Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR
PROMOTING ANGIOGENESIS
Background of the Invention
This application claims priority to U.S. Provisional Patent Application Serial
No.
60/264,457, filed on January 26, 2001, the contents of which are hereby
incoporated
herein.
Background of the Invention
The depletion of oxygen supply to due to obstructed or inadequate blood supply
is the common pathological state associated with various tissue ischemias,
including
myocardial ischemia, ischaemic bowel disease, and peripheral ischemia. The
alleviation
of tissue ischemia is critically dependent upon angiogenesis, the process by
which new
capillaries are generated from existing vasculature and tissue. The
spontaneous growth
of new blood vessels provide collateral circulation in and around an ischemic
area,
improves blood flow, and alleviates the symptoms caused by the ischemia.
Although
surgery or angioplasty may help to revascularize ischemic regions in some
cases, the
extent, complexity and location of the arterial lesions which cause the
occlusion often
prohibits such treatment.
Alternative methods for the treatment of chronic ischemia have focused on the
delivery of angiogenic growth factors, over twenty of which are known. To
date,
modest but significant angiogenesis has been achieved following administration
(e.g., by
local injection) of exogenous factors to animal models. For example, purified
recombinant VEGF-A has been demonstrated to elicit a modest but significant
vascularization following injection into ischemic skeletal muscle tissue in a
rabbit model
of chronic limb ischemia [Takeshita et al., Circulation 90, 228:1994]. In
addition,
direct injection of vectors containing cDNA encoding VEGF-A has also been
shown to
induce a modest stimulation of angiogenesis in ischemia animal models in both
skeletal
and cardiac muscle [Takeshita et al., Biochemical and Biophysical Research
Communications 227, 628:1996; and MacGovern et al., Human Gene Therapy 8, 215:
1997]. However, such local application often results in diffusion of the
factors away
from the desired site, thus diminishing angiogenic effect. Moreover, it is
known that
such bolus delivery of angiogenic factors generally results in a local and
disorganized
hodge-podge of new blood vessels, only a fraction of which (if any) contribute
to
3 5 amelioration of a blockage.
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An additional limitation associated with current approaches for promoting
angiogenesis and effective treatment of ischemia is the inability of stable
blood vessels
to form by application of single agents using known techniques. In past
studies, when
vessels were followed for several months after treatment of ischemic animal
models, the
vast majority of new vessels were observed to regress after the applied growth
factor had
become depleted. Thus, current approaches for ischemia therapy require
repeated
applications of factors to maintain newly formed vasculature.
Other related therapy methods attempt to circumvent the need for multiple
applications by relying on the transplantation of autologous or non-autologous
cells
which can produce sustained levels of angiogenic proteins. In one such
approach, a
subject's endogenous cells are isolated, cultured, and transfected with
expression vectors
encoding angiogenic proteins. Following in vitro manipulations, these cells
are injected
back into the patient at the site of tissue ischemia. However, the drawbacks
of this
approach include the time and effort required to isolate, culture and
transfect target cells
from each individual patient, as well as difficulties in securing sustained
expression of
angiogenic proteins. In addition, sub-optimal cell survival and
differentiation states of
the cells following injection also limit the efficacy and of this approach.
To avoid these problems, cells have been obtained from non-patient (e.g.,
allogeneic), even non-human, sources and manipulated in the manner described
above.
However, the modified non-patient or non-human cells are often rejected by the
patient's
own immune system, making this approach impractical too.
Accordingly, improved therapies for promoting tissue angiogenesis and
generating stable vasculature in a safe, reliable, and non-invasive manner are
needed to
treat tissue ischemia and other related conditions.
Summary of the Invention
The present invention provides novel methods and compositions for promoting
angiogenesis to treat a variety of tissue ischemias, including peripheral and
myocardial
ischemia. Selected angiogenic factors or synergistic combinations of factors,
functional
analogues of such factors or combinations of factors, or nucleic acids
encoding such
factors or combinations of factors, are delivered to a localized area of
tissue in an
amount effective to induce angiogenesis within the area of tissue.
The invention further includes improved methods and vehicles for delivering
such
factors or combinations of factors, functional analogues of such factors or
combinations
of factors, and nucleic acids encoding such factors or combinations of
factors.
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In one embodiment, the invention provides a. method of promoting angiogenesis
comprising delivering PDGF-BB to a localized area of tissue in an amount
effective to
induce angiogenesis within the area of tissue. The PDGF-BB can be delivered
either
alone or in combination with another angiogenesis-promoting factor,
particularly bFGF
and/or VEGF-A. The angiogenesis-promoting factor or combination of factors can
be
administered in the form of a protein composition or an expression plasmid
encoding the
protein(s). The angiogenesis-promoting factor or combination of factors can
also be
administered in the form of functional analogues of the factor or combination
of factors.
For example, anti-idiotypic antibodies of PDGF-BB, VEGF-A and/or bFGF can be
administered in accordance with the invention.
When administering the angiogenesis promoting factors of the invention in the
form of an expression plasmid, suitable vectors include, but are not limited
to,
adenoviral vectors, retroviral vectors, adeno-associated viral vectors, RNA
vectors,
liposomes, cationic lipids, lentiviral vectors and transposons.
In another embodiment, the invention provides a method for promoting
angiogenesis by delivering angiogenic factors, such as those described above,
to a
localized area of tissue using heparin sepharose-containing microcapsules in
an amount
effective to induce angiogenesis within the area of tissue. The angiogenic
factors or
expression plasmids encoding the factors are incorporated into the
microcapsules as
described in the working examples provided below for slow, sustained release
into
localized areas of tissue.
In a particular embodiment, the microcapsules are made up of uncoated heparin
sepharose beads, heparin sepharose beads coated with a single layer of
alginate polymer,
heparin sepharose beads coated with poly-ethylene glycol (PEG) polymer or
heparin
sepharose beads coated with alternating layers of alginate and PEG. Typically,
the
microcapsules range in size from 1-200 microns.
Suitable angiogenic factors for incorporating into the microcapsules include,
for
example, M-CSF, GM-CSF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E,
neuropilin, FGF-1 , FGF-2(bFGF), FGF-3, FGF-4, FGF-5, FGF-6, PDGF-BB, PDGF-
AA, Angiopoietin l, Angiopoietin 2, erythropoietin, BMP-2, BMP-4, BMP-7, TGF-
beta, IGF-1, Osteopontin, Pleiotropin, Activin, Endothelin-1, and combinations
thereof
or an expression vectors encoding such angiogenic factors. The angiogenic
factors can
be purified from their native sources or produced by recombinant expression.
The microcapsules are contacted with the localized area of tissue generally by
injection or surgical implantation. For example, injection can be performed
using a
catheter based trans-myocardial injection technology, such as the NOGA
technology.
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In yet another embodiment, the present invention provides a method for
promoting angiogenesis by contacting a localized area of tissue with a
gradient of one or
more angiogenic factors or a nucleic acid encoding one or more angiogenic
factors, such
that directed vascular growth along the gradient is achieved. Such directed
vascular
growth can be used to achieve interconnection and/or intraconnection of blood
vessels
(e.g., to circumvent blood flow around a blockage within a blood vessel).
In a particular embodiment, the angiogenic factor or nucleic acid is released
in a
gradient using a biocompatible material which is contacted with (e.g.,
implanted within)
the localized area of tissue. The angiogenic factor is associated with the
biocompatible
material (e.g., absorbed onto the biocompatible material) such that it is
released onto
surrounding tissue. This can be achieved by treating the biocompatible
material with the
angiogenic factor prior to contact with (e.g., implantation into) a selected
area of tissue.
The angiogenic factor is then released from the biocompatible material onto
the
surrounding tissue in a directed gradient determined by the placement of the
biocompatible polymer
Suitable biocompatible materials include, for example, polymers or threads
which incorporate the angiogenic factor. In a preferred embodiment, the
biocompatible
material is an absorbable thread, such as polyglyconate monofilament,
poliglecaprone
25-(Monocryl), polydiaxonone (PDS II), polyglactin 910, polyglycolic acid,
Biodyn
glycomer 631, chromic surgical gut or plain surgical gut.
These and other embodiments of the invention are described in the following
figure detailed description, examples and figures.
Brief Description of the Figures
Figure 1 is a graph comparing levels of angiogenesis in the Matrigel model
using a low dose of transduced cells encoding GFP alone (control), VEGF-A,
VEGF-C,
VEGF-D bFGF or PDGF-BB. C57B1/10 mice were each injected subcutaneously into
the abdominal with a low dose of 3 x 105 retrovirally transduced autologous
myoblast
cells, suspended in 0.4m1 of Matrigel. Mice were sacrificed 13 days later and
the
matrigel pellet and a section of the abdominal muscle adjacent to the pellet
was
removed. Samples were sectioned and the number of microvessels in the
abdominal
muscle was quantif ed by visual inspection of sections under the microscope.
Shown is
the number of microvessels per 10 high power fields counted. The most potent
angiogenic effect was observed with VEGF-A, PDGF-BB and bFGF. Analysis of the
dose response curve for PDGF-BB and VEGF-A transduced cells showed that PDGF-
BB was more potent than VEGF-A at lower doses.
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Figure 2 is a ,graph comparing levels of angiogenesis in the Matrigel model
using a high dose of cells transduced to express bFGF, VEGF-A and PDGF-BB.
C57B1/10 mice were each injected with a high cell dose of 2 x 106 retrovirally
transduced autologous myoblast cells suspended in 0.4m1 of Matrigel. Mice were
sacrificed 13 days later, the pellets were recovered, sectioned and the number
of
microvessels counted by visual inspection. Shown are the number of
microvessels per
high power fields. At this cell dose, PDGF-BB was as potent as either bFGF or
VEGF-A at stimulating angiogenesis.
10 Figure 3 shows photographs of mouse corneas 6 days following the
implantation
of pellets coated with control saline (A), PDGF-BB (B), VEGF-A (C) or bFGF (D)
alone. Bottom panels: Quantification of the angiogenic effect elicited by each
factor.
Vessel length (E), clock hours (F) and area (G) are shown.
Figure 4 (A) shows photographs of mouse corneas 6 days following the
implantation of pellets coated with VEGF-A alone (left panel), bFGF (middle
panel) or
both factors combined (right panel). (B) shows the quantification of the
angiogenic
effect elicited by each growth factor in terms of clock hours (left panel),
vessel length
(middle panel) and area (right panel).
Figure 5 (top panels) shows photographs of mouse corneas 6 days post-
transplantation of pellets coated with bFGF alone (left panel) or bFGF
combined with
PDGF-BB (middle and right panels). Bottom panels show photographs of mouse
corneas 6 days post-transplantation of pellets coated with either VEGF-A alone
(left
panel) or VEGF-A combined with PDGF-BB (right panel).
Figure 6 is a graph comparing the quantification of angiogenesis in the mouse
cornea model using PDGF-BB, VEGF-A or bFGF either alone or in combination.
Corneal micropockets were created with a cataract knife in the eyes of 8-week
old
C57B1/6 mice. Into this pocket, aluminum sulfate pellets coated with between
80 and
160ng of recombinant human PDGF-BB, VEGF-A, bFGF or combinations thereof were
implanted and mice were monitored daily. A total of S mice were transplanted
per
group. The area of newly grown vessels was assessed 5 days post implantation.
Mice
implanted with control pellets showed no evidence of angiogenesis. When tested
alone,
bFGF stimulated the highest level of angiogenesis followed by VEGF-A and PDGF-
BB.
The level of angiogenesis stimulated by VEGF-A in combination with PDGF-BB was
equivalent to that observed for bFGF alone. Unexpectedly, the most potent
combination
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was PDGF-BB and bFGF. Of all combinations tested, PDGF-BB and bFGF together
stimulated the greatest level of angiogenesis, significantly greater than that
observed for
VEGF-A and bFGF.
Figure 7 is a schematic illustration of the experimental strategy to make
heparin
sepharose/alginate microcapsules. Heparin sephaxose beads (Pharmacia: 50-150~m
in
size) are mixed with a solution of sodium alginate to a final concentration of
200mg/ml.
The heparin sepharoselalginate solution is then loaded into a Sml syringe and
slowly
injected into a coaxial airflow system constructed at Genetix. The coaxial air
flow
creates a mist of the heparin sepharose/alginate solution which drops into a
1.5%
calcimn chloride bath. Once the alginate hits the calcium solution the
alginate becomes
cross-linked, forming a solid gel capsule roughly in the shape of a sphere.
The size of
the microcapsules can vary greatly from 50 - 400~m. Large microcapsules
(greater than
200~m in size) are removed from the capsule mixture using a 200~m sieve. Once
formed the capsules are washed twice in sterile water and stored in buffer
composed of
0.9% sodium chloride and 1mM calcium chloride. Capsules are loaded with
recombinant human PDGF-BB by incubation in binding buffer (0.9% NaCI, 1mM
CaCl2 and 0.05% gelatin) at 4°C overnight (~16 hours) with gentle
shaking. The next
day the capsules are removed, washed twice in binding buffer and either
cultured in vitro
to determine the kinetics of PDGF-BB release or injected in vivo to assess
angiogenesis.
The efficiency of PDGF-BB uptake is quantified by ELISA of the binding buffer
following removal of the capsules.
Figure 8 is a graph showing that hepaxin sepharose/alginate capsules bind
large
amounts of recombinant human PDGF-BB. Shown is the amount of PDGF-BB
absorbed by 3000 capsules following incubation with various quantities of
growth
factor. The amount of PDGF-BB remaining in the binding buffer following
incubation
with capsules was quantified by ELISA. Three thousand capsules were able to
absorb at
least 35 ~,g of PDGF-BB representing 13 ng of PDGF-BB per capsule.
Figure 9 is a graph showing that heparin sepharose/alginate microcapsules
provide slow, high level and long term release of bound PDGF-BB in vitro. Ten
wg of
recombinant human PDGF-BB was incubated with three different types of test
samples.
The first test sample was composed of non-encapsulated heparin sepharose beads
while
the second and third groups were composed of alginate encapsulated heparin
sepharose
beads made using either a 1.2% or a 1.6% alginate solution. Three thousand
beads/microcapsules were incubated with PDGF-BB at 4°C overnight with
gentle
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shaking. ELISA analysis of the binding buffer the next day showed absorption
of 90%
(9~g) of the PDGF by the capsules. Following incubation with PDGF-BB, the
beads/microcapsules were washed, resuspended in Smls of serum free medium and
incubated at 37°C. Every 24 hours the medium was changed and the amount
of PDGF-
BB present in the medium quantified by ELISA. A slow, sustained release of
approximately 0.5-3% of the total bound PDGF-BB (representing 125-250ng) was
detected each day for a minimum of 14 days, the longest time point tested.
Importantly,
the proportion of PDGF-BB released per day is equivalent to the amount of PDGF-
BB
that was estimated to be secreted by muscle cells transduced with the PDGF-BB
retrovirus in the foregoing Matrigel experiments. The release kinetics for non-
encapsulated heparin sepharose beads was better than those observed for the
alginate
encapsulated heparin sepharose.
Figure 10 is a graph showing that PDGF-BB microcapsules potently stimulate
angiogensis in vivo in the stringent Matrigel model. Three thousand
microcapsules
loaded with 1 ~,g or l Owg of PDGF-BB were mixed with 400w1 of Matrigel and
subcutaneously injected into the abdominal region of C57B1/10 mice. Thirteen
days later
mice were sacrificed, the pellets and a section of the adjacent abdominal
muscle was
removed, fixed, sectioned and the number of microvessels quantified by visual
inspection of the sections under the microscope. The results showed that the
number of
microvessels in mice receiving microcapsules loaded with I Omg of PDGF-BB was
2.5-
fold greater than that of control mice.
Figure 11 is a graph showing that PDGF-BB microcapsules stimulate
angiogenesis in infarcted rat hearts 3 weeks post-injection. Infarcted rat
hearts were
injected with 1600 microcapsules containing ~g (control) or 18~.g of PDGF-BB
in a
volume of 201. Three weeks post injection rats were sacrificed, hearts were
removed,
fixed, sectioned and the number of microvessels within the infarct region
quantified by
visual inspection under a microscope. Shown is the number of microvessels per
5 high
power fields for recipients of control and PDGF-BB microcapsules. Rats
injected with
PDGF-BB microvessels showed an approximate 2-fold increase in the number of
microvessels as compared to control rats.
Figure 12 shows an analysis of cardiac function in rats injected with control
vs.
PDGF-BB microcapsules following myocardial infarction. Left ventricular
pressure
(LVP), dP/dT, neg dP/dT and tau were measured prior to sacrifice at 3 weeks
post
injection. Left ventricular pressure (LVP) is the maximum pressure in the left
ventricle
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during contraction. The dPldT variable is the first derivative of the pressure
wave and is
separately viewed for the upstroke (dP/dT) and the downstroke (neg dP/dT). The
upstroke (dP/dT) is a measure of contractility and reflects the condition of
the muscle
independent of the pressure. Neg dP/dT reflects the relaxation of the muscle,
which
together with the relaxation constant, tau, provides information on the
stiffness of the
ventricular wall following infarction. A significant improvement in all
parameters was
detected in rats injected with PDGF-BB microcapsules. Rats injected with PDGF-
BB
microcapsules showed a 25% increase in left ventricular pressure, a 2-3 fold
increase in
cardiac contractility/relaxation and a 2.5-3 fold decrease in the relaxation
constant tau.
Figure 13 is a graph showing that PDGF-BB and bFGF delivered by slow
release microcapsules potently synergize to stimulate angiogensis in vivo in
the stringent
Matrigel model. Three thousand microcapsules loaded with 1 ~g of bFGF were
mixed
with 4001 of Matrigel and subcutaneously injected into the abdominal region of
C57B1/10 mice. Thirteen days later mice were sacrificed, the pellets and a
section of the
adjacent abdominal muscle was removed, fixed, sectioned and the number of
microvessels quantified by visual inspection of the sections under the
microscope. The
results showed that the number of microvessels in mice receiving bFGF + PDGF-
BB
microcapsules was 4-fold greater than that of mice implanted with either
growth factor
alone.
Figure 14 is a schematic illustration of the structure of various angiogenic
expression plasmids. All vectors were constructed using the pCI vector
backbone from
Promega. All vectors contained the Cytomegalovirus immediate-early
enhancer/promoter region, a chimeric intron and the late poly adenylation
signal from
SV40. The cDNA encoding either human PDGF-BB, VEGF-A or bFGF was inserted
into this vector downstream of the chimeric intron. A cDNA encoding for the
mature
PDGF-BB protein was cis-linked to the secretory signal from the marine
IgGkappa
immunoglobulin light chain gene while the VEGF-A cDNA utilized its endogenous
secretory signal. Since endogenous bFGF is not secreted by the usual Golgi
pathway
and prior groups have had difficulty in obtaining high level secretion of bFGF
from cells
transduced with the bFGF cDNA, the bFGF cDNA was linked in cis to the
secretory
signal from the human Interleukin-2 cDNA. The level of angiogenic protein
secreted
from transiently transfected 293T cells, as assessed by ELISA, is shown to the
right. To
analyze cardiac function in rats injected with control vs. PDGF-BB expression
plasmids
following myocardial infarction, test animals are anesthetized and intubated.
The chest
wall is opened and a myocardial infarct is created by tying off the anterior
descending
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artery. 180~g of control or test expression plasmid is injected into the heart
wall in a
volmne of 20q,1. Cardiac function is assessed 3 weeks post injection. Animals
are
sacrificed, the heart is removed and efficiency of plasmid uptake is assessed
by staining
with X-gal. The size of the infarct and the extent of angiogenesis is
quantified.
Detailed Description of the Invention
While growth promoting factors have been described as an angiogenic agents,
the efficacy of such proteins as a therapeutic agents for the treatment of
peripheral
and/or myocardial ischemia has not yet been demonstrated. The present
invention
provides, for the first time, methods for promoting angiogenesis and treating
such
ischemia using particular angiogenic factors, such as PDGF-BB, as well as
particular
combinations of factors (e.g., combinations which include PDGF-BB). The
present
invention also provides improved methods for delivering the factors to
increase their
efficacy, for example, by enabling directed and/or controlled release of the
angiogenic
factors onto surrounding tissue (e.g., ischemic myocardium).
ANGIOGENIC FACTORS
As used herein, the term "angiogenic factor" xefers to any knovtm protein
factor
capable of promoting growth of new blood vessels from existing vasculature
("angiogenesis"). Suitable angiogenic factors for use in the invention
include, for
example, PDGF-BB, PDGF-AA, M-CSF, GM-CSF, VEGF-A, VEGF-B, VEGF-C,
VEGF-D, VEGF-E, neuropilin, FGF-1 , FGF-2(bFGF), FGF-3, FGF-4, FGF-5, FGF-6,
Angiopoietin 1, Angiopoietin 2, erythropoietin, BMP-2, BMP-4, BMP-7, TGF-beta,
IGF-1, Osteopontin, Pleiotropin, Activin, Endothelin-l and combinations
thereof.
The term "angiogenic factor" also refers to functional analogues of the above-
mentioned factors. Such functional analogues include, for example, functional
peptides
or portions of the factors. Functional analogues also include anti-idiotypic
antibodies
which bind to the receptors of the factors and, thus, mimic the activity of
the factors in
promoting angiogenesis. Methods for generating such anti-idiotypic antibodies
are well
known in the art and are described, for example, in WO 97/23510, the contents
of which
are incorporated by reference herein.
Antigens have specific epitopes to which certain antibodies will bind. The
region
of the antibody that specifically interacts with the epitopes on the antigen
is called the
antigen combining site. The antigen combining site is composed of a collection
of
idiotopes which are unique sequences on the antibody that specifically
interact with the
epitopes on the antigen. The specific collection of idiotopes that interact
with the
epitopes on the antigen is defined as an antibody's "idiotype". Accordingly,
an anti-
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idiotype antibody is an antibody that is directed against the antigen
combining site of the
first antibody. Anti-idiotype antibodies combine with those specific sequences
and may
resemble or act as the epitope to which the first antibody reacts. For
example, one can
have an antibody that binds to specific epitopes on bFGF. One can then make a
second
antibody (an anti-idiotype antibody) that specifically interacts with antigen
combining
site of the first antibody. This anti-idiotype antibody may then mimic the
biological
activity of bFGF itself by binding to the bFGF receptor and activating it.
The use of anti-idiotype antibodies (as growth factor analogues) in the
methods
of the present invention can be advantageous in that many growth factors have
a very
short half life and therefore most of the factor that is given to a patient is
not utilized. In
contrast, antibodies have much greater half lives and therefore their potency
is
maintained for a greater length of time in vivo. In addition the levels of
growth factors
that are required to achieve a biological effect can, in certain instances,
produce adverse
reactions such as toxicity and hypotension. Since lower levels of anti-
idiotype
antibodies may be required to produce the same biological effect these adverse
side
effects may be prevented.
Angiogenic factors used in the present invention can be purified from their
native
sources or produced by recombinant expression and subsequently administered to
patients as a protein composition. Alternatively, the factors can be
administered in the
form of an expression plasmid encoding the factors, as is described in further
detail
below. The construction of suitable expression plasmids is well known in the
art.
Particular angiogenic expression plasmids for use in the invention are shown
in Figure
14 and are described in Example 1. Suitable vectors for constructing
expression
plasmids include, for example, adenoviral vectors, retroviral vectors, adeno-
associated
viral vectors, RNA vectors, liposomes, cationic lipids, lentiviral vectors and
transposons.
Accordingly, in one embodiment, the present invention provides novel methods
and compositions for promoting angiogenesis to promote angiogenesis and to
treat a
variety of tissue ischemias. Selected angiogenic factors or synergistic
combinations of
factors, functional analogues of such factors or combinations of factors, or
nucleic acids
encoding such factors or combinations of factors, are delivered to a localized
area of
tissue in an amount effective to induce angiogenesis within the area of
tissue.
In a preferred embodiment, the invention provides a method of promoting
angiogenesis comprising delivering PDGF-BB to a localized area of tissue in an
amount
effective to induce angiogenesis within the area of tissue. The PDGF-BB can be
delivered either alone or in combination with another angiogenesis-promoting
factor.
Particularly preferred combinations include PDGF-BB combined with bFGF and/or
VEGF-A.
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DELIVERY VEHICLES FOR ANGIOGENIC FACTORS
I. In another embodiment, the present invention provides a means for
delivering
angiogenic factors to a localized area of tissue in a controlled, sustained
fashion. One
problem in using purified (e.g., recombinant) angiogenic proteins to stimulate
angiogenesis for the treatment of myocardial and peripheral tissue ischemia
can be the
short half life of the protein upon injection in vivo. This problem is
addressed by way of
the present invention using slow-release heparin sepharose-containing
microcapsules in
an amount effective to induce angiogenesis within the area of tissue.
One property that,many angiogenic factors share is the ability to bind to
heparin,
a highly sulfated glycosaminoglycan that plays a role in anti-coagulation in
vivo. This
property is exploited in making the heparin sepharose containing microcapsules
of the
present invention which bind angiogenic factors and provide slow release of
the factors
when contacted (e.g., implanted) with localized tissue. The experimental
process for
making the microcapsules is described in detail in Example 3 and shown
schematically
in Figure 7.
In one embodiment, the microcapsules are injected or surgically implanted into
localized areas of tissue. In another embodiment, the microcapsules are
delivered to
localized areas of tissue using the NOGA system (Biosense). NOGA is~a 3
dimensional
catheter based transmyocardial injection system. A catheter is inserted into a
major
veinlartery and is snaked up into either of the ventricals of the heart. The
end of the
catheter contains a needle and a space in which therapeutic agents can be
inserted and
subsequently injected intra-myocardially into the damaged areas of the heart
muscle.
This delivery method is much easier and safer for the patient and is much more
efficacious than other methods of delivery including intracoronary injections
or placing
therapeutic agents against the wall of the heart. Due to the nature of the
system, only
therapeutic agents which can be physically delivered through a 25-27 gauge
needle can
be used. Thus macrocapsules described in the prior art cannot be used with
such a
system, whereas microcapsules of the present invention can be used with the
system.
The microcapsules are composed of single heparin sepharose beads which
optionally can be coated with a thin layer of alginate polymer. In a
particular
embodiment of the invention, the microcapsules are made up of uncoated heparin
sepharose beads, heparin sepharose beads coated with a single layer of
alginate polymer,
heparin sepharose beads coated with poly-ethylene glycol (PEG) polymer or
heparin
sepharose beads coated with alternating layers of alginate and PEG.
Using, for example, the coaxial air flow technology shown in Figure 7 and
described in Example l, the microcapsules can also be made small enough for
use in the
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NOGA delivery system. For example, the microcapsules typically range from 1-
200
microns in size. Moreover, they are able to absorb large quantities of
angiogenic factors,
such as FGF-2, VEGF-A and PDGF-BB, and slowly release the factors over
extended
periods of time at levels which axe able to stimulate the growth of new blood
vessels in
vivo.
II. In another embodiment, the present invention provides a means for
delivering
angiogenic factors to a localized area of tissue using a gradient of one or
more
angiogenic factors or a nucleic acid encoding one or more angiogenic factors,
such that
directed vascular growth along the gradient is achieved. Angiogenic factors,
such as
those described in the preceding examples, work by providing a gradient of
angiogenic
factor that stimulates the chemotaxis arid proliferation of endothelial cells,
and their
supporting cells towards the source of the factor. Regulating the growth of
new vessels
from existing vasculature (angiogenesis) that effectively bypass an arterial
lesion
requires strict spatial and temporal control. Accordingly, by forming a
directed gradient
of angiogenic factors in accordance with the present invention,
interconnection and/or
intraconnection of blood vessels (e.g., to circumvent blood flow around a
blockage
within a blood vessel) can be achieved.
In a particular embodiment, the angiogenic factor (or a nucleic acid encoding
the
factor) is released in a gradient using a biocompatible material which
contains the factor
such that the factor is released onto surrounding tissue when the
biocompatible material
is contacted with (e.g., implanted within) the tissue. This can be achieved by
treating
the biocompatible material with the angiogenic factor prior to contact with
(e.g.,
implantation into) a selected area of tissue in a manner which allows for
release of the
factor from the material in vivo. The biocompatible material is then contacted
with
(e.g., implanted into) a localized area of tissue in a configuration which
provides a
directed gradient of the angiogenic factor once it is released from the
material.
Suitable biocompatible materials for use in the invention include, for
example,
polymers or threads which incorporate the angiogenic factor. In a preferred
embodiment, the biocompatible material is an absorbable thread, such as
polyglyconate
monofilament, poliglecaprone 25-(Monocryl), polydiaxonone (PDS II),
polyglactin 910,
polyglycolic acid, Biodyn glycomer 631, chromic surgical gut or plain surgical
gut. The
biocompatible material can be coated with one or several angiogenic factors to
allow
delivery of growth factor or growth factor combinations that provide the
optimal
angiogenic stimulus. The biocompatible material can also be engineered to
release
certain growth factors at specific rates and at specific times that may help
mimic the
natural angiogenic process more closely.
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The invention is further illustrated by the following examples which should
not
be construed as limiting.
EQUIVALENTS
Although the invention has been described with reference to its preferred
embodiments, other embodiments can achieve the same results. Those skilled in
the art
will recognize or be able to ascertain using no more than routine
experimentation,
numerous equivalents to the specific embodiments described herein. Such
equivalents
are considered to be within the scope of this invention and are encompassed by
the
following claims.
INCORPORATION BY REFERENCE
The contents of all references and patents cited herein are hereby
incorporated by
reference in their entirety.
EXAMPLES
The following examples were perfomed to demonstrate and quantify the
angiogenic potential of PDGF-BB alone and in combination with other angiogenic
factors. A variety of assays were used, including the stringent Matrigel
assay, the mouse
cornea assay, and the ischemic rat heart assay, all as described in detail
below.
EXAMPLE 1 - Construction of Retroviral Vectors Containing Angiogenic cDNAs ,
Analysis of Virus Titer and Assessment of Stable Gene Transfer
In order to provide stable, high-level delivery of PDGF-BB in the Matrigel
model, primary myoblasts from C57B1/10 mice were transduced with retroviral
vectors
encoding human PDGF-BB. To compare the angiogenic potential of PDGF-BB to
other
known angiogenic agents, retroviral vectors encoding human VEGF-A165, VEGF-C,
VEGF-A, VEGF-D, PDGF-BB, or bFGF also were constructed and tested. All vectors
are shown schematically in Figure 14. Since VEGF-C, VEGF-D and PDGF-BB cDNAs
encode proteins which are inactive (or less active in the case of PDGF-BB) in
their non-
processed form, vectors containing cDNAs encoding the mature forms of the
aforementioned proteins linked to the powerful secretory signal from the
marine IgG
kappa immunoglobulin gene were constructed.
All vectors also contained the gene encoding the green fluorescent protein
(GFP)
to enable the fast efficient and non-toxic selection of transduced target
cells by
fluorescence activated cell sorting (FACS). FACS sorting is used to isolate
the brightest
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10% of GFP positive retrovirally transduced cells. Since both GFP and the
angiogenic
cDNA are translated from the same mRNA molecule, this ensures that the sorted
cells
also express high levels of the angiogenic protein. A strong correlation
between the
fraction of top GFP expressing cells and the amount of angiogenic protein
secreted by
the sorted cells exists. This result, in combination with data from Southern
blot analysis
of transduced cells which showed 3-6 proviral copies per genome in sorted GFP
positive
cells, indicates that the levels of angiogenic protein production and
secretion are at their
maximal level using this system.
All vectors were tested for stability of gene transfer and virus titer.
Vectors
demonstrated a virus titer ranging from approximately 5 x 105 - 1.2 x 106
infectious virus
particles per ml and all vectors showed stable transfer of the angiogenic cDNA
to target
' primary skeletal muscle myoblasts from C57B1/10 mice. To enable the easy
localization
and quantification of transduced myoblasts following injection in vivo, all
myoblast
samples were also marked by infection with a retroviral vector encoding a (3-
galactosidase/neomycin ((3-GEO) resistance fusion gene.
I. Characterization of the Protein Expression and Secretion Properties of
Retrovirally Transduced C57B1/10 Myoblasts
High-Ievel expression and secretion of the encoded angiogenic proteins from
transduced myoblast cells was demonstrated by either Western blot or ELISA
analysis
of supernatants from virally transduced myoblasts.
Angiogenic Factor Amount of Protein Secreted (ng/1 x 106 cells/24 hours
VEGF-A 88
VEGF-C 790
VEGF-D 580
PDGF-BB 56
bFGF 20
EXAMPLE 2 - Quantification of Angiogenic Stimulation by Transduced Myoblasts
Using the Stringent Matrigel Assay
The potency of the angiogenic proteins secreted from the transduced myoblasts
described in Example 1 was assessed in vivo using the stringent Matrigel
assay. In
brief, 3 x 105 - 2 x 106 transduced myoblasts were suspended in Matrigel and
injected
subcutaneously into the dorsal abdominal region of C57B1/10 mice. The Matrigel
pellets, in addition to a section of abdominal muscle adjacent to the Matrigel
pellet, were
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recovered 13 days post-injection. Following harvesting of the Matrigel pellet
and the
adjacent abdominal muscle 13 days post-injection, Matrigel pellets were
stained with X-
gal and analyzed for the presence of blue, retrovirally transduced myoblasts.
In addition,
the number of microvessels in the adjacent abdominal muscle was quantified by
visual
inspection.
A significant (p< 0.05) angiogenic response was observed for VEGF-A, VEGF-
C, PDGF-BB, and bFGF (see Figures 1 and 2). Transplantation transduced
myoblasts
secreting the aforementioned growth factors resulted in approximately a 4-5-
fold
increase in the number of microvessels observed. The most potent angiogenic
response
was observed for PDGF-BB, followed by VEGF-A and bFGF (Figures l and 2).
EXAMPLE 3 - Assessment of the Angiogenic Potential of PDGF-BB using the
Mouse Cornea Model
The potential of factors, such as PDGF-BB, as angiogenic agents was also
investigated using the mouse conlea model. Corneal micropockets were created
with a
cataract knife in the eye of 8-week old C57B1/6 mice. Into this pocket, a
0.34mm X
0.34mm sucrose aluminum sulfate pellet coated with hydron polymer containing
160ng
of recombinant human PDGF-BB, 160ng of human VEGF-A, or 80 ng of human bFGF
was implanted and mice were monitored daily. While those mice implanted with
control
pellets showed no evidence of angiogenesis, all mice receiving PDGF-BB coated
pellets
showed evidence of potent angiogenesis (see Figure 3). Thus, recombinant PDGF-
BB
protein can potently stimulate the growth of new vessels in both the mouse
Matrigel and
corneal assays. In contrast to the results obtained using the Matrigel assay,
PDGF-BB by
itself, although inducing a clear and potent angiogenic response, was less
potent than
either VEGF-A or bFGF (Figure 3). The differences observed for the Matrigel
and
mouse corneas models could be explained by the production of additional
endogenous
angiogenic factors by transduced myoblasts that synergize more readily with
PDGF-BB
compared to VEGF-A. Alternatively, the levels of recombinant VEGF-A and PDGF-
BB produced in vivo may differ to the levels produced in vitro.
Unexpectedly, the combination of bFGF and PDGF-BB proved to be, by fax, the
most potent combination (Figure 5), producing an angiogenic effect many times
greater
than the any factor alone (Figure 3) or bFGF and VEGF-A combined (Figure 4).
Moreover, the level of this synergistic effect appeared to be specific to PDGF-
BB and
bFGF since PDGF-BB combined with VEGF-A did not elicit nearly as potent an
effect
(see Figure 6). Therefore, the most potent combination of angiogenic factors
observed
was PDGF-BB and bFGF.
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EXAMPLE 4 - Therapeutic Effect of PDGF-BB Delivered Via Slow Release
Microcapsules for the Treatment of Myocardial Ischemia
The following studies were performed to assess the ability of PDGF-BB to
promote angiogenesis in vivo in the stringent Matrigel model and to improve
cardiac
function in ischemic rat models having myocardial infarction, using a slow
release
delivery system employing heparin-sepharose/alginate microcapsules. This
delivery
approach was based upon the ability of certain factors, such as bFGF and PDGF-
BB, to
bind strongly to heparin molecules both in vitro and in vivo.
Sepharose beads coated with heparin (approximately 50-150~,m in size) were
purchased from Pharmacia. The beads were sterilized using UV irradiation and
mixed
v~ith a 1.6% solution of alginate polymer. This polymer is able to form gels
through
chemical cross-linking with multivalent cations such as calcium. The procedure
for
making heparin-sephaxose/alginate capsules, shown in Figure 7, was as follows.
Sterilized heparin-sepharose beads were mixed with a 1.6% alginate solution
and the
mixture was loaded into a Sml syringe. The mixture was then extruded through a
needle
and a mist of hepaxin sepharose/alginate, produced using a coaxial air flow
system,
dropped into a wash bath of 1.5% calcium chloride solution. Once the alginate
hit the
calcium solution, the alginate became cross-linked, forming a solid gel
capsule in the
shape of a sphere. Once formed, the capsules were forced through a 250~,m
sieve,
washed twice in sterile water and stored in buffer composed of 0.9% sodium
chloride
and 1mM calcium chloride. Visual analysis of the capsules under the microscope
showed that the vast majority of microcapsules were composed of individual
heparin
sepharose beads coated with a thin layer of alginate.
Heparin-sepharose/alginate microcapsules were incubated overnight at 4 degrees
Celcius in binding buffer composed of 0.9% sodium chloride, 1mM calcium
chloride,
0.05% gelatin and 10~,g of recombinant PDGF-BB for 16 hours. The next day, the
binding buffer was removed from the microcapsules and analyzed by ELISA to
quantify
the amount of PDGF-BB absorbed by the capsules. In a typical experiment,
approximately 75-90% of the PDGF-BB protein is absorbed by the microcapsules
(see
Figure 8). Next, the heparin-sepharoselalginate microcapsules were washed
twice in
fresh binding buffer and either placed in vitro to assess release kinetics or
injected into
the myocardium of rats that had undergone surgically induced myocardial
infarction.
To assess the release kinetics of the bound PDGF-BB in vitro, three thousand
heparin-sepharose/alginate microcapsules or three thousand non-alginate
encapsulated
heparin sepharose beads containing 9~,g of bound PDGF-BB were placed in serum
free
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medium and incubated at 37 degrees Celcius. Every 24 hours the medium was
changed
and the amount of PDGF-BB present in the medium quantified by ELISA. The
results
showed a slow, sustained release of approximately 0.5-3% of the total bound
PDGF-BB
for a minimum of 14 days, the longest time point analyzed (see Figure 9).
Importantly,
the proportion of PDGF-BB released per day was equivalent to the amount of
PDGF-BB
that we estimated to be secreted by muscle cells transduced with the PDGF-BB
retrovirus in the Matrigel experiments described in Example 1.
The ability of the heparin-sepharose/alginate microcapsules to stimulate
angiogenesis in vivo was assessed using the stringent Matrigel assay. Three
thousand
microcapsules loaded with 1 ~,g or 10~g of PDGF-BB were mixed with 4001 of
Matrigel and subcutaneously injected into the abdominal region of C57B1/10
mice.
Thirteen days later mice were sacrificed, the pellets and a section of the
adjacent
abdominal muscle was removed, fixed, sectioned and the number of microvessels
quantified by visual inspection of the sections under the microscope. As shown
in
Figure 10, the number of microvessels in mice receiving microcapsules loaded
with
l Omg of PDGF-BB was 2.5-fold greater than that of control mice.
In addition, as shown in Figure 13, PDGF-BB and bFGF delivered by slow
release microcapsules synergize to stimulate angiogensis in vivo in the
stringent
Matrigel model. Three thousand microcapsules loaded with 1 ~,g of bFGF were
mixed
with 400.1 of Matrigel and subcutaneously injected into the abdominal region
of
C57B1/10 mice. Thirteen days later mice were sacrificed, the pellets and a
section of the
adjacent abdominal muscle was removed, fixed, sectioned and the number of
microvessels quantified by visual inspection of the sections under the
microscope.
Figure 13 shows that the number of microvessels in mice receiving bFGF + PDGF-
BB
' microcapsules was 4-fold greater than that of mice implanted with either
growth factor
alone.
PDGF-BB microcapsules were also tested for their ability to stimulate
angiogenesis in infaxcted rat hearts 3 weeks post-injection. Infarcted rat
heaxts were
injected with 1600 microcapsules containing ~,g (control) or 18~,g of PDGF-BB
in a
volume of 201. Three weeks post injection rats were sacrificed, hearts were
removed,
fixed, sectioned and the number of microvessels within the infarct region
quantified by
visual inspection under a microscope (i.e., number of microvessels per 5 high
power
fields for recipients of control and PDGF-BB microcapsules). As shown in
Figure 11,
rats injected with PDGF-BB microvessels showed an approximate 2-fold increase
in the
number of microvessels as compared to control rats.
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The ability of the heparin-sepharose/alginate microcapsules to stimulate
angiogenesis in vivo was also assessed using the ischemic rat heart model as
follows.
Adult male rats were anesthetized, intubated and ventilated with a Harvard
respirator.
Under sterile conditions, a left lateral thoractomy was performed. The heart
was exposed
and the left descending coronary artery was ligated with a 8-0 Prolene suture.
Immediately after infarction each heart was injected twice intramyocaridally
with 10,1
of a buffer suspension containing approximately 800 heparin sepharose/alginate
microcapsules with 9~g of absorbed recombinant human PDGF-BB protein. Thus, a
total of 1600 microcapsules containing 18~,g of human PDGF-BB protein were
injected
into each rat heart. The lungs were then inflated and the wound was closed in
layers.
Three weeks later, cardiac function was assessed using a variety of parameters
including left ventricular pressure (LVP), dP/dT (a measure of cardiac
contractility),
negative dP/dT (a measure of relaxation of the cardiac muscle) and tau (the
relaxation
constant) (see Figure 12). In rats injected with PDGF-BB microcapsules, a 25%
increase in left ventricular pressure was detected (see Figure 12). Moreover,
cardiac
contractility/relaxation increased 2.5 - 3-fold while the relaxation constant,
tau, was
decreased by approximately 3-fold (Figure 12). Thus, a significant improvement
in all
parameters was detected in rats injected with PDGF-BB microcapsules.
EXAMPLE 5 - Directed Delivery of Angiogenic Factors Using Biocompatible
Threads
Regulating the growth of new vessels from existing vasculature (angiogenesis)
that effectively bypass an arterial lesion requires strict spatial and
temporal control.
Angiogenic factors, such as those described in the preceding examples, work by
providing a gradient of angiogenic factor that stimulates the chemotaxis and
proliferation of endothelial cells, and their supporting cells towards the
source of the
factor.
Today, most efforts to stimulate the growth of new vessels involve the
injection
of proteins into or near affected areas. Such injections can result in the
spread of the
angiogenic factor over a large area, greatly diminishing their efficacy, and
can dilute the
angiogenic stimulus over a large region resulting in an unorganized hodge-
podge of new
vessels that do not provide any therapeutic benefit.
To solve this problem, biocompatible absorbable threads containing angiogenic
factors can be employed to provide a small, highly localized and orderly
gradient of
angiogenic factors in the appropriate and crucial areas. This enables the
creation of a
"molecular road map" that directs the growth of new vessels from around the
site of the
arterial occlusion to join again at a point below or downstream of the
blockage. To
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achieve this, absorbable surgical threads can be coated with the appropriate
angiogenic
factors (e.g., PDGF-BB, FGF-2, VEGF-A and PDGF-B). Such threads can then be
surgically placed at the site of arterial occlusion such that they provide a
clear spatial
direction and gradient of angiogenic factors) to direct the generation of new
vessels
around the block.
The biocompatible threads can be coated with one or several angiogenic factors
to allow delivery of growth factor or growth factor combinations that provide
the
optimal angiogenic stimulus. Moreover, such threads can be engineered to
release
certain growth factors at specific rates and at specific times that may help
mimic the
natural angiogenic process more closely.
EXAMPLE 6 - Treatment of Ischemia Using Angiogenic Expression Plasmids
Figure 14 shows a variety of expression plasmids encoding angiogenic factors
that can be administered directly to localized areas of tissue to promote
angiogenesis.
To show that the plasmids encode biologically active PDGF-BB protein,
supernatant from 293T cells transiently transfected with the PDGF-BB
expression
plasmid was added to NIH 3T3 cells growing under serum free conditions.
Seventy two
hours later cells were trypsinized, spun down and counted using a
hemocytometer. The
results showed that the PDGF-BB supernatant specifically and potently induced
the
proliferation of NIH3T3 cells.
To analyze cardiac function following administration of expression plasmids in
vivo, test animals (e.g., rats) can be injected with control vs. angiogenic
plasmids (e.g.,
PDGF-BB expression plasmids) following myocardial infarction. To achieve this,
test
animals can be anesthetized and intubated. The chest wall is opened and a
myocardial
infarct is created by tying off the anterior descending artery. 180~g of
control or test
expression plasmid is injected into the heart wall in a volume of 20,1.
Cardiac function
is assessed 3 weeks post injection. Animals are sacrificed, the heart is
removed and
efficiency of plasmid uptake is assessed by staining with X-gal. The size of
the infarct
and the extent of angiogenesis is quantified.
