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Diabetes Care 29:2028-2033, 2006
DOI: 10.2337/dc06-0556
© 2006 by the American Diabetes Association
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Pathophysiology/Complications
Original Article

Erythropoietin Is Expressed in the Human Retina and It Is Highly Elevated in the Vitreous Fluid of Patients With Diabetic Macular Edema

Cristina Hernández, MD1, Alex Fonollosa, PHD2, Marta García-Ramírez, MD1, Mónica Higuera, PHD1, Roberto Catalán, MD3, Adela Miralles, PHD4, José García-Arumí, MD2 and Rafael Simó, MD1

1 Diabetes Research Unit, Endocrinology Division, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain
2 Ophthalmology Department, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain
3 Biochemistry Department, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain
4 Tissue Bank and Cell Therapy Center, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain

Address correspondence and reprint requests to Dr. Rafael Simó, Diabetes Research Unit, Endocrinology Division, Hospital Universitari Vall d’Hebron, Pg. Vall d’Hebron 119-129, 08035 Barcelona, Spain. E-mail: rsimo{at}ir.vhebron.net


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 CONCLUSIONS
 References
 
OBJECTIVE—Erythropoietin has been recently found to be increased in the vitreous fluid from ischemic retinal diseases such as proliferative diabetic retinopathy (PDR). The aims of the present study were 1) to measure erythropoietin levels in the vitreous fluid from patients with diabetic macular edema (DME), a condition in which the ischemia is not a predominat event, and 2) to compare erythropoietin mRNA expression between human retinas from nondiabetic and diabetic donors without retinopathy.

RESEARCH DESIGN AND METHODS—Vitreous samples from 12 type 2 diabetic patients with DME without significant retinal ischemia and 12 PDR patients were prospectively analyzed. Ten nondiabetic patients with macular holes served as the control group. Erythropoietin was assessed by radioimmunoassay (milliunits per milliliter). Erythropoietin mRNA expression was measured by quantitative real-time RT-PCR analysis in the retina from eight nondiabetic and eight age-matched diabetic donors without diabetic retinopathy

RESULTS—Intravitreal erythropoietin concentration was higher in both PDR and DME patients than in nondiabetic control subjects (PDR vs. control subjects: median 302 [range 117–1,850] vs. 30 mU/ml [10–75], P < 0.01; DME vs. control subjects: 430 [41–3,000] vs. 30 mU/ml [10–75], P < 0.01). However, no significant differences were found between DME and PDR patients. Erythropoietin mRNA expression was detected in the human retina, and it was higher in the retina from diabetic than from nondiabetic donors.

CONCLUSIONS—As occurs in PDR, intravitreous erythropoietin concentrations are strikingly higher in DME. Erythropoietin is expressed in the human retina, and it is upregulated in diabetic patients even without retinopathy. These findings suggest that other factors apart from ischemia are involved in the overexpression of erythropoietin in diabetic retinopathy.

Abbreviations: BBB, blood-brain barrier • BRB, blood-retinal barrier • DME, diabetic macular edema • PDR, proliferative diabetic retinopathy • RPE, retinal pigment epithelium • VEGF, vascular endothelial growth factor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESEARCH DESIGN AND METHODS
 RESULTS
 CONCLUSIONS
 References
 
Diabetic retinopathy is the primary cause of blindness in working-age individuals in developed countries (1). Visual loss primarily occurs from either proliferation of new retinal vessels (proliferative diabetic retinopathy [PDR]) or increased permeability of retinal vessels (diabetic macular edema [DME]) (2). DME is the main cause of visual loss in type 2 diabetic patients (3); therefore, it is a relevant contributor to diabetic eye disease. However, the pathogenic mechanisms of DME have been studied much less than PDR.

Reduction of visual acuity in DME results from accumulation of fluid due to a breakdown of the blood-retinal barrier (BRB) (3,4). Vascular endothelial growth factor (VEGF), also called vascular permeability factor, and several proinflammatory cytokines have been involved in the pathogenesis of DME (5–9). However, the knowledge of permeability modulators of BRB in the setting of DME is still very limited.

Erythropoietin was first described as a glycoprotein produced exclusively in fetal liver and adult kidney that acts as a major regulator of erythropoiesis (10). However, recent experimental evidence suggests that erythropoietin has a potent neuroprotective effect in the brain (11) and in the retina (12). Erythropoietin expression has also been found in the human brain (13) and the human fetal retina (14), but it is not known whether its expression persists in the adult retina.

Hypoxia is a major stimulus for both systemic (10) and intraocular erythropoietin production (15). In fact, high intravitreous levels of erythropoietin have recently been reported in ischemic retinal diseases such as PDR (16–18). In addition, it has been reported that erythropoietin has an angiogenic potential equivalent to VEGF (18,19). Therefore, erythropoietin could be an important factor involved in stimulating retinal angiogenesis in PDR. However, intravitreal levels of erythropoietin have not been measured in patients with DME, a condition in which the hypoxia is not a predominant event.

The aim of the present study was to evaluate intravitreal erythropoietin concentration in patients with DME in comparison with PDR and nondiabetic subjects. In addition, retinal erythropoietin expression in both diabetic and nondiabetic human retinas has also been investigated for the first time.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
RESULTS
 CONCLUSIONS
 References
 
The present study included 24 type 2 diabetic patients (12 consecutive patients with DME and 12 consecutive patients with PDR, age 61.6 ± 15.3 years) in whom a classic three-port pars plana vitrectomy was performed. Vitreous from 10 age-matched nondiabetic patients (age 59.6 ± 10.7 years) with macular hole requiring vitrectomy served as the control group (n = 10). The exclusion criteria were 1) photocoagulation in the preceding 3 months or previous vitreoretinal surgery, 2) recent vitreous hemorrhage (<3 months before vitrectomy) or intravitreous hemoglobin >5 mg/ml, 3) renal failure (creatinine ≥120 µmol/l), and 4) presence of anemia (hemoglobin <12 g/dl).

Fluorescein angiography was performed in all patients with DME in order to assess the degree of retinal ischemia denoted by nonperfusion of the retinal capillaries. For this purpose, we used the method developed by the Central Retinal Vein Occlusion Study Group (20). The photographic protocol included fluorescein angiographic views of posterior pole and midperiphery. Capillary nonperfusion was measured using a transparent disc area template placed over the angiogram. The number of disc areas was counted by the same ophthalmologist in every case, who was unaware of the clinical status of the patients. According to this protocol, eyes with <10 disc areas of nonperfusion were classified as having little or no evidence of ischemia.

The protocol for sample collection was approved by the hospital’s ethics committee, and informed consent was obtained from the patients.

Vitrectomy and collection of specimens
In all cases, a classic three-port pars plana vitrectomy was performed. For visualization of the vitreous cavity, we used a wide-field system with a precorneal Volk lens of 130° and inversion image system by Moeller-Wedel (Hamburg, Germany).

Undiluted vitreous samples (~1 ml) were obtained at the onset of vitrectomy by aspiration into a 1-ml syringe attached to the vitreous cutter (Alcoon Model; Ten-Thousand Ocutome, Irvine, CA) before starting intravitreal infusion of balanced salt solution. The vitreous samples were transferred to a tube, placed immediately on ice, and centrifuged at 16,000g for 5 min at 4°C. Supernatants were frozen at –80°C until assayed. For serum determinations, blood samples were collected simultaneously with the vitrectomy, then centrifuged at 3,000g for 10 min at 4°C to obtain serum, aliquoted, and stored at –80°C until assayed.

Human postmortem eyes were obtained from 16 donors: 8 nondiabetic and 8 age-matched diabetic donors without diabetic retinopathy (age 73 ± 2.3 vs. 71 ± 8 years, respectively; P = 0.23). Although erythropoietin mRNA has extraordinary stability for postmortem degradation (21), the time elapsed from death to enucleation was <4 h in all cases. After enucleation, the eyes were snap frozen in liquid nitrogen and stored at –80°C.

Erythropoietin assessment
Erythropoietin was assessed by radioimmunoassay (Incstar-Diasorin). The intra- and interassay coefficients of variation were 4.8 and 10.6%, respectively. The lowest limit of detection was 4.4 mU/ml.

Intravitreous hemoglobin
Vitreous hemoglobin levels were measured by spectophotometry (Uvikon 860; Kontron Instruments, Zurich, Switzerland), which permits to measure hemoglobin in micromolar concentration. The lowest limit of detection was 0.03 mg/ml.

Protein assay
Vitreal proteins were measured by a previously validated microturbidimetric method with an autoanalyzer (Hitachi 917; Boehringer, Mannheim, Germany). This method, based on the benzetonium chloride reaction, is a highly specific method for the detection of proteins and has a higher sensibility and reproducibility than the classic method of Lowry. The lowest level of proteins detected was 0.02 mg/ml. Intra- and interassay coefficients of variation were 2.9 and 3.7%, respectively.

RNA extraction and erythropoietin mRNA quantification
Human neuretina and retinal pigment epithelium (RPE) were harvested under the microscopic dissection of isolated eye cups from donors. Total RNA was extracted from isolated retinal tissues using the RNeasy Mini Kit with DNAse digestion (Quiagen Distributors, IZASA, Barcelona, Spain) according to the manufacturer’s instructions. A 1-µg aliquot of total RNA was used directly for reverse transcription, which was carried out using random hexanucleotide priming and TaqMan Reverse Transcription Reagents (Applied Biosystems, Madrid, Spain) in a 50-µl reaction according to the manufacturer’s protocol.

Quantitative real time using erythropoietin-specific primers (TaqMan premade gene expression assay Hs0017267; Applied Biosystems; Gene Bank RfSeq NM 000799.2) was performed taking 2 µl of the reverse-transcription reaction as template in a PCR setup with the TaqMan Universal Mastermix. The reaction was conducted as follows: 95°C for 10 min and 50 cycles of 15 s at 95°C and 1 min at 60°C in Applied Biosystems 7000 equipment. Each sample was assayed in duplicate, and control negative samples were included in each experiment. Automatic Relative Quantification data were obtained with ABI Prism 7000 SDS software (Applied Biosystems) using ß-actin as endogenous gene expression control (Hs9999903_m1; Applied Biosystems).

In addition, standard PCR was performed with specific primers designed using Primer3 software in an Applied Biosystems 2720 Thermal Cycler. A ß-actin 350-bp product was obtained with forward 5'-AAACTGGAACGGTGAAGGT-3' and reverse 5'-TCAAGTTGGGGGACAAAAA-3' primers, and a 210-bp erythropoietin product was obtained with forward 5'-CTTCTCCTGTCCCTGCTGTC-3' and reverse 5'-TCC ATC CTC TTC CAG GCA TA-3' primers. PCR products were electrophorezed on 2% agarose gel and stained with ethidium bromure.

We conducted this study in accordance with the Declaration of Helsinki and received approval from the ethics committee of Vall d’Hebron University Hospital.

Statistical analysis
The Kolmogorov-Smirnov test was utilized to confirm the assumption of the normality of the variables. Student’s t test and ANOVA were used to compare continous variables and the {chi}2 test for categorical variables. Because of their skewed distribution, erythropoietin concentrations were displayed as median and range, and the statistical comparisons were performed using a nonparametric test (Mann-Whitney U test). Correlations were examined by Spearman’s rank correlation. Levels of statistical significance were set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
CONCLUSIONS
 References
 
The main clinical characteristics of subjects included in the study are summarized in Table 1. We did not observe significant differences in serum erythropoietin among groups (DME: median 12.4 mU/ml [range 7.8–46.7]; PDR: 10.6 mU/ml [2.5–19.2]; and control subjects: 8.7 mU/ml [4.1–20.8]; P = NS). Fluorescein angiography demonstrated a lack of significant retinal ischemia in all patients with DME (mean of disc areas of retinal nonperfusion 0.8 [range 0–4]).


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  		Table 1— Main clinical features of subjects included in the study

 
Intravitreous protein concentration was significantly higher in diabetic patients than in control subjects (3.1 [range 1.1–7.2] vs. 0.75 mg/ml [0.4–1.7]; P < 0.001). However, no significant differences were detected between DME and PDR patients (3.9 [1.9–7.2] vs. 3.1 mg/ml [1.1–5.1]; P = 0.22).

The intravitreal erythropoietin concentration was significantly higher in the whole group of diabetic patients than in control subjects in absolute terms (326 [range 41–3,000] vs. 30 mU/ml [10–75]; P < 0.0001) and also after correcting for intravitreal proteins (103 [13–1,027] vs. 50 mU/mg [13–107]; P = 0.03). The differences remained significant when DME and PDR patients were compared separately with the control group in absolute terms (430 [41–3,000] vs. 30 mU/ml [10–75], P < 0.0001 and 302 [117–850] vs. 30 mU/ml [10–75]; P < 0.0001) and after correcting for intravitreal proteins (105 [13–967] vs. 50 mU/mg [13–107], P = 0.02 and 65 [32–1,027] vs. 50 mU/mg [13–107]; P = 0.03).

Higher intravitreous erythropoietin concentration was detected in subjects with DME in comparison with PDR patients, but the difference was not statistically significant either in absolute terms or after adjusting for intravitreal proteins (430 [range 41–3,000] vs. 302 mU/ml [117–850]; P = 0.21 and 105 [13–967] vs. 65 mU/mg [32–1,027]; P = 0.53, respectively; Fig. 1).


Figure 1
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  		Figure 1— Intravitreous and serum concentrations of erythropoietin (mU/ml) in patients with DME and PDR and in control subjects. Values are expressed as median (range).

 
Erythropoietin was 30-fold higher in vitreous fluid than in serum from diabetic patients (326 [range 41–3,000] vs. 11.2 mU/ml [2.5–46.7]; P < 0.0001), whereas, in control subjects, the difference was only 3.5-fold higher (30 [10–55] vs. 8.7 mU/ml [4.1–20.8]; P = 0.012). No correlation between intravitreal and serum erythropoietin concentration was detected either in diabetic patients or in the control group.

Erythropoietin mRNA was detected in human retina from both nondiabetic control donors and diabetic donors (Fig. 2). A significantly higher expression was observed in RPE cells than in neuroretina in the whole group (1.62 ± 0.61 vs. 0.39 ± 0.13; P = 0.02). Higher expression was also observed in RPE cells than in neuroretina when analyzing diabetic donors and nondiabetic donors separately, but the differences were not statistically significant (2.21 ± 1.08 vs. 0.54 ± 0.23; P = 0.18 and 0.93 ± 0.24 vs. 0.21 ± 0.06; P = 0.07) (Fig. 2). In addition, higher expression of erythropoietin mRNA was detected in RPE cells (2.4-fold) and in the neuroretina (2.6-fold) from diabetic donors in comparison with nondiabetic donors.


Figure 2
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  		Figure 2— Expression of mRNA erythropoietin in the human retina. A: Agarose gel electrophoresis of RT-PCR products from a representative nondiabetic donor. 1, brain (positive control); 2, pancreas (negative control); 3, neuroretina; 4, retinal pigment epithelium; 5, marker 100 bp. B: Erythropoietin (Epo) mRNA measured by real-time RT-PCR (TaqMan Assay) in RPE and neuroretina (R) from nondiabetic and diabetic donors. Kidney (K) was used as positive control. Erythropoietin gene expression data ({Delta}{Delta}Ct) are shown after normalizing with ß-actin. Results are presented as the means ± SE.

 

   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 CONCLUSIONS
References
 
In the present study, we have found that intravitreal erythropoietin concentrations were markedly higher in patients with DME than in control subjects. Furthermore, intravitreous erythropoietin levels in DME were in the same range as that obtained in PDR. After correcting for vitreal proteins, the differences were not so evident but remained at significant levels. In this regard, it should be mentioned that the rationale for performing the correction for intravitreous proteins is to explore the source (intraocular synthesis versus blood borne) of the specific peptide that we want to analyze rather than to obtain a better index of its activity. In the present study, erythropoietin serum concentration was also determined and it was strikingly lower than that obtained in the vitreous, not only in diabetic patients with DME and PDR (~30 times less) but also in control subjects (~3.5 times less). Therefore, it seems clear that the elevated concentrations of erythropoietin obtained in the vitreous fluid of diabetic patients are not serum derived, and in consequence, the crude concentration of erythropoietin rather than the ratio of erythropoietin to vitral proteins better reflects the production of erythropoietin by the retina. In addition, our results strongly suggest not only that erythropoietin is synthesized by the retina but also that its production is upregulated in diabetic subjects. Confirming this point, in the present study, erythropoietin RNA expression was detected for the first time in the adult human retina, and its expression was significantly higher in diabetic than in nondiabetic donors. In addition, in both diabetic and nondiabetic donors, erythropoietin expression was found to be more abundant in RPE than in the neuroretina.

Unlike PDR, DME is a condition in which the ischemia is not a predominant event. In this regard, it should be noted that fluorescein angiography confirmed the lack of significant retinal ischemia in all patients with DME included in the study. Therefore, our results strongly suggest that stimulating agents other than ischemia are involved in the increased intravitreous levels of erythropoietin found in DME. Watanabe et al. (18) observed an increase in the vitreous erythropoietin levels in patients with inflammatory eye diseases. Several data indicate that inflammation is involved in the pathogenesis of both PDR (22) and DME (6–9,23). Therefore, inflammation might contribute to the high levels of erythropoietin observed in patients with DME. Hyperglycemia could be another factor that induces erythropoietin production. Although there are no studies evaluating the effect of glucose on erythropoietin expression in retinal cells, a direct relationship has been shown between glucose and erythropoietin concentrations in a Chinese hamster ovary cell line (24). In addition, we have found higher expression of erythropoietin mRNA in the retinas from diabetic donors without diabetic retinopathy in comparison with retinas from nondiabetic donors. Therefore, it seems that erythropoietin overexpression is an early event in the retina of diabetic patients, and it is unrelated to the ischemic process. Furthermore, this finding supports the concept that ischemia is not necessary to induce erythropoietin expression in the diabetic eye.

It has been suggested that erythropoietin plays an important role in PDR development, and its blockade could be beneficial for the treatment of PDR (18). However, whether the increase of erythropoietin concentration observed in DME has a pathogenic action or, by contrast, might be part of a self-regulated physiological protection mechanism to prevent retinal damage needs to be clarified. From the data available so far, the second hypothesis seems the most likely (13). Martínez-Estrada et al. (25), using an in vitro model of bovine blood-brain barrier (BBB), provided evidence that erythropoietin protects against the VEGF-induced permeability of the BBB and restores the tight junction proteins. Erythropoietin treatment also prevents an increase in BBB permeability in a rat model of induced seizures (26). Since BRB is structurally and functionally similar to the BBB (27), it is possible that erythropoietin could act as an antipermeability factor in the retina. In this regard, a substantive improvement of DME after treating azotemia-induced anemia with erythropoietin has been reported (28). In addition, there is growing evidence that erythropoietin is a neurotrophic factor (11,12). In this regard, it has been shown that erythropoietin protects cultured neurons from hypoxia and glutamate toxicity (29–31), and its systemic administration reduces neuronal injury in animal models of focal ischemic stroke and inflammation (32–34). Retinal neurodegeneration is an early event in diabetic retinopathy, and therefore, it might be possible that higher production of erythropoietin is needed as a neuroprotective factor. In fact, Junk et al. (32) demonstrated that erythropoietin administration is associated with both histopathological and functional protection of retinal neurons in a model of transient global retinal ischemia. Grimm et al. (15) reported that erythropoietin expression induced in the hypoxic mouse retina protects against light-induced retinal degeneration. Tsai et al. (35) demonstrated that intravitreal administration of erythropoietin has a protective effect on the viability of retinal ganglion cells in a rat model of glaucoma. Layton et al. (36) demonstrated in primary retinal cultures that erythropoietin’s neurothrophic function is attenuated at glucose concentrations similar to those which occur in diabetes. Apart from its antipermeability and neuroprotective actions, erythropoietin exerts an anti-inflammatory effect on the brain (37,38), and this action might also be extrapolated to the diabetic retina. Finally, it has been demonstrated that erythropoietin protects against high-glucose–induced apoptosis as well as the deleterious effect of free radicals (39,40). For all these reasons, it seems that erythropoietin is crucial for retinal homeostasis and its enhancement in DME could be a physiological consequence rather than a pathogenic contributor.

The strikingly high intravitreous erythropoietin levels detected in patients with DME raises the question of why these patients do not develop neovascularization as occurs in PDR patients. At present, we do not have any reliable explanation for this. However, it should be considered that the development of neovessels, rather than depending on a single angiogenic factor, is mediated by the balance between angiogenic and antiangiogenic factors. Therefore, in patients with DME, other factors counteracting the angiogenic effect of erythropoietin and favoring the permeability of BRB must exist.

In conclusion, we have demonstrated that intravitreal erythropoietin levels are increased in patients with DME at the same range as that observed in PDR subjects. In addition, we have demonstrated mRNA erythropoietin expression in the human retina and that its upregulation in diabetic subjects is an early event in the development of diabetic retinopathy. These findings suggest that other factors unrelated to the ischemia might be crucial in the upregulation of erythropoietin. Further studies are needed to investigate the precise role of erythropoietin, not only in the setting of DME but also in the normal human retina.


   Acknowledgments
 
This study was supported by grants from Novo Nordisk Pharma (01/0066), the Instituto de Salud Carlos III (G03/212 and C03/08), and the Ministerio de Ciencia y Tecnología (SAF2003-00550).


   Footnotes
 
C.H. and A.F. contributed equally to this work.

A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances.

Received for publication March 13, 2006. Accepted for publication May 30, 2006.


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 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 CONCLUSIONS
 References
 

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