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Ischemic heart disease is a leading cause of death worldwide. Ubiquitin (UB), an evolutionary conserved protein, is found in all eukaryotic cells. Previous work has shown that treatment of mice with exogenous UB (eUB) reduces inflammatory response and preserves heart function 3 days following ischemia/reperfusion injury (I/R). This study investigated the long-term (28 days post-I/R) cardioprotective potential of eUB using a mouse model of myocardial I/R; and tested the hypothesis that eUB modulates phenotype and function of macrophages (key cells involved in inflammation post-I/R) using thioglycolate-elicited mouse peritoneal macrophages. Heart function measured at 3, 7, 14 and 28 days post-I/R using echocardiography showed that eUB improves heart function throughout the observation period, and decreases I/R-mediated increase in left ventricular dilation at 3, 14 and 28 days timepoints. Myocardial fibrosis, hypertrophy and apoptosis were lower in eUB-treated hearts 28 days post-I/R. These changes in the heart associated with decreased expression of fibrosis-related proteins (collagen-1α1 and MMP-2) and hypertrophy-related protein (MYH-7B) in UB-treated hearts. Activation of GSK3β (pro-apoptotic kinase) was lower (vs. Sham), while activation of anti-apoptotic kinases, ERK1/2 (vs. I/R) and Akt (vs. Sham), was higher in eUB-treated hearts 28 days post-I/R. Serum levels of IL-6, IL-2 and G-CSF were lower in I/R+UB vs. I/R group 28 days post-I/R. In peritoneal macrophages, eUB induced cytoskeleton reorganization in M1-polarized (IFNγ treatment for 72 hours; 100U/mL) and M2-polarized (IL-4 treatment for 72 hours; 20ng/mL) cells. eUB decreased secretion of IL-1β and TNFα in M1-polarized macrophages, while it decreased secretion of TNFα, IL-10 and GM-CSF in M2-polarized macrophages. Efferocytosis was lower in eUB-treated M2-polarized macrophages, which was reversed by CXCR4 receptor antagonist (AMD3100). eUB enhanced migration of M1-polarized macrophages, while it decreased the migration of M2-polarized macrophages. AMD3100 negated the effects of eUB on M1-polarized macrophage migration. eUB decreased activation of STAT1 and FAK, while increasing activation of ERK1/2 in M1-polarized macrophages. In M2-polarized macrophages, eUB decreased Akt activation. Thus, UB treatment preserves heart function and decreases adverse cardiac remodeling 28 days post-I/R. In polarized macrophages, eUB reduces secretion of inflammatory cytokines, and alters phenotype and function of M1- and M2-polarized macrophages.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25189号 / 医博第5075号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 江木 盛時, 教授 後藤 慎平 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25500号 / 医博第5100号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 松田 秀一, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Insulin resistance and chronic hyperglycemia promote vascular damage, increase circulating levels of inflammatory cytokines and lead to increased morbidity and mortality. MicroRNAs (miRs) -103/107 have been shown to negatively regulate insulin sensitivity and glucose homeostasis. Based on complimentary binding profiles, the downstream target gene of miR-103/107 is caveolin-1 (Cav-1). We hypothesized that daily administration of the phosphodiesterase-5 inhibitor tadalafil (TAD) ± the curcumin analogue (HO-3867) will attenuate inflammation, improve metabolic parameters and reduce infarct size after ischemia/reperfusion injury (IRI). Furthermore, we propose that TAD therapy will reduce myocardial expression of miR-103/107 and increase mRNA and protein levels of its target gene, Cav-1. Leptin receptor null mice were randomized to receive daily injections of TAD (1mg/kg), HO-3867 (25mg/Kg), combination therapy, or control for 12weeks with weight and fasting glucose monitored weekly. Upon completion, cardiomyocytes were isolated from each group and were subjected to simulated ischemia and reoxygenation (SI/RO) for cell viability and reactive oxygen species (ROS) measurement. Another set were subjected to IRI in a Langendorff model. Plasma samples were taken to measure plasma concentrations of cytokines. For miR expression, total RNA was isolated from TAD and DMSO treated mice and was subjected to reverse transcription and real time PCR using miR assay probes to determine expression. TAD, HO-3867 and the combination of both attenuated fasting glucose levels, reduced myocardial infarct size after IRI and inflammatory cytokines when compared to control (p<0.05 for each vs. control). Cardiomyocytes isolated from each treatment groups and subjected to SI/RO demonstrated reduced necrosis as shown by trypan blue exclusion assay, ROS generation, and improved mitochondrial membrane potential as compared to DMSO (control). Likewise, both mRNA and protein expression of Cav-1 were reduced in diabetic hearts but were significantly increased in TAD treated diabetic mice, which may be a mechanism to improve insulin signaling through downregulation of miR-103/107 and upregulation of Cav-1. These studies suggest that TAD alone or in combination may be a unique strategy to improve metabolic parameters and precondition diabetic hearts against IRI.

Le récepteur éboueur CD36 facilite l’internalisation des acides gras libres non estérifiés (AGNE) au niveau des tissus cardiaque et périphériques. Lors d’une ischémie-reperfusion du myocarde (MI/R), les dommages produits sont en partie liés à l’internalisation des AGNE et à la production d’espèces réactives de l’oxygène, contrairement à ce qui est observé chez des souris déficientes en CD36 (CD36-/-). Nous avons émis l’hypothèse selon laquelle le CP-3(iv), un ligand synthétique du récepteur CD36, exercerait un effet cardioprotecteur en réduisant la taille de la zone myocardique infarcie lors d’une ischémie transitoire du myocarde. Nos objectifs étaient 1) de déterminer l’effet cardioprotecteur du CP-3(iv) et 2) de définir son mécanisme. Pour cela, des études in vivo et ex vivo ont été faites. Des souris de type sauvage ont été traitées avec le CP-3(iv) (289 nmol/kg) par voie sous-cutanée pendant 14 jours avant d’être soumises à 30 minutes d’ischémie suivant la ligature de l’artère coronaire gauche descendante et de sa reperfusion pendant une période de 6 ou 48 heures. De plus, des coeurs isolés de souris ont été perfusés 30 minutes, suivi de 40 minutes à faible débit (10%) et de 30 minutes de reperfusion pendant laquelle le coeur est perfusé avec le CP-3(iv) à une concentration de 10-6 M. Nos travaux ont montré que l’effet cardioprotecteur d’un traitement préventif par le CP-3(iv) permet de diminuer la taille de l’infarctus et préserve l’hémodynamie cardiaque de façon dépendante du CD36 puisque cet effet est non visible chez les souris CD36-/-. De plus, le CP-3(iv) exerce non seulement un effet systémique, mais aussi un effet cardioprotecteur direct sur le coeur isolé. / The scavenger receptor CD36 facilitates the internalization of non-esterified fatty acids (NEFA) on cardiac and peripheral tissues. During myocardial ischemia and reperfusion (MI/R), the damage induced is in part related to the internalization of NEFA and the production of reactive oxygen species, in opposition to what is observed in CD36-deficient mice (CD36-/-). We hypothesized that CP-3(iv), a synthetic ligand of the CD36 receptor, provides a cardioprotective effect by reducing the infarct area during a transient myocardial ischemia. Our objectives were 1) to determine the cardioprotective effect of CP-3(iv) and 2) to define its mechanism. For this, in vivo and ex vivo studies have been done. Wild-type mice were treated with CP-3(iv) (289 nmol/kg) subcutaneously during 14 days before being submitted to 30 minutes of ischemia following left anterior descending coronary artery ligature and reperfusion for a period of 6 to 48 hours. In addition, isolated mouse hearts were perfused 30 minutes, followed by 40 minutes with low flow (10%) and 30 minutes of reperfusion during which the heart is perfused with CP-3(iv) at a concentration of 10-6 M. Our work has shown that the cardioprotective effect of preventive treatment with CP-3(iv) reduces the infarct size and preserves cardiac hemodynamics in a CD36-dependent manner because this effect is not visible in CD36-/- mice. In addition, CP-3(iv) not only exerts a systemic effect, but also a direct cardioprotective effect on the isolated heart.