Martin F. Reiner

Post-ischaemic silencing of p66Shc reduces ischaemia/reperfusion brain injury and its expression correlates to clinical outcome in stroke

AIM 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. METHODS AND RESULTS 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. CONCLUSION 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.

Genetic deletion of the adaptor protein p66Shc increases susceptibility to short-term ischaemic myocardial injury via intracellular salvage pathways

AIMS Several intracellular mediators have been implicated as new therapeutic targets against myocardial ischaemia and reperfusion injury. However, clinically effective salvage pathways remain undiscovered. Here, we focused on the potential role of the adaptor protein p66(Shc) as a regulator of myocardial injury in a mouse model of cardiac ischaemia and reperfusion. METHODS AND RESULTS Adult male p66(Shc) deficient (p66(Shc) (-/-)) and C57Bl/6 wild-type (WT) mice were exposed to 30, 45, or 60 min of ischaemia and reperfusion (5, 15 min, or 24 h). Infarct size, systemic and intracardiac inflammation and oxidants, as well as cytosolic and mitochondrial apoptotic pathways were investigated. Following 30, but not 45 or 60 min of ischaemia, genetic p66(Shc) deficiency was associated with larger infarcts. In WT mice, in vivo p66(Shc) knock down by siRNA with transient protein deficiency confirmed these findings. P66(Shc) inhibition was not associated with any modification in post-infarction inflammation, oxidative burst nor cardiac vessel density or structure. However, in p66(Shc) (-/-) mice activation of the protective and anti-apoptotic Reperfusion Injury Salvage Kinases and Survivor Activating Factor Enhancement pathways were blunted and mitochondrial swelling and cellular apoptosis via the caspase-3 pathway increased compared with WT. CONCLUSIONS Genetic deletion of p66(Shc) increased susceptibility to myocardial injury in response to short-term ischaemia and reperfusion in mice. Still, additional studies are needed for assessing the role of this pathway in acute coronary syndrome patients.

Reduction of albumin urinary excretion is associated with reduced cardiovascular events in hypertensive and/or diabetic patients. A meta-regression analysis of 32 randomized trials

BACKGROUND The association between renal dysfunction and risk of cardiovascular (CV) events and mortality has been reported in several studies. However, it is unclear whether reduction in urinary albumin excretion (UAE) is associated with reduced risk of clinical events. Therefore, we sought to investigate, in a meta-regression analysis of randomized studies enrolling hypertensive and/or diabetic patients, whether changes in UAE are associated with changes in CV outcomes and all-cause mortality. METHODS MEDLINE, ISI Web of Science, Cochrane Database and Scopus were searched for randomized trials enrolling more than 200 diabetic and/or hypertensive patients, reporting UAE at baseline and at end of follow-up and CV events [CV death, myocardial infarction (MI), and stroke], as well all-cause mortality. RESULTS Thirty-two trials enrolling 80,812 participants were included in analyses. Meta-regression analysis showed that each 10% reduction of UAE was significantly associated with 13% reduction of MI (Regression Coefficient [RC]:0.0055; 95% Confidence Interval [CI]:0.0014 to 0.0095; p=0.010), with 29% reduction of stroke (RC:0.0124; CI:0.0030 to 0.0218; p=0.013) and with 14% reduction of the composite outcome (CV death, MI, stroke)(RC:0.0059; CI:0.0027 to 0.0090; p=0.001), whereas not significantly associated with all-cause (RC:0.0028; CI:-0.0047 to 0.0103; p=0.486) and CV mortality (RC:0.0028; CI:-0.0047 to 0.0103; p=0.447). Results were mostly confirmed by sensitivity analysis. No heterogeneity or publication bias was detected. CONCLUSIONS Reduction in UAE is associated with reduced risk of MI and stroke in diabetic and/or hypertensive patients. These findings suggest that UAE changes may represent a valuable intermediate end-point for CV risk evaluation in clinical practice.

High-density lipoprotein from patients with coronary heart disease loses anti-thrombotic effects on endothelial cells: impact on arterial thrombus formation.

Thrombus formation is determined by the balance between pro- thrombotic mediators and anti-thrombotic factors.High-density lipoprotein (HDL) from healthy subjects exerts anti-thrombotic properties. Whether this is also the case for HDL from patients with stable coronary heart disease (CHD) or acute coronary syndrome (ACS) is unknown.In human aortic endothelial cells in culture,HDL (50 µg/ml) from healthy subjects (HS) inhibited thrombin-induced tissue factor (TF) expression and activity, while HDL (50 µg/ml) from CHD and ACS patients did not. Similarly, only healthy HDL increased endothelial tissue factor pathway inhibitor (TFPI) expression and tissue plasminogen activator (tPA) release, while HDL from CHD and ACS patients had no effect. Healthy HDL inhibited thrombin-induced plasminogen activator inhibitor type 1 (PAI-1) expression, while HDL from ACS patients enhanced endothelial PAI-1 expression. Inhibition of nitric oxide (NO) formation with L-NAME (100 µmol/l) abolished the anti-thrombotic effects of healthy HDL on TF, TFPI, and tPA expression. The exogenous nitric oxide donor, DETANO, mimicked the effects of healthy HDL and counterbalanced the loss of anti-thrombotic effects of HDL from CHD and ACS patients in endothelial cells. In line with this observation, healthy HDL, in contrast to HDL from CHD and ACS patients, increased endothelial NO production. In the laser-injured carotid artery of the mouse, thrombus formation was delayed in animals treated with healthy HDL compared with mice treated with vehicle or HDL from patients with CHD or ACS. In conclusion, HDL from CHD and ACS patients loses the ability of healthy HDL to suppress TF and to increase TFPI and t-PA and instead enhances PAI-1 and arterial thrombus formation.

Amotosalen/ultraviolet A pathogen inactivation technology reduces platelet activatability, induces apoptosis and accelerates clearance

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.

Dietary α-linolenic acid increases the platelet count in ApoE−/− mice by reducing clearance

Previously we reported that dietary intake of alpha-linolenic acid (ALA) reduces atherogenesis and inhibits arterial thrombosis. Here, we analyze the substantial increase in platelet count induced by ALA and the mechanisms of reduced platelet clearance. Eight-week-old male apolipoprotein E knockout (ApoE(-/-)) mice were fed a 0.21g% cholesterol diet complemented by either a high- (7.3g%) or low-ALA (0.03g%) content. Platelet counts doubled after 16 weeks of ALA feeding, whereas the bleeding time remained similar. Plasma glycocalicin and glycocalicin index were reduced, while reticulated platelets, thrombopoietin, and bone marrow megakaryocyte colony-forming units remained unchanged. Platelet contents of liver and spleen were substantially reduced, without affecting macrophage function and number. Glycoprotein Ib (GPIb) shedding, exposure of P-selectin, and activated integrin αIIbβ3 upon activation with thrombin were reduced. Dietary ALA increased the platelet count by reducing platelet clearance in the reticulo-endothelial system. The latter appears to be mediated by reduced cleavage of GPIb by tumor necrosis factor-α-converting enzyme and reduced platelet activation/expression of procoagulant signaling. Ex vivo, there was less adhesion of human platelets to von Willebrand factor under high shear conditions after ALA treatment. Thus, ALA may be a promising tool in transfusion medicine and in high turnover/high activation platelet disorders.

Endothelial LOX-1 activation differentially regulates arterial thrombus formation depending on oxLDL levels: role of the Oct-1/SIRT1 and ERK1/2 pathways

Aims The lectin-like oxLDL receptor-1 (LOX-1) promotes endothelial uptake of oxidized low-density lipoprotein (oxLDL) and plays an important role in atherosclerosis and acute coronary syndromes (ACS). However, its role in arterial thrombus formation remains unknown. We investigated whether LOX-1 plays a role in arterial thrombus formation in vivo at different levels of oxLDL using endothelial-specific LOX-1 transgenic mice (LOX-1TG) and a photochemical injury thrombosis model of the carotid artery. Methods and results In mice fed a normal chow diet, time to arterial occlusion was unexpectedly prolonged in LOX-1TG as compared to WT. In line with this, tissue factor (TF) expression and activity in carotid arteries of LOX-1TG mice were reduced by half. This effect was mediated by activation of octamer transcription factor 1 (Oct-1) leading to upregulation of the mammalian deacetylase silent information regulator-two 1 (SIRT1) via binding to its promoter and subsequent inhibition of NF-&kgr;B signaling. In contrast, intravenous injection of oxLDL as well as high cholesterol diet for 6 weeks led to a switch from the Oct-1/SIRT1 signal transduction pathway to the ERK1/2 pathway and in turn to an enhanced thrombotic response with shortened occlusion time. Conclusions Thus, LOX-1 differentially regulates thrombus formation in vivo depending on the degree of activation by oxLDL. At low oxLDL levels LOX-1 activates the protective Oct-1/SIRT1 pathway, while at higher levels of the lipoprotein switches to the thrombogenic ERK1/2 pathway. These findings may be important for arterial thrombus formation in ACS and suggest that SIRT1 may represent a novel therapeutic target in this context.

Dietary omega-3 alpha-linolenic acid does not prevent venous thrombosis in mice

Venous thromboembolism (VTE) is a leading cause of cardiovascular death. Omega-3 fatty acids (n-3 FA) exhibit protective effects against cardiovascular disease. Others and our group have reported that the plant-derived n-3 FA alpha-linolenic acid (ALA) displays antiinflammatory, anticoagulant and antiplatelet effects, thereby reducing atherosclerosis and arterial thrombosis in mice fed a high ALA diet. Since procoagulant factors such as tissue factor and fibrin as well as platelets and leukocytes are crucially involved in the development of VTE, we investigated possible protective effects of dietary ALA on venous thrombus formation in a mouse model of stenosis- and furthermore, in a mouse model of endothelial injury-induced venous thrombosis. Four week old C57BL/6 mice underwent four weeks of high (7.3g%) or low ALA (0.03g%) treatment before being exposed to inferior vena cava (IVC) stenosis for 48 hours or laser injury of the endothelium of the internal jugular vein (IJV). Thrombus generation frequency, thrombus size and composition (IVC stenosis group) and time to thrombus formation (endothelial injury group) were assessed. In addition, plasma glycocalicin, a marker of platelet activation, platelet P-selectin and activated integrin expression as well as plasma thrombin generation was determined, but did not reveal any significant differences between the groups. Despite its protective properties against arterial thrombus formation, dietary ALA did not protect against venous thrombosis neither in the IVC stenosis nor the endothelial injury model, further indicating that the biological processes involved in arterial and venous thrombosis are different.

Ticagrelor, but not clopidogrel, reduces arterial thrombosis via endothelial tissue factor suppression

Aims The P2Y12 antagonist ticagrelor reduces mortality in patients with acute coronary syndrome (ACS), compared with clopidogrel, and the mechanisms underlying this effect are not clearly understood. Arterial thrombosis is the key event in ACS; however, direct vascular effects of either ticagrelor or clopidogrel with focus on arterial thrombosis and its key trigger tissue factor have not been previously investigated. Methods and results Human aortic endothelial cells were treated with ticagrelor or clopidogrel active metabolite (CAM) and stimulated with tumour necrosis factor-alpha (TNF-&agr;); effects on procoagulant tissue factor (TF) expression and activity, its counter-player TF pathway inhibitor (TFPI) and the underlying mechanisms were determined. Further, arterial thrombosis by photochemical injury of the common carotid artery, and TF expression in the murine endothelium were examined in C57BL/6 mice treated with ticagrelor or clopidogrel. Ticagrelor, but not CAM, reduced TNF-&agr;-induced TF expression via proteasomal degradation and TF activity, independently of the P2Y12 receptor and the equilibrative nucleoside transporter 1 (ENT1), an additional target of ticagrelor. In C57BL/6 mice, ticagrelor prolonged time to arterial occlusion, compared with clopidogrel, despite comparable antiplatelet effects. In line with our in vitro results, ticagrelor, but not clopidogrel, reduced TF expression in the endothelium of murine arteries. Conclusion Ticagrelor, unlike clopidogrel, exhibits endothelial-specific antithrombotic properties and blunts arterial thrombus formation. The additional antithrombotic properties displayed by ticagrelor may explain its greater efficacy in reducing thrombotic events in clinical trials. These findings may provide the basis for new indications for ticagrelor.

Reduction of C-reactive protein is not associated with reduced cardiovascular risk and mortality in patients treated with statins. A meta-analysis of 22 randomized trials

BACKGROUND The association between C-reactive protein (CRP) levels and risk of cardiovascular (CV) events has been reported in several studies. However, it is unclear whether a reduction in CRP is associated with a reduction in risk of clinical events. Therefore we sought to investigate, in a meta-regression analysis of randomized studies enrolling patients treated by statins, whether changes in CRP are associated with changes in risk of CV events or overall survival. METHODS Randomized trials enrolling patients treated by statins, reporting CRP at baseline and at end of follow-up, CV events [myocardial infarction (MI) and stroke], CV and all-cause mortality were selected. RESULTS Twenty-two trials enrolling 54,213 participants were included in the analysis. Meta-analysis showed that active treatment significantly reduced risk of all-cause death by 8%, myocardial infarction by 11%, stroke by 10.3% and the composite outcome (including CV death, MI and stroke) by 8%, whereas risks of CV mortality was not significantly reduced. Meta-regression analysis revealed that reduction in CRP levels was significantly associated only with the reduction of MI, whereas no relationship was identified between changes in CRP and risk of stroke, CV and all-cause mortality, and the composite outcome. CONCLUSIONS These findings demonstrate that statin-induced changes in CRP do not correlate with major CV events apart from the risk of MI nor with overall survival in high-risk patients. These data suggest that although CRP may be a surrogate marker for coronary risk, it should not be used for predicting the effectiveness of statin therapy.