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Retinal Vascular Endothelial Growth Factor Induces Intercellular Adhesion Molecule-1 and Endothelial Nitric Oxide Synthase Expression and Initiates Early Diabetic Retinal Leukocyte Adhesion in Vivo

2002; Elsevier BV; Volume: 160; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)64869-9

1525-2191

Antonia M. Joussen, Vassiliki Poulaki, Wenying Qin, Bernd Kirchhof, Nicholas Mitsiades, Stanley J. Wiegand, John S. Rudge, George D. Yancopoulos, Anthony P. Adamis,

Nitric Oxide and Endothelin Effects

Leukocyte adhesion to the diabetic retinal vasculature results in early blood-retinal barrier breakdown, capillary nonperfusion, and endothelial cell injury and death. Previous work has shown that intercellular adhesion molecule-1 (ICAM-1) and CD18 are required for these processes. However the relevant in vivo stimuli for ICAM-1 and CD18 expression in diabetes remain unknown. The current study investigated the causal role of endogenous vascular endothelial growth factor (VEGF) and nitric oxide in initiating these events. Diabetes was induced in Long-Evans rats with streptozotocin, resulting in a two- to threefold increase in retinal leukocyte adhesion. Confirmed diabetic animals were treated with a highly specific VEGF-neutralizing Flt-Fc construct (VEGF TrapA40). Retinal ICAM-1 mRNA levels in VEGF TrapA40-treated diabetic animals were reduced by 83.5% compared to diabetic controls (n = 5, P < 0.0001). VEGF TrapA40 also potently suppressed diabetic leukocyte adhesion in retinal arterioles (47%, n = 11, P < 0.0001), venules (36%, n = 11, P < 0.0005), and capillaries (36%, n = 11, P < 0.001). The expression of endothelial nitric oxide synthase (eNOS), a downstream mediator of VEGF activity, was increased in diabetic retina, and was potently suppressed with VEGF TrapA40 treatment (n = 8, P < 0.005). Further, VEGF TrapA40 reduced the diabetes-related nitric oxide increases in the retinae of diabetic animals. The inhibition of eNOS with N-ω-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. Leukocyte adhesion to the diabetic retinal vasculature results in early blood-retinal barrier breakdown, capillary nonperfusion, and endothelial cell injury and death. Previous work has shown that intercellular adhesion molecule-1 (ICAM-1) and CD18 are required for these processes. However the relevant in vivo stimuli for ICAM-1 and CD18 expression in diabetes remain unknown. The current study investigated the causal role of endogenous vascular endothelial growth factor (VEGF) and nitric oxide in initiating these events. Diabetes was induced in Long-Evans rats with streptozotocin, resulting in a two- to threefold increase in retinal leukocyte adhesion. Confirmed diabetic animals were treated with a highly specific VEGF-neutralizing Flt-Fc construct (VEGF TrapA40). Retinal ICAM-1 mRNA levels in VEGF TrapA40-treated diabetic animals were reduced by 83.5% compared to diabetic controls (n = 5, P < 0.0001). VEGF TrapA40 also potently suppressed diabetic leukocyte adhesion in retinal arterioles (47%, n = 11, P < 0.0001), venules (36%, n = 11, P < 0.0005), and capillaries (36%, n = 11, P < 0.001). The expression of endothelial nitric oxide synthase (eNOS), a downstream mediator of VEGF activity, was increased in diabetic retina, and was potently suppressed with VEGF TrapA40 treatment (n = 8, P < 0.005). Further, VEGF TrapA40 reduced the diabetes-related nitric oxide increases in the retinae of diabetic animals. The inhibition of eNOS with N-ω-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 adhesion of leukocytes to the retinal vasculature is one of the earliest events in experimental diabetes.1Miyamoto K Khosrof S Bursell SE Rohan R Murata T Clermont AC Aiello LP Ogura Y Adamis AP Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition.Proc Natl Acad Sci USA. 1999; 96: 10836-10841Crossref PubMed Scopus (658) Google Scholar Enhanced vascular permeability, endothelial cell damage, and capillary nonperfusion are some of the pathological consequences of diabetic retinal leukocyte adhesion.1Miyamoto K Khosrof S Bursell SE Rohan R Murata T Clermont AC Aiello LP Ogura Y Adamis AP Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition.Proc Natl Acad Sci USA. 1999; 96: 10836-10841Crossref PubMed Scopus (658) Google Scholar, 2Joussen AM Murata T Tsujikawa A Kirchhof B Bursell SE Adamis AP Leukocyte-mediated endothelial cell injury and death in the diabetic retina.Am J Pathol. 2001; 158: 147-152Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar When neutralizing anti-intercellular adhesion molecule-1 (ICAM-1) antibodies are administered to newly diabetic animals, the leukocyte-related pathologies are dramatically reduced.1Miyamoto K Khosrof S Bursell SE Rohan R Murata T Clermont AC Aiello LP Ogura Y Adamis AP Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition.Proc Natl Acad Sci USA. 1999; 96: 10836-10841Crossref PubMed Scopus (658) Google Scholar, 2Joussen AM Murata T Tsujikawa A Kirchhof B Bursell SE Adamis AP Leukocyte-mediated endothelial cell injury and death in the diabetic retina.Am J Pathol. 2001; 158: 147-152Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar Similarly, when the bioactivity of the ICAM-1 counterreceptor CD18 is inhibited, diabetic retinal leukocyte adhesion is potently suppressed.3Barouch FC Miyamoto K Allport JR Fujita K Bursell SE Aiello LP Luscinskas FW Adamis AP Integrin-mediated neutrophil adhesion and retinal leukostasis in diabetes.Invest Ophthalmol Vis Sci. 2000; 41: 1153-1158PubMed Google Scholar Diabetic retinopathy in rodents recapitulates much of the pathology of human diabetic retinopathy. In both species, the diabetic retinal vasculature up-regulates ICAM-1, contains increased numbers of leukocytes, and develops blood-retinal barrier breakdown and capillary nonperfusion1Miyamoto K Khosrof S Bursell SE Rohan R Murata T Clermont AC Aiello LP Ogura Y Adamis AP Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition.Proc Natl Acad Sci USA. 1999; 96: 10836-10841Crossref PubMed Scopus (658) Google Scholar, 2Joussen AM Murata T Tsujikawa A Kirchhof B Bursell SE Adamis AP Leukocyte-mediated endothelial cell injury and death in the diabetic retina.Am J Pathol. 2001; 158: 147-152Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar, 3Barouch FC Miyamoto K Allport JR Fujita K Bursell SE Aiello LP Luscinskas FW Adamis AP Integrin-mediated neutrophil adhesion and retinal leukostasis in diabetes.Invest Ophthalmol Vis Sci. 2000; 41: 1153-1158PubMed Google Scholar, 4Schröder S Palinski W Schmid-Schönbein GW Activated monocytes and granulocytes, capillary nonperfusion, and neovascularization in diabetic retinopathy.Am J Pathol. 1991; 139: 81-100PubMed Google Scholar, 5McLeod DS Lefer DJ Merges C Lutty GA Enhanced expression of intracellular adhesion molecule-1 and P-selectin in the diabetic human retina and choroid.Am J Pathol. 1995; 147: 642-653PubMed Google Scholar Moreover, the leukocyte-related pathologies observed in early diabetes persist into established diabetes1Miyamoto K Khosrof S Bursell SE Rohan R Murata T Clermont AC Aiello LP Ogura Y Adamis AP Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition.Proc Natl Acad Sci USA. 1999; 96: 10836-10841Crossref PubMed Scopus (658) Google Scholar, 4Schröder S Palinski W Schmid-Schönbein GW Activated monocytes and granulocytes, capillary nonperfusion, and neovascularization in diabetic retinopathy.Am J Pathol. 1991; 139: 81-100PubMed Google Scholar (Joussen and colleagues, unpublished data). 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.6Tolentino MJ Miller JW Gragoudas ES Jakobiec FA Flynn E Chatzistefanou K Ferrara N Adamis AP Intravitreous injections of vascular endothelial growth factor produce retinal ischemia and microangiopathy in an adult primate.Ophthalmology. 1996; 103: 1820-1828Abstract Full Text PDF PubMed Scopus (449) Google Scholar, 7Detmar M Brown LF Schon MP Elicker BM Velasco P Richard L Fukamura D Monsky D Claffey KP Jain RK Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice.J Invest Dermatol. 1998; 111: 1-6Crossref PubMed Scopus (461) Google Scholar, 8Lu M Perez V Ma N Miyamoto K Peng HB Liao JK Adamis AP VEGF increases retinal vascular ICAM-1 expression in vivo.Invest Ophthalmol Vis Sci. 1999; 40: 1808-1812PubMed Google Scholar, 9Miyamoto K Khosrof S Bursell S-E Moromizato Y Aiello LP Ogura Y Adamis AP Vascular endothelial growth factor-induced retinal vascular permeability is mediated by intercellular adhesion molecule-1 (ICAM-1).Am J Pathol. 2000; 156: 1733-1739Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar Although VEGF is expressed in the early diabetic retina,10Murata T Nakagawa K Khalil A Ishibashi T Inomata H Sueishi K The relation between expression of vascular endothelial growth factor and breakdown of the blood retinal barrier in diabetic rat retinas.Lab Invest. 1996; 74: 819-825PubMed Google Scholar, 11Amin RH Frank RN Kennedy A Eliott D Puklin JE Abrams GW Vascular endothelial growth factor is present in glial cells of the retina and optic nerve of human subjects with nonproliferative diabetic retinopathy.Invest Ophthalmol Vis Sci. 1997; 38: 36-47PubMed Google Scholar 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).12Radisavljevi Z Avraham H Avraham S Vascular endothelial growth factor up-regulates ICAM-1 expression via the phosphatidylinositol 3 OH-kinase/AKT/nitric oxide pathway and modulates migration of brain microvascular endothelial cells.J Biol Chem. 2000; 275: 20770-20774Crossref PubMed Scopus (193) Google Scholar 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,13Zochodne DW Verge VM Cheng C Hoke A Jolley C Thomsen K Rubin I Lauritzen M Nitric oxide synthase activity and expression in experimental diabetic neuropathy.J Neuropathol Exp Neurol. 2000; 59: 798-807Crossref PubMed Scopus (49) Google Scholar 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. 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. 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. 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 Eco RI/Bam HI 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 Eco RI, 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, 1× 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 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 ε 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-6Rα (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. 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).2Joussen AM Murata T Tsujikawa A Kirchhof B Bursell SE Adamis AP Leukocyte-mediated endothelial cell injury and death in the diabetic retina.Am J Pathol. 2001; 158: 147-152Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar 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. 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. 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. 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. 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. The monoclonal antibodies (mAbs) used in the flow cytometry experiments were purified IgG of murine origin. The fluorescein isothiocyanate-conjugated mAbs LFA-1α 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.3Barouch FC Miyamoto K Allport JR Fujita K Bursell SE Aiello LP Luscinskas FW Adamis AP Integrin-mediated neutrophil adhesion and retinal leukostasis in diabetes.Invest Ophthalmol Vis Sci. 2000; 41: 1153-1158PubMed Google Scholar 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 × 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. 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. 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). Leukocyte adhesion was quantified using the lectin perfusion technique (Figure 2A).2Joussen AM Murata T Tsujikawa A Kirchhof B Bursell SE Adamis AP Leukocyte-mediated endothelial cell injury and death in the diabetic retina.Am J Pathol. 2001; 158: 147-152Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar 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,1Miyamoto K Khosrof S Bursell SE Rohan R Murata T Clermont AC Aiello LP Ogura Y Adamis AP Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition.Proc Natl Acad Sci USA. 1999; 96: 10836-10841Crossref PubMed Scopus (658) Google Scholar, 2Joussen AM Murata T Tsujikawa A Kirchhof B Bursell SE Adamis AP Leukocyte-mediated endothelial cell injury and death in the diabetic retina.Am J Pathol. 2001; 158: 147-152Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar 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). 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). 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). Leukocyte adhesion was quantified using the lectin perfusion technique2Joussen AM Murata T Tsujikawa A Kirchhof B Bursell SE Adamis AP Leukocyte-mediated endothelial cell injury and death in the diabetic retina.Am J Pathol. 2001; 158: 147-152Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar (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.