endothelin-1 and Hypertrophy

Aldosterone exerts direct effects on the vascular system by inducing oxidative stress, inflammation, hypertrophic remodeling, fibrosis, and endothelial dysfunction. Aldosterone exerts its effects through genomic and nongenomic pathways in a mineralocorticoid receptor (MR)-dependent or independent manner. Other aldosterone receptors such as GPR30 have been identified. A tight relation exists between the aldosterone and angiotensin II pathways, as well as with the endothelin-1 system. There is a correlation between plasma levels of aldosterone and cardiovascular risk. Recently, an increasing body of evidence has underlined the importance of aldosterone in cardiovascular complications associated with the metabolic syndrome, such as arterial remodeling and endothelial dysfunction. Blockade of MR is an increasingly used evidence-based therapy for many forms of cardiovascular disease, including hypertension, heart failure, chronic kidney disease, and diabetes mellitus.

Topics: Adipocytes; Aldosterone; Angiotensin II; Animals; Antihypertensive Agents; Blood Vessels; Cardiovascular Diseases; Endothelin A Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Fibrosis; Gene Expression Regulation; Humans; Hypertension; Hypertrophy; Metabolic Syndrome; Mineralocorticoid Receptor Antagonists; Mineralocorticoids; Muscle, Smooth, Vascular; Oxidative Stress; Receptor, Endothelin A; Receptors, Mineralocorticoid; Renin-Angiotensin System; Signal Transduction; Sodium; Vasculitis; Vasoconstriction

ET (endothelin)-1 was first described as a potent vasoconstrictor. Since then, many other deleterious properties mediated via its two receptors, ETA and ETB, have been described, such as inflammation, fibrosis and hyperplasia. These effects, combined with a wide tissue distribution of the ET system, its up-regulation in pathological situations and a local autocrine/paracrine activity due to a high tissue receptor binding, make the tissue ET system a key local player in end-organ damage. Furthermore, ET-1 interacts in tissues with other systems such as the RAAS (renin-angiotensin-aldosterone system) to exert its effects. In numerous genetically modified animal models, non-specific or organ-targeted ET-1 overexpression causes intense organ damage, especially hypertrophy and fibrosis, in the absence of haemodynamic changes, confirming a local activity of the ET system. ET receptor antagonists have been shown to prevent and sometimes reverse these tissue alterations in an organ-specific manner, leading to long-term benefits and an improvement in survival in different animal models. Potential for such benefits going beyond a pure haemodynamic effect have also been suggested by clinical trial results in which ET receptor antagonism decreased the occurrence of new digital ulcers in patients with systemic sclerosis and delayed the time to clinical worsening in patients with PAH (pulmonary arterial hypertension). The tissue ET system allows therapeutic interventions to provide organ selectivity and beneficial effects in diseases associated with tissue inflammation, hypertrophy or fibrosis.

Topics: Animals; Cell Communication; Disease Models, Animal; Endothelin Receptor Antagonists; Endothelin-1; Fibrosis; Humans; Hypertrophy; Inflammation; Vasoconstriction

A proteasome-dependent proteolytic pathway serves important functions in cell cycle control and transcriptional regulation; however, its pathophysiological role in cardiovascular diseases is still unclear. We have recently obtained evidence that proteasome inhibitors are capable of preventing the development of deoxycorticosterone acetate (DOCA)-salt-induced hypertension or hypertrophy and of ischemic acute renal failure (ARF). Beneficial effects of the proteasome inhibitors were accompanied by a decrease in endothelin-1 (ET-1) content in the aorta and kidney of DOCA-salt and ischemic ARF animals, respectively. In addition, there is evidence showing that the reduction of nuclear factor-kappaB (NF-kappaB) activation is involved in the mechanisms for suppressive effects of proteasome inhibitors on ET-1 gene transcription and the consequent decrease in ET-1 mRNA expression in the cultured vascular endothelial cells. These findings suggest that a proteasome-dependent proteolytic pathway has a crucial role in the pathogenesis of ET-1-related cardiovascular diseases, probably through the activation of NF-kappaB, and also that the use of proteasome inhibitors may be a novel approach to the treatment of cardiovascular diseases.

Topics: Acute Kidney Injury; Animals; Blood Vessels; Cardiovascular Diseases; Endothelin-1; Humans; Hypertension; Hypertrophy; Ischemia; Kidney; Proteasome Endopeptidase Complex; Proteasome Inhibitors

The endothelin system has been implicated in the pathogenesis of arterial hypertension and renal disorders. Endothelin-1, the predominant isoform of the endothelin peptide family, regulates vasoconstriction and cell proliferation in tissues both within and outside the cardiovascular system through activation of Gi-protein-coupled ET(A) and ET(B) receptors. Endothelin synthesis is regulated through autocrine mechanisms by endothelin converting enzymes, chymases, and non-endothelin converting enzyme metalloproteases. In-vitro experiments have demonstrated that endothelin-1 stimulates growth in vascular smooth muscle and in the kidney. Recent studies indicate that endothelin mRNA and protein are also increased in vivo in the kidney and vasculature in hypertension and renal disease. Studies using molecular or pharmacological inhibition of the endothelin system demonstrate that endothelin-1 contributes to the functional and structural changes associated with arterial hypertension and glomerulosclerosis, and that these effects are only in part dependent on blood pressure. These experimental studies and first clinical trials suggest that endothelin antagonists may offer therapeutic potential to reduce end-organ damage in diseases associated with vascular remodeling and renal injury.

Topics: Amino Acid Sequence; Animals; Blood Pressure; Blood Vessels; Endothelin-1; Endothelins; Humans; Hypertension; Hypertension, Renal; Hypertrophy; Kidney Diseases; Molecular Sequence Data; Rats; Receptors, Endothelin

The survival rates of women with breast cancer have improved significantly over the last four decades due to advances in breast cancer early diagnosis and therapy. However, breast cancer survivors have an increased risk of cardiovascular complications following chemotherapy. While this increased risk of later occurring structural cardiac remodeling and/or dysfunction has largely been attributed to the cardiotoxic effects of breast cancer therapies, the effect of the breast tumor itself on the heart prior to cancer treatment has been largely overlooked. Thus, the objectives of this study were to assess the cardiac phenotype in breast cancer patients prior to cancer chemotherapy and to determine the effects of human breast cancer cells on cardiomyocytes.. We investigated left ventricular (LV) function and structure using cardiac magnetic resonance imaging in women with breast cancer prior to systemic therapy and a control cohort of women with comparable baseline factors. In addition, we explored how breast cancer cells communicate with the cardiomyocytes using cultured human cardiac and breast cancer cells.. Our results indicate that even prior to full cancer treatment, breast cancer patients already exhibit relative LV hypertrophy (LVH). We further demonstrate that breast cancer cells likely contribute to cardiomyocyte hypertrophy through the secretion of soluble factors and that at least one of these factors is endothelin-1.. Overall, the findings of this study suggest that breast cancer cells play a greater role in inducing structural cardiac remodeling than previously appreciated and that tumor-derived endothelin-1 may play a pivotal role in this process.

Topics: Breast Neoplasms; Case-Control Studies; Cell Communication; Cell Line, Tumor; Cells, Cultured; Culture Media, Conditioned; Endothelin-1; Female; Humans; Hypertrophy; Hypertrophy, Left Ventricular; Magnetic Resonance Imaging; Middle Aged; Myocytes, Cardiac; Paracrine Communication; Retrospective Studies; Tumor Cells, Cultured; Ventricular Remodeling

Prevalence of major depression in people with chronic heart failure is higher than in normal populations. Depression in heart failure has become a major issue. Psilocybin-containing mushrooms commonly known as magic mushrooms, have been used since ancient times for their mind healing properties. Their safety in cardiovascular disease conditions is not fully known and may pose as a risk for users suffering from these illnesses. Study investigates the effects and safety of Psilocybe cubensis and Panaeolus cyanescens magic mushrooms use from genus Psilocybe and Panaeolus respectively, in a pathological hypertrophy conditions in which endothelin-1 disorder is a contributor to pathogenesis. We examined the effects of the mushrooms extracts on endothelin-1-induced hypertrophy and tumor necrosis factor-α (TNF- α)-induced cell injury in H9C2 cardiomyocytes. Mushrooms were oven dried and extracted with cold and boiling-hot water. H9C2 cardiomyocytes were induced with endothelin-1 prior to treatment with extracts over 48 h. Cell injury was stimulated with TNF-α. Results proposed that the water extracts of Panaeolus cyanescens and Psilocybe cubensis did not aggravate the pathological hypertrophy induced by endothelin-1 and also protected against the TNF-α-induced injury and cell death in concentrations used. Results support medicinal safe use of mushrooms under controlled conditions and cautioned use of higher concentrations.

Topics: Agaricales; Animals; Endothelin A Receptor Antagonists; Endothelin-1; Hallucinogens; Heart Failure; Humans; Hypertrophy; Myocytes, Cardiac; Phenylpropionates; Psilocybe; Psilocybin; Pyridazines; Rats; Receptor, Endothelin A; Tumor Necrosis Factor-alpha

Endothelin-1 (ET-1), a potent vasoconstrictor normally active in maintaining vascular tone, may mediate significant pathogenic effects, contributing to several serious diseases when aberrantly expressed or regulated. The present study evaluates the capacity of ET-1 to affect endothelin-1-associated hypertrophic activity and decreased expression of heme oxygenase-1 by H9c2 rat cardiomyoblasts in vitro, corresponding to in vivo processes underlying cardiovascular diseases (CVDs). Beta estradiol (β-E) is tested for its capacity to alter the effects of ET-1. H9c2 cells, cultured 48 h, were stimulated with 100-10,000 nM of ET-1 and evaluated for changes in cell size, cell viability, and expression of the cytoprotective heat shock protein heme oxygenase-1 (HO-1), with 200 nM of β-E included in selected cultures to evaluate its effect on ET-1-mediated changes. The application of 100 to 10,000 nM of ET-1 resulted in a significant increase in average cell size and decreases in both cell viability and HO-1 protein content (p < 0.05). Moreover, 200 nM of β-E was observed to significantly counteract these effects by cardiomyoblasts stimulated with 1000 nM of ET-1 (p < 0.05). Sprague-Dawley rats treated intravenously with 1000 ng/kg of ET-1 demonstrated reduced HO-1 expression in peripheral blood and left ventricular tissue, which was counteracted by injection of 200 ng/kg β-E-demonstrating a possible correspondence between in vitro and in vivo effects. An outcome of particular value for clinical use of β-E, in the management of cardiac hypertrophy, is the observed capacity of the drug to abate ET-1-mediated suppression of HO-1 expression. It has been previously demonstrated that HO-1 inducers exhibit potent cardioprotective properties, thus offering the promise of combining them with β-E, allowing lower effective dosage of the drug and concomitantly lower adverse side effects associated with its clinical use. Major findings of this investigation are that pretreatment of cardiomyoblasts with β-E inhibited their hypertrophic response to ET-1 and counteracts the decrease of cell viability. These effects were associated with a restoration of HO-1 protein expression in both under in vitro and in vivo conditions.

Topics: Animals; Cell Line; Endothelin-1; Estradiol; Estrogens; Heme Oxygenase (Decyclizing); Hypertrophy; Male; Myoblasts, Cardiac; Rats, Sprague-Dawley

ET-1 was used to stimulate primary rat cardiomyocytes and establish a cardiomyocyte hypertrophy model. Different. Rat cardiomyocyte hypertrophy model was established successfully.

Topics: Animals; Apoptosis; Astragalus Plant; Cells, Cultured; Drugs, Chinese Herbal; Endothelin-1; Humans; Hypertrophy; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Receptors, Calcitriol; Saponins; Vitamin D

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Cardiomyopathies; Endothelin-1; Humans; Hypertrophy; rho-Associated Kinases

Peroxisome proliferator-activated receptor

Topics: Adiponectin; Blotting, Western; Cells, Cultured; Electrophoretic Mobility Shift Assay; Endothelin-1; Fenofibrate; Fluorescent Antibody Technique; Humans; Hypertrophy; Immunoprecipitation; Myocytes, Cardiac; NF-kappa B; PPAR alpha

Despite the increasing prevalence of heart failure with preserved ejection fraction (HFpEF) in humans, there remains no evidence-based therapies for HFpEF. Endothelin-1 (ET-1) antagonists are a possibility because elevated ET-1 levels are associated with adverse cardiovascular effects, such as arterial and pulmonary vasoconstriction, impaired left ventricular (LV) relaxation, and stimulation of LV hypertrophy. LV hypertrophy is a common phenotype in HFpEF, particularly when associated with hypertension.. In the present study, we found that ET-1 levels were significantly elevated in patients with chronic stable HFpEF. We then sought to investigate the effects of chronic macitentan, a dual ET-A/ET-B receptor antagonist, on cardiac structure and function in a murine model of HFpEF induced by chronic aldosterone infusion. Macitentan caused LV hypertrophy regression independent of blood pressure changes in HFpEF. Although macitentan did not modulate diastolic dysfunction in HFpEF, it significantly reduced wall thickness and relative wall thickness after 2 weeks of therapy. In vitro studies showed that macitentan decreased the aldosterone-induced cardiomyocyte hypertrophy. These changes were mediated by a reduction in the expression of cardiac myocyte enhancer factor 2a. Moreover, macitentan improved adverse cardiac remodeling, by reducing the stiffer cardiac collagen I and titin n2b expression in the left ventricle of mice with HFpEF.. These findings indicate that dual ET-A/ET-B receptor inhibition improves HFpEF by abrogating adverse cardiac remodeling via antihypertrophic mechanisms and by reducing stiffness. Additional studies are needed to explore the role of dual ET-1 receptor antagonists in patients with HFpEF.

Topics: Aged; Animals; Case-Control Studies; Collagen Type I; Connectin; Diastole; Echocardiography; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Female; Heart; Heart Failure; Humans; Hypertrophy; Hypertrophy, Left Ventricular; In Vitro Techniques; Male; MEF2 Transcription Factors; Mice; Middle Aged; Myocytes, Cardiac; Pyrimidines; RNA, Messenger; Stroke Volume; Sulfonamides; Ventricular Dysfunction, Left; Ventricular Remodeling

The purpose of this study was to explore the effect of polydatin on ventricular remodeling after myocardial infarction in coronary artery ligation rats and to elucidate the underlying mechanisms. A rat model of ventricular remodeling after myocardial infarction was established by left coronary artery ligation. Rats with coronary artery ligation were randomly divided into five groups: control, plus 40 mg/kg captopril, plus 25 mg/kg polydatin, plus 50 mg/kg polydatin, and plus 100 mg/kg polydatin. The sham-operated group was used as a negative control. Rats were administered intragastrically with the corresponding drugs or drinking water for seven weeks. At the end of the treatment, the left ventricular weight index and heart weight index were assessed. The cross-sectional size of cardiomyocytes was measured by staining myocardium tissue with hematoxylin and eosin. Collagen content was counted by Sirius red in aqueous saturated picric acid. The concentrations of angiotensin I, angiotensin II, aldosterone, and endothelin 1 in myocardium or serum were determined by radioimmunoassay. Hydroxyproline and nitric oxide concentrations and glutathione peroxidase and catalase activities in serum were measured by ultraviolet spectrophotometry. Our results showed that seven weeks of polydatin treatment resulted in a significantly reduced left ventricular weight index, heart weight index, serum concentrations of hydroxyproline and aldosterone, an increased concentration of nitric oxide as well as enhanced activities of glutathione peroxidase and catalase. Myocardial angiotensin I, angiotensin II, and endothelin 1 levels were also reduced. The cardiomyocyte cross-sectional area and collagen deposition diminished. This study suggests that polydatin may attenuate ventricular remodeling after myocardial infarction in coronary artery ligation rats through restricting the excessive activation of the renin-angiotensin-aldosterone system and inhibiting peroxidation.

Topics: Aldosterone; Animals; Antioxidants; Captopril; Collagen; Coronary Occlusion; Coronary Vessels; Endothelin-1; Glucosides; Heart Ventricles; Hydroxyproline; Hypertrophy; Male; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Nitric Oxide; Phytotherapy; Plant Extracts; Polygonum; Rats, Sprague-Dawley; Renin-Angiotensin System; Stilbenes; Ventricular Remodeling

The signaling cascades of the mitogen activated protein kinase (MAPK) family, calcineurin/NFATc4, and PI3K/Akt/GSK3, are believed to participate in endothelin-1 (ET-1)-induced cardiac hypertrophy. The aim of this study was to investigate whether KMUP-1, a synthetic xanthine-based derivative, prevents cardiomyocyte hypertrophy induced by ET-1 and to elucidate the underlying mechanisms. We found that in H9c2 cardiomyocytes, stimulation with ET-1 (100 nM) for 4 days induced cell hypertrophy and enhanced expressions of hypertrophic markers, including atrial natriuretic peptide and brain natriuretic peptide, which were all inhibited by KMUP-1 in a dose-dependent manner. In addition, KMUP-1 prevented ET-1-induced intracellular reactive oxygen species generation determined by the DCFH-DA assay in cardiomyocytes. KMUP-1 also attenuated phosphorylation of ERK1/2 and Akt/GSK-3β, and activation of calcineurin/NFATc4 and RhoA/ROCK pathways induced by ET-1. Furthermore, we found that the expression of heme oxygenase-1 (HO-1), a stress-response enzyme implicated in cardio-protection, was up-regulated by KMUP-1. Finally, KMUP-1 attenuated ET-1-stimulated activator protein-1 DNA binding activity. In conclusion, KMUP-1 attenuates cardiomyocyte hypertrophy induced by ET-1 through inhibiting ERK1/2, calcineurin/NFATc4 and RhoA/ROCK pathways, with associated cardioprotective effects via HO-1 activation. Therefore, KMUP-1 may have a role in pharmacological therapy of cardiac hypertrophy.

Topics: Animals; Calcineurin; Endothelin-1; Enzyme Activation; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Heme Oxygenase-1; Hypertrophy; Mitogen-Activated Protein Kinases; Models, Biological; Myocytes, Cardiac; NFATC Transcription Factors; Piperidines; Protein Binding; Proto-Oncogene Proteins c-akt; Rats; Reactive Oxygen Species; rho-Associated Kinases; rhoA GTP-Binding Protein; Signal Transduction; Transcription Factor AP-1; Xanthines

Emerging evidence supports a key role for endothelin-1 (ET-1) and the transactivation of the epidermal growth factor receptor (EGFR) in angiotensin II (Ang II) action. We aim to determine the potential role played by endogenous ET-1, EGFR transactivation and redox-dependent sodium hydrogen exchanger-1 (NHE-1) activation in the hypertrophic response to Ang II of cardiac myocytes. Electrically paced adult cat cardiomyocytes were placed in culture and stimulated with 1 nmol l(-1) Ang II or 5 nmol l(-1) ET-1. Ang II increased ~45 % cell surface area (CSA) and ~37 % [(3)H]-phenylalanine incorporation, effects that were blocked not only by losartan (Los) but also by BQ123 (AT1 and ETA receptor antagonists, respectively). Moreover, Ang II significantly increased ET-1 messenger RNA (mRNA) expression. ET-1 similarly increased myocyte CSA and protein synthesis, actions prevented by the reactive oxygen species scavenger MPG or the NHE-1 inhibitor cariporide (carip). ET-1 increased the phosphorylation of the redox-sensitive ERK1/2-p90(RSK) kinases, main activators of the NHE-1. This effect was prevented by MPG and the antagonist of EGFR, AG1478. Ang II, ET-1 and EGF increased myocardial superoxide production (187 ± 9 %, 149 ± 8 % and 163.7 ± 6 % of control, respectively) and AG1478 inhibited these effects. Interestingly, Los inhibited only Ang II whilst BQ123 cancelled both Ang II and ET-1 actions, supporting the sequential and unidirectional activation of AT1, ETA and EGFR. Based on the present evidence, we propose that endogenous ET-1 mediates the hypertrophic response to Ang II by a mechanism that involves EGFR transactivation and redox-dependent activation of the ERK1/2-p90(RSK) and NHE-1 in adult cardiomyocytes.

Topics: Angiotensin II; Animals; Blotting, Western; Cardiomegaly; Cats; Disease Models, Animal; Electric Stimulation; Endothelin-1; ErbB Receptors; Hypertrophy; Myocytes, Cardiac; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Signal Transduction; Sodium-Hydrogen Exchangers; Transcriptional Activation

Vascular Gqα signaling has been shown to contribute to cardiac hypertrophy. In addition, angiotensin II (ANG II) was shown to induce vascular smooth muscle cell (VSMC) hypertrophy through Gqα signaling; however, the studies on the role of Gqα and PLC-β1 proteins in VSMC hypertrophy in animal model are lacking. The present study was therefore undertaken to examine the role of Gqα/PLC-β1 proteins and the signaling pathways in VSMC hypertrophy using spontaneously hypertensive rats (SHR). VSMC from 16-wk-old SHR and not from 12-wk-old SHR exhibited enhanced levels of Gqα/PLC-β1 proteins compared with age-matched Wistar-Kyoto (WKY) rats as determined by Western blotting. However, protein synthesis as determined by [(3)H]leucine incorporation was significantly enhanced in VSMC from both 12- and 16-wk-old SHR compared with VSMC from age-matched WKY rats. Furthermore, the knockdown of Gqα/PLC-β1 in VSMC from 16-wk-old SHR by antisense and small interfering RNA resulted in attenuation of protein synthesis. In addition, the enhanced expression of Gqα/PLC-β1 proteins, enhanced phosphorylation of ERK1/2, and enhanced protein synthesis in VSMC from SHR were attenuated by the ANG II AT1 and endothelin-1 (ET-1) ETA receptor antagonists losartan and BQ123, respectively, but not by the ETB receptor antagonist BQ788. In addition, PD98059 decreased the enhanced expression of Gqα/PLC-β1 and protein synthesis in VSMC from SHR. These results suggest that the enhanced levels of endogenous ANG II and ET-1 through the activation of AT1 and ETA receptors, respectively, and MAP kinase signaling, enhanced the expression of Gqα/PLC-β1 proteins in VSMC from 16-wk-old SHR and result in VSMC hypertrophy.

Topics: Angiotensin II; Animals; Cells, Cultured; Disease Models, Animal; Endothelin-1; Extracellular Signal-Regulated MAP Kinases; GTP-Binding Protein alpha Subunits, Gq-G11; Hypertension; Hypertrophy; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NF-kappa B; Phospholipase C beta; Phosphorylation; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptor, Angiotensin, Type 1; Receptor, Endothelin A; RNA Interference; Signal Transduction; Transcription Factor AP-1; Transfection; Up-Regulation

Cardiomyocyte hypertrophy is the cellular response that mediates pathologic enlargement of the heart. This maladaptation is also characterized by cell behaviors that are typically associated with apoptosis, including cytoskeletal reorganization and disassembly, altered nuclear morphology, and enhanced protein synthesis/translation. Here, we investigated the requirement of apoptotic caspase pathways in mediating cardiomyocyte hypertrophy. Cardiomyocytes treated with hypertrophy agonists displayed rapid and transient activation of the intrinsic-mediated cell death pathway, characterized by elevated levels of caspase 9, followed by caspase 3 protease activity. Disruption of the intrinsic cell death pathway at multiple junctures led to a significant inhibition of cardiomyocyte hypertrophy during agonist stimulation, with a corresponding reduction in the expression of known hypertrophic markers (atrial natriuretic peptide) and transcription factor activity [myocyte enhancer factor-2, nuclear factor kappa B (NF-κB)]. Similarly, in vivo attenuation of caspase activity via adenoviral expression of the biologic effector caspase inhibitor p35 blunted cardiomyocyte hypertrophy in response to agonist stimulation. Treatment of cardiomyocytes with procaspase 3 activating compound 1, a small-molecule activator of caspase 3, resulted in a robust induction of the hypertrophy response in the absence of any agonist stimulation. These results suggest that caspase-dependent signaling is necessary and sufficient to promote cardiomyocyte hypertrophy. These results also confirm that cell death signal pathways behave as active remodeling agents in cardiomyocytes, independent of inducing an apoptosis response.

Topics: Angiotensin II; Animals; Animals, Newborn; Apoptosis; Bronchodilator Agents; Cardiomegaly; Caspase 3; Caspase 9; Cells, Cultured; Cysteine Proteinase Inhibitors; Endothelin-1; Enzyme Activation; Fluorescent Antibody Technique; Hypertrophy; In Vitro Techniques; Isoproterenol; Membrane Potential, Mitochondrial; Myocardium; Myocytes, Cardiac; Oligopeptides; Phenylephrine; Rats; Rats, Sprague-Dawley; Signal Transduction; Vasoconstrictor Agents

The study objective was to determine whether the vasculopathy seen in nonobstructed lung regions in chronic thromboembolic pulmonary hypertension is induced by the local blood flow increase or by factors released by the ischemic lung.. Three groups of 10 piglets were studied 5 weeks after right pulmonary artery ligation, right pneumonectomy, or right pulmonary artery dissection (sham). Pulmonary vascular resistance, pulmonary arterial vasoreactivity, and morphometry were measured, and gene expressions of factors involved in vascular smooth muscle cell proliferation were quantified.. Left lung blood flow was similarly increased after right pneumonectomy and right pulmonary artery ligation. Compared with right pneumonectomy, right pulmonary artery ligation resulted in left lung vasculopathy with increased pulmonary vascular resistance (P = .0009), medial hypertrophy of the distal pulmonary artery (P < .0001), and decreases in maximal relaxation to acetylcholine (P = .013) and endothelial nitric oxide synthase gene expression (P = .041). These values were similar after sham and right pneumonectomy. In the left lung, right pulmonary artery ligation increased the gene expressions for insulin-like growth factor (P = .034), platelet-derived growth factor (P = .0006), and vascular endothelial growth factor (P = .0105) compared with right pneumonectomy and sham. Whereas endothelin-1 gene expression was not affected, expressions of endothelin-1 receptors A and B were downregulated after right pneumonectomy (P = .048 and P = .039, respectively) and right pulmonary artery ligation (P = .033 and P = .028, respectively).. Pulmonary vasculopathy was absent in the remaining lung 5 weeks after right pneumonectomy but developed in the nonobstructed lung regions 5 weeks after right pulmonary artery ligation, suggesting that factors released by the ischemic lung induced vascular remodeling in the contralateral lung. This endocrine process may involve the release of factors involved in vascular smooth muscle cell proliferation.

Topics: Animals; Cell Proliferation; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelin-1; Gene Expression Regulation; Hypertrophy; Ischemia; Ligation; Lung; Nitric Oxide Synthase Type III; Platelet-Derived Growth Factor; Pneumonectomy; Pulmonary Artery; Pulmonary Circulation; Receptors, Endothelin; Regional Blood Flow; RNA, Messenger; Somatomedins; Swine; Time Factors; Vascular Endothelial Growth Factor A; Vascular Resistance; Vasodilation; Vasodilator Agents

Medial hypertrophy of pulmonary arterioles during pulmonary arterial hypertension (PAH) in humans is associated with enhanced proliferation of smooth muscle cells (SMCs). Elevated matrix metalloproteinase (MMP)-2 has been found in pulmonary artery SMCs (PA-SMCs) in humans with idiopathic PAH, leading to the hypothesis that MMP-2 contributes to the proliferation and migration of vascular SMCs in the pathogenesis of PAH. Rapidly growing meat-type (broiler) chickens provide a model of spontaneous PAH. The present study was conducted to determine whether MMP-2 is involved in the medial hypertrophy of pulmonary arterioles in this model. Cultured PA-SMCs from normal birds were used to evaluate the effect of MMPs on cell proliferation. Gelatin zymography showed that endothelin (ET)-1-induced proliferation of PA-SMCs was concomitant with increased pro- and active MMP-2 production. Reverse transcription PCR demonstrated upregulation of MMP-2 mRNA. However, PA-SMC proliferation was inhibited by the MMP inhibitors doxycycline and cis-9-octadecenoyl-N-hydroxylamide. In vivo experiments revealed a significant increase of MMP-2 expression in hypertrophied pulmonary arterioles of PAH broiler chickens, which was positively correlated with wall thickness and medial hypertrophy. MMP-2 may contribute to medial hypertrophy in pulmonary arterioles during PAH in broiler chickens by enhancing the proliferation of vascular SMCs.

Topics: Animals; Arterioles; Chickens; Doxycycline; Electrophoresis, Polyacrylamide Gel; Endothelin-1; Familial Primary Pulmonary Hypertension; Gene Expression Regulation, Enzymologic; Hydroxamic Acids; Hypertension, Pulmonary; Hypertrophy; Lung; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; Myocytes, Smooth Muscle; Poultry Diseases; Pulmonary Artery; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Chloride

We recently identified leptin as a downstream factor mediating the hypertrophic effects of both angiotensin II and endothelin-1 in cardiomyocytes, an effect dependent on increased leptin biosynthesis, however, the mechanism for such increased leptin production is not known. This study was designed to elucidate the mechanisms underlying angiotensin II- and endothelin-1-stimulated synthesis in cultured ventricular myocytes. The hypertrophic effects of both angiotensin II (100 nM) and endothelin-1 (10 nM) were associated with increased leptin secretion and gene expression by 40 and 50 %, and 86 and 68 %, respectively. These effects were associated with significantly increased nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation by 34 and 52 %, as well as enhanced translocation of NF-κB into nuclei and also the NF-κB-DNA binding activity by 35 and 31 % induced by angiotensin II and endothelin-1, respectively. On their own, 24 h treatment with either angiotensin II or endothelin-1 increased cell surface area by 30 and 40 %, protein synthesis by 30 % and the α-skeletal actin gene by 53 and 68 %, respectively, indicating a robust hypertrophic effect whereas this was completely prevented by NF-κB inhibition. In addition, NF-κB inhibition significantly attenuated angiotensin II and endothelin-1-induced p38 MAPK activation whereas inhibition of p38 MAPK blocked both angiotensin II- and endothelin-1-induced increases in leptin secretion. The ability of both angiotensin II- and endothelin-1 to increase leptin production in cardiomyocytes and the resultant hypertrophic response are mediated by NF-κB and dependent on p38 MAPK activation.

Topics: Active Transport, Cell Nucleus; Angiotensin II; Animals; Cell Enlargement; Cell Nucleus; Cell Size; Cells, Cultured; Endothelin-1; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Hypertrophy; Leptin; Myocytes, Cardiac; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Primary Cell Culture; Rats; Rats, Sprague-Dawley

The SS-16(BN)/Mcwi consomic rat was produced by the introgression of chromosome 16 from the Brown Norway (BN/NHsdMcwi) rat onto the genetic background of the Dahl salt-sensitive (SS/Mcwi) rat by marker-assisted breeding. We have previously shown that the normotensive SS-16(BN)/Mcwi consomic strain is better protected from developing left ventricular dysfunction and fibrosis with aging than the hypertensive SS/Mcwi parental strain; however, the mechanism of this protection was not clear since the SS-16(BN)/Mcwi had both lowered blood pressure and an altered genetic background compared with SS/Mcwi. Microarray analysis of SS-16(BN)/Mcwi and SS/Mcwi left ventricle tissue and subsequent protein pathway analysis were used to identify alterations in gene expression in signaling pathways involved with the observed cardioprotection on the SS background. The SS-16(BN)/Mcwi rats exhibited much higher mRNA levels of expression of transcription factor JunD, a gene found on chromosome 16. Additionally, high levels of differential gene expression were found in pathways involved with angiogenesis, oxidative stress, and growth factor signaling. We tested the physiological relevance of these pathways by experimentally determining the responsiveness of neonatal cardiomyocytes to factors from identified pathways and found that cells isolated from SS-16(BN)/Mcwi rats had a greater growth response to epidermal growth factor and endothelin-1 than those from parental SS/Mcwi. We also demonstrate that the SS-16(BN)/Mcwi is better protected from developing fibrosis with surgically elevated afterload than other normotensive strains, indicating that gene-gene interactions resulting from BN chromosomal substitution confer specific cardioprotection. When combined with our previous findings, these data suggest that that SS-16(BN)/Mcwi may have an increased angiogenic potential and better protection from oxidative stress than the parental SS/Mcwi strain. Additionally, the early transient idiopathic left ventricular hypertrophy in the SS-16(BN)/Mcwi may be related to altered myocyte sensitivity to growth factors.

Topics: Animals; Antioxidants; Cardiotonic Agents; Chromosomes, Mammalian; Endothelin-1; Epidermal Growth Factor; Heart Ventricles; Hypertrophy; Male; Myocytes, Cardiac; Oligonucleotide Array Sequence Analysis; Rats; Rats, Inbred BN; Rats, Inbred Dahl; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Up-Regulation; Vascular Endothelial Growth Factor A

Diabetes mellitus is the leading cause of vascular complications such as atherosclerosis. This study was designed to investigate whether Prunella vulgaris (APV) would inhibit diabetic atherosclerosis in db/db mice with type 2 diabetes. The db/db mice were treated with high fat/high cholesterol (HFHC) diet and an aqueous extract of APV (100 and 200 mg/kg/day) for eight weeks to examine the long-term effect on metabolic abnormalities and diabetic atherosclerosis. APV treatment markedly lowered blood glucose and systolic blood pressure. The db/db mice experienced an increase in blood urea nitrogen as well as a decrease of creatinine clearance, the latter of which was restored by treatment with APV. Treatment with APV markedly decreased total plasma cholesterol, triglyceride, and LDL-cholesterol and also increased the HDL-cholesterol. In addition, malondialdehyde and TGF-β1 were decreased by treatment of APV. On the other hand, total NO level was decreased in db/db mice. However, the NO level was increased by treatment with APV, suggesting an association with vascular dysfunction. Vascular relaxation of aortic rings by acetylcholine or SNP-inducement was ameliorated by APV in a dose-dependent manner. Damage of vascular intima and hypertrophic of media were observed in db/db mice; however its dysfunction was improved by the treatment of APV. APV treatment significantly reduced the aortic expressions of ICAM-1, VCAM-1, ET-1, and nitrotyrosine. Furthermore, expression of eNOS in aortic was remarkably increased by APV treatment. Taken together, APV suppressed hyperglycemia and diabetic vascular dysfunction in HFHC diet-db/db mice. The present data suggest that Prunella vulgaris may prevent development of diabetic atherosclerosis.

Topics: Acetylcholine; Animals; Aorta; Atherosclerosis; Blood Glucose; Blood Pressure; Blood Urea Nitrogen; Cholesterol, Dietary; Creatinine; Diabetes Complications; Diabetes Mellitus, Type 2; Diet, High-Fat; Dose-Response Relationship, Drug; Endothelin-1; Hyperglycemia; Hypertrophy; Hypoglycemic Agents; Intercellular Adhesion Molecule-1; Lipids; Male; Malondialdehyde; Mice; Mice, Inbred Strains; Mice, Knockout; Nitric Oxide; Phytotherapy; Plant Extracts; Prunella; Transforming Growth Factor beta1; Tunica Intima; Tunica Media; Tyrosine; Vascular Cell Adhesion Molecule-1; Vasodilation

Hyperleptinemia is known to participate in cardiac hypertrophy and hypertension, but the relationship between pressure overload and leptin is poorly understood. We therefore examined the expression of leptin (ob) and the leptin receptor (ob-R) in the pressure-overloaded rat heart. We also examined gene expressions in culture cardiac myocytes to clarify which hypertension-related stimulus induces these genes.. Pressure overload was produced by ligation of the rat abdominal aorta, and ob and ob-R isoform mRNAs were measured using a real-time polymerase chain reaction (PCR). We also measured these gene expressions in neonatal rat cardiac myocytes treated with angiotensin II (ANGII), endothelin-1 (ET-1), or cyclic mechanical stretch. Leptin and the long form of the leptin receptor (ob-Rb) gene were significantly increased 4 weeks after banding, but expression of the short form of the leptin receptor (ob-Ra) was unchanged. ob-Rb protein expression was also detected by immunohistochemistry in hypertrophied cardiac myocytes after banding. Meanwhile, plasma leptin concentrations were not different between the control and banding groups. In cultured myocytes, ANGII and ET-1 increased only ob mRNA expression. However, mechanical stretch activated both ob and ob-Rb mRNA expression in a time-dependent manner, but ob-Ra mRNA was unchanged by any stress.. We first demonstrated that both pressure mediated hypertrophy and mechanical stretch up-regulate ob-Rb gene expression in heart and cardiac myocytes, which are thought to be important for leptin action in cardiac myocytes. These results suggest a new local mechanism by which leptin affects cardiac remodeling in pressure-overloaded hearts.

Topics: Angiotensin II; Animals; Cells, Cultured; Endothelin-1; Hypertrophy; Leptin; Male; Myocytes, Cardiac; Pressure; Protein Isoforms; Rats; Rats, Wistar; Receptors, Leptin; RNA, Messenger; STAT3 Transcription Factor; Stress, Mechanical; Up-Regulation

We examined the contribution of p70 ribosomal S6 kinase (p70S6K) to airway smooth muscle hypertrophy, a structural change found in asthma. In human airway smooth muscle cells, transforming growth factor (TGF)-beta, endothelin-1, and cardiotrophin-1 each induced phosphorylation of p70S6K and ribosomal protein S6 while increasing cell size, total protein synthesis, and relative protein abundance of alpha-smooth muscle actin and SM22. Transfection of myocytes with siRNA against either p70S6K or S6, or infection with retrovirus encoding a kinase-dead p70S6K, reduced cell size and protein synthesis but had no effect on contractile protein expression per mg total protein. Infection with a retrovirus encoding a constitutively active, rapamycin-resistant (RR) p70S6K increased cell size but not contractile protein expression. siRNA against S6 decreased cell size in myocytes expressing RR p70S6K. Finally, TGF-beta treatment, but not RR p70S6K expression, increased KCl-induced fractional shortening. Together, these data suggest that p70S6K activation is both required and sufficient for airway smooth muscle cell size enlargement but not contractile protein expression. Further, ribosomal protein S6 is required for p70S6K-mediated cell enlargement. Finally, we have shown for the first time in a functional cell system that p70S6K-mediated myocyte enlargement alone, without preferential contractile protein expression, is insufficient for increased cell shortening.

Topics: Airway Remodeling; Animals; Asthma; Cell Enlargement; Cells, Cultured; Contractile Proteins; Cytokines; Disease Models, Animal; Endothelin-1; Enzyme Activation; Humans; Hypertrophy; Lung; Mice; Mice, Inbred BALB C; Microfilament Proteins; Muscle Contraction; Muscle Proteins; Muscle, Smooth; Mutation; Myocytes, Smooth Muscle; Ovalbumin; Phenotype; Phosphorylation; Potassium Chloride; Ribosomal Protein S6; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; Transduction, Genetic; Transforming Growth Factor beta

The mixed-lineage kinases (MLKs) act upstream of mitogen-activated protein kinases, but their role in cardiac biology and pathology is largely unknown.. We investigated the effect of a MLK1-3 inhibitor CEP-11004 on G protein-coupled receptor agonist-induced stress response in neonatal rat cardiac myocytes in culture.. CEP-11004 administration dose-dependently attenuated phenylephrine and endothelin-1 (ET-1)-induced c-Jun N-terminal kinase activation. MLK inhibition also reduced ET-1- and phenylephrine-induced phosphorylation of p38 mitogen-activated protein kinase. In contrast, phenylephrine-induced extracellular signal-regulated kinase phosphorylation was further up-regulated by CEP-11004. ET-1 increased activator protein-1 binding activity 3.5-fold and GATA-binding protein 4 (GATA-4) binding activity 1.8-fold, both of which were attenuated with CEP-11004 administration by 59% and 63% respectively. Phenylephrine induced activator protein-1 binding activity by 2.6-fold, which was decreased by 81% with CEP-11004 administration. Phenylephrine also induced a 3.7-fold increase in the transcriptional activity of B-type natriuretic peptide (BNP), which was attenuated by 41% with CEP-11004 administration. In agreement, MLK inhibition also reduced hypertrophic agonist-induced secretion of immunoreactive atrial natriuretic peptide and BNP.. These results showed that inhibition of the MLK1-3 signalling pathway was sufficient for suppressing the activity of key nuclear effectors (GATA-4 and activator protein-1 transcription factors) in cardiac hypertrophy, and attenuated the agonist-induced atrial natriuretic peptide secretion and activation of BNP gene transcription.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Carbazoles; Cardiomegaly; Cell Nucleus; Endothelin-1; Genes, jun; Heart; Hypertrophy; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase Kinase Kinase 11; Mitogen-Activated Protein Kinases; Myocytes, Cardiac; Natriuretic Peptide, Brain; p38 Mitogen-Activated Protein Kinases; Phenylephrine; Phosphorylation; Rats; Rats, Sprague-Dawley; Signal Transduction; Transcription Factor AP-1; Transcription Factors

Transgenic overexpression of human endothelin-2 in rats was used to characterize the contribution of endothelin to diabetic cardiomyopathy.. Diabetes mellitus was induced by streptozotocin in transgenic rats and transgene-negative controls. Nondiabetic animals were included as well to form a 4-group study design. Heart morphological and molecular alterations were analysed following 6 months of hyperglycaemia.. Plasma endothelin concentrations were significantly higher in both transgenic groups than in wild-type groups (nondiabetic: 3.5 +/- 0.4 vs. 2.1 +/- 0.2, P < 0.05; diabetic: 4.5 +/- 0.4 vs. 2.5 +/- 0.4 fmol mL(-1), P < 0.01). Diabetes induced cardiac hypertrophy in both wild-type and transgenic rats and showed the highest myocardial interstitial tissue volume density in diabetic transgenic rats (1.5 +/- 0.07%) as compared with nondiabetic transgenic (1.1 +/- 0.03%), nondiabetic wild-type (0.8 +/- 0.01%) and diabetic wild-type rats (1.1 +/- 0.03%; P < 0.01 for all comparisons). A similar pattern with the most severe changes in the enothelin-2 transgenic, diabetic animals was observed for hypertrophy of the large coronary arteries and the small intramyocardial arterioles respectively. Cardiac mRNA expression of endothelin-1, endothelin receptors type A and B were altered in some degree by diabetes or transgenic overexpression of endothelin-2, but not in a uniform manner. Blood pressure did not differ between any of the four groups.. Overexpression of the human endothelin-2 gene in rats aggravates diabetic cardiomyopathy by more severe coronary and intramyocardial vessel hypertrophy and myocardial interstitial fibrosis. This transgenic intervention provides further and independent support for a detrimental, blood pressure-independent role of endothelins in diabetic cardiac changes.

Topics: Animals; Arterioles; Blood Pressure; Cardiomyopathies; Coronary Vessels; Diabetes Complications; Diabetes Mellitus, Experimental; Disease Models, Animal; Endothelin-1; Endothelin-2; Hypertrophy; Myocardium; Rats; Rats, Transgenic; Receptors, Endothelin; RNA, Messenger; Streptozocin

Increased medial arterial thickness is a structural change in pulmonary arterial hypertension (PAH). The role of smooth muscle hypertrophy in this process has not been well studied. Bone morphogenetic proteins (BMPs), transforming growth factor (TGF)-beta1, serotonin (or 5-hydroxytryptamine; 5-HT), and endothelin (ET)-1 have been implicated in PAH pathogenesis. We examined the effect of these mediators on human pulmonary artery smooth muscle cell size, contractile protein expression, and contractile function, as well on the roles of glycogen synthase kinase (GSK)-3beta and p70 ribosomal S6 kinase (p70S6K), two proteins involved in translational control, in this process. Unlike epidermal growth factor, BMP-4, TGF-beta1, 5-HT, and ET-1 each increased smooth muscle cell size, contractile protein expression, fractional cell shortening, and GSK-3beta phosphorylation. GSK-3beta inhibition by lithium or SB-216763 increased cell size, protein synthesis, and contractile protein expression. Expression of a non-phosphorylatable GSK-3beta mutant blocked BMP-4-, TGF-beta1-, 5-HT-, and ET-1-induced cell size enlargement, suggesting that GSK-3beta phosphorylation is required and sufficient for cellular hypertrophy. However, BMP-4, TGF-beta1, 5-HT, and ET-1 stimulation was accompanied by an increase in serum response factor transcriptional activation but not eIF2 phosphorylation, suggesting that GSK-3beta-mediated hypertrophy occurs via transcriptional, not translational, control. Finally, BMP-4, TGF-beta1, 5-HT, and ET-1 treatment induced phosphorylation of p70S6K and ribosomal protein S6, and siRNAs against p70S6K and S6 blocked the hypertrophic response. We conclude that mediators implicated in the pathogenesis of PAH induce pulmonary arterial smooth muscle hypertrophy. Identification of the signaling pathways regulating vascular smooth muscle hypertrophy may define new therapeutic targets for PAH.

Topics: Actins; Bone Morphogenetic Protein 4; Endothelin-1; Epidermal Growth Factor; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Hypertension; Hypertrophy; Indoles; Lithium Chloride; Maleimides; Muscle Contraction; Potassium Chloride; Pulmonary Artery; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Messenger; Serotonin; Signal Transduction; Transforming Growth Factor beta1

Cardiomyocyte hypertrophy is associated with changes in gene expression. Extracellular signal-regulated kinases 1/2 (ERK1/2) and RhoA [activated by hypertrophic agonists (e.g. endothelin-1)] regulate gene expression and are implicated in the response, but their relative significance in regulating the cardiomyocyte transcriptome is unknown. Our aim was to establish the significance of ERK1/2 and/or RhoA in the early cardiomyocyte transcriptomic response to endothelin-1.. Cardiomyocytes were exposed to endothelin-1 (1 h) with/without PD184352 (to inhibit ERK1/2) or C3 transferase (C3T, to inhibit RhoA). RNA expression was analyzed using microarrays and qPCR. ERK1/2 signaling positively regulated approximately 65% of the early gene expression response to ET-1 with a small (approximately 2%) negative effect, whereas RhoA signaling positively regulated approximately 10% of the early gene expression response to ET-1 with a greater (approximately 14%) negative contribution. Of RNAs non-responsive to endothelin-1, 66 or 448 were regulated by PD184352 or C3T, respectively, indicating that RhoA had a more significant effect on baseline RNA expression. mRNAs upregulated by endothelin-1 encoded a number of receptor ligands (e.g. Ereg, Areg, Hbegf) and transcription factors (e.g. Abra/Srf) that potentially propagate the response.. ERK1/2 dominates over RhoA in the early transcriptomic response to endothelin-1. RhoA plays a major role in maintaining baseline RNA expression but, with upregulation of Abra/Srf by endothelin-1, RhoA may regulate changes in RNA expression over longer times. Our data identify ERK1/2 as a more significant node than RhoA in regulating the early stages of cardiomyocyte hypertrophy.

Topics: Animals; Animals, Newborn; Cells, Cultured; Endothelin-1; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation; Hypertrophy; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocytes, Cardiac; Rats; rhoA GTP-Binding Protein; RNA, Messenger; Signal Transduction; Time Factors

In patients with chronic thromboembolic pulmonary hypertension, high flow in unobstructed lung regions may induce small-vessel damage responsible for persistent pulmonary hypertension after pulmonary thromboendarterectomy. In piglets, closure of an experimental aortopulmonary shunt reverses the flow-induced vascular lesions and diminishes the elevated levels of messenger RNA (mRNA) expression for endothelin-1 and endothelin receptor A (ETA). We wanted to study the effect of the ETA antagonist TBC 3711 on reversal of flow-induced pulmonary vascular lesions.. Twenty piglets were studied. In 15 piglets, pulmonary vasculopathy was induced by creating an aortopulmonary shunt. After 5 weeks of shunting, some animals were studied (n = 5); others underwent shunt closure for 1 week with (n = 5) or without (n = 5) TBC3711 treatment. Anti-ETA treatment started 1 week before and ended 1 week after the shunt closure. The controls were sham-operated animals (n = 5).. High blood flow led to medial hypertrophy of the distal pulmonary arteries (54.9% +/- 1.3% vs 35.3% +/- 0.9%; P < .0001) by stimulating smooth muscle cell proliferation (proliferating cell nuclear antigen) and increased the expression of endothelin-1, ETA or endothelin receptor type A or endothelin receptor A, angiopoietin 1, and Tie2 (real-time polymerase chain reaction). One week after shunt closure, gene expression levels were normal and smooth muscle cells showed increased apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) without proliferation. However, pulmonary artery wall thickness returned to control values only in the group given TBC3711 (33.2% +/- 8% with and 50.3% +/- 1.3% without; P < .05).. Anti-ETA therapy accelerated the reversal of flow-induced pulmonary arterial disease after flow correction. In patients with chronic thromboembolic pulmonary hypertension and severe distal pulmonary vasculopathy, anti-ETA agents may prove useful for preventing persistent pulmonary hypertension after pulmonary thromboendarterectomy.

Topics: Angiopoietin-1; Animals; Animals, Newborn; Antihypertensive Agents; Aorta; Apoptosis; Cell Proliferation; Disease Models, Animal; Endothelin A Receptor Antagonists; Endothelin-1; Gene Expression Regulation; Hemodynamics; Hypertension, Pulmonary; Hypertrophy; Isoxazoles; Muscle, Smooth, Vascular; Pulmonary Artery; Pulmonary Circulation; Pulmonary Embolism; Receptor, Endothelin A; Receptor, TIE-2; RNA, Messenger; Sulfones; Swine; Time Factors

We investigated the role of leptin and its interaction with endothelin 1 (ET-1) in fibronectin (FN) synthesis and cardiomyocyte hypertrophy, two characteristic features of diabetic cardiomyopathy.. Endothelial cells [human umbilical vein endothelial cells (HUVECs)] were examined for FN production and neonatal rat cardiomyocytes for hypertrophy, following incubation with glucose, ET-1, leptin and specific blockers. FN, ET-1, leptin and leptin receptors mRNA expression and FN protein were measured. Myocytes were also morphometrically examined. Furthermore, hearts from streptozotocin-diabetic rats were analysed.. Glucose caused increased FN mRNA and protein expression in HUVECs and cardiomyocytes hypertrophy along with upregulation of ET-1 mRNA, leptin mRNA and protein. Glucosemimetic effects were seen with leptin and ET-1. Leptin receptor antagonist (leptin quadruple mutant) and dual endothelin A endothelin B (ETA/ETB) receptor blocker bosentan normalized such abnormalities. Hearts from the diabetic animals showed hypertrophy and similar mRNA changes.. These data indicate that in diabetes increased FN production and cardiomyocyte hypertrophy may be mediated through leptin with its interaction with ET-1.

Topics: Analysis of Variance; Animals; Cells, Cultured; Diabetic Angiopathies; Endothelial Cells; Endothelin-1; Extracellular Matrix Proteins; Fibronectins; Humans; Hypertrophy; Leptin; Male; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Receptors, Leptin; RNA, Messenger; Statistics, Nonparametric; Umbilical Veins

A potential link between tissue-type transglutaminase (tTG) and cardiac hypertrophy was suggested recently. However, whether tTG is implicated in hypertrophic agonist-induced cardiac hypertrophy is not yet known. The purpose of this study was to investigate the effects of tTG on cardiomyocyte hypertrophy induced by endothelin (ET) 1. Real-time quantitative RT-PCR and Western blot analysis demonstrated that ET-1 increased the expression of tTG mRNA and protein in cardiomyocytes by activating ET(A) receptors. ET-1 failed to cause increases in cell size and [(3)H]leucine uptake, sarcomere reorganization, and gene induction of the atrial natriuretic factor when cardiomyocytes were treated with monodansylcadaverine, a competitive inhibitor of tTG. Furthermore, the effects of ET-1 on multifunctional activities of tTG were determined by evaluating the incorporation of [(3)H]putrescine into N,N'-dimethylated casein and charcoal absorption, respectively. The results showed that ET-1 did not influence the basal transglutaminase activity of cardiomyocytes but significantly inhibited the 0.1-mmol/L Ca(2+)-stimulated transglutaminase activity. Otherwise, ET-1 elevated the activity of GTPase in a concentration- and time-dependent manner. In vivo, right ventricular hypertrophy induced by 2 weeks of chronic hypoxia was depressed by the tTG inhibitor cystamine (10 to 30 mg/kg, 2 times per day, IP) in a dose-dependent manner. Taken together, our data strongly supported the notion that tTG may act as a positive regulator of the hypertrophic program in response to ET-1. This is probably attributable to the signaling activity of tTG rather than transglutaminase activity.

Topics: Animals; Animals, Newborn; Cadaverine; Cells, Cultured; Cystamine; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelin-1; Enzyme Inhibitors; GTP Phosphohydrolases; GTP-Binding Proteins; Hypertrophy; Myocytes, Cardiac; Protein Glutamine gamma Glutamyltransferase 2; Rats; Rats, Wistar; Receptor, Endothelin A; RNA, Messenger; Transglutaminases

In cardiomyocytes, certain extracellular stimuli that activate heterotrimeric G protein-coupled receptors (GPCRs) can induce hypertrophy by regulating gene expression and increasing protein synthesis. We investigated if rat embryonic cardiomyocytes (H9c2) underwent variations in the expression levels and subcellular distribution of key components of GPCR-activated signaling pathways during endothelin-1 (ET-1)-induced hypertrophic response. A significant increase of p115RhoGEF protein level was evident in ET-1-treated cells. Real-time quantitative PCR showed RhoGEF mRNA levels were significantly increased. Inhibition of the Rho-associated kinase (ROCK) caused a significant decrease of p115RhoGEF protein in the nuclear fraction, whereas an inhibitor of PKC induced a redistribution of the protein between membrane/organelle and nuclear fractions. The ROCK inhibitor also decreased H9c2 cell hypertrophic response. These results indicate that ROCK and its downstream target molecules, which are involved in inducing the hypertrophic response, are also implicated in signaling the up-regulation of the p115RhoGEF protein.

Topics: Animals; Cell Line; Embryo, Mammalian; Endothelin-1; GTP-Binding Protein alpha Subunits, G12-G13; Guanine Nucleotide Exchange Factors; Hypertrophy; Myocytes, Cardiac; Protein Kinase C; Protein Subunits; Protein Transport; Rats; Rho Guanine Nucleotide Exchange Factors; rho-Associated Kinases; rhoA GTP-Binding Protein; Signal Transduction; Subcellular Fractions; Up-Regulation

Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of adrenomedullin (AM) and intermedin (IMD) and their receptor activity modifying proteins (RAMPs 1-3) is augmented in cardiomyocytes, indicating that the myocardial AM/ IMD system may be activated in response to pressure loading and ischemic insult. The aim was to examine effects on (i) parameters of cardiomyocyte hypertrophy and on (ii) expression of AM and IMD and their receptor components in NO-deficient cardiomyocytes of an intervention chosen specifically for ability to alleviate pressure loading and ischemic injury concurrently.. The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 35 mg.kg(-1).day(-1)) was given to rats for 8 weeks, with/ without concurrent administration of beta-adrenoceptor antagonist, atenolol (25 mg.kg(-1).day(-1)) / calcium channel blocker, nifedipine (20mg.kg(-1).day(-1)).. In L-NAME treated rats, atenolol / nifedipine abolished increases in systolic blood pressure and plasma AM and IMD levels and in left ventricular cardiomyocytes: (i) normalized increased cell width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP, BNP, ET) genes; (ii) normalized augmented membrane protein oxidation; (iii) normalized mRNA expression of AM, IMD, RAMP1, RAMP2 and RAMP3.. normalization of blood pressure and membrane oxidant status together with prevention of hypertrophy and normalization of the augmented expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes by atenolol / nifedipine supports involvement of both pressure loading and ischemic insult in stimulating cardiomyocyte hypertrophy and induction of these counter-regulatory peptides and their receptor components. Attenuation of augmented expression of IMD in this model cannot however be explained simply by prevention of cardiomyocyte hypertrophy.

Topics: Adrenomedullin; Animals; Atenolol; Atrial Natriuretic Factor; Blood Pressure; Endothelin-1; Gene Expression Regulation; Hypertrophy; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Myocytes, Cardiac; Natriuretic Peptide, Brain; Neuropeptides; NG-Nitroarginine Methyl Ester; Nifedipine; Nitric Oxide; Oxidative Stress; Peptides; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptor Activity-Modifying Protein 1; Receptor Activity-Modifying Protein 2; Receptor Activity-Modifying Protein 3; Receptor Activity-Modifying Proteins; Receptors, Cell Surface; Systole

Conjugated linoleic acid (CLA) refers to a naturally occurring mixture of positional and geometric isomers of linoleic acid. Evidence suggests that CLA is a dietary constituent and nutraceutical with anti-cancer, insulin-sensitizing, immunomodulatory, weight-partitioning, and cardioprotective properties. The aim of this study was to evaluate the effects of intervention with CLA on cardiac hypertrophy. In vitro, CLA prevented indicators of cardiomyocyte hypertrophy elicited by endothelin-1, including cell size augmentation, protein synthesis, and fetal gene activation. Similar anti-hypertrophic effects of CLA were observed in hypertrophy induced by angiotensin II, fibroblast growth factor, and mechanical strain. CLA may inhibit hypertrophy through activation of peroxisome proliferator-activated receptors (PPARs). CLA stimulated PPAR activity in cardiomyocytes, and the anti-hypertrophic effects of CLA were blocked by genetic and pharmacological inhibitors of PPAR isoforms alpha and gamma. CLA may disrupt hypertrophic signaling by stimulating diacylglycerol kinase zeta, which decreases availability of diacylglycerol and thereby inhibits the protein kinase Cepsilon pathway. In vivo, dietary CLA supplementation significantly reduced blood pressure and cardiac hypertrophy in spontaneously hypertensive heart failure rats. These data suggest that dietary supplementation with CLA may be a viable strategy to prevent pathological cardiac hypertrophy, a major risk factor for heart failure.

Topics: Angiotensin II; Animals; Dietary Supplements; Diglycerides; Endothelin-1; Fibroblast Growth Factors; Hypertrophy; Linoleic Acids, Conjugated; Models, Biological; Myocytes, Cardiac; PPAR alpha; PPAR gamma; Protein Kinase C-epsilon; Rats; Rats, Sprague-Dawley

The transcription factor PPARgamma is expressed in endothelium and vascular muscle where it may exert antiinflammatory and antioxidant effects. We tested the hypothesis that PPARgamma plays a protective role in the vasculature by examining vascular structure and function in heterozygous knockin mice expressing the P465L dominant negative mutation in PPARgamma (L/+). In L/+ aorta, responses to the endothelium-dependent agonist acetylcholine (ACh) were not affected, but there was an increase in contraction to serotonin, PGF(2alpha), and endothelin-1. In cerebral blood vessels both in vitro and in vivo, ACh produced dilation that was markedly impaired in L/+ mice. Superoxide levels were elevated in cerebral arterioles from L/+ mice and responses to ACh were restored to normal with a scavenger of superoxide. Diameter of maximally dilated cerebral arterioles was less, whereas wall thickness and cross-sectional area was greater in L/+ mice, indicating cerebral arterioles underwent hypertrophy and remodeling. Thus, interference with PPARgamma signaling produces endothelial dysfunction via a mechanism involving oxidative stress and causes vascular hypertrophy and inward remodeling. These findings indicate that PPARgamma has vascular effects which are particularly profound in the cerebral circulation and provide genetic evidence that PPARgamma plays a critical role in protecting blood vessels.

Topics: Acetylcholine; Animals; Aorta; Arterioles; Cerebrovascular Circulation; Dinoprost; Endothelin-1; Female; Gene Expression Profiling; Genes, Dominant; Hypertension; Hypertrophy; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; PPAR gamma; Serotonin; Serotonin Agents; Signal Transduction; Vasodilator Agents

Cardiac hypertrophy is a key structural feature of diabetic cardiomyopathy. Previous studies have shown that diabetes-induced endothelin-1 (ET-1) and sodium hydrogen exchanger-1 (NHE-1) mediate structural and functional deficits in the heart. In order to gain a mechanistic understanding of the role of ET-1 and NHE-1 in cardiomyocyte hypertrophy, we have utilized an in vitro endothelial-myocyte co-culture system to reveal cellular interactions that may arbitrate cardiomyocyte deficits in diabetes.. Rat ventricular cardiomyocytes were cultured in high glucose levels, which caused cellular hypertrophy. Hypertrophic markers, atrial natruritic peptide (ANP) and angiotensinogen (Agt), as well as inducible nitric oxide synthase (iNOS) were upregulated by high glucose. Treatment of cells with ET antagonist bosentan and NHE-1 inhibitor cariporide prevented glucose-induced cardiomyocyte hypertrophy and expression of ANP, Agt, and iNOS. Bosentan and cariporide treatment of cardiomyocytes co-cultured with endothelial cells produced a more pronounced normalization of glucose-induced changes as compared to cardiomyocyte cultured alone. To further explore the signaling mechanisms involved, we investigated the mitogen activated protein kinase (MAPK) pathway and its cross-interaction with signaling proteins known to be altered in diabetes. Our results indicate that MAPK activation is associated with cardiomyocyte hypertrophy and is inhibited by bosentan, cariporide, as well as protein kinase C inhibiton. Furthermore, MAPK activation was found to be upstream of the transcription factors, nuclear factor-kappaB and activating protein-1.. These results demonstrate that ET-1 and NHE-1 may mediate cardiomyocyte hypertrophy via MAPK activation and provide an insight into the pathogenesis of diabetic cardiomyopathy.

Topics: Animals; Animals, Newborn; Cells, Cultured; Endothelin-1; Endothelium, Vascular; Glucose; Humans; Hypertrophy; Mitogen-Activated Protein Kinases; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Sodium-Hydrogen Exchangers; Umbilical Veins

Cardiomyocyte hypertrophy and extracellular matrix remodeling, primarily mediated by inflammatory cytokine-stimulated cardiac fibroblasts, are critical cellular events in cardiac pathology. The molecular components governing these processes remain nebulous, and few genes have been linked to both hypertrophy and matrix remodeling. Here we show that p8, a small stress-inducible basic helix-loop-helix protein, is required for endothelin- and alpha-adrenergic agonist-induced cardiomyocyte hypertrophy and for tumor necrosis factor-stimulated induction, in cardiac fibroblasts, of matrix metalloproteases (MMPs) 9 and 13-MMPs linked to general inflammation and to adverse ventricular remodeling in heart failure. In a stimulus-dependent manner, p8 associates with chromatin containing c-Jun and with the cardiomyocyte atrial natriuretic factor (anf) promoter and the cardiac fibroblast mmp9 and mmp13 promoters, established activator protein 1 effectors. p8 is also induced strongly in the failing human heart by a process reversed upon therapeutic intervention. Our results identify an unexpectedly broad involvement for p8 in key cellular events linked to cardiomyocyte hypertrophy and cardiac fibroblast MMP production, both of which occur in heart failure.

Topics: Animals; Atrial Natriuretic Factor; Basic Helix-Loop-Helix Transcription Factors; Chromatin; Endothelin-1; Enzyme Induction; Fibroblasts; Gene Expression Regulation, Enzymologic; Heart Failure; HeLa Cells; Humans; Hypertrophy; Matrix Metalloproteinase 13; Matrix Metalloproteinase 9; Myocardium; Myocytes, Cardiac; Neoplasm Proteins; Phenylephrine; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-jun; Rats; RNA, Messenger; Transcription Factor AP-1; Tumor Necrosis Factor-alpha

Numerous neurohumoral factors such as endothelin (ET)-1 and angiotensin (Ang) II as well as the stretch stimulus act concertedly in the in vivo overloaded heart in inducing hypertrophy and failure. The primary culture of rat neonatal cardiomyocytes is the only in vitro model that allows the comparative analysis of growth responses and signaling events in response to different stimuli. In the present study, we examined stretched rat cardiomyocytes grown on flexible bottomed culture plates for hypertrophic growth responses (protein synthesis, protein/DNA ratio, and cell volume), F-actin filaments rearrangement (by confocal laser scanning microscopy), and for signaling events (activation of phospholipase C [PLC]-beta, protein kinase C [PKC], mitogenactivated protein [MAP] kinases) and compared these responses with ET-1 (10-8 M)-stimulated cells. Cyclic stretch for 48 h induced hypertrophic growth in cardiomyocytes indicated by increases in the rate of protein synthesis, cell volume, and diameter, which were less pronounced in comparison to stimulation by ET-1. During cyclic stretch, we observed disoriented F-actin, particularly stress-fibers whereas during ET-1 stimulation, Factins rearranged clearly in alignment with sarcomeres and fibers. The upstream part of signaling by cyclic stretch did not follow the PLCbeta-PKC cascade, which, in contrast, was strongly activated during ET-1 stimulation. Cyclic stretch and, to greater extent, ET-1 stimulated downstream signaling through ERK, p38 MAP kinase, and JNK pathways, but the involvement of tyrosine kinase and PI3 kinase-Akt signaling during cyclic stretch could not be proven. Taken together, our results demonstrate that both cyclic stretch and ET-1 induce hypertrophic responses in cardiomyocytes with different effects on organization of F-actin stress fibers in case of stretch. Furthermore, on the short-term basis, cyclical stretch, unlike ET-1, mediates its hypertrophic response not through activation of PLC-beta and PKC but more likely through integrin-linked pathways, which both lead to downstream activation of the MAP kinase family.

Topics: Actins; Animals; Animals, Newborn; DNA; Endothelin-1; Fluorescent Dyes; Hypertrophy; L-Lactate Dehydrogenase; MAP Kinase Signaling System; Microscopy, Confocal; Myocytes, Cardiac; Protein Transport; Rats; Signal Transduction; Tetrazolium Salts; Thiazoles

Endothelin-1 (ET1) is a vasoactive peptide that stimulates hypertrophy of vascular smooth muscle cells (VSMC) through diverse signaling pathways mediated by G(q)/G(i)/G(13) heterotrimeric G proteins. We have found that ET1 stimulates the activity of cAMP-dependent protein kinase (PKA) in VSMC as profoundly as the G(s)-linked beta-adrenergic agonist, isoproterenol (ISO), but in a transient manner. PKA activation by ET1 was mediated by type-A ET1 receptors (ETA) and recruited an autocrine signaling mechanism distinct from that of ISO, involving G(i)-coupled betagamma subunits of heterotrimeric G proteins, extracellular signal-regulated kinases ERK1/2, cyclooxygenase COX-1 (but not COX-2) and prostacyclin receptors. In the functional studies, inhibition of PKA or COX-1 attenuated ET1-induced VSMC hypertrophy, suggesting the positive role of PKA in this response to ET1. Furthermore, we found that ET1 stimulates a Gbetagamma-mediated, PKA-dependent phosphorylation and inactivation of glycogen synthase kinase-3 (GSK3), an enzyme that regulates cell growth. Together, this study describes that (i) PKA can be transiently activated by G(i)-coupled agonists such as ET1 by an autocrine mechanism involving Gbetagamma/calcium/ERK/COX-1/prostacyclin signaling, and (ii) this PKA activation promotes VSMC hypertrophy, at least in part, through PKA-dependent phosphorylation and inhibition of GSK3.

Topics: Adrenergic beta-Agonists; Animals; Autocrine Communication; Cyclic AMP-Dependent Protein Kinases; Cyclooxygenase 1; Endothelin-1; Enzyme Activation; Epoprostenol; Glycogen Synthase Kinase 3; GTP-Binding Protein beta Subunits; GTP-Binding Protein gamma Subunits; Hypertrophy; Isoproterenol; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphorylation; Rats; Rats, Inbred WKY; Signal Transduction; Vasoconstrictor Agents

Nitric oxide (NO), in addition to its vasodilator action, has also been shown to antagonize the mitogenic and hypertrophic responses of growth factors and vasoactive peptides such as endothelin-1 (ET-1) in vascular smooth muscle cells (VSMCs). However, the mechanism by which NO exerts its antimitogenic and antihypertrophic effect remains unknown. Therefore, the aim of this study was to determine whether NO generation would modify ET-1-induced signaling pathways involved in cellular growth, proliferation, and hypertrophy in A-10 VSMCs. Treatment of A-10 VSMCs with S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP), two NO donors, attenuated the ET-1-enhanced phosphorylation of several key components of growth-promoting and hypertrophic signaling pathways such as ERK1/2, PKB, and Pyk2. On the other hand, inhibition of the endogenous NO generation with N(G)-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, increased the ET-1-induced phosphorylation of these signaling components. Since NO mediates its effect principally through a cGMP-soluble guanylyl cyclase (sGC) pathway, we investigated the role of these molecules in NO action. 8-Bromoguanosine 3',5'-cyclic monophosphate, a nonmetabolizable and cell-permeant analog of cGMP, exhibited a effect similar to that of SNAP and SNP. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of sGC, reversed the inhibitory effect of NO on ET-1-induced responses. SNAP treatment also decreased the protein synthesis induced by ET-1. Together, these data demonstrate that NO, in a cGMP-dependent manner, attenuated ET-1-induced phosphorylation of ERK1/2, PKB, and Pyk2 and also antagonized the hypertrophic effects of ET-1. It may be suggested that NO-induced generation of cGMP contributes to the inhibition of ET-1-induced mitogenic and hypertrophic responses in VSMCs.

Topics: Animals; Aorta, Thoracic; Cell Proliferation; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Endothelin-1; Enzyme Inhibitors; Focal Adhesion Kinase 2; Guanylate Cyclase; Hypertrophy; Leucine; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Oxadiazoles; Phosphorylation; Protein Biosynthesis; Proto-Oncogene Proteins c-akt; Quinoxalines; Rats; Receptors, Cytoplasmic and Nuclear; S-Nitroso-N-Acetylpenicillamine; Signal Transduction; Soluble Guanylyl Cyclase

We studied the effect of chronic endothelin A receptor blockade by atrasentan on the pulmonary endothelin-1 system and vascular endothelial growth factor (VEGF) expression in piglets with high pulmonary blood flow. Twenty-five 4-week-old piglets with high pulmonary blood flow were randomized to three groups: sham operated (n = 8), placebo (water) (n = 7), or treatment with atrasentan (2 mg/kg per day) (n = 10). After 3 months, mean pulmonary arterial pressure (PAP) was higher in the placebo group than in the sham group [18 +/- 2 mm Hg versus 14 +/- 1 mm Hg; P < 0.05 (ANOVA)]. Atrasentan treatment was associated with lower cardiac output, PAP (14 +/- 1 mm Hg), and medial wall thickness of pulmonary arteries (diameter: 50-150 microM) compared with placebo [13.6 +/- 3.0% versus 18.1 +/- 4.2%; P < 0.05 (ANOVA)]. Quantitative real-time polymerase chain reaction for endothelin-1, endothelin B receptor, and endothelin-converting enzyme-1 mRNA in lung tissue did not differ. However, immunostaining as well as mRNA for VEGF were lower in atrasentan-treated animals (relative gene expression: atrasentan versus placebo: 0.8 +/- 0.3 versus 1.5 +/- 0.3; P = 0.009). Atrasentan treatment effectively reduces medial hypertrophy in piglets with chronic pulmonary hyperperfusion. Chronic endothelin A receptor blockade by atrasentan may interfere with the expression of VEGF.

Topics: Animals; Atrasentan; Blood Pressure; Cardiac Output; Chronic Disease; Disease Models, Animal; Endothelin A Receptor Antagonists; Endothelin-1; Gene Expression Regulation; Hypertension, Pulmonary; Hypertrophy; Immunohistochemistry; Lung; Pulmonary Artery; Pulmonary Circulation; Pyrrolidines; Random Allocation; Receptor, Endothelin B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Swine; Vascular Endothelial Growth Factor A

The cardiovascular benefit of fish oil, including eicosapentaenoic acid (EPA), in humans and experimental animals has been reported. The role of endothelin-1 (ET-1) in cardiac hypertrophy is well known. Endothelin-1 stimulates prepro-ET-1 mRNA expression in cardiomyocytes, and the autocrine/paracrine system of ET-1 is important for cardiomyocyte hypertrophy. Although many studies link EPA to cardiac protection, the effect of EPA on cardiac hypertrophy has yet to be clarified. Recently, we demonstrated that ET-1-induced cardiomyocytic change could be prevented by pretreatment with EPA. The present study investigated the changes of different components of the ET system at the mRNA level in ET-1-administered cardiomyocytes, and examined the effect of EPA pretreatment. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats, cultured in Dulbecco's modified Eagle's medium and Ham F12 supplemented with 0.1% fatty acid-free bovine serum albumin for 3 days. At Day 4 of culture, the cardiomyocytes were divided into 3 groups: control group, ET-1-treated (0.1 nM) group, and ET-1-treated group pretreated with EPA (10 microM). Twenty-four hours after treatment, the gene expressions of different components of the endothelin system in three experimental groups were evaluated by real-time polymerase chain reaction. Prepro-ET-1 mRNA expression was 53% upregulated in ET-1-induced hypertrophied cardiomyocytes and suppressed in the EPA-pretreated group. Endothelin-converting enzyme-1 (ECE-1) was also increased in ET-1-administered cardiomyocytes by 42% compared with the control group and was reversed in the EPA-pretreated group. The two receptors of ET system, ET(A) and ET(B), tended to be increased in the ET-1-treated group, but no statistical significance was seen among study groups. Endothelin-1 increased prepro-ET-1 and ECE-1 mRNA expression in hypertrophied-neonatal cardiomyocytes, and this was reversed with EPA pretreatment. Thus, EPA may play a crucial role in the regression of ET-1-induced cardiomyocyte hypertrophy, partly through the suppression of ET-1 and ECE-1 expression.

Topics: Animals; Animals, Newborn; Aspartic Acid Endopeptidases; Cells, Cultured; Eicosapentaenoic Acid; Endothelin-1; Endothelin-Converting Enzymes; Gene Expression Regulation; Heart Ventricles; Hypertrophy; Metalloendopeptidases; Myocytes, Cardiac; Rats; RNA, Messenger

Cardiomyocytes release (or metabolize) several diffusible agents (e.g., nitric oxide [NO], endothelin-1 [ET-1], and angiotensin II) that exert direct effects on myocyte function under various pathologic conditions. Although cardiac hypertrophy is a compensatory mechanism in response to different cardiovascular diseases, there can be a pathologic transition in which the myocardium becomes dysfunctional. Recently, NO has been found to be an important regulator of cardiac remodeling. Specifically, NO has been recognized as a potent antihypertrophic and proapoptotic mediator in cultured cardiomyocytes. We demonstrated that ET-1-induced hypertrophic remodeling in neonatal cardiomyocytes was arrested by pretreatment with eicosapentaenoic acid (EPA), a major component of fish oil. In some recent studies, EPA has demonstrated cardioprotective effects by modulating NO. This study investigated the changes in NO synthase (NOS) in ET-1-induced hypertrophied cardiomyocytes and in total levels of nitrates and nitrites. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats and were cultured in D-MEM/Ham F12 supplemented with 0.1% fatty acid-free bovine serum albumin for 3 days. At Day 4 of culture, the cardiomyocytes were divided into three groups: control group, ET-1 (0.1 nM) group, and ET-1 pretreated with EPA (10 microM) group. NOS gene expression was evaluated 24 hrs after treatment using real-time polymerase chain reaction. Endothelial NOS (eNOS) mRNA expression was decreased in the ET-1 group compared with controls and was unchanged by pretreatment with EPA. mRNA expression of inducible NOS (iNOS) was significantly increased in ET-1-treated cardiomyocytes and was suppressed by EPA pretreatment. Neuronal NOS gene expression and total NO level did not exhibit a statistically significant change in any of the groups. There may be some interaction between ET-1, eNOS, and iNOS in ET-1-induced and EPA-regressed hypertrophied cardiomyocytes that suppress iNOS expression without modulating total NO level or eNOS gene expression.

Topics: Animals; Animals, Newborn; Cells, Cultured; Eicosapentaenoic Acid; Endothelin-1; Gene Expression Regulation; Hypertrophy; Myocytes, Cardiac; Nitric Oxide; Nitric Oxide Synthase Type III; Nitrites; Rats; Rats, Sprague-Dawley; RNA, Messenger

Human heart failure is preceded by a process called cardiac remodeling, in which heart chambers progressively enlarge and contractile function deteriorates. Programmed cell death (apoptosis) of cardiac muscle cells has been identified as an essential process in the progression to heart failure. The execution of the apoptotic program entails complex interactions between and execution of multiple molecular subprograms. Endothelin (ET)-1, a potent vasoconstrictor peptide, is synthesized and secreted by cardiomyocytes and induces hypertrophy of cardiomyocytes. The cardiovascular benefit of fish oil containing eicosapentaenoic acid (EPA) in humans and experimental animals was reported. Recently, we found that ET-1-induced cardiomyocytic remodeling could be prevented by pretreatment with EPA. The aim of the present study is to investigate whether there would be any alteration in the expression of important apoptosis-related molecules in ET-1-administered hypertrophied cardiomyocytes. We also sought to determine, if there are alterations in apoptotic molecules, what type of role for EPA would then exist. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats and were cultured for 3 days. At Day 4 of culture, the cardiomyocytes were divided into three groups: control, the ET-1 (0.1 nM)-treated group, and the ET-1 group pretreated with EPA (10 microM). Twenty-four hours after the treatment, the gene expressions of three important molecules related to apoptosis (caspase-3, Bax, and Bcl-2) in three experimental groups were evaluated by real-time polymerase chain reaction. The present study could not demonstrate any significant or representative alteration in any of the above three apoptosis-related important markers in either ET-1-induced hypertrophied cardiomyocytes with or without EPA pretreatment. The present study would at least be able to exclude the involvement of some representative molecules related to apoptosis in ET-1-induced hypertrophied cardiomyocytes. In addition, the present study demonstrates that the antihypertrophic effect of EPA to ET-1-administered cardiomyocytes appears not to modulate the apoptosis signaling cascade.

Topics: Animals; Animals, Newborn; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspases; Cells, Cultured; Eicosapentaenoic Acid; Endothelin-1; Heart Ventricles; Hypertrophy; Myocytes, Cardiac; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley

To investigate the role of endothelin-1 and its receptors on hypertrophy or proliferation of cultured cardial cells.. Cardiomyocytes and cardiac fibroblasts were isolated by trypsin digestion method, DNA and protein synthesis were measured by 3H-dexyribonucleotidethymine (3H-TdR) and 3H-Leucine (3H-Leu) incorporation, while protein content was measured by Bradford method. Atrial natriuretic peptide (ANP) mRNA expression of cardiomyocyte was measured by reverse transcripted-polymerase chain reaction. Selective endothelin (ET) receptor subtype antagonists BQ123 and BQ788 were used to block ET(A) receptors (ET(A)R) and ET(B)R respectively and to observe the effects of the two receptors during cardiac hypertrophy.. ET-1 significantly increased the 3H-TdR and 3H-Leu incorporation rate of cardiomyocytes and cardiac fibroblasts in a dose-dependent manner and increased protein content. Furthermore, ET-1 promoted the ANP mRNA expression of cardiomyocyte. ET(A)R antagonist remarkably blocked these effects, while ET(B)R antagonist had no obvious effect.. ET-1 can induce the hypertrophy for cardiomyocytes and the proliferation for cardiac fibroblasts. These effects are mediated by ET(A)R.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cell Proliferation; Cells, Cultured; Endothelin-1; Fibroblasts; Hypertrophy; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; RNA, Messenger

Chronic administration of thiazolidinediones might predispose to cardiac hypertrophy. The aim was to investigate direct effects of rosiglitazone in rat ventricular cardiomyocytes maintained in vitro (24 h). Rosiglitazone (< or =10(-5) M) did not increase protein synthesis and produced small inconsistent increases in cellular protein. In the presence of serum (10% v/v), but not insulin-like growth factor (IGF-1, 10(-8) M) or insulin (1 U/ml), an interaction with rosiglitazone to stimulate protein synthesis was observed. The hypertrophic responses to noradrenaline (5x10(-6) M), PMA (10(-7) M) and ET-1 (10(-7) M) were not attenuated by rosiglitazone. Rosiglitazone (10(-7) M) did not influence protein synthesis in response to insulin (1 U/ml) and elevated glucose (2.5x10(-2) M) alone or in combination, but attenuated the increase in protein mass observed in response to elevated glucose alone. In re-differentiated cardiomyocytes, a model of established hypertrophy, rosiglitazone (10(-8) M-10(-6) M) increased protein synthesis. Together, these data indicate that rosiglitazone does not initiate cardiomyocyte hypertrophy directly in vitro. However, during chronic administration, the interaction of rosiglitazone with locally-derived growth-regulating factors may make a modest contribution to cardiac remodelling and influence the extent of compensatory hypertrophy of the compromised rat heart.

Topics: Animals; Cell Survival; Cells, Cultured; Culture Media; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Drug Interactions; Endothelin-1; Glucose; Heart Ventricles; Hypertrophy; Insulin; Insulin-Like Growth Factor I; Isoproterenol; Male; Myocytes, Cardiac; Norepinephrine; Phenylalanine; Rats; Rats, Sprague-Dawley; Rosiglitazone; Tetradecanoylphorbol Acetate; Thiazolidinediones

Diacylglycerol (DAG) is a lipid second messenger that transiently accumulates in cells stimulated by endothelin-1 (ET-1) and other Galphaq protein-coupled receptor agonists. Diacylglycerol kinase (DGK) is thought to be an enzyme that controls the cellular levels of DAG by converting it to phosphatidic acid; however, the functional role of DGK has not been examined in cardiomyocytes. Because DGK inactivates DAG, a strong activator of protein kinase C (PKC), we hypothesized that DGK inhibited ET-1-induced activation of a DAG-PKC signaling cascade and subsequent cardiomyocyte hypertrophy.. Real-time reverse transcription-polymerase chain reaction demonstrated a significant increase of DGK-zeta mRNA by ET-1 in cardiomyocytes. To determine the functional role of DGK-zeta, we overexpressed DGK-zeta in cardiomyocytes using a recombinant adenovirus encoding rat DGK-zeta (Ad-DGKzeta). ET-1-induced translocation of PKC-epsilon was blocked by Ad-DGKzeta (P<0.01). Ad-DGKzeta also inhibited ET-1-induced activation of extracellular signal-regulated kinase (P<0.01). Luciferase reporter assay revealed that ET-1-mediated increase of activator protein-1 (AP1) DNA-binding activity was significantly inhibited by DGK-zeta (P<0.01). In cardiomyocytes transfected with DGK-zeta, ET-1 failed to cause gene induction of atrial natriuretic factor, increases in [3H]-leucine uptake, and increases in cardiomyocyte surface area.. We demonstrated for the first time that DGK-zeta blocked ET-1-induced activation of the PKC-epsilon-ERK-AP1 signaling pathway, atrial natriuretic factor gene induction, and resultant cardiomyocyte hypertrophy. DGK-zeta might act as a negative regulator of hypertrophic program in response to ET-1, possibly by controlling cellular DAG levels.

Topics: Adenoviridae; Animals; Atrial Natriuretic Factor; Cell Enlargement; Cells, Cultured; Diacylglycerol Kinase; Diglycerides; Endothelin-1; Gene Expression Regulation; Hypertrophy; Myocytes, Cardiac; Protein Kinase C; Protein Kinase C-epsilon; Rats; Rats, Sprague-Dawley; RNA, Messenger; Second Messenger Systems; Transcriptional Activation; Transduction, Genetic

Endothelin-1 (ET-1) has been found to increase cardiac beta-myosin heavy chain (beta-MyHC) gene expression and induce hypertrophy in cardiomyocytes. ET-1 has been demonstrated to increase intracellular reactive oxygen species (ROS) in cardiomyocytes. The exact molecular mechanism by which ROS regulate ET-1-induced beta-MyHC gene expression and hypertrophy in cardiomyocytes, however, has not yet been fully described. We aim to elucidate the molecular regulatory mechanism of ROS on ET-1-induced beta-MyHC gene expression and hypertrophic signaling in neonatal rat cardiomyocytes. Following stimulation with ET-1, cultured neonatal rat cardiomyocytes were examined for 3H-leucine incorporation and beta-MyHC promoter activities. The effects of antioxidant pretreatment on ET-1-induced cardiac hypertrophy and mitogen-activated protein kinase (MAPKs) phosphorylation were studied to elucidate the redox-sensitive pathway in cardiomyocyte hypertrophy and beta-MyHC gene expression. ET-1 increased 3H-leucine incorporation and beta-MyHC promoter activities, which were blocked by the specific ET(A) receptor antagonist BQ-485. Antioxidants significantly reduced ET-1-induced 3H-leucine incorporation, beta-MyHC gene promoter activities and MAPK (extracellular signal-regulated kinase, p38, and c-Jun NH2 -terminal kinase) phosphorylation. Both PD98059 and SB203580 inhibited ET-1-increased 3H-leucine incorporation and beta-MyHC promoter activities. Co-transfection of the dominant negative mutant of Ras, Raf, and MEK1 decreased the ET-1-induced beta-MyHC promoter activities, suggesting that the Ras-Raf-MAPK pathway is required for ET-1 action. Truncation analysis of the beta-MyHC gene promoter showed that the activator protein-2 (AP-2)/specificity protein-1 (SP-1) binding site(s) were(was) important cis-element(s) in ET-1-induced beta-MyHC gene expression. Moreover, ET-1-induced AP-2 and SP-1 binding activities were also inhibited by antioxidant. These data demonstrate the involvement of ROS in ET-1-induced hypertrophic responses and beta-MyHC expression. ROS mediate ET-1-induced activation of MAPK pathways, which culminates in hypertrophic responses and beta-MyHC expression.

Topics: Animals; Animals, Newborn; Cardiac Myosins; Cells, Cultured; Endothelin-1; Enzyme Inhibitors; Gene Expression Regulation; Genes, Reporter; Humans; Hypertrophy; MAP Kinase Signaling System; Myocytes, Cardiac; Myosin Heavy Chains; Oxidation-Reduction; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptor, Endothelin A

Evidence from in vivo studies suggests that some inputs to cardiac hypertrophy are opposed by the actions of estrogen. However, the mechanisms of E2 action in this respect are mainly unknown. An important pathway that is utilized by multiple hypertrophic stimuli involves the activation of the tyrosine phosphatase, calcineurin (PP2B). Here we show that 17beta-estradiol (E2) significantly prevents angiotensin II (AngII)- or endothelin-1 (ET-1)-induced new protein synthesis, skeletal muscle actin expression, and increased surface area in cultured rat cardiomyocytes. ET-1 stimulated calcineurin phosphatase activity, resulting in new protein synthesis, and both were prevented by E2. E2 induced the MCIP1 gene, an inhibitor of calcineurin activity, via phosphatidylinositol 3-kinase, transcriptional, and mRNA stability mechanisms. Small interfering RNA for MCIP1 significantly reversed both the E2 restraint of protein synthesis and the inhibition of AngII-induced calcineurin activity. AngII-induced the translocation of the hypertrophic transcription factor, NF-AT, to the nucleus of the cardiomyocyte and stimulated NF-AT transcriptional activity. Both were prevented by E2. AngII also stimulated the activation of ERK and protein kinase C, contributing to cardiac hypertrophy. E2 inhibited these pathways, related to the stimulation of atrial natriuretic peptide production and secretion. Thus, restraint of calcineurin and kinase signaling to the hypertrophic program underlie these important effects of E2.

Topics: Angiotensin II; Animals; Animals, Newborn; Calcineurin; Cell Nucleus; Cells, Cultured; DNA-Binding Proteins; Endothelin-1; Estradiol; Estrogens; Extracellular Signal-Regulated MAP Kinases; Hypertrophy; Intracellular Signaling Peptides and Proteins; Leucine; Microscopy, Fluorescence; Models, Biological; Muscle Proteins; Myocytes, Cardiac; Nitric Oxide Synthase; Phosphatidylinositol 3-Kinases; Protein Kinase C; Rats; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Time Factors; Transcription, Genetic

Various cardiovascular pathologies are associated with vascular smooth muscle cell (VSMC) hypertrophy and elevated plasma leptin levels. We used the rat portal vein (RPV) cultured for three days to investigate the effect of mechanical stretch on autocrine secretion of leptin and the effect of exogenous leptin (3.1 nM) on VSMC. Stretching the RPV significantly up-regulated leptin production by greater than 100-fold and leptin receptor expression by up to 10-fold. In addition, stretch increased tissue weight by 23 +/- 1.3 and 30 +/- 1% (P < 0.05), respectively, in the absence or presence of leptin, although this was significantly attenuated by an antileptin antibody (166 ng/ml). Unstretched RPV weight decreased by 7.5 +/- 1.8% in the absence of leptin, whereas in the presence of leptin, weight increased by 6.5 +/- 1.8% (P < 0.05). VSMC size and [3H]leucine incorporation rates were significantly increased by leptin in stretched and unstretched tissues. Leptin-induced hypertrophy was associated with significant extracellular signal-regulated kinase (ERK1/2) activation as well as increased expression of angiotensinogen, the angiotensin type 1 receptor as well as preproendothelin-1, and the endothelin type A receptor, whereas ERK inhibition or inhibition of either the angiotensin II or endothelin-1 systems at both the synthesis and receptor levels blocked the hypertrophic response. The effects of leptin were also completely blocked by the cholesterol-chelating agent methyl-beta-cyclodextrin. Therefore, our study demonstrates stretch-dependent leptin release and a direct hypertrophic effect of leptin on RPV, the latter likely dependent on intact cholesterol-rich membrane microdomains and locally produced paracrine factors.

Topics: Angiotensin II; Animals; Cell Culture Techniques; Cells, Cultured; Culture Media; Endothelin-1; Hypertrophy; Leptin; Male; Models, Biological; Muscle, Smooth, Vascular; Organ Size; Portal Vein; Rats; Rats, Sprague-Dawley; Receptors, Cell Surface; Receptors, Leptin; Stress, Mechanical; Up-Regulation

Various Gq protein-coupled receptor agonists such as the alpha1 adrenoceptor agonist phenylephrine, angiotensin II, and endothelin-1 are potent hypertrophic factors. There is evidence of potential cross talk between these agents, particularly in terms of endothelin-1 as playing a central role in mediating the actions of other hypertrophic factors. Using cultured rat neonatal ventricular myocytes, we assessed the potential cross talk between these factors and sought to examine the potential underlying mechanisms. Twenty-four-hour exposure to either agent produced significant hypertrophy as determined by cell size and molecular markers. Although the hypertrophic effects of phenylephrine and angiotensin II were expectedly prevented by alpha1 and AT1 receptor antagonists, respectively, these effects were also blocked by the ETA receptor antagonist BQ123 [cyclo(D-Asp-Pro-D-Val-Leu-D-Trp)] but not by the ETB antagonist BQ788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine). Both phenylephrine and angiotensin II significantly increased protein expression of both endothelin receptor subtypes. Both phenylephrine and angiotensin II produced significant activation of p38 as well as extracellular signal-regulated protein kinase and c-Jun NH2-terminal kinase, although this was unaffected by endothelin receptor blockade. Further studies revealed that the effects of phenylephrine and angiotensin II were mediated by stimulated endothelin-1 production occurring via two separate mechanisms: angiotensin II by increasing the levels of the endothelin-1 precursor prepro endothelin-1 and phenylephrine by upregulating endothelin-converting enzyme 1. Our results indicate that the endothelin-1 system plays an obligatory role in the hypertrophic response to both phenylephrine and angiotensin II in cultured myocytes through a mechanism independent of mitogenactivated protein kinase activation.

Topics: Angiotensin II; Animals; Animals, Newborn; Aspartic Acid Endopeptidases; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Gene Expression; Heart Ventricles; Hypertrophy; Metalloendopeptidases; Mitogen-Activated Protein Kinases; Muscle Cells; Myocardium; Phenylephrine; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin

The effects of endothelin-1 (ET-1) and other drugs on the reactive oxygen species (ROS) generation and cardiomyocyte hypertrophy were examined in experiments on the cultured neonatal rat cardiomyocytes. The role of ROS on neonatal rat cardiomyocyte hypertrophy induced by ET-1 was studied and the relationship of PKC activation and ROS generation was investigated. The level of intracellular ROS was measured by the ROS-specific probe 2',7'-dichlorofluorescin diacetate (DCF-DA). Cardiomyocyte hypertrophy was determined by the RNA content, the total protein of cells and the cell surface area. The results are as follows. The fluorescence intensity of intracellular DCF-DA increased by 77% in cultured neonatal rat cardiac myocytes treated with ET-1 (10 nmol/L) vs control group. Compared with control group, the fluorescence intensity of intracellular PI, protein content and cell surface area increased by 128%, 87% and 151% respectively (all P<0.01) in cardiac myocytes treated with ET-1 (10 nmol/L). ABT-627, CC, or CAT inhibited the ET-1-induced increase in fluorescence intensity of intracellular DCF-DA by 62%,60% and 51% respectively (all P<0.01), and also attenuated the cardiac hypertrophy. The fluorescence intensity of intracellular DCF-DA increased by 74% (P<0.01) in myocytes treated with PMA (1 micromol/L) vs control group. Therefore, in the course of cardiomyocyte hypertrophy, ET-1 increases intracellular ROS in the cultured neonatal rat cardiac myocytes and inhibits cardiomyocyte hypertrophy induced by ROS. The ET(A) and PKC activation mediate the ROS production and cardiomyocyte hypertrophy induced by ET-1. ROS is necessary in the ET-1-induced cardiomyocyte hypertrophy.

Topics: Animals; Animals, Newborn; Cell Enlargement; Cells, Cultured; Endothelin-1; Female; Hypertrophy; Male; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

Aldosterone is produced not only in the adrenal gland but also in the extra-adrenal tissues, including failing human heart. This study examined the production of dehydroepiandrosterone (DHEA) in human heart and elucidated the possible physiological significance. Method and Results- Using left ventricular tissues obtained at autopsy, reverse transcription-polymerase chain reaction followed by Southern blot analysis revealed the gene expressions of CYP17. By measuring plasma aldosterone and DHEA levels at the coronary sinuses and aortic roots during cardiac catheterization, we found that DHEA but not aldosterone was secreted from control subjects (P<0.0001 and P=0.74, respectively), whereas aldosterone but not DHEA was secreted from patients with heart failure (P=0.0017 and P=0.67, respectively). To examine the significance of DHEA, we measured myocyte cell sizes and the gene expression of B-type natriuretic peptide (BNP), using a neonatal rat cardiocyte culture system. We found that DHEA (10(-8) mol/L) significantly inhibited the increase in myocyte cell sizes and BNP mRNA levels upregulated by endothelin-1 (P=0.031 and P<0.0001, respectively).. CYP17 gene expression and production of DHEA were demonstrated in human control heart. Also, we found that cardiac production of DHEA was suppressed in failing heart. We postulated that DHEA and/or its metabolites exert a cardioprotective action through antihypertrophic effects.

Topics: Adult; Aged; Aldosterone; Animals; Blotting, Southern; Cardiac Catheterization; Cell Size; Cells, Cultured; Dehydroepiandrosterone; Endothelin-1; Female; Heart Failure; Heart Ventricles; Humans; Hypertrophy; Male; Middle Aged; Myocytes, Cardiac; Natriuretic Peptide, Brain; Neoplasms; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Steroid 11-beta-Hydroxylase; Steroid 17-alpha-Hydroxylase

Endothelin (ET)-1 is a potent vasoconstrictor that contributes to vascular remodeling in hypertension and other cardiovascular diseases. Endogenous ET-1 is produced predominantly by vascular endothelial cells. To directly test the role of endothelium-derived ET-1 in cardiovascular pathophysiology, we specifically targeted expression of the human preproET-1 gene to the endothelium by using the Tie-2 promoter in C57BL/6 mice.. Ten-week-old male C57BL/6 transgenic (TG) and nontransgenic (wild type; WT) littermates were studied. TG mice exhibited 3-fold higher vascular tissue ET-1 mRNA and 7-fold higher ET-1 plasma levels than did WT mice but no significant elevation in blood pressure. Despite the absence of significant blood pressure elevation, TG mice exhibited marked hypertrophic remodeling and oxidant excess-dependent endothelial dysfunction of resistance vessels, altered ET-1 and ET-3 vascular responses, and significant increases in ET(B) expression compared with WT littermates. Moreover, TG mice generated significantly higher oxidative stress, possibly through increased activity and expression of vascular NAD(P)H oxidase than did their WT counterparts.. In this new murine model of endothelium-restricted human preproET-1 overexpression, ET-1 caused structural remodeling and endothelial dysfunction of resistance vessels, consistent with a direct nonhemodynamic effect of ET-1 on the vasculature, at least in part through the activation of vascular NAD(P)H oxidase.

Topics: Animals; Antioxidants; Endothelin-1; Endothelin-3; Endothelium, Vascular; Humans; Hypertrophy; Male; Mesenteric Arteries; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Transgenic; NADPH Oxidases; NG-Nitroarginine Methyl Ester; Nitric Oxide; Oxidative Stress; Reactive Oxygen Species; Receptor, Endothelin A; Receptor, Endothelin B; Receptor, TIE-2; Recombinant Fusion Proteins; RNA, Messenger; Vascular Resistance; Vasoconstriction; Vasoconstrictor Agents; Vasodilator Agents

Endothelin-1 (ET-1) is synthesized and secreted by cardiomyocytes and induces cardiac hypertrophy. Peroxisome proliferator-activated receptor-alpha (PPAR-alpha) is a lipid-activated nuclear receptor that negatively regulates the vascular inflammatory gene response by interacting with transcription factors, such as nuclear factor-kappaB and activator protein-1 (AP-1). We reported that PPAR-alpha activator, fenofibrate (10 microM), and PPAR-alpha overexpression markedly inhibited the ET-1-induced increase in protein synthesis in cultured neonatal rat cardiomyocytes. Activation of protein kinase C and one or more of the mitogen-activated protein kinase cascades by ET-1 induces many of the features of hypertrophy. We demonstrated that PPAR-alpha activation significantly inhibits ET-1-induced cardiac hypertrophy through negative regulation of AP-1 binding activity partly secondary to inhibition of the JNK pathway. Zechner et al. demonstrated a significant role of p38 mitogen-activated protein kinase (p38) in myocardial cell hypertrophic growth and gene expression. Therefore, we investigated the effect of fenofibrate on ET-1-induced p38 activation in cardiomyocytes. The phosphorylation of p38 was transiently increased after 15 and 30 minutes of stimulation with ET-1, which was significantly inhibited by fenofibrate (10 microM). Neither application of ET-1 nor fenofibrate treatment affected the expression level of p38 in cardiomyocytes. These results suggest that the negative effect of the PPAR-alpha activator, fenofibrate, on ET-1-induced cardiac hypertrophy may be partly due to inhibition of the p38 signaling pathway.

Topics: Animals; Animals, Newborn; Cell Size; Cells, Cultured; Endothelin-1; Fenofibrate; Hypertrophy; Myocytes, Cardiac; p38 Mitogen-Activated Protein Kinases; Phosphorylation; PPAR alpha; Rats; Rats, Sprague-Dawley; Signal Transduction; Time Factors

Hypokalemia causes renal tubulointerstitial injury with an elevation in renal endothelin-1 (ET-1). It was hypothesized that hypokalemic tubulointerstitial injury is ameliorated by the blockade of ET-A receptors (ETA), whereas ET-B receptor (ETB) antagonism may exacerbate the injury, because ETB is thought to mediate vasodilation. Rats were fed a K(+)-deficient diet alone (LC) or with an ETA-selective antagonist ABT-627 (LA) or an ETB-selective antagonist A-192621 (LB) for 8 wk. Control rats were on a normal K(+) diet alone or with the ETA-selective or ETB-selective antagonists. The severity of hypokalemia was not significantly different among LA, LB, and LC. LC developed tubulointerstitial injury with an elevation of renal preproET-1 mRNA level. There was an increase in tubular osteopontin expression, macrophage infiltration, collagen accumulation, and tubular cell hyperplasia. ETA blockade significantly ameliorated all parameters for renal injury in the cortex without suppressing local ET-1 and ETA expression. By contrast, ETB blockade significantly reduced local ET-1 and ETA expression and improved the injury to a similar extent in the cortex. In the medulla, ETA or ETB blockade only partially blocked renal injury. ETA blockade did not affect BP in normokalemic or hypokalemic rats. ETB blockade induced a BP elevation with a decrease in urinary Na(+) excretion in normokalemic but not in hypokalemic rats. These results indicate that ET-1 can mediate hypokalemic renal injury in two different ways: by directly stimulating ETA and by locally promoting endogenous ET-1 production via ETB. Thus, ETA as well as ETB blockade may be renoprotective in hypokalemic nephropathy.

Topics: Albuminuria; Animals; Atrasentan; Endothelin Receptor Antagonists; Endothelin-1; Hypertension; Hypertrophy; Hypokalemia; Kidney; Kidney Diseases; Kidney Tubules; Male; Potassium; Proteinuria; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptor, Endothelin B

beta-Catenin is a transcriptional activator that regulates embryonic development as part of the Wnt pathway and also plays a role in tumorigenesis. The mechanisms leading to Wnt-induced stabilization of beta-catenin, which results in its translocation to the nucleus and activation of transcription, have been an area of intense interest. However, it is not clear whether stimuli other than Wnts can lead to important stabilization of beta-catenin and, if so, what factors mediate that stabilization and what biologic processes might be regulated. Herein we report that beta-catenin is stabilized in cardiomyocytes after these cells have been exposed to hypertrophic stimuli in culture or in vivo. The mechanism by which beta-catenin is stabilized is distinctly different from that used by Wnt signaling. Although, as with Wnt signaling, inhibition of glycogen synthase kinase-3 remains central to hypertrophic stimulus-induced stabilization of beta-catenin, the mechanism by which this occurs involves the recruitment of activated PKB to the beta-catenin-degradation complex. PKB stabilizes the complex and phosphorylates glycogen synthase kinase-3 within the complex, inhibiting its activity directed at beta-catenin. Finally, we demonstrate via adenoviral gene transfer that beta-catenin is both sufficient to induce growth in cardiomyocytes in culture and in vivo and necessary for hypertrophic stimulus-induced growth. Thus, in these terminally differentiated cells, beta-catenin is stabilized by hypertrophic stimuli acting via heterotrimeric G protein-coupled receptors. The stabilization occurs via a unique Wnt-independent mechanism and results in cellular growth.

Topics: Animals; beta Catenin; Cell Division; Cells, Cultured; Cytoskeletal Proteins; Drug Stability; Endothelin-1; Gene Transfer Techniques; Glycogen Synthase Kinase 3; Heterotrimeric GTP-Binding Proteins; Hypertrophy; Myocytes, Cardiac; Phenylephrine; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Receptors, Cell Surface; Signal Transduction; Trans-Activators; Wnt Proteins; Zebrafish Proteins

Cardiac hypertrophy refers to the abnormal growth of cardiomyocytes, and is often caused by valvular heart disease and hypertension. It involves the activation of growth, including increased protein synthesis and changes in gene expression. Transforming growth factor-beta1 (TGF-beta1) may play a central role in protecting the heart during the hypertrophic response by helping to restore normal functions of the affected myocardium. We tested the hypothesis that cardiomyocytes respond to stretch-induced paracrine hypertrophic stimuli with increased expression of TGF-beta1. To that purpose, we investigated whether angiotensin II (All), endothelin- I (ET-1) and TGF-beta, secreted by stretched cardiac and vascular cells, are involved in the paracrine mechanisms of stretch-induced changes of TGF-beta1 mRNA expression in stationary (i.e. non-stretched) cardiomyocytes. Our results indicated that TGF-beta1 mRNA expression in stationary cardiomyocytes was increased by AII release from cardiomyocytes that had been stretched for 30-60 min. Furthermore, it is likely that ET-1 and TGF-beta were released by stretched cardiac fibroblasts and endothelial cells to induce TGF-beta1 mRNA expression in stationary cardiomyocytes. Stretched vascular smooth muscle cells did not influence TGF-beta1 mRNA expression in stationary cardiomyocytes. These results indicate that AII, ET-I and TGF-beta, released by cardiac cell types, act as paracrine mediators of TGF-beta1 mRNA expression in cardiomyocytes. Therefore, we conclude that in stretched myocardium the cardiomyocytes, cardiac fibroblasts and endothelial cells take part in intercellular interactions contributing to cardiomyocyte hypertrophy.

Topics: Angiotensin II; Animals; Blotting, Northern; Cells, Cultured; Endothelin-1; Fibroblasts; Hypertrophy; Male; Muscle, Smooth; Myocardium; Paracrine Communication; Rats; Rats, Wistar; RNA; RNA, Messenger; Stress, Mechanical; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

The purpose of the study was to assess whether increased pulmonary flow and subsequent development of pulmonary vascular remodelling could alter the expression of endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) in the rat lung. Nine 42-day-old Wistar rats underwent abdominal aortocaval shunt to increase pulmonary blood flow for 12 weeks. The shunt resulted in significant medial hypertrophy of pulmonary artery without significant alterations in pulmonary or systemic blood pressure. Using competitive reverse transcription-PCR, significant increases in the preproET-1 mRNA expression and eNOS mRNA expression in the lungs of rats with abdominal aortocaval shunt were detected. Increased eNOS protein in the lung of shunt rats was also found by Western blot analysis. However, the plasma ET-1 concentration in the pulmonary artery (sham: 5+/-0.7 pg/ml; shunt: 6+/-0.8 pg/ml) or the lung ET-1 content (sham: 218+/-41 ng/g protein; shunt: 224+/-40 ng/g protein) was unchanged. There was an elevated immunohistochemical expression of eNOS, but not ET-1, in the pulmonary vascular endothelium in rats with the shunt. These results suggest that eNOS and ET-1 may be involved in remodelling prior to the development of pulmonary hypertension.

Topics: Animals; Endothelin-1; Endothelins; Endothelium, Vascular; Hypertension, Pulmonary; Hypertrophy; Immunohistochemistry; Lung; Models, Animal; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Protein Precursors; Pulmonary Artery; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Cysteine-rich protein (Cyr61) and connective tissue growth factor (CTGF) are key immediate early growth factors with functions in cell proliferation, differentiation, and extracellular matrix synthesis. Studies were performed to assess the gene expression profile of Cyr61 and CTGF in rat urinary bladder during growth in response to partial outlet obstruction. The mRNA levels of Cyr61 as determined by ribonuclease protection assay increased sharply after 1 day and remained elevated throughout the time period of the obstruction. This correlates well with increased bladder weight. The CTGF mRNA levels seemed to peak within the second week of the urethral obstruction and correlate well with increased type I collagen mRNA. The expression pattern of either Cyr61 or CTGF proteins corroborated that of their respective mRNAs. Immunohistochemical analyses showed that immunoreactivity of Cyr61 was confined to detrusor smooth muscle and that of CTGF was detected within both detrusor muscle and lamina propria layers. These data strongly indicate the involvement of Cyr61 and CTGF in bladder wall remodeling as a result of the outlet obstruction.

Topics: Angiotensin II; Animals; Biomarkers; Connective Tissue Growth Factor; Cysteine-Rich Protein 61; Endothelin-1; Gene Expression; Growth Substances; Hypertrophy; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Male; Parathyroid Hormone-Related Protein; Proteins; Rats; Rats, Sprague-Dawley; RNA, Messenger; Urinary Bladder; Urinary Bladder Neck Obstruction; Vasoconstrictor Agents

In chronic diseases such as diabetes mellitus, continuous stress stimuli trigger a persistent, self-reinforcing reprogramming of cellular function and gene expression that culminates in the pathological state. Late-onset, stable changes in gene expression hold the key to understanding the molecular basis of chronic diseases. Renal failure is a common, but poorly understood complication of diabetes. Diabetic nephropathy begins with mesangial cell hypertrophy and hyperplasia, combined with excess matrix deposition. The vasoactive peptide endothelin promotes the mesangial cell hypertophy characteristic of diabetic nephropathy. In this study, we examined the signaling pathways and changes in gene expression required for endothelin-induced mesangial cell hypertrophy. Transcriptional profiling identified seven genes induced with slow kinetics by endothelin. Of these, p8, which encodes a small basic helix-loop-helix protein, was most strongly and stably induced. p8 is also induced in diabetic kidney. Mesangial cell hypertrophy and p8 induction both require activation of the ERK, JNK/SAPK and PI-3-K pathways. Small interfering RNA (siRNA)-mediated RNA interference indicates that p8 is required for endothelin-induced hypertrophy. Thus, p8 is a novel marker for diabetic renal hypertrophy.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cells, Cultured; Diabetic Nephropathies; DNA-Binding Proteins; Endothelin-1; Gene Expression Profiling; Gene Expression Regulation; Genetic Markers; Glomerular Mesangium; Growth Substances; Humans; Hypertrophy; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Rats; RNA Interference; Signal Transduction; Transcriptional Activation

Increased endothelin-1 (ET-1) or aldosterone may be associated with promotion of cardiovascular hypertrophy and fibrosis. We evaluated whether the selective ETA receptor-antagonist BMS 182874 (BMS) prevents cardiac and vascular collagen deposition and hypertrophy in aldosterone-infused rats. Rats received subcutaneous aldosterone (0.75 microg/h) and 1% sodium chloride in drinking water +/- BMS (40 mg/kg per day in food) for 6 weeks. Heart and aorta were cross-sectioned and stained with Sirius red. Heart weight did not change with either aldosterone infusion or BMS treatment. Cardiac and aortic interstitial and perivascular collagen were quantified with videomorphometry. Aortic collagen and media cross-sectional area were significantly increased 3.5-fold (P < .01) and 1.13-fold (P < .05), respectively, with aldosterone infusion and decreased in BMS-treated rats (P < .05, P < .001, respectively). Aldosterone infusion increased interstitial and perivascular collagen in the left (1.6- and 2.7-fold, P < .05 and P < .01, respectively) and right ventricle (1.5- and 1.7-fold, P > .05 and P < .05, respectively). BMS prevented collagen deposition except for interstitial collagen in the right ventricle. Cardiac and aortic fibrosis were significantly increased in aldosterone-infused hypertensive rats. The ETA receptor antagonist prevented cardiac and aortic collagen deposition and aortic hypertrophy. This suggests a role for ET-1 in fibrosis of heart and large vessels in conditions associated with mineralocorticoid excess.

Topics: Aldosterone; Animals; Antihypertensive Agents; Aorta; Aortic Diseases; Blood Pressure; Body Weight; Cardiomyopathies; Collagen; Dansyl Compounds; Endothelin Receptor Antagonists; Endothelin-1; Fibrosis; Heart; Hypertrophy; Male; Myocardium; Organ Size; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A

Topics: Animals; Animals, Newborn; Apoptosis; Blotting, Western; Carcinogens; Carrier Proteins; Chromones; Electrophoresis, Gel, Two-Dimensional; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Eukaryotic Initiation Factor-4E; Flavonoids; Hypertrophy; Insulin; Intracellular Signaling Peptides and Proteins; Morpholines; Myocardium; Oxidative Stress; Peptide Initiation Factors; Phorbol Esters; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Precipitin Tests; Protein Binding; Protein Biosynthesis; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Tetradecanoylphorbol Acetate; Time Factors

Increased endothelin-1 may be associated with elevation of blood pressure (BP) and promotion of vascular hypertrophy, especially in salt-sensitive hypertension. Mineralocorticoid hypertension has been associated with activation of the endothelin system. We evaluated whether in aldosterone-infused rats the selective endothelin type A receptor-antagonist BMS 182874 prevents BP elevation and vascular hypertrophy. Rats were infused with aldosterone (0.75 microg/h) subcutaneously via a mini-osmotic pump and were offered 1% NaCl in the drinking water+/-BMS 182874 (40 mg/kg in food) for 6 weeks. Systolic BP was monitored by the tail-cuff method, and vascular changes of mesenteric arteries were evaluated using a pressurized myograph. Aldosterone-infusion significantly increased BP to 151+/-7 mm Hg compared with controls (108+/-4 mm Hg, P<0.01). BMS 182874 normalized BP (117+/-4 mm Hg). Media cross-sectional area of aorta was significantly increased by aldosterone infusion (P<0.05), and BMS treatment normalized it (P<0.001). Aldosterone infusion increased media width and media-to-lumen ratio of mesenteric resistance arteries (17.6+/-0.4 microm and 7.5+/-0.4%) compared with controls (14.2+/-0.5 microm, P<0.01, and 5.9+/-0.1%, P<0.05). BMS 182874 normalized media and media-to-lumen ratio (15.1+/-0.6 microm and 5.7+/-0.1%, both P<0.01). In conclusion, the endothelin type A receptor antagonist attenuated BP elevation and prevented vascular remodeling or hypertrophy of aorta and mesenteric resistance arteries in aldosterone-infused rats. These results suggest a role for endothelin-1 in BP elevation and structural alterations of large and small vessels in aldosterone and salt-induced hypertension.

Topics: Aldosterone; Animals; Antihypertensive Agents; Aorta; Blood Pressure; Body Weight; Dansyl Compounds; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Hypertension; Hypertrophy; Infusions, Parenteral; Male; Mesenteric Arteries; Potassium; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; RNA, Messenger; Vasoconstriction; Vasodilation

We investigated the potential of natural occurring antioxidant alpha-lipoic acid to prevent hypertension and hypertensive tissue injury induced by deoxycorticosterone acetate (DOCA) and salt in rats. Two weeks after the start of DOCA-salt treatment, the rats were given alpha-lipoic acid (10 or 100 mg/kg/day, s.c.) or its vehicle for 2 weeks. Uninephrectomized rats without DOCA-salt treatment served as sham-operated controls. In vehicle-treated DOCA-salt rats, systolic blood pressure increased markedly after 3-4 weeks. Daily administration of 100 mg/kg alpha-lipoic acid for 2 weeks suppressed the increase in systolic blood pressure, whereas 10 mg/kg alpha-lipoic acid did not affect the progression of DOCA-salt-induced hypertension. When the degree of vascular hypertrophy of the aorta was morphometrically evaluated at 4 weeks, there were significant increases in media cross-sectional area in vehicle-treated DOCA-salt rats compared with sham-operated rats. The development of vascular hypertrophy was markedly suppressed by alpha-lipoic acid at 100 mg/kg but not at 10 mg/kg. Histopathological examination of the kidney in vehicle-treated DOCA-salt rats revealed fibrinoid-like necrosis in glomeruli and thickening of small arteries. In these animals, creatinine clearance decreased, and fractional excretion of Na(+), urinary excretion of protein and N-acetyl-beta-glucosaminidase increased. Such renal lesions and dysfunctions were ameliorated in DOCA-salt rats given alpha-lipoic acid. In addition, a marked increase in endothelin-1 content in both the aorta and kidney was evident in vehicle-treated DOCA-salt rats compared with findings in sham-operated rats. Significant attenuation of this increase occurred in alpha-lipoic acid-treated DOCA-salt rats. These results suggest that administration of alpha-lipoic acid to DOCA-salt hypertensive rats lessens the increased blood pressure and protects against renal and vascular injuries, possibly through the suppression of renal and vascular endothelin-1 overproduction.

Topics: Animals; Aorta; Blood Pressure; Body Weight; Desoxycorticosterone; Dose-Response Relationship, Drug; Endothelin-1; Hypertension; Hypertrophy; Kidney; Kidney Diseases; Male; Organ Size; Rats; Rats, Sprague-Dawley; Thioctic Acid

This study was designed to evaluate the capacity of norepinephrine (NE) to induce hypertrophic remodeling of small arteries in rats, and to determine the involvement of endothelin (ET) to initiate and maintain it.. Treatment with NE (2.5 microg/kg per min) for 14 or 28 days produced a similar inward hypertrophic remodeling, characterized by a smaller lumen, but increased media thickness and cross-sectional area. Arterial stiffness was reduced. Histological evaluation confirmed the hypertrophic nature of remodeling. Concomitant administration of LU135252 (ET-receptor antagonist) for the first 14 days of NE administration prevented the development of hypertrophy, without altering arterial mechanics. Treatment with the same antagonist from day 14 to day 28 of NE or angiotensin II (Ang II) treatment failed to regress established vascular hypertrophy. In contrast, normalization of arterial structure was observed with prazosin, an alpha-adrenergic blocker. Endothelin content in small mesenteric arteries showed a transient elevation following chronic NE administration.. Increased circulating NE levels are associated with hypertrophic remodeling of small arteries, in which ET plays an initiating role. However, the maintenance of vascular hypertrophy is ET-independent, either in the presence of augmented circulating levels of NE or Ang II. Thus, early rather than late treatment with ET-receptor antagonists may be a preferable approach to limit small artery-mediated end-organ damage in cardiovascular diseases.

Topics: Angiotensin II; Animals; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Hypertrophy; Mesenteric Arteries; Norepinephrine; Phenylpropionates; Pyrimidines; Rats; Rats, Sprague-Dawley; Time Factors; Vascular Resistance

Heparan sulfate (HS) is one of the components of extracellular matrix and a potent anti-growth factor in various cells. Heparin has a similar structure to HS and is demonstrated to inhibit myocardial cell hypertrophy. We examined the intracellular signal mechanisms linking to the inhibitory effects of heparin and HS on endothelin-1 (ET-1)-induced hypertrophy in cultured rat neonatal myocardial cells (MCs). Heparin inhibited ET-1-induced c-fos mRNA expression. Heparin and HS inhibited ET-1-induced activation of c-fos promoter/enhancer in MCs. Although heparin and HS inhibited ET-1-induced activation of the wild-type c-fos serum response element (SRE), the activation of a mutated c-fos SRE that contains an intact binding site for the serum response factor (SRF) but lacks the ternary complex factor (TCF) binding site, was not inhibited. In addition, heparin and HS inhibited the activation of TPA response element (TRE). However, heparin did not inhibit the activation of cyclic AMP response element (CRE). Furthermore, heparin and HS inhibited ET-1-induced activation of extracellular signal-regulated kinase (ERK) and phosphorylation of Elk-1, which is one of the TCFs. These results indicate that heparin and HS inhibited ET-1-induced ERK activation, resulting in suppression of Elk-1 phosphorylation, and lead to inhibition of c-fos gene expression through SRF-independent manner. Moreover, heparin and HS inhibited ET-1-induced [3H] leucine incorporation. These results suggest that heparin and HS inhibit ET-1 induced myocardial cell hypertrophy through the inhibition of gene expression and protein synthesis.

Topics: Amino Acids; Animals; Cyclic AMP Response Element-Binding Protein; DNA-Binding Proteins; Endothelin-1; Enhancer Elements, Genetic; Enzyme Activation; Enzyme Inhibitors; ets-Domain Protein Elk-1; Gene Expression; Heparin; Heparitin Sulfate; Hypertrophy; Mitogen-Activated Protein Kinases; Myocardium; Phosphorylation; Promoter Regions, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Response Elements; RNA, Messenger; Transcription Factors

Chronic administration of troglitazone might predispose to cardiac hypertrophy. The aims of the study were to determine if troglitazone could (i) initiate a trophic response directly in ventricular cardiomyocytes and (ii) modify responses to other trophic stimuli. After 24 h, troglitazone (10 nM-10 microM) (i) did not increase cellular protein mass and decreased incorporation of [14C]phenylalanine, a marker of protein synthesis, (ii) interacted with serum (10% v/v) and insulin-like growth factor-1 (10 nM) to produce small trophic responses, (iii) increased cellular protein mass but not protein synthesis with insulin (1 unit/ml). Troglitazone (1 microM) attenuated responses to phorbol-12-myristate-13-acetate (PMA) (100 nM), and noradrenaline (5 microM) and endothelin-1 (100 nM), which also activate protein kinase C. In summary, troglitazone does not initiate cardiomyocyte growth directly in vitro, and can inhibit protein kinase C-mediated growth mechanisms. However, the interaction of troglitazone with serum growth factors may contribute modestly to the development of hypertrophy. As troglitazone produced a moderate hypertrophic effect per se in re-differentiated cardiomyocytes, it may directly increase the severity of established hypertrophy.

Topics: Animals; Cattle; Cell Differentiation; Cell Survival; Chromans; Culture Media; Dose-Response Relationship, Drug; Drug Interactions; Endothelin-1; Fetal Blood; Glucose; Heart Ventricles; Hydrogen-Ion Concentration; Hypertrophy; Hypoglycemic Agents; Insulin; Insulin-Like Growth Factor I; Male; Myocardium; Norepinephrine; Phenylalanine; Protein Kinase C; Rats; Rats, Sprague-Dawley; Tetradecanoylphorbol Acetate; Thiazoles; Thiazolidinediones; Troglitazone

The transcriptional regulation of an isolated rat phospholamban (PL) promoter fragment in rat cardiomyocytes was analyzed by applying a new method to reach substantially higher transfection efficiencies: gene gun biolistics. The gene gun transfection method was optimized for application to primary cultures of rat neonatal cardiomyocytes. Cells, cultured at different densities (0.75-1.50x10(5)cells/cm(2)) in serum-free medium, were transfected with DNA coated gold particles. A transfection efficiency of up to 10% could be achieved (compared to <1% with other methods) by the gene gun as checked using a RSV- beta-Gal construct. Cardiomyocytes were stimulated by endothelin-1 (ET-1) (10(-8)M) to induce hypertrophy, thereby yielding the characteristic changes in gene expression (upregulation of Atrial Natriuretic Factor (ANF) and downregulation of PL). The basal activity of an ANF promoter fragment (increasing from the lowest to highest density 2.6-fold) and its ET-1 inducibility (only significant upregulation of 2.6-fold, at lowest density) appeared to be dependent on the plating density of the cardiomyocytes. A PL promoter fragment was isolated, sequenced and 1.4 kb was subcloned in a luciferase reporter vector. The basal activity of the PL promoter fragment was not dependent on the plating density. ET-1 did not downregulate the PL promoter, rather a significant upregulation (1.4-fold) was found at the highest plating density. In conclusion, plating density of the cardiomyocytes can influence promoter activity as shown with an ANF promoter fragment. A newly isolated and sequenced rat PL promoter fragment did not direct gene expression as expected on basis of downregulation of the PL gene by ET-1 observed in this model.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Base Sequence; Biolistics; Calcium-Binding Proteins; Cells, Cultured; Culture Media, Serum-Free; Endothelin-1; Gene Expression Regulation; Genes, Reporter; Heart; Hypertrophy; Luciferases; Molecular Sequence Data; Muscle Proteins; Myocardium; Promoter Regions, Genetic; Rats; Recombinant Fusion Proteins; Transcription, Genetic; Transfection

Urinary bladder hypertrophy and hyperplasia are common features of bladder outlet obstruction (BOO). The urinary bladder is known to synthesize endothelin-1 (ET-1), which is a potent vasoconstrictor peptide with mitogenic properties. Using an animal model of partial BOO, we investigated the potential role of ET-1 and its receptor subtypes (ET(A) and ET(B)) in bladder smooth muscle cell (SMC) proliferation.. Partial BOO was produced in adult male New Zealand White rabbits. After 3 weeks, the bladder was removed and SMCs from the dome and bladder neck were grown using standard explant methodology. At passage 2, the cells were made quiescent and then further incubated in foetal calf serum (FCS), control age-matched rabbit serum (CRS) or partial BOO serum (BRS) in the presence or absence of ET(A)-antagonist (BQ123) or ET(B)-antagonist (BQ788). SMC proliferation was then measured 24 h later with 5-bromo-2'deoxy-uracil and by cell counting using a haemocytometer at 48 h. Immunostaining for alpha-actin was performed on detrusor and bladder neck cells to confirm the presence of smooth muscle cells.. BQ123 and BQ788 did not influence detrusor or bladder neck SMC proliferation in FCS or CRS. However, in the presence of BRS, BQ123 and BQ788 (100 nmol/L) significantly (p = 0.008) inhibited detrusor and bladder neck SMC proliferation. Cell counts were significantly reduced from the detrusor (p = 0.03, p = 0.01 with BQ123 and BQ788, respectively) and bladder neck (p = 0.01 for both BQ123 and BQ78).. These results suggest that ET antagonists may have a role in preventing SMC hyperplasia associated with partial BOO.

Topics: Animals; Cell Division; Endothelin-1; Hyperplasia; Hypertrophy; Male; Rabbits; Receptors, Endothelin; Urinary Bladder; Urinary Bladder Neck Obstruction

This study investigated the role of renal nitric oxide synthase (NOS), endothelin, and possible mechanisms of renovascular dysfunction in salt-sensitive hypertension. Salt-sensitive (DS) and salt-resistant (DR) Dahl rats were treated for 8 wk with high salt diet (4% NaCl) alone or in combination with the ET(A) receptor antagonist LU135252 (60 mg/kg per d). Salt loading markedly increased NOS activity (pmol citrulline/mg protein per min) in renal cortex and medulla in DR but not in DS rats by 270 and 246%, respectively. Hypertension in DS rats was associated with renal artery hypertrophy, increased vascular and renal endothelin-1 (ET-1) protein content, and glomerulosclerosis. In the renal artery but not in the aorta of hypertensive DS rats, endothelium-dependent relaxation to acetylcholine was unchanged; however, endothelial dysfunction due to enhanced prostanoid-mediated, endothelium-dependent contractions and attenuation of basal nitric oxide release was present. Treatment with LU135252 reduced hypertension in part, but completely prevented activation of tissue ET-1 without affecting ET-3 levels. This was associated with a slight increase of renal NOS activity, normalization of endothelial dysfunction and renal artery hypertrophy, and marked attenuation of glomerulosclerosis. Thus, DS rats fail to increase NOS activity in response to salt loading. This abnormality may predispose to activation of the tissue ET-1 system, abnormal renal vasoconstriction, and renal injury. Chronic ET(A) receptor blockade normalized salt-induced changes in the renal artery and reduced glomerular injury, suggesting therapeutic potential for ET antagonists in salt-sensitive forms of hypertension.

Topics: Acetylcholine; Animals; Endothelin Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Enzyme Inhibitors; Glomerulosclerosis, Focal Segmental; Hypertension; Hypertrophy; Kidney; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Phenylpropionates; Pyrimidines; Rats; Rats, Inbred Dahl; Renal Artery; Sodium Chloride, Dietary; Vasodilator Agents

Effects of sitaxsentan (TBC11251), an orally active, highly selective antagonist of endothelin A receptors, were examined on the development and maintenance of pulmonary hypertension, pulmonary vascular remodeling, and cardiac hypertrophy in the rat. The pulmonary vasoconstrictor response to acute hypoxia (10% O(2)for 90 min) was prevented with sitaxsentan (5 mg/kg infused iv 10 min prior to the onset of hypoxia) while BQ-788 (a specific endothelin B receptor antagonist) was without effect. The same dose of sitaxsentan delivered iv 50 min after the onset of hypoxia reversed the established pulmonary vasoconstriction. In a 2-week model of hypoxia using 10% O(2), treatment with sitaxsentan (15 mg/kg per day in drinking water) attenuated pulmonary hypertension and the associated right ventricular hypertrophy, and prevented the remodeling of small pulmonary arteries (50-100 microM) without affecting systemic arterial blood pressure or heart rate. Institution of sitaxsentan treatment (15 and 30 mg/kg per day in drinking water) for 4 weeks after 2 weeks of untreated hypoxia produced a significant, dose dependent reversal of the established pulmonary hypertension, right heart hypertrophy, and pulmonary vascular remodeling despite continued hypoxic exposure. Sitaxsentan blocked increased plasma endothelin levels in the prevention protocol but did not affect the established elevated levels in the intervention study. Sitaxsentan dose dependently (10 and 50 mg/kg per day in the drinking water) attenuated right ventricular systolic pressure, right heart hypertrophy, and pulmonary vascular remodeling observed 3 weeks after a single subcutaneous injection of monocrotaline. These findings support the hypothesis that endothelin-1 plays a significant role in the development of pulmonary hypertension, pulmonary vascular remodeling, and the associated cardiac hypertrophy, and further suggest that specific endothelin-A receptor blockade may be useful in the treatment of pulmonary hypertension of diverse etiologies.

Topics: Animals; Cardiomegaly; Disease Models, Animal; Endothelin Receptor Antagonists; Endothelin-1; Hypertension, Pulmonary; Hypertrophy; Hypoxia; Isoxazoles; Male; Monocrotaline; Oxygen; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Thiophenes; Vasoconstriction; Weight Gain

The tunica propria of seminiferous tubules contains a particular type of smooth muscle cell (myoid cells) arranged in a contractile epithelioid layer that is responsible for sperm and tubular fluid flow. Unlike other types of smooth muscle (SM) cells, highly purified populations of peritubular smooth muscle cells (PSMC) survive and maintain their contractile phenotype in primary cultures in controlled conditions. We used this culture model to investigate the response of the SM contractile phenotype to prolonged exposure to platelet-derived growth factor (PDGF), one of the main factors involved in vascular SM pathologies. We observed that 4-day continuous exposure of PSMC to PDGF-BB at nanomolar concentrations in plain medium enhances contractile phenotype traits and induces cell hypertrophy without inducing proliferation. In Northern and Western blotting experiments, SM-alpha-actin transcript and protein were found to be markedly increased in the PDGF-BB-treated samples, which is in line with the formation of conspicuous SM-alpha-actin-containing stress fibers. Moreover, binding sites for endothelin-1 were increased, and the calcium response to the contractile agonist, determined in single fura-2-loaded cells, was enhanced. In response to PDGF-BB, the cells underwent immediate, transient contraction, as seen in a scanning electron microscope, followed by a gradual increase in size, as evaluated by cytofluorometry, and enhancement of protein synthesis. The observed pattern of response to PDGF-BB was not accompanied by cell proliferation, as assessed by [3H]thymidine incorporation and direct cell counts. Unlike other SM cell types, in which proliferation and loss of contractile traits are induced by PDGF, chronic treatment of PSMC with this growth factor results in hypertrophy rather than hyperplasia.

Topics: Actins; Animals; Becaplermin; Binding Sites; Calcium; Cell Division; Cell Size; Cells, Cultured; Endothelin-1; Hypertrophy; Male; Microscopy, Electron; Muscle Contraction; Muscle, Smooth; Phenotype; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Rats; Rats, Wistar; Seminiferous Tubules; Testis

The cardiopulmonary profile of three rat strains (Sprague-Dawley, Wistar and High altitude-sensitive) was compared upon exposure to hypoxia (9% O2) for 0, 7 or 14 days. No differences were observed among the in vitro contractile (ET-1) and relaxant (carbachol) responses of pulmonary artery isolated from the three strains during normoxia. Chronic hypoxia decreased ET-1 contractile responses and diminished relaxant responses to carbachol similarly in all strains. In Sprague-Dawley, Wistar and High altitude-sensitive rats, pulmonary arterial pressure rose time-dependently and was elevated by 108%, 116% and 167%, respectively, after 14 days of hypoxia compared to normoxic controls. Right ventricular hypertrophy was increased by 51%, 93% and 55%, respectively, at 14 days. Hypoxia-induced hypertrophy and medial thickening in the pulmonary vasculature were more pronounced in High altitude-sensitive rats. Sprague-Dawley exhibited hypoxia-induced airway hyperresponsiveness to intravenous methacholine, but there were no hypoxia- or strain-related differences in in vitro tracheal contractility. Although each strain exhibited greater sensitivity for a particular hypoxia-induced parameter, pulmonary vascular functional and structural changes suggest that High altitude-sensitive rats represent a choice model of hypoxia-induced pulmonary hypertension.

Topics: Altitude; Animals; Carbachol; Disease Models, Animal; Endothelin-1; Endothelins; Hypertension, Pulmonary; Hypertrophy; Hypertrophy, Right Ventricular; Hypoxia; In Vitro Techniques; Male; Methacholine Chloride; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Rats, Wistar; Species Specificity; Trachea; Vasoconstriction; Vasodilation

We investigated whether angiotensin II (Ang II) and endothelin-1(ET-1) are involved in submandibular hypertrophy in response to repeated treatment with isoproterenol (ISO) in rats. The immunoreactive Ang II (IRAng II) and immunoreactive ET-1 (IRET-1) contents of ISO-induced hypertrophy were significantly higher than those of control glands. Treatment of isolated gland tissues with ISO (1 microM) or dobutamine (1 microM) caused significant increases in the IRAng II and IRET- 1 contents of the glands compared with controls. These increases were suppressed by pretreatment with enalapril (3 microM) or captopril (3 microM). Treatment with Ang II (10 microM) also caused an increase in IRET-1 content. Our findings suggest that Ang II and ET-1 are involved in the submandibular gland hypertrophy that develops in rats repeatedly treated with ISO, and that these biologically active peptides may act as growth factors. They also imply that the tissue renin-angiotensin system and Ang II specific receptors are present in the submandibular glands.

Topics: Adrenergic beta-Agonists; Angiotensin II; Animals; Dobutamine; Enalapril; Endothelin-1; Hypertrophy; Isoproterenol; Male; Rats; Rats, Wistar; Submandibular Gland

Cultured neonatal rat cardiac myocytes have been used extensively to study cellular and molecular mechanisms of cardiac hypertrophy. However, there are only a few studies in cultured mouse myocytes despite the increasing use of genetically engineered mouse models of cardiac hypertrophy. Therefore, we characterized hypertrophic responses in low-density, serum-free cultures of neonatal mouse cardiac myocytes and compared them with rat myocytes. In mouse myocyte cultures, triiodothyronine (T3), norepinephrine (NE) through a beta-adrenergic receptor, and leukemia inhibitory factor induced hypertrophy by a 20% to 30% increase in [(3)H]phenylalanine-labeled protein content. T3 and NE also increased alpha-myosin heavy chain (MyHC) mRNA and reduced beta-MyHC. In contrast, hypertrophic stimuli in rat myocytes, including alpha(1)-adrenergic agonists, endothelin-1, prostaglandin F(2alpha), interleukin 1beta, and phorbol 12-myristate 13-acetate (PMA), had no effect on mouse myocyte protein content. In further contrast with the rat, none of these agents increased atrial natriuretic factor or beta-MyHC mRNAs. Acute PMA signaling was intact by extracellular signal-regulated kinase (ERK1/2) and immediate-early gene (fos/jun) activation. Remarkably, mouse but not rat myocytes had hypertrophy in the absence of added growth factors, with increases in cell area, protein content, and the mRNAs for atrial natriuretic factor and beta-MyHC. We conclude that mouse myocytes have a unique autonomous hypertrophy. On this background, T3, NE, and leukemia inhibitory factor activate hypertrophy with different mRNA phenotypes, but certain Gq- and protein kinase C-coupled agonists do not.

Topics: Adrenergic alpha-Agonists; Animals; Animals, Newborn; Atrial Natriuretic Factor; Cells, Cultured; Dinoprost; Endothelin-1; Fetus; Growth Inhibitors; Growth Substances; Heart; Hypertrophy; Interleukin-1; Interleukin-6; Leukemia Inhibitory Factor; Lymphokines; Mice; Microscopy, Phase-Contrast; Models, Animal; Myocardium; Myosin Heavy Chains; Myosins; Norepinephrine; Phenotype; Phenylalanine; Protein Biosynthesis; Proteins; Rats; RNA, Messenger; Tetradecanoylphorbol Acetate; Triiodothyronine

Accumulating evidence suggests that the local synthesis of endothelin-1 (ET-1) plays a role in the development of heart failure in vivo. We investigated the role of endothelin-converting enzyme-1 (ECE-1), which mediates the conversion of big ET-1 to mature ET-1, in the development of alpha1-adrenergic-stimulated hypertrophy in cultured neonatal rat cardiac myocytes.. Phenylephrine (PE) induced the expression of ET-1 in rat cardiac myocytes and accelerated the conversion of big ET-1 to ET-1. The ECE-1 mRNA levels were markedly increased 3 hours after PE stimulation (3.6-fold compared with saline stimulation, P<0.005). A specific ECE-1 antagonist, FR901533, inhibited the PE-stimulated increase in protein synthesis rate by 45% (P<0.05). As genetic markers for the hypertrophic response, FR901533 inhibited the PE-stimulated transcriptional activities of the 3.5-kb beta-myosin heavy chain promoter by 79% (P<0.01) but did not affect that of the 3.4-kb atrial natriuretic factor (ANF) promoter. In Bio14.6 Syrian cardiomyopathic hamsters, ventricular ET-1 and ANF mRNA levels did not correlate at 2 different stages.. ET-1-independent pathways may mediate activation of the ANF gene program in ventricular myocytes both in vitro and in vivo. These results also indicate that the conversion of big ET-1 to ET-1 in rat cardiac myocytes is required for the development of alpha1-adrenergic-stimulated hypertrophy and beta-myosin heavy chain gene transcription.

Topics: Adrenergic alpha-Agonists; Animals; Animals, Newborn; Aspartic Acid Endopeptidases; Atrial Natriuretic Factor; Cells, Cultured; Cricetinae; Endothelin-1; Endothelin-Converting Enzymes; Hypertrophy; Mesocricetus; Metalloendopeptidases; Myocardium; Myosin Heavy Chains; Phenylephrine; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tetracyclines; Transcription, Genetic

The sphingolipid metabolites, sphingosine (SPH), SPH 1-phosphate (S1P), and sphingosylphosphorylcholine (SPC), can act as intracellular as well as extracellular signaling molecules. These compounds have been implicated in the regulation of cell growth, differentiation, and programmed cell death in nonmyocytes, but the effects of sphingolipid metabolites in cardiac myocytes are not known. Cultured neonatal rat cardiac myocytes were stimulated with SPH (1 to 10 micromol/L), S1P (1 to 10 micromol/L), or SPC (0.1 to 10 micromol/L) for 24 hours to determine the effects of sphingolipid metabolites on the rates of protein synthesis and degradation. Stimulation with SPC led to an increase in the total amount of protein, an accelerated rate of total protein synthesis, and a decrease in protein degradation in a dose-dependent manner. However, S1P had little effect and SPH had no effect on total protein synthesis. In addition, stimulation with SPC led to a 1.4-fold increase in myocardial cell size and enhanced atrial natriuretic factor gene expression. Pretreatment of the cardiac myocytes with pertussis toxin or PD98059 attenuated the SPC-induced hypertrophic growth response. Further, stimulation with SPC increased phosphorylation of mitogen-activated protein kinase (MAPK) and stimulated MAPK enzyme activity. Finally, endothelin-1 stimulated the generation of SPC in cardiac myocytes. The observation that SPC induces a hypertrophic growth response in cardiac myocytes suggests that SPC may play a critical role in the development of cardiac hypertrophy. The effects of SPC could be mediated, in part, by activation of a G protein-coupled receptor and a MAPK signaling cascade.

Topics: Adrenergic beta-Agonists; Animals; Animals, Newborn; Cell Size; Endothelin-1; Enzyme Inhibitors; Flavonoids; GTP-Binding Proteins; Heart; Hypertrophy; Isoproterenol; MAP Kinase Signaling System; Muscle Proteins; Myocardium; Pertussis Toxin; Phosphorylation; Phosphorylcholine; Protein Processing, Post-Translational; Rats; Sphingosine; Virulence Factors, Bordetella

Bladder outlet obstruction (BOO) is associated with altered bladder structure and function. Endothelin-1 (ET-1) has mitogenic and potent contractile properties. There are two ET receptors: ET(A) and ET(B). Nitric oxide synthase (NOS) is the enzyme responsible for the synthesis of nitric oxide (NO) which is involved in smooth muscle relaxation. We investigated whether there are any changes in the density of ET-receptors and NOS in the detrusor and bladder neck in a rabbit model of BOO. Partial BOO was induced in adult male New Zealand White rabbits. Sham operated age-matched rabbits acted as controls. After six weeks the urinary bladders were excised and detrusor and bladder neck sections incubated with radioligands for ET-1, ET(A) and ET(B) receptors and with [3H]-1-NOARG (a ligand for NOS). NADPH histochemistry was also performed. BOO bladder weights were significantly increased (P = 0.002). ET-1 binding and ETA receptor binding sites were significantly increased in the BOO detrusor smooth muscle (P = 0.04, P = 0.03 respectively) and urothelium (P = 0.002, P = 0.02 respectively). ET(B) receptor binding sites were also significantly increased in the BOO detrusor smooth muscle (P = 0.04). However, there was no change in the BOO bladder neck. NOS was significantly decreased in the detrusor smooth muscle (P = 0.003) and urothelium (P = 0.0002). In the bladder neck NOS was also significantly reduced in the urothelium (P = 0.003). NADPH staining was decreased in the detrusor and bladder neck. The up-regulation of ET receptors along with the down-regulation of NOS in the detrusor may contribute to the symptoms associated with BOO. Since ET-1 has a mitogenic role, especially via its ETA receptors, the increase in ETA receptors may also be involved in detrusor hyperplasia and hypertrophy in BOO. ET antagonists may therefore have a role in the treatment of patients with BOO.

Topics: Animals; Autoradiography; Binding Sites; Disease Models, Animal; Down-Regulation; Endothelin-1; Humans; Hyperplasia; Hypertrophy; Male; Muscle, Smooth; NADPH Dehydrogenase; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroarginine; Prostatic Hyperplasia; Rabbits; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Up-Regulation; Urinary Bladder Neck Obstruction

Sodium plays an important role in the pathogenesis and therapy of hypertension, a major risk factor for cardiovascular disease. This study investigated the involvement of endothelin in vascular alterations in salt-induced Dahl hypertension. Salt-sensitive (DS) and salt-resistant (DR) Dahl rats were treated with a high-sodium diet (NaCl 4%) with or without ET(A) receptor antagonist LU135252 for two months, and effects of treatments on systolic blood pressure, vascular endothelin-1 (ET-1) protein content, aortic hypertrophy, and vascular reactivity of isolated aortic rings were studied. In DS rats, a high-sodium diet increased systolic pressure (190+/-4 versus 152+/-2 mm Hg, P<.05) and aortic ET-1 protein content (4.2-fold, P<.0001) and induced aortic hypertrophy as assessed by tissue weight (P<.0001). Sodium diet markedly reduced NO-mediated endothelium-dependent relaxations to acetylcholine (49+/-4% versus 81+/-4%, P<.0001) and contractions to ET-1 (92+/-7 versus 136+/-8% of KCl, P=.0011). ET-1 tissue levels were highly and inversely correlated with endothelium-dependent relaxations (r=0.931, P<.0001) and contractions to ET (r=0.77, P=.0007). LU135252 treatment reduced systolic blood pressure only in part (168+/-3 versus 190+/-4 mm Hg, P<.05) but normalized sodium-induced changes of vascular reactivity, tissue ET-1 protein content, and vascular structure (P<.001 versus sodium). None of these effects were observed in DR rats. These results suggest that ET-1 acts as a local mediator of vascular dysfunction and aortic hypertrophy in Dahl salt-induced hypertension. ET(A) receptor antagonism may have therapeutic potential for lowering vascular ET-1 content, improving endothelial function, and preventing structural changes in salt-sensitive hypertension.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Blood Pressure; Endothelin Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Hypertension; Hypertrophy; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth, Vascular; Nitroprusside; Phenylpropionates; Potassium Chloride; Pyrimidines; Rats; Rats, Inbred Strains; Receptor, Endothelin A; Sodium, Dietary; Systole; Vasodilation

We examined the activation of the p38 mitogen-activated protein kinase (p38-MAPK) pathway by the G protein-coupled receptor agonists, endothelin-1 and phenylephrine in primary cultures of cardiac myocytes from neonatal rat hearts. Both agonists increased the phosphorylation (activation) of p38-MAPK by approximately 12-fold. A p38-MAPK substrate, MAPK-activated protein kinase 2 (MAPKAPK2), was activated approximately fourfold and 10 microM SB203580, a p38-MAPK inhibitor, abolished this activation. Phosphorylation of the MAPKAPK2 substrate, heat shock protein 25/27, was also increased. Using selective inhibitors, activation of the p38-MAPK pathway by endothelin-1 was shown to involve protein kinase C but not Gi/Go nor the extracellularly responsive kinase (ERK) pathway. SB203580 failed to inhibit the morphological changes associated with cardiac myocyte hypertrophy induced by endothelin-1 or phenylephrine between 4 and 24 h. However, it decreased the myofibrillar organization and cell profile at 48 h. In contrast, inhibition of the ERK cascade with PD98059 prevented the increase in myofibrillar organization but not cell profile. These data are not consistent with a role for the p38-MAPK pathway in the immediate induction of the morphological changes of hypertrophy but suggest that it may be necessary over a longer period to maintain the response.

Topics: Animals; Animals, Newborn; Calcium-Calmodulin-Dependent Protein Kinases; Cardiomegaly; Cell Size; Cell Survival; Cells, Cultured; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; GTP-Binding Proteins; Heart Ventricles; Hypertrophy; Intracellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinases; Myocardium; p38 Mitogen-Activated Protein Kinases; Phenylephrine; Phosphorylation; Protein Kinase C; Protein Serine-Threonine Kinases; Rats; Receptors, Cell Surface; Virulence Factors, Bordetella

In vitro studies demonstrated a relationship between ET-1 and basic Fibroblast Growth Factor (bFGF), and of bFGF with Platelet Derived Growth Factor (PDGF). The present study was carried out to investigate in vivo the behaviour after vascular stress of circulating ET-1, bFGF and PDGF, and catecholamines, and their relationship. In 12 healthy normotensives (NTs) and 15 essential hypertensives (Ehs) venous blood samples to determine circulating ET-1, bFGF and PDGF, and catecholamine (EPI and NE) levels were drawn before and at the third minute of a handgrip test. Blood pressures (BP) and heart rate were automatically recorded before starting, and at 1, 2, and 3 minutes during the test. The NTs showed, in basal condition, lower values than the EHs of all the examined parameters; later, the handgrip test induced significant increases in circulating levels of ET-1, bFGF and catecholamine. In the EHs at the third minute of the exercise significant increases in plasma ET-1 (p < 0.002), bFGF (p < 0.006), and EPI and NE (p < 0.0005) levels were observed. Systolic and diastolic BP significantly increased after handgrip test in NTs and EHs. Plasma ET-1 correlated with bFGF both before (p < 0.01) and at the acme (p < 0.05) of the isometric exercise. Our results show that in EHs plasma ET-1 and bFGF correlate each other, indicating that in human hypertension a linkage between ET-1 and bFGF exists.

Topics: Catecholamines; Endothelin-1; Endothelium, Vascular; Fibroblast Growth Factor 2; Hand Strength; Humans; Hypertension; Hypertrophy; Muscle Contraction; Muscle, Smooth, Vascular; Platelet-Derived Growth Factor; Stress, Physiological; Vasoconstriction

In cardiac hypertrophy, both excessive enlargement of cardiac myocytes and progressive interstitial fibrosis are well known to occur simultaneously. In the present study, to investigate the interaction between ventricular myocytes (MCs) and cardiac nonmyocytes (NMCs), mostly fibroblasts, during cardiocytes hypertrophy, we examined the change in cell size and gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in cultured MCs as markers for hypertrophy in the neonatal rat ventricular cardiac cell culture system.. The size of cultured MCs significantly increased in the MC-NMC coculture. Concomitantly, secretions of ANP and BNP into culture media were significantly increased in the MC-NMC coculture compared with in the MC culture (with the possible contamination of NMC <1% of MC). Moreover, in the MC culture, enlargement of MC and an increase in ANP and BNP secretions were induced by treatment with conditioned media of the NMC culture. A considerable amount of endothelin (ET)-1 production was detected in the NMC-conditioned media. BQ-123, an ET-A receptor antagonist, and bosentan, a nonselective ET receptor antagonist, significantly blocked the hypertrophic response of MCs induced by treatment with NMC-conditioned media. Angiotensin II (Ang II) (10(-10) to 10(-6) mol/L) and transforming growth factor-beta1 (TGF-beta1) (10(-13) to 10(-9) mol/L), both of which are known to be cardiac hypertrophic factors, did not induce hypertrophy in MC culture, but both Ang II and TGF-beta1 increased the size of MCs and augmented ANP and BNP productions in the MC-NMC coculture. This hypertrophic activity of Ang II and TGF-beta1 was associated with the potentiation of ET-1 production in the MC-NMC coculture, and the effect of Ang II or TGF-beta1 on the secretions of ANP and BNP in the coculture was significantly suppressed by pretreatment with BQ-123.. These results demonstrate that NMCs regulate MC hypertrophy at least partially via ET-1 secretion and that the interaction between MCs and NMCs plays a critical role during the process of Ang II- or TGF-beta1-induced cardiocyte hypertrophy.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Cardiomegaly; Cattle; Coculture Techniques; Culture Media, Conditioned; Endothelin Receptor Antagonists; Endothelin-1; Hypertrophy; Lipoproteins, LDL; Myocardium; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Paracrine Communication; Rats; Rats, Wistar; Receptors, Angiotensin; Receptors, Endothelin; RNA, Messenger; Transforming Growth Factor beta; Ventricular Function

The present study was designed to assess how the renal expression of mRNA for endothelin (ET) families is regulated during compensatory renal hypertrophy in rats. ET family gene expression was studied in the contralateral kidney of uninephrectomized and sham-operated rats. Rats were sacrificed at 0, 1, 3, 6, 12, adn 24 h after unilateral nephrectomy of the sham operation. Three hours after the left nephrectomy, ET-1 and ET receptor B mRNA levels in the renal cortex increased significantly (ET-1, 10.6-fold compared to those in sham-operated rats, p < 0.001, and ET receptor B, 6.8-fold, p < 0.001), and then decreased gradually to the control level after 24 h; in contrast, ET-3 and ET receptor A mRNA levels demonstrated little change throughout the experiment. We additionally measured the plasma ET concentration and renal ET-1 production following unilateral nephrectomy. ET-1 levels in the renal cortex increased gradually, with a peak 6 h after nephrectomy (3.6-fold compared to those in sham-operated rats, p < 0.01), and then decreased to the control level after 24 h. However, plasma ET-1 levels demonstrated little change until after 24 h. The glomerular expression of ET-1 and ET receptors A and B mRNA demonstrated minimal change throughout the experimental period. Glomerular ET-3 mRNA expression were detected in neither the unilateral nephrectomy nor the sham-operated rats. Our results indicate that the time course of the mRNA expression of ET-1 and ET receptor B differs from that of ET-3 and ET receptor A in the renal cortex, and that glomerular mRNA levels for ET families are not associated with renal hypertrophy in the early stages following unilateral nephrectomy.

Topics: Animals; Autoradiography; Blotting, Northern; Endothelin-1; Gene Expression Regulation; Hypertrophy; Kidney Cortex; Kidney Diseases; Kidney Glomerulus; Male; Organ Size; Rats; Rats, Inbred F344; Receptors, Endothelin; RNA, Messenger

The effect of the combined ETA/ETB endothelin receptor antagonist bosentan on blood pressure, vascular hypertrophy, and pathologic renal changes was investigated in a model of malignant hypertension, severe vascular hypertrophy, and enhanced vascular expression of endothelin-1, the deoxycorticosterone acetate (DOCA), and salt-treated spontaneously hypertensive rat (SHR). DOCA-salt treated SHR received 100 mg bosentan per kilogram weight per day mixed with their food. Systolic blood pressure of untreated DOCA-salt SHR rose to 241 +/- 1.5 mm Hg, whereas that of bosentan-treated rats rose to 221 +/- 5.1 mm Hg (P < .01). Cardiac and conduit artery mass were not affected by treatment. Small arteries from the coronary, renal, and mesenteric circulations showed a smaller media width and cross-sectional area of the media in rats treated with bosentan than in untreated rats. The kidneys showed the presence of fibrinoid necrosis in a high percentage of afferent arterioles and glomeruli of untreated DOCA-SHR. Some kidneys of treated rats exhibited less severe vascular hypertrophy and lesser extent of vascular or glomerular fibrinoid necrosis, but the renal injury score of bosentan-treated DOCA-SHR was only at the limit of significance from that of untreated rats (P = .06). These results suggest a role for endothelin-1 in blood pressure elevation and the severe vascular hypertrophy of small arteries of the coronary, renal, and mesenteric vasculature, but not of the heart or larger conduit vessels in the malignant hypertension that SHR develop after treatment with DOCA and salt. Although some bosentan-treated rats showed fewer renal lesions, a significant effect on renal pathology could not be unambiguously demonstrated. Further studies will be necessary to determine whether endothelin antagonists may indeed offer some degree of renal protection and have therapeutic potential in severe or malignant hypertension.

Topics: Animals; Arteries; Blood Pressure; Bosentan; Desoxycorticosterone; Endothelin Receptor Antagonists; Endothelin-1; Hemodynamics; Hypertension, Malignant; Hypertrophy; Kidney; Organ Size; Rats; Rats, Inbred SHR; Renin; Sulfonamides

Topics: Amino Acids; Angiotensin II; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cardiovascular System; Endothelin Receptor Antagonists; Endothelin-1; Enzyme Activation; Gene Expression; Hypertrophy; Mice; Mice, Knockout; Myocardium; Peptides, Cyclic; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; Stress, Mechanical

1. Enhanced endothelin-1 gene expression has been found in blood vessels of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. In this study, the effects of salt, DOCA and the development of hypertension in DOCA-salt hypertensive rats on the expression of the endothelin-1 gene in blood vessels and on vascular hypertrophy were compared in Sprague-Dawley (SD) rats and spontaneously hypertensive rats (SHR). 2. Increased endothelin-1 mRNA was found by northern blot analysis in the mesenteric arterial bed of DOCA-salt hypertensive rats and DOCA-salt SHR, but not in DOCA or salt-treated SD rats or in SHR, even when blood pressure reached a mean of 211 mmHg in DOCA-treated SHR. 3. Vascular structure was studied in small mesenteric arteries mounted on a wire myograph. The media width to lumen diameter ratio showed a close correlation with systolic blood pressure except in DOCA-salt hypertensive rats and DOCA-salt SHR, in which it was greater than accounted for by the level of blood pressure. Treatment of DOCA-salt hypertensive rats with the combined ETA/ETB endothelin antagonist bosentan lowered blood pressure slightly, but vascular hypertrophy regressed almost completely and any hypertrophy remaining could be explained by the residual elevated blood pressure. 4. In conclusion, SHR do not exhibit enhanced expression of endothelin-1 in blood vessels. DOCA, salt and elevated blood pressure interact to induce increased arterial expression of endothelin-1. Vascular overexpression of the endothelin-1 gene may produce vascular hypertrophy independently of blood pressure elevation.

Topics: Animals; Blood Pressure; Blood Vessels; Blotting, Northern; Desoxycorticosterone; Endothelin-1; Gene Expression; Hypertension; Hypertrophy; Male; Mesenteric Arteries; Rats; Rats, Inbred SHR; Rats, Sprague-Dawley; RNA, Messenger; Sodium Chloride