Background Keratoconus (KC) is associated with oxidative stress and hypoxia and as
several times discussed, potentially with inflammatory components. Inflammation, hypoxia,
and oxidative stress may result in metabolic dysfunction and are directly linked to
each other. In the current study, we investigate the effect of hypoxia through NF-kappa
B signaling pathways on iNOS, hypoxia-induced factors (HIF), ROS, and proliferation
of normal and KC human corneal fibroblasts (HCFs), in vitro. Methods Primary human
KC-HCFs and normal HCFs were isolated and cultured in DMEM/Ham's F12 medium supplemented
with 5% fetal calf serum. Hypoxic conditions were generated and quantitative PCR and
Western blot analysis were performed to examine NF-kappa B, iNOS, HIF, and PHD2 expression
in KC and normal HCFs. ROS level was analyzed using flow cytometry and proliferation
by BrdU-ELISA. Results Hypoxia increased NF-kappa B mRNA and protein expression in
normal HCFs, but in KC-HCFs NF-kappa B mRNA and protein expression remained unchanged.
Hypoxic conditions upregulated iNOS mRNA expression of normal HCFs, but iNOS mRNA
expression of KC-HCFs and iNOS protein expression of both HCF types remained unchanged.
Hypoxia downregulated HIF-1 alpha and HIF-2 alpha mRNA expression in normal and KC-HCFs.
PHD2 mRNA expression is upregulated under hypoxia in KC-HCFs, but not in normal HCFs.
PHD2 protein expression was upregulated by hypoxia in both HCF types. Total ROS concentration
is downregulated in normal and KC-HCFs under hypoxic conditions. Proliferation rate
of KC-HCFs was upregulated through hypoxia, but did not change in normal HCFs. Conclusions
Hypoxia increases NF-kappa B and iNOS mRNA expression in normal HCFs, but there does
not seem to be enough capacity in KC-HCFs to increase NF-kappa B and iNOS mRNA expression
under hypoxia, maybe due to the preexisting oxidative stress. HIF and PHD2 do not
show altered iNOS regulation under hypoxic conditions in KC-HCFs, and therefore do
not seem to play a role in keratoconus pathogenesis. An increased proliferation of
cells may refer to compensatory mechanisms under hypoxia in KC. Understanding the
mechanism of the altered regulation of NF-kappa B and iNOS in KC-HCFs will provide
better insight into the potential inflammatory component of the KC pathogenesis.
