The Secretome of Preconditioned Mesenchymal Stem Cells Drives Polarization and Reprogramming of M2a Macrophages toward an IL-10-Producing Phenotype.

The preconditioning of mesenchymal stem cells (MSCs) has been recognized as an attractive tool to improve their regenerative and immunomodulatory capacities based on their paracrine effects. In this study, we examined the potential of an MSC-conditioned medium (MSC-CM) to alter the phenotype of murine macrophages and to drive reprogramming toward an anti-inflammatory, M2-like state in vitro. We further explored the impact of MSC cytokine preconditioning on the immunosuppressive properties of the MSC secretome. The MSC-CM suppressed the expression of proinflammatory genes in murine M1 macrophages, but only the CM from preconditioned MSCs (preMSC-CM) downregulated their expression during M1 polarization. Remarkably, only the preMSC-CM significantly increased the expression of M2a-, M2b- and M2c-specific genes and proteins during M2a polarization. Further, macrophages were found to secrete high levels of anti-inflammatory IL-10. Similarly, M2a macrophages cultured in the presence of the preMSC-CM displayed an enhanced expression of M2b/M2c-specific markers, suggesting that the secretome of preMSC promotes the repolarization of M2a-like macrophages to M2b/M2c subtypes. The preMSC-CM was found to be enriched in molecules involved in M2 polarization. Additionally, a unique downregulation of extracellular matrix components was observed. Altogether, the preMSC-CM may provide an attractive strategy to dampen inflammation by suppressing the expression of proinflammatory mediators and promoting the polarization and phenotype switch of M2a cells to IL-10-secreting M2b/M2c-like macrophages.

Impaired non-canonical transforming growth factor-ß signalling prevents profibrotic phenotypes in cultured peptidylarginine deiminase 4-deficient murine cardiac fibroblasts.

Transforming growth factor-ß (TGF-ß) becomes rapidly activated in the infarcted heart. Hence, TGF-ß-mediated persistent activation of cardiac fibroblasts (CFs) and exaggerated fibrotic responses may result in adverse cardiac remodelling and heart failure. Additionally, peptidylarginine deiminase 4 (PAD4) was described to be implicated in organ fibrosis. Here, we investigated the impact of PAD4 on CF function and myofibroblast transdifferentiation in vitro. The expression of fibrosis-related genes was largely similar in cultured WT and PAD4-/- CFs of passage 3, although collagen III was reduced in PAD4-/- CFs. Exposure to TGF-ß inhibited proliferation and increased contractile activity and migration of WT CFs, but not of PAD4-/- CFs. However, under baseline conditions, PAD4-/- CFs showed comparable functional characteristics as TGF-ß-stimulated WT CFs. Although the SMAD-dependent TGF-ß pathway was not disturbed in PAD4-/- CFs, TGF-ß failed to activate protein kinase B (Akt) and signal transducer and activator of transcription 3 (STAT3) in these cells. Similar results were obtained in WT CFs treated with the PAD4 inhibitor Cl-amidine. Abrogated Akt activation was associated with diminished levels of phosphorylated, inactive glycogen synthase kinase-3ß (GSK-3ß). Consequently, PAD4-/- CFs did not upregulate collagen I and α-smooth muscle actin (α-SMA) expression after TGF-ß treatment. Thus, PAD4 is substantially involved in the regulation of non-canonical TGF-ß signalling and may represent a therapeutic target for the treatment of adverse cardiac remodelling.

Recovery of neutrophil apoptosis by ectoine: a new strategy against lung inflammation.

The life span of neutrophilic granulocytes has a determining impact on the intensity and duration of neutrophil driven lung inflammation. Based on the compatible solute ectoine, we aimed to prevent anti-apoptotic reactions in neutrophils triggered by the inflammatory microenvironment in the lung. Neutrophils from chronic obstructive pulmonary disease patients and control individuals were exposed to inflammatory mediators and xenobiotics in the presence or absence of ectoine. The in vivo relevance of this approach was tested in xenobiotic-induced lung inflammation in rats. The reduction of apoptosis rates of ex vivo-exposed neutrophils from all study groups was significantly restored in the presence of ectoine. However, natural apoptosis rates not altered by inflammatory stimuli were not changed by ectoine. Mechanistic analyses demonstrated the preventive effect of ectoine on the induction of anti-apoptotic signalling. Neutrophilic lung inflammation induced by single or multiple expositions of animals to environmental particles was reduced after the therapeutic intervention with ectoine. Analyses of neutrophils from bronchoalveolar lavage indicate that the in vivo effect is due to the restoration of neutrophil apoptosis. Ectoine, a compound of the highly compliant group of compatible solutes, demonstrates a reproducible and robust effect on the resolution of lung inflammation.

Molecular mechanisms underlying delayed apoptosis in neutrophils from multiple trauma patients with and without sepsis.

Delayed neutrophil apoptosis and overshooting neutrophil activity contribute to organ dysfunction and subsequent organ failure in sepsis. Here, we investigated apoptotic signaling pathways that are involved in the inhibition of spontaneous apoptosis in neutrophils isolated from major trauma patients with uneventful outcome as well as in those with sepsis development. DNA fragmentation in peripheral blood neutrophils showed an inverse correlation with the organ dysfunction at d 10 after trauma in all patients, supporting the important role of neutrophil apoptosis regulation for patient's outcome. The expression of the antiapoptotic Bcl-2 protein members A1 and Mcl-1 were found to be diminished in the septic patients at d 5 and d 10 after trauma. This decrease was also linked to an impaired intrinsic apoptosis resistance, which has been previously shown to occur in neutrophils during systemic inflammation. In patients with sepsis development, delayed neutrophil apoptosis was found to be associated with a disturbed extrinsic pathway, as demonstrated by reduced caspase-8 activity and Bid truncation. Notably, the expression of Dad1 protein, which is involved in protein N-glycosylation, was significantly increased in septic patients at d 10 after trauma. Taken together, our data demonstrate that neutrophil apoptosis is regulated by both the intrinsic and extrinsic pathway, depending on patient's outcome. These findings might provide a molecular basis for new strategies targeting cell death pathways in apoptosis-resistant neutrophils during systemic inflammation.

Dermal application of nitric oxide releasing acidified nitrite-containing liniments significantly reduces blood pressure in humans.

Vascular ischemic diseases, hypertension, and other systemic hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). NO but also its active derivates like nitrite or nitroso compounds are important effector and signal molecules with vasodilating properties. Our previous findings point to a therapeutical potential of cutaneous administration of NO in the treatment of systemic hemodynamic disorders. Unfortunately, no reliable data are available on the mechanisms, kinetics and biological responses of dermal application of nitric oxide in humans in vivo. The aim of the study was to close this gap and to explore the therapeutical potential of dermal nitric oxide application. We characterized with human skin in vitro and in vivo the capacity of NO, applied in a NO-releasing acidified form of nitrite-containing liniments, to penetrate the epidermis and to influence local as well as systemic hemodynamic parameters. We found that dermal application of NO led to a very rapid and significant transepidermal translocation of NO into the underlying tissue. Depending on the size of treated skin area, this translocation manifests itself through a significant systemic increase of the NO derivates nitrite and nitroso compounds, respectively. In parallel, this translocation was accompanied by an increased systemic vasodilatation and blood flow as well as reduced blood pressure. We here give evidence that in humans dermal application of NO has a therapeutic potential for systemic hemodynamic disorders that might arise from local or systemic insufficient availability of NO or its bio-active NO derivates, respectively.

Depletion of neutrophil extracellular traps in vivo results in hypersusceptibility to polymicrobial sepsis in mice.

INTRODUCTION: Although the formation of neutrophil (PMN) extracellular traps (NETs) has been detected during infection and sepsis, their role in vivo is still unclear. This study was performed in order to evaluate the influence of NETs depletion by administration of recombinant human (rh)DNase on bacterial spreading, PMN tissue infiltration and inflammatory response in a mouse model of polymicrobial sepsis. METHODS: In a prospective controlled double-armed animal trial, polymicrobial sepsis was induced by cecal ligation and puncture (CLP). After CLP, mice were treated with rhDNase or phosphate buffered saline, respectively. Survival, colony forming unit (CFU) counts in the peritoneal cavity, lung, liver and blood were determined. PMN and platelet counts, IL-6 and circulating free (cf)-DNA/NETs levels were monitored. PMN infiltration, as well as organ damage, was analyzed histologically in the lungs and liver. Capability and capacity of PMN to form NETs were determined over time. RESULTS: cf-DNA/NETs were found to be significantly increased 6, 24, and 48 hours after CLP when compared to the levels determined in sham and naïve mice. Peak levels after 24 hours were correlated to enhanced capacity of bone marrow-derived PMN to form NETs after ex vivo stimulation with phorbol-12-myristate-13-acetate at the same time. rhDNase treatment of mice resulted in a significant reduction of cf-DNA/NETs levels 24 hours after CLP (P < 0.001). Although overall survival was not affected by rhDNase treatment, median survival after 24 hours was significantly lower when compared with the CLP group (P < 0.01). In mice receiving rhDNase treatment, CFU counts in the lung (P < 0.001) and peritoneal cavity (P < 0.05), as well as serum IL-6 levels (P < 0.001), were found to be already increased six hours after CLP. Additionally, enhanced PMN infiltration and tissue damage in the lungs and liver were found after 24 hours. In contrast, CFU counts in mice without rhDNase treatment increased later but more strongly 24 hours after CLP (P < 0.001). Similarly, serum IL-6 levels peaked after 24 hours (P < 0.01). CONCLUSIONS: This study shows, for the first time, that depletion of NETs by rhDNase administration impedes the early immune response and aggravates the pathology that follows polymicrobial sepsis in vivo.

Deoxyribonuclease is a potential counter regulator of aberrant neutrophil extracellular traps formation after major trauma.

INTRODUCTION: Neutrophil extracellular traps (NET) consist of a DNA scaffold that can be destroyed by Deoxyribonuclease (DNase). Thus DNases are potential prerequisites for natural counter regulation of NETs formation. In the present study, we determined the relationship of NETs and DNase after major trauma. METHODS: Thirty-nine major trauma patients, 14 with and 25 without sepsis development were enrolled in this prospective study. Levels of cell-free (cf)-DNA/NETs and DNase were quantified daily from admission until day 9 after admission. RESULTS: Levels of cf-DNA/NETs in patients who developed sepsis were significantly increased after trauma. In the early septic phase, DNase values in septic patients were significantly increased compared to patients without sepsis (P < 0.05). cf-DNA/NETs values correlated to values of DNase in all trauma patients and patients with uneventful recovery (P < 0.01) but not in septic patients. Recombinant DNase efficiently degraded NETs released by stimulated neutrophils in a concentration-dependent manner in vitro. CONCLUSIONS: DNase degrades NETs in a concentration-dependent manner and therefore could have a potential regulatory effect on NET formation in neutrophils. This may inhibit the antibacterial effects of NETs or protect the tissue from autodestruction in inadequate NETs release in septic patients.

Peptidylarginine deiminase 4 deficiency in bone marrow cells prevents plaque progression without decreasing atherogenic inflammation in apolipoprotein E-knockout mice.

Introduction: Despite multiple studies in the past, the role of peptidylarginine deiminase 4 (PAD4) in atherosclerosis is currently insufficiently understood. In this regard, PAD4 deletion or inhibition of enzymatic activity was previously reported to ameliorate disease progression and inflammation. Besides, strong influence of neutrophil extracellular traps (NETs) on atherosclerosis burden has been proposed. Here, we studied the role of PAD4 for atherogenesis and plaque progression in a mouse model of atherosclerosis. Methods and results: Lethally irradiated ApoE -/- mice were reconstituted with ApoE -/-/Pad4 -/- bone marrow cells and fed a high-fat diet (HFD) for 4 and 10 weeks, respectively. PAD4 deficiency did not prevent the development of atherosclerotic lesions after 4 weeks of HFD. However, after 10 weeks of HFD, mice with bone marrow cells-restricted PAD4 deficiency displayed significantly reduced lesion size, impaired lipid incorporation, decreased necrotic core area and less collagen when compared to ApoE -/- bone marrow-transplanted mice as demonstrated by histological staining. Moreover, flow cytometric analysis and quantitative real-time PCR revealed different macrophage subsets in atherosclerotic lesions and higher inflammatory response in these mice, as reflected by increased content of M1-like macrophages and upregulated aortic expression of the pro-inflammatory genes CCL2 and iNOS. Notably, diminished oxLDL uptake by in vitro-polarized M1-like macrophages was evidenced when compared to M2-like cells. Conclusion: These results suggest that pharmacological inhibition of PAD4 may impede lipid accumulation and lesion progression despite no beneficial effects on vascular inflammation.

Concomitant Intra-Aortic Balloon Pumping Significantly Reduces Left Ventricular Pressure during Central Veno-Arterial Extracorporeal Membrane Oxygenation-Results from a Large Animal Model.

(1) Introduction: Simultaneous ECMO and IABP therapy is frequently used. Haemodynamic changes responsible for the success of the concomitant mechanical circulatory support system approach are rarely investigated. In a large-animal model, we analysed haemodynamic parameters before and during ECMO therapy, comparing central and peripheral ECMO circulation with and without simultaneous IABP support. (2) Methods: Thirty-three female pigs were divided into five groups: (1) SHAM, (2) (peripheral)ECMO(-)IABP, (3) (p)ECMO(+)IABP, (4) (central)ECMO(-)IABP, and (5) (c)ECMO(+)IABP. Pigs were cannulated in accordance with the group and supported with ECMO (±IABP) for 10 h. Systemic haemodynamics, cardiac index (CI), and coronary and carotid artery blood flow were determined before, directly after, and at five and ten hours on extracorporeal support. Systemic inflammation (IL-6; IL-10; TNFα; IFNγ), immune response (NETs; cf-DNA), and endothelial injury (ET-1) were also measured. (3) Results: IABP support during antegrade ECMO circulation led to a significant reduction of left ventricular pressure in comparison to retrograde flow in (p)ECMO(-)IABP and (p)ECMO(+)IABP. Blood flow in the left anterior coronary and carotid artery was not affected by extracorporeal circulation. (4) Conclusions: Concomitant central ECMO and IABP therapy leads to significant reduction of intracavitary cardiac pressure, reduces cardiac work, and might therefore contribute to improved recovery in ECMO patients.

Trefoil factor family peptide 2 acts pro-proliferative and pro-apoptotic in the murine retina.

Although expression of trefoil factor family (TFF) peptides has been reported in the brain, nothing is known about TFF expression in the retina. The aim of this study was to test whether TFF peptides are expressed in the murine retina and have any function here. In contrast to most tissues studied, where TFF1 and TFF3 are the predominant peptides, TFF2 is the only peptide expressed in the murine retina. Immunohistochemical studies on murine retinal sections indicate that cells of the ganglion cell layer are the retinal source for murine TFF2 (Tff2). In organotypic murine retina cell cultures recombinant TFF2 exerted a strong pro-apoptotic and pro-proliferative rather than an anti-apoptotic and anti-proliferating effect described in most human cancer cell lines investigated so far. In blockage experiments we were able to demonstrate that the pro-apoptotic effect of TFF2 is caspase-dependent. Western blot analysis of TFF2 treated retinal wholemount homogenates revealed significant reductions in the phosphorylation level of ERK and STAT3 proteins compared to basal conditions, suggesting that in the developing murine retina survival mechanism are down-regulated upon TFF2 administration. Our results suggest that during retinal cell death periods, requiring a tightly regulated balance between cell survival and cell death, TFF2 acts pro-proliferative and pro-apoptotic at least in developing mouse retinae cultured in vivo.

Whole body UVA irradiation lowers systemic blood pressure by release of nitric oxide from intracutaneous photolabile nitric oxide derivates.

RATIONALE: Human skin contains photolabile nitric oxide derivates like nitrite and S-nitroso thiols, which after UVA irradiation, decompose and lead to the formation of vasoactive NO. OBJECTIVE: Here, we investigated whether whole body UVA irradiation influences the blood pressure of healthy volunteers because of cutaneous nonenzymatic NO formation. METHODS AND RESULTS: As detected by chemoluminescence detection or by electron paramagnetic resonance spectroscopy in vitro with human skin specimens, UVA illumination (25 J/cm(2)) significantly increased the intradermal levels of free NO. In addition, UVA enhanced dermal S-nitrosothiols 2.3-fold, and the subfraction of dermal S-nitrosoalbumin 2.9-fold. In vivo, in healthy volunteers creamed with a skin cream containing isotopically labeled (15)N-nitrite, whole body UVA irradiation (20 J/cm(2)) induced significant levels of (15)N-labeled S-nitrosothiols in the blood plasma of light exposed subjects, as detected by cavity leak out spectroscopy. Furthermore, whole body UVA irradiation caused a rapid, significant decrease, lasting up to 60 minutes, in systolic and diastolic blood pressure of healthy volunteers by 11+/-2% at 30 minutes after UVA exposure. The decrease in blood pressure strongly correlated (R(2)=0.74) with enhanced plasma concentration of nitrosated species, as detected by a chemiluminescence assay, with increased forearm blood flow (+26+/-7%), with increased flow mediated vasodilation of the brachial artery (+68+/-22%), and with decreased forearm vascular resistance (-28+/-7%). CONCLUSIONS: UVA irradiation of human skin caused a significant drop in blood pressure even at moderate UVA doses. The effects were attributed to UVA induced release of NO from cutaneous photolabile NO derivates.

Mcl-1-mediated impairment of the intrinsic apoptosis pathway in circulating neutrophils from critically ill patients can be overcome by Fas stimulation.

The systemic inflammatory response syndrome and subsequent organ failure are mainly driven by activated neutrophils with prolonged life span, which is believed to be due to apoptosis resistance. However, detailed underlying mechanisms leading to neutrophil apoptosis resistance are largely unknown, and possible therapeutic options to overcome this resistance do not exist. Here we report that activated neutrophils from severely injured patients exhibit cell death resistance due to impaired activation of the intrinsic apoptosis pathway, as evidenced by limited staurosporine-induced mitochondrial membrane depolarization and decreased caspase-9 activity. Moreover, we found that these neutrophils express high levels of antiapoptotic Mcl-1 and low levels of proapoptotic Bax protein. Mcl-1 up-regulation was dependent on elevated concentrations of GM-CSF in patient serum. Accordingly, increased Mcl-1 protein stability and GM-CSF serum concentrations were shown to correlate with staurosporine-induced apoptosis resistance. However, cross-linking of neutrophil Fas by immobilized agonistic anti-Fas IgM resulted in caspase-dependent mitochondrial membrane depolarization and apoptosis induction. In conclusion, the observed impairment of the intrinsic pathway and the resulting apoptosis resistance may be overcome by immobilized agonistic anti-Fas IgM. Targeting of neutrophil Fas by immobilized agonistic effector molecules may represent a new therapeutic tool to limit neutrophil hyperactivation and its sequelae in patients with severe immune disorders.

Increased serum soluble Fas after major trauma is associated with delayed neutrophil apoptosis and development of sepsis.

INTRODUCTION: Deregulated apoptosis and overshooting neutrophil functions contribute to immune and organ dysfunction in sepsis and multiple organ failure (MOF). In the present study, we determined the role of soluble Fas (sFas) in the regulation of posttraumatic neutrophil extrinsic apoptosis and the development of sepsis. METHODS: Forty-seven major trauma patients, 18 with and 29 without sepsis development during the first 10 days after trauma, were enrolled in this prospective study. Seventeen healthy volunteers served as controls. Blood samples from severely injured patients were analyzed at day 1, day 5 and day 9 after major trauma. sFas levels, plasma levels of neutrophil elastase (PMNE) and levels of interleukin (IL)-6 were quantified by enzyme-linked immunosorbent assay and related to patients' Sequential Organ Failure Assessment (SOFA) score and Multiple Organ Dysfunction Score (MODS). Neutrophil apoptosis was determined by propidium iodide staining of fragmented DNA and flow cytometry. sFas-mediated effects on neutrophil apoptosis were investigated in cells cultured with agonistic anti-Fas antibodies in the presence of recombinant sFas, sFas-depleted serum or untreated serum from septic patients. RESULTS: Serum levels of sFas in patients who later developed sepsis were significantly increased at day 5 (P < 0.01) and day 9 (P < 0.05) after trauma compared with patients with uneventful recovery. Apoptosis of patient neutrophils was significantly decreased during the observation period compared with control cells. Moreover, Fas-mediated apoptosis of control neutrophils was efficiently inhibited by recombinant sFas and serum from septic patients. Depletion of sFas from septic patient sera diminished the antiapoptotic effects. In septic patients, sFas levels were positively correlated with SOFA at day 1 (r = 0.7, P < 0.001), day 5 (r = 0.62, P < 0.01) and day 9 (r = 0.58, P < 0.01) and with PMNE and leukocyte counts (r = 0.49, P < 0.05 for both) as well as MODS at day 5 (r = 0.56, P < 0.01) after trauma. CONCLUSIONS: Increased sFas in patients with sepsis development impairs neutrophil extrinsic apoptosis and shows a positive correlation with the organ dysfunction scores and PMNE. Therefore, sFas might be a therapeutic target to prevent posttrauma hyperinflammation and sepsis.

Neutrophil extracellular traps modulate inflammatory markers and uptake of oxidized LDL by human and murine macrophages.

Neutrophil extracellular traps (NETs) are web-like structures, which are released upon neutrophil activation. It has previously been demonstrated that NETs are present in atherosclerotic lesions of both humans and animal models thus playing a decisive role in atherosclerosis. Besides, macrophages have a crucial role in disease progression, whereby classically activated M1 macrophages sustain inflammation and alternatively activated M2 macrophages display anti-inflammatory effects. Although NETs and macrophages were found to colocalize in atherosclerotic lesions, the impact of NETs on macrophage function is not fully understood. In the present study, we aimed to investigate the effect of NETs on human and murine macrophages in respect to the expression of pro-inflammatory cytokines, matrix metalloproteinases (MMPs) and uptake of oxidized LDL (oxLDL) in vitro. Human THP-1 and murine bone marrow-derived macrophages were cultured under M1 (LPS + IFN-γ)- and M2a (IL-4)-polarizing culture conditions and treated with NETs. To mimic intraplaque regions, cells were additionally cultured under hypoxic conditions. NETs significantly increased the expression of IL-1ß, TNF-α and IL-6 in THP-M1 macrophages under normoxia but suppressed their expression in murine M1 macrophages under hypoxic conditions. Notably, NETs increased the number of oxLDL-positive M1 and M2 human and murine macrophages under normoxia, but did not influence formation of murine foam cells under hypoxia. However, oxLDL uptake did not strongly correlate with the expression of the LDL receptor CD36. Besides, upregulated MMP-9 expression and secretion by macrophages was detected in the presence of NETs. Again, hypoxic culture conditions dampened NETs effects. These results suggest that NETs may favor foam cell formation and plaque vulnerability, but exert opposite effects in respect to the inflammatory response of human and murine M1 macrophages. Moreover, effects of NETs on macrophages' phenotype are altered under hypoxia.

VEGF Contributes to Mesenchymal Stem Cell-Mediated Reversion of Nor1-Dependent Hypertrophy in iPS Cell-Derived Cardiomyocytes.

Myocardial hypertrophy is present in many heart diseases, representing a strong predictor of adverse cardiovascular outcomes. Regarding therapeutic intervention, mesenchymal stem cells (MSCs) have been suggested to significantly reduce cardiac hypertrophy and progression to heart failure. Preconditioning of MSCs was previously demonstrated to highly improve their paracrine activity resulting in modulation of immune responses and the progression of diseases. Here, we studied the effects of bone marrow-derived preconditioned MSCs on hypertrophied induced pluripotent stem cell-derived cardiomyocytes (iPS-CM) and also sought to identify MSC-derived antihypertrophic molecules. Phenylephrine (PE) was used to induce hypertrophy in murine iPS-CM, and markers of hypertrophy were identified by microarray analysis. Murine MSCs were treated with IFN-γ and IL-1ß to enhance their paracrine activity, and transcriptional profiling was performed by microarray analysis. Hypertrophied iPS-CM were subsequently cocultured with preconditioned MSCs or MSC-conditioned medium (CM), respectively. Effects on hypertrophied iPS-CM were studied by cell area quantification, real-time PCR, and western blot. In some experiments, cells were incubated with fractions of MSC-CM obtained by ultrafiltration or by MSC-CM supplemented with inhibitory antibodies. Intracellular and extracellular levels of vascular endothelial growth factor (VEGF) were evaluated by western blot and ELISA. PE-induced hypertrophy in iPS-CM was associated with an upregulation of neuron-derived orphan receptor (Nor1) expression, activation of Akt, and inhibition of both strongly prevented hypertrophy induction in iPS-CM. VEGF secreted by preconditioned MSCs provoked hypertrophy regression in iPS-CM, and a negative correlation between Nor1 expression and hypertrophic growth could be evidenced. Our results demonstrate that Nor1 expression strongly supports hypertrophy in iPS-CM. Moreover, the secretome of preconditioned MSCs triggered regression of hypertrophy in iPS-CM in a VEGF-dependent manner. We suggest that the delivery of the MSC-derived secretome may represent a therapeutic strategy to limit cardiac hypertrophy. However, additional in vivo studies are needed to prove this hypothesis.

In vitro hemo- and cytocompatibility of bacterial nanocelluose small diameter vascular grafts: Impact of fabrication and surface characteristics.

OBJECTIVE: There is an increasing need for small diameter vascular grafts with superior host hemo- and cytocompatibilities, such as low activation of platelets and leukocytes. Therefore, we aimed to investigate whether the preparation of bacterial nanocellulose grafts with different inner surfaces has an impact on in vitro host cytocompatibility. METHODS: We have synthesized five different grafts in a bioreactor, namely open interface surface (OIS), inverted (INV), partially air dried (PAD), surface formed in air contact (SAC) and standard (STD) that were characterized by a different surface roughness. The grafts (length 55 mm, inner diameter 5 mm) were attached to heparinized polyvinyl chloride tubes, loaded with human blood and rotated at 37°C for 4 hours. Then, blood was analyzed for frequencies of cellular fractions, oxidative products, soluble complement and thrombin factors. The results were compared to clinically approved grafts made of polyethylene terephthalate and expanded polytetrafluoroethylene. Additionally, blood platelets were labelled with 111Indium-oxine to visualize the distribution of adherent platelets in the loop by scintigraphy. RESULTS: SAC nanocellulose grafts with the lowest surface roughness exhibited superior performance with <10% leukocyte and <50% thrombocyte loss in contrast to other grafts that exhibited >65% leukocyte and >90% thrombocyte loss. Of note, SAC nanocellulose grafts showed lowest radioactivity with scintigraphy analyses, indicating reduced platelet adhesion. Although the levels of reactive oxygen species and cell free DNA did not differ significantly, the levels of thrombin-antithrombin complexes were lowest in SAC grafts. However, all nanocellulose grafts exhibited enhanced complement activation. CONCLUSION: The systematic variation of the inner surfaces of BNC vascular grafts significantly improves biocompatibility. Especially, SAC grafts exhibited the lowest loss of platelets as well as leukocytes and additionally significantly diminished activation of the coagulation system. Further animal studies are needed to study in vivo biocompatibilities.

High levels of cell-free DNA accurately predict late acute kidney injury in patients after cardiac surgery.

Use of cardiopulmonary bypass in cardiac surgery triggers systemic inflammation by neutrophil activation leading to neutrophil extracellular traps (NETs) release. Hence, nuclear DNA released by necrotic and apoptotic cells might contribute to an increase in circulating cell-free DNA (cfDNA). cfDNA/NETs might induce endothelial damage and organ dysfunction. This study focuses on the accuracy of cfDNA to predict acute kidney injury (AKI) after on-pump surgery. 58 cardiac patients undergoing on-pump surgery were prospectively enrolled. Blood samples were taken preoperatively, immediately after surgery, at day 1, 2, 3 and 5 from patients with (n = 21) or without (n = 37) postoperative AKI development. Levels of cfDNA, neutrophil gelatinase-associated lipocalin (NGAL) and creatinine in patients' plasma were quantified. ROC curves were used to assess the predictive value of the biomarkers for AKI. Further baseline characteristics and perioperative variables were analyzed.cfDNA and NGAL levels highly increased in AKI patients and significant intergroup differences (vs. non-AKI) were found until day 3 and day 5 after surgery, respectively. cfDNA levels were significantly elevated in patients who developed late AKI (>24 hours), but not in those with AKI development during the first 24 hours (early AKI). NGAL and creatinine, which were highest in patients with early AKI, accurately predicted during the first 24 postoperative hours (early AKI). At day 3, at a threshold of 260.53 ng/ml cfDNA was the best predictor for AKI (AUC = 0.804) compared to NGAL (AUC = 0.699) and creatinine (AUC = 0.688). NGAL, but not cfDNA, was strongly associated with AKI stages and mortality. Monitoring of cfDNA levels from the first postoperative day might represent a valuable tool to predict late AKI after on-pump surgery.

Compromised Anti-inflammatory Action of Neutrophil Extracellular Traps in PAD4-Deficient Mice Contributes to Aggravated Acute Inflammation After Myocardial Infarction.

Innate immune responses and rapid recruitment of leukocytes, which regulate inflammation and subsequent healing, play a key role in acute myocardial infarction (MI). Peptidylarginine deiminase 4 (PAD4) is critically involved in chromatin decondensation during the release of Neutrophil Extracellular Traps (NETs) by activated neutrophils. Alternatively, activated macrophages (M2) and accurate collagen deposition determine the repair of the infarcted heart. In this study, we investigated the impact of NETs on macrophage polarization and their role for acute cardiac inflammation and subsequent cardiac healing in a mouse model of acute MI. NETs were found to promote in vitro macrophage polarization toward a reparative phenotype. NETs suppressed pro-inflammatory macrophages (M1) under hypoxia and diminished IL-6 and TNF-α expression. Further on, NETs strongly supported M2b polarization and IL-10 expression. In cardiac fibroblasts, NETs increased TGF-ß expression under hypoxic culture conditions. PAD4-/- mice subjected to permanent ligation of the left anterior descending artery suffered from overwhelming inflammation in the acute phase of MI. Noteworthy, PAD4-/- neutrophils were unable to release NETs upon ex vivo stimulation with ionomycin or PMA, but produced significantly higher amounts of reactive oxygen species (ROS). Increased levels of circulating cell-free DNA, mitochondrial DNA and cardiac troponin were found in PAD4-/- mice in the acute phase of MI when compared to WT mice. Reduced cardiac expression of IL-6, IL-10, and M2 marker genes, as well as increased TNF-α expression, suggested a pro-inflammatory state. PAD4-/- mice displayed significantly increased cardiac MMP-2 expression under baseline conditions. At day 1, post-MI, PAD4-/- mice showed increased end-diastolic volume and increased thinning of the left ventricular wall. Interestingly, improved cardiac function, as demonstrated by significantly increased ejection fraction, was found at day 21. Altogether, our results indicate that NETs support macrophage polarization toward an M2 phenotype, thus displaying anti-inflammatory properties. PAD4 deficiency aggravates acute inflammation and increases tissue damage post-MI, partially due to the lack of NETs.

Targeting of cell-free DNA by DNase I diminishes endothelial dysfunction and inflammation in a rat model of cardiopulmonary bypass.

The use of cardiopulmonary bypass (CPB) results in the activation of leukocytes, release of neutrophil extracellular traps (NETs) and severe inflammation. We hypothesize that targeting of circulating cell-free DNA (cfDNA) by DNases might represent a feasible therapeutic strategy to limit CPB-associated side effects. Male Wistar rats (n = 24) underwent CPB with deep hypothermic circulatory arrest (DHCA) and were divided into 3 groups: control (group 1), one i.v. bolus DNase I before CPB start (group 2) and a second DNase I dose before reperfusion (group 3). We found a positive correlation between plasma cfDNA/NETs levels and compromised endothelial vasorelaxation after CPB. DNase I administration significantly diminished plasma cfDNA/NETs levels. Further, a dose-dependent improvement in endothelial function accompanied by significant reduction of circulating intercellular adhesion molecule (ICAM)-1 was observed. Rats of group 3 had significantly reduced plasma IL-6 levels and downregulated expression of adhesion molecules resulting in impaired leukocyte extravasation and reduced MPO activity in lungs. Mechanistically, digestion of NETs by DNase I significantly diminished NETs-dependent upregulation of adhesion molecules in human endothelial cells. Altogether, systemic DNase I administration during CPB efficiently reduced cfDNA/NETs-mediated endothelial dysfunction and inflammation and might represents a promising therapeutic strategy for clinical practice.

Preconditioning of bone marrow-derived mesenchymal stem cells highly strengthens their potential to promote IL-6-dependent M2b polarization.

BACKGROUND: During the last decade, mesenchymal stem cells (MSCs) have gained much attention in the field of regenerative medicine due to their capacity to differentiate into different cell types and to promote immunosuppressive effects. However, the underlying mechanism of MSC-mediated immunoregulation is not fully understood so far. Macrophages are distinguished in classical activated, pro-inflammatory M1 and alternatively activated M2 cells, which possess different functions and transcriptional profiles with respect to inflammatory responses. As polarization is not fixed, macrophage functional plasticity might be modulated by the microenvironment allowing them to rapidly react to danger signals and maintaining tissue homeostasis. METHODS: Murine MSCs were preconditioned with IL-1ß and IFN-É£ to enhance their immunosuppressive capacity regarding macrophage polarization under M1- and M2a-polarizing conditions. Macrophage polarization was analyzed by real-time PCR, flow cytometry, and cytokine detection in culture supernatants. The role of MSC-derived nitric oxide (NO), prostaglandin E2 (PGE2), and IL-6 in this process has been evaluated using siRNA transfection and IL-6 receptor-deficient macrophages, respectively. RESULTS: Preconditioned, but not unprimed, MSCs secreted high levels of NO, IL-6, and PGE2. Co-culture with macrophages (M0) in the presence of M1 inducers (LPS + IFN-É£) led to significant reduction of CD86 and iNOS protein in macrophages and diminished TNF-α secretion. Additionally, CD86 and iNOS protein expression as well as NO and IL-10 secretion were markedly increased under M2a-polarizing culture conditions (IL-4). MSC-dependent macrophage polarization did not depend on direct cell-cell contact. Co-culturing in the presence of LPS and IFN-É£ resulted in the upregulation of M2a, M2b, and M2c marker genes, whereas in the presence of IL-4 only M2b markers were significantly increased. In turn, IL-10-producing regulatory M2b cells significantly inhibited IFN-É£ expression in CD4+ T lymphocytes. Finally, we show that MSC-mediated macrophage polarization strongly depends on IL-6, whereas a minor role for NO and PGE2 was found. CONCLUSIONS: Preconditioning of MSCs highly strengthens their capacity to regulate macrophage features and to promote immunosuppression. Repression of M1 polarization during inflammation and M2b polarization under anti-inflammatory conditions strongly depend on functional IL-6 signaling in macrophages. The potential benefit of preconditioned MSCs and IL-6 should be considered for future clinical treatment.