Functional Differences Between Typical and Multinucleated Endothelial Cells Under Low-Density Lipoprotein Exposure

TL;DR

This study compares how typical and multinucleated endothelial cells respond to LDL, finding that multinucleated cells accumulate more cholesterol and show increased inflammation.

Contribution

The study reveals functional differences in lipid handling and inflammation between typical and multinucleated endothelial cells under LDL exposure.

Findings

MVECs accumulate more cholesterol than TECs after LDL exposure.

MVECs show elevated proinflammatory gene expression and reduced antioxidant and barrier-related gene expression.

Secretome analysis shows altered protein secretion in MVECs, including reduced vWF and increased t-PA.

Abstract

Endothelial cells are key regulators of vascular homeostasis, and their dysfunction plays a central role in the development of atherosclerosis and other cardiovascular diseases. Multinucleated variant endothelial cells (MVECs) have been described in pathological vascular regions; however, their functional properties remain poorly characterized. The aim of the present study was to compare lipid handling, inflammatory activation, barrier-associated features, and secretory profiles of typical endothelial cells (TECs, EA.hy926 line) and MVECs under low-density lipoprotein (LDL) exposure. MVECs were generated by polyethylene glycol-induced fusion of EA.hy926 cells and incubated with LDL under standardized conditions. Intracellular cholesterol accumulation was assessed biochemically, cytokine secretion was quantified by ELISA, gene expression of inflammatory, endothelial, junctional, and vasoactive markers was analyzed by quantitative real-time PCR, and the endothelial secretome was characterized using data-independent acquisition liquid chromatography–tandem mass spectrometry (DIA-LC-MS). MVECs demonstrated enhanced cholesterol accumulation compared with TECs following LDL exposure. At the transcriptional level, MVECs were characterized by elevated basal expression of proinflammatory markers, including IL1B, IL6, and NFKB1, and showed a markedly amplified IL6 and IL8 response to LDL. In parallel, MVECs exhibited reduced expression of genes associated with antioxidant defense (SOD1), barrier integrity (TJP1), and hemostatic function (VWF). Consistent with transcriptional data, mass spectrometry-based secretome analysis revealed decreased secretion of von Willebrand factor (vWF), vascular endothelial growth factor C (VEGFC), and endothelin-1 (EDN1) by MVECs, accompanied by increased secretion of tissue-type plasminogen activator (t-PA). Functional enrichment analysis of secretome-associated proteins highlighted pathways related to extracellular matrix–receptor interaction, focal adhesion, cell adhesion molecules, complement and coagulation cascades, and leukocyte transendothelial migration. In contrast, TECs demonstrated a more pronounced transcriptional response in EDN1, consistent with their role in vascular tone regulation. Immunocytochemical analysis further revealed altered subcellular distribution of the tight junction protein ZO-1 in MVECs, indicating junctional destabilization. Taken together, these results indicate that MVECs represent a distinct endothelial phenotype characterized by enhanced lipid accumulation, sustained proinflammatory activation, altered secretory signaling, and reduced barrier and hemostatic potential. Such features suggest that MVECs may contribute to the maintenance of chronic endothelial dysfunction and vascular inflammation under conditions of lipid overload.

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