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1.
Ann Neurol ; 85(1): 32-46, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30525223

RESUMO

OBJECTIVE: Neurotrophin-3 (NT3) plays a key role in the development and function of locomotor circuits including descending serotonergic and corticospinal tract axons and afferents from muscle and skin. We have previously shown that gene therapy delivery of human NT3 into affected forelimb muscles improves sensorimotor recovery after stroke in adult and elderly rats. Here, to move toward the clinic, we tested the hypothesis that intramuscular infusion of NT3 protein could improve sensorimotor recovery after stroke. METHODS: Rats received unilateral ischemic stroke in sensorimotor cortex. To simulate a clinically feasible time to treatment, 24 hours later rats were randomized to receive NT3 or vehicle by infusion into affected triceps brachii for 4 weeks using implanted catheters and minipumps. RESULTS: Radiolabeled NT3 crossed from the bloodstream into the brain and spinal cord in rodents with or without strokes. NT3 increased the accuracy of forelimb placement during walking on a horizontal ladder and increased use of the affected arm for lateral support during rearing. NT3 also reversed sensory impairment of the affected wrist. Functional magnetic resonance imaging during stimulation of the affected wrist showed spontaneous recovery of peri-infarct blood oxygenation level-dependent signal that NT3 did not further enhance. Rather, NT3 induced neuroplasticity of the spared corticospinal and serotonergic pathways. INTERPRETATION: Our results show that delayed, peripheral infusion of NT3 can improve sensorimotor function after ischemic stroke. Phase I and II clinical trials of NT3 (for constipation and neuropathy) have shown that peripheral high doses are safe and well tolerated, which paves the way for NT3 as a therapy for stroke. ANN NEUROL 2019;85:32-46.


Assuntos
Neurotrofina 3/administração & dosagem , Recuperação de Função Fisiológica/efeitos dos fármacos , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Feminino , Injeções Intramusculares , Distribuição Aleatória , Ratos , Recuperação de Função Fisiológica/fisiologia , Córtex Sensório-Motor/diagnóstico por imagem , Córtex Sensório-Motor/efeitos dos fármacos , Córtex Sensório-Motor/fisiologia , Acidente Vascular Cerebral/fisiopatologia , Fatores de Tempo
2.
Cardiovasc Res ; 77(3): 570-9, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18032393

RESUMO

AIMS: We investigated the role of src family kinases (srcFK) in agonist-mediated Ca2+-sensitization in pulmonary artery and whether this involves interaction with the rho/rho-kinase pathway. METHODS AND RESULTS: Intra-pulmonary arteries (IPAs) and cultured pulmonary artery smooth muscle cells (PASMC) were obtained from rat. Expression of srcFK was determined at the mRNA and protein levels. Ca2+-sensitization was induced by prostaglandin F(2 alpha) (PGF(2 alpha)) in alpha-toxin-permeabilized IPAs. Phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light-chain-20 (MLC20) and translocation of rho-kinase in response to PGF(2 alpha) were also determined. Nine srcFK were expressed at the mRNA level, including src, fyn, and yes, and PGF(2 alpha) enhanced phosphorylation of three srcFK proteins at tyr-416. In alpha-toxin-permeabilized IPAs, PGF(2 alpha) enhanced the Ca2+-induced contraction (pCa 6.9) approximately three-fold. This enhancement was inhibited by the srcFK blockers SU6656 and PP2 and by the rho-kinase inhibitor Y27632. Y27632, but not SU6656 or PP2, also inhibited the underlying pCa 6.9 contraction. PGF(2 alpha) enhanced phosphorylation of MYPT-1 at thr-697 and thr-855 and of MLC20 at ser-19. This enhancement, but not the underlying basal phosphorylation, was inhibited by SU6656. Y27632 suppressed both basal and PGF(2 alpha)-mediated phosphorylation. The effects of SU6656 and Y27632, on both contraction and MYPT-1 and MLC20 phosphorylation, were not additive. PGF(2 alpha) triggered translocation of rho-kinase in PASMC, and this was inhibited by SU6656. CONCLUSIONS: srcFK are activated by PGF(2 alpha) in the rat pulmonary artery and may contribute to Ca2+-sensitization and contraction via rho-kinase translocation and phosphorylation of MYPT-1.


Assuntos
Cálcio/metabolismo , Artéria Pulmonar/metabolismo , Quinases Associadas a rho/fisiologia , Quinases da Família src/fisiologia , Animais , Células Cultivadas , Dinoprosta/farmacologia , Masculino , Cadeias Leves de Miosina/metabolismo , Fosforilação , Proteína Fosfatase 1/metabolismo , Ratos , Ratos Wistar , Tirosina/metabolismo
3.
Free Radic Biol Med ; 45(10): 1468-76, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18805479

RESUMO

Reactive oxygen species are implicated in pulmonary hypertension and hypoxic pulmonary vasoconstriction. We examined the effects of low concentrations of peroxide on intrapulmonary arteries (IPA). IPAs from Wistar rats were mounted on a myograph for recording tension and estimating intracellular Ca2+ using Fura-PE3. Ca2+ sensitization was examined in alpha-toxin-permeabilized IPAs, and phosphorylation of MYPT-1 and MLC(20) was assayed by Western blot. Peroxide (30 microM) induced a vasoconstriction with transient and sustained components and equivalent elevations of intracellular Ca2+. The transient constriction was strongly suppressed by indomethacin, the TP-receptor antagonist SQ-29584, and the Rho kinase inhibitor Y-27632, whereas sustained constriction was unaffected. Neither vasoconstriction nor elevation of intracellular Ca2+ was affected by removal of extracellular Ca2+, whereas dantrolene suppressed the former and ryanodine abolished the latter. Peroxide-induced constriction of permeabilized IPAs was unaffected by Y-27632 but abolished by PKC inhibitors; these also suppressed constriction in intact IPAs. Peroxide caused translocation of PKCalpha, but had no significant effect on MYPT-1 or MLC(20) phosphorylation. We conclude that in IPAs peroxide causes transient release of vasoconstrictor prostanoids, but sustained constriction is associated with release of Ca2+ from ryanodine-sensitive stores and a PKC-dependent but Rho kinase- and MLC(20)-independent constrictor mechanism.


Assuntos
Cálcio/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteína Quinase C/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiologia , Vasoconstrição/efeitos dos fármacos , Animais , Masculino , Ratos , Ratos Wistar , Fatores de Tempo
4.
Lancet Psychiatry ; 5(1): 79-92, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28781208

RESUMO

Blood-brain barrier pathology is recognised as a central factor in the development of many neurological disorders, but much less is known about the role of the blood-brain barrier in psychiatric disorders. We review post-mortem, serum-biomarker, CSF-biomarker, and neuroimaging studies that have examined blood-brain barrier structure and function in schizophrenia and related psychoses. We consider how blood-brain barrier dysfunction could relate to glutamatergic and inflammatory abnormalities, which are increasingly understood to play a part in the pathogenesis of psychosis. Mechanisms by which the blood-brain barrier and its associated solute transporters moderate CNS availability of antipsychotic drugs are summarised. We conclude that the complex nature of blood-brain barrier dysfunction in psychosis might be relevant to many aspects of disrupted neuronal and synaptic function, increased permeability to inflammatory molecules, disrupted glutamate homoeostasis, impaired action of antipsychotics, and development of antipsychotic resistance. Future research should address the longitudinal course of blood-brain barrier alterations in psychosis, to determine whether blood-brain barrier dysfunction is a cause or consequence of the pathology associated with the disorder.


Assuntos
Antipsicóticos/farmacocinética , Barreira Hematoencefálica , Encéfalo , Transtornos Psicóticos , Barreira Hematoencefálica/diagnóstico por imagem , Barreira Hematoencefálica/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Humanos , Transtornos Psicóticos/diagnóstico , Transtornos Psicóticos/tratamento farmacológico , Transtornos Psicóticos/metabolismo
5.
Sci Transl Med ; 8(355): 355ra118, 2016 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-27605553

RESUMO

Lysosomal storage diseases (LSDs) often manifest with severe systemic and central nervous system (CNS) symptoms. The existing treatment options are limited and have no or only modest efficacy against neurological manifestations of disease. We demonstrate that recombinant human heat shock protein 70 (HSP70) improves the binding of several sphingolipid-degrading enzymes to their essential cofactor bis(monoacyl)glycerophosphate in vitro. HSP70 treatment reversed lysosomal pathology in primary fibroblasts from 14 patients with eight different LSDs. HSP70 penetrated effectively into murine tissues including the CNS and inhibited glycosphingolipid accumulation in murine models of Fabry disease (Gla(-/-)), Sandhoff disease (Hexb(-/-)), and Niemann-Pick disease type C (Npc1(-/-)) and attenuated a wide spectrum of disease-associated neurological symptoms in Hexb(-/-) and Npc1(-/-) mice. Oral administration of arimoclomol, a small-molecule coinducer of HSPs that is currently in clinical trials for Niemann-Pick disease type C (NPC), recapitulated the effects of recombinant human HSP70, suggesting that heat shock protein-based therapies merit clinical evaluation for treating LSDs.


Assuntos
Proteínas de Choque Térmico/uso terapêutico , Esfingolipidoses/tratamento farmacológico , Administração Intravenosa , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Proteínas Morfogenéticas Ósseas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Doença de Fabry/tratamento farmacológico , Doença de Fabry/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Glicoesfingolipídeos/metabolismo , Proteínas de Choque Térmico/farmacologia , Humanos , Hidroxilaminas/farmacologia , Hidroxilaminas/uso terapêutico , Injeções Intraperitoneais , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/efeitos dos fármacos , Lisossomos/patologia , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Proteínas/metabolismo , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Esfingolipidoses/patologia , Distribuição Tecidual
6.
Methods Mol Biol ; 814: 415-30, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22144323

RESUMO

In vitro blood-brain barrier (BBB) models using primary cultured brain endothelial cells are important for establishing cellular and molecular mechanisms of BBB function. Co-culturing with BBB-associated cells especially astrocytes to mimic more closely the in vivo condition leads to upregulation of the BBB phenotype in the brain endothelial cells. Rat brain endothelial cells (RBECs) are a valuable tool allowing ready comparison with in vivo studies in rodents; however, it has been difficult to obtain pure brain endothelial cells, and few models achieve a transendothelial electrical resistance (TEER, measure of tight junction efficacy) of >200 Ω cm(2), i.e. the models are still relatively leaky. Here, we describe methods for preparing high purity RBECs and neonatal rat astrocytes, and a co-culture method that generates a robust, stable BBB model that can achieve TEER >600 Ω cm(2). The method is based on >20 years experience with RBEC culture, together with recent improvements to kill contaminating cells and encourage BBB differentiation.Astrocytes are isolated by mechanical dissection and cell straining and are frozen for later co-culture. RBECs are isolated from 3-month-old rat cortices. The brains are cleaned of meninges and white matter and enzymatically and mechanically dissociated. Thereafter, the tissue homogenate is centrifuged in bovine serum albumin to separate vessel fragments from other cells that stick to the myelin plug. The vessel fragments undergo a second enzyme digestion to separate pericytes from vessels and break down vessels into shorter segments, after which a Percoll gradient is used to separate capillaries from venules, arterioles, and single cells. To kill remaining contaminating cells such as pericytes, the capillary fragments are plated in puromycin-containing medium and RBECs grown to 50-60% confluence. They are then passaged onto filters for co-culture with astrocytes grown in the bottom of the wells. The whole procedure takes ∼2 weeks, using pre-frozen astrocytes, from isolation of RBECs to generation of high-resistance/low-permeability RBEC monolayers.


Assuntos
Astrócitos/citologia , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/fisiologia , Encéfalo/citologia , Técnicas de Cultura de Células/métodos , Células Endoteliais/citologia , Animais , Impedância Elétrica , Ratos , Junções Íntimas/fisiologia
7.
Free Radic Biol Med ; 46(5): 633-42, 2009 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19103285

RESUMO

Reactive oxygen species play a key role in vascular disease, pulmonary hypertension, and hypoxic pulmonary vasoconstriction. We investigated contractile responses, intracellular Ca(2+) ([Ca(2+)](i)), Rho-kinase translocation, and phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light chain (MLC(20)) in response to LY83583, a generator of superoxide anion, in small intrapulmonary arteries (IPA) of rat. LY83583 caused concentration-dependent constrictions in IPA and greatly enhanced submaximal PGF(2alpha)-mediated preconstriction. In small femoral or mesenteric arteries of rat, LY83583 alone was without effect, but it relaxed a PGF(2)alpha-mediated preconstriction. Constrictions in IPA were inhibited by superoxide dismutase and tempol, but not catalase, and were endothelium and guanylate cyclase independent. Constrictions were also inhibited by the Rho-kinase inhibitor Y27632 and the Src-family kinase inhibitor SU6656. LY83583 did not raise [Ca(2+)](i), but caused a Y27632-sensitive constriction in alpha-toxin-permeabilized IPA. LY83583 triggered translocation of Rho-kinase from the nucleus to the cytosol in pulmonary artery smooth muscle cells and enhanced phosphorylation of MYPT-1 at Thr-855 and of MLC(20) at Ser-19 in IPA. This enhancement was inhibited by superoxide dismutase and abolished by Y27632. Hydrogen peroxide did not activate Rho-kinase. We conclude that in rat small pulmonary artery, superoxide triggers Rho-kinase-mediated Ca(2+) sensitization and vasoconstriction independent of hydrogen peroxide.


Assuntos
Broncoconstrição/fisiologia , Hipertensão Pulmonar/enzimologia , Miócitos de Músculo Liso/fisiologia , Superóxidos/farmacologia , Quinases Associadas a rho/fisiologia , Quinases da Família src/fisiologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Amidas/farmacologia , Aminoquinolinas/farmacologia , Animais , Broncoconstrição/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Guanilato Ciclase/antagonistas & inibidores , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/prevenção & controle , Indóis/farmacologia , Masculino , Miócitos de Músculo Liso/patologia , Cadeias Leves de Miosina/metabolismo , Fosforilação , Proteína Fosfatase 1/metabolismo , Artéria Pulmonar/patologia , Piridinas/farmacologia , Ratos , Ratos Wistar , Sulfonamidas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases da Família src/antagonistas & inibidores
8.
Cardiovasc Res ; 80(3): 453-62, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-18682436

RESUMO

AIMS: We investigated the role of src-family kinases (srcFKs) in hypoxic pulmonary vasoconstriction (HPV) and how this relates to Rho-kinase-mediated Ca(2+) sensitization and changes in intracellular Ca(2+) concentration ([Ca(2+)](i)). METHODS AND RESULTS: Intra-pulmonary arteries (IPAs) were obtained from male Wistar rats. HPV was induced in myograph-mounted IPAs. Auto-phosphorylation of srcFKs and phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and myosin light-chain (MLC(20)) in response to hypoxia were determined by western blotting. Translocation of Rho-kinase and effects of siRNA knockdown of src and fyn were examined in cultured pulmonary artery smooth muscle cells (PASMCs). [Ca(2+)](i) was estimated in Fura-PE3-loaded IPA. HPV was inhibited by two blockers of srcFKs, SU6656 and PP2. Hypoxia enhanced phosphorylation of three srcFK proteins at Tyr-416 (60, 59, and 54 kDa, corresponding to src, fyn, and yes, respectively) and enhanced srcFK-dependent tyrosine phosphorylation of multiple target proteins. Hypoxia caused a complex, time-dependent enhancement of MYPT-1 and MLC(20) phosphorylation, both in the absence and presence of pre-constriction. The sustained component of this enhancement was blocked by SU6656 and the Rho-kinase inhibitor Y27632. In PASMCs, hypoxia caused translocation of Rho-kinase from the nucleus to the cytoplasm, and this was prevented by anti-src siRNA and to a lesser extent by anti-fyn siRNA. The biphasic increases in [Ca(2+)](i) that accompany HPV were also inhibited by PP2. CONCLUSION: Hypoxia activates srcFKs and triggers protein tyrosine phosphorylation in IPA. Hypoxia-mediated Rho-kinase activation, Ca(2+) sensitization, and [Ca(2+)](i) responses are depressed by srcFK inhibitors and/or siRNA knockdown, suggesting a central role of srcFKs in HPV.


Assuntos
Hipóxia/metabolismo , Artéria Pulmonar/metabolismo , Vasoconstrição/fisiologia , Quinases da Família src/metabolismo , Amidas/farmacologia , Animais , Cálcio/metabolismo , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Masculino , Modelos Animais , Cadeias Leves de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosforilação , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Piridinas/farmacologia , Ratos , Ratos Wistar , Sulfonamidas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Quinases da Família src/antagonistas & inibidores
9.
J Neurochem ; 93(4): 825-33, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15857386

RESUMO

Aquaporins (AQPs) are a family of proteins that mediate water transport across cells, but the extent to which they are involved in water transport across endothelial cells of the blood-brain barrier is not clear. Expression of AQP1 and AQP4 in rat brain microvessel endothelial cells was investigated in order to determine whether these isoforms were present and, in particular, to examine the hypothesis that brain endothelial expression of AQPs is dynamic and regulated by astrocytic influences. Reverse-transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry showed that AQP1 mRNA and protein are present at very low levels in primary rat brain microvessel endothelial cells, and are up-regulated in passaged cells. Upon passage, endothelial cell expression of mdr1a mRNA is decreased, indicating loss of blood-brain barrier phenotype. In passage 4 endothelial cells, AQP1 mRNA levels are reduced by coculture above rat astrocytes, demonstrating that astrocytic influences are important in maintaining the low levels of AQP1 characteristic of the blood-brain barrier endothelium. Reverse-transcriptase-PCR revealed very low levels of AQP1 mRNA present in the RBE4 rat brain microvessel endothelial cell line, with no expression detected in primary cultures of rat astrocytes or in the C6 rat glioma cell line. In contrast, AQP4 mRNA is strongly expressed in astrocytes, but no expression is found in primary or passaged brain microvessel endothelial cells, or in RBE4 or C6 cells. Our results support the concept that expression of AQP1, which is seen in many non-brain endothelia, is suppressed in the specialized endothelium of the blood-brain barrier.


Assuntos
Aquaporinas/metabolismo , Encéfalo/citologia , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Regulação da Expressão Gênica/fisiologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Aquaporina 1 , Aquaporina 4 , Aquaporinas/genética , Northern Blotting/métodos , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Células Cultivadas , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica/métodos , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Tempo
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