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International audience; Renal ischaemia reperfusion (I/R) triggers a cascade of events including oxidative stress, apoptotic body and microparticle (MP) formation as well as an acute inflammatory process that may contribute to organ failure. Macrophages are recruited to phagocytose cell debris and MPs. The tyrosine kinase receptor MerTK is a major player in the phagocytosis process. Experimental models of renal I/R events are of major importance for identifying I/R key players and for elaborating novel therapeutical approaches. A major aim of our study was to investigate possible involvement of MerTK in renal I/R. We performed our study on both natural mutant rats for MerTK (referred to as RCS) and on wild type rats referred to as WT. I/R was established by of bilateral clamping of the renal pedicles for 30′ followed by three days of reperfusion. Plasma samples were analysed for creatinine, aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH), kidney injury molecule -1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL) levels and for MPs. Kidney tissue damage and CD68-positive cell requirement were analysed by histochemistry. monocyte chemoattractant protein-1 (MCP-1), myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), and histone 3A (H3A) levels in kidney tissue lysates were analysed by western blotting. The phagocytic activity of blood-isolated monocytes collected from RCS or WT towards annexin-V positive bodies derived from cultured renal cell was assessed by fluorescence-activated single cell sorting (FACS) and confocal microscopy analyses. The renal I/R model for RCS rat described for the first time here paves the way for further investigations of MerTK-dependent events in renal tissue injury and repair mechanisms.

International audience; Malignant gliomas are the most common primary brain tumors. Due to both their invasive nature and resistance to multimodal treatments, these tumors have a very high percentage of recurrence leading in most cases to a rapid fatal outcome. Recent data demonstrated that neural stem/progenitor cells possess an inherent ability to migrate towards glioma cells, track them in the brain and reduce their growth. However, mechanisms involved in these processes have not been explored in-depth. In the present report, we investigated interactions between glioma cells and neural stem/progenitor cells derived from the subventricular zone, the major brain stem cell niche. Our data show that neural stem/pro-genitor cells are attracted by cultured glioma-derived factors. Using multiple approaches, we demonstrate for the first time that the vitamin K-dependent factor protein S produced by glioma cells is involved in tumor tropism through a mechanism involving the tyrosine kinase receptor Tyro3 that, in turn, is expressed by neural stem/ progenitor cells. Neural stem/progenitor cells decrease the growth of both glioma cell cultures and clonogenic population. Cultured neural stem/progenitor cells also engulf, by phagocytosis, apoptotic glioma cell-derived fragments and this mechanism depends on the exposure of phosphatidylserine eat-me signal and is stimulated by protein S. The disclosure of a role of protein S/Tyro3 axis in neural stem/progenitor cell tumor-tropism and the demonstration of a phagocytic activity of neural stem/progenitor cells towards dead glioma cells that is regulated by protein S open up new perspectives for both stem cell biology and brain physiopathology.

(IF: 3,67); International audience; Transferrin is well known as an iron transport glycoprotein. Dimeric or tetrameric transferrin forms have recently been reported to modulate phagocytosis by human leukocytes. It is mainly synthesized by the liver, and also by other sources, such as Sertoli cells of the testis. Sertoli cells show a strong phagocytic activity toward apoptotic germ cells and residual bodies. Here, we provide evidence that purified human dimeric transferrin from commercial sources decreased residual body phagocytosis, unlike monomeric transferrin. The presence of iron appeared essential for dimeric transferrin inhibitory activity. Importantly, dimeric transferrin could be visualized by immunoblotting in Sertoli cell lysates as well as in culture media, indicating that dimeric transferrin could be physiologically secreted by Sertoli cells. By siRNA-mediated knockdown, we show that endogenous transferrin significantly inhibited residual body ingestion by Sertoli cells. These results are the first to identify dimeric transferrin in Sertoli cells and to demonstrate its implication as a physiological modulator of residual body phagocytosis by Sertoli cells.

In the adult mammalian brain, neural stem cells persist in the subventricular zone (SVZ) of lateral ventricles. It is well established that cortical damage leads to SVZ cell proliferation and neuronal differentiation. We have previously demonstrated in rat that, when treated with the apoptosis-inducing agent staurosporine, cortex explants release heat-labile factors that promote SVZ cell culture proliferation. In the present report, we investigated in vitro mechanisms involved in cortex injury-triggered neurogenesis in the rat. We demonstrated, using immunoblotting analysis and fibroblast growth factor (FGF)-2 enzyme-linked sandwich immunosorbent assay, that treatment of cortex explants with apoptosis-inducing agents increases the release of FGF-2. We next determined the effects of apoptotic cortex-released factors in regulating SVZ cell proliferation and neuronal differentiation by using bromodeoxyuridine incorporation and microtubule-associated protein 2 immunostaining assays, respectively. We found that conditioned media derived from staurosporine-treated cortex explants enhanced SVZ cell culture proliferation and differentiation by over 50 and 80%, respectively. Finally, we showed that immunodepletion of FGF-2 or pharmacological blockade of FGF-2 receptor by SU5402 completely abolished staurosporine-treated cortex mitogenic activity on SVZ cultures but did not alter its activity on neuronal cell differentiation. Altogether, the present report establishes that the release of endogenous FGF-2 by apoptotic cortex explants plays a major role in the induction of SVZ cell proliferation but not neuronal differentiation, which probably depends on the release of other as yet unidentified cortical factors.

The gamma-carboxyglutamate-containing proteins are a family of secreted vitamin K-dependent proteins in which some glutamyl residues are post-translationally modified to gamma-carboxyglutamic acid residues. A vitamin K-dependent gamma-glutamyl carboxylase enzyme catalyses this post-translational modification. The gamma-carboxylase reaction requires vitamin K in its reduced form, vitamin K hydroquinone, and generates gamma-carboxyglutamate and vitamin K 2,3,-epoxide which is then recycled back to the hydroquinone form by a vitamin K reductase system. Warfarin blocks the vitamin K cycle and hence inhibits the gamma-carboxylase reaction, and this property of Warfarin has led to its wide use in anticoagulant therapy. Until recently, interest in vitamin K-dependent proteins was mostly restricted to the field of hematology. However, the discovery that the anti-coagulant factor protein S and its structural homologue Gas6 (growth arrest-specific gene 6), two vitamin K-dependent proteins, are ligands for the Tyro3/Axl/Mer family of related tyrosine kinase receptors has opened up a new area of research. Moreover, the phenotypes associated with the invalidation of genes encoding vitamin K-dependent proteins or their receptors revealed their implication in regulating phagocytosis during many cell differentiation phenomena such as retinogenesis, neurogenesis, osteogenesis, and spermatogenesis. Additionally, protein S was identified as the major factor responsible for serum-stimulated phagocytosis of apoptotic cells. Therefore, the elucidation of the molecular mechanisms underlying the role of vitamin K-dependent proteins in regulating apoptotic cell phagocytosis may lead to a better understanding of the physiopathology of cell differentiation and could form the framework of new therapeutic strategies aiming at a selective targeting of cell phagocytosis associated pathologies.

In rodents, the subventricular zone (SVZ) harbours neural stem cells that proliferate and produce neurons throughout life. Previous studies showed that factors released by the developing cortex promote neurogenesis in the embryonic ventricular zone. In the present report, we studied in the rat the possible involvement of endogenous factors derived from the embryonic cortex in the regulation of the development of postnatal SVZ cells. To this end, SVZ neurospheres were maintained with explants or conditioned media (CM) prepared from embryonic day (E) 13, E16 or early postnatal cortex. We demonstrate that early postnatal cortex-derived factors have no significant effect on SVZ cell proliferation or differentiation. In contrast, E13 and E16 cortex release diffusible, heat-labile factors that promote SVZ cell expansion through increased proliferation and reduced cell death. In addition, E16 cortex-derived factors stimulate neuronal differentiation in both early postnatal and adult SVZ cultures. Fibroblast growth factor (FGF)-2- but not epidermal growth factor (EGF)-immunodepletion drastically reduces the mitogenic effect of E16 cortex CM, hence suggesting a major role of endogenous FGF-2 released by E16 cortex in the stimulation of SVZ cell proliferation. The evidence we provide here for the regulation of SVZ cell proliferation and neuronal differentiation by endogenous factors released from embryonic cortex may be of major importance for brain repair research.

Neural stem cells, whose major reservoir in the adult mammalian brain is the subventricular zone (SVZ), ensure neuropoiesis, a process during which many generated cells die. Removal of dead cells and debris by phagocytes is necessary for tissue homeostasis. Using confocal and electron microscopy, we demonstrate that cultured SVZ cells phagocytose both 1 and 2 µm latex beads and apoptotic cell-derived fragments. We determine by flow cytometry that phagocytic cells represent more than 10% of SVZ cultured cells. Phenotyping of SVZ cells using nestin, GFAP, Sox2, or LeX/SSEA and quantification of aldehyde dehydrogenase (ALDH) activity, reveals that cells with neural stem-cell features phagocytose and represent more than 30% of SVZ phagocytic cells. In vivo, nestin-, Sox2-, and ALDH-expressing neural stem-like cells engulfed latex beads or apoptotic cell-derived fragments that were injected into mice lateral brain ventricles. We show also that SVZ cell phagocytic activity is an active process, which depends both on cytoskeleton dynamic and on recognition of phosphatidylserine eat-me signal, and is stimulated by the vitamin K-dependent factor protein S (ProS). ProS neutralizing antibodies inhibit SVZ cell phagocytic activity and exposure of SVZ cells to apoptotic cell-derived fragments induces a transient Mer tyrosine kinase receptor (MerTK) phosphorylation. Conversely, MerTK blocking antibodies impair both basal and ProS-stimulated SVZ cell phagocytic activity. By revealing that neural stem-like cells act within the SVZ neurogenic niche as phagocytes and that the ProS/MerTK path represents an endogenous regulatory mechanism for SVZ cell phagocytic activity, the present report opens-up new perspectives for both stem cell biology and brain physiopathology. Stem Cells 2015;33:515–525

Vascular Endothelial Growth Factor (VEGF) has been typically considered to be an endothelial-specific growth factor. However, it was recently demonstrated that VEGF can interact with non endothelial cells. In this study, we tested whether vascular smooth muscles cells (VSMCs) can express VEGF receptors, such as flk-1, flt-1, and neuropilin (NP)-1, and respond to VEGF in vitro. In cultured VSMCs, flk-1 and flt-1 expression was inversely related to cell density. The expression of flk-1 was down-regulated with increasing passage numbers. However, NP-1 levels were not affected by cell density or passage numbers. Flk-1, Flt-1, and NP-1 protein levels were confirmed by Western Blotting. Although the functional mature form of Flk-1 protein is expressed at low levels in VSMCs, phosphorylation of Flk-1 following VEGF(165) stimulation was still observed. SMCs migrated significantly in response to VEGF(165) and VEGF-E, whereas Placenta Growth Factor (PlGF) induced migration only at higher concentrations. Since VEGF-E is a specific activator of flk-1 while PlGF specifically activates only flt-1, SMC migration induced by VEGF(165) is likely to be mediated primarily through the flk-1 receptor. VSMCs did not significantly proliferate in response to VEGF(165), PlGF, and VEGF-E. In conclusion, our studies demonstrate the presence of VEGF receptors on VSMCs that are functional. These studies also indicate that in vivo, VEGF may play a role in modulating the response of VSMCs.

The anticoagulant factor protein S is a secreted vitamin K-dependent γ-carboxylated protein that is mainly made in the liver. Protein S is homologous to the growth arrest specific protein, Gas6, the expression of which is up-regulated in cultured fibroblasts upon serum withdrawal. We report here the synthesis and secretion of protein S by cultured human vascular smooth muscle cells (HVSMCs). Western blot analysis revealed that similar amounts of protein S are secreted by both growing and growth-arrested HVSMCs. HVSMC-derived protein S was found to be γ-carboxylated as it was precipitated by barium citrate and was shown to possess protein C cofactor activity. Treatment with the vitamin K antagonist warfarin led to the accumulation of intracellular undercarboxylated protein S forms that were rapidly secreted upon the reintroduction of vitamin K. Northern blotting analysis showed that cultured HVSMCs express a protein S transcript. The expression of protein S messenger RNA was unaffected by either warfarin, growth arrest, or various VSMC mitogens, such as platelet-derived growth factor-BB, basic fibroblast growth factor, transforming growth factor-β, or hepatocyte growth factor. Thrombin, however, induced an up-regulation of protein S expression at both messenger RNA and protein levels. The evidence we provide for protein S secretion by cultured HVSMCs and its up-regulation by thrombin, together with earlier reports showing that protein S acts as a mitogen for these cells, suggests that, in addition to its known role in regulating blood clotting, protein S may also be an important autocrine factor in the pathophysiology of the vasculature.

Thrombin, a serine protease, is an important effector of many cellular processes and has been shown to up-regulate the expression of several genes. The mechanisms underlying thrombin-mediated regulation of gene transcription remain poorly understood. The original aim of this work was to study the effects of thrombin on the activation of transcription factors, Sp1, NF-kappaB, and CREB by means of electrophoretic mobility-shift assays (EMSA). However, an inconsistent pattern of results was observed. We raised the possibility that some EMSA results may have been erroneous by the fact that during the nuclear protein extraction and EMSA procedure, transcription factors are dephosphorylated by cellular phosphatases and hence their DNA-binding capacities are modified. Therefore, we have altered the original nuclear extraction protocol by including a mixture of phosphatase inhibitors during protein extraction and subsequent EMSA steps. We show here that this simple measure led to significant changes in both basal and thrombin-induced levels of activation of Sp1 and CREB, but not of NF-kappaB. In light of the data presented here, it would be important to reexamine the conclusions of many reports in which EMSA was used to assess the basal and agonist-induced levels of transcription factor DNA-binding activities.

Thromb Haemost 2008; 100: 527–529 Vitamin K or vitamin “Koagulation” (German spelling for coagulation) was discovered by the 1943 Nobel Prize winners Henrik Dam and Edward A. Doisy, as a fat soluble substance, the deficiency of which caused bleeding disorders.Vitamin K in its reduced form is required as a cofactor for the γ-glutamyl carboxylase enzyme that catalyses the γ-carboxylation of specific glutamic acid (Glu) residues of a subclass of proteins (1). This subclass of proteins was then termed vitamin K-dependent proteins (VKDP), or γ-carboxylated proteins or simply Gla-proteins. The enzymatic reaction generates γ-carboxyglutamate (Gla) and vitamin K 2,3,-epoxide which is then recycled back to the hydroquinone form by a reductase enzyme (1). Warfarin (3-[α-acetonyl-benzyl-4-hydroxycoumarin]) inhibits the activity of the vitamin K epoxide reductase blocking the vitamin K cycle.This property of warfarin has led to its widespread use in anticoagulant therapy (2). In 2000, warfarin was ranked among the top-selling drugs with a turnover estimated at 500 million dollars. The γ-carboxylation process appears to be required for the activities of all Gla-proteins studied to date. Carboxylase substrates synthesised in the presence of warfarin are undercarboxylated and have impaired biological activities (3). In the present theme issue, Shearer and Newman stress the importance of exploring vitamin K metabolism and catabolism pathways for a better understanding of pathologies linked to vitamin K deficiency (4). The occurrence of such pathologies is established in bone and arteries, two tissues that express many VKDP, whereby expression is hypothesised in others. The various molecular forms of vitamin K are different not only with regard to their co-factor activities but also with regard to their absorption, transport, cellular uptake, tissue distribution, turnover and catabolism.This paper provides a clear description of the nomenclature and chemical structure of K vitamins, namely phylloquinone (vitamin K1) and menaquinones (vitamins K2) as well as their dietary sources. Further research is certainly needed in this area to determine to what extent bacterially fermented food as well as intestinal flora contribute to the maintenance of vitamin K status. The effects of other putative non-cofactor functions of vitamin K, which potentially include the suppression of inflammation, prevention of brain oxidative damage and a role in sphingolipid synthesis as well as the possible existence of a receptor for vitamin K, are important issues that require long-term basic research to be clarified. The full consequences of dietary vitamin K deficiency on tissue distribution and metabolism of K vitamins as well as the consequences of oral anticoagulants on the metabolism of K vitamins remains to be assessed in the light of new findings on VKDP. Only few proteins are known to contain Gla residues. These include: (i) certain proteins of the blood coagulation system: prothrombin, factors VII, IX and X, protein C, protein S and protein Z; (ii) the protein encoded by the growth arrest specific gene GAS6, which presents strong structural homology with protein S; (iii) Gla proteins expressed in mineralised tissues: osteocalcin and matrix Gla-protein (MGP); (iv) Gla-containing snake venom proteins and conotoxins; (v) four transmembrane Gla-proteins PRGP1, PRGP2, TMG3 and TMG4 whose functions are as yet unknown; (vi) some members of the connexin family of proteins (5). The precise role of the γ-carboxylation modification for connexin protein function is not as yet known. Except for protein S and protein Z, VKDP of the blood-clotting cascade are serine protease zymogens and are activated by serine proteases. The role of protein Z in blood clotting is not fully clarified and neither cellular effects nor a specific pathology have been as yet associated with protein Z. The review by Vasse et al. (6) in the present issue covers this topic. Two further contributions in the present theme issue relate to the therapeutic functions of VKDP in haemostasis. In her concise review Ulla Hedner concentrates on the therapeutic use of factor VIIa in bleeding associated pathologies (7). The paper by Paul Monahan reviews the data obtained from factor IX knockout mice and other haemophilia B mouse models (8). Such mouse models have not only enabled a better understanding of the role of factor IX in haemostasis, thrombosis and wound healing but also pro

Alpha-thrombin, a key enzyme of the coagulation cascade, is also a potent mitogen for human vascular smooth muscle cells (HVSMC). Here it is demonstrated that the alpha-thrombin-mediated reduction of intracellular cAMP levels is sensitive to pertussis toxin (PTX). cAMP-elevating agents inhibited alpha-thrombin- and serum-induced mitogenesis, thus cAMP confers an anti-mitogenic signal on HVSMC. The PTX-dependent ADP-ribosylation of a 41 kDa Gi alpha protein(s) was significantly inhibited (up to 55%) by thrombin. HVSMC membranes had an intrinsic GTP'ase activity which was significantly increased (up to 36%) by thrombin. PTX treatment did not alter thrombin-induced elevation of GTP'ase activity. Thrombin stimulated phosphatidyl inositol (PI) turnover in a PTX-insensitive manner. This suggested that PTX insensitive G proteins such as Gq are also activated by thrombin. This study on HVSMC provides additional evidence for the involvement of different families of G proteins in thrombin signalling.

SummaryThe monoclonal theory of atherosclerosis postulates that the initial vascular smooth muscle cell (VSMC) proliferative event involves the expansion of a single cell or a sub-population of cells thus implying differences in the replicative potential of VSMC. Using the technique of limited dilution, VSMC clones derived from animal tissues have been previously isolated and shown to be morphologically heterogeneous. However, the same technique applied to human VSMC (HVSMC) has been unsuccessful, possibly because HVSMC do not grow when plated at very low densities. In this report, the anchorage-independent growth of HVSMC in semi-solid medium was studied. Platelet-derived growth factor-BB (PDGF-BB) and to a lesser extent PDGF-AB and basic fibroblast growth factor (bFGF) induced colony formation. This assay provided a tool for the isolation of HVSMC clones. In terms of their growth characteristics and responsiveness to several growth factors, isolated HVSMC clones and the original parental cell population exhibited marked heterogeneity.

alpha-Thrombin, a key enzyme of the coagulation cascade, is also a potent mitogen for many cell types. In the present study, the responsiveness to alpha-thrombin of cultured human vascular smooth muscle cells (HVSMC) derived from either vein or normal and atherosclerotic arteries was investigated. All HVSMC populations examined responded mitogenically to alpha-thrombin. However, the extent of this response varied between different cell populations. No significant differences were observed between HVSMC derived from vein versus artery or atherosclerotic versus normal tissues. The responsiveness of a specific HVSMC culture to alpha-thrombin was not affected by cell density and remained constant over several passages. Unlike platelet-derived growth factor BB (PDGF-BB), alpha-thrombin did not exhibit any significant chemotactic effects on HVSMC or induce their anchorage independent growth in semi-solid medium. The hypothesis that the observed variability in HVSMC responsiveness to alpha-thrombin is due to the heterogeneity of cultured HVSMC is raised and discussed.

Human alpha-thrombin is a known human vascular smooth muscle cell (HVSMC) mitogen. We have previously reported that gamma-thrombin, in which the anion binding exosite is disrupted, but not catalytically inactivated PPACK-thrombin is mitogenic on HVSMC. Here, the structural requirements for thrombin's mitogenic activity on HVSMC were further investigated. Total inhibition of thrombin-induced DNA synthesis was achieved by AT-III and hirudin. AT-III, added 1 h after exposure of cells to thrombin, failed to alter thrombin-induced mitogenicity. Modification of thrombin's anion binding exosite with peptides derived from the C-terminal sequence of hirudin resulted in a partial loss of thrombin's mitogenic activity. PPACK-inactivated thrombin failed to induce a significant expression of the immediate early gene, c-fos. Unlike PPACK-inactivated thrombin, alpha-thrombin and gamma-thrombin induced a significant increase in the level of the PDGF-A gene expression. A correlation between PDGF-A gene induction and thrombin-induced mitogenicity was suggested by the fact that the mitogenic forms of thrombin also stimulated PDGF-A gene expression. Together, these results indicate that the mitogenic activity of thrombin on HVSMC requires the integrity of the enzyme's active site and is altered by modifications of its anion binding exosite.

Protein S is a vitamin K–dependent glycoprotein, which, besides its anticoagulant function, acts as an agonist for the tyrosine kinase receptors Tyro3, Axl, and Mer. The endothelium expresses Tyro3, Axl, and Mer and produces protein S. The interaction of protein S with endothelial cells and particularly its effects on angiogenesis have not yet been analyzed. Here we show that human protein S, at circulating concentrations, inhibited vascular endothelial growth factor (VEGF) receptor 2–dependent vascularization of Matrigel plugs in vivo and the capacity of endothelial cells to form capillary-like networks in vitro as well as VEGF-A–induced endothelial migration and proliferation. Furthermore, protein S inhibited VEGF-A–induced endothelial VEGFR2 phosphorylation and activation of mitogen-activated kinase-Erk1/2 and Akt. Protein S activated the tyrosine phosphatase SHP2, and the SHP2 inhibitor NSC 87877 reversed the observed inhibition of VEGF-A–induced endothelial proliferation. Using siRNA directed against Tyro3, Axl, and Mer, we demonstrate that protein S-mediated SHP2 activation and inhibition of VEGF-A–stimulated proliferation were mediated by Mer. Our report provides the first evidence for the existence of a protein S/Mer/SHP2 axis, which inhibits VEGFR2 signaling, regulates endothelial function, and points to a role for protein S as an endogenous angiogenesis inhibitor.

Rodent fibroblastic cells transformed by ras oncogenes can grow in serum-free (S−) medium. We have studied clonal lines of mouse NIH3T3 fibroblasts transfected with the EJ-H-ras oncogene, and observed that practically all become independent of exogenous growth and attachment factors shortly after transfection. Moreover, all the clones tested soon form anchorage-independent (Al) colonies in S− medium, and most give rise to spheroids able to grow in suspension. The cell-conditioned S−medium of the transformed (TR) cells stimulates autocrinally the Al and anchored growth of these cells, in the absence of serum, and it contains growth factors related to TGF-α (or EGF), PDGF and bFGF, and other uncharacterized factors. Some of these factors are not found, or are found only in very small amounts, in the S− medium of non-transformed NIH3T3 cells, which also stimulates the growth of the TR cells, in the absence of serum. In addition, the TR cells contain 4-6 times more cell-associated bFGF than the non-transformed cells and release more latent TGF-β activatable by acid treatments. However, no active TGF-β is secreted by either cell type. Activated TGF-β and pure TGF-β I stimulate the growth of the anchored TR and NIH3T3 cells, but inhibit the Al growth of the TR cells. Another inhibitor of this growth is also found in the concentrated medium of the NIH3T3 cells. © 1992 Wiley-Liss, Inc.

TGF-beta 1 stimulates thymidine incorporation and the growth rate of mouse NIH3T3 fibroblasts and of those cells transformed by the EJ-H-ras oncogene (TR15 cells), in the presence and the absence of serum. Thymidine incorporation, in serum-deprived cells, is stimulated to a higher degree by 0.1-1 ng/ml of TGF-beta in NIH3T3 than in TR15 cells, which have a 10-fold higher basal level of incorporation. In both cell types TGF-beta 1 is as active, or more active than other mitogens (TGF-alpha, PDGF-AB, bFGF) at the same concentration. The growth rate of NIH3T3 cells, in low serum or serum-free (S-) medium, is stimulated by only 10 picograms/ml of TGF-beta 1, and that of TR15 cells, in S- medium, by only 1 picogram/ml. In contrast, TGF-beta 1 inhibits mitogenically unestablished mouse embryo fibroblasts and these fibroblasts immortalized spontaneously and able to grow in S- medium. It also inhibits the anchorage-independent growth of TR15 cells. NIH3T3 and TR15 cells respond, similarly, to TGF-beta activated by acification of their culture medium. The kinetics of thymidine incorporation and of activation of the c-myc proto-oncogene, observed already after 1 hr, in treated NIH3T3 and TR15 cells, suggests a direct mitogenic stimulation. The level of activated c-myc RNA is 2-fold higher at 2 hr, and subsequently decreases relatively less in the TR15 cells.

Neural stem cells persist in the adult mammalian brain, within the subventricular zone (SVZ). The endogenous mechanisms underpinning SVZ neural stem cell proliferation, self-renewal, and differentiation are not fully elucidated. In the present report, we describe a growth-stimulatory activity of liver explant-conditioned media on SVZ cell cultures and identify hepatocyte growth factor (HGF) as a major player in this effect. HGF exhibited a mitogenic activity on SVZ cell cultures in a mitogen-activated protein kinase (MAPK) (ERK1/2)-dependent manner as U0126, a specific MAPK inhibitor, blocked it. Combining a functional neurosphere forming assay with immunostaining for c-Met, along with markers of SVZ cells subtypes, demonstrated that HGF promotes the expansion of neural stem-like cells that form neurospheres and self-renew. Immunostaining, HGF enzyme-linked immunosorbent assay and Madin-Darby canine kidney cell scattering assay indicated that SVZ cell cultures produce and release HGF. SVZ cell-conditioned media induced proliferation on SVZ cell cultures, which was blocked by HGF-neutralizing antibodies, hence implying that endogenously produced HGF accounts for a major part in SVZ mitogenic activity. Brain sections immunostaining revealed that HGF is produced by nestin-expressing cells and c-Met is expressed within the SVZ by immature cells. HGF intracerebroventricular injection promoted SVZ cell proliferation and increased the ability of these cells exposed in vivo to HGF to form neurospheres in vitro, whereas intracerebroventricular injection of HGF-neutralizing antibodies decreased SVZ cell proliferation. The present study unravels a major role, both in vitro and in vivo, for endogenous HGF in SVZ neural stem cell growth and self-renewal.

Extracts of the Drosophila cell line KCo contain a TGF-beta-like activity. This bioactivity, like mammalian TGF-beta, induces the anchorage-independent growth of rat NRK-49F cells in the presence of EGF, inhibits 3H-thymidine incorporation in the mink epithelial cell line CCL-64 and is acid and heat stable but destroyed by dithiothreitol. On a reverse phase column a single bioactive peak eluted at 37-40% acetonitrile. This TGF-beta-like activity in KCo cells inhibits the growth of the homotypic Drosophila cell line 14-11-23, whereas porcine TGF-beta 1 had no effect.

The tubulin-binding agent combretastatin A-4-phosphate (CA-4-P), rapidly disrupts the vascular network of tumors leading to secondary tumor cell death. In vitro, CA-4-P destabilizes microtubules and causes endothelial cell death. In this study we analyze the mechanisms by which CA-4-P induces the death of proliferating endothelial cells. We demonstrate that at/=7.5 nmol/L, CA-4-P damages mitotic spindles, arrests cells at metaphase, and leads to the death of mitotic cells with characteristic G(2)/M DNA content. Mitotic arrest was associated with elevated levels of cyclin B1 protein and p34(cdc2) activity. Inhibition of p34(cdc2) activity by purvalanol A caused mitotic-arrested cells to rapidly exit mitosis, suggesting that sustained p34(cdc2) activity was responsible for metaphase arrest. Pharmacological prevention of entry into mitosis protected cells from undergoing cell death, further establishing the link between mitosis and cell death induction by CA-4-P. CA-4-P-mediated cell death shared characteristics of apoptosis but was independent of caspase activation suggesting the involvement of a non-caspase pathway(s). These data suggest that induction of apoptosis in endothelial cells by CA-4-P is associated with prolonged mitotic arrest. Therefore, by activating cell death pathways, CA-4-P, in addition to being an effective anti-vascular agent, may also interfere with regrowth of blood vessels in the tumor.

Activated protein C (APC) exerts endothelial protein C receptor (EPCR)-dependent neuroprotective effects in a brain focal ischemia model and direct cellular effects on human umbilical vein endothelial cells (HUVECs) via protease-activated receptor-1 (PAR-1). Because PAR receptors are expressed in brain endothelium and mediate intracellular calcium concentration ([Ca2+]i) signaling, we hypothesized that APC may regulate intracellular [Ca2+] flux in human brain endothelial cells (BECs) via EPCR and PAR-1. Primary cortical BECs derived from human autopsies (early passage) and HUVECs were used for [Ca2+]i imaging fluorometry. Cells were exposed for 1 minute to APC, protein C zymogen, or mutant Ser360Ala-APC, and [Ca2+]i was monitored in the presence or absence of antibodies against PAR-1, PAR-2, PAR-3, or EPCR. APC, but not protein C zymogen or the active site mutant Ser360Ala-APC, induced dose-dependent [Ca2+]i release in human BECs (Delta[Ca2+]i max = 278.3 +/- 19.5 nM; EC50 for APC = 0.23 +/- 0.02 nM, n = 70 measurements). APC-induced [Ca2+]i signaling was abolished by a cleavage site blocking anti-PAR-1 antibody, whereas anti-PAR-2 and -PAR-3 antibodies were without effect. Antibody RCR252 that ablates APC binding to EPCR blocked APC-mediated [Ca2+]i signaling, whereas anti-EPCR antibody RCR92 that does not block APC binding did not abolish the APC-induced [Ca2+]i response. Experiments using HUVECs confirmed the findings for BECs. Thapsigargin inhibited the APC-induced [Ca2+]i signal, implicating the endoplasmic reticulum as a major source for the APC-induced [Ca2+]i release. These data suggest that APC regulates [Ca2+]i in human brain endothelium and in HUVECs by binding to EPCR and signaling via PAR-1.

The anticoagulant factor protein S is a secreted vitamin K-dependent γ-carboxylated protein that is mainly synthesised in the liver but is also made by endothelial cells and megakaryocytes in culture. In previous studies we have shown that protein S acts as a mitogen for cultured human vascular smooth muscle cells. The synthesis and secretion of protein S by endothelial cells suggests that in addition to its role in the coagulation cascade, protein S may be an important autocrine factor implicated in the pathophysiology of the vascular system. The effects of protein S on hVSMC proliferation, migration and survival are discussed. The activation of the components of the MAP kinase pathway, ERK1/2, JNK/SAPK and p38 is also summarised. Binding and chemical cross-linking experiments provided evidence for the existence of a cell surface protein S receptor(s). By virtue of its many cellular effects, it is suggested here that the anticoagulant factor protein S plays an important role in the pathophysiology of the vasculature.

SummaryThe synthetic peptide SFLLRNPNDKYEPF, identical in sequence to the new amino-terminus of the thrombin receptor generated following cleavage by thrombin, acts as a thrombin receptor agonist/ activating peptide (TRAP). In this study, Northern blot analysis showed that cultured human vascular smooth muscle cells (HVSMC) express a thrombin receptor transcript. TRAP, in contrast to thrombin was shown to be a weak mitogen for HVSMC. A combination of TRAP and enzymatically-inactivated thrombin (PPACK-thrombin) which provides receptor occupancy, did not potentiate TRAP-induced mitogenesis, indicating that TRAP and PPACK-thrombin do not reproduce the mitogenic effect of enzymatically-active thrombin. Both thrombin and TRAP, induced the expression of c-fos and the PDGF-A gene in a pertussis toxin (PTX)-insensitive manner. Examination of thrombin and TRAP-treated cells by immunofluorescence staining followed by computer assisted image analysis revealed that thrombin and to a lesser extent TRAP induced PDGF-AA protein expression. Antibodies to PDGF-AA partially inhibited thrombin but not TRAP-induced mitogenesis in HVSMC. This study indicates that in addition to the common signalling pathways utilised by thrombin and TRAP, enzymatically-active thrombin activates other signalling pathways and hence TRAP does not mimic fully the biological effect of thrombin on HVSMC.

The effects of cyclic AMP (cAMP)-elevating agents on the activity of cis-acting gene promoter sequences are frequently studied using the luciferase-reporter-gene system. The aim of the present study was to assess whether cAMP-elevating agents induce any changes in the level of luciferase activity independently of a transcriptional activation of promoter elements. Chloramphenicol acteyltransferase (CAT) and luciferase reporter genes under the control of the same promoter elements were transiently expressed in primary cultures of human vascular smooth-muscle cells. Transfected cells were treated with a cell-permeable and non-hydrolysable cAMP analogue, 2′-O-monobutyryl-8-bromo cAMP, or with the cAMP-elevating agents forskolin and prostaglandin E1 (PGE1). Forskolin and PGE1 induced a significant increase in the level of luciferase activity, but had no effect on CAT activity. Conclusions based solely on the use of the luciferase-reporter-gene system in studies involving promoter activation by cAMP-elevating agents could therefore be misleading.

Abstract The urokinase-type plasminogen activator (UPA) and its receptor are expressed in the vasculature and are involved in cell migration and remodeling of the extracellular matrix in the neointima. Vessels with atherosclerosis or neointimal hyperplasia, when compared with normal vessels, contain high UPA activity as well as increased levels of UPA receptor. In this study, we have identified the stimulation of vascular smooth muscle cell proliferation as a novel activity for UPA in the vessel wall. High-molecular-weight-UPA (12-200 nmol/L range) stimulated DNA synthesis and cell proliferation, which was half that induced by fetal calf serum or by platelet-derived growth factor-BB. UPA did not induce growth of endothelial cells, and tissue-type plasminogen activator showed no activity on either cell type. Induction of proliferation required the complete UPA molecule but was independent of the proteolytic activity of UPA, whereas neither the amino-terminal fragment nor the catalytic domain by itself was mitogenic. UPA also stimulated c -fos /c -myc mRNA expression and mitogen-activated protein kinase activity in smooth muscle cells. Blocking monoclonal antibodies against the UPA receptor and the enzymatic removal of receptors were ineffective in inhibiting the mitogenic effect of UPA, suggesting a UPA receptor–independent mechanism. Thus, we provide evidence for a novel function of UPA on vascular smooth muscle cell proliferation that, together with its previously documented involvement in regulating pericellular proteolysis-related events and cell migration, provides additional evidence for a role in the pathogenesis of atherosclerosis/restenosis.

The presence of specific binding sites for the coagulation factor protein S (PS) on the surface of human vascular smooth muscle cells (HVSMC) is described. The binding characteristics of 125I-PS to HVSMC were studied and found to be saturable, reversible and, as described by the Hill equation, co-operative (h 1.74; Kd 0.33 nM). Autoradiographic analysis of detergent extracts of HVSMC chemically cross-linked with 125I-PS and separated by SDS/PAGE revealed radioactivity associated with two proteins with apparent molecular masses of 220 and 230 kDa respectively. The mitogenic activity of PS on HVSMC was also investigated. Protein S was shown to stimulate DNA synthesis of growth-arrested HVSMC and to support their proliferation under low-serum conditions in a sustained and dose-dependent manner.