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From the Laboratory for Surgical Research,*
Children’s
Hospital, Harvard Medical School, Boston, Massachusetts; the Department
of Ophthalmology,
Massachusetts Eye and Ear
Infirmary, Harvard Medical School, Boston, Massachusetts; the
Department of Ophthalmology,
RWTH Aachen,
Aachen, Germany; and the Joslin Diabetes
Center,§
Harvard Medical School,
Boston, Massachusetts
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Leukocytes adhere to the retinal vascular endothelium early in experimental diabetic retinopathy.7 Leukocyte adhesion, like endothelial cell death, has its onset before any clinical pathology is apparent. Further, its occurrence coincides with the development of blood-retinal barrier breakdown and capillary nonperfusion.7 Intercellular adhesion molecule-1 (ICAM-1) and CD18 have been shown to be operative in these events.7,8 The expression of both molecules is increased in diabetes, and the specific inhibition of ICAM-1 or CD18 prevents diabetic retinal leukocyte adhesion and blood-retinal barrier breakdown.
Previous work in non-ophthalmic tissues has demonstrated that adherent leukocytes can mediate endothelial cell and parenchymal injury.9,10 Studies have also colocalized leukocytes with dead and dying endothelial cells in the diabetic retina.4 However, the primacy of leukocytes in the development of diabetic retinal endothelial cell death is in doubt.5,6 To address this issue, the current study directly examined the causal role of leukocytes in the development of diabetic retinal endothelial cell injury and death. The temporal association between leukocyte adhesion and endothelial cell injury and death was assessed, and leukocyte adhesion was then disrupted using antibody-based ICAM-1- and CD18-neutralizing reagents. The effect of leukocyte adhesion blockade on retinal endothelial cell injury and death was then determined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All animal experiments followed the Association for Research in Vision and Ophthalmology guidelines and were approved by the Animal Care and Use Committees of the Children’s Hospital and the Joslin Diabetes Center. After an overnight fast, pathogen-free Long-Evans rats weighing 200 to 250 g (Charles River) received single 60 mg/kg intraperitoneal (i.p.) injections of streptozotocin (Sigma, St. Louis, MO) in 0.05 mol/L citrate buffer (pH 4.5). Control nondiabetic animals received citrate buffer alone. Animals with blood glucose levels greater than 250 mg/dl 48 hours after streptozotocin injection were deemed diabetic. Before each experiment and sacrifice, the diabetic state was reconfirmed. The animals selected for study possessed blood glucose levels >250 mg/dl at all time points tested. Blood glucose was measured with an automated test strip employing the glucose oxidase method (One Touch Meter, Lifescan, Milpitas, CA). The rats were fed standard laboratory chow and were allowed free access to food and water in an air-conditioned room with a 12-hour light/12-hour dark cycle. All animals were sacrificed at the conclusion of the experiment, 9 days after streptozotocin injection.
Lectin Labeling of Adherent Retinal Leukocytes
The animals were anesthetized with intramuscular xylazine hydrochloride (8 mg/kg; Phoenix Pharmaceutical, St. Joseph, MO) and ketamine hydrochloride (50 mg/kg; Parke-Davis, Morris Plains, NJ). The chest cavity was carefully opened and a 14-gauge perfusion cannula was introduced into the left ventricle. Drainage was achieved using a 16-gauge needle placed in the right atrium. The animals were perfused with 250 ml phosphate buffered saline (PBS) per kg body weight (BW) over 2 minutes to remove erythrocytes and non-adherent leukocytes. After PBS perfusion, fixation with 1% paraformaldehyde and 0.5% glutaraldehyde was achieved using 200 ml/kg perfusate over approximately 3 minutes. The PBS perfusion was performed at a physiological pressure, since the pumping heart provided the motive force. All subsequent perfusions were postmortem and were performed at 100 mmHg pressure. Nonspecific binding was blocked with 1% albumin in PBS (total volume 100 ml/kg BW) followed by perfusion with fluorescein-isothiocyanate (FITC)-coupled Concanavalin A lectin (20 µg/ml in PBS, pH 7.4, 5 mg/kg BW; Vector Labs, Burlingame, CA). Concanavalin A was used to label adherent leukocytes and vascular endothelial cells. Residual unbound lectin was removed with a 1% albumin in PBS perfusion for 1 minute followed by a PBS perfusion for 4 minutes. The retinae were carefully removed and flat mounts prepared using a fluorescence anti-fading medium (Southern Biotechnology, Birmingham, AL). The retinae were then imaged using a fluorescence microscope (Zeiss Axiovert, Oberkochen, Germany; FITC filter). Retinae in which the peripheral collecting vessels of the ora serrata were not visible were discarded. The total number of leukocytes in the retinal arterioles, venules, and capillaries was then determined.
In Vivo Leukocyte Imaging in the Diabetic Retina
Leukocyte dynamics in the retinal microcirculation were evaluated using carboxyfluorescein diacetate succinimidyl ester (CFDASE; Molecular Probes, Eugene, OR) labeling and a scanning laser ophthalmoscope (SLO; Rodenstock Instrument, Munich, Germany). Immediately before the observation of leukocyte dynamics, each rat was anesthetized with intramuscular xylazine hydrochloride (4 mg/kg; Phoenix Pharmaceuticals, St. Joseph, MO) and ketamine hydrochloride (25 mg/kg; Parke-Davis). The pupil of the left eye was dilated with 1% tropicamide (Alcon, Humancao, Puerto Rico). CFDASE was dissolved in dimethylsulfoxide (20 mg/ml), and 10 mg/kg of this solution was injected through a jugular vein catheter at a rate of 0.25 ml/minute. The fundus was observed with the SLO using the argon blue laser as the illumination source and the standard fluorescein angiography filter in the 20° or 40° field setting. The images were recorded on videotape at the rate of 30 frames/second. The video recordings were analyzed on a computer equipped with a video digitizer (Radius, San Jose, CA). The digitized video images were viewed in real time at 640 x 480 pixels with an intensity resolution of 256 steps. Leukocytes were defined as being adherent to the vascular endothelium if they remained stationary for at least 30 seconds. The adherent leukocytes were expressed as the number of cells within a circle around the optic disk measuring three disk diameters in diameter.
CD18 Immunofluorescence
To confirm the identity of the sticking cells within the vasculature, CD18 immunofluorescence was performed. CD18 is a leukocyte marker expressed on monocytes and neutrophils.11 Sticking leukocytes were labeled with fluorescein isothiocyanate-linked Concanavalin A as described above. Flat mounts were permeabilized with 1% Triton X-100 (Sigma) in PBS for 24 hours and nonspecific binding was blocked with 1% albumin in PBS. The retinae were then incubated with a biotinylated anti-rat CD18 antibody (1:100; Pharmingen, San Diego, CA) overnight at 4°C. After five washes with PBS, the tissues were exposed to streptavidin-coupled CyChrome (1:500; Pharmingen) for 2 hours at 25°C, and mounted with an anti-fading reagent (Southern Biotechnology, Irvine, CA) after three PBS washes.
Propidium Iodide (PI) Labeling
Dead and injured endothelial cells were labeled in vivo using PI (Molecular Probes). After the induction of deep anesthesia with 50 mg/kg i.p. sodium pentobarbital, PI (1 mg/ml in PBS) was injected intravenously via the tail vein at a concentration of 5 µmol/kg (0.668 ml/200 mg BW). The solution was allowed to circulate for 20 minutes, after which it was followed by fixation via whole body perfusion and lectin labeling as described above. Retinal flat mounts were examined by fluorescence microscopy as described above. Labeled endothelial cells were distinguished from surrounding cells, especially pericytes, by focusing through the tissue to discern the distinct cellular outline and nuclear shape of the endothelial cells.
Systemic ICAM-1 and CD18 Blockade
Confirmed diabetic animals were randomized to receive i.p. injections of 1 mg/kg of a mouse anti-rat ICAM-1 neutralizing antibody (clone 1A29; R&D Systems, Minneapolis, MN) or 1 mg/kg of an isotype control non-immune mouse IgG1 (R&D Systems). In separate experiments, diabetic rats received either 1 mg/kg of a mouse anti-rat CD18 F(ab')2 (WT.3) (Associates of Cape Cod Inc., Falmouth, MA) or a purified mouse anti-human non-immune F(ab')2 (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). All blocking reagents were injected at a final concentration of 1 mg/ml in sterile PBS. The animals were treated 3 times weekly for 1 week. Cell death and retinal leukostasis were determined 1 week after the first injection of the blocking reagent.
Statistical Analysis
All results are expressed as 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 the P values < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adherent retinal leukocytes were labeled in situ with
FITC-linked Concanavalin A. Preliminary experiments using various
FITC-linked lectins revealed that Concanavalin A gave the most
homogeneous staining in the retinal vasculature. After leukocyte
labeling, whole body perfusion was performed under physiological
perfusion pressure to remove non-adherent blood elements from the
retinal circulation. Retinal flat mounts were prepared and the adherent
leukocytes were counted in the arterioles, venules, and capillaries.
Compared to the nondiabetic control retinae, a 2.2-fold
(n = 9, P < 0.005), 3.8-fold
(n = 9, P < 0.001) and 3.6-fold
(n = 8, P < 0.001) increase in
adherent leukocytes was seen in the diabetic retinal arterioles,
venules, and capillaries, respectively (Figure 1)
.
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. A quantitative
assessment of leukocyte adhesion was completed using the SLO. The
adherent leukocyte density was increased in the arterioles and venules
of 1-week diabetic rats. The arterioles contained 4.4 ± 1.9
vs. 1.5 ± 0.9 adherent leukocytes in the diabetic
versus nondiabetic retinae, respectively (n = 9,
P < 0.01). The venules contained 2.9 ± 1.2
vs. 1.2 ± 1.0 adherent leukocytes in the diabetic and
non-diabetic retinae, respectively (n = 9, P <
0.01). The -fold increases observed with the two methods were
comparable.
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.
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Endothelial cell injury and death were investigated using PI, a
molecule that is excluded from uninjured viable cells. PI fluoresces
when it leaks through injured cell membranes and binds to DNA and RNA,
primarily identifying cells undergoing either necrosis or
apoptosis.13
PI was injected intravenously and endothelial
cell injury and death were quantified in retinal flat mounts. Little or
no PI staining was observed in nondiabetic retinae. In contrast,
endothelial PI staining was markedly increased in the retinal
arterioles (23.0 ± 10.8 cells/retina), venules (31.8 ± 7.6
cells/retina), and capillaries (27.6 ± 6.1 cells/retina) of
diabetic eyes (Figure 4)
. In both small
and large caliber vessels, PI-positive cells were often found in
clusters (Figure 4)
. A cohort of 2-month diabetic animals was also
studied and revealed a similar level of leukocyte adhesion and
endothelial cell injury (data not shown). These data confirmed that
endothelial cell injury and death are ongoing during the course of
diabetes, a result previously shown by others.3,4
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To assess the role of leukocyte adhesion in retinal endothelial
cell injury and death, in vivo adhesion blockade experiments
were performed. Diabetic animals were randomized to receive either 1
mg/kg of an ICAM-1-neutralizing antibody or 1 mg/kg of a
CD18-neutralizing F(ab')2. For each experiment, a
separate cohort of diabetic animals received 1 mg/kg of a corresponding
non-immune isotype control. The reagents were administered 3 times
weekly following the onset of diabetes. All animals were analyzed 1
week after the first injection of the blocking reagent. The ICAM-1
antibody reduced diabetes-associated leukocyte adhesion 48.6%
(n = 10, P < 0.001) in
arterioles, 41.2% (n = 10, P <
0.001) in venules, and 70% (n = 10,
P < 0.001) in capillaries (Figure 5)
. Similarly, when compared to the
controls, the ICAM-1 antibody reduced endothelial cell PI staining
90.1% (n = 10, P < 0.001) in
venules, 90.8% (n = 10, P <
0.001) in arterioles, and 89.9% (n = 10,
P < 0.001) in capillaries.
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Treatment with CD-18 F(ab')2 reduced leukocyte
adhesion 39.6% in venules (n = 12,
P < 0.05) and 42.3% (n = 12,
P < 0.005) in capillaries. CD18 blockade also potently
reduced endothelial cell PI staining 48.1% (n =
12, P < 0.005) in venules and 42.5%
(n = 12, P < 0.005) in
capillaries. Unlike the ICAM-1 blockade experiment, arteriolar
leukocyte adhesion and endothelial cell PI staining were not affected
by CD18 blockade.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Leukocyte adhesion and endothelial cell death are ongoing in diabetes.3,4,7 The current data represent a snapshot in time, but the cumulative number of injured and dying endothelial cells in the diabetic retina likely increases with time and becomes sizable. Given the presumably larger total capillary area in the retina, the proportion of injured and dying capillary endothelial cells is smaller than that for the arterioles and venules. This fact does not seem to fit with the preferential development of acellular capillaries in the diabetic retina. We propose that the clustered damage to capillaries, where little endothelial reserve exists, contributes to the formation of acellular capillaries. Moreover, because a single endothelial cell can form a retinal capillary lumen, and only a few endothelial cells are needed to form a retinal capillary,14 the death of a relatively small number of capillary endothelial cells can take on outsized importance. In contrast, we speculate that discrete and focal damage to larger vessels is less likely to impede flow and cause the death of a vessel, given the relatively smaller scale of the injury and the larger number of surrounding endothelial cells available for repair.
The methods used to assess leukocyte adhesion and endothelial cell death are novel for the retina. Because leukocytes were observed to adhere to the arterioles of the diabetic retina, an unusual result, confirmation was required. The SLO and perfusion methods mutually confirmed the increased leukocyte adhesion in diabetic arterioles, as well as the capillaries7 and venules. Although the -fold increases were comparable, the differences in absolute numbers were likely due to the optics of the SLO, which precluded visualization of the retinal periphery and mid-periphery.
Although endothelial cell death was previously observed to occur 4 to 6 months after the onset of experimental diabetes3,4 the current data demonstrate that endothelial cell injury and death can be detected within 1 week of diabetes. Furthermore, the number of injured and dying endothelial cells appears to be much greater than previously recognized. The numerical disparity may be due to the fact that both necrotic and apoptotic cells are quantified with PI labeling. Previous work quantified endothelial cell apoptosis alone.3 Moreover, because the PI method cannot discriminate between lethal and sublethal injury, it is conceivable that some of the injured PI-stained cells observed in the current study are still viable. Lastly, it is important to note that different time points were examined in the two studies. Nevertheless, it cannot be ruled out that some of the PI staining observed in these studies was artifactual in nature.
Finally, we speculate, as others have before us, that acellular capillaries develop when retinal endothelial cells reach replicative senescence.3 Acellular capillaries were not observed in our 1-week diabetic rats. Replicative senescence requires time, potentially explaining the delay between the onset of endothelial cell death and the appearance of acellular capillaries. Endothelial cell death and proliferation are known to proceed simultaneously in the diabetic human retina.15 We speculate that replicative senescence eventually tips the balance in favor of endothelial cell death.
Taken together, the data identify a new pathogenic phenomenon in diabetic retinopathy: leukocyte-mediated endothelial cell injury and death. The results of this study suggest that the inhibition of ICAM-1 and CD18 may avert or suppress acellular capillary formation via the suppression of endothelial cell injury and death. If so, the retinal ischemia and VEGF up-regulation that characterize diabetic retinopathy may be preventable.
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Footnotes |
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Supported by the Roberta W. Siegel Fund (to A. P. A.), Deutsche Forschungsgemeinschaft DFG Jo 324/2–1 (to A. M. J.), the Juvenile Diabetes Foundation International (to A. M. J. and A. P. A.), National Institutes of Health grants EY11627 and EY12611 (to A. P. A.), and the Massachusetts Lions (to A. P. A.).
Accepted for publication September 28, 2000.
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B. Miljanovic, R. J. Glynn, D. M. Nathan, J. E. Manson, and D. A. Schaumberg A Prospective Study of Serum Lipids and Risk of Diabetic Macular Edema in Type 1 Diabetes Diabetes, November 1, 2004; 53(11): 2883 - 2892. [Abstract] [Full Text] [PDF]
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C. Zhang, J. Yang, and L. K. Jennings Leukocyte-Derived Myeloperoxidase Amplifies High-Glucose--Induced Endothelial Dysfunction Through Interaction With High-Glucose--Stimulated, Vascular Non--Leukocyte-Derived Reactive Oxygen Species Diabetes, November 1, 2004; 53(11): 2950 - 2959. [Abstract] [Full Text] [PDF]
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L. Zheng, C. Szabo, and T. S. Kern Poly(ADP-Ribose) Polymerase Is Involved in the Development of Diabetic Retinopathy via Regulation of Nuclear Factor-{kappa}B Diabetes, November 1, 2004; 53(11): 2960 - 2967. [Abstract] [Full Text] [PDF]
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B. M. Ben-Mahmud, G. E. Mann, A. Datti, A. Orlacchio, E. M. Kohner, and R. Chibber Tumor Necrosis Factor-{alpha} in Diabetic Plasma Increases the Activity of Core 2 GlcNAc-T and Adherence of Human Leukocytes to Retinal Endothelial Cells: Significance of Core 2 GlcNAc-T in Diabetic Retinopathy Diabetes, November 1, 2004; 53(11): 2968 - 2976. [Abstract] [Full Text] [PDF]
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J R MacKinnon, R M Knott, and J V Forrester Altered L-selectin expression in lymphocytes and increased adhesion to endothelium in patients with diabetic retinopathy Br J Ophthalmol, September 1, 2004; 88(9): 1137 - 1141. [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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A. Canton, E. M. Martinez-Caceres, C. Hernandez, C. Espejo, J. Garcia-Arumi, and R. Simo CD4-CD8 and CD28 Expression in T Cells Infiltrating the Vitreous Fluid in Patients With Proliferative Diabetic Retinopathy: A Flow Cytometric Analysis Arch Ophthalmol, May 1, 2004; 122(5): 743 - 749. [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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V. Poulaki, N. Mitsiades, F. E. Kruse, S. Radetzky, E. Iliaki, B. Kirchhof, and A. M. Joussen Activin A in the Regulation of Corneal Neovascularization and Vascular Endothelial Growth Factor Expression Am. J. Pathol., April 1, 2004; 164(4): 1293 - 1302. [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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J M Hughes, A Brink, A N Witmer, M Hanraads-de Riemer, I Klaassen, and R O Schlingemann Vascular leucocyte adhesion molecules unaltered in the human retina in diabetes Br J Ophthalmol, April 1, 2004; 88(4): 566 - 572. [Abstract] [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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T. C. B. Moore, J. E. Moore, Y. Kaji, N. Frizzell, T. Usui, V. Poulaki, I. L. Campbell, A. W. Stitt, T. A. Gardiner, D. B. Archer, et al. The Role of Advanced Glycation End Products in Retinal Microvascular Leukostasis Invest. Ophthalmol. Vis. Sci., October 1, 2003; 44(10): 4457 - 4464. [Abstract] [Full Text] [PDF]
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 S. Ishida, T. Usui, K. Yamashiro, Y. Kaji, S. Amano, Y. Ogura, T. Hida, Y. Oguchi, J. Ambati, J. W. Miller, et al. VEGF164-mediated Inflammation Is Required for Pathological, but Not Physiological, Ischemia-induced Retinal Neovascularization J. Exp. Med., August 4, 2003; 198(3): 483 - 489. [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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R. Chibber, B. M. Ben-Mahmud, G. E. Mann, J. J. Zhang, and E. M. Kohner Protein Kinase C {beta}2-Dependent Phosphorylation of Core 2 GlcNAc-T Promotes Leukocyte-Endothelial Cell Adhesion: A Mechanism Underlying Capillary Occlusion in Diabetic Retinopathy Diabetes, June 1, 2003; 52(6): 1519 - 1527. [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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K. Koizumi, V. Poulaki, S. Doehmen, G. Welsandt, S. Radetzky, A. Lappas, N. Kociok, B. Kirchhof, and A. M. Joussen Contribution of TNF-{alpha} to Leukocyte Adhesion, Vascular Leakage, and Apoptotic Cell Death in Endotoxin-Induced Uveitis In Vivo Invest. Ophthalmol. Vis. Sci., May 1, 2003; 44(5): 2184 - 2191. [Abstract] [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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M. Gotte, A. M. Joussen, C. Klein, P. Andre, D. D. Wagner, M. T. Hinkes, B. Kirchhof, A. P. Adamis, and M. Bernfield Role of Syndecan-1 in Leukocyte-Endothelial Interactions in the Ocular Vasculature Invest. Ophthalmol. Vis. Sci., April 1, 2002; 43(4): 1135 - 1141. [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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A. M. Joussen, V. Poulaki, W. Qin, B. Kirchhof, N. Mitsiades, S. J. Wiegand, J. Rudge, G. D. Yancopoulos, and A. P. Adamis 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 Am. J. Pathol., February 1, 2002; 160(2): 501 - 509. [Abstract] [Full Text] [PDF]
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A. M. Joussen, S. Huang, V. Poulaki, K. Camphausen, W.-D. Beecken, B. Kirchhof, and A. P. Adamis In Vivo Retinal Gene Expression in Early Diabetes Invest. Ophthalmol. Vis. Sci., November 1, 2001; 42(12): 3047 - 3057. [Abstract] [Full Text] [PDF]
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A. M. Joussen, W.-D. Beecken, Y. Moromizato, A. Schwartz, B. Kirchhof, and V. Poulaki Inhibition of Inflammatory Corneal Angiogenesis by TNP-470 Invest. Ophthalmol. Vis. Sci., October 1, 2001; 42(11): 2510 - 2516. [Abstract] [Full Text] [PDF]
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T. Qaum, Q. Xu, A. M. Joussen, M. W. Clemens, W. Qin, K. Miyamoto, H. Hassessian, S. J. Wiegand, J. Rudge, G. D. Yancopoulos, et al. VEGF-initiated Blood-Retinal Barrier Breakdown in Early Diabetes Invest. Ophthalmol. Vis. Sci., September 1, 2001; 42(10): 2408 - 2413. [Abstract] [Full Text] [PDF]
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