Chen Jun Zhu, Zhang Fu Rong, Tao Qian Min, Wang Xing Xiang, Zhu Jian Hua, Zhu Jun Hui
Department of Cardiovascular, the First Affiliated Hospital, Medical School of Zhejiang University, Hangzhou 310003, People's Republic of China.
Clin Sci (Lond). 2004 Sep;107(3):273-80. doi: 10.1042/CS20030389.
Hypercholesterolaemia contributes to atherosclerosis and coronary artery diseases by inducing endothelial cell injury and dysfunction. Recent studies have provided increasing evidence that EPCs (endothelial progenitor cells) participate in ongoing endothelial repair and postnatal neovascularization. However, the changes in EPCs in patients with hypercholesterolaemia have not been elucidated to date. Therefore we investigated the number and functional activity of EPCs in patients with hypercholesterolemia. Total MNCs (mononuclear cells) were isolated from 20 patients with hypercholesterolaemia and 20 matched control subjects. EPCs were characterized as adherent cells double-positive for DiI-LDL (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanide percholate-labelled low-density lipoprotein) uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope, and were characterized further by demonstrating the expression of KDR (kinase insert domain-containing receptor), CD34 and AC133 by flow cytometry. Proliferation, migration and in vitro vasculogenesis activity of EPCs were assayed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay, modified Boyden chamber assay and an in vitro vasculogenesis kit respectively. EPC adhesion assay was performed by replating cells on fibronectin-coated dishes and then counting the adherent cells. As a result, the number of EPCs was significantly reduced in patients with hypercholes-terolaemia compared with that in control subjects (41.8 +/- 8.7 compared with 64.5 +/- 16.6 EPCs/x 200 field respectively; P < 0.05). The number of EPCs was inversely correlated with total cholesterol (r = -0.659, P < 0.001) and LDL-cholesterol (r = -0.611, P < 0.001) levels. In addition, the functional activities of isolated EPCs, such as proliferative, migratory, adhesive and in vitro vasculogenesis capacity, were also impaired. In conclusion, the results of the present study may state a novel pathophysiological mechanism of hypercholesterolaemia: the reduction of EPCs with decreased functional activity.
高胆固醇血症通过诱导内皮细胞损伤和功能障碍，促进动脉粥样硬化和冠状动脉疾病的发生。最近的研究提供了越来越多的证据表明，内皮祖细胞（EPCs）参与正在进行的内皮修复和出生后的新血管形成。然而，迄今为止，高胆固醇血症患者体内EPCs的变化尚未阐明。因此，我们研究了高胆固醇血症患者EPCs的数量和功能活性。从20例高胆固醇血症患者和20例匹配的对照受试者中分离出外周血单个核细胞（MNCs）。通过激光扫描共聚焦显微镜下的直接荧光染色，将EPCs鉴定为对DiI-LDL（1,1'-二油酰基-3,3,3',3'-四甲基吲哚羰花青标记的低密度脂蛋白）摄取和凝集素结合呈双阳性的贴壁细胞，并通过流式细胞术检测KDR（含激酶插入结构域受体）、CD34和AC133的表达进一步鉴定。分别使用MTT [3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-2H-四唑溴盐] 法、改良的Boyden小室法和体外血管生成试剂盒检测EPCs的增殖、迁移和体外血管生成活性。通过将细胞重新接种到纤连蛋白包被的培养皿上，然后计数贴壁细胞来进行EPC黏附试验。结果显示，与对照组相比，高胆固醇血症患者的EPCs数量显著减少（分别为41.8±8.7个/200视野和64.5±16.6个/200视野；P<0.05）。EPCs数量与总胆固醇（r = -0.659，P<0.001）和低密度脂蛋白胆固醇（r = -0.611，P<0.001）水平呈负相关。此外，分离的EPCs的功能活性，如增殖、迁移、黏附及体外血管生成能力也受到损害。总之，本研究结果可能揭示了高胆固醇血症一种新的病理生理机制：EPCs数量减少且功能活性降低。
