Biomedical subjects

Daniela Virgintino

Publications and source records attributed to Daniela Virgintino.

10 recordsLinked to original sources

PubMed2007

An intimate interplay between precocious, migrating pericytes and endothelial cells governs human fetal brain angiogenesis.

In order to better understand the process of angiogenesis in the developing human brain, we have examined the spatial relationship and relative contributions of endothelial cells and pericytes, the two primary cell types involved in vessel growth, together with their relation with the vascular basement membrane. Pericytes were immunolocalized through use of the specific markers nerve/glial antigen 2 (NG2) proteoglycan, endosialin (CD248) and the platelet-derived growth factor receptor beta (PDGFR-beta), while endothelial cells were identified by the pan-endothelial marker CD31 and the blood brain barrier (BBB)-specific markers claudin-5 and glucose transporter isoform 1 (GLUT-1). The quantitative analysis demonstrates that microvessels of the fetal human telencephalon are characterized by a continuous layer of activated/angiogenic NG2 pericytes, which tightly invest endothelial cells and participate in the earliest stages of vessel growth. Immunolabelling with anti-active matrix metalloproteinase-2 (aMMP-2) and anti-collagen type IV antibodies revealed that aMMP-2 producing endothelial cells and pericytes are both associated with the vascular basement membrane during vessel sprouting. Detailed localization of the two vascular cell types during angiogenesis suggests that growing microvessels of the human telencephalon are formed by a pericyte-driven angiogenic process in which the endothelial cells are preceded and guided by migrating pericytes during organization of the growing vessel wall.

Basement Membrane↗

PubMed2005

In vivo assessment of epichlorohydrin effects: the chorioallantoic membrane model.

BACKGROUND: Previous studies on the effects of the epichlorohydrin (ECH) epoxide demonstrated this compound's toxicity and mutagenicity and suggested a carcinogenic activity also in humans. To gain a better understanding of ECH effects in vivo, the substance was tested on developing tissues utilizing the chick embryo chorioallantoic membrane (CAM) assay. MATERIAL/METHODS: Gelatin sponges adsorbed with ECH were implanted onto nine-day CAMs. After five days the membranes were fixed, cut in serial sections, and stained with toluidine blue. Sections of the ECH-treated CAMs were also submitted to immunocytochemistry for the basal lamina glycoprotein laminin and the gap junction protein connexin 43 (Cx43). Control CAMs were treated with saline solution and submitted to identical procedures. RESULTS: ECH-treated CAMs displayed proliferation of both the epithelial layers and the mesenchyme cells and vessels. The laminin immunolabeling was interrupted beneath the ectoderm thickenings, which penetrated the mesenchyme. The endoderm showed papilloma-like formations and its laminin-positive basal membrane protruded toward the mesenchyme, together with clusters of endodermal cells. The mesenchyme showed increased numbers of cells and microvessels. These reactions were restricted to regions corresponding to the implant. Cx43 expression was strongly decreased in the ECH-treated CAMs compared with the controls, where the connexin punctate pattern regularly decorated the epithelial cell contours. CONCLUSIONS: The study confirms that ECH elicits tissue proliferation at the contact site and corroborates the suggestion of an ECH carcinogenic effect due to hallmarks of tumoral growth, such as angiogenesis, basal membrane alterations, and loss of intercellular communication via gap junctions.

Animals↗

PubMed2004

Involvement of metalloprotease-2 in the development of human brain microvessels.

The involvement of the metalloprotease-2 (MMP-2) in vessel development was investigated in the human telencephalon by double immunoreactions with antibodies to the enzyme, latent (proMMP-2) and active (aMMP-2) forms, and an antibody against collagen type IV, a constitutive component of the extracellular matrix (ECM) of the vessel basal lamina. MMP-2 is expressed in both 12- and 18-week telencephalic vessels, the proenzyme being mainly localised in endothelial cells and the active form prevailing in alpha-actin-reactive periendothelial cells identified as pericytes. Endothelial cells intensely positive for aMMP-2 were revealed in some microvessels and appeared locally associated with discontinuities of the collagen basal lamina. No detectable expression of MMP-2 was observed in perivascular glial processes revealed by vimentin/glial fibrillary acidic protein immunostainings. Double immunoreactions performed to further investigate telencephalon angiogenesis have demonstrated that both the endothelial cells and pericytes strongly express vascular endothelial growth factor (VEGF). Taken together, the results indicate that MMP-2 is largely involved in human brain angiogenesis and suggest that endothelial cells and pericytes tightly interplay in both angiogenesis mechanisms, by ECM proteolysis, and angiogenesis regulation, by local (autocrine/juxtacrine) VEGF action.

Actins↗

PubMed2004

Immunolocalization of tight junction proteins in the adult and developing human brain.

The formation of endothelial tight junctions (TJs) is crucial in blood-brain barrier (BBB) differentiation, and the expression and targeting of TJ-associated proteins mark the beginning of BBB functions. Using confocal microscopy, this study analyzed endothelial TJs in adult human cerebral cortex and the fetal telencephalon and leptomeninges in order to compare the localization of two TJ-associated transmembrane proteins, occludin and claudin-5. In the arterioles and microvessels of adult brain, occludin and claudin-5 form continuous bands of endothelial immunoreactivity. During fetal development, occludin and claudin-5 immunoreactivity is first detected as a diffuse labeling of endothelial cytoplasm. Later, at 14 weeks, the immunosignal for both proteins shifts from the cytoplasm to the interface of adjacent endothelial cells, forming a linear, widely discontinuous pattern of immunoreactivity that achieves an adult-like appearance within a few weeks. These results demonstrate that occludin and claudin-5 expression is an early event in human brain development, followed shortly by assembly of both proteins at the junctional areas. This incremental process suggests more rapid establishment of the human BBB, consistent with its specific function of creating a suitable environment for neuron differentiation and neurite outgrowth during neocortical histogenesis.

Aged↗

PubMed2003

Effects of 6-aminonicotinamide gliotoxin on blood-brain barrier differentiation in the chick embryo cerebellum.

The hypothesis of astroglial cell involvement in prenatal setting up of the blood-brain barrier (BBB) has been examined by producing glial degeneration in cerebellum of chicken embryos submitted to the action of gliotoxin 6-aminonicotinamide (6-AN), which was applied onto the embryo chorioallantoic membrane during both early and late embryonic development. The effects of 6-AN on the cerebellum astroglial cells and microvessels were analysed under the light microscope by immunostaining for 3CB2 (chick-specific glial marker) and HT7 (chick-specific marker of BBB-provided brain endothelia), under the electron microscope, as well as by the vascular permeability tracer horseradish peroxidase. The results, showing good suitability of the 6-AN model also when applied in early embryonic development, demonstrated a correlation between perivascular glia depletion and endothelial barrier impairment and suggested that astroglia play a role in the BBB prenatal differentiation.

6-Aminonicotinamide↗

PubMed2003

VEGF expression is developmentally regulated during human brain angiogenesis.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor working as an endothelial cell-specific mitogen and exerting a trophic effect on neurons and glial cells, both these activities being essential during central nervous system vascularisation, development and repair. The vascularisation of human telencephalon takes place by means of an angiogenic mechanism, which starts at the beginning of corticogenesis and actively proceeds up to the last neuronal migration, when the basic scheme of the vascular network has been drawn. Our study focused on VEGF during this critical developmental period with the aim of identifying the cells that express VEGF and of correlating the events of angiogenesis with the main events of cerebral cortex formation. The results show that in fetal human brain VEGF protein is located on multiple cell types, cells proper to the nervous tissue, neuroepithelial cells, neuroblasts and radial glia cells, and non-neuronal cells, endothelial and periendothelial cells. In these cells VEGF expression appears developmentally regulated and is correlated with angiogenesis, which in turn responds to the high metabolic demands of the differentiating neocortex.

Brain↗

PubMed2003

Transgenic mice expressing F3/contactin from the TAG-1 promoter exhibit developmentally regulated changes in the differentiation of cerebellar neurons.

F3/contactin (CNTN1) and TAG-1 (CNTN2) are closely related axonal glycoproteins that are differentially regulated during development. In the cerebellar cortex TAG-1 is expressed first as granule cell progenitors differentiate in the premigratory zone of the external germinal layer. However, as these cells begin radial migration, TAG-1 is replaced by F3/contactin. To address the significance of this differential regulation, we have generated transgenic mice in which F3/contactin expression is driven by TAG-1 gene regulatory sequences, which results in premature expression of F3/contactin in granule cells. These animals (TAG/F3 mice) display a developmentally regulated cerebellar phenotype in which the size of the cerebellum is markedly reduced during the first two postnatal weeks but subsequently recovers. This is due in part to a reduction in the number of granule cells, most evident in the external germinal layer at postnatal day 3 and in the inner granular layer between postnatal days 8 and 11. The reduction in granule cell number is accompanied by a decrease in precursor granule cell proliferation at postnatal day 3, followed by an increase in the number of cycling cells at postnatal day 8. In the same developmental window the size of the molecular layer is markedly reduced and Purkinje cell dendrites fail to elaborate normally. These data are consistent with a model in which deployment of F3/contactin on granule cells affects proliferation and differentiation of these neurons as well as the differentiation of their synaptic partners, the Purkinje cells. Together, these findings indicate that precise spatio-temporal regulation of TAG-1 and F3/contactin expression is critical for normal cerebellar morphogenesis.

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

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↗

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↗