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F. Zhang, H. Yang, J. Yuan, P. Gjorstrup, P. Reinach; Resolvin Ameliorates Hypertonicity-Induced Inflammation and Protects Against Losses in Human Corneal Epithelial Cell Barrier Function. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1950.
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© ARVO (1962-2015); The Authors (2016-present)
Dry eye can be associated with tear film hyperosmolarity leading to persistent inflammation and corneal epithelial barrier disruption. Current therapeutic options are limited to mitigate these sequealae. Resolvins are a family of local endogenous lipid mediators released by injury. We used a scratch wound assay and found in human corneal epithelial cells (HCEC) that the resolvin E1 (RvE1) and its analog RX-10008 stimulate cell migration. The ability of resolvins to promote these responses prompted us to determine in HCEC the cell signaling pathways underlying them. Accordingly, we provide here evidence in hypertonic challenged HCEC that RvE1 induces through a GTP binding protein interaction and epidermal growth receptor (EGFR) transactivation: a) restoration of barrier function; b) declines in proinflammatory mediator release.
SV-40 immortalized HCEC were maintained in DMEM/F12. Medium tonicity was changed from 300 mOsm to 450 mOsm by adding NaCl. To resolve possible roles of EGFR and GPCR, the HCEC were pre-exposed for 30 min to the EGFR tyrosine kinase inhibitor AG1478 or pertussis toxin, to block the GTP binding protein Gi, and then challenged by hypertonicity for 20 hours in the absence or presence of RvE1 (0.1 µM). Proinflammatory chemokine IL-8 release was determined using an enzyme-linked immunosorbent assay (ELISA). IL-8 was normalized to the total amount of lysed cellular protein. Trans-epithelial electrical resistance (TEER) monitored barrier function. HCEC grow on air-lifted inserts with a pore size of 0.4 µm. Resistance was measured with a volt-ohm meter (EVOM).
Hypertonicity (450 mOsm) induced an increase in IL-8 release from approximately 1000 ng/mg cell protein (basal release) to 5500 ng/mg cell protein. Such an increase was reduced to 2550 ng/mg cell protein by preincubation with RvE1 (0.1 µM). Preincubation with either AG1478 (1 µM), or pertussis toxin (100 ng/ml) blocked the suppression by RvE1 of hypertonicity-induced increases in IL-8 release. Hypertonicity (375 mOsm) reduced after 24 hours the TEER from 433.8 ± 15.9 (ohms.cm2) to 228.9 ± 19.8 (ohms.cm2) which is indicative of barrier function disruption (n=3). However, if RvE1 (0.1 µM) was present during this challenge this decline in TEER did not occur.
A resolvin, RvE1, ameliorates hypertonicity-induced inflammation by stimulating Gi and EGFR transactivation. This analogue also protects against losses in HCEC barrier function induced by exposure to hypertonicity.
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