Association between endotoxemia and blood no in the portal circulation of cirrhotic patients: results of a pilot study.

Pathophysiology of portal vein thrombosis (PVT) in cirrhosis is still not entirely understood. Elevated levels of lipopolysaccharides (LPS) in portal circulation are significantly associated with hypercoagulation, increased platelet activation and endothelial dysfunction. The aim of the study was to investigate if LPS was associated with reduced portal venous flow, the third component of Virchow's triad, and the underlying mechanism. Serum nitrite/nitrate, as a marker of nitric oxide (NO) generation, and LPS were measured in the portal and systemic circulation of 20 patients with cirrhosis undergoing transjugular intrahepatic portosystemic shunt (TIPS) procedure; portal venous flow velocity (PVV) was also measured in each patient and correlated with NO and LPS levels. Serum nitrite/nitrate and LPS were significantly higher in the portal compared to systemic circulation; a significant correlation was found between LPS and serum nitrite/nitrate (R = 0.421; p < 0.01). Median PVV before and after TIPS was 15 cm/s (6-40) and 31 cm/s (14-79), respectively. Correlation analysis of PVV with NO and LPS showed a statistically significant negative correlation of PVV with portal venous NO concentration (R = - 0.576; p = 0.020), but not with LPS. In vitro study with endothelial cells showed that LPS enhanced endothelial NO biosynthesis, which was inhibited by L-NAME, an inhibitor of NO synthase, or TAK-242, an inhibitor of TLR4, the LPS receptor; this effect was accomplished by up-regulation of eNOS and iNOS. The study shows that in cirrhosis, endotoxemia may be responsible for reduced portal venous flow via overgeneration of NO and, therefore, contribute to the development of PVT.

Impact of heat-not-burn cigarette passive smoking on children's oxidative stress, endothelial and platelet function.

Growing global use of heat-not-burn cigarettes (HNBC) prompts investigation. Prior studies assessed HNBC's effects on cardiovascular health, revealing heightened oxidative stress, platelet activation, and endothelial dysfunction. However, limited understanding exists regarding passive smoking's impact on children exposed to HNBC. This study aims to assess levels of oxidative stress, endothelial and platelet function among children exposed to passive smoke from HNBC, traditional tobacco (TT) cigarettes and unexposed subjects. Seventy-eight children (2-18 years) were divided into three groups: HNBC passive smokers (n = 26), TT cigarette exposed (n = 26), and control (CNT) group (n = 26, unexposed). Oxidative stress was evaluated by serum NADPH oxidase-2 (NOX2) activity, assessed by soluble Nox2-derived peptide (sNOX2-dp), isoprostanes, hydrogen peroxide (H2O2) production, hydrogen break-down activity (HBA) and NO bioavailability. Endothelial function was assessed by brachial flow-mediated dilation (FMD). Platelet function was evaluated by soluble CD40 ligand (sCD40L), soluble P-selectin (sP-selectin) and thrombus formation by T-TAS analysis. Passive smoking-exposed children (both HNBC and TT) exhibited significantly increased serum sNOX2-dp, isoprostanes, H2O2, sCD40L sP-selectin and thrombus formation versus controls. Conversely, exposed children displayed reduced brachial FMD and serum NO bioavailability. No significant differences were found between children exposed to passive smoking of HNBC vs TT. Multivariable regression linked sNOX2 (standardized coefficient ß: 0.284; SE: 0.040; p = 0.01) and H2O2 (standardized coefficient ß: 0.243; SE: 0.0; p = 0.02) as independent predictors of FMD, and isoprostanes (standardized coefficient ß:0.388; SE: 0.022; p < 0.001) and serum cotinine (standardized coefficient ß:0.270; SE: 0.048; p = 0.01) with sNOX2-dp levels. Exposure to HNBC smoke heightened oxidative stress, endothelial dysfunction, platelet activation, and thrombus formation in children. Findings suggest avenues for interventions to curb childhood passive smoking exposure.

Low-grade endotoxemia is associated with cardiovascular events in community-acquired pneumonia.

OBJECTIVES: Community-acquired pneumonia (CAP) is associated with low-grade endotoxemia but its relationship with cardiovascular events (CVE) has not been investigated. METHODS: We evaluated the incidence of CVE including myocardial infarction, stroke, and cardiovascular death in 523 adult patients hospitalized for CAP. Serum lipopolysaccharide (LPS) and zonulin, a marker of gut permeability, were analyzed in the cohort, that was followed-up during hospitalization and up to 43 months thereafter. RESULTS: During the hospital-stay, 55 patients experienced CVE with a progressive increase from the lowest (0.6%) to highest LPS tertile (23.6%, p < 0.001). Logistic regression analyses showed that higher LPS tertile was independently associated with CVE; LPS significantly correlated with age, hs-CRP and zonulin. In a sub-group of 23 CAP patients, blood E. coli DNA was higher in patients compared to 24 controls and correlated with LPS. During the long-term follow-up, 102 new CVE were registered; the highest tertile of LPS levels was associated with incident CVE; Cox regression analysis showed that LPS tertiles, age, history of CHD, and diabetes independently predicted CVE. CONCLUSIONS: In CAP low-grade endotoxemia is associated to short- and long-term risk of CVE. Further study is necessary to assess if lowering LPS by non-absorbable antibiotics may result in improved outcomes.

Dark Chocolate Intake Positively Modulates Gut Permeability in Elite Football Athletes: A Randomized Controlled Study.

Gut barrier disruption can lead to enhanced intestinal permeability, which allows endotoxins, pathogens, and other proinflammatory substances to move through the intestinal barrier into circulation. Intense exercise over a prolonged period increases intestinal permeability, which can be further worsened by the increased production of reactive oxygen species (ROS) and pro-inflammatory cytokines. The aim of this study was to assess the degree of intestinal permeability in elite football players and to exploit the effect of cocoa polyphenols on intestinal permeability induced by intensive physical exercise. Biomarkers of intestinal permeability, such as circulating levels of zonulin, a modulator of tight junctions, occludin, a tight junction protein, and LPS translocation, were evaluated in 24 elite football players and 23 amateur athletes. Moreover, 24 elite football players were randomly assigned to either a dark chocolate (>85% cocoa) intake (n = 12) or a control group (n = 12) for 30 days in a randomized controlled trial. Biochemical analyses were performed at baseline and after 30 days of chocolate intake. Compared to amateur athletes, elite football players showed increased intestinal permeability as indicated by higher levels of zonulin, occludin, and LPS. After 30 days of dark chocolate intake, decreased intestinal permeability was found in elite athletes consuming dark chocolate. In the control group, no changes were observed. In vitro, polyphenol extracts significantly improved intestinal damage in the human intestinal mucosa cell line Caco-2. These results indicate that chronic supplementation with dark chocolate as a rich source of polyphenols positively modulates exercise-induced intestinal damage in elite football athletes.

Aging and Antithrombotic Treatment.

Significance: Several aging-related pathophysiological mechanisms have been described to contribute to increased thrombotic risk in the elderly, including oxidative stress, endothelial dysfunction, and platelet and coagulation cascade activation. Antithrombotic treatment in the elderly should be individualized. Recent Advances: Recent studies have clarified some pathophysiological mechanisms of enhanced oxidative stress and thrombotic alterations in older adults. In the last decade, randomized trials have evaluated different antithrombotic strategies to reduce the risk of cardiovascular events in these patients. Critical Issues: The proportion of elderly patients included in clinical trials is generally low, thus not reflecting the daily clinical practice. There is no consensus on the most appropriate antithrombotic treatment in the elderly, also considering that bleeding risk management may be challenging in this high-risk subgroup of patients. Routine antiplatelet treatment is not a valid strategy for the primary prevention of cardiovascular events given the associated high risk of bleeding. In elderly patients with acute coronary syndrome, low-dose prasugrel or clopidogrel, shorter dual antiplatelet therapy, and no pretreatment before stent placement should be considered. Advanced age should not be the only reason for the underuse of oral anticoagulation in patients with atrial fibrillation, with direct oral anticoagulants preferred over warfarin for stroke prevention. Instead, a case-by-case clinical evaluation is warranted based on patient's bleeding risk also. Future Directions: There is a need for a structured tailored approach to manage thrombotic risk in elderly patients. The choice of the most appropriate antithrombotic treatment should balance efficacy and safety to reduce the risk of bleeding.

Diverticular Disease Worsening Is Associated with Increased Oxidative Stress and Gut Permeability: New Insights by Circulating Biomarkers.

Diverticular disease (DD) management is impaired by its pathogenesis, which is still not completely defined, with an unmet clinical need for improved therapies. Ex vivo DD human models demonstrated the presence of a transmural oxidative imbalance that supports an ischemic pathogenesis. This study aimed to assess, with the use of circulating biomarkers, insights into DD pathogenesis and possible therapeutic targets. Nox2-derived peptide, H2O2, antioxidant capacity, isoprostanes, thromboxanes, TNF-α, LPS and zonulin were evaluated by ELISA in healthy subjects (HS) and asymptomatic and symptomatic DD patients. Compared to HS, DD patients presented low antioxidant capacity and increase in sNox2-dp, H2O2 and isoprostanes paralleled to a TNFα increase, lower than that of oxidative markers. TxB2 production correlated to Nox2 and isoprostanes, suggesting platelet activation. An increase in zonulin and LPS highlighted the role of gut permeability and LPS translocation in DD pathogenesis. The increase of all the markers statistically correlated with DD severity. The present study confirmed the presence of a main oxidative imbalance in DD and provides evidence of platelet activation driven by LPS translocation. The use of circulating biomarkers could represent a new clinical tool for monitoring disease progression and validate therapeutic strategies never tested in DD as antioxidant supplementation.

PAD4-Induced NETosis Via Cathepsin G-Mediated Platelet-Neutrophil Interaction in ChAdOx1 Vaccine-Induced Thrombosis-Brief Report.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare syndrome characterized by platelet anti-PF4 (platelet-activating antiplatelet factor 4)-related thrombosis. Platelet-neutrophil interaction has been suggested to play a role, but the underlying mechanism has not been fully elucidated. METHODS: The study included 10 patients with VITT after ChAdOx1 (chimpanzee adenovirus Oxford 1) nCoV-19 (Oxford-AstraZeneca) vaccine administration, 10 patients with ischemic stroke (IS), 10 patients with acute deep vein thrombosis, and 10 control subjects in whom blood levels of neutrophil extracellular traps (NETs), soluble TF (tissue factor), and thrombin generation were examined. Furthermore, we performed in vitro studies comparing the effect of serum from patients and controls on NETs formation. Finally, immunohistochemistry was performed in cerebral thrombi retrieved from a patients with VITT and 3 patients with IS. RESULTS: Compared with patients with IS, patients with deep vein thrombosis, controls, and patients with VITT had significantly higher blood values of CitH3 (citrullinated histone H3), soluble TF, D-dimer, and prothrombin fragment 1+2 (P<0.0001). Blood CitH3 significantly correlated with blood soluble TF (Spearman rank correlation coefficient=0.7295; P=0.0206) and prothrombin fragment 1+2 (Spearman rank correlation coefficient=0.6809; P<0.0350) in patients with VITT. Platelet-neutrophil mixture added with VITT plasma resulted in higher NETs formation, soluble TF and thrombin generation, and platelet-dependent thrombus growth under laminar flow compared with IS and deep vein thrombosis plasma; these effects were blunted by PAD4 (protein arginine deiminase 4) and cathepsin G inhibitors, anti-FcγRIIa (Fc receptor for IgG class IIa), and high doses of heparin. Immunohistochemistry analysis showed a more marked expression of PAD4 along with more diffuse neutrophil infiltration and NETs formation as well as TF and cathepsin expression in VITT thrombus compared with thrombi from patients with IS. CONCLUSIONS: Patients with VITT display enhanced thrombogenesis by PAD4-mediated NETs formation via cathepsin G-mediated platelet/neutrophil interaction.

Endothelial dysfunction, oxidative stress and low-grade endotoxemia in COVID-19 patients hospitalised in medical wards.

BACKGROUND: Endothelial dysfunction, assessed by flow-mediated dilation (FMD), is related to poor prognosis in patients with COVID-19 pneumonia (CP). In this study, we explored the interplay among FMD, NADPH oxidase type 2 (NOX-2) and lipopolysaccharides (LPS) in hospitalised patients with CP, community acquired pneumonia (CAP) and controls (CT). METHODS: We enrolled 20 consecutive patients with CP, 20 hospitalised patients with CAP and 20 CT matched for sex, age, and main cardiovascular risk factors. In all subjects we performed FMD and collected blood samples to analyse markers of oxidative stress (soluble Nox2-derived peptide (sNOX2-dp), hydrogen peroxide breakdown activity (HBA), nitric oxide (NO), hydrogen peroxide (H2O2)), inflammation (TNF-α and IL-6), LPS and zonulin levels. RESULTS: Compared with controls, CP had significant higher values of LPS, sNOX-2-dp, H2O2,TNF-α, IL-6 and zonulin; conversely FMD, HBA and NO bioavailability were significantly lower in CP. Compared to CAP patients, CP had significantly higher levels of sNOX2-dp, H2O2, TNF-α, IL-6, LPS, zonulin and lower HBA. Simple linear regression analysis showed that FMD inversely correlated with sNOX2-dp, H2O2, TNF-α, IL-6, LPS and zonulin; conversely FMD was directly correlated with NO bioavailability and HBA. Multiple linear regression analysis highlighted LPS as the only predictor of FMD. CONCLUSION: This study shows that patients with COVID-19 have low-grade endotoxemia that could activate NOX-2, generating increased oxidative stress and endothelial dysfunction.

Gut dysbiosis-derived low-grade endotoxemia: A common soil for liver and cardiovascular disease.

Gut dysbiosis is characterized by bacteria overgrowth that ultimately leads to increased intestinal barrier permeability and bacteria or bacteria product translocation such as lipopolysaccharide (LPS) in the portal and eventually systemic circulation. Intestinal epithelial cells and hepatocytes encompass enzymatic armamentarium to counteract the LPS toxic effect, however impaired degradation results in LPS accumulation in hepatocytes and endothelial wall. Experimental and clinical study documented that in patients with liver disease, such as non-alcoholic fatty acid liver disease (NAFLD), low-grade endotoxemia by LPS is implicated in liver inflammation and thrombosis via interaction with its Toll-like receptor 4 (TLR4) expressed by hepatocytes and platelets. Furthermore, studies in patients with severe atherosclerosis documented that LPS localizes into atherosclerotic plaque in close association with activated macrophages expressing TLR4 suggesting a role for LPS in vascular inflammation, atherosclerotic progression, and thrombosis. Finally, LPS may directly interact with myocardial cells to induce electric and functional changes leading to atrial fibrillation or heart failure. This review will focus on experimental and clinical evidence suggesting low-grade endotoxemia as mechanism potentially accounting for vascular damage occurring at level of hepatic and systemic circulation and myocardial cells.

Low Grade Endotoxemia and Oxidative Stress in Offspring of Patients with Early Myocardial Infarction.

Background and aims: Offspring of patients with early myocardial infarction are at higher cardiovascular risk, but the underlying physio-pathological mechanism is unclear. NADPH oxidase-type 2 (NOX-2) plays a pivotal role as mediator of oxidative stress and could be involved in activating platelets in these patients. Furthermore, altered intestinal permeability and serum lipopolysaccharide (LPS) could be a trigger to promote NOX-2 activation and platelet aggregation. This study aims to evaluate the behavior of low grade endotoxemia, oxidative stress and platelet activation in offspring of patients with early myocardial infarction. Methods: We enrolled, in a cross-sectional study, 46 offspring of patients with early myocardial infarction and 86 healthy subjects (HS). LPS levels and gut permeability (assessed by zonulin), oxidative stress (assessed by serum NOX-2-derived peptide (sNOX2-dp) release, hydrogen peroxide (H2O2) production and isoprostanes), serum nitric oxide (NO) bioavailability and platelet activation (by serum thromboxane B2 (TXB2) and soluble P-Selectin (sP-Selectin)) were analyzed. Results: Compared to HS, offspring of patients with early myocardial infarction had higher values of LPS, zonulin, serum isoprostanes, sNOX2-dp H2O2, TXB2, p-selectin and lower NO bioavailability. Logistic regression analysis showed that the variables associated with offspring of patients with early myocardial infarction were LPS, TXB2 and isoprostanes. The multiple linear regression analysis confirmed that serum NOX-2, isoprostanes, p-selectin and H2O2 levels were significantly associated to LPS. Furthermore, serum LPS, isoprostanes and TXB2 levels were significantly associated with sNOX-2-dp. Conclusions: Offspring of patients with early myocardial infarction have a low grade endotoxemia that could generate oxidative stress and platelet activation increasing their cardiovascular risk. Future studies are needed to understand the role of dysbiosis in this population.

Association of proprotein convertase subtilisin/kexin type 9 (PCSK9) levels with abnormally high ankle-brachial index in atrial fibrillation.

BACKGROUND: High ankle-brachial index (ABI) has been associated with increased risk of worse outcomes in the general population. Few data on atrial fibrillation (AF) exist. Experimental data suggest that proprotein convertase subtilisin/kexin type 9 (PCSK9) contributes to vascular calcification but clinical data on this association are lacking. AIMS: We wanted to investigate the relationship between circulating PCSK9 levels and an abnormally high ABI in patients suffering from AF. METHODS: We analyzed data from 579 patients included in the prospective ATHERO-AF study. An ABI≥1.4 was considered high. PCSK9 levels were measured coincidentally with ABI measurement. We used optimized cut-offs of PCSK9 for both ABI and mortality obtained from Receiver Operator Characteristic (ROC) curve analysis. All-cause mortality according to the ABI value was also analyzed. RESULTS: One hundred and fifteen patients (19.9%) had an ABI ≥1.4. The mean (standard deviation [SD]) age was 72.1 (7.6) years, and 42.1% of patients were women. Patients with ABI ≥1.4 were older, more frequently male, and diabetic. Multivariable logistic regression analysis showed an association between ABI ≥1.4 and serum levels of PCSK9 > 1150 pg/ml (odds ratio [OR], 1.649; 95% confidence interval [CI], 1.047-2.598; P = 0.031). During a median follow-up of 41 months, 113 deaths occurred. In multivariable Cox regression analysis, an ABI ≥1.4 (hazard ratio [HR], 1.626; 95% CI, 1.024-2.582; P = 0.039), CHA2DS2-VASc score (HR, 1.249; 95% CI, 1.088-1.434; P = 0.002), antiplatelet drug use (HR, 1.775; 95% CI, 1.153-2.733; P = 0.009), and PCSK9 > 2060 pg/ml (HR, 2.200; 95% CI, 1.437-3.369; P < 0.001) were associated with all-cause death. CONCLUSIONS: In AF patients, PCSK9 levels relate to an abnormally high ABI ≥1.4. Our data suggest PCSK9 role in contributing to vascular calcification in AF patients.

Structure, Activation, and Regulation of NOX2: At the Crossroad between the Innate Immunity and Oxidative Stress-Mediated Pathologies.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is a multisubunit enzyme complex that participates in the generation of superoxide or hydrogen peroxide (H2O2) and plays a key role in several biological functions. Among seven known NOX isoforms, NOX2 was the first identified in phagocytes but is also expressed in several other cell types including endothelial cells, platelets, microglia, neurons, and muscle cells. NOX2 has been assigned multiple roles in regulating many aspects of innate and adaptive immunity, and human and mouse models of NOX2 genetic deletion highlighted this key role. On the other side, NOX2 hyperactivation is involved in the pathogenesis of several diseases with different etiologies but all are characterized by an increase in oxidative stress and inflammatory process. From this point of view, the modulation of NOX2 represents an important therapeutic strategy aimed at reducing the damage associated with its hyperactivation. Although pharmacological strategies to selectively modulate NOX2 are implemented thanks to new biotechnologies, this field of research remains to be explored. Therefore, in this review, we analyzed the role of NOX2 at the crossroads between immunity and pathologies mediated by its hyperactivation. We described (1) the mechanisms of activation and regulation, (2) human, mouse, and cellular models studied to understand the role of NOX2 as an enzyme of innate immunity, (3) some of the pathologies associated with its hyperactivation, and (4) the inhibitory strategies, with reference to the most recent discoveries.

Natural Activators of Autophagy Reduce Oxidative Stress and Muscle Injury Biomarkers in Endurance Athletes: A Pilot Study.

BACKGROUND: Oxidative stress and impaired autophagy are directly and indirectly implicated in exercise-mediated muscle injury. Trehalose, spermidine, nicotinamide, and polyphenols possess pro-autophagic and antioxidant properties, and could therefore reduce exercise-induced damage to skeletal muscle. The aim of this study was to investigate whether a mixture of these compounds was able to improve muscle injury biomarkers in endurance athletes through the modulation of oxidative stress and autophagic machinery. METHODS AND RESULTS: sNOX2-dp; H2O2 production; H2O2 breakdown activity (HBA); ATG5 and p62 levels, both markers of autophagic process; and muscle injury biomarkers were evaluated in five endurance athletes who were allocated in a crossover design study to daily administration of 10.5 g of an experimental mixture or no treatment, with evaluations conducted at baseline and after 30 days of mixture consumption. Compared to baseline, the mixture intake led to a remarkable reduction of oxidative stress and positively modulated autophagy. Finally, after the 30-day supplementation period, a significant decrease in muscle injury biomarkers was found. CONCLUSION: Supplementation with this mixture positively affected redox state and autophagy and improved muscle injury biomarkers in athletes, allowing for better muscle recovery. Moreover, it is speculated that this mixture could also benefit patients suffering from muscle injuries, such as cancer or cardiovascular patients, or elderly subjects.

Toll-Like Receptor 4-Dependent Platelet-Related Thrombosis in SARS-CoV-2 Infection.

BACKGROUND: SARS-CoV-2 is associated with an increased risk of venous and arterial thrombosis, but the underlying mechanism is still unclear. METHODS: We performed a cross-sectional analysis of platelet function in 25 SARS-CoV-2 and 10 healthy subjects by measuring Nox2 (NADPH oxidase 2)-derived oxidative stress and thromboxane B2, and investigated if administration of monoclonal antibodies against the S protein (Spike protein) of SARS-CoV-2 affects platelet activation. Furthermore, we investigated in vitro if the S protein of SARS-CoV-2 or plasma from SARS-CoV-2 enhanced platelet activation. RESULTS: Ex vivo studies showed enhanced platelet Nox2-derived oxidative stress and thromboxane B2 biosynthesis and under laminar flow platelet-dependent thrombus growth in SARS-CoV-2 compared with controls; both effects were lowered by Nox2 and TLR4 (Toll-like receptor 4) inhibitors. Two hours after administration of monoclonal antibodies, a significant inhibition of platelet activation was observed in patients with SARS-CoV-2 compared with untreated ones. In vitro study showed that S protein per se did not elicit platelet activation but amplified the platelet response to subthreshold concentrations of agonists and functionally interacted with platelet TLR4. A docking simulation analysis suggested that TLR4 binds to S protein via three receptor-binding domains; furthermore, immunoprecipitation and immunofluorescence showed S protein-TLR4 colocalization in platelets from SARS-CoV-2. Plasma from patients with SARS-CoV-2 enhanced platelet activation and Nox2-related oxidative stress, an effect blunted by TNF (tumor necrosis factor) α inhibitor; this effect was recapitulated by an in vitro study documenting that TNFα alone promoted platelet activation and amplified the platelet response to S protein via p47phox (phagocyte oxidase) upregulation. CONCLUSIONS: The study identifies 2 TLR4-dependent and independent pathways promoting platelet-dependent thrombus growth and suggests inhibition of TLR4. or p47phox as a tool to counteract thrombosis in SARS-CoV-2.

Gut-derived low-grade endotoxaemia, atherothrombosis and cardiovascular disease.

Systemic inflammation has been suggested to have a pivotal role in atherothrombosis, but the factors that trigger systemic inflammation have not been fully elucidated. Lipopolysaccharide (LPS) is a component of the membrane of Gram-negative bacteria present in the gut that can translocate into the systemic circulation, causing non-septic, low-grade endotoxaemia. Gut dysbiosis is a major determinant of low-grade endotoxaemia via dysfunction of the intestinal barrier scaffold, which is a prerequisite for LPS translocation into the systemic circulation. Experimental studies have demonstrated that LPS is present in atherosclerotic arteries but not in normal arteries. In atherosclerotic plaques, LPS promotes a pro-inflammatory status that can lead to plaque instability and thrombus formation. Low-grade endotoxaemia affects several cell types, including leukocytes, platelets and endothelial cells, leading to inflammation and clot formation. Low-grade endotoxaemia has been described in patients at risk of or with overt cardiovascular disease, in whom low-grade endotoxaemia was associated with atherosclerotic burden and its clinical sequelae. In this Review, we describe the mechanisms favouring the development of low-grade endotoxaemia, focusing on gut dysbiosis and changes in gut permeability; the plausible biological mechanisms linking low-grade endotoxaemia and atherothrombosis; the clinical studies suggesting that low-grade endotoxaemia is a risk factor for cardiovascular events; and the potential therapeutic tools to improve gut permeability and eventually eliminate low-grade endotoxaemia.

The Sodium-Glucose Co-Transporter-2 (SGLT2) Inhibitors Reduce Platelet Activation and Thrombus Formation by Lowering NOX2-Related Oxidative Stress: A Pilot Study.

Sodium−glucose co-transporter-2 inhibitors or gliflozins, the newest anti-hyperglycemic class, induce cardioprotective benefits in patients with type 2 diabetes (T2D). As platelet activation and oxidative stress play a key role in atherothrombotic-related complications, we hypothesized that gliflozins might modulate oxidative stress, platelet activation and thrombus formation. We performed an interventional open-label single-arm before-after study in 32 T2D patients on top of their ongoing metformin therapy. The population was divided into two groups: treatment with GLP-1 receptor agonists (GLP-1RA, Group A) and gliflozins (Group B). Oxidative stress, platelet activation and thrombus growth were assessed before and after 15 days of treatment. Compared to the baseline, gliflozins treatment significantly decreased sNOX2-dp (−45.2%, p < 0.001), H2O2 production (−53.4%, p < 0.001), TxB2 (−33.1%, p < 0.001), sP-selectin (−49.3%, p < 0.001) and sCD40L levels (−62.3%, p < 0.001) as well as thrombus formation (−32%, p < 0.001), whereas it potentiated anti-oxidant power (HBA, +30.8%, p < 0.001). Moreover, a significant difference in oxidative stress, platelet activation and thrombus formation across groups A and B was found. In addition, an in vitro study on stimulated platelets treated with gliflozins (10−30 µM) showed a reduction in oxidative stress, platelet activation and thrombus growth. Our results showed that gliflozins have antiplatelet and antithrombic activity related to an NOX2 down-regulation, suggesting a new mechanism responsible for cardiovascular protection.

Natural Activators of Autophagy Increase Maximal Walking Distance and Reduce Oxidative Stress in Patients with Peripheral Artery Disease: A Pilot Study.

Trehalose, spermidine, nicotinamide, and polyphenols have been shown to display pro-autophagic and antioxidant properties, eventually reducing cardiovascular and ischemic complications. This study aimed to investigate whether a mixture of these components improves maximal walking distance (MWD) in peripheral artery disease (PAD) patients. Nitrite/nitrate (NOx), endothelin-1, sNOX2-dp, H2O2 production, H2O2 break-down activity (HBA), ATG5 and P62 levels, flow-mediated dilation (FMD), and MWD were evaluated in 20 PAD patients randomly allocated to 10.5 g of mixture or no-treatment in a single-blind study. The above variables were assessed at baseline and 60 days after mixture ingestion. Compared with baseline, mixture intake significantly increased MWD (+91%; p < 0.01) and serum NOx (+96%; p < 0.001), whereas it significantly reduced endothelin-1 levels (−30%, p < 0.01). Moreover, mixture intake led to a remarkable reduction in sNOX2dp (−31%, p < 0.05) and H2O2 (−40%, p < 0.001) and potentiated antioxidant power (+110%, p < 0.001). Finally, mixture ingestion restored autophagy by increasing ATG5 (+43%, p < 0.01) and decreasing P62 (−29%, p < 0.05). No changes in the above-mentioned variables were observed in the no-treatment group. The treatment with a mixture of trehalose, spermidine, nicotinamide, and polyphenols improves MWD in PAD patients, with a mechanism possibly related to NOX2-mediated oxidative stress downregulation and autophagic flux upregulation. Clinical Trial Registration unique identifier: NCT04061070.

Sex-Related Differences in Oxidative, Platelet, and Vascular Function in Chronic Users of Heat-not-Burn vs. Traditional Combustion Cigarettes.

Smoking is still a major cardiovascular risk factor, despite many public awareness campaigns and dedicated interventions. Recently, modified risk products (MRP), e.g., heat-not-burn cigarettes (HNBCs), have been introduced as surrogates of traditional combustion cigarettes (TCCs). Although these products are promoted as healthier than TCCs, few studies have been conducted to assess it. This work is a sex-focused sub-study of a prospective observational study in which apparently healthy chronic TCC smokers were age-matched with regular HNBC users. Blood samples were collected for biochemical assays and blood pressure and flow-mediated dilation (FMD) were measured. Out of 60 subjects, 33 (55%) were women, and 27 (45%) men, with 11 (33%) vs. 9 (33%) non-smokers, respectively, 10 (30%) vs. 10 (37%) TCC smokers, and 12 (36%) vs. 8 (30%) HNBC smokers (p = 0.946). Bivariate and multivariable analyses showed no statistically significant between-sex differences in NO, H2O2, sCD40L, sNox2-dp, sP-selectin, platelet aggregation, cotinine or FMD, overall, in non-smokers, in TCC smokers, or in HNBC smokers (all p > 0.05). HNBCs appeared safer than TCCs when focusing on Nox2-dp (p = 0.026) and sP-selectin (p = 0.050) but had similar levels of the other measured markers. In conclusion, HNBCs have similar detrimental effects on women and men's oxidative stress (H2O2: p = 0.49; sNox2-dp: p = 0.31) and platelet activation (sP-selectin: p = 0.33; platelet aggregation p = 0.87).

Proprotein convertase subtilisin/kexin type 9 (PCSK9) levels in primary antiphospholipid syndrome. The multicenter ATHERO-APS study.

BACKGROUND: The proprotein convertase subtilisin/kexin type 9 (PCSK9) is emerging as a novel cardiovascular risk factor. Levels of PCSK9 in thrombotic primary antiphospholipid syndrome (PAPS) have never been investigated. METHODS: Cross sectional comparison of baseline characteristics of 91 PAPS patients enrolled in the multicenter prospective ATHERO-APS cohort study. PCSK9 levels were categorized into tertiles and the association with arterial and recurrent thrombosis were assessed by univariable and multivariable logistic regression analysis. RESULTS: Median age was 51 years and 71.4% (n = 65) were women. Overall, 33% (n = 30) experienced an arterial event while 31% (n = 28) had recurrent thrombotic events. Median PCSK9 levels were 1243 (1100-1650) pg/ml. Patients in the third PCSK9 tertile (>1458 pg/ml) showed a higher prevalence of dyslipidemia, lupus anticoagulant positivity and a history of previous arterial and recurrent thrombosis than patients in the first and second tertile. PCSK9 levels were higher in arterial than venous thrombosis (1502 vs. 1180 pg/ml, p = 0.002), and in patients with recurrent vs isolated thrombosis (1680 vs. 1150 pg/m, p < 0.001). High plasma PCSK9 levels were associated with a 4-fold increase risk for arterial events and with a 10-fold increase risk for recurrent thrombosis after adjustment for confounding factors. CONCLUSION: These preliminary data suggest that PCSK9 levels are increased in PAPS patients with arterial and recurrent thrombosis. Its role as a possible therapeutic target in PAPS needs further studies.

Low-grade endotoxemia and NOX2 in patients with coronary microvascular angina.

BACKGROUND: Endothelial dysfunction and oxidative stress were hypothesized to be involved in the pathogenesis of coronary microvascular angina (MVA). NADPH oxidase-2 (NOX2) activation could provoke increased oxidative stress and endothelial dysfunction, but data on MVA have not been provided yet. AIMS: This study aimed to evaluate the interaction among NOX2 activation, serum lipopolysaccharide (LPS) levels, as well as oxidative stress production as potential causes of endothelial dysfunction in MVA patients. METHODS: In this study, we wanted to compare serum levels of soluble NOX2-dp (sNOX2-dp), H2O2 production, hydrogen peroxide breakdown activity (HBA), nitric oxide (NO) bioavailability, endothelin 1 (ET-1), serum zonulin (as intestinal permeability assay), and LPS in 80 consecutive subjects, including 40 MVA patients and 40 controls (CT), matched for age and sex. RESULTS: Compared with CT, MVA patients had significantly higher values of sNOX2-dp, H2O2, ET-1, LPS, and zonulin. Conversely HBA and NO bioavailability were significantly lower in MVA patients. Simple linear regression analysis showed that sNOX2 was associated with serum LPS, serum zonulin, H2O2, and ET-1. Furthermore, an inverse correlation between sNOX2, HBA, and nitric oxide bioavailability was observed. Multiple linear regression analysis showed that LPS and zonulin emerged as the only independent predictive variables associated with sNOX2. CONCLUSIONS: This study provides the first report attesting that patients with MVA have high LPS levels, NOX2 activation, and an imbalance between pro-oxidant and antioxidant systems, in favor of the oxidizing molecules that could be potentially implicated in the endothelial dysfunction and vasoconstriction of this disease.