The interaction between CD40 and its ligand (CD40L) plays a crucial role in the pro-inflammatory activation of endothelial cells (EC) in the early phase of atherosclerosis. Homozygosity for the C allele of the T-1C single nucleotide polymorphism (SNP) in the Kozak sequence of the CD40 gene significantly increases the risk for developing coronary heart disease (CHD) in the Caucasian population. The CC genotype provokes a pro-inflammatory EC phenotype compensated by enhanced CD40 shedding and release of soluble CD40 (sCD40) attributed to the disintegrin metalloprotease ADAM17. This study investigates the association between sCD40 shedding/release and the T-1C SNP of the CD40 gene in human endothelial cells on the molecular level. The role of sCD40 as a potential biomarker of CHD will be evaluated, too.

Comparative transcriptome analysis of CD40L-stimulated human umbilical vein endothelial cells (HUVEC) with CC and TT genotype was performed. For functional studies, genotyped HUVEC were stimulated with CD40L and tumor necrosis factor alpha (TNF-α) as well. TAPI-0 optionally inhibited ADAM17. The expression of relevant gene products was investigated using RT-PCR, Western blot, and ELISA. Differences in surface expression of ADAM17 and CD40 were detected by flow cytometry. Levels of high sensitive c-reactive protein (hs-CRP), interleukine-6 (IL-6) and sCD40 in plasma samples from patients with CHD were recorded using ELISA. Receiver operating characteristic (ROC) analyses were performed on stored control and CHD samples.

ADAM17 was selected for further investigations based on the transcriptome analysis. Stimulation of HUVEC with TNF-α increased the release of sCD40 into the supernatant (36%, p=0.0005). Inhibition of ADAM17 prevented this release and enhanced surface expression of CD40 (20%, p=0.04). Stimulation of HUVEC with CD40L after previous inhibition of ADAM17 upregulated monocyte chemoattractant protein-1 (MCP-1) mRNA and protein levels more strongly than without inhibition (80%, p=0.005; 44%, p=0.04). ADAM17 surface expression was elevated following stimulation with CD40L and TNF-α (12%, p=0.03; 25%, p=0.007), just as its regulator IRhom2 (53%, p=0.002;, 133%, p=0.02). A similar mechanism for ADAM17-dependent release of sCD40 was demonstrated for the soluble vascular cell adhesion molecule  (sVCAM). sVCAM and sCD40 levels correlated highly in the supernatant of TNF-α stimulated HUVEC (r=0.77, p=0.01). Genotype-specific ROC analyses of sCD40 as a biomarker for CHD demonstrated an excellent discrimination ability (AUC=0.93, all genotypes; n=45-49). Presence of the C allele enhanced the quality of discrimination (AUC=0.95, CC genotype; n=27-34). In plasma samples from CHD patients (n=75-93), we observed a medium to small correlation of sCD40 with the inflammatory markers hs-CRP (r=0.41), IL-6 (r=0.34), and high sensitive troponin T (r=0.23). Moreover, we observed an age-related increase in sCD40 and an increase in patients with impaired renal function.

We provide a mechanism by which membrane-bound CD40 is shedded from the endothelial surface by the protease ADAM17, generating the soluble isoform sCD40 and limiting CD40 signalling in HUVEC. Our findings highlight the role of ADAM17 in maintaining endothelial homeostasis through negative feedback. The shedded sCD40 represents a robust biomarker for CHD, especially in combination with homozygosity for the C allele as a genetic risk factor.