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M Presta

Publications and source records attributed to M Presta.

At least 91 records · Page 5Linked to original sources
PubMed1993

Internalization of basic fibroblast growth factor (bFGF) in cultured endothelial cells: role of the low affinity heparin-like bFGF receptors.

We have shown (Presta et al., Cell Regul., 2:719-726, 1991) that a long-lasting interaction of basic fibroblast growth factor (bFGF) with endothelial GM 7373 cells is required to induce cell proliferation. In the present work, we have investigated the interaction of 125I-bFGF with GM 7373 cells, its pathway of internalization, and its intracellular fate under the same experimental conditions previously utilized to assess the mitogenic activity of the growth factor. Cell cultures were incubated with 10 ng/ml 125I-bFGF for 2 h at 4 degrees C. Then, cells were shifted to 37 degrees C without changing the medium. A rapid down-regulation of high affinity sites, paralleled by a rapid internalization of 125I-bFGF, was observed during the first 1-2 h at 37 degrees C. After 6-8 h, also low affinity sites down-regulate. This was paralleled by a continuous internalization of 125I-bFGF and by a slow disappearance of the growth factor from the culture medium. This suggests that GM 7373 cells activate, when exposed to bFGF for a long period of time, a late internalization pathway mediated by low affinity sites. This hypothesis was supported by the following experimental evidence: 1) soluble heparin inhibited the prolonged internalization of 125I-bFGF and its binding to low affinity sites with the same potency; 2) treatment of GM 7373 cells with heparinase, which removes most of the low affinity sites, also inhibited the prolonged internalization of 125I-bFGF. 125I-bFGF internalized via low affinity sites was partially protected from lysosomal degradation. This was the case also when 125I-bFGF was internalized in the presence of soluble heparin, suggesting that the complexes bFGF-cell surface glycosaminoglycan and bFGF-soluble heparin are maintained during the internalization of the growth factor. Moreover, the capacity of soluble heparin to inhibit the mitogenic activity of bFGF also when added to cell cultures several hours after the growth factor indicates that the requirement for a prolonged interaction of bFGF with GM 7373 cells in order to induce cell proliferation might be related to the late internalization of the growth factor via low affinity sites.

Animals↗
PubMed1993

Estro-progestinic replacement therapy modulates the levels of basic fibroblast growth factor (bFGF) in postmenopausal endometrium.

The levels of basic fibroblast growth factor (bFGF) in human endometrium do not vary during the ovulatory cycle but they increase significantly after menopause (M. Rusnati et al., Growth Factors, 3, 299-307, 1990). Here we report that cyclic estro-progestinic replacement therapy reduces the levels of bFGF in human postmenopausal endometrium to those measured in ovulatory cycle endometrium. Also, we have observed that the levels of bFGF in well-differentiated adenocarcinomas of the endometrium from postmenopausal patients are lower than those detected in their normal counterpart and are comparable to those measured in the normal cycling tissue. The data indicate that the stimulation of cell proliferation induced in postmenopausal endometrium by hormonal therapy or by neoplastic transformation is paralleled by a decrease of endometrial levels of bFGF. This may be due to an increase of the turnover rate of the growth factor in the proliferating versus the quiescent tissue and suggests a role of bFGF in the growth of normal and neoplastic human endometrium. Our data represent the first evidence that sex hormones modulate the levels of biologically active bFGF in vivo.

Adenocarcinoma↗
PubMed1993

Basic fibroblast growth factor modulates integrin expression in microvascular endothelial cells.

During angiogenesis capillary endothelial cells undergo a coordinated set of modifications in their interactions with extracellular matrix components. In this study we have investigated the effect of the prototypical angiogenic factor basic fibroblast growth factor (bFGF) on the expression and function of several integrins in microvascular endothelial cells. Immunoprecipitation experiments with antibodies to individual subunits indicated that microvascular cells express at their surface several integrins. These include the alpha 1 beta 1, alpha 2 beta 1, and alpha 3 beta 1 laminin/collagen receptors; the alpha 6 beta 1 laminin receptor; the alpha 5 beta 1 and alpha v beta 1 fibronectin receptors; the alpha 6 beta 4 basement membrane receptor; and the alpha v beta 3 and alpha v beta 5 vitronectin receptors. Treatment with bFGF caused a significant increase in the surface expression of the alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha 6 beta 4, and alpha v beta 5 integrins. In contrast, the level of expression of the alpha 1 beta 1 and alpha v beta 3 integrins was decreased in bFGF-treated cells. Immunoprecipitation of metabolically labeled cells indicated that bFGF increases the biosynthesis of the alpha 3, alpha 5, alpha 6, beta 4, and beta 5 subunits and decreases the production of the alpha v and beta 3 subunits. These results suggest that bFGF modulates integrin expression by altering the biosynthesis of individual alpha or beta subunits. In accordance with the upregulation of several integrins observed in bFGF-treated cells, these cells adhered better to fibronectin, laminin, vitronectin, and type I collagen than did untreated cells. The largest differences in beta 1 integrin expression occurred approximately 72 h after exposure to bFGF, at a time when the expression of the endothelial cell-to-cell adhesion molecule endoCAM was also significantly upregulated. In contrast, a shorter exposure to bFGF (24-48 h) was required for the maximal induction of plasminogen activator production in the same cells. Taken together, these results show that bFGF causes significant changes in the level of expression and function of several integrins in microvascular endothelial cells.

Adrenal Cortex↗
PubMed1993

Subcellular localization and biological activity of M(r) 18,000 basic fibroblast growth factor: site-directed mutagenesis of a putative nuclear translocation sequence.

Residues 27-31 (Lys-Asp-Pro-Lys-Arg) of the 155-amino acid form of basic fibroblast growth factor (bFGF) are in good agreement with a consensus sequence for nuclear translocation. To evaluate the role of this sequence in mediating the intracellular localization and biological activity of bFGF, basic residues Lys-27, Lys-30, and Arg-31 were changed to neutral glutamine residues by site-directed mutagenesis of the human bFGF cDNA. The bFGF mutant (M1Q-bFGF) was expressed in eukaryotic cells and in prokaryotic cells, from which it was purified to homogeneity. Transient expression of bFGF cDNA and of M1Q-bFGF cDNA in simian COS-1 cells followed by immunolocalization and by subcellular fractionation indicated that both molecules localize in the nucleus, as well as in the cytoplasm of transfected cells, and interact with nuclear chromatin and with eukaryote DNA in a similar manner. Prokaryotic expression of M1Q-bFGF cDNA yields a polypeptide endowed with a receptor-binding capacity and mitogenic activity similar to that exerted by wild-type bFGF. However, recombinant M1Q-bFGF showed a drastically reduced capacity to induce the production of urokinase-type plasminogen activator (uPA) in endothelial cells. The uPA-inducing activity of M1Q-bFGF was fully restored by the presence of soluble heparin in the culture medium. In conclusion, the sequence bFGF(27-31) does not appear to represent a nuclear translocation and/or retention sequence for bFGF. However, neutralization of its basic residues seems to modify the tertiary structure of the growth factor, thus affecting some of its biological properties.

3T3 Cells↗
PubMed1993

The NH2-terminal extension of high molecular weight forms of basic fibroblast growth factor (bFGF) is not essential for the binding of bFGF to nuclear chromatin in transfected NIH 3T3 cells.

Immunolocalization of basic fibroblast growth factor (bFGF) was investigated in NIH 3T3 cells transfected with a cDNA encoding for the 18 kD form of human bFGF (18 kD-bFGF) or with a bFGF cDNA encoding for both 18 kD-bFGF and NH2-terminal extended high molecular weight forms of bFGF (HMW-bFGFs). Nuclear and cytoplasmic bFGF-immunoreactivity was observed in both transfectants. Nuclear bFGF immunoreactivity was evenly distributed during interfase and associated with condensed chromosomes throughout the mitotic cycle. Cell fractionation, followed by Western blot analysis, confirmed the presence of 18 kD-bFGF and of HMW-bFGFs in the nucleus of transfected cells. Also, both 18-kD bFGF and HMW-bFGFs copurified with nuclear chromatin. After trypsin digestion, chromatin-bound bFGFs showed a rapid degradation of the nuclear-targeting NH2-terminal extension of HMW-bFGFs which were converted to the 18 kD form. On the contrary, 18 kD-bFGF appeared to be trypsin-resistant when bound to nuclear chromatin or to isolated eukaryote DNA. Thus, our data indicate that: i) both 18 kD-bFGF and HMW-bFGFs localize into the nucleus of transfected NIH 3T3 cells and bind to nuclear chromatin; ii) the interaction of all bFGF isoforms with nuclear chromatin is mediated by one or more sequences present within the 18 kD form; iii) the chromatin-binding domain of HMW-bFGFs is distinct from their nuclear-targeting domain.

3T3 Cells↗
PubMed1993

Cerebellar vermis hypoplasia in a case of cri-du-chat syndrome.

We describe a 6-year-old child who presented the phenotype of cri-du-chat disease. The study of her caryotype confirmed an interstitial deletion of the short arm of chromosome 5. The neurological examination showed mental retardation, behavioral disturbances and features of cerebellar and cortico-spinal impairment. The MRI scan of the brain showed hypoplasia of the vermis associated with dysgenesia of the corpus callosum. This is the first report of vermian hypoplasia in cri-du-chat disease. We suggest that the most likely pathogenesis of this malformation is a midline dysraphia.

Cerebellum↗
PubMed1992

Structure-function relationship of basic fibroblast growth factor: site-directed mutagenesis of a putative heparin-binding and receptor-binding region.

Basic residues Arg-118, Lys-119, Lys-128, and Arg-129 within a putative heparin-binding and receptor-binding region of the 155-amino acid form of basic fibroblast growth factor (bFGF) have been changed to neutral glutamine residues by site-directed mutagenesis of the human bFGF cDNA. The bFGF mutant (M6B-bFGF) was expressed in E. coli and purified to homogeneity. When compared to wild type bFGF, M6B-bFGF showed in cultured endothelial cells a similar receptor-binding capacity and mitogenic activity, but a reduced affinity for heparin-like low affinity binding sites, a reduced chemotactic activity, and a reduced capacity to induce the production of urokinase-type plasminogen activator. In vivo, M6B-bFGF lacked a significant angiogenic activity. Modifications of both the primary and the tertiary structure of bFGF appear to be responsible for the modified biological properties of M6B-bFGF, thus confirming the possibility to dissociate at the structural level some of the biological activities exerted by bFGF on endothelial cells.

Amino Acid Sequence↗
PubMed1991

A six-amino acid deletion in basic fibroblast growth factor dissociates its mitogenic activity from its plasminogen activator-inducing capacity.

A recombinant deletion mutant of the 155-amino acid form of human basic fibroblast growth factor (bFGF), lacking amino acid residues 27-32 (Lys-Asp-Pro-Lys-Arg-Leu), was expressed in Escherichia coli and purified to homogeneity by heparin-Sepharose affinity chromatography. When maintained in the presence of an equimolar concentration of soluble heparin, the bFGF mutant (M1-bFGF) is as potent as bFGF in stimulating cell proliferation in normal and transformed fetal bovine aortic endothelial cells, in adult bovine aortic endothelial cells, and in NIH 3T3 fibroblasts. However, under the same experimental conditions, M1-bFGF is at least 100 times less efficient than bFGF in stimulating plasminogen activator (PA) production in endothelial cells, as assayed by chromogenic PA assay, SDS/PAGE zymography, and Northern blot analysis of urokinase-type PA mRNA. In the presence of heparin, M1-bFGF binds to bFGF plasma membrane receptors present on endothelial cells in a manner undistinguishable from bFGF. It also induces the same tyrosine phosphorylation pattern when added to NIH 3T3 cells. The data suggest that the PA-inducing activity of bFGF may depend upon a functional domain that differs from those involved in the mitogenic activity of the growth factor and that the binding of bFGF to its plasma membrane receptor may not be sufficient to induce urokinase-type PA production in endothelial cells.

Amino Acid Sequence↗
PubMed1991

Biologically active synthetic fragments of human basic fibroblast growth factor (bFGF): identification of two Asp-Gly-Arg-containing domains involved in the mitogenic activity of bFGF in endothelial cells.

Synthetic peptides derived from the amino acid sequence of human basic fibroblast growth factor (bFGF) have been assayed for the capacity to exert bFGF agonist and antagonist activities in cultured endothelial cells. bFGF fragments A and C, which correspond to the sequences bFGF (38-61) and bFGF (82-101), induce a limited but statistically significant increase in cell number when administered to cultures of fetal bovine aortic endothelial GM 7373 cells and adult bovine aortic endothelial cells. The two peptides also exert a partial antagonist activity when GM 7373 cells are stimulated to proliferate by bFGF, but they do not affect cell proliferation induced by serum, epidermal growth factor (EGF), phorbol ester (TPA), or 1,2-diacylglycerol (diC8). Moreover, antibodies raised against peptides A and C specifically quench the mitogenic activity of bFGF. Peptides A and C contain the amino acid sequence Asp-Gly-Arg (DGR), which is the inverse of the cell adhesion signal sequence RGD recognized by integrins. DGR- and RGD-containing tetra- and heptapeptides inhibit the mitogenic activity exerted by bFGF and by the two active bFGF fragments. They do not affect cell proliferation induced by acidic FGF, EGF, serum, TPA, and diC8. However, neither peptides A and C, their corresponding antibodies, nor DGR-and RGD-containing peptides inhibit the binding of 125I-bFGF to its low and high affinity binding sites. The data suggest that amino acid residues 38-61 and 82-101, both containing a core DGR sequence, represent two "activation" domains of bFGF. Both domains are involved in the modulation of the mitogenic activity of bFGF without interacting directly with the bFGF receptor.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Nuclear localization of endogenous basic fibroblast growth factor in cultured endothelial cells.

Indirect immunofluorescence using anti-human placental bFGF antibodies demonstrates the presence of bFGF-like reactivity in the cytoplasm and in the nucleus of adult bovine aortic endothelial cells and of normal and transformed fetal bovine aortic endothelial AG 7680 and GM 7372 cells. Biologically active immunoreactive Mr 18,000 bFGF can be isolated by heparin-Sepharose affinity chromatography from the extract of GM 7372 cell nuclei. Quantitation of bFGF content by biological and immunological methods indicates that 100,000 bFGF molecules per nucleus are present in GM 7372 cells, with nuclear bFGF corresponding to 25-30% of total cellular bFGF. Immunoprecipitation experiments demonstrate that the nuclear localization of newly synthesized bFGF occurs when GM 7372 cells are biosynthetically labeled both in the absence and in the presence of suramin, a molecule that inhibits the binding of bFGF to its plasma membrane receptor. Thus the data indicate that endogenous bFGF undergoes an intracellular sorting to the nucleus of the endothelial cell.

Animals↗
PubMed1991

Basic fibroblast growth factor requires a long-lasting activation of protein kinase C to induce cell proliferation in transformed fetal bovine aortic endothelial cells.

Basic fibroblast growth factor (bFGF) induces a protein kinase C (PKC)-dependent mitogenic response in transformed fetal bovine aortic endothelial GM 7373 cells. A long-lasting interaction of bFGF with the cell is required to induce cell proliferation. bFGF-treated cells are in fact committed to proliferate only after they have entered the phase S of the cell cycle, 12-14 h after the beginning of bFGF treatment. Before that time, the mitogenic response to bFGF is abolished by 1) removal of extracellular bFGF by suramin, 2) addition of neutralizing anti-bFGF antibodies to the culture medium, 3) inhibition of PKC activity by the protein kinase inhibitor H-7, and 4) down-regulation of PKC by cotreatment with phorbol ester. Thus the requirement for a prolonged interaction of bFGF with the cell reflects the requirement for a prolonged activation of PKC. Similar conclusions can be drawn for the PKC activators 12-O-tetradecanoyl phorbol 13-acetate and 1,2-dioctanoyl-sn-glycerol. The two molecules require 16 and 6 h, respectively, of activation of PKC to induce 50% of maximal cell proliferation. The requirement for a long-lasting activation of PKC appears to be a mechanism for the control of cell proliferation capable of discriminating among transient nonmitogenic stimuli and long-lasting mitogenic stimuli.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Liver genotoxic activity of an epoxide derivative of the hepatocarcinogenic beta-blocker DL-ZAMI 1305.

A single administration of the sex-dependent hepatocarcinogenic beta-blocker DL-1-(2-nitro-3-methyl-phenoxy)-3-tert-butylamino-propan-2-ol (DL-ZAMI 1305) induces dose-dependent liver DNA damage, as evaluated by alkaline sucrose gradient analysis, in female but not in male Fisher 344 rats. A single administration of the direct mutagenic epoxide-derivative of DL-ZAMI 1305 3-methyl-2-nitro-1-(2,3-epoxypropoxy)-benzene induces dose-dependent DNA damage in the liver of animals of both sexes. However, also in this case, the genotoxic activity of the compound appears to be significantly higher in female than in male rats. A DNA-damaging capacity similar in the two sexes is instead exerted by DL-ZAMI 1305-unrelated direct mutagens, like N-methyl-N-nitrosourea (MNU) and methyl-methanesulfonate (MMS). The data confirm the sex-dependent susceptibility of rat liver to the genotoxic activity of DL-ZAMI 1305-related molecules, also in the absence of an absolute requirement for a metabolic activation of the compound.

Animals↗
PubMed1990

Basic fibroblast growth factor in neuronal cultures of human fetal brain.

The presence of basic fibroblast growth factor (bFGF) was investigated in neuronal cells derived from 12 and 18 week-old human fetal brain cultures. To this purpose, the ability of bFGF to stimulate plasminogen activator (PA) production in fetal bovine aortic endothelial GM 7373 cells was used as an assay for this molecule in neuronal cell extracts. The identity of the PA-stimulating activity of neuronal cell extract with bFGF was confirmed by its high affinity for heparin and by its cross-reactivity with polyclonal antibodies to human placental bFGF. These antibodies recognized a Mr 18,000 cell-associated protein both in Western blot and in immuno-precipitation experiments. All the neurons showed bFGF immunoreactivity, as demonstrated by immunocytochemical staining, while nonneuronal cells were unstained. The data demonstrate for the first time that cultured human fetal brain neurons contain and synthesize bFGF.

Animals↗