Role of Toll-Like Receptor Signaling in Cardiac Ischemia

描述（由申请人提供）：心血管疾病是美国的头号杀手。Toll样受体（TLR）介导的先天性免疫和炎症反应在心肌缺血/再灌注（I/R）损伤的病理生理机制中起重要作用。众所周知，TLR 4介导的NF-κ B信号通路的激活在心肌I/R损伤中起有害作用，而PI 3 K/Akt信号通路的激活保护心肌I/R损伤。我们已经报道了TLR 4缺陷或TLR 2调节通过PI 3 K/Akt依赖性机制保护心肌免受I/R损伤。我们已经发现TLR 4缺乏增加了心肌中microRNA-146 a（miR-146 a）和miR-486的表达。我们还发现，TLR 2调节增加了心肌、循环外泌体和骨髓间充质干细胞分泌的外泌体中miR-146 a和miR-486的量。我们已经证明，心肌中miR-146 a表达增加可减少心肌梗死面积，并改善心肌I/R后的心功能。miR-146 a靶向IRAK 1和TRAF 6，它们是TLR介导的NF-κ B活化的重要中间体，因此减弱I/R诱导的NF-κ B活化。此外，我们发现miR-486抑制PTEN表达，导致PI 3 K/Akt信号转导的激活。我们的研究结果表明，无论是TLR 4缺陷或TLR 2调节诱导心肌保护通过上调microRNA表达，抑制NF-κ B激活和激活PI 3 K/Akt信号。综合考虑，我们的数据和文献清楚地表明，TLR 4介导的NF-κ B激活与心肌I/R损伤的疾病进展密切相关，而PI 3 K/Akt信号通路的激活与心肌损伤的减轻密切相关。目前，对NF-κ B和PI 3 K/Akt在I/R损伤中的差异调节机制尚不完全清楚。本申请旨在破译在I/R诱导的心肌损伤中差异调节这些信号通路的细胞和分子机制。然后，我们将以实际的方式应用这些机械知识来开发新的和新颖的心脏病发作疗法。具体而言，我们将确定microRNA在心肌I/R损伤中NF-kB和PI 3 K/Akt的差异调节中的作用。我们还将尝试将这些知识应用于开发新的基于miR的心脏病发作疗法。我们假设i）miRNAs差异激活PI 3 K/Akt信号传导并抑制心肌I/R损伤中的NF-kB激活，ii）miRNAs被携带在被心肌细胞有效摄取的外泌体中，通过保护性与有害的细胞内信号传导的差异激活而导致心脏保护。具体目标1将阐明NF-κ B和PI 3 K/Akt调节心肌细胞中miR-146 a和miR-486表达的机制。具体目标2将定义miR-146 a和miR-486对心肌损伤和功能恢复的作用。具体目标3将研究合成的作用， 携带miR-146 a和miR-486的外泌体作为心肌I/R损伤的治疗剂。

DESCRIPTION (provided by applicant): Cardiovascular disease is the number one killer in the United States. Toll-like receptor (TLR)-mediated innate immune and inflammatory responses play a central role in the pathophysiologic mechanisms of myocardial ischemia/reperfusion (I/R) injury. It is well known that activation of TLR4-mediated NF-kB signaling plays a deleterious role in myocardial I/R injury, while activation of the PI3K/Akt signaling pathway protects against myocardial I/R injury. We have reported that either TLR4 deficiency or TLR2 modulation protects the myocardium from I/R injury via a PI3K/Akt dependent mechanism. We have discovered that TLR4 deficiency increases the expression of microRNA-146a (miR-146a) and miR-486 in the myocardium. We have also found that TLR2 modulation increases the amount of miR-146a and miR-486 in the myocardium, in circulating exosomes, and in exosomes secreted by bone marrow mesenchymal stem cells. We have demonstrated that increased expression of miR-146a in the myocardium decreases myocardial infarct size and improves cardiac function following myocardial I/R. MiR-146a targets IRAK1 and TRAF6 which are the important intermediates in TLR-mediated NF-kB activation, thus, attenuating I/R-induced NF-kB activation. In addition, we have found that miR-486 inhibits PTEN expression, leading to activation of PI3K/Akt signaling. Our findings suggest that either TLR4 deficiency or TLR2 modulation induce cardioprotection via up-regulation of microRNA expression that inhibits NF-kB activation and activates PI3K/Akt signaling. When considered together, our data and the literature clearly show that TLR4-mediated NF-kB activation is closely linked to disease progression in myocardial I/R injury, while activation of PI3K/Akt signaling pathways is closely associated with attenuation of myocardial injury. At present, we do not fully understand the mechanisms of differential regulation of NF-kB and PI3K/Akt in I/R injury. This application is directed at deciphering the cellular and molecular mechanisms that differentially regulate these signaling pathways in I/R induced myocardial injury. We will then apply that mechanistic knowledge in a practical fashion to develop new and novel therapies for heart attack. Specifically, we will define the role of microRNA in differential regulation of NF-kB and PI3K/Akt in myocardial I/R injury. We will also attempt to apply this knowledge for the development of new and novel miR based therapies for heart attack. We hypothesize that i) miRNAs differentially activate PI3K/Akt signaling and inhibit NF-kB activation in myocardial I/R injury and ii) miRNAs are carried in exosomes which are efficiently uptaken by cardiac myocytes, resulting in cardioprotection via differential activation f protective versus deleterious intracellular signaling. Specific Aim 1 will elucidate the mechanisms by which modulation of NF-kB and PI3K/Akt regulates the expression of miR-146a and miR-486 in cardiac myocytes. Specific Aim 2 will define the effect of miR-146a and miR-486 on myocardial injury and functional recovery. Specific Aim 3 will investigate the role of synthetic exosomes carrying miR-146a and miR-486 as therapeutics in myocardial I/R injury.