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¶
From the Massachusetts Eye and Ear Infirmary,*
Boston,Massachusetts; the Surgical ResearchLaboratory,
Children’s Hospital, Boston,Massachusetts; the Dana-Farber CancerInstitute,
Boston, Massachusetts; RegeneronPharmaceuticals,
Tarrytown, New York; and theDepartment of Vitreoretinal Surgery,¶
Center forOphthalmology and Zentrum für Molekulare Medizin, University ofCologne, Köln, Germany
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-nitro-L-arginine methyl ester
also potently reduced retinal leukocyte adhesion. Although neutrophil
CD11a, CD11b, and CD18 levels were increased in 1-week
diabetic animals, VEGF TrapA40 did not alter the
expression of these integrin adhesion molecules. Taken
together, these data demonstrate that VEGF induces retinal
ICAM-1 and eNOS expression and initiates early diabetic retinal
leukocyte adhesion in vivo. The inhibition of VEGF
bioactivity may prove useful in the treatment of the early diabetic
retinopathy.
The injection of vascular endothelial growth factor (VEGF) into normal nondiabetic eyes recapitulates many of the retinal vascular changes triggered by diabetes, including ICAM-1 up-regulation, leukocyte adhesion, vascular permeability, and capillary nonperfusion.6-9 Although VEGF is expressed in the early diabetic retina,10,11 it is not known if it triggers the retinal ICAM-1 up-regulation and leukocyte adhesion seen early in the disease. Nor is it known if VEGF alters the expression of surface integrins required for neutrophil adhesion to the diabetic retinal vasculature.
In brain endothelium, VEGF-induced ICAM-1 up-regulation is mediated by nitric oxide (NO).12 NO, a molecule with both cytotoxic and signaling capabilities, is generated by various NO synthases (NOS). Although the endothelial isoform, eNOS, is up-regulated in diabetic neural ganglia,13 its expression and regulation by VEGF in another neural tissue, the diabetic retina, remains unknown.
In the current studies, the role of endogenous VEGF in the induction of retinal ICAM-1 and leukocyte adhesion was studied in vivo. The regulation of retinal eNOS and NO by VEGF was also examined, as was the effect of VEGF on the expression of the neutrophil integrins CD11a, CD11b, and CD18. Overall, these experiments investigated the direct causal role of VEGF and NO in the initiation of the earliest stages of diabetic retinopathy.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Male Long-Evans rats weighting 
200 g were used in these
experiments. All protocols abided by the Association for Research in
Vision and Ophthalmology (ARVO) statement on the Use of Animals in
Ophthalmology and Vision Research and were approved by the Animal Care
and Use Committee of the Children’s Hospital. The animals were fed
standard laboratory chow and allowed free access to water in an
air-conditioned room with a 12-hour light-dark cycle. Except as noted
below, the animals were anesthetized with ketamine (80 mg/kg; Ketalar,
Parke-Davis, Morris Plains, NJ) and xylazine (4 mg/kg; Rompun,
Harver-Lockhart, Morris Plains, NJ) before all experimental
manipulations.
Induction of Diabetes
After 12 hours of fasting, the animals received a single 60-mg/kg intraperitoneal injection of streptozotocin (Sigma, St. Louis, MO) in 10 mmol/L of sodium citrate buffer, pH 4.5. Control nondiabetic animals were fasted and received citrate buffer alone. Twenty-four hours later, animals with blood glucose levels >250 mg/dl were considered diabetic. All experiments were performed 1 week after the induction of diabetes. The diabetic state was confirmed a second time before analysis.
ICAM-1 RNase Protection Assay
Retinae were gently dissected free and cut at the optic disk immediately after enucleation, and frozen in liquid nitrogen. Total RNA was isolated according to the acid guanidinium thiocyanate-phenol-chloroform extraction method. A 425-bp EcoRI/BamHI fragment of rat ICAM-1 cDNA was prepared by the reverse transcriptase-polymerase chain reaction. The polymerase chain reaction product was cloned into pBluescript II KS vector. After linearization by digestion with EcoRI, transcription was performed with T7 RNA polymerase in the presence of [32P]dUTP generating a 225-bp riboprobe. Sequencing verified the identity of the cloned cDNA. Ten µg of total cellular RNA was used for the ribonuclease protection assay. All samples were simultaneously hybridized with an 18S riboprobe (Ambion, Austin, TX) to normalize for variations in loading and recovery of RNA. Protected fragments were separated on a gel of 5% acrylamide, 8 mol/L urea, 1x Tris-borate-ethylenediaminetetraacetic acid, and quantified on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Quantitation was completed within the linear range of signal.
VEGF TrapA40 and Interleukin (IL)-6R Trap Reagents
VEGF TrapA40 and IL-6R Trap were synthesized at Regeneron Pharmaceuticals Inc. (Tarrytown, NY). VEGF TrapA40 consisted of immunoglobulin repeats 1 to 3 of the extracellular domain of human Flt-1 fused to the Fc portion of human IgG1. The protein was expressed in Chinese hamster ovary cells and purified via protein A affinity chromatography and size exclusion chromatography. The recombinant Flt-Fc chimera was then chemically modified to improve the pharmacokinetic profile of the parent molecule, without affecting its ability to bind VEGF with high affinity (Rudge, SJ Wiegand, GD Yancopoulos, unpublished data).
In detail, Chinese hamster ovary-derived parental Flt(1-3Ig)Fc was
incubated with sulfo-NHS-acetate (Pierce, Rockford, IL) in
phosphate-buffered saline (PBS)/5% glycerol, pH 7.2, such that the
acetylation reagent was present in 40-fold molar excess. The
acetylation reaction specifically modifies the 
amino group of
lysines present in the parental molecule. The mixture was placed on a
rocker and incubated overnight at room temperature. The
acetate-modified flt(1-3Ig)Fc termed FltFc A40, was then extensively
dialyzed against PBS/5% glycerol (25-kd molecular weight cut-off
(MWCO)) tubing. After dialysis, the concentration was checked
spectrophotometrically (absorbance at 280 nm) and modification was
assessed by isoelectric focusing analysis. With this
modification the pI shifts from 9.5 for parental Flt(1-3Ig)Fc to 5.8 to
6.5 for FltFc A40. The purity of the modified recombinant protein was
determined to be >95% by Coomassie-stained sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. The protein was filter
sterilized and stored in PBS, pH 7.2, containing 5% glycerol at
-20°C. VEGF TrapA40 bioactivity was confirmed
in endothelial cell proliferation assays before its use (data not
shown). IL-6R Trap was made from the extracellular domain of human
IL-6R
(the low-affinity IL-6 receptor) fused to the Fc domain of
human IgG1. IL-6R Trap binds only human IL-6 with
low affinity, and not mouse or rat IL-6. IL-6R Trap was Chinese hamster
ovary cell derived, purified via protein A and size exclusion
chromatography, and was >95% pure on Coomassie-stained gels. VEGF
TrapA40 and IL-6R Trap were dissolved in sterile
Tris-BisTris-Cl-sodium acetate (TBA) buffer (15 mg/ml). On day 7 of
diabetes, diabetic rats were randomized to receive a single 25 mg/kg
intraperitoneal injection of either VEGF TrapA40
or IL-6R Trap.
Retinal Leukostasis Quantitation
Deep anesthesia was induced with 50 mg/kg of sodium pentobarbital. The chest cavity was carefully opened and the left ventricle was entered with a 14-gauge perfusion cannula fixed to a vessel clamp, carefully avoiding ventricular obstruction. The right atrium was opened with a 12-gauge needle to achieve outflow. With the heart providing the motive force, 250 ml/kg of PBS was perfused to clear erythrocytes and nonsticking leukocytes. Fixation was then achieved via perfusion with 1% paraformaldehyde and 0.5% glutaraldehyde at a pressure of 100 mmHg. At this point the heart stopped. A systemic blood pressure of 120 mmHg was maintained by perfusing a total volume of 200 ml/kg for 3 minutes. The inhibition of nonspecific binding with 1% albumin in PBS (total volume 100 ml/kg) was followed by perfusion with fluorescein isothiocyanate-coupled Concanavalin A lectin (20 µg/ml in PBS, pH 7.4, total concentration 5 mg/kg body weight) (Vector Laboratories, Burlingame, CA). The latter stained adherent leukocytes and the vascular endothelium. Lectin staining was followed by 1% bovine serum albumin/PBS perfusion for 1 minute, and PBS perfusion alone for 4 minutes, to remove excess Concanavalin A (Vector Laboratories).2
The retinae were flat-mounted in a water-based fluorescence anti-fading medium (Southern Biotechnology, Birmingham, Alabama) and imaged via fluorescence microscopy (Zeiss Axiovert, fluorescein isothiocyanate filter; Zeiss, Lakewood, NJ). Only whole retinae in which the peripheral collecting vessels of the ora serrata were visible were used for analysis. Leukocyte location was scored as being either arteriolar, venular, or capillary. The large vessels emanating from the optic nerve and their first grade branches were defined as being either arterioles or venules. Arterioles were differentiated from venules by virtue of their smaller diameter. Vessels between these major vessels have a diameter approximating the size of an adherent leukocyte. These vessels were considered as capillaries. The total number of adherent leukocytes per retina was counted.
Quantification of Retinal NO
NO was converted to nitrite and the total nitrite concentration of each retina was estimated using the Total Nitric Oxide assay (R&D Systems, Minneapolis, MN). Briefly, each retina was placed in 100 µl of 40 mmol/L Tris buffer (pH 7.8) supplemented with 3 mmol/L dithiothreitol, 1 mmol/L L-arginine, 1 mmol/L NADH, and 4 µmol/L each of FAD, FMN, and H4 biopterin (Sigma), and homogenized with mechanical homogenization. The samples were subsequently cleared by centrifugation and retinal protein levels estimated using a commercial assay (BCA kit; Bio-Rad, Hercules, CA). Equal amounts of protein per sample were ultrafiltered through a 10,000 molecular weight cutoff filter to eliminate proteins. The samples were subsequently incubated with nitric reductase to convert NO to nitrite, according to the manufacturer’s instructions, and incubated with the Griess reagent (1% sulfonamide, 0.1% naphthylethylene diamine dishydrochloride, 2.5% H3PO4) (Sigma) at room temperature for 10 minutes. Nitrite was determined at 550 nm using a microplate reader and the concentration was calculated using sodium nitrite standards.
Enzyme-Linked Immunosorbent Assay for eNOS
Each retina was placed in 100 µl of solution (4°C) consisting of 20 mmol/L imidazole hydrochloride, 100 mmol/L KCl, 1 mmol/L MgCl, 1 mmol/L EGTA, 1% Triton, 10 mmol/L NaF, 1 mmol/L sodium molybdinate, and 1 mmol/L ethylenediaminetetraacetic acid supplemented with protease inhibitors (Complete Mini; Roche, Basel, Switzerland). Samples were centrifuged for 10 minutes at 13,000 rpm. Two µl of the supernatant was used for protein determination via the mini BCA assay (Pierce Scientific, CA). A commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems) was used to quantitate eNOS levels according to the manufacturer’s instructions. The reaction was stopped and the absorption measured in an ELISA reader at 450 nm. All measurements were performed in duplicate. The tissue sample concentration was calculated from a standard curve and corrected for protein concentration.
Enzyme-Linked Immunosorbent Assay for ICAM-1
Each retina was homogenized in 100 µl of solution consisting of 20 mmol/L imidazole hydrochloride, 100 mmol/L KCl, 1 mmol/L MgCl, 1 mmol/L EGTA, 1% Triton, 10 mmol/L NaF, 1 mmol/L sodium molybdinate, and 1 mmol/L ethylenediaminetetraacetic acid. The solution was supplemented with a cocktail of protease inhibitors (Complete, Roche) before use. Samples were cleared via centrifugation for 10 minutes at 13,000 rpm and assessed for protein concentration with the BCA assay (Mini BCA kit, Pierce Scientific). Flat-bottom 96-well microtiter plates (Immuno-Plate I 96-F; Nunc, Naperville, IL) were coated with 50 µl/well (1 ng/ml) of the specific rabbit anti-ICAM-1 antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) in coating buffer (0.6 mol/L NaCl, 0.26 mol/L H3PO4, and 0.08 N NaOH, pH 9.6) for 16 to 24 hours at 4°C. Nonspecific sites were then blocked with 2% bovine serum albumin in PBS for 60 minutes at 37°C, followed by sample addition of a 50-µl aliquot in duplicate, and incubated for 60 minutes at 37°C. After washes, 50 µl of a biotinylated rabbit polyclonal antibody (3.5 µg/ml in PBS, pH 7.5, 0.05% Tween-20, and 2% fetal calf serum) was added and incubated for 45 minutes at 37°C. The plates were washed, streptavidin-peroxidase conjugate (1/1000, R&D Systems) was added, and the plates were incubated for 30 minutes at 37°C. The plates were again washed, the substrate TMB (3,3',5,5'-tetramethylbenzidine; Kirkegaard & Perry, Gaithersburg, MD) was added for color development, and the reaction was quenched with 100 µl of 1 mol/L H2PO4. The plates were then read at 450 nm with an automated microplate reader.
Treatment with N--Nitro-L-Arginine Methyl Ester
(L-NAME)
The NO synthase inhibitor L-NAME (Sigma) was dissolved in saline and filter- sterilized. A freshly prepared solution (30 mg/ml) was administered via intraperitoneal injection at a dose of 30 mg/kg every other day to diabetic and nondiabetic animals. Control animals received injections of solvent alone. Eight days after the induction of diabetes, each animal had received four treatment doses. Control and L-NAME-treated animals were analyzed for retinal leukocyte adhesion on day 8 as described above.
Flow Cytometry
The monoclonal antibodies (mAbs) used in the flow cytometry
experiments were purified IgG of murine origin. The fluorescein
isothiocyanate-conjugated mAbs LFA-1 chain (anti-rat CD11a), WT.5
(anti-rat CD11b), WT.3 (anti-rat CD18), and phycoerythrin-conjugated
mAb OX-1 (anti-rat CD45) were all obtained from Pharmingen (San Diego,
CA).
The surface expression of CD11a, CD11b, and CD18 on rat neutrophils from nondiabetic, diabetic, and diabetic VEGF TrapA40-treated animals was determined via flow cytometry as previously described.3 Briefly, whole blood anti-coagulated with ethylenediaminetetraacetic acid (Life Technologies, Inc., Grand Island, NY) was obtained from the hearts of deeply anesthetized rats. Neutrophils were isolated from whole blood by density gradient centrifugation with NIM2 (Neutrophil Isolation Media; Cardinal Associates, Santa Fe, NM) according to the manufacturer’s instructions. Red blood cells were lysed via hypotonic lysis. The preparations contained >91% neutrophils as determined by eosin and methylene blue staining (Leukostat Staining System; Fisher Scientific, Pittsburgh, PA).
The cells were resuspended in 5% Dulbecco’s modified Eagle’s medium containing 20 µg/ml of fluorescein isothiocyanate-labeled mAb to CD11a, CD11b, and CD18 (Pharmingen), incubated for 15 minutes at 25°C, and counterstained with phycoerythrin-coupled mAb to CD45. The cells were then washed with PBS and incubated with 1 mg/ml propidium iodide (Molecular Probes, Eugene, OR) to identify dead cells. After centrifugation for 5 minutes at 500 x g, the cells were resuspended in 300 ml of PBS and surface fluorescence was analyzed with a FACScan (Becton Dickinson, San Jose, CA). Vital neutrophils were gated manually on the basis of their characteristic foreword and side light-scattering properties. The surface expression is presented as the percentage of positive neutrophils.
Statistics
All results are expressed as the mean ± SD. The data were analyzed by Whitney-Mann U-test with post hoc comparisons tested using Fisher’s protected least significant difference procedure. Differences were considered statistically significant when P values were <0.05. For the comparison of CD11a, CD11b, and CD18 expression with flow cytometry, a nonparametric one-way analysis of variance (Friedmann’s test) was used.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Twenty-four hours after treatment with 25 mg/kg of VEGF
TrapA40, ICAM-1 mRNA levels were quantified via
ribonuclease protection assay. When normalized to 18S, retinal ICAM-1
mRNA levels in diabetic animals treated with VEGF
TrapA40 were 3.83 ± 2.52 (optical density
units) (n = 11) versus 23.3 ±
8.84 (n = 13) in the untreated diabetic animals
(Figure 1
, P < 0.0001).
Compared to nondiabetic control animals, diabetic animals showed a
threefold increase in ICAM-1 protein levels (0.35 ± 0.035
versus 1.007 ± 0.09 pg/mg, P <
0.0001, n = 6). Treatment with VEGF
TrapA40 reduced the ICAM-1 protein to those of
the nondiabetic animals (1.007 ± 0.09 to 0.42 ± 0.03 pg/mg,
P < 0.0005, n = 8). There were no
differences between the untreated diabetic controls and those that
received IL-6 (from 1.007 ± 0.09 to 1.01 ± 0.17,
n = 6). The ICAM-1 levels of isolated leukocytes from
nondiabetic and diabetic animals were assessed by immunoassay and were
found to lack detectable ICAM-1 expression (data not shown).
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Leukocyte adhesion was quantified using the lectin perfusion
technique (Figure 2A)
.2
The
total number of retinal leukocytes in the nondiabetic animals was
32.16 ± 7.16 in arterioles, 34.83 ± 6.52 in venules, and
33.66 ± 7.86 in capillaries, resulting in a lower density of
leukocytes per vessel length in the capillaries. As was previously
observed,1,2
leukocyte adhesion in the diabetic retinae
was increased two- to threefold as compared to the nondiabetic animals
(Figure 2B)
. VEGF TrapA40 significantly reduced
leukocyte adhesion in the retinal arterioles (47%, n =
11, P < 0.0001), venules (36%, n =
11, P < 0.0005), and capillaries (36%,
n = 11, P < 0.001) when compared to
the IL-6R Trap-treated diabetic animals (Figure 2C)
.
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The total nitrite concentration was estimated from retinal tissue
using a total NO assay based on the conversion of NO to nitrate and its
subsequent quantification (Figure 3)
.
Compared to the retinae of nondiabetic animals, the retinae of diabetic
animals demonstrated a 4.25-fold increase in normalized NO levels
(31.69 ± 3.27 µmol/L versus 134.92 ±
7.83 µmol/L; P < 0.0001, n = 10).
Treatment with VEGF TrapA40 reduced the retinal
NO levels almost to nondiabetic levels (50.15 ± 4.85 µmol/L
retinal weight; P > 0.05 versus nondiabetic
controls, n = 8).
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The enzyme eNOS was quantified from retinal protein extracts using
a sensitive ELISA (Figure 4)
. Compared to
the retinae of nondiabetic animals, the retinae of diabetic animals
demonstrated a 1.6-fold increase in normalized eNOS levels (1.23
± 0.11 pg/mg versus 2.03 ± 0.22 pg/mg;
P < 0.005, n = 10). Pretreatment with
VEGF TrapA40 reduced the retinal eNOS levels to
nondiabetic levels (1.20 ± 0.08 pg/mg retinal weight,
P > 0.05 versus nondiabetic controls,
n = 8). The eNOS levels in diabetic animals treated
with IL-6R Trap did not differ significantly from the untreated
diabetic animals (n = 6, P >
0.05).
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Leukocyte adhesion was quantified using the lectin perfusion
technique2
(Figure 5)
.
Systemical treatment with the NO-synthase inhibitor L-NAME
significantly reduced leukocyte adhesion in the retinal arterioles
(56%, n = 10, P < 0.0001), venules
(68%, n = 10, P < 0.0001), and
capillaries (52%, n = 10, P < 0.0001)
when compared to nontreated diabetic animals. Nondiabetic animals
treated with L-NAME showed a trend toward a reduction in leukostasis in
the retinal arterioles (11%, n = 11, P
> 0.05), venules (27%, n = 11, P <
0.0005), and capillaries (18%, n = 11,
P < 0.001) when compared to untreated nondiabetic
animals.
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The expression of surface integrins CD11a, CD11b, and CD18 was
measured on neutrophils isolated from nondiabetic rats, diabetic rats,
and diabetic rats treated with VEGF TrapA40. In
agreement with previously published results,3
neutrophil
CD11a, CD11b, and CD18 surface expression levels were 2.1-fold
(n = 3, P < 0.05), 2.6-fold
(n = 3, P < 0.05), and 2.7-fold
(n = 3, P < 0.05) greater in
leukocytes from 1-week diabetic animals versus nondiabetic
controls (Table 1)
. Treatment with 25
mg/kg VEGF TrapA40 did not alter neutrophil
surface integrin expression under the conditions tested
(P > 0.05 for all).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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There is evidence to causally link VEGF to inflammation. We previously demonstrated that exogenous VEGF causes leukocyte adhesion in the retinal vasculature.9 We have also shown that VEGF up-regulation in diabetes correlates with the increased surface integrin expression and integrin-mediated adhesion. Specifically, we showed that the ß2-integrin heterodimers LFA-1 (CD11a/CD18) and Mac-1 (CD11a/CD18) are expressed at higher levels on diabetic rat neutrophils. The increased leukocyte adhesion was blocked in vivo and in vitro with neutralizing antibodies to CD11b, CD18, and ICAM-1. Skin-7 and tumor-derived15 VEGF has also been shown to trigger leukocyte adhesion in experimental models. In the eye, Flt-1-based VEGF inhibition was demonstrated to inhibit inflammation-related choroidal neovascularization.16 In the latter study, inflammation, in addition to neovascularization, was reduced after the systemic administration of a soluble Flt-1-based reagent. Similar anti-inflammatory effects after VEGF inhibition were described in a model of rheumatoid arthritis.17 The results of the current study, which show a reduction in inflammation after VEGF inhibition, are consistent with these published reports. Taken together, the evidence suggests that VEGF-induced inflammation might not be limited to the eye, but rather represents a common mechanism in disease.
In the current study, we choose to study neutrophil surface integrin expression, because neutrophils represent the majority of leukocytes in the diabetic retinae of rats18 and humans.19 However, we cannot exclude the possibility that monocytes are adhering to the vascular endothelium. In our earlier studies, we have shown that the adherent population is positive for the integrin CD18, a molecule that is present on neutrophils and monocytes. The enhanced neutrophil integrin expression that characterizes diabetes was not affected by the inhibition of VEGF in the current study. This finding is in general agreement with recent data showing that VEGF inhibition does not alter neutrophil CD11b and CD18 expression induced by conditioned tumor medium.20 Nevertheless, retinal VEGF may still serve to attract leukocytes to the retinal vasculature. Monocytes, a leukocyte class operative in human diabetic retinopathy,4,5 migrate in response to VEGF.21 Moreover, via their own VEGF, leukocytes may serve to increase the local concentration of VEGF when they adhere to endothelium. By example, VEGF has been documented in neutrophils,22 monocytes,23 eosinophils,24 lymphocytes,25 and platelets.26
The current data also show that endogenous VEGF is a stimulus for retinal eNOS expression. The serine/threonine protein kinase AKT/PKB was recently shown to be activated by VEGF and to mediate the induction of eNOS, a cascade leading to increased NO production, and in turn, ICAM-1 up-regulation.12 Based on these data, we hypothesized that the inhibition of endogenous VEGF would lead to the down-regulation of retinal ICAM-1 through a reduction in eNOS expression. The results obtained in this study are consistent with this hypothesis. Retinal nitrite levels were up-regulated in the diabetic animals and were normalized via the inhibition of endogenous VEGF. Moreover, the direct inhibition of eNOS via the systemic administration of L-NAME reduced diabetic leukocyte adhesion. Diabetes-related increases in retinal NO have been described before.27 However, the literature concerning the effect on NO on the expression of inflammatory molecules is controversial and the effects might be tissue-dependent.12,28,29 A down-regulation of ICAM-1 expression via NO has been shown in two models of ischemia-reperfusion.28,29 Our data, however, are in agreement with those of Radisavljevi and colleagues12 demonstrating the action of VEGF and NO on ICAM-1 up-regulation.
Taken together, the current data causally link VEGF with the inflammatory events that characterize the earliest stages of diabetic retinopathy. As such, VEGF may serve an important potential therapeutic target for the prevention and treatment of early diabetic retinopathy.
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Acknowledgements |
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Footnotes |
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Supported by The Roberta W. Siegel Fund (to A. P. A.), the Juvenile Diabetes Foundation (to A. M. J. and A. P. A.), the National Eye Institute (grants R01 EY12611 and EY11627 to A. P. A.), the Deutsche Forschungsgemeinschaft (DFG Jo324/2-1 to A. M. J.), the Ernst und Berta Grimmke Stiftung Düsseldorf (to A. M. J.), and a Massachusetts Lions Eye Research Fund grant (to A. P. A.).
Accepted for publication October 22, 2001.
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S. Satofuka, A. Ichihara, N. Nagai, T. Koto, H. Shinoda, K. Noda, Y. Ozawa, M. Inoue, K. Tsubota, H. Itoh, et al. Role of Nonproteolytically Activated Prorenin in Pathologic, but Not Physiologic, Retinal Neovascularization Invest. Ophthalmol. Vis. Sci., January 1, 2007; 48(1): 422 - 429. [Abstract] [Full Text] [PDF]
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 A. S. Motiwala, H. K. Janagama, M. L. Paustian, X. Zhu, J. P. Bannantine, V. Kapur, and S. Sreevatsan Comparative Transcriptional Analysis of Human Macrophages Exposed to Animal and Human Isolates of Mycobacterium avium Subspecies paratuberculosis with Diverse Genotypes. Infect. Immun., November 1, 2006; 74(11): 6046 - 6056. [Abstract] [Full Text] [PDF]
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K. Muranaka, Y. Yanagi, Y. Tamaki, T. Usui, N. Kubota, A. Iriyama, Y. Terauchi, T. Kadowaki, and M. Araie Effects of Peroxisome Proliferator-Activated Receptor {gamma} and Its Ligand on Blood-Retinal Barrier in a Streptozotocin-Induced Diabetic Model. Invest. Ophthalmol. Vis. Sci., October 1, 2006; 47(10): 4547 - 4552. [Abstract] [Full Text] [PDF]
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P. E. van Eeden, L. B. G. Tee, S. Lukehurst, C.-M. Lai, E. P. Rakoczy, L. D. Beazley, and S. A. Dunlop Early vascular and neuronal changes in a VEGF transgenic mouse model of retinal neovascularization. Invest. Ophthalmol. Vis. Sci., October 1, 2006; 47(10): 4638 - 4645. [Abstract] [Full Text] [PDF]
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 A. B. El-Remessy, M. Al-Shabrawey, Y. Khalifa, N.-T. Tsai, R. B. Caldwell, and G. I. Liou Neuroprotective and Blood-Retinal Barrier-Preserving Effects of Cannabidiol in Experimental Diabetes Am. J. Pathol., January 1, 2006; 168(1): 235 - 244. [Abstract] [Full Text] [PDF]
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W. Chen, W. J. Esselman, D. B. Jump, and J. V. Busik Anti-inflammatory Effect of Docosahexaenoic Acid on Cytokine-Induced Adhesion Molecule Expression in Human Retinal Vascular Endothelial Cells Invest. Ophthalmol. Vis. Sci., November 1, 2005; 46(11): 4342 - 4347. [Abstract] [Full Text] [PDF]
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G B Arden, R L Sidman, W Arap, and R O Schlingemann Spare the rod and spoil the eye Br. J. Ophthalmol., June 1, 2005; 89(6): 764 - 769. [Abstract] [Full Text] [PDF]
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S. Y. Kim, M. A. Johnson, D. S. McLeod, T. Alexander, B. C. Hansen, and G. A. Lutty Neutrophils Are Associated With Capillary Closure in Spontaneously Diabetic Monkey Retinas Diabetes, May 1, 2005; 54(5): 1534 - 1542. [Abstract] [Full Text] [PDF]
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H. Tamura, K. Miyamoto, J. Kiryu, S. Miyahara, H. Katsuta, F. Hirose, K. Musashi, and N. Yoshimura Intravitreal Injection of Corticosteroid Attenuates Leukostasis and Vascular Leakage in Experimental Diabetic Retina Invest. Ophthalmol. Vis. Sci., April 1, 2005; 46(4): 1440 - 1444. [Abstract] [Full Text] [PDF]
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N. Nagai, K. Noda, T. Urano, Y. Kubota, H. Shinoda, T. Koto, K. Shinoda, M. Inoue, T. Shiomi, E. Ikeda, et al. Selective Suppression of Pathologic, but Not Physiologic, Retinal Neovascularization by Blocking the Angiotensin II Type 1 Receptor Invest. Ophthalmol. Vis. Sci., March 1, 2005; 46(3): 1078 - 1084. [Abstract] [Full Text] [PDF]
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 B. Stratmann and D. Tschoepe Pathobiology and cell interactions of platelets in diabetes Diabetes and Vascular Disease Research, February 1, 2005; 2(1): 16 - 23. [Abstract] [PDF]
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 A. B. El-Remessy, M. Bartoli, D. H. Platt, D. Fulton, and R. B. Caldwell Oxidative stress inactivates VEGF survival signaling in retinal endothelial cells via PI 3-kinase tyrosine nitration J. Cell Sci., January 1, 2005; 118(1): 243 - 252. [Abstract] [Full Text] [PDF]
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S. X. Zhang, J.-x. Ma, J. Sima, Y. Chen, M. S. Hu, A. Ottlecz, and G. N. Lambrou Genetic Difference in Susceptibility to the Blood-Retina Barrier Breakdown in Diabetes and Oxygen-Induced Retinopathy Am. J. Pathol., January 1, 2005; 166(1): 313 - 321. [Abstract] [Full Text] [PDF]
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K. V. Ramana, B. Friedrich, S. Srivastava, A. Bhatnagar, and S. K. Srivastava Activation of Nulcear Factor-{kappa}B by Hyperglycemia in Vascular Smooth Muscle Cells Is Regulated by Aldose Reductase Diabetes, November 1, 2004; 53(11): 2910 - 2920. [Abstract] [Full Text] [PDF]
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V. Poulaki, A. M. Joussen, N. Mitsiades, C. S. Mitsiades, E. F. Iliaki, and A. P. Adamis Insulin-Like Growth Factor-I Plays a Pathogenetic Role in Diabetic Retinopathy Am. J. Pathol., August 1, 2004; 165(2): 457 - 469. [Abstract] [Full Text] [PDF]
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D. C. Yeh, D. V. Bula, J. W. Miller, E. S. Gragoudas, and J. G. Arroyo Expression of Leukocyte Adhesion Molecules in Human Subfoveal Choroidal Neovascular Membranes Treated with and without Photodynamic Therapy Invest. Ophthalmol. Vis. Sci., July 1, 2004; 45(7): 2368 - 2373. [Abstract] [Full Text] [PDF]
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 J. C. Mamputu and G. Renier Advanced glycation end-products increase monocyte adhesion to retinal endothelial cells through vascular endothelial growth factor-induced ICAM-1 expression: inhibitory effect of antioxidants J. Leukoc. Biol., June 1, 2004; 75(6): 1062 - 1069. [Abstract] [Full Text] [PDF]
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S. Miyahara, J. Kiryu, K. Yamashiro, K. Miyamoto, F. Hirose, H. Tamura, H. Katsuta, K. Nishijima, A. Tsujikawa, and Y. Honda Simvastatin Inhibits Leukocyte Accumulation and Vascular Permeability in the Retinas of Rats with Streptozotocin-Induced Diabetes Am. J. Pathol., May 1, 2004; 164(5): 1697 - 1706. [Abstract] [Full Text] [PDF]
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M R Stanford The pathogenesis of diabetic retinopathy Br. J. Ophthalmol., April 1, 2004; 88(4): 444 - 445. [Full Text] [PDF]
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W. Chen, D. B. Jump, M. B. Grant, W. J. Esselman, and J. V. Busik Dyslipidemia, but Not Hyperglycemia, Induces Inflammatory Adhesion Molecules in Human Retinal Vascular Endothelial Cells Invest. Ophthalmol. Vis. Sci., November 1, 2003; 44(11): 5016 - 5022. [Abstract] [Full Text] [PDF]
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 V. Poulaki, C. S. Mitsiades, C. McMullan, D. Sykoutri, G. Fanourakis, V. Kotoula, S. Tseleni-Balafouta, D. A. Koutras, and N. Mitsiades Regulation of Vascular Endothelial Growth Factor Expression by Insulin-Like Growth Factor I in Thyroid Carcinomas J. Clin. Endocrinol. Metab., November 1, 2003; 88(11): 5392 - 5398. [Abstract] [Full Text] [PDF]
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K. Yamashiro, A. Tsujikawa, S. Ishida, T. Usui, Y. Kaji, Y. Honda, Y. Ogura, and A. P. Adamis Platelets Accumulate in the Diabetic Retinal Vasculature Following Endothelial Death and Suppress Blood-Retinal Barrier Breakdown Am. J. Pathol., July 1, 2003; 163(1): 253 - 259. [Abstract] [Full Text] [PDF]
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S. Ishida, T. Usui, K. Yamashiro, Y. Kaji, E. Ahmed, K. G. Carrasquillo, S. Amano, T. Hida, Y. Oguchi, and A. P. Adamis VEGF164 Is Proinflammatory in the Diabetic Retina Invest. Ophthalmol. Vis. Sci., May 1, 2003; 44(5): 2155 - 2162. [Abstract] [Full Text] [PDF]
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T. Abiko, A. Abiko, A. C. Clermont, B. Shoelson, N. Horio, J. Takahashi, A. P. Adamis, G. L. King, and S.-E. Bursell Characterization of Retinal Leukostasis and Hemodynamics in Insulin Resistance and Diabetes: Role of Oxidants and Protein Kinase-C Activation Diabetes, March 1, 2003; 52(3): 829 - 837. [Abstract] [Full Text] [PDF]
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 J. Holash, S. Davis, N. Papadopoulos, S. D. Croll, L. Ho, M. Russell, P. Boland, R. Leidich, D. Hylton, E. Burova, et al. VEGF-Trap: A VEGF blocker with potent antitumor effects PNAS, August 20, 2002; 99(17): 11393 - 11398. [Abstract] [Full Text] [PDF]
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J. T. Rosenbaum Sugar Creates a Sticky Business: Round Up the Usual Suspects Am. J. Pathol., May 1, 2002; 160(5): 1547 - 1550. [Full Text] [PDF]
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A. M. Joussen, V. Poulaki, A. Tsujikawa, W. Qin, T. Qaum, Q. Xu, Y. Moromizato, S.-E. Bursell, S. J. Wiegand, J. Rudge, et al. Suppression of Diabetic Retinopathy with Angiopoietin-1 Am. J. Pathol., May 1, 2002; 160(5): 1683 - 1693. [Abstract] [Full Text] [PDF]
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A P Adamis Is diabetic retinopathy an inflammatory disease? Br. J. Ophthalmol., April 1, 2002; 86(4): 363 - 365. [Full Text] [PDF]
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