Hypertension is a risk factor for myocardial ischemia via the promotion of endothelial dysfunction and atherosclerosis (Ogita et al., 2004). Hypertension not only a predisposes the heart to ischemic events, but as shown by clinical and experimental studies, exacerbates the heart’s susceptibility to ischemia-reperfusion injury (Golden et al., 1994; Besík et al., 2007; Snoeckx et al., 1986) due to impairments in regulation of calcium handling, ion homeostasis, and energy metabolism (Galiñanes et al., 2004). Nutraceuticals have demonstrated beneficial and protective effects on both hypertension and ischemia reperfusion injury. Resveratrol, a naturally occurring polyphenol which is present in grapes and wine, acts as a cardioprotective agent and can be used to protect the heart against ischemia reperfusion injury. Here we investigate the effectiveness of chronic dietary resveratrol intake in normotensive and hypertensive rats on protection against ischemia-reperfusion injury, assessed in an isolated perfused Langendorff heart model. Rats ingested either a High dose of 2.7mg/day-which mimicked the resveratrol content in daily supplemental intake levels, a Low dose of 0.027mg/day- which mimicked the resveratrol content in moderate red wine intake, or no resveratrol in the drinking water for 28 days, at which point hearts were excised and mounted on a Langendorff apparatus. Once stable conditions were established all hearts were subjected to 30 minutes of no flow ischemia followed by 2 hours of reperfusion. Interestingly, SHR animals did not exhibit reduced recovery, or increased infarct size as compared to WKY. Infarct size as measured by triphenyltetrazolium chloride staining after 2 hours of reperfusion was significantly reduced in High and Low groups (combined WKY and SHR) as compared to Controls (19.9±0.9 and 19.4±0.8 vs 27.7±0.7 % of baseline, p<0.0001). Left ventricular developed pressure was significantly improved 2 hours post ischemia in both High and Low groups (combined SHR and WKY) compared to Controls (83.1±4.1 and 78.6±3.4 vs.67.9±3.2% of baseline, p<0.01). A higher rate of maximal pressure development was maintained in High and Low groups (combined SHR and WKY) compared to Controls (90.5±4.7 and 95.6±5.0 vs.73.5±4.8% of baseline, p<.05). Resveratrol treatment at a High and Low dose reduced contracture of the myocardium as compared to Control (7.2±3.0 and 6.9±2.9 vs. 20.1±2.9 mmHg- LVEDP, p<0.01). In conclusion resveratrol treatment at both a High and Low dose protects against a decline in cardiac function, and reduces infarct size post ischemia reperfusion. Additionally, tolerance to ischemia reperfusion injury in SHR is not reduced as compared to WKY.

Hypertension is a risk factor for myocardial ischemia via the promotion of endothelial dysfunction and atherosclerosis (Ogita et al., 2004). Hypertension not only a predisposes the heart to ischemic events, but as shown by clinical and experimental studies, exacerbates the heart’s susceptibility to ischemia-reperfusion injury (Golden et al., 1994; Besík et al., 2007; Snoeckx et al., 1986) due to impairments in regulation of calcium handling, ion homeostasis, and energy metabolism (Galiñanes et al., 2004). Nutraceuticals have demonstrated beneficial and protective effects on both hypertension and ischemia reperfusion injury. Resveratrol, a naturally occurring polyphenol which is present in grapes and wine, acts as a cardioprotective agent and can be used to protect the heart against ischemia reperfusion injury. Here we investigate the effectiveness of chronic dietary resveratrol intake in normotensive and hypertensive rats on protection against ischemia-reperfusion injury, assessed in an isolated perfused Langendorff heart model. Rats ingested either a High dose of 2.7mg/day-which mimicked the resveratrol content in daily supplemental intake levels, a Low dose of 0.027mg/day- which mimicked the resveratrol content in moderate red wine intake, or no resveratrol in the drinking water for 28 days, at which point hearts were excised and mounted on a Langendorff apparatus. Once stable conditions were established all hearts were subjected to 30 minutes of no flow ischemia followed by 2 hours of reperfusion. Interestingly, SHR animals did not exhibit reduced recovery, or increased infarct size as compared to WKY. Infarct size as measured by triphenyltetrazolium chloride staining after 2 hours of reperfusion was significantly reduced in High and Low groups (combined WKY and SHR) as compared to Controls (19.9±0.9 and 19.4±0.8 vs 27.7±0.7 % of baseline, p<0.0001). Left ventricular developed pressure was significantly improved 2 hours post ischemia in both High and Low groups (combined SHR and WKY) compared to Controls (83.1±4.1 and 78.6±3.4 vs.67.9±3.2% of baseline, p<0.01). A higher rate of maximal pressure development was maintained in High and Low groups (combined SHR and WKY) compared to Controls (90.5±4.7 and 95.6±5.0 vs.73.5±4.8% of baseline, p<.05). Resveratrol treatment at a High and Low dose reduced contracture of the myocardium as compared to Control (7.2±3.0 and 6.9±2.9 vs. 20.1±2.9 mmHg- LVEDP, p<0.01). In conclusion resveratrol treatment at both a High and Low dose protects against a decline in cardiac function, and reduces infarct size post ischemia reperfusion. Additionally, tolerance to ischemia reperfusion injury in SHR is not reduced as compared to WKY.

Myocardial ischemia-reperfusion (I/R) injury is an important health concern in myocardial infarction and situations such as angioplasty and cardiac surgeries. Therefore, patients and physicians need therapeutic interventions that are applicable at the time of surgery. Flavonoids and ascorbate (vitamin C) are known for their antioxidant activity and may be involved in the currently known health benefits of plant based foods and drinks. The objectives of this study were to 1) determine the extent to which ascorbate or catechin alone at levels which could be in blood after dietary supplementation, can protect myocardial tissue in the reperfusion phase of I/R injury, and 2) evaluate the possible cooperative or synergistic protective effect of ascorbate and catechin when given together. Isolated rat hearts (n=48) were perfused in the retrograde mode with modified Krebs-Henseleit buffer, and following the induction of 30 min global ischemia, ascorbate (150 µM) and/or catechin (5 µM) were added directly into the perfusate during 90 min reperfusion. To determine the histopathological features, hematoxylin and eosin (H&E) stain was used in one heart per condition; while to assess the biochemical analysis, the heart tissues were assessed for apoptosis (caspase-3 activity), oxidative stress (thiobarbituric acid reactive substances (TBARS) and total malondialdehyde (MDA) levels), and redox status (reduced and oxidized glutathione tissue levels). A comparison of IR hearts with two controls, sham (perfused for a 15 min stabilization period) and continuous perfusion (perfused for 135 min), showed in most but not all measurements that this was a suitable model of IR injury. The treatment experiments showed that 150 µM ascorbate protected the heart against lipid peroxidation and cell apoptosis by 100%, while 5 µM catechin protected by 67% and 90% respectively. No cooperative protective effect could be observed when ascorbate and catechin were used together. None of the treatments significantly affected either reduced or oxidized glutathione levels. In conclusion, this study showed strong protection by ascorbate, which could be used in clinically relevant situations, and is the first to report the protection by catechin at this dose under conditions of myocardial ischemia-reperfusion injury.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

The treatment of asthma still relies on primary therapy with bronchodilators; in particular β adrenergic receptor (bAR) agonists with a diverse range of short acting and long acting βARs available. An increase in the number of cardiovascular events with the use of bronchodilators have recently been reported including hypertrophy, heart failure, myocardial ischaemia and infarction. Several subtypes of βAR receptors exist including the β1 Adrenergic Receptor (β1AR) and β2 Adrenergic Receptor (β2AR), both located in the heart. The effects of selective β2AR agonists were investigated in the Langendorff model of myocardial ischaemia reperfusion injury, isolated perfused rat hearts underwent 35 minutes of ischaemia and 120 minutes of reperfusion. The selective β2AR long acting β agonists Formoterol and Salmeterol had no significant effect on infarct to risk ratio or time taken to depolarisation and hypercontracture in isolated cardiomyocytes. The non-selective β1AR agonist Isoproterenol has been show to induce myocardial ischaemia and infarction in rat hearts previously, here we demonstrated Isoproterenol (0.5μM) significantly decreased time taken to depolarisation and hypercontracture in isolated cardiomyocytes. The short acting β2AR agonist Salbutamol (0.01μ-1μM) significantly increased infarct to risk ratio in the Langendorff in addition to significantly decreasing time to hypercontracture in cardiomyocytes in the oxidative stress model highlighting a potential role of the mitochondrial permeability transition pore (mPTP). Activation of phosphorylated Akt and phosphorylated Erk1/2 via the PI3K/Akt signalling pathway and p44/p42 MAPK pathway were investigated by western blot analysis. Salbutamol significantly elevated expression of p-Akt in rat hearts exposed to reperfusion for 20 and 120 minutes whilst reducing expression of p-Erk. Recorded elevated cleaved caspase 3 expression in Salbutamol treated hearts can be associated as a marker of increased in cardiomyocyte cell death. The β1AR antagonist CGP 20712 was administered in the presence of Salbutamol with minimal reduction in infarct size in rat hearts recorded and no significant change in time taken to hypercontracture in isolated cardiomyocytes suggesting that Salbutamol mediated toxicity is via β2AR activation. Confirmation of this was verified with the β2AR antagonist ICI 118, 551. Significant decrease in infarct size was recorded in addition to a significant increase in time to hypercontracture in the oxidative stress model. Further to this, caspase 3 expression was significantly reduced in addition with p-Akt expression. With a potential role of the mitochondria and the mPTP contributing to Salbutamol induced myocardial injury, the Cyclophilin D inhibitor Cyclosporin A was administered in hearts and cardiomyocytes in the presence of Salbutamol. Infarct size was significantly reduced whilst time taken to hypercontracture significantly increased, suggesting that CsA treatment inhibits Salbutamol mediated injury via Cyclophilin D inhibition of the mPTP. To conclude, our results demonstrated that Salbutamol caused cardiotoxicity at tissue, cellular and protein level in conditions of ischaemia reperfusion injury. Further to this, inhibition of Cyclophilin D by CsA, or the use of the β2AR antagonist ICI 118, 551 inhibits Salbutamol induced toxicity.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21415号 / 医博第4405号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 坂井 義治, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Statins can act as preconditioning agents against ischemia reperfusion (IR)-injury through a mechanism involving cyclooxygenase (COX)-2 and the upregulation of prostaglandin synthesis. The following study investigated the effect of selective and non-selective COX inhibition on rosuvastatin-mediated protection against IR-induced endothelial dysfunction in the human forearm vasculature. Healthy volunteers were randomized to drugs with different COX-inhibiting properties: 81mg aspirin (OD), 325mg aspirin (OD), 400mg ibuprofen (QID), 200mg celecoxib (BID) or placebo. A single dose of 40mg rosuvastatin was also administered 24-hours prior to IR. Endothelial function before and after IR was assessed by measuring flow-mediated dilation of the radial artery. Our results show that 81mg and 325mg aspirin (more COX-1 selective), 400mg ibuprofen (similar selectivity for COX-1/2) and 200mg celecoxib (COX-2 selective) all effectively abolished statin-mediated protection against IR-induced endothelial dysfunction in the forearm (2-way ANOVA, p<0.05). These findings indicate that even partial COX-2 inhibition is sufficient to attenuate statin-induced preconditioning.