Yuh-Lien Chen

High Glucose Impairs Early and Late Endothelial Progenitor Cells by Modifying Nitric Oxide–Related but Not Oxidative Stress–Mediated Mechanisms

OBJECTIVE—Endothelial progenitor cells (EPCs) are impaired in diabetes. This study aimed to investigate the direct effects of high glucose on EPCs. RESEARCH DESIGN AND METHODS—Mononuclear cells isolated from healthy subjects were incubated with glucose/mannitol or drugs for EPC study. After 4 days of culture, attached early EPCs appeared. The monolayer late EPCs with cobblestone shape appeared at 2–4 weeks. Various immunofluroscence stainings were used to characterize the early and late EPCs. Senescence assay and the activity of endothelial nitric oxide synthase (eNOS) were determined. Migration and tube formation assay were done to evaluate the capacity for vasculogenesis in late EPCs. RESULTS—Chronic incubation with high glucose but not mannitol (osmotic control) dose-dependently reduced the number and proliferation of early and late EPCs, respectively. High glucose enhanced EPC senescence and impaired the migration and tube formation of late EPCs. High glucose also decreased eNOS, FoxO1, and Akt phosphorylation and bioavailable nitric oxide (NO) in both EPCs. The effects of high glucose could be ameliorated by coincubation with NO donor sodium nitroprusside or p38 mitogen–activated protein kinase inhibitor and deteriorated by eNOS inhibitor or PI3K (phosphatidylinositol 3′-kinase) inhibitor. Antioxidants including vitamin C, N-acetylcysteine–and polyethylene glycol (PEG)-conjugated superoxide dismutase, and PEG-catalase had no effects, whereas pyrrolidine dithiocarbamate, diphenyleneiodonium, apocynin, and rotenone even deteriorated the downregulation of both EPCs. CONCLUSIONS—High glucose impaired the proliferation and function of early and late EPCs. NO donor but not antioxidants reversed the impairments, suggesting the role of NO-related rather than oxidative stress–mediated mechanisms in hyperglycemia-caused EPC downregulation.

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 (77.2 ± 3.2% and 80.0 ± 2.2%, respectively); and with Sal B (1, 2.5, 5, 10, and 20 μg/ml), 84.5 ± 1.9%, 78.8 ± 1.2%, 58.9 ± 0.4%, 58.7 ± 0.9%, and 57.4 ± 0.3%, 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 (45.7 ± 2.5% and 55.8 ± 1.2%, 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.

The cytotoxicity of corrosion products of nitinol stent wire on cultured smooth muscle cells.

Although nitinol is one of most popular materials of intravascular stents, there are still few confirmative biocompatibility data available, especially in vascular smooth muscle cells. In this report, the nitinol wires were corroded in Dulbeccos modified Eagles medium with constant electrochemical breakdown voltage and the supernatant and precipitates of corrosion products were prepared as culture media. The dose and time effects of different concentrations of corrosion products on the growth and morphology of smooth muscle cells were evaluated with [(3)H]-thymidine uptake ratio and cell cycle sorter. Both the supernatant and precipitate of the corrosive products of nitinol wire were toxic to the primary cultured rat aortic smooth muscle cells. The growth inhibition was correlated well with the increased concentrations of the corrosion products. Although small stimulation was found with released nickel concentration of 0.95 +/- 0.23 ppm, the growth inhibition became significant when the nickel concentration was above 9 ppm. The corrosion products also altered cell morphology, induced cell necrosis, and decreased cell numbers. The cell replication was inhibited at the G0-G1 to S transition phase. This was the first study to demonstrate the cytotoxicity of corrosion products of current nitinol stent wire on smooth muscle cells, which might affect the postimplantation neointimal hyperplasia and the patency rate of cardiovascular stents.

Increased ferritin gene expression in atherosclerotic lesions

To identify genes potentially implicated in atherogenesis, a cDNA library was constructed from human atherosclerotic aorta and differentially screened with 32P-labeled-cDNAs prepared from human normal and atherosclerotic aortas. Two cDNA clones exhibiting higher hybridization to the 32P-labeled cDNAs from atherosclerotic vessels were isolated and identified to be genes encoding L-ferritin and H-ferritin, respectively. Northern blot analysis confirmed that the expression of both ferritin genes was notably higher in human and rabbit atherosclerotic aortas than in their normal counterparts. A time-course study illustrated that both L- and H-ferritin mRNAs were markedly increased in aortas of rabbits after feeding with a high cholesterol diet for 6 wk, which was also the time period after which the formation of lesions became evident. In situ hybridization revealed that both L- and H-ferritin mRNAs were induced in endothelial cells and macrophages of human early lesions. The signals were also detected in the smooth muscle cells of advanced lesions. Immunostaining further identified the presence of ferritin protein in atherosclerotic lesions. On the other hand, Prussian blue stain revealed the presence of iron deposits in advanced lesions but not in early human or rabbit lesions. Further experiments with cultured human monocytic THP-1 cells and aortic smooth muscle cells demonstrated that ferritin mRNAs were subjected to up-regulation by treatment with IL-1 or TNF, while TGF, PDGF, and oxidized LDL did not affect the expression of either ferritin gene in both cell lines. Collectively, these results clearly demonstrate that ferritin genes are susceptible to induction in the course of plaque formation.

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.

Overexpression of HO-1 Protects against TNF-α-Mediated Airway Inflammation by Down-Regulation of TNFR1-Dependent Oxidative Stress

Oxidative stresses are believed to play an important role in the induction of both cell adhesion molecules and pro-inflammatory cytokines, a key event in a variety of inflammatory processes. The enzyme heme oxygenase-1 (HO-1) functions as an antioxidant and serves to protect against tissue injury. In this study, we report that HO-1 was induced in cultured human tracheal smooth muscle cells after either treatment with a potent inducer of HO-1 activity, cobalt protoporphyrin IX, or infection with a recombinant adenovirus that carries the human HO-1 gene. Overexpression of HO-1 protected against tumor necrosis factor (TNF)-alpha-mediated airway inflammation via the down-regulation of oxidative stress, adhesion molecules, and interleukin-6 in both cultured human tracheal smooth muscle cells and the airways of mice. In addition, HO-1 overexpression inhibited TNF-alpha-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression, adherence of THP-1 cells, generation of interleukin-6, p47(phox) translocation, and nuclear factor-kappaB activation. HO-1 overexpression also attenuated TNF-alpha-induced oxidative stress, which was abrogated in the presence of both the HO-1 inhibitor, zinc protoporphyrin IX, as well as a carbon monoxide scavenger. In addition, HO-1 overexpression reduced the formation of a TNFR1/c-Src/p47(phox) complex. These results suggest that HO-1 functions as a suppressor of TNF-alpha signaling, not only by inhibiting the expression of adhesion molecules and generation of interleukin-6, but also by diminishing intracellular reactive oxygen species production and nuclear factor-kappaB activation in both cultured human tracheal smooth muscle cells and the airways of mice.

Superoxide Dismutase Inhibits the Expression of Vascular Cell Adhesion Molecule-1 and Intracellular Cell Adhesion Molecule-1 Induced by Tumor Necrosis Factor-α in Human Endothelial Cells Through the JNK/p38 Pathways

Objective— Expression of adhesion molecules on endothelial cells and subsequent leukocyte recruitment are critical early events in the development of atherosclerosis. We tried to study possible effects of Cu/Zn superoxide dismutase (SOD) on adhesion molecule expression and its underlying mechanism in the prevention and treatment of cardiovascular disorders. Methods and Results— Human aortic endothelial cells (HAECs) were transfected with adenovirus carrying the human SOD gene (AdSOD) to investigate whether SOD expression in HAECs attenuated tumor necrosis factor (TNF)-&agr;–induced reactive oxygen species production and adhesion molecule expression and to define the mechanisms involved. SOD expression significantly suppressed TNF-&agr;–induced expression of vascular cell adhesion molecule-1 and intercellular cell adhesion molecule-1 and reduced the binding of the human neutrophils to TNF-&agr;–stimulated HAECs. SOD expression suppressed c-JUN N-terminal kinase and p38 phosphorylation. It also attenuated intracellular superoxide anion production and NADPH oxidase activity in TNF-&agr;–treated HAECs. Conclusions— These results provide evidence that SOD expression in endothelial cells attenuates TNF-&agr;–induced superoxide anion production and adhesion molecule expression, and that this protective effect is mediated by decreased JNK and p38 phosphorylation and activator protein-1 and nuclear factor &kgr;B inactivation. These results suggest that SOD has antiinflammatory properties and may play important roles in the prevention of atherosclerosis and inflammatory response.

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 (5 μM) 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 10 μM) 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.

Increased corrosion resistance of stent materials by converting current surface film of polycrystalline oxide into amorphous oxide

Current efforts of new stent technology have been aimed largely at the improvement of intravascular stent biocompatibility. Among the chemical characteristics of metallic stents, surface oxide corrosion properties are paramount. Using our unique technique, the currently marketed 316 L stainless steel and nitinol stent wires covered with polycrystalline oxide were chemically etched and then passivated to form amorphous oxide. Excellent metallic-stent corrosion resistance with an amorphous oxide surface was demonstrated in our previous in vitro study. For in vivo validation, we compared the corrosion behavior of different oxide surfaces on various forms of test wires in the abdominal aorta of mongrel dogs using open-circuit potential and cyclic anodic polarization measurements. After conduction, the retrieved test wires were observed under scanning electron microscope. No passivity breakdown was found for wires covered with amorphous oxide, while wires with polycrystalline oxide showed breakdown at potentials between +0.2 to + 0.6 V. It has been proven that severe pitting or crevice corrosion occurred on the surface of polycrystalline oxide, while the surface of amorphous oxide was free of degradations in our experiment. We have demonstrated that this amorphous oxide coating on metallic material provides better corrosion resistance, not only in vitro but also in vivo, and it is superior not only in strength safety but also in medical device biocompatibility.

Anti-Inflammatory Effects of Different Drugs/Agents with Antioxidant Property on Endothelial Expression of Adhesion Molecules

Atherosclerosis is a chronic inflammatory process. The adhesion of leukocytes to the vascular endothelium, mediated by endothelial cell adhesion molecules including vascular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin, is the pivotal early event in atherogenesis. Inflammatory cytokines could activate redox-sensitive transcription factors and induce endothelial expression of adhesion molecules, which could be inhibited to various degrees by different antioxidants suggesting the potential role of endogenous reactive oxygen species (ROS) in atherogenesis. Many clinical drugs that against cardiovascular diseases have exhibited antioxidant effects; these drugs simultaneously inhibit endothelial adhesion molecule expression, such as aspirin, probucol, HMG-CoA reductase inhibitors, angiotensin receptor blockers, angiotensin converting enzyme inhibitors, peroxisome proliferator-activated receptor alpha and gamma ligands, calcium channel blockers, beta-adrenergic blockers, etc. In addition, we have previously demonstrated that Ginkgo biloba extract, a Chinese herb with antioxidant activity, could significantly suppress inflammatory cytokine-stimulated endothelial adhesiveness to human monocytic cells by attenuating intracellular ROS formation, redox-senstive transcription factor activation, and VCAM-1 as well as ICAM-1 expression in human aortic endothelial cells. The similar anti-atherosclerosis effects have been also shown in other Chinese herbs or dietary supplements with antioxidant activity such as magnolol and salvianolic acid B either in vitro or in vivo. Thus, oxidative stress is critical to endothelial adhesiveness in atherogenesis. The inhibition of endothelial adhesion molecule expression by drugs/agents with antioxidant activity may serve as a potential therapeutic strategy for clinical atherosclerosis.