Papel de los esfingolípidos en las alteraciones vasculares asociadas al daño pulmonar agudo

El síndrome de distrés respiratorio agudo (SDRA) se caracteriza por edema pulmonar y colapso alveolar que conduce a hipoxemia arterial grave. Las causas más frecuentes de SDRA son la sepsis y los traumatismos. Aunque las estrategias protectoras de soporte ventilatorio y hemodinámico han permitido mejorar el pronóstico, la mortalidad asociada se mantiene intolerablemente elevada por lo que el descubrimiento de nuevos tratamientos efectivos tendría un gran impacto en la supervivencia de los pacientes. Además, la existencia de disfunción vascular pulmonar es un factor independiente asociado a un peor pronóstico en estos pacientes. Los esfingolípidos son componentes estructurales de las membranas, que regulan la dinámica de éstas y forman parte de los microdominios de membrana denominados balsas lipídicas de membrana (“lipid rafts”). Los esfingolípidos actúan también como segundos mensajeros intracelulares implicados en la regulación de procesos celulares clave como la diferenciación, el crecimiento, la apoptosis o la inmunidad innata y adquirida. Estudios previos sugieren que la ceramida producida por la esfingomielinasa (SMasa) neutra (nSMasa) está implicada en la regulación del tono vascular pulmonar. Además, la esfingomielinasa ácida (aSMasa) se encuentra elevada en pacientes en estado crítico. Las evidencias acumuladas durante los últimos años sugieren que los esfingolípidos podrían desempeñar un papel en el SDRA. La imipramina o su análogo desipramina y el D609 son compuestos no relacionados químicamente que tienen en común su capacidad de inhibir la esfingomielinasa ácida..

The Flavonoid Quercetin Reverses Pulmonary Hypertension in Rats

Quercetin is a dietary flavonoid which exerts vasodilator, antiplatelet and antiproliferative effects and reduces blood pressure, oxidative status and end-organ damage in humans and animal models of systemic hypertension. We hypothesized that oral quercetin treatment might be protective in a rat model of pulmonary arterial hypertension. Three weeks after injection of monocrotaline, quercetin (10 mg/kg/d per os) or vehicle was administered for 10 days to adult Wistar rats. Quercetin significantly reduced mortality. In surviving animals, quercetin decreased pulmonary arterial pressure, right ventricular hypertrophy and muscularization of small pulmonary arteries. Classic biomarkers of pulmonary arterial hypertension such as the downregulated expression of lung BMPR2, Kv1.5, Kv2.1, upregulated survivin, endothelial dysfunction and hyperresponsiveness to 5-HT were unaffected by quercetin. Quercetin significantly restored the decrease in Kv currents, the upregulation of 5-HT2A receptors and reduced the Akt and S6 phosphorylation. In vitro, quercetin induced pulmonary artery vasodilator effects, inhibited pulmonary artery smooth muscle cell proliferation and induced apoptosis. In conclusion, quercetin is partially protective in this rat model of PAH. It delayed mortality by lowering PAP, RVH and vascular remodeling. Quercetin exerted effective vasodilator effects in isolated PA, inhibited cell proliferation and induced apoptosis in PASMCs. These effects were associated with decreased 5-HT2A receptor expression and Akt and S6 phosphorylation and partially restored Kv currents. Therefore, quercetin could be useful in the treatment of PAH.This work was supported by grants and fellowships by the Spanish Ministerio de Economia y Competitividad (SAF2011-28150 to F.P-V, SAF2010-22066-C02-01 to JD, and −02 to AC); Instituto de Salud Carlos III Red HERACLES RD06/0009 to JD; Miguel Servet Program CP12/03304 to LM; predoctoral grants BES-2012-051904 to DMS, CM, JMS, and PG; and Junta de Andalucia (Proyecto de excelencia, P12-CTS-2722)

Impact of a TAK-1 inhibitor as a single or as an add-on therapy to riociguat on the metabolic reprograming and pulmonary hypertension in the SUGEN5416/hypoxia rat model.

Background: Despite increasing evidence suggesting that pulmonary arterial hypertension (PAH) is a complex disease involving vasoconstriction, thrombosis, inflammation, metabolic dysregulation and vascular proliferation, all the drugs approved for PAH mainly act as vasodilating agents. Since excessive TGF-β signaling is believed to be a critical factor in pulmonary vascular remodeling, we hypothesized that blocking TGFβ-activated kinase 1 (TAK-1), alone or in combination with a vasodilator therapy (i.e., riociguat) could achieve a greater therapeutic benefit. Methods: PAH was induced in male Wistar rats by a single injection of the VEGF receptor antagonist SU5416 (20 mg/kg) followed by exposure to hypoxia (10%O2) for 21 days. Two weeks after SU5416 administration, vehicle, riociguat (3 mg/kg/day), the TAK-1 inhibitor 5Z-7-oxozeaenol (OXO, 3 mg/kg/day), or both drugs combined were administered for 7 days. Metabolic profiling of right ventricle (RV), lung tissues and PA smooth muscle cells (PASMCs) extracts were performed by magnetic resonance spectroscopy, and the differences between groups analyzed by multivariate statistical methods. Results: In vitro, riociguat induced potent vasodilator effects in isolated pulmonary arteries (PA) with negligible antiproliferative effects and metabolic changes in PASMCs. In contrast, 5Z-7-oxozeaenol effectively inhibited the proliferation of PASMCs characterized by a broad metabolic reprogramming but had no acute vasodilator effects. In vivo, treatment with riociguat partially reduced the increase in pulmonary arterial pressure (PAP), RV hypertrophy (RVH), and pulmonary vascular remodeling, attenuated the dysregulation of inosine, glucose, creatine and phosphocholine (PC) in RV and fully abolished the increase in lung IL-1β expression. By contrast, 5Z-7-oxozeaenol significantly reduced pulmonary vascular remodeling and attenuated the metabolic shifts of glucose and PC in RV but had no effects on PAP or RVH. Importantly, combined therapy had an additive effect on pulmonary vascular remodeling and induced a significant metabolic effect over taurine, amino acids, glycolysis, and TCA cycle metabolism via glycine-serine-threonine metabolism. However, it did not improve the effects induced by riociguat alone on pulmonary pressure or RV remodeling. None of the treatments attenuated pulmonary endothelial dysfunction and hyperresponsiveness to serotonin in isolated PA. Conclusion: Our results suggest that inhibition of TAK-1 induces antiproliferative effects and its addition to short-term vasodilator therapy enhances the beneficial effects on pulmonary vascular remodeling and RV metabolic reprogramming in experimental PAH.This work was supported by the Instituto de Salud Carlos III-ISCIII (Grant numbers: PI15/01100 and PI19/01616 to LM), the Spanish Ministry of Science and Innovation MCIN (Grant numbers: PID 2019-107363RB-I00 to FP-V, PID 2020-117939RBI00 to AC and PID 2021-123238OB-I00, PDC 2021-121696-I00 to JRC and PID2019-106564RJ-I00 to JI-G), the Comunidad de Madrid-CAM (CM S2017/BMD-3727 to AC and LM and B2017/ BMD3875 to JI-G) and, as appropriate, by “ERDF A way of making Europe”, co-funded by the “European Union”. FP-V received funding from Fundación Contra la Hipertensión Pulmonar (Empathy grant) and JR-C from La Caixa Foundation (Health Research Call 2020: HR20-00075). This work was performed under the Maria de Maeztu Units of Excellence Programme–Grant MDM-2017-0720 funded by MCIN/AEI/10.13039/501100011033.S

Effects of Quercetin in a Rat Model of Hemorrhagic Traumatic Shock and Reperfusion

Background: We hypothesized that treatment with quercetin could result in improved hemodynamics, lung inflammatory parameters and mortality in a rat model of hemorrhagic shock. Methods: Rats were anesthetized (80 mg/kg ketamine plus 8 mg/kg xylazine i.p.). The protocol included laparotomy for 15 min (trauma), hemorrhagic shock (blood withdrawal to reduce the mean arterial pressure to 35 mmHg) for 75 min and resuscitation by re-infusion of all the shed blood plus lactate Ringer for 90 min. Intravenous quercetin (50 mg/kg) or vehicle were administered during resuscitation. Results: There was a trend for increased survival 84.6% (11/13) in the treated group vs. the shock group 68.4% (13/19, p > 0.05 Kaplan–Meier). Quercetin fully prevented the development of lung edema. The activity of aSMase was increased in the shock group compared to the sham group and the quercetin prevented this effect. However, other inflammatory markers such as myeloperoxidase activity, interleukin-6 in plasma or bronchoalveolar fluid were similar in the sham and shock groups. We found no bacterial DNA in plasma in these animals. Conclusions: Quercetin partially prevented the changes in blood pressure and lung injury in shock associated to hemorrhage and reperfusion.Supported by FundaciónMutuaMadrileña (AP102962012), SpanishMINECO(SAF 2011-28150; SAF2014-55399R; SAF2014-58920) and ISCIII (CP12/03304, FIS 15/1492)

Impact of a TAK-1 inhibitor as a single or as an add-on therapy to riociguat on the metabolic reprograming and pulmonary hypertension in the SUGEN5416/hypoxia rat model

Background: Despite increasing evidence suggesting that pulmonary arterial hypertension (PAH) is a complex disease involving vasoconstriction, thrombosis, inflammation, metabolic dysregulation and vascular proliferation, all the drugs approved for PAH mainly act as vasodilating agents. Since excessive TGF-β signaling is believed to be a critical factor in pulmonary vascular remodeling, we hypothesized that blocking TGFβ-activated kinase 1 (TAK-1), alone or in combination with a vasodilator therapy (i.e., riociguat) could achieve a greater therapeutic benefit.Methods: PAH was induced in male Wistar rats by a single injection of the VEGF receptor antagonist SU5416 (20 mg/kg) followed by exposure to hypoxia (10%O2) for 21 days. Two weeks after SU5416 administration, vehicle, riociguat (3 mg/kg/day), the TAK-1 inhibitor 5Z-7-oxozeaenol (OXO, 3 mg/kg/day), or both drugs combined were administered for 7 days. Metabolic profiling of right ventricle (RV), lung tissues and PA smooth muscle cells (PASMCs) extracts were performed by magnetic resonance spectroscopy, and the differences between groups analyzed by multivariate statistical methods.Results:In vitro, riociguat induced potent vasodilator effects in isolated pulmonary arteries (PA) with negligible antiproliferative effects and metabolic changes in PASMCs. In contrast, 5Z-7-oxozeaenol effectively inhibited the proliferation of PASMCs characterized by a broad metabolic reprogramming but had no acute vasodilator effects. In vivo, treatment with riociguat partially reduced the increase in pulmonary arterial pressure (PAP), RV hypertrophy (RVH), and pulmonary vascular remodeling, attenuated the dysregulation of inosine, glucose, creatine and phosphocholine (PC) in RV and fully abolished the increase in lung IL-1β expression. By contrast, 5Z-7-oxozeaenol significantly reduced pulmonary vascular remodeling and attenuated the metabolic shifts of glucose and PC in RV but had no effects on PAP or RVH. Importantly, combined therapy had an additive effect on pulmonary vascular remodeling and induced a significant metabolic effect over taurine, amino acids, glycolysis, and TCA cycle metabolism via glycine-serine-threonine metabolism. However, it did not improve the effects induced by riociguat alone on pulmonary pressure or RV remodeling. None of the treatments attenuated pulmonary endothelial dysfunction and hyperresponsiveness to serotonin in isolated PA.Conclusion: Our results suggest that inhibition of TAK-1 induces antiproliferative effects and its addition to short-term vasodilator therapy enhances the beneficial effects on pulmonary vascular remodeling and RV metabolic reprogramming in experimental PAH

Effetti degli antiossidanti sulla funzionalita e sulla sopravvivenza di isole pancreatiche umane: ruolo nella modulazione di molecole pro-infiammatorie.

Il diabete mellito di tipo 1 (T1DM) è causato dalla distruzione autoimmune progressiva e specifica di beta-cellule pancreatiche secernenti insulina. Una volta che il T1DM si è manifestato, l’unica forma di trattamento del paziente diventa la somministrazione di insulina esogena, un approccio che richiede più iniezioni quotidiane ed un controllo costante della glicemia, legata a sua volta ad innumerevoli variabili come l’età o lo stile di vita. La malattia ha un impatto socio-sanitario di estrema rilevanza non solo per la sua notevole diffusione, ma anche perchè associata a complicanze acute (coma ipoglicemico e iperglicemico) e croniche (danni a carico di occhi, reni, sistema nervoso, apparato cardio-circolatorio). Gli obbiettivi principali della terapia del T1DM sono l'eliminazione dei sintomi dovuti all'iperglicemia e la prevenzione dell'insorgenza delle complicanze croniche della malattia. In tale contesto si inserisce il trapianto di pancreas endocrino, spesso associato al trapianto di rene, che tuttavia rimane una complessa procedura chirurgica invasiva. Il trapianto di isole pancreatiche, cioè solamente della parte endocrina del pancreas, è diventato un approcio terapeutico di notevole interesse. Negli ultimi anni sono stati fatti enormi progressi in questo settore, anche se rimangono tuttavia alcuni ostacoli che ne precludono una sua ampia applicazione. In particolare il ridotto numero di donatori e la ridotta funzionalità delle isole dopo trapianto rappresentano ancora dei problemi irrisolti. Quest’ultima è la probabile conseguenza di uno stress sulle isole stesse, legato a danni meccanici (isolamento) e biologici (apoptosi, necrosi e/o produzione di molecole pro-infiammatorie) che causano la perdita precoce e rapida di una parte significativa della massa beta-cellulare. Inoltre, è stato anche dimostrato che le isole pancreatiche umane sono in grado di produrre molecole pro-infiammatorie, fra le quali il “monocyte chemoattractant protein-1” (MCP-1), confermando probabilmente l’osservazione che una delle cause principali della ridotta sopravvivenza delle isole pancreatiche dopo trapianto sia il fenomeno noto come "inflammatory blood mediated reaction" (IBMIR). Alcuni studi hanno anche evidenziato come lo stress ossidativo possa agire come causa scatenante del processo infiammatorio cronico, non necessariamente iniziando la risposta infiammatoria ex-novo, ma inasprendo le risposte infiammatorie già in atto. L’uso, durante l’isolamento, di antiossidanti naturali o prodotti sinteticamente sembra possa migliorare la sopravvivenza e la funzionalità dell’isola pancreatica umana. Poiché non ci sono molte informazioni disponibili sull’eventuale ruolo delle molecole antiossidanti nella preparazione delle isole pancreatiche e nella modulazione delle molecole pro-infiammatorie, lo scopo del mio internato di tesi è stato quello di separare le isole pancreatiche da pancreas di donatori multiorgano in presenza o meno di una molecola antiossidante di nuova generazione, la bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)-decandioate (Iacvita, IAC) alla concentrazione 100 µmol/L e di valutare la resa, la morfologia, le caratteristiche molecolari e la funzione in vitro e in vivo delle isole ottenute. Per tali finalità, dopo l’isolamento, la quantità delle isole è stata determinata mediante conta al microscopio ottico, in relazione al diametro delle isole stesse. La morfologia è stata valutata con tecniche di microscopia ottica ed elettronica e la vitalità utilizzando metodi fluorimetrici. Per gli studi molecolari, è stata utilizzata la metodica di RT-PCR quantitativa RT-PCR. La funzione in vitro è stata determinata con studi di secrezione insulinica in risposta al glucosio, quantificata mediante I.R.M.A e infine la funzione in vivo è stata studiata mediante trapianto sotto la capsula renale in roditori con diabete indotto chimicamente. I risultati ottenuti in questa tesi sembrano supportare l’ipotesi che l’utilizzo di Iacvita possa prevenire lo stress ossidativo indotto sia durante la procedura di isolamento che di coltura pre-trapianto delle isole pancreatiche umane e possa migliorare la funzionalità e la vitalità beta-cellulare. La riduzione dello stress ossidativo osservata in questo studio sembra essere associata ad una ridotta espressione della molecola pro-infiammatoria CCL2/MCP-1 e del suo recettore CCR2. Tuttavia, il ruolo della molecola e delle sue proprietà nel trapianto dovrà essere ulteriormente approfondita

Kv7 channels critically determine coronary artery reactivity: left-right differences and down-regulation by hyperglycaemia

Aims Voltage-gated potassium channels encoded by KCNQ genes (Kv7 channels) are emerging as important regulators of vascular tone. In this study, we analysed the contribution of Kv7 channels to the vasodilation induced by hypoxia and the cyclic AMP pathway in the coronary circulation. We also assessed their regional distribution and possible impairment by diabetes. Methods and results We examined the effects of Kv7 channel modulators on K+ currents and vascular reactivity in rat left and right coronary arteries (LCAs and RCAs, respectively). Currents from LCA were more sensitive to Kv7 channel inhibitors (XE991, linopirdine) and activators (flupirtine, retigabine) than those from RCA. Accordingly, LCAs were more sensitive than RCAs to the relaxation induced by Kv7 channel enhancers. Likewise, relaxation induced by the adenylyl cyclase activator forskolin and hypoxia, which were mediated through Kv7 channel activation, were greater in LCA than in RCA. KCNQ1 and KCNQ5 expression was markedly higher in LCA than in RCA. After incubation with high glucose (HG, 30 mmol/L), myocytes from LCA, but not from RCA, were more depolarized and showed reduced Kv7 currents. In HG-incubated LCA, the effects of Kv7 channel modulators and forskolin were diminished, and the expression of KCNQ1 and KCNQ5 was reduced. Finally, vascular responses induced by Kv7 channel modulators were impaired in LCA, but not in RCA, from type 1 diabetic rats. Conclusion Our results reveal that the high expression and function of Kv7 channels in the LCA and their down-regulation by diabetes critically determine the sensitivity to key regulators of coronary tone.Depto. de Farmacología y ToxicologíaFac. de MedicinaTRUEpu

Kv7 channels critically determine coronary artery reactivity: left-right differences and down-regulation by hyperglycaemia

Aims Voltage-gated potassium channels encoded by KCNQ genes (Kv7 channels) are emerging as important regulators of vascular tone. In this study, we analysed the contribution of Kv7 channels to the vasodilation induced by hypoxia and the cyclic AMP pathway in the coronary circulation. We also assessed their regional distribution and possible impairment by diabetes. Methods and results We examined the effects of Kv7 channel modulators on K+ currents and vascular reactivity in rat left and right coronary arteries (LCAs and RCAs, respectively). Currents from LCA were more sensitive to Kv7 channel inhibitors (XE991, linopirdine) and activators (flupirtine, retigabine) than those from RCA. Accordingly, LCAs were more sensitive than RCAs to the relaxation induced by Kv7 channel enhancers. Likewise, relaxation induced by the adenylyl cyclase activator forskolin and hypoxia, which were mediated through Kv7 channel activation, were greater in LCA than in RCA. KCNQ1 and KCNQ5 expression was markedly higher in LCA than in RCA. After incubation with high glucose (HG, 30 mmol/L), myocytes from LCA, but not from RCA, were more depolarized and showed reduced Kv7 currents. In HG-incubated LCA, the effects of Kv7 channel modulators and forskolin were diminished, and the expression of KCNQ1 and KCNQ5 was reduced. Finally, vascular responses induced by Kv7 channel modulators were impaired in LCA, but not in RCA, from type 1 diabetic rats. Conclusion Our results reveal that the high expression and function of Kv7 channels in the LCA and their down-regulation by diabetes critically determine the sensitivity to key regulators of coronary tone.Depto. de Farmacología y ToxicologíaFac. de MedicinaTRUEpu

Fas activation alters tight junction proteins in acute lung injury

Background:The acute respiratory distress syndrome (ARDS) is characterized by protein-rich oedema in the alveolar spaces, a feature in which Fas-mediated apoptosis of the alveolar epithelium has been involved. Objective:To determine whether Fas activation increases protein permeability by mechanisms involving disruption of the paracellular tight junction (TJ) proteins in the pulmonary alveoli. Methods: Protein permeability and the expression of TJ proteins were assessed in vivo in wild-type and Fas-deficient lpr mice 16 hours after the intratracheal instillation of recombinant human soluble Fas ligand (rh-sFasL), and at different time points in vitro in human pulmonary alveolar epithelial cells (HPAEpiC) exposed to rh-sFasL Results:Activation of the Fas pathway increased protein permeability in mouse lungs and altered the expression of the TJ proteins occludin and zonula occludens-1 in the alveolar-capillary membrane in vivo and in human alveolar epithelial cell monolayers in vitro. Blockade of caspase-3, but not inhibition of tyrosine kinase dependent pathways, prevented the alterations in TJ protein expression and permeability induced by the Fas/FasL system in human alveolar cell monolayers in vitro. We also observed that both the Fas-induced increase of protein permeability and disruption of TJ proteins occurred before cell death could be detected in the cell monolayers in vitro. Conclusion:Targeting caspase pathways could prevent the disruption of TJs and reduce the formation of lung oedema in the early stages of ARDS.Sin financiación10.844 JCR (2109) Q1, 5/64 Respiratory System3.248 SJR (2019) Q1, 6/149 Pulmonary and Respiratory MedicineNo data IDR 2019UE

Activation of PPARβ/δ prevents hyperglycaemia-induced impairment of Kv7 channels and cAMP-mediated relaxation in rat coronary arteries

PPARβ/δ activation protects against endothelial dysfunction in diabetic models. Elevated glucose is known to impair cAMP-induced relaxation and Kv channel function in coronary arteries (CA). Herein, we aimed to analyse the possible protective effects of the PPARβ/δ agonist GW0742 on the hyperglycaemic-induced impairment of cAMP-induced relaxation and Kv channel function in rat CA. As compared with low glucose (LG), incubation under high glucose (HG) conditions attenuated the relaxation induced by the adenylate cyclase activator forskolin in CA and this was prevented by GW0742. The protective effect of GW0742 was supressed by a PPARβ/δ antagonist. In myocytes isolated from CA under LG, forskolin enhanced Kv currents and induced hyperpolarization. In contrast, when CA were incubated with HG, Kv currents were diminished and the electrophysiological effects of forskolin were abolished. These deleterious effects were prevented by GW0742. The protective effects of GW0742 on forskolin-induced relaxation and Kv channel function were confirmed in CA from type-1 diabetic rats. In addition, the differences in the relaxation induced by forskolin in CA incubated under LG, HG or HG + GW0742 were abolished by the Kv7 channel inhibitor XE991. Accordingly, GW0742 prevented the down-regulation of Kv7 channels induced by HG. Finally, the preventive effect of GW0742 on oxidative stress and cAMP-induced relaxation were overcome by the pyruvate dehydrogenase kinase 4 (PDK4) inhibitor dichloroacetate (DCA). Our results reveal that the PPARβ/δ agonist GW0742 prevents the impairment of the cAMP-mediated relaxation in CA under HG. This protective effect was associated with induction of PDK4, attenuation of oxidative stress and preservation of Kv7 channel functionDepto. de Farmacología y ToxicologíaFac. de MedicinaTRUEpu