Biomedical subjects

Luisa Roncali

Publications and source records attributed to Luisa Roncali.

11 recordsLinked to original sources

PubMed2003

Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice.

In this study, we investigated the involvement of the blood-brain barrier (BBB) in the brain of the dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy (DMD). To this purpose, we used two tight junction markers, the Zonula occludens (ZO-1) and claudin-1 proteins, and a glial marker, the aquaporin-4 (AQP4) protein, whose expression is correlated with BBB differentiation and integrity. Results showed that most of the brain microvessels in mdx mice were lined by altered endothelial cells that showed open tight junctions and were surrounded by swollen glial processes. Moreover, 18% of the perivascular glial endfeet contained electron-dense cellular debris and were enveloped by degenerating microvessels. Western blot showed a 60% reduction in the ZO-1 protein content in mdx mice and a similar reduction in AQP4 content compared with the control brain. ZO-1 immunocytochemistry and claudin-1 immunofluorescence in mdx mice revealed a diffuse staining of microvessels as compared with the control ones, which displayed a banded staining pattern. ZO-1 immunogold electron microscopy showed unlabeled tight junctions and the presence of gold particles scattered in the endothelial cytoplasm in the mdx mice, whereas ZO-1 gold particles were exclusively located at the endothelial tight junctions in the controls. Dual immunofluorescence staining of alpha-actin and ZO-1 revealed colocalization of these proteins. As in ZO-1 staining, the pattern of immunolabeling with anti-alpha-actin antibody was diffuse in the mdx vessels and pointed or banded in the controls. alpha-actin immunogold electron microscopy showed gold particles in the cytoplasms of endothelial cells and pericytes in the mdx mice, whereas alpha-actin gold particles were revealed on the endothelial tight junctions and the cytoskeletal microfilaments of pericytes in the controls. Perivascular glial processes of the mdx mice appeared faintly stained by anti-AQP4 antibody, while in the controls a strong AQP4 labeling of glial processes was detected at light and electron microscope level. The vascular permeability of the mdx brain microvessels was investigated by means of the horseradish peroxidase (HRP). After HRP injection, extensive perivascular areas of marker escape were observed in mdx mice, whereas HRP was exclusively intravascularly localized in the controls. Inflammatory cells, CD4-, CD8-, CD20-, and CD68-positive cells, were not revealed in the perivascular stroma of the mdx brain. These findings indicate that dystrophin deficiency in the mdx brain leads to severe injury of the endothelial and glial cells with disturbance in alpha-actin cytoskeleton, ZO-1, claudin-1, and AQP4 assembly, as well as BBB breakdown. The BBB alterations suggest that changes in vascular permeability are involved in the pathogenesis of the neurological dysfunction associated with DMD.

Actins↗

PubMed2002

Vascular endothelial growth factor and vascular endothelial growth factor receptor-2 expression in mdx mouse brain.

Recent data have demonstrated that vascular endothelial growth factor (VEGF) is expressed by subsets of neurons, coincident with angiogenesis within its developing cerebral cortex. In this study, with the aim of elucidating the mechanisms of vascular involvement during brain impairment in Duchenne muscular distrophy (DMD), we have correlated the vascular density with VEGF and VEGF receptor-2 (VEGFR-2) expression in the brain cortex of normal and mdx mouse, an animal model with a genetic defect in a region homologous with the human DMD gene. Results showed that in mdx mouse, tissue area occupied by microvessels positive to factor VIII related antigen and VEGFR-2 increased in parallel to the tissue area occupied by neurons positive to VEGF. Our data suggest that increased vascularity in the brain of mdx mouse may be due, at least in part, to proliferation of endothelial cells in response to VEGF secreted by neuronal cells.

Animals↗

PubMed2002

Aquaporin-1 expression in the chick embryo chorioallantoic membrane.

The chick embryo chorioallantoic membrane (CAM) is commonly used in vivo to study both angiogenesis and anti-angiogenesis. Rapid membrane water transport is mediated by a family of molecular water channels, called aquaporins (AQPs), which have been identified in the epithelial and endothelial cells of higher vertebrates. AQP1, expressed in adsorptive and secretory epithelia, is also expressed in endothelial cells of capillaries and arteries. Its mRNA has been found in vascular smooth muscle cells (VSMCs) of arteries and capillaries, as well as in a subset of VSMCs of human atherosclerotic plaques. This study investigated the developmental expression of AQP1 in the chick CAM by Western blot and immunohistochemistry. Western blot results show that a major nonglycosylated band was observed with electrophoretic mobility of approximately 28 kDa in the three developmental stages examined. Immunohistochemistry data demonstrate that AQP1 was clearly expressed in the ectodermal and endodermal epithelia, the vascular endothelium, and the VSMCs. Because little information is available on the behavior of microvessel AQP1 during angiogenesis in normal and pathological conditions, our data relative to the pattern of expression of AQP1 in CAM blood vessels in normal conditions may be considered a useful tool to further investigate its modifications in several experimental conditions implying a stimulation or an inhibition of angiogenesis in the CAM assay.

Animals↗

PubMed2002

Developmental changes of HT7 expression in the microvessels of the chick embryo brain.

The neurothelin/HT7 antigen is a chick-specific, cell-surface glycoprotein expressed by the brain endothelial cells and widely utilized in experimental studies as a marker of barrier-provided vessels. Previous immunohistochemical studies have demonstrated that HT7 is already expressed in the embryonic brain vessels and that it is first detectable on embryonic day 10 and developmentally regulated. In the present study, the vascular expression of HT7 was investigated in different regions of the central nervous system from the 5th day right up to the latest stage of the chick embryo development. The study was carried out utilizing a monoclonal antibody anti-HT7, which was detected by both enzymatic and fluorescent immunohistochemical methods. The observations demonstrated the presence of HT7-stained vessels as early as embryonic day 6 in the rhombencephalon and mesencephalon, and at embryonic day 9 in the prosencephalon. Regional differences were also evidenced within the rhombencephalon and mesencephalon, since the endothelial antigen HT7 was expressed earlier in the brain stem (tegmentum of the medulla oblongata, pons and mesencephalon) than in the cerebellum and optic tectum. The caudo-cranial and ventro-dorsal gradients of HT7 expression were temporally and spatially related to the development of the brain vessels. The early detection of HT7 staining in the choroid plexus epithelium and perineural vessel endothelium, sites of the blood-cerebrospinal fluid barrier and pial barrier, respectively, has confirmed this antigen to be a precocious marker for all the barrier systems in the brain.

Animals↗

PubMed2002

In situ hybridization and immunogold localization of vascular endothelial growth factor receptor-2 on the pericytes of the chick chorioallantoic membrane.

This paper describes the expression of VEGF and of VEGFR-2 in the vasculature of the chorioallantoic membrane (CAM) as revealed by in situ hybridization and immunoelectron microscopy. Results showed that VEGFR-2 is expressed in both the endothelial cells and the pericytes, while VEGF in the chorionic epithelial cells. VEGF may therefore be released to promote both angiogenesis, by initiating an angiogenic response by endothelial cells expressing VEGFR-2, and the recruitment of pericytes along the capillary wall, playing also a crucial role in maturation and stabilization of the CAM blood vessels.

Animals↗

PubMed2002

Aquaporin-4 expression during development of the cerebellum.

Aquaporins (AQPs) are small integral membrane proteins that provide a major pathway for water transport in many epithelial and endothelial cell types of several tissues. Aquaporin-4 (AQP4) is prevalently expressed in the brain and here we summarize data from our findings and from other literature concerning AQP4 expression in the cerebellum and the relationships between its expression and blood-brain barrier differentiation and integrity. The role of AQP4 in blood-brain barrier pathological conditions associated with an increased vascular permeability is also discussed. Overall, the data clearly indicate a close relationship between blood-brain barrier functioning and the control of water flux by astroglial cells and the crucial role played by AQP4 in this setting.

Animals↗

PubMed2002

Endothelial cell heterogeneity and organ specificity.

Endothelial cells consist of a heterogeneous population covering the entire inner surface of blood vessels. This review will focus on the factors influencing this heterogeneity including: (1) morphological and functional differences between large and small vessels and between cells derived from various microvascular endothelial beds; (2) the microenvironment and extracellular matrix modulating the phenotype; (3) different response to growth factors; (4) organ specificity reflecting the cumulative expression of post-translation modifications and also the expression of unique genes under the control of organ-specific regulatory elements; and (5) pathological conditions, such as tumor growth, which is accompanied by the development of a characteristic tumor vasculature and tumors formed by endothelial cells.

Animals↗

PubMed2002

Expression of P-glycoprotein in human cerebral cortex microvessels.

P-Glycoprotein (P-gp) is an ATP-dependent efflux transporter that extrudes non-polar molecules, including cytotoxic substances and drugs, from the cells. It was initially found in cancer cells and then was shown to be a normal component of complex transport systems working at the blood-brain barrier (BBB). Previous studies have demonstrated that, in the brain, P-gp is localized on the luminal plasmalemma of BBB endothelial cells and that it may interact with the caveolar compartment of these cells. The aim of this study was to identify the site of cellular expression of P-gp in human brain in situ and to morphologically determine whether an association may exist between P-gp and caveolin-1, a structural and functional protein of the caveolar frame. The study was carried out on human cerebral cortex by immunoconfocal microscopy with antibodies to both P-gp and caveolin-1. The results show that P-gp marks the microvessels of the cortex and that the transporter is localized in the luminal endothelial compartment, where it co-localizes with caveolin-1. The demonstration of this co-localization of P-gp with caveolin-1 contributes a morphological backing to biochemical studies on P-gp/caveolin-1 relationships and leads us to suggest that interactions between these molecules may occur at the BBB endothelia.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Angiogenic activity of rat mast cells in the chick embryo chorioallantoic membrane is down-regulated by treatment with recombinant human alpha-2a interferon and partly mediated by fibroblast growth factor-2.

BACKGROUND AND OBJECTIVES: Many data suggest that the density of mast cells (MC) is strongly correlated with the extent of both normal and pathologic angiogenesis, such as the vessel formation that occurs in chronic inflammatory diseases and tumors. We have previously demonstrated that isolated MC and their secretory granules, but not degranulated MC, induce an angiogenic response in the chick embryo chorioallantoic membrane (CAM) assay. DESIGN AND METHODS: The aim of this study was to investigate whether pre-treatment of MC with an anti-angiogenic molecule, namely recombinant human interferon-alpha2a (rhIFN-alpha2a), reduced the angiogenic activity of their conditioned media (CM) in the CAM assay. RESULTS: Our data indicate that rhIFN-alpha2a at 500-1000 IU is able to reduce the angiogenic activity of CM significantly. When MC were treated with rhIFN-alpha2a at 25-250 IU they retained their angiogenic activity. Addition of anti-fibroblast growth factor-2 (FGF-2) antibodies (but not anti-vascular endothelial growth factor) substantially reduced the angiogenic activity of CM treated with sub-optimal concentrations of rhIFN-alpha2a. INTERPRETATION AND CONCLUSIONS: FGF-2 may be the main angiogenic factor secreted by MC and higher concentrations of rhIFN-alpha2a possibly inhibit angiogenesis by blocking the actions of FGF-2 produced by MC. Finally, the morphologic features of MC treated with rhIFN-alpha2a, characterized by an atypical secretory pathway, are compatible with a slow release of the angiogenic cytokines stored in MC granules.

Allantois↗

PubMedDate not supplied

Differential expression of connexin43 in foetal, adult and tumour-associated human brain endothelial cells.

Connexin43 (Cx43), the main protein constituting the gap junctions between astrocytes, has previously been demonstrated in endothelial cells of somatic vessels where the intercellular coupling that it provides plays a role in endothelial proliferation and migration. In this study, Cx43 expression was analysed in human brain microvascular endothelial cells of the cortical plate of 18-week foetal telencephalon, in adult cerebral cortex and glioma (astrocytomas). The study was carried out by immunocytochemistry utilizing a Cx43 monoclonal antibody and a polyclonal antibody anti-GLUT1 (glucose transporter isoform 1) to identify the endothelial cells and to localize Cx43. Endothelial Cx43 is differently expressed in the cortical plate, cerebral cortex and astrocytoma. Within the cortical plate and tumour, Cx43 is highly expressed in microvascular endothelial cells whereas it is virtually absent in the cerebral cortex microvessels. The high expression of the gap junction protein in developing brain, as well as in brain tumours, may be related to the growth status of the microvessels during brain and tumour angiogenesis. The lack of endothelial Cx43 in the cerebral cortex is in agreement with the characteristics of the mature brain endothelial cells that are sealed by tight junctions. In conclusion, the results indicate that endothelial Cx43 expression is developmentally regulated in the normal human brain and suggest that it is controlled by the microenvironment in both normal and tumour-related conditions.

Adult↗

PubMedDate not supplied

Angiogenesis: basic and clinical aspects.

The cardiovascular system is the first functional organ system to develop in the vertebrate embryo. A widely accepted view is that blood vessels arise through two mechanisms during development, vasculogenesis and angiogenesis. New vessels in the adult arise mainly through angiogenesis, although vasculogenesis also may occur. The existence of a postnatal vasculogenesis is also supported by the evidence that both endothelial cells and endothelial precursor cells co-exist in the circulation. 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. Considerable benefit can be derived in the clinical setting from manipulating angiogenesis, either positively or negatively. There is a variety of important clinical situations in which it would be desiderable to promote angiogenic processes, such as situations in which it would be desiderable to promote angiogenic processes, such as for the induction of collateral vascularization in an ischemic heart or limb. Conversely, there are pathologic conditions in which preventing angiogenic processes could be useful in the treatment of a growing tumor or a chronic inflammatory process.

Angiogenesis Inhibitors↗