Abstract Body: Introduction: Vascular remodeling is a critical pathological process in atherosclerosis or after vascular injury due to angioplasty or stent placement. Smooth muscle cell (SMC) phenotypic switching is a key factor, but how mitochondrial activities contribute to SMC phenotypes is not well understood.
Hypothesis: We hypothesized that mitochondrial respiration is essential in SMC proliferation and migration.
Methods: To study SMC mitochondrial respiration in vitro, we inactivated mitochondrial respiratory Complex I (CI) by Cre-loxP targeting of the critical NDUFS4 subunit or by using CI inhibitors. In vivo, we studied mice homozygous for a Ndufs4^loxP allele, with SMC-specific Tagln-Cre for developmental studies and SMA-Cre^ERT2 for post-developmental inactivation. For the latter, we treated with tamoxifen at age 6 weeks to yield NDUFS4 iSMKO mice. Carotid artery ligation was performed to induce vascular injury.
Results: In cultured Ndufs4^-/- SMCs, we found lower overall CI expression and activity (P=0.0027), plus decreased respirasome assembly, ATP-linked oxygen consumption (P<0.0001), respiratory capacity, NAD⁺/NADH ratio (P=0.0035), aspartate levels, cell proliferation (P<0.0001), and migration (P= 0.0027). Ndufs4^-/- SMCs showed increased mitochondrial fragmentation (P<0.0001); interestingly, inhibiting mitochondrial fission or promoting mitochondrial fusion restored both proliferation and migration, and similar effects were noted in CI inhibitor-treated human SMCs. In vivo, NDUFS4 was highly expressed in the arterial wall during embryogenesis and in cells forming the neointima after arterial injury in adulthood. Mice lacking SMC NDUFS4 were born in Mendelian ratios, reached adulthood, and lacked an obvious phenotype up to 20 weeks of age; by electron microscopy, however, SMCs in the aortic media had altered mitochondrial ultrastructure. NDUFS4 iSMKO mice showed decreased neointima formation after vascular injury (intima/media ratio 57% lower; control, n=13; NDUFS4 iSMKO, n=14; P=0.02), with increased mitochondrial fragmentation in neointimal SMCs.
Conclusion: CI-dependent mitochondrial respiration is essential in SMC proliferation, migration, normal mitochondrial ultrastructure, and vascular remodeling in adulthood. Mechanistic studies suggest that mitochondrial respiratory CI function promotes SMC proliferation and migration by supporting the biosynthesis of aspartate and ATP, maintaining NAD⁺/NADH balance, and limiting mitochondrial fragmentation.