Role of Toll-Like Receptor Signaling in Cardiac Ischemia

描述(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发挥重要作用。众所周知，TLR4介导的NF-kB信号通路的激活在心肌I/R损伤中起着有害的作用，而PI3K/Akt信号通路的激活对心肌I/R损伤具有保护作用。我们已报道TLR4缺乏或TLR2调节通过PI3K/Akt依赖机制保护心肌免受I/R损伤。我们发现，TLR4缺乏增加了心肌中microRNA-146a(miR-146a)和miR-486的表达。我们还发现，TLR2调节增加了心肌、循环外体和骨髓间充质干细胞分泌的外体中miR-146a和miR-486的数量。我们已经证明，在心肌I/R后，miR-146a在心肌中的表达增加可以缩小心肌梗死范围，改善心功能。miR-146a靶向TLR介导的NF-kB激活的重要中间产物IRAK1和TRAF6，从而减弱I/R诱导的NF-kB激活。此外，我们还发现miR-486抑制PTEN的表达，导致PI3K/Akt信号的激活。我们的发现表明，无论是TLR4缺乏还是TLR2调节都通过上调microRNA的表达来诱导心脏保护，该表达抑制了NF-kB的激活，并激活了PI3K/Akt信号。综合考虑，我们的数据和文献清楚地表明，TLR4介导的NF-kB激活与心肌I/R损伤的疾病进展密切相关，而PI3K/Akt信号通路的激活与心肌损伤的减轻密切相关。目前，我们还不完全清楚NF-kB和PI3K/Akt在I/R损伤中的差异性调控机制。这项应用旨在破译在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制知识来开发心脏病发作的新疗法。具体地说，我们将确定microRNA在心肌I/R损伤中对NF-kB和PI3K/Akt的差异调控中的作用。我们还将尝试将这一知识应用于开发基于miR的心脏病发作新疗法。我们假设，在心肌I/R损伤中，miRNAs差异性地激活PI3K/Akt信号并抑制核因子-kB的激活；ii)miRNAs携带在被心肌细胞有效摄取的外体中，从而通过差异性激活保护性和伤害性细胞内信号而起到心脏保护作用。具体目的1阐明核因子-kB和PI3K/Akt调控心肌细胞miR-146a和miR-486表达的机制。具体目标2将确定miR-146a和miR-486在心肌损伤和功能恢复中的作用。具体目标3将调查合成蛋白的作用 携带miR-146a和miR-486的外切体治疗心肌I/R损伤

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.