Antisense oligonucleotide targeting c-fos mRNA limits retinal pigment epithelial cell proliferation: a key step in the progression of proliferative vitreoretinopathy

Exp Eye Res. 2006 Dec;83(6):1405-11. doi: 10.1016/j.exer.2006.07.020. Epub 2006 Sep 12.

Abstract

The purpose of this work was to investigate the effect of c-fos antisense oligonucleotide (c-fos-AS-ON) on proliferative vitreoretinopathy (PVR). Cultures of human retinal pigment epithelial (hRPE) cells were established from adult human corneal donors. These cells were positively stained for cytokeratins. C-fos-AS-ON effect on serum-stimulated cell proliferation was estimated by evaluating the incorporation of 5-bromo-2'-deoxy-uridine (BrdU) into cellular DNA. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were respectively performed to quantify the serum-stimulated c-fos gene mRNA and protein expression in hRPE cells. Eight rabbits (16 eyes) were divided into c-fos-AS-ON treatment group and control group. 2.5 x 10(5) cultured hRPE cells were injected into the vitreous cavity of eyes to establish a PVR model. Prevalence of PVR and retinal detachment were determined by indirect ophthalmoscopy on days 1, 3, 7, 14, 21 and 28 post-injection and by pathological study on days 28 post-injection. The results showed that blocking the expression of c-fos by the addition of c-fos-AS-ON to the culture medium significantly inhibited the hRPE cells proliferation. This effect of c-fos-AS-ON was found to be sequence specific (the use of a sense or a mismatch sense oligonucleotide had no such an effect) and dose-dependent (0.375 microM was the lowest effective dose tested). Growth inhibition by c-fos-AS-ON remained for at least 72 h. By using RT-PCR and Western blotting, we found that the c-fos-AS-ON could specifically inhibit c-fos mRNA and protein synthesis in cultured hRPE cells. Though the eyes injected with c-fos-AS-ON also developed features of PVR, the severities of days 14, 21 and 28 post-injection were significantly lower than those in the control eyes (P<0.05). We conclude that c-fos-AS-ON can inhibit cultured hRPE cell proliferation, which mechanism may relate to blocking the expression of c-fos and can reduce the prevalence of experimental PVR. These findings establish a rationale for investigating the potential use of a c-fos-AS-ON as a novel therapeutical tool in the treatment of PVR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Cell Proliferation
  • Cells, Cultured
  • Disease Progression
  • Gene Targeting / methods
  • Genes, fos / genetics*
  • Genetic Therapy / methods*
  • Humans
  • Keratins / metabolism
  • Male
  • Oligonucleotides, Antisense*
  • Pigment Epithelium of Eye / pathology*
  • Proto-Oncogene Proteins c-fos / metabolism
  • RNA, Messenger / genetics
  • Rabbits
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Transfection
  • Vitreoretinopathy, Proliferative / pathology*
  • Vitreoretinopathy, Proliferative / prevention & control

Substances

  • Oligonucleotides, Antisense
  • Proto-Oncogene Proteins c-fos
  • RNA, Messenger
  • Keratins