Central to the aetiology of ARDS (Acute Respiratory Distress Syndrome) is the over-production of superoxide (O₂^▪-) (Muzaffar et al., 2003; 2004a; 2004b). Apart from eliciting direct pathological effects, O₂^▪- reacts with nitric oxide (NO) to produce reactive nitrogen species, thereby reducing the bioavailability of NO (Muzaffar et al., 2003). A principal source for O₂^▪- is NAD[P]H oxidase, an enzyme that is upregulated by inflammogens. Since O₂^▪- triggers the expression of many proteins, it is reasonable to suggest that O₂^▪- may directly elicit an up-regulation of NAD[P]H oxidase, which would result in a self-perpetuating inflammatory cascade. In order to test this novel hypothesis, the effect of prolonged exposure to O₂^▪- on the expression of gp91^phox (a catalytic subunit of NADPH oxidase) and subsequent O₂^▪- formation in primary cultures if porcine pulmonary arterial endothelial cells (PAEC) was investigated.

PAECs were incubated with xanthine / xanthine oxidase system (X / XO; generates O 2^▪), or tumour necrosis factor- (TNF-) or thromboxane A₂ analogue, U46619 (± superoxide dismutase [SOD] or catalase) for 16 hrs. Both TNF- and U46619, have previously been shown to upregulate gp91^phox expression in PAECs (Muzaffar et al., 2003). Cells were washed and O₂^▪- then measured spectrophometrically and gp91^phox expression assessed using Western blotting. Post-incubation, the role of NAD[P]H oxidase was also studied using apocynin, an NAD[P]H oxidase inhibitor.

X / XO elicited a concentration-dependent (non-cumulative) increase in the formation of O₂^▪- in PAECs (maximal response at 0.02 U/ml of XO; 8.6 ± 0.3 µmoles / h compared to 4.1 ± 0.6 µmoles / h in controls) following a 16 hour exposure and washing. The X / XO -induced increase in O₂^▪- .- release (after 16 hour incubation) was blocked by the presence of SOD (3.1 ± 1.2 µmoles / h) but not catalase (7.6 ± 1.4 µmoles / h) over the 16 hr incubation phase. The formation of O₂^▪- induced with TNF- and U46619 was also inhibited by continual presence of SOD over the 16-hour incubation phase (4.4 ± 0.6 µmoles / h). Apocynin (1 µM), an inhibitor of NADPH oxidase activity, completely inhibited O₂^▪- formation induced with X / XO, TNF- or U46619 (5.1 ± 0.4 m moles / h) after the 16 hour incubation phase. Rotenone and allopurinol were without effect. Finally, XO, TNF- or U46619 all induced gp91^phox expression in PAECs after a 16 hr incubation, an effect blocked by the continual presence of SOD.

These data demonstrate that O₂^▪- directly upregulates gp91^phox expression and activity in PAECs and augments O₂^▪- formation. This constitutes a novel and potentially important pathological mechanism by which excess vascular O₂^▪- formation creates a self-perpetuating cascade through increased NAD[P]H oxidase expression and the formation of O 2^▪-. Although the intracellular mechanisms involved remain to be elucidated, the study highlights the therapeutic importance of preventing intravascular oxidative stress reactions.

Muzaffar, S., et al. (2003) Thorax 58 : 598-604.
Muzaffar, S., et al. (2004a) Br. J. Pharmacol. 141: 488-496.
Muzaffar, S., et al. (2004b) Circulation 110: 1140-1147.