Constitutive genetic deletion of the adaptor protein p66^Shc was shown to protect from ischaemia/reperfusion injury. Here, we aimed at understanding the molecular mechanisms underlying this effect in stroke and studied p66^Shc gene regulation in human ischaemic stroke. Ischaemia/reperfusion brain injury was induced by performing a transient middle cerebral artery occlusion surgery on wild-type mice. After the ischaemic episode and upon reperfusion, small interfering RNA targeting p66^Shc was injected intravenously. We observed that post-ischaemic p66^Shc knockdown preserved blood–brain barrier integrity that resulted in improved stroke outcome, as identified by smaller lesion volumes, decreased neurological deficits, and increased survival. Experiments on primary human brain microvascular endothelial cells demonstrated that silencing of the adaptor protein p66^Shc preserves claudin-5 protein levels during hypoxia/reoxygenation by reducing nicotinamide adenine dinucleotide phosphate oxidase activity and reactive oxygen species production. Further, we found that in peripheral blood monocytes of acute ischaemic stroke patients p66^Shc gene expression is transiently increased and that this increase correlates with short-term neurological outcome. Post-ischaemic silencing of p66^Shc upon reperfusion improves stroke outcome in mice while the expression of p66^Shc gene correlates with short-term outcome in patients with ischaemic stroke.

Background and Purpose—: Inflammation is a major pathogenic component of ischemia/reperfusion brain injury, and as such, interventions aimed at inhibiting inflammatory mediators promise to be effective strategies in stroke therapy. JunD—a member of the AP-1 (activated protein-1) family of transcription factors—was recently shown to regulate inflammation by targeting IL (interleukin)-1β synthesis and macrophage activation. The purpose of the present study was to assess the role of JunD in ischemia/reperfusion-induced brain injury. Methods—: WT (wild type) mice randomly treated with either JunD or scramble (control) siRNA were subjected to 45 minutes of transient middle cerebral artery occlusion followed by 24 hours of reperfusion. Stroke size, neurological deficit, plasma/brain cytokines, and oxidative stress determined by 4-hydroxynonenal immunofluorescence staining were evaluated 24 hours after reperfusion. Additionally, the role of IL-1β was investigated by treating JunD siRNA mice with an anti–IL-1β monoclonal antibody on reperfusion. Finally, JunD expression was assessed in peripheral blood monocytes isolated from patients with acute ischemic stroke. Results—: In vivo JunD knockdown resulted in increased stroke size, reduced neurological function, and increased systemic inflammation, as confirmed by higher neutrophil count and lymphopenia. Brain tissue IL-1β levels were augmented in JunD siRNA mice as compared with scramble siRNA, whereas no difference was detected in IL-6, TNF-α (tumor necrosis factor-α), and 4-hydroxynonenal levels. The deleterious effects of silencing of JunD were rescued by treating mice with an anti–IL-1β antibody. In addition, JunD expression was decreased in peripheral blood monocytes of patients with acute ischemic stroke at 6 and 24 hours after onset of stroke symptoms compared with sex- and age-matched healthy controls. Conclusions—: JunD blunts ischemia/reperfusion-induced brain injury via suppression of IL-1β.

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.

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/m² 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.

Sirtuin 3 (Sirt3) is a mitochondrial, nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylase that reduces oxidative stress by activation of superoxide dismutase 2 (SOD2). Oxidative stress enhances arterial thrombosis. This study investigated the effects of genetic Sirt3 deletion on arterial thrombosis in mice in an inflammatory setting and assessed the clinical relevance of these findings in patients with ST-elevation myocardial infarction (STEMI). Using a laser-induced carotid thrombosis model with lipopolysaccharide (LPS) challenge, in vivo time to thrombotic occlusion in Sirt3^−/− mice (n = 6) was reduced by half compared to Sirt3^+/+ wild-type (n = 8, P < 0.01) controls. Ex vivo analyses of whole blood using rotational thromboelastometry revealed accelerated clot formation and increased clot stability in Sirt3^−/− compared to wild-type blood. rotational thromboelastometry of cell-depleted plasma showed accelerated clotting initiation in Sirt3^−/− mice, whereas overall clot formation and firmness remained unaffected. Ex vivo LPS-induced neutrophil extracellular trap formation was increased in Sirt3^−/− bone marrow-derived neutrophils. Plasma tissue factor (TF) levels and activity were elevated in Sirt3^−/− mice, whereas plasma levels of other coagulation factors and TF expression in arterial walls remained unchanged. SOD2 expression in bone marrow -derived Sirt3^−/− neutrophils was reduced. In STEMI patients, transcriptional levels of Sirt3 and its target SOD2 were lower in CD14⁺ leukocytes compared with healthy donors (n = 10 each, P < 0.01). Sirt3 loss-of-function enhances experimental thrombosis in vivo via an increase of neutrophil extracellular traps and elevation of TF suggesting thrombo-protective effects of endogenous Sirt3. Acute coronary thrombosis in STEMI patients is associated with lower expression levels of SIRT3 and SOD2 in CD14⁺ leukocytes. Therefore, enhancing SIRT3 activity by pan-sirtuin activating NAD⁺-boosters may provide a novel therapeutic target to prevent or treat thrombotic arterial occlusion in myocardial infarction or stroke.

Rheumatoid Arthritis (RA) is a chronic inflammatory disorder where incidence and severity of myocardial infarction are increased. Data on the incidence and outcome of stroke are conflicting. Thus, we investigated outcome after Ischemia/Reperfusion (I/R) brain injury in a mouse model of RA and assessed for the role of the tumour necrosis factor-α (TNF-α) inhibitor Infliximab herein. We used a TNF-α reliant mouse model of RA. RA and wildtype (WT) animals were treated with vehicle (RA/WT) or Infliximab (RA Infliximab) for 4 weeks, before undergoing I/R brain injury. RA-animals displayed larger strokes and poorer neurological performance. Immunohistochemistry on brain sections revealed increased numbers of resident and peripheral innate immune cells (microglia and macrophages); increased Blood-Brain-Barrier (BBB)-disruption; decreased levels of the tight junction proteins (TJPs) claudin-5 and occludin; increased expression of matrix-metalloproteinases (MMP)-3 and -9 and enhanced lipid peroxidation. Treatment with Infliximab corrected these alterations. We show that RA associates to worse stroke-outcome via exacerbated BBB degradation by decrease of the TJPs claudin-5 and occludin. We identified MMPs-3 and -9 and increased oxidative stress as potential mediators thereof. Increased numbers of resident and peripheral innate immune cells (microglia and macrophages) may in turn contribute to all these effects. Infliximab-treatment restored the phenotype of RA-mice to baseline. Our data provide evidence clearly linking RA to adverse stroke-outcome in mice and indicate an approved TNF-α inhibitor as a potential strategy to reduce stroke-burden in this setting.