RESUMEN
Brain endothelial cells are unique among endothelial cells in that they express apical junctional complexes, including tight junctions, which quite resemble epithelial tight junctions both structurally and functionally. They form the blood-brain-barrier (BBB) which strictly controls the exchanges between the blood and the brain compartments by limiting passive diffusion of blood-borne solutes while actively transporting nutrients to the brain. Accumulating experimental and clinical evidence indicate that BBB dysfunctions are associated with a number of serious CNS diseases with important social impacts, such as multiple sclerosis, stroke, brain tumors, epilepsy or Alzheimer's disease. This review will focus on the implication of brain endothelial tight junctions in BBB architecture and physiology, will discuss the consequences of BBB dysfunction in these CNS diseases and will present some therapeutic strategies for drug delivery to the brain across the BBB.
Asunto(s)
Barrera Hematoencefálica/fisiología , Encéfalo/fisiología , Homeostasis , Enfermedades del Sistema Nervioso/fisiopatología , Uniones Estrechas/fisiología , Movimiento Celular/fisiología , Células Endoteliales/fisiología , Células Endoteliales/ultraestructura , Humanos , Leucocitos/fisiología , Meningitis Bacterianas/fisiopatología , Meningitis Viral/fisiopatología , Esclerosis Múltiple/fisiopatología , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Enfermedades Neurodegenerativas/fisiopatología , Permeabilidad , Accidente Cerebrovascular/fisiopatologíaRESUMEN
Systemically injected neural precursor cells (NPCs) were unexpectedly shown to reach the cerebral parenchyma and induce recovery in various diffuse brain pathologies, including animal models of multiple sclerosis. However, the molecular mechanisms supporting NPC migration across brain endothelium remain elusive. Brain endothelium constitutes the blood-brain barrier, which uniquely controls the access of drugs and trafficking of cells, including leukocytes, from the blood to the brain. Taking advantage of the availability of in vitro models of human and rat blood-brain barrier developed in our laboratory and validated by us and others, we show here that soluble hyaluronic acid, the major ligand of the adhesion molecule CD44, as well as anti-CD44 blocking antibodies, largely prevents NPC adhesion to and migration across brain endothelium in inflammatory conditions. We present further evidence that NPCs, surprisingly, induce the formation of apical cups at the surface of brain endothelial cells, enriched in CD44 and other adhesion molecules, thus hijacking the endothelial signaling recently shown to be involved in leukocyte extravasation. These results demonstrate the pivotal role of CD44 in the trans-endothelial migration of NPCs across brain endothelial cells: we propose that they may help design new strategies for the delivery of therapeutic NPCs to the brain by systemic administration.
Asunto(s)
Encéfalo/metabolismo , Células Endoteliales/metabolismo , Receptores de Hialuranos/biosíntesis , Receptores de Hialuranos/fisiología , Neuronas/citología , Células Madre/citología , Animales , Encéfalo/embriología , Adhesión Celular , Movimiento Celular , Células Endoteliales/citología , Humanos , Ácido Hialurónico/química , Ácido Hialurónico/metabolismo , Leucocitos/citología , Ratones , Ratas , Transducción de SeñalRESUMEN
Physiological studies of the blood-brain barrier (BBB) are often performed in rats. We describe the functional characterization of a reproducible in vitro model of the rat BBB and its validation for investigating mechanisms involved in BBB regulation. Puromycin-purified primary cultures of brain endothelial cells, co-cultured with astrocytes in the presence of hydrocortisone (HC) and cAMP, presented low sucrose permeability (< or =0.1 x 10(-3) cm/min) and high transendothelial electrical resistance (> or =270 Omega cm(2)). Expression of specific BBB markers and their transcripts was detected by immunostaining and RT-PCR, respectively: tight junction proteins (claudin-3 and -5, ZO-1 and occludin) and transporters (P-gp, Bcrp and Oatp-2). RT-PCR experiments demonstrated a role of treatment by astrocytes, HC and cAMP in regulation of the transcript level of tight junction proteins (claudin-5 and ZO-1) as well as transporters (Mdr1a, Mrp3, Mrp4, Bcrp, Glut-1), while transcript level of Mdr1b was significantly decreased. The functionality of efflux pumps (P-gp, Mrps and Bcrp) was demonstrated in the presence of specific inhibitors (PSC833, MK571 or Ko143, respectively) by (i) assessing the uptake of the common substrates rhodamine 123 and daunorubicin and (ii) evaluating apical to basolateral and basolateral to apical polarized transport of daunorubicin. In addition, a good correlation (R=0.94) was obtained between the permeability coefficients of a series of compounds of various lipophilicity and their corresponding in vivo rodent blood-brain transfer coefficients. Taken together, our results provide compelling evidence that puromycin-purified rat brain endothelial cells constitute a reliable model of the rat BBB for physiological and pharmacological characterization of BBB transporters.
Asunto(s)
Astrocitos/fisiología , Barrera Hematoencefálica/fisiología , Permeabilidad Capilar/fisiología , Células Endoteliales/fisiología , Regulación de la Expresión Génica/fisiología , Análisis de Varianza , Animales , Animales Recién Nacidos , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/citología , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo/métodos , AMP Cíclico/farmacología , Impedancia Eléctrica , Regulación de la Expresión Génica/efectos de los fármacos , Hidrocortisona/farmacología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Animales , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosAsunto(s)
Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/fisiología , Estructuras de la Membrana Celular/fisiología , Migración Transendotelial y Transepitelial/fisiología , Transporte Biológico/genética , Transporte Biológico/fisiología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/fisiología , Estructuras de la Membrana Celular/química , Estructuras de la Membrana Celular/genética , Estructuras de la Membrana Celular/metabolismo , Movimiento Celular/genética , Movimiento Celular/fisiología , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Molécula 1 de Adhesión Intercelular/fisiología , Modelos BiológicosRESUMEN
It is now well established that both normal and pathological (or scrapie) isoforms of prion protein, PrPc and PrPsc respectively, are involved in the development and progression of various forms of neurodegenerative diseases, including scrapie in sheep, bovine spongiform encephalopathy (or "mad cow disease") and Creutzfeldt-Jakob disease in human, collectively known as prion diseases. The protein PrPc is highly expressed in the central nervous system in neurons and glial cells, and also present in non-brain cells, such as immune cells or epithelial and endothelial cells. Identification of the physiological functions of PrPc in these different cell types thus appears crucial for understanding the progression of prion diseases. Recent studies highlighted several major roles for PrPc that may be considered in two major domains : (1) cell survival (protection against oxidative stress and apoptosis) and (2) cell adhesion. In association with cell adhesion, distinct functions of PrPc were observed, depending on cell types : neuronal differentiation, epithelial and endothelial barrier integrity, transendothelial migration of monocytes, T cell activation. These observations suggest that PrPc functions may be particularly relevant to cellular stress, as well as inflammatory or infectious situations.
Asunto(s)
Proteínas PrPC/fisiología , Animales , Adhesión Celular , Humanos , Modelos Moleculares , Neuronas/patología , Neuronas/fisiología , Oxidación-Reducción , Proteínas PrPC/química , Enfermedades por Prión/patología , Scrapie/patologíaRESUMEN
Endothelin-1 (ET-1) and angiotensin II (AngII), two potent vasoactive peptides involved in the regulation of cardiovascular homeostasis, also induce mitogenic and pro-angiogenic responses in vitro and in vivo. Both peptides are produced by cleavage of inactive precursors by metalloproteases (endothelin-converting enzyme and angiotensin-converting enzyme, respectively) and activate two subtypes of membrane receptors (ETA-R and ETB-R for ET-1, AT1R and AT2R for AngII) that all belong to the superfamily of G-protein coupled receptors. There is increasing evidence that ETA-R, ETB-R and AT1R are expressed in a variety of cancer cells and tissues, and may play a role on tumor growth, angiogenesis and invasion in vivo. This review summarizes the similarities and differences between the ET-1 and AngII systems with regard to their reported effects on various aspects of cancer. In addition to being expressed on vascular endothelium, ET-1 and AngII receptors participate in tumor angiogenesis through the production of the angiogenic factor VEGF. Furthermore, recent clinical studies indicate that a selective ETA-R antagonist has beneficial effects in prostate cancer, suggesting that a similar approach using ETB-R and AT1R blockers might be envisioned. Experimental data presented here suggest that a combined therapy targeting both ET-1 and AngII systems may prove valuable for future treatments of highly angiogenic tumors.
Asunto(s)
Angiotensina II/fisiología , Endotelina-1/fisiología , Neoplasias/fisiopatología , Neovascularización Patológica/fisiopatología , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Angiotensina II/efectos de los fármacos , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Bencimidazoles/farmacología , Bencimidazoles/uso terapéutico , Compuestos de Bifenilo , Ensayos Clínicos como Asunto , Endotelina-1/efectos de los fármacos , Humanos , Invasividad Neoplásica/fisiopatología , Neoplasias/irrigación sanguínea , Receptores de Angiotensina/efectos de los fármacos , Receptores de Angiotensina/fisiología , Receptores de Endotelina/efectos de los fármacos , Receptores de Endotelina/fisiología , Tetrazoles/farmacología , Tetrazoles/uso terapéuticoRESUMEN
Neural precursor (NPC) based therapies are used to restore neurons or oligodendrocytes and/or provide neuroprotection in a large variety of neurological diseases. In multiple sclerosis models, intravenously (i.v) -delivered NPCs reduced clinical signs via immunomodulation. We demonstrated recently that NPCs were able to cross cerebral endothelial cells in vitro and that the multifunctional signalling molecule, CD44 involved in trans-endothelial migration of lymphocytes to sites of inflammation, plays a crucial role in extravasation of syngeneic NPCs. In view of the role of CD44 in NPCs trans-endothelial migration in vitro, we questioned presently the benefit of CD44 overexpression by NPCs in vitro and in vivo, in EAE mice. We show that overexpression of CD44 by NPCs enhanced over 2 folds their trans-endothelial migration in vitro, without impinging on the proliferation or differentiation potential of the transduced cells. Moreover, CD44 overexpression by NPCs improved significantly their elongation, spreading and number of filopodia over the extracellular matrix protein laminin in vitro. We then tested the effect of CD44 overexpression after i.v. delivery in the tail vein of EAE mice. CD44 overexpression was functional invivo as it accelerated trans-endothelial migration and facilitated invasion of HA expressing perivascular sites. These in vitro and in vivo data suggest that CD44 may be crucial not only for NPC crossing the endothelial layer but also for facilitating invasion of extravascular tissues.
Asunto(s)
Movimiento Celular , Endotelio Vascular/metabolismo , Receptores de Hialuranos/metabolismo , Células-Madre Neurales/metabolismo , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Endotelio Vascular/citología , Citometría de Flujo , Inmunohistoquímica , Ratones , Células-Madre Neurales/citología , Reacción en Cadena de la PolimerasaRESUMEN
It has been recently shown that systemically injected neural precursor cells (NPCs) could cross brain endothelium and favor functional recovery in animal models of multiple sclerosis (MS). Here we show that human NPCs express receptors of the chemokines IL8 and CXCL13 (CXCR1 and CXCR5, respectively) and migrate across brain endothelial cells in vitro, in response to these chemokines. Considering that these chemokines have been found overexpressed in MS in active, but not inactive areas of demyelination, our data suggest that systemically injected human NPCs may be considered for targeting active areas of demyelination in therapeutic approaches of MS.
Asunto(s)
Encéfalo/inmunología , Quimiocina CXCL13/fisiología , Quimiotaxis de Leucocito/inmunología , Células Madre Embrionarias/inmunología , Células Endoteliales/inmunología , Interleucina-8/fisiología , Neuronas/inmunología , Encéfalo/metabolismo , Células Cultivadas , Quimiocina CXCL13/biosíntesis , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/trasplante , Células Endoteliales/metabolismo , Células Endoteliales/trasplante , Humanos , Interleucina-8/biosíntesis , Neuronas/metabolismo , Neuronas/trasplanteRESUMEN
BACKGROUND: Breast cancer is a heterogeneous disease that is not totally eradicated by current therapies. The classification of breast tumors into distinct molecular subtypes by gene profiling and immunodetection of surrogate markers has proven useful for tumor prognosis and prediction of effective targeted treatments. The challenge now is to identify molecular biomarkers that may be of functional relevance for personalized therapy of breast tumors with poor outcome that do not respond to available treatments. The Mitochondrial Tumor Suppressor (MTUS1) gene is an interesting candidate whose expression is reduced in colon, pancreas, ovary and oral cancers. The present study investigates the expression and functional effects of MTUS1 gene products in breast cancer. METHODS AND FINDINGS: By means of gene array analysis, real-time RT-PCR and immunohistochemistry, we show here that MTUS1/ATIP3 is significantly down-regulated in a series of 151 infiltrating breast cancer carcinomas as compared to normal breast tissue. Low levels of ATIP3 correlate with high grade of the tumor and the occurrence of distant metastasis. ATIP3 levels are also significantly reduced in triple negative (ER- PR- HER2-) breast carcinomas, a subgroup of highly proliferative tumors with poor outcome and no available targeted therapy. Functional studies indicate that silencing ATIP3 expression by siRNA increases breast cancer cell proliferation. Conversely, restoring endogenous levels of ATIP3 expression leads to reduced cancer cell proliferation, clonogenicity, anchorage-independent growth, and reduces the incidence and size of xenografts grown in vivo. We provide evidence that ATIP3 associates with the microtubule cytoskeleton and localizes at the centrosomes, mitotic spindle and intercellular bridge during cell division. Accordingly, live cell imaging indicates that ATIP3 expression alters the progression of cell division by promoting prolonged metaphase, thereby leading to a reduced number of cells ungergoing active mitosis. CONCLUSIONS: Our results identify for the first time ATIP3 as a novel microtubule-associated protein whose expression is significantly reduced in highly proliferative breast carcinomas of poor clinical outcome. ATIP3 re-expression limits tumor cell proliferation in vitro and in vivo, suggesting that this protein may represent a novel useful biomarker and an interesting candidate for future targeted therapies of aggressive breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma/genética , Proteínas Portadoras/biosíntesis , Cromosomas Humanos Par 8/ultraestructura , Regulación Neoplásica de la Expresión Génica , Mitosis , Proteínas Supresoras de Tumor/biosíntesis , Animales , Proteínas Portadoras/genética , Proliferación Celular , Mapeo Cromosómico , Femenino , Células HeLa , Humanos , Ratones , Ratones SCID , Invasividad Neoplásica , Pronóstico , Proteínas Supresoras de Tumor/genéticaRESUMEN
The conversion of prion protein (PrP(C)) to its protease-resistant isoform is involved in the pathogenesis of prion diseases. Although PrP(C) is highly expressed in neurons and other cell types, its physiological function still remains elusive. Here, we describe how we evaluated its expression, subcellular localization and putative function in brain endothelial cells, which constitute the blood-brain barrier. We detected its expression in microvascular endothelium in mouse brain sections and at intercellular junctions of freshly isolated brain microvessels and cultured brain endothelial cells of mouse, rat and human origin. PrP(C) co-localized with the adhesion molecule platelet endothelial cell adhesion molecule-1 (PECAM-1); moreover, both PrP(C) and PECAM-1 were present in raft membrane microdomains. Using mixed cultures of wild-type and PrP(C)-deficient mouse brain endothelial cells, we observed that PrP(C) accumulation at cell-cell contacts was probably dependent on homophilic interactions between adjacent cells. Moreover, we report that anti-PrP(C) antibodies unexpectedly inhibited transmigration of U937 human monocytic cells as well as freshly isolated monocytes through human brain endothelial cells. Significant inhibition was observed with various anti-PrP(C) antibodies or blocking anti-PECAM-1 antibodies as control. Our results strongly support the conclusion that PrP(C) is expressed by brain endothelium as a junctional protein that is involved in the trans-endothelial migration of monocytes.