Abstract Body: Immune system activation has been shown to play an important role in the pathogenesis of hypertension and substantial evidence has been highlighting the importance of inflammasomes in vascular disease progression. Mitochondria damage leads to mitochondrial DNA (mtDNA) releasing into the cytoplasm, which, in turn, triggers the activation of leucine-rich-containing (NLR) family pyrin domain containing 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasomes, thereby exacerbating inflammation. However, whether mtDNA release-dependent exacerbation of inflammasomes drives endothelial cell activation and vascular damage remains to be elucidated. We hypothesize that cytosolic mtDNA release triggers endothelial cell activation and inflammation via Toll-Like Receptor 9 (TLR9) and inflammasome pathway activation, leading to vascular dysfunction. To assess this hypothesis, a concentration-response curve to endothelin-1 (ET-1) was performed in mesenteric resistance arteries (MRAs) isolated from wild-type and TLR9 knockout C57 mice in the presence or absence of mtDNA (4ug/mL) for one hour. To test inflammasome pathway activation, vascular smooth muscle cells (VSMC) and endothelial cells (EC) were incubated with TLR9 agonist (ODN2395, 3uM) or mtDNA (4ug/mL) and co-incubated with TLR9/AIM2 inhibitor (ODN2088, 2uM) or NLRP3 inhibitor (MCC950, 10uM) for 24 hours. Protein expression was assessed via Western blot and immunophenotyping was performed by flow cytometry. Data are presented as mean ± S.E.M and significance was set at p<0.05. After incubation with mtDNA, MRAs from wild-type mice presented an increased contractile response to ET-1 which was prevented in TLR9 KO mice. In VSMC, TLR9 expression increased after incubation with ODN2395 (vehicle: 1.2 ± 0.2 vs ODN2395: 4 ± 1.5) or mtDNA (vehicle: 1.2 ± 0.2 vs mtDNA: 4.2 ± 1.2) while co-incubation with ODN2088 reversed this effect. Additionally, mtDNA promoted increased TNF-alpha (vehicle: 1 ± 0.1 vs mtDNA: 2.6 ± 0.5) and IL-6 (vehicle: 1 ± 0.1 vs mtDNA: 4.6 ± 0.3) expression in VSMC which was abolished in the presence of ODN2088. In EC, flow cytometry indicated that incubation with mtDNA promoted a 4-fold increase in the expression of CD31+ and a 2-fold increase in CD54+ cells while co-incubation with ODN2088 or MCC950 reversed this effect. Altogether these data suggest that cell-free mtDNA triggers the innate immune response via TLR9 and inflammasomes driving EC activation, vascular dysfunction, and inflammation.