PURPOSE: The human corneal endothelium has a very low mitotic rate, and with aging there is a decrease in the number of cells. 15-epi-LXA4 is an anti-inflammatory, bioactive lipid formed when aspirin acetylates cyclooxygenease-2 and redirects cyclooxygenease-2 catalytic activity away from prostaglandins. The purpose of the current study was to evaluate the action of 15-epi-LXA4 in the endothelium viability of human corneas stored in Optisol-GS. METHODS: Human corneal endothelial (HCE) cells along with the Descemet's membrane were isolated from fresh human eyes obtained from National Disease Research Interchange. Cell phenotype was identified by using the tight junctions cell marker ZO-1. LXA4 receptor (FPR2/ALX) was detected by immunostaining of HCE cells and human corneal tissue using a polyclonal antibody. Cell proliferation was evaluated with Ki-67 antibody. To measure cell migration, confluent HCE cells were wounded by a linear scraping with a sterile pipette tip in the center of the well and incubated for 24 h with or without 15-epi-LXA4. To evaluate the reparative capacity of 15-epi-LXA4, 7 pairs of human corneas were incubated in Dulbecco's modified Eagle's medium/F12 media at 37°C with or without 100 nM 15-epi-LXA4 for 24 h and then stored at 4°C in Optisol-GS for 12 days. Endothelial viability was assessed by 2 staining techniques: a viability/cytotoxicity kit and trypan blue combined with alizarin red S. RESULTS: HCE cells and the endothelium of human corneal sections strongly expressed the LXA4 receptor. There was a 3-fold increase in cell proliferation when HCE cells were incubated with 100 nM 15-epi-LXA4 for 24 h. No significant migration was observed after 24 h incubation with 15-epi-LXA4. Corneas incubated for 24 h in Dulbecco's modified Eagle's medium/F12 media in the presence of 15-epi-LXA4 and then stored for 12 days in Optisol-GS had a 36% to 56% increase in viability compared with controls without 15-epi-LXA4. CONCLUSIONS: 15-epi-LXA4 is an important mediator that protects the integrity of the human endothelium during corneal preservation in Optisol-GS.
PURPOSE: The human corneal endothelium has a very low mitotic rate, and with aging there is a decrease in the number of cells. 15-epi-LXA4 is an anti-inflammatory, bioactive lipid formed when aspirin acetylates cyclooxygenease-2 and redirects cyclooxygenease-2 catalytic activity away from prostaglandins. The purpose of the current study was to evaluate the action of 15-epi-LXA4 in the endothelium viability of human corneas stored in Optisol-GS. METHODS:Human corneal endothelial (HCE) cells along with the Descemet's membrane were isolated from fresh human eyes obtained from National Disease Research Interchange. Cell phenotype was identified by using the tight junctions cell marker ZO-1. LXA4 receptor (FPR2/ALX) was detected by immunostaining of HCE cells and human corneal tissue using a polyclonal antibody. Cell proliferation was evaluated with Ki-67 antibody. To measure cell migration, confluent HCE cells were wounded by a linear scraping with a sterile pipette tip in the center of the well and incubated for 24 h with or without 15-epi-LXA4. To evaluate the reparative capacity of 15-epi-LXA4, 7 pairs of human corneas were incubated in Dulbecco's modified Eagle's medium/F12 media at 37°C with or without 100 nM 15-epi-LXA4 for 24 h and then stored at 4°C in Optisol-GS for 12 days. Endothelial viability was assessed by 2 staining techniques: a viability/cytotoxicity kit and trypan blue combined with alizarin red S. RESULTS: HCE cells and the endothelium of human corneal sections strongly expressed the LXA4 receptor. There was a 3-fold increase in cell proliferation when HCE cells were incubated with 100 nM 15-epi-LXA4 for 24 h. No significant migration was observed after 24 h incubation with 15-epi-LXA4. Corneas incubated for 24 h in Dulbecco's modified Eagle's medium/F12 media in the presence of 15-epi-LXA4 and then stored for 12 days in Optisol-GS had a 36% to 56% increase in viability compared with controls without 15-epi-LXA4. CONCLUSIONS:15-epi-LXA4 is an important mediator that protects the integrity of the human endothelium during corneal preservation in Optisol-GS.
Authors: Justin T English; Paul C Norris; Robin R Hodges; Darlene A Dartt; Charles N Serhan Journal: Prostaglandins Leukot Essent Fatty Acids Date: 2017-01-19 Impact factor: 4.006