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Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.
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Regeneración Nerviosa , Humanos , Neoplasias/tratamiento farmacológico , Regeneración Nerviosa/efectos de los fármacos , Isoformas de Proteínas/agonistas , Transducción de Señal/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I/química , Fosfatidilinositol 3-Quinasa Clase I/efectos de los fármacos , Cardiotónicos/farmacología , Animales , Biocatálisis/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Neuritas/efectos de los fármacos , Daño por Reperfusión/prevención & control , Compresión Nerviosa , Proliferación Celular/efectos de los fármacosRESUMEN
Preconditioning, postconditioning, and remote conditioning of the myocardium enhance the ability of the heart to withstand a prolonged ischemia/reperfusion insult and the potential to provide novel therapeutic paradigms for cardioprotection. While many signaling pathways leading to endogenous cardioprotection have been elucidated in experimental studies over the past 30 years, no cardioprotective drug is on the market yet for that indication. One likely major reason for this failure to translate cardioprotection into patient benefit is the lack of rigorous and systematic preclinical evaluation of promising cardioprotective therapies prior to their clinical evaluation, since ischemic heart disease in humans is a complex disorder caused by or associated with cardiovascular risk factors and comorbidities. These risk factors and comorbidities induce fundamental alterations in cellular signaling cascades that affect the development of ischemia/reperfusion injury and responses to cardioprotective interventions. Moreover, some of the medications used to treat these comorbidities may impact on cardioprotection by again modifying cellular signaling pathways. The aim of this article is to review the recent evidence that cardiovascular risk factors as well as comorbidities and their medications may modify the response to cardioprotective interventions. We emphasize the critical need for taking into account the presence of cardiovascular risk factors as well as comorbidities and their concomitant medications when designing preclinical studies for the identification and validation of cardioprotective drug targets and clinical studies. This will hopefully maximize the success rate of developing rational approaches to effective cardioprotective therapies for the majority of patients with multiple comorbidities. SIGNIFICANCE STATEMENT: Ischemic heart disease is a major cause of mortality; however, there are still no cardioprotective drugs on the market. Most studies on cardioprotection have been undertaken in animal models of ischemia/reperfusion in the absence of comorbidities; however, ischemic heart disease develops with other systemic disorders (e.g., hypertension, hyperlipidemia, diabetes, atherosclerosis). Here we focus on the preclinical and clinical evidence showing how these comorbidities and their routine medications affect ischemia/reperfusion injury and interfere with cardioprotective strategies.
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Poscondicionamiento Isquémico , Precondicionamiento Isquémico Miocárdico , Isquemia Miocárdica , Daño por Reperfusión Miocárdica , Animales , Humanos , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/prevención & control , Daño por Reperfusión Miocárdica/metabolismo , Isquemia Miocárdica/tratamiento farmacológico , Isquemia Miocárdica/prevención & control , Factores de Riesgo , Factores de Riesgo de Enfermedad Cardiaca , IsquemiaRESUMEN
The use of biomarkers is undisputed in the diagnosis of primary myocardial infarction (MI), but their value for identifying MI is less well studied in the postoperative phase following coronary artery bypass grafting (CABG). To identify patients with periprocedural MI (PMI), several conflicting definitions of PMI have been proposed, relying either on cardiac troponin (cTn) or the MB isoenzyme of creatine kinase, with or without supporting evidence of ischaemia. However, CABG inherently induces the release of cardiac biomarkers, as reflected by significant cTn concentrations in patients with uncomplicated postoperative courses. Still, the underlying (patho)physiological release mechanisms of cTn are incompletely understood, complicating adequate interpretation of postoperative increases in cTn concentrations. Therefore, the aim of the current review is to present these potential underlying mechanisms of cTn release in general, and following CABG in particular (Graphical Abstract). Based on these mechanisms, dissimilarities in the release of cTnI and cTnT are discussed, with potentially important implications for clinical practice. Consequently, currently proposed cTn biomarker cut-offs by the prevailing definitions of PMI might warrant re-assessment, with differentiation in cut-offs for the separate available assays and surgical strategies. To resolve these issues, future prospective studies are warranted to determine the prognostic influence of biomarker release in general and PMI in particular.
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Puente de Arteria Coronaria , Infarto del Miocardio , Humanos , Puente de Arteria Coronaria/efectos adversos , Infarto del Miocardio/etiología , Troponina I , Troponina T , BiomarcadoresRESUMEN
Heart transplantation with donation after circulatory death (DCD) provides excellent patient outcomes and increases donor heart availability. However, unlike conventional grafts obtained through donation after brain death, DCD cardiac grafts are not only exposed to warm, unprotected ischemia, but also to a potentially damaging pre-ischemic phase after withdrawal of life-sustaining therapy (WLST). In this review, we aim to bring together knowledge about changes in cardiac energy metabolism and its regulation that occur in DCD donors during WLST, circulatory arrest, and following the onset of warm ischemia. Acute metabolic, hemodynamic, and biochemical changes in the DCD donor expose hearts to high circulating catecholamines, hypoxia, and warm ischemia, all of which can negatively impact the heart. Further metabolic changes and cellular damage occur with reperfusion. The altered energy substrate availability prior to organ procurement likely plays an important role in graft quality and post-ischemic cardiac recovery. These aspects should, therefore, be considered in clinical protocols, as well as in pre-clinical DCD models. Notably, interventions prior to graft procurement are limited for ethical reasons in DCD donors; thus, it is important to understand these mechanisms to optimize conditions during initial reperfusion in concert with graft evaluation and re-evaluation for the purpose of tailoring and adjusting therapies and ensuring optimal graft quality for transplantation.
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Trasplante de Corazón , Humanos , Trasplante de Corazón/métodos , Preservación de Órganos/métodos , Obtención de Tejidos y Órganos/métodos , Animales , Perfusión/métodos , Donantes de Tejidos , Metabolismo EnergéticoRESUMEN
Ischaemic heart disease, which often manifests clinically as myocardial infarction (MI), remains a major cause of mortality worldwide. Despite the development of effective pre-clinical cardioprotective therapies, clinical translation has been disappointing. Nevertheless, the 'reperfusion injury salvage kinase' (RISK) pathway appears to be a promising target for cardioprotection. This pathway is crucial for the induction of cardioprotection by numerous pharmacological and non-pharmacological interventions, such as ischaemic conditioning. An important component of the cardioprotective effects of the RISK pathway involves the prevention of mitochondrial permeability transition pore (MPTP) opening and subsequent cardiac cell death. Here, we will review the historical perspective of the RISK pathway and focus on its interaction with mitochondria in the setting of cardioprotection.
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Precondicionamiento Isquémico Miocárdico , Isquemia Miocárdica , Daño por Reperfusión Miocárdica , Humanos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Daño por Reperfusión Miocárdica/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial/farmacología , Isquemia Miocárdica/prevención & control , Isquemia Miocárdica/metabolismo , Mitocondrias/metabolismo , Mitocondrias Cardíacas/metabolismoRESUMEN
Aspirin loading (chewable or intravenous) as soon as possible after presentation is a class I recommendation by current ST elevation myocardial infarction (STEMI) guidelines. Earlier achievement of therapeutic antiplatelet effects by aspirin loading has long been considered the standard of care. However, the effects of the loading dose of aspirin (alone or in addition to a chronic maintenance oral dose) have not been studied. A large proportion of myocardial cell death occurs upon and after reperfusion (reperfusion injury). Numerous agents and interventions have been shown to limit infarct size in animal models when administered before or immediately after reperfusion. However, these interventions have predominantly failed to show significant protection in clinical studies. In the current review, we raise the hypothesis that aspirin loading may be the culprit. Data obtained from animal models consistently show that statins, ticagrelor, opiates, and ischemic postconditioning limit myocardial infarct size. In most of these studies, aspirin was not administered. However, when aspirin was administered before reperfusion (as is the case in the majority of studies enrolling STEMI patients), the protective effects of statin, ticagrelor, morphine, and ischemic postconditioning were attenuated, which can be plausibly attributable to aspirin loading. We therefore suggest studying the effects of aspirin loading before reperfusion on the infarct size limiting effects of statins, ticagrelor, morphine, and/ or postconditioning in large animal models using long reperfusion periods (at least 24 h). If indeed aspirin attenuates the protective effects, clinical trials should be conducted comparing aspirin loading to alternative antiplatelet regimens without aspirin loading in patients with STEMI undergoing primary percutaneous coronary intervention.
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Inhibidores de Hidroximetilglutaril-CoA Reductasas , Intervención Coronaria Percutánea , Infarto del Miocardio con Elevación del ST , Animales , Infarto del Miocardio con Elevación del ST/terapia , Aspirina , Ticagrelor , Intervención Coronaria Percutánea/efectos adversos , Derivados de la Morfina , Resultado del TratamientoRESUMEN
PURPOSE: Patients hospitalized with COVID-19 may develop a hyperinflammatory, dysregulated cytokine "storm" that rapidly progresses to acute respiratory distress syndrome, multiple organ dysfunction, and even death. Remote ischaemic conditioning (RIC) has elicited anti-inflammatory and cytoprotective benefits by reducing cytokines following sepsis in animal studies. Therefore, we investigated whether RIC would mitigate the inflammatory cytokine cascade induced by COVID-19. METHODS: We conducted a prospective, multicentre, randomized, sham-controlled, single-blind trial in Brazil and South Africa. Non-critically ill adult patients with COVID-19 pneumonia were randomly allocated (1:1) to receive either RIC (intermittent ischaemia/reperfusion applied through four 5-min cycles of inflation (20 mmHg above systolic blood pressure) and deflation of an automated blood-pressure cuff) or sham for approximately 15 days. Serum was collected following RIC/sham administration and analyzed for inflammatory cytokines using flow cytometry. The endpoint was the change in serum cytokine concentrations. Participants were followed for 30 days. RESULTS: Eighty randomized participants (40 RIC and 40 sham) completed the trial. Baseline characteristics according to trial intervention were overall balanced. Despite downward trajectories of all cytokines across hospitalization, we observed no substantial changes in cytokine concentrations after successive days of RIC. Time to clinical improvement was similar in both groups (HR 1.66; 95% CI, 0.938-2.948, p 0.08). Overall RIC did not demonstrate a significant impact on the composite outcome of all-cause death or clinical deterioration (HR 1.19; 95% CI, 0.616-2.295, p = 0.61). CONCLUSION: RIC did not reduce the hypercytokinaemia induced by COVID-19 or prevent clinical deterioration to critical care. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04699227.
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PURPOSE: Coronavirus disease 19 (COVID-19) has, to date, been diagnosed in over 130 million persons worldwide and is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Several variants of concern have emerged including those in the United Kingdom, South Africa, and Brazil. SARS-CoV-2 can cause a dysregulated inflammatory response known as a cytokine storm, which can progress rapidly to acute respiratory distress syndrome (ARDS), multi-organ failure, and death. Suppressing these cytokine elevations may be key to improving outcomes. Remote ischemic conditioning (RIC) is a simple, non-invasive procedure whereby a blood pressure cuff is inflated and deflated on the upper arm for several cycles. "RIC in COVID-19" is a pilot, multi-center, randomized clinical trial, designed to ascertain whether RIC suppresses inflammatory cytokine production. METHODS: A minimum of 55 adult patients with diagnosed COVID-19, but not of critical status, will be enrolled from centers in the United Kingdom, Brazil, and South Africa. RIC will be administered daily for up to 15 days. The primary outcome is the level of inflammatory cytokines that are involved in the cytokine storm that can occur following SARS-CoV-2 infection. The secondary endpoint is the time between admission and until intensive care admission or death. The in vitro cytotoxicity of patient blood will also be assessed using primary human cardiac endothelial cells. CONCLUSIONS: The results of this pilot study will provide initial evidence on the ability of RIC to suppress the production of inflammatory cytokines in the setting of COVID-19. TRIAL REGISTRATION: NCT04699227, registered January 7th, 2021.
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COVID-19 , Adulto , Cuidados Críticos , Síndrome de Liberación de Citoquinas/prevención & control , Citocinas , Células Endoteliales , Humanos , Proyectos Piloto , SARS-CoV-2 , Resultado del TratamientoRESUMEN
A substantial number of chronic coronary syndrome (CCS) patients undergoing percutaneous coronary intervention (PCI) experience periprocedural myocardial injury or infarction. Accurate diagnosis of these PCI-related complications is required to guide further management given that their occurrence may be associated with increased risk of major adverse cardiac events (MACE). Due to lack of scientific data, the cut-off thresholds of post-PCI cardiac troponin (cTn) elevation used for defining periprocedural myocardial injury and infarction, have been selected based on expert consensus opinions, and their prognostic relevance remains unclear. In this Consensus Document from the ESC Working Group on Cellular Biology of the Heart and European Association of Percutaneous Cardiovascular Interventions (EAPCI), we recommend, whenever possible, the measurement of baseline (pre-PCI) cTn and post-PCI cTn values in all CCS patients undergoing PCI. We confirm the prognostic relevance of the post-PCI cTn elevation >5× 99th percentile URL threshold used to define type 4a myocardial infarction (MI). In the absence of periprocedural angiographic flow-limiting complications or electrocardiogram (ECG) and imaging evidence of new myocardial ischaemia, we propose the same post-PCI cTn cut-off threshold (>5× 99th percentile URL) be used to define prognostically relevant 'major' periprocedural myocardial injury. As both type 4a MI and major periprocedural myocardial injury are strong independent predictors of all-cause mortality at 1 year post-PCI, they may be used as quality metrics and surrogate endpoints for clinical trials. Further research is needed to evaluate treatment strategies for reducing the risk of major periprocedural myocardial injury, type 4a MI, and MACE in CCS patients undergoing PCI.
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Enfermedad de la Arteria Coronaria , Lesiones Cardíacas , Infarto del Miocardio , Intervención Coronaria Percutánea , Biomarcadores , Consenso , Humanos , Infarto del Miocardio/etiología , Intervención Coronaria Percutánea/efectos adversos , Factores de Riesgo , Resultado del TratamientoRESUMEN
Myocardial infarction requires urgent reperfusion to salvage viable heart tissue. However, reperfusion increases infarct size further by promoting mitochondrial damage in cardiomyocytes. Exosomes from a wide range of different cell sources have been shown to activate cardioprotective pathways in cardiomyocytes, thereby reducing infarct size. Yet, it is currently challenging to obtain highly pure exosomes in quantities enough for clinical studies. To overcome this problem, we used exosomes isolated from CTX0E03 neuronal stem cells, which are genetically stable, conditionally inducible and can be produced on an industrial scale. However, it is unknown whether exosomes from neuronal stem cells may reduce cardiac ischaemia/reperfusion injury. In this study, we demonstrate that exosomes from differentiating CTX0E03 cells can reduce infarct size in mice. In an in vitro assay, these exosomes delayed cardiomyocyte mitochondrial permeability transition pore opening, which is responsible for cardiomyocyte death after reperfusion. The mechanism of MPTP inhibition was via gp130 signalling and the downstream JAK/STAT pathway. Our results support previous findings that exosomes from non-cardiomyocyte-related cells produce exosomes capable of protecting cardiomyocytes from myocardial infarction. We anticipate our findings may encourage scientists to use exosomes obtained from reproducible clinical-grade stocks of cells for their ischaemia/reperfusion studies.
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Receptor gp130 de Citocinas/metabolismo , Exosomas/fisiología , Janus Quinasa 1/metabolismo , Janus Quinasa 2/metabolismo , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/complicaciones , Células-Madre Neurales/fisiología , Sustancias Protectoras/administración & dosificación , Animales , Receptor gp130 de Citocinas/genética , Regulación de la Expresión Génica , Janus Quinasa 1/genética , Janus Quinasa 2/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/etiología , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Células-Madre Neurales/citologíaRESUMEN
The benefits of remote ischaemic conditioning (RIC) have been difficult to translate to humans, when considering traditional outcome measures, such as mortality and heart failure. This paper reviews the recent literature of the anti-inflammatory effects of RIC, with a particular focus on the innate immune response and cytokine inhibition. Given the current COVID-19 pandemic, the inflammatory hypothesis of cardiac protection is an attractive target on which to re-purpose such novel therapies. A PubMed/MEDLINE™ search was performed on July 13th 2020, for the key terms RIC, cytokines, the innate immune system and inflammation. Data suggest that RIC attenuates inflammation in animals by immune conditioning, cytokine inhibition, cell survival and the release of anti-inflammatory exosomes. It is proposed that RIC inhibits cytokine release via a reduction in nuclear factor kappa beta (NF-κB)-mediated NLRP3 inflammasome production. In vivo, RIC attenuates pro-inflammatory cytokine release in myocardial/cerebral infarction and LPS models of endotoxaemia. In the latter group, cytokine inhibition is associated with a profound survival benefit. Further clinical trials should establish whether the benefits of RIC in inflammation can be observed in humans. Moreover, we must consider whether uncomplicated MI and elective surgery are the most suitable clinical conditions in which to test this hypothesis.
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Citocinas/fisiología , Inmunidad Innata , Inflamación/terapia , Precondicionamiento Isquémico Miocárdico , Daño por Reperfusión Miocárdica/prevención & control , Animales , COVID-19/complicaciones , Supervivencia Celular , Vesículas Extracelulares/fisiología , Humanos , Inmunidad Humoral , Inflamación/sangre , Daño por Reperfusión Miocárdica/inmunologíaRESUMEN
Stroke remains one of the most common causes of death and disability worldwide. Several preclinical studies demonstrated that the brain can be effectively protected against ischaemic stroke by two seemingly distinct treatments: remote ischaemic conditioning (RIC), involving cycles of ischaemia/reperfusion applied to a peripheral organ or tissue, or by systemic administration of glucagon-like-peptide-1 (GLP-1) receptor (GLP-1R) agonists. The mechanisms underlying RIC- and GLP-1-induced neuroprotection are not completely understood. In this study, we tested the hypothesis that GLP-1 mediates neuroprotection induced by RIC and investigated the effect of GLP-1R activation on cerebral blood vessels, as a potential mechanism of GLP-1-induced protection against ischaemic stroke. A rat model of ischaemic stroke (90 min of middle cerebral artery occlusion followed by 24-h reperfusion) was used. RIC was induced by 4 cycles of 5 min left hind limb ischaemia interleaved with 5-min reperfusion periods. RIC markedly (by ~ 80%) reduced the cerebral infarct size and improved the neurological score. The neuroprotection established by RIC was abolished by systemic blockade of GLP-1R with a specific antagonist Exendin(9-39). In the cerebral cortex of GLP-1R reporter mice, ~ 70% of cortical arterioles displayed GLP-1R expression. In acute brain slices of the rat cerebral cortex, activation of GLP-1R with an agonist Exendin-4 had a strong dilatory effect on cortical arterioles and effectively reversed arteriolar constrictions induced by metabolite lactate or oxygen and glucose deprivation, as an ex vivo model of ischaemic stroke. In anaesthetised rats, Exendin-4 induced lasting increases in brain tissue PO2, indicative of increased cerebral blood flow. These results demonstrate that neuroprotection against ischaemic stroke established by remote ischaemic conditioning is mediated by a mechanism involving GLP-1R signalling. Potent dilatory effect of GLP-1R activation on cortical arterioles suggests that the neuroprotection in this model is mediated via modulation of cerebral blood flow and improved brain perfusion.
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Arteriolas/efectos de los fármacos , Circulación Cerebrovascular/efectos de los fármacos , Miembro Posterior/irrigación sanguínea , Incretinas/farmacología , Infarto de la Arteria Cerebral Media/prevención & control , Precondicionamiento Isquémico , Accidente Cerebrovascular Isquémico/prevención & control , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Animales , Arteriolas/metabolismo , Arteriolas/fisiopatología , Modelos Animales de Enfermedad , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/fisiopatología , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/fisiopatología , Masculino , Ratas Sprague-Dawley , Flujo Sanguíneo RegionalRESUMEN
Acute myocardial infarction (AMI) and the heart failure (HF) which may follow are among the leading causes of death and disability worldwide. As such, new therapeutic interventions are still needed to protect the heart against acute ischemia/reperfusion injury to reduce myocardial infarct size and prevent the onset of HF in patients presenting with AMI. However, the clinical translation of cardioprotective interventions that have proven to be beneficial in preclinical animal studies, has been challenging. One likely major reason for this failure to translate cardioprotection into patient benefit is the lack of rigorous and systematic in vivo preclinical assessment of the efficacy of promising cardioprotective interventions prior to their clinical evaluation. To address this, we propose an in vivo set of step-by-step criteria for IMproving Preclinical Assessment of Cardioprotective Therapies ('IMPACT'), for investigators to consider adopting before embarking on clinical studies, the aim of which is to improve the likelihood of translating novel cardioprotective interventions into the clinical setting for patient benefit.
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Insuficiencia Cardíaca , Infarto del Miocardio , Daño por Reperfusión , Animales , Insuficiencia Cardíaca/prevención & control , HumanosRESUMEN
Mesenchymal stromal/stem cells (MSCs) have been widely tested against many diseases, with more than 1000 registered clinical trials worldwide. Despite many setbacks, MSCs have been approved for the treatment of graft-versus-host disease and Crohn disease. However, it is increasingly clear that MSCs exert their therapeutic functions in a paracrine manner through the secretion of small extracellular vesicles (sEVs) of 50-200 nm in diameter. Unlike living cells that can persist long-term, sEVs are non-living and non-replicative and have a transient presence in the body. Their small size also renders sEV preparations highly amenable to sterilization by filtration. Together, acellular MSC-sEV preparations are potentially safer and easier to translate into the clinic than cellular MSC products. Nevertheless, there are inherent challenges in the development of MSC-sEV drug products. MSC-sEVs are products of living cells, and living cells are sensitive to changes in the external microenvironment. Consequently, quality control metrics to measure key identity and potency features of MSC-sEV preparations have to be specified during development of MSC-sEV therapeutics. The authors have previously described quantifiable assays to define the identity of MSC-sEVs. Here the authors discuss requirements for prospective potency assays to predict the therapeutic effectiveness of the drug substance in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Although potency assays should ideally reflect the mechanism of action (MoA), this is challenging because the MoA for the reported efficacy of MSC-sEV preparations against multiple diseases of diverse underlying pathology is likely to be complex and different for each disease and difficult to fully elucidate. Nevertheless, robust potency assays could be developed by identifying the EV attribute most relevant to the intended biological activity in EV-mediated therapy and quantifying the EV attribute. Specifically, the authors highlight challenges and mitigation measures to enhance the manufacture of consistent and reproducibly potent sEV preparations, to identify and select the appropriate EV attribute for potency assays despite a complex "work-in-progress" MoA and to develop assays likely to be compliant with regulatory guidance for assay validation.
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Vesículas Extracelulares , Enfermedad Injerto contra Huésped , Células Madre Mesenquimatosas , Humanos , Estudios ProspectivosRESUMEN
Acute myocardial infarction causes lethal injury to cardiomyocytes during both ischaemia and reperfusion (IR). It is important to define the precise mechanisms by which they die in order to develop strategies to protect the heart from IR injury. Necrosis is known to play a major role in myocardial IR injury. There is also evidence for significant myocardial death by other pathways such as apoptosis, although this has been challenged. Mitochondria play a central role in both of these pathways of cell death, as either a causal mechanism is the case of mitochondrial permeability transition leading to necrosis, or as part of the signalling pathway in mitochondrial cytochrome c release and apoptosis. Autophagy may impact this process by removing dysfunctional proteins or even entire mitochondria through a process called mitophagy. More recently, roles for other programmed mechanisms of cell death such as necroptosis and pyroptosis have been described, and inhibitors of these pathways have been shown to be cardioprotective. In this review, we discuss both mitochondrial and mitochondrial-independent pathways of the major modes of cell death, their role in IR injury and their potential to be targeted as part of a cardioprotective strategy. This article is part of a special Issue entitled 'Mitochondria as targets of acute cardioprotection' and emerged as part of the discussions of the European Union (EU)-CARDIOPROTECTION Cooperation in Science and Technology (COST) Action, CA16225.
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Mitocondrias/genética , Infarto del Miocardio/genética , Daño por Reperfusión Miocárdica/genética , Miocardio/metabolismo , Apoptosis/genética , Autofagia/genética , Muerte Celular/genética , Humanos , Mitocondrias/patología , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Miocardio/patología , Necrosis/genética , Necrosis/patología , Transducción de Señal/genéticaRESUMEN
Small extracellular vesicles (EVs) are novel players in vascular biology. However, a thorough understanding of their production and function remains elusive. Endothelial senescence is a key feature of vascular ageing and thus, is an attractive therapeutic target for the treatment of vascular disease. In this study, we sought to characterize the EV production of senescent endothelial cells. To achieve this, Human Umbilical Vascular Endothelial Cells (HUVECs) were replicated until they reached senescence, as determined by measurement of Senescence-Associated ß-Galactosidase activity via microscopy and flow cytometry. Expression of the endosomal marker Rab7 and the EV marker CD63 was determined by immunofluorescence. Small EVs were isolated by ultracentrifugation and characterized using electron microscopy, nanoparticle tracking analysis and immunoassays to assess morphology, size, concentration and expression of exosome markers CD9 and CD81. Migration of HUVECs in response to EVs was studied using a transwell assay. The results showed that senescent endothelial cells express higher levels of Rab7 and CD63. Moreover, senescent endothelial cells produced higher levels of CD9- and CD81-positive EVs. Additionally, small EVs from both young and senescent endothelial cells promoted HUVEC migration. Overall, senescent endothelial cells produce an increased number of functional small EVs, which may have a role in vascular physiology and disease.
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Senescencia Celular/genética , Células Endoteliales/metabolismo , Exosomas/genética , Vesículas Extracelulares/genética , Biomarcadores/metabolismo , Células Endoteliales/citología , Citometría de Flujo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Tetraspanina 29/genética , Tetraspanina 30/genética , beta-Galactosidasa/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7RESUMEN
Treatment of acute myocardial infarct patients (AMI) includes rapid restoration of coronary blood flow and pharmacological therapy aimed to prevent pain and maintain vessel patency. Many interventions have been investigated to offer additional protection. One such intervention is remote ischaemic conditioning (RIC) involving short-episodes of ischaemia of the arm with a blood pressure cuff, followed by reperfusion to protect the heart organs from subsequent severe ischaemia. However, the recent CONDI2-ERIC-PPCI multicentre study of RIC in STEMI showed no benefit in clinical outcome in low risk patients. It could also be argued that these patients were already in a partially protected state, highlighting the disconnect between animal- and clinical-based outcome studies. To improve potential translatability, we developed an animal model using pharmacological agents similar to those given to patients presenting with an AMI, prior to PPCI. Rats underwent MI on a combined background of an opioid agonist, heparin and a platelet-inhibitor thereby allowing us to assess whether additional cardioprotective strategies had any effect over and above this "cocktail". We demonstrated that the "background drugs" were protective in their own right, reducing MI from 57.5 ± 3.7% to 37.3 ± 2.9% (n = 11, p < 0.001). On this background of drugs, RIC did not add any further protection (38.0 ± 3.4%). However, using a caspase inhibitor, which acts via a different mechanistic pathway to RIC, we were able to demonstrate additional protection (20.6 ± 3.3%). This concept provides initial evidence to develop models which can be used to evaluate future animal-to-clinical translation in cardioprotective studies.
Asunto(s)
Analgésicos Opioides/farmacología , Anticoagulantes/farmacología , Inhibidores de Caspasas/farmacología , Circulación Coronaria/efectos de los fármacos , Extremidades/irrigación sanguínea , Precondicionamiento Isquémico Miocárdico , Infarto del Miocardio/prevención & control , Miocardio/patología , Inhibidores de Agregación Plaquetaria/farmacología , Animales , Modelos Animales de Enfermedad , Masculino , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Ratas Sprague-Dawley , Flujo Sanguíneo Regional , Supervivencia Tisular , Investigación Biomédica TraslacionalRESUMEN
Atherosclerotic plaques impair vascular function and can lead to arterial obstruction and tissue ischaemia. Rupture of an atherosclerotic plaque within a coronary artery can result in an acute myocardial infarction, which is responsible for significant morbidity and mortality worldwide. Prompt reperfusion can salvage some of the ischaemic territory, but ischaemia and reperfusion (IR) still causes substantial injury and is, therefore, a therapeutic target for further infarct limitation. Numerous cardioprotective strategies have been identified that can limit IR injury in animal models, but none have yet been translated effectively to patients. This disconnect prompts an urgent re-examination of the experimental models used to study IR. Since coronary atherosclerosis is the most prevalent morbidity in this patient population, and impairs coronary vessel function, it is potentially a major confounder in cardioprotective studies. Surprisingly, most studies suggest that atherosclerosis does not have a major impact on cardioprotection in mouse models. However, a major limitation of atherosclerotic animal models is that the plaques usually manifest in the aorta and proximal great vessels, and rarely in the coronary vessels. In this review, we examine the commonly used mouse models of atherosclerosis and their effect on coronary artery function and infarct size. We conclude that none of the commonly used strains of mice are ideal for this purpose; however, more recently developed mouse models of atherosclerosis fulfil the requirement for coronary artery lesions, plaque rupture and lipoprotein patterns resembling the human profile, and may enable the identification of therapeutic interventions more applicable in the clinical setting.
Asunto(s)
Enfermedades de la Aorta/complicaciones , Aterosclerosis/complicaciones , Enfermedad de la Arteria Coronaria/complicaciones , Infarto del Miocardio/etiología , Miocardio/patología , Placa Aterosclerótica , Animales , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/patología , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Ratones Noqueados para ApoE , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Fenotipo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Rotura Espontánea , Receptores Depuradores de Clase B/deficiencia , Receptores Depuradores de Clase B/genética , Especificidad de la EspecieRESUMEN
Mesenchymal stromal cells (MSCs) exhibit antiapoptotic and proangiogenic functions in models of myocardial infarction which may be mediated by secreted small extracellular vesicles (sEVs). However, MSCs have frequently been harvested from aged or diseased patients, while the isolated sEVs often contain high levels of impurities. Here, we studied the cardioprotective and proangiogenic activities of size-exclusion chromatography-purified sEVs secreted from human foetal amniotic fluid stem cells (SS-hAFSCs), possessing superior functional potential to that of adult MSCs. We demonstrated for the first time that highly pure (up to 1.7 × 1010 particles/µg protein) and thoroughly characterised SS-hAFSC sEVs protect rat hearts from ischaemia-reperfusion injury in vivo when administered intravenously prior to reperfusion (38 ± 9% infarct size reduction, p < 0.05). SS-hAFSC sEVs did not protect isolated primary cardiomyocytes in models of simulated ischaemia-reperfusion injury in vitro, indicative of indirect cardioprotective effects. SS-hAFSC sEVs were not proangiogenic in vitro, although they markedly stimulated endothelial cell migration. Additionally, sEVs were entirely responsible for the promigratory effects of the medium conditioned by SS-hAFSC. Mechanistically, sEV-induced chemotaxis involved phosphatidylinositol 3-kinase (PI3K) signalling, as its pharmacological inhibition in treated endothelial cells reduced migration by 54 ± 7% (p < 0.001). Together, these data indicate that SS-hAFSC sEVs have multifactorial beneficial effects in a myocardial infarction setting.
Asunto(s)
Líquido Amniótico/citología , Cardiotónicos/metabolismo , Movimiento Celular , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Daño por Reperfusión/metabolismo , Animales , Quimiotaxis , Humanos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Fosfatidilinositol 3-Quinasa/metabolismo , Ratas , Daño por Reperfusión/patologíaRESUMEN
Despite an increase in the rates of survival in patients suffering myocardial infarction, as yet there is no therapy specifically targeting ischaemia and reperfusion injury of the myocardium. With a greater understanding of immune activation during infarction, more potential treatment targets are now being identified. The innate immune system is believed to play an important role in the myocardium after ischaemia-driven cardiomyocyte death. The release of intracellular contents including DNA into the extracellular space during necrosis and cell rupture is now believed to create a pro-inflammatory milieu which propagates the inflammatory process. DNA and DNA fragments have been shown to activate the innate immune system by acting as Danger-Associated Molecular Patterns (DAMPs), which act as ligands on toll-like receptors (TLRs). Stimulation of TLRs, in turn, can activate intracellular cell death pathways such as pyroptosis. Here, we review the role of DNA fragments during ischaemia and reperfusion, and assess their potential as a target in the quest to preserve cardiomyocyte viability following myocardial infarction.