[Show abstract][Hide abstract] ABSTRACT: Levosimendan is a positive inotropic drug for the treatment of acute decompensated heart failure (HF). Clinical trials showed that levosimendan was particularly effective in HF due to myocardial infarction. Myocardial necrosis induces a strong inflammatory response, involving chemoattractants guiding polymorphonuclear neutrophils (PMN) into the infarcted myocardial tissue. Our aim was to examine whether levosimendan exhibits anti-inflammatory effects on human adult cardiac myocytes (HACM) and human heart microvascular endothelial cells (HHMEC). Cardiac myocytes and endothelial cells were stimulated with interleukin-1β (IL)-1β (200 U/ml) and treated with levosimendan (0.1-10 µM) for 2-48 hours. IL-1β strongly induced expression of IL-6 and IL-8 in HACM and E-selectin and intercellular adhesion molecule-1 (ICAM-1) in HHMEC and human umbilical vein endothelial cells (HUVEC). Treatment with levosimendan strongly attenuated IL-1β-induced expression of IL-6 and IL-8 in HACM as well as E-selectin and ICAM-1 in ECs. Levosimendan treatment further reduced adhesion of PMN to activated endothelial cells under both static and flow conditions by approximately 50 %. Incubation with 5-hydroxydecanoic acid, a selective blocker of mitochondrial ATP-dependent potassium channels, partly abolished the above seen anti-inflammatory effects. Additionally, levosimendan strongly diminished IL-1β-induced reactive oxygen species and nuclear factor-κB (NF-κB) activity through inhibition of S536 phosphorylation. In conclusion, levosimendan exhibits anti-inflammatory effects on cardiac myocytes and endothelial cells in vitro. These findings could explain, at least in part, the beneficial effects of levosimendan after myocardial infarction.
Thrombosis and Haemostasis 10/2014; 113(1). DOI:10.1160/TH14-06-0549 · 5.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Binding of TNF to its receptor (TNFR1) elicits the spatiotemporal assembly of two signaling complexes that coordinate the
balance between cell survival and cell death. We have shown previously that, following TNF treatment, the mRNA decay protein
tristetraprolin (TTP) is Lys-63-polyubiquitinated by TNF receptor-associated factor 2 (TRAF2), suggesting a regulatory role
in TNFR signaling. Here we demonstrate that TTP interacts with TNFR1 in a TRAF2-dependent manner, thereby initiating the MEKK1/MKK4-dependent
activation of JNK activities. This regulatory function toward JNK activation but not NF-κB activation depends on lysine 105
of TTP, which we identified as the corresponding TRAF2 ubiquitination site. Disabling TTP polyubiquitination results in enhanced
TNF-induced apoptosis in cervical cancer cells. Together, we uncover a novel aspect of TNFR1 signaling where TTP, in alliance
with TRAF2, acts as a balancer of JNK-mediated cell survival versus death.
[Show abstract][Hide abstract] ABSTRACT: Urokinase-type plasminogen activator (u-PA) plays pivotal role in extracellular proteolysis and is also thought to be critically involved in the modulation of angiogenesis. Interleukin (IL)-33 is a member of the IL-1 cytokine family, which is thought to act as danger signal that is released from cells after injury. IL-33 is involved in the pathogenesis of various inflammatory diseases and was previously shown to induce angiogenesis and inflammatory activation of endothelial cells.
We investigated the impact of IL-33 on u-PA in endothelial cells as a new possible function for IL-33.
We could demonstrate that IL-33 upregulated u-PA mRNA expression and protein production in human coronary artery and human umbilical vein endothelial cells in a time- and concentration-dependent manner via interaction with its receptor ST2 and activation of the NF-κB pathway but independent of autocrine IL-1-induced effects. The hydroxymethylglutaryl-coenzyme A reductase inhibitor simvastatin abrogated the IL-33-induced increase in u-PA, thus providing further evidence for pleiotropic effects of statins. IL-33 induced u-PA-dependent capillary-like tube formation and vessel sprouting. In human carotid atherosclerotic plaques (n=16), u-PA mRNA positively correlated with IL-33 mRNA expression (r=0.780, p<0.001). Furthermore, IL-33 and u-PA protein were detected in endothelial cells in these samples using fluorescence immunohistochemistry.
We hypothesize that IL-33, representing a danger signal that is released following tissue damage, in addition to its role in the inflammatory activation of endothelial cells, is also involved in u-PA-driven angiogenesis, a process that has been shown before to be linked to inflammation in various pathologies. This article is protected by copyright. All rights reserved.
Journal of Thrombosis and Haemostasis 04/2014; 12(6). DOI:10.1111/jth.12581 · 5.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transcription factor Sox18 plays a role in angiogenesis, including lymphangiogenesis, where it is upregulated by growth factors and directs the expression of genes encoding, e.g., guidance molecules and a matrix metalloproteinase. Conversely, we found that in human umbilical vein endothelial cells (HUVEC) Sox18 is repressed by the pro-inflammatory mediator TNFα (as well as IL-1 and LPS). Since a common feature of these mediators is the activation of the NF-κB signaling pathway, we investigated whether Sox18 downregulation is dependent on this transcription factor. Transduction of HUVEC with an adenoviral vector directing the expression of the NF-κB inhibitor IκBα prevented the downregulation of Sox18. Transient transfections of Sox18 promoter reporter genes revealed that the downregulation takes place on the level of transcription, and that the p65/RelA subunit of NF-κB was operative. Furthermore, the responsible promoter region of Sox18 is located within -1.0 kb from the transcriptional start site. The repression of Sox18 and its target genes may lead to altered formation of vessels in inflamed settings.
Biochemical and Biophysical Research Communications 11/2013; 442(3-4). DOI:10.1016/j.bbrc.2013.11.030 · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metastases destroy the function of infested organs and are the main reason of cancer-related mortality. Heteronemin, a natural product derived from a marine sponge, was tested in vitro regarding its properties to prevent tumour cell intravasation through the lymph-endothelial barrier. In three-dimensional (3D) cell cultures consisting of MCF-7 breast cancer cell spheroids that were placed on lymph-endothelial cell (LEC) monolayers, tumour cell spheroids induce "circular chemorepellent-induced defects" (CCIDs) in the LEC monolayer; 12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE) and NF-κB activity are major factors inducing CCIDs, which are entry gates for tumour emboli intravasating the vasculature. This 3D co-culture is a validated model for the investigation of intravasation mechanisms and of drugs preventing CCID formation and hence lymph node metastasis. Furthermore, Western blot analyses, NF-κB reporter, EROD, SELE, 12(S)-HETE, and adhesion assays were performed to investigate the properties of heteronemin. Five micromolar heteronemin inhibited the directional movement of LECs and, therefore, the formation of CCIDs, which were induced by MCF-7 spheroids. Furthermore, heteronemin reduced the adhesion of MCF-7 cells to LECs and suppressed 12(S)-HETE-induced expression of the EMT marker paxillin, which is a regulator of directional cell migration. The activity of CYP1A1, which contributed to CCID formation, was also inhibited by heteronemin. Hence, heteronemin inhibits important mechanisms contributing to tumour intravasation in vitro and should be tested in vivo.
Archives of Toxicology 03/2013; 87(10). DOI:10.1007/s00204-013-1045-1 · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Health beneficial effects of xanthohumol have been reported, and basic research provided evidence for anti-cancer effects. Furthermore, xanthohumol was shown to inhibit the migration of endothelial cells. Therefore, this study investigated the anti-metastatic potential of xanthohumol. MCF-7 breast cancer spheroids which are placed on lymphendothelial cells (LECs) induce "circular chemorepellent-induced defects" (CCIDs) in the LEC monolayer resembling gates for intravasating tumour bulks at an early step of lymph node colonisation. NF-κB reporter-, EROD-, SELE-, 12(S)-HETE- and adhesion assays were performed to investigate the anti-metastatic properties of xanthohumol. Western blot analyses were used to elucidate the mechanisms inhibiting CCID formation. Xanthohumol inhibited the activity of CYP, SELE and NF-kB and consequently, the formation of CCIDs at low micromolar concentrations. More specifically, xanthohumol affected ICAM-1 expression and adherence of MCF-7 cells to LECs, which is a prerequisite for CCID formation. Furthermore, markers of epithelial-to-mesenchymal transition (EMT) and of cell mobility such as paxillin, MCL2 and S100A4 were suppressed by xanthohumol. Xanthohumol attenuated tumour cell-mediated defects at the lymphendothelial barrier and inhibited EMT-like effects thereby providing a mechanistic explanation for the anti-intravasative/anti-metastatic properties of xanthohumol.
Archives of Toxicology 03/2013; 87(7). DOI:10.1007/s00204-013-1028-2 · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metastasis is associated with poor prognosis for melanoma responsible for about 90% of skin cancer-related mortality. To metastasize, melanoma cells must escape keratinocyte control, invade across the basement membrane and survive in the dermis by resisting apoptosis before they can intravasate into the circulation. α-Catulin (CTNNAL1) is a cytoplasmic molecule that integrates the crosstalk between nuclear factor-kappa B and Rho signaling pathways, binds to β-catenin and increases the level of both α-catenin and β-catenin and therefore has potential effects on inflammation, apoptosis and cytoskeletal reorganization. Here, we show that α-catulin is highly expressed in melanoma cells. Expression of α-catulin promoted melanoma progression and occurred concomitantly with the downregulation of E-cadherin and the upregulation of expression of mesenchymal genes such as N-cadherin, Snail/Slug and the matrix metalloproteinases 2 and 9. Knockdown of α-catulin promoted adhesion to and inhibited migration away from keratinocytes in an E-cadherin-dependent manner and decreased the transmigration through a keratinocyte monolayer, as well as in Transwell assays using collagens, laminin and fibronectin coating. Moreover, knockdown promoted homotypic spheroid formation and concomitantly increased E-cadherin expression along with downregulation of transcription factors implicated in its repression (Snail/Slug, Twist and ZEB). Consistent with the molecular changes, α-catulin provoked invasion of melanoma cells in a three-dimensional culture assay by the upregulation of matrix metalloproteinases 2 and 9 and the activation of ROCK/Rho. As such, α-catulin may represent a key driver of the metastatic process, implicating potential for therapeutic interference.
International Journal of Cancer 02/2013; 132(3). DOI:10.1002/ijc.27698 · 5.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Investigating the bioactivity of traditional medical remedies under the controlled conditions of a laboratory is an option to find additional applications, novel formulations or lead structures for the development of new drugs. The present work analysed the anti‑neoplastic activity of increasing polar extracts of the rainforest plant Critonia morifolia (Asteraceae) that has been successfully used as traditional remedy to treat various inflammatory conditions in the long-lasting medical tradition of the Central American Maya, which was here also confirmed in vitro. The apolar petroleum ether extract exhibited the most potent anti‑proliferative and pro‑apoptotic effects in HL‑60 cells and triggered down-regulation of Cdc25C and cyclin D1 within 30 min followed by the inhibition of c-Myc expression and the onset of caspase-3 activation within 2 h. Subsequent to these very rapid molecular responses Chk2 and H2AX became phosphorylated (γ‑H2AX) after 4 h. Analysis of the cell cycle distribution showed an accumulation of cells in the G2-M phase within 8 h and after 24 h in S-phase. This was temporally paralleled by the down-regulation of Cdc25A, Cdc25B, Wee1 and Akt. Therefore, the attenuation of cell cycle progression in the G2-M phase was consistent with the known role of Chk2 for G2-M arrest and with the role of Cdc25B in S-phase progression. These findings suggest the presence of two distinct active principles in the petroleum ether extract of C. moriflia. These facilitated the strong apoptotic response evidenced by the rapid activation of caspase-3 that was later enforced by the inhibition of the survival kinase Akt. Importantly, the efficient down-regulation of Akt, which is successfully tested in current clinical trials, is a unique property of C. morifolia.
International Journal of Oncology 03/2012; 40(6):2131-9. DOI:10.3892/ijo.2012.1412 · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations in the transcription factor SOX18 are responsible for specific cardiovascular defects in humans and mice. In order to gain insight into the molecular basis of its action, we identified target genes of SOX18 and analyzed one, MMP7, in detail.
SOX18 was expressed in HUVEC using a recombinant adenoviral vector and the altered gene expression profile was analyzed using microarrays. Expression of several regulated candidate SOX18 target genes was verified by real-time PCR. Knock-down of SOX18 using RNA interference was then used to confirm the effect of the transcription factor on selected genes that included the guidance molecules ephrin B2 and semaphorin 3G. One gene, MMP7, was chosen for further analysis, including detailed promoter studies using reporter gene assays, electrophoretic mobility shift analysis and chromatin-immunoprecipitation, revealing that it responds directly to SOX18. Immunohistochemical analysis demonstrated the co-expression of SOX18 and MMP7 in blood vessels of human skin.
The identification of MMP7 as a direct SOX18 target gene as well as other potential candidates including guidance molecules provides a molecular basis for the proposed function of this transcription factor in the regulation of vessel formation.
PLoS ONE 01/2012; 7(1):e30982. DOI:10.1371/journal.pone.0030982 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acute versus chronic inflammation is controlled by the accurate activation and regulation of interdependent signaling cascades. TNF-receptor 1 engagement concomitantly activates NF-κB and JNK signaling. The correctly timed activation of these pathways is the key to account for the balance between NF-κB-mediated cell survival and cell death, the latter fostered by prolonged JNK activation. Tristetraprolin (TTP), initially described as an mRNA destabilizing protein, acts as negative feedback regulator of the inflammatory response: it destabilizes cytokine-mRNAs but also acts as an NF-κB inhibitor by interfering with the p65/RelA nuclear import pathway. Our biochemical studies provide evidence that TTP contributes to the NF-κB/JNK balance. We find that the MAP 3-kinase MEKK1 acts as a novel TTP kinase that, together with the TNF receptor-associated factor 2 (TRAF2), constitutes not only a main determinate of the NF-κB-JNK cross-talk but also facilitates "TTP hypermodification": MEKK1 triggers TTP phosphorylation as prerequisite for its Lys-63-linked, TRAF2-mediated ubiquitination. Consequently, TTP no longer affects NF-κB activity but promotes the activation of JNK. Based on our data, we suggest a model where upon TNFα induction, TTP transits a hypo- to hypermodified state, thereby contributing to the molecular regulation of NF-κB versus JNK signaling cascades.
[Show abstract][Hide abstract] ABSTRACT: Acute versus chronic inflammation is controlled by the accurate activation and regulation of interdependent signaling cascades.
TNF-receptor 1 (TNFR1) engagement concomitantly activates nuclear factor κB (NF-κB) and c-Jun N-terminal kinase (JNK) signaling.
The correctly timed activation of these pathways is the key to account for the balance between NF-κB-mediated cell survival
and cell death, the latter fostered by prolonged JNK activation. Tristetraprolin (TTP), initially described as an mRNA destabilizing
protein, acts as negative feedback regulator of the inflammatory response: it destabilizes cytokine-mRNAs but also acts as
an NF-κB inhibitor by interfering with the p65/RelA nuclear import pathway.
Our biochemical studies provide evidence that TTP contributes to the NF-κB/JNK balance. We find that the mitogen-activated-protein-3-kinase
(MAP3K) MEKK1 acts as a novel TTP kinase which, together with the TNF-receptor associated factor-2 (TRAF2), constitutes not
only a main determinate of the NF-κB-JNK crosstalk, but also facilitates TTP-hypermodification: MEKK1 triggers TTP phosphorylation
as prerequisite for its K63-linked, TRAF2-mediated ubiquitination. Consequently, TTP no longer affects NF-κB activity but
promotes the activation of JNK.
Based on our data we suggest a model where upon TNFα induction, TTP transits a hypo- to hypermodified state, thereby contributing
to the molecular regulation of NF-κB versus JNK signaling cascades.
Journal of Biological Chemistry 09/2011; · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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-κB 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 IκB kinase 2 or IκBα 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. CONCLUSIONS: 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.
[Show abstract][Hide abstract] ABSTRACT: The aim of the present study was to investigate the changes in absolute myocardial blood flow (AMF) after intracoronary injections of mesenchymal SC (MSC) and compared to controls in closed-chest reperfused acute myocardial infarction (AMI) in pigs. Male MSCs, transiently transfected with Luciferase (Luc-MSC) were delivered (9.7 ± 1.2 x 10(6)) intracoronary in the open infarct-related artery one-week post-AMI in female pigs (group MSC), while saline was injected with the same injection rate in controls (group C). The AMF was measured immediately after, and 3, 12 and 24 h post-intracoronary Luc-MSC or saline injections. In vitro bioluminescence images and quantitative real-time TaqMan PCR measurements were performed to quantify the sex-mismatched MSCs. No difference between the groups was observed regarding the weight, heart rate, blood pressure and global ejection fraction 1-week post-AMI. The baseline AMF were similar in the groups (61.3 ± 15. vs 61.1 ± 12.0 ml/min). AMF was decreased significantly immediately after intracoronary MSC delivery (42.0 ± 12.4 vs 57.7 ± 15.7 ml/min p = 0.013), and remained low at 3 h (40.9 ± 13.4 vs 55.8 ± 4.9 ml/min, p = 0.004), 12 h (43.0 ± 3.7 vs 57.8 ± 5.4 ml/min, p = 0.001) with incomplete recovery at 24 h (47.2 ± 5.5 vs 62.1 ± 14.1 ml/min, p = 0.038) as compared to controls, respectively. In vitro bioluminescence displayed transfected Luc-MSCs along the proximal and mid part of the LAD, with limited number (295 ± 101 sry copied/million cardiac cells) of Y-chromosome-MSCs in the infarcted area. Intracoronary injection of SCs results in immediate decrease of AMF, with delayed recovery. The delivery of the SC into the injured myocardium might be hindered by the altered coronary pressure and flow conditions.
[Show abstract][Hide abstract] ABSTRACT: I-kappa B kinase 2 (IKK2 or IKK-beta) is one of the most crucial signaling kinases for activation of NF-kappa B, a transcription factor that is important for inflammation, cell survival and differentiation. Since many NF-kappa B activating pathways converge at the level of IKK2, molecular interactions of this kinase are pivotal for regulation of NF-kappa B signaling.
We searched for proteins interacting with IKK2 using the C-terminal part (amino acids 466-756) as bait in a yeast two-hybrid system and identified the N-terminal part (amino acids 1-228) of the TNF-receptor associated factor TRAF1 as putative interaction partner. The interaction was confirmed in human cells by mammalian two-hybrid and coimmunoprecipitation experiments. The IKK2/TRAF1 interaction seemed weaker than the interaction between TRAF1 and TRAF2, an important activating adapter molecule of NF-kappa B signaling. Reporter gene and kinase assays using ectopic expression of TRAF1 indicated that it can both activate and inhibit IKK2 and NF-kappa B. Co-expression of fluorescently tagged TRAF1 and TRAF2 at different ratios implied that TRAF1 can affect clustering and presumably the activating function of TRAF2 in a dose dependent manner.
The observation that TRAF1 can either activate or inhibit the NF-kappa B pathway and the fact that it influences the oligomerization of TRAF2 indicates that relative levels of IKK2, TRAF1 and TRAF2 may be important for regulation of NF-kappa B activity. Since TRAF1 is an NF-kappa B induced gene, it might act as a feedback effector molecule.
PLoS ONE 09/2010; 5(9):e12683. DOI:10.1371/journal.pone.0012683 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: More than 60% of conventional drugs are derived from natural compounds, some of the most effective pharmaceuticals (e.g. aspirin, quinine and various antibiotics) originate from plants or microbes, and large numbers of potentially valuable natural substances remain to be discovered. Plants with considerable medicinal potential include members of the genus Acalypha. Notably, extracts of A. platyphilla, A. fruticosa, A. siamensis, A. guatemalensis and A. wilkesiana have been recently shown to have antioxidant, antimicrobial and cytotoxic effects. In the study presented here we investigated the anti-inflammatory, anti-proliferative and pro-apoptotic activities of A. alopecuroidea, which is endemic in parts of Central America and is traditionally used by the Mopan- and Itza-Maya in the form of decoctions to treat skin conditions, and as a tea to treat stomach and urinary complaints. We demonstrate here that extracts of A. alopecuroidea can inhibit TNFalpha-induced E-selectin production, providing a mechanistic validation of its traditional use against inflammatory diseases. Furthermore, a fraction of A. alopecuroidea root extracts purified by solid phase extraction and separated by HPLC displayed strong cell cycle inhibitory activity by down-regulating and inactivating two proto-oncogenes (cyclin D1 and Cdc25A), and simultaneously inducing cyclin A, thereby disturbing orchestrated cell cycle arrest, and thus (presumably) triggering caspase 3-dependent apoptosis. The results of this study indicate that there are high prospects for purifying an active principle from A. alopecuroidea for further in vivo and preclinical studies.
International Journal of Oncology 11/2009; 35(4):881-91. DOI:10.3892/ijo_00000403 · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Long-wavelength ultraviolet (UVA-1) radiation causes oxidative stress that modifies cellular molecules. To defend themselves against noxious oxidation products, skin cells produce detoxifying enzymes and antioxidants. We have recently shown that UVA-1 oxidized the abundant membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), which then induced the stress-response protein heme oxygenase 1 (HO-1) in dermal fibroblasts. Here we examined the effects of UVA-1- and UV-oxidized phospholipids on global gene expression in human dermal fibroblasts and keratinocytes. We identified a cluster of genes that were coinduced by UVA-1-oxidized PAPC and UVA-1 radiation. The cluster included HO-1, glutamate-cysteine ligase modifier subunit, aldo-keto reductases-1-C1 and -C2, and IL-8. These genes are members of the cellular stress response system termed "antioxidant response." Accordingly, the regulatory regions of all of these genes contain binding sites for NF-E2-related factor 2 (NRF2), a major regulator of the antioxidant response. Both UVA-1 irradiation and treatment with oxidized lipids led to increased nuclear accumulation and DNA binding of NRF2. Silencing and deficiency of NRF2 suppressed the antioxidant response. Taken together, our data show that UVA-1-mediated lipid oxidation induces expression of antioxidant response genes, which is dependent on the redox-regulated transcription factor NRF2. Our findings suggest a different view on UV-generated lipid mediators that were commonly regarded as detrimental
The FASEB Journal 09/2009; 24(1):39-48. DOI:10.1096/fj.09-133520 · 5.48 Impact Factor