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INTRODUCTION AND HYPOTHESIS: In randomized clinical trials onabotulinumtoxinA was demonstrated to be an effective and well-tolerated treatment for overactive bladder (OAB) with urinary incontinence (UI). However, data reporting onabotulinumtoxinA use in everyday clinical practice are limited. Here, we present the results from a large, first-of-its-kind real-world study in patients with OAB. METHODS: This was a prospective, observational, multinational study (GRACE; ClinicalTrials.gov , NCT02161159) performed in four European countries. Patients (N = 504) aged ≥ 18 years with OAB inadequately managed with ≥ 1 anticholinergic received onabotulinumtoxinA per their physician's normal clinical practice. RESULTS: Physicians primarily used rigid cystoscopes for onabotulinumtoxinA injection; anesthesia/analgesia was utilized during most treatment procedures. Significant reductions in UI episodes/day from baseline to weeks 1 and 12 were observed as well as in micturition, urgency, and nocturia episodes/day. These improvements in urinary symptoms corresponded to higher scores on the treatment benefit scale at week 12. The use of other OAB medications dropped from baseline to weeks 1 and 12 and was sustained to week 52, which paralleled a reduction in the number of incontinence products used during that time frame. Adverse reactions were reported in 2.6% of patients throughout the study. CONCLUSIONS: In this real-world study, significant improvements in urinary symptoms were seen following onabotulinumtoxinA treatment as early as week 1 and sustained to at least week 12. This was accompanied by a reduced reliance upon incontinence products and reduction in concomitant OAB medication use. OnabotulinumtoxinA was well tolerated with no new safety signals.
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Toxinas Botulínicas Tipo A , Vejiga Urinaria Hiperactiva , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Toxinas Botulínicas Tipo A/efectos adversos , Europa (Continente) , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Calidad de Vida , Resultado del Tratamiento , Vejiga Urinaria Hiperactiva/tratamiento farmacológico , Adulto JovenRESUMEN
BACKGROUND: Extracellular matrix (ECM) proteins play critical functions regulating vascular formation and function. Laminin is a major component of the vascular basal lamina, and transgenic mice deficient in astrocyte or pericyte laminin show defective blood-brain barrier (BBB) integrity, indicating an important instructive role for laminin in cerebral blood vessels. As previous work shows that in the normal brain, vascular expression of the laminin receptor α6ß4 integrin is predominantly restricted to arterioles, but induced on all vessels during neuroinflammation, it is important to define the role of this integrin in the maintenance of BBB integrity. METHODS: α6ß4 integrin expression was analyzed using dual immunofluorescence (dual-IF) of brain sections taken from the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). To investigate the role of endothelial α6ß4 integrin, transgenic mice lacking ß4 integrin in endothelial cells (ß4-EC-KO) and wild-type (WT) littermates were subject to EAE, and clinical score and various neuropathological parameters were examined by immunofluorescence. In addition, ß4 integrin null brain endothelial cells (BECs) were examined in culture for expression of tight junction proteins using immunocytochemistry and flow cytometry. RESULTS: Cerebrovascular expression of ß4 integrin was markedly upregulated during EAE progression, such that by the acute stage of EAE (day 21), the vast majority of blood vessels expressed ß4 integrin. In the EAE model, while the ß4-EC-KO mice showed the same time of disease onset as the WT littermates, they developed significantly worse clinical disease over time, resulting in increased clinical score at the peak of disease and maintained elevated thereafter. Consistent with this, the ß4-EC-KO mice showed enhanced levels of leukocyte infiltration and BBB breakdown and also displayed increased loss of the endothelial tight junction proteins claudin-5 and ZO-1. Under pro-inflammatory conditions, primary cultures of ß4KO BECs also showed increased loss of claudin-5 and ZO-1 expression. CONCLUSIONS: Taken together, our data suggest that α6ß4 integrin upregulation is an inducible protective mechanism that stabilizes the BBB during neuroinflammatory conditions.
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Barrera Hematoencefálica/patología , Encefalomielitis Autoinmune Experimental/patología , Endotelio Vascular/patología , Integrina alfa6beta4/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Encefalomielitis Autoinmune Experimental/metabolismo , Endotelio Vascular/metabolismo , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones Transgénicos , Uniones Estrechas/metabolismo , Uniones Estrechas/patologíaRESUMEN
Vascular remodeling involves a highly coordinated break-down and build-up of the vascular basal lamina and inter-endothelial tight junction proteins. In light of the important role of matrix metalloproteinases (MMPs) in tissue remodeling, the goal of this study was to examine the role of MMP-9 in remodeling of cerebral blood vessels, both in hypoxia-induced angiogenesis and in the vascular pruning that accompanies the switch from hypoxia back to normoxia. In a chronic mild hypoxia model of cerebrovascular remodeling, gel zymography revealed that MMP-9 levels were increased, both during hypoxic-induced angiogenesis and in the post-hypoxic pruning response. Interestingly, compared to wild-type mice, MMP-9 KO mice showed no alteration in hypoxic-induced angiogenesis, but did show marked delay in post-hypoxic vascular pruning. In wild-type mice, vascular pruning was associated with fragmentation of vascular laminin and the tight junction protein claudin-5, while this process was markedly attenuated in MMP-9 KO mice. In vitro experiments showed that hypoxia stimulated MMP-9 expression in brain endothelial cells but not pericytes. These results show that while MMP-9 is not essential for hypoxic-induced cerebral angiogenesis, it plays an important role in post-hypoxic vascular pruning by degrading laminin and claudin-5.
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Claudina-5/genética , Hipoxia , Laminina/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Remodelación Vascular , Animales , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Circulación Cerebrovascular , Claudina-5/metabolismo , Células Endoteliales/citología , Inmunohistoquímica , Laminina/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pericitos/citología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
OBJECTIVE: Laminin is a major component of the vascular basal lamina, implying that laminin receptors, such as α6ß1 and α6ß4 integrins, may regulate vascular remodeling and homeostasis. Previous studies in the central nervous system have shown that ß4 integrin is expressed by only a fraction of cerebral vessels, but defining the vessel type and cellular source of ß4 integrin has proved controversial. The goal of this study was to define the class of vessel and cell type expressing ß4 integrin in cerebral vessels and to examine its potential role in vascular remodeling. APPROACH AND RESULTS: Dual-immunofluorescence showed that ß4 integrin is expressed predominantly in arterioles, both in the central nervous system and in peripheral organs. Cell-specific knockouts of ß4 integrin revealed that ß4 integrin expression in cerebral vessels is derived from endothelial cells, not astrocytes or smooth muscle cells. Lack of endothelial ß4 integrin had no effect on vascular development, integrity, or endothelial proliferation, but in the hypoxic central nervous system, its absence led to defective arteriolar remodeling and associated transforming growth factor-ß signaling. CONCLUSIONS: These results define high levels of ß4 integrin in arteriolar endothelial cells and demonstrate a novel link among ß4 integrin, transforming growth factor-ß signaling, and arteriolar remodeling in cerebral vessels.
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Arteriolas/metabolismo , Células Endoteliales/patología , Hipoxia Encefálica/patología , Factores de Iniciación de Péptidos/fisiología , Actinas/análisis , Animales , Arteriolas/patología , Astrocitos/metabolismo , Células Endoteliales/metabolismo , Ratones , Ratones Endogámicos C57BL , Factores de Iniciación de Péptidos/análisis , Transducción de Señal , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Neuropathic pain is a common cause of pain after nerve injury, but its molecular basis is poorly understood. In a post-gene chip microarray effort to identify new target genes contributing to neuropathic pain development, we report here the characterization of a novel neuropathic pain contributor, thrombospondin-4 (TSP4), using a neuropathic pain model of spinal nerve ligation injury. TSP4 is mainly expressed in astrocytes and significantly upregulated in the injury side of dorsal spinal cord that correlates with the development of neuropathic pain states. TSP4 blockade by intrathecal antibodies, antisense oligodeoxynucleotides, or inactivation of the TSP4 gene reverses or prevents behavioral hypersensitivities. Intrathecal injection of TSP4 protein into naive rats is sufficient to enhance the frequency of EPSCs in spinal dorsal horn neurons, suggesting an increased excitatory presynaptic input, and to cause similar behavioral hypersensitivities. Together, these findings support that injury-induced spinal TSP4 may contribute to spinal presynaptic hypersensitivity and neuropathic pain states. Development of TSP4 antagonists has the therapeutic potential for target-specific neuropathic pain management.
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Neuralgia/metabolismo , Umbral del Dolor/fisiología , Médula Espinal/metabolismo , Trombospondinas/metabolismo , Regulación hacia Arriba/fisiología , 6-Ciano 7-nitroquinoxalina 2,3-diona , Análisis de Varianza , Animales , Anticuerpos/uso terapéutico , Modelos Animales de Enfermedad , Antagonistas de Aminoácidos Excitadores/farmacología , Proteínas Fluorescentes Verdes/genética , Humanos , Hiperalgesia/metabolismo , Hiperalgesia/patología , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/genética , Inyecciones Espinales , Masculino , Ratones , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Neuralgia/tratamiento farmacológico , Neuralgia/etiología , Oligodesoxirribonucleótidos Antisentido/administración & dosificación , Dimensión del Dolor , Umbral del Dolor/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Bloqueadores de los Canales de Sodio/farmacología , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Médula Espinal/fisiopatología , Nervios Espinales/lesiones , Tetrodotoxina/farmacología , Trombospondinas/deficiencia , Trombospondinas/genética , Regulación hacia Arriba/efectos de los fármacos , Valina/análogos & derivados , Valina/farmacologíaRESUMEN
BACKGROUND: There is increasing evidence to suggest that pericytes play a crucial role in regulating the remodeling state of blood vessels. As cerebral pericytes are embedded within the extracellular matrix (ECM) of the vascular basal lamina, it is important to understand how individual ECM components influence pericyte remodeling behavior, and how cytokines regulate these events. METHODS: The influence of different vascular ECM substrates on cerebral pericyte behavior was examined in assays of cell adhesion, migration, and proliferation. Pericyte expression of integrin receptors was examined by flow cytometry. The influence of cytokines on pericyte functions and integrin expression was also examined, and the role of specific integrins in mediating these effects was defined by function-blocking antibodies. Expression of pericyte integrins within remodeling cerebral blood vessels was analyzed using dual immunofluorescence (IF) of brain sections derived from the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). RESULTS: Fibronectin and collagen I promoted pericyte proliferation and migration, but heparan sulfate proteoglycan (HSPG) had an inhibitory influence on pericyte behavior. Flow cytometry showed that cerebral pericytes express high levels of α5 integrin, and lower levels of α1, α2, and α6 integrins. The pro-inflammatory cytokine tumor necrosis factor (TNF)-α strongly promoted pericyte proliferation and migration, and concomitantly induced a switch in pericyte integrins, from α1 to α2 integrin, the opposite to the switch seen when pericytes differentiated. Inhibition studies showed that α2 integrin mediates pericyte adhesion to collagens, and significantly, function blockade of α2 integrin abrogated the pro-modeling influence of TNF-α. Dual-IF on brain tissue with the pericyte marker NG2 showed that while α1 integrin was expressed by pericytes in both stable and remodeling vessels, pericyte expression of α2 integrin was strongly induced in remodeling vessels in EAE brain. CONCLUSIONS: Our results suggest a model in which ECM constituents exert an important influence on pericyte remodeling status. In this model, HSPG restricts pericyte remodeling in stable vessels, but during inflammation, TNF-α triggers a switch in pericyte integrins from α1 to α2, thereby stimulating pericyte proliferation and migration on collagen. These results thus define a fundamental molecular mechanism in which TNF-α stimulates pericyte remodeling in an α2 integrin-dependent manner.
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Cerebro/citología , Cerebro/metabolismo , Integrina alfa1/biosíntesis , Integrina alfa2/biosíntesis , Pericitos/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Células Cultivadas , Cerebro/efectos de los fármacos , Femenino , Ratones , Ratones Endogámicos C57BL , Pericitos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Pericytes play critical roles in the development, maturation and remodeling of blood vessels, and in the central nervous system (CNS), evidence suggests that pericytes also regulate blood flow and form an integral part of the blood-brain barrier. The study of this important cell type has been hampered by the lack of any pericyte-specific marker and by the difficulty of culturing pericytes in adequate numbers to high purity. Here we present a novel yet simple approach to isolate and culture large numbers of pericytes from the mouse CNS that nevertheless leads to very pure pericyte cultures. In our method, vascular cells obtained from adult mice brains are cultured initially under conditions optimized for endothelial cells, but after two passages switched to a medium optimized for pericyte growth. After growing the cells for 1-2 additional passages we obtained a largely homogeneous population of cells that expressed the pericyte markers NG2, PDGFß-receptor, and CD146, but were negative for markers of endothelial cells (CD31), microglia (Mac-1) and astrocytes (GFAP). Under these conditions, pericytes could be grown to high passage number, and were maintained highly pure and largely undifferentiated, as determined by antigen expression profile and low levels of α-SMA expression, a marker of pericyte differentiation. Furthermore, switching the cells from pericyte medium into DMEM containing 10% FBS promoted α-SMA expression, demonstrating that high passage pericytes could still differentiate. Thus, we provide an alternative approach to the culture of CNS pericytes that is easy to establish and provides large numbers of highly pure pericytes for extended periods of time. This system should provide others working in the pericyte field with a useful additional tool to study the behavior of this fascinating cell type.
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Encéfalo/irrigación sanguínea , Técnicas de Cultivo de Célula/métodos , Pericitos/citología , Animales , Antígenos/metabolismo , Biomarcadores/metabolismo , Antígeno CD146/metabolismo , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Ratones , Ratones Endogámicos C57BL , Pericitos/metabolismo , Proteoglicanos/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas y Péptidos Salivales/metabolismoRESUMEN
BACKGROUND: As the primary resident immune cells, microglia play a central role in regulating inflammatory processes in the CNS. The extracellular matrix (ECM) protein vitronectin promotes microglial activation, switching microglia into an activated phenotype. We have shown previously that microglia express two vitronectin receptors, αvß3 and αvß5 integrins. As these integrins have well-defined roles in activation and phagocytic processes in other cell types, the purpose of the current study was to investigate the contribution of these two integrins in microglial activation. METHODS: Microglial cells were prepared from wild-type, ß3 integrin knockout (KO), ß5 integrin KO or ß3/ß5 integrin DKO mice, and their interactions and activation responses to vitronectin examined in a battery of assays, including adhesion, expression of activation markers, MMP-9 expression, and phagocytosis. Expression of other αv integrins was examined by flow cytometry and immunoprecipitation. RESULTS: Surprisingly, when cultured on vitronectin, microglia from the different knockout strains showed no obvious defects in adhesion, activation marker expression, MMP-9 induction, or phagocytosis of vitronectin-coated beads. To investigate the reason for this lack of effect, we examined the expression of other αv integrins. Flow cytometry showed that ß3/ß5 integrin DKO microglia expressed residual αv integrin at the cell surface, and immunoprecipitation confirmed this finding by revealing the presence of low levels of the αvß1 and αvß8 integrins. ß1 integrin blockade had no impact on adhesion of ß3/ß5 integrin DKO microglia to vitronectin, suggesting that in addition to αvß1, αvß3, and αvß5, αvß8 also serves as a functional vitronectin receptor on microglia. CONCLUSIONS: Taken together, this demonstrates that the αvß3 and αvß5 integrins are not essential for mediating microglial activation responses to vitronectin, but that microglia use multiple redundant receptors to mediate interactions with this ECM protein.
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Integrina alfaVbeta3/metabolismo , Microglía/metabolismo , Receptores de Vitronectina/metabolismo , Vitronectina/metabolismo , Animales , Adhesión Celular/fisiología , Técnicas de Cultivo de Célula , Células Cultivadas , Fibronectinas/metabolismo , Integrina alfaVbeta3/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/citología , Receptores de Vitronectina/genéticaRESUMEN
OBJECTIVE: In the central nervous system (CNS), ß4 integrin is predominantly expressed by endothelial cells lining arterioles. As ß4 integrin plays an essential role in epithelial tissues, organizing structural proteins into specialized adhesive structures called hemidesmosomes (HD), the aim of this study was to determine whether it plays a similar role in CNS endothelium. METHODS: Dual-immunofluorescence was used to examine the relationship between ß4 integrin expression and co-expression of the HD proteins plectin and CD151 in frozen sections of mouse brain, both under normoxic (control) conditions and following chronic mild hypoxia. The requirement of ß4 integrin for the localization of HD proteins was examined in transgenic mice lacking ß4 integrin expression specifically in endothelial cells (ß4-EC-KO mice). RESULTS: Immunofluorescence revealed that in the normal adult CNS, plectin and CD151 strongly co-localized with ß4 integrin in arterioles. However, in the chronic mild hypoxia model, in which extensive cerebrovascular remodeling is observed, plectin and CD151 were strongly upregulated on all cerebral vessels, but surprisingly, in capillaries, this occurred in a ß4 integrin-independent manner. Unexpectedly, absence of endothelial ß4 integrin (in ß4-EC-KO mice) had no impact on the expression level or distribution pattern of plectin and CD151 within stable or remodeling cerebral vessels. CONCLUSIONS: These results demonstrate that the HD proteins plectin and CD151 are closely associated with ß4 integrin on arterioles in normal brain, and are strongly upregulated on remodeling blood vessels. However, unlike its described role in the epidermis, ß4 integrin is not essential for localization or regulation of expression of plectin and CD151 in cerebral vessels.
RESUMEN
Activated protein C (APC) is a serine protease that promotes favorable changes in vascular barrier integrity and post-ischemic angiogenic remodeling in animal models of ischemic stroke, and its efficacy is currently being investigated in clinical ischemic stroke trials. Interestingly, application of sub-clinical chronic mild hypoxia (CMH) (8% O2) also promotes angiogenic remodeling and increased tight junction protein expression, suggestive of enhanced blood-brain barrier (BBB) integrity, though the role of APC in mediating the influence of CMH has not been investigated. To examine this potential link, we studied CMH-induced cerebrovascular remodeling after treating mice with two different reagents: (i) a function-blocking antibody that neutralizes APC activity, and (ii) exogenous recombinant murine APC. While CMH promoted endothelial proliferation, increased vascular density, and upregulated the angiogenic endothelial integrins α5ß1 and αvß3, these events were almost completely abolished by functional blockade of APC. Consistent with these findings, addition of exogenous recombinant APC enhanced CMH-induced endothelial proliferation, expansion of total vascular area and further enhanced the CMH-induced right-shift in vessel size distribution. Taken together, our findings support a key role for APC in mediating physiological remodeling of cerebral blood vessels in response to CMH.
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Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Encéfalo/patología , Regulación de la Expresión Génica/fisiología , Neovascularización Fisiológica/fisiología , Proteína C/metabolismo , Animales , Anticuerpos/farmacología , Modelos Animales de Enfermedad , Endotelio Vascular/metabolismo , Integrina alfa5/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Proteína C/inmunología , Factores de TiempoRESUMEN
In animal models, hypoxic pre-conditioning confers protection against subsequent neurological insults, mediated in part through an extensive vascular remodeling response. In light of the therapeutic potential of this effect, the goal of this study was to establish the dose-response relationship between level of hypoxia and the extent of cerebrovascular modeling, and to define the mildest level of hypoxia that promotes remodeling. Mice were exposed to different levels of continuous hypoxia (8-21% O2) for seven days before several aspects of vascular remodeling were evaluated, including endothelial proliferation, total vascular area, arteriogenesis, and fibronectin/α5ß1 integrin expression. For most events, the threshold level of hypoxia that stimulated remodeling was 12-13% O2. Interestingly, many parameters displayed a biphasic dose-response curve, with peak levels attained at 10% O2, but declined thereafter. Further analysis in the 12-13% O2 range revealed that vascular remodeling occurs by two separate mechanisms: (i) endothelial hyperplasia, triggered by a hypoxic threshold of 13% O2, which leads to increased capillary growth, and (ii) endothelial hypertrophy, triggered by a more severe hypoxic threshold of 12% O2, which leads to expansion of large vessels and arteriogenesis. Taken together, these results define the hypoxic thresholds for vascular remodeling in the brain, and point to two separate mechanisms mediating this process.
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Encéfalo/irrigación sanguínea , Hipoxia Encefálica/fisiopatología , Precondicionamiento Isquémico/métodos , Neovascularización Fisiológica/fisiología , Remodelación Vascular/fisiología , Animales , Proliferación Celular , Células Endoteliales/fisiología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BLRESUMEN
The goal of this chapter is to highlight techniques used to determine the role of molecular mechanisms involved in remodeling of cerebral blood vessels. Enhanced vascularization in the central nervous system (CNS) is seen in many diseases including stroke, cancer, and multiple sclerosis (MS). However, despite the prevalence of this phenomenon in these different pathological conditions, the exact nature of how it occurs still remains unclear. To better understand the process of cerebrovascular remodeling, we use the chronic hypoxia model, in which a vigorous and robust angiogenic remodeling response takes place. In this model, mice are placed in a hypoxic chamber (8 % O2 for up to 14 days), which results in strong vascular remodeling and increased vessel density within the CNS. Using an immunofluorescent (IF)-based approach, different aspects of this vascular remodeling response can be examined. By employing this method, we have shown that chronic mild hypoxia triggers both angiogenic (capillary sprouting) and arteriogenic (widening of arterial vessels) responses. Furthermore, we have used this system to define both the expression pattern and potential role of candidate adhesion molecules in this vascular remodeling process. Thus, the techniques described in this chapter can be used to define the importance of different molecular mechanisms in vascular remodeling in the CNS.
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Encéfalo/irrigación sanguínea , Neovascularización Fisiológica , Animales , Hipoxia de la Célula , Proliferación Celular , Circulación Cerebrovascular , Células Endoteliales/fisiología , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Fibronectinas/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Secciones por Congelación , Integrina alfaV/metabolismo , Ratones , Microscopía Fluorescente , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismoRESUMEN
Blood vessels in the central nervous system (CNS) are unique in forming the blood-brain barrier (BBB), which confers high electrical resistance and low permeability properties, thus protecting neural cells from potentially harmful blood components. Endothelial cells, which form the inner cellular lining of all blood vessels, play a critical role in this process by forming tight adhesive interactions between each other. To study the properties of primary brain endothelial cells (BECs), a number of different methods have been described. In this chapter, we present a relatively simple method that produces high numbers of primary mouse BECs that are highly pure (greater than 99 % CD31-positive). In addition, we also describe an immunocytochemical approach to demonstrate the endothelial purity of these cultures.
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Células Endoteliales/fisiología , Animales , Encéfalo/irrigación sanguínea , Separación Celular , Células Cultivadas , Arterias Cerebrales/citología , Venas Cerebrales/citología , Endotelio Vascular/citología , Ratones , Cultivo Primario de CélulasRESUMEN
Pericytes are perivascular cells that play an important role in the development, maturation, and remodeling of blood vessels. However, studies of this important cell type on vascular remodeling have been hindered due to the difficulty of culturing pericytes in adequate numbers to high purity. In this chapter, we present a novel yet simple method to isolate and culture large numbers of pure pericytes from the mouse central nervous system (CNS). In our approach, vascular cells obtained from adult mice brains are cultured initially under conditions optimized for endothelial cells. Following two passages, the medium is switched over to optimize pericyte growth. After growing the cells for 2-3 additional passages, this approach produces a largely homogeneous population of cells that express the pericyte markers NG2, PDGFß receptor, and CD146 but are negative for markers of endothelial cells (CD31), astrocytes (GFAP), and microglia (Mac-1), demonstrating a highly pure pericyte culture. Thus, our technique provides an effective method to culture CNS pericytes that is easy to establish and provides large numbers of highly pure pericytes for extended periods of time. This system provides a useful tool for those wishing to study pericyte behavior.
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Microvasos/citología , Pericitos/fisiología , Animales , Biomarcadores/metabolismo , Encéfalo/irrigación sanguínea , Separación Celular , Células Cultivadas , Inmunohistoquímica , Ratones , Cultivo Primario de CélulasRESUMEN
As well as being a central regulator of inflammatory and immune-mediated events, TNF-α also influences vascular remodeling, resulting in alterations in the structure and function of blood vessels. In addition to endothelial cells, pericytes are another type of vascular cell that significantly contribute to the development, maturation, stabilization, and remodeling of blood vessels. To investigate the regulatory influence of different factors on pericyte behavior, we recently described a novel yet simple approach of isolating and culturing highly pure, high density cultures of mouse brain pericytes. In this chapter, we briefly describe this culture system, as well as methods for examining different aspects of pericyte behavior, including cell adhesion, cell migration, and cell proliferation. These assays can be used to examine the influence of TNF-α or any other factor on pericyte behavior.
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Encéfalo/citología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Endotelio Vascular/citología , Pericitos/citología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Encéfalo/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Ratones , Pericitos/efectos de los fármacosRESUMEN
Alterations in vascular structure and function are a central component of demyelinating disease. In addition to blood-brain barrier (BBB) breakdown, which occurs early in the course of disease, recent studies have described angiogenic remodeling, both in multiple sclerosis tissue and in the mouse demyelinating model, experimental autoimmune encephalomyelitis (EAE). As the precise timing of vascular remodeling in demyelinating disease has yet to be fully defined, the purpose of the current study was to define the time-course of these events in the MOG35-55 EAE model. Quantification of endothelial cell proliferation and vessel density revealed that a large part of angiogenic remodeling in cervical spinal cord white matter occurs during the pre-symptomatic phase of EAE. At the height of vascular remodeling, blood vessels in the cervical spinal cord showed strong transient upregulation of fibronectin and the α5ß1 integrin. In vitro experiments revealed that α5 integrin inhibition reduced brain endothelial cell proliferation under inflammatory conditions. Interestingly, loss of vascular integrity was evident in all vessels during the first 4-7days post-immunization, but after 14days, was localized predominantly to venules. Taken together, our data demonstrate that extensive vascular remodeling occurs during the pre-symptomatic phase of EAE and point to a potential role for the fibronectin-α5ß1 integrin interaction in promoting vascular remodeling during demyelinating disease.
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Encéfalo/irrigación sanguínea , Encefalomielitis Autoinmune Experimental/patología , Fibronectinas/biosíntesis , Integrina alfa5beta1/biosíntesis , Médula Espinal/irrigación sanguínea , Animales , Encefalomielitis Autoinmune Experimental/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BLRESUMEN
Chronic cerebral hypoxia leads to a strong vascular remodeling response, though little is known about which part of the vascular tree is modified, or whether this response includes formation of new arterial vessels. In this study, we examined this process in detail, analyzing how hypoxia (8% O(2) for 14 days) alters the size distribution of vessels, number of arteries/arterioles, and expression pattern of endoglin (CD105), a marker of angiogenic endothelial cells in tumors. We found that cerebral hypoxia promoted the biggest increase in the number of medium to large size vessels, and this correlated with increased numbers of alpha smooth muscle actin (α-SMA)-positive arterial vessels. Surprisingly, hypoxia induced a marked reduction in CD105 expression on brain endothelial cells (BECs) within remodeling arterial vessels, and these BECs also displayed an angiogenic switch in ß1 integrins (from α6 to α5), previously described for developmental angiogenesis. In vitro, transforming growth factor (TGF)-ß1 also promoted this switch of BEC ß1 integrins. Together, these results show that cerebral hypoxia promotes arteriogenesis, and identify reduced CD105 expression as a novel marker of arteriogenesis. Furthermore, our data suggest a mechanistic model whereby BECs in remodeling arterial vessels downregulate CD105 expression, which alters TGF-ß1 signaling, to promote a switch in ß1 integrins and arteriogenic remodeling.
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Arterias Cerebrales/patología , Hipoxia Encefálica/metabolismo , Integrina beta1/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neovascularización Fisiológica/fisiología , Actinas/biosíntesis , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Células Cultivadas , Enfermedad Crónica , Endoglina , Células Endoteliales/metabolismo , Citometría de Flujo , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Integrina alfa5beta1/metabolismo , Integrina alfa6beta1/metabolismo , Integrina beta1/genética , Integrina beta1/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/fisiología , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/metabolismo , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
Spinal cord injury-induced pain is a common clinical problem affecting adversely the quality of daily lives of spinal cord injured patients. Management with current pain medications can only lead to partial pain relief in some spinal cord injured patients, which is usually associated with unfavorable side effects. Development of specific medications for spinal cord injury-induced pain states relies on identification of new targets and/or pathways that contribute to chronic pain development post injury. We describe here the generation of a spinal cord contusion injury model that mimics the etiology and phenotypes of chronic pain states in spinal cord injured patients. Therefore, this model can be a useful tool for studying spinal cord injury mechanisms, functional recovery, research, and development of new medications for better functional and symptomatic improvements, including pain management.
Asunto(s)
Dolor Crónico/etiología , Modelos Animales de Enfermedad , Traumatismos de la Médula Espinal/complicaciones , Médula Espinal/fisiopatología , Animales , Contusiones/complicaciones , Contusiones/fisiopatología , Ratones , Manejo del Dolor/métodos , Ratas , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
Fibronectin is a critical regulator of vascular modelling, both in development and in the adult. In the hypoxic adult central nervous system (CNS), fibronectin is induced on angiogenic vessels, and endothelial cells show strong induction of the two fibronectin receptors α5ß1 and αvß3 integrins. In a previous study, we found that the αvß3 integrin is dispensable for hypoxic-induced cerebral angiogenesis, but a role for the endothelial α5ß1 integrin was suggested. To directly investigate the role of endothelial α5 integrin in cerebral angiogenesis, wild-type mice and mice lacking α5 integrin expression in endothelial cells (α5-EC-KO) were subject to hypoxia (8% O(2)) for 0, 2, 4, 7 or 14 days. Quantification of cerebral vessel density and endothelial-specific proteins claudin-5 and Glut-1 revealed that α5-EC-KO mice displayed an attenuated angiogenic response, which correlated with delayed endothelial proliferation. α5-EC-KO mice showed no defect in the ability to organize a cerebrovascular fibronectin matrix, and no compensatory increase in vascular αvß3 integrin expression. Consistent with these findings, primary α5KO brain endothelial cells (BEC) in culture exhibited delayed growth and proliferation. Taken together, these studies demonstrate an important angiogenic role for the α5ß1 integrin in promoting BEC proliferation in response to cerebral hypoxia.
Asunto(s)
Proliferación Celular , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Hipoxia Encefálica/etiología , Integrina alfa5/fisiología , Integrina beta1/fisiología , Neovascularización Patológica/etiología , Neovascularización Patológica/patología , Animales , Células Cultivadas , Hipoxia Encefálica/genética , Hipoxia Encefálica/patología , Integrina alfa5/biosíntesis , Integrina alfa5/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neovascularización Patológica/genéticaRESUMEN
Spinal cord injury (SCI) commonly results in the development of neuropathic pain, which can dramatically impair the quality of life for SCI patients. SCI-induced neuropathic pain can be manifested as both tactile allodynia (a painful sensation to a non-noxious stimulus) and hyperalgesia (an enhanced sensation to a painful stimulus). The mechanisms underlying these pain states are poorly understood. Clinical studies have shown that gabapentin, a drug that binds to the voltage-gated calcium channel alpha-2-delta-1 subunit (Ca(v)α2δ-1) proteins is effective in the management of SCI-induced neuropathic pain. Accordingly, we hypothesized that tactile allodynia post SCI is mediated by an upregulation of Ca(v)α2δ-1 in dorsal spinal cord. To test this hypothesis, we examined whether SCI-induced dysregulation of spinal Ca(v)α2δ-1 plays a contributory role in below-level allodynia development in a rat spinal T9 contusion injury model. We found that Ca(v)α2δ-1 expression levels were significantly increased in L4-6 dorsal, but not ventral, spinal cord of SCI rats that correlated with tactile allodynia development in the hind paw plantar surface. Furthermore, both intrathecal gabapentin treatment and blocking SCI-induced Ca(v)α2δ-1 protein upregulation by intrathecal Ca(v)α2δ-1 antisense oligodeoxynucleotides could reverse tactile allodynia in SCI rats. These findings support that SCI-induced Ca(v)α2δ-1 upregulation in spinal dorsal horn is a key component in mediating below-level neuropathic pain states, and selectively targeting this pathway may provide effective pain relief for SCI patients. Spinal cord contusion injury caused increased calcium channel Ca(v)α2δ-1 subunit expression in dorsal spinal cord that contributes to neuropathic pain states.