Endoplasmic Reticulum Stress, NRF2 Signalling and Cardiovascular Diseases in a Nutshell.

PURPOSE OF REVIEW: This short review is intended primarily to summarize the understanding of the interrelated roles of endoplasmic reticulum (ER) stress, oxidative stress and inflammation in cardiovascular diseases. RECENT FINDINGS: Insults interfering with ER function lead to the accumulation of unfolded and misfolded proteins in the ER. An excess of proteins folding in the ER is known as ER stress. This condition initiates the unfolded protein response (UPR). When the UPR fails to control the level of unfolded and misfolded proteins, ER-initiated apoptotic signalling is induced. Moreover, the role of the protective nuclear erythroid-related factor 2 (Nrf2)/antioxidant-related element (ARE) and the activation of the pro-inflammatory nuclear factor-kappa B (NF-kB) are analysed. Authors summarize evidence that oxidative stress, inflammation and ER stress are closely entwined phenomena. They are involved in the pathogenesis of different cardiovascular diseases. Current literature data are presented, focusing on three topics of related pathologies: atherosclerotic plaque, coronary artery disease and diabetes. This review will provide a basic platform for study and application to several other conditions in which oxidative stress, ER stress and inflammation are key features. Future studies in this area may identify the most promising molecules to be investigated as common targets for cardiovascular diseases.

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.

Effects of nebivolol on endothelial gene expression during oxidative stress in human umbilical vein endothelial cells.

The endothelium plays a key role in the development of atherogenesis and its inflammatory and proliferative status influences the progression of atherosclerosis. The aim of this study is to compare the effects of two beta blockers such as nebivolol and atenolol on gene expression in human umbilical vein endothelial cells (HUVECs) following an oxidant stimulus. HUVECs were incubated with nebivolol or atenolol (10 micromol/L) for 24 hours and oxidative stress was induced by the addition of oxidized (ox)-LDL. Ox-LDL upregulated adhesion molecules (ICAM-1, ICAM-2, ICAM-3, E-selectin, and P-selectin); proteins linked to inflammation (IL-6 and TNFalpha), thrombotic state (tissue factor, PAI-1 and uPA), hypertension such as endothelin-1 (ET-1), and vascular remodeling such as metalloproteinases (MMP-2, MMP-9) and protease inhibitor (TIMP-1). The exposure of HUVECs to nebivolol, but not to atenolol, reduced these genes upregulated by oxidative stress both in terms of protein and RNA expression. The known antioxidant properties of the third generation beta blocker nebivolol seem to account to the observed differences seen when compared to atenolol and support the specific potential protective role of this beta blocker on the expression of a number of genes involved in the initiation and progression of atherosclerosis.

Nuclear factor kappa B in patients with a history of unstable angina: case re-opened.

This study aims at assessing NF-kB activity in unstable angina (UA) patients free of symptoms after a 1 year follow-up (1YFU). Plasma oxidized low-density lipoproteins (oxLDL), circulating NF-kB, Interleukin 6 (IL-6) and Interleukin 1ß (IL-1ß), high-sensitivity C-reactive protein (hs-CRP), as markers of oxidative stress and inflammation and plasma double-stranded DNA (ds-DNA), as marker of Neutrophil Extracellular Traps (NETs), were measured in 23 of the previously enrolled 27 UA patients. These measurements were compared to the UA data at baseline, and then compared to the data derived from the stable angina (SA) and controls (C) enrolled in our previous study (we demonstrated that UA had higher levels of NF-kB compared to SA and C). After a 1YFU, UA patients show a significant decrease in NF-kB, IL-6, hs-CRP, oxLDL, and ds-DNA plasma levels (p < 0.001) and in IL-1ß and White Blood Cells (WBC) (p < 0.005), without differences in lipid and glucose assessment. If compared to SA and C, UA after a 1YFU have higher levels of NF-kB, IL-6, ds-DNA, WBC, and oxLDL compared to C (p < 0.001), but only IL-6 is higher than SA (p < 0.001). No differences are found in lipid and glucose assessment. After a 1YFU, patients with a history of UA improve their oxidative and inflammatory status, such as the levels of circulating ds-DNA, without achieving the status of C. They become comparable to SA subjects. This study provides new insight on the multiple and apparently contradictory facets of NF-kB in UA and on its possible role as mediator in NETs' formation.

Lung ultrasound in internal medicine efficiently drives the management of patients with heart failure and speeds up the discharge time.

Lung ultrasound (LUS) is a valid tool for the assessment of heart failure (HF) through the quantification of the B-lines. This study in HF patients aims to evaluate if LUS: (1) can accelerate the discharge time; (2) can efficiently drive diuretic therapy dosage; and (3) may have better performance compared to the amino-terminal portion of B type natriuretic peptide (NT-proBNP) levels in monitoring HF recovery. A consecutive sample of 120 HF patients was admitted from the Emergency Department (ED) to the Internal Medicine Department (Verona University Hospital). The Chest X-ray (CXR) group underwent standard CXR examination on admission and discharge. The LUS group underwent LUS on admission, 24, 48 and 72 h later, and on discharge. The Inferior Cava Vein Collapsibility Index, ICVCI, and the NT-proBNP were assessed. LUS discharge time was significantly shorter if compared to CXR group (p < 0.01). During hospitalization, the LUS group underwent an increased number of diuretic dosage modulations compared to the CXR group (p < 0.001). There was a stronger association between partial pressure of oxygen in arterial blood (PaO2) and B-lines compared to the association between PaO2 and NT-proBNP both on admission and on discharge (p < 0.001). The B-lines numbers were significantly higher on admission in patients with more severe HF, and the ICVCI was inversely associated with B-lines number (p < 0.001). The potential of LUS in tailoring diuretic therapy and accelerating the discharge time in HF patients is confirmed. Until the technique comes into common use in different departments, it is plausible that LUS will evolve with different facets.

Physical Exercise Reduces Cytotoxicity and Up-Regulates Nrf2 and UPR Expression in Circulating Cells of Peripheral Artery Disease Patients: An Hypoxic Adaptation?

AIM: Ischemia-reperfusion (I-R) produces reactive oxygen species (ROS) that damage cells and favour cytotoxicity and apoptosis in peripheral artery disease (PAD) patients. Since brief episodes of I-R (ischemic conditioning) protect cells against ischemic harms, we evaluated whether a short-course of supervised treadmill training, characterized by repeated episodes of I-R, makes peripheral blood mononuclear cells (PBMCs) from PAD patients with intermittent claudication more resistant to I-R injuries by reducing oxidative stress and by inducing an adaptative response of unfolded protein response (UPR) and nuclear factor-E2-related factor (Nrf2) pathway expression. METHODS: 24 PAD patients underwent 21 sessions of treadmill training and a treadmill test as indicator of acute response to I-R. RESULTS: Maximal and pain free walking distance improved (p＜0.01), whereas LDH leakage and apoptosis of PBMCs decreased (p＜0.01); plasma malondialdehyde and ROS generation in PBMCs declined, while plasma glutathione augmented (p＜0.01). Moreover we demonstrated an up-regulation of UPR and Nrf2 expression in PBMCs (p＜0.01). To understand whether treadmill training may act as a trigger of ischemic conditioning, we examined the effect of repeated episodes of I-R on adaptative response in PBMCs derived from the patients. We showed an up-regulation of UPR and Nrf2 gene expression (p＜0.01), while oxidative stress and cytotoxicity, after an initial increase, declined (p＜0.01). This positive effect on cytotoxicity was reduced after inhibition of UPR and Nrf2 pathways. CONCLUSIONS: Treadmill training in PAD patients through UPR and Nrf2 up-regulation may trigger hypoxic adaptation similar to conditioning, thus modifying cell survival.

Effect of sulfhydryl and non-sulfhydryl angiotensin-converting enzyme inhibitors on endothelial function in essential hypertensive patients.

BACKGROUND: Oxidative inactivation of nitric oxide (NO) is regarded as an important cause of reduced endothelium-dependent vasodilation in essential hypertension. Because zofenopril, an angiotensin-converting enzyme (ACE) inhibitor with a sulfhydryl (SH) group, has demonstrated antioxidant properties and to reduce adhesion molecule expression in vitro, in this study we evaluated the effect of this drug in comparison with the carboxylic ACE inhibitor ramipril and the beta-adrenoreceptor blocker atenolol on (1) circulating adhesion molecules and some oxidative stress parameters and (2) endothelium-dependent vasodilation in essential mildly hypertensive patients. METHODS: A total of 45 healthy subjects and 45 matched hypertensive patients participated in the study. Hypertensive patients were randomly treated with zofenopril (15 to 30 mg/d), ramipril (2.5 to 5 mg/d), and atenolol (50 to 100 mg/d). At baseline and after an 8-week therapy we evaluated blood pressure (BP) values, plasma and LDL hydroperoxides, plasma 8-isoprostanes, circulating levels of oxidized-(ox)LDL and of adhesion molecules (intercellular cell adhesion molecule-1 [ICAM-1], and vascular cell adhesion molecule-1 [VCAM-1], and E-selectin). Furthermore, all patients underwent ultrasound detection of brachial artery reactivity and endothelium-dependent dilation (flow-mediated dilation, FMD) was evaluated. RESULTS: All the treatments determined similar significant (P < .001) reduction of both systolic and diastolic BP values. Plasma (P < .01) and LDL hydroperoxides (P < .01), plasma 8-isoprostanes (P < .05), circulating oxLDL (P < .05), and adhesion molecules (P < .05) were significantly reduced only in patients receiving zofenopril. Similarly FMD was significantly increased (P < .001) in the zofenopril-treated group. CONCLUSIONS: Our results suggest that in mildly hypertensive patients without organ damage zofenopril, beyond its BP-lowering effects and through its sustained antioxidant activity, offers important advantages in reducing endothelial activation.

Non-Exertional Heatstroke: A Case Report and Review of the Literature.

BACKGROUND Heatstroke (HS) is a life-threatening condition characterized by an elevation of the core body temperature above 40°C, central nervous system dysfunction, and possible multi-organ failure. HS can trigger systemic inflammation, disseminated intravascular coagulation (DIC), rhabdomyolysis, cerebral edema and seizures, pulmonary edema, heart dysfunctions, and renal and hepatic failure. CASE REPORT We report the case of a 41-year-old Romanian woman with a history of alcoholism who developed HS after arriving by bus in Verona, Italy in June 2016. The patient developed consecutive multi-organ dysfunction, including liver and renal failure, rhabdomyolysis, DIC, and arrhythmia. The patient was successfully treated with conservative measures. After 17 days, she recovered completely. CONCLUSIONS The exact mechanism of HS-related multiple organ dysfunction is not completely understood and its pathogenesis is complex. It involves inflammation, oxidative stress, endoplasmic reticulum (ER) stress, and mitochondrial dysfunction. Development of a model in which chronic alcohol abuse alters oxidative, inflammatory, and ER stress response could also be a conceivable solution to the positive prognosis of severe HS patients, in which liver failure has a prominent role.

An exploratory look at NETosis in atherosclerosis.

Current evidence suggests the likelihood of a link between venous thromboembolism (VTE) and atherosclerosis, although they have been traditionally considered as different pathological entities. The contribution of neutrophils to human atherogenesis has been underestimated, if compared to their contribution established in VTE. This is due to the major importance attributed to macrophages in plaque destabilization. Nevertheless, the role of neutrophils in atherogenesis deserves increasing attention. In particular, neutrophil extracellular traps (NETs) are net-like chromatin fibres that are released from dying neutrophils. The death of neutrophils with NETs formation is called NETosis. During activation, neutrophils produce reactive oxygen species (ROS), through the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The main function of NETs is trapping and killing pathogens. Nevertheless, NETs formation has been observed in various chronic inflammatory diseases, autoimmune diseases, vasculitis, lung diseases, cancer and VTE. Recent studies suggest that NETs formation might contribute also to atherosclerosis progression. New data report the presence of NETs in the luminal portion of human atherosclerotic vessels and coronary specimens obtained from patients after acute myocardial infarction. Programmed death mechanisms in atherosclerosis such as apoptosis, efferocytosis and also NETosis, share common features and triggers. If defective, they can lead the cells to a switch from programmed death to necrosis, resulting in the release of pro-atherogenic factors, accumulation of cell debris and progression of the disease. This review provides evidence on the emerging role of neutrophils focusing on NETosis and oxidative stress burden in orchestrating common mechanisms in atherosclerosis and thrombosis.

Enhanced plasma levels of oxidized low-density lipoprotein increase circulating nuclear factor-kappa B activation in patients with unstable angina.

OBJECTIVES: The purpose of this study was to investigate the effect of circulating levels of oxidized low-density lipoprotein (ox-LDL) on nuclear factor-kappa B (NF-kB) activation in peripheral blood mononuclear cells (PBMC) of patients with unstable angina (UA) or stable angina (SA) and control subjects. BACKGROUND: Nuclear factor-kB might be involved in atherosclerosis, as is suggested by the presence of activated NF-kB in human atherosclerotic lesions. METHODS: Levels of plasma ox-LDL and circulating NF-kB in PBMC (and in separated lymphocytes and monocytes) were measured in 27 control subjects and 29 SA and 27 UA patients. In in vitro studies, the effect of ox-LDL and of the sera derived from a subgroup of UA patients and control subjects on monocytic NF-kB activation was also evaluated. RESULTS: The UA and SA patients had higher levels of circulating ox-LDL and NF-kB in PBMC than control subjects (p < 0.001). The increase in circulating NF-kB was mainly due to the activation of monocytes. In the in vitro studies, ox-LDL dose-dependently increased the activation of NF-kB in monocytes, but not in lymphocytes derived from healthy volunteers. This increase was related to the expression of lectin-like ox-LDL receptor-1 on monocytes. The incubation of monocytes with the sera derived from the UA patients induced a significant increase in NF-kB activation compared with the sera derived from the control subjects. CONCLUSIONS: The data suggest that the activation of NF-kB in monocytes of UA patients is, at least in part, induced by circulating molecules such as ox-LDL, which has been found to be particularly elevated in UA patients.

Plasma levels of oxidized-low-density lipoproteins are higher in patients with unstable angina and correlated with angiographic coronary complex plaques.

OBJECTIVES: To measure circulating levels of oxidized-low-density lipoproteins (ox-LDL) in patients with stable and unstable angina and controls, and to investigate their correlation with the extent of coronary artery disease (CAD) and the presence of complex plaques at coronary angiography. METHODS AND RESULTS: Circulating ox-LDL were assessed, using ELISA, in patients with unstable angina (UA, n=26), stable angina (SA, n=29) and in controls (C, n=27). All patients underwent coronary angiography. The extent of CAD was evaluated using a quantitative score, while the presence of complex, vulnerable plaques was angiographically assessed. Ox-LDL were higher in UA patients than in SA patients and in C subjects, and in SA patients than in C subjects (C, 45.6+/-12.8 U/L; SA, 58.8+/-11.0 U/L; UA, 73.7+/-13.6 U/L; p<0.001). No correlation was found with the extent of atherosclerotic disease in the coronary tree. Patients with angiographic complex lesions showed significantly higher levels of ox-LDL (68.4+/-13.9 U/L versus 55.2+/-16.4 U/L, p<0.001). Multiple regression analysis showed that ox-LDL were independent predictors of the presence of complex plaques (p<0.023). CONCLUSIONS: Ox-LDL levels are higher in unstable patients and correlate with the presence of angiographically documented complex plaques. Ox-LDL might be markers of destabilization of CAD.

Lung ultrasound in internal medicine: training and clinical practice.

BACKGROUND: Lung ultrasound (LUS) represents an emerging technique for bedside chest imaging in different clinical settings. A standardized approach allows the diagnosis, the quantification, and the follow-up of different conditions for which acute respiratory failure is the main clinical presentation. The aim of this study was to test what skill targets could be achieved in LUS, with a short-training course offered to 19 Medical Doctors attending the certification board school in Internal Medicine at the University of Verona, Italy. METHODS: The training course (theoretical and practical) consisted of 9 h subdivided in 4 days. Each trainee examined three healthy volunteers during the first day that was also the day of the theoretical lessons. Moreover, they examined nine patients per day (a total of 27 patients). Trainees were tested in the recognition of the basic signs in LUS, the managing of the Bedside Lung Ultrasound Evaluation (the BLUE protocol), and the recognition of the broad clinical scenarios recognized by the LUS. Kappa statistic was used to calculate the inter-observer agreement (trainees/tutor). RESULTS: Twenty-seven patients were examined by the 19 trainees (ten trainees had previous limited experience in general ultrasound). The agreement among the trainees and the tutor in the recognition of the LUS basic signs and in the recognition of the BLUE protocol profiles ranged from "fair" to "excellent". In particular, the agreement among the trainees and the tutor in the final LUS diagnosis was "excellent" for the recognition of the interstitial syndrome and the pleural effusion, "substantial" for the recognition of the normal lung, and "moderate" for the recognition of consolidation and pneumothorax. LUS outcome gave useful information and drove change in therapy in 16 patients. It affected immediate management in nine patients. The concordance between the previous X chest ray and LUS was observed in 21 patients. CONCLUSIONS: A short training in LUS provided good proficiency in the recognition only of the main signs of the BLUE protocol, but allowed a correct LUS diagnosis in the Internal Medicine most frequent clinical settings of acute respiratory failure. This study supports incorporating LUS into Internal Medicine fellowship training programs.

Nrf2 expression is increased in peripheral blood mononuclear cells derived from mild-moderate ex-smoker COPD patients with persistent oxidative stress.

Inadequacy of antioxidant nuclear factor-E2-related factor 2 (Nrf2) and endoplasmic reticulum stress-mediated unfolded protein response has been implicated in severe chronic obstructive pulmonary disease (COPD) and cigarette smoking-induced emphysema. As evidence suggests that the ability to upregulate Nrf2 expression may influence the progression of COPD and no data exist up to now in ex-smokers with mild-moderate COPD, this study was first aimed to evaluate Nrf2 and unfolded protein response expression in peripheral blood mononuclear cells (PBMC) of mild-moderate ex-smokers with COPD compared to smoking habit-matched non-COPD subjects. Then, we tested whether oxidative stress persists after cigarette smoking cessation and whether the concentrations of oxidized phospholipids (oxidation products of the phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine [oxPAPC]) in the PBMC of the same subjects may have a causative role in determining the upregulation of Nrf2. The expression (mRNA and protein) of Nrf2 and of its related gene heme oxygenase-1 was significantly increased in COPD group without differences in the unfolded protein response. Plasma malondialdehyde, the circulating marker of oxidative stress, and oxPAPC in PBMC were significantly higher in COPD than in non-COPD subjects. The fact that the expression of p47phox, a subunit of NADPH oxidase, was increased in PBMC of COPD patients and that it was directly correlated with oxPAPC may indicate that oxPAPC may be one of the determinants of oxidative stress-induced Nrf2 upregulation. Finally, we also demonstrated that lung function inversely correlated with plasma malondialdehyde and with Nrf2 and heme oxygenase-1 mRNA expression in all subjects. Our results indicate that mild-moderate ex-smokers with COPD may be able to counteract oxidative stress by increasing the expression of Nrf2/antioxidant-response elements. Because Nrf2 failure significantly contributes to the development of COPD, our findings suggest that the possibility to prevent Nrf2 reduction may open a new scenario in helping to prevent the oxidative stress-associated lung function decline.

The platelet-endothelium interaction mediated by lectin-like oxidized low-density lipoprotein receptor-1 reduces the intracellular concentration of nitric oxide in endothelial cells.

OBJECTIVES: To address the potential role of the endothelial lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in the thrombotic system, in this study we first examined whether platelet interaction with LOX-1 generated reactive oxygen species (ROS) and superoxide (O2.-) and then investigated the relationship between the intracellular production of O2.- and the availability of nitric oxide (NO). BACKGROUND: Oxidative inactivation of NO is regarded as an important cause of its decreased biologic activity which may favor platelet-dependent arterial thrombosis. METHODS: Bovine aortic endothelial cells (BAECs) and Chinese hamster ovary-K1 cells stably expressing bovine LOX-1 (BLOX-1-CHO) were incubated at different times with human platelets. The ROS, O2.-, and NO were measured in cells by flow cytometry. RESULTS: The incubation of BAECs and BLOX-1-CHO cells with human platelets induced a sharp and dose-dependent increase in intracellular concentration of ROS and O2.- (p from <0.01 to <0.001). The increase in intracellular concentration of O2.- was followed by a dose-dependent reduction in basal and bradykinin-induced intracellular NO concentration (p from <0.01 to <0.001). The increase in O2.- and the reduction of NO were inhibited by the presence of vitamin C and anti-LOX-1 monoclonal antibody (p < 0.001). CONCLUSIONS: The results of this study show that one of the pathophysiologic consequences of platelet binding to LOX-1 may be the inactivation of NO through an increased cellular production of O2.-.

Nebivolol and its 4-keto derivative increase nitric oxide in endothelial cells by reducing its oxidative inactivation.

OBJECTIVES: The objective of the present study was to elucidate the vasodilator mechanisms of nebivolol, a high selective beta(1)-receptor antagonist with antioxidant properties. BACKGROUND: Oxidative inactivation of nitric oxide (NO) is regarded as an important cause of its decreased biological activity. METHODS: Oxidative stress was induced through the binding of oxidized (ox)-low-density lipoprotein (LDL) to its specific endothelial receptor, called "lectin-like oxidized LDL receptor-1" (LOX-1), in bovine and human endothelial cells and in Chinese hamster ovary cells stably expressing bovine LOX-1 (BLOX-1-CHO cells). Reactive oxygen species (ROS), superoxide (O(2)(*-)), and NO were measured in cells by flow cytometry. RESULTS: Nebivolol and its 4-keto derivative prevented in a dose-dependent manner the increase of ROS (p < 0.001) and O(2)(*-) (p < 0.001) in bovine aortic endothelial cells (BAECs), human umbilical vein endothelial cells (HUVECs), and BLOX-1-CHO cells stimulated with ox-LDL. Atenolol had no effect. The incubation of HUVECs and BAECs with ox-LDL reduced basal and bradykinin-induced NO and nitrite concentration (p from <0.001 to <0.01). Nebivolol and its 4-keto derivative prevented the reduction of basal and stimulated NO and nitrite concentration (p from <0.001 to <0.01) while atenolol had no effect. The preincubation of BAECs with blocking anti-LOX-1 monoclonal antibody (LOX-1 mAb) significantly counteracted the effect of ox-LDL on stimulated generation of NO (p < 0.001), but the effect was significantly lower than that of nebivolol and its 4-keto derivative alone (p < 0.01). CONCLUSIONS: In conclusion, the findings of the present study indicate that nebivolol increases NO also by decreasing its oxidative inactivation.

Nebivolol decreases oxidative stress in essential hypertensive patients and increases nitric oxide by reducing its oxidative inactivation.

OBJECTIVE: To obtain further insight into the mechanism underlying the vasodilator effect of nebivolol. Since oxidative inactivation of nitric oxide (NO) is regarded as an important cause of its decreased biological activity, we studied (1) the effect of nebivolol on some oxidative parameters in essential hypertensive patients; (2) the effect of plasma of nebivolol-treated patients on reactive oxygen species production and NO availability in endothelial cells. METHODS: A total of 20 healthy subjects and 20 matched essential hypertensive patients treated with atenolol or nebivolol according to a double-blind, randomized design participated in the study. We measured low-density lipoprotein (LDL) and plasma hydroperoxides, 8-isoprostanes, oxidized LDL, susceptibility of LDL to oxidation (lag phase) and LDL vitamin E and the effect of plasma of nebivolol- and atenolol-treated patients on reactive oxygen species production and NO availability in endothelial cells exposed to oxidative stress. RESULTS: In hypertensive patients, nebivolol and atenolol significantly reduced blood pressure values after 4 weeks of treatment. Plasma and LDL hydroperoxides, plasma 8-isoprostanes, plasma ox-LDL and LDL lag phase were significantly improved only in the patients receiving nebivolol compared with the atenolol group. Similarly there was a reduction of reactive oxygen species (ROS) and O2*- concentration in endothelial cells exposed to oxidative stress after incubation of the cells with plasma of the patients enrolled in the trial only in the patients receiving nebivolol compared to atenolol group. Furthermore, the reduction of basal and stimulated NO induced by oxidative stress in endothelial cells was significantly lower in the patients receiving nebivolol compared to atenolol group. CONCLUSIONS: The findings of the present study indicate that nebivolol, through its antioxidant properties, increases NO also by decreasing its oxidative inactivation.

Short training in focused cardiac ultrasound in an Internal Medicine department: what realistic skill targets could be achieved?

The importance of focused cardiac ultrasound (FCU) in Internal Medicine care has been recognized by the American Society of Echocardiography. The aim of this study was to test what realistic skill targets could be achieved in FCU, with a relatively short training (theoretical and practical) of 9 h offered to Internal Medicine certification board attending students, and if the addition of further 9 h of training could significantly improve the level of competence. Kappa statistic was used to calculate the inter-observer agreement (trainees/tutor). The agreement between the trainees (who completed the entire training) and the tutor was, respectively, "substantial" (k = 0.71) for the identification of pericardial effusion, "moderate" (k = 0.56-0.54) for the identification of marked right ventricular and left ventricular enlargement, "substantial" (k = 0.77) for the assessment of global cardiac systolic function by visual inspection and "fair" (k = 0.35) for the assessment of size and respiratory change in the diameter of the inferior cave vein (IVC). 18 h training in FCU provided proficiency in obtaining adequate images from the parasternal window without providing the ability to correctly master the apical and subcostal windows. As concerns the interpretative skills, only pericardial effusion and visual estimation of global systolic function could be correctly identified, while ventricular enlargement and IVC prove to be more difficult to evaluate. This study supports incorporating FCU into Internal Medicine fellowship training programs, and should facilitate the design of other similar training courses.

Endoplasmic reticulum stress and Nrf2 signaling in cardiovascular diseases.

Various cellular perturbations implicated in the pathophysiology of human diseases, including cardiovascular and neurodegenerative diseases, diabetes mellitus, obesity, and liver diseases, can alter endoplasmic reticulum (ER) function and lead to the abnormal accumulation of misfolded proteins. This situation configures the so-called ER stress, a form of intracellular stress that occurs whenever the protein-folding capacity of the ER is overwhelmed. Reduction in blood flow as a result of atherosclerotic coronary artery disease causes tissue hypoxia, a condition that induces protein misfolding and ER stress. In addition, ER stress has an important role in cardiac hypertrophy mainly in the transition to heart failure (HF). ER transmembrane sensors detect the accumulation of unfolded proteins and activate transcriptional and translational pathways that deal with unfolded and misfolded proteins, known as the unfolded protein response (UPR). Once the UPR fails to control the level of unfolded and misfolded proteins in the ER, ER-initiated apoptotic signaling is induced. Furthermore, there is considerable evidence that implicates the presence of oxidative stress and subsequent related cellular damage as an initial cause of injury to the myocardium after ischemia/reperfusion (I/R) and in cardiac hypertrophy secondary to pressure overload. Oxidative stress is counterbalanced by complex antioxidant defense systems regulated by a series of multiple pathways, including the UPR, to ensure that the response to oxidants is adequate. Nuclear factor-E2-related factor (Nrf2) is an emerging regulator of cellular resistance to oxidants; Nrf2 is strictly interrelated with the UPR sensor called pancreatic endoplasmic reticulum kinase. A series of studies has shown that interventions against ER stress and Nrf2 activation reduce myocardial infarct size and cardiac hypertrophy in the transition to HF in animals exposed to I/R injury and pressure overload, respectively. Finally, recent data showed that Nrf2/antioxidant-response element pathway activation may be of importance also in ischemic preconditioning, a phenomenon in which the heart is subjected to one or more episodes of nonlethal myocardial I/R before the sustained coronary artery occlusion.

The atherosclerotic plaque vulnerability: focus on the oxidative and endoplasmic reticulum stress in orchestrating the macrophage apoptosis in the formation of the necrotic core.

Although the understanding the pathophysiology of atherogenesis and atherosclerosis progression has been one of the major goals of cardiovascular research during the last decades, the precise mechanisms underlying plaque destabilization are still unknown. The disruption of the plaque and the thrombosis in the lumen that are mostly determined by the expansion of the necrotic core (NC) are driven by various mechanisms, including accelerated macrophage apoptosis and defective phagocytic clearance (defective efferocytosis). Oxidative stress is implicated in the expansion of the NC: in fact, many oxidized compounds and processes contribute to the macrophage apoptosis; in addition, the oxidized derivatives of polyunsatured fatty acids promote defective efferocytosis, with the final result of NC expansion. In the last years the role of the endoplasmic reticulum (ER) stress is under investigation to better define its possible contribution in affecting the NC expansion. The abnormal amount of apoptotic cells in the vulnerable plaque has been demonstrated to be related both to the sustained ER stress and to the expression of survival and protective genes, such as the unfolded protein response or/and the nuclear erytroid- related factor 2. In this review the authors focus on the promising results of the oxidative and ER stress in contributing to triggering and orchestrating the atherosclerotic plaque vulnerability.

Zofenopril inhibits the expression of adhesion molecules on endothelial cells by reducing reactive oxygen species.

Hypertension and coronary artery disease are intimately connected. The migration of circulating monocytes into the subendothelial occurs through the expression of some adhesion molecules on endothelial cells. The nuclear factor (NF)-kappaB, a redox-sensitive element, plays a key role in adhesion molecule gene induction. In this study we have compared the effects of two different angiotensin converting enzyme (ACE) inhibitors, one possessing an active sulfhydryl group (zofenopril) and one lacking this group (enalapril) on the cellular redox state (monitored by measuring intracellular reactive oxygen species and thiol status), expression of adhesion molecules, and activation of NF-kappaB in human umbilical vein endothelial cells (HUVECs). Zofenoprilat, the active form of zofenopril, significantly and dose dependently reduced the intracellular reactive oxygen species (ROS) and superoxide formation induced by oxidized low-density lipoprotein (ox-LDL) (P <.001) and tumor necrosis factor-alpha (TNF-alpha) (P <.001). Enalaprilat, the active form of enalapril, was ineffective. Zofenoprilat but not enalaprilat also decreased the consumption of the intracellular GSH induced by ox-LDL (P <.01) and TNF-alpha (P <.01). Although zofenoprilat significantly and dose dependently reduced the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), and E-selectin induced by ox-LDL (P <.01) and TNF-alpha (P <.01) on HUVECs, enalaprilat did not. Ox-LDL and TNF-alpha increased the activation of NF-kappaB and the preincubation of HUVECs with zofenoprilat, but not with enalaprilat, dose dependently reduced its activation (P <.001). The conclusion is that the sulfhydryl (SH)-containing ACE inhibitors may be useful in inhibiting foam cell formation and thus slow the development of atherosclerosis.