The effect of lipoproteins on endothelial nitric oxide synthase is modulated by lipoperoxides

BACKGROUND AND AIM: The effect of low-density lipoproteins (LDLs) on endothelial nitric oxide synthase (eNOS) is debated. By coupling in vivo and in vitro experiments we evaluated the role of oxidized lipid substrates in the modulation of eNOS activity by LDLs. MATERIALS AND METHODS: Plasma lipids, nitrite/nitrates (NO2/NO3), and malondialdehyde (MDA) were measured in 14 controls, and in 13 patients with familial hypercholesterolemia (FH) before and after 12 weeks of treatment with atorvastatin (20 mg u.i.d.). Nitric oxide synthase in cell lysate and NO2/NO3 into the medium were measured in human microvascular (HMEC-1) and umbilical vein (HUVEC) endothelial cells after 24 h of incubation with increasing concentrations of mildly oxidized LDLs with and without atorvastatin and in HMEC-1 with and without vitamin C. In HMEC-1, NO2/NO3 was also determined after exposure to more intensively oxidized LDLs. RESULTS: At baseline, plasma NO2/NO3 (56 +/- 7 vs. 35 +/- 3 micro M) and MDA (5.6 +/- 0.7 vs. 2.9 +/- 0.3 micro M), were significantly (P < 0.02 for both) higher in the FH patients. In the whole study group, NO2/NO3 was more strongly correlated with plasma MDA (Rho = 0.70) than LDL-cholesterol (Rho = 0.57). In the FH patients, atorvastatin induced a significant decline in plasma total and LDL-cholesterol (-3.1 +/- 0.5 and -2.9 +/- 0.5 mM, respectively), NO2/NO3 (-35 +/- 8 microM) and MDA (-3.4 +/- 0.7 microM) (P < 0.001 for all). Changes in plasma NO2/NO3 were related to the concomitant changes in plasma MDA (Rho = 0.79, P < 0.006) and not to changes in LDL-cholesterol. In HMEC-1 and in HUVEC, mildly oxidized LDLs stimulated both e-NOS and NO2/NO3 accumulation; the effect on e-NOS was potentiated by vitamin C in HMEC-1. Atorvastatin had no effect in HMEC-1 while it stimulated eNOS but not NO2/NO3 in HUVEC. The accumulation of NO2/NO3 in HMEC exposed to increasing concentrations of more intensively oxidized-LDLs showed a nonlinear dose-response curve. CONCLUSIONS: In uncomplicated patients with FH, plasma NO2/NO3 concentrations are elevated; the cross-sectional data, intervention study and in vitro experiments indicate that oxidized lipids exert a tonic stimulatory action on e-NOS and NO2/NO3 generation not mediated through superoxide anion formation. Atorvastatin amplify this eNOS response in HUVEC, but not in HMEC, and this effect is not associated with a parallel increased NO2/NO3 generation.