RESUMO
OBJECTIVE: There are currently no effective treatments for the prevention of dementia associated with vascular cognitive impairment. MicroRNAs regulate gene expression at the post-transcriptional level and play key roles in vascular disorders. TNFα (tumor necrosis factor-α) regulates blood-brain barrier breakdown through modification of cerebral tight junctions. Here, we sought key TNFα-responsive microRNAs that might influence blood-brain barrier breakdown via cerebral tight junction disruption in vascular cognitive impairment. APPROACH AND RESULTS: Using a mouse model of vascular cognitive impairment, chronic cerebral hypoperfusion within the white matter was induced with bilateral common carotid artery stenosis (BCAS) surgery. TNFα gene expression was increased in white matter post-BCAS surgery, and TNFα stimulation decreased claudin-5, ZO-1 (tight-junction protein 1), and occludin gene expression in murine brain endothelial cells. In silico analysis predicted 8 candidate microRNAs as regulators of claudin-5, ZO-1, and occludin gene expression. Of these, only miR-501-3p was upregulated by TNFα in vitro and was upregulated in the white matter after BCAS surgery. Further, miR-501-3p directly bound to the 3'-untranslated region of human ZO-1 and downregulated transendothelial electric resistance. In vivo administration of a locked nucleic acid -modified antisense oligonucleotide versus miR-501-3p suppressed BCAS-induced reduction of ZO-1 gene expression and blood-brain barrier disruption within the white matter and significantly ameliorated working memory deficits after BCAS surgery. CONCLUSIONS: We here provide the first evidence that the TNFα-miR-501-3p-ZO-1 axis plays an important role in the pathogenesis of cerebral hypoperfusion-induced working memory deficits and white matter lesions, as a result of blood-brain barrier breakdown via tight junction disruption. Therapeutic manipulation of miR-501-3p holds promise for limiting vascular cognitive impairment progression.
Assuntos
Comportamento Animal , Barreira Hematoencefálica/fisiopatologia , Permeabilidade Capilar , Transtornos Cerebrovasculares/terapia , Transtornos Cognitivos/terapia , Cognição , Terapia Genética/métodos , MicroRNAs/genética , Oligonucleotídeos Antissenso/administração & dosagem , Regiões 3' não Traduzidas , Animais , Sítios de Ligação , Barreira Hematoencefálica/metabolismo , Transtornos Cerebrovasculares/genética , Transtornos Cerebrovasculares/fisiopatologia , Transtornos Cerebrovasculares/psicologia , Claudina-5/genética , Claudina-5/metabolismo , Transtornos Cognitivos/genética , Transtornos Cognitivos/fisiopatologia , Transtornos Cognitivos/psicologia , Modelos Animais de Doenças , Impedância Elétrica , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Ocludina/genética , Ocludina/metabolismo , Oligonucleotídeos Antissenso/genética , Junções Íntimas/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
We investigated the effects of variations in anesthesia exposure time prior to conducting anxiety response behavioral testing in sham controls from an experimental murine model. The staying time in the center area of the Open Field test in the "long exposure" group was significantly decreased compared to that of the "short exposure" group. Significant correlation was found between anesthesia time and the duration of staying time in the center area. We conclude that anesthesia time may have a significant impact on behavioral anxiety testing in this context, and advise careful control of this parameter in protocol optimization in related surgical animal models.
Assuntos
Anestesia por Inalação , Anestésicos Inalatórios/farmacologia , Ansiedade , Comportamento Animal/efeitos dos fármacos , Isoflurano/farmacologia , Animais , Masculino , Camundongos Endogâmicos C57BL , Modelos Anatômicos , Modelos Animais , Fatores de TempoRESUMO
Backgrounds: Many factors are involved in cellular aging, and senescence induction requires complex regulation of various signaling networks and processes. Specifically, in the area of aging-related vascular cognitive impairment, laboratory-based findings have not yet yielded agents of practical use for clinical settings. One possible reason is that the physiologic elements of aging have been insufficiently considered. We sought to establish techniques to better model cellular aging using modulation of microRNAs, aiming to identify key microRNAs capable of fine-tuning aging-associated genes, and thereby regulating the senescence of vascular endothelial cells. Methods: We utilized expression microRNA arrays to evaluate control and senescent vascular endothelial cells in order to identify testable candidates. Bioinformatic analysis was used to select key microRNAs. These candidates were then modulated in vitro using microRNA mimics and inhibitors in endothelial cells, and senescence-associated gene expression patterns were evaluated by qPCR. Results: Seventeen microRNAs were found to be significantly increased more than 2-fold in senescent cells. Of those, bioinformatic analysis concluded that miR-181a-5p, miR-30a-5p, miR-30a-3p, miR-100-5p, miR-21-5p, and miR-382-5p were likely associated with regulation of cellular senescence. We evaluated the potential targets of these six microRNAs by comparing them with cell-cycling and apoptosis-related genes from published mRNA transcriptional array data from aged tissues, and found that miR-181a-5p, miR-30a-5p and miR-30a-3p were enriched in overlapping targets compared with the other candidates. Modulation of these microRNAs in vascular endothelial cells revealed that over-expression of miR-30a-5p, and inhibition of both miR-30a-3p and miR-181a-5p, induced senescence. Conclusion: miR-181a-5p, miR-30a-5p and miR-30a-3p likely contribute to aging-associated vascular endothelial cell senescence.
RESUMO
The recently discovered peptide apelin is known to be involved in the maintenance of insulin sensitivity. However, questions persist regarding its precise role in the chronic setting. Fasting glucose, insulin, and adiponectin levels were determined on mice with generalized deficiency of apelin (APKO). Additionally, insulin (ITT) and glucose tolerance tests (GTT) were performed. To assess the impact of exogenously delivered apelin on insulin sensitivity, osmotic pumps containing pyroglutamated apelin-13 or saline were implanted in APKO mice for 4 wk. Following the infusion, ITT/GTTs were repeated and the animals euthanized. Soleus muscles were harvested and homogenized in lysis buffer, and insulin-induced Akt phosphorylation was determined by Western blotting. Apelin-13 infusion and ITTs/GTTs were also performed in obese diabetic db/db mice. To probe the underlying mechanism for apelin's effects, apelin-13 was also delivered to cultured C2C12 myotubes. 2-[3H]deoxyglucose uptake and Akt phosphorylation were assessed in the presence of various inhibitors. APKO mice had diminished insulin sensitivity, were hyperinsulinemic, and had decreased adiponectin levels. Soleus lysates had decreased insulin-induced Akt phosphorylation. Administration of apelin to APKO and db/db mice resulted in improved insulin sensitivity. In C2C12 myotubes, apelin increased glucose uptake and Akt phosphorylation. These events were fully abrogated by pertussis toxin, compound C, and siRNA knockdown of AMPKalpha1 but only partially diminished by LY-294002 and not at all by L-NAME. We conclude that apelin is necessary for the maintenance of insulin sensitivity in vivo. Apelin's effects on glucose uptake and Akt phosphorylation are in part mediated by a G(i) and AMPK-dependent pathway.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Resistência à Insulina/fisiologia , Obesidade/fisiopatologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Adipocinas , Animais , Apelina , Células Cultivadas , Cromonas/farmacologia , Desoxiglucose/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Sacarose Alimentar/farmacologia , Inibidores Enzimáticos/farmacologia , Hiperinsulinismo/metabolismo , Hiperinsulinismo/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfolinas/farmacologia , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Obesidade/metabolismo , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores para Leptina/genética , Receptores para Leptina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , TrítioRESUMO
Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease.
Assuntos
Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/patologia , Expressão Gênica , Heme Oxigenase-1/genética , Animais , Aneurisma da Aorta Abdominal/tratamento farmacológico , Aneurisma da Aorta Abdominal/metabolismo , Peso Corporal , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Ativação Enzimática , Genes Reporter , Heme/metabolismo , Heme/farmacologia , Heme Oxigenase-1/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Mediadores da Inflamação/metabolismo , Lipídeos/sangue , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , SuínosRESUMO
The release of free fatty acids (FFAs) from adipocytes (i.e. lipolysis) is increased in obesity and is a contributory factor to the development of insulin resistance. A recently identified adipokine, apelin, is up-regulated in states of obesity. Although apelin is secreted by adipocytes, its functions in them remain largely unknown. To determine whether apelin affects lipolysis, FFA, glycerol, and leptin levels, as well as abdominal adiposity, were measured at baseline and after reintroduction of exogenous apelin in apelin-null mice. To examine apelin's effects in vitro, isoproterenol-induced FFA/glycerol release, and hormone-sensitive lipase (HSL) and acetyl CoA carboxylase phosphorylation were investigated in 3T3-L1 cells and isolated wild-type adipocytes. Serum FFA, glycerol, and leptin concentrations, as well as abdominal adiposity, were significantly increased in apelin-null vs. wild-type mice; these changes were ameliorated in response to exogenous apelin. Apelin also reduced isoproterenol-induced FFA release in adipocytes isolated from wild-type but not APJ-null mice. In 3T3-L1 cells and isolated adipocytes, apelin attenuated isoproterenol-induced FFA/glycerol release. Apelin's inhibition was reversed by pertussis toxin, the G(q) inhibitor glycoprotein antagonist 2A, and the AMP-activated protein kinase inhibitors compound C and dorsomorphin. Apelin increased HSL phosphorylation at Ser-565 and also abrogated isoproterenol-induced HSL phosphorylation at Ser-563. Notably, apelin increased acetyl CoA carboxylase phosphorylation, suggesting AMPK activation. In conclusion, apelin negatively regulates lipolysis. Its actions may be mediated by pathways involving G(q), G(i), and AMP-activated protein kinase.