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.

PROJECT NARRATIVE - PROJECT 2 Asthma is a chronic inflammation of the airways in which the local resident cells of the airway play a dominant role in the initiation and perpetuation of the inflammatory response. The chronic inflammation can lead to progressive airway changes over time that may interfere with routine activities of daily living. Our project will determine the effects of local mediators on the development of asthma and identify possible targets for future therapeutic interventions.