Hyperphosphataemia increases cardiovascular mortality in patients with kidney disease. Direct effects of high inorganic phosphate (Pi) concentrations have previously been demonstrated on endothelial cells (ECs), including generation of procoagulant endothelial microvesicles (MVs). However, no mechanism directly sensing elevated intracellular Pi has ever been described in mammalian cells. Here, we investigated the hypothesis that direct inhibition by Pi of the phosphoprotein phosphatase PP2A fulfils this sensing role in ECs, culminating in cytoskeleton disruption and MV generation. ECs were treated with control (1 mM [Pi]) vs. high (2.5 mM [Pi]), a condition that drives actin stress fibre depletion and MV generation demonstrated by confocal microscopy of F-actin and NanoSight Nanoparticle tracking, respectively. Immuno-blotting demonstrated that high Pi increased p-Src, p-PP2A-C and p-DAPK-1 and decreased p-TPM-3. Pi at 100 μM directly inhibited PP2A catalytic activity. Inhibition of PP2A enhanced inhibitory phosphorylation of DAPK-1, leading to hypophosphorylation of Tropomyosin-3 at S284 and MV generation. p-Src is known to perform inhibitory phosphorylation on DAPK-1 but also on PP2A-C. However, PP2A-C can itself dephosphorylate (and therefore inhibit) p-Src. The direct inhibition of PP2A-C by Pi is, therefore, amplified by the feedback loop between PP2A-C and p-Src, resulting in further PP2A-C inhibition. These data demonstrated that PP2A/Src acts as a potent sensor and amplifier of Pi signals which can further signal through DAPK-1/Tropomyosin-3 to generate cytoskeleton disruption and generation of potentially pathological MVs.

Drug-induced cardiotoxicity may be modulated by endogenous arachidonic acid (AA)–derived metabolites known as epoxyeicosatrienoic acids (EETs) synthesized by cytochrome P450 2J2 (CYP2J2). The biologic effects of EETs, including their protective effects on inflammation and vasodilation, are diverse because, in part, of their ability to act on a variety of cell types. In addition, CYP2J2 metabolizes both exogenous and endogenous substrates and is involved in phase 1 metabolism of a variety of structurally diverse compounds, including some antihistamines, anticancer agents, and immunosuppressants. This review addresses current understanding of the role of CYP2J2 in the metabolism of xenobiotics and endogenous AA, focusing on the effects on the cardiovascular system. In particular, we have promoted here the hypothesis that CYP2J2 influences drug-induced cardiotoxicity through potentially conflicting effects on the production of protective EETs and the metabolism of drugs.

Systemic inflammation, induced by disease or experimental intervention, is well established to result in elevated levels of circulating triglycerides, and reduced levels of high-density lipoprotein-cholesterol (HDL-C), in most mammalian species. However, the relationship between inflammation and low-density lipoprotein-cholesterol (LDL-C) concentrations is less clear. Most reports indicate that systemic inflammation, as observed during sepsis or following high dose experimental endotoxaemia, lowers total, and LDL-C in man. However, isolated reports have suggested that certain inflammatory conditions are associated with increased LDL-C. In this review, we summarize the emerging evidence that low-grade inflammation specifically of intestinal origin may be associated with increased serum LDL-C levels. Preliminary insights into potential mechanisms that may mediate these effects, including those connecting inflammation to trans-intestinal cholesterol efflux (TICE), are considered. We conclude that this evidence supports the potential downregulation of major mediators of TICE by inflammatory mediators in vitro and during intestinal inflammation in vivo. The TICE-inflammation axis therefore merits further study in terms of its potential to regulate serum LDL-C, and as a readily druggable target for hypercholesterolaemia.

The NLRP3 inflammasome complex undergoes priming and activation leading to pro-inflammatory IL-1β and IL-18 synthesis. This study investigated the potential role of mitochondrial redox modulation in activation of the NLRP3 inflammasome in human cells. THP-1 cells were differentiated to macrophages with PMA for 24 hours and the NLRP3 inflammasome primed with LPS (0.1µg/ml) followed by activation with bzATP. NLRP3 and pro-IL1β were detected by Western blotting and ASC speck formation by immunofluorescence as markers of NLRP3 inflammasome priming. IL-1β and IL-18 in cell media were quantified by ELISA as markers of inflammasome activation. Under the conditions employed, intracellular oxidant generation (mitoparaquat and paraquat; Pq) assessed by mitoSOX fluorescence, was not sufficient to prime macrophages and did not affect priming mediated by LPS. At a relatively high extracellular concentration (5 µM) mitoPq, but not Pq, was able to increase IL-1β production in LPS-primed macrophages, suggesting promotion of inflammatory responses; IL-18 production was not affected. In LPS-primed and bzATP activated cells, the non-targeted slow release H2S compound, GYY4137, slightly reduced IL-1β production but was able to reduce IL-18 synthesis by approximately 50%. Although the mitochondria-targeted, slow release H2S compound AP39 did not affect inflammasome priming, at 300 nM (final media concentration) it reduced IL-1β and IL-18 synthesis in primed and activated cells. These data support a role for redox active oxygen and sulfur species, particularly when targeted to mitochondria, in modulating inflammatory cytokine synthesis in human macrophages with pro- and anti-inflammatory activities respectively.

Background: Anti-inflammatory targeting of IL-1β in people with previous MI is beneficial in terms of recurrent cardiovascular events. Mechanisms for modulation of IL-1β synthesis by macrophages is therefore also of potential value. The NLRP3 inflammasome complex plays a crucial role in IL-1β synthesis and is activated in a two-step process – “Signal 1” and “Signal 2”. This study investigated the possible role of mitochondrial and cytosolic superoxide and slow-release hydrogen sulfide generation in activation of the NLRP3 inflammasome in human THP-1 monocytes.

Endothelial cells play roles in regulating blood flow and in control of inflammation, both of which play important roles in atherogenesis. It is known that the NLRP3 inflammasome is involved in innate defence mechanisms in monocytes and macrophages and requires two steps for activation, signal 1 and signal 2, which result in the production of active IL-1β and IL-18. The aim of this study was to investigate NLRP3 inflammasome activation in human endothelial cells. Phorbol 12-myristate 13-acetate (PMA; 5 ng/ml) differentiated THP-1 cells (positive control), human umbilical vein endothelial cells (HUVEC) and EA.hy926 cells were ‘primed’ for signal 1 with lipopolysaccharide (LPS) (0.1–5 µg/ml) for 24 hours and then activated (signal 2) by exposure to ATP (300 µM) for 1 hour. Using Western blotting, NLRP3 protein, pro-IL-1β, pro-IL-18, active IL-1β and active IL-18 were observed in THP-1 cells demonstrating priming and activation of the inflammasome as expected. EA.hy926 cells but not HUVEC expressed pro-IL-1β. Inflammasome activation was confirmed by ELISA which detected any active end products in the cell culture supernatant. THP-1 cells secreted IL-1β and IL-18 as expected. However, for endothelial cells, only EA.hy926 cells showed some low level expression of active IL-1β. In conclusion, although there was evidence of NLRP3 inflammasome priming for both endothelial cell types, only EA.hy926 cells showed extracellular IL-1β production. Other end products of NLRP3 inflammasome activation may be worthy of investigation in order to fully describe the response of endothelial cells to innate stimuli.

Cytochrome P450 2J2 (CYP2J2), a multifunctional enzyme that is abundant in cardiac tissue, metabolises endogenous arachidonic acid to epoxyeicosatrienoic acids (EETs) which protect the heart. EETs dilate blood vessels increasing blood flow to heart muscle cells, and reduce inflammation which may protect against development of coronary artery disease. Moreover, it is reported that EETs mitigate the harmful effects of the cancer chemotherapeutic drug, doxorubicin, which causes cardiotoxicity in some patients. In vivo, EETs are rapidly metabolised by soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs) which are less biologically active. Several studies have shown that sEH may play an influential role in the development of atherosclerosis and cardiovascular disease, however, its role in cardiotoxicity has not been well reported. The current study aimed to investigate in vitro the role of EETs in the cardiotoxicity of a drug known to interact with CYP2J2, astemizole.Human cardiac myocytes (HCM) and human umbilical vein endothelial cell‐derived EA.hy926 cells, were incubated with astemizole in the absence or presence of inhibitors which were hypothesised to either enhance or diminish EET production. Changes to gene and protein expression were determined and toxicity was assessed.mRNA and protein expression of CYP2J2 and sEH was established in both cell models with higher expression in HCM in line with literature reports. Investigation of sEH expression showed the apparent presence of multiple mRNA isoforms but the predominant isoform was assessed. Treatment of cells with astemizole showed a toxic effect in both cell types accompanied by increased mRNA expression of sEH and CYP2J2 in EA.hy926 cells and HCM. Addition of the sEH inhibitor trans‐4‐[4‐3‐adamantan‐1‐yl‐ureido)‐cyclohexyloxyl]‐benzoic acid, (t‐AUCB), which increases levels of EETs in cells and exogenous 11,12‐EET protected cells against the damaging effects of six hours exposure to astemizole. In response to t‐AUCB in astemizole treated cells sEH expression was increased and CYP2J2 mRNA and protein was decreased; presumably a feedback mechanism exists following sEH inhibition that involves both CYP2J2 and sEH.Taken together, these data show that enhancing EET levels protected against astemizole toxicity in cardiovascular cells in vitro, indicating that the balance between endogenous CYP2J2 and sEH activities may play key role in counteracting damage to the heart by certain drugs. Agents that promote EETs in the heart may be useful adjuncts to counteract the cardiotoxicity due to certain drugs.Support or Funding InformationThis research was supported by Biotechnology and Biosciences Research Council, UK (ref: BB/M503368/1) and AstraZeneca.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Extracellular vesicles (EVs), including microparticles (MPs) and exosomes (EXOs), are derived from a wide range of mammalian cells including blood platelets, endothelial cells, and kidney cells and can be detected in body fluids including blood and urine. While EVs are well established as diagnostic markers under pathophysiological and stress conditions, there is also mounting evidence of their functional significance as vehicles for communication between cells mediated by the presence of nucleic acids, especially microRNAs (miRs), encapsulated in the EVs. miRs regulate gene expression, are transported both in MPs and EXOs, and exert profound effects in the kidney. Here we review current understanding of the links between EVs and miRs, discuss the importance of miRs in kidney disease, and shed light on the role of EVs in transferring miRs through the circulation among the renal, vascular, and inflammatory cell populations that are functionally important in patients with chronic kidney disease.

There is accumulating evidence that makes the link between the circadian variation in blood pressure and circadian variations in vascular contraction. The importance of vascular endothelium-derived redox-active and redox-derived species in the signalling pathways involved in controlling vascular smooth muscle contraction are well known, and when linked to the circadian variations in the processes involved in generating these species, suggests a cellular mechanism for the circadian variations in blood pressure that links directly to the peripheral circadian clock. Relaxation of vascular smooth muscle cells involves endothelial-derived relaxing factor (EDRF) which is nitric oxide (NO) produced by endothelial NO synthase (eNOS), and endothelial-derived hyperpolarising factor (EDHF) which includes hydrogen peroxide (H2O2) produced by NADPH oxidase (Nox). Both of these enzymes appear to be under the direct control of the circadian clock mechanism in the endothelial cells, and disruption to the clock results in endothelial and vascular dysfunction. In this review, we focus on EDRF and EDHF and summarise the recent findings on the influence of the peripheral circadian clock mechanism on processes involved in generating the redox species involved and how this influences vascular contractility, which may account for some of the circadian variations in blood pressure and peripheral resistance. Moreover, the direct link between the peripheral circadian clock and redox-signalling pathways in the vasculature, has a bearing on vascular endothelial dysfunction in disease and aging, which are both known to lead to dysfunction of the circadian clock.

Introduction: Senescence comprises the cellular and molecular changes leading to compromised functionality of organs. The presence of senescent endothelial cells in human atherosclerotic lesions suggests a contribution to vascular pathology. Better understanding of endothelial senescence will help identify its role in endothelial dysfunction. In this study gene expression changes were assessed in human endothelial cells following induction of senescence either by replicative or peroxide-induced stress and genes identified that were differentially expressed to be used as biomarkers of endothelial aging. Methods: Replicative senescent endothelial cells (REPS) were established by passaging human umbilical vein endothelial cells (HUVECs) up to 25 population doublings and stress-induced premature senescence (SIPS) was induced by treating HUVECs with tertiary butyl hydrogen peroxide (t-BHP) for 1 hr for consecutive 3 days. Senescence was confirmed by staining with senescence marker SA β-gal and p53 expression through Western blotting. Gene expression changes were confirmed by qPCR using Taqman probes. Results: REPS and SIPS cells were found to be 71 % (912 of 1285) and 81 % (980 of 1210) positive for SA-beta gal staining respectively, compared to young endothelial cells (10 % (130 of 1308); Fig1). CST1 was identified as highly expressed gene (90 fold) in REPS but not SIPS of endothelial cells in microarray gene expression analysis. Further validation using qPCR showed a 164 fold increase in CST1 expression (fig2) in REPS endothelial cells only. Conclusion: In conclusion, this study confirms that CST1 as a new marker of replicative senescence in human endothelial cells.

There is a need for robust in vitro models to sensitively capture skeletal muscle adverse toxicities early in the research and development of novel xenobiotics. To this end, an in vitro rat skeletal muscle model (L6) was used to study the translation of transcriptomics data generated from an in vivo rat model. Novel sulfonyl isoxazoline herbicides were associated with skeletal muscle toxicity in an in vivo rat model. Gene expression pathway analysis on skeletal muscle tissues taken from in vivo repeat dose studies identified enriched pathways associated with mitochondrial dysfunction, oxidative stress, energy metabolism, protein regulation and cell cycle. Mitochondrial dysfunction and oxidative stress were further explored using in vitro L6 metabolic models. These models demonstrated that the sulfonyl isoxazoline compounds induced mitochondrial dysfunction, mitochondrial superoxide production and apoptosis. These in vitro findings accurately concurred with the in vivo transcriptomics data, thereby confirming the ability of the L6 skeletal muscle models to identify relevant in vivo mechanisms of xenobiotic-induced toxicity. Moreover, these results highlight the sensitivity of the L6 galactose media model to study mitochondrial perturbation associated with skeletal muscle toxicity; this model may be utilised to rank the potency of novel xenobiotics upon further validation.

The NLRP3 inflammasome may be involved in atherosclerosis by activation of inflammatory processes in response to danger-associated molecular patterns and pattern-associated molecular patterns. NLRP3 inflammasome requires two steps for activation: signal 1 or priming and signal 2 for activation. This process results in active caspase-1 which in turn cleaves the pro-forms of IL-1β and IL-18 to their active pro-inflammatory forms and also mediates pyroptosis. The aim of this study is to investigate NLRP3 inflammasome activation in human endothelial cells. Differentiated THP-1 cells (positive control), EA.hy926 and human umbilical vein endothelial (HUVEC) were ‘primed’ with lipopolysaccharide (LPS) for 24 hours and then activated by exposure to ATP (300 µM) for 1 hour. Phorbol 12-myristate 13-acetate (PMA) (100 nM) which was used to differentiate THP-1 cells was also investigated as an EA.hy926 cell priming agent. Using Western blotting, NLRP3, pro-IL-1β and processed IL-1β were observed in THP-1 cells following LPS and PMA demonstrating priming and activation of the inflammasome as expected. NLRP3 protein was also upregulated in human endothelial cells following LPS priming but pro-IL-1β expression was not readily observed. However, following PMA treatment of EA.hy926 cells, a substantial synthesis of pro-IL-1β was detected suggesting effective inflammasome priming. Interestingly, pro-IL-1β expression was reduced by high levels of LPS (5 µg/ml) Further work is aimed at defining whether active IL-1β is produced in endothelial cells and defining the pyroptotic response in these cells.

Hyperphosphatemia in patients with advanced CKD is thought to be an important contributor to cardiovascular risk, in part because of endothelial cell (EC) dysfunction induced by inorganic phosphate (Pi). Such patients also have an elevated circulating concentration of procoagulant endothelial microparticles (MPs), leading to a prothrombotic state, which may contribute to acute occlusive events. We hypothesized that hyperphosphatemia leads to MP formation from ECs through an elevation of intracellular Pi concentration, which directly inhibits phosphoprotein phosphatases, triggering a global increase in phosphorylation and cytoskeletal changes. In cultured human ECs (EAhy926), incubation with elevated extracellular Pi (2.5 mM) led to a rise in intracellular Pi concentration within 90 minutes. This was mediated by PiT1/slc20a1 Pi transporters and led to global accumulation of tyrosine- and serine/threonine-phosphorylated proteins, a marked increase in cellular Tropomyosin-3, plasma membrane blebbing, and release of 0.1- to 1-μm-diameter MPs. The effect of Pi was independent of oxidative stress or apoptosis. Similarly, global inhibition of phosphoprotein phosphatases with orthovanadate or fluoride yielded a global protein phosphorylation response and rapid release of MPs. The Pi-induced MPs expressed VE-cadherin and superficial phosphatidylserine, and in a thrombin generation assay, they displayed significantly more procoagulant activity than particles derived from cells incubated in medium with a physiologic level of Pi (1 mM). These data show a mechanism of Pi-induced cellular stress and signaling, which may be widely applicable in mammalian cells, and in ECs, it provides a novel pathologic link between hyperphosphatemia, generation of MPs, and thrombotic risk.

Silybum marianum (milk thistle) is a medicinal plant used for the treatment of various liver disorders. This study examined whether the main flavonolignans from S. marianum (i.e. silybin, silychristin, silydianin) and their 2,3-dehydro derivatives (i.e. 2,3-dehydrosilybin, 2,3-dehydrosilychristin, 2,3-dehydrosilydianin) activate the Nrf2 pathway, which regulates the expression of genes encoding many cytoprotective enzymes, including NAD(P)H:quinone oxidoreductase 1 (NQO1). After 48 h of exposure, 2,3-dehydrosilydianin at concentrations of 25 μM and higher significantly elevated the activity of NQO1 in murine hepatoma Hepa1c1c7 cells. In contrast, other tested compounds at non-cytotoxic concentrations had a mild or negligible effect on the NQO1 activity. Using a luciferase reporter assay, 2,3-dehydrosilydianin was found to significantly activate transcription via the antioxidant response element in stably transfected human AREc32 reporter cells. Moreover, 2,3-dehydrosilydianin caused the accumulation of Nrf2 and significantly induced the expression of the Nqo1 gene at both the mRNA and protein levels in Hepa1c1c7 cells. We found that 2,3-dehydrosilydianin also increased to some extent the expression of other Nrf2 target genes, namely of the heme oxygenase-1 gene (Hmox1) and the glutamate-cysteine ligase modifier subunit gene (Gclm). We conclude that 2,3-dehydrosilydianin activates Nrf2 and induces Nrf2-mediated gene expression in Hepa1c1c7 cells.

Introduction Protease activated receptors (PARs) form a distinct group of G-protein coupled receptors with key roles in several pathophysiological processes including inflammation and atherothrombotic disease. This study aimed to investigate the effects of oxidative stress on PAR biology in human endothelial cells. EA.hy926 cells were investigated as an endothelial cell model and subsequently the effect of tert-butylhydroperoxide (tert-BHP) as an oxidative stressor was investigated in terms of PAR expression and activation. Methods Effects of thrombin, PAR-activating peptide and tert-BHP treatments on EA.hy926 PAR expression were determined by qRT-PCR and activity was assessed by Western blotting for pERK1/2. Results Both primary HUVECs and EA.hy926 expressed PAR1–3 and thrombin caused a rapid increase in PAR1 and PAR3 gene expression. PAR1 and PAR2 activating peptides caused significant increases in PAR1 and, to a lesser extent, PAR3. Induction of PAR2 via PAR2 activating peptide was not observed.PAR4 activating peptide caused time-dependent increases in PAR1–3. PAR activity increased with PAR activating peptide treatment, however there was no clear relationship between activity and expression of PARs mRNA. Repeated exposure to sub-cytotoxic concentrations of tert-BHP, 1 hr daily for 3 days, caused significant increases in PAR1 and PAR3 expression in EA.hy926 cells. Conclusions HUVEC PAR expression could be modelled in EA.hy926s. Induction of PAR gene expression in EA.hy926 cells was induced by activating peptides and thrombin. Increased PAR1 expression was often mirrored by increased PAR3 expression suggesting possible crosstalk. EA.hy926 cells treated with tert-BHP under conditions that induce stress-induced premature senescence, displayed significant increases in PAR1 and PAR3 gene expression. Further investigation is required to determine if this increased expression is due to redox changes alone and/or due to induction of a senescence phenotype.