Clinical Pathology - Theses

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Author: Al-Sarray, Sameer A. Jadaan × Type: PhD thesis × Subject: lysine methyltransferase × Subject: cyproheptadine × Subject: R-PFI-2 ×

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    Characterising the role of selective Set7 inhibitors in transcriptional regulation and cytokine production in human microvascular endothelial cells
    Al-Sarray, Sameer A. Jadaan ( 2019)
    Set7 is a lysine methyltransferase which catalyses the transfer of a monomethyl group to lysine 4 of histone H3 (H3K4me1). Set7 is also associated with regulation of different non-histone proteins including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Recent reports have implicated Set7 in the activation of pro- inflammatory genes in human vascular endothelial cells in response to hyperglycemia. In addition, several studies have demonstrated that Set7 is required for NF-κB dependent tumor necrosis factor alpha (TNF-α) induced inflammation. Vascular inflammation can drive endothelial dysfunction, and is involved in the development of cardiovascular disease (CVD). Manipulation of Set7 methyltransferase activity by genetic deletion or pharmacological inhibition has been reported to regulate cytokine signalling in human microvascular endothelial cells (HMEC-1). Selective Set7 inhibitors have recently been developed, but their impact on vascular inflammation remains unknown. In this project, HMEC-1 cells were used to assess the Set7 inhibitors, PFI-2 and cyproheptadine hydrochloride mediated regulation under basal conditions and during TNF-α induced inflammation. Using RNA sequencing and gene set enrichment analysis (GSEA), Set7 inhibitors upregulated the expression of genes involved in the control of cholesterol Sterol Regulatory Element-Binding Proteins (SREBP), cholesterol and fatty acyl biosynthesis. On the other hand, pharmacological inhibition of Set7 by PFI-2 and cyproheptadine hydrochloride reduced the expression of genes implicated in cell cycle and smooth muscle cell contraction. Given its contribution to pro-inflammatory gene activation, we hypothesized that targeting Set7 activity may reduce the burden of vascular inflammation. Transcriptome profiling by RNA sequencing and GSEA studies showed PFI-2 and cyproheptadine hydrochloride can attenuate TNF-α induced gene expression. Set7 inhibitors suppressed the expression of genes implicated in cytokine and interferon signalling and NF-κB activation. Having shown that pharmacological inhibition of Set7 activity attenuates TNF-α driven gene expression, we proposed that PFI-2 and cyproheptadine hydrochloride could suppress TNF-α induced cytokine release. To test this hypothesis, inflammatory insult was induced and the impact of Set7 inhibitors was assessed by cytokine array. We detected robust attenuation in the secretion of various pro-inflammatory cytokines including IL-1b, IL-6, TNF-α and IFN- ɣ. Moreover, pharmacological Set7 inhibitors attenuated TNF-α stimulated production of key chemokines such as IL-8, MCP-1 and RANTES. PFI-2 and cyproheptadine hydrochloride also decreased release of growth factors and cytokines associated with allergic inflammation. We show, for the first time, Set7 inhibition attenuates induced cytokine expression and secretion. This work will lead to new therapeutic opportunities to address vascular inflammation.