RESUMEN
Clinical data implicate fluctuations of high levels of plasma glucose in cardiovascular diseases. Endothelial cells (EC) are the first cells of the vessel wall exposed to them. Our aim was to evaluate the effects of oscillating glucose (OG) on EC function and to decipher new molecular mechanisms involved. Cultured human ECs (EA.hy926 line and primary cells) were exposed to OG (5/25 mM alternatively at 3 h), constant HG (25 mM) or physiological concentration (5 mM, NG) for 72 h. Markers of inflammation (Ninj-1, MCP-1, RAGE, TNFR1, NF-kB, and p38 MAPK), oxidative stress (ROS, VPO1, and HO-1), and transendothelial transport proteins (SR-BI, caveolin-1, and VAMP-3) were assessed. Inhibitors of ROS (NAC), NF-kB (Bay 11-7085), and Ninj-1 silencing were used to identify the mechanisms of OG-induced EC dysfunction. The results revealed that OG determined an increased expression of Ninj-1, MCP-1, RAGE, TNFR1, SR-B1, and VAMP-3 andstimulated monocyte adhesion. All of these effects were induced bymechanisms involving ROS production or NF-kB activation. NINJ-1 silencing inhibited the upregulation of caveolin-1 and VAMP-3 induced by OG in EC. In conclusion, OG induces increased inflammatory stress, ROS production, and NF-kB activation and stimulates transendothelial transport. To this end, we propose a novel mechanism linking Ninj-1 up-regulation to increased expression of transendothelial transport proteins.
Asunto(s)
Proteínas Portadoras , Células Endoteliales , Humanos , Células Endoteliales/metabolismo , Regulación hacia Arriba , Proteínas Portadoras/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/farmacología , Caveolina 1/genética , Caveolina 1/metabolismo , Glucosa/farmacología , Glucosa/metabolismo , FN-kappa B/metabolismo , Estrés OxidativoRESUMEN
Myocardial infarction is one of the leading causes of death worldwide, despite numerous efforts to find efficient prognostic biomarkers and treatment targets. In the present study, we aimed to assess the potential of six microRNAs known to be involved in cardiovascular diseases, cell-free DNA (cfDNA), and mitochondrial DNA (mtDNA) circulating in plasma to be used as prognostic tools for the occurrence of unfavorable outcomes such as major adverse cardiovascular events (MACE) after acute ST-segment elevation myocardial infarction (STEMI). Fifty STEMI patients were enrolled and monitored for 6 months for the occurrence of MACE. Plasma was collected at three time points: upon admission to hospital (T0), at discharge from hospital (T1), and 6 months post-STEMI (T6). Plasma levels of miR-223-3p, miR-142-3p, miR-155-5p, miR-486-5p, miR-125a-5p, and miR-146a-5p, as well as of cfDNA and mtDNA, were measured by RT-qPCR. Results showed that the levels of all measured miRNAs, as well as of cfDNA and mtDNA, were the most increased at T1, compared to the other two time points. In the plasma of STEMI patients with MACE compared to those without MACE, we determined increased levels of miRNAs, cfDNA, and mtDNA at T1. Hence, we used the levels of all measured parameters at T1 for further statistical analysis. Statistical analysis demonstrated that all six miRNAs and cfDNA plus mtDNA levels, respectively, were associated with MACE. The minimal statistical model that could predict MACE in STEMI patients was the combination of mtDNA and miR-142-3p levels, as evidenced by ROC analysis (AUC = 0.97, p < 0.001). In conclusion, the increased plasma levels of mtDNA, along with miR-142-3p, could be used to predict unfavorable outcomes in STEMI patients.
Asunto(s)
Ácidos Nucleicos Libres de Células , MicroARNs , Infarto del Miocardio , Infarto del Miocardio con Elevación del ST , Biomarcadores , ADN Mitocondrial/genética , Humanos , MicroARNs/genética , Infarto del Miocardio/genéticaRESUMEN
Peripheral artery disease (PAD) is an atherosclerotic disorder affecting arteries of the lower limbs, the major risk factors including dyslipidemia and diabetes mellitus (DM). We aimed to identify alterations of the proteins in high-density lipoproteins (HDL) associated with HDL dysfunction in PAD patients. HDL2 and HDL3 were isolated from plasma of PAD patients with/without DM (PAD-DM/PAD) and healthy subjects (N). Apolipoprotein AI (ApoAI), ApoAII, ApoCIII, clusterin (CLU), paraoxonase 1 (PON1), myeloperoxidase (MPO), and ceruloplasmin (CP) were measured in HDL2 /HDL3 and plasma. Oxidation and glycation of the analyzed proteins were assessed as malondialdehyde-protein adducts (MDA) and advanced glycation end-products (AGE), respectively. The anti-inflammatory effect of HDL3 was estimated as its potential to reduce monocyte adhesion to tumor necrosis factor α-activated endothelial cells. We show that in PAD patients compared to N subjects: (i) HDL2 presented increased levels of MDA-PON1, AGE-PON1, AGE-ApoAI, ApoAII, ApoCIII, and CP levels, and decreased PON1 levels; (ii) HDL3 had increased levels of MDA- and AGE-CLU and -ApoAI, MDA-PON1, ApoCIII, CLU, MPO, CP, and reduced PON1 levels. All these alterations were exacerbated by DM. These changes were more pronounced in HDL3 , which had reduced anti-inflammatory potential in PAD and became pro-inflammatory in PAD-DM. In PAD patients' plasma, CLU levels and MPO specific activity increased, while PON1 specific activity decreased. In conclusion, HDL function is altered in PAD patients due to multiple modifications of associated proteins that are aggravated by DM. Plasma CLU, MPO, and PON1 could constitute indicators of HDL dysfunction and contribute to risk stratification in PAD patients.
Asunto(s)
Arildialquilfosfatasa , Clusterina , Diabetes Mellitus Tipo 2 , Enfermedad Arterial Periférica , Peroxidasa , Arildialquilfosfatasa/genética , Arildialquilfosfatasa/metabolismo , Clusterina/genética , Clusterina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliales/metabolismo , Humanos , Lipoproteínas HDL , Peroxidasa/genética , Peroxidasa/metabolismoRESUMEN
Atherosclerosis is the main cause of cardiovascular diseases with high prevalence worldwide. A promising therapeutic strategy to reverse atherosclerotic process is to improve the athero-protective potential of high-density lipoproteins (HDL). Since the small intestine is a source of HDL, we aimed to activate transcription of the endogenous HDL major proteins, apolipoprotein AI (ApoAI) and paraoxonase 1 (PON1), in enterocytes, and to evaluate their potential to correct the pro-inflammatory status of endothelial cells (EC). Caco-2 enterocytes were transfected with CRISPR activation plasmids targeting ApoAI or PON1, and their gene and protein expression were measured in cells and conditioned medium (CM). ATP binding cassette A1 and G8 transporters (ABCA1, ABCG8), scavenger receptor BI (SR-BI), and transcription regulators peroxisome proliferator-activated receptor γ (PPARγ), liver X receptors (LXRs), and sirtuin-1 (SIRT1) were assessed. Anti-inflammatory effects of CM from transfected enterocytes were estimated through its ability to inhibit tumor necrosis factor α (TNFα) activation of EC. Transcriptional activation of ApoAI or PON1 in enterocytes induces: (i) increase of their gene and protein expression, and secretion in CM; (ii) stimulation of ABCA1/G8 and SR-BI; (iii) upregulation of PPARγ, LXRs, and SIRT1. CM from transfected enterocytes attenuated the TNFα-induced inflammatory and oxidative stress in EC, by decreasing TNF receptor 1, monocyte chemoattractant protein-1, and p22phox. In conclusion, transcriptional activation of endogenous ApoAI or PON1 in enterocytes by CRISPR/dCas9 system is a realistic approach to stimulate biogenesis and function of major HDL proteins which can regulate cholesterol efflux transporters and reduce the inflammatory stress in activated EC.
Asunto(s)
Apolipoproteína A-I/genética , Arildialquilfosfatasa/genética , Células Endoteliales/citología , Enterocitos/citología , Apolipoproteína A-I/metabolismo , Arildialquilfosfatasa/metabolismo , Sistemas CRISPR-Cas , Células CACO-2 , Medios de Cultivo Condicionados/química , Células Endoteliales/metabolismo , Enterocitos/metabolismo , Regulación de la Expresión Génica , Humanos , Lipoproteínas HDL/metabolismo , Estrés Oxidativo , Activación Transcripcional , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Uptake of modified lipoproteins by macrophages turns them into foam cells, the hallmark of the atherosclerotic plaque. The initiation and progression of atherosclerosis have been associated with mitochondrial dysfunction. It is known that aggregated low-density lipoproteins (agLDL) induce massive cholesterol accumulation in macrophages in contrast with native LDL (nLDL) and oxidized LDL (oxLDL). In the present study we aimed to assess the effect of agLDL on the mitochondria and ER function in macrophage-derived foam cells, in an attempt to estimate the potential of these cells, known constituents of early fatty streaks, to generate atheroma in the absence of oxidative stress. Results show that agLDL induce excessive accumulation of free (FC) and esterified cholesterol in THP-1 macrophages and determine mitochondrial dysfunction expressed as decreased mitochondrial membrane potential and diminished intracellular ATP levels, without generating mitochondrial reactive oxygen species (ROS) production. AgLDL did not stimulate intracellular ROS (superoxide anion or hydrogen peroxide) production, and did not trigger endoplasmic reticulum stress (ERS) or apoptosis. In contrast to agLDL, oxLDL did not modify FC levels, but stimulated the accumulation of 7-ketocholesterol in the cells, generating oxidative stress which is associated with an increased mitochondrial dysfunction, ERS and apoptosis. Taken together, our results reveal that agLDL induce foam cells formation and mild mitochondrial dysfunction in human macrophages without triggering oxidative or ERS. These data could partially explain the early formation of fatty streaks in the intima of human arteries by interaction of monocyte-derived macrophages with non-oxidatively aggregated LDL generating foam cells, which cannot evolve into atherosclerotic plaques in the absence of the oxidative stress.
Asunto(s)
Estrés del Retículo Endoplásmico/efectos de los fármacos , Células Espumosas/efectos de los fármacos , Lipoproteínas LDL/química , Lipoproteínas LDL/farmacología , Mitocondrias/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Agregado de Proteínas , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Colesterol/metabolismo , Células Espumosas/citología , Células Espumosas/metabolismo , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Mitocondrias/metabolismoRESUMEN
AIMS: The objectives of the present study were to investigate the mechanisms of Ninj-1 regulation in TNFα-activated human endothelial cells (HEC), and to test if Amlodipine (AML) ameliorates the inflammatory stress by decreasing Ninj-1 expression. MAIN METHODS: TNFα-activated HEC with/without AML (0.1 µM and 1 µM) were used. TNFα-receptor 1 (TNFR1) was silenced and inhibitors for oxidative stress (N-acetyl cysteine), endoplasmic reticulum stress (salubrinal, 4-phenyl butyric acid), or NF-kB (Bay 11-7085) and p38 MAPK (SB203580) were used. Levels of Ninj-1, TNFR1, monocyte adhesion, endoplasmic reticulum stress (ERS) sensors, NADPH oxidase- and mitochondria-derived oxidative species were evaluated. KEY FINDINGS: The novel findings that we report here are: (i) silencing the endothelial TNFR1 leads to decreased Ninj-1 expression and diminished monocyte adhesion; (ii) increased oxidative stress, ERS and NF-kB activation enhance Ninj-1 expression and monocyte adhesion; (iii) up-regulation of endothelial Ninj-1 expression stimulates monocytes adhesion to TNFα - activated HEC; (iv) AML diminishes monocyte adhesion by reducing Ninj-1 expression through mechanisms involving the decrease of NADPH oxidase and mitochondria-dependent oxidative stress, ERS and NF-kB. In addition, AML alleviates apoptosis by reducing the pro-apoptotic CHOP expression and re-establishing the mitochondrial transmembrane potential. SIGNIFICANCE: The results of the present study suggest that Ninj-1 and the proteins involved in its regulation can be considered therapeutic targets for the alleviation of inflammation- dependent disorders. In addition, we demonstrate that some of the benefic effects of AML can be achieved through regulation of Ninj-1.
Asunto(s)
Amlodipino/farmacología , Moléculas de Adhesión Celular Neuronal/fisiología , Adhesión Celular/fisiología , Monocitos/citología , Factores de Crecimiento Nervioso/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Regulación hacia Arriba , Vasodilatadores/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Silenciador del Gen , Células Endoteliales de la Vena Umbilical Humana , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , NADPH Oxidasas/metabolismo , FN-kappa B/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/genéticaRESUMEN
There is a stringent need to find means for risk stratification of coronary artery diseases (CAD) patients. We aimed at identifying alterations of plasma high-density lipoproteins (HDL) components and their validation as dysfunctional HDL that could discriminate between acute coronary syndrome (ACS) and stable angina (SA) patients. HDL2 and HDL3 were isolated from CAD patients' plasma and healthy subjects. ApolipoproteinAI (apoAI), apoAII, apoCIII, malondialdehyde (MDA), myeloperoxidase (MPO), ceruloplasmin and paraoxonase1 (PON1) were assessed. The anti-inflammatory potential of HDL subfractions was tested by evaluating the secreted inflammatory molecules of tumor necrosis factor α-activated endothelial cells (EC) upon co-incubation with HDL2 or HDL3. We found in ACS versus SA patients: 40% increased MPO, MDA, apoCIII in HDL2 and HDL3, 35% augmented apoAII in HDL2, and in HDL3 increased ceruloplasmin, decreased apoAII (40%) and PON1 protein and activity (15% and 25%). Co-incubation of activated EC with HDL2 or HDL3 from CAD patients induced significantly increased levels of secreted inflammatory molecules, 15-20% more for ACS versus SA. In conclusion, the assessed panel of markers correlates with the reduced anti-inflammatory potential of HDL subfractions isolated from ACS and SA patients (mostly for HDL3 from ACS) and can discriminate between these two groups of CAD patients.
Asunto(s)
Síndrome Coronario Agudo/sangre , Síndrome Coronario Agudo/diagnóstico , Antiinflamatorios/sangre , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/diagnóstico , Lipoproteínas HDL/sangre , Síndrome Coronario Agudo/terapia , Adulto , Biomarcadores , Estudios de Casos y Controles , Enfermedad de la Arteria Coronaria/terapia , Diagnóstico Diferencial , Células Endoteliales/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Type 2 diabetes mellitus is a worldwide epidemic and its atherosclerotic complications determine the high morbidity and mortality of diabetic patients. Caffeic acid (CAF), a phenolic acid present in normal diets, is known for its antioxidant properties. The aim of this study was to investigate CAF's anti-inflammatory properties and its mechanism of action, using cultured human endothelial cells (HEC) incubated with glycated low-density lipoproteins (gLDL). Levels of the receptor for advanced glycation end-products (RAGE), inflammatory stress markers (C reactive protein, CRP; vascular cell adhesion molecule-1, VCAM-1; monocyte chemoattractant protein-1, MCP-1), and oxidative stress and endoplasmic reticulum stress (ERS) markers were evaluated in gLDL-exposed HEC, in the presence/absence of CAF. RAGE silencing or blocking, specific inhibitors for oxidative stress (apocynin, N-acetyl-cysteine), and ERS (salubrinal) were used. The results showed that: (i) gLDL induced CRP synthesis and secretion through mechanisms involving NADPH oxidase-dependent oxidative stress and ERS in HEC; (ii) gLDL-RAGE interaction, oxidative stress, and ERS stimulated the secretion of VCAM-1 and MCP-1 in HEC; and (iii) CAF reduced the secretion of CRP, VCAM-1, and MCP-1 in gLDL-exposed HEC by inhibiting RAGE expression, oxidative stress, and ERS. In conclusion, CAF might be a promising alternative to ameliorate a wide spectrum of disorders due to its complex mechanisms of action resulting in anti-inflammatory and antioxidative properties. © 2017 BioFactors, 43(5):685-697, 2017.
Asunto(s)
Antioxidantes/farmacología , Ácidos Cafeicos/farmacología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Proteína C-Reactiva/genética , Diabetes Mellitus Tipo 2/patología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Productos Finales de Glicación Avanzada/genética , Humanos , Inflamación/metabolismo , Inflamación/patología , Lipoproteínas LDL/farmacología , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Receptor para Productos Finales de Glicación Avanzada/genética , Receptores CCR2/genética , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
Oxidatively modified low-density lipoproteins (oxLDL) alter the proper function of the endoplasmic reticulum (ER), inducing ER stress (ERS), which consequently activates inflammatory pathways in macrophages. Matrix metalloproteinase-9 (MMP-9) is the main protease acting on the degradation of the extracellular matrix and the ensuing destabilization of the atherosclerotic plaque. We aimed to investigate whether ERS induced by oxLDL or tunicamycin (TM) in human macrophages is associated with the stimulation of MMP-9 expression and secretion. The results showed that oxLDL induced in THP-1 macrophages: (i) increase of MMP-9 gene expression and its pro-form secretion, (ii) intracellular accumulation of 7-ketocholesterol, (iii) ERS activation (increased eIF2α phosphorylation, XBP1 and CHOP mRNA levels, and Grp78 protein expression), and (iv) oxidative stress (increased levels of reactive oxygen species and NADPH oxidase activity). Incubation of macrophages with ERS inducer, TM determined the secretion of both pro- and active-form of MMP-9 and oxidative stress. Treatment of oxLDL or TM-incubated cells with ERS inhibitor, sodium phenylbutyrate decreased MMP-9 gene expression, secretion, and activity. The inhibitor of NADPH oxidase, apocynin, decreased XBP-1 and CHOP mRNA levels, and MMP-9 gene expression and secretion in oxLDL-exposed cells. In conclusion, oxLDL stimulate MMP-9 expression and secretion in human macrophages by mechanisms involving ERS. J. Cell. Biochem. 118: 661-669, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Acetofenonas/farmacología , Aterosclerosis/etiología , Aterosclerosis/genética , Aterosclerosis/metabolismo , Línea Celular , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Humanos , Cetocolesteroles/metabolismo , Lipoproteínas LDL/toxicidad , Macrófagos/efectos de los fármacos , NADPH Oxidasas/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Tunicamicina/toxicidadRESUMEN
We aimed to determine the levels of microRNAs (miRNAs) in sera and HDL of acute coronary syndrome (ACS) compared to stable angina (SA) patients with/without hyperglycemia, and evaluate comparatively the functional effect of these sera on the processing machinery proteins (Drosha, DGCR8, Dicer) and miRNAs production in human macrophages. MiRNAs levels in sera and HDL from 35 SA and 72 ACS patients and 30 healthy subjects were measured by using microRNA TaqMan assays. MiR-223, miR-92a, miR-486, miR-122, miR-125a and miR-146a levels were higher in the hyperglycemic ACS compared to normoglycemic sera. MiR-223 and miR-486 prevailed in HDL2, while miR-92a predominated in HDL3, all three miRNAs discriminating between ACS and SA patients; their levels were increased in HDL from hyperglycemic ACS patients versus normoglycemic ones. The incubation of human macrophages with sera from ACS and SA patients showed that all patients' sera induced an increase of Drosha, DGCR8 and Dicer expressions and of selected miRNAs levels compared to control sera, the effect being higher in the case of hyperglycemic versus normoglycemic ACS sera. The addition of glucose to SA and ACS sera increased Drosha, DGCR8 and Dicer expression and miRNAs levels in the exposed macrophages. In conclusion, hyperglycemia is associated with increased miR-223, miR-92a, miR-486 levels in HDL, which discriminate between ACS and SA patients. Exposure of human macrophages to ACS compared to SA sera determines the upregulation of Drosha, DGCR8 and Dicer expression and the increase of selected miRNAs production, the effect being augmented by an increased glucose concentration.
Asunto(s)
Síndrome Coronario Agudo/sangre , Angina Estable/sangre , Hiperglucemia/fisiopatología , Lipoproteínas HDL/sangre , Macrófagos/metabolismo , MicroARNs/genética , Síndrome Coronario Agudo/diagnóstico , Síndrome Coronario Agudo/epidemiología , Adulto , Anciano , Angina Estable/diagnóstico , Angina Estable/epidemiología , Estudios de Casos y Controles , Femenino , Humanos , Macrófagos/patología , Masculino , MicroARNs/sangre , Persona de Mediana Edad , Prevalencia , Rumanía/epidemiologíaRESUMEN
Type 2 Diabetes Mellitus is a worldwide epidemic, and its atherosclerotic complications produce morbidity and mortality in affected patients. It is known that the vascular cell adhesion molecule-1 (VCAM-1) levels are increased in the sera of diabetic patients. Our aim was to investigate the impact of the endoplasmic reticulum stress (ERS) in VCAM-1 expression and secretion in human endothelial cells (HEC) exposed to glycated low-density lipoproteins (gLDL). The results showed that 24 h incubation of HEC with gLDL induces (i) stimulation of VCAM-1 expression and secretion, determining increased monocyte adhesion to HEC; (ii) RAGE up-regulation and free cholesterol loading; (iii) ERS activation (increased eIF2α phosphorylation and CHOP mRNA levels, and decreased GRP78 protein expression); and (iv) oxidative stress [increased levels of reactive oxygen species (ROS) and glutamate cysteine ligase catalytic unit gene expression]. Treatment of gLDL-exposed HEC with ERS inhibitors, salubrinal (Sal) and sodium phenylbutyrate (PBA), decreased intracellular ROS. Incubation of gLDL-exposed cells with the anti-oxidant N-acetyl-cysteine (NAC) reduced ERS, revealed by decreased eIF2α phosphorylation and CHOP gene expression and increased GRP78 expression, thus validating the interconnection between ERS and oxidative stress. Sal, PBA, NAC and inhibitors of p38 MAP kinase and NF-kB induced the decrease of VCAM-1 expression and of the ensuing monocyte adhesion induced by gLDL. In conclusion, in HEC, gLDL stimulate the expression of cellular VCAM-1, the secretion of soluble VCAM-1, and the adhesion of monocytes through mechanisms involving p38 MAP kinase and NF-kB signalling pathways activated by RAGE, ERS and oxidative stress, thus contributing to diabetic atherosclerosis.
Asunto(s)
Estrés del Retículo Endoplásmico/efectos de los fármacos , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Lipoproteínas LDL/farmacología , Monocitos/metabolismo , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Adhesión Celular/efectos de los fármacos , Línea Celular , Chaperón BiP del Retículo Endoplásmico , Productos Finales de Glicación Avanzada , Humanos , Lipoproteínas LDL/metabolismoRESUMEN
Small non-coding microRNAs (miRNAs) are implicated in gene regulation, including those involved in coronary artery disease (CAD). Our aim was to identify whether specific serum miRNAs present in the circulating lipoproteins (Lp) are associated with stable or vulnerable CAD patients. A cardiovascular disease-focused screening array was used to assess miRNAs distribution in sera collected from 95 CAD patients: 30 with stable angina (SA), 39 with unstable angina (UA), 26 at one month after myocardial infarction (MI) and 16 healthy control subjects. We found that miR-486, miR-92a and miR-122 presented the highest expression in CAD sera. These miRNA together with miR-125a, miR-146a and miR-33a were further individually analyzed by TaqMan assays. The results were consistent with PCR-array screening data that all of these miRNAs were significantly increased in CAD patients compared to controls. Using a binary logistic regression model, we established that miR-486 and miR-92a in association with some high-density lipoprotein (HDL) components can designate vulnerable CAD patients. Further, all classes of Lp were isolated from sera by density gradient ultracentrifugation. Analysis of the selected miRNAs in each Lp class showed that they were associated mainly with HDL, miR-486 and miR-92a having the highest levels. In UA and MI patients, miR-486 prevailed in HDL2, while miR-92a prevailed in HDL3, and their levels discriminate between stable and vulnerable CAD patients. We identified two circulating miRNAs that in association with some lipid metabolism biomarkers can be used as an additional tool to designate vulnerable CAD patients.
Asunto(s)
Angina Estable/diagnóstico , Angina Inestable/diagnóstico , Enfermedad de la Arteria Coronaria/diagnóstico , Lipoproteínas HDL/sangre , MicroARNs/metabolismo , Infarto del Miocardio/diagnóstico , Adulto , Anciano , Angina Estable/metabolismo , Angina Inestable/metabolismo , Biomarcadores/metabolismo , Enfermedad de la Arteria Coronaria/metabolismo , Diagnóstico Diferencial , Femenino , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , Infarto del Miocardio/metabolismo , Adulto JovenRESUMEN
SCOPE: We aimed at investigating the mechanisms linking hyperlipidemia (HL) with dysfunctional HDL and its main antioxidant enzyme, paraoxonase1 (PON1). PON1 expression and activity was determined in the small intestine, liver, and sera of normal and HL hamsters and associated with the ER stress (ERS) and the development of aortic valve lesions. METHODS AND RESULTS: Male Golden Syrian hamsters were fed standard chow (N) or standard diet with 3% cholesterol and 15% butter for 16 weeks. All hamsters on fat diet developed HL, 50% also hyperglycemia (HLHG) and a fourfold increased homeostasis model assessment of insuline resistance. PON1 expression was reduced in the small intestine and liver (N > HL > HLHG) along with the increased extent of ERS, oxidized lipids, and decreased expression of liver X receptors beta (LXRß) in the small intestine, peroxisome proliferator-activated receptor-γ (PPARγ) in the liver, and of the glucose transporter 4 in the myocardium. Serum PON1 levels decreased along with the increase of oxidized LDL and lesion areas of the aortic valves (N > HL > HLHG). CONCLUSION: The fat diet activates the ERS and oxidative stress, decreases LXRß, PPARγ, and PON1 in the small intestine, liver, and sera of all HL animals, in parallel with the appearance of atherosclerotic lesions in the aortic valves.
Asunto(s)
Arildialquilfosfatasa/metabolismo , Estrés del Retículo Endoplásmico , Hiperlipidemias/metabolismo , Intestino Delgado/metabolismo , Lipoproteínas HDL/fisiología , Hígado/metabolismo , Animales , Arildialquilfosfatasa/genética , Cricetinae , Dieta Alta en Grasa , Transportador de Glucosa de Tipo 2/análisis , Masculino , Mesocricetus , Miocardio/metabolismoRESUMEN
MicroRNAs (miRNAs) are small non-coding RNA sequences that regulate gene expression post-transcriptionally by translation inhibition or mRNA degradation. The aim of the present study was to analyze serum miRNAs modulated by hyperlipidemia and/or hyperglycemia and to correlate them with biochemical parameters within lipid metabolism. Five selected circulating miRNAs (miR-125a-5p, miR-146a, miR-10a, miR-21 and miR-33a) were individually analyzed by TaqMan miRNA assays along with lipid and inflammation parameters in sera from 20 hyperlipidemic (HL) and/or hyperglycemic (HG) patients, and compared with data from five normolipidemic/normoglycemic subjects. Results showed: (1) the levels of all the analyzed circulating miRNA were increased in HL sera and correlated positively with sera's lipid and inflammatory parameters; (2) circulating miR-125a-5p and miR-146a levels were increased in HG and/or HL sera; (3) all selected miRNAs were detected in α-lipoprotein fraction from sera, and miR-33a was also present in ß-lipoprotein fraction; (4) miRNA concentrations were increased in the α-lipoprotein fraction from HL sera. These data show a statistically significant correlation of the analyzed miRNA with increased lipids, specifically with α- and ß-lipoproteins, and CRP and IL-1ß levels in HL and/or HG sera, suggesting a contribution of these miRNAs to the atherosclerotic process.
Asunto(s)
Hiperglucemia/sangre , Hiperlipidemias/sangre , MicroARNs/sangre , Adulto , Anciano , Apolipoproteína B-100/sangre , Apolipoproteínas E/sangre , Glucemia/metabolismo , Proteína C-Reactiva/metabolismo , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Femenino , Humanos , Interleucina-1beta/sangre , Modelos Lineales , Masculino , Persona de Mediana Edad , Triglicéridos/sangreRESUMEN
The role of HDL in the modulation of endoplasmic reticulum (ER) stress in macrophage-derived foam cells is not completely understood. Therefore, we aimed to investigate whether HDL may inhibit ER stress in correlation with the secretion of apoE and CETP from lipid-loaded macrophages. To this purpose, THP-1 macrophages were loaded with lipids by incubation with human oxidized LDL (oxLDL) and then exposed to human HDL3. ER stress signaling markers, protein kinase/Jun-amino-terminal kinase (SAPK/JNK p54/p46) and eukaryotic initiation factor-2α (eIF2α), as well as the secreted apoE and CETP, were evaluated by immunoblot analysis. Out of the many different bioactive lipids of oxLDL, we tested the effect of 9-hydroxy-octadecadienoic acid (9-HODE) and 4-hydroxynonenal (4-HNE) on ER stress. Tunicamycin was used as positive control for ER stress induction. Results showed that oxLDL, 9-HODE and 4-HNE induce ER stress in human macrophages by activation of eIF-2α and SAPK/JNK (p54/p46) signaling pathways. OxLDL stimulated apoE and CETP secretion, while tunicamycin determined a reduction of the secreted apoE and CETP, both in control and lipid-loaded macrophages. The addition of HDL3 to the culture medium of tunicamycin-treated cells induced: (i) the reduction of ER stress, expressed as decreased levels of eIF-2α and SAPK/JNK, and (ii) a partial recovery of the secreted apoE and CETP levels in lipid-loaded macrophages. These data suggest a new mechanism by which HDL3 diminish ER stress and stimulate cholesterol efflux from lipid-loaded macrophages.
Asunto(s)
Apolipoproteínas E/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Regulación hacia Abajo/fisiología , Estrés del Retículo Endoplásmico/fisiología , Metabolismo de los Lípidos/fisiología , Lipoproteínas HDL3/metabolismo , Lipoproteínas HDL3/fisiología , Macrófagos/metabolismo , Aldehídos/farmacología , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Humanos , Ácidos Linoleicos Conjugados/fisiología , Lípidos/administración & dosificación , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Cholesteryl ester transfer protein (CETP) and apolipoprotein E (apoE) are secreted by macrophages. Apolipoprotein A-I (apoA-I) is a potent inducer of apoE secretion from lipid-loaded macrophages, but its effect on CETP is not known. We aimed to identify the signaling pathways involved in apoA-I and HDL-mediated regulation of CETP and apoE secretion from lipid-loaded macrophages. THP-1 macrophages were loaded with lipids by incubation with human copper-oxidized LDL. The cells were subsequently exposed to human purified apoA-I or HDL(3) with/without inhibitors of NF-κB (TPCK) or PKA (H89). CETP and apoE in the cultured cells and media were quantified by real-time PCR and Western blot. Results showed that in lipid-loaded macrophages: (i) CETP and apoE gene expression and secretion were increased in the presence of apoA-I, and further increased by inhibition of NF-kB with TPCK; (ii) CETP and apoE gene expression and secretion were reduced by the inhibition of PKA with H89; (iii) PKA-gamma subunit was activated by oxidized LDL and moreover by apoA-I. We also showed that: (i) siRNA-mediated CETP gene silencing diminished apoE secretion from both non-loaded and lipid-loaded macrophages; (ii) addition of apoA-I partially restored apoE secretion from lipid-loaded macrophages with the silenced CETP gene. In conclusion, our data suggest a new mechanism by which apoA-I stimulates CETP secretion, in addition to apoE, from lipid loaded macrophages, a process involving NF-κB inhibition and/or PKA pathway activation.
Asunto(s)
Apolipoproteína A-I/metabolismo , Apolipoproteínas E/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , HDL-Colesterol/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Macrófagos/metabolismo , FN-kappa B/metabolismo , Apolipoproteína A-I/farmacología , Proteínas de Transferencia de Ésteres de Colesterol/genética , HDL-Colesterol/farmacología , Expresión Génica , Silenciador del Gen , Humanos , Metabolismo de los Lípidos , Macrófagos/efectos de los fármacos , ARN Interferente Pequeño/genética , Transducción de SeñalRESUMEN
Amlodipine, alone or in combination with other drugs, was successfully used to treat hypertension. Our aim was to evaluate the potential of amlodipine (Am) to restore endothelial dysfunction induced by irreversibly glycated low density lipoproteins (AGE-LDL), an in vitro model mimicking the diabetic condition. Human endothelial cells (HEC) from EA.hy926 line were incubated with AGE-LDL in the presence/absence of Am and the oxidative and inflammatory status of the cells was evaluated along with the p38 MAPK and NF-κB signalling pathways. The cellular NADPH activity, 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine levels in the culture medium and the adhesion of human monocytes to HEC were measured by chemiluminescence, UHPLC, Western Blot and spectrofluorimetric techniques. The gene expression of NADPH subunits (p22(phox), NOX4), eNOS and inflammatory molecules (MCP-1, VCAM-1) were determined by Real Time PCR, while the protein expression of p22(phox), MCP-1, iNOS, phospho-p38 MAPK and phospho-p65 NF-κB subunit were measured by Western Blot. Results showed that in HEC incubated with AGE-LDL, Am led to: (i) decrease of the oxidative stress: by reducing p22(phox), NOX4, iNOS expression, NADPH oxidase activity, 4-HNE and 3-nitrotyrosine levels; (ii) decrease of the inflammatory stress: by the reduction of MCP-1 and VCAM-1 expression, as well as of the number of monocytes adhered to HEC; (iii) inhibition of ROS-sensitive signalling pathways: by decreasing phosphorylation of p38 MAPK and p65 NF-κB subunits. In conclusion, the reported data demonstrate that amlodipine may improve endothelial dysfunction in diabetes through anti-oxidant and anti-inflammatory mechanisms.