Lubrano V, Vassalle C, Blandizzi C, Del Tacca M, Palombo C, L'Abbate A, Baldi S, Natali A
C.N.R, Institute of Clinical Physiology, University of Pisa, Pisa, Italy.
Eur J Clin Invest. 2003 Feb;33(2):117-25. doi: 10.1046/j.1365-2362.2003.01083.x.
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.
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.
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.
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.
低密度脂蛋白（LDL）对内皮型一氧化氮合酶（eNOS）的影响存在争议。通过体内和体外实验相结合，我们评估了氧化脂质底物在LDL调节eNOS活性中的作用。
在14名对照者以及13名家族性高胆固醇血症（FH）患者中，于阿托伐他汀（20mg，每日一次）治疗12周前后，检测血浆脂质、亚硝酸盐/硝酸盐（NO2/NO3）以及丙二醛（MDA）。在人微血管（HMEC-1）和脐静脉（HUVEC）内皮细胞中，用不同浓度的轻度氧化LDL，分别在有和没有阿托伐他汀的情况下孵育24小时后，以及在有和没有维生素C的HMEC-1中孵育24小时后，测量细胞裂解物中的一氧化氮合酶以及培养基中的NO2/NO3。在HMEC-1中，暴露于更高度氧化的LDL后也测定了NO2/NO3。
基线时，FH患者的血浆NO2/NO3（56±7对35±3μM）和MDA（5.6±0.7对2.9±0.3μM）显著更高（两者P均<0.02）。在整个研究组中，NO2/NO3与血浆MDA的相关性（Rho = 0.70）比与LDL胆固醇的相关性（Rho = 0.57）更强。在FH患者中，阿托伐他汀使血浆总胆固醇和LDL胆固醇显著下降（分别为-3.1±0.5和-2.9±0.5mM）、NO2/NO3下降（-35±8μM）以及MDA下降（-3.4±0.7μM）（所有P均<0.001）。血浆NO2/NO3的变化与血浆MDA的伴随变化相关（Rho = 0.79，P<0.006），而与LDL胆固醇的变化无关。在HMEC-1和HUVEC中，轻度氧化的LDL刺激了e-NOS和NO2/NO3的积累；在HMEC-1中，维生素C增强了对e-NOS的作用。阿托伐他汀在HMEC-1中无作用，而在HUVEC中刺激了eNOS但未刺激NO2/NO3。暴露于浓度不断增加的更高度氧化LDL的HMEC中，NO2/NO3的积累呈现非线性剂量反应曲线。
在无并发症的FH患者中，血浆NO2/NO3浓度升高；横断面数据、干预研究和体外实验表明，氧化脂质对e-NOS和NO2/NO3的生成具有持续性刺激作用，且并非通过超氧阴离子的形成介导。阿托伐他汀增强了HUVEC中的这种eNOS反应，但在HMEC中未增强，且这种作用与NO2/NO3生成的平行增加无关。
