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1.
Methods Mol Biol ; 2206: 143-150, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754816

RESUMO

Pericytes are integral part of neurovascular unit and play a role in the maintenance of blood-brain barrier integrity, angiogenesis, and cerebral blood flow regulation. Despite their important functional roles, a univocal phenotypic identification is still emerging also for the lack of a "pan-pericyte" marker. In the present study, we describe in detail the method for performing fluorescence immunohistochemistry on thick free-floating sections from human fetal brain in high resolution laser confocal microscopy. This method enables to obtain three-dimensional images of pericytes and provides insights about their distribution and localization in the microvessels of human developing brain.

2.
IBRO Rep ; 9: 164-182, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32803016

RESUMO

Vanadium, a transition series metal released during some industrial activities, induces oxidative stress and lipid peroxidation. Ameliorative effect of a pure compound from the methanolic extract of Moringa oleifera leaves, code-named MIMO2, in 14-day old mice administered with vanadium (as sodium metavanadate 3 mg/kg) for 2 weeks was assessed. Results from body weight monitoring, muscular strength, and open field showed slight reduction in body weight and locomotion deficit in vanadium-exposed mice, ameliorated with MIMO2 co-administration. Degeneration of the Purkinje cell layer and neuronal death in the hippocampal CA1 region were observed in vanadium-exposed mice and both appeared significantly reduced with MIMO2 co-administration. Demyelination involving the midline of the corpus callosum, somatosensory and retrosplenial cortices was also reduced with MIMO2. Microglia activation and astrogliosis observed through immunohistochemistry were also alleviated. Immunohistochemistry for myelin, axons and oligodendrocyte lineage cells were also carried out and showed that in vanadium-treated mice brains, oligodendrocyte progenitor cells increased NG2 immunolabelling with hypertrophy and bushy, ramified appearance of their processes. MIMO2 displayed ameliorative and antioxidative effects in vanadium-induced neurotoxicity in experimental murine species. This is likely the first time MIMO2 is being used in vivo in an animal model.

3.
Curr Pharm Des ; 26(13): 1428-1437, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32186270

RESUMO

P-Glycoprotein (P-gp) is a 170-kDa transmembrane glycoprotein that works as an efflux pump and confers multidrug resistance (MDR) in normal tissues and tumors, including nervous tissues and brain tumors. In the developing telencephalon, the endothelial expression of P-gp, and the subcellular localization of the transporter at the luminal endothelial cell (EC) plasma membrane are early hallmarks of blood-brain barrier (BBB) differentiation and suggest a functional BBB activity that may complement the placental barrier function and the expression of P-gp at the blood-placental interface. In early fetal ages, P-gp has also been immunolocalized on radial glia cells (RGCs), located in the proliferative ventricular zone (VZ) of the dorsal telencephalon and now considered to be neural progenitor cells (NPCs). RG-like NPCs have been found in many regions of the developing brain and have been suggested to give rise to neural stem cells (NSCs) of adult subventricular (SVZ) neurogenic niches. The P-gp immunosignal, associated with RG-like NPCs during cortical histogenesis, progressively decreases in parallel with the last waves of neuroblast migrations, while 'outer' RGCs and the deriving astrocytes do not stain for the efflux transporter. These data suggest that in human glioblastoma (GBM), P-gp expressed by ECs may be a negligible component of tumor MDR. Instead, tumor perivascular astrocytes may dedifferentiate and resume a progenitor-like P-gp activity, becoming MDR cells and contribute, together with perivascular P-gpexpressing glioma stem-like cells (GSCs), to the MDR profile of GBM vessels. In conclusion, the analysis of Pgp immunolocalization during brain development may contribute to identify the multiple cellular sources in the GBM vessels that may be involved in P-gp-mediated chemoresistance and can be responsible for GBM therapy failure and tumor recurrence.

4.
Int J Med Sci ; 17(2): 153-160, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32038098

RESUMO

Aims: Systemic Lupus Erythematosus (SLE) is a connective tissue disease characterized by a wide range of pleomorphic pictures, including mucocutaneous, renal, musculoskeletal and neurological symptoms. It involves oral tissues, with hyposalivation, tooth decay, gingivitis, angular cheilitis, ulcers and glossitis. Temporomandibular disorders represent a heterogeneous group of inflammatory or degenerative diseases of the stomatognatic system, with algic and/or dysfunctional clinical features involving temporomandibular joint (TMJ) and related masticatory muscles. The aim of this study was to investigate the prevalence of oral manifestations and temporomandibular disorders (TMD) in SLE patients (Lp) compared with a control group. Methods: Fifty-five patients (9 men and 46 women) with diagnosed Lupus were recruited in the study group. A randomly selected group of 55 patients, matched by sex and age, served as control group. The examination for TMD symptoms and signs was based on the standardized Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) through a questionnaire and clinical examination. Results: Lupus patients complained more frequently (95.8%) of oral and TMJ symptoms (dysgeusia, stomatodynia, masticatory muscle pain during function, neck and shoulder muscles pain and presence of tinnitus) but only xerostomia (χ2=4,1548 p=0,0415), temple headache (χ2=4,4542 p=0,035) and the sensation of a stuck jaw (Mid-p-test p=0,043) were significant. About signs, cheilitis (p=0,0284) oral ulcers (χ2=4,0104 p=0,045) and fissured tongue are significantly more frequent in study group. The salivary flow was significantly decreased in the study group respect to the control one (p<0.0001). As regard to the oral kinematics, restricted movements (RM) in protrusion and left lateral movement were significantly different between study group and controls. In particular, 85,2% of Lp showed limited protrusion versus 56,4% of controls (χ2= 10,91 p<0,001); 59,3% of Lp had also a limitation during left lateral movement versus 47,3% of controls (T=2,225 p=0,0282). About bruxism, only the indentations on the lateral edges of the tongue were found in Lp group (72,7%), with a significant difference respect to controls (χ2=7,37 p=0,007). Conclusions: While masticatory muscles have an overlapping behavior in both groups, the findings collected show a more severe TMJ kinematic impairment in Lp than in controls, with protrusion and left lateral movements significantly different. In addition, a remarkable reduction of salivary flow has been detected in Lp compared to controls. In conclusion, this autoimmune disease seems to play a role in oral manifestations and TMJ disorders, causing an increase in orofacial pain and an altered chewing function.


Assuntos
Bruxismo/fisiopatologia , Dor Facial/fisiopatologia , Lúpus Eritematoso Sistêmico/fisiopatologia , Transtornos da Articulação Temporomandibular/fisiopatologia , Adulto , Bruxismo/complicações , Bruxismo/diagnóstico , Dor Facial/complicações , Dor Facial/diagnóstico , Feminino , Cefaleia/complicações , Cefaleia/fisiopatologia , Humanos , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/diagnóstico , Masculino , Mastigação , Músculos da Mastigação/fisiopatologia , Pessoa de Meia-Idade , Amplitude de Movimento Articular/fisiologia , Inquéritos e Questionários , Articulação Temporomandibular/fisiopatologia , Transtornos da Articulação Temporomandibular/complicações , Transtornos da Articulação Temporomandibular/diagnóstico , Doenças Dentárias/complicações , Doenças Dentárias/diagnóstico , Doenças Dentárias/fisiopatologia , Xerostomia/complicações , Xerostomia/diagnóstico , Xerostomia/fisiopatologia
5.
Neurobiol Dis ; 139: 104821, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32088380

RESUMO

BACKGROUND AND AIM: Patients with Parkinson's disease (PD) are often characterized by functional gastrointestinal disorders. Such disturbances can occur at all stages of PD and precede the typical motor symptoms of the disease by many years. However, the morphological alterations associated with intestinal disturbances in PD are undetermined. This study examined the remodelling of colonic wall in 6-hydroxydopamine (6-OHDA)-induced PD rats. METHODS: 8 weeks after 6-OHDA injection animals were sacrificed. Inflammatory infiltrates, collagen deposition and remodelling of intestinal epithelial barrier and tunica muscularis in the colonic wall were assessed by histochemistry, immunohistochemistry, immunofluorescence and western blot analysis. RESULTS: 6-OHDA rats displayed significant alterations of colonic tissues as compared with controls. Signs of mild inflammation (eosinophil infiltration) and a transmural deposition of collagen fibres were observed. Superficial colonic layers were characterized by severe morphological alterations. In particular, lining epithelial cells displayed a reduced claudin-1 and transmembrane 16A/Anoctamin 1 (TMEM16A/ANO1) expression; goblet cells increased their mucin expression; colonic crypts were characterized by an increase in proliferating epithelial cells; the density of S100-positive glial cells and vimentin-positive fibroblast-like cells was increased as well. Several changes were found in the tunica muscularis: downregulation of α-smooth muscle actin/desmin expression and increased proliferation of smooth muscle cells; increased vimentin expression and proliferative phenotype in myenteric ganglia; reduction of interstitial cells of Cajal (ICCs) density. CONCLUSIONS: A pathological remodelling occurs in the colon of 6-OHDA rats. The main changes include: enhanced fibrotic deposition; alterations of the epithelial barrier; activation of mucosal defense; reduction of ICCs. These results indicate that central nigrostriatal denervation is associated with histological changes in the large bowel at mucosal, submucosal and muscular level. These alterations might represent morphological correlates of digestive symptoms in PD.

6.
J Bone Miner Res ; 35(4): 766-775, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31826311

RESUMO

Previous results showed that intermittently administered irisin improves bone mass in normal mice and prevents the development of disuse-induced osteoporosis and muscular atrophy in hindlimb-suspended mice, a murine model able to mimic the absence of mechanical loading. A recent study showed that irisin increases survival of osteocytes acting through integrin αV/ß5 receptors. To better understand the action of irisin on these cells, we investigated the downstream signaling cascades in osteocyte-like cells (MLO-Y4) treated with recombinant irisin (rec-irisin) in vitro and we analyzed survival of osteocytes and caspase activation in cortical bone of osteoporotic mice treated with rec-irisin in vivo. Our results revealed that rec-irisin activated the MAP kinases Erk1 and Erk2 and increased the expression of the transcription factor Atf4 (2.5-fold, p < .05) through an Erk-dependent pathway in osteocytes. Some key genes expressed by MLO-Y4 cells were modulated by long-term irisin treatment, either continuously administered or given with intermittent short pulses. Interestingly, Sost mRNA was severely downregulated only upon intermittent irisin administration (10-fold, p < .001). Furthermore, rec-irisin upregulated Tfam mRNA (fourfold, p < .05) and Bcl2/Bax ratio (twofold, p < .05) in MLO-Y4 cells. By detecting caspase-9 and caspase-3, we also found that rec-irisin inhibited apoptosis induced by hydrogen peroxide and dexamethasone, respectively. In cortical bone of unloading C57BL6 mice treated with vehicle (unload-veh), irisin prevented disuse-induced reduction of viable osteocytes (+30% versus unload-veh, p < .05) and increase of empty lacunae (+110% versus unload-veh, p < .05), as well as caspase-9 (threefold, p < .05) and caspase-3 (twofold, p < .05) activations. Our findings revealed underlying mechanisms of irisin action on osteocytes, which increases their functions and exerts anti-apoptotic effects, confirming that mechanosensor cells of bone are sensitive to the exercise-mimetic myokine irisin. © 2019 American Society for Bone and Mineral Research.

7.
PLoS One ; 14(3): e0213508, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30870435

RESUMO

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 wild-type (WT) and NG2 knock-out (NG2KO) mice, and in the cortex of adult naïve 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 naïve 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-ß (TGF-ß) 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.


Assuntos
Antígenos/biossíntese , Barreira Hematoencefálica/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Microvasos/metabolismo , Oligodendroglia/metabolismo , Proteoglicanas/biossíntese , Células-Tronco/metabolismo , Animais , Antígenos/genética , Barreira Hematoencefálica/patologia , Movimento Celular/genética , Proliferação de Células/genética , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Claudina-5/genética , Claudina-5/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Camundongos , Camundongos Knockout , Microvasos/patologia , Oligodendroglia/patologia , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteoglicanas/genética , Células-Tronco/patologia , Junções Íntimas/genética , Junções Íntimas/metabolismo , Junções Íntimas/patologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
8.
Stem Cell Res Ther ; 10(1): 29, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30646960

RESUMO

Cognitive deficit has been identified in one third of patients affected by Duchenne Muscular Dystrophy, primarily attributed to loss of the short Dp71 dystrophin, the major brain dystrophin isoform. In this study, we investigated for the first time the Dp71 and Dp71-associated proteins cellular localization and expression in human neurons obtained by differentiation from induced pluripotent stem cell line of a patient affected by cognitive impairment. We found structural and molecular alterations in both pluripotent stem cell and derived neurons, reduced Dp71 expression, and a Ca2+ cytoplasmic overload in neurons coupled with increased expression of the SERCA2 pump in the dystrophic neurons. These results suggest that the reduction of Dp71 protein in the Duchenne muscular dystrophy neurons leads to alterations in SERCA2 and to elevated cytosolic Ca2+ concentration with consequent potential disruption of the dystrophin proteins and Dp71-associated proteins.


Assuntos
Disfunção Cognitiva/genética , Distrofina/genética , Distrofina/metabolismo , Distrofia Muscular de Duchenne/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Adulto , Diferenciação Celular , Humanos , Masculino , Distrofia Muscular de Duchenne/metabolismo , Neurônios , Adulto Jovem
9.
Transl Oncol ; 12(3): 545-549, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30639963

RESUMO

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma. Signal transducer and activator of transcription 3 (STAT3) is a cytoplasmic transcription with many important functions, including regulation of cell proliferation, differentiation, survival, angiogenesis, and immune response. MATERIALS AND METHODS: In this study, we have compared by means of RNAscope technology STAT3 RNA expression in human DLBCL in a selected group of activated B-cell-like DLBCL (ABC-DLBCL) patients with another group of germinal center B-cell-like DLBCL (GBC-DLBCL) patients. RESULTS: The results have shown that ABC DLBCL tissue samples contained a significantly higher number of STAT3-positive cells as compared with GCB tissue samples. Moreover, by means of confocal immunofluorescence analysis, we have observed that tumor vessels in ABC samples appeared lined by endothelial cells expressing both FVIII and STAT3 signals, while in GCB samples, only few vessels coexpressed FVIII and STAT3. CONCLUSIONS: These data confirm other reports showing that STAT3 is highly expressed and activated in ABC-DLBCL and our previously published data demonstrating that, in primary central nervous system lymphoma, tumor vessels appeared lined by endothelial cells expressing both FVIII and STAT3.

10.
Fluids Barriers CNS ; 15(1): 28, 2018 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-30290761

RESUMO

BACKGROUND: Nanotubular structures, denoted tunneling nanotubes (TNTs) have been described in recent times as involved in cell-to-cell communication between distant cells. Nevertheless, TNT-like, long filopodial processes had already been described in the last century as connecting facing, growing microvessels during the process of cerebral cortex vascularization and collateralization. Here we have investigated the possible presence and the cellular origin of TNTs during normal brain vascularization and also in highly vascularized brain tumors. METHODS: We searched for TNTs by high-resolution immunofluorescence confocal microscopy, applied to the analysis of 20-µm, thick sections from lightly fixed, unembedded samples of both developing cerebral cortex and human glioblastoma (GB), immunolabeled for endothelial, pericyte, and astrocyte markers, and vessel basal lamina molecules. RESULTS: The results revealed the existence of pericyte-derived TNTs, labeled by proteoglycan NG2/CSPG4 and CD146. In agreement with the described heterogeneity of these nanostructures, ultra-long (> 300 µm) and very thin (< 0.8 µm) TNTs were observed to bridge the gap between the wall of distant vessels, or were detected as short (< 300 µm) bridging cables connecting a vessel sprout with its facing vessel or two apposed vessel sprouts. The pericyte origin of TNTs ex vivo in fetal cortex and GB was confirmed by in vitro analysis of brain pericytes, which were able to form and remained connected by typical TNT structures. CONCLUSIONS: None of the multiple roles described for TNTs can be excluded from a possible involvement during the processes of both normal and pathological vessel growth. A possible function, suggested by the pioneering studies made during cerebral cortex vascularization, is in cell searching and cell-to-cell recognition during the processes of vessel collateralization and vascular network formation. According to our results, it is definitely the pericyte-derived TNTs that seem to actively explore the surrounding microenvironment, searching for (site-to-site recognition), and connecting with (pericyte-to-pericyte and/or pericyte-to-endothelial cell communication), the targeted vessels. This idea implies that TNTs may have a primary role in the very early phases of both physiological and tumor angiogenesis in the brain.


Assuntos
Neoplasias Encefálicas/fisiopatologia , Córtex Cerebral/fisiopatologia , Células Endoteliais/fisiologia , Glioblastoma/fisiopatologia , Nanotubos , Neovascularização Patológica , Neovascularização Fisiológica , Pericitos/fisiologia , Adulto , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Comunicação Celular , Células Cultivadas , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/citologia , Células Endoteliais/citologia , Feminino , Glioblastoma/irrigação sanguínea , Glioblastoma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Pericitos/citologia
11.
Oncotarget ; 8(19): 31254-31269, 2017 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-28415725

RESUMO

With the aim of elucidating the relationship between Stat3 expression and tumor vessels abnormalities in the PCNLs, in this study we evaluated Stat3 and pStat3 expression by Real-time PCR and by immunohistochemistry in biopsy sections from PCNSL patients. Correlations of the expression levels with the presence of aberrant vessels were analyzed by confocal laser microscopy analysis, using FVIII as endothelial cell marker, CD133 and nestin as cancer stem cell (CSC) marker, CD20 as tumor cell marker, and Stat3. In addition, we investigated Stat3 mutations in lymphoma cells to clarify the role of the constitutive expression of Stat3 and of its phosphorylated forms. Results showed that in PCNSL, putative endothelial cells lining the vessels are heterogeneous, expressing FVIII/ pStat3/CD133 (presumably originally they are vascular progenitor cells), as well as FVIII/CD20/CD133 (presumably originally they are tumor cells). Finally, we detected a fraction of the FVIII+ endothelial cell that co-expressed Stat3 bearing a tetraploid karyotype, while no amplification signal for the Stat3 gene was detected.


Assuntos
Neoplasias do Sistema Nervoso Central/metabolismo , Neoplasias do Sistema Nervoso Central/patologia , Linfoma/metabolismo , Linfoma/patologia , Neovascularização Patológica/metabolismo , Fator de Transcrição STAT3/metabolismo , Antígeno AC133/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Neoplasias do Sistema Nervoso Central/genética , Células Endoteliais/metabolismo , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Antígeno Ki-67/metabolismo , Linfoma/genética , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Neovascularização Patológica/genética , Nestina/metabolismo , Fosforilação , Fator de Transcrição STAT3/genética
12.
Fluids Barriers CNS ; 13(1): 17, 2016 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-27655189

RESUMO

Annexin A1 is a potent anti-inflammatory molecule that has been extensively studied in the peripheral immune system, but has not as yet been exploited as a therapeutic target/agent. In the last decade, we have undertaken the study of this molecule in the central nervous system (CNS), focusing particularly on the primary interface between the peripheral body and CNS: the blood-brain barrier. In this review, we provide an overview of the role of this molecule in the brain, with a particular emphasis on its functions in the endothelium of the blood-brain barrier, and the protective actions the molecule may exert in neuroinflammatory, neurovascular and metabolic disease. We focus on the possible new therapeutic avenues opened up by an increased understanding of the role of annexin A1 in the CNS vasculature, and its potential for repairing blood-brain barrier damage in disease and aging.

14.
Acta Neuropathol ; 132(1): 23-42, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27026411

RESUMO

In adult CNS, nerve/glial-antigen 2 (NG2) is expressed by oligodendrocyte progenitor cells (OPCs) and is an early marker of pericyte activation in pathological conditions. NG2 could, therefore, play a role in experimental autoimmune encephalomyelitis (EAE), a disease associated with increased blood-brain barrier (BBB) permeability, inflammatory infiltrates, and CNS damage. We induced EAE in NG2 knock-out (NG2KO) mice and used laser confocal microscopy immunofluorescence and morphometry to dissect the effect of NG2 KO on CNS pathology. NG2KO mice developed milder EAE than their wild-type (WT) counterparts, with less intense neuropathology associated with a significant improvement in BBB stability. In contrast to WT mice, OPC numbers did not change in NG2KO mice during EAE. Through FACS and confocal microscopy, we found that NG2 was also expressed by immune cells, including T cells, macrophages, and dendritic cells (DCs). Assessment of recall T cell responses to the encephalitogen by proliferation assays and ELISA showed that, while WT and NG2KO T cells proliferated equally to the encephalitogenic peptide MOG35-55, NG2KO T cells were skewed towards a Th2-type response. Because DCs could be responsible for this effect, we assessed their expression of IL-12 by PCR and intracellular FACS. IL-12-expressing CD11c+ cells were significantly decreased in MOG35-55-primed NG2KO lymph node cells. Importantly, in WT mice, the proportion of IL-12-expressing cells was significantly lower in CD11c+ NG2- cells than in CD11c+ NG2+ cells. To assess the relevance of NG2 at immune system and CNS levels, we induced EAE in bone-marrow chimeric mice, generated with WT recipients of NG2KO bone-marrow cells and vice versa. Regardless of their original phenotype, mice receiving NG2KO bone marrow developed milder EAE than those receiving WT bone marrow. Our data suggest that NG2 plays a role in EAE not only at CNS/BBB level, but also at immune response level, impacting on DC activation and thereby their stimulation of reactive T cells, through controlling IL-12 expression.


Assuntos
Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Animais , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/patologia , Células da Medula Óssea/imunologia , Transplante de Medula Óssea , Células Dendríticas/patologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Índice de Gravidade de Doença , Medula Espinal/imunologia , Medula Espinal/patologia , Linfócitos T/imunologia , Linfócitos T/patologia
15.
Exp Cell Res ; 343(2): 190-207, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-27015747

RESUMO

The blood-brain barrier (BBB) is altered in mdx mouse, an animal model to study Duchenne muscular dystrophy (DMD). Our previous work demonstrated that perivascular glial endfeet control the selective exchanges between blood and neuropil as well as the BBB development and integrity; the alterations of dystrophin and dystrophin-associated protein complex (DAPs) in the glial cells of mdx mouse, parallel damages of the BBB and increase in vascular permeability. The aim of this study was to improve our knowledge about brain cellular components in the mdx mouse through the isolation, for the first time, of the adult neural stem cells (ANSCs). We characterized them by FACS, electron microscopy, confocal immunofluorescence microscopy, Real Time-PCR and western blotting, and we studied the expression of the DAPs aquaporin-4 (AQP4), potassium channel Kir4.1, α- and ß-dystroglycan (αDG, ßDG), α-syntrophin (αSyn), and short dystrophin isoform Dp71 proteins. The results showed that the mdx ANSCs expressed CD133 and Nestin receptor as the control ones, but showed a reduction in Notch receptor and altered cell proliferation with an increment in the apoptotic nuclei. Ultrastructurally, they appeared 50% size reduced compared to control ones, with a few cytoplasmic organelles. Moreover, the mdx ANSCs are devoid in full length dystrophin 427, and they expressed post-transcriptional reduction in the Dp71 in parallel with the ubiquitin proteasome activation, and decrement of DAPs proteins which appeared diffused in the cytoplasm and not polarized on the stem cells plasmamembrane, as prevalently observed in the controls. Overall, these results indicate that structural and molecular alterations affect the neural stem cells in the dystrophic brain, whose increased apoptosis and reduced Dp71 and DAPs proteins expression, together with loss in Dp427 dystrophin, could be responsible of the altered mdx glial maintenance and differentiation and consequent failure in the vessels barrier control occurring in the adult dystrophic brain.


Assuntos
Separação Celular/métodos , Distrofia Muscular Animal/patologia , Células-Tronco Neurais/citologia , Antígeno AC133/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Aquaporina 4/metabolismo , Western Blotting , Proteínas de Ligação ao Cálcio , Diferenciação Celular , Distroglicanas/metabolismo , Distrofina/metabolismo , Citometria de Fluxo , Imunofluorescência , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Membrana , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Proteínas Musculares , Distrofia Muscular Animal/genética , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/ultraestrutura , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Esferoides Celulares/citologia , Esferoides Celulares/ultraestrutura , Ubiquitina/metabolismo
16.
J Crohns Colitis ; 10(10): 1194-204, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26995183

RESUMO

BACKGROUND AND AIMS: Intestinal 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. METHODS: Surgical samples of left colon from non-stenotic SL [≤ 3 years, n = 9] and LL [≥ 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 [α-SMA], desmin, vimentin], and proliferation [proliferating nuclear antigen [PCNA]] by western blot and/or immunolabelling. RESULTS: 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. CONCLUSIONS: 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.


Assuntos
Colite Ulcerativa/patologia , Colo/patologia , Remodelação Vascular , Doença Aguda , Adulto , Idoso , Biomarcadores/metabolismo , Western Blotting , Estudos de Casos e Controles , Colo/irrigação sanguínea , Colo/metabolismo , Progressão da Doença , Feminino , Fibrose , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Método Simples-Cego
17.
J Cell Mol Med ; 19(2): 485-500, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25521239

RESUMO

Bowel inflammatory fibrosis has been largely investigated, but an integrated assessment of remodelling in inflamed colon is lacking. This study evaluated tissue and cellular changes occurring in colonic wall upon induction of colitis, with a focus on neuromuscular compartment. Colitis was elicited in rats by 2,4-dinitrobenzenesulfonic acid (DNBS). After 6 and 21 days, the following parameters were assessed on paraffin sections from colonic samples: tissue injury and inflammatory infiltration by histology; collagen and elastic fibres by histochemistry; HuC/D, glial fibrillar acidic protein (GFAP), proliferating cell nuclear antigen (PCNA), nestin, substance P (SP), von Willebrand factor, c-Kit and transmembrane 16A/Anoctamin1 (TMEM16A/ANO1) by immunohistochemistry. TMEM16A/ANO1 was also examined in isolated colonic smooth muscle cells (ICSMCs). On day 6, inflammatory alterations and fibrosis were present in DNBS-treated rats; colonic wall thickening and fibrotic remodelling were evident on day 21. Colitis was associated with both an increase in collagen fibres and a decrease in elastic fibres. Moreover, the neuromuscular compartment of inflamed colon displayed a significant decrease in neuron density and increase in GFAP/PCNA-positive glia of myenteric ganglia, enhanced expression of neural SP, blood vessel remodelling, reduced c-Kit- and TMEM16A/ANO1-positive interstitial cells of Cajal (ICCs), as well as an increase in TMEM16A/ANO1 expression in muscle tissues and ICSMCs. The present findings provide an integrated view of the inflammatory and fibrotic processes occurring in the colonic neuromuscular compartment of rats with DNBS-induced colitis. These morphological alterations may represent a suitable basis for understanding early pathophysiological events related to bowel inflammatory fibrosis.


Assuntos
Colite/patologia , Miócitos de Músculo Liso/patologia , Animais , Colo/patologia , Inflamação/patologia , Masculino , Ratos , Ratos Sprague-Dawley
18.
Front Neurosci ; 8: 324, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25360079

RESUMO

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.

19.
Cancer Lett ; 353(1): 41-51, 2014 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-25038272

RESUMO

Bcl-6 translocation is a genetic alteration that is commonly detected in Primary Central Nervous System Lymphoma. The role of this protein in cerebral tumors is unclear. In this study we investigated Bcl-6 translocation and its transcriptional and translational levels in formalin-fixed, paraffin-embedded cerebral tissue sections from glioblastoma (GBM), low-grade glioma (Astrocytoma grade II and III), and meningioma patients, and correlated them with apoptotic processes and p53 and caspase-3 expression. The results showed a frequency of 36.6% of Bcl-6 translocation in GBM patients and a decreased expression in low-grade glioma patients, correlated with the severity of the disease. Bcl-6 translocation induced an overexpression of both Bcl-6 protein and messenger in GBM, inhibiting apoptotic processes and caspases 3 expression. On the contrary, in low-grade gliomas and meningiomas Bcl-6 expression was reduced, resulting in an increase of apoptotic processes. Finally, p53 expression levels in brain tumors were comparable to Bcl-6 levels. Overall, these data demonstrate, for the first time, that the Bcl-6 gene translocates in GBM patients and that its translocation and expression are correlated with apoptosis inhibition, indicating a key role for this gene in the control of cellular proliferation. This study offers further insights into glioblastoma biology, and supports Bcl-6 as a new diagnostic marker to evaluate the disease severity.


Assuntos
Biomarcadores Tumorais/genética , Proteínas de Ligação a DNA/genética , Glioblastoma/genética , Translocação Genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Biomarcadores Tumorais/análise , Neoplasias Encefálicas/química , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Caspase 3/análise , Proliferação de Células , Proteínas de Ligação a DNA/análise , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/química , Glioblastoma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Proteínas Proto-Oncogênicas c-bcl-6 , RNA Mensageiro/análise , Estudos Retrospectivos , Proteína Supressora de Tumor p53/análise
20.
J Inherit Metab Dis ; 36(3): 455-66, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23344887

RESUMO

This study investigates glio-vascular interactions in human fetal brain at midgestation, specifically examining the expression and immunolocalization of the CXCL12/CXCR4/CXCR7 ligand-receptor axis and its possible role in the vascular patterning of the developing brain. At midgestation, the telencephalic vesicles are characterized by well developed radial glia cells (RGCs), the first differentiated astrocytes and a basic vascular network mainly built of radial vessels. RGCs have been recognized to contribute to cerebral cortex neuro-vascular architecture and have also been demonstrated to act as a significant source of neural cells (Rakic, Brain Res 33:471-476, 1971; Malatesta et al, Development 127:5253-5263, 2000). According to our hypothesis CXCL12, a potent migration and differentiation chemokine released by RGCs, may act as a linking factor coordinating neuroblast migration with vessel growth and patterning through the activation of different ligand/receptor axes. The obtained results support this hypothesis showing that together with CXCR4/CXCR7-reactive neuroblasts, which migrate in close association with CXCL12 RGCs, layer-specific subsets of CXCL12 RGCs and astrocytes specifically contact the microvessel wall. Moreover, the CXCL12/CXCR4/CXCR7 system appears to be directly involved in microvessel growth, its members being differentially expressed in angiogenically activated microvessels and vascular sprouts.


Assuntos
Encéfalo/irrigação sanguínea , Encéfalo/embriologia , Comunicação Celular/fisiologia , Quimiocina CXCL12/fisiologia , Receptores CXCR4/fisiologia , Receptores CXCR/fisiologia , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/fisiologia , Encéfalo/metabolismo , Encéfalo/patologia , Quimiocina CXCL12/metabolismo , Feto/metabolismo , Feto/patologia , Idade Gestacional , Humanos , Imuno-Histoquímica , Ligantes , Neovascularização Fisiológica/fisiologia , Neuroglia/metabolismo , Neuroglia/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Receptores CXCR/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais/fisiologia
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