Exogenous vascular endothelial growth factor induces malformed and hyperfused vessels during embryonic neovascularization.

Vascular endothelial growth factor (VEGF) is a potent and specific endothelial mitogen that is able to induce angiogenesis in vivo [Leung, D. W., Cachianes, G., Kuang, W.-J., Goeddel, D. V. & Ferrara, N. (1989) Science 246 1306-1309]. To determine if VEGF also influences the behavior of primordial endothelial cells, we used an in vivo vascular assay based on the de novo formation of vessels. Japanese quail embryos injected with nanomolar quantities of the 165-residue form of VEGF at the onset of vasculogenesis exhibited profoundly altered vessel development. In fact, the overall patterning of the vascular network was abnormal in all VEGF-injected embryos. The malformations were attributable to two specific endothelial cell activities: (i) inappropriate neovascularization in normally avascular areas and (ii) the unregulated, excessive fusion of vessels. In the first instance, supernumerary vessels directly linked the inflow channel of the heart to the aortic outflow channel. The second aberrant activity led to the formation of vessels with abnormally large lumens. Ultimately, unregulated vessel fusion generated massive vascular sacs that obliterated the identity of individual vessels. These observations show that exogenous VEGF has an impact on the behavior of primordial endothelial cells engaged in vasculogenesis, and they strongly suggest that endogenous VEGF is important in vascular patterning and regulation of vessel size (lumen formation).

Appearance of insulin-like growth factor mRNA in the liver and pyloric ceca of a teleost in response to exogenous growth hormone.

Augmentation of vertebrate growth by growth hormone (GH) is primarily due to its regulation of insulin-like growth factor I (IGF I) and IGF II levels. To characterize the effect of GH on the levels of IGF I and IGF II mRNA in a teleost, 10 micrograms of bovine GH (bGH) per g of body weight was administered to juvenile rainbow trout (Oncorhynchus mykiss) through i.p. injection. The levels of IGF I and IGF II mRNA were determined simultaneously, by using RNase protection assays, in the liver, pyloric ceca, kidney, and gill at 0, 1, 3, 6, 12, 24, 48, and 72 hr after injection. In the liver, IGF I mRNA levels were significantly elevated at 6 and 12 hr (approximately 2- to 3-fold, P < or = 0.01), while IGF II mRNA levels were significantly elevated at 3 and 6 hr (approximately 3-fold, P < or = 0.01). In the pyloric ceca, IGF II mRNA levels were significantly elevated at 12, 24, and 48 hr (approximately 3-fold, P < or = 0.01), while IGF I mRNA was below the limits of assay accuracy. GH-dependent IGF mRNA appearance was not detected in the gill and kidney. Serum bGH levels, determined by using a radioimmunoassay, were significantly elevated at 3 and 6 hr (P < 0.005). In primary hepatocyte culture, IGF I and IGF II mRNA levels increased in a bGH dose-dependent fashion, with ED50 values of approximately 45 and approximately 6 ng of bGH per ml, respectively. The GH-dependent appearance of IGF II mRNA in the liver and pyloric ceca suggests important roles for this peptide hormone exclusive of IGF I.

Tissue factor gene expression in the adipose tissues of obese mice

Altered expression of proteins of the fibrinolytic and coagulation cascades in obesity may contribute to the cardiovascular risk associated with this condition. We previously reported that plasminogen activator inhibitor 1 (PAI-1) is dramatically up-regulated in the plasma and adipose tissues of genetically obese mice. This change may disturb normal hemostatic balance and create a severe hypofibrinolytic state. Here we show that tissue factor (TF) gene expression also is significantly elevated in the epididymal and subcutaneous fat pads from ob/ob mice compared with their lean counterparts, and that its level of expression in obese mice increases with age and the degree of obesity. Cell fractionation and in situ hybridization analysis of adipose tissues indicate that TF mRNA is increased in adipocytes and in unidentified stromal vascular cells. Transforming growth factor β (TGF-β) is known to be elevated in the adipose tissue of obese mice, and administration of TGF-β increased TF mRNA expression in adipocytes in vivo and in vitro. These observations raise the possibility that TF and TGF-β may contribute to the increased cardiovascular disease that accompanies obesity and related non-insulin-dependent diabetes mellitus, and that the adipocyte plays a key role in this process. The recent demonstration that TF also influences angiogenesis, cell adhesion, and signaling suggests that its exact role in adipose tissue physiology/pathology, may be complex.

Removal of LIF (leukemia inhibitory factor) results in increased vitamin A (retinol) metabolism to 4-oxoretinol in embryonic stem cells

Retinoids, vitamin A (retinol) and its metabolic derivatives, are required for normal vertebrate development. In murine embryonic stem (ES) cells, which remain undifferentiated when cultured in the presence of LIF (leukemia inhibitory factor), little metabolism of exogenously added retinol takes place. After LIF removal, ES cells metabolize exogenously added retinol to 4-hydroxyretinol and 4-oxoretinol and concomitantly differentiate. The conversion of retinol to 4-oxoretinol is a high-capacity reaction because most of the exogenous retinol is metabolized rapidly, even when cells are exposed to physiological (≈1 μM) concentrations of retinol in the medium. No retinoic acid or 4-oxoRA synthesis from retinol was detected in ES cells cultured with or without LIF. The cytochrome P450 enzyme CYP26 (retinoic acid hydroxylase) is responsible for the metabolism of retinol to 4-oxoretinol, and CYP26 mRNA is greatly induced (>15-fold) after LIF removal. Concomitant with the expression of CYP26, differentiating ES cells grown in the absence of LIF activate the expression of the differentiation marker gene FGF-5 whereas the expression of the stem cell marker gene FGF-4 decreases. The strong correlation between the production of polar metabolites of retinol and the differentiation of ES cells upon removal of LIF suggests that one important action of LIF in these cells is to prevent retinol metabolism to biologically active, polar metabolites such as 4-oxoretinol.

Superoxide-mediated clastogenesis and anticlastogenic effects of exogenous superoxide dismutase

Superoxide-mediated clastogenesis is characteristic for various chronic inflammatory diseases with autoimmune reactions and probably plays a role in radiation-induced clastogenesis and in the congenital breakage syndromes. It is consistently prevented by exogenous superoxide dismutase (SOD), but not by heat-inactivated SOD, indicating that the anticlastogenic effect is related to the catalytic function of the enzyme. Increased superoxide production by activated monocytes/macrophages is followed by release of more long-lived metabolites, so-called clastogenic factors, which contain lipid peroxidation products, unusual nucleotides of inosine, and cytokines such as tumor necrosis factor α. Since these components are not only clastogenic, but can stimulate further superoxide production by monocytes and neutrophils, the genotoxic effects are self-sustaining. It is shown here that anticlastogenic effects of exogenous SOD are preserved despite extensive washing of the cells and removal of all extracellular SOD. Using flow cytometry and confocal laser microscopy, rapid adherence of the fluorescently labeled enzyme to the cell surface could be observed with slow uptake into the cell during the following hours. The degree of labeling was concentration and time dependent. It was most important for monocytes, compared with lymphocytes, neutrophils, and fibroblasts. The cytochrome c assay showed significantly diminished O2− production by monocytes, pretreated with SOD and washed thereafter. The preferential and rapid binding of SOD to monocytes may be of importance not only for the superoxide-mediated genotoxic effects, described above, but also from a therapeutic standpoint. It can explain the observation that beneficial effects of injected SOD lasted for weeks and months despite rapid clearance of the enzyme from the blood stream according to pharmacodynamic studies.

Defects in transforming growth factor-β signaling cooperate with a Ras oncogene to cause rapid aneuploidy and malignant transformation of mouse keratinocytes

Genetic inactivation of the transforming growth factor-β (TGF-β) signaling pathway can accelerate tumor progression in the mouse epidermal model of multistage carcinogenesis. By using an in vitro model of keratinocyte transformation that parallels in vivo malignant conversion to squamous cell carcinoma, we show that v-rasHa transduced primary TGF-β1−/− keratinocytes and keratinocytes expressing a TGF-β type II dominant-negative receptor transgene have significantly higher frequencies of spontaneous transformation than control genotypes. Malignant transformation in the TGF-β1−/− keratinocytes is preceded by aneuploidy and accumulation of chromosomal aberrations. Similarly, transient inactivation of TGF-β signaling with a type II dominant-negative receptor adenovirus causes rapid changes in ploidy. Exogenous TGF-β1 can suppress aneuploidy, chromosome breaks, and malignant transformation of the TGF-β1−/− keratinocytes at concentrations that do not significantly arrest cell proliferation. These results point to genomic instability as a mechanism by which defects in TGF-β signaling could accelerate tumor progression in mouse multistage carcinogenesis.

T cell functions in granulocyte/macrophage colony-stimulating factor deficient mice

Immunological functions were analyzed in mice lacking granulocyte/macrophage colony-stimulating factor (GM-CSF). The response of splenic T cells to allo-antigens, anti-mouse CD3 mAb, interleukin 2 (IL-2), or concanavalin A was comparable in GM-CSF +/+ and GM-CSF −/− mice. To investigate the responses of CD8+ and CD4+ T cells against exogenous antigens, mice were immunized with ovalbumin peptide or with keyhole limpet hemocyanin (KLH). Cytotoxic CD8+ T cells with specificity for ovalbumin peptide could not be induced in GM-CSF −/− mice. After immunization with KLH, there was a delay in IgG generation, particularly IgG2a, in GM-CSF −/− mice. Purified CD4+ T cells from GM-CSF −/− mice immunized with KLH showed impaired proliferative responses and produced low amounts of interferon-γ (IFN-γ) and IL-4 when KLH-pulsed B cells or spleen cells were used as antigen presenting cells (APC). When enriched dendritic cells (DC) were used as APC, CD4+ T cells from GM-CSF −/− mice proliferated as well as those from GM-CSF +/+ mice and produced high amounts of IFN-γ and IL-4. To analyze the rescue effect of DC on CD4+ T cells, supernatants from (i) CD4+ T cells cultured with KLH-pulsed DC or (ii) DC cultured with recombinant GM-CSF were transferred to cultures of CD4+ T cells and KLH-pulsed spleen cells from GM-CSF −/− mice. Supernatants from both DC sources contained a factor or factors that restored proliferative responses and IFN-γ production of CD4+ T cells from GM-CSF −/− mice.

Interaction of Fibroblast Growth Factor-2 (FGF-2) with Free Gangliosides: Biochemical Characterization and Biological Consequences in Endothelial Cell Cultures

Exogenous gangliosides affect the angiogenic activity of fibroblast growth factor-2 (FGF-2), but their mechanism of action has not been elucidated. Here, a possible direct interaction of sialo-glycolipids with FGF-2 has been investigated. Size exclusion chromatography demonstrates that native, but not heat-denatured, 125I-FGF-2 binds to micelles formed by gangliosides GT1b, GD1b, or GM1. Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency being GT1b > GD1b > GM1 = GM2 = sulfatide > GM3 = galactosyl-ceramide, whereas asialo-GM1, neuraminic acid, and N-acetylneuramin-lactose were ineffective. Scatchard plot analysis of the binding data of fluorochrome-labeled GM1 to immobilized FGF-2 indicates that FGF–2/GM1 interaction occurs with a Kd equal to 6 μM. This interaction is inhibited by the sialic acid-binding peptide mastoparan and by the synthetic fragments FGF-2(112–129) and, to a lesser extent, FGF-2(130–155), whereas peptides FGF-2(10–33), FGF-2(39–59), FGF-2(86–96), and the basic peptide HIV-1 Tat(41–60) were ineffective. These data identify the COOH terminus of FGF-2 as a putative ganglioside-binding region. Exogenous gangliosides inhibit the binding of 125I-FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations. The order of relative potency was GT1b > GD1b > GM1 > sulfatide a = sialo-GM1. Accordingly, GT1b,GD1b, GM1, and GM2, but not GM3 and asialo-GM1, prevent the binding of 125I-FGF-2 to a soluble, recombinant form of extracellular FGFR-1. Conversely, the soluble receptor and free heparin inhibit the interaction of fluorochrome-labeled GM1 to immobilized FGF-2. In agreement with their FGFR antagonist activity, free gangliosides inhibit the mitogenic activity exerted by FGF-2 on endothelial cells in the same range of concentrations. Also in this case, GT1b was the most effective among the gangliosides tested while asialo-GM1, neuraminic acid, N-acetylneuramin-lactose, galactosyl-ceramide, and sulfatide were ineffective. In conclusion, the data demonstrate the capacity of exogenous gangliosides to interact with FGF-2. This interaction involves the COOH terminus of the FGF-2 molecule and depends on the structure of the oligosaccharide chain and on the presence of sialic acid residue(s) in the ganglioside molecule. Exogenous gangliosides act as FGF-2 antagonists when added to endothelial cell cultures. Since gangliosides are extensively shed by tumor cells and reach elevated levels in the serum of tumor-bearing patients, our data suggest that exogenous gangliosides may affect endothelial cell function by a direct interaction with FGF-2, thus modulating tumor neovascularization.

Binding of Insulin-like Growth Factor (IGF)–Binding Protein-5 to Smooth-Muscle Cell Extracellular Matrix Is a Major Determinant of the Cellular Response to IGF-I

Insulin-like growth factor–binding protein-5 (IGFBP-5) has been shown to bind to fibroblast extracellular matrix (ECM). Extracellular matrix binding of IGFBP-5 leads to a decrease in its affinity for insulin-like growth factor-I (IGF-I), which allows IGF-I to better equilibrate with IGF receptors. When the amount of IGFBP-5 that is bound to ECM is increased by exogenous addition, IGF-I’s effect on fibroblast growth is enhanced. In this study we identified the specific basic residues in IGFBP-5 that mediate its binding to porcine smooth-muscle cell (pSMC) ECM. An IGFBP-5 mutant containing alterations of basic residues at positions 211, 214, 217, and 218 had the greatest reduction in ECM binding, although three other mutants, R214A, R207A/K211N, and K202A/R206N/R207A, also had major decreases. In contrast, three other mutants, R201A/K202N/R206N/R208A, and K217N/R218A and K211N, had only minimal reductions in ECM binding. This suggested that residues R207 and R214 were the most important for binding, whereas alterations in K211 and R218, which align near them, had minimal effects. To determine the effect of a reduction in ECM binding on the cellular replication response to IGF-I, pSMCs were transfected with the mutant cDNAs that encoded the forms of IGFBPs with the greatest changes in ECM binding. The ECM content of IGFBP-5 from cultures expressing the K211N, R214A, R217A/R218A, and K202A/R206N/R207A mutants was reduced by 79.6 and 71.7%, respectively, compared with cells expressing the wild-type protein. In contrast, abundance of the R201A/K202N/R206N/R208A mutant was reduced by only 14%. Cells expressing the two mutants with reduced ECM binding had decreased DNA synthesis responses to IGF-I, but the cells expressing the R201A/K202N/R206N/R208A mutant responded well to IGF-I. The findings suggest that specific basic amino acids at positions 207 and 214 mediate the binding of IGFBP-5 to pSMC/ECM. Smooth-muscle cells that constitutively express the mutants that bind weakly to ECM are less responsive to IGF-I, suggesting that ECM binding of IGFBP-5 is an important variable that determines cellular responsiveness.

The Fibrinogen Globe of Tenascin-C Promotes Basic Fibroblast Growth Factor-induced Endothelial Cell Elongation

To investigate the potential role of tenascin-C (TN-C) on endothelial sprouting we used bovine aortic endothelial cells (BAECs) as an in vitro model of angiogenesis. We found that TN-C is specifically expressed by sprouting and cord-forming BAECs but not by nonsprouting BAECs. To test whether TN-C alone or in combination with basic fibroblast growth factor (bFGF) can enhance endothelial sprouting or cord formation, we used BAECs that normally do not sprout and, fittingly, do not express TN-C. In the presence of bFGF, exogenous TN-C but not fibronectin induced an elongated phenotype in nonsprouting BAECs. This phenotype was due to altered actin cytoskeleton organization. The fibrinogen globe of the TN-C molecule was the active domain promoting the elongated phenotype in response to bFGF. Furthermore, we found that the fibrinogen globe was responsible for reduced cell adhesion of BAECs on TN-C substrates. We conclude that bFGF-stimulated endothelial cells can be switched to a sprouting phenotype by the decreased adhesive strength of TN-C, mediated by the fibrinogen globe.

Fibroblast Growth Factor (FGF) Soluble Receptor 1 Acts as a Natural Inhibitor of FGF2 Neurotrophic Activity during Retinal Degeneration

Fibroblast growth factors (FGF) 1 and 2 and their tyrosine kinase receptor (FGFR) are present throughout the adult retina. FGFs are potential mitogens, but adult retinal cells are maintained in a nonproliferative state unless the retina is damaged. Our work aims to find a modulator of FGF signaling in normal and pathological retina. We identified and sequenced a truncated FGFR1 form from rat retina generated by the use of selective polyadenylation sites. This 70-kDa form of soluble extracellular FGFR1 (SR1) was distributed mainly localized in the inner nuclear layer of the retina, whereas the full-length FGFR1 form was detected in the retinal Muller glial cells. FGF2 and FGFR1 mRNA levels greatly increased in light-induced retinal degeneration. FGFR1 was detected in the radial fibers of activated retinal Muller glial cells. In contrast, SR1 mRNA synthesis followed a biphasic pattern of down- and up-regulation, and anti-SR1 staining was intense in retinal pigmented epithelial cells. The synthesis of SR1 and FGFR1 specifically and independently regulated in normal and degenerating retina suggests that changes in the proportion of various FGFR forms may control the bioavailability of FGFs and thus their potential as neurotrophic factors. This was demonstrated in vivo during retinal degeneration when recombinant SR1 inhibited the neurotrophic activity of exogenous FGF2 and increased damaging effects of light by inhibiting endogenous FGF. This study highlights the significance of the generation of SR1 in normal and pathological conditions.

Exogenous Administration of Gangliosides Displaces GPI-anchored Proteins from Lipid Microdomains in Living Cells

Exogenous application of gangliosides to cells affects many cellular functions. We asked whether these effects could be attributed to the influence of gangliosides on the properties of sphingolipid–cholesterol microdomains on the plasma membrane, also termed rafts. The latter are envisaged as lateral assemblies of sphingolipids (including gangliosides), cholesterol, and a specific set of proteins. Rafts have been implicated in processes such as membrane trafficking, signal transduction, and cell adhesion. Recently, using a chemical cross-linking approach with Madin-Darby canine kidney (MDCK) cells permanently expressing a GPI-anchored form of growth hormone decay accelerating factor (GH-DAF) as a model system, we could show that GPI-anchored proteins are clustered in rafts in living cells. Moreover, this clustering was dependent on the level of cholesterol in the cell. Here we show that incubation of MDCK cells with gangliosides abolished subsequent chemical cross-linking of GH-DAF. Furthermore, insertion of gangliosides into the plasma membrane of MDCK GH-DAF cells renders GH-DAF soluble when subjected to extraction with Triton X-114 at 4°C. Our data suggest that exogenous application of gangliosides displaces GPI-anchored proteins from sphingolipid–cholesterol microdomains in living cells.

Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa

Protease-activated receptor 2 (PAR2) is expressed by vascular endothelial cells and other cells in which its function and physiological activator(s) are unknown. Unlike PAR1, PAR3, and PAR4, PAR2 is not activatable by thrombin. Coagulation factors VIIa (FVIIa) and Xa (FXa) are proteases that act upstream of thrombin in the coagulation cascade and require cofactors to interact with their substrates. These proteases elicit cellular responses, but their receptor(s) have not been identified. We asked whether FVIIa and FXa might activate PARs if presented by their cofactors. Co-expression of tissue factor (TF), the cellular cofactor for FVIIa, together with PAR1, PAR2, PAR3, or PAR4 conferred TF-dependent FVIIa activation of PAR2 and, to lesser degree, PAR1. Responses to FXa were also observed but were independent of exogenous cofactor. The TF/FVIIa complex converts the inactive zymogen Factor X (FX) to FXa. Strikingly, when FX was present, low picomolar concentrations of FVIIa caused robust signaling in cells expressing TF and PAR2. Responses in keratinocytes and cytokine-treated endothelial cells suggested that PAR2 may be activated directly by TF/FVIIa and indirectly by TF/FVIIa-generated FXa at naturally occurring expression levels of TF and PAR2. These results suggest that PAR2, although not activatable by thrombin, may nonetheless function as a sensor for coagulation proteases and contribute to endothelial activation in the setting of injury and inflammation. More generally, these findings highlight the potential importance of cofactors in regulating PAR function and specificity.

Metalloprotease-mediated ligand release regulates autocrine signaling through the epidermal growth factor receptor

Ligands that activate the epidermal growth factor receptor (EGFR) are synthesized as membrane-anchored precursors that appear to be proteolytically released by members of the ADAM family of metalloproteases. Because membrane-anchored EGFR ligands are thought to be biologically active, the role of ligand release in the regulation of EGFR signaling is unclear. To investigate this question, we used metalloprotease inhibitors to block EGFR ligand release from human mammary epithelial cells. These cells express both transforming growth factor α and amphiregulin and require autocrine signaling through the EGFR for proliferation and migration. We found that metalloprotease inhibitors reduced cell proliferation in direct proportion to their effect on transforming growth factor α release. Metalloprotease inhibitors also reduced growth of EGF-responsive tumorigenic cell lines and were synergistic with the inhibitory effects of antagonistic EGFR antibodies. Blocking release of EGFR ligands also strongly inhibited autocrine activation of the EGFR and reduced both the rate and persistence of cell migration. The effects of metalloprotease inhibitors could be reversed by either adding exogenous EGF or by expressing an artificial gene for EGF that lacked a membrane-anchoring domain. Our results indicate that soluble rather than membrane-anchored forms of the ligands mediate most of the biological effects of EGFR ligands. Metalloprotease inhibitors have shown promise in preventing spread of metastatic disease. Many of their antimetastatic effects could be the result of their ability to inhibit autocrine signaling through the EGFR.

Conservation of fibroblast growth factor function in lens regeneration

In urodele amphibians, lens induction during development and regeneration occurs through different pathways. During development, the lens is induced from the mutual interaction of the ectoderm and the optic vesicle, whereas after lentectomy the lens is regenerated through the transdifferentiation of the iris-pigmented epithelial cells. Given the known role of fibroblast growth factors (FGFs) during lens development, we examined whether or not the expression and the effects of exogenous FGF during urodele lens regeneration were conserved. In this paper, we describe expression of FGF-1 and its receptors, FGFR-2 (KGFR and bek variants) and FGFR-3, in newts during lens regeneration. Expression of these genes was readily observed in the dedifferentiating pigmented epithelial cells, and the levels of expression were high in the lens epithelium and the differentiating fibers and lower in the retina. These patterns of expression implied involvement of FGFs in lens regeneration. To further elucidate this function, we examined the effects of exogenous FGF-1 and FGF-4 during lens regeneration. FGF-1 or FGF-4 treatment in lentectomized eyes resulted in the induction of abnormalities reminiscent to the ones induced during lens development in transgenic mice. Effects included transformation of epithelial cells to fiber cells, double lens regeneration, and lenses with abnormal polarity. These results establish that FGF molecules are key factors in fiber differentiation, polarity, and morphogenesis of the lens during regeneration even though the regenerating lens is induced by a different mechanism than in lens development. In this sense, FGF function in lens regeneration and development should be regarded as conserved. Such conservation should help elucidate the mechanisms of lens regeneration in urodeles and its absence in higher vertebrates.