Min-Ho Oak

Red Wine Polyphenolic Compounds Inhibit Vascular Endothelial Growth Factor Expression in Vascular Smooth Muscle Cells by Preventing the Activation of the p38 Mitogen-Activated Protein Kinase Pathway

Objective—Moderate consumption of red wine has a beneficial effect on the cardiovascular system. This study examines whether red wine polyphenolic compounds (RWPCs) affect vascular endothelial growth factor (VEGF) expression, a major angiogenic and proatherosclerotic factor in vascular smooth muscle cells (VSMCs). Methods and Results—VEGF mRNA expression was assessed by Northern blot analysis and the release of VEGF by immunoassay in cultured VSMCs. Short-term and long-term exposure of VSMCs to RWPCs inhibited VEGF mRNA expression and release of VEGF in response to platelet-derived growth factor AB (PDGFAB), transforming growth factor-&bgr;1, or thrombin. The PDGFAB-induced expression of VEGF was markedly reduced by SB203580 (inhibitor of p38 mitogen-activated protein kinase [MAPK]), antioxidants, and diphenylene iodonium (inhibitor of flavin-dependent enzymes), slightly reduced by PD98059 (inhibitor of MEK), and not significantly affected by wortmannin (inhibitor of PI-3-kinase) and L-JNKI (inhibitor of JNK). Short-term and long-term treatment of VSMCs with RWPCs markedly reduced PDGFAB-induced production of reactive oxygen species and phosphorylation of p38 MAPK. Conclusions—These data indicate that RWPCs strongly inhibit growth factor–induced VEGF expression in VSMCs by preventing the redox-sensitive activation of the p38 MAPK pathway. The potential antiangiogenic and antiatherosclerotic properties of RWPCs are likely to contribute to cardiovascular protection by preventing the development of atherosclerotic lesions.

Red Wine Polyphenolic Compounds Strongly Inhibit Pro-Matrix Metalloproteinase-2 Expression and Its Activation in Response to Thrombin via Direct Inhibition of Membrane Type 1–Matrix Metalloproteinase in Vascular Smooth Muscle Cells

Background—Regular consumption of moderate amounts of red wine is associated with a reduced risk of coronary disease. Matrix metalloproteinases (MMPs) that participate in extracellular matrix degradation have been involved in atherosclerotic plaque growth and instability. The present study examined whether red wine polyphenolic compounds (RWPCs) inhibit activation of MMP-2, a major gelatinase, in vascular smooth muscle cells (VSMCs). Methods and Results—Expression of pro-MMP-2 was assessed by Western and Northern blot analyses; MMP-2 activity was assessed by zymography and cell invasion by a modified Boyden’s chamber assay. High levels of pro-MMP-2 and low levels of MMP-2 activity were found in conditioned medium from unstimulated VSMCs. Thrombin induced cell-associated pro-MMP-2 protein expression and MMP-2 activity in conditioned medium of VSMCs. The stimulatory effect of thrombin on MMP-2 activation was prevented by RWPCs in a concentration-dependent and reversible manner. Thrombin markedly increased cell-associated membrane type 1 (MT1)–MMP activity, the physiological activator of pro-MMP-2, and this response was not affected by RWPCs. However, addition of RWPCs directly to MT1-MMP abolished its metalloproteinase activity in a reversible manner. Finally, matrix invasion of VSMCs was stimulated by thrombin, and this response was prevented by RWPCs as efficiently as a broad-spectrum MMP inhibitor. Conclusions—The present findings demonstrate that RWPCs effectively inhibit thrombin-induced matrix invasion of VSMCs, most likely by preventing the expression and activation of MMP-2 via direct inhibition of MT1-MMP activity. The inhibitory effect of RWPCs on the activation of pro-MMP-2 and matrix degradation might contribute to their beneficial effects on the cardiovascular system.

Catechin prevents endothelial dysfunction in the prediabetic stage of OLETF rats by reducing vascular NADPH oxidase activity and expression.

OBJECTIVES In the prediabetic stage, hyperinsulinemia or insulin resistance is thought to be closely associated with oxidative stress, which is also the main contributor of endothelial dysfunction. Clinical studies have indicated that regular intake of green tea reduces the risk of cardiovascular diseases. This study examined whether catechin prevents endothelial dysfunction and hyperglycemia in the prediabetic stage of a type 2 diabetic (T2D) rat, the Otsuka Long-Evans Tokushima Fatty (OLETF) model. METHODS AND RESULTS 30 OLETF rats, 13 week-old, were randomized into two equal groups for daily treatment with either catechin (30 mg/kg/day), Catechin-OLETF) or saline for 12 weeks. Intraperitoneal glucose tolerance tests and blood pressure measurements were performed at 13 and 25 weeks. Ex vivo vascular relaxation was assessed in organ chambers, vascular superoxide anion production by dihydroethidine, vascular NADPH oxidase activity by lucigenin, and expression by immunohistochemistry. Catechin significantly reduced blood pressure (OLETF vs. Catechin-OLETF; 138+/-16 mmHg vs. 126+/-16 mmHg, p=0.013), fasting sugar (129+/-11 mg/dL vs. 118+/-9 mg/dL, p=0.02) and the insulin level (2.13+/-1.29 ng/mL vs. 0.53+/-0.27 ng/mL, p=0.004). In the aorta of Catechin-OLETF at 25 weeks, endothelium-dependent relaxations were significantly improved and NADPH oxidase activity in aortic rings was markedly decreased compared with those of OLETF. Catechin reduced vascular reactive oxygen species formation in the aorta and suppressed the expression of p22phox and p47phox NADPH oxidase subunits. CONCLUSIONS Intake of catechin normalized blood pressure and prevented endothelial dysfunction and insulin resistance in the prediabetic stage. Prevention of vascular oxidative stress by inhibiting NADPH oxidase expression and activity is likely to contribute to the beneficial effect on the vascular system.

Cocoa Procyanidins Inhibit Expression and Activation of MMP-2 in Vascular Smooth Muscle Cells by Direct Inhibition of MEK and MT1-MMP Activities

AIMS Expression and activation of matrix metalloproteinase (MMP)-2 play pivotal roles in the migration and invasion of human aortic vascular smooth muscle cells (VSMC) originating from normal human tissue, which is strongly linked to atherosclerosis. The present study investigated the possible inhibitory effects of cocoa procyanidin on thrombin-induced expression and activation of MMP-2 in VSMC. METHODS AND RESULTS Cocoa procyanidin fraction (CPF) and procyanidin B2, one of major procyanidins in cocoa (3 microg/mL and 5 microM, respectively), strongly inhibited thrombin-induced activation and expression of pro-MMP-2 in VSMC, as determined by zymography. The thrombin-induced invasion and migration of VSMC were inhibited by CPF or procyanidin B2 (P < 0.05), as assessed by a modified Boyden chamber and wound healing assays, respectively. An enzymatic assay data demonstrated that CPF and procyanidin B2 directly inhibited membrane type-1 (MT1)-MMP activity (P < 0.05), and this inhibition of CPF was greater than those of red wine polyphenols. Western blot data showed that CPF and procyanidin B2 inhibited thrombin-induced phosphorylation of extracellular signal-regulated protein kinase but not mitogen-activated protein kinase kinase (MEK) in VSMC. Kinase and pull-down data revealed that CPF and procyanidin B2 inhibited MEK1 activity and directly bound with glutathione-S-transferase-MEK1. In addition, the thrombin-induced invasion and migration and the activation and expression of pro-MMP-2 in VSMC were attenuated by U0126 (a well-known inhibitor of MEK1). CONCLUSION Cocoa procyanidins are potent inhibitors of MEK and MT1-MMP, and subsequently inhibit the expression and activation of pro-MMP-2, and also the invasion and migration of VSMC, which may in part explain the molecular action of antiatherosclerotic effects of cocoa.

Decaffeinated green tea extract improves hypertension and insulin resistance in a rat model of metabolic syndrome

BACKGROUND Oxidative stress and endothelial dysfunction are closely associated with hypertension and insulin resistance (IR) in metabolic syndrome (MetS). It is still controversial whether green tea extract (GTE) may have blood pressure (BP) lowering effect. Decaffeinated GTE might be presumed to have strong antioxidative effect and BP-lowering effect as compared with catechins. Thus we investigated whether decaffeinated-GTE could attenuate hypertension and IR by improving endothelial dysfunction and reducing oxidative stress in a rat model of MetS. METHODS AND RESULTS 20 Otsuka Long-Evans Tokushima Fatty (OLETF) rats at 13 weeks old, MetS rats, were randomized into a saline treated group (OLETF; n = 10) and a group treated with decaffeinated-GTE (25 mg/kg/day) (GTE-OLETF; n = 10). Intraperitoneal glucose tolerance tests and BP measurements were performed at 13 and 25 weeks. Decaffeinated-GTE significantly reduced BP (OLETF vs. GTE-OLETF; 130 ± 7 vs. 121 ± 3 mmHg, p = 0.01), fasting/postprandial 2 h glucose (141 ± 18/159 ± 13 vs. 115 ± 7/132 ± 16 mg/dL, p = 0.009/0.002) and insulin levels (4.8 ± 2.3 vs. 2.4 ± 1.3 ng/mL, p < 0.001). Decaffeinated-GTE significantly reduced vascular reactive oxygen species (ROS) formation and NADPH oxidase activity, and improved endothelium dependent relaxation in the thoracic aorta of OLETF rats. Decaffeinated-GTE also suppressed the expression of p47 and p22phox (NADPH oxidase subunits) in the immunohistochemical staining, and stimulated phosphorylation of endothelial nitric oxide synthase (eNOS) and Akt in the immunoblotting of aortas. CONCLUSIONS Decaffeinated-GTE reduced the formation of ROS and NADPH oxidase activity and stimulated phosphorylation of eNOS and Akt in the aorta of a rat model of MetS, which resulted in improved endothelial dysfunction and IR, and eventually lowered BP.

Anti-Allergic Prenylated Flavonoids from the Roots of Sophora flavescens

The bioassay-guided fractionation of the MeOH extract from the root of Sophora flavescens led to the isolation of eight known prenylated flavonoids ( 1 - 8) responsible for the IN VITRO anti-allergic activity. Among them, kushenol N ( 3), sophoraflavanone G ( 6), and leachianone A ( 7) demonstrated significant inhibition of the release of beta-hexosaminidase from cultured RBL-2H3 cells with IC (50) values ranging from 15 to 30 muM.

Selectivity of commonly used inhibitors of clathrin-mediated and caveolae-dependent endocytosis of G protein-coupled receptors.

Among the multiple G protein-coupled receptor (GPCR) endocytic pathways, clathrin-mediated endocytosis (CME) and caveolar endocytosis are more extensively characterized than other endocytic pathways. A number of endocytic inhibitors have been used to block CME; however, systemic studies to determine the selectivity of these inhibitors are needed. Clathrin heavy chain or caveolin1-knockdown cells have been employed to determine the specificity of various chemical and molecular biological tools for CME and caveolar endocytosis. Sucrose, concanavalin A, and dominant negative mutants of dynamin blocked other endocytic pathways, in addition to CME. In particular, concanavalin A nonspecifically interfered with the signaling of several GPCRs tested in the study. Decreased pH, monodansylcadaverine, and dominant negative mutants of epsin were more specific for CME than other treatments were. A recently introduced CME inhibitor, Pitstop2™, showed only marginal selectivity for CME and interfered with receptor expression on the cell surface. Blockade of receptor endocytosis by epsin mutants and knockdown of the clathrin heavy chain enhanced the β2AR-mediated ERK activation. Overall, our studies show that previous experimental results should be interpreted with discretion if they included the use of endocytic inhibitors that were previously thought to be CME-selective. In addition, our study shows that endocytosis of β2 adrenoceptor through clathrin-mediated pathway has negative effects on ERK activation.

Lysimachia clethroides extract promote vascular relaxation via endothelium-dependent mechanism.

Lysimachia clethroides is widely used in traditional herbal medicine for many purposes, especially for blood vessel-related diseases in Korea and East Asia. Experiments were undertaken to determine whether hydro-alcoholic extract obtained from L. clethroides (LCE) has vasorelaxant activity in the rat aorta rings and, if so, to elucidate the underlying mechanism. Rat aorta rings were suspended in organ chambers for the measurement of changes in isometric tension in the presence or absence of several inhibitors. LCE induced endothelium-dependent vasodilation (ED50 = 6.1 μg/mL) and that was abolished by nitric oxide synthase inhibitor, Nω-nitro-L-arginine, and guanylyl cyclase inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one, PI3-kinase inhibitor, wortmannin, and cell permeable superoxide dismutase. In addition, LCE decreased vessels contraction by phenylephrine. Prostaglandin synthesis inhibitor, indometacin, and inhibitors of endothelium-derived hyperpolarizing factor, charybdotoxin plus apamin, did not affect vasodilatory effect of LCE. In cultured endothelial cells, LCE-induced phosphorylation of serine 1177-endothelial nitric oxide synthase and serine 473-Akt. LCE inhibited strongly nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in smooth muscle cells and angiotensin II-induced contraction of rat aorta. Finally, increased oxidative stress in rat aorta-induced by angiotensin II is ameliorated by LCE. Taken together, LCE induces an endothelium-dependent vasodilation and might be involved, at least in part, the activation of the nitric oxide-cyclic guanosine monophosphate pathway. In addition, LCE decreases oxidative stress in aorta by inhibition of NADPH oxidase activity. The present findings indicate that LCE could be a candidate of herbal medicine for cardiovascular diseases associated with disturbed NO production and endothelial dysfunction.

Enhanced IL-12p40 production in LPS-stimulated macrophages by inhibiting JNK activation by artemisinin

Artemisinin can be isolated from Artemisia annua L. In addition to its well-known anti-malarial activity, artemisinin has antitumor and anti-microbial effects. In this study, we investigated the effect of artemisinin on the production of IL-12p40, which is important in the generation of T helper 1 responses. Artemisinin significantly induced IL-12p40 production in LPS-stimulated RAW264.7 macrophage cells. To elucidate the signaling molecules regulated by artemisinin in induced IL-12p40 production, the DNA-binding activity of several transcription factors and activation of mitogen-activated protein kinase (MAPK)s were investigated. The band intensities of NF-κB, AP-1, and SP1, and the activation of p38 MAPK and ERK were not changed by artemisinin. However, the induced phosphorylation of JNK was significantly decreased by artemisinin, and inhibition of the JNK signaling pathway further increased IL-12p40 production in LPS-stimulated RAW264.7 macrophage cells. Taken together, these data suggest that artemisinin induces the production of IL-12p40 in LPS-stimulated macrophage cells by inhibiting JNK activity.

Agonist-induced changes in RalA activities allows the prediction of the endocytosis of G protein-coupled receptors.

GTP binding proteins are classified into two families: heterotrimeric large G proteins which are composed of three subunits, and one subunit of small G proteins. Roles of small G proteins in the intracellular trafficking of G protein-coupled receptors (GPCRs) were studied. Among various small G proteins tested, GTP-bound form (G23V) of RalA inhibited the internalization of dopamine D2 receptor independently of the previously reported downstream effectors of RalA, such as Ral-binding protein 1 and PLD. With high affinity for GRK2, active RalA inhibited the GPCR endocytosis by sequestering the GRK2 from receptors. When it was tested for several GPCRs including an endogenous GPCR, lysophosphatidic acid receptor 1, agonist-induced conversion of GTP-bound to GDP-bound RalA, which presumably releases the sequestered GRK2, was observed selectively with the GPCRs which have tendency to undergo endocytosis. Conversion of RalA from active to inactive state occurred by translocation of RGL, a guanine nucleotide exchange factor, from the plasma membrane to cytosol as a complex with Gβγ. These results suggest that agonist-induced Gβγ-mediated conversion of RalA from the GTP-bound form to the GDP-bound form could be a mechanism to facilitate agonist-induced internalization of GPCRs.