Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Pharmacological targeting of the RAGE-NFκB signalling axis impedes monocyte activation under diabetic conditions through the repression of SHP-2 tyrosine phosphatase expression
M. Dorenkamp1, M. Nasiry1, S. Koch1, D. Semo1, I. Löffler2, G. Wolf2, H. Reinecke1, R. Godfrey1
1Klinik für Kardiologie I: Koronare Herzkrankheit, Herzinsuffizienz und Angiologie, Universitätsklinikum Münster, Münster; 2Klinik für Innere Medizin III, Universitätsklinikum Jena, Jena;
Purpose: Monocytes play a vital role in the development of cardiovascular diseases. Type 2 diabetes mellitus (T2DM) is a major CVD risk factor by inducing monocyte activation. T2DM-induced aberrant activation and enhanced migration of monocytes is a vital pathomechanism that leads to atherogenesis, thereby promoting CAD and PAD. The tyrosine phosphatase SHP-2 was found to be upregulated in diabetic monocytes.  However, the exact mechanisms leading to SHP-2 upregulation in hyperglycemic monocytes are unknown and was, therefore, investigated in the present study.

Methods: Primary human monocytes were isolated from non T2DM, T2DM patients, wt and T2DM mice. Non T2DM 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. The release of inflammatory cytokines was detected using ELISA. To understand the functional relevance of the RAGE-NFκB-SHP-2 pathway in mediating monocyte activation, pharmacological inhibitors for SHP-2, RAGE or NFκB were used.

Results: Monocytes from T2DM patients revealed an elevated SHP-2 expression (1.29-fold, p=0.012). Under normoglycemic conditions, the serum from T2DM patients strongly induced SHP-2 expression, indicating that the T2DM serum itself contains critical factors that directly regulate SHP-2 expression. Interestingly, incubation with TNFa potentiated SHP-2 expression in monocytes (2.9-fold, p=0.0014). In line with this, we detected a positive correlation between TNFα and SHP-2 transcript levels in T2DM monocytes. Monocytes exposed to methylglyoxal (2.3-fold, p<0.001) or AGE mimetic, AGE-BSA (3.9-fold, p=0.003), revealed an elevated SHP-2 expression. Co-treatment of these molecules with an NFκB inhibitor or genetic inhibition of p65 blocked SHP-2 upregulation. The pharmacological inhibition of RAGE was sufficient to block MG- or AGE-BSA-induced SHP-2 expression and activity in monocytes. Confirming the importance of RAGE-NFκB signalling in regulating SHP-2 expression, elevated binding of NFκB to the SHP-2 promoter, induced by MG or AGE-BSA, was reversed by RAGE and NFκB inhibition. Besides, we also detected elevated RAGE levels in human (1.32-fold, p=0.024)  and murine (1.16 fold, p=0.014) T2DM monocytes Importantly, MG (1.7-fold, p=0.003) and RAGE-ligand AGE-BSA (1.9-fold, p=0.012) treatment of non-T2DM monocytes phenocopied the aberrant pro-migratory phenotype of T2DM monocytes (1.43-fold, p<0.001), which was entirely reversed by either SHP-2- or RAGE inhibitor.

Conclusions: This study identified the upstream signalling mediators contributing to SHP-2 dependent monocyte activation in T2DM conditions. We found that the glucose metabolite (MG) or RAGE ligand (AGE-BSA) alone were sufficient to induce a pro-migratory phenotype in monocytes by upregulating SHP-2 via the RAGE-NFκB signalling axis. Moreover, pro-inflammatory cytokine TNFα expression was correlated with enhanced SHP-2 levels in T2DM monocytes and could robustly induce SHP-2 activation in vitro. Finally, our data reveal the pharmacological inhibition of the identified RAGE-NFκB-SHP-2 axis as a potential new therapeutic approach to impede the T2DM driven, SHP-2-dependent pro-inflammatory monocyte activation.

https://dgk.org/kongress_programme/jt2022/aP462.html