The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22357号 / 医博第4598号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 伊佐 正, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Sepsis is dysregulated host immune response to infection causing life-threatening organ dysfunction. Endothelial cell dysfunction and uncontrolled inflammatory responses are two contributors for sepsis-induced mortality. The crosstalk between endothelial and immune cells plays a critical role in the pathophysiology of sepsis. Therefore, understanding the mechanism of interaction between endothelial and immune cells will provide novel information to develop therapeutic strategies for sepsis. Pathogen associated moleculear patterns (PAMPs) and/or damage associated molecular patterns (DAMPs) produced during sepsis, activate endothelial cells to increase the expression of adhesion molecules, attracting immune cell infiltration into the tissues. Uncontrolled inflammatory responses during the early phase of sepsis contribute to organ failure and lethality. Over 100 clinical trials, targeting inflammatory responses in sepsis, have failed in the past three decades. Thereby, developing novel therapeutic strategies for sepsis are urgent. Heat shock protein A12B (HSPA12B), as one member of HSP70 family, predominately expressed in the endothelial cells, plays important roles in many pathophysiological processes. Currently, we observed endothelial cell specific HSPA12B deficiency (HSPA12B-/-) exacerbates mortality in sepsis induced by cecal ligation puncture (CLP). HSPA12B-/- septic mice exhibits increased expressions of adhesion molecule and infiltrated macrophages in the myocardium and activated macrophages in the peritoneal cavity. In vitro studies show that HSPA12B could be secreted from endothelial cells via exosome. HSPA12B carried by exosomes can be uptaken by macrophages to downregulate macrophage NF-kB activation and pro-inflammatory cytokine production. Trained immunity, induced by β-glucan, causes immune memory in innate immune cells, with an altered response towards another challenge. We have found that mice received β-glucan seven days before CLP sepsis exhibit attenuated mortality with decreased pro-inflammatory responses. We found that β-glucan significantly increased the levels of HSPA12B in endothelial cells and endothelial exosomes. β-glucan induced endothelial exosomes markedly suppress macrophage NF-kB activation and pro-inflammatory responses. The current data suggests that HSPA12B plays a novel role in the regulation of immune and inflammatory responses and that HSPA12B could be an important mediator for the crosstalk between endothelial cells and macrophages during sepsis. β-glucan regulates endothelial cell functions and immune/inflammatory responses, thus improving survival outcome in CLP sepsis.

Although the RING finger protein 121 (RNF121) is a highly conserved E3 ubiquitin ligase from Caenorhabditis elegans to human, its function is poorly understood and in higher eukaryotes it has been studied only at in vitro level. RNF121 has been described to have various functions: i) it was ascribed to function as a broad regulator of NF-κB activation, ii) it was shown to control intracellular trafficking of various membrane proteins, and iii) its downregulation leads to apoptosis. Moreover, RNF121 might have a role in cancer as its expression was found to be 16.4-fold higher in patients suffering from Barrett esophagus (precancerous lesion of esophageal adenocarcinoma) and was even more increased in esophageal adenocarcinoma comparing to healthy population. In addition, RNF121 gene is localized in the candidate region containing breast cancer susceptibility genes. To gain insight into physiological functions of RNF121, Rnf121 knockout mice (Rnf121tm1b(EUCOMM)Hmgu ) were generated in the Czech Centre for Phenogenomics and further studied in our laboratory. Rnf121+ /- intercross breedings showed a prenatal lethal phenotype of Rnf121-/- embryos, which were dying prior embryonic day (E) 11.5. Preliminary experiments carried out in our laboratory showed numerous vascular defects in null mutant embryo,...

Induction of C-reactive protein (CRP) synthesis in hepatocytes by cytokines occurs at the transcriptional level. In Hep3B cells, the transcription factors C/EBPβ, STAT3, and Rel p50 have been shown to participate in this process. A C/EBP binding site centered at -53 and an overlapping nonconsensus κB site on the promoter are critical for CRP expression. We have previously found that an oligonucleotide containing a κB site diminished binding of C/EBPβ to the C/EBP site, suggesting that unidentified Rel proteins present in Hep3B nuclei facilitate the formation of C/EBPβ-complexes. The current studies were undertaken to determine which of the five Rel proteins, p50/p65/p52/c-Rel/RelB, play such a role. Mutation of the nonconsensus κB site did not abolish binding of C/EBPβ to its binding site, indicating that this site was not necessary for the formation of C/EBPβ-complexes. Depletion of Rel proteins from Hep3B nuclei led to decreased formation of C/EBPβ-complexes on a CRP promoter-derived oligonucleotide that contained only the intact C/EBP binding site but not the nonconsensus κB site. This finding indicates that Rel proteins are involved in the binding of C/EBPβ to its binding site by a κB site-independent mechanism. Electrophoretic mobility shift assays (EMSAs) revealed that it was c-Rel that facilitated formation of C/EBPβ-complexes and that c-Rel bound directly to C/EBPβ-complexes formed on the C/EBP site. Cotransfection of c-Rel enhanced the induction of CRP promoter-driven luciferase activity and enhanced endogenous CRP expression in cells transfected with C/EBPβ. We conclude that c-Rel regulates CRP expression without the requirement of binding to a κB site, and binds directly to C/EBPβ to facilitate the binding of C/EBPβ to the CRP promoter.

AimsWe have reported that either toll-like receptor 4 deficiency (TLR4 -/-) or TLR2 modulation protects against myocardial ischaemia/reperfusion (I/R) injury. The mechanisms involve attenuation of I/R-induced nuclear factor KappaB (NF-κB) activation. MicroRNA-146a (miR-146a) has been reported to target interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), resulting in inhibiting NF-κB activation. This study examined the role of microRNA-146a in myocardial I/R injury.Methods and resultsWe constructed lentivirus expressing miR-146a (LmiR-146a). LmiR-146a was transfected into mouse hearts through the right common carotid artery. The lentivirus vector (LmiR-Con) served as vector control. Untransfected mice served as I/R control. Sham operation served as sham control. Seven days after transfection, the hearts were subjected to ischaemia (60 min) followed by reperfusion (4 h). Myocardial infarct size was analysed by triphenyltetrazolium chloride (TTC) staining. In separate experiments, the hearts were subjected to ischaemia (60 min) followed by reperfusion for up to 7 days. Cardiac function was measured by echocardiography prior to I/R, 3 and 7 days after myocardial I/R. LmiR-146a transfection significantly decreased I/R-induced myocardial infarct size by 55% and prevented I/R-induced decreases in ejection fraction (EF%) and fractional shortening (%FS). LmiR-146a transfection attenuated I/R-induced myocardial apoptosis and caspase-3/7 and-8 activities. LmiR-146a transfection suppresses IRAK1 and TRAF6 expression in the myocardium. In addition, transfection of LmiR-146a prevented I/R-induced NF-κB activation and inflammatory cytokine production.ConclusionsMicroRNA-146a protects the myocardium from I/R injury. The mechanisms may involve attenuation of NF-κB activation and inflammatory cytokine production by suppressing IRAK1 and TRAF6.

AimsWe have reported that either toll-like receptor 4 deficiency (TLR4 -/-) or TLR2 modulation protects against myocardial ischaemia/reperfusion (I/R) injury. The mechanisms involve attenuation of I/R-induced nuclear factor KappaB (NF-κB) activation. MicroRNA-146a (miR-146a) has been reported to target interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), resulting in inhibiting NF-κB activation. This study examined the role of microRNA-146a in myocardial I/R injury.Methods and resultsWe constructed lentivirus expressing miR-146a (LmiR-146a). LmiR-146a was transfected into mouse hearts through the right common carotid artery. The lentivirus vector (LmiR-Con) served as vector control. Untransfected mice served as I/R control. Sham operation served as sham control. Seven days after transfection, the hearts were subjected to ischaemia (60 min) followed by reperfusion (4 h). Myocardial infarct size was analysed by triphenyltetrazolium chloride (TTC) staining. In separate experiments, the hearts were subjected to ischaemia (60 min) followed by reperfusion for up to 7 days. Cardiac function was measured by echocardiography prior to I/R, 3 and 7 days after myocardial I/R. LmiR-146a transfection significantly decreased I/R-induced myocardial infarct size by 55% and prevented I/R-induced decreases in ejection fraction (EF%) and fractional shortening (%FS). LmiR-146a transfection attenuated I/R-induced myocardial apoptosis and caspase-3/7 and-8 activities. LmiR-146a transfection suppresses IRAK1 and TRAF6 expression in the myocardium. In addition, transfection of LmiR-146a prevented I/R-induced NF-κB activation and inflammatory cytokine production.ConclusionsMicroRNA-146a protects the myocardium from I/R injury. The mechanisms may involve attenuation of NF-κB activation and inflammatory cytokine production by suppressing IRAK1 and TRAF6.

Cardiovascular collapse is the major factor contributing to the mortality of trauma-hemorrhage (T-H) patients. Toll-like receptors (TLRs) play a critical role in T-H-induced cardiac dysfunction. This study evaluated the role of TLR9 agonist, CpG-oligodeoxynucleotide (ODN) 1826, in cardiac functional recovery after T-H. Trauma-hemorrhage was induced in a murine model by soft tissue injury and blood withdrawals from the jugular vein to a mean arterial pressure of 35 ± 5 mmHg. Mice were treated with CpG-ODN 1826 (10 μg/30 g body weight) by intraperitoneal injection 1 h before T-H (n = 5-8/group). Hemodynamic parameters were measured before, during hemorrhage, and at 60 min after T-H. Trauma-hemorrhage significantly decreased the mean arterial pressure and left ventricular pressure compared with sham controls. In contrast, CpG-ODN administration significantly attenuated the decrease in arterial pressure and left ventricular pressure due to T-H. Trauma-hemorrhage markedly decreased myocardial levels of phosphorylated Akt by 57.9%. However, CpG-ODN treatment significantly blunted the decrement in phospho-Akt by activating the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. The PI3K inhibitor LY294002 partially abolished CpG-induced cardioprotection, indicating that additional signaling pathways are involved in the protective effect of CpG-ODN after T-H. We observed that CpG-ODN treatment also significantly attenuated the decrease in myocardial phospho-ERK levels after T-H. Inhibition of ERK by U0126 also partially abolished the cardioprotective effect of CpG-ODN after T-H. Our data suggest that CpG-ODN significantly attenuates T-H-induced cardiac dysfunction. The mechanisms involve activation of both PI3K/Akt and ERK signaling pathways. The TLR9 agonist, CpG-ODN 1826, may provide a novel treatment strategy for preventing or managing cardiac dysfunction and enhancing recovery in T-H patients.

Doxorubicin (Dox) is a major anticancer chemotherapeutic agent. However, it causes cardiomyopathy due to the side effect of cardiomyocyte apoptosis. We have previously reported that angiopoietin-1 significantly reduced myocardial infarction after ischemic injury and protected cardiomyocytes from oxidative stress-induced apoptosis. It is hypothesized that angiopoietin-1 may protect cardiomyocytes from Dox-induced apoptosis. Cardiomyocytes H9C2 were transfected with adenovirus expressing angiopoietin-1 (Ad5-Ang-1) 24 h before the cells were challenged with Dox at a concentration of 2 μmol/L. Ad5-GFP served as the vector control. Cardiomyocyte apoptosis was evaluated using Annexin V-FITC staining and caspase-3 and caspase-8 activity was determined by Western blotting. The results showed that Dox treatment significantly induced cardiomyocyte apoptosis as evidenced by the greater number of Annexin V-FITC stained cells and increases in caspase-3 and caspase-8 activity. In contrast, overexpression of angiopoietin-1 significantly prevented Dox-induced cardiomyocyte apoptosis. To elucidate the mechanisms by which angiopoietin-1 protected cells from Dox-induced apoptosis, we analyzed both extrinsic and intrinsic apoptotic signaling pathways. We observed that angiopoietin-1 prevented Dox-induced activation of both extrinsic and intrinsic apoptotic signaling pathways. Specifically, angiopoietin-1 prevented DOX-induced in-creases in FasL and Bax levels and cleaved caspase-3 and caspase-8 levels in H9C2 cells. In addition, overexpression of angiopoietin-1 also activated the pro-survival phosphoinositide-3 kinase (PI3K)/Akt signaling pathway and decreased Dox-induced nuclear factor-kappaB (NF-κB) activation. Our data suggest that promoting the expression of angiopoietin-1 could be a potential approach for reducing Dox-induced cardiomyocyte cytoxicity.

Activation of NF-κB contributes to cardiac hypertrophy and the interleukin-1 receptor (IL-1R)-mediated MyD88-dependent signaling pathway predominately activates NF-κB. Recent studies have shown that the TIR/ BB-Loop mimetic (AS-1) disrupted the interaction of MyD88 with the IL-1R, resulting in blunting of NF-κB activation. We have examined the effects of AS-1 on the IL-1b-induced hypertrophic response using cultured neonatal cardiac myocytes in vitro and transverse aortic constriction (TAC) pressure overload-induced cardiac hypertrophy in vivo. Neonatal cardiac myocytes were treated with AS-1 15 min prior to IL-1β stimulation for 24 h. AS-1 treatment significantly attenuated IL-1β-induced hypertrophic responses of cardiac myocytes. In vivo experiments showed that AS-1 administration prevented cardiac hypertrophy and dysfunction induced by pressure overload. AS-1 administration disrupted the interaction of IL-1R with MyD88 in the pressure overloaded hearts and prevented activation of NF-κB. In addition, AS-1 prevented increases in activation of the MAPK pathway (p38 and p-ERK) in TAC-induced hypertrophic hearts. Our data suggest that the IL-1R-mediated MyD88-dependent signaling pathway plays a role in the development of cardiac hypertrophy and AS-1 attenuation of cardiac hypertrophy is mediated by blocking the interaction between IL-1R and MyD88, resulting in decreased NF-κB binding activity and decreased MAPK activation.