Submitted on November 3, 2006
Accepted on June 8, 2007
R(+)-
-lipoic acid inhibits endothelial apoptosis and proliferation - involvement of Akt and Retinoblastoma protein / E2F-1
Michaela Artwohl1, Kathrin Muth1, Karin Kosulin2, Rainer de Martin3, Thomas Holzenbein4, Georg Rainer5, Angelika Freudenthaler6, Nicole Huttary7, Leopold Schmetterer8, Werner K Waldhausl1, and Sabina Monika Baumgartner-Parzer1*
1 Division of Endocrinology and Metabolism, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria
2 Division of Endocrinology and Metabolism, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria; Division of Molecular Microbiology and Development of Genetic Diagnostics, Children's Cancer Research Institute, Vienna, Austria
3 Center for Biomolecular Medicine and Pharmacology, Institute of Vascular Biology and Thrombosis Research, Medical University of Vienna, Vienna, Austria
4 Department of Surgery, Division of Vascular Surgery, Medical University of Vienna, Vienna, Austria
5 Department of Ophthalmology, Medical University of Vienna, Vienna, Austria
6 Division of Endocrinology and Metabolism, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria; Vienna, Austria
7 Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria
8 Department of Medical Physics, Medical University of Vienna, Vienna, Vienna, Austria
* To whom correspondence should be addressed. E-mail: sabina.baumgartner-parzer{at}meduniwien.ac.at.
-Lipoic acid was recently demonstrated to improve endothelial dysfunction or retinopathy not only in rats, but also in diabetic patients. This study tested the hypothesis that R(+)--lipoic acid (LA) directly affects human endothelial cell function (apoptosis, proliferation, and protein expression) independent of the cells' vascular origin.
Macrovascular endothelial cells (macECs), isolated from umbilical- (HUVECs) and adult saphenous veins, and from aortae, as well as microvascular endothelial cells (micECs) from retinae, skin, and uterus were exposed to LA (1µmol/l-1mmol/l) with/without different stimuli (30mmol/l glucose, TNF-, VEGF, Wortmannin, LY294002). Apoptosis, proliferation, cell cycle distribution, and protein expression were determined by DNA-fragmentation assays, 3H-thymidine incorporation, FACS- and Western blot analyses, respectively.
In macro- and microvascular endothelial cells, LA (1mmol/l) reduced (p<0.05) basal (macECs: -36±4%; micECs: -46±6%) and stimulus-induced (TNF-: macECs: -75±11%; micECs: -68±13%) apoptosis. In HUVECs, inhibition of apoptosis by LA (500µmol/l) was paralleled by reduction of NF-
B. Additionally, LA's anti-apoptotic activity was reduced by phosphatidylinositol 3-kinase (PI-3kinase) inhibitors (Wortmannin, L294002), being in line with LA-induced Akt phosphorylation (Ser437: +159±43%; Thr308: +98±25%; p<0.01). LA (500µmol/l) inhibited (p<0.001) proliferation of macrovascular- (-29±3%) and microvascular endothelial cells (-29±3%) by arresting the cells at the G1/S-transition due to an increased ratio of cyclin E/p27Kip (4.2-fold), to upregulation of p21WAF-1/Cip1 (+104±21%), and to reduced expression of cyclin A (-32±11%), of hyper-phosphorylated Retinoblastoma protein (macECs: -51±7%, micECs: -50±15%) and of E2F-1 (macECs: -48±3%, micECs: -31±10%).
LA's ability to inhibit apoptosis and proliferation of endothelial cells could beneficially affect endothelial dysfunction, which precedes manifestation of late diabetic vascular complications.
