Lipid-Induced Endothelial Dysfunction: Pro-Atherogenic Properties of Multinucleated Variant Endothelial Cells

Endothelial dysfunction is an early event in the development of cardiovascular diseases and is characterized by impaired barrier function, inflammatory activation of endothelial cells (ECs), and alterations in lipid metabolism. In addition to typical (mononuclear) endothelial cells (TECs), multinucleated variant endothelial cells (MVECs) are present within the vascular wall; however, their functional role remains poorly understood. The aim of the present study was to investigate the molecular and functional characteristics of MVECs and their potential contribution to the development of endothelial dysfunction. Primary human umbilical vein endothelial cells (HUVECs) were used, and multinucleated cells were generated by polyethylene glycol-induced fusion. Cells were incubated under control conditions or exposed to low-density lipoproteins (LDL; 100 µg/mL, 24 h). A comprehensive analysis was performed, including transcriptomic and proteomic (secretome) profiling using gene set enrichment analysis (GSEA), as well as functional assays assessing transendothelial LDL transport, intracellular cholesterol accumulation, macrophage migration, and the expression and secretion of pro-inflammatory cytokines (IL-6, IL-8). MVECs exhibited pronounced differences compared to TECs. GSEA revealed reduced enrichment of pathways related to canonical nuclear factor kappa B (NF-κB) signaling and negative regulation of NF-κB transcription factor activity, actin cytoskeleton organization, focal adhesion assembly, basement membrane organization, and vesicle-mediated transport in MVECs relative to TECs, indicating impaired cytoskeletal integrity, altered cell–matrix interactions, dysregulated inflammatory signaling, and reduced vesicular trafficking activity. Functionally, MVECs demonstrated an increased capacity for cholesterol accumulation and enhanced transendothelial migration of macrophages. Notably, transendothelial LDL transport across the MVEC monolayer was not increased, suggesting a predominance of intracellular lipid accumulation. MVECs also exhibited a pronounced pro-inflammatory phenotype, characterized by elevated expression and secretion of IL-6 and IL-8. Taken together, these findings indicate that MVECs represent a functionally altered endothelial phenotype with impaired barrier function, dysregulated lipid metabolism, and enhanced inflammatory activity. Local accumulation of MVECs within the vascular wall may contribute to the formation of pro-atherogenic regions and play a role in the initiation and progression of endothelial dysfunction.