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Biomedical subjects

L Claesson-Welsh

Publications and source records attributed to L Claesson-Welsh.

At least 37 records · Page 2Linked to original sources
PubMed1999

Platelet-derived growth factor beta receptor regulates interstitial fluid homeostasis through phosphatidylinositol-3' kinase signaling.

Platelet-derived growth factor (PDGF) isoforms lead to mitogenic, survival, and chemotactic responses in a variety of mesenchymal cell types during development and in the adult. We have studied the importance of phosphatidylinositol-3' kinase (PI3K) signaling in these responses by mutating the PI3K-binding sites in the PDGF-beta receptor by gene targeting in embryonic stem cells. Homozygous mutant mice developed normally; however, cells derived from the mutants were less chemotactic and had largely lost their ability to contract collagen gels in response to PDGF. Injection of a mast cell degranulating agent in mice led to a decrease in interstitial fluid pressure resulting in edema formation. In contrast to wild-type mice, mutant mice were unable to normalize the pressure after treatment with PDGF. Taken together, these observations suggest a function for PDGF signaling through PI3K in interstitial fluid homeostasis by modulating the tension between cells and extracellular matrix structures.

3T3 Cells↗
PubMed1999

Fibroblast growth factor receptor-1-mediated endothelial cell proliferation is dependent on the Src homology (SH) 2/SH3 domain-containing adaptor protein Crk.

Stimulation of fibroblast growth factor receptor-1 (FGFR-1) expressed on endothelial cells leads to cellular migration and proliferation. We have examined the role of the Src homology (SH) 2/SH3 domain-containing adaptor protein Crk in these processes. Transient tyrosine phosphorylation of Crk in fibroblast growth factor-2-stimulated endothelial cells was dependent on the juxtamembrane tyrosine residue 463 in FGFR-1, and a Crk SH2 domain precipitated FGFR-1 via phosphorylated Tyr-463, indicating direct complex formation between Crk and FGFR-1. Furthermore, Crk SH2 and SH3 domains formed ligand-independent complexes with Shc, C3G, and the Crk-associated substrate (Cas). Tyrosine phosphorylation of C3G and Cas increased as a consequence of growth factor treatment. We examined the role of Crk in FGFR-1-mediated cellular responses by use of cells expressing chimeric platelet-derived growth factor receptor-alpha/FGFR-1 (alphaR/FR) wild type and mutant Y463F receptors. The kinase activity of alphaR/FR Y463F was intact, but both Crk and the adaptor FRS-2 were no longer tyrosine-phosphorylated in the mutant cells. Both wild type and mutant receptor cells migrated efficiently, whereas cells expressing the mutant alphaR/FR Y463F failed to proliferate and Erk2 and Jun kinase activities were suppressed in these cells. In wild type alphaR/FR cells transiently expressing an SH2 domain mutant of Crk, Erk and Jun kinase activities as well as DNA synthesis were attenuated. Our data indicate that Crk participates in signaling complexes downstream of FGFR-1, which propagate mitogenic signals.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Contribution of Src and Ras pathways in FGF-2 induced endothelial cell differentiation.

We have examined fibroblast growth factor (FGF) receptor-1 mediated signal transduction in differentiation of endothelial cells (EC). The activated FGFR-1 couples to Ras through two adaptor proteins, FRS2 and Shc. In FGF-2 treated proliferating EC, FRS2 as well as Shc are tyrosine phosphorylated and interact with Grb2. In contrast, in FGF-2 treated differentiating cells, Shc, but not FRS2, is engaged in Grb2-interactions. Sustained MAP kinase activity has previously been implicated in differentiation. In FGF stimulated proliferating and differentiating endothelial cells, the MAP kinase Erk2 is activated in a sustained manner. Inhibition of MEK and MAP kinase activity by PD98059 treatment of cells, still allows EC tube formation. The FGFR-1 mediates activation of protein kinase C (PKC) through direct binding and activation of phospholipase C-gamma (PLC-gamma), and has also been shown to activate the cytoplasmic tyrosine kinase Src. Treatment of the cells with the PKC inhibitor bisindolylmaleimide does not prevent tube formation. In contrast, Src kinase activity is a prerequisite for EC differentiation, since treatment of the cells with PP1, a Src family specific inhibitor, abrogates tube formation. In differentiating EC, FGF-2 induces complex formation between Src and focal adhesion kinase (FAK). These data indicate that the Ras pathway is initiated via Shc or FRS2, dependent on the cellular program. Blocking the function of Src family kinases, attenuates differentiation.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Tyrosine phosphatase SHP-2 is involved in regulation of platelet-derived growth factor-induced migration.

SHP-2 is a ubiquitously expressed Src homology-2-containing cytosolic tyrosine phosphatase that binds to and becomes tyrosine-phosphorylated by the activated platelet-derived growth factor receptor-beta (PDGFR-beta). Removal of the binding site on the receptor, by mutation of Tyr1009 to Phe1009 (denoted Y1009F), led to loss of PDGF-stimulated phosphatase activity in cells expressing the mutated receptor, and these cells failed to form membrane edge ruffles and to migrate toward PDGF. Furthermore, treatment with phosphatase inhibitors phenylarsine oxide (PAO) and orthovanadate led to loss of PDGF-stimulated phosphatase activity and attenuated PDGF-stimulated migration of wild type PDGFR-beta cells. Treatment of wild type PDGFR-beta cells with combinations of PAO or orthovanadate and phosphatidylinositol 3-kinase inhibitors wortmannin or LY294002 resulted in a synergistic inhibition of PDGFR-beta-mediated cell migration. PDGF stimulation of wild type PDGFR-beta cells led to induction of p125 focal adhesion kinase (FAK) activity at low concentrations of the growth factor and a decrease at higher concentrations. In the mutant Y1009F cells and in wild type PDGFR-beta cells treated with PAO and orthovanadate, FAK activity was not increased in response to PDGF. These results suggest that SHP-2 activity is involved in regulation of FAK activity and thereby of cell migration through PDGFR-beta, independently of phosphatidylinositol 3-kinase.

Animals↗
PubMed1999

Signaling via fibroblast growth factor receptor-1 is dependent on extracellular matrix in capillary endothelial cell differentiation.

Differentiation of endothelial cells, i.e., formation of a vessel lumen, is a prerequisite for angiogenesis. The underlying molecular mechanisms are ill defined. We have studied a brain capillary endothelial cell line (IBEC) established from H-2Kb-tsA58 transgenic mice. These cells form hollow tubes in three-dimensional type I collagen gels in response to fibroblast growth factor-2 (FGF-2). Culture of IBEC on collagen gels in the presence of FGF-2 protected cells from apoptosis and allowed tube formation (i.e., differentiation) but not growth of the cells. FGF-induced differentiation, but not cell survival, was inhibited by treatment of the cells with an anti-beta1-integrin IgG. Changes in integrin expression in the collagen-gel cultures could not be detected. Rather, cell-matrix interactions critical for endothelial cell differentiation were created during the culture, as indicated by the gradual increase in tyrosine phosphorylation of focal adhesion kinase in the collagen-gel cultures. Inclusion of laminin in the collagen gels led to FGF-2-independent formation of tube structures, but cells were not protected from apoptosis. These data indicate that FGF receptor-1 signal transduction in this cell model results in cell survival. Through mechanisms dependent on cell-matrix interactions, possibly involving the alpha3beta1-integrin and laminin produced by the collagen-cultured IBE cells, FGF stimulation also leads to differentiation of the cells.

Amino Acid Sequence↗
PubMed1999

Signal transduction by fibroblast growth factor receptors.

The fibroblast growth factor family, with its prototype members acidic FGF (FGF-1) and basic FGF (FGF-2), binds to four related receptor tyrosine kinases, expressed on most types of cells in tissue culture. In many respects, the FGF receptors appear similar to other growth factor receptors. Thus, dimerization of receptor monomers upon ligand binding is likely to be a requisite for activation of the kinase domains, leading to receptor trans phosphorylation. FGF receptor-1 (FGFR-1), which shows the broadest expression pattern of the four FGF receptors contains at least seven tyrosine phosphorylation sites. A number of signal transduction molecules are affected by binding with different affinities to these phosphorylation sites. The potential roles of these signal transduction molecules in FGF-induced biological responses and in pathological processes are discussed.

Alternative Splicing↗
PubMed1999

Dual effects of heparin on VEGF binding to VEGF receptor-1 and transduction of biological responses.

Vascular endothelial growth factor (VEGF) regulates blood vessel formation by binding to the receptor tyrosine kinases VEGF receptor-1 (Flt-1) or VEGF receptor-2 (KDR) and to the structurally unrelated neuropilins. As exon 7-containing isoforms of VEGF bind to heparin, angiogenesis may be modulated by heparin/heparan sulfate. We analyzed the effect of heparin on VEGF(165)-binding and activation of VEGF receptor-1 in porcine aortic endothelial cells, which lack expression of VEGF receptor-2 and neuropilins. Heparin decreased binding of (125)I-VEGF to 50% at 5 microg/ml and cross-linking of (125)I-VEGF to VEGF receptor-1 on intact cells was similarly decreased. Schatchard analyses showed that the affinity for binding of (125)I-VEGF to VEGF receptor-1 was decreased in the presence of heparin. In contrast, VEGF receptor-1 kinase activity was elevated when cells were treated simultaneously with VEGF and heparin. In accordance, VEGF-induced tyrosine phosphorylation of phospholipase Cgamma (PLCgamma) and DNA synthesis were augmented by heparin. However, basal PLCgamma tyrosine phosphorylation and DNA synthesis levels were to some extent increased by incubation of cells with heparin alone. We conclude that although heparin decreases binding of VEGF to VEGF receptor-1, the remaining binding results in more efficient kinase activation. Taken together, there is no loss of VEGF/VEGF receptor-1 function in the presence of heparin.

Journal Article↗
PubMed1999

Phosphoinositide 3 kinase is critical for survival, mitogenesis and migration but not for differentiation of endothelial cells.

Angiogenesis involves endothelial cell invasion and migration into the surrounding tissue where cells differentiate, to form new lumen-containing vessels. We have investigated the role of phosphoinositide 3-kinase (PI3-kinase) in vascular endothelial growth factor (VEGF)- and fibroblast growth factor (FGF)-induced angiogenesis. Angiogenesis in vivo in chick embryos was inhibited by treatment with the PI3-kinase inhibitors wortmannin and LY294002. Stimulation of primary bovine capillary endothelial (BCE) cells with FGF-2, VEGF-A(165), or a combination of the two induced PI3-kinase activity in vitro and subsequent activation of the serine/threonine kinase Akt. The combination of FGF-2 and VEGF-A(165) led to an additive response. Activation of PI3-kinase was strictly required for FGF-2- and VEGF-A(165)-induced migration and DNA synthesis of BCE cells. Tubular morphogenesis was unaffected by treatment with wortmannin or LY294002, but survival of the tubular structures was dependent on PI3-kinase activity. VEGF-A(165) and FGF-2 induced increased stability of the tubular structures in a synergistic manner. These data indicate that PI3-kinase activity is required for migration, mitogenicity and survival but not for differentiation of endothelial cells during angiogenesis.

Journal Article↗
PubMed1998

Vascular endothelial growth factor C induces angiogenesis in vivo.

Vascular endothelial growth factor C (VEGF-C) recently has been described to be a relatively specific growth factor for the lymphatic vascular system. Here we report that ectopic application of recombinant VEGF-C also has potent angiogenic effects in vivo. VEGF-C is sufficiently potent to stimulate neovascularization from limbal vessels in the mouse cornea. Similar to VEGF, the angiogenic response of corneas induced by VEGF-C is intensive, with a high density of new capillaries. However, the outgrowth of microvessels stimulated by VEGF-C was significantly longer than that induced by VEGF. In the developing embryo, VEGF-C was able to induce branch sprouts from the established blood vessels. VEGF-C also induced an elongated, spindle-like cell shape change and actin reorganization in both VEGF receptor (VEGFR)-2 and VEGFR-3-overexpressing endothelial cells, but not in VEGFR-1-expressing cells. Further, both VEGFR-2 and VEGFR-3 could mediate proliferative and chemotactic responses in endothelial cells on VEGF-C stimulation. Thus, VEGF-C may regulate physiological angiogenesis and participate in the development and progression of angiogenic diseases in addition to lymphangiogenesis.

Allantois↗
PubMed1998

Identification of vascular endothelial growth factor receptor-1 tyrosine phosphorylation sites and binding of SH2 domain-containing molecules.

Receptor tyrosine phosphorylation is crucial for signal transduction by creating high affinity binding sites for Src homology 2 domain-containing molecules. By expressing the intracellular domain of Flt-1/vascular endothelial growth factor receptor-1 in the baculosystem, we identified two major tyrosine phosphorylation sites at Tyr-1213 and Tyr-1242 and two minor tyrosine phosphorylation sites at Tyr-1327 and Tyr-1333 in this receptor. This pattern of phosphorylation of Flt-1 was also detected in vascular endothelial growth factor-stimulated cells expressing intact Flt-1. In vitro protein binding studies using synthetic peptides and immunoblotting showed that phospholipase C-gamma binds to both Y(p)1213 and Y(p)1333, whereas Grb2 and SH2-containing tyrosine protein phosphatase (SHP-2) bind to Y(p)1213, and Nck and Crk bind to Y(p)1333 in a phosphotyrosine-dependent manner. In addition, unidentified proteins with molecular masses around 74 and 27 kDa bound to Y(p)1213 and another of 75 kDa bound to Y(p)1333 in a phosphotyrosine-dependent manner. SHP-2, phospholipase C-gamma, and Grb2 could also be shown to bind to the intact Flt-1 intracellular domain. These results indicate that a spectrum of already known as well as novel phosphotyrosine-binding molecules are involved in signal transduction by Flt-1.

3T3 Cells↗
PubMed1998

2'-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain.

Vascular endothelial growth factor (VEGF) has been implicated in the pathological induction of new blood vessel growth in a variety of proliferative disorders. Using the SELEX process (systematic evolution of ligands by exponential enrichment), we have isolated 2'-F-pyrimidine RNA oligonucleotide ligands (aptamers) to human VEGF165. Representative aptamers from three distinct sequence families were truncated to the minimal sequence capable of high affinity binding to VEGF (23-29 nucleotides) and were further modified by replacement of 2'-O-methyl for 2'-OH at all ribopurine positions where the substitution was tolerated. Equilibrium dissociation constants for the interaction of VEGF with the truncated, 2'-O-methyl-modified aptamers range between 49 and 130 pM. These aptamers bind equally well to murine VEGF164, do not bind to VEGF121 or the smaller isoform of placenta growth factor (PlGF129), and show reduced, but significant affinity for the VEGF165/PlGF129 heterodimer. Cysteine 137 in the exon 7-encoded domain of VEGF165 forms a photo-inducible cross-link to a single uridine residue in each of the three aptamers. The aptamers potently inhibit the binding of VEGF to the human VEGF receptors, KDR and Flt-1, expressed by transfected porcine aortic endothelial cells. Furthermore, one of the aptamers is able to significantly reduce intradermal VEGF-induced vascular permeability in vivo.

Amino Acid Sequence↗
PubMed1998

PDGF induces reorganization of vimentin filaments.

In this study we demonstrate that stimulation with platelet-derived growth factor (PDGF) leads to a marked reorganization of the vimentin filaments in porcine aortic endothelial (PAE) cells ectopically expressing the PDGF beta-receptor. Within 20 minutes after stimulation, the well-spread fine fibrillar vimentin was reorganized as the filaments aggregated into a dense coil around the nucleus. The solubility of vimentin upon Nonidet-P40-extraction of cells decreased considerably after PDGF stimulation, indicating that PDGF caused a redistribution of vimentin to a less soluble compartment. In addition, an increased tyrosine phosphorylation of vimentin was observed. The redistribution of vimentin was not a direct consequence of its tyrosine phosphorylation, since treatment of cells with an inhibitor for the cytoplasmic tyrosine kinase Src, attenuated phosphorylation but not redistribution of vimentin. These changes in the distribution of vimentin occurred in conjunction with reorganization of actin filaments. In PAE cells expressing a Y740/751F mutant receptor that is unable to bind and activate phosphatidylinositol 3'-kinase (PI3-kinase), the distribution of vimentin was virtually unaffected by PDGF stimulation. Thus, PI3-kinase is important for vimentin reorganization, in addition to its previously demonstrated role in actin reorganization. The small GTPase Rac has previously been shown to be involved downstream of PI3-kinase in the reorganization of actin filaments. In PAE cells overexpressing dominant negative Rac1 (N17Rac1), no change in the fine fibrillar vimentin network was seen after PDGF-BB stimulation, whereas in PAE cells overexpressing constitutively active Rac1 (V12Rac1), there was a dramatic change in vimentin filament organization independent of PDGF stimulation. These data indicate that PDGF causes a reorganization of microfilaments as well as intermediate filaments in its target cells and suggest an important role for Rac downstream of PI3-kinase in the PDGF stimulated reorganization of both actin and vimentin filaments.

Actin Cytoskeleton↗
PubMed1998

Fibroblast growth factor receptor-1 mediates chemotaxis independently of direct SH2-domain protein binding.

Endothelial cells expressing fibroblast growth factor receptor-1 (FGFR-1) migrate and proliferate in response to treatment with FGF. We analysed ligand-induced migration and proliferation of porcine aortic endothelial cells expressing wild-type FGFR-1, point-mutated Y766F FGFR-1, unable to activate phospholipase C-gamma1 (PLC-gamma1), or carboxyl-terminally truncated FGFR-1, lacking either 48 (from amino acid 774 in the FGFR-1 sequence) or 63 (from amino acid 759) amino acid residues of the C-terminal tail. The truncated CT63 FGFR-1 mutant failed to mediate chemotaxis, but was in response to ligand stimulation capable of mediating proliferation of the cells, stimulation of MAP kinase activity and tyrosine phosphorylation of FRS2, an FGFR-1 specific signaling molecule. The defect in migration-capacity of CT63 was not due to loss of Y766, and thereby PLC-gamma1 activation, since cells expressing the mutant Y766F FGFR-1 migrated as efficiently as the wild-type receptor cells. Induction of phospholipase A2 (PLA2) activity by the activated FGFR-1 was dependent on the presence of Y766, and was therefore also not critical for the chemotactic response. Although the FGFR-1 only very inefficiently mediates activation of phosphatidylinositol 3' kinase (PI 3-kinase), the PI 3-kinase inhibitor wortmannin suppressed wild-type FGFR-1 mediated migration. We conclude that the signal transduction pathway for FGFR-1 mediated migration is independent of phosphotyrosine residues in the receptor and requires activation of a wortmannin-sensitive enzyme.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Angiostatin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif RGD.

Angiostatin, a fragment of plasminogen, has been identified and characterized as an endogenous inhibitor of neovascularization. We show that angiostatin treatment of endothelial cells in the absence of growth factors results in an increased apoptotic index whereas the proliferation index is unchanged. Angiostatin also inhibits migration and tube formation of endothelial cells. Angiostatin treatment has no effect on growth factor-induced signal transduction but leads to an RGD-independent induction of the kinase activity of focal adhesion kinase, suggesting that the biological effects of angiostatin relate to subversion of adhesion plaque formation in endothelial cells.

Angiostatins↗
PubMed1998

Activation of Stat5 by platelet-derived growth factor (PDGF) is dependent on phosphorylation sites in PDGF beta-receptor juxtamembrane and kinase insert domains.

Signal transducers and activators of transcription (Stats) are known to transduce signals from the cell surface to the nucleus in cytokine receptor signaling. We examined the capacity of platelet-derived growth factor (PDGF) receptor to interact with and activate Stat molecules. Activation of the PDGF beta-receptor led to tyrosine phosphorylation of Stat1, Stat3 and Stat5, which was accompanied by specific DNA-binding activities. These events were only weakly stimulated by the activated PDGF alpha-receptor. In cells expressing PDGF beta-receptors mutated at Tyr579, Tyr581 or Tyr775, tyrosine phosphorylation as well as DNA-binding activity of Stat5 was reduced. Immobilized peptides containing phosphorylated Tyr579, Tyr581 or Tyr775 bound Stat5, suggesting direct binding of Stat5 to these tyrosine residues of the PDGF beta-receptor. Members of the Janus kinase family were also shown to interact with the PDGF beta-receptor, and to a lesser extent with the alpha-receptor, but their importance for PDGF-induced Stat activation remains to be determined.

3T3 Cells↗
PubMed1998

Placenta growth factor stimulates MAP kinase and mitogenicity but not phospholipase C-gamma and migration of endothelial cells expressing Flt 1.

Vascular endothelial growth factor (VEGF) and placenta growth factor (PIGF) are structurally related growth factors for endothelial cells. VEGF binds to the related receptor tyrosine kinases Flt 1 and KDR/Flk 1 with high affinity, whereas PlGF binds only to Flt 1. Ligand-stimulated KDR is known to transduce signals for cellular activity such as proliferation and migration, whereas weak or no responses have been recorded for Flt 1. We examined VEGF and PlGF for their capacity to stimulate signal transduction in porcine aortic endothelial cells expressing Flt 1 or KDR. VEGF had essentially no effect on Flt 1 expressing cells, but induced DNA synthesis and migration of KDR expressing cells. PIGF on the other hand induced DNA synthesis but not migration of the Flt 1 cells. In agreement, MAP kinase, examined as a marker for DNA synthesis, was activated both by VEGF-stimulation of the KDR cells and by PlGF-stimulation of the Flt 1 cells. In contrast, phospholipase C-gamma (PLC-gamma), was tyrosine phosphorylated only in VEGF stimulated KDR cells, and not in the PlGF-stimulated Flt 1 cells, which is in agreement with a role for PLC-gamma in cellular migration. We furthermore examined induction of protein levels of plasminogen activator (PA), which was evident in the PlGF-stimulated Flt 1 cells, but not in the VEGF-stimulated KDR cells. These data show that Flt 1 is able to mediate an array of biological signals when appropriately stimulated and that the pattern of responses of PlGF-stimulation of Flt 1 is distinct from the pattern of responses to VEGF-stimulation of KDR.

Animals↗
PubMed1998

Platelet-derived growth factor (PDGF)-induced actin rearrangement is deregulated in cells expressing a mutant Y778F PDGF beta-receptor.

Platelet-derived growth factor-stimulated actin rearrangement and edge ruffle formation have previously been shown to be dependent on activation of phosphatidylinositol 3'-kinase, the activity of which also is important for directed migration of cells. This lipid kinase binds to phosphorylated tyrosine residues Y740 and Y751 in the kinase insert of the human platelet-derived growth factor ss-receptor. We examined the role of two other tyrosine residues in the kinase insert of this receptor, Y775 and Y778, for ligand-induced actin rearrangement. Both were shown to be phosphorylation sites; Y775 was only marginally phosphorylated in cells expressing the wild-type ss-receptor, whereas Y778 was phosphorylated at higher stoichiometry. Mutant receptors Y775F, Y778F and Y775/778F were active kinases and mediated proliferative responses when expressed in porcine aortic endothelial cells. Fluorescence staining of actin in platelet-derived growth factor-stimulated PAE cells revealed that Y778 is involved in regulation of the actin cytoskeleton since the cells contained, apart from edge ruffles and circular ruffles, a novel type of giant ruffle on the dorsal side of the cell, which consisted of irregular multilayered actin structures. Mutation at Y778 had no effect on activation of phosphatidylinositol 3'-kinase, nor on the GTPase activating protein of Ras and phospholipase C(gamma), and the extent of directed migration towards platelet-derived growth factor of these cells was not changed. We conclude that actin rearrangement is regulated in part by Y778 in the platelet-derived growth factor ss-receptor, potentially through binding of a novel signaling molecule to this site.

Actins↗