Comparative Analysis of Endothelial Cell Culture Models Under Altered Mechanical Conditions and Glucose Variations

Endothelial dysfunction is a defining feature of diabetic vascular disease and is characterized by impaired nitric oxide signaling, inflammatory activation, altered mechanotransduction, and disturbed angiogenic responses. The present study investigated whether hyperglycemia modulates endothelial phenotype in a model-dependent manner under distinct structural and mechanical culture conditions. EA.hy926 endothelial cells were cultured for 7 days under normoglycemic (5.5 mM) or high-glucose (25 mM) conditions as static monolayers (G-force = 1 g), adherent clinorotated cells, multicellular spheroids (MCSs) generated during clinorotation, and Matrigel-derived endothelial structures. Gene expression was assessed by qPCR using marker panels related to nitric oxide signaling, PI3K CA-AKT-mTOR signaling, inflammatory adhesion, angiogenesis, and structural adhesion, whereas protein abundance and spatial distribution of eNOS, AKT1, VCAM1, vinculin, and VEGFA together with CDH5/VE-cadherin were analyzed by Western blotting and confocal microscopy. Clinorotation generated both adherent endothelial cells and MCSs. High glucose reduced eNOS-related expression in most models, with the strongest decreases in adherent clinorotated cells and MCSs, whereas Matrigel cultures showed a divergent transcriptional response. PI3K CA expression was markedly suppressed by high glucose in clinostat-derived populations, while mTOR was differentially regulated in spheroids and Matrigel cultures. VCAM1 expression was most prominent in MCSs, whereas ICAM1 was highest in Matrigel cultures. VEGF-related signaling differed substantially among models, and Matrigel cultures showed the strongest VEGFA-associated protein signal and the clearest angiogenic organization under normoglycemic conditions, which became less distinct under high glucose. Vinculin protein abundance was highest in MCSs and Matrigel cultures, reflecting pronounced differences in structural organization. Overall, these findings show that endothelial responses to hyperglycemia are strongly shaped by mechanical and structural context and support the use of complementary in vitro models for studying diabetic endothelial dysfunction.