endothelin-1 and Hyperplasia

Topics: Angiotensin II; Animals; Aorta; Diphosphonates; Endothelin-1; Human Umbilical Vein Endothelial Cells; Humans; Hyperplasia; Male; Myocytes, Smooth Muscle; Naphthalenesulfonates; Nitric Oxide; Nitric Oxide Synthase Type III; Purinergic P2 Receptor Agonists; Rats; Rats, Wistar; Receptors, Purinergic P2; Reperfusion Injury; Tunica Intima; Vasodilation; Water

Vein graft restenosis has an adverse impact on bridge vessel circulation and patient prognosis after coronary artery bypass grafting.. We used the extravascular supporter α-cyanoacrylate (α-CA), the local application rapamycin/sirolimus (RPM), and a combination of the two (α-CA-RPM) in rat models of autogenous vein graft to stimulate vein graft change. The aim of our study was to observe the effect of α-CA, RPM, and α-CA-RPM on vein hyperplasia.. Fifty healthy Sprague Dawley (SD) rats were randomized into the following 5 groups: sham, control, α-CA, RPM, and α-CA-RPM. Operating procedure as subsequently described was used to build models of grafted rat jugular vein on carotid artery on one side. The level of endothelin-1 (ET-1) was determined by enzyme-linked immunosorbent assay (ELISA). Grafted veins were observed via naked eye 4 weeks later; fresh veins were observed via microscope and image-processing software in hematoxylin-eosin (HE) staining and immunohistochemistry after having been fixed and stored" (i.e. First they were fixed and stored, and second they were observed); α-Smooth Muscle Actin (αSMA) and von Willebrand factor (vWF) were measured with reverse transcription-polymerase chain reaction (RT-PCR). Comparisons were made with single-factor analysis of variance and Fisher's least significant difference test, with p < 0.05 considered significant.. We found that intimal thickness of the α-CA, RPM, and α-CA-RPM groups was lower than that of the control group (p < 0.01), and the thickness of the α-CA-RPM group was notably lower than that of the α-CA and RPM groups (p < 0.05).. RPM combined with α-CA contributes to inhibiting intimal hyperplasia in rat models and is more effective for vascular patency than individual use of either α-CA or RPM.

Topics: Actins; Animals; Carotid Arteries; Cell Proliferation; Coronary Artery Bypass; Cyanoacrylates; Disease Models, Animal; Drug Combinations; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Female; Graft Occlusion, Vascular; Hyperplasia; Jugular Veins; Male; Random Allocation; Rats, Sprague-Dawley; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Sirolimus; Time Factors; Treatment Outcome; Tunica Intima

Giant-cell arteritis (GCA) is an inflammatory disease of large/medium-sized arteries, frequently involving the temporal arteries (TA). Inflammation-induced vascular remodelling leads to vaso-occlusive events. Circulating endothelin-1 (ET-1) is increased in patients with GCA with ischaemic complications suggesting a role for ET-1 in vascular occlusion beyond its vasoactive function.. To investigate whether ET-1 induces a migratory myofibroblastic phenotype in human TA-derived vascular smooth muscle cells (VSMC) leading to intimal hyperplasia and vascular occlusion in GCA.. Immunofluorescence/confocal microscopy showed increased ET-1 expression in GCA lesions compared with control arteries. In inflamed arteries, ET-1 was predominantly expressed by infiltrating mononuclear cells whereas ET receptors, particularly ET-1 receptor B (ET. ET-1 is upregulated in GCA lesions and, by promoting VSMC migration towards the intimal layer, may contribute to intimal hyperplasia and vascular occlusion in GCA.

Topics: Actins; Aged; Blotting, Western; Case-Control Studies; Cell Movement; Endothelin Receptor Antagonists; Endothelin-1; Female; Fluorescent Antibody Technique; Focal Adhesion Kinase 1; Giant Cell Arteritis; Humans; Hyperplasia; In Vitro Techniques; Leukocytes, Mononuclear; Male; Microscopy, Confocal; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Receptor, Endothelin A; Receptor, Endothelin B; Reverse Transcriptase Polymerase Chain Reaction; src-Family Kinases; Tunica Intima; Vascular Remodeling

The aim of this study was to investigate the regulatory mechanism of endothelin-1 (ET-1), an endothelium-derived vasoconstrictor, on adipogenesis in vitro and in vivo.. 3T3-L1 preadipocytes were used to explore the mechanisms mediating ET-1 actions on preadipocyte proliferation and adipocyte differentiation. To investigate the in vivo effect of ET-1, male Sprague-Dawley rats were infused with ET-1 or saline for 4 weeks via intraperitoneally implanted osmotic pumps, and the fat pad weight and adipocyte size of adipose tissues were measured.. ET-1 stimulated preadipocyte proliferation and increased the cell number at the mitotic clonal expansion stage of adipocyte differentiation via the endothelin A receptor (ETAR) and activation of the protein kinase C (PKC) pathway. ET-1, via ETAR, inhibited adipocyte differentiation partially through an ERK-dependent pathway. Furthermore, no significant difference in the body weight and fat pad weight was observed in either ET-1- or saline-infused rats. Compared with saline-infused rats, the adipocyte cell number was significantly increased but the adipocyte size was significantly decreased in ET-1-infused rats.. Chronic ET-1 infusion increased the number of small adipocytes without the change of white adipose tissue mass in rats, which were associated with ET-1-stimulated preadipocyte proliferation, but not ET-1-suppressed adipocyte differentiation.

Topics: 3T3-L1 Cells; Adipocytes; Adipogenesis; Adipose Tissue; Animals; Body Weight; Cell Differentiation; Dose-Response Relationship, Drug; Endothelin-1; Hyperplasia; Male; Mice; Obesity; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A

Pathological proliferation of human airway smooth muscle cells (HASMCs) causes hyperplasia in chronic lung diseases. Signaling pathways that link airway inflammation to HASMC proliferation might provide therapeutic targets for the prevention of airway remodeling and chronic lung diseases. Endothelin-1 (ET-1) signals via endothelin-A- and B-receptors (ETAR, ETBR) to perpetuate HASMC-associated and TNFα-dependent inflammatory processes.. endothelin receptor antagonists (ERAs) suppress HASMC proliferation induced by inflammatory cytokines. HASMCs were stimulated ex vivo with cytokines in the presence or absence of ERAs (ETAR-specific/selective: BQ123, ambrisentan; ETBR-specific: BQ788; non-selective: bosentan, macitentan, ACT-132577) or cytokine-blocking antibodies. Cell counts, DNA-synthesis (BrdU-incorporation assay), cytokine production (ELISA) and ETBR expression (whole-genome microarray data, western blot) were analyzed. ET-1-induced HASMC proliferation and DNA-synthesis were reduced by protein kinase inhibitors and ETAR-specific/selective ERAs but not by BQ788. TNFα-induced HASMC proliferation and DNA-synthesis were reduced by all ERAs. TNFα induced ET-1 and ETBR expression. TNFα- and ET-1-induced GM-CSF releases were both reduced by BQ123 and BQ788. TNFα- and ET-1-induced IL-6 releases were both reduced by BQ123 but not by BQ788. Combined but not single blockade of GM-CSF-receptor-α-chain and IL-6 reduced TNFα- and ET-1-induced HASMC proliferation and DNA-synthesis. Combined but not single treatment with GM-CSF and IL-6 induced HASMC proliferation and DNA-synthesis in the presence of ET-1. In conclusion, TNFα induces HASMC proliferation via ET-1/GM-CSF/IL-6. ETBR requires up-regulation by TNFα to mediate ET-1 effects on HASMC proliferation. This signaling cascade links airway inflammation to HASMC-associated remodeling processes and is sensitive to ERAs. Therefore, ERAs could prevent inflammation-induced airway smooth muscle hyperplasia.

Topics: Antibodies, Blocking; Biomarkers; Bronchi; Bronchial Neoplasms; Carcinoma; Cell Proliferation; Cells, Cultured; DNA Replication; Endothelin Receptor Antagonists; Endothelin-1; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Hyperplasia; Interleukin-6; Muscle, Smooth; Protein Kinase Inhibitors; Receptor, Endothelin A; Receptor, Endothelin B; Recombinant Proteins; Signal Transduction; Tumor Necrosis Factor-alpha

To investigate the effects of Batroxobin (BX) on the intimal hyperplasia of graft veins.. Twenty dogs were evenly divided into 2 groups. The femoral veins were grafted into the femoral artery by microsurgery, and the experimental group was treated with BX (0.1 BU/kg/48 hours). The serum level of endothelin-1 (ET-1) was detected 2 weeks after operation. Computer image analysis system was performed to calculate the cross-sectional area of neointima and media in the vein grafts 8 weeks after operation. Immunohistochemistry method was performed to identify proliferating cell nuclear antigen (PCNA) and c-Myc.. The experimental group had a lower level of serum ET-1 than the control group (P < .01), and both intimal hyperplasia and media thickness of graft veins were reduced by BX in comparison with the control group (P < .05). C-Myc expression was higher in the control group than in the experimental group (P < .01). The PCNA expression in the experimental group was significantly lower than the control group (P < .05).. These findings suggested that BX could inhibit intimal hyperplasia through suppressing cell proliferation activity.

Topics: Animals; Batroxobin; Cell Proliferation; Dogs; Endothelin-1; Femoral Artery; Femoral Vein; Fibrinolytic Agents; Hyperplasia; Immunohistochemistry; Microsurgery; Models, Animal; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-myc; Random Allocation; Transplantation, Autologous; Tunica Intima; Tunica Media

We studied the impact of diabetes mellitus (DM) on the radial artery (RA) in 30 patients with DM and 30 non-diabetic patients undergoing coronary artery bypass grafting with autologous RA. RAs were recorded as normal if there was no cellular or stromal tissue between the endothelium and the internal elastic lamina. The RA was normal in 26.7% of diabetic and 76.7% of non-diabetic patients (p = 0.000298). Intimal thickness index and intima:media ratio were higher in the former than in the latter (p < 0.05; p < 0.05), with no significant difference in luminal narrowing (p > 0.05). Electron microscopy scores were lower in the non-diabetic group (p < 0.001); endothelial nitric oxide synthase (eNOS) protein expression and optical density were higher (p < 0.001). Von Willebrand factor and endothelin-1 messenger RNA (mRNA) levels were higher in the DM patients (p < 0.001). The quality of the RA in patients with DM was thus inferior to that in non-diabetic patients. Care should be taken when selecting RA as a conduit in patients with DM.

Topics: Aged; Carotid Intima-Media Thickness; Carotid Stenosis; Coronary Artery Bypass; Coronary Artery Disease; Diabetic Angiopathies; Endothelin-1; Endothelium, Vascular; Female; Humans; Hyperplasia; Male; Microscopy, Electron, Transmission; Middle Aged; Nitric Oxide Synthase Type III; Radial Artery; RNA, Messenger; Transplantation, Autologous; Up-Regulation; von Willebrand Factor

In this study, we examined the synergistic effects of the two potent pathogenic factors, platelet-derived growth factor-BB (PDGF-BB) and endothelin-1 (ET-1) to induce vascular hyperplasia using ex vivo organ-culture system.. In organ-cultured rat tail arteries, concomitant treatment with 100 ng/ml PDGF-BB and 300 nM ET-1 for 4 days induced medial hyperplasia with increased smooth muscle cell proliferation. Concomitant treatment with PDGF-BB (10-300 nM) and ET-1 (30 nM-1 μM) dose-dependently suppressed contractile responses to high K(+) and norepinephrine. This dyscontractility was accompanied by decreased α-actin protein expression. In all series of experiments, concomitant treatment with PDGF-BB and ET-1 exhibited stronger effects than sole treatment with PDGF-BB (100 ng/ml) or ET-1 (300 nM). Western blot analysis revealed that concomitant treatment with PDGF-BB and ET-1 synergistically phosphorylated extracellular signal-regulated kinase 1 and 2 (ERK1/2), Akt, and a downstream target of mammalian target of rapamycin (mTOR), p70 ribosomal S6 kinase in cultured artery. Consistently, a MAPK/ERK kinase (MEK) inhibitor, PD98059 (30 μM), a phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, and an mTOR inhibitor, rapamycin (30 nM), partially restored PDGF-BB and ET-1-induced hyperplastic changes.. We evidenced for the first time at tissue level that PDGF-BB and ET-1 synergistically accelerate vascular smooth muscle hyperplastic changes and lose its contractility, at least partially through ERK1/2, Akt, and mTOR activation.

Topics: Animals; Arteries; Becaplermin; Blotting, Western; Cell Proliferation; Dose-Response Relationship, Drug; Drug Synergism; Endothelin-1; Humans; Hyperplasia; Immunohistochemistry; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Organ Culture Techniques; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-sis; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Ribosomal Protein S6 Kinases, 70-kDa; Tail; Time Factors; TOR Serine-Threonine Kinases; Vasoconstriction; Vasoconstrictor Agents

β(1)-Integrin mediates cardiomyocyte growth and survival and its proper regulation is essential for the structural and functional integrity of the heart. β(1)-Integrin expression is enhanced in hypertrophy, but the mechanism and significance of its up-regulation are unknown. Because reactive oxygen species (ROS) are important mediators of myocardial remodeling we examined their role in regulated β(1)-integrin expression. Hypertrophy was induced in neonatal cardiomyocytes by endothelin-1 (ET-1), which activated the regulatory NADPH oxidase subunit Rac1, evoked ROS, and enhanced fetal gene expression and cardiomyocyte size. ET-1 also enhanced cell adhesion and FAK phosphorylation and inhibited oxidative stress-induced cardiomyocyte apoptosis. Further, ET-1 increased β(1)-integrin mRNA and protein expression via Rac1-ROS-dependent MEK/ERK and EGF receptor-PI3K/Akt activation as shown by adenoviral dominant-negative Rac1 or overexpression of copper/zinc-superoxide dismutase. The relevance of regulated β(1)-integrin expression was examined in cardiomyocytes, in which targeting siRNA impeded the ET-1-induced β(1)-integrin up-regulation. In these cells, ET-1-induced cell adhesion, FAK phosphorylation, and hypertrophic response were significantly blunted, whereas its antiapoptotic effect was predominantly unchanged, suggesting at least partial dissociation of prohypertrophic and prosurvival signaling elicited by ET-1. In conclusion, β(1)-integrin up-regulation in response to ET-1 is mediated via Rac1-ROS-dependent activation of prohypertrophic pathways and is mandatory for ET-1-induced FAK activation, cell adhesion, and hypertrophic response.

Topics: Animals; Apoptosis; Cell Adhesion; Cells, Cultured; Endothelin-1; Gene Expression Regulation, Developmental; Hyperplasia; Integrin beta1; Mutation; Myocardium; Myocytes, Cardiac; rac1 GTP-Binding Protein; Rats; Rats, Wistar; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; Transgenes; Up-Regulation; Ventricular Remodeling

We showed previously that vascular smooth muscle cells (VMSC) from spontaneously hypertensive rats (SHR) exhibit increased proliferation. The present study was undertaken to examine whether the enhanced levels of endogenous angiotensin (ANG) II and endothelin (ET)-1 contribute to the enhanced proliferation of VSMC from SHR and to further investigate the underlying mechanisms responsible for this response. The enhanced proliferation of VSMC from SHR compared with Wistar-Kyoto (WKY) rats was attenuated by losartan, BQ-123, BQ-788, and AG-1478, inhibitors of AT(1), ET(A), ET(B) and epidermal growth factor (EGF-R) receptors, respectively. In addition, BQ-123 and BQ-788 also attenuated the enhanced production of superoxide anion (O(2)(-)) and NADPH oxidase activity. Furthermore, diphenyleneiodonium (DPI, inhibitor of NADPH oxidase), N-acetyl-L-cysteine (NAC, O(2)(-) scavenger), and PP2 (inhibitor of c-Src) also inhibited the augmented proliferation of VSMC from SHR to WKY levels. In addition, the enhanced phosphorylation of EGF-R in VSMC from SHR compared with WKY was also attenuated by inhibitors of AT(1), ET(A), ET(B), and EGF-R but not by inhibitors of platelet-derived growth factor receptor or insulin-like growth factor receptor. Furthermore, the enhanced phosphorylation of ERK1/2 in VSMC from SHR was also attenuated by AT(1), ET(A), ET(B), c-Src, and EGF-R inhibitors. The phosphorylation of c-Src was significantly augmented in VSMC from SHR compared with VSMC from WKY and was attenuated by DPI and NAC. These data suggest that endogenous vasoactive peptides, through increased oxidative stress and resultant activation of c-Src, transactivate EGF-R, which through mitogen-activated protein (MAP) kinase signaling may contribute to the hyperproliferation of VSMC from SHR.

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Cell Proliferation; Cells, Cultured; CSK Tyrosine-Protein Kinase; Disease Models, Animal; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Free Radical Scavengers; Hyperplasia; Hypertension; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NADPH Oxidases; Oxidative Stress; Phosphorylation; Protein-Tyrosine Kinases; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptor, Angiotensin, Type 1; Receptor, Endothelin A; Receptor, Endothelin B; Signal Transduction; src-Family Kinases; Superoxides

Although endothelin-1 (ET-1) has been suggested to contribute to the pathogenesis of neointima formation and atherosclerosis, the individual roles of ET-1 derived from certain cell types in this disease remain unclear. In this study, we determined the role of vascular endothelial ET-1 on vascular inflammation and neointima formation using vascular endothelial ET-1-knockout [ET-1(f/f); Tie2-Cre (+)] mice.. Intimal hyperplasia was induced by complete ligation of the left carotid artery in 12-week-old male ET-1(f/f);Tie2-Cre (+) mice (n = 35) and the wild-type (WT) littermates (n = 34). Following this intervention, neointima formation was reduced in ET-1(f/f);Tie2-Cre (+) mice compared with the WT mice, independent of the difference in blood pressure. This reduction was associated with a decrease in inflammatory cell recruitment to the vessel wall, which was accompanied by reduced expression levels of endothelial adhesion molecules as well as chemokines and a decrease in vascular smooth muscle cell proliferation.. The results of our study provide direct evidence for the role of vascular endothelial ET-1 in mediating vascular inflammation and neointima formation following vascular injury in addition to promoting vasoconstriction and cell proliferation. Furthermore, this study suggests a strategy for the efficient design of ET receptor antagonists with targeted inhibition of ET-1 signalling in vascular endothelial cells.

Topics: Animals; Blood Pressure; Carotid Arteries; Carotid Artery Injuries; Cell Adhesion Molecules; Cell Proliferation; Chemotaxis, Leukocyte; Disease Models, Animal; Endothelial Cells; Endothelin-1; Heart Rate; Hyperplasia; Inflammation; Integrases; Ligation; Macrophages; Male; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Receptor, TIE-2; Receptors, Endothelin; Regional Blood Flow; Time Factors; Tunica Intima

Medial thickening and vascular hypertrophy of resistance arteries can lead to cardiovascular complications associated with diabetes. While previous studies have established a role of type 1 diabetes in vascular remodeling, we recently extended these observations to type 2 diabetes and reported increased collagen deposition due to alterations in matrix metalloproteinase expression and activity in mesenteric resistance arteries. These studies also showed that remodeling response was mediated by endothelin-1 (ET-1) via activation of ET(A) receptors, whereas blockade of ET(B) receptors exacerbated the remodeling. However, the effectiveness of glycemic control strategies in preventing these vascular changes, including activation of the ET system still remained unclear. Also, very little is known about whether and to what extent reorganization of the extracellular matrix (ECM) affects vascular compliance and vasomotor tone. Accordingly, this study assessed structural remodeling of mesenteric microvessels, vascular compliance, and myogenic tone, as well as the role of matrix metalloproteinases (MMP) in mediating these processes. Spontaneously diabetic, non-obese Goto-Kakizaki (GK) rats, a model for type 2 diabetes, and normoglycemic Wistar rats were used for the studies. A subset of GK rats were administered metformin to achieve euglycemia. Glycemic control normalized the increased media-to-lumen ratios (M/L) and myogenic tone seen in diabetes, as well as normalizing plasma ET-1 levels and mesenteric ET(A) receptor expression. There was increased collagen synthesis in diabetes paralleled by decreased collagenase MMP-13 activity, while glycemic control attenuated the process. These findings and our previous study taken together suggest that hyperglycemia-mediated activation of ET-1 and ET(A) receptors alter vascular structure and mechanics in type 2 diabetes.

Topics: Animals; Blood Glucose; Collagen; Diabetes Mellitus, Type 2; Disease Models, Animal; Endothelin-1; Hyperplasia; Hypoglycemic Agents; Male; Matrix Metalloproteinase 13; Mesenteric Arteries; Metformin; Microvessels; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Vasoconstriction

Endothelin-1 (ET)/ET(A) receptor system has been known to play an important role in the pathogenesis of neointimal hyperplasia after endothelial injury. However, the pathological role of endothelin ET(B) receptors on neointimal hyperplasia remains to be elucidated. In the present study, we investigated the pathological role of ET(B) receptors on neointimal hyperplasia in balloon-injured rat carotid arteries by pharmacological blockade with use of 2R-(4-propoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N-(2,6-diethylphenyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid (A-192621), a selective ET(B) receptor antagonist, 2R-(4-methoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N,N-di(n-butyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid (ABT-627), a selective ET(A) receptor antagonist, and (+)-(5S,6R,7R)-2-butyl-7-[2-((2S)-2-carboxypropyl)-4-methoxyphenyl]-5-(3,4-methylenedioxyphenyl)cyclopenteno[1,2-b]pyridine-6-carboxylic acid (J-104132), an ET(A)/ET(B) dual receptor antagonist. Moreover, the spotting-lethal rats, which carry a naturally occurring deletion in the endothelin ET(B) receptor gene, were used to examine the effects of genetic deficiency for this receptor subtype. Two weeks after balloon injury, the ratio of the neointimal to the medial area (neointima/media ratio) was determined. Treatment with A-192621 (30 mg/kg/day) for 2 weeks after injury significantly increased the neointima/media ratio in the injured artery. In contrast, ABT-627 (10 mg/kg/day) and J-104132 (10 mg/kg/day) markedly decreased the neointima/media ratio to the same extent. Furthermore, the neointima/media ratio in the injured artery of the ET(B)-deficient rat was significantly increased compared with that of the wild-type rat, and this increase was abolished by treatment with J-104132. These findings suggest that the inhibition of the ET(B) receptor system leads to an aggravation of neointimal hyperplasia after balloon injury, and the augmentation of ET(A)-mediated actions are responsible for the neointimal hyperplasia aggravated by the pharmacological blockade of ET(B) receptor or by its genetic deficiency. The antagonism of the ET(A) receptor system is essential for preventing restenosis after angioplasty.

Topics: Angioplasty, Balloon; Animals; Carotid Arteries; Carotid Artery Injuries; Disease Models, Animal; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Hyperplasia; Male; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Receptor, Endothelin B; Tunica Intima

Mesangial cell growth constitutes a key feature of progressive glomerular injury. Vasopressin (AVP), a potent peptide vasoconstrictor, acts on mesangial cells through the V(1A) receptors, inducing contraction and cell proliferation. This study examined the effects of YM218, a nonpeptide AVP V(1A) receptor-selective antagonist, on the mitogenic and hypertrophic effects of AVP in rat mesangial cells. When added to mesangial cells whose growth was arrested, AVP concentration-dependently induced hyperplasia and hypertrophy. YM218 potently prevented AVP-induced hyperplasia and hypertrophy of these cells. Furthermore, AVP stimulated endothelin (ET)-1 secretion from mesangial cells in a concentration-dependent manner and this effect was potently inhibited by YM218. ET-1 also induced hyperplasia and hypertrophy in mesangial cells and this effect was completely abolished by ET(A) receptor-selective antagonist YM598. In addition, AVP-induced hyperplasia and hypertrophy were partly inhibited by YM598. These results suggest that AVP may modulate mesangial cell growth not only by its direct action but also through the stimulation of ET-1 secretion. YM218 displays high potency in inhibiting the AVP-induced physiologic responses of mesangial cells via the V(1A) receptors and is a potent pharmacologic probe for investigating the physiologic and pathophysiologic roles of AVP in several renal diseases.

Topics: Animals; Antidiuretic Hormone Receptor Antagonists; Arginine Vasopressin; Benzazepines; Cell Proliferation; Cell Size; Cells, Cultured; DNA Replication; Dose-Response Relationship, Drug; Endothelin-1; Hyperplasia; Male; Mesangial Cells; Piperidines; Protein Biosynthesis; Pyrimidines; Rats; Rats, Wistar; Receptor, Endothelin A; Receptors, Vasopressin; Sulfonamides

Angiopoietins are involved in blood vessel maturation and remodeling.. One consequence of endothelium-specific tyrosine kinase-2 (Tie2) receptor activation by angiopoietin-1 (Ang1) is the release of endothelium-derived growth factors that recruit vascular wall cells. We investigated this process in idiopathic pulmonary arterial hypertension (iPAH).. Ang1, Ang2, and total and phosphorylated Tie2 expression (mRNA and protein) was evaluated in human lung specimens and in cultured pulmonary artery smooth muscle cells (PA-SMCs) and pulmonary endothelial cells (P-ECs) isolated from patients with iPAH and control subjects. Media collected from Ang1-treated P-ECs were assessed for their PA-SMC growth-promoting effect.. Tie2 receptor was fourfold higher in lungs and P-ECs from patients with iPAH than in those from control subjects, with a parallel increase in phosphorylated lung Tie2 receptor. In contrast, Ang1 and Ang2 expression in lungs, P-ECs, and PA-SMCs did not differ. Incubation of PA-SMCs with medium collected from P-EC cultures induced marked proliferation, and this effect was stronger when using P-ECs from patients with iPAH than from control subjects. Ang1 pretreatment of P-ECs from either patients or control subjects induced a further increase in PA-SMC proliferation. Fluoxetine, an inhibitor of the mitogenic action of serotonin, reduced the growth-promoting effect of P-EC media. Ang1 added to P-ECs from patients with iPAH increased the production of endothelin-1 (ET-1) and serotonin, but not of platelet-derived growth factor-BB or epidermal growth factor, and increased the amount of mRNA encoding tryptophan hydroxylase-1 (the rate-limiting enzyme of serotonin synthesis), preproET-1, and ET-1-converting enzyme.. The Ang1/Tie2 pathway is potentiated in iPAH, contributing to PA-SMC hyperplasia via increased stimulation of endothelium-derived growth factors synthesis by P-ECs.

Topics: Angiopoietin-1; Aspartic Acid Endopeptidases; Blotting, Western; Endothelial Cells; Endothelin-1; Endothelin-Converting Enzymes; Endothelium, Vascular; Female; Fluoxetine; Humans; Hyperplasia; Hypertension, Pulmonary; Immunohistochemistry; Male; Metalloendopeptidases; Muscle, Smooth, Vascular; Receptor, TIE-2; Selective Serotonin Reuptake Inhibitors

Newborn rats exposed to 60% O2 for 14 days develop endothelin (ET)-1-dependent pulmonary hypertension with vascular remodeling, characterized by increased smooth muscle cell (SMC) proliferation and medial thickening of pulmonary resistance arteries. Using immunohistochemistry and Western blot analyses, we examined the effect of exposure to 60% O2 on expression in the lung of receptors for the platelet-derived growth factors (PDGF), which are implicated in the pathogenesis of arterial smooth muscle hyperplasia. We observed a marked O2-induced upregulation of PDGF-alpha and -beta receptors (PDGF-alphaR and -betaR) on arterial smooth muscle. This led us to examine pulmonary vascular PDGF receptor expression in 60% O2-exposed rats given SB-217242, a combined ET receptor antagonist, which we found prevented the O2-induced upregulation of PDGF-betaR, but not PDGF-alphaR, on arterial smooth muscle. PDGF-BB, a major PDGF-betaR ligand, was found to be a potent in vitro inducer of hyperplasia and DNA synthesis in cultured pulmonary artery SMC from infant rats. A critical role for PDGF-betaR ligands in arterial SMC proliferation was confirmed in vivo using a truncated soluble PDGF-betaR intervention, which attenuated SMC proliferation induced by exposure to 60% O2. Collectively, these data are consistent with a major role for PDGF-betaR-mediated SMC proliferation, acting downstream of increased ET-1 in a newborn rat model of 60% O2-induced pulmonary hypertension.

Topics: Animals; Animals, Newborn; Becaplermin; Carboxylic Acids; Cell Proliferation; Endothelin Receptor Antagonists; Endothelin-1; Female; Hyperplasia; Hypertension, Pulmonary; Indans; Ligands; Models, Animal; Muscle, Smooth, Vascular; Oxygen; Platelet-Derived Growth Factor; Pregnancy; Proto-Oncogene Proteins c-sis; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Receptor, Platelet-Derived Growth Factor alpha; Receptor, Platelet-Derived Growth Factor beta; Receptors, Endothelin; Up-Regulation

The phosphodiesterase type-5 (PDE-5) inhibitor sildenafil has been reported to improve pulmonary arterial hypertension (PAH), but the mechanisms that account for this effect are incompletely understood. Severe pulmonary hypertension has been characterized by defects in a signaling pathway involving angiopoietin-1 and the bone morphogenetic receptor-2 (BMPR-2). We investigated the effects of sildenafil on hemodynamics and signaling molecules in a piglet overcirculation-induced model of early PAH.. Thirty 3-week-old piglets were randomized to placebo or sildenafil therapy 0.75 mg/kg TID after anastomosis of the left subclavian artery to the pulmonary arterial trunk or after a sham operation. Three months later, the animals underwent a hemodynamic evaluation followed by pulmonary tissue sampling for morphometry, immunohistochemistry or radioimmunoassay, and real-time quantitative-polymerase chain reaction. Chronic systemic-to-pulmonary shunting increased pulmonary mRNA for angiopoietin-1, endothelin-1 (ET-1), angiotensin II, inducible nitric oxide synthase, vascular endothelial growth factor, and PDE-5. Pulmonary messenger RNA for BMPR-1A and BMPR-2 decreased. Pulmonary angiotensin II, ET-1, and vascular endothelial growth factor proteins increased. Pulmonary artery pressure increased from 20+/-2 to 33+/-1 mm Hg, and arteriolar medial thickness increased by 91%. The expressions of angiopoietin-1, ET-1, and angiotensin II were tightly correlated to pulmonary hypertension. Sildenafil prevented the increase in pulmonary artery pressure, limited the increase in medial thickness to 41%, and corrected associated biological perturbations except for the angiopoietin-1/BMPR-2 pathway, PDE-5, and angiotensin II.. Sildenafil partially prevents overcirculation-induced PAH and associated changes in signaling molecules. Angiotensin II, PDE-5, and angiopoietin-1/BMPR-2 signaling may play a dominant role in the early stages of the disease.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Anastomosis, Surgical; Angiopoietin-1; Angiotensin II; Animals; Arterioles; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Protein Receptors, Type II; Cyclic Nucleotide Phosphodiesterases, Type 5; Drug Evaluation, Preclinical; Endothelin-1; Gene Expression Regulation; Hyperplasia; Hypertension, Pulmonary; Models, Animal; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Piperazines; Protein Serine-Threonine Kinases; Pulmonary Artery; Purines; Random Allocation; Receptors, Growth Factor; RNA, Messenger; Signal Transduction; Sildenafil Citrate; Subclavian Artery; Sulfones; Sus scrofa; Vascular Endothelial Growth Factor A

Pulmonary hypertension (PH) is a progressive disease characterized by raised pulmonary vascular resistance, thought to be curable only through lung transplantation. Pathophysiologically, proliferation of pulmonary artery smooth muscle cells triggers pulmonary arterial stenosis and/or regurgitation, especially in advanced PH.. Using a rat model of advanced pulmonary vascular disease produced by injecting monocrotaline, we show that hepatocyte growth factor (HGF) targets pulmonary arterioles and blocks the progression of PH. In these rats, endogenous HGF production was dramatically downregulated during developing experimental PH, but c-Met/HGF receptor was abundant in the medial layers of pulmonary arterioles. HGF gene transfection 2 weeks after the monocrotaline injection resulted in milder medial hyperplasia in lung arterioles and inhibited overgrowth of pulmonary artery smooth muscle cells. Notably, exogenous HGF reduced lung expression levels of endothelin-1 and transforming growth factor-beta, which are critically involved in PH-linked fibrogenic events. Overall, medial wall thickening of pulmonary arteries was almost completely prevented by HGF, and the total collagen deposition in the lung decreased; both effects contributed to the suppression of pulmonary artery hypertension.. Our results suggest that the loss of endogenous HGF may be a feature of the pathogenesis of PH and that HGF supplementation may minimize pathological lung conditions, even advanced PH.

Topics: Animals; Arterioles; Collagen; Endothelin-1; Extracellular Matrix; Fibrosis; Genetic Therapy; Hepatocyte Growth Factor; Humans; Hyperplasia; Hypertension, Pulmonary; Male; Monocrotaline; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Proto-Oncogene Proteins c-met; Rats; Rats, Wistar; Transfection; Transforming Growth Factor beta; Tunica Media

We have investigated the protective effect of YM598, a selective endothelin type A receptor antagonist, against an endothelin-1-induced proliferation of rat mesangial cells and renal function in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of type II diabetes. YM598, but not K-8794, a selective endothelin type B receptor antagonist, inhibited the endothelin-1-induced proliferation of cultured mesangial cells derived from intact Wistar rats in a concentration-dependent manner. YM598 (0.1 or 1 mg/kg), enalapril (5 mg/kg), an angiotensin- converting enzyme inhibitor, or vehicle was administered once daily by gastric gavage to 22-week-old male OLETF rats for 32 weeks. YM598 blunted the development of albuminuria in a dose-dependent manner. A higher dose of YM598 reduced albuminuria comparable with enalapril. Urinary endothelin-1 excretion was greater in the diabetic rats than in the control rats, and was not substantially influenced by the agents. Enalapril, but not YM598, mildly lowered the blood pressure in the diabetic rats, indicating that blood pressure reduction is not involved in the major mechanism of the renoprotective effect of YM598 in OLETF rats. These data suggest that endothelin is involved in the progression of diabetic nephropathy in OLETF rats, and an endothelin type A antagonist is promising for the treatment of diabetic nephropathy.

Topics: Administration, Oral; Albuminuria; Angiotensin-Converting Enzyme Inhibitors; Animals; Cell Proliferation; Cells, Cultured; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Models, Animal; Disease Progression; Dose-Response Relationship, Drug; Enalapril; Endothelin A Receptor Antagonists; Endothelin-1; Hyperplasia; Male; Mesangial Cells; Pyrimidines; Rats; Rats, Inbred OLETF; Rats, Wistar; Receptor, Endothelin A; Sulfonamides

Topics: Animals; Arteriovenous Fistula; Chemokine CCL2; Disease Models, Animal; Endothelin-1; Humans; Hyperplasia; Intercellular Adhesion Molecule-1; Plasminogen Activator Inhibitor 1; Tunica Intima; Veins

We hypothesized that the venous limb of an arteriovenous (AV) fistula would evince up-regulation of genes relevant to vascular remodeling along with neointimal hyperplasia and relevant histological changes. Using the aorto-caval model of an AV fistula model in the rat, we demonstrate marked up-regulation in such proinflammatory genes as monocyte chemoattractant protein-1, plasminogen activator inhibitor-1, and endothelin-1, 2 weeks after the creation of the fistula. Neointimal hyperplasia occurred in variable degrees by 5 weeks after establishing the fistula, and by 16 weeks, such neointimal hyperplasia was progressive and pronounced; at this time point, abundant extracellular matrix was also observed. Smooth muscle cells were present in the hyperplastic neointima as evidenced by staining for alpha-smooth muscle actin; ultrastructurally, smooth muscle cells with a synthetic as well as a contractile phenotype were readily observed. Accumulation of extracellular matrix in the model at 16 weeks was accompanied by increased expression of transforming growth factor-beta1 mRNA, the latter finding contrasting with the suppression of transforming growth factor-beta1 mRNA observed in this model at 2 weeks. In summary, we describe marked up-regulation in proinflammatory genes and progressive neointimal formation in the venous vasculature in an AV fistula model in the rat. We suggest that such alteration in gene expression and histological injury, in conjunction with the relative simplicity of this model, offer a new approach in the study of such timely biological and clinically relevant phenomena as differential gene expression in response to hemodynamic forces, processes involved in vascular remodeling, mechanisms of injury in venous bypass grafts, and mechanisms of dysfunction of AV fistulae used in hemodialysis.

Topics: Animals; Arteriovenous Fistula; Blotting, Northern; Chemokine CCL2; Disease Models, Animal; Endothelin-1; Gene Expression; Hyperplasia; Inflammation; Inflammation Mediators; Microscopy, Electron; Plasminogen Activator Inhibitor 1; Rats; RNA, Messenger; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tunica Intima; Vena Cava, Inferior

Topics: Bosentan; Cell Division; Endothelin-1; Humans; Hyperplasia; Models, Biological; Organ Culture Techniques; Saphenous Vein; Sulfonamides; Tunica Intima

Endothelin, a peptide with strong vasoconstrictive and mitogenic properties, has been found to increase after cardiac transplantation. We therefore assessed the association between its precursor peptide, big endothelin-1, and intimal hyperplasia and coronary flow reserve after heart transplantation.. Thirty-five patients without hemodynamically significant coronary artery disease after heart transplantation were investigated: Average peak flow velocity in the left anterior descending artery (LAD) was assessed by intracoronary Doppler at baseline as well as after injection of adenosine; coronary flow reserve was calculated as a ratio of both and was corrected for patient age and baseline average peak flow velocity. Lumen, intima + media and total vessel area were measured by intracoronary ultrasound. The plasma concentration of big endothelin-1 in venous blood was determined by radioimmunoassay.. Patients with elevated big endothelin-1 levels (>2 fmol/ml) tended to have a decreased corrected coronary flow reserve (2.60 +/- 0.9 vs 3.21 +/- 1.0, p = 0.078). They also had a significantly larger intima + media area (5.82 +/- 2.9 vs 2.37 +/- 2.9 mm(2), p = 0.004) and total vessel area (18.36 +/- 5.8 vs 12.81 +/- 4.8 mm(2), p = 0.012) than those with normal plasma concentrations.. Our study suggests an association between elevated big endothelin-1 plasma levels and the development of intimal hyperplasia and reduction of coronary flow reserve after cardiac transplantation.

Topics: Adult; Aged; Biomarkers; Cohort Studies; Coronary Angiography; Coronary Circulation; Coronary Disease; Endothelin-1; Female; Heart Transplantation; Hemodynamics; Humans; Hyperplasia; Male; Middle Aged; Predictive Value of Tests; Prevalence; Probability; Radioimmunoassay; Risk Assessment; Sensitivity and Specificity; Severity of Illness Index; Tunica Intima; Ultrasonography, Doppler; Ultrasonography, Interventional

Endothelin (ET)-1, which had been discovered as the most potent vasoconstrictive peptide, plays active roles in the various biological responses. However, it is controversial how is ET-1 involved in the vascular remodeling process. Therefore, present experiments were performed to investigate the role of ET-1 for the initiation/progression of intimal hyperplasia. Intimal hyperplasia was caused with the time to peak of 4 to 6 weeks after endothelial removal of the rabbit carotid artery and accompanied by increased ET-1 content as well as ET receptor density within the hyperplastic vessel wall. ET-1 receptors could be classified into three subtypes of ETA, ETB and tentative nonETA/nonETB. TUNEL- and Ki-67-positive cells were detectable with the time to peak of 1 to 2 weeks, whereas all positive cells disappeared within 6 weeks. ATZ1993 as a mixed type antagonist for ET-A, ET-B and nonETA/nonETB receptor subtypes inhibited the intimal hyperplasia, of which inhibition was accompanied by increased TUNEL- and p53-positive cells and decreased Bcl-2-positive cells. ET-1 accelerated the [3H]-thymidine incorporation by cultured vascular smooth muscle cells and, on the other hand, reduced TUNEL-positive cells, which was caused by the serum deprivation. The reduction of TUNEL-positive cells with ET-1 was blocked by ATZ1993 or BQ123 as an antagonist for ETA receptor, but unaffected by BQ788 as an antagonist for ETB receptor. These results strongly suggest that ET-1 plays crucial roles as a mitogen and an inhibitory factor of apoptosis in the vascular remodeling process after endothelial removal.

Topics: Animals; Apoptosis; Carotid Artery, Common; Cell Division; Cells, Cultured; DNA; Endothelin Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Hyperplasia; Indazoles; Rabbits; Receptor, Endothelin A; Receptors, Endothelin; Regeneration; Time Factors; Tunica Intima

The present experiments were designed to investigate the ability to produce nitric oxide, concentrations of N(G)-monomethyl-L-arginine (L-NMMA), and asymmetric N(G), N(G)-dimethyl-L-arginine (ADMA) in endothelial cells, endothelin-1 within the vessel wall, and the degree of intimal hyperplasia (intima/media ratio) in perimenopausal human uterine arteries. According to the tentative classification based on basal cyclic GMP levels, 16 arteries could be grouped into groups I and II consisting of eight each. Net production of the nucleotide was significantly higher in group I than that in group II. Concentration of L-NMMA plus ADMA and endothelin-1 content were significantly higher in group II. All specimens from group I were histologically normal, whereas mild to severe intimal hyperplasia was observed in group II specimens. Although considerable individual variations were detectable in the intima/media ratio, L-NMMA plus ADMA and endothelin-1 (n = 35 each), there were significant and positive correlations between three parameters, indicating that intimal hyperplasia became greater as L-NMMA plus ADMA and endothelin-1 were increased. These results suggest that endogenous nitric oxide synthase inhibitors in endothelial cells and endothelin-1 within the vessel wall are important markers of intimal hyperplasia.

Topics: Adult; Arginine; Arteries; Chromatography, High Pressure Liquid; Climacteric; Cyclic GMP; Endothelin-1; Endothelium, Vascular; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Female; Humans; Hyperplasia; In Vitro Techniques; Middle Aged; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Tunica Intima; Tunica Media; Uterus

The pathological role of oxidative stress in patients treated by hemodialysis has gained increasing recognition in recent years. Because complications related to vascular access are a major source of morbidity, immunohistochemical evidence of oxidative stress and activation of growth factors were examined in native arteriovenous (AV) fistulae (n = 11) and expanded polytetrafluoroethylene (ePTFE) grafts (n = 15) recovered from hemodialysis patients at the time of surgical revision or resection. To show the presence of oxidative stress in tissues, three markers were chosen: N(epsilon)(carboxymethyl)lysine, a structurally identified advanced glycation end product; 4-hydroxy-2,3-nonenol, a lipid peroxidation product; and redox-active transition metals bound to proteins, a source of Fenton chemistry-generated free radicals. Markers of cell growth and proliferation were endothelin-1 (ET-1), a potent mitogenic peptide implicated in the formation of intimal hyperplasia; transforming growth factor-beta (TGF-beta), a stimulus to vascular cell growth and matrix production; and platelet-derived growth factor (PDGF), a mediator of intimal hyperplasia. All specimens studied showed significant intimal hyperplasia. In general, the neointima close to the vascular lumen of the AV fistula and the pseudointima close to the lumen of the ePTFE graft were positive for oxidative stress markers. At sites of injury, especially in the presence of histological evidence of inflammation and healing, expression of oxidative markers was particularly intense. Prominent staining of PDGF was shown at sites of anastomotic hyperplasia and in neovasculature. TGF-beta was associated with proliferation or repair in both AV fistulae and ePTFE grafts. ET-1 staining was most intense in the neointima and pseudointima. This study showed histochemical colocalization of markers of oxidative stress with growth factors known to contribute to intimal hyperplasia.

Topics: Adult; Aged; Arteriovenous Anastomosis; Arteriovenous Fistula; Arteriovenous Shunt, Surgical; Biomarkers; Constriction, Pathologic; Endothelin-1; Female; Growth Substances; Humans; Hyperplasia; Iron; Kidney Failure, Chronic; Lipid Peroxidation; Lysine; Male; Middle Aged; Oxidation-Reduction; Oxidative Stress; Platelet-Derived Growth Factor; Polytetrafluoroethylene; Renal Dialysis; Transforming Growth Factor beta; Tunica Intima; Vascular Patency

Administration of exogenous endothelin-1 (ET-1) has been shown to stimulate neointimal hyperplasia following arterial balloon angioplasty (BA). However, the specific effects of ET-1 on the cellular and extracellular matrix response of the vessel wall after balloon injury and the persistence of these ET-1 effects have not been studied. The objectives of this study were to determine the acute (1 week) and long term (10 weeks) effects of administering exogenous ET-1 after arterial BA on neointimal hyperplasia, collagen synthesis and content, cellular proliferation, and ET(A) and ET(B) receptor expression. Thirty-one rabbits were randomized to receive subcutaneous ET-1 (500 pmol/kg/day for 1 week) or placebo time-release pellets and sacrificed at either 1 or 10 weeks after BA. At 1 week, there was a significant two-fold increase in intimal cross-sectional area (CSA) in ET-1 treated animals compared with placebo. ET-1 treated animals showed significant increases in collagen synthesis (ten-fold) and collagen content (three-fold) compared to placebo treated animals. ET-1 treated animals also had a significant increase (two-fold) in proliferation rates. In addition, ET(A) and ET(B) receptor expression were significantly upregulated in ET-1 treated animals. By 10 weeks these stimulatory effects on intimal CSA and collagen content were no longer evident with a 'catch up' phenomenon observed in the placebo treated animals. Similarly, ET(A) and ET(B) mRNA levels had declined significantly in both groups. Therefore, exogenous ET-1 acutely stimulates extracellular and cellular processes including increased expression of ET(A) and ET(B) receptors contributing to intimal hyperplasia. However, these effects are transient and not maintained long term after withdrawal of exogenous ET-1 stimulation.

Topics: Analysis of Variance; Angioplasty, Balloon; Animals; Cell Division; Collagen; Drug Implants; Endothelin-1; Extracellular Matrix; Gene Expression; Hyperplasia; Male; Microscopy, Confocal; Models, Animal; Rabbits; Random Allocation; Receptors, Endothelin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics, Nonparametric; Stimulation, Chemical; Time Factors; Tunica Intima

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

Urinary bladder hypertrophy and hyperplasia is a common feature of bladder outlet obstruction (BOO). The urinary bladder is known to synthesize endothelin-1 (ET-1). ET-1 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 [endothelin-A and -B (ET(A) and ET(B))] in bladder vascular smooth muscle cells (SMC) proliferation. In the presence of 3-week-old BOO serum, ET(A) and ET(B) antagonists 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 ET(A) and ET(B) antagonists, respectively) and bladder neck (p = 0.01 for both ET(A) and ET(B) antagonists). These results suggest that ET-1 antagonists may prevent SMC hyperplasia associated with partial BOO.

Topics: Animals; Bromodeoxyuridine; Endothelin-1; Hyperplasia; Male; Oligopeptides; Peptides, Cyclic; Piperidines; Rabbits; Urinary Bladder; Urinary Bladder Neck Obstruction

In situ hybridization and immunohistochemical examinations of surgically resected specimens of human hyperplastic tonsils showed that macrophages in the germinal center and mast cells in the parafollicular and interfollicular areas expressed the transcript and protein of endothelin (ET)-1, but not ET-2 and ET-3. The macrophages appeared to be activated, since they possessed significant amounts of inducible nitric oxide synthase. None of these expressions was observed in normal tonsil. Our results suggest that the over-production of ET-1 by macrophages and mast cells may be involved in the pathogenesis of hyperplastic tonsils.

Topics: Adult; Antibodies, Monoclonal; Endothelin-1; Female; Humans; Hyperplasia; Immunohistochemistry; Macrophages; Male; Mast Cells; Middle Aged; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Palatine Tonsil; Reference Values; RNA, Messenger

The present experiments were designed to investigate the effect of ATZ1993 [3-carboxy-4,5-dihydro-1-[1-(3-ethoxyphenyl)propyl]-7-(5-pyrimidinyl)met hoxy-[1H]-benz[g]indazole] on the intimal hyperplasia after balloon endothelial denudation of the rabbit carotid artery. ATZ1993 inhibited the specific [125I]endothelin (ET)-1 binding not only to ET-receptor subtype A (ET(A)) with a pKi value of 8.69+/-0.02, but also to ET-receptor subtype B (ET(B)) with a pKi value of 7.20+/-0.03. Counterscreening in the binding assay (30 different receptors) confirmed that ATZ1993 had a high selectivity for ET receptors. Increases in intima:media ratio and DNA content in the vessel wall were significantly (P < 0.005) inhibited by ATZ1993 in a daily dose of 30 mg x 200 ml(-1) x kg(-1) for 1 week before and 6 weeks after balloon denudation. Inhibition of the intimal hyperplasia with ATZ1993 was determined as approximately 77% for increases in intima:media ratio and DNA content. Plasma concentrations of ATZ1993 ranged between 121.6+/-26.6 and 131.7+/-20.9 nM throughout experimental periods. Mean arterial blood pressure, heart rate and body weight gain remained unaffected by administering ATZ1993. These results demonstrate that ATZ1993 is a novel nonpeptide and nonselective ET(A)/ET(B)-receptor antagonist, and the agent when administered orally inhibits effectively intimal hyperplasia after balloon denudation of the rabbit carotid artery.

Topics: Administration, Oral; Angioplasty, Balloon; Animals; Blood Pressure; Body Weight; Carotid Artery Injuries; Carotid Artery, Common; Endothelin Receptor Antagonists; Endothelin-1; Heart Rate; Humans; Hyperplasia; Indazoles; Male; Muscle, Smooth, Vascular; Rabbits; Receptors, Endothelin; Swine; Tunica Intima

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

Endothelin-1 (ET-1) is a powerful vasoconstrictor and a potent mitogen for vascular smooth muscle cells. Excessive smooth muscle cell proliferation is a feature of intimal hyperplasia, the pathological lesion of vein graft stenosis. This study investigates the role of ET-1 in isolated human saphenous vein smooth muscle cells and also in an organ culture of the human saphenous vein.. Growth-arrested human saphenous vein smooth muscle cells were stimulated with ET-1 and proliferation quantified by [3H]thymidine uptake in these cells compared with unstimulated control cells. Organ cultures of the human saphenous vein were established with endothelium intact, with endothelium denuded, and with endothelium denuded and the culture medium supplemented with ET-1.. ET-1 stimulated DNA synthesis in isolated smooth muscle cells in a dose-dependent manner with half-maximal stimulation at 1 nmol/l. Addition of ET-1 to denuded vein caused a significant increase in median (range) neointimal thickness, from 4 (0-19) to 20 (4-30) microns (P < 0.05). In veins cultured with ET-1 a parallel increase in median (range) neointimal proliferation index, from 21 (0-31) to 33 (23-46) per cent (P < 0.05), was also observed.. These results demonstrate that ET-1 is a mediator of intimal hyperplasia in human saphenous vein in vitro. Endothelin receptor antagonists may therefore be of therapeutic value in the modulation of vein graft intimal hyperplasia.

Topics: Cell Division; Cells, Cultured; Endothelin-1; Humans; Hyperplasia; Muscle, Smooth, Vascular; Saphenous Vein

1. The present experiments were designed to investigate the effect of long-term oral nicotine (10 mg/200 ml/kg/day for 7 weeks) on the intimal hyperplasia after endothelial removal of the rabbit carotid artery. 2. The plasma concentrations of nicotine were determined to be 11.7-12.5 ng/ml during the term of administration and corresponded to the plasma levels in human smokers. 3. Six weeks after the endothelial removal, light microscopy revealed a marked intimal hyperplasia. Administration of nicotine tended to accelerate the intimal hyperplasia, which was estimated by comparing the histological findings, DNA content and wet weight of the vessel wall. 4. Acetylcholine- and A23187-induced endothelium-dependent relaxations were greatly impaired in the hyperplastic artery strips. The impairment of relaxations tended to be accelerated in the nicotine group. Sodium nitroprusside-induced relaxation was not different between the control and the hyperplastic artery strips and remained unaffected in the nicotine group. 5. The concentrations of endogenous nitric oxide (NO) synthesis inhibitors, NG-monomethyl-L-arginine (L-NMMA) and asymmetrical NG,NG-dimethyl-L-arginine (ADMA) were significantly more increased in the regenerated endothelial cells compared with those in the control endothelial cells. The concentrations of L-NMMA and ADMA in the regenerated endothelial cells were significantly increased by as much as 1.3 x 10(-6) and 5.6 x 10(-7) M, respectively, in the nicotine group. 6. Immunoreactive endothelin-1 was significantly increased in the hyperplastic vessel wall (2.4 times that of the control) in 6 weeks. Administration of nicotine tended to increase the level. 7. It seems possible to assume from these results that, although, under the present experimental conditions, nicotine exhibited a tendency to accelerate the intimal hyperplasia after endothelial removal, the longer exposure to nicotine or a higher dose of the agent or both would significantly accelerate the intimal hyperplasia through the enhanced impairment of endothelium-derived relaxing factor/ NO production, which might be brought about by the enhanced increases in L-NMMA and ADMA concentrations, and the enhanced increase in endothelin-1 in the vessel wall.

Topics: Animals; Arginine; Body Weight; Carotid Arteries; Endothelin-1; Endothelium, Vascular; Hyperplasia; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nicotine; Nitric Oxide Synthase; omega-N-Methylarginine; Rabbits; Tunica Intima

Balloon angioplasty has become an important intervention in clinical cardiology; however, the technique is associated with a high incidence of restenosis, requiring repeated procedures. Endothelin-1 (ET-1), specifically through its action on ET(A) receptors, has been implicated in the cell proliferation and subsequent neointimal formation that leads to restenosis. Therefore we examined a potent antagonist of the ET(A) receptor, A127722.5, in a pig model of balloon angioplasty in iliac and carotid arteries. Ten pigs received A-127722.5 (7.5 mg/kg b.i.d.) orally, starting 3 days before angioplasty and continuing for 4 weeks; 10 additional pigs were treated with the same dosing regimen of the angiotensin-converting enzyme (ACE) inhibitor captopril (3.0 mg/kg b.i.d.), while a third group of 10 animals received placebo. At 2 and 4 weeks after the start of treatment, these doses of the ET(A) receptor antagonist and ACE inhibitor blocked the presser responses induced by big ET-1 and angiotensin I, respectively. In the iliac arteries, neointimal formation, neointimal/medial ratio, and maximal neointimal thickness were all significantly reduced, and the residual lumen area was significantly increased in pigs treated with the ET(A) receptor antagonist compared with placebo and captopril-treated groups. Medial collagen content, collagen deposition, and medial growth also were significantly reduced relative to the placebo group. Beneficial effects also were observed in the carotid arteries, although the results were less striking. Captopril was ineffective in protecting against the effects of balloon angioplasty in both vessels. Our results indicate that an orally active and potent antagonist of the ET(A) receptor inhibits cell proliferation and synthesis of extracellular matrix in pigs and may provide an important therapeutic approach to the prevention of restenosis.

Topics: Angioplasty, Balloon; Angiotensin I; Angiotensin-Converting Enzyme Inhibitors; Animals; Atrasentan; Blood Pressure; Captopril; Carotid Artery Injuries; Carotid Artery, Common; Collagen; Endothelin Receptor Antagonists; Endothelin-1; Hyperplasia; Iliac Artery; Male; Muscle, Smooth, Vascular; Pyrrolidines; Receptor, Endothelin A; Swine; Swine, Miniature; Tunica Intima

Topics: Cells, Cultured; Endothelin-1; Humans; Hyperplasia; Saphenous Vein

Endothelins (ETs) are 21 amino acid peptides with vasoactive and mitogenic properties. The three isopeptides (ET-1, -2, and -3) and their receptors (E1A and ETB subtypes) display expression in numerous tissues and possibly mediate autocrine/paracrine actions. The present investigation shows that ET-1 triggers biphasic increases of the concentration of cytoplasmic Ca2+ ([Ca2+]i) in pathological human parathyroid cells. Both the peak and sustained [Ca2+]i increase, as well as the proportion of responding cells, are dose-dependent in the 10(-10)-10(-7) mol/L range of ET-1. In absence of external Ca2+, the ET-1-induced [Ca2+]i peak is attenuated. ET-3 has no effect on [Ca2+]i indicating functional dominance of the ETA receptor subtype. ET-1 (10 nmol/L) lowers parathyroid hormone secretion in 0.5 mmol/L but not in higher external Ca2+ concentrations, and parathyroid cell ET release is inhibited by increases of external Ca2+. Fibroblasts overgrowing the parathyroid chief cells during monolayer culture respond to ET-1 with biphasic [Ca2+]i increases or repetitive [Ca2+]i spikes, but show no response to elevation of external Ca2+. These findings imply that ET secretion and ET receptor expression may constitute an autocrine/paracrine mechanism in the regulation of human PTH secretion.

Topics: Adenoma; Calcium; Cells, Cultured; Endothelin-1; Endothelin-3; Epithelial Cells; Fibroblasts; Humans; Hyperparathyroidism; Hyperplasia; Multiple Endocrine Neoplasia Type 1; Parathyroid Glands; Parathyroid Hormone; Parathyroid Neoplasms; Signal Transduction; Tumor Cells, Cultured