Ginkgolide B Reduces Atherogenesis and Vascular Inflammation in ApoE−/− Mice

To investigate whether ginkgolide B (a platelet-activating factor inhibitor) affects vascular inflammation in atherosclerosis-prone apolipoprotein E-deficient (ApoE(-/-)) mice.Human platelets were used to evaluate the effects of ginkgolide B on platelet aggregation and signal transduction. Ginkgolide B attenuated platelet aggregation and inhibited phosphatidylinositol 3 kinase (PI3K) activation and Akt phosphorylation in thrombin- and collagen-activated platelets. ApoE(-/-) mice were administered a high-cholesterol diet for 8 weeks. Plasma platelet factor 4 (PF4) and RANTES (regulated upon activation, normal T-cell expressed, and secreted protein) were then measured using an enzyme-linked immunosorbent assay. Scanning electron microscopy and immunohistochemistry were used to determine atherosclerotic lesions. Ginkgolide B decreased plasma PF4 and RANTES levels in ApoE(-/-) mice. Scanning electron microscopic examination showed that ginkgolide B reduced aortic plaque in ApoE(-/-) mice. Immunohistochemistry analysis demonstrated that ginkgolide B diminished P-selectin, PF4, RANTES, and CD40L expression in aortic plaque in ApoE(-/-) mice. Moreover, ginkgolide B suppressed macrophage and vascular cell adhesion protein 1 (VCAM-1) expression in aorta lesions in ApoE(-/-) mice. Similar effects were observed in aspirin-treated ApoE(-/-) mice.Ginkgolide B significantly reduced atherosclerotic lesions and P-selectin, PF4, RANTES, and CD40L expression in aortic plaque in ApoE-/- mice. The efficacy of ginkgolide B was similar to aspirin. These results provide direct evidence that ginkgolide B inhibits atherosclerosis, which may be associated with inhibition of the PI3K/Akt pathway in activated platelets

Ginkgolide B Inhibits JAM-A, Cx43, and VE-Cadherin Expression and Reduces Monocyte Transmigration in Oxidized LDL-Stimulated Human Umbilical Vein Endothelial Cells

Aim. To investigate the effect of ginkgolide B on junction proteins and the reduction of monocyte migration in oxidized low-density lipoprotein- (ox-LDL-) treated endothelial cells. Methods. Human umbilical vein endothelial cells (HUVECs) were used in the present study. Immunofluorescence and Western blot were performed to determine the expression of junctional adhesion molecule-A (JAM-A), connexin 43 (Cx43), and vascular endothelial cadherin (VE-cadherin). Monocyte migration was detected by the Transwell assay. Results. ox-LDL stimulation increased JAM-A expression by 35%, Cx43 expression by 24%, and VE-cadherin expression by 37% in HUVECs. Ginkgolide B (0.2, 0.4, and 0.6 mg/mL) dose-dependently abolished the expression of these junction proteins. The monocyte transmigration experiments showed that the level of monocyte migration was sixfold higher in the ox-LDL-treated group than in the control group. Ginkgolide B (0.6 mg/mL) nearly completely abolished monocyte migration. Both ginkgolide B and LY294002 suppressed Akt phosphorylation and the expression of these junction proteins in ox-LDL-treated endothelial cells. These results suggest that the ginkgolide B-induced inhibition of junction protein expression is associated with blockade of the PI3K/Akt pathway. Conclusion. Ginkgolide B suppressed junction protein expression and reduced monocyte transmigration that was induced by ox-LDL. Ginkgolide B may improve vascular permeability in atherosclerosis

Role of Trx in nuclear translocation of pSmad3.

<p>(<b>A</b>) <i>Top</i>, Nuclear pSmad3 and Trx proteins in Ad-GFP, Ad-Trx, and Ad-TD cells. <i>Bottom</i>, Cytosolic Trx expression in Ad-GFP, Ad-Trx, and Ad-TD cells. The cells were treated with or without ox-LDL (100 µg/ml) stimulation for 6 h as indicated. <i>Left column</i>, Quantitative nuclear pSmad3 protein is shown. <i>Right column</i>, Nuclear and cytosolic Trx expression was normalized to that of the unstimulated Ad-GFP group. The numbers above the columns indicate the relative expression ratio of nuclear Trx to cytosolic Trx. The data from three separate experiments are expressed as mean ±SEM. *<i>p</i><0.05, compared with unstimulated Ad-GFP cells; ^#<i>p</i><0.05, compared with ox-LDL-stimulated Ad-GFP cells. (<b>B</b>) Phosphorylated Smad3 expression in Ad-GFP, Ad-Trx, and Ad-TD cells was assessed by immunofluorescent analysis.</p

Thioredoxin downregulates VCAM-1 and ICAM-1 expression in HUVECs.

<p>(<b>A</b>) <i>Top</i>, Immunoblot of Trx1 in HUVECs infected by GFP adenovirus (Ad-GFP), Trx adenovirus (Ad-Trx), and TD adenovirus (Ad-TD). <i>Bottom</i>, Trx1 activity in Ad-GFP, Ad-Trx, and Ad-TD cells, determined by insulin reduction-based assay. (<b>B</b>) Reactive oxygen species production in HUVECs. After 2 h stimulation of ox-LDL (100 µg/ml), ROS production was analyzed by measuring the mean fluorescence intensity using flow cytometry. (<b>C</b>, <b>D</b>) Immunoblot of VCAM-1 and ICAM-1 in Ad-GFP, Ad-Trx, and Ad-TD cells under basal conditions and after 4 h ox-LDL (100 µg/ml) stimulation. Relative VCAM-1 and ICAM-1 expression was determined by densitometric analysis. In all of the histograms, each value represents the mean ±SEM (<i>n</i> = 3 independent measurements). *<i>p</i><0.05, compared with unstimulated Ad-GFP cells; ^#<i>p</i><0.05, compared with ox-LDL-stimulated Ad-GFP cells. (<b>E</b>) U937 monocyte adhesion assay in ox-LDL-stimulated Ad-GFP, Ad-Trx, and Ad-TD cells. U937 cells that adhered to HUVECs were observed under a fluorescent microscope at 200× magnification. (<b>F</b>) The intensity of fluorescence-labeled adherent U937 monocytes was measured with a fluorometer (excitation wavelength, 485 nm; emission wavelength, 530 nm). The intensity was normalized to that of control cells in each group. The data are expressed as mean ±SEMs (<i>n</i> = 5). *<i>p</i><0.05, compared with ox-LDL-treated Ad-GFP group; ^#<i>p</i><0.05, compared with unstimulated Ad-GFP group. (<b>G</b>) <i>Top</i>, Immunoblot of Trx1, VCAM-1, and ICAM-1 in wildtype HUVECs (Wt) and HUVECs transfected by negative control siRNA (NC) or Trx siRNA (si-Trx). <i>Bottom</i>, Relative Trx, VCAM-1, and ICAM-1 expression was determined by densitometric analysis. In all of the histograms, each value represents the mean ±SEM (<i>n</i> = 3 independent measurements). *<i>p</i><0.05, compared with Wt cell.</p

Smad3 is a newly recognized interaction partner of Trx.

<p>Reciprocal immunoprecipitation was performed using an anti-Trx antibody. Smad3 and pSmad3 were co-immunoprecipitated with Trx. Rabbit IgG served as a negative control. (<b>A, B</b>) Immunoblot with anti-Trx, anti-Smad3, or anti-pSmad3 antibody in wildtype HUVECs under basal or ox-LDL-stimulated conditions. The data from three separate experiments are expressed as mean ±SEM. *<i>p</i><0.05, compared with unstimulated HUVECs. (<b>C, D</b>) Immunoblot with anti-Trx, anti-Smad3, or anti-pSmad3 antibody in Ad-GFP (1, 2, 5, 6), Ad-Trx (3, 7), and Ad-TD (4, 8) cells under basal or ox-LDL-stimulated conditions. The data from three separate experiments are expressed as mean ±SEM. *<i>p</i><0.05, compared with unstimulated Ad-GFP cells; ^#<i>p</i><0.05, compared with unstimulated Ad-Trx cells; ^$<i>p</i><0.05, compared with unstimulated Ad-TD cells.</p

Effect of Trx on the expression of pSmad3 and Smad3 in HUVECs stimulated with ox-LDL and nLDL.

<p>(<b>A</b>) Immunoblot of pSmad3 and Smad3 expression in ox-LDL-stimulated Ad-GFP, Ad-Trx, and Ad-TD HUVECs. (<b>B</b>) Immunoblot of pSmad3 and Smad3 expression in nLDL-treated Ad-GFP, Ad-Trx, and Ad-TD HUVECs. Relative pSmad3 and Smad3 content was determined by densitometric analysis. The data from three separate experiments are expressed as mean ±SEM. *<i>p</i><0.05, compared with unstimulated Ad-GFP cells (Smad3/β-actin); ^#<i>p</i><0.05, compared with ox-LDL-stimulated Ad-GFP cells (pSmad3/Smad3); ^&<i>p</i><0.05, unstimulated cells compared with ox-LDL stimulated cells (Smad3/β-actin).</p

Role of SIS3 in VCAM-1 and ICAM-1 expression in HUVECs stimulated with ox-LDL.

<p>(<b>A</b>) Overexpression of Trx suppressed ox-LDL-induced VCAM-1 expression. The pretreatment of Ad-Trx cells with SIS3 for 1 h completely reversed the inhibitory effect of Trx on VCAM-1 expression. (<b>B</b>) The overexpression of Trx inhibited ox-LDL-induced ICAM-1 expression. The pretreatment of Ad-Trx cells with SIS3 reversed the inhibitory effect of Trx on ICAM-1 expression. The relative expression of VCAM-1 and ICAM-1 was determined by densitometric analysis. The data from three separate experiments are expressed as mean ±SEM. *<i>p</i><0.05, compared with unstimulated Ad-GFP cells; ^#<i>p</i><0.05, compared with ox-LDL-stimulated Ad-GFP cells; ^$<i>p</i><0.05, compared with ox-LDL-stimulated Ad-Trx cells.</p

Ginkgolide B inhibits platelet and monocyte adhesion in TNFα-treated HUVECs under laminar shear stress

Abstract Background Endothelial cells are sensitive to changes in both blood components and mechanical stimuli. Endothelial cells may undergo phenotypic changes, such as changes in adhesion protein expression, under different shear stress conditions. Such changes may impact platelet and monocyte adhesion to endothelial cells. This phenomenon is linked to chronic vascular inflammation and the development of atherosclerosis. In the present study, we investigated the effects of ginkgolide B on platelet and monocyte adhesion to human umbilical vein endothelial cells (HUVECs) under different conditions of laminar shear stress. Methods Platelet and monocyte adhesion to endothelial cells was determined by the Bioflux 1000. HUVECs were incubated with ginkgolide B or aspirin for 12 h, and then TNFα was added for 2 h to induce the inflammatory response under conditions of 1 and 9 dyn/cm2 laminar shear stress. The protein expression was analyzed by Western blot. Results The number of platelets that adhered was greater under conditions of 1 dyn/cm2 than under conditions of 9 dyn/cm2 of laminar shear stress (74.8 ± 19.2 and 59.5 ± 15.1, respectively). Ginkgolide B reduced the tumor necrosis factor α (TNFα)-induced increase in platelet and monocyte adhesion to HUVECs at 1 and 9 dyn/cm2 of laminar shear stress. In TNFα-treated HUVECs, the number of monocytes that adhered was greater under conditions of 1 dyn/cm2 of laminar shear stress compared with 9 dyn/cm2 (29.1 ± 4.9 and 22.7 ± 3.7, respectively). Ginkgolide B inhibited the TNFα-induced expression of vascular cell adhesion molecule-1(VCAM-1), VE-cadherin, and Cx43 in HUVECs at 1 and 9 dyn/cm2. The expression of these proteins was not different between 1 and 9 dyn/cm2. Conclusions Ginkgolide B suppressed platelet and monocyte adhesion under different conditions of laminar shear stress. Moreover, ginkgolide B reduced VCAM-1, VE-cadherin and Cx43 expression in TNFα-treated HUVECs under laminar shear stress. This suggested that ginkgolide B might shed light on the treatment of inflammation in atherosclerosis