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RS Berk, Z Dong, M Katar, I Vlodavsky, HQ Miao, P Kussie, E Navarro, P Bohlen; Heparanase Expression in Murine Corneas Infected with Pseudonomas Aeruginosa . Invest. Ophthalmol. Vis. Sci. 2002;43(13):35.
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Purpose: This study was designed to establish the presence of heparanase in the mouse cornea before and during infection with Pseudomonas aeruginosa. Heparan sulfate is an important component of the extracellular matrix (ECM) and the vascular basal laminar which functions as a barrier to the extravasation of metastatic and inflammatory cells. Cleavage of heparan sulfate by heparanase may play a role in the disassembly of the ECM and basal laminar. Methods: C57BL/6J mice were intracorneally infected with P. aeruginosa. Corneal samples were harvested, washed and pooled for various time samples ranging from 0 to 8 days post-infection. Gene and protein expression was temporally detected by semi-quantitative RT-PCR and immunoblot analysis. In situ hybridization was used to localize gene expression of heparanase in both uninfected and infected tissues. Sequence homology confirmed the identity of the enzyme. Enzyme assays utilized tritium labeled heparan sulfate as substrate and activity was based on the release of radiolabeled sulfate fractions. Results: Semi-quantitative RT-PCR on corneal extracts indicated gene expression in both uninfected and infected naïve and immunized mice. Immuoblotting characteristically demonstrated several protein bands including the 65 kDa precursor and activated 50 kDa enzyme. In situ hybridization indicated gene expression in the corneal epithelium of both uninfected and infected corneas. Enzymatic assays demonstrated the expression of the enzyme. Conclusion: Gene, protein and enzymatic expression for corneal heparanase expression was demonstrated in both uninfected and infected murine corneas and was reflection of the inflammatory response in infected mice.
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