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Importance: Here, we demonstrate that the direct binding of p53 on the IL-18 promoter region regulates its gene expression. However, the presence of E6 and E7 from human papillomavirus type 38 impairs this mechanism via a new inhibitory complex formed by DNA methyltransferase 1 (DNMT1)/PKR/ΔNp73α, which binds to the region formerly occupied by p53 in primary keratinocytes., Competing Interests: The authors declare no conflict of interest.

Emerging evidence highlights the potential prognostic relevance of circulating lipids in metastatic castration-resistant prostate cancer (mCRPC), with a proposed 3-lipid signature. This study aims to analyze the lipidomic profiles of individuals with mCRPC to identify lipid species that could serve as predictive indicators of prognosis and therapeutic response. Plasma samples were collected from mCRPC patients initiating first-line treatment (1 L) (n = 29) and those previously treated with at least two lines of therapy (> 2 L) (n = 19), including an androgen-receptor signaling inhibitor and a taxane. Employing an untargeted lipidomic approach, lipids were extracted from the plasma samples and subjected to analysis. A comprehensive identification and quantification of 789 plasma lipids was achieved. Notably, 75 species displayed significant dysregulation in > 2 L patients in comparison to the 1 L group. Among these, 63 species exhibited elevated levels, while 12 were reduced. Patients included in > 2 L cohort showed elevated levels of acylcarnitines (CAR), diacylglycerols (DG), phosphatidylethanolamines (PE), triacylglycerols (TG), and ceramides (Cer). Notably, some upregulated lipids, including CAR 14:0, CAR 24:1, Cer d18:1/16:0, Cer d18:1/18:0 (C18 Cer), Cer d18:2/18:0, Cer d18:1/24:1, and Cer d20:1/24:1, showed significant associations with overall survival (OS) in univariate models. Specifically, increased levels of C18 Cer remained significantly associated with poorer OS in the multivariate model, even after adjusting for treatment line and PSA levels (Hazard Ratio: 3.59 [95% Confidence Interval 1.51-8.52], p = 0.004). Employing quantitative mass spectrometry, our findings underscore the independent prognostic significance of C18 Cer in individuals with mCRPC. This discovery opens avenues for further studies within this field., (© 2023. Springer Nature Limited.)

Introduction: Arterial thrombosis is the main underlying mechanism of acute atherothrombosis. Combined antiplatelet and anticoagulant regimens prevent thrombosis but increase bleeding rates. Mast cell-derived heparin proteoglycans have local antithrombotic properties, and their semisynthetic dual AntiPlatelet and AntiCoagulant (APAC) mimetic may provide a new efficacious and safe tool for arterial thrombosis. We investigated the in vivo impact of intravenous APAC (0.3-0.5 mg/kg; doses chosen according to pharmacokinetic studies) in two mouse models of arterial thrombosis and the in vitro actions in mouse platelets and plasma., Materials and Methods: Platelet function and coagulation were studied with light transmission aggregometry and clotting times. Carotid arterial thrombosis was induced either by photochemical injury or surgically exposing vascular collagen after infusion of APAC, UFH or vehicle. Time to occlusion, targeting of APAC to the vascular injury site and platelet deposition on these sites were assessed by intra-vital imaging. Tissue factor activity (TF) of the carotid artery and in plasma was captured., Results: APAC inhibited platelet responsiveness to agonist stimulation (collagen and ADP) and prolonged APTT and thrombin time. After photochemical carotid injury, APAC-treatment prolonged times to occlusion in comparison with UFH or vehicle, and decreased TF both in carotid lysates and plasma. Upon binding from circulation to vascular collagen-exposing injury sites, APAC reduced the in situ platelet deposition., Conclusions: Intravenous APAC targets arterial injury sites to exert local dual antiplatelet and anticoagulant actions and attenuates thrombosis upon carotid injuries in mice. Systemic APAC provides local efficacy, highlighting APAC as a novel antithrombotic to reduce cardiovascular complications., Competing Interests: Declaration of competing interest The corresponding and shared last author of the manuscript, Riitta Lassila has worked with this concept for 15 years and is the CSO of the Aplagon OY. A. Jouppila and B. C. Cooley received research funding from Aplagon Ltd. L. Liberale, and G. G. Camici are coinventors on the International Patent WO/2020/226993 filed in April 2020. The patent relates to the use of antibodies which specifically bind IL-1α to reduce various sequelae of ischemia-reperfusion injury to the central nervous system. G. G. Camici is scientific consultant to Sovida. N. R. Bonetti; S.S. Saeedi Saravi; L. Pasterk; S. Gobbato; T. F. Lüscher and J. H. Beer have no conflicts of interest to declare., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Sickle cell disease (SCD) is a genetic hemoglobinopathy associated with high morbidity and mortality. The primary cause of hospitalization in SCD is vaso-occlusive crisis (VOC), mediated by alteration of red blood cells, platelets, immune cells and a pro-adhesive endothelium., Objectives: We investigated the potential therapeutic use of the plant-derived omega-3 alpha-linolenic acid (ALA) in SCD., Methods: Berkeley mice were fed a low- or high-ALA diet for 4 weeks, followed by analysis of liver fibrosis, endothelial activation, platelet activation and formation of platelet-neutrophils aggregates. Aggregation of platelets over collagen under flow after high-ALA was compared to a blocking P-selectin Fab., Results: Dietary high-ALA was able to reduce the number of sickle cells in blood smear, liver fibrosis, and the expression of adhesion molecules on the endothelium of aorta, lungs, liver and kidneys (VCAM-1, ICAM-1 and vWF). Specific parameters of platelet activation were blunted after high-ALA feeding, notably P-selectin exposure and the formation of neutrophil-platelet aggregates, along with a correspondingly reduced expression of PSGL-1 on neutrophils. By comparison, in vivo treatment of SCD mice with the anti-P-selectin Fab was able to similarly reduce the formation of neutrophil-platelet aggregates, but did not reduce GpIbα shedding nor the activation of the α IIb β 3 integrin in response to thrombin. Both ALA feeding and P-selectin blocking significantly reduced collagen-mediated cell adhesion under flow., Conclusions: Dietary ALA is able to reduce the pro-inflammatory and pro-thrombotic state occurring in the SCD mouse model and may represent a novel, inexpensive and readily available therapeutic strategy for SCD., (© 2021 International Society on Thrombosis and Haemostasis.)

Aging is a major risk factor for cardiovascular diseases, including thrombotic events. The gut microbiota has been implicated in the development of thrombotic risk. Plant-derived omega-3 fatty acid ɑ-linolenic acid (ALA) confers beneficial anti-platelet and anti-inflammatory effects. Hence, antithrombotic activity elicited by ALA may be partly dependent on its interaction with gut microbiota during aging. Here, we demonstrate that lifelong dietary ALA decreases platelet hyperresponsiveness and thrombus formation in aged mice. These phenotypic changes can be partly attributed to alteration of microbial composition and reduction of its metabolite trimethylamine N-oxide and inflammatory mediators including TNF-α, as well as the upregulated production of short-chain fatty acid acetate. ALA-rich diet also dampens secretion of increased procoagulant factors, tissue factor and plasminogen activator inhibitor-1, in aged mice. Our results suggest long-term ALA supplementation as an attractive, accessible, and well-tolerated nutritional strategy against age-associated platelet hyperreactivity and thrombotic potential., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Background and Aims: Early revascularization -the gold standard therapy for ischemic stroke- is often withheld in the elderly population due to high risk of complications. Thus, safe and effective preventive and therapeutic options are needed. The plant-derived omega-3-fatty-acid alpha-linolenic-acid (ALA) has emerged as a novel cardiovascular-protective agent. As of yet, little is known about its potential therapeutic effects on stroke. We hereby aimed to investigate the impact of a clinically relevant long-term dietary intervention with ALA on stroke outcome., Methods: Six month-old C57BL/6 wildtype males were either fed an ALA-rich (high ALA) or a control diet (low ALA) for 12 months. At 18 months, brain ischemia/reperfusion was induced by transient middle cerebral artery occlusion (tMCAO). Stroke size and neurological function were assessed. Functional blood-brain-barrier-(BBB) permeability and protein expression were assessed by immunohistochemistry. Baseline inflammatory markers were measured at 18 months., Results: High ALA-fed animals displayed decreased circulating TNF-α levels and Neutrophil-to-Lymphocyte Ratios at 18 months. Stroke size and neurological dysfunction were significantly reduced in high ALA-fed animals. Coherently to the reduced stroke size, functional BBB integrity and occludin endothelial expression were maintained by high ALA supplementation. Additionally, ALA reduced endothelial activation and thus recruitment and activation of macrophages and resident microglia. Finally, high ALA diet reduced the expression of BBB-degrading and neurotoxic MMP-3 and MMP-9., Conclusions: We demonstrate the beneficial effects of a clinically relevant and feasible dietary intervention with a safe and readily available compound in the setting of stroke. The protective effects observed with ALA supplementation may relate to blunting of inflammation and might pave the way for novel stroke treatments., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Platelet adhesion to the sub-endothelial matrix and damaged endothelium occurs through a multi-step process mediated in the initial phase by glycoprotein Ib binding to von Willebrand factor (vWF), which leads to the subsequent formation of a platelet plug. The plant-derived ω-3 fatty acid α-linolenic acid is an abundant alternative to fish-derived n-3 fatty acids and has anti-inflammatory and antithrombotic properties. In this study, we investigated the impact of α-linolenic acid on human platelet binding to vWF under high-shear flow conditions (mimicking blood flow in stenosed arteries). Pre-incubation of fresh human blood from healthy donors with α-linolenic acid at dietary relevant concentrations reduced platelet binding and rolling on vWF-coated microchannels at a shear rate of 100 dyn/cm 2 Depletion of membrane cholesterol by incubation of platelet-rich plasma with methyl-β cyclodextrin abrogated platelet rolling on vWF. Analysis of glycoprotein Ib by applying cryo-electron tomography to intact platelets revealed local clusters of glycoprotein Ib complexes upon exposure to shear force: the formation of these complexes could be prevented by treatment with α-linolenic acid. This study provides novel findings on the rapid local rearrangement of glycoprotein Ib complexes in response to high-shear flow and highlights the mechanism of in vitro inhibition of platelet binding to and rolling on vWF by α-linolenic acid., (Copyright© 2020 Ferrata Storti Foundation.)

Introduction: Gut microbiota-dependent trimethylamine-N-oxide (TMAO) correlates with arterial thrombotic events including myocardial infarction and stroke, and mortality. However, the association of TMAO with recurrent venous thromboembolism (VTE) and mortality remains unknown., Methods: TMAO plasma levels were assessed by high performance liquid chromatography in 859 patients aged ≥65 years with acute VTE and categorized into low (<2.28 μmol/L), medium (2.28-6.57 μmol/L), and high levels (>6.57 μmol/L) based on the 25th and 75th percentile. Associations of TMAO with recurrent VTE, major or non-major bleeding, and mortality were investigated., Results: During a mean follow-up of 28 months, 106 patients developed recurrent VTE, 259 had major or non-major bleeding events, and 179 patients died. The risk of recurrent VTE did not differ significantly between patients with low, medium (adjusted subhazard ratio [SHR], 1.38; 95% confidence interval [CI], 0.81 to 2.36; p = 0.232) and high TMAO levels (SHR, 1.44; 95% CI, 0.80 to 2.58, p = 0.221). Compared with low TMAO levels, the adjusted hazard ratio [HR] for mortality was 0.68 (95% CI, 0.47 to 0.98, p = 0.039) in patients with medium TMAO levels and 1.02 (95% CI, 0.68 to 1.52, p = 0.922) in patients with high TMAO levels. Fractional polynomial Cox-regression confirmed a U-shaped association (adjusted p = 0.045), with the lowest mortality risk in patients with TMAO around 4 μmol/L. TMAO was not associated with major or non-major bleeding., Conclusion: TMAO showed a U-shaped association with mortality in elderly patients with acute VTE and was not associated with recurrent VTE and major or non-major bleeding., (Copyright © 2018. Published by Elsevier Ltd.)

Background: Omega-3 fatty acids (n - 3 FA) may have blood pressure (BP)-lowering effects in untreated hypertensive and elderly patients. The effect of n - 3 FA on BP in young, healthy adults remains unknown. The Omega-3 Index reliably reflects an individuals' omega-3 status. We hypothesized that the Omega-3 Index is inversely associated with BP levels in young healthy adults., Methods: The current study (n = 2036) is a cross-sectional study investigating the baseline characteristics of a cohort, which includes healthy adults, age 25-41 years. Individuals with cardiovascular disease, known diabetes or a BMI higher than 35 kg/m were excluded. The Omega-3 Index was determined in whole blood using gas chromatography. Association with office and 24-h BP was assessed using multivariable linear regression models adjusted for potential confounders., Results: Median Omega-3 Index was 4.58% (interquartile range 4.08; 5.25). Compared with individuals in the lowest Omega-3 Index quartile, individuals in the highest had a SBP and DBP that was 4 and 2 mmHg lower, respectively (P < 0.01). A significant linear inverse relationship of the Omega-3 Index with 24-h and office BP was observed. Per 1-U increase in log-transformed Omega-3 Index the lowering in BP (given as multivariable adjusted β coefficients; 95% confidence interval) was -2.67 mmHg (-4.83; -0.51; P = 0.02) and -2.30 mmHg (-3.92; -0.68; P = 0.005) for 24-h SBP and DBP, respectively., Conclusion: A higher Omega-3 Index is associated with statistically significant, clinically relevant lower SBP and DBP levels in normotensive young and healthy individuals. Diets rich in n - 3 FA may be a strategy for primary prevention of hypertension.

Amotosalen and ultraviolet A (UVA) photochemical-based pathogen reduction using the Intercept™ Blood System (IBS) is an effective and established technology for platelet and plasma components, which is adopted in more than 40 countries worldwide. Several reports point towards a reduced platelet function after Amotosalen/UVA exposure. The study herein was undertaken to identify the mechanisms responsible for the early impairment of platelet function by the IBS. Twenty-five platelet apheresis units were collected from healthy volunteers following standard procedures and split into 2 components, 1 untreated and the other treated with Amotosalen/UVA. Platelet impedance aggregation in response to collagen and thrombin was reduced by 80% and 60%, respectively, in IBS-treated units at day 1 of storage. Glycoprotein Ib (GpIb) levels were significantly lower in IBS samples and soluble glycocalicin correspondingly augmented; furthermore, GpIbα was significantly more desialylated as shown by Erythrina Cristagalli Lectin (ECL) binding. The pro-apoptotic Bak protein was significantly increased, as well as the MAPK p38 phosphorylation and caspase-3 cleavage. Stored IBS-treated platelets injected into immune-deficient nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice showed a faster clearance. We conclude that the IBS induces platelet p38 activation, GpIb shedding and platelet apoptosis through a caspase-dependent mechanism, thus reducing platelet function and survival. These mechanisms are of relevance in transfusion medicine, where the IBS increases patient safety at the expense of platelet function and survival., (Copyright© 2017 Ferrata Storti Foundation.)

The aim of the present study was to investigate the methylation status in the promoter region of Dipeptidyl peptidase-IV (Dpp4) gene, in livers from obese Zucker rats with different patterns of immunohistochemical positivity. Molecular analysis was carried-out on DNA obtained from livers of obese and lean Zucker rats and of control Wistar rats using the bisulfite conversion method and DNA sequencing. Our study focused on the genomic region of 1,000 bp, which includes the final part of 680 bp of the Dpp4 gene promoter and a small stretch of 320 bp at the beginning of the gene. The results indicate that the different immunohistochemical pattern of DPP4 observed in obese (fa/fa) and lean (fa/-) Zucker rats is not correlated to DNA methylation of its promoter. This is in agreement with the results of other studies carried-out on visceral and subcutaneous adipose tissue with varying levels of enzyme expression, in which differences in the methylation pattern of the Dpp4 promoter region were not observed., (Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)