Tanja Stachon1, Lorenz Latta1, Krasimir Kolev2, Berthold Seitz1, Achim Langenbucher3, Nóra Szentmáry1. 1. Klinik für Augenheilkunde, Universitätsklinikum des Saarlandes UKS, Homburg/Saar. 2. Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary. 3. Institut für Experimentelle Ophthalmologie, Universität des Saarlandes, Homburg/Saar.
Abstract
PURPOSE: In recent years, there has been increasing evidence of an inflammatory component in keratoconus. A key gene in inflammatory processes is the nuclear factor kappa B (NF-κB). NF-κB is a transcription factor for the enzyme nitric oxide synthase (NOS), which is involved with the competing enzyme arginase (Arg) in inflammatory processes. The aim of this study was to analyze the isotypes of NOS and arginase, the expression of NF-κB, NOS and arginase, and the regulatory mechanism of NOS and arginase in keratocytes of keratoconus patients using the inhibitor 1400W in vitro. METHODS: Human keratocytes were isolated from surgically removed corneas of 8 KC patients and 8 normal human corneal buttons and were cultured to confluence, in vitro. Quantitative PCR and Western blot analysis were performed to examine NF-κB, NOS and arginase expression in keratocytes. Nitrite and urea concentrations in the supernatant of the cells were analyzed using 0 - 40 µM 1400W iNOS inhibitor concentrations. RESULTS: Only the isotypes iNOS and Arg-II were detected in the keratocytes. The mRNA expression of NF-κB and iNOS were higher in KC keratocytes than in normal cells (p = 0.0135 and p = 0.0001), whereas no differences were measurable in Arg-II expression. In the WB, a higher band intensity was measurable in NF-κB (p = 0.0012), and in iNOS, no differences in band intensity could be detected. In the supernatant of the KC keratocytes, lower concentrations of nitrite and urea were measured after the addition of the inhibitor 1400W (p ≤ 0.014), but not in normal cells (p ≥ 0.178). CONCLUSION: Due to the increased expression of NF-κB and iNOS, an inflammatory component in keratoconus must be assumed. The different regulation of the KC keratocytes by the iNOS inhibitor 1400W suggests an altered metabolic activity which can be caused by inflammatory processes. Thieme. All rights reserved.
PURPOSE: In recent years, there has been increasing evidence of an inflammatory component in keratoconus. A key gene in inflammatory processes is the nuclear factor kappa B (NF-κB). NF-κB is a transcription factor for the enzyme nitric oxide synthase (NOS), which is involved with the competing enzyme arginase (Arg) in inflammatory processes. The aim of this study was to analyze the isotypes of NOS and arginase, the expression of NF-κB, NOS and arginase, and the regulatory mechanism of NOS and arginase in keratocytes of keratoconus patients using the inhibitor 1400W in vitro. METHODS: Human keratocytes were isolated from surgically removed corneas of 8 KC patients and 8 normal human corneal buttons and were cultured to confluence, in vitro. Quantitative PCR and Western blot analysis were performed to examine NF-κB, NOS and arginase expression in keratocytes. Nitrite and urea concentrations in the supernatant of the cells were analyzed using 0 - 40 µM 1400W iNOS inhibitor concentrations. RESULTS: Only the isotypes iNOS and Arg-II were detected in the keratocytes. The mRNA expression of NF-κB and iNOS were higher in KC keratocytes than in normal cells (p = 0.0135 and p = 0.0001), whereas no differences were measurable in Arg-II expression. In the WB, a higher band intensity was measurable in NF-κB (p = 0.0012), and in iNOS, no differences in band intensity could be detected. In the supernatant of the KC keratocytes, lower concentrations of nitrite and urea were measured after the addition of the inhibitor 1400W (p ≤ 0.014), but not in normal cells (p ≥ 0.178). CONCLUSION: Due to the increased expression of NF-κB and iNOS, an inflammatory component in keratoconus must be assumed. The different regulation of the KC keratocytes by the iNOS inhibitor 1400W suggests an altered metabolic activity which can be caused by inflammatory processes. Thieme. All rights reserved.