Robert Fürst

Inhibition of p38 MAPK activation via induction of MKP-1 : atrial natriuretic peptide reduces TNF-alpha-induced actin polymerization and endothelial permeability

The atrial natriuretic peptide (ANP) is a cardiovascular hormone possessing antiinflammatory potential due to its inhibitory action on the production of inflammatory mediators, such as tumor necrosis factor-&agr; (TNF-&agr;). The aim of this study was to determine whether ANP is able to attenuate inflammatory effects of TNF-&agr; on target cells. Human umbilical vein endothelial cells (HUVECs) were treated with TNF-&agr; in the presence or absence of ANP. Changes in permeability, cytoskeletal alterations, phosphorylation of p38 MAPK and HSP27, and expression of MKP-1 were determined by macromolecule permeability assay, fluorescence labeling, RT-PCR, and immunoblotting. Antisense studies were done by transfecting cells with MKP-1 antisense oligonucleotides. Activation of HUVECs with TNF-&agr; lead to a significant increase of macromolecule permeability and formation of stress fibers. Treatment of cells with ANP (10−8 to 10−6 mol/L) significantly reduced the formation of stress fibers and elevated permeability. Both TNF-&agr;–induced effects were shown to be mediated via the activation of p38 using SB203580, a specific inhibitor of p38. ANP significantly reduced the TNF-&agr;–induced activation of p38 and attenuated the phosphorylation of HSP27, a central target downstream of p38. ANP showed no effect on p38 upstream kinases MKK3/6. However, a significant induction of the MAPK phosphatase MKP-1 mRNA and protein could be observed in ANP-treated cells. Antisense experiments proved a causal role for MKP-1 induction in the ANP-mediated inhibition of p38. These data show the inhibitory action of ANP on TNF-&agr;–induced changes in endothelial cytoskeleton and macromolecule permeability involving an MKP-1–induced inactivation of p38 MAPK. These effects point to an antiinflammatory and antiatherogenic potential of this cardiovascular hormone.

Atrial Natriuretic Peptide Induces Mitogen-Activated Protein Kinase Phosphatase-1 in Human Endothelial Cells via Rac1 and NAD(P)H Oxidase/Nox2-Activation

The cardiovascular hormone atrial natriuretic peptide (ANP) exerts anti-inflammatory effects on tumor necrosis factor-&agr;–activated endothelial cells by inducing mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). The underlying mechanisms are as yet unknown. We aimed to elucidate the signaling pathways leading to an induction of MKP-1 by ANP in primary human endothelial cells. By using antioxidants, generation of reactive oxygen species (ROS) was shown to be crucially involved in MKP-1 upregulation. ANP was found to increase ROS formation in cultured cells as well as in the endothelium of intact rat lung vessels. We applied NAD(P)H oxidase (Nox) inhibitors (apocynin and gp91ds-tat) and revealed this enzyme complex to be crucial for superoxide generation and MKP-1 expression. Moreover, by performing Nox2/4 antisense experiments, we identified Nox2 as the critically involved Nox homologue. Pull-down assays and confocal microscopy showed that ANP activates the small Rho-GTPase Rac1. Transfection of a dominant-negative (RacN17) and constitutively active Rac1 mutant (RacV12) indicated that ANP-induced superoxide generation and MKP-1 expression are mediated via Rac1 activation. ANP-evoked production of superoxide was found to activate c-Jun N-terminal kinase (JNK). Using specific inhibitors, we linked ANP-induced JNK activation to MKP-1 expression and excluded an involvement of protein kinase C, extracellular signal-regulated kinase, and p38 MAPK. MKP-1 induction was shown to depend on activation of the transcription factor activator protein-1 (AP-1) by using electrophoretic mobility shift assay and AP-1 decoys. In summary, our work provides insights into the mechanisms by which ANP induces MKP-1 and shows that ANP is a novel endogenous activator of endothelial Rac1 and Nox/Nox2.

MAPK phosphatase-1 represents a novel anti-inflammatory target of glucocorticoids in the human endothelium

Glucocorticoids are well‐established anti‐inflammatory drugs thought to mainly act by inhibition of proinflammatory transcription factors like NFKB. In recent years, however, transcription factor‐independent mechanisms of glucocorticoid action have been proposed, namely the influence on MAPK pathways. Here we identify MAPK phosphatase‐1 (MKP‐1) as a pivotal mediator of the anti‐inflammatory action of glucocorticoids in the human endothelium. We applied dexamethasone (Dex) to TNF‐a‐activated human endothelial cells and used the adhesion molecule E‐selectin as inflammatory read‐out parameter. Dex is known to reduce the expression of E‐selectin, which is largely regulated by NF‐κB. Here, we communicate that Dex at low concentrations (1–100 nM) markedly attenuates E‐selectin expression without affecting NF‐κB. Importantly, Dex is able to increase the expression of MKP‐1, which causes an inactivation of TNF‐α‐induced p38 MAPK and mediates inhibition of E‐selectin expression. In endothelial MKP‐1‐/‐cells differentiated from MKP‐1‐/‐embryonic stem cells and in MKP‐1‐silenced human endothelial cells, Dex did not inhibit TNF‐α‐evoked E‐selectin expression. Thus, our findings introduce MKP‐1 as a novel and crucial mediator of the anti‐inflammatory action of glucocorticoids at low concentrations in the human endothelium and highlight MKP‐1 as an important and promising anti‐inflammatory drug target. Fürst, R., Schroeder, T., Eilken, H. M., Bubik, M. F., Kiemer, A. K., Stefan Zahler, S., Vollmar, A. M. MAPK phosphatase‐1 represents a novel anti‐inflammatory target of glucocorticoids in the human endothelium FASEB J. 21, 74–80 (2007)

Ginkgo biloba extract EGb 761 increases endothelial nitric oxide production in vitro and in vivo

Abstract.Beneficial effects of Ginkgo biloba on peripheral arterial occlusive disease have been repeatedly shown in clinical trials, especially after use of EGb® 761, a standardized special extract. Since the underlying mechanisms are widely unknown, we aimed to elucidate the molecular basis on which EGb® 761 protects against endothelial dysfunction in vitro and in vivo. Application of therapeutically feasible doses of EGb® 761 for 48 h caused endothelial nitric oxide (NO) production by increasing endothelial nitric oxide synthase (eNOS) promoter activity and eNOS expression in vitro. Phosphorylation of eNOS at a site typical for Akt (Ser 1177) was acutely enhanced by treatment with EGb® 761, as was Akt phosphorylation at Ser 478. Furthermore, the extract caused acute relaxation of isolated aortic rings and NO-dependent reduction of blood pressure in vivo in rats. These influences on eNOS represent a putative molecular basis for the protective cardiovascular properties of EGb® 761.

Cyclin-dependent Kinase 5 Regulates Endothelial Cell Migration and Angiogenesis

Angiogenesis contributes to various pathological conditions. Due to the resistance against existing antiangiogenic therapy, an urgent need exists to understand the molecular basis of vessel growth and to identify new targets for antiangiogenic therapy. Here we show that cyclin-dependent kinase 5 (Cdk5), an important modulator of neuronal processes, regulates endothelial cell migration and angiogenesis, suggesting Cdk5 as a novel target for antiangiogenic therapy. Inhibition or knockdown of Cdk5 reduces endothelial cell motility and blocks angiogenesis in vitro and in vivo. We elucidate a specific signaling of Cdk5 in the endothelium; in contrast to neuronal cells, the motile defects upon inhibition of Cdk5 are not caused by an impaired function of focal adhesions or microtubules but by the reduced formation of lamellipodia. Inhibition or down-regulation of Cdk5 decreases the activity of the small GTPase Rac1 and results in a disorganized actin cytoskeleton. Constitutive active Rac1 compensates for the inhibiting effects of Cdk5 knockdown on migration, suggesting that Cdk5 exerts its effects in endothelial cell migration via Rac1. Our work elucidates Cdk5 as a pivotal new regulator of endothelial cell migration and angiogenesis. It suggests Cdk5 as a novel, pharmacologically accessible target for antiangiogenic therapy and provides the basis for a new therapeutic application of Cdk5 inhibitors as antiangiogenic agents.

Plant-Derived Anti-Inflammatory Compounds: Hopes and Disappointments regarding the Translation of Preclinical Knowledge into Clinical Progress

Many diseases have been described to be associated with inflammatory processes. The currently available anti-inflammatory drug therapy is often not successful or causes intolerable side effects. Thus, new anti-inflammatory substances are still urgently needed. Plants were the first source of remedies in the history of mankind. Since their chemical characterization in the 19th century, herbal bioactive compounds have fueled drug development. Also, nowadays, new plant-derived agents continuously enrich our drug arsenal (e.g., vincristine, galantamine, and artemisinin). The number of new, pharmacologically active herbal ingredients, in particular that of anti-inflammatory compounds, rises continuously. The major obstacle in this field is the translation of preclinical knowledge into evidence-based clinical progress. Human trials of good quality are often missing or, when available, are frequently not suitable to really prove a therapeutical value. This minireview will summarize the current situation of 6 very prominent plant-derived anti-inflammatory compounds: curcumin, colchicine, resveratrol, capsaicin, epigallocatechin-3-gallate (EGCG), and quercetin. We will highlight their clinical potential and/or pinpoint an overestimation. Moreover, we will sum up the planned trials in order to provide insights into the inflammatory disorders that are hypothesized to be beneficially influenced by the compound.

Perspective of Cyclin-dependent kinase 9 (CDK9) as a Drug Target

Deregulation of cyclin-dependent kinases (CDKs) has been associated with many cancer types and has evoked an interest in chemical inhibitors with possible therapeutic benefit. While most known inhibitors display broad selectivity towards multiple CDKs, recent work highlights CDK9 as the critical target responsible for the anticancer activity of clinically evaluated drugs. In this review, we discuss recent findings provided by structural biologists that may allow further development of highly specific inhibitors of CDK9 towards applications in cancer therapy. We also highlight the role of CDK9 in inflammatory processes and diseases.

The contribution of the capillary endothelium to blood clearance and tissue deposition of anionic quantum dots in vivo

The increasing interest in biomedical applications of semiconductor quantum dots (QDs) is closely linked to the use of surface modifications to target specific sites of the body. The immense surface area of vascular endothelium is a possible interaction platform with systemically administered QDs. Therefore, the aim of this study was to investigate the microvascular distribution of neutral, cationic, and anionic QDs in vivo. QDs with carboxyl-, amine- and polyethylene glycol surface coatings were injected into the blood circulation of mice. In vivo microscopy of the cremaster muscle, two-photon microscopy of skeletal and heart muscle, as well as quantitative fluorescence measurements of blood, excreta, and tissue samples were performed. Transmission electron microscopy was used to detect QDs at the cellular level. The in vitro association of QDs with cultured endothelial cells was investigated by flow cytometry and confocal microscopy. Anionic QDs exhibited a very low residence time in the blood stream, preferably accumulated in organs with a prominent mononuclear phagocytic component, but were also found in other tissues with low phagocytic properties where they were predominantly associated with capillary endothelium. This deposition behavior was identified as a new, phagocyte-independent principle contributing to the rapid clearance of anionic QDs from the circulation.

Vasoprotective actions of the atrial natriuretic peptide.

The Natriuretic Peptide (NP) family, especially its best-characterized member Atrial Natriuretic Peptide (ANP), plays an important role in the regulation of blood pressure homeostasis and salt and water balance. Besides their action in cardiovascular physiology, NPs have been described as anti-inflammatory regulators of macrophage function: they have been reported to inhibit the induction of inflammatory mediators, such as iNOS, COX-2, and TNF-alpha. In the following review we will focus on a rather novel aspect of NP action: NPs, especially ANP, will be presented as vasoprotective agents. We will specifically focus on ANPs interaction with the complex intracellular signalling networks responsible for proliferation, vascular permeability, attraction and adhesion of leukocytes, and the induction of cytoprotective proteins. We will also discuss the critical mediator systems involved in mediating ANPs beneficial actions. Recently, ANP as well as BNP, another member of the NP family, have been introduced as cardiovascular therapeutics. In this context, we will highlight the physiological and pharmacological relevance of NPs, particularly ANP, as endogenous vasoprotective agents.

Investigation of the marine compound spongistatin 1 links the inhibition of PKCα translocation to nonmitotic effects of tubulin antagonism in angiogenesis

The aims of the study were to meet the demand of new tubulin antagonists with fewer side effects by characterizing the antiangiogenic properties of the experimental compound spongistatin 1, and to elucidate nonmitotic mechanisms by which tubulin antagonists inhibit angiogenesis. Although tubulin‐inhibiting drugs and their antiangiogenic properties have been investigated for a long time, surprisingly little is known about their underlying mechanisms of action. Antiangiogenic effects of spongistatin 1 were investigated in endothelial cells in vitro, including functional cell‐based assays, live‐cell imaging, and a kinome array, and in the mouse cornea pocket assay in vivo. Spongistatin 1 inhibited angiogenesis at nanomolar concentrations (IC50: cytotoxicity> 50 nM, proliferation 100 pM, migration 1.0 nM, tube formation 1.0 nM, chemotaxis 1.0 nM, aortic ring sprouting 500 pM, neovascularization in vivo 10 µg/kg). Further, a kinome array and validating data showed that spongistatin 1 inhibits the phosphorylation activity of protein kinase Cα (PKCα), an essential kinase in angiogenesis, and its translocation to the membrane. Thus, we conclude that PKCα might be an important target for the antiangiogenic effects of tubulin antagonism. In addition, the data from the kinase array suggest that different tubulin antagonists might have individual intracellular actions.—Rothmeier, A. S., Ischenko, I., Joore, J., Garczarczyk, D., Fürst, R., Bruns, C. J., Vollmar, A. M., Zahler, S. Investigation of the marine compound spongistatin 1 links the inhibition of PKCα translocation to nonmitotic effects of tubulin antagonism in angiogenesis. FASEB J. 23, 1127–1137 (2009)