Background Although vascular endothelial growth factor (VEGF)is a primary mediator of retinal angiogenesis, VEGF inhibitionalone is insufficient to prevent retinal neovascularization.Hence, it is postulated that there are other potent ischemia-inducedangiogenic factors. Erythropoietin possesses angiogenic activity,but its potential role in ocular angiogenesis is not established.
Methods We measured both erythropoietin and VEGF levels in thevitreous fluid of 144 patients with the use of radioimmunoassayand enzyme-linked immunosorbent assay. Vitreous proliferativepotential was measured according to the growth of retinal endothelialcells in vitro and with soluble erythropoietin receptor. Inaddition, a murine model of ischemia-induced retinal neovascularizationwas used to evaluate erythropoietin expression and regulationin vivo.
Results The median vitreous erythropoietin level in 73 patientswith proliferative diabetic retinopathy was significantly higherthan that in 71 patients without diabetes (464.0 vs. 36.5 mIUper milliliter, P<0.001). The median VEGF level in patientswith retinopathy was also significantly higher than that inpatients without diabetes (345.0 vs. 3.9 pg per milliliter,P<0.001). Multivariate logistic-regression analyses indicatedthat erythropoietin and VEGF were independently associated withproliferative diabetic retinopathy and that erythropoietin wasmore strongly associated with the presence of proliferativediabetic retinopathy than was VEGF. Erythropoietin and VEGFgene-expression levels are up-regulated in the murine ischemicretina, and the blockade of erythropoietin inhibits retinalneovascularization in vivo and endothelial-cell proliferationin the vitreous of patients with diabetic retinopathy in vitro.
Conclusions Our data suggest that erythropoietin is a potentischemia-induced angiogenic factor that acts independently ofVEGF during retinal angiogenesis in proliferative diabetic retinopathy.
Pathologic growth of new blood vessels is a common final pathwayin ocular neovascular diseases, such as proliferative diabeticretinopathy, that often result in catastrophic loss of vision.Vascular endothelial growth factor (VEGF) is a primary angiogenicfactor that mediates such ischemia-induced retinal neovascularization.VEGF levels are elevated in the vitreous fluid of patients withproliferative diabetic retinopathy, and VEGF induces proliferationin vascular endothelial cells in vitro.1 Although inhibitionof VEGF reduces retinal neovascularization,2,3 it does not completelyinhibit ischemia-driven retinal neovascularization. Thus, theinvolvement of other angiogenic factors in this process seemslikely.
The glycoprotein erythropoietin stimulates the formation ofred cells by enhancing both their proliferation and their differentiationand by preventing apoptotic death of erythropoietin-responsiveerythroid precursor cells.4,5,6 A major signal that regulatesthe production of erythropoietin in these tissues is hypoxia,and the brain has a paracrine system involving erythropoietinand erythropoietin receptors, suggesting that erythropoietincontributes to the survival of neurons by protecting them fromischemic damage.7,8,9 Furthermore, erythropoietin shows angiogenicactivity in vascular endothelial cells, stimulating proliferation,migration, and angiogenesis in vitro, probably by means of theerythropoietin receptor expressed in those cells.10,11 Suchangiogenic activity involves several signal-transduction cascadessuch as extracellular signal-regulated kinase, Janus kinase2 (known as JAK2), and signal transducer and activator of transcription5 (STAT5).12,13,14,15 Moreover, the inhibition of erythropoietinby soluble erythropoietin receptor abrogates angiogenesis invivo.16,17
Since erythropoietin is an ischemia-induced paracrine factorthat promotes angiogenesis, we wished to identify its potentialrole during retinal angiogenesis in proliferative diabetic retinopathy.Therefore, we examined in vitro the expression and functionof erythropoietin in the vitreous fluid of patients with proliferativediabetic retinopathy and evaluated the role of erythropoietinin an in vivo experimental model of retinal angiogenesis. Ourdata provide strong evidence that erythropoietin is a potentretinal angiogenic factor independent of VEGF and is capableof stimulating ischemia-induced retinal angiogenesis in proliferativediabetic retinopathy.
Methods
Preparation of Vitreous-Fluid Samples and Analysis of Erythropoietin and VEGF Levels
We conducted this study in accordance with the Declaration ofHelsinki and received institutional approval from the reviewcommittees of Kyoto University Hospital and Otsu Red Cross Hospital.Patients with proliferative diabetic retinopathy or nondiabeticocular diseases who were seen consecutively for treatment withpars plana vitrectomy at these centers were approached for participationfrom June 1997 through September 2004. All patients providedwritten informed consent. Samples of undiluted vitreous fluidwere harvested from the eyes of participating patients who hadproliferative diabetic retinopathy or nondiabetic ocular diseases.None of the patients with nondiabetic ocular diseases had diabetesmellitus. Proliferative diabetic retinopathy was classifiedclinically as active neovascularization if there were perfused,multibranching iridic or preretinal capillaries and as quiescentif previously documented active proliferation had regressedfully or if there were only nonperfused, gliotic vessels orfibrosis.1
Blood samples were collected if available. Erythropoietin levelswere measured with the use of radioimmunoassay (Recombigen Epokit, Diagnostic Products Corporation). VEGF levels were measuredwith the use of an enzyme-linked immunosorbent assay (ELISA)(R&D Systems).
Cell Culture, Western Blot Analysis, and Assays of Cell Growth
Total protein levels were obtained from primary cultures ofbovine retinal microvascular endothelial cells (BRECs),18 andthe expression of specific antigens was assessed by Westernblot analysis19 with primary antibodies against phospho-p44/p42(New England Biolabs), extracellular signal-regulated kinase1 (Santa Cruz Biotechnology), phospho-STAT5 (Upstate Biotechnology),and STAT5 (Santa Cruz Biotechnology).
BRECs were plated separately (700 cells per well) in 96-wellculture plates coated with collagen type I.1 The next day, humanrecombinant erythropoietin (0.1 to 50 IU per milliliter) (Kirin),human recombinant VEGF (10 ng per milliliter) (Genzyme), orvitreous fluid (final volume, 20 percent) was added in conjunctionwith phosphate-buffered saline, soluble erythropoietin receptor(25 µg per milliliter), soluble Flt-1-Fc chimera (2.5µg per milliliter) (Genzyme), both proteins, or heat-denaturedsoluble erythropoietin receptor (25 µg per milliliter).Soluble erythropoietin receptor was prepared as previously described.20After four days, cell growth was assayed with the use of 5-bromo-2'-deoxyuridineduring DNA synthesis. The absorbance of each sample was measuredwith an ELISA reader and compared with the absorbance of controlsamples stimulated with phosphate-buffered saline. We performedthe assay of cell growth in triplicate for the samples fromeach patient.
Real-Time Polymerase Chain Reaction
Total RNA was prepared from the retinas of mice with the useof an acid guanidinium thiocyanate–phenol–chloroformmethod. This was followed by synthesis of complementary DNAand a real-time polymerase chain reaction (PCR).21
Ischemia-Induced Retinal Neovascularization in a Murine Model
All animals were handled according to the Association for Researchin Vision and Ophthalmology Statement for the Use of Animalsin Ophthalmic and Vision Research (www.arvo.org/AboutARVO/animalst.asp).Litters of seven-day-old C57BL/6J mice were exposed to oxygen(mean [±SD], 75±2 percent) for five days and thenreturned to room air on day 12.22 Mice of the same age, keptin room air, served as controls. A 0.5-µl solution containingvarious concentrations of either soluble erythropoietin receptor,soluble Flt-1-Fc, both proteins, or heat-denatured soluble erythropoietinreceptor was injected into the vitreous of one eye on day 12and day 14.2 As a control, an equivalent volume and concentrationof human IgG was injected into the contralateral eye on thesame days. On day 19, neovascular quantification was performedin a double-masked manner.2
Statistical Analysis
The primary objective of this study was to determine whethererythropoietin is an angiogenic factor independent of VEGF inpatients with proliferative diabetic retinopathy. For primaryanalyses, we performed a two-sample t-test to compare the levelof erythropoietin in the group of patients who had proliferativediabetic retinopathy with the level in the group with nondiabeticocular diseases. We calculated the Pearson correlation coefficientbetween the erythropoietin and VEGF levels in the proliferativediabetic retinopathy group; data on erythropoietin and VEGFlevels were transformed with the use of common base-10 logarithms.We also performed the Mann–Whitney rank-sum test and calculatedthe Spearman rank-correlation coefficient to confirm the resultsof parametric analyses. To demonstrate and quantify the independentassociations of erythropoietin and VEGF with proliferative diabeticretinopathy, we performed multivariate logistic-regression analyseswith disease status (proliferative diabetic retinopathy or nondiabeticocular diseases) as the response variable and with four covariates— erythropoietin, VEGF, age, and sex. In vitro data wereanalyzed with the use of one-way analysis of variance followedby Fisher's least-significant-difference test. For evaluationof in vivo retinal angiogenesis, the paired t-test or the Mann–Whitneyrank-sum test was used for quantitative data. All reported Pvalues are two-sided.
To detect an approximately 100 percent mean difference in erythropoietinbetween the group of patients with proliferative diabetic retinopathyand the group with nondiabetic ocular diseases with the useof a two-sample t-test on log-transformed data, with a two-sidedalpha of 0.05 and a power of 95 percent, a sample size of 72was required for each group. The variance of the log-transformederythropoietin value was set to be 0.24, which was suggestedas a result of preliminary data from five patients with proliferativediabetic retinopathy. To analyze the correlation between erythropoietinand VEGF for 72 patients with proliferative diabetic retinopathy,we had a power of 0.94 when we used a test of no correlationon the basis of the Fisher transformation of the Pearson correlationcoefficient, resulting in a true correlation value of 0.4.
Results
Vitreous Erythropoietin and VEGF Levels
Samples of undiluted vitreous fluid were harvested from theeyes of 73 patients with proliferative diabetic retinopathyand 71 patients with nondiabetic ocular diseases, includingidiopathic epiretinal membrane, proliferative vitreoretinopathy,uveitis, idiopathic macular hole, rhegmatogenous retinal detachment,retinal macroaneurysms, Terson's syndrome, and choroidal neovascularmembrane (Table 1). Samples of aqueous humor harvested fromseven patients with uveitis were included.
The vitreous erythropoietin and VEGF levels were significantlyhigher in the patients with proliferative diabetic retinopathythan in those with nondiabetic ocular diseases (Figure 1A).The median erythropoietin level was 464.0 mIU per milliliter(range, 64.5 to 3720.0) in the patients with proliferative diabeticretinopathy and 36.5 mIU per milliliter (range, 0.4 to 698.0)in the patients with nondiabetic ocular diseases (P<0.001).The median VEGF level was 345.0 pg per milliliter (range, 0to 10,600.0) in the group with proliferative diabetic retinopathyand 3.9 pg per milliliter (range, 0 to 2130.0) in the nondiabeticgroup (P<0.001). Clearly, the proportion of patients withproliferative diabetic retinopathy, which corresponds to theresponse variable in the multivariate logistic-regression analyses,was higher in higher quartiles of erythropoietin and VEGF levels(Table 2).
Figure 1. Vitreous Levels of Erythropoietin and Vascular Endothelial Growth Factor (VEGF).
In Panel A, box-and-whisker plots show the levels of erythropoietin and VEGF for patients with proliferative diabetic retinopathy (PDR) and for patients with nondiabetic ocular diseases. The plots also show the levels in the subgroups with active and quiescent proliferative diabetic retinopathy and those in the following subgroups of nondiabetic ocular diseases: idiopathic macular hole, rhegmatogenous retinal detachment, inflammation, idiopathic epiretinal membrane, or other diseases. The lower and upper whiskers correspond to the 10th and 90th percentiles, respectively. The line in the box corresponds to the median value, and the plus sign (+) represents the mean value. Panel B shows a scatter plot for the correlation between log-transformed vitreous VEGF levels and vitreous erythropoietin levels in patients with proliferative diabetic retinopathy.
Table 2. The Proportion of Patients with Proliferative Diabetic Retinopathy in Each Quartile of the Vitreous Levels of Erythropoietin and Vascular Endothelial Growth Factor (VEGF).
For exploratory purposes, we also examined the erythropoietinand VEGF levels in subgroups of patients with various stagesof proliferative diabetic retinopathy as compared with subgroupsof patients with various nondiabetic ocular diseases (Figure 1A).The level of erythropoietin was significantly higher inthe patients with active as compared with quiescent proliferativediabetic retinopathy (P=0.001). The level of erythropoietinwas significantly higher in the patients with proliferativediabetic retinopathy than in the patients in the nondiabeticsubgroups (P<0.001). Among the patients with nondiabeticocular diseases, those with inflammation had slightly higherlevels of erythropoietin than did those with the other diseases.
A scatter plot of log-transformed levels of erythropoietin andVEGF in the patients with proliferative diabetic retinopathyindicated a weak correlation (Figure 1B). The Pearson correlationcoefficient was 0.29 (P=0.01). The corresponding value for patientswith active proliferative diabetic retinopathy was 0.14 (P=0.32),and that for patients with quiescent proliferative diabeticretinopathy was 0.28 (P=0.23). Similar results were obtainedfrom rank-based nonparametric analyses (data not shown).
Blood samples were collected from 36 patients with proliferativediabetic retinopathy and 42 with nondiabetic ocular diseases.The median plasma level of erythropoietin was 18.7 mIU per milliliter(range, 7.0 to 43.4) in the group with proliferative diabeticretinopathy and was slightly lower than that in the nondiabeticgroup (22.4 mIU per milliliter; range, 5.6 to 63.6). The Pearsoncorrelation coefficient for the plasma and the vitreous erythropoietinlevels in patients with proliferative diabetic retinopathy was–0.16 (P=0.14).
In multivariate logistic-regression analyses, the associationof erythropoietin and VEGF with proliferative diabetic retinopathywas initially assessed with the use of untransformed valuesbut was found to fit substantially better with the use of logtransformations to make the erythropoietin and VEGF distributionssymmetric. Neither age (P=0.39) nor sex (P=0.74) was significantlyassociated with proliferative diabetic retinopathy (Table 3),and the two covariates were thus dropped from the model. Whenwe fitted only log-transformed erythropoietin and VEGF, we observedthat both were significantly associated with proliferative diabeticretinopathy, but erythropoietin more strongly (Table 3).
Table 3. Logistic-Regression Analyses Showing the Association of Erythropoietin and VEGF with Proliferative Diabetic Retinopathy.
Treatment with Soluble Erythropoietin Receptor to Attenuate Erythropoietin Intracellular Signaling
Erythropoietin (10 IU per milliliter) increased phosphorylationof intracellular signaling substrates of STAT5 and 42/44 mitogen-activatedprotein kinase 15 minutes after stimulation of BRECs. This increasewas inhibited by the addition of soluble erythropoietin receptorat a concentration 2500 times as great as that of erythropoietin(Figure 2A).
Figure 2. Immunoblots of Phosphorylation (Phospho) of Signal Transducer and Activator of Transcription 5 (STAT5) and Extracellular Regulated Kinase (ERK) Induced by Erythropoietin (Panel A), Growth of Bovine Retinal Microvascular Endothelial Cells (BRECs) after Treatment with Recombinant Erythropoietin or VEGF (Panel B), Growth of BRECs in Response to Erythropoietin and VEGF in Vitreous Fluid (Panel C), and the Mean Inhibitory Effects of Erythropoietin and VEGF Blockade on Cellular Growth (Panel D).
In Panel A, BRECs were treated with either human recombinant erythropoietin (10 IU per milliliter) or erythropoietin preincubated with soluble erythropoietin receptor at a concentration 2500 times as great as that of erythropoietin for 15 minutes, and total proteins were assessed by Western blot analysis, of which a representative blot is shown. Similar data were obtained from other Western blots (data not shown). In Panel B, recombinant erythropoietin and VEGF were added to the growth cultures. Growth stimulation was assessed by measuring at 405 nm the amount of erythropoietin and VEGF that the treated cells absorbed as compared with the control, with a reference wavelength of 490 nm. The determinations were performed in triplicate, and experiments were repeated three times. P values are for the comparison with control samples stimulated with phosphate-buffered saline. Data are means ±SD. In Panel C, control samples of phosphate-buffered saline or samples of vitreous fluid from eight patients with proliferative diabetic retinopathy were added to cultures of BRECs. The level of cell growth that was stimulated by vitreous fluid of each patient is shown. Soluble erythropoietin receptor (25 µg per milliliter), soluble Flt-1-Fc chimera (2.5 µg per milliliter), or a combination of both proteins was added to the cultures. Heat-denatured soluble erythropoietin receptor (25 µg per milliliter) was added as a control. We compared the four groups to which we added human vitreous according to the ratio of cell growth in each group with that in the control group, to which phosphate-buffered saline was added in each experiment. All P values are for the comparison with samples from each patient stimulated only with vitreous fluid. Panel D shows the mean inhibitory effect of erythropoietin and VEGF blockade on cellular growth in each sample, expressed as a percentage of the level induced by vitreous-fluid stimulation. Data are means ±SD.
Effect of Erythropoietin, VEGF, and Vitreous Fluid on the Growth of Retinal Endothelial Cells
The growth of BRECs was stimulated in a dose-dependent mannerafter exposure to erythropoietin, with a maximal cell growthobserved at 20 IU of erythropoietin per milliliter (absorbance,1.63±0.19 times as high as that of control samples stimulatedwith phosphate-buffered saline; P<0.001) (Figure 2B). VEGF(10 ng per milliliter) also stimulated cell growth (absorbance,1.90±0.29; P<0.001). Vitreous samples from eight patientswith proliferative diabetic retinopathy stimulated the growthof BRECs, which was significantly inhibited by soluble erythropoietinreceptor (25 µg per milliliter) in all cases (range ofinhibition, 22 to 74 percent). Soluble Flt-1-Fc also inhibitedcellular growth in each case (range of inhibition, 22 to 85percent) (Figure 2C), and the mean inhibitory effect of erythropoietinand VEGF blockade on cellular growth, expressed as a percentageof the level induced by the vitreous fluid from each of theeight patients, was 56, 54, and 16 percent after the additionof soluble erythropoietin receptor, soluble Flt-1-Fc, and both,respectively. Heat-denatured soluble erythropoietin receptordid not affect the proliferation of BRECs induced by vitreoussamples from four patients with proliferative diabetic retinopathy(Figure 2D).
Expression of Erythropoietin and VEGF in Ischemic Retinas of Mice
Retinal levels of erythropoietin messenger RNA (mRNA) in miceincreased dramatically when they were 17 days old, then decreasedsomewhat on day 19. The levels of VEGF mRNA were also increasedon day 17 and paralleled the changes to erythropoietin transcriptlevels (Figure 3A).
Figure 3. Expression of Erythropoietin mRNA and VEGF mRNA in Ischemic Retinas of Mice.
Real-time PCR was performed to analyze mRNA levels of erythropoietin and VEGF from the retinas of mice (from 12 days, 0 hours, of age to 19 days). After normalization to ribosomal protein S18 mRNA concentrations as an internal control, the increases in the expression of each gene at each time point, as compared with expression in age-matched controls at day 12, 0 hours, are shown. The light blue bar denotes age-matched controls, and the blue bar experimental mice. Panel B shows the inhibitory effects of erythropoietin and VEGF blockades on ischemia-induced retinal angiogenesis. The inhibition of retinal neovascularization is dose-dependent on the extent of erythropoietin inhibition. Intraocular injections of soluble erythropoietin receptor, soluble Flt-1-Fc chimera, a combination of these proteins, or heat-denatured soluble erythropoietin receptor were performed on days 12 and 14 in one eye. An equivalent dose of human IgG was injected into the contralateral eye as a control. Retinal neovascularization, as determined from neovascular nuclei counts per 6-µm histologic section per eye, is expressed as a percentage of the counts in the contralateral eye. Data are means ±SE.
Soluble Erythropoietin Receptor and Soluble Flt-1-Fc in an in Vivo Model of Ischemia-Induced Retinal Neovascularization
Intraocular injections of soluble erythropoietin receptor reducedhistologically evident retinal neovascularization in 19-day-oldmice in a dose-dependent manner as compared with equivalentinjections of human IgG or heat-denatured soluble erythropoietinreceptor. The mean percentage inhibition, as compared with valuesin the contralateral eye injected with IgG, was 65, 59, and55 percent after injection of 25, 62.5, and 250 ng of solubleerythropoietin receptor, respectively. The observed maximuminhibition of retinal neovascularization by soluble erythropoietinreceptor treatment was found to be at least as great as thatwith soluble Flt-1-Fc (52 percent), with no statistically significantdifference (P=0.81). The mean percentage inhibition of neovascularizationwas 30 percent when a combination of soluble erythropoietinreceptor and soluble Flt-1-Fc was injected (Figure 3B).
Discussion
The present study indicates that the level of erythropoietinin the vitreous fluid of patients with proliferative diabeticretinopathy is strikingly higher than the level in nondiabeticpatients. With the use of multivariate logistic-regression analyses,we observed that erythropoietin and VEGF were each independentlyassociated with proliferative diabetic retinopathy. Indeed,erythropoietin was more strongly associated with proliferativediabetic retinopathy than was VEGF. No significant correlationwas observed between the vitreous and plasma levels of erythropoietin,suggesting that increased erythropoietin levels in the vitreousfluid are probably due to increased local production in theretina, as we have shown in the murine model of ischemia-inducedretinal neovascularization.
The presence of erythropoietin in the vitreous is probably notdue to the breakdown of the blood–retinal barrier. However,we cannot rule out the possibility of an intraocular mechanismfor controlling levels of erythropoietin, much like the mechanismthat increases ascorbate in the aqueous humor (i.e., saturationkinetics).23,24 Ascorbate protects ocular tissues such as thelens against free radicals produced mainly by ultraviolet radiation.
Although we observed dramatic and concomitant up-regulationof both VEGF and erythropoietin in an experimental murine modelof ischemic retinas, we observed only a weak correlation betweenthe vitreous levels of erythropoietin and VEGF in patients withproliferative diabetic retinopathy. It is well documented thaterythropoietin is up-regulated in situations of hypoxia by amolecular mechanism similar to that of VEGF, including transcriptionalactivation by hypoxia-inducible factor25 and increased mRNAstability.26 We also observed a slight increase in the vitreouserythropoietin level in patients with inflammatory eye diseases.Although erythropoietin, like VEGF, is an ischemia-induced localretinal factor, the present data suggest that a stimulant otherthan ischemia, such as high glucose levels, oxidative stress,intraocular inflammation, or the presence of other cytokines,may also differentially affect erythropoietin expression.
Other evidence also supports the concept that erythropoietinis involved in proliferative diabetic retinopathy. Erythropoietinstimulates proliferation of BRECs in a dose-dependent way. However,erythropoietin may have a biphasic effect on endothelial proliferationmuch as VEGF does.27,28,29 Erythropoietin in the vitreous fluidof patients with proliferative diabetic retinopathy is bioactiveand stimulates proliferation of BRECs. The blockade of erythropoietininhibited the stimulation of cell growth in vitro as efficientlyas did VEGF, suggesting that erythropoietin might have angiogenicpotency equivalent to that of VEGF in patients with proliferativediabetic retinopathy. The combination of soluble erythropoietinreceptor and soluble Flt-1-Fc reduced the proliferation of BRECs,though the inhibition with both proteins was still incompletewhen samples from Patients 2, 4, and 8 were used. These in vitroresults suggest that although VEGF and erythropoietin seem tohave major roles in the pathogenesis of proliferative diabeticretinopathy, other growth factors may also be involved. Ourresults with the in vivo model are consistent with this hypothesis.
Several reports show the efficacy of treatment with erythropoietinfor various diseases. Correcting anemia with erythropoietintherapy may slow the progression of renal failure30 and mayreduce the progression of tumors.31 Erythropoietin treatmentcan counter neural damage in patients who have had strokes.32Small case studies of patients with diabetic nephropathy indicatethat treating anemia with erythropoietin improves diabetic retinopathy.33,34However, patients in those studies were also being aggressivelytreated for other coexisting conditions that can affect retinopathy(e.g., hypertension, hyperlipidemia, and azotemia). Furthermore,both of those reports lacked comparison groups of untreatedpatients.33,34 In addition, erythropoietin is used to treatanemia, and this may confound observations, since anemia isa risk factor for diabetic retinopathy.35
Erythropoietin blockade is likely to be beneficial for the treatmentof proliferative diabetic retinopathy. However, erythropoietinblockade may be hazardous for retinal diseases that involveapoptosis of retinal photoreceptors.36 In fact, this strategymight worsen diabetic neuropathy if administered indiscriminately,37because erythropoietin is a survival factor for retinal photoreceptors38,39and acts as a neurologic protection factor in diabetic neuropathy.37,40Conversely, the possibility that erythropoietin might have abeneficial effect on neuronal damage may be counterbalancedby risk for patients who also have retinal vasoproliferativediseases. Local as opposed to systemic therapy might potentiallyovercome these problems. Further studies, including the analysisof neuronal side effects, will be necessary to determine whethererythropoietin blockade would work as an approach to the treatmentof proliferative diabetic retinopathy.
Supported by grants-in-aid for scientific research from theJapanese Ministry of Education, Science, and Culture and Ministryof Health and Welfare.
We are indebted to Dr. Tetsuji Yamashita (Mitsubishi KagakuBio-clinical Laboratory) for his technical advice and assistancewith ELISA and radioimmunoassay techniques and to Dr. Lois E.H.Smith (Department of Ophthalmology, Children's Hospital andHarvard Medical School) and Dr. Nobuyuki Takakura (Cancer ResearchCenter, Kanazawa University) for their helpful discussion.
Source Information
From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto (D.W., K.S., M. Kurimoto, J.K., M. Kita, I.S., H.O., T.O., T.M., N.Y., H.T.); the Department of Pharmacoepidemiology, Kyoto University Graduate School of Public Health, Kyoto (S. Matsui); the Laboratory of Biosignals and Response, Kyoto University Graduate School of Biostudies, Kyoto (T.K., S. Masuda, M.N.); the Department of Ophthalmology, Otsu Red Cross Hospital, Otsu (M. Kita); and the Department of Ophthalmology, Hyogo Prefectural Amagasaki Hospital, Amagasaki (H.T.) — all in Japan.
Address reprint requests to Dr. Takagi at the Department of Ophthalmology, Hyogo Prefectural Amagasaki Hospital, Higashidaimotsu-cho, 1-1-1, Amagasaki 660-0828, Japan, or at hitoshi{at}kuhp.kyoto-u.ac.jp.
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