The Vitamin E Analog Gamma-Tocotrienol (GT3) and Statins Synergistically Up-Regulate Endothelial Thrombomodulin (TM)

Abstract

Statins; a class of routinely prescribed cholesterol-lowering drugs; inhibit 3-hydroxy-3-methylglutaryl-coenzymeA reductase (HMGCR) and strongly induce endothelial thrombomodulin (TM); which is known to have anti-inflammatory; anti-coagulation; anti-oxidant; and radioprotective properties. However; high-dose toxicity limits the clinical use of statins. The vitamin E family member gamma-tocotrienol (GT3) also suppresses HMGCR activity and induces TM expression without causing significant adverse side effects; even at high concentrations. To investigate the synergistic effect of statins and GT3 on TM; a low dose of atorvastatin and GT3 was used to treat human primary endothelial cells. Protein-level TM expression was measured by flow cytometry. TM functional activity was determined by activated protein C (APC) generation assay. Expression of Kruppel-like factor 2 (KLF2), one of the key transcription factors of TM, was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). TM expression increased in a dose-dependent manner after both atorvastatin and GT3 treatment. A combined treatment of a low-dose of atorvastatin and GT3 synergistically up-regulated TM expression and functional activity. Finally; atorvastatin and GT3 synergistically increased KLF2 expression. These findings suggest that combined treatment of statins with GT3 may provide significant health benefits in treating a number of pathophysiological conditions; including inflammatory and cardiovascular diseases.

Keywords: Kruppel-like transcription factors; activated protein C; endothelial cells; gamma-tocotrienol; statins; thrombomodulin.

Figure 1

Effect of various doses of atorvastatin and gamma-tocotrienol (GT3) on thrombomodulin (TM) expression. Human umbilical vein endothelial cells (HUVECs) were either treated with vehicle or different concentrations (1, 2.5, and 5 μM) of atorvastatin (AT) (A) or various doses (1, 2.5, and 5 μM) of GT3 (B) for 24 h. TM expression was measured by flow cytometry using an anti-TM antibody, CD141 (an alternate name of TM). TM protein expression was measured as mean florescence intensity (MFI) against its respective isotype control. The data represent mean ± standard deviation (SD) of three independent experiments and each experiment consisted of three replicates per sample. Differences in TM expression between vehicle-treated and drug-treated groups were analyzed by Student’s t-test; p values less than 0.05 were considered statistically significant.

Figure 2

Combined effect of atorvastatin and GT3 on TM expression. TM expression as detected by flow cytometry in HUVECs pretreated with either vehicle (Veh), 1 μM GT3, 1 μM atorvastatin (AT), or 1 μM AT plus 1 μM GT3 for 24 h (A) and with either vehicle; 2.5 μM GT3, 2.5 μM AT, or 2.5 μM AT plus 2.5 μM GT3 for 24 h (B). TM expression was measured by flow cytometry using an anti-TM antibody, CD141. TM protein expression was measured as mean florescence intensity (MFI) against its respective isotype control. The data represent mean ± SD of three independent experiments and each experiment consisted of three replicates per sample. Differences in TM expression between vehicle-treated and drug-treated groups were analyzed by Student’s t-test; p values less than 0.05 were considered statistically significant.

Figure 3

Combined effect of atorvastatin and GT3 on the functional activity of TM. Functional activity of TM as detected by activated protein C (APC) generation in HUVECs pretreated with either vehicle, 1 μM GT3, 1 μM atorvastatin (AT), and 1 μM GT3 plus 1 μM AT (A); or vehicle, 2.5 μM GT3, 1 μM AT, and 2.5 μM GT3 plus 1 μM AT (B); or vehicle, 5 μM GT3, 1 μM AT, and 5 μM GT3 plus 1 μM AT (C); or vehicle, 2.5 μM GT3, 2.5 μM AT, or 2.5 μM GT3 plus 2.5 μM AT for 24 h (D). The data are presented as mean ± SD of optical density from three independent experiments, each consisting of three to six replicates per sample.

Figure 4

Combined effect of atorvastatin and GT3 on KLF2 mRNA expression. Kruppel-like factor 2 (KLF2) expression as detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) at 6 h (A); 12 h (B); and 24 h (C) in HUVECs pretreated with either vehicle, 2.5 μM GT3, 2.5 μM atorvastatin (AT), or 2.5 μM AT plus 2.5 μM GT3 (AT + GT3) for 24 h. KLF2 levels are expressed as fold change over the basal expression after normalization to 18 s. The data are presented as the mean ± SD of three independent experiments. NS, not statistically significant.

Figure 5

A schematic representation of the combined effect of atorvastatin and GT3 on TM expression and function as well as KLF2 expression. A more pronounced effect on TM expression and function and KLF2 expression was observed after combined treatment with atorvastatin (AT) and GT3 (middle column, indicated by two arrows) than after treatment with each drug alone (left and right panel, represented by single arrow).