High Altitude Adaptation: A Model for Chronic Hypoxia

描述（由申请人提供）：慢性缺氧是许多疾病的中心特征，包括缺血性心脏病、脑血管疾病和慢性阻塞性肺病。了解慢性缺氧的细胞和生理反应将为治疗这些广泛流行的疾病提供基础。缺氧的中心转录反应是由脯氨酸羟化酶结构域（PHD）：缺氧诱导因子（HIF）途径介导的。在这一途径中，PHD（由三种异构体组成）脯丙基羟基化HIF（由三种异构体组成）的α亚基，并将后者作为降解目标。在缺氧条件下，PHD活性减弱，从而使HIF-α稳定，并激活一系列参与缺氧适应的基因，例如促进从有氧代谢向无氧代谢转变的基因。有人可能会认为，激活这一途径将对这些疾病有直接的好处。然而，很明显，这一途径的慢性激活会导致两种潜在的严重不良反应，肺动脉高压和红细胞增多。对这一途径的治疗性操作要求确定缓和这些不良反应的方法。对藏族人口的研究，他们已经适应了高海拔和慢性缺氧，提供了一个独特的机会来追求这一点。引人注目的是，这个人群避免了肺动脉高压和红细胞增多症，这些疾病折磨着那些在高海拔地区生活的低海拔居民。因此，如果人们能够确定发生这种情况的机制，就可以采用可以改善这些后果的方法。最近，大量对西藏人群的独立全基因组研究为这种适应的遗传基础提供了令人信服的证据，他们一致指出两个基因，PHD2（也称为EGLN1）和HIF2A（也称为EPAS1）基因。在本应用中，我们将重点关注PHD2基因。上述研究发现了一系列在藏族人群中富集的内含子和外显子单核苷酸多态性（snp）。在拟议项目的初始R21阶段，我们将首先通过一系列体外检测确定功能重要的SNP，包括报告基因、蛋白质：蛋白质相互作用和基于细胞培养的检测。在该项目的后续R33阶段，我们将生成一个小鼠敲入系来模拟西藏SNP。然后，我们将检查这种SNP改善肺动脉高压和红细胞增多症的能力，这在两种独立的慢性缺氧模型中可见。在一个模型中，我们将这些小鼠暴露在缺氧中三周。在第二个模型中，我们将这些小鼠与最近产生的携带敲入Hif2a突变的小鼠系杂交，该突变表现出高度渗透的红细胞增多和肺动脉高压。我们预计，拟议的研究将确定一条途径，通过该途径可以参与缺氧反应，同时最大限度地减少其最严重的不良影响。

DESCRIPTION (provided by applicant): Chronic hypoxia is a central feature of many diseases, including ischemic heart disease, cerebrovascular disease, and chronic obstructive pulmonary disease. Understanding the cellular and physiologic responses to chronic hypoxia will provide the basis for therapies for these widely prevalent diseases. The central transcriptional response to hypoxia is mediated by the Prolyl Hydroxylase Domain (PHD):Hypoxia Inducible Factor (HIF) pathway. In this pathway, PHD (which consists of three isoforms) prolyl hydroxylates the α subunit of HIF (which consists of three isoforms) and targets the latter for degradation. Under hypoxia, PHD activity is attenuated, allowing for the stabilization of HIF-α and the activation of a broad range of genes involved in hypoxic adaptation, such as ones that promote a shift from aerobic to anaerobic metabolism. One might presume that activation of this pathway would be of direct benefit in these diseases. However, it is clear that chronic activation of this pathway leads to two potentially serious adverse effects, pulmonary hypertension and erythrocytosis. Therapeutic manipulation of this pathway mandates identifying means of tempering these adverse effects. Study of the Tibetan population, who have adapted to high altitudes and chronic hypoxia, offers a unique opportunity to pursue this. Strikingly, this population has avoided the pulmonary hypertension and erythrocytosis that afflict low altitude dwellers who ascend to high altitudes. Hence, if one were able to identify the mechanisms by which this occurs, this would allow approaches that could ameliorate these consequences. A large number of independent genome wide studies of the Tibetan population have recently provided convincing evidence for a genetic basis for this adaptation, and they consistently point to two genes, the PHD2 (also known as EGLN1) and HIF2A (also known as EPAS1) genes. In this application, we will focus on the PHD2 gene. The above referenced studies have identified a series of intronic and exonic single nucleotide polymorphisms (SNPs) that are enriched in the Tibetan population. In the initial R21 phase of the proposed project, we will first identify the functionally important SNP through a series of in vitro assays that will include reporter gene, protein:protein interaction, and cell culture-based assays. In the subsequent R33 phase of the proposed project, we will generate a mouse knockin line to model the Tibetan SNP. We will then examine the capacity of this SNP to ameliorate the pulmonary hypertension and erythrocytosis that is seen in two independent models of chronic hypoxia. In one model, we will expose these mice to hypoxia for three weeks. In the second model, we will cross these mice with a recently generated mouse line bearing a knockin Hif2a mutation that displays highly penetrant erythrocytosis and pulmonary hypertension. We anticipate that the proposed studies will identify a pathway by which the hypoxic response can be engaged while minimizing its most serious adverse effects.