https://doi.org/10.1007/s00392-025-02625-4
1Department of Cardiology I - Coronary and Peripheral Vascular Disease, Heart Failure, University Hospital Münster, Vascular Signalling, Molecular Cardiology Münster, Deutschland; 2Department of Anaesthesiology, Intensive Care and Pain Medicine, University Hospital Münster Münster, Deutschland
Introduction Type 2 diabetes mellitus (T2DM) remains one of the main cardiovascular risk factors for atherosclerosis. Not only is its prevalence increasing drastically worldwide, but it also increases cardiovascular mortality. DM plays a major role in the pathobiology of atherosclerosis by driving chronic inflammation and the subsequent accumulation of leukocytic cells. It remains unclear how the accumulation of monocytes in atherosclerotic lesions is driven and what the impact of endothelial junctional adhesion molecules (JAM) is. We aimed to investigate the impact of DM on JAM-C and whether JAM-C could contribute to monocyte accumulation.
Methods: Human coronary artery endothelial cells (HCAEC) were stimulated with serum of non-diabetic (non-DM) patients or serum of patients with T2DM. Moreover, HCAECs were pre-conditioned in hyperglycemia (HG) or metabolic stress condition (HG/oxLDL). Afterwards, the expression of endothelial surface JAM-C and soluble JAM-C was assessed by FACS and ELISA. Functional analyses were carried out using flow assays. Either a knockdown (KD) of JAM-C using a silencing RNA approach or overexpression (OX) of JAM-C by plasmid DNA transfection was performed in HCAEC. Cells were seeded as confluent monolayer and exposed to T2DM patient serum. Afterwards, the endothelium was perfused with primary human CD14++ monocytes obtained from healthy donors under physiological flow conditions found in microvessels. Monocytic-endothelial interaction was monitored in real-time by phase contrast microscopy and analysed by single-cell tracking. Furthermore, we investigated the levels of JAM-C in CD14++monocytes after hyperglycemic pre-conditioning using FACS and qPCR approaches.
Results: In our study, we detected a significant elevation of JAM-C on HCAEC after exposure to T2DM patient serum (2-fold, p=0.0052). We were able to mimic these effects by pre-conditioning HCAEC with cell culture medium containing 25 mM glucose (HG) and 50 µM oxLDL (1.6-fold, p=0.0449). Cleavage of JAM-C from the surface of HCAEC was not affected by DM-conditioning. HG did not alter monocytic JAM-C. Observation of EC-monocytic interaction under physiological flow conditions after KD of endothelial JAM-C revealed no effect of JAM-C on adhesion or transendothelial migration (TEM) rates. KD of JAM-C resulted in a significantly prolonged transendothelial migration phase (Ctrl: mean 4.3 minutes, JAM-C KD 4.9 minutes; p<0.001). KD of JAM-C shortened the time phase of monocytes in the abluminal compartment before reverse migration (Ctrl: mean 105 min, JAM-C kd 79 min, p<0.0001). Furthermore, KD led to a faster monocytic reverse migration phenotype (Ctrl: mean 3.3 minutes, JAM-C KD: 1.4 min, p<0.0001). We obtained opposite results by overexpressing JAM-C. OX resulted in a prolonged TEM phase (Ctrl: 4.3 minutes, JAM-C OX: 6.2 minutes, p=0.006) and time in the abluminal compartiment phase (Ctrl: 85 minutes, JAM-C ovex: 151 minutes, p<0.0001). Furthermore, the reverse migration phase itself was prolonged (Ctrl: 1,9 minutes, JAM-C OX: 4,8 minutes, p=0.0003).
Conclusions: Our study demonstrates for the first time that T2DM increases endothelial JAM-C, which directly results in a dysfunctional reverse migration phenotype in CD14++ monocytes. Given the known role of monocyte migration in atherosclerosis, it is likely that elevated JAM-C levels in DM promote an increase in monocyte accumulation, thereby further driving local inflammation and atherosclerosis.