Ryuzo Abe

Olive oil polyphenol oleuropein inhibits smooth muscle cell proliferation.

OBJECTIVESnThe Mediterranean diet, with a high content of olive oil, is associated with a reduced risk of coronary artery disease. The aim of this study was to determine the effect of oleuropein, one of the polyphenols in olive oil, on vascular smooth muscle cell (SMC) proliferation in vitro.nnnDESIGNnThis was an experimental study.nnnMATERIALS AND METHODSnBovine vascular SMCs were cultured in the presence of 100 μM of oleuropein. On days 1, 3 and 5, cell number was counted. Cell cycle analysis was performed by flow cytometry. Cell cycle regulators were assessed by immunoblotting.nnnRESULTSnCell proliferation in the presence of oleuropein was significantly inhibited by 92%. Cell cycle analysis revealed that oleuropein treatment blocked cells in the G1-S phase compared with the non-treated group. Among G1 phase regulators, retinoblastoma protein, cyclinD, p21 and p27 were not affected by oleuropein, but extracellular signal-regulated kinase 1/2 (ERK1/2) activation was inhibited. Growth of SMC treated with 100 μM of the mitogen-activated protein (MAP) kinase/ERK kinase 1 (MEK1) inhibitor PD98059 was also significantly inhibited by 70%.nnnCONCLUSIONSnOleuropein inhibited SMC proliferation through a cell cycle block between the G1 and the S phases, which may be regulated by ERK1/2. These results suggest a mechanism by which olive oil consumption may have beneficial effects on cardiovascular mortality by inhibiting SMC proliferation.

Laminar shear, but not orbital shear, has a synergistic effect with thrombin stimulation on tissue factor expression in human umbilical vein endothelial cells

INTRODUCTIONnHigh levels of tissue factor (TF) have been associated with atherosclerotic plaques. The specific pathways linked to TF expression in endothelial cells (ECs) have not been well defined. This study compared TF expression in human umbilical vein ECs (HUVECs) exposed to laminar shear stress (LSS) using a parallel flow chamber and to orbital shear stress (OSS) using an orbital shaker. We also compared the effects of thrombin (TH) stimulation of ECs exposed to different shear forces on the expression of TF and investigated the role that second messengers, p38 and extracellular signal-regulated kinase 1 and 2 (ERK1/2), had in the EC response.nnnMETHODSnHUVECs were subjected to 2, 4, or 6 hours of LSS or OSS in the presence or absence of 4 U/mL of TH. Western blot analysis of ERK1/2 and p38 activation and polymerase chain reaction analysis of TF in the presence of inhibitors to these second messengers was performed in HUVECs subjected to OSS or LSS in the presence or absence of TH.nnnRESULTSnTF expression was increased and peaked at 2 hours in all HUVECs exposed to LSS or TH. Stimulation of static HUVECs with TH resulted in an increase in TF expression of 5.68 ± 1.58-, 3.80 ± 1.21-, and 2.54 ± 0.38-fold at 2, 4, and 6 hours, respectively (n = 6 experiments). In the absence of TH, HUVECs exposed to LSS demonstrated a 9.51 ± 0.62-, 7.31 ± 1.43-, and 4.39 ± 1.32-fold increase in TF expression at 2, 4, and 6 hours, respectively (n = 6 experiments). TF was increased significantly more when exposed to LSS in the presence of TH (18.85 ± 1.43-, 15.05 ± 0.95-, and 8.91 ± 1.06-fold increases at 2, 4, and 6 hours, respectively [n = 6 experiments], P < .01). Between-group analysis showed a significant difference between groups (P < .001). OSS did not significantly increase TF expression in the presence or absence of TH. ERK1/2 and p38 activation was increased in LSS and LSS + TH but not in OSS or OSS + TH (n = 3 experiments).nnnCONCLUSIONnLSS and TH independently increased TF expression, but OSS did not. LSS + TH stimulation showed a synergistic effect, which suggests that these mechanical and chemical stimuli work through different pathways or that an intracellular interaction between TH and LSS may be present that does not occur in OSS.

Pulsatile to-fro flow induces greater and sustained expression of tissue factor RNA in HUVEC than unidirectional laminar flow

Tissue factor (TF) is expressed in atherosclerotic lesions. Since mechanical forces influence endothelial cell (EC) function and are thought to account for the unique distribution of atherosclerosis in areas exposed to disturbed flow, we hypothesized that disturbed to-fro flow (TFF) and unidirectional pulsatile forward flow (PFF) would have different effects on TF expression in EC. TF RNA expression in HUVEC exposed to mechanical stress in the presence or absence of chemical stimulation with thrombin was determined. TFF induced a significantly higher TF expression than PFF that was sustained for 8 h. Combination of mechanical and chemical stimuli induced significantly higher TF expression than only mechanical stresses, and this effect was synergistic in both TFF and PFF. The MAPK p38 inhibitor SB-203580 significantly inhibited TF expression induced by mechanical and chemical stimulations, but the MEK inhibitor PD-98059 did not inhibit TF induced by TFF. Immunoblotting revealed that ERK1/2 phosphorylation induced by TFF was sustained for 120 min, whereas that induced by PFF was not. We conclude that disturbed flow induced greater and sustained amplification of TF expression, and this synergistic effect may be regulated by p38 MAPK and ERK1/2. These results provide added insight into the mechanism of atherosclerosis in areas of disturbed flow.

Olive Oil Polyphenols Differentially Inhibit Smooth Muscle Cell Proliferation through a G1/S Cell Cycle Block Regulated by ERK1/2.

We hypothesized that polyphenols contained in olive oil play a role in reducing the risk of atherosclerosis. The aim of this study was to determine if the polyphenols in olive oil, oleuropein (Ole), hydroxytyrosol (HT), and tyrosol (Tyr) could inhibit smooth muscle cell (SMC) proliferation through its influence on cell cycle regulation. Bovine vascular SMC were cultured in the presence of Ole, HT, or Tyr at concentration of 1, 10, or 100 μmol/L. On days 1, 3, and 5, numbers of cells were counted. Cell cycle analysis was performed by flow cytometry on day 1 after SMC were stained with propidium iodide. Cell populations grown in the presence of Ole or HT at 100 μmol/L concentration were significantly inhibited after 5 days of exposure. Tyr had a similar tendency but it did not attain significance. Cell cycle analysis revealed that 66% of cells were in G1 phase in Ole group, compared with 48% in control group. To examine the cell cycle block between G1 and S phases, we performed Western blotting and found that ERK1/2 activation was inhibited by Ole or HT. We conclude that olive oil polyphenols could inhibit SMC proliferation through a cell cycle block between G1 and S phases which may be regulated by ERK1/2. These results demonstrate a mechanism by which olive oil consumption may be atheroprotective by inhibiting SMC proliferation.

Varying Effects of Hemodynamic Forces on Tissue Factor RNA Expression in Human Endothelial Cells

BACKGROUNDnAtherosclerotic lesions predominantly localize in areas exposed to distinct hemodynamic conditions. In such lesions, tissue factor (TF) is over-expressed. Therefore, we hypothesized that varying types of mechanical forces may induce different effects on TF expression in endothelial cell, and may also influence the effects of chemical stimuli.nnnMATERIALS AND METHODSnTF RNA expression in human umbilical vein endothelial cells (HUVEC) exposed to mechanical stress in the presence or absence of chemical stimulation with thrombin (Th) was determined. The forces examined were: steady unidirectional laminar flow (LF), pulsatile unidirectional laminar flow (PF), constant oscillatory flow (OF), pulsatile to-fro flow (TFF), and cyclic strain (CS).nnnRESULTSnMechanical stimulation of HUVEC with LF for 2 h induced an 8.7 ± 0.7-fold increase in TF RNA expression, while PF induced 4.7 ± 0.9 and TFF induced 8.6 ± 1.7-fold, respectively. These responses were significantly higher than static controls. Exposure to OF or CS did not result in any significant increase, whereas chemical stimulation with Th led to significant TF expression (4.9 ± 0.3-fold). The combination of mechanical-chemical stimuli induced significantly higher TF expression than mechanical stresses alone, and this effect was synergistic. Combination of LF+Th for 2 h induced significantly increased TF expression (16.6 ± 1.7-fold), as did PF+Th (14.8 ± 2.4) and TFF+Th (17.4 ± 1.0). Furthermore, after 6 h exposure, only TFF demonstrated significantly higher TF expression both with and without Th.nnnCONCLUSIONSnWhile uniform laminar flow resulted in transient TF expression, disturbed flow induced sustained amplification of TF expression. Further investigation is needed to elucidate the mechanism of localized atherosclerosis in areas exposed to disturbed flow.

Cyclic strain delays the expression of tissue factor induced by thrombin in human umbilical vein endothelial cells.

Most studies of tissue factor (TF) expression in endothelial cells (EC) are performed under stationary culture conditions. The purpose of this study was to determine the influence of mechanical stimuli such as cyclic strain (CS) on the expression of TF in EC exposed to thrombin (Thr). Human umbilical vein endothelial cells (HUVEC) were exposed to 4 U·mL(-1) Thr in the presence or absence of 10% average CS at 60 cycles·min(-1) and then TF expression was measured. TF messenger RNA (mRNA) expression peaked at 2 hours in HUVEC exposed to Thr, but at 4 hours in HUVEC exposed to both Thru2009+u2009CS. TF expression was inhibited by p38 and extracellular signal-regulated protein kinase (ERK) inhibitors. For both Thr or Thru2009+u2009CS stimuli, p38 and ERK activity peaked at 5 minutes (pu2009<u20090.05). Nuclear factor-kappa B levels remained high in the Thr group but not in the Thru2009+u2009CS group, while Egr-1 levels were elevated in the Thru2009+u2009CS group. We demonstrated CS-delayed, Thr-induced TF mRNA expression in HUVEC, which may be modulated by p38 and ERK inhibitors.