Fig 1: Corneal stromal cell‐derived IL‐1β and CCL20 are involved in LPA‐induced proliferation of corneal endothelial cells (CECs). A, Mitomycin C‐pretreated human corneal stromal cells (HCSCs) were maintained in TC medium with or without 20 μmol/L LPA for 2 d before CM collection. ELISA showed that the concentrations of secreted IL‐1β and CCL20 were both significantly greater in the LPA‐treated group. B, The dose‐dependence of IL‐1β‐ and CCL20‐induced B4G12 cell proliferation was determined by cell counts. The number of cells was significantly higher after treatment of recombinant IL‐1β alone for 2 d. In contrast, no difference was observed in the CCL20‐treated group. C, The involvement of IL‐1β in LPA‐induced proliferation of CECs was investigated via neutralization assay of the CM. Immortalized HCECs (B4G12) co‐cultured with mitomycin C‐pretreated HCSCs in TC medium for 5 d were greater in cell number in the LPA‐treated group (20 μmol/L). Subsequent IL‐1β neutralization with 2 ng/mL IL‐1β‐specific antibody (IL‐1β Ab, clone AS10) significantly attenuated the LPA‐induced proliferation in co‐cultures. D, The proliferation‐promoting effect of IL‐1β on RCECs was also examined 2 d later. The fraction of RCECs in the co‐culture increased significantly with increasing concentrations of IL‐1β (20, 200 or 1000 pg/mL). E, The morphology and ECD of the 200 pg/mL IL‐1β‐treated tissue‐cultured rabbit corneal endothelium was examined via immunostaining for ZO‐1 on Day 2. The corneal ECD was significantly higher in the LPA‐treated cells than that in controls and had the normal hexagonal phenotype typical of RCECs. (n = 3; *P < .5, **P < .05)
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