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

项目总结/摘要 虽然许多人认为一氧化碳（CO）严格地说是一种有毒气体，但CO也是从自然界中产生的。 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， 中等水平的CO将引发针对缺氧和缺血事件的有效细胞保护作用。这些特性导致 然而，最佳的CO水平，通过它的最安全和最 有效的治疗效果仍然是未知的。模型系统表现出增加的内源性CO作为 保护策略，而不是病理副作用，将提供安全有效的CO暴露水平的见解。 我们的初步工作揭示了两个人类种群和一个潜水哺乳动物，都适应了慢性缺氧， 表达增加的CO产生或涉及HO-2基因的阳性选择。我们之前对西藏基因组的研究 揭示了HO-2位点的正选择，表明HO/CO途径在高海拔地区的重要作用。 适应同样，我们对秘鲁土著人的初步潮气末CO测量表明，高海拔土著人 与低海拔当地人相比，潮气末CO增加。同样，我的论文工作表明，海象是 已知的唯一一种哺乳动物产生和维持CO在相同的中等水平，最近被认为是治疗和保护性的， 上面提到的人类和实验室动物研究。象海豹表现出重复的、自愿的睡眠呼吸暂停事件（~ 10-15分钟），在陆地上时，已知它们经常经历一定程度的缺氧和组织缺血， 对其他哺乳动物产生有害影响。根据这些初步证据，我认为高海拔地区的土著人和大象 海豹代表了理想的模型，以提高我们对HO/CO自然上调背后的机制的理解 途径减轻缺氧诱导的损伤。具体而言，该提案概述了一种多学科方法， HO/CO途径自然上调背后的细胞和遗传机制的调查，并探讨 相关的组织特异性保护特性。人类和海象将在慢性疾病期间被采样。 低氧和常氧。将评价血液中HO-1、HO-2和胆绿素还原酶（BVR）的数量和活性 (plus海象的骨骼肌）。前体（血红蛋白和血红素）和产物（即CO，铁， 胆绿素和胆红素）的HO和BVR活性。CO的去除率为 通过潮气末CO值和胆红素分解产物（粪胆素和尿胆素）的排泄来确定 在粪便和尿液中测量。血红素储存清除将通过研究红细胞寿命来测量。评价 这种途径的遗传调控，来自血液样本（以及骨骼和肝脏组织）的RNA的转录组学， 在低氧和常氧期后服用的象海豹）将显示特定的 基因的HO/CO途径的活性和氧的可用性在两个国家。抗炎、抗- 将在血液和组织样品中测量细胞凋亡、抗增殖和抗氧化作用，并将在 组和基因表达值和HO/CO途径的活性。

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.