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 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-kappaB) 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 IkappaBalpha (IkappaBalpha) expression, in a time-dependent manner within 1 hr. Ginsenoside Rg2 prevented the decrease of IkappaBalpha 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.

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-3beta, was increased by mechanical stretching and completely blocked by wortmannin, a PI3K inhibitor. Treatment with lithium or SB216763 (GSK-3beta 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-3beta. 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 / 한국실험동물학회지

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.

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 [3H]-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.

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.

Inhibition of Angiotensin II-Induced Vascular Smooth Muscle Cell Hypertrophy by Different Catechins

A cumulative evidence indicates that consumption of tea catechin, flavan-3-ol derived from green tea leaves, lowers the risk of cardiovascular diseases. However, a precise mechanism for this cardiovascular action has not yet been fully understood. In the present study, we investigated the effects of different green tea catechins, such as epigallocatechin-3 gallate (EGCG), epigallocatechin (EGC), epicatechin-3 gallate (ECG), and epicatechin (EC), on angiotensin II (Ang II) -induced hypertrophy in primary cultured rat aortic vascular smooth muscle cell (VSMC). [3H]-leucine incorporation was used to assess VSMC hypertrophy, protein kinase assay, and western blot analysis were used to assess mitogen-activated protein kinase (MAPK) activity, and RT-PCR was used to assess c-jun or c-fos transcription. Ang II increased [3H]-leucine incorporation into VSMC. However, EGCG and ECG, but not EGC or EC, inhibited [3H]-leucine incorporation increased by Ang II. Ang II increased phosphorylation of c-Jun, extracellular-signal regulated kinase (ERK) 1/2 and p38 MAPK in VSMC, however, EGCG and ECG, but not EGC or EC, attenuated c-Jun phosphorylation increased by Ang II. ERK 1/2 and p38 MAPK phosphorylation induced by Ang II were not affected by any catechins. Ang II increased c-jun and c-fos mRNA expression in VSMC, however, EGCG inhibited c-jun but not c-fos mRNA expression induced by Ang II. ECG, EGC and EC did not affect c-jun or c-fos mRNA expression induced by Ang II. Our findings indicate that the galloyl group in the position 3 of the catechin structure of EGCG or ECG is essential for inhibiting VSMC hypertrophy induced by Ang II via the specific inhibition of JNK signaling pathway, which may explain the beneficial effects of green tea catechin on the pathogenesis of cardiovascular diseases observed in several epidemiological studies.

Increased Catalase Activity by All-trans Retinoic Acid and Its Effect on Radiosensitivity in Rat Glioma Cells / 대한방사선종양학회지

PURPOSE: It has been reported that all-trans retinoic acid (ATRA) can inhibit glioma growing in vitro. However, clinical trials with ATRA alone in gliomas revealed modest results. ATRA has been shown to increase radiosensitivity in other tumor types, so combining radiation and ATRA would be one of alternatives to increase therapeutic efficacy in malignant gliomas. Thus, we intended to know the role of catalase, which is induced by ATRA, for radiosensitivity. If radiation-reduced reactive oxygen species (ROS) is removed by catalase, the effect of radiation will be reduced. MATERIALS AND METHODS: A rat glioma cell line (36B10) was used for this study. The change of catalase activity and radiosensitivity by ATRA, with or without 3-amino-1, 2, 4-triazole (ATZ), a chemical inhibitor of catalase were measured. Catalase activity was measured by the decomposition of H2O2 spectrophotometrically. Radiosensitivity was measured with clonogenic assay. Also ROS was measured using a 2, 7-dichlorofluorescein diacetate spectrophotometrically. RESULTS: When 36B10 cells were exposed to 10, 25 and 50microM of ATRA for 48 h, the expression of catalase activity were increased with increasing concentration and incubation time of ATRA. Catalase activity was decreased with increasing the concentration of AT (1, 10 mM) dose-dependently. ROS was increased with ATRA and it was augmented with the combination of ATRA and radiation. ATZ decreased ROS production and increased cell survival in combination of ATRA and radiation despite the reduction of catalase. CONCLUSION: The increase of ROS is one of the reasons for the increased radiosensitivity in combination with ATRA. The catalase that is induced by ATRA doesn`t decrease ROS production and radiosensitivity.

Effect of Paclitaxel Local Delivery on Neointimal Formation after Endothelial Denudation of the Rat Carotid Artery

BACKGROUND AND OBJECTIVES: Mechanisms of restenosis following successful coronary angioplasty (PTCA) are knownasvascularsmoothmuscle cells(VSMCs)proliferationandmigration, elastic recoil or vascular wall remodeling. Paclitaxel whose effect on the stabilization of microtubles leads to cell death is highly lipophilic, permitting easy pass through cell membrane, and has a long-term antiproliferative effect. This study was performed to evaluate effect of paclitaxel on VSMCs proliferation and whether locally delivered paclitaxel can prevent stenosis and neointimal formation in rat carotid artery injury model. MATERIALS AND METHODS: Cultured VSMCs were exposed to sequential concentrations of paclitaxel in vitro, and proliferation inhibition was analyzed with 3H-thymidine incorporation. Paclitaxel of a suitable concentration was applied to the endothelium-denuded carotid artery of Fisher 344 inbred rats for 20 minutes. Angiogram and morphometric analysis of carotid artery was performed after 2 weeks. RESULTS: 3H-thymidine incorporation in cultured VSMCs was decreased dose-dependently from the concentration of 0.1 micromol/L (2,454+/-149cpm/ microgram protein) to 100 micromol/L (1,323+/-69cpm/ microgram protein) of paclitaxel by single and 20-minute exposure in the presence of platelet-derived growth factor (p<0.005). In the absence of platelet-derived growth factor, the decrement of 3H-thymidine incorporation was evident above the concentration of 5 micromol/L of paclitaxel. To evaluate in vivo effect, paclitaxel (0.1 or 1 micromol/L) was administered into the endothelium-denuded carotid artery by balloon injury and incubated for 20 minutes. Percent stenoses (32.2+/-9.8%) of paclitaxel-treated group was less than those (46.3+/-7.5%) of control group on histologic analysis (p<0.01). Paclitaxel-treated group also had wider lumen on carotid angiogram and less neointimal thickening than control on histologic examination (p<0.005). CONCLUSION: Proliferation of VSMCs was effectively inhibited and neointimal formation and luminal stenosis was prevented in rat carotid artery injury model by single, brief and local delivery of low-dose paclitaxel. This strategy could be applied to clinical settings for the prevention of restenosis after PTCA.

Tyrosine phosphorylation of paxillin may be involved in vascular smooth muscle contraction

Paxillin is a regulatory component of the complex of cytoskeletal proteins that link the actin cytoskeleton to the plasma membrane. However, the role of paxillin during smooth muscle contraction is unclear. We investigated a possible role for the membrane-associated dense plaque protein paxillin in the regulation of contraction in rat aortic vascular smooth muscle. The tyrosine phosphorylation of paxillin, which was increased by norepinephrine, reached a peak level after 1 min stimulation and then decreased with time. However, norepinephrine induced a sustained contraction that reached a steady state 30 min after application. Pretreatment with tyrphostin, an inhibitor of tyrosine kinase, inhibited the tyrosine phosphorylation of paxillin and also the contraction stimulated by norepinephrine. Both inhibitions were concentration-dependent, and the degree of correlation between them was high. These results show that, in rat aortic smooth muscle, tyrosine kinase(s) activated by norepinephrine may phosphorylate the tyrosine residues of paxillin, thereby providing a source of regulation during vascular smooth muscle contraction.

The transfection of caldesmon DNA into primary cultured rat aortic vascular smooth muscle

Caldesmon (CaD), one of microfilament-associated proteins, plays a key role in microfilament assembly in mitosis. We have investigated the effects of overexpression of the high molecular weight isoform of CaD (h-CaD) on the physiology of vascular smooth muscle cells (VSMCs). Rat aortic VSMCs were stably transfected with plasmids carrying a full length human h-CaD cDNA under control of cytomegalovirus promoter. The majority of the overexpressed h-CaD appears to be localized predominantly on cytoskeleton structures as determined by detergent lysis. The overexpression of h-CaD, however, does not decrease the level of endogenous low molecular weight isoform of CaD. h-CaD overexpressing VSMCs (h-CaD/VSMCs) show a decreased growth rate than that of vector-only transfected cells when determined by (3H)thymidine uptake and cell counting after fetal bovine serum (FBS) stimulation. h-CaD/VSMCs were smaller than vector-transfected cells by 18% in cell diameter. These data suggest that overexpression of h-CaD can inhibit the poliferation and the cell volume of VSMCs stimulated by growth factors and that the gene therapy with h-CaD may be helpful to prevent the conditions associated with hypertrophy and/or hyperplasia of VSMCs after arterial injuries.

Inhibitory effects of ginseng saponins on c-fos mRNA expression and the proliferation of rat aortic vascular smooth muscle cells stimulated by angiotensin II

To evaluate the possibility that the ginseng saponins could be developed as an anti-arteriosclerotic agent, we examined the inhibitory effects of ginseng saponins (total saponin(TS), panaxatriol(PT), panaxadiol(PD)) on the expression of c-fos mRNA and the proliferation of cultured rat aortic vascular smooth muscle cells (VSMCs) stimulated by angiotensin II (Ang II). TS and PT (1.0 mg/ml) suppressed c-fos mRNA induction in VSMCs stimulated by 10-5 M Ang II. The order of inhibitory potency was PT>TS. Ginseng saponins (0.01~1.0 mg/ml) inhibited the proliferation of VSMCs stimulated by Ang II in a concentration dependent manner, the inhibitory potency was TS> PT> PD at 0.1~1.0 mg/ml. These results suggest that ginseng saponins may suppress Ang II-stimulated proliferation of aortic VSMCs which can be seen in atherosclerosis, hypertension and restenosis.

Gene Therapy Using Retroviral Vector Containing Rat Erythropoietin / 대한혈액학회지

BACKGROUND: Erythropoietin (EPO) is crucial to red blood cell regulation. Since the recombinant EPO was clinically available, it has been widely used in the treatment of anemia associated with disorders such as chronic renal failure, cancer and acquired immunodeficiency syndrome. Ex vivo therapy of recombinant EPO, however, requires large dose and frequent administration, which gives a financial impact to the patients. To make in vivo delivery system of EPO will be valuable to the patients who need EPO for a long time. METHODS: We have tried to make an in vivo EPO delivery system using transduced vascular smooth muscle cells with EPO gene in a rat model. Recombinant retroviral vector containing EPO gene was made employing LXSN and LNFX plasmid. Recombinant retrovirus was produced from PA317 packaging cell. Infection of the vascular smooth muscle cells with the virus and selection with G418 was done in vitro. These transduced cells were transplanted to the balloon-injured carotid artery, and hemoglobin and hematocrit as well as reticulocyte were measured in sequence. RESULTS: The virus titer was ten times greater in case of LNFEp than LEpSN, whereas EPO production from infected vascular smooth muscle cell was similar between LEpSN and LNFEp vectors (67mU/106cell/day and 68mU/106cell/day, respectively). The levels of hemoglobin and hematocrit increased gradually after transplantation of the transduced cells with LEpSN, reached to peak after 3 weeks (18.4+/-0.63gm/dL in case of hemoglobin and 50.7+/-0.62% in case of hematocrit) and remained thereafter. The percentage of reticulocyte was elevated from the 4th day to the 14th day after transplantation and returned to normal. CONCLUSION: The low dose required to the elevation of RBC mass and long sustained effect of transduced smooth muscle cell could make this gene therapy feasible to the clinical conditions.