U.S. patent application number 11/651334 was filed with the patent office on 2008-01-24 for prevention and treatment of retinal ischemia and edema.
This patent application is currently assigned to The Children's Medical Center Corporation. Invention is credited to Anthony P. Adamis.
Application Number | 20080019977 11/651334 |
Document ID | / |
Family ID | 26811943 |
Filed Date | 2008-01-24 |
United States Patent
Application |
20080019977 |
Kind Code |
A1 |
Adamis; Anthony P. |
January 24, 2008 |
Prevention and treatment of retinal ischemia and edema
Abstract
The present invention relates to methods of treating
retinopathy, retinal ischemia and/or retinal edema comprising
administering an integrin or integrin subunit antagonist, leukocyte
adhesion-inducing cytokine antagonist or growth factor antagonist,
a selectin antagonist or adhesion molecule antagonist.
Inventors: |
Adamis; Anthony P.; (Jamaica
Plain, MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
The Children's Medical Center
Corporation
Boston
MA
|
Family ID: |
26811943 |
Appl. No.: |
11/651334 |
Filed: |
January 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10364922 |
Feb 11, 2003 |
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11651334 |
Jan 9, 2007 |
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09474523 |
Dec 29, 1999 |
6524581 |
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10364922 |
Feb 11, 2003 |
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09248752 |
Feb 12, 1999 |
6670321 |
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09474523 |
Dec 29, 1999 |
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60114221 |
Dec 30, 1998 |
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Current U.S.
Class: |
424/158.1 ;
514/44A; 514/789 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 2039/505 20130101; A61P 27/00 20180101; A61K 31/7088 20130101;
A61K 33/10 20130101; C07K 16/2821 20130101; A61K 33/08 20130101;
A61P 27/02 20180101; A61K 9/0048 20130101; C07K 16/2845
20130101 |
Class at
Publication: |
424/158.1 ;
514/044; 514/789 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/7088 20060101 A61K031/7088; A61K 45/00
20060101 A61K045/00; A61P 27/00 20060101 A61P027/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was supported, in whole or in part, by a grant
2P01 HL32262-15 from the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1.-69. (canceled)
70. A method for reducing or preventing retinal injury in a mammal,
comprising administering to the mammal a vascular endothelial
growth factor (VEGF) antagonist, wherein said VEGF antagonist
inhibits leukocyte interaction, thereby reducing or preventing
retinal injury.
71. The method of claim 70, wherein the antagonist is an antibody
or antibody fragment specific for VEGF.
72. The method of claim 70, wherein the antagonist is an antisense
molecule that hybridizes to the nucleic acid sequence that encodes
VEGF, or a peptide mimetic molecule, ribozyme, an aptamer or small
molecule antagonist that inhibits VEGF.
73. The method of claim 70, wherein the VEGF antagonist is
administered in a pharmaceutically acceptable carrier.
74. The method of claim 70, wherein a decrease of at least one
condition selected from the group consisting of retinal edema and
retinal ischemia occurs.
75. The method of claim 74 wherein said decrease is between about
10% and about 90%.
76. The method of claim 70, wherein the mammal is a human and
wherein said human has diabetic retinopathy.
77. A method for treating an individual having diabetic retinopathy
comprising administering to said individual a VEGF antagonist,
wherein leukocyte interaction is reduced or inhibited.
78. The method of claim 77, wherein at least one additional
antagonist that inhibits the binding of a leukocyte to an
endothelial cell or to another leukocyte is administered to said
individual.
79. The method of claim 78, wherein the additional antagonist is at
least one antagonist selected from the group consisting of: an
integrin antagonist, selectin antagonist, leukocyte
adhesion-inducing cytokine antagonist or another growth factor
antagonist and an adhesion molecule antagonist.
80. The method of claim 79, wherein the integrin is selected from a
group consisting of: LFA-1, Mac-1 and p150.95.
81. The method of claim 80, wherein the integrin antagonist
comprises an integrin subunit antagonist.
82. The method of claim 81, wherein the integrin subunit antagonist
comprises a CD18 antagonist, CD11a antagonist or CD11b
antagonist.
83. The method of claim 79, wherein the selectin is selected from a
group consisting of: P-selectin, E-selectin and L-selectin.
84. The method of claim 79, wherein the leukocyte adhesion-inducing
cytokine antagonist or growth factor antagonist is selected from a
group consisting of: TNF-1.alpha., IL-1.beta., MCP-1 and another
VEGF antagonist.
85. The method of claim 79, wherein the adhesion molecule
antagonist is selected from the group consisting of: a PCAM
antagonist, a VCAM antagonist, an ICAM-1 antagonist, an ICAM-2
antagonist and an ICAM-3 antagonist.
86. A method for treating an individual with at least one condition
selected from the group consisting of retinal edema and retinal
ischemia comprising administering to the individual a VEGF
antagonist, wherein a decrease in retinal edema, retinal ischemia
or retinal edema and retinal ischemia occurs.
87. The method of claim 86, wherein said decrease is between about
10% and about 90%.
88. A method of preventing or reducing retinal leukocyte adhesion
in a mammal, comprising administering to the mammal an effective
amount of a VEGF antagonist.
89. The method of claim 88, wherein retinal leukocyte adhesion is
reduced by between about 10% and about 90%.
90. A method of treating or preventing neovascularization in a
mammal comprising administering to the mammal a VEGF antagonist,
wherein said neovascularization is reduced.
91. The method of claim 90, wherein the mammal has a disease,
condition or disorder selected from the group consisting of:
age-related macular degeneration, choroidal neovascularization,
sickle cell retinopathy, retina vein occlusion, diabetic
retinopathy, a condition associated with limbal injury, a condition
associated with increased neovascularization, traumatic alkali
injury, Stevens Johnson syndrome and ocular cicatricial
pemphagoid.
92. The method of claim 90, wherein the neovascularization is
reduced in the cornea, the retina or the choroid.
Description
RELATED APPLICATION
[0001] This application is a Continuation of U.S. application Ser.
No. 10/364,922, filed Feb. 11, 2003, which is a Continuation of
U.S. application Ser. No. 09/474,523 filed Dec. 29, 1999, which is
a Continuation-in-Part of U.S. application Ser. No. 09/248,752,
filed Feb. 12, 1999, which claims the benefit of U.S. Provisional
Application No. 60/114,221, filed Dec. 30, 1998. The entire
teachings of the above applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] Diabetes affects over 16 million Americans. The World Health
Organization indicates that diabetes afflicts 120 million people
worldwide, and estimates that this number will increase to 300
million by the year 2025. Diabetics are faced with numerous
complications including kidney failure, non-traumatic amputations,
an increase in the incidence of heart attack or stroke, nerve
damage, and loss of vision. Diabetic retinopathy is a form of
visual impairment suffered by diabetics.
[0004] In particular, diabetic retinopathy is responsible for 13.1%
and 18.2% newly reported cases of blindness for men and women,
respectively. Kohner E. M., et al. Diabetic Retinopathy Metabolism,
25:1985-1102 (1975). The prevalence of blind diabetics in the
population is about 100 people per million. Id.
[0005] Less than optimal methods of treatment for diabetic
retinopathy exist. For example, laser treatment may be used to slow
the progression of edema, but it cannot be used to reverse the
symptoms of diabetes. Accordingly, a need exists to develop
effective methods of treatment to reduce or impede vision loss
and/or diabetic retinopathy.
SUMMARY OF THE INVENTION
[0006] The present invention relates to methods for inhibiting the
binding of a leukocyte to an endothelial cell or another leukocyte
in the retinal vasculature. The present invention pertains to
methods of treating (e.g., reducing or preventing) retinal injury
in a mammal (e.g., human, individual, patient) wherein the injury
involves retinal edema or retinal ischemia, comprising
administering a compound that inhibits the binding of a leukocyte
to endothelium or to another leukocyte wherein a reduction in edema
or ischemia (e.g., non-perfusion) occurs. The compound comprises an
integrin antagonist (e.g., lymphocyte function associated
molecule-1 (LFA-1), Mac-1 or p150.95), a selectin (e.g.,
P-selectin, E-selectin and L-selectin) antagonist, an adhesion
molecule antagonist (e.g., Intercellular Adhesion Molecule
(ICAM)-1, ICAM-2, ICAM-3, Platelet Endothelial Adhesion Molecule
(PCAM), Vascular Cell Adhesion Molecule (VCAM)), or a leukocyte
adhesion-inducing cytokine or growth factor antagonist (e.g., Tumor
Neucrosis Factor-.alpha. (TNF-.alpha.), Interleukin-1 .beta. (IL-1
.beta.), Monocyte Chemotatic Protein-1 (MCP-1) and a Vascular
Endothelial Growth Factor (VEGF)). The integrin antagonist can be
an integrin subunit (e.g., CD18 or a CD11b) antagonist. The
antagonist can be administered with or without a carrier (e.g.,
pharmaceutically acceptable carrier).
[0007] In particular, the invention pertains to methods of treating
or preventing retinal injury in a mammal comprising administering
to the mammal an adhesion molecule antagonist and/or an integrin
antagonist, wherein the adhesion molecule antagonist and/or the
integrin antagonist inhibits leukocyte interaction, thereby
reducing or preventing retinal injury. The antagonist can be
administered in a carrier (e.g., a pharmaceutically acceptable
carrier). The antagonist for adhesion molecule can be a VCAM, PCAM,
ICAM-2 or ICAM-3 antagonist or, preferably, an ICAM-1 antagonist.
In particular, the antagonist can be an antibody or an antibody
fragment which is specific for ICAM-1, an antisense molecule that
hybridizes to the nucleic acid sequence which encodes ICAM-1, or a
peptide mimetics molecule, a ribozyme, an aptamer, or a small
molecule antagonist that inhibits ICAM-1. The integrin antagonist
can be a LFA-1 antagonist, Mac-1 antagonist or p150.95 antagonist.
The integrin antagonist also comprises an integrin subunit
antagonist (e.g., a CD18 antagonist and/or a CD11b antagonist). The
antagonist can be an antibody or antibody fragment specific for
CD18 and/or CD11b, an antisense molecule that hybridizes to the
nucleic acid sequence that encodes CD18 and/or CD11b, or a peptide
mimetic molecule, a ribozyme, an aptamer or a small molecule
antagonist that inhibits CD18 or CD11b.
[0008] Another aspect of the invention includes a method for
preventing or treating an individual having retinal injury (e.g.,
injury caused by diabetic retinopathy), wherein the injury is
associated with retinal edema and/or retinal ischemia, comprising
administering to the individual a compound that inhibits Mac-1 or a
pathway thereof. The compound inhibits ICAM-1, CD18, CD11b, and/or
VEGF, and causes a decrease of ischemia and/or edema (e.g., between
about 10% and about 90%). Leukocyte interaction can also be
reduced. The compound can be an antibody, an antibody fragment, a
peptide mimetic molecule, an antisense molecule, a ribozyme, an
aptamer and/or a small molecule antagonist. Examples for such a
compound are ICAM-1, CD18, CD11b, and/or VEGF.
[0009] The invention also pertains to a method of treating an
individual having retinopathy or at risk for retinopathy (e.g.,
diabetic retinopathy) comprising administering an antagonist (e.g.,
ICAM-1, CD18, CD11b and/or VEGF), as described herein. The
antagonist can optionally be administered in a suitable carrier
(e.g., pharmaceutically acceptable carrier). Administration of this
antagonist results in a decrease in retinal ischemia and/or retinal
edema. Preferably, a decrease in ischemia and/or edema occurs by at
least about 10%, and more preferably, by about 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95% (e.g., between 10% and 95%). Accordingly,
the present invention also relates to methods for treating or
preventing retinal edema and/or retinal ischemia comprising
administering an ICAM antagonist (e.g., ICAM-1), a CD18 antagonist,
a CD11b antagonist and/or a VEGF antagonist, wherein a decrease in
the edema and/or ischemia occurs.
[0010] The present invention also relates to methods of treating
diabetic retinopathy by administering an ICAM-1, CD18, CD11b and/or
VEGF antagonist and at least one additional antagonist that
inhibits the binding of a leukocyte to an endothelial cell or to
another leukocyte. The additional antagonist can be an integrin
antagonist (e.g., an integrin subunit antagonist such as CD18
and/or CD11b), a selectin antagonist, a leukocyte adhesion-inducing
or growth factor antagonist, or adhesion molecule antagonist. The
additional antagonist can be, for example, another ICAM antagonist
(e.g., an antagonist that is specific for a different portion or
epitope of the ICAM-1 molecule), a PCAM antagonist or a VCAM
antagonist. The types of integrin antagonists, selectin
antagonists, and leukocyte adhesion-inducing or growth factor
antagonists are described herein.
[0011] The invention also encompasses a method of inhibiting
leukocyte interaction, comprising contacting a leukocyte, an
endothelial cell or a leukocyte adhesion-inducing cytokine, with a
compound or antagonist, as defined herein. The compound can be an
integrin antagonist (e.g., an integrin sub-unit antagonist such as
CD18 and/or CD11b), a selectin antagonist, an adhesion molecule
antagonist or a leukocyte adhesion-inducing cytokine or growth
factor antagonist. In particular, the invention relates to a method
of inhibiting leukocyte interaction, comprising contacting an
endothelial cell with an adhesion molecule antagonist (e.g., ICAM-1
specific antagonist), an integrin subunit antagonist (e.g., CD18
and/or CD11b specific antagonist), or a leukocyte adhesion-inducing
cytokine antagonist or growth factor antagonist (e.g.,
TNF-1.alpha., IL-1.beta., MCP-1 and VEGF antagonist).
[0012] The invention also pertains to a method of preventing or
reducing retinal leukostasis an a mammal comprising administering
to the mammal an effective amount of an ICAM, CD18, CD11b and/or
VEGF antagonist. The types of antagonist is described herein. The
method results in retinal leukostasis reduction by between about
10% and 90%.
[0013] Another aspect of the invention is a method of decreasing
retinal leukocyte adhesion in a mammal, comprising administering to
the mammal an effective amount of an antagonist that is specific
for CD11b, CD18 or a combination thereof. The retinal leukocyte
adhesion is decreased between about 10% and 90%.
[0014] Yet another aspect of the invention is a method of treating
or preventing neovascularization in a mammal, comprising
administering to the mammal a CD18 antagonist and an ICAM-1
antagonist, or a CD18 antagonist. The types of antagonists are
described herein. The method is applicable to diseases or
conditions associated with neovascularization including, but not
limited to, age-related macular degeneration, choroidal
neovascularization, sickle cell retinopathy, retina vein occlusion,
diabetic retinopathy, a condition associated with limbal injury, a
condition associated with increased neovascularization, traumatic
alkali injury, Stevens Johnson syndrome and ocular cicatricial
pemphagoid. The neovascularization can be reduced in the cornea,
the retina or the choroid.
[0015] Advantages of the present invention include effective
treatment for retinopathy, retinal edema, retinal ischemia,
neovascularization and other associated disease. Treatment of these
diseases and/or conditions have been ineffective until the
discovery of the present invention. For the first time, the present
invention provides useful methods of treatment which target
molecules that are involved in these diseases.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1A is a graph showing the density of trapped leukocytes
as measured on 0, 3, 7, 14, 21 and 28 days after diabetes
induction. The graph shows a time course of diabetic retinal
leukostasis. All data show the mean .+-. the standard deviation
(SD).
[0017] FIG. 1B is a graph showing the retinal vascular [.sup.125I]
albumin permeation measured 0, 3, 7, 14, 21 and 28 days after
diabetes induction. The graph shows a time course of vascular
leakage. All data show the mean .+-. the standard deviation
(SD).
[0018] FIGS. 2A-D show four photographs of the same retinal area.
FIG. 2A and FIG. 2B show the retinal area after seven days of
diabetes. FIG. 2C and FIG. 2D show the retinal area after eight
days of diabetes. FIG. 2A and FIG. 2C are photographs from orange
leukocyte fluorography (AOLF) and FIG. 2B and FIG. 2D are
photographs from fluorescein angiography. Scale bar denotes 100
.mu.m (3.2 pixel=1 .mu.m).
[0019] FIG. 3A-F show six photographs of a retinal area. FIGS. 3A
and 3B show the retinal area after one week, FIGS. 3C and 3D show
the retinal area after two weeks, and FIGS. 3E and 3F show the
retinal area after four weeks. FIGS. 3A, 3C, and 3E are photographs
from AOLF and FIGS. 3B, 3D and 3F are photographs from fluorescein
angiography. Scale bar denotes 100 .mu.m (3.2 pixel=1 .mu.m).
[0020] FIG. 4A is a photograph of ribonuclease protection assay
results showing ICAM-1 mRNA levels from controls and a diabetic rat
three days following diabetes induction. Each lane is the signal
from the two retinas of a single animal. The lane labeled "Probes"
shows a hundred-fold dilution of the full-length ICAM-1 and 18S
riboprobes. The lanes labeled "RNase-(0.1)" and "RNase-(0.01)" show
the ten-fold and hundred-fold dilutions, respectively, of the full
length riboprobes without sample or RNase.
[0021] FIG. 4B is a bar graph showing units of normalized ICAM-1
mRNA for controls, three days and seven days after diabetes
induction.
[0022] FIGS. 5A-D are photographs of the retinal area. The
representative retinal leukostasis is shown in non-diabetic test
subjects (FIG. 5A), diabetic test subjects (FIG. 5B), diabetic test
subjects given 5 mg/kg mouse control IgG1 (FIG. 5C) and diabetic
test subjects treated with 5 mg/kg anti-ICAM-1 mAb-treated animals
(FIG. 5D). Scale Bars=100 .mu.m; 3.2 pixel=1 .mu.m.
[0023] FIG. 6A is a bar graph showing the density of trapped
leukocytes (.times.10.sup.-5 cells/pixel.sup.2) for control,
diabetic test subjects not given anything, diabetic test subjects
given 5 mg/kg mouse IgG1, diabetic test subjects treated with 3
mg/kg anti-ICAM-1 antibody, and diabetic test subjects treated with
5 mg/kg anti-ICAM-1 antibody. NS=Not Significant.
[0024] FIG. 6B is a bar graph showing the retinal vascular
.sup.125I albumin permeation (.mu.g plasma xg tissue wet
weight.sup.-1.times.min.sup.-1) for control, diabetic test subjects
not given anything, diabetic test subjects given 5 mg/kg mouse
IgG1, diabetic test subjects treated with 3 mg/kg anti-ICAM-1
antibody, and diabetic test subjects treated with 5 mg/kg
anti-ICAM-1 antibody. NS=Not Significant.
[0025] FIG. 7 is a bar graph showing the amount of adherent
neutrophils to endothelium in vitro (thousands per mm.sup.2) from
control rats and rats having Diabetes Mellitus (DM). All data shown
are means .+-. Standard Deviation (SD).
[0026] FIG. 8 is a bar graph showing the amount of adherent
neutrophils (thousands per mm.sup.2) for untreated, CD11a, CD11b,
CD18, or CD11a/CD11b/CD18 cocktail treated for control and DM rats.
All data shown are means .+-. SD.
[0027] FIGS. 9A-D are photographs of leukostasis in from AOLF
retinas in non-diabetic rat (FIG. 9A), diabetic rat (FIG. 9B),
diabetic rat treated with the control F(ab').sub.2 (FIG. 9C) and
anti-CD 18 F(ab').sub.2 fragments treated rats (FIG. 9D).
[0028] FIG. 10 is a bar graph showing the density fo trapped
leukocytes (.times.10.sup.-5 cells/pixel.sup.2) for control, DM, DM
and F(ab').sub.2, and DM and anti-CD18 F(ab').sub.2 fragment
treated rats.
[0029] FIGS. 11A-B are photographs of AOLF retina before (FIG. 11A)
and 48 hours after a 50 ng Vascular Endothelial Growth Factor
(VEGF) injection (FIG. 11B). Scale bar denotes 100 .mu.m (3.2
pixel=1 .mu.m).
[0030] FIG. 12 is a bar graph showing the density of trapped
leukocytes (.times.10.sup.-5 cells/pixel.sup.2, mean=SD) in the
retina using AOLF for rats injected intravitreously with 0, 5, 10,
50, 100 ng of VEGF after 48 hours.
[0031] FIG. 13 is a bar graph showing the density of trapped
leukocytes (.times.10.sup.-5 cells/pixel.sup.2, mean=SD) in the
retina using AOLF for rats injected intravitreously with the
vehicle alone or with 50 ng of VEGF after 6, 24, 48, 72, or 120
hours.
[0032] FIG. 14 is a bar graph showing the density of trapped
leukocytes (.times.10.sup.-5 cells/pixel.sup.2, mean=SD) in the
retina using AOLF for rats injected intravitreously with the
vehicle alone or 50 ng of VEGF with and without anti-VEGF mAb
treatment after 48 hours.
[0033] FIGS. 15A-B are photographs of retina 48 hours after rats
were injected intravitreously with 50 ng (FIG. 15A) followed by
fluorescein angiography (FIG. 15B). Arrows indicate areas of
capillary non-perfusion downstream from static leukocytes. Scale
bar denotes 100 .mu.m (3.2 pixel=1 .mu.m).
[0034] FIGS. 16A-B show VEGF-induced retinal ICAM-1 gen expression.
FIG. 16A is a photograph showing results of a ribonuclease
protection that demonstrated that retinal ICAM-1 levels were
significantly increased 20 h following the intravitreous delivery
of 50 ng VEGF. Control animals received 5 .mu.l of PBS solvent
alone. Each lane shows the signal from one retina of one animal.
The lane labeled "Probes" shows a hundred-fold dilution of the
full-length ICAM-1 and 18S riboprobes. The lanes labeled
"RNase-(0.1)" and "RNase-(0.01)" show the ten-fold and hundred-fold
dilutions, respectively, of the full-length riboprobes without
sample or RNase. The lane labeled "RNase+" shows the full-length
riboprobes with RNase, but without sample. FIG. 16B is a bar graph
showing the amount of normalized ICAM-1 mRNA in the retina
(arbitrary units, mean +SD) for rats injected with the vehicle
alone and with 50 ng of VEGF. NS=not significant.
[0035] FIGS. 17A-B are bar graphs showing the effect of anti-ICAM-1
mAb on permeability and leukostasis following intravitreous VEGF
injection. FIG. 17A is a bar graph showing the retinal vascular
[.sup.125I] albumin permeation (.mu.g plasma.times.g tissue wet
weight.sup.-1.times.min.sup.-1, mean +SD) for rats that were
untreated, or treated with the vehicle alone, 50 ng VEGF, 50 VEGF
and mouse IgG1, or 50 ng VEGF and an anti-ICAM-1 antibody. FIG. 17B
is a bar graph showing the density of trapped leukocytes
(.times.10.sup.-5 cells/pixel.sup.2, mean=SD) in the retina using
AOLF for untreated rats or rats treated with treated with the
vehicle alone, 50 ng VEGF, 50 VEGF and mouse IgG1, or 50 ng VEGF
and an anti-ICAM-1 antibody. ICAM-1 bioactivity was inhibited via
intravenous administration of ICAM-1 neutralizing antibody and
retinal permeability (FIG. 17A) or leukostasis (FIG. 17B) were
evaluated, respectively. NS=not significant.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The invention relates to methods of treating and/or
preventing retinal injury in a mammal by administering to the
mammal a compound that inhibits leukocyte interaction which is the
binding of a leukocyte to an endothelial cell or to another
leukocyte. Several antagonists inhibit leukocyte interaction and
include an integrin antagonist, a selectin antagonist, an adhesion
molecule antagonist, or a leukocyte adhesion-inducing cytokine
antagonist or growth factor antagonist. In particular, an
intercellular adhesion molecule-1 antagonist, a CD18 antagonist, a
CD11b antagonist or a VEGF antagonist are encompassed by the
present method. Administration of such antagonists results in a
significant decrease in retinal edema and/or retinal ischemia. The
retinal injury can be caused by retinopathy or a visually-related
disease that involves leukocyte occlusion in blood vessels (e.g.,
capillaries) and their destruction (e.g., atrophy). In particular,
the present methods pertain to treating diabetic retinopathy.
[0037] Diabetic retinopathy is a progressive degeneration of
retinal blood vessels and is a consequence of diabetes, in
particular, diabetes mellitus. One important aspect of the disease
is retinal edema. Fluid build up from deteriorating blood vessels
and capillaries causes edema. As the disease progresses, the damage
proliferates and large hemorrhages and retinal detachment can
result.
[0038] The term "retinopathy" also refers to noninflammatory
degenerative diseases of the retina. The methods of the present
invention encompass retinopathy or a visually-related disease that
is characterized by one or more of the following retinal signs:
capillary obstruction, nonperfusion, leukostasis, formation of
vascular lesions and/or proliferation of new blood vessels in
association with ischemic areas of the retina. Leukostasis refers
to the stasis or non-movement of white blood cells (e.g.,
leukocytes) in the vasculature. Other disorders or diseases
implicated by the invention involve diseases which result in
retinal edema and/or retinal ischemia. Examples of such diseases
include vein occlusions, sickle cell retinopathy, radiation
retinopathy, diabetic retinopathy, VEGF-induced diseases and
retinopathy prematurity.
[0039] Capillary occlusions constitute a characteristic pathologic
feature in diabetic retinopathy, and, when widespread, initiate
neovascularization. Neovascularization (e.g., angiogenesis) refers
to the formation or growth of new blood vessels. Microaneurysms,
intraretinal microvascular abnormalities and vasodilation also are
commonly found in early stages of diabetic retinopathy and have
been correlated to capillary occlusions. Schroder, S. et al.,
American Journal of Pathology, 139 (81), 81-100 (1991). Leukocytes
cause capillary obstruction that is involved in diabetic
retinopathy via two mechanisms. This obstruction is the result of
the leukocytes' large cells volume and high cytoplasmic rigidity.
Leukocytes can become trapped in capillaries under conditions of
reduced perfusion pressure (e.g., caused by vasoconstriction) or in
the presence of elevated adhesive stress between leukocytes and the
endothelium, endothelial swelling, or narrowing of the capillary
lumen by perivascular edema. Id. Examples of leukocytes include
granulocytes, lymphocytes, monocytes, neutrophils, eosinophils, and
basophils. Elevated adhesive stress can result from release of
chemotactic factors or expression of adhesion molecules on
leukocytes or endothelial cells. Secondly, leukocytes injures
capillaries leading to capillary death, also known as "capillary
dropout."
[0040] A number of glycoproteins are involved in the adhesion of
leukocytes. In the case of neutrophils and monocytes, a family of
glycoproteins, known as O.sub.2 integrins, have been identified.
This family of integrins include Lymphocyte Function Associated
Antigen-1 (LFA-1), Mac-1, and p150.95. Some integrins are made up
of molecules referred to as "subunits" or "integrin subunits." The
LFA-1 integrin is comprised of 2 subunits, CD11a and CD18, Mac-1
integrin is comprised of CD11b and CD18, and p150.95 is made up of
CD11c and CD18.
[0041] A corresponding family of glycoproteins, referred to as
selectins, are expressed in endothelial cells or can be induced by
stimulation with endotoxins or cytokines. The selectins include
P-selectin, E-selectin, and L-selectin. The selectin family is
involved in endothelial interaction. Firm adhesion of activated
polymorphonuclear neutrophils (PMN) to the endothelial cells occur
through the interaction between integrins (e.g., LFA-1, MAC-1 and
p150.95) expressed on the PMNs and members of the immunoglobulin
superfamily of proteins, referred to as Intercellular Adhesion
Molecule-1 (ICAM-1), Platelet Endothelial Adhesion Molecule (PCAM),
and Vascular Cell Adhesion Molecule (VCAM), expressed by the
endothelium. Additionally, cytokines such as Tumor Necrosis
Factor-a (TNF-.alpha.), Interleukin-1 .beta. (IL-1 .beta.),
Monocyte Chemotatic Protein-1 (MCP-1), and growth factors (VEGF)
can induce the surface expression of ICAM-1, VCAM-1, and E-selectin
on endothelial cells.
[0042] Intercellular adhesion molecules are involved in and are
important for inflammation responses. Mediators of inflammation
cause an induction of ICAM-1 expression on various cell types and
sites of inflammation. Both soluble and membrane forms of ICAM-1
exist. Roep, B. O. et al., Lancet 343, 1590-1593, 1590 (1994).
ICAM-1 is an inducible cell surface ligand for LFA-1. Larson, R.
S., et al., Immunological Reviews, 114, 181-217, 192 (1990). ICAM-1
is a single chain glycoprotein with a peptide backbone of 55 kD.
ICAM-1 is a member of immunoglobulin super family consisting of 5
immunoglobulin-like domains. ICAM-1 is expressed or induced by
inflammatory mediators on many cell types including endothelial
cells, epithelial cells, keratinocytes, synovial cells,
lymphocytes, and monocytes. The LFA-1 binding site is the first
immunoglobulin domain of ICAM-1. ICAM-1 also binds with Mac-1, an
important mechanism in retinal edema and retinal ischemia. Various
forms of ICAM-1 can be used to generate antagonists, such as
antibodies or antisense molecules.
[0043] Retinal leukostasis is a very early event in diabetic
retinopathy with important functional consequences. Both retinal
vascular leakage and non-perfusion follow its development. The
inhibition of ICAM-1 activity blocks diabetic retinal leukostasis
and potently prevents blood-retinal barrier breakdown. Leukostasis
is associated with the development of vascular nonperfusion and
thus its inhibition can also prevent capillary dropout. Indeed,
activated leukocytes are increased in diabetes and leukocytes have
been associated with capillary loss in the diabetic choroid. The
data described herein demonstrate that ICAM-1-mediated leukostasis
is increased in the retinal vasculature very early in diabetes and
accounts for the majority of diabetes-associated retinal vascular
leakage. Thus, these data, described herein, indicate ICAM-1 as a
new therapeutic target for the prevention of many of the
sight-threatening retinal abnormalities, especially those
associated with diabetes. See Example 1.
[0044] The data described herein also show that CD11a, CD11b, and
CD18 .beta..sub.2 integrin levels were increased on the surface of
neutrophils from diabetic rats. The increases correlated with the
enhanced functional adhesiveness of diabetic neutrophils to rat
endothelial cell monolayers. Similarly, in an in vivo model of
experimentally-induced diabetes, use of anti-CD18 F(ab').sub.2
fragments significantly decreased diabetic retinal leukostasis by
62%, confirming the relevance of the in vitro findings. The data
described herein indicate that the Mac-1 integrin complex is
operative in the adhesion of diabetic neutrophils to the retinal
capillary endothelium. Since a major ligand for Mac-1 is ICAM-1,
these results are consistent with data, shown in Example 1, that
ICAM-1 blockade prevents diabetic retinal leukostasis and
blood-retinal barrier breakdown. See also Example 2.
[0045] Based on the data described herein, it is reasonable to
believe that the leukocyte adhesive changes in this model of
diabetes are of a systemic nature. The assayed neutrophils were
isolated from the peripheral blood, and therefore reflected
systemic neutrophil adhesion molecule expression and bioactivity.
The causes of the surface integrin changes remain unknown, however
they are likely to be linked to hyperglycemia. For example,
hyperglycemia directly impacts TNF.alpha. expression, a cytokine
known to activate integrin adhesion molecules on leukocytes. In
vitro work has also shown that hyperglycemia promotes increased
leukocyte adhesion to endothelium via ICAM-1 and CD18. Thus,
hyperglycemia, either directly or indirectly, is a proximal
stimulus for the ICAM-1 and CD18 upregulation seen in diabetes.
[0046] Also, these data show that a low-level retinal leukostasis
occurs in the normal state. The same molecules that are operative
in the diabetic state also mediate this presumably normal
phenomenon. If the low-level leukostasis in the non-diabetic state
is physiologic, then the specificity of an anti-integrin therapy
can be compromised.
[0047] The results, described herein, also provide additional
evidence of leukocyte involvement in the pathogenesis of diabetic
retinopathy. The aggregate data indicate that diabetic retinopathy
should be, in one sense, redefined as an inflammatory disease. Very
early in diabetes leukocytes adhere to the vascular endothelium,
trigger breakdown of the blood-retinal barrier, impede flow, and in
some instances, extravasate into the retinal parenchyma. The
identification of Mac-1 as a functional adhesive molecule in
diabetic retinopathy provides a target for the prevention and/or
treatment of the disease.
[0048] Data described herein also show that VEGF induces retinal
vascular permeability and leukostasis through ICAM-1. Retinal
leukostasis was also spatially linked to capillary non-perfusion.
The vitreous concentration at which VEGF begins to induce these
changes (12.5 nM) is within the range of vitreous VEGF
concentrations observed in human eyes with diabetic retinopathy.
The leukostasis observed in these studies was specific to VEGF
because co-injection of a neutralizing antibody abrogated the
response. Finally, these findings are consistent with our data
showing VEGF-induced ICAM-1 expression in the retinal vasculature.
See Example 3.
[0049] Leukocytes, via their own VEGF, serve to amplify the direct
effects of VEGF when they bind to endothelium. VEGF has been
demonstrated in neutrophils, monocytes, eosinophils, lymphocytes
and platelets. The fact that some leukocytes possess high affinity
VEGF receptors and migrate in response to VEGF makes this scenario
even more likely.
[0050] The data also show that VEGF-induced capillary non-perfusion
occurs downstream from areas of leukocyte adhesion.
Leukocyte-mediated non-perfusion characterizes experimental
diabetic retinopathy. In diabetes, patent capillaries become
occluded downstream from newly arrived static leukocytes. Later,
following the disappearance of the leukocytes, the capillaries
reopen. Since neutrophil and monocyte diameters can exceed those of
retinal capillary lumens, leukocyte-mediated flow impedance is a
likely mechanism.
[0051] Taken together, these data indicate that VEGF-induced
vascular permeability is mediated by ICAM-1-mediated retinal
leukostasis. These data are the first to show that a
non-endothelial cell type contributes to VEGF-induced vascular
permeability. They are also the first to provide a mechanism for
the capillary non-perfusion induced by VEGF. Given these findings,
targeting ICAM-1 proves useful in the treatment of diseases
characterized by VEGF-induced vascular changes, such as diabetic
retinopathy.
[0052] The invention takes advantage of the surprising discovery
that inhibiting integrins, and in particular the Mac-1 or a pathway
thereof, results in a reduction in retinal edema and/or retinal
ischemia. This reduction in both retinal edema and/or retinal
ischemia provides an effective treatment for various ocular
diseases, including retinopathy. In one aspect of the invention, an
antagonist's biological activity refers to a compound that inhibits
the Mac-1 integrin adhesion or a pathway thereof. Inhibition can
occur directly (e.g., by inhibiting binding of the Mac-1 molecule
or a subunit thereof such as CD18 or CD11b), or indirectly (e.g.,
by inhibiting a molecule that affects Mac-1 such as by inhibiting
ICAM-1 expression or Vascular Endothelial Growth Factor (VEGF)
expression). The surprising results of directly inhibiting Mac-1 by
inhibiting Mac-1 subunits are shown in Example 2. Several molecules
indirectly impact on Mac-1's biological activity (e.g., its ability
to bind to ICAM-1, induce leukocyte adhesion, induce leukostasis,
cause edema and/or cause ischemia). For example, ICAM-1 directly
binds to Mac-1. Inhibiting ICAM-1 reduces retinal edema and
ischemia. See Example 1. Similarly, VEGF mediates ICAM-1 expression
in the retinal vasculature, and induces vascular permeability and
non-perfusion. Inhibiting VEGF results in decreased expression of
ICAM-1, and a reduction in both retinal edema and retinal ischemia.
See Example 3. Additionally, TNF-.alpha., a cytokine, induces
ICAM-1 expression, which, in turn, can stimulate and increase
leukocyte adhesion. Inhibiting the TNF-.alpha. pathway,
significantly reduces leukocyte adhesion. See Example 2. Inhibiting
Mac-1 and molecules that affect the Mac-1 pathway (e.g., ICAM-1
expression) unexpectedly results in reductions of retinal edema and
ischemia.
[0053] Inhibition of a molecule encompassed by the invention (e.g.,
Mac-1, CD18, CD11b, ICAM-1, VEGF or TNF-.alpha.) can be
accomplished in several ways. A molecule can be made inactive or
its action disrupted. For example, the expression of these
molecules can be inhibited prior to the molecule exiting the cell
using, for example, antisense technology, etc. The molecule can
also be made inactive by inhibiting its binding to a receptor after
it exits the cell or is exposed on the membrane of the cell, e.g.,
with an antibody or antibody fragment. Additionally, the action of
these molecules can be inhibited by disrupting the signaling
downstream from the receptor (e.g. alterations in phosphorylation).
These and other methods can be used so long as the activity or
action of one or more of the molecule described herein is inhibited
or disrupted.
[0054] The invention relates to preventing or treating retinal
injury wherein the retinal injury involves retinal edema and/or
retinal ischemia, comprising administering a compound that inhibits
the binding of a leukocyte to an endothelial cell or another
leukocyte in, for example, a blood vessel or capillary, which
results in the reduction of retinal edema and/or retinal ischemia.
The term "retinal injury" is defined herein as a decreased ability
for the retina to function normally as measured, for example, by
the patient's vision, electrical signal potential, fluorescein
angiograms or other known methods or methods developed in the
future. The compound has the ability to inhibit or reduce leukocyte
occlusion in the retinal vasculature. As described herein, the
compound can be an integrin antagonist (e.g., Mac-1 antagonist), an
integrin subunit antagonist (e.g., CD18 antagonist or a CD11b
antagonist), a selectin antagonist, a leukocyte adhesion-inducing
cytokine antagonist or growth factor antagonist (e.g., TNF-.alpha.,
IL-1.beta., MCP-1 and VEGF antagonist), or an adhesion molecule
antagonist (e.g., an ICAM-1, ICAM-2, ICAM-3, PCAM or VCAM
antagonist). In particular, the invention also pertains to
administering an ICAM-1 antagonist, a VEGF antagonist, a Mac-1
antagonist, a CD18 antagonist, or a CD11b antagonist, to treat
retinal edema, retinal ischemia, and/or diabetic retinopathy. The
various forms of the antagonists are described herein.
[0055] The methods described herein can be used for treating ocular
tissue that experiences leukostasis, edema and/or ischemia. Such
tissue includes the retina, and the choroid. For example, the
invention includes a method of treating or reducing leukostasis,
edema and/or ischemia in the retina or the choroid of an affected
mammal by administering to the mammal one or a combination of any
one of the antagonists described herein.
[0056] The invention includes methods of inhibiting leukocyte
interaction, comprising contacting a leukocyte or endothelial cell
with an antagonist. For example, using the various antagonists
described herein, one can contact a leukocyte with an integrin
antagonist, an endothelial cell with an adhesion molecule
antagonist or a selectin antagonist, or subject the cytokines that
induce surface expression of ICAM-1, VCAM-1, and E-selectin to a
leukocyte adhesion-inducing cytokine antagonist.
[0057] The invention further comprises the use of an ICAM-1, a
CD18, a CD11b or a VEGF antagonist in conjunction with a second
antagonist. Genetic variability that exists among various patient
populations and/or additional mechanisms can warrant administering
more than one antagonist. Any combination of the above antagonists
can be used. For example, the present methods include administering
an ICAM-1 antagonist, which is specific to a particular epitope of
ICAM-1, and an additional ICAM-1 antagonist, which is specific to a
different epitope or genetic variation. Similarly, an ICAM-1
antagonist can be administered with any one of the antagonists
described herein. Administering a combination of antagonists to
prevent the leukocyte adhesion to endothelial cells and/or
leukocytes results in even more effective treatment of diabetic
retinopathy or a more dramatic reduction in retinal edema and/or
ischemia. See Example 2 in which both a CD18 and CD11b antagonist
was used to reduce leukocyte adhesion. Given the causal effect of
leukocyte adhesion on retinal edema and/or ischemia, as proven by
the data, administration of a CD18 and/or CD11b antagonist is
expected to reduce retinal edema and/or retinal ischemia. The
combination of antagonists can be administered at substantially the
same time, or sequentially, with suitable intervals between
administration of the antagonists to confer the desired effect.
[0058] The invention also relates to decreasing or reducing the
amount of ischemia and/or edema present in an individual by
administering an effective amount of an ICAM-1 a CD18, a CD11b or a
VEGF antagonist. Ischemia refers to tissue which lacks proper or
suitable blood flow. Ischemia refers to an inadequate circulation
of blood flow which can be the result of a mechanical obstruction
(e.g., trapped leukocyte) of the blood supply or damage to the
blood supplying vessel which results in a reduction of the blood
flow. Inadequate blood flow results in reduced tissue oxygenation.
Hence, ischemia can be a function of leukostasis, and can be
measured by determining the density of trapped leukocytes, and
other methods known in the art or developed in the future as
described herein.
[0059] Edema refers to the build up of excess fluid caused by
vasculature leakage (e.g., vascular permeability). Edema also
refers to the build up or accumulation of fluid when the fluid is
not timely or properly cleared. As described herein, leukocytes
become trapped in the capillaries in the conditions of reduced
perfusion pressure (e.g., caused by constriction as seen in early
stages of diabetes) or in the presence of an elevated adhesive
stress between leukocytes and endothelium, endothelial swelling or
narrowing of the capillary lumen by perivascular edema. The
leukocyte build up can cause leakage from the blood vessel. Thus,
edema can be measured by determining the amount of retinal vascular
albumin permeation, as referred to as "vascular permeability," as
described herein.
[0060] The methods of treatment described herein include reducing
or decreasing the amount of ischemia and/or edema by administering
antagonist that inhibits leukocyte and endothelial cell
interaction, as described herein. The decrease in ischemia is at
least about 10% and can be greater, such as at least about 20%,
30%, 40%, 50% 60%, 70%, 80%, 90%, or 95%. The decrease in edema is
at least about 10%, and can be greater, such as at least about 20%,
30%, 40%, 60%, 70%, 80%, 90%, and preferably at least about
95%.
[0061] The reduction or decrease of the retinal edema and/or
retinal ischemia can be determined, as compared to a control,
standard, or baseline. For example, a measure of edema or ischemia
can be made, in a mammal, prior to administering one of the
compounds described herein, and one or more times subsequent to
administration. A percentage change between two or more
measurements, or a value reflecting the change in the measurements
can be determined. The level of edema and/or ischemia can be
quantified using methods known in the art, and a decrease, as
compared with a control, standard, or baseline, indicates
successful treatment. The quantified amounts of edema and/or
ischemia can be compared with a suitable control to determine if
the levels are decreased. The sample to be tested can be compared
with levels for the specific individual from previous time points
(e.g., before having diabetic retinopathy, or during various phases
of treatment for the diabetic retinopathy), or with levels in
normal individuals (e.g., an individual without the disease) or
suitable controls. An individual who is being treated for diabetic
retinopathy can be monitored by determining the levels of edema
and/or ischemia at various time points. Such levels of edema and/or
ischemia can be determined before treatment, during treatment, and
after treatment. A decrease in the level of ischemia and/or edema,
as described herein, indicates successful treatment. Ischemia
and/or edema can be measured using methods now known or those
developed in the future. See Kohner E. M., et al. Diabetic
Retinopathy Metabolism, 25:1985-1102 (1975). For example, ischemia
and edema can be measured using a fluorescein angiogram or by
measuring the vision loss in a patient. Edema can also be assessed
by measuring electrical signals or potential, visualizing the
retina using a slit lamp, fluorescein angiogram, or by using a
sensitive isotope dilution method.
[0062] Another aspect of the invention includes method for treating
or preventing neovascularization. One of the more difficult
problems in opthalmology is treating the ocular surface
abnormalities that accompany limbal cell injury. The limbus is a
specialized tissue that marks the transition between cornea and
conjunctiva. Stem cells reside in this area and give rise to the
normal corneal epithelium. When the limbus is sufficiently
destroyed, an inflammatory corneal neovascularization ensues and a
conjunctiva-like epithelium covers the cornea. The latter lacks the
smoothness and cohesion of the normal corneal epithelium, making it
optically inferior and prone to erosions. Corneal
neovascularization, and the serum it delivers via leaky vessels,
supports the abnormal conjunctiva-like surface that covers the
cornea. The selective injury of corneal vessels produced a
reversion to a more normal corneal epithelial phenotype. Huang, A.
et al Ophthal. 95:228 (1988). Unlike the experimental model, the
laser injury of corneal vessels has not seen long-term success in
humans. Thus, an effective treatment for the corneal
neovascularization that follows limbal injury has previously
remained an elusive goal.
[0063] Corneal neovascularization secondary to limbal injury
requires, in part, vascular endothelial growth factor (VEGF). VEGF
induces intercellular adhesion molecule-1 (ICAM-1) expression in
the vasculature of various tissues. Further, exogenous VEGF induces
the adhesion of leukocytes to the endothelium of ocular surface
vessels, a process that can be partially blocked with anti-ICAM-1
antibodies. The effect of ICAM-1 and its common ligand the
.beta..sub.2 integrin CD18 was tested, on limbal injury-associated
corneal neovascularization and inflammation in a
pathophysiologically-relevant model.
[0064] Corneal neovascularization leads to vision loss in eyes that
have undergone extensive injury to the limbus. This situation
characterizes a number of conditions, including traumatic alkali
injury, Stevens Johnson syndrome and ocular cicatricial pemphagoid.
Other conditions that involve neovascularization are diseases such
as age-related macular degeneration, choroidal neovascularization,
sickle cell retinopathy, retina vein occlusion, diabetic
retinopathy, a condition associated with limbal injury and a
condition associated with increased neovascularization. To date, no
treatments have proven effective at preventing the
neovascularization associated with these conditions. A
pathophysiologically-relevant mouse model of limbal injury was
utilized to test the role of CD18 and intercellular adhesion
molecule-1 (ICAM-1) in the production of corneal
neovascularization. The data described herein show that CD18 and
ICAM-1 deficient mice have 39% (n=5, p=0.0054) and 33% (n=5,
p=0.013) less neovascularization, respectively, when compared to
strain-specific normal controls. Corneal neutrophil counts were
reduced by 66% (n=5, p=0.0019) and 39% (n=5, p=0.0016) in the CD18
and ICAM-1 deficient mice, respectively. Taken together, these data
identify CD-18 and ICAM-1 as important mediators of the
inflammation-associated neovascularization that follows limbal
injury. CD18 and ICAM-1 also serve as therapeutic targets for the
treatment of the corneal neovascularization associated with limbal
injury.
[0065] Hence, another embodiment of the invention includes methods
of treating or preventing ocular (e.g., corneal, retinal or
choroid) neovascularization in a mammal (e.g., an individual) by
administering to the mammal a CD18 antagonist and an ICAM-1
antagonist or CD18 antagonist. The inhibition of both CD18 and
ICAM-1, or CD18, result in significantly less neovascularization,
or as compared to a control, as defined herein. See Example 4.
[0066] Hence, the present methods utilize various forms of
antagonists. An antagonist, as defined herein, means a compound
that can inhibit, either partially or fully, the binding of a
leukocyte to an endothelial cell or to another leukocyte. An
antagonist's biological activity also refers to a compound that can
reduce or lessen the interaction between a leukocyte and an
endothelial cell, or another leukocyte.
[0067] The terms, "antagonist" or "antibody," include proteins and
polypeptides that are integrin (e.g., LFA-1, Mac-1 or p150.95)
antagonists, integrin subunit (CD18, CD11a or CD11b) antagonists,
adhesion molecule (e.g., ICAM, PCAM or VCAM) antagonists, selectin
(e.g., P-selectin, L-selectin or E-selectin) antagonists, or
leukocyte adhesion-inducing cytokine antagonists or growth factor
antagonists (e.g. antagonists to TNF-.alpha., IL-1.beta., MCP-1 or
VEGF). These terms also include proteins and polypeptides that have
amino acid sequences analogous to the amino acid sequence of the
protein, as described herein, and/or functional equivalents
thereof. These terms also encompass various analogues, homologues,
or derivatives thereof. Analogous amino acid sequences are defined
to mean amino acid sequences with sufficient identity to the
antagonist's amino acid sequence so as to possess its biological
activity. For example, an analogous peptide can be produced with
"silent" changes in amino acid sequence wherein one, or more, amino
acid residues differ from the amino acid residues of the protein,
yet still possess its biological activity. Examples of such
differences include additions, deletions, or substitutions of
residues of the amino acid sequence of the protein or polypeptide.
Also encompassed by these terms, are analogous polypeptides that
exhibit greater, or lesser, biological activity of the
antagonist.
[0068] Antagonists also include antibody or antibody fragments,
peptide mimetics molecules, antisense molecules, ribozymes,
aptamers (nucleic acid molecules), and small molecule antagonists.
Soluble forms of molecules (e.g., soluble ICAM) can also act as an
antagonist because it can bind to the leukocyte, thereby preventing
the membrane bound form from binding.
[0069] The term "antagonist" and "nucleic acid sequence" include
homologues, as defined herein. The homologous proteins and nucleic
acid sequences can be determined using methods known to those of
skill in the art. Initial homology searches can be performed at
NCBI against the GenBank (release 87.0), EMBL (release 39.0), and
SwissProt (release 30.0) databases using the BLAST network service.
Altshul, S F, et al, J. Mol. Biol. 215: 403 (1990); Altschul, S F.,
Nucleic Acids Res. 25:3389-3402 (1998). the teachings of both are
incorporated herein by reference. Computer analysis of nucleotide
sequences can be performed using the MOTIFS and the FindPatterns
subroutines of the Genetics Computing Group (GCG, version 8.0)
software. Protein and/or nucleotide comparisons can also be
performed according to Higgins and Sharp (Higgins, D. G. and P. M.
Sharp, "Description of the method used in CLUSTAL," Gene, 73:
237-244 (1988)). Homologous proteins and/or nucleic acid sequences
are defined as those molecules with greater than 70% sequences
identity and/or similarity (e.g., 75%, 80%, 85%, 90%, or 95%
homology).
[0070] Biologically active derivatives or analogs of the
antagonists described herein also include peptide mimetics. Peptide
mimetics can be designed and produced by techniques known to those
of skill in the art. (see e.g., U.S. Pat. Nos. 4,612,132; 5,643,873
and 5,654,276, the teachings of which are incorporated herein by
reference). These mimetics can be based, for example, on the
protein's specific amino acid sequence and maintain the relative
position in space of the corresponding amino acid sequence. These
peptide mimetics possess biological activity similar to the
biological activity of the corresponding peptide compound, but
possess a "biological advantage" over the corresponding amino acid
sequence with respect to one, or more, of the following properties:
solubility, stability and susceptibility to hydrolysis and
proteolysis.
[0071] Methods for preparing peptide mimetics include modifying the
N-terminal amino group, the C-terminal carboxyl group, and/or
changing one or more of the amino linkages in the peptide to a
non-amino linkage. Two or more such modifications can be coupled in
one peptide mimetic molecule. Modifications of peptides to produce
peptide mimetics are described in U.S. Pat. Nos. 5,643,873 and
5,654,276, the teachings of which are incorporated herein by
reference. Other forms of the proteins, polypeptides and antibodies
described herein and encompassed by the present invention, include
those which are "functionally equivalent." This term, as used
herein, refers to any nucleic acid sequence and its encoded amino
acid which mimics the biological activity of the protein,
polypeptide or antibody and/or functional domains thereof.
[0072] The term, "ICAM-1 antagonist" includes antagonists that
directly (e.g., by inhibiting the ICAM-1 molecules itself) or
indirectly inhibit ICAM-1 (e.g., by inhibiting a molecules that
affects induction of ICAM-1 such as a VEGF antagonist or a
TNF-.alpha. antagonist). Such antagonist are those which lead to a
reduction in edema and/or ischemia. Antagonists also include other
integrin antagonists (e.g., a LFA-1 or p150.95 antagonists),
selectin antagonists (e.g., P-selectin, E-selectin or L-selectin
antagonist) and other adhesion molecule antagonists (e.g., ICAM-2,
ICAM-3, PCAM or VCAM antagonist) or a leukocyte adhesion-inducing
cytokine antagonist or growth factor antagonist (an antagonist for
TNF-1.alpha., IL-1.beta., MCP-1 or VEGF).
[0073] An ICAM-1 antagonist is also a composition that inhibits the
binding of ICAM-1 to a receptor or has the ability to decrease or
affect the function of ICAM-1. Such antagonists include antibodies
to ICAM-1 (e.g., the IA29 antibody), antisense molecules that
hybridize to nucleic acid which encodes ICAM-1. ICAM-1 antagonists
also include ribozymes, aptimers, or small molecule inhibitors that
are specific for ICAM-1 or the nucleic acid that encodes ICAM-1.
Antagonists of ICAM-1 include compounds which inhibit the binding
between LFA-1 or Mac-1 and ICAM-1, or compounds that reduce the
biological activity or function of ICAM-1. The biological activity
of ICAM-1 refers to the ability to bind to LFA-1 or, in particular,
to Mac-1, the ability to induce leukocyte adhesion, the ability to
cause ischemia and/or the ability to cause edema.
[0074] The terms "antibody" or "immunoglobulin" refer to an
immunoglobulin or fragment thereof having specificity to a molecule
involved in leukocyte-leukocyte interaction or
leukocyte-endothelium interaction. Examples of such antibodies
include anti-integrin antibodies (e.g., antibodies specific to
LFA-1, Mac-1 or p150.35), anti-integrin subunit antibodies (e.g.,
antibodies specific to CD18, CD11b or a combination thereof),
anti-selectin antibodies (e.g., antibodies specific to P-selection,
E-selection and L-selectin), antibodies to leukocyte
adhesion-inducing cytokine antagonists or growth factor antagonists
(e.g., TNF-.alpha., IL-1.beta., MCP-1 and VEGF antibodies), and
adhesion molecule antibodies (e.g., ICAM-1, ICAM-2, ICAM-3, PCAM or
VCAM antibodies). For example, the terms "ICAM-1 antibody," or
"ICAM-1 immunoglobulin" refer to immunoglobulin or fragment thereof
having specificity for ICAM-1.
[0075] The term, "antibody" is also intended to encompass both
polyclonal and monoclonal antibodies including transgenically
produced antibodies. The terms polyclonal and monoclonal refer to
the degree of homogeneity or an antibody preparation and are not
intended to be limited to particular methods of production. An
antibody can be raised against an appropriate immunogen, such as an
isolated and/or recombinant polypeptide (e.g., ICAM-1, CD18, CD11b,
VEGF, or TNF-.alpha.) or portion thereof (including synthetic
molecules such as synthetic peptides). In one embodiment,
antibodies can be raised against an isolated and/or recombinant
antigen or portion thereof (e.g., a peptide) or against a host cell
which expresses recombinant antigen or a portion thereof. In
addition, cells expressing recombinant antigen (e.g., ICAM-1, CD18,
CD11b, VEGF, or TNF-.alpha.), such as transfected cells, can be
used as immunogens or in a screening for an antibody which binds
the receptor.
[0076] Preparation of immunizing antigen, and polyclonal and
monoclonal antibody production, can be performed using any suitable
technique. A variety of methods have been described (see e.g.,
Kohler et al., Nature, 256:495-497 (1975) and Eur. J. Immunol. 6:
511-519 (1976); Milstein et al., Nature 266:550-552 (1977);
Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D. Lane,
1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory: Cold Spring Harbor, N.Y.); Current Protocols In
Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F.
M. et al., Eds., (John Wiley & Sons: New York, N.Y.), Chapter
11, (1991)).
[0077] Following immunization, anti-peptide antisera can be
obtained from the immunized animal, and if desired, polyclonal
antibodies can be isolated from the serum. As described herein,
purified recombinant proteins generated in E. coli were used to
immunize rabbits to generate specific antibodies directed against
the antigen. These antibodies recognize the recombinant protein
expressed in E. coli. Monoclonal antibodies can also be produced by
standard techniques which are well known in the art (Kohler and
Milstein, Nature 256:495-497 (1975); Kozbar et al., Immunology
Today 4:72 (1983); and Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)). Generally, a
hybridoma is produced by fusing a suitable immortal cell line
(e.g., a myeloma cell line such as SP2/0) with antibody producing
cells. The antibody producing cell, preferably those of the spleen
or lymph nodes, can be obtained from animals immunized with the
antigen of interest. The fused cells (hybridomas) can be isolated
using selective culture conditions, and cloned by limiting
dilution. Cells which produce antibodies with the desired
specificity can be selected by a suitable assay (e.g., ELISA).
[0078] Other suitable methods of producing or isolating antibodies
of the requisite specificity can be used, including, for example,
methods which select recombinant antibody from a library, by PCR,
or which rely upon immunization of transgenic animals (e.g., mice)
capable of producing a full repertoire of human antibodies (see
e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551-2555
(1993); Jakobovits et al., Nature, 362: 255-258 (1993); Lonberg et
al., U.S. Pat. No. 5,545,806; Surani et al., U.S. Pat. No.
5,545,807).
[0079] For example, the monoclonal antibody, IA29 can be used as
described herein. The IA29 antibody is specific for ICAM-1, and can
be purchased from R and D Systems, Minneapolis, Minn. Similarly,
the anti-CD11a, anti-CD18, and the anti-CD11b antibodies utilized
in the experiments described herein are the WT.1 mAb, 6G2 mAb and
the MRC OX-42 mAb, respectively, and can be obtained from Serotec,
Inc. (Raleigh, N.C.).
[0080] Functional fragments of antibodies, including fragments of
chimeric, humanized, primatized, veneered or single chain
antibodies, can also be produced. Functional fragments or portions
of the foregoing antibodies include those which are reactive with
the antigen (e.g., ICAM-1, CD18, CD11b, VEGF, or TNF-.alpha.). For
example, antibody fragments capable of binding to the antigen or
portion thereof, including, but not limited to, Fv, Fab, Fab' and
F(ab').sub.2 fragments are encompassed by the invention. Such
fragments can be produced by enzymatic cleavage or by recombinant
techniques. For instance, papain or pepsin cleavage can generate
Fab or F(ab').sub.2 fragments, respectively. Antibodies can also be
produced in a variety of truncated forms using antibody genes in
which one or more stop codons has been introduced upstream of the
natural stop site. For example, a chimeric gene encoding a
F(ab').sub.2 heavy chain portion can be designed to include DNA
sequences encoding the CH.sub.1 domain and hinge region of the
heavy chain.
[0081] It will be appreciated that the antibody can be modified,
for example, by incorporation of or attachment (directly or
indirectly (e.g., via a linker)) of a detectable label such as a
radioisotope, spin label, antigen (e.g., epitope label such as a
FLAG tag) or enzyme label, flourescent or chemiluminescent group
and the like, and such modified forms are included within the term
"antibody."
[0082] A suitable antagonist is also an antisense molecule that can
hybridize to the nucleic acid which encodes the target polypeptide
(e.g., ICAM-1, CD18, CD11b, VEGF, or TNF-.alpha.). The
hybridization inhibits transcription and/or synthesis of the
protein. Antisense molecules can hybridize to all, or a portion of
the nucleic acid. Producing such antisense molecules can be done
using techniques well-known to those of skill in the art. For
example, antisense molecules or constructs can be made using method
known in the art. DeMesmaeker, Alain, et al., Acc Chem. Res.
28:366-374 (1995), Setlow, Jane K., Genetic Engineering, 20:143-151
(1998); Dietz, U.S. Pat. No. 5,814,500, filed Oct. 31, 1996,
entitled, "Delivery Construct for Antisense Nucleic Acids and
Method of Use," the teachings all of which are incorporated by
reference in their entirety. In particular, constructing an
antisense molecule for an ICAM-1 antagonist is described in detail
in WO 97/46671, entitled, "Enhanced Efficacy of Liposomal
Anti-sense Delivery," the teachings of which are incorporated by
reference in their entirety. Additionally, developing an antisense
molecule to inhibit a retinal disorder (e.g., retinopathy) is
described in Robinson, G. S., et al., Proc. Natl. Acad. Sci.
93:4851-4856 (1996).
[0083] Administration and Dosages:
[0084] The terms "pharmaceutically acceptable carrier" or a
"carrier" refer to any generally acceptable excipient or drug
delivery device that is relatively inert and non-toxic. The
antagonist can be administered with or without a carrier. A
preferred embodiment is to administer the antagonist (e.g., ICAM-1
antagonist) to the retinal area or the vasculature around or
leading to the retina. Exemplary carriers include calcium
carbonate, sucrose, dextrose, mannose, albumin, starch, cellulose,
silica gel, polyethylene glycol (PEG), dried skim milk, rice flour,
magnesium stearate, and the like. Suitable formulations and
additional carriers are described in Remington's Pharmaceutical
Sciences, (17th Ed., Mack Pub. Co., Easton, Pa.), the teachings of
which are incorporated herein by reference in their entirety. The
antagonist can be administered systemically or locally (e.g., by
injection or diffusion).
[0085] Suitable carriers (e.g., pharmaceutical carriers) also
include, but are not limited to sterile water, salt solutions (such
as Ringer's solution), alcohols, polyethylene glycols, gelatin,
carbohydrates such as lactose, amylose or starch, magnesium
stearate, talc, silicic acid, viscous paraffin, fatty acid esters,
hydroxymethylcellulose, polyvinyl pyrolidone, etc. Such
preparations can be sterilized and, if desired, mixed with
auxiliary agents, e.g., lubricants, preservatives, stabilizers,
wetting agents, emulsifiers, salts for influencing osmotic
pressure, buffers, coloring, and/or aromatic substances and the
like which do not deleteriously react with the active compounds.
They can also be combined where desired with other active
substances, e.g., enzyme inhibitors, to reduce metabolic
degradation. A carrier (e.g., a pharmaceutically acceptable
carrier) is preferred, but not necessary to administer an
antagonist (e.g., an ICAM-1 antagonist).
[0086] For parenteral application, particularly suitable are
injectable, sterile solutions, preferably oily or aqueous
solutions, as well as suspensions, emulsions, or implants,
including suppositories. In particular, carriers for parenteral
administration include aqueous solutions of dextrose, saline, pure
water, ethanol, glycerol, propylene glycol, peanut oil, sesame oil,
polyoxyethylene-polyoxypropylene block polymers, and the like.
Ampules are convenient unit dosages.
[0087] Preferably, the antagonist is administered locally to the
eye, retinal area, choroid area or associated vasculature. The
antagonist can also be administered to the cornea of the eye. The
antagonist diffuses into the eye and contacts the retina or
surrounding vasculature (e.g., eye drops, creams or gels).
[0088] One or more antagonists described herein can be
administered. When administering more than one antagonist, the
administration of the antagonists can occur simultaneously or
sequentially in time. The antagonists can be administered before
and after one another, or at the same time. Thus, the term
"co-administration" is used herein to mean that the antagonists
will be administered at times to reduce leukostasis, edema and/or
ischemia. The methods of the present invention are not limited to
the sequence in which the various antagonists are administered, so
long as the antagonists are administered close enough in time to
produce the desired effect. The methods also include
co-administration with other drugs that used to treat retinopathy
or other diseases described herein.
[0089] The compositions of the present invention can be
administered intravenously, parenterally, orally, nasally, by
inhalation, by implant, by injection, or by suppository. The
composition can be administered in a single dose or in more than
one dose over a period of time to confer the desired effect.
[0090] The actual effective amounts of drug of the present
invention can vary according to the specific drug being utilized,
the particular composition formulated, the mode of administration
and the age, weight and condition of the patient, for example. As
used herein, an effective amount of an ICAM antagonist is an amount
of the drug which is capable of reducing the edema and/or ischemia
levels. Dosages for a particular patient can be determined by one
of ordinary skill in the art using conventional considerations,
(e.g. by means of an appropriate, conventional pharmacological
protocol).
EXEMPLIFICATION
Example 1
Prevention of Leukostasis and Vascular Leakage Diabetic Retinopathy
via ICAM-1 Inhibition
[0091] Diabetic retinopathy is a leading cause of adult vision loss
and blindness. Much of the retinal damage that characterizes the
disease results from retinal vascular leakage and non-perfusion.
This study demonstrates that diabetic retinal vascular leakage and
non-perfusion are temporally and spatially associated with retinal
leukocyte stasis (leukostasis) in the rat model of
streptozotocin-induced diabetes. Retinal leukostasis increases
within days of developing diabetes and correlates with the
increased expression of retinal intercellular adhesion molecule-1
(ICAM-1). ICAM-1 blockade with a monoclonal antibody prevents
diabetic retinal leukostasis (e.g., resulting in ischemia) and
vascular leakage (e.g., resulting in edema) by 48.5% and 85.6%,
respectively. These data identify the causal role of leukocytes in
the pathogenesis of diabetic retinopathy and demonstrate the
important utility of ICAM-1 inhibition as a therapeutic strategy
for the prevention of diabetic retinopathy.
[0092] While retinal vascular leakage and non-perfusion are
recognized as two major complications of diabetes, their
pathogenesis remains poorly understood. Leukocytes may be involved
in the genesis of these complications. Diabetic retinopathy is not
generally considered an inflammatory disease, yet the retinal
vasculature of humans and rodents with diabetes mellitus contains
increased numbers of leukocytes. Many of these leukocytes are
static. The causes and consequences of this phenomenon are largely
unknown. Intercellular adhesion molecule-1 (ICAM-1) is a peptide
that mediates leukocyte adhesion and transmigration. ICAM-1 may be
operative in the stasis observed in diabetic retinopathy because
ICAM-1 immunoreactivity is increased in the diabetic retinal
vasculature of humans. However, little is known about the direct
pathogenetic role of ICAM-1 in diabetic retinopathy. This study
investigated the mechanisms of diabetic retinal leukocyte stasis
(leukostasis) and the role leukocytes play in the development of
two sight-threatening complications, vascular leakage and capillary
non-perfusion.
Experimental Procedures
[0093] Animals and Experimental Diabetes. Long-Evans rats weighing
approximately 200 g received a single 60 mg/kg injection of
streptozotocin (Sigma, St. Louis, Mo.) in 10 mM citrate buffer, pH
4.5, after an overnight fast. Control non-diabetic animals received
citrate buffer alone. Animals with blood glucose levels greater
than 250 mg/dl 24 hours later were considered diabetic. Blood
pressure was measured using a noninvasive cuff sensor and
monitoring system (Ueda Electronics, Tokyo, Japan). Blood
anticoagulated with EDTA was drawn from the abdominal aorta of each
rat after the experiment. The blood sample was analyzed using a
hematology analyzer. The rats were fed on standard laboratory chow
and were allowed free access to water in an air-conditioned room
with a 12-hour light-12-hour dark cycle until they were used for
the experiments.
[0094] Acridine Orange Leukocyte Fluorography (AOLF) and
Fluorescein Angiography. Leukocyte dynamics in the retina were
studied with AOLF (Miyamoto, K., et al., Invest. Opthalmol. Vis.
Sci., 39:2190-2194 (1998); Nishiwaki, H., et al., Invest.
Opthalmol. Vis. Sci., 37:1341-1347 (1996); Miyamoto, K., et al.,
Invest. Opthalmol. Vis. Sci., 37:2708-2715 (1996)). Intravenous
injection of acridine orange causes leukocytes and endothelial
cells to fluoresce through the non-covalent binding of the molecule
to double stranded nucleic acid. When a scanning laser
opthalmoscope is utilized, retinal leukocytes within blood vessels
can be visualized in vivo. Twenty minutes after acridine orange
injection, static leukocytes in the capillary bed can be observed.
Immediately after observing and recording the static leukocytes,
fluorescein angiography was performed to study the relationship
between static leukocytes and retinal vasculature.
[0095] Twenty-four hours before AOLF and fluorescein angiography
were performed, all rats had a heparin-lock catheter surgically
implanted in the right jugular vein for the administration of
acridine orange or sodium fluorescein dye. The catheter was
subcutaneously externalized to the back of the neck. The rats were
anesthetized for this procedure with xylazine hydrochloride (4
mg/kg) (Phoenix Pharmaceutical, St. Joseph, Mo.) and ketamine
hydrochloride (25 mg/kg) (Parke-Davis, Morris Plains, N.J.).
Immediately before AOLF, each rat was again anesthetized, and the
pupil of the left eye was dilated with 1% tropicamide (Alcon,
Humancao, Puerto Rico) to observe leukocyte dynamics. A focused
image of the peripapillary fundus of the left eye was obtained with
a scanning laser opthalmoscope (SLO; Rodenstock Instrument, Munich,
Germany). Acridine orange (Sigma, St. Louis, Mo.) was dissolved in
sterile saline (1.0 mg/ml) and 3 mg/kg was injected through the
jugular vein catheter at a rate of 1 ml/min. The fundus was
observed with the SLO using the argon blue laser as the
illumination source and the standard fluorescein angiography filter
in the 40o field setting for 1 minute. Twenty minutes later, the
fundus was again observed to evaluate leukostasis in the retina.
Immediately after evaluating retinal leukostasis, 20 .mu.l of 1%
sodium fluorescein dye was injected into the jugular vein catheter.
The images were recorded on a videotape at the rate of 30
frames/sec. The video recordings were analyzed on a computer
equipped with a video digitizer (Radius, San Jose, Calif.) that
digitizes the video image in real time (30 frames/sec) to
640.times.480 pixels with an intensity resolution of 256 steps. For
evaluating retinal leukostasis, an observation area around the
optic disc measuring ten disc diameters in diameter was determined
by drawing a polygon surrounded by the adjacent major retinal
vessels. The area was measured in pixels and the density of trapped
leukocytes was calculated by dividing the number of trapped
leukocytes, which were recognized as fluorescent dots, by the area
of the observation region. The densities of leukocytes were
calculated generally in eight peripapillary observation areas and
an average density was obtained by averaging the eight density
values.
[0096] Isotope Dilution Technique. Vascular leakage was quantified
using an isotope dilution technique based on the injection of
bovine serum albumin (BSA) labeled with two different iodine
isotopes, .sup.125I and .sup.131I. Briefly, purified monomer BSA (1
mg) was iodinated with 1 mCi of .sup.131I or .sup.125I using the
iodogen method. Polyethylene tubing (0.58 mm internal diameter) was
used to cannulate the right jugular vein and the left or right
iliac artery. The tubing was filled with heparinized saline. The
right jugular vein cannula was used for tracer injection. The iliac
artery cannula was connected to a one ml syringe attached to a
Harvard Bioscience model PHD 2000 constant-withdrawal pump preset
to withdraw at a constant rate of 0.055 ml/min. At time 0,
[.sup.125I]BSA (50 million cpm in 0.3 ml of saline) was injected
into the jugular vein and the withdrawal pump started. At the
eight-minute mark, 0.2 ml (50 million cpm) of [.sup.131I]BSA was
injected. At the ten-minute mark, the heart was excised, the
withdrawal pump was stopped, and the retina was quickly dissected
and sampled for g-spectrometry. Tissue and arterial samples were
weighed and counted in a g-spectrometer (Beckman 5500, Irvine,
Calif.). The data were corrected for background and a quantitative
index of [.sup.125I]BSA tissue clearance was calculated as
previously described and expressed as .mu.g plasma.times.g tissue
wet weight-1.times.min-1. Briefly, [.sup.125I] BSA tissue activity
was corrected for [.sup.125I] BSA contained within the tissue
vasculature by multiplying [125I]BSA activity in the tissue by the
ratio of [.sup.125I]BSA/[.sup.131I]BSA in the arterial plasma
sample obtained at the end of the experiment. The
vascular-corrected [.sup.125I]BSA activity was divided by the
time-averaged [.sup.125I]BSA plasma activity (obtained from a
well-mixed sample of plasma taken from the withdrawal syringe) and
by the tracer circulation time (10 minutes) and then normalized per
gram tissue wet weight.
[0097] Ribonuclease Protection Assay. The retinas were gently
dissected free and cut at the optic disc after enucleation, and
frozen immediately in liquid nitrogen. Total RNA was isolated from
rat retinas according to the acid guanidinium
thiocyanate-phenol-chloroform extraction method. A 425-base pair
EcoRI/BamHI fragment of rat ICAM-1 cDNA was prepared by reverse
transcription-polymerase chain reaction and cloned into pBluescript
II KS vector. A 472 nucleotide antisense riboprobe was prepared by
in vitro transcription (Promega, Madison, Wis.) of linearized
plasmid DNA with T7 RNA polymerase in the presence of
[.sup.32P]dUTP. The sequence of the cloned cDNA was verified by DNA
sequencing. Twenty micrograms of total cellular RNA were used for
ribonuclease protection assays. All samples were simultaneously
hybridized with an 18S riboprobe (Ambion, Austin, Tex.) to
normalize for variations in loading and recovery of RNA. Protected
fragments were separated on a gel of 5% acrylamide, 8M urea,
1.times. Tris-borate-EDTA, and quantified with a PhosphorImager
(Molecular Dynamics, Sunnyvale, Calif.).
[0098] ICAM-1 Blockade. Twenty four hours following streptozotocin
injection, confirmed diabetic animals received intraperitoneal
injections of 3 mg/kg or 5 mg/kg rat ICAM-1 neutralizing antibody
(1A29; R&D Systems, Minneapolis Minn.) or 5 mg/kg normal mouse
IgG1 (R&D Systems) in sterile phosphate buffered saline. The
animals were treated three times per week. Retinal leukostasis and
vascular leakage were studied one week following diabetes
induction.
[0099] Statistical Analysis. All results are expressed as means
.+-. SD. The data were compared by analysis of variance (ANOVA)
with post-hoc comparisons tested using Fisher's protected least
significant difference (PLSD) procedure. Differences were
considered statistically significant when P values were less than
0.05.
Results and Discussion
[0100] Time-Course Changes of Retinal Leukostasis and Vascular
Leakage after Diabetes Induction. Retinal leukostasis was
quantified in Long-Evans rats. Diabetic rats, like humans with
diabetes, develop retinal non-perfusion and increased vascular
permeability. FIG. 1 shows the time course of diabetic retinal
leukostasis and vascular leakage. In FIG. 1A, leukostasis was
serially quantified using AOLF. Non-diabetic animals (day 0) and
animals with streptozotocin-induced diabetes of varying duration
were studied. Using AOLF, a time course analysis showed that
retinal leukostasis increased 1.9-fold as early as three days
following diabetes induction (n=5, p<0.05) (FIG. 1A). After one
week of diabetes, retinal leukostasis was 3.2-fold higher than in
non-diabetic controls (n=5, p<0.0001). This finding remained
unchanged in degree for three additional weeks (n=5, p<0.0001)
(FIG. 1A). Reliable leukostasis quantitation beyond the four-week
time point was precluded by cataract formation.
[0101] Leukocyte adhesion to endothelial cells can trigger the
disorganization of endothelial cell adherens and tight junctions
and vascular leakage. To determine if diabetic retinal leukostasis
was correlated with blood-retinal barrier breakdown, retinal
albumin permeation was quantified (FIG. 1B). In FIG. 1B,
radioactive albumin permeation into retinal tissue was quantitated
at the same time points using the isotope dilution technique.
Retinal albumin permeation characterizes human and rodent diabetic
retinopathy and can be sensitively quantified using the isotope
dilution technique (Tilton, R. G., et al., Diabetes 42:221-232
(1993); Tilton, R. G., et al., J. Clin. Invest. 99:2192-2202
(1997); and Vinores, S. A., et al., Am. J. Pathol., 134:231-235
(1989). A time course analysis in diabetic rats revealed a 2.9-fold
(n=8, p<0.0001) and 10.7-fold (n=8, p<0.0001) increase in
albumin permeation following one and four weeks of diabetes (FIG.
1B). The breakdown of the blood-retinal barrier followed the onset
of diabetes-associated retinal leukostasis.
[0102] Leukocyte-Induced Non-perfusion and Reperfusion in Retinal
Capillaries. To further characterize the diabetic retinal
leukostasis, serial AOLF and fluorescein angiography studies were
performed. FIG. 2 shows that static leukocytes are in flux, block
capillary flow and transmigrate. Serial AOLF of static leukocytes
in the same retinal area after seven (FIG. 2A) and eight (FIG. 2C)
days of diabetes shows their complete replacement within a 24 hour
period. The arrow points to a static leukocyte (FIGS. 2A and 2B)
that appears to have transmigrated (FIG. 2B). One day later, AOLF
and fluorescein angiography show that the leukocyte has disappeared
(FIGS. 2C and 2D). The arrowhead shows a patent capillary (FIG. 2B)
that subsequently becomes obstructed by a static leukocyte 24 hours
later (FIGS. 2C and 2D). These studies revealed that the individual
leukocytes observed with AOLF are in flux, even though the overall
degree of leukostasis is constant (FIG. 2). The static retinal
leukocytes observed seven days following the induction of diabetes
are topographically distinct from those observed 24 hours later.
Furthermore, a fraction of the leukocytes are in the extravascular
space, a result consistent with their rapid transmigration
following-dye labeling.
[0103] Fluorescein angiography and AOLF were also used to study
retinal non-perfusion. These studies identified static leukocytes
directly associated with areas of downstream non-perfusion (FIGS. 2
and 3). FIG. 3 shows leukocyte-induced non-perfusion and
reperfusion. Serial studies were completed one (FIGS. 3A and 3B),
two (FIGS. 3C and 3D) and four (FIGS. 3E and 3F) weeks following
diabetes induction using both AOLF (FIGS. 3A, 3C, and 3E) and
fluorescein angiography (FIGS. 3B, 3D, and 3F). The arrow shows a
patent capillary (FIG. 3B) that subsequently becomes occluded
downstream from a static leukocyte (FIGS. 3C and 3D), and then
opens up when the leukocyte disappears (FIGS. 3E and 3F). The
arrowhead shows a patent capillary (FIG. 3B) that becomes occluded
downstream from a static leukocyte (FIGS. 3C and 3D) and then
remains closed after the leukocyte has disappeared (FIGS. 3E and
3F). The non-perfused capillaries were patent prior to the onset of
the leukostasis, indicating a causal relationship. As the
leukocyte(s) disappeared, the capillaries either reperfused or
remained closed (FIG. 3). Reperfusion has been observed in human
diabetic retinopathy, but the mechanism, until now, has remained
unexplained.
[0104] ICAM-1 Gene Expression in Diabetic Retina. To determine if
retinal ICAM-1 expression increases in association with diabetic
retinal leukostasis, ICAM-1 mRNA levels were quantified using the
ribonuclease protection assay. FIG. 4 shows ICAM-1 gene expression
in diabetic retina. FIG. 4A shows results from a ribonuclease
protection assay, which demonstrates that retinal ICAM-1 levels
were significantly increased seven days following diabetes
induction. Each lane shows the signal from the two retinas of a
single animal. The lane labeled "Probes" shows a hundred-fold
dilution of the full-length ICAM-1 and 18S riboprobes. The lanes
labeled "RNase-(0.1)" and "RNase-(0.01)" show the ten-fold and
hundred-fold dilutions, respectively, of the full-length riboprobes
without sample or RNase. When normalized to 18S RNA, the retinal
ICAM-1 levels after seven days of diabetes were 2.2-fold higher
(n=4, p<0.05) than in the non-diabetic controls (FIG. 4B).
Retinas analyzed three days following diabetes induction
demonstrated that retinal ICAM-1 mRNA levels were 1.5-fold higher
than non-diabetic controls, but this increase was not statistically
significant (n=5, p>0.05) (FIG. 4). After one week of diabetes,
the retinal ICAM-1 levels were 2.2-fold greater, a significant
increase when compared to non-diabetic controls (n=4, p<0.05).
The ICAM-1 increase coincided temporally with the development of
diabetic retinal leukostasis and blood-retinal barrier
breakdown.
[0105] An Anti-ICAM-1 Monoclonal Antibody (mAb) Prevents
Leukostasis and Vascular Leakage in Diabetic Retina. To assess
whether ICAM-1 mediates diabetic retinal leukostasis, a well
characterized ICAM-1 neutralizing antibody (1A29) was used for in
vivo adhesion blockade experiments. Tamatani, T. et al., Int.
Immunol. 2, 165-171 (1990); Kawasaki, K., et al., J. Immunol. 150,
1074-1083 (1993); Kelly, K. et al., Proc. Natl. Acad. Sci. USA 91,
812-816 (1994). Animals received either 3 or 5 mg/kg
intraperitoneal injections of the ICAM-1 antibody three times
weekly. Control diabetic animals received an equivalent amount of a
non-immune isotype control antibody. All animals were analyzed one
week following diabetes induction. The results showed that the
ICAM-1 antibody blocked diabetes-induced leukostasis by 40.9% (3
mg/kg, n=5, p<0.01) and 48.5% (5 mg/kg, n=5, p<0.001) (FIGS.
5 and 6A). The peripheral leukocyte counts at one week increased by
40% (5 mg/kg, n=5, p<0.05) compared to the control antibody
treated animals, a result consistent with successful systemic
ICAM-1 blockade (Table 1). Body weight, plasma glucose and blood
pressure were unchanged in all diabetic groups (Table 1).
TABLE-US-00001 TABLE 1 Characteristics of control, diabetic, mouse
IgG1-treated diabetic, and anti-ICAM-1 mAb- treated diabetic rats
Diabetes + Diabetes + anti-ICAM-1 5 mg/kg mAb Control Diabetes
mouse IgG1 3 mg/kg 5 mg/kg n 6 CR 7 5 5 5 Body weight (g) 271 .+-.
12 240 .+-. 12 * 235 .+-. 9 * 238 .+-. 6 * 239 .+-. 12 * Plasma
glucose (mg/dl) 123 .+-. 19 332 .+-. 35 * 316 .+-. 61 * 351 .+-. 83
* 373 .+-. 68 * Blood Pressure (mmHg) 111 .+-. 6 104 .+-. 12 109
.+-. 14 105 .+-. 9 105 .+-. 10 Leukocyte count
(.times.10.sup.3/.mu.l) 6.1 .+-. 1.6 5.0 .+-. 1.5 .diamond-solid.
5.3 .+-. 0.8 .diamond-solid. 6.9 .+-. 1.4 .diamond-solid. 7.4 .+-.
2.3 Values are means .+-. SD. * P < 0.0001 vs. control rats;
.diamond-solid. P < 0.05 vs 5 mg/kg anti-ICAM-1 mAb-treated
diabetic rats. All results are expressed as means .+-. SD. Unpaired
groups of two were compared using two sample t-test or two sample
t-test with Welch's correction. To compare three or more groups,
analysis or variance was followed by the post hoc test with
Fisher's PLSD procedure. Differences were considered statistically
significant when P values were less than 0.05.
Values are means .+-. SD. * P<0.0001 vs. control rats;
.diamond-solid. P<0.05 vs. 5 mg/kg anti-ICAM-1 mAb-treated
diabetic rats. All results are expressed as means .+-. SD. Unpaired
groups of two were compared using two sample t-test or two sample
t-test with Welch's correction. To compare three or more groups,
analysis of variance was followed by the post hoc test with
Fisher's PLSD procedure. Differences were considered statistically
significant when P values were less than 0.05.
[0106] The effect of the ICAM-1 inhibition on blood-retinal barrier
breakdown was tested using the same antibody. Animals receiving 3
and 5 mg/kg of the anti-ICAM-1 antibody had 63.5% (3 mg/kg, n=4,
p<0.0001) and 85.6% (5 mg/kg, n=4, p<0.0001) less retinal
albumin permeation at one week (FIG. 6B). The results suggest that
the ICAM-1-dependent component of the leukostasis is largely
responsible for the blood-retinal barrier breakdown.
Example 2
Integrin-Mediated Neutrophil Adhesion and Retinal Leukostasis in
Diabetes
Introduction:
[0107] Leukocyte-endothelial cell interactions in tissues are
mediated by adhesion molecules expressed on the surface of
leukocytes and endothelial cells. Immunoglobulin superfamily
molecules such as ICAM-1 are expressed on endothelial cells and
bind to .beta..sub.2-integrins expressed on leukocytes. The
integrins are transmembrane receptors that consist of noncovalently
bound heterodimers composed of .alpha.- and .beta.-chains. The
2-integrins are operative in leukocyte adhesion and include LFA-1
(lymphocyte function associated antigen, CD11a/CD18), Mac-1
(leukocyte adhesion receptor, CD11b/CD18) and p150/95 (CD11c/CD18).
Each of the .beta..sub.2-integrins has a common .alpha.-chain in
combination with a unique .alpha.-chain. CD18 is required for the
firm attachment of healthy human neutrophils to human umbilical
vein endothelial cells.
[0108] In vivo studies from our laboratory have investigated the
role of leukocytes in diabetic retinopathy. Utilizing acridine
orange leukocyte fluorography, the density of static leukocytes in
the retinas of streptozotocin-induced diabetic rats was
demonstrated to be increased. Retinal leukocyte stasis
(leukostasis) was observed within three days of diabetes induction,
and was temporally and spatially correlated with capillary
non-perfusion and blood-retinal barrier breakdown. The onset of
retinal leukostasis coincided with the upregulation of retinal
ICAM-1 expression. Causality was demonstrated when an anti-ICAM-1
antibody prevented the diabetes-associated increases in retinal
leukostasis and vascular leakage by 48.5% and 85.6%, respectively.
However the identities and bioactivities of the neutrophil adhesion
molecules mediating diabetic retinal leukostasis are less well
understood.
[0109] The aim of the current study was to investigate in greater
detail the role of neutrophils in early diabetic retinal
leukostasis. A time point of one week of diabetes was chosen in
this study because steady-state increases in diabetic retinal
leukostasis and ICAM-1 expression are achieved in one week. Since
adhesion can occur in the absence of increased adhesion molecule
expression, both adhesion molecule expression and bioactivity were
examined. Finally, the role of CD18 in the development of diabetic
retinal leukostasis was examined in vivo using acridine orange
leukocyte fluorography and neutralizing anti-CD18 F(ab').sub.2
fragments.
Methods:
[0110] Diabetes was induced in Long Evans rats with streptozotocin.
The expression of the surface integrin subunits CD11a, CD11b, and
CD18 on rat neutrophils isolated from peripheral blood was
quantitated with flow cytometry. In vitro neutrophil adhesion was
studied using quantitative endothelial cell-neutrophil adhesion
assays. The adhesive role of the integrin subunits CD11a, CD11b and
CD18 was tested using specific neutralizing monoclonal antibodies.
CD18 bioactivity was blocked in vivo with anti-CD18 F(ab').sub.2
fragments and the effect on retinal leukocyte adhesion was
quantitated with acridine orange leukocyte fluorography (AOLF).
Animals
[0111] Male Long-Evans rats weighing approximately 200 g were used
for these experiments. The rats were fed standard laboratory chow
and allowed free access to water in an air-conditioned room with a
12-hour light-12-hour dark cycle.
Induction of Diabetes
[0112] Rats received a single 60 mg/kg intraperitoneal injection of
streptozotocin (Sigma, St. Louis, Mo.) in 10 mM sodium citrate
buffer, pH 4.5, after an overnight fast. Control non-diabetic
animals received citrate buffer alone. Animals with blood glucose
levels greater than 250 mg/dl 24 hours after injection were
considered diabetic. All experiments were performed one week
following the induction of diabetes.
Monoclonal Antibodies and F(ab')2 Fragments
[0113] The monoclonal antibodies (mAb) were murine in origin and
were used as purified IgG. For the in vitro studies, mAbs WT.1
(anti-rat CD11a), 6G2 (anti-rat CD18), and MRC OX-42 (anti-rat
CD11b) were obtained from Serotec Inc. (Raleigh, N.C.).
FITC-conjugated mouse IgG.sub.1 mAb isotype control was obtained
from PharMingen (San Diego, Calif.). Fluorescein isothiocyanate
(FITC)-conjugated goat anti-mouse IgG.sub.1 Ab was obtained from
Caltag Laboratories (Burlingame, Calif.). For the in vivo studies,
WT.3 anti-rat LFA-1 beta chain (CD18) F(ab').sub.2 fragments were
obtained from Seikagaku America (Division of Associates of Cape
Cod, Inc., Falmouth, Mass.). Purified mouse anti-human IgG
F(ab').sub.2 fragments were obtained from Jackson ImmunoResearch
Laboratories Inc. (West Grove, Pa.).
Flow Cytometry
[0114] The surface expression of CD11a, CD11b, and CD18 on rat
neutrophils was determined using flow cytometry as previously
described. Allport J R, et al., J Immunol., 158:4365-4372 (1997).
Briefly, whole blood anticoagulated with EDTA was obtained from the
hearts of rats anesthetized with inhaled isofluorane. Leukocytes
were isolated by dextran sedimentation and hypotonic lysis of
contaminating erythrocytes. Aliquots of 5.times.10.sup.5 cells in
100 .mu.l RPMI 1640 medium (BioWhittaker, Walkersville, Md.)
containing 5% fetal bovine serum (RPMI-5%) were incubated on ice
for 10 min. The tubes were centrifuged at 400.times.g for 5 min at
4.degree. C. The cell pellets were resuspended in 100 .mu.l RPMI-5%
containing 20 .mu.g/ml primary mAb to CD11a, CD11b, CD18 or isotype
control and incubated for 45 min on ice. Primary mAb were detected
with FITC-conjugated goat anti-mouse IgG.sub.1 Ab as previously
detailed. The fluorescence of 10.sup.4 cells was measured on a
FACScan (Becton Dickinson, San Jose, Calif.). Neutrophils were
manually gated on the basis of their characteristic forward and
side light scattering properties. The surface expression is
presented as the mean channel fluorescence on a logarithmic
scale.
Endothelial Cell-Neutrophil Adhesion Assays
[0115] Peripheral blood was obtained from rats anesthetized with
inhaled isofluorane via heart puncture with a 16-gauge EDTA flushed
needle. Neutrophils were isolated from whole blood by density
gradient centrifugation with NIM.circle-solid.2.TM. (Neutrophil
Isolation Media; Cardinal Associates, Santa Fe, N. Mex.) according
to the manufacturer's instructions. Preparations contained >94%
neutrophils as determined by eosin and methylene blue staining
(Leukostat staining system; Fischer Scientific, Pittsburgh, Pa.).
There was no red blood cell contamination. The cells were used
immediately after collection.
[0116] The adhesion of unstimulated neutrophils to confluent
monolayers of rat prostate endothelial cells (RPEC) was determined
under static conditions as previously described. (Luscinskas F W,
et al., J Immunol., 149:2163 (1992); Kiely J M, et al., "Methods in
Molecular Biology, Adhesion Protein Protocols,"
Leukocyte-endothelial monolayer adhesion assay (static conditions),
131-136 (1999). RPEC were obtained from the American Type Culture
Collection (ATCC; Manassas, Va.) and cultured in Eagle's minimum
essential media (ATCC) supplemented with 5% fetal bovine serum
(FBS; GIBCO, Gaithersburg, Md.) and 0.3 ng/ml porcine intestinal
heparin (Sigma, St. Louis, Mo.). RPEC were grown to confluence on
tissue culture-treated plastic microtiter 96-well plates,
stimulated for 24-hours with 30 ng/ml recombinant human TNF-.alpha.
(Genzyme Corp., Cambridge, Mass.), and incubated for 15 minutes
with RPMI-5%. TNF-.alpha. stimulation of ICAM-1 surface expression
was utilized for all experiments. Neutrophils were resuspended at
2.times.10.sup.6 cells/ml in RPMI-5% and incubated for 10 min at
37.degree. C. with 1 .mu.M of the fluorescent marker,
2',7'-bis-(2-carboxyethyl)-5 (and 6) carboxyfluorescein,
acetoxymethyl ester (Molecular Probes, Eugene, Oreg.) in DMSO
(vehicle). Fluorescent labeled neutrophils were washed once and
then incubated in RPMI-5% alone or RPMI-5% with a saturating
concentration of mAb (30 .mu.g/ml) to CD11a, CD11b, or CD18 for 10
min at room temperature. The neutrophils were washed and then
incubated (2.times.10.sup.6 neutrophils/ml, 50 .mu.l per well) with
RPEC for 10 min at 37.degree. C. Non-adherent cells were removed
and the content of the wells lysed with 10 mM Tris-HCl, pH 8.4
containing 0.1% SDS. Fluorescence was determined in a microtiter
plate fluorimeter (excitation 485 nm, emission 530-540 nm) and the
adhesion reported as the number of adherent
neutrophils/mm.sup.2.
Acridine Orange Leukocyte Fluorography (AOLF)
[0117] Leukocyte dynamics in the retina were studied with AOLF.
(Miyamoto, K., et al., "In vivo demonstration of increased
leukocyte entrapment in retinal microcirculation of diabetic rats,"
Invest Opthalmol Vis Sci., 39:2190-2194 (1998); Miyamoto, K., et
al., Proc Natl Acad Sci USA., 96(19):10836-41 (1999); Nishiwaki,
H., et al., Invest Opthalmol Vis Sci., 36:123-130 (1995);
Nishiwaki, H., et al., Invest Opthalmol Vis Sci., 37:1341-1347
(1997)). Rats were anesthetized with 4 mg/kg xylazine hydrochloride
(Phoenix Pharmaceutical, St. Joseph, Mo.) and 25 mg/kg ketamine
hydrochloride (Parke-Davis, Morris Plains, N.J.). The day before
leukocyte dynamics were observed, a heparin-lock catheter was
surgically implanted in the right jugular vein of each rat. The
catheter was subcutaneously externalized to the back of the neck.
Rats received intravenous injections of 5 mg/kg anti-rat beta chain
(CD18, WT.3) F(ab').sub.2 fragments or 5 mg/kg anti-human IgG
isotype control F(ab').sub.2 fragments in sterile phosphate
buffered saline 24 hours before AOLF was performed. The experiments
were carried out in a masked fashion.
[0118] Immediately before AOLF, each rat was again anesthetized,
and the pupil of the left eye was dilated with 1% tropicamide
(Alcon, Humancao, Puerto Rico) to observe leukocyte dynamics. A
focused image of the peripapillary fundus of the left eye was
obtained with a scanning laser opthalmoscope (SLO; Rodenstock
Instruments, Munich, Germany). Acridine orange (Sigma, St. Louis,
Mo.) was dissolved in sterile saline (1.0 mg/ml) and 3 mg/kg was
injected through the jugular vein catheter at a rate of 1 ml/min.
The fundus was observed with the SLO using the argon blue laser as
the illumination source and the standard fluorescein angiography
filter in the 400 field setting for 1 min. Twenty min later, the
fundus was again observed to evaluate leukostasis in the retina.
The images were recorded on videotape at the rate of 30 frames/sec.
The video recordings were analyzed on a computer equipped with a
video digitizer (Radius, San Jose, Calif.) that digitizes the video
image in real time (30 frames/sec) to 640.times.480 pixels with an
intensity resolution of 256 steps. For evaluating retinal
leukostasis, an observation area around the optic disc measuring
five disc diameters in radius was determined by drawing a polygon
bordered by the adjacent major retinal vessels. The density of
trapped leukocytes was calculated by dividing the number of static
leukocytes (recognized as fluorescent dots) by the area of the
observation region (in pixels). The density of static leukocytes
was calculated in 8-10 peripapillary observation areas and an
average density (.times.10.sup.-5 cells/pixel.sup.2) was
obtained.
[0119] Blood pressures and heart rates were measured using a
noninvasive cuff sensor and monitoring system (Ueda Electronics,
Tokyo, Japan). Blood anticoagulated with EDTA was drawn from the
abdominal aorta of each rat after the experiment to determine the
leukocyte count using a hematology analyzer. The leukocyte count
was determined using a hematology analyzer.
Statistical Analysis
[0120] All results are expressed as means .+-. SD. The data were
compared by analysis of variance (ANOVA) with post-hoc comparisons
tested using Fisher's protected least significant difference (PLSD)
procedure. Differences were considered statistically significant
when p values were less than 0.05.
Results
[0121] Neutrophil CD11a, CD11b, and CD18 surface integrin levels
were 62% (n=5, p=0.006), 54% (n=5, p=0.045) and 38% (n=5, p=0.009)
greater in diabetic vs. non-diabetic animals, respectively.
Seventy-five percent more neutrophils from diabetic vs.
non-diabetic animals adhered to rat endothelial cell monolayers
(n=6, p=0.02). Pre-treatment of leukocytes with either anti-CD11b
or anti-CD18 antibodies lowered the proportion of adherent diabetic
neutrophils by 41% (n=6, p=0.01 for each treatment), while
anti-CD11a antibodies had no significant effect (n=6, p=0.5). In
vivo, systemic administration of anti-CD18 F(ab').sub.2 fragments
decreased diabetic retinal leukostasis by 62% (n=5, p=0.001).
Increased Surface Integrin Expression on Diabetic Neutrophils
[0122] Integrin expression was measured on the surface of
neutrophils from normal and diabetic rats. As shown in Table 2, the
flow cytometric analyses demonstrated statistically significant
increases in the diabetic leukocyte CD11a, CD11b, and CD18 levels,
as evidenced by the increases in mean channel fluorescence.
Neutrophil CD11a, CD11b, and CD18 levels were 62% (n=5, p=0.006),
54% (n=5, p=0.045), and 38% (n=5, p=0.009) greater, respectively,
on the one week-diabetic leukocytes vs. the non-diabetic
leukocytes. Integrin expression was similarly increased on two
week-diabetic neutrophils with CD11a, CD11b, and CD18 levels being
53%, 24%, and 38% greater, respectively. TABLE-US-00002 TABLE 2
Flow-cytometric analysis of integrin molecule expression on
neutrophils. Diabetes Diabetes + + anti-CD18 Control Diabetes
control F(ab;).sub.2 F(ab').sub.2 n 5 5 5 5 Body Weight 268 .+-. 10
236 .+-. 4* 233 .+-. 7* 237 .+-. 17* (g) Plasma glucose 122 .+-. 21
327 .+-. 40* 357 .+-. 60* 351 .+-. 28* (mg/dl) Blood pressure 110
.+-. 7 106 .+-. 13 103 .+-. 8 103 .+-. 7 (mmHg) Leukocyte 6.4 .+-.
1.4 4.9 .+-. 1.6.dagger. 5.0 .+-. 1.3 6.7 .+-. 0.9 count
(.times.10.sup.3 .mu.L) Values are means .+-. SD. *P < 0.001 vs.
control rats .dagger.P < 0.05 vs. anti-CD18 F(ab').sup.2-treated
diabetic rats.
[0123] Diabetic Neutrophils Exhibit Increased Adhesion to
TNF.alpha.-Activated Endothelial Cell Monolayers In Vitro
[0124] The functional adhesion of purified neutrophils to cultured
endothelial cell monolayers was investigated. Adhesion assays were
performed by adding diabetic or non-diabetic neutrophils to
TNF-a-stimulated rat endothelial cell monolayers under static
conditions. TNF-.alpha. was added to maximize endothelial cell
ICAM-1 expression. Preliminary experiments demonstrated a 2.7-fold
increase in endothelial cell ICAM-1 expression with TNF-.alpha.
(n=4, p<0.0001). FIG. 7 shows that adhesion of control and
diabetic rat neutrophils to confluent TNF-activated rat endothelial
cell monolayers under static conditions. Neutrophils isolated from
diabetic rats demonstrated significantly increased adhesion to rat
endothelial cell monolayers. FIG. 7 shows that adhesion of control
and diabetic rat neutrophils to confluent TNF-activated rat
endothelial cell monolayers under static conditions. Neutrophils
isolated from diabetic rats demonstrated significantly increased
adhesion to rat endothelial cell monolayers. As shown in FIG. 7,
75% more neutrophils from the diabetic rats adhered to the
endothelial cell monolayers than neutrophils isolated from
non-diabetic rats (n=6, p=0.02).
[0125] The .beta..sub.2-integrin molecules mediating neutrophil
adhesion in vitro were examined. FIG. 8 shows the effect of
anti-integrin antibodies on neutrophil adhesion in vitro.
Neutrophils were pre-incubated with anti-CD11a, anti-CD11b,
anti-CD18 (30 .mu.g/ml of each mAb), or an equimolar mixture of
anti-CD11a/CD11b/CD18 antibodies prior to their use in the adhesion
studies. In a representative experiment shown in FIG. 8, untreated
diabetic neutrophils exhibited increased adhesion to
TNF.alpha.-activated endothelial cell monolayers under all
treatment conditions. Pretreatment with anti-CD11b or anti-CD18
antibodies each decreased diabetic neutrophil adhesion by 41% (n=6,
p=0.01 for each treatment). In contrast, pretreatment with the
anti-CD11a antibody did not significantly affect diabetic
neutrophil adhesion (n=6, p=0.5 vs. untreated diabetic
neutrophils). Moreover, treatment with an equimolar mixture of
anti-CD11a, anti-CD11b, and anti-CD18 monoclonal antibodies
significantly reduced diabetic neutrophil adhesion by 72% (n=6,
p<0.0001 vs untreated diabetic neutrophils). Non-diabetic
neutrophil adhesion was also reduced with the anti-CD11a, anti-CD
11b and anti-CD18 antibodies, as well as with the
anti-CD11a/CD11b/CD18 antibody cocktail. The decreases were 39%,
49%, 53%, and 52%, respectively (n=6, p<0.05 for each treatment
vs. untreated non-diabetic neutrophils).
In Vivo CD18 Blockade Decreases Leukostasis in Diabetic Rat
Retinas
[0126] Retinal leukostasis in living animals was measured with
AOLF. Intravenous injection of acridine orange causes leukocytes
and endothelial cells to fluoresce through the non-covalent binding
of the molecule to double stranded DNA. When a scanning laser
opthalmoscope is utilized, retinal leukocytes within blood vessels
can be visualized in vivo. Twenty minutes after acridine orange
injection, static leukocytes in the capillary bed can be observed
as fluorescent dots. These labeled cells are leukocytes because
blocking CD18, expressed on leukocytes but not on endothelial
cells, causes them to disappear (see below).
[0127] Leukocyte dynamics in the retina were observed after CD18
F(ab').sub.2 blockade as shown in the representative photos of FIG.
9. FIG. 9 shows retinal leukostasis following CD18 blockade.
Representative photos from acridine orange leukocyte fluorography
revealed static fluorescent leukocytes in the retinas of control
and diabetic rats. The leukostasis in non-diabetic rat retina (FIG.
9A), was increased in diabetic rat retina (FIG. 9B), and unchanged
following treatment with the control F(ab').sub.2 (FIG. 9C),
however retinal leukostasis was reduced in diabetic rats treated
with anti-CD18 F(ab').sub.2 fragments (FIG. 9D). As expected,
retinal leukostasis was increased in the diabetic vs. non-diabetic
rat retinae (FIG. 9B vs. 9A). Treatment of the diabetic rats with
the isotype control F(ab').sub.2 fragments did not lead to
detectable changes in the degree of leukostasis (FIG. 9C vs. 9B).
However, treatment with the anti-CD18 F(ab').sub.2 fragments led to
a striking decrease in retinal leukostasis (FIG. 9D vs. 9C).
Measurements of leukostasis were obtained throughout the entire
retinae to avoid any potential sampling error and the means and
standard deviations from independent experiments were compared
(FIG. 10). FIG. 10 shows the quantitation of retinal leukostasis
following CD18 blockade. When CD18 bioactivity was inhibited via
systemic administration of 5 mg/kg of the anti-CD18 neutralizing
F(ab').sub.2 (clone WT.3), retinal leukostasis was inhibited in
diabetic rat retinas. This confirmed that anti-CD18 blockade
significantly decreased leukostasis in diabetic rats by 62% (n=5,
p=0.001 vs. animals receiving control F(ab').sub.2) (FIG. 10). The
body weight, plasma glucose level, blood pressure, and leukocyte
counts for the control and diabetic animals are shown in Table 3.
The diabetic animals all had significantly elevated blood glucose
levels and decreased body weight as compared with the normal rats,
as is the norm. Blood pressure was similar among groups. The
peripheral leukocyte counts in the diabetic anti-CD18
F(ab')2-treated animals were increased compared to the untreated
diabetic animals, a result consistent with successful CD18
blockade. TABLE-US-00003 TABLE 3 Characteristics of control,
diabetic, control mAb treated diabetic, and anti-CD18
F(ab').sub.2-treated diabetic rats Control Diabetes p-value n CD11a
115.0 .+-. 12.8 185.9 .+-. 18.5 0.006 5 CD11b 182.6 .+-. 39.2 281.9
.+-. 84.9 0.045 5 CD18 193.2 .+-. 34.2 267.1 .+-. 34.3 0.009 5
Values are means .+-. SD of mean channel fluorescence.
[0128] The results of the blocking adhesion studies indicate that
Mac-1 is the predominant CD18 integrin involved in diabetic
neutrophil adhesion to activated RPEC monolayers. At present, the
reason for a lack of a CD11a-dependent component in diabetic vs.
non-diabetic neutrophil adhesion is not known. The residual
non-CD18-dependent neutrophil adhesion may be due to the
VLA.sub.4-VCAM adhesion pathway because rat neutrophils
constitutively express VLA.sub.4 on their surface.
[0129] Conclusion: Neutrophils from diabetic animals exhibit higher
levels of surface integrin expression and integrin-mediated
adhesion. In vivo, CD18 blockade significantly decreases
leukostasis in the diabetic retinal microvasculature. Integrin
adhesion molecules serve as therapeutic targets for the treatment
and/or prevention of early diabetic retinopathy.
Example 3
Vascular Endothelial Growth Factor (VEGF)-Induced Retinal Vascular
Permeability is Mediated by ICAM-1
SUMMARY
[0130] Two prominent VEGF-induced retinal effects are vascular
permeability and capillary non-perfusion. The mechanisms by which
these effects occur are not completely known. Using a rat model, it
is shown that intravitreous injections of VEGF precipitate an
extensive retinal leukocyte stasis (leukostasis) that coincides
with enhanced vascular permeability and capillary non-perfusion.
The leukostasis is accompanied by the upregulation of intercellular
adhesion molecule-1 (ICAM-1) expression in the retina. The
inhibition of ICAM-1 bioactivity with a neutralizing antibody
prevents the permeability and leukostasis increases by 79% and 54%,
respectively. These data are the first to demonstrate that a
non-endothelial cell type contributes to VEGF-induced vascular
permeability. Additionally, they identify a potential mechanism for
VEGF-induced retinal capillary non-perfusion.
[0131] In experimental diabetes, the increased presence of static
leukocytes in the retinal circulation is correlated with increased
vascular permeability. The leukostasis and vascular permeability
changes coincide with the upregulation of retinal ICAM-1. When
ICAM-1 bioactivity is blocked with an antibody, retinal leukostasis
and vascular permeability are reduced by 49% and 86%,
respectively.
[0132] When the retina is bathed in pathophysiologic concentrations
of vascular endothelial growth factor (VEGF), enhanced vascular
permeability and capillary non-perfusion are among the vascular
changes induced. The mechanisms by which these changes occur are
largely unknown. The current studies examined the mechanisms
underlying VEGF-induced retinal permeability and non-perfusion.
Given the ability of VEGF to increase ICAM-1 expression in the
retinal vasculature, the role of ICAM-1 in VEGF-induced vascular
permeability and non-perfusion was examined in vivo.
Methods:
[0133] Animals. Long-Evans rats weighing approximately 200 g were
used for these experiments. They were allowed free access to food
and water in an air-conditioned room with a 12-hour light/12-hour
dark cycle until they were used for the experiments.
[0134] Intravitreous Injection Procedure. The rats were
anesthetized with xylazine hydrochloride (4 mg/kg) (Phoenix
Pharmaceutical, St. Joseph, Mo.) and ketamine hydrochloride (25
mg/kg) (Parke-Davis, Morris Plains, N.J.). Intravitreous injections
were performed by inserting a 30-gauge needle into the vitreous at
a site 1 mm posterior to the limbus of the eye. Insertion and
infusion were performed and directly viewed through an operating
microscope. Care was taken not to injure the lens or the retina.
The head of the needle was positioned over the optic disc, and a 5
.mu.l volume was slowly injected into the vitreous. Any eyes that
exhibited damage to the lens or retina were discarded and not used
for the analyses.
[0135] Acridine Orange Leukocyte Fluorography (AOLF) and
Fluorescein Angiography. Leukocyte dynamics were evaluated using
acridine orange leukocyte fluorography (AOLF). Nishiwaki H, et al.,
Invest Opthalmol Vis Sci, 37:1341-1347 (1996); Miyamoto K, et al.,
Invest Opthalmol Vis Sci 39:2190-2194 (1998). Intravenous injection
of acridine orange causes leukocytes and endothelial cells to
fluoresce through the non-covalent binding of the molecule to
double stranded nucleic acid. When a scanning laser opthalmoscope
is utilized, retinal leukocytes and blood vessels can be visualized
in vivo. Twenty minutes following acridine orange injection, static
leukocytes in the capillary bed are observed.
[0136] Twenty-four hours before leukocyte dynamics were observed, a
heparin-lock catheter was surgically implanted in the right jugular
vein for the administration of acridine orange and sodium
fluorescein dye. The catheter was subcutaneously externalized to
the back of the neck. The rats were anesthetized for this procedure
with xylazine hydrochloride (4 mg/kg) and ketamine hydrochloride
(25 mg/kg).
[0137] Immediately before AOLF, each rat was again anesthetized,
and the pupil of the left eye was dilated with 1% tropicamide
(Alcon, Humancao, Puerto Rico) to observe leukocyte dynamics. A
focused image of the peripapillary fundus of the left eye was
obtained with a scanning laser opthalmoscope (SLO; Rodenstock
Instrument, Munich, Germany). Acridine orange (Sigma, St. Louis,
Mo.) was dissolved in sterile saline (1.0 mg/ml) and 3 mg/kg was
injected through the jugular vein catheter at a rate of 1 ml/min.
The fundus was observed with the SLO using the argon blue laser as
the illumination source and the standard fluorescein angiography
filter in the 40.degree. field setting for 1 minute. Twenty minutes
later, the fundus was again observed to evaluate retinal
leukostasis. The images were recorded on videotape at the rate of
30 frames/sec. The recordings were analyzed on a computer equipped
with a video digitizer (Radius, San Jose, Calif.) that digitizes
video images in real time (30 frames/sec) at 640.times.480 pixels
with an intensity resolution of 256 steps. For evaluating retinal
leukostasis, an observation area around the optic disc measuring
five disc diameters in radius was outlined by drawing a polygon
bordered by the adjacent major retinal vessels. The area was
measured in pixels and the density of trapped leukocytes was
calculated by dividing the number of static leukocytes, which were
recognized as fluorescent dots, by the area of the observation
region. The density of leukocytes was calculated in eight
peripapillary observation areas and an average density was obtained
by averaging the eight density values.
[0138] Immediately after observing and recording the static
leukocytes, fluorescein angiography was performed to study the
relationship between static leukocytes and the retinal vasculature.
Twenty .mu.l of 1% sodium fluorescein was injected into the jugular
vein catheter and the images were captured using the SLO as
described above.
[0139] Quantitation of Retinal ICAM-1 mRNA Levels. Retinas were
gently dissected free and cut at the optic disc immediately after
enucleation and frozen in liquid nitrogen. Total RNA was isolated
from rat retinas according to the acid guanidinium
thiocyanate-phenol-chloroform extraction method. A 425-base pair
EcoRI/BamHI fragment of rat ICAM-1 cDNA was prepared by reverse
transcription-polymerase chain reaction. The PCR product was cloned
into pBluescript II KS vector. After linearization by digestion
with EcoNI, transcription was performed with T7 RNA polymerase in
the presence of [.sup.32P]dUTP generating a 225-base pair
riboprobe. An automated DNA sequencer verified the sequence of the
cloned cDNA. Ten micrograms of total cellular RNA was used for the
ribonuclease protection assay. All samples were simultaneously
hybridized with an 18S riboprobe (Ambion, Austin, Tex.) to
normalize for variations in loading and recovery of RNA. Protected
fragments were separated on a gel of 5% acrylamide, 8M urea,
1.times. Tris-borate-EDTA, and quantified with a PhosphorImager
(Molecular Dynamics, Sunnyvale, Calif.).
[0140] Quantitation of Retinal Vascular Permeability. Vascular
leakage was quantified using the isotope dilution technique. Tilton
R G, et. al., J Clin Invest 99:2192-2202 (1997). Briefly, purified
monomer bovine serum albumin (BSA; Sigma, St. Louis, Mo.) (1 mg)
was iodinated with 1 mCi of .sup.131I or .sup.125I using the
iodogen method. Polyethylene tubing (0.58 mm internal diameter) was
used to cannulate the right jugular vein and the left or right
iliac artery. The tubing was filled with heparinized saline (400 U
heparin/ml). The right jugular vein cannula was used for tracer
injection. The iliac artery cannula was connected to a one ml
syringe attached to a Harvard Bioscience model PHD 2000 constant
withdrawal pump preset to withdraw at a constant rate of 0.055
ml/min. At time 0, [.sup.125I] albumin (50 million cpm in 0.3 ml
saline) was injected into the jugular vein and the withdrawal pump
started. At the eight minute mark, 0.2 ml (50 million cpm in 0.3 ml
saline) of [.sup.131I]BSA was injected into the jugular vein. At
the ten-minute mark, the heart was excised, the withdrawal pump was
stopped, and the retina was quickly dissected and sampled for
.gamma.-spectrometry. Tissue and arterial samples were weighed and
counted in a .gamma.-spectrometer (Beckman 5500, Irvine, Calif.).
The data were corrected for background and a quantitative index of
[.sup.125I] tissue clearance was calculated as previously described
and expressed as .mu.g plasma.times.g tissue wet
weight.sup.-1.times.min.sup.-1. Briefly, [.sup.125I] BSA tissue
activity was corrected for [.sup.125I] BSA contained within the
tissue vasculature by multiplying [.sup.125I]BSA activity in the
tissue by the ratio of [.sup.125I]BSA/[.sup.131I]BSA in an arterial
plasma sample. The vascular-corrected [.sup.125I]BSA activity was
divided by the time-averaged [.sup.125I]BSA plasma activity
(obtained from a well-mixed sample of plasma taken from the
withdrawal syringe) and by the tracer circulation time (10 min) and
then normalized per gram tissue wet weight.
[0141] Anti-ICAM-1 Antibody Inhibition of Retinal Vascular
Permeability and Leukostasis. To study the in vivo effect of ICAM-1
blockade on VEGF-induced retinal vascular permeability and
leukostasis, a well characterized rat ICAM-1 neutralizing
monoclonal antibody (mAb) was used utilized (1A29; R&D Systems,
Minneapolis, Minn.). Tamatani T, et al., Int Immunol, 165-171
(1990); Kawasaki K, et al., J Immunol, 150:1074-1083 (1993); Kelly
K J, et al., Proc Natl Acad Sci USA, 91:812-816 (1994). The animals
were randomly divided into five groups. The first group received no
treatment. The second group received 5 .mu.l of phosphate-buffered
saline (PBS) injected into the vitreous of the left eye. The third
group received 50 ng VEGF.sub.165 in 511 PBS injected into the
vitreous of the left eye (12.5 nM final concentration). The fourth
group received 50 ng VEGF in PBS injected into the vitreous of the
left eye plus 5 mg/kg isotype-matched normal mouse IgG1 (R&D
Systems) given intravenously. The fifth group received 50 ng VEGF
in PBS injected into the vitreous of the left eye plus 5 mg/kg of
the anti-ICAM-1 mAb given intravenously. Twenty-four hours later,
retinal leukocyte dynamics and vascular permeability were
quantified.
[0142] Statistical Analysis. All results are expressed as the mean
.+-. SD. Unpaired groups of two were compared using the two sample
t-test or the two sample t-test with Welch's correction. To compare
three or more groups, analysis of variance (ANOVA) followed by the
post hoc test with Fisher's protected least significant difference
(PLSD) procedure was used. Differences were considered
statistically significant when P values were less than 0.05.
Results:
[0143] VEGF-induced Retinal Leukostasis. FIG. 11A shows AOLF
appearance of a normal retinal prior to injection of 50 ng VEGF.
FIG. 11B shows AOLF appearance of the same retinal area 48 h
following intravitreous VEGF injection. Numerous static leukocytes
are visible, as well as vessel dilation and tortuosity. A single 50
ng intravitreous injection of VEGF.sub.165 (R& D Systems,
Minneapolis, Minn.) in 5 .mu.l PBS was able to induce marked
retinal leukostasis 48 h later (FIG. 11). Vessel dilation and
tortuosity were also evident. A dose-response study demonstrated
that a 2.6-fold increase in leukostasis could be induced with as
little as 10 ng VEGF (2.5 nM) (FIG. 12, n=5, p<0.05). A plateau
was reached with 50-100 ng VEGF (.about.4-5-fold, n=5, p=<0.001
to 0.0001). Based on these data, the 50 ng dose was chosen for the
time course experiments. Intravitreous injections of 50 ng VEGF
were followed by AOLF 6, 24, 48, 72, and 120 h later. Twenty-four
hours following intravitreous injection, VEGF increased retinal
leukostasis 4.8-fold (FIG. 13, n=5, p<0.01 vs. vehicle control).
The VEGF-induced leukostasis increases peaked 48 h post-injection
and persisted for at least 120 h (n=5, p<0.01).
[0144] To confirm that this effect was due to VEGF alone, four rats
received a mixture of VEGF with a 50:1 molar excess of a previously
characterized VEGF neutralizing monoclonal antibody (A4.6.1,
Genentech, South San Francisco, Calif.) (FIG. 14). Co-injection of
the anti-VEGF antibody completely abrogated the VEGF-induced
leukostasis 48 h later (n=4, p<0.001).
[0145] VEGF-induced Retinal Capillary Perfusion. FIG. 15 shows
leukocyte-induced capillary non-perfusion. FIG. 15A shows the
retina 48 hours after fifty ng VEGF was delivered via intravitreous
injection as measured with AOLF. AOLF was immediately followed by
fluorescein angiography and FIG. 15B shows areas of capillary
non-perfusion downstream from static leukocytes. Fluorescein
angiography performed 20 minutes following AOLF revealed relatively
large areas of downstream capillary non-perfusion associated with
some of the static leukocytes (FIG. 15). The majority of the
leukocytes observed appeared to be in the intravascular space.
Normal and vehicle injected eyes did not exhibit non-perfusion.
[0146] VEGF-induced Retinal ICAM-1 Gene Expression. Twenty hours
following intravitreous injection of 50 ng VEGF or PBS vehicle
alone, total RNA was isolated from each rat retina and ICAM-1 gene
expression was quantitated using the ribonuclease protection assay
(FIG. 16A). When normalized to 18S, retinal ICAM-1 levels in the
VEGF-injected eyes were 2.8-fold greater than in the eyes injected
with vehicle alone (FIG. 16B, n=5, p<0.02).
[0147] ICAM-1 Blockade of VEGF-induced Vascular Permeability and
Leukostasis. Animals receiving intravitreous VEGF had a 3.2-fold
increase in vascular permeability 24 h following injection (FIG.
17A, n=4, p<0.0001 vs. vehicle control). Similarly, there was a
4.3-fold increase in retinal leukostasis (FIG. 17B, n=5,
p<0.0001 vs. vehicle control). Intravenous treatment with the
non-immune control antibody did not significantly alter the degree
of VEGF-induced permeability (FIG. 17A, n=3, p>0.05) or
leukostasis (FIG. 17B, n=4, p>0.05). However, the animals
receiving intravenous anti-ICAM mAb had a 79% reduction in
VEGF-induced retinal vascular permeability (FIG. 17A, n=4,
p<0.0001 vs. untreated) and a 54% reduction in VEGF-induced
retinal leukostasis (FIG. 17B, n=4, p<0.01 vs. untreated).
Example 4
CD18 and ICAM-1 Dependent Corneal Neovascularization and
Inflammation Following Limbal Injury
Materials and Methods.
Corneal Neovascularization Model
[0148] Male CD 18-deficient and ICAM-1-deficient mice were used
(Jackson Labs, Bar Harbor, Me.) and strain-specific normal male
C57BL/6 mice served as controls. The mice were anesthetized with 50
mg/kg intraperitoneal pentobarbital sodium and a drop of
proparicaine was instilled into the left eye. A number 15
Bard-Parker blade (vendor and city) was used to debride the corneal
epithelium. Two microliters of 0.15 M NaOH was then applied
topically and the limbal epithelium was removed with a Tooke
Corneal Knife, 2.5.times.15 mm Dissecting Blade (Arista, N.Y.) A
rotary motion parallel to limbus was utilized. Erythromycin
ophthalmic ointment was instilled postoperatively.
Measurement of Corneal Neovascularization
[0149] For measurement of neovascularization, mice were injected
approximately 8 .mu.g of the endothelial cell-specific marker BS-1
lectin conjugated to FITC (Vector Laboratory) per 1 g of body
weight on day 7 after scraping or day 2 after implantation of VEGF.
In 30 minutes after the injection of the dye, the eyes were
harvested and fixed with 10% neutral buffered formalin, and then
the cornea was flat-mounted on slide glasses. Fluorescence in the
flat-mounted cornea was captured using CCD camera attached to a
Leica Fluorescence microscope and saved to Macintosh 6500 (Apple
computer) as a .tif image file. The images were taken with the same
settings including exposure time on both study group and control
one. The digital images were processed using OpenLab Software and
integrated optical density in the images was measured.
Peripheral Leukocyte Counts
[0150] Peripheral blood samples were collected from tail vessels
into Eppendorf tube with EDTA when cornea that was served for
confirming infiltration of PMN was enucleated. For total leukocyte
count blood was incubated with Turk solution and then counted
manually using Hemocytemeter. The preparation of a thin, air-dried
edge smear was made to perform the microscopic manual differential
and stained with Giemsa solution. PMN count was then calculated
from the differential.
Corneal Leukocyte Counts
[0151] To determine the counts of PMN infiltration in cornea the
eyes were enucleated on day 2 after scraping or implantation of
VEGF, and stored in 10% neutral buffered formalin. The tissue was
embedded in paraffin, and 5-.mu.m-thick sections were cut and then
transferred to slide glasses. The tissue sections were stained with
Giemsa stain. The slides were then observed microscopically, and
the number of PMNs was counted in 5 fields (2 of periphery, 2 of
midperiphery and 1 of center) in the cornea from inflammatory
models and in 1 field between VEGF pellet and corneal limbus in the
cornea from VEGF-induced Neovascularization models.
Statistical Analysis
[0152] Student t-test and ANOVA were used for the comparison.
Probability less than 0.05 was considered significant.
Results:
Corneal Neovascularization in CD18 KO, ICAM-1 KO and Normal
Mice
[0153] To determine if CD18 and ICAM-1 were important in the
development of the corneal neovascularization associated with
limbal injury, limbal injury was followed by quantitation of
corneal neovascularization 7 d later. Compared to the
strain-specific controls, the CD18 null mice 39% fewer vessels
(n=5, p=0.0054). Similarly, the ICAM-1 null mice has 33% less
neovascularization that the control mice (n=5, p=0.013).
Corneal PMN Density in CD18 KO, ICAM-1 KO and Normal Mice
[0154] To determine if the inhibition of corneal neovascularization
was associated with the decreased transmigration of PMN into the
cornea, corneal PMN counts were performed 2 d following limbal
injury. This time point was chosen because it manifested maximum
corneal opacity and corneal leukocyte infiltration. Compared to the
strain-specific controls, the CD null mice had 66% fewer PMN (n=5,
p=0.0016). The ICAM-1 null mice had 65% fewer PMN (n=5, p=0.0019)
compared to the strain-specific controls.
Peripheral Blood PMN Counts in CD18 KO, ICAM-1 KO and Normal
Mice
[0155] To determine if peripheral PMN cell counts were altered in
the animals, standard PMN counts were calculated from the
differential. The average count in the C57BL6/J controls was
6263.+-.2313.18 vs. counts of 9315.+-.1486 and 10,794.+-.2199 in
the CD18 and ICAM knockout mice, respectively.
Discussion:
[0156] The data indicate that CD18 and ICAM-1 amplify the corneal
neovascularization that occurs following limbal injury. The process
is associated with higher corneal leukocyte counts, and the latter
are likely causal, in part, for the increased neovascularization.
The data also indicate that the CD18 and ICAM-1 KO mice have a
higher proportion of circulating leukocytes, a result consistent
with absence of CD18 and ICAM-1 systemically. It also confirms that
the corneal leukocytes likely transmigrated, and are not the result
of systemic leukocytopenia. Taken together, these data identify
CD18 and ICAM-1 mediators of the inflammatory corneal
neovascularization in a clinically relevant model of limbal
injury.
[0157] Experiments described herein show that limbal injury
upregulates VEGF. When VEGF is inhibited, corneal
neovascularization is reduced. VEGF is known to act directly on the
endothelial cells and the vasculature, resulting in
neovascularization. However, leukocytes augment this process. The
mechanism involves VEGF. Leukocytes, via their own VEGF, serve to
amplify the direct effects of non-leukocyte VEGF on the
vasculature. VEGF has been demonstrated in neutrophils, monocytes,
eosinophils, lymphocytes and platelets. It has also been identified
in the neutrophils and monocytes that infiltrate the cornea
following limbal injury. The fact that some leukocytes possess high
affinity VEGF receptors and migrate in response to VEGF is
consistent with this scenario. Endogenous VEGF triggers leukocyte
adhesion, transmigration and further VEGF release, producing a
positive feedback loop.
EQUIVALENTS
[0158] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims.
[0159] The relevant teachings of all the references, patents and/or
patent applications cited herein are incorporated herein by
reference in their entirety.
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