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Toshinari Funaki, Akira Matsuda, Nobuyuki Ebihara, Akira Murakami, Sunil Chauhan, Ula V. Jurkunas, Reza Dana; Atp Contributes Corneal Endothelial Wound Healing Via P2x7 Receptor. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6436.
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When corneal endothelial cells (CECs) are injured, the resulting defect in the monolayer is repaired through enlargement, migration and spreading of neighboring CECs. Since CECs possess abundant mitochondria and are highly metabolically active, we hypothesize that release of adenosine triphosphate (ATP) from CECs is involved in the CEC homeostasis. In this study, we investigated the role of ATP and P2 receptor in CEC wound healing process.
For the in vitro assay, culture human corneal endothelial cells were confluent in 10% DMEM, and then a small scrape wound was performed across a monolayer of cells. Various concentration of ATP (10µM~3mM), ecto-ATPase inhibitor (ARL67156) and P2X7 receptor antagonist (oxidized ATP: oATP) were added in 1% DMEM. After 20 hours, cells were visualized by a phase-contrast microscopy. The wounded (denuded) area was measured by the Image J 1.37v software. In vivo assay: Syngenic mice (Balb/c) corneal bottons were incubated with or without ARL67156 and oATP for 30 minutes at 37°C in DMEM before transplantation. At post transplant day 1, corneal endothelial morphology was assessed by ZO-1 staining, and then the wound areas were measured by the Image J 1.37v software.
Low dose (10µM, 30µM) concentration of ATP promoted CEC wound closure compared with control (10µM p=0.25, 30µM p=0.17), however high dose (1mM, 3mM) significantly delayed compared with control (1mM p=0.045, 3mM p=0.0005). Interestingly ARL67156 accelerated corneal wound closure compared with low dose concentration of ATP (p=0.031), but oATP delayed compared with high dose concentration of ATP (p=0.056). Moreover ARL67156 accelerated endothelial wound healing through enlargement and spreading in mice corneal transplant (% of wounded area: Control 45.5%, ARL67156 5.7%, oATP 80.7%).
Exogenous ATP contributes corneal endothelial wound healing process by interacting with P2X7 receptor and improves wound healing.
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