Mitochondrial transfer via endothelial-derived extracellular vesicles as a potential therapy for vascular disease in MELAS - PROJECT SUMMARY Mitochondrial diseases are common inherited metabolic diseases affecting an estimated 1 in 5,000 people. The Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes (MELAS) syndrome is the most prev- alent mitochondrial disease; its cardinal symptom is stroke-like episodes (SLE). Vascular endothelial cell (EC) dysfunction is believed to cause SLE in MELAS. Currently there are no effective therapies for SLE. Cell-derived Extracellular Vesicles (EVs) constitute an important mechanism of intercellular communication, and lately have been implicated in horizontal transfer of mitochondria. Perturbations in numbers and content of circulating En- dothelial Cell-derived EVs (EC-EVs) have been reported in various cardiovascular disorders. In vitro studies have demonstrated either detrimental or restorative effects of EC-EVs on recipient cells. We hypothesize that the EV effects on target cells may be, at least in part, determined by the quality of their mitochondrial cargo. Preliminary data showed a significant percentage of medium/large EC-EVs to contain mitochondria (mitoEVs); mitoEVs from cytokine-treated ECs contain mostly depolarized mitochondria; and pretreatment of cytokine- treated ECs with a mitochondria-targeted antioxidant to result in mitoEVs with more polarized mitochondria. Upon incubation with naïve ECs, EVs from cytokine-stimulated ECs (not from control ECs) upregulated inflam- matory pathways. Our goal is to assess the contribution of mitoEVs in EC dysfunction, and the potential of mitoEVs derived from healthy ECs to ameliorate the inflammatory MELAS EC phenotype. In Aim 1, we will characterize the mitoEVs released from cytokine-stimulated ECs (including the quality/polarization of mitochon- drial cargo), the mechanisms that lead to mitoEV production, and the EV paracrine effects. The role of mitochon- drial cargo quality in EV paracrine effects will be assessed by either depolarizing the EV mitochondrial cargo, depolarizing the mitochondria in source ECs, or pretreating cytokine-stimulated ECs with mitoprotective agents, and, in either case, studying the EC-EV paracrine effects on target naïve ECs. By employing a MELAS iPSC- derived iEC model (different levels of heteroplasmy) in Aim 2, we will characterize the mitoEVs released from MELAS iECs, and the potential of mitochondrial transfer via EVs released from healthy iECs to improve function and ameliorate the inflammatory phenotype of MELAS iECs with high levels of heteroplasmy. In Aim 3, we will investigate the mitochondrial cargo quality in EC-EVs isolated from blood samples of MELAS patients and their contribution to EC dysfunction ex vivo. Our study will bridge benchtop to clinical practice by applying our in vitro findings on EV-mediated mitochondrial transfer towards understanding EC dysfunction and SLE pathogenesis, potentially suggesting a new therapeutic strategy for MELAS patients. The proposed work will take place under the supervision of co-sponsors Drs. Alevriadou, a vascular bioengineer (University at Buffalo-SUNY) and Kozicz, an expert in stem cell models of mitochondrial diseases (Mayo Clinic). Training is tailored for development as a physician-scientist in the field of clinical genetics, reflecting on the basic science research goals of the proposal.
