Note: Descriptions are shown in the official language in which they were submitted.
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to WOUND TREATMENT THROUGH INHIBITION OF
ADENOSINE DIPHOSPHATE RIBOSYL TRANSFERASE
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention pertains to a method for healing a wound in a
mammal which comprises the steps of (A) providing a therapeutic wound healing
composition comprising a therapeutically effective amount of an inhibitor of
mono-
adenosine diphosphate-ribosyl transferase to inhibit adenosine
diphosphate-ribosylation of vascular endothelial growth factor; and (B)
contacting the
therapeutic wound healing composition with a wound in a mammal. This invention
also pertains to wound healing compositions and to methods for preparing and
using
the wound healing compositions and the pharmaceutical products in which the
therapeutic compositions may be used. This invention further pertains to
therapeutic
dermatological-wound healing compositions useful to minimize and treat diaper
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2
dermatitis and to methods for preparing and using the therapeutic
dermatological-
wound healing compositions.
Description of the Background
The disclosures referred to herein to illustrate the background of the
invention and to provide additional detail with respect to its practice are
incorporated
Io herein by reference and, for convenience, are referenced in the following
text and
respectively grouped in the appended bibliography.
Wounds are internal or external bodily injuries or lesions caused by
physical means, such as mechanical, chemical, viral, bacterial, or thermal
means, which
disrupt the normal continuity of structures. Such bodily injuries include
contusions,
wounds in which the skin is unbroken, incisions, wounds in which the skin is
broken
by a cutting instrument, and lacerations, wounds in which the skin is broken
by a dull
or blunt instrument. Wounds may be caused by accidents or by surgical
procedures.
Patients who suffer major or chronic wounds could benefit from an enhancement
in
2o the wound healing process. Wound healing consists of a series of processes
whereby
injured tissue is repaired, specialized tissue is regenerated, and new tissue
is
reorganized. Wound healing consists of three major phases: a) an inflammation
phase
(0-3 days), b) a cellular proliferation phase (3-12 days), and (c} a
remodeling phase
(3 days-6 months). During the inflammation phase, platelet aggregation and
clotting
form a matrix which traps plasma proteins and blood cells to induce the influx
of
various types of cells. During the cellular proliferation phase, new
connective or
granulation tissue and blood vessels are formed. During the remodeling phase,
granulation tissue is replaced by a network of collagen and elastin fibers
leading to the
formation of scar tissue.
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Macrophages play a key role in the induction of angiogenesis in
fibroproliferative states, including wound repair, rheumatoid arthritis, and
solid tumor
development (1-5). Production of angiogenic activity by macrophages depends on
the
balance of production of positive angiogenic regulators and inhibitors of
angiogenesis
(6,7,8). Positive angiogenic regulators previously shown to be produced by
monocytes and macrophages include the cytokines TNFa and II-8 (9,10,11 );
negative
regulators include thrombospondin-1, Ifny -inducible protein-10 (~yIP-10) and
other as
yet uncharacterized protein inhibitors ( 12,13,14). The mechanisms controlling
the
balance of positive and negative angiogenesis regulators are not well
understood.
1o Non-activated monocytes and macrophages exhibit a non-angiogenic phenotype
(1,4).
Following activation with agents such as interferon-'y and/or endotoxin (LPS),
macrophages express angiogenic activity, characterized by the expression of
angiogenic cytokines, as well as of inhibitors of angiogenesis ( 15,16,17,18).
Activated cells also produce and release oxygen radicals, nitric oxide (NO)
and their
derivatives (17,19). These radicals have been shown to play an important role
in
regulating the angiogenic phenotype of activated macrophages (20,21). Agents
such
as Ifny and LPS, as well as reduced oxygen tension (hypoxia) and elevated
lactate
levels, induce macrophages to express angiogenic activity (1-3,9,22).
Recently,
macrophages in vivo have been shown to express vascular endothelial growth
factor
(VEGF), an endothelial-specific mitogen that is potently angiogenic (18,23-
30).
United States patent no. 5,510,391 (Elson) discloses a method of
treating blood vessel disorders of the skin and skin disorders caused by photo-
aging
comprising: a) coformulating a pharmaceutical composition wherein the
composition
contains from 0.01 % to 50% vitamin K; and b) applying the pharmaceutical
composition topically to treat blood vessel disorders of the skin and skin
disorders
caused by photo-aging. The blood vessel disorders of the skin and skin
disorders
caused by photo-aging includes actinic and iatrogenic purpura, lentigines,
telangiectasias of the face, spider angiomas, spider veins of the face and
leg.
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SUMMARY OF THE INVENTION
The present invention pertains to a method for healing a wound in a
mammal which comprises the steps of
(A) providing a therapeutic wound healing composition comprising a
therapeutically ei~ective amount of an inhibitor of mono-adenosine
diphosphate-ribosyl transferase to inhibit adenosine diphosphate-ribosylation
of
vascular endothelial growth factor; and
(B) contacting the therapeutic wound healing composition with a wound in a
mammal.
In a preferred embodiment, the mammal is man. In another preferred
embodiment, the inhibitor of mono-adenosine diphosphate-ribosyl transferase is
selected from the group consisting of Vitamin K1, Vitamin K2, Vitamin K3,
Vitamin
K4, Vitamin KS, Vitamin K6, Novobiocin, m-iodo benzyl guanidine, nicotinamide,
coumermycin, dicoumarol, and silybin. More preferred inhibitors of mono-
adenosine
diphosphate-ribosyl transferase are Vitamin K1, Vitamin K3, Novobiocin, and
silybin.
2o The inhibitor of mono-adenosine diphosphate-ribosyl transferase is present
in the
therapeutic wound healing composition in an amount from about 0.1% to about
10%,
by weight of the therapeutic wound healing composition. The wound may be
selected
from the group consisting of pressure ulcers, decubitus ulcers, diabetic
ulcers, and
burn injuries. The therapeutic wound healing composition may further comprise
a
pharmaceutically acceptable carrier.
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The present invention also pertains to a wound healing composition
which comprises:
(A) a therapeutically effective amount of an inhibitor of mono-adenosine
diphosphate-ribosyl transferase to inhibit adenosine diphosphate-ribosylation
of
5 vascular endothelial growth factor; and
(B) a pharmaceutically acceptable Garner.
The present invention further pertains to a method for treating diaper
dermatitis in a human which comprises the steps of:
(A) providing a therapeutic diaper dermatitis wound healing composition
comprising:
(a) a therapeutically effective amount of an inhibitor of mono-adenosine
diphosphate-ribosyl transferase to inhibit adenosine diphosphate-ribosylation
of
vascular endothelial growth factor;
(b) a buffering agent to maintain the pH of dermatitis in a range from about 5
to about 8; and
(c) an anti-inflammatory agent; and
(B) contacting the therapeutic diaper dermatitis wound healing composition
with diaper dermatitis in a human.
The present invention further pertains to a therapeutic dermatological-
wound healing composition useful to minimize and treat diaper dermatitis which
comprises a therapeutically effective amount of
(1) a therapeutic wound healing composition comprising an inhibitor of mono-
adenosine diphosphate-ribosyl transferase to inhibit adenosine
diphosphate-ribosylation of vascular endothelial growth factor;
(2) a buffering agent to maintain the pH of dermatitis in a range from about 5
to about 8; and
(3) an anti-inflammatory agent.
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BRIEF DESCRIPTION OF THE FIGURES
Figure 1 illustrates the nitrite production by MPMs.
Figure 2 illustrates VEGF production by A) RAW264.7 cells, and B)
MPMs.
to Figure 3 illustrates competitive RT-PCR analysis of VEGF mRNA
levels in control (non-stimulated) MPMs 24 hours following plating.
Figure 4 illustrate RT-PCR analysis of VEGF isoforms produced by
Ifny/LPS-activated MPMs, with or without AG treatment.
Figure 5 illustrates TNFa production by MPMs.
Figure 6 illustrates ADP-Ribosylation of rVEGF165 bY bacterial toxins
and by macrophage cytosolic extract.
DETAILED DESCRIPTION OF THE INVENTION
Production of macrophage-dependent angiogenic activity (MDAA)
requires activation by factors such as Interferon-~y and/or endotoxin, hypoxia
or high
concentrations of lactate (Jensen et al. Lab. Invest. 54, 574, 1986). Previous
work
has demonstrated that the inducible nitric oxide synthase (iNOS) pathway in
macrophages regulates MDAA, with inhibition of iNOS down-regulating expression
of MDAA (Leibovich et al, PNAS USA 91, 4190, 1994). It has now been found that
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7
although non-activated macrophages are non-angiogenic, they nevertheless
express
significant levels of the angiogenic growth factor VEGF. This constitutive
expression
of VEGF is not hypoxia or lactate dependent. The VEGF produced constitutiveIy
by
normoxic, non-activated macrophages is found to be in a non-angiogenic form,
due to
post-translational modification by the process of arginine-specific ADP-
ribosylation.
In contrast, VEGF produced by LPS-activated, hypoxic, or lactate-treated
macrophages is in the non-ADP-ribosylated form, and is angiogenic. Inhibition
of the
iNOS pathway in LPS-activated macrophages abrogates MDAA expression by a dual
mechanism. First, VEGF reverts to the ADP-ribosylated, non-angiogenic state;
1o second, iNOS-inhibited macrophages express an anti-angiogenic factor that
blocks the
angiogenic activity of several angiogenic factors, including VEGF, TNFa and
bFGF.
In mice where the iNOS gene has been specifically deleted (iNOS knockout mice,
iNOS-/-), wound repair is markedly inhibited (Yamasaki et al., J. Clin.
Invest. 101,
967, 1998). This inhibition is manifested in delayed wound closure, and a
delay in the
formation of granulation tissue. Macrophages from iNOS-/- mice express reduced
levels of 1VIDAA in comparison to iNOS+/+ mice, although total VEGF production
is
not markedly altered. The role of the iNOS pathway and ADP-ribosylation of
VEGF
in regulating angiogenesis in wound repair and the modalities for
pharmacologically
modulating macrophage-dependent angiogenic activity and wound repair by
targeting
2o iNOS and ADP-ribosylation pathways are under investigation.
Murine thioglycolate-induced peritoneal macrophages (MPMs) and the
murine RAW264.7 macrophage-like cell line (RAW cells) constitutively produce
vascular endothelial growth factor (VEGF). VEGF production is increased under
hypoxic conditions or following cell activation with interferon-y (Ifny) and
endotoxin
(LPS). In contrast, TNFa, is produced only by Ifriy/LPS-activated cells.
Lactate
(20~ does not increase VEGF production by these cells. However, hypoxia,
lactate, and Ifny/LPS-activated MPMs express angiogenic activity, while
normoxic,
non-activated MPMs do not. Lack of angiogenic activity is not due to an anti-
3o angiogenic factors) in the medium of these cells. Angiogenic activity
produced by
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hypoxia and lactate-treated MPMs is neutralized by anti-VEGF antibody, which
also
neutralizes most of the angiogenic activity produced by Ifny/LPS-activated
MPMs.
The inducible nitric oxide synthase (iNOS) inhibitors Ng vitro-L-arginine-
methyl ester
(L-NAME) ( 1. SmM) and aminoguanidine (AG)( 1 mM) block production of
angiogenic activity by MPMs and RAW cells. In RAW cells, L-NAME and AG block
Ifny/LPS-activated, but not constitutive VEGF production, while in MPMs,
neither
constitutive nor Ifny/LPS-activated VEGF synthesis is affected. Synthesis of
TNFa is
also unaffected. In contrast to normoxic, non-activated MPMs, iNOS-inhibited,
Ifny/LPS-activated MPMs produce an anti-angiogenic factor(s). Accordingly,
VEGF
1o is a major contributor to macrophage-derived angiogenic activity, and that
activation
by hypoxia, lactate or Ifny/LPS switches macrophage-derived VEGF from a non-
angiogenic to an angiogenic state. This switch may involve a post-
translational
modification of VEGF, possibly by the process of ADP-ribosylation. ADP-
ribosylation by MPM cytosolic extracts or by cholera toxin switches rVEGF165
from
1s an angiogenic to a non-angiogenic state. In Ifny/LPS-activated MPMs, the
iNOS-
dependent pathway also regulates the expression of an anti-angiogenic factors)
that
antagonizes the bio-activity of VEGF and provides an additional regulatory
pathway
controlling the angiogenic phenotype of macrophages.
2o In accord with the present invention, the expression of the angiogenic
growth factor VEGF by MPMs and RAW cells was examined, and compared with
that of TNFa. The effects of hypoxia, lactate and the L-arginine-dependent
inducible
NO-synthase (iNOS) pathway on the production of VEGF and TNFa by these cells
was also examined. VEGF production was found to be regulated both
25 transcriptionally and translationally by hypoxia and the iNOS pathway, and
post-
translational modification may play an important role in regulating the bio-
activity of
VEGF as an angiogenic factor. In addition, the iNOS pathway in Ifny/LPS-
activated
macrophages regulates the expression of anti-angiogenic factor that
antagonizes the
angiogenic effects of VEGF, providing an additional regulatory pathway to
control
3o the angiogenic phenotype of macrophages.
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Vascular endothelial growth factor (VEGF), a polypeptide growth
factor that is potently angiogenic (induces the growth of new blood vessels),
is
chemically modified by macrophages (a cell that plays a key role in regulating
angiogenesis) in wound repair. This chemical modification involves the
ADP-ribosylation of VEGF by enzymes (ADP-ribosyl transferases) in macrophages.
In particular, the cytoplasmic mono-ADP-ribosyl transferases are involved in
ADP-ribosylation of VEGF, and this modification results in a change in the
properties
of the VEGF from being angiogenic to being non-angiogenic. Macrophages make
to VEGF constitutively, and it seems that macrophages regulate the angiogenic
activity
of VEGF by this ADP-ribosylation reaction. Inhibitors of mono-ADP-ribosylation
such as: Vitamin K1, Vitamin K2, Vitamin K3, Novobiocin, m-iodo benzyl
guanidine,
and nicotinamide change the phenotype of macrophages from a non-angiogenic to
an
angiogenic phenotype by inhibiting the ADP-ribosylation of VEGF. Since
angiogenesis induction is a key event in normal wound repair, VEGF production
by
macrophages, the key cells that control angiogenesis by producing angiogenic
factors,
must require a switch from the non-angiogenic to the angiogenic phenotype,
that is,
from the ADP-ribosylated to the non-modified form.
2o Since the inhibitors of mono-ADP-ribosylation can block the
ADP-ribosylation of VEGF, these inhibitors, and their derivatives and analogs
are
valuable in the treatment of chronic, non-healing wounds, where angiogenesis
is
deficient. In many chronic wounds, including but not limited to, pressure
ulcers,
decubitus ulcers, diabetic ulcers, and certain burn injuries, wounds fail to
heal, at least
in part due to failures in angiogenesis. The macrophage phenotype in these
wounds
may be non-angiogenic, with VEGF being produced in the non-angiogenic,
ADP-ribosylated form. In this case, treatment of these wounds with inhibitors
of
ADP-ribosylation would block the ADP-ribosylation of VEGF, and thus result in
the
production of non-modified, angiogenic VEGF. This VEGF should then participate
in
3o stimulating angiogenesis in the wounds, and help promote repair.
Accordingly, the
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present invention is directed to a formulation of ADP-ribosyl transferase
inhibitors in
an appropriate vehicle suitable for local application to wounds.
Present technology for the treatment of chronic wounds generally
5 involves intensive wound care, debridement, use of antiseptics, antibiotics,
and the use
of occlusive dressings. Technologies in development include the use of growth
factors, usually prepared by genetic engineering using recombinant DNA
technology.
Growth factor therapy is currently in clinical trials. Growth factors are
extremely
expensive, and their efficacy is still in doubt. The advantages of the use of
ADP-
to ribosylation inhibitors for the treatment of chronic wounds are: a) the
compounds are
low molecular weight, well characterized, available, and relatively cheap; b)
the
compounds modulate the bio-activity of the wound's own biological mediators,
shifting them from being non-angiogenic to being angiogenic, rather than
attempting
to introduce an exogenous growth factor activity; c) formulation of low
molecular
weight inhibitors for delivery to wounds should be a relatively simple
exercise,
certainly compared to the formulation of growth factors; and d) vitamin-K
compounds, which constitute one of the major groups of mono-ADP ribosylation
inhibitors, have been available for other purposes for many years, and have
FDA
approval.
As set out above, the present invention is directed to a method for
healing a wound in a mammal which comprises the steps of (A) providing a
therapeutic wound healing composition comprising a therapeutically effective
amount
of an inhibitor of mono-adenosine diphosphate-ribosyl transferase to inhibit
adenosine
diphosphate-ribosylation of vascular endothelial growth factor; and (B)
contacting the
therapeutic wound healing composition with a wound in a mammal.
The inhibitor of mono-adenosine diphosphate-ribosyl transferase may
be any inhibitor, including active derivatives and analogs, which inhibits
3o ADP-ribosylation of vascular endothelial growth factor, thereby switching
vascular
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endothelial growth factor from the non-angiogenic form to the angiogenic
phenotype,
that is, from the ADP-ribosylated form to the non-ADP-ribosylated form.
Preferably,
the inhibitor of mono-adenosine diphosphate-ribosyl transferase is selected
from the
group consisting of Vitamin K1, Vitamin K2, Vitamin K3, Vitamin K4, Vitamin
K5,
Vitamin K6, Novobiocin, m-iodo benzyl guanidine, nicotinamide, coumermycin,
dicoumarol, and silybin. More preferably, the inhibitor of mono-adenosine
diphosphate-ribosyl transferase is selected from the group consisting of
Vitamin K1,
Vitamin K3, Novobiocin, and silybin.
to The amount of inhibitor of mono-adenosine diphosphate-ribosyl
transferase present in the therapeutic wound healing compositions of the
present
invention is a therapeutically effective amount. A therapeutically ei~ective
amount of
inhibitor of mono-adenosine diphosphate-ribosyl transferase is that amount of
inhibitor
of mono-adenosine diphosphate-ribosyl transferase necessary for the inventive
composition to switch the vascular endothelial growth factor from the non-
angiogenic
form to the angiogenic phenotype, that is, from the ADP-ribosylated form to
the
non-ADP-ribosylated form, and thereby promote wound healing. The exact amount
of inhibitor of mono-adenosine diphosphate-ribosyl transferase is a matter of
preference subject to such factors as the type of condition being treated as
well as the
other ingredients in the composition. In a preferred embodiment, inhibitor of
mono-
adenosine diphosphate-ribosyl transferase is present in the therapeutic wound
healing
composition in an amount from about 0.1% to about 10%, preferably from about
0.2% to about 8%, and more preferably from about 0.3% to about 5%, by weight
of
the therapeutic wound healing composition.
The types of wounds which may be healed using the wound healing
compositions of the present invention are those which result from an injury
which
causes epidermal damage such as incisions, wounds in which the skin is broken
by a
cutting instrument, and lacerations, wounds in which the skin is broken by a
dull or
3o blunt instrument. The therapeutic compositions may be used to treat
pressure ulcers,
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decubitus ulcers, diabetic ulcers, and burn injuries. The therapeutic
compositions may
also be used to treat various dermatological disorders such as hyperkeratosis,
burns,
donor site wounds from skin transplants, ulcers (cutaneous, decubitis, venous
stasis,
and diabetic), psoriasis, skin rashes, and sunburn photoreactive processes.
The wound
healing compositions can be used for the following indications: a) Healing of
cuts and
scrapes; b) Burns (heals burns with less scaring and scabbing); c) Decubitus
ulcers; d)
Bed sores, pressure ulcers; e) Fissures, Hemorrhoids; f) Use in combination
with
immunostimulators (simulated healing in healing deficient people); g) Post
surgical
wounds; h) Bandages; i) Diabetic ulcers; j) Venous ulceration; and k) Use in
1o combination with wound cleansing agents. Preferably, the therapeutic
compositions
may be used to treat pressure ulcers, decubitus ulcers, diabetic ulcers, and
burn
injuries.
In another embodiment, the present invention is directed to a wound
healing composition which comprises (A) a therapeutically effective amount of
an
inhibitor of mono-adenosine diphosphate-ribosyl transferase to inhibit
adenosine
diphosphate-ribosylation of vascular endothelial growth factor; and (B) a
pharmaceutically acceptable carrier, wherein the amount and type of inhibitor
of
mono-adenosine diphosphate-ribosyl transferase to inhibit adenosine
2o diphosphate-ribosylation of vascular endothelial growth factor are set out
above.
In a specific embodiment, the present invention is directed to diaper
dermatitis. Diaper dermatitis, or diaper rash, is an irritant contact
dermatitis localized
to the skin area in contact with the diaper in infants. Diaper dermatitis
occurs in about
65% of infants ranging from one to 20 months of age. The manifestations of
diaper
dermatitis vary from diffuse erythema to nodular lesions. Prolonged contact of
the
skin with urine-soaked diapers results in maceration of the epidermis.
Occlusive
rubber or plastic pants further aggravates the injury. Diaper dermatitis is
caused by
ammonia from the urine raising the pH of the skin and combining with
constituents of
3o skin oil to form irritants. Bacterial or yeast infections may further
complicate diaper
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dermatitis by causing persistent and severe inflammation. Diaper dermatitis is
generally treated by keeping the skin dry by changing diapers frequently and
applying
talcum powder to the irntated area. In severe cases, rubber pants and plastic
disposable diaper coverings should be avoided.
s
In accord with the present invention, a method for treating diaper
dermatitis in a human is provided which comprises the steps of (A) providing a
therapeutic diaper dermatitis wound healing composition comprising: (a) a
therapeutically effective amount of an inhibitor of mono-adenosine
1o diphosphate-ribosyl transferase to inhibit adenosine diphosphate-
ribosylation of
vascular endothelial growth factor; (b) a buffering agent to maintain the pH
of
dermatitis in a range from about 5 to about 8; and (c) an anti-inflammatory
agent; and
(B) contacting the therapeutic diaper dermatitis wound healing composition
with
diaper dermatitis in a human. Buffering agents can help prevent diaper
dermatitis by
is neutralizing ammonia but do not heal injured mammalian cells. Anti-
inflammatory
agents can reduce inflammation (erythema) in a patient but do not promote the
wound
healing process. Wound healing compositions can increase the resuscitation
rate of
injured mammalian cells and the proliferation rate of new manunalian cells to
replace
dead cells. Applicants have found that the combination of a buffering agent,
an anti-
2o inflammatory agent, and a wound healing composition results in a
therapeutic
dermatological-wound healing compositions useful for minmizing and treating
diaper
dermatitis. The dermatological-wound healing compositions may optionally
contain a
therapeutically effective amount of a topical antiseptic to further reduce the
duration
and severity of diaper dermatitis.
2s
Buffering agents are solute compounds which will form a solution to
which moderate amounts of either a strong acid or base may be added without
causing
a large change in the pH value of the solution. In Bronsted's terminology, a
buffering
agent contains both a weak acid and its conjugate weak base. Buffering
solutions
3o usually contain (a) a weak acid and a salt of the weak acid, (b) a mixture
of an acid
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salt with the normal salt, or (c) a mixture of two acid salts, for example
NaH2P04 and
Na2HP04. A weak acid becomes a buffering agent when alkali is added and a weak
base becomes a buffering agent when acid is added. The buffering agents in the
dermatological-wound healing compositions of the present invention may be
selected
from a wide range of therapeutic agents and mixtures of therapeutic agents.
Buffering
agents which occur in nature include phosphates, carbonates, ammonium salts,
proteins of plant and animal tissues, and the carbonic-acid-bicarbonate system
in
blood. Noniimiting illustrative specific examples of buffering agents include
citric
acid-sodium citrate solution, phosphoric acid-sodium phosphate solution, and
acetic
to acid-sodium acetate solution. Preferably, the buffering agent is phosphoric
acid-
sodium phosphate.
The amount of buffering agent used in the present invention is an
effective amount and may vary depending upon the dosage recommended or
permitted
for the particular buffering agent. In general, the amount of buffering agent
present is
the ordinary dosage required to obtain the desired result. Such dosages are
known to
the skilled practitioner in the medical arts and are not a part of the present
invention.
In a preferred embodiment, the buffering agent in the dermatological-wound
healing
composition is present in an amount to maintain the pH of the dermatitis in a
range
2o from about 5 to about 8, preferably from about 5.5 to about 7.5, and more
preferably
from about 6 to about 7.
Anti-inflammatory agents are compounds that counteract or suppress
the inflammatory process. The anti-inflammatory agents in the dermatological-
wound
healing compositions of the present invention may be selected from a wide
variety of
steroidal, non-steroidal, and salicylate water-soluble and water-insoluble
drugs and
their acid addition or metallic salts. Both organic and inorganic salts may be
used
provided the anti-inflammatory agent maintains its medicament value. The anti-
inflammatory agents may be selected from a wide range of therapeutic agents
and
3o mixtures of therapeutic agents which may be administered in sustained
release or
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prolonged action form. Nonlimiting illustrative specific examples of non-
steroidal
anti-inflammatory agents include the following medicaments: ibuprofen,
naproxen,
sulindac, diflunisal, piroxicam, indomethacin, etodoiac, meclofenamate sodium,
fenoproben calcium, ketoprofen, mefenamic acid, nabumetone, ketorolac
5 tromethamine, diclofenac, and evening primrose oil (containing about 72%
linoleic
acid and about 9% gamma-linolenic acid). Nonlimiting illustrative specific
examples
of salicylate anti-inflammatory agents include the following medicaments:
acetylsalicylic acid, mesalamine, salsalate, diflunisal, salicylsalicylic
acid, and choline
magnesium trisalicylate. Nonlimiting illustrative specific examples of
steroidal anti-
1o inflammatory agents include the following medicaments: flunisolide,
triamcinoline,
triamcinoline acetonide, beclomethasone diproprionate, betamethasone
diproprionate,
hydrocortisone, cortisone, dexamethasone, predinisone, methyl prednisolone,
and
prednisolone.
15 Preferred anti-inflammatory agents to be employed may be selected
from the group consisting of ibuprofen, naproxen, sulindac, diflunisal,
piroxicam,
indomethacin, etodolac, meclofenamate sodium, fenoproben calcium, ketoprofen,
mefenamic acid, nabumetone, ketorolac tromethamine, diclofenac, evening
primrose
oil, acetylsalicylic acid, mesalamine, salsalate, diflunisal, salicylsalicylic
acid, choline
2o magnesium trisalicylate, flunisolide, triamcinoline, triamcinoline
acetonide,
beclomethasone diproprionate, betamethasone diproprionate, hydrocortisone,
cortisone, dexamethasone, predinisone, methyl prednisolone, and prednisolone.
In a
preferred embodiment, the anti-inflammatory agent is selected from the group
consisting of ibuprofen, naproxen, sulindac, diflunisal, piroxicam,
indomethacin,
etodolac, meclofenamate sodium, fenoproben calcium, ketoprofen, mefenamic
acid,
nabumetone, ketorolac tromethamine, diclofenac, and evening primrose oil. In a
more
preferred embodiment, the anti-inflammatory agent is evening primrose oil.
The anti-inflammatory agent of the present invention may be used in
3o many distinct physical forms well known in the pharmaceutical art to
provide an initial
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16
dosage of the anti-inflammatory agent and/or a further time-release form of
the anti-
inflammatory agent. Without being limited thereto, such physical forms include
free
forms and encapsulated forms, and mixtures thereof.
The amount of anti-inflammatory agent used in the present invention is
a therapeutically effective amount and may vary depending upon the therapeutic
dosage recommended or permitted for the particular anti-inflammatory agent. In
general, the amount of anti-inflammatory agent present is the ordinary dosage
required to obtain the desired result. Such dosages are known to the skilled
1o practitioner in the medical arts and are not a part of the present
invention. In a
preferred embodiment, the anti-inflammatory agent in the dermatological-wound
healing composition is present in an amount from about 0.01 % to about 10%,
preferably from about 0.1% to about 5%, and more preferably from about 1% to
about 3%, by weight.
In another specific embodiment, the present invention is directed to a
therapeutic dermatological-wound healing composition useful to minimize and
treat
diaper dermatitis which comprises a therapeutically effective amount of
(1) a therapeutic wound healing composition comprising an inhibitor of mono-
2o adenosine diphosphate-ribosyl transferase to inhibit adenosine
diphosphate-ribosylation of vascular endothelial growth factor;
(2) a buffering agent to maintain the pH of dermatitis in a range from about 5
to about 8; and
(3) an anti-inflammatory agent.
Once prepared, the inventive therapeutic wound healing compositions
may be stored for future use or may be formulated in effective amounts with
pharmaceutically acceptable carriers to prepare a wide variety of
pharmaceutical
compositions. Examples of pharmaceutically acceptable carriers are
pharmaceutical
3o appliances and topical vehicles. Examples of pharmaceutical appliances are
sutures,
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WO 99/63982 ~ ~ PCTlUS99/13264
staples, gauze, bandages, burn dressings, artificial skins, liposome or micell
formulations, microcapsules, aqueous vehicles for soaking gauze dressings, and
the
like, and mixtures thereof. Topical compositions employ topical vehicles, such
as
creams, gels formulations, foams, ointments and sprays, salves, and films,
which are
intended to be applied to the skin or body cavity and are not intended to be
taken by
mouth. Oral topical compositions employ oral vehicles, such as mouthwashes,
rinses,
oral sprays, suspensions, and dental gels, which are intended to be taken by
mouth but
are not intended to be ingested. The preferred topical vehicles are water and
pharmaceutically acceptable water-miscible organic solvents such as ethyl
alcohol,
isopropyl alcohol, propylene glycol, glycerin, and the like, and mixtures of
these
solvents. Water-alcohol mixtures are particularly preferred and are generally
employed in a weight ratio from about 1:1 to about 20:1, preferably from about
3:1 to
about 20:1, and most preferably from about 3:1 to about 10:1, respectively.
A variety of traditional ingredients may optionally be included in the
pharmaceutical composition in ei~'ective amounts such as buffers,
preservatives,
tonicity adjusting agents, antioxidants, polymers for adjusting viscosity or
for use as
extenders, and excipients, and the like. Other conventional additives include
humectants, emollients, lubricants, stabilizers, dyes, and perfumes, providing
the
2o additives do not interfere with the therapeutic properties of the
therapeutic wound
healing composition. Specific illustrative examples of such traditional
ingredients
include acetate and borate buffers; thimerosol, sorbic acid, methyl and propyl
paraben
and chlorobutanol preservatives; sodium chloride and sugars to adjust the
tonicity;
and excipients such as mannitol, lactose and sucrose. Other conventional
pharmaceutical additives known to those having ordinary skill in the
pharmaceutical
arts may also be used in the pharmaceutical composition. The ultimate
pharmaceutical
compositions are readily prepared using methods generally known in the
pharmaceutical arts.
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18
In accordance with this invention, therapeutically effective amounts of
the therapeutic wound healing compositions of the present invention may be
employed
in the pharmaceutical appliance. These amounts are readily determined by those
skilled in the art without the need for undue experimentation. The exact
amount of
the therapeutic wound healing composition employed is subject to such factors
as the
type and concentration of the therapeutic wound healing composition and the
type of
pharmaceutical appliance employed. Thus, the amount of therapeutic wound
healing
composition may be varied in order to obtain the result desired in the final
product
and such variations are within the capabilities of those skilled in the art
without the
1o need for undue experimentation. In a preferred embodiment, the
pharmaceutical
composition will comprise the therapeutic wound healing composition in an
amount
from about 0.1% to about 10%, by weight of the pharmaceutical composition. In
a
more preferred embodiment, the pharmaceutical composition will comprise the
therapeutic wound healing composition in an amount from about 0.2% to about
8%,
by weight of the pharmaceutical composition. In a most preferred embodiment,
the
pharmaceutical composition will comprise the therapeutic wound healing
composition
in an amount from about 0.3% to about 5%, by weight of the pharmaceutical
composition.
2o The present invention extends to methods for making the
pharmaceutical compositions. In general, a pharmaceutical composition is made
by
contacting a therapeutically effective amount of a therapeutic wound healing
composition with a pharmaceutically acceptable Garner and the other
ingredients of
the final desired pharmaceutical composition. The therapeutic wound healing
composition may be in a solvent and may be absorbed onto a pharmaceutical
appliance.
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19
RESULTS
Production of Nitrite by RAW264.7 Cells and MPMs
Figure 1 shows the production of nitrite by MPMs. Nitrite was not
produced by non-activated cells, either with or without lactate. Following
challenge
with Ifny/LPS, nitrite production was strongly induced, with nitrite
accumulating over
the 48 hr. incubation period. L-NAME (I.SmM) blocked nitrite production by
about
70-80%; AG (1mM) blocked nitrite production by >95%. RAW264.7 cells produced
to nitrite in a similar manner, and L-NAME and AG blocked nitrite synthesis by
RAW264.7 cells to a similar extent (data not shown).
Production of VEGF by RAW264.7 Cells and Murine Peritoneal Macrophages
The production of VEGF by RAW cells is shown in Figure 2A. Non-
stimulated RAW cells produced VEGF in an apparently constitutive manner over
the
48 hour incubation period. This spontaneous production of VEGF was similar in
regular culture plates and in gas-permeable Permanox plates. Stimulation of
cells with
Ifny and LPS increased the production of VEGF by RAW cells over the
constitutive
level produced by non-stimulated cells by about 3-4 fold by 18 hours. By 48
hours,
the stimulated VEGF levels were only 2 fold increased over the constitutive
level.
The iNOS inhibitors AG (I.OmM) and L-NAME (I.SmM) did not block the
constitutive production of VEGF by non-stimulated RAW cells, but reduced the
production of VEGF by Ifny/LPS-activated RAW cells, to a level markedly below
that
of the non-stimulated cells. Sodium lactate {25mM) did not alter the
production of
VEGF by these cells, either with or without Ifny/LPS activation. RAW cells
cultured
under hypoxic conditions produced increased amounts of VEGF. After 18 hours,
VEGF levels in the media of cells cultured under hypoxic conditions were about
3
fold greater than those in the media of control, normoxic cells. This
differential was
less marked by 48 hours. Analyses of the dissolved oxygen levels in the
conditioned
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media directly following harvesting indicated clearly that under normoxic
conditions,
oxygen levels were consistently high (p02 > 145). After 24 and 48 hours
incubation
under hypoxic conditions (95% N2/5%C02), the p02 was 7lmm and 46 mm
respectively.
The production of VEGF by MPMs was similar to that of RAW cells,
with constitutive production occurring over 48 hours (Figure 2B). Increased
production was induced by Ifny/LPS. However, in contrast to RAW cells, iNOS
inhibitors did not significantly reduce the production of VEGF by Ifny/LPS-
activated
1o MPMs. As was observed for RAW cells, sodium lactate did not modulate the
production of VEGF by these cells. Culture of MPMs under hypoxic conditions
resulted in an increase in VEGF production in the first 18 hours; after 48
hours,
however, constitutive production of VEGF was only slightly higher than that of
hypoxic cells. Oxygen levels determined in the conditioned media of MPMs were
similar to those found in RAW cell media.
Quantitative RT-PCR Analysis of VEGF mRNA Levels
A typical example of a quantitative RT-PCR dilution series using the
2o VEGF RNA minigene as internal standard is shown in Figure 3. The PCR
amplification product of the minigene is 293 by in size. The native mRNA PCR
amplification band is 362 by in size. The point of equivalence for the
amplified
minigene and the amplified native mRNA is readily determined from the dilution
series. The values determined from these analyses were normalized to the
levels of
G3PDH mRNA determined in parallel samples, although little variation in the
G3PDH
mRNA levels were in fact observed between samples. On this basis, the relative
amounts of VEGF mRNA in the various macrophage preparations are shown in Table
1. Both hypoxia and Ifny/LPS activation upregulated VEGF steady state mRNA
levels in MPMs at 4 and 10 hours. By 24 hours, however, the levels of VEGF
3o mRNA were similar in all the groups. In RAW cells, VEGF mRNA levels
remained
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21
elevated at 24 hours. Aminoguanidine treatment of Ifny/LPS-treated MPMs did
not
significantly reduce their steady-state VEGF mRNA levels at any time point; in
RAW
cells, however, the VEGF mRNA levels were reduced by 70-80% at 4, 10 and 24
hours.
RT-PCR Analysis of VEGF mRNA Isoforms
Three isoforms of VEGF were found to be produced by both non-
activated and Ifny/LPS-activated MPMs. These isoforms corresponded to VEGF-1
to (652bp), VEGF-2 {580bp) and VEGF-3 (448 bp)(45). The relative proportions
of the
VEGF isoforms expressed by MPMs at each time point following Ifny/LPS
activation
were only slightly modulated by Ifny/LPS-activation and by inhibition of iNOS
with
AG (Figure 4). In RAW cells, VEGF mRNA isoforms were similarly unaffected by
Ifiry/LPS activation and by AG treatment.
Production of TNFa by MPMs and RAW264.7 Cells
TNFa was not produced by either non-stimulated MPMs or by
RAW264.7 cells over the 48 hour test period. Production of TNFa by MPMs is
2o shown in Figure 5. Following stimulation with Ifny/LPS, TNFa expression was
strongly induced, with increased TNFa in the conditioned media being apparent
by 8
hours following challenge. There was no significant difference in TNFa
production in
cells treated with or without sodium lactate. Similarly, culture of cells in
Permanox
dishes, under either normoxic or hypoxic conditions, did not modulate TNFa
production. The iNOS inhibitors L-NAME and AG had no significant effect on the
production of TNFa by MPMs. Production of TNFa by RAW cells was similar to
that observed in MPMs (data not shown).
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22
ADP-Ribosylation of VEGF by Bacterial Toxins and Macrophage Extracts
Labeling of rVEGF with 32P-NAD was observed using cholera toxin
and macrophage cytosolic extracts (Figure 6). Labeling with cholera toxin
resulted in
a single 32P-labeled band corresponding to the size of rVEGF165 standard
(Figure
6C). Labeling with macrophage cytosolic extracts resulted in the 32P-labeling
of a
large number of bands, due to the endogenous labeling of macrophage cytosolic
proteins (Figure 6A). To clearly demonstrate labeling of rVEGF165 in this
mixture,
immunoprecipitation of the macrophage cytosolic labeling mixture with anti-
VEGF
1o antibody was necessary. Following immunoprecipitation, a prominent labeled
band
corresponding to rVEGF165 was clearly visible (Figure 6B). This band was not
present in control reactions carried out in the absence of rVEGF 165 ~
Labeling of
VEGF using Pertussis toxin was not observed (Figure 6E).
Angiogenic and Anti-Angiogenic Responses in Rat Corneas
The angiogenic responses induced in rat corneas by the concentrated
conditioned media from the MPMs cultured under various conditions are shown in
Table 2. Medium from non-activated MPMs cultured under normoxic conditions did
2o not induce angiogenesis. This medium did not contain anti-angiogenic
activity, as the
angiogenic effects of VEGF (25ng) were unaffected by this medium. Medium from
Ifny/LPS-activated MPMs was potently angiogenic, while medium from iNOS-
inhibited IfnylLPS-activated MPMs showed markedly reduced angiogenic activity.
In
contrast to medium from normoxic, non-activated MPM, this medium was found to
contain anti-angiogenic activity, as we have reported previously (36). Medium
from
normoxic, lactate-treated non-activated MPMs showed significant angiogenic
activity.
Similarly, medium from non-activated MPMs cultured under hypoxic conditions
showed significant angiogenic activity. In both these cases, a polyclonal
antibody to
VEGF neutralized the angiogenic activity in the conditioned media. Angiogenic
3o responses induced by rVEGF 165 were neutralized by anti-VEGF antibody in
control
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23
experiments, while those induced by bFGF (20ng/implant) and TNFa
(20ng/implant)
were unaffected.
The angiogenic responses induced by rVEGF 165 that was ADP-
s ribosylated using cholera toxin or MPM cytosolic extract are shown in Table
3. While
control VEGF (taken through a sham labeling procedure in the absence of
cholera
toxin and MPM cytoplasmic extracts) strongly induced angiogenesis, both
cholera
toxin-mediated and MPM cytoplasmic extract-mediated ADP-ribosylated VEGF
showed greatly reduced angiogenic responses, indicating that the ADP-
ribosylation
to abrogated the angiogenic activity of the VEGF. Since the VEGF was purified
from
the reaction mixtures using heparin-Sepharose binding and elution, we also
tested
eluates from control VEGF-free reactions prepared with cholera toxin or
macrophage
cytosolic extract, to determine first if these extracts contained angiogenic
activity in
their own right, and second, if any anti-angiogenic activity might be enriched
in the
15 eluates through this procedure, and interfere with the angiogenic activity
of the
VEGF. The eluates were therefore tested alone, and then with the post-reaction
addition of rVEGF165. The sham eluates did not exhibit direct angiogenic
activity,
nor did they exhibit anti-angiogenic activity when combined with VEGF.
2o DISCUSSION
In this study, it has been shown that murine macrophages (MPMs)
produce VEGF, a potent, endothelial cell specific, angiogenic growth factor
(23,24).
VEGF production by MPMs does not require activation, with significant VEGF
levels
25 being released into the conditioned media over 18-48 hours without the
addition of
external stimulants. This constitutive level of VEGF production was, however,
markedly increased by stimulation of the cells with IfnY/LPS (Figure 2). In
contrast,
the production of TNFa was strictly dependent on macrophage activation with
Ifny
and LPS, as has been shown in many previous studies (Figure 4)(37-39).
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24
VEGF expression has been shown to be regulated by oxygen tension
both in vivo and in vitro (40-44), with low levels of oxygen (hypoxia)
resulting in the
upregulation of VEGF expression. This increased expression has been shown to
be
regulated both at the transcriptional level and at the level of mRNA
stability,
depending upon the cell type. In our studies, oxygen concentrations were
measured in
the conditioned media of macrophages cultured in both normal and Permanox
culture
dishes. These measurements indicated that under these conditions, the media on
the
MPMs and RAW cells were normoxic, suggesting that the constitutive VEGF
production observed was not due to induction of VEGF gene expression by low
oxygen tension. However, when cells were specifically incubated under hypoxic
conditions, significant upregulation of VEGF, but not of TNFa or nitrite
production,
was observed in both cell types. This upregulation of VEGF expression was
apparent
both at the mRNA and the protein level. These observations suggest that the
expression of the VEGF gene is regulated by oxygen tension in macrophages, as
observed in other cell types. It is not yet clear, however, whether this
regulation
occurs at the level of transcription or at the level of mRNA stability.
Knighton and coworkers have shown previously that the expression of
angiogenic activity by rabbit bone-marrow-derived macrophages is regulated by
2o hypoxia, and that the high levels of lactate that accumulate in the
conditioned media of
hypoxic macrophages are important in regulating the expression of macrophage-
derived angiogenic activity (5). In MPMs and RAW cells, culture in the
presence of
high lactate concentrations (25mM), under normoxic conditions, did not
.modulate the
level of expression of VEGF mRNA or protein. However, it is important to note
that
2s while non-stimulated MPMs express significant levels of VEGF, the
conditioned
media from these cells is non-angiogenic (4,8,22). Following lactate or
hypoxia
treatments, the media exhibit angiogenic activity (Table I). This raises the
important
question of how the angiogenic activity of VEGF is regulated. First, VEGF may
operate in synergy with TNFa to stimulate the microvasculature in the
conditioned
3o media from Ifny/LPS-activated macrophages. However, the fact that medium
from
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lactate-treated or hypoxia-treated non-activated macrophages, which do not
contain
TNFa, express potent angiogenic activity suggests that under the appropriate
conditions, VEGF can be angiogenic in the absence of TNFa. This is supported
by
the fact that the angiogenic activity in these media is neutralized by anti-
VEGF
s antibodies (Table 1). A second possibility tested was that medium from
normoxic,
non-activated MPMs might contain an anti-angiogenic factors) that blocks the
angiogenic effects of VEGF. This hypothesis was tested using the rat corneal
bio-
assay, by combining concentrated conditioned medium from these cells with
rVEGF 165, to determine if the angiogenic effects of the VEGF were inhibited.
No
to inhibition of the effects of VEGF were in fact observed in this system,
clearly
indicating that anti-angiogenic factors) were not present in this conditioned
medium.
This is in contrast to the conditioned medium from iNOS-inhibited, Ifny/LPS-
activated
macrophage medium, as discussed further below.
1s It was then hypothesized that the VEGF produced by non-stimulated
MPMs may differ structurally from the VEGF produced by stimulated MPMs. This
structural difference could relate to alternatively spliced isoforms of VEGF
with
differing angiogenic activities, or to post-translational modification of VEGF
by, for
example, ADP ribosylation-dependent mechanisms (32,4s,46). Our results using
RT-
2o PCR indicate that the isoforms of VEGF are not markedly changed during
macrophage activation, by lactate, or by inhibition of iNOS. VEGFI, 2 and 3
mRNA
isoforms are produced in similar proportions under all conditions tested. It
thus
seems that the most likely mechanism for regulation of VEGF angiogenic
activity
might involve post-translational mechanisms, as has been suggested recently by
2s Hussain et al (32,47). In support of this hypothesis, rVEGF was shown to be
a
substrate for ADP-ribosylation, and ADP-ribosylation was shown to abrogate the
angiogenic activity of rVEGF. Since macrophages are impermeable to NAD+,
metabolic labeling of endogenously synthesized VEGF by macrophages using 32p_
labeled NAD+, is not possible (32,33). However, we demonstrated labeling of
3o rVEGF165 in vitro, using cytosolic extracts of macrophages, as well as by
the
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26
bacterial arginine-specific ADP-ribosyl transferase, cholera toxin subunit A
(Figure
6)(34,35). Pertussis toxin, on the other hand, which is a cysteine-specific
ADP-ribosyl
transferase, did not modify rVEGF 165 (48). In addition, we showed that ADP-
ribosylation of VEGF abrogates its angiogenic activity. In contrast to
unmodified
rVEGF 165, rVEGF 165 derivatized using either cholera toxin or macrophage
cytosolic extract was found to be non-angiogenic (Table 3).
It has been shown that the production of angiogenic activity by human
monocytes and by murine macrophages is induced by activation of the cells with
1o Ifiry/LPS (1-3,20,21,49). In addition, the L-arginine-dependent inducible
nitric oxide
synthase (iNOS)-dependent pathway plays an important role in regulating the
expression of angiogenic activity by Ifny/LPS-activated macrophages (21).
Inhibitors
of iNOS, such as L-NAME, Ng-monomethyl-L-arginine (L-NMMA),
diphenyleneiodonium (DPI) and AG block the production of angiogenic activity
by
activated macrophages, without inhibiting the production of the angiogenic
cytokines
TNFa and Il-8 (21,49,50). In this study, we show that the iNOS inhibitors L-
NAME
and AG markedly inhibit the production of VEGF by Ifny/LPS-activated RAW cells
(>70% inhibition), but have little effect on the constitutive (non-stimulated)
production of VEGF by these cells. Interestingly, in Ifny/LPS-activated RAW
cells,
2o L-NAME and AG inhibit VEGF production to a level significantly below that
of non-
stimulated cells. This suggests that the pathways involved in the regulation
of VEGF
production in non-activated and activated RAW cells are different, with only
the
activated pathway being sensitive to iNOS products. This might relate to the
nature
of the transcriptional promotors involved in the expression of the VEGF gene
under
constitutive and activated conditions. In MPMs, on the other hand, the iNOS
inhibitors had no significant effect on the production of either the
constitutive or
Infy/LPS-stimulated VEGF. However, it is again important to note that the
angiogenic activity of the MPM conditioned media was markedly down-regulated
by
the iNOS inhibitors. Our results suggest that two mechanisms are involved in
the
3o regulation of expression of angiogenic activity by the iNOS-inhibited,
Ifny/LPS-
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27
activated MPMs. The first is analogous to that observed in the activation of
macrophages by hypoxia and lactate; namely the regulation of the ADP-
ribosylation of
VEGF, and hence of its angiogenic activity. Inf~y/LPS activation switches the
production of VEGF from the ADP-ribosylated, non-angiogenic form to the
unmodified, angiogenic form. Second, the iNOS-dependent pathway regulates the
expression of an inhibitor of angiogenesis. When the iNOS pathway is active
and NO
is produced, the inhibitor is inactive or absent; when the iNOS pathway is
blocked
with AG or L-NAME, the inhibitor is active. We have previously reported that
this
anti-angiogenic activity is present in the conditioned medium of iNOS-
inhibited
1o Ifny/LPS-activated MPMs (36). The nature of this inhibitor is not yet
clear; however
it is not neutralized by specific antibodies to thrombospondin-1 or yIP-10,
both of
which are potent anti-angiogenic agents that may be produced by macrophages
(51,52). Specific antibodies to TNFa and TGF~i also do not neutralize the anti
angiogenic activity. The inhibitor binds weakly to heparin-Sepharose and has
an
apparent molecular weight >100kDa (36).
Hussain and coworkers (32,47) have suggested that ADP-ribosylation-
dependent mechanisms may be involved in the post-translational modification of
angiogenic factors, resulting in non-angiogenic forms. Our results suggest
that this
2o may indeed be one of the mechanisms regulating the production of angiogenic
activity
by macrophages. We suggest that VEGF produced by the constitutive pathway is
normally in the ADP-ribosylated, non-angiogenic form, while VEGF produced by
Ifny/LPS-activated MPMs is in the unribosylated, angiogenic form. Activation
may
thus regulate the post-transcriptional modification of VEGF from the ADP-
ribosylated
non-angiogenic form to the unmodified angiogenic form. In addition, the iNOS
pathway in activated MPMs appears to regulate the production (or bio-activity)
of an
anti-angiogenic factor, that is apparent only in Ifny/LPS-activated, iNOS-
inhibited
MPM medium.
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These results clearly indicate that VEGF is a substrate for ADP-
ribosylation, and that ADP-ribosylation of VEGF abrogates its angiogenic
activity.
Preliminary results (manuscript in preparation) also indicate that vitamin-K3
and
novobiocin, both inhibitors of mono-ADP-ribosylation reactions (34,53), result
in the
production of angiogenically active VEGF by non-activated normoxic
macrophages,
without affecting the level of VEGF production or the production of TNFa,
suggesting the involvement of mono-ADP-ribosylation in the regulation of
angiogenic
activity in macrophages. Ultimate proof, however, of the role of mono-ADP-
ribosylation in the regulation of VEGF bio-activity by macrophages, will
require the
to direct demonstration that VEGF is differentially ADP-ribosylated in
macrophages
under conditions that modify oxygen tension or Ifny/LPS-induced macrophage
activation and the iNOS-dependent pathway.
In summary, on the basis of these observations, it appears that VEGF
is an important contributor to macrophage-dependent angiogenic activity. VEGF
production in macrophages is regulated at several levels. Constitutively
expressed
VEGF is normally angiogenically inactive. Hypoxia and Ifny/LPS activation
increase
the absolute amount of VEGF produced, but also result in the expression of
angiogenic VEGF. High lactate does not increase the amount of VEGF produced,
but
2o also results in the production of angiogenic VEGF. The change in the
angiogenic
phenotype of VEGF may be due to post-translational modification, perhaps by
the
process of ADP-ribosylation, that modulates VEGF bio-activity. rVEGF 165 is a
substrate for ADP-ribosylation by cholera toxin and by MPM cytoplasmic
extracts,
and ADP-ribosylation of rVEGF165 was shown to abrogate its angiogenic
activity.
In hypoxic and Ifn~y/LPS-activated MPMs, activation upregulated VEGF mRNA
expression, and also shifted the balance of post-translational modiftcation of
VEGF
from the non-angiogenic to the angiogenic form. In RAW264.7 cells, the
Ifny/LPS
activation-dependent modulation of VEGF mRNA levels is regulated in part by
the
iNOS pathway, but the constitutive production of VEGF in non-activated cells
is not.
3o In MPMs on the other hand, the regulation of VEGF mRNA level by Ifny/LPS
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29
activation is not significantly dependent on the iNOS pathway. VEGF angiogenic
activity in these cells appears to be regulated at the level of post-
translational
modification. Finally, when the iNOS pathway is inhibited in Ifny/LPS-
activated
MPMs, an anti-angiogenic factor is expressed that blocks the angiogenic
activity of
VEGF. Together, regulation of VEGF bio-activity by post-translational
modification,
and iNOS-dependent regulation of the expression an anti-angiogenic factor,
provide
novel mechanisms for controlling the angiogenic phenotype of macrophages, and
may
play a key role in the regulation of macrophage-dependent angiogenic activity
in vivo,
in wound repair, fibroproliferation, and possibly in solid tumor development.
to
The present invention is further illustrated by the following examples
which are not intended to limit the effective scope of the claims. All parts
and
percentages in the examples and throughout the specification and claims are by
weight
of the final composition unless otherwise specified.
EXAMPLES
Materials & Methods
2o Murine Peritoneal Macrophages (MPMs) and RAW264.7 cells
Balb-c mice {male, 6-8 weeks, Taconic, Germantown, N~ were
injected intraperitoneally with 2.5 ml sterile Brewer s thioglycollate broth
(3% w/v)
(Difco Labs., Detroit, MI). Five days later, the mice were sacrified and MPMs
were
harvested using PBS containing 100 U/ml of heparin. Cells were centrifuged at
3008
for 5 mins. at 4oC., washed twice with serum-free DMEM, and resuspended in
DMEM containing 10% FCS and SOpg/ml gentamycin (DMEM-10%FCS). Cells
were seeded into 60 mm tissue culture dishes (Costar, Cambridge, MA)(4x106
cells/dish) and incubated at 37oC in a humidified incubator in 95% air/S% C02
for 4
3o hrs to allow the cells to adhere. In some experiments, cells were seeded in
Contur
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WO 99/63982 PCT/US99/13Z64
Permanox gas-permeable dishes (Miles, Naperville, IL) rather than regular
tissue
culture dishes, to increase the availability of ambient gases to the cells on
the base of
the dishes. Non-adherent cells were removed by washing with serum free DMEM,
and the cells were refed with DMEM/1% FCS. MPMs were activated using 100U/ml
5 murine Ifny (Sigma Chemical Co., St. Louis, MO) and 100ng/ml of LPS (E.coli
serotype OSS:BS, Sigma) either in the presence or absence of the iNOS
inhibitors L-
NAME ( 1. SmM) or AG ( 1 mM). To test the effects of lactate on MPMs, sodium
lactate (25 mM) was added to the cultures at the start of the incubation
period. To
test the effects of hypoxia, MPMs were incubated in Permanox dishes, either
under
to normoxic conditions (95% air, 5%C02) or under hypoxic conditions (95%N2,
5%C02). Media and cells were harvested at the indicated time points following
addition of Ifn-y/LPS and/or lactate. Aliquots of media were sampled
immediately
following incubation, and analyzed in a Blood Gas Analyzer (Instrumentation
Lab.,
Lexington, MA). The remaining media were centrifuged at 4oC for 5 rains at
15,000
~5 g to remove cellular debris, and stored at -80oC. prior to analysis.
RAW264.7 cells were obtained from ATTC, and routinely maintained
in DMEM-10%FCS. Cells were passaged by scraping, and plated in either regular
or
Permanox dishes, with or without IfnyILPS, with or without sodium lactate, and
2o under hypoxic conditions, as described above. The effects of L-NAME and AG
on
the production of VEGF and TNFa by these cells were also tested. Media and
cells
were harvested and treated as described above.
Isolation of Total Cellular RNA
Total cellular RNA was isolated from macrophage cell cultures using
TRI REAGENT (Molecular Research Center, Inc., Cincinnati, OI-~. Medium was
removed from the cells, TRI REAGENT added directly to the culture dishes, and
the
cell lysate passed several times through a 21 gauge syringe needle. Samples
were
3o stored at RTo for 5 rains., 0.2m1 chloroform was then added per milliliter
lysis
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31
reagent, the mixture vortexed for 15 secs. and then incubated at RTO for 10
mins.
The resultant mixture was centrifuged at 12,OOOg for 15 rains. at 40C. The
aqueous
(upper) phase was transferred to a fresh microfuge tube, and RNA precipitated
by
adding O. Sml isopropanol per l ml TRI REAGENT used for the original
extraction.
Samples were incubated at RTo for 5 rains. and then centrifuged at 12,OOOg for
10
rains. at 40C. The RNA pellets were washed with 75% ethanol, air dried for 5
rains.
and dissolved in RNAase-free water.
Quantitative RT-PCR Analysis of VEGF mRNA levels
VEGF mRNA levels were determined by RT-PCR using an internal
minigene RNA standard that is present through both the RT and the PCR reaction
stages. The 293 by VEGF minigene RNA standard, containing a 69 by gene
deletion,
was prepared as follows: Total RNA from MPMs was subjected to RT and PCR
through 3 S cycles, using the following primers:
Sense minigene primer: (18-mer) in exon 1 (positions 41-58): 5'
GGACCCTGGCTTTACTGC.3'
Anti-sense minigene primer (39mer), starting in exon 5, spanning an intron,
and
continuing into exon 4 to position 387, deleting 69 by of the gene to position
318, and
2o continuing to position 300. The primer thus spans an intron, and contains a
69 by
deletion.
5'TTGGTCTGCATTCACATCGGC-GTGATGTTGCTCTCTGAC3'.
The PCR band was purified from primers by ethanol precipitation, and blunt end
ligated into the pCR-Script AmpSK(+) vector (Stratagene, La Jolla, CA). The
orientation of the minigene fragment in the vector was determined by dideoxy
sequence analysis. A clone containing the minigene insert in an antisense
orientation
was used for subsequent in vitro transcription for the preparation of the RNA
minigene. The vector was linearized with Notl, treated with proteinase-K
(4p,g/ml)
for 1 hr. at 370C., and purified by phenol extraction and ethanol
precipitation. The
linearized plasmid was then transcribed in vitro using a VTRAN-7 transcription
kit
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(Sigma), using T7 RNA polymerise, yielding sense RNA. The reaction product was
treated with RNAase-free DNAase-1 (l0u/mg DNA in the transcription reaction)
(Promega, Madison, WI) for 2 hours at 370C. The reaction mixture was then
heated
to 900C. for 5 rains., cooled, and lOx transcription stop solution (SM.
ammonium
s acetate, O.1M. EDTA) were added, followed by phenol extraction and
isopropanol
precipitation. The RNA concentration was determined spectrophotometrically.
VEGF RNA minigene {2.Spg per reaction) was then incorporated into the RT-PCR
reactions. Total RNA from macrophages treated under various conditions was
added
to the RT-PCR reactions in amounts ranging from 1-200ng/reaction. The
oligonucleotide primers used for the competitive RT-PCR reaction were 18-mers
nested into the initial primers used to prepare the minigene:
Sense primer in exon 1: 5' ACCCTGGCTTTACTGCTG 3'
Antisense primer {intron spanning): s' GGTCTGCATTCACATCGG.3'
The antisense primer was used for the initial RT reaction; the reverse
transcriptase
1s was inactivated at 99oC. for s rains., and added to a PCR mix containing an
equivalent amount of sense primer. PCR was then carried out for 25 cycles. The
reactions were analyzed by electrophoresis on l.s% agarose gels in TAE buiTer,
stained with ethidium bromide, and scanned using the Molecular Dynamics
FIuorImage Analyzer. The concentrations of input RNA that gave bands of equal
2o intensity to that of the internal VEGF RNA minigene were then determined.
Although
intron-spanning primers were used throughout, controls for genomic DNA
contamination of total RNA preparations were routinely carried out. These
controls
involved the performance of parallel reactions in the absence of reverse
transcriptase.
25 As a control for a housekeeping gene that is not markedly modulated
by the various culture conditions used, an RT-PCR procedure for the enzyme
glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was also developed (details
not
shown). Parallel reactions for G3PDH mRNA levels were performed on the various
macrophage RNA samples, and the VEGF mRNA levels determined by RT-PCR were
3o normalized to the G3PDH levels.
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RT-PCR Analysis of VEGF mRNA Isoforms
For reverse transcription, l.Opg of total RNA was reverse transcribed
using 100 ng of the reverse VEGF specific primer indicated below, using SO U
MuLv
reverse transcriptase with an RNA PCR Kit (Perkin Elmer, Foster City, CA),
following the manufacturer's protocol. Following the initial RT reaction step,
the 20
p,l reaction volumes were boiled for 5 mins. to inactivate the reverse
transcriptase.
lOOng forward primer (see below) were added, together with 80~t1 of a PCR
master
to mix, to give a final concentration of 1 mM MgCl2, 1X PCR buffer II, and 2.5
U Taq
polymerase (Perkin-Elmer) per reaction. PCR primers were selected to enable
the
amplification of the three differentially spliced murine isoforms of VEGF mRNA
formed from the VEGF gene. These VEGF mRNA isoforms are derived from a gene
containing 8 exons (45). The largest, VEGF-l, is formed using all 8 exons.
VEGF-2
lacks exon 7, and VEGF-3 lacks exons 6 and 7. By using PCR primers in exons 3
and
8, the three different isoforms of VEGF generate PCR amplification products of
different sizes, and since they amplify from the same primers, the ratio of
intensities of
the three bands gives an estimate of the relative abundance of the three
differentially
spliced mRNA isoforms.The primers selected for the PCR amplifications were:
2o Forward primer, located in exon 3: 5'GATGAAGCCCTGGAGTGC3'
Reverse Primer, located in exon 8: 5'TCCCAGAAACAACCCTAA3'
The following cycling program for PCR was used: Denaturation at 940C for 1
min.,
annealing at 540C for lmin., and extension for 2 rains. at 720C, for 25
cycles, with a
final extension at 720C for 15 rains. PCR reactions were then analv~P.~ ~".
electrophoresis on 1.5% agarose gels using TAE buffer, and stained with
ethidium
bromide. Gels were scanned using a Molecular Dynamics FluorImage analyzer, and
the staining intensities of the PCR-amplified VEGF isoform bands were analyzed
using the ImageQuant image analysis software package (Molecular Dynamics).
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34
Assay of VEGF Protein Levels by ELISA
VEGF in conditioned media was assayed using a sandwich ELISA kit
(Quantikine M, R & D Systems, Minneapolis, MN.), following the manufacturer's
protocol. This assay detects murine VEGF with sensitivity in the range of 3-
500
pg/ml. Samples with VEGF concentrations above this range were diluted with
RPMI
and re-assayed. All samples were assayed in triplicate. Results are presented
as
means +/- standard deviations of the mean (S.D.).
1o Assay of TNFa by ELISA
Murine TNFa was assayed using a sandwich ELISA kit (TNF-A
Minikit, Endogen, Woburn, MA), following the procedure of the manufacturer.
All
samples were assayed in triplicate. Results are presented as means +/- S.D.
Assay of Nitrite
To determine the production of nitric oxide (NO) by the cells under the
various conditions tested, the media were analyzed for nitrite using the
Griess
2o reaction, as described previously. Briefly, SOp,I culture medium were
placed in a 96-
well plate, followed by SOpL, of cold 350mM ammonium chloride, pH 9.6. 100 p.l
of a
mixture of 1 part SmM sulfaniiic acid, 1 part SmM N-(1-Naphthyl)
ethylenediamine
and 3 parts glacial acetic acid was added. After 10 minutes of incubation in
the dark
at room temperature, absorbance at 570nm was determined using a microplate
scanner (BioTek Instruments, Burlington. VT). The system was calibrated using
freshly-prepared standard nitrite solutions. A linear regression line was
determined
from the standards, and the experimental nitrite concentrations calculated.
Results are
means + S.D.
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Assay of Angiogenic and AntiAngiogenic Activity
Conditioned media from MPM cultures were concentrated 20 fold and
diafiltered using Amicon centrifugal spin filters (3KDa cut-of~(Beverly, MA).
Five p,l
5 concentrated media were incorporated into equal volumes of slow-release
Hydron
(12% w/v in 95% ethanol) (Interferon Sciences, New Brunswick, Nn and allowed
to
dry. Hydron pellets were implanted aseptically into pockets within rat corneal
stromas, 2mm from the Timbal vasculature, as described previously (1,2,4,9).
Corneas
were examined daily for seven days using a stereomicroscope and perfused with
1o colloidal carbon at the end of the observation period to provide a
permanent record of
the angiogenic responses. Corneas were examined histologically for any
evidence of
non-specific inflammation. Angiogenic responses were assessed on a graded
scale as
follows: No response, or slight budding of the Timbal vasculature that
regresses rapidly
= 0; Formation of a few capillary buds and sprouts that progress less the
0.2mm from
15 the limbus, and start to regress = 1; Persistent growth of a network of
capillary buds
and sprouts that grow at least lmm towards the implant, but do not reach and
invade
the implant = 2; strong growth of a dense network of capillary buds and
sprouts that
reaches and surrounds the implant = 3. Four corneal implants were prepared per
test
sample, and the responses summed. A maximal response thus has a score of 12,
while
2o a minimal response has a score of 0. For the assay of anti-angiogenic
activity, test
conditioned media (20x concentrated) were combined with 20ng recombinant human
VEGF165 (gift of Dr. Napoleone Ferrara, Genentech Inc., S. San Francisco, CA).
The effects of the test media on the angiogenic activity of the rVEGF were
then
determined using the corneal bio-assay.
Effects of Anti-VEGF Antibodies on Macrophage Angiogenic Activity
To determine the contribution of VEGF to the angiogenic activity of
the MPM conditioned media, an affinity purified neutralizing polyclonal
antibody to
3o VEGF (gift of Dr. Napoleone Ferrara) was used. Concentrated conditioned
media
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36
prepared as described above were incubated with anti-VEGF antibody at a final
concentration of 10 ~g/ml at 370C. for 2 hours. Controls were incubated with
pre-
immune IgG at the same concentration. These treated media were then assayed
for
angiogenic activity in the rat corneal bio-assay.
ADP-Ribosytation of rVEGF
Initial attempts to metabolically label VEGF endogenously synthesized
in MPMs, using 32P-NAD+ were unsuccesful, as macrophages are impermeable to
1o NAD+, which cannot enter the cells and provide a substrate for the
cytoplasmic ADP-
ribosyl transferases (32,33). We therefore used either permeabilized MPMs
(data not
shown) or macrophage cytoplasmic extracts to determine whether exogenous rVEGF
is a substrate for macrophage ADP-ribosyl transferases. Similarly, rVEGF was
tested
as a substrate for cholera toxin (an arginine-specific ADP-ribosyl
transferase) and for
pertussis toxin (a cysteine-specific ADP-ribosyl transferase) (34,35).
i) Cytosolic extracts of MPMs were prepared as follows: MPMs were
plated in 100mm culture dishes ( 10 x 106 cells per dish in 1 Oml medium) in
RPMI1640 medium containing 10% fetal calf serum, and incubated at 37oC.
overnight. The medium was then removed, and the cells were washed (x2) with
cold
PBS. The cells were then harvested by scraping into cold PBS (lml/dish ). The
cells
were spun down at 300g and resuspended on ice in 20mM Tris-HCl pH7.5, 1mM
EDTA, 5mM MgCl2, 1mM DTT, 2mM mercaptoethanol, 1mM PMSF, l~tg/ml
leupeptin, lp,g/ml aprotinin, and 0.25M sucrose (Iml/SOx106 cells) and
sonicated
briefly. The extract was centrifuged in the cold for 15 rains. at 1100g to
remove
nuclei and insoluble debris. The protein content of the extracts was
determined using
the Bradford method (BioRad, Richmond, CA), and the extracts were stored at -
80oC. until use. To determine whether these extracts were able to ADP-
ribosylate
rVEGF 165, labeling reactions were set up containing: 500ng VEGF 165, l Op.g
3o macrophage protein extract, 20mM Tris-HCI pH7.8, 20mM isoniazid, 120mM
CA 02329160 2000-11-28
WO 99/63982 ' PCT/US99/13264
37
MgCl2, IOmM NaF, 0.02% leupeptin, 0.54mM NADP, 0.4mM isobutyl-
methylxanthine, 0.1% lubrol, 2mM DTT, IOmM thymidine, and 7p,Ci 32P-labelled
NAD+ (800Ci/mmol)(DuPont-NEN, Wilmington, DE). After 2 hours incubation at
30oC., the reaction mixture was placed on ice, and pre-cleared for 30 rains.
with 101
of Protein-A/G-agarose (Santa Cruz Biotech,, Santa Cruz, CA). lOp,g of a
murine
anti-VEGF monoclonal antibody (gift of Texas Biotechnology, Inc., Dallas, T3~,
was
added to the supernatant, and the mixture was incubated on ice for 2 hours.
lOp,l
Protein-A/G-agarose beads were then added, and the mixture was further
incubated
for 2 hours. at 4oC. with gentle rocking. The beads were harvested by
centrifugation,
1o and washed (x3) with cell lysis buffer. The beads were then incubated in an
equal
volume of 2x electrophoresis sample buffer (final concentration of 100mM DTT),
and
heated at 95oC. for 10 rains. to elute bound VEGF from the beads. The samples
were
then separated using 0.1% SDS-15% PAGE, and the fractionated proteins were
transferred to a nitrocellulose membrane by semi-dry electrophoretic transfer.
The
filters were then immunostained using anti-VEGF antibody, and the VEGF bands
were detected using enhanced fluorecence detection reagents (Amersham Vistra
reagents) and a Fluorimage Analyzer (Molecular Dynamics). The nitrocellulose
blots
were then analyzed using a PhosphorImage analyzer (Molecular Dynamics,
Sunnyvale, CA), to determine the localization of 32P-labeled bands.
ii) rVEGF165 was incubated for up to 2 hours at 30oC. with cholera
toxin as follows: SOOng rVEGF, 250p,g cholera toxin (A-subunit, Sigma Chemical
Co., St. Louis, MO), in the reaction buffer described above. The reaction was
terminated by the addition of an equal volume of cold 10% TCA. The
precipitated
protein was washed (x3) with water-saturated chloroform, and finally
resuspended in
an equal volume of 2x PAGE sample buffer, as above. The samples were separated
by SDS-PAGE, and transferred to a nitrocellulose membrane as described above.
iii) SOOng rVEGF165 was incubated for up to 2 hours at 30oC. with
25~g pertuseis toxin (Sigma, cat. no. P-0317) in the reaction mixture
described above.
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38
Pertussis toxin was pre-activated by incubation for 30 rains. with IOmM ATP
and
20mM DTT prior to addition to the VEGF reaction mixture. The reaction was
terminated and analyzed as described above.
Effects of ADP-Ribosylation on the Angiogenic Activity of VEGF
To determine whether ADP-ribosylation of VEGF modulates its bio-
activity as an angiogenic factor, rVEGFI6$ was treated as described above with
either
cholera toxin, or macrophage cytosolic extract, but in the presence of
unlabeled
1o NAD+. To facilitate the recovery of rVEGF from the reaction mixture, rather
than
using immunoprecipitation for the recovery of VEGF, which requires the use of
harsh,
denaturing conditions for the recovery of VEGF from the Protein-A/G-agarose
beads,
heparin-Sepharose binding was used to recover the VEGF. Following the labeling
reaction, lOpl washed heparin-Sepharose beads were added, and the mixture was
incubated at 4oC. for 4 hours with gentle agitation. The beads were then
washed (x3)
with 100p,1 20mM Tris-HCl pH7.8 containing 0.4M. NaCI. VEGF was eluted from
the beads by incubation with 20p1 Tris-HCl containing 1.SM. NaCI. Recovery of
VEGF was determined by specific ELISA. Control reactions were carned out in
the
absence of bacterial toxins and macrophage extract. To ensure that anti-
angiogenic
2o activity was not present in the macrophage extracts or the cholera toxin
preparations,
similar labeling reactions were carried out in the absence of VEGF, and the
heparin-
Sepharose eluates from these reactions were tested in the anti-angiogenesis
assay.
Figure 1 illustrates the nitrite production by MPMs. Cells were
incubated in DMEM/1%FCS, with or without sodium lactate (25mM), Ifny (100u/ml)
and LPS (100ng/ml), L-NAME (l.SmM), or AG (ImM), as indicated. Media were
harvested 8, 24 and 48 hours after challenge with ifn~y/LPS. Results are means
+/-
S.D. of triplicate determinations in a typical experiment. Similar results
were found in
at least three separate experiments.
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39
Figure 2 illustrates VEGF production by A) RAW264.7 cells, and B)
MPMs. Cells were incubated in DMEM/1%FCS, with or without sodium lactate
(25mM), IFny ( I OOu/ml) and LPS ( 1 OOng/ml), L-NAME ( 1. SmM), or AG ( 1
mM), as
indicated. Media were harvested 18 and 48 hours after challenge with Ifny/LPS.
Results are means +/- S.D. of triplicate determinations in a typical
experiment.
Similar results were found in at least three separate experiments.
Figure 3 illustrates competitive RT-PCR analysis of VEGF mRNA
levels in control (non-stimulated) MPMs 24 hours following plating. Varying
1o amounts of total RNA (1-200ng) isolated from MPMs were reverse transcribed
and
amplified by PCR through 25 cycles in the presence of a VEGF RNA minigene
(2.Spg) that amplifies using the same primers as the native VEGF mRNA, as
described in Methods. The RNA minigene yields an amplified PCR product of
293bp,
the native VEGF mRNA yields a 362bp fragment. The amount of total RNA that
yields an amplification band of the same intensity as the minigene is
determined from
these analyses.
Figure 4 illustrate RT-PCR analysis of VEGF isoforms produced by
Ifny/LPS-activated MPMs, with or without AG treatment. Total RNA isolated from
2o MPMs was reverse transcribed and amplified by PCR, as described in Methods.
PCR
primers were located in exons 3 and 8, resulting in the amplification of 3 PCR
products corresponding to 652, 580 and 448bp.
Figure 5 illustrates TNFa production by MPMs. Cells were incubated
in DMEM/1%FCS, with or without sodium lactate (25mM), Ifny (100u/ml) and LPS
(100ng/ml), L-NAME (l.SmM), or AG (1mM), as indicated. Media were harvested
8, 24 and 48 hours after challenge with Ifn~y/LPS. Results are means +/- S.D.
of
triplicate determinations in a typical experiment. Similar results were found
in at least
three separate experiments.
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Figure 6 illustrates ADP-R,ibosylation of rVEGF165 bY bacterial toxins
and by macrophage cytosolic extract. A. rVEGF (SOOng) was incubated with
macrophage cytosolic extract (see Methods) in the presence of 32P-NAD+. The
total
labeling reaction was analyzed on the 0.1% SDS-15% PAGE gel. B. The rVEGF165
5 -macrophage cytosolic extract labeling mixture was immuno-precipitated with
anti-
VEGF antibody, and the immunoprecipitated VEGF was analyzed by SDS-PAGE. A
dominant 32P-labeled band migrating in the same position as rVEGF165
(determined
by Western analysis of the same blot) is indicated. C. rVEGF165 was incubated
with
cholera toxin subunit A and 32P-NAD+ as decribed in Methods. D. Cholera to~cin
1o was incubated with 32P-NAD+ in the absence of rVEGF165~ E. rVEGF165 was
incubated with pertussis toxin and 32P-NAD+, as described in Methods.
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Table 1
Relative VEGF mRNA Levels* in Macrophages Determined by Competitive RT-PCR
Time (hoursl
4 10 24
Control (unstimulated) MPMs I 1 1
Hypoxic MPMs 2.8 5 I .4
1o Ifny/LPS-activated MPMs 2.2 4.8 1
Ifny/LPS-activated MPMs 2 4.7 1
+ AG ( 1 mM)
Control (unstimulated) RAW cells1 1.3 1.2
Hypoxic RAW cells ' 3 5.8 2.5
Ifny/LPS-activated RAW cells2.4 5.4 2.2
Ifny/LPS-activated RAW cells 0.9 1.4 1.2
+ AG ( 1 mM)
* VEGF mRNA levels for each group are compared with the G3PDH mRNA level in
the same RNA samples..
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Table 2
Angiogenic and Anti-Angiogenic Responses Induced in Rat Corneas by Conditioned
Media from Mouse Peritoneal Macrophages (MPMs) Cultured Under Various
Conditions In Vitro
Macrophage Culture Conditions) An i~o,enic Score2
1. Normoxia 1
2. Hypoxia 9
l0 3. Normoxia + Lactate (25mM) 8
4. Ifny ( 1 OOU/ml))/LP S ( 1 OOng/ml)11
5. Ifny/LPS + Aminoguanidine (1mM) 2
6. Group 2 + anti-VEGF Ab ( 1 Opg/ml)1
7. Group 3 + anti-VEGF Ab (lOp.g/ml)2
8. Group 4 + anti-VEGF Ab (lOp.g/ml)4
9. rVEGF 165 (20ng) 11
10. bFGF (20ng) 12
11. TNFa (20ng) 10
12. Group 9 + anti-VEGF Ab (lOpg/ml) 2
13. Group 10 + anti-VEGF Ab (10~g/ml)11
14. Group 11 + anti-VEGF Ab (10~g/ml)11
15. Group 1 + rVEGF 165 (20ng) 11
16. Group 5 + rVEGF 165 (20ng) 2
1 Macrophages were incubated for 48 hours under the indicated conditions,
concentrated (x20) and diaflltered using Centricon 3 (3000 M.Wt. cut-ofd
filters
(Amicon). Samples were then combined with equal volumes of Hydron (Interferon
Sciences, Inc.)(12% w/v in 95% ethanol). 101 droplets were then allowed to dry
on
3o the cut ends of 2mm diameter Teflon rods. These pellets were then implanted
aseptically in the corneas of rats.
2 Angiogenic responses were assessed 7 days following implantation. The
angiogenic
score represents the sum of the graded angiogenic responses from 4 individual
corneas
for each test sample. A maximal response would score 12; a minimal response 0
(see
Methods).
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43
Table 3
Effects of ADP-Ribosylation# on the Angiogenic Activity of rVEGF 165
Test Material Angiogenic score
1 Sham-reacted rVEGF 165 (20ng) 11
2. Cholera toxin-treated rVEGF 165 (20ng)
to (Heparin-Sepharose eluate)
3. Cholera toxin control 1
(Heparin-Sepharose eluate)
4. rVEGF 165 (20ng) + Cholera toxin control 10
5. Macrophage cytosolic extract-treated rVEGF1653
(20ng)
(Heparin-Sepharose eluate)
6. Macrophage cytosolic extract control
7. rVEGF 165 (20ng) + Macrophage cytosolic 11
extract control
# rVEGF165 was treated in a reaction mixture with either cholera toxin or
macrophage cytosolic extracts, as described in the Methods section. Controls
of
VEGF treated in the absence of cholera toxin or macrophage cytosolic extract,
were
performed to determine the effects of the buffers on VEGF. Controls of the
cholera
toxin and macrophage cytosolic.extract incubated without VEGF were also
performed, to determine whether extraneous angiogenic or anti-angiogenic
factors
were present in these reagents. All reactions were treated with heparin-
Sepharose as
described in Methods, to recover the VEGF from the reaction mixtures.
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44
Throughout this application, various publications have been
referenced. The disclosures in these publications are incorporated herein by
reference
in order to more fully describe the state of the art.
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Throughout this disclosure, applicant will suggest various theories or
mechanisms by which applicant believes the components in the therapeutic wound
healing compositions function to inhibit adenosine diphosphate-ribosylation of
1o vascular endothelial growth factor. While applicant may offer various
mechanisms to
explain the present invention, applicant does not wish to be bound by theory.
These
theories are suggested to better understand the present invention but are not
intended
to limit the effective scope of the claims.
While the invention has been particularly described in terms of specific
embodiments, those skilled in the art will understand in view of the present
disclosure
that numerous variations and modifications upon the invention are now enabled,
which variations and modifications are not to be regarded as a departure from
the
spirit and scope of the invention. Accordingly, the invention is to be broadly
2o construed and limited only by the scope and spirit of the following claims.
