Pulsatile Stretch Stimulates Superoxide Production in Human Aortic Endothelial Cells

BACKGROUND: Free radicals such as superoxide and nitric oxide (NO) play a key role in the pathophysiology of atherosclerosis. Mechanical forces such as pulsatile stretch may be involved in free radical production. We studied superoxide production by pulsatile stretch in human endothelial cells. METHODS AND RESULTS: Human cultured aortic endothelial cells were exposed to pulsatile stretch up to 24 hours, and superoxide production was examined. Short-term stretch for 1 hour (10% average elongation, 50 cycles per minute) increased superoxide production 2.2-fold. This effect was reduced by diphenyleneiodonium chloride, an NADPH oxidase inhibitor, but not by the xanthine oxidase inhibitor oxypurinol or the cyclooxygenase inhibitor indomethacin. Prolonged stretch up to 6 hours increased superoxide production, but it returned to near the control level after 24 hours of stretch. However, after blockade of NO production, 24 hours of stretch did increase superoxide production 2.4-fold compared with 24 hours of stretch alone. Moreover, 24-hour stretch doubled NO synthase (NOS) (III) protein and mRNA expression. The tetrahydrobiopterin synthesis inhibitor 2,4-diamino-6-hydroxypyrimidine had no effect on unstretched cells but doubled superoxide production compared with 24-hour stretch alone; this increase was halved by cotreatment with 6-methyl-5,6,7,8-tetrahydropterine, a lipid-soluble form of tetrahydrobiopterine. CONCLUSIONS: Short-term stretch increased superoxide production from human aortic endothelial cells via NADPH oxidase and NOS (III), whereas prolonged stretch increased both superoxide and NO production. The increase in NOS (III) protein with prolonged stretch acts as a scavenger mechanism whereby NO inactivates superoxide. Tetrahydrobiopterin determines the balance of superoxide and NO production from NOS (III) after prolonged stretch in which NOS (III) level is upregulated.