ABSTRACT: BACKGROUND Intimal hyperplasia (IH) significantly limits the long-term patency of saphenous vein grafts following bypass surgery, with no reliable human-relevant models available to fully understand its complex pathogenesis. Previously, we established a static ex vivo culture model using human saphenous vein segments to study IH, demonstrating its translational value for evaluating potential therapeutic interventions. METHODS Human saphenous vein segments were cultured ex vivo for 7 days under static conditions. Histological and immunohistochemical analyses were conducted to evaluate endothelial dysfunction, vascular smooth muscle cell (VSMC) phenotype switching, extracellular matrix (ECM) remodeling, inflammation, and apoptosis. Spatial transcriptomics (GeoMx) were utilized to characterize the localized transcriptional alterations, which were subsequently validated using targeted qPCR, western blotting, and additional immunostaining techniques. RESULTS Cultured vein segments developed characteristic features of IH, including marked endothelial dysfunction, significant VSMC dedifferentiation toward synthetic and inflammatory phenotypes, increased apoptosis and proliferation, and extensive ECM remodeling. Spatial transcriptomics revealed distinct transcriptional signatures in different vascular wall regions, highlighting localized activation of inflammatory (IL-6, IL-1β, CXCL2), oxidative stress, and ECM remodeling pathways (MMP9, TIMP1). Importantly, we also observed evidence of osteochondrogenic differentiation of human VSMCs during IH, with significant upregulation of osteogenic markers such as RunX2 and osteopontin. CONCLUSIONS Our ex vivo human saphenous vein model accurately captures the complex molecular and cellular dynamics of IH, offering critical insights into endothelial dysfunction, VSMC plasticity, and osteochondrogenic transitions. This translational model holds significant promise for evaluating novel therapeutic strategies targeting graft IH.
