Jaw-Wen Chen

Regulation of Cardiomyocyte Apoptotic Signaling by Insulin-like Growth Factor I

Apoptosis is regulated by specific intracellular signaling pathways. The development of cardiomyopathy involves the apoptosis of cardiomyocytes; however, the details of their apoptotic signaling are not yet known. Insulin-like growth factor I (IGF I) is an important survival growth factor for myocardium and other tissues, but the effects of IGF I on apoptotic signaling remain largely unknown. To study apoptotic signaling pathways in cardiomyocytes and to understand IGF I actions on the apoptotic signaling of cardiac muscle cells, we have defined the effects of IGF I on Bcl-2, Bax, caspase 3, DNA fragmentation, and cell survival in primary cardiomyocytes. Compared with Bax levels, the levels of Bcl-2 were found to be quite low in these cells. Serum withdrawal and doxorubicin reduced cell viability, increased fragmentation of DNA, increased cellular contents of Bax, and activated caspase 3. IGF I enhanced cell viability, suppressed DNA fragmentation, attenuated Bax induction, and suppressed caspase 3 activation. The levels of Bcl-2-associated Bax were increased after serum withdrawal and incubation with doxorubicin and were reduced by IGF I. Thus, cardiomyocyte apoptosis induced by serum withdrawal and doxorubicin likely results, in part, from the induction of Bax and activation of caspase 3, but IGF I may inhibit cardiomyocyte apoptosis by attenuating Bax induction and caspase 3 activation. These findings provide new insight into the mechanisms of cardiomyocytes apoptosis and may help elucidate how IGF I modulates apoptotic signaling in cardiac muscle.

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.

Long-term angiotensin-converting enzyme inhibition reduces plasma asymmetric dimethylarginine and improves endothelial nitric oxide bioavailability and coronary microvascular function in patients with syndrome X

Angiotensin-converting enzyme (ACE) inhibition has been shown to improve clinical myocardial ischemia in patients with syndrome X (angina pectoris, positive treadmill exercise test, normal coronary angiograms, and no evidence of coronary spasm). This study was conducted to investigate the effects of long-term ACE inhibitors on endothelial nitric oxide (NO) metabolism and coronary microvascular function in patients with syndrome X. After a 2-week washout period, 20 patients with syndrome X were randomized to receive either enalapril, an ACE inhibitor, 5 mg twice daily (n = 10) or placebo (n = 10) in a double-blind design for 8 weeks. Another 6 age- and gender-matched subjects with negative treadmill exercise tests were also studied as controls. Compared with control subjects, patients with syndrome X had significantly reduced coronary flow reserve, reduced plasma levels of nitrate and nitrite (NOx), and a reduced plasma L-arginine to asymmetric dimethylarginine (ADMA) ratio (an index of systemic NO metabolism), as well as reduced endothelial function. These patients also had increased plasma levels of ADMA, which is an endogenous inhibitor of NO synthase and of von Willebrand factor, a marker of endothelial injury. Baseline characteristics including exercise performance and coronary flow reserve were similar between enalapril and placebo groups. After an 8-week treatment period, exercise duration (p = 0.001) and coronary flow reserve (p = 0.001) significantly improved with enalapril but not with placebo. Enalapril treatment, but not placebo, reduced plasma von Willebrand factor (p = 0.03) and ADMA levels (p = 0.01) and increased NOx levels (p = 0.01) and the ratio of L-arginine to ADMA (p <0.01). In patients with syndrome X, the plasma NOx level was positively and ADMA level inversely correlated with coronary flow reserve before and after the treatment. In conclusion, long-term ACE inhibitor treatment with enalapril improved coronary microvascular function as well as myocardial ischemia in patients with syndrome X. This may be related to the improvement of endothelial NO bioavailability with the reduction of plasma ADMA levels.

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.

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.

Matrix Metalloproteinase-9 Is Essential for Ischemia-Induced Neovascularization by Modulating Bone Marrow–Derived Endothelial Progenitor Cells

Objective—Both matrix metalloproteinases (MMPs) and endothelial progenitor cells (EPCs) have been implicated in the process of neovascularization. Here we show that the impaired neovascularization in mice lacking MMP-9 is related to a defect in EPC functions in vasculogenesis. Methods and Results—Hindlimb ischemia surgery was conducted in MMP-9−/− mice and wild-type (MMP-9+/+) mice. Blood flow recovery was markedly impaired in MMP-9−/− mice when compared with that in wild-type mice as determined by laser Doppler imaging. Flow cytometry demonstrated that the number of EPC-like cells (Sca-1+/Flk-1+) in peripheral blood increased in wild-type mice after hindlimb ischemia surgery and exogenous vascular endothelial growth factor stimulation, but not in MMP-9−/− mice. Plasma levels and bone marrow concentrations of soluble Kit-ligand (sKitL) were significantly elevated in wild-type mice in response to tissue ischemia, but not in MMP-9−/− mice. C-kit positive bone marrow cells of MMP-9−/− mice have attenuated adhesion and migration than those isolated from wild-type mice. In in vitro studies, incubation with selective MMP-9 inhibitor suppressed the colony formation, migration, and tube formation capacities of EPC. Transplantation of bone marrow cells from wild-type mice restored collateral flow formation in MMP-9−/− mice. Conclusions—These findings suggest that MMP-9 deficiency impairs ischemia-induced neovascularization, and these effects may occur through a reduction in releasing the stem cell-active cytokine, and EPC mobilization, migration, and vasculogenesis functions.

Intake of Red Wine Increases the Number and Functional Capacity of Circulating Endothelial Progenitor Cells by Enhancing Nitric Oxide Bioavailability

Objective—Red wine (RW) consumption has been associated with a reduction of cardiovascular events, but limited data are available on potential mediating mechanisms. This study tested the hypothesis that intake of RW may promote the circulating endothelial progenitor cell (EPC) level and function through enhancement of nitric oxide bioavailability. Methods and Results—Eighty healthy, young subjects were randomized and assigned to consume water (100 mL), RW (100 mL), beer (250 mL), or vodka (30 mL) daily for 3 weeks. Flow cytometry was used to quantify circulating EPC numbers, and in vitro assays were used to evaluate EPC functions. After RW ingestion, endothelial function determined by flow-mediated vasodilation was significantly enhanced; however, it remained unchanged after water, beer, or vodka intake. There were significantly increased numbers of circulating EPC (defined as KDR+CD133+, CD34+CD133+, CD34+KDR+) and EPC colony-forming units only in the RW group (all P<0.05). Only RW ingestion significantly enhanced plasma levels of nitric oxide and decreased asymmetrical dimethylarginine (both P<0.01). Incubation of EPC with RW (but not beer or ethanol) and resveratrol in vitro attenuated tumor necrosis factor-α–induced EPC senescence and improved tumor necrosis factor-α–suppressed EPC functions and tube formation. Incubation with nitric oxide donor sodium nitroprusside significantly ameliorated the inhibition of tumor necrosis factor-α on EPC proliferation, but incubation with endothelial nitric oxide synthase inhibitor l-NAME and PI3K inhibitor markedly attenuated the effect of RW on EPC proliferation. Conclusion—The intake of RW significantly enhanced circulating EPC levels and improved EPC functions by modifying nitric oxide bioavailability. These findings may help explain the beneficial effects of RW on the cardiovascular system. This study demonstrated that a moderate intake of RW can enhance circulating levels of EPC in healthy subjects by increasing nitric oxide availability. Direct incubation of EPC with RW and resveratrol can modify the functions of EPC, including attenuation of senescence and promotion of EPC adhesion, migration, and tube formation. These data suggest that RW ingestion may alter the biology of EPC, and these alterations may contribute to its unique cardiovascular-protective effect.

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.

INSULIN-LIKE GROWTH FACTOR I IMPROVES CARDIOVASCULAR FUNCTION AND SUPPRESSES APOPTOSIS OF CARDIOMYOCYTES IN DILATED CARDIOMYOPATHY

To investigate how insulin-like growth factor I (IGF-I) modulates cardiovascular function and myocardial apoptosis in heart failure, the therapeutic effects of IGF-I were determined in a canine model of dilated cardiomyopathy. The animals were paced at 220 beats/min, and the left ventricular (LV) chamber became dilated after 2 weeks. A subset of paced dogs was treated with sc injections of IGF-I from week 3 to week 4. After 4 weeks of pacing, untreated paced dogs developed significant ventricular dysfunction. IGF-I-treated paced dogs showed better cardiac output, stroke volume, LV end-systolic pressure, and LV end-diastolic pressure. Moreover, pulmonary wedge pressure and systemic vascular resistance were increased in the untreated group and decreased in the IGF-I-treated group. IGF-I treatment was associated with less thinning of the ventricular wall. Compared with the controls, untreated paced dogs showed increased apoptosis of cardiac muscle cells, which was partially suppressed by IGF-I treatment. The...

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.