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Tina B McKay, Shrestha Priyadarsini, Jesper Hjortdal, Dimitrios Karamichos; Self-Assembled System Recapitulates Keratoconus Hypoxic Condition In Vitro. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
Keratoconus (KC) is a progressive corneal ectasia linked to thinning of the corneal stroma. Hard contact lenses, rigid gas permeable lenses, and scleral lenses are the primary treatment modality for early stages of KC. Unfortunately, contact lenses may lead to hypoxic conditions within the cornea. The effect of hypoxia on extracellular matrix (ECM) assembly in the KC pathobiology has yet to be explored. We have previously shown that primary cells isolated from KC corneas have altered metabolism that favors aerobic glycolysis. We hypothesize that KC-derived cells are more susceptible to hypoxia-induced oxidative stress compared to healthy controls leading to exacerbation of the KC pathology.
Primary cells of human corneal fibroblasts isolated from healthy controls (HCFs) and human keratoconus patients (HKCs) were cultured in 3D constructs and maintained or transferred to normoxic (21 percent O2) or hypoxic (2 percent O2) conditions, respectively, for one week. Conditioned media was collected daily and probed for collagen and matrix metalloproteinases (MMPs) secretion by Western blot. At the four week time-point, constructs were isolated and probed for Collagen I, III, and V and MMP-1, -2, -3, -9, and -13, as well as hypoxia markers. Thickness of the ECM assembled by HCFs and HKCs was also measured using immunofluorescence microscopy.
HKCs showed elevated basal hypoxia-inducible factor-1α and aryl hydrocarbon receptor expression, suggesting increased stabilization of hypoxia-related proteins in KC. Hypoxia significantly reduced Collagen I secretion at a much earlier time-point (t 48 hours) in HKCs compared to HCFs (t 120 hours). Hypoxia also upregulated the expression of MMP-1 and -2 with no effect on MMP-3, -9, or -13, suggesting differential effects on regulation of ECM degradation. ECM thickness was reduced in both cell types following one week in hypoxia.
These results show that HKCs have increased responsiveness to hypoxia-induced oxidative stress, which leads to immediate reduction of collagen secretion. This study suggests that the use of treatment modalities that limit oxygen levels in the human corneal stroma may have consequential effects on ECM assembly in progressive KC patients.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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