Purpose: The number of patients suffering from Diabetes mellitus type 2 (T2DM) is still rising which is directly related to cardiovascular pathologies like coronary (CAD) and peripheral artery disease (PAD). Diabetes-induced aberrant activation of monocytes is an important pathomechanism leading to impaired arteriogenesis and enhanced atherosclerosis, thereby, promoting CAD and PAD. The tyrosine phosphatase SHP-2 was found to be upregulated in diabetic monocytes. However, the pathway regulating SHP-2 expression in diabetic monocytes is not explored and was, therefore, investigated in the present study.

Methods: Primary human monocytes were isolated via negative immunological magnetic isolation from peripheral blood of T2DM patients and healthy individuals. Monocytes were incubated with Methylglyoxal (MG), a highly reactive side product of glycolysis, Receptor for advanced glycation end product (RAGE) ligand AGE-bovine serum (AGE-BSA) or TNFα for 24 hours. Transwell migration assays were used to study monocyte migratory potential. Western Blot, RT-qPCR and FACS were performed to quantify the expression of relevant molecules. To understand the functional relevance of the RAGE-NFκB-SHP-2 pathway, pharmacological inhibitors for SHP-2, RAGE or NFκB were used.

Results: Significantly enhanced SHP-2 expression was detected via Western Blot in monocytes, which were incubated with TNFα (2.9-fold, p=0.0014), MG (2.3-fold, p<0.001) or AGE-BSA (3.9-fold, p=0.003), respectively.  Co-incubation of these molecules with NFκB-inhibitor blocked SHP-2 upregulation. Moreover, the pharmacological inhibition of RAGE reversed the MG or AGE-BSA induced SHP-2 expression and activity in monocytes. Of note, RAGE expression on monocytes was enhanced after incubation with MG (1.5-fold, p=0.026) or AGE-BSA (1.2-fold). Consistent with this, we detected elevated RAGE mRNA levels in T2DM monocytes (1.32-fold, p=0.024). Besides, we detected augmented levels of SHP-2 mRNA in monocytes of T2DM patients (1.26-fold, p=0.032) which was more pronounced in monocytes with elevated TNFα expression. Furthermore, MG (1.7-fold, p=0.003) and RAGE-ligand AGE-BSA (1.9-fold, p=0.012) provoked the enhanced migration of monocytes which could be significantly reduced after the application of SHP-2 inhibitor. Interestingly, pharmacological inhibition of RAGE in these conditions alone was sufficient to block enhanced monocyte migration. Moreover, monocytes from T2DM patients revealed a comparable pro-migratory phenotype ex vivo, which was completely restored after the pharmacological inhibition of SHP-2.