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

项目概要/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，但一氧化碳也在体内从自然环境中产生。 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，暴露于 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。这些属性导致 研究 CO 的治疗潜力。然而，最安全、最有效的最佳 CO 水平 能否达到强效治疗效果仍是未知数。模型系统表现出增加的内源CO作为 保护策略，而不是病理副作用，将提供安全有效的二氧化碳暴露水平的见解。 我们的初步工作揭示了两个人类群体和一种潜水哺乳动物都适应了慢性缺氧， 表达 CO 产量增加或涉及 HO-2 基因的正选择。我们之前对西藏基因组的研究 揭示了 HO-2 基因座的正选择，表明 HO/CO 途径在高海拔地区发挥着重要作用 适应。同样，我们对秘鲁当地人进行的潮末二氧化碳初步测量表明，高海拔地区的当地人 与低海拔地区相比，潮气末二氧化碳含量增加。同样，我的论文工作表明象海豹是 唯一已知能够产生并维持中等水平二氧化碳的哺乳动物，最近被认为具有治疗和保护作用 上面提到的人类和实验动物研究。象海豹表现出反复的、自愿的睡眠呼吸暂停事件（〜 10-15 分钟）在陆地上时，众所周知，它们经常经历一定程度的缺氧和组织缺血，这会导致 对其他哺乳动物造成有害影响。由于这一初步证据，我建议高海拔地区的当地人和大象 密封代表了加深我们对 H2O/CO 自然上调背后机制的理解的理想模型 缓解缺氧引起的损伤的途径。具体来说，该提案概述了一种多学科方法 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。将在慢性病期间对人类和象海豹进行采样 缺氧和常氧。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）和产物（即 CO、铁、 胆绿素和胆红素）的 HO 和 BVR 活性将在相同的血液样本中进行测量。 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 在粪便和尿液中进行测量。血红素储存的去除将通过研究红细胞寿命来测量。评估 该途径的遗传调控、血液样本（以及骨骼和肝脏组织）中 RNA 的转录组学 象海豹）在缺氧和常氧期后进行的检测将显示特定的上调或下调 两种状态下与 HO/CO 途径活性和氧气可用性相关的基因。抗炎、抗炎标志物 将在血液和组织样本中测量细胞凋亡、抗增殖和抗氧化作用，并在不同细胞之间进行比较 组和基因表达值以及 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.