Corneal endothelial cell survival in organ cultures under acute oxidative stress: effect of VIP.
OBJECTIVE
Human corneal endothelium, a neural crest-derived tissue, has a very limited regenerative capacity and may depend on trophic factors for its survival throughout life, as well as after injury and during storage before transplantation. The purpose of this study was to determine whether vasoactive intestinal peptide (VIP), a 28-amino acid neurotrophic factor present in human aqueous humor, promotes the survival of corneal endothelium in corneal organ cultures, and whether VIP is produced by the corneal endothelium.


METHODS
Thirteen viable human donor corneas that had been received from the Central Florida Lions Eye Bank and stored in preservation medium (Optisol-GS; Chiron Vision, Irvine, CA) at 4 degrees C for 8 to 17 days were bisected. Each half was treated with either 0 or 10 nM VIP (15 minutes) and subjected to H(2)O(2) (1.4 mM, 30 minutes) treatment at 37 degrees C. The numbers of live and dead corneal endothelial (CE) cells isolated from the corneas were then determined under fluorescence microscopy using a live-dead viability-cytotoxicity assay conducted by an observer uninformed of the treatment. The effect of VIP (10(-16) to 10(-6) M) on CE cell survival was also studied in fresh bovine corneas in situ, by using the same assay. The presence of VIP in the corneal endothelium in fresh human donor and bovine eyes was examined by immunocytochemistry, in situ hybridization, and Western blot analysis, whereas VIP in the bovine aqueous humor was assessed by radioimmunoassay.


RESULTS
VIP (10 nM) significantly increased CE survival in 10 of 13 human corneas. The mean survival of CE cells (+/-SEM) was 42% +/- 3% in control corneas versus 59% +/- 3% in VIP-treated corneas (P < 0.001). In bovine corneas, VIP at concentrations as low as 10(-10) M demonstrated a significant protective effect. The mean number of dead CE cells on bovine corneas was maximally decreased by 10(-6) M VIP from 46 +/- 5 to 18 +/- 3 per field. In CE cells from fresh human and bovine corneas, VIP immunoreactivity and mRNA were detected. VIP was also present in bovine aqueous humor at 40 +/- 8 pM.


CONCLUSIONS
VIP may be an autocrine trophic factor that protects CE cells from H(2)O(2) in normal aqueous humor and possibly from other oxidative insults.
Koh
S W
SW
Department of Ophthalmology, University of Maryland at Baltimore, USA. skoh@umaryland.edu
Waschek
J A
JA
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism; D015153:Blotting, Western; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D019610:Cytoprotection / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004728:Endothelium, Corneal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005260:Female; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D017403:In Situ Hybridization; D008297:Male; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D009924:Organ Culture Techniques; D018384:Oxidative Stress; D011863:Radioimmunoassay; D018005:Receptors, Vasoactive Intestinal Peptide / Q000378:metabolism; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism / Q000494:pharmacology*
D018005:Receptors, Vasoactive Intestinal Peptide; D014660:Vasoactive Intestinal Peptide; D006861:Hydrogen Peroxide