Endothelial JAM-A upregulation induced by diabetes mellitus leads to an enhanced and rapid transmigration of CD14++ monocytes

Dilvin Semo (Münster)1, S. Vieth (Münster)1, M. Dorenkamp (Münster)1, I. Löffler (Jena)2, H. Reinecke (Münster)1, A. Zarbock (Münster)3, R. Godfrey (Münster)1

1University Hospital Münster Vascular Signalling, Molecular Cardiology, Department of Cardiology I - Coronary and Peripheral Vascular Disease, Heart Failure Münster, Deutschland; 2University Hospital Jena Department of Internal Medicine III Jena, Deutschland; 3University Hospital Münster Department of Anaesthesiology, Intensive Care and Pain Medicine Münster, Deutschland


Diabetes mellitus is one of the most costly cardiovascular risk factors due to its high prevalence, with more than 540 million people worldwide suffering from it and higher rates of cardiovascular disease and death in this cohort.
Diabetes mellitus leads to endothelial dysfunction that appears to be oxidative stress-dependent. Monocytic recruitment and accumulation are enhanced in diabetes mellitus; among many other cells and molecules involved in atherosclerosis, monocytic accumulation drives local inflammation and, thus, atherosclerosis development. Since previous studies described a role of the interplay between endothelial JAM-A (junctional adhesion molecule A) and leukocyte LFA-1 (lymphocyte function-associated antigen 1) for leukocytic transendothelial migration (TEM), we investigated the potential functional role of JAM-A for monocytes in the context of diabetes mellitus.

Human coronary artery endothelial cells (HCAEC) were exposed to the serum of non-diabetic (nonDM) patients or the serum of patients with type II diabetes mellitus (T2DM) for 48 hours. The expression levels of endothelial surface JAM-A and soluble JAM-A were measured by FACS and ELISA. HCAEC were also pre-conditioned in hyperglycemia (HG).
Flow assays were performed for functional analysis. After the knockdown of JAM-A using a silencing RNA approach, a confluent HCAEC monolayer was exposed to T2DM patient serum. The monolayer was then perfused with primary human monocytes (CD14++ monocytes) isolated from healthy controls under physiological flow conditions found in microvessels. In a second approach, the surface expression of JAM-A was enhanced by plasmid DNA transfection into HCAEC. The confluent HCAEC monolayer was perfused with monocytes, and cell-cell interaction during the flow assay was examined by phase contrast microscopy and analyzed by single-cell tracking using Image J software.

Exposure of HCAEC to T2DM patient serum resulted in a twofold increase in surface JAM-A expression and increased soluble JAM-A (by 70%). Mimicking an HG milieu by HG pre-conditioning of HCAEC increased JAM-A expression.
Knockdown of endothelial JAM-A resulted in up to 70% reduced transmigration capacity of CD14++ monocytes, although adhesion rates were unaffected. Single cell tracking revealed a delayed initiation of transmigration after adhesion in JAM-A knockdown. Furthermore, reduced endothelial JAM-A expression directly affected TEM itself, resulting in a significantly prolonged TEM phase. Similar data were obtained by preincubation of monocytes with recombinant human JAM-A.
Overexpression of JAM-A induced a faster paracellular TEM of monocytes along HCAEC. Not only was TEM initiated earlier after adhesion, but the TEM period itself was also shortened. Blocking JAM-A on the endothelium reversed the observed effects. 

Our study demonstrates for the first time an association between DM-induced JAM-A upregulation and the quality of transendothelial migration of CD14++ monocytes. JAM-A upregulation enhances the transmigration capacity of monocytes and induces rapid monocyte transmigration. Because of the known impact of monocyte migration on atherosclerosis, it could be considered that the elevated JAM-A levels in DM promote faster and increased monocyte accumulation, thereby further promoting atherosclerosis.


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