Pech-Amsellem M A, Myara I, Storogenko M, Demuth K, Proust A, Moatti N
Laboratoire de Biochimie Appliquée, Faculté de Pharmacie, Université Paris XI, Châtenay-Malabry, France.
Cardiovasc Res. 1996 Jun;31(6):975-83.
The aim of the study was to investigate LDL modifications by cultured human umbilical vein endothelial cells (HUVEC) from women smokers and non-smokers.
Modifications of LDL by HUVEC were studied by determining the values of thiobarbituric acid-reactive substances (TBARS) and the percentage of the most electronegative oxidized LDL fraction (fraction C) by using an ion-exchange chromatographic method based on fast protein liquid chromatography (FPLC). We also studied the cellular production of superoxide anion, the effect of various inhibitors and cysteine, and determined total intracellular glutathione content and cell growth.
LDL exposed to HUVEC from smokers for 48 h showed significantly greater modifications than LDL exposed to HUVEC from non-smokers, as assessed by TBARS determination (19.4 +/- 1.2, mean +/- s.e.m., n = 20 versus 15.4 +/- 0.7 nmol/mg LDL, n = 19; P < 0.01) and by FPLC (percentage of fraction C: 39 +/- 7, n = 29 versus 14 +/- 3, n = 34; P < 0.001). Moreover, HUVEC from smokers produced significantly more superoxide anion than those from non-smokers (0.46 +/- 0.13 nmol/10(5) cell/min, n = 9 versus 0.22 +/- 0.05, n = 10; P < 0.05). Superoxide production, like cell-induced modification of LDL, was strongly dependent on the presence of cysteine in the medium. Furthermore, HUVEC from smokers had a significantly (P < 0.05) higher total intracellular glutathione content than those from non-smokers (39.9 +/- 3.1 nmol/mg, n = 9 versus 31.8 +/- 2.2, n = 7). Finally, HUVEC from smokers and non-smokers showed similar growth at 48 h.
HUVEC from smokers converted significantly more LDL into an atherogenic form than HUVEC from non-smokers, a phenomenon that was not due to altered cell growth. HUVEC-mediated LDL modifications were strongly thiol-dependent, as both LDL modifications and superoxide anion production were inhibited in cysteine-free medium. Stimulation of cystine uptake by HUVEC, reflected by the enhanced total glutathione content, could account for the enhanced superoxide anion production. All these observations may be relevant to the pathophysiology of smoking-related cardiovascular disease.
本研究旨在调查来自吸烟女性和非吸烟女性的培养人脐静脉内皮细胞（HUVEC）对低密度脂蛋白（LDL）的修饰作用。
采用基于快速蛋白质液相色谱（FPLC）的离子交换色谱法，通过测定硫代巴比妥酸反应性物质（TBARS）的值以及最具电负性的氧化LDL组分（组分C）的百分比，研究HUVEC对LDL的修饰作用。我们还研究了超氧阴离子的细胞产生、各种抑制剂和半胱氨酸的作用，并测定了细胞内总谷胱甘肽含量和细胞生长情况。
通过TBARS测定（19.4±1.2，平均值±标准误，n = 20，而15.4±0.7 nmol/mg LDL，n = 19；P < 0.01）和FPLC（组分C的百分比：39±7，n = 29，而14±3，n = 34；P < 0.001）评估，暴露于来自吸烟者的HUVEC 48小时的LDL显示出比暴露于来自非吸烟者的HUVEC的LDL明显更大的修饰。此外，来自吸烟者的HUVEC产生的超氧阴离子明显多于来自非吸烟者的HUVEC（0.46±0.13 nmol/10⁵细胞/分钟，n = 9，而0.22±0.05，n = 10；P < 0.05）。超氧阴离子的产生，如同细胞诱导的LDL修饰一样，强烈依赖于培养基中半胱氨酸的存在。此外，来自吸烟者的HUVEC的细胞内总谷胱甘肽含量明显高于来自非吸烟者的HUVEC（P < 0.05）（39.9±3.1 nmol/mg，n = 9，而31.8±2.2，n = 7）。最后，来自吸烟者和非吸烟者的HUVEC在48小时时显示出相似的生长情况。
来自吸烟者的HUVEC比来自非吸烟者的HUVEC将更多的LDL转化为致动脉粥样硬化形式，这一现象并非由于细胞生长改变所致。HUVEC介导的LDL修饰强烈依赖于硫醇，因为在无半胱氨酸的培养基中LDL修饰和超氧阴离子产生均受到抑制。HUVEC对胱氨酸摄取的刺激，表现为总谷胱甘肽含量增加，可能解释了超氧阴离子产生的增加。所有这些观察结果可能与吸烟相关心血管疾病的病理生理学有关。
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