Evidence for a major role of endogenous fibroblast growth factor-2 in apoptotic cortex-induced subventricular zone cell proliferation.

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.

Expression of vascular endothelial growth factor receptors in smooth muscle cells.

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.

Cellular effects and signalling pathways activated by the anti-coagulant factor, protein S, in vascular cells protein S cellular effects.

The anticoagulant factor protein S is a secreted vitamin K-dependent gamma-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.

The anticoagulant factor, protein S, is produced by cultured human vascular smooth muscle cells and its expression is up-regulated by thrombin.

The anticoagulant factor protein S is a secreted vitamin K-dependent gamma-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 gamma-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-beta, 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. (Blood. 2000;95:2008-2014)

Thrombin activates transcription factors sp1, NF-kappaB, and CREB: importance of the use of phosphatase inhibitors during nuclear protein extraction for the assessment of transcription factor DNA-binding activities.

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.

Induction of vascular SMC proliferation by urokinase indicates a novel mechanism of action in vasoproliferative disorders.

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.

Evidence for cultured human vascular smooth muscle cell heterogeneity: isolation of clonal cells and study of their growth characteristics.

The 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 factor-BB (PDGF-BB) and to 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.

Involvement of pertussis toxin-sensitive and -insensitive G proteins in alpha-thrombin signalling on cultured human vascular smooth muscle cells.

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.

Variability in the proliferative responsiveness of cultured human vascular smooth muscle cells to alpha-thrombin.

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.

Prothrombin cleavage by human vascular smooth muscle cells: a potential alternative pathway to the coagulation cascade.

Thrombin is a potent mitogen for human vascular smooth muscle cells (HVSMC) and its enzymatic activity is required for this function. The present study demonstrates that prothrombin is also mitogenic for HVSMC due to the generation of enzymatically active thrombin which occurs upon incubation of prothrombin with the cells. Analysis by SDS-PAGE, immunoblotting, and amino acid sequencing revealed that prothrombin incubated with HVSMC undergoes limited proteolysis. Prethrombin 1 was formed through cleavage at R155-S156. Cleavage at R271-T272 generated fragment 1.2 and prethrombin 2 whilst cleavage at R284-T285 yielded truncated prothrombin 2 (prethrombin 2'). However, cleavage at R320-I321 which, during prothrombin activation produces two-chain alpha-thrombin, was not detectable. Studies on HVSMC-conditioned medium revealed that a similar pattern of prothrombin cleavage occurred by a cell-secreted factor(s). Amidolytic activity analysis indicated that 1-3% catalytically active thrombin-like activity was generated upon incubation of prothrombin with HVSMC-conditioned medium. By treating conditioned medium with various classes of proteinase inhibitors or hirudin, it was determined that prothrombin is cleaved by a cell-derived serine proteinase-like factor(s) at R271-S272 and by alpha-thrombin at R155-S156 and R284-T285. Antibodies neutralising the activity of either urokinase, tissue plasminogen activator, or factor Xa failed to alter the prothrombin cleaving activity of conditioned medium. This activity which may catalyse an alternative pathway for the generation of thrombin, was eluted from a gel filtration column as a single peak with apparent molecular mass of 30-40 kDa.

Thrombin receptor activating peptide (TRAP) stimulates mitogenesis, c-fos and PDGF-A gene expression in human vascular smooth muscle cells.

The synthetic peptide SFLLRNPNDKYEPF, identical in sequence to the new amino-terminus of the thrombin receptor generated following cleavage of thrombin, acts 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.

Structural domains of thrombin involved in the induction of mitogenesis in cultured human vascular smooth muscle cells.

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.

Long-term chemotaxis studies on adherent cells: effect of platelet-derived growth factor-BB on human vascular smooth muscle cell migration.

Several chemotaxis methods have been developed which allow the study of different aspects of cell migration. The major limitation of such methods is the lack of a sustained chemotactic signal. Long-term chemotaxis phenomena which are known to take place in vivo have remained largely uninvestigated. Ways to maintain sustained chemotactic signals were sought and the used to investigate the long-term chemotactic effect of platelet-derived growth factor BB (PDGF-BB) on human vascular smooth muscle cells (HVSMC). PDGF-BB was adsorbed onto microcarrier beads and then embedded in agar. PDGF-BB diffusion was slow and a high and sustained local concentration was maintained in the agar. When PDGF-BB-loaded beads embedded in agar were placed at the edge of a tissue culture dish with HVSMC plated in the center, preferential movement was observed in the direction of the PDGF-BB source. This method was subsequently used to study directional movement of HVSMC arising from explants. This report demonstrates that PDGF-BB if present in an anisotropic concentration induces directional cell movement from such explants. By allowing the study of the effect of sustained chemotactic signals upon cultured cells or cells arising from explants, this method may provide a suitable model for investigating in vivo chemotaxis phenomena.

Evaluation of the use of the luciferase-reporter-gene system for gene-regulation studies involving cyclic AMP-elevating agents.

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.

Evidence for a protein S receptor(s) on human vascular smooth muscle cells. Analysis of the binding characteristics and mitogenic properties of protein S on human vascular smooth muscle cells.

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.

[Transforming Growth Factor Beta: structure, biologic effects and mechanism of action]. / Transforming Growth Factor Beta: structure, effets biologiques et mécanismes d'action.

For more than a decade, Transforming Growth Factor Beta (TGF beta) has been intensively studied. Such studies have led to the identification of many related polypeptides which constitute the TGF beta family. These factors modulate many animal cell processes such as cell cycle progression, differentiation, adhesion, migration, extracellular matrix production, tissue development and repair processes. Recent studies have shown that TGF beta has also a cardioprotective effect and is implicated in inflammatory process. The mechanism by which cytokines of the TGF beta family initiate their many biological effects are not yet fully understood. However, the recent molecular cloning of various TGF beta receptor genes has provided many clues to address these questions. These findings have led to a proposition of an original and specific model for the mechanism of action of TGF beta.

Growth in serum-free medium of NIH3T3 cells transformed by the EJ-H-ras oncogene: evidence for multiple autocrine growth factors.

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 (AI) 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 AI and anchored growth of these cells, in the absence of serum, and it contains growth factors related to TGF-alpha (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-beta activatable by acid treatments. However, no active TGF-beta is secreted by either cell type. Activated TGF-beta and pure TGF-beta 1 stimulate the growth of the anchored TR and NIH3T3 cells, but inhibit the AI growth of the TR cells. Another inhibitor of this growth is also found in the concentrated medium of the NIH3T3 cells.

Transforming growth factor beta stimulates mitogenically mouse NIH3T3 fibroblasts and those cells transformed by the EJ-H ras oncogene.

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.

Drosophila cell extracts contain a TGF-beta-like activity.

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.