Nox2 Is Determinant for Ischemia-Induced Oxidative Stress and Arterial Vasodilatation: A Pilot Study in Patients With Hereditary Nox2 Deficiency
To the Editor:
Reactive oxidant species (ROS) are a family of molecules that are involved in the modulation of arterial tone via rapid degradation of nitric oxide (NO).1 NADPH oxidase is a predominant cellular source of O2−-producing enzymes.1 Four homologs of gp91phox (Nox 2) named Nox1, Nox3, Nox4, and Nox5 have been identified as components of nonphagocyte-type NADPH oxidase.2 Recent studies performed in Nox1 and Nox2 knock-out animals suggested that these Nox isoforms may be implicated in controlling vascular function via modulation of NO bioactivity.3,4
X-chronic granulomatous disease (X-CGD) is a rare primary immunodeficiency affecting the innate immunologic system; it is caused by mutations in any of the 4 genes encoding subunits of the O2− generating NADPH oxidase, resulting in defective O2− generation and intracellular killing.5 We speculated that in patients with downregulation of NADPH oxidase the reduction of oxidative stress could result in increased NO bioavailability and eventually enhanced arterial vasodilatation. To explore this hypothesis we studied 3 male patients (age 41±7.0 years) with hereditary deficiency of Nox2, in whom oxidative stress as well as flow-mediated vasodilatation (FMD) were determined. Twenty male healthy subjects (HS; age 38±6.0 years) were used as control.
None of the HS and patients had risk factors for atherosclerosis or previous cardiovascular disease.
HS had significantly higher oxidative stress, as assessed by 8-hydroxy-2′-deoxyguanosine (8-OHdG) serum levels (1.3±0.7 versus 0.40±0.10 ng/mL, P<0.001) and urinary isoprostanes (247±14 versus 50±42 pg/mg of creatinine, P<0.001) than Nox2-deficient patients.
Consistently with the burst of ROS usually occurring during the reperfusion phase,6 after FMD HS showed an increase of oxidative stress that, however, was not detected in patients, suggesting that activation of Nox2 has a pivotal role in the ROS formation occurring during FMD (Figure, A and B).
Resting platelets of HS expressed iNOS; an upregulation of platelet iNOS was detected in Nox-2 deficient patients (Figure, C).
Despite that oxidative stress seems to be implicated in inhibiting NO-mediated human arterial dilatation, the role of NADPH oxidase has not been investigated.7 To explore this issue FMD was measured in Nox2-deficient patients and HS. Patients and HS had similar baseline brachial artery diameter (3.40±0.52 mm versus 3.66±0.61 mm, respectively, t test, P>0.05). FMD was higher and lasted longer in patients compared with HS (16.0±1.0% versus 10.6±2.3%, U test, P<0.001 and 17.3±2.8 minutes versus 5.6±1.5 minutes, t test, P=0.003, respectively) (Figure, D).
To investigate whether in patients NO was responsible for the prolonged arterial vasodilatation, the experiment was repeated after the iv injection of L-NAME, which fully blunted FMD (Figure, D). L-NAME infusion in HS elicited similar findings (not shown). No side effects were observed during the injection of L-NAME. Administration of a single dose of 0.4 mg nitroglycerin induced similar dilatation in patients and HS (not shown).
Even if FMD of each patient was analyzed by the same operator in 3 separate occasions and had an acceptable reproducibility (<3%), the small sample size and the large variability of the method8 limit definite conclusion. The fact that in patients platelet iNOS was upregulated could indirectly support the hypothesis that NADPH-generating O2− attenuates eNOS expression,9 but other mechanism cannot be excluded. For instance, because of the short half-life of NO, persistent arterial dilatation could also depend on other mechanism including ROS interaction with vasoconstrictor molecules.10
In conclusion, this pilot study provides the first evidence that in human Nox2 activity may play an important role in enhancing systemic and local oxidative stress and modulating NO-mediated arterial dilatation. This finding may help to develop strategies that prevent ROS formation and ultimately arterial dysfunction.
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