PROJECT SUMMARY/ABSTRACT There is substantial evidence that hypertensive glaucoma dramatically alters the capacity of trabecular meshwork (TM) cells to cope with mechanical stress, which in turn contributes to impaired drainage of aqueous humor from the anterior eye. Functional loss is associated with stiffening of TM cells and their matrix, and with loss of homeostatic mechanosensing that adjusts the resistance of the outflow pathway to the intensity of experienced pressure and dynamics of aqueous humor flow. Fibrotic remodeling that underlies the increase in flow resistance can be caused by chronic elevations in intraocular pressure (IOP) or by the cytokine TGF which induces it in the absence of mechanical stress. There are currently no conceptual tools to link IOP stress, stages of TM damage, TGF modulation and mechanotransduction in healthy and glaucomatous eyes into a unified coherent mechanistic model. The goal of this competing renewal is to test hypotheses about how TM cells navigate their response to pressure, shear, and strain, how these mechanisms are reorganized in glaucoma and how matrix stiffness and TGF act through mechanosensitive channels to serve as triggers for fibrotic remodeling that leads to functional loss in glaucoma. Aim 1 will establish the roles of TRPV4 and Piezo1 channels in IOP regulation in conditional knockout mouse models and ex vivo studies of conventional outflow. Aim 2 investigates the molecular foundation of abnormal mechanotransduction in glaucomatous TM cells, studies the role of extracellular matrix as a central determinant of mechanosensitivity, dissects the paradoxical dissonance between TRPV4 gene/protein expression and function, and tests hypotheses about involvement of mechanochannels in intracellular stress signaling, contractility, epithelial mesenchymal transition and regulation of cells’ proliferative potential. The goal of Aim 3 is to bring together mechanotransduction and TGF signaling into a unified framework that would accounts for biophysical (mechanical) and biochemical induction of many types of glaucoma. Upon completion of the study we hope to expand our understanding of the complexity of interlocked molecular mechanisms through which the TM regulates outflow facility, define the molecular triggers through which glaucoma hijacks homeostatic force sensing by mechanosensitive ion channels, integrins and the cytoskeleton, and identify strategies to mitigate injury by restoring the cells ability to autoregulate the mechanoresponse.