Flow-based imaging reveals dissociated monocyte adhesion and transmigration patterns between TNF-α and diabetes-induced vascular inflammation.

Monocyte-endothelial interactions drive both acute inflammation and chronic vascular disease in type 2 diabetes mellitus (T2DM), yet whether TNF-α-induced and diabetes-associated monocyte trafficking engage similar or distinct molecular programs remains unclear. Using a physiological flow-based imaging system, we quantified CD14+ monocyte adhesion, transendothelial migration (TEM), and abluminal residence on HUVEC monolayers activated with TNF-α or T2DM serum under controlled shear stress. Both stimuli induced comparable monocyte adhesion (~40 cells by 3 min), but TEM efficiency diverged markedly: TNF-α promoted robust TEM (~18% by 5 min) whereas T2DM conditions showed severely impaired TEM (~5%). Under T2DM conditions, monocytes exhibited prolonged abluminal retention (median 85 min vs. 25 min; p<0.0001), a phenotype recapitulated by endothelial hyperglycemic exposure alone. Comparative RNA-seq analysis of T2DM (GSE92724) and TNF-α-stimulated (GSE134489) endothelial cells revealed near-zero transcriptional correlation (r = 0.018) between conditions. TNF-α drove coordinate NF-κB-dependent upregulation of VCAM1, ICAM1, E-selectin, and junctional molecules with organized junctional remodeling, whereas T2DM produced VCAM1-biased adhesion with ICAM1 downregulation (0.61-fold), claudin suppression, and junctional disorganization. KEGG pathway mapping confirmed organized endothelial-leukocyte integrin co-activation under TNF-α versus discoordination junctional loss under T2DM. qPCR validation identified selective RAGE and JAM3 upregulation under T2DM-consistent with AGE-RAGE signaling and JAM-3-MAC-1 monocyte trapping-without classical NF-κB activation. These findings define two fundamentally distinct paradigms: a TNF-α "recruitment model" enabling efficient trafficking and resolution, versus a T2DM "retention model" characterized by VCAM1-biased adhesion, junctional disorganization, and impaired reverse transmigration driving chronic monocyte accumulation, identifying AGE-RAGE signaling and JAM-3-MAC-1 interactions as potential therapeutic targets for diabetic vascular inflammation.