Friday, March 14, 2025
3/14/2025
PROJECT SUMMARY Vascular occlusive disease remains a critical cardiovascular health issue and results in about 500,000 percutaneous-coronary angioplasties annually in the US. Restenosis due to neointimal hyperplasia occurs after 10-30% of angioplasties and remains a critical problem in cardiovascular medicine. No new approaches to improve the standard treatment, the insertion of mTOR inhibitor-or taxol-eluting stents, have been developed in a decade. Addressing this critical problem will require novel strategies to inhibit proliferation of vascular smooth muscle cells (VSMCs), the main driver of neointimal hyperplasia. Our long-term goal is to determine how mitochondrial function modulates cytosolic signaling events in VSMCs and endothelial cells in vascular disease. The overall objective of the proposed research is to determine how the highly conserved GTPase MIRO1, which resides in the outer mitochondrial membrane, regulates VSMC proliferation and neointima formation and test new mechanistic therapies. MIRO1 is known to control intracellular trafficking of mitochondria. Nascent data suggest that MIRO1 may have additional functions beyond mitochondrial trafficking. MIRO1 associates with the mitochondria-ER contact sites (MERCS) that facilitate mitochondrial Ca2+ entry and maintains the organization of mitochondrial cristae that is pivotal for electron transport chain (ETC) activity. Yet, the functional consequences of MIRO1 binding are not fully understood, and the implications for human disease, in particular in vascular disease, have remained unknown. We previously reported that mitochondrial Ca2+ uptake and energy production are required for cell- cycle progression in VSMCs in G1/S transition and beyond. Our pilot studies reveal that proliferation in VSMCs with MIRO1 deletion is abolished starting at G1/S transition. Thus, we speculate that MIRO1 controls VSMC proliferation via two hitherto unrecognized processes. Thus, our central hypothesis is that MIRO1 is required for VSMC proliferation, and thus for neointimal hyperplasia, by controlling the mitochondrial-ER Ca2+ transit and ETC activity. This is supported by our strong pilot data demonstrating that MIRO1 deletion blocks neointimal hyperplasia, VSMC proliferation and ATP production. Our novel tools and assays put us in a perfect position to test our hypothesis, including VSMC lines that lack MIRO1 or express MIRO1 mutants, and innovative RNA-aptamer-based tools for targeting of VSMCs in vivo after vascular injury. Our specific aims are 1. to determine the extent to which MIRO1 regulation of Ca2+ transport at MERCS controls cell-cycle progression, 2. to establish the extent to which the control of cristae organization by MIRO1 regulates ETC activity and proliferation, and 3. to test whether VSMC-specific aptamers that deliver siMIRO1 effectively reduce neointima hyperplasia. The anticipated outcomes of the proposed study are knowledge of the mechanisms by which MIRO1 promotes VSMC proliferation, and of the potential of RNA-based aptamers with siMIRO1 activity in preventing restenosis after vascular injury.
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