Biomedical subjects

L Roncali

Publications and source records attributed to L Roncali.

At least 37 records · Page 2Linked to original sources

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↗

PubMed2001

Developmental effects of lead acetate on the chick embryo metanephros.

The developmental effects of lead acetate were studied in the chick embryo metanephros, the third renal rudiment that acquires morphological characteristics of functioning kidney already during the prenatal life. Lead exposure was obtained by applying a lead acetate solution on the chick embryo chorioallantoic membrane at the days 9, 10 and 11 of incubation. Quantitative evaluation of the lead concentration assessed by furnace atomic absorption spectrophotometry at the days 14 and 21 of incubation demonstrated metal presence both in the chorioallantoic membrane (CAM) and in metanephros (MN). The lead concentration was higher in CAM than in MN; the metal amount was similar in the CAM of 14 and 21 day embryos, but significantly higher in the 14day embryo MN than in the 21 day embryo MN. Morphological observations on metanephros tissue of control and lead-treated embryos were performed under light, electron transmission and electron scanning microscopes. Peculiar attention was devoted to the expression of the junctional protein connexin 43, the major component of the gap junctions in the renal cells. The results indicated that lead treatment does not intervene in the general differentiation of the metanephric nephrons. The lead is reabsorbed by the proximal tubule cells that are engulfed by endocytotic vacuoles and metal deposits and show long term degenerative changes. Expression of Cx43 protein and ultrastructure of gap junctions between proximal tubule cells appeared to be unchanged. The morphological aspects of the MN corpuscles and tubules agree with the suggestion of a lead cytotoxic effect but do not corroborate, at least in this experimental model, the view of primary damage exerted by lead on the gap junctions of the renal epithelial cells.

Abnormalities, Drug-Induced↗

PubMed2000

Immunogold cytochemistry of the blood-brain barrier glucose transporter GLUT1 and endogenous albumin in the developing human brain.

The blood-brain barrier (BBB) glucose transporter, GLUT1, was detected by immunogold electron microscopy on the microvascular compartment of the human foetus telencephalon at the 12th and 18th weeks of gestation. By computerized morphometry, the cellular and subcellular localization of the immunosignal for GLUT1 was quantitatively evaluated. The study showed that the glucose transporter is strongly expressed by endothelial cells while a very low signal is detected on vascular pericytes. The GLUT1 antigenic sites are preferentially associated to the ablumenal and junctional plasma membranes of the endothelial cells and tend to increase significantly with age. A parallel study carried out by the endogenous serum protein albumin demonstrated that already at the 12th week the endothelial routes are hindered to the protein as happens at the blood-endothelium interface of mature brain. The results demonstrate that in the human foetus the brain microvessels express BBB-specific functional activities early.

Blood-Brain Barrier↗

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↗

PubMed2000

Interferon beta-1a prevents the effects of lipopolysaccharide on embryonic brain microvessels.

By means of light and electron microscopy we have studied the effect of interferon beta-1a (IFNbeta-1a) in the optic tecta of 20-day-old chick embryos under normal conditions and after exposure to lipopolysaccharide (LPS) which mimics the blood-brain barrier (BBB) disruption in meningoencephalitis. Optic tecta were examined for: (i) ultrastructure by means of transmission electron microscopy; (ii) the immunohistochemical localization of HT7 antigen, a specific marker of differentiation of the brain microvessels; (iii) the brain microvessel permeability, by means of horseradish peroxidase (HRP) tracer; (iv) the expression of microvessel glycoconjugates, by means of lectin histochemistry, using Ricinus communis agglutinin-I (RCA-I), specific for beta-D-galactosyl moieties and Wheat Germ agglutinin (WGA) specific for sialyl and N-acetylglucosaminyl moieties. A morphometric evaluation of brain microvessel permeability and of glycoconjugate expression was also performed. In control- and in IFNbeta-1a-treated embryos, HRP was confined to the vessel lumina which were sealed by the interendothelial tight junctions. RCA-I binding sites were recognizable both in the basal membranes and in the tight junctions, while WGA sites were present on the luminal side of the endothelial cells. HRP was blocked in the vessels lumina by the interendothelial tight junctions. After LPS treatment, HRP showed an extravascular localization and the labeling of microvessels by anti-HT7 antibodies disappeared. RCA-I binding was only found ultrastructurally and appeared as irregularly clustered gold particles, in the cleft of damaged tight junctions, but were no longer detectable in the endothelial basement membranes. After pretreatment of LPS-treated embryos with IFNbeta-1a, the vessel permeability to HRP strongly decreased and the vessels showed the normal pattern of HT7 protein and of the RCA-I binding sites. These results indicate that the changes induced by LPS in the endothelial cells are prevented by IFNbeta-1a.

Animals↗

PubMed2000

Angiogenesis and mast cell density with tryptase activity increase simultaneously with pathological progression in B-cell non-Hodgkin's lymphomas.

Node biopsies of 16 benign lymphadenopathies and 72 B-cell non-Hodgkin's lymphomas (B-NHLs) were investigated for counts of microvessels, total metachromatic mast cells (MCs) and MCs expressing tryptase, an angiogenesis-inducing molecule. Counts were higher in B-NHLs. When grouped according to the Working Formulation (WF) malignancy grades, they were significantly higher in low-grade B-NHLs vs. lymphadenopathies and intermediate-grade vs. low-grade tumors and there was a further increase in the high-grade tumors. A high correlation was demonstrated in all groups of tissues between microvessel counts and both total metachromatic and tryptase-reactive MCs. These results suggest that angiogenesis in B-NHLs increases with their progression, and that MCs cooperate in its induction via the tryptase contained in their secretory granules.

Chymases↗

PubMed2000

Aquaporin-4-containing astrocytes sustain a temperature- and mercury-insensitive swelling in vitro.

In order to understand the molecular mechanism underlying astroglial swelling, we studied primary astrocyte cultures from newborn mouse and analyzed them for expression of functional water channels. Immunocytochemical analysis of mouse brain confirms the presence of AQP4 location in astrocytic endfeet with a polarized pattern, as found in rat. Using Southern blot PCR and Western blot analysis, we demonstrate that primary astrocyte cultures from mouse express the AQP4 water channel at both the RNA and protein levels. Two polypeptides, of 30 kDa and 32 kDa, were identified in the astrocytes. Densitometric analysis demonstrates that the 32-kDa form represents 25% of the total AQP4 protein. Moreover, immunofluorescence experiments show strong surface membrane expression of AQP4 protein in cultured cells, even though the polarity of the expression is not maintained. Furthermore, functional studies indicate that cultured astrocytes manifest rapid and temperature-independent volume changes in response to osmotic gradients, in agreement with a channel-mediated water transport. Water movement was found to be HgCl(2) insensitive, suggesting AQP4 and AQP7 as putative water channels. Using Western blot and PCR experiments, we exclude the presence of AQP7 in astrocytes, indicating that only AQP4 is responsible for the rapid water movement. Altogether, the results indicate that primary astrocyte cultures are a valid cell model for further investigation of the molecular mechanism of water movement in the brain and its physiological regulation.

Animals↗

PubMed2000

An experimental study in the chick embryo chorioallantoic membrane of the anti-angiogenic activity of cyclosporine in rheumatoid arthritis versus osteoarthritis.

OBJECTIVE AND DESIGN: Angiogenesis plays an important role in the pathogenesis of rheumatoid arthritis (RA) and correlates with clinical score, synovial hyperplasia and infiltration of inflammatory cells. Many of the available treatments for RA have been shown to possess some degree of anti-angiogenic activity. Here, we studied the effect of cyclosporine, which exerts anti-angiogenic activity in vitro and in vivo [1] on angiogenesis induced in vivo in the chick embryo chorioallantoic membrane (CAM) by synovial RA and osteoarthritis (OA) tissues. MATERIAL AND METHODS: Wet synovial biopsies from 10 RA and 6 OA patients were treated with vehicle alone or with cyclosporine and implanted on the CAM at day 8 of incubation. On day 12, CAM tissues were assessed for the extent of angiogenesis and mononuclear cell infiltration. RESULTS: Cyclosporine inhibited angiogenesis and reduced the number of mononuclear cells in the CAM extracellular matrix only in RA implants. CONCLUSIONS: These data provide further evidence for a central role of new-formed blood vessels in RA. Moreover, cyclosporine on account of both its immunosuppressive and its anti-angiogenic activity can be proposed for the treatment of RA.

Allantois↗

PubMed2000

Immunolocalization of glutamic acid decarboxylase in postmortem human cerebellar cortex. A light microscopy study.

The distribution of glutamic acid decarboxylase (GAD), the gamma-aminobutyric acid (GABA) synthesizing enzyme, was examined in the postmortem human cerebellar cortex by immunocytochemistry. The results, obtained on nervous tissues taken at autopsy and fixed within 24-36 h after death, enabled the authors to precisely reveal the topographical distribution of GAD-containing neurons and axon terminals in the human cerebellar cortex. Labeled neurons, corresponding to different neuronal cytotypes of the cerebellar cortex, showed a diffuse cytoplasmic immunoreactivity in both bodies and processes. Labeled axon terminals appeared as immunoreactive puncta. The use of immunocytochemistry in the detection of GAD in the postmortem human brain greatly increases the possibility of carrying out morphological studies on the GABAergic system, both in normal and in pathological conditions.

Adult↗

PubMed2000

Glutamic acid decarboxylase-positive neuronal cell bodies and terminals in the human cerebellar cortex.

The distribution of gamma-aminobutyric acid (GABA) in the human cerebellar cortex was studied using immunohistochemistry for glutamic acid decarboxylase (GAD), the enzyme that catalyses GABA synthesis. Observations by light microscopy revealed, in all layers of the cerebellar cortex, strong, punctate positivity for GAD, related to putative GABAergic nerve terminals, as well as a diffuse cytoplasmic immunoreactivity within neuronal cell bodies. GAD-positive nerve terminals were found in close relationship with the walls of the cerebellar cortex microvessels. Observations by electron microscopy revealed positive nerve terminals in contact with the astrocyte perivascular sheath of capillaries. GAD immunoreactivity was also detected within astroglial perivascular endfeet and endothelial cells. The findings provide further insights into the GABAergic synapses of the circuitry of the human cerebellar cortex. The detection of 'vascular' GAD immunoreactivities suggests that GABAergic mechanisms may regulate cerebellar microvessel function.

Aged↗

PubMed2000

Hematopoiesis and angiogenesis: a link between two apparently independent processes.

In early ontogeny, hematopoiesis is closely associated with angiogenesis. This article reviews recent studies on the role of angiogenic factors that regulate the proliferation and differentiation of endothelial cells in promoting hematopoietic cell growth and studies on the ability of hematopoietic cytokines to affect several endothelial cell functions. The findings in all these studies support the hypothesis formulated at the beginning of this century that a common ancestral cell, the hemangioblast, gives rise to cells of both the endothelial and the hematopoietic lineages.

Animals↗

PubMed2000

The chick embryo chorioallantoic membrane as a model for in vivo research on anti-angiogenesis.

Anti-angiogenesis, i.e. inhibition of blood vessel growth, is being investigated as a way to prevent the growth of tumors and other angiogenesis-dependent diseases. Pharmacological inhibition interferes with the angiogenic cascade or the immature neovasculature with synthetic or semi-synthetic substances, endogenous inhibitors or biological antagonists. The chick embryo chorioallantoic membrane (CAM) is an extraembryonic membrane commonly used in vivo to study both new vessel formation and its inhibition in response to tissues, cells, or soluble factors. Angiogenesis or anti-angiogenesis is evaluated quantitatively or semiquantitatively. The fields of application of CAM in the study of anti-angiogenesis, including our personal experience, are illustrated in this paper.

Allantoin↗

PubMed2000

Cholinergic nerve fibres associated with the microvessels of the human cerebral cortex: a study based on monoclonal immunocytochemistry for choline acetyltrasferase.

The distribution of cholinergic nerve fibres associated with the microvasculature of the human parietal cerebral cortex was investigated by immunocytochemistry, employing monoclonal antibodies against choline acetyl-transferase, the acetylcholine-synthesizing enzyme. The results revealed strongly immunoreactive nerve fibres in the tunica adventitia of arterioles penetrating the superficial cortical layers from the pial vasculature. Networks of stained nerve fibres were seen within the tunica muscularis of the radially directed arterioles that cross the intermediate and deep cortical laminae, and of their transverse and recurrent branches. Tiny positive nerve fibres were also seen around the cortex capillaries, some reaching the endothelial cells. The morphological data support the involvement of acetylcholine in microvasculature local regulation, possibly with a differentiated role in the arterioles and capillaries.

Antibodies, Monoclonal↗

PubMed1999

Developmental expression of ZO-1 antigen in the mouse blood-brain barrier.

Tight junction biogenesis during blood-brain barrier development (BBB) in mesencephalon microvessels of mouse embryos of day 9, foetuses of day 15 and 19 and new-born (2-day-old) mice was examined by light and electron microscopy, using monoclonal antibodies recognizing the tight junction peripheral membrane protein ZO-1. A faint spot-like staining began to be recognizable under the light microscope in day 15 vessels in which the endothelial cells showed isolated fusion points between the external plasma membrane leaflets under the electron microscope. A stronger labelling was present in microvessels of day 19 foetuses and new-born animals when the endothelial tight junction appeared completely differentiated. In the immunogold study, gold particles were seen scattered throughout the cytoplasm of endothelial cells of day 15 foetuses. In day 19 foetuses and in the new-born mice, gold particles were located only at the cytoplasmic surfaces of the tight junctions. The results indicate that the ZO-1 protein is a specific molecular marker in the developing brain endothelial tight junctions and that its expression takes place parallel to BBB morphofunctional maturation.

Aging↗

PubMed1999

Human erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo.

Hematopoietic and endothelial cell lineages share common progenitors. Accordingly, cytokines formerly thought to be specific for the hematopoietic system have been shown to affect several functions in endothelial cells, including angiogenesis. In this study, we investigated the angiogenic potential of erythropoietin (Epo), the main hormone regulating proliferation, differentiation, and survival of erythroid cells. Epo receptors (EpoRs) have been identified in the human EA.hy926 endothelial cell line by Western blot analysis. Also, recombinant human Epo (rHuEpo) stimulates Janus Kinase-2 (JAK-2) phosphorylation, cell proliferation, and matrix metalloproteinase-2 (MMP-2) production in EA.hy926 cells and significantly enhances their differentiation into vascular structures when seeded on Matrigel. In vivo, rHuEpo induces a potent angiogenic response in the chick embryo chorioallantoic membrane (CAM). Accordingly, endothelial cells of the CAM vasculature express EpoRs, as shown by immunostaining with an anti-EpoR antibody. The angiogenic response of CAM blood vessels to rHuEpo was comparable to that elicited by the prototypic angiogenic cytokine basic fibroblast growth factor (FGF2), it occurred in the absence of a significant mononuclear cell infiltrate, and it was not mimicked by endothelin-1 (ET-1) treatment. Taken together, these data demonstrate the ability of Epo to interact directly with endothelial cells and to elicit an angiogenic response in vitro and in vivo and thus act as a bona fide direct angiogenic factor.

Allantois↗

PubMed1999

Alterations of blood vessel development by endothelial cells overexpressing fibroblast growth factor-2.

A close relationship exists between angiogenesis and the formation of vascular lesions. The development of the vascular system in the chick embryo chorioallantoic membrane (CAM) may thus represent a model to study the effects of the deregulation of endothelial cell behaviour. Alterations of the developing vascular tree of the CAM were observed after exposure to murine aortic endothelial (MAE) cells overexpressing human fibroblast growth factor-2 (FGF2) cDNA (pZipFGF2 MAE cells), or to their conditioned medium (CM). pZipFGF2 MAE cells injected into the allantoic sac or applied on to the CAM of day 8-9 chick embryos induce neovascularization and the appearance of haemangioma-like lesions. This activity was not prevented by anti-FGF2 antibodies. The CM from pZipFGF2 MAE cells was also active when adsorbed into a gelatin sponge and applied on to the CAM, both in the absence and in the presence of anti-FGF2 antibodies. No effects on vessel development were exerted by parental MAE cells, FGF2-transfected NIH 3T3 fibroblasts, or their conditioned media. In vitro, pZipFGF2 MAE cell CM caused parental MAE cells to invade fibrin gels and to undergo morphogenesis on Matrigel. This activity was not mimicked by recombinant FGF2 nor affected by anti-FGF2 antibodies, and depended on a M (r) approximately 45 000 heat-labile heparin-binding factor. Size exclusion chromatography of pZipFGF2 MAE cell CM demonstrated that the in vitro activity co-purified with an in vivo angiogenic capacity. Thus, FGF2 overexpression in mouse endothelial cells induces the production of an angiogenic activity distinct from FGF2, which may contribute to the genesis of angioproliferative lesions.

Allantois↗

PubMed1999

Immunohistochemical and ultrastructural characterization of cortical plate microvasculature in the human fetus telencephalon.

The blood-brain barrier (BBB) differentiation was investigated by immunohistochemistry and electron microscopy in the radial microvasculature of the telencephalon cortical plate (CP) of 12- and 18-week human fetuses. The BBB-specific glucose transporter isoform 1 (GLUT1) is expressed in both stages, with a main localization on the ablumenal and lateral plasma membranes of the endothelial cells. The endothelial cells are welded by short junctions with fusion points of the plasma membranes at 12 weeks and by extensive tight junctions at 18 weeks. The basal lamina is discontinuous beneath the endothelium-pericyte layer at 12 weeks and splits into two continuous layers circumscribing the pericytes in the later stage. The expression of laminin, a basal lamina glycoprotein, is continuous already at 12 weeks. The CP microvessels are tightly surrounded by processes of glial cells. Immunodetection of the cytoskeletal filament proteins, vimentin (VIM), and glial fibrillary acidic protein (GFAP), demonstrates that at 12 weeks the perivascular glial processes are mostly represented by VIM-stained fibers of the radial glia. At 18 weeks, GFAP-stained radial glia fibers, processes of VIM-stained astroblasts, and GFAP-positive astrocytes also build the perivascular envelopes. The results indicate that the vessel differentiation is already under way in the human CP at the midgestational age and entails the establishment of some barrier devices. The early relationship between perivascular glia coverage formation and endothelial barrier maturation suggests that also immature astroglial cells are involved in the setting up of the BBB.

Adult↗