Nasal polyp-derived superoxide anion: dose-dependent inhibition by nitric oxide and pathophysiological implications.

The epithelium of the paranasal sinuses produces nitric oxide (NO), which probably plays a major role in the nonspecific defense of these cavities through its bacteriostatic and cilia motility stimulation properties. Abundant eosinophils of nasal polyps potentially generate superoxide anion (O2-*), but NO and O2-* inactivate reciprocally. The purpose of the present work was to evaluate the relationship between NO concentrations and nasal polyp production of O2-*. Polyp fragments from 24 patients were studied using histological examination and lucigenin-enhanced chemiluminescence (to assess O2-* production). The effect of various concentrations of exogenous NO on chemiluminescent signals was assessed. Basal and phorbol ester-stimulated O2-* production varied largely among patients, but both were highly related to eosinophilic infiltration. A slow releasing NO donor DETA NONOate (DETA/NO NOC-18) dose dependently inhibited lucigenin-enhanced chemiluminescence from phorbol ester-stimulated polyp fragments, with an EC50 of 1.5 mM. The NO concentration in normal maxillary sinus was estimated about 10 ppm (i.e., 0.5 microM in aqueous phase) (Lundberg, et al. Nature Med 1995;1:370). Calculations revealed that the DETA NONOate 0.75 mM and 1.5 mM generate steady-state concentrations of NO of 0.5 microM and 2.5 microM, respectively. In conclusion, the NO concentration present in paranasal sinuses appears to partially suppress (approximately 20-40%) O2-* production from polyp eosinophils. Conversely, phagocytic-derived O2-* could contribute to decrease sinus NO concentration, further altering this natural local defense. Together, these events could participate in chronic inflammation and contribute to the pathophysiology of nasal polyps.

Detection of superoxide anion released extracellularly by endothelial cells using cytochrome c reduction, ESR, fluorescence and lucigenin-enhanced chemiluminescence techniques.

Endothelium produces oxygen-derived free radicals (nitric oxide, NO&z.rad;; superoxide anion, O(2)(*-)) which play a major role in physiology and pathology of the vessel wall. However, little is known about endothelium-derived O(2)(*-) production, particularly due to the difficulty in assessing O(2)(*-) when its production is low and to controversies recently raised about the use of lucigenin-enhanced chemiluminescence. We compared four techniques of O(2)(*-) assessment when its production is low. In the present study, we have compared ferricytochrome c reduction, electron spin resonance (ESR) spectroscopy using DMPO as spin trap, hydroethidine fluorescence, and lucigenin-enhanced chemiluminescence to assess O(2)(*-) production in cultured bovine aortic endothelial cells (BAEC). We focused our study on extracellular O(2)(*-) production because the specificity of the signal is provided by the use of superoxide dismutase, and this control cannot be obtained intracellularly. We found that the calcium ionophore A23187 dose-dependently stimulated O(2)(*-) production, with a good correlation between all four techniques. The signals evoked by postconfluent BAEC were increased 2- to 7-fold in comparison to just-confluent BAEC, according to the technique used. Ferricytochrome c 20 microm rather than at 100 microm appears more suitable to detect O(2)(*-). However, in the presence of electron donors such as NADH or NADPH, lucigenin-enhanced chemiluminescence generated high amounts of O(2)(*-). Thus, ferricytochrome c reduction, electron spin resonance (ESR), and hydroethidine fluorescence appear as adequate tools for the detection of extracellular endothelium-derived O(2)(*-) production, whereas lucigenin may be artifactual, even when a low concentration of lucigenin is employed.

The mitogenic effect of 17beta-estradiol on in vitro endothelial cell proliferation and on in vivo reendothelialization are both dependent on vascular endothelial growth factor.

In addition to their actions on reproductive function, estrogens have important effects on endothelial cells. The present study was designed to evaluate the mechanism(s) by which 17beta-estradiol (E2) promotes endothelial cell proliferation. The potential involvement of vascular endothelial growth factor (VEGF) was investigated by the coadministration of polyclonal anti-VEGF antibody. First, the effect of E2 on the proliferation of cultured foetal bovine aortic endothelial cells (FBAEC) was studied. E2 stimulated this proliferation with an EC50 between 10(-11) and 10(-10) M and this effect was inhibited by the anti-VEGF antibody. The effect of a physiological dose of E2 was then studied in the rat model of carotid injury. After deendothelializing balloon injury, reendothelialization of the denuded surface may influence the growth of the underlying smooth muscle cells. Male Sprague-Dawley rats were castrated and then received E2 from subcutaneously implanted pellets that released 3.2 microg/kg/day. Endothelial regrowth (Evans blue staining) and neointimal thickening were evaluated 2 weeks after the carotid injury. In comparison to the placebo group, E2 increased the extent of reendothelialization (p = 0.0002) and reduced neointimal thickening (p = 0.0007). Anti-VEGF antibody abolished the effect of E2 on reendothelialization as well as on neointimal thickening. Thoracic aorta VEGF content was increased in E2-treated rats compared to control rats. In conclusion, the present study demonstrates that E2 increases endothelial cell proliferation in vitro and reendothelialization in vivo by means of a mechanism dependent on endogenous VEGF. This effect could contribute to the antiatherogenic effect of a physiological dose of E2.

Estradiol increases rat aorta endothelium-derived relaxing factor (EDRF) activity without changes in endothelial NO synthase gene expression: possible role of decreased endothelium-derived superoxide anion production.

OBJECTIVES: Estradiol is known to exert a protective effect against atherosclerosis, but the mechanism(s) whereby this protection is mediated is/are unclear. However, estradiol-treated castrated animals exhibit increased activity of endothelium-derived relaxing factor (EDRF), which could contribute to vasculoprotection. In the present work, we investigated the molecular mechanism(s) of the enhancement of EDRF activity in the thoracic aorta of oophorectomized female rats given 17 beta-estradiol (E2, 2 or 40 micrograms/kg/day) compared to those given a placebo. METHODS AND RESULTS: The abundance in the thoracic aorta of NO synthase I, II and III mRNA (using RT-PCR) and of NO synthase I, II and III immunoreactive protein (using Western blotting) was unaltered by E2. NO synthase activity (based on arginine/citrulline conversion) in thoracic aorta homogenates did not differ significantly among the three groups, suggesting that NO production was not enhanced by E2. In contrast, lucigenin-enhanced chemiluminescence of aorta from the E2 group was decreased compared to that of the placebo group. Desendothelialization and exogenously added superoxide dismutase suggested that this difference was due to a decrease in extracellular endothelium-derived production of superoxide anion (O2-.). Experiments in cultured bovine aortic endothelial cells confirmed a decreased extracellular production of O2-. in response to ethinylestradiol (1 nM) using both lucigenin-enhanced chemiluminescence and ESR spectroscopy. Luminol-enhanced chemiluminescence revealed that ethinylestradioltreated cultured endothelial cells generated less peroxynitrite (the byproduct of NO-. and O2-. interaction) than control cells. CONCLUSION: Estradiol increases rat aorta EDRF activity in the absence of changes in endothelial NO synthase gene expression. The decreased endothelium-derived generation of O2-. in response to estrogens could account for enhanced EDRF-NO bioactivity and decreased peroxynitrite release. All of these effects could contribute to the vascular protective properties of estrogens.

Electron spin resonance detection of extracellular superoxide anion released by cultured endothelial cells.

OBJECTIVE AND METHODS: Endothelium produces oxygen-derived free radicals which play a major role in vessel wall physiology and pathology. Whereas NO* production from endothelium has been extensively characterized, little is known about endothelium-derived O2-*. In the present study, we determined the O2-* production of bovine aortic endothelial cells (BAEC) using the spin trap 5,5-dimethyl-1 pyrroline-N-oxide (DMPO) and electron spin resonance (ESR) spectroscopy. RESULTS: An ESR adduct DMPO-OH detected in the supernatant of BAEC after stimulation with the calcium ionophore A23187 originated from the trapping of extracellular O2-*, because coincubation with superoxide dismutase (30 U/ml) completely suppressed the ESR signal, whereas catalase (2000 U/ml) had no effect. A23187 stimulated extracellular O2-* production in a time- and dose-dependent manner. The coenzymes NADH and NADPH both increased the ESR signal, whereas a flavin antagonist, diphenylene iodonium, abolished the ESR signal. Phorbol myristate acetate potentiated, whereas bisindolylmaleimide I inhibited the A23187-stimulated O2-* production, suggesting the involvement of protein kinase C. These signals were not altered L-NAME, a NO-synthase inhibitor, suggesting that the endogenous production of NO* did not alter O2-* production. Finally, the amount of O2-* generated by A23187-stimulated post-confluent BAEC was one order of magnitude higher than that evoked by rat aortic smooth muscle cells stimulated under the same conditions.