Muscarinic acetylcholine receptors (mAChRs) are G-protein coupled receptors that mediate various actions of Acetylcholine (ACh) in the central nervous system and peripheral nervous system. In mammals, five distinct mAChR subtypes (M1-M5) have been recognised with the M2 subtype being predominantly present in the heart. The mAChR antagonists are routinely used for the treatment of various pathophysiological conditions including respiratory conditions. However, it has been postulated that mAChR antagonists may increase morbidity and mortality in chronic obstructive pulmonary disorder (COPD) and asthma patients with underlying cardiovascular disease, raising concerns regarding the cardiovascular safety of these agents. The current study was therefore undertaken to investigate the effects of individual mAChR antagonists in the setting of myocardial ischaemia reperfusion injury and oxidative stress models. We also investigated whether the inhibition of the mitochondrial permeability transition pore (MPTP) with cyclosporine-A (CsA) in the presence and absence of individual mAChR antagonists provided protection against ischaemia reperfusion injury. Furthermore, we also aimed to investigate the intracellular signalling pathway associated with mAChRs antagonists mediated myocardial injury under the stress conditions. Langendorff results showed that the non-selective M1-M3 mAChR antagonist, ipratropium bromide, the M2 mAChR antagonist, AF-DX 116 and the M3 mAChR antagonist, DAU 5884 significantly increased the infarct size to risk ratio of the heart in conditions of ischaemia and reperfusion. Detrimental effects of AF-DX 116 and DAU 5884 were abrogated by co-treatment of these drugs with mAChR agonist, acetylcholine (ACh) and/or CsA. Cell viability data of isolated cardiac myocytes revealed that AF-DX 116 and DAU 5884 caused a concentration dependent decrease in the viability of cardiac myocytes as well as causing a reduction in the time taken to depolarisation and hypercontracture under oxidative stress. AF-DX 116 and DAU 5884 significantly increased the levels of p-SAPK/JNK and decreased the levels of p-Akt and p-ERK. In addition, ACh and CsA showed to activate p-Akt and p-ERK. To conclude, the data suggest that AF-DX 116 and DAU 5884 caused cardiotoxicity at cellular, tissue and protein level in conditions of ischaemia reperfusion injury and oxidative stress. Furthermore, inhibition of the mitochondrial transition pore with CsA protected against the AF-DX 116 and DAU 5884 induced injury via activation of the pro-survival proteins, p-Akt and p-ERK.

Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. Toll-like receptor (TLR)-mediated signalling plays a role in cerebral ischaemia/reperfusion (I/R) injury. Modulation of TLRs has been reported to protect against cerebral I/R injury. This study examined whether modulation of TLR3 with poly (I:C) will induce protection against cerebral I/R injury. Mice were treated with or without Poly (I:C) (n = 8/group) 1 hr prior to cerebral ischaemia (60 min.) followed by reperfusion (24 hrs). Poly (I:C) pre-treatment significantly reduced the infarct volume by 57.2% compared with untreated I/R mice. Therapeutic administration of Poly (I:C), administered 30 min. after cerebral ischaemia, markedly decreased infarct volume by 34.9%. However, Poly (I:C)-induced protection was lost in TLR3 knockout mice. In poly (I:C)-treated mice, there was less neuronal damage in the hippocampus compared with untreated I/R mice. Poly (I:C) treatment induced IRF3 phosphorylation, but it inhibited NF-κB activation in the brain. Poly (I:C) also decreased I/R-induced apoptosis by attenuation of Fas/FasL-mediated apoptotic signalling. In addition, Poly (I:C) treatment decreased microglial cell caspase-3 activity. In vitro data showed that Poly (I:C) prevented hypoxia/reoxygenation (H/R)-induced interaction between Fas and FADD as well as caspase-3 and -8 activation in microglial cells. Importantly, Poly (I:C) treatment induced co-association between TLR3 and Fas. Our data suggest that Poly (I:C) decreases in cerebral I/R injury via TLR3 which associates with Fas, thereby preventing the interaction of Fas and FADD, as well as microglial cell caspase-3 and -8 activities. We conclude that TLR3 modulation by Poly (I:C) could be a potential approach for protection against ischaemic stroke.

Aims Toll-like receptor (TLR)-mediated signalling pathways have been implicated in myocardial ischaemia/reperfusion (I/R) injury. Activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway protects the myocardium from ischaemic injury. We hypothesized that the modulation of TLR2 would induce cardioprotection against I/R injury via activation of the PI3K/Akt signalling. Methods and results Mice were treated with TLR2 ligands, peptidoglycan (PGN) or Pam3CSK4, respectively, 1 h before the hearts were subjected to ischaemia (1 h), followed by reperfusion (4 h). Infarct size was determined by triphenyltetrazolium chloride staining. Cardiac function and haemodynamic performance were evaluated. Infarct size was significantly reduced in PGN-or Pam3CSK4-treated mice compared with untreated I/R mice. Administration of TLR2 ligands improved cardiac function following I/R. PGN treatment increased the levels of phospho-Akt and phospho-GSK-3β (glycogen synthase kinase-3β), compared with untreated I/R hearts. PGN stimulation increased TLR2 tyrosine phosphorylation and association of the p85 subunit of PI3K with TLR2. To investigate the role of PI3K/Akt signalling in PGN-induced cardioprotection, we administered the PI3K inhibitor, Wortmannin, to the mice 15 min before PGN treatment. We also administered PGN to kinase-deficient Akt (kdAkt) transgenic mice 1 h before myocardial I/R. Both PI3K inhibition and kdAkt mice abolished the cardioprotection induced by PGN. To examine the role of TLR2 in PGN-induced cardioprotection, we administrated PGN to TLR2 knockout mice 1 h before the hearts were subjected to I/R. PGN-induced cardioprotection was lost in TLR2-deficient mice. Conclusion These results demonstrate that TLR2 ligands induced cardioprotection, which is mediated through a TLR2/PI3K/Akt-dependent mechanism.

Aims: Bifunctional apoptosis regulator (BAR) is an endoplasmic reticulum protein that interacts with both the extrinsic and intrinsic apoptosis pathways. We hypothesize that over-expression of BARΔRING prevents apoptosis and injury following ischaemia/reperfusion (I/R) and attenuates doxorubicin (DOX)-induced cardiotoxicity. Methods and results: We generated a line of transgenic mice that carried a human BARΔRING transgene under the control of the mouse α-myosin heavy chain promoter. The RING domain, which binds ubiquitin conjugating enzymes, was deleted to prevent auto-ubiquitination of BAR and allow accumulation of the BAR protein, which binds apoptosis-regulating proteins. High levels of human BARΔRING transcripts and 42 KDa BARΔRING protein were expressed in the hearts of transgenic mice. When excised hearts were reperfused ex vivo for 45 min as Langendorff preparations after 45 min of global ischaemia, the functional recovery of the hearts, expressed as left ventricular developed pressure x heart rate, was 23 ± 1.7% in the non-transgenic hearts compared with 51.5 ± 4.3% in the transgenic hearts (P < 0.05). For in vivo studies, mice were subjected to 50 min of ligation of the left descending anterior coronary artery followed by 4 h of reperfusion. The infarct sizes following I/R injury, expressed as the percentage of the area at risk, were significantly smaller in the transgenic mice than in the non-transgenic mice (29 ± 4 vs. 55 ± 4%, P < 0.05). In hearts of mice subjected to cardiac I/R injury, BAR transgenic hearts had significantly fewer in situ oligo-ligation-positive cardiac cells (5.0 ± 0.4 vs. 13.4 ± 0.5%, P < 0.05). Over-expression of BARΔRING also significantly attenuated DOX-induced cardiac dysfunction and apoptosis. Conclusion: Our results demonstrate that over-expression of BARΔRING renders the heart more resistant to I/R injury and DOX-induced cardiotoxicity, and this protection correlates with reduced cardiomyocyte apoptosis.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Obesity has reached epidemic proportions worldwide and is currently a serious health problem. It is associated with metabolic abnormalities, oxidative stress, hypertension, insulin resistance and an increased disposition for the development of cardiovascular disease. Elucidation of the pathophysiological mechanisms underlying obesity and its relationship with metabolic and cardiovascular diseases is essential for prevention and management of these disorders. Melatonin, the pineal gland hormone, is a powerful antioxidant and has been shown to protect the myocardium against ischaemia/reperfusion (I/R) injury. Long- as well as shortterm melatonin treatment also reversed several of the harmful effects of obesity in an animal model of hyperphagia-induced obesity (DIO). However, its effects on myocardial I/R injury and intracellular signalling in obesity induced by a high fat diet (HFD) are still unknown. Aims of study: (i) To evaluate the ability of a high fat diet (HFD) to induce obesity in rats. Apart from evaluating its effects on the biometric parameters and resistance to ischaemia/reperfusion injury (as indicated by infarct size in regional ischaemia and functional recovery after global ischaemia), special attention will be given on the interplay between adiponectin, AMPK, leptin, and FFA in this model. (ii) To evaluate the effect of daily oral administration of melatonin to rats on the HFD as well as their littermate controls, on the parameters listed above as well as on the development of obesity. In this study melatonin will be administered from the onset of the feeding of the high fat diet. Methods: Male Wistar rats were divided into 4 groups: (i) control rats (receiving normal rat chow) (C); (ii) control rats receiving melatonin (CM); (iii) obese rats (receiving HFD) (HFD); (iv) obese rats receiving melatonin (HM). Animals were kept on the diet for 16 weeks and melatonin treatment (10mg/kg/day, added to the drinking water) started at the onset of the feeding. Following feeding and treatment, the animals were grouped into fasted/ non-fasted of which biometric parameters were recorded and blood collected at the time of sacrifice for metabolic and biochemical assays. Hearts were perfused in the working mode for evaluation of myocardial function and infarct size determination after exposure to 35min regional ischaemia/60min reperfusion. For study of intracellular signaling, hearts were perfused in the working mode, subjected to 20min global ischaemia/10min reperfusion and freeze-clamped for Western blotting. Plasma leptin, adiponectin, free fatty acid, triglycerides, total cholesterol, phospholipids, conjugated dienes and thiobarbituric reactive substances (TBARS) levels were determined. Several kinases were investigated including, the RISK (reperfusion injury salvage kinase) (PKB/Akt and ERK p44/42) and SAFE (survivor activating factor enhancement) (STAT-3) pathways, AMPK and JNK under baseline conditions or following 10 min reperfusion. In addition, expression of UCP-3 and PGC1-α was determined. Results: Significant increases in body weight, visceral fat, blood glucose, insulin, HOMA index and leptin and a reduction in adiponectin levels were observed in the fasted high fat diet (HFD) group when compared with controls (C). Significant increases in free fatty acid and triglyceride levels were also noted the HFD group while other serum lipid parameters, including TBARS, remained unchanged. No differences in functional recovery during reperfusion or infarct size after exposure to 35 min regional ischaemia, as well as functional recovery during reperfusion after 20 min global ischaemia were observed between the control and HFD groups. Baseline and 10 min reperfusion data were similar for the RISK and SAFE pathway kinases for the control vs HFD groups. The HFD also had no effect on the expression and phosphorylation of myocardial AMPK and JNK, as well as on the expression of UCP-3 and PGC1-α, when compared to the controls. Treatment with melatonin significantly reduced body weight, visceral fat, blood glucose, HOMA index and serum leptin levels in HFD treated groups, while having no effect on the lipid profile. Although melatonin significantly reduced infarct size in both control [% of area at risk: 20.59 ± 2.29 (CM) vs 38.08 ± 2.77 (C)] and high-fat diet groups [% of area at risk: 11.43 ± 2.94 (HM) vs 38.06 ± 3.59 (H)], it was without effect on myocardial functional recovery during reperfusion. Melatonin had no effect on the intracellular signaling pathways studied. Conclusions: The HFD proved to be a useful model of diet-induced obesity with a more pronounced impact on biometric and metabolic changes compared to the DIO model. Long-term melatonin treatment successfully prevented the development of metabolic abnormalities associated with the high fat diet and obesity as well as significantly reduced myocardial infarct size. The mechanisms involved in melatonin-induced cardioprotection in obesity have not been fully elucidated in this study and require further investigation. However, the anti-obesogenic and cardioprotective properties of melatonin were very significant indeed and support the suggestion of this hormone as a potential tool in the treatment of obesity and associated cardiovascular complications. / AFRIKAANSE OPSOMMING: Inleiding: Vetsug (obesiteit) het wêreldwyd epidemiese afmetings aangeneem en word tans as ‘n ‘n ernstige gesondheidsprobleem beskou. Vetsug word geassosieer met metaboliese afwykings, oksidatiewe stres, hipertensie, insulienweerstandigheid en is‘n belangrike risikofaktor vir die ontwikkeling van kardiovaskulêre siekte. Ten spyte hiervan, het onlangse studies ‘n gunstige effek van vetsug op die uitkomste van miokardiale infarksie in pasiënte gerapporteer, die sg obesiteitsparadoks. Kennis van die patofisiologiese meganismes onderliggend aan vetsug en die ontstaan van metaboliese afwykinge en hartsiekte is noodsaaklik vir die voorkoming en behandeling van hierdie toestande. Melatonien, die hormoon afgeskei deur die pineaalklier, is ‘n kragtige antioksidant en vry radikaal opruimer. Dit is voorheen aangetoon dat dit die hart teen iskemie/herperfusie (I/H) besering kan beskerm en sommige van die skadelike gevolge van vetsug in diermodelle kan omkeer. Die effek van melatonien op miokardiale I/H besering en intrasellulêre seintransduksie prosesse in vetsug geïduseer deur ‘n hoë vet dieet is egter nog onbekend. Doelstellings: (i) Die ontwikkeling en karakterisering van ‘n nuwe model van vetsug en insulienweerstandigheid geïnduseer deur 'n hoë vet dieet (HVD) en die evaluering van die effek daarvan op miokardiale I/H besering en die gepaardgaande intrasellulêre seintransduksieprosesse; (ii) Bepaling van die effek van daaglikse toediening van melatonien aan rotte op die HVD sowel as aan kontroles op ‘n standard dieet, op die ontwikkeling van dieet-geïnduseerde metaboliese veranderinge, miokardiale infarktgrootte en funksionele herstel na koronêre arterie afbinding, sowel as intrasellulêre seintransduksie. Metodiek: Vier groepe van manlike Wistar rotte is bestudeer: (i) kontrole rotte (op‘n standaard dieet) (K); (ii) kontrole rotte op ‘n standard dieet plus melatonien (KM); (iii) dieetrotte (op‘n HVD); (iv) HVD rotte wat melatonien ontvang (HM). Die HVD en melatonien (10mg/kg/dag in die drinkwater) is vir 16 weke toegedien. Na die periode van behandeling, is die diere in vastende en nie-vastende groepe verdeel, die biometriese parameters genoteer en bloedmonsters vir metaboliese en biochemiese bepalings versamel, tydens verwydering van die harte. Harte is geperfuseer volgens die werkhartmodel vir bepaling van miokardiale funksie en infarktgrootte na blootstelling aan 35min streeksiskemie. Vir evaluering van intrasellulêre seintransduksie, is geperfuseerde werkende rotharte blootgestel aan 15min globale iskemie/10 min herperfusie en gevriesklamp vir latere analises volgens die Western kladtegniek.hart. Serum leptien, adiponektien, vryvetsure, trigliseried, totale cholesterol, fosfolipiede, gekonjugeerde diene en tiobarbituursuur reaktiewe stowwe (TBARS) is bepaal. Met gebruik van Western kladtegniek, is die aktivering en/of uitdrukking van die RISK (PKB/ Akt en ERK p44/42) en SAFE (STAT-3) seintransduksiepaaie, AMPK, JNK, UCP-3 en PGC1-α, onder basislyn toestande of na 10 min herperfusie bestudeer. Resultate:‘n Beduidende toename in liggaamsgewig, visserale vet, die HOMA indeks, insulien en leptien vlakke is in die HVD groep waargeneem vergeleke met die kontrole (K) rotte. Adiponektien vlakke was laer in die HVD groep. Die HVD groep is ook gekenmerk deur ‘n beduidende styging in serum vryvetsuur en trigliseried vlakke, terwyl die ander lipied parameters, insluitende die TBARS vlakke, onveranderd was. Infarktgrootte en funksionele herstel tydens herperfusie na blootstelling aan 35 min streeksiskemie, asook funksionele herstel tydens herperfusie na 20 min globale iskemie het nie verskil tussen harte van die kontrole en HVD rotte nie. Aktivering van PKB/Akt, ERK p44/p42, STAT3, AMPK en JNK by basislyn en na 10 min herperfusie was soortgelyk in die kontrole en HFD groepe. Die HVD het ook geen effek op die uitdrukking van UCP-3 en PGC1-α in vergelyking met die kontrole gehad nie. Behandeling met melatonien het die liggaamsgewig, visserale vet, bloedglukose, HOMA indeks en serum leptien vlakke in die HVD groepe statisties beduidend verlaag, terwyl dit geen invloed op die lipiedprofiel gehad het nie. Melatonien behandeling het die miokardiale infarktgrootte beduidend en tot dieselfde mate verminder in beide kontrole [20.59 ± 2.29 (KM) vs 38.08 ± 2.77% (K)] en HVD groepe [11.43 ± 2.94 (HM) vs 38.06 ± 3.59% (HVD)]. Geen verskille is egter tussen die funksionele herstel gedurende herperfusie van die behandelde en onbehandelde kontrole en HVD groepe waargeneem nie. Melatonien het ook geen uitwerking op die intrasellulêre seintransduksiepaaie gehad nie. Gevolgtrekkings: Die resultate het getoon dat die HFD 'n goeie model van dieetgeïnduseerde vetsug en insulien weerstandigheid ontlok, met 'n meer uitgesproke impak op biometriese en metaboliese veranderinge as die voorheen gebruikte hoë-sukrose dieet. Langtermyn melatonien- behandeling het die ontwikkeling van metaboliese abnormaliteite geassosieer met die HVD, voorkom, asook miokardiale infarktgrootte na koronêre afbinding beduidend verminder. Die meganismes betrokke in melatonien-geïnduseerde miokardiale beskerming moet egter in meer detail ondersoek word. Die resultate verkry steun die voorstel dat melatonientoediening voordelig sal wees in die behandeling van vetsug en sy kardiovaskulêre komplikasies.

Surgical myocardial revascularization remains the standard of care for patients with multi-vessel coronary artery disease. A growing body of evidence indicates that systemic inflammation and myocardial oxidative stress are associated with the development of postoperative atrial fibrillation (POAF) and low cardiac output syndrome in patients undergoing cardiac surgery. Statins have been shown to exert rapid anti-inflammatory and antioxidant effects by inhibiting myocardial NOX2 oxidases and by increasing the bioavailability of nitric oxide (NO). However, whether these so-called pleiotropic effects of statins result in improved patient outcomes remains to be established. To provide further insights into the mechanisms of action and impact on clinical outcomes of peri-operative statin treatment in patients undergoing cardiac surgery, I studied the molecular mechanisms underlying the myocardial nitroso-redox balance in samples of the right atrial appendages (RAA) obtained before (PRE) and after cardiopulmonary bypass (CPB) and reperfusion (POST) and setup two double-blind randomised placebo-controlled trials: 1) STARR (Statin Treatment on Atrial Refractoriness and Reperfusion injury), which tested the effect of Atorvastatin (80 mg once daily for up to 6 days before surgery and 5 days after) on the atrial effective refractory period (AERP, over 4 post-operative days) and superoxide production in paired PRE- and POST- RAA samples from 60 patients 2) STICS (Statin Treatment In Cardiac Surgery), which assessed the effects of peri-operative treatment with Rosuvastatin (20mg od) on POAF (assessed by continuous holter ECG monitoring for 5 days postoperatively) and myocardial injury (assessed by serial troponin I measurements) in 1922 patients undergoing elective cardiac surgery. I observed that atrial superoxide production increased significantly after reperfusion due to increased mitochondrial and NOX2 oxidase activity and to uncoupling of NOS activity. NOS activity in RAA samples decreased significantly after reperfusion (by 60&percnt;), but this reduction was not prevented by BH4 supplementation (10 &mu;M) or NOX2 inhibition. Instead, I identified increased endothelial NOS S-glutathionylation as the main mechanism responsible for NOS uncoupling after reperfusion. In STARR, atorvastatin prevented increase in RAA superoxide production, maintained the functionally coupled status of NOS and NO bioavailability after reperfusion but had no measurable effect on postoperative AERP. In STICS, treatment with rosuvastatin significantly reduced LDL-C concentration by 48 hours after surgery but had no effect on the incidence of POAF (203 (21&percnt;) of the Rosuvastatinallocated patients vs. 197 (20&percnt;) of the placebo-allocated patients) or on perioperative myocardial damage (P = 0.80). Pre-defined subgroup analyses (age, sex, prior statin use, baseline troponin concentration, duration of randomized treatment before surgery, type of cardiac surgery, and postoperative use of anti-inflammatory drugs) did not identify any category of patient who benefited from perioperative rosuvastatin treatment. Nor were there beneficial effects on any of the other in-hospital clinical outcomes that were assessed. In conclusion, cardiac surgery on CPB is associated with myocardial nitroso redox imbalance that is reversed by perioperative intensive therapy with statins. However, these effects have no beneficial effects on common in-hospital complications after elective cardiac surgery. Although the benefits of long-term statin therapy in patients requiring myocardial revascularization are well established, the work presented in this thesis does not support routine use of perioperative intensive therapy with statins for the prevention of postoperative complications in patients undergoing elective cardiac surgery.

[ES] La lesión por isquemia-reperfusión (I/R) hepática (IRI) es una causa importante de mortalidad y morbilidad en la resección hepática y el trasplante de hígado. Durante la hipoxia, el hígado permanece sin oxígeno, cambiando su metabolismo y parando la síntesis de ATP. Paradójicamente, el restablecimiento del flujo de oxígeno causa más daño, activando el sistema inmunitario que generará una gran cantidad de especies reactivas de oxígeno (ROS) causando daño celular y tisular. La ciclooxigenasa-2 (COX-2) es una enzima clave en la biosíntesis de prostaglandinas y su importancia en la IRI es controvertida. La PGE2, prostaglandina E2, es el principal producto de la COX-2, y participa en la mediación de procesos patológicos como la inflamación, la fiebre y el dolor. El uso de AINEs, inhibidores específicos de la COX, apunta a un efecto beneficioso en la resolución del proceso inflamatorio, pero cada vez más estudios apoyan el papel antiinflamatorio de la COX-2. De hecho, estudios previos han demostrado que la sobreexpresión de COX-2 en hepatocitos protege a los ratones de la apoptosis y el estrés celular, además de reducir la respuesta inflamatoria en diferentes modelos de enfermedad hepática. En esta tesis doctoral, se utilizó un ratón transgénico que sobreexpresa COX-2 en los hepatocitos (h-COX-2 Tg) para dilucidar el papel y la implicación de la COX-2 en la IRI. Los animales de tipo silvestre (Wt) y h-COX-2 Tg fueron sometidos a 90 min de isquemia, seguidos de 4 o 24 h de reperfusión. Comparando los animales h-COX-2 Tg con sus hermanos Wt, el daño celular y tisular se atenúa tras la IRI. Entre las distintas vías modificadas, la cascada inflamatoria está menos activada, con menor liberación de citoquinas pro-inflamatorias, menor reclutamiento hepático, e infiltración de neutrófilos. Las vías de necrosis y apoptosis también se atenúan así como se reduce del estrés del retículo endoplásmico, y aumenta la autofagia. La respuesta antioxidante se potencia en el contexto de la sobreexpresión de COX-2 y la producción total de ROS es menor, lo que contribuye a un menor daño tisular. Cuando los animales Wt se someten un pre-condicionamiento (PC), la COX-2 endógena se induce a niveles más altos que sin PC, mostrando menos daño, una inflamación atenuada, y una respuesta antioxidante mejorada. Además, se muestra que el papel de la COX-2 en esta protección es específico, ya que su inhibición con DFU revierte los efectos observados e iguala el daño causado a los animales Wt. Las mitocondrias son actores centrales en la fisiopatología de la IRI. En este sentido, la función mitocondrial está preservada en los hígados que sobreexpresan COX-2, con un potencial de membrana mitocondrial conservado y una tasa respiratoria preservada. Estos efectos pueden explicarse por una estabilización de las crestas mitocondriales, invaginaciones de la membrana mitocondrial interna (IMM) que se mantienen mediante interacciones de varias isoformas de la proteína OPA1. Su procesamiento está mediado por proteasas, como OMA1. En ratones h-COX-2 Tg hay un menor procesamiento de OPA1, que se correlaciona con una actividad atenuada de OMA1. Por otro lado, se realizó un estudio retrospectivo en pacientes que habían sido sometidos a un trasplante hepático. Se analizaron los niveles de PGE2 y se correlacionaron con las funciones hepáticas tras el trasplante. Este análisis muestra que la presencia de PGE2 en el plasma de los pacientes receptores se correlaciona con un mejor pronóstico, mientras que unos niveles más bajos de PGE2 se asocian con una disfunción precoz del injerto. Todos estos resultados presentan a la COX-2 como un nuevo actor en la protección del hígado tras I/R, mostrando un papel antiinflamatorio y antioxidante, así como reduciendo el daño mitocondrial, el estrés celular y la muerte celular. Además, se demuestra cómo las prostaglandinas derivadas de la COX-2 en condiciones fisiológicas pueden desempeñar un papel protector en casos de trasplante hepático. / [CA] La lesió per isquèmia-reperfusió (I/R) hepàtica (IRI) és una causa important de mortalitat i morbiditat en la resecció hepàtica i el trasplantament de fetge. Durant la hipòxia, el fetge roman sense oxigen, canviant el seu metabolisme i aturant la síntesi d'ATP. Paradoxalment, el restabliment del flux d'oxigen causa més danys, activant el sistema immunitari que genera una gran quantitat d'espècies reactives d'oxigen (ROS) causant dany cel·lular i tissular. La ciclooxigenasa-2 (COX-2) és un enzim clau en la biosíntesi de prostaglandines i la seva importància a l'IRI és controvertida. La PGE2, prostaglandina E2, és el principal producte de la COX-2, i participa en la mediació de processos patològics com la inflamació i la febre. Mentre que l'ús d'AINEs, inhibidors específics de la COX-2, apunta a un efecte beneficiós en la resolució del procés inflamatori, cada cop més estudis donen suport a un paper antiinflamatori de la COX-2. De fet, estudis previs han demostrat que la sobreexpressió de COX-2 en hepatòcits protegeix els ratolins de l'apoptosi i l'estrès cel·lular, a més de reduir la resposta inflamatòria, en diferents models de malaltia hepàtica. En aquesta tesi, s'ha utilitzat un ratolí transgènic que sobreexpressa la COX-2 en els hepatòcits (h-COX-2 Tg) per dilucidar el paper i la implicació de la COX-2 a l'IRI. Els animals de tipus silvestre (Wt) i h-COX-2 Tg van ser sotmesos a 90 min d'isquèmia, seguits de 4 o 24 h de reperfusió. Comparant els animals h-COX-2 Tg amb els seus germans Wt, el dany cel·lular i tissular s'atenua després de l'IRI. Entre les diferents vies modificades, la cascada inflamatòria està menys activada, s'alliberen menys citocines proinflamatòries , hi ha un menor reclutament hepàtic i menor infiltració de neutròfils. Les vies de necrosi i apoptosi també s'atenuen, així com es redueix l'estrès del reticle endoplasmàtic, i l'autofàgia augmenta. La resposta antioxidant es potencia i la producció total de ROS també és menor, fet que contribueix a un menor dany tissular. Quan els animals Wt se sotmeten a un precondicionament (PC), la COX-2 endògena s'indueix a nivells més alts que sense PC, i aquests fetges mostren menys dany, una inflamació atenuada i una resposta antioxidant millorada. A més, es mostra que el paper de la COX-2 en aquesta protecció és específic, ja que la seva inhibició amb DFU, reverteix els efectes observats i iguala el dany causat als animals Wt. Els mitocondris són actors centrals en la fisiopatologia de l'IRI. En aquest sentit, la funció mitocondrial és preservada als fetges que sobreexpressen COX-2, com es pot demostrar per un potencial de membrana mitocondrial conservat i una taxa respiratòria preservada. Aquests efectes es poden explicar per una estabilització de les crestes mitocondrials, invaginacions de la membrana mitocondrial interna (IMM) que es mantenen mitjançant interaccions de diverses isoformes d'OPA1, una proteïna de la IMM. El seu processament està mediat per proteasas, com OMA1. En ratolins h-COX-2 Tg hi ha un menor processament d'OPA1, que es correlaciona amb una activitat atenuada d'OMA1, mostrant una estabilització de les crestes. D'altra banda, es va fer un estudi retrospectiu amb pacients que havien estat sotmesos a un trasplantament hepàtic. Es van analitzar els nivells de PGE2 i es van correlacionar amb les funcions hepàtiques després del trasplantament. Aquesta anàlisi mostra que la presència de PGE2 en el plasma dels pacients receptors es correlaciona amb un millor pronòstic, mentre que uns nivells més baixos de PGE2 s'associen amb una disfunció precoç de l'empelt. Tots aquests resultats presenten a la COX-2 com un nou actor en la protecció del fetge després d'I/R, mostrant un paper antiinflamatori i antioxidant, així com reduint la lesió mitocondrial, l'estrès cel·lular i la mort cel·lular. A més, es demostra com les prostaglandines derivades de la COX-2 en condicions fisiològiques poden exercir un paper protector en casos de trasplantament hepàtic. / [EN] Hepatic ischemia-reperfusion (I/R) injury (IRI) is a major cause of mortality and morbidity in liver resection and liver transplantation. During the hypoxia, the liver remains without oxygen supply, shifting its metabolism and stopping the ATP synthesis. Paradoxically, the restoration of oxygen flow causes the most damage with an activation of the immune system that will generate a burst of reactive species of oxygen (ROS) that will cause cell and tissue damage. Cyclooxygenase-2 (COX-2) is a key enzyme in prostaglandin biosynthesis and its importance in IRI is controversial. PGE2, prostaglandin E2, is the main product of COX-2, and is mainly involved in mediating pathological processes such as inflammation, fever and pain. While the use of NSAIDs, specific COX inhibitors, points to a beneficial effect in the resolution of the inflammatory process, several studies support the idea of an anti-inflammatory role of COX-2. In fact, previous studies have shown that COX-2 overexpression in hepatocytes protects mice from apoptosis and cellular stress, as well as reducing the inflammatory response, in different liver disease models. In this PhD thesis, a hepatocyte-specific COX-2 transgenic mouse (h-COX-2 Tg) was used to elucidate the role and involvement of COX-2 in IRI. Wild type (Wt) and h-COX-2 Tg animals were subjected to 90 min of ischemia, followed by 4 or 24 h of reperfusion. Comparing h-COX-2 Tg animals with their Wt littermates, cellular and tissue damage resulting from IRI is attenuated. Among these pathways, the inflammatory cascade is less activated, with less pro-inflammatory cytokine release, less hepatic recruitment and neutrophil infiltration. Necrosis and apoptosis pathways are also attenuated such as reduced endoplasmic reticulum stress, and increased autophagy. The antioxidant response appears to be enhanced in the context of COX-2 overexpression and total ROS production is also lower, contributing to less tissue damage. When Wt animals are subjected to preconditioning (PC), endogenous COX-2 is induced at higher levels than without PC, and these livers show less damage, attenuated inflammation, and an enhanced antioxidant response. Furthermore, the role of COX-2 in this observed protection has been shown to be specific, as its inhibition with DFU, reverses the observed effects, and matched the damage caused to Wt animals. Mitochondria are central players in the pathophysiology of IRI. In this regard, mitochondrial function is preserved in COX-2-overexpressing livers, as can be demonstrated by a conserved mitochondrial membrane potential and a preserved respiratory rate. These results can be explained by a stabilisation of mitochondrial cristae, invaginations of the inner mitochondrial membrane (IMM) that maintained through interactions of various isoforms of OPA1. Its processing is mediated by proteases, such as OMA1, which acts under certain stimuli. In h-COX-2 Tg mice, there is a reduced OPA1 processing that correlates with attenuated OMA1 activity, showing a stabilisation of cristae in the context of COX-2 overexpression after I/R. On the other hand, a retrospective study was conducted in patients who had undergone liver transplantation. In this part of the study, PGE2 levels were analysed and correlated with liver functions after transplantation. This analysis shows that the presence of PGE2 in the plasma of recipients correlates with a better prognosis, while lower PGE2 levels are associated with early graft dysfunction. All these results present COX-2 as a new player in liver protection after I/R, showing an anti-inflammatory and antioxidant role, as well as reducing mitochondrial damage, cell stress and cell death. Furthermore, it is shown how COX-2-derived prostaglandins under physiological conditions can play a protective role in cases of liver transplant. / This work has been carried out with the financial support of the Spanish Ministry of Science and Innovation (SAF2016-75004R and PID2019-108977RB-100), the CIBERehd (Centro de Investigaciones Biomédicas En Red de Enfermedades Hepáticas y Digestivas) and the COST Action (CA15203 - Mitochondrial mapping: Evolution - Age - Gender - Lifestyle - Environment (MITOEAGLE)). Marina Fuertes Agudo benefited from a pre-doctoral FPI contract (BES-2017- 081928) associated with the SAF2016-75004R project. She spent 3 months in the laboratory of Dr. Pau Sancho Bru at the Institut d’Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS, Barcelona, Spain) funded by a short stay grant awarded by the CIBERehd and 3 months in the laboratory of Dr. Anne Dubart Kupperschmitt and Dr. Jean Charles Duclos Vallée at the Institut Nationale de la Santé et la Recherche Médicale (INSERM, Villejuif, France) funded by a short stay grant awarded by the European Molecular Biology Organisation (EMBO, SEG_9771). / Fuertes Agudo, M. (2023). Role of Cyclooxygenase-2 in Ischemia-Reperfusion Injury in the Liver [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/196602

Le zinc est un métal, membre des éléments trace dont le rôle protecteur contre l’ischémie-reperfusion myocardique a été suggéré. Dans ce travail de thèse, nous avons étudié le mécanisme par lequel le zinc procure cette protection. Nous avons utilisé des cultures cellulaires de cardiomyoblastes de rat, des cardiomyocytes isolés de rat, des mitochondries isolées ainsi qu’un modèle de cœur isolé perfusé de rat. Nous avons montré que l'administration de zinc au début de la reperfusion permet de diminuer la mort cellulaire sur un modèle d’ischémie-reperfusion simulée. Nous avons montré que l’administration de zinc entraine l’activation de la cascade des kinases de survie (PI-3K/Akt), l’inhibition de GSK-3β et celle de l’ouverture du pore de transition de perméabilité mitochondrial. Nous avons ensuite montré qu’en présence de zinc, l’activation d’Akt provenait d’une inhibition de certaines phosphatases et d’une activation du domaine tyrosine kinase du récepteur IGF-1. Nos derniers travaux ont eu pour but de préciser le rôle du zinc suite à l'activation des récepteurs à adénosine. / Zinc is a member of the family of the metal trace elements and its role in the protection against lethal reperfusion injury has been strongly suggested. The aim of the present work was to determine the molecular mechanisms involved in zinc cardioprotection. We have used cultured rat cardiomyoblasts, isolated rat cardiomyocytes, isolated mitochondria and isolated perfused rat heart. We demonstrated that zinc given at reperfusion, limits cell necrosis in a model of simulated ischemia-reperfusion. Administration of zinc results in activation of the reperfusion-injury salvage kinases pathway (PI-3K/Akt), inhibition of GSK-3β and of the opening of the mitochondrial permeability transition pore. We then showed that, in the presence of zinc, activation of Akt is mediated by the inhibition of various phosphatases and by activation of the tyrosine kinase domain of the IGF-1 receptor. Our latest experiments report the action of zinc following activation of adenosine receptors

Despite the development and use of specialised cardiac preservation solutions, the quality of the donor heart may still be compromised by its obligatory exposure to periods of ischaemia (both cold and warm) followed by reperfusion upon reintroduction of the recipient circulation. This is reflected in Transplant Registry data showing increased primary allograft failure as a function of increasing ischaemic time. The research described in this thesis is designed to further the understanding of the mechanisms by which the donor heart may be adapted to these prolonged periods of ischaemia and reperfusion by the activation of endogenous pro-survival signalling pathways by the addition of pharmacological agents to Celsior, a clinical preservation solution. Studies were conducted in an isolated working rat heart model of donor heart preservation. The first study investigated the cardioprotective effects of a novel inhibitor of poly(ADP-ribose) polymerase 1, INO-1153. Maximum protective effect (after a 6 hour storage period) was observed when the PARP inhibitor was administered prior to cardiac arrest and storage and when the agent was added to the Celsior cardioplegic / storage solution. This protective affect was associated with activation of the Akt signalling pathway and could be prevented by inhibition of Akt phosphorylation and activation. The second study examined functional protection and pro-survival signalling pathway activation in hearts arrested and stored for 6 hours in Celsior supplemented with glyceryl trinitrate (an exogenous source of nitric oxide) and Cariporide (an inhibitor of sodium hydrogen exchange). Here, cardiac protection was accompanied by activation of the ERK 1/2 pro-survival pathway as well as a decrease in apoptosis. The third study examined the cardioprotective effect of supplementation of Celsior with all three agents after an extended (10 hour) period of hypothermic storage. Significant recovery of function was only observed in the triply supplemented hearts, being accompanied by activation of both the Akt and ERK pathways. These studies demonstrate for the first time the feasibility of recruitment of endogenous pro-survival pathways as an approach to increasing the post-storage function of the donor heart. Importantly, for the logistics of clinical transplantation, these pathways can be recruited by addition of appropriate pharmacological agents to the arresting and storage solution.

Die extrakorporale Zirkulation unter Einsatz einer Herz-Lungen-Maschine kann postoperativ zu kontraktilen ventrikulären Funktionsstörungen führen, die Morbidität und Mortalität für betroffene Patienten erhöht. Diese kardiale Dysfunktion bezeichnet man als myokardiales Stunning, welche durch die globale Ischämie ausgelöst wird. Das Phänomen der reversiblen kontraktilen Dysfunktion weißt eine hohe klinische Relevanz auf und ist somit in den vergangenen Jahrzehnten sowohl klinisch als auch experimentell intensiv erforscht worden. Dabei kamen unterschiedlichste Spezies, Methoden und Modelle zum Einsatz. Ziel der vorliegenden Arbeit ist die Etablierung eines neuartigen akut instrumentierten Tiermodells, anhand dessen Folgen des kardiopulmonalen Bypasses und Wirkung des endogenen Opioidsystems auf myokardiales Stunning untersucht werden können. Mit Hilfe des entwickelten Versuchsmodells können die Auswirkungen applizierter Opioidrezeptorantagonisten und die Effekte der kardiopulmonalen Zirkulation auf die kontraktile myokardiale Dysfunktion valide untersucht werden. Als Versuchstiere wurden 50 männlich kastrierte Schweine der Rassenkreuzung „Deutsche Landrasse“ und „Yorkshireschwein“ eingesetzt. In Allgemeinanästhesie wurden die Tiere über eine Thorakotomie instrumentiert und anschließend elektrisch Kammerflimmern induziert. Nach Erreichen einer stabilen extrakorporalen Zirkulation unter der Herz-Lungen-Maschine wurde nach Ablauf der ischämischen Phase eine standardisierte Reaninmation und Weaning durchgeführt. Alle 50 Tiere konnten den Versuch erfolgreich durchlaufen. Die Analyse und Auswertung sämtlicher archivierter Daten und Proben der Versuchstiere wurde zu einem späteren Zeitpunkt durchgeführt.