THE DINUCLEOTIDE UP4A INDUCE THE PRODUCTION OF REACTIVE OXYGEN SPECIES IN VSMCS VIA NADPH OXIDASE: 1C.02:

Objective: There is a growing body of evidence that nucleotides and purinergic signaling play a crucial role in the development of vascular diseases such as atherosclerosis. Uridine adenosine tetraphosphate (Up4A), an endothelium-derived dinucleoside polyphosphate showed inflammatory promoting effects. The inflammatory response is triggered by NADPH oxidase by influencing the production of chemokines. Here, we investigated the effect of Up4A on activation of NADPH oxidase and the production of reactive oxygen species in vascular smooth muscle cells. Methods: Rat vascular smooth muscle cells (VSMCs) were used for experiments. MCP-1 and Nox1/4 expression was measured by real-time PCR. Rac1 phosphorylation was measured by ELISA. Translocation of p47phox was detected by Western Blot technique in membrane fraction and cytosolic protein fraction of the cells. H2O2 production was examined by loading VSMCs with 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein-dieacetate-acetylester (CM-H2DCFDA). Results: Previous work proposed that the MCP-1 expression in VSMCs is controlled by the intracellular redox status. Thus, we investigated the effect of tiron, a vitamin E analog, which is able to significantly diminish the Up4A-induced MCP-1 expression (48 ± 23% decrease, n = 5). Furthermore, incubation of CM-H2DCFDA-labeled VSMCs with Up4A resulted in a significant and dose-dependent increase in DCF fluorescence intensity (n = 6). Thrombin (8 IE/mL) was used as positive control. NADPH oxidase is composed of different subunits. The expression of Nox1 is increased after Up4A stimulation of the cells, whereas Nox4 expression is decreased. The activation of NADPH oxidase requires phosphorylation of Rac1 and translocation of p47phox to the plasma membrane. Up4A induced Rac1 phosphorylation in a significant and time-dependent manner. P47phox amount in membrane fraction increased time-dependently after Up4A stimulation, whereas the amount in the cytosolic protein fraction decreased, respectively. Conclusions: In this study we could show that Up4A is influencing the vascular ROS production in a NADPH oxidase dependent manner. Therefore, the endothelial-derived factor Up4A is not only a potent vasoconstrictor but in addition a potent inductor of pro-inflammatory response in the vascular wall.