Oxygen Sensitive Signaling in Primary Cardiac Fibroblast

描述（由申请人提供）：在常氧条件下，哺乳动物器官中的氧浓度范围为12至0.5%以下，动脉血中的氧浓度约为14%，心肌中的氧浓度<10%。轻度缺氧时，心肌氧降至约1-3%。作为对慢性中度缺氧的反应，细胞调整其常氧设定点，使pO2的再氧化依赖性升高导致“感知高氧”。氧传感已被积极研究用于缺氧。然而，再氧化的研究大多是在氧化损伤的背景下进行的。心脏成纤维细胞（CF）协调生理（修复）和病理（纤维化）组织重塑。这一建议基于一项惊人的观察，即从成年小鼠心室分离出来的CF，在10%或21%的O2（“升高的O2”，相对于细胞在体内调节的pO2）中培养，与3%（轻度缺氧）相比，表现出可逆性的生长抑制和指示分化的表型。工作假设是，即使细胞调节的pO2相对过量，O2也会导致特定信号转导途径的激活，从而改变CF的表型和生物学功能。为了验证这一假设并确定其在复氧心脏中的意义，我们提出了以下四个具体目标：目的1：确定环境O2诱导的CF生长抑制升高（10-21%）是否与分化表型有关。目的2：检查p21Wafl/Cip1/ sdi1诱导在升高（10-21%）环境O2诱导的CF生长抑制和分化中的意义。目的3：表征暴露于升高（10-21%）环境O2的细胞中tgf β和p38MAPK活化在赋予目标1和2中概述的细胞反应中的作用。AIM 4：描述缺血再充氧（I-R）心脏的感知高氧（定义见Aims 1-3）。这一建议具有三个根本重要的意义。首先，它为缺血-再氧化生物学提供了一个新的视角，这在许多心脏疾病中都很重要。与慢性中度缺氧期间细胞调节的pO2相比，pO2的边际相对升高可能作为触发心脏成纤维细胞分化和组织重塑的信号，这是一个与再灌注损伤相关的纤维化和组织修复相关的新概念。其次，该建议假设从心脏分离的CF对pO2从<10%O2（体内正常氧）到21% O2（被广泛解释为体外正常氧）的变化并非“盲目”。这就提出了一个重要的基础广泛的问题，即在细胞培养过程中控制环境氧。最后，建立了慢性中度缺氧后再充氧的细胞培养模型。

DESCRIPTION (provided by applicant): Oxygen concentration ranges from 12 to below 0.5% in mammalian organs under normoxic conditions with approximately 14% in arterial blood and <10% in the myocardium. During mild hypoxia, myocardial O2 drops to about 1-3%. In response to chronic moderate hypoxia, cells adjust their normoxia set-point such that reoxygenation- dependent elevation of pO2 results in "perceived hyperoxia". O2 sensing has been actively studied for hypoxia. Reoxygenation, however, has been mostly studied in the context of oxidative injury. Cardiac fibroblasts (CF) orchestrate physiological (repair) and pathological (fibrosis) tissue remodeling. This proposal rests on a striking observation that CF, isolated from adult murine ventricle, cultured in 10% or 21% O2 ("elevated O2", relative to pO2 to which cells are adjusted in vivo), compared to 3% (mildly hypoxic), exhibit reversible growth inhibition and a phenotype indicative of differentiation. The working hypothesis is that O2, even in marginal relative excess of the pO2 to which cells are adjusted, results in activation of specific signal transduction pathways that alter the phenotype and biological function of CF. To test this hypothesis and establish its significance in the reoxygenated heart, we propose the following four specific aims: AIM 1:Determine whether elevated (10-21%) ambient O2-induced growth inhibition of CF is associated with differentiation phenotype. AIM 2: Examine the significance of p21Wafl/Cip1/Sdi1-induction in elevated (10-21%) ambient O2-induced growth inhibition and differentiation of CF. AIM 3: Characterize the role of TGFbeta and p38MAPK activation in cells exposed to elevated (10-21%) ambient O2 in conferring the cellular responses outlined in Aims 1 & 2. AIM 4: Characterize perceived hyperoxia (as defined in Aims 1-3) in the ischemia-reoxygenated (I-R) heart. This proposal has three fundamentally important significances. First, it offers a fresh look at ischemia-reoxygenation biology, centrally important in numerous cardiac disorders. That marginal relative elevation in pO2, compared to pO2 to which cells are adjusted during chronic moderate hypoxia, may serve as a signal to trigger cardiac fibroblast differentiation and tissue remodeling is a novel concept relevant to fibrosis and tissue repair related to reperfusion injury. Second, this proposal postulates that CF isolated from the heart are not "blind" to a change of pO2 from <10%O2 (in vivo normoxia) to 21% O2 (widely interpreted as in vitro normoxia). This raises a significant broad-based issue of controlling ambient O2 during cell culture. Finally, an isolated cell culture model to study chronic moderate hypoxia followed by reoxygenation is offered.