Abstract
Purpose: :
Previous studies have shown that low concentrations of aspirin-triggered Lipoxin A4 (epi-LxA4) stimulate wound closure in rabbit corneal endothelial cells (ARVO 2008). Human endothelium has a very low mitotic rate, and with aging there is a decrease in the number of cells. Epi-LxA4 is a anti-inflammatory bioactive lipid formed when aspirin acetylates cyclooxygenease-2 (COX-2) and redirects COX-2 catalytic activity away from prostaglandins. Epi-LxA4 acts through specific G-protein coupled receptors. The purpose of the current study is to investigate the action of epi-LxA4 in human corneal endothelium.
Methods: :
Human corneal endothelial cells (HCEC) along with the Descemet’s membrane were isolated from fresh human eyes obtained from NDRI. The cells were suspended in DMEN/F12 supplemented with 15% FBS. Cell phenotype was identified using ZO-1 and -SMA antibodies. LxA4 receptor was detected in HCEC and human corneal tissue using polyclonal antibody FPRL1. HCEC were starved for 24 h, then treated with 100 nM epi-LxA4 for 24 h and cell proliferation was evaluated with Ki-67 antibody. To measure wound closured in vitro, confluent HCEC were wounded by a linear scraping with a sterile pipette tip in the center of the well and incubated for 24 h and 48 h with or without epi-LxA4. Human corneas were suspended in Corneal Storage Media (Optisol GS, Bausch & Lomb) in presence of epi-LxA4 (100 nM ) and incubated at 4ºC for 10 days. The viability of the endothelial cells were assayed using LIVE/DEAD Viability/Cytotoxicity kit (Molecular Probes).
Results: :
HCEC strongly expressed the FPRL1 receptor in the plasma membrane and in the nuclei. In human corneal tissue, strong positive staining was found in the endothelium. There was a three-fold increase in cell proliferation when HCEC were incubated with epi-LxA4 for 24 h. Significant endothelial wound healing was observed after 48 h incubation with epi-LxA4. Human corneas incubated for 10 days with Optisol GS in the presence of epi- LxA4 showed no toxic effects to the endothelium.
Conclusions: :
Epi-LxA4 could be an important mediator for protecting the integrity of the human endothelium during corneal preservation.
Keywords: cornea: endothelium • wound healing • lipids