Dissertations / Theses on the topic 'Myocardial ischemia/chemically induced'

Os modelos experimentais de falência do ventrículo direito (VD) descritos são de difícil execução, por envolverem procedimentos cirúrgicos complexos e dispendiosos, com alta mortalidade e baixa reprodutividade. Também são raros aqueles que promovem falência ventricular exclusiva do ventrículo direito. Com o objetivo de simular a situação clínica de falência ventricular exclusiva do VD, desenvolvemos um modelo experimental de falência crônica isolada do VD, feito através da injeção de etanol intramiocárdico. A disfunção do VD foi induzida em treze cães adultos, através de múltiplas injeções de etanol a 96% (dose total 1ml/kg), na via de entrada e na porção trabecular do VD. Foram analisados parâmetros hemodinâmicos, metabólicos e ecocardiográficos, em condições basais e após indução da falência. Os animais foram mantidos vivos por um período de 14 dias, após o qual foram reoperados e mortos para análise morfológica dos corações. As pressões foram aferidas na artéria femoral, átrio esquerdo (AE), átrio direito (AD), tronco pulmonar (TP) e VD. O fluxo sanguíneo foi medido na aorta e no tronco pulmonar. Na primeira operação, os dados hemodinâmicos foram registrados antes e imediatamente após a indução da falência do VD. Estes parâmetros também foram coletados na reoperação (14 dias pós-indução da falência). Amostras de sangue arterial e venoso foram colhidas simultaneamente ao registro dos parâmetros hemodinâmicos para avaliação dos gases sanguíneos e dosagem de lactato. Foram feitas avaliações ecocardiográficas no pré-operatório, no sexto e no 130 dia pós-operatório (PO). Houve uma mortalidade de 15,4% (2/13). Observou-se uma queda significativa no débito cardíaco pulmonar após a indução da falência e na reoperação (14º PO), quando comparado aos valores medidos no momento basal (p=0,002). Esta queda do desempenho ventricular direito ocasionou um aumento nos valores do lactato venoso, quando comparados os valores basais contra aqueles do pós infusão de etanol (p=0,0001) e com os da reoperação (p=0,0003). Os dados ecocardiográficos revelaram um aumento no volume diastólico do VD, quando comparados os dados basais com aqueles da primeira semana de seguimento (p=0,0001) e com aqueles medidos duas semanas após a indução da falência (p=0,0084). Houve uma queda significativa da fração de ejeção do VD na primeira e segunda semanas em comparação aos valores basais (p=0,0001). A avaliação microscópica feita a partir de amostras retiradas do VD revelou infartos transmurais, com características histológicas compatíveis com infarto de duas semanas de evolução. A injeção de etanol intramiocárdio permitiu a criação de um modelo simples, econômico e reprodutível de falência crônica isolada do VD. A avaliação dos dados hemodinâmicos, ecocardiográficos e bioquímicos medidos nos diferentes momentos do experimento é compatível com a indução de falência grave do VD. Este modelo pode ser útil no estudo da fisiopatologia da falência isolada do VD, bem como na avaliação de assistência ventricular.<br>Isolated chronic right ventricle (RV) dysfunction is difficult to achieve in experimental models, and sometimes very expensive. Some models require complex surgical procedures, with a high mortality rate and low reproducibility. In order to simulate the clinical status of RV failure, we have developed an experimental model of chronic RV failure by injecting local intramyocardial ethanol 96%. A severe RV dysfunction was induced in 13 mongrel dogs by means of multiple intramyocardial injections of ethanol 96% (total dose of 1ml/kg), in the inlet and trabecular portions of the RV. The hemodynamics, metabolic and ecocardiografic parameters were evaluated at baseline condition and after RV failure. The animals were followed for a 14-day period. After that, they were reoperated for pressure measurements, taken from femoral artery, left atrium (LA), right atrium (RA), RV and main pulmonary artery (PA). Blood flow was measured in the aorta (AO) and PA. Metabolic parameters were assessed simultaneously to pressure measurements, with arterial and venous blood samples withdrawn for gas and lactate analysis, and then, they were killed for morphological evaluation of the heart. All the animals were submitted to echocardiographic evaluation before the first operation, on sixth and on de 13th postoperative day. The mortality rate was 15,4% (2/13). There was a significant decrease in the pulmonary blood flow after RV failure induction (p=0,0018), as well as in the reoperation (p=0,002), as compared to baseline. This decrease in RV performance caused an increase in the venous lactate level, when baseline values are compared with pos infarction values (p=0,001) and reoperation (p=0003). The echocardiografic findings revealed an increased RV diastolic volume on the 6th (p=0,0001) and 13th postoperative day (p=0,0084), as compared to baseline values. Conversely, there was a significant decrease in the RV ejection fraction on the 6th and 13th postoperative day, when compared with baseline values (p=0,0001). Microscopic evaluation of RV samples showed transmural infarctions with histological characteristics aged of two weeks. Intra myocardial ethanol injection has allowed the creation of a simple, inexpensive and reproducible model of chronic RV failure. The hemodynamic, metabolic and echocardiographic parameters assessed in different moments of the protocol are compatible with severe RV failure. It may be useful for RV physiology studies of isolated right sided heart failure, as well as in the assessment of ventricular assist devices.

Mental stress ischemia (MSI) is associated with poor prognosis for coronary artery disease (CAD) and is amenable to treatment, yet no easily administered test exists to diagnose it. Given the known increase in systemic vascular tone in response to stress, we studied the ability of peripheral arterial tonometry (PAT), a noninvasive functional measure of arterial tone, to predict those vulnerable to MSI. Seventy-seven patients with chronic stable CAD were subjected to mental stress with concomitant assessment of myocardial perfusion and pulse wave amplitude. Nuclear perfusion imaging was used to document MSI, and PAT was used to measure pulse wave and microarterial tone. A ratio of PAT measurements during stress to those before stress was used to characterize vascular responses. Serum catecholamines and endothelin-1 (ET-1) were simultaneously measured. Subjects who experienced MSI had a lower average PAT ratio than those who did not (0.76 ¡À 0.04 vs. 0.91 ¡À 0.05, P = 0.03). A receiver operating characteristics curve for PAT ratio predicting MSI had an area under the curve of 0.613 (standard error, 0.065, one-sided P = 0.04). Maxima of sensitivity and specificity were observed at a threshold of 0.78 to define an abnormal PAT ratio. Cross-tabulation of groups above and below this threshold with groups of subjects with and without MSI showed a significant predictive relationship between PAT ratio and MSI (P = 0.03). Subjects at or below this threshold (¡Ü0.78) displayed a significant increase in norepinephrine levels during mental stress (235 pg/ml at baseline, 259 pg/ml during mental stress, P = 0.007). Subjects above this threshold (>0.78) displayed a significant decline in their ET-1 levels 24 hours after mental stress (1.15 pg/ml after mental stress, 0.93 pg/ml 24 hours later, P = 0.01), while those at or below threshold had a continued increase. PAT ratio is a complex functional measure of peripheral arterial tone that significantly predicts the occurrence of MSI. It may have clinical value as an easily administered screening test for MSI.

[ES] Las intervenciones actuales utilizadas en el ámbito clínico durante el infarto agudo de miocardio (IAM) se centran en la revascularización de la zona isquémica. Entre dichas estrategias, la angioplastia coronaria, procedimiento por el cual se utiliza un catéter para desobstruir la arteria ocluida, es el método más utilizado. Sin embargo, se ha descrito este proceso (conocido como reperfusión) desencadena un daño adicional en el miocardio, por lo que la combinación de dicha intervención con moléculas cardioprotectoras resulta de gran interés para tratar de reducir el tamaño del infarto. El presente trabajo propone dos nuevas moléculas con el fin de precondicionar el área isquémica antes de la reperfusión en el contexto del IAM. La primera estrategia propuesta se ha basado en el aporte de un ácido graso (diDHA) en la zona isquémica antes de la reperfusión para tratar de reducir el estrés de los cardiomiocitos y el número de células muertas antes de la reperfusión. Además, se han sintetizado nanoconjugados basados en la unión covalente de diDHA a un unido covalentemente a un esqueleto polimérico (ácido poli-L-glutámico, PGA) con el fin de incrementar la estabilidad del diDHA y conseguir una liberación controlada de la molécula. Los resultados obtenidos mostraron que la formulación PGA-diDHA6.4 fue la más optimizada, mostrando un mejor efecto en el precondicionamiento de los cardiomiocitos antes de la reperfusión en términos de reducción de apoptosis, generación de especies reactivas de oxígeno y mantenimiento de la función mitocondrial in vitro. Además, dicho nanoconjugado también mostró un modesto efecto terapéutico cuando se administró en modelos in vivo de isquemia-reperfusión en ratas y cerdos, reduciendo el tamaño final de infarto respecto a los grupos control. La segunda estrategia terapéutica propuesta se ha centrado en aumentar el potencial terapéutico de las vesículas celulares de pequeño tamaño (SEV o exosomas) procedentes de medio condicionado de células madre estromales (MSC). Numerosos estudios han descrito el papel terapéutico de factores paracrinos secretados por las MSC, donde se incluyen tanto factores solubles como vesículas extracelulares (EV) y, en especial, SEV. Diversas estrategias, como la modificación genética o precondicionamiento de estas células, han sido utilizadas para aumentar el potencial terapéutico de las mismas. En este trabajo se ha propuesto la modificación genética de las MSC con el objetivo de enriquecer las SEV en proteínas de interés que pudiesen potenciar el efecto terapéutico de las SEV nativas. En base a estudios previos, donde se ha visto que la oncostatina-M (OSM) podría jugar un papel anti-fibrótico en el contexto del IAM, se decidió incorporar dicha proteína en la superficie de las SEV derivadas de MSC mediante su fusión con proteínas presentes de forma natural en la superficie de las SEV, con el objetivo de desencadenar una respuesta en las células diana. La modificación de la secuencia de la OSM y su fusión con la tetraspanina CD81 permitieron cargar de manera efectiva la OSM en la superficie de las SEV, y los resultados preliminares en fibroblastos ventriculares cardíacos mostraron un efecto funcional beneficioso con respecto a los SEV control y los enriquecidos en CD81, reduciendo la tasa de proliferación de las células en condiciones de ayuno, y modificando la expresión y la liberación de la proteína telo-Col1α1 en las células después de ser estimuladas con TGFβ-1, α-dextrano y ácido ascórbico-L-sulfato En resumen, dos nuevas estrategias terapéuticas avanzadas libres de células han sido propuestas en el presente trabajo, donde se han mostrado resultados preliminares prometedores para reducir el daño en el miocardio tras el IAM en términos de reducción de apoptosis de cardiomiocitos y de activación de fibroblastos car<br>[CA] Les intervencions actuals utilitzades en l'àmbit clínic durant l'infart agut de miocardi (IAM) se centren en la revascularització de la zona isquèmica. Entre aquestes estratègies, l'angioplàstia coronària, procediment pel qual s'utilitza un catèter per a desobstruir l'artèria oclosa, és el procés més utilitzat. No obstant això, s'ha descrit que aquest procés (conegut com a reperfusió) desencadena un mal addicional en el miocardi. En conseqüència, la combinació d'aquesta intervenció amb molècules cardioprotectores resulta de gran interés per a tractar de reduir la grandària de l'infart. El present treball proposa dues noves molècules amb potencial cardioprotector en el context del IAM. Com a primera estratègia terapèutica, s'ha proposat l'aportació d'un àcid gras (diDHA) a la zona isquèmica del miocardio abans de la reperfusió per a tractar de reduir l'estrés dels cardiomiocitos i el nombre de cèl·lules mortes abans de la reperfusió. A més, s'han sintetitzat nanoconjugats basats en la unió covalent de diDHA a un esquelet polimèric (àcid poli-L-glutàmic, PGA) amb la finalitat d'incrementar l'estabilitat del diDHA i aconseguir un alliberament controlat de la molècula. Els resultats obtinguts van mostrar que la formulació PGA-diDHA6.4 va ser la més efectiva, mostrant un millor efecte en el precondicionament dels cardiomiocitos abans de la reperfusió en termes de reducció d'apoptosi, generació d'espècies reactives d'oxigen i manteniment de la funció mitocondrial in vitro. A més, el nanoconjugat PGA-diDHA6.4 també va mostrar un modest efecte terapèutic quan es va administrar en models in vivo d'isquèmia-reperfusió en rates i porcs, reduint la grandària final d'infart respecte als grups control. La segona estratègia proposada s'ha centrat en potenciar l'efect terapèutic de vesícules extracelul·lars de xicoteta grandària (SEV o exosomes) que son secretades per cèl·lules mare estromales. Nombrosos estudis han descrit el paper terapèutic de factors paracrinos secretats per les MSC, on s'inclouen tant factors solubles com vesícules extracelul·lars (EV) i, especialment, les SEV. Diverses estratègies, com la modificació genètica o el precondicionament de les MSC, s'han estudiat per augmentar el potencial terapèutic d'aquestes cèl·lules. En aquest treball, es va pensar en la modificació genètica de les MSC amb l'objectiu d'enriquir les SEV en proteïnes d'interés que pogueren potenciar l'efecte terapèutic de les SEV natives. Sobre la base d'estudis previs, on s'ha vist que la oncostatina-M (OSM) podria jugar un paper anti-fibròtic en el context del IAM, es va decidir incorporar aquesta proteïna en la superfície de les SEV derivades de MSC mitjançant la seua fusió amb proteïnes presents de manera natural en la superfície de les SEV, amb l'objectiu de desencadenar una resposta en les cèl·lules diana. La modificació de la seqüència de la OSM i la seua fusió amb la tetraspanina CD81 van permetre carregar de manera efectiva la OSM en la superfície de les SEV, i els resultats preliminars en fibroblastos ventriculars cardíacs van mostrar un efecte funcional respecte als SEV control i els enriquits en CD81, reduint la taxa de proliferació de les cèl·lules en condicions de dejuni, i modificant l'expressió i la secreció de la proteïna telo-Col1α1 en les cèl·lules després de ser estimulades amb TGFβ-1, α-dextran i àcid ascòrbic-L-sulfat, simulant una activació dels fibroblastos in vitro. En resum, dues noves estratègies terapèutiques avançades lliures de cèl·lules han sigut proposades en el present treball, on s'han mostrat resultats preliminars prometedors per a reduir el mal en el miocardi després del IAM en termes de reducció d'apoptosi de cardiomiocitos i d'activació de fibroblastos cardíacs.<br>[EN] Current therapeutic approaches against acute myocardial infarction (AMI) are focused on myocardial ischemic zone revascularization. The most common strategy is called primary angioplasty, in which a catheter is introduced to unblock the affected artery and restore blood flux, in a process called reperfusion. Nevertheless, an additional injury on cardiac tissue is caused after reperfusion, and the combination of primary angioplasty with the use of cardioprotective molecules has emerged as a potential strategy to reduce cardiac tissue injury. Two new cell-free therapeutic strategies to preconditionate myocardial ischemic area before reperfusion have been proposed to reduce cardiac injury after AMI. The first therapeutic strategy proposed consisted on the input of a free fatty acid (di-docosahexaenoic acid, diDHA) covalently bound to a polymeric backbone (poly-L-glutamic acid, PGA) in order to increase diDHA solubility and stability and modulate its effect on target cells. Results showed that PGA-diDHA6.4 conjugate administration during ischemia protected cardiomyocytes from reperfusion-induced injury, as apoptotic number of cells and oxidative stress was reduced, and mitochondrial function was less affected when compared to untreated cells. In addition to this, PGA-diDHA6.4 also showed therapeutic effects when locally administered in an ischemia-reperfusion in vivo model in rats and pigs, where a modest reduction of area at risk was observed compared to control groups. The second cell-free strategy proposed in this work was focused on enhancing the therapeutic potential of small extracellular vesicles (SEV or exosomes) isolated form mesenchymal stromal cells (MSC) conditioned media. Previous studies have described the therapeutic potential of paracrine factors released by MSC, where both soluble factors and vesicular components are included. In particular, SEV have gained special attention. Several stretegies, such as genetic modification or cell preconditioning, have been tested to enhance the MSC therapeutic potential. In this work, it was proposed MSC genetic modification in order to load proteins of interest on SEV and potentiate its native therapeutic potential. Based on previous findings, where it has been described a potential anti-fibrotic role of oncostatin-M (OSM) in AMI context, we decided to incorporate OSM on SEV surface by its fusion to CD81 tetraspanin, a protein naturally loaded on SEV surface, in order to trigger functional effects on target cells. OSM sequence modification was necessary in order to load the protein on SEV surface efficiently, and preliminary data showed that modified OSM-CD81 loaded on SEV had a functional effect on human ventricular cardiac fibroblasts. Concretely, decrease of proliferation rate after starvation and telo-Collagen1α1 location pattern modification was observed after stimulation with a pro-fibrotic cocktail (containing TGFβ-1, α-dextran and ascorbic-L-acid sulphate) in vitro when cells were treated with modified OSM-CD81- SEV compared to ctrl and CD81-loaded SEV treatments. Overall, two new advanced cell-free therapies with preliminary promising results have been proposed in order to reduce myocardial injury after AMI in terms of cardiomyocytes apoptosis reduction and fibrosis mitigation.<br>Tejedor Gascón, S. (2021). Development of new advanced therapies to mitigate ischemia-reperfusion-induced injury during acute myocardial infarction [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/171487<br>TESIS

Submitted by Fabíola Silva (fabiola.silva@famerp.br) on 2016-09-27T15:19:20Z No. of bitstreams: 1 marcosaureliobdeoliveira_tese.pdf: 6679652 bytes, checksum: cda6b164bdebbe6a1f4cfdb403d7ea04 (MD5)<br>Made available in DSpace on 2016-09-27T15:19:20Z (GMT). No. of bitstreams: 1 marcosaureliobdeoliveira_tese.pdf: 6679652 bytes, checksum: cda6b164bdebbe6a1f4cfdb403d7ea04 (MD5) Previous issue date: 2014-12-09<br>Introduction: Solutions that cause elective cardiac arrest are often evolving, but the ideal compound has not yet been found. The authors compare a new cardioplegic solution with histidine-tryptophan-glutamate (HTG) with histidine-tryptophan-acetoglutarato (HTK) in a model of isolated rat heart. Objective: Benchmarking HTK and HTG solutions regarding the activity of caspase, KI -67, IL - 8, Shannon entropy and fractal dimension. Materials and Methods: Twenty male rats, Wistar race, were analyzed and heparinized. The chest was opened, held and cardiectomia infused 40 mL/kg of appropriate cardioplegia. The hearts were kept for 2 hours at 4°C in the same solution, and afterwards placed in the Langendorff apparatus for 30 min with Ringer-Locke solution. The immunohistochemical analyses were performed for caspase, IL-8, KI-67, Shannon entropy and fractal dimension. Results: The concentration of caspase was lower in group 2, and KI-67 was higher in group 2, both P<0.05. There was no statistical difference between the values of IL-8 between the groups. Both the Shannon entropy and the fractal dimension were not different in the two groups. Conclusions: The HTG solution was better than with HTK because they had reduced caspase (apoptosis) increased KI-67 (cell proliferation), they did not show different values of IL-8 (inflammation, necrosis). Additionally they did not show change in amount or distribution of the information (observed by the Shannon entropy and fractal dimension) in histological slices.<br>Introdução: As soluções que provocam parada cardíaca eletiva estão em constante evolução, porém o composto ideal ainda não foi encontrado. Os autores comparam uma nova solução cardioplégica com histidina-triptofano-glutamato (HTG) com histidinatriptofano- acetoglutarato (HTK) em modelo de coração isolado de rato. Objetivo: Avaliar comparativamente as soluções HTK e HTG no que se refere à atividade de caspase, KI-67, IL-8, entropia de Shannon e dimensão fractal. Materiais e Métodos: Vinte ratos machos de raça Wistar foram anestesiados e heparinizados. O tórax foi aberto, realizada cardiectomia e infundido 40 mL/Kg de solução cardioplégica apropriada. Os corações foram mantidos por 2 horas na mesma solução a 4oC e, após esse período, colocados em aparato de Langendorff por 30 minutos com solução de Ringer Locke. Foram feitas análises imuno-histoquímicas para caspase, IL-8, KI-67, entropia de Shannon e dimensão fractal. Resultados: A concentração de caspase estava menor no grupo 2, e da KI-67 estava mais elevada no grupo 2, ambos com P<0,05. Não houve diferença estatística entre os valores de IL-8 entre os grupos. Tanto a entropia de Shannon quanto a dimensão fractal não foram diferentes nos dois grupos. Conclusões: A solução com HTG foi mais eficaz que a com HTK, pois reduziu a caspase (apoptose), aumentou o KI-67 (proliferação celular); não apresentou valores diferentes de IL-8 (inflamação e necrose) que no grupo 1 e não apresentou alteração da quantidade ou distribuição das informações (observadas pela entropia de Shannon e dimensão fractal) nas lâminas histológicas.

L'hypoxie intermittente (HI), induite par les apnées du sommeil, conduit à des altérations de la sensibilité à l'insuline et de l'homéostasie glucidique mais les mécanismes impliqués restent mal connus. L'objectif de ce travail était d'étudier les effets et les mécanismes sous jacents d'une exposition chronique à l'HI sur l'homéostasie glucidique. L'HI induit une résistance à l'insuline à la fois systémique et tissulaire, ainsi qu'une amélioration de la tolérance au glucose associée à une activation de l'AMPK musculaire. L'HI cause également des altérations du foie et du tissu adipeux associées à un changement du pattern d'expression des gènes dans ces tissus et à un risque accru de développement de pathologies vasculaires comme l'athérosclérose. Enfin, la délétion de PHD1, une des protéines régulatrices de HIF-1, entraîne une résistance à l'insuline associée une stéatose hépatique, faisant de HIF-1 une cible potentielle impliquée dans les altérations metaboliques induites par l'HI<br>Intermittent hypoxia (IH), induced by sleep apnea, leads to alterations in insulin sensitivity and glucose homeostasis but the mechanisms involved remains poorly understood. The objective of this work was to study the effects and the underlying mechanisms of chronic exposure to IH on glucose homeostasis. IH induces both systemic and tissue-specific insulin resistance , as well as improved glucose tolerance associated with an activation of muscle AMPK. IH also causes a change in the pattern of gene expression in liver and adipose tissue and an increased risk of vascular pathologies such as atherosclerosis development. Finally, the deletion of PHD1, a regulatory protein of HIF-1, leads to insulin resistance associated with hepatic steatosis, making HIF-1 a possible target involved in the metabolic changes induced by IH

碩士<br>長庚大學<br>基礎醫學研究所<br>92<br>Background: Resveratrol (trans-3,4’,5-trihydroxystilbene), a natural antioxidant derived from grapes, has been found to have a protective effect against myocardial ischemia/reperfusion (I/R) injury. However, its underlying mechanisms are largely unknown. The present study was to investigate the molecule mechanisms underlying the protective effect of resveratrol against I/R injuries of the rat hearts. Methods: The left main coronary artery was occluded for 60 min followed by 5 hr of reperfusion in anesthetized rats. Animals were treated with or without resveratrol 24 hr before occlusion and the severity of I/R-induced arrhythmias, mortality and myocardial infarction were compared. Results: Pretreatment of resveratrol reduced I/R-induced arrhythmia and mortality, including reduced incidence and duration of ventricular tachycardia (VT) and ventricular fibrillation (VF). It also significantly decreased the infarct size. Resveratrol dose-dependently suppressed I/R-induced the expression of tumor necrosis factor-alpha (TNF-) in the left ventricular tissue. Conclusions: Resveratrol is a potent anti-arrhythmia and anti-infarction agent in I/R rat hearts. Suppression of TNF- expression in occluded ventricular tissue may be implicated in its protective effect.

博士<br>國立陽明大學<br>臨床醫學研究所<br>105<br>Acute kidney injury (AKI) is a common and severe complication of acute myocardial infarction and cardiac surgery. It results in increased mortality, morbidity, and duration of hospitalization. Because its underlying mechanisms are unclear, there is no specific therapy to prevent or treat it. A regional transient ischemia and reperfusion (I/R) may provide protection for distant tissue or organs, a phenomenon known as remote preconditioning. Myocardial ischemia and reperfusion, induced by a 40-minute occlusion and a 3-hour reperfusion of the left anterior descending coronary artery, significantly increased blood urea nitrogen and creatinine levels in addition to causing histological changes in the kidneys. Kidney apoptosis was also significantly increased, as evidenced by the increase in the terminal deoxynucleotidyl transferase-mediated 20-deoxyuridine 50-triphosphate (dUTP) nick-end labelling (TUNEL)-positive nuclei. Furthermore, myocardial ischemia and reperfusion significantly increased the serum levels of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6) as well as the tumor necrosis factor- α levels in the kidneys. Myocardial preconditioning (MPC) was elicited by two 10-min coronary artery occlusions and two 10-min reperfusions. MPC significantly made myocardial infarct size smaller, and reduced the serum creatinine level, renal histological damage and apoptosis. The increase in the serum levels of TNF-α,IL-1 and IL-6, and of TNF-α in the kidneys, was significantly inhibited. Western blot analysis found that MPC significantly increased Bcl-2 and decreased Bax in the kidneys. Phosphorylation of Akt and extracellular signal-regulated kinases 1 and 2 (ERK1/2) were significantly increased. Hemodynamics, area at risk and mortality did not differ significantly among the groups. Baicalein is a component of the root of Scutellaria baicalensis, which has traditionally been used to treat cardiovascular and liver diseases in Asia, we investigated whether baicalein can attenuate kidney, liver and lung injury induced by myocardial ischemia and reperfusion in rats. The induced myocardial I/R significantly increased the serum levels of aspartate transaminase (AST) and alanine transaminase (ALT), indicating the presence of liver injury. Hepatic apoptosis was significantly increased. Myocardial I/R also significantly increased histological damage and wet/dry weight ratio of lung. Serum and bronchoalveolar lavaged fluid (BALF) levels of TNF-α, IL-1, and IL-6 were significantly elevated, as were TNF-α levels in the lung. In our study, intravenous pretreatment with baicalein (in doses of 3, 10, or 30mg/kg) 10 min before myocardial I/R significantly reduced the increases in the creatinine level, renal and pulmonary histological damage, and triple apoptosis induced by myocardial ischemia and reperfusion. In addition, the increases in the serum levels of TNF-α, IL-1, and IL-6, and of TNF-α in the kidneys, livers and lungs were significantly reduced. Western blot analysis revealed that baicalein significantly increased Bcl-2 and reduced Bax in the kidney, liver and lung. The phosphorylation of Akt and ERK1/2 was also significantly increased. Furthermore, baicalein significantly reduced the serum level increase of AST and ALT, wet/dry weight ratio of lung and BALF levels of TNF-α. Moreover, baicalein decreased p53 and cytochrome c in the lungs, while phosphorylation of p38 and c-Jun N-terminal kinase (JNK) was decreased. MPC significantly reduced kidney injury and apoptosis induced by myocardial I/R; in addition, baicalein can also reduce the kidney, liver and lung injury induced by myocardial I/R. The mechanisms might be related to limitation of apoptosis, possibly via inhibition of both the extrinsic and intrinsic pathways of apoptosis, including inhibition of TNF-α production and modulation of pro- and anti-apoptotic signaling elements. This project may provide a novel pathway in both health care and clinical organ damage therapies.

碩士<br>國立中央大學<br>生命科學系<br>102<br>Chest pain is the hallmark of myocardial ischemia, that often occurs in heart when sufficient oxygen for the needs is not acquired. Myocardial ischemia induces production of proton or bradykinin, which activates cardiac primary afferents, specifically Aδ- and C-fibre afferents, to transduce cardiac nociception to the central nervous system, leading to pain. In previous studies, acid-sensing ion channel 3 (ASIC3) on cardiac sensory afferents to sense protons and some regulations, participate in the regulation of myocardial ischemia. Transient myocardial ischemia induces prolonged ST-segment depression and more severe cardiac fibrosis in ASIC3-/-, but not in ASIC3+/+ or TRPV1-/-. However, molecular involved in ischemia-induced pain is not clear. In this study, I used low-dose isoproterenol to induce transient myocardial ischemia in ASIC3 or TRPV1 knockout mice. Isoproterenol injection caused transient hypoxia in cardiac muscle. Expression of proton-sensing G-Proton-coupled receptor(GPCR) G2A,was increased after isoproterenol injection. In ASIC3-/- mice, transient hypoxia occurred early and G2A expression was inhibited. Therefore, G2A may participate in ASIC3-mediated pathway to protect cardiac cells from severe ischemic insults.

博士<br>國立臺灣大學<br>藥理學研究所<br>106<br>Snake venom proteins have been broadly investigated and applied in the numerous areas of life science. These compounds can directly interact with cells in eliciting cellular responses including the activation or blockade of cellular physiological functions. However, some snakes contain some specific components which bind to their respective receptors in regulating cellular biological functions. According to the studies of their structure and activity relationship, we can not only use them as the research tool but also develop the new application and therapeutic strategy. In this study, we investigated the effect of trimucrin, a disintegrin isolated from the venom of Trimeresurus mucrosquamatus which originally was found to inhibit platelet aggregation. We further study its mechanisms of anti-inflammation and attenuating myocardial I-R injury. As we know, sepsis always causes lots of death due to septic shock and multiple organs failure, but the development of therapeutic strategy for sepsis has been restricted. In the anti-inflammatory study, we used endotoxin such as LPS to induce the acute inflammatory responses, and evaluated the effects of trimucrin in anti-inflammatory responses and investigated the mechanism on LPS-stimulated phagocytes. However, in addition to antiplatelet activity, disintegrin has been demonstrated to exert the anti-inflammatory effect but the possible mechanism is still unclear. In this study, we investigated if trimucrin exhibits anti-inflammatory activity on LPS-induced responses in THP-1 and RAW 264.7 cells. Trimucrin decreases the release of pro-inflammatory cytokines including TNF-α, IL-6, IL-1β and IL-8, nitric oxide, reactive oxygen species (ROS) as well as inhibiting adhesion and migration of LPS-stimulated phagocytes. Trimucrin also significantly attenuates the expression of NFκB-related downstream inducible enzymes like iNOS and COX-2. In addition, we observed that the molecular mechanism of trimucrin-mediated anti-inflammation is associated with decreasing phosphorylation of MAPK molecules including ERK1/2, JNK and p38. Furthermore, trimucrin also inhibits LPS-induced phosphorylation of FAK, PI3K and Akt in a concentration- dependent manner. Trimucrin also reverses NF-κB DNA binding activity via suppressing LPS-induced translocation of p65 into nucleus and cytosolic release of IκB. In cellular binding assay, the flowcytometric analysis showed that FITC-trimucrin bound to cells in a concentration-dependent manner. The anti-αVβ3 mAb also specifically reduced the binding of FITC-conjugated trimucrin. Binding assays demonstrated that integrin αVβ3 was the binding site for trimucrin on THP-1 and RAW 264.7 cells. In conclusion, we showed that trimucrin reduces the inflammatory reaction through inhibition of iNOS expression and NO production by blockade of MAP Kinase and NF-kB activation in LPS-stimulated phagocytes. Platelet-induced thrombus formation plays an important role in the pathological responses of acute myocardial infarction (AMI). Platelets also induce thrombotic occlusion of the coronary artery in microcirculation leading to exacerbating myocardial ischemia following ischemia reperfusion (I-R) injury. Disintegrins are a group of R(K)GD-containing snake venom proteins which inhibit platelet aggregation by blocking αIIbβ3 integrin. The previous reports found that hemorrhagic venom proteins have various pathological responses by influencing blood cells, plasma proteins and vessel wall in cardiovascular system. Furthermore, disintegrins can specifically affect cell-cell and cell-matrix interactions, some disintegrin mimetics have been applied for the anti-platelet, anti-thrombotic and anti-angiogenic agents. However, trimucrin, a novel small-mass RGD-containing disintegrin, has been demonstrated to possess anti-platelet and anti-inflammatory effect through blockade of platelet αIIbβ3 and phagocyte αVβ3 integrin. In this study, we found that the platelet-rich plasma prepared from trimucrin-treated rats showed to diminish platelet aggregation in response to ADP. We tried to determine whether trimucrin is cardioprotective in rats subjected to myocardial ischemia-reperfusion (I-R) injury. The left anterior descending coronary artery of rats was subjected to 1 h occlusion and 3 h reperfusion. The animals were treated with trimucrin intravenously, and the severities of I-R-induced arrhythmia and infarction were compared. Trimucrin significantly reduced I-R-induced arrhythmias and reduced mortality, as well as infarct volume, troponin-I levels, creatine kinase, and lactate dehydrogenase activity. Trimucrin also improved cardiac function and survival rates after I-R injury. In addition, trimucrin concentration-dependently inhibited platelet adhesion on collagen- and fibrinogen-coated surfaces. Trimucrin also significantly reduced neutrophil infiltration into heart tissues after I-R injury. Furthermore, trimucrin treatment caused significant downregulation of Bax, Caspase-3 apoptotic proteins and upregulation of anti-apoptotic Bcl-2 protein. These results demonstrate that trimucrin exerts cardioprotective property against myocardial I-R injury mediated through antiplatele, anti-inflammatory, anti-apoptotic mechanism, as well as improvements in cardiac function. In summary, based on the anti-inflammatory effects of anti-thrombotic venom proteins on phagocytes and myocardial I-R injury, these findings may provide a potentially pharmacological approach for the development of anti-inflammatory and cardiovascular protective agents.