Oxygen radical inhibition of nitric oxide-dependent vascular function in sickle cell disease

Abstract

Plasma xanthine oxidase (XO) activity was defined as a source of enhanced vascular superoxide (O(2)( *-)) and hydrogen peroxide (H(2)O(2)) production in both sickle cell disease (SCD) patients and knockout-transgenic SCD mice. There was a significant increase in the plasma XO activity of SCD patients that was similarly reflected in the SCD mouse model. Western blot and enzymatic analysis of liver tissue from SCD mice revealed decreased XO content. Hematoxylin and eosin staining of liver tissue of knockout-transgenic SCD mice indicated extensive hepatocellular injury that was accompanied by increased plasma content of the liver enzyme alanine aminotransferase. Immunocytochemical and enzymatic analysis of XO in thoracic aorta and liver tissue of SCD mice showed increased vessel wall and decreased liver XO, with XO concentrated on and in vascular luminal cells. Steady-state rates of vascular O(2)( *-) production, as indicated by coelenterazine chemiluminescence, were significantly increased, and nitric oxide (( *)NO)-dependent vasorelaxation of aortic ring segments was severely impaired in SCD mice, implying oxidative inactivation of ( *)NO. Pretreatment of aortic vessels with the superoxide dismutase mimetic manganese 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin markedly decreased O(2)( small middle dot-) levels and significantly restored acetylcholine-dependent relaxation, whereas catalase had no effect. These data reveal that episodes of intrahepatic hypoxia-reoxygenation associated with SCD can induce the release of XO into the circulation from the liver. This circulating XO can then bind avidly to vessel luminal cells and impair vascular function by creating an oxidative milieu and catalytically consuming (*)NO via O(2)( small middle dot-)-dependent mechanisms.

Figure 1

Red cell production of reactive oxygen species. (A) Rates of superoxide release by HbA and HbS red cells. Values are mean ± SEM (n = 3–9). Statistical analysis was by two-way ANOVA with the Tukey post hoc test. *, P < 0.05. (B) Aminotriazole-mediated catalase inactivation by HbA and HbS red cells. Values at each time point represent mean ± SD with n = 5. (C) Rates of HbA and HbS red cell NO consumption. Values represent mean ± SEM (n = 4–14). Statistical analysis was by two-way ANOVA with the Tukey post hoc test. *, P < 0.05 compared with basal.

Figure 2

Immunocytochemical analysis of XOR in C57BL/6J control and sickle cell mouse tissues. (A) Western blot analysis of plasma and liver XO in SCD mice. (B) Descending thoracic aortic segments from knockout-transgenic SC mice display intense immunofluorescent staining for XO (red) that is associated with the endothelium and, to a lesser extent, smooth muscle cells (L, lumen). Liver sections from SC mice show decreased XOR staining in the pericentral hepatocytes when compared with controls (CV, central vein). Nuclei were counterstained with Hoechst in all experiments. (C) Hematoxylin and eosin staining of liver sections from control and sickle cell mouse tissues.

Figure 3

Superoxide production by C57BL/6J control and sickle cell mouse vessels. Values represent mean ± SD (n = 4). Statistical analysis was by two-way ANOVA with the Duncan's post hoc test. *, P < 0.05 compared with control. +, P < 0.05 compared with xanthine-treated sickle cell vessels. **, P < 0.05 compared with control and all treated vessel groups.

Figure 4

ACh-dependent vascular relaxation in C57BL/6J control and sickle cell mouse vessels. Values represent mean ± SEM (n = 4–8). All statistical analyses were by one-way ANOVA with Student–Newman Keuls pairwise multiple comparison. *, P < 0.05 compared with sickle cell mouse vessel response.