MECHANISMS OF RESPIRATORY ENDOTHELIAL INJURY BY XO

XO 损伤呼吸内皮细胞的机制

• 批准号: 2222274
• 负责人: JOHN E REPINE
• 金额: $ 32.63万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-10 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2222274
• 关键词: MECHANISMS; RESPIRATORY; ENDOTHELIAL; INJURY; XO

Xanthine oxidase (XO) appears to participate in a variety of vascular injury syndromes including pulmonary, cardiac, renal, cerebral, and intestinal ischemia-reperfusion or hypoxia/reoxygenation. This possibility is consistent with (a) the high concentrations of XO in capillary endothelial cells, (b) the capacity of XO to generate toxic O2 metabolites and chemotaxins for neutrophils in vitro, and (c) the ability of XO inhibitors to decrease injury in animals. The problem is that the role of XO in human vascular disease is still unproven because XO has been implicated only by using non-specific or incomplete inhibitors of XO activity in animals rather than more accurate modern approaches in relevant human systems. Indeed, the mechanisms which govern regulation of expression of XO and its precursor, xanthine dehydrogenase (XD), the effects of this regulation on pathologic conditions, and the molecular basis for formation of XO and from XD following ischemia and other vascular insults are unclear. Our preliminary data indicate involvement of XO in many diseases. To address definitively the contribution of XO to inflammation and vascular damage, we chose to clone and sequence human XD/XO. Since our prior submission, we have 1) cloned and partially sequenced two candidate DNA probes specific for XO, 2) developed a monoclonal antibody specific for mammalian XO, 3) determined that our human liver cDNA library contains the human XO gene, 4) developed a yeast expression system for human XD, and 5) analyzed new sequence data which supports our hypothesis for XD to XO conversion. Our goal is to test definitively the premise that XO contributes to human disease and to explore the structural basis for XD to XO conversion. Our major objectives are: (a) to construct and test an XD/XO null mutant to produce definitive evidence for the hypothesis that XO dependent mechanisms contribute to cell injury and neutrophil adherence following exposure to hypoxia and reoxygenation; (b) to determine the structural difference between XD and XO activities and the basis for conversion of XD to XO in vitro; and (c) to determine if parameters of ischemia/reperfusion regulate XD/XO gene expression. These critical objectives require cloning and sequencing a human XD/XO cDNA clone. The significance of this project is that it will precisely define the role in ischemic vascular disease of XO in light of its native regulatory mechanisms. This approach will generate new understanding of fundamental mechanisms of tissue injury and inflammation.

黄嘌呤氧化酶 (XO) 似乎参与多种血管生成 损伤综合征包括肺、心、肾、脑等 肠道缺血再灌注或缺氧/复氧。 这 可能性与 (a) 中 XO 浓度高一致 毛细血管内皮细胞，(b) XO 产生有毒 O2 的能力 体外中性粒细胞的代谢物和趋化因子，以及（c） XO抑制剂减少动物损伤的能力。 问题是XO在人类血管疾病中的作用仍然存在 未经证实，因为 XO 仅通过使用非特定或 动物中XO活性的不完全抑制剂而不是更多 相关人类系统中准确的现代方法。 确实， XO及其表达调节的机制 前体，黄嘌呤脱氢酶（XD），这种调节的影响 病理条件以及 XO 形成的分子基础 缺血和其他血管损伤后的 XD 尚不清楚。 我们的初步数据表明 XO 与许多疾病有关。 到 明确阐述 XO 对炎症和血管的贡献 损伤，我们选择克隆并测序人类 XD/XO。 自从我们之前 提交后，我们有 1) 克隆并部分测序了两个候选 DNA XO 特异性探针，2) 开发了一种特异性单克隆抗体 哺乳动物 XO，3) 确定我们的人类肝脏 cDNA 文库包含 人类 XO 基因，4) 开发了人类 XD 的酵母表达系统， 5) 分析了支持我们 XD 假设的新序列数据 到 XO 的转换。 我们的目标是明确测试 XO 贡献的前提 人类疾病并探索XD到XO的结构基础 转换。 我们的主要目标是： (a) 构建并测试 XD/XO 无效突变体为以下假设提供明确的证据： XO 依赖性机制导致细胞损伤和中性粒细胞损伤 暴露于缺氧和复氧后的坚持； (b) 至 确定 XD 和 XO 活性之间的结构差异 XD在体外转化为XO的基础； (c) 确定是否 缺血/再灌注参数调节 XD/XO 基因表达。 这些关键目标需要对人类 XD/XO 进行克隆和测序 cDNA 克隆。 该项目的意义在于它将精确定义 XO 在缺血性血管疾病中的作用 监管机制。 这种方法将产生新的理解 组织损伤和炎症的基本机制。