title: Diminished heme oxygenase-1 mRNA expression in RPE cells from diabetic donors as quantitated by competitive RT/PCR.
abstract: OBJECTIVE
  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.
authors.affiliation: Rockefeller University, New York 10021, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: da Silva
authors.forename: R A
authors.initials: RA
authors.lastname: Stoltz
authors.forename: M W
authors.initials: MW
authors.lastname: Dunn
authors.forename: N G
authors.initials: NG
authors.lastname: Abraham
authors.forename: S
authors.initials: S
authors.lastname: Shibahara
mesh_terms: D003920:Diabetes Mellitus / Q000378:metabolism* / Q000473:pathology; D018628:Gene Dosage; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics*; D051547:Heme Oxygenase-1; D006801:Humans; D006973:Hypertension / Q000378:metabolism / Q000473:pathology; D008565:Membrane Proteins; D010857:Pigment Epithelium of Eye / Q000378:metabolism* / Q000473:pathology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D012016:Reference Values; D014019:Tissue Donors; D014158:Transcription, Genetic
chemical_list: D008565:Membrane Proteins; D012333:RNA, Messenger; C496541:HMOX1 protein, human; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1