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J. Yuan, F. Zhang, H. Yang, P. Reinach, I. Polikarpov; PPAR Mediates Hypertonicity-Induced Increases in Proinflammatory Release in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5895.
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© ARVO (1962-2015); The Authors (2016-present)
Dry eye disease can be associated with increased tear film osmolarity leading to elevated inflammatory cytokine and matrix metalloproteinases levels in ocular surface epithelial cells. As peroxisome proliferator-activated receptor γ (PPARγ) gene overexpression is implicated in inhibiting injury-induced corneal inflammation in mice, we determined if changes in the PPARγ activation status of human corneal epithelial cells (HCEC) affect the increases in proinflammatory cytokine release induced by a hypertonic challenge.
Immunocytochemistry probed for PPARγ expression in SV40-immortalized human corneal epithelial cells (HCEC). To monitor changes in IL-8 release in triplicate, ELISA was used. HCEC were preexposed for 30 min to either a PPARγ agonist or an antagonist, (1 µM ciglitazone or 1 µM GW9662) under isotonic conditions. Subsequently, they were maintained for an additional 20 h in sucrose supplemented hypertonic (450 mOsm) DMEM/F12 medium, respectively. In the other three groups, HCEC were maintained in an isotonic DMEM/F12 medium (i.e.300 mOsm) throughout and were exposed to either: a) ciglitazone; b) GW9662. Supernatants were collected and centrifuged at 1,000 rpm for 5 min to remove cell debris. The amount of IL-8 in the culture medium was normalized to the total amount of lysed cellular protein.
PPARγ expression was detected in the nuclear membrane. In isotonic medium (300 mOsm), PPARγ activation by ciglitazone decreased IL-8 release by 26% from its isotonic level of 2053 ± 67 pg/mg protein to 1522±52 pg/mg protein whereas PPARγ inhibition by GW9662 elevated this control IL-8 level by 51% to 3107±178 pg/mg protein. Under the hypertonic condition (450 mOsm), IL-8 release instead increased 3.83-fold from the isotonic control to 9911± 52 pg/mg protein. On the other hand, ciglitazone preexposure suppressed this rise by 81%. IL-8 release declined from 9911± 52 to 1929± 58 pg/mg protein. Conversely, GW9662 preexposure enhanced the hypertonicity-induced IL-8 increase by 68% to 16647 ± 453 pg/mg protein.
Ciglitazone blocked essentially a large amount of the rise in IL-8 release induced by hypertonicity whereas GW9662 enhanced the increase in IL-8 induced by this challenge. These opposing effects on PPARγ activation point to the possibility that ligand-induced PPARγ activation in a clinical setting may reduce in some cases anterior ocular surface inflammation.
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