Background: 

Interaction of CD40 ligand (CD154) with its receptor CD40 not only plays a role in cell-mediated immune responses but also activates endothelial cells and promotes inflammatory cell recruitment during the initiation and progression of atherosclerosis. CD40 is preferentially expressed on endothelial cells at atherosclerosis predilection sites whereas activated platelets present CD154 on their surface before being shed (sCD154) into the plasma. The aim of this study was to investigate the pathophysiological relevance of CD154-CD40-mediated platelet-endothelial cell-interactions in the early phase of atherosclerosis.

Methods and Results: 

Increasing amounts of recombinant trimeric sCD154 or CD154-expressing mouse myeloma cells were added to human umbilical vein endothelial cells (HUVEC) under oscillatory flow (ibidi m-slides) or static conditions, respectively. The amount of von Willebrand factor (vWF) released into the medium was measured by ELISA and visualized by immunohistochemical staining of ultra large vWF fibres on the endothelial cell surface. Using confocal fluorescence or two photon microscopy, CD154-induced formation and elongation of ultra large vWF fibres was confirmed by ex vivo perfusion of carotid arteries from wild type mice, and this was largely absent in arteries of CD40 knockout mice. In fact, these ultra large vWF fibres formed a sticky grid-like network to which fluorescence dye-labelled murine blood platelets readily attach to. Moreover, vWF release from the cultured HUVEC was abolished using the specific calcium chelator BAPTA. There was a rise in intracellular [Ca²⁺] upon stimulation of these cells with sCD154 under flow (Fura-2 ratio imaging) as well as with the CD154-positive myeloma cells under static conditions (Fluo-4 imaging) with a somewhat delayed kinetic as compared to a receptor-dependent agonist such as histamine. Further investigations with pharmacological inhibitors revealed that phospholipase C, a protein tyrosine kinase of the Src family, and the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) were implicated in the CD154-mediated vWF release.

Conclusions: 

CD154 induces a phospholipase C/calcium-dependent release of vWF from the Weibel-Palade bodies accompanied by formation of ultra large vWF fibres on the endothelial surface under flow. The enhanced adhesiveness of these cells in turn may lead to an increased recruitment of platelets that subsequently may reinforce endothelial cell-monocyte interaction during the initiation of atherosclerosis.