EZETIMIBE PROTECTS THP-1 CELLS FROM ISCHEMIA-REPERFUSION INJURY REDUCING OXIDATIVE STRESS AND UP-REGULATING NRF2/ ARE GENE EXPRESSION

Background and Aims: We demonstrated that physical training, characterized by repeated ischemia-reperfusion (I-R) episodes (ischemic conditioning, IC), protects circulating cells from peripheral artery disease (PAD) patients against ischemic harms by reducing oxidative stress (OS) and by up-regulating nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway expression. Ezetimibe (Eze) has been shown to alleviate OS enhancing Nrf2 nuclear translocation in an AMPK/p62-dependent manner. In a cellular I-R and IC model, we aimed to investigate: 1) the effect of Eze on OS and Nrf2/ARE gene expression 2) whether Eze could have a synergistic effect on IC. Methods: THP-1 cells were treated with or without Eze (50mM) overnight, then subjected to 1 or 6 repetitive I-R cycles using EVOS FL Auto Imaging System. Reactive oxygen species (ROS) formation was evaluated with DCF in cytofluorimetry. Nrf2/ARE and p62 gene expression were evaluated by RT-PCR and western blotting. Results: When THP-1 cells were exposed to 1 I-R cycle, the preincubation with Eze significantly reduced ROS formation (p<0.01) and up-regulated Nrf2/ARE pathway expression and p62 phosphorylation (p<0.001). Multiple I-R cycles, acting as IC, significantly reduced ROS formation and upregulated Nrf2/ARE gene expression (p<0.001); in these conditions, Eze preincubation was able not only to almost abolish ROS formation (p<0.01) but also further up-regulate Nrf2/ARE expression. Conclusions: In our I-R model, Eze not only restores I-R-induced oxidative damages through Nrf2/ARE signaling up-regulation but also has a synergistic effect on IC. This new \u201cpleiotropic\u201d effect, if confirmed in vivo, may strengthen the use of Eze in PAD patien

Enhanced Levels of Oxidized Low-Density Lipoprotein Prime Monocytes to Cytokine Overproduction via Upregulation of CD14 and Toll-Like Receptor 4 in Unstable Angina

Objectives— The purpose of this study was to establish whether oxidized low-density lipoprotein (oxLDL) contributes to cytokine overproduction via upregulation of CD14 and toll-like receptor-4 (TLR-4) expression on circulating monocytes of unstable angina (UA) patients. Methods and Results— Expression of CD14 and TLR-4 on circulating monocytes, and the concentration of plasma oxLDL, (interleukin [IL])-6, IL-1 beta, IL-8, tumor necrosis factor (TNF)-alpha, monocyte chemoattractant protein-1 (MCP-1) were measured in 27 control (C) subjects, 29 patients with stable angina (SA), and 27 with UA. CD14 and TLR-4 expression on monocytes and circulating IL-6, IL-1 beta, and oxLDL were higher in UA than in SA and C subjects ( P <0.001). In in vitro experiments, oxLDL increased CD14 and TLR-4 expression ( P <0.001) in control monocytes as well as IL-6, IL-1 beta, and at a lower extent TNF-α and MCP-1 levels in the supernatant ( P from <0.05 to <0.001). The preincubation of sera derived from UA patients but with control monocytes also induced a significant increase of CD14 and TLR-4 expression ( P <0.001) and of IL-6 and IL-1 beta production ( P <0.001) in the supernatant. Conclusions— In UA patients oxLDL may contribute to monocyte overproduction of some cytokines by upregulating CD14 and TLR-4 expression

Ezetimibe prevents ischemia/reperfusion-induced oxidative stress and up-regulates Nrf2/ARE and UPR signaling pathways

BACKGROUND: While reperfusion is crucial for survival after an episode of ischemia, it also causes oxidative stress. Nuclear factor-E2-related factor 2 (Nrf2) and unfolded protein response (UPR) are protective against oxidative stress and endoplasmic reticulum (ER) stress. Ezetimibe, a cholesterol absorption inhibitor, has been shown to activate the AMP-activated protein kinase (AMPK)/Nrf2 pathway. In this study we evaluated whether Ezetimibe affects oxidative stress and Nrf2 and UPR gene expression in cellular models of ischemia-reperfusion (IR). METHODS: Cultured cells were subjected to simulated IR with or without Ezetimibe. RESULTS: IR significantly increased reactive oxygen species (ROS) production and the percentage of apoptotic cells without the up-regulation of Nrf2, of the related antioxidant response element (ARE) gene expression or of the pro-survival UPR activating transcription factor 6 (ATF6) gene, whereas it significantly increased the pro-apoptotic CCAAT-enhancer-binding protein homologous protein (CHOP). Ezetimibe significantly decreased the cellular ROS formation and apoptosis induced by IR. These effects were paralleled by the up-regulation of Nrf2/ARE and ATF6 gene expression and by a down-regulation of CHOP. We also found that Nrf2 activation was dependent on AMPK, since Compound C, a pan inhibitor of p-AMPK, blunted the activation of Nrf2. CONCLUSIONS: Ezetimibe counteracts IR-induced oxidative stress and induces Nrf2 and UPR pathway activation

Do dogs and cats passively carry sars-cov-2 on hair and pads?

The epidemiological role of domestic animals in the spread and transmission of SARS-CoV-2 to humans has been investigated in recent reports, but some aspects need to be further clarified. To date, only in rare cases have dogs and cats living with COVID-19 patients been found to harbour SARS-CoV-2, with no evidence of pet-to-human transmission. The aim of the present study was to verify whether dogs and cats act as passive mechanical carriers of SARS-CoV-2 when they live in close contact with COVID-19 patients. Cutaneous and interdigital swabs collected from 48 dogs and 15 cats owned by COVID-19 patients were tested for SARS-CoV-2 by qRT-PCR. The time elapsed between owner swab positivity and sample collection from pets ranged from 1 to 72 days, with a median time of 23 days for dogs and 39 days for cats. All samples tested negative, suggesting that pets do not passively carry SARS-CoV-2 on their hair and pads, and thus they likely do not play an important role in the virus transmission to humans. This data may contribute to confirming that the direct contact with the hair and pads of pets does not represent a route for the transmission of SARS-CoV-2

Increased endoplasmic reticulum stress and Nrf2 repression in peripheral blood mononuclear cells of patients with stable coronary artery disease.

Endoplasmic reticulum (ER) stress is involved in the pathophysiology of atherosclerosis. Insults interfering with ER function lead to the accumulation of unfolded and misfolded proteins in the ER that initiates the unfolded protein response (UPR). When the UPR fails to control the level of unfolded and misfolded proteins, ER-initiated apoptotic signaling is induced. We evaluated: (1) the UPR and ER-initiated apoptotic signaling in peripheral blood mononuclear cells (PBMCs) of stable coronary artery disease (CAD) patients; (2) PBMC content of oxidation products of phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC); (3) the possible origin of oxPAPC in PBMCs; and (4) the expression of nuclear erythroid-related factor 2 (Nrf2)/antioxidant-related element (ARE), a cellular defense mechanism. Twenty-nine CAD patients and 28 matched controls were enrolled. Expression of glucose-regulated protein 78kDa (GRP78/BiP), as a representative of the UPR, and of C/EBP homologous protein (CHOP), as a representative of ER apoptosis, was significantly higher in CAD than in controls (p<0.01). Concentrations of oxPAPC in PBMCs, in plasma, and in low-density lipoprotein (LDL) were significantly higher in CAD compared to controls (p<0.01). The oxPAPC in PBMCs may derive from circulating ox-LDL. Nrf2/ARE gene expression and circulating and cellular glutathione were significantly lower in CAD compared to controls (p<0.01). In in vitro studies, increasing amounts of oxPAPC induced a dose-dependent increase in CHOP and apoptosis-related protein expression (p<0.01) and a progressive decrease in Nrf2/ARE gene expression (p<0.01). In PBMCs of CAD patients there is an activation of the UPR and ER-initiated apoptotic signaling, possibly related to an abnormal concentration of oxPAPC in PBMCs

Detection and genetic characterization of domestic cat hepadnavirus in cats with cavitary effusions

: After the identification of the novel domestic cat hepadnavirus (DCH) in 2018, its potential pathogenetic role in feline hepatic diseases has been suggested. Following the detection of DCH in a cat's serum and peritoneal effusion, the aim of this study was to retrospectively investigate the presence of DCH in cats with and without cavitary effusions along with DCH presence in effusions. Stored serum and effusion samples from cats with and without effusions admitted to the Veterinary Teaching Hospital of Lodi (Italy) in 2020-2022 were included based on results of hematobiochemical parameters. Effusions were classified based on cytological and physicochemical findings. The likelihood of liver damage was estimated based on clinical and laboratory findings. Samples were tested for DCH presence by quantitative PCR (qPCR). Positive samples were subjected to whole genome sequencing and phylogenetic analysis. DCH was detected in both serum and peritoneal effusion samples of 2/72 (2.8%) enrolled cats, included in the group with effusions (2/33; 6.1%), with one cat showing inflammatory and the other non-inflammatory effusion. Both DCH-positive cats belonged to the group with a likelihood of liver damage (2/22, 9.1%). Phylogeny showed that the DCH sequences from this study clustered with the prototypic Australian strain but were not included in the clade with other Italian DCH sequences. Results suggest the circulation of different DCH variants in Italy and show the presence of DCH in effusion samples from DCH-positive cats, mirroring the presence of HBV in body fluids from HBV-infected humans. Further studies are still recommended to define the pathogenic role of DCH in cats

Endoplasmic reticulum stress and Nrf2 repression in circulating cells of type 2 diabetic patients without the recommended glycemic goals

Endoplasmic reticulum (ER) stress plays a role in the pathogenesis of type 2 diabetes mellitus (T2DM), with activation of the unfolded protein response (UPR) and ER apoptosis in \u3b2-cells. The aim of the study is investigating the role of the prolonged glycemic, inflammatory, and oxidative impairment as possible UPR and ER apoptosis inductors in triggering the ER stress response and the protective nuclear erythroid-related factor 2 (Nrf2)/antioxidant-related element (ARE) activation in peripheral blood mononuclear cells (PBMC) of T2DM patients without glycemic target. Oxidative stress markers (oxidation product of phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine [oxPAPC], and malondialdehyde [MDA]), the UPR and ER apoptosis, the activation of the pro-inflammatory nuclear factor-kappa B (NF-kB) with its inhibitory protein inhibitor-kB\u3b1, and the expression of the protective Nrf2 and heme oxygenase-1 (HO-1) were evaluated in PBMC of 15 T2DM patients and 15 healthy controls (C). OxPAPC concentrations (in PBMC and plasma), MDA levels (in plasma), the expressions of the glucose-regulated protein 78 kDa (or BiP) as representative of UPR, and of the CCAAT/enhancer-binding protein homologous protein as representative of ER apoptosis were significantly higher (p < 0.01) in T2DM with respect to C. IkB\u3b1 expression was significantly lower (p < 0.01) in T2DM as well as Nrf2 and HO-1. In vitro experiments demonstrated that hyperglycemic conditions, if prolonged, were NF-kB inductors, without a corresponding Nrf2/ARE response. In PBMC of T2DM without glycemic target achievement, there is an activation of the UPR and of the ER apoptosis, which may be related to the chronic exposure to hyperglycemia, to the augmented inflammation, and to the augmented oxidative stress, without a corresponding Nrf2/ARE defense activation

Serum oxidative stress-induced repression of Nrf2 and GSH depletion: a mechanism potentially involved in endothelial dysfunction of young smokers

AbstractBackground: Although oxidative stress plays a major role in endothelial dysfunction (ED), the role of glutathione (GSH), ofnuclear erythroid-related factor 2 (Nrf2) and of related antioxidant genes (ARE) are yet unknown. In this study we combined an in vivo with an in vitro model to assess whether cigarette smoking affects flow-mediated vasodilation (FMD), GSHconcentrations and the Nrf2/ARE pathway in human umbilical vein endothelial cells (HUVECs).Methods and Results: 52 healthy subjects (26 non-smokers and 26 heavy smokers) were enrolled in this study. In smokerswe demonstrated increased oxidative stress, i.e., reduced concentrations of GSH and increased concentrations of oxidationproducts of the phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC) in serum and inperipheral blood mononuclear cells (PBMC), used as in vivo surrogates of endothelial cells. Moreover we showedimpairment of FMD in smokers and a positive correlation with the concentration of GSH in PBMC of all subjects. In HUVECsexposed to smokers\u2019 serum but not to non-smokers\u2019 serum we found that oxidative stress increased, whereas nitric oxideand GSH concentrations decreased; interestingly the expression of Nrf2, of heme oxygenase-1 (HO-1) and of glutamatecysteineligase catalytic (GCLC) subunit, the rate-limiting step of synthesis of GSH, was decreased. To test the hypothesisthat the increased oxidative stress in smokers may have a causal role in the repression of Nrf2/ARE pathway, we exposedHUVECs to increasing concentrations of oxPAPC and found that at the highest concentration (similar to that found insmokers\u2019 serum) the expression of Nrf2/ARE pathway was reduced. The knockdown of Nrf2 was associated to a significantreduction of HO-1 and GCLC expression induced by oxPAPC in ECs.Conclusions: In young smokers with ED a novel further consequence of increased oxidative stress is a repression of Nrf2/ARE pathway leading to GSH depletion

Evidence of exposure to SARS-CoV-2 in cats and dogs from households in Italy

SARS-CoV-2 emerged from animals and is now easily transmitted between people. Sporadic detection of natural cases in animals alongside successful experimental infections of pets, such as cats, ferrets and dogs, raises questions about the susceptibility of animals under natural conditions of pet ownership. Here, we report a large-scale study to assess SARS-CoV-2 infection in 919 companion animals living in northern Italy, sampled at a time of frequent human infection. No animals tested PCR positive. However, 3.3% of dogs and 5.8% of cats had measurable SARS-CoV-2 neutralizing antibody titers, with dogs from COVID-19 positive households being significantly more likely to test positive than those from COVID-19 negative households. Understanding risk factors associated with this and their potential to infect other species requires urgent investigation