Fuchs endothelial corneal dystrophy (FECD) decreases vision quality and can progress to vision loss, for which 17,000 individuals/year require corneal transplantation in the United States. The hallmarks of FECD are the progressive accumulation of deposits (guttae) on the basement (Descemet’s) membrane of the corneal endothelium and progressive loss of corneal endothelial cells (CEnCs). These changes are present in all forms of FECD regardless of genetic background. But it is unknown how guttae form or how this contributes to CEnC dysfunction. Novel preliminary data in support of this project show that guttae-like structures can be developed in cell culture. Long-term culture of primary bovine CEnCs (BCEnCs) leads to guttae development in the extracellular matrix (ECM) secreted by the cells. The guttae are most prominent under conditions of high glucose and/or low oxygen, which are environments that favor glycolysis over oxidative respiration. The pathological ECM developed under these conditions does not favorably support the growth of non-diseased human CEnCs. The central hypothesis of this proposal is that a common pathway leading to the development of guttae in the ECM of CEnCs is glycolytic metabolism from mitochondrial dysfunction. Furthermore, the composition of the pathological ECM does not support the growth and metabolism needs of healthy CEnCs. The objectives of the proposed studies are to identify the metabolic pathways that contribute to guttae formation and to understand how pathological ECM disrupts CEnC function. The objectives will be achieved with three Specific Aims. Aim 1 is to identify alterations in ECM composition resulting from oxygen stress in CEnCs, and to compare those alterations to changes in FECD patient specimens. The composition of BCEnC- secreted ECM, non-diseased human Descemet’s membrane, and patient FECD specimens will be compared with quantitative proteomics analyses. Aim 2 is to discover CEnC stressors that lead to the formation