Health position paper and redox perspectives on reactive oxygen species as signals and targets of cardioprotection.

The present review summarizes the beneficial and detrimental roles of reactive oxygen species in myocardial ischemia/reperfusion injury and cardioprotection. In the first part, the continued need for cardioprotection beyond that by rapid reperfusion of acute myocardial infarction is emphasized. Then, pathomechanisms of myocardial ischemia/reperfusion to the myocardium and the coronary circulation and the different modes of cell death in myocardial infarction are characterized. Different mechanical and pharmacological interventions to protect the ischemic/reperfused myocardium in elective percutaneous coronary interventions and coronary artery bypass grafting, in acute myocardial infarction and in cardiotoxicity from cancer therapy are detailed. The second part keeps the focus on ROS providing a comprehensive overview of molecular and cellular mechanisms involved in ischemia/reperfusion injury. Starting from mitochondria as the main sources and targets of ROS in ischemic/reperfused myocardium, a complex network of cellular and extracellular processes is discussed, including relationships with Ca2+ homeostasis, thiol group redox balance, hydrogen sulfide modulation, cross-talk with NAPDH oxidases, exosomes, cytokines and growth factors. While mechanistic insights are needed to improve our current therapeutic approaches, advancements in knowledge of ROS-mediated processes indicate that detrimental facets of oxidative stress are opposed by ROS requirement for physiological and protective reactions. This inevitable contrast is likely to underlie unsuccessful clinical trials and limits the development of novel cardioprotective interventions simply based upon ROS removal.

Anthracyclines and Biomarkers of Myocardial Injury: The Effect of Remote Ischemic Conditioning.

Background: Remote ischemic conditioning (RIC) has been beneficial in laboratory studies of anthracycline cardiotoxicity, but its effects in patients is not established. Objectives: The authors studied the effect of RIC on cardiac biomarkers and function during and after anthracycline chemotherapy. Methods: The ERIC-Onc study (Effect of Remote Ischaemic Conditioning in Oncology Patients; NCT02471885) was a randomized, single-blind, sham-controlled study of RIC at each chemotherapy cycle. The primary endpoint was troponin T (TnT) during chemotherapy and up to 1 year. Secondary outcomes included cardiac function, major adverse cardiovascular events (MACE), and MACE or cancer death. Cardiac myosin-binding-protein C (cMyC) was investigated in parallel with TnT. Results: The study was prematurely halted after the evaluation of 55 patients (RIC n = 28, sham n = 27). Biomarkers increased from baseline to cycle 6 of chemotherapy for all patients (median TnT 6 [IQR: 4-9] ng/L to 33 [IQR: 16-36)] ng/L; P ≤ 0.001; cMyC 3 (IQR: 2-5) ng/L to 47 (IQR: 18-49) ng/L; P ≤ 0.001). Mixed-effects regression analysis for repeated measures showed no difference in TnT between the 2 groups (RIC vs sham, mean difference 3.15 ng/L; 95% CI: -0.04 to 6.33; P = 0.053), or cMyC (RIC vs sham, mean difference 4.17 ng/L; 95% CI: -0.12 to 8.45; P = 0.056). There were more MACE and cancer deaths in the RIC group (11 vs 3; HR: 0.25; 95% CI: 0.07-0.90; P = 0.034), with more cancer deaths (8 vs 1; HR: 0.21; 95% CI: 0.04-0.95; P = 0.043) at 1 year. Conclusions: TnT and cMyC significantly increased during anthracycline chemotherapy with 81% having a TnT ≥14 ng/L at cycle 6. RIC did not affect the rise in biomarkers, but there was a small increase in early cancer deaths, possibly related to the greater proportion of patients with metastatic disease randomized to the RIC group (54%vs 37%). (Effect of Remote Ischaemic Conditioning in Oncology Patients [ERIC-ONC]; NCT02471885).

Defining Peri-Operative Myocardial Injury during Cardiac Surgery Using High-Sensitivity Troponin T.

OBJECTIVE: Cut-offs for high-sensitivity troponin (hs-Tn) elevations to define prognostically significant peri-operative myocardial injury (PMI) in cardiac surgery is not well-established. We evaluated the associations between peri-operative high-sensitivity troponin T (hs-TnT) elevations and 1-year all-cause mortality in patients undergoing cardiac surgery. METHODS: The prognostic significance of baseline hs-TnT and various thresholds for post-operative hs-TnT elevation at different time-points on 1-year all-cause mortality following cardiac surgery were assessed after adjusting for baseline hs-TnT and EuroSCORE in a post-hoc analysis of the ERICCA trial. RESULTS: 1206 patients met the inclusion criteria. Baseline elevation in hs-TnT >x1 99th percentile upper reference limit (URL) was significantly associated with 1-year all-cause mortality (adjusted hazard ratio 1.90, 95% confidence interval 1.15-3.13). In the subgroup with normal baseline hs-TnT (n = 517), elevation in hs-TnT at all post-operative time points was associated with higher 1-year mortality, reaching statistical significance for elevations above: ≥100 × URL at 6 h; ≥50 × URL at 12 and 24 h; ≥35 × URL at 48 h; and ≥30 × URL at 72 h post-surgery. Elevation in hs-TnT at 24 h ≥ 50 × URL had the optimal sensitivity and specificity (73% and 75% respectively). When the whole cohort of patients was analysed, including those with abnormal baseline hs-TnT (up to 10 × URL), the same threshold had optimal sensitivity and specificity (66% and 70%). CONCLUSIONS: Both baseline and post-operative hs-TnT elevations are independently associated with 1-year all-cause mortality in patients undergoing cardiac surgery. The optimal threshold to define a prognostically significant PMI in our study was ≥50 × URL elevation in hs-TnT at 24 h.

Remote Ischemic Conditioning: Challenges and Opportunities.

Remote ischemic conditioning (RIC) has been investigated as a promising, safe, and well-tolerated nonpharmacological therapy for cardio-cerebrovascular disease over the past 3 decades; variable results have been found when it is used in cerebrovascular versus cardiovascular disease. For patients with cardiovascular disease, milestone studies suggest that the roles of RIC may be limited. Recently, however, 2 large trials investigating RIC in patients with cerebrovascular disease found promising results, which may reignite the field's research prospects after its setbacks in the cardiovascular field. This perspectives article highlights several important clinical trials of RIC in the cardio-cerebrovascular disease and describes the many challenges of RIC in clinical translation. Finally, based on the available evidence, several promising research directions such as chronic RIC, early initiation in target population, improvement of compliance, better understanding of dosing, and identification of specific biomarkers are proposed and should be investigated before RIC can become applied into clinical practice for patient benefit.

The RISK pathway leading to mitochondria and cardioprotection: how everything started.

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.

A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.

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.

Remote Ischaemic Conditioning in STEMI Patients in Sub-Saharan AFRICA: Rationale and Study Design for the RIC-AFRICA Trial.

PURPOSE: Despite evidence of myocardial infarct size reduction in animal studies, remote ischaemic conditioning (RIC) failed to improve clinical outcomes in the large CONDI-2/ERIC-PPCI trial. Potential reasons include that the predominantly low-risk study participants all received timely optimal reperfusion therapy by primary percutaneous coronary intervention (PPCI). Whether RIC can improve clinical outcomes in higher-risk STEMI patients in environments with poor access to early reperfusion or PPCI will be investigated in the RIC-AFRICA trial. METHODS: The RIC-AFRICA study is a sub-Saharan African multi-centre, randomized, double-blind, sham-controlled clinical trial designed to test the impact of RIC on the composite endpoint of 30-day mortality and heart failure in 1200 adult STEMI patients without access to PPCI. Randomized participants will be stratified by whether or not they receive thrombolytic therapy within 12 h or arrive outside the thrombolytic window (12-24 h). Participants will receive either RIC (four 5-min cycles of inflation [20 mmHg above systolic blood pressure] and deflation of an automated blood pressure cuff placed on the upper arm) or sham control (similar protocol but with low-pressure inflation of 20 mmHg and deflation) within 1 h of thrombolysis and applied daily for the next 2 days. STEMI patients arriving greater than 24 h after chest pain but within 72 h will be recruited to participate in a concurrently running independent observational arm. CONCLUSION: The RIC-AFRICA trial will determine whether RIC can reduce rates of death and heart failure in higher-risk sub-optimally reperfused STEMI patients, thereby providing a low-cost, non-invasive therapy for improving health outcomes.

Effect of Remote Ischaemic Conditioning on the Inflammatory Cytokine Cascade of COVID-19 (RIC in COVID-19): a Randomized Controlled Trial.

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.

Fasting increases susceptibility to acute myocardial ischaemia/reperfusion injury through a sirtuin-3 mediated increase in fatty acid oxidation.

Fasting increases susceptibility to acute myocardial ischaemia/reperfusion injury (IRI) but the mechanisms are unknown. Here, we investigate the role of the mitochondrial NAD+-dependent deacetylase, Sirtuin-3 (SIRT3), which has been shown to influence fatty acid oxidation and cardiac outcomes, as a potential mediator of this effect. Fasting was shown to shift metabolism from glucose towards fatty acid oxidation. This change in metabolic fuel substrate utilisation increased myocardial infarct size in wild-type (WT), but not SIRT3 heterozygous knock-out (KO) mice. Further analysis revealed SIRT3 KO mice were better adapted to starvation through an improved cardiac efficiency, thus protecting them from acute myocardial IRI. Mitochondria from SIRT3 KO mice were hyperacetylated compared to WT mice which may regulate key metabolic processes controlling glucose and fatty acid utilisation in the heart. Fasting and the associated metabolic switch to fatty acid respiration worsens outcomes in WT hearts, whilst hearts from SIRT3 KO mice are better adapted to oxidising fatty acids, thereby protecting them from acute myocardial IRI.

Negative interaction between nitrates and remote ischemic preconditioning in patients undergoing cardiac surgery: the ERIC-GTN and ERICCA studies.

Remote ischaemic preconditioning (RIPC) using transient limb ischaemia failed to improve clinical outcomes following cardiac surgery and the reasons for this remain unclear. In the ERIC-GTN study, we evaluated whether concomitant nitrate therapy abrogated RIPC cardioprotection. We also undertook a post-hoc analysis of the ERICCA study, to investigate a potential negative interaction between RIPC and nitrates on clinical outcomes following cardiac surgery. In ERIC-GTN, 185 patients undergoing cardiac surgery were randomized to: (1) Control (no RIPC or nitrates); (2) RIPC alone; (3); Nitrates alone; and (4) RIPC + Nitrates. An intravenous infusion of nitrates (glyceryl trinitrate 1 mg/mL solution) was commenced on arrival at the operating theatre at a rate of 2-5 mL/h to maintain a mean arterial pressure between 60 and 70 mmHg and was stopped when the patient was taken off cardiopulmonary bypass. The primary endpoint was peri-operative myocardial injury (PMI) quantified by a 48-h area-under-the-curve high-sensitivity Troponin-T (48 h-AUC-hs-cTnT). In ERICCA, we analysed data for 1502 patients undergoing cardiac surgery to investigate for a potential negative interaction between RIPC and nitrates on clinical outcomes at 12-months. In ERIC-GTN, RIPC alone reduced 48 h-AUC-hs-cTnT by 37.1%, when compared to control (ratio of AUC 0.629 [95% CI 0.413-0.957], p = 0.031), and this cardioprotective effect was abrogated in the presence of nitrates. Treatment with nitrates alone did not reduce 48 h-AUC-hs-cTnT, when compared to control. In ERICCA there was a negative interaction between nitrate use and RIPC for all-cause and cardiovascular mortality at 12-months, and for risk of peri-operative myocardial infarction. RIPC alone reduced the risk of peri-operative myocardial infarction, compared to control, but no significant effect of RIPC was demonstrated for the other outcomes. When RIPC and nitrates were used together they had an adverse impact in patients undergoing cardiac surgery with the presence of nitrates abrogating RIPC-induced cardioprotection and increasing the risk of mortality at 12-months post-cardiac surgery in patients receiving RIPC.

Cardiovascular outcomes associated with treatment of type 2 diabetes in patients with ischaemic heart failure.

AIM: The optimal strategy for diabetes control in patients with heart failure (HF) following myocardial infarction (MI) remains unknown. Metformin, a guideline-recommended therapy for patients with chronic HF and type 2 diabetes mellitus (T2DM), is associated with reduced mortality and HF hospitalizations. However, worse outcomes have been reported when used at the time of MI. We compared outcomes of patients with T2DM and HF of ischaemic aetiology according to antidiabetic treatment. METHODS AND RESULTS: This study used linked data from primary care, hospital admissions, and death registries for 4.7 million inhabitants in England, as part of the CALIBER resource. The primary endpoint was a composite of cardiovascular mortality and HF hospitalization. The secondary endpoints were the individual components of the primary endpoint and all-cause mortality. To evaluate the effect of temporal changes in diabetes treatment, antidiabetic medication was included as time-dependent covariates in survival analyses. The study included 1172 patients with T2DM and prior MI and incident HF between 3 January 1998 and 26 February 2010. Five hundred and ninety-six patients had the primary outcome over median follow-up of 2.53 (IQR: 0.98-4.92) years. Adjusted analyses showed a reduced hazard of the composite endpoint for exposure to all antidiabetic medication with hazard ratios (HRs) of 0.50 [95% confidence interval (CI): 0.42-0.59], 0.66 (95% CI: 0.55-0.80), and 0.53 (95% CI: 0.43-0.65), respectively. A similar effect was seen for all-cause mortality [HRs of 0.43 (95% CI: 0.35-0.52), 0.57 (95% CI: 0.46-0.70), and 0.34 (95% CI: 0.27-0.43), respectively]. CONCLUSIONS: When considering changes in antidiabetic treatment over time, all drug classes were associated with reduced risk of cardiovascular mortality and HF hospitalization.

Do We Really Need Aspirin Loading for STEMI?

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.

RIC in COVID-19-a Clinical Trial to Investigate Whether Remote Ischemic Conditioning (RIC) Can Prevent Deterioration to Critical Care in Patients with COVID-19.

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.

Hydralazine protects the heart against acute ischaemia/reperfusion injury by inhibiting Drp1-mediated mitochondrial fission.

AIMS: Genetic and pharmacological inhibition of mitochondrial fission induced by acute myocardial ischaemia/reperfusion injury (IRI) has been shown to reduce myocardial infarct size. The clinically used anti-hypertensive and heart failure medication, hydralazine, is known to have anti-oxidant and anti-apoptotic effects. Here, we investigated whether hydralazine confers acute cardioprotection by inhibiting Drp1-mediated mitochondrial fission. METHODS AND RESULTS: Pre-treatment with hydralazine was shown to inhibit both mitochondrial fission and mitochondrial membrane depolarisation induced by oxidative stress in HeLa cells. In mouse embryonic fibroblasts (MEFs), pre-treatment with hydralazine attenuated mitochondrial fission and cell death induced by oxidative stress, but this effect was absent in MEFs deficient in the mitochondrial fission protein, Drp1. Molecular docking and surface plasmon resonance studies demonstrated binding of hydralazine to the GTPase domain of the mitochondrial fission protein, Drp1 (KD 8.6±1.0 µM), and inhibition of Drp1 GTPase activity in a dose-dependent manner. In isolated adult murine cardiomyocytes subjected to simulated IRI, hydralazine inhibited mitochondrial fission, preserved mitochondrial fusion events, and reduced cardiomyocyte death (hydralazine 24.7±2.5% vs. control 34.1±1.5%, P=0.0012). In ex vivo perfused murine hearts subjected to acute IRI, pre-treatment with hydralazine reduced myocardial infarct size (as % left ventricle: hydralazine 29.6±6.5% vs. vehicle control 54.1±4.9%, P=0.0083), and in the murine heart subjected to in vivo IRI, the administration of hydralazine at reperfusion, decreased myocardial infarct size (as % area-at-risk: hydralazine 28.9±3.0% vs. vehicle control 58.2±3.8%, P<0.001). CONCLUSION: We show that, in addition to its antioxidant and anti-apoptotic effects, hydralazine, confers acute cardioprotection by inhibiting IRI-induced mitochondrial fission, raising the possibility of repurposing hydralazine as a novel cardioprotective therapy for improving post-infarction outcomes.

Effect of remote ischaemic conditioning on infarct size and remodelling in ST-segment elevation myocardial infarction patients: the CONDI-2/ERIC-PPCI CMR substudy.

The effect of limb remote ischaemic conditioning (RIC) on myocardial infarct (MI) size and left ventricular ejection fraction (LVEF) was investigated in a pre-planned cardiovascular magnetic resonance (CMR) substudy of the CONDI-2/ERIC-PPCI trial. This single-blind multi-centre trial (7 sites in UK and Denmark) included 169 ST-segment elevation myocardial infarction (STEMI) patients who were already randomised to either control (n = 89) or limb RIC (n = 80) (4 × 5 min cycles of arm cuff inflations/deflations) prior to primary percutaneous coronary intervention. CMR was performed acutely and at 6 months. The primary endpoint was MI size on the 6 month CMR scan, expressed as median and interquartile range. In 110 patients with 6-month CMR data, limb RIC did not reduce MI size [RIC: 13.0 (5.1-17.1)% of LV mass; control: 11.1 (7.0-17.8)% of LV mass, P = 0.39], or LVEF, when compared to control. In 162 patients with acute CMR data, limb RIC had no effect on acute MI size, microvascular obstruction and LVEF when compared to control. In a subgroup of anterior STEMI patients, RIC was associated with lower incidence of microvascular obstruction and higher LVEF on the acute scan when compared with control, but this was not associated with an improvement in LVEF at 6 months. In summary, in this pre-planned CMR substudy of the CONDI-2/ERIC-PPCI trial, there was no evidence that limb RIC reduced MI size or improved LVEF at 6 months by CMR, findings which are consistent with the neutral effects of limb RIC on clinical outcomes reported in the main CONDI-2/ERIC-PPCI trial.

Glucagon-like peptide-1 (GLP-1) receptor activation dilates cerebral arterioles, increases cerebral blood flow, and mediates remote (pre)conditioning neuroprotection against ischaemic stroke.

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.

Exosomes from neuronal stem cells may protect the heart from ischaemia/reperfusion injury via JAK1/2 and gp130.

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.