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BackgroundSalt sensitivity of blood pressure is an independent predictor of cardiovascular morbidity and mortality. The exact mechanism by which salt intake increases blood pressure and cardiovascular risk is unknown. We previously found that sodium entry into antigen-presenting cells (APCs) via the amiloride-sensitive epithelial sodium channel EnaC (epithelial sodium channel) leads to the formation of IsoLGs (isolevuglandins) and release of proinflammatory cytokines to activate T cells and modulate salt-sensitive hypertension. In the current study, we hypothesized that ENaC-dependent entry of sodium into APCs activates the NLRP3 (NOD [nucleotide-binding and oligomerization domain]-like receptor family pyrin domain containing 3) inflammasome via IsoLG formation leading to salt-sensitive hypertension.MethodsWe performed RNA sequencing on human monocytes treated with elevated sodium in vitro and Cellular Indexing of Transcriptomes and Epitopes by Sequencing analysis of peripheral blood mononuclear cells from participants rigorously phenotyped for salt sensitivity of blood pressure using an established inpatient protocol. To determine mechanisms, we analyzed inflammasome activation in mouse models of deoxycorticosterone acetate salt-induced hypertension as well as salt-sensitive mice with ENaC inhibition or expression, IsoLG scavenging, and adoptive transfer of wild-type dendritic cells into NLRP3 deficient mice.ResultsWe found that high levels of salt exposure upregulates the NLRP3 inflammasome, pyroptotic and apoptotic caspases, and IL (interleukin)-1β transcription in human monocytes. Cellular Indexing of Transcriptomes and Epitopes by Sequencing revealed that components of the NLRP3 inflammasome and activation marker IL-1β dynamically vary with changes in salt loading/depletion. Mechanistically, we found that sodium-induced activation of the NLRP3 inflammasome is ENaC and IsoLG dependent. NLRP3 deficient mice develop a blunted hypertensive response to elevated sodium, and this is restored by the adoptive transfer of NLRP3 replete APCs.ConclusionsThese findings reveal a mechanistic link between ENaC, inflammation, and salt-sensitive hypertension involving NLRP3 inflammasome activation in APCs. APC activation via the NLRP3 inflammasome can serve as a potential diagnostic biomarker for salt sensitivity of blood pressure.

Aims The lymphocyte adaptor protein (LNK) is a negative regulator of cytokine and growth factor signalling. The rs3184504 variant in SH2B3 reduces LNK function and is linked to cardiovascular, inflammatory, and haematologic disorders, including stroke. In mice, deletion of Lnk causes inflammation and oxidative stress. We hypothesized that Lnk−/− mice are susceptible to atrial fibrillation (AF) and that rs3184504 is associated with AF and AF-related stroke in humans. During inflammation, reactive lipid dicarbonyls are the major components of oxidative injury, and we further hypothesized that these mediators are critical drivers of the AF substrate in Lnk−/− mice. Methods and results Lnk−/− or wild-type (WT) mice were treated with vehicle or 2-hydroxybenzylamine (2-HOBA), a dicarbonyl scavenger, for 3 months. Compared with WT, Lnk−/− mice displayed increased AF duration that was prevented by 2-HOBA. In the Lnk−/− atria, action potentials were prolonged with reduced transient outward K+ current, increased late Na+ current, and reduced peak Na+ current, pro-arrhythmic effects that were inhibited by 2-HOBA. Mitochondrial dysfunction, especially for Complex I, was evident in Lnk−/− atria, while scavenging lipid dicarbonyls prevented this abnormality. Tumour necrosis factor-α (TNF-α) and interleukin-1 beta (IL-1β) were elevated in Lnk−/− plasma and atrial tissue, respectively, both of which caused electrical and bioenergetic remodelling in vitro. Inhibition of soluble TNF-α prevented electrical remodelling and AF susceptibility, while IL-1β inhibition improved mitochondrial respiration but had no effect on AF susceptibility. In a large database of genotyped patients, rs3184504 was associated with AF, as well as AF-related stroke. Conclusion These findings identify a novel role for LNK in the pathophysiology of AF in both experimental mice and humans. Moreover, reactive lipid dicarbonyls are critical to the inflammatory AF substrate in Lnk−/− mice and mediate the pro-arrhythmic effects of pro-inflammatory cytokines, primarily through electrical remodelling. [ABSTRACT FROM AUTHOR]

The epithelial Na+ channel (ENaC) promotes the absorption of Na+ in the aldosterone-sensitive distal nephron, colon, and respiratory epithelia. Deletion of genes encoding subunits of ENaC results in early postnatal mortality. Here, we present the initial characterization of a mouse with dramatically suppressed expression of the ENaC γ-subunit. We used this hypomorphic (γmt) allele to explore the importance of this subunit in homeostasis of electrolytes and body fluid volume. At baseline, γ-subunit expression in cmt/mt mice was markedly suppressed in the kidney and lung, whereas electrolytes resembled those of littermate controls. Aldosterone levels in γmt/mt mice exceeded those seen in littermate controls. Quantitative magnetic resonance measurement of body composition revealed similar baseline body water, lean tissue mass, and fat tissue mass in cmt/mt mice and controls. γmt/mt mice exhibited a more rapid decline in body water and lean tissue mass in response to a low-Na+ diet than the controls. Replacement of drinking water with 2% saline selectively and transiently increased body water and lean tissue mass in γmt/mt mice relative to the controls. Lower blood pressures were variably observed in γmt/mt mice on a high-salt diet compared with the controls. γmt/mt also exhibited reduced diurnal blood pressure variation, a "nondipping" phenotype, on a high-Na+ diet. Although ENaC in the renal tubules and colon works to prevent extracellular fluid volume depletion, our observations suggest that ENaC in other tissues may participate in regulating extracellular fluid volume and blood pressure. [ABSTRACT FROM AUTHOR]

Aims Prior studies have focused on the role of the kidney and vasculature in salt-induced modulation of blood pressure; however, recent data indicate that sodium accumulates in tissues and can activate immune cells. We sought to examine mechanisms by which salt causes activation of human monocytes both in vivo and in vitro. Methods and results To study the effect of salt in human monocytes, monocytes were isolated from volunteers to perform several in vitro experiments. Exposure of human monocytes to elevated Na+ ex vivo caused a co-ordinated response involving isolevuglandin (IsoLG)-adduct formation, acquisition of a dendritic cell (DC)-like morphology, expression of activation markers CD83 and CD16, and increased production of pro-inflammatory cytokines tumour necrosis factor-α, interleukin (IL)-6, and IL-1β. High salt also caused a marked change in monocyte gene expression as detected by RNA sequencing and enhanced monocyte migration to the chemokine CC motif chemokine ligand 5. NADPH-oxidase inhibition attenuated monocyte activation and IsoLG-adduct formation. The increase in IsoLG-adducts correlated with risk factors including body mass index, pulse pressure. Monocytes exposed to high salt stimulated IL-17A production from autologous CD4+ and CD8+ T cells. In addition, to evaluate the effect of salt in vivo , monocytes and T cells isolated from humans were adoptively transferred to immunodeficient NSG mice. Salt feeding of humanized mice caused monocyte-dependent activation of human T cells reflected by proliferation and accumulation of T cells in the bone marrow. Moreover, we performed a cross-sectional study in 70 prehypertensive subjects. Blood was collected for flow cytometric analysis and 23Na magnetic resonance imaging was performed for tissue sodium measurements. Monocytes from humans with high skin Na+ exhibited increased IsoLG-adduct accumulation and CD83 expression. Conclusion Human monocytes exhibit co-ordinated increases in parameters of activation, conversion to a DC-like phenotype and ability to activate T cells upon both in vitro and in vivo sodium exposure. The ability of monocytes to be activated by sodium is related to in vivo cardiovascular disease risk factors. We therefore propose that in addition to the kidney and vasculature, immune cells like monocytes convey salt-induced cardiovascular risk in humans. [ABSTRACT FROM AUTHOR]

Dynein-mediated autophagosome (AP) trafficking was recently demonstrated to contribute to the formation of autophagolysosomes (APLs) and autophagic flux process in coronary arterial myocytes (CAMs). However, it remains unknown how the function of dynein as a motor protein for AP trafficking is regulated under physiological and pathological conditions. The present study tested whether the dynein-mediated autophagy maturation is regulated by a redox signalling associated with lysosomal Ca2+ release machinery. In primary cultures of CAMs, reactive oxygen species (ROS) including H2O2 and O2-. (generated by xanthine/xanthine oxidase) significantly increased dynein ATPase activity and AP movement, which were accompanied by increased lysosomal fusion with AP and APL formation. Inhibition of dynein activity by (erythro-9-(2-hydroxy-3-nonyl)adenine) (EHNA) or disruption of the dynein complex by dynamitin (DCTN2) overexpression blocked ROS-induced dynein activation, AP movement and APL formation, and resulted in an accumulation of AP along with a failed breakdown of AP. Antagonism of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca2+ signalling with NED-19 and PPADS abolished ROS-enhanced lysosomal Ca2+ release and dynein activation in CAMs. In parallel, all these changes were also enhanced by overexpression of NADPH oxidase-1 (Nox1) gene in CAMs. Incubation with high glucose led to a marked O2-. production compared with normoglycaemic CAMs, while Nox1 inhibitor ML117 abrogated this effect. Moreover, ML117 and NED-19 and PPADS significantly suppressed dynein activity and APL formation caused by high glucose. Taken together, these data suggest that ROS function as important players to regulate dynein-dependent AP trafficking leading to efficient autophagic maturation in CAMs. [ABSTRACT FROM AUTHOR]

Inflammasomes are intracellular machinery turning on inflammatory response to danger factors. Recent studies reported that activation of inflammasomes also directly produce injurious action on cells or tissues independent of canonical inflammation. The present study hypothesized that activation of endothelial Nlrp3 inflammsome directly induces endothelial dysfunction at early stage of obesity without canonical inflammation yet. It was demonstrated that Nlrp3+/+ mice under 6-week high fat diet (HFD) had increased formation and activation of Nlrp3 inflammasome in coronary arterial endothelium compared to normal diet (ND)-treated mice, as shown by confocal microscopic detection of inflammasome aggregation and active caspase-1 formation. Ultrasound Doppler measurement showed that HFD markedly reduced bradykinin-induced increases in coronary blood flow by 95% in Nlrp3+/+ mice, but only by 45% in Nlrp3-/- mice. Vascular reactivity studies confirmed that HFD impaired endothelium-dependent vasodilation to acetylcholine (10-9-10-5 M) in freshly isolated coronary arteries of Nlrp3+/+ mice (maximum dilation: 53±3% vs. 84±3% of ND), but not in Nlrp3-/- mice (maximum dilation: 81±3% vs. 85±2% of ND). Similarly, visfatin (an injurious adipokine) simulates HFD-induced injurious action on endothelial dysfunction in normal Nlrp3+/+coronary arteries, which was attenuated by Nlrp3 gene deletion. In Nlrp3+/+ mice, HFD markedly increased the number of circulating endothelial progenitor cells (EPCs) in peripheral blood (1.73±0.69% vs. 0.46±0.05% of ND) and adhesion molecule VCAM-1 levels in coronary arterial endothelium. Nlrp3-/- mice fed HFD exibited higher level of circulating EPCs (4.57±1.27% vs. 0.32±0.04% of ND), but similar VCAM-1 expression. Together, these results suggest that endothelial Nlrp3 inflammasome activation in response to obesity or injurious adipokine produces endothelial dysfunction and inhibits EPC mobilization for endothelial repair, which are distinct from its canonical inflammatory responses such as inflammtory cell recruitment and infiltration in the vasculature. It is proposed that Nlrp3 inflammasome activation may trigger endothelial injury resulting in ultimate vascular inflammation and schlerosis. [ABSTRACT FROM AUTHOR]

Retinoic acid-inducible gene-I (RIG-I) is a putative RNA helicase and recently identified as a cytosolic RNA receptor in mammalian cells. The role of RIG-I in the regulation of vascular function under physiological and pathological conditions is unknown. The present study tested whether RIG-I activation triggers inflammasome formation, turning on inflammation in mouse endothelial cells (EOMA cell line). By real time RT-PCR and Western blot analysis, transfection of mouse ECs with RIG-I specific agonist, 5’-triphosphate double-stranded RNA (3pRNA, 0.5 mg/L) increased RIG-I mRNA level by 106% and protein level by 81% compared to those in control double-stranded RNA (dsRNA) transfected ECs. ELISA analyses showed that 3pRNA significantly increased release of type I IFN alpha by 31 folds and IL-1 beta (a prototype cytokine from inflammasome activation) by 8 folds in these ECs. Proatherogenic stimulation of mouse ECs with cholesterol crystals or 7-ketocholesterol also markedly increased protein expression of RIG-I, but had no effect on RIG-I mRNA levels. Measurements of active caspase-1, an inflammasome activation marker using FLICA fluorescent probe that specifically binds to cleaved caspase-1, demonstrated that 3pRNA doubled FLICA positive cells compared to that in control dsRNA transfected ECs. Interestingly, cholesterol crystals significantly increased FLICA positive cells by 3 folds. This activation of caspase-1 in ECs by cholesterol crystals was further confirmed by increase in cleaved caspase-1 (p10) using Western blot analysis and by enhanced IL-1 beta release as detected by ELISA. In the presence of 3pRNA, cholesterol crystal-induced inflammasome activation was not further augmented. These data indicate that increased expression and activity of RIG-I activate IL-1 beta producing inflammasomes in ECs, which may represent an early molecular mechanism mediating vascular inflammation or injury upon atherogenic stimulations. [ABSTRACT FROM AUTHOR]

Aims: Prior studies have focused on the role of the kidney and vasculature in salt-induced modulation of blood pressure; however, recent data indicate that sodium accumulates in tissues and can activate immune cells. We sought to examine mechanisms by which salt causes activation of human monocytes both in vivo and in vitro., Methods and Results: To study the effect of salt in human monocytes, monocytes were isolated from volunteers to perform several in vitro experiments. Exposure of human monocytes to elevated Na+ex vivo caused a co-ordinated response involving isolevuglandin (IsoLG)-adduct formation, acquisition of a dendritic cell (DC)-like morphology, expression of activation markers CD83 and CD16, and increased production of pro-inflammatory cytokines tumour necrosis factor-α, interleukin (IL)-6, and IL-1β. High salt also caused a marked change in monocyte gene expression as detected by RNA sequencing and enhanced monocyte migration to the chemokine CC motif chemokine ligand 5. NADPH-oxidase inhibition attenuated monocyte activation and IsoLG-adduct formation. The increase in IsoLG-adducts correlated with risk factors including body mass index, pulse pressure. Monocytes exposed to high salt stimulated IL-17A production from autologous CD4+ and CD8+ T cells. In addition, to evaluate the effect of salt in vivo, monocytes and T cells isolated from humans were adoptively transferred to immunodeficient NSG mice. Salt feeding of humanized mice caused monocyte-dependent activation of human T cells reflected by proliferation and accumulation of T cells in the bone marrow. Moreover, we performed a cross-sectional study in 70 prehypertensive subjects. Blood was collected for flow cytometric analysis and 23Na magnetic resonance imaging was performed for tissue sodium measurements. Monocytes from humans with high skin Na+ exhibited increased IsoLG-adduct accumulation and CD83 expression., Conclusion: Human monocytes exhibit co-ordinated increases in parameters of activation, conversion to a DC-like phenotype and ability to activate T cells upon both in vitro and in vivo sodium exposure. The ability of monocytes to be activated by sodium is related to in vivo cardiovascular disease risk factors. We therefore propose that in addition to the kidney and vasculature, immune cells like monocytes convey salt-induced cardiovascular risk in humans., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.)