Transcriptome Analysis of Gene Expression Provides New Insights into the Effect of Mild Therapeutic Hypothermia on Primary Human Cortical Astrocytes Cultured under Hypoxia

Mootaz M Salman, Philip Kitchen, M Nicola Woodroofe, Roslyn M Bill, Alex C Conner, Paul R Heath, Matthew T Conner

Research output: Contribution to journalArticle

Abstract

Hypothermia is increasingly used as a therapeutic measure to treat brain injury. However, the cellular mechanisms underpinning its actions are complex and are not yet fully elucidated. Astrocytes are the most abundant cell type in the brain and are likely to play a critical role. In this study, transcriptional changes and the protein expression profile of human primary cortical astrocytes cultured under hypoxic conditions for 6 h were investigated. Cells were treated either with or without a mild hypothermic intervention 2 h post-insult to mimic the treatment of patients following traumatic brain injury (TBI) and/or stroke. Using human gene expression microarrays, 411 differentially expressed genes were identified following hypothermic treatment of astrocytes following a 2 h hypoxic insult. KEGG pathway analysis indicated that these genes were mainly enriched in the Wnt and p53 signaling pathways, which were inhibited following hypothermic intervention. The expression levels of 168 genes involved in Wnt signaling were validated by quantitative real-time-PCR (qPCR). Among these genes, 10 were up-regulated and 32 were down-regulated with the remainder unchanged. Two of the differentially expressed genes (DEGs), p38 and JNK, were selected for validation at the protein level using cell based ELISA. Hypothermic intervention significantly down-regulated total protein levels for the gene products of p38 and JNK. Moreover, hypothermia significantly up-regulated the phosphorylated (activated) forms of JNK protein, while downregulating phosphorylation of p38 protein. Within the p53 signaling pathway, 35 human apoptosis-related proteins closely associated with Wnt signaling were investigated using a Proteome Profiling Array. Hypothermic intervention significantly down-regulated 18 proteins, while upregulating one protein, survivin. Hypothermia is a complex intervention; this study provides the first detailed longitudinal investigation at the transcript and protein expression levels of the molecular effects of therapeutic hypothermic intervention on hypoxic human primary cortical astrocytes. The identified genes and proteins are targets for detailed functional studies, which may help to develop new treatments for brain injury based on an in-depth mechanistic understanding of the astrocytic response to hypoxia and/or hypothermia.

Original languageEnglish
Pages (from-to)386
JournalFrontiers in Cellular Neuroscience
Volume11
Early online date14 Dec 2017
DOIs
Publication statusPublished - 14 Dec 2017

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Induced Hypothermia
Gene Expression Profiling
Astrocytes
Gene Expression
Proteins
Hypothermia
Genes
Brain Injuries
Hypoxia
Wnt Signaling Pathway
Therapeutic Uses
Proteome
Therapeutics
Real-Time Polymerase Chain Reaction
Down-Regulation
Enzyme-Linked Immunosorbent Assay
Stroke
Phosphorylation
Apoptosis

Bibliographical note

Copyright © 2017 Salman, Kitchen, Woodroofe, Bill, Conner, Heath and Conner. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Funding: HCED grant number GD-13-3 (M Salman), BMRC Sheffield Hallam University, University of Sheffield and RIHS School of Sciences University of Wolverhampton.

Keywords

  • hypothermia
  • astrocyte
  • Wnt signalling pathway
  • p53 signalling
  • apoptosis
  • hypoxia
  • MAPK

Cite this

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abstract = "Hypothermia is increasingly used as a therapeutic measure to treat brain injury. However, the cellular mechanisms underpinning its actions are complex and are not yet fully elucidated. Astrocytes are the most abundant cell type in the brain and are likely to play a critical role. In this study, transcriptional changes and the protein expression profile of human primary cortical astrocytes cultured under hypoxic conditions for 6 h were investigated. Cells were treated either with or without a mild hypothermic intervention 2 h post-insult to mimic the treatment of patients following traumatic brain injury (TBI) and/or stroke. Using human gene expression microarrays, 411 differentially expressed genes were identified following hypothermic treatment of astrocytes following a 2 h hypoxic insult. KEGG pathway analysis indicated that these genes were mainly enriched in the Wnt and p53 signaling pathways, which were inhibited following hypothermic intervention. The expression levels of 168 genes involved in Wnt signaling were validated by quantitative real-time-PCR (qPCR). Among these genes, 10 were up-regulated and 32 were down-regulated with the remainder unchanged. Two of the differentially expressed genes (DEGs), p38 and JNK, were selected for validation at the protein level using cell based ELISA. Hypothermic intervention significantly down-regulated total protein levels for the gene products of p38 and JNK. Moreover, hypothermia significantly up-regulated the phosphorylated (activated) forms of JNK protein, while downregulating phosphorylation of p38 protein. Within the p53 signaling pathway, 35 human apoptosis-related proteins closely associated with Wnt signaling were investigated using a Proteome Profiling Array. Hypothermic intervention significantly down-regulated 18 proteins, while upregulating one protein, survivin. Hypothermia is a complex intervention; this study provides the first detailed longitudinal investigation at the transcript and protein expression levels of the molecular effects of therapeutic hypothermic intervention on hypoxic human primary cortical astrocytes. The identified genes and proteins are targets for detailed functional studies, which may help to develop new treatments for brain injury based on an in-depth mechanistic understanding of the astrocytic response to hypoxia and/or hypothermia.",
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Transcriptome Analysis of Gene Expression Provides New Insights into the Effect of Mild Therapeutic Hypothermia on Primary Human Cortical Astrocytes Cultured under Hypoxia. / Salman, Mootaz M; Kitchen, Philip; Woodroofe, M Nicola; Bill, Roslyn M; Conner, Alex C; Heath, Paul R; Conner, Matthew T.

In: Frontiers in Cellular Neuroscience, Vol. 11, 14.12.2017, p. 386.

Research output: Contribution to journalArticle

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AU - Bill, Roslyn M

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AU - Heath, Paul R

AU - Conner, Matthew T

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