The pleiotropic effects of metformin: time for prospective studies

The global prevalence of diabetes has risen to epidemic proportions and the trend is predicted to continue. The consequent burden of cardiovascular morbidity and mortality is a major public health concern and new treatments are required to mitigate the deleterious effects of cardiovascular disease in diabetic patients. Ischaemia-reperfusion injury is well known to exacerbate the harmful effects of acute myocardial infarction and subsequent therapeutic reperfusion, and several mechanical and pharmacological approaches to mitigating this injury have been investigated. Metformin, which is cheap, relatively safe and widely used in type 2 diabetes, is one such pharmacotherapy with considerable pre-clinical evidence for cardioprotective utility beyond its glucose-lowering effect. However, despite convincing basic evidence its translation to clinical application has largely been limited to studies of cardiovascular risk. There are several barriers to prospective randomized assessment in the context of acute myocardial infarction, not least the accessibility and already widespread use of metformin among patients with type 2 diabetes at high risk of cardiovascular events. In the place of class 1 evidence, well-designed prospective cohort studies of the potential pleiotropic utility of metformin in cardiovascular disease, and particularly its benefit in ischaemia-reperfusion injury, are needed. Given the availability of metformin worldwide, this is particularly true in low- and middle-income countries where the optimal therapy for acute myocardial infarction, primary percutaneous coronary intervention, may not be available, and instead patients are managed with thrombolysis. As this is less effective, metformin as an adjunct to thrombolysis (or PPCI) could represent an effective, cheap means of cardioprotection with global relevance

The potential role of stromal derived factor 1α in remote ischaemic conditioning

Background Alleviating the injury associated with ST-elevation myocardial infarction is central to improving the global burden of coronary heart disease. The chemokine stromal cell-derived factor 1α (SDF-1α) and its receptor, CXCR4, have dual potential benefit in this regard: acutely protecting the heart from lethal ischaemia-reperfusion injury (IRI) whilst mitigating adverse ventricular remodelling by recruiting progenitor cells to the site of injury. This project hypothesised that SDF-1α mediates the acute cardioprotection conferred by remote ischaemic conditioning (RIC), the phenomenon whereby brief cycles of non-lethal tissue ischaemia and reperfusion remote from the heart protects against myocardial IRI. Methods and Results This thesis defines a paradigm for evidencing a role in RIC that includes induction of cardioprotection by exogenous administration of SDF-1α at the time of reperfusion, abolition of cardioprotection by specific antagonism of CXCR4, increased production of SDF-1α as a direct effect of RIC, and absence of cardioprotection in CXCR4-deficient mice. A murine in vivo model of myocardial IRI and a novel ELISA for active SDF-1α were established and used to investigate this paradigm. This thesis provides the first description of cardioprotection against myocardial IRI as a result of exogenous SDF-1α administered prior to reperfusion. Moreover, AMD3100, a highly specific inhibitor of CXCR4, abolishes the beneficial effect of RIC in vivo. Next, SDF-1α cleavage and inactivation was unexpectedly demonstrated to increase after RIC, which may be attributable to up-regulation of dipeptidyl peptidase-4. Finally, inducible cardiomyocyte-specific CXCR4 deletion unexpectedly conferred protection against myocardial IRI. The protective mechanism was not established and, furthermore, it prohibited the use of these mice in experiments to validate the role of CXCR4 signalling in RIC. Conclusions The intrinsic role of SDF-1α in RIC remains equivocal. However, modulation of the SDF-1α-CXCR4 axis with other approaches, including exogenous SDF-1α, has potential utility in cardioprotection against myocardial IRI

Therapeutic strategies utilising SDF-1α in ischaemic cardiomyopathy

Heart failure is rapidly increasing in prevalence and will redraw the global landscape for cardiovascular health. Alleviating and repairing cardiac injury associated with myocardial infarction (MI) is key to improving this burden. Homing signals mobilise and recruit stem cells to the ischaemic myocardium where they exert beneficial paracrine effects. The chemoattractant cytokine SDF-1α and its associated receptor CXCR4 are upregulated after MI and appear to be important in this context. Activation of CXCR4 promotes both cardiomyocyte survival and stem cell migration towards the infarcted myocardium. These effects have beneficial effects on infarct size, and left ventricular remodelling and function. However, the timing of endogenous SDF-1α release and CXCR4 upregulation may not be optimal. Furthermore, current ELISA-based assays cannot distinguish between active SDF-1α, and SDF-1α inactivated by dipeptidyl peptidase 4 (DPP4). Current therapeutic approaches aim to recruit the SDF-1α-CXCR4 pathway or prolong SDF-1α life-time by preventing its cleavage by DPP4. This review assesses the evidence supporting these approaches and proposes SDF-1α as an important confounder in recent studies of DPP4 inhibitors

A novel recombinant antibody specific to full-length stromal derived factor-1 for potential application in biomarker studies

Background: Stromal derived factor-1α (SDF-1α/CXCL12) is a chemokine that is up-regulated in diseases characterised by tissue hypoxia, including myocardial infarction, ischaemic cardiomyopathy and remote ischaemic conditioning (RIC), a technique of cyclical, non-injurious ischaemia applied remote from the heart that protects the heat from lethal ischaemia-reperfusion injury. Accordingly, there is considerable interest in SDF-1α as a potential biomarker of such conditions. However, SDF-1α is rapidly degraded and inactivated by dipeptidyl peptidase 4 and other peptidases, and the kinetics of intact SDF-1α remain unknown. Methods & results: To facilitate investigation of full-length SDF-1α we established an ELISA using a novel recombinant human antibody we developed called HCI.SDF1. HCI.SDF1 is specific to the N-terminal sequence of all isoforms of SDF-1 and has a comparable KD to commercially available antibodies. Together with a detection antibody specific to the α-isoform, HCI.SDF1 was used to specifically quantify full-length SDF-1α in blood for the first time. Using RIC applied to the hind limb of Sprague-Dawley rats or the arms of healthy human volunteers, we demonstrate an increase in SDF-1α using a commercially available antibody, as previously reported, but an unexpected decrease in full-length SDF-1α after RIC in both species. Conclusions: We report for the first time the development of a novel recombinant antibody specific to fulllength SDF-1. Applied to RIC, we demonstrate a significant decrease in SDF-1α that is at odds with the literature and suggests a need to investigate the kinetics of full-length SDF-1α in conditions characterised by tissue hypoxia

Remote ischaemic conditioning reduces infarct size in animal in vivo models of ischaemia-reperfusion injury: a systematic review and meta-analysis

AIMS: The potential of remote ischaemic conditioning (RIC) to ameliorate myocardial ischaemia-reperfusion injury (IRI) remains controversial. We aimed to analyse the pre-clinical evidence base to ascertain the overall effect and variability of RIC in animal in vivo models of myocardial IRI. Furthermore, we aimed to investigate the impact of different study protocols on the protective utility of RIC in animal models and identify gaps in our understanding of this promising therapeutic strategy. METHODS AND RESULTS: Our primary outcome measure was the difference in mean infarct size between RIC and control groups in in vivo models of myocardial IRI. A systematic review returned 31 reports, from which we made 22 controlled comparisons of remote ischaemic preconditioning (RIPreC) and 21 of remote ischaemic perconditioning and postconditioning (RIPerC/RIPostC) in a pooled random-effects meta-analysis. In total, our analysis includes data from 280 control animals and 373 animals subject to RIC. Overall, RIPreC reduced infarct size as a percentage of area at risk by 22.8% (95% CI 18.8-26.9%), when compared with untreated controls (P < 0.001). Similarly, RIPerC/RIPostC reduced infarct size by 22.2% (95% CI 17.1-25.3%; P < 0.001). Interestingly, we observed significant heterogeneity in effect size (T2 = 92.9% and I2 = 99.4%; P < 0.001) that could not be explained by any of the experimental variables analysed by meta-regression. However, few reports have systematically characterized RIC protocols, and few of the included in vivo studies satisfactorily met study quality requirements, particularly with respect to blinding and randomization. CONCLUSIONS: RIC significantly reduces infarct size in in vivo models of myocardial IRI. Heterogeneity between studies could not be explained by the experimental variables tested, but studies are limited in number and lack consistency in quality and study design. There is therefore a clear need for more well-performed in vivo studies with particular emphasis on detailed characterization of RIC protocols and investigating the potential impact of gender. Finally, more studies investigating the potential benefit of RIC in larger species are required before translation to humans

Stromal cell-derived factor-1α signals via the endothelium to protect the heart against ischaemia-reperfusion injury

AIMS: The chemokine stromal derived factor-1α (SDF-1α) is known to protect the heart acutely from ischaemia-reperfusion injury via its cognate receptor, CXCR4. However, the timing and cellular location of this effect, remains controversial. METHODS AND RESULTS: Wild type male and female mice were subjected to 40 min LAD territory ischaemia in vivo and injected with either saline (control) or SDF-1α prior to 2 h reperfusion. Infarct size as a proportion of area at risk was assessed histologically using Evans blue and triphenyltetrazolium chloride. Our results confirm the cardioprotective effect of exogenous SDF-1α in mouse ischaemia-reperfusion injury and, for the first time, show protection when SDF-1α is delivered just prior to reperfusion, which has important therapeutic implications. The role of cell type was examined using the same in vivo ischaemia-reperfusion protocol in cardiomyocyte- and endothelial-specific CXCR4-null mice, and by Western blot analysis of endothelial cells treated in vitro. These experiments demonstrated that the acute infarct-sparing effect is mediated by endothelial cells, possibly via the signalling kinases Erk1/2 and PI3K/Akt. Unexpectedly, cardiomyocyte-specific deletion of CXCR4 was found to be cardioprotective per se. RNAseq analysis indicated altered expression of the mitochondrial protein co-enzyme Q10b in these mice. CONCLUSIONS: Administration of SDF-1α is cardioprotective when administered prior to reperfusion and may, therefore, have clinical utility. SDF-1α-CXCR4-mediated cardioprotection from ischaemia-reperfusion injury is contingent on the cellular location of CXCR4 activation. Specifically, cardioprotection is mediated by endothelial signalling, while cardiomyocyte-specific deletion of CXCR4 has an infarct-sparing effect per se

Ammonium tetrathiomolybdate following ischemia/reperfusion injury: Chemistry, pharmacology, and impact of a new class of sulfide donor in preclinical injury models

Background: Early revascularization of ischemic organs is key to improving outcomes, yet consequent reperfusion injury may be harmful. Reperfusion injury is largely attributed to excess mitochondrial production of reactive oxygen species (ROS). Sulfide inhibits mitochondria and reduces ROS production. Ammonium tetrathiomolybdate (ATTM), a copper chelator, releases sulfide in a controlled and novel manner, and may offer potential therapeutic utility. Methods and findings: In vitro, ATTM releases sulfide in a time-, pH-, temperature-, and thiol-dependent manner. Controlled sulfide release from ATTM reduces metabolism (measured as oxygen consumption) both in vivo in awake rats and ex vivo in skeletal muscle tissue, with a superior safety profile compared to standard sulfide generators. Given intravenously at reperfusion/resuscitation to rats, ATTM significantly reduced infarct size following either myocardial or cerebral ischemia, and conferred survival benefit following severe hemorrhage. Mechanistic studies (in vitro anoxia/reoxygenation) demonstrated a mitochondrial site of action (decreased MitoSOX fluorescence), where the majority of damaging ROS is produced. Conclusions: The inorganic thiometallate ATTM represents a new class of sulfide-releasing drugs. Our findings provide impetus for further investigation of this compound as a novel adjunct therapy for reperfusion injury

The 10th Biennial Hatter Cardiovascular Institute workshop: cellular protection—evaluating new directions in the setting of myocardial infarction, ischaemic stroke, and cardio-oncology

Due to its poor capacity for regeneration, the heart is particularly sensitive to the loss of contractile cardiomyocytes. The onslaught of damage caused by ischaemia and reperfusion, occurring during an acute myocardial infarction and the subsequent reperfusion therapy, can wipe out upwards of a billion cardiomyocytes. A similar program of cell death can cause the irreversible loss of neurons in ischaemic stroke. Similar pathways of lethal cell injury can contribute to other pathologies such as left ventricular dysfunction and heart failure caused by cancer therapy. Consequently, strategies designed to protect the heart from lethal cell injury have the potential to be applicable across all three pathologies. The investigators meeting at the 10th Hatter Cardiovascular Institute workshop examined the parallels between ST-segment elevation myocardial infarction (STEMI), ischaemic stroke, and other pathologies that cause the loss of cardiomyocytes including cancer therapeutic cardiotoxicity. They examined the prospects for protection by remote ischaemic conditioning (RIC) in each scenario, and evaluated impasses and novel opportunities for cellular protection, with the future landscape for RIC in the clinical setting to be determined by the outcome of the large ERIC-PPCI/CONDI2 study. It was agreed that the way forward must include measures to improve experimental methodologies, such that they better reflect the clinical scenario and to judiciously select combinations of therapies targeting specific pathways of cellular death and injury