SHP2 tyrosine phosphatase mediates diabetes-induced pro-atherosclerotic activation of monocytes through upregulation of the adhesion molecule CX3CR1

M. Dorenkamp (Münster)¹, W. Wang (Münster)², S. Vieth (Münster)², D. Semo (Münster)², M. Schwietzer (Münster)², M. Nasiry (Münster)², I. Löffler (Jena)³, G. Wolf (Jena)³, H. Reinecke (Münster)², R. Godfrey (Münster)^2
1Department of Cardiology I - Coronary and Peripheral Vascular Disease, Heart Failure, University Hospital Münster, Vascular Signalling, Molecular Cardiology Münster, Deutschland; ²Universitätsklinikum Münster Klinik für Kardiologie I: Koronare Herzkrankheit, Herzinsuffizienz und Angiologie Münster, Deutschland; ³Universitätsklinikum Jena Klinik für Innere Medizin III Jena, Deutschland

Purpose: Type II diabetes mellitus (T2DM) is an increasingly prevalent disease whose morbidity and mortality are mainly determined by the cardiovascular sequelae. These sequelae, such as myocardial infarction and stroke, are primarily caused by atherosclerotic processes, which are accelerated in type II diabetic patients. A pro-atherosclerotic activation of monocytes appears to be an essential pathophysiological component. Decoding the underlying signalling processes is the basis of this work.

Methods: Primary human monocytes were obtained from patients with and without T2DM using negative immunological magentic isolation (Milteny Biotec). Murine monocytes were isolated from the bone marrow of leptin-deficient, T2DM mice (db/db). Cell biological methods such as Western blot, FACS and qPCR were used to investigate relevant signalling pathways. Functional studies to determine monocyte migration and adhesion were performed using transwell migration assays and interactions of labelled monocytes with immobilised CX3CL1. Pharmacological inhibition of SHP2 tyrosine phosphatase (SHP2) was carried out using SHP099.

Results: Our studies demonstrated a significant increase of the adhesion molecule CX3CR1 on monocytes from T2DM patients as well as on monocytes from T2DM mice compared to healthy patients or wild-type mice. Interestingly, this effect could be induced in vitro solely by incubation with methylglyoxal (MG), a highly reactive by-product of glycolysis that is accumulated at elevated levels in diabetic patients. We further demonstrated that this MG effect is mediated by augmented expression and activation of SHP2 tyrosine phosphatase in monocytes. Thus, pharmacological inhibition of SHP2 prevented the increased expression of CX3CR1 on monocytes despite incubation with methylglyoxal. Interestingly, this was also be demonstrated at a functional level, as the elevated migration of diabetic monocytes towards CX3CL1, the cognate ligand for CX3CR1, was completely reversed by blocking SHP2. Furthermore, it was also shown that the enhanced adhesion of diabetic monocytes to immobilised CX3CL1 could also be completely inhibited by blocking SHP2. In addition, we were able to show that under additional inflammatory conditions, such as stimulation with TNFa or LPS, significantly enhanced SHP2 expression and consecutively increased CX3CR1 expression can be detected on monocytes. This effect of augmented adhesion potential of these inflammatory monocytes was also significantly attenuated by pharmacological inhibition of SHP2.

Conclusions: In the present study we demonstrate for the first time that the elevated expression and activity of SHP2 tyrosine phosphatase in diabetic monocytes directly leads to increased expression of the adhesion molecule CX3CR1 on diabetic monocytes. Excitingly, pharmacological inhibition of SHP2 alone can reverse the increased migration and adhesion potential of diabetic monocytes. These findings reveal a new potential therapeutic target to prevent the accelerated development of atherosclerosis and subsequent adverse cardiovascular diseases in patients with diabetes mellitus.