The contribution of CXCL12-expressing radial glia cells to neuro-vascular patterning during human cerebral cortex development

This study was conducted on human developing brain by laser confocal and transmission electron microscopy (TEM) to make a detailed analysis of important features of blood-brain barrier (BBB) microvessels and possible control mechanisms of vessel growth and differentiation during cerebral cortex vascularization. The BBB status of cortex microvessels was examined at a defined stage of cortex development, at the end of neuroblast waves of migration, and before cortex lamination, with BBB-endothelial cell markers, namely tight junction (TJ) proteins (occludin and claudin-5) and influx and efflux transporters (Glut-1 and P-glycoprotein), the latter supporting evidence for functional effectiveness of the fetal BBB. According to the well-known roles of astroglia cells on microvessel growth and differentiation, the early composition of astroglia/endothelial cell relationships was analyzed by detecting the appropriate astroglia, endothelial, and pericyte markers. GFAP, chemokine CXCL12, and connexin 43 (Cx43) were utilized as markers of radial glia cells, CD105 (endoglin) as a marker of angiogenically activated endothelial cells (ECs), and proteoglycan NG2 as a marker of immature pericytes. Immunolabeling for CXCL12 showed the highest level of the ligand in radial glial (RG) fibers in contact with the growing cortex microvessels. These specialized contacts, recognizable on both perforating radial vessels and growing collaterals, appeared as CXCL12-reactive en passant, symmetrical and asymmetrical, vessel-specific RG fiber swellings. At the highest confocal resolution, these RG varicosities showed a CXCL12-reactive dot-like content whose microvesicular nature was confirmed by ultrastructural observations. A further analysis of RG varicosities reveals colocalization of CXCL12 with Cx43, which is possibly implicated in vessel-specific chemokine signaling

Neural crest cell-derived pericytes act as pro-angiogenic cells in human neocortex development and gliomas

Central nervous system diseases involving the parenchymal microvessels are frequently associated with a 'microvasculopathy', which includes different levels of neurovascular unit (NVU) dysfunction, including blood-brain barrier alterations. To contribute to the understanding of NVU responses to pathological noxae, we have focused on one of its cellular components, the microvascular pericytes, highlighting unique features of brain pericytes with the aid of the analyses carried out during vascularization of human developing neocortex and in human gliomas. Thanks to their position, centred within the endothelial/glial partition of the vessel basal lamina and therefore inserted between endothelial cells and the perivascular and vessel-associated components (astrocytes, oligodendrocyte precursor cells (OPCs)/NG2-glia, microglia, macrophages, nerve terminals), pericytes fulfil a central role within the microvessel NVU. Indeed, at this critical site, pericytes have a number of direct and extracellular matrix molecule- and soluble factor-mediated functions, displaying marked phenotypical and functional heterogeneity and carrying out multitasking services. This pericytes heterogeneity is primarily linked to their position in specific tissue and organ microenvironments and, most importantly, to their ontogeny. During ontogenesis, pericyte subtypes belong to two main embryonic germ layers, mesoderm and (neuro)ectoderm, and are therefore expected to be found in organs ontogenetically different, nonetheless, pericytes of different origin may converge and colonize neighbouring areas of the same organ/apparatus. Here, we provide a brief overview of the unusual roles played by forebrain pericytes in the processes of angiogenesis and barriergenesis by virtue of their origin from midbrain neural crest stem cells. A better knowledge of the ontogenetic subpopulations may support the understanding of specific interactions and mechanisms involved in pericyte function/dysfunction, including normal and pathological angiogenesis, thereby offering an alternative perspective on cell subtype-specific therapeutic approaches

Emerging roles of CXCL12-bearing microvesicles in glio-vascular communication during human brain development

The CXC chemokine axis formed by CXCL12 and its receptors CXCR4/CXCR7 is involved in CNS development enhancing migration and differentiation of neuronal precursors [1, 2]. According to these data, our recent studies have demonstrated that during human cerebral cortex development, radial glia (RG) cells express high levels of CXCL12, which finds its receptor on migrating postmitotic neuroblasts [3]. Moreover, during this first survey we have also revealed the ligand concentrated in RG processes and in astrocyte endfeet in contact with the microvessel wall. Features and distribution of these glio-vascular contacts have been further studied to ascertain the possible involvement of the glial CXC ligand/receptor system in vessel growth and differentiation. The study was carried out on human telencephalon by laser confocal and transmission electron microscopy to detect the expression of chemokine CXCL12 together with specific glio-vascular markers and reveal subcellular details of the identified cell structures. Immunolabelling for CXCL12 showed the highest level of RG CXCL12-enriched vascular contacts in the subcortical layers, where the chemokine concentrated in small swellings that appeared at intervals along the RG fibres. These RG varicosities formed en passant vascular contacts or asymmetrical enlargements that were seen to specifically bent and come in contact with the vessel wall. At the highest confocal resolution, both symmetrical and asymmetrical RG varicosities appeared filled with CXCL12 labelled dot-like structures that have been regarded as cell microvesicles (MVs). Their vesicular nature was confirmed by ultrastructural observations that allowed to recognize small membrane-bound MVs gathered in RG perivascular varicosities. Aspects of CXCL12 MV shedding also appeared a common feature of these specialized glio-vascular contacts confirming the hypothesis that RG-derived CXCL12 can be conveyed to the recipient vascular cells participating in glio-vascular communication and coordinating neuro-vascular interactions during cerebral cortex development and vascularization

Cell-to-cell communication within the neurovascular unit (NVU) in a model of cerebral cortex demyelination

The concept of neurovascular unit (NVU) emphasizes the critical role of cell-to-cell interaction and communication between glial, neuronal, and vascular cell components during blood-brain barrier (BBB) development, and in adult normal and pathological conditions. In this study we have analysed the involvement of the nerve glial antigen 2, NG2, a chondroitin sulphate proteoglycan, highly expressed in developing and adult CNS, in cell cross-talk within the NVU. During CNS development NG2 is expressed by activated pericyte and appears downregulated as these cells undergo terminal differentiation. NG2 has also been identified on the surface of oligodendrocyte precursor cells, OPCs, evenly distributed throughout the CNS already by the end of the first postnatal week in mice and throughout adulthood. In a previous study on cerebral cortex experimental autoimmune encephalomyelitis (EAE) in mice, we firstly observed and described the glia-limitans-like position of NG2-bearing OPCs that during neuroinflammation extend processes to the pial surface and acquire a perivascular arrangement, coming in contact with the wall of EAE cortex microvessels. With the aim of understanding if a subset of OPCs specifically contributes to the cell composition of the NVU during EAE, we have explored, by morphometric analyses applied to laser confocal microscopy, OPCs distribution and vascular relationships in the cerebral cortex of WT controls and naïve NG2KO and in EAE WT and EAE NG2KO mice, at both early (20 dpi) and late (40 dpi) disease stages. In EAE WT mice, juxtavascular (JV) and perivascular (PV) OPCs were identified in a higher number compared to healthy mice. On the contrary, absence of NG2 in EAE NG2 KO mice seemed to affect the proliferative response of OPCs, specifically inhibiting the emergence of the JV and PV OPC subsets. The results indicate that in WT mice during EAE, the NVU microenvironment, classically formed by perivascular astrocytes, receives the insertion of OPCs as a specific vascular subset and suggest NG2 as the molecule involved in the observed NVU damage

BBB-endothelial tight junction response to NG2 in murine model of chronic EAE

The blood brain barrier (BBB) phenotype of brain endothelial cells (ECs) is the result of the influence and interaction from all the cell components of neurovascular unit (pericytes, astrocytes, oligodendrocytes, microglia, neurons) and basal lamina molecules. Pericyte-associated NG2, a transmembrane chondroitin sulphate proteoglycan, modulates EC proliferation and migration through its interaction with the involved cell growth factors and receptors (Fukushi et al., 2004). Our previous studies carried out on a model of cerebral cortex EAE (experimental autoimmune encephalomyelitis), induced by MOG in C57BL/6 mice, demonstrated the impairment of BBB-microvessels with dismantled tight junction (TJ) strands and scarce perivascular infiltrations (Errede et al., 2012). Interestingly, the datum of a minimal inflammatory infiltrate has been also reported in a model of EAE induced in knout-out mice for the proteoglycan NG2 (Kucharova et al., 2011). On the basis of these data, the present study was carried out on the same model of EAE to clarify the role of NG2 on ECs of brain microvessels, utilizing two groups of mice, wild type (WT) and homozygous NG2 KO (NG2-/-). The expression of two integral proteins of the endothelial TJs, claudin-5 and occludin, the relevance of IBA1 reactive microglia cells and the level of BBB leakage by an exogenous permeability tracer, FITC-Dextran have been analyzed by immunohistochemistry and high resolution confocal microscopy. The results on the junctional staining pattern showed that unlike WT EAE, NG2 KO EAE microvessels were characterized by TJs continuous junctional strands with an unusual distribution of junctional proteins organized in honeycomb-like meshes. These findings suggest that NG2 proteoglycan can be directly implicated in pericyte/EC relations, including the mutual organization of TJ proteins in BBB- microvessels during neurological disease

Fibrotic and Vascular Remodelling of Colonic Wall in Patients with Active Ulcerative Colitis

open16noIntestinal fibrosis is a complication of inflammatory bowel disease [IBD]. Although fibrostenosis is a rare event in ulcerative colitis [UC], there is evidence that a fibrotic rearrangement of the colon occurs in the later stages. This is a retrospective study aimed at examining the histopathological features of the colonic wall in both short-lasting [SL] and long-lasting [LL] UC. Surgical samples of left colon from non-stenotic SL [a parts per thousand currency sign 3 years, n = 9] and LL [a parts per thousand yen 10 years, n = 10] UC patients with active disease were compared with control colonic tissues from cancer patients without UC [n = 12] to assess: collagen and elastic fibres by histochemistry; vascular networks [CD31/CD105/nestin] by immunofluorescence; parameters of fibrosis [types I and III collagen, fibronectin, RhoA, alpha-smooth muscle actin [alpha-SMA], desmin, vimentin], and proliferation [proliferating nuclear antigen [PCNA]] by western blot and/or immunolabelling. Colonic tissue from both SL-UC and LL-UC showed tunica muscularis thickening and transmural activated neovessels [displaying both proliferating CD105-positive endothelial cells and activated nestin-positive pericytes], as compared with controls. In LL-UC, the increased collagen deposition was associated with an up-regulation of tissue fibrotic markers [collagen I and III, fibronectin, vimentin, RhoA], an enhancement of proliferation [PCNA] and, along with a loss of elastic fibres, a rearrangement of the tunica muscularis towards a fibrotic phenotype. A significant transmural fibrotic thickening occurs in colonic tissue from LL-UC, together with a cellular fibrotic switch in the tunica muscularis. A full-thickness angiogenesis is also evident in both SL- and LL-UC with active disease, as compared with controls.openIppolito, Chiara; Colucci, Rocchina; Segnani, Cristina; Errede, Mariella; Girolamo, Francesco; Virgintino, Daniela; Dolfi, Amelio; Tirotta, Erika; Buccianti, Piero; Di Candio, Giulio; Campani, Daniela; Castagna, Maura; Bassotti, Gabrio; Villanacci, Vincenzo; Blandizzi, Corrado; Bernardini, NunziaIppolito, Chiara; Colucci, ROCCHINA LUCIA; Segnani, Cristina; Errede, Mariella; Girolamo, Francesco; Virgintino, Daniela; Dolfi, Amelio; Tirotta, Erika; Buccianti, Piero; Di Candio, Giulio; Campani, Daniela; Castagna, Maura; Bassotti, Gabrio; Villanacci, Vincenzo; Blandizzi, Corrado; Bernardini, Nunzi

BBB-endothelial cell response to cerebral cortex demyelination in a mouse model of chronic EAE

Changes in blood-brain barrier (BBB) function have been implicated in demyelinating diseases. This study aimed to investigate the response of cerebral cortex microvessels to nerve fibre demyelination in a chronic model of murine experimental autoimmune encephalomyelitis (EAE) characterized by areas of extensive subpial demyelination along with well-demarcated lesions extended to deeper cortex layers. These cortices showed activation of microglia and astrogliosis with absence of typical perivascular inflammatory infiltrates. On the basis of these data, we have analyzed the expression of two integral proteins of endothelial tight junctions, claudin-5 and occludin, a structural protein of caveolae, caveolin-1, as well as the BBB-specific endothelial transporter, Glut1 in the cerebral cortex of EAE-affected mice by immunofluorescence confocal microscopy. Microvascular endothelial cells showed an increased expression of caveolin-1 and a coincident decrease of both claudin-5 and occludin junctional staining pattern. At a very early disease stage, claudin-5 molecules formed aggregates and vacuoles that also stained for Glut 1, whereas occludin pattern became diffusely cytoplasmic at advanced stages of the disease. Internalization/dismantling and loss of tight junction proteins and impairment of BBB function were confirmed by coexpression of claudin-5 whit the autophagosomal marker MAP1LC3A and by FITC-dextran experiments that showed leakage of the tracer into the perivascular neuropil. Overall, these observations indicate that in the cerebral cortex of EAE mice, during demyelination and independently from the inflammatory involvement of the cortex, a ‘microvascular disease’ characterized by a differential involvement of claudin-5 and occludin occurs, thereby possibly contributing to demyelinating disease progression

Characterization of oligodendrocyte lineage precursor cells in the mouse cerebral cortex: a confocal microscopy approach to demyelinating diseases

The identification of stem cells resident in the adult central nervous system has redirected the focus of research into demyelinating diseases, such as multiple sclerosis, mainly affecting the brain white matter. This immunocytochemical and morphometrical study was carried out by confocal microscopy in the adult mouse cerebral cortex, with the aim of analysing, in the brain grey matter, the characteristics of the oligodendrocyte lineage cells, whose capability to remyelinate is still controversial. The observations demonstrated the presence in all the cortex layers of glial restricted progenitors, reactive to A2B5 marker, oligodendrocyte precursor cells, expressing the NG2 proteoglycan, and pre­oligodendrocytes and pre­myelinating oligodendrocytes, reactive to the specific marker O4. NG2 expressing cells constitute the major immature population of the cortex, since not only oligodendrocyte precursor cells and pre-oligodendrocytes but also a part of the glial restrict progenitors express the NG2 proteoglycan. Together with the popula­ tion of these immature cells, a larger population of mature oligodendrocytes was revealed by the classical oligodendrocyte and myelin markers, 2’,3’-cyclic nucleotide 3’­phosphodiesterase, myelin basic protein and myelin oligodendrocyte glycoprotein. The results indicate that oligodendrocyte precursors committed to differentiate into myelin forming oligodendrocytes are present through all layers of the adult cortex and that their phenotypic features exactly recall those of the oligodendroglial lineage cells during development