VASCULAR BIOLOGY Tyrosine nitration limits stretch-induced CD40 expression and disconnects CD40 signaling in human endothelial cells

Hemodynamic forces are important effec-tors of endothelial cell phenotype and function. Because CD40-CD154 interac-tions between endothelial cells and mono-nuclear leukocytes or activated platelets play an important role in vascular dys-function, we investigated the effects of cyclic stretch on CD40 expression in hu-man cultured endothelial cells. Short-term stretch transiently up-regulated CD40 expression while long-term stretch resulted in a distinct decline in CD40 protein which was prevented by inhibi-tion of the 20S proteasome or scavenging of peroxynitrite. Tyrosine nitration of CD40 also occurred under static conditions on addition of authentic peroxynitrite, and according to mass spectrometry analysis Tyr-82 but not Tyr-31 was its target in the native protein. Immunofluorescence anal-ysis of endothelial cells transduced with a control or Tyr-82 to Ala mutated AAV9-CD40-eGFP expression construct confirmed a peroxynitrite-dependent re-distribution of the protein from the cell membrane to the cytoplasm, which was prevented by methyl--cyclodextrin. Moreover, CD154-stimulated IL-12p40 and E-selectin expression markedly de-creased after exposure to authentic per-oxynitrite or cyclic stretch, respectively. Coimmunoprecipitation demonstrated a decreased binding of TRAF2 and TRAF6 to the CD40 protein after tyrosine nitra-tion. Through this posttranslational oxidative modification of an important costimulatory molecule, endothelial cells are able to quickly adapt to unfavorable hemodynamics and maintain their anti-inflammatory phenotype. (Blood. 2011;118(13):3734-3742