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 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.

Phenotypic changes in low-density lipoprotein particles as markers of adverse clinical outcomes in COVID-19.

BACKGROUND AND AIMS: Low-density lipoprotein (LDL) plasma concentration decline is a biomarker for acute inflammatory diseases, including coronavirus disease-2019 (COVID-19). Phenotypic changes in LDL during COVID-19 may be equally related to adverse clinical outcomes. METHODS: Individuals hospitalized due to COVID-19 (n = 40) were enrolled. Blood samples were collected on days 0, 2, 4, 6, and 30 (D0, D2, D4, D6, and D30). Oxidized LDL (ox-LDL), and lipoprotein-associated phospholipase A2 (Lp-PLA2) activity were measured. In a consecutive series of cases (n = 13), LDL was isolated by gradient ultracentrifugation from D0 and D6 and was quantified by lipidomic analysis. Association between clinical outcomes and LDL phenotypic changes was investigated. RESULTS: In the first 30 days, 42.5% of participants died due to Covid-19. The serum ox-LDL increased from D0 to D6 (p < 0.005) and decreased at D30. Moreover, individuals who had an ox-LDL increase from D0 to D6 to over the 90th percentile died. The plasma Lp-PLA2 activity also increased progressively from D0 to D30 (p < 0.005), and the change from D0 to D6 in Lp-PLA2 and ox-LDL were positively correlated (r = 0.65, p < 0.0001). An exploratory untargeted lipidomic analysis uncovered 308 individual lipids in isolated LDL particles. Paired-test analysis from D0 and D6 revealed higher concentrations of 32 lipid species during disease progression, mainly represented by lysophosphatidyl choline and phosphatidylinositol. In addition, 69 lipid species were exclusively modulated in the LDL particles from non-survivors as compared to survivors. CONCLUSIONS: Phenotypic changes in LDL particles are associated with disease progression and adverse clinical outcomes in COVID-19 patients and could serve as a potential prognostic biomarker.

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.

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.

Remote Ischemic Preconditioning Protects Against Endothelial Dysfunction in a Human Model of Systemic Inflammation: A Randomized Clinical Trial.

OBJECTIVE: Inflammation, oxidative stress, and endothelial dysfunction are known to contribute to ischemia-reperfusion injury. Remote ischemic preconditioning (RIPC) protects from endothelial dysfunction and the damage induced by ischemiareperfusion. Using intensive periodontal treatment (IPT), an established human model of acute systemic inflammation, we investigated whether RIPC prevents endothelial dysfunction and modulates systemic levels of inflammation and oxidative stress. APPROACH AND RESULTS: Forty-nine participants with periodontitis were randomly allocated to receive either 3 cycles of ischemia-reperfusion on the upper limb (N=24, RIPC) or a sham procedure (N=25, control) before IPT. Endothelial function assessed by flow-mediated dilatation of the brachial artery, inflammatory cytokines, markers of vascular injury, and oxidative stress were evaluated at baseline, day 1, and day 7 after IPT. Twenty-four hours post-IPT, the RIPC group had lower levels of IL-10 (interleukin-10) and IL-12 (interleukin-12) compared with the control group (P<0.05). RIPC attenuated the IPTinduced increase in IL-1ß (interleukin-1ß), E-selectin, sICAM-3 (soluble intercellular adhesion molecule 3), and sTM (soluble thrombomodulin) levels between the baseline and day 1 (P for interaction <0.1). Conversely, oxidative stress was differentially increased at day1 in the RIPC group compared with the control group (P for interaction <0.1). This was accompanied by a better flow-mediated dilatation (mean difference 1.75% [95% CI, 0.428­3.07], P=0.011). After 7 days from IPT, most of the inflammatory markers, endothelial-dependent and -independent vasodilation, were similar between groups. CONCLUSIONS: RIPC prevented acute endothelial dysfunction by modulation of inflammation and oxidation processes in patients with periodontitis following exposure to an acute inflammatory stimulus. REGISTRATION: URL: https://clinicaltrials.gov/ct2/show/NCT03072342; Unique identifier: NCT03072342.

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.

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.

Cardio-oncology Issues in Lymphoma Patients.

BACKGROUND: Advances in Lymphoma management have resulted in significant improvements in patient outcomes over the last 50 years. Despite these developments, cardiotoxicity from lymphoma treatments remains an important cause of mortality and morbidity in this cohort of patients. We outlined the most common cardiotoxicities associated with lymphoma treatments and their respective investigation and management strategies, including the role of cardiac pre-assessment and late effects monitoring.

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.

Does remote ischaemic conditioning reduce inflammation? A focus on innate immunity and cytokine response.

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.

Translation of experimental cardioprotective capability of P2Y12 inhibitors into clinical outcome in patients with ST-elevation myocardial infarction.

We studied the translational cardioprotective potential of P2Y12 inhibitors against acute myocardial ischemia/reperfusion injury (IRI) in an animal model of acute myocardial infarction and in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). P2Y12 inhibitors may have pleiotropic effects to induce cardioprotection against acute myocardial IRI beyond their inhibitory effects on platelet aggregation. We compared the cardioprotective effects of clopidogrel, prasugrel, and ticagrelor on infarct size in an in vivo rat model of acute myocardial IRI, and investigated the effects of the P2Y12 inhibitors on enzymatic infarct size (48-h area-under-the-curve (AUC) troponin T release) and clinical outcomes in a retrospective study of STEMI patients from the CONDI-2/ERIC-PPCI trial using propensity score analyses. Loading with ticagrelor in rats reduced infarct size after acute myocardial IRI compared to controls (37 ± 11% vs 52 ± 8%, p < 0.01), whereas clopidogrel and prasugrel did not (50 ± 11%, p > 0.99 and 49 ± 9%, p > 0.99, respectively). Correspondingly, troponin release was reduced in STEMI patients treated with ticagrelor compared to clopidogrel (adjusted 48-h AUC ratio: 0.67, 95% CI 0.47-0.94). Compared to clopidogrel, the composite endpoint of cardiac death or hospitalization for heart failure within 12 months was reduced in STEMI patients loaded with ticagrelor (HR 0.63; 95% CI 0.42-0.94) but not prasugrel (HR 0.84, 95% CI 0.43-1.63), prior to PPCI. Major adverse cardiovascular events did not differ between clopidogrel, ticagrelor, or prasugrel. The cardioprotective effects of ticagrelor in reducing infarct size may contribute to the clinical benefit observed in STEMI patients undergoing PPCI.

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.

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.

Increased production of functional small extracellular vesicles in senescent endothelial cells.

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.

Effect of remote ischaemic conditioning on clinical outcomes in patients with acute myocardial infarction (CONDI-2/ERIC-PPCI): a single-blind randomised controlled trial.

BACKGROUND: Remote ischaemic conditioning with transient ischaemia and reperfusion applied to the arm has been shown to reduce myocardial infarct size in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). We investigated whether remote ischaemic conditioning could reduce the incidence of cardiac death and hospitalisation for heart failure at 12 months. METHODS: We did an international investigator-initiated, prospective, single-blind, randomised controlled trial (CONDI-2/ERIC-PPCI) at 33 centres across the UK, Denmark, Spain, and Serbia. Patients (age >18 years) with suspected STEMI and who were eligible for PPCI were randomly allocated (1:1, stratified by centre with a permuted block method) to receive standard treatment (including a sham simulated remote ischaemic conditioning intervention at UK sites only) or remote ischaemic conditioning treatment (intermittent ischaemia and reperfusion applied to the arm through four cycles of 5-min inflation and 5-min deflation of an automated cuff device) before PPCI. Investigators responsible for data collection and outcome assessment were masked to treatment allocation. The primary combined endpoint was cardiac death or hospitalisation for heart failure at 12 months in the intention-to-treat population. This trial is registered with ClinicalTrials.gov (NCT02342522) and is completed. FINDINGS: Between Nov 6, 2013, and March 31, 2018, 5401 patients were randomly allocated to either the control group (n=2701) or the remote ischaemic conditioning group (n=2700). After exclusion of patients upon hospital arrival or loss to follow-up, 2569 patients in the control group and 2546 in the intervention group were included in the intention-to-treat analysis. At 12 months post-PPCI, the Kaplan-Meier-estimated frequencies of cardiac death or hospitalisation for heart failure (the primary endpoint) were 220 (8·6%) patients in the control group and 239 (9·4%) in the remote ischaemic conditioning group (hazard ratio 1·10 [95% CI 0·91-1·32], p=0·32 for intervention versus control). No important unexpected adverse events or side effects of remote ischaemic conditioning were observed. INTERPRETATION: Remote ischaemic conditioning does not improve clinical outcomes (cardiac death or hospitalisation for heart failure) at 12 months in patients with STEMI undergoing PPCI. FUNDING: British Heart Foundation, University College London Hospitals/University College London Biomedical Research Centre, Danish Innovation Foundation, Novo Nordisk Foundation, TrygFonden.

The importance of clinically relevant background therapy in cardioprotective studies.

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.

Mouse models of atherosclerosis and their suitability for the study of myocardial infarction.

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.