R-(+)-alpha-lipoic acid inhibits endothelial cell apoptosis and proliferation: involvement of Akt and retinoblastoma protein/E2F-1.
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.
Artwohl
Michaela
M
Department of Internal Medicine III, Division of Endocrinology and Metabolism, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria.
Muth
Kathrin
K
Kosulin
Karin
K
de Martin
Rainer
R
Hölzenbein
Thomas
T
Rainer
Georg
G
Freudenthaler
Angelika
A
Huttary
Nicole
N
Schmetterer
Leopold
L
Waldhäusl
Werner K
WK
Baumgartner-Parzer
Sabina M
SM
D017209:Apoptosis / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D050687:E2F1 Transcription Factor / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D008063:Thioctic Acid / Q000008:administration & dosage*
D050687:E2F1 Transcription Factor; D008063:Thioctic Acid