Search

PRINCIPLES: The morbidity and mortality of infectious diseases due to smoking are not widely appreciated by physicians. However, cigarette smoking appears to be a major risk factor for respiratory tract and other systemic infections. Only limited data are available on the association between smoking and Mycoplasma (M) pneumoniae. We raise the question of whether smoking increases the presence of M pneumoniae in circulating leukocytes. Furthermore we have studied whether the combination of smoking and M pneumoniae infection affects circulating levels of inflammatory biomarkers. METHODS: Prevalence of latent M pneumoniae infection was analysed in a total of 122 subjects by polymerase chain reaction. Smoking status was documented at presentation. Circulating levels of c-reactive protein, macrophage chemoattractant protein-1 and complement factor 5a were determined by commercial enzyme linked immuno-sorbent assays. RESULTS: We found a significant association between smoking and latent M pneumoniae infection (p = 0.009). This association remained significant after correction for age, gender and diabetes (OR 3.4; 95% CI 1.3–9.4; p = 0.017). There was no correlation between circulating levels of the inflammatory biomarkers studied and smoking or M pneumoniae infection, respectively. CONCLUSIONS: Our data indicate that smoking is associated with the presence of M pneumoniae in circulating leukocytes. This could contribute to the pro-inflammatory effects of smoking. Despite the low number of subjects included in this study, this is an interesting finding demanding further investigation.

Background: Venous thromboembolism is a major health problem. After thrombus formation, its resolution is essential to re-establish blood flow, which is crucially mediated by infiltrating neutrophils and monocytes in concert with activated platelets and endothelial cells. Thus, we aimed to modulate leukocyte function during thrombus resolution post-thrombus formation by blocking P-selectin/CD62P-mediated cell interactions., Methods: Thrombosis was induced by inferior vena cava stenosis through ligation in mice. After 1 day, a P-selectin-blocking antibody or isotype control was administered and thrombus composition and resolution were analyzed., Results: Localizing neutrophils and macrophages in thrombotic lesions of wild-type mice revealed that these cells enter the thrombus and vessel wall from the caudal end. Neutrophils were predominantly present 1 day and monocytes/macrophages 3 days after vessel ligation. Blocking P-selectin reduced circulating platelet-neutrophil and platelet-Ly6C high monocyte aggregates near the thrombus, and diminished neutrophils and Ly6C high macrophages in the cranial thrombus part compared with isotype-treated controls. Depletion of neutrophils 1 day after thrombus initiation did not phenocopy P-selectin inhibition but led to larger thrombi compared with untreated controls. In vitro, P-selectin enhanced human leukocyte function as P-selectin-coated beads increased reactive oxygen species production by neutrophils and tissue factor expression of classical monocytes. Accordingly, P-selectin inhibition reduced oxidative burst in the thrombus and tissue factor expression in the adjacent vessel wall. Moreover, blocking P-selectin reduced thrombus density determined by scanning electron microscopy and increased urokinase-type plasminogen activator levels in the thrombus, which accelerated caudal fibrin degradation from day 3 to day 14. This accelerated thrombus resolution as thrombus volume declined more rapidly after blocking P-selectin., Conclusions: Inhibition of P-selectin-dependent activation of monocytes and neutrophils accelerates venous thrombosis resolution due to reduced infiltration and activation of innate immune cells at the site of thrombus formation, which prevents early thrombus stabilization and facilitates fibrinolysis., Competing Interests: Disclosures None.

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.

Background: Plg-R KT , 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-R KT 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-R KT . Mice genetically deficient in Plg-R KT and littermate controls fed a HFD or control low fat diet (LFD) were used to determine the role of Plg-R KT in insulin resistance, glucose tolerance, type 2 diabetes, and associated mechanisms including adipose inflammation, fibrosis, and ectopic lipid storage. The role of Plg-R KT in adipogenesis was determined using 3T3-L1 preadipocytes and primary cultures established from Plg-R KT -deficient and littermate control mice., Results: Plg-R KT was highly expressed in both human and mouse AT, and its levels dramatically increased during adipogenesis. Plg-R KT -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-R KT regulated the expression of PPARγ and other adipogenic molecules, suggesting a novel role for Plg-R KT in the adipogenic program., Conclusions: Plg-R KT coordinately regulates multiple aspects of adipose function that are important to maintain efficient metabolic homeostasis., (© 2021 International Society on Thrombosis and Haemostasis.)

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.)

Multifunctionality, interference-free signal readout, and quantum effect are important considerations for flexible sensors equipped within a single unit towards further miniaturization. To address these criteria, we present the slotted carbon nanotube (CNT) junction features tunable Fano resonance driven by flexoelectricity, which could serve as an ideal multimodal sensory receptor. Based on extensive ab initio calculations, we find that the effective Fano factor can be used as a temperature-insensitive extrinsic variable for sensing the bending strain, and the Seebeck coefficient can be used as a strain-insensitive intrinsic variable for detecting temperature. Thus, this dual-parameter permits simultaneous sensing of temperature and strain without signal interference. We further demonstrate the applicability of this slotted junction to ultrasensitive chemical sensing which enables precise determination of donor-type, acceptor-type, and inert molecules. This is due to the enhancement or counterbalance between flexoelectric and chemical gating. Flexoelectric gating would preserve the electron-hole symmetry of the slotted junction whereas chemical gating would break it. As a proof-of-concept demonstration, the slotted CNT junction provides an excellent quantum platform for the development of multistimuli sensation in artificial intelligence at the molecular scale., Competing Interests: The authors declare that there is no conflict of interest., (Copyright © 2021 Jinlong Ren et al.)

Currently available synthetic small diameter vascular grafts reveal low patency rates due to thrombosis and intimal hyperplasia. Biofunctionalized grafts releasing nitric oxide (NO) in situ may overcome these limitations. In this study, a drug-eluting vascular graft was designed by blending polycaprolactone (PCL) with S-nitroso-human-serum-albumin (S-NO-HSA), a nitric oxide donor with prolonged half-life. PCL-S-NO-HSA grafts and patches were fabricated via electrospinning. The fabrication process was optimized. Patches were characterized in vitro for their morphology, drug release, biomechanics, inflammatory effects, cell proliferation, and expression of adhesion molecules. The selected optimized formulation (8%PCL-S-NO-HSA) had superior mechanical/morphological properties with high protein content revealing extended NO release (for 28 days). 8%PCL-S-NO-HSA patches significantly promoted endothelial cell proliferation while limiting smooth muscle cell proliferation. Expression of adhesion molecules (ICAM-1, VCAM-1) and pro-inflammatory macrophage/cytokine markers (CD80, IL-1α, TNF-α) was significantly reduced. 8%PCL-S-NO-HSA patches had superior immunomodulatory properties by up-regulating anti-inflammatory cytokines (IL-10) and M2 macrophage marker (CD163) at final time points. Grafts were further evaluated in a small rodent model as aortic implants up to 12 weeks. Grafts were assessed by magnetic resonance imaging angiography (MRI) in vivo and after retrieval by histology. All grafts remained 100 % patent with no signs of thrombosis or calcification. 8%PCL-S-NO-HSA vascular grafts supported rapid endothelialization, whereas smooth muscle cell proliferation was hampered in earlier phases. This study indicates that 8%PCL-S-NO-HSA grafts effectively support long-term in situ release of bioactive NO. The beneficial effects observed can be promising features for long-term success of small diameter vascular grafts. STATEMENT OF SIGNIFICANCE: Despite extensive research in the field of small diameter vascular graft replacement, there is still no appropriate substitute to autografts yet. Various limitations are associated with currently available synthetic vascular grafts such as thrombogenicity and intimal hyperplasia. Therefore, developing new generations of such conduits has become a major focus of research. One of the most significant signaling molecules that are involved in homeostasis of the vascular system is nitric oxide. The new designed nitric-oxide eluting vascular grafts described in this study induce rapid surface endothelialization and late migration of SMCs into the graft wall. These beneficial effects have potential to improve current limitations of small diameter vascular grafts., Competing Interests: Declaration of Competing Interest We declare that there is no conflict of interests regarding the publication of this paper entitled, “S-nitroso human serum albumin as a nitric oxide donor in drug-eluting vascular grafts; biofunctionality and preclinical evaluation”., (Copyright © 2021. Published by Elsevier Ltd.)

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.

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.

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., (Copyright © 2020. Published by Elsevier Ltd.)

Aims: Tenascin-C (TN-C) is suggested to be detrimental in cardiac remodelling after myocardial infarction (MI). The aim of this study is to reveal the effects of TN-C on extracellular matrix organization and its haemodynamic influence in an experimental mouse model of MI and in myocardial cell culture during hypoxic conditions., Methods and Results: Myocardial infarction was induced in TN-C knockout (TN-C KO) and wild-type mice. Six weeks later, cardiac function was studied by magnetic resonance imaging and under isolated working heart conditions. Myocardial mRNA levels and immunoreactivity of TN-C, TIMP-1, TIMP-3, and matrix metalloproteinase (MMP)-9, as well as serum and tissue activities of angiotensin-converting enzyme (ACE), were determined at 1 and 6 weeks after infarction. Cardiac output and external heart work were higher, while left ventricular wall stress and collagen expression were decreased (P < 0.05) in TN-C KO mice as compared with age-matched controls at 6 weeks after infarction. TIMP-1 expression was down-regulated at 1 and 6 weeks, and TIMP-3 expression was up-regulated at 1 week (P < 0.01) after infarction in knockout mice. MMP-9 level was lower in TN-C KO at 6 weeks after infarction (P < 0.05). TIMP-3/MMP-9 ratio was higher in knockout mice at 1 and 6 weeks after infarction (P < 0.01). ACE activity in the myocardial border zone (i.e. between scar and free wall) was significantly lower in knockout than in wild-type mice 1 week after MI (P < 0.05)., Conclusions: Tenascin-C expression is induced by hypoxia in association with ACE activity and MMP-2 and MMP-9 elevations, thereby promoting left ventricular dilatation after MI., (© 2020 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

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.

Background: Percutaneous coronary intervention represents the most important treatment modality of coronary artery stenosis. In-stent restenosis (ISR) is still a limitation for the long-term outcome despite the introduction of drug eluting stents. It has been shown that adipokines directly influence vessel wall homeostasis by influencing the function of endothelial cells and arterial smooth muscle cells. Visceral adipose tissue-derived serpin vaspin was recently identified as a member of serine protease inhibitor family and serveral studies could demonstrate a relation to metabolic diseases. The aim of this study was to investigate a role of vaspin in the development of in-stent restenosis in vivo and on migration of smooth muscle cells and endothelial cells in vitro., Methods: We studied 85 patients with stable coronary artery disease who underwent elective and successful PCI with implatation of drug eluting stents. Blood samples were taken directly before PCI. Vaspin plasma levels were measured by specific ELISA. ISR was evaluated eight months later by coronary angiography. Human coronary artery smooth muscle cells (HCASMC) and human umbilical vein endothelial cells (HUVEC) migration was analyzed by an in-vitro migration assay with different concentrations (0.004ng/mL up to 40ng/mL) of vaspin as well as by an scratch assay. For proliferation an impedance measurement with specialiced E-Plates was performed., Results: During the follow up period, 14 patients developed ISR. Patients with ISR had significantly lower vaspin plasma levels compared to patients without ISR (0.213 ng/ml vs 0.382 ng/ml; p = 0.001). In patients with plasma vaspin levels above 1.35 ng/ml we could not observe any restenosis. There was also a significant correlation of plasma vaspin levels and late lumen loss in the stented coronary segments. Further we could demonstrate that vaspin nearly abolishes serum induced migration of HCASMC (100% vs. 9%; p<0.001) in a biphasic manner but not migration of HUVEC. Proliferation of HCASMC and HUVEC was not modulated by vaspin treatment., Conclusion: We were able to show that the adipokine vaspin selectively inhibits human coronary SMC migration in vitro and has no effect on HUVEC migration. Vaspin had no effect on proliferation of HUVEC which is an important process of the healing of the stented vessel. In addition, the occurrence of ISR after PCI with implantation of drug eluting stents was significantly associated with low vaspin plasma levels before intervention. Determination of vaspin plasma levels before PCI might be helpful in the identification of patients with high risk for development of ISR after stent implantation. In addition, the selective effects of vaspin on smooth muscle cell migration could potentially be used to reduce ISR without inhibition of re-endothelialization of the stented segment., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2019 European Society for Vascular Surgery. Published by Elsevier B.V. All rights reserved.)

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.

Background: Epidural-related maternal fever (ERMF) is an adverse effect of epidural analgesia during labor and is associated with perinatal and neonatal morbidity. Local anesthetics have been proposed to trigger ERMF via sterile inflammation. Ropivacaine is currently the most frequently used epidural anesthetic and considered least toxic. This study investigates molecular effects of ropivacaine on human umbilical vein endothelial cells (HUVECs) as model system for endothelial cells and human placental trophoblasts (TBs), compares the effects to the putative anti-inflammatory lidocaine and investigates the partially alleviating impact of the anti-inflammatory corticosteroid dexamethasone., Methods: HUVECs and TBs were exposed to ropivacaine (35 μM-7 mM) or lidocaine (21 mM) with or without dexamethasone (1 μM). AnnexinV/propidium iodide staining and lactate dehydrogenase release were used to analyze apoptosis and cytotoxicity. Proinflammatory interleukins-6 (IL-6) and IL-8 as well as prostaglandin E2 (PGE2) were measured by enzyme-linked immunosorbent assay (ELISA), while activation of signaling pathways was detected by Western blotting. Oxidative stress was visualized by live cell imaging and quantification of antioxidant proteins, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, platelet endothelial cell adhesion molecule 1, cyclooxygenase 2, and mitochondrial deoxyribonucleic acid by real-time polymerase chain reaction. Dissipation of the mitochondrial membrane potential was assessed with cytofluorimetric analysis using the J-Aggregate (JC-1 staining [cytofluorimetric analysis using the J-Aggregate])., Results: Ropivacaine exposure dose-dependently induced apoptosis and an increased release of IL-6, IL-8, and PGE2 from HUVECs and TBs. Furthermore, caspase-3, nuclear factor-κB, and p38 mitogen-activated protein kinase pathways were activated, while extracellular signal-regulated kinase 1/2 and protein kinase B (Akt) were dephosphorylated. Downregulation of antioxidative proteins induced oxidative stress and upregulation of ICAM1, VCAM1, and PECAM1 possibly facilitate leukocyte transmigration. Mitochondrial effects included increased release of the proinflammatory mitochondrial DNA damage-associated molecular patterns, but no significant dissipation of the mitochondrial membrane potential. Conversely, lidocaine exhibited repression of IL-6 and IL-8 release over all time points, and early downregulation of COX2 and cell adhesion molecules, which was followed by a late overshooting reaction. Dexamethasone reduced especially inflammatory effects, but as an inducer of mitophagy, had negative long-term effects on mitochondrial function., Conclusions: This study suggests that ropivacaine causes cellular injury and death in HUVECs and TBs via different signaling pathways. The detrimental effects induced by ropivacaine are only partially blunted by dexamethasone. This observation strengthens the importance of inflammation in ERMF.

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.

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.)

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.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The aim of this study was to describe the potential associations of the expression of matricellular components in adverse post-infarction remodeling of the geriatric heart. In male geriatric (OM, age: 18 months) and young (YM, age: 11 weeks) OF1 mice myocardial infarction (MI) was induced by permanent ligation of the left anterior descending coronary artery. Cardiac function was evaluated by MRI. Plasma and myocardial tissue samples were collected 3d, 7d, and 32d post-MI. Age and MI were associated with impaired cardiac function accompanied by left-ventricular (LV) dilatation. mRNA expression of MMP-2 (7d: p < 0.05), TIMP-1 (7d: p < 0.05), TIMP-2 (7d: p < 0.05), Collagen-1 (3d and 7d: p < 0.05) and Collagen-3 (7d: p < 0.05) in LV non-infarcted myocardium was significantly higher in YM than in OM after MI. MMP-9 activity in plasma was increased in OM after MI (3d: p < 0.01). Tenascin-C protein levels assessed by ELISA were decreased in OM as compared to YM after MI in plasma (3d: p < 0.001, 7d: p < 0.05) and LV non-infarcted myocardium (7d: p < 0.01). Dysregulation in ECM components in non-infarcted LV might be associated and contribute to adverse LV remodeling and impaired cardiac function. Thus, targeting ECM might be a potential therapeutic approach to enhance cardiac function in geriatric patients following MI., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.)

Small diameter vascular grafts from human placenta, decellularized with either Triton X-100 (Triton) or SDS and crosslinked with heparin were constructed and characterized. Graft biochemical properties, residual DNA, and protein composition were evaluated to compare the effect of the two detergents on graft matrix composition and structural alterations. Biocompatibility was tested in vitro by culturing the grafts with primary human macrophages and in vivo by subcutaneous implantation of graft conduits (n = 7 per group) into the flanks of nude rats. Subsequently, graft performance was evaluated using an aortic implantation model in Sprague Dawley rats (one month, n = 14). In situ graft imaging was performed using MRI angiography. Retrieved specimens were analyzed by electromyography, scanning electron microscopy, histology and immunohistochemistry to evaluate cell migration and the degree of functional tissue remodeling. Both decellularization methods resulted in grafts of excellent biocompatibility in vitro and in vivo, with low immunogenic potential. Proteomic data revealed removal of cytoplasmic proteins with relative enrichment of ECM proteins in decelluarized specimens of both groups. Noteworthy, LC-Mass Spectrometry analysis revealed that 16 proteins were exclusively preserved in Triton decellularized specimens in comparison to SDS-treated specimens. Aortic grafts showed high patency rates, no signs of thrombus formation, aneurysms or rupture. Conduits of both groups revealed tissue-specific cell migration indicative of functional remodeling. This study strongly suggests that decellularized allogenic grafts from the human placenta have the potential to be used as vascular replacement materials. Both detergents produced grafts with low residual immunogenicity and appropriate mechanical properties. Observed differences in graft characteristics due to preservation method had no impact on successful in vivo performance in the rodent model., (Copyright © 2018 Elsevier Ltd. 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.

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 1β 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.

Background: Intracoronary (IC) injection of mesenchymal stem cells (MSCs) results in a prompt decrease of absolute myocardial blood flow (AMF) with late and incomplete recovery of myocardial tissue perfusion. Here, we investigated the effect of decreased AMF on oxidative stress marker matrix metalloproteinase-2 (MMP-2) and its influence on the fate and homing and paracrine character of MSCs after IC or intramyocardial cell delivery in a closed-chest reperfused myocardial infarction (MI) model in pigs., Methods: Porcine MSCs were transiently transfected with Ad-Luc and Ad-green fluorescent protein (GFP). One week after MI, the GFP-Luc-MSCs were injected either IC (group IC, 11.00 ± 1.07 × 10 6 ) or intramyocardially (group IM, 9.88 ± 1.44 × 10 6 ). AMF was measured before, immediately after, and 24 h post GFP-Luc-MSC delivery. In vitro bioluminescence signal was used to identify tissue samples containing GFP-Luc-MSCs. Myocardial tissue MMP-2 and CXCR4 receptor expression (index of homing signal) were measured in bioluminescence positive and negative infarcted and border, and non-ischemic myocardial areas 1-day post cell transfer. At 7-day follow-up, myocardial homing (cadherin, CXCR4, and stromal derived factor-1alpha) and angiogenic [fibroblast growth factor 2 (FGF2) and VEGF] were quantified by ELISA of homogenized myocardial tissues from the bioluminescence positive and negative infarcted and border, and non-ischemic myocardium. Biodistribution of the implanted cells was quantified by using Luciferase assay and confirmed by fluorescence immunochemistry. Global left ventricular ejection fraction (LVEF) was measured at baseline and 1-month post cell therapy using magnet resonance image., Results: AMF decreased immediately after IC cell delivery, while no change in tissue perfusion was found in the IM group (42.6 ± 11.7 vs. 56.9 ± 16.7 ml/min, p  = 0.018). IC delivery led to a significant increase in myocardial MMP-2 64 kD expression (448 ± 88 vs. 315 ± 54 intensity × mm 2 , p  = 0.021), and decreased expression of CXCR4 (592 ± 50 vs. 714 ± 54 pg/tissue/ml, p  = 0.006), with significant exponential decay between MMP-2 and CXCR4 ( r  = 0.679, p  < 0.001). FGF2 and VEGF of the bioluminescence infarcted and border zone of homogenized tissues were significantly elevated in the IM goups as compared to IC group. LVEF increase was significantly higher in IM group (0.8 ± 8.4 vs 5.3 ± 5.2%, p  = 0.046) at the 1-month follow up., Conclusion: Intracoronary stem cell delivery decreased AMF, with consequent increase in myocardial expression of MMP-2 and reduced CXCR4 expression with lower level of myocardial homing and angiogenic factor release as compared to IM cell delivery.

Aims: Left ventricular (LV) hypertrophy is characterized by cardiomyocyte hypertrophy and interstitial fibrosis ultimately leading to increased myocardial stiffness and reduced contractility. There is substantial evidence that the altered expression of matrix metalloproteinases (MMP) and Tenascin-C (TN-C) are associated with the progression of adverse LV remodeling. However, the role of TN-C in the development of LV hypertrophy because of chronic pressure overload as well as the regulatory role of TN-C on MMPs remains unknown., Methods and Results: In a knockout mouse model of TN-C, we investigated the effect of 10 weeks of pressure overload using transverse aortic constriction (TAC). Cardiac function was determined by magnetic resonance imaging. The expression of MMP-2 and MMP-9, CD147 as well as myocardial fibrosis were assessed by immunohistochemistry. The expression of TN-C was assessed by RT-qPCR and ELISA. TN-C knockout mice showed marked reduction in fibrosis (P < 0.001) and individual cardiomyocytes size (P < 0.01), in expression of MMP-2 (P < 0.05) and MMP-9 (P < 0.001) as well as preserved cardiac function (P < 0.01) in comparison with wild-type mice after 10 weeks of TAC. In addition, CD147 expression was markedly increased under pressure overload (P < 0.01), irrespectively of genotype. TN-C significantly increased the expression of the markers of hypertrophy such as ANP and BNP as well as MMP-2 in H9c2 cells (P < 0.05, respectively)., Conclusion: Our results are pointed toward a novel signaling mechanism that contributes to LV remodeling via MMPs upregulation, cardiomyocyte hypertrophy as well as myocardial fibrosis by TN-C under chronic pressure overload.

The BCR/ABL1 inhibitor Nilotinib is increasingly used to treat patients with chronic myeloid leukemia (CML). Although otherwise well-tolerated, Nilotinib has been associated with the occurrence of progressive arterial occlusive disease (AOD). Our objective was to determine the exact frequency of AOD and examine in vitro and in vivo effects of Nilotinib and Imatinib on endothelial cells to explain AOD-development. In contrast to Imatinib, Nilotinib was found to upregulate pro-atherogenic adhesion-proteins (ICAM-1, E-selectin, VCAM-1) on human endothelial cells. Nilotinib also suppressed endothelial cell proliferation, migration and tube-formation and bound to a distinct set of target-kinases, relevant to angiogenesis and atherosclerosis, including angiopoietin receptor-1 TEK, ABL-2, JAK1 and MAP-kinases. Nilotinib and siRNA against ABL-2 also suppressed KDR expression. In addition, Nilotinib augmented atherosclerosis in ApoE-/- mice and blocked reperfusion and angiogenesis in a hindlimb-ischemia model of arterial occlusion, whereas Imatinib showed no comparable effects. Clinically overt AOD-events were found to accumulate over time in Nilotinib-treated patients. After a median observation-time of 2.0 years, the AOD-frequency was higher in these patients (29.4%) compared to risk factor- and age-matched controls (<5%). Together, Nilotinib exerts direct pro-atherogenic and anti-angiogenic effects on vascular endothelial cells, which may contribute to development of AOD in patients with CML.

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.

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-1β, 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.

Background: Perioperative oxygen (O2) therapy can cause hyperoxia. Extreme hyperoxia can injure the cardiovascular system and remote organs., Objective: Our primary objective was to test the hypothesis that exposure to moderate hyperoxia will induce injury to human umbilical vein endothelial cells (HUVECs), a model for studying the vascular endothelium under controlled conditions., Design: In-vitro cell culture study., Setting: Department of Anaesthesia, General Intensive Care and Pain Management, Medical University of Vienna, Austria. Study period from the beginning of October 2013 to the end of July 2014., Cells: HUVECs were isolated from fresh umbilical cords., Interventions: HUVECs were exposed to constant hyperoxia (40% O2), cyclic hyperoxia/anoxia (40%/0% O2, average 20% O2), constant normoxia (21% O2) and constant anoxia (0% O2) using a cell culture bioreactor., Main Outcome Measures: Cell growth, viability and release of IL-6, IL-8 and macrophage migration inhibitory factor were assessed at baseline and after 6, 12, 24 and 48 h of treatment. A phosphokinase array was performed after 60 min of treatment to identify activated cellular signalling pathways., Results: Constant hyperoxia and cyclic hyperoxia/anoxia impeded cell growth, reduced viability, triggered a proinflammatory response, proven by IL-6, IL-8 and migration inhibitory factor release, and induced apoptosis and necrosis. The inflammatory and cytotoxicity responses were highest in the constant hyperoxia group. Phosphokinase arrays revealed that different O2 concentrations activated distinct sets of cytoprotective and cell death-associated kinases, including mitogen-activated protein kinases, Src kinases, p53, Akt, mitogen-activated and stress-activated kinase, Lyn, Lck, p70S6, signal transducers and activators of transcription 5b and 6, glycogen synthase kinase 3a/b and 5' AMP-activated protein kinases 1/2., Conclusion: Continuous moderate hyperoxia and cyclic moderate hyperoxia/anoxia-induced endothelial inflammation, apoptosis and necrosis. Given the large surface area of the vascular endothelium, moderately elevated O2 levels may contribute to cardiovascular inflammation and injury., Trial Registration: This in-vitro study was not registered in a database.

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.)

Background. Mobilization of bone marrow-origin CD34+ cells was investigated 3 days (3d) after acute myocardial infarction (AMI) with/without ischemic preconditioning (IP) in relation to stromal-derived factor-1 (SDF-1α)/ chemokine receptor type 4 (CXCR4) axis, to search for possible mechanisms behind insufficient cardiac repair in the first days post-AMI.  Methods. Closed-chest reperfused AMI was performed by percutaneous balloon occlusion of the mid-left anterior descending (LAD) coronary artery for 90min, followed by reperfusion in pigs. Animals were randomized to receive either IP initiated by 3x5min cycles of re-occlusion/re-flow prior to AMI (n=6) or control AMI (n=12). Blood samples were collected at baseline, 3d post-AMI, and at 1-month follow-up to analyse chemokines and mobilized CD34+ cells. To investigate the effect of acute hypoxia, SDF-1α and matrix metalloproteinase (MMP)-2 in vitro were assessed, and a migration assay of CD34+ cells toward cardiomyocytes was performed.  Results. Reperfused AMI induced significant mobilisation of CD34+ cells (baseline: 260±75 vs. 3d: 668±180; P<0.001) and secretion of MMP-2 (baseline: 291.83±53.40 vs. 3d: 369.64±72.89; P=0.011) into plasma, without affecting the SDF-1α concentration. IP led to the inhibition of MMP-2 (IP: 165.67±47.99 vs. AMI: 369.64±72.89; P=0.004) 3d post-AMI, accompanied by increased release of SDF-1α (baseline: 23.80±12.36 vs. 3d: 45.29±11.31; P=0.05) and CXCR4 (baseline: 0.59±0.16 vs. 3d: 2.06±1.42; P=0.034), with a parallel higher level of mobilisation of CD34+ cells (IP: 881±126 vs. AMI: 668±180; P=0.026), compared to non-conditioned AMI. In vitro , CD34+ cell migration toward cardiomyocytes was enhanced by SDF-1α, which was completely abolished by 90min hypoxia and co-incubation with MMP-2.  Conclusions . Non-conditioned AMI induces MMP-2 release, hampering the ischemia-induced increase in SDF-1α and CXCR4 by cleaving the SDF-1α/CXCR4 axis, with diminished mobilization of the angiogenic CD34+ cells. IP enforces CD34+ cell mobilization via inhibition of MMP-2., Competing Interests: Competing interests: No competing interests were disclosed.

Following the implantation of biodegradable vascular grafts, macrophages and fibroblasts are the major two cell types recruited to the host-biomaterial interface. In-vitro biocompatibility assessment usually involves one cell type, predominantly macrophages. In this study, macrophage and fibroblast mono- and co-cultures, in paracrine and juxtacrine settings, were used to evaluate a new biodegradable thermoplastic polyurethane (TPU) vascular graft. Expanded-polytetrafluoroethylene (ePTFE) grafts served as controls. Pro/anti-inflammatory gene expression of macrophages and cytokines was assessed in vitro and compared to those of an in vivo rat model. Host cell infiltration and the type of proliferated cells was further studied in vivo. TPU grafts revealed superior support in cell attachment, infiltration and proliferation compared with ePTFE grafts. Expression of pro-inflammatory TNF-α/IL-1α cytokines was significantly higher in ePTFE, whereas the level of IL-10 was higher in TPU. Initial high expression of pro-inflammatory CCR7 macrophages was noted in TPU, however there was a clear transition from CCR7 to anti-inflammatory CD163 expression in vitro and in vivo only in TPU, confirming superior cell-biomaterial response. The co-culture models, especially the paracrine model, revealed higher fidelity to the immunomodulatory/biocompatibility behavior of degradable TPU grafts in vivo. This study established an exciting approach developing a co-culture model as a tool for biocompatibility evaluation of degradable biomaterials.

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-1β, 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.

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.)