Abstract
Purpose: :
The corneal endothelium is fundamental for corneal transparency. This monolayer of cells has a very low mitotic rate in humans, and during aging, the number of cells decrease resulting in edema and loss of vision. Lipoxin A4 and aspirin-triggered lipoxin-A4 (epi-LxA4) are bioactive eicosanoids that act through specific receptors and protect tissues from inflammation. Recently, we have found that human corneal endothelium express the LxA4 receptor. The current study is to investigate the effect of epi-LxA4 on corneal endothelial proliferation and wound healing.
Methods: :
Rabbit corneal endothelial cells (RCEC) along with the Decemet’s membrane were isolated from rabbit eyes. Cell phenotype was identified by using the endothelial cell marker ZO-1. LxA4 receptor in RCEC was detected with the polyclonal antibody FPRL1. Cell proliferation was evaluated with Ki-67 antibody. To measure in vitro wound closure, confluent RCEC were wounded by a linear scraping with a sterile plastic tip in the center of the well. Cells were incubated for 24 hours in DMEM/F12 with or without LxA4. Wound healing was determined by phase contrast images collected by a camera attached to the microscope and the width of the wound was calculated using Metavue Image software.
Results: :
When RCECs were cultured with serum, they co-expressed ZO-1 and α-SMA as identified by immunofluorescence with correspondent monoclonal antibodies. LxA4 receptor was localized in both the plasma membrane and cytoplasm of RCECs. Epi-LxA4 promoted cell proliferation in a dose-dependent fashion with optimal concentrations at 50 to 100 nM. In the in vitro model of endothelial wound healing, treatment with epi-LxA4 (100 nM) significantly enhanced endothelial wound closure as compared to the control (p< 0.001).
Conclusions: :
These results show that epi-LxA4, at very low concentrations, stimulates proliferation of endothelial cells and significantly increases wound closure. Therefore, LxA4 could be an important component to add to corneal preservation media or during surgeries that damage the endothelium.
Keywords: cornea: endothelium • lipids • wound healing