TROPHIC EFFECT VALIDATION OF PACAP ON HUMAN CORNEAL ENDOTHELIUM BY USING CONFOCAL MICROSCOPY

Purpose: To investigate the trophic effect of autocrine factor, pituitary adenylate cyclase-activating polypeptide (PACAP), on human corneal endothelial cells (HCECs) isolated from the peripheral corneal–scleral donor tissue using confocal microscopy. Methods: Human corneal endothelial cells (HCECs) were isolated from the peripheral corneal–scleral donor tissue (n=xx). Undifferentiated cells were grown in DMEM-F12 (1:1) medium supplemented with 10% fetal bovine serum (FBS), 1 mM L-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin. Then, they were switched gradually in differentiating serum-free medium containing DMEM/F12 supplemented with cell culture media (GlutaMAX; Life Technologies, Inc.), basic fibroblast growth factor (bFGF; 10 ng/mL), epidermal growth factor (EGF; 10 ng/mL), B-27 supplement and penicillin/streptomycin in the following 6 culturing passages. PAC1/VPAC receptors expression on HCECs has been evaluated through immunofluorescence analysis. Protective effect of PACAP (100 nM) on endothelial cell monolayer integrity following growth factors deprivation has been assessed by analyzing apoptotic death with nuclear dye Hoechst 33342, and Tight-Junction (TJ) proteins (ZO-1 and Claudin-1) expression using confocal microscopy. Results: PAC1, VPAC1 and VPAC2 receptors have been detected on HCECs monolayer culture. PACAP addition has enhanced corneal endothelium barrier integrity after deprivation of EGF/bFGF in growth medium, as revealed by analyzing apoptotic nuclei, ZO-1, claudin-1 and integrin α3 expression using confocal microscopy. Conclusions: Our data suggest that PACAP could represent an interesting trophic factor maintaining human corneal endothelium functionality