HEME OXYGENASE-1 IN HEPATIC ISCHEMIA/REPERFUSION INJURY

描述(由申请人提供)： 缺血再灌注(I/R)损伤仍然是原位肝移植(OLT)的主要限制因素之一。这一建议是建立在我们对诱导OLTS中血红素加氧酶-1(HO-1)表达的新方法的研究中获得的见解的基础上的。我们假设：(I)HO-I促进全身炎症反应，通过STAT6途径导致一个主要的“细胞保护型”2型T细胞亚群的出现；(Ii)HO-1抑制凋亡机制并触发2型抗凋亡细胞蛋白机制；(Iii)透明质酸(HA)提供“危险”信号以激活Kupffer细胞上的Toll样受体4(TLR4)复合体；HO-1-TLR4串扰通过抑制Kupffer cetl的激活来防止L/R损伤。我们提出了以下具体目标：(1)。为了评估局部和系统对HO-1促进的细胞保护的需求；通过使用HO-1TG小鼠，我们将确定局部(内皮/实质)和系统(淋巴细胞)HO-1表达的影响。我们将分析哪些肝脏成分与脾T细胞亚群在HO-1效应中起作用。将评估STAT6在主要依赖HO-1的细胞保护型-2T细胞出现中的作用。(2)。为了剖析HO-1促进的细胞保护机制中的凋亡/抗凋亡途径：我们计划确定HO-1介导的细胞保护的分子基础：在肝移植受者和原代培养的小鼠肝细胞中，基于死亡信号机制(肿瘤坏死因子/FasL)和凋亡刺激(ROS)。这将包括caspase的激活，细胞色素c的释放，以及抗/促凋亡基因的表达。我们将分析HO-1介导的抑制肝细胞凋亡机制和上调内皮细胞抗凋亡分子是否代表局部肝或全身性T细胞依赖的特征。(3)。探讨HO-1和TLR4复合体-I/R损伤与先天免疫的相关性？我们将研究从受损的肝窦内皮细胞脱落的HA如何与Kupffer细胞相互作用，在多大程度上触发TLR4突变/缺陷与WT小鼠Kupffer细胞的表达/激活。探讨沙门冬氨酸对TLR4突变型/TLR4KO小鼠肝脏I/R损伤的影响。我们将分析HO活性抑制是通过什么机制影响先天免疫平衡，并触发表型/功能性TLR4激活。

DESCRIPTION (provided by applicant): lschemia/reperfusion (I/R) insult remains one of the major limitations of orthotopic liver transplantation (OLT). This proposal is built upon the insights gained from our studies on a novel approach of inducing heme oxygenase-1 (HO-1) expression in OLTs. We hypothesize that: (i) HO-I facilitates a systemic mileu, which via STAT6 pathway leads to emergence of a dominant "cytoprotectlve" type-2 T cell subset; (ii) HO-1 depresses apoptotic machinery and triggers type-2 anti-apoptotic cytoprotactive mechanisms; (iii) hyaluronic acid (HA) provides a "danger" signal to activate toll-like receptor 4 (TLR 4) complex on Kupffer cells; HO-1 - TLR 4 cross-talk prevents l/R insult by keeping Kupffer cetl activation in check. We propose following specific aims: (1). To evaluate local vs. systemic ceil requirements for HO-1-facilitated cytoprotection; By using HO-1 Tg mice, we will determine impact of local (endothelial/parenchymal) vs. systemic (lymphocyte) HO-1 expression. We will analyze which hepatic components vs. spleen T cell subsets are instrumental in HO-1 effects. The role of STAT6 in the emergence of a dominant HO-1-dependent cytoprotective type-2 T cell will be assessed. (2). To dissect apoptotic/anti-apoptotic pathways in the mechanism of HO-1-facilitated cytoprotection: We plan to determine the molecular basis of HO-1 mediated cytoprotection in OLT recipients and in primary mouse hepatocyte cultures upon the death signaling machinery (TNF/FasL), and apoptotic stimuli (ROS). This will include activation of caspases, cytochrome c release, and expression of anti-/pro-apoptotic genes. We will analyze whether HO-1-mediated depression of apoptotic machinery in hepatocytes and upregulation of endothelial anti-apoptotic molecules represents local hepatic or systemic T cell-dependent feature. (3). To explore cross-talk between HO-1 and TLR 4 complex - I/R injury a case for innate immunity? We will study how HA, shed from damaged sinusoidai endothelial cells, interacts with Kupffer cells, to what extent HA triggers expression/activation of Kupffer ceils in TLR 4 mutant/deficient vs. WT mice. The ability of sHA to affect hepatic I/R insult in TLR 4 mutant/TLR 4 KO mice will be probed. We will analyze by which mechanism depression of HO activity affects innate immunity balance, and triggers phenotypic/functional TLR4 activation.