Marion Gröger

Dimethylfumarate Inhibits TNF-Induced Nuclear Entry of NF-κB/p65 in Human Endothelial Cells

Fumaric acid esters, mainly dimethylfumarate (DMF), have been successfully used to treat psoriasis. Based on previous observations that DMF inhibited expression of several TNF-induced genes in endothelial cells, we wished to explore the molecular basis of DMF function in greater detail. In first experiments we analyzed DMF effects on tissue factor expression in human endothelial cells in culture, because tissue factor is expressed by two independent sets of transcription factors, by NF-κB via TNF and by early gene response-1 transcription factor via vascular endothelial growth factor (VEGF). We show that DMF inhibits TNF-induced tissue factor mRNA and protein expression as well as TNF-induced DNA binding of NF-κB proteins, but not VEGF-induced tissue factor protein, mRNA expression, or VEGF-induced early gene response-1 transcription factor/DNA binding. To determine where DMF interferes with the TNF/NF-κB signaling cascade, we next analyzed DMF effects on IκB and on the subcellular distribution of NF-κB. DMF does not inhibit TNF-induced IκBα phosphorylation and IκB degradation; thus, NF-κB is properly released from IκB complexes even in the presence of DMF. Importantly, DMF inhibits the TNF-induced nuclear entry of NF-κB proteins, and this effect appears selective for NF-κB after the release from IκB, because the constitutive shuttling of inactive NF-κB/IκB complexes into and out from the nucleus is not blocked by DMF. Moreover, DMF does not block NF-κB/DNA binding. In conclusion, DMF appears to selectively prevent the nuclear entry of activated NF-κB, and this may be the basis of its beneficial effect in psoriasis.

The fibrin-derived peptide Bβ 15-42 protects the myocardium against ischemia-reperfusion injury

In the event of a myocardial infarction, current interventions aim to reopen the occluded vessel to reduce myocardial damage and injury. Although reperfusion is essential for tissue salvage, it can cause further damage and the onset of inflammation. We show a novel anti-inflammatory effect of a fibrin-derived peptide, Bβ15–42. This peptide competes with the fibrin fragment N-terminal disulfide knot-II (an analog of the fibrin E1 fragment) for binding to vascular endothelial (VE)-cadherin, thereby preventing transmigration of leukocytes across endothelial cell monolayers. In acute or chronic rat models of myocardial ischemia-reperfusion injury, Bβ15–42 substantially reduces leukocyte infiltration, infarct size and subsequent scar formation. The pathogenic role of fibrinogen products is further confirmed in fibrinogen knockout mice, in which infarct size was substantially smaller than in wild-type animals. Our findings conclude that the interplay of fibrin fragments, leukocytes and VE-cadherin contribute to the pathogenesis of myocardial damage and reperfusion injury. The naturally occurring peptide Bβ15–42 represents a potential candidate for reperfusion therapy in humans.NOTE: In the HTML version of this paper originally published online, the name of an author was given incorrectly. The correct name for Peter Fried is Peter Friedl. Also, the first affiliation was given incorrectly. The correct affiliation is Department of General Dermatology, Medical University of Vienna, 18-20 Waehringer Guertel, Vienna, 1090, Austria. These errors have been corrected in the HTML version of the article.

Plasmacytoid Dendritic Cell Recruitment by Immobilized CXCR3 Ligands

Plasmacytoid dendritic cells (pDCs) recognize microbes, viruses in particular, and provide unique means of innate defense against them. The mechanism of pDC tissue recruitment remained enigmatic because the ligands of CXCR3, the cardinal chemokine receptor on pDCs, have failed to induce in vitro chemotaxis of pDCs in the absence of additional chemokines. In this study, we demonstrate that CXCR3 is sufficient to induce pDC migration, however, by a migratory mechanism that amalgamates the features of haptotaxis and chemorepulsion. To mediate “haptorepulsion” of pDCs, CXCR3 requires the encounter of its cognate ligands immobilized, optimally by heparan sulfate, in a form of a negative gradient. This is the first report of the absolute requirement of chemokine immobilization and presentation for its in vitro promigratory activity. The paradigmatic example of pDC haptorepulsion described here may represent a new pathophysiologically relevant migratory mechanism potentially used by other cells in response to other chemokines.

IL-3 Induces Expression of Lymphatic Markers Prox-1 and Podoplanin in Human Endothelial Cells

Factors determining lymphatic differentiation in the adult organism are not yet well characterized. We have made the observation that mixed primary cultures of dermal blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) grown under standard conditions change expression of markers during subculture: After passage 6, they uniformly express LEC-specific markers Prox-1 and podoplanin. Using sorted cells, we show that LEC but not BEC constitutively express IL-3, which regulates Prox-1 and podoplanin expression in LEC. The addition of IL-3 to the medium of BEC cultures induces Prox-1 and podoplanin. Blocking IL-3 activity in LEC cultures results in a loss of Prox-1 and podoplanin expression. In conclusion, endogenous IL-3 is required to maintain the LEC phenotype in culture, and the addition of IL-3 to BEC appears to induce transdifferentiation of BEC into LEC.

Exercise training increases endothelial progenitor cells and decreases asymmetric dimethylarginine in peripheral arterial disease: A randomized controlled trial

BACKGROUND Supervised exercise training (SET) is recommended as initial treatment to improve walking capacity in peripheral arterial disease (PAD) patients with intermittent claudication. Various mechanisms by which SET yields beneficial effects are postulated, however data regarding its influence on angiogenesis are scarce. Thus, we designed a prospective randomized controlled trial to study the impact of SET on markers of angiogenesis and endothelial function in PAD. METHODS Forty PAD patients were randomized to SET on top of best medical treatment (SET+BMT) for 6 months versus best medical treatment (BMT) only. Endothelial progenitor cells (EPC) were assessed by whole-blood flow cytometry (co-expression of CD34+ CD133+ KDR+) and cell culture assays (endothelial cell-colony forming units, circulating angiogenic cells, migration assay) at baseline, 3, 6 and 12-months after inclusion. Changes of plasma levels of asymmetric dimethylarginine (ADMA), vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1) and maximum walking distance were determined. RESULTS EPC - measured by flow cytometric and cell culture techniques - increased significantly upon training paralleled by a significant decrease of ADMA when compared to the BMT group (p<0.05). Six months after training cessation, the beneficial effect of SET on EPC diminished, but maximum walking distance was significantly improved compared to baseline and controls (p<0.05). No significant changes were observed for VEGF and SDF-1 plasma levels in time course. CONCLUSIONS SET increases circulating EPC counts and decreases ADMA levels reflecting enhanced angiogenesis and improved endothelial function, which might contribute to cardiovascular risk reduction.

Interleukin-33 Induces Expression of Adhesion Molecules and Inflammatory Activation in Human Endothelial Cells and in Human Atherosclerotic Plaques

Objective— Interleukin (IL)-33 is the most recently described member of the IL-1 family of cytokines and it is a ligand of the ST2 receptor. While the effects of IL-33 on the immune system have been extensively studied, the properties of this cytokine in the cardiovascular system are much less investigated. Methods/Results— We show here that IL-33 promoted the adhesion of human leukocytes to monolayers of human endothelial cells and robustly increased vascular cell adhesion molecule-1, intercellular adhesion molecule-1, endothelial selectin, and monocyte chemoattractant protein-1 protein production and mRNA expression in human coronary artery and human umbilical vein endothelial cells in vitro as well as in human explanted atherosclerotic plaques ex vivo. ST2-fusion protein, but not IL-1 receptor antagonist, abolished these effects. IL-33 induced translocation of nuclear factor-&kgr;B p50 and p65 subunits to the nucleus in human coronary artery endothelial cells and human umbilical vein endothelial cells and overexpression of dominant negative form of I&kgr;B kinase 2 or I&kgr;B&agr; in human umbilical vein endothelial cells abolished IL-33-induced adhesion molecules and monocyte chemoattractant protein-1 mRNA expression. We detected IL-33 and ST2 on both protein and mRNA level in human carotid atherosclerotic plaques. Conclusion— We hypothesize that IL-33 may contribute to early events in endothelial activation characteristic for the development of atherosclerotic lesions in the vessel wall, by promoting adhesion molecules and proinflammatory cytokine expression in the endothelium.

Components of the interleukin-33/ST2 system are differentially expressed and regulated in human cardiac cells and in cells of the cardiac vasculature

Interleukin-33 (IL-33) is a recently described member of the IL-1 family of cytokines, which was identified as a ligand for the ST2 receptor. Components of the IL-33/ST2 system were shown to be expressed in normal and pressure overloaded human myocardium, and soluble ST2 (sST2) has emerged as a prognostic biomarker in myocardial infarction and heart failure. However, expression and regulation of IL-33 in human adult cardiac myocytes and fibroblasts was not tested before. In this study we found that primary human adult cardiac fibroblasts (HACF) and human adult cardiac myocytes (HACM) constitutively express nuclear IL-33 that is released during cell necrosis. Tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-1β significantly increased both IL-33 protein and IL-33 mRNA expression in HACF and HACM as well as in human coronary artery smooth muscle cells (HCASMC). The nuclear factor-κB (NF-κB) inhibitor dimethylfumarate inhibited TNF-α- and IL-1β-induced IL-33 production as well as nuclear translocation of p50 and p65 NF-κB subunits in these cells. Mitogen-activated protein/extracellular signal-regulated kinase inhibitor U0126 abrogated TNF-α-, IFN-γ-, and IL-1β-induced and Janus-activated kinase inhibitor I reduced IFN-γ-induced IL-33 production. We detected IL-33 mRNA in human myocardial tissue from patients undergoing heart transplantation (n = 27) where IL-33 mRNA levels statistically significant correlated with IFN-γ (r = 0.591, p = 0.001) and TNF-α (r = 0.408, p = 0.035) mRNA expression. Endothelial cells in human heart expressed IL-33 as well as ST2 protein. We also reveal that human cardiac and vascular cells have different distribution patterns of ST2 isoforms (sST2 and transmembrane ST2L) mRNA expression and produce different amounts of sST2 protein. Both human macrovascular (aortic and coronary artery) and heart microvascular endothelial cells express specific mRNA for both ST2 isoforms (ST2L and sST2) and are a source for sST2 protein, whereas cardiac myocytes, cardiac fibroblasts and vascular SMC express only minor amounts of ST2 mRNA and do not secrete detectable amounts of sST2 antigen. In accordance with the cellular distribution of ST2 receptor, human cardiac fibroblasts and myocytes as well as HCASMC did not respond to treatment with IL-33, as recombinant human IL-33 did not induce NF-κB p50 and p65 subunits nuclear translocation or increase IL-6, IL-8, and monocyte chemoattractant protein (MCP-1) level in HACF, HACM and HCASMC. In summary, we found that endothelial cells seem to be the source of sST2 and the target for IL-33 in the cardiovascular system. IL-33 is expressed in the nucleus of human adult cardiac fibroblasts and myocytes and released during necrosis. Proinflammatory cytokines TNF-α, IFN-γ and IL-1β increase IL-33 in these cells in vitro, and IL-33 mRNA levels correlated with TNF-α and IFN-γ mRNA expression in human myocardial tissue.

Activation of Fas inhibits heat-induced activation of HSF1 and up-regulation of hsp70

Activation of heat shock factor (HSF) 1‐DNA binding and inducible heat shock protein (hsp) 70 (also called hsp72) expression enables cells to resist various forms of stress and survive. Fas, a membrane‐bound protein, is a central proapoptotic factor; its activation leads to a cascade of events, resulting in programmed cell death. These two mechanisms with contradictory functions, promoting either cell survival or death, were examined for their potential to inhibit each others activation. Induction of FAS‐mediated signaling was followed by a rapid decrease in HSF1‐DNA binding and inducible hsp70 expression. Inhibition of HSF1‐DNA binding was demonstrated to be based on absent hyperphosphorylation of HSF1 during FAS signaling. These effects of FAS activation on the HSF1/hsp70 stress response were blocked by ICE (caspase 1) inhibitors, suggesting an ICE‐mediated process. Furthermore, inhibition of HSF1/hsp70 was accompanied by an increase in apoptosis rates from 20% to 50% in response to heat stress. When analyzing the effects of HSF1/hsp70 activation on Fas‐mediated apoptosis, protection from apoptosis was seen in cells with induced hsp70 protein levels, but not in cells that were just induced for HSF1‐DNA binding. Thus, we conclude that inhibition of HSF1/hsp70 stress response during Fas‐mediated apoptosis and vice versa may facilitate a cell to pass a previously chosen pathway, stress resistance or apoptosis, without the influence of inhibitory signals.—Schett, G., Steiner, C.‐W., Gröger, M., Winkler, S., Graninger, W., Smolen, J., Xu, Q., Steiner, G. Activation of Fas inhibits heat‐induced activation of HSF1 and up‐regulation of hsp70. FASEB J. 13, 833–842 (1999)

Overexpression of transcription factor Ets‐1 in rheumatoid arthritis synovial membrane: Regulation of expression and activation by interleukin‐1 and tumor necrosis factor α

OBJECTIVE To investigate the expression of the transcription factor Ets-1 in synovial tissue and cultured synovial fibroblasts from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to study the regulation of Ets-1 expression and activation in synovial fibroblasts by proinflammatory cytokines. METHODS In situ expression of Ets-1 in synovial tissue from RA and OA patients was examined by double immunohistochemistry. The effects of interleukin-1 (IL-1) or tumor necrosis factor alpha (TNFalpha) on Ets-1 expression and activation (DNA binding) in cultured synovial fibroblasts were analyzed by Western blotting and DNA gel shift assay, respectively. In addition, the intracellular location of Ets-1 in synovial fibroblasts was determined by immunofluorescence. RESULTS Pronounced expression of Ets-1 was detected in synovial tissues from all RA patients evaluated, particularly in the synovial lining layer and the sublining areas. Ets-1 was expressed by both fibroblasts and macrophages as well as by endothelial cells, while only a few T cells stained positive for Ets-1. In synovial specimens from OA patients, Ets-1 expression was much less frequently observed and was largely restricted to vascular cells. Ets-1 was expressed to a similar degree in cultured synovial fibroblasts from RA and OA patients, as demonstrated by reverse transcriptase-polymerase chain reaction and Western blotting. Both IL-1 and TNFalpha induced pronounced up-regulation of Ets-1 in synovial fibroblasts. Moreover, binding of Ets-1 to its specific DNA binding site was induced by both cytokines, although with different time courses. Immunofluorescence staining revealed a dominant nuclear localization of Ets-1 in IL-1- or TNFalpha-stimulated synovial fibroblasts. CONCLUSION The overexpression of Ets-1 observed in RA synovial tissue appears to be caused by TNFalpha and IL-1, suggesting that Ets-1 may be an important factor in the cytokine-mediated inflammatory and destructive cascade characteristic of RA.

Peptide Bβ15-42 Preserves Endothelial Barrier Function in Shock

Loss of vascular barrier function causes leak of fluid and proteins into tissues, extensive leak leads to shock and death. Barriers are largely formed by endothelial cell-cell contacts built up by VE-cadherin and are under the control of RhoGTPases. Here we show that a natural plasmin digest product of fibrin, peptide Bß15-42 (also called FX06), significantly reduces vascular leak and mortality in animal models for Dengue shock syndrome. The ability of Bß15-42 to preserve endothelial barriers is confirmed in rats i.v.-injected with LPS. In endothelial cells, Bß15-42 prevents thrombin-induced stress fiber formation, myosin light chain phosphorylation and RhoA activation. The molecular key for the protective effect of Bß15-42 is the src kinase Fyn, which associates with VE-cadherin-containing junctions. Following exposure to Bß15-42 Fyn dissociates from VE-cadherin and associates with p190RhoGAP, a known antagonists of RhoA activation. The role of Fyn in transducing effects of Bß15-42 is confirmed in Fyn−/− mice, where the peptide is unable to reduce LPS-induced lung edema, whereas in wild type littermates the peptide significantly reduces leak. Our results demonstrate a novel function for Bß15-42. Formerly mainly considered as a degradation product occurring after fibrin inactivation, it has now to be considered as a signaling molecule. It stabilizes endothelial barriers and thus could be an attractive adjuvant in the treatment of shock.