CD40L-CD40-TRAF signaling plays a role in the progression of atherosclerosis and influences the pathogenesis of coronary heart diseases. According to our own previous data, we tested further the hypothesis that CD40L-CD40-TRAF signaling is a potential therapeutic target in hyperlipidemia, diabetes, and hypertension. In a mouse model of hyperlipidemia and diabetes (db/db mice), TRAF6 inhibitor treatment (2,5 mg/kg/d for 7 days via subcutaneously implanted osmotic pumps) led to a significant decrease of cardiac CD40L, p-47 Phox, NOX2, and eNOS protein expression compared to untreated db/db mice. In a second mouse model of arterial hypertension (C57BL/6 mice treated with 1 mg/kg/d angiotensin-II for 7 days), TRAF6 inhibitor treatment caused a significant reduction of MDA- and 3NT-positive proteins in plasma compared to TRAF6 inhibitor untreated hypertensive animals. Our results indicate that TRAF6 inhibition could be a therapeutic target in hyperlipidemia and hypertension by reducing the expression of pro-inflammatory and oxidative stress markers. The translational aspect of our study is based on the characterization of the CD40L-CD40-TRAF cascade and major markers of thrombosis, endothelial cell activation, oxidative stress, and inflammation in vascular bypass material and sera from patients with coronary artery disease (CAD) with diabetes (fasting blood glucose >150  mg/dl) and/or hypertension (resting systolic blood pressure >160 mg/dl, collaborative DZHK project). Our initial Olink targeted plasma proteomic analysis using the IMMUNO-ONCOLOGY panel enriched for components of the CD40-CD40L-TRAF signaling cascade showed a stepwise increase in markers of atherothrombosis and endothelial cell activation with each additional comorbidity for 24 protein targets, which represents a third of all detected proteins. In addition, specific gene clusters that correlate with the presence of the comorbidities were identified in isolated aortic mRNA of CAD patients through next-generation sequencing. Upon bioinformatical analysis of these omics data, key markers like NOX2, CD40L, CD68, and 3NT were validated on a quantitative basis using qRT-PCR and immunoblotting analysis. A stepwise increase of these markers was observed with each additional comorbidity in CAD patients' plasma. Analysis of the patient's sera and vascular bypass material for pro-oxidative, pro-inflammatory, and pro-thrombotic parameters were employed to understand the function of the CD40-CD40L-TRAF axis in diabetes and hypertension-associated cardiovascular disease.

Funding: S.D. was supported by a vascular biology research grant on "CD40L and inflammation in hypertension" of the Else-Kröner-Fresenius Foundation (2019_A110). C.K., E.L., and A.D. were supported by shared expertise grants of the DZHK (German Center for Cardiovascular Research), Partner Site Berlin, Partner Site Munich Heart Alliance and Partner Site Rhine-Main, Mainz, Germany. Christian Weber, Philipp Wild, Thomas Münzel are PIs of the DZHK (German Center for Cardiovascular Research), Partner Site Munich and Rhine-Main, Mainz, Germany.