Oxygen Sensitive Signaling in Primary Cardiac Fibroblast

描述（由申请人提供）：在含氧量正常的条件下，哺乳动物器官中的氧浓度范围为12%至0.5%以下，动脉血中约为14%，心肌中<10%。在轻度缺氧期间，心肌O2下降至约1- 3%。响应于慢性中度缺氧，细胞调节其常氧设定点，使得pO 2的再氧合依赖性升高导致“感知的高氧”。氧传感已积极研究缺氧。然而，复氧主要是在氧化损伤的背景下研究的。心脏成纤维细胞（CF）协调生理（修复）和病理（纤维化）组织重塑。该提议基于一个惊人的观察结果，即从成年鼠心室分离的CF，在10%或21%O2（“升高的O2”，相对于细胞在体内调节到的pO 2）中培养，与3%（轻度缺氧）相比，表现出可逆的生长抑制和指示分化的表型。工作假设是，O2，即使在边际相对过量的pO 2细胞调整，导致激活特定的信号转导途径，改变CF的表型和生物学功能。为了检验这一假设，并建立其意义，在再氧心脏，我们提出了以下四个具体目标：目的1：确定是否升高（10-21%）环境O2诱导的CF生长抑制与分化表型。目的2：检测p21 Wafl/Cip 1/Sdi 1-诱导在高氧（10-21%）诱导的CF生长抑制和分化中的意义。目标3：描述TGF β和p38 MAPK活化在暴露于升高的（10-21%）环境O2的细胞中赋予目标1和2中概述的细胞反应的作用。目的4：表征缺血-再氧合（I-R）心脏中感知的高氧（如目的1-3所定义）。这一建议有三个根本性的重要意义。首先，它提供了一个新的缺血-复氧生物学，在许多心脏疾病的中心重要。与慢性中度缺氧期间细胞调整至的pO 2相比，pO 2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，该提议假定从心脏分离的CF对pO 2从<10%O2（体内常氧）到21%O2（广泛解释为体外常氧）的变化不是“盲”的。这就提出了一个重要的广泛的问题，在细胞培养过程中控制周围的O2。最后，一个孤立的细胞培养模型，研究慢性中度缺氧，然后复氧。

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.