Thymic medullar conduits-associated podoplanin promotes natural regulatory T cells.

Podoplanin, a mucin-like plasma membrane protein, is expressed by lymphatic endothelial cells and responsible for separation of blood and lymphatic circulation through activation of platelets. Here we show that podoplanin is also expressed by thymic fibroblastic reticular cells (tFRC), a novel thymic medulla stroma cell type associated with thymic conduits, and involved in development of natural regulatory T cells (nTreg). Young mice deficient in podoplanin lack nTreg owing to retardation of CD4(+)CD25(+) thymocytes in the cortex and missing differentiation of Foxp3(+) thymocytes in the medulla.

The urokinase receptor (CD87) represents a central mediator of growth factor-induced endothelial cell migration.

Angiogenesis, the sprouting of blood vessels form pre-existing vasculature after injury or in neoplastic diseases, is initiated by growth factor-induced endothelial cell migration. Recently, the major angiogenic growth factor VEGF165 has become the target of therapeutic interventions. However, this approach has been clinically proven to be of limited efficacy, which might be due to the fact that tumour angiogenesis is not only induced by VEGF, but also by a variety of other growth factors.

VEGF-induced endothelial cell migration requires urokinase receptor (uPAR)-dependent integrin redistribution.

Aims: Vascular endothelial growth factor (VEGF)-initiated angiogenesis requires coordinated proteolytic degradation of extracellular matrix provided by the urokinase plasminogen activator/urokinase receptor (uPA/uPAR) system and regulation of cell migration provided by integrin-matrix interaction. In this study, we investigated the mechanisms underlying the uPAR-dependent modulation of VEGF-induced endothelial migration.

Methods And Results: We used flow cytometry to quantify integrins at the cell surface.

Constant darkness induces IL-6-dependent depression-like behavior through the NF-κB signaling pathway.

Substantial experimental evidence indicates a major role for the circadian system in mood disorders. Additionally, proinflammatory cytokines have been proposed to be involved in the pathogenesis of depression. However, the molecular elements determining the functional interplay between these two systems in depression have not been described as yet.

Signal integration and coincidence detection in the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) cascade: concomitant activation of receptor tyrosine kinases and of LRP-1 leads to sustained ERK phosphorylation via down-regulation of dual specificity phosphatases (DUSP1 and -6).

Diverse stimuli can feed into the MAPK/ERK cascade; this includes receptor tyrosine kinases, G protein-coupled receptors, integrins, and scavenger receptors (LDL receptor-related protein (LRP)). Here, we investigated the consequence of concomitant occupancy of the receptor tyrosine kinases (by EGF, basic FGF, VEGF, etc.) and of LRP family members (by LDL or lactoferrin).

Density enhanced phosphatase-1 down-regulates urokinase receptor surface expression in confluent endothelial cells.

VEGF(165), the major angiogenic growth factor, is known to activate various steps in proangiogenic endothelial cell behavior, such as endothelial cell migration and invasion, or endothelial cell survival. Thereby, the urokinase-type plasminogen activator (uPA) system has been shown to play an essential role not only by its proteolytic capacities, but also by induction of intracellular signal transduction. Therefore, expression of its cell surface receptor uPAR is thought to be an essential regulatory mechanism in angiogenesis.

Soluble M6P/IGF2R released by TACE controls angiogenesis via blocking plasminogen activation.

Rationale: The urokinase plasminogen activator (uPA) system is among the most crucial pericellular proteolytic systems associated with the processes of angiogenesis. We previously identified an important regulator of the uPA system in the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R).

Objective: Here, we wanted to clarify whether and how did the soluble form of M6P/IGF2R (sM6P/IGF2R) contribute to modulation of the uPA system.

Oxidized phospholipids are more potent antagonists of lipopolysaccharide than inducers of inflammation.

Polyunsaturated fatty acids are precursors of multiple pro- and anti-inflammatory molecules generated by enzymatic stereospecific and positionally specific insertion of oxygen, which is a prerequisite for recognition of these mediators by cellular receptors. However, nonenzymatically oxidized free and esterified polyunsaturated fatty acids also demonstrate activities relevant to inflammation. In particular, phospholipids containing oxidized fatty acid residues (oxidized phospholipids; OxPLs) were shown to induce proinflammatory changes in endothelial cells but paradoxically also to inhibit inflammation induced via TLR4.

Involvement of CK2 in activation of electrophilic genes in endothelial cells by oxidized phospholipids.

Oxidized phospholipids (OxPLs) are increasingly recognized as pleiotropic lipid mediators demonstrating a variety of biological activities. In particular, OxPLs induce electrophilic stress response and stimulate expression of NF-E2-related factor 2 (NRF2)-dependent genes. The mechanisms of NRF2 upregulation in response to OxPLs, however, are incompletely understood.

Anti-inflammatory properties of the PI3K pathway are mediated by IL-10/DUSP regulation.

Resolution of inflammation is an important hallmark in the course of infectious diseases. Dysregulated inflammatory responses may have detrimental consequences for the affected organism. Therefore, tight regulation of inflammation is indispensable.

Indirubin-3'-monoxime blocks vascular smooth muscle cell proliferation by inhibition of signal transducer and activator of transcription 3 signaling and reduces neointima formation in vivo.

Objective: Our goal was to examine the influence of indirubin-3'-monoxime (I3MO), a natural product-derived cyclin-dependent kinase inhibitor, on vascular smooth muscle cell (VSMC) proliferation in vitro, experimentally induced neointima formation in vivo, and related cell signaling pathways.

Methods And Results: I3MO dose-dependently inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation by arresting cells in the G(0)/G(1) phase of the cell cycle as assessed by 5-bromo-2'-deoxyuridine incorporation and flow cytometry. PDGF-induced activation of the kinases Akt, Erk1/2, and p38(MAPK) was not affected.

Analysis of oxidized phospholipids by MALDI mass spectrometry using 6-aza-2-thiothymine together with matrix additives and disposable target surfaces.

6-Aza-2-thiothymine (ATT) is introduced as novel matrix system for the analysis of oxidized phospholipids (OxPLs) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). A systematic evaluation comparing different established and novel matrix substances, especially 2,4,6-THAP matrix (Stubiger, G.; Belgacem O.

Myeloid PTEN promotes inflammation but impairs bactericidal activities during murine pneumococcal pneumonia.

Phosphatidylinositol 3-kinase has been described as an essential signaling component involved in the chemotactic cell influx that is required to eliminate pathogens. At the same time, PI3K was reported to modulate the immune response, thus limiting the magnitude of acute inflammation. The precise role of the PI3K pathway and its endogenous antagonist phosphatase and tensin homolog deleted on chromosome 10 (PTEN) during clinically relevant bacterial infections is still poorly understood.

Oxidized phospholipids regulate expression of ATF4 and VEGF in endothelial cells via NRF2-dependent mechanism: novel point of convergence between electrophilic and unfolded protein stress pathways.

Objective: The ATF4 arm of the unfolded protein response is increasingly recognized for its relevance to pathology, and in particular to angiogenic reactions. Oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, were shown to upregulate vascular endothelial growth factor (VEGF) and induce angiogenesis via an ATF4-dependent mechanism. In this study, we analyzed the mechanism of ATF4 upregulation by OxPLs and more specifically the involvement of NRF2, the major transcriptional mediator of electrophilic stress response.

Novel function for blood platelets and podoplanin in developmental separation of blood and lymphatic circulation.

During embryonic development, lymph sacs form from the cardinal vein, and sprout centrifugally to form mature lymphatic networks. Separation of the lymphatic from the blood circulation by a hitherto unknown mechanism is essential for the homeostatic function of the lymphatic system. O-glycans on the lymphatic endothelium have recently been suggested to be required for establishment and maintenance of distinct blood and lymphatic systems, primarily by mediating proper function of podoplanin.

Targeting of VEGF-dependent transendothelial migration of cancer cells by bevacizumab.

Cancer progression is often associated with the formation of malignant effusions. Vascular endothelial growth factor (VEGF) is a major regulator of vascular permeability and has been implicated as mediator of tumor progression. We examined the production and secretion of VEGF(165) in various primary cancer cells derived from malignant effusions, and the role of exogenous VEGF(165) as a mediator of effusion formation.

Crosstalk between the NF-kappaB activating IKK-complex and the CSN signalosome.

A great variety of signalling pathways regulating inflammation, cell development and cell survival require NF-kappaB transcription factors, which are normally inactive due to binding to inhibitors, such as IkappaBalpha. The canonical activation pathway of NF-kappaB is initiated by phosphorylation of the inhibitor by an IkappaB kinase (IKK) complex triggering ubiquitination of IkappaB molecules by SCF-type E3-ligase complexes and rapid degradation by 26S-proteasomes. The ubiquitination machinery is regulated by the COP9 signalosome (CSN).

Multi-hit inhibition of circulating and cell-associated components of the toll-like receptor 4 pathway by oxidized phospholipids.

Objective: Oxidized phospholipids (OxPLs) that are abundant in atherosclerotic lesions are increasingly recognized as context-dependent lipid mediators demonstrating both pro- and antiinflammatory activities. Molecular mechanisms of their effects are largely unknown. Here we present novel information on the mechanisms whereby OxPLs modulate activation of TLR4 by lipopolysaccharide (LPS).

Podoplanin deficient mice show a RhoA-related hypoplasia of the sinus venosus myocardium including the sinoatrial node.

We investigated the role of podoplanin in development of the sinus venosus myocardium comprising the sinoatrial node, dorsal atrial wall, and primary atrial septum as well as the myocardium of the cardinal and pulmonary veins. We analyzed podoplanin wild-type and knockout mouse embryos between embryonic day 9.5-15.

Mannose 6-phosphate/insulin-like growth factor 2 receptor limits cell invasion by controlling alphaVbeta3 integrin expression and proteolytic processing of urokinase-type plasminogen activator receptor.

The multifunctional mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) is considered a tumor suppressor. We report here that RNA interference with M6P/IGF2R expression in urokinase-type plasminogen activator (uPA)/urokinase-type plasminogen activator receptor (uPAR) expressing human cancer and endothelial cells resulted in increased pericellular plasminogen activation, cell adhesion, and higher invasive potential through matrigel. M6P/IGF2R silencing led also to the cell surface accumulation of urokinase and plasminogen and enhanced expression of alphaV integrins.

Urokinase mediates endothelial cell survival via induction of the X-linked inhibitor of apoptosis protein.

Urokinase-type plasminogen activator (uPA) additionally elicits a whole array of pro-angiogenic responses, such as differentiation, proliferation, and migration. In this study, we demonstrate that in endothelial cells uPA also protects against apoptosis by transcriptional up-regulation and partially by mRNA stabilization of inhibitor of apoptosis proteins, most prominently the X-linked inhibitor of apoptosis protein (XIAP). The antiapoptotic activity of uPA was dependent on its protease activity, the presence of uPA receptor (uPAR) and low-density lipoprotein receptor-related protein (LRP), but independent of the phosphatidylinositol 3 (PI3) kinase pathway, whereas vascular endothelial growth factor (VEGF)-induced antiapoptosis was PI3 kinase dependent.

Pulmonary vein, dorsal atrial wall and atrial septum abnormalities in podoplanin knockout mice with disturbed posterior heart field contribution.

The developing sinus venosus myocardium, derived from the posterior heart field, contributes to the atrial septum, the posterior atrial wall, the sino-atrial node, and myocardium lining the pulmonary and cardinal veins, all expressing podoplanin, a coelomic and myocardial marker. We compared development and differentiation of the myocardium and vascular wall of the pulmonary veins (PV), left atrial dorsal wall, and atrial septum in wild type with podoplanin knockout mouse embryos (E10.5-E18.

The plasminogen activator inhibitor "paradox" in cancer.

Proteolysis in general and specifically the plasminogen activating system regulated by urokinase (uPA) its specific receptor, the GPI membrane anchored urokinase receptor (uPAR) and the specific plasminogen activator inhibitor 1 (PAI-1) plays a major role in tumorigenesis, tumor progression, tumor invasion and metastasis formation. This is exemplified by a body of published work showing a positive correlation between the expression of uPA or uPAR in several tumors and their malignancy. It is generally assumed that such a "pro-malignant" effect of the uPA-uPAR system is mediated by increased local proteolysis thus favoring tumor invasion, by a pro-angiogenic effect of this system and also by uPA-uPAR signaling towards the tumor thereby shifting the tumor phenotype to a more "malignant" one.