A metabolomics study on the ischaemic heart identifies succinate as a metabolite that drives the production of reactive oxygen species and contributes to ischaemia-reperfusion injury; pharmacological inhibition of succinate accumulation ameliorates ischaemia-reperfusion injury in a mouse model of heart attack and a rat model of stroke. In this metabolomics study of the ischaemic heart, Michael Murphy and colleagues identify a metabolite that drives the production of reactive oxygen species and contributes to ischaemia reperfusion injury. They show that succinate is a conserved metabolic signature of ischaemia in several tissues. Succinate accumulates during ischaemia due to a reversal of the enzyme succinate dehydrogenase. Upon reperfusion the accumulated succinate is rapidly oxidized and drives reactive oxygen species production through reverse electron transport at mitochondrial complex I. Pharmacological blockade of succinate accumulation ameliorates ischaemia reperfusion injury in mouse models of heart attack and stroke. Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS)1,2,3,4,5. Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion1,3. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies.

Transient or permanent interruption of cerebral blood flow by occlusion of a cerebral artery gives rise to an ischaemic stroke leading to irreversible damage or dysfunction to the cells within the affected tissue along with permanent or reversible neurological deficit. Extensive research has identified excitotoxicity, oxidative stress, inflammation and cell death as key contributory pathways underlying lesion progression. The cornerstone of treatment for acute ischaemic stroke remains reperfusion therapy with recombinant tissue plasminogen activator (rt-PA). The downstream sequelae of events resulting from spontaneous or pharmacological reperfusion lead to an imbalance in the production of harmful reactive oxygen species (ROS) over endogenous anti-oxidant protection strategies. As such, anti-oxidant therapy has long been investigated as a means to reduce the extent of injury resulting from ischaemic stroke with varying degrees of success. Here we discuss the production and source of these ROS and the various strategies employed to modulate levels. These strategies broadly attempt to inhibit ROS production or increase scavenging or degradation of ROS. While early clinical studies have failed to translate success from bench to bedside, the combination of anti-oxidants with existing thrombolytics or novel neuroprotectants may represent an avenue worthy of clinical investigation. Clearly, there is a pressing need to identify new therapeutic alternatives for the vast majority of patients who are not eligible to receive rt-PA for this debilitating and devastating disease.

Adeno-associated virus type 2 (AAV-2) targeting vectors have been generated by insertion of ligand peptides into the viral capsid at amino acid position 587. This procedure ablates binding of heparan sulfate proteoglycan (HSPG), AAV-2's primary receptor, in some but not all mutants. Using an AAV-2 display library, we investigated molecular mechanisms responsible for this phenotype, demonstrating that peptides containing a net negative charge are prone to confer an HSPG nonbinding phenotype. Interestingly, in vivo studies correlated the inability to bind to HSPG with liver and spleen detargeting in mice after systemic application, suggesting several strategies to improve efficiency of AAV-2 retargeting to alternative tissues.

Adenovirus type 5 (Ad) based vectors transduce vascular endothelial cells (EC) and have been widely used for vascular gene transfer. However, many cell types express the Ad receptor (cox-sackievirus adenovirus receptor; CAR), preventing selective EC infection and precluding clinical use. We previously isolated the human EC-binding peptides SIGYPLP and LSNFHSS by phage display and demonstrated by means of a bispecific antibody that SIGYPLP directs efficient, high-level, EC-selective Ad-mediated gene transfer. We now generate genetically modified Ad fiber proteins with selective EC tropism by engineering these peptides into the HI loop of the Ad fiber. SIGYPLP, but not LSNFHSS, enhanced EC selectivity, demonstrating maintenance of peptide–cell binding fidelity upon incorporation into virions. Combining fiber mutations that block CAR binding (detargeting) with SIGYPLP insertion (retargeting) generated a novel Ad vector, AdKO1SIG, in a single component system. AdKO1SIG demonstrated efficient and selective tropism for EC compared with control Ad vectors. This is the first demonstration of genetic incorporation of a novel, mammalian, cell-selective ligand that retains its targeting fidelity in the Ad fiber HI loop, in combination with point mutations that abolish fiber–CAR interaction. This study demonstrates the potential for improving the cell-selectivity and safety of adenoviral vectors.

Virus-mediated gene delivery is restricted by the infectivity profile of the chosen vector. Targeting the vascular endothelium via systemic delivery has been attempted using peptides isolated in vitro (using either phage or vector display) and implicit reliance on target receptor expression in vivo. This has limited application since endothelial cells in vitro and in vivo differ vastly in receptor profiles and because of the existence of complex endothelial "zip codes" in vivo. We therefore tested whether in vivo phage display combined with adeno-associated virus (AAV) capsid modifications would allow in vivo homing to the endothelium residing in defined organs. Extensive in vivo biopanning in rats identified four consensus peptides homing to the lung or brain. Each was incorporated into the VP3 region of the AAV-2 capsid to display the peptide at the virion surface. Peptides that conferred heparan independence were shown to retarget virus to the expected vascular bed in vivo in a preferential manner, determined 28 days post-systemic injection by both virion DNA and transgene expression profiling. Our findings significantly impact the design of viral vectors for targeting individual vascular beds in vivo.