Improving Induced Pluripotent Stem Cell-Derived Endothelial Cells as Therapy for Peripheral Arterial Disease

Veterans experience peripheral arterial disease (PAD) onset at an earlier age than their non-Veteran counterparts and are at increased risk of experiencing adverse outcomes from this disease. Advanced PAD is a significant cause of morbidity, including major amputation. While endovascular and surgical treatment options have advanced, often patients are limited to palliation consisting of pain control and major amputation, both of which lead to significant morbidity and disability. There remains a great need for improved therapeutic modalities to prevent limb loss. Advanced PAD is a disease of aging, driven by age-related mitochondrial dysfunction, or mitochondriopathy, and resulting loss of innate vascular regenerative function. For patients facing major amputation, vascular regenerative therapies from combined pharmacologic treatments and exogenous stem cells present a novel avenue for therapy to improve function and prevent major amputation. Prior work demonstrated the potency of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) in accelerating vascular regeneration in murine ischemic hindlimbs. Our prior work demonstrated improved function of iPSC-ECs with Rg2-mediated stimulation of mitophagy. The renewability and scalability of iPSC-based technology provides an advantageous platform for cell therapy to treat ischemic PAD limbs. In Aim 1, we hypothesize that Rg2 treatment of aged murine aortic ECs will act through the HIF-1α/VEGF-A pathway to similarly reverse native EC mitochondriopathy and support sprouting angiogenesis. We will use aged (22-25 month old) and young (5 month old) MtKeima mice, expressing the pH-sensitive dual-excitation fluorescent protein Keima, to track mitochondrial recycling in lysosomes in isolated cells and tissues. We will use harvested ECs from these mice to explore the impact of Rg2 on angiogenesis in vitro, and we will use the hindlimb ischemia (HLI) model on live aged mice to explore the impact of Rg2 on vascular regeneration in vivo. This aim will evaluate the effect of stimulating mitophagy to reverse mitochondriopathy and to restore regenerative capacity in native ECs. In Aim 2, we hypothesize that iPSC-EC survival in an ischemic foot will require a primed implantation bed, as well as embedded exogenous support, to achieve functional angiogenesis. To test the minimum necessary support, we will utilize an in vitro model of angiogenic sprouting with varying levels of nutritional support to assess iPSC-EC performance. We will then use the HLI technique in aged mtKeima mice and embed optimized iPSC- ECs in Rg2-primed and unprimed limbs. This aim will evaluate the combined requirements of pharmacologic and cellular therapy to treat advanced limb ischemia in a murine model of age-related ischemia. This CDA2 will provide me with the training and experience to apply advanced in vitro and murine models to optimize regenerative approaches for human clinical trials, to ultimately address this critically unmet clinical need among Veterans. This training will equip me to achieve VA Merit funding and further my career as a VA vascular surgeon-scientist.