Thymosin-␤4 Inhibits Corneal Epithelial Cell Apoptosis after Ethanol Exposure In Vitro

PURPOSE. The purpose of this study was to determine the effect of thymosin beta 4 (T␤ 4 ) treatment on human corneal epithelial cells exposed to ethanol in vitro. The efficacy of T␤ 4 in preventing mitochondrial disruption and in inhibiting caspasemediated apoptosis was examined. METHODS. Nontransformed human corneal epithelial cells (HCECs) at passage 4 were untreated or treated with ethanol (20% for 20 seconds) or a combination of ethanol and T␤ 4 . The cells were allowed to recover from ethanol treatment for 24 hours. Mitochondrial membrane integrity and the release of cytochrome c to the cytoplasm were assessed using microscopy, Western blot, and ELISA. Bcl-2 expression and cell proliferation were measured using ELISA. Colorimetric activity assays were completed for caspase-2, -3, -8, and -9. RESULTS. T␤ 4 treatment decreased deleterious mitochondrial alterations, significantly decreased cytochrome c release from mitochondria, and increased Bcl-2 expression in ethanol-exposed human corneal epithelial cells. In ethanol-exposed corneal epithelium T␤ 4 treatment inhibited caspase-2, -3, -8, and -9 activity, with caspase-8 showing the most significant inhibition. T␤ 4 treatment resulted in no significant effect on the proliferation of human corneal epithelial cells after ethanol exposure. CONCLUSIONS. T␤ 4 plays an antiapoptotic role under conditions of epithelial cell challenge with an external stress such as exposure to ethanol. T␤ 4 may function as an antiapoptotic agent by inhibiting the release of cytochrome c from mitochondria and by suppressing the activation of caspases. (Invest Ophthalmol Vis Sci

Pseudomonas aeruginosa Binds to Extracellular Matrix Deposited by Human Corneal Epithelial Cells

PURPOSE. To measure the effect of extracellular matrix substrate, pH, and O 2 on Pseudomonas aeruginosa binding. METHODS. Extracellular matrix substrates were prepared from human corneal epithelial cells cultured in 2% or 20% O 2 . P. aeruginosa strains ATCC 19660 or PAO1 (suspended in pH 7.0 or 7.5 buffer) were cultured on extracellular matrix substrates in 2% or 20% O 2 . The mean number of adherent bacteria per counted per field Ϯ SEM (n ϭ 15) was determined for combinations of bacteria, extracellular matrix substrate, pH, and O 2 . Binding in the presence of antibodies directed against laminin-5 was also measured. RESULTS. Extracellular matrix substrates produced by cells cultured in 20% O 2 , combined with an environment of pH 7.0, provided the least favorable conditions for binding of strain 19660. In contrast, extracellular matrix substrates produced by cells cultured in 2% O 2 , combined with an environment of pH 7.0, provided the most favorable conditions for binding of strain 19660. Binding of PAO1, however, as a function of extracellular matrix substrate and pH, did not similarly compare with binding of strain 19660. Antibodies against laminin-5 chains served to increase the number of strain 19660 bacteria bound to extracellular matrix substrates compared with the control. CONCLUSIONS. The extracellular matrix secreted by hypoxic corneal epithelial cells is a substrate for binding of P. aeruginosa. Results in previous studies have shown that hypoxic extracellular matrix contains less laminin-5 protein than normoxic matrix. The antibody studies in this report suggest that the decrease in laminin-5 content in hypoxic matrix, relative to matrix secreted by normoxic corneal epithelium, may be responsible for increased bacterial adhesion. (Invest Ophthalmol Vis Sci. 2002;43:3654 -3659) A n intact layer of epithelium firmly attached to the underlying basement membrane creates a barrier that is essential for the cornea to maintain proper vision. The epithelial barrier, along with elements provided by the tear film, plays a role in preventing bacterial binding, colonization, and infectivity of the corneal surface and stromal spread of the bacteria. Microbial infections that disrupt the integrity of corneal epithelial cell-extracellular matrix, if left untreated, will result in cellular loss, inflammatory reactions, and subsequent decreased visual acuity. Pseudomonas aeruginosa is a virulent opportunistic pathogen responsible for a particularly destructive corneal disease