Cell Type-Specific Roles of NF-κB Linking Inflammation and Thrombosis

The transcription factor NF-κB is a central mediator of inflammation with multiple links to thrombotic processes. In this review, we focus on the role of NF-κB signaling in cell types within the vasculature and the circulation that are involved in thrombo-inflammatory processes. All these cells express NF-κB, which mediates important functions in cellular interactions, cell survival and differentiation, as well as expression of cytokines, chemokines, and coagulation factors. Even platelets, as anucleated cells, contain NF-κB family members and their corresponding signaling molecules, which are involved in platelet activation, as well as secondary feedback circuits. The response of endothelial cells to inflammation and NF-κB activation is characterized by the induction of adhesion molecules promoting binding and transmigration of leukocytes, while simultaneously increasing their thrombogenic potential. Paracrine signaling from endothelial cells activates NF-κB in vascular smooth muscle cells and causes a phenotypic switch to a “synthetic” state associated with a decrease in contractile proteins. Monocytes react to inflammatory situations with enforced expression of tissue factor and after differentiation to macrophages with altered polarization. Neutrophils respond with an extension of their life span—and upon full activation they can expel their DNA thereby forming so-called neutrophil extracellular traps (NETs), which exert antibacterial functions, but also induce a strong coagulatory response. This may cause formation of microthrombi that are important for the immobilization of pathogens, a process designated as immunothrombosis. However, deregulation of the complex cellular links between inflammation and thrombosis by unrestrained NET formation or the loss of the endothelial layer due to mechanical rupture or erosion can result in rapid activation and aggregation of platelets and the manifestation of thrombo-inflammatory diseases. Sepsis is an important example of such a disorder caused by a dysregulated host response to infection finally leading to severe coagulopathies. NF-κB is critically involved in these pathophysiological processes as it induces both inflammatory and thrombotic responses

Untersuchung zur Funktion adipöser Triglycerid-Lipase in kultivierten Endothelzellen

Im Jahr 2004 wurde die adipöse Triglycerid-Lipase (ATGL) von drei unabhängigen Forschungsgruppen als Schlüsselenzym des Triglyceridmetabolismus identifiziert. Systemischer Verlust des ATGL-Gens führt zu massiver Akkumulation von neutralen Lipiden in einer Vielzahl von Geweben. Daneben zeigen Aorten von ATGL(-/-) Mäusen eine verminderte Acetylcholin-vermittelte Relaxation. Diese überraschende Beobachtung deutet auf eine gestörte Funktion des Endothels als Folge der ATGL-Defizienz hin. Ziel meiner Diplomarbeit war es, die Rolle von ATGL in der NO/cGMP-Signalkaskade sowohl in mikrovaskulären (HMECs-1) als auch in makrovaskulären (HUVECs) humanen Endothelzellen zu charakterisieren. Hierzu wurde die Expression der ATGL durch RNA-Interferenz für 24 h blockiert. Anschließend wurden die Effekte der ATGL-Defizienz auf die L-Citrullinbildung, die Phosphorylierung der eNOS am Serin-1177 und auf die endotheliale cGMP-Bildung untersucht. Weder auf L-Citrullin- noch auf cGMP-Ebene konnten Unterschiede zwischen ATGL-siRNA und scramble-siRNA-Zellen festgestellt werden. Versuche mit verlängerter Inkubation der Endothelzellen mit siRNA deuteten auf eine mögliche Verminderung der endothelialen cGMP-Bildung in ATGL-siRNA-Zellen hin. Diese vorläufigen Ergebnisse müssen aber in künftigen Experimenten bestätigt werden.Eindeutige Reduktion von eNOS-Aktivität und cGMP-Bildung wurde durch Vorinkubation mit NM-221, einem noch nicht vollständig charakterisierten Hemmstoff der ATGL, beobachtet. Diese Ergebnisse sind jedoch mit Vorsicht zu betrachten, da NM-221 nicht ATGL-spezifisch ist und möglicherweise andere Signalkaskaden in der Zelle beeinflusst.Zusammenfassend kann man sagen, dass 24-stündige RNA-Interferenz der ATGL keine nennenswerten Auswirkungen auf die NO/cGMP-Signalkaskade in Endothelzellen hatte. Ziel weiterer Forschung sollte es sein, das Zeitfenster der RNA-Interferenz zu verlängern und so mögliche langfristige Effekte beobachten zu können.Adipose triglyceride lipase (ATGL) was identified in 2004 as a key enzyme in triglyceride metabolism. Systemic deletion of the ATGL gene in mice leads to massive accumulation of neutral lipids in adipose and non-adipose tissues. In addition, endothelium-dependent aortic relaxation is virtually abolished in ATGL-deficient aortas, indicating a severe endothelial dysfunction.In view of these findings, it was the aim of the present work to study the role of ATGL in NO/cGMP signal transduction. Human umbilical vein endothelial cells (HUVECs) and a microvascular endothelial cell line (HMEC-1) were treated with small interfering RNA (siRNA) targeted against ATGL. After incubation with siRNA for 24 h enzyme activity of endothelial NO synthase (eNOS) was measured by monitoring the conversion of [3H]L-arginine into [3H]L-citrulline. Additionally, eNOS phosphorylation at Serin-1177 and cGMP formation were determined by western blot analysis and radioimmunoassay, respectively.The results demonstrate that eNOS activity, cGMP formation and eNOS phosphorylation at Serin-1177 were not affected by ATGL knockdown. By contrast, preincubation with NM-221, an inhibitor of ATGL, led to a decrease in eNOS activity and cGMP formation, indicating a potential effect of ATGL on NO/cGMP signaling. However, the Zechner laboratory showed that NM-221 additionally affects lipolytic activity in an ATGL-independent way. Concerning these findings, the data obtained with NM-221 should be considered critically. Interestingly, prolonged incubation with siRNA (48 h and 72 h) resulted in decreased cGMP formation in endothelial cells treated with ATGL siRNA. These data suggest that longer periods of ATGL knockdown are required to determine possible effects of ATGL on NO/cGMP signaling. Further studies are necessary to confirm and clarify this issue.vorgelegt von Marion MussbacherGraz, Univ., Dipl.-Arb., 2010(VLID)21317

Antagonistic Functions of Androgen Receptor and NF-κB in Prostate Cancer—Experimental and Computational Analyses

Prostate cancer is very frequent and is, in many countries, the third-leading cause of cancer related death in men. While early diagnosis and treatment by surgical removal is often curative, metastasizing prostate cancer has a very bad prognosis. Based on the androgen-dependence of prostate epithelial cells, the standard treatment is blockade of the androgen receptor (AR). However, nearly all patients suffer from a tumor relapse as the metastasizing cells become AR-independent. In our study we show a counter-regulatory link between AR and NF-κB both in human cells and in mouse models of prostate cancer, implying that inhibition of AR signaling results in induction of NF-κB-dependent inflammatory pathways, which may even foster the survival of metastasizing cells. This could be shown by reporter gene assays, DNA-binding measurements, and immune-fluorescence microscopy, and furthermore by a whole set of computational methods using a variety of datasets. Interestingly, loss of PTEN, a frequent genetic alteration in prostate cancer, also causes an upregulation of NF-κB and inflammatory activity. Finally, we present a mathematical model of a dynamic network between AR, NF-κB/IκB, PI3K/PTEN, and the oncogene c-Myc, which indicates that AR blockade may upregulate c-Myc together with NF-κB, and that combined anti-AR/anti-NF-κB and anti-PI3K treatment might be beneficial

Neutrophil-Specific STAT4 Deficiency Attenuates Atherosclerotic Burden and Improves Plaque Stability via Reduction in Neutrophil Activation and Recruitment Into Aortas of Mice

BACKGROUND AND AIMS: Neutrophils drive atheroprogression and directly contribute to plaque instability. We recently identified signal transducer and activator of transcription 4 (STAT4) as a critical component for bacterial host defense in neutrophils. The STAT4-dependent functions of neutrophils in atherogenesis are unknown. Therefore, we investigated a contributory role of STAT4 in neutrophils during advanced atherosclerosis. METHODS: We generated myeloid-specific , neutrophil-specific , and control mice. All groups were fed a high-fat/cholesterol diet (HFD-C) for 28 weeks to establish advanced atherosclerosis. Aortic root plaque burden and stability were assessed histologically by Movat pentachrome staining. Nanostring gene expression analysis was performed on isolated blood neutrophils. Flow cytometry was utilized to analyze hematopoiesis and blood neutrophil activation. homing of neutrophils to atherosclerotic plaques was performed by adoptively transferring prelabeled and bone marrow cells into aged atherosclerotic mice and detected by flow cytometry. RESULTS: STAT4 deficiency in both myeloid-specific and neutrophil-specific mice provided similar reductions in aortic root plaque burden and improvements in plaque stability via reduction in necrotic core size, improved fibrous cap area, and increased vascular smooth muscle cell content within the fibrous cap. Myeloid-specific STAT4 deficiency resulted in decreased circulating neutrophils via reduced production of granulocyte-monocyte progenitors in the bone marrow. Neutrophil activation was dampened in HFD-C fed mice via reduced mitochondrial superoxide production, attenuated surface expression of degranulation marker CD63, and reduced frequency of neutrophil-platelet aggregates. Myeloid-specific STAT4 deficiency diminished expression of chemokine receptors CCR1 and CCR2 and impaired neutrophil trafficking to atherosclerotic aorta. CONCLUSIONS: Our work indicates a pro-atherogenic role for STAT4-dependent neutrophil activation and how it contributes to multiple factors of plaque instability during advanced atherosclerosis in mice

Optimized plasma preparation is essential to monitor platelet-stored molecules in humans

<div><p>Platelets store a plethora of different molecules within their granules, modulating numerous pathways, not only in coagulation, but also in angiogenesis, wound healing, and inflammatory diseases. These molecules get rapidly released upon activation and therefore represent an easily accessible indirect marker for platelet activation. Accurate analysis of platelet-derived molecules in the plasma requires appropriate anticoagulation to avoid <i>in vitro</i> activation and subsequent degranulation of platelets, potentially causing artificially high levels and masking biologically relevant differences within translational research studies. However, there is still enormous heterogeneity among anticoagulants used to prevent unwanted platelet activation, so that plasma levels reported for platelet granule contents range over several orders of magnitude. To address this problem and to define the most robust method of plasma preparation to avoid <i>in vitro</i> platelet activation during processing, we compared plasma concentrations of the three platelet-stored factors thrombospondin (TSP-1), platelet factor 4 (PF4), and soluble P-selectin (sCD62P) between human blood samples anticoagulated with either citrate-theophylline-adenosine-dipyridamole (CTAD), acid-citrate-dextrose (ACD), citrate, ethylenediaminetetraacetic acid (EDTA) or heparin. Additionally, we assessed the effect of storage temperature and time between blood drawing and sample processing within the differentially anticoagulated samples. Our data strongly support the use of CTAD as anticoagulant for determining plasma concentrations of platelet-stored molecules, as anticoagulation with heparin or EDTA led to a 12.4- or 8.3-fold increase in plasma levels of PF4, respectively. Whereas ACD was similar effective as CTAD, citrate only showed comparable PF4 plasma levels when plasma was kept at 4°C. Moreover, blood sampling with CTAD as anticoagulant resulted in the most reproducible values, even when samples were processed at ambient temperature or after storage over 6 hours. In the latter case, anticoagulation with heparin or EDTA led to artificially high plasma levels indicative of <i>in vitro</i> platelet activation. Therefore, we want to raise scientific awareness for choosing CTAD as optimal anticoagulant for the detection of platelet-stored molecules in plasma.</p></div

Fold-increase of platelet-stored factors due to suboptimal plasma generation.

<p>Plasma concentrations of PF4 (A, C) and TSP-1 (B, D) were calculated as fold of plasma levels relative to CTAD plasma 30 min 4°C (8 healthy donors). Fold-increase is depicted for ACD (dark grey bar), citrate (grey bar), heparin (white bar) or EDTA (patterned bar) plasma at 0.5 h, 2 h, 6 h, and 24 h. Samples were either processed at 4°C (A-B) or at ambient temperature (C-D). Significant differences were analyzed using one-way ANOVA with Dunnett correction and were depicted as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 (in comparison to CTAD 30 min 4°C).</p

Effect of <i>in vitro</i> platelet activation on concentration of plasma factors.

<p>Blood of 8 healthy donors was anticoagulated with CTAD, ACD, citrate, heparin or EDTA (grey bar) and stimulated with the platelet activator thrombin (dark grey bar) at a submaximal dose for 0.5 h and 6 h at room temperature. Subsequently, plasma was prepared and analyzed for PF4 (A, C) and TSP-1 (B, D) levels. Significant differences were analyzed using two-way ANOVA with Bonferroni correction and were depicted as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 (in comparison to untreated).</p

Time-dependent differences in plasma levels of platelet-stored factors.

<p>Blood from 8 healthy donors was anticoagulated with CTAD (black bar), ACD (dark grey bar), citrate (grey bar), heparin (white bar) or EDTA (patterned bar) and stored at 4°C (A-B) or at room temperature (C-D) for 0.5 h, 2 h, 6 h or 24 h until plasma preparation. Concentrations of PF4 (A, C) and TSP-1 (B, D) were determined for each time point. Significant differences were analyzed using two-way ANOVA with Dunnett correction (with CTAD as reference) and were depicted as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.</p