The NLRP3 inflammasome has been reported to be activated by atherogenic factors, whereby endothelial injury and consequent atherosclerotic lesions are triggered in the arterial wall. However, the mechanisms activating and regulating NLRP3 inflammasomes remain poorly understood. The present study tested whether acid sphingomyelinase (ASM) and ceramide associated membrane raft (MR) signaling platforms contribute to the activation of NLRP3 inflammasomes and atherosclerotic lesions during hypercholesterolemia. We found that 7-ketocholesterol (7-Keto) or cholesterol crystal (ChC) markedly increased the formation and activation of NLRP3 inflammasomes in mouse carotid arterial endothelial cells (CAECs), as shown by increased colocalization of NLRP3 with ASC or caspase-1, enhanced caspase-1 activity and elevated IL-1β levels, which were markedly attenuated by mouse Asm siRNA, ASM inhibitor- amitriptyline, and deletion of mouse Asm gene. In CAECs with NLRP3 inflammasome formation, membrane raft (MR) clustering with NADPH oxidase subunits was found remarkably increased as shown by CTXB (MR marker) and gp91^phox aggregation indicating the formation of MR redox signaling platforms. This MR clustering was blocked by MR disruptor (MCD), ROS scavenger (Tempol) and TXNIP inhibitor (verapamil), accompanied by attenuation of 7-Keto or ChC-induced increase in caspase-1 activity. In animal experiments, Western diet fed mice with partially ligated left carotid artery (PLCA) were found to have significantly increased neointimal formation, which was associated with increased NLRP3 inflammasome formation and IL-1β production in the intima of Asm^+/+ mice but not in Asm^-/- mice. These results suggest that Asm gene and ceramide associated MR clustering are essential to endothelial inflammasome activation and dysfunction in the carotid arteries, ultimately determining the extent of atherosclerotic lesions.

The intestinal microbe-derived metabolite trimethylamine N-oxide (TMAO) is implicated in the pathogenesis of cardiovascular diseases (CVDs). The molecular mechanisms of how TMAO induces atherosclerosis and CVDs’ progression are still unclear. In this regard, high-mobility group box protein 1 (HMGB1), an inflammatory mediator, has been reported to disrupt cell–cell junctions, resulting in vascular endothelial hyper permeability leading to endothelial dysfunction. The present study tested whether TMAO associated endothelial dysfunction results via HMGB1 activation. Biochemical and RT-PCR analysis showed that TMAO increased the HMGB1 expression in a dose-dependent manner in endothelial cells. However, prior treatment with glycyrrhizin, an HMGB1 binder, abolished the TMAO-induced HMGB1 production in endothelial cells. Furthermore, Western blot and immunofluorescent analysis showed significant decrease in the expression of cell–cell junction proteins ZO-2, Occludin, and VE-cadherin in TMAO treated endothelial cells compared with control cells. However, prior treatment with glycyrrhizin attenuated the TMAO-induced cell–cell junction proteins’ disruption. TMAO increased toll-like receptor 4 (TLR4) expression in endothelial cells. Inhibition of TLR4 expression by TLR4 siRNA protected the endothelial cells from TMAO associated tight junction protein disruption via HMGB1. In conclusion, our results demonstrate that HMGB1 is one of the important mediators of TMAO-induced endothelial dysfunction.

Genetic defects of anion exchanger 1 (AE1) may lead to spherocytic erythrocyte morphology, severe hemolytic anemia, and/or cation leak. In normal erythrocytes, osmotic shock, Cl⁻ removal, and energy depletion activate Ca²⁺-permeable cation channels with Ca²⁺-induced suicidal erythrocyte death, i.e., surface exposure of phosphatidylserine, cell shrinkage, and membrane blebbing, all features typical for apoptosis of nucleated cells. The present experiments explored whether AE1 deficiency favors suicidal erythrocyte death. Peripheral blood erythrocyte numbers were significantly smaller in gene-targeted mice lacking AE1 ( AE1^−/− mice) than in their wild-type littermates ( AE1^+/+ mice) despite increased percentages of reticulocytes ( AE1^−/−: 49%, AE1^+/+: 2%), an indicator of enhanced erythropoiesis. Annexin binding, reflecting phosphatidylserine exposure, was significantly larger in AE1^−/−erythrocytes/reticulocytes (∼10%) than in AE1^+/+ erythrocytes (∼1%). Osmotic shock (addition of 400 mM sucrose), Cl⁻ removal (replacement with gluconate), or energy depletion (removal of glucose) led to significantly stronger annexin binding in AE1^−/− erythrocytes/reticulocytes than in AE1^+/+ erythrocytes. The increase of annexin binding following exposure to the Ca²⁺ ionophore ionomycin (1 μM) was, however, similar in AE1^−/− and in AE1^+/+ erythrocytes. Fluo3 fluorescence revealed markedly increased cytosolic Ca²⁺ permeability in AE1^−/− erythrocytes/reticulocytes. Clearance of carboxyfluorescein diacetate succinimidyl ester-labeled erythrocytes/reticulocytes from circulating blood was more rapid in AE1^−/− mice than in AE1^+/+ mice and was accelerated by ionomycin treatment in both genotypes. In conclusion, lack of AE1 is associated with enhanced Ca²⁺ entry and subsequent scrambling of cell membrane phospholipids.

The intestinal microbe derived metabolite trimethylamine N‐oxide (TMAO) is implicated in the pathogenesis of chronic kidney diseases (CKD). However, the molecular mechanisms of how TMAO induces CKD and progression of renal diseases are still unclear. Hence, the present study tested whether high mobility group box 1 (HMGB1) contributes to TMAO‐induced podocyte injury and glomerular sclerosis. In in vitro , biochemical analysis showed that TMAO treatment significantly increased the HMGB1 expression compared to vehicle treated podocytes. However, prior treatment with glycyrrhizin, an HMGB1 binder abolished the TMAO‐induced HMGB1 expression in podocytes. Furthermore, western blot and immunofluorescent analysis showed that significant decrease in the expression of the nephrin and podocin compared to control cells. However, prior treatment with glycyrrhizin attenuated the TMAO‐induced nephrin and podocin reduction. In addition, TMAO treatment significantly increased the toll like receptor 4 (TLR4) expression and cell permeability compared to vehicle treated podocytes. However, prior treatment with glycyrrhizin attenuated the TMAO induced TLR4 expression and cell permeability. In in vivo studies, Western blot and biochemical analysis showed that TMAO treatment significantly increased the HMGB1, TLR4, desmin, urinary protein, urinary albumin, glomerular damage index and significantly decreased the podocin expression in TMAO treated mice compared to control mice. However, such TMAO‐induced increase of HMGB1, TLR4, desmin expression, glomerular damage index, urinary protein, urinary albumin were attenuated in glycyrrhizin treated mice. Based on these results, it is concluded that HMGB1 is one of the important mediators of TMAO‐induced podocyte injury and glomerular sclerosis.