Interleukin-13-mediated mechanisms of pulmonary vascular remodeling

项目总结 肺动脉高压(PAH)是一种由血管阻力升高引起的致命疾病，原因是 小肺动脉内的腔-闭塞突起。为了改善临床结果，更好地理解 关于PAH的发病机制亟待研究。最近，我们报道了慢性暴露于 使用转基因方法(IL-13 TG)的小鼠肺中的IL-13诱导了令人印象深刻的平滑 肌细胞驱动的肺血管重构(PVR)导致肺动脉高压(PH)。我们进一步 研究表明，IL-13在体内和体外诱导血管平滑肌细胞增殖是通过 精氨酸酶2(Arg2)通过IL-13受体α2(IL-13Ra2)表达。此外，我们还观察到IL-13增加了该基因的表达。 和低氧诱导因子1a(HIF1a)通过IL-13Ra2的活性，以及IL-13诱导的细胞增殖。 HIF1a基因敲除后人肺动脉平滑肌细胞(HPASMC)数量显著减少 放下。此外，HIF1a在转录水平上调节Arg2的表达。这些发现表明 HIF1a在IL-13诱导的血管平滑肌细胞增殖中的重要作用鉴于HIF1a是一种 代谢从氧化磷酸化转换到糖酵解，这些发现提示IL-13可能诱导 Warburg类型效应(癌细胞有氧糖酵解增加和氧化磷酸化减少 导致增殖)以刺激血管平滑肌细胞的增殖。 具体目标 我们提出以下假设： 1.IL-13通过IL-13Ra2刺激hPASMC增殖。 2.HIF1a在IL-13通过IL-13Ra2刺激的hPASMC增殖中起重要作用。 3.IL-13通过IL-13Ra2、HIF1a调节代谢从氧化磷酸化向糖酵解的转变 和Arg2，从而诱导PASMC增殖和PH。 我们的目标是： 目的1.明确IL-13Ra2在体内外IL-13诱导的PH形成中的作用。 目的2.明确HIF1a在IL-13-IL-13Ra2诱导的小鼠骨髓细胞增殖表型形成中的作用。 PASMC的体外和体内实验。 目的3.明确IL-13在代谢从氧化磷酸化到 体外和体内PASMC的糖酵解。 实验方法 我们将使用hPASMC和IL-13转基因小鼠的体内和体外方法。为了解决目标1， 比较IL-13Ra2缺失突变体与IL-13转基因小鼠的肺血管表型 至IL-13Tg。进一步转染IL-13Ra2特异性siRNA的hPASMC将用IL-13和 将进行细胞增殖分析。结果将与转染对照的hPASMC的结果进行比较 SiRNA。为了解决AIM 2，HIF1a在IL-13中的表达将被检测和比较。 IL-13Ra2基因敲除或不下调hPSAMC后，IL-13 TG和IL-13 TG与 IL-13Ra2缺失突变小鼠。探讨IL-13刺激下AIM 3、乳酸水平和氧耗率的变化 将对hPASMC进行评估。一种代谢调节剂将被给予IL-13转基因小鼠，以观察代谢是否 IL-13TG通过HIF1a的改变改变了这些动物的肺血管表型。 这项工作将为我们提供一个新的洞察力，使我们能够更好地理解 免疫和代谢过程之间的复杂相互作用以及一种新的调控途径 HIF1a。 这笔赠款提议在耶鲁大学开展一项研究指导计划，该计划的主要赞助人是Dr。 世界著名的肺部炎症和免疫生物学研究人员Jack Elias和Hyung Joon Chun博士， 血管信号和药理学方面的专家，作为共同导师。我们还邀请了耶鲁大学的专家 调查人员Gerald Shadel、Patty Lee、William Sessa、Frank Giordano和Chun Geun Lee博士担任顾问 为委员会成员提供科学和职业咨询。拟议的职业和研究计划 将提供一个非凡的科学环境，赵博士可以在其中启动她的未来 作为一名独立的内科科学家职业。

PROJECT SUMMARY Pulmonary Arterial Hypertension (PAH) is a fatal disease caused by elevated vascular resistance due to lumen-obliterating processes in small pulmonary arteries. To improve clinical outcome, a better understanding regarding the pathogenesis of PAH is desperately needed. Recently we reported that chronic exposure of interleukin-13 (IL-13) in murine lungs, using a transgenic approach (IL-13 Tg), induced impressive smooth muscle cell-driven pulmonary vascular remodeling (PVR) leading to pulmonary hypertension (PH). We further showed that IL-13-induced proliferation of vascular smooth muscle cells in vivo and in vitro is mediated through Arginase2 (Arg2) via IL-13 receptor alpha2 (IL-13Ra2). Also, we observed that IL-13 increased the expression and activity of hypoxic inducible factor1a (HIF1a) via IL-13Ra2, as well, and that IL-13-induced proliferation of human pulmonary artery smooth muscle cell (hPASMC) is significantly decreased with knocking HIF1a gene down. Furthermore, HIF1a regulates Arg2 expression at transcriptional level. These findings implicate the important role of HIF1a in IL-13-induced proliferation of vascular smooth muscle cells. Given that HIF1a is a metabolic switch from oxidative phosphorylation to glycolysis, these findings suggest that IL-13 might induce the Warburg type effect (increased aerobic glycolysis and decreased oxidative phosphorylation in cancer cells leading to proliferation) to stimulate proliferation of vascular smooth muscle cells. Specific Aims We propose the following hypotheses: 1. IL-13 stimulates the proliferation of hPASMC through IL-13Ra2. 2. HIF1a plays a critical role in IL-13-stimulated proliferation of hPASMC via IL-13Ra2. 3. IL-13 regulates the metabolic shift from oxidative phosphorylation to glycolysis via IL-13Ra2, HIF1a and Arg2, thereby inducing PASMC proliferation and PH. Our Aims are to: AIM 1. Define the role of IL-13Ra2 in the development of IL-13-induced PH in vitro and in vivo. AIM 2. Define the role of HIF1a in the generation of the IL-13-IL-13Ra2-induced proliferative phenotypes in PASMC in vitro and in vivo. AIM 3. Define the role of IL-13 in the development of the metabolic shift from oxidative phosphorylation to glycolysis in PASMC in vitro and in vivo. Experimental Approach We will use both in vivo and in vitro approaches using hPASMC and IL-13 Tg mice. To address AIM 1, the pulmonary vascular phenotype will be assessed in IL-13 Tg mice crossed with IL-13Ra2 null mutant compared to IL-13 Tg. Further hPASMC transfected with the specific siRNA of IL-13Ra2 will be treated with IL-13 and proliferation assays will be done. The results will be compared to those from hPASMC transfected with control siRNA. To address AIM 2, HIF1a expression will be examined and compared using Western blot in IL-13- treated hPSAMC with or without knocking the IL-13Ra2 gene down, and IL-13 Tg and IL-13 Tg crossed with IL-13Ra2 null mutant mice. To address AIM 3, lactate levels and oxygen consumption rates in IL-13-stimulated hPASMC will be assessed. A metabolic modifier will be given to IL-13 Tg mice to see whether the metabolic shift in IL-13 Tg through HIF1a alters the pulmonary vascular phenotype in these animals. This work will provide us with a novel insight into the pathogenesis of pulmonary hypertension induced by a complex interaction between immune and metabolic processed in addition to a novel regulatory pathway of HIF1a. This grant proposes a research mentorship program at Yale University under the primary sponsorship of Dr. Jack Elias, a world-renowned investigator in lung inflammation and immunobiology, and Dr. Hyung Joon Chun, an expert in vascular signaling and pharmacology, as a co-mentor. We have also enlisted the expertise of Yale investigators Drs. Gerald Shadel, Patty Lee, William Sessa, Frank Giordano and Chun Geun Lee as advisory committee members to provide scientific and career counseling. The proposed career and research program as outlined will provide an extraordinary scientific environment wherein Dr. Cho can launch her future independent career as a physician-scientist.