Posttranslational control of VSMC identity and vascular health

The phenotypic switching of vascular smooth muscle cells (VSMCs) from a contractile to a proliferative state is critical in the development of occlusive vascular diseases, such as coronary artery disease (CAD). This process is traditionally understood to be regulated by transcriptional mechanisms involving the serum response factor/myocardin (SRF/MYOCD) transcriptional complex. However, the role of protein post-translational modifications in this process remains largely unexplored. This application aims to determine the significance of neddylation, an emerging ubiquitin-like protein modification, in VSMC homeostasis and vascular health. Neddylation involves the attachment of one or more NEDD8 moieties to protein substrates through a NEDD8- specific enzymatic cascade, with NEDD8 activating enzyme 1 (NAE1) playing an indispensable role. Recent preclinical and animal studies have demonstrated neddylation's importance in embryonic development, organ homeostasis, and tumorigenesis. Despite this, the role of NAE1-mediated neddylation in smooth muscle biology remains poorly understood. Preliminary data reveal associations between NAE1 variants and cardiovascular events in humans. Deleting Nae1 in pan-SMCs of neonatal or adult mice results in severe visceral myopathy and premature death due to a pseudo-obstructive intestinal condition, suggesting the importance of neddylation in visceral SMCs. To further investigate neddylation in adult VSMCs, we developed inducible VSMC-specific NAE1 knockout mice. These mice exhibited significant vascular remodeling at baseline, including aneurysmal dilation, elastin lamina distension and breaks, collagen deposition, and spontaneous coronary artery remodeling. Additionally, under hypercholesterolemic conditions, the mutant mice developed advanced lesions with thin caps in coronary arteries and thoracic aortae-areas typically resistant to lesion formation, ultimately leading to death a few months after high-fat diet feeding. These striking vascular phenotypes underscore the previously unrecognized role of VSMC neddylation in vascular health and disease and highlight the imperative need to elucidate the underlying mechanisms. Guided by our preliminary data, we hypothesize that protein neddylation preserves VSMC homeostasis by maintaining the contractile gene program and preventing cell death. To test this, we propose two aims. Aim 1 will explore how neddylation regulates the SRF/MYOCD axis to establish and maintain the VSMC contractile phenotype at baseline. Aim 2 will investigate how neddylation controls VSMC metabolism and suppresses VSMC ferroptosis to prevent lesion formation in both large and small arteries under hypercholesterolemic conditions. Unique genetic mouse models, pharmacological inhibitors, multi-omics, lineage tracing reporters, and sophisticated biochemical and molecular analyses will be used to rigorously examine the proposed mechanisms in vitro and in vivo. Completing the proposed studies will uncover the mechanisms controlling VSMC phenotypic plasticity and create opportunities to target VSMC neddylation to prevent and treat vascular diseases.