FIBRONECTION IN HEPATIC ISCHEMIA REPERFUSION INJURY

肝缺血再灌注损伤中的纤维连接

• 批准号: 7078515
• 负责人: Ana J. Coito
• 金额: $ 33.95万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7078515
• 关键词: biological signal transduction; cell adhesion; cell migration; cryopreservation; cytokine; enzyme activity; fibronectins; free radical oxygen; gene expression; genetically modified animals; inflammation; integrins; laboratory mouse; laboratory rat; leukocyte activation /transformation; leukocytes; liver function; liver ischemia /hypoxia; liver transplantation; metalloendopeptidases; nitric oxide synthase; oxidative stress; protein structure function; reperfusion; tumor necrosis factor alpha

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.

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（奥尔特）的主要限制因素，特别是边缘移植。关于白细胞募集到肝脏炎症刺激部位的机制知之甚少。基于我们先前研究的证据，我们假设纤连蛋白（FN）是导致肝I/R损伤的抗原非依赖性级联反应的组成部分，可能提供适当的共刺激信号可以启动白细胞信号传导和募集的空间地址。我们希望这一建议将揭示新的机制和潜在的目标，对I/R损伤。因此，我们建议达到以下具体目标：1 .分析不同的FN-α 4 β 1/α 5 β 1相互作用对肝I/R损伤中白细胞募集和细胞因子释放的作用：将来自遗传性肥胖Zucker和非脂肪变性SD大鼠的肝脏在移植到同系瘦Zucker/SD大鼠中之前在4 ℃下储存在UW中，并将三种不同的FN（EIIIA）-α 4 β 1，FN（CS 1）-α 4 β 1，和FN（RGD）-α 5 β 1相互作用将被特异性肽和功能阻断mAb靶向。将探讨对（i）肝功能和存活率;（ii）白细胞粘附/迁移（体内和体外）和（iii）细胞因子表达的不同治疗操作。 2.为了剖析FN-α 4 β 1/α 5 β 1整合素对肝I/R损伤中金属蛋白酶-2（MMP-2）和金属蛋白酶-9（MMP-9）表达的作用机制：我们计划（i）确定不同FN细胞结合位点对MMP-2和MMP-9表达/活化的贡献，以及各自的抑制剂（二）确定MMP-2和MMP-9在肝移植物中的个体来源;（iii）评估MMP-2和MMP-9表达在肝脏I/R损伤后FN-整合素相互作用机制中的功能意义。 3.探讨FN在肝I/R损伤中诱导型一氧化氮合酶（iNOS）、基质金属蛋白酶（MMPs）和细胞因子之间的相互作用：首先，用特异性iNOS抑制剂1400 W在体内探讨iNOS表达的功能意义。第二，我们将剖析TNF-α或其他相关细胞因子之间的相互作用，和iNOS FN调节MMP的表达/活化。第三，我们将揭示细胞内的机制，如MAP激酶转导途径对白细胞粘附到纤连蛋白和MMP的表达。