Yung Hsiang Chen

Salvianolic acid B attenuates VCAM-1 and ICAM-1 expression in TNF-α-treated human aortic endothelial cells

Attachment to, and migration of leukocytes into the vessel wall is an early event in atherogenesis. Expression of cell adhesion molecules by the arterial endothelium may play a major role in atherosclerosis. It has been suggested that antioxidants inhibit the expression of adhesion molecules and may thus attenuate the processes leading to atherosclerosis. In the present study, the effects of a potent water‐soluble antioxidant, salvianolic acid B (Sal B), and an aqueous ethanolic extract (SME), both derived from a Chinese herb, Salvia miltiorrhiza, on the expression of endothelial‐leukocyte adhesion molecules by tumor necrosis factor‐α (TNF‐α)‐treated human aortic endothelial cells (HAECs) were investigated. When pretreated with SME (50 and 100 μg/ml), the TNF‐α‐induced expression of vascular adhesion molecule‐1 (VCAM‐1) was notably attenuated (77.2u2009±u20093.2% and 80.0u2009±u20092.2%, respectively); and with Sal B (1, 2.5, 5, 10, and 20 μg/ml), 84.5u2009±u20091.9%, 78.8u2009±u20091.2%, 58.9u2009±u20090.4%, 58.7u2009±u20090.9%, and 57.4u2009±u20090.3%, respectively. Dose‐dependent lowering of expression of intercellular cell adhesion molecule‐1 (ICAM‐1) was also seen with SME or Sal B. In contrast, the expression of endothelial cell selectin (E‐selectin) was not affected. SME (50 μg/ml) or Sal B (5 μg/ml) significantly reduced the binding of the human monocytic cell line, U937, to TNF‐α‐stimulated HAECs (45.7u2009±u20092.5% and 55.8u2009±u20091.2%, respectively). SME or Sal B significantly inhibited TNF‐α‐induced activation of nuclear factor kappa B (NF‐κB) in HAECs (0.36‐ and 0.48‐fold, respectively). These results demonstrate that SME and Sal B have anti‐inflammatory properties and may explain their anti‐atherosclerotic properties. This new mechanism of action of Sal B and SME, in addition to their previously reported inhibition of LDL, may help explain their efficacy in the treatment of atherosclerosis. J. Cell. Biochem. 82:512–521, 2001.

Ginkgo biloba Extract Inhibits Tumor Necrosis Factor-α–Induced Reactive Oxygen Species Generation, Transcription Factor Activation, and Cell Adhesion Molecule Expression in Human Aortic Endothelial Cells

Objective—This study was conducted to examination whether Ginkgo biloba extract (GBE), a Chinese herb with antioxidant activity, could reduce cytokine-induced monocyte/human aortic endothelial cell (HAEC) interaction, a pivotal early event in atherogenesis. Methods and Results—Pretreatment of HAECs with GBE (50 and 100 &mgr;g/mL for 18 hours) significantly suppressed cellular binding between the human monocytic cell line U937 and tumor necrosis factor-&agr; (TNF-&agr;)-stimulated HAECs by using in vitro binding assay (68.7% and 60.1% inhibitions, respectively). Cell enzyme–linked immunosorbent assay and immunoblot analysis showed that GBE (50 &mgr;g/mL for 18 hours) significantly attenuated TNF-&agr;–induced cell surface and total protein expression of vascular cellular adhesion molecule-1 and intracellular adhesion molecule-1 (63.5% and 69.2%, respectively; P <0.05). However, pretreatment with probucol (5 &mgr;mol/L for 18 hours) reduced the expression of vascular cellular adhesion molecule-1 but not intracellular adhesion molecule-1. Preincubation of HAECs with GBE or probucol significantly reduced intracellular reactive oxygen species formation induced by TNF-&agr; (76.8% and 68.2% inhibitions, respectively; P <0.05). Electrophoretic mobility shift assay demonstrated that both GBE and probucol inhibited transcription factor nuclear factor-&kgr;B activation in TNF-&agr;–stimulated HAECs (55.2% and 65.6% inhibitions, respectively) but only GBE could inhibit the TNF-&agr;–stimulated activator protein 1 activation (45.1% inhibition, P <0.05). Conclusions—GBE could reduce cytokine-stimulated endothelial adhesiveness by downregulating intracellular reactive oxygen species formation, nuclear factor-&kgr;B and activator protein 1 activation, and adhesion molecule expression in HAECs, supporting the notion that the natural compound Ginkgo biloba may have potential implications in clinical atherosclerosis disease.

Magnolol attenuates VCAM‐1 expression in vitro in TNF‐α‐treated human aortic endothelial cells and in vivo in the aorta of cholesterol‐fed rabbits

In a previous study, we showed that magnolol, a potent antioxidant derived from a Chinese herb, attenuates monocyte chemotactic protein‐1 (MCP‐1) expression and intimal hyperplasia in the balloon‐injured aorta of cholesterol‐fed rabbits. Expression of cell adhesion molecules by the arterial endothelium and the attachment of leukocytes to the endothelium may play a major role in atherosclerosis. In the present study, the effects of magnolol on the expression of endothelial‐leukocyte adhesion molecules and the activation of nuclear factor kappa B (NF‐κB) in tumour necrosis factor‐α (TNF‐α)‐treated human aortic endothelial cells (HAECs) were investigated. Pretreatment of HAECs with magnolol (5u2003μM) significantly suppressed the TNF‐α‐induced expression of vascular cell adhesion molecule‐1 (VCAM‐1) (64.8±1.9%), but had no effect on the expression of intercellular cell adhesion molecule‐1 and endothelial cell selectin. Magnolol (5 and 10u2003μM) significantly reduced the binding of the human monocytic cell line, U937, to TNF‐α‐stimulated HAECs (58.4 and 56.4% inhibition, respectively). Gel shift assays using the 32P‐labelled NF‐κB consensus sequence as probe showed that magnolol pretreatment reduced the density of the shifted bands seen after TNF‐α‐induced activation. Immunoblot analysis and immunofluorescence staining of nuclear extracts demonstrated a 58% reduction in the amount of NF‐κB p65 in the nuclei in magnolol‐treated HAECs. Magnolol also attenuated intracellular H2O2 generation in both control and TNF‐α treated HAECs. Furthermore, in vivo, magnolol attenuates the intimal thickening and TNF‐α and VCAM‐1 protein expression seen in the thoracic aortas of cholesterol‐fed rabbits. Taken together, these data demonstrate that magnolol inhibits TNF‐α‐induced nuclear translocation of NF‐κB p65 and thereby suppresses expression of VCAM‐1, resulting in reduced adhesion of leukocytes. These results suggest that magnolol has anti‐inflammatory properties and may play important roles in the prevention of atherosclerosis and inflammatory responses in vivo.

Endotoxin Induces Toll-Like Receptor 4 Expression in Vascular Smooth Muscle Cells via NADPH Oxidase Activation and Mitogen-Activated Protein Kinase Signaling Pathways

Objective—Toll-like receptor 4 (TLR4) plays a major role mediating endotoxin-induced cellular inflammation and regulates vascular smooth muscle cell (VSMC) proliferation, which is related to atherogenesis and restenosis. This study was conducted to investigate the mechanisms involved in lipopolysaccharide (LPS)-induced TLR4 expression in VSMCs. Methods and Results—Stimulation of human aortic smooth muscle cells (HASMCs) with LPS significantly increased TLR4 expression. The increase was regulated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (including the activation of subunits p47phox and Rac1), which mediates the production of reactive oxygen species and the activation of intracellular mitogen-activated protein kinase signaling pathways. Treatment with polyethylene-glycol-conjugated superoxide dismutase, N-acetylcysteine (NAC), diphenylene iodonium (DPI), or apocynin significantly decreased LPS-induced TLR4 expression. An actinomycin D chase experiment showed that LPS increased the half-life of TLR4 mRNA. Inhibition of NADPH oxidase activity by DPI, apocynin, or NAC significantly decreased TLR4 mRNA stability, as did the knock-down of RAC1 gene expression by RNA interference. We also demonstrated in an animal model that LPS administration led to a significant elevation of balloon-injury–induced neointimal hyperplasia, and of TLR4 expression, in rabbit aorta. Conclusion—These findings suggest that NADPH oxidase activation, mRNA stabilization, and MAPK signaling pathways play critical roles in LPS-enhanced TLR4 expression in HASMCs, which contributes to vascular inflammation and cardiovascular disorders.

Salvianolic acid B attenuates MMP-2 and MMP-9 expression in vivo in apolipoprotein-E-deficient mouse aorta and in vitro in LPS-treated human aortic smooth muscle cells.

Salvianolic acid B (Sal B), a water‐soluble antioxidant derived from a Chinese medicinal herb, is believed to have multiple therapeutic and preventive against human vascular diseases, including atherosclerosis and restenosis. To elucidate the underlying cellular mechanisms, we produced hypercholesterolemia by feeding apo‐E‐deficient mice a 0.15% cholesterol diet and inflammation in human aortic smooth muscle cells (HASMCs) with the endotoxin lipopolysaccharide (LPS), focusing on the metallopreteinases MMP‐2 and MMP‐9, the relevant signal transduction pathways and the effects of Sal B. Immunohistochemical analyses indicated apo‐E‐deficient mice fed a 0.15% cholesterol diet for 12 weeks exhibited thickened intima and elevated levels of MMP‐2 and MMP‐9 in aortic sections, both of which were attenuated by 0.3% Sal B dietary supplement. Western blotting and zymography analyses indicated that unstimulated HASMCs exhibited basal levels of protein and activity levels for MMP‐2 and barely detectable levels for MMP‐9, both of which were markedly upregulated by LPS, which also induced cell migration. Sal B significantly attenuated upregulations of both MMPs as well as the LPS‐induced cell migration through the inactivation of MMP‐2 and MMP‐9 protein synthesis as well as the downregulation of the extracellular‐signal‐regulated kinase 1/2 (ERK1/2) and c‐Jun NH2‐terminal kinase (JNK). These results demonstrate that Sal B has anti‐migration properties on smooth muscle cells and may explain its anti‐atherosclerotic properties. This novel mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis. J. Cell. Biochem. 100: 372–384, 2007.

Salvianolic acid B attenuates cyclooxygenase-2 expression in vitro in LPS-treated human aortic smooth muscle cells and in vivo in the apolipoprotein-E-deficient mouse aorta.

Inflammation plays an essential role in atherosclerosis and post‐angioplasty restenosis and the synthesis and release of inflammatory cytokines from vascular smooth muscle cells is an important contributor to these pathologies. It is assumed that drugs that prevent the overproduction of inflammatory cytokines may inhibit cardiovascular disorders. In the present study, the effects of a water‐soluble antioxidant, salvianolic acid B (Sal B), derived from a Chinese herb, on the expression of cyclooxygenase (COX) in lipopolysaccharide (LPS)‐treated human aortic smooth muscle cells (HASMCs) and in the aortas of cholesterol‐fed apoE deficient mice were investigated. In unstimulated HASMCs, COX‐2 mRNA and protein were almost undetectable, but were strongly upregulated in response to LPS. In contrast, HASMCs with or without LPS treatment showed constitutive expression of COX‐1 mRNA and protein. The activation of COX‐2 protein synthesis in LPS‐stimulated HASMCs was shown to involve the activation of the extracellular‐signal‐regulated kinase 1/2 (ERK1/2), c‐Jun NH2‐terminal kinase (JNK), and p38 mitogen‐activated protein kinase pathway. Incubation of HASMCs with Sal B before LPS stimulation resulted in pronounced downregulation of COX‐2 expression. Sal B treatment suppressed ERK1/2 and JNK phosphorylation and attenuated the increase in prostaglandin E2 production and NADPH oxidase activity in LPS‐treated HASMCs. When apoE‐deficient mice were fed a 0.15% cholesterol diet with or without supplementation with 0.3% Sal B for 12 weeks, the intima/media area ratio in the thoracic aortas was significantly reduced in the Sal B group (0.010u2009±u20090.009%) compared to the apoE‐deficient group (0.114u2009±u20090.043%) and there was a significant reduction in COX‐2 protein expression in the thickened intima. These results demonstrate that Sal B has anti‐inflammatory properties and may explain its anti‐atherosclerotic properties. This new mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis. J. Cell. Biochem. 98: 618–631, 2006.

The role of human antigen R, an RNA-binding protein, in mediating the stabilization of toll-like receptor 4 mRNA induced by endotoxin: A novel mechanism involved in vascular inflammation

Objective—Lipopolysaccharide (LPS) interacts with toll-like receptor 4 (TLR4) and induces proliferation of vascular smooth muscle cells (VSMCs) which plays a causal role in atherogenesis. The role of TLR4 expression and regulation in LPS-stimulated VSMCs remains unclear. TLR4 mRNAs often contain AU-rich elements (AREs) in their 3′ untranslated regions (3′UTR) which have a high affinity for RNA-binding proteins. It is not know whether the RNA-binding protein, human antigen R (HuR), regulates TLR4 expression in human aortic smooth muscle cells (HASMCs). Methods and Results—Stimulation of HASMCs with LPS significantly increased the cytosolic HuR level in vitro. Immunoprecipitation and RT-PCR demonstrated that LPS markedly increased the interaction of HuR and 3′UTR of TLR4 mRNA. The reporter plasmid, which contains the 3′UTR of TLR4 mRNA, significantly increased luciferase reporter gene expression in LPS-induced HASMCs. These data suggest that the 3′UTR of TLR4 mRNA confers LPS responsiveness and that HuR modulates 3′UTR-mediated gene expression. Knock-down of HuR inhibited LPS-induced TLR4 mRNA stability in HASMCs and luciferase reporter gene expression in CMV-Luciferase-TLR4 3′UTR-transfected HASMCs. In addition, inhibition of NADPH oxidase activity by diphenylene iodonium, knock-down of Rac1 gene expression by siRNA, and decrease of p38 MAPK activity by SB203580 significantly decreased the cytosolic HuR level, which mediates TLR4 mRNA stability. Conclusion—Activation of NADPH oxidase and the MAPK-signaling pathway contribute to HuR-mediated stabilization of TLR4 mRNA induced by LPS in HASMCs. In the balloon injured rabbit aorta model, systemic inflammation induced by LPS caused intimal hyperplasia and increased TLR4 and HuR expression.

Expression of interleukin‐1β and interleukin‐1 receptor antagonist in oxLDL‐treated human aortic smooth muscle cells and in the neointima of cholesterol‐fed endothelia‐denuded rabbits

The migration of vascular smooth muscle cells (VSMCs) from the media to the intima and the proliferation of intimal VSMCs are key events in restenotic lesion development. These events, which are preceded and accompanied by inflammation, are modulated by the proinflammatory cytokine, interleukin‐1β (IL‐1β), which induces vascular smooth muscle cells to express adhesion molecules and to proliferate. IL‐1β action is complex and regulated, in part, by its naturally occurring inhibitor, the IL‐1 receptor antagonist (IL‐1ra). Whether there was a temporal and spatial correlation between IL‐1β and IL‐1ra expression in, and release by, oxidized low density lipoproteins (oxLDL)‐stimulated human aortic smooth muscle cells (HASMCs) was determined by using ELISA and Western blot. In addition, IL‐1β and IL‐1ra expression was detected in the neointima of endothelia‐denuded cholesterol‐fed New Zealand white rabbits by immunohistochemistry and Western blot. In HASMCs, oxLDL induced IL‐β and IL‐1ra expression and release in a dose‐ and time‐dependent manner. Treatment with 20 μg/ml oxLDL resulted in increased IL‐1β release after 6 h, which peaked at 24 h, and in increased IL‐1ra release, first seen after 12 h, but continuing to increase for at least 48 h. In the cells, IL‐β expression showed a similar pattern to release, whereas IL‐1ra expression was seen in unstimulated cells and was not increased by oxLDL treatment. Confocal microscopy showed colocalization of IL‐β and IL‐1ra expression in oxLDL‐stimulated HASMCs. oxLDL caused significant induction of nuclear factor kappa B and activator protein‐1 DNA binding activity in HASMCs (6.6‐ and 3.3‐fold, respectively). In cholesterol‐fed endothelia‐denuded rabbits, the notably thickened intima showed significant IL‐1β and IL‐1ra expression. These results provide further support for the role of IL‐1 system in the pathogenesis of restenosis. This is the first demonstration of IL‐1β and IL‐1ra expression and secretion of oxLDL‐treated HASMCs and their expression in the rabbit neointima, suggesting that the smooth muscle cells of the intima are an important source of these factors.

Carvedilol Inhibits Tumor Necrosis Factor-α–Induced Endothelial Transcription Factor Activation, Adhesion Molecule Expression, and Adhesiveness to Human Mononuclear Cells

Objective—We tested the hypothesis that carvedilol, a &bgr;-adrenoceptor and &agr;-adrenoceptor antagonist with potent antioxidant property, could inhibit tumor necrosis factor-&agr; (TNF-&agr;)–induced endothelial adhesiveness to human mononuclear cells (MNCs), an early sign of atherogenesis. Methods and Results—Circulating MNCs were isolated from the peripheral blood of healthy subjects. Compared with control condition, pretreatment of carvedilol (10 &mgr;mol/L for 18 hours) or probucol (5 &mgr;mol/L for 18 hours), but not propanolol, prazosin, or both propanolol and prazosin significantly decreased TNF-&agr;–stimulated adhesiveness of cultured human aortic endothelial cells (HAECs) to MNCs. Carvedilol inhibited TNF-&agr;–stimulated endothelial vascular cell adhesion molecule-1 (VCAM-1) and E-selectin (66.0±2.0% and 55.60±1.0% of control, P<0.05, respectively) expression, whereas probucol inhibited only VCAM-1 expression (79.0±5.0% of control, P<0.05). Propanolol, prazosin, or both did not alter the expression of adhesion molecules. Further, pretreatment with carvedilol significantly inhibited TNF-&agr;–stimulated intracellular reactive oxygen species (ROS) production and the activation of redox sensitive nuclear factor kappa B and activator protein-1 transcription pathways. Conclusions—Carvedilol reduced TNF-&agr;–stimulated endothelial adhesiveness to human MNCs by inhibiting intracellular ROS production, transcription factor activation, and VCAM-1 as well as E-selectin expression, suggesting its potential role in clinical atherosclerosis disease.

Effects of Ginkgo biloba extract on the proliferation of vascular smooth muscle cells in vitro and on intimal thickening and interleukin‐1β expression after balloon injury in cholesterol‐fed rabbits in vivo

Restenosis may develop in response to cytokine activation and smooth muscle cell proliferation. Ginkgo biloba extract (EGb) has been used to treat cardiovascular and cerebrovascular diseases. In the present study, the effects of EGb on the growth of cultured vascular smooth muscle cells (VSMC), as well as on the expression of interleukin‐1β (IL‐1β) and the intimal response in balloon‐injured arteries of cholesterol‐fed rabbits, were investigated. Using bromodeoxyuridine incorporation as an index of cell proliferation, EGb was found to inhibit serum‐induced mitogenesis of cultured rat aorta VSMC in a dose‐dependent manner. In vivo, EGb and probucol (u2009positive control) reduced the atheroma area in thoracic aortas of male New Zealand white rabbits fed a 2% cholesterol diet for 6 weeks with balloon denudation of the abdominal aorta being performed at the end of the third week. Intimal hyperplasia, expressed as the intimal/medial area ratio, in the abdominal aortas was significantly inhibited in the both the EGb group (0.61u2009±u20090.06) and the probucol group (0.55u2009±u20090.03) compared to the C group (0.87u2009±u20090.02). In the balloon‐injured abdominal aorta, both EGb and probucol significantly reduced IL‐1β mRNA and protein expression and the percentage of proliferating cells. The inhibitory effects of EGb on the intimal response might be attributed to its antioxidant capacity. EGb may have therapeutic potential for the prevention of restenosis after angioplasty. J. Cell. Biochem. 85: 572–582, 2002.