Search

Background: Inflammation is a key feature of aldosterone-induced vascular damage and dysfunction, but molecular mechanisms by which aldosterone triggers inflammation remain unclear. The NLRP3 inflammasome is a pivotal immune sensor that recognizes endogenous danger signals triggering sterile inflammation. Methods: We analyzed vascular function and inflammatory profile of wild-type (WT), NLRP3 knockout ( NLRP3 −/− ), caspase-1 knockout ( Casp-1 −/− ), and interleukin-1 receptor knockout ( IL-1R −/− ) mice treated with vehicle or aldosterone (600 µg·kg −1 ·d −1 for 14 days through osmotic mini-pump) while receiving 1% saline to drink. Results: Here, we show that NLRP3 inflammasome plays a central role in aldosterone-induced vascular dysfunction. Long-term infusion of aldosterone in mice resulted in elevation of plasma interleukin-1β levels and vascular abnormalities. Mice lacking the IL-1R or the inflammasome components NLRP3 and caspase-1 were protected from aldosterone-induced vascular damage. In vitro, aldosterone stimulated NLRP3-dependent interleukin-1β secretion by bone marrow–derived macrophages by activating nuclear factor-κB signaling and reactive oxygen species generation. Moreover, chimeric mice reconstituted with NLRP3-deficient hematopoietic cells showed that NLRP3 in immune cells mediates aldosterone-induced vascular damage. In addition, aldosterone increased the expression of NLRP3, active caspase-1, and mature interleukin-1β in human peripheral blood mononuclear cells. Hypertensive patients with hyperaldosteronism or normal levels of aldosterone exhibited increased activity of NLRP3 inflammasome, suggesting that the effect of hyperaldosteronism on the inflammasome may be mediated through high blood pressure. Conclusions: Together, these data demonstrate that NLRP3 inflammasome, through activation of IL-1R, is critically involved in the deleterious vascular effects of aldosterone, placing NLRP3 as a potential target for therapeutic interventions in conditions with high aldosterone levels.

Background: The mechanisms of blood pressure (BP) regulation by endothelin (ET)-1 produced by endothelial cells are complex and remain unclear. Long-term exposure to endothelial human ET-1 overexpression causes sustained blood pressure elevation via ETA receptors. ET-1 has been shown to stimulate the release of aldosterone from the adrenal cortex. Whether aldosterone plays a role in ET-1 endothelium overexpression-induced BP elevation is still unknown. Methods and Results: Nine to 12-week-old male ieET-1 mice and control ieCre mice expressing a tamoxifen-inducible Cre recombinase (CreER T2 ) under the control of EC-specific Tie2 promoter, were treated with tamoxifen (1 mg/kg/day, SC) for 5 days and studied 3 months later. Plasma aldosterone level measured by ELISA was higher in ieET-1 compared with ieCre mice (1.21±0.14 vs. 0.70±0.09 ng/mL, P Scnn1a ), TSC22 domain family member 3 ( Rsc22d3 ), serum- and glucocorticoid-induced kinase 1 ( Sgk1 ), and ET type A and B receptors in the kidney or adrenal glands. The mRNA expression of aldosterone synthase ( Cyp11b2 , fold change: 0.52±0.06 vs 1.00±0.16, P Hsd3b1) and 6 (Hsd3b6) or steroidogenic acute regulatory protein ( Star ), were decreased in the adrenal cortex of ieET-1 compared with ieCre mice. CYP11B2 and HSD3B1 protein levels measured by Western Blotting were unchanged. Treatment of ieET-1 mice with MR antagonist eplerenone (100 mg/kg per day) during the last 2 weeks decreased systolic BP more during the day (128±2 vs. 134±3 mm Hg) than during the night (137±2 vs. 140±2 mm Hg) compared to untreated ieET-1 mice. Conclusions: These results showed that aldosterone contributes at least in part to the BP elevation caused by endothelial human ET-1 overexpression.

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, pvs. Veh [E max , 46.6±4.0 vs . 91.5±2.8, p-/- T1D mice. Diabetes increased vascular caspase-1 [arbitrary units (a.u.), 1.2±0.1 vs. 0.8±0.1, pvs. 0.8±0.5 p -/- 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-/- 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.

Diabetic patients and animal models of type 2 diabetes (DM2) display increased plasma aldosterone (aldo) levels. Aldo induces vascular inflammation and endothelial dysfunction. NOD-like receptors, which are pattern recognition receptors involved in a variety of host innate immune responses, promote vascular inflammation. We hypothesized that aldo via mineralocorticoid receptors (MR) activates the inflammasome platform in the vasculature of DM2 mice. Control (db/+) and diabetic (db/db) mice were treated with vehicle or spironolactone (spiro - MR antagonist, 50 mg/Kg/day). Mesenteric resistance arteries (MA) from db/db mice exhibited reduced acetylcholine (ACh) dilation, which was reversed by spiro [Emax (% of relaxation): db/+: 78.5±4.1; db/db: 40.5±6.4; db/+spiro: 77.0±3.8; db/db+spiro: 62.8±5.9 n=3-6 pIn vitro, aldo increased mature IL-1β in vascular smooth muscle cells (VSMC) (cont: 0.9±0.01 ; LPS+Nigericine: 6.1±2.1 ; Aldo 4h: 9.7±2.6; LPS+Aldo 4h: 12.8±1.9 n=3-5, pin vivo directly activates NLRP3/inflammasome in the vasculature and whether NLRP3 activation contributes to aldo-induced vascular injury, aldo was infused (600 ug/Kg/day for 14 days) in wild type (WT) and NLRP3 knockout mice ( NLRP3-/- ) after bone marrow transplantation from WT donor. The groups were constituted: WT->WT, WT->WT+aldo and WT-> NLRP3 -/-+aldo. NLRP3 -/- mice were protected against aldo-induced endothelial dysfunction [Emax: WT: 89.3±2.9; WT+aldo: 39.8±1.8; NLRP3-/- +aldo: 87.7±4.2, pWT mice, but WT-> NLRP3 -/- mice were protected from aldo-induced endothelial dysfunction [Emax: WT->WT: 95.1±3.1; WT->WT+aldo: 57.1±4.7; WT->NLRP3-/-+aldo: 85.3±3.1 p

Aldosterone (Aldo) excess aggravates endothelial dysfunction in diabetes. Aldo exerts its effects via activation of both mineralocorticoid receptors (MR) and G protein-coupled estrogen receptors (GPER). Considering that GPER activation has beneficial effects in the vasculature, we hypothesized that GPER-mediated vascular effects of aldosterone are decreased/abrogated in diabetes. Second-order mesenteric arteries from control (B6BKS-Leprdb/+) and diabetic (db/db) female mice were incubated with 10 nM Aldo, in the presence of either vehicle (veh), the MR antagonist eplerenone (Eple, 10 μM) or the GPER antagonist G15 (1μM), and the effects on phenylephrine (Phe) vascular reactivity were determined. Aldo increased Phe maximal response (Emax, % of KCl contraction) in arteries from control (veh: 112.5±3.2 vs. Aldo: 129.1±2.8 p0.05). In control vessels, Eple did not alter Phe Emax either in the presence of Aldo or veh (p>0.05), whereas G15 abrogated Aldo-induced increase in Phe Emax (Aldo: 129.1±2.8 vs. G15+Aldo: 110.3±3.6 p

Fluoxetine, a selective serotonin reuptake inhibitor (SSRI) has properties that go beyond its antidepressant effects and alters mechanisms involved in the regulation of vasomotor tone. While there are many studies demonstrating the acute effects of fluoxetine in the vasculature, studies on the chronic effects of this SSRI are still limited. Here we postulated that chronic treatment with fluoxetine enhances vascular reactivity to vasodilator stimuli by increasing nitric oxide (NO) signaling. The effects of chronic treatment with fluoxetine on vascular reactivity were determined in resistance mesenteric arteries from Wistar rats, which were treated with (I) vehicle (water for 21 days) or (II) fluoxetine (10 mg/kg/day for 21 days in the drinking water). Fluoxetine treatment increased endothelium-dependent (pEC50, Veh = -7.08±0.07; Fluox = -7.4±0.11, p0.05). In conclusion, chronic treatment with fluoxetine increases endothelium-dependent and -independent relaxation response in resistance mesenteric arteries by mechanisms that involve increased NOS activity, NO generation and KATP channels activation. These effects may contribute to the cardiovascular side effects associated with fluoxetine treatment.