Acute and short-term fluctuations in gravity are associated with changes in circulatory plasma protein levels.

Gravitational changes between micro- and hypergravity cause several adaptations and alterations in the human body. Besides muscular atrophy and immune system impairment, effects on the circulatory system have been described, which can be associated with a wide range of blood biomarker changes. This study examined nine individuals (seven males, two females) during a parabolic flight campaign (PFC). Thirty-one parabolas were performed in one flight day, resulting in ~22 s of microgravity during each parabola. Each participant was subjected to a single flight day with a total of 31 parabolas, totaling 11 min of microgravity during one parabolic flight. Before and after (1 hour (h) and 24 h), the flights blood was sampled to examine potential gravity-induced changes of circulating plasma proteins. Proximity Extension Assay (PEA) offers a proteomic solution, enabling the simultaneous analysis of a wide variety of plasma proteins. From 2925 unique proteins analyzed, 251 (8.58%) proteins demonstrated a differential regulation between baseline, 1 h and 24 h post flight. Pathway analysis indicated that parabolic flights led to altered levels of proteins associated with vesicle organization and apoptosis up to 24 h post microgravity exposure. Varying gravity conditions are associated with poorly understood physiological changes, including stress responses and fluid shifts. We provide a publicly available library of gravity-modulated circulating protein levels illustrating numerous changes in cellular pathways relevant for inter-organ function and communication.

Application and challenges of TCR and BCR sequencing to investigate T- and B-cell clonality in elastase-induced experimental murine abdominal aortic aneurysm.

Background: An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease. Although its pathogenesis is still poorly understood, recent evidence suggests that AAA displays autoimmune disease characteristics. Particularly, T cells responding to AAA-related antigens in the aortic wall may contribute to an initial immune response. Single-cell RNA (scRNA) T cell receptor (TCR) and B cell receptor (BCR) sequencing is a powerful tool for investigating clonality. However, difficulties such as limited numbers of isolated cells must be considered during implementation and data analysis, making biological interpretation challenging. Here, we perform a representative single-cell immune repertoire analysis in experimental murine AAA and show a reliable bioinformatic processing pipeline highlighting opportunities and limitations of this approach. Methods: We performed scRNA TCR and BCR sequencing of isolated lymphocytes from the infrarenal aorta of male C57BL/6J mice 3, 7, 14, and 28 days after AAA induction via elastase perfusion of the aorta. Sham-operated mice at days 3 and 28 and non-operated mice served as controls. Results: Comparison of complementarity-determining region (CDR3) length distribution of 179 B cells and 796 T cells revealed neither differences between AAA and control nor between the disease stages. We found no clonal expansion of B cells in AAA. For T cells, we identified several clones in 11 of 16 AAA samples and one of eight control samples. Immune receptor repertoire comparison indicated that only a few clones were shared between the individual AAA samples. The most frequently used V-genes in the TCR beta chain in AAA were TRBV3, TRBV19, and the splicing variant TRBV12-2 + TRBV13-2. Conclusion: We found no clonal expansion of B cells but evidence for clonal expansion of T cells in elastase-induced AAA in mice. Our findings imply that a more precise characterization of TCR and BCR distribution requires a more extensive number of lymphocytes to prevent undersampling and potentially detect rare clones. Thus, further experiments are necessary to confirm our findings. In summary, this paper examines TCR and BCR sequencing results, identifies limitations and pitfalls, and offers guidance for future studies.

Quantitative assessment of angioplasty-induced vascular inflammation with 19F cardiovascular magnetic resonance imaging.

BACKGROUND: Macrophages play a pivotal role in vascular inflammation and predict cardiovascular complications. Fluorine-19 magnetic resonance imaging (19F MRI) with intravenously applied perfluorocarbon allows a background-free direct quantification of macrophage abundance in experimental vascular disease models in mice. Recently, perfluorooctyl bromide-nanoemulsion (PFOB-NE) was applied to effectively image macrophage infiltration in a pig model of myocardial infarction using clinical MRI scanners. In the present proof-of-concept approach, we aimed to non-invasively image monocyte/macrophage infiltration in response to carotid artery angioplasty in pigs using 19F MRI to assess early inflammatory response to mechanical injury. METHODS: In eight minipigs, two different types of vascular injury were conducted: a mild injury employing balloon oversize angioplasty only (BA, n = 4) and a severe injury provoked by BA in combination with endothelial denudation (BA + ECDN, n = 4). PFOB-NE was administered intravenously three days after injury followed by 1H and 19F MRI to assess vascular inflammatory burden at day six. Vascular response to mechanical injury was validated using X-ray angiography, intravascular ultrasound and immunohistology in at least 10 segments per carotid artery. RESULTS: Angioplasty was successfully induced in all eight pigs. Response to injury was characterized by positive remodeling with predominantly adventitial wall thickening and concomitant infiltration of monocytes/macrophages. No severe adverse reactions were observed following PFOB-NE administration. In vivo 19F signals were only detected in the four pigs following BA + ECDN with a robust signal-to-noise ratio (SNR) of 14.7 ± 4.8. Ex vivo analysis revealed a linear correlation of 19F SNR to local monocyte/macrophage cell density. Minimum detection limit of infiltrated monocytes/macrophages was estimated at approximately 410 cells/mm2. CONCLUSIONS: In this proof-of-concept study, 19F MRI enabled quantification of monocyte/macrophage infiltration after vascular injury with sufficient sensitivity. This may provide the opportunity to non-invasively monitor vascular inflammation with MRI in patients after angioplasty or even in atherosclerotic plaques.

Modulation of mTOR Signaling in Cardiovascular Disease to Target Acute and Chronic Inflammation.

Inflammation is a key component in the pathogenesis of cardiovascular diseases causing a significant burden of morbidity and mortality worldwide. Recent research shows that mammalian target of rapamycin (mTOR) signaling plays an important role in the general and inflammation-driven mechanisms that underpin cardiovascular disease. mTOR kinase acts prominently in signaling pathways that govern essential cellular activities including growth, proliferation, motility, energy consumption, and survival. Since the development of drugs targeting mTOR, there is proven efficacy in terms of survival benefit in cancer and allograft rejection. This review presents current information and concepts of mTOR activity in myocardial infarction and atherosclerosis, two important instances of cardiovascular illness involving acute and chronic inflammation. In experimental models, inhibition of mTOR signaling reduces myocardial infarct size, enhances functional remodeling, and lowers the overall burden of atheroma. Aside from the well-known effects of mTOR inhibition, which are suppression of growth and general metabolic activity, mTOR also impacts on specific leukocyte subpopulations and inflammatory processes. Inflammatory cell abundance is decreased due to lower migratory capacity, decreased production of chemoattractants and cytokines, and attenuated proliferation. In contrast to the generally suppressed growth signals, anti-inflammatory cell types such as regulatory T cells and reparative macrophages are enriched and activated, promoting resolution of inflammation and tissue regeneration. Nonetheless, given its involvement in the control of major cellular pathways and the maintenance of a functional immune response, modification of this system necessitates a balanced and time-limited approach. Overall, this review will focus on the advancements, prospects, and limits of regulating mTOR signaling in cardiovascular disease.

The collagen receptor glycoprotein VI promotes platelet-mediated aggregation of ß-amyloid.

Cerebral amyloid angiopathy (CAA) and ß-amyloid (Aß) deposition in the brain parenchyma are hallmarks of Alzheimer's disease (AD). We previously reported that platelets contribute to Aß aggregation in cerebral vessels by secreting the factor clusterin upon binding of Aß40 to the fibrinogen receptor integrin αIIbß3 Here, we investigated the contribution of the collagen receptor GPVI (glycoprotein VI) in platelet-induced amyloid aggregation. Using platelets isolated from GPVI-wild type and GPVI-deficient human donors and mice, we found that Aß40 bound to GPVI, which induced the release of ATP and fibrinogen, resulting in platelet aggregation. Binding of Aß40 to integrin αIIbß3, fibrinogen, and GPVI collectively contributed to the formation of amyloid clusters at the platelet surface. Consequently, blockade of αIIbß3 or genetic loss of GPVI reduced amyloid fibril formation in cultured platelets and decreased the adhesion of Aß-activated platelets to injured carotid arteries in mice. Application of losartan to inhibit collagen binding to GPVI resulted in decreased Aß40-stimulated platelet activation, factor secretion, and platelet aggregation. Furthermore, the application of GPVI- or integrin-blocking antibodies reduced the formation of platelet-associated amyloid aggregates. Our findings indicate that Aß40 promotes platelet-mediated amyloid aggregation by binding to both GPVI and integrin αIIbß3 Blocking these pathways may therapeutically reduce amyloid plaque formation in cerebral vessels and the brain parenchyma of patients.

Syndecan-1 Predicts Outcome in Patients with ST-Segment Elevation Infarction Independent from Infarct-related Myocardial Injury.

Syndecan-1 (sdc1) is a surface protein part of the endothelial glycocalyx (eGC). Soluble sdc1 is derived from shedding and indicates damaged eGC. We assessed the predictive value of plasma sdc1 concentrations for future cardiovascular events in acute reperfused ST-segment elevation myocardial infarction (STEMI) patients. A total of 206 patients admitted for STEMI were included in this study (29% female; age 65 ± 12 years) and followed-up for six months. Plasma samples were obtained post-intervention and analyzed for sdc1 by Enzyme-linked Immunosorbent Assay (ELISA). Primary outcome was six-month-mortality. Sdc1 did not correlate with biomarkers such as creatine kinase (CK) (r = 0.11; p = 0.01) or troponin (r = -0.12; p = 0.09), nor with infarct size (r = -0.04; p = 0.67) and myocardial salvage index (r = 0.11; p = 0.17). Sdc-1 was associated with mortality (changes per 100 ng/mL sdc-1 concentration; HR 1.08 95% 1.03-1.12; p = 0.001). An optimal cut-off was calculated at >120 ng/mL. After correction for known risk factors sdc1 >120 ng/mL was independently associated with mortality after 6 months. In our study, sdc1 is independently associated with six-month-mortality after STEMI. Combining clinical evaluation and different biomarkers assessing both infarct-related myocardial injury and systemic stress response might improve the accuracy of predicting clinical prognosis in STEMI patients.

Phagocytosis of a PFOB-Nanoemulsion for 19F Magnetic Resonance Imaging: First Results in Monocytes of Patients with Stable Coronary Artery Disease and ST-Elevation Myocardial Infarction.

Fluorine-19 magnetic resonance imaging (19F MRI) with intravenously applied perfluorooctyl bromide-nanoemulsions (PFOB-NE) has proven its feasibility to visualize inflammatory processes in experimental disease models. This approach is based on the properties of monocytes/macrophages to ingest PFOB-NE particles enabling specific cell tracking in vivo. However, information on safety (cellular function and viability), mechanism of ingestion and impact of specific disease environment on PFOB-NE uptake is lacking. This information is, however, crucial for the interpretation of 19F MRI signals and a possible translation to clinical application. To address these issues, whole blood samples were collected from patients with acute ST-elevation myocardial infarction (STEMI), stable coronary artery disease (SCAD) and healthy volunteers. Samples were exposed to fluorescently-labeled PFOB-NE and particle uptake, cell viability and migration activity was evaluated by flow cytometry and MRI. We were able to show that PFOB-NE is ingested by human monocytes in a time- and subset-dependent manner via active phagocytosis. Monocyte function (migration, phagocytosis) and viability was maintained after PFOB-NE uptake. Monocytes of STEMI and SCAD patients did not differ in their maximal PFOB-NE uptake compared to healthy controls. In sum, our study provides further evidence for a safe translation of PFOB-NE for imaging purposes in humans.

IL-1 family cytokines in cardiovascular disease.

The interleukin (IL)-1 family is a group of cytokines crucially involved in regulating immune responses to infectious challenges and sterile insults. The family consists of the eponymous pair IL-1α and IL-1ß, IL-18, IL-33, IL-37, IL-38, and several isoforms of IL-36. In addition, two endogenous inhibitors of functional receptor binding, IL-1R antagonist (IL-1Ra) and IL-36Ra complete the family. To gain biological activity IL-1ß and IL-18 require processing by the protease caspase-1 which is associated with the multi-protein complex inflammasome. Numerous clinical association studies and experimental approaches have implicated members of the IL-1 family, their receptors, or component of the processing machinery in underlying processes of cardiovascular diseases (CVDs). Here we summarize the current state of knowledge regarding the pro-inflammatory and disease-modulating role of the IL-1 family in atherosclerosis, myocardial infarction, aneurysm, stroke, and other CVDs. We discuss clinical evidence, experimental approaches and lastly lend a perspective on currently developing therapeutic strategies involving the IL-1 family in CVD.

CD36-triggered cell invasion and persistent tissue colonization by tumor microvesicles during metastasis.

Tumor microvesicles are a peculiar type of extracellular vesicles that circulate in the blood of patients with metastatic cancer. The itineraries and immune cell interactions of tumor microvesicles during the intravascular and extravascular stages of metastasis are largely unknown. We found that the lipid receptor CD36 is a major mediator of the engulfment of pancreatic tumor microvesicles by myeloid immune cells in vitro and critically samples circulating tumor microvesicles by resident liver macrophages in mice in vivo. Direct nanoscopic imaging of individual tumor microvesicles shows that the microvesicles rapidly decay during engulfment whereby their cargo is targeted concomitantly to the plasma membrane and the cytoplasm excluding lysosomal compartments. CD36 also promotes internalization of blood cell (nontumor) microvesicles, which involves endolysosomal pathways. A portion of tumor microvesicles circulating in the liver microcirculation traverses the vessel wall in a CD36-dependent way. Extravasated microvesicles colonize distinct perivascular Ly6C- macrophages for at least 2 wk. Thus, the microvesicles are increasingly integrated into CD36-induced premetastatic cell clusters and enhance development of liver metastasis. Hence, promotion of metastasis by pancreatic tumor microvesicles is associated with CD36-regulated immune cell invasion and extravasation of microvesicles and persistent infiltration of specific tissue macrophages by microvesicle cargo.-Pfeiler, S., Thakur, M., Grünauer, P., Megens, R. T. A., Joshi, U., Coletti, R., Samara, V., Müller-Stoy, G., Ishikawa-Ankerhold, H., Stark, K., Klingl, A., Fröhlich, T., Arnold, G. J., Wörmann, S., Bruns, C. J., Algül, H., Weber, C., Massberg, S., Engelmann, B. CD36-triggered cell invasion and persistent tissue colonization by tumor microvesicles during metastasis.

Distinct Pathogenesis of Pancreatic Cancer Microvesicle-Associated Venous Thrombosis Identifies New Antithrombotic Targets In Vivo.

OBJECTIVE: Cancer patients are at high risk of developing deep venous thrombosis (DVT) and venous thromboembolism, a leading cause of mortality in this population. However, it is largely unclear how malignant tumors drive the prothrombotic cascade culminating in DVT. APPROACH AND RESULTS: Here, we addressed the pathophysiology of malignant DVT compared with nonmalignant DVT and focused on the role of tumor microvesicles as potential targets to prevent cancer-associated DVT. We show that microvesicles released by pancreatic adenocarcinoma cells (pancreatic tumor-derived microvesicles [pcMV]) boost thrombus formation in a model of flow restriction of the mouse vena cava. This depends on the synergistic activation of coagulation by pcMV and host tissue factor. Unlike nonmalignant DVT, which is initiated and propagated by innate immune cells, thrombosis triggered by pcMV was largely independent of myeloid leukocytes or platelets. Instead, we identified externalization of the phospholipid phosphatidylethanolamine as a major mechanism controlling the prothrombotic activity of pcMV. Disrupting phosphatidylethanolamine-dependent activation of factor X suppressed pcMV-induced DVT without causing changes in hemostasis. CONCLUSIONS: Together, we show here that the pathophysiology of pcMV-associated experimental DVT differs markedly from innate immune cell-promoted nonmalignant DVT and is therefore amenable to distinct antithrombotic strategies. Targeting phosphatidylethanolamine on tumor microvesicles could be a new strategy for prevention of cancer-associated DVT without causing bleeding complications.

Propagation of thrombosis by neutrophils and extracellular nucleosome networks.

Neutrophils, early mediators of the innate immune defense, are recruited to developing thrombi in different types of thrombosis. They amplify intravascular coagulation by stimulating the tissue factor-dependent extrinsic pathway via inactivation of endogenous anticoagulants, enhancing factor XII activation or decreasing plasmin generation. Neutrophil-dependent prothrombotic mechanisms are supported by the externalization of decondensed nucleosomes and granule proteins that together form neutrophil extracellular traps. These traps, either in intact or fragmented form, are causally involved in various forms of experimental thrombosis as first indicated by their role in the enhancement of both microvascular thrombosis during bacterial infection and carotid artery thrombosis. Neutrophil extracellular traps can be induced by interactions of neutrophils with activated platelets; vice versa, these traps enhance adhesion of platelets via von Willebrand factor. Neutrophil-induced microvascular thrombus formation can restrict the dissemination and survival of blood-borne bacteria and thereby sustain intravascular immunity. Dysregulation of this innate immune pathway may support sepsis-associated coagulopathies. Notably, neutrophils and extracellular nucleosomes, together with platelets, critically promote fibrin formation during flow restriction-induced deep vein thrombosis. Neutrophil extracellular traps/extracellular nucleosomes are increased in thrombi and in the blood of patients with different vaso-occlusive pathologies and could be therapeutically targeted for the prevention of thrombosis. Thus, during infections and in response to blood vessel damage, neutrophils and externalized nucleosomes are major promoters of intravascular blood coagulation and thrombosis.

Distinct surveillance pathway for immunopathology during acute infection via autophagy and SR-BI.

The mechanisms protecting from immunopathology during acute bacterial infections are incompletely known. We found that in response to apoptotic immune cells and live or dead Listeria monocytogenes scavenger receptor BI (SR-BI), an anti-atherogenic lipid exchange mediator, activated internalization mechanisms with characteristics of macropinocytosis and, assisted by Golgi fragmentation, initiated autophagic responses. This was supported by scavenger receptor-induced local increases in membrane cholesterol concentrations which generated lipid domains particularly in cell extensions and the Golgi. SR-BI was a key driver of beclin-1-dependent autophagy during acute bacterial infection of the liver and spleen. Autophagy regulated tissue infiltration of neutrophils, suppressed accumulation of Ly6C+ (inflammatory) macrophages, and prevented hepatocyte necrosis in the core of infectious foci. Perifocal levels of Ly6C+ macrophages and Ly6C- macrophages were unaffected, indicating predominant regulation of the focus core. SR-BI-triggered autophagy promoted co-elimination of apoptotic immune cells and dead bacteria but barely influenced bacterial sequestration and survival or inflammasome activation, thus exclusively counteracting damage inflicted by immune responses. Hence, SR-BI- and autophagy promote a surveillance pathway that partially responds to products of antimicrobial defenses and selectively prevents immunity-induced damage during acute infection. Our findings suggest that control of infection-associated immunopathology can be based on a unified defense operation.

Biological basis and pathological relevance of microvascular thrombosis.

Microvascular thrombosis indicates a pathological occlusion of microvessels by fibrin- and/or platelet-rich thrombi. It is observed during systemic infections, cancer, myocardial infarction, stroke, neurodegenerative diseases and in thrombotic microangiopathies. Microvessel thrombosis can cause greatly differing symptoms that range from limited changes in plasma coagulation markers to severe multi-organ failure. Because microvessel thrombi are difficult to detect and often occur only transiently, their importance for disease development and host biology is likely markedly under-appreciated. Recently, clear indications for a biological basis of microvascular thrombosis have been obtained. During systemic infections microvessel thrombosis can mediate an intravascular innate immune response (immunothrombosis). This biological form of thrombosis is based on the generation of fibrin inside blood vessels and is critically triggered by neutrophils and their interactions with platelets which result in the release of neutrophil extracellular traps (extracellular nucleosomes). Immunothrombosis is critically supported by neutrophil elastase and the activator molecules of blood coagulation tissue factor and factor XII. Identification of the biological driving forces of microvascular thrombosis should help to elucidate the mechanisms promoting pathological vessel occlusions in both microvessels and large vessels.

Functional relevance of the cannabinoid receptor 2 - heme oxygenase pathway: a novel target for the attenuation of portal hypertension.

AIMS: In liver cirrhosis, inflammation triggers portal hypertension. Kupffer cells (KC) produce vasoconstrictors upon activation by bacterial constituents. Here, we hypothesize that the anti-inflammatory action of the cannabinoid receptor 2 (CB2) agonists JWH-133 and GP 1a attenuate portal hypertension. MAIN METHODS: In vivo measurements of portal pressures and non-recirculating liver perfusions were performed in rats 4weeks after bile duct ligation (BDL). Zymosan (150µg/ml, isolated liver perfusion) or LPS (4mg/kgb.w., in vivo) was infused to activate the KC in the absence or presence of JWH-133 (10mg/kgb.w.), GP 1a (2.5mg/kgb.w.) or ZnPP IX (1µM). Isolated KC were treated with Zymosan (0.5mg/ml) in addition to JWH-133 (5µM). The thromboxane (TX) B2 levels in the perfusate and KC media were determined by ELISA. Heme oxygenase-1 (HO-1) and CB2 were analyzed by Western blot or confocal microscopy. KEY FINDINGS: JWH-133 or GP 1a pre-treatment attenuated portal pressures following KC activation in all experimental settings. In parallel, HO-1 expression increased with JWH-133 pre-treatment. However, the inhibition of HO-1 enhanced portal hypertension, indicating the functional role of this novel pathway. In isolated KC, the expression of CB2 and HO-1 increased with Zymosan, LPS and JWH-133 treatment while TXB2 production following KC activation was attenuated by JWH-133 pre-treatment. SIGNIFICANCE: JWH-133 or GP 1a treatment attenuates portal hypertension. HO-1 induction by JWH-133 plays a functional role. Therefore, the administration of JWH-133 or GP 1a represents a promising new treatment option for portal hypertension triggered by microbiological products.

oxLDL downregulates the dendritic cell homing factors CCR7 and CCL21.

INTRODUCTION: Dendritic cells (DCs) and oxLDL play an important role in the atherosclerotic process with DCs accumulating in the plaques during plaque progression. Our aim was to investigate the role of oxLDL in the modulation of the DC homing-receptor CCR7 and endothelial-ligand CCL21. METHODS AND RESULTS: The expression of the DC homing-receptor CCR7 and its endothelial-ligand CCL21 was examined on atherosclerotic carotic plaques of 47 patients via qRT-PCR and immunofluorescence. In vitro, we studied the expression of CCR7 on DCs and CCL21 on human microvascular endothelial cells (HMECs) in response to oxLDL. CCL21- and CCR7-mRNA levels were significantly downregulated in atherosclerotic plaques versus non-atherosclerotic controls [90% for CCL21 and 81% for CCR7 (P < 0.01)]. In vitro, oxLDL reduced CCR7 mRNA levels on DCs by 30% and protein levels by 46%. Furthermore, mRNA expression of CCL21 was significantly reduced by 50% (P < 0.05) and protein expression by 24% in HMECs by oxLDL (P < 0.05). CONCLUSIONS: The accumulation of DCs in atherosclerotic plaques appears to be related to a downregulation of chemokines and their ligands, which are known to regulate DC migration. oxLDL induces an in vitro downregulation of CCR7 and CCL21, which may play a role in the reduction of DC migration from the plaques.

Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo.

Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features.

Immunoregulatory effects of the flavonol quercetin in vitro and in vivo.

PURPOSE: Atherosclerosis is known to be an inflammatory disease. Dendritic cells (DCs) are essential for the regulation of the immune system. Up to 10% of the cells in atherosclerotic plaques are DCs. The cardiovascular protective effects of flavonoids (tea, wine) may be mediated by anti-inflammatory mechanisms that affect DC regulation. We aimed to characterize the impact of the flavonol quercetin on DC activity and differentiation in vitro and in vivo. METHODS: For the in vitro experiments, we used murine DCs and endothelial cells to study adhesion properties. For all other experiments (DC phagocytosis capacity, DC maturation, DC differentiation (BDCA-1/-2) and NF-kB-activation), human monocyte-derived DCs were used. The cells were incubated with quercetin (10 µmol/L) ± oxLDL (10 µg/mL) between 24 and 48 h. For in vivo experiments, eight healthy male volunteers took 500 mg of quercetin twice daily over 4 weeks, five healthy male volunteers served as control. Before and after intake, blood samples were collected. Peripheral blood leukocytes were isolated (analyses of DC differentiation), and plasma was immediately frozen. RESULTS: Quercetin reduced DC adhesion (-42%; p < 0.05) and expression of CD11a (-21%; p < 0.05). OxLDL-induced DC differentiation was partially inhibited by quercetin (BDCA-1-29%; BDCA-2-33%; p < 0.05). These effects were achieved by compensation of oxLDL-induced up-regulation of NF-kB by quercetin. The 4-week treatment with quercetin resulted in relevant plasma levels (2.47 µmol/L) and reduced BDCA-2 + DCs in the peripheral blood by 42% (p < 0.05) as well as systemic levels of the NO-synthase inhibitor asymmetric dimethylarginine (-31%, p < 0.05). CONCLUSION: In vitro, quercetin reduced DC adhesion and oxLDL-induced DC differentiation. In vivo, quercetin reduced circulating plasmacytoid DCs and systemic ADMA-levels. The immunoregulatory effects of quercetin may contribute to the anti-atherosclerotic potential of flavonols.

Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases.

Blood neutrophils provide the first line of defense against pathogens but have also been implicated in thrombotic processes. This dual function of neutrophils could reflect an evolutionarily conserved association between blood coagulation and antimicrobial defense, although the molecular determinants and in vivo significance of this association remain unclear. Here we show that major microbicidal effectors of neutrophils, the serine proteases neutrophil elastase and cathepsin G, together with externalized nucleosomes, promote coagulation and intravascular thrombus growth in vivo. The serine proteases and extracellular nucleosomes enhance tissue factor- and factor XII-dependent coagulation in a process involving local proteolysis of the coagulation suppressor tissue factor pathway inhibitor. During systemic infection, activation of coagulation fosters compartmentalization of bacteria in liver microvessels and reduces bacterial invasion into tissue. In the absence of a pathogen challenge, neutrophil-derived serine proteases and nucleosomes can contribute to large-vessel thrombosis, the main trigger of myocardial infarction and stroke. The ability of coagulation to suppress pathogen dissemination indicates that microvessel thrombosis represents a physiological tool of host defense.

Treatment with the leukotriene inhibitor montelukast for 10 days attenuates portal hypertension in rat liver cirrhosis.

UNLABELLED: The mechanisms underlying intrahepatic vasoconstriction are not fully elucidated. Here we investigated the Kupffer cell (KC)-dependent increase in portal pressure by way of actions of vasoconstrictive cysteinyl leukotrienes (Cys-LTs). Liver cirrhosis was induced in rats by bile duct ligation (BDL for 4 weeks; controls: sham-operation) and thioacetamide application (18 weeks). Infusion of leukotriene (LT) C(4) or LTD(4) in isolated perfused livers (20 nM, BDL and sham) demonstrated that LTC(4) is a more relevant vasoconstrictor. In BDL animals the Cys-LT(1) receptor inhibitor montelukast (1 microM) reduced the maximal portal perfusion pressure following LTC(4) or LTD(4) infusion. The infusion of LTC(4) or D(4) in vivo (15 microg/kg b.w.) confirmed LTC(4) as the more relevant vasoconstrictor. Activation of KCs with zymosan (150 microg/mL) in isolated perfused BDL livers increased the portal perfusion pressure markedly, which was attenuated by LT receptor blockade (Ly171883, 20 microM). Cys-LTs in the effluent perfusate increased with KC activation but less with additional blockade of KCs with gadolinium chloride (10 mg/kg body weight, 48 and 24 hours pretreatment). KCs were isolated from normal rat livers and activated with zymosan or lipopolysaccharide at different timepoints. This resulted in an increase in Cys-LT production that was not influenced by preincubation with montelukast (1 microM). Infusion of LTC(4) (20 nM) and the thromboxane analog U46619 (0.1 microM) further enhanced portal pressure, indicating additive effects. Treatment with montelukast for 10 days resulted in an impressive reduction in the basal portal pressure and an attenuation of the KC-dependent increase in portal pressure. CONCLUSION: Activation of isolated KCs produced Cys-LTs. Infusion of Cys-LTs increased portal pressure and, vice versa, treatment with montelukast reduced portal pressure in rat liver cirrhosis. Therefore, montelukast may be of therapeutic benefit for patients with portal hypertension.