JP2003529554A - Survivin promotes angiogenesis - Google Patents

Abstract

(57) Abstract The present invention discloses a method of promoting angiogenesis using an agent that increases the active function and / or expression of survivin. The present invention also discloses a method of inhibiting angiogenesis using an agent that inhibits the activity, function and / or expression of survivin.

Description

Detailed Description of the Invention

(Related Application) This application is filed on Dec. 21, 1999 in US Provisional Patent Application No. 60 /
No. 172,991, which is hereby incorporated by reference in its entirety. This application is related to US Application No. 08 / 975,080, filed November 20, 1997, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION The present invention relates generally to the modulation of survivin to induce or inhibit angiogenesis.

BACKGROUND OF THE INVENTION Gene control of cell death / viability (apoptosis) maintains tissue and organ homeostasis by eliminating aging or damaged cells (Vaux).
Et al., 1999). This process involves inhibitors and stimulators of cell death of various gene families, culminating in the activation of intracellular cysteine proteases known as caspases (Salvesen et al., 1997). Abnormalities in the apoptotic moiety are associated with cancer (Thompson, 1995) and vascular disease (Rudin et al., 199).
It is known to contribute to human diseases, including 7). In particular, an abnormal increase in cell death is associated with atherosclerotic plaque instability (Bjorkerud et al. 1996), congestive heart failure (Olivetti et al. 1997), coronary disease (Olivetti et al.
1996) and ischemic neuronal depletion (Chen et al., 1998).

The endothelium is one of the most important sites for controlling apoptosis in vascular injury and remodeling (Karsan et al., 1996). In inflammation, a heterogeneous group of protective genes activated by nuclear factor κB is the cytokine, tumor necrosis factor α (
It counteracts endothelial cell (EC) cell death and pre-inflammatory changes induced by TNFα) (Bach et al., 1997). Inhibition of apoptosis may also be obligatory during vascular remodeling and neovascularization (Risau, 1997). In this context, EC-specific mitogens, including vascular endothelial cell growth factor (VEGF) or basic fibroblast growth factor (bFGF), are critical in maintaining EC viability in vivo. Transduce signals (Benjamin et al., 1997;
Alon et al., 1995; Yuan et al., 1996). However, ECs have not completely elucidated the downstream effector genes that couple mitogen-dependent survival to anti-apoptotic machinery.

Angiopoietin-1 (Ang-1) has been shown to be an endothelium-specific ligand essential for embryonic vascular stabilization, branched morphogenesis, and postnatal angiogenesis. During angiogenesis, ECs receive cues from growth factors to initiate endothelial cell mitosis, migration and organization into primitive blood vessels and patent vascular networks (Risau 19).
97; Hanahan 1997). These processes are critically dependent on retention of endothelial cell viability. Disruption of endothelial cell-matrix contacts, or interference with extracellular survival signals, is sufficient to initiate caspase-dependent apoptosis in the endothelium, peaked by rapid regression of the vasculature (Brooks et al., 1
994; O'Reilly et al., 1996). Unlike most angiogenic regulators, including fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF), Ang-1 does not stimulate endothelial cell proliferation in vivo and in vitro. Promotes tube network stabilization and branched morphogenesis (Davis et al., 199).
6; Koblizek et al., 1998; Papapetropoulos et al., 19
99; Witzenbichler et al., 1998). Little is known about the signaling requirements of these responses, and it is unclear whether the role of Ang-1 in angiogenesis involves protecting ECs from apoptosis (Pa.
papetropoulos et al., 1999; Kontos et al., 1998).

[0006] Numerous apoptosis inhibitors characterized by their anti-apoptotic function (
IAP) has been identified. These molecules are evolutionarily highly conserved.
That is, these molecules contain two or three amino-terminal Cys of about 70 amino acids.
/ His baculovirus IAP repeats (BIR) and shares a similar organization organized into carboxy terminal zinc binding domains called RING fingers (Duckett et al., 1996; Hay et al., 1995; Liston et al., 1996). Rothe, M et al., 1995; Roy et al., 1995). Recombinant expression of the IAP protein blocks apoptosis induced by various stimuli in vitro (Duckett et al., 1996; Liston et al., 1996) and abnormally prolongs in vivo in a developmentally regulated Drosophila eye model. Promoted cell viability (Hay et al., 1995).

Survivin has recently been identified as a new member of the IAP family. Survivin is a partially conserved BIR domain and a highly charged alternative to the RING finger that inhibits growth factor (IL-3) withdrawal-induced apoptosis when translocated to B cell precursors. Is a 16.5-kDa cytoplasmic protein containing a carboxy-terminal coiled-coil region (Ambrosini).
Et al., 1997). Based on global sequence conservation, the absence of the carboxy-terminal RING finger and the presence of one partially conserved BIR domain, survivin is the most distally related member of the IAP family and is highly associated with NAIP. Of similarities (Roy et al., 1995). Furthermore, unlike other IAP proteins, survivin is undetectable in adult tissues, but in vivo, in all of the most common human cancers, lung, colon, breast, pancreas and prostate, and, It is prominently expressed in about 50% of the high grade non-Hodgkin's lymphomas. Furthermore, survivin does not bind to caspases in cell-free systems (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 was not previously discovered.

Angiogenesis, the formation of new blood vessels from existing blood vessels (Risau, W. 19).
97) is an essential process for organ and tissue development, and genetic dysregulation of these mechanisms is associated with embryonic lethality (Hanahan, D. 1997). In adult organisms, angiogenesis provides a beneficial compensatory mechanism that increases blood supply in response to hypoxia or during tissue repair and / or remodeling (Carme).
Liet, P, 2000). But the same process can have disastrous consequences for cancer,
Here, increased angiogenesis due to neovascularization contributes to tumor progression and metastatic spread (Hanahan et al., 1996). Cell-cell (Stromblad et al., 1996) and cell-matrix interactions (Brooks et al., 1998).
) Or by a receptor-initiated intracellular signal (Lin et al.,
1998; Claeson-Welsh et al., 1998) targeted inhibition of angiogenesis leads to rapid regression of newly formed blood vessels in vitro and in vivo, and the appearance of classical morphological features of apoptosis in targeted endothelium. (Alon et al., 1995; Yuan et al., 1996; Lin et al., 1998). Thus, the continuous suppression of EC apoptosis (Bach et al., 1997) may constitute one of the crucial requirements for angiogenesis, which is bcl-2 in endothelium stimulated by VEGF or Ang-1. (Gerber et al., 1998a; Nor et al., 1999) or IAP (O'Connor et al., 2000a; Tran et al., 1
999; Papapetropoulos et al., 2000) consistent with upregulation of protective genes of the gene family.

DISCLOSURE OF THE INVENTION The present invention is based on the discovery that angiogenic stimulation potently induces survivin expression in endothelium during vascular remodeling and angiogenesis in vitro and in vivo. Survivin expression and function influences endothelial cell viability during the proliferative and stable phases of angiogenesis. Therapeutic engineering of survivin expression / function on the endothelium can affect compensatory or pathological (tumor) angiogenesis.

The present invention is also based on the finding that Ang-1 prevents endothelial cell apoptosis by activating the critical survival messenger Akt and by upregulating survivin. Activation of anti-apoptotic pathways mediated by Akt and survivin in endothelial cells may contribute to Ang-1 stabilization of vasculature during angiogenesis in vivo. Therefore, Ang-1 / Akt
Targeted engineering of / survivin may be used to improve endothelial cell viability and support therapeutic angiogenesis in vivo.

Based on these observations, the present invention provides methods of promoting or inhibiting angiogenesis and treating conditions by inducing compensatory angiogenesis. 1
In one embodiment, the disclosed methods are useful in treating ischemic disease caused by myocardial infarction, peripheral vascular occlusion, cerebral ischemia, or stroke. In another embodiment, the disclosed methods of inhibiting angiogenesis are useful for treating vascular proliferative disorders such as cancer, restenosis, graft occlusion with vascular bypass, or coronary vascular injury with transplant.

The disclosed methods include providing a cell or tissue with an apoptosis-modulating agent, wherein the agent is a survivin polypeptide, a survivin transgene, a survivin antisense molecule, a survivin peptidomimetic, or , Ang-1 or Akt, or an agent that modulates survivin expression or activity in cells or tissues. In one embodiment, the agent is provided in the implant. The implant can be a stent and the implant can be coated or impregnated with a survivin transgene operably linked to the expression control elements of the vector. In another embodiment, a survivin transgene or antisense molecule is included in a transfection facilitating composition, eg, a transfection facilitating lipid or transfection facilitating particle.

In one embodiment, the present invention comprises a method of inhibiting angiogenesis comprising administering an agent that inhibits survivin. Examples of agents that inhibit survivin include, but are not limited to, survivin antibodies, survivin antisense molecules, Akt phosphorylation inhibitors, and other inhibitors of survivin function or expression. In another embodiment, the method inhibits tumor angiogenesis or cancer cell metastasis and the agent is a survivin antisense molecule. Preferably, the method inhibits VEGF-induced functions such as ceramide or TNFα-induced apoptosis and capillary formation and maintenance. Most preferably, the method comprises
Inhibits VEGF-induced capillary formation and maintenance.

As shown in the examples below, survivin is involved not only in the proliferative phase of angiogenesis, but also in the remodeling and stabilizing phases of angiogenesis. Survivin antisense molecules are sufficient to induce endothelial cell apoptosis and regression of capillary-like structures.

In one aspect, the invention contemplates the use of inhibitors of Survivin expression and function as agents that inhibit angiogenesis, such as antagonists. In another aspect, the invention contemplates the use of survivin and agents that induce the expression and function of survivin as agents that promote angiogenesis, such as agonists.

Detailed Description of the Preferred Embodiments I. GENERAL DESCRIPTION The 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-regulation of survivin up to about 16-fold. Mitogen stimulation rapidly increases survivin RNA expression in endothelial cells,
It peaked after 6 to 10 hours of culture and decreased by 24 hours. Inflammatory cytokines, tumor necrosis factor alpha or interleukin-1 did not induce survivin expression in endothelial cells. The formation of three-dimensional vasculature in vitro was accompanied by a stronger induction of survivin in endothelial cells compared to two-dimensional culture. By immunohistochemistry, survivin was minimally expressed in the endothelium of non-proliferating capillaries of normal skin, but was highly upregulated in newly formed blood vessels of granular tissue in vivo. Recombinant expression of green fluorescent protein-survivin in endothelial cells was demonstrated by caspase-3
It reduced activity and antagonized apoptosis induced by tumor necrosis factor α / 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 finding that Ang-1, which acts through the Tie-2 receptor, induces phosphorylation of the live serine / threonine kinase Akt (or protein kinase B). This involves upregulation of survivin on endothelial cells and protection of the endothelium from death-induced stimuli. In addition, dominant negative mutant genes counteract Ang-1's ability to protect cells from undergoing apoptosis. Activation of anti-apoptotic pathways mediated by Akt and survivin in endothelial cells may contribute to Ang-1 stabilization of vasculature during angiogenesis in vivo.

Further, the present invention is based on the finding that antisense oligonucleotides against the apoptosis inhibitor survivin suppressed the expression of survivin in endothelial cells induced by vascular endothelial growth factor (VEGF). In contrast, survivin antisense oligonucleotides did not affect anti-apoptotic bcl-2 levels in endothelium. Antisense targeting of survivin as assessed by cell death assay indicates VEG for TNFα- or ceramide-induced cell death.
It disappears the anti-apoptotic function of F, enhances the caspase-3 activity, and is about 17 kDa.
Promoted the production of the active caspase-3 subunit of Escherichia coli and increased the cleavage of the caspase substrate poly-ADP ribose polymerase. In contrast, survivin antisense oligonucleotides had no effect on endothelial cell viability in the absence of VEGF. Platelet-endothelial cell adhesion molecule-1 (PECAM-
1, CD31), lymphocyte function-related molecule-3 (LFA-3, CD58), or intracellular adhesion molecule-1 (ICAM-1, CD54) reduces anti-apoptotic function of VEGF in endothelium I didn't let it. VE
When tested for other angiogenic functions of GF, antisense survivin targeting induced rapid regression of the three-dimensional capillary network, but did not affect endothelial cell migration / chemotacticity. These data suggest that the anti-apoptotic function of VEGF during angiogenesis is largely mediated by inducible 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. To do.

II. Specific Embodiments A. Survivin molecule 1. Survivin Polypeptides The present invention uses survivin proteins, as well as allelic variants of survivin proteins and conservative amino acid substitutions of survivin proteins. As used herein, the term "survivin protein" or "survivin" refers in part to a protein having the amino acid sequence of human survivin. The term also includes allelic variants of naturally-occurring survivin, which include naturally-occurring proteins having the slightly different amino acid sequences specifically cited above. Allelic variants have slightly different amino acid sequences than those listed above, but will still have the necessary ability to inhibit cell apoptosis. The proteins of the survivin family, as used herein, also refer to survivin 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 described herein. Conservative variants, as used herein, include amino acid sequences that do not deleteriously affect the ability of the survivin protein to bind to survivin binding or signaling partners and / or inhibit cell apoptosis. Means change. Substitutions, insertions or deletions may be made in the survivin protein if the altered sequence prevents the survivin protein from associating with the survivin binding or signaling partner and / or preventing the survivin protein from inhibiting cell apoptosis. It is said to have harmful effects. For example, the overall charge, structure or hydrophobic / hydrophilic properties of survivin can be changed without detrimentally affecting the activity of survivin. Thus, the amino acid sequence of survivin can be altered, eg, by making the peptide more hydrophobic or hydrophilic without adversely affecting the activity of survivin.

Allelic variants, conservative substitution variants and members of the family of proteins survivin have the ability to inhibit cell apoptosis. The protein is
Generally, it has an amino acid sequence which has at least about 75%, more preferably at least about 80%, even more preferably at least about 90%, and most preferably at least about 95% amino acid sequence identity with the human survivin sequence. The identity or homology with respect to the sequences is determined herein by aligning the sequences, introducing gaps if necessary for maximum homology, and including any conservative substitutions that are homologous. It is defined as the ratio of amino acid residues in the candidate sequence that are identical to the known peptide. N-terminal, C-terminal or internal extensions, deletions, or insertions into the peptide sequence are
It should not be construed as affecting homology.

Thus, the survivin protein of the present invention is a molecule having the full length amino acid sequence of a native protein; a fragment thereof, or at least about 3, 5, 10, 15 or more amino acid residues of the survivin protein. A peptide having a contiguous sequence of; amino acid sequence variants of the sequence, wherein the amino acid residue has an inserted N or C terminus in or within the sequence of the native survivin protein.
An amino acid sequence variant of the disclosed survivin sequence substituted by another residue,
Or a fragment thereof as described above. Possible variants further include those containing mutations predetermined by, for example, homologous recombination, site-directed or PCR mutagenesis, and rabbit, rat, murine, pig, sheep, horse and non-human primate species. Corresponding survivin proteins of other animal species, including but not limited to alleles or other natural variants of the protein survivin family; and survivin proteins containing moieties other than natural amino acids, such as enzymes or radioisotopes. Detectable moieties), which are covalently modified by substitution, chemical, enzymatic, or other suitable means.

The present invention also includes or uses 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 can have significant advantages over polypeptide embodiments, including, for example, more economical manufacturing; higher chemical stability; enhanced pharmacological properties (half-life, absorption, strength). , Potency, etc.); changed specificity (
For example, broad spectrum biological activity); diminished antigenicity; and others.

2. Survivin Nucleic Acids The invention also uses nucleic acid molecules or transgenes that encode survivin and related survivin proteins. A nucleic acid, as used herein, encodes a peptide as defined above, or is complementary to a nucleic acid sequence encoding the peptide, or hybridizes to the nucleic acid and its string It remains stably attached to it under mild conditions or is at least 75%, preferably at least 80%, more preferably at least 85% with the peptide sequence.
RNA or DN encoding polypeptides that share% RNA sequence identity
Define as A. Particularly contemplated are genomic DNA, cDNA, mRNA and antisense molecules, and nucleic acids that are based on different backbones or that contain different bases (whether derived from natural sources or synthetic).

“Stringent conditions” as used herein are conditions under which hybridization will result in a clear and detectable sequence. Stringent conditions are (1) low ionic strength and high temperature used for washing (eg, 0.0
5M NaCl / 0.0015M Sodium Titanate / 0.1% SDS
(0 ° C.), or (2) during hybridization, at 42 ° C., denaturant, formamide, etc. (eg 0.1% bovine serum albumin / 0.1% ficoll / 0.1).
% Polyvinylpyrrolidone / 50% with 50 mM sodium phosphate buffer (pH 6.5) containing 75 mM NaCl, 75 mM sodium citrate (v / v)
) Formamide) is used. Another example is 0.2 × SSC at 42 ° C. and 0.
50% formamide at 42 ° C, 5x SSC (0.
75M NaCl, 0.075M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhard solution, sonicated salmon sperm DNA (50 g / ml), 0.1 % SDS, and 10%
Use of dextran sulfate. One of ordinary skill in the art can readily determine and modify the appropriate stringent conditions to obtain a clear and detectable hybridization signal.

The invention further uses fragments of nucleic acid molecules encoding survivin. 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 to encode an active portion of a survivin protein, such as a C-terminal coil or an IAP motif, the fragment must be large enough to encode the functional region (s) of the survivin protein. is there.

Modifications to the primary structure itself, such as deletions, additions, or changes in amino acids that are incorporated into the protein sequence during translation, can be done without destroying the activity of the protein. Such substitutions or other changes result in a protein having an amino acid sequence encoded by the DNA within the contemplation of the invention.

B. Survivin Antisense Molecules Antisense survivin molecules are complementary to the RNA encoded by the survivin gene (“sense gene”) and can hybridize or anneal.
Antisense survivin molecules are used to inhibit expression of the survivin gene, thereby inhibiting angiogenesis and treating diseases associated with angiogenesis.

The antisense nucleic acid preferably reverses the coding region of the sense gene with respect to its normal presentation for transcription, allowing transcription of its complement and thus is encoded by these DNAs. It is created by allowing the complementarity of each RNA. To block the production of mRNA produced by the sense gene, the antisense DNA should preferably be expressed at about the same time as the sense gene if the antisense nucleic acid is desired to be expressed in the cell. The timing is that antisense RNA blocks the function of RNA encoded by the sense gene,
They must be similar in the sense that they must be inside the cell. To achieve this result, the coding region of antisense DNA is often placed under the control of the same promoter found in the sense gene, thus causing transcription in both, simultaneously.

For a review of design considerations and use of antisense oligonucleotides, see U.
See hlmann et al. (1990) and Milligan et al. (1993), the disclosure of which is incorporated herein by reference.

In principle, antisense nucleic acids having a sequence complementary to any region of the survivin gene may be useful in the method of inhibiting angiogenesis of the present invention, although survivin mRNA transcripts containing translation initiation codons Particular preference is given to nucleic acid molecules which are complementary in part. Also,
Nucleic acid molecules complementary to a portion of the survivin mRNA located about 40 nucleotides upstream (5 'direction) or about 40 nucleotides downstream (3' direction) from the translation initiation codon are also preferred.

In another embodiment, antisense oligonucleotides that hybridize or anneal to at least a portion of survivin mRNA in cells can be used in the methods of the invention. The oligonucleotides are typically short in length and can diffuse into cells fairly easily. The antisense oligonucleotide is a polydeoxynucleotide containing 2'-deoxy-D-ribose, a polyribonucleotide containing D-ribose, any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or Polymers include, but are not limited to, non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing non-standard linkages, including but not limited to DNA and RNA. As such, it includes nucleotides in a configuration that allows base pairing and base stacking. They may include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA and DNA and DNA: RNA hybrids,
Also, unmodified forms of polynucleotides or oligonucleotides, known types of modifications, such as labels known in the art, "caps", methylations, substitution of one or more natural nucleotides by analogs, internucleotide modifications, For example, those with uncharged chains (eg methylphosphonates, phosphorotriesters, phosphoramidates, carbamates, etc.) and those with charged or sulfur-containing chains (eg phosphorothioates, phosphorodithioates, etc.), pendants Having a moiety, for example, proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) and saccharides (eg, monosaccharides), inserts (eg, acridine, psoralen, etc.) Things, chelating agents (eg metals,
Those having radioactive metals, boron, metal oxides, etc.), those having alkylating agents,
And those having a modified chain (for example, α-anomeric nucleic acid) are also included.

“Nucleoside” as used with respect to survivin antisense nucleic acid molecules
The terms "nucleotide" and "nucleic acid" include moieties that include not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. The modification includes methylated purines and pyrimidines, acylated purines and pyrimidines,
Or includes other heterocycles. Modified nucleosides or nucleotides also include modifications on the sugar moiety, eg, one or more of the hydroxyl groups are substituted with halogens, aliphatic groups, or functionalized as ethers, amines, or otherwise. .

C. Angiogenesis Angiogenesis is the process by which new blood vessels are formed (Folkman et al., 199).
2). Thus, angiogenesis is essential for regeneration, development and wound repair. But,
Inappropriate angiogenesis can have severe consequences. For example, many parenchymal tumors only become vascularized as a result of angiogenesis before they begin to grow and metastasize rapidly. Angiogenesis is so important for these functions that it must be carefully regulated to maintain health. The angiogenic process is thought to begin with the degradation of basement membrane by proteases secreted from ECs activated by mitogens such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). There is. Cells migrate and proliferate, forming solid endothelial cell buds into the interstitial space,
Thereafter, vascular loops are formed and capillaries develop with the formation of tight junctions and the deposition of new basement membranes.

The rate of angiogenesis involves changes in the local equilibrium between positive and negative regulators of microvascular proliferation. Abnormal angiogenesis occurs when an organism loses its angiogenic control, causing excessive or inadequate blood vessel growth. Conditions such as myocardial infarction, peripheral vascular occlusion, cerebral ischemia, or stroke may result from the lack of angiogenesis normally required for natural healing. In contrast, excessive blood vessel growth may support tumor growth and spread, blindness, psoriasis and rheumatoid arthritis.

It has recently been demonstrated that gene therapy can be performed to modulate angiogenesis.
For example, enhanced angiogenesis in the treatment of ischemia has been demonstrated in rabbit models and human clinical trials with VEGF using hydrogel-coated angioplasty balloons as the gene delivery system. Successful introduction and sustained expression of the VEGF gene in the vessel wall subsequently increased angiogenesis in the ischemic limb (Takes).
hita et al. (1996); Isner et al., 1996).

Alternative methods of regulating angiogenesis are still desirable for many reasons. For example, native endothelial cell (EC) number and / or viability are believed to decrease over time. Thus, in a patient population, such as the elderly, a population of ECs that is compatible to respond to administered angiogenic cytokines,
Can be limited. In addition, agents that promote or inhibit angiogenesis may be useful in one location, but they may not be desirable in another. Therefore, a means to more accurately regulate angiogenesis at a location is desirable.

The present invention provides a method of modulating angiogenesis by providing cells or tissues with survivin or a modulator of survivin expression. Modulators of survivin expression are molecules that alter the expression of survivin in cells.

1. Method of Inducing Angiogenesis In one embodiment, the present invention provides a method of inducing angiogenesis. The present invention provides that the inhibitor of apoptosis survivin is expressed by EC during angiogenesis and vascular remodeling, Ang-1 prevents EC apoptosis by phosphorylation of Akt and by upregulating survivin, And it is based on the finding that phosphorylation of Akt is necessary for the expression of survivin. Since inhibition of apoptosis may be required during vascular remodeling and angiogenesis, survivin and agents that increase survivin expression or survivin activity are useful to induce angiogenesis. The term "survivin activity" as used herein refers to activity associated with inhibition of apoptosis by survivin, eg, survivin. Thus, survivin, Ang-1, Akt, or any other agent that increases the expression of survivin or enhances the functional activity of survivin, can be used to induce angiogenesis. Administering an amount of the molecule effective to inhibit apoptosis may be sufficient to induce angiogenesis. Similarly, apoptosis-inhibiting amounts of survivin, Ang-1, Akt, and other agents that increase expression of survivin are effective in treating diseases and conditions that require induction of compensatory angiogenesis. Examples of diseases and conditions that can be treated with these molecules include myocardial infarction, peripheral vascular occlusion, cerebral ischemia and stroke.

2. Method of Preventing Angiogenesis In another embodiment, the present invention provides a method of inhibiting angiogenesis using an agent that inhibits expression of survivin. An example of an inhibitor of survivin expression is an antisense molecule. Alternatively, modulators that inhibit the expression of survivin or survivin dominant negative mutants, such as the C84A mutant (Li et al. (19
99), which is incorporated herein by reference in its entirety). Agents that inhibit the expression of antisense survivin molecules or survivin include restenosis, graft obstruction due to vascular bypass, coronary vasculopathy due to transplantation, rheumatoid arthritis, psoriasis, ocular neovascularization, diabetic retinopathy, neovascular glaucoma. , Is useful for preventing diseases or conditions such as angiogenesis-dependent tumors, and tumor metastases.

Agents that inhibit the function of survivin are also useful in inhibiting angiogenesis, especially in cancer cells to prevent metastasis. Examples of such agents include, but are not limited to, survivin antibodies, Akt phosphorylation inhibitors, and inhibitors of survivin function.

D. Methods of Delivering Survivin Transgenes or Antisense Molecules Gene therapy is the method of delivering functionally active therapeutic or other forms of genes to targeted cells. Early efforts in gene transfer into somatic tissues rely on an indirect means called in vitro gene therapy, in which target cells are removed from the body and transfected or infected with a vector carrying the recombinant gene. ,
Implant again in the living body. Techniques currently used to introduce DNA into cells in vitro include calcium phosphate-DNA precipitation, DEAE-dextran transfection, electroporation, liposome-mediated DNA transfer or transduction with recombinant viral vectors. Including. These transfection protocols refer to epithelial cells (US Pat. No. 4,868,116; Morga
n et al., 1987), endothelial cells (WO89 / 05345), hepatocytes (Ledl.
ey et al., 1987; Wilson et al., 1990), fibroblasts (Rosenbe).
rg et al., 1988; US Pat. No. 4,963,489), lymphocytes (US Pat. No. 5,399,346; Blaese et al., 1995) and hematopoietic stem cells (Lim).
Et al., 1989; U.S. Pat. No. 5,399,346).
Used to introduce A.

Direct in vivo gene transfer is described by liposomes (Ledley et al., 1987).
) Or a proteoliposome containing a virus envelope receptor protein (N
has been carried out with a DNA formulation encapsulated in icolau et al., 1983) and with DNA bound to a polylysine-glycoprotein carrier complex. In addition, a "gene gun" has been used for gene delivery to cells (EP 9068).
389). Finally, naked DNA, or DNA associated with liposomes, is
Can be formulated in a liquid carrier solution for injection into cells and for injection into the interstitial space.

Viral vectors are often the most efficient gene therapy system, and recombinant replication-defective viral vectors have been used to transduce (ie, infect) cells both in vitro and in vivo. The vectors include retrovirus, adenovirus, and adeno-associated and herpesvirus vectors. Thus, in one embodiment, the survivin transgene or survivin antisense molecule can be subcloned into a suitable vector and introduced into cells or tissues by the gene transfer techniques discussed above.

In another embodiment, the survivin transgene or survivin antisense molecule can be provided to cells or tissues using a composition that facilitates transfection, eg, a cationic liposome containing the desired polynucleotide. The desired polynucleotide is a survivin transgene or survivin antisense molecule.

E. Methods of Delivering Cells Expressing Survivin The present invention provides methods of delivering endothelial cells engineered to express an apoptosis-inhibiting amount of survivin to a patient. Genetically engineered, preferably autologous endothelial cells can be directly implanted in the patient, where they produce and deliver survivin. In one preferred embodiment, autologous endothelial cells have been 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 known. Typically, autologous cells obtained from a patient are stably transfected with a nucleic acid encoding the desired protein. They are collected from tissue culture dishes and placed in implant devices to be subcutaneous, intraperitoneal, intraspleen, intraomental, cavernous, intrathecal, intraventricular, and intramuscular sites, and lymph nodes or fat. Implant in tissue.

Methods for obtaining cells stably transfected with a nucleic acid encoding survivin are discussed above.

F. Implantable Devices As noted above, local induction of angioplasty using hydrogel-coated angioplasty balloons as a gene delivery system has been successfully demonstrated (Takeshi).
ta et al., 1996; Isner et al., 1996). These studies demonstrated sustained expression of the VEGF gene in the vessel wall followed by increased angiogenesis in the ischemic limb. Thus, the delivery system may be used to provide a survivin-encoding nucleic acid molecule or transgene to a patient in need thereof. The system may also be used to locally deliver agents that modulate survivin expression in contact cells.
In one preferred embodiment, a survivin-encoding nucleic acid molecule and VE
Both GF genes may be delivered in combination or sequentially to induce local 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 is carried out with the support suitable for placement in contact with cells and, if necessary, the various components present, under conditions that preserve their basic structural and functional properties. , Requires various factors that facilitate the attachment of these cells to the support. Examples of suitable implant materials are fibrous collagen and type II collagen. The implant material is coated or impregnated with an agent that provides cells or tissues. Another implant device consists of a solid, single piece of collagen gel (“collagen matrix”) in which cells are embedded (US Pat. No. 4,485,096). Other agents such as polytetrafluoro-ethylene (PTFE) fibers (Moullier)
Et al., 1993; WO 94/24298) may be included in collagen implants to impart strength or other desired characteristics to collagen gels. The matrix can be implanted at various sites. A surgical incision is made at the appropriate site, the matrix is inserted, and the incision is closed.

In a further embodiment, the implant can be provided to cells or tissues as a stent. Implantable stents are small tubes, several millimeters in diameter and several centimeters in length, used to deliver therapeutic agents directly to a target site. Stents are suitable for local delivery of viral vectors encoding survivin nucleic acid molecules or survivin-derived molecules to a target site and are often designed to be degradable into products that are non-toxic to cells of the implanted vessel wall. There is (Riasubram
anian et al., 1994). Examples of biodegradable polymeric materials for making stents are poly-L-lactic acid (PLLA) and poly-E-caprolactone (PL).
C) (Raiasubramanian et al., 1994) and poly-β-hydroxybutanoic acid (US Pat. No. 5,935,506). Stents can be impregnated with survivin nucleic acid molecules, survivin antisense molecules, or agents that induce survivin expression for delivery and surgical implantation at the target site.

G. Methods of Delivery of Pharmaceutical Compositions and Agents Agents that promote or inhibit angiogenesis can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal or buccal routes. Alternatively or concurrently, administration can be by the oral route. The dosage administered depends on the age, health, and weight of the recipient, the type of concurrent treatment, in some cases, the frequency of treatment, and the nature of the effect desired. For example, a survivin inhibitor, such as a survivin antibody, an inhibitor of survivin expression or function, or an Akt phosphorylation inhibitor, is administered systemically or locally to the treated individual as a means of blocking angiogenesis in tumor cells. . Alternatively, as a means of promoting angiogenesis, survivin itself, Ang-1, Akt, or any other agent that increases survivin expression or induces survivin function, is administered systemically or locally to the individual. Administer. As described below, there are many methods that can be readily adapted to the administration of the agent.

The present invention contemplates compositions that include one or more agents that promote or inhibit angiogenesis. Individual requirements will vary, but determining the optimum range for the effective amount of each ingredient in the composition is within the skill of the art. Typical dosages include 0.1 to 100 mg / kg body weight. Preferred dosages include 0.1 to 10 mg / kg body weight. The most preferred dosage comprises 0.1 to 1 mg / kg body weight.

In addition to pharmacologically active agents, the compositions of the invention facilitate processing of the active compound into a preparation that can be used pharmaceutically to deliver to the site of action. Suitable pharmaceutically acceptable carriers may be included, including excipients and adjuvants.

Formulations suitable for parenteral administration include aqueous solutions of the active compounds in water form, eg, 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 such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides.
Aqueous injection suspensions may contain agents which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol and / or dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate agents for delivery to cells.

As mentioned above, the pharmaceutical formulations for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. In fact, all three types of formulations can be used simultaneously for systemic administration of the active ingredient.

Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets including coated tablets, elixirs, suspensions, syrups or inhaled and controlled release forms thereof.

In practicing the methods of the present invention, 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 agent may be co-administered with other compounds, such as chemotherapeutic agents, that are typically prescribed for these conditions, according to generally accepted medical practices.

In one embodiment, the invention provides agents that modulate survivin expression or function in combination with immunomodulatory agents in immunosuppressive therapy, such as those used in the treatment of patients undergoing grafts or grafts. Think of using. Transplantation of healthy organs or cells into a patient suffering from a disease is often rejected by the organism due to an immune response initiated in response to foreign tissue or cells. Currently, the only way to block this immune response is to administer chronic, non-specific immunosuppressive drugs. Agents that modulate the expression or function of survivin can inhibit or induce angiogenesis depending on the condition, and immunomodulators can suppress unwanted immune responses. The combination facilitates recovery of patients undergoing transplantation.

Without further explanation, one of ordinary skill in the art would be able to make and use the compounds of the invention and practice the claimed methods using the foregoing description and the following illustrative examples. The following working examples therefore, in particular, point out preferred embodiments of the invention and are not to be construed as limiting the remaining disclosure in any way.

Examples Example 1 Materials and Methods Cells and Cell Culture Human umbilical vein ECs were mixed with 20% fetal calf serum (FCS), 50 μg / ml endothelial cell growth supplement (ECGS), 100 μg / ml heparin, 100 μg. / Ml
Penicillin, 5% in M199 medium supplemented with 100 μg / ml streptomycin (all from Life Technologies, Grand Island, NY)
Maintained at 37 ° C. in CO ₂ . Bovine aortic ECs were isolated and analyzed by De Luca et al.
1994) and maintained in culture medium. Sub-confluent ECs are M199 and 0.
It was made stationary by culturing in 1% FCS for 24 hours. Cells were detached with 0.05% trypsin / 0.02% EDTA and seeded in C6 well plates (M
Seedlings (Costar, New Bedford, A.) were grown to 70% confluency and used at passage 2 to 3.

Modulation of Survivin Expression in EC Resting subconfluent EC was tested for VEGF (Collaborative Biomedical Products, Bedford, MA; 10-100 ng / ml), basic fibroblast growth factor (bFGF, La Jolla, CA). Calbiochem; 5 ng / ml,
) 10% FCS, or recombinant IL-1 (R &D; Minneapolis, MN; 2n)
g / ml, 200 U / ml) or TNFα (10 ng / ml, Endogen, Woburn, MA) for 14 hours at 37 ° C. in M199 in 0.1% FCS. Cells were washed and collected by trypsin / EDTA, 4% SD
Extracted in S and protease inhibitors. A protein-normalized aliquot of cell extract was electrophoresed on a 13.5% SDS polyacrylamide gel, transferred to a nylon membrane (Millipore) for 1 hour at 1 A, and immunoblotted with 1 μg / ml rabbit antibody against survivin. Then, chemiluminescence (Amersham, Arlington Heights, IL) was performed. Eighteen samples were analyzed with equal protein loading by immunoblotting with a mouse antibody against β-actin. For Northern hybridization, serum starved EC were stimulated with 100 ng / ml VEGF and collected at increasing time intervals at 37 ° C. for 1.5-24 hours. All RN
A was extracted using TRI reagent (10 ⁶ cells / 0.2 ml, Molecular Research Center, Cincinnati, OH) and further as described (Ambro.
Sini et al., 1997), ³² Pα-dCTP-random primed survivin cDNA or control β-actin probe for Northern hybridization.

Three-dimensional EC culture medium EC was added to rat tail type I collagen (1.5 mg) in M199 (pH 7.5).
/ Ml) and human plasma-derived fibronectin (0.15 mg / ml) in a liquefied matrix at a density of 3 × 10 ⁶ / ml. 1 ml of EC suspension was transferred to each well of rat tail type I coated C6 wells and warmed to 37 ° C to polymerize the matrix. M199 and 20% FCS, 50 μg / ml ECGS, 100 μg
/ 3 heparin, 100 μg / ml penicillin and 100 μg / ml streptomycin for 24 hours at 37 ° C. after incubation at 37 ° C.
They were placed in T and embedded in paraffin for immunohistochemical analysis. Separately, 2 or 3
Dimensional EC cultures were homogenized in a tissue polisher and immunoblotted for survivin expression. ECs were observed in the gel during the incubation period to extend and form multicellular tubule structures as described (Sierra-Honiman et al., 1998).

Immunohistochemistry Four skin biopsies containing granular tissue or normal, non-inflamed skin by hematoxylin-eosin staining were collected from Ale New Haven Hospital. 5 μm section,
Prepared from paraffin-embedded tissues, deparaffinized in xylene and dehydrated in graded alcohol with quenching of endogenous peroxidase in 2% H ₂ O ₂ in methanol. Immunological localization of survivin was performed using 0.01M citrate buffer (p
After antigen recovery in a pressure cooker in H6.0) for 5 minutes, as previously described (Amb.
Rosini et al., 1997). The binding of the primary antibody is 3, 3, as a substrate.
3'diaminobenzidine, or separately 3-amino-9-ethylcarbazole (
AEC, vector). Control experiments were performed 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 into the EcoRI site of pEGFPc1 (Clontech, San Francisco), a plasmid encoding green fluorescent protein (GFP). pEGF
The correct orientation and reading frame of the Pc1 fusion plasmid was confirmed by DNA sequencing. Bovine aortic EC were seeded in C6-well plates at 40-50% confluence and transfected with GFP-vector or GFP-survivin by lipofectin for 6 hours at 37 ° C. After removal of the DNA-lipid mixture, EC monolayers were placed in complete growth medium at 37 ° C for 35 hours with 5 ng / ml T.
Incubated with NFα and 5 μg / ml cycloheximide for an additional 8 hours at 37 ° C. Fix cells (suspended cells and adherent cells) in 70% ethanol and
Staining with μg / ml propidium iodide, 100 μg / ml RNAse A and 0.05% Triton X-100 (pH 7.4) in PBS, GFP expressing cells were analyzed for DNA content by flow cytometry. In other experiments, GFP
Bovine ECs transfected with vector or GFP-survivin were treated with control medium or 5-10 ng / ml TNFα and 10 μg / ml cycloheximide for 8 hours at 37 ° C. Cells are collected and fluorogenic substrate Ac-DEV
D-AMC (N-acetyl-Asp-Glu-Val-Asp-aldehyde, C
Caspase-3 activity was analyzed in the presence or absence of the caspase-3 inhibitor Ac-DEVD-CHO by hydrolysis of (Pharmingen, San Diego, A.). Fluorescence emission was quantified with a spectrofluorometer at an excitation wavelength of 360 nm and an emission wavelength of 460 nm.

Example 1.1 Induction of Survivin by Mitogen Stimulation in ECs Expression of an endogenous survivin of approximately 16.5 kD in quiescent serum-depleted endothelium was consistent with previous observations in immunoassays. It was minimally detectable by blot (Figure 1A) (Ambrosini et al., 1997). EC stimulation with serum or specific mitogens VEGF or bFGF up-regulated survivin expression 8-16 fold by immunoblot (FIG. 1A). Induction of survivin by VEGF was concentration-dependent with a maximum at about 50 ng / ml (FIG. 1B).
). EC stimulation with the cytokines TNFα or IL-1 did not increase survivin expression, which was reduced below background levels in untreated cells (FIG. 1).
A). In control experiments by flow cytometry, TNFα or IL
-1 stimulated a strong upregulation of intracellular adhesion molecule-1 in EC, whereas VEGF had no effect (not shown). By Northern hybridization, the major 1.9 kb survivin message and a faint 3.4 kb survivin transcript were minimally detected in quiescent EC (FIG. 1C). VEGF
Treatment resulted in rapid upregulation of survivin RNA in ECs in response peaking 6 to 10 hours after stimulation, decreasing 24 hours post treatment, and approaching background levels.

Example 1.2 Survivin expression in 3D EC cultures Survivin was expressed at very low levels in 2D EC cultures by immunohistochemistry (Fig. 2A). In contrast, survivin was strongly expressed in EC due to the formation of three-dimensional blood vessels in the collagen / fibronectin matrix (Fig. 2B). No staining of the three-dimensional EC culture was observed with the control unbound antibody (Fig. 2C). By immunoblotting, a remarkable survivin band of about 16.5 kD was significantly induced in the EC extract of the three-dimensional blood vessels as compared to the two-dimensional EC culture medium.

Example 1.3 Expression of survivin in EC during growth in vivo In 4 of 4 cases survivin was potent in the cytoplasm of newly formed capillaries of skin granule tissue by immunohistochemistry. (Fig. 3A). Abundant survivin expression was also demonstrated in large vessel EC of granular tissue at the dermis / subcutaneous junction (FIG. 3C). In contrast, no staining of granular tissue was observed in the absence of primary antibody (Fig. 3B, D) or with a control preimmune antibody (not shown). Pre-immune I analysis by analysis of non-proliferating capillaries in non-inflamed normal skin
Minimal detectable expression of survivin was found in EC (Fig. 3E) compared to control staining with gG (Fig. 3F).

Example 1.4 Anti-apoptotic effect of survivin on EC Treatment with TNFα / cycloheximide induced EC apoptosis and development of hypodiploid group by propidium iodide staining and flow cytometry (FIG. 4A). Expression of GFP-survivin inhibited TNFα-induced apoptosis in EC and reduced the proportion of hypodiploid cells to control levels in untreated cultures (Fig. 4A). In contrast, transfection with GFP-vector alone did not affect TNFα-induced EC apoptosis (Fig. 4A).
Furthermore, GFP-survivin expression in EC strongly inhibited caspase-3 activity in TNFα-treated EC, as determined by DEVD analysis, while
The GFP-vector alone had no effect (Fig. 4B). In the control experiment,
Preincubation of TNFα-treated EC extracts with the caspase-3 inhibitor DEVD-CHO abolished DEVD hydrolysis (FIG. 4B).

Example 2 Materials and Methods Example 2.1 Effect of Ang-1 on Akt Bovine lung microvascular endothelial cells (MVEC, Vectek, Albany, NY) were treated with 10% fetal bovine serum (FBS), L-glutamine. And Dulbecco's modified Eagles medium containing antibodies (penicillin and streptomycin).
Cells (up to 12 passages) were used in experiments; cultures had a typical paving stone-like morphology and stained uniformly for von Willebrand factor as assessed by indirect immunofluorescence.

The recombinant form of Ang-1 was used for all experiments. This form of Ang-1 is Cys
It differs from the native Tie2 ligand in that it has a modified NH2-terminal sequence and mutation at 245, which makes it easier to manufacture 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 by Ang-1 was inhibited by serine 473 (Fig. 5A) in a response suppressed by the PI3 kinase inhibitor wortmannin (WM; Fig. 5B).
), Increased Akt phosphorylation at threonine 308 (not shown) and upregulated Akt kinase activity (FIG. 5B). Ang-1 stimulated Akt phosphorylation in a time-dependent manner with maximal activation occurring within 15-30 minutes and sustained phosphorylation lasting up to 2 hours (FIG. 5C). Ang-1 stimulated phosphorylation of Akt on serine 473 is antagonized by preincubating Ang-1 with soluble Tie2 receptor, but by incubation with soluble Tie1 receptor body (5D). I wasn't antagonized. Furthermore, Ang-1-induced Akt phosphorylation was partially blocked by the physiological antagonist of Ang-1, angiopoietin-2 (Ang-2; Maisonpierre et al., 1997). Interestingly, Ang-2 only weakly activated Akt in MVEC. Hence (Bill, do you need the previous sentence). Taken together, these findings indicate that Ang-1 is mediated by PI-3 through the Tie2 receptor.
We demonstrate that it stimulates Akt activation through a kinase-dependent mechanism.

Example 2.2 Effect of Ang-1 on Endothelial Cell Apoptosis Induced by Desorption from Matrix, Anoikis (Frisch et al., 1997) MVEC were placed in serum-free medium and seeded in Petri dishes for 18 hours. And underwent sufficient apoptosis, which was determined by propidium iodide staining and flow cytometry to show the appearance of a hypodiploid cell population (~ 25% vs. 2% for control).
, Adhesion culture) (FIG. 6A). Incubation of MVECs cultured with Ang-1 under these conditions resulted in 75% inhibition of apoptosis in the WM-reversed reaction (FIG. 6A). To determine whether Akt is required for Ang-1 cell protection, MVEC were infected with adenovirus β-galactosidase or activation deficient Akt (AA-Akt; Fujio et al. 1999) and the degree of apoptosis was determined. . When MVEC was transduced with AA-Akt,
The cytoprotective effect of Ang-1 on anoikis disappeared, whereas the control adenovirus encoding β-galactosidase had no effect. MVEC was β-galactosidase, HA-tagged with activation-deficient phosphate mutant Akt (A
A-Akt, Fujio et al., 1999) were infected with 100 MOI of adenovirus. After 4 hours, virus was removed and cells were allowed to recover overnight in complete medium. In preliminary experiments with β-galactosidase virus, these conditions were optimal for infecting 95% of the culture. Infected cells were placed in bacteriological dishes for apoptosis experiments or lysed in buffer for immunoblotting.

WM is also Ser-473 induced by Ang-1 in suspended endothelial cells
The above Akt phosphorylation was prevented (Fig. 6B). In summary, these data
Ang-1 has been shown to mediate endothelial cell protection through an integrin-independent PI-3-kinase / Akt-dependent pathway.

Example 2.3 Ang-1 and two known anti-apoptotic genes survivin and Bc
Possible linkage between 1-2 (Ambrosini et al., 1997 and Gerbe
r et al., 1998) MVECs treated with Ang-1 rapidly induced a time-dependent increase in survivin RNA levels (Li et al., 1998), which peaked 12 h after stimulation and up to 24 h. It persisted (Figure 7A). On the other hand, Ang-1 is M
It did not upregulate bcl-2 RNA expression in VEC (Fig. 5A).
). Consistent with the receptor-mediated response, by preincubating Ang-1 with soluble Tie-2 receptor, the Survivin RNA An in MVEC
The g-1 induction disappeared (FIG. 7B). When MVECs were transfected with the survivin-luciferase promoter construct (Li et al., 1999a), Ang-
1 up-regulates survivin transcriptional activity 3-7 fold, which is 2 of the stimulus.
It persisted until 4 hours (Fig. 7C). VEGF or Ang-1 strongly induce the expression of survivin protein in HUVEC, and its effect is
Alternatively, it disappeared by transduction with AA-Akt (FIG. 7D). Human umbilical vein endothelial cells (HUVEC) were used in these experiments due to the higher sensitivity of survivin antibodies with human survivin. HUVECs were isolated from the umbilical vein and 2
0% fetal bovine serum (FBS), 50 μg / ml EC growth supplement (ECGS, commercial preparation containing mainly acidic fibroblast growth factor), 100 μg / ml porcine heparin, 10 U / ml penicillin and 100 μg. Cultured in gelatin-coated tissue culture flasks in M199 containing 1 / ml streptomycin. Two to three individual donors were pooled at passage 1 and used up to passage 3. The culture had a typical paving stone-like morphology and stained uniformly for von Willebrand factor as assessed by indirect immunofluorescence. Identical results were obtained using MVEC.

In contrast, VEGF weakly induced bcl-2 protein expression in MVEC, whereas Ang-1 had no effect (FIG. 7D). These data show that the distinct angiogenic factors Ang-1 and VEGF are associated with PI-3 kinase /
It is demonstrated to stimulate survivin expression in endothelial cells via an Akt-dependent mechanism.

[0077] Example 2.4 induced survivin expression, a MVEC to mediate anti-apoptotic function of Ang-1, wild-type survivin (GFP-survivin) or dominant-negative Cys ⁸⁴ -> Ala survivin mutant (GFP-C84A survivin) was transfected with cDNA containing green fluorescent protein (GFP). Cytoprotection in response to apoptosis-inducing stimuli (Li et al., 1999b) was determined. The fusion of survivin and GFP does not interfere with its biological activity (Li et al., 19
99b). The survivin-GFP (Cys85-Ala) construct is a mutation in the BIR1 domain. It is targeted to the spindle, which resembles endogenous survivin, but lacks anti-apoptotic function.

Expression with Ang-1, or with GFP-survivin alone or in combination with Ang-1, was found in MVECs with hypodiploid DNA content induced by TNFα / cycloheximide or by anoikis. Suppressed appearance (Fig. 8A
And B). In contrast, transfection of MVEC with GFP-C84A survivin abolished the cytoprotective effect of Ang-1 on TNFα / cycloheximide- or anoikis-induced cell death (FIGS. 8A and B).
These data identify survivin as a novel PI-3 kinase, an Akt-dependent target gene for Ang-1, demonstrating that survivin is required for the anti-apoptotic effects of Ang-1.

Example 3 Materials and Methods Survivin-Mediated Angiogenesis In Vivo in Animal Models Plasmid DNA Survivin-encoding DNA was associated with mammalian expression vectors, including cytomegalovirus vectors (Takeshita et al., 1996). The biological activity of survivin obtained from cells transfected with this construct ph survivin is confirmed before performing arterial gene transfer. The plasmid pGSVLacZ, containing a nuclear-targeted β-galactosidase sequence linked to the simian virus 40 early promoter, is used for control transfection experiments (Ta.
Keshita et al., 1996).

Animal Models Animal studies are performed in a hindpaw ischemia animal model (Takeshita et al., 199).
6). Rabbits weighing 4 to 4.5 kg were premedicated with xylazine (2.5 mg / kg) followed by ketamine (50 mg / kg) and acepromazine (0.8 mg).
/ Kg) for anesthesia. A longitudinal incision is made on one limb, extending down from the inguinal ligament and pointing just proximal to the patella. With this incision, the femoral artery is dissected free along its entire length using a surgical magnifier; all branches of the artery are also dissected free. After dissection of the popliteal and saphenous arteries, the iliac artery and all of the above arteries are connected. Finally, the femoral artery is completely cut from its proximal origin as a branch of the iliac artery to the distant point where it bifurcates into the saphenous and popliteal arteries. Once the femoral artery is severed, a thrombotic occlusion of the external iliac artery extends back from the common popliteal artery to its origin. Consequently, the blood supply to the distal limb depends on the collateral arteries, which can originate from the internal iliac arteries. By this operation procedure, severe limb ischemia (Takashita)
Et al., 1996). Postoperative anesthesia (levorphanol tartrate 60 mg / kg
) Is given subcutaneously if necessary, and a prophylactic antibiotic (enrofloxacin 2.5
(mg / kg) is subcutaneously administered for 5 days.

Percutaneous Arterial Gene Transfer A 10-day interval between surgery and gene transfer time allows postoperative recovery and development of endogenous collateral vessels in rabbits. Baseline angiography is performed on day 10 (day 0) after surgery. The internal iliac arteries of the ischemic limb of many animals were cut through a 2 mm hydrogel-coated balloon catheter (Slider®, Boston Scientific,
Transfect transdermally with ph Survivin using Watertown Massachusetts. The angioplasty balloon was replaced with 5 Fr. Advance the deflated balloon through a Teflon sheet (Boston Scientific); apply the plasmid DNA solution from a conventional pipette to the 20 μm layer of hydrogel coating the outer surface of the inflated balloon; and Finally, deflate the balloon; prepare it by deflating it into a protective sheet and inflating the balloon again to prevent circulation of blood back into the sheet (and onto the coated balloon) after introduction into the circulation ( In vitro). The sheet and angioplasty catheter were then introduced through the right carotid artery and a 0.014 inch guide wire (HiTorqu) under fluoroscopic guidance.
e Floppy II®; Advanced Cardiovascular System, Temecula, Calif.) is used to advance to the lower abdominal aorta.
Then, a balloon catheter is advanced to the internal iliac artery of the ischemic limb, inflated at 4 to 6 atmospheres for 1 minute, and air is extracted. Using the same protocol, plasmid p
The internal iliac arteries of control animals carrying GSVLacZ are transfected. Heparin is not administered during transfection or angioplasty.

RT-PCR gene expression was performed by RT-PCR in rabbits in which the iliac arteries were transfected using a hydrogel balloon catheter as described above.
Evaluate with. Transfected arterial segments are obtained 7, 14, 21 and 30 days after transfection. Remote tissues such as brain, heart, liver, lung, spleen, and testis are also post-transfection for analysis of survivin mRNA?
Take out in 7 days. Total cellular RNA is isolated using TRI reagent (Molecular Research Center, Cincinnati, Ohio) according to the manufacturer's instructions.
The extracted DNA is treated with DNase I (0.5 μl, 10 U / μl, Rnase-free, Message Clean Kit; Gen Hunter, Boston, Mass.) For 30 minutes at 37 ° C. to eliminate DNA contamination. The yield of extracted RNA is determined spectrophotometrically by UV absorption at 260 nm. To check that the RNA is not degraded and the ribosomal bands are intact, each RNA sample is subjected to 1% nondenaturing small agarose gel electrophoresis. Reactions containing deoxynucleotides, RNasin (Promega, Madison, Wis.), Random hexanucleotide primers (Promega), and Moloney murine leukemia virus reverse transcriptase (Gibco BRL, Gaithersburg, MD) using each RNA sample To create cDNA. The reaction is terminated by incubating the reaction at 42 ° C for 1 hour and then at 95 ° C for 5 minutes to terminate the reaction. PCR amplification is carried out for 30 cycles at 94 ° C for 20 seconds and ends at 72 ° C for 5 minutes. A region or fragment of survivin is amplified using an oligonucleotide primer selected from a particular region of the nucleic acid encoding survivin. RT-PCR products are analyzed by 2% agarose gel electrophoresis.

Transfection Efficiency To evaluate the efficiency of arterial gene transfer in vivo in an animal model, LacZ
-Tf arteries were collected on day 5 and the β-galactosidase activity was determined as previously described (Takeshita et al. 1996) 5-bromo-4-chloro-3-indolyl-β-D-galactoside chromogen. (X-Gal; Sigma Chemical Company, St. Louis, MO). X
-After staining with Gal solution, the tissue was embedded in paraffin, sectioned, and hematoxylin-
Counterstain with eosin. Nuclear-localized β-galactosidase expression of the plasmid pGSVLacZ could not result from endogenous β-galactosidase activity; thus histochemical identification of β-galactosidase in the cell nucleus was successful for successful gene transfer and gene expression. Interpreted as evidence. Cytoplasmic or other staining is considered non-specific for purposes of the present study.

Evaluation of angiogenesis in the ischemic limb Development of collateral blood vessels in the ischemic limb was measured by calf blood pressure measurement and internal iliac arteries immediately before transfection (day 0) and 30 days post transfection. Continuous evaluation by contrast. In addition to these two parameters, limb blood flow and capillary density in the ischemic limb muscle are evaluated 30 days after transfection.

Calf Blood Pressure Ratio Calf blood pressure was measured immediately before transfection using a Doppler flowmeter (Model 1059, Parks Medical Electronics, Aloha, Oreg.).
On both day 0) and day 30, measurements are taken on both hind limbs. In each case, shave the hind legs,
Cleaned, posterior tibial artery pulses are identified using a Doppler probe and systolic blood pressure in both limbs is determined using standard techniques (Takeshita et al., 1996). Calf blood pressure ratio is defined as the ratio of ischemic limb systolic pressure to normal limb systolic pressure for each rabbit.

Selective Iliac Angiography Selective internal iliac angiography is performed on day 0 (immediately before transfection) and again 30 days after transfection (Takeshita et al., 1).
996). 3 Fr. Infusion Catheter (Tracker-18, Target Therapois, San Jose, CA) was introduced into the common carotid artery through a small phlebotomy and a 0.014 inch guide wire (Hi-torque f) was used.
lopyII) is used to advance into the internal iliac artery of the ischemic limb under fluoroscopic guidance.
The tip of the catheter is placed in the internal iliac artery at the level of the interspace between the 7th lumbar spine and the 1st sacrum. After intraarterial infusion of 0.26 mg of nitroglycerin, 5 ml of non-ionic contrast agent (Isovue-370, Squibb Diagnostics, Pittsburgh, NJ) was programmed to reproducibly deliver a flow rate of 1 ml / sec. Inject using an automated angiographic injector (Medlad, Pittsburgh, PA). After that, consecutive angiographic images (1 per second
Times, 10 seconds) on a 105 mm spot photograph. Morphometric angiographic analysis of collateral vessel development is performed using a grid overlay consisting of 2.5 mm circles arranged in rows spaced apart by 5 mm. The overlay is applied to the 4 second angiogram recorded at the level of the medial thigh. The number of contrast-opaque arteries intersecting on the circles, as well as the total number of circles encompassing the medial femoral region, are measured in a single-blind fashion. The angiographic score is calculated for each photograph as the ratio of intersecting opaque arteries divided by the total number of circles in the basal region of the ischemic thigh.

Blood Flow Measurement A 0.018 inch guide wire with a 12 MHz piezoelectric transducer at the distal tip (
FloMap: Cardiometrics, Mountain View, California)
Is used to measure blood flow velocity (Takeshita et al., 1996). Doppler wire records real-time spectral analysis of Doppler signals,
From this, the average peak velocity (APV, time average of the instantaneous peak velocity waveform) was calculated and shown online. Wire placed in common iliac artery origin, 3Fr
． It is advanced through a drip catheter to the proximal segment of the internal iliac artery that feeds the ischemic limb. A steady speed of 2 minutes is required before recording a static APV. Maximum AP
V through papaverine (Sigma Chemical Company), 2 via infusion catheter
Record after bolus injection of mg / 0.4 ml saline. Then, the Doppler wire is pulled out from the internal iliac artery and advanced again to the iliac artery of the normal limb; 3Fr. The distal tip of the infusion catheter is repositioned at the origin of the common iliac artery. Again, blood flow velocity is recorded after rest and papaverine infusion.

After all Doppler measurements, 3 Fr. The drip 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 tube diameters of the internal iliac artery in the ischemic limb and the external artery of the normal limb were determined as described by an automatic edge detection system (Quantum 20001; QCS, An.
n Arbor Michigan). Photos selected for analysis are scanned using a high resolution video camera, the signal produced by the video camera is digitized, and presented on a video monitor (laser scan; Imagecom, Santa Clara, Calif.). The centerline is traced manually for the 10 mm segment starting shortly at the tip of the Doppler wire.
The appearance is then automatically detected based on the weighted sum of the first and second derivatives applied to the digitized luminance information. The vessel diameter is then measured at the site of the Doppler sample volume (5 mm distal from the wire tip). The cross-sectional area is calculated by estimating the circular lumen.

The Doppler-derived flow rate is Q _D = (πd ² /4)(0.5XAPV), where Q _D = Doppler-derived time-averaged flow, d = vessel diameter, and APV = time of spectral peak velocity. Calculate as the average). The average velocity is estimated as 0.5XAPV by estimating the time average parabolic velocity profile across the blood vessel.
Angiographic lumen diameter measurements from an angiogram recorded just prior to Doppler recording are used for static and maximum flow rate calculations.

The Effect of Survivin Gene Transfer on the Anatomical Evidence of Capillary-to-Muscular Fiber Collateral Arteriogenesis was further examined by identifying the capillaries in light microscopic sections taken from the ischemic hindlimb (
Takeshita et al., 1996). Tissue specimens are obtained from ischemic hind limbs at the time of death (30 days after transfection) as transverse sections. Muscle samples are embedded in optimal cutting temperature compounds (Miles, Elkhart, Ind.) And immediately frozen in liquid nitrogen. Multiple frozen sections (5 μm thick) are then cut from each specimen on a cryostat (Miles), the muscle fibers are laid sideways, after which two sections are placed on glass slides. Tissue sections are stained for alkaline phosphatase using the indoxyl-tetrazolium method to detect capillary endothelial cells (Takeshita et al., 1996), then counterstained with eosin. Assess capillaries identified at necropsy for muscle fibers to ensure that analysis of capillary density is not underestimated due to muscle atrophy or underestimated due to interstitial edema Randomly choose a total of 20 different fields and count the number of capillaries and muscle fibers with a 20 × objective to determine the capillary to muscle fiber ratio.

Assessment of tissue sites distal to the ischemic limb 14 rabbits are randomly selected from those undergoing survivin arterial gene transfer. Tissue sections are collected from gonadal, liver, heart, lung, brain and contralateral (non-transfected) lower limb skeletal muscle and examined by light microscopy for evidence of neovascularization and evidence of immune-related inflammatory cell infiltration. ..

Morphometric analysis of gene transfer sites Representative tissue sections are collected on day 30 from gene transfer sites in a large number of randomly selected rabbits. These included several transfected with ph survivin and several transfected with LacZ. In each case, the gene transfer site was identified by the fact that the gene transfer is carried out in the origin of the internal iliac artery; therefore, an arterial segment about 5 mm long is located just at the end of the common iliac bifurcation. Remove from the origin of this artery. Sections were stained with hematoxylin and eosin and then morphometrically evaluated for intimal and medial thickness by light microscopy, which gives the ratio of intimal to media (Takeshit).
a et al., 1996).

Example 3.1 Survivin-Mediated Angiogenesis In Vivo Plasmid DNA The plasmid phSurvivin consists of a eukaryotic pUC118 expression vector into which a cDNA encoding survivin has been inserted. A 763 base pair cytomegalovirus promoter / enhancer is used to drive expression of survivin. The pUC118 vector contains the polyadenylation sequence of SV40, the E. coli origin of replication, and the β-lactamase gene required for ampicillin resistance. Plasmids are prepared from a culture of E. coli transformed with phSurvivin, purified using a Qiagen chip 2500 column, precipitated with isopropanol, washed with 70% ethanol and dried in a SpeedVac. The purified plasmid was redissolved in sterile saline and
Stored in vials and pooled for quality control analysis (1.75-1
． Absorbance at wavelengths of 260 nm and 280 nm showing a ratio between 85; Gel coagulation assay with amoebic cell lysates of horseshoe crab to reveal that the level of bacterial endotoxin is less than 5 endotoxin units / kg body weight [Bio -Whittaker]; bacterial culture; Southern blot for contamination levels of E. coli genomic DNA; and ethidium bromide staining after agarose gel electrophoresis to confirm> 90% of the nucleic acid is in closed circular supercoiled form ). To confirm the identity of the prepared plasmids, the survivin coding region from each pool batch is re-sequenced (App
Lied Biosystem 373A).

Percutaneous Arterial Gene Transfer Arterial gene transfer is performed on patients with ischemic legs. Arterial gene transfer is performed using a hydrogel-coated balloon angioplasty catheter (Boston Scientific). Done. Using a sterile pipette, 2000 g of plasmid DNA is applied to the outer hydrogel coating of the inflated angioplasty balloon at 10.3 μg / μl in 194.2 μl of sterile saline. Deflate the balloon, 2280mmH
It is placed back into the protective sheath which has been reinflated to g and advanced under fluoroscope guidance to the gene transfer site with the sheath covering the 45.7 mm guidewire. The balloon is then deflated, the sheath replaced, and the balloon reinflated at nominal pressure for 4-5 minutes. The balloon is deflated to remove all catheters and wires, and a final angiogram is recorded to ensure satisfactory patency of the site.

Intravascular ultrasound may impair transfection efficiency at the intended site (distal popliteal artery) atherosclerotic plaques (Isner et al., 19).
96) just before gene transfer to show the absence of Ultrasound diagnosis is repeated 4 and 12 weeks post gene transfer to ensure the absence of neointimal thickening resulting from inflation of the hydrogel-coated angioplasty balloon catheter.

Digitally processed differential angiography is performed 4 weeks after gene therapy and magnetic resonance angiography is performed 4 and 12 weeks after gene therapy. Both of these are done to detect angiogenesis promoted by gene transfer.

Example 4 Preservation of vascular homeostasis during inflammation, immune response and transplant adaptation
It relies on the ability of endothelial cells (EC) to continuously counteract a cell's suicide program (ie, apoptosis) (Karsan et al., 1996). This process involves the continuous activation of intracellular cysteine proteases (ie, caspases) after mitochondrial damage, initiated by cell surface death receptor binding or by cytoplasmic assembly of cell death initiation factors (ie, apoptosomes). With cascade (Hengartner, 2000). Inhibiting EC apoptosis is also due to the large number of receptor-ligand interactions on the EC surface.
Proliferation, migration and re-repair of C are stimulated and a new network of blood vessels is an essential prerequisite for angiogenesis (Risau, 1997). In this context, vascular endothelial growth factor (VEGF) (Alon et al., 1995; Yuan et al., 1996) or the angiopoietin-1 (Ang-1) receptor Tie-2 (Lin et al., 199).
Antibody or adenovirus targeting of key angiogenic regulators, including 8), resulted in regression of the vascular network with morphological and biochemical features of EC apoptosis. Survival signals mediated by adhesion molecules (ie, integrins), intrusions (Ruegg et al., 1998; Isik et al., 1998), and activation of the phosphoinositide 3 / Atk pathway (Fujio et al., 1999; Gerber et al.,
In addition to 1998b), angiogenesis is accompanied by de novo expression in endothelial cells of a heterogeneous group of anti-apoptotic "protective genes", some of which are induced via signal transduction of NF-6β (Stehlik et al., 1998). Related (Bach et al.,
1997). Specifically, stimulation of EC angiogenesis by VEGF or Ang-1 is associated with anti-apoptotic upregulation of bcl-2 and A1 molecules (Gerber et al., 1998a; Nor et al., 1999), apoptosis inhibitory factor (IAP) protein. Expression (Devereaux et al., 1999), and expression of survivin and XIAP (O'Connor et al., 2000a; Tran et al., 1999; Papapetropoulos et al., 2000).

In the examples below, an antisense targeting method was used to confirm the relative contribution of survivin to the anti-apoptotic function of VEGF in endothelial cells.

Materials and Methods EC Culture Human umbilical vein ECs were cultured in 20% fetal calf serum (FCS), 50 μg / ml endothelial cell growth supplement (EC) as described by O'Connor et al. (2000).
GS), 100 μg / ml heparin, 100 μg / ml penicillin and 100
M199 medium containing μg / ml streptomycin (all of these are Lif
e Technologies (obtained from Grand Island, NY)) at 37 ° C. under 5% CO ₂ . Subconfluent ECs were rested by culturing in M199 + 0.1% FCS for 18 hours.
Cells were detached with 0.05% trypsin / 0.02% EDTA, seeded in C6 well plates (Costar Corp., New Bedford, MA), grown to 70% confluence, and passage 2 and passage. Used between generation 3

Antisense Gene Targeting Resting EC monolayers were treated with 50 ng / ml recombinant VEGF (Collaborative Biomedical Pr) in M199 + 0.1% FCS.
incubation at 37 ° C. for 24 hours with products, Bedford, Mass.). When the incubation is complete, wash the EC and trypsin /
Collected by EDTA and 0.5% Triton X-100, 0.5% NP-4
Dissolved in 0, 0.05 M Tris-HCl, 0.15 M NaCl and protease inhibitors. A protein-normalized portion of the cell extract was electrophoresed on an SDS polyacrylamide gradient gel to yield a nylon membrane (Millip).
ore Corp. 1A for 1 hour and a rabbit antibody to survivin and a mouse monoclonal antibody to bcl-2 (Transduct).
immunoblotting with 2 μg / ml of Ion Laboratories, CA) followed by chemiluminescence (Amersham, Arlington Height).
ts, IL) and autoradiography. The samples were subsequently analyzed by Western blotting using a mouse antibody against β-actin to confirm equal protein loading. E mediated by VEGF
To reveal the contribution of survivin to C protection, a 2'-Omethoxyethyl chimeric phosphorothioate oligonucleotide was utilized. Sequence 5'-T
Survivin antisense oligonucleotides with GTGCTATTCTGTGAATT-3 '(SEQ ID NO: 1) were used in T24 bladder epithelial cancer cells and HeLa.
It was previously characterized for its ability to suppress endogenous survivin mRNA expression in epithelial cancer cells (Li et al., 1996b). Sequence 5'-TAAGC
A scrambled oligonucleotide with TGTTCTATGTGTTT-3 '(SEQ ID NO: 2) was used as a control, which was also characterized in previous cell culture assays (Li et al., 1999b). Various oligonucleotides with a uniform phosphorothioate linkage were synthesized. The underlined nucleoside corresponds to 2'-O-methoxyethyl nucleoside. Platelet endothelial cell adhesion molecule-
1 (PECAM-1, CD31), lymphocyte function-related molecule-3 (LFA-3, C
D58) and antisense oligonucleotides against intercellular adhesion molecule-1 (ICAM-1, CD54) were synthesized as described above and these were characterized in previous studies (Baker et al., 1997). For transfection experiments, control scrambled oligonucleotides or various antisense oligonucleotides were added at increasing concentrations (50-500 nM).
1 ml according to manufacturer's instructions (Life Technologies, MD)
Of OPTI-MEM and 6 μl of lipofectin and incubated with serum starved EC for 8 hours. 1 more transfection medium
Replacement with M199 + 0.1% FCS for 8 hours, followed by VEGF stimulation for 24 hours. Transfection efficiency was monitored by fluorescence microscopy using FITC-conjugated oligonucleotides and was always> 85%. To demonstrate the effect of antisense targeting on survivin mRNA expression in proliferating endothelial cells, ECs were transfected with control oligonucleotides or survivin antisense oligonucleotides and incubated at 37 ° C for 24 hours. Later collected and total RNA was quiagen
Extracted using Rneasy reagent according to the manufacturer's instructions. Sample 1
%% agarose-formaldehyde gel, transferred to a Hybond nylon membrane, hybridized with ³² P random prime labeled survivin cDNA, and visualization of radioactive bands was performed by autoradiography. Northern blots were reprobed with random prime ³² P-labeled human G3PDH cDNA to confirm equal loading of various RNA samples.

EC Apoptosis Determination ECs were transfected with control oligonucleotides or various antisense oligonucleotides at increasing concentrations to 50 ng / m 2.
Stimulated with 1 VEGF for 16 hours at 37 ° C. and in the presence of 25 μM C-6 ceramide or TNFα (10 ng / ml, Endogen, Woburn,
MA) + cycloheximide (10 μg / ml, Sigma) in the presence of
Incubated at 37 ° C for an additional 12 hours. At the end of the incubation period, EC (floating cells + adherent cells) were collected, fixed with 70% ethanol and fixed at 70 μg / ml propidium iodide + 100 μg / ml RNase A as described (O'Connor et al., 2000). And 0.05% Triton X-10
The cells were stained with phosphate buffered saline containing 0 (pH 7.4) and analyzed for DNA content by flow cytometry. In another experiment, transfected EC cells stimulated with VEGF and incubated with C-6 ceramide for 12 hours at 37 ° C were collected and collected in PBS (pH 7.4).
) And fixed with 4% paraformaldehyde containing 0.25% Triton X-100 for 10 minutes at 22 ° C. The cell nuclei were treated with 6.5 μg / ml of 4,
6-diamidino-2-phenylindole (DAPI, Sigma), 16% polyvinyl alcohol (Air Products and Chemical)
s, Allentown, PA) and 40% glycerol. Cells were independently scored for morphological signs of apoptosis (chromatin condensation, DNA fragmentation) using Zeiss fluorescence microscopy.

Activation of Caspases Transfected ECs were transfected with 50 ng / ml VEGF as described above.
The solubilized cell extract was collected after stimulating with and following incubation with 25 μg / ml C-6 ceramide and the solubilized cell extract was collected with the fluorogenic substrate Ac-DEVD-AM.
C (N-acetyl-Asp-Glu-Val-Asp-aldehyde, Pharm
Ingen, San Diego) was assayed for caspase-3-dependent hydrolysis. Fluorescence emission was quantified on a spectrofluorometer using an excitation wavelength of 360 nm and emission of 460 nm. For biochemical markers of caspase activation, transfection EC treated with VEGF + ceramide was 0.25%.
Triton X-100, 10 mM KCl, 1.5 mM MgCl2, 1 mM E
Dissolved in DTA, 1 mM DTT, 20 mM HEPES + protease inhibitor. Protein-normalized aliquots of various cell extracts were separated by SDS gel electrophoresis and subjected to nylon membrane (Millipore Corp.).
Rabbit antibody to Caspase 3 (Transduction
Laboratories) 1: 5000 dilution or mouse antibody to poly ADP ribose polymerase (PARP, Pharmingen, San.
Immunoblot with 1: 1000 dilution of Diego, followed by chemiluminescence (A
Mersham, Arlington Heights, IL).

EC migration Migration assays were performed using a Boyden chamber (Neuropr).
ob; Morales-Ruiz et al., 2000). Briefly, quiescent ECs were transfected with control oligonucleotides or survivin antisense oligonucleotides, stimulated with VEGF, and
． Peeled off using 05% trypsin and 0.53 mM EDTA. 230
00 cells were suspended in M199 medium containing 0.1% BSA and added to the lower chamber. Polycarbonate filter (8μm diameter) 100μg /
Coated with ml of type I collagen. The upper half of the chamber was attached and the chamber was incubated in an inverted position for 2 hours at 37 ° C. VE
GF or D-erythro-sphingosine-1-phosphate (SPP-1, C
albiochem) was added separately to the upper chamber increasing its concentration (1-500 ng / ml) and incubated at 37 ° C. for a further 5 hours.
At the end of incubation, cells were fixed with 70% ethanol to remove non-migrated EC present on the upper surface of the filter. The migrated cells were stained with Giemsa and counted at 400x magnification in 3 random fields per well (Mo
Rales-Ruiz et al., 2000). Each experiment was performed in triplicate,
Expressed as total cells counted per well.

Three-dimensional Capillary Formation Monolayers of quiescent EC in C6 well plates (80% confluence)
Were transfected with 500 nM control oligonucleotides or survivin antisense oligonucleotides. After incubation for 8 hours, the transfection medium was replaced with M199 medium containing 0.1% FCS at 37 ° C. for a further 18 hours. Rat type I collagen (3 mg / ml, Becton Dickinson, Bedford, MA)
Were kept on ice neutralized with sterile 1 M NaOH in 1/10 volume of 10 × DMEM. The suspended EC was added to the collagen suspension to a final concentration of 1 × 10 ⁶ cells / ml collagen. Ten drops of EC-collagen mixture (0.1 ml each) were added to a 35 mm plate. The plate was placed in a 37 ° C humidified incubator to gel the EC-collagen mixture for 10 minutes, after which
20% FCS, 50 μg / ml ECGS, 100 μg / ml heparin, 10
3 ml of M199 medium containing 0 μg / ml penicillin and 100 μg / ml streptomycin was added to each plate. ECs were allowed to form capillary-like blood vessels by incubation for 24 hours in the presence of 16 nM phorbol myristate acetate (PMA, Sigma). After an additional 24 hours of incubation, ECs were placed in phosphate buffered saline (PBS, pH 7.2).
Washed three times with fresh M in the presence or absence of 50 ng / ml VEGF.
199 growth medium was supplemented. Description (Papapetropoulos et al., 199
As in 9), the cultures were examined by phase contrast microscopy for the presence of capillary-like blood vessels when incubated at 37 ° C. for a further 48 hours.

Example 4.1 Inhibition of VEGF-induced Survivin Expression in ECs by Antisense Targeting Previous studies have shown that 2'-Omethoxyethyl chimeric phosphorothioate antisense oligonucleotides of survivin (5'- TGTGCTATTTCG
TGAATT-3 '; SEQ ID NO: 1) was shown to inhibit survivin mRNA and protein expression in HeLa and T24 cancer cell lines (Li et al., 1999b). Consistent with these observations, Northern blotting (FIG. 9A) suppressed survivin mRNA expression in proliferating ECs in a dose-dependent manner by increasing the concentration of survivin antisense oligonucleotides. . In contrast, a control scrambled oligonucleotide (5'-TAAGCTGTTCTATGTGTT-3 '
The compared concentrations of SEQ ID NO: 2) did not reduce survivin mRNA levels in ECs (FIG. 9A). In parallel experiments, stimulation with VEGF resulted in approximately 4-fold increased expression of survivin in quiescent endothelial cells (Fig. 9).
B). This is based on previous observations (O'Connor et al., 2000; Tran et al., 1
999; Papapetropoulos et al., 2000). EC
Was pretreated with antisense to survivin at increasing concentrations, but not with the control oligonucleotide, and Western blotting suppressed the induction of survivin by VEGF in a dose-dependent manner (FIG. 9B). In contrast, transfection with control oligonucleotides or survivin antisense oligonucleotides did not reduce anti-apoptotic bcl-2 expression in endothelial cells by Western blotting (Gerber et al., 1998; Nor). Et al., 1999) (Fig. 9)
C).

Example 4.2 Antisense targeting of survivin suppresses the anti-apoptotic function of VEGF in ECs DAPI nuclear staining by chromatin condensation and DNA fragmentation by exposing quiescent ECs to C-6 ceramide. Apoptosis was induced as revealed by (Fig. 10A, B). Addition of VEGF attenuated EC apoptosis induced by ceramide and restored normal nuclear morphology (FIGS. 10A, B).
. Under these experimental conditions, transfection of EC with survivin antisense oligonucleotides completely reversed the anti-apoptotic function of VEGF against ceramide-induced apoptosis, whereas the control oligonucleotides. Had no effect (FIGS. 10A, B). Similarly, treatment with ceramide or the combination of TNFα + cycloheximide is revealed by propidium iodide staining and flow cytometry by the appearance of cell fractions with hypodiploid (ie, sub-G1) DNA content. Thus, there was an approximately 7-fold increase in EC apoptosis (Fig. 10C, D). Addition of VEGF counteracted both ceramide-induced and TNFα-induced apoptosis in ECs by about 45% (Fig. 10C, D). However, consistent with the data presented above, transfection of EC with survivin antisense, but not control oligonucleotides, suppressed VEGF protection against both cell death-induced stimuli, and reduced EC apoptosis by VEGF. Was restored to the level observed in the absence of (Fig. 10C, D). Next, it was determined whether suppression of VEGF cytoprotection by survivin targeting was associated with biochemical features of apoptosis in EC. Treatment of quiescent EC with ceramide resulted in increased caspase as measured by hydrolysis of the fluorogenic substrate DEVD-AMC and in a reaction that was completely suppressed by the caspase-3 inhibitor DEVD-CHO. A catalytic activity of −3 was produced (FIG. 11A).
. This results in proteolytic cleavage of the pro-form caspase-3 of approximately 32 kDa and generation of active subunits of approximately 19 kDa and approximately 17 kDa (FIG. 11B).
(Salvesen et al., 1997), and cleavage of the caspase substrate poly ADP ribose polymerase (PARP) of approximately 115 kDa into an apoptotic fragment of approximately 85 kDa (FIG. 11C). Under these experimental conditions,
By adding VEGF, the ceramide-induced caspase-3 activity was reduced and the active caspase-3 subunit of approximately 17 kDa and approximately 8 kDa.
The production of the 5 kDa PARP fragment was almost completely inhibited (Fig. 11A-C).
). In contrast, transfection of ECs with antisense to survivin, but not control oligonucleotides, restored production of active caspase-3 and apoptotic fragments of PARP of approximately 17 kDa (FIGS. 11A-C).

Example 4.3 Antisense Targeting Specificity of Survivin Next, it was elucidated whether antisense targeting of survivin affects EC viability upon VEGF stimulation. E with 0% serum over 24 hours
By incubating C, D compared to continuously growing cultures
Hydrolysis of EDV results in increased caspase-3 activity (Figure 12A).
, And apoptotic cell fraction with low diploid DNA content appeared by propidium iodide staining and flow cytometry (FIG. 12B). By adding ceramide to these cells, caspase-3 activity, and hypodiploid DN
The production of EC with A content was further increased (Fig. 12A, B). But VE
In the absence of GF, transfection of EC with control oligonucleotides or survivin antisense oligonucleotides did not further enhance caspase-3 activity or apoptotic cell production in the presence or absence of ceramide ( 12A, B).

In other experiments, transfection of EC with antisense oligonucleotides against PECAM-1 (CD31), LFA-3 (CD58) or ICAM-1 (CD54) was performed as previously described (Baker et al., 1997). Thus, Northern blotting resulted in concentration-dependent suppression of these various targeted mRNAs. However, expression of these various antisense oligonucleotides when analyzed for nuclear morphology by DAPI staining revealed
It did not significantly reduce the anti-apoptotic function of VEGF against ceramide-induced EC death (Fig. 13). In contrast, consistent with the data presented above, transfection of EC with survivin antisense but not control oligonucleotide resulted in V.sub.1 in ceramide-treated cultures.
It blocked the cytoprotective effect of EGF (Fig. 13).

Example 4.4 Role of Survivin in VEGF-induced EC Migration and Re-repair Other VEGF-induced angiogenic responses (ie EC migration and stabilization of the three-dimensional vascular network (Risau) Et al., 1997)), the potential role of survivin targeting was next investigated. First, stimulation with VEGF or SPP-1 resulted in EC chemotaxis and migration in a specific and concentration-dependent manner, consistent with previous observations (Morales-Ruiz et al., 2000) (Fig. 15).
Transfection of VEGF-stimulated EC with control oligonucleotides or inhibitory concentrations of survivin antisense oligonucleotides
It did not increase EC migration in response to GF or SPP-1 (FIG. 15). In another series of experiments, the effect of antisense survivin targeting on VEGF-induced angiogenesis during the non-proliferative re-repair phase was investigated. Addition of VEGF to PMA-stimulated and control oligonucleotide-transfected EC-collagen gels assisted in the generation of viable three-dimensional capillary-like structures, which were incubated at 37 ° C for 3 days. Persisted throughout the culture (Fig. 15)
(Ilan et al., 1998). In contrast, in the absence of PMA, viable capillaries were not formed (not shown), and disruption of VEGF resulted in earlier observations (Il.
Consistent with An et al., 1998), rapid retraction of the three-dimensional vascular network occurred in 3 days of culture (FIG. 15). Under these experimental conditions, transfection of EC with survivin antisense oligonucleotides completely reversed the protective effect of VEGF on the formation and maintenance of capillaries, and in three days of culture, the three-dimensional vascular network. It resulted in complete regression (Fig. 15).

Although the present invention has been described in detail with reference to the above examples, 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 appended claims. All cited patents, patent applications and publications referred to in this application are incorporated herein by reference in their entirety.

[0111]     (References)   The following references are incorporated herein by reference in their entireties. Ackermann et al., J Biol Chem 1999, 274: 11245-11252. Adams et al., Science 1998, 281: 1322-1326 Alon et al., Nat Med 1995, 1: 1024-1028 Altieri et al., Lab Invest 1999, 79: 1327-1333 Ambrosini et al., Nat Med 1997, 3: 917-921 Bach et al., Immunol Today 1997, 18: 483-486. Baker et al., J Biol Chem 1997, 272: 11994-12000. Benjamin et al., Proc Natl Acad Sci U S A 1997, 94: 8761- 8766 Bjorkerud S et al., Am J Pathol 1996, 149: 367-380. Blaese et al., Science 1995, 270: 475480 Brooks et al., Cell 1998, 92: 391-400 Carmeliet, Nat Med 2000, 6: 389-395 Chen et al., J Neurosci 1998, 18: 4914-4928 Chen et al., Neoplasia 2000, 2: 236-242 Claesson-Welsh et al., Proc Natl Acad Sci U S A 1998, 95: 5579-5583 Davis et al., Cell 1996, 87: 1161-9 De Luca et al., J. Biol. Chem. 1994, 269: 19193-19196 Deveraux et al., Genes Dev 1999, 13: 239-252 Duckett et al., EMBO J 1996, 15: 2685-2694 Folkman et al., J. Biol. Chem. 1992, 267 (16), 1093110934 Fraser et al., Curr Biol 1999, 9: 292-301 Frisch et al., Curr Opin Cell Biol 1997, 9: 701-6 Fujio et al., J. Biol. Chem. 1999, 274: 16349-54 Gerber et al., J. Biol. Chem. 1998a, 273: 13313-13316 Gerber et al., J. Biol. Chem. 1998b, 273: 30336-30343 Hanahan et al., Cell 1996, 86: 353-364 Hanahan, Science 1997, 277: 48-50 Hay et al., Cell 1995, 83: 1253-1262 Hengartner, Nature 2000, 407: 770-776 Ilan et al., J Cell Sci 1998, 111: 3621-3631 Isik et al., J Cell Physiol 1998, 175: 149-155 Isner et al., Lancet 1996, 348: 370 Karsan et al., J Atheroscler Thromb 1996, 3: 75-80 Kobayashi et al., Proc Natl Acad Sci U S A 1999, 96: 1457-1462 Koblizek et al., Curr. Biol. 1998, 8: 529-532. Kontos et al., Mol Cell Biol 1998, 18: 4131-40. Ledley et al., J. Pediatrics 1987, 110: 1 Ledley et al., Proc. Natl. Acad. Sci. 1987, 84: 53355339 Li et al., Nature 1998, 396: 580-4 Li et al., Biochem. J. 1999a, 344: 305-11. Li et al., Nat Cell Biolog 1999b, 1: 461-466 Lin et al., Proc Natl Acad Sci U S A 1998, 95: 8829-8834 Lim et al., Proc. Natl. Acad. Sci. USA 1989, 86: 88928896 Liston, P. et al., Nature 1996, 379: 349-353 Maisonpierre et al., Science 1997, 277: 55-60 Milligan et al., J. Med. Chem. 1993, 36 (14): 19231937 Morales-Ruiz et al., Circ Res 2000, 86: 892-896 Morgan et al., Science 1987, 237: 14761479 Moullier et al., Nature Genetics 1993, 4: 154 Nicolau et al., Proc. Natl. Acad. Sci. U.S.A. 1983, 80: 1068 Nor et al., Am J Pathol 1999, 154: 375-384. O'Connor et al., Am J Pathol 2000a, 156: 393-398 O'Connor et al., Proc Natl Acad Sci USA 2000b, 97: 13103-13107 Olie et al., Cancer Res 2000, 60: 2805-2809 Olivetti et al., J Mol Cell Cardiol 1996, 28: 2005-2016 Olivetti et al., N Engl J Med 1997, 336: 1131-1141 O'Reilly et al., Nat Med 1996, 2: 689-92 Papapetropoulos et al., Lab Invest 1999, 79: 213-23. Papapetropoulos et al., J Biol Chem 2000, 275: 9102-9105 Raiasubramanian et al., ASAIO Journal 1994, 40 (3): M584-9 Reed, J Clin Oncol 1999, 17: 2941-2953 Risau, Nature 1997, 386: 671-674 Rosenberg et al., Science 1988, 242: 15751578 Rothe, M. et al., Cell 1995, 83: 1243-1252 Roy et al., Cell 1995, 80: 167-178 Roy et al., Blood 1997, 595: 2645 Rudin et al., Annu Rev Med 1997, 48: 267-281 Ruegg et al., Nat Med 1998, 4: 408-414. Salvesen et al., Cell 1997, 91: 443-446. Sierra-Honigman et al., Science 1998, 281: 1683-1686 Stehlik et al., J Exp Med 1998, 188: 211-216 Stromblad et al., Chem Biol 1996, 3: 881-885 Takeshita et al., Laboratory Investigation 1996, 75: 487502 Tamm et al., Cancer Res 1998, 58: 5315-5320 Thompson, Science 1995, 267: 1456-1462 Tran et al., Biochem Biophys Res Commun 1999, 264: 781-788. Uhlmann et al., Chemical Reviews 1990, 90: 543584 Uren et al., Proc Natl Acad Sci U S A 1999, 96: 10170-10175 Vaux DL et al., Cell 1999, 96: 245-254 Velculescu et al., Nat Genet 1999, 23: 387-388 Wilson et al., Proc. Natl. Acad. Sci. 1990, 87: 84378441 Witzenbichler et al., J Biol Chem 1998, 273: 18514-21. Yuan et al., Proc Natl Acad Sci U S A 1996, 93: 14765-14770 Zhao et al., J Cell Sci 2000, 113: 4363-4371.

[Brief description of drawings]

1 (A)-(C) show modulation of survivin expression in EC. A. Resting EC, medium or serum (10% FCS), VEGF (100 ng / ml)
, BFGF (5 ng / ml), TNFα (10 ng / ml) or IL-2 (2
ng / ml) for 16 hours at 37 ° C. Collect cells and SD
S-extracted and analyzed for expression of survivin or β-actin by immunoblot. B. Controls or ECs with specified increasing concentrations of VEGF 1
It was stimulated for 6 hours at 37 ° C. and analyzed for expression of survivin or β-actin by immunoblot. C. 100 ng / m of total RNA at specified time intervals
It was extracted from 1 VEGF-stimulated EC, separated on an agarose-formaldehyde denaturing gel and hybridized to survivin or β-actin using a probe.

FIG. 2 (A)-(D) shows the expression of survivin in a three-dimensional EC culture medium. ECs were grown in 3D fibronectin-collagen gels, embedded in paraffin and analyzed for survivin expression by immunohistochemistry. A. Survivin expression in control two-dimensional EC cultures. B. Survivin expression in 3D EC cultures. C. Control staining of 3D EC cultures with preimmune antibody. D.
Two (2D) or three (3D) dimensional EC cultures were harvested, homogenized in a tissue polisher and analyzed for survivin expression by immunoblot.

3 (A)-(F) show expression of survivin in proliferating and non-proliferating skin capillaries. Formalin-fixed and paraffin-embedded skin biopsy 5 μm sections containing granular tissue and normal skin were analyzed for survivin expression by immunohistochemistry after antigen recovery in a pressure cooker. A. Strong survivin cytoplasmic expression of survivin in EC of dermal capillaries in granular tissue. Inset, detailed illustration of skin capillaries stained for survivin expression. C. Expression of survivin in the macrovascular endothelium in the granular tissue at the dermis / subcutaneous junction. B, D. Control staining in 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 of panel E in the presence of preimmune antibody. Magnification 200 times (A, E, F) and 400 times (B, C, D).

4 (A)-(B) show the anti-apoptotic function of survivin in EC. A. Subconfluent bovine aortic ECs were transfected with GFP-vector or GFP-survivin by lipofectin, cultured for 35 hours at 37 ° C., 5 ng
/ Ml TNFα / 5 μg / ml cycloheximide for an additional 8 hours 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 shown in brackets for each condition tested. B. Untreated or G
ECs transfected with FP-vector or GFP-survivin were incubated with the indicated concentrations of TNFα / 10 μg / ml cycloheximide, collected and collected in the presence or absence of the caspase-3 inhibitor DEVD-CHO. Caspase-3 activity was analyzed by hydrolysis of the sexual substrate DEVD-AMC. Data are means ± SD of duplicates of a representative experiment.

FIG. 5 (A)-(D) show that Ang-1 stimulates Akt phosphorylation and kinase activity. A. MVEC were incubated with Ang-1 (250 ng / ml) for 15 minutes and analyzed by Western blot for Akt phosphorylation (serine 473, upper panel) or total Akt expression (lower panel). Cells were processed as described and cell lysates prepared for Akt kinase assay (see B). 20 μg of protein was separated on SDS-polyacrylamide gel electrophoresis gel (SD-PAGE) and transferred to polyvinylidene difluoride membrane (Millipore). After blocking with T-PBS containing 5% milk (PBS containing 0.2% Tween 20) for 1 hour, the membrane was incubated with anti-Akt antibody (Santa Cruz), phosphorus-specific Akt antibody (New England Biolabs). . ECL (Amersham) was used for detection. B. Akt was immunoprecipitated from MVEC and analyzed for kinase 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 mM NaCl, 20 mM Tris-HCl, pH 7
． 4, 20 mM NaF, 2 μg / ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). Lysate with protein G-agarose at 4 ° C
Were precleared for 30 minutes and immunoprecipitated with anti-Akt antibody for 2 hours in the presence of 2 mg / ml bovine serum albumin with or without 16 μg / ml competing peptide (Santa Cruz). The immunoprecipitates were washed twice with cell lysis buffer, once with water and once with kinase buffer (20 mM HEPES, pH 7.2, 10).
Washed with mM MgCl ₂ , 10 mM MnCl ₂ ). The immunoprecipitated proteins were treated with 3 μl of 50 μl of kinase buffer containing 2 μg of histone H2B (Roche Molecular Biochemicals) and [ ³² P-] ATP (5 μM, 10 μCi).
Incubated for 0 minutes at room temperature. The kinase reaction was stopped by the addition of SDS sample buffer and the sample was subjected to Cerekenov counting and SDS-PAGE,
Then, it was subjected to 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 amounts of time and Akt activation determined as above. D. Ang-1 induced Akt phosphorylation is blocked by soluble Tie-2 and Ang-2 but not soluble Tie1.
MVEC can be either vehicle (TBS and CHAPS) or Ang-1 (250n
g / ml), Ang-2 (alone (2.5 μg / ml), soluble Tie1 or T
Incubation with various designated combinations of ie2 receptors (2.5 μg / ml) was followed by Akt phosphorylation or total Akt expression determined by Western blot. Ang-1 induced Akt phosphorylation is blocked by soluble Tie-2 but not Tie1 or Ang-2. In all panels, the data are representative of at least 3 experiments.

6 (A)-(C) show that Ang-1 inhibits endothelial cell apoptosis via the PI-3 kinase / Akt pathway. A. MVEC to Ang-1 (
250 ng / ml) or wortmannin (WM, 200 nM) for 18 hours in a serum-free medium of bacteriophage dishes, then
Apoptosis was determined by propidium iodide staining and flow cytometry. (MVEC is vehicle (TBS containing CHAPS) or Ang-1 (
250 ng / ml) in the presence of serum-free medium of bacteriophage dishes). Cells were incubated for 18 hours and both floating and adherent cells were collected. To determine the number of hypodiploid cells, MVECs were fixed in 70% ethanol for 1 hour, stained with a solution containing 500 μg / ml RNAase H and 50 μg / ml propidium iodide, and fluorescence activated cell sorter ( FACS) for analysis. At least 5000 events were analyzed and the percentage of cells in the sub-G1 population was calculated. B. Experimental conditions are the same as A, except that MVEC were infected with adenovirus encoding β-galactosidase or dominant negative AA-Akt for 24 hours, placed in suspension and treated with Ang-1 for 18 hours. For each panel, the percentage of MVEC with hypodiploid (apoptotic) DNA content is shown. C. Ang-1 activates Akt in an integrin-dependent manner. MVECs seeded in serum-free medium of bacteriophage dishes were treated with vehicle or Ang-1 (250 ng / ml) for 15 minutes in the absence or presence of WM (200 nM) and phosphorylated Akt (above). Panel) or whole Akt (bottom panel). For all panels, data are representative of 3 independent experiments.

FIGS. 7 (A)-(D) show that Ang-1 induces survivin expression via the PI-3 kinase / Akt pathway. A. Time-dependent expression of survivin RNA. Serum starved MVECs were treated with Ang-1 at designated time intervals and survivin, GAPDH and bcl-2 RNA expression was examined by Northern hybridization. B. Soluble Tie2 prevents Ang-1 induction of survivin RNA. The experimental conditions were: Ang-1 was pre-incubated for 24 hours in the absence or presence of soluble Tie2 receptor, then added to MVEC, survivin, G
APDH or bcl-2 RNA expression was determined. C. Ang-1 stimulates survivin promoter activity. MVECs were cotransfected with a promoterless luciferase cassette (pLUC-42) or a plasmid encoding the 1.2 kb survivin promoter fragment (pLUC-cyc1.2) with β-galactosidase to determine relative luciferase activity. . D.
VEGF and Ang-1 increase survivin protein expression. HUV
ECs were treated with VEGF (50 ng / ml) or Ang-1 (250 ng / ml) under the various conditions tested, collected after 18 hours and analyzed by Western blot for survivin, actin and bcl-2 protein expression. did.
The numbers below the survivin panel indicate relative levels based on the densitometer. For all panels, the data represent a representative of 2-4 experiments.

8 (A)-(B) show that survivin mediates the anti-apoptotic effect of Ang-1. A. TNFα / cycloheximide. B. Anoikis. MVE
C is a GFP vector, GFP-survivin (survivin) or GFP-C8
Transfection with 4A survivin (C84A survivin) followed by TNF
Whether it is treated with α (5 ng / ml) and cycloheximide (5 μg / ml) (A),
Alternatively, they were seeded in serum-free medium on bacteriological dishes in the absence or presence of Ang-1 (250 ng / ml) (B). 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 represent representatives of two replicates.

9 (A)-(C) show inhibition of survivin expression by antisense in EC. A. EC was designated as increasing concentrations of mixed control (control) or survivin antisense oligonucleotides (survivin AS)
CDNA for survivin or GAPDH
Was hybridized with. The densitometric quantification of hybridizing bands under the various conditions tested is shown in the panel below. B. EC is 0.1% FCS
Were serum-starved for 18 h in the presence of VEGF and stimulated with VEGF (50 ng / ml) in the presence of the indicated oligonucleotide concentrations. Protein-normalized aliquots of detergent-solubilized EC extract were immunoblotted with antibodies to survivin or β-actin, followed by chemiluminescence. C. The experimental conditions are the same as B except that EC extracts treated with control or survivin antisense oligonucleotides are analyzed by Western blot with an antibody against bcl-2. In panels B and C, molecular weight markers at kDa are shown on the left. WB,
Western blot.

(A)-(D) show inhibition of VEGF cell protection by survivin targeting. A. ECs were transfected with control or survivin antisense oligonucleotides, treated with VEGF (50 ng / ml) and exposed to C-6 ceramide (25 μg / ml). Cell nuclei were stained with 6.5 μg / ml 4,6-diamidino-2-phenylindole (DAPI, Sigma), 16% polyvinyl alcohol and 40% glycerol and morphologically examined for apoptosis using Zeiss fluorescence microscopy. Evaluation (chromatin condensation, DNA fragmentation). Phase contrast or DAPI staining for each field is obtained from a representative experiment from at least 3 independent determinations. B. The experimental conditions are the same as A. Data are expressed as apoptotic rate as determined by nuclear morphology by DAPI staining and represent the mean ± SEM of 3 independent experiments. C and D. ECs transfected with control or survivin antisense oligonucleotides were transfected with C-6 ceramide (25
μg / ml, C) or TNFα (10 ng / ml) and cycloheximide (10
Incubated with the combination of μg / ml, D) for 12 hours at 37 ° C. Cells were analyzed for DNA content by propidium iodide staining and flow cytometry. The percentage of hypodiploid, apoptotic cells with sub-G1 DNA content is shown for each condition tested. Data represent at least 2 independent decision experiments. FITC with comparable transfection efficiency
-Demonstrated by fluorescence microscopy of ECs transfected with conjugated oligonucleotides.

FIGS. 11 (A)-(C) show modulation of caspase activity by survivin targeting. A. The conditions are as described in FIG. Ceramide-treated ECs stimulated with VEGF and transfected with control or survivin antisense oligonucleotides were hydrolyzed to the fluorogenic substrate DEVD-AMC in the presence or absence of the caspase inhibitor DEVD-CHO. Was analyzed for caspase-3 activity. Data are means ± SD of two independent determinations. B. EC extracts under various specified conditions were analyzed by Western blot for caspase-
Caspase-3 proteolytic cleavage by antibodies to 3 was analyzed. An inactive caspase of about 32 kDa, an intermediate product of about 24 kDa, and about 1
The positions of the 7 and approximately 19 kDa active subunits are indicated. C. EC extracts were analyzed by Western blot for proteolytic cleavage of the caspase-3 substrate PAPP. Densitometric quantification under the various conditions tested was approximately 17
Active caspase-3 subunit (B) of kDa or apoptosis about 85kD
was performed on the PARP fragment (a) of a.

FIG. 12 (A)-(B) shows the effect of survivin targeting on quiescent EC. Starved ECs were transfected with the indicated oligonucleotides and incubated in the absence (-ceramide) or presence (+ ceramide) of ceramide. Cell extracts were subjected to hydrolysis of the luminogenic substrate DEVD-AMC in the presence or absence of DEVD-CHO (A) or by DNA content by propidium iodide staining and flow cytometry (B). It was analyzed for caspase-3 activity. Data are expressed as the mean ± SD of three independent experiments. In B, the percentage of apoptotic cells with hypodiploid sub-G1 DNA content is shown.

FIG. 13 shows the effect of antisense oligonucleotides on EC adhesion molecules on VEGF cell protection. The experimental conditions are the same as in FIG. 10, except that quiescent ECs were transfected with designated antisense oligonucleotides, stimulated with VEGF and exposed to C-6 ceramide. Cells were incubated for 12 hours at 37 ° C. and then analyzed for nuclear morphology by DAPI staining. Data are means ± SEM of 3 independent transfection experiments.

FIG. 14 shows the effect of survivin targeting on VEGF-induced EC migration / chemotaxis. ECs were transfected with control or survivin antisense oligonucleotides, stimulated with VEGF and exposed to designated increasing concentrations of VEGF or control SPP-1 in Boyden chambers. After incubating for 5 hours at 37 ° C., migrating cells were counted microscopically by Giemsa. Data is,
Mean ± SEM of a representative of 3 replicate experiments out of 3 independent determinations.

FIG. 15 shows the effect of survivin targeting on capillary formation. ECs transfected with control or survivin antisense oligonucleotides
Were cultured in collagen gel in the presence of PMA and in the absence of VEGF (none)
Or stabilized in the presence. The three-dimensional capillary network was analyzed by phase contrast microscopy during 3 days of culture at 37 ° C. Statue is one of at least three independent decisions
Represents one experiment. Magnification 100 times.

[Sequence list]

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission Date] July 19, 2001 (2001.7.19)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Contents of correction]

[Brief description of drawings]

1 (A)-(C) show modulation of survivin expression in EC. A. Resting EC, medium or serum (10% FCS), VEGF (100 ng / ml)
, BFGF (5 ng / ml), TNFα (10 ng / ml) or IL-2 (2
ng / ml) for 16 hours at 37 ° C. Collect cells and SD
S-extracted and analyzed for expression of survivin or β-actin by immunoblot. B. Controls or ECs with specified increasing concentrations of VEGF 1
It was stimulated for 6 hours at 37 ° C. and analyzed for expression of survivin or β-actin by immunoblot. C. 100 ng / m of total RNA at specified time intervals
It was extracted from 1 VEGF-stimulated EC, separated on an agarose-formaldehyde denaturing gel and hybridized to survivin or β-actin using a probe.

FIG. 2 (A)-(D) shows the expression of survivin in a three-dimensional EC culture medium. ECs were grown in 3D fibronectin-collagen gels, embedded in paraffin and analyzed for survivin expression by immunohistochemistry. A. Survivin expression in control two-dimensional EC cultures. B. Survivin expression in 3D EC cultures. C. Control staining of 3D EC cultures with preimmune antibody. D.
Two (2D) or three (3D) dimensional EC cultures were harvested, homogenized in a tissue polisher and analyzed for survivin expression by immunoblot.

3 (A)-(F) show expression of survivin in proliferating and non-proliferating skin capillaries. Formalin-fixed and paraffin-embedded skin biopsy 5 μm sections containing granular tissue and normal skin were analyzed for survivin expression by immunohistochemistry after antigen recovery in a pressure cooker. A. Strong survivin cytoplasmic expression of survivin in EC of dermal capillaries in granular tissue. Inset, detailed illustration of skin capillaries stained for survivin expression. C. Expression of survivin in the macrovascular endothelium in the granular tissue at the dermis / subcutaneous junction. B, D. Control staining in 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 of panel E in the presence of preimmune antibody. Magnification 200 times (A, E, F) and 400 times (B, C, D).

4 (A)-(B) show the anti-apoptotic function of survivin in EC. A. Subconfluent bovine aortic ECs were transfected with GFP-vector or GFP-survivin by lipofectin, cultured for 35 hours at 37 ° C., 5 ng
/ Ml TNFα / 5 μg / ml cycloheximide for an additional 8 hours 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 shown in brackets for each condition tested. B. Untreated or G
ECs transfected with FP-vector or GFP-survivin were incubated with the indicated concentrations of TNFα / 10 μg / ml cycloheximide, collected and collected in the presence or absence of the caspase-3 inhibitor DEVD-CHO. Caspase-3 activity was analyzed by hydrolysis of the sexual substrate DEVD-AMC. Data are means ± SD of duplicates of a representative experiment.

FIG. 5 (A)-(D) show that Ang-1 stimulates Akt phosphorylation and kinase activity. A. MVEC were incubated with Ang-1 (250 ng / ml) for 15 minutes and analyzed by Western blot for Akt phosphorylation (serine 473, upper panel) or total Akt expression (lower panel). Cells were processed as described and cell lysates prepared for Akt kinase assay (see B). 20 μg of protein was separated on SDS-polyacrylamide gel electrophoresis gel (SD-PAGE) and transferred to polyvinylidene difluoride membrane (Millipore). After blocking with T-PBS containing 5% milk (PBS containing 0.2% Tween 20) for 1 hour, the membrane was incubated with anti-Akt antibody (Santa Cruz), phosphorus-specific Akt antibody (New England Biolabs). . ECL (Amersham) was used for detection. B. Akt was immunoprecipitated from MVEC and analyzed for kinase 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 mM NaCl, 20 mM Tris-HCl, pH 7
． 4, 20 mM NaF, 2 μg / ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). Lysate with protein G-agarose at 4 ° C
Were precleared for 30 minutes and immunoprecipitated with anti-Akt antibody for 2 hours in the presence of 2 mg / ml bovine serum albumin with or without 16 μg / ml competing peptide (Santa Cruz). The immunoprecipitates were washed twice with cell lysis buffer, once with water and once with kinase buffer (20 mM HEPES, pH 7.2, 10).
Washed with mM MgCl ₂ , 10 mM MnCl ₂ ). The immunoprecipitated proteins were treated with 3 μl of 50 μl of kinase buffer containing 2 μg of histone H2B (Roche Molecular Biochemicals) and [ ³² P-] ATP (5 μM, 10 μCi).
Incubated for 0 minutes at room temperature. The kinase reaction was stopped by the addition of SDS sample buffer and the sample was subjected to Cerekenov counting and SDS-PAGE,
Then, it was subjected to 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 amounts of time and Akt activation determined as above. D. Ang-1 induced Akt phosphorylation is blocked by soluble Tie-2 and Ang-2 but not soluble Tie1.
MVEC can be either vehicle (TBS and CHAPS) or Ang-1 (250n
g / ml), Ang-2 (alone (2.5 μg / ml), soluble Tie1 or T
Incubation with various designated combinations of ie2 receptors (2.5 μg / ml) was followed by Akt phosphorylation or total Akt expression determined by Western blot. Ang-1 induced Akt phosphorylation is blocked by soluble Tie-2 but not Tie1 or Ang-2. In all panels, the data are representative of at least 3 experiments.

6 (A)-(C) show that Ang-1 inhibits endothelial cell apoptosis via the PI-3 kinase / Akt pathway. A. MVEC to Ang-1 (
250 ng / ml) or wortmannin (WM, 200 nM) for 18 hours in a serum-free medium of bacteriophage dishes, then
Apoptosis was determined by propidium iodide staining and flow cytometry. (MVEC is vehicle (TBS containing CHAPS) or Ang-1 (
250 ng / ml) in the presence of serum-free medium of bacteriophage dishes). Cells were incubated for 18 hours and both floating and adherent cells were collected. To determine the number of hypodiploid cells, MVECs were fixed in 70% ethanol for 1 hour, stained with a solution containing 500 μg / ml RNAase H and 50 μg / ml propidium iodide, and fluorescence activated cell sorter ( FACS) for analysis. At least 5000 events were analyzed and the percentage of cells in the sub-G1 population was calculated. B. Experimental conditions are the same as A, except that MVEC were infected with adenovirus encoding β-galactosidase or dominant negative AA-Akt for 24 hours, placed in suspension and treated with Ang-1 for 18 hours. For each panel, the percentage of MVEC with hypodiploid (apoptotic) DNA content is shown. C. Ang-1 activates Akt in an integrin-dependent manner. MVECs seeded in serum-free medium of bacteriophage dishes were treated with vehicle or Ang-1 (250 ng / ml) for 15 minutes in the absence or presence of WM (200 nM) and phosphorylated Akt (above). Panel) or whole Akt (bottom panel). For all panels, data are representative of 3 independent experiments.

FIGS. 7 (A)-(D) show that Ang-1 induces survivin expression via the PI-3 kinase / Akt pathway. A. Time-dependent expression of survivin RNA. Serum starved MVECs were treated with Ang-1 at designated time intervals and survivin, GAPDH and bcl-2 RNA expression was examined by Northern hybridization. B. Soluble Tie2 prevents Ang-1 induction of survivin RNA. The experimental conditions were: Ang-1 was pre-incubated for 24 hours in the absence or presence of soluble Tie2 receptor, then added to MVEC, survivin, G
APDH or bcl-2 RNA expression was determined. C. Ang-1 stimulates survivin promoter activity. MVECs were cotransfected with a promoterless luciferase cassette (pLUC-42) or a plasmid encoding the 1.2 kb survivin promoter fragment (pLUC-cyc1.2) with β-galactosidase to determine relative luciferase activity. . D.
VEGF and Ang-1 increase survivin protein expression. HUV
ECs were treated with VEGF (50 ng / ml) or Ang-1 (250 ng / ml) under the various conditions tested, collected after 18 hours and analyzed by Western blot for survivin, actin and bcl-2 protein expression. did.
The numbers below the survivin panel indicate relative levels based on the densitometer. For all panels, the data represent a representative of 2-4 experiments.

8 (A)-(B) show that survivin mediates the anti-apoptotic effect of Ang-1. A. TNFα / cycloheximide. B. Anoikis. MVE
C is a GFP vector, GFP-survivin (survivin) or GFP-C8
Transfection with 4A survivin (C84A survivin) followed by TNF
Whether it is treated with α (5 ng / ml) and cycloheximide (5 μg / ml) (A),
Alternatively, they were seeded in serum-free medium on bacteriological dishes in the absence or presence of Ang-1 (250 ng / ml) (B). 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 represent representatives of two replicates.

9 (A)-(C) show inhibition of survivin expression by antisense in EC. A. EC was designated as increasing concentrations of mixed control (control) or survivin antisense oligonucleotides (survivin AS)
CDNA for survivin or GAPDH
Was hybridized with. The densitometric quantification of hybridizing bands under the various conditions tested is shown in the panel below. B. EC is 0.1% FCS
Were serum-starved for 18 h in the presence of VEGF and stimulated with VEGF (50 ng / ml) in the presence of the indicated oligonucleotide concentrations. Protein-normalized aliquots of detergent-solubilized EC extract were immunoblotted with antibodies to survivin or β-actin, followed by chemiluminescence. C. The experimental conditions are the same as B except that EC extracts treated with control or survivin antisense oligonucleotides are analyzed by Western blot with an antibody against bcl-2. In panels B and C, molecular weight markers at kDa are shown on the left. WB,
Western blot.

(A)-(D) show inhibition of VEGF cell protection by survivin targeting. A. ECs were transfected with control or survivin antisense oligonucleotides, treated with VEGF (50 ng / ml) and exposed to C-6 ceramide (25 μg / ml). Cell nuclei were stained with 6.5 μg / ml 4,6-diamidino-2-phenylindole (DAPI, Sigma), 16% polyvinyl alcohol and 40% glycerol and morphologically examined for apoptosis using Zeiss fluorescence microscopy. Evaluation (chromatin condensation, DNA fragmentation). Phase contrast or DAPI staining for each field is obtained from a representative experiment from at least 3 independent determinations. B. The experimental conditions are the same as A. Data are expressed as apoptotic rate as determined by nuclear morphology by DAPI staining and represent the mean ± SEM of 3 independent experiments. C and D. ECs transfected with control or survivin antisense oligonucleotides were transfected with C-6 ceramide (25
μg / ml, C) or TNFα (10 ng / ml) and cycloheximide (10
Incubated with the combination of μg / ml, D) for 12 hours at 37 ° C. Cells were analyzed for DNA content by propidium iodide staining and flow cytometry. The percentage of hypodiploid, apoptotic cells with sub-G1 DNA content is shown for each condition tested. Data represent at least 2 independent decision experiments. FITC with comparable transfection efficiency
-Demonstrated by fluorescence microscopy of ECs transfected with conjugated oligonucleotides.

FIGS. 11 (A)-(C) show modulation of caspase activity by survivin targeting. A. The conditions are as described in FIG. Ceramide-treated ECs stimulated with VEGF and transfected with control or survivin antisense oligonucleotides were hydrolyzed to the fluorogenic substrate DEVD-AMC in the presence or absence of the caspase inhibitor DEVD-CHO. Was analyzed for caspase-3 activity. Data are means ± SD of two independent determinations. B. EC extracts under various specified conditions were analyzed by Western blot for caspase-
Caspase-3 proteolytic cleavage by antibodies to 3 was analyzed. An inactive caspase of about 32 kDa, an intermediate product of about 24 kDa, and about 1
The positions of the 7 and approximately 19 kDa active subunits are indicated. C. EC extracts were analyzed by Western blot for proteolytic cleavage of the caspase-3 substrate PAPP. Densitometric quantification under the various conditions tested was approximately 17
Active caspase-3 subunit (B) of kDa or apoptosis about 85kD
was performed on the PARP fragment (a) of a.

FIG. 12 (A)-(B) shows the effect of survivin targeting on quiescent EC. Starved ECs were transfected with the indicated oligonucleotides and incubated in the absence (-ceramide) or presence (+ ceramide) of ceramide. Cell extracts were subjected to hydrolysis of the luminogenic substrate DEVD-AMC in the presence or absence of DEVD-CHO (A) or by DNA content by propidium iodide staining and flow cytometry (B). It was analyzed for caspase-3 activity. Data are expressed as the mean ± SD of three independent experiments. In B, the percentage of apoptotic cells with hypodiploid sub-G1 DNA content is shown.

FIG. 13 shows the effect of antisense oligonucleotides on EC adhesion molecules on VEGF cell protection. The experimental conditions are the same as in FIG. 10, except that quiescent ECs were transfected with designated antisense oligonucleotides, stimulated with VEGF and exposed to C-6 ceramide. Cells were incubated for 12 hours at 37 ° C. and then analyzed for nuclear morphology by DAPI staining. Data are means ± SEM of 3 independent transfection experiments.

14 (A)-(B) show the effect of survivin targeting on VEGF-induced EC migration / chemotactic. ECs were transfected with control or survivin antisense oligonucleotides, stimulated with VEGF and exposed to designated increasing concentrations of VEGF or control SPP-1 in Boyden chambers.
After incubating for 5 hours at 37 ° C., migrating cells were counted microscopically by Giemsa. Data are means ± SEM of three representative replicate experiments out of three independent determinations.

FIG. 15 shows the effect of survivin targeting on capillary formation. ECs transfected with control or survivin antisense oligonucleotides
Were cultured in collagen gel in the presence of PMA and in the absence of VEGF (none)
Or stabilized in the presence. The three-dimensional capillary network was analyzed by phase contrast microscopy during 3 days of culture at 37 ° C. Statue is one of at least three independent decisions
Represents one experiment. Magnification 100 times.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 45/00 A61L 31/00 Z 4C087 48/00 A61P 9/00 A61L 31/00 9/10 A61P 9 / 00 43/00 105 9/10 A61K 37/24 43/00 105 37/547 C12N 15/09 ZNA C12N 15/00 ZNAA // C12N 5/10 5/00 B (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA ( M, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Sessa, William, C. NY Haven, Connecticut, United States Congress Avenue 295, Yale University School of Medicine, Boyer Center for Molecular Meditation, Room 436 D-F Term (Reference) 4B024 AA01 BA80 CA04 DA03 EA02 EA04 GA11 GA18 HA17 4B065 AA93 AC14 BA02 CA24 CA44 4C081 AB13 AC10 BA12 CD34 4C084 AA01 AA02 AA13 AA16 DB55 DC03 MA52 MA55 MA56 MA63 MA66 NA14 ZA362 ZA452 ZC412 4C086 AA01 AA02 AA03 EA16 MA01 A51 MA53 A36 A03 A45 MA31 A63 A01 MA45 MA63 MA63 MA66 MA14 NA14 MA63 MA66 NA14 ZA36 ZA45 ZC41

Claims (49)

[Claims] 1. A method of promoting angiogenesis comprising the step of providing a cell or tissue with an apoptosis-inhibiting concentration of survivin or survivin activity. 2. An agent selected from the group consisting of a survivin polypeptide, a survivin transgene, a survivin peptidomimetic, and an agent that modulates the expression of survivin in a cell or tissue. The method according to claim 1, which is increased by: 3. The agent is angiopoietin-1 or VEGF,
The method of claim 2. 4. The method of claim 2, wherein the agent is activated serine-threonine kinase Akt. 5. The method of claim 1 or 2 wherein the agent is provided in an implant.
The method according to any one of 1. 6. The method of claim 5, wherein the implant is coated or impregnated with a survivin transgene. 7. The method of claim 6, wherein the transgene is operably linked to an expression control element. 8. The method of claim 7, wherein the transgene is contained within a vector. 9. The method of claim 7, wherein the transgene is contained within a composition that facilitates transfection. 10. The method of claim 9, wherein the transfection facilitating composition is a transfection facilitating lipid or transfection facilitating particle. 11. The method of any one of claims 1 or 2, wherein the method treats the condition by inducing compensatory angiogenesis. 12. The method of claim 11, wherein the condition is ischemic disease. 13. The method of claim 12, wherein the ischemic disease is caused by myocardial infarction, peripheral vascular occlusion, cerebral ischemia or stroke. 14. A method of promoting endothelial cell angiogenesis, comprising providing a subject with endothelial cells engineered to express an apoptosis-inhibiting amount of survivin. 15. The method of claim 14, wherein the cells are provided in an implant. 16. The method of claim 15, wherein the implant is a stent. 17. The method of claim 15, wherein the implant is coated or impregnated with a survivin transgene. 18. The method of claim 17, wherein the transgene is operably linked to an expression control element. 19. The method of claim 18, wherein the transgene is contained within a vector. 20. The method of claim 17, wherein the transgene is contained within a composition that facilitates transfection. 21. The method of claim 20, wherein the transfection facilitating composition is a transfection facilitating lipid or transfection facilitating particle. 22. The method of claim 14, wherein the method treats a condition by inducing compensatory angiogenesis. 23. The method of claim 22, wherein the condition is ischemic disease. 24. The method of claim 23, wherein the ischemic disease is caused by myocardial infarction, peripheral vascular occlusion, cerebral ischemia or stroke. 25. A method of preventing a vascular proliferative disorder, comprising providing to a patient an agent that modulates survivin expression or activity. 26. The method of claim 25, wherein the agent downregulates survivin expression. 27. The method of claim 26, wherein the agent is a survivin antisense molecule. 28. The method of claim 26, wherein the agent is provided in an implant. 29. The method of claim 28, wherein the implant is a stent. 30. The method of claim 29, wherein the stent is coated or impregnated with survivin antisense molecules. 31. The method of claim 30, wherein the survivin antisense molecule is included in a composition that facilitates transfection. 32. The method of claim 31, wherein the transfection facilitating composition is a transfection facilitating lipid or transfection facilitating particle. 33. The method of claim 25, wherein the vascular proliferative disorder is restenosis, graft occlusion by vascular bypass surgery, or coronary vascular disorder due to transplantation. 34. The method of any one of claims 11 or 22, wherein the condition is selected from the group consisting of tissue injury or wound. 35. A device suitable for implantation in a patient, said device being coated or impregnated with a survivin transgene or survivin antisense molecule. 36. A device suitable for implantation in a patient, the device comprising endothelial cells engineered to express an apoptosis-inhibiting amount of survivin. 37. A method of inhibiting angiogenesis of cells comprising administering an agent that inhibits survivin. 38. The method of claim 37, wherein the cells are tumorigenic. 39. A VE comprising administering an agent that inhibits survivin.
A method of inhibiting GF-induced angiogenesis. 40. A VE comprising administering an agent that inhibits survivin.
A method of inhibiting GF-inducing activity. 41. The VEGF-inducing activity is capillary formation.
The method described in 0. 42. The method of any one of claims 37, 38, 39, 40 or 41, wherein the agent is an inhibitor of survivin function or an inhibitor of survivin expression. 43. The method of claim 42, wherein the agent is a survivin antibody, a survivin antisense molecule, or an inhibitor of Akt phosphorylation. 44. A method of treating a patient in need thereof with an agent that modulates survivin expression or function and an immunomodulatory agent. 45. The agent inhibits the expression or function of survivin,
The method of claim 44. 46. The agent promotes the expression or function of survivin,
The method of claim 44. 47. The method of claim 44, wherein the agent is an immunosuppressant agent. 48. The method of claim 44, wherein the patient has undergone a graft. 49. The method of claim 45, wherein the patient has received a transplant.