Abstract Body: Heart attacks caused by a blocked coronary artery typically require surgery to either re-open the artery (stenting) or completely bypass the blockage with a graft. Vascular surgery damages the thin endothelial cell lining of the blood vessel, resulting in local inflammation and neointima formation from proliferating vascular smooth muscle cells (VSMC). This ultimately reduces long term surgical success rates. Immune cells, such as macrophages (Φ), are recruited early after blood vessel damage following stent placement, preceding neointima formation. However, little is known about the function of Φ during vascular repair following surgery. We aimed to determine if cell-to-cell interactions form between Φ and VSMC in vivo and identify if gap junctions, formed by connexin 43 (Cx43), play a key role in initiating VSMC proliferation and neointima formation. Permanent carotid ligations were performed in C57BL/6 mice to induce vascular injury and neointima formation. Carotids were harvested at 1-14 days post-injury to measure Φ accumulation and interactions with SMC in the vessel wall via histology, en face immunofluorescence, and electron microscopy (Transmission (TEM), and Serial Block Face Scanning (SBFSEM)). Interactions between Φ and VSMC were assessed in vitro using human coronary artery SMC co-cultured with THP-1 monocyte-derived Φ in single-well and transwell systems. Our in vivo investigation revealed that Φ primarily accumulated within 200-500μM of the carotid ligation 3 days post-injury, coinciding with areas of the greatest neointima burden observed 14 days post-injury. Confocal microscopy revealed Cx43 gap junctions at breaks in the inner elastic laminar between Φ and VSMC 3 days post-injury, which was similarly confirmed by TEM and SBFSEM. Direct VSMC-to-Φ gap junction communication was demonstrated in vitro by cell-to-cell calcein dye transfer in our Φ-VSMC co-culture model. Bulk RNA sequencing and 5-ethynyl-2'-deoxyuridine (EDU) incorporation in VSMC revealed induction of a proliferative phenotype when co-cultured under direct contact with Φ. Preliminary data suggest that CCR2⁺ macrophage-specific knockout of Cx43 may prevent neointima formation in mice. Overall, our data suggest that Φ accumulation occurs early in vascular injury and initiates VSMC proliferation and neointimal formation through direct Φ-VSMC gap junction communication.