Interleukin-4 receptor alpha signaling regulates monocyte homeostasis.

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-4Rαf/f LysMcre ), 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-4Rαf/f LysMcre mice without changes in overall migratory function. Thus, we identified a homeostatic role of IL-4Rα in regulating the lifespan of monocytes in vivo.

Innate Immune Training with Bacterial Extracts Enhances Lung Macrophage Recruitment to Protect from Betacoronavirus Infection.

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.

The plasminogen receptor Plg-RKT regulates adipose function and metabolic homeostasis.

BACKGROUND: Plg-RKT , a unique transmembrane plasminogen receptor, enhances the activation of plasminogen to plasmin, and localizes the proteolytic activity of plasmin on the cell surface. OBJECTIVES: We investigated the role of Plg-RKT in adipose function, metabolic homeostasis, and obesity. METHODS: We used adipose tissue (AT) sections from bariatric surgery patients and from high fat diet (HFD)-induced obese mice together with immunofluorescence and real-time polymerase chain reaction to study adipose expression of Plg-RKT . Mice genetically deficient in Plg-RKT and littermate controls fed a HFD or control low fat diet (LFD) were used to determine the role of Plg-RKT in insulin resistance, glucose tolerance, type 2 diabetes, and associated mechanisms including adipose inflammation, fibrosis, and ectopic lipid storage. The role of Plg-RKT in adipogenesis was determined using 3T3-L1 preadipocytes and primary cultures established from Plg-RKT -deficient and littermate control mice. RESULTS: Plg-RKT was highly expressed in both human and mouse AT, and its levels dramatically increased during adipogenesis. Plg-RKT -deficient mice, when fed a HFD, gained more weight, developed more hepatic steatosis, and were more insulin resistant/glucose intolerant than HFD-fed wild-type littermates. Mechanistically, these metabolic defects were linked with increased AT inflammation, AT macrophage and T-cell accumulation, adipose and hepatic fibrosis, and decreased insulin signaling in the AT and liver. Moreover, Plg-RKT regulated the expression of PPARγ and other adipogenic molecules, suggesting a novel role for Plg-RKT in the adipogenic program. CONCLUSIONS: Plg-RKT coordinately regulates multiple aspects of adipose function that are important to maintain efficient metabolic homeostasis.

MiRNA Let-7a and Let-7d Are Induced by Globotriaosylceramide via NF-kB Activation in Fabry Disease.

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.

Pharmacological inhibition of fatty acid oxidation reduces atherosclerosis progression by suppression of macrophage NLRP3 inflammasome activation.

BACKGROUND: Inflammation is a key process during atherosclerotic lesion development and propagation. Recent evidence showed clearly that especially the inhibition of interleukin (IL)-1ß 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-1ß 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-1ß 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-1ß and cleaved caspase-1 in the atherosclerotic lesion as well as of IL-1ß 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-1ß.

Alternative activation of human macrophages enhances tissue factor expression and production of extracellular vesicles.

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.

Assessment of a long-term in vitro model to characterize the mechanical behavior and macrophage-mediated degradation of a novel, degradable, electrospun poly-urethane vascular graft.

An assessment tool to evaluate the degradation of biodegradable materials in a more physiological environment is still needed. Macrophages are critical players in host response, remodeling and degradation. In this study, a cell culture model using monocyte-derived primary macrophages was established to study the degradation, macro-/micro-mechanical behavior and inflammatory behavior of a new designed, biodegradable thermoplastic polyurethane (TPU) scaffold, over an extended period of time in vitro. For in vivo study, the scaffolds were implanted subcutaneously in a rat model for up to 36 weeks. TPU scaffolds were fabricated via the electrospinning method. This technique provided a fibrous scaffold with an average fiber diameter of 1.39 ± 0.76 µm and an average pore size of 7.5 ± 1.1 µm. The results showed that TPU scaffolds supported the attachment and migration of macrophages throughout the three-dimensional matrix. Scaffold degradation could be detected in localized areas, emphasizing the role of adherent macrophages in scaffold degradation. Weight loss, molecular weight and biomechanical strength reduction were evident in the presence of the primary macrophage cells. TPU favored the switch from initial pro-inflammatory response of macrophages to an anti-inflammatory response over time both in vitro and in vivo. Expression of MMP-2 and MMP-9 (the key enzymes in tissue remodeling based on ECM modifications) was also evident in vitro and in vivo. This study showed that the primary monocyte-derived cell culture model represents a promising tool to characterize the degradation, mechanical behavior as well as biocompatibility of the scaffolds during an extended period of observation.

Neutrophil Extracellular Trap Degradation by Differently Polarized Macrophage Subsets.

OBJECTIVE: Macrophages are immune cells, capable to remodel the extracellular matrix, which can harbor extracellular DNA incorporated into neutrophil extracellular traps (NETs). To study the breakdown of NETs we studied the capability of macrophage subsets to degrade these structures in vitro and in vivo in a murine thrombosis model. Furthermore, we analyzed human abdominal aortic aneurysm samples in support of our in vitro and in vivo results. Approach and Results: Macrophages were seeded onto blood clots or isolated NETs and polarized. All macrophages were capable to degrade NETs. For initial breakdown, macrophages relied on extracellular deoxyribonucleases. Proinflammatory polarization enhanced NET degradation. The boost in degradation was because of increased macropinocytosis, as inhibition by imipramine diminished their NET breakdown. Inhibition of macropinocytosis in a murine thrombosis model led to increased NET burden and reduced thrombus resolution in vivo. When analyzing abdominal aortic aneurysm samples, macrophage density furthermore corresponded negatively with the amount of local NETs in the intraluminal thrombi as well as in the vessel wall, as increased macrophage density was associated with a reduction in NET burden. CONCLUSIONS: We provide evidence that macrophages degrade NETs by extracellular predigestion and subsequent uptake. Furthermore, we show that proinflammatory macrophages increase NET degradation through enhanced macropinocytosis, priming them for NET engulfment. Based on our findings, that inhibition of macropinocytosis in mice corresponded to increased NET amounts in thrombi and that local macrophage density in human abdominal aortic aneurysm is negatively associated with surrounding NETs, we hypothesize, that macrophages are able to degrade NETs in vivo.

The pro-inflammatory marker soluble suppression of tumorigenicity-2 (ST2) is reduced especially in diabetic morbidly obese patients undergoing bariatric surgery.

BACKGROUND: High soluble suppression of tumorigenicity-2 (sST2) is a marker of poor prognosis in chronic inflammatory conditions. ST2 and its ligand interleukin (IL)-33 are elevated in adipose tissue of obese individuals. We aimed to evaluate circulating sST2 and IL-33 as possible markers of metabolic benefit in morbidly overweight patients after Roux-en-Y gastric bypass (RYGB) bariatric surgery. METHODS: sST2, IL-33, high sensitive IL-6, high sensitive C-reactive protein (hsCRP), leptin, cholesterol metabolism and liver parameters were measured in 80 morbidly obese individuals before and 1 year after bariatric surgery. RESULTS: sST2 was higher (P = 0.03) in diabetics as compared to individuals without diabetes. Baseline sST2 was also higher in males than in females (P= 0.0002). One year after bariatric surgery, sST2 levels were decreased (median 120, IQR 59-176 pg/mL) as compared to sST2 before surgery (median 141, IQR 111-181, P = 0.0024), and the diabetic group showed most pronounced reduction in sST2 (P = 0.0016). An association was found between sST2 and liver function parameters before and after bariatric surgery, and between baseline sST2 and total cholesterol, triglyceride, total low density lipoprotein (LDL), small dense LDL, Apolipoprotein B as well as with small dense high density lipoproteins (HDL). In the subgroup of diabetic patients positive correlation between IL-33 and sST2 (r = 0.44, P = 0.05) was noticed. CONCLUSIONS: Circulating sST2 is associated with markers of liver functions and lipid metabolism in severely obese patients and a reduction of sST2 was shown after successful bariatric surgery, most prominently in diabetic patients.

Long Term Evaluation of Nanofibrous, Bioabsorbable Polycarbonate Urethane Grafts for Small Diameter Vessel Replacement in Rodents.

OBJECTIVE: Biodegradable materials for in situ vascular tissue engineering could meet the increasing clinical demand for sufficient synthetic small diameter vascular substitutes in aortocoronary bypass and peripheral vascular surgery. The aim of this study was to design a new degradable thermoplastic polycarbonate urethane (dPCU) with improved biocompatibility and optimal biomechanical properties. Electrospun conduits made from dPCU were evaluated in short and long term follow up and compared with expanded polytetrafluoroethylene (ePTFE) controls. METHODS: Both conduits were investigated prior to implantation to assess their biocompatibility and inflammatory potential via real time polymerase chain reaction using a macrophage culture. dPCU grafts (n = 28) and ePTFE controls (n = 28) were then implanted into the infrarenal abdominal aorta of Sprague-Dawley rats. After seven days, one, six, and 12 months, grafts were analysed by histology and immunohistochemistry (IHC) and assessed biomechanically. RESULTS: Anti-inflammatory signalling was upregulated in dPCU conduits and increased significantly over time in vitro. dPCU and ePTFE grafts offered excellent long and short term patency rates (92.9% in both groups at 12 months) in the rat model without dilatation or aneurysm formation. In comparison to ePTFE, dPCU grafts showed transmural ingrowth of vascular specific cells resulting in a structured neovessel formation around the graft. The graft material was slowly reduced, while the compliance of the neovessel increased over time. CONCLUSION: The newly designed dPCU grafts have the potential to be safely applied for in situ vascular tissue engineering applications. The degradable substitutes showed good in vivo performance and revealed desirable characteristics such as biomechanical stability, non-thrombogenicity, and minimal inflammatory response after long term implantation.

Differential expression of Plg-RKT and its effects on migration of proinflammatory monocyte and macrophage subsets.

Membrane-bound plasmin is used by immune cells to degrade extracellular matrices, which facilitates migration. The plasminogen receptor Plg-RKT 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-RKT by monocyte and macrophage subsets and whether potential differential expression might have functional consequences for cell migration. Proinflammatory CD14++CD16+ human monocytes and Ly6Chigh mouse monocytes expressed the highest levels of Plg-RKT 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-RKT 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-RKT monoclonal antibody, ε-amino-caproic acid, aprotinin, and the aminoterminal fragment of urokinase-type plasminogen activator. In an in vivo peritonitis model, significantly less Ly6Chigh monocyte recruitment was observed in Plg-RKT -/- compared with Plg-RKT +/+ mice. Immunohistochemical analysis of human carotid plaques and adipose tissue showed that proinflammatory macrophages also exhibited high levels of Plg-RKT in vivo. Our data demonstrate higher expression of Plg-RKT on proinflammatory monocyte and macrophage subsets that impacts their migratory capacity.

Mitochondria Are a Subset of Extracellular Vesicles Released by Activated Monocytes and Induce Type I IFN and TNF Responses in Endothelial Cells.

RATIONALE: Extracellular vesicles, including microvesicles, are increasingly recognized as important mediators in cardiovascular disease. The cargo and surface proteins they carry are considered to define their biological activity, including their inflammatory properties. Monocyte to endothelial cell signaling is a prerequisite for the propagation of inflammatory responses. However, the contribution of microvesicles in this process is poorly understood. OBJECTIVE: To elucidate the mechanisms by which microvesicles derived from activated monocytic cells exert inflammatory effects on endothelial cells. METHODS AND RESULTS: LPS (lipopolysaccharide)-stimulated monocytic cells release free mitochondria and microvesicles with mitochondrial content as demonstrated by flow cytometry, quantitative polymerase chain reaction, Western Blot, and transmission electron microscopy. Using RNAseq analysis and quantitative reverse transcription-polymerase chain reaction, we demonstrated that both mitochondria directly isolated from and microvesicles released by LPS-activated monocytic cells, as well as circulating microvesicles isolated from volunteers receiving low-dose LPS-injections, induce type I IFN (interferon), and TNF (tumor necrosis factor) responses in endothelial cells. Depletion of free mitochondria significantly reduced the ability of these microvesicles to induce type I IFN and TNF-dependent genes. We identified mitochondria-associated TNFα and RNA from stressed mitochondria as major inducers of these responses. Finally, we demonstrated that the proinflammatory potential of microvesicles and directly isolated mitochondria were drastically reduced when they were derived from monocytic cells with nonrespiring mitochondria or monocytic cells cultured in the presence of pyruvate or the mitochondrial reactive oxygen species scavenger MitoTEMPO. CONCLUSIONS: Mitochondria and mitochondria embedded in microvesicles constitute a major subset of extracellular vesicles released by activated monocytes, and their proinflammatory activity on endothelial cells is determined by the activation status of their parental cells. Thus, mitochondria may represent critical intercellular mediators in cardiovascular disease and other inflammatory settings associated with type I IFN and TNF signaling.

Changes of Circulating Extracellular Vesicles from the Liver after Roux-en-Y Bariatric Surgery.

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.

The Relevance of Vascular Endothelial Growth Factor, Hypoxia Inducible Factor-1 Alpha, and Clusterin in Carotid Plaque Instability.

BACKGROUND: Stroke is one of the leading causes of morbidity and mortality. Thromboembolism, as a major cause of carotid artery-related stroke, can be caused by plaque rupture which is associated with neoangiogenesis within the carotid plaque. AIM: We sought to investigate a possible correlation between angiogenesis-related factors and preoperative neurological manifestations in patients with internal carotid artery stenosis, for a better understanding of thromboembolism in internal carotid artery stenosis-related stroke. METHODS: This study included 54 patients (asymptomatic, n = 20 and symptomatic, n = 34) undergoing carotid endarterectomy for high-grade internal carotid artery stenosis. In the retrieved carotid plaques, angiogenesis-related factors (vascular endothelial growth factor [VEGF], hypoxia inducible factor-1 alpha [HIF-1α], and Clusterin) were measured by immunohistochemistry and quantified by real-time polymerase chain reaction. RESULTS: We demonstrated the expression of VEGF, HIF-1α, and Clusterin by endothelial cells and smooth muscle cells in the carotid plaques. Noteworthy, mRNA VEGF levels were .7-fold higher in symptomatic patients (P = .017) compared to asymptomatic patients. In contrast, mRNA Clusterin levels were 1.8-fold lower (P = .021). Levels of mRNA HIF-1α were 1.5-fold higher in asymptomatic patients, but no statistical significance was reached between the 2 groups. CONCLUSIONS: Our results show an association between VEGF and Clusterin and neurological symptoms of patients with high-grade carotid artery stenosis.

Bariatric surgery in morbidly obese individuals affects plasma levels of protein C and thrombomodulin.

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.

Pretreatment With Argon Protects Human Cardiac Myocyte-Like Progenitor Cells from Oxygen Glucose Deprivation-Induced Cell Death by Activation of AKT and Differential Regulation of Mapkinases.

BACKGROUND: The noble gas argon induces cardioprotection in a rabbit model of myocardial ischemia and reperfusion. However, no studies in human primary cells or subjects have been performed so far. We used human cardiac myocyte-like progenitor cells (HCMs) to investigate the protective effect on the cellular level. METHODS: HCMs were pretreated with 30% or 50% argon before oxygen-glucose deprivation (OGD) and reperfusion. We evaluated apoptotic states by flow cytometry and the activation of mitogen-activated protein kinase (MAPKs) members extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), p38 MAPkinase, and protein kinase B (Akt) by Westernblot analysis and by activity assays of downstream transcription factors. Specific inhibitors were used to proof a significant participation of these pathways in the protection by argon. Beneficial effects were further assessed by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay, lactate dehydrogenase (LDH), mitochondrial deoxyribonucleic acid (mtDNA), and cytokine release. RESULTS: Pretreatment with 30% or 50% argon for 90 min before OGD resulted in a significant protection of HCMs against apoptosis. This effect was reversed by the application of MAPK and Akt inhibitors during argon exposure. Argon 30% reduced the release of LDH by 33% and mtDNA by 45%. The release of interleukin 1ß was reduced by 44% after OGD and more than 90% during reperfusion. CONCLUSIONS: Pretreatment with argon protects HCMs from apoptosis under ischemic conditions via activation of Akt, Erk, and biphasic regulation of JNK. Argon gas is cheap and easily administrable, and might be a novel therapy to reduce myocardial ischemia-reperfusion injury.

Urokinase plasminogen activator protects cardiac myocytes from oxidative damage and apoptosis via hOGG1 induction.

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 H2O2. 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.

Hyperoxia Induces Inflammation and Cytotoxicity in Human Adult Cardiac Myocytes.

Supplemental oxygen (O2) is used as adjunct therapy in anesthesia, emergency, and intensive care medicine. We hypothesized that excessive O2 levels (hyperoxia) can directly injure human adult cardiac myocytes (HACMs). HACMs obtained from the explanted hearts of transplantation patients were exposed to constant hyperoxia (95% O2), intermittent hyperoxia (alternating 10 min exposures to 5% and 95% O2), constant normoxia (21% O2), or constant mild hypoxia (5% O2) using a bioreactor. Changes in cell morphology, viability as assessed by lactate dehydrogenase (LDH) release and trypan blue (TB) staining, and secretion of vascular endothelial growth factor (VEGF), macrophage migration inhibitory factor (MIF), and various pro-inflammatory cytokines (interleukin, IL; chemokine C-X-C motif ligand, CXC; granulocyte-colony stimulating factor, G-CSF; intercellular adhesion molecule, ICAM; chemokine C-C motif ligand, CCL) were compared among treatment groups at baseline (0 h) and after 8, 24, and 72 h of treatment. Changes in HACM protein expression were determined by quantitative proteomic analysis after 48 h of exposure. Compared with constant normoxia and mild hypoxia, constant hyperoxia resulted in a higher TB-positive cell count, greater release of LDH, and elevated secretion of VEGF, MIF, IL-1ß, IL-6, IL-8, CXCL-1, CXCL-10, G-CSF, ICAM-1, CCL-3, and CCL-5. Cellular inflammation and cytotoxicity gradually increased and was highest after 72 h of constant and intermittent hyperoxia. Quantitative proteomic analysis revealed that hypoxic and hyperoxic O2 exposure differently altered the expression levels of proteins involved in cell-cycle regulation, energy metabolism, and cell signaling. In conclusion, constant and intermittent hyperoxia induced inflammation and cytotoxicity in HACMs. Cell injury occurred earliest and was greatest after constant hyperoxia, but even relatively brief repeating hyperoxic episodes induced a substantial inflammatory response.

Interleukin-33 stimulates GM-CSF and M-CSF production by human endothelial cells.

Interleukin (IL)-33, a member of the IL-1 family of cytokines, is involved in various inflammatory conditions targeting amongst other cells the endothelium. Besides regulating the maturation and functions of myeloid cells, granulocyte macrophage-colony stimulating factor (GM-CSF) and macrophage-CSF (M-CSF) have been shown to play a role in such pathologies too. It was the aim of our study to investigate a possible influence of IL-33 on GM-CSF and M-CSF production by human endothelial cells. IL-33, but not IL-18 or IL-37, stimulated GM-CSF and M-CSF mRNA expression and protein production by human umbilical vein endothelial cells (HUVECs) and human coronary artery ECs (HCAECs) through the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway in an IL-1-independent way. This effect was inhibited by the soluble form of ST2 (sST2), which is known to act as a decoy receptor for IL-33. The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor fluvastatin could also be shown to moderately reduce the IL-33-mediated effect on M-CSF, but not on GM-CSF expression. In addition, IL-33, IL-1ß, GM-CSF and M-CSF were detected in endothelial cells of human carotid atherosclerotic plaques using immunofluorescence. Upregulation of GM-CSF and M-CSF production by human endothelial cells, an effect that appears to be mediated by NF-κB and to be independent of IL-1, may be an additional mechanism through which IL-33 contributes to inflammatory activation of the vessel wall.

Tissue factor is induced by interleukin-33 in human endothelial cells: a new link between coagulation and inflammation.

Tissue factor (TF) is the primary trigger of coagulation. Elevated levels of TF are found in atherosclerotic plaques, and TF leads to thrombus formation when released upon plaque rupture. Interleukin (IL)-33 was previously shown to induce angiogenesis and inflammatory activation of endothelial cells (ECs). Here, we investigated the impact of IL-33 on TF in human ECs, as a possible new link between inflammation and coagulation. IL-33 induced TF mRNA and protein in human umbilical vein ECs and coronary artery ECs. IL-33-induced TF expression was ST2- and NF-κB-dependent, but IL-1-independent. IL-33 also increased cell surface TF activity in ECs and TF activity in ECs-derived microparticles. IL-33-treated ECs reduced coagulation time of whole blood and plasma but not of factor VII-deficient plasma. In human carotid atherosclerotic plaques (n = 57), TF mRNA positively correlated with IL-33 mRNA expression (r = 0.691, p < 0.001). In this tissue, IL-33 and TF protein was detected in ECs and smooth muscle cells by immunofluorescence. Furthermore, IL-33 and TF protein co-localized at the site of clot formation within microvessels in plaques of patients with symptomatic carotid stenosis. Through induction of TF in ECs, IL-33 could enhance their thrombotic capacity and thereby might impact on thrombus formation in the setting of atherosclerosis.