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Unravelling the Impact of Cyclic Mechanical Stretch in Keratoconus—A Transcriptomic Profiling Study

Biomechanical and molecular stresses may contribute to the pathogenesis of keratoconus (KC). We aimed to profile the transcriptomic changes in healthy primary human corneal (HCF) and KC-derived cells (HKC) combined with TGFβ1 treatment and cyclic mechanical stretch (CMS), mimicking the pathophysiolo...

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Autores principales: Akoto, Theresa, Cai, Jingwen, Nicholas, Sarah, McCord, Hayden, Estes, Amy J., Xu, Hongyan, Karamichos, Dimitrios, Liu, Yutao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10139219/
https://www.ncbi.nlm.nih.gov/pubmed/37108600
http://dx.doi.org/10.3390/ijms24087437
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author Akoto, Theresa
Cai, Jingwen
Nicholas, Sarah
McCord, Hayden
Estes, Amy J.
Xu, Hongyan
Karamichos, Dimitrios
Liu, Yutao
author_facet Akoto, Theresa
Cai, Jingwen
Nicholas, Sarah
McCord, Hayden
Estes, Amy J.
Xu, Hongyan
Karamichos, Dimitrios
Liu, Yutao
author_sort Akoto, Theresa
collection PubMed
description Biomechanical and molecular stresses may contribute to the pathogenesis of keratoconus (KC). We aimed to profile the transcriptomic changes in healthy primary human corneal (HCF) and KC-derived cells (HKC) combined with TGFβ1 treatment and cyclic mechanical stretch (CMS), mimicking the pathophysiological condition in KC. HCFs (n = 4) and HKCs (n = 4) were cultured in flexible-bottom collagen-coated 6-well plates treated with 0, 5, and 10 ng/mL of TGFβ1 with or without 15% CMS (1 cycle/s, 24 h) using a computer-controlled Flexcell FX-6000T Tension system. We used stranded total RNA-Seq to profile expression changes in 48 HCF/HKC samples (100 bp PE, 70–90 million reads per sample), followed by bioinformatics analysis using an established pipeline with Partek Flow software. A multi-factor ANOVA model, including KC, TGFβ1 treatment, and CMS, was used to identify differentially expressed genes (DEGs, |fold change| ≥ 1.5, FDR ≤ 0.1, CPM ≥ 10 in ≥1 sample) in HKCs (n = 24) vs. HCFs (n = 24) and those responsive to TGFβ1 and/or CMS. PANTHER classification system and the DAVID bioinformatics resources were used to identify significantly enriched pathways (FDR ≤ 0.05). Using multi-factorial ANOVA analyses, 479 DEGs were identified in HKCs vs. HCFs including TGFβ1 treatment and CMS as cofactors. Among these DEGs, 199 KC-altered genes were responsive to TGFβ1, thirteen were responsive to CMS, and six were responsive to TGFβ1 and CMS. Pathway analyses using PANTHER and DAVID indicated the enrichment of genes involved in numerous KC-relevant functions, including but not limited to degradation of extracellular matrix, inflammatory response, apoptotic processes, WNT signaling, collagen fibril organization, and cytoskeletal structure organization. TGFβ1-responsive KC DEGs were also enriched in these. CMS-responsive KC-altered genes such as OBSCN, CLU, HDAC5, AK4, ITGA10, and F2RL1 were identified. Some KC-altered genes, such as CLU and F2RL1, were identified to be responsive to both TGFβ1 and CMS. For the first time, our multi-factorial RNA-Seq study has identified many KC-relevant genes and pathways in HKCs with TGFβ1 treatment under CMS, suggesting a potential role of TGFβ1 and biomechanical stretch in KC development.
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spelling pubmed-101392192023-04-28 Unravelling the Impact of Cyclic Mechanical Stretch in Keratoconus—A Transcriptomic Profiling Study Akoto, Theresa Cai, Jingwen Nicholas, Sarah McCord, Hayden Estes, Amy J. Xu, Hongyan Karamichos, Dimitrios Liu, Yutao Int J Mol Sci Article Biomechanical and molecular stresses may contribute to the pathogenesis of keratoconus (KC). We aimed to profile the transcriptomic changes in healthy primary human corneal (HCF) and KC-derived cells (HKC) combined with TGFβ1 treatment and cyclic mechanical stretch (CMS), mimicking the pathophysiological condition in KC. HCFs (n = 4) and HKCs (n = 4) were cultured in flexible-bottom collagen-coated 6-well plates treated with 0, 5, and 10 ng/mL of TGFβ1 with or without 15% CMS (1 cycle/s, 24 h) using a computer-controlled Flexcell FX-6000T Tension system. We used stranded total RNA-Seq to profile expression changes in 48 HCF/HKC samples (100 bp PE, 70–90 million reads per sample), followed by bioinformatics analysis using an established pipeline with Partek Flow software. A multi-factor ANOVA model, including KC, TGFβ1 treatment, and CMS, was used to identify differentially expressed genes (DEGs, |fold change| ≥ 1.5, FDR ≤ 0.1, CPM ≥ 10 in ≥1 sample) in HKCs (n = 24) vs. HCFs (n = 24) and those responsive to TGFβ1 and/or CMS. PANTHER classification system and the DAVID bioinformatics resources were used to identify significantly enriched pathways (FDR ≤ 0.05). Using multi-factorial ANOVA analyses, 479 DEGs were identified in HKCs vs. HCFs including TGFβ1 treatment and CMS as cofactors. Among these DEGs, 199 KC-altered genes were responsive to TGFβ1, thirteen were responsive to CMS, and six were responsive to TGFβ1 and CMS. Pathway analyses using PANTHER and DAVID indicated the enrichment of genes involved in numerous KC-relevant functions, including but not limited to degradation of extracellular matrix, inflammatory response, apoptotic processes, WNT signaling, collagen fibril organization, and cytoskeletal structure organization. TGFβ1-responsive KC DEGs were also enriched in these. CMS-responsive KC-altered genes such as OBSCN, CLU, HDAC5, AK4, ITGA10, and F2RL1 were identified. Some KC-altered genes, such as CLU and F2RL1, were identified to be responsive to both TGFβ1 and CMS. For the first time, our multi-factorial RNA-Seq study has identified many KC-relevant genes and pathways in HKCs with TGFβ1 treatment under CMS, suggesting a potential role of TGFβ1 and biomechanical stretch in KC development. MDPI 2023-04-18 /pmc/articles/PMC10139219/ /pubmed/37108600 http://dx.doi.org/10.3390/ijms24087437 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Akoto, Theresa
Cai, Jingwen
Nicholas, Sarah
McCord, Hayden
Estes, Amy J.
Xu, Hongyan
Karamichos, Dimitrios
Liu, Yutao
Unravelling the Impact of Cyclic Mechanical Stretch in Keratoconus—A Transcriptomic Profiling Study
title Unravelling the Impact of Cyclic Mechanical Stretch in Keratoconus—A Transcriptomic Profiling Study
title_full Unravelling the Impact of Cyclic Mechanical Stretch in Keratoconus—A Transcriptomic Profiling Study
title_fullStr Unravelling the Impact of Cyclic Mechanical Stretch in Keratoconus—A Transcriptomic Profiling Study
title_full_unstemmed Unravelling the Impact of Cyclic Mechanical Stretch in Keratoconus—A Transcriptomic Profiling Study
title_short Unravelling the Impact of Cyclic Mechanical Stretch in Keratoconus—A Transcriptomic Profiling Study
title_sort unravelling the impact of cyclic mechanical stretch in keratoconus—a transcriptomic profiling study
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10139219/
https://www.ncbi.nlm.nih.gov/pubmed/37108600
http://dx.doi.org/10.3390/ijms24087437
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