Cargando…

Acute hypoxia influences collagen and matrix metalloproteinase expression by human keratoconus cells in vitro

Keratoconus (KC) is a progressive corneal ectasia linked to thinning of the central cornea. Hard contact lenses, rigid gas permeable lenses, and scleral lenses are the primary treatment modalities for early to mid- stages of KC to correct refractive error and astigmatism that develops as a result of...

Descripción completa

Detalles Bibliográficos
Autores principales: McKay, Tina B., Hjortdal, Jesper, Priyadarsini, Shrestha, Karamichos, Dimitrios
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5398580/
https://www.ncbi.nlm.nih.gov/pubmed/28426715
http://dx.doi.org/10.1371/journal.pone.0176017
_version_ 1783230490525827072
author McKay, Tina B.
Hjortdal, Jesper
Priyadarsini, Shrestha
Karamichos, Dimitrios
author_facet McKay, Tina B.
Hjortdal, Jesper
Priyadarsini, Shrestha
Karamichos, Dimitrios
author_sort McKay, Tina B.
collection PubMed
description Keratoconus (KC) is a progressive corneal ectasia linked to thinning of the central cornea. Hard contact lenses, rigid gas permeable lenses, and scleral lenses are the primary treatment modalities for early to mid- stages of KC to correct refractive error and astigmatism that develops as a result of an irregular corneal structure. These treatments are associated with significant drawbacks, including reduced availability of the tear film and oxygen to the corneal epithelium and stroma. However, it remains unknown whether hypoxia affects corneal integrity in the KC pathobiology. A number of studies have associated elevated oxidative stress with KC both in vitro and ex vivo. We hypothesized that KC-derived corneal fibroblasts are more susceptible to hypoxia-induced oxidative stress compared to healthy controls leading to exacerbation of corneal thinning in KC. This study investigated the effects of hypoxia on ECM secretion, assembly, and matrix metalloproteinase (MMP) expression in human corneal fibroblasts from healthy controls (HCFs) and KC patients (HKCs) in vitro. HCFs and HKCs were cultured in 3D constructs for 3 weeks and maintained or transferred to normoxic (21% O(2)) or hypoxic (2% O(2)) conditions, respectively, for 1 additional week. At the 4 week time-point, constructs were isolated and probed for Collagen I, III, and V, keratocan and MMP-1, -2, -3, -9, and -13, as well as hypoxia markers, hypoxia inducible factor-1α and lactoferrin. Conditioned media was also collected and probed for Collagen I, III, and V by Western blot. Thickness of the ECM assembled by HCFs and HKCs was measured using immunofluorescence microscopy. Results showed that hypoxia significantly reduced Collagen I secretion in HKCs, as well as upregulated the expression of MMP-1 and -2 with no significant effects on MMP-3, -9, or -13. ECM thickness was reduced in both cell types following 1 week in a low oxygen environment. Our study shows that hypoxia influences collagen and MMP expression by HKCs, which may have consequential effects on ECM structure in the context of KC.
format Online
Article
Text
id pubmed-5398580
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-53985802017-05-04 Acute hypoxia influences collagen and matrix metalloproteinase expression by human keratoconus cells in vitro McKay, Tina B. Hjortdal, Jesper Priyadarsini, Shrestha Karamichos, Dimitrios PLoS One Research Article Keratoconus (KC) is a progressive corneal ectasia linked to thinning of the central cornea. Hard contact lenses, rigid gas permeable lenses, and scleral lenses are the primary treatment modalities for early to mid- stages of KC to correct refractive error and astigmatism that develops as a result of an irregular corneal structure. These treatments are associated with significant drawbacks, including reduced availability of the tear film and oxygen to the corneal epithelium and stroma. However, it remains unknown whether hypoxia affects corneal integrity in the KC pathobiology. A number of studies have associated elevated oxidative stress with KC both in vitro and ex vivo. We hypothesized that KC-derived corneal fibroblasts are more susceptible to hypoxia-induced oxidative stress compared to healthy controls leading to exacerbation of corneal thinning in KC. This study investigated the effects of hypoxia on ECM secretion, assembly, and matrix metalloproteinase (MMP) expression in human corneal fibroblasts from healthy controls (HCFs) and KC patients (HKCs) in vitro. HCFs and HKCs were cultured in 3D constructs for 3 weeks and maintained or transferred to normoxic (21% O(2)) or hypoxic (2% O(2)) conditions, respectively, for 1 additional week. At the 4 week time-point, constructs were isolated and probed for Collagen I, III, and V, keratocan and MMP-1, -2, -3, -9, and -13, as well as hypoxia markers, hypoxia inducible factor-1α and lactoferrin. Conditioned media was also collected and probed for Collagen I, III, and V by Western blot. Thickness of the ECM assembled by HCFs and HKCs was measured using immunofluorescence microscopy. Results showed that hypoxia significantly reduced Collagen I secretion in HKCs, as well as upregulated the expression of MMP-1 and -2 with no significant effects on MMP-3, -9, or -13. ECM thickness was reduced in both cell types following 1 week in a low oxygen environment. Our study shows that hypoxia influences collagen and MMP expression by HKCs, which may have consequential effects on ECM structure in the context of KC. Public Library of Science 2017-04-20 /pmc/articles/PMC5398580/ /pubmed/28426715 http://dx.doi.org/10.1371/journal.pone.0176017 Text en © 2017 McKay et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
McKay, Tina B.
Hjortdal, Jesper
Priyadarsini, Shrestha
Karamichos, Dimitrios
Acute hypoxia influences collagen and matrix metalloproteinase expression by human keratoconus cells in vitro
title Acute hypoxia influences collagen and matrix metalloproteinase expression by human keratoconus cells in vitro
title_full Acute hypoxia influences collagen and matrix metalloproteinase expression by human keratoconus cells in vitro
title_fullStr Acute hypoxia influences collagen and matrix metalloproteinase expression by human keratoconus cells in vitro
title_full_unstemmed Acute hypoxia influences collagen and matrix metalloproteinase expression by human keratoconus cells in vitro
title_short Acute hypoxia influences collagen and matrix metalloproteinase expression by human keratoconus cells in vitro
title_sort acute hypoxia influences collagen and matrix metalloproteinase expression by human keratoconus cells in vitro
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5398580/
https://www.ncbi.nlm.nih.gov/pubmed/28426715
http://dx.doi.org/10.1371/journal.pone.0176017
work_keys_str_mv AT mckaytinab acutehypoxiainfluencescollagenandmatrixmetalloproteinaseexpressionbyhumankeratoconuscellsinvitro
AT hjortdaljesper acutehypoxiainfluencescollagenandmatrixmetalloproteinaseexpressionbyhumankeratoconuscellsinvitro
AT priyadarsinishrestha acutehypoxiainfluencescollagenandmatrixmetalloproteinaseexpressionbyhumankeratoconuscellsinvitro
AT karamichosdimitrios acutehypoxiainfluencescollagenandmatrixmetalloproteinaseexpressionbyhumankeratoconuscellsinvitro