FIBRONECTION 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，尤其是边缘移植物。目前对白细胞聚集到肝脏炎症刺激部位的机制知之甚少。根据我们先前研究的证据，我们假设纤维连接蛋白(FN)是导致肝脏I/R损伤的抗原非依赖性级联反应的组成部分，可能提供了适当的共刺激信号启动白细胞信号和募集的空间地址。我们预计这项提案将揭示对抗I/R损伤的新机制和潜在靶点。因此，我们建议解决以下具体目标：1 。为了分析不同的Fn-α4beta1/alpha5beta1相互作用在肝脏I/R损伤中对白细胞募集和细胞因子释放的作用：来自遗传脂肪Zucker和非脂肪变性SD大鼠的肝脏在移植到同基因瘦肉Zucker/SD大鼠之前将被保存在UW的4C下，并且三种不同的Fn(EIIIA)-alpha4beta1、Fn(CS1)-alpha4beta1和Fn(RGD)-alpha5beta1相互作用将与特定的多肽和功能阻断单抗靶向。对(I)肝功能和存活；(Ii)白细胞黏附/迁移(体内和体外)和(Iii)细胞因子表达的不同治疗方法进行探讨。 2.探讨FN-α4beta1/alpha5beta1整合素在肝I/R损伤中对基质金属蛋白酶-2(MMP一2)和MMP一9(MMP一9)表达的影响机制：(I)研究不同FN细胞结合部位对MMP一2和MMP一9表达/激活的影响及其各自的抑制物(TIMP-2和TIMP-1)；(Ii)明确移植肝中MMP一2和MMP一9的来源；(3)评价基质金属蛋白酶-2和基质金属蛋白酶-9在FN-整合素相互作用肝I/R损伤机制中的功能意义。 3.探讨诱导型一氧化氮合酶(INOS)、基质金属蛋白酶(MMPs)和细胞因子在肝I/R损伤FN中的相互作用：首先，用特异性iNOS抑制剂1400W在体内探讨iNOS表达的功能意义。其次，我们将剖析肿瘤坏死因子-α或其他相关细胞因子与诱导型一氧化氮合酶在FN调节基质金属蛋白酶表达/活化中的相互作用。第三，我们将揭示细胞内机制，如MAPKinase转导途径对白细胞与纤维连接蛋白黏附和基质金属蛋白酶表达的影响。

DESCRIPTION (provided by applicant): lschemia reperfusion (I/R) injury remains a major limitation in orthotopic liver transplantation (OLT), particularly with marginal grafts. Very little is known about the mechanisms involved on leukocyte recruitment into sites of inflammatory stimulation in liver. Based upon evidence from our previous studies, we hypothesize that fibronectin (FN) is an integral part of the antigen independent cascade leading to liver I/R injury, likely providing a spatial address at which appropriate co-stimulatory signals can initiate leukocyte signaling and recruitment. We expect this proposal will unveil novel mechanisms and potential targets against I/R injury. Therefore, we propose to address the following specific aims: 1 . To analyze the function of distinct FN-alpha4beta1/alpha5beta1interactions upon leukocyte recruitment and cytokine release in liver I/R injury: Livers from genetically fat Zucker and non-steatotic SD rats will be stored at 4C in UW before being transplanted into syngeneic lean Zucker/SD rats, and the three distinct FN (EIIIA)-alpha4beta1, FN (CS1)-alpha4beta1,and FN (RGD)-alpha5beta1 interactions will be targeted with specific peptides and function-blocking mAbs. The distinct therapeutic manipulation upon (i) hepatic function and survival; (ii) leukocyte adhesion/migration (in vivo and in-vitro), and (iii) cytokine expression, will be probed. 2. To dissect mechanisms of FN-alpha4beta1/alpha5beta1 integrin action upon metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) expression in liver I/R Injury: We plan to (i) determine the contribution of distinct FN cell binding sites upon MMP-2 and MMP-9 expression/activation, and respective inhibitors (TIMP-2 and TIMP-1) in rat OLT; (ii) identify the individual sources of MMP-2 and MMP-9 in liver grafts; and (iii) evaluate the functional significance of MMP-2 and MMP-9 expression in the mechanisms of FN-integrin interactions upon liver I/R injury. 3. To explore the interplay between inducible nitric oxide synthase (iNOS), MMPs, and cytokines in the context of FN in liver I/R injury: First, the functional significance of iNOS expression will be probed in vivo with 1400W, a specific iNOS inhibitor. Second, we will dissect the interplay between TNF-alpha or other relevant cytokines, and iNOS upon FN regulation of MMP expression/activation. Third, we will unveil intracellular mechanisms, such as MAPKinase transduction pathways upon leukocyte adhesion to fibronectin and MMP expression.