RESUMEN
Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have proven to delay diabetic kidney disease (DKD) progression on top of the standard of care with the renin-angiotensin system (RAS) blockade. The molecular mechanisms underlying the synergistic effect of SGLT2i and RAS blockers is poorly understood. We gave a SGLT2i (empagliflozin), an angiotensin-converting enzyme inhibitor (ramipril), or a combination of both drugs for 8 weeks to diabetic (db/db) mice. Vehicle-treated db/db and db/m mice were used as controls. At the end of the experiment, mice were killed, and the kidneys were saved to perform a differential high-throughput proteomic analysis by mass spectrometry using isobaric tandem mass tags (TMT labeling) that allow relative quantification of the identified proteins. The differential proteomic analysis revealed 203 proteins differentially expressed in one or more experimental groups (false discovery rate < 0.05 and Log2 fold change ≥ ±1). Fourteen were differentially expressed in the kidneys from the db/db mice treated with empagliflozin with ramipril. Among them, MAP17 was up-regulated. These findings were subsequently validated by Western blot. The combined therapy of empagliflozin and ramipril up-regulated MAP17 in the kidney of a diabetic mice model. MAP17 is a major scaffolding protein of the proximal tubular cells that places transporters together, namely SGLT2 and NHE3. Our results suggest that SGLT2i on top of RAS blockade may protect the kidney by boosting the inactivation of NHE3 via the up-regulation of key scaffolder proteins such as MAP17.
Asunto(s)
Diabetes Mellitus Experimental , Nefropatías Diabéticas , Ratones , Animales , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/metabolismo , Sistema Renina-Angiotensina , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Ramipril/farmacología , Ramipril/uso terapéutico , Proteómica , Intercambiador 3 de Sodio-Hidrógeno/metabolismoRESUMEN
Treatments with sodium-glucose 2 cotransporter inhibitors (SGLT2i) or endothelin receptor antagonists (ERA) have shown cardiorenal protective effects. The present study aimed to evaluate the cardiorenal beneficial effects of the combination of SGLT2i and ERA on top of renin-angiotensin system (RAS) blockade. Type 2 diabetic mice (db/db) were treated with different combinations of an SGLT2i (empagliflozin), an ERA (atrasentan), and an angiotensin-converting enzyme inhibitor (ramipril) for 8 weeks. Vehicle-treated diabetic mice and non-diabetic mice were included as controls. Weight, blood glucose, blood pressure, and kidney and heart function were monitored during the study. Kidneys and heart were collected for histological examination and to study the intrarenal RAS. Treatment with empagliflozin alone or combined significantly decreased blood glucose compared to vehicle-treated db/db. The dual and triple therapies achieved significantly greater reductions in diastolic blood pressure than ramipril alone. Compared to vehicle-treated db/db, empagliflozin combined with ramipril or in triple therapy significantly prevented GFR increase, but only the triple combination exerted greater protection against podocyte loss. In the heart, empagliflozin alone or combined reduced cardiac isovolumetric relaxation time (IVRT) and left atrium (LA) diameter as compared to vehicle-treated db/db. However, only the triple therapy was able to reduce cardiomyocyte area. Importantly, the add-on triple therapy further enhanced the intrarenal ACE2/Ang(1-7)/Mas protective arm of the RAS. These data suggest that triple therapy with empagliflozin, atrasentan and ramipril show synergistic cardiorenal protective effects in a type 2 diabetic mouse model.
Asunto(s)
Diabetes Mellitus Tipo 2 , Sistema Renina-Angiotensina , Ratones , Animales , Transportador 2 de Sodio-Glucosa , Atrasentán/farmacología , Antagonistas de los Receptores de Endotelina/farmacología , Glucemia , Ramipril/farmacología , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Receptores de EndotelinaRESUMEN
IL-17A is overexpressed in the lung during acute neutrophilic inflammation. Acetylcholine (ACh) increases IL-8 and Muc5AC production in airway epithelial cells. We aimed to characterize the involvement of nonneuronal components of cholinergic system on IL-8 and Muc5AC production in bronchial epithelial cells stimulated with IL-17A. Bronchial epithelial cells were stimulated with recombinant human IL-17A (rhIL-17A) to evaluate the ChAT expression, the ACh binding and production, the IL-8 release, and the Muc5AC production. Furthermore, the effectiveness of PD098,059 (inhibitor of MAPKK activation), Bay11-7082 (inhibitor of IkBα phosphorylation), Hemicholinium-3 (HCh-3) (choline uptake blocker), and Tiotropium bromide (Spiriva®) (anticholinergic drug) was tested in our in vitro model. We showed that rhIL-17A increased the expression of ChAT, the levels of ACh binding and production, and the IL-8 and Muc5AC production in stimulated bronchial epithelial cells compared with untreated cells. The pretreatment of the cells with PD098,059 and Bay11-7082 decreased the ChAT expression and the ACh production/binding, while HCh-3 and Tiotropium decreased the IL-8 and Muc5AC synthesis in bronchial epithelial cells stimulated with rhIL-17A. IL-17A is involved in the IL-8 and Muc5AC production promoting, via NFκB and ERK1/2 pathway activation, the synthesis of ChAT, and the related activity of autocrine ACh in bronchial epithelial cells.
Asunto(s)
Acetilcolina/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Interleucina-17/farmacología , Interleucina-8/metabolismo , Mucina 5AC/metabolismo , FN-kappa B/metabolismo , Comunicación Autocrina/efectos de los fármacos , Bronquios/citología , Línea Celular , Citometría de Flujo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The induction of nitric oxide synthase (iNOS) expression via the signal transducer and activator of transcription 1 (STAT-1) is involved in the mechanism of oxidative/nitrosative stress. We investigated whether acetylcholine (ACh) generates oxidative/nitrosative stress in bronchial epithelial cells during airway inflammation of COPD and evaluated the effects of Tiotropium, a once-daily antimuscarinic drug, and Olodaterol, a long-acting ß2-agonist on these mechanisms. Human bronchial epithelial cells (16-HBE) were stimulated (4h, 37°C) with induced sputum supernatants (ISSs) from healthy controls (HC) (n=10), healthy smokers (HS) (n=10) or COPD patients (n=10), as well as with ACh (from 1µM to 100µM). The activation of STAT-1 pathway (STAT-1Ser727 and STAT-1Tyr701) and iNOS was evaluated in the cell lysates by Western blot analysis as well as nitrotyrosine levels by ELISA, while reactive oxygen species (ROS) were evaluated by flow cytometry. Finally, the effect of Tiotropium (Spiriva®) (100nM), alone or in combination with Olodaterol (1nM), was tested in this model. ISSs from COPD patients significantly increased the phosphorylation of STAT-1Ser727 and STAT-1Tyr701, iNOS and ROS/Nitrotyrosine when compared with ISSs from HC or HS subjects in 16-HBE cells. Furthermore, synthetic ACh increased all these parameters in stimulated 16HBE when compared with untreated cells. Tiotropium and Olodaterol reduced the oxidative/nitrosative stress generated by ACh and ISSs. We concluded that ACh mediated the oxidative/nitrosative stress involving the STAT-1 pathway activation in human bronchial epithelial cells during COPD. ß2-Long acting and antimuscarinic drugs, normally used in the treatment of COPD as bronchodilator, might be able to control these cellular events.
Asunto(s)
Acetilcolina/farmacología , Bronquios/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Enfermedad Pulmonar Obstructiva Crónica/patología , Factor de Transcripción STAT1/metabolismo , Western Blotting , Bronquios/citología , Bronquios/metabolismo , Células Cultivadas , Agonistas Colinérgicos/farmacología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo II/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT1/antagonistas & inhibidores , Factor de Transcripción STAT1/genética , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
AIMS: Atrial fibrillation (AF), the most common cardiac arrhythmia favouring ischemic stroke and heart failure involves left atrial remodelling, fibrosis and a complex interplay between cardiovascular risk factors. This study examined whether activated factor X (FXa) induces pro-remodelling and pro-fibrotic responses in atrial endothelial cells (AECs) and human atrial tissues and determined the underlying mechanisms. METHODS AND RESULTS: AECs collected from porcine hearts and human right atrial appendages (RAA) from patients undergoing heart surgery. Protein expression levels were assessed by Western blot and immunofluorescence staining, mRNA levels by RT-qPCR, formation of reactive oxygen species (ROS) and NO using fluorescent probes, thrombin and angiotensin II generation by specific assays, fibrosis by Sirius red staining and senescence by senescence-associated beta-galactosidase (SA-ß-gal) activity. In AECs, FXa increased ROS formation, senescence (SA-ß-gal activity, p53, p21), angiotensin II generation and the expression of pro-inflammatory (VCAM-1, MCP-1), pro-thrombotic (tissue factor), pro-fibrotic (TGF-ß and collagen-1/3a) and pro-remodelling (MMP-2/9) markers whereas eNOS levels and NO formation were reduced. These effects were prevented by inhibitors of FXa but not thrombin, protease-activated receptors antagonists (PAR-1/2) and inhibitors of NADPH oxidases, ACE, AT1R, SGLT1/SGLT2. FXa also increased expression levels of ACE1, AT1R, SGLT1/2 proteins which were prevented by SGLT1/2 inhibitors. Human RAA showed tissue factor mRNA levels that correlated with markers of endothelial activation, pro-remodelling and pro-fibrotic responses and SGLT1/2 mRNA levels. They also showed protein expression levels of ACE1, AT1R, p22phox, SGLT1/2, and immunofluorescence signals of nitrotyrosine and SGLT1/2 colocalized with those of CD31. FXa increased oxidative stress levels which were prevented by inhibitors of the AT1R/NADPH oxidases/SGLT1/2 pathway. CONCLUSION: FXa promotes oxidative stress triggering premature endothelial senescence and dysfunction associated with pro-thrombotic, pro-remodelling and pro-fibrotic responses in AECs and human RAA involving the AT1R/NADPH oxidases/SGLT1/2 pro-oxidant pathway. Targeting this pathway may be of interest to prevent atrial remodelling and the progression of atrial fibrillation substrate.
Asunto(s)
Apéndice Atrial , Células Endoteliales , Fibrosis , NADPH Oxidasas , Especies Reactivas de Oxígeno , Receptor de Angiotensina Tipo 1 , Transducción de Señal , Humanos , Animales , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales/efectos de los fármacos , NADPH Oxidasas/metabolismo , NADPH Oxidasas/genética , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 1/genética , Especies Reactivas de Oxígeno/metabolismo , Células Cultivadas , Apéndice Atrial/metabolismo , Apéndice Atrial/patología , Apéndice Atrial/enzimología , Remodelación Atrial/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Fibrilación Atrial/metabolismo , Fibrilación Atrial/enzimología , Fibrilación Atrial/patología , Fibrilación Atrial/fisiopatología , Fibrilación Atrial/genética , Estrés Oxidativo/efectos de los fármacos , Sus scrofa , Angiotensina II , Óxido Nítrico/metabolismo , Atrios Cardíacos/metabolismo , Atrios Cardíacos/patología , Atrios Cardíacos/enzimologíaRESUMEN
BACKGROUND: COVID-19 is associated with an increased risk of cardiovascular complications. Although cytokines have a predominant role in endothelium damage, the precise molecular mechanisms are far from being elucidated. OBJECTIVES: The present study hypothesized that inflammation in patients with COVID-19 contributes to endothelial dysfunction through redox-sensitive SGLT2 overexpression and investigated the protective effect of SGLT2 inhibition by empagliflozin. METHODS: Human plasma samples were collected from patients with acute, subacute, and long COVID-19 (n = 100), patients with non-COVID-19 and cardiovascular risk factors (n = 50), and healthy volunteers (n = 25). Porcine coronary artery endothelial cells (ECs) were incubated with plasma (10%). Protein expression levels were determined using Western blot analyses and immunofluorescence staining, mRNA expression by quantitative reverse transcription-polymerase chain reaction, and the level of oxidative stress by dihydroethidium staining. Platelet adhesion, aggregation, and thrombin generation were determined. RESULTS: Increased plasma levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, monocyte chemoattractant protein-1, and soluble intercellular adhesion molecule-1 were observed in patients with COVID-19. Exposure of ECs to COVID-19 plasma with high cytokines levels induced redox-sensitive upregulation of SGLT2 expression via proinflammatory cytokines IL-1ß, IL-6, and tumor necrosis factor-α which, in turn, fueled endothelial dysfunction, senescence, NF-κB activation, inflammation, platelet adhesion and aggregation, von Willebrand factor secretion, and thrombin generation. The stimulatory effect of COVID-19 plasma was blunted by neutralizing antibodies against proinflammatory cytokines and empagliflozin. CONCLUSION: In patients with COVID-19, proinflammatory cytokines induced a redox-sensitive upregulation of SGLT2 expression in ECs, which in turn promoted endothelial injury, senescence, platelet adhesion, aggregation, and thrombin generation. SGLT2 inhibition with empagliflozin appeared as an attractive strategy to restore vascular homeostasis in COVID-19.
Asunto(s)
COVID-19 , Enfermedades Vasculares , Animales , Humanos , COVID-19/metabolismo , Citocinas/metabolismo , Células Endoteliales/metabolismo , Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Síndrome Post Agudo de COVID-19 , Especies Reactivas de Oxígeno/metabolismo , Transportador 2 de Sodio-Glucosa/metabolismo , Transportador 2 de Sodio-Glucosa/farmacología , Porcinos , Trombina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We quantified TGF-ß1 and acetylcholine (ACh) concentrations in induced sputum supernatants (ISSs) from 18 healthy controls (HC), 22 healthy smokers (HS) and 21 COPDs. ISSs from HC, HS and COPD as well as rhTGF-ß1 were also tested in neutrophil adhesion and in mAChR2, mAChR3 and ChAT expression experiments in human bronchial epithelial cells (16-HBE). Finally, we evaluated the effects of Olodaterol (a novel inhaled ß(2)-adrenoceptor agonist) and Tiotropium Spiriva®, alone or in combination, on neutrophil adhesion and mAChRs and ChAT expression in stimulated 16-HBE. The results showed that 1) TGF-ß1 and ACh concentrations are increased in ISSs from COPD in comparison to HC and HS, and TGF-ß1 in HS is higher than in HC; 2) ISSs from COPD and HS caused increased neutrophil adhesion to 16-HBE when compared to ISSs from HC. The effect of ISSs from COPD was significantly reduced by TGF-ß1 depletion or by the pretreatment with Olodaterol or Tiotropium alone or in combination, while the effect of ISSs from HS was significantly reduced by the pretreatment with Olodaterol alone; 3) mAChR2, mAChR3 and ChAT expression was increased in 16-HBE stimulated with ISSs from COPD and TGF-ß1 depletion significantly reduced this effect on mAChR3 and ChAT expression; 4) rhTGF-ß1 increased mAChR2, mAChR3 and ChAT expression in 16-HBE; 5) Olodaterol did not affect the expression of mAChRs and ChAT in 16-HBE. Our findings support the use of ß2 long-acting and anticholinergic drugs to control the bronchoconstriction and TGF-ß1-mediated neutrophilic inflammation in COPD.
Asunto(s)
Acetilcolina/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/uso terapéutico , Antagonistas Colinérgicos/uso terapéutico , Neutrófilos/efectos de los fármacos , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Factor de Crecimiento Transformador beta1/metabolismo , Acetilcolina/análisis , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Anciano , Análisis de Varianza , Benzoxazinas/farmacología , Benzoxazinas/uso terapéutico , Broncoconstricción/efectos de los fármacos , Broncodilatadores/farmacología , Broncodilatadores/uso terapéutico , Estudios de Casos y Controles , Adhesión Celular/efectos de los fármacos , Línea Celular Transformada , Colina O-Acetiltransferasa/metabolismo , Antagonistas Colinérgicos/farmacología , Quimioterapia Combinada , Células Epiteliales/efectos de los fármacos , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/metabolismo , Derivados de Escopolamina/farmacología , Derivados de Escopolamina/uso terapéutico , Fumar/efectos adversos , Esputo/química , Bromuro de Tiotropio , Factor de Crecimiento Transformador beta1/análisisRESUMEN
Empagliflozin, a sodium-glucose co-transporter 2 inhibitor developed, has been shown to reduce cardiovascular events in patients with type 2 diabetes and established cardiovascular disease. Several studies have suggested that empagliflozin improves the cardiac energy state which is a partial cause of its potency. However, the detailed mechanism remains unclear. To address this issue, we used a mouse model that enabled direct measurement of cytosolic and mitochondrial ATP levels. Empagliflozin treatment significantly increased cytosolic and mitochondrial ATP levels in the hearts of db/db mice. Empagliflozin also enhanced cardiac robustness by maintaining intracellular ATP levels and the recovery capacity in the infarcted area during ischemic-reperfusion. Our findings suggest that empagliflozin enters cardiac mitochondria and directly causes these effects by increasing mitochondrial ATP via inhibition of NHE1 and Nav1.5 or their common downstream sites. These cardioprotective effects may be involved in the beneficial effects on heart failure seen in clinical trials.
Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Ratones , Animales , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Experimental/tratamiento farmacológico , Mitocondrias , Adenosina TrifosfatoRESUMEN
Acetylcholine (ACh), synthesized by choline acetyltransferase (ChAT), and muscarinic M(1), M(2), and M(3) receptors (MRs) are involved in fibroblast proliferation. We evaluated ChAT, MRs, and extracellular signal-regulated kinase (ERK) 1/2 and nuclear factor (NF) kappaB activation in lung fibroblasts from patients with chronic obstructive pulmonary disease (COPD), control smokers, and controls. Human fetal lung fibroblasts (HFL-1) stimulated with interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and cigarette smoke extracts (CSEs) were evaluated for ChAT and MR expression. We tested the effects of ACh on fibroblast proliferation and its ability to bind fibroblasts from patients with COPD, control smokers, controls, and HFL-1 stimulated with IL-1beta, TNF-alpha, and CSE. ChAT, M(1), and M(3) expression and ERK1/2 and NFkappaB activation were increased, whereas M(2) was reduced, in COPD and smoker subjects compared with controls. IL-1beta increased the ChAT and M(3), TNF-alpha down-regulated M(2), and CSE increased ChAT and M(3) expression while down-regulating the expression of M(2) in HFL-1 cells. ACh stimulation increased fibroblast proliferation in patients with COPD, control smokers, and controls, with higher effect in control smokers and patients with COPD and increased HFL-1 proliferation only in CSE-treated cells. The binding of ACh was higher in patients with COPD and in control smokers than in controls and in CSE-treated than in IL-1beta- and TNF-alpha-stimulated HFL-1 cells. Tiotropium (Spiriva; [1alpha,2beta,4beta,5alpha,7beta-7-hydroxydi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatrcyclo[3.3.1.0(24)], C(19)H(22) NO(4)S(2)Br.H(2)O), gallamine triethiodide (C(19)H(22)N(4)O(2)S.2HCl.H(2)O), telenzepine [4,9-d-dihydro-3-methyl-4-[(4-methyl-1piperazinyl) acetyl]-10H-thieno [3,4-b][1,5]benzodiazepine-10-one dihydrobromide, C(30)H(60)I(3)N(3)O(3)], 4-diphenylacetoxy-N-methylpiperidine, PD098059 [2-(2-amino-3methoxyphenyl)-4H-1benzopyran-4-one, C(16)H(13)NO(3)], and BAY 11-7082 [(E)-3-(4-methylphenylsulfonyl)-2-propenetrile, C(10)H(9)NO(2)C], down-regulated the ACh-induced fibroblast proliferation, promoting the MRs and ERK1/2 and NFkappaB pathways involvement in this phenomenon. These results suggest that cigarette smoke might alter the expression of ChAT and MRs, promoting airway remodeling in COPD and that anticholinergic drugs, including tiotropium, might prevent these events.
Asunto(s)
Proliferación Celular , Colina O-Acetiltransferasa/biosíntesis , Fibroblastos/metabolismo , Pulmón/patología , Enfermedad Pulmonar Obstructiva Crónica/patología , Receptores Muscarínicos/biosíntesis , Fumar/efectos adversos , Anciano , Western Blotting , Estudios de Casos y Controles , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Fibroblastos/enzimología , Fibroblastos/patología , Humanos , Interleucina-1beta/farmacología , Pulmón/enzimología , Pulmón/metabolismo , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/enzimología , Enfermedad Pulmonar Obstructiva Crónica/etiología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Nicotiana/efectos adversos , Factor de Necrosis Tumoral alfa/farmacologíaAsunto(s)
Compuestos de Bencidrilo , Glucósidos , Linagliptina , Nefritis Hereditaria , Humanos , Linagliptina/uso terapéutico , Linagliptina/farmacología , Glucósidos/uso terapéutico , Glucósidos/farmacología , Compuestos de Bencidrilo/uso terapéutico , Compuestos de Bencidrilo/farmacología , Nefritis Hereditaria/tratamiento farmacológico , Masculino , Riñón/efectos de los fármacos , Riñón/metabolismo , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Femenino , AdultoRESUMEN
AIMS: IL-17A plays a key role in the persistence of airway inflammation, oxidative stress, and reduction of steroid-sensitivity in COPD. We studied the effect of IL-17A on chromatin remodeling and IL-8 production. MAIN METHODS: We measured the levels of IL-8 and IL-17A in induced sputum supernatants (ISS) from healthy controls (HCs), healthy smokers (HSs), and COPD patients by enzyme-linked immunosorbent assay (ELISA). A human bronchial epithelial cell line (16HBE) was stimulated with ISS from HCs, HSs, or COPD subjects. IL-8 was evaluated in 16HBE by Western blot and real-time polymerase chain reaction (PCR). Histone deacetylase 2 (HDAC2), acetyl histone H3 (Ac-His H3) (k9) and inhibitor kappa kinase alpha (IKKα) levels were evaluated in the nuclear extract by Western blot. Finally, we evaluated the effect of IL-17A depletion in ISS, the silencing of IKKα, and the anti-inflammatory effects of Tiotropium Spiriva® (100nM) on 16HBE. KEY FINDINGS: IL-8 and IL-17A levels were higher in ISS from COPD patients and HSs than from HCs. IL-8 protein and messenger RNA (mRNA) levels were increased in 16HBE stimulated with ISS from COPD patients compared with untreated cells. Furthermore, ISS from COPD patients reduced the nuclear levels of HDAC2 while increasing the activity of both Ac-His H3 (k9) and IKKα in stimulated 16HBE. IL-17A depletion in ISS and the IKKα silencing in 16HBE significantly increased the nuclear levels of HDAC2, reduced Ac-His H3 (k9), and promoted IL-8 synthesis in stimulated 16HBE. Tiotropium controls the proinflammatory activity generated by ISS from COPD patients in 16HBE. SIGNIFICANCE: IL-17A present in the airway of COPD patients, which induces chromatin remodeling, promotes the release of IL-8 in the bronchial epithelium. Tiotropium is able to control this proinflammatory activity.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Células Epiteliales/metabolismo , Interleucina-17/metabolismo , Interleucina-8/metabolismo , Bromuro de Tiotropio/farmacología , Bronquios/citología , Bronquios/efectos de los fármacos , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Histona Desacetilasa 2/metabolismo , Histonas/metabolismo , Humanos , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Fumar/metabolismo , Esputo/metabolismoRESUMEN
T-lymphocytes, including Th17-cells and T-cells expressing acetylcholine (ACh), are key components of systemic inflammation in chronic obstructive pulmonary disease (COPD). We investigated whether ACh promotes Th17 cells in COPD. ACh, IL-17A, IL-22, RORγt, FOXP3 expression and AChIL-17A, AChIL-22, AChRORγt coexpression was evaluated in peripheral blood mononuclear cells (PBMC) from COPD patients (n=16), healthy smokers (HS) (n=12) and healthy control subjects (HC) (n=13) (cultured for 48 h with PMA) by flow cytometry. Furthermore, we studied the effect of Tiotropium (Spiriva®) (100 nM) and Olodaterol (1nM) alone or in combination, and of hemicholinium-3 (50 µM) on AChIL-17A, AChIL-22, AChRORγt, and FOXP3 expression in CD3+PBT-cells of PBMC from COPD patients (n=6) cultured for 48 h with PMA. CD3+PBT-cells expressing ACh, IL-17A, IL-22 and RORγt together with CD3+PBT-cells co-expressing AChIL-17A, AChIL-22 and AChRORγt were significantly increased in COPD patients compared to HC and HS subjects with higher levels in HS than in HC without a significant difference. CD3+FOXP3+PBT-cells were increased in HS than in HC and COPD. Tiotropium and Olodaterol reduced the percentage of CD3+PBT-cells co-expressing AChIL-17A, AChIL-22, and AChRORγt while increased the CD3+FOXP3+PBT-cells in PBMC from COPD patients, cultured in vitro for 48 h, with an additive effect when used in combination. Hemicholnium-3 reduced the percentage of ACh+IL-17A+, ACh+IL-22+, and ACh+RORγt+ while it did not affect FOXP3+ expression in CD3+PBT-cells from cultured PBMC from COPD patients. We concluded that ACh might promote the increased levels of Th17-cells in systemic inflammation of COPD. Long-acting ß2-agonists and anticholinergic drugs might contribute to control this event.
Asunto(s)
Acetilcolina/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Acetilcolina/farmacología , Anciano , Anciano de 80 o más Años , Benzoxazinas/farmacología , Antagonistas Colinérgicos/farmacología , Femenino , Factores de Transcripción Forkhead/metabolismo , Humanos , Interleucina-17/metabolismo , Interleucinas/metabolismo , Espacio Intracelular/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Factores de Riesgo , Derivados de Escopolamina/farmacología , Células Th17/efectos de los fármacos , Bromuro de Tiotropio , Interleucina-22RESUMEN
Cigarette smoke extract (CSE) affects the expression of Choline Acetyl-Transferase (ChAT), muscarinic acetylcholine receptors, and mucin production in bronchial epithelial cells. Mucin 5AC (MUC5AC), muscarinic acetylcholine receptor M3, ChAT expression, acetylcholine levels and acetylcholine binding were measured in a human pulmonary mucoepidermoid carcinoma cell line (H292) stimulated with CSE. We performed ChAT/RNA interference experiments in H292 cells stimulated with CSE to study the role of ChAT/acetylcholine in MUC5AC production. The effects of Hemicholinium-3 (HCh-3) (50 µM) (a potent and selective choline uptake blocker) and Tiotropium bromide (Spiriva(®)) (100 nM), alone or in combination with Salmeterol (SL) and Fluticasone propionate (FP), were tested in this model. MUC5AC, muscarinic acetylcholine receptor M3, ChAT, acetylcholine expression and acetylcholine binding significantly increased in H292 cells stimulated with CSE (5%) compared to untreated cells. HCh-3 reduced acetylcholine binding and MUC5AC production in H292 cells stimulated with CSE. ChAT/RNA interference eliminated the effect of CSE on MUC5AC production. FP reduced ChAT and acetylcholine binding in unstimulated cells, while showing a partial effect in CSE stimulated cells. SL increased the ChAT expression and acetylcholine binding in H292 cells stimulated with or without CSE. Tiotropium, alone or together with FP and SL, reduced acetylcholine binding and MUC5AC production in H292 cells stimulated with CSE. CSE affects the ChAT/acetylcholine expression, increasing MUC5AC production in H292 cells. Pharmacological treatment with anticholinergic drugs reduces the secretion of MUC5AC generated by autocrine acetylcholine activity in airway epithelial cells.
Asunto(s)
Acetilcolina/metabolismo , Colina O-Acetiltransferasa/metabolismo , Mezclas Complejas/farmacología , Mucina 5AC/metabolismo , Nicotiana , Humo , Albuterol/análogos & derivados , Albuterol/farmacología , Androstadienos/farmacología , Bronquios/citología , Broncodilatadores/farmacología , Línea Celular Tumoral , Colina O-Acetiltransferasa/genética , Antagonistas Colinérgicos/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Fluticasona , Hemicolinio 3/farmacología , Humanos , Inhibidores de la Captación de Neurotransmisores/farmacología , Interferencia de ARN , Receptor Muscarínico M3/metabolismo , Xinafoato de Salmeterol , Derivados de Escopolamina/farmacología , Bromuro de TiotropioRESUMEN
The parasympathetic nervous system is a key regulator of the human organism involved in the pathophysiology of various disorders through cholinergic mechanisms. In the lungs, acetylcholine (ACh) released by vagal nerve endings stimulates muscarinic receptors thereby increasing airway smooth muscle tone. Contraction of airway smooth muscle cells leads to increased respiratory resistance and dyspnea. An additional branch of the cholinergic system is the non-neuronal cholinergic system expressed in nearly all cell types present in the airways. Activation of this system may contribute to an increased cholinergic tone in the lungs, inducing pathophysiological processes like inflammation, remodeling, mucus hypersecretion and chronic cough. Selective muscarinic receptor antagonists specifically inhibit acetylcholine at the receptor inducing bronchodilation in patients with obstructive airway diseases. This paper reviews preclinical pharmacological research activities on anticholinergics including experimental models of asthma and chronic obstructive pulmonary disease, COPD. It discloses various options to follow up the non-neuronal cholinergic system as a novel drug target for the treatment of key aspects of obstructive airway diseases, in particular those of a chronic nature.
Asunto(s)
Asma/tratamiento farmacológico , Terapia Molecular Dirigida/métodos , Antagonistas Muscarínicos/uso terapéutico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Receptores Muscarínicos/metabolismo , Acetilcolina/inmunología , Acetilcolina/metabolismo , Animales , Asma/inmunología , Asma/metabolismo , Humanos , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/inervación , Pulmón/metabolismo , Antagonistas Muscarínicos/farmacología , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Receptores Muscarínicos/inmunologíaRESUMEN
Novel pharmacological strategies are aimed at the resolution of systemic inflammation in COPD potentiating peripheral blood T-cell (PBT-cell) apoptosis. Although muscarinic acetylcholine receptors (mAChRs) M(3) and choline-acetyltransferase (ChAT) participate in the airway inflammation of COPD, their role in PBT-cell apoptosis remains unexplained. We evaluated in PBT-cells from COPD patients, smoker (S) and control (C) subjects: (1) apoptosis (by annexin V binding), (2) mAChR M(3) and ChAT expression, acetylcholine (ACh)-binding; (3) choline levels in serum and PBT-cells extracts. We tested the effects of Tiotropium (Spiriva(®)) and hemicholinium-3 (HCh-3) on apoptosis, NFκB pathway, caspases 3 and 8 activity and choline levels, in PBT-cells from COPD patients. We showed that: (1) apoptosis, mAChR M(3) and ChAT expression and the CD3+ and CD8+ ACh-binding are increased in PBT-cells from COPD patients when compared to C subjects, while CD4+/CD8+ ratio of ACh-binding to PBT cells was reduced in COPD; (2) choline levels are higher in serum and PBT-cells extracts from COPD patients than in S and C; (3) Tiotropium and HCh-3 reduced CD4+ and increased CD8+ apoptosis via caspases 3 and 8 activities and via IκB mediated mechanisms in COPD patients. This study suggests the involvement of non-neuronal components of cholinergic system in the regulation of PBT-cell apoptosis in COPD and demonstrates that Tiotropium regulates CD4+ and CD8+ PBT-cell apoptosis. It provides novel putative pharmacological targets for the resolution of systemic inflammation in COPD.
Asunto(s)
Apoptosis/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Antagonistas Colinérgicos/farmacología , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Anciano , Linfocitos T CD8-positivos/metabolismo , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Colina/sangre , Colina O-Acetiltransferasa/metabolismo , Antagonistas Colinérgicos/metabolismo , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , FN-kappa B/metabolismo , Unión Proteica , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Receptor Muscarínico M3/metabolismo , Derivados de Escopolamina/farmacología , Transducción de Señal/efectos de los fármacos , Bromuro de TiotropioRESUMEN
AIMS: Acetylcholine (ACh) is synthesized by Choline Acetyl-Transferase (ChAT) that exerts its physiological effects in airway epithelial cells via muscarinic receptor (MR) activation. We evaluate the effect of ACh stimulation on human bronchial epithelial cells (16-HBE) and test whether cigarette smoke extract (CSE) can modify the basal cellular response to ACh affecting the non-neuronal cholinergic system signalling. MAIN METHODS: ACh stimulated 16-HBE were tested for ACh-binding, Leukotriene B(4) (LTB(4)) release and ERK1/2 and NFkB pathway activation. Additionally, we investigated all the aforementioned parameters as well as ChAT and MR proteins and mRNA expression and endogenous ACh production in CSE-treated 16-HBE. KEY FINDINGS: We showed that ACh induced in 16-HBE, in a concentration-dependent manner, LTB(4) release via the activation of ERK1/2 and NFkB pathways. The addition of Tiotropium (Spiriva®), Gallamine, Telenzepine and 4-DAMP (muscarinic receptor antagonists), as well as of PD 098059 (MAPKK inhibitor) and BAY117082 (inhibitor of IkBα phosphorilation), down-regulated the ACh-induced effects. Additionally, CSE treatment of 16-HBE increased the binding of ACh, and shifted the LTB4 release from the concentration ACh 1µM to 10nM. Finally, we observed that the treatment of 16-HBE with CSE increased the expression of ChAT, M(2) and M(3) and of endogenous ACh production in 16-HBE. Tiotropium regulated the LTB4 release and ACh production in CSE treated 16-HBE. SIGNIFICANCE: CSE increases the pro-inflammatory activity of human bronchial epithelial cells, and promotes the cellular response to lower concentrations of ACh, by affecting the expression of ChAT and MRs. Tiotropium might prevent pro-inflammatory events generated by ACh together with CSE.