Abstract P630: Nlrp3 Inflammasome Activation By Mitochondrial Dna Contributes To Oxidative Stress And Inflammation In The Vasculature Of Type 1 Diabetic Mice

Abstract

NLRP3 Inflammasome is a platform that regulates inflammatory responses by caspase-1 activation and processing of pro-IL-1β and pro-IL-18 to mature cytokines. NLRP3 is activated by several mechanisms including mitochondrial DNA (mitDNA). Circulating mitDNA is increased in diabetes, a condition associated with NLRP3 activation. We tested the hypothesis that mitDNA release is increased in type 1 diabetes (T1D) leading to NLRP3 activation and contributing to vascular inflammatory and oxidative processes. Wild type (WT) and NLRP3-deficient (NLRP3 -/- ) mice were treated with vehicle or streptozotocin (40 mg/kg), i.p. for 5 days. Vascular reactivity was determined in mesenteric resistance arteries (MA). Cultured vascular smooth muscle cells (VSMC) were stimulated with mitDNA of T1D (dmDNA) and control (cmDNA) mice. Caspase-1 and IL-1β activation was evaluated by western blot analysis and reactive oxygen species (ROS) by fluorescence to DHE. DNA was extracted, purified and amplified by real-time-PCR. Data are presented as mean ± standard error of mean, Veh vs. T1D. NLRP3 -/- T1D mice exhibited attenuated hyperglycemia vs. WT T1D mice [mg/dL, 241.0±27.7 vs. 337.6±18.1, p<0.05]. MA from T1D mice exhibited decreased ACh-induced dilatation vs. Veh [E max , 46.6±4.0 vs . 91.5±2.8, p<0.05], which was not observed in NLRP3 -/- T1D mice. Diabetes increased vascular caspase-1 [arbitrary units (a.u.), 1.2±0.1 vs. 0.8±0.1, p<0.05)] and IL-1β activation [4.8±1.1 vs. 0.8±0.5 p <0.05], but this activation was attenuated in NLRP3 -/- T1D. T1D mice exhibited increased NLRP3 activation and mitDNA release in pancreatic cells and increased circulating mitDNA. dmDNA, but not cmDNA, increased NLRP3 activation in VSMC (i.e. activated caspase-1 and increased IL-1β levels) [a.u., 4.2±0.1 vs. 1.9±0.1; 2.3±0.1 vs. 0.7±0.1, p<0.05]. NLRP3 activation was attenuated in NLRP3 -/- VMSC, but not in WT VSMC incubated with a TLR-9 antagonist. Increased ROS generation was observed in response to dmDNA, which was prevented by a mitochondrial uncoupler. Our data show that T1D increases mitDNA release, which promotes vascular NLRP3 activation via mitochondrial superoxide production, contributing to T1D-associated vascular dysfunction. Financial Support: FAPESP, CNPq.