Abstract P075: Angiotensin II Induces Endothelial Dysfunction And Vascular Remodeling Dependent Of Nlrp3 Inflammasome

Abstract

The NLRP3 inflammasome is a multimeric protein complex constituted by NLRP3, Asc and Capase-1 (Casp1). It triggers an inflammatory response by releasing the pro-inflammatory cytokines IL-1β and IL-18. NLRP3 inflammasome is expressed in different cells and its activation has been associated with several diseases including atherosclerosis and hypertension. Herein we tested the hypothesis that angiotensin II (AngII) induces vascular damage by activating the NLPR3 inflammasome in the vasculature. C57BL/6J male mice (Ctrl) and Casp-1 deficient mice (Casp1-/-) were treated with AngII (490 ng/min/kg/14 days by osmotic mini pump). In Ctrl mice, AngII treatment impaired the vascular relaxation to acetylcholine in mesenteric arteries, increased aorta media thickness [Ctrl: 49.4 ± 2.5 vs AngII: 62.3 ± 2.3* (μm), *P<0.05] and cross-sectional area [Ctrl: 0.11 ± 0.1 vs AngII: 0.15 ± 0.2* (mm), *P<0.05] and triggered NLRP3 inflammasome activation in aorta and mesenteric arteries, analyzed by caspase-1 cleavage and IL-1B maturation via western blot and casp1 activity - FAM-FLICA assay. Fascinatingly, Casp1-/- mice were protected from AngII-induced endothelial dysfunction and vascular remodeling. Furthermore, AngII (0.1uM) incubation, combined or not with lipopolysaccharide (500 ng.ml –1 ultrapure) or Nigericin (20 μM), elevated Casp1 cleavage and IL-1B maturation in Rat Aortic Smooth Muscle Cells (RASMC). Moreover, AngII elevated PCNA (~2.5-fold) and CyclinD1 (~2.1-fold) protein expression and induced vascular migration and proliferation measured by scratch assay and cell counting kit-8 (CCK-8) assay respectively. Interestingly NLRP3 antagonist incubation (MCC950, 1uM) abolished PCNA expression and attenuated the vascular migration and proliferation produced by AngII incubation. Our data suggest that AngII induces vascular damage by activating NLPR3 inflammasome directly in the vasculature. We place this innate immune receptor as a master regulator of the vascular phenotype and as a target for therapeutic strategies for vascular diseases. Future studies will be helpful providing a better understanding into the molecular mechanism of NLRP3 inflammasome activation and regulation in the control of vascular diseases.