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Diabetes Care 25:1060-1065, 2002
© 2002 by the American Diabetes Association, Inc.

Pathophysiology/Complications
Original Article

Neurotrophic Factor Receptors in Epiretinal Membranes After Human Diabetic Retinopathy

Takayuki Harada, MD, PHD1,2, Chikako Harada, MD, PHD1,2, Yoshinori Mitamura, MD, PHD3,4, Chihiro Akazawa, MD, PHD5, Kenji Ohtsuka, MD, PHD4, Shigeaki Ohno, MD, PHD2, Shinobu Takeuchi, MD, PHD3 and Keiji Wada, MD, PHD1

1 Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan
2 Department of Ophthalmology and Visual Sciences, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan
3 Toho University Sakura Hospital, Sakura, Chiba, Japan
4 Sapporo Medical University, Sapporo, Hokkaido, Japan
5 Department of Neurochemistry, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 CONCLUSIONS
 References
 
OBJECTIVE—Formation of epiretinal membranes (ERMs) in the posterior fundus results in progressive deterioration of vision. ERMs have been associated with numerous clinical conditions, including proliferative diabetic retinopathy (PDR), but its pathogenic mechanisms are still unknown. This study was conducted to determine whether neurotrophic factor receptors (tyrosine kinase receptors trkA, trkB, and trkC; low-affinity neurotrophin [NT] receptor p75 [p75NTR]; glial cell line–derived neurotrophic factor receptor-{alpha}1 [GFR{alpha}1] and GFR{alpha}2; and Ret) are involved in the formation of ERMs after PDR.

RESEARCH DESIGN AND METHODS—ERM samples were obtained by vitrectomy from 19 subjects with PDR aged 57 ± 8 years with 17 ± 8 years of diabetes and 15 subjects with idiopathic ERM. They were processed for RT-PCR analysis. In addition, 11 ERM samples from PDR patients aged 47 ± 18 years with 13 ± 4 years of diabetes were processed for immunohistochemical analysis.

RESULTS—Expressions of trkA, trkB, trkC, p75NTR, and Ret mRNAs were similar in both groups. In contrast, GFR{alpha}2 expression levels were significantly higher (17 of 19 vs. 2 of 15 subjects in idiopathic ERM, P < 0.0001) in PDR subjects. Accordingly, immunohistochemical analysis revealed expression of GFR{alpha}2 protein in all of the 11 ERMs derived from PDR patients, and that region was double-labeled with glial cell-specific markers. On the other hand, GFR{alpha}1 expression was lower (8 of 19 vs. 12 of 15 subjects with idiopathic ERM, P = 0.0258) in PDR subjects.

CONCLUSIONS—These results suggest a possibility that glial cell line-derived neurotrophic factor receptor (GDNF) subtypes are differently involved in the formation of ERMs.

Abbreviations: bFGF, basic fibroblast growth factor • CNS, central nervous system • ERM, epiretinal membrane • GFR • glial cell line–derived neurotrophic factor receptor • GDNF, glial cell line–derived neurotrophic factor • G3PDH, glyceraldehyde-3-phosphate dehydrogenase • NGF, nerve growth factor • NT, neurotrophin • NTN, neurturin • p75NTR, low-affinity NT receptor p75 • PDR, proliferative diabetic retinopathy • PVR, proliferative vitreoretinopathy • TGF-ß, transforming growth factor-ß • trk, tyrosine kinase receptor.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESEARCH DESIGN AND METHODS
 RESULTS
 CONCLUSIONS
 References
 
Epiretinal membranes (ERMs) involving the macular or perimacular regions can cause a reduction in vision, metamorphopsia, micropsia, or occasionally monocular diplopia. These are unfortunately relatively common diseases. The incidence of ERMs increases with age and may approach 20% of the total population by age 70 (1). The presence of ERMs has been associated with various clinical conditions, including postretinal detachment repair, nonproliferative retinovascular disorders, trauma, ocular inflammatory disorders, proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), vitreous hemorrhage, congenital disorders, and idiopathic or spontaneous developments (2). The prevalence of ERMs in PDR is reported to be ~20% in type 1 diabetes and ~5% in type 2 diabetes (3,4). Histopathological studies have shown that ERMs are composed of various cell types such as glial cells, fibroblasts, and endothelial cells (1,5–7), but their pathogenic mechanisms are still unknown. On the other hand, different trophic factors and cytokines, such as basic fibroblast growth factor (bFGF) and interleukin-6, have been detected in ERMs and vitreous fluid, coincident with ocular diseases such as PDR and PVR (5,6,8–12). Because some peptide factors are soluble mediators of angiogenesis, it seems possible that diabetic neovascularization and PVR are caused or aggravated by these factors (9).

In recent years, many trophic factors have been shown to act in neural cell survival and differentiation mediated by their specific receptors (13–16). For example, biological activity of the nerve growth factor (NGF) family of neurotrophins (NTs; e.g., NGF, brain-derived neurotrophic factor, and NT-3) is mediated by two types of transmembrane glycoproteins, the tyrosine kinase receptors (trkA, trkB, and trkC), and the low-affinity NT receptor p75 (p75NTR) (13–16). Glial cell line–derived neurotrophic factor (GDNF) and neurturin (NTN) are other trophic factors that are structurally related and are distant members of the transforming growth factor-ß (TGF-ß) superfamily, with prominent capacities to promote survival of distinct populations of central and peripheral neurons (17–19). They mediate their actions through the multicomponent receptor system, comprising a high-affinity ligand-binding coreceptor GDNF receptor family {alpha}-component (GFR{alpha}) and the transmembrane protein tyrosine kinase Ret (20–22). Although the preferred interactions appear to be GDNF-GFR{alpha}1 and NTN-GFR{alpha}2 in vivo, there is the prevalence of cross-interaction between NTN-GFR{alpha}1 (23). The distribution of GDNF, NTN, and their receptors in the central nervous system (CNS) have previously been described (21–23). These studies showed overlapping patterns of expression for GDNF, NTN, and their receptors, indicating multiple mechanisms of GDNF and NTN action in the CNS. Interestingly, the latest studies have shown that GDNF functions are not restricted to neurons but instead are implicated in glial development (24). Together with the previous findings that the glial cell is one of the main components of ERMs, these results suggest a possibility that GDNF receptors may be involved in the formation of ERMs.

In the present study, we examined the expression of various trophic factor receptor genes (trkA, trkB, trkC, p75NTR, GFR{alpha}1, GFR{alpha}2, and Ret) in ERMs obtained from PDR and control patients, and we found high expression levels of GFR{alpha}2 mRNAs in PDR. In addition, immunohistochemical analysis revealed the GFR{alpha}2 protein in the glial component of ERMs. We also proposed a possible relationship between GFR{alpha} expressions and ERM formation.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
RESULTS
 CONCLUSIONS
 References
 
This study was carried out in accordance with the tenets of the Helsinki Declaration. Informed consent was obtained from each patient for the collection of samples. Criteria for inclusion in the study were age <80 years, absence of renal or hematological diseases or uremia, no administration of chemotherapy or life-support measures, and the fewest possible chronic pathologies other than diabetes. The ERMs were surgically removed from consecutive eyes with secondary ERM after PDR (30 eyes) or idiopathic ERM (control subjects, 15 eyes) undergoing pars plana vitrectomy and membrane peeling. Membranes were dissected from the retinal surface with horizontal scissors or membrane pick. Samples derived from 19 of the PDR patients (aged 57 ± 8 years, duration of diabetes 17 ± 8 years) and 15 control subjects (aged 68 ± 7 years) were processed for RT-PCR analysis. The remainder of the PDR samples (from subjects aged 47 ± 18 years, duration of diabetes 13 ± 4 years) were processed for immunohistochemistry. These samples were embedded in optimum cutting temperature compound (Miles Laboratories, Naperile, IL), flash-frozen in liquid nitrogen, and then stored at -80°C.

RNA extraction and amplification by RT-PCR
Total cellular RNA was prepared using Isogen reagent (Nippon Gene, Tokyo) according to the manufacturer’s protocol. Surgical materials were immediately homogenized in 800 µl of isogen, and 200 µl of chloroform was then added. After centrifugation at 4°C, the aqueous phase was collected, and total RNA was precipitated with an equal volume of isopropanol. RNA was then dissolved in 12 µl of water treated with diethyl pyrocarbonate. Next, 0.1 µg of RNA extracted from each sample was reverse-transcribed into first-strand cDNA using the Superscript Preamplification System (Gibco, Paisley, Scotland, U.K.) and oligo-dT primers.

RT-PCR analysis was performed as previously described (25). A volume of 1 µl of each cDNA product from the reverse transcription procedure was used as the template for PCR amplification in a reaction mixture containing PCR buffer (10 mmol/l Tris-HCl, pH 8.3, 50 mmol/l KCl, 1.5 mmol/l MgCl2), 0.2 mmol/l dNTPs, a 0.2 mmol/l set of oligonucleotide primers, and 2.5 units Taq DNA polymerase in a final volume of 50 µl. Complementary DNA reverse-transcribed from total RNA was amplified by using primers specific for trkA (sense: 5'-GCA TCT GGA GCT CCG TGA TC-3'; antisense: 5'-CTC TGC CCA GCA CGT CAA GT-3'), trkB (sense: 5'-AAG ACC CTG AAG GAT GCC AG-3'; antisense: 5'-AGT AGT CAG TGC TGT ACA CG-3'), trkC (sense: 5'-CTA CAA CCT CAG CCC GAC CA-3'; antisense: 5'-GCT GTA GAC ATC TCT GGA CA-3'), p75NTR (sense: 5'-TGG ACA GCG TGA CGT TCT CC-3'; antisense: 5'-GAT CTC CTC GCA CTC GGC GT-3'), GFR{alpha}1 (sense: 5'-AAG CAC AGC TAC GGG ATG CT-3'; antisense: 5'-GGT CAC ATC TGA GCC ATT GC-3'), GFR{alpha}2 (sense: 5'-ACG AGA CCC TCC GCT CTT TG-3'; antisense: 5'-GGG AGG CTT CGT AGA ACT CCT C-3'), Ret (sense: 5'-TGG CAA TTG AAT CCC TTT TT-3'; antisense: 5'-ATG CCA TAG AGT TTG TTT TC-3'), bFGF (sense: 5'-GAG AAG AGC GAC CCT CAC A-3'; antisense: 5'-TAG CTT TCT GCC CAG GTC C-3'), and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (sense: 5'-ACC ACA GTC CAT GCC ATC AC-3'; antisense: 5'-TCC ACC ACC CTG TTG CTG TA-3').

PCR was performed after initial denaturation at 94°C for 3 min. Each cycle consisted of a heat-denaturation step at 94°C for 15 s; annealing of primers at either 55°C (bFGF and G3PDH), 56°C (trkB and trkC), 58°C (trkA, p75NTR, and GFR{alpha}2), or 60°C (GFR{alpha}1 and Ret) for 2 min; and then polymerization at 72°C for 2 min. The expected sizes of the amplified cDNA fragments of trkA, trkB, trkC, p75NTR, GFR{alpha}1, GFR{alpha}2, Ret, bFGF, and G3PDH were 565, 406, 448, 371, 441, 339, 439, 277, and 452 bp, respectively. Negative controls for PCR were performed using "templates" derived from RT reactions lacking either reverse transcriptase or total RNA. After 35 cycles, 15 µl of each reaction mixture were electrophoresed on a 2% Tris borate–EDTA agarose gel and stained with ethidium bromide. In some experiments, human cDNA acquired from skeletal muscle (26) or testis (Takara, Kyoto, Japan) was used as positive control.

Immunohistochemistry
Frozen sections (7-µm thick) were cut by a cryostat, mounted on 3-aminopropyltriethoxysilane–coated glass slides, and air-dried at room temperature. For immunohistochemical analysis, the sections were fixed in ice-cold acetone and then washed with PBS. The sections were incubated with normal donkey serum for 30 min to block nonspecific staining. They were stained with a goat polyclonal antibody against GFR{alpha}2 (1.0 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA). For double-labeling immunofluorescence studies, they were then incubated with a mouse monoclonal antibody against vimentin (1x; Zymed, San Francisco, CA) or glutamine synthetase (1.0 µg/ml; Chemicon, Temecula, CA) (27). GFR{alpha}2 was visualized with fluorescein isothiocyanate–conjugated donkey anti-goat IgG (200x; Jackson Immunoresearch, West Groove, PA), and vimentin or glutamine synthetase was visualized with Cy3-conjugated donkey anti-mouse IgG (200x; Jackson Immunoresearch). The sections were examined by a confocal laser scanning microscope (Olympus, Tokyo). GFR{alpha}2 primary antibody preabsorbed by blocking peptide (Santa Cruz Biotechnology) was used for negative controls. The adjacent section of each specimen was stained with hematoxylin and eosin.

Statistics
Data are presented as the means ± SEM, except as noted. The {chi}2 test was used to test for significance of the difference between presence of various mRNAs examined in PDR versus control subjects. Statistical significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
CONCLUSIONS
 References
 
Expression of receptors for trophic factors in ERMs was examined by RT-PCR analysis. As shown in Tables 1 and 2, mRNAs encoding high-affinity (trkA, trkB, and trkC) and low-affinity (p75NTR) NT receptors were expressed at similar levels in both PDR and idiopathic ERM (control) patients. Next, we examined the expression of GDNF receptors (Fig. 1). Surprisingly, GFR{alpha}2 mRNA (339 bp) was detected in 17 of 19 (89%) PDR patients, but in only 2 of 15 (13%) control subjects (P < 0.0001). On the other hand, GFR{alpha}1 mRNA (441 bp) was detected in 12 of 15 (80%) control subjects, but in only 8 of 19 (42%) PDR patients (P = 0.0258). Ret mRNA (439 bp) was detected in 6 of 19 (32%) PDR patients and 5 of 15 (33%) control subjects (P = 0.9135). We also examined the relationship between GFR{alpha} and Ret mRNA expressions in both PDR and control patients. Interestingly, GFR{alpha}2-positive and Ret-negative patients were found among 12 of 19 (63%) of those with PDR, but in none (0%) of the control subjects. In addition, GFR{alpha}1-positive and Ret-negative subjects were found among 8 of 15 (53%) control subjects, but in only 2 of 19 (11%) PDR patients.


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  		Table 1— Patients and results of RT-PCR on the PDR and idiopathic ERMs

 

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  		Table 2— Patients that were positive for presence of mRNA

 


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  		Figure 1— RT-PCR analysis of GDNF receptors in ERMs derived from patients summarized in Table 1. cDNA derived from ERMs with PDR (patients 1–19) and idiopathic ERMs (patients 20–34) was analyzed by PCR using primers specific for 441-bp GFR{alpha}1, 339-bp GFR{alpha}2, and 439-bp Ret, respectively. As controls for cDNA integrity, the cDNA was also amplified with primers for 452-bp G3PDH. After 35 cycles, 15 µl of each sample was electrophoresed on a 2% Tris borate–EDTA agarose gel and stained with ethidium bromide. Note the high expression levels of GFR{alpha}2 as well as the low expression levels of GFR{alpha}1 in ERMs derived from PDR patients. P, positive controls using human cDNA acquired from skeletal muscle.

 
Immunohistochemical analysis was performed to identify the GFR{alpha}2 protein expression in ERMs after PDR. GFR{alpha}2 protein was detected in all of the 11 samples examined (example in Fig. 2A). The immunoreactivity was completely abolished when primary antibody was preabsorbed with blocking peptide (Fig. 2D). GFR{alpha}2 may be involved in the formation of glial components in ERMs (10,28), so we examined whether GFR{alpha}2 is coexpressed with the glial cell–specific marker vimentin. ERMs contained a large area composed of glial cells (Fig. 2B), and many cells in that area were double-labeled with GFR{alpha}2 (Fig. 2C). Similar results were obtained in experiments using glutamine synthetase, another glial cell marker (data not shown).



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  		Figure 2— Expression of GFR{alpha}2 (A) and vimentin (B) in the ERM derived from a PDR patient aged 52 years with a 13-year history of diabetes. C: Double-labeling method demonstrates the expression of GFR{alpha}2 protein in glial regions. D: Negative control stained with GFR{alpha}2 antibody preabsorbed with blocking peptide. E: Hematoxylin and eosin staining of the adjacent section. Bar represents 200 µm.

 
Previous studies have shown the high expression level of bFGF in secondary ERMs, coincident with ocular diseases such as PDR and PVR (3,10,16,17,19). We also examined the bFGF mRNAs and detected them in 13 of 19 (68%) PDR patients and 10 of 15 (67%) control subjects (P = 0.9135) (Tables 1 and 2). In 6 of 11 (55%) PDR samples, bFGF immunoreactivity was observed in the large area and partially double-labeled with vimentin or GS (data not shown), as well as GFR{alpha}2. Although it was not specific for PDR, we could also determine high expression levels of bFGF mRNA and protein in ERMs.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 CONCLUSIONS
References
 
This study was the first to demonstrate high expression levels of GFR{alpha}2 mRNA in ERMs after PDR. Such a high expression level was specific for GFR{alpha}2 and was not observed in other NT receptors and Ret. In addition, we showed that the GFR{alpha}2 protein is coexpressed with glial cell–specific markers. Together with the recent findings that GDNF functions are not restricted to neurons only, but also implicated in glial differentiation (29), our results suggest the possibility that GFR{alpha}2 may be involved in the abnormal glial proliferation that leads to the formation and development of PDR membranes. The mechanism of ERM formation in PDR is still unknown, but its first step is thought to be the neovascularization after the interaction of retinal vessels with the vitreous. In this process, many angiogenic factors (e.g., angiogenin, platelet-derived endothelial cell growth factor, vascular endothelial cell growth factor, and prostaglandins) play an important role (9,30–32). Thus, blocking these angiogenic factors may lead to the inhibition of pathological angiogenesis and ERM formation.

However, ERM is composed of many cell types, such as retinal pigment epithelial cells, glial cells, fibroblasts, myofibroblasts, fibrous astrocytes, macrophages, and endothelial cells (1,5–7). Because these cells surround newly formed vessels, PDR membranes may be stimulated by trophic factors released from vascular components. In turn, because glial cells produce and/or store growth factors, which activate vascular cells (29,33–35), they may further stimulate neovascularization as well as the proliferation of other cell types. Thus, inhibiting the enlargement of the glial component may be one of the promising strategies to prevent the development of ERMs. On the other hand, expression of another GFR{alpha} receptor, GFR{alpha}1, was found to be significantly higher in idiopathic ERMs. Idiopathic ERMs are thought to be nonangiogenic "fibroglial membrane," and the glial cells appear to derive from the underlying retina, traversing the inner limiting membrane of the retina through surface breaks (1,5–7). Thus, the retina may be the important endogenous supplier of GDNF and/or NTN in idiopathic ERMs. In fact, though not determined in the human retina, GDNF and NTN are widely localized to the rodent retina (28,36,37). Such a different route and condition of trophic factor supply may upregulate different GFR{alpha} receptor subtypes in PDR and idiopathic ERM. Recent studies have shown that GFR{alpha} receptors can deliver the intracellular GDNF signaling in the absence of Ret (38). Because GFR{alpha} receptors are usually coexpressed with and mediate GDNF binding to Ret (39,40), imbalance in the expressions of these two receptors may represent one of the causes of ERM development.

Finally, although bFGF is reported to play a key role in angiogenesis after PDR and PVR (5,6,8–12), high expression levels of bFGF were detected in both primary and secondary ERMs (Table 1). These results may suggest that bFGF is a general and important player in the formation of preretinal tissues under various conditions (10,11). On the other hand, because high GFR{alpha}2 expression levels were specific to PDR membranes (Table 1 and Fig. 1), GFR{alpha}2 may be involved in the pathogenesis of ERMs after PDR. If so, inhibiting biological activity of GFR{alpha}2 by specific blocker, antisense oligonucleotide, or neutralizing antibodies may be useful in preventing the development of PDR membranes, especially in avoiding membrane recurrence. Further investigations on the precise role of GFR{alpha}2 in the process of membrane formation will be needed.


   Acknowledgments
 
This study was supported by Ministry of Health, Labour and Welfare of Japan; the Ministry of Education, Culture, Sports, Science and Technology of Japan; a Human Frontier Science Program long-term fellowship (LT00170–2001/B to T.H.); and a postdoctoral fellowship from Uehara Memorial Foundation (to C.H.).

We thank Pia Lippincott for critical reading of the manuscript.


   Footnotes
 
Address correspondence and reprint requests to Takayuki Harada, MD, PhD, Department of Molecular Neuroscience, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. E-mail: harada.aud{at}mri.tmd.ac.jp.

Received for publication 3 November 2001 and accepted in revised form 13 March 2002.

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


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 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 CONCLUSIONS
 References
 

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