Superoxide auto-augments superoxide formation and upregulates gp91phox expression in porcine pulmonary artery endothelial cells: Inhibition by iloprost

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Central to the aetiology of Acute Respiratory Distress Syndrome (ARDS) is superoxide, the principal source of which is nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase). To test whether superoxide may influence NADPH oxidase expression directly, the effect of incubation of superoxide with porcine pulmonary arterial endothelial cells on the expression of gp91phox (a catalytic subunit of NADPH oxidase) and superoxide formation was investigated. Since iloprost has been purported to be potentially effective in treating ARDS, the effect of iloprost on superoxide-mediated effects was also studied.

Pulmonary artery endothelial cells were incubated with xanthine/xanthine oxidase which generates superoxide, or tumour necrosis factor α (TNFα) or thromboxane A2 analogue, U46619 (± superoxide dismutase [SOD] or catalase or iloprost) for 16 h. Cells were then washed and superoxide formation assessed spectrophometrically and gp91phox expression using Western blotting. The role of NADPH oxidase was also studied in the above settings using apocynin, an NADPH oxidase inhibitor.

Superoxide, TNFα and U46619 elicited an increase in the formation of superoxide and induced gp91phox expression in pulmonary artery endothelial cells following a 16 h incubation an effect blocked by the continual presence of SOD and apocynin but not catalase. Apocynin completely inhibited superoxide formation induced with xanthine/xanthine oxidase after the 16 h incubation. Rotenone and allopurinol were without effect. Iloprost inhibited the formation of superoxide and gp91phox expression.

These data demonstrate that superoxide upregulates gp91phox expression in pulmonary artery endothelial cells and thus augments superoxide formation, an effect blocked by iloprost. This constitutes a novel mechanism by which vascular superoxide creates a self-perpetuating cascade that may be of importance to the etiology of ARDS and other vasculopathies.

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