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.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.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).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.
