Project Summary: This grant has focused in the past on determining the potential role of factors induced by mechanical stress in trabecular meshwork (TM) cells as modulators of Schlemm's canal (SC) endothelium permeability and overall outflow facility. A major accomplishment towards this purpose was the success in growing SC cells in culture. Our studies have documented the activation of the transforming growth factor beta 1 (TGF-beta1) and interleukin 6 (IL-6) promoters, as well as the increased production of these cytokines in TM cells in response to mechanical stress both in human TM (HTM) cells in culture and perfused porcine anterior segments. We have also shown that a subpopulation of HTM cells is characterized by high levels of expression of the gene encoding for chitinase 3-like 1 (CHI3L1), a protein known to inhibit the production of proinflammatory cytokines. Both TGF-beta1&IL-6 are known to increase vascular endothelial permeability, and we have documented that perfusion of IL-6 increases outflow facility. We therefore hypothesize that both TGF-beta1 and IL-6 can act to increase the permeability of the endothelium of SC. An earlier study supported by this grant showed that certain kinds of mechanical stress could influence the mitogen-activated protein kinase (MARK) pathways. Since there are known relationships, direct and indirect, between IL-6, TGF-beta1, and CHI3L1 and the MARK pathway, we hypothesize that a molecular mechanism mediating these responses includes the involvement of an autocrine amplification loop between TGF-beta1 and MAPKs, which leads to increased expression of certain cytokines including IL-6;and that CHI3L1 expression acts to negatively modulate the stress-induced induction of TGF-beta1 and IL-6. We further hypothesize that unbalanced increased expression of CHI3L1 in the outflow pathway might result in an increase in outflow resistance with implication for glaucoma pathogenesis. To test our hypothesis, we propose the following 3 specific aims: SA1) determine whether activation of TGF-beta1and IL-6 in TM cells results in increased outflow facility in situ through effects on SC endothelium;SA2) evaluate whether the induction of IL-6 by mechanical stress in outflow pathway cells involves an amplifying loop between TGF-beta1 and MAPKs;and SA3) evaluate if the CHI3L1 acts to negatively modulate the stress-induced induction of TGF-beta1 and IL-6, thereby causing an increase in aqueous humor outflow resistance. Relevance: This project has the potential to increase our understanding of both normal and abnormal outflow pathway function and to lead to new treatments for glaucoma.