RESUMO
Patients with Takotsubo syndrome displayed endothelial dysfunction, but underlying mechanisms have not been fully clarified. This study aimed to explore molecular signalling responsible for catecholamine excess induced endothelial dysfunction. Human cardiac microvascular endothelial cells were challenged by epinephrine to mimic catecholamine excess. Patch clamp, FACS, ELISA, PCR, and immunostaining were employed for the study. Epinephrine (Epi) enhanced small conductance calcium-activated potassium channel current (ISK1-3) through activating α1 adrenoceptor. Phenylephrine enhanced edothelin-1 (ET-1) and reactive oxygen species (ROS) production, and the effects involved contribution of ISK1-3. H2O2 enhanced ISK1-3 and ET-1 production. Enhancing ISK1-3 caused a hyperpolarization, which increases ROS and ET-1 production. BAPTA partially reduced phenylephrine-induced enhancement of ET-1 and ROS, suggesting that α1 receptor activation can enhance ROS/ET-1 generation in both calcium-dependent and calcium-independent ways. The study demonstrates that high concentration catecholamine can activate SK1-3 channels through α1 receptor-ROS signalling and increase ET-1 production, facilitating vasoconstriction.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 1 , Células Endoteliais , Epinefrina , Espécies Reativas de Oxigênio , Receptores Adrenérgicos alfa 1 , Transdução de Sinais , Canais de Potássio Ativados por Cálcio de Condutância Baixa , Vasoconstrição , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Adrenérgicos alfa 1/genética , Espécies Reativas de Oxigênio/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Vasoconstrição/efeitos dos fármacos , Células Cultivadas , Epinefrina/farmacologia , Peróxido de Hidrogênio/metabolismo , Potenciais da Membrana , Fenilefrina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Canais de Potássio Éter-A-Go-GoRESUMO
Endothelial dysfunction may contribute to pathogenesis of Takotsubo cardiomyopathy, but mechanism underlying endothelial dysfunction in the setting of catecholamine excess has not been clarified. The study reports that D1/D5 dopamine receptor signaling and small conductance calcium-activated potassium channels contribute to high concentration catecholamine induced endothelial cell dysfunction. For mimicking catecholamine excess, 100 µM epinephrine (Epi) was used to treat human cardiac microvascular endothelial cells. Patch clamp, FACS, ELISA, PCR, western blot and immunostaining analyses were performed in the study. Epi enhanced small conductance calcium-activated potassium channel current (ISK1-3) without influencing the channel expression and the effect was attenuated by D1/D5 receptor blocker. D1/D5 agonists mimicked the Epi effect, suggesting involvement of D1/D5 receptors in Epi effects. The enhancement of ISK1-3 caused by D1/D5 activation involved roles of PKA, ROS and NADPH oxidases. Activation of D1/D5 and SK1-3 channels caused a hyperpolarization, reduced NO production and increased ROS production. The NO reduction was membrane potential independent, while ROS production was increased by the hyperpolarization. ROS (H2O2) suppressed NO production. The study demonstrates that high concentration catecholamine can activate D1/D5 and SK1-3 channels through NADPH-ROS and PKA signaling and reduce NO production, which may facilitate vasoconstriction in the setting of catecholamine excess.
Assuntos
Células Endoteliais , Epinefrina , Espécies Reativas de Oxigênio , Transdução de Sinais , Humanos , Transdução de Sinais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Óxido Nítrico/metabolismo , Catecolaminas/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Endotélio Vascular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , NADPH Oxidases/metabolismo , Receptores de Dopamina D5/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores Dopaminérgicos/metabolismoRESUMO
Arterial occlusion-induced ischemic stroke (IS) is a highly frequent stroke subtype. Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that modulates antioxidant genes. Its role in IS is still unelucidated. The current study focused on constructing a transient middle cerebral artery occlusion (tMCAO) model for investigating the NRF2-related mechanism underlying cerebral ischemia/reperfusion (I/R) injury. Each male C57BL/6 mouse was injected with/with no specific NRF2 activator post-tMCAO. Changes in blood-brain barrier (BBB)-associated molecule levels were analyzed using western-blotting, PCR, immunohistochemistry, and immunofluorescence analysis. NRF2 levels within cerebral I/R model decreased at 24-h post-ischemia. NRF2 activation improved brain edema, infarct volume, and neurological deficits after MCAO/R. Similarly, sulforaphane (SFN) prevented the down-regulated tight junction proteins occludin and zonula occludens 1 (ZO-1) and reduced the up-regulated aquaporin 4 (AQP4) and matrix metalloproteinase 9 (MMP9) after tMCAO. Collectively, NRF2 exerted a critical effect on preserving BBB integrity modulating ferroptosis and inflammation. Because NRF2 is related to BBB injury regulation following cerebral I/R, this provides a potential therapeutic target and throws light on the underlying mechanism for clinically treating IS.
Assuntos
Ferroptose , AVC Isquêmico , Traumatismo por Reperfusão , Acidente Vascular Cerebral , Animais , Masculino , Camundongos , Barreira Hematoencefálica , Infarto Cerebral , Inflamação , Isquemia , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2 , Acidente Vascular Cerebral/tratamento farmacológicoRESUMO
Myeloid differentiation primary response gene 88 (MyD88), a downstream molecule directly linked to Toll-like receptor (TLRs) and IL1 receptor, has been implicated in ischemia-reperfusion injury across various organs. However, its role in cerebral ischemia-reperfusion injury (CIRI) remains unclear. Five transient middle cerebral artery occlusion (tMCAO) microarray datasets were obtained from the Gene Expression Omnibus (GEO) database. We screened these datasets for differentially expressed genes (DEGs) using the GSE35338 and GSE58720 datasets and performed weighted gene co-expression network analysis (WGCNA) using the GSE30655, GSE28731, and GSE32529 datasets to identify the core module related to tMCAO. A protein-protein interaction (PPI) network was constructed using the intersecting DEGs and genes in the core module. Finally, we identified Myd88 was the core gene. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) validated that TNFα, IL17, and MyD88 signaling pathways were significantly enriched in tMCAO. Subsequently, we investigated the mechanistic role of MyD88 in the tMCAO model using male C57BL/6 mice. MyD88 expression increased significantly 24 h after reperfusion. After intraperitoneal administration of TJ-M2010-5, a MyD88-specific inhibitor, during reperfusion, the infarction volumes in the mice were ameliorated. TJ-M2010-5 inhibits the activation of microglia and astrocytes. Moreover, it attenuates the upregulation of inflammatory cytokines TNFα, IL17, and MMP9 while preserving the expression level of ZO1 after tMCAO, thereby safeguarding against blood-brain barrier (BBB) disruption. Finally, our findings suggest that MyD88 regulates the IRAK4/IRF5 signaling pathway associated with microglial activation. MyD88 participates in CIRI by regulating the inflammatory response and BBB damage following tMCAO.
Assuntos
Barreira Hematoencefálica , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide , Traumatismo por Reperfusão , Fator 88 de Diferenciação Mieloide/metabolismo , Animais , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Masculino , Camundongos , Infarto da Artéria Cerebral Média/metabolismo , Inflamação/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Microglia/metabolismo , Microglia/efeitos dos fármacos , Mapas de Interação de Proteínas , Piperazinas , TiazóisRESUMO
In the current study, we aimed to investigate whether disulfiram (DSF) exerts a neuroprotective role in cerebral ischemiareperfusion (CI-RI) injury by modulating ferredoxin 1 (FDX1) to regulate copper ion (Cu) levels and inhibiting inflammatory responses. To simulate CI-RI, a transient middle cerebral artery occlusion (tMCAO) model in C57/BL6 mice was employed. Mice were administered with or without DSF before and after tMCAO. Changes in infarct volume after tMCAO were observed using TTC staining. Nissl staining and hematoxylin-eosin (he) staining were used to observe the morphological changes of nerve cells at the microscopic level. The inhibitory effect of DSF on initial inflammation was verified by TUNEL assay, apoptosis-related protein detection and iron concentration detection. FDX1 is the main regulatory protein of copper death, and the occurrence of copper death will lead to the increase of HSP70 stress and inflammatory response. Cuproptosis-related proteins and downstream inflammatory factors were detected by western blotting, immunofluorescence staining, and immunohistochemistry. The content of copper ions was detected using a specific kit, while electron microscopy was employed to examine mitochondrial changes. We found that DSF reduced the cerebral infarction volume, regulated the expression of cuproptosis-related proteins, and modulated copper content through down regulation of FDX1 expression. Moreover, DSF inhibited the HSP70/TLR-4/NLRP3 signaling pathway. Collectively, DSF could regulate Cu homeostasis by inhibiting FDX1, acting on the HSP70/TLR4/NLRP3 pathway to alleviate CI/RI. Accordingly, DSF could mitigate inflammatory responses and safeguard mitochondrial integrity, yielding novel therapeutic targets and mechanisms for the clinical management of ischemia-reperfusion injury.
Assuntos
Cobre , Dissulfiram , Homeostase , Inflamação , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão , Animais , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/patologia , Dissulfiram/farmacologia , Camundongos , Cobre/metabolismo , Homeostase/efeitos dos fármacos , Masculino , Inflamação/metabolismo , Inflamação/tratamento farmacológico , Inflamação/patologia , Regulação para Baixo/efeitos dos fármacos , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Modelos Animais de Doenças , Proteínas Ferro-Enxofre/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Apoptose/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptor 4 Toll-Like/metabolismoRESUMO
Endothelial dysfunction contributes to the pathogenesis of Takotsubo syndrome (TTS). However, the exact mechanism underlying endothelial dysfunction in the setting of TTS has not been completely clarified. This study aims to investigate the roles of angiotensin II (Ang II) and intermediate-conductance Ca2+-activated K+ (SK4) channels in catecholamine-induced endothelial dysfunction. Human cardiac microvascular endothelial cells (HCMECs) were exposed to 100⯵M epinephrine (Epi), mimicking the setting of TTS. Epi treatment increased the ET-1 concentration and reduced NO levels in HCMECs. Importantly, the effects of Epi were found to be mitigated in the presence of Ang II receptor blockers. Furthermore, Ang II mimicked Epi effects on ET-1 and NO production. Additionally, Ang II inhibited tube formation and increased cell apoptosis. The effects of Ang II could be reversed by an SK4 activator NS309 and mimicked by an SK4 channel blocker TRAM-34. Ang II also inhibited the SK4 channel current (ISK4) without affecting its expression level. Ang II could depolarize the cell membrane potential. Ang II promoted ROS release and reduced protein kinase A (PKA) expression. A ROS blocker prevented Ang II effect on ISK4. The PKA activator Sp-8-Br-cAMPS increased SK4 channel currents. Epinephrine enhanced the activity of ACE by activating the α1 receptor/Gq/PKC signal pathway, thereby promoting the secretion of Ang II. The study suggested that high-level catecholamine can increase Ang II release from endothelial cells by α1 receptors/Gq/PKC signal pathway. Ang II can inhibit SK4 channel current by increasing ROS generation and reducing PKA expression, thereby contributing to endothelial dysfunction.
Assuntos
Angiotensina II , Catecolaminas , Células Endoteliais , Espécies Reativas de Oxigênio , Angiotensina II/farmacologia , Humanos , Catecolaminas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Epinefrina/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Apoptose/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células CultivadasRESUMO
The emergence of acute ischemic stroke (AIS) induced cardiovascular dysfunctions as a bidirectional interaction has gained paramount importance in understanding the intricate relationship between the brain and heart. Post AIS, the ensuing cardiovascular dysfunctions encompass a spectrum of complications, including heart attack, congestive heart failure, systolic or diastolic dysfunction, arrhythmias, electrocardiographic anomalies, hemodynamic instability, cardiac arrest, among others, all of which are correlated with adverse outcomes and mortality. Mounting evidence underscores the intimate crosstalk between the heart and the brain, facilitated by intricate physiological and neurohumoral complex networks. The primary pathophysiological mechanisms contributing to these severe cardiac complications involve the hypothalamic-pituitary-adrenal (HPA) axis, sympathetic and parasympathetic hyperactivity, immune and inflammatory responses, and gut dysbiosis, collectively shaping the stroke-related brain-heart axis. Ongoing research endeavors are concentrated on devising strategies to prevent AIS-induced cardiovascular dysfunctions. Notably, labetalol, nicardipine, and nitroprusside are recommended for hypertension control, while ß-blockers are employed to avert chronic remodeling and address arrhythmias. However, despite these therapeutic interventions, therapeutic targets remain elusive, necessitating further investigations into this complex challenge. This review aims to delineate the state-of-the-art pathophysiological mechanisms in AIS through preclinical and clinical research, unraveling their intricate interplay within the brain-heart axis, and offering pragmatic suggestions for managing AIS-induced cardiovascular dysfunctions.