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Arterial thrombosis may be initiated by endothelial inflammation or denudation, activation of blood-borne elements or the coagulation system. Tissue factor (TF), a central trigger of the coagulation cascade, is regulated by the pro-inflammatory NF-κB-dependent pathways. Sirtuin 6 (SIRT6) is a nuclear member of the sirtuin family of NADUsing a laser-induced carotid thrombosis model, in vivo arterial occlusion occurred 45% faster in 12-week-old male endothelial-specific Sirt6Endothelium-specific Sirt6 deletion promotes arterial thrombosis in mice. In cultured human aortic endothelial cells, SIRT6 silencing enhances TF expression and activates pro-inflammatory pathways including TNF-α, cleaved PARP-1, VCAM-1 and COX-2. Hence, endogenous endothelial SIRT6 exerts a protective role in experimental arterial thrombosis.

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., Atherosclerosis, 325, ISSN:0021-9150

Summary 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., Graphical abstract, Highlights • Gut microbiota-derived TMAO and SCFAs associate with thrombotic potential in aging • Lifelong dietary ALA suppresses thrombus formation in aged mice • ALA-rich diet regulates aged gut microbiota and its metabolites TMAO and acetate • ALA-rich diet dampens inflammatory state in aged mice, Hematology; Nutrition; Microbiome

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 (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.

Background Stroke is the second leading cause of death and the number one cause of adult disability worldwide. As a strongly age-dependant disease, its prevalence is expected to rise along with the average age in western populations. While the epidemiological evidence linking stroke to age is non-refutable, the specific and independent effects of age on stroke remain elusive. This presents an important missing link for developing targeted treatments tailored to the growing elderly population. A potential mechanism pertinent to stroke outcome in the elderly is a chronic low-grade inflammatory state, coined “inflamm-aging”. Such a phenomenon could not only increase the risk for stroke, but also negatively affect its outcome and thus offers both preventive and therapeutic value. Purpose To determine the specific effects of age on the outcome after stroke in mice and delineate culprit molecular pathways with a focus on inflammatory mediators and to assess the efficacy of specific anti-inflammatory treatment with the TNF-α antibody Infliximab in this setting. Methods Old (18–20 months) C57BL/6 wildtype mice were compared to young (12 weeks) controls. Baseline levels of inflammatory cytokines were assayed in plasma and brain homogenates by ELISA. Ischemic stroke was induced by transient middle cerebral artery occlusion (30 minutes/48 h). Neurological function was assessed by a Bederson based score and the RotaRod test. Anti-inflammatory treatment with Infliximab was administered to a subset of old mice via weekly intraperitoneal injections (10 mg/kg) for 4 weeks prior to stroke induction. Young and old control animals received vehicle. Results At baseline (prior to stroke), old animals showed significantly higher plasma levels of TNF-α compared to young (Fig. 1A), while IL-6 and IL-1β remained below detection level in both groups. In brain homogenates of healthy old and young animals, TNF-α and IL-1β did not differ, while IL-6 was below detection level. Old mice showed significantly larger stroke sizes (Fig.1B), performed worse neurologically (Fig. 1C) and suffered from higher post-stroke mortality compared to young (Fig. 1D). Pre-treatment with the TNF-α inhibitor Infliximab significantly decreased stroke size, neurological impairment and mortality in old animals (Fig1B-D). Figure 1 Conclusions In a model lacking additional confounding factors, we demonstrate a direct adverse effect of age per se on stroke outcome and mortality. Elevated TNF-α plasma levels in old mice outline the mechanism of “inflamm-aging” as a possible culprit. This concept is strongly supported by the beneficial effect of Infliximab on stroke outcome in old animals. Further investigation of the downstream mediators of the observed effect could help in tailoring treatments to the particularly vulnerable and growing elderly population.

Background A fundamental determinant of cardio- and cerebrovascular diseases is vascular aging, characterized by arterial stiffness. Arterial stiffness is an independent predictor of adverse cardio- and cerebrovascular events and mortality. Fish-derived omega-3 fatty acids (n3-FA) have been described to decrease cardiovascular events in high risk populations. Little is known on the effects of the plant-derived n3 FA alpha-linolenic acid (ALA). More insight is urgently needed, because of the low costs and abundant global supply of ALA. Thus, we aimed to investigate the effects of a long-term dietary intervention with ALA on age-dependent arterial stiffness and the magnitude of these effects on a specific vascular endpoint – ischemic stroke – in a mouse model of aging. Methods C57BL/6 wildtype males were either fed an ALA-rich (high ALA, 7.3 g%) or a respective control (0.3 g%) diet for 12 months, starting from 6 months of age. At 9, 15 and 18 months, arterial stiffness was assessed by measuring pulse wave velocity (PWV) in the right common carotid artery using a Vevo 3100 system (VisualSonics, Fig. 1A). At 18 months, ischemic stroke was induced by transient middle cerebral artery occlusion (30 mins/48 h). Stroke size was assessed by triphenyl tetrazolium chloride staining and neurological function by a Bederson based score. Results Arterial stiffness steadily and significantly increased in controls over time, while ALA clearly and effectively prevented it (PWV at 9 vs. 18 months: controls + 95%; p Stroke size at 18 months was significantly decreased in ALA-fed animals compared to controls (28.39 mm3 vs. 51.77 mm3 p=0.0017) (Fig. 1B). In line with the morphological changes, controls performed significantly worse neurologically (Fig. 1C). Additionally, post-stroke survival at 48 h was improved in ALA-fed animals compared to controls, with 85% survival compared to 57% (Fig. 1D). Figure 1 Conclusion We demonstrate that long-term dietary supplementation with the plant-derived ALA fully prevents the development of age-dependent arterial stiffness. The magnitude of this effect is clearly reflected in biologically relevant decreased stroke size, improved neurological performance and even post-stroke survival. This study not only demonstrates vasoprotective effects of ALA, but also links them to improved outcome of a specific clinical endpoint. Future analyses will aim at delineating the molecular basis of the observed benefits. This will result in a better understanding of some ambiguous results from clinical trials and likely define the population which benefits from ALA.

High-density lipoproteins (HDL) are important endogenous inhibitors of inflammatory responses. Functional impairment of HDL might contribute to the excess mortality experienced by patients with liver disease, but the effect of cirrhosis on HDL metabolism and function remain elusive. To get an integrated measure of HDL quantity and quality, we assessed several metrics of HDL function using apolipoprotein (apo) B-depleted sera from patients with compensated cirrhosis, patients with acutely decompensated cirrhosis and healthy controls. We observed that sera of cirrhotic patients showed reduced levels of HDL-cholesterol and profoundly suppressed activities of several enzymes involved in HDL maturation and metabolism. Native gel electrophoresis analyses revealed that cirrhotic serum HDL shifts towards the larger HDL2 subclass. Proteomic assessment of isolated HDL identified several proteins, including apoA-I, apoC-III, apoE, paraoxonase 1 and acute phase serum amyloid A to be significantly altered in cirrhotic patients. With regard to function, these alterations in levels, composition and structure of HDL were strongly associated with metrics of function of apoB-depleted sera, including cholesterol efflux capability, paraoxonase activity, the ability to inhibit monocyte production of cytokines and endothelial regenerative activities. Of particular interest, cholesterol efflux capacity appeared to be strongly associated with liver disease mortality. Our findings may be clinically relevant and improve our ability to monitor cirrhotic patients at high risk.

Background/Aims: High-density lipoproteins (HDLs) are important endogenous inhibitors of inflammatory responses. Dysfunctional HDL might contribute to the serious complications experienced by patients with acute and chronic liver failure, but the effect of cirrhosis on several metrics of HDL function is not understood. Methods: We measured metrics of HDL function in an explorative cross sectional study of 59 patients with compensated cirrhosis, 21 patients with acutely decompensated cirrhosis and 20 healthy controls. HDL particle size was assessed using native gel electrophoresis. HDL cholesterol efflux potential was assessed using [ 3 H]-cholesterol labeled macrophages. Paraoxonase activity was determined by photometry using phenylacetate as substrate. Nuclear factor-κB activation in monocytes and cytokines were assessed by flow cytometry. Endothelial regenerative activity was determined using an electric cell-substrate impedance sensing system. Results: Sera of cirrhotic patients showed low HDL-cholesterol levels and profoundly suppressed activities of several serum enzymes involved in HDL maturation and metabolism. Native gel electrophoresis analyses revealed that cirrhotic HDL shifts toward the larger HDL 2 subclass. We observed that (i) HDL cholesterol efflux capability, (ii) paraoxonase activity, (iii) the ability to inhibit monocyte production of pro-inflammatory cytokines and (iv) endothelial regenerative activities were significantly reduced in stable cirrhotic patients, and markedly suppressed in acutely decompensated patients. Of particular interest, HDL cholesterol efflux capacity appeared to be strongly associated with mortality of cirrhotic patients, independently of HDL cholesterol levels. Conclusion: Cirrhosis affects HDL metabolism and several metrics of HDL function. HDL cholesterol efflux capacity appears to predict 1-year mortality independent of HDL-cholesterol levels.

Background/Aims: End-stage renal disease (ESRD) is associated with a prothrombotic phenotype and substantial activation of platelets can occur in the course of hemodialysis. However, the underlying mechanisms for increased platelet reactivity remain unclear. Increased levels of oxidized albumin, termed “advanced oxidation protein products (AOPPs)” accumulate in subjects with renal disease. Methods: Platelet aggregation was assessed with platelet aggregometry. Platelet activation, formation of reactive oxygen species and tissue factor expression on the surface of platelets and endothelial cells was measured by flow cytometry. Platelet adherence was assessed under flow using the Cellix system. The oxidative status of albumin isolated from ESRD patients was determined by photometric analysis. Tissue factor levels in serum samples were determined using a commercially available ELISA kit. Results: Albumin isolated from hemodialysis patients as well as in vitro generated AOPP-albumin promoted platelet activation via CD36, dependent on the AOPP-content of albumin. AOPP-albumin mediated platelet activation was prevented by scavenging superoxide anions and inhibitors of phospholipase C and protein kinase C. Furthermore, AOPP and serum tissue factor levels were considerably increased in ESRD patients on hemodialysis and a significant correlation of AOPP and serum tissue factor was found. Conclusion: Interaction of platelet CD36 with endogenous oxidized albumin may link oxidative stress with a prothrombotic phenotype in ESRD.

Sirtuin 3 (Sirt3) is an NAD+-dependent mitochondrial deacetylase associated with superoxide dismutase 2 (SOD2)-mediated protection from oxidative stress. We have reported accelerated weight gain and impaired metabolic flexibility in atherosclerotic Sirt3−/− mice. Oxidative stress is a hallmark of endothelial dysfunction. Yet, the role of Sirt3 in this context remains unknown. Thus, we aimed to unravel the effects of endogenous Sirt3 on endothelial function and oxidative stress. Knockdown of Sirt3 in human aortic endothelial cells (HAEC) increased intracellular mitochondrial superoxide accumulation, as assessed by electron spin resonance spectroscopy and fluorescence imaging. Endothelium-dependent relaxation of aortic rings from Sirt3−/− mice exposed to a normal diet did not differ from wild-type controls. However, following 12 weeks of high-cholesterol diet and increasing oxidative stress, endothelial function of Sirt3−/− mice was mildly impaired compared with wild-type controls. Relaxation was restored upon enhanced superoxide scavenging using pegylated superoxide dismutase. Knockdown of Sirt3 in cultured HAEC diminished SOD2 specific activity, which was compensated for by a CCAAT/enhancer binding protein beta (C/EBP-β)-dependent transcriptional induction of SOD2. Abrogation of this feedback regulation by simultaneous knockdown of C/EBP-β and Sirt3 exacerbated mitochondrial superoxide accumulation and culminated into endothelial cell death upon prolonged culture. Taken together, Sirt3 deficiency induces a mild, superoxide-dependent endothelial dysfunction in mice fed a high-cholesterol diet. In cultured endothelial cells, a novel C/EBP-β-dependent rescue mechanism maintains net SOD2 activity upon transient knockdown of Sirt3. Electronic supplementary material The online version of this article (doi:10.1007/s00395-016-0552-7) contains supplementary material, which is available to authorized users.

Endothelial dysfunction is a hallmark of inflammatory conditions. We recently demonstrated that prostaglandin (PG)E(2) enhances the resistance of pulmonary endothelium in vitro and counteracts lipopolysaccharide (LPS)-induced pulmonary inflammation in vivo via EP4 receptors. The aim of this study was to investigate the role of the EP1/EP3 receptor agonist 17-phenyl-trinor-(pt)-PGE(2) on acute lung inflammation in a mouse model. In LPS-induced pulmonary inflammation in mice, 17-pt-PGE(2) reduced neutrophil infiltration and inhibited vascular leakage. These effects were unaltered by an EP1 antagonist, but reversed by EP4 receptor antagonists. 17-pt-PGE(2) increased the resistance of pulmonary microvascular endothelial cells and prevented thrombin-induced disruption of endothelial junctions. Again, these effects were not mediated via EP1 or EP3 but through activation of the EP4 receptor, as demonstrated by the lack of effect of more selective EP1 and EP3 receptor agonists, prevention of these effects by EP4 antagonists and EP4 receptor knock-down by siRNA. In contrast, the aggregation enhancing effect of 17-pt-PGE(2) in human platelets was mediated via EP3 receptors. Our results demonstrate that 17-pt-PGE(2) enhances the endothelial barrier in vitro on pulmonary microvascular endothelial cells, and accordingly ameliorates the recruitment of neutrophils, via EP4 receptors in vivo. This suggests a beneficial effect of 17-pt-PGE(2) on pulmonary inflammatory diseases.

Plasma advanced oxidation protein products (AOPPs), a class of pro-inflammatory pathogenic mediators, accumulate in subjects with chronic kidney disease. Whether AOPPs contribute to coagulation abnormalities, which are frequently seen in uremic patients, is unknown. Here we report that AOPPs activate platelets via a CD36-mediated signaling pathway. Activation of signaling pathways by AOPP-platelet interaction resulted in the expression of several platelet activation markers and rapidly induced the expression of CD40 ligand, triggering platelet adhesion to endothelial cells and promoting endothelial tissue factor expression. AOPPs and serum tissue factor levels were considerably increased in end stage renal disease patients on hemodialysis and a significant correlation of AOPPs and serum tissue factor was found. Interestingly, serum levels of AOPPs and tissue factor were substantially lower in stable kidney transplant patients when compared with hemodialysis patients. Given that CD36 is known to transduce the effects of oxidized lipids into platelet hyperactivity, our findings reveal previously unknown pro-thrombotic activities of oxidized plasma albumin via a CD36 dependent pathway.

Platelets express the EP2, EP3 and EP4 receptors. Prostaglandin (PG) E2 has a biphasic effect on platelets. Low concentrations of PGE2 enhance platelet aggregation through the activation of the EP3 receptors, while at high concentrations it attenuates aggregation via the EP4 receptor. Consequently, EP3 receptor inhibition was shown to inhibit artherothrombosis, but had no influence on bleeding time in vivo. In this study, we investigated the role of the EP3 receptor in adhesion and thrombus formation under flow conditions in vitro. The EP3 agonist sulprostone caused an increase in the adhesion of washed platelets to fibrinogen as well as to collagen under low shear stress, an effect that was blocked by the EP3 antagonist L-798106. In contrast, when whole blood was perfused over collagen-coated surfaces, sulprostone did not enhance binding and thrombus formation of platelets on collagen; at high concentrations it even attenuated this response. We conclude that in more physiological models of thrombus formation, the role for EP3 receptors is limited, indirectly suggesting that the primary action of PGE2 in haemostasis might be an inhibitory one.

Secretory phospholipase A2 (sPLA2) generates bioactive lysophospholipids implicated in acute and chronic inflammation, but the pathophysiologic role of sPLA2 is poorly understood. Given that high-density lipoprotein (HDL) is the major substrate for sPLA2 in plasma, we investigated the effects of sPLA2-mediated modification of HDL (sPLA2-HDL) on neutrophil function, an essential arm of the innate immune response and atherosclerosis. Treatment of neutrophils with sPLA2-HDL rapidly prevented agonist-induced neutrophil activation, including shape change, neutrophil extracellular trap formation, CD11b activation, adhesion under flow and migration of neutrophils. The cholesterol-mobilizing activity of sPLA2-HDL was markedly increased when compared to native HDL, promoting a significant reduction of cholesterol-rich signaling microdomains integral to cellular signaling pathways. Moreover, sPLA2-HDL effectively suppressed agonist-induced rise in intracellular Ca²⁺ levels. Native HDL showed no significant effects and removing lysophospholipids from sPLA2-HDL abolished all anti-inflammatory activities. Overall, our studies suggest that the increased cholesterol-mobilizing activity of sPLA2-HDL and suppression of rise in intracellular Ca²⁺ levels are likely mechanism that counteracts agonist-induced activation of neutrophils. These counterintuitive findings imply that neutrophil trafficking and effector responses are altered by sPLA2-HDL during inflammatory conditions.

The use of the lipid lowering agent niacin is hampered by a frequent flush response which is largely mediated by prostaglandin (PG) D2. Therefore, concomitant administration of the D-type prostanoid (DP) receptor antagonist laropiprant has been proposed to be a useful approach in preventing niacin-induced flush. However, antagonizing PGD2, which is a potent inhibitor of platelet aggregation, might pose the risk of atherothrombotic events in cardiovascular disease. In fact, we found that in vitro treatment of platelets with laropiprant prevented the inhibitory effects of PGD2 on platelet function, i.e. platelet aggregation, Ca flux, P-selectin expression, activation of glycoprotein IIb/IIIa and thrombus formation. In contrast, laropiprant did not prevent the inhibitory effects of acetylsalicylic acid or niacin on thrombus formation. At higher concentrations, laropiprant by itself attenuated platelet activation induced by thromboxane (TP) and E-type prostanoid (EP)3 receptor stimulation, as demonstrated in assays of platelet aggregation, Ca flux, P-selectin expression, and activation of glycoprotein IIb/IIIa. Inhibition of platelet function exerted by EP4 or I-type prostanoid (IP) receptors was not affected by laropiprant. These in vitro data suggest that niacin/laropiprant for the treatment of dyslipidemias might have a beneficial profile with respect to platelet function and thrombotic events in vascular disease. Citation: Philipose S, Konya V, Lazarevic M, Pasterk LM, Marsche G, et al. (2012) Laropiprant Attenuates EP3 and TP Prostanoid Receptor-Mediated Thrombus Formation. PLoS ONE 7(8): e40222. doi:10.1371/journal.pone.0040222 Editor: Christian Schulz, Heart Center Munich, Germany Received April 17, 2012; Accepted June 2, 2012; Published August 1, 2012 Copyright: 2012 Philipose et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Dr. Philipose was funded by the PhD Program in Molecular Medicine of the Medical University of Graz. This study was supported by the Jubilaumsfonds of the Austrian National Bank (OeNB, grants 13487 and 14263) and the Austrian Science Fund (FWF; grants P22521-B18, P19473-B05, P21004-B02 and P22976-B18. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: akos.heinemann@medunigraz.at

Psoriasis is a chronic inflammatory disorder associated with increased cardiovascular mortality. Psoriasis affects high-density lipoprotein (HDL) composition, generating dysfunctional HDL particles. However, data regarding the impact of anti-psoriatic therapy on HDL composition and function are not available. HDL was isolated from 15 psoriatic patients at baseline and after effective topical and/or systemic anti-psoriatic therapy and from 15 ageand sex-matched healthy controls. HDL from psoriatic patients showed a significantly impaired capability to mobilize cholesterol from macrophages (6.4 vs. 8.0% [ 3 H]cholesterol efflux, Po0.001), low paraoxonase (217 vs. 350mM � 1 minute � 1 mg � 1 protein, P ¼0.011) and increased Lp-PLA2 activities (19.9 vs. 12.1nM � 1 minute � 1 mg � 1 protein, P ¼0.028). Of particular interest, the anti-psoriatic therapy significantly improved serum lecithincholesterol acyltransferase activity and decreased total serum lipolytic activity but did not affect serum levels of HDL-cholesterol. Most importantly, these changes were associated with a significantly improved HDLcholesterol efflux capability. Our results provide evidence that effective anti-psoriatic therapy recovers HDL composition and function, independent of serum HDL-cholesterol levels, and support to the emerging concept that HDL function may be a better marker of cardiovascular risk than HDL-cholesterol levels.