Background: 

CD40-CD40 ligand (CD40L) mediated endothelial cell-leukocyte-platelet-interactions may play an important role in the early phase of atherosclerosis. CD40 has been shown to be expressed in capillaries and venules as well as at arterial bifurcations but not by endothelial cells of straight arteries. Despite being constantly subjected to enhanced oxidative stress induced by constant stretching, these endothelial cells are capable of maintaining their anti-atherosclerotic phenotype hence pointing to an adaptive compensatory mechanism.

Methods and Results: 

We previously reported that short-term stretch transiently up-regulates CD40 expression while long-term stretch resulted in a distinct decline in CD40 protein and tyrosine nitration which was prevented by scavenging of peroxynitrite or inhibition of the 20S proteasome. Further investigations revealed that tyrosine nitration of CD40 also occur under static conditions in the presence of authentic peroxynitrite. Mass spectrometry analysis of a recombinant CD40 chimeric protein comprising the N-terminal part of the molecule revealed that only the extracellular tyrosine residue 82 but not 31 is the target of peroxynitrite in the native protein. As compared to Tyr-31, which is buried in the polypeptide chain, Tyr-82 is freely accessible by peroxynitrite as demonstrated by way of computer-assisted molecular modelling. Immunofluorescence analysis of endothelial cells transduced with a control or Tyr-82 to Ala mutated AAV9-CD40-eGFP expression construct confirmed a peroxynitrite-dependent redistribution of the protein from the cell membrane to the cytoplasm which was prevented by methyl-b-cyclodextrin. Interestingly, Tyr-82 has been shown to be critical for CD40L binding. Thus, CD40L-stimulated interleukin-12p40 and E-selectin expression markedly decreased following exposure to authentic peroxynitrite or cyclic stretch, respectively. Moreover, co-immunoprecipitation analyses demonstrated a decreased binding of the intracellular adaptor proteins TRAF2 and TRAF6 to CD40 following nitration.

Conclusion: 

Through this post-translational oxidative modification of CD40, endothelial cells seem capable of quickly adapting to an unfavourable hemodynamic situation and maintain their anti-atherosclerotic phenotype. Previously, it has been demonstrated that CD40-CD40L interaction itself can lead to an increased formation of peroxynitrite. CD40 nitration may thus as well constitute a self-inhibiting mechanism by which excessive stimulation of this immunologically important co-stimulatory pathway balances itself.