OBJECTIVETo investigate the effects of evodiamine (Evo), a component of Evodiaminedia rutaecarpa (Juss.) Benth, on cardiomyocyte hypertrophy induced by angiotensin II (Ang II) and further explore the potential mechanisms.

METHODSCardiomyocytes from neonatal Sprague Dawley rats were isolated and characterized, and then the cadiomyocyte cultures were randomly divided into control, model (Ang II 0.1 μmol/L), and Evo (0.03, 0.3, 3 μmol/L) groups. The cardiomyocyte surface area, protein level, intracellular free calcium ([Ca]) concentration, activity of nitric oxide synthase (NOS) and content of nitric oxide (NO) were measured, respectively. The mRNA expressions of atrial natriuretic factor (ANF), calcineurin (CaN), extracellular signal-regulated kinase-2 (ERK-2), and endothelial nitric oxide synthase (eNOS) of cardiomyocytes were analyzed by real-time reverse transcriptionpolymerase chain reaction. The protein expressions of calcineurin catalytic subunit (CnA) and mitogen-activated protein kinase phosphatase-1 (MKP-1) were detected by Western blot analysis.

RESULTSCompared with the control group, Ang II induced cardiomyocytes hypertrophy, as evidenced by increased cardiomyocyte surface area, protein content, and ANF mRNA expression; increased intracellular free calcium ([Ca]) concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but decreased MKP-1 protein expression (P<0.05 or P<0.01). Compared with Ang II, Evo (0.3, 3 μmol/L) significantly attenuated Ang II-induced cardiomyocyte hypertrophy, decreased the [Ca] concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but increased MKP-1 protein expression (P<0.05 or P<0.01). Most interestingly, Evo increased the NOS activity and NO production, and upregulated the eNOS mRNA expression (P<0.05).

CONCLUSIONEvo signifificantly attenuated Ang II-induced cardiomyocyte hypertrophy, and this effect was partly due to promotion of NO production, reduction of [Ca]i concentration, and inhibition of CaN and ERK-2 signal transduction pathways.

Angiotensin II ; Animals ; Atrial Natriuretic Factor ; metabolism ; Calcineurin ; genetics ; metabolism ; Calcium ; metabolism ; Dual Specificity Phosphatase 1 ; genetics ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Hypertrophy ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Quinazolines ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley

BACKGROUND/AIMS: We investigated whether angiotensin III (Ang III) is involved in monocyte recruitment through regulation of the chemokine monocyte chemoattractant protein-1 (MCP-1) in cultured human proximal tubular epithelial cells (HK-2 cells). METHODS: We measured MCP-1 levels in HK-2 cells that had been treated with various concentrations of Ang III and Ang II type-1 (AT1) receptor antagonists at various time points. The phosphorylation states of p38, c-Jun N-terminal kinases (JNK), and extracellular-signal-regulated kinases were measured in Ang III-treated cells to explore the mitogen-activated protein kinase (MAPK) pathway. MCP-1 levels in HK-2 cell-conditioned media were measured after pre-treatment with the transcription factor inhibitors curcumin or pyrrolidine dithiocarbamate. RESULTS: Ang III increased MCP-1 protein production in dose- and time-dependent manners in HK-2 cells, which was inhibited by the AT1 receptor blocker losartan. p38 MAPK activity increased significantly in HK-2 cells exposed to Ang III for 30 minutes, and was sustained at higher levels after 60 minutes (p < 0.05). Total phosphorylated JNK protein levels tended to increase 20 minutes after stimulation with Ang III. Pre-treatment with a p38 inhibitor, a JNK inhibitor, or curcumin significantly inhibited Ang III-induced MCP-1 production. CONCLUSIONS: Ang III increases MCP-1 synthesis via stimulation of intracellular p38 and JNK MAPK signaling activity and subsequent activated protein-1 transcriptional activity in HK-2 cells.
Angiotensin II Type 1 Receptor Blockers/pharmacology ; Angiotensin III/*pharmacology ; Cell Line ; Chemokine CCL2/*metabolism ; Dose-Response Relationship, Drug ; Epithelial Cells/*drug effects/metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors/metabolism ; Kidney Tubules, Proximal/*drug effects/metabolism ; Phosphorylation ; Protein Kinase Inhibitors/pharmacology ; Signal Transduction/drug effects ; Time Factors ; Transcription Factor AP-1/metabolism ; Up-Regulation ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/metabolism

OBJECTIVETo evaluate the effects and possible mechanisms of rutaecarpine on angiotensin II (Ang II)-induced proliferation in cultured rat vascular smooth muscle cells (VSMCs).

METHODSVSMCs were isolated from Male Sprague-Dawley rat aorta, and cultured by enzymic dispersion method. Experiments were performed with cells from passages 3-8. The cultured VSMCs were randomly divided into control, model (Ang II 0.1 μmol/L), and rutaecarpine (0.3-3.0 μmol/L) groups. VMSC proliferation was induced by Ang II, and was evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and cell counting. To examine the mechanisms involved in anti-proliferative effects of rutaecarpine, nitric oxide (NO) levels and NO synthetase (NOS) activity were determined. Expressions of VSMC proliferation-related genes including endothelial nitric oxide synthase (eNOS), and c-myc hypertension related gene-1 (HRG-1) were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSRutaecarpine (0.3-3.0 μmol/L) inhibited Ang II-induced VSMC proliferation and the best effects were achieved at 3.0 μmol/L. The Ang II-induced decreases in cellular NO contents and NOS activities were antagonized by rutaecarpine (P <0.05). Ang II administration suppressed the expressions of eNOS and HRG-1, while increased c-myc expression (P <0.05). All these effects were attenuated by 3.0 μmol/L rutaecarpine (P <0.05).

CONCLUSIONRutaecarpine is effective against Ang II-induced rat VSMC proliferation, and this effect is due, at least in part, to NO production and the modulation of VMSC proliferation-related gene expressions.

Angiotensin II ; pharmacology ; Animals ; Base Sequence ; Cell Proliferation ; drug effects ; Cells, Cultured ; DNA Primers ; Hemeproteins ; metabolism ; Indole Alkaloids ; pharmacology ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Nitric Oxide Synthase Type III ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; Quinazolines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo observe the vasodilating effect of adrenomedullin 2 (ADM2) by antagonizing angiotensin 1 (Ang II), and to explore its mechanism.

METHODSEighteen male, 180-200 g SD rats were randomly divided into 3 groups (n = 6): control group, Ang II (150 ng/(kg x min)) group and Ang II (150 ng/(kg x min)) + ADM2(500 ng/(kg x h)) group. Mini-osmotic pumps filled with peptide were implanted in the back of rats subcutaneously. After two weeks, the blood pressure was measured by the way of carotid intubation. The plasma was collected for the detection of nitric oxide (NO) content and the activity of endothelial nitric oxide synthase (eNOS). The in situ oxidation of fluorescent dye dihydroethidium (DHE) was used for detecting superoxide in rat arteries. The rat isolated arterial rings were made for studying the vasodilating effect of ADM2. Human umbilical vein endothelial cell line EA. hy 926 cells were cultured and their intracellular reactive oxygen species (ROS) were evaluated by probe DCFH-DA.

RESULTSADM2 dramatically decreased the blood pressure in angiotensin II-induced hypertension rat model, enhanced plasma NO content and the activity of eNOS and reduced superoxide formation in vessel walls. ADM2 also induced relaxation of the vascular rings preconstricted by Ang II in a concentration-dependent and endothelium-dependent manner. In cultured vascular endothelium, ADM2 ameliorated the ROS generation induced by Ang II.

CONCLUSIONAdrenomedullin 2 relaxed blood vessels by antagonizing angiotensin II-induced oxidative stress and improving the vascular endothelial function.

Adrenomedullin ; pharmacology ; Angiotensin II ; pharmacology ; Animals ; Antihypertensive Agents ; pharmacology ; Blood Pressure ; drug effects ; Drug Antagonism ; Endothelium, Vascular ; drug effects ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Male ; Nitric Oxide ; blood ; Nitric Oxide Synthase Type III ; blood ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Vasodilation ; drug effects

This study aims to investigate the effect of total flavones of Fructus Chorspondiatis (TFFC) on the mRNA and protein expression of collagen type I and III of rat cardiac fibroblasts (CFs) induced by angiotensin II (Ang II), and explore its anti-myocardial fibrosis molecular mechanism. Neonatal rat CFs were prepared from Sprague-Dawley rats (1-3 d after birth). The expression of collagen type I and III mRNA and protein were measured by RT-PCR and Western blotting, respectively. The study showed that stimulation of neonatal rat CFs with 100 nmol.L-1 of Ang II for 72 h resulted in a significant increase of the expression of collagen type I and III mRNA and protein. The changes on the expression level were blocked by TFFC. The results demonstrated that TFFC can inhibit myocardial fibrosis induced by Ang II in rats, which is probably associated with the collagen type I and III mRNA and protein levels up-regulated by Ang II, and TFFC was shown to decrease the expression levels of collagen type I and III mRNA and protein.
Anacardiaceae ; chemistry ; Angiotensin II ; pharmacology ; Animals ; Animals, Newborn ; Cells, Cultured ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Fibroblasts ; cytology ; metabolism ; Flavones ; administration & dosage ; isolation & purification ; pharmacology ; Fruit ; chemistry ; Myocardium ; cytology ; metabolism ; Plants, Medicinal ; chemistry ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of an anti-fibrotic tetra peptide Ac-SDKP on vascular fibrosis by regulating extracellular regulated protein kinase (ERK1/2) activity through Ang II.

METHODSRat vascular adventitial fibroblasts were cultured in vitro. They were randomly divided into control group, Ang II (10(-6) mmol/L) group, Ang II and Ac-SDKP joint action group, PD98059 group. Type I, III collagen contents in adventitia fibroblasts were measured by RT-PCR and the expressions of matrix metalloproteinases (MMP-2) and transforming growth factor-beta1 (TGF-beta1) were determined by Western blot.

RESULTSAc-SDKP could reduced Ang II-induced expression of type I, III collagen secretion and TGF-beta1 at mRNA,and increase MMP-2 expression, PD98059 could inhibit the above effect.

CONCLUSIONThe results suggested that Ac-SDKP could inhibit the formation and development of vascular fibrosis through blocking ERK1/2 pathway mediated by Ang II. Ac-SDKP therefore served as an antifibrotic factor in vascular fibrosis.

Angiotensin II ; adverse effects ; Animals ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Collagen Type III ; biosynthesis ; Fibroblasts ; cytology ; drug effects ; metabolism ; Flavonoids ; pharmacology ; MAP Kinase Signaling System ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Oligopeptides ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo investigate the effect of losartan on prostatic hyperplasia in spontaneous hypertension rats (SHRs) and its pathophysiological mechanism.

METHODSWe randomly divided 36 male SHRs into three groups of equal number to be treated intragastrically with high-dose losartan (30 mg per kg per d), low-dose losartan (15 mg per kg per d) and distilled water (control group). After 6 weeks of intervention, we measured the body weight and tail artery blood pressure of the rats and compared them with the baseline data. We collected blood from the heart for determination of the levels of serum angiotensin II (Ang II), insulin-like growth factor-1 (IGF-1) and interleukin-6 (IL-6) by enzyme-linked immunosorbent assay (ELISA), and harvested their prostates for measurement of their weight, observation of the tissue ultrastructures under the electron microscope and detection of the expression of endothelial nitric oxide synthase (eNOS) in the prostate tissue by immunohistochemistry.

RESULTSCompared with the control group, the low- and high-dose losartan groups showed significant decreases in systolic blood pressure ([203.75 +/- 10.28] vs [184.54 +/- 16.90] mmHg, P = 0.013; [203.75 +/- 10.28] vs [166.88 +/- 14.74] mmHg, P = 0.001) and diastolic blood pressure ([151.58 +/- 9.96] vs [136.71 +/- 14.28] mmHg, P = 0.022; [151.58 +/- 9.96] vs [122.71 +/- 11.56] mmHg, P < 0.001) of the lower tail artery after treatment, as well as in the prostate weight ([0.73 +/- 0.08] vs [0.64 +/- 0.10] mg, P = 0.011; [0.73 +/- 0.08 ] vs [0.50 +/- 0.17] mg, P < 0.001). Electron microscopy revealed edema of the basal and columnar epithelial cells, concentrated and marginated heterochromatin and widened nuclear gap of interstitial fibroblast nuclei, and reduced mitochondria and endoplasmic reticula in the low-dose losartan group, and even more obvious in the high-dose group. The level of serum Ang II was remarkably higher in the low- and high-dose losartan groups than in the control ([61.32 +/- 2.49] vs [54.85 +/- 7.20] pg/ml, P = 0.021; [65.49 +/- 6.78] vs [54.85 +/- 7.20] pg/ml, P < 0.001]) , that of serum IGF-1 was lower in high-dose losartan than in the control group ([1.50 +/- 0.11] vs [1.60 +/- 0.10] ng/ml, P = 0.03), but the serum IL-6 levels exhibited no significant differences among the three groups. The expression of eNOS in the prostate tissue was significantly higher in the losartan groups than in the controls (P = 0.022), even higher in the high-dose than in the low-dose group.

CONCLUSIONLosartan can suppress the progression of prostate hyperplasia in spontaneous hypertension rats by inhibiting RAS, IGF-1 and angiogenesis.

Angiotensin II ; blood ; Animals ; Antihypertensive Agents ; pharmacology ; therapeutic use ; Hypertension ; drug therapy ; metabolism ; pathology ; Insulin-Like Growth Factor I ; metabolism ; Interleukin-6 ; blood ; Losartan ; pharmacology ; therapeutic use ; Male ; Nitric Oxide Synthase Type III ; metabolism ; Prostate ; drug effects ; metabolism ; pathology ; Prostatic Hyperplasia ; drug therapy ; metabolism ; pathology ; Rats ; Rats, Inbred SHR

OBJECTIVETo evaluate the effect of clearance of superoxide anion by catechin on the expression of nitrogen monoxidum (NO) and endothelial nitricoxide synthase (eNOS) and apoptosis in endothelial progenitor cells (EPCs) induced by angiotensin II (Ang II).

METHODSThe marrow endothelial progenitor cells of Sprague-Dawley rats were isolated and assigned to control (no treatment), Ang II treatment and Ang II + catechin treatment groups. After 48 hrs of culture, the concentration of O2*- in the supernate was measured by the NBT method, and NO concentration in the supernate was measured by the nitrate reductase method; the apoptosis rate of EPCs was detected by the TUNEL method; the mRNA expression of eNOS was detected by RT-PCR; the protein expression of eNOS was detected by Western blot analysis.

RESULTSAng II of 10-6 mol/L was determined as the suitable concentration for cell induction by the MTT test. Catechin of 400 mg/L was determined as an advisable intervention dosage. The apoptosis rate of EPCs in the control, the Ang II and the Ang II+catechin treatment groups were 2.48+/-0.12%, 54.18+/-0.77% and 16.87+/-0.35%, respectively, and there were significant differences among the three groups (P<0.01). The O2*- concentration in the Ang II and the Ang II+catechin treatment groups (81.7+/- 3.6 and 62.3+/- 2.2 U/L respectively) was significantly higher than that in the control group (33.7+/- 2.8 U/L) (P<0.01). An increased NO concentration was also found in the Ang II (189. 8+/- 9.0 micromol/L) and the Ang II+catechin treatment groups (276.4+/- 10.1 micromol/L) compared with that in the control group (105.8+/- 9.8 micromol/L) (P<0.01). There were significant differences in the concentrations of O2*- and NO between the Ang II and the Ang II+catechin treatment groups (P<0.05). The mRNA (P<0.05) and protein expression (P<0.01) of eNOS in the Ang II and the Ang II+catechin treatment groups increased significantly compared with those in the control group. The Ang II+catechin treatment group showed increased eNOS protein expression compared with the Ang II group (P<0.05).

CONCLUSIONSAng II may induce the generation of O2*-, inactivate NO and increase gene and protein expression of eNOS in EPCs. Catechin might decrease the apoptosis of EPCs through the effective clearance of O2*-and the reduction of NO inactivation and of eNOS protein uncoupling.

Angiotensin II ; pharmacology ; Animals ; Apoptosis ; drug effects ; Catechin ; pharmacology ; Cell Survival ; drug effects ; Endothelial Cells ; drug effects ; metabolism ; Female ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type III ; analysis ; genetics ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; drug effects ; metabolism ; Superoxides ; metabolism

OBJECTIVETo investigate the changes in angiotensinogen (AGT), angiotensin II type 1 receptor (AT(1)R), endothelial nitric oxide synthase (eNOS) mRNA and protein expressions and nitric oxide (NO) content in the rat glomeruli in response to leptin stimulation.

METHODSThe glomeruli isolated from male SD rats were stimulated with 3 nmol/L leptin for 2 h. Real-time PCR and Western blotting were performed to analyze the mRNA and protein expressions of AGT, AT(1)R and eNOS in the glomeruli, and nitrite concentration in the glomeruli was measured by nitrate reductase assay.

RESULTSIn comparison with the control group, exposure to leptin increased the mRNA levels of AGT, ATR(1) and eNOS in the isolated glomeruli by 2.69-/+0.17, 3.77-/+0.16 and 2.56-/+0.29 folds (P=0.024, 0.018 and 0.044), and their protein levels by 2.06-/+0.10, 2.67-/+0.08 and 1.61-/+0.13 folds (P=0.021, 0.015 and 0.032), respectively. The NO production in the glomeruli was also increased by 2.77-/+0.14 folds (P=0.000) following leptin exposure.

CONCLUSIONLeptin exposure of isolated rat glomeruli directly causes activation of the internal renal renin-angiotensin system and enhanced NO production, suggesting that leptin plays a role in the pathogenesis of maladaptation in renal hemodynamics in obesity.

Animals ; Gene Expression Regulation ; drug effects ; Kidney Glomerulus ; drug effects ; metabolism ; Leptin ; pharmacology ; Male ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Angiotensin, Type 1 ; genetics ; metabolism ; Renin-Angiotensin System ; drug effects

BACKGROUNDCurrently, there are still divergent opinions about the mechanisms of the impaired neovascularization in diabetic subjects. Due to the remarkable therapeutic effect of angiotensin-converting enzyme inhibititors (ACEIs) on the reduction of blood pressure and the protection of target organs, the clinical application of this kind of drugs is very widespread. However, it is still not clear about the role and related molecular pathway of this kind of drugs in the limbs' postischemic revascularization. It is of major therapeutic importance to resolve these questions. This study aimed to investigate the reasons of the impaired angiogenesis in the hind limbs of rats with diabetic ischemia, the role and related molecular mechanisms of ACEI in postischemic revascularization.

METHODSHind limbs ischemia was induced in diabetic rats by right femoral artery excision. Diabetic rats were randomly allocated to one of the following treatments for 4 weeks: ACEI by perindopril; perindopril in combination with a nitric oxide synthase (NOS) inhibitor; perindopril in combination with bradykinin (BK)-B1 receptor (B1R) antagonist or saline. The differences of angiogenesis, the mRNA and protein expression of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF) and basic fibroblast (bFGF), constitutive nitric oxide synthase (cNOS) activity and nitric oxide (NO) content were observed after treatment.

RESULTSIn non-ischemic hind limbs, no significant changes in capillary density, or the mRNA and protein expression of eNOS, VEGF and bFGF, or the NO content and the cNOS activity were observed among all groups. On the contrary, in ischemic hind limbs, the capillary density in diabetic rats decreased by 27% when compared with the control rats, so did the mRNA and protein expression of eNOS, VEGF and bFGF, or the NO content and the cNOS activity (P < 0.05). The capillary density was increased by 1.65-fold in the perindopril treatment group in reference to untreated diabetic rats. Moreover, administration of perindopril enhanced the mRNA expression of eNOS, VEGF, and bFGF by 1.45-, 1.44-, and 1.33-fold, increased the protein content of the above indices by 1.55-, 1.30- and 1.50-fold compared with the untreated diabetic rats respectively. Perindopril also increased NO content and cNOS activity to 1.33- and 1.38-fold of that in untreated diabetic rats. The combination of BK-B1R antagonist significantly decreased the above indices (P < 0.05). In contrast, the combination of NOS inhibitor decreased the expression of eNOS and bFGF, the NO content and the cNOS activity, while the expression of VEGF did not change.

CONCLUSIONSDiabetes mellitus reduces the neovascularization, related growth factors expression and activity in the diabetic rat ischemic legs model. Treatment of perindopril improves postischemic revascularization. This effect is mediated, at least in part, by the BK-B1R-related pathway, and the activation of VEGF/eNOS/bFGF signals may be involved in the pro-angiogenic effect.

Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; Animals ; Blood Glucose ; analysis ; Diabetes Mellitus, Experimental ; physiopathology ; Extremities ; blood supply ; Fibroblast Growth Factor 2 ; genetics ; Neovascularization, Physiologic ; drug effects ; Nitric Oxide ; analysis ; Nitric Oxide Synthase Type III ; metabolism ; Perindopril ; pharmacology ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Streptozocin ; Vascular Endothelial Growth Factor A ; genetics