Puromycin aminonucleoside increases podocyte permeability by modulating ZO-1 in an oxidative stress-dependent manner.

Puromycin aminonucleoside (PAN)-induced nephrosis is a widely studied animal model of human idiopathic nephrotic syndrome because PAN injection into rats results in increased glomerular permeability with the characteristic ultrastructural changes in podocytes similar to human nephrosis. To investigate the role of zonula occludens (ZO)-1 and oxidative stress on PAN-induced podocyte phenotypical changes and hyperpermeability in vitro, we cultured rat and mouse podocytes and treated with various concentrations of PAN. PAN treatment increased oxidative stress level of podocytes significantly with the induction of Nox4. In addition, PAN changed the ultrastructure of podocytes, such as shortening and fusion of microvilli, and the separation of intercellular gaps, which were improved by anti-oxidative vitamin C and Nox4 siRNA. PAN also disrupted the intercellular linear ZO-1 staining and induced inner cytoplasmic re-localization of ZO-1 protein, resulting in increased podocyte intercellular permeability. PAN reduced ZO-1 protein amount and mRNA expression in a dose-dependent manner, which means that PAN could also modulate ZO-1 protein transcriptionally. However, the decreased ZO-1 protein of podocytes by PAN was improved by Nox4 siRNA transfection. Furthermore, vitamin C mitigated the quantitative and distributional disturbances of ZO-1 protein caused by PAN. Our results demonstrate that the phenotypical changes of intercellular ZO-1 by oxidative stress via Nox4 likely contribute to the glomerular hyperpermeability caused by PAN.

Angiotensin II Modulates p130Cas of Podocytes by the Suppression of AMP-Activated Protein Kinase.

Angiotensin II (Ang II) induces the pathological process of vascular structures, including renal glomeruli by hemodynamic and nonhemodynamic direct effects. In kidneys, Ang II plays an important role in the development of proteinuria by the modification of podocyte molecules. We have previously found that Ang II suppressed podocyte AMP-activated protein kinase (AMPK) via Ang II type 1 receptor and MAPK signaling pathway. In the present study, we investigated the roles of AMPK on the changes of p130Cas of podocyte by Ang II. We cultured mouse podocytes and treated them with various concentrations of Ang II and AMPK-modulating agents and analyzed the changes of p130Cas by confocal imaging and western blotting. In immunofluorescence study, Ang II decreased the intensity of p130Cas and changed its localization from peripheral cytoplasm into peri-nuclear areas in a concentrated pattern in podocytes. Ang II also reduced the amount of p130Cas in time and dose-sensitive manners. AMPK activators, metformin and AICAR, restored the suppressed and mal-localized p130Cas significantly, whereas, compound C, an AMPK inhibitor, further aggravated the changes of p130Cas. Losartan, an Ang II type 1 receptor antagonist, recovered the abnormal changes of p130Cas suppressed by Ang II. These results suggest that Ang II induces the relocalization and suppression of podocyte p130Cas by the suppression of AMPK via Ang II type 1 receptor, which would contribute to Ang II-induced podocyte injury.

Ginsenoside Rg3 inhibits lipopolysaccharide-induced adhesion molecule expression in human umbilical vein endothelial cell and C57BL/6 mice.

Intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), P- and E-selectin play a key role for initiation of vascular inflammation. Ginsenoside, a class of steroid glycosides, is abundant in Panax ginseng root, which has been used for health promotion in Korea. In this study, we investigated the mechanism by which ginsenoside Rg3 may inhibit ICAM-1 and VCAM-1 expressions stimulated with lipopolysaccharide (LPS) in human umbilical vein endothelial cell (HUVEC) and C57BL/6 mice. LPS increased ICAM-1 and VCAM-1 expression. Ginsenoside Rg3 prevented LPS-mediated increase of ICAM-1 and VCAM-1 expression. LPS induced IkappaBα (IκBα) degradation within 1 hr. Ginsenoside Rg3 prevented the IκBα degradation stimulated with LPS. Moreover, ginsenoside Rg3 reduced LPS-mediated THP-1 monocyte adhesion to HUVEC, in a concentration-dependent manner. In C57BL/6 mice, injection of LPS increased aortic ICAM-1 and VCAM-1 expression, which was prevented by ginsenoside Rg3. These data provide a novel mechanism where the ginsenoside Rg3 may provide direct vascular benefits with inhibition of leukocyte adhesion into vascular wall thereby providing prevention against vascular inflammatory disease.

Angiotensin II suppresses adenosine monophosphate-activated protein kinase of podocytes via angiotensin II type 1 receptor and mitogen-activated protein kinase signaling.

BACKGROUND: Adenosine monophosphate (AMP)-activated protein kinase (AMPK), as a sensor of cellular energy status, has been known to play an important role in the pathophysiology of diabetes and its complications. As AMPK is also expressed in podocytes, it is possible that podocyte AMPK would be an important contributing factor in the development of diabetic proteinuria. We investigated the roles of AMPK in the pathological changes of podocytes induced by angiotensin II (Ang II), a major injury inducer in diabetic proteinuria. METHODS: Mouse podocytes were incubated in media containing various concentrations of Ang II and AMPK-modulating agents. The changes of AMPKα were analyzed by confocal imaging and Western blotting in response to Ang II. RESULTS: Ang II changed the localization of AMPKα from peripheral cytoplasm into internal cytoplasm and peri- and intranuclear areas in podocytes. Ang II also reduced AMPKα (Thr172) phosphorylation in time- and dose-sensitive manners. In particular, 10(-7 )M Ang II reduced phospho-AMPKα significantly and continuously at 6, 24, and 48 h. AMPK activators, metformin and 5-aminoimidazole-4-carboxamide-1ß-riboside, restored the suppressed AMPKα (Thr172) phosphorylation. Losartan, an Ang II type 1 receptor antagonist, also recovered the suppression and the mal-localization of AMPKα, which were induced by Ang II. PD98059, a mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor, also restored the AMPKα (Thr172) phosphorylation suppressed by Ang II. CONCLUSION: We suggest that Ang II induces the relocation and suppression of podocyte AMPKα via Ang II type 1 receptor and MAPK signaling pathway, which would be an important mechanism in Ang II-induced podocyte injury.

Ginsenoside rg2 inhibits lipopolysaccharide-induced adhesion molecule expression in human umbilical vein endothelial cell.

Vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), P- and E-selectin play a pivotal role for initiation of atherosclerosis. Ginsenoside, a class of steroid glycosides, is abundant in Panax ginseng root, which has been used for prevention of illness in Korea. In this study, we investigated the mechanism(s) by which ginsenoside Rg2 may inhibit VCAM-1 and ICAM-1 expressions stimulated with lipopolysaccharide (LPS) in human umbilical vein endothelial cell (HUVEC). LPS increased VCAM-1 and ICAM-1 expression. Ginsenoside Rg2 prevented LPS-mediated increase of VCAM-1 and ICAM-1 expression. On the other hand, JSH, a nuclear factor kappa B (NF-κB) inhibitor, reduced both VCAM-1 and ICAM-1 expression stimulated with LPS. SB202190, inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), and wortmannin, phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, reduced LPS-mediated VCAM-1 but not ICAM-1 expression. PD98059, inhibitor of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) did not affect VCAM-1 and ICAM-1 expression stimulated with LPS. SP600125, inhibitor of c-Jun N-terminal kinase (JNK), reduced LPS-mediated ICAM-1 but not VCAM-1 expression. LPS reduced IkappaBα (IκBα) expression, in a time-dependent manner within 1 hr. Ginsenoside Rg2 prevented the decrease of IκBα expression stimulated with LPS. Moreover, ginsenoside Rg2 reduced LPS-mediated THP-1 monocyte adhesion to HUVEC, in a concentration-dependent manner. These data provide a novel mechanism where the ginsenoside Rg2 may provide direct vascular benefits with inhibition of leukocyte adhesion into vascular wall thereby providing protection against vascular inflammatory disease.

Regulation of type IV collagen alpha 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 alpha chains of type IV collagen has not been well studied. To investigate the regulation of alpha(IV) chains in diabetic conditions, we examined whether glucose and advanced glycosylation endproduct (AGE) regulate the metabolism of each alpha(IV) chains in the diabetic tissue and glomerular epithelial cells (GEpC). Glomerular collagen alpha3(IV) and alpha5(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 [(3)H]-proline incorporation assay and each alpha(IV) chain proteins including alpha1(IV), alpha3(IV), and alpha5(IV) in time-dependent and subchain-specific manners. However, the changes of each alpha(IV) chains mRNA expression was not well correlated to the those of each chain proteins. The present findings suggest that the metabolism of individual alpha(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.

Quercetin prevents cardiac hypertrophy induced by pressure overload in rats.

Inhibition of cardiac hypertrophy leads to a significant reduction in cardiovascular mortality and morbidity. Quercetin is by far the most abundant flavonoid and believed to ameliorate cardiovascular disease. Therefore, we investigated whether quercetin supplementation could attenuate the development of cardiac hypertrophy induced by pressure overload. Three weeks after suprarenal transverse abdominal aortic constriction, heart to body weight (HW/BW) ratio increased compared to the sham group (3.40 +/- 0.06 mg/g versus 2.83 +/- 0.02 mg/g, P<0.001). The quercetin administered group showed complete inhibition of cardiac hypertrophy (2.85 +/- 0.01 mg/g, P<0.001). Malonyldialdehyde production induced by pressure overload was suppressed by quercetin. The activities of extracellular signal-regulated kinase (ERK1/2), p38 MAP kinase, Akt and GSK-3beta were significantly increased with pressure overload and attenuated by quercetin treatment. We conclude that quercetin appears to block the development of cardiac hypertrophy induced by pressure overload in rats and that these effects may be mediated through reduced oxidant status and inhibition of ERK1/2, p38 MAP kinase, Akt and GSK-3beta activities.

Epigallocatechin-3-O-gallate inhibits TNFalpha-induced monocyte chemotactic protein-1 production from vascular endothelial cells.

Monocyte chemotactic protein-1 (MCP-1) plays a pivotal role in the recruitment of monocytes and thus in the development of inflammatory cardiovascular diseases. Epigallocatechin-3-O-gallate (EGCG), the major catechin derived from green tea, has multiple beneficial effects to reduce cardiovascular disease but the effects of EGCG on vascular endothelial MCP-1 production is not known. In this study, we investigated the mechanisms by which EGCG may inhibit tumor necrosis factor-alpha (TNFalpha)-induced MCP-1 production in bovine coronary artery endothelial cells. TNFalpha increased MCP-1 production in both a concentration and time-dependent manner. Inhibitors of phosphatidylinositol-3-OH kinase (PI-3 kinase), LY294002 and wortmannin, decreased TNFalpha-induced MCP-1 production. EGCG prevented TNFalpha-mediated MCP-1 production and reduced phosphorylation of Akt (Ser473). In addition, EGCG attenuated TNFalpha mediated down-regulation of TNFalpha receptor 1 (TNFR1), but not TNFR2. In conclusion, EGCG inhibited TNFalpha-induced MCP-1 production. Moreover, EGCG inhibited Akt phosphorylation as well as TNF activation of TNFR1, which subsequently resulted in reduced MCP-1 production. These data provide a novel mechanism where the green tea flavonoid, EGCG, could provide direct vascular benefits in inflammatory cardiovascular diseases.

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 overloadmediated 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.

(-)-Epigallocatechin-3-O-gallate (EGCG) attenuates the hemodynamics stimulated by caffeine through decrease of catecholamines release.

A human study of the effects on hemodynamics of caffeine and epigallocatechin-3-O-gallate (EGCG) was performed. Caffeine tablets (200 mg) were orally administered to healthy males aged between 25 and 35 years 30 min after oral administration of EGCG tablets (100 and 200 mg). The increase in BP induced by caffeine was inhibited when co-administrated with EGCG. We found that caffeine slightly decreased heart rate (HR) in the volunteers. Although EGCG enhanced HR reduction, the effect was not significant. In addition, caffeine increased blood catecholamine levels, but EGCG inhibited the increase in noradrenaline, adrenaline and dopamine levels induced by caffeine. Whether EGCG decreases the elevated HR and systolic perfusion pressure, and ventricular contractility induced by adrenergic agonists in the isolated rat heart was investigated. The modified Krebs-Henseleit solution was perfused through a Langendorff apparatus to the isolated hearts of rats. HR, systolic perfusion pressure, and developed maximal rates of contraction (+dP/dtmax) and relaxation (-dP/dtmax) were increased by epinephrine (EP) and isoproterenol (IP). In contrast, EGCG decreased the elevated HR, systolic perfusion pressure, and left ventricular ±dp/dtmax induced by EP and/or IP. In conclusion, EGCG could attenuate the hemodynamics stimulated by caffeine through decreasing catecholamine release.

Inhibition of the PDGF beta-receptor tyrosine phosphorylation and its downstream intracellular signal transduction pathway in rat and human vascular smooth muscle cells by different catechins.

Abnormal proliferation of vascular smooth muscle cells (VSMC) as well as the platelet-derived growth factor (PDGF) plays an important role in the development of proliferative cardiovascular diseases. In this study, we show that treatment of rat and human aortic VSMC with 50 microM 2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3,5,7-triol (catechin) and epicatechin (EC) fails to inhibit the PDGF-Rb-activated intracellular signal transduction pathway and VSMC growth. In contrast, 10-50 microM epigallocatechin-3 gallate (EGCG), epicatechin-3 gallate (ECG), and catechin-3 gallate (CG), which all have a galloyl group in the 3-position of the catechin structure, effectively inhibit tyrosine-phosphorylation of PDGF-Rb, PI 3'-K, and PLC-gamma1 as well as the PDGF-BB-induced increase in [Ca2+]i. The PDGF-BB-induced increase in DNA synthesis and cell number was inhibited by ECG, EGCG, and CG, but not by catechin and EC. Epigallocatechin (EGC) that has a galloyl group in the 2-position effectively inhibited VSMC growth without affecting the PDGF-Rb signal pathway. A reduction of 45% and 70% of the intimal and medial cell number in the S-phase, respectively, has been observed in the catheter-injured left carotid artery 7 days after treatment of Wistar Kyoto rats with 10 mg/day EGCG. These results suggest that the galloyl group in the P3-position of the catechin structure is essential for inhibiting the PDGF-Rbeta-mediated intracellular signal transduction pathway.

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) alpha-receptor (PDGF-Ralpha) or the PDGF beta-receptor (PDGF-Rbeta), 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-Rbeta. 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-Ralpha and -beta, 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 microM 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.

Angiotensin II induces endoplasmic reticulum stress in podocyte, which would be further augmented by PI3-kinase inhibition.

INTRODUCTION: Angiotensin II (Ang II) contributes to the pathological process of vascular structures, including renal glomeruli by hemodynamic and nonhemodynamic direct effects. On renal effects, Ang II plays an important role in the development of proteinuria and glomerulosclerosis by the modification of podocyte molecules and cell survival. In the present study, we investigated the effect of Ang II on endoplasmic reticulum (ER) stress in podocytes. METHODS: We cultured mouse podocytes with increasing doses of Ang II and evaluated ER stress markers by Western blotting. RESULTS: Ang II increased Bip protein, an ER chaperone, in a dose-dependent manner at 24 h, which was ameliorated by losartan, an angiotensin II type 1 receptor antagonist. Ang II also increased ER stress markers, such as phospho-PERK, phospho-eIF2α, and ATF4 proteins of podocyte, significantly in a dose-dependent manner at 24 h. Increased phospho-PERK and ATF4 proteins were further augmented by phosphoinositide 3 (PI3)-kinase inhibitor, LY294002, which suggested that Ang II could induce podocyte ER stress of PERK-eIF2α-ATF4 axis via PI3-kinase pathway. DISCUSSION: These studies suggest that Ang II could induce podocyte ER stress of PERK-eIF2α-ATF4 axis via PI3-kinase pathway, which would contribute to the development of podocyte injury induced by Ang II, and the augmentation of PI3-kinase would be a therapeutic target.

Tyrosine kinase participates in vasoconstriction through a Ca(2+)- and myosin light chain phosphorylation-independent pathway.

This study was undertaken to determine the role of tyrosine kinase on intracellular Ca(2+) ([Ca(2+)](i)), myosin light chain (MLC) phosphorylation, and contraction caused by norepinephrine (NE) in rat aorta. NE induced a sustained contraction with an increase of [Ca(2+)](i). On the other hand, NE increased the phosphorylation of the 20 kDa MLC transiently. Pretreatment with genistein and tyrophostin 25, tyrosine kinase inhibitors, significantly inhibited NE-induced contraction, but did not affect the increase of [Ca(2+)](i) and MLC phosphorylation. These results suggest that tyrosine kinase may regulate the NE-mediated contraction without altering [Ca(2+)](i) and MLC phosphorylation in rat aorta.

Mitogen-activated protein kinases partially regulate endothelin-1-induced contractions through a myosin light chain phosphorylation-independent pathway.

Endothelin (ET), derived from the endothelium of blood vessels, is a potent vasoactive peptide. Although it has been reported to be involved in cardiovascular diseases, such as hypertension, the mechanism by which ET evokes vasoconstriction is still unclear. On the other hand, p42/p44 mitogen-activated protein kinase (MAPK) and p38 MAPK are activated by a variety of growth factors and cellular stresses, respectively. However, the role of p42/p44 MAPK and p38 MAPK on the ET-1-induced vasoconstriction is not fully understood. This study was undertaken to determine whether p42/p44 MAPK and p38 MAPK participate in the regulation of vascular smooth muscle contraction by ET-1. The isometric vasoconstriction and intracellular Ca(2+) ([Ca(2+)](i)) were simultaneously measured using CAF-100. Phosphorylation of myosin light chain (MLC) and p42/p44 MAPK, p38 MAPK were determined by Western blots. In rat thoracic aorta, ET-1 induced a sustained contraction. In contrast, [Ca(2+)](i) was decreased with time. Both PD98059, an inhibitor of p42/p44 MAPK, and SB203580, an inhibitor of p38 MAPK, partially attenuated ET-1-induced contractions in concentration-dependent manners. ET-1 increased phosphorylation of both p42/p44 MAPK and p38 MAPK, and PD98059 and SB203580 completely decreased phosphorylation of p42/p44 MAPK and p38 MAPK in response to ET-1 stimulation, respectively. On the other hand, PD98059 and SB203580 did not affect MLC phosphorylation in response to ET-1 stimulation. These results indicate that p38 MAPK, as well as p42/p44 MAPK, may partially regulate the ET-1-induced contraction through a MLC phosphorylation-independent pathway.

Differential expression of αB-crystallin causes maturation-dependent susceptibility of oligodendrocytes to oxidative stress.

Oligodendrocyte precursor cells (OPCs) are most susceptible to oxidative stress in the brain. However, the cause of differences in susceptibility to oxidative stress between OPCs and mature oligodendrocytes (mOLs) remains unclear. Recently, we identified in vivo that αB-crystallin (aBC) is expressed in mOLs but not in OPCs. Therefore, we examined in the present study whether aBC expression could affect cell survival under oxidative stress induced by hydrogen peroxide using primary cultures of OPCs and mOLs from neonatal rat brains. Expression of aBC was greater in mOLs than in OPCs, and the survival rate of mOLs was significantly higher than that of OPCs under oxidative stress. Suppression of aBC by siRNA transfection resulted in a decrease in the survival rate of mOLs under oxidative stress. These data suggest that higher susceptibility of OPCs than mOLs to oxidative stress is due, at least in part, to low levels of aBC expression.

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 postischemic cardiac function compared to the control group. Phosphorylation of Akt and its downstream substrate, GSK-3ß, was increased by mechanical stretching and completely blocked by wortmannin, a PI3K inhibitor. Treatment with lithium or SB216763 (GSK-3ß 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-3ß. 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.

Epigallocatechin-3-gallate Regulates Inducible Nitric Oxide Synthase Expression in Human Umbilical Vein Endothelial Cells.

Inducible nitric oxide synthase (iNOS) is a main enzyme producing nitric oxide during inflammation and thus contributes to the initiation and development of inflammatory cardiovascular diseases such as atherosclerosis. Epigallocatechin-3-gallate (EGCG), the major catechin derived from green tea, has multiple beneficial effects for treating cardiovascular disease, but the effect of EGCG on the expression of vascular iNOS remains unknown. In this study, we investigated (i) whether EGCG inhibits the expression of vascular iNOS induced by angiotensin II in human umbilical vein endothelial cells and, if it does inhibit, (ii) mechanisms underlying the inhibition. Angiotensin II increased expression levels of vascular iNOS; EGCG counteracted this effect. EGCG increased the production of reactive oxygen species. Moreover, EGCG did not affect the production of reactive oxygen species induced by angiotensin II. These data suggest a novel mechanism whereby EGCG provides direct vascular benefits for treating inflammatory cardiovascular diseases.

Epigallocatechin-3-gallate Regulates NADPH Oxidase Expression in Human Umbilical Vein Endothelial Cells.

Vascular NADPH oxidase plays a pivotal role in producing superoxide in endothelial cells and thus acts in the initiation and development of inflammatory cardiovascular diseases such as atherosclerosis. Epigallocatechin-3-gallate (EGCG), the major catechin derived from green tea, has multiple beneficial effects for treating cardiovascular disease but the effect of EGCG on the expression of vascular NADPH oxidase remains unknown. In this study, we investigated the mechanism(s) by which EGCG might inhibit the expression of subunits of NADPH oxidase, namely p47(phox), p67(phox) and p22(phox), induced by angiotensin II (Ang II) in human umbilical vein endothelial cells. Ang II increased the expression levels of p47(phox), p67(phox), and p22(phox), but EGCG counteracted this effect on p47(phox). Moreover, EGCG did not affect the production of reactive oxygen species induced by Ang II. These data suggest a novel mechanism whereby EGCG might provide direct vascular benefits for treating inflammatory cardiovascular diseases.

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.