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Biomedical subjects

D Ribatti

Publications and source records attributed to D Ribatti.

At least 73 records · Page 4Linked to original sources
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

Postnatal vasculogenesis.

It is generally accepted that vasculogenesis is limited to early embryogenesis and is believed not to occur in adult, whereas angiogenesis occurs in both the developing embryo and postnatal life. However, the distinction between them is not absolute, because both require endothelial cell proliferation and migration and three-dimensional reorganization of newly formed blood vessels, nor are they mutually exclusive, inasmuch as angioblasts can be incorporated into expanding pre-existing blood vessels. Recent observations indicate that vasculogenesis may not be restricted to early embryogenesis, but may also have a physiological role or contribute to the pathology of vascular diseases in adults. The major evidence in favor of this new view comes from: (i) demonstration of the presence of circulating endothelial cells and endothelial precursor cells; (ii) newly described mechanisms of blood vessel formation in tumor growth. The potential biomedical applications of endothelial precursor cells and the new opportunities for the development of new forms of tumor-targeted treatments are discussed.

Animals↗
PubMed2001

Vascular endothelial growth factor and vascular endothelial growth factor receptor-2 expression in the chick embryo area vasculosa.

Vascular endothelial growth factor is an angiogenic factor in vivo and in vitro that plays a crucial role in the control of blood vessel development and in pathological angiogenesis. The vascularized extraembryonic membranes of the chick embryo include the area vasculosa and the chorioallantoic membrane. In this study, we investigated the expression of vascular endothelial growth factor and of its receptor-2, specifically expressed by the endothelial cells, in the chick area vasculosa at days 6, 10 and 14 of incubation. Our results indicate that, in all the three developmental stages examined, vascular endothelial growth factor is clearly expressed in the endodermal cells immediately adjacent to the mesodermal endothelial cells which, in turn, expressed vascular endothelial growth factor receptor-2. These observations suggest that during the development of the vascular system, endodermal cells, expressing vascular endothelial growth factor, initiate angiogenesis by stimulating directly mesodermal cells, which express vascular endothelial growth factor receptor-2. Moreover, our data demonstrate that vascular endothelial growth factor receptor-2 expression is also maintained by endothelial cells in the later stages of development, until day 14 of incubation. In accord with other literature data, this suggests that vascular endothelial growth factor is required not only for proliferation, but also for the survival of endothelial cells.

Allantois↗
PubMed2001

Microvessel overexpression of aquaporin 1 parallels bone marrow angiogenesis in patients with active multiple myeloma.

The erythrocyte water channel aquaporin 1 (AQP1) is expressed in multiple absorptive and secretory epithelia including the capillary endothelia. Immunoblot analysis showed that bone marrow biopsies of patients with active multiple myeloma (MM) display significantly higher levels of AQP1 than those from patients with non-active MM, whose values are higher, but to a lesser extent, than those of patients with monoclonal gammopathies of undetermined significance (MGUS). Values of MGUS overlapped those of patients with anaemia as a result of iron or vitamin B12 deficiencies (called 'benign anaemias'). Immunohistochemistry and computerized image analysis of AQP1 highlighted bone marrow microvessels whose area per microscopic field was significantly greater in patients with active MM, and always larger than and closely correlated with the microvessel area when assessed with factor VIII-related antigen/von Willebrand's factor (FVIII-VWF). The intensity of AQP1 expression by microvessels evaluated using image analysis was significantly greater in active than non-active MM and in the latter over MGUS or benign anaemias. It is suggested that, among plasma cell tumours, AQP1 expression is preferentially associated with microvessels of MM and that the highest degree of expression occurs in active MM in step with enhanced angiogenesis, in which AQP1 recognizes more immature neovessels than FVIII-VWF. It may, perhaps, favour angiogenesis in a positive loop and, hence, MM progression, and thus be applied for therapeutic vascular targeting.

Adult↗
PubMed2001

Mast cells and their secretory granules are angiogenic in the chick embryo chorioallantoic membrane.

BACKGROUND: Many data suggest that the density of mast cells is highly correlated with the extent of both normal and pathological angiogenesis. OBJECTIVE: In this study we have compared in an in vivo assay, the chick embryo chorioallantoic membrane, the angiogenic potential of mast cell suspensions isolated from rats, degranulated mast cells and their secretory granules. METHODS: Gelatin sponges adsorbed with cell suspensions of rat mast cells, degranulated mast cells and their secretory granules were implanted on the top of the chorioallantoic membrane at day 8 of incubation. At day 12 the angiogenic response was evaluated macroscopically, microscopically and by a morphometric method of 'point counting'. RESULTS: Isolated mast cells and their secretory granules, but not degranulated mast cells, induced an angiogenic response in the chorioallantoic membrane. The addition of antifibroblast growth factor-2 or antivascular endothelial growth factor antibodies reduced the angiogenic response of both mast cells and their secretory granules by 50% and 30%, respectively. CONCLUSION: These data support the evidence that the angiogenic properties of mast cells depend on the angiogenic molecules contained in their secretory granules and indicate that fibroblast growth factor-2 and vascular endothelial growth factor are the angiogenic cytokines primarily and perhaps synergistically responsible for this vasoproliferative activity.

Allantois↗
PubMed2001

In vivo absence of synergism between fibroblast growth factor-2 and vascular endothelial growth factor.

Fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) are potent angiogenesis inducers in vivo and in vitro and may act in synergy. This possibility has been investigated by their simultaneous administration in the chick embryo chorioallantoic membrane (CAM) assay. Macroscopic and microscopic quantification of the angiogenic response 4 days after administration clearly demonstrated the absence of synergism. When FGF-2 or VEGF concentration was fixed at 0.25 microg/embryo, the simultaneous addition of increasing concentration (0.25, 0.50, 1.0 microg/embryo) of VEGF or FGF-2 did not stimulate a synergistic dose-dependent angiogenic response. In both conditions, the angiogenic response overlapped that induced by the two growth factors administered alone. It is suggested that exogenous administration of FGF-2 and VEGF in the CAM assay may induce an activation of endogenous angiogenic factors, such as FGF-2, and endogenous inhibitors of angiogenesis, such as nitric oxide, normally expressed in the CAM during the development of its vascular tree. Thus, in an in vivo system, evaluation of synergistic action between two cytokines and discrimination of their specific activity are more difficult than in an in vitro assay.

Animals↗
PubMed2001

Role of hematopoietic growth factors in angiogenesis.

In early ontogeny, hematopoiesis is closely associated with angiogenesis. This article reviews recent studies of the effect of hematopoietic growth factors on several endothelial cell functions together with recent findings about angiogenesis and antiangiogenic therapies in hematopoietic malignancies such as leukemia, lymphoma and myeloma.

Animals↗
PubMed2001

Bone marrow angiogenesis and plasma cell angiogenic and invasive potential in patients with active multiple myeloma.

Factor VIII-related antigen-positive microvessel areas were measured by both immunohistochemistry and computerized image analysis in patients with active multiple myeloma (MM), nonactive MM and monoclonal gammopathies of undetermined significance (MGUS). A 5- to 6-fold larger area was found in patients with active MM compared to the other two groups. The conditioned medium (CM) of their bone marrow plasma cells stimulated endothelial cell proliferation and chemotaxis, monocyte chemotaxis and angiogenesis in vivo [chick embryo chorioallantoic membrane (CAM) system] more strongly and frequently than the CM of patients with nonactive MM and MGUS. An immunoassay of plasma cell lysates gave significantly higher levels of fibroblast growth factor-2 (FGF-2) in patients with active MM than in the other two groups, and a neutralizing anti-FGF-2 antibody inhibited by 54-68% the biological activity exerted by the CM in vitro and in the CAM. In situ hybridization of bone marrow plasma cells and gelatin zymography of CM showed that patients with active MM express higher levels of matrix metalloproteinase-2 (MMP-2) mRNA and protein than those with nonactive MM and MGUS, whereas MMP-9 expression and secretion overlapped in all groups. Overall data suggest that patients with active MM represent the vascular phase of plasma cell tumors that is triggered by bone marrow plasma cells, at least partly, through FGF-2 and MMP-2. Both angiogenesis and MMP-2 secretion can account for intramedullary and extramedullary spreading of plasma cells during the active MM.

Bone Marrow↗
PubMed2001

Tissue distribution and membrane localization of aquaporin-9 water channel: evidence for sex-linked differences in liver.

Aquaporin-9 (AQP9) is a water channel membrane protein also permeable to small solutes such as urea, glycerol, and 5-fluorouracil, a chemotherapeutic agent. With the aim of understanding the pathophysiological role of AQP9, we performed an extensive analysis by Western blotting, RT-PCR, and immunolocalization in rat tissues. Western blotting analysis revealed a major band of approximately 32 kD in testis, liver, and brain. Immunofluorescence showed strong expression of AQP9 in the plasma membrane of testis Leydig cells. In liver, AQP9 expression was found to be sex-linked. Male rats had higher levels of AQP9 than female in terms of both protein and mRNA. Moreover, in female livers the expression of AQP9 was mostly confined to perivascular hepatocytes, whereas males showed a more homogeneous hepatocyte staining. No differences in AQP9 expression level related to the age or to protein content of the diet were found, indicating that differences in the liver may be gender-dependent. In the brain, AQP9 expression was found in tanycytes mainly localized in the areas lacking a blood-brain barrier (BBB), such as the circumventricular organs (CVOs) of the third ventricles, the subfornical organ, the hypothalamic regions, and the glial processes of the pineal gland. AQP9 expression in the osmosensitive region of the brain suggests a role in the mechanism of central osmoreception. All these findings show a unique tissue distribution of AQP9 compared to the other known aquaporins.

Animals↗
PubMed2001

Role of aquaporin-4 water channel in the development and integrity of the blood-brain barrier.

In this study, we have investigated the expression of aquaporin 4 during blood-brain barrier development in the optic tectum of chick embryos and newly hatched chicks, by means of western-blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and freeze-fracture and high-resolution immunogold electron microscopy. In the optic tecta of day-14 embryos, western blot analysis revealed an approx. 30 kDa band, immunoreactive for aquaporin-4, which was increased in day-20 embryos and in chicks. Semi-quantitative reverse transcriptase chain reaction experiments showed that there was already a high level of aquaporin-4 mRNA in day-9 embryos as well as in the subsequent stages and in newly hatched chicks. Immunohistochemically, reactivity for aquaporin-4 was detected in the optic tectum of day-14 embryos; similar results were obtained in telencephalon and cerebellum. Ultrastructurally, the microvessels of the tectum showed immunoreactivity for aquaporin-4 on the astroglial endfeet, which discontinuously surrounded endothelial cells joined by immature tight junctions. In the tectum, telencephalon and cerebellum of 20-day embryos and chicks, aquaporin-4 strongly labeled the ependymal cells and the subpial glial membranes, as well as the bodies and processes of astroglial cells. A continuous aquaporin-4 staining was found around the microvessel endothelial cells, which were sealed off from one another by extensive tight junctions. A complete astrocytic sheath, labeled by anti-aquaporin-4 gold particles, enveloped the endothelium-pericyte layer. Orthogonal arrays of particles were observed on fractured astrocytic membranes, starting from embryonic day 14 when the aquaporin-4 immunogold staining revealed clusters of gold particles, often forming square or rectangular clusters. The results showed that aquaporin-4 expression and organization of the intramembrane particles in orthogonal arrays followed the same temporal sequence. Finally, the lipopolysaccharide, a substance that induces blood-brain barrier disruption, determines a remarkable reduction in aquaporin-4 labeling, expressed by a few aquaporin-4 gold particles attached on swollen perivascular glial membranes. All these data show that aquaporin-4 expression occurs in the chick embryonic brain, in parallel with maturation and functioning of the blood-brain barrier and suggest that there is a close relationship between water transport regulation and brain development.

Animals↗
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↗
PubMed2001

Coordinate immunoreactivity to vascular endothelial growth factor receptor-2 and its ligand suggests a paracrine regulation during the development of the vascular system in the chick embryo bursa of Fabricius.

The bursa of Fabricius is a lymphoid organ of the chick which plays an important role in the development of the immune system. The role of angiogenic factors in the development of the vascular system of this organ has been poorly investigated. Vascular endothelial growth factor (VEGF) is a major regulator of endothelial cell proliferation, angiogenesis and vascular permeability, and its activities are mediated by two receptors, VEGFR-1 and VEGFR-2. In this study we have investigated by immunohistochemistry the VEGF and VEGFR-2 immunoreactivity in developing bursa of Fabricius. Starting from day 10 of incubation, the endodermal epithelium reacts with VEGF and gives rise to the lymphoid follicles, while the vascular endothelium reacts with VEGFR-2. These data support the view that VEGF acts as a paracrine stimulator of angiogenesis in the avian embryo and confirm the requirement of the endodermal layer for the normal formation of blood vessels by mesodermal cells.

Animals↗
PubMed2001

Angiogenic activity of leptin in the chick embryo chorioallantoic membrane is in part mediated by endogenous fibroblast growth factor-2.

Recently, it has been demonstrated that leptin, the product of the ob gene, playing a key role in the regulation of body weight, is angiogenic in vitro and in vivo. In this study we investigated the angiogenic potential of human leptin in vivo by using the chick embryo chorioallantoic membrane (CAM) assay, with the aim to establish whether this angiogenic activity is partly dependent on endogenous fibroblast growth factor-2 (FGF-2), which is normally expressed during CAM development. Results showed that leptin is able to stimulate angiogenesis and that the angiogenic response is similar to that obtained with FGF-2. The stimulating property of leptin is specific, as the application of anti-leptin antibodies onto the CAM significantly inhibits the angiogenic response. Moreover, this angiogenic activity is in part due to the activation of endogenous FGF-2. The application of anti-FGF-2 antibodies reduces the angiogenic response to leptin by 40%. Our study confirms that leptin is angiogenic in vivo and suggests that, at least in the chick CAM, its activity is in part mediated by the activation of endogenous FGF-2.

Allantois↗
PubMed2000

I-309 binds to and activates endothelial cell functions and acts as an angiogenic molecule in vivo.

Several chemokines have been shown to act as angiogenic molecules or to modulate the activity of growth factors such as fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor (VEGF). The detection of the CC chemokine receptor (CCR) 8 message in human umbilical vein endothelial cells (HUVECs) by reverse transcription- polymerase chain reaction (RT-PCR) and RNase protection assay (RPA), prompted us to investigate the potential role exerted by the CC chemokine I-309, a known ligand of such receptor, in both in vitro and in vivo angiogenesis assays. We show here that I-309 binds to endothelial cells, stimulates chemotaxis and invasion of these cells, and enhances HUVEC differentiation into capillary-like structures in an in vitro Matrigel assay. Furthermore, I-309 is an inducer of angiogenesis in vivo in both the rabbit cornea and the chick chorioallantoic membrane assay (CAM).

Allantois↗
PubMed2000

Inhibition of protein kinase C counteracts TNFalpha-induced intercellular adhesion molecule 1 expression and fluid phase endocytosis on brain microvascular endothelial cells.

TNFalpha (100 U/ml, 24 h) upregulated intercellular adhesion molecule 1 (ICAM1) expression and fluid phase endocytosis (FPE) of horseradish peroxidase on brain microvascular endothelial cell (BMEC) culture. The protein kinase C (PKC) inhibitor staurosporin (0. 5-10 nM) antagonized ICAM1 expression and FPE due to TNFalpha, whereas the protein kinase A inhibitor H89 (0.5-10 nM) did not. These findings indicate that a PKC-dependent mechanism may affect TNFalpha signalling on different barrier properties of BMECs.

Animals↗