.g/ml propidium iodide plus
100 ~g/ml
RNAse A and 0.05% Triton X-100 in PBS, pH 7.4, and GFP-expressing cells were
analyzed for DNA content by flow cytometry. In other experiments, bovine EC
transfected with GFP-vector or GFP-survivin were treated with control medium
or 5-10
ng/ml TNFa plus 10 g/ml cycloheximide for 8-h at 37°C. Cells were
harvested, and
analyzed for caspase-3 activity by hydrolysis. of the fluorogenic substrate Ac-
DEVD-
AMC (N-Acetyl- Asp-Glu-Val-Asp-aldehyde, Pharmingen, San Diego, CA), in the
presence or in the absence of the caspase-3 inlubitor Ac-DEVD-CHO.
Fluorescence
emissions were quantitated on a spectrofluorometer with excitation wavelength
of 360
nm and emission of 460 nm.
Example 1.1
Mitogen-Stimulated Induction of Survivin in EC.
Expression of 16.5 kD endogenous survivin in quiescent, serum-deprived
endothelium was minimally detectable by inununoblotting (Figure 1A), in
agreement
with previous observations (Ambrosini et al. 1997). EC stimulation with serum,
or
specific mitogens, VEGF or bFGF, resulted in an 8-16-fold up regulation of
survivin
expression, by immunoblotting (Figure 1A). Survivin induction by VEGF was
concentration-dependent and maximal at ~50 ng/ml (Figure 1B). EC stimulation
with
cytolcines TNFa or IL-1 did not increase smvivin expression, which was reduced
below
background levels of untreated cells (Figure 1A). In control experiments by
flow
cytometry, TNFcc or IL-1 stimulated strong up regulation of intercellular
adhesion
molecule-I in EC, whereas VEGF was ineffective (not shown). By Northern
hybridization, a main 1.9-kb survivin message and a fainter 3.4-kb survivin
transcript
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were minimally detected in quiescent EC (Figure 1 C). VEGF treatment resulted
in rapid
up regulation of survivin RNA in EC, in a response that peaked 6- to 10-h
after
stimulation, and decreased to approach background levels 24 h after treatment
(Figure
1 C).
Example 1.2
Survivin Expression in Three-Dimensional EC Cultures.
Survivin was expressed at very low levels in two-dimensional EC cultures, by
immunohistochemistry (Figure 2A). In contrast, formation of three-dimensional
vascular
tubes in collagen/fibronectin matrix resulted in strong expression of survivin
in EC
(Figure 2B). No staining of three-dimensional EC cultures was observed with
control
non-binding antibody (Figure 2C). By immunoblotting, a prominent 16.5 kD
survivin
band was prominently induced in EC extracts of three-dimensional vascular
tubes, as
compaxed with two-dimensional EC cultures (Figure 2D).
Example 1.3
Survivin Expression in Proliferating EC. Ih Tlivo.
In four out of four cases, survivin was strongly expressed in the cytoplasm of
EC
of newly formed capillaries of skin granulation tissue, by
immunohistochemistry (Figure
3A). Abundant expression of survivin was also demonstrated in EC of large
vessels of
granulation tissue at the dermis/hypodermis junction (Figure 3C). In contrast,
no staining
of granulation tissue was observed in the absence of primary antibody (Figure
3B, D), or
with control preimmune antibody (not shown). Analysis of non-proliferating
capillaries
of non-inflamed normal skin revealed miumally detectable expression of
survivin in EC
(Figure 3E), as compared with control staining with preimmune IgG (Figure 3F).
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Example 1.4
Anti-Apoptotic EfFect of Survivin in EC.
Treatment with TNFa/cycloheximide induced EC apoptosis and generation of an
hypodiploid population by propidium iodide staining and flow cytometry (Figure
4A).
Expression of GFP-survivin inhibited TNFa-induced apoptosis in EC, and reduced
the
percentage of hypodiploid cells to control levels of untreated cultures
(Figure 4A). In
contrast, fransfection of GFP-vector alone did not affect TNFa-induced EC
apoptosis
(Figure 4A). Moreover, expression of GFP-survivin in EC strongly inhibited
caspase-3
activity in TNFa-treated EC, as determined by DEVD hydrolysis, whereas GFP-
vector
alone was ineffective (Figure 4B). In control experiments, preincubation of
TNFa-
treated EC extracts with the caspase-3 inhibitor DEVD-CHO abrogated DEVD
hydrolysis (Figure 4B).
Example 2
Materials and Methods
Example 2.1
Effect of Ang--1 on Akt.
Bovine lung microvasculax endothelial cells (MVEC, Vectelc, Albany, NY) were
cultured in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine
serum
(FBS), L-glutamine and antibiotics (penicillin and streptomycin). Cells (up to
passage
12) were used for the experiments; cultures had typical cobblestone morphology
and
stained uniformly for von Willebrand factor, as assessed by indirect
immunofluorescence.
A recombinant form of Ang-1 was used in all of the experiments. This form of
Ang-1 differs from the native Tie2 ligand in that it possesses a modified NH2-
terminal
sequence and a mutation in Cys245 that make it easier to produce and purify.
Microvascular endothelial cells (MVEC) were treated with Ang-1. Changes in
the anti-apoptotic serine/threonine kinase, Akt (or protein kinase B) were
analyzed.
Stimulation of MVEC with Ang-1 increased Akt phosphorylation on serine 473
(Fig.
5A), threonine 308 (not shown) and up-regulated Akt kinase activity (Fig. 5B),
in a
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reaction suppressed by the PI3 kinase inhibitor, wortmannin (WM; Fig. 5B). Ang-
1
stimulated Akt phosphorylation in a time-dependent manner with maximal
activation
occurring within 15-30 min, and sustained phosphorylation lasting for up to 2
h (Fig.
SC). Ang-1 stimulated phosphorylation of Akt on Ser 473 was antagonized by
preincubation of Ang-1 with soluble Tie 2 receptor, but not by incubation with
soluble
Tiel receptor bodies (SD). In addition, Ang-1 induced Akt phosphorylation was
partially
blocked by the physiological antagonist of Ang-l, angiopoeitin-2 (Ang-2;
Maisonpierre
et al., 1997). Interestingly, Ang-2 alone weakly activated Alct in MVEC.
Therefore,
.,
Ang-1 via the Tie 2 receptor stimulates Akt activation through a PI-3 kinase
dependent
mechanism.
Example 2.2
Effect of Ana-1 on Endothelial Cell A~optosis Induced by Detachment from the
Matrix,
i.e. Anoikis (Frisch et al. 1997)
MVEC in serum free media and plated onto petri dishes for 18 h underwent
extensive apoptosis as determined by appearance of a hypodiploid cell
population (~25%
versus 2% of control, adherent cultures) by propidium iodide staining and flow
cytometry
(Fig. 6A). Incubation of MVEC cultured under these conditions with Ang-1
inhibited
apoptosis by 75%, in a reaction reversed by WM (Fig. 6A). To examine if Akt
was
required for Ang-1 cytoprotection, MVEC were infected with adenoviral (3-
galactosidase
or activation deficient Akt (AA-Akt; Fujio et al., 1999) and determined the
degree of
apoptosis. Transduction of MVEC with AA-Akt abrogated the cytoprotective
effect of
Ang-1 against anoikis, whereas a control adenovirus encoding (3-galactosidase
was
ineffective. MVEC were infected with 100 MOI of adenovirus containing the (3-
galactosidase, HA-tagged activation deficient phosphorylation mutant Alct (AA-
Akt,
Fujio et al., 1999). After 4 ~hr, the virus was removed and cells left to
recover overnight in
complete medium. In preliminary experiments with the /3-galactosidase virus,
these
conditions were optimal for infecting 95%.of the cultures. Infected cells were
plated in
bacteriological dishes for apoptosis experiments or lysed in buffer for
immunoblotting.
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WM also prevented Akt phosphorylation on Ser 473 induced by Ang-1 in
suspended endothelial cells (Fig. 6B). Collectively, these data indicate that
Ang-1
mediates endothelial cell protection through an integrin independent, PI-3-
kinase /Akt-
dependent pathway.
Example 2.3
The Potential Link between Ana-l and Two Known Anti-Apoptotic Genes, Survivin
and
Bcl-2 lAmbrosini et al. 1997 and Gerber et al. 1998
Treatment of MVEC with Ang-1 rapidly induced a time-dependent increase in
survivin RNA levels (Li et al., 1998), which peaked 12 h after stimulation and
remained
sustained for up to 24 h (Fig 7A). In contrast, Ang-1 did not up-regulate bcl-
2 RNA
expression in MVEC (Fig. 5A). Consistent with a receptor-mediated response,
preincubation of Ang-1 with soluble Tie-2 receptor abolished Ang-1 induction
of
survivin RNA in MVEC (Fig. 7B). When MVEC were transfected with a survivin-
luciferase promoter construct (Li et al., 1999a), Ang-1 stimulated a 3-7-fold
up-
regulation of survivin transcriptional activity, which persisted for up to 24
h after
stimulation (Fig. 7C). VEGF or Ang-1 strongly induced expression of survivin
protein in
HUVEC, an effect abrogated by WM, or by transduction with AA-Akt (Fig. 7D).
Human
umbilical vein endothelial cells (HUVEC ) were used in these experiments due
to greater
sensitivity of the survivin antibody with human survivin. HUVEC were isolated
from
umbilical veins and cultured on gelatin coated tissue culture flasks in M199
containing
20% fetal bovine serum (FBS), 50 ~,g/ml EC growth supplement (ECGS, a
commercial
preparation that contains mainly acidic fibroblast growth factor), 100 ~,g/ml
porcine
heparin, l0U/ml penicillin and 100 ~,g/ml streptomycin. Two to three
individual donors
were pooled at passage one and used up to passage three. Cultures had typical
cobblestone morphology and stained uniformly for von Willebrand factor, as
assessed by
indirect immunofluoresence. Identical results were obtained using MVEC.
In contrast, VEGF weakly induced bcl-2 protein expression in MVEC, whereas
Ang-1 was ineffective (Fig. 7D). These data demonstrate that distinct
angiogenic factors,
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Ang-1 and VEGF, stimulate survivin expression in endothelial cells via a PI-3
kinase/Akt
dependent mechanism.
Example 2.4
Induced Survivin Expression Mediates the Anti-Anoptotic Function of Ang-1
MVEC was transfected with cDNAs containing green fluorescent protein (GFP)
fused to wild-type-survivin (GFP-survivin), or to a dominant negative Cys84--
>Ala
survivin mutant (GFP-C84A survivin). Cytoprotection in response to apoptosis-
inducing
stimuli (Li et al., 1999b) was determined. Fusion of survivin with GFP does
interfere
with its biological activity (Li et al., 1999b). The survivin-GFP (Cys84-Ala)
construct is
a mutation in the BIRl domain. It is targeted to the mitotic spindle similar
to
endogenous survivin but is devoid of its anti-apoptotic function.
Treatment with Ang-1, or expression of GFP-survivin, alone or in combination
with Ang-1, suppressed the appearance of MVEC with hypodiploid DNA content
induced by TNFalcycloheximide. or by anoikis (Figs. 8A and B). In contrast,
transfection
of MVEC with GFP-C84A survivin abrogated the cytoprotective effect of Ang-1
against
TNFcc/cycloheximide- or anoikis-induced cell death (Figs. 8A and B). These
data
identify survivin as a novel PI-3-lcinase, Akt-dependent target gene for Ang-
l, and
demonstrate that survivin is necessary for the anti-apoptotic effect of Ang-1.
Example 3
Materials and Methods
Survivin Mediated In Vivo Angiogenesis in Animal Model
Plasmid DNA
DNA encoding survivin is assembled into a mammalian expression vector
containing the cytomegalovirus promoter (Talceshita et al., 1996). The
biologic activity
of survivin obtained from cells transfected with this construct, phSurvivin,
is confirmed
before performing arterial gene transfer. The plasmid pGSVLacZ containing a
nuclear-
targeted -galactosidase sequence coupled to the simian virus 40 early promoter
is used
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for control transfection experiments (Takeshita et al., 1996).
Animal Model
Animal studies are performed in an animal model of hindlimb ischemia
(Talceshita et al., 1996). Rabbits weighing 4 to 4.5 kg are anesthetized with
ketamine (50
mg/kg) and acepromazine (0.8 mg/kg) after premedication with xylazine (2.5
mg/kg). A
longitudinal incision is performed in one limb, extending inferiorly from the
inguinal
ligament to a point just proximal to the patella. Through this incision, the
femoral artery
is dissected free along its entire length using a surgical loupe; all branches
of the artery
axe also dissected free. After dissection of the popliteal and saphenous
arteries, the
external iliac arter as well as all of the above arteries are ligated.
Finally, the femoral
artery is completely excised from its proximal origin as a branch of the
external iliac
artery, to the point distally at which it bifurcates into the saphenous and
popliteal arteries.
Once the femoral artery is excised, thrombotic occlusion of the external iliac
artery
extends retrograde to its origin from the common iliac artery. Consequently,
blood
supply to the distal limb is dependent on the collateral arteries, which may
originate from
the internal iliac artery. This operative procedure results in severe limb
ischemia
(Takashita et al., 1996). Postoperative analgesia (levorphanol tartrate 60
mg/kg) is
administered subcutaneously as required and prophylactic antibiotics
(enrofloxancin 2.5
mg/kg) are aclininistered subcutaneously for 5 days.
Percutaneous Arterial Gene Transfer
An interval of 10 days between the time of surgery and gene transfer is
allowed for
postoperative recovery of the rabbits and development of endogenous collateral
vessels.
At 10 days postoperatively (Day 0), a baseline angiogram is performed. The
internal iliac
artery of the ischemic limb of a number of aumals is transfected with
phSurvivin
percutaneously using a 2mm hydrogel-coated balloon catheter (Slider, Boston
Scientific, Watertown massachusetts). The angioplasty balloon is prepared (ex
vivo) by
advancing the deflated balloon through a 5 Fr. Teflon sheath (Boston
Scientific);
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applying the solution of plasmid DNA from a conventional pipette to the 20 ~,m
layer of
hydrogel coating the external surface of the inflated balloon; and finally,
deflating the
balloon, retracting same into the protective sheath, and re-inflating the
balloon to prevent
backflow of blood into the sheath (and onto the coated balloon) after
introduction into the
circulation. The sheath and angioplasty catheter are then introduced via the
right carotic
artery and advanced to the lower abdominal aorta using a 0.014-inch guide-wire
(Hi-
Torque Floppy IITM; Advanced Cardiovascular System, Temecula, California)
under
fluoroscopic guidance. The balloon catheter is then advanced into the internal
iliac artery
of the ischemic limb, inflated for 1 minute at 4 to 6 atmospheres, deflated
and withdrawn.
An identical protocol is used to transfect the internal iliac arter of control
animals with
the plasmid pGSVLacZ. Heparin is not administered at the time of transfection
or
angiography.
RT-PCR
Gene expression is evaluated at the mRNA level by RT-PCR in rabbits in which
the iliac artery was transfected-using the hydrogel balloon catheter, as
described above.
Transfected arterial segments are obtained at 7, 14, 21, and 30 days
posttransfection.
Remote tissues such as brain, heart, liver, lung, spleen, testes, are also
retrieved _< 7 days
posttransfection for analysis of survivin mRNA. Total cellular RNA is isolated
using
TRI reagent (Molecular Research Center, Cincinnati, Ohio) according to the
manufacturer's instructions. Extracted DNA is treated with DNase I (0.5 ~,1,
10 U/~.1,
Rnase-free, Message Clean kit; GenHunter, Boston, Massachusetts) at
37°C for 30
minutes to eliminate DNA contamination. The yield of extracted RNA is
determined
spectrophotometrically by ultraviolet absorbance at 260 nm. To check that the
RNA is
not degraded and that the ribosomal bands are intact, each RNA sample is
subjected to
1% nondenaturing mini-agarose gel electrophoresis. Each RNA sample is used to
make
cDNA in a reaction containing deoxynucleotides, RNasin (Promega, Madison,
Wisconsin), random hexanucleotide primers (Promega), and Moloney marine
leukemia
virus reverse transcriptase (GIBCO BRL, Gaithersburg, Maryland). Reactions are
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incubated at 42°C for 1 hour, then at 95°C for 5 minutes to
terminate the reaction. The
PCR amplification is performed for 30 cycles at 94°C for 20 seconds,
ending with 5
minutes at 72°C. Oligonucleotide primers selected from a specific
region of the nucleic
acid encoding survivin is used to amplify that region or fragment of survivin.
RT-PCR
products are analyzed by 2% agaose gel electrophoresis.
Transfection Efficienc
To assess the efficiency of ih vivo arterial gene transfer in the animal
model,
LacZ-Tf arteries are harvested on Day 5, and (3-galactosidase activity is
determined by
incubation with 5-bomo-4-chloro-3-indolyl-~3-D-galactoside chromogen (X-Gal;
Sigma
Chemical Company, St. Louis Missouri) as previously described (Takeshita et
al., 1996).
After staining with X-Gal solution, tissues are paraffin-embedded, sectioned,
and
counterstained with hematoxylin-eosin. Nuclear localized (3-galactosidase
expression of
the plasmid pGSVLacZ could not result from endogenous ~i-galactosidase
activity;
accordingly, histochemical identification of ~i-galactosidase within the cell
nucleus is
interpreted as evidence for successful gene transfer and gene expression.
Cytoplasmic or
other staining is considered nonspecific for the purpose of the present study.
Evaluation of Angio,genesis in the Ischemic Limb
Development of collateral vessels in the ischemic limb is serially evaluated
by
calf blood pressure measurement and internal iliac arteriography immediately
before
transfection (Day 0) and again on Day 30 posttransfection. In addition to
these two
parameters, limb blood flow and capillary density in the ischemic limb muscles
are
evaluated on Day 30 posttranfection.
Calf Blood Pressure Ratio
Calf blood pressure is measured in both hindlimbs using a Doppler flowmeter
(model 1059, Parks Medical Electonics, Aloha, Oregon) immediately before
transfection
(Day 0) as well as on Day 30. On each occasion, the hindlimbs are shaved and
cleaned,
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the pulse of the posterior tibial artery is identified using a Doppler probe,
and the systolic
blood pressure in both~limbs is determined using standard techniques
(Takeshita et al.,
1996). The calf blood pressure ratio is defined for each rabbit as the ratio
of systolic
pressure of the ischemic limb to systolic pressure of the normal limb.
Selective Iliac AngiographX
Selective internal iliac arteriography is performed on Day 0 (immediately
before
transfection) and again on Day 30 posttransfection (Takeshita et al., 1996). A
3 Fr.
Infusion catheter (Tracker-18, Target Therapeutic, San Jose, California) is
introduced
into a common carotid artery through a small cutdown and advanced to the
internal iliac
artery of the ischemic limb using 0.014 inch guidewire (Hi-torque floppy II)
under
fluoroscopicguidance.. The tip of catheter is positioned in the internal iliac
artery at the
livel of the interspace between the seventh lumbar and the first sacral
vertebrae. After
intraarterial injection of 0.26 mg of nitroglycerin, 5 ml of nonionic contrast
media
(Isovue-370, Squibb Diagnostics, New Brunswick, New Jersey) is injected using
an
automated angiographic injector (Medrad, Pittsburgh, Pennsylvania) programmed
to
reproducibly deliver a flow rate of lml/second. Serial angiographic images (1
per second
for 10 seconds) are then recorded on 105-mm spot film. Morphometric
angiographic
analysis of collateral vessel development is performed, using a grid overlay
comprised of
2.5-min circles arranged in rows spaced 5 mm apart. The overlay is applied to
the 4-
second angiogram recorded at the level of the medial thigh. A defined area is
chosen in
which the number of contrast-opacified arteries crossing over circles as well
as the total
number of circles encompassing the medial thigh area are counted in single
blind fashion.
An angiographic score is calculated for each film as the ratio of crossing
opacified
arteries divided by the total number of circles in the defined area of the
ischemic thigh.
Blood Flow Measurement
A 0.018-inch guidewire with a 12-MHz piezoelectric transducer at the distal
tip
(FloMap: Cardiometrics, Mountainview, California) is used to measure blood
flow
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velocity (Takeshita et al., 1996). The Doppler wire records a real-time
spectral analysis
of the Doppler signal from which the average peak velocity (APV, temporal
average fo
the instantaneous peak velocity waveform) is calculated and displayed on line.
The wire
is advanced through the 3 Fr. infusion catheter positioned at the origin of
the common
iliac artery to the proximal segment of the internal iliac artery supplying
the ischemic
limb. A stabilized velocity of 2 minutes before recording resting APV is
required.
Maximum APV is recorded after bolus injection of papaverine (Sigma Chemical
Company), 2mg/0.4 ml saline, via the infusion catheter. The Doppler wire is
then pulled
back from the internal iliac artery and readvanced to the iliac artery of the
normal limb;
the distal tip of the 3 Fr. infusion catheter is repositioned at the origin of
the common
iliac artery. Blood flow velocity is again recorded at rest and after
papaverine injection.
After completing all Doppler measurements, the 3 Fr. infusion catheter is
redirected to the proximal segment of the internal iliac artery of the
ischemic limb, and
selective internal iliac angiography is performed as described. The
angiographic luminal
diameter of the internal iliac artery in the ischemic limb and of the external
artery in the
normal limb are determined using an automated edge-detection system (Quantum
20001;
QCS, Ann Arbor Michigan) as described. The film selected for analysis is
scanned with
a lugh resolution video camera, and the signal produced by the video camera is
digitized
and displayed on a video monitor (Laser Scan; ImageComm, Santa Clara,
California).
Center lines axe traced manually for a 10-mm segment beginning immediately
distal to
the tip of the Doppler wire. The contours are subsequently detected
automatically on the
basis of the weighted sum of first and second derivative functions applied to
the digitized
brightness information. The vascular diameter is then measured at the site of
the Doppler
sample volume (S mm distal to the wire tip). Cross Sectional area is
calculated assuming
a cirular lumen.
Doppler-derived flow is calculated as QD= ( d2/4)(O.S X APV) where QD
Doppler-derived time average flow, d =vesseh diameter, and APV = time average
of the
spectral peak velocity. The mean velocity is estimated as O.S X APV by
assuming a
time-averaged parabolic velocity profile across the vessel. Angiographic
Iuminal
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diameter measurements from the angiogram recorded immediately before Doppler
recording are used for calculation of rest and maximum flow.
Capillary to Muscle Fiber Ratio
The effect of survivin gene transfer upon anatomic evidence of collateral
artery
formation is further examined by identifying capillaries in light microscopic
sections
taken from the ischemic hindlimbs (Takeshita et al. 1996). Tissue specimens
are
obtained as transverse sections from the ischemic hindlimbs at the time of
death (Day 30
posttransfection). Muscle samples are embedded in optimal cutting temperature
compound (Miles, Elkhart, Indiana) and snap-frozen in liquid nitrogen.
Multiple frozen
sections (5 ~m in thickness) are then cut from each specimen on a cryostat
(Miles) so that
the muscle fibers are oriented in a transverse fashion, and two sections are
then placed on
glass slides. Tissue sections are stained for alkaline phosphatase using an
indoxyl-
tetrazolium method to detect capillary endothelial cells (Takeshita et al.
1996) and then
counterstained with eosin. To ensure that analysis of capillary density is not
overestimated because of muscle atrophy or underestimated because of
interstitial edema,
capillaries identified at necropsy axe evaluated in relation to muscle fibers;
a total number
of 20 different fields is randomly selected and the number of capillaries and
muscle fibers
axe counted under a 20X objective to determine the capillary to muscle fiber
ratio.
Evaluation of Tissue Sites Remote from the Ischemic Limb
Fourteen rabbits axe selected at random from those undergoing survivin
arterial
gene transfer. Tissue sections are systematically retrieved from gonads,
liver, heart, lung
brain, and contralateral (nontransfected) lower limb skeletal muscle and
examined by
light microscopy for evidence of neoangiogenesis as well as evidence of immune-
related
inflammtory cell infiltrates.
Morahometric Analysis of the Site of Gene Transfer.
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Representative tissue sections are harvested on Day 30 from the site of gene
transfer in a
number of rabbits selected at random. These included a few transfected with
phSurvivin
and a few transfected with LacZ. In each case, the site of gene transfer is
identified by
the fact that gene transfer is performed at the origin of the internal iliac
artery;
accordingly, an arterial segment approximately 5 mfn in length is retrieved
from the
origin of this artery just distal to the bifurcation of the common iliac
artery. The section
is stained with hematoxylin and eoxin and then morphometrically evaluated by
light
microscopy for intimal and medial thickness, from which the intima to media
ratio is
derived (Takeshita et al., 1996).
Example 3.1
Survivin Mediated Ih T~ivo An;=io eg nesis
Plasmid DNA
Plasmid phSurvivin consists of a eucaryotic pUC 118 expression vector into
which cDNA encoding survivin has been inserted. A 763 basepair cytomegalovirus
promoter/enhancer is used to drive Survivin expression. The PUC 118 vector
includes an
SV40 polyadenylation sequence,, an Escherichia coli origin of replication, and
the (3-
lactamase gene for ampicillin resistance. The plasmid is prepared from
cultures of
phSurvivin transformed E coli, purified with a Qiagen-tip 2500 column,
precipitated with
isopropanol washed with 70%, ethanol, and dried on a Speed Vac. The purified
plasmid
is reconstituted in sterile saline; stored in vials, and pooled for quality
control analyses
(absorbance at wavelengths of 260 and 280 nm to document ratio between 1.75
and 1.85;
limulus amoebocyte lysate gel-clot assay [Bio-Whittaker] to establish
bacterial endotoxin
levels below 5 endotoxin units per kg bodyweight; microbial cultures; southern
blot for
level of contaminating genomic E coli DNA; and ethidium bomide staining after
agarose-
gel electrophoresis to confirm that over 90% of the nucleic acid was in the
closed,
circular supercoiled form). To confirm the identity of the prepared plasmid,
the survivin
coding region from each pooled batch is resequenced (Applied Biosystem 373A).
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Percutaneous Arterial Gene Transfer .
Arterial gene transfer is performed on a patient with an ischemic leg.
Arterial gene transfer is done with a hydrogel-coated balloon-angioplasty-
catheter
(Boston Scientific). A sterile pipette is used to apply 2000 g plasmid DNA at
10.3 ~,g/~,1
in 194.2 ~,1 sterile saline to external hydrogel coat of the inflated
angiophasty balloon.
The balloon is deflated, retracted into a protective sheath reintlated to 2280
mm Hg, and
advanced along with the sheath over a 45.7 mm guidewire under floroscopic
guidance to
the site of gene transfer. The balloon is then deflated, the sheath retracted,
and the
balloon reinflated at nominal pressures for 4-5 min. The balloon is deflated,
all catheters
and wires removed, and a final angiogram recorded to ensure satisfactory
patency of the
site.
Intravascular ultrasound is done immediately before gene transfer to show that
the
intended site, the distal popliteal artery is free of atherosclerotic plaque
that might
compromise transfection efficiency (Isner et al., 1996). Repeat ultrasound at
4 weeks
and 12 weeks after gene transfer is done to ensure that there is no neointimal
thickening
resulting from inflation of the hydrogel-coated angioplasty-balloon-catheter.
Digital substraction angiography is performed 4 weelcs after gene therapy, and
magnetic resonance angiography is performed 4 and 12 weeks after gene therapy.
Both
are performed to detect gene transfer promoted angiogenesis.
Example 4
The preservation of vascular homeostasis during inflammation, immune response
and transplant accommodation depends' on the ability of endothelial cells (EC)
to
continuously counteract a cellular suicide program, i.e. apoptosis (Karsan et
al., 1996).
This process involves a sequential cascade activation of intracellular
cysteine proteases,
i. e., caspases, initiated by ligation of cell surface death receptors or by
cytoplasmic
assembly of cell death initiators, i. e., apoptosome, after mitochondria)
damage
(Hengartner, 2000). Inhibition of EC apoptosis is also an obligatory
prerequisite of
angiogenesis, in which multiple receptor-ligand interactions at the EC surface
stimulate
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proliferation, migration and remodeling of EC to generate new vascular
networks (Risau,
1997). In this context, antibody or adenoviral targeting of critical
angiogenesis
regulators, including vascular endothelial cell growth factor (VEGF) (Alon et
al., 1995;
Yuan et al., 1996), or the angiopoietin-1 (Ang-1) receptor, Tie-2 (Lin et al.,
1998),
resulted in involution of vascular networks accompanied by morphological and
biochemical hallmarks of EC apoptosis. Iri addition to survival signals
mediated by
adhesion molecule, i. e., integrin; engagement (Ruegg et al., 1998; Isilc et
al., 1998), and
activation of the phophoinositide 3/Akt pathways (Fujio et al., 1999; Gerber
et al.,
1998b), angiogenesis has been associated with de hovo expression of an
heterogeneous
set of anti-apoptotic "protective genes" in the endothelium (Bath et al.,
1997), some of
which become induced via NF-KB signaling (Stehlik et al., 1998). Specifically,
stimulation of EC angiogenesis by VEGF or Ang-1 resulted in up-regulation of
anti-
apoptotic bcl-2 and A1 molecules (Gerber et al., 1998a; Nor et al., 1999) and
expression
of Inhibitor of Apoptosis (IAP) proteins (Devereaux et al., 1999), survivin
and XIAP
(O'Connor et al., 2000a; Tran et al., 1,999; Papapetropoulos et al., 2000).
In the following Examples, an antisense targeting strategy was used to
identify the
relative contribution of survivin to the anti-apoptotic function of VEGF in
endothelium.
Materials and Methods
EC Culture.
Human umbilical vein EC were maintained in M199 medium containing
20% fetal calf serum (FCS), 50 ~,ghnl endothelial cell growth supplement
(ECGS), 100
~,g/ml heparin, 100 ~,g/ml penicillin, and 100 ~,g/ml streptomycin (all from
Life
Technologies, Grand Island, NY) in 5% COa at 37°C, as described by
O"Connor et al.
(2000). Subconfluent EC were rendered quiescent by a 18 h culture in M199 plus
0.1%
FCS. Cells were detached with 0.05% trypsin/0.02% EDTA, seeded in C6-well
plates
(Costar Corp., New Bedford, MA), grown to 70% confluency, and used between
passages 2 and 3.
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Antisense Gene Tar eting_
Quiescent EC monolayers were incubated with 50 ng/ml of recombinant VEGF
(Collaborative Biomedical Products, Bedford, MA) for 24 h at 37°C in
M199 plus 0.1%
FCS. At the end of the incubation, EC were washed, harvested by trypsin/EDTA,
and
lysed in 0.5% Triton X-100, 0.5% NP-40, 0.05 M Tris-Hcl, 0.15 M NaCI plus
protease
inhibitors. Protein-normalized aliquots of cell extracts were electrophoresed
on SDS
polyacrylamide gradient gels, transferred to nylon membranes (Millipore Corp.)
for 1 h at
1 A, and immunoblotted with 2 ~,g/ml of a rabbit antibody to survivin or a
mouse
monoclonal antibody to bcl-2 (Transduction Laboratories, CA) followed by
chemiluminescence (Amersham, Arlington Heights, IL) and autoradiography.
Samples
were sequentially analyzed by Western blotting with a mouse antibody to (3-
actin to
confirm equivalent protein loading. To determine the contribution of survivin
to EC
protection mediated by VEGF, 2'-O methoxyethyl chimeric phosphorothioate
oligonucleotides were utilized. A survivin antisense oligonucleotide with the
sequence
5'-TGTGCTATTCTGTGAATT-3' (SEQ ID NO: 1) was characterized previously for its
ability to suppress endogenous survivin mRNA expression in T24 bladder and
HeLa
epithelial carcinoma cells (Li et al., 1999b). A scrambled oligonucleotide
with the
sequence 5'TAAGCTGTTCTATGTGTT-3' (SEQ ID NO: 2) was used as a control, and
also characterized in previous cell culture assays (Li et al., 1999b).
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 (Baker
et al., 1997). For transfection experiments, increasing concentrations of
control
scrambled or the various antisense oligonucleotide (50-500 nM) were mixed with
1 ml of
OPTI-MEM and 6 ~1 Lipofectin according to manufacturer instructions (Life
Technologies, MD), and incubated with serum-starved EC for 8 h. The
transfection
medium was replaced with M199 plus 0.1% FCS for an additional 18 h followed by
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46
VEGF stimulation for 24 h. Transfection efficiency was monitored by
fluorescence
microscopy using FITC-conjugated oligonucleotides and was always >85%. To
determine the effect of antisense targeting on survivin mRNA expression in
proliferating
endothelitun, EC were transfected with control or the survivin antisense
oligonucleotide,
harvested after a 24 h culture at 37°C and total RNA was extracted
using the Quiagen
Rneasy reagent, according to the manufacturer's recommendations. Samples were
separated on 1 % agarose-formaldehyde gels, transferred to Hybond nylon
membranes
and hybridized with a 3zP-random primed labeled survivin cDNA with
visualization of
radioactive bands by autoradiography. Northern blots were re-probed with
random
primed 32P-labeled human G3PDH cDNA to confirm equal loading of the various
RNA
samples.
Determination of EC Apoptosis.
EC were transfected with increasing concentrations of control or the various
antisense oligonucleotides, stimulated with 50 ng/ml VEGF for 16 h at
37°C, and
incubated in the presence of 25 ~,M C-6 ceramide or the combination of TNFa
(10 ng/ml
Endogen, Woburn, MA) plus cycloheximide (I0 ~g/mI, Sigma) for an additional 12
h at
37°C. At the end of the incubation, EC (floaters plus attached cells)
were harvested,
fixed in 70% ethanol, stained with 10 ~,g/ml propidium iodide plus 100 ~g/ml
RNase A
and 0.05% Triton X-100 in phosphate-buffered saline, pH 7.4, and analyzed for
DNA
content by flow cytometry, as~ described (O' Connor et al., 2000). In other
experiments,
transfected EC stimulated with VEGF and incubated with C-6 ceramide for 12 h
at 37°C
were harvested, washed in PBS, pH 7.4, and fixed in 4% paraformaldehyde
containing
0.25% Triton X-100 for 10 min at 22°C. Cell nuclei were stained with
6.5 ~,g/ml 4,6-
diamidino-2-phenylindole (DAPI, Sigma), 16% polyvinyl alcohol (Air Products
and
Chemicals, Allentown, PA), and 40% glycerol. Cells were independently scored
for
morphologic signs of apoptosis (chromatin condensation, DNA fragmentation)
using a
Zeiss fluorescent microscope.
Caspase Activation.
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Transfected EC, stimulated with 50 ng/ml VEGF and incubated with 25 ~,g/ml C-
6 ceramide as described above, were harvested, and 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). 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
EC
treated with VEGF plus ceramide were lysed in 0.25% Triton X-100, 10 mM KCI,
1.5
mM MgCl2, 1 mM EDTA, 1 mM DTT, 20 mM HEPES plus protease inhibitors.
Protein-normalized aliquots of the various cell extracts were separated by SDS
gel
electrophoresis, transferred to nylon membranes (Millipore Corp.), and
innnunoblotted
with a 1:5000 dilution of a rabbit antibody to caspase 3 (Transduction
Laboratories), or a
1:1000 dilution.of a mouse antibody to Poly-ADP ribose polymerase (PARP,
Pharmingen, San Diego, CA) followed by chemiluminescence (Amersham, Arlington
Heights, IL).
EC migration.
Migration assays were performed using a Boyden chamber (Neuroprobe;
Morales-Ruiz et al., 2000). Briefly, quiescent EC were transfected with
control or the
survivin antisense oligonucleotide, stimulated with VEGF, and detached using
0.05%
trypsin and 0.53 mM EDTA. Twenty thousand cells were suspended in M199 medium
containing 0.1% BSA and added to the lower chamber. Polycarbonate filters (8-
~,m
diameter) were coated with 100 ~g/ml type I collagen. The top half of the
chamber was
attached and the chamber was incubated in an inverted position at 37°C
for 2 h.
Increasing concentrations (1-500 ng/ml) of VEGF or D-erythro-sphyngosine-1-
phosphate (SPP-1, Calbiocheni) were separately added to the upper chamber and
incubated for an additional 5 h at 37°C. At the end of the incubation,
cells were fixed in
70% ethanol and non-migrating EC on the upper surface of the filter were
removed.
Migrated cells were stained with Giemsa and counted at 400x magnification in 3
random
fields per well (Morales-Ruiz et al., 2000). Each experiment was performed in
triplicate
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48
and migration was expressed as the number of total cells counted per well.
Three Dimensional Canillarv Formation.
Monolayers of quiescent EC (80% confluency) in C6-well plates were transfected
with 500 nM of control or the survivin antisense oligonucleotide. After an 8-h
incubation, the transfection medium was replaced with M199 medium containing
0.1%
FCS for an additional 18 h at 37°C. Rat tail type I collagen (3 mg/ml,
Becton Dickinson
Bedford, MA) in 1/10 volume of 10x DMEM was neutralized with sterile 1 M NaOH
and
kept on ice. Suspended EC were added to the collagen suspension to a final
concentration
of 1x106 cells/ml 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 min, after which 3 ml of M199
medium
containing 20% FCS, 50 ~,g/ml ECGS, 100 ~g/ml heparin, 100 ~,g/ml penicillin,
and 100
~,g/ml streptomycin were added to each plate. EC were allowed to form
capillary-like
vascular tubes over a 24-h incubation.in the presence of 16 nM phorbol
myristate acetate
(PMA, Sigma). After additional 24 h incubation, EC were washed three times in
phosphate buffered saline (PBS), pH 7.2, 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 additional 48 h incubation at 37°C as described (Papapetropoulos
et al., 1999).
Example 4.1
Inhibition of VEGF-Induced Survivin Expression in EC by Antisense Targeting.
Previous studies demonstrated that a survivin 2'-O methoxyethyl chimeric
phosphorothioate antisense oligonucleotide (5'-TGTGCTATTCTGTGAATT-3'; SEQ ID
NO: 1 ) inhibited survivin mRNA and protein expression in HeLa and T24 cancer
cell
lines (Li et al., 1999b). Consistent with these observations, increasing
concentrations of
the survivin antisense oligonucleotide suppressed survivin mRNA expression in
proliferating EC in a dose-dependent manner, by Northern blotting (Figure 9A).
In
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49
contrast, comparable concentrations of a control scrambled oligonucleotide (5'-
TAAGCTGTTCTATGTGTT-3'; SEQ ID NO: 2), did not decrease survivin mRNA levels
in EC (Figure 9A). In parallel experiments, VEGF stimulation resulted in a ~4-
fold
increased survivin expression in quiescent endothelium (Figure 9B), and in
agreement
with previous observations (O'Connor et al., 2000; Tran et al., 1999;
Papapetropoulos et
al., 2000). Pre-treatment of EC with increasing concentrations of the survivin
antisense,
but not control oligonucleotide, suppressed VEGF-induction of survivin in a
dose-
dependent manner by Western blotting (Figure 9B). In contrast, transfection
with control
or the survivin antisense oligonucleotide did not reduce anti-apoptotic bcl-2
expression in
endothelium, by Western blotting (Gerber et al., 1998; Nor et al., 1999)
(Figure 9C).
Example 4.2
Antisense Targeting of Survivin Suppresses the Anti-Apoptotic Function of VEGF
in EC.
Exposure of quiescent EC to C-6 ceramide resulted in induction of apoptosis as
determined by chromatin condensation and DNA fragmentation, by DAPI nuclear
staining (Figure 10A, B). Addition of VEGF attenuated ceramide-induced EC
apoptosis
and restored normal nuclear morphology (Figure 10A, B). Under these
experimental
conditions, transfection of EC with the survivin antisense oligonucleotide
completely
reversed the anti-apoptotic function of VEGF against ceramide-induced
apoptosis,
whereas the control oligonucleotide was ineffective (Figure 10A, B).
Similarly,
treatment with ceramide or the combination of TNFa plus cycloheximide resulted
in a
~7-fold increase in EC apoptosis, as determined by appearance of a cell
fiaction with
hypodiploid, i. e. sub-G1, DNA content, by propidium iodide staining and flow
cytometry
(Figure 10C, D). Addition of VEGF counteracted both ceramide- and TNFa -
induced
apoptosis in EC by ~45% (Figure 10C, D). However, and consistent with the data
presented above, EC transfection with the survivin antisense, but not the
control
oligonucleotide, suppressed VEGF protection against both cell death-inducing
stimuli
and restored EC apoptosis to levels observed in the absence of VEGF (Figure l
OC, D).
Next, it was determined whether suppression of VEGF cytoprotection by survivin
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targeting was associated with biochemical hallmarks of apoptosis in EC.
Treatment of
quiescent EC with ceramide resulted in increased caspase-3 catalytic activity,
as
determined by hydrolysis of the fluorogenic substrate DEVD-AMC, and in a
reaction
entirely suppressed by the caspase-3 inhibitor, DEVD-CHO (Figure 11A). This
was
associated with proteolytic cleavage of ~32 kDa proform caspase-3 and
generation of
active subunits of ~19 and ~17 lcDa (Figure 11B) (Salvesen et al., 1997), and
cleavage of
the N115 kDa caspase substrate poly ADP ribose polymerase (PARP) to an ~85 kDa
apoptotic fragment (Figure 11 C). Under these experimental conditions,
addition of
VEGF reduced ceramide-induced caspase-3 activity, nearly completely inhibited
the
generation of ~17 kDa active caspase-3 subunit, and of ~85 lcDa PARP fragment
(Figure
11A-C). In contrast, EC transfection with the survivin antisense, but not
control
oligonucleotide, restored the proteolytic generation of ~17 kD active caspase-
3 and
apoptotic fiagmentation of PARP (Figure 11A-C).
Example 4.3
Specificity of Antisense Taraetin;~ of Survivin.
It was next determined whether antisense survivin targeting exclusively
affected
EC viability during VEGF stimulation. Incubation of EC in 0% serum for 24 h
resulted
in increased caspase-3 activity by DEVD hydrolysis as compared with
continuously
growing cultures (Figure 12A), and appearance 'of an apoptotic cell fraction
with
hypodiploid DNA content by propidium iodide staining and flow cytometry
(Figure
12B). Addition of ceramide to these cells further increased caspase-3 activity
and
generation of EC with hypodiploid DNA content (Figure 12A, B). However, in the
absence of VEGF, transfection of EC with control or survivin antisense
oligonucleotide
did not further enhance caspase-3 activity or generation of apoptotic cells in
the presence
or in the absence of ceramide (Figure 12A, B).
In other experiments, EC ~transfection with antisense oligonucleotides to
PECAM-
1(CD31), LFA-3 (CD58), or ICAM-1 (CD54) resulted in concentration-dependent
suppression of the various targeted mRNAs, by Northern blotting, as described
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51
previously (Baker et al., 1997). However, when analyzed for nuclear morphology
by
DAPI staining, expression of the various antisense oligonucleotides did not
significantly
reduce the anti-apoptotic function of VEGF against ceramide-induced EC death
(Figure
13). In contrast, and in agreement with the data presented above, EC
transfection with
the survivin antisense, but not control oligonucleotide blocked the
cytoprotective effect
of VEGF in ceramide-treated cultures (Figure 13).
Example 4.4
Role of Survivin in VEGF-Induced EC Migration and Remodeling,
The potential role of survivin targeting on other angiogenic responses induced
by
VEGF, i. e. EC migration and stabilization of three-dimensional vasculax
networks (Risau
et al., 1997) was next investigated: 'First, stimulation with VEGF or SPP-1
resulted in
EC chemotaxis and migration in a specific and concentration-dependent manner
(Figure
15), in agreement with previous observations (Morales-Ruiz et al., 2000).
Transfection
of VEGF-stimulated EC with inhibitory concentrations of control or the
survivin
antisense oligonucleotide failed to decrease EC migration in response to VEGF
or SPP-1
(Figure 15). In a second series of experiments, the effect of antisense
survivin targeting
on the non-proliferative, remodeling phase of VEGF-induced angiogenesis was
investigated. Addition of VEGF to EC-collagen gels primed with PMA and
transfected
with control oligonucleotide supported the generation of viable three-
dimensional
capillary-like structures, which persisted throughout a 3-day culture at
37°C (Figure 15)
(Ilan et al. 1998). 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 over a 3-day culture (Figure 15), in agreement with previous
observations (Ilan et al., 1998). Under these experimental conditions,
transfection of EC
with the survivin antisense oligonucleotide completely reversed the protective
effect of
VEGF on capillary formation and maintenance and resulted in complete
involution ~f
three dimensional vascular networks during a 3-day culture (Figure 15).
Although the present invention has been described in detail with reference to
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52
examples above, it is understood that various modifications can be made
without
departing from the spirit of the invention. Accordingly, the invention is
limited only by
the following claims. All cited patents, patent applications and publications
referred to in
this application are herein incorporated by reference in their entirety.
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SEQUENCE LISTING
<110> Altieri, Dario C.
Sessa, William C.
Yale University
<120> Survivin Promotion of Angiogenesis
<130> 44574-5056-WO
<140>
<141>
<150> US 60/172,991
<151> 1999-12-21
<160> 2
<170> Patentln Ver. 2.1
<210> 1
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Survivin
antisense oligonucleotide
<400> 1
tgtgctattc tgtgaatt 18
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Control
(scrambled) oligonucleotide
<400> 2
taagctgttc tatgtgtt 18
1
