Blood-brain barrier pericyte importance in malignant gliomas: What we can learn from stroke and Alzheimer's disease

The pericyte, a constitutive component of the central nervous system, is a poorly understood cell type that envelops the endothelial cell with the intended purpose of regulating vascular flow and endothelial cell permeability. Previous studies of pericyte function have been limited to a small number of disease processes such as ischemic stroke and Alzheimer's disease. Recently, publications have postulated a link between glioma stem cell differentiation and pericyte function. These studies suggest that there may be an important interaction of pericytes with tumor cells and other components of the tumor microenvironment in malignant primary glial neoplasms, most notably glioblastoma. This potential cellular interaction underscores the need to pursue more investigations of pericytes in malignant brain tumor biology. In this review, we summarize the functional roles of pericytes, particularly focusing on changes in pericyte biology during response to immune cells, inflammation, and hypoxic conditions. The information presented is based on the available data from studies of pericyte function in other central nervous system diseases but will serve as a foundation for research investigations to further understand the role of pericytes in malignant gliomas

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

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

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

Defining the role of NG2-expressing cells in experimental models of multiple sclerosis. A biofunctional analysis of the neurovascular unit in wild type and NG2 null mice

During experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis associated with blood-brain barrier (BBB) disruption, oligodendrocyte precursor cells (OPCs) overexpress proteoglycan nerve/glial antigen 2 (NG2), proliferate, and make contacts with the microvessel wall. To explore whether OPCs may actually be recruited within the neurovascular unit (NVU), de facto intervening in its cellular and molecular composition, we quantified by immunoconfocal morphometry the presence of OPCs in contact with brain microvessels, during postnatal cerebral cortex vascularization at postnatal day 6, in wildtype (WT) and NG2 knock-out (NG2KO) mice, and in the cortex of adult naive and EAE-affected WT and NG2KO mice. As observed in WT mice during postnatal development, a higher number of juxtavascular and perivascular OPCs was revealed in adult WT mice during EAE compared to adult naive WT mice. In EAE-affected mice, OPCs were mostly associated with microvessels that showed altered claudin-5 and occludin tight junction (TJ) staining patterns and barrier leakage. In contrast, EAE-affected NG2KO mice, which did not show any significant increase in vessel-associated OPCs, seemed to retain better preserved TJs and BBB integrity. As expected, absence of NG2, in both OPCs and pericytes, led to a reduced content of vessel basal lamina molecules, laminin, collagen VI, and collagen IV. In addition, analysis of the major ligand/receptor systems known to promote OPC proliferation and migration indicated that vascular endothelial growth factor A (VEGF-A), platelet-derived growth factor-AA (PDGF-AA), and the transforming growth factor-beta (TGF-beta) were the molecules most likely involved in proliferation and recruitment of vascular OPCs during EAE. These results were confirmed by real time-PCR that showed Fgf2, Pdgfa and Tgfb expression on isolated cerebral cortex microvessels and by dual RNAscope-immunohistochemistry/ in situ hybridization (IHC/ISH), which detected Vegfa and Vegfr2 transcripts on cerebral cortex sections. Overall, this study suggests that vascular OPCs, in virtue of their developmental arrangement and response to neuroinflammation and growth factors, could be integrated among the classical NVU cell components. Moreover, the synchronized activation of vascular OPCs and pericytes during both BBB development and dysfunction, points to NG2 as a key regulator of vascular interactions

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