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

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 datasets, 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≤0.05. RESULTS Pulpitis gene signatures were created using differential gene expression analysis at cutoff points p=0.0001 and 0.000018. Top upregulated genes were selected as potential pulpitis biomarkers. Among these, IL8, IL6 and MMP9 were previously identified as pulpitis biomarkers. Novel up-regulated 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=0.0001). Fluvastatin reduced IL8, IL6, CCL21, AQP9 (p<0.001) and MMP9 (p<0.05) in the ex-vivo pulpitis model, while dequalinium chloride reduced AQP9 (p<0.001) 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 licensed drugs is warranted.