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N. Guo, Y. Du, W. W.-Y. Kao, Y. Yamaguchi, J. L. Funderburgh; Hyaluronan Expression by Keratocytes Mediates Stromal Inflammatory Response to LPS. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4313.
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© ARVO (1962-2015); The Authors (2016-present)
Hyaluronan (HA) is not present in healthy corneal stroma but appears in healing wounds and in a variety of corneal pathologies. Keratocytes produce HA in vitro by upregulation of hyaluronan synthase 2 (Has2) gene expression. In other tissues HA, as well as fragments of HA, activate inflammatory cells via TLR2 and TLR4 receptors. This study tests the hypothesis that in corneal stroma expression of Has2 gene by keratocytes serves to activate or enhance inflammatory response upon injury.
Mice lacking Has2 expressed by keratocytes were generated by mating mice in which Has2 gene was modified by LoxP (Has2f/f) with Kera-Cre transgenic mice expressing Cre recombinase in keratocytes driven by keratocan promoter. Stromal inflammation was induced by direct intrastromal injection of bacterial endotoxin (LPS) in Has2Δk/Δk (homozygous ablation of Has2 in keratocytes), Has2Δk/f (heterozygous ablation of Has2), and in wild type mice. Stromal HA was stained using biotin-HA binding protein. Inflammatory cells were identified by immunostaining with antibodies to CD45 and CD11c.
Has2Δk/Δk mice lacking HAS2 in keratocan-expressing tissues exhibit normal overall size, gross morphology and behavior. Corneas are transparent and of normal size. Two days and 7 days after intrastromal injection of LPS, both normal and heterozygous mice exhibited extensive HA deposited in peripheral stroma and marked corneal edema. An infiltrate of CD45+cells which were largely CD11c-positive co-localized with the HA-containing regions of the stroma. In the Has2Δk/Δk mice, HA was essentially absent from the stroma, edema was markedly reduced, as were the numbers of inflammatory cells.
This study shows Has2 expression by keratocytes to be unnecessary for normal corneal development or for maintenance of corneal transparency. The biosynthetic source of HA in pathological corneal stromal tissue was confirmed to be the HAS2 enzyme in keratocytes. The results suggest that corneal edema during inflammation is related to accumulation of HA in the tissue and also strongly support the hypothesis that infiltration of CD11c+ (dendritic) cells after inflammatory stimulus is dependent on the presence of HA in the tissue.
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