Altered regulation of arginine metabolism during hypoxia in keratoconus human corneal fibroblasts

Abstract

Purpose : Keratoconus (KC) is a corneal disease, which leads to conical corneal shape and loss of vision in later stages. Recent studies discuss its potential inflammatory origin. Inflammatory processes are often accompanied by hypoxia, which result in oxidative stress and mitochondrial dysfunction.
In a previous study we determined changes in arginine metabolism, increased inducible nitric oxide synthase (iNOS) expression and a decreased arginase (Arg-II) activity in KC human corneal fibroblasts (HCF). Hypoxia inducible factor-1α (HIF-1α) and HIF-2α are transcription factors, e.g. for iNOS. The hypoxic response is mediated by HIFs, which are stabilized under hypoxic conditions by prolyl-hydroxylases (PHD), which have different isoforms, with cytoplasmatic or nuclear localization. The purpose of our present study was to evaluate iNOS, Arg-II, HIF-1α, HIF-2α, transmembane-P4H (P4H-TM) and hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) expression in normal and KC HCFs, under hypoxic conditions.

Methods : Primary human KC-HCFs (n=8) and normal HCFs (n=8) were isolated by digestion in collagenase A (1 mg/ml) from human corneal buttons, and cultured in DMEM/Ham’s F12 medium supplemented with 5% fetal calf serum. iNOS, Arg-II, HIF-1α, HIF-2α, P4H-TM and PHD2 were evaluated using qPCR. Hypoxic conditions were generated for 48h using CoCl2 for iNOS, Arg-II, HIF-1α and HIF-2α expression measurements and generating 1% O₂milieu in a hypoxic chamber for P4H-TM and PHD2 expression measurements.

Results : Hypoxia upregulated iNOS expression in normal HCFs (p=0.044), but not in KC-HCFs (p=0.863). Arg-II expression decreased through hypoxia in KC-HCFs (p=0.003), but not in normal HCFs (p=0.210). HIF-1α and HIF-2α expression was downregulated in both HCF types under hypoxia (p<0.014). Using 1% O₂, P4H-TM expression increased in normal (p=0.022), but not in KC-HCFs (p=0.232) and PHD2 expression increased in KC (p=0.009), but not in normal HCFs (p=0.496).

Conclusions : Under hypoxia, iNOS, Arg-II, P4H-TM and PHD2 regulation differs between KC and normal HCFs. The altered regulation may be related to the increased oxidative damage and mitochondrial dysfunction in KC-HCFs.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.