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1.
Aging Cell ; 18(5): e13000, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31267651

RESUMO

Dual-specificity tyrosine phosphorylation-regulated kinase-1A (DYRK1A) is known to phosphorylate the microtubule-associated tau protein. Overexpression is correlated with tau hyperphosphorylation and neurofibrillary tangle (NFT) formation in Alzheimer's disease (AD). This study assessed the potential of SM07883, an oral DYRK1A inhibitor, to inhibit tau hyperphosphorylation, aggregation, NFT formation, and associated phenotypes in mouse models. Exploratory neuroinflammatory effects were also studied. SM07883 specificity was tested in a kinase panel screen and showed potent inhibition of DYRK1A (IC50  = 1.6 nM) and GSK-3ß (IC50  = 10.8 nM) kinase activity. Tau phosphorylation measured in cell-based assays showed a reduction in phosphorylation of multiple tau epitopes, especially the threonine 212 site (EC50  = 16 nM). SM07883 showed good oral bioavailability in multiple species and demonstrated a dose-dependent reduction of transient hypothermia-induced phosphorylated tau in the brains of wild-type mice compared to vehicle (47%, p < 0.001). Long-term efficacy assessed in aged JNPL3 mice overexpressing the P301L human tau mutation (3 mg/kg, QD, for 3 months) exhibited significant reductions in tau hyperphosphorylation, oligomeric and aggregated tau, and tau-positive inclusions compared to vehicle in brainstem and spinal cord samples. Reduced gliosis compared to vehicle was further confirmed by ELISA. SM07883 was well tolerated with improved general health, weight gain, and functional improvement in a wire-hang test compared to vehicle-treated mice (p = 0.048). SM07883, a potent, orally bioavailable, brain-penetrant DYRK1A inhibitor, significantly reduced effects of pathological tau overexpression and neuroinflammation, while functional endpoints were improved compared to vehicle in animal models. This small molecule has potential as a treatment for AD.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Isoquinolinas/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas tau/metabolismo , Administração Oral , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Isoquinolinas/administração & dosagem , Isoquinolinas/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/química , Proteínas tau/genética , Proteínas tau/toxicidade , Quinases Dyrk
2.
Am J Physiol Cell Physiol ; 312(4): C428-C437, 2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28148497

RESUMO

Mechanochemical signal transduction occurs when mechanical forces, such as fluid shear stress, are converted into biochemical responses within the cell. The molecular mechanisms by which endothelial cells (ECs) sense/transduce shear stress into biological signals, including the nature of the mechanosensor, are still unclear. G proteins and G protein-coupled receptors (GPCRs) have been postulated independently to mediate mechanotransduction. In this study, we used in situ proximity ligation assay (PLA) to investigate the role of a specific GPCR/Gαq/11 pair in EC shear stress-induced mechanotransduction. We demonstrated that sphingosine 1-phosphate (S1P) stimulation causes a rapid dissociation at 0.5 min of Gαq/11 from its receptor S1P3, followed by an increased association within 2 min of GPCR kinase-2 (GRK2) and ß-arrestin-1/2 with S1P3 in human coronary artery ECs, which are consistent with GPCR/Gαq/11 activation and receptor desensitization/internalization. The G protein activator AlF4 resulted in increased dissociation of Gαq/11 from S1P3, but no increase in association between S1P3 and either GRK2 or ß-arrestin-1/2. The G protein inhibitor guanosine 5'-(ß-thio) diphosphate (GDP-ß-S) and the S1P3 antagonist VPC23019 both prevented S1P-induced activation. Shear stress also caused the rapid activation within 7 s of S1P3/Gαq/11 There were no increased associations between S1P3 and GRK2 or S1P3 and ß-arrestin-1/2 until 5 min. GDP-ß-S, but not VPC23019, prevented dissociation of Gαq/11 from S1P3 in response to shear stress. Shear stress did not induce rapid dephosphorylation of ß-arrestin-1 or rapid internalization of S1P3, indicating no GPCR activation. These findings suggest that Gαq/11 participates in the sensing/transducing of shear stress independently of GPCR activation in ECs.


Assuntos
Células Endoteliais/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Lisofosfolipídeos/metabolismo , Mecanotransdução Celular/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Esfingosina/análogos & derivados , Células Cultivadas , Humanos , Masculino , Receptores de Lisoesfingolipídeo , Resistência ao Cisalhamento/fisiologia , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato , Estresse Mecânico
3.
J Cell Biochem ; 115(1): 121-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23913776

RESUMO

Endothelial cells undergo a rapid cell-cell junction inclination following exposure to atheroprotective unidirectional flow. In contrast, atherosclerotic lesions correlate with a heterogeneous distribution of the junctional wall inclination in cells exposed to time-varying, reversing, and oscillatory flow as well as to low mean shear stress. However, the underlying biochemical events by which endothelial cells distinctively respond to unidirectional versus flow reversal remain unclear. Here, we show that the subcellular distribution of flow-induced Akt-1 phosphorylation in endothelial cells lining the mouse aorta varies depending on local hemodynamics. Activated Akt-1 accumulated in perinuclear areas of cells in regions predisposed to disturbed flow but were localized at the cell-cell junction in regions of high unidirectional laminar shear stress. In flow-adapted human endothelial cells, reversal in flow direction was associated within minutes with a subcellular concentration of phosphorylated Akt-1 at the upstream edge of cells. Interestingly, oscillatory flow (with a zero mean shear stress) failed to activate Akt-1, whereas a unidirectional pulsatile flow of similar amplitude induced an increase in Akt-1 phosphorylation. Finally, silencing of the G protein αq/11 subunit abrogated both flow-induced Akt-1 and GSK-3ß activation. Together, these results characterize the existence of a Gαq/11-mediated Akt-1 signaling pathway that is dynamically responsive to flow direction, thereby offering a novel approach to regulating EC dysfunctions in regions subjected to flow reversal.


Assuntos
Células Endoteliais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Aorta/citologia , Células Cultivadas , Células Endoteliais/citologia , Endotélio Vascular/citologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Estresse Mecânico
4.
Antimicrob Agents Chemother ; 57(11): 5462-71, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23979751

RESUMO

Cerebral malaria (CM) is associated with low nitric oxide (NO) bioavailability, cerebrovascular constriction, occlusion, and hypoperfusion. Administration of exogenous NO partially prevents the neurological syndrome and associated vascular pathology in an experimental CM (ECM) mouse model. In this study, we evaluated the effects of transdermal glyceryl trinitrate in preventing ECM and, in combination with artemether, rescuing late-stage ECM mice from mortality. The glyceryl trinitrate and/or artemether effect on survival and clinical recovery was evaluated in C57BL/6 mice infected with P. berghei ANKA. NO synthase (NOS) expression in mouse brain was determined by Western blots. Mean arterial pressure (MAP) and pial arteriolar diameter were monitored using a tail-cuff blood pressure system and a cranial window preparation, respectively. Preventative administration of glyceryl trinitrate at 0.025 mg/h decreased ECM mortality from 67 to 11% and downregulated inducible NOS expression in the brain. When administered as adjunctive rescue therapy with artemether, glyceryl trinitrate increased survival from 47 to 79%. The adjunctive therapy caused a sustained reversal of pial arteriolar vasoconstriction in ECM mice, an effect not observed with artemether alone. Glyceryl trinitrate induced a 13% decrease in MAP in uninfected mice but did not further affect MAP in hypotensive ECM mice. Glyceryl trinitrate, when combined with artemether, was an effective adjunctive rescue treatment for ECM. This treatment ameliorated pial arteriolar vasospasm and did not significantly affect MAP. These results indicate that transdermal glyceryl trinitrate has potential to be considered as a candidate for adjunctive therapy for CM.


Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Encéfalo/efeitos dos fármacos , Malária Cerebral/tratamento farmacológico , Nitroglicerina/farmacologia , Vasodilatadores/farmacologia , Administração Cutânea , Animais , Artemeter , Pressão Arterial , Encéfalo/irrigação sanguínea , Encéfalo/parasitologia , Sinergismo Farmacológico , Feminino , Expressão Gênica/efeitos dos fármacos , Malária Cerebral/mortalidade , Malária Cerebral/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/patogenicidade , Análise de Sobrevida , Resultado do Tratamento , Vasoconstrição/efeitos dos fármacos
5.
PLoS Pathog ; 9(6): e1003444, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23818850

RESUMO

Cerebrovascular dysfunction plays a key role in the pathogenesis of cerebral malaria. In experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA, cerebrovascular dysfunction characterized by vascular constriction, occlusion and damage results in impaired perfusion and reduced cerebral blood flow and oxygenation, and has been linked to low nitric oxide (NO) bioavailability. Here, we directly assessed cerebrovascular function in ECM using a novel cranial window method for intravital microscopy of the pial microcirculation and probed the role of NOS isoforms and phosphorylation patterns in the impaired vascular responses. We show that pial arteriolar responses to endothelial NOS (eNOS) and neuronal NOS (nNOS) agonists (Acetylcholine (ACh) and N-Methyl-D-Aspartate (NMDA)) were blunted in mice with ECM, and could be partially recovered by exogenous supplementation of tetrahydrobiopterin (BH4). Pial arterioles in non-ECM mice infected by Plasmodium berghei NK65 remained relatively responsive to the agonists and were not significantly affected by BH4 treatment. These findings, together with the observed blunting of NO production upon stimulation by the agonists, decrease in total NOS activity, augmentation of lipid peroxidation levels, upregulation of eNOS protein expression, and increase in eNOS and nNOS monomerization in the brain during ECM development strongly indicate a state of eNOS/nNOS uncoupling likely mediated by oxidative stress. Furthermore, the downregulation of Serine 1176 (S1176) phosphorylation of eNOS, which correlated with a decrease in cerebrovascular wall shear stress, implicates hemorheological disturbances in eNOS dysfunction in ECM. Finally, pial arterioles responded to superfusion with the NO donor, S-Nitroso-L-glutathione (GSNO), but with decreased intensity, indicating that not only NO production but also signaling is perturbed during ECM. Therefore, the pathological impairment of eNOS and nNOS functions contribute importantly to cerebrovascular dysfunction in ECM and the recovery of intrinsic functionality of NOS to increase NO bioavailability and restore vascular health represents a target for ECM treatment.


Assuntos
Circulação Cerebrovascular , Malária Cerebral , Microcirculação , Óxido Nítrico/metabolismo , Plasmodium berghei/metabolismo , Acetilcolina/farmacologia , Animais , Biopterinas/análogos & derivados , Biopterinas/farmacologia , Agonistas Colinérgicos , Agonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Malária Cerebral/metabolismo , Malária Cerebral/parasitologia , Malária Cerebral/fisiopatologia , Camundongos , N-Metilaspartato/farmacologia , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo
6.
Biochem Biophys Res Commun ; 434(3): 641-6, 2013 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-23583373

RESUMO

Although several potential mechanosensors/mechanotransducers have been proposed, the precise mechanisms by which ECs sense and respond to mechanical forces and translate them into biochemical signals remains unclear. Here, we report that two major ligand-dependent tyrosine autophosphorylation sites of VEGFR2, Y1175 and Y1214, are rapidly activated by shear stress in human coronary artery endothelial cells (HCAECs). Neutralizing antibody against VEGFR2 not only abrogates flow-induced phosphorylation of these tyrosine residues, but also has a marked inhibitory effect on downstream eNOS activation. In situ proximity ligation assay revealed that VEGF and VEGFR2 are closely associated in HCAECs, and more importantly, this association is increased with flow. Finally, we show that flow-induced VEGFR2 activation is attenuated in the presence of the broad spectrum matrix metalloproteinase (MMP) inhibitor, GM6001. Taken together, our results suggest that a ligand-dependent mechanism involving the activity of MMPs plays a key role in the early, shear stress-induced activation of VEGFR2.


Assuntos
Metaloproteinases da Matriz/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Anticorpos Neutralizantes/imunologia , Western Blotting , Células Cultivadas , Vasos Coronários/citologia , Vasos Coronários/enzimologia , Vasos Coronários/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Endotélio Vascular/metabolismo , Humanos , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologia
7.
Am J Physiol Cell Physiol ; 303(4): C467-73, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22700794

RESUMO

Disturbed flow patterns, including reversal in flow direction, are key factors in the development of dysfunctional endothelial cells (ECs) and atherosclerotic lesions. An almost immediate response of ECs to fluid shear stress is the increase in cytosolic calcium concentration ([Ca(2+)](i)). Whether the source of [Ca(2+)](i) is extracellular, released from Ca(2+) intracellular stores, or both is still undefined, though it is likely dependent on the nature of forces involved. We have previously shown that a change in flow direction (retrograde flow) on a flow-adapted endothelial monolayer induces the remodeling of the cell-cell junction along with a dramatic [Ca(2+)](i) burst compared with cells exposed to unidirectional or orthograde flow. The heterotrimeric G protein-α q and 11 subunit (Gα(q/11)) is a likely candidate in effecting shear-induced increases in [Ca(2+)](i) since its expression is enriched at the junction and has been previously shown to be activated within seconds after onset of flow. In flow-adapted human ECs, we have investigated to what extent the Gα(q/11) pathway mediates calcium dynamics after reversal in flow direction. We observed that the elapsed time to peak [Ca(2+)](i) response to a 10 dyn/cm(2) retrograde shear stress was increased by 11 s in cells silenced with small interfering RNA directed against Gα(q/11). A similar lag in [Ca(2+)](i) transient was observed after cells were treated with the phospholipase C (PLC)-ßγ inhibitor, U-73122, or the phosphatidylinositol-specific PLC inhibitor, edelfosine, compared with controls. Lower levels of inositol 1,4,5-trisphosphate accumulation seconds after the onset of flow correlated with the increased lag in [Ca(2+)](i) responses observed with the different treatments. In addition, inhibition of the inositol 1,4,5-trisphosphate receptor entirely abrogated flow-induced [Ca(2+)](i). Taken together, our results identify the Gα(q/11)-PLC pathway as the initial trigger for retrograde flow-induced endoplasmic reticulum calcium store release, thereby offering a novel approach to regulating EC dysfunctions in regions subjected to the reversal of blood flow.


Assuntos
Cálcio/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Sinalização do Cálcio/fisiologia , Citosol/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Humanos , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Fosfatos de Inositol/metabolismo , Fosfoinositídeo Fosfolipase C/metabolismo , Interferência de RNA , Sistemas do Segundo Mensageiro , Estresse Mecânico
8.
ASN Neuro ; 2(3): e00037, 2010 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-20640189

RESUMO

Vaccine-based autoimmune (anti-amyloid) treatments are currently being examined for their therapeutic potential in Alzheimer's disease. In the present study we examined, in a transgenic model of amyloid pathology, the expression of two molecules previously implicated in decreasing the severity of autoimmune responses: TREM2 (triggering receptor expressed on myeloid cells 2) and the intracellular tolerance-associated transcript, Tmem176b (transmembrane domain protein 176b). In situ hybridization analysis revealed that both molecules were highly expressed in plaque-associated microglia, but their expression defined two different zones of plaque-associated activation. Tmem176b expression was highest in the inner zone of amyloid plaques, whereas TREM2 expression was highest in the outer zone. Induced expression of TREM2 occurred co-incident with detection of thioflavine-S-positive amyloid deposits. Transfection studies revealed that expression of TREM2 correlated negatively with motility, but correlated positively with the ability of microglia to stimulate CD4(+) T-cell proliferation, TNF (tumour necrosis factor) and CCL2 (chemokine ligand 2) production, but not IFNgamma (interferon gamma) production. TREM2 expression also showed a positive correlation with amyloid phagocytosis in unactivated cells. However, activating cells with LPS (lipopolysaccharide), but not IFNgamma, reduced the correlation between TREM2 expression and phagocytosis. Transfection of Tmem176b into both microglial and macrophage cell lines increased apoptosis. Taken together, these data suggest that, in vivo, Tmem176b(+) cells in closest apposition to amyloid may be the least able to clear amyloid. Conversely, the phagocytic TREM2(+) microglia on the plaque outer zones are positioned to capture and present self-antigens to CNS (central nervous system)-infiltrating lymphocytes without promoting pro-inflammatory lymphocyte responses. Instead, plaque-associated TREM2(+) microglia have the potential to evoke neuroprotective immune responses that may serve to support CNS function during pro-inflammatory anti-amyloid immune therapies.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/prevenção & controle , Amiloide/genética , Amiloide/metabolismo , Imunoterapia Ativa , Glicoproteínas de Membrana/biossíntese , Receptores Imunológicos/biossíntese , Doença de Alzheimer/metabolismo , Amiloide/fisiologia , Animais , Linhagem Celular Transformada , Células Cultivadas , Regulação da Expressão Gênica/imunologia , Humanos , Imunoterapia Ativa/métodos , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fagocitose/fisiologia , Placa Amiloide/genética , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Receptores Imunológicos/genética , Receptor Gatilho 1 Expresso em Células Mieloides
9.
Am J Physiol Cell Physiol ; 299(3): C621-9, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20554908

RESUMO

Atheroprone regions of the arterial circulation are characterized by time-varying, reversing, and oscillatory wall shear stress. Several in vivo and in vitro studies have demonstrated that flow reversal (retrograde flow) is atherogenic and proinflammatory. The molecular and structural basis for the sensitivity of the endothelium to flow direction, however, has yet to be determined. It has been hypothesized that the ability to sense flow direction is dependent on the direction of inclination of the interendothelial junction. Immunostaining of the mouse aorta revealed an inclination of the cell-cell junction by 13 degrees in direction of flow in the descending aorta where flow is unidirectional. In contrast, polygonal cells of the inner curvature where flow is disturbed did not have any preferential inclination. Using a membrane specific dye, the angle of inclination of the junction was dynamically monitored using live cell confocal microscopy in confluent human endothelial cell monolayers. Upon application of shear the junctions began inclining within minutes to a final angle of 10 degrees in direction of flow. Retrograde flow led to a reversal of junctional inclination. Flow-induced junctional inclination was shown to be independent of the cytoskeleton or glycocalyx. Additionally, within seconds, retrograde flow led to significantly higher intracellular calcium responses than orthograde flow. Together, these results show for the first time that the endothelial intercellular junction inclination is dynamically responsive to flow direction and confers the ability to endothelial cells to rapidly sense and adapt to flow direction.


Assuntos
Células Endoteliais/fisiologia , Endotélio Vascular/fisiologia , Junções Intercelulares/fisiologia , Animais , Aorta/citologia , Aorta/fisiologia , Cálcio/fisiologia , Células Cultivadas , Endotélio Vascular/citologia , Humanos , Técnicas In Vitro , Espaço Intracelular/metabolismo , Mecanotransdução Celular , Camundongos , Camundongos Endogâmicos C57BL , Reologia , Estresse Mecânico , Veias Umbilicais/citologia
10.
J Physiol ; 587(Pt 10): 2365-73, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19332487

RESUMO

It has been recently shown that endothelial platelet endothelial cell adhesion molecule-1 (PECAM-1) expression is pro-atherogenic. PECAM-1 is involved in sensing rapid changes in fluid shear stress but the mechanisms for activating signalling complexes at the endothelial cell junction have yet to be elucidated. Additional studies suggest the activation of membrane-bound G proteins G alpha(q/11) also mediate flow-induced responses. Here, we investigated whether PECAM-1 and G alpha(q/11) could act in unison to rapidly respond to fluid shear stress. With immunohistochemistry, we observed a co-localization of G alpha(q/11) and PECAM-1 at the cell-cell junction in the atheroprotected section of mouse aortae. In contrast, G alpha(q/11) was absent from junctions in atheroprone areas as well as in all arterial sections of PECAM-1 knockout mice. In primary human endothelial cells, temporal gradients in shear stress led to a rapid dissociation of the G alpha(q/11)-PECAM-1 complex within 30 s and a partial relocalization of the G alpha(q/11) staining to perinuclear areas within 150 min, whereas transitioning fluid flow devoid of temporal gradients did not disrupt the complex. Inhibition of G protein activation eliminated temporal gradient flow-induced G alpha(q/11)-PECAM-1 dissociation. These results allow us to conclude that G alpha(q/11)-PECAM-1 forms a mechanosensitive complex and its localization suggests the G alpha(q/11)-PECAM-1 complex is a critical mediator of vascular diseases.


Assuntos
Endotélio Vascular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Hemorreologia/fisiologia , Mecanotransdução Celular/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Estresse Mecânico , Animais , Aorta/citologia , Aorta/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Inibidores Enzimáticos/farmacologia , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Humanos , Junções Intercelulares/metabolismo , Espaço Intracelular/metabolismo , Mecanotransdução Celular/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Ligação Proteica/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Tionucleotídeos/farmacologia
11.
J Neurochem ; 109 Suppl 1: 117-25, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19393017

RESUMO

Two different macrophage populations contribute to CNS neuroinflammation: CNS-resident microglia and CNS-infiltrating peripheral macrophages. Markers distinguishing these two populations in tissue sections have not been identified. Therefore, we compared gene expression between LPS (lipopolysaccharide)/interferon (IFN)gamma-treated microglia from neonatal mixed glial cultures and similarly treated peritoneal macrophages. Fifteen molecules were identified by quantative PCR (qPCR) as being enriched from 2-fold to 250-fold in cultured neonatal microglia when compared with peritoneal macrophages. Only three of these molecules (C1qA, Trem2, and CXCL14) were found by qPCR to be also enriched in adult microglia isolated from LPS/IFNgamma-injected CNS when compared with infiltrating peripheral macrophages from the same CNS. The discrepancy between the in vitro and in vivo qPCR data sets was primarily because of induced expression of the 'microglial' molecules (such as the tolerance associated transcript, Tmem176b) in CNS-infiltrating macrophages. Bioinformatic analysis of the approximately 19000 mRNAs detected by TOGA gene profiling confirmed that LPS/IFNgamma-activated microglia isolated from adult CNS displayed greater similarity in total gene expression to CNS-infiltrating macrophages than to microglia isolated from unmanipulated healthy adult CNS. In situ hybridization analysis revealed that nearly all microglia expressed high levels of C1qA, while subsets of microglia expressed Trem2 and CXCL14. Expression of C1qA and Trem2 was limited to microglia, while large numbers of GABA+ neurons expressed CXCL14. These data suggest that (i) CNS-resident microglia are heterogeneous and thus a universal microglia-specific marker may not exist; (ii) the CNS micro-environment plays significant roles in determining the phenotypes of both CNS-resident microglia and CNS-infiltrating macrophages; (iii) the CNS microenvironment may contribute to immune privilege by inducing macrophage expression of anti-inflammatory molecules.


Assuntos
Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/fisiologia , Macrófagos/metabolismo , Microglia/metabolismo , Animais , Northern Blotting , Células Cultivadas , Biologia Computacional , Células Dendríticas/metabolismo , Perfilação da Expressão Gênica , Hibridização In Situ , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Modelos Neurológicos , RNA/biossíntese , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Reversa
12.
Dis Model Mech ; 1(2-3): 175-81; discussion 179, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19048083

RESUMO

Atherosclerosis is a chronic inflammatory disease of large arteries in which lesion development preferentially occurs at vessel sites exposed to rapid changes in flow. We have previously shown that platelet endothelial cell adhesion molecule (PECAM-1), a surface receptor of the immunoglobulin superfamily, is involved in mechanosensing of rapid changes in flow. We wondered whether apolipoprotein E deficient (ApoE(-/-)) mice, predisposed to development of atheromas, would be protected from atherosclerosis by deficiency in PECAM-1. Using double knockout (DKO) mice for both PECAM-1 and ApoE (ApoE(-/-)/PECAM-1(-/-)) we found a significant reduction of sudanophilic lesions in their aortae compared to single knockout (SKO) (ApoE(-/-)/PECAM-1(+/+)) mice maintained on a high-fat Western diet. Immunostaining of aortic sinus cross sections demonstrated significantly lower ICAM-1 expression in DKO lesions compared with SKO lesions, and en face preparations of vessel regions subjected to disturbed and laminar flow showed less disruption of junctional connexin 43 in DKO than in SKO mice. Thus, PECAM-1 deficiency reduced the extent of lesions at the aortic arch and the aortic sinus, and lowered atherogenic indices. These results suggest that PECAM-1 is an important factor in the atherogenic changes seen in the ApoE-deficient mouse model and thus should be considered as a potential target for protection against atherosclerosis.


Assuntos
Aterosclerose/fisiopatologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , Animais , Aorta/metabolismo , Aterosclerose/metabolismo , Conexina 43/metabolismo , Feminino , Masculino , Camundongos , Camundongos Knockout , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética
13.
Neurotherapeutics ; 4(4): 571-9, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17920538

RESUMO

Microglial activation and macrophage infiltration into the CNS are common features of CNS autoimmune disease and of chronic neurodegenerative diseases. Because these cells largely express an overlapping set of common macrophage markers, it has been difficult to separate their respective contributions to disease onset and progression. This problem is further confounded by the many types of macrophages that have been termed microglia. Several approaches, ranging from molecular profiling of isolated cells to the generation of irradiation chimeric rodent models, are now beginning to generate rudimentary definitions distinguishing the various types of microglia and macrophages found within the CNS and the potential roles that these cells may play in health and disease.


Assuntos
Doenças do Sistema Nervoso Central/patologia , Microglia/fisiologia , Animais , Doenças Autoimunes/patologia , Humanos , Macrófagos/fisiologia
14.
Immunol Rev ; 213: 48-65, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16972896

RESUMO

Central nervous system (CNS) immune privilege is an experimentally defined phenomenon. Tissues that are rapidly rejected by the immune system when grafted in sites, such as the skin, show prolonged survival when grafted into the CNS. Initially, CNS immune privilege was construed as CNS isolation from the immune system by the blood-brain barrier (BBB), the lack of draining lymphatics, and the apparent immunoincompetence of microglia, the resident CNS macrophage. CNS autoimmunity and neurodegeneration were presumed automatic consequences of immune cell encounter with CNS antigens. Recent data have dramatically altered this viewpoint by revealing that the CNS is neither isolated nor passive in its interactions with the immune system. Peripheral immune cells can cross the intact BBB, CNS neurons and glia actively regulate macrophage and lymphocyte responses, and microglia are immunocompetent but differ from other macrophage/dendritic cells in their ability to direct neuroprotective lymphocyte responses. This newer view of CNS immune privilege is opening the door for therapies designed to harness autoreactive lymphocyte responses and also implies (i) that CNS autoimmune diseases (i.e. multiple sclerosis) may result as much from neuronal and/or glial dysfunction as from immune system dysfunctions and (ii) that the severe neuronal and glial dysfunction associated with neurodegenerative disorders (i.e. Alzheimer's disease) likely alters CNS-specific regulation of lymphocyte responses affecting the utility of immune-based therapies (i.e. vaccines).


Assuntos
Sistema Nervoso Central/imunologia , Tolerância Imunológica/fisiologia , Animais , Células Apresentadoras de Antígenos/imunologia , Humanos , Microglia/imunologia
15.
Neurochem Int ; 49(2): 145-53, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16759751

RESUMO

Microglial activation is one of the earliest and most prominent features of nearly all CNS neuropathologies often occurring prior to other indicators of overt neuropathology. Whether microglial activation in seemingly healthy CNS tissue during the early stages of several is a response to early stages of neuronal or glial distress or an early sign of microglial dysfunction causing subsequent neurodegeneration is unknown. Here we characterize and discuss how changes in the CNS microenvironment (neuronal activity/viability, glial activation) lead to specific forms of microglial activation. Specifically, we examine the potential role that TREM-2 expressing microglia may play in regulating the effector function of autoreactive T cell responses. Thus, we suggest that ubiquitous suppression of microglial activation during CNS inflammatory disorders rather than targeted manipulation of microglial activation, may in the end be maladaptive leading to incomplete remission of symptoms.


Assuntos
Encefalopatias/complicações , Encefalite/imunologia , Gliose/imunologia , Microglia/imunologia , Linfócitos T/imunologia , Animais , Apresentação de Antígeno/imunologia , Autoimunidade/imunologia , Encefalopatias/fisiopatologia , Encefalite/fisiopatologia , Gliose/fisiopatologia , Humanos , Imunoterapia/efeitos adversos , Imunoterapia/normas , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Degeneração Neural/imunologia , Degeneração Neural/fisiopatologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia
16.
Cell Transplant ; 14(8): 527-36, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16355564

RESUMO

Xenografts of fetal porcine mesencephalic cells implanted into the rat striatum are generally rejected within several weeks. The fetal donor mesencephalon predominantly consists of neurons, but also contains microglial and endothelial cells, which are more immunogenic. In the present work, we investigated the occurrence of donor endothelial cells in grafts of porcine mesencephalic cells implanted into the rat striatum. Pig endothelial cells were monitored by immunochemical methods, using a monoclonal antibody (mAb) that recognizes a peptidic epitope of the porcine beta1 integrin, and isolectin IB4, for the staining of the Galalpha1,3Gal epitope. The analysis also involved the detection of the pig hyaluronate receptor CD44, and the cell adhesion molecule CD31. The anti-beta1 integrin mAb revealed endothelial-like cells in grafts of porcine mesencephalic cells as soon as 1 week after implantation. A similar staining pattern was obtained with the IB4 lectin. Unlike aortic endothelial cells, these pig brain-derived endothelial-like cells were not recognized by the anti-CD44 antibody. They also failed to express the CD31 adhesion molecule, a fact which suggests that they remained poorly mature, even in grafts maintained during 45 days in immunosuppressed rats. Interestingly, a strong expression of beta1 integrin immunoreactivity was noticed in a large proportion (80%) of the cells freshly dissociated from the fetal pig mesencephalic tissue. The immunoreactivity decreased progressively after transplantation of the cells into the rat brain. This observation suggests that dissociated neuroblasts are capable of a temporary expression of beta1 integrin. This molecule is known to participate in the process of cell sorting and migration in the developing brain. Hence, its expression could be the hallmark of a rescue mechanism triggered by the disruption of the cell/matrix interactions during the dissociation of the fetal mesencephalon. This disruption might account for part of the dramatic cell death process that occurs during the manipulation of the donor tissue.


Assuntos
Antígenos Heterófilos/imunologia , Integrina beta1/imunologia , Mesencéfalo/citologia , Animais , Anticorpos Monoclonais/metabolismo , Antígenos Heterófilos/metabolismo , Astrócitos/imunologia , Astrócitos/metabolismo , Transplante de Tecido Encefálico/imunologia , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais/transplante , Transplante de Tecido Fetal/imunologia , Receptores de Hialuronatos/metabolismo , Imuno-Histoquímica , Integrina beta1/metabolismo , Mesencéfalo/imunologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Ratos , Suínos
17.
Transgenic Res ; 14(4): 373-84, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16201404

RESUMO

The transplantation of fetal porcine neurons is a potential therapeutic strategy for the treatment of human neurodegenerative disorders. A major obstacle to xenotransplantation, however, is the immune-mediated rejection that is resistant to conventional immunosuppression. To determine whether genetically modified donor pig neurons could be used to deliver immunosuppressive proteins locally in the brain, transgenic pigs were developed that express the human T cell inhibitory molecule hCTLA4-Ig under the control of the neuron-specific enolase promoter. Expression was found in various areas of the brain of transgenic pigs, including the mesencephalon, hippocampus and cortex. Neurons from 28-day old embryos secreted hCTLA4-Ig in vitro and this resulted in a 50% reduction of the proliferative response of human T lymphocytes in xenogenic proliferation assays. Transgenic embryonic neurons also secreted hCTLA4-Ig and had developed normally in vivo several weeks after transplantation into the striatum of immunosuppressed rats that were used here to study the engraftment in the absence of immunity. In conclusion, these data show that neurons from our transgenic pigs express hCTLA4-Ig in situ and support the use of this material in future pre-clinical trials in neuron xenotransplantation.


Assuntos
Encéfalo/imunologia , Transplante de Tecido Fetal/imunologia , Imunoconjugados/genética , Neurônios/imunologia , Transgenes , Transplante Heterólogo/imunologia , Abatacepte , Animais , Animais Geneticamente Modificados , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Rejeição de Enxerto/prevenção & controle , Humanos , Imunoconjugados/metabolismo , Imuno-Histoquímica , Terapia de Imunossupressão/métodos , Neurônios/transplante , Ratos/genética , Suínos/genética , Linfócitos T , Imunologia de Transplantes
18.
Exp Neurol ; 192(2): 373-83, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15755555

RESUMO

Xenograft rejections of embryonic pig neural cells implanted into the adult rat striatum occurs within 3-4 weeks, following a dramatic T cell infiltration. Little is known about the cross-talk between the brain and peripheral lymphoid tissues which results in this recruitment and lymphocyte homing. To better characterize the dynamics of the T cell response against xenogeneic neural cells implanted into the brain parenchyma, we used both qualitative and quantitative methods to follow the alterations of the CDR3 length distribution (CDR3-LD) of the TCR (T cell receptor) beta chain in the transplanted striatum and compared this response to that observed in the deep cervical lymph nodes, spleen, and blood. Data showed that the T cell repertoire diversity was highly altered in the recipient brain during xenograft rejection. Comparison of the alterations of the CDR3-LD between several animals revealed a single public alteration in the Vbeta20 family, and many private alterations of the CDR3-LD which differed from one infiltrated brain to another. Alterations of the T cell repertoire were also observed in lymphocytes homed into the deep cervical lymph nodes. However, they differed from the alterations detected in the infiltrated brains. Conversely, no significant alteration of the CDR3-LD was detected in the spleen or in the blood. These data suggest that the deep cervical lymph nodes play an active role in the process of xenograft recognition or/and rejection. However, they also indicate that the fate of T cells homed in the brain and deep cervical lymph nodes differs.


Assuntos
Transplante de Tecido Encefálico/imunologia , Rejeição de Enxerto/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transplante Heterólogo , Animais , Regiões Determinantes de Complementaridade/metabolismo , Corpo Estriado/imunologia , Corpo Estriado/metabolismo , Corpo Estriado/transplante , Embrião de Mamíferos , Feminino , Expressão Gênica , Rejeição de Enxerto/fisiopatologia , Ratos , Ratos Endogâmicos Lew , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Suínos , Linfócitos T/imunologia
19.
Brain ; 127(Pt 5): 981-95, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-14985265

RESUMO

Multiple sclerosis is an inflammatory demyelinating disease of the CNS associated with T cells autoreactive for myelin components. In this study, we analysed the T-cell receptor (TCR) usage of the variable beta (Vbeta) chain transcriptome in the blood of multiple sclerosis patients at various stages of the disease using a global and quantitative comparison of the complementarity-determining region 3 length distribution (CDR3-LD) of transcripts of the 26 Vbeta genes. We investigated 35 patients: 12 with a high risk of multiple sclerosis, 10 with clinically definite multiple sclerosis, 13 with a relapsing-remitting worsening and active multiple sclerosis and 13 healthy individuals. Cells bearing the TCR transcripts with altered CDR3-LD were sorted and studied for CD4 or CD8 phenotype, cytokine transcript accumulation and response to human myelin basic protein (MBP). We show that patients from all the groups have a significantly skewed blood T-cell repertoire. Vbeta transcriptome patterns were more altered in patients from the clinically definite multiple sclerosis group and the worsening and active multiple sclerosis group than in the high risk group. The T cells sorted from Vbeta families with altered CDR3-LD concerned both CD4 and CD8 T cells, with a more pronounced skewing in the CD8 compartment. These cells displayed a significantly increased level of interferon-gamma, interleukin-2 and tumour necrosis factor-alpha transcripts compared with their counterparts from the healthy individual group. Furthermore, using interferon-gamma enzyme-linked immunospot (ELISPOT) assays, T cells from four out of seven altered Vbeta families tested from multiple sclerosis patients responded to human MBP, whereas no response was observed with human albumin or with altered Vbeta families from healthy individuals. Our data support the concept of an early autoimmune component in the disease and emphasize the possible involvement of CD8-positive T cells in multiple sclerosis.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Genes de Imunoglobulinas , Esclerose Múltipla/sangue , Complexo Receptor-CD3 de Antígeno de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta/sangue , Adulto , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Criança , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Interferon gama/sangue , Interleucina-2/sangue , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Reação em Cadeia da Polimerase/métodos , Estatísticas não Paramétricas , Transcrição Gênica
20.
Exp Neurol ; 183(2): 367-78, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14552878

RESUMO

A recombinant adeno-associated virus (rAAV) was used to investigate the impact of an ectopic expression of the NGF high-affinity receptor in adult neurons. The rat TrkA cDNA cloned in a pCMX vector was first tagged with a human c-Myc sequence. The resulting vector was shown to encode a functional receptor which promoted the expression of TrkA immunoreactivity upon transfection of 293 fibroblasts or nnr5 cells, a TrkA-defective variant of PC12 cells. These cells also accumulate TrkA transcripts upon transfection and extended neurites in the presence of NGF. Therefore, the TrkA(myc) cassette was inserted into the pSSV9 plasmid. The new vectors shared properties similar to pCMX TrkA(myc) in 293 and nnr5 cells and enabled the preparation of rAAV TrkA(myc) viruses. Unilateral injection of this rAAV into the substantia nigra (SN) resulted in a protracted expression of TrkA (or c-Myc) immunoreactivity in numerous cell bodies, including tyrosine-hydroxylase (TH)-positive dopaminergic neurons. The presence of TrkA receptors in corresponding striatal dopaminergic endings was demonstrated by the advent of a striato-nigral retrograde axonal transport of (125)I-NGF. Likewise, ectopic expression of TrkA in neurons of the parafascicular thalamic nucleus promoted a striatofuge transport of NGF toward this structure. To investigate whether ectopic expression of TrkA in SN neurons may confer neuroprotection, lesions were induced by 6-hydroxydopamine in striata located ipsilateral to the virus injection site. NGF or vehicle were next delivered dorsally to the virus-treated SN for 2 weeks, before sacrifice and processing of brains for TH-immunohistochemistry. NGF treatment, in contrast to treatment with vehicle, significantly enhanced the number of dopaminergic neurons counted in the lesioned SN. These data suggest that ectopic TrkA can mediate the trophic actions of NGF and influence neuronal plasticity in vivo.


Assuntos
Dopamina/metabolismo , Mesencéfalo/metabolismo , Fator de Crescimento Neural/metabolismo , Neurônios/metabolismo , Receptor trkA/biossíntese , Animais , Transporte Axonal/efeitos dos fármacos , Transporte Axonal/fisiologia , Linhagem Celular , Dependovirus/genética , Feminino , Expressão Gênica/fisiologia , Humanos , Mesencéfalo/citologia , Fator de Crescimento Neural/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Células PC12 , Ratos , Ratos Sprague-Dawley , Receptor trkA/genética , Substância Negra/citologia , Substância Negra/metabolismo
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