Identifying protective roles of the heme oxygenase/carbon monoxide pathway in hypoxia-tolerant model systems

项目摘要/摘要 虽然许多人认为一氧化碳(CO)严格来说是一种有毒气体，但一氧化碳也是在人体内从天然气体中产生的 通过血红素加氧酶(HO-1和HO-2)分解血红素。最近的实验室研究表明，接触到 适度的一氧化碳水平将对缺氧和缺血事件产生强大的细胞保护作用。这些属性导致了 对一氧化碳治疗潜力的研究。然而，最安全和最安全的最佳CO水平 能否达到有效的治疗效果仍是个未知数。内生CO增加的模型系统 保护策略，而不是病理副作用，将提供对安全有效的一氧化碳暴露水平的洞察。 我们的初步工作揭示了两种人类种群和一种潜水哺乳动物，它们都适应慢性低氧， 表达CO产生增加或涉及HO-2基因的正选择。我们之前对藏族基因组的研究 显示HO-2基因的正选择，提示HO/CO途径在高海拔地区具有重要作用 适应。同样，我们在秘鲁原住民中进行的初步潮气末CO测量表明，高海拔原住民有 与低海拔原住民相比，潮气末CO增加。同样，我的论文工作表明，象海豹是 只有已知的哺乳动物能产生并保持与最近被认为具有治疗和保护作用的中等水平的一氧化碳 以上提到的人类和实验动物研究。海象反复表现出自愿的睡眠呼吸暂停事件(~ 10-15分钟)，在陆地上，已知他们经常经历程度的缺氧和组织缺血，这将 在其他哺乳动物身上引发有害影响。根据这一初步证据，我认为高海拔原住民和大象 海豹模型是提高我们对HO/CO自然上调背后机制的理解的理想模型 减轻缺氧性损伤的途径。具体地说，这项提案概述了一种多学科的方法来研究 研究HO/CO途径自然上调背后的细胞和遗传机制，并探索 相关的组织特异性保护特性。人类和象海豹将在慢性病时期进行采样 低氧和常氧。将评估血液中HO-1、HO-2和胆绿素还原酶(BVR)的数量和活性 (加上象海豹的骨骼肌)来自所有患者。前体(血红蛋白和血红素)及其产物(即一氧化碳、铁、 HO和BVR活性的胆绿素和胆红素)将在相同的血液样本中测量。CO的去除率将为 通过呼气末CO值和胆红素分解产物(斯特胆林和尿胆碱)的排泄量确定的将是 在粪便和尿液中测量。血红素库的清除将通过调查红细胞寿命来衡量。评估 这一途径的遗传调节，从血液样本(加上骨骼和肝脏组织)的RNA转录 在低氧和常氧期后采集的海豹)将显示特定的上调或下调 在这两种状态下与HO/CO途径活性和氧气利用率相关的基因。抗炎、抗炎、抗炎的标志物 将在血液和组织样本中测量细胞凋亡、抗增殖和抗氧化能力，并将其与 并对HO/CO途径的基因表达水平和活性进行了研究。

Project Summary/Abstract While many see carbon monoxide (CO) as strictly a toxic gas, CO is also produced in the body from the natural breakdown of heme by heme oxygenase enzymes (HO-1 and HO-2). Recent laboratory studies have shown that exposure to moderate levels of CO will elicit potent cytoprotective effects against hypoxic and ischemic events. These properties have led to the investigation of the therapeutic potential of CO. However, the optimal CO levels through which the safest and the most potent therapeutic effect can be achieved is still unknown. Model systems which exhibit increased endogenous CO as a protective strategy, rather than pathological side effect, will provide insight into CO exposure levels that are safe and effective. Our preliminary work has revealed two human populations and one diving mammal, all adapted to chronic hypoxia, that express increased CO production or positive selection involving the HO-2 gene. Our previous work with Tibetan genomes revealed positive selection at the HO-2 locus, suggesting an important role of the HO/CO pathway in high-altitude adaptation. Similarly, our preliminary end-tidal CO measurements in Peruvian natives show that high-altitude natives have increased end-tidal CO compared to low altitude natives. Likewise, my dissertation work has shown that elephant seals are the only mammal known to produce and maintain CO at the same moderate levels recently deemed therapeutic and protective in the human and laboratory animal studies mentioned above. Elephant seals exhibit repeated, voluntary sleep apnea events (~ 10-15 min) when on land, where they are known to regularly experience degrees of hypoxia and tissue ischemia which would elicit detrimental effects in other mammals. Due to this preliminary evidence, I propose that high-altitude natives and elephant seals represent ideal models to improve our understanding on the mechanisms behind the natural upregulation of the HO/CO pathway in alleviating hypoxia-induced injuries. Specifically, this proposal outlines a multidisciplinary approach into the investigation of the cellular and genetic mechanisms behind the natural upregulation of the HO/CO pathway, and explores the associated tissue-specific protective properties. Humans and elephant seals will be sampled during periods of chronic hypoxia and normoxia. The quantity and activity of HO-1, HO-2 and biliverdin reductase (BVR) will be evaluated in the blood (plus skeletal muscle in elephant seals) from all patients. The precursors (hemoglobin and heme) and products (i.e. CO, iron, biliverdin and bilirubin) of HO and BVR activity will be measured in the same blood samples. The removal rates of CO will be determined through end-tidal CO values and the excretion of bilirubin breakdown products (stercobilin and urobilin) will be measured in the feces and urine. The heme store removal will be measured by investigating red blood cell lifespan. To evaluate the genetic regulation of this pathway, transcriptomics on RNA from the blood samples (plus skeletal and liver tissue in elephant seals) taken after hypoxic and normoxic periods will demonstrate the upregulation or downregulation of specific genes in relation to the HO/CO pathway activity and oxygen availability in the two states. Markers of anti-inflammation, anti- apoptosis, anti-proliferation, and anti-oxidation will be measured in blood and tissue samples and will be compared between groups and to gene expression values and the activity of the HO/CO pathway.