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1 From the Department of Surgical Research, Children’s Hospital, Harvard Medical School, Boston, Massachusetts; 2 Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston; 4 Department of Ophthalmology, Affiliated Hospital of Jining Medical College, Jining, Shandong, P.R. China; 5 Department of Vitreoretinal Surgery, Center for Ophthalmology, University of Cologne, Germany; 6 Pennsylvania State College of Medicine, Hershey, Pennsylvania; 7 Centre de recherche Guy-Bernier, Hopital Maisonneuve-Resemont, Montreal, Canada; 8 Department of Ophthalmology, Universite de Montreal, Canada; and 9 Regeneron Pharmaceuticals, Tarrytown, New York.
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Abstract |
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METHODS. Retinal VEGF mRNA levels were quantified in 1-week diabetic rats using the RNase protection assay. VEGF bioactivity was blocked via the systemic administration of a highly specific VEGF-neutralizing soluble Flt/Fc construct (VEGF TrapA40). An inactive IL6 receptor/Fc construct (IL6R Trap) was used as an isotype control. Blood–retinal barrier breakdown was quantified using the Evans blue technique and was spatially localized with fluorescent microspheres.
RESULTS. Retinal VEGF mRNA levels in 1-week diabetic animals were 3.2-fold higher than in nondiabetic controls (P < 0.0001). Similarly, retinal vascular permeability in 8-day diabetic animals was 1.8-fold higher than in normal nondiabetic controls (P < 0.05). Diabetes-induced blood–retinal barrier breakdown was dose-dependently inhibited with VEGF TrapA40, with 25 mg/kg producing complete inhibition of the diabetes-induced increases (P < 0.05). Blood–retinal barrier breakdown in diabetic animals treated with solvent alone or IL6R Trap did not differ significantly from untreated diabetic animals (P > 0.05). Spatially, early blood–retinal barrier breakdown was localized to the retinal venules and capillaries of the superficial retinal vasculature.
CONCLUSIONS. Early blood–retinal barrier breakdown in experimental diabetes is VEGF dependent and is restricted, in part, to the venules and capillaries of the superficial inner retinal vasculature. VEGF inhibition should prove a useful therapeutic approach in the treatment of early diabetic blood–retinal barrier breakdown.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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VEGF, a potent vasopermeability factor,3 4 may be operative in the pathogenesis of diabetic blood–retinal barrier breakdown. Retinal VEGF levels are upregulated in diabetes and coincide with blood–retinal barrier breakdown in rodents5 6 and humans.7 Flt-1 and Flk-1, the major high-affinity VEGF receptors, are similarly upregulated in the diabetic retina8 and localize to the retinal vasculature.8 9 10 Although VEGF is required for experimental retinal neovascularization,11 its definitive role in diabetic blood–retinal barrier breakdown is unknown.
The vessel phenotype(s) involved in blood–retinal barrier breakdown have not yet been determined. The blood–retinal barrier consists of two spatially distinct monolayers of cells: the retinal pigment epithelium (outer barrier) and the retinal vascular endothelium (inner barrier). Both monolayers possess tight junctions, which are presumably operative in the maintenance of the barrier. Although each monolayer exhibits increased permeability in diabetes, the retinal vasculature is the predominant site of leakage in early experimental diabetes12 and human diabetic retinopathy.13 Of note, VEGF receptors are present in the retinal vasculature but have not been detected in retinal pigment epithelium in vivo.14 At the cellular level, blood–retinal barrier breakdown is associated with endocytic vesicle formation and, to a lesser extent, degenerative endothelial changes.15
In the present study, the direct causal role of VEGF in diabetic blood–retinal barrier breakdown was analyzed. VEGF TrapA40, a high-affinity soluble VEGF receptor/Fc chimera, was administered systemically to diabetic animals to inhibit VEGF bioactivity and to assess its effect on blood–retinal barrier breakdown. In a separate set of experiments, the specific vasculature and vessel phenotype(s) responsible for diabetic blood–retinal barrier breakdown were identified using 0.1-µm red fluorescent microspheres. These microspheres label hyperpermeable blood vessels because they pass through the endothelial cell monolayer but are trapped in the vessel wall by the basal lamina (basement membrane). They do not, however, readily cross the endothelium of intact vessels.16
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents
Evans blue dye (Sigma, St. Louis, MO) was dissolved in normal
saline (30 mg/ml), sonicated for 5 minutes in an ultrasonic cleaner
(G1125P1T; Laboratory Supplies, Hicksville, NY), filtered through a
5-µm filter (Millipore, Bedford, MA), and stored at 4°C.
VEGF TrapA40 and IL6R Trap were synthesized by Regeneron Pharmaceuticals Inc. (Tarrytown, NY). VEGF TrapA40 was made from immunoglobulin repeats 1 to 3 of the extracellular domain of human Flt-1 fused to the Fc portion of human IgG. The protein was expressed in CHO cells and purified via protein A affinity chromatography, followed by size exclusion chromatography. The recombinant VEGF TrapA40 was then chemically modified to improve the pharmacokinetic profile of the parent molecule, without affecting its ability to bind VEGF with high affinity (Rudge J, Wiegand S, Yancopoulos GD, personal communication, 2000). The purity of the modified recombinant protein was determined to be >95% by Coomassie-stained SDS PAGE. The protein was filter sterilized and stored at 3.25 mg/ml in PBS, pH 7.2, containing 5% glycerol at -20°C.
IL6R Trap was similarly generated, using the extracellular domain of human IL6R alpha (the low-affinity IL6 receptor) fused to the Fc domain of human IgG1. IL6R Trap only binds human IL6 with low affinity and does not bind to rat IL6. Like VEGF TrapA40, it was CHO cell derived, purified over protein A, and was >95% pure on Coomassie-stained gels. It was filter sterilized and stored at 3.25 mg/ml in PBS, pH 7.2, containing 5% glycerol at -20°C.
Experimental Diabetes
After a 24-hour fast, diabetes was induced with a single 60
mg/kg intraperitoneal injection of streptozotocin (Sigma) in 10 mM
citrate buffer, pH 4.5. Animals that served as nondiabetic controls
received an equivalent amount of citrate buffer alone. Twenty-four
hours later, rats with blood glucose levels >250 mg/dl were
deemed diabetic. Streptozotocin-injected rats with blood glucose
levels < 120 mg/dl were deemed "streptozotocin nondiabetic
controls." Just before experimentation, blood glucose levels
were measured again to confirm diabetic status.
VEGF mRNA Protection Assay
Three normal nondiabetic and three 1-week diabetic rats were
anesthetized with ketamine (80 mg/kg, Ketalar; Parke-Davis, Morris
Plains, NJ) and xylazine (4 mg/kg, Rompun; Harver-Lockhart, Morris
Plains, NJ). Both eyes were enucleated and bisected at the equator
before they were killed. The retinas were then carefully dissected away
and immediately frozen in liquid nitrogen until later use, at which
time the tissue RNA was extracted.
Previously described templates for the transcription of rat VEGF and 18S riboprobes using RT-PCR were used.17 The RNase protection assay was performed as previously described.17 Briefly, a riboprobe for 18S rRNA (Ambion, Austin TX) was included in each sample to control for variable loading and recovery of tissue RNA. Fifteen micrograms of total cellular RNA from both nondiabetic and 1-week diabetic rats was hybridized with 32P-labeled antisense VEGF and 18S rRNA riboprobes (200,000 cpm/probe) overnight at 42°C in 30 µl hybridization buffer. The riboprobe identified three major VEGF isoforms: VEGF120, VEGF164, and VEGF188.17 Hybridized RNA was digested with nuclease A (20 µg/ml; Ambion, Austin, TX) and RNase T1 (2 µg/ml; Ambion) for 1 hour at 25°C in 300 µl digestion buffer. The digestions were terminated via the addition of 20 µl of 10% SDS and 50 µg proteinase K (Ambion) for 15 minutes at 37°C. After acid guanidinium thiocyanate-phenol-chloroform extraction and ethanol precipitation, the protected fragments were resolved on 6% polyacrylamide, 7 M urea gels (Ambion) and visualized with autoradiography. Densitometry was performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Administration of VEGF TrapA40
Ten 1-week diabetic rats received either 10 mg/kg
(n = 5 animals) or 25 mg/kg (n = 5
animals) VEGF TrapA40 via tail vein injection. Ten 1-week diabetic
controls received 25 mg/kg IL6R Trap (n = 5 animals) or
an equivalent volume of solvent alone (PBS + 5% glycerol,
n = 5 animals). Additional controls consisted of
untreated diabetic (n = 3 animals), normal nondiabetic
(n = 5 animals), and streptozotocin-treated nondiabetic
animals (n = 5 animals).
Blood–Retinal Barrier Quantitation
Twenty-four hours after treatment, on day 8 of diabetes, the
animals were anesthetized and the right jugular vein and right iliac
artery were cannulated with 0.28- and 0.58-mm internal diameter
polyethylene tubing (Becton Dickinson, Sparks, MD), respectively, and
filled with heparinized saline (400 units heparin/ml saline). Evans
blue was injected through the jugular vein over 10 seconds at a dosage
of 45 mg/kg. Immediately after Evans blue infusion, the rats turned
visibly blue, confirming their uptake and distribution of the dye.
Subsequently, at 15-minute intervals, 0.1 ml blood was drawn from the
iliac artery for 2 hours to obtain the time-averaged plasma Evans blue
concentration, as described previously.18
After the dye
had circulated for 120 minutes, the chest cavity was opened, and rats
were perfused for 2 minutes via the left ventricle at 37°C with 0.05
M, pH 3.5, citrate-buffered paraformaldehyde (1% wt/vol; Sigma). The
perfusion was at a physiological pressure of 120 mm Hg. A pH of 3.5 was
used to optimize binding of Evans blue to albumin,19
20
and the perfusion solution was warmed to 37°C to prevent
vasoconstriction. Immediately after perfusion, both eyes were
enucleated and bisected at the equator. The retinas were carefully
dissected away under an operating microscope and thoroughly dried in a
Speed-Vac (Savant, St. Paul, MN) for 5 hours. The dry weight was used
to normalize the quantitation of Evans blue leakage. Evans blue was
extracted by incubating each retina in 120 µl formamide (Sigma) for
18 hours at 70°C. The supernatant was filtered through Ultrafree-MC
tubes (30,000 NMWL UFC3LTK00; Millipore, Bedford, MA) at 3000 rpm for 2
hours, and 60 µl of the filtrate was used for triplicate
spectrophotometric measurements (Du-640; Beckman, Fullerton, CA). Each
measurement occurred over a 5-second interval, and all sets of
measurements were preceded by known standards. The
background-subtracted absorbance was determined by measuring each
sample at both 620 nm, the absorbance maximum for Evans blue in
formamide, and 740 nm, the absorbance minimum. The concentration of dye
in the extracts was calculated from a standard curve of Evans blue in
formamide. Blood–retinal barrier breakdown was calculated using the
following equation, with results being expressed in µl plasma·g
retina dry
weight-1·h-1.
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Spatial Localization of Diabetic Blood–Retinal Barrier Breakdown
Using Fluorescent Microspheres
One-week diabetic rats and age-matched controls were
anesthetized as described above and received a 0.2 ml tail vein
injection of 0.1-µm red fluorescent microspheres (Duke Scientific,
Palo Alto, CA), diluted fourfold in normal saline. Twenty minutes
later, the chest cavity was opened under deep anesthesia with 50 mg/kg
sodium pentobarbital. Lectin counterstaining of the vasculature was
performed as described previously.21
Briefly, fixation was
achieved via perfusion with 1% paraformaldehyde and 0.5%
glutaraldehyde (Sigma) followed by perfusion with 5 mg/kg FITC-coupled
Con A lectin (20 µg/ml in PBS, pH 7.4; Vector Laboratories, Irvine,
CA). The lectin perfusion was followed by perfusion with PBS for 4
minutes. The retinas were then carefully dissected free and
flat-mounted in a water-based fluorescence antifading medium (Southern
Biotechnology, Birmingham, AL). The tissues were imaged with
fluorescence microscopy (Zeiss Axiovert FITC filter; Zeiss, Wetzlar,
Germany). Localization of leakage was performed by focusing up and down
within the retinal flat mounts.
Statistics
Normally distributed data in two groups were analyzed with a
Student’s t-test. For data that were not normally
distributed, a Wilcoxon rank-sum test was used if there were two
groups, and a Kruskal–Wallis test was used if there were three or more
groups. All multiple pairwise comparisons used Duncan’s multiple range
test. Differences were considered statistically significant if
P < 0.05. All numerical results are expressed as
means ± SE.
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Results |
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Increased VEGF mRNA Levels in 1-Week Diabetic Retina
VEGF mRNA levels were 3.2-fold higher in 1-week diabetic rats than
in normal nondiabetic rats (Fig. 1)
. The normalized VEGF mRNA levels (means ± SE) in nondiabetic
animals and 1-week diabetic animals were 10.1 ± 0.33optical
density (OD) units (n = 5 retinas) and 32.2 ±
1.24 OD units (n = 6 retinas) , respectively
(P < 0.0001, Student’s t-test).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The data indicate that VEGF is causal for early blood–retinal barrier breakdown in diabetes. These results must be reconciled with a recent report showing an 86% reduction in early diabetic blood–retinal barrier breakdown after ICAM-1 inhibition.22 The VEGF and ICAM-1 pathways are likely mechanistically linked. Exogenous VEGF stimulates ICAM-1 expression in the retinal vasculature,23 24 and VEGF recently was demonstrated to be an important endogenous inducer of ICAM-1 expression in the diabetic retina (Joussen AM, Poulaki V, Qin W, et al., personal communication, 2000). Moreover, ICAM-1 mediates leukocyte adhesion to the diabetic retinal vasculature,22 a process that initiates blood–retinal barrier breakdown. The mechanisms that underlie leukocyte-mediated vascular permeability are still not known. However, retinal endothelial cell injury and death may play a role, even after only 1 week of hyperglycemia.21 As noted above, diabetic blood–retinal barrier breakdown coincides with degenerative endothelial cell changes.13 If endothelial cell death is specifically prevented with an anti-FasL antibody, diabetic blood–retinal barrier dysfunction is also inhibited (Joussen AM, Poulaki V, Qin W, et al., personal communication, 2000). Leukocytes also have the ability to trigger the disorganization of endothelial cell-to-cell adherens25 and tight junctions26 in nonocular systems. Moreover, via their own VEGF, leukocytes may serve to amplify the direct permeability effects of VEGF. VEGF has been identified in neutrophils,27 monocytes,28 eosinophils,29 lymphocytes30 and platelets.31
Fluorescent microspheres were used in these studies to localize leakage in the diabetic retina. These microspheres do not readily cross the endothelium of intact vessels but do label hyperpermeable blood vessels because they pass through the endothelial cell basal lamina but are trapped in the vessel wall by the basal lamina. In early diabetes, the venules and capillaries of the superficial inner retinal vasculature were demonstrated to be the principal sites of blood–retinal barrier breakdown. We speculate that the venular side of the vasculature is preferentially affected because the smooth muscle layer of the arteriolar circulation impedes the diffusion of VEGF made in the retinal parenchyma. Although McDonald and colleagues16 32 localized leakage in the tracheal mucosa after only 1 to 3 minutes of microsphere circulation, the present study required 20 minutes for optimal localization of retinal vascular leakage. The prolonged circulation time may be partially explained by the fact that unlike tracheal mucosa, the blood–retinal barrier has tight junctions, although they are breached in diabetes.33 The breached diabetic blood–retinal barrier is still less permeable than leaky vessels elsewhere in the body.18
Although fluorescent microspheres have no resemblance to any biological macromolecules, their usage was chosen over immunostaining for leakage of endogenous plasma proteins such as albumin, because unlike endogenous plasma proteins, particulate intravascular tracers, such as fluorescent microspheres, are trapped in the vessel wall and thus accumulate with the passage of time. This property has enabled fluorescent microspheres to be used successfully in the localization of sites of leakage in other systems.16 32 Admittedly, however, because the microspheres are larger than caveolae, they would be incapable of marking vascular leakage mediated by the caveolae membrane system.
Blood–retinal barrier breakdown in newly diabetic animals was also shown to be a consequence of diabetes itself and not an acute toxic effect of the diabetogenic drug streptozotocin. Retinal Evans blue content in normal nondiabetic animals was compared with that of STZ-treated nondiabetic animals, that is, rats that had received STZ but remained nondiabetic, and the results were no different from normal nondiabetic animals. These data discount the notion of an acute toxic effect of STZ on retinal vascular permeability.
In conclusion, these experiments demonstrate that the inhibition of VEGF suppresses blood–retinal barrier breakdown in the superficial venules and capillaries of the inner retina, the principal site of vascular permeability in early diabetes. Taken together, the data identify a new molecular target for the treatment of early diabetic blood–retinal barrier breakdown.
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Acknowledgements |
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Footnotes |
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Supported by the Roberta W. Siegel Fund (APA), Medical Research Council of Canada (HH), Heart and Stroke Foundation of Canada (HH), E. A. Baker Foundation (HH), National Institutes of Health Grants EY12611 and EY11627 (APA), the Juvenile Diabetes Foundation (APA, AMJ), the Falk Foundation (APA), and the Iacocca Foundation (APA), and the Leopold Schepp Foundation for fellowship support (TQ). HH is a scholar of the fonds de la Recherche en Sante du Quebec.
Submitted for publication March 8, 2001; revised May 11, 2001; accepted May 31, 2001.
Commercial relationships policy: E (SJW, JR, GDY); F,C (APA); N (all others).
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, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114. tony_adamis{at}meei.harvard.edu
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