RESUMEN
Acute lung injury (ALI) including acute respiratory distress syndrome (ARDS) is a major complication and increase the mortality of patients with cardiac surgery. We previously found that the protein cargoes enriched in circulating extracellular vesicles (EVs) are closely associated with cardiopulmonary disease. We aimed to evaluate the implication of EVs on cardiac surgery-associated ALI/ARDS. The correlations between "oncoprotein-induced transcript 3 protein (OIT3) positive" circulating EVs and postoperative ARDS were assessed. The effects of OIT3-overexpressed EVs on the cardiopulmonary bypass (CPB) -induced ALI in vivo and inflammation of human bronchial epithelial cells (BEAS-2B) were detected. OIT3 enriched in circulating EVs is reduced after cardiac surgery with CPB, especially with postoperative ARDS. The "OIT3 positive" EVs negatively correlate with lung edema, hypoxemia and CPB time. The OIT3-overexpressed EVs can be absorbed by pulmonary epithelial cells and OIT3 transferred by EVs triggered K48- and K63-linked polyubiquitination to inactivate NOD-like receptor protein 3 (NLRP3) inflammasome, and restrains pro-inflammatory cytokines releasing and immune cells infiltration in lung tissues, contributing to the alleviation of CPB-induced ALI. Overexpression of OIT3 in human bronchial epithelial cells have similar results. OIT3 promotes the E3 ligase Cbl proto-oncogene B associated with NLRP3 to induce the ubiquitination of NLRP3. Immunofluorescence tests reveal that OIT3 is reduced in the generation from the liver sinusoids endothelial cells (LSECs) and secretion in liver-derived EVs after CPB. In conclusion, OIT3 enriched in EVs is a promising biomarker of postoperative ARDS and a therapeutic target for ALI after cardiac surgery.
RESUMEN
Ferroptosis is a novel cell death mechanism that is mediated by iron-dependent lipid peroxidation. It may be involved in atherosclerosis development. Products of phospholipid oxidation play a key role in atherosclerosis. 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) is a phospholipid oxidation product present in atherosclerotic lesions. It remains unclear whether PGPC causes atherosclerosis by inducing endothelial cell ferroptosis. In this study, human umbilical vein endothelial cells (HUVECs) were treated with PGPC. Intracellular levels of ferrous iron, lipid peroxidation, superoxide anions (O2â¢-), and glutathione were detected, and expression of fatty acid binding protein-3 (FABP3), glutathione peroxidase 4 (GPX4), and CD36 were measured. Additionally, the mitochondrial membrane potential (MMP) was determined. Aortas from C57BL6 mice were isolated for vasodilation testing. Results showed that PGPC increased ferrous iron levels, the production of lipid peroxidation and O2â¢-, and FABP3 expression. However, PGPC inhibited the expression of GPX4 and glutathione production and destroyed normal MMP. These effects were also blocked by ferrostatin-1, an inhibitor of ferroptosis. FABP3 silencing significantly reversed the effect of PGPC. Furthermore, PGPC stimulated CD36 expression. Conversely, CD36 silencing reversed the effects of PGPC, including PGPC-induced FABP3 expression. Importantly, E06, a direct inhibitor of the oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine IgM natural antibody, inhibited the effects of PGPC. Finally, PGPC impaired endothelium-dependent vasodilation, ferrostatin-1 or FABP3 inhibitors inhibited this impairment. Our data demonstrate that PGPC impairs endothelial function by inducing endothelial cell ferroptosis through the CD36 receptor to increase FABP3 expression. Our findings provide new insights into the mechanisms of atherosclerosis and a therapeutic target for atherosclerosis.
Asunto(s)
Aterosclerosis , Ciclohexilaminas , Ferroptosis , Fenilendiaminas , Animales , Ratones , Humanos , Fosfolípidos , Fosforilcolina , Éteres Fosfolípidos/metabolismo , Éteres Fosfolípidos/farmacología , Ratones Endogámicos C57BL , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Endotelio/metabolismo , Glutatión/metabolismo , Hierro/metabolismo , Proteína 3 de Unión a Ácidos GrasosRESUMEN
Endothelial-to-mesenchymal transition (EndMT), the process by which an endothelial cell (EC) undergoes a series of molecular events that result in a mesenchymal cell phenotype, plays an important role in atherosclerosis. 1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), derived from the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine, is a proinflammatory lipid found in atherosclerotic lesions. Whether POVPC promotes EndMT and how simvastatin influences POVPC-mediated EndMT remains unclear. Here, we treated human umbilical vein ECs with POVPC, simvastatin, or both, and determined their effect on EC viability, morphology, tube formation, proliferation, and generation of NO and superoxide anion (O2â¢-). Expression of specific endothelial and mesenchymal markers was detected by immunofluorescence and immunoblotting. POVPC did not affect EC viability but altered cellular morphology from cobblestone-like ECs to a spindle-like mesenchymal cell morphology. POVPC increased O2- generation and expression of alpha-smooth muscle actin, vimentin, Snail-1, Twist-1, transforming growth factor-beta (TGF-ß), TGF-ß receptor II, p-Smad2/3, and Smad2/3. POVPC also decreased NO production and expression of CD31 and endothelial NO synthase. Simvastatin inhibited POVPC-mediated effects on cellular morphology, production of O2â¢- and NO, and expression of specific endothelial and mesenchymal markers. These data demonstrate that POVPC induces EndMT by increasing oxidative stress, which stimulates TGF-ß/Smad signaling, leading to Snail-1 and Twist-1 activation. Simvastatin inhibited POVPC-induced EndMT by decreasing oxidative stress, suppressing TGF-ß/Smad signaling, and inactivating Snail-1 and Twist-1. Our findings reveal a novel mechanism of atherosclerosis that can be inhibited by simvastatin.
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FosforilcolinaRESUMEN
We previously demonstrated that circulating extracellular vesicles (EVs) from patients with valvular heart disease (VHD; vEVs) contain inflammatory components and inhibit endothelium-dependent vasodilation. Neutrophil chemotaxis plays a key role in renal dysfunction, and dexmedetomidine (DEX) can reduce renal dysfunction in cardiac surgery. However, the roles of vEVs in neutrophil chemotaxis and effects of DEX on vEVs are unknown. Here, we investigated the impact of vEVs on neutrophil chemotaxis in kidneys and the influence of DEX on vEVs. Circulating EVs were isolated from healthy subjects and patients with VHD. The effects of EVs on chemokine generation, forkhead box protein O3a (FOXO3a) pathway activation and neutrophil chemotaxis on cultured human umbilical vein endothelial cells (HUVECs) and kidneys in mice and the influence of DEX on EVs were detected. vEVs increased FOXO3a expression, decreased phosphorylation of Akt and FOXO3a, promoted FOXO3a nuclear translocation, and activated the FOXO3a signaling pathway in vitro. DEX pretreatment reduced vEV-induced CXCL4 and CCL5 expression and neutrophil chemotaxis in cultured HUVECs via the FOXO3a signaling pathway. vEVs were also found to suppress Akt phosphorylation and activate FOXO3a signaling to increase plasma levels of CXCL4 and CCL5 and neutrophil accumulation in kidney. The overall mechanism was inhibited in vivo with DEX pretreatment. Our data demonstrated that vEVs induced CXCL4-CCL5 to stimulate neutrophil infiltration in kidney, which can be inhibited by DEX via the FOXO3a signaling. Our findings reveal a unique mechanism involving vEVs in inducing neutrophils chemotaxis and may provide a novel basis for using DEX in reducing renal dysfunction in valvular heart surgery.
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Quimiotaxis de Leucocito/inmunología , Vesículas Extracelulares/inmunología , Enfermedades de las Válvulas Cardíacas/inmunología , Células Endoteliales de la Vena Umbilical Humana/inmunología , Riñón/inmunología , Neutrófilos/inmunología , Insuficiencia Renal/inmunología , Agonistas de Receptores Adrenérgicos alfa 2/farmacología , Adulto , Animales , Estudios de Casos y Controles , Quimiocina CCL5/efectos de los fármacos , Quimiocina CCL5/inmunología , Quimiocina CCL5/metabolismo , Quimiotaxis de Leucocito/efectos de los fármacos , Dexmedetomidina/farmacología , Vesículas Extracelulares/efectos de los fármacos , Vesículas Extracelulares/metabolismo , Femenino , Proteína Forkhead Box O3/efectos de los fármacos , Proteína Forkhead Box O3/inmunología , Proteína Forkhead Box O3/metabolismo , Enfermedades de las Válvulas Cardíacas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inflamación , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Neutrófilos/efectos de los fármacos , Fosforilación , Factor Plaquetario 4/efectos de los fármacos , Factor Plaquetario 4/inmunología , Factor Plaquetario 4/metabolismo , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Insuficiencia Renal/metabolismo , VasodilataciónRESUMEN
PURPOSE OF REVIEW: This review summarizes the effects of microparticles and exosomes in the progression of atherosclerosis and the prospect for their diagnostic and therapeutic potentials. RECENT FINDINGS: Microparticles and exosomes can induce endothelial dysfunction, vascular inflammation, coagulation, thrombosis, and calcification via their components of proteins and noncoding RNAs, which may promote the progression of atherosclerosis. The applications of microparticles and exosomes become the spotlight of clinical diagnosis and therapy. Microparticles and exosomes are members of extracellular vesicles, which are generated in various cell types by different mechanisms of cell membrane budding and multivesicular body secretion, respectively. They are important physiologic pathways of cell-to-cell communication in vivo and act as messengers accelerating or alleviating the process of atherosclerosis. Microparticles and exosomes may become diagnostic biomarkers and therapeutic approaches of atherosclerosis.
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Aterosclerosis/metabolismo , Comunicación Celular , Micropartículas Derivadas de Células/metabolismo , Exosomas/metabolismo , Animales , Biomarcadores/metabolismo , Progresión de la Enfermedad , Endotelio Vascular/metabolismo , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Trombosis/metabolismo , Calcificación Vascular/metabolismoRESUMEN
Ischemia postconditioning (PTC) can reduce myocardial ischemia/reperfusion injury. However, the effectiveness of PTC cardioprotection is reduced or lost in diabetes and the mechanisms are largely unclear. Hyperglycemia can induce overexpression of inducible nitric oxide synthesis (iNOS) in the myocardium of diabetic subjects. However, it is unknown whether or not iNOS especially its overexpression plays an important role in the loss of cardioprotection of PTC in diabetes. C57BL6 and iNOS-/- mice were treated with streptozotocin to induce diabetes. Part of diabetic C57BL6 mice were also treated with an iNOS specific inhibitor, 1400â¯W. Mice were subjected to myocardial ischemia/ reperfusion with/without PTC. The hemodynamic parameters, plasma levels of cardiac troponin T (cTnT), TNF-α, IL-6 and nitric oxide (NO) were monitored. The myocardial infarct size, superoxide anion (O2-) generation, nitrotyrosine production and apoptosis were measured. The expression of phosphorylated Akt, endothelial NOS (eNOS), iNOS and Erk1/2 in ischemic heart were detected by immunoblot analysis. In diabetic C57BL6 and iNOS-/- mice, the post-ischemic hemodynamics were impaired, the cTnT, TNF-α, IL-6 level, myocardial infarct size, apoptotic index, O2- and nitrotyrosine generation were increased and the Akt/eNOS signal pathways were inhibited. PTC improved hemodynamic parameters, reduced cTnT level, myocardial infarct size, apoptotic index, O2- and nitrotyrosine generation and activated Akt/eNOS and Erk1/2 signal pathways in both non-diabetic C57BL6 and iNOS-/- mice as well as diabetic iNOS-/- mice, but not in diabetic C57BL6 mice. PTC also increased NO production in both non-diabetic and diabetic C57BL6 and iNOS-/- mice, and enhanced iNOS expression in non-diabetic C57BL6 mice. 1400â¯W restored the cardioprotection of PTC in diabetic C57BL6 mice. Our data demonstrated that PTC reduced myocardial ischemia/reperfusion injury in non-diabetic mice but not C57BL6 diabetic mice. Deletion of iNOS restored the cardioprotection of PTC in diabetic mice. Our findings suggest that iNOS plays a key role in the reduction of cardioprotection of PTC in diabetes and may provide a therapeutic target for diabetic patients.
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Diabetes Mellitus Experimental/enzimología , Poscondicionamiento Isquémico , Miocardio/enzimología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Animales , Apoptosis , Glucemia/metabolismo , Peso Corporal , Citocinas/metabolismo , Diabetes Mellitus Experimental/fisiopatología , Mediadores de Inflamación/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones Endogámicos C57BL , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Troponina T/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Función VentricularRESUMEN
Simvastatin treatment is cardioprotective in patients undergoing noncoronary artery cardiac surgery. However, the mechanisms by which simvastatin treatment protects the myocardium under these conditions are not fully understood. Seventy patients undergoing noncoronary cardiac surgery, 35 from a simvastatin treatment group and 35 from a control treatment group, were enrolled in our clinical study. Simvastatin (20 mg/d) was administered preoperatively for 5-7 days. Myocardial tissue biopsies were taken before and after surgery. Apoptosis was detected by TUNEL staining. The expressions of Bcl-2 and Bak in myocardial tissue were detected by immunoblotting. The expressions of miRNA and Bcl-2 mRNA were detected by quantitative real-time polymerase chain reaction assays. Cardiomyocytes were isolated from rat and cultured cells. MiR-15a-5p mimic was transfected into cardiomyocytes, and the Bcl-2 was detected by immunoblotting. TUNEL staining showed significantly less myocardial apoptosis in the simvastatin treatment group when compared with the control treatment group. Protein expression of Bcl-2 was increased in the simvastatin treatment group before surgery, and Bak expression was increased in the control treatment group after surgery. Further comparisons showed that Bcl-2/Bak ratios were reduced in the control treatment group but were not significantly changed in the simvastatin treatment group after surgery. Furthermore, microarray assays revealed that miR-15a-5p was significantly decreased by simvastatin treatment. This was validated by quantitative real-time polymerase chain reaction analysis. MiR-15a-5p was predicted to target Bcl-2 mRNA at nucleotide positions 2529-2536. This was validated by luciferase binding assays. Coincident with the change in miR-15a-5p, the mRNA expression of Bcl-2 was increased in the simvastatin treatment group. MiR-15a-5p mimic significantly inhibited Bcl-2 expression in cardiomyocytes. Our findings strongly suggest that simvastatin treatment preoperatively protected the myocardium in patients undergoing noncoronary artery cardiac surgery, at least in part, by inhibiting apoptosis via suppressing miR-15a-5p expression, leading to increasing expression of Bcl-2 and decreasing expression of Bak.
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Apoptosis/efectos de los fármacos , Procedimientos Quirúrgicos Electivos/efectos adversos , Cardiopatías/prevención & control , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , MicroARNs/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Simvastatina/administración & dosificación , Adulto , Animales , Células Cultivadas , China , Esquema de Medicación , Femenino , Cardiopatías/genética , Cardiopatías/metabolismo , Cardiopatías/patología , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/efectos adversos , Masculino , MicroARNs/genética , Persona de Mediana Edad , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Simvastatina/efectos adversos , Resultado del Tratamiento , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismoRESUMEN
Endothelial dysfunction is an early stage of atherosclerosis. We recently have shown that 25-hydroxycholesterol found in atherosclerotic lesions could impair endothelial function and vasodilation by uncoupling and inhibiting endothelial nitric oxide synthase (eNOS). 1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), the oxidation product of oxidized low-density lipoprotein, is another proinflammatory lipid and has also been found in atherosclerotic lesions. However, whether POVPC promotes atherosclerosis like 25-hydroxycholesterol remains unclear. The purpose of this study was to explore the effects of POVPC on endothelial function and vasodilation. Human umbilical vein endothelial cells (HUVECs) were incubated with POVPC. Endothelial cell proliferation, migration and tube formation were measured. Nitric oxide (NO) production and superoxide anion generation (O2-) were determined. The expression and phosphorylation of endothelial nitric oxide synthase (eNOS), AKT, PKC-ßII and P70S6K as well as the association of eNOS and heat shock protein 90 (HSP90) were detected by immunoblotting and immunoprecipitation. Endothelial cell apoptosis was monitored by TUNEL staining. The expression of Bcl-2, Bax, and Cleaved Caspase 3 were detected by immunoblotting. Finally, aortic ring from C57BL6 mice were isolated and treated with POVPC and the endothelium-dependent vasodilation was evaluated. POVPC significantly inhibited HUVECs proliferation, migration, tube formation, decreased NO production but increased O2- generation. POVPC inhibited the phosphorylation of Akt and eNOS at Ser1177, increased activation of PKC-ßII, P70S6K and the phosphorylation of eNOS at Thr495, reduced the association of HSP90 with eNOS. Meanwhile, POVPC induced endothelial cell apoptosis by inhibiting Bcl-2 expression, increasing Bax and cleaved caspase-3 expressions as well as caspase-3 activity and impaired endothelium-dependent vasodilation. These data demonstrated that POVPC impaired endothelial function by uncoupling and inhibiting eNOS as well as by inducing endothelial cell apoptosis. Therefore, POVPC may play an important role in the development of atherosclerosis and may be considered as a potential therapeutic target for atherosclerosis.
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Células Endoteliales de la Vena Umbilical Humana/patología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Éteres Fosfolípidos/farmacología , Vasodilatación/efectos de los fármacos , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Neovascularización Fisiológica/efectos de los fármacos , Óxido Nítrico/metabolismo , Oxidación-Reducción , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C beta/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Superóxidos/metabolismoRESUMEN
Increased evidence has showed that normal high density lipoprotein (HDL) could convert to dysfunctional HDL in diseases states including coronary artery disease (CAD), which regulated vascular endothelial cell function differently. Long non-coding RNAs (lncRNAs) play an extensive role in various important biological processes including endothelial cell function. However, whether lncRNAs are involved in the regulation of HDL metabolism and HDL-induced changes of vascular endothelial function remains unclear. Cultured human umbilical vein endothelial cells (HUVECs) were treated with HDL from healthy subjects and patients with CAD and hypercholesterolemia for 24 h, then the cells were collected for lncRNA-Seq and the expressions of lncRNAs, genes and mRNAs were identified. The bioinformatic analysis was used to evaluate the relationship among lncRNAs, encoding genes and miRNAs. HDL from healthy subjects and patients with CAD and hypercholesterolemia leaded to different expressions of lncRNAs, genes and mRNAs, and further analysis suggested that the differentially expressed lncRNAs played an important role in the regulation of vascular endothelial function. Thus, HDL from patients with CAD and hypercholesterolemia could cause abnormal expression of lncRNAs in vascular endothelial cells to affect vascular function.
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Enfermedad de la Arteria Coronaria/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Lipoproteínas HDL/metabolismo , ARN Largo no Codificante/genética , Células Cultivadas , Femenino , Humanos , Lipoproteínas HDL/administración & dosificación , Masculino , Persona de Mediana Edad , ARN Largo no Codificante/metabolismoRESUMEN
BACKGROUND: We previously demonstrated that endothelial microparticles (EMPs) are increased in mitral valve diseases and impair valvular endothelial cell function. Perioperative systemic inflammation is an important risk factor and complication of cardiac surgery. In this study, we investigate whether EMPs increase in congenital heart diseases to promote inflammation and endothelial dysfunction. METHODS: The level of plasma EMPs in 20 patients with atrial septal defect (ASD), 23 patients with ventricular septal defect (VSD), and 30 healthy subjects were analyzed by flow cytometry. EMPs generated from human umbilical vascular endothelial cells (HUVECs) were injected into C57BL6 mice, or cultured with HUVECs without or with siRNAs targeting P38 MAPK. The expression and/or phosphorylation of endothelial nitric oxide synthase (eNOS), P38 MAPK, and caveolin-1 in mouse heart and/or in cultured HUVECs were determined. We evaluated generation of nitric oxide (NO) in mouse hearts, and levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in cultured HUVECs and in mice. RESULTS: EMPs were significantly elevated in patients with ASD and VSD, especially in those with pulmonary hypertension when compared with controls. EMPs increased caveolin-1 expression and P38 MAPK phosphorylation and decreased eNOS phosphorylation and NO production in mouse hearts. EMPs stimulated P38 MAPK expression, TNF-α and IL-6 production, which were all inhibited by siRNAs targeting P38 MAPK in cultured HUVECs. CONCLUSIONS: EMPs were increased in adult patients with congenital heart diseases and may contribute to increased inflammation leading to endothelial dysfunction via P38 MAPK-dependent pathways. This novel data provides a potential therapeutic target to address important complications of surgery of congenial heart disease.
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Micropartículas Derivadas de Células/metabolismo , Células Endoteliales/metabolismo , Cardiopatías Congénitas/patología , Cardiopatías Congénitas/fisiopatología , Adulto , Animales , Caveolina 1/metabolismo , Demografía , Ecocardiografía Doppler , Endotelio Vascular/diagnóstico por imagen , Endotelio Vascular/patología , Endotelio Vascular/fisiopatología , Femenino , Cardiopatías Congénitas/sangre , Cardiopatías Congénitas/diagnóstico por imagen , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Interleucina-6/sangre , Masculino , Ratones , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , Factor de Necrosis Tumoral alfa/sangre , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Cold cardioplegia is used to induce heart arrest during cardiac surgery. However, endothelial function may be compromised after this procedure. Accordingly, interventions such as adenosine, that mimic the effects of preconditioning, may minimize endothelial injury. Herein, we investigated whether adenosine prevents cold-induced injury to the endothelium. Cultured human cardiac microvascular endothelial cells were treated with adenosine for different durations. Phosphorylation and expression of endothelial nitric oxide synthase (eNOS), p38MAPK, ERK1/2, and p70S6K6 were measured along with nitric oxide (NO) production using diaminofluorescein-2 diacetate (DAF-2DA) probe. Cold-induced injury by hypothermia to 4°C for 45 minutes to mimic conditions of cold cardioplegia during open heart surgery was induced in human cardiac microvascular endothelial cells. Under basal conditions, adenosine stimulated NO production, eNOS phosphorylation at serine 1177 from 5 minutes to 4 hours and inhibited eNOS phosphorylation at threonine 495 from 5 minutes to 6 hours, but increased phosphorylation of ERK1/2, p38MAPK, and p70S6K only after exposure for 5 minutes. Cold-induced injury inhibited NO production and the phosphorylation of the different enzymes. Importantly, adenosine prevented these effects of hypothermic injury. Our data demonstrated that adenosine prevents hypothermic injury to the endothelium by activating ERK1/2, eNOS, p70S6K, and p38MAPK signaling pathways at early time points. These findings also indicated that 5 minutes after administration of adenosine or release of adenosine is an important time window for cardioprotection during cardiac surgery.
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Adenosina/administración & dosificación , Frío/efectos adversos , Crioprotectores/administración & dosificación , Células Endoteliales/efectos de los fármacos , Hipotermia Inducida/efectos adversos , Lesiones del Sistema Vascular/prevención & control , Células Cultivadas , Citoprotección , Esquema de Medicación , Células Endoteliales/enzimología , Células Endoteliales/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Lesiones del Sistema Vascular/enzimología , Lesiones del Sistema Vascular/etiología , Lesiones del Sistema Vascular/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Endothelial dysfunction is a key early step in atherosclerosis. 25-Hydroxycholesterol (25-OHC) is found in atherosclerotic lesions. However, whether 25-OHC promotes atherosclerosis is unclear. Here, we hypothesized that 25-OHC, a proinflammatory lipid, can impair endothelial function, which may play an important role in atherosclerosis. Bovine aortic endothelial cells were incubated with 25-OHC. Endothelial cell proliferation, migration, and tube formation were measured. Nitric oxide (NO) production and superoxide anion generation were determined. The expression and phosphorylation of endothelial NO synthase (eNOS) and Akt as well as the association of eNOS and heat shock protein (HSP)90 were detected by immunoblot analysis and immunoprecipitation. Endothelial cell apoptosis was monitored by TUNEL staining and caspase-3 activity, and expression of Bcl-2, Bax, cleaved caspase-9, and cleaved caspase-3 were detected by immunoblot analysis. Finally, aortic rings from Sprague-Dawley rats were isolated and treated with 25-OHC, and endothelium-dependent vasodilation was evaluated. 25-OHC significantly inhibited endothelial cell proliferation, migration, and tube formation. 25-OHC markedly decreased NO production and increased superoxide anion generation. 25-OHC reduced the phosphorylation of Akt and eNOS and the association of eNOS and HSP90. 25-OHC also enhanced endothelial cell apoptosis by decreasing Bcl-2 expression and increasing cleaved caspase-9 and cleaved caspase-3 expressions as well as caspase-3 activity. 25-OHC impaired endothelium-dependent vasodilation. These data demonstrated that 25-OHC could impair endothelial function by uncoupling and inhibiting eNOS activity as well as by inducing endothelial cell apoptosis. Our findings indicate that 25-OHC may play an important role in regulating atherosclerosis.
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Endotelio/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hidroxicolesteroles/farmacología , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Desacopladores/farmacología , Vasodilatación/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Bovinos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Técnicas In Vitro , Mediadores de Inflamación/metabolismo , Óxido Nítrico/biosíntesis , Ratas , Ratas Sprague-Dawley , Superóxidos/metabolismoRESUMEN
Normal high density lipoprotein (HDL) protects vascular function; however these protective effects of HDL may absent in valvular heart disease (VHD). Because vascular function plays an important role in maintaining the circulation post-cardiac surgery and some patients are difficult to stabilize, we hypothesized that a deleterious vascular effect of HDL may contribute to vascular dysfunction in VHD patients following surgery. HDL was isolated from age-match 28 healthy subjects and 84 patients with VHD and during cardiac surgery. HDL pro-inflammation index was measured and the effects of HDL on vasodilation, protein interaction, generation of nitric oxide (NO) and superoxide were determined. Patients with VHD received either simvastatin (20mg/d) or routine medications, and endothelial effects of HDL were characterized. HDL inflammation index significantly increased in VHD patients and post-cardiac surgery. HDL from VHD patients and post-cardiac surgery significantly impaired endothelium-dependent vasodilation, inhibited both Akt and endothelial nitric oxide synthase (eNOS) phosphorylation at S1177, eNOS associated with heat shock protein 90 (HSP90), NO production and increased eNOS phosphorylation at T495 and superoxide generation. Simvastatin therapy partially reduced HDL inflammation index, improved the capacity of HDL to stimulate eNOS and Akt phosphorylation at S1177, eNOS associated with HSP90, NO production, reduced eNOS phosphorylation at T495 and superoxide generation, and improved endothelium-dependent vasodilation. Our data demonstrated that HDL from VHD patients and cardiac surgery contributed to endothelial dysfunction through uncoupling of eNOS. This deleterious effect can be reversed by simvastatin, which improves the vasoprotective effects of HDL. Targeting HDL may be a therapeutic strategy for maintaining vascular function and improving the outcomes post-cardiac surgery.
Asunto(s)
Enfermedades de las Válvulas Cardíacas/metabolismo , Válvulas Cardíacas/metabolismo , Lipoproteínas HDL/metabolismo , Adulto , Anciano , Animales , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/patología , Estudios de Casos y Controles , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Femenino , Regulación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Enfermedades de las Válvulas Cardíacas/tratamiento farmacológico , Enfermedades de las Válvulas Cardíacas/genética , Enfermedades de las Válvulas Cardíacas/patología , Válvulas Cardíacas/efectos de los fármacos , Válvulas Cardíacas/patología , Humanos , Hipolipemiantes/uso terapéutico , Lipoproteínas HDL/farmacología , Masculino , Ratones , Persona de Mediana Edad , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Simvastatina/uso terapéutico , Superóxidos/antagonistas & inhibidores , Superóxidos/metabolismo , Vasodilatación/efectos de los fármacosRESUMEN
We previously demonstrated that normal high-density lipoprotein (nHDL) can promote angiogenesis, whereas HDL from patients with coronary artery disease (dHDL) is dysfunctional and impairs angiogenesis. Autophagy plays a critical role in angiogenesis, and HDL regulates autophagy. However, it is unclear whether nHDL and dHDL regulate angiogenesis by affecting autophagy. Endothelial cells (ECs) were treated with nHDL and dHDL with or without an autophagy inhibitor. Autophagy, endothelial nitric oxide synthase (eNOS) expression, miRNA expression, nitric oxide (NO) production, superoxide anion (O2â¢-) generation, EC migration, and tube formation were evaluated. nHDL suppressed the expression of miR-181a-5p, which promotes autophagy and the expression of eNOS, resulting in NO production and the inhibition of O2â¢- generation, and ultimately increasing in EC migration and tube formation. dHDL showed opposite effects compared to nHDL and ultimately inhibited EC migration and tube formation. We found that autophagy-related protein 5 (ATG5) was a direct target of miR-181a-5p. ATG5 silencing or miR-181a-5p mimic inhibited nHDL-induced autophagy, eNOS expression, NO production, EC migration, tube formation, and enhanced O2â¢- generation, whereas overexpression of ATG5 or miR-181a-5p inhibitor reversed the above effects of dHDL. ATG5 expression and angiogenesis were decreased in the ischemic lower limbs of hypercholesterolemic low-density lipoprotein receptor null (LDLr-/-) mice when compared to C57BL/6 mice. ATG5 overexpression improved angiogenesis in ischemic hypercholesterolemic LDLr-/- mice. Taken together, nHDL was able to stimulate autophagy by suppressing miR-181a-5p, subsequently increasing eNOS expression, which generated NO and promoted angiogenesis. In contrast, dHDL inhibited angiogenesis, at least partially, by increasing miR-181a-5p expression, which decreased autophagy and eNOS expression, resulting in a decrease in NO production and an increase in O2â¢- generation. Our findings reveal a novel mechanism by which HDL affects angiogenesis by regulating autophagy and provide a therapeutic target for dHDL-impaired angiogenesis.
Asunto(s)
MicroARNs , Humanos , Ratones , Animales , MicroARNs/metabolismo , Células Endoteliales/metabolismo , Angiogénesis , Ratones Endogámicos C57BL , Autofagia/genéticaRESUMEN
Diabetic foot ulcers (DFUs) are a serious vascular disease. Currently, no effective methods are available for treating DFUs. Pro-protein convertase subtilisin/kexin type 9 (PCSK9) regulates lipid levels to promote atherosclerosis. However, the role of PCSK9 in DFUs remains unclear. In this study, we found that the expression of PCSK9 in endothelial cells (ECs) increased significantly under high glucose (HG) stimulation and in diabetic plasma and vessels. Specifically, PCSK9 promotes the E3 ubiquitin-protein ligase NEDD4 binding to vascular endothelial growth factor receptor 2 (VEGFR2), which led to the ubiquitination of VEGFR2, resulting in its degradation and downregulation in ECs. Furthermore, PCSK9 suppresses the expression and activation of AKT, endothelial nitric oxide synthase (eNOS), and ERK1/2, leading to decreased nitric oxide (NO) production and increased superoxide anion (O2._) generation, which impairs vascular endothelial function and angiogenesis. Importantly, using evolocumab to limit the increase in PCSK9 expression blocked the HG-induced inhibition of NO production and the increase in O2._ production, as well as inhibited the phosphorylation and expression of AKT, eNOS, and ERK1/2. Moreover, evolocumab improved vascular endothelial function and angiogenesis, and promoted wound healing in diabetes. Our findings suggest that targeting PCSK9 is a novel therapeutic approach for treating DFUs.
RESUMEN
Cardiopulmonary bypass has been speculated to elicit systemic inflammation to initiate acute lung injury (ALI), including acute respiratory distress syndrome (ARDS), in patients after cardiac surgery. We previously found that post-operative patients showed an increase in endothelial cell-derived extracellular vesicles (eEVs) with components of coagulation and acute inflammatory responses. However, the mechanism underlying the onset of ALI owing to the release of eEVs after cardiopulmonary bypass, remains unclear. Plasma plasminogen-activated inhibitor-1 (PAI-1) and eEV levels were measured in patients with cardiopulmonary bypass. Endothelial cells and mice (C57BL/6, Toll-like receptor 4 knockout (TLR4-/-) and inducible nitric oxide synthase knockout (iNOS-/-)) were challenged with eEVs isolated from PAI-1-stimulated endothelial cells. Plasma PAI-1 and eEVs were remarkably enhanced after cardiopulmonary bypass. Plasma PAI-1 elevation was positively correlated with the increase in eEVs. The increase in plasma PAI-1 and eEV levels was associated with post-operative ARDS. The eEVs derived from PAI-1-stimulated endothelial cells could recognize TLR4 to stimulate a downstream signaling cascade identified as the Janus kinase 2/3 (JAK2/3)-signal transducer and activator of transcription 3 (STAT3)-interferon regulatory factor 1 (IRF-1) pathway, along with iNOS induction, and cytokine/chemokine production in vascular endothelial cells and C57BL/6 mice, ultimately contributing to ALI. ALI could be attenuated by JAK2/3 or STAT3 inhibitors (AG490 or S3I-201, respectively), and was relieved in TLR4-/- and iNOS-/- mice. eEVs activate the TLR4/JAK3/STAT3/IRF-1 signaling pathway to induce ALI/ARDS by delivering follistatin-like protein 1 (FSTL1), and FSTL1 knockdown in eEVs alleviates eEV-induced ALI/ARDS. Our data thus demonstrate that cardiopulmonary bypass may increase plasma PAI-1 levels to induce FSTL1-enriched eEVs, which target the TLR4-mediated JAK2/3/STAT3/IRF-1 signaling cascade and form a positive feedback loop, leading to ALI/ARDS after cardiac surgery. Our findings provide new insight into the molecular mechanisms and therapeutic targets for ALI/ARDS after cardiac surgery.
Asunto(s)
Lesión Pulmonar Aguda , Vesículas Extracelulares , Proteínas Relacionadas con la Folistatina , Síndrome de Dificultad Respiratoria , Animales , Humanos , Ratones , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/metabolismo , Células Endoteliales/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas Relacionadas con la Folistatina/metabolismo , Proteínas Relacionadas con la Folistatina/uso terapéutico , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Pulmón/metabolismo , Ratones Endogámicos C57BL , Inhibidor 1 de Activador Plasminogénico/metabolismo , Inhibidor 1 de Activador Plasminogénico/uso terapéutico , Síndrome de Dificultad Respiratoria/etiología , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/uso terapéuticoRESUMEN
The mechanism by which aging induces aortic aneurysm and dissection (AAD) remains unclear. A total of 430 participants were recruited for the screening of differentially expressed plasma microRNAs (miRNAs). We found that miR-1204 is significantly increased in both the plasma and aorta of elder patients with AAD and is positively correlated with age. Cell senescence induces the expression of miR-1204 through p53 interaction with plasmacytoma variant translocation 1, and miR-1204 induces vascular smooth muscle cell (VSMC) senescence to form a positive feedback loop. Furthermore, miR-1204 aggravates angiotensin II-induced AAD formation, and inhibition of miR-1204 attenuates ß-aminopropionitrile monofumarate-induced AAD development in mice. Mechanistically, miR-1204 directly targets myosin light chain kinase (MYLK), leading to the acquisition of a senescence-associated secretory phenotype (SASP) by VSMCs and loss of their contractile phenotype. MYLK overexpression reverses miR-1204-induced VSMC senescence, SASP and contractile phenotypic changes, and the decrease of transforming growth factor-ß signaling pathway. Our findings suggest that aging aggravates AAD via the miR-1204-MYLK signaling axis.
Asunto(s)
Envejecimiento , Aneurisma de la Aorta , Disección Aórtica , Senescencia Celular , MicroARNs , Músculo Liso Vascular , Quinasa de Cadena Ligera de Miosina , Transducción de Señal , Animales , Femenino , Humanos , Masculino , Ratones , Envejecimiento/genética , Envejecimiento/metabolismo , Angiotensina II/metabolismo , Aneurisma de la Aorta/metabolismo , Aneurisma de la Aorta/genética , Aneurisma de la Aorta/patología , Disección Aórtica/metabolismo , Disección Aórtica/genética , Disección Aórtica/patología , Proteínas de Unión al Calcio , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/genética , Factor de Crecimiento Transformador beta/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Mitral valve endothelial cells are important for maintaining lifelong mitral valve integrity and function. Plasma endothelial microparticles (EMPs) increased in various pathological conditions related to activation of endothelial cells. However, whether EMPs will increase in mitral valve disease and their relationship remains unclear. Here, 81 patients with mitral valve disease and 45 healthy subjects were analyzed for the generation of EMPs by flow cytometry. Human mitral valve endothelial cells (HMVECs) were treated with EMPs. The phosphorylation of Akt and endothelial nitric oxide synthase (eNOS), the association of eNOS and heat shock protein 90 (HSP90), and the generation of nitric oxide (NO) and superoxide anion (O(2)(â-)) were measured. EMPs were increased significantly in patients with mitral valve disease compared with those in healthy subjects. EMPs were negatively correlated with mitral valve area in patients with isolated mitral stenosis. EMPs were significantly higher in the group with severe mitral regurgitation than those in the group with mild and moderate mitral regurgitation. Furthermore, EMPs were decreased dramatically in both Akt and eNOS phosphorylation and the association of HSP90 with eNOS in HMVECs. EMPs decreased NO production but increased O(2)(â-) generation in HMVECs. Our data demonstrated that EMPs were significantly increased in patients with mitral valve disease. The increase of EMPs can in turn impair HMVEC function by inhibiting the Akt/eNOS-HSP90 signaling pathway. These findings suggest that EMPs may be a therapeutic target for mitral valve disease.
Asunto(s)
Micropartículas Derivadas de Células/metabolismo , Células Endoteliales , Insuficiencia de la Válvula Mitral/metabolismo , Estenosis de la Válvula Mitral/metabolismo , Válvula Mitral , Óxido Nítrico Sintasa de Tipo III/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Adulto , Estudios de Casos y Controles , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Femenino , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Válvula Mitral/citología , Válvula Mitral/metabolismo , Óxido Nítrico/metabolismo , Fosforilación , Transducción de Señal , Superóxidos/metabolismoRESUMEN
Normal high-density lipoprotein (nHDL) can induce angiogenesis in healthy individuals. However, HDL from patients with coronary artery disease undergoes various modifications, becomes dysfunctional (dHDL), and loses its ability to promote angiogenesis. Here, we identified a long non-coding RNA, HDRACA, that is involved in the regulation of angiogenesis by HDL. In this study, we showed that nHDL downregulates the expression of HDRACA in endothelial cells by activating WW domain-containing E3 ubiquitin protein ligase 2, which catalyzes the ubiquitination and subsequent degradation of its transcription factor, Kruppel-like factor 5, via sphingosine 1-phosphate (S1P) receptor 1. In contrast, dHDL with lower levels of S1P than nHDL were much less effective in decreasing the expression of HDRACA. HDRACA was able to bind to Ras-interacting protein 1 (RAIN) to hinder the interaction between RAIN and vigilin, which led to an increase in the binding between the vigilin protein and proliferating cell nuclear antigen (PCNA) mRNA, resulting in a decrease in the expression of PCNA and inhibition of angiogenesis. The expression of human HDRACA in a hindlimb ischemia mouse model inhibited the recovery of angiogenesis. Taken together, these findings suggest that HDRACA is involved in the HDL regulation of angiogenesis, which nHDL inhibits the expression of HDRACA to induce angiogenesis, and that dHDL is much less effective in inhibiting HDRACA expression, which provides an explanation for the decreased ability of dHDL to stimulate angiogenesis.
Asunto(s)
Lipoproteínas HDL , ARN Largo no Codificante , Ratones , Animales , Humanos , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Antígeno Nuclear de Célula en Proliferación , ARN Largo no Codificante/genética , Células Endoteliales/metabolismo , Neovascularización Fisiológica/genéticaRESUMEN
An apolipoprotein A-I mimetic peptide, D-4F, has been shown to improve vasodilation and inhibit atherosclerosis in hypercholesterolemic low-density lipoprotein receptor-null (LDLr(-/-)) mice. To study the metabolic variations of D-4F ininhibiting atherosclerosis, metabonomics, a novel system biological strategy to investigate the pathogenesis, was developed. Female LDLr(-/-) mice were fed a Western diet and injected with or without D-4F intraperitoneally. Atherosclerotic lesion formation was measured, whereas plasma metabolic profiling was obtained on the basis of ultra-high-performance liquid chromatography in tandem with time-of-flight mass spectrometry operating in both positive and negative ion modes. Data were processed by multivariate statistical analysis to graphically demonstrate metabolic changes. The partial least-squares discriminate analysis model was validated with cross-validation and permutation tests to ensure the model's reliability. D-4F significantly inhibited the formation of atherosclerosis in a time-dependent manner. The metabolic profiling was altered dramatically in hypercholesterolemic LDLr(-/-) mice, and a significant metabolic profiling change in response to D-4F treatment was observed in both positive and negative ion modes. Thirty-six significantly changed metabolites were identified as potential biomarkers. A series of phospholipid metabolites, including lysophosphatidylcholine (LysoPC), lysophosphatidylethanolamine (LysoPE), phosphatidylcholine (PC), phatidylethanolamine (PE), sphingomyelin (SM), and diacylglycerol (DG), particularly the long-chain LysoPC, was elevated dramatically in hypercholesterolemic LDLr(-/-) mice but reduced by D-4F in a time-dependent manner. Quantitative analysis of LysoPC, LysoPE, PC, and DG using HPLC was chosen to validate the variation of these potential biomarkers, and the results were consistent with the metabonomics findings. Our findings demonstrated that D-4F may inhibit atherosclerosis by regulating phospholipid metabolites specifically by decreasing plasma long-chain LysoPC.