FIBRONECTION IN HEPATIC ISCHEMIA REPERFUSION INJURY

肝缺血再灌注损伤中的纤维连接

• 批准号: 6887678
• 负责人: Ana J. Coito
• 金额: $ 34.76万
• 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/6887678
• 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（OLT）的主要限制，特别是边缘移植物。关于白细胞募集到肝脏炎症刺激部位的机制知之甚少。根据我们之前研究的证据，我们假设纤连蛋白 (FN) 是导致肝 I/R 损伤的抗原独立级联的一个组成部分，可能提供一个空间地址，在该地址上适当的共刺激信号可以启动白细胞信号传导和招募。我们预计该提案将揭示针对 I/R 损伤的新机制和潜在目标。因此，我们建议实现以下具体目标：1 。为了分析不同的 FN-α4β1/α5β1 相互作用对肝脏 I/R 损伤中白细胞募集和细胞因子释放的功能：来自遗传性脂肪 Zucker 和非脂肪变性 SD 大鼠的肝脏将在移植到同系瘦 Zucker/SD 大鼠之前在 4°C 下保存在 UW 中，并且三种不同的 FN (EIIIA)-alpha4beta1、FN (CS1)-alpha4beta1 和 FN (RGD)-alpha5beta1 相互作用将通过特定肽和功能阻断 mAb 进行靶向。针对 (i) 肝功能和存活率的独特治疗操作；将探测 (ii) 白细胞粘附/迁移（体内和体外）和 (iii) 细胞因子表达。 2. 剖析 FN-α4β1/α5β1 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/激活以及各自抑制剂（TIMP-2 和 TIMP-1)在大鼠OLT中； (ii) 确定肝移植物中 MMP-2 和 MMP-9 的单独来源； (iii) 评估 MMP-2 和 MMP-9 表达在肝 I/R 损伤时 FN-整合素相互作用机制中的功能意义。 3. 探讨诱导型一氧化氮合酶 (iNOS)、MMP 和细胞因子在肝 I/R 损伤中 FN 背景下的相互作用：首先，将使用 1400W（一种特异性 iNOS 抑制剂）在体内探讨 iNOS 表达的功能意义。其次，我们将剖析 TNF-α 或其他相关细胞因子与 iNOS 在 FN 调节 MMP 表达/激活后的相互作用。第三，我们将揭示细胞内机制，例如白细胞粘附纤连蛋白和 MMP 表达时的 MAPK 转导途径。