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Background: During infection, neutrophil extracellular traps (NETs) are associated with severity of pulmonary diseases such as acute respiratory disease syndrome. NETs induce subsequent immune responses, are directly cytotoxic to pulmonary cells, and are highly procoagulant. Anticoagulation treatment was shown to reduce in-hospital mortality, indicating thromboinflammatory complications. However, data are sparsely available on the involvement of NETs in secondary events after virus clearance, which can lead to persistent lung damage and postacute sequelae with chronic fatigue and dyspnea., Objectives: This study focuses on late-phase events using a murine model of viral lung infection with postacute sequelae after virus resolution., Methods: C57BL/6JRj mice were infected intranasally with the betacoronavirus murine coronavirus (MCoV, strain MHV-A95), and tissue samples were collected after 2, 4, and 10 days. For NET modulation, mice were pretreated with OM-85 or GSK484 and DNase I were administered intraperitoneally between days 2 to 5 and days 4 to 7, respectively., Results: Rapid, platelet-attributed thrombus formation was followed by a second, late phase of thromboinflammation. This phase was characterized by negligible virus titers but pronounced tissue damage, apoptosis, oxidative DNA damage, and presence of NETs. Inhibition of NETs during the acute phase did not impact virus burden but decreased lung cell apoptosis by 67% and oxidative stress by 94%. Prevention of neutrophil activation by immune training before virus infection reduced damage by 75%, NETs by 31%, and pulmonary thrombi by 93%., Conclusion: NETs are detrimental inducers of tissue damage during respiratory virus infection but do not contribute to virus clearance., Competing Interests: Declaration of competing interests M.S., P.G., P.H., M.B., R.P., T.F., R.N., B.H., J.W., C.H., B.K.P., J.B.K-P. declare no conflict of interest. P.J.H. is a consultant for OM Pharma., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Aims: Ischaemia-reperfusion injury (IRI) following myocardial infarction remains a challenging topic in acute cardiac care and consecutively arising heart failure represents a severe long-term consequence. The extent of neutrophil infiltration and neutrophil-mediated cellular damage are thought to be aggravating factors enhancing primary tissue injury. Toll-like receptor 9 was found to be involved in neutrophil activation as well as chemotaxis and may represent a target in modulating IRI, aspects we aimed to illuminate by pharmacological inhibition of the receptor., Methods and Results: Forty-nine male adult Sprague-Dawley rats were used. IRI was induced by occlusion of the left coronary artery and subsequent snare removal after 30 min. Oligonucleotide (ODN) 2088, a toll-like receptor 9 (TLR9) antagonist, control-ODN, or DNase, were administered at the time of reperfusion and over 24 h via a mini-osmotic pump. The hearts were harvested 24 h or 4 weeks after left coronary artery occlusion and immunohistochemical staining was performed. Echocardiography was done after 1 and 4 weeks to determine ventricular function. Inhibition of TLR9 by ODN 2088 led to left ventricular wall thinning (P = 0.003) in association with drastically enhanced neutrophil infiltration (P = 0.005) and increased markers of tissue damage. Additionally, an up-regulation of the chemotactic receptor CXCR2 (P = 0.046) was found after TLR9 inhibition. No such effects were observed in control-ODN or DNase-treated animals. We did not observe changes in monocyte content or subset distribution, hinting towards neutrophils as the primary mediators of the exerted tissue injury., Conclusions: Our data indicate a TLR9-dependent, negative regulation of neutrophil infiltration. Blockage of TLR9 appears to prevent the down-regulation of CXCR2, followed by an uncontrolled migration of neutrophils towards the area of infarction and the exertion of disproportional tissue injury resulting in potential aneurysm formation. In comparison with previous studies conducted in TLR -/- mice, we deliberately chose a transient pharmacological inhibition of TLR9 to highlight effects occurring in the first 24 h following IRI., (© 2023 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Atherosclerosis is a chronic, inflammatory disease of the vessel wall where triggered immune cells bind to inflamed endothelium, extravasate and sustain local inflammation. Leukocyte adhesion and extravasation are mediated by adhesion molecules expressed by activated endothelial cells, like intercellular adhesion molecule 1 (ICAM-1). Extracellular adherence protein (Eap) from Staphylococcus aureus binds to a plethora of extracellular matrix proteins, including ICAM-1 and its ligands macrophage-1 antigen (Mac-1, α M β 2 ) and lymphocyte function-associated antigen 1 (LFA-1, α L β 2 ), thereby disrupting the interaction between leukocytes and endothelial cells. We aimed to use Eap to inhibit the interaction of leukocytes with activated endothelial cells in settings of developing and established atherosclerosis in apolipoprotein E (ApoE) deficient mice on high-fat diet. In developing atherosclerosis, Eap treatment reduced circulating platelet-neutrophil aggregates as well as infiltration of T cells and neutrophils into the growing plaque, accompanied by reduced formation of neutrophil extracellular traps (NETs). However, plaque size did not change. Intervention treatment with Eap of already established plaques did not result in cellular or morphological plaque changes, whereas T cell infiltration was increased and thereby again modulated by Eap. We conclude that although Eap leads to cellular changes in developing plaques, clinical implications might be limited as patients are usually treated at a more advanced stage of disease progression. Hence, usage of Eap might be an interesting mechanistic tool for cellular infiltration during plaque development in basic research but not a clinical target., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Interleukin-4 (IL-4) and its receptors (IL-4R) promote the proliferation and polarization of macrophages. However, it is unknown if IL-4R also influences monocyte homeostasis and if steady state IL-4 levels are sufficient to affect monocytes. Employing full IL-4 receptor alpha knockout mice (IL-4Rα -/- ) and mice with a myeloid-specific deletion of IL-4Rα (IL-4Rα f/f LysM cre ), we show that IL-4 acts as a homeostatic factor regulating circulating monocyte numbers. In the absence of IL-4Rα, murine monocytes in blood were reduced by 50% without altering monocytopoiesis in the bone marrow. This reduction was accompanied by a decrease in monocyte-derived inflammatory cytokines in the plasma. RNA sequencing analysis and immunohistochemical staining of splenic monocytes revealed changes in mRNA and protein levels of anti-apoptotic factors including BIRC6 in IL-4Rα -/- knockout animals. Furthermore, assessment of monocyte lifespan in vivo measuring BrdU + cells revealed that the lifespan of circulating monocytes was reduced by 55% in IL-4Rα -/- mice, whereas subcutaneously applied IL-4 prolonged it by 75%. Treatment of human monocytes with IL-4 reduced the amount of dying monocytes in vitro. Furthermore, IL-4 stimulation reduced the phosphorylation of proteins involved in the apoptosis pathway, including the phosphorylation of the NFκBp65 protein. In a cohort of human patients, serum IL-4 levels were significantly associated with monocyte counts. In a sterile peritonitis model, reduced monocyte counts resulted in an attenuated recruitment of monocytes upon inflammatory stimulation in IL-4Rα f/f LysM cre mice without changes in overall migratory function. Thus, we identified a homeostatic role of IL-4Rα in regulating the lifespan of monocytes in vivo., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Changes in Ca2 + influx during proinflammatory stimulation modulates cellular responses, including the subsequent activation of inflammation. Whereas the involvement of Ca 2+ has been widely acknowledged, little is known about the role of Na + . Ranolazine, a piperazine derivative and established antianginal drug, is known to reduce intracellular Na + as well as Ca2 + levels. In stable coronary artery disease patients ( n = 51) we observed reduced levels of high-sensitive C-reactive protein (CRP) 3 mo after the start of ranolazine treatment ( n = 25) as compared to the control group. Furthermore, we found that in 3,808 acute coronary syndrome patients of the MERLIN-TIMI 36 trial, individuals treated with ranolazine (1,934 patients) showed reduced CRP values compared to placebo-treated patients. The antiinflammatory effects of sodium modulation were further confirmed in an atherosclerotic mouse model. LDL -/- mice on a high-fat diet were treated with ranolazine, resulting in a reduced atherosclerotic plaque burden, increased plaque stability, and reduced activation of the immune system. Pharmacological Na + inhibition by ranolazine led to reduced express of adhesion molecules and proinflammatory cytokines and reduced adhesion of leukocytes to activated endothelium both in vitro and in vivo. We demonstrate that functional Na + shuttling is required for a full cellular response to inflammation and that inhibition of Na + influx results in an attenuated inflammatory reaction. In conclusion, we demonstrate that inhibition of Na + -Ca 2+ exchange during inflammation reduces the inflammatory response in human endothelial cells in vitro, in a mouse atherosclerotic disease model, and in human patients.

Quantitative and functional analysis of mononuclear leukocyte populations is an invaluable tool to understand the role of the immune system in the pathogenesis of a disease. Cryopreservation of mononuclear cells (MNCs) is routinely used to guarantee similar experimental conditions. Immune cells react differently to cryopreservation, and populations and functions of immune cells change during the process of freeze-thawing. To allow for a setup that preserves cell number and function optimally, we tested four different cryopreservation media. MNCs from 15 human individuals were analyzed. Before freezing and after thawing, the distribution of leukocytes was quantified by flow cytometry. Cultured cells were stimulated using lipopolysaccharide, and their immune response was quantified by flow cytometry, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). Ultimately, the performance of the cryopreservation media was ranked. Cell recovery and viability were different between the media. Cryopreservation led to changes in the relative number of monocytes, T cells, B cells, and their subsets. The inflammatory response of MNCs was altered by cryopreservation, enhancing the basal production of inflammatory cytokines. Different cryopreservation media induce biases, which needs to be considered when designing a study relying on cryopreservation. Here, we provide an overview of four different cryopreservation media for choosing the optimal medium for a specific task.

Training of the innate immune system with orally ingested bacterial extracts was demonstrated to have beneficial effects on infection clearance and disease outcome. The aim of our study was to identify cellular and molecular processes responsible for these immunological benefits. We used a murine coronavirus (MCoV) A59 mouse model treated with the immune activating bacterial extract Broncho-Vaxom (BV) OM-85. Tissue samples were analysed with qPCR, RNA sequencing, histology, and flow cytometry. After BV OM-85 treatment, interstitial macrophages accumulated in lung tissue leading to a faster response of type I interferon (IFN) signalling after MCoV infection resulting in overall lung tissue protection. Moreover, RNA sequencing showed that lung tissue from mice receiving BV OM-85 resembled an intermediate stage between healthy and viral infected lung tissue at day 4, indicating a faster return to normal tissue homoeostasis. The pharmacologic effect was mimicked by adoptively transferring naive lung macrophages into lungs from recipient mice before virus infection. The beneficial effect of BV OM-85 was abolished when inhibiting initial type I IFN signalling. Overall, our data suggest that BV OM-85 enhances lung macrophages allowing for a faster IFN response towards a viral challenge as part of the oral-induced innate immune system training., (© 2021 The Author(s) Published by S. Karger AG, Basel.)

Recent data suggest that the disease-associated microenvironment, known as the leukemic stem cell (LSC) niche, is substantially involved in drug resistance of LSC in BCR-ABL1 + chronic myeloid leukemia (CML). Attacking the LSC niche in CML may thus be an effective approach to overcome drug resistance. We have recently shown that osteoblasts are a major site of niche-mediated LSC resistance against second- and third-generation tyrosine kinase inhibitors (TKI) in CML. In the present study, we screened for drugs that are capable of suppressing the growth and viability of osteoblasts and/or other niche cells and can thereby overcome TKI resistance of CML LSC. Proliferation was analyzed by determining 3 H-thymidine uptake in niche-related cells, and apoptosis was measured by Annexin-V/DAPI-staining and flow cytometry. We found that the dual PI3 kinase (PI3K) and mTOR inhibitor BEZ235 and the selective pan-PI3K inhibitor copanlisib suppress proliferation of primary osteoblasts (BEZ235 IC 50 : 0.05 μM; copanlisib IC 50 : 0.05 μM), the osteoblast cell line CAL-72 (BEZ235 IC 50 : 0.5 μM; copanlisib IC 50 : 1 μM), primary umbilical vein-derived endothelial cells (BEZ235 IC 50 : 0.5 μM; copanlisib IC 50 : 0.5 μM), and the vascular endothelial cell line HMEC-1 (BEZ235 IC 50 : 1 μM; copanlisib IC 50 : 1 μM), whereas no comparable effects were seen with the mTOR inhibitor rapamycin. Furthermore, we show that BEZ235 and copanlisib cooperate with nilotinib and ponatinib in suppressing proliferation and survival of osteoblasts and endothelial cells. Finally, BEZ235 and copanlisib were found to overcome osteoblast-mediated resistance against nilotinib and ponatinib in K562 cells, KU812 cells and primary CD34 + /CD38 - CML LSC. Together, targeting osteoblastic niche cells through PI3K inhibition may be a new effective approach to overcome niche-induced TKI resistance in CML. Whether this approach can be translated into clinical application and can counteract drug resistance of LSC in patients with CML remains to be determined in clinical trials., Competing Interests: The authors declare that they have no study-specific conflict of interest (COI) to disclose. COI to disclose outside of this study: K.V.G. received honoraria from Novartis, Incyte, BMS, Pfizer and Abbvie. P.V. received grant support from Celgene/BMS, Pfizer, and Incyte, and consultancy honoraria from Novartis, Pfizer, Incyte, Celgene/BMS, and OAP Orphan Pharmaceuticals., (AJCR Copyright © 2021.)

We have previously shown that treatment with third-generation antisense oligonucleotides against miR-494-3p (3GA-494) reduces atherosclerotic plaque progression and stabilizes lesions, both in early and established plaques, with reduced macrophage content in established plaques. Within the plaque, different subtypes of macrophages are present. Here, we aimed to investigate whether miR-494-3p directly influences macrophage polarization and activation. Human macrophages were polarized into either proinflammatory M1 or anti-inflammatory M2 macrophages and simultaneously treated with 3GA-494 or a control antisense (3GA-ctrl). We show that 3GA-494 treatment inhibited miR-494-3p in M1 macrophages and dampened M1 polarization, while in M2 macrophages miR-494-3p expression was induced and M2 polarization enhanced. The proinflammatory marker CCR2 was reduced in 3GA-494-treated atherosclerosis-prone mice. Pathway enrichment analysis predicted an overlap between miR-494-3p target genes in macrophage polarization and Wnt signaling. We demonstrate that miR-494-3p regulates expression levels of multiple Wnt signaling components, such as LRP6 and TBL1X. Wnt signaling appears activated upon treatment with 3GA-494, both in cultured M1 macrophages and in plaques of hypercholesterolemic mice. Taken together, 3GA-494 treatment dampened M1 polarization, at least in part via activated Wnt signaling, while M2 polarization was enhanced, which is both favorable in reducing atherosclerotic plaque formation and increasing plaque stability., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Background: Inflammation is a key process during atherosclerotic lesion development and propagation. Recent evidence showed clearly that especially the inhibition of interleukin (IL)-1β reduced atherosclerotic adverse events in human patients. Fatty acid oxidation (FAO) was previously demonstrated to interact with the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) pathway which is required for mature IL-1β secretion. To understand possible anti-inflammatory properties of FAO inhibition, we tested the effect of pharmacological FAO inhibition using the inhibitor for long-chain 3-ketoacyl coenzyme A thiolase trimetazidine on atherosclerotic plaque development and inflammation., Experimental Approach: The effect of FAO inhibition was determined in LDL-R -/- male mice on a C57/BL6 background. In vitro effects of trimetazidine treatment were analyzed in human umbilical vein endothelial cells and human monocyte derived macrophages., Key Results: We were able to demonstrate that inhibition of FAO reduced atherosclerotic plaque growth. We did not find direct anti-inflammatory properties of trimetazidine in endothelial cells or macrophages in vitro. However, we found that the activation of the NLRP3 system and the secretion of IL-1β were significantly reduced in macrophages after FAO inhibition. These results were confirmed in atherosclerotic lesions of mice treated with trimetazidine as they showed a significant reduction of IL-1β and cleaved caspase-1 in the atherosclerotic lesion as well as of IL-1β and IL-18 in the circulation., Conclusion: Overall, we therefore suggest that the main mechanism of reducing inflammation of trimetazidine and FAO inhibition is the reduction of the NLRP-3 activation leading to reduced levels of the proinflammatory cytokine IL-1β., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Fabry disease is a hereditary genetic defect resulting in reduced activity of the enzyme α-galactosidase-A and the accumulation of globotriaosylceramide (Gb3) in body fluids and cells. Gb3 accumulation was especially reported for the vascular endothelium in several organs., Methods: Three Fabry disease patients were screened using a micro-RNA screen. An in vitro approach in human endothelial cells was used to determine miRNA regulation by Gb3., Results: In a micro-RNA screen of three Fabry patients undergoing enzyme replacement therapy, we found that miRNAs let-7a and let-7d were significantly increased after therapy. We demonstrate in vitro in endothelial cells that Gb3 induced activation of NF-κB and activated downstream targets. In addition, NF-κB activity directly reduced let-7a and let-7d miRNA expression as inhibiting NF-kB nuclear entry abolished the Gb3 effects., Conclusion: We suggest that let-7a and let-7d are potential markers for enzyme activity and inflammation in Fabry disease patients.

After successful cardiopulmonary resuscitation (CPR), many patients show signs of an overactive immune activation. Monocytes are a heterogeneous cell population that can be distinguished into 3 subsets by flow cytometry (classical monocytes [CM: CD14 ++ CD16 - ], intermediate monocytes [IM: CD14 ++ CD16 + CCR2 + ] and non-classical monocytes [NCM: CD14 + CD16 ++ CCR2 - ]). Fifty-three patients admitted to the medical intensive care unit (ICU) after cardiac arrest were included. Blood was taken on admission and after 72 h. The primary endpoint of this study was survival at 6 months and the secondary endpoint was neurological outcome as determined by cerebral performance category (CPC)-score at 6 months. Median age was 64.5 (49.8-74.3) years and 75.5% were male. Six-month mortality was 50.9% and survival with good neurological outcome was 37.7%. Monocyte subset distribution upon admission to the ICU did not differ according to survival. Seventy-two hours after admission, patients who died within 6 months showed a higher percentage of the pro-inflammatory subset of IM (8.3% [3.8-14.6]% vs. 4.1% [1.5-8.2]%; P = 0.025), and a lower percentage of CM (87.5% [79.9-89.0]% vs. 90.8% [85.9-92.7]%; P = 0.036) as compared to survivors. In addition, IM were predictive of outcome independent of time to ROSC and witnessed cardiac arrest, and correlated with CPC-score at 6 months (R = 0.32; P = 0.043). These findings suggest a possible role of the innate immune system in the pathophysiology of post cardiac arrest syndrome., (© 2020 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals LLC on behalf of Society for Leukocyte Biology.)

Macrophages are versatile cells that can be polarized by the tissue environment to fulfill required needs. Proinflammatory polarization is associated with increased tissue degradation and propagation of inflammation whereas alternative polarization within a Th2 cytokine environment is associated with wound healing and angiogenesis. To understand if polarization of macrophages can lead to a procoagulant macrophage subset we polarized human monocyte derived macrophages to a proinflammatory and an alternative activation state. Alternative polarization with interleukin-4 and IL-13 led to a macrophage phenotype characterized by increased tissue factor (TF) production and release and by an increase in extracellular vesicle production. In addition, also TF activity was enhanced in extracellular vesicles of alternatively polarized macrophages. This TF induction was dependent on signal transducer and activator of transcription-6 signaling and poly ADP ribose polymerase activity. In contrast to monocytes, human macrophages did not show increased tissue factor expression upon stimulation with lipopolysaccharide and interferon-γ. Previous polarization to either a proinflammatory or an alternative activation subset does not change the subsequent stimulation of TF. The inability of proinflammatory activated macrophages to respond to lipopolysaccharide and interferon-γ with an increase in TF production seems to be due to an increase in TF promoter methylation and was reversible when treating these macrophages with a demethylation agent. In conclusion, we provide evidence that proinflammatory polarization of macrophages does not lead to enhanced procoagulatory function, whereas alternative polarization of macrophages leads to an increased expression of TF and increased production of TF bearing extracellular vesicles by these cells suggesting a procoagulatory phenotype of alternatively polarized macrophages.

Nicorandil, a balanced vasodilator, is used in the second-line therapy of angina pectoris. In this study, we aimed to illuminate the effects of nicorandil on inflammation, apoptosis, and atherosclerotic plaque progression. Twenty-five LDL-R -/- mice were fed a high-fat diet for 14 weeks. After 6 weeks mice were randomly allocated to treatment with nicorandil (10 mg/kg/day) or tap water. Nicorandil treatment led to a more stable plaque phenotype, displaying an increased thickness of the fibrous cap ( p = 0.014), a significant reduction in cholesterol clefts ( p = 0.045), and enhanced smooth muscle cell content ( p = 0.009). In endothelial cells nicorandil did not reduce the induction of adhesion molecules or proinflammatory cytokines. In H 2 O 2 challenged endothelial cells, pretreatment with nicorandil significantly reduced the percentage of late apoptotic/necrotic cells ( p = 0.016) and the ratio of apoptotic to living cells ( p = 0.036). Atherosclerotic lesions of animals treated with nicorandil exhibited a significantly decreased content of cleaved caspase-3 ( p = 0.034), lower numbers of apoptotic nuclei ( p = 0.040), and reduced 8-oxogunanine staining ( p = 0.039), demonstrating a stabilizing effect of nicorandil in established atherosclerotic lesions. We suggest that nicorandil has a positive effect on atherosclerotic plaque stabilization by reducing apoptosis.

Aim: Current guidelines suggest the use of epinephrine in patients with cardiac arrest (CA). However, evidence for increased survival in good neurological condition is lacking. In experimental settings, epinephrine-induced impairment of microvascular flow was shown. The aim of our study was to analyze the association between epinephrine treatment and intestinal injury in patients after CA., Methods: We have included 52 patients with return of spontaneous circulation (ROSC) after CA admitted to our medical intensive care unit (ICU). Blood was taken on admission and levels of circulating intestinal fatty acid binding protein (iFABP) were analyzed., Results: Patients were 64 (49.8-73.8) years old and predominantly male (76.9%). After six months, 50% of patients died and 38.5% of patients had a cerebral performance category (CPC)-score of 1-2. iFABP levels were lower in survivors (234 IQR 90-399 pg/mL) as compared to non-survivors (283, IQR 86-11500 pg/mL; p < 0.05). Plasma levels of iFABP were not associated with time to ROSC but correlated with epinephrine-dose (R = 0.32; p < 0.05). 40% of patients receiving ≥3 mg of epinephrine as compared to 10.5% of patients treated with <3 mg (p < 0.05) developed iFABP plasma levels >1500 pg/mL, which was associated with dramatically increased mortality (HR4.87, 95%CI 1.95-12.1; p < 0.001). iFABP levels predicted mortality independent from time to ROSC and the disease severity score SAPS II. In contrast to mortality, iFABP plasma levels were not associated with neurological outcome., Conclusions: In this small, single centre study, cumulative dose of epinephrine used in cardiac arrest patients was associated with an increase in biomarker indicative of intestinal injury and 6-month mortality., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Obesity is closely linked to increased markers of metabolic syndrome and development of diabetes. Roux-en-Y bariatric surgery reduces hyperinsulinemia and improves insulin sensitivity and hence benefits morbidly obese patients., Aim: To determine changes in markers of metabolic syndrome, pancreatic function, and hepatic insulin sensitivity in patients before and 1 year after undergoing Roux-en-Y gastric bypass surgery., Methods: We enrolled 43 consecutive patients in a single center. Markers for metabolic syndrome included proinsulin, insulin, C-peptide, liver enzymes, and serum levels of selected microRNAs hsa-miR-122, hsa-miR-130, hsa-miR-132, and hsa-miR-375., Results: After surgery, all patients showed a significant 37% drop of body mass index (p < 0.001). Furthermore, proinsulin (59% reduction, p < 0.001), insulin (76% reduction, p < 0.001), and C-peptide (56% reduction, p < 0.001) were all reduced 1 year after surgery. Using the hepatic insulin clearance score, we determined a significant increase in hepatic insulin clearance after surgery (76% increase, p < 0.001). Especially diabetic patients showed a marked 2.1-fold increase after surgery. Hepatic enzymes ALT (35% reduction, p = 0.002) and γGT (48% reduction, p < 0.001) were significantly reduced in all patients with similar improvement in diabetic and non-diabetic patients. miRNAs hsa-miR-122, hsa-miR-130, and hsa-miR-132 were all significantly reduced whereas hsa-miR-375 was increased after gastric bypass surgery (p < 0.001 for all miRNAs)., Conclusion: Both liver and pancreatic stress parameters were reduced significantly 1 year after Roux-en-Y gastric bypass surgery suggesting an overall amelioration of the metabolic syndrome in all patients regardless of previous health status.

Monocytes are activated in inflammatory conditions via a variety of cytokine receptors as well as in a procoagulatory setting through thrombin, acting upon protease-activated receptors (PARs). This study investigated the expression pattern of PAR1 and PAR3 on human monocyte subsets. Furthermore, a possible regulation of the expression of PAR1 and PAR3 in these cells by inflammatory activation were studied. CD16 + monocytes showed significantly higher levels of PAR1 and PAR3 as compared with CD16 - monocytes. Ex vivo treatment of whole blood with lipopolysaccharide (LPS) increased PAR1 and PAR3 messenger ribonucleic acid (mRNA) in human monocytes. In addition, increase of PAR1 was seen in all three subsets upon LPS treatment, whereas PAR3 increased significantly only in CD16 - monocytes and nonclassical CD16 + monocytes. Protein levels of PAR1 and PAR3 significantly increased on monocytes in vivo in human endotoxemia 1 hour after LPS infusion. PAR1 increased significantly in CD16 - monocytes and nonclassical CD16 + monocytes. In this in vivo model, PAR3 was also significantly elevated in CD16 - monocytes and increased slightly albeit not significantly in CD16 + monocytes. Endotoxemia increased plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) expression in monocytes in humans. Pretreatment of healthy volunteers with the PAR1 antagonist vorapaxar blocked the increase in PAI-1 but not the increase in TF. We here provide new evidence for a critical role for monocytes as cellular mediators that contribute to the activation of coagulation in diseases characterized by an inflammatory state., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Membrane-bound plasmin is used by immune cells to degrade extracellular matrices, which facilitates migration. The plasminogen receptor Plg-R KT is expressed by immune cells, including monocytes and macrophages. Among monocytes and macrophages, distinct subsets can be distinguished based on cell surface markers and pathophysiological function. We investigated expression of Plg-R KT by monocyte and macrophage subsets and whether potential differential expression might have functional consequences for cell migration. Proinflammatory CD14 ++ CD16 + human monocytes and Ly6C high mouse monocytes expressed the highest levels of Plg-R KT and bound significantly more plasminogen compared with the other respective subsets. Proinflammatory human macrophages, generated by polarization with lipopolysaccharide and interferon-γ, showed significantly higher expression of Plg-R KT compared with alternatively activated macrophages, polarized with interleukin-4 and interleukin-13. Directional migration of proinflammatory monocytes was plasmin dependent and was abolished by anti-Plg-R KT monoclonal antibody, ε-amino-caproic acid, aprotinin, and the aminoterminal fragment of urokinase-type plasminogen activator. In an in vivo peritonitis model, significantly less Ly6C high monocyte recruitment was observed in Plg-R KT -/- compared with Plg-R KT +/+ mice. Immunohistochemical analysis of human carotid plaques and adipose tissue showed that proinflammatory macrophages also exhibited high levels of Plg-R KT in vivo. Our data demonstrate higher expression of Plg-R KT on proinflammatory monocyte and macrophage subsets that impacts their migratory capacity., (© 2019 by The American Society of Hematology.)

Aims: We have shown that 14q32 microRNAs are highly involved in vascular remodelling and cardiovascular disease. However, the 14q32 locus also encodes 41 'orphan' small nucleolar RNAs (snoRNAs). We aimed to gather evidence for an independent role for 14q32 snoRNAs in human cardiovascular disease., Methods and Results: We performed a lookup of the 14q32 region within the dataset of a genome wide association scan in 5244 participants of the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER). Single nucleotide polymorphisms (SNPs) in the snoRNA-cluster were significantly associated with heart failure. These snoRNA-cluster SNPs were not linked to SNPs in the microRNA-cluster or in MEG3, indicating that snoRNAs modify the risk of cardiovascular disease independently. We looked at expression of 14q32 snoRNAs throughout the human cardio-vasculature. Expression profiles of the 14q32 snoRNAs appeared highly vessel specific. When we compared expression levels of 14q32 snoRNAs in human vena saphena magna (VSM) with those in failed VSM-coronary bypasses, we found that 14q32 snoRNAs were up-regulated. SNORD113.2, which showed a 17-fold up-regulation in failed bypasses, was also up-regulated two-fold in plasma samples drawn from patients with ST-elevation myocardial infarction directly after hospitalization compared with 30 days after start of treatment. However, fitting with the genomic associations, 14q32 snoRNA expression was highest in failing human hearts. In vitro studies show that the 14q32 snoRNAs bind predominantly to methyl-transferase Fibrillarin, indicating that they act through canonical mechanisms, but on non-canonical RNA targets. The canonical C/D-box snoRNA seed sequences were highly conserved between humans and mice., Conclusion: 14q32 snoRNAs appear to play an independent role in cardiovascular pathology. 14q32 snoRNAs are specifically regulated throughout the human vasculature and their expression is up-regulated during cardiovascular disease. Our data demonstrate that snoRNAs merit increased effort and attention in future basic and clinical cardiovascular research., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.)

Circulating extracellular vesicles are small particles enclosed by a phospholipid bilayer. Vesicles deriving directly from the cellular membrane by an active budding process retain cell origin specific proteins and RNA. These vesicles carry pathophysiological information from their parental cell and hold the potential to allow analysis of organs without the need for a biopsy. We included in our study 27 patients undergoing bariatric surgery. Hepatic extracellular vesicles were determined by flow cytometry. mRNA specific for hepatic cellular origin was determined in the extracellular vesicle fraction using qPCR. Surgery led to a massive reduction of weight and overall hepatic stress as determined by alanine transaminase (ALT), aspartate transaminase (AST) and γ-glutamyltransferase (GGT). Total extracellular vesicle numbers were reduced after bariatric surgery. Liver specific vesicles identified by HepPar1 or asialoglycoprotein receptor (ASGPR) were significantly reduced after bariatric surgery in both AnnexinV + and AnnexinV - subgroups. When analyzing circulating liver-specific mRNAs, we found reduced levels of these mRNAs after surgery even though total circulating RNA remained unchanged. We conclude that circulating hepatic extracellular vesicles are detectable in samples from patients undergoing gastric bypass surgery. These vesicles are reduced after a reduction of hepatic stress also observed with classic liver enzyme measurements. We conclude that ASGPR or HepPar positive vesicles hold the potential to serve as liver specific vesicle markers.

Obesity is associated with a prothrombotic milieu and an increased risk for thrombotic events. Bariatric surgery is the most effective treatment for obesity resulting in dramatic weight loss and reduced inflammation and extrinsic coagulation pathway activation. Blood samples were drawn from 60 patients undergoing Roux-en-Y gastric bypass surgery before and 1 year after the intervention. Protein C (PC), activated PC (APC), soluble thrombomodulin (TM), soluble E-selectin (E-Sel), prothrombin time (PT) and activated partial thromboplastin time (aPTT) were evaluated. Both PC (187.4 ± 64.5% before surgery to 118.1 ± 48% 1 year after surgery, p < 0.001) and APC (138.7 ± 64.4% before surgery to 69.1 ± 65.7% after surgery, p < 0.001) were reduced following surgical intervention. TM showed a similar behavior with a reduction of soluble TM after the procedure from 5.7 ± 2.6 to 3.2 ± 1.4 ng/ml (p < 0.001). Similarly, soluble E-Sel was reduced after surgery from 26.6 ± 12.7 to 5.5 ± 4.1 ng/ml (p < 0.001). In contrast, aPTT was not shortened but slightly increased from 29.1 ± 4.8 s. before surgery to 31 ± 4.4 s. (p = 0.001) after surgery and levels of PT were reduced after surgery to 89.6 ± 15.5% from an initial 97.5 ± 13.5% (p < 0.001). In conclusion, we demonstrate a reduction of PC and APC 1 year after bariatric surgery accompanied by a reduction in soluble TM and soluble E-Sel. The reduction of PC and APC is not paralleled by a reduction but in contrast by a prolongation of aPTT suggesting a compensatory upregulation of PC during obesity. The reduction of TM and E-Sel might hint towards an improved endothelial function in this cohort of patients.

Interleukin (IL)-33 is a member of the IL-1 family and is able to act cardioprotective. The aim of this study was to investigate the regulation of IL-33 by 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) reductase inhibitors (statins) and bisphosphonates (BPs) in human cardiac tissue. The lipophilic fluvastatin, simvastatin, atorvastatin, and lovastatin as well as the nitrogenous BPs alendronate and ibandronate, but not hydrophilic pravastatin increased IL-33 mRNA and intracellular IL-33 protein levels in both human adult cardiac myocytes (HACM) and fibroblasts (HACF). Additionally, fluvastatin reduced soluble ST2 secretion from HACM. IL-33 was also up-regulated by the general inhibitor of prenylation perillic acid, a RhoA kinase inhibitor Y-27632, and by latrunculin B, but statin-induced IL-33 expression was inhibited by mevalonate, geranylgeranyl pyrophosphate (GGPP) and RhoA activator U-46619. The IL-33 promoter was 2.3-fold more accessible in statin-treated HACM compared to untreated cells (P = 0.037). In explanted hearts of statin-treated patients IL-33 protein was up-regulated as compared with the hearts of non-statin-treated patients (P = 0.048). As IL-33 was previously shown to exert cardioprotective effects, one could speculate that such up-regulation of IL-33 expression in human cardiac cells, which might happen mainly through protein geranylgeranylation, could be a novel mechanism contributing to known cardioprotective effects of statins and BPs., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background and Aims: Macrophages are versatile immune cells involved in tissue degradation and remodeling. Proinflammatory macrophages have the highest capacity of matrix degradation and proteolysis. Within atherosclerotic lesions, proinflammatory macrophages are associated with unstable plaques. Statins have been demonstrated to increase plaque stability. Possible changes of polarized macrophage tissue degradation behavior under statin treatment are currently unknown., Methods: Polarized macrophages were tested in vitro for matrix degradation capacity with or without statin treatment., Results: Proinflammatory macrophages show high matrix degradation capacity, which is lost after statin treatment. Statin concentrations were within a physiological range and did not influence overall macrophage polarization. Proinflammatory macrophages showed however a loss of filopodia where activators of MMPs are located. Loss of matrix degradation in proinflammatory macrophages was associated with changes of MMP14 activation and loss of uPAR localization at filopodia. Supplementation of mevalonate restored localization of uPAR to cellular protrusions and matrix degradation capacity., Conclusion: Statins reduce the matrix degradation potential of proinflammatory macrophages by reducing uPAR localization to cellular filopodia and reducing intracellular MMP14 activation., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Obesity is considered to be a major comorbidity. Obese patients suffer from an increased proinflammatory state associated with a premature aging phenotype including increased secretion of senescence-associated secretory proteins (SASP) and reduced telomere length. Micro-ribonucleic acids (miRNAs) are non-coding RNA molecules that could modify the post-transcriptional process. Several studies have reported associations between miRNAs and metabolic unhealthy conditions., Aim: To determine if bariatric surgery and the resulting weight loss could reverse the premature aging phenotype., Methods: We enrolled 58 morbidly obese patients undergoing bariatric surgery. Markers of premature aging including the SASP IL-6, CRP and PAI-1, 7 miRNAs, as well as telomere length and telomere oxidation in mononuclear cells were evaluated., Results: Patients showed a significant drop of body mass index (BMI; 43.98 ± 3.5 versus 28.02 ± 4.1, p < 0.001). We observed a significant reduction in SASP including a reduction of 55% of plasma IL-6 levels (p = 0 < 0.001), 83% of CRP levels (p = 0.001) and 15% of plasma PAI-1 levels (p < 0.001). Telomere length doubled in the patient cohort (p < 0.001) and was accompanied by a reduction in the telomere oxidation index by 70% (p < 0.001). Telomere length was inversely correlated with telomere oxidation. The aging-associated miRNA miR10a&#95;5p was upregulated significantly (p = 0.039), while the other tested miRNAs showed no difference., Conclusion: Our data indicate a significant reduction of the proinflammatory SASP after bariatric surgery. We observed an increase in telomere length and reduced oxidative stress at telomeres. miR10a&#95;5p which is downregulated during aging was upregulated after surgery. Overall, bariatric surgery ameliorated the premature aging phenotype.

Objective: Macrophages are versatile immune cells capable of polarizing into functional subsets depending on environmental stimulation. In atherosclerotic lesions, proinflammatory polarized macrophages are associated with symptomatic plaques, whereas Th2 (T-helper cell type 2) cytokine-polarized macrophages are inversely related with disease progression. To establish a functional cause for these observations, we analyzed extracellular matrix degradation phenotypes in polarized macrophages., Approach and Results: We provide evidence that proinflammatory polarized macrophages rely on membrane-bound proteases including MMP-14 (matrix metalloproteinase-14) and the serine protease uPA (urokinase plasminogen activator) together with its receptor uPAR for extracellular matrix degradation. In contrast, Th2 cytokine alternatively primed macrophages do not show different proteolytic activity in comparison to unpolarized macrophages and lack increased localization of MMP-14 and uPA receptor to the cell membrane. Nonetheless, they express the highest amount of the serine protease uPA. However, uPA activity is blocked by similarly increased expression of its inhibitor PAI-1 (plasminogen activator inhibitor 1). When inhibiting PAI-1 or when analyzing macrophages deficient in PAI-1, Th2 cytokine-polarized macrophages display the same matrix degradation capability as proinflammatory-primed macrophages. Within atherosclerotic lesions, macrophages positive for the alternative activation marker CD206 express high levels of PAI-1. In addition, to test changed tissue remodeling capacities of alternatively activated macrophages, we used a bleomycin lung injury model in mice reconstituted with PAI-1 -/- bone marrow. These results supported an enhanced remodeling phenotype displayed by increased fibrosis and elevated MMP activity in the lung after PAI-1 loss., Conclusions: We were able to demonstrate matrix degradation dependent on membrane-bound proteases in proinflammatory stimulated macrophages and a forced proteolytical quiescence in alternatively polarized macrophages by the expression of PAI-1., (© 2017 The Authors.)

The role of uPA in tissue remodeling and cell migration is already well established. In addition, uPA was reported to stabilize p53, a key cell cycle control, DNA repair and apoptosis initiation protein. We aimed to determine the role of uPA-uPAR signaling towards cell survival or apoptosis in human adult cardiac myocytes (HACM). HACM were stimulated with uPA and DNA damage was inflicted by incubating cells with 200 µM H 2 O 2 . To analyze for apoptotic cells we applied TUNEL staining. Oxidative damage foci were analyzed by staining for 8-oxoguanine base pairs. In vivo qPCR analysis from RNA extracted from failing human hearts demonstrated a close relation of uPA with apoptosis and the p53 pathway. Furthermore, we observed a close correlation of uPA and p53 protein in homogenized tissue lysates. In vitro studies revealed that uPA preincubation protected HACM from oxidative damage induced cell death and reduced oxidative damage foci. uPA protection is independent of its catalytic activity, as the amino terminal fragment of uPA showed similar protection. A key enzyme for repairing oxidative DNA damage is the p53 target hOGG1. We found a significant increase of hOGG1 after pretreatment of HACM with uPA. Knockdown of hOGG1 completely abrogated the protective effect of uPA. We conclude that uPA might have a tissue protective role in human hearts besides its role in tissue remodeling. Tissue protection is mediated by the DNA repair protein hOGG1. This might be beneficial during tissue remodeling and thus could be a target for therapeutic approaches in the diseased heart.

Aims: Levosimendan is an inodilator for the treatment of acute decompensated heart failure (HF). Data from clinical studies suggest that levosimendan is particularly effective in HF due to myocardial infarction. After acute revascularization, no reflow-phenomenon is a common complication that may lead to pump failure and cardiogenic shock. Our aim was to examine whether levosimendan interferes with the pro-thrombotic phenotype of activated endothelial cells in vitro., Methods: Human heart microvascular endothelial cells (HHMEC) and human umbilical vein endothelial cells (HUVEC) were treated with interleukin-1β (IL-1β) (200U/mL) or thrombin (5U/mL) and co-treated with or without levosimendan (0.1-10μM) for 2-24h. In addition, flow experiments were performed. Effects on plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) expression and activity were measured by rt-PCR, specific ELISA and flow cytometry., Results: Treatment with IL-1β or thrombin significantly increased the expression of PAI-1 and TF in endothelial cells. Co-treatment with levosimendan strongly attenuated the effects of IL-1β and thrombin on PAI-1 and TF mRNA by up to 50% and 45%, in a dose- and time-dependent manner. Similar results were obtained under flow conditions. Furthermore, co-treatment with levosimendan dampened the antigen production of PAI-1 and the surface expression of TF by 35% and 45%, respectively. Additionally, levosimendan diminished both TF and PAI-1 activity., Conclusion: Levosimendan down-regulates the expression of the pro-thrombotic and anti-fibrinolytic biomolecules TF and PAI-1 in activated human endothelial cells. Our findings may, at least in part, explain some of the beneficial effects of levosimendan after myocardial reperfusion., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Human monocytes are a heterogeneous cell population, which can be divided into a classical (CD14++CD16-), a non-classical (CD14+CD16+), and an intermediate (CD14++CD16+) subset. We hypothesized that low-grade inflammation may differentially affect monocyte subsets. We used a human lipopolysaccharide (LPS) infusion model to mimic low-grade inflammation to identify, which monocyte subsets are preferentially activated under these conditions. Monocyte subsets were identified by staining for CD14 and CD16, activation status of monocytes was analyzed by staining for CD11b and a novel in situ mRNA hybridization approach to detect IL-6 and IL-8 specific mRNA at the single-cell level by flow cytometry. After LPS challenge, cell numbers of monocyte subsets dropped after 2 h with cell numbers recovering after 6 h. Distribution of monocyte subsets was skewed dramatically towards the intermediate subset after 24 h. Furthermore, intermediate monocytes displayed the largest increase of CD11b expression after 2 h. Finally, IL-6 and IL-8 mRNA levels increased in intermediate and non-classical monocytes after 6 h whereas these mRNA levels in classical monocytes changed only marginally. In conclusion, our data indicates that the main responding subset of monocytes to standardized low-grade inflammation induced by LPS in humans is the CD14++CD16+ intermediate subset followed by the CD14+CD16+ non-classical monocyte subset. Circulating classical monocytes showed comparably less reaction to LPS challenge in vivo.

Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aging is a major factor predisposing for multiple diseases. Telomeres at the ends of chromosomes protect the integrity of chromosomal DNA. A specialized six-protein complex termed shelterin protects the telomere from unwanted interaction with DNA damage pathways. The aim of our study was to evaluate the integrity of telomeres and the stability of telomere protection during aging in endothelial cells (EC). We describe that aging EC can be characterized by an increased cell size (40%, p=0.02) and increased expression of PAI 1 (4 fold, p=0.02), MCP1 (10 fold, p=0.001) and GMCSF (15 fold, p=0.004). Telomeric state in aging cells is defined by an increased telomere oxidation (27%, p=0.01), reduced telomere length (62%, p=0.02), and increased DNA damage foci formation (5% in young EC versus 16% in aged EC, p=0.003). This telomeric dysfunction is accompanied by a reduction in the shelterin component TRF1 (33% mRNA, p=0.001; 24% protein, p=0.007). Overexpression of TRF1 in aging EC reduced telomere-associated DNA damage foci to 5% (p=0.02) and reduced expression levels of MCP1 (18% reduction, p=0.008). Aged EC have increased telomere damage and an intrinsic loss of telomere protection. Reestablishing telomere integrity could therefore be a target for rejuvenating endothelial cell function., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objectives: Despite underlying pathologies leading to ICU admittance are heterogeneous, many patients develop a systemic inflammatory response syndrome often in the absence of microbial pathogens. Mitochondrial DNA that shows similarities to bacterial DNA may be released after tissue damage and activates the innate immune system by binding to toll-like receptor-9 on immune cells. The aim of this study was to analyze whether levels of mitochondrial DNA are associated with 30-day survival and whether this predictive value is modified by the expression of its receptor toll-like receptor-9., Design: Single-center, prospective, observational study., Setting: A tertiary ICU in a university hospital., Patients: Two hundred twenty-eight consecutive patients admitted to a medical ICU between August 2012 and August 2013., Interventions: None., Measurements and Main Results: Blood was taken within 24 hours after ICU admission, and the levels of circulating mitochondrial DNA were quantified by real-time polymerase chain reaction. Toll-like receptor-9 expression in monocytes was measured by flow cytometry. Median acute physiology and chronic health evaluation II score was 20, and 30-day mortality was 25%. Median mitochondrial DNA levels at admission were significantly higher in nonsurvivors when compared with survivors (26.9, interquartile range = 11.2-60.6 ng/mL vs 19.7, interquartile range = 9.5-34.8 ng/mL; p < 0.05). Patients with plasma levels of mitochondrial DNA in the highest quartile (mitochondrial DNA > 38.2 ng/mL) had a 2.6-fold higher risk (p < 0.001) of dying, independently of age, gender, diagnosis, and acute physiology and chronic health evaluation II score. Mitochondrial DNA improved the c-statistic of acute physiology and chronic health evaluation II score (p < 0.05) and showed enhancement in individual risk prediction indicated by a net reclassification improvement of 32.3% (p < 0.05). Stratification of patients according to toll-like receptor-9 expression above/below median demonstrated that only patients with high expression of toll-like receptor-9 showed an increased risk associated with increased mitochondrial DNA levels (odds ratio, 2.7; p < 0.01), whereas circulating mitochondrial DNA was not associated with mortality in patients with low toll-like receptor-9 expression (odds ratio, 1.1; p = 0.98)., Conclusions: Circulating levels of mitochondrial DNA at ICU admission predict mortality in critically ill patients. This association was in particular present in patients with elevated toll-like receptor-9 expression.

Many of the aging-related morbidities, including cancer, cardiovascular disease, neurodegenerative disease, and infectious susceptibility, are linked to a decline in immune competence with a concomitant rise in proinflammatory immunity, placing the process of immune aging at the center of aging biology. Immune aging affects individuals older than 50 years and is accelerated in patients with the autoimmune disease rheumatoid arthritis. Immune aging results in a marked decline in protective immune responses and a parallel increase in tissue inflammatory responses. By studying immune cells in patients with rheumatoid arthritis, several of the molecular underpinnings of the immune aging process have been delineated, such as the loss of telomeres and inefficiencies in the repair of damaged DNA. Aging T cells display a series of abnormalities, including the unopposed up-regulation of cytoplasmic phosphatases and the loss of glycolytic competence, that alter their response to stimulating signals and undermine their longevity. Understanding the connection between accelerated immune aging and autoimmunity remains an area of active research. With increasing knowledge of the molecular pathways that cause immunosenescence, therapeutic interventions can be designed to slow or halt the seemingly inevitable deterioration of protective immunity with aging., (Copyright © 2014 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.)

Background: Accurate risk prediction is important for an adequate management of heart failure (HF) patients. We assessed the incremental prognostic ability of a multi-biomarker approach in advanced HF., Methods: In 349 patients with advanced HF (median 75 years, 66% male) we investigated the incremental prognostic value of 12 novel biomarkers involved in different pathophysiological pathways including inflammation, immunological activation, oxidative stress, cell growth, remodeling, angiogenesis and apoptosis., Results: During a median follow-up of 4.9 years 55.9% of patients died. Using multivariable Cox regression and bootstrap variable-selection age, chronic obstructive pulmonary disease, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and the following 5 novel biomarkers were retained in the best mortality prediction model: the chemokine fractalkine, the angiogenic and mitogenic hepatocyte growth factor (HGF), the growth differentiation factor 15 (GDF-15) influencing cardiac remodeling and apoptosis, and the 2 pro-apoptotic molecules soluble apoptosis-stimulating fragment (sFAS) and soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). This multi-biomarker score had strong discriminatory power for 5-year mortality (area under the Receiver Operating Characteristic curve [AUC]=0.81) and improved risk prediction beyond the prognostic power of a comprehensive conventional risk score including known clinical predictors and NT-proBNP (AUC=0.77). Net reclassification confirmed a significant improvement of individual risk prediction (p=0.003)., Conclusions: Risk prediction by a multi-biomarker score is superior to a conventional risk score including clinical parameters and NT-proBNP. Additional predictive information from different biological pathways reflects the multisystemic character of HF., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Immunological processes are implicated in the multifactorial pathophysiology of heart failure (HF). The multifunctional chemokine fractalkine (CX3CL1) promotes the extravasation of cytotoxic lymphocytes into tissues. We aimed to assess the prognostic value of fractalkine in HF. Fractalkine plasma levels were determined in 349 patients with advanced systolic HF (median 75 years, 66% male). During a median follow-up of 4.9 years (interquartile range: 4.6-5.2), 55.9% of patients died. Fractalkine was a significant predictor of all-cause mortality (p<0.001) with a hazard ratio of 2.78 (95% confidence interval: 1.95-3.95) for the third compared to the first tertile. This association remained significant after multivariable adjustment for demographics, clinical predictive variables and N-terminal pro-B-type natriuretic peptide (NT-proBNP, p=0.008). The predictive value of fractalkine did not significantly differ between patients with ischaemic and non-ischaemic HF aetiology (p=0.79). The predictive value of fractalkine tertiles was not significantly modified by tertiles of NT-proBNP (p=0.18) but was more pronounced in the first and third tertile of NT-proBNP. Fractalkine was also an independent predictor of cardiovascular mortality (p=0.015). Fractalkine levels were significantly lower in patients on angiotensin-converting enzyme inhibitor therapy (p<0.001). In conclusion, circulating fractalkine with its pro-inflammatory and immunomodulatory effects is an independent predictor of mortality in advanced HF patients. Fractalkine improves risk prediction beyond NT-proBNP and might therefore help to identify high risk patients who need special care. Our data indicate the implication of immune modulation in HF pathology.

Immune aging is associated with loss of critical immune functions, such as host protection from infection and malignancy. Unexpectedly, immunosenescence also renders the host susceptible to inflammation, which may translate into tissue-damaging disease as the senescent immune system loses its ability to maximize inflammatory protection while minimizing inflammatory injury. On the other hand, chronic inflammation associated with immune-mediated disease represents a profound stress factor for the immune system, affecting cellular turn-over, replication and exhaustion. Immune cell longevity is tightly connected to the functional integrity of telomeres which are regulated by cell multiplication, exposure to oxidative stress and DNA repair mechanisms. Lymphocytes are amongst the few cell types that can actively elongate telomeres through the action of telomerase. In patients with the autoimmune disease rheumatoid arthritis (RA), telomerase deficiency is associated with prematurity of immune aging. Patients with RA have other defects in DNA repair mechanisms, including the kinase Ataxia telangiectasia mutated (ATM), critically involved in the repair of DNA double strand breaks. ATM deficiency in RA shortens lymphocyte survival. Dynamics of telomeric length and structure are beginning to be understood and have distinct patterns in different autoimmune diseases, suggesting a multitude of molecular mechanisms defining the interface between chronic immune stimulation and progressive aging of the immune system.

Objective: To assess the prognostic value of the mitogenic, antiapoptotic, angiogenic and antifibrotic hepatocyte growth factor (HGF) in heart failure (HF)., Design: Prospective cohort study., Setting/patients: Assessment of HGF levels at inclusion in 351 patients with advanced HF (median 75 years, interquartile range (IQR) 63-82, 66% male)., Main Outcome Measures: All-cause mortality, cardiovascular mortality., Results: During a median follow-up of 16&emsp14;months, 26% of patients died. HGF tertiles were associated with an increasing risk for all-cause mortality (p < 0.001) with a hazard ratio (HR) of 3.06 (95% confidence interval (CI) 1.69 to 5.53) for the third compared with the first tertile. This association remained significant after multivariable adjustment for B-type natriuretic peptide (BNP) and other risk factors (p = 0.002). Subgroup analysis revealed that HGF was a strong predictor of the secondary end point cardiovascular mortality in ischaemic HF (p = 0.009) with an adjusted HR of 6.2 (95% CI 1.76 to 21.8) comparing the third with the first tertile but not in non-ischaemic HF (HR = 1.47, 95% CI 0.48 to 4.49, p = 0.5). Patients with high HGF but low BNP had a comparable survival rate to those with elevated BNP but low HGF (p=0.66). Of note, the dose of angiotensin converting enzyme (ACE) inhibitors inversely correlated with HGF concentrations (r = -0.25, p < 0.001)., Conclusions: HGF is a strong and independent predictor of mortality in advanced HF and, in particular, in ischaemic HF. These results indicate that HGF with its multiple effects on myocardial function exerts an overall deleterious effect in advanced HF. HGF may be of special interest for risk prediction and tailoring of HF treatment.

Purpose of Review: Inflammation is a key component in cardiovascular disease. Controlling inflammatory events and their subsequent processes holds the potential for novel therapeutic treatment options. Cytokines are the propagators of inflammation. In this review we will discuss important cytokines including IL-6, TNF-α, MCP-1, fractalkine, M-CSF and GDF-15, and their effect on cardiac outcome., Recent Findings: Recent studies have shed light on the role of IL-6 in cardiovascular disease. Long-term IL-6 levels are highly associated with coronary heart disease. Molecular studies indicate that a permanent prolongation of STAT signaling in cardiac myocytes might be a potential reason for the detrimental effects of IL-6. TNF-α was long considered to have detrimental effects on myocardial function but recent studies show cardioprotective mechanisms for TNF-α. Macrophage modulating cytokines emerge as interesting molecular targets to treat cardiovascular disease. Especially, the two different subtypes of monocytes, a pro-inflammatory and a reparative subset, and their different chemotactic properties might be possible drug targets. Finally, we discuss GDF-15, which emerges as a novel biomarker in cardiovascular disease reflecting information from several pathological pathways., Summary: Cytokines are the main proximal mediators of inflammation and hold the potential of being good molecular targets for novel treatment regimes. Cytokines might be valuable biomarkers, adding information about the pathologic pathways in cardiovascular disease.

The pleiotropic cytokine oncostatin M (OSM), a member of the glycoprotein (gp)130 ligand family, plays a key role in inflammation and cardiovascular disease. As inflammation precedes and accompanies pathological angiogenesis, we investigated the effect of OSM and other gp130 ligands on vascular endothelial growth factor (VEGF) production in human vascular smooth muscle cells (SMC). Human coronary artery SMC (HCASMC) and human aortic SMC (HASMC) were treated with different gp130 ligands. VEGF protein was determined by ELISA. Specific mRNA was detected by RT-PCR. Western blotting was performed for signal transducers and activators of transcription1 (STAT1), STAT3, Akt and p38 mitogen-activated protein kinase (p38 MAPK). OSM mRNA and VEGF mRNA expression was analyzed in human carotid endaterectomy specimens from 15 patients. OSM increased VEGF production in both HCASMC and HASMC derived from different donors. OSM upregulated VEGF and OSM receptor-specific mRNA in these cells. STAT3 inhibitor WP1066, p38 MAPK inhibitors SB-202190 and BIRB 0796, extracellular signal-regulated kinase1/2 (Erk1/2) inhibitor U0126, and phosphatidylinositol 3-kinase (PI3K) inhibitors LY-294002 and PI-103 reduced OSM-induced VEGF synthesis. We found OSM expression in human atherosclerotic lesions where OSM mRNA correlated with VEGF mRNA expression. Interferon-γ (IFN-γ), but not IL-4 or IL-10, reduced OSM-induced VEGF production in vascular SMC. Our findings that OSM, which is present in human atherosclerotic lesions and correlates with VEGF expression, stimulates production of VEGF by human coronary artery and aortic SMC indicate that OSM could contribute to plaque angiogenesis and destabilization. IFN-γ reduced OSM-induced VEGF production by vascular SMC.

Objective: To assess the prognostic value of the pro-apoptotic, but also cell growth-inducing molecule soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) in heart failure (HF)., Methods: We assayed sTWEAK levels in 351 patients with advanced HF (non-ischemic: 130, ischemic: 221). During a median follow-up of 4.9 years, 195 patients (56%) died., Results: sTWEAK concentrations were associated with extended survival in patients with non-ischemic (P=0.022), but not with ischemic HF (P=0.82). The inverse association in non-ischemic HF remained significant in a multivariable Cox regression model (P=0.025) with a hazard ratio of 0.40 (95% confidence interval: 0.21-0.77) comparing the third to the first tertile (P=0.007)., Conclusion: Low sTWEAK levels independently predict mortality in advanced non-ischemic HF. sTWEAK-induced proliferation of cardiomyocytes may explain its impact on suvival. The different prognostic value of sTWEAK in ischemic and non-ischemic HF may point towards distinct pathogenic pathways determining the course of disease., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Objective: Whereas angiogenesis, the formation of new blood vessels from preexisting vessels, may be beneficial in restoring failing myocardium, apoptosis may contribute to the progression of heart failure (HF). We investigated the role of pigment epithelium-derived factor (PEDF), a recently discovered antiangiogenic factor with additional proapoptotic effects, in patients with advanced HF., Methods: We assayed PEDF levels in 351 patients with advanced HF at baseline. During the median follow-up time of 16 months, 50% of patients experienced the composite end point of rehospitalization and/or death., Results: The risk of a clinical event increased with concentrations of the antiangiogenic marker PEDF, with a 1.94-fold higher risk in the third tertile compared with the first tertile (95% CI, 1.33-2.84). This association remained significant after adjustment for B-type natriuretic peptide (BNP) and other risk factors in a Cox regression model (P = .015). Experimental data revealed that PEDF may contribute to the progression of HF by inducing apoptosis in human cardiac myocytes and fibroblasts via activation of caspase 3., Conclusions: We suggest a role of PEDF in the progression of HF by inducing apoptosis of human cardiac myocytes and fibroblasts. Our clinical data suggest that PEDF concentrations may have the potential to become a valuable marker of the prognosis of HF, in addition to BNP.

Objectives: Members of the glycoprotein 130 (gp130) receptor-gp130 ligand family play a role in angiogenesis in different tissues. We tested the effect of this cytokine family on the angiopoietin (Ang)-Tie system, which is involved in blood vessel maturation, stabilization, and regression., Results: Oncostatin M (OSM) increased Ang2 expression in human umbilical vein endothelial cells via Janus kinase/signal transducer and activator of transcription (JAK/STAT) and mitogen-activated protein (MAP) kinase activation. Furthermore, OSM induced Ang2 expression in macrovascular endothelial cells isolated from the human aorta and in microvascular endothelial cells isolated from human heart. Our in vivo experiments revealed that mRNA expression of Ang2 in hearts of mice injected with OSM increased significantly, and levels of OSM mRNA significantly correlated with mRNA levels of Ang2 in human hearts. In addition, OSM increased the expression of its own receptors, gp130 and OSM receptor, in endothelial cells in vitro and in mice in vivo, and levels of OSM mRNA significantly correlated with mRNA levels of gp130 and OSM receptor in human hearts., Conclusion: Our data, showing the effects of OSM on the Ang-Tie system in endothelial cells, in hearts of mice, and in human heart tissue, provide yet another link between inflammation and angiogenesis.

Cytokines regulating the mobilisation, recruitment and survival of mononuclear cells may play an important role in progression of heart failure. Therefore, we investigated the role of granulocyte colony stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1) and macrophage colony stimulating factor (M-CSF) in patients with advanced heart failure. G-CSF, MCP-1 and M-CSF were determined in plasma of 351 patients with advanced heart failure by specific ELISAs. During a median follow up period of 16 months (95% confidence interval [CI]: 15-17 months) 175 patients (50%) experienced the composite endpoint rehospitalisation and all-cause mortality. M-CSF tertiles were associated with a gradually increasing risk with hazard ratios (HR) of 2.2 (95% CI: 1.5-3.2; for trend, p<0.001) for the composite endpoint and 2.6 (95% CI: 1.5-4.6; for trend, p=0.002) for all-cause mortality comparing third and first tertile. These associations remained significant in a multivariable Cox regression model after adjustment for BNP and other known risk factors (p=0.043 and p=0.024). High MCP-1 concentrations were associated with an increased risk of all-cause mortality with an adjusted HR of 1.9 (third vs. first tertile, 95% CI: 1.1-3.3; for trend, p=0.034). In contrast, G-CSF tertiles were not significantly associated with the composite endpoint or all-cause mortality in multivariable Cox regression. In conclusion, the independent and concentration-dependent association of macrophage-modulating cytokines and in particular of M-CSF with adverse outcome in advanced HF patients suggests that these cytokines may play an important pathophysiological role in progression of cardiomyopathy.

Cardiac diseases such as myocardial infarction and heart failure are among the leading causes of death in western societies. Therapeutic angiogenesis has been suggested as a concept to combat these diseases. The biology of angiogenic factors expressed in the heart such as vascular endothelial growth factor (VEGF) is well studied, whereas data on anti-angiogenic mediators in the heart are scarce. Here we study the expression of the anti-angiogenic factor pigment epithelium-derived factor (PEDF) in the human heart and in human cardiac cells. PEDF expression could be detected in human cardiac tissue on the protein and mRNA levels. PEDF mRNA levels were significantly lower in explanted human ischemic hearts as compared to healthy hearts. Our in vitro experiments showed that human adult cardiac myocytes and fibroblasts constitutively secrete PEDF. In addition to anoxic conditions, cobalt chloride, 2,2'dipyridyl and dimethoxally glycine, which stabilize hypoxia inducible factor-alpha decreased PEDF expression. Furthermore we show that PEDF inhibits VEGF-induced sprouting. We have identified PEDF in healthy and ischemic human hearts and we show that PEDF expression is down-regulated by low oxygen levels. Therefore, we suggest a role for PEDF in the regulation of angiogenesis in the heart and propose PEDF as a possible therapeutic target in heart disease.

Although the nervous and vascular systems are functionally different, they show a high degree of anatomic parallelism and cross-talk. They also share similar mechanisms and molecular cues that regulate their development and maintenance. Malfunctioning of this cross-talk can cause or influence several vascular and neuronal disorders. In this review, we first provide a brief overview of the molecular and cellular mechanisms that govern the neurovascular link. Second, we focus on two neurodegenerative diseases, Alzheimer's disease and amyotrophic lateral sclerosis, to illustrate how a defective neurovascular link might contribute to their pathogenesis. Finally, we briefly discuss some therapeutic implications of the neurovascular link for designing strategies to treat these diseases.

Filopodia, "the fingers that do the walking," have been identified on endothelial cells at the tip of sprouting vessels for half a century, but the key role of the tip cell in vessel branching has been recognized only in the past few years. A model is emerging, whereby tip cells lead the way in a branching vessel, stalk cells elongate the sprout, and a very recently discovered phalanx cell ensures quiescence and perfusion of the newly formed branch. Recent genetic studies have shed light on the molecular signature of these distinct endothelial phenotypes; this provides a novel conceptual framework of how vessel morphogenesis occurs. Here, we will discuss the molecular candidates that participate in the decision of endothelial cells to adapt these distinct fates and highlight the emerging insights on how these cells send out filopodia while navigating.

Aims: Apoptosis plays an important role in the progression of heart failure (HF). The purpose of this study was to assess whether the pro-apoptotic molecules apoptosis-stimulating fragment (FAS, CD95/APO-1) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) predict event-free survival of HF patients., Methods and Results: We assayed soluble (s)FAS and sTRAIL levels in 351 patients with advanced HF. During the median follow-up time of 16 months, 175 patients (50%) experienced the composite endpoints: rehospitalization and death. The hazard increased with sFAS concentrations, with a hazard ratio of 2.3 comparing fourth and first quartiles. This association remained significant after adjustment for B-type natriuretic peptide (BNP) and other risk factors in a Cox regression model (P = 0.014). Patients with high sFAS but low BNP had a comparable event-free survival rate with those with elevated BNP only (P = 0.78). Conversely, high sTRAIL concentrations were related to a better prognosis. Particularly, the risk of mortality dropped by 70% in the fourth quartile of sTRAIL (P = 0.001, multivariable Cox regression model)., Conclusion: sFAS is an independent risk predictor in advanced HF patients. It may be of particular value for the identification of high-risk patients in addition to BNP. Conversely, sTRAIL appears to be protective and could be an interesting therapeutic agent.

Stromal derived factor 1 (SDF-1) is a CXC chemokine important in the homing process of stem cells to injured tissue. It has been implicated in healing and tissue repair. Growing evidence suggests that the glycoprotein-130 (gp130) ligand family is involved in repair processes in the heart. The aim of our study was to determine whether gp130 ligands could affect SDF-1 expression in cardiac cells. Human adult cardiac myocytes (HACMs) and fibroblasts (HACFs) were treated with gp130 ligands. Protein and mRNA levels of SDF-1 were determined using ELISA and RT-PCR, respectively. mRNA levels of SDF-1 were determined in human and mouse heart samples by RT-PCR. HACMs and HACFs constitutively express SDF-1, which was significantly up-regulated by the gp130 ligand oncostatin M (OSM). This effect was counteracted by a p38 inhibitor and to a lesser extent by a PI3K inhibitor. mRNA expression of SDF-1 in hearts of mice injected with OSM increased significantly. Levels of OSM and SDF-1 mRNA correlated significantly in human failing hearts. Our data, showing that OSM induces SDF-1 protein secretion in human cardiac cells in vitro and murine hearts in vivo, suggest that OSM via the induction of SDF-1 might play a key role in repair and tissue regeneration.

Introduction: Macrophage colony stimulating factor (M-CSF) is a key factor for monocyte and macrophage survival and proliferation. M-CSF has been implicated in cardiac healing and repair after myocardial infarction., Methods and Results: We show by immunohistochemistry and Western blotting analysis that M-CSF protein is present in human heart tissue. Cultured human adult cardiac myocytes (HACM) and human adult cardiac fibroblasts (HACF) isolated from human myocardial tissue constitutively express M-CSF. When HACM and HACF were treated with tumor necrosis factor-alpha (TNF-alpha) M-CSF protein production and M-CSF mRNA expression, determined by ELISA or by using RT-PCR, respectively, was significantly increased. To determine a possible role of nuclear factor kappaB (NF-kappaB) and activating protein 1 (AP-1) in M-CSF regulation, blockers to both pathways and an adenovirus overexpressing a dominant negative (dn) form of IkappaB kinase 2 (IKK2) were used. Only the NF-kappaB blocker dimethylfumarate and the dn IKK2, but not januskinase inhibitor-1 (JNK-I), were able to block the TNF-alpha-induced increase in M-CSF production in these cells, suggesting that the induction of M-CSF through TNF-alpha is mainly dependent on the activation of the NF-kappaB pathway. The monocyte activation marker CD11b was significantly increased after incubating U937 cells with conditioned medium from HACM or HACF as determined by FACS analysis., Conclusions: Our in vitro data taken together with our immunohistochemistry data suggest that human cardiac cells constitutively express M-CSF. This expression of M-CSF in the human heart and its upregulation by TNF-alpha might contribute to monocyte and macrophage survival and differentiation.