PC238. TBK1 as a Promising Druggable Target to Interrupt Interferon γ Atherogenic Signals and to Prevent or to Treat Obstructive Vascular Disease

Abstract

Interferon γ is a recognized culprit of atherogenesis. Blockade of interferon γ signaling in endothelial and smooth muscle cells should positively affect intimal hyperplasia. Expression of signal transducer and activator of transcription 1 (STAT1), the key transcriptional regulator of interferon γ signaling, is regulated by autocrine interferon β. In turn, interferon β is downstream to the noncanonical IκB kinase TANK-binding kinase 1 (TBK1; Fig 1). We checked whether TBK1 inhibition, using the clinically safe drug amlexanox as well as another inhibitor, MRT67307, reduces STAT1 expression and interferon γ signaling in vascular cells. Human coronary artery endothelial and smooth muscle cells were treated with poly I:C (100 μg/mL), a double-stranded RNA mimetic that stimulates TBK1 phosphorylation, or with human recombinant interferon γ (500 U/mL). Cells were pretreated with amlexanox (50-100 μM) for 1 hour or 24 to 48 hours before poly I:C or interferon γ, respectively, and harvested for RNA and protein isolation at 1 hour, 7 hours, and 24 hours after treatment. Samples were analyzed by Western blot and reverse transcription quantitative polymerase chain reaction. Treatment of endothelial and smooth muscle cells with poly I:C for 1 hour was optimal to induce TBK1 phosphorylation. Poly I:C upregulated interferon γ and STAT1 messenger RNA levels in both cell types. Phosphorylated TBK1 levels were increased by mere addition of amlexanox, consistent with its mechanism of action. Preincubation with amlexanox decreased basal STAT1 expression and inhibited poly I:C-induced upregulation of interferon γ and STAT1 messenger RNA, confirmed at the protein level for STAT1. Importantly, pretreatment of cells with amlexanox for 24 hours to 48 hours blunted the upregulation of the interferon γ-dependent gene IDO (Fig 2). Altogether our data validate the critical role of TBK1 in mediating interferon γ atherogenic signaling and set the basis for the pursuit of TBK1 inhibitors as novel therapies to prevent and to treat obstructive vascular disease. Future studies are aimed at checking the effectiveness of amlexanox in curbing intimal hyperplasia in a rat carotid artery model of post-balloon angioplasty stenosis and in a canine model of vein interposition graft failure.Fig 2View Large Image Figure ViewerDownload Hi-res image Download (PPT)