Hee-Yul Ahn

Green tea compounds inhibit tyrosine phosphorylation of PDGF β-receptor and transformation of A172 human glioblastoma.

The effect of the green tea compounds 2‐(3,4‐dihydroxyphenyl)‐3,4‐dihydro‐2H‐1‐benzopyran‐3,5,7‐triol (catechin), epicathechin (EC), epigallocathechin‐3 gallate (EGCG), epicathechin‐3 gallate (ECG) and catechin‐3 gallate (CG) on the tyrosine phosphorylation of PDGF β‐receptor (PDGF‐Rβ) and on the anchorage‐independent growth of A172 glioblastoma cells in semisolid agar has been investigated. Treatment of A172 glioblastoma with 50 μM CG, ECG, EGCG and 25 μM Tyrphostin 1296 resulted in an 82±17%, 77±21%, 75±8% and 55±11%, respectively (mean±S.D., n=3) inhibition of the PDGF‐BB‐induced tyrosine phosphorylation of PDGF‐Rβ. The PDGF‐Rβ downstream intracellular transduction pathway including tyrosine phosphorylation of phospholipase C‐γ1 (PLC‐γ1) and phosphatidylinositol 3′‐kinase (PI 3′‐K) was also inhibited. Spheroid formation was completely inhibited by 50 μM ECG, CG, EGCG and by 25 μM Tyrphostin 1296. We conclude that catechins of the green tea possessing the gallate group in their chemical structure act as anticancer agents probably partly via their ability to suppress the tyrosine kinase activity of the PDGF‐Rβ.

Mechanisms of the inhibitory effects of epigallocatechin-3 gallate on platelet-derived growth factor-BB-induced cell signaling and mitogenesis

An enhanced activity of receptor tyrosine kinases (RTKs), such as the platelet‐derived growth factor (PDGF) α‐receptor (PDGF‐Rα) or the PDGF β‐receptor (PDGF‐Rβ), is involved in the development of proliferative diseases. We have previously demonstrated that green tea catechins containing a galloyl group in the third position of the catechin structure interfere with PDGF‐BB‐ induced mitogenic signaling pathways by inhibiting tyrosine phosphorylation of the PDGF‐Rβ. However, the underlying cellular and molecular mechanisms are unknown. Using human vascular smooth muscle cells (VSMC) and porcine endothelial cells (AEC) stably transfected with PDGF‐Rα and ‐β, respectively, we demonstrate that EGCG preferably inhibited PDGF‐BB isoform‐mediated signal transduction pathways and cell proliferation. To elucidate cellular and molecular mechanisms of the inhibitory effects of EGCG, we studied the distribution of incorporated EGCG into cellular compartments after subcellular fractionation. Interestingly, most (85%) of the EGCG was found in the cytoplasmic fraction, whereas only ∼2% was found within the cell plasma membranes. However, no alteration of membrane fluidity has been observed after treatment of VSMC with 50 µM EGCG. Binding studies with [125I]‐PDGF‐BB on EGCG‐treated VSMC demonstrated that the specific binding of PDGF‐BB was completely abolished. Moreover, when [125I]‐PDGF‐BB was incubated with VSMC in the presence of EGCG, a 50% reduction of cellular [125I]‐PDGF‐BB binding was observed. Our findings suggest that plasma membrane incorporated EGCG or soluble EGCG directly interacts with PDGF‐BB, thereby preventing specific receptor binding.

Vasodilating and hypotensive effects of fangchinoline and tetrandrine on the rat aorta and the stroke-prone spontaneously hypertensive rat

Comparative studies of the effects of tetrandrine (TET) and fangchinoline (FAN), two major components of the Radix of Stephannia tetrandrae, on vasodilations and on calcium movement in vascular smooth muscle, and studies of hypotensive effects on stroke-prone spontaneously hypertensive rats (SHRSP) were performed in the following experiments. TET and FAN inhibited high K+ (65.4 mM) and induced sustained contraction in the rat aorta smooth muscle strips. IC50 values for TET and FAN were 0.27 +/- 0.05 microM (n = 6) and 9.53 +/- 1.57 microM (n = 6), respectively, and this inhibition was antagonized by increasing the Ca2+ concentration in the medium. The IC50 of TET for norepinephrine (NE)-induced contraction (0.86 +/- 0.04 g) was 3.08 +/- 0.05 microM (n = 4), and the IC50 of FAN for NE-induced contraction (0.88 +/- 0.07 g) was 14.20 +/- 0.40 microM (n = 4). At the molecular level, radiolabelled 45Ca2+ uptake tests revealed that TET and FAN also inhibited high K+ (65.4 mM) and 1 microM NE-stimulated Ca2+ influx in rat aorta strips at the maximal concentration was needed to inhibit the contraction. TET (3 mg/kg) and FAN (30 mg/kg) administered by intravenous (i.v.) bolus injection also lowered the mean arterial pressure (MAP) significantly during the period of observation in conscious SHRSP, respectively. These results showed that TET was more potent than FAN in blocking calcium channels and antihypertensive activity.

Epigallocatechin-3 gallate prevents cardiac hypertrophy induced by pressure overload in rats.

Pressure overload diseases, such as valvular stenosis and systemic hypertension, manifest morphologically in patients as cardiac concentric hypertrophy. Prevention of cardiac remodeling due to increased pressure overload is important to reduce morbidity and mortality. Epigallocatechin-3 gallate (EGCG) is a major bioactive polyphenol present in green tea which has been found to be a nitric oxide-mediated vasorelaxant and to be cardioprotective in myocardial ischemia-reperfusion injury. Therefore, we investigated whether EGCG supplementation could reduce in vivo pressure overload-mediated cardiac hypertrophy. Cardiac hypertrophy was induced by suprarenal transverse abdominal aortic constriction (AC) in rats. Three weeks after AC surgery, heart to body weight ratio increased in the AC group by 34% compared to the sham group. EGCG administration suppressed the load-induced increase in heart weight by 69%. Attenuation of cardiac hypertrophy by EGCG was associated with attenuation of the increase in myocyte cell size and fibrosis induced by aortic constriction. Despite abolition of hypertrophy by EGCG, transstenotic pressure gradients did not change. Echocardiogram revealed that increased left ventricular systolic dimensions and deteriorated systolic function were relieved by EGCG. These results suggest that EGCG prevents the development of left ventricular concentric hypertrophy by pressure overload and may be a useful therapeutic modality to prevent cardiac remodeling in patients with pressure overload myocardial diseases.

Local delivery of green tea catechins inhibits neointimal formation in the rat carotid artery injury model

It has been shown that green tea catechins (GTC) suppress proliferation of vascular smooth muscle cells (VSMCs) and that epigallocatechin-3-gallate (EGCG), which is a major constituent of GTC, selectively inhibits the platelet-derived growth factor-BB (PDGF-BB)-induced intracellular signaling transduction pathway. Vascular smooth muscle cell proliferation is one of major mechanisms of restenosis following percutaneous coronary intervention. This study tested whether GTC can inhibit VSMC proliferation and prevent neointimal formation in a rat carotid artery injury model. Vascular smooth muscle cell proliferation inhibition was analyzed with [3H]thymidine incorporation. Green tea catechins were applied to the endothelium-denuded carotid arteries of rats for 20 min. Angiography and morphometric analysis was performed after 2 weeks. Green tea catechins decreased [3H]thymidine incorporation stimulated with PDGF-BB dose dependently. In the absence of PDGF-BB, the decrement of [3H]thymidine incorporation was evident above a concentration of 10 µg/ml of GTC. Carotid arteriographic evaluation showed that the minimum luminal diameter in the GTC-treated group (n = 12) was 5.9 ± 1.6 arbitrary units (a.u.) and was significantly larger than in the control group (4.3 ± 1.4 a.u., n = 10) (P < 0.05). The GTC-treated group also showed a significant reduction in neointimal formation compared with the control group (0.29 ± 0.11 vs 0.42 ± 0.10 mm2, P < 0.05). To identify the active ingredients, we performed a similar experiment using EGCG. The effects of EGCG were similar to those of GTC. Green tea catechins effectively inhibited VSMC proliferation. Neointimal formation was prevented in the rat carotid artery injury model by local delivery of GTC. As EGCG showed similar effects, it may be one of the major constituents of GTC having these effects.

Mechanical Stretch-Induced Protection against Myocardial Ischemia-Reperfusion Injury Involves AMP-Activated Protein Kinase.

AMP-activated protein kinase (AMPK) protects various tissues and cells from ischemic insults and is activated by many stimuli including mechanical stretch. Therefore, this study investigated if the activation of AMPK is involved in stretch-induced cardioprotection (SIC). Intraventricular balloon and aorto-caval shunt (ACS) were used to stretch rat hearts ex vivo and in vivo, respectively. Stretch preconditioning reduced myocardial infarct induced by ischemia-reperfusion (I/R) and improved post-ischemic functional recovery. Phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase (ACC) were increased by mechanical stretch and ACC phosphorylation was completely blocked by the AMPK inhibitor, Compound C. AMPK activator (AICAR) mimicked SIC. Gadolinium, a blocker of stretch-activated ion channels (SACs), inhibited the stretch-induced phosphorylation of AMPK and ACC, whereas diltiazem, a specific L-type calcium channel blocker, did not affect AMPK activation. Furthermore, SIC was abrogated by Compound C and gadolinium. The in vivo stretch induced by ACS increased AMPK activation and reduced myocardial infarct. These findings indicate that stretch preconditioning can induce the cardioprotection against I/R injury, and activation of AMPK plays an important role in SIC, which might be mediated by SACs.

Involvement of phospholipase D in oxidative stress‐induced necrosis of vascular smooth muscle cells

Phospholipase D (PLD) has been associated with necrosis. However, it is not clear whether PLD plays a causative role in this cellular process. We investigated the role of PLD in oxidative stress‐induced necrosis of vascular smooth muscle cells (VSMCs). Pervanadate (hydrogen peroxide plus orthovanadate) but not hydrogen peroxide alone activated PLD in a dose‐ and time‐dependent manner. Exposure of VSMCs to pervanadate resulted in necrosis. Pretreatment with butan‐1‐ol, a PLD inhibitor, attenuated both pervanadate‐induced necrosis and increase of intracellular Ca2+. Removal of extracellular Ca2+ inhibited pervanadate‐induced necrosis by 50%. These results suggest that PLD activation mediates pervanadate‐induced necrosis of VSMCs, which is at least partly due to Ca2+ toxicity.

Tyrosine kinase participates in vasoconstriction through a Ca2+- and myosin light chain phosphorylation-independent pathway

This study was undertaken to determine the role of tyrosine kinase on intracellular Ca2+ ([Ca2+]i), myosin light chain (MLC) phosphorylation, and contraction caused by norepinephrine (NE) in rat aorta. NE induced a sustained contraction with an increase of [Ca2+]i. On the other hand, NE increased the phosphorylation of the 20 kDa MLC transiently. Pretreatment with genistein and tyrphostin 25, tyrosine kinase inhibitors, significantly inhibited NE‐induced contraction, but did not affect the increase of [Ca2+]i and MLC phosphorylation. These results suggest that tyrosine kinase may regulate the NE‐mediated contraction without altering [Ca2+]i and MLC phosphorylation in rat aorta.

Regulation of Type IV Collagen α Chains of Glomerular Epithelial Cells in Diabetic Conditions

An early feature of diabetic nephropathy is the alteration of the glomerular basement membrane (GBM), which may result in microalbuminuria, subsequent macroproteinuria, and eventual chronic renal failure. Although type IV collagen is the main component of thickened GBM in diabetic nephropathy, cellular metabolism of each α chains of type IV collagen has not been well studied. To investigate the regulation of α(IV) chains in diabetic conditions, we examined whether glucose and advanced glycosylation endproduct (AGE) regulate the metabolism of each α(IV) chains in the diabetic tissue and glomerular epithelial cells (GEpC). Glomerular collagen α3(IV) and α5(IV) chains protein were higher and more intense in immunofluorescence staining according to diabetic durations compared to controls. In vitro, mainly high glucose and partly AGE usually increased total collagen protein of GEpC by [3H]-proline incorporation assay and each α(IV) chain proteins including α1(IV), α3(IV), and α5(IV) in time-dependent and subchain-specific manners. However, the changes of each α(IV) chains mRNA expression was not well correlated to the those of each chain proteins. The present findings suggest that the metabolism of individual α(IV) chains of GBM is differentially regulated in diabetic conditions and those changes might be induced not only by transcriptional level but also by post-translational modifications.

Activation of Akt/protein kinase B mediates the protective effects of mechanical stretching against myocardial ischemia-reperfusion injury.

Akt/protein kinase B is a well-known cell survival factor and activated by many stimuli including mechanical stretching. Therefore, we evaluated the cardioprotective effect of a brief mechanical stretching of rat hearts and determined whether activation of Akt through phosphatidylinositol 3-kinase (PI3K) is involved in stretch-induced cardioprotection (SIC). Stretch preconditioning reduced infarct size and improved post-ischemic cardiac function compared to the control group. Phosphorylation of Akt and its downstream substrate, GSK-3β, was increased by mechanical stretching and completely blocked by wortmannin, a PI3K inhibitor. Treatment with lithium or SB216763 (GSK-3β inhibitors) before ischemia induction mimicked the protective effects of SIC on rat heart. Gadolinium (Gd3+), a blocker of stretch-activated ion channels (SACs), inhibited the stretch-induced phosphorylation of Akt and GSK-3β. Furthermore, SIC was abrogated by wortmannin and Gd3+. In vivo stretching induced by an aorto-caval shunt increased Akt phosphorylation and reduced myocardial infarction; these effects were diminished by wortmannin and Gd3+ pretreatment. Our results showed that mechanical stretching can provide cardioprotection against ischemia-reperfusion injury. Additionally, the activation of Akt, which might be regulated by SACs and the PI3K pathway, plays an important role in SIC.