Does pyrroloqinoline quinone have potential as a novel therapy to prevent oxidative stress and mitochondrial dysfunction under conditions of sepsis? Helen F. Galley, Damon A. Lowes and Nigel R. Webster. Division of Applied Medicine School of Medicine and Dentistry University of Aberdeen. Funded by the Anaesthetic Research Society Heath Family Project Grant Background Around 37,000 people die from sepsis each year in the UK. Despite the the Surviving Sepsis Campaign, the mortality rate remains at 31% for sepsis overall and 70% in patients who go on to develop multiple organ failure. Sepsis is essentially a dysregulated massive inflammatory response with release of cytokines, oxidative stress and mitochondrial dysfunction. Reactive oxygen species (ROS) are generated as by-products of the four electron reduction of molecular oxygen to water during the production of ATP. ROS can be damaging so their activity is normally tightly regulated by a collaborative network of antioxidants. When antioxidant defences are overwhelmed, oxidative stress results, which can cause damage to lipids, proteins, and nucleic acids, both within mitochondria and cells. Oxidative stress, inflammation and mitochondrial dysfunction are the hallmarks of sepsis [1] and the potential for benefit from antioxidants acting specifically in mitochondria has been recognised in several recent reviews [2,3,4]. We have shown that antioxidants which act within mitochondria are more effective at reducing oxidative damage caused by sepsis than antioxidants which do not specifically protect mitochondria [5,6,7]. Melatonin, for example, is highly lipophilic and can reach all cellular compartments with the highest levels in mitochondria. We have shown that melatonin and its metabolites reduce cytokine levels, oxidative stress and mitochondrial dysfunction in cells treated with lipopolysaccharide (LPS) and peptidoglycan G (PepG) to mimic conditions similar to those seen in sepsis [6]. In an LPS/PepG rat model of sepsis we also showed that melatonin reduced organ dysfunction and interleukin (IL)-6 release, improved mitochondrial dysfunction and decreased oxidative stress [5]. Melatonin treatment also resulted in lower mortality in animal models of sepsis [reviewed in 8]. PQQ is a water-soluble quinone which is an oxidoreductase co-enzyme synthesised by bacteria and is present in foods of plant origin [9]. Although PQQ is not synthesised in mammals it is rapidly absorbed by the large intestine after oral administration, and the trace levels of PQQ found in human tissues are thought to be derived from the diet. PQQ is a potent antioxidant, scavenging superoxide and hydroxyl radicals and inhibiting lipid peroxidation [10]. In animal models, PQQ protected tissues against hypoxic/ischaemic injury [11], carrageenan-induced inflammation [10], liver damage caused by ethanol [12] or carbon tetrachloride [13]. These protective effects of PQQ seem to be generally attributed to its antioxidant properties [10,14], but the precise mechanisms are unclear. Animals devoid of PQQ in the diet have substantially lower mitochondrial numbers than animals fed high doses of PQQ [15] and administration of PQQ was reported to stimulate mitochondrial biogenesis in in vivo disease models of oxidative stress [16], enhance mitochondrial complex I activity and prevent the effects of complex I inhibitors [17]. The balance between autophagy –the ‘recycling’ pathway which removes damaged mitochondria, and biogenesis of new mitochondria is crucial for restoration of cellular homeostasis during sepsis. Mitochondrial biogenesis is controlled via the action of the transcription factor pCREB on the peroxisome proliferator-activated receptor-- coactivator-1(PGC-1) [18]. PGC-1 is a transcriptional co-activator that induces mitochondrial biogenesis, up-regulates metabolic function independent of biogenesis, and enhances mitochondrial antioxidant defences by binding to the nuclear respiratory factors that activate mitochondrial transcription factors. Previous studies have shown that PQQ can activate pCREB/PGC-1[16]. However it is not known whether PQQ specifically protects within mitochondria and whether PQQ may represent a more effective alternative to melatonin under conditions of sepsis. The aim of this study was to assess the effect of a range of concentrations of PQQ on mitochondrial function and PCG1 expression under conditions mimicking sepsis. An endothelial ex vivo model of sepsis was established using human plasma which had been exposed to a range of inflammatory mediators. Methods After obtaining approval from the University of Aberdeen College of Life Sciences and Medicine Ethics Review Board, healthy volunteers were invited to donate blood by advertising. Subjects were excluded if they were smokers or took any regular medication or had an existing chronic health condition. A single venous blood sample (50ml) was collected into lithium-heparin tubes then centrifuged. The plasma layer was removed and replaced with an equal volume of pre-warmed RPMI 1640 cell culture medium with and without 2µg/mL lipopolysaccharide (LPS, E.coli, strain O55:B5), 20µg/mL peptidoglycan (PepG, S. Aureus, strain 6571), 10 µg/mL lipoteichochic acid (LTA, S. Aureus), 7ng/ml flagellin (S. Typhimurium) and 10 µg/mL bacterial DNA (E. coli, strain B) to mimic sepsis like conditions. Plasma was incubated for 3h at 37oC before the addition of 2mM ATP, pH7.4 and a further 3h incubation. Cellular components were then separated by centrifugation and the supernatant (termed ‘septic plasma’) was removed and used immediately for downstream cellular assays or frozen for subsequent cytokine/nitrite analysis to assess the extent of the inflammatory status. Cytokine profiles were obtained using the Proteome profiler human cytokine array panel A (R&D Systems, Paisley, UK) and nitrite formation was measured using the Greiss assay. HUVEC-C human endothelial cells (ATCC/LGC Standards Ltd., UK) were maintained in RPMI 1640 medium containing 10% foetal bovine serum (FBS, Biosera, UK), 50 μg/mL gentamycin (Lonza, UK) and 2.5 μg/mL amphotericin B (Sigma, UK) in a humidified atmosphere of 5% CO2 / 95% air at 37°C. HUVEC were treated with untreated or ‘septic plasma’ with a range of PQQ concentrations (1-30M) for up to 3 days. HUVEC cell viability was determined using the acid phosphatase activity assay. Mitochondrial membrane potential was assessed using the fluorescent dye 5,5,6,6-tetrachloro-1,1,3,3- tetraethyl-benzimidazolcarbocyanine iodide (JC-1). Mitochondrial metabolic activity was measured using AlamarBlueTM. and mitochondrial number was established using Mitotracker green. Total glutathione was measured using monochlorobimane as a measure of total oxidative stress. Protein expression of PGC1 was determined using standard western blotting techniques. Six independent experiments were performed using pooled plasma from 4 donors (n=6). Data are presented as box and whisker plots showing median, interquartile range (IQR) and full range. Individual data points are shown when n<6. No assumptions were made about data distribution and statistical analysis was performed using Kruskal-Wallis analysis with post hoc Mann Whitney U test as appropriate. A p-value of <0.05 was considered to be significant. Results Septic plasma Characterisation of the ‘septic plasma’ using cytokine array analysis revealed increased concentrations of a large number of cytokines and chemokines (Figure 1). Nitrite formation was also markedly increased (Figure 2). Figure 1. Cytokine array data from human endothelial cells exposed to ‘septic plasma’. Individual data points showing signal intensity relative to control (non-septic) plasma, n=4. Figure 2. Nitrite production as an indication of nitric oxide production in human endothelial cells exposed to ‘septic plasma’. Individual data points shown, n=3. Oxidative stress Exposure of endothelial cells to ‘septic plasma’ resulted in significant loss of cellular reduced glutathione (0.18 [0.13-0.22] units compared with 0.60 [0.53-0.66], median [range], n=6, p<0.0001, Mann Whitney U test). Treatment with PQQ at any concentration had no effect on glutathione and levels remained low (data not shown). Mitochondrial function Mitochondrial membrane potential was significantly lower when endothelial cells were exposed to the ‘septic plasma in the absence of PQQ. There was a dose dependent restoration of membrane potential in cells treated with PQQ (Figure 3). Figure 3. Mitochondrial membrane potential in human endothelial cells exposed to ‘septic plasma’. Median, interquartile and full range shown, n=6. P value shown is Kruskall Wallis across PQQ groups. * = significantly lower than control (non-septic plasma), Mann Whitney U test. Mitochondrial metabolic activity was unchanged when endothelial cells were exposed to the ‘septic plasma’ in the absence of PQQ and there was further dose dependent decrease in metabolic activity in cells treated with PQQ (Figure 4). Figure 4. Mitochondrial metabolic activity in human endothelial cells exposed to ‘septic plasma’. Median, interquartile and full range shown, n=6. P value shown is Kruskall Wallis across PQQ groups. * = significantly lower than control (non-septic plasma), Mann Whitney U test. Mitochondrial density was unchanged in endothelial cells exposed to the ‘septic plasma’ in the absence of PQQ compared to control (cells treated with non-septic plasma). In cells treated with the highest concentrations of PQQ, mitochondrial density was higher than control cells (Figure 5). Figure 5. Mitochondrial