Activator protein-1 in epiretinal membranes of patients with proliferative diabetic retinopathy

Abstract

To the Editor: Angiogenesis is the most important event in proliferative diabetic retinopathy. Previous studies indicated that activator protein-1 (AP-1) is involved in angiogenesis [1, 2]. AP-1 is a transcription factor and a homodimer or heterodimer mainly composed of Jun-Jun and Jun-Fos. The gene encoding Jun was originally identified in avian sarcoma virus 17, and the abbreviation ‘Jun’ is derived from the Japanese ‘ju-nana’, which means 17. cJun (encoded by JUN) is a member of the Jun family and is a principal component of AP-1. The aim of the present study was to assess the potential role of AP-1 in the pathogenesis of proliferative diabetic retinopathy. For this purpose, we examined the expression of JUN mRNA in epiretinal membranes secondary to proliferative diabetic retinopathy. Because perivascular glial cells are essential for the regulation of retinal angiogenesis [3], we also examined levels of phosphorylated c-Jun in glial cells from epiretinal membranes. Epiretinal membranes were surgically removed from consecutive patients with proliferative diabetic retinopathy (23 type 2 diabetic patients) or idiopathic epiretinal membranes (controls, 16 non-diabetic patients) during pars plana vitrectomy. The epiretinal membrane in proliferative diabetic retinopathy is mainly composed of neovascular vessels. On the other hand, idiopathic epiretinal membrane is a non-angiogenic fibroglial membrane that is not associated with diabetes mellitus. Samples derived from 19 of the 23 patients with proliferative diabetic retinopathy and the 16 control patients were processed for RT-PCR analysis. The remaining samples from the diabetic patients were processed for immunohistochemistry. Informed consent was obtained from each patient. The present investigations were carried out in accordance with the principles of the Declaration of Helsinki as revised in 2000. RT-PCR analysis was performed as previously described [4, 5]. The primer sequences used to target the genes in PCR were as follows: JUN: 5′-GCATGA GGA ACC GCA TCG CTG CCT CCA AG-3′ and 5′-GAC CAA GTC CTT CCC ACT CGT GCA CAC TG-3′; and GAPDH: 5′-ACC ACA GTC CAT GCC ATC AC-3′ and 5′-TCC ACC ACC CTG TTG CTG TA-3′. Reactions were conducted under the following conditions: precycling at 94°C for 3 min, then 35 cycles consisting of denaturation at 94°C for 30 s, annealing at either 62°C (JUN) or 55°C (GAPDH) for 30 s and polymerisation at 72°C for 1 min. The expected sizes of the amplified cDNA fragments of JUN and GAPDH were 409 and 452 bp, respectively. Immunohistochemical analysis was performed as previously described [4, 5]. The following antibodies were used to detect antigens: a goat polyclonal antibody against phosphorylated c-Jun (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a mouse monoclonal antibody against vimentin (1:500 dilution; Nichirei, Tokyo, Japan). The antibodies were detected with Cy3-conjugated donkey anti-goat IgG (1:1,000 dilution; Jackson Immunoresearch, West Grove, PA, USA) and Cy2-conjugated donkey anti-mouse IgG (1:200 dilution; Jackson Immunoresearch), respectively. To visualise all nuclei, the Y. Mitamura (*) . A. Tashimo . K. Ohtsuka Department of Ophthalmology, School of Medicine, Sapporo Medical University, S-1, W-16, Chuo-ku, Sapporo, 060-8543 Japan e-mail: ymita@sapmed.ac.jp Tel.: +81-11-611-2111 Fax: +81-11-613-6575