CD40 Ligand–Dependent Tyrosine Nitration of Prostacyclin Synthase In Vivo

Abstract

Background-Cells in human atherosclerotic lesions express the immune mediator CD40 and its ligand, CD40L, but the mechanisms and the mediators by which CD40L contributes to atherosclerosis are poorly defined. Here, we show how CD40L increases vascular inflammation and thrombosis via tyrosine nitration and inhibition of prostacyclin synthase (PGIS), an enzyme with antithrombotic, antiproliferative, and dilatory functions in the normal vasculature. Methods and Results-Exposure of cultured human aortic endothelial cells to clinically relevant concentrations of CD40L (20 to 80 ng/mL) dose-dependently increased the production of superoxide (O 2 ⅐ Ϫ ), decreased nitric oxide (NO) bioactivity, and increased PGIS nitration. Furthermore, inhibition of CD40 expression by small interfering RNA blocked the effects of CD40L on O 2 ⅐ Ϫ , NO bioactivity, and PGIS nitration, which indicates a specific effect of CD40L. In addition, either depletion of mitochondria ( 0 cells, ie, mitochondria-depleted cells, to prevent mitochondrial O 2 ⅐ Ϫ ) or adenoviral overexpression of superoxide dismutase, as well as inhibition of NO synthase, abolished the CD40L-enhanced PGIS nitration, which implies that the mitochondria might be the source of O 2 ⅐ Ϫ and thus peroxynitrite (ONOO Ϫ ). Furthermore, SQ29548, a thromboxane A 2 /prostaglandin H 2 receptor antagonist, significantly reduced CD40L-enhanced expression of intercellular adhesion molecule-1. Finally, administration of CD40L resulted in PGIS inhibition and nitration in the aortas of C57BL6 mice but less in mice overexpressing human superoxide dismutase, which suggests that ONOO Ϫ might be required for CD40L-enhanced PGIS nitration in vivo. Conclusions-We

Endothelial Nitric Oxide Synthase–Dependent Tyrosine Nitration of Prostacyclin Synthase in Diabetes In Vivo

“…We have shown (22)(23)(24) that in BCAs, PGI 2 is responsible for ϳ80% of angiotensin II-induced relaxation, whereas NO accounts for ϳ20%. Stimulation of BCAs with angiotensin II (50 nmol/l) triggered a rapid rise of tension (constriction) followed by a PGI 2 -mediated relaxation (22)(23)(24). Thus, BCAs appeared to be an ideal system for studying the effects of high glucose on PGI 2 -dependent relaxation.…”

“…Isolation and isometric measurement of tension in BCAs. BCAs of the left ventricle were isolated and assayed as described previously (22)(23)(24). Briefly, experiments were started by obtaining, from each spiral, a reference response of vasoconstriction relaxation and prostanoid release after addition of angiotensin II (50 nmol/l) over a time period of 30 min.…”

“…Assay of PGIS activity. PGIS activity was assayed by the stable metabolite of PGI 2 , 6-keto-PGF1␣, after incubating cells with its substrate, PGH 2 (10 Ϫ5 mol/l for 3 min), as described previously (22)(23)(24)(25). Detection of aortic O 2 .؊ .…”

“…Aortic proteins were isolated and hydrolyzed by pronase digestion (72 h at 56°C). 3-nitrotyrosine was quantified by a high-performance liquid chromatography/ultraviolet detector coupled with electrochemical detections, as previously described (22)(23)(24). Immunoprecipation and Western blots.…”

“…Immunoprecipation and Western blots. Immunoprecipitation and Western blots were performed as described previously (21)(22)(23)(24)(25). Quantification of Western blot.…”