Studies on the biological effects of ozone: 11. Release of factors from human endothelial cells

Background: Empirical observations have shown that ozonated autohemotherapy markedly improves the symptoms of chronic limb ischemia (muscular pain at rest, intermittent claudication, etc) in atherosclerotic patients, but mech anisms of action remain unclear. Aims : Human endothelial cells (HUVECs) are known to release nitrogen monox ide (NO) and we investigated the biological effects of human ozonated serum on HUVECs in culture. Methods : We assessed the relevance of peroxidation, the release of NO as n itrite and of three classical cytokines. Results : The treatment of HUVECs with ozonated serum yields a dose depende nt increase of thiobarbituric acid reactive substances (TBARS) and of hydro gen peroxide (H2O2) and a decrease of protein thiol groups (PTG). Concomita ntly, in comparison to either the control or the oxygenated sample, there i s a significant and steady increase of nitric oxide (NO) production; this i s markedly enhanced by the addition of L-arginine (20 muM) and inhibited in the presence of the NO inhibitor, L-NAME (20 mM). The main mediator of ozo ne action is H2O2 as it has been shown either after its direct measurement or by the addition of 20, 40 and 100 muM. Moreover, during 24 hours incubat ion we have investigated the production of endothelin 1 (ET-1), E-selectin and Interleukin 8 (IL-8) and it appears that ozonation enhances IL-8, inhib its E-selectin and hardly modifies ET-1 production. Conclusions : It appears that reinfusion of ozonated blood, by enhancing re lease of NO, may induce vasodilation in ischemic areas and reduce hypoxia.