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A. H. Kakazu, J. He, N. G. Bazan, H. E. P. Bazan; Aspirin-Triggered Lipoxin A4 (epi-LxA4) Is an Important Mediator for Maintaining the Integrity of Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1818.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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).
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.
Epi-LxA4 could be an important mediator for protecting the integrity of the human endothelium during corneal preservation.
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