Abstract 524: Deletion of Methionine Sulfoxide Reductase A in Mice Increases Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia

Abstract

Introduction: Neointima formation is the leading cause of restenosis after balloon angioplasty and surgical endarterectomy. ROS production promotes neointimal hyperplasia and causes methionine oxidation. Recent data suggest that oxidation of methionines can alter specific protein activity and induce cell signaling. Methionine Sulfoxide Reductase A (MsrA) reverses methionine oxidation. In humans, two independent genome-wide association studies have found MsrA polymorphisms to correlate with increased ischemic cardiovascular disease. Hypothesis: MsrA protects against neointimal formation. Methods: Immunofluorescence for MsrA was performed in autopsy specimen of arteries with and without neointimal hyperplasia. 12-week old WT and MsrA-/- mice underwent ligation of the left common carotid artery and received two injections of BrdU prior to sacrifice. Fourteen days after ligation, the neointimal area was determined using Image J. Cell proliferation and apoptosis in the neointima were quantified by BrdU and TUNEL staining. Proliferation of vascular smooth muscle cells (VSMC) from MsrA-/- and WT mice was determined by cell counts and FACS analysis. Levels of cell cycle proteins, pAkt, pGSK3β were analyzed by immunoblot in MsrA-/- and WT VSMC, mRNA levels by qrtPCR. Results: MsrA was detected in all layers of the vascular wall and in neointima in humans. Neointimal area was significantly increased in MsrA-/- compared to WT mice 14 days post-ligation (n=5-9, p<0.05). Additionally, neointimas from MsrA-/- mice contained a higher percentage of proliferating cells (0.92±1.42% vs 8.89±8.89%, p=0.007). MsrA-/- VSMC displayed significantly increased proliferation compared to WT VSMC (p<0.05). Moreover, the progression to S phase was accelerated and protein levels of the cell cycle regulators Cyclin D and CDK4 were upregulated in MsrA-/- VSMC. However, cyclin D mRNA levels are not increased (p=0.24). This implies that MsrA post-translationally regulates cyclin D. GSK3β, which is inhibited by Akt, promotes cyclin D degradation. An increase in pAkt and pGSK3β was identified in MsrA-/- VSMC. Conclusion: These data indicate that MsrA regulates neointima formation after vascular injury and support a role for MsrA in the regulation of Akt signaling.