Project Summary / Abstract The primary mechanism responsible for many acute vascular disorders, including in-stent restenosis, vein graft disease, and cardiac allograft arteriopathy, is pathological vascular smooth muscle cell (VSMC) activation. VSMC activation leads to neointimal formation and re-occlusion of injured blood vessels. Strategies to prevent abnormal VSMC remodeling, such as cell growth inhibitors used in drug eluting stents, have the undesired effect of impairing re-endothelialization. This attenuation of endothelial healing increases the risk of neoatherosclerosis and late stent thrombosis. In order to prevent pathological VSMC growth, but not re- endothelialization, there is a critical need to identify cell-surface proteins that may can be targeted by agents to distinguish between VSMCs and endothelial cells (ECs) and modulate cellular processes utilized by VSMCs during pathological remodeling that do not affect endothelium healing. The objective of this proposal is to utilize VSMC-targeting aptamers that specifically modulate VSMC migration, proliferation and apoptosis to define 1) define the cell-surface proteins and mechanism of action by which VSMC-targeting aptamers modulate VSMC but not EC processes; and 2) determine the impact of these cell- and process-specific ligands on neointimal formation and re-endothelialization following acute vascular injury. We recently identified a VSMC-specific anti- migratory aptamer that prevents VSMC migration with no effect on EC migration. We determined that the VSMC anti-migratory aptamer operates by antagonizing PDGFR-β activation, but has no effect on PDGF-BB- mediated VSMC proliferation. These data suggest the novel concept that PDGFR-β migration and proliferation signaling may be dissociated. We have now identified two additional VSMC-targeting aptamers that modulate VSMC, but not EC, proliferation and apoptosis through unknown mechanisms originating at the cell surface. We will test the overall hypothesis that VSMC cell surface proteins and signaling pathways necessary for migration, proliferation and apoptosis following acute vascular injury may be cell-specifically modulated to prevent neointimal formation without altering EC re-endothelialization. In Aim 1, we will define the mechanism by which VSMC PDGFR-β dependent migration is dissociated from PDGFR-β dependent proliferation and may be inhibited to prevent neointimal formation without interfering with re-endothelialization. Aim 2 will identify the cell-surface protein, using a novel application of a whole-genome CRISPR library, and define the mechanisms where VSMC proliferation may be specifically inhibited in VSMCs to prevent neointimal formation without impacting re-endothelialization. Aim 3 will determine the mechanism and cell surface-proteins by which VSMC apoptosis may be induced to limit neointimal formation while preserving re-endothelialization. Completion of this study will result in a mechanistic understanding of modu...