IBE Cells Research Articles

Fibroblast growth factor-2 induces the activation of Src through Fes, which regulates focal adhesion disassembly

Cell migration is regulated by focal adhesion (FA) turnover. Fibroblast growth factor-2 (FGF-2) induces FA disassembly in the murine brain capillary endothelial cell line IBE, leading to FGF-2-directed chemotaxis. We previously showed that activation of Src and Fes by FGF-2 was involved in chemotaxis of IBE cells. In this study, we examined the interplay between Src and Fes. FGF-2 treatment decreased the number of FA in IBE cells, but not in cells expressing dominant-negative Fes (denoted KE5-15 cells). FGF-2 induced the activation of Src and subsequent binding to and phosphorylation of Cas in IBE cells, but not in KE5-15 cells. Focal adhesion kinase (FAK) activation and tyrosine phosphorylation by Src were also delayed in KE5-15 cells compared to parental cells. FGF-2 induced activation of Src within FA in IBE cells, but not in KE5-15 cells. Downregulation of Fes or FAK using small interfering RNA diminished Src activation by FGF-2 within FA. These findings suggest that activation of Fes by FGF-2 enhances FAK-dependent activation of Src within FA, promoting FGF-2-induced disassembly of focal adhesions.

Angiopoietin 1 Is Mitogenic for Cultured Endothelial Cells

The angiopoietin (Ang)/Tie2 system is implicated in blood vessel formation and maturation. However, the mitogenic effects of angiopoietins remain to be elucidated. Here, we show that Ang1 is mitogenic for cultured endothelial cells. Ang1 dose-dependently induced the proliferation and increased the labeling index of a murine brain capillary endothelial cell line, IBE cells. Ang1 also increased the labeling index of human umbilical vein endothelial cells (HUVEC). Ang1 up-regulated the expression of cyclin D1 in both of these cells. Ang1 activated mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) in IBE cells and HUVECs. Activated PI3K was associated with c-Fes protein tyrosine kinase in these cells, but not with Tie2. p70 S6 kinase (p70 S6K) was activated by Ang1-treatment, although this activation was blocked by a PI3K inhibitor, LY294002. Simultaneous treatment of cells with PD98059 (MAPK/extracellular regulated kinase kinase inhibitor) and rapamycin (mTOR inhibitor) completely blocked Ang1-induced mitogenic activity for IBE cells and HUVECs. Although Ang2 at high concentration weakly activated Tie2 and p70 S6K, it failed to activate Ras and MAPK, or to induce cell proliferation. Taken together, these findings indicate that Ang1 exerts mitogenic activity on endothelial cells, which requires activation of both MAPK and p70 S6K.

Pigment epithelium-derived factor inhibits fibroblast-growth-factor-2-induced capillary morphogenesis of endothelial cells through Fyn

Pigment epithelium-derived factor (PEDF) exerts anti-angiogenic actions. However, the signal-transduction pathways regulated by PEDF remain to be elucidated. We show here that PEDF inhibited fibroblast growth factor 2 (FGF-2) induced capillary morphogenesis of a murine brain capillary endothelial cell line (IBE cells) and of human umbilical-vein endothelial cells (HUVECs) cultured on growth-factor-reduced Matrigel. We previously showed that FGF-2-mediated capillary morphogenesis was blocked by the Src-kinase inhibitor PP2 and that expression of dominant negative Fyn in IBE cells inhibited capillary morphogenesis. We examined the effect of PEDF on kinase activity of Fyn and found that PEDF downregulated FGF-2-promoted Fyn activity by tyrosine phosphorylation at the C-terminus in a Fes-dependent manner. In a stable IBE cell line expressing kinase-inactive Fes (KE5-15 Fes cells), PEDF failed to inhibit FGF-2-induced capillary morphogenesis or Fyn activity. PEDF induced the colocalization of Fyn and Fes in IBE cells expressing wild-type Fes, but not in KE5-15 Fes cells. In addition, wild-type Fes increased the tyrosine phosphorylation of Fyn in vitro, suggesting that Fes might directly phosphorylate Fyn. Expression of constitutively active Fyn (Y531F) in IBE cells exhibited capillary morphogenesis in the absence of FGF-2 and was resistant for PEDF treatment. Our results suggest that PEDF downregulates Fyn through Fes, resulting in inhibition of FGF-2-induced capillary morphogenesis of endothelial cells.

Effect of low adapted temperature and medium composition on growth and erythropoietin (EPO) production by Chinese hamster ovary cells

Temperature and medium composition were changed with the aim of increasing growth and erythropoietin (EPO) production in EPO-producing Chinese hamster ovary (CHO) cells. We used the CHO cell line, IBE, and its derivative, CO5, which over-expresses the first two enzymes of the urea cycle, carbamoyl phosphate synthetase I (CPS I) and ornithine transcarbamoylase (OTC). When supplements were added to the medium at 33 degrees C, the growth of IBE and CO5 cells increased by 27% and 26%, respectively and the maximum yield of EPO was increased by 40% in both cell lines. The absolute EPO concentration in the CO5 cells was always 55-60% higher than in the IBE cells. In addition, when the two cell lines were continuously cultured with supplements at 33 degrees C until their growth rates approached those at 37 degrees C, the growth rates of both IBE and CO5 cells increased by 54% and their maximum EPO levels increased by up to 73% and 56%, respectively. Therefore, the growth and EPO expression levels of CO5 cells increased 2.2-fold and 2.6-fold, respectively, compared to those of the IBE cells. These results indicate that adaptation to lower temperature as well as medium supplementation could be important for improving cell growth and EPO production.

T‐cell factor‐4‐dependent up‐regulation of fibronectin is involved in fibroblast growth factor‐2‐induced tube formation by endothelial cells

Deletion of fibronectin or its receptor, alpha(5) integrin, interferes with the formation of a functional circulation in mice. We hypothesized that alpha(5)beta(1) integrin/fibronectin interaction may be involved in differentiation of endothelial cells during angiogenesis. We examined the effect of blocking antibody against alpha(5)beta(1) integrin in fibroblast growth factor-2 (FGF-2)-induced angiogenesis by Matrigel plug assay. Although the antibody did not inhibit the recruitment of endothelial cells into plugs, it inhibited organization of lumen-containing capillaries. The antibody also inhibited FGF-2-induced tube formation by murine brain capillary endothelial cells (IBE cells) cultured in type I collagen gels. We previously showed that FGF-2 failed to induce tube formation by IBE cells expressing kinase-dead c-Fyn (KDFyn cells). Association with beta-catenin enhances the transcriptional activity of T-cell factor-4 (TCF-4), which up-regulates the expression of fibronectin. FGF-2 induced association of beta-catenin with TCF-4 and up-regulation of fibronectin in IBE cells, but not in KDFyn cells. Expression of mutant TCF-4, which does not associate with beta-catenin, inhibited FGF-2-induced tube formation and expression of fibronectin in IBE cells. FGF-2-induced tyrosine phosphorylation of beta-catenin, and association with TCF-4 was increased in IBE cells, but not in KDFyn cells. Taken together, interaction of alpha(5)beta(1) integrin and fibronectin is involved in FGF-2-induced tube formation by endothelial cells and up-regulation of fibronectin through TCF-4 seemed to be involved in this process.

Role of focal adhesion formation in migration and morphogenesis of endothelial cells

Cell motility and morphogenesis are regulated by a balance between formation and disassembly of stress fibers and focal adhesions. To understand the mechanisms underlying these cellular responses in angiogenesis, we studied the Rho family protein-driven pathways in FGF-2-induced chemotaxis and capillary morphogenesis of murine brain capillary endothelial cell line, IBE cells. Cells seeded onto fibronectin-coated surface migrated toward FGF-2. Expression of dominant negative Rho A (DNRho) or kinase-dead p21-activated kinase 1 (KDPAK1), or treatment with Y27632 inhibited chemotaxis in association with the lack of FGF-2-induced decrease in focal adhesions. On Matrigel, DNRho and Y27632 induced FGF-2-independent capillary morphogenesis despite loss of stress fiber formation. KDPAK1 cells formed stress fibers and showed capillary morphogenesis in response to FGF-2. Increase in focal adhesions was closely associated with capillary morphogenesis. Our results suggest that formation or disassembly of focal adhesions seems to determine the motility or morphogenesis of endothelial cells.

Fibroblast Growth Factor-2-mediated Capillary Morphogenesis of Endothelial Cells Requires Signals via Flt-1/Vascular Endothelial Growth Factor Receptor-1: POSSIBLE INVOLVEMENT OF c-Akt

Capillary morphogenesis is a crucial angiogenic response of endothelial cells. Although fibroblast growth factor-2 (FGF-2) potently induces capillary morphogenesis, the contribution of vascular endothelial growth factor-A (VEGF-A) in this response has not been clarified well. Here we examined the role of VEGF signaling in FGF-2-induced capillary morphogenesis by murine brain capillary endothelial cells (IBE cells) and human umbilical vein endothelial cells. FGF-2-treated IBE cells rapidly extended on Matrigel in association with actin reorganization. Chimeric protein, of which the extracellular domain of VEGF receptor-1 (VEGFR-1) fused to immunoglobulin Fc, inhibited FGF-2-induced cell extension, resulting in decreased capillary morphogenesis. Blocking antibody against VEGFR-1 inhibited FGF-2-induced capillary formation. Also, anti-VEGF-A antibody inhibited FGF-2-induced capillary morphogenesis, which was restored by the addition of placental growth factor-1. Similar results were obtained by the experiments with human umbilical vein endothelial cells. Expression of kinase-inactive c-Akt in IBE cells showed impaired capillary morphogenesis promoted by FGF-2. Conversely, stable cell lines expressing activated c-Akt demonstrated ligand-independent capillaries, which were resistant to the treatment with anti-VEGFR-1 blocking antibody. Upstream of c-Akt, calmodulin-dependent signals seemed to be involved. Taken together, signals via VEGFR-1 were required for FGF-2-induced capillary morphogenesis by endothelial cells, and c-Akt activity seemed to be involved in this process.

Sonic Hedgehog Induces Capillary Morphogenesis by Endothelial Cells through Phosphoinositide 3-Kinase

Sonic hedgehog (Shh) acts as a morphogen in many cell types. Recent studies have shown that hedgehog signaling is involved in vascular development as well as postnatal angiogenesis. However, the direct action of Shh on cultured endothelial cells has not been clearly shown. To address this issue, we examined the effect of Shh on morphological changes by murine brain capillary endothelial cells (IBE cells) and human umbilical endothelial cells (HUVECs). Shh induced capillary morphogenesis by these cells. The effect was inhibited by cyclopamine or pertussis toxin. Shh-induced capillary morphogenesis was also blocked by LY294002, a phosphoinositide 3-kinase (PI3-kinase) inhibitor. Shh rapidly increased PI3-kinase activity in IBE cells and HUVECs; this activity was inhibited by cyclopamine. Nuclear localization of Gli1 was increased in Shh-treated IBE cells, which was not affected by LY294002. Actinomycin D and cycloheximide inhibited Shh-induced capillary morphogenesis. In IBE cells expressing kinase-inactive c-Fes, Shh failed to stimulate PI3-kinase activity and capillary morphogenesis. Considered collectively, Shh induced capillary morphogenesis of endothelial cells through both rapid activation of c-Fes/PI3-kinase pathways and transcriptionally regulated pathways.

Negative regulation of urokinase-type plasminogen activator production through FGF-2-mediated activation of phosphoinositide 3-kinase.

Activation of phosphoinositide 3-kinase (PI3-kinase) is involved in many cellular responses. FGF-2 is one of the potent inducers of urokinase-type plasminogen activator (uPA) production in endothelial cells. However, little is known about the molecular mechanisms underlying FGF-2-mediated uPA production. Here we examined the signal transduction pathways involved in the regulation of uPA production by FGF-2-treatment. FGF-2 potently upregulated uPA production in murine brain capillary endothelial cells (IBE cells), as well as porcine aortic endothelial (PAE) cells and L6 myoblasts ectopically expressing FGFR1. PI3-kinase inhibitors, wortmannin and LY294002, both enhanced FGF-2-dependent uPA production by these cells. Stable expression of activated mutant p110alpha catalytic subunit of PI3-kinase into IBE cells decreased FGF-2-mediated uPA production, suggesting that PI3-kinase exhibited the negative regulatory effect on uPA production. No increase in FGF-2-induced PI3-kinase activity was observed in proteins immunoprecipitated by anti-phosphotyrosine antibody. Although stable expression of deleted mutant p85alpha regulatory subunit, which lacks association with p110 catalytic subunit, in IBE cells showed no dominant negative effect, transient expression of dominant negative Ras inhibited FGF-2-mediated PI3-kinase activation. These results suggest that only activated Ras contributed the FGF-2-mediated PI3-kinase activation. In cells stably expressing mutant p85alpha subunit, FGF-2 efficiently induced uPA production. Taken together, activation of PI3-kinase by FGF-2 is Ras-dependent and results in down-regulation of uPA production.

Effects of vitamin D(3)-binding protein-derived macrophage activating factor (GcMAF) on angiogenesis.

The vitamin D(3)-binding protein (Gc protein)-derived macrophage activating factor (GcMAF) activates tumoricidal macrophages against a variety of cancers indiscriminately. We investigated whether GcMAF also acts as an antiangiogenic factor on endothelial cells. The effects of GcMAF on angiogenic growth factor-induced cell proliferation, chemotaxis, and tube formation were examined in vitro by using cultured endothelial cells (murine IBE cells, porcine PAE cells, and human umbilical vein endothelial cells [HUVECs]) and in vivo by using a mouse cornea micropocket assay. Blocking monoclonal antibodies to CD36, a receptor for the antiangiogenic factor thrombospondin-1, which is also a possible receptor for GcMAF, were used to investigate the mechanism of GcMAF action. GcMAF inhibited the endothelial cell proliferation, chemotaxis, and tube formation that were all stimulated by fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor-A, or angiopoietin 2. FGF-2-induced neovascularization in murine cornea was also inhibited by GcMAF. Monoclonal antibodies against murine and human CD36 receptor blocked the antiangiogenic action of GcMAF on the angiogenic factor stimulation of endothelial cell chemotaxis. In addition to its ability to activate tumoricidal macrophages, GcMAF has direct antiangiogenic effects on endothelial cells independent of tissue origin. The antiangiogenic effects of GcMAF may be mediated through the CD36 receptor.

Role of c-Fyn in FGF-2-Mediated Tube-like Structure Formation by Murine Brain Capillary Endothelial Cells

Tube formation of endothelial cells is an important step of angiogenesis. However, little is known about the molecular mechanisms underlying growth factor-mediated tube formation by endothelial cells. FGF-2 stimulates tube formation by a murine brain capillary endothelial cell line, IBE cells, when cultured on collagen gels (differentiation-associated culture condition), whereas cells proliferate and migrate without forming tube on fibronectin-coated surface (proliferation/migration-associated condition). To elucidate FGF-2-mediated signal transduction pathways leading to tube formation by endothelial cells, we focused on the contribution of Src family kinases. Src family kinase inhibitor PP2 attenuated FGF-2-induced tube formation. Stable expression of kinase-inactive c-Src in IBE cells demonstrated no dominant negative effect on FGF-2-induced tube formation. In vitro kinase assay revealed that c-Fyn was activated by FGF-2 only in cells cultured on collagen gels. Three independent cell lines, expressing kinase-inactive c-Fyn, all exhibited attenuation of FGF-2-mediated tube formation. However, FGF-2-mediated proliferation or migration was not clearly perturbed in these cells. These results show the first time that c-Fyn plays a pivotal role in tube formation by endothelial cells.

Angiopoietin 2 stimulates migration and tube-like structure formation of murine brain capillary endothelial cells through c-Fes and c-Fyn.

The angiopoietin (Ang)/Tie2 system is exclusively involved in vasculogenesis and angiogenesis. Ang2 is known to inhibit Ang1-mediated phosphorylation of Tie2 as well as cellular responses during embryonic development. Recent studies have demonstrated that Ang2 has angiogenic activities in adult tissues and cultured endothelial cells. In the present study, we examined the downstream signaling pathways involved in Ang2-mediated cellular responses by murine brain capillary cell line, IBE cells. Tie2 was tyrosine phoshorylated by Ang2. Ang2 showed no effect on proliferation, but stimulated chemotaxis and tube-like structure formation. Phosphoinositide 3-kinase (PI 3-kinase) was activated by Ang2 through c-Fes and was involved in chemotaxis toward Ang2. Ang2 also activated c-Fyn in IBE cells. Cells expressing kinase-inactive c-Fyn attenuated Ang2-induced tube formation, suggesting that c-Fyn was responsible for Ang-2-mediated tube formation. Collecting these data, Ang2 activates c-Fes and c-Fyn, leading to migration and tube formation by murine capillary endothelial cells.

Signals via FGF Receptor 2 Regulate Migration of Endothelial Cells

Fibroblast growth factors (FGFs) stimulate angiogenesis, of which signals are transduced via FGF receptor (FGFR) tyrosine kinases. Although FGFR1 is a major receptor in endothelial cells, FGFR2 is frequently detectable in endothelial cells. We have previously demonstrated that the intracellular domain of FGFR1 sufficiently transduced signals leading to proliferation, migration, urokinase secretion, and tube formation. However, little is known about the roles of signaling via FGFR2 alone in endothelial cells. Murine brain capillary endothelial cells, denoted IBE cells, express small amounts of IIIc FGFR2, which is not activated by keratinocyte growth factor (KGF). We then transfected the IIIb FGFR2 in these cells. Three stable cell lines expressing IIIb FGFR2 demonstrated chemotaxis toward KGF, but never proliferated, secreted urokinase, or formed tube-like structure by KGF treatment. Weak but sustained activation of mitogen-activated protein kinase (MAPK) was observed in these cells. Chemotaxis toward KGF was significantly attenuated by treatment with PD98059. This is the first demonstration that signaling solely via FGFR2 in endothelial cells only contributes to motility through MAPK.

Inhibition of FGF-2-Mediated Chemotaxis of Murine Brain Capillary Endothelial Cells by Cyclic RGDfV Peptide through Blocking the Redistribution of c-Src into Focal Adhesions

The αvβ3 integrin is essential for fibroblast growth factor (FGF)-induced angiogenesis in vivo. However, the role of this integrin in FGF-2-mediated cellular responses by cultured endothelial cells is largely unknown. Cyclic RGDfV (cRGDfV) peptide is widely used to inhibit the binding of αvβ3 integrin to vitronectin. To investigate the role of this integrin in FGF-2-mediated cellular responses, we used immortalized murine brain capillary endothelial cells, denoted IBE cells. Because IBE cells proliferate and migrate in response to FGF-2-treatment, when cultured on fibronectin-coated surface, we first examined the inhibitory activity of this peptide on the binding of αvβ3 integrin to fibronectin as well as vitronectin. Solid phase binding assay revealed that cRGDfV peptide strongly inhibited the binding of purified αvβ3 integrin to vitonectin- and fibronectin-coated plastic surfaces at a concentration of 50 μM. cRGDfV peptide at 50 μM inhibited spreading as well as adhesion of IBE cells on vitronectin-coated plastic surface but not on fibronectin. On fibronectin-coated substrata, cRGDfV at 50 μM attenuated FGF-2-mediated chemotaxis, but not FGF-2-induced proliferation, of IBE cells. We have previously demonstrated that mitogen-activated protein kinase (MAPK) activation within focal adhesions through c-Src activity was involved in FGF-2-induced chemotaxis of IBE cells. Treatment of cells with cRGDfV peptide was associated with reduced c-Src activity without tyrosine dephosphorylation. Immunofluorescent staining showed that cRGDfV inhibited redistribution of c-Src into focal adhesions. MAPK activation by FGF-2 within focal adhesions was also attenuated in the presence of cRGDfV peptide. Our results indicated that cRGDfV peptide inhibited redistribution of c-Src into focal adhesions, leading to impaired MAPK activation within focal adhesions and motility in FGF-2-treated endothelial cells.

The Role of Mitogen-Activated Protein Kinase Activation within Focal Adhesions in Chemotaxis toward FGF-2 by Murine Brain Capillary Endothelial Cells

Fibroblast growth factors (FGFs) regulate a number of angiogenic cellular responses such as migration of endothelial cells. To examine the role of mitogen-activated protein kinase (MAPK) in endothelial cell migration, chemotaxis toward FGF-2 was determined in murine brain capillary endothelial cells, denoted IBE cells. PD98059, a specific inhibitor for MAPK/Erk kinase, inhibited FGF-2-induced chemotaxis of IBE cells. It has been reported that c-Src tyrosine kinase phosphorylates focal adhesion kinase at tyrosine 925 within focal adhesions, which in turn creates the binding site for Grb2, leading to MAPK activation. The Src family tyrosine kinase inhibitor, PP1, as well as overexpression of kinase-inactive c-Src, attenuated chemotaxis toward FGF-2. To investigate the signaling events involved in FGF-2-induced chemotaxis, MAPK activation was monitored in IBE cells by indirect immunofluorescence staining. Activated MAPK was initially observed in the cytoplasm and gradually moved into nuclei. A fraction of MAPK was activated by FGF-2 within focal adhesions, where FGF receptor-1 and Src family kinases were also colocalized. MAPK activation within focal adhesions was remarkably decreased in kinase-inactive c-Src-expressing IBE cells. Our data suggest that activation of MAPK by FGF-2 within focal adhesions may depend on c-Src activity and is crucial for FGF-2-induced migration of IBE cells.

Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis

AbstractEndostatin, which corresponds to the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis. Fibroblast growth factor-2 (FGF-2)–induced angiogenesis in the chicken chorioallantoic membrane was inhibited by endostatin, but not by an endostatin mutant R158/270A, lacking heparin-binding ability. Endostatin was internalized by endothelial cells, but not by mouse fibroblasts. Treatment of murine brain endothelial (IBE) cells with endostatin reduced the proportion of cells in S phase, whereas growth-arrested IBE cells in collagen gels treated with endostatin displayed enhanced tubular morphogenesis. IBE cells overexpressing Shb, an adaptor protein implicated in angiostatin-induced apoptosis, displayed elevated apoptosis and decreased tubular morphogenesis in collagen gels in response to endostatin when added together with FGF-2. Induction of apoptosis was dependent on the heparin-binding ability of endostatin and the expression of Shb with a functional Src homology 2 (SH2)-domain. Endostatin treatment for 10 minutes or 24 hours induced tyrosine phosphorylation of Shb and formation of multiprotein complexes. An Shb SH2 domain fusion protein precipitated a 125-kd phosphotyrosyl protein in endostatin-treated cells. The 125-kd component either contained intrinsic tyrosine kinase activity or occurred in complex with a tyrosine kinase. In conclusion, our data show that endostatin induces tyrosine kinase activity and enhanced apoptosis in FGF-treated endothelial cells.

Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis

Endostatin, which corresponds to the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis. Fibroblast growth factor-2 (FGF-2)–induced angiogenesis in the chicken chorioallantoic membrane was inhibited by endostatin, but not by an endostatin mutant R158/270A, lacking heparin-binding ability. Endostatin was internalized by endothelial cells, but not by mouse fibroblasts. Treatment of murine brain endothelial (IBE) cells with endostatin reduced the proportion of cells in S phase, whereas growth-arrested IBE cells in collagen gels treated with endostatin displayed enhanced tubular morphogenesis. IBE cells overexpressing Shb, an adaptor protein implicated in angiostatin-induced apoptosis, displayed elevated apoptosis and decreased tubular morphogenesis in collagen gels in response to endostatin when added together with FGF-2. Induction of apoptosis was dependent on the heparin-binding ability of endostatin and the expression of Shb with a functional Src homology 2 (SH2)-domain. Endostatin treatment for 10 minutes or 24 hours induced tyrosine phosphorylation of Shb and formation of multiprotein complexes. An Shb SH2 domain fusion protein precipitated a 125-kd phosphotyrosyl protein in endostatin-treated cells. The 125-kd component either contained intrinsic tyrosine kinase activity or occurred in complex with a tyrosine kinase. In conclusion, our data show that endostatin induces tyrosine kinase activity and enhanced apoptosis in FGF-treated endothelial cells.

Constitutive expression of the DNA-binding domain of Ets1 increases endothelial cell adhesion and stimulates their organization into capillary-like structures.

We previously reported that the Ets1 transcription factor is expressed in endothelial cells during angiogenesis both in normal and pathological development. We analyse here the effects of the stable expression of an Ets transdominant negative mutant (Ets1-DB), consisting in an Ets1 protein lacking its transactivation domain. A retrovirus containing the Ets1-DB sequence fused to an IRES-Neo sequence was designed and used to infect brain capillary (IBE) and aorta (MAE) mouse endothelial cell lines. Cells expressing this Ets1 mutant were examined for proliferation, migration and adhesion. Consistent changes were observed on cell morphology, with increased spreading and modifications in the organization of the cytoskeleton, and increased cell adhesion. We investigated the ability of endothelial cells to organise into capillary-like structures using three-dimensional gels. On Matrigel, all endothelial cell lines formed a cord-like network within 24 h, with an increased ability of Ets1-DB cells to spread on this substrate. In long term cultures, IBE cells expressing Ets1-DB showed a higher capacity to form branched structures; this effect was potentiated by FGF2. These results demonstrate a role of the Ets transcription factors in the regulation of the adhesive and morphogenetic properties of endothelial cells.

Role of Thrombospondin-1-Derived Peptide, 4N1K, in FGF-2-Induced Angiogenesis

Angiogenesis involves proliferation of capillary endothelial cells and formation of lumen-containing tube-like structures. A recently established murine brain capillary endothelial cell line, IBE, can either proliferate or form tube-like structures (i.e., differentiate) in response to fibroblast growth factor-2 (FGF-2), dependent on the culture conditions. The 4N1K peptide (KRFYVVMWKK), which is derived from the C-terminal cell-binding domain of thrombospondin-1 (TSP-1), inhibited tube formation, but not proliferation of IBE cells. Polyclonal antibodies against 4N1K blocked TSP-1-induced inhibition of tube formation by IBE cells. 4N1K inhibited tyrosine phosphorylation of focal adhesion kinase and FGF-2-stimulated tyrosine phosphorylation of phospholipase C-γ in tube-forming, but not proliferating, IBE cells. The peptide also inhibited FGF-2-induced neovascularization in mouse cornea. Our results indicate that TSP-1 may exert its inhibitory effects on angiogenesis via the C-terminal cell-binding domain containing the 4N1K sequence by inhibiting tube formation by endothelial cells.

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-β1-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 α3β1-integrin and laminin produced by the collagen-cultured IBE cells, FGF stimulation also leads to differentiation of the cells.