FIG. 5.

CCN2(IV) administration into normal mice increases oxidative stress. C57BL/6 mice received a single i.p. injection of recombinant CCN2(IV) (2.5 ng/g body weight) or saline and were sacrificed after 24 h. Measurements were performed in aortic samples from saline and CCN2(IV)-injected mice. (A) O₂^•− production was evaluated by an increase in 2-OH-E⁺ generation by HPLC analysis of DHE fluorescence. A representative HPLC chromatogram (left) and the quantification of the data (right). The arrows show 2-OH-E⁺ peak. (B) NAD(P)H oxidase activity was evaluated by the lucigenin method. (C, D) Nox1 gene and protein expression was determined by real-time PCR and Western blot, respectively. (E) NO production was analyzed by diaminofluorescein fluorescence in aortic samples. (F) Protein nitrosylation levels were evaluated in paraffin-embedded aortic sections. (G) eNOS protein expression was analyzed by Western blot. (H) iNOS gene expression was performed by real-time PCR. (I) Concentration–response curve to ACh (1 nM–10 μM) in aortic segments. Data are expressed as mean±SEM of fold increase over saline of 8–10 animals per group. *p<0.05 versus saline. **p<0.01 versus control. ACh, acetylcholine; eNOS, endothelial NO synthase; iNOS, inducible NO synthase; NO, nitric oxide. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars