R-(+)-α-硫辛酸抑制内皮细胞凋亡和增殖：Akt及视网膜母细胞瘤蛋白/E2F-1的作用
R-(+)-alpha-lipoic acid inhibits endothelial cell apoptosis and proliferation: involvement of Akt and retinoblastoma protein/E2F-1.
作者信息
Artwohl Michaela, Muth Kathrin, Kosulin Karin, de Martin Rainer, Hölzenbein Thomas, Rainer Georg, Freudenthaler Angelika, Huttary Nicole, Schmetterer Leopold, Waldhäusl Werner K, Baumgartner-Parzer Sabina M
机构信息
Department of Internal Medicine III, Division of Endocrinology and Metabolism, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria.
出版信息
Am J Physiol Endocrinol Metab. 2007 Sep;293(3):E681-9. doi: 10.1152/ajpendo.00584.2006. Epub 2007 Jun 12.
Lipoic acid was recently demonstrated to improve endothelial dysfunction or retinopathy not only in rats but also in diabetic patients. We tested the hypothesis that R-(+)-alpha-lipoic acid (LA) directly affects human endothelial cell (EC) function (e.g., apoptosis, proliferation, and protein expression), independent of the cells' vascular origin. Macrovascular EC (macEC), isolated from umbilical (HUVEC) and adult saphenous veins and from aortae, as well as microvascular EC (micEC) from retinae, skin, and uterus, were exposed to LA (1 mumol/l-1 mmol/l) with/without different stimuli (high glucose, TNF-alpha, VEGF, wortmannin, LY-294002). Apoptosis, proliferation, cell cycle distribution, and protein expression were determined by DNA fragmentation assays, [(3)H]thymidine incorporation, FACS, and Western blot analyses, respectively. In macro- and microvascular EC, LA (1 mmol/l) reduced (P < 0.05) basal (macEC, -36 +/- 4%; micEC, -46 +/- 6%) and stimulus-induced (TNF-alpha: macEC, -75 +/- 11%; micEC, -68 +/- 13%) apoptosis. In HUVEC, inhibition of apoptosis by LA (500 mumol/l) was paralleled by reduction of NF-kappaB. LA's antiapoptotic activity was reduced by PI 3-kinase inhibitors (wortmannin, LY-294002), being in line with LA-induced Akt phosphorylation (Ser(437), +159 +/- 43%; Thr(308), +98 +/- 25%; P < 0.01). LA (500 mumol/l) inhibited (P < 0.001) proliferation of macEC (-29 +/- 3%) and micEC (-29 +/- 3%) by arresting the cells at the G(1)/S transition due to an increased ratio of cyclin E/p27(Kip) (4.2-fold), upregulation of p21(WAF-1/Cip1) (+104 +/- 21%), and reduction of cyclin A (-32 +/- 11%), of hyperphosphorylated retinoblastoma protein (macEC: -51 +/- 7%; micEC: -50 +/- 15%), and of E2F-1 (macEC: -48 +/- 3%; micEC: -31 +/- 10%). LA's ability to inhibit apoptosis and proliferation of ECs could beneficially affect endothelial dysfunction, which precedes manifestation of late diabetic vascular complications.
最近有研究表明，硫辛酸不仅能改善大鼠的内皮功能障碍或视网膜病变，对糖尿病患者也有同样效果。我们验证了以下假设：R-(+)-α-硫辛酸（LA）可直接影响人内皮细胞（EC）功能（如凋亡、增殖和蛋白表达），且与细胞的血管来源无关。从脐静脉（HUVEC）、成人隐静脉和主动脉分离得到的大血管EC（macEC），以及从视网膜、皮肤和子宫分离得到的微血管EC（micEC），分别在有/无不同刺激因素（高糖、TNF-α、VEGF、渥曼青霉素、LY-294002）的情况下，暴露于LA（1 μmol/l - 1 mmol/l）。分别通过DNA片段化分析、[³H]胸腺嘧啶掺入法、流式细胞术（FACS）和蛋白质印迹分析来测定细胞凋亡、增殖、细胞周期分布和蛋白表达。在大血管和微血管EC中，LA（1 mmol/l）可降低（P < 0.05）基础状态下（macEC，-36 ± 4%；micEC，-46 ± 6%）以及刺激诱导下（TNF-α：macEC，-75 ± 11%；micEC，-68 ± 13%）的细胞凋亡。在HUVEC中，LA（500 μmol/l）抑制细胞凋亡的同时，NF-κB也减少。PI 3-激酶抑制剂（渥曼青霉素、LY-294002）可降低LA的抗凋亡活性，这与LA诱导的Akt磷酸化一致（Ser(437)，+159 ± 43%；Thr(308)，+98 ± 25%；P < 0.01）。LA（500 μmol/l）通过使细胞停滞在G(1)/S期来抑制（P < 0.001）macEC（-29 ± 3%）和micEC（-29 ± 3%）的增殖，这是由于细胞周期蛋白E/p27(Kip)比值增加（4.2倍）、p21(WAF-1/Cip1)上调（+104 ± 21%）、细胞周期蛋白A减少（-32 ± 11%）、视网膜母细胞瘤蛋白过度磷酸化减少（macEC：-51 ± 7%；micEC：-50 ± 15%）以及E2F-1减少（macEC：-48 ± 3%；micEC：-31 ± 10%）。LA抑制EC凋亡和增殖的能力可能对内皮功能障碍产生有益影响，而内皮功能障碍是糖尿病晚期血管并发症发生的先兆。
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