1. P2Y2 Agonist Promotes Corneal Epithelial Wound Healing ID: 14359 Yong-Soo Byun, MD, PhD; Jongsoo Joo, MD; Sung A. Lim, MD; Woong-Joo Whang, MD; Choun-Ki Joo, MD, PhD The authors have no financial interests to disclose WCCVII, San Diego 2015

2. Purinergic receptors P2Y2 receptors are a family of purinergic G protein-coupled receptors, stimulated by nucleotides, ATP or UTP.

3. P2Y2 agonist used in the ophthalmology A stabilized derivative of UTP is commercially available for dry eye treatment in limited country. mainly act on the conjunctival epithelium & goblet cell Adv Ther (2012) 29(7):579–589.

4. Purpose The aim of this study was to evaluate the effect of P2Y2 agonist on corneal epithelium, especially wound healing process Methods & Materials Materials P2Y2 agonist: diquafosol tetrasodium 3% In vivo wound healing study SD Rats treated with P2Y2 agonist or vehicle qid n=9 in each group Ethanol-assisted 3 mm round epithelial wound In vitro study Simian virus (SV)40 stably transfected human corneal epithelial (THCE) cells WST-8 cell proliferation assay EGFR/ERK/AKT signaling pathway Intracellular calcium assay with Fluo-4/AM

5. Figure 1. The effect of P2Y2 agonist on epithelial wound healing in vivo. (A) After making 3 mm diameter epithelial wound, percentage of wound closure was assessed on 0, 12, 24, and 36 hour. (B) Six and 12 hours after wound making, wound closure percentage of P2Y2 agonist treated eyes was significantly higher, or faster wound healing than the control eyes. (*; p<0.001) S1. The histologic findings of the central epithelium after completely healed. scale bar = 20μm Control P2Y2 agonist ControlP2Y2 agonist ControlP2Y2 agonist

6. Wounded epitheliumUnwounded epithelium stroma epi stroma epi P2Y2 Merge Figure 2. P2Y2 immunostaining in unwounded epithelium and wounded epithelium of rat cornea. P2Y2 receptor (Green) was strongly stained near the margin (white arrow) of wounded cornea epithelium was in wound margin, while there was no positive stain in normal unwounded epithelium. DAPI (Blue) and EGFR (Red) was used for nuclear and epithelium counterstaining. Scale bars = 20μm

7. Time (hour) Absorbance (450nm) Figure 3. The effect of P2Y2 agonist on cell proliferation in vitro. THCE cells (1x10 4 ) were seeded into 96-well plates and starved for 12 h. Cells were then incubated under the various concentration of P2Y2 agonist over time and cell proliferation was assayed using WST-8. The absorbance values at 450 nm are given on the y axis. After 24 h, the absorbance value was significantly higher at P2Y2 agonist concentrations of 20, 50, 100, and 200 μM, compared to control with only KSFM (*; p < 0.05). P2Y2 agonist promoted cell proliferation at concentration from 20 to 200 μM, whereas it inhibited cell proliferation above 2 mM. * * * *

8. Figure 4. P2Y2 agonist induced cell proliferation depends on intracellular Ca 2+ & EGFR mediated ERK activation. (A) The phosphorylation of EGFR, ERK, and AKT at indicated times were assessed by Western blot analysis. Serum- and growth-factor- starved THCE cells were treated with 200 μM suramin (B), 1 μM AG1478 (C), or 10 μM BAPTA/AM (D) for 30 min and further incubated with or without 100 μM P2Y2 agonist for 5 min. Cell lysates were immunoblotted with phospho-ERK1/2, ERK1/2, phosphor-AKT, AKT, and P2Y2 antibody. GAPDH was used as a probe to normalize protein loading. The results are representative of three independent experiments. A B CD

9. Figure 5. Changes in [Ca 2+ ] after application of P2Y2 agonist (100 μM) in cultured, monolayered THCE cells. Cells were incubated with 5 μM Fluo-4/AM in KSFM with or without calcium ion for 30 minutes. Fluo-4/AM fluorescence was measured at 490 nm (fluorescence intensity = F/F0). (A) Fluorescence images were obtained at 0.5 second intervals. Ca 2+ currents activated by P2Y2 agonist (100 μM) in KSFM with calcium ion (B), and in KSFM without calcium ion (C).

10. Calcium indicator: Fluo-4/AM (5 μM) Media: KSFM (Ca 2+ conc. = 0.09 mM) Loading soln.: Diaquafosol 200 μM in KSFM

11. Summary In vivo animal study, topical P2Y2 agonist promoted the corneal epithelial wound healing. And P2Y2 was highly expressed on the wound margin. In vitro study, P2Y2 agonist (20 to 200 μM) promoted THEC cell proliferation, Which was mediated by activation of ERK pathway via increase of intracellular calcium and EGFR activation. We presented for the first time that P2Y2 agonist has the additive effect to promote the corneal epithelial wound healing, in addition to well known effect to improve dry eye syndrome.