Investigating Murine Double Minute-2 Pro-Angiogenic Function in Health and Disease: An Integrative Approach from Rodent and Human Skeletal Muscle to Primary Endothelial Cells

Capillaries are essential for overall skeletal muscle health, transporting nutrients and oxygen as well as removing waste from muscle cells. Angiogenesis is the process of capillary growth from pre-existing capillaries, and is regulated by a balance of pro- and anti-angiogenic factors. Our laboratory has demonstrated that Murine Double Minute-2 (Mdm2) is essential for skeletal muscle capillary maintenance as well as for exercise-induced angiogenesis. The aim of my research was to further investigate Mdm2 function in both physiological (exercise) and pathological (diabetes) contexts at the tissue level of the skeletal muscle and in primary endothelial cells. I first contributed to show that Mdm2 protein levels are elevated following a prolonged exercise training program in human skeletal muscle (academic research paper 1). Next, I investigated the response of Mdm2 to an acute bout of exercise, which itself represents a powerful pro-angiogenic stimulus (academic research paper 2). Muscle contractile activity stimulates Mdm2 phosphorylation on its serine 166 concomitant with increased pro-angiogenic vascular endothelial growth factor-A (VEGF-A). Mdm2 phosphorylation was found to be dependent on VEGF-A signaling, demonstrating for the first time that Mdm2 and VEGF-A can interact in a complex regulatory loop. In vitro experiments show that VEGF-A-dependent activation of Mdm2 leads to increased migratory activity of endothelial cells. This effect appeared to be a result of enhanced Mdm2-FoxO1 binding, resulting in the inhibition of FoxO1-dependent regulation of thrombospondin-1 expression, a potent anti-angiogenic molecule. I further explored this relationship between VEGF-A and Mdm2 in academic research paper 3, demonstrating that VEGF-driven Mdm2 phosphorylation was dependent on extracellular signal related kinases 1/2 (ERK1/2) - p90 ribosomal s6 kinase (p90RSK) in primary human endothelial cells. Finally, academic research paper 4 provided evidence that Mdm2 is a clinically relevant protein in type 1 diabetes (T1D), a highly understudied disease in the context of skeletal muscle angio-regulation. Mdm2 protein abundance was lowered in muscles from T1D animals alongside increases in key anti-angiogenic Mdm2 targets and significant capillary regression. Taken together, my dissertation research clearly identifies Mdm2 as a master regulator of angio-adaptation at the level of the skeletal muscle and endothelial cells in both health and disease.