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

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 425–8,000 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.