PROJECT 4 SUMMARY Mechanisms of pyrophosphate dysregulation. Hydroxyapatite deposition in bones and teeth is a carefully orchestrated biochemical process aimed at balancing the concentrations of inorganic pyrophosphate (PPi), a potent calcification inhibitor, and the local concentrations of phosphate (Pi), a calcification promoter, to establish a proper PPi/Pi ratio to enable regulated tissue mineralization to take place. While we have a good understanding of what molecules control physiological skeletal and dental mineralization, limited data exists as to whether alterations in those same mechanisms lead to ectopic calcification in the eyes, brain, and vasculature in diseases of aging such as age-related macular degeneration (AMD), Alzheimer's disease (AD) and atherosclerosis. AMD is clinically characterized by the presence of extracellular deposits known as drusen, located between the retinal pigment epithelium (RPE) and the choroidal microvasculature in the area known as the Bruch’s membrane in the macular region. Drusen is also observed in the normal aging eye associated with local inflammation, and the presence of a high number of large drusen in the macular region is a significant risk factor for developing AMD. While there are no murine models that fully reflect the pathophysiology of human AMD, the Abcc6-/- and the Enpp1-/- mouse models of pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) respectively, manifest not only vascular and brain calcification but also calcifications in the eyes and therefore these mouse models can be used as surrogates to understand drusen formation. In this PO1 component we will test the overarching hypothesis that dysregulation of the PPi/Pi ratio underlies the pathophysiology of ectopic calcification in diseases of aging, focusing our studies primarily on eye calcification. Given that tissue-nonspecific alkaline phosphatase (TNAP) is the major pyrophosphatase controlling the PPi/Pi ratio, TNAP may be a useful druggable target for the prevention/amelioration of drusen formation. Thus, the goals of this Project 4, designed to be mutually supportive with the other PO1 components, are to probe the causative role of PPi dysregulation in eye calcification by genetically manipulating the production and degradation of PPi in vivo. We will investigate if the PPi/Pi ratio is being controlled (either in the choroid endothelium, RPE cells or both compartments) via the production and function of a special type of extracellular vesicles known as matrix vesicles (MVs), where hydroxyapatite starts to form during physiological skeletal/dental mineralization, or if this ratio is controlled via the pathophysiological upregulation of TNAP activity alone. We will also conduct pharmacological proof-of-concept studies aimed at affecting calcification in the eye via modulating the production and degradation of systemic and local PPi concentrations. Completion of these studies, within the context...