Identifying potential regulatory mechanisms controlling aqueous humor outflow resistance has important implications for understanding glaucoma and developing effective therapies. We hypothesize that Rho/Rho kinase signaling pathway regulated cytoskeletal tension and cell extracellular matrix interactions of trabecular meshwork (TM) cells play a critical role in the synthesis and turnover of extracellular matrix, and contribute to the homeostasis of aqueous humor outflow resistance. Abnormal activation of Rho/Rho kinase signaling within the outflow pathway is therefore predicted to contribute to the increased aqueous outflow resistance in glaucomatous eyes. Compelling evidence for this hypothesis derives from our recent studies of agonist and RhoA induced changes in TM cell contractile tone and aqueous humor outflow facility in perfused models. Contractile agonists (lysophospholipids and thrombin), TGF beta, extracellular matrix (ECM) proteins and increased intraocular pressure, all stimulated myosin II phosphorylation and actomyosin assembly in TM cells, which are recognized characteristics of increased cellular cytoskeletal tension. These effects were further associated with increased Rho GTPase activation in TM cells, and with significant decreases in aqueous outflow facility in perfused porcine and human eyes. Moreover, expression of constitutively active RhoA significantly decreased aqueous outflow facility in organ cultured porcine eye anterior segments and increased expression of genes encoding various ECM proteins and cytokines in human TM cells. Taken together, these observations indicate the existence of a potential interaction between Rho GTPase signaling, cytoskeletal tension, ECM synthesis and aqueous outflow resistance. Given the convincing evidence in support of a role for Rho GTPase induced cytoskeletal tension and cell ECM interactions in regulation of aqueous humor outflow resistance we propose in this application to investigate: 1. the association between Rho GTPase induced cytoskeletal tension and ECM synthesis and the expression of cytokines in human TM cells; 2. in vivo effects of sustained activation of Rho GTPase on intraocular pressure and ECM synthesis in the outflow pathway in a live rodent model, and 3. the expression and activation status of the Rho/Rho kinase signaling components in the outflow pathway of eyes subjected to elevated intraocular pressure, and in hypertensive open angle glaucomatous human donor eyes. Completion of these studies should unravel the role of TM cytoskeletal tension and cell ECM interactions in the homeostasis of aqueous humor outflow resistance, define the regulation of ECM synthesis by the Rho/Rho kinase signaling pathway, and clarify the significance of these events in increased intraocular pressure in glaucomatous eyes. Relevance to public health: Better understanding of the molecular mechanisms regulating homeostasis of aqueous humor outflow resistance could provide novel avenues for designing targeted therapies for treatment of glaucoma. Understanding the etiology of primary open angle glaucoma and developing effective therapies requires identification of regulatory mechanisms which control aqueous humor outflow. In the proposed study we explore the novel paradigm that there is a strong functional connection between the cytoskeletal signaling and the homeostasis of aqueous humor outflow resistance. Exploring this connection will provide important insights into our understanding of physiological and pathological regulation of aqueous outflow resistance in normal and ocular hypertensive glaucoma eyes and have an important bearing on translational application to novel therapies for glaucoma. ? ? ?
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