RESUMO
BACKGROUND/AIMS: Endothelial cell dysfunction is the principal pathological process underlying atherosclerotic cardiovascular disease. G protein-coupled receptor 124 (GPR124), an orphan receptor in the adhesion GPCR subfamily, promotes angiogenesis in the brain. In the present study, we explored the role of endothelial GPR124 in the development and progression of atherosclerosis in adult mice. METHODS: Using tetracycline-inducible transgenic systems, we generated mice expressing GPR124 specifically under control of the Tie-2 promoter. The animal model of atherosclerosis was constructed by intravenously injecting AAV-PCSK9DY into tetracycline-regulated mice and feeding the mice a high-fat diet for 16 consecutive weeks. Biochemical analysis and immunohistochemistry methods were used to address the role and mechanism of GPR124 in the pathological process of atherosclerosis. RESULTS: Higher TC (total cholesterol) and LDL-C (low density lipoprotein cholesterol) levels in serum and greater lipid deposition in the aortic sinus were found in atherosclerotic mice with GPR124 overexpression, coincident with the elevated proliferation of smooth muscle cells. We observed an elevation of ONOO- in the aortic sinus in this model by using immunofluorescence, and the experiments showed that the specific overexpression of GPR124 in the endothelium induced the up-regulation of CD68, NLRP3 and caspase-1 levels in the aortic sinus. CONCLUSION: The above results indicate that manipulating GPR124 in the endothelium may contribute to delayed pathological progression of atherosclerosis.
Assuntos
Aterosclerose/patologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Aterosclerose/metabolismo , Caspase 1/metabolismo , Colesterol/sangue , LDL-Colesterol/sangue , Dieta Hiperlipídica , Modelos Animais de Doenças , Humanos , Inflamação/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ácido Peroxinitroso/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Receptores Acoplados a Proteínas G/genética , Seio Aórtico/metabolismo , Seio Aórtico/patologiaRESUMO
Accumulating evidence suggests that formation of peroxynitrite (ONOO(-)) in the cerebral vasculature contributes to the progression of ischemic damage, while the underlying molecular mechanisms remain elusive. To fully understand ONOO(-) biology, efficient tools that can realize the real-time tracing of endogenous ONOO(-) fluxes are indispensable. While a few ONOO(-) fluorescent probes have been reported, direct visualization of ONOO(-) fluxes in the cerebral vasculature of live mice remains a challenge. Herein, we present a fluorescent switch-on probe (NP3) for ONOO(-) imaging. NP3 exhibits good specificity, fast response, and high sensitivity toward ONOO(-) both in vitro and in vivo. Moreover, NP3 is two-photon excitable and readily blood-brain barrier penetrable. These desired photophysical and pharmacokinetic properties endow NP3 with the capability to monitor brain vascular ONOO(-) generation after injury with excellent temporal and spatial resolution. As a proof of concept, NP3 has enabled the direct visualization of neurovascular ONOO(-) formation in ischemia progression in live mouse brain by use of two-photon laser scanning microscopy. Due to these favorable properties, NP3 holds great promise for visualizing endogenous peroxynitrite fluxes in a variety of pathophysiological progressions in vitro and in vivo.
Assuntos
Traumatismo Cerebrovascular/metabolismo , Células Endoteliais/metabolismo , Corantes Fluorescentes/química , Ácido Peroxinitroso/metabolismo , Animais , Traumatismo Cerebrovascular/patologia , Células Endoteliais/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacocinética , Camundongos , Estrutura Molecular , Ácido Peroxinitroso/químicaRESUMO
Lower global cognitive function scores are a common symptom of autism spectrum disorders (ASDs). This study investigates the effects of melatonin on hippocampal serine/threonine kinase signaling in an experimental ASD model. We found that chronic melatonin (1.0 or 5.0 mg/kg/day, 28 days) treatment significantly rescued valproic acid (VPA, 600 mg/kg)-induced decreases in CaMKII (Thr286), NMDAR1 (Ser896), and PKA (Thr197) phosphorylation in the hippocampus without affecting total protein levels. Compared with control rats, the immunostaining of pyramidal neurons in the hippocampus revealed a decrease in immunolabeling intensity for phospho-CaMKII (Thr286) in the hippocampus of VPA-treated rats, which was ameliorated by chronic melatonin treatment. Consistent with the elevation of CaMKII/PKA/PKC phosphorylation observed in melatonin-treated rat, long-term potentiation (LTP) was enhanced after chronic melatonin (5.0 mg/kg) treatment, as reflected by extracellular field potential slopes that increased from 56 to 60 min (133.4 ± 3.9% of the baseline, P < 0.01 versus VPA-treated rats) following high-frequency stimulation (HFS) in hippocampal slices. Accordingly, melatonin treatment also significantly improved social behavioral deficits at postnatal day 50 in VPA-treated rats. Taken together, the increased phosphorylation of CaMKII/PKA/PKC signaling might contribute to the beneficial effects of melatonin on autism symptoms.
Assuntos
Transtorno Autístico , Comportamento Animal/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Melatonina/farmacologia , Análise de Variância , Animais , Antioxidantes/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/análise , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Modelos Animais de Doenças , Feminino , Hipocampo/química , Imuno-Histoquímica , Masculino , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Ácido Valproico/farmacologiaRESUMO
The present study was designed to investigate the role of autophagy-lysosome signaling in the brain after application of nanoparticles. Here, lipid nanoparticles (LNs) induced elevations of Atg5, P62, LC3 and cathepsin B in mice brain. The transmission electron microscopy revealed a dramatic elevation of lysosome vacuoles colocalized with LNs cluster inside the neurons in mice brain. Immunoblot data revealed abnormal expression of cathepsin B in brain cortex following LNs injection, whereas its expression was further elevated in Atg5(+/-) mice. The importance of Atg5 in the LNs-induced autophagy-lysosome cascade was further supported by our finding that neurovascular response was exaggerated in Atg5(+/-) mice. In addition, the siRNA knockdown of Atg5 significantly blunted the increasing of LC3 and P62 in LNs-treated Neuro-2a cells. Taken together, we propose that LNs induce autophagy-lysosome signaling and neurovascular response at least partially via an Atg5-dependent pathway. FROM THE CLINICAL EDITOR: These authors investigated autophagy-lysosome signaling in the mouse brain after application of lipid nanoparticles and report that these nanoparticles induce autophagy-lysosome signaling and neurovascular response at least partially via an Atg5-dependent pathway.
Assuntos
Encéfalo/metabolismo , Catepsina B/metabolismo , Lipídeos/química , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/deficiência , Nanopartículas/química , Animais , Proteína 5 Relacionada à Autofagia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas Associadas aos Microtúbulos/genéticaRESUMO
AIMS: Autism spectrum disorder (ASD) is a wide range of neurodevelopmental disorders involving deficits in social interaction and communication. Unfortunately, autism remains a scientific and clinical challenge owing to the lack of understanding the cellular and molecular mechanisms underlying it. This study aimed to investigate the pathophysiological mechanism underlying leukocyte-endothelial adhesion in autism-related neurovascular inflammation. METHODS: Male BTBR T+tf/J mice were used as an autism model. The dynamic pattern of leukocyte-endothelial adhesion in mouse cerebral vessels was detected by two-photon laser scanning microscopy (TPLSM). Using FACS, RT-PCR, and Western blotting, we explored the expression of cell adhesion molecules, the mRNA expression of endothelial chemokine, the protein levels of cathepsin B, and inflammatory mediators. RESULTS: We found a significant increase in leukocyte-endothelial adhesion in BTBR mice, accompanied by elevated expression of the adhesion molecule neutrophils CD11b and endothelial ICAM-1. Our data further indicate that elevated neutrophil cathepsin B levels contribute to elevated endothelial chemokine CXCL7 levels in BTBR mice. The pharmacological inhibition of cathepsin B reverses the enhanced leukocyte-endothelial adhesion in the cerebral vessels of autistic mice. CONCLUSION: Our results revealed the prominent role of cathepsin B in modulating leukocyte-endothelial adhesion during autism-related neurovascular inflammation and identified a promising novel approach for autism treatment.
Assuntos
Transtorno Autístico/tratamento farmacológico , Catepsina B/antagonistas & inibidores , Adesão Celular/efeitos dos fármacos , Dipeptídeos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Animais , Transtorno Autístico/metabolismo , Catepsina B/metabolismo , Adesão Celular/fisiologia , Dipeptídeos/uso terapêutico , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Leucócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Prolonged occlusion of multiple microvessels causes microvascular injury. G protein-coupled receptor 124 (GPR124) has been reported to be required for maintaining central nervous system (CNS) angiogenesis and blood-brain barrier integrity. However, the molecular mechanisms by which GPR124 regulates pericytes during ischemia have remained elusive. Methods: A microsphere embolism-induced ischemia model was used to evaluate the expression of GPR124 following microsphere embolism. Immunocytochemistry and stochastic optical reconstruction microscopy imaging were used to assess the expression and distribution of GPR124 in human brain vascular pericytes (HBVPs) and after the treatment with 3-morpholino-sydnonimine (SIN-1) or oxygen-glucose deprivation (OGD). The effect of GPR124 knockdown or overexpression on HBVP migration was analyzed in vitro using wound healing assays and a microfluidic device. GPR124 loss-of-function studies were performed in HBVPs and HEK293 cells using CRISPR-Cas9-mediated gene deletion. Time-lapse imaging was used to assess dynamic changes in the formation of filopodia in an individual cell. Finally, to explore the functional domains required for GPR124 activity, deletion mutants were constructed for each of the N-terminal domains. Results: GPR124 expression was increased in pericytes following microsphere embolism. Morphological analysis showed localization of GPR124 to focal adhesions where GPR124 bound directly to the actin binding protein vinculin and upregulated Cdc42. SIN-1 or OGD treatment redistributed GPR124 to the leading edges of HBVPs where GPR124 signaling was required for pericyte filopodia formation and directional migration. Partial deletion of GPR124 domains decreased SIN-1-induced filopodia formation and cell migration. Conclusion: Taken together, our results provide the first evidence for a role of GPR124 in pericyte migration under ischemic conditions and suggest that GPR124 was essential for Cdc42 activation and filopodia formation.
Assuntos
Isquemia Encefálica/metabolismo , Polaridade Celular/fisiologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Pericitos/citologia , Pericitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Western Blotting , Linhagem Celular , Polaridade Celular/genética , Adesões Focais/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica , Imunoprecipitação , Lentivirus/genética , Masculino , Camundongos , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas G/genética , Cicatrização/genética , Cicatrização/fisiologiaRESUMO
Valproate exposure is associated with increased risks of autism spectrum disorder. To date, the mechanistic details of disturbance of melatonin receptor subtype 1 (MTNR1A) internalization upon valproate exposure remain elusive. By expressing epitope-tagged receptors (MTNR1A-EGFP) in HEK-293 and Neuro-2a cells, we recorded the dynamic changes of MTNR1A intracellular trafficking after melatonin treatment. Using time-lapse confocal microscopy, we showed in living cells that valproic acid interfered with the internalization kinetics of MTNR1A in the presence of melatonin. This attenuating effect was associated with a decrease in the phosphorylation of PKA (Thr197) and ERK (Thr202/Tyr204). VPA treatment did not alter the whole-cell currents of cells with or without melatonin. Furthermore, fluorescence resonance energy transfer imaging data demonstrated that valproic acid reduced the melatonin-initiated association between YFP-labeled ß-arrestin 2 and CFP-labeled MTNR1A. Together, we suggest that valproic acid influences MTNR1A intracellular trafficking and signaling in a ß-arrestin 2-dependent manner.
Assuntos
Espaço Intracelular/metabolismo , Receptor MT1 de Melatonina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ácido Valproico/farmacologia , beta-Arrestinas/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endocitose/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Melatonina/farmacologia , Camundongos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Septic encephalopathy (SE) is a critical factor determining sepsis mortality. Vascular inflammation is known to be involved in SE, but the molecular events that lead to the development of encephalopathy remain unclear. Using time-lapse in vivo two-photon laser scanning microscopy, we provide the first direct evidence that cecal ligation and puncture in septic mice induces microglial trafficking to sites adjacent to leukocyte adhesion on inflamed cerebral microvessels. Our data further demonstrate that septic injury increased the chemokine CXCL1 level in brain endothelial cells by activating endothelial P2RX7 and eventually enhanced the binding of Mac-1 (CD11b/CD18)-expressing leukocytes to endothelial ICAM-1. In turn, leukocyte adhesion upregulated endothelial CX3CL1, thereby triggering microglia trafficking to the injured site. The sepsis-induced increase in endothelial CX3CL1 was abolished in CD18 hypomorphic mutant mice. Inhibition of the P2RX7 pathway not only decreased endothelial ICAM-1 expression and leukocyte adhesion but also prevented microglia overactivation, reduced brain injury, and consequently doubled the early survival of septic mice. These results demonstrate the role of the P2RX7 pathway in linking neurovascular inflammation to brain damage in vivo and provide a rationale for targeting endothelial P2RX7 for neurovascular protection during SE.
Assuntos
Lesões Encefálicas/metabolismo , Células Endoteliais/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/metabolismo , Antígeno de Macrófago 1/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Encefalopatia Associada a Sepse/metabolismo , Animais , Lesões Encefálicas/patologia , Adesão Celular , Células Endoteliais/patologia , Leucócitos/patologia , Camundongos , Camundongos Mutantes , Encefalopatia Associada a Sepse/patologiaRESUMO
AIMS: Diabetes mellitus increases the risk of stroke, but the mechanisms are unclear. The present study tested the hypothesis that diabetes mellitus disturbs the brain microcirculation and increases the susceptibility to cerebral damage in a middle cerebral artery occlusion (MCAO) model of ischemia. METHODS: Diabetes was induced by streptozocin in mice expressing green fluorescent protein in endothelial cells (Tie2-GFP mice). Four weeks later, they were subjected to transient (20 min) MCAO. In vivo blood flow was measured by two-photon laser-scanning microscopy (TPLSM) in cerebral arteries, veins, and capillaries. RESULTS: There was a significant decrease in red blood cell (RBC) velocity in capillaries in diabetic mice as assessed by TPLSM, yet the regional cerebral blood flow, as assessed by laser Doppler flowmetry, was maintained. Brain capillary flow developed turbulence after MCAO only in diabetic mice. These mice sustained increased neurological deficits after MCAO which were accompanied by an exaggerated degradation of tight junction proteins and blunted CaMKII phosphorylation in cerebral tissues indicating disruption of the blood-brain barrier and disturbed cognitive potential. CONCLUSION: Diabetic mice are more susceptible to disturbances of cerebral capillary blood flow which may predispose them to neurovascular defects following ischemia.
Assuntos
Circulação Cerebrovascular/fisiologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Análise de Variância , Animais , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Velocidade do Fluxo Sanguíneo/fisiologia , Barreira Hematoencefálica/fisiopatologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Eritrócitos/fisiologia , Proteínas de Fluorescência Verde/genética , Fluxometria por Laser-Doppler , Camundongos , Camundongos Transgênicos , Receptor TIE-2/genética , Receptores de AMPA/metabolismoRESUMO
AIMS: Although there is accumulating evidence that increased formation of reactive nitrogen species in cerebral vasculature contributes to the progression of ischemic damage, but the underlying molecular mechanisms remain elusive. Peroxiredoxin 1 (Prx1) can initiate the antioxidant response by scavenging free radicals. Therefore, we tested the hypothesis that Prx1 regulates the susceptibility to nitrosative stress damage during cerebral ischemia in vitro and in vivo. RESULTS: Proteomic analysis in endothelial cells revealed that Prx1 was upregulated after stress-related oxygen-glucose deprivation (OGD). Although peroxynitrite upregulated Prx1 rapidly, this was followed by its polyubiquitination within 6 h after OGD mediated by the E3 ubiquitin ligase E6-associated protein (E6AP). OGD colocalized E6AP with nitrotyrosine in endothelial cells. To assess translational relevance in vivo, mice were studied after middle cerebral artery occlusion (MCAO). This was accompanied by Prx1 ubiquitination and degradation by the activation of E6AP. Furthermore, brain delivery of a lentiviral vector encoding Prx1 in mice inhibited blood-brain barrier leakage and neuronal damage significantly following MCAO. INNOVATION AND CONCLUSIONS: Nitrosative stress during ischemic insult activates E6AP E3 ubiquitin ligase that ubiquitinates Prx1 and subsequently worsens cerebral damage. Thus, targeting the Prx1 antioxidant defense pathway may represent a novel treatment strategy for neurovascular protection in stroke.
Assuntos
Células Endoteliais/metabolismo , Peroxirredoxinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Imuno-Histoquímica , Infarto da Artéria Cerebral Média/metabolismo , Masculino , Camundongos , Peroxirredoxinas/genética , Proteômica , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/fisiologiaRESUMO
The translation of experimental stroke research from the laboratory to successful clinical practice remains a formidable challenge. We previously reported that PEGylated-lipid nanoparticles (PLNs) effectively transport across the blood-brain barrier along with less inflammatory responses. In the present study, PLNs conjugated to Fas ligand antibody that selectively present on brain ischaemic region were used for therapeutic targeting. Fluorescent analysis of the mice brain show that encapsulated 3-n-Butylphthalide (dl-NBP) in PLNs conjugated with Fas ligand antibody effectively delivered to ipsilateral region of ischaemic brain. Furthermore, the confocal immunohistochemical study demonstrated that brain-targeted nanocontainers specifically accumulated on OX42 positive microglia cells in ischaemic region of mice model. Finally, dl-NBP encapsulated nano-drug delivery system is resulted in significant improvements in brain injury and in neurological deficit after ischaemia, with the significantly reduced dosages versus regular dl-NBP. Overall, these data suggests that PLNs conjugated to an antibody specific to the Fas ligand constituted an ideal brain targeting drug delivery system for brain ischaemia.
Assuntos
Anticorpos/administração & dosagem , Isquemia Encefálica/terapia , Proteína Ligante Fas/imunologia , Lipídeos/química , Nanopartículas , Polietilenoglicóis/química , Animais , Anticorpos/química , Modelos Animais de Doenças , CamundongosRESUMO
Nanocarrier-based drug delivery systems have attracted wide interest for the treatment of brain disease. However, neurotoxicity of nanoparticle has limited their therapeutic application. Here we demonstrated that lipid nanoparticles (LNs) accumulated in the brain parenchyma within 3 h of intravenous injection to mice and persisted for more than 24 weeks, coinciding with a dramatic activation of brain microglia. Morphological characteristic of microglial activation also observed in LNs-treated Cx3cr1GFP/+ mice. In vivo study with two-photon confocal microscopy revealed abnormal Ca²âº waves in microglia following LNs injection. The correlated activation of caspase-1, IL-1ß and neurovascular damage following LNs injection was attenuated in P2X7-/- mice. PEGylation of LNs reduced correlated nanoparticles aggregation. Moreover, PEGylation of LNs ameliorated the P2X7/caspase-1/IL-1ß signalling-dependent microglia activation and neurovascular damage. In conclusion, PEGylation of LNs is a promising biomaterial for brain-targeted therapy that inhibits P2X77-dependent neuroinflammatory response.