Question: 

The co-stimulatory CD40-CD40 ligand (CD154) dyad is well-known to play an important role in the development of atherosclerosis. Herein, we hypothesised that CD40 ligation induces von Willebrand factor (vWF) release from structurally and functionally intact endothelial cells, thereby enabling platelet adhesion which in turn facilitates the recruitment of circulating monocytes.

Methods: 

Recombinant trimeric soluble CD154 or CD154-expressing mouse myeloma cells were used to stimulate human umbilical vein endothelial cells (HUVECs). vWF was quantified by ELISA and visualized by immunofluorescence staining. CD154-induced signalling was evaluated by a combination of patch-clamp technology, calcium imaging and pharmacological inhibition. Mouse carotid arteries were perfusedex vivoperfused and stimulated with soluble CD154. vWF multimer formation and fluorescence dye-labelled mouse platelets and/or monocytes were visualized by fluorescence microscopy.

Results: 

Cultured HUVECs released vWF upon CD40 ligation and this was inhibited by the calcium chelator BAPTA. Monitoring CD154-induced calcium transients revealed a time-delayed kinetic in comparison with histamine. Voltage clamp recordings excluded an involvement of plasma membrane cation channels in this intracellular calcium rise. Further evaluation of CD40 signalling in the endothelial cells pointed towards an involvement of a tyrosine kinase of the Src family, phospholipase Cg1, the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and the inositol trisphosphate receptor (IP₃R). An involvement of protein kinase A in CD154-stimulated vWF release, on the other hand, could be excluded. Moreover,ex vivoperfused carotid arteries of wild type mice showed a luminal release of vWF as ultra-large vWF multimers (ULVWF) upon stimulation with soluble CD154 which was absent in blood vessels of CD40 knockout mice. Subsequent perfusion with washed calcein-labelled murine platelets resulted in their increased adherence to these multimers and activation as judged by positive immunoreactivity to P-selectin. Additional perfusion with isolated dye-labelled murine monocytes resulted in their firm adhesion to the vessel wall where they co-localised with both ULVWF multimers and platelets.

Conclusions: 

CD40 ligation leads to vWF release from intact endothelial cells. This in turn facilitates the adhesion and subsequent activation of circulating platelets which seem to amplify endothelial cell-monocyte interaction, thus possibly initiating or promoting early atherosclerotic lesion formation.