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

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

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

Inhibition of lectin-like oxidized low-density lipoprotein receptor-1 expression: is it right now a safe and promising therapeutic approach for atherosclerosis?

Lectin -like oxidized(ox) LDL receptor-1 (LOX-1) is the major receptor for oxLDL; several studies in vitro and in animal models have shown that LOX-1 is involved in almost all proatherogenic effects of oxLDL. Growing evidence also demonstrated that LOX-1 plays a role not only in the initiation and formation, but also in plaque destabilization and rupture and strengthen the role of this receptor in cardiovascular disease. As several studies gathered from animal models, as well as genetic studies, support the hypothesis that the binding and internalization of oxLDL by scavenger receptors such as LOX-1 triggers a series of mechanisms leading to endothelial dysfunction and foam cell formation, modulation of LOX-1 levels has been suggested to be one therapeutic route in attenuating early events in atherosclerosis. This is an editorial comment to a paper in which the authors designed a pyrrole\u2013imidazole polyamide to bind to a proximal site of the AP-1 binding region in the LOX-1 gene promoter. This molecule interfered with AP-1 protein binding to the LOX-1 gene promoter and inhibited the phorbol myristate acetate-mediated enhancement of LOX-1 gene expression in human umbilical vein endothelial cells and decreased apoptosis induced by Angiotensin II and oxLDL in HUVECs. As pyrrole\u2013imidazole polyamide was efficacious in inhibition of LOX-1 expression and apoptosis, which has been shown to be associated with the vulnerability of plaques, the authors propose the use of this agent for the study and treatment of atherosclerotic disease. Although silencing LOX-1 expression opens an appealing scenario on treatment of atherosclerosis, some important questions remain to be elucidated before translating fascinating results, obtained in vitro and in animal models, to patients. Further studies on inhibition of LOX-1 expression or drugs targeting LOX-1 may help to enlighten these open issue

Nd:YAG laser versus polidocanol injection for palliation of esophageal malignancy: a prospective, randomized study.

Palliation is often the only treatment that can be offered to patients affected by esophageal malignancy. This prospective study was carried out in order to compare two endoscopic palliative treatments: Nd:YAG laser and local injection of 3% polidocanol. We randomized 34 patients with inoperable malignancies to one of the two treatments. After the first course, 88.8% of the patients in the laser group and 81.5% in the polidocanol group were able to swallow a normal oral caloric intake. Only one major complication (esophageal perforation) was observed (polidocanol group) and was successfully treated with endoscopic placement of a prosthesis. We believe that both techniques are safe and effective for the palliation of esophageal malignant strictures but that polidocanol injection is cheap, simple, and more widely available

Cardiovascular function and comorbidities in elderly subjects with COPD

Purpose: The mechanisms underlying pathogenetic interactions between chronic obstructive pulmonary disease (COPD) and cardiovascular disease (CVD) are largely unknown despite a clinical mutual association in causing mortality and morbidity. Besides smoking, obesity and systemic inflammation share the complicity of being causative factors for both CVD and COPD. The present study aims to investigate, in an unselected, community-dwelling, elderly population, possible relationships between lung and cardiovascular impairements. Methods: A screening questionnaire was administered to 500 subjects aged from 65 to 84 years, randomly selected from the general population of Verona. All consenting participants underwent conventional pulmonary function test and diagnostic cardiovascular study (Echocardiografy, carotid Echo-Color-Doppler and Ankle-Brachial-Index). Blood pressure (BP), body mass index (BMI) and biochemical metabolic blood data were routinely obtained from all the participants. Results: COPD vs not-COPD patients differ for age (mean age: 70 vs 67 ys) but not for BP (mean: 130/80 vs 130/82 mmHg), BMI (mean: 28 vs 27 g/m2), total cholesterol (mean: 192 vs 220 mg/dL) and glycaemia (mean: 102 vs 97 mg/dL). In COPD patient forced expiratory volume in the first second (FEV1) was linearly related to reductions in left ventricular end-diastolic volume (p<0.05) and stroke volume (p<0.05) but not with ejection fraction. Left ventricular mass is related to pO2 value (p<0.001). COPD patients related to control patients present: pathological Intima-Media-Thickness (mean: 0.12 vs 0.08 cm, p<0.001) and ABI (mean: 0.87 vs 1.05, p<0.001), a grater extension of atherosclerotic burden (mean number of carotid plague: 2.5 vs 1.2) and higher level of plaque calcification. Conclusions: Magnitude of changes in the cardiac structure and function is related to the severity of COPD. COPD patients show a great prevalence of peripheral vasculopathy and a typical pattern of cardiac and vascular remodelling that expose them to high cardiovascular risk. In particular the mechanical reduction of cardiac outflow linked to hyperinflation state, vascular tone regulation and stiffness due to O2 blood pressure variation and the great atherosclerotic tendency have to be taken into account in the evaluation of COPD patients

Systemic lupus erythematosus and thrombotic thrombocytopenic purpura. Report of three cases and review of the literature.

We describe three female patients affected by both systemic lupus erythematosus and thrombotic thrombocytopenic purpura, one with a fatal outcome. The literature about this disease association is reviewed