MECHANISMS OF RESPIRATORY ENDOTHELIAL INJURY BY XO

黄嘌呤氧化酶（XO）似乎参与多种血管 损伤综合征，包括肺、心、肾、脑和 肠缺血-再灌注或缺氧/复氧。 这 可能性是一致的（a）高浓度的XO在 毛细血管内皮细胞，（B）XO产生毒性O2的能力 体外中性粒细胞的代谢物和趋化因子，和（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是 未经证实，因为XO仅通过使用非特异性或 XO活性的不完全抑制剂，而不是更多 在相关的人类系统中使用精确的现代方法。 持续增 调节XO表达的机制及其 前体，黄嘌呤脱氢酶（XD），这种调节的影响 病理条件和XO形成的分子基础 以及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参与许多疾病。 到 明确阐述XO对炎症和血管 由于损伤，我们选择克隆和测序人类XD/XO。 自从我们的前任 提交，我们已经1）克隆和部分测序两个候选DNA XO特异性探针，2）开发了特异性单克隆抗体， 哺乳动物XO，3）确定我们的人肝cDNA文库含有 人XO基因，4）开发了人XD的酵母表达系统， 5）分析新的序列数据，支持我们对XD的假设 转换成XO 我们的目标是明确地测试XO有助于 人类疾病，并探讨XD到XO的结构基础 转换. 我们的主要目标是：（a）建立和测试一个 XD/XO无效突变体，为以下假设提供明确证据： XO依赖性机制有助于细胞损伤和中性粒细胞 暴露于缺氧和复氧后的粘附;（B） 确定XD和XO活性之间的结构差异， 体外XD转化为XO的基础;以及（c）确定 缺血/再灌注参数调节XD/XO基因表达。 这些关键目标需要克隆和测序人XD/XO 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。

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.