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

Abstract

IntroductionCD40-CD154 costimulation among APCs, Th cells, and activated platelets is of critical importance for correct functioning of the cellular and humoral immune response. On the other hand, exaggerated CD40-CD154 costimulation has been implicated in chronic inflammatory and autoimmune diseases like psoriasis or rheumatoid arthritis. 1,2 Moreover, CD40 expression by nonimmune cells such as endothelial cells, fibroblasts, or vascular smooth muscle cells, and the proinflammatory response of these cells to CD40 ligation, suggest an important role for CD40-CD154 costimulation in atherosclerosis and other vasculopathies. 3,4 Endothelial cells are constantly exposed to mechanical forces exerted by the flowing blood. While laminar shear stress, because of the resulting increase in NO formation, is considered to principally exert anti-atherosclerotic effects, 5,6 cyclic stretch upregulates proinflammatory gene expression in these cells through activation of NAD(P)H oxidase(s) and secondary formation of reactive oxygen species (ROS), namely superoxide anions (O 2 Ϫ ) and hydrogen peroxide (H 2 O 2 ). 7,8 Moreover, NO and O 2 Ϫ rapidly react with each other to produce peroxynitrite. This reaction, which is only limited by diffusion, not only weakens the NO blockade of proinflammatory gene expression 9 but, because of secondary tyrosine nitration of different target proteins, may exert a plethora of additional potentially deleterious effects. 10 In native murine blood vessels, CD40 is strongly expressed in capillaries and postcapillary venules as well as at arterial bifurcations like the aortic arch but not by endothelial cells of straight unbranched arteries. 11 Despite being constantly subjected to enhanced stretch and thus oxidative stress, these endothelial cells are capable of maintaining their anti-atherogenic phenotype hence pointing to a compensatory mechanism. 12 Thus far, it is not known whether CD40 expression in human vascular cells is mechanosensitive. Therefore, we here have investigated the effects of cyclic stretch on CD40 expression, the putative role of ROS therein and potential consequences thereof in human cultured endothelial cells. MethodsA full description of the methods can be found in supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Cell cultureIn brief, human primary umbilical vein endothelial cells were kept under static conditions or exposed to cyclic stretch (FlexCell FX-3000 tension system). For CD40-eGFP fusion protein expression, human immortalized umbilical vein endothelial cells were transduced with an adeno-associated virus serotype 9 (AAV9)-based expression construct. CD40 stimulation was achieved by using the mouse myeloma cell line P3xTB.A7 stably transfected with human CD154. Human premonocytic THP-1 cells were maintained under standard conditions. Hydrogen peroxide formation in The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by p...

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