Identification and validation of novel biomarkers and therapeutics for pulpitis using connectivity mapping

Banan Al‐Natour; Robby Rankin; Robyn McKenna; Hayley McMillan; Shu‐Dong Zhang; Imad About; Asma A. Khan; Johnah C. Galicia; Fionnuala T. Lundy; Ikhlas A. El‐Karim

doi:10.1111/iej.13547

Identification and validation of novel biomarkers and therapeutics for pulpitis using connectivity mapping

Banan Al‐Natour, Robby Rankin, Robyn McKenna, Hayley McMillan, Shu‐Dong Zhang, Imad About, Asma A. Khan, Johnah C. Galicia, Fionnuala T. Lundy, Ikhlas A. El‐Karim

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Abstract

AIM: To create an irreversible pulpitis gene signature from microarray data of healthy and inflamed dental pulps, followed by a bioinformatics approach using connectivity mapping to identify therapeutic compounds that could potentially treat pulpitis.

METHODOLOGY: The Gene Expression Omnibus (GEO) database, an international public repository of genomics data sets, was searched for human microarray datasets assessing pulpitis. An irreversible pulpitis gene expression signature was generated by differential expression analysis. The statistically significant connectivity map (ssCMap) method was used to identify compounds with a highly correlating gene expression pattern. qPCR was used to validate novel pulpitis genes. An ex vivo pulpitis model was used to test the effects of the compounds identified, and the level of inflammatory cytokines was measured with qPCR, ELISA and multiplex array. Means were compared using the t-test or ANOVA with the level of significance set at p ≤ .05.

RESULTS: Pulpitis gene signatures were created using differential gene expression analysis at cutoff points p = .0001 and .000018. Top upregulated genes were selected as potential pulpitis biomarkers. Among these, IL8, IL6 and MMP9 were previously identified as pulpitis biomarkers. Novel upregulated genes, chemokine (C-C motif) ligand 21 (CCL21), metallothionein 1H (MT1H) and aquaporin 9 (AQP9) were validated in the pulp tissue of teeth clinically diagnosed with irreversible pulpitis using qPCR. ssCMap analysis identified fluvastatin (Statin) and dequalinium chloride (Quaternary ammonium) as compounds with the strongest correlation to the gene signatures (p = .0001). Fluvastatin reduced IL8, IL6, CCL21, AQP9 (p < .001) and MMP9 (p < .05) in the ex vivo pulpitis model, while dequalinium chloride reduced AQP9 (p < .001) but had no significant effect on the other biomarkers.

CONCLUSIONS: AQP9, MT1H and CCL21 were identified and validated as novel biomarkers for pulpitis. Fluvastatin and dequalinium chloride identified by the ssCMap as potential therapeutics for pulpitis reduced selected pulpitis biomarkers in an ex vivo pulpitis model. In vivo testing of these licenced drugs is warranted.

Original language	English
Pages (from-to)	1571-1580
Number of pages	10
Journal	International Endodontic Journal
Volume	54
Issue number	9
Early online date	17 Jun 2021
DOIs	https://doi.org/10.1111/iej.13547
Publication status	Published (in print/issue) - Sept 2021

Bibliographical note

Funding Information:
The authors would like to thanks Dr Bettina Schock Wellcome-Wolfson institute for Experimental Medicine, Queen?s University Belfast for advice on ssCMap and review of the manuscript. We also thank Ms Catherine Fulton for technical expertise.

Publisher Copyright:
© 2021 The Authors. International Endodontic Journal published by John Wiley & Sons Ltd on behalf of British Endodontic Society

Keywords

ORIGINAL SCIENTIFIC ARTICLE
ORIGINAL SCIENTIFIC ARTICLES
dequalinium chloride
fluvastatin
irreversible pulpitis
pulp capping
ssCMap
vital pulp treatment

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Cite this

@article{e1b0f80415b148e6b22755c72868d506,

title = "Identification and validation of novel biomarkers and therapeutics for pulpitis using connectivity mapping",

abstract = "AIM: To create an irreversible pulpitis gene signature from microarray data of healthy and inflamed dental pulps, followed by a bioinformatics approach using connectivity mapping to identify therapeutic compounds that could potentially treat pulpitis.METHODOLOGY: The Gene Expression Omnibus (GEO) database, an international public repository of genomics data sets, was searched for human microarray datasets assessing pulpitis. An irreversible pulpitis gene expression signature was generated by differential expression analysis. The statistically significant connectivity map (ssCMap) method was used to identify compounds with a highly correlating gene expression pattern. qPCR was used to validate novel pulpitis genes. An ex vivo pulpitis model was used to test the effects of the compounds identified, and the level of inflammatory cytokines was measured with qPCR, ELISA and multiplex array. Means were compared using the t-test or ANOVA with the level of significance set at p ≤ .05.RESULTS: Pulpitis gene signatures were created using differential gene expression analysis at cutoff points p = .0001 and .000018. Top upregulated genes were selected as potential pulpitis biomarkers. Among these, IL8, IL6 and MMP9 were previously identified as pulpitis biomarkers. Novel upregulated genes, chemokine (C-C motif) ligand 21 (CCL21), metallothionein 1H (MT1H) and aquaporin 9 (AQP9) were validated in the pulp tissue of teeth clinically diagnosed with irreversible pulpitis using qPCR. ssCMap analysis identified fluvastatin (Statin) and dequalinium chloride (Quaternary ammonium) as compounds with the strongest correlation to the gene signatures (p = .0001). Fluvastatin reduced IL8, IL6, CCL21, AQP9 (p < .001) and MMP9 (p < .05) in the ex vivo pulpitis model, while dequalinium chloride reduced AQP9 (p < .001) but had no significant effect on the other biomarkers.CONCLUSIONS: AQP9, MT1H and CCL21 were identified and validated as novel biomarkers for pulpitis. Fluvastatin and dequalinium chloride identified by the ssCMap as potential therapeutics for pulpitis reduced selected pulpitis biomarkers in an ex vivo pulpitis model. In vivo testing of these licenced drugs is warranted.",

keywords = "ORIGINAL SCIENTIFIC ARTICLE, ORIGINAL SCIENTIFIC ARTICLES, dequalinium chloride, fluvastatin, irreversible pulpitis, pulp capping, ssCMap, vital pulp treatment",

author = "Banan Al‐Natour and Robby Rankin and Robyn McKenna and Hayley McMillan and Shu‐Dong Zhang and Imad About and Khan, {Asma A.} and Galicia, {Johnah C.} and Lundy, {Fionnuala T.} and El‐Karim, {Ikhlas A.}",

note = "Funding Information: The authors would like to thanks Dr Bettina Schock Wellcome-Wolfson institute for Experimental Medicine, Queen?s University Belfast for advice on ssCMap and review of the manuscript. We also thank Ms Catherine Fulton for technical expertise. Publisher Copyright: {\textcopyright} 2021 The Authors. International Endodontic Journal published by John Wiley & Sons Ltd on behalf of British Endodontic Society",

year = "2021",

month = sep,

doi = "10.1111/iej.13547",

language = "English",

volume = "54",

pages = "1571--1580",

journal = "International Endodontic Journal",

issn = "0143-2885",

publisher = "Wiley-Blackwell Publishing Ltd",

number = "9",

}

TY - JOUR

T1 - Identification and validation of novel biomarkers and therapeutics for pulpitis using connectivity mapping

AU - Al‐Natour, Banan

AU - Rankin, Robby

AU - McKenna, Robyn

AU - McMillan, Hayley

AU - Zhang, Shu‐Dong

AU - About, Imad

AU - Khan, Asma A.

AU - Galicia, Johnah C.

AU - Lundy, Fionnuala T.

AU - El‐Karim, Ikhlas A.

N1 - Funding Information: The authors would like to thanks Dr Bettina Schock Wellcome-Wolfson institute for Experimental Medicine, Queen?s University Belfast for advice on ssCMap and review of the manuscript. We also thank Ms Catherine Fulton for technical expertise. Publisher Copyright: © 2021 The Authors. International Endodontic Journal published by John Wiley & Sons Ltd on behalf of British Endodontic Society

PY - 2021/9

Y1 - 2021/9

N2 - AIM: To create an irreversible pulpitis gene signature from microarray data of healthy and inflamed dental pulps, followed by a bioinformatics approach using connectivity mapping to identify therapeutic compounds that could potentially treat pulpitis.METHODOLOGY: The Gene Expression Omnibus (GEO) database, an international public repository of genomics data sets, was searched for human microarray datasets assessing pulpitis. An irreversible pulpitis gene expression signature was generated by differential expression analysis. The statistically significant connectivity map (ssCMap) method was used to identify compounds with a highly correlating gene expression pattern. qPCR was used to validate novel pulpitis genes. An ex vivo pulpitis model was used to test the effects of the compounds identified, and the level of inflammatory cytokines was measured with qPCR, ELISA and multiplex array. Means were compared using the t-test or ANOVA with the level of significance set at p ≤ .05.RESULTS: Pulpitis gene signatures were created using differential gene expression analysis at cutoff points p = .0001 and .000018. Top upregulated genes were selected as potential pulpitis biomarkers. Among these, IL8, IL6 and MMP9 were previously identified as pulpitis biomarkers. Novel upregulated genes, chemokine (C-C motif) ligand 21 (CCL21), metallothionein 1H (MT1H) and aquaporin 9 (AQP9) were validated in the pulp tissue of teeth clinically diagnosed with irreversible pulpitis using qPCR. ssCMap analysis identified fluvastatin (Statin) and dequalinium chloride (Quaternary ammonium) as compounds with the strongest correlation to the gene signatures (p = .0001). Fluvastatin reduced IL8, IL6, CCL21, AQP9 (p < .001) and MMP9 (p < .05) in the ex vivo pulpitis model, while dequalinium chloride reduced AQP9 (p < .001) but had no significant effect on the other biomarkers.CONCLUSIONS: AQP9, MT1H and CCL21 were identified and validated as novel biomarkers for pulpitis. Fluvastatin and dequalinium chloride identified by the ssCMap as potential therapeutics for pulpitis reduced selected pulpitis biomarkers in an ex vivo pulpitis model. In vivo testing of these licenced drugs is warranted.

AB - AIM: To create an irreversible pulpitis gene signature from microarray data of healthy and inflamed dental pulps, followed by a bioinformatics approach using connectivity mapping to identify therapeutic compounds that could potentially treat pulpitis.METHODOLOGY: The Gene Expression Omnibus (GEO) database, an international public repository of genomics data sets, was searched for human microarray datasets assessing pulpitis. An irreversible pulpitis gene expression signature was generated by differential expression analysis. The statistically significant connectivity map (ssCMap) method was used to identify compounds with a highly correlating gene expression pattern. qPCR was used to validate novel pulpitis genes. An ex vivo pulpitis model was used to test the effects of the compounds identified, and the level of inflammatory cytokines was measured with qPCR, ELISA and multiplex array. Means were compared using the t-test or ANOVA with the level of significance set at p ≤ .05.RESULTS: Pulpitis gene signatures were created using differential gene expression analysis at cutoff points p = .0001 and .000018. Top upregulated genes were selected as potential pulpitis biomarkers. Among these, IL8, IL6 and MMP9 were previously identified as pulpitis biomarkers. Novel upregulated genes, chemokine (C-C motif) ligand 21 (CCL21), metallothionein 1H (MT1H) and aquaporin 9 (AQP9) were validated in the pulp tissue of teeth clinically diagnosed with irreversible pulpitis using qPCR. ssCMap analysis identified fluvastatin (Statin) and dequalinium chloride (Quaternary ammonium) as compounds with the strongest correlation to the gene signatures (p = .0001). Fluvastatin reduced IL8, IL6, CCL21, AQP9 (p < .001) and MMP9 (p < .05) in the ex vivo pulpitis model, while dequalinium chloride reduced AQP9 (p < .001) but had no significant effect on the other biomarkers.CONCLUSIONS: AQP9, MT1H and CCL21 were identified and validated as novel biomarkers for pulpitis. Fluvastatin and dequalinium chloride identified by the ssCMap as potential therapeutics for pulpitis reduced selected pulpitis biomarkers in an ex vivo pulpitis model. In vivo testing of these licenced drugs is warranted.

KW - ORIGINAL SCIENTIFIC ARTICLE

KW - ORIGINAL SCIENTIFIC ARTICLES

KW - dequalinium chloride

KW - fluvastatin

KW - irreversible pulpitis

KW - pulp capping

KW - ssCMap

KW - vital pulp treatment

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U2 - 10.1111/iej.13547

DO - 10.1111/iej.13547

M3 - Article

C2 - 33964033

SN - 0143-2885

VL - 54

SP - 1571

EP - 1580

JO - International Endodontic Journal

JF - International Endodontic Journal

IS - 9

ER -

Identification and validation of novel biomarkers and therapeutics for pulpitis using connectivity mapping

Abstract

Bibliographical note

Keywords

UN SDGs

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