Tendinopathies are common injuries that typically result from accumulation of sub-rupture fatigue damage. We have developed an in vivo model of sub-rupture fatigue damage accumulation using the rat patellar tendon to investigate the onset and pathogenesis of tendinopathy. We found that just one bout of fatigue loading results in collagen matrix damage and a 20% stiffness loss that is not recovered out to at least 10-weeks. We also found that running exercise leads to repair when initiated 2-weeks after onset of sub-rupture fatigue injury but promotes further degeneration when initiated 1-day after onset of injury, uniquely positioning us to determine the underlying mechanisms necessary to develop therapeutics to transform everyday activity into a reparative stimulus. We identified glycosaminoglycans (GAGs), specifically hyaluronan (HA) and dermatan sulfate (DS), to be increased prior to initiation of therapeutic but not degenerative exercise. Postmortem depletion of GAGs showed that their increase after sub-rupture fatigue injury reduces matrix shear strain and increases dynamic modulus which are properties that are associated with modulation of proliferation, apoptosis, and αSMA differentiation. Investigation of the repair response from therapeutic exercise identified an increase in population of αSMA+ cells and integrin α5 (α5+/tenocytes and α5+/αSMA+ cells); an integrin that enhances the capacity of cells to withstand loads thereby preventing cell death. Our inhibition of αSMA+ cells in therapeutic exercise increased the area of high severity matrix damage. Blocking integrin α5 altered the morphology of αSMA and tenocytes and decreased damage area, further enhancing the therapeutic effect of exercise. We will test the hypothesis that (1) the increase in GAGs after onset of fatigue injury modulates the stressful mechanical environment of cells in damaged tendons resulting from subsequent loading, leading to an increase in population of αSMA+ cells and integrin α5 (Aim 1); (2) the increase in αSMA+ cells will largely decrease the area of high matrix damage and that αSMA+ cells mediate tissue repair (Aim 2); and (3) that integrin α5 protects cells form apoptosis in response to higher loading but promotes a catabolic response from the surviving α5+/tenocytes while enhancing the functionality of α5+/αSMA+ cells (Aim 3). We will deplete HA and DS in vivo prior to initiation of therapeutic exercise to determine their role in transforming loading into a reparative stimulus (Aim 1). Pharmaceuticals will be used to inhibit the population αSMA+ cells (using Simvastatin in Aim 2) and to block integrin α5 (using ATN- 161 in Aim 3) to interrogate their role in promoting repair of fatigue damaged tendons. scRNAseq will be used to compare the cell populations that are associated with repair versus degeneration of fatigue damaged tendons and determine the effect of inhibition of αSMA+ cells and blocking of integrin α5 on these cell populations. The proposed st...