published_or_final_version / abstract / Medicine / Master / Master of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Myocardial infarction (MI) is primarily caused by blockade of the coronary circulation, resulting in ischemic insult. The only available remedy is reperfusion, which induces oxidative stress and activates inflammatory responses at the site of injury. Cysteinyl leukotrienes (cysLTs) are potent pro-inflammatory mediators that exert their effects through two classical receptors: cysLT receptor 1 (CysLT1R) and cysLT receptor 2 (CysLT2R), the latter of which is prevalent in the heart and circulatory system and has been implicated in cardiovascular disease. However, although endothelial CysLT2R overexpression exacerbates MI damage and induces vascular hyperpermeability, understanding of CysLT2R activation-induced mechanisms is poor, as isolating CysLT2R-specific effects has proven difficult due to a lack of appropriate pharmacological agents. We investigate herein the role of CysLT2R activation in myocardial ischemia/reperfusion injury. We have characterized a novel CysLT2R-selective antagonist BayCysLT2 in both in vitro and in vivo systems, and establish that CysLT2R-selective antagonism attenuates exacerbated MI injury, adhesion molecule gene regulation, and myocardial neutrophil presence observed in CysLT2R overexpressing (EC) mice. We also examined effects of CysLT2R antagonism in long-term cardiac remodeling post-myocardial infarction, and found that blockade of CysLT2R post-reperfusion, regardless of whether CysLT2R is overexpressed or not, elicits a mild pathological cardiac hypertrophic response despite mitigating infarction damage to the apical ventricular wall. Finally, we created a novel mouse model (EC/KO) that expresses CysLT2R predominantly in vascular endothelium in order to identify tissue-specific mechanisms of CysLT2R activation. Surprisingly, MI injury was attenuated in EC/KO mice, indicating that both endothelial and non-endothelial CysLT2R expression subsets have roles in mediating infarction injury. Indeed, EC/KO mice demonstrated hyperpermeability in cremaster venules only when leukotrienes are applied, in contrast to EC mice. In addition, endothelial CysLT2R activation facilitates leukocyte transmigration, whereas non-endothelial CysLT2Rs regulate basal rolling leukocyte flux in microvasculature. Although much work remains to be done, the characterization of a CysLT2R-selective antagonist provides a vital tool for CysLT2R research moving forward, and our investigation of CysLT2R activation reveal the existence of a complicated and multi-faceted pathway resulting in activation of pro-inflammatory mechanisms. / Thesis (Ph.D, Physiology) -- Queen's University, 2013-09-26 10:29:03.466

Inflammation has modulatory effects on the brain, particularly during development. These plastic changes can hold severe functional consequences. Perinatal hypoxia-ischemia (HI)-induced inflammation can result in cerebral palsy and cognitive impairment. In an attempt to reduce inflammation in the brain, we assessed the probiotic Bifidobacterium (B.) infantis as an HI intervention, using a rat model. Rat pups, developmentally equivalent to preterm infants, were exposed to chronic hypoxia from postnatal (PND) 3 –PND 10. Inflammation was assessed through hippocampal concentrations of the cytokine interleukin-6 (IL-6). Tissue was collected from pups on PND 10 and analyzed via enzyme-linked immunosorbent assay (ELISA). Results showed lower IL-6 concentrations in hypoxic groups , regardless of B. infantis administration. Qualitative observations suggested poor gut health in association with hypoxia and probiotic exposure. These preliminary findings support the chronic hypoxia exposure model of HI and suggest the association with IL-6 and HI events is less straightforward than expected. / October 2016

Pressure ischemia is a medical condition characterised by the necrosis of the skin and underlying tissues in body areas exposed to prolonged pressure. This condition leads to the development of bedsores and affects 9% of hospitalised patients, costing the NHS between £1.4 and £2.1 billion per year. The severity of pressure ischemia has been linked to the concentration of sweat lactate, a product of sweat gland metabolism under anaerobic conditions, such as hypoxia. Normal levels of lactate in human sweat are 20±7 mM, but under ischemic conditions these can rise up to approximately 70 mM. This project presents the development of a novel flexible electrochemical enzyme-based biosensor for the continuous and non-invasive monitoring of sweat lactate with the potential for becoming a body-worn device for the early detection of pressure ischemia onset. The core of the recognition system is a flexible laminate, comprising two highly porous polycarbonate membranes, which provide support for the lactate oxidase enzyme, immobilised via covalent cross-linking. Oxidation of lactate produces H2O2, which is subsequently determined electrochemically. The transducer comprises a two-electrode system on a single flexible polycarbonate membrane, sputter-coated with gold (CE/RE) and platinum (WE) to render it conductive. The developed design has been improved through investigation into different factors regarding the immobilisation method of the enzyme in the laminate and the lowering of interferences from oxidising compounds present in sweat. The sensing system exhibits lactate selectivity at physiologically relevant concentrations in sweat for pressure ischemia (0–70 mM), with good reproducibility (7.2–12.2% RSD) for a hand-manufactured device. The reliability of the sensor’s performance and the capability to detect lactate fluctuations on human sweat samples has been demonstrated. The sensing system showed excellent operational and mechanical stability. The application of Nafion® on the WE lowered interferences from ascorbic acid and uric acid by 96.7 and 81.7% respectively. These results show promise towards the further development of a body-­‐worn monitoring device for determining lactate levels in undiluted human sweat samples in a reproducible, fast and accurate manner.

Ischemic preconditioning is an endogenous cardioprotective mechanism in which short periods of ischemia and reperfusion provide protection when given before a subsequent ischemic event. Early mechanistic studies showed ATP-sensitive potassium (KATP) channels to play an important role in ischemic preconditioning. KATP channels link intracellular energy metabolism to membrane excitability and contractility. It is thought that KATP channels provide a cardioprotective role during ischemia by inducing action potential shortening, reducing an excessive Ca^2+ influx, and by preventing arrhythmias. However, the mechanisms by which KATP channels protect during ischemic preconditioning are not known. In this study, we investigated a novel potential mechanism in which alterations in subcellular KATP channel trafficking during ischemia and ischemic preconditioning may result in altered levels of surface channel density, and therefore, a greater degree of cardioprotection. In the optimization of our experiments, we compared various antibodies for their specificity and sensitivity for channel subunit detection in immunoblotting. In addition, we examined the effects of varying salt concentrations during tissue homogenization in order to determine the optimal conditions for protein isolation. Furthermore, we examined the effect of heating the samples prior to SDS-PAGE for improved detection of channel proteins by immunoblotting. The subcellular trafficking of some membrane proteins is altered by ischemia. For example, the glucose transporter, Glut4, translocates from endosomal compartments to the sarcolemma (Sun, Nguyen, DeGrado, Schwaiger, & Brosius, 1994). Conflicting data exists regarding the effects of ischemia on KATP channel subcellular trafficking and the regulation of KATP channel surface density (Edwards et al., 2009 and Bao, Hadjiolova, Coetzee, & Rindler, 2011). We therefore, sought to test our hypothesis that KATP channels are internalized from the surface of cardiomyocytes to endosomal compartments during ischemia, and this internalization can be reduced and/or reversed by ischemic preconditioning. We subjected isolated Langendorff-perfused mouse hearts to ischemia, reperfusion, or ischemic preconditioning events and measured the density of KATP channels in the sarcolemmal and endosomal compartments. We also determined the degree of injury by staining heart slices with triphenyltetrazolium chloride and compared infarct sizes between hearts subjected to ischemia and ischemic preconditioning. Our data demonstrated that KATP channels are, in fact, internalized during ischemia and that reperfusion led to a slow recovery of surface KATP channel density. Interestingly, ischemic preconditioning reduced the size of infarcts induced by ischemia and also prevented the ischemia-induced decrease of KATP channel surface density, thereby, contributing to cardioprotection.

Despite years of research, an effective therapy for treatment of ischemic stroke has yet to be found. Survivors of stroke may suffer debilitating and permanent motor dysfunction for the remainder of their lives. Current treatments are limited to physical therapy and tissue plasminogen factor (tPA), a thrombolytic medication with a time- window of efficacy up to only three hours after symptom onset. Clinical studies and animal models have shown that partial recovery of motor function occurs with or without pharmacological interventions due to adaptive plasticity and reorganization in the brain. The precise mechanisms, though unclear, have become a major focus of stroke research. In the following study, we investigated inosine, a naturally occurring purine nucleoside that stimulates axonal growth, as a potential long-term stroke treatment. Following controlled cortical ischemia in the motor cortex of rhesus monkeys, recovery of dominant hand function was monitored through NHP Upper Extremity Motor Dysfunction Scale ratings for two weeks post-operation and through performance on two motor tasks, the Hand Dexterity Task (HDT) and the Digit Coordination Task (DCT). Results of cage- side assessment ratings demonstrated a trend towards greater recovery in the group treated with inosine for functional strength in the dominant hand on 12-14 days after surgery. The suggested trend is enough evidence to pursue research on the use of inosine as a therapeutic agent in post-stroke functional recovery.

No description available.

Hydrogen sulfide (H2S) is the newest member of the gasotransmitter family and is becoming well known for its cardioprotective effects in preclinical trials. Many recent studies have shown the benefits of exogenous H2S in the setting of acute myocardial infarction (AMI) and pressure overload-induced heart failure, but current formulations are derived from inorganic salts which have shortcomings in the precision and control of release of H2S. The main objective of this thesis was to determine if the novel, orally active, slow-releasing compound, SG1002, can attenuate the severity of damage and adverse remodeling caused by ischemia/reperfusion injury through an induction of angiogenesis. A traditional sodium salt, Na2S, which has been previously shown to be cardioprotective, was used as a positive control. SG1002 improved overall left ventricular function as measured by increased ejection fraction from echocardiography and decreased QRS interval from electrocardiography compared to untreated animals following MI. SG1002 therapy was also associated with an induction of angiogenesis, which was determined through qRT-PCR, western blot, and histological methods. SG1002 increased VEGF protein levels, which was paralleled with an increase in capillary density in the infarct region. SG1002 also upregulated microRNA-126, which is thought to repress the inhibitor of VEGF, Spred-1. It is possible that this “angiomiR” plays a key role in the angiogenesis-related cardioprotection of H2S. The combination of increased pro-angiogenic factors along with greater vascular density resulting from SG1002 therapy indicates the therapeutic potential for this drug in the prevention and/or treatment of ischemic heart failure.

Primary graft non-function is one of the major limitations of organ transplantation increasing the risk of rejection and early graft failure. A major cause of primary non-function is ischemia reperfusion injury (IRI), an obligatory insult in transplantation. During procurement, the donor is subjected to a period of ischemia inducing the release of tissue-damaging factors such as nitric oxide and reactive oxygen species. Upon engraftment and reperfusion with the recipient blood, these ischemia-induced factors cause rapid cell death and amplification of the inflammatory response leading to further tissue damage. / CD39 is an integral vascular and immune ectonucleotidase. CD39 hydrolyses extracellular nucleotides ATP and ADP into AMP, which is then hydrolysed into adenosine by CD73. Extracellular adenosine produced by the concerted action of CD39 and CD73 has potent anti-inflammatory and anti-coagulation effects acting principally via the purinergic adenosine receptor A2a. / NKT cells have only recently been recognised and constitute an important subset of T lymphocytes that display both effector and suppressive functions. NKT cells are found in high proportion in the liver of mice and are implicated by depletion studies in protection against hepatic IRI. / We have generated mice transgenic for human CD39 (hCD39) and have shown they have an anti-coagulant phenotype. As CD39 is also critical to immune regulation we hypothesised that transgenic expression of hCD39 would modify lymphocyte development and/or function and consequently impact on ischemia reperfusion injury. / Flow cytometric analysis was used to assess the number and phenotype of lymphocytes within the thymus and in the periphery of hCD39 transgenic mice. In vitro and in vivo assays were used to test the function of CD4+ T cells and invariant NKT cells from hCD39 transgenic mice. Bone marrow adoptive transfers experiments defined the role of hCD39 expression on bone marrow progenitor cells in comparison to tissue expression. The importance of adenosine signalling through the A2a receptor was studied by crossing hCD39 transgenic mice with A2a receptor knock-out (KO) mice. The effect of hCD39 expression on ischemia reperfusion injury was evaluated in a model of murine liver transplantation / A high level of hCD39 expression in the transgenic thymus resulted in lymphocyte maturation blockade and peripheral lymphopenia of CD4+ T cells and invariant NKT cells. Both lymphocyte populations were functionally deficient. The observed phenotype resulted from the expression of hCD39 on bone marrow progenitor cells but was independent of A2a receptor signalling. Over-expression of hCD39 in transgenic livers was protective against ischemia reperfusion injury induced by cold storage and liver transplantation.