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Biomedical subjects

M Presta

Publications and source records attributed to M Presta.

At least 19 recordsLinked to original sources
PubMed2004

Recombinant human erythropoietin induces intussusceptive microvascular growth in vivo.

The role of erythropoietin (Epo) in angiogenesis has not been completely clarified. Epo induces endothelial cell proliferation and migration and stimulates angiogenesis on rat aortic rings in vitro and in vivo in the chick embryo chorioallantoic membrane (CAM) assay. The aim of the present study was to evaluate the ultrastructural aspects of angiogenesis in the CAM vasculature after recombinant human Epo (rHuEpo) exposure. The results demonstrated that after rHuEpo stimulation, the generation of new blood vessels occurred more frequently following an intussusceptive microvascular growth (IMG) mechanism. We have performed our experiments between days 8 and 12 of incubation, that is, when in the normal condition the capillary network expands mainly by IMG, and because it is generally accepted that implants made from days 8 to 10 are strongly angiogenic. This response is peculiar of rHuEpo, because it is abolished when an Epo-blocking antibody was coadministered with Epo.

Animals↗
PubMed2003

Overexpression of cytosolic sialidase Neu2 induces myoblast differentiation in C2C12 cells.

Cytosolic sialidase Neu2 has been implicated in myoblast differentiation. Here we observed a significant upregulation of Neu2 expression during differentiation of murine C2C12 myoblasts. This was evidenced both as an increase in Neu2 mRNA steady-state levels and in the cytosolic sialidase enzymatic activity. To understand the biological significance of Neu2 upregulation in myoblast differentiation, C2C12 cells were stably transfected with the rat cytosolic sialidase Neu2 cDNA. Neu2 overexpressing clones were characterized by a marked decrement of cell proliferation and by the capacity to undergo spontaneous myoblast differentiation also when maintained under standard growth conditions. This was evidenced by the formation of myogenin-positive myotubes and by a significant decrease in the nuclear levels of cyclin D1 protein. No differentiation was on the contrary observed in parental and mock-transfected cells under the same experimental conditions. The results indicate that Neu2 upregulation per se is sufficient to trigger myoblast differentiation in C2C12 cells.

Animals↗
PubMed2003

Erythropoietin as an angiogenic factor.

Erythropoietin (Epo) is produced by the fetal liver and adult kidney and is an essential stimulator of erythropoiesis. It has, however, been shown to modulate host cellular signal transduction pathway to perform many other functions. New sites of Epo production have been found, such as the female reproductive organs and central nervous system. This review summarizes the involvement of Epo in the regulation of angiogenesis in both normal and pathological conditions.

Angiogenesis Inducing Agents↗
PubMed2003

Heparin derivatives as angiogenesis inhibitors.

Angiogenesis is the process of generating new capillary blood vessels. Uncontrolled endothelial cell proliferation is observed in tumor neovascularization and in angioproliferative diseases. Tumors cannot growth as a mass above few mm(3) unless a new blood supply is induced. It derives that the control of the neovascularization process may affect tumor growth and may represent a novel approach to tumor therapy. Angiogenesis is controlled by a balance between proangiogenic and antiangiogenic factors. The angiogenic switch represents the net result of the activity of angiogenic stimulators and inhibitors, suggesting that counteracting even a single major angiogenic factor could shift the balance towards inhibition. Heparan sulfate proteoglycans are involved in the modulation of the neovascularization that takes place in different physiological and pathological conditions. This modulation occurs through the interaction with angiogenic growth factors or with negative regulators of angiogenesis. Thus, the study of the biochemical bases of this interaction may help to design glycosaminoglycan analogs endowed with angiostatic properties. The purpose of this review is to provide an overview of the structure/function of heparan sulfate proteoglycans in endothelial cells and to summarize the angiostatic properties of synthetic heparin-like compounds, chemically modified heparins, and biotechnological heparins.

Angiogenesis Inducing Agents↗
PubMed2002

Anti-angiogenic activity of the purine analog 6-thioguanine.

The antimetabolite 6-thioguanine (6-TG) is utilized in the management of acute myelogenous leukemia (AML). Angiogenesis is a possible therapeutic target in hematologic tumors. Thus, we addressed the possibility that 6-TG may also act as an anti-angiogenic molecule. 6-TG inhibited endothelial cell proliferation triggered by fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) and delayed the repair of a mechanically wounded endothelial cell monolayer. Also, 6-TG inhibited sprouting within fibrin gel, morphogenesis on Matrigel, and collagen gel invasion by endothelial cells. 2-Aminopurine was ineffective. In vivo, 6-TG inhibited basal, VEGF-induced, and FGF2-induced vascularization in the chick embryo chorioallantoic membrane and prevented neovascularization triggered by leukemia LIK cells or their conditioned medium. Finally, bone marrow vascularization in AML patients was decreased to control values in the early remission phase and persisted unvaried after 8-12 months of maintenance therapy with 6-TG. Thus, 6-TG inhibits different steps of the angiogenesis process in vitro and exerts a potent anti-angiogenic activity in vivo. Its anti-angiogenic activity, together with its antimetabolite activity towards tumor cells, may contribute to its action during maintenance therapy in AML. These results suggest a new rationale for the use of purine analogs in the management of AML.

2-Aminopurine↗
PubMed2001

Fibroblast growth factor-2 antagonist activity and angiostatic capacity of sulfated Escherichia coli K5 polysaccharide derivatives.

The angiogenic basic fibroblast growth factor (FGF2) interacts with tyrosine kinase receptors (FGFRs) and heparan sulfate proteoglycans (HSPGs) in endothelial cells. Here, we report the FGF2 antagonist and antiangiogenic activity of novel sulfated derivatives of the Escherichia coli K5 polysaccharide. K5 polysaccharide was chemically sulfated in N- and/or O-position after N-deacetylation. O-Sulfated and N,O-sulfated K5 derivatives with a low degree and a high degree of sulfation compete with heparin for binding to 125I-FGF2 with different potency. Accordingly, they abrogate the formation of the HSPG.FGF2.FGFR ternary complex, as evidenced by their capacity to prevent FGF2-mediated cell-cell attachment of FGFR1-overexpressing HSPG-deficient Chinese hamster ovary (CHO) cells to wild-type CHO cells. They also inhibited 125I-FGF2 binding to FGFR1-overexpressing HSPG-bearing CHO cells and adult bovine aortic endothelial cells. K5 derivatives also inhibited FGF2-mediated cell proliferation in endothelial GM 7373 cells and in human umbilical vein endothelial (HUVE) cells. In all these assays, the N-sulfated K5 derivative and unmodified K5 were poorly effective. Also, highly O-sulfated and N,O-sulfated K5 derivatives prevented the sprouting of FGF2-transfected endothelial FGF2-T-MAE cells in fibrin gel and spontaneous angiogenesis in vitro on Matrigel of FGF2-T-MAE and HUVE cells. Finally, the highly N,O-sulfated K5 derivative exerted a potent antiangiogenic activity on the chick embryo chorioallantoic membrane. These data demonstrate the possibility of generating FGF2 antagonists endowed with antiangiogenic activity by specific chemical sulfation of bacterial K5 polysaccharide. In particular, the highly N,O-sulfated K5 derivative may provide the basis for the design of novel angiostatic compounds.

Angiogenesis Inhibitors↗
PubMed2001

Inhibition of fibroblast growth factor-2-induced vascular tumor formation by the acyclic nucleoside phosphonate cidofovir.

Cidofovir [(S)-HPMPC; (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] is an antiviral drug that has been approved for the treatment of cytomegalovirus retinitis in AIDS patients. Cidofovir also possesses potent inhibitory activity against various human papillomavirus-induced tumors in animal models and patients. In addition, cidofovir inhibits the development of murine polyomavirus-induced hemangiomas in rats by an as-yet-uncharacterized, antivirus-independent mechanism. Here we report the inhibitory effect of cidofovir on the development of virus-independent vascular tumors originated by basic fibroblast growth factor (FGF2)-overexpressing endothelial cells (FGF2-T-MAE cells). In vitro, cidofovir was cytostatic to FGF2-T-MAE cells at a 50% cytostatic concentration of 6.7 microg/ml. Cidofovir concentrations >25 microg/ml resulted in cytotoxicity because of induction of apoptosis. Cidofovir did not affect FGF2-T-MAE cell sprouting in three-dimensional fibrin gel and morphogenesis on Matrigel at noncytotoxic concentrations. In vivo, cidofovir (100 microg/egg) completely suppressed hemangioma formation on the chick chorioallantoic membrane (CAM) induced by intra-allantoic injection of FGF2-T-MAE cells, without affecting the formation of normal CAM vessels. Accordingly, cidofovir applied locally at 200 microg/disc, reduced neovascularization on the CAM by only 35%. Intratumoral or systemic administration of cidofovir caused a significant inhibition of the growth of s.c., i.p., or intracerebral FGF2-T-MAE xenografts in nude mice and severe combined immunodeficient mice. Drug-induced apoptosis was observed in FGF2-T-MAE tumors as soon as 2 days after the beginning of treatment. In conclusion, cidofovir appears to inhibit the growth of endothelium-derived tumors via induction of apoptosis without exerting a direct antiangiogenic activity.

Animals↗
PubMed2001

Paracrine and autocrine effects of fibroblast growth factor-4 in endothelial cells.

Recombinant Fibroblast Growth Factor-4 (FGF4) and FGF2 induce extracellular signal-regulated kinase-1/2 activation and DNA synthesis in murine aortic endothelial (MAE) cells. These cells co-express the IIIc/Ig-3 loops and the novel glycosaminoglycan-modified IIIc/Ig-2 loops isoforms of FGF receptor-2 (FGFR2). The affinity of FGF4/FGFR2 interaction is 20-30 times lower than that of FGF2 and is enhanced by heparin. Overexpression of FGF2 or FGF4 cDNA in MAE cells results in a transformed phenotype and increased proliferative capacity, more evident for FGF2 than FGF4 transfectants. Both transfectants induce angiogenesis when applied on the top of the chick embryo chorioallantoic membrane. However, in contrast with FGF2-transfected cells, FGF4 transfectants show a limited capacity to growth under anchorage-independent conditions and lack the ability to invade 3D fibrin gel and to undergo morphogenesis in vitro. Also, they fail to induce hemangiomas when injected into the allantoic sac of the chick embryo. In conclusion, although exogenous FGF2 and FGF4 exert a similar response in MAE cells, significant differences are observed in the biological behavior of FGF4 versus FGF2 transfectants, indicating that the expression of the various members of the FGF family can differently affect the behavior of endothelial cells and, possibly, of other cell types, including tumor cells.

3T3 Cells↗
PubMed2001

Pentosan polysulfate as an inhibitor of extracellular HIV-1 Tat.

HIV-1 Tat protein, released from HIV-infected cells, may act as a pleiotropic heparin-binding growth factor. From this observation, extracellular Tat has been implicated in the pathogenesis of AIDS and of AIDS-associated pathologies. Here we demonstrate that the heparin analog pentosan polysulfate (PPS) inhibits the interaction of glutathione S-transferase (GST)-Tat protein with heparin immobilized to a BIAcore sensor chip. Competition experiments showed that Tat-PPS interaction occurs with high affinity (K(d) = 9.0 nm). Also, GST.Tat prevents the binding of [(3)H]heparin to GST.Tat immobilized to glutathione-agarose beads. In vitro, PPS inhibits GST.Tat internalization and, consequently, HIV-1 long terminal repeat transactivation in HL3T1 cells. Also, PPS inhibits cell surface interaction and mitogenic activity of GST.Tat in murine adenocarcinoma T53 Tat-less cells. In all assays, PPS exerts its Tat antagonist activity with an ID(50) equal to approximately 1.0 nm. In vivo, PPS inhibits the neovascularization induced by GST.Tat or by Tat-overexpressing T53 cells in the chick embryo chorioallantoic membrane. In conclusion, PPS binds Tat protein and inhibits its cell surface interaction, internalization, and biological activity in vitro and in vivo. PPS may represent a prototypic molecule for the development of novel Tat antagonists with therapeutic implications in AIDS and AIDS-associated pathologies, including Kaposi's sarcoma.

Animals↗
PubMed2001

Inhibition of angiogenesis and tumor growth by SCH221153, a dual alpha(v)beta3 and alpha(v)beta5 integrin receptor antagonist.

New blood vessel formation is essential for tumor growth and metastatic spread. Integrins alpha(v)beta3 and alpha(v)beta5 are arginine-glycine-aspartic acid-dependent adhesion receptors that play a critical role in angiogenesis. Hence, selective dual alpha(v)beta3 and alpha(v)beta5 antagonists may represent a novel class of angiogenesis and tumor-growth inhibitors. Here, an arginine-glycine-aspartic acid-based peptidomimetic library was screened to identify alpha(v)beta3 antagonists. Selected compounds were then modified to generate potent and selective dual inhibitors of alpha(v)beta3 and alpha(v)beta5 receptors. One of these compounds, SCH 221153, inhibited the binding of echistatin to alpha(v)beta3 (IC50 = 3.2 nM) and alpha(v)beta5 (IC50 = 1.7 nM) with similar potency. Its IC50 values for related alpha(IIb)beta3 and alpha5beta1 receptors were 1294 nM and 421 nM, respectively, indicating that SCH 221153 is highly selective for alpha(v)beta3 and alpha(v)beta5 receptors. In cell-based assays, SCH 221153 inhibited the binding of echistatin to alpha(v)beta3- and alpha(v)beta5-expressing 293 cells and blocked the adhesion of endothelial cells to immobilized vitronectin and fibroblast growth factor 2 (FGF2). SCH 221153, but not the inactive analogue SCH 216687, was effective in inhibiting FGF2 and vascular endothelial growth factor-induced endothelial cell proliferation in vitro with an IC50 equal to 3-10 microM. Angiogenesis induced by FGF2 in the chick chorioallantoic membrane assay was also inhibited by SCH 221153. Finally, SCH 221153 exerted a significant inhibition on tumor growth induced by intradermal or s.c. injection of human melanoma LOX cells in severe combined immunodeficient mice.

Allantois↗
PubMed2001

Modulation of tumor angiogenesis by conditional expression of fibroblast growth factor-2 affects early but not established tumors.

Fibroblast growth factor-2 (FGF2) is a pleiotropic heparin-binding growth factor endowed with a potent angiogenic activity in vitro and in vivo. To investigate the impact of the modulation of FGF2 expression on the neovascularization at different stages of tumor growth, we generated stable transfectants (Tet-FGF2) from the human endometrial adenocarcinoma HEC-1-B cell line in which FGF2 expression is under the control of the tetracycline-responsive promoter (Tet-off system). After transfection, independent clones were obtained in which FGF2 mRNA and protein were up-regulated compared with parental cells. Also, the conditioned medium of Tet-FGF2 transfectants caused proliferation, urokinase-type plasminogen activator up-regulation, migration, and sprouting of cultured endothelial cells. A 3-day treatment of Tet-FGF2 cell cultures with tetracycline abolished FGF2 overexpression and the biological activity of the conditioned medium without affecting their proliferative capacity. Tet-FGF2 cells formed tumors when nude mice received s.c. injections. The administration of 2.0 mg/ml tetracycline in the drinking water before cell transplantation, continued throughout the whole experiment, inhibited FGF2 expression in Tet-FGF2 tumor lesions. This was paralleled by a significant decrease in the rate of tumor growth and vascularization to values similar to those observed in lesions generated by parental HEC-1-B cells. Tetracycline administration 20 days after tumor cell implant, although equally effective in reducing FGF2 expression and inhibiting tumor vascularity, only minimally impaired the growth of established Tet-FGF2 tumors. The results indicate that FGF2 expression deeply affects the initial tumor growth and neovascularization of HEC-1-B human endometrial adenocarcinoma in nude mice. On the contrary, the growth of established tumors appears to be independent of the inhibition of FGF2 expression and decreased vascular density. The possibility that a significant reduction of angiogenesis may not affect the progression of large tumors points to the use of antiangiogenic therapy in early tumor stage.

Adenocarcinoma↗
PubMed2001

alpha(v)beta(3) integrin engagement modulates cell adhesion, proliferation, and protease secretion in human lymphoid tumor cells.

OBJECTIVE: The mechanisms used by human lymphoproliferative diseases to invade locally and metastasize are thought to be similar to those developed by solid tumors, including cell proliferation and secretion of extracellular matrix-degrading enzymes following adhesion to extracellular matrix proteins. Hence, the ability of Namalwa (Burkitt's lymphoma), U266 (multiple myeloma), and CEM (T-cell lymphoblastic leukemia) cells to interact with the extracellular matrix components vitronectin and fibronectin was determined. Fresh bone marrow plasma cells from patients with multiple myeloma also were studied. MATERIALS AND METHODS: Engagement of alpha(v)beta(3) integrin, formation and protein composition of focal adhesion contacts on the cell surface, phosphorylation of several signal transduction proteins in the contacts, cell proliferation, and enzyme secretion were studied following adhesion to vitronectin and fibronectin. RESULTS: All three lines adhered to immobilized vitronectin and fibronectin. Adhesion was fully prevented by neutralizing monoclonal anti-alpha(v)beta(3) integrin antibody. Integrin engagement caused the formation of phosphorylated pp60(src)/focal adhesion kinase complexes and the aggregation of focal adhesion plaques containing the beta(3) integrin subunit, the cytoskeletal proteins vinculin, cortactin, and paxillin, the tyrosine kinases focal adhesion kinase and pp60(src), the adapter protein Grb-2, and the mitogen-activated protein kinase ERK-2. Free and immobilized vitronectin and fibronectin stimulated the proliferation of cells under serum-free conditions and the production and release of urokinase-type plasminogen activator, and increased the release of the activated forms of matrix metalloproteinase-2 and matrix metalloproteinase-9 in an alpha(v)beta(3) integrin-dependent manner. Similar results were obtained in myeloma plasma cells. CONCLUSIONS: The demonstrated ability of lymphoid tumor cells to interact with the extracellular matrix components vitronectin and fibronectin via alpha(v)beta(3) integrin can be interpreted as evidence of a novel mechanism for their invasion and spreading. This interaction allows them to adhere to the substratum and enhances their proliferation and protease secretion.

Antibodies, Monoclonal↗
PubMed2001

Activation of endothelial cell mitogen activated protein kinase ERK(1/2) by extracellular HIV-1 Tat protein.

Extracellular Tat protein, the transactivating factor of the human immunodeficiency virus type 1 (HIV-1), modulates gene expression, growth, and angiogenic activity in endothelial cells by interacting with the vascular endothelial growth factor (VEGF) receptor-2 (Flk-1/KDR). Recombinant Tat protein, produced as glutathione-S-transferase chimera (GST-Tat), activates mitogen-activated protein kinase (MAPK) ERK(1/2) in human, murine, and bovine endothelial cells whereas GST is ineffective. In bovine aortic endothelial cells, GST-Tat and the 165 amino acid VEGF isoform (VEGF165) induce transient ERK(1/2) phosphorylation with similar potency and kinetics. The synthetic peptide Tat(41-60), but not peptides Tat(1-21) and Tat(71-86), causes ERK(1/2) phosphorylation, thus implicating Tat/KDR interaction in the activation of this signalling pathway. Accordingly, GST-Tat induces ERK(1/2) phosphorylation in KDR-transfected porcine aortic endothelial cells but not in parental cells. MAPK kinase inhibitors PD098059 and U0126 prevent ERK(1/2) phosphorylation by Tat. However, they do not affect the angiogenic activity exerted by Tat in the murine Matrigel plug and chick embryo chorioallantoic membrane assays. Blocking of MAPK kinase activity impairs instead the angiogenic response to VEGF165 and to fibroblast growth factor-2 (FGF-2). Our data demonstrate that ERK(1/2) activation following the interaction of HIV-1 Tat protein with endothelial cell Flk-1/KDR receptor does not represent an absolute requirement for a full angiogenic response to this growth factor that appears to utilize mechanism(s) at least in part distinct from those triggered by other prototypic angiogenic growth factors.

Animals↗
PubMed2000

Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans.

Tat, the transactivator protein of human immunodeficiency virus-1, has the unusual capacity of being internalized by cells when present in the extracellular milieu. This property can be exploited for the cellular delivery of heterologous proteins fused to Tat both in cell culture and in living animals. Here we provide genetic and biochemical evidence that cell membrane heparan sulfate (HS) proteoglycans act as receptors for extracellular Tat uptake. Cells genetically defective in the biosynthesis of fully sulfated HS are selectively impaired in the internalization of recombinant Tat fused to the green fluorescent protein, as evaluated by both flow cytometry and functional assays. In wild type cells, Tat uptake is competitively inhibited by soluble heparin and by treatment with glycosaminoglycan lyases specifically degrading HS chains. Cell surface HS proteoglycans also mediate physiological internalization of Tat green fluorescent protein released from neighboring producing cells. In contrast to extracellular Tat uptake, both wild type cells and cells genetically impaired in proteoglycan synthesis are equally proficient in the extracellular release of Tat, thus indicating that proteoglycans are not required for this process. The ubiquitous distribution of HS proteoglycans is consistent with the efficient intracellular delivery of heterologous proteins fused with Tat to different mammalian cell types.

Animals↗
PubMed2000

Nerve growth factor induces sphingomyelin accumulation in pheochromocytoma cells.

The pheochromocytoma cells are a well-known model for studying the nerve growth factor (NGF)-induced molecular changes during the differentiation process. The involvement of sphingomyelin (SM) was studied using the fluorescent analogue of ceramide, i.e. N-lissamine rhodaminyl-(12-aminododecanoyl) D-erythro-sphingosine (C12-LRh-Cer). This fluorescent analogue is metabolically active and can be used to follow the biosynthesis of SM in intact cells. NGF induces a 4-fold increase of fluorescent SM content in PC12 cells, when loaded with C12-LRh-Cer. Treatment of PC12 cells with actinomycin D or cycloheximide completely abolishes the NGF-induced elevation of SM. Inhibition of p140(trkA) receptor by AG-879 prevents extracellular signal-regulated kinase 1/2 phosphorylation and suppresses the increase of SM. Inhibition of protein kinase C (PKC), protein kinase A (PKA) and phosphatidylinositol 3-kinase does not have any effect on NGF-induced C12-LRh-SM accumulation. On the other hand, activation of PKA or PKC with simultaneous treatment with NGF has a synergistic effect on increase of SM content. The NGF-induced SM increase in PC12 cells is an effect promoted by other differentiating agents like dibutyryl cyclic AMP or fibroblast growth factor-2 but not by a mitogenic agent like epidermal growth factor.

Animals↗
PubMed2000

Examining new models for the study of autocrine and paracrine mechanisms of angiogenesis through FGF2-transfected endothelial and tumour cells.

Angiogenesis is the process of generating new capillary blood vessels. Uncontrolled endothelial cell proliferation is observed in tumour neovascularization. Several growth factors and cytokines have been shown to stimulate endothelial cell proliferation in vitro and in vivo and among them FGF2 was one of the first to be characterised. FGF2 is a Mr 18,000 heparin-binding cationic polypeptide that induces proliferation, migration, and protease production in endothelial cells in culture and neovascularization in vivo. FGF2 interacts with endothelial cells through two distinct classes of receptors, the high affinity tyrosine-kinase receptors (FGFRs) and low affinity heparan sulfate proteoglycans (HSPGs) present on the cell surface and in the extracellular matrix. Besides experimental evidence for paracrine mode of action for FGF2, some observations raise the hypothesis that FGF2 may also play an autocrine role in endothelial cells. FGF2 may therefore represent a target for anti-angiogenic therapies. In order to assess the angiostatic potential of different classes of compounds, novel experimental models have been developed based on the autocrine and/or the paracrine capacity of FGF2.

Angiogenesis Inhibitors↗
PubMed2000

The discovery of angiogenic factors: a historical review.

Angiogenesis is a biological process by which new capillaries are formed and it occurs in many physiological and pathological conditions. It is controlled by the net balance between molecules that have positive and negative regulatory activity and this concept had led to the notion of the "angiogenic switch," depending on an increased production of one or more of the positive regulators of angiogenesis. Numerous inducers of angiogenesis have been identified and this review offers a historical account of the relevant literature concerning the discovery of the best-characterized angiogenic factors.

Angiogenesis Inducing Agents↗