Vascular Endothelial Growth Factor in Ocular Fluid of Patients with Diabetic Retinopathy and Other Retinal Disorders
Lloyd Paul Aiello, Robert L. Avery, Paul G. Arrigg, Bruce A. Keyt, Henry D. Jampel, Sabera T. Shah, Louis R. Pasquale, Hagen Thieme, Mami A. Iwamoto, John E. Park, Hung V. Nguyen, Lloyd M. Aiello, Napoleone Ferrara, and George L. King
Background Retinal ischemia induces intraocular neovascularization,which often leads to glaucoma, vitreous hemorrhage, and retinaldetachment, presumably by stimulating the release of angiogenicmolecules. Vascular endothelial growth factor (VEGF) is an endothelial-cell-specificangiogenic factor whose production is increased by hypoxia.
Methods We measured the concentration of VEGF in 210 specimensof ocular fluid obtained from 164 patients undergoing intraocularsurgery, using both radioimmunoassays and radioreceptor assays.Vitreous proliferative potential was measured with in vitroassays of the growth of retinal endothelial cells and with VEGF-neutralizingantibody.
Results VEGF was detected in 69 of 136 ocular-fluid samplesfrom patients with diabetic retinopathy, 29 of 38 samples frompatients with neovascularization of the iris, and 3 of 4 samplesfrom patients with ischemic occlusion of the central retinalvein, as compared with 2 of 31 samples from patients with noneovascular disorders (P<0.001, P<0.001, and P = 0.006,respectively). The mean (±SD) VEGF concentration in 70samples of ocular fluid from patients with active proliferativediabetic retinopathy (3.6 ±6.3 ng per milliliter) washigher than that in 25 samples from patients with nonproliferativediabetic retinopathy (0.1 ±0.1 ng per milliliter, P =0.008), 41 samples from patients with quiescent proliferativediabetic retinopathy (0.2 ±0.6 ng per milliliter, P<0.001),or 31 samples from nondiabetic patients (0.1 ±0.2 ngper milliliter, P = 0.003). Concentrations of VEGF in vitreousfluid (8.8 ±9.9 ng per milliliter) were higher than thosein aqueous fluid (5.6 ±8.6 ng per milliliter, P = 0.033)in all 10 pairs of samples obtained simultaneously from thesame patient; VEGF concentrations in vitreous fluid declinedafter successful laser photocoagulation. VEGF stimulated thegrowth of retinal endothelial cells in vitro, as did vitreousfluid containing measurable VEGF. Stimulation was inhibitedby VEGF-neutralizing antibodies.
Conclusions Our data suggest that VEGF plays a major part inmediating active intraocular neovascularization in patientswith ischemic retinal diseases, such as diabetic retinopathyand retinal-vein occlusion.
Intraocular neovascularization occurs in numerous ischemic retinaldisorders, including diabetic retinopathy, ischemic retinal-veinocclusion, and retinopathy of prematurity1,2. This proliferationoften results in vitreous hemorrhage, retinal detachment, andneovascular glaucoma, with subsequent visual loss. Althoughnumerous growth factors stimulate angiogenesis in vivo,3,4,5,6these factors are not consistently increased in proliferativeretinopathies5,6 or secreted, as would be expected if they causedintraocular neovascularization at sites distant from those ofretinal ischemia.
In neovascular retinopathies such as proliferative diabeticretinopathy, there is initially extensive active proliferationof new vessels1,7. Visual loss at this time results from vitreoushemorrhage or fluid exudation from fragile new vessels. Withtime, or after laser photocoagulation, the proliferating vesselsbecome fibrotic and involute, causing traction on the retinathat may lead to complications such as retinal detachment8,9.Eventually the disease becomes inactive and further visual lossceases10. The timely inhibition of factors that cause activevascular proliferation may prevent visual loss.
To account for the clinical manifestations of neovascular diseaseinduced by ocular ischemia, it has been hypothesized that acausative substance is produced in the retina. The hypotheticalsubstance is induced by retinal ischemia and is angiogenic,diffusible, more concentrated in vitreous than in aqueous humor,increased during active proliferation, reduced during quiescentproliferation, and diminished after successful laser therapy.Vascular endothelial growth factor (VEGF) is a candidate forsuch a substance. It is an endothelial-cell-specific angiogenic11and vasopermeable12 factor that binds to high-affinity, membrane-boundreceptors with tyrosine kinase activity13,14. Its expressionis induced by hypoxia in tumors12,15,16,17. VEGF is secretedin four homodimeric forms resulting from alternative RNA splicing18.In ocular tissues its production is increased by hypoxia inretinal pigment epithelial cells19 and retinal pericytes,20,21and by ischemia-induced neovascularization of the iris in primateeyes22. In our study we attempted to ascertain whether intraocularconcentrations of VEGF correlated with active neovascularizationand to determine whether VEGF fulfilled the other criteria23hypothesized for an angiogenic factor in ischemic ocular disorders.
Methods
Study Subjects
Undiluted samples of aqueous, vitreous, and subretinal fluidwere obtained from patients undergoing intraocular surgery.The protocol for sample collection was approved by the institutionalreview board at each institution, and all patients gave informedconsent. The surgical procedures included cataract extraction,trabeculectomy, anterior-chamber paracentesis, pars plana vitrectomy,scleral buckling, and pneumatic retinopexy. The patients' primarydiagnoses included cataract, chronic open-angle glaucoma, traumaticglaucoma, phacolytic glaucoma, proliferative diabetic retinopathy,neovascular glaucoma, stage 5 retinopathy of prematurity, ischemiccentral-retinal-vein occlusion, vitreous hemorrhage, tractionor rhegmatogenous retinal detachment, macular pucker, endophthalmitis,leukemic retinopathy, epiretinal membrane, choroidal neovascularmembrane, uveitis, and retinitis.
Clinical data, including determinations of ocular neovascularactivity, were obtained from the surgeon at the time of surgerywith standardized forms and were confirmed by standardized fundusphotography whenever possible. Neovascularization was consideredto be active if there were perfused, multibranching iridic orpreretinal capillaries (Figure 1A) and to be quiescent if previouslydocumented active proliferation had regressed fully or if onlynonperfused, gliotic vessels or fibrosis was present (Figure 1B).
Figure 1. Classification of Active and Quiescent Intraocular Neovascularization.
Panel A shows typical active neovascularization as defined in the Methods section (vitreous VEGF concentration, 1.9 ng per milliliter). There is extensive retinal neovascularization (black arrows) arising from the optic-nerve head, vitreous hemorrhage (white arrow), and scarring from panretinal laser photocoagulation (arrowhead). Panel B shows quiescent neovascularization with no detectable vitreous VEGF concentration. There are numerous nonperfused, gliotic vessel remnants (arrows).
The results in patients with retinal neovascularization werecompared with those in patients with the same underlying disordersbut no retinal neovascularization and with those in patientswith no known underlying potentially neovascularizing disorder.We therefore excluded from this group patients with diabetesmellitus, rubeosis iridis, retinal detachment, choroidal neovascularmembranes, retinopathy of prematurity, central-retinal-veinocclusion, uveitis, and retinitis. The indications for surgeryin the control group included cataract, chronic open-angle glaucoma,angle-recession glaucoma, and idiopathic epiretinal membrane.
Sample Collection
The samples of ocular fluid were collected in sterile tubes(Thomas Scientific, Swedesboro, N.J.), placed immediately onice, clarified by centrifugation at 16,000 x g for five minutesat 4 °C, and rapidly frozen at -80 °C. The samples wereshipped by overnight mail in dry ice with accompanying dataon the patient to the Joslin Diabetes Center, where they werecatalogued and labeled in an anonymous fashion. The specimenswere then shipped without identifying clinical information toGenentech for the VEGF immunologic and receptor-binding assays.
Radioimmunoassay
Concentrations of VEGF in ocular fluid were measured by radioimmunometricassay with two monoclonal antibodies that bound to differentepitopes on VEGF165 and longer isoforms24. The assays were performedin 96-well immunoplates (Immunlon-1, Dynatech, Chantilly, Va.);each well was coated with 100 microl of a solution containing10 µg of anti-VEGF monoclonal antibody 5F8 (similar toantibody B2.6.224) per milliliter of solution in 50-mM carbonatebuffer (pH 9.6) for 16 hours at 4 °C. The plates were washedthree times, blocked for one hour at 25 °C with 300 microlof 0.5 percent bovine serum albumin and 0.03 percent Tween 80in phosphate-buffered saline (pH 7.4) per well, and washed subsequentlywith 0.03 percent Tween 80 in phosphate-buffered saline (pH7.4) before the addition of diluted samples of ocular fluid(in duplicate measurements of three to six serial twofold dilutions)or standard solutions of VEGF165 (range, 20 pg per milliliterto 50 µg per milliliter).
The plates were incubated for two hours at 25 °C with gentleagitation, the supernatants were discarded, and the wells washed.One hundred microls of a solution containing 2 x 104 cpm of125I-labeled anti-VEGF165 monoclonal antibody 2E3 (similar toantibody A4.6.124) was added, and the plates were incubatedfor two hours at 25 °C with gentle agitation. The supernatantswere discarded, the plates washed, and the wells counted bygamma scintigraphy (LKB gamma counter, Gaithersburg, Md.). Concentrationsof VEGF were quantitated from a standard curve for VEGF witha four-parameter fit (Kaleidagraph, Synergy Software, Reading,Pa.). The sensitivity of the assay was 0.05 ng per milliliter.The intraassay and interassay coefficients of variation were5 and 6 percent, respectively. Ocular-fluid samples with undetectableVEGF concentrations in this assay or the receptor-binding assay(described below) were assigned values of 0.05 ng per milliliter.
Receptor-Binding Assay
The procedure for the receptor-binding assay was similar tothat used in the radioimmunoassay, except that 125I-labeledfms-like tyrosine kinase (Flt-1) receptor-IgG chimeric proteinwas used instead of 125I-labeled anti-VEGF monoclonal antibody2E3. The 5F8 monoclonal anti-VEGF antibody used to coat theplates does not interfere with ligand-receptor binding. Flt-1is a high-affinity VEGF receptor25. Its entire extracellulardomain (758 residues) was cloned by the polymerase chain reactionwith Pfu polymerase and fused to the coding sequence for aminoacids 216 through 443 of an IgG1 heavy-chain clone,26 as describedby Park et al27. The chimeric receptor has the same affinityfor VEGF as does the full-length VEGF receptor. The sensitivityand variability of this assay were similar to those of the radioimmunoassay.
Assay of Retinal Vascular Endothelial-Cell Growth
Bovine endothelial cells from the retinal microvasculature wereisolated by homogenization and a series of filtration stepsas described elsewhere28. Primary cultures were grown in fibronectin-coateddishes (NYBen Reagents, New York Blood Center, New York) containingDulbecco's modified Eagle's medium with 5.5 mM glucose, 10 percenthorse serum (Wheaton, Pipersville, Pa.), 50 mg of heparin perliter, and 50 units of endothelial-cell growth factor per liter(Boehringer Mannheim, Indianapolis). After the cultures wereconfluent, the medium was changed to include 5 percent fetal-calfserum (Hyclone, South, Utah). The cells were fed every threedays. Endothelial-cell homogeneity was confirmed with anti-factorVIII antibodies. For the assay, the cells were plated sparsely(2500 cells per well) in 24-well dishes (Costar, Cambridge,Mass.) and incubated overnight in Dulbecco's modified Eagle'smedium containing 10 percent calf serum (GIBCO, Grand Island,N.Y.). The mediums were changed the next day, and either VEGFor vitreous fluid (final volume, 15 to 25 percent) was added,with either phosphate-buffered saline or a 1- to 10-fold excessof VEGF-neutralizing rabbit polyclonal antibody. After incubationat 37 °C for four days, the cells were lysed in 0.1 percentsodium dodecyl sulfate, and the DNA content was measured withdye (Hoechst 33258, Hoefer Scientific Instruments, San Francisco)and a fluorometer (model TKO-100, Hoefer). A statistically significantgrowth response could be detected at concentrations of VEGFas low as 0.1 ng per milliliter. The plates were assayed intriplicate, and each experiment was repeated at least threetimes unless otherwise indicated.
Statistical Analysis
Categorical data were compared by Fisher's exact test. The unpairedStudent's t-test was used to compare quantitative data withnormal distributions. Log-normal transformed data were analyzedby the paired t-test for comparisons with unequal variance beforeand after laser treatment. Immunoreactivity and receptor-bindingcorrelations were determined with linear regression analysis(sum of squares), and significance was calculated from the mean-squareF ratio. Ocular-gradient results were analyzed with the pairedStudent's t-test. Values are reported as means ±SD. Two-tailedtest results were considered statistically significant at P 0.05.
Results
We obtained 210 samples of ocular fluid from 171 eyes of 164patients (88 women and 76 men) undergoing intraocular surgery.Sixty-five percent of the patients were white, 15 percent wereblack, 5 percent were Hispanic, and 15 percent were of otheror undetermined descent. The mean age of the 31 patients withno known neovascular disorders was 64 years (range, 10 to 89),and the mean age of the 113 patients with proliferative diseaseswas 57 years (range, 5 months to 89 years). These groups didnot differ with regard to sex or ethnic background. The meanages of the patients with nonproliferative and proliferativediabetic retinopathy were 69 and 53 years, respectively, butthere was no difference in mean age among the patients withactive proliferative diabetic retinopathy, quiescent proliferativediabetic retinopathy, or neovascularization of the iris (51,55, and 51 years, respectively). The patients with no neovasculardisorders and those with nonproliferative diabetic retinopathywere slightly older than those with proliferative diabetic retinopathyor neovascularization of the iris, probably because the firsttwo of these groups primarily undergo surgery for conditions(e.g., cataract) associated with advancing age. Only two patientswith neovascularization had not received retinal laser photocoagulationbefore the collection of ocular fluids. There was no correlationof VEGF concentrations with age, sex, or ethnic background inany subgroup.
Detection of VEGF in Ocular Fluid
The number and percentage of samples of aqueous and vitreousfluid with detectable concentrations of immunoreactive VEGFare shown in Table 1; results for subretinal-fluid samples arepresented in a footnote to Table 1. VEGF was detected in 2 of31 samples from patients with no neovascular disorder, 69 of136 samples from patients with diabetes mellitus, 29 of 38 samplesfrom patients with neovascularization of the iris, 5 of 9 samplesfrom patients with chronic retinal detachment, and 3 of 4 samplesfrom patients with central-retinal-vein occlusion. Among patientswith diabetes mellitus, VEGF was detectable in 58 of 70 samplesfrom patients with active proliferative retinopathy, as comparedwith 9 of 41 samples from patients with quiescent proliferativeretinopathy and 2 of 25 samples from patients with nonproliferativediabetic retinopathy. VEGF was detected in similar proportionsin both aqueous and vitreous fluids among the various groupsof patients. It was not detected in any sample collected frompatients with endophthalmitis, leukemic retinopathy, uveitis,retinitis, epiretinal membrane, or choroidal neovascular membrane.VEGF was detected in 47 percent of 68 patients with retinaldetachment, although 39 had concurrent proliferative diabeticretinopathy. When patients with diabetes mellitus were excluded,VEGF was detected in 5 of 9 samples from patients with chronicretinal detachment (i.e., lasting more than three weeks), ascompared with 1 of 31 samples from patients without neovasculardisorders (P = 0.003). Mean concentrations of VEGF were higherin samples from patients with chronic retinal detachment (2.8±4.7 ng per milliliter) than patients with acute retinaldetachment (0.1 ±0.1 ng per milliliter, P = 0.024).
Table 1. Concentrations of Immunoreactive VEGF in Ocular Fluids of Patients with Retinal Disorders.
VEGF Concentrations and Ocular Neovascularization
The VEGF concentrations in samples of aqueous and vitreous humorfrom patients without neovascular disorders were compared withthose in samples from patients with active or quiescent neovascularization(Figure 2). VEGF was detectable in only 13 of 96 samples frompatients with no active neovascularization (mean, 0.1 ±0.4ng per milliliter), and when detectable, the concentrationswere low (0.7 ±0.9 ng per milliliter). The mean VEGFconcentration in 70 samples from patients with active proliferativediabetic retinopathy (3.6 ±6.3 ng per milliliter) wassignificantly higher than that in 31 samples from nondiabeticpatients with no proliferative disorder (0.1 ±0.2 ngper milliliter, P = 0.003) and that in 41 samples from patientswith quiescent proliferative diabetic retinopathy (0.2 ±0.6ng per milliliter, P<0.001). The concentrations of VEGF werealso high in samples from patients with active proliferationfrom ischemic central-retinal-vein occlusion, retinopathy ofprematurity, or rubeosis iridis. The concentrations were similarwhen samples of aqueous and vitreous humor were considered individually(Table 2).
Figure 2. Concentrations of Immunoreactive VEGF in Ocular Fluids from Patients Undergoing Intraocular Surgery.
Aqueous (squares), vitreous (diamonds), and mean (arrowheads) VEGF concentrations are shown. Values of zero or below on the y axis denote concentrations below 0.05 ng per milliliter. PDR denotes proliferative diabetic retinopathy, and CRVO central-retinal-vein occlusion.
Table 2. Concentrations of Immunoreactive VEGF in Aqueous and Vitreous Fluids.
Determination of Intraocular Gradient of VEGF
Simultaneously collected samples of fluid from the anteriorsegment (aqueous humor) and the posterior segment (either vitreoushumor or subretinal fluid) were tested for VEGF in 14 patients,10 of whom had active ocular neovascularization and detectableVEGF in the posterior segment. In each of these patients, theposterior-segment VEGF concentration exceeded the aqueous concentration.The mean posterior-segment VEGF concentration was almost 60percent higher (8.8 ±9.9 vs. 5.6 ±8.6 ng per milliliter,P = 0.033).
Response of VEGF after Treatment of Ocular Neovascularization
Collecting ocular fluids before and after treatment for neovascularizationis difficult, because patients who undergo surgery rarely requiresubsequent procedures. We studied six patients with active neovascularizationand one patient with quiescent neovascularization, from eachof whom two samples of ocular fluid were collected. The subsequentprocedures involved were trabeculectomy (one patient), secondvitrectomy (five patients), and anterior-chamber paracentesis(one patient). All the patients had also undergone panretinallaser photocoagulation for neovascularization in the intervalbetween the two operations, and the extent of proliferationhad decreased. The intraocular concentrations of VEGF were reducedby an average of 75 percent (from 5.6 ±6.5 to 1.4 ±2.0ng per milliliter, P = 0.008) in all the patients after successfultreatment.
Receptor-Binding Capacity of Ocular VEGF
Among the 19 vitreous-fluid samples randomly tested, all 16samples with detectable immunoreactive VEGF also had receptor-bindingactivity, as compared with none of the 3 samples without detectableimmunoreactive VEGF. The receptor-binding concentration was45 percent of the immunoreactivity concentration, and the correlationbetween the two types of activity was statistically significant(R2 = 0.93, P<0.001).
Stimulation of Retinal Vascular Endothelial-Cell Growth by Ocular VEGF
The number of retinal endothelial cells was increased in vitroin a dose-dependent manner after exposure to 5 or 10 ng of VEGFper milliliter. VEGFneutralizing antibodies blocked this stimulatoryeffect (Figure 3). The maximally effective VEGF concentrationwas 25 ng per milliliter, and the half-maximally effective concentrationwas 1 ng per milliliter (data not shown).
Figure 3. Growth of Retinal Endothelial Cells in Response to VEGF.
Recombinant human VEGF was added to triplicate cultures of bovine retinal endothelial cells in the presence of a fourfold excess of VEGF-neutralizing antibodies, as shown. Growth stimulation was assessed after four days by measuring the total cellular DNA content. Plus and minus signs indicate that antibody was used and not used, respectively. Values are expressed as means ±SD. NS denotes not significant.
To determine whether VEGF in human vitreous was bioactive, theendothelial proliferative activity of pooled samples of vitreoushumor from five patients with active proliferative diabeticretinopathy, a single patient with active proliferative diabeticretinopathy, and pooled samples from six patients with quiescentproliferative diabetic retinopathy was measured. The concentrationsof VEGF in these samples were 4.0, 3.3, and <0.05 ng permilliliter, respectively. All the samples stimulated retinalendothelial-cell growth (Figure 4). The addition of VEGF-neutralizingantibody did not significantly reduce the response to vitreousfluid from patients with quiescent retinopathy, but it inhibitedby 65 percent the response to vitreous fluid from patients withactive proliferative diabetic retinopathy.
Figure 4. Growth of Retinal Endothelial Cells in Response to VEGF in Vitreous Fluid.
Control samples of phosphate-buffered saline (bars 1 and 2) or vitreous-fluid samples from patients with quiescent (bars 3 and 4) or active (bars 5, 6, 7, and 8) neovascularization were added to cultures of retinal endothelial cells (final volume, 25 percent). The samples used in bars 3 and 4 were pooled from six patients, those used in bars 7 and 8 were pooled from five patients, and that used in bars 5 and 6 was from a single patient. VEGF-neutralizing antibody was added to the cultures shown in the even-numbered bars, and phosphate-buffered saline to the remaining cultures. The maximal VEGF-neutralizing capacity of the antibody was either 5.0 ng per milliliter (bars 2, 4, and 8) or 50 ng per milliliter (bar 6). Growth stimulation was assessed after four days by measuring the total cellular DNA content. The results are expressed as mean (±SD) percentages of the control value (bar 1) for triplicate wells obtained in either multiple experiments (bars 1, 2, 3, 4, 7, and 8) or a single experiment (bars 5 and 6). NS denotes not significant.
Discussion
We found detectable concentrations of VEGF in ocular fluid frompatients with active retinal and anterior-segment neovascularizationassociated with several ocular diseases with underlying retinalischemia. In the patients with active neovascularization, VEGFconcentrations in the aqueous and vitreous fluid were higherthan those that have stimulated endothelial-cell proliferationin vitro and in vivo22. In patients with quiescent neovascularizationresulting from retinopathy of prematurity, central-retinal-veinocclusion, or diabetes mellitus, concentrations of VEGF werelow or undetectable.
The clinical observation that anterior-segment neovascularizationcommonly arises in conjunction with ischemic retinal diseasesuggests a gradient-driven diffusion of angiogenic factors fromthe posterior to the anterior segment of the eye. Among allthe samples, concentrations of VEGF were higher in aqueous thanin vitreous fluid, but in simultaneously collected samples theconcentrations of VEGF in vitreous fluid were always higher.This gradient may result from the rapid clearance of VEGF fromthe anterior chamber or from its increased use or more rapiddegradation in that chamber. Nevertheless, a vitreous-to-aqueousgradient would promote the anterior diffusion of VEGF, potentiallyaccounting for the clinically observed anterior-segment neovascularizationassociated with retinal ischemia.
Panretinal photocoagulation induces the regression of clinicallyactive ocular neovascularization8,9. We found that concentrationsof VEGF declined in all patients who had decreased neovascularactivity after laser photocoagulation. Presumably, the reductionin retinal ischemia after laser therapy reduces the productionof angiogenic factors, suppressing neovascularization. Indeed,ischemia-induced VEGF production is reversible by the reinstitutionof a normal oxygen supply in vitro21,29.
Vitreous fluid from patients without active retinal neovascularizationstimulated the growth of retinal endothelial cells, as haveretinal extracts from normal eyes30. In samples of vitreousfrom patients with active proliferative diabetic retinopathyand high concentrations of VEGF, anti-VEGF antibodies reducedin vitro stimulation of growth by more than 65 percent, to valuessimilar to those in samples from patients with quiescent neovascularizationand undetectable concentrations of VEGF. The incomplete inhibitionof growth-stimulatory activity suggests the simultaneous activityof other proliferative factors. Indeed, vitreous concentrationsof basic fibroblast growth factor,31 insulin-like growth factors,and insulin-like growth factor-binding proteins are increasedin patients with diabetic retinopathy and rubeosis iridis5.
VEGF is present in the ocular membranes of patients with proliferativediabetic retinopathy,32 hypoxia stimulates the secretion ofVEGF in retinal pigment epithelial cells,29 and VEGF productionincreases with neovascularization of the iris in primates22.We have reported that retinal endothelial cells have large numbersof high-affinity VEGF receptors14 and that hypoxia increasesthe VEGF content of messenger RNA (mRNA) in retinal pericytes,retinal endothelial cells, and retinal pigment epithelial cells-- an effect reversed by the reinstitution of normal oxygenation21.These observations suggest a plausible scenario for the initiationand control of the ischemic ocular neovascular response. Alterationsin retinal perfusion arising from decreased blood flow (e.g.,central-retinal-vein occlusion or carotid occlusive disease33,34,35),retinal-capillary loss (resulting from diabetic retinopathyor radiation retinopathy, for example), peripheral-vasculatureagenesis or obliteration (with, e.g., retinopathy of prematurityor sickle cell disease), or separation of the choroidal bloodsupply from the retinal pigment epithelium (with, e.g., retinaldetachment) can all result in relative retinal ischemia. Thisischemia stimulates the synthesis and secretion of VEGF in retinalpericytes, endothelial cells, the retinal pigment epithelium,and possibly other cell types. Depending on the particular variantof mRNA splicing,18 secreted VEGF is either bound to cell-surfaceor basement-membrane proteoglycans containing heparin (VEGF189,286)or is freely diffusible within the vitreous cavity (VEGF121,165)36.Diffusible VEGF follows its concentration gradient from thevitreous to the anterior segment and is ultimately cleared throughthe trabecular meshwork. Neovascularization induced by the directaction of VEGF on endothelial cells can arise anywhere alongthis course, especially in areas of high exposure, such as thepupillary border and the trabecular meshwork. The angiogenicpotential of VEGF is enhanced by the synergistic activity offibroblast growth factor liberated by cellular disruption ordeath4,37,38. Cell death without ischemia would have less vasoproliferativepotential, since increased VEGF production would not be possible.Indeed, the risk of ocular neovascularization in anoxic retinaldisorders, such as retinal-artery occlusion, is lower than inischemic diseases, such as retinal-vein occlusion33. The reductionin relative retinal ischemia as a result of reperfusion, laserphotocoagulation, or extensive cell death may reduce the productionof VEGF and result in neovascular regression and quiescence.
VEGF meets the criteria hypothesized for an ischemia-inducedocular angiogenic factor,23 and we suggest that it has an importantrole in mediating the neovascular response of diabetic retinopathyand other ischemic retinal disorders. Deciphering the mechanismsunderlying the expression of hypoxia-induced VEGF and its biologiceffects should increase our understanding of numerous ocularneovascular diseases and provide new therapeutic approachesto preserving vision.
Supported in part by a grant (EY05110) (to Dr. King) and a pilotand feasibility grant (DERC36836-08) (to Dr. L.P. Aiello), bothfrom the National Institutes of Health; a Capps Scholarshipin Diabetes (to Dr. L.P. Aiello); a Heed/Knapp Fellowship fromthe Heed Ophthalmic Foundation (to Dr. L.P. Aiello); and a Diabetesand Endocrinology Research Center Grant (36836) from the NationalInstitutes of Health (to the Joslin Diabetes Center).
We are indebted to Ms. Leslie Balmat, Sven E. Bursell, Ph.D.,Peter A. Campochiaro, M.D., Jerry D. Cavallerano, O.D., Ph.D.,Leo T. Chylack, M.D., Eugene DeJuan, M.D., Mr. Dennis Fleming,Julia A. Haller, M.D., Jin Kim, Ph.D., Ms. Kimberly Keville,Mr. Ji Lu, Jeffrey Luttrull, M.D., and Pearl Yazbek, R.N., fortheir assistance in the acquisition of samples, the productionof antibody, and the preparation of the manuscript.
Source Information
From the Department of Ophthalmology, Beetham Eye Institute (L.P.A., P.G.A., S.T.S., L.M.A.), and the Division of Research (L.P.A., H.T., G.L.K.), Joslin Diabetes Center; the Departments of Medicine and Ophthalmology, Brigham and Women's Hospital (L.R.P., M.A.I., G.L.K.); and Harvard Medical School (L.P.A., P.G.A., S.T.S., L.R.P., H.T., M.A.I., L.M.A., G.L.K.) -- all in Boston; the Neuroscience Research Institute, University of California, Santa Barbara (R.L.A.); Genentech, Inc., San Francisco (B.A.K., J.E.P., H.V.N., N.F.); and the Department of Ophthalmology, Wilmer Ophthalmological Institute, Johns Hopkins Hospital, Baltimore (H.D.J.).
Address reprint requests to Dr. L.P. Aiello at the Beetham Eye Institute, Joslin Diabetes Center, 1 Joslin Place, Boston, MA 02215.
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(2007). Suppression of Diabetes-Induced Retinal Inflammation by Blocking the Angiotensin II Type 1 Receptor or Its Downstream Nuclear Factor-{kappa}B Pathway. IOVS
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Maines, L. W., French, K. J., Wolpert, E. B., Antonetti, D. A., Smith, C. D.
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Harhaj, N. S., Felinski, E. A., Wolpert, E. B., Sundstrom, J. M., Gardner, T. W., Antonetti, D. A.
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Kuiper, E. J., de Smet, M. D., van Meurs, J. C., Tan, H. S., Tanck, M. W. T., Oliver, N., van Nieuwenhoven, F. A., Goldschmeding, R., Schlingemann, R. O.
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(2005). Erythropoietin as a Retinal Angiogenic Factor in Proliferative Diabetic Retinopathy. NEJM
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(2005). The RAGE Axis in Early Diabetic Retinopathy. IOVS
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(2005). Effect of Insulin on Plasma Vascular Endothelial Growth Factor in Children with New-Onset Diabetes. J. Clin. Endocrinol. Metab.
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Yokoi, M, Yamagishi, S-i, Takeuchi, M, Ohgami, K, Okamoto, T, Saito, W, Muramatsu, M, Imaizumi, T, Ohno, S
(2005). Elevations of AGE and vascular endothelial growth factor with decreased total antioxidant status in the vitreous fluid of diabetic patients with retinopathy. Br J Ophthalmol
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(2005). Minocycline Reduces Proinflammatory Cytokine Expression, Microglial Activation, and Caspase-3 Activation in a Rodent Model of Diabetic Retinopathy. Diabetes
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(2005). Nonhuman Primate Models for Diabetic Ocular Neovascularization Using AAV2-Mediated Overexpression of Vascular Endothelial Growth Factor. Diabetes
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(2005). Preclinical Pharmacokinetics of Ranibizumab (rhuFabV2) after a Single Intravitreal Administration. IOVS
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[Abstract][Full Text]
1 heavy-chain clone,26 as described by Park et al27. The chimeric receptor has the same affinity for VEGF as does the full-length VEGF receptor. The sensitivity and variability of this assay were similar to those of the radioimmunoassay. 
0.05. 
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