Background: Transgenic mice with tamoxifen-inducible endothelium-restricted human endothelin-1 overexpression (ieET-1) exhibited blood pressure (BP) elevation 3 weeks or 3 months after induction, and vascular injury was observed after 3 months. We hypothesized that 3-week or 3-month exposure to ET-1 overexpression leads to gene dysregulation in mesenteric arteries (MAs). Methods: Ten to 12-week old male ieET-1 mice and control ieCre mice expressing a tamoxifen-inducible Cre recombinase under the control of the endothelium-specific Tie2 promoter were treated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and euthanized 16 days or 3 months later. RNA was extracted from MAs and used for total RNA-sequencing using Illumina HiSeq-2500. Differentially expressed (DE) genes were identified with fold change >1.3 and P <0.005. DE genes were validated by reverse transcription-quantitative PCR (RT-qPCR) using another set of mice. Alternative splicing was revealed by Spladder and validated by RT-qPCR in MAs. Results: RNA-sequencing revealed DE genes after 3-week (54) and 3-month ET-1 overexpression (7). Alternative splicing changes in mRNAs were revealed after 3-week (39) and 3-month ET-1 overexpression (21). One of the 3 genes validated by RT-qPCR, Khdrbs3, encodes KH domain containing RNA binding signal transduction associated 3 that regulates exon retention of some mRNAs including exon 7 of vascular endothelial growth factor A ( Vegfa ). Khdrbs3 was up-regulated after 3-week (fold change: 2.4±0.1 vs 1.0±0.1, P <0.05) and 3-month ET-1 overexpression (2.3±0.3 vs 1.3±0.1, P <0.05). Vegfa exon 7 retention was validated by demonstration of up-regulation of Vegfa 164 (1.3±0.1 vs 0.9±0.1, P<0.05) and Vegfa 188 isoforms (1.1±0.1 vs 0.9±0.1, P<0.05) after 3-month ET-1 overexpression ( P <0.05). Neuropilin 2 ( Nrp2 ) that interacts with VEGFA receptors was one of DE genes with alternative splicing in exon 17 ( Nrp2 a isoforms). Predicted Nrp2 a down-regulation ( P <0.05) was validated by RT-qPCR showing a decrease after 3-month ET overexpression (0.6±0.1 vs 1.1±0.2, P <0.05). Conclusions: This study demonstrated that 3-month ET-1 overexpression up-regulated Khdrbs3 and alternative splicing of Vegfa and Nrp2 , which may play a role in vascular injury in hypertension.
Introduction NLRP3 inflammasome is a molecular platform of innate immune system that regulates the inflammatory response through the activation of caspase‐1 and IL‐1β. It can be activated by several ligands, for example, mitochondrial DNA (mDNA). Some studies have shown that circulating mDNA is increased in patients with diabetes and play a role in the development of the disease; however, its role in vascular changes is still unknown. In this sense, we tested the hypothesis that mDNA contributes to vascular inflammatory/oxidative processes associated to type 1 diabetes (T1D) via NLRP3 inflammasome activation. Methods Wild‐type and NLRP3‐deficient (NLRP3 −/− ) mice were treated with vehicle (Veh) or streptozotocin (40 mg/kg) (T1D) for 5 days. Vascular reactivity was determined in mesenteric arteries. Caspase‐1 and IL‐1β expression were evaluated by western blot. Pancreatic mDNA was extracted from control (cmDNA) and diabetic animals (dmDNA) for endothelial cells stimulation. ROS generation was determined by chemiluminescence. Hydrogen peroxide (H 2 O 2 ) production and calcium influx were determined by fluorescence. Data are presented as mean ± standard error of the mean. Results Diabetes increased vascular caspase‐1 and IL‐1β activation [arbitrary units (a.u.), 1.2±0.1 vs. 0.8±0.1; 4.8±1.1 vs. 0.8±0.5 vs . the Veh, respectively, p <0.05]. However, this activation was attenuated in T1D NLRP3 −/− . Mesenteric arteries from T1D exhibited decreased ACh‐induced vasodilatation vs. Veh [(E max ), 46.6±4.0 vs . 91.5±2.8, n=4–5, p<0.05], which was not observed in T1D NLRP3 −/− . The NLRP3 inhibitor MCC950 acutely improved endothelium‐dependent vasodilation in T1D [(E max ), 62.5±3.8 vs . 85.6±3.7, n=4–5, p<0.05]. ROS generation [(Relative luminescence unit, RLU), 1.5×10 7 ±2.3 vs. 48.9×10 3 ±1.1, n=4–5, p<0.05] as well as H 2 O 2 production [(Relative fluorescence unit, RFU), 1082±242.6 vs. 232.0±42.8, n=4–5, p<0.05] were lower in mesenteric bed from T1D NLRP3 −/− than T1D. Only incubation with dmDNA reduced ACh‐induced vasodilation [(E max ), 48.4 ± 4.1 vs. 90.7 ± 3.4, n=4–5, p<0.05], which was attenuated by the presence of an antioxidant [(E max ), 48.4 ± 4.1 vs. 71.5 ± 3.1, n=4–5, p<0.05]. Similarly, only LPS‐primed cells incubated with dmDNA had significant NLRP3 activation (caspase‐1 and IL‐1β activation, respectively) [(u.a.), 1.6 ± 0.2 vs. 0.9 ± 0.1; and 1.6 ± 0.2 vs. 1.0 ± 0.1, n=4–5, p<0.05]. The dmDNA induced in a time‐dependent way both calcium influx and ROS generation in endothelial cells, being the last reversed by the presence of an antioxidant. Likewise, T1D patients had increased circulating mDNA vs. healthy volunteers and also higher NLRP3 serum expression, and caspase‐1/IL‐1β activation. Conclusion The T1D increases mDNA release which in turn stimulates increased calcium influx, induces ROS generation, and triggers vascular NLRP3 inflammasome activation contributing to inflammatory response and endothelial dysfunction in diabetes. Support or Funding Information Financial Support: FAPESP and CAPES. Ethics Committee number (026/2015). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
Introduction Aldosterone levels were increased in diabetic patients and animals with type 2 diabetes mellitus (DM2). The excess of aldosterone (aldo) aggravates endothelial dysfunction in diabetes by promoting insulin resistance, fibrosis, oxidative stress and inflammation. The pro‐inflammatory cytokines IL‐1β was release by activation of NOD‐like receptors that comprise a group of pattern recognition receptors involved in a variety of host innate immune responses. Recently, our group demonstrated the involvement of NLRP3 in aldosterone‐induced vascular dysfunction. So, considering that we hypothesized which aldosterone activates the inflammasome platform in the vasculature of DM2 mice. Methods Mesenteric arteries from control (db/+) and diabetic (db/db) mice treated with vehicle or spironolactone (spiro – mineralocorticoid receptor (MR) antagonist, 50 mg/Kg/day) or NLRP3 antagonist (MCC950, 10 mg/Kg/day) were used to determine vascular reactivity and aldosterone‐induced inflammasome activation. The plasma of animals was used to evaluated IL‐1β and aldosterone levels. Results Db/db mice exhibited increased IL‐1β levels, increased vascular expression/activation of caspase‐1, as well reduced acetylcholine (ACh) vasodilation, compared to control (Cont) mice. Spironolactone and MCC950 treatment decreased plasma IL‐1β levels [(pg/mL) (Cont: 44.4±9.9 n=7; Cont+Spiro: 27.2±8.3 n=5, db/db: 85.4±21.9 n=7; db/db+Spiro: 11.6±3.2 n=6, p<0.05) and ( Cont: 2.0±2.0 n=6; Cont+MCC950: 0.0±0.0 n=3, db/db: 318.2±97.6 n=5; db/db+MCC950: 37.5±28.8 n=5, p<0.05)]. Spiro treatment but not MCC950 reduced caspase‐1 activity/expression in mesenteric arteries from db/db mice (arbitrary units (a.u.) Cont: 1.0±0.1 n=6; Cont+Spiro: 0.8±0.2 n=5, db/db: 2.4±0.6 n=4; db/db+Spiro: 0.5±0.2 n=6, p<0.05) and ( Cont: 1.0±0.4 n=4; Cont+MCC950: 0.8±0.2 n=4, db/db: 2.0±0.9 n=4; db/db+MCC950: 1.1±0.2 n=5, p>0.05)]. Spiro treatment reduced glucose levels in db/db mice [Glucose (mg/dL), Cont: 156.1±5.7 n=10; Cont+spiro: 150.2±7.3 n=9; db/db: 265.1±17.4 n=12; db/db+spiro: 185.5±21.6 n=8, p<0.05] and improved Ach vasodilation in diabetic mice [Emax (% of relaxation) Cont: 78.5±4.1 n=7; control+spiro: 77.04±3.83 n=3; db/db: 40.5±6.4 n=8; db/db+spiro: 62.83±5.89 n=6, p<0.05]. Despite treatment with MCC950 does not reduce the aldosterone plasma levels [Aldo (pg/mL), Cont: 222.0±29.3 n=6; Cont+MC9950:183.7±81.0 n=3, db/db:552.7±85.4 n=5; db/db+MCC950:434.2±73.8 n=5, p>0.05] and glucose levels in db/db mice [Glucose (mg/dL), Cont: 178.3±9.1 n=6; Cont+MCC950: 166.3±4.8 n=3, db/db: 402.0±36.1 n=4; db/db+MCC950: 317.8±36.5 n=5, p>0.05], MCC950 improve the maximum response (Emax) of ACh [(Emax (% of relaxation) Cont: 78.5±4.1 n=6; Cont+MCC950: 77.0±3.8 n=3; db/db: 40.5±6.4 n=5; db/db+MCC950: 62.8±5.9 n=5, p<0.05]. Conclusion These results suggest that aldosterone activates the inflammasome platform in the vasculature of DM2 mice. The MR and NLRP3 receptors activation plays a crucial role on aldosterone‐induced in diabetes‐associated vascular dysfunction and inflammation. Support or Funding Information CAPES, CNPq, FAPESP This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
Introduction: We previously demonstrated a role for γδ T cells in hypertension and vascular injury. In inflammatory conditions γδT17 are a prominent producer of IL-17A, which has been shown to contribute to hypertension. The development and expansion of γδT17 cells is regulated in part through IL-23 receptors (IL-23R). Hypothesis: We hypothesized that angiotensin (Ang) II-induced blood pressure (BP) elevation and vascular injury would be blunted in mice with dysfunctional IL-23R. Methods: Wild-type (WT) and Il23r knock-in ( Il23r gfp/gfp ) mice were infused or not with Ang II (490ng/kg/min, SC) for 7 or 14 days. BP was monitored via telemetry, mesenteric artery function and remodeling using pressurized myography, and T cell profiling in mesenteric artery perivascular adipose tissue (PVAT) by flow cytometry. Results: Il-23r gfp/gfp mice exhibited a greater BP elevation in response to Ang II than WT by day 3 (152±5 vs 144±7 mm Hg, P <0.05), which was sustained through day 9 (169±2 vs 155±5 mm Hg, P <0.05), but eventually similar to that of WT mice by the end of the Ang II infusion (167±2 vs 167±4 mm Hg). Il-23r gfp/gfp mice were not protected from vascular dysfunction and remodeling after 14 days of Ang II. Il23r gfp/gfp mice had less γδT17 cells in PVAT than WT mice (40±8 vs 108±15 cells/PVAT, P <0.05). Ang II increased interferon-γ producing γδ T cells in WT (13±3 vs 5±1 cells/PVAT, P <0.05) and Il23r gfp/gfp mice (14±3 vs 3±1 cells/PVAT, P <0.05), and interferon-γ producing CD4 + (125±27 vs 38±13 cells/PVA, P <0.05) and CD8 + T cells (76±13 vs 33±8 cells/PVA, P <0.05) only in Il23r gfp/gfp mice. Conclusion: Functional IL-23R deficiency exaggerated BP elevation during the initiation of Ang II-induced hypertension, potentially due to the increased number of interferon-γ producing T cells.
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.
Introduction: Innate and adaptive immune cells contribute to hypertension and end-organ damage. High blood pressure (BP) causes cardiovascular injury and the release of damage-associated molecule patterns such as adenosine triphosphate (ATP). ATP can bind to the purinergic receptor P2X7 (P2RX7) on innate immune cells triggering interleukin-1β release, which drives further immune activation. Hypothesis: We hypothesized that P2rx7 knockout or P2RX7 antagonism would blunt angiotensin II (AngII)-induced BP elevation and cardiovascular injury through decreased immune activation. Methods: Ten-to-12-week-old male C57BL/6J wild-type (WT) and P2rx7 -/- mice were infused or not with AngII (1000 ng/kg/min) for 14 days. A second group of AngII-infused WT mice was also infused with the P2RX7 antagonist AZ10606120 (694 ng/kg/min) or vehicle. BP was determined by telemetry, plasma ATP using a bioluminescence assay, mesenteric artery function using pressurized myography, cardiac left ventricle (LV) function and mass by ultrasound and activated immune T cell infiltration in aortic perivascular adipose tissue (PVAT) by flow cytometry. Results: AngII increased plasma ATP in WT mice (4.4±1.2 vs 2.0±0.9 μM, P <0.05). AngII-induced systolic BP elevation was reduced by P2rx7 deficiency (164±3 vs 176±2 mm Hg, P <0.05) or P2RX7 antagonism (143±5 vs 170±5 mm Hg, P <0.01). AngII decreased LV fractional shortening (FS, 32.5±3.1% vs 43.8±2.4%, P <0.05) and increased LV mass/body weight (BW) in WT mice (LVmass/BW, 6.3±0.2 vs 4.2±0.2 mg/g, P <0.001), which were exaggerated in P2rx7 -/- (FS: 20.2±3.1% and LVmass/BW: 7.2±0.5 mg/g, P <0.05), but not in mice receiving AZ10606120. AngII reduced the dilatation response of mesenteric arteries to acetylcholine in WT (61±7 vs 83±4%, P <0.05), but not in P2rx7 -/- or AZ10606120-treated mice. AngII increased CD69 + CD8 + T cell infiltration in aortic PVAT of WT (60±16 vs 16±3 cells/aortic PVAT, P <0.001), but not in P2rx7 -/- or AZ10606120-treated mice. Conclusion: P2rx7 knockout or antagonism attenuates AngII-induced BP elevation, vascular injury, and infiltration of activated CD8 + T cells into aortic PVAT. P2rx7 knockout exacerbated AngII-induced cardiac dysfunction and hypertrophy, whereas P2RX7 antagonism did not.
O diabetes mellitus tipo 2 (DM2), uma doença que afeta milhões de pessoas em todo o mundo, é marcado pela presença de complicações micro e macrovasculares, as quais estão associadas à disfunção endotelial, inflamação e fibrose.A aldosterona, cujos níveis plasmáticos estão elevados em pacientes e modelos experimentais de DM2, aumenta a geração de espécies reativas de oxigênio (ERO) e a expressão de marcadores inflamatórios.As citocinas IL-1β e IL-18 são liberadas principalmente após ativação de plataformas moleculares denominadas inflamassomas, as quais incluem os receptores NLRP3.Recentemente, demonstramos que o receptor NLRP3 contribui para a disfunção vascular induzida pela aldosterona.Considerando a existência de evidências que a aldosterona, via receptor mineralocorticoide (MR) e NLRP3, induz a produção de mediadores inflamatórios e, consequentemente, ativação do inflamassoma, podendo assim contribuir para o processo inflamatório no diabetes, nós hipotetizamos que o bloqueio de receptores MR e NLRP3 previne a ativação do inflamassoma e reduz o desenvolvimento das alterações vasculares funcionais associadas ao DM2.Observamos que artérias mesentéricas de animais diabéticos apresentam aumento da expressão/ativação de caspase-1 e IL-11β, aumento dos níveis plasmáticos de IL-1β, aumento da atividade da caspase-1 em macrófagos do lavado peritoneal e prejuízo no relaxamento dependente de endotélio, comparativamente a artérias do grupo controle.Os tratamentos com espironolactona (antagonista MR) e MCC950 (inibidor de receptor NLRP3) atenuam a disfunção vascular, reduzem a expressão e a atividade da caspase-1 e diminuem os níveis plasmáticos de IL-1β.Células de músculo liso vascular e macrófagos derivados da medula estimulados com aldosterona também apresentam aumento da expressão dos componentes do inflamassoma, o que poderia contribuir para as alterações observadas em animais diabéticos.Pacientes com DM2 apresentam correlação positiva entre os níveis de aldosterona e de IL-1β e/ou glicemia.Em conclusão, nosso estudo demonstra que a aldosterona induz disfunção vascular e processo inflamatório no diabetes tipo 2 através da ativação de MR e do inflamassoma NLRP3.
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