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

The Caspase 1 Inhibitor VX-765 Protects the Isolated Rat Heart via the RISK Pathway

PURPOSE: Protecting the heart from ischaemia-reperfusion (IR) injury is a major goal in patients presenting with an acute myocardial infarction. Pyroptosis is a novel form of cell death in which caspase 1 is activated and cleaves interleukin 1β. VX-785 is a highly selective, prodrug caspase 1 inhibitor that is also clinically available. It has been shown to be protective against acute IR in vivo rat model, and therefore might be a promising possibility for future cardioprotective therapy. However, it is not known whether protection by VX-765 involves the reperfusion injury salvage kinase (RISK) pathway. We therefore investigated whether VX-765 protects the isolated, perfused rat heart via the PI3K/Akt pathway and whether protection was additive with ischaemic preconditioning (IPC). METHODS: Langendorff-perfused rat hearts were subject to ischaemia and reperfusion injury in the presence of 30 μM VX-765, with precedent IPC, or the combination of VX-765 and IPC. RESULTS: VX-765 reduced infarct size (28 vs 48% control; P < 0.05) to a similar extent as IPC (30%; P < 0.05). The PI3 kinase inhibitor, wortmannin, abolished the protective effect of VX-765. Importantly in the model used, we were unable to show additive protection with VX-765 + IPC. CONCLUSIONS: The caspase 1 inhibitor, VX-765, was able to reduce myocardial infarction in a model of IR injury. However, the addition of IPC did not demonstrate any further protection

Cardioprotection mediated by exosomes is impaired in the setting of type II diabetes but can be rescued by the use of non-diabetic exosomes in vitro

Many patients with ischaemic heart disease also have diabetes. As myocardial infarction is a major cause of mortality and morbidity in these patients, treatments that increase cell survival in response to ischaemia and reperfusion are needed. Exosomes-nano-sized, lipid vesicles released from cells-can protect the hearts of non-diabetic rats. We previously showed that exosomal HSP70 activates a cardioprotective signalling pathway in cardiomyocytes culminating in ERK1/2 and HSP27 phosphorylation. Here, we investigated whether the exosomal cardioprotective pathway remains intact in the setting of type II diabetes. Exosomes were isolated by differential centrifugation from non-diabetic and type II diabetic patients, from non-diabetic and Goto Kakizaki type II diabetic rats, and from normoglycaemic and hyperglycaemic endothelial cells. Exosome size and number were not significantly altered by diabetes. CD81 and HSP70 exosome markers were increased in diabetic rat exosomes. However, exosomes from diabetic rats no longer activated the ERK1/2 and HSP27 cardioprotective pathway and were no longer protective in a primary rat cardiomyocytes model of hypoxia and reoxygenation injury. Hyperglycaemic culture conditions were sufficient to impair protection by endothelial exosomes. Importantly, however, exosomes from non-diabetic rats retained the ability to protect cardiomyocytes from diabetic rats. Exosomes from diabetic plasma have lost the ability to protect cardiomyocytes, but protection can be restored with exosomes from non-diabetic plasma. These results support the concept that exosomes may be used to protect cardiomyocytes against ischaemia and reperfusion injury, even in the setting of type II diabetes

Unique morphological characteristics of mitochondrial subtypes in the heart: the effect of ischemia and ischemic preconditioning.

RATIONALE: Three subsets of mitochondria have been described in adult cardiomyocytes - intermyofibrillar (IMF), subsarcolemmal (SSM), and perinuclear (PN). They have been shown to differ in physiology, but whether they also vary in morphological characteristics is unknown. Ischemic preconditioning (IPC) is known to prevent mitochondrial dysfunction induced by acute myocardial ischemia/reperfusion injury (IRI), but whether IPC can also modulate mitochondrial morphology is not known. AIMS: Morphological characteristics of three different subsets of adult cardiac mitochondria along with the effect of ischemia and IPC on mitochondrial morphology will be investigated. METHODS: Mouse hearts were subjected to the following treatments (N=6 for each group): stabilization only, IPC (3x5 min cycles of global ischemia and reperfusion), ischemia only (20 min global ischemia); and IPC and ischemia. Hearts were then processed for electron microscopy and mitochondrial morphology was assessed subsequently. RESULTS: In adult cardiomyocytes, IMF mitochondria were found to be more elongated and less spherical than PN and SSM mitochondria. PN mitochondria were smaller in size when compared to the other two subsets. SSM mitochondria had similar area to IMF mitochondria but their sphericity measures were similar to PN mitochondria. Ischemia was shown to increase the sphericity parameters of all 3 subsets of mitochondria; reduce the length of IMF mitochondria, and increase the size of PN mitochondria. IPC had no effect on mitochondrial morphology either at baseline or after ischemia. CONCLUSION: The three subsets of mitochondria in the adult heart are morphologically different. IPC does not appear to modulate mitochondrial morphology in adult cardiomyocytes

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

Ischaemic injuries

Professor Derek Yellon and Dr Sean Davidson discuss their research on preventing injury to the heart from ischaemia-reperfusion and the potentially protective role of exosome