NO Modulates NADPH Oxidase Function Via Heme Oxygenase-1 in Human Endothelial Cells

Abstract

Abstract-NO is known to induce expression of heme oxygenase-1, an antioxidant enzyme in blood vessels. We tested whether NO might modulate the endothelial NADPH oxidase function via heme oxygenase-1. In human microvascular endothelial cells, the NO donor DETA-NONOate (0.1 to 1 mmol/L) strongly induced expression of heme oxygenase-1 but not Cu/Zn superoxide dismutase. This was associated with a reduction of the superoxide-generating capacity of NADPH oxidase, an effect that depended on de novo gene transcription and heme oxygenase-1 activity. Activation of NADPH oxidase by tumor necrosis factor (TNF) ␣ increased generation of reactive oxygen species. DETA-NONOate alone had little effect on TNF-stimulated reactive oxygen species, but it enhanced the TNF response when: (1) heme oxygenase-1 expression was blocked with specific small-interfering RNA; (2) heme oxygenase-1 activity was blocked by zinc-protoporphyrin; or (3) NADPH oxidase activity was blocked by diphenyleneiodonium. Moreover, the heme oxygenase-1 end product bilirubin directly inhibited fully functional NADPH oxidase and seemed to interrupt the assembly and activation of the oxidase. In conclusion, NO may modulate superoxide production by NADPH oxidase in human vascular endothelial cells, at least partly by inducing heme oxygenase-1. Our results indicate that suppression of NADPH oxidase-dependent reactive oxygen species formation may represent a novel mechanism underlying the cardiovascular protective actions of heme oxygenase-1 and bilirubin. Key Words: bilirubin Ⅲ endothelium Ⅲ heme oxygenase-1 Ⅲ NADPH oxidase Ⅲ nitric oxide Ⅲ oxidant stress N itric oxide is crucial for maintaining normal vascular function, and inactivation of NO by excessive production of reactive oxygen species (ROS) is implicated in several cardiovascular diseases. 1,2 There is increasing evidence that NADPH oxidase expressed in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) is a major source of superoxide. 3 Five isoforms of the Nox subunit of NADPH oxidase have been identified (named Nox1 to Nox5), of which Nox1, Nox2, and Nox4 are found in vascular tissues. 4 -6 Increased NADPH oxidase activation is associated with conditions such as endothelial dysfunction, intimal hyperplasia, atherosclerosis, and hypertension 7 and has been linked to impairment of endothelial NO function in patients with coronary artery disease. 8 Recently, we found that treatment of human microvascular ECs (HMECs) with NO donors inhibited the superoxidegenerating capacity of NADPH oxidase, which may involve S-nitrosylation of the p47phox subunit. 9 In addition, previous studies have revealed that exposure of ECs to exogenous NO induces Nrf2-dependent expression of heme oxygenase-1 (HO-1), 10 the rate-limiting enzyme in heme degradation. Several lines of evidence have indicated that induction of HO-1 has remarkable antioxidant effects, because: (1) bilirubin is a free radical scavenger and has chain-breaking antioxidant activity which may prevent lipid peroxidation 11 ;(2) HO-1 expression ...

Heme Oxygenase-1 Inhibition of Nox Oxidase Activation Is a Microvascular Endothelial Antioxidant Effect of NO

“…However, in the present study it was observed that cellular levels of the key heme-containing Nox subunits present in endothelium, other subunits thought to regulate the Nox oxidases investigated, and Cu,Zn-SOD were not altered by the increased HO-1 expression elicited by exposure to NO. 1 These data on the absence of detectible changes in Nox oxidase expression also rule out another recently reported mechanism 5 involving NO donors inhibiting the expression of mRNA for Nox1. Thus, the superoxide-depressing effects of exposure of endothelium to NO seemed to be associated with an increased HO-1 activity and not with an alternative antioxidant action of NO or a change in the expression of Cu,Zn-SOD or key components of Nox oxidases present in endothelium.…”

“…Regulation by protein kinase C-p47 phox is likely to activate Nox oxidases containing Nox1 or Nox2 but not the Nox4 subunits 8 of the oxidases, which are observed to be present in the endothelial cells studied. 1 Overall, the results of the study of Jiang et al 1 support a mechanism shown in the Figure according to which prolonged exposure to NO elicits increased expression of HO-1, and the generation of bilirubin, resulting from the metabolism of heme by this enzyme, attenuates Nox oxidase-derived superoxide production by preventing oxidase activation by p47 phoxdependent pathways. Based on previous work in the literature demonstrating the potential importance of NO x in increasing the expression of HO-1 and the redox stress-associated regulation of transcriptional activation of this gene, 2,3 NO x derived from the reaction of superoxide with NO or the oxidation of NO are likely to mediate the increase in HO-1 expression caused by prolonged exposure of endothelium to NO.…”

Redox Control of Renal Function and Hypertension

“…Phycorubins are similar in activity to bilirubin, which is ubiquitously found in mammalian systems and actively inhibits the translocation of p47 phox to the plasma membrane (144,210). The obvious advantage of using compounds that are converted to bilirubin is their oral bioavailability.…”

DHA May Have a Profoundly Protective Impact on the Lungs of Smokers

“…The antioxidant properties of this enzyme are mediated largely via intracellular generation of free bilirubin, which functions physiologically to inhibit certain NADPH oxidase complexes [43][44][45][46]. Intriguingly, in a massive prospective study in the UK, serum levels of bilirubin at baseline were found to correlate inversely with risk for both COPD and lung cancer, after controlling for smoking habit [47].…”