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1From the Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts; the 2Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan; the 4Department of Ophthalmology, Faculty of Medicine, University of Tokyo, Tokyo, Japan; the 5Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan; 6Eyetech Research Center, Woburn, Massachusetts; and the 7Kyorin Eye Center, Mitaka, Japan.
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Abstract |
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METHODS. Retinal leukostasis and BRB breakdown were simultaneously quantified by combining concanavalin A lectin (ConA) perfusion labeling with a fluorophotometric dextran leakage assay. CD45 immunohistochemistry was performed to confirm that ConA-stained cells within the vasculature were leukocytes. Retinal leukostasis and BRB breakdown were compared in nondiabetic rats receiving intravitreous injections of VEGF120 or VEGF164. Retinal intercellular adhesion molecule (ICAM)-1 and VEGF protein levels were studied by Western blot and ELISA, respectively. An anti-VEGF164(165) aptamer (EYE001) was administered by intravitreous injection to 2-week and 3-month diabetic rats, and the effect on retinal leukostasis and BRB breakdown was quantified.
RESULTS. Compared with VEGF120, VEGF164 more potently increased retinal ICAM-1 levels (2.2-fold), leukostasis (1.9-fold), and BRB breakdown (2.1-fold, P < 0.01 for all), despite negligible differences in vitreoretinal VEGF levels at the time of evaluation (P > 0.05). Retinal leukostasis and leakage increased with the duration of diabetes (P < 0.01) and correlated closely (P < 0.01, r = 0.889). The isoform-specific blockade of endogenous VEGF164 with EYE001 resulted in a significant suppression of retinal leukostasis and BRB breakdown in both early (72.4% and 82.6%, respectively) and established (48.5% and 55.0%, respectively) diabetes (P < 0.01).
CONCLUSIONS. On an equimolar basis, VEGF164 is at least twice as potent as VEGF120 at inducing ICAM-1–mediated retinal leukostasis and BRB breakdown in vivo. The inhibition of diabetic retinal leukostasis and BRB breakdown with EYE001 in early and established diabetes indicates that VEGF164 is an important isoform in the pathogenesis of early diabetic retinopathy.
Vascular endothelial growth factor (VEGF) is a hypoxia-induced angiogenic factor2 3 and a vasopermeability factor.4 5 In patients with diabetic retinopathy, the VEGF levels in intraocular fluids increase not only during the proliferative stage,6 7 which is characterized by ischemia-initiated retinal angiogenesis, but also during the nonproliferative stage,8 9 during which hypoxia is less well documented. Furthermore, based on VEGF localization studies in surgically excised tissues from human diabetic eyes, VEGF has been suggested to play a crucial role both in fibrovascular proliferation10 11 and BRB breakdown.12 The upstream stimuli for expression of VEGF in early retinopathy remain unknown.
Recently, it has been shown that leukocyte adhesion is operative in the pathogenesis of vascular leakage.13 14 15 VEGF increases the expression of intercellular adhesion molecule (ICAM)-1 on endothelial cells in vitro.16 17 In vivo, intravitreous injections of VEGF164 induce ICAM-1 expression in the murine retinal vasculature.17 In a separate study, VEGF-induced BRB breakdown was shown to be leukocyte dependent in part, when the inhibition of ICAM-1 prevented BRB breakdown in VEGF165-injected rat eyes.18
In the human retinal vasculature, leukocyte counts and ICAM-1 immunoreactivity are both increased in eyes with diabetic retinopathy.19 Similarly, experimental rat diabetes results in increased levels of retinal VEGF20 21 22 and ICAM-1,23 coincident with increased retinal leukocyte stasis (leukostasis),23 24 and BRB breakdown.20 23 When ICAM-1 bioactivity is inhibited with a neutralizing antibody, retinal leukostasis and BRB breakdown are both suppressed.23 In experimental diabetes, the inhibition of VEGF suppresses retinal ICAM-1expression,25 leukostasis,25 and BRB breakdown.22
VEGF has at least five isoforms generated through the alternative splicing of mRNA arising from a single gene. The human proteins are one residue longer than the murine homologues. The two major prevalent isoforms in the retina are VEGF121(120) and VEGF165(164).26 RNase protection assays have identified VEGF164 as the predominant VEGF isoform expressed in the diabetic retina, accounting for at least 80% of total VEGF in experimental diabetes.22
In the present study, the differential potency of VEGF164 and VEGF120 in inducing retinal leukostasis and BRB breakdown was compared. The current data demonstrate that on an equimolar basis, VEGF164 was significantly more potent at inducing these pathologic responses when administered exogenously. Moreover, the inhibition of endogenous VEGF164 potently inhibited diabetic retinal leukostasis and BRB breakdown, both in early and established diabetes. Taken together, these data indicate that VEGF164 is an appropriate target for the inhibition of several important diabetic retinal disorders.
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Intravitreous Administration of VEGF and the Anti-VEGF165 Aptamer EYE001
Animals were anesthetized with intramuscular xylazine hydrochloride (6 mg/kg; Phoenix Pharmaceutical, St. Joseph, MO) and ketamine hydrochloride (40 mg/kg; Parke-Davis, Morris Plains, NJ). Intravitreous injections were performed by inserting a 33-gauge double-caliber needle (Ito Corp., Fuji, Japan) into the vitreous 1 mm posterior to the corneal limbus. Insertion and infusion were directly viewed through an operating microscope, taking care not to injure the lens or the retina. Any eyes that exhibited damage to the lens or retina were discarded and not used for the analyses.
Rats were randomized to receive intravitreous injections of 5 µL of sterile phosphate-buffered saline (PBS) containing 1.7 pmol murine VEGF120, VEGF164 (R&D Systems, Minneapolis, MN), or vehicle alone. The dosage was determined from a previous report describing leukocyte adhesion to the rat retinal vasculature after intravitreous injections of VEGF165.18 The retinas were analyzed 24, 48, and 72 hours after injection.
In separate experiments, diabetic animals received 5 µL intravitreous injections of sterile PBS containing 2.5 nmol of the anti-VEGF165 aptamer (EYE001; Eyetech Pharmaceuticals, New York, NY), 40-kDa polyethylene glycol (PEG; Eyetech), or vehicle alone. All reagents were injected 48 hours before evaluation. The anti-VEGF165 aptamer is an oligonucleotide (28 ribonucleotide bases) that binds to the exon-7–encoded domain of human VEGF165 with high specificity and affinity (200 pM).27 It does not bind to VEGF120(121). The oligonucleotide is conjugated to a 40-kDa PEG moiety to increase its half-life. The anti-VEGF165 aptamer efficiently neutralizes VEGF164 in rats,28 29 consistent with the fact that the exon-7–encoded VEGF domain is completely conserved between the rat and human. The dose and the evaluation time points were determined from a previous report.30
Retinal Leukostasis Quantification
The retinal vasculature and adherent leukocytes were imaged with fluorescein-isothiocyanate (FITC)- or rhodamine-coupled concanavalin A lectin (ConA) (Vector Laboratories, Burlingame, CA). A perfusion labeling technique previously reported24 was used, with slight modification. Animals were deeply anesthetized with intramuscular xylazine hydrochloride and ketamine hydrochloride. The chest cavity was carefully opened, and a 14-gauge perfusion cannula was introduced into the aorta. After drainage was achieved from the right atrium, the animals were perfused with 500 mL of PBS per kg body weight (BW) to remove erythrocytes and nonadherent leukocytes. Perfusion with ConA (40 µg/mL in PBS [pH 7.4], 5 mg/kg BW) was then performed to label adherent leukocytes and vascular endothelial cells, followed by removal of residual unbound lectin with PBS perfusion. The retinas were carefully removed, fixed with 1% paraformaldehyde, and flatmounted in a mounting medium for fluorescence (Vector Laboratories). Each retina was imaged with an epifluorescence microscope (DM RXA; Leica, Deerfield, IL), and the total number of adherent leukocytes per retina was determined.
CD45 Immunofluorescence
Adherent leukocytes were labeled with FITC-coupled ConA, as just described. The flatmounted retinas were permeabilized with 0.5% Triton X (Sigma) in PBS for 24 hours, and nonspecific binding was blocked with 5% normal goat serum. The retinas were then incubated with a mouse anti-rat CD45 antibody (clone OX-1, 1:500; BD Pharmingen, San Diego, CA) overnight at 4°C, followed by incubation with a Texas red–conjugated goat antibody against mouse immunoglobulins (1:200; Jackson ImmunoResearch, West Grove, PA). The flatmounts were prepared with a mounting medium for fluorescence and then imaged with an epifluorescence microscope.
BRB Breakdown Quantification
After deep anesthesia with xylazine hydrochloride and ketamine hydrochloride, rats received intravenous injection of FITC-conjugated dextran (4.4 kDa, 50 mg/mL in PBS, 50 mg/kg BW; Sigma). After 10 minutes, the chest cavity was opened, and a 14-gauge perfusion cannula was introduced into the aorta. A blood sample was collected immediately before perfusion. After drainage was achieved from the right atrium, each rat was perfused with PBS (500 mL/kg BW) to clear the remaining intravascular dextran. The blood sample was centrifuged at 7000 rpm for 20 minutes at 4°C, and the supernatant was diluted at 1:1000. Immediately after perfusion, the retinas were carefully removed, weighed, and homogenized to extract the FITC-dextran in 0.4 mL of water. The extract was processed through a 30,000 molecular weight filter (Ultrafree-MC; Millipore, Bedford, MA) at 7000 rpm for 90 minutes at 4°C. The fluorescence in each 300-µL sample was measured (excitation, 485 nm; emission, 538 nm), using a spectrofluorometer (SpectrAmax Gemini XS; Molecular Devices, Sunnyvale, CA) with water as a blank. Corrections were made by subtracting the autofluorescence of retinal tissue from rats without FITC-dextran injection. The amount of FITC-dextran in each retina was calculated from a standard curve of FITC-dextran in water. For normalization, the retinal FITC-dextran amount was divided by the retinal weight and by the concentration of FITC-dextran in the plasma. BRB breakdown was calculated using the following equation, with the results being expressed in microliters per gram per hour
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The use of FITC-dextran in the quantification of BRB breakdown enabled the simultaneous assessment of retinal leukostasis. Perfusion labeling with rhodamine-ConA was used instead of PBS perfusion and was performed 10 minutes after the injection of FITC-dextran. Before homogenization, each retina was flatmounted, and the number of leukocytes counted under an epifluorescence microscope.
Western Blot Analysis for ICAM-1
Animals were killed with an overdose of anesthesia, and the retinas were immediately isolated. The retinas were subsequently homogenized in lysis buffer and centrifuged at 4°C for 10 minutes. The supernatants were collected and mixed with sample buffer. Each sample, containing 100 µg of total protein, was then boiled for 3 minutes, separated by SDS-PAGE, and electroblotted to a polyvinylidene difluoride (PVDF) membrane (BioRad, Hercules, CA). After nonspecific binding was blocked with 5% normal goat serum, the membranes were incubated with a mouse anti-human ICAM-1 monoclonal antibody (1:200; Santa Cruz Biotechnologies, Santa Cruz, CA) at room temperature for 60 minutes, followed by incubation with a horseradish peroxidase–conjugated goat antibody directed against mouse immunoglobulin (1:20,000; Amersham Pharmacia, Piscataway, NJ). The signals were visualized with an enhanced chemiluminescence kit (ECL Plus; Amersham Pharmacia), according to the manufacturer’s protocol.
Enzyme-Linked Immunosorbent Assay for VEGF
The animals were killed with an overdose of anesthesia, and the eyes were immediately enucleated. The retina-vitreous-lens capsule complex was carefully isolated and placed in 150 µL of lysis buffer (20 mM imidazole HCl, 10 mM KCl, 1 mM MgCl2, 10 mM EGTA, 1% Triton, 10 mM NaF, 1 mM sodium molybdate, 1 mM EDTA [pH 6.8]) supplemented with a protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN) and sonicated. The lysate was centrifuged at 14,000 rpm for 15 minutes at 4°C, and the VEGF levels in the supernatant were determined with the mouse VEGF kit (Quantikine; R&D Systems), according to the manufacturer’s protocol. The assay also recognizes rat VEGF. Total protein was determined using the bicinchoninic acid (BCA) kit (Bio Rad) and was used to normalize the VEGF protein levels.
Statistical Analyses
All results are expressed as the mean ± SD. The data were processed for statistical analyses with the Mann-Whitney test, Spearman rank correlation, Kruskal-Wallis test, and Dunn procedure. Differences were considered statistically significant when P < 0.05. The percentage of inhibition was calculated, with the diabetes-induced increases representing 100%.
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Differential Potency of VEGF120 and VEGF164 in Inducing Retinal ICAM-1 Expression, Retinal Leukostasis, and BRB Breakdown
To examine the differential potency of the two major VEGF isoforms, exogenous murine VEGF120 or VEGF164 was injected into eyes of nondiabetic rats. VEGF ELISA measurements demonstrated that VEGF120- and VEGF164-injected eyes contained 99.4 ± 17.4 pg/mg versus 100.6 ± 15.4 pg/mg total VEGF at 24 hours after injection, 51.3 ± 4.2 pg/mg versus 51.6 ± 5.5 pg/mg at 48 hours, and 22.7 ± 5.4 pg/mg versus 32.3 ± 4.1 pg/mg at 72 hours, respectively (n = 4 each VEGF at each time point). The vitreoretinal VEGF protein levels measured 18.5 ± 1.3 pg/mg (n = 4) in the untreated eyes. The vitreoretinal VEGF protein levels did not differ at the 24- and 48-hour time points between the VEGF120- and VEGF164-injected eyes (P > 0.05, Mann-Whitney test).
As shown in Table 2 , retinal leukostasis and BRB breakdown were quantified in normal rat eyes injected with vehicle, VEGF120, or VEGF164 at 24, 48, and 72 hours after injection, together with untreated eyes. Because both VEGF isoforms showed maximal effect on the retinal parameters at 48 hours, this was the time point chosen to compare the differential potencies of the two isoforms.
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Discussion |
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The ability to measure both leukostasis and leakage in the same retina provided the opportunity to demonstrate that these two pathologic conditions correlated positively. Both also worsened as diabetes progressed. The data from the new combined procedure compare favorably and are in agreement with the data obtained using alternative methods, including scanning laser ophthalmoscopy,34 FITC-ConA perfusion labeling,24 the isotope dilution method,23 and the Evans blue technique.31 Previous studies have used morphometry to assess dextran leakage and BRB breakdown in tissue sections.35 36 The current method extends those capabilities by providing a level of overall sensitivity and quantitation comparable to that of the Evans blue dye leakage assay.22 25 31 32 33
Previous work has shown that VEGF165 is a more potent endothelial cell mitogen than VEGF121.37 38 This is explained in part by the recent evidence that neuropilin-1, a VEGF165 receptor that binds to the exon-7–encoded domain lacking VEGF121, enhances VEGF receptor (R)-2 signal transduction,39 40 which is responsible for VEGF-induced mitogenesis.41 42 VEGF165 is also thought to facilitate angiogenesis in human diseases when it is coexpressed with VEGFR-2 and neuropilin-1.11 43 The present study demonstrates for the first time that VEGF164 is more potent at inducing BRB breakdown than VEGF120. This result may be explained by the recent finding that VEGFR-2, but not VEGFR-1, stimulation is responsible for vascular permeability44 and endothelial cell mitogenicity.41 42
The present study documents that VEGF164 induces retinal leukostasis and BRB breakdown more potently than VEGF120. A previous report showed that VEGF-induced BRB breakdown is mediated, in part, through ICAM-1–dependent retinal leukostasis.18 New in vitro and in vivo data also show that VEGF165 more potently induces endothelial ICAM-1 expression, as well as leukocyte adhesion and migration (Usui T, Ishida S, Yamashiro K, et al., manuscript submitted, 2002). In eyes with early diabetes, the expression of retinal VEGF164 is 11 times higher than VEGF120.22 The current data show that in addition to this differential expression, VEGF164, on an equimolar basis, was approximately two times more potent in inducing leukostasis and BRB breakdown than VEGF120. Thus, VEGF164 is more proinflammatory in the retina than is VEGF120.
The current data also show that an anti-VEGF165 aptamer can suppress retinal leukostasis and BRB breakdown in both early and established diabetes. The efficacy in established diabetes was diminished but still notable. We speculate that this is attributable, in part, to the accumulation of advanced glycation end products (AGEs). A significant increase in retinal AGE levels is seen at 3 months of diabetes and is not present at 2 weeks (Kaji Y, Ishida S, Yamashiro K, et al., unpublished data, 2002). AGEs can directly induce vascular endothelial ICAM-1, both in vivo45 and in vitro.46 This direct stimulation of ICAM-1 gene expression by AGEs probably dilutes the stimulatory effect of VEGF over time.
A soluble VEGFR-1/Fc fusion protein that inhibits all VEGF isoforms has been shown to suppress retinal leukostasis25 and leakage efficiently22 in early diabetes. The degree of inhibition observed with the anti-VEGF165 aptamer in the present study was comparable to the inhibition achieved with the VEGFR-1/Fc fusion protein. The comparability of the results with the two VEGF inhibitors is consistent with our hypothesis that VEGF165, but not VEGF121, plays a major role in diabetic retinal leukostasis and BRB breakdown. As the disease progresses to proliferative diabetic retinopathy, VEGF165-specific signaling is thought to accelerate fibrovascular proliferation.11 Considering that VEGF and its receptors are constitutively expressed in the normal retina,26 the complete blockade of VEGF bioactivity may therefore not be desirable.47 Several reports have recently demonstrated the neuroprotective effects of VEGF both in vivo47 and in vitro.48 49 50 Thus, blocking VEGF165 alone, a more pathogenic isoform in the retina, may be more desirable.
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Footnotes |
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Submitted for publication August 8, 2002; revised November 8, 2002; accepted November 24, 2002.
Disclosure: S. Ishida, Eyetech Pharmaceuticals (F); T. Usui, Eyetech Pharmaceuticals (F); K. Yamashiro, Eyetech Pharmaceuticals (F); Y. Kaji, Eyetech Pharmaceuticals (F); E. Ahmed, Eyetech Pharmaceuticals (F); K.G. Carrasquillo, Eyetech Pharmaceuticals (E); S. Amano, None; T. Hida, None; Y. Oguchi, None; A.P. Adamis, Eyetech Pharmaceuticals (E)
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Anthony P. Adamis, Eyetech Research Center, 42 Cummings Park, Woburn, MA 01801; tony.adamis{at}eyetk.com.
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L. Wang, J. H. Kwak, S. I. Kim, Y. He, and M. E. Choi Transforming Growth Factor-{beta}1 Stimulates Vascular Endothelial Growth Factor 164 via Mitogen-activated Protein Kinase Kinase 3-p38{alpha} and p38{delta} Mitogen-activated Protein Kinase-dependent Pathway in Murine Mesangial Cells J. Biol. Chem., August 6, 2004; 279(32): 33213 - 33219. [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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T. Usui, S. Ishida, K. Yamashiro, Y. Kaji, V. Poulaki, J. Moore, T. Moore, S. Amano, Y. Horikawa, D. Dartt, et al. VEGF164(165) as the Pathological Isoform: Differential Leukocyte and Endothelial Responses through VEGFR1 and VEGFR2 Invest. Ophthalmol. Vis. Sci., February 1, 2004; 45(2): 368 - 374. [Abstract] [Full Text] [PDF]
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