Diminished heme oxygenase-1 mRNA expression in RPE cells from diabetic donors as quantitated by competitive RT/PCR

Jean Louis Da Silva, Robert A. Stoltz, Michael W. Dunn, Nader G. Abraham, Shigaki Shibahara

Research output: Contribution to journalArticlepeer-review

24 Citations (Scopus)

Abstract

Purpose. The RPE is essential in the maintenance of retinal vasculature homeostasis, since increased cellular expression of heme oxygenase-1 (HO-1) has been implicated as a defense mechanism against oxidative stress. This study was done in an effort to determine the levels of the stress protein (32 kD), HO-1, in retinal pigment epithelium (RPE) cells obtained from diabetic and normal eyes. Methods. We measured the levels of HO-1 in the RPE from eight normal and six diabetic donor eyes. For comparison, HO-1 levels were assayed in RPE cells from four donor eyes with long-standing hypertension. Heme oxygenase-1 mRNA copy number was determined by competitive RT/PCR on various ex vivo samples and on RPE cultured from the same donors. Total RNA (1-200 ng) was reverse-transcribed and then amplified by PCR in the same tube as an internal standard obtained by deleting a 50 bp restriction site from the native HO-1 gene. Results. Relative to the RPE obtained from control donor eyes, RPE from diabetic donors exhibited significantly decreased levels of HO-1 mRNA. In contrast, no significant difference in the levels of HO-1 mRNA was observed in RPE samples derived from hypertensive donor eyes. The diabetic group showed a range of 340-450 HO-1 mRNA copies/ng of total RNA, as compared to 425-8000 HO-1 mRNA copies/ng of total RNA in RPE from normal donors and 460-7605 copies/ng in hypertensive donor eyes. Conclusions. This study represents initial studies exploring the quantitative expression of heme oxygenase in the RPE human eyes. The decreased expression of HO-1, a stress/heat shock protein, may in the RPE contribute to the vulnerability of the neuroretina to significant metabolic alterations encountered in the diabetic state. This was a limited study; additional screening from different donor eyes will be done in order to establish the relationship between RPE, HO-1 expression and eye diseases in which oxidative stress is believed to be a determinant in the pathophysiology.

Original languageEnglish
Pages (from-to)380-386
Number of pages7
JournalCurrent Eye Research
Volume16
Issue number4
DOIs
Publication statusPublished - 1997

Keywords

  • Competitive RT/PCR
  • Diabetes mellitus
  • Heme oxygenase
  • Retinal pigment epithelium (RPE) cells

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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