Expression of Erythropoietin and Its Receptor in the ... - Diabetes Care

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Expression of Erythropoietin and Its Receptor in the Human Retina A comparative study of diabetic and nondiabetic subjects MARTA GARCÍA-RAMÍREZ, PHD CRISTINA HERNA´ NDEZ, MD, PHD RAFAEL SIMO´ , MD, PHD

OBJECTIVE — The purpose of this study was to evaluate erythropoietin (Epo) and Epo receptor (EpoR) expression in the retina and in vitreous fluid from diabetic and nondiabetic donors. To gain insight into the mechanisms responsible for the regulation of Epo production in the retina, we also assessed retinal expression of hypoxia-inducible factors (HIF-1␣ and HIF-2␣). RESEARCH DESIGN AND METHODS — Eighteen postmortem eyes from 9 diabetic patients without clinically detectable retinopathy were compared with 18 eyes from 9 nondiabetic donors. mRNA of Epo, HIF-1␣, and HIF-2␣ (quantitative RT-PCR) were measured separately in neuroretina and retinal pigment epithelium (RPE). Epo and EpoR were assessed in the retina (immunofluorescence by confocal laser microscopy) and in the vitreous fluid (radioimmunoassay and enzyme-linked immunosorbent assay, respectively). RESULTS — Epo and EpoR mRNAs were significantly higher in the RPE than in the neuroretina. Higher expression of Epo was detected in the retinas (both in the RPE and in the neuroretina) from diabetic donors. By contrast, EpoR expression was similar in both groups. We did not find any difference in HIF-1␣ and HIF-2␣ mRNA expression between diabetic and nondiabetic donors (both in RPE and neuroretina). Intravitreal Epo concentration was higher in diabetic donors than in nondiabetic control subjects. However, EpoR concentrations were similar in both groups. CONCLUSIONS — Epo overexpression is an early event in the retina of diabetic patients, and this is not associated with any change in EpoR. At this early stage, other factors apart from hypoxia seem to be more important in accounting for the Epo upregulation that exists in the diabetic retina. Diabetes Care 31:1189–1194, 2008

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rythropoietin (Epo) was first described as a glycoprotein produced exclusively in fetal liver and adult kidney that acts as a major regulator of erythropoiesis (1). However, Epo expression has been found in the human brain (2), and, recently, we have demonstrated that Epo mRNA also exists in the adult human retina (3). Epo has a potent neuroprotective effect in the brain as well as in the retina (4,5).

The retina is the most metabolically active tissue in the human body and, therefore, is highly sensitive to reductions in oxygen tension. Hypoxia-inducible factor (HIF) is the primary hypoxic signaling protein in cells for regulating angiogenesis and is able to induce the transcription of more than 70 genes, such as Epo (6). Indeed, HIF was discovered during studies of the regulation of Epo (7). Hypoxia is a major stimulus for both

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From the CIBERDEM (Centro de Investigacioń Biome´dica en Red de Diabetes y Enfermedades Metabo´licas) (Instituto de Salud Carlos III) and Diabetes Research Unit, Institut de Recerca Hospital Universitari Vall d’Hebron, Universitat Auto`noma de Barcelona, Barcelona, Spain. Corresponding author: Dr. Rafael Simo´, Diabetes Research Unit. Endocrinology Division, Hospital Universitari Vall d’Hebron, Pg. Vall d’Hebron 119-129, 08035 Barcelona, Spain. E-mail: [email protected]. Received for publication 29 October 2007 and accepted in revised form 4 March 2008. Published ahead of print at http://care.diabetesjournals.org on 10 March 2008. DOI: 10.2337/dc07-2075. Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/ dc07-2075. Abbreviations: BBB, blood-brain barrier; Epo, erythropoietin; EpoR, erythropoietin receptor; GFAP, glial fibrillar acidic protein; HIF, hypoxia-inducible factor; RPE, retinal pigment epithelium. © 2008 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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systemic (1) and intraocular Epo production (8), and high intravitreous levels of Epo have been reported in ischemic retinal diseases such as proliferative diabetic retinopathy (9 –11). In addition, it has been reported that Epo has an angiogenic potential equivalent to that of vascular endothelial growth factor (11,12). However, we also found elevated intravitreal levels of Epo in patients with diabetic macular edema, a condition in which neither hypoxia nor angiogenesis is a predominant event (3). Therefore, factors other than ischemia must be involved in the primary regulation of Epo production in the human retina. Regarding the Epo receptor (EpoR), its expression has been detected in the mouse retina (8,13) and in the human fetal retina (14), but until now it has not been investigated in the adult human retina. In a previous study, we found a higher expression of Epo mRNA in the retinas from diabetic than from nondiabetic subjects (3). In the present study, we have evaluated not only Epo but also EpoR expression (mRNA and protein) in the early stages of diabetic retinopathy. In addition, intravitreal levels of Epo and EpoR have also been measured. Furthermore, to gain insight into the mechanisms responsible for the regulation of Epo production in the retina, we also assessed the retinal expression of HIF-1␣ and HIF-2␣. RESEARCH DESIGN AND METHODS Human retinas Eighteen human postmortem eyes were obtained from nine diabetic donors (aged 64.1 ⫾ 8.1 years) free of funduscopic abnormalities in the ophthalmological examinations performed during the preceding 2 years. Eighteen eye cups obtained from nine nondiabetic donors matched by age (aged 66.9 ⫾ 6.8 years) were used as the control group. The time elapsed from death to eye enucleation was 3.4 ⫾ 1.9 h. After enucleation, one eye of each donor was snap-frozen in liquid nitrogen and stored at ⫺80°C until they 1189

Erythropoietin and its receptor in human retina 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. Quantification and quality of total mRNA were determined with a bioanalyzer in a LabChip (Agilent Technologies, Palo Alto, CA). One microgram of total RNA was directly used for reverse transcription, which was performed with TaqMan Reverse Transcription Reagents (Applied Biosystems, Madrid, Spain) in a 50-␮l reaction according to the manufacturer’s instructions. Quantitative realtime PCR was performed using 60 ng of the reverse transcription reaction as a template with TaqMan Universal MasterMix (Applied Biosystems). All reactions were conducted as follows: 95°C for 10 min and 50 cycles of 15 s at 95°C and 1 min at 60°C in an Applied Biosystems 7000 system. Each sample was assayed in duplicate and control water samples were included in each experiment. Automatic relative quantification data were obtained in an ABI Prism 7000 (SDS software; Applied Biosystems) using ␤-actin gene as the endogenous expression reference gene (Hs9999903_m1; Applied Biosystems). TaqMan premade gene expression assays (Applied Biosystems) were used to amplify human Epo (Hs00171267_m1, GenBank accession number NM 000799.2) and EpoR (Hs00181092_m1 Exon Boundary 6 –7, GenBank accession number NM 000121.2).

Figure 1—A: Real-time quantitative RT-PCR analysis of Epo mRNA in human retinas. Bars represent the mean ⫾ SD of the values obtained in the nine diabetic and the nine nondiabetic donors studied. Epo mRNA gene expression was calculated after normalizing with ␤-actin. B: Real-time quantitative RT-PCR analysis of EpoR mRNA in human retinas.

were assayed for mRNA analysis. The other eye was fixed in 4% paraformaldehyde and embedded in paraffin for the immunohistochemical study. The clinical features of diabetic and nondiabetic donors included in the study are shown in Table 1 of the online appendix (available at http://dx.doi.org/10.2337/dc072075). Three patients who were being treated with diet and/or oral agents were changed to insulin after admission, and the two patients who were being treated with insulin plus metformin were treated 1190

with insulin alone. All patients died within 15 days after admission. All ocular tissues were used in accordance with applicable laws and with the Declaration of Helsinki for research involving human tissue. In addition, this study was approved by the ethics committee of our hospital. RNA extraction and Epo mRNA quantification Human neuroretina and retinal pigment epithelium (RPE) were harvested using

HIF mRNA expression HIF-1␣ and HIF-2␣ mRNA transcripts were studied in the same samples with the TaqMan premade gene expression assays (Hs00153153_m1 and Hs01026138_m1, respectively; Applied Biosystems). Glial fibrillar acidic protein mRNA expression Glial fibrillar acidic protein (GFAP) mRNA was assessed in neuroretinas using a TaqMan premade gene expression assay (Hs00157674_m1; Applied Biosystems). Epo and EpoR immunofluorescence Paraffinized eyes were serially cut at 7-␮m thickness. Sections were deparaffinized with xylene and rehydrated in ethanol. Sections were placed in antigen-retrieval solution (Dako A/S, Glostrup, Denmark) at 95°C. To eliminate the autofluorescence of RPE due to melanin and lipofuscin we used a method described DIABETES CARE, VOLUME 31, NUMBER 6, JUNE 2008

Garcıá-Ramı´rez, Hernańdez, and Simo´ sponding to 10 field frame images (⫻40, numerical aperture 0.9) of each retina sample were measured. These results were then normalized, taking into account the area analyzed. All these calculations were made using specific software (FluoView ASW 1.4; Olympus). GFAP immunofluorescence Tissue sections were incubated overnight at 4°C with the primary antibody, antihuman GFAP (1:200; Sigma, Madrid, Spain). After washing, sections were incubated with Alexa Fluor 488 (Molecular Probes) secondary antibody for 1 h. GFAP immunofluorescence in the neuroretina was quantified using a laser confocal scanning microscope. The procedure was the same as that mentioned above for Epo and EpoR. Epo and EpoR assessment in vitreous fluid Epo was assessed by radioimmunoassay (Incstar/DiaSorin). The lowest limit of detection was 4.4 mU/ml. EpoR was measured by ELISA (R&D Systems, Minneapolis, MN). The lowest limit of detection was 30 pg/ml.

Figure 2—A: Comparison of Epo immunofluorescence (green) in the human retina between representative samples from a diabetic donor (a) and a nondiabetic donor (b). RPE, retinal pigment epithelium; PR, photoreceptors; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. The bar represents 20 ␮m. B: Quantification of Epo immunofluorescence in nondiabetic and diabetic retinas. N, neuroretina.

elsewhere (15). Sections were then incubated for 1 h with 1% BSA in 0.3% Triton X-100 in PBS to block unspecific binding of the antibodies and then incubated overnight at 4°C with a specific primary antibody to human Epo (1:100, N-19; Santa Cruz Biotechnology, Heidelberg, Germany) and EpoR (1:200, M-20; Santa Cruz Biotechnology). After washing, sections were incubated with Alexa Fluor 488 and 594 secondary antibodies, respectively (Molecular Probes, Eugene, OR), at room temperature for 1 h. Slides were cover-slipped with a drop of mounting medium containing 4,6-diamidino-2phenylindole for visualization of cell DIABETES CARE, VOLUME 31, NUMBER 6, JUNE 2008

nuclei (Vector Laboratories, Burlingame, CA). Image acquisition Images were acquired with a confocal laser scanning microscope (FV1000; Olympus, Hamburg, Germany), using a 488-nm laser line for Epo and a 594-nm laser for EpoR. Each image was saved at a resolution of 1,024 ⫻ 1,024 pixel image size. Image analysis To quantify Epo and EpoR immunofluorescence in RPE and the neuroretina, the total fluorescence intensity values corre-

Intravitreous proteins Vitreal proteins were measured by a previously validated microturbidimetric method with an autoanalyzer (Hitachi 917; Boehringer Mannheim). This method, based on the benzethonium 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. Intraand interassay coefficients of variation were 2.9 and 3.7%, respectively. Statistical analysis Student’s t test was used to compare the variables. Correlations were examined by Spearman’s rank correlation. Levels of statistical significance were set at P ⬍ 0.05. RESULTS Epo and EpoR mRNAs in the human retina: comparison between diabetic and nondiabetic donors ␤-Actin mRNA expression was similar in both the RPE and the neuroretina (NS). In addition, no differences were observed in ␤-actin mRNA expression between dia1191

Erythropoietin and its receptor in human retina P ⫽ 0.04) and nondiabetic donors (r ⫽ 0.90; P ⫽ 0.001) HIF-1␣ and HIF-2␣ mRNA expressions were similar for diabetic and nondiabetic donors in both RPE (HIF-1␣ 0.44 ⫾ 0.26 vs. 0.42 ⫾ 0.37; P ⫽ 0.89 and HIF-2␣ 0.43 ⫾ 0.26 vs. 0.48 ⫾ 0.41; P ⫽ 0.82) and the neuroretina (HIF-1␣ 0.46 ⫾ 0.24 vs. 0.36 ⫾ 0.31; P ⫽ 0.36 and HIF-2␣ 0.29 ⫾ 0.16 vs. 0.24 ⫾ 0.21; P ⫽ 0.58). HIF-2␣ mRNA correlated with Epo mRNA in diabetic donors (r ⫽ 0.62; P ⫽ 0.03) but not in nondiabetic donors (r ⫽ 0.26; P ⫽ 0.33). We did not find any significant differences in Epo and EpoR mRNA levels between patients who were changed to insulin treatment after admission and patients who continued to receive oral treatment.

Figure 3—A: Comparison of EpoR immunofluorescence (red) in the human retina between representative samples from a diabetic donor (a) and a nondiabetic donor (b). PR, photoreceptors; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. The bar represents 20 ␮m. B: Quantification of EpoR immunofluorescence in nondiabetic and diabetic retinas. N, neuroretina.

betic and nondiabetic retinas (NS). Thus, we have calculated mRNA gene expression after normalization with ␤-actin. Significantly higher Epo mRNA expression was observed in RPE than in the neuroretina in the whole group (1.13 ⫾ 1.01 vs. 0.26 ⫾ 0.31; P ⫽ 0.003). Higher expression was also observed in RPE than in the neuroretina when diabetic donors and nondiabetic donors were analyzed separately (1.63 ⫾ 1.27 vs. 0.43 ⫾ 0.41; P ⫽ 0.03 and 0.68 ⫾ 0.38 vs. 0.13 ⫾ 0.08; P ⫽ 0.002). In addition, a higher expression of Epo mRNA was detected in the retina from diabetic donors compared with nondiabetic donors (Fig. 1A). 1192

A significantly higher level of mRNA EpoR was observed in RPE than in the neuroretina in the whole group (1.48 ⫾ 1.24 vs. 0.37 ⫾ 0.41; P ⫽ 0.002). Higher expression was also detected in RPE than in the neuroretina when diabetic donors and nondiabetic donors were analyzed separately (1.62 ⫾ 1.16 vs. 0.33 ⫾ 0.24; P ⫽ 0.01 and 1.35 ⫾ 1.36 vs. 0.41 ⫾ 0.55; P ⫽ 0.07). However, we did not find any difference between EpoR mRNA levels in diabetic and nondiabetic donors in either RPE or the neuroretina (Fig. 1B). In the neuroretina but not in RPE, a direct correlation between EPO and EpoR mRNA was detected in diabetic (r ⫽ 0.73;

Epo content in the human retina: comparison between diabetic and nondiabetic donors Laser scanning confocal images of Epo and EpoR immunofluorescence are displayed in Figs. 2 and 3. Epo immunofluorescence intensity was higher in diabetic donors than in nondiabetic donors in both RPE (5,770 ⫾ 1,491 vs. 3,933 ⫾ 1,530; P ⫽ 0.05) and in the neuroretina (5,644 ⫾ 3,671 vs. 3,671 ⫾ 1,401; P ⫽ 0.06) (Fig. 2). EpoR immunofluorescence was higher in RPE than in the neuroretina in both diabetic donors (8,883 ⫾ 2,171 vs. 4,734 ⫾ 2,474; P ⬍ 0.001) and nondiabetic donors (9,079 ⫾ 6,260 vs. 4,763 ⫾ 3,680; P ⫽ 0.07). However, no differences in EpoR were detected between diabetic and nondiabetic donors either in the RPE or in the neuroretina (Fig. 3). A direct correlation was observed between Epo and EpoR in diabetic donors (RPE: r ⫽ 0.87; P ⫽ 0.002; neuroretina: r ⫽ 0.86; P ⫽ 0.01) and nondiabetic donors (RPE: r ⫽ 0.94; P ⫽ 0.005; neuroretina: r ⫽ 0.24; P ⫽ 0.5). Neuroglial activation GFAP mRNA expression was higher in the neuroretina from diabetic donors than from nondiabetic donors (0.40 ⫾ 0.21 vs. 0.13 ⫾ 0.14; P ⫽ 0.04). Confocal microscopy revealed a higher immunofluorescence intensity in the neuroretinas from diabetic donors (10148 ⫾ 3,909 vs. 6,798 ⫾ 1,544; P ⫽ 0.04) (Fig. 4), thus confirming that neuroglial activation does exist. DIABETES CARE, VOLUME 31, NUMBER 6, JUNE 2008

Garcıá-Ramı´rez, Hernańdez, and Simo´

Figure 4—A: Comparison of GFAP immunofluorescence (green) in the human retina between representative samples from a nondiabetic donor (a) and a diabetic donor (b). PR, photoreceptors; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. In the diabetic retina, the endfeet of the Mu¨ller cells show abundant GFAP immunofluorescence, and the radial processes stain intensely throughout both the inner and outer retina. The bar represents 20 ␮m. B: Quantification of GFAP immunofluorescence in nondiabetic and diabetic retinas.

Epo and EpoR assessment in vitreous fluid: comparison between diabetic and nondiabetic retinas Intravitreal Epo concentration was higher in diabetic donors than in nondiabetic control subjects (124 ⫾ 92 vs. 36 mU/ ml ⫾ 13; P ⫽ 0.03). However, no differences in intravitreal EpoR concentration were detected between diabetic donors and nondiabetic control subjects (99 ⫾ 58 vs. 96 ⫾ 86 pg/ml; P ⫽ 0.95). Because intravitreal proteins were similar in diabetic and in nondiabetic donors, the results were not influenced by this confounding factor. DIABETES CARE, VOLUME 31, NUMBER 6, JUNE 2008

CONCLUSIONS — In the present study we have found a higher expression of Epo (both mRNA and protein) in the retinas from diabetic donors without clinically detectable diabetic retinopathy in comparison with the retinas from nondiabetic donors. In addition, intravitreal Epo concentrations were significantly higher in diabetic donors than in nondiabetic donors. Therefore, our results suggest that Epo overexpression is an early event in the retina of diabetic patients. It is important to note that in the retinas from diabetic donors, reactive changes in Mu¨ller cells such as upregulation of GFAP

were present. Therefore, although the retinas from diabetic donors were without overt vascular abnormalities, they were actually already being damaged by the diabetic milieu. In fact, glial activation and neurodegeneration have been described as early events in the pathogenesis of diabetic retinopathy (16). The complete mechanisms that regulate Epo expression in the human retina remain to be elucidated. The hypoxiainduced upregulation of Epo expression is modulated at the transcriptional level by HIF and in particular by HIF-2␣ (17). In the present study, no differences between HIF-1␣ and HIF-2␣ were detected between diabetic and nondiabetic donors. These findings suggest not only that Epo overexpression is an early event in the retina of diabetic patients but also that at this stage it is unrelated to a hypoxicischemic stimulus. In support of this concept, it should be stressed that EpoR expression, which is upregulated by hypoxia in rat brains (18) and in rat retinas (8), was similar in diabetic and in nondiabetic retinas. It should also be noted that EpoR expression has not been described previously in human retina. Apart from hypoxia, other factors could regulate Epo expression. Watanabe et al. (11) observed an increase in the vitreous Epo levels in patients with inflammatory eye diseases. Given that inflammation has been involved in the pathogenesis of diabetic retinopathy (19), it might be a contributing factor to the high levels of Epo observed in diabetic patients. Hyperglycemia could be another factor that induces Epo production. Although there are no studies evaluating the effect of glucose on Epo expression in retinal cells, a direct relationship has been shown between glucose and Epo concentrations in a Chinese hamster ovary cell line (20). A reduction in Epo catabolism could also contribute to the higher Epo levels detected in the retina and the vitreous fluid from diabetic donors. In this regard, the glycosylation of Epo reduces its affinity for EpoR (21). Because Epo is degraded only by EpoR-expressing cells and their receptor binding determines the rate of intracellular degradation (22), it is possible that a higher degree of Epo glycosylation is associates with lower clearance of Epo. The mechanisms causing the increase in Epo in the diabetic retina cannot be identified in the present study. However, there are many reasons for thinking that in these early stages of diabetic retinopa1193

Erythropoietin and its receptor in human retina thy Epo might have beneficial rather than pathogenic actions. First, it has been demonstrated using an in vitro model of the bovine blood-brain barrier (BBB) that Epo protects against the vascular endothelial growth factor–induced permeability of the BBB and restores the tight junction proteins (23). Epo treatment also prevents an increase in BBB permeability in a rat model of induced seizures (24). Because the blood-retinal barrier is structurally and functionally similar to the BBB, it is possible that Epo could act as an antipermeability factor in the retina. In fact, Epo was able to improve diabetic macular edema when it was administered for treatment of anemia in diabetic patients with renal failure (25). Second, there is growing evidence that Epo is a neurotrophic factor not only in the brain but also in the retina (4,5). Third, Epo exerts an anti-inflammatory effect on the brain (26), and this action might also be extrapolated to the diabetic retina. Finally, Epo is a potent physiological stimulus for the mobilization of endothelial progenitor cells (27), and, therefore, it could play a relevant role in regulating the traffic of circulating endothelial progenitor cells toward injured retinal sites. We conclude that Epo overexpression is an early event in the retina of diabetic patients and that at this stage it is not mediated by HIF. This finding suggests that other factors beside hypoxia might be crucial in the upregulation of Epo in the initial stages of diabetic retinopathy. EpoR is also expressed in the retina from diabetic patients, but it is similar in nondiabetic subjects. Further studies are needed to investigate not only the precise regulation of Epo/EpoR in the retina in physiological and pathological conditions but also its potential role in the therapeutic armamentarium. Acknowledgments — This study was supported by grants from the Instituto de Salud Carlos III (CIBERDEM), the Ministerio de Ciencia y Tecnologıá (SAF2006-05284), and the Fundacioń para la Diabetes and the Generalitat de Catalunya (2005SGR-00030).

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