Mechanisms and Consequences of Hyaluronan Production in Asthma

摘要--项目2 在之前的获奖周期中，我们确定了原代呼吸道上皮细胞(AECs) 合成重链(HC)修饰的透明质酸(HA)顶端支架和气道平滑肌细胞(ASMC) 合成一种透明质酸电缆的细胞外基质，这种电缆具有白细胞粘附性，或者我们也称之为 病理性--HA。我们在该项目中的研究揭示了这种病理-HA基质在发病中的重要性 哮喘的炎症、呼吸道重塑和高反应性。在呼吸道内，有恒定的 紧密结合的ASMC和上覆上皮之间的相互作用，以及越来越多的数据表明 上皮细胞和平滑肌在早期炎症发生中的主要作用 调解人。在这个项目2的继续中，我们建议调查呼吸道上皮细胞产生的NO和 在哮喘中，细胞因子促进病理性透明质酸基质的形成。在……里面 初步数据显示，呼吸道上皮对典型的Th2细胞因子或混合细胞因子有反应。 与HA的产生和诱导型一氧化氮合酶的诱导有关。利用我们的新型共培养系统，我们证明了 ASMC可以通过产生白细胞黏附的HA电缆来响应上皮源性的NO。我们也 表明透明质酸合成的主要贡献者是通过透明质酸合成酶2(HAS2)。协作性 本计划其他成员对NO调节和信号机制的研究， 支持我们的实验，以确定病理性HA的形成机制，包括NO代谢(项目 1)、特定蛋白硫醇亚硝化(项目3)和NO激活途径(项目4)。因此，在这方面 继续，我们建议检验由哮喘呼吸道产生的NO和Hcs的假设 上皮通过以下途径促进ASMCs形成病理性的、白细胞黏附的HC-HA基质 这些机制似乎是sGC非依赖的NO识别途径。为了检验这一假设，我们 计划确定导致产生HA的呼吸道上皮细胞的途径[目标1]。我们还计划确定 ASMC产生白细胞粘附性HA基质的途径[目标2]。最后，我们将确定 NO和HA在体内炎症、AHR和重塑发展中的作用[目标3]。项目2 取决于计划的协作互动和互补技能、专业知识和资源 来检验假设。程序核心支持新的小鼠模型，具有技术挑战性的分析，并共享 临床样本，以促进项目2中的平移和机械实验，而不是其他 有可能。

ABSTRACT - PROJECT 2 During the previous award cycle, we identified mechanisms by which primary airway epithelial cells (AECs) synthesize heavy chain (HC) modified hyaluronan (HA) apical rafts, and airway smooth muscle cells (ASMCs) synthesize an extracellular matrix of HA cables, which are leukocyte-adhesive or what we have also termed as pathologic-HA. Our studies in the Program revealed the importance of this pathologic-HA matrix in the genesis of inflammation, airway remodeling and hyperresponsiveness of asthma. Within the airway, there is constant interaction between the closely coupled ASMCs and overlying epithelium, and increasing data indicate a primary role for epithelium and smooth muscle in the origins of inflammation through production of early mediators. In this continuation of Project 2, we propose to investigate if airway epithelial production of NO and cytokines promotes the accumulation of a pathologic-HA matrix by the smooth muscle in asthma. In preliminary data, we show that the airway epithelium responds to prototypic Th2 cytokines or cytokine mixes with the production of HA and induction of iNOS. Utilizing our novel co-culture system, we show that the ASMCs can respond to epithelial derived NO with the production of leukocyte-adhesive HA cables. We also show that the major contributor to HA production is via the hyaluronan synthase 2 (HAS2). Collaborative studies of NO regulation and signaling mechanisms, which are investigated by other members of this Program, support our experiments to define mechanisms of pathological HA formation, including NO metabolism (Project 1), specific protein thiol nitrosylation (Project 3), and NO activation pathways (Project 4). Thus, in this continuation, we propose to test the hypothesis that NO and HCs produced by the asthmatic airway epithelium promote formation of a pathologic, leukocyte-adhesive HC-HA matrix by ASMCs, through mechanisms that appear to be sGC-independent NO recognition pathways. To test this hypothesis, we plan to identify pathways leading to airway epithelial cell production of HA [Aim 1]. We also plan to identify pathways of ASMC production of a leukocyte-adhesive HA matrix [Aim 2]. Finally, we will determine the contribution of NO and HA on the development of inflammation, AHR, and remodeling in vivo [Aim 3]. Project 2 depends on the collaborative interactions and complementary skills, expertise, and resources of the Program to test hypotheses. Program Cores support novel murine models, technically challenging assays, and shared clinical samples in order to facilitate translational and mechanistic experiments in Project 2 not otherwise possible.