Endocardial endothelial celsl stimulate proliferation and collagen synthesis of cardiac fibroblasts

Given that vascular endothelial cells play an important role in the modulation of vascular structure and function, we hypothesized that endocardial endothelial cells (EECs) may have a modulator role in regulating the cardiac interstitial cells. Endocardial endothelial cells were isolated from freshly collected pig hearts and cardiac fibroblasts were isolated from 3- to 4-d-old Wistar rats. Fibroblasts were cultured in the presence or absence of conditioned medium from EECs. Proliferation of cardiac fibroblasts was measured by the incorporation of [³H]-Thymidine and collagen synthesis was assayed by the incorporation of [³H]-proline. To determine the involvement of signaling mediators, in separate experiments, cardiac fibroblasts were incubated with BQ123 (selective ET_A receptor antagonist), PD142893 (nonselective ET_A/ET_B receptor antagonist), Bis-indolylmaleimide (PKC inhibitor), PD 098059 (MEK inhibitor), or neutralizing anti-transforming growth factor (TGF)-β-antibody. Endocardial endothelium-derived factors endothelin (ET)-1, TGF-β, and Angiotensin (Ang)-II in the conditioned medium were assayed by enzyme-linked immunosorbent assay using commercially available kits. We report here evidence that suggest that endocardial endothelial cells stimulate both proliferation and collagen synthesis of cardiac fibroblasts. The response seems to be mediated by endothelin through its ET_A receptor. Our results also indicate that protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) pathways are essential for the EEC-induced proliferation of cardiac fibroblasts.     

Angiotensin-(1-7) abrogates mitogen-stimulated proliferation of cardiac fibroblasts

Previous studies showed that angiotensin-(1-7) [Ang-(1-7)] attenuates cardiac remodeling by reducing both interstitial and perivascular fibrosis. Although a high affinity binding site for Ang-(1-7) was identified on cardiac fibroblasts, the molecular mechanisms activated by the heptapeptide hormone were not identified. We isolated cardiac fibroblasts from neonatal rat hearts to investigate signaling pathways activated by Ang-(1-7) that participate in fibroblast proliferation. Ang-(1-7) reduced (3)H-thymidine, -leucine and -proline incorporation into cardiac fibroblasts stimulated with serum or the mitogen endothelin-1 (ET-1), demonstrating that the heptapeptide hormone decreases DNA, protein and collagen synthesis. The reduction in DNA synthesis by Ang-(1-7) was blocked by the AT((1-7)) receptor antagonist [d-Ala(7)]-Ang-(1-7), showing specificity of the response. Treatment of cardiac fibroblasts with Ang-(1-7) reduced the Ang II- or ET-1-stimulated increase in phospho-ERK1 and -ERK2. In contrast, Ang-(1-7) increased dual-specificity phosphatase DUSP1 immunoreactivity and mRNA, suggesting that the heptapeptide hormone increases DUSP1 to reduce MAP kinase phosphorylation and activity. Incubation of cardiac fibroblasts with ET-1 increased cyclooxygenase 2 (COX-2) and prostaglandin synthase (PGES) mRNAs, while Ang-(1-7) blocked the increase in both enzymes, suggesting that the heptapeptide hormone alters the concentration and the balance between the proliferative and anti-proliferative prostaglandins. Collectively, these results indicate that Ang-(1-7) participates in maintaining cardiac homeostasis by reducing proliferation and collagen production by cardiac fibroblasts in association with up-regulation of DUSP1 to reduce MAP kinase activities and attenuation of the synthesis of mitogenic prostaglandins. Increased Ang-(1-7) or agents that enhance production of the heptapeptide hormone may prevent abnormal fibrosis that occurs during cardiac pathologies.     

Arrhythmogenic action of endothelin-1(1-31) through conversion to endothelin-1(1-21)

Endothelin (ET)-1(1-21) is known to play an important role in the pathogenesis of acute ischemic arrhythmia. In the present study, we attempted to determine whether administration of ET-1(1-31) would result in arrhythmia in perfused isolated rat hearts. Forty-eight Sprague-Dawley rats weighing approximately 250-350 g were randomized into 6 groups. Heart was isolated and perfused in a Langendorff mode. The effects of ET-1(1-31) on arrhythmia, heart rate, coronary flow, and heart function were analyzed. Perfusion with 1 nM ET-1(1-31) resulted in frequent ventricular ectopic beats (VEBs) and ventricular tachycardia (VT). Overall VEB was 128.0 (approximately 66.0-1015.0), and the arrhythmia score (AS) was 2.18 +/- 0.87; both were significantly higher than those of the control group (P < 0.01). Pretreatment with perfusion of 10 nM of the ETA-receptor antagonist BQ(123) markedly attenuated the occurrence of VEB and VT induced by ET-1(1-31). AS in 10 nM BQ123 group was significantly lower than that in 1 nM ET-1(1-31) group (P < 0.01). The arrhythmia induced by 1 nM ET-1(1-31) was partially but significantly reduced by phosphoramidon (1 microM), a neutral endopeptidase/ET-converting enzyme inhibitor. ET-1(1-31) per se caused arrhythmia in perfused isolated rat hearts. This arrhythmogenic action is in part mediated by ET(A) receptor and may be attributed mainly to the conversion of ET-1(1-31) to ET-1(1-21.).     

Hexarelin suppresses cardiac fibroblast proliferation and collagen synthesis in rat

Abnormal growth of cardiac fibroblasts is critically involved in the pathophysiology of cardiac hypertrophy/remodeling. Hexarelin is a synthetic growth hormone secretagogue (GHS), which possesses a variety of cardiovascular protective activities mediated via the GHS receptor (GHSR), including improving cardiac dysfunction and remodeling. The cellular and molecular mechanisms underlying the effect of GHS on cardiac fibrosis are, however, not clear. In this report, cultured cardiac fibroblasts from 8-day-old rats were stimulated with ANG II or FCS to induce proliferation. The fibroblast proliferation and DNA and collagen synthesis were evaluated utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, (3)H-thymidine incorporation, and (3)H-proline incorporation. The level of mRNA of transforming growth factor (TGF)-beta was evaluated by RT-PCR, and the active TGF-beta1 release from cardiac fibroblasts was evaluated by ELISA. The level of cellular cAMP was measured by radioimmunoassay. In addition, the effects of 3,7-dimethyl-l-propargylxanthine (DMPX; a specific adenosine receptor A(2)R antagonist) and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; a specific A(1)R antagonist) were tested. It was found that incubation with 10(-7) mol/l hexarelin for 24 h 1) inhibited the ANG II-induced proliferation and collagen synthesis and the 5% FCS- and TGF-beta-induced increase of DNA synthesis in cardiac fibroblast and 2) reduced ANG II-induced upregulation of TGF-beta mRNA expression and active TGF-beta1 release from fibroblasts. Hexarelin increased the cellular level of cAMP in cardiac fibroblasts. DMPX (10(-8) mol/l) but not DPCPX abolished the effect of hexarelin on cardiac fibroblast DNA synthesis. It is concluded that hexarelin inhibits DNA and collagen synthesis and proliferation of cardiac fibroblasts through activation of both GHSR and A(2)R and diminishment of ANG II-induced increase in TGF-beta expression and release.     

Molecular modeling of the complex of endothelin-1 (ET-1) with the endothelin type A (ET(A)) receptor and the rational design of a peptide antagonist

ET-1 is the most potent vasoconstrictor known to date, causing vasoconstriction when bound to the ET(A) receptor. Inhibitors of the binding of ET-1 to the ET(A) receptor would be of immense value as potential therapeutic agents in the treatment of cardiovascular disorders such as angina and hypertension. We present here the rational design of such an inhibitor, which is arrived at on the basis of a model of the ET-1/ET(A) receptor complex proposed by us. The model is found to be consistent with binding and mutagenesis studies of ET-1 as well as of BQ123, a known, potent ET(A)-selective antagonist which competes with ET-1 for receptor binding. BQ123 is a peptidic antagonist which is constrained to adopt a definite conformation on account of its cyclic nature. The noncyclic peptide antagonist designed by us also has a unique conformation because it contains two dehydro-Alanine (deltaAla) residues which, on account of their planarity, cause the peptide backbone to bend in a specific and predictable manner. The folding rules for peptides containing deltaAla were derived in our earlier studies. Energy minimization and modelling of the complex of the designed peptide with the ET(A) receptor indicate that the antagonist is ET(A)-selective and the binding is more stable and more specific as compared to that of BQ123.     

Functional endothelin receptors are present on nuclei in cardiac ventricular myocytes

Endothelins are thought to act through two specific, plasmalemmal G protein-coupled receptor subtypes, ETAR and ETBR. However, in subfractionated cardiac membranes, ETAR immunoreactivity was detected only in the plasma membrane whereas ETBR immunoreactivity was detected predominantly in membranes of intracellular origin. Confocal microscopy demonstrated the presence of intracellular ETAR and ETBR in ventricular myocytes. ETAR were primarily on plasma membrane (surface membranes and transverse-tubules) and to a lesser extent on the nucleus while ETBR localized primarily to the nuclei. Western blot analysis of nuclei isolated from the heart indicated the presence of endothelin receptors: both ETAR and ETBR copurified with nucleoporin 62, whereas markers of endoplasmic reticulum and Golgi membranes were depleted. Radioligand binding studies revealed that isolated nuclei contain specific [125I]ET-1 binding sites. Specific [125I]ET-1 binding was reduced by 70-80% using the ETAR-selective antagonist BQ610 and 20-30% using the ETBR-specific antagonist BQ788. IRL-1620, an ETBR-specific agonist, also reduced [125I]ET-1 binding. Furthermore, ET-1 and IRL-1620 altered the incorporation of 32P into nuclear proteins and caused a transient increase in nuclear Ca2+ concentration. Hence, cardiac nuclei possess both ETAR and ETBR subtypes, which are functional with respect to ligand binding and are coupled to signaling mechanisms within the nuclear membrane.     

Molecular Modeling of the Complex of Endothelin-1(ET-1) with the Endothelin Type A (ETA) Receptor and the Rational Design of a Peptide Antagonist

Abstract

ET-1 is the most potent vasoconstrictor known to date, causing vasoconstriction when bound to the ET_a receptor. Inhibitors of the binding of ET-1 to the ET_A receptor would be of immense value as potential therapeutic agents in the treatment of cardiovascular disorders such as angina and hypertension. We present here the rational design of such an inhibitor, which is arrived at on the basis of a model of the ET-1/ET_A receptor complex proposed by us. The model is found to be consistent with binding and mutagenesis studies of ET-1 as well as of BQ123, a known, potent ET_A-selective antagonist which competes with ET-1 for receptor binding. BQ123 is a peptidic antagonist which is constrained to adopt a definite conformation on account of its cyclic nature. The noncyclic peptide antagonist designed by us also has a unique conformation because it contains two dehydro-Alanine (δAla) residues which, on account of their planarity, cause the peptide backbone to bend in a specific and predictable manner. The folding rules for peptides containing δAla were derived in our earlier studies. Energy minimization and modelling of the complex of the designed peptide with the ET_A receptor indicate that the antagonist is ET_A -selective and the binding is more stable and more specific as compared to that of BQ123.     

心室成纤维细胞条件培养液促进成纤维细胞胶原合成和增殖

采用心室成纤维细胞条件培养液培养心室成纤维细胞,通过测定[^3H]－脯氨酸（[^3H]－proline）的掺入率来了解心室成纤维细胞总胶原合成速率,通过测定[^3H]－胸腺嘧啶核苷（[^3H]－TdR）的掺入率以及c-fos基因的表达丰度来了解心室成纤维细胞的增殖速率。结果显示：心室成纤维细胞条件培养液（FCGM）能增加细胞自身的[^3H]－proline的掺入率和[^3H]－TdR的掺入率,并具有剂量依赖性;FCGM也能促进细胞自身c-fos基因的表达,刺激后1h达高峰。ETA受体拮抗剂BQ123能部分阻断FCGM增加成纤维细胞胶原合成的增殖作用,而AT1受体拮抗剂CV11974和α肾上腺素受体拮抗剂regitin无此效果。结果提示：心室成纤维细胞具有自分泌功能,能分泌内皮素等生物活性物质,促进成纤维细胞胶原的合成和增殖。     

A novel ETA antagonist (BQ-123) inhibits endothelin-1-induced phosphoinositide breakdown and DNA synthesis in rat vascular smooth muscle cells.

The effects of a novel cyclic pentapeptide (BQ-123), an endothelin (ET) antagonist selective for the ETA receptor subtype, on phosphoinositide breakdown and DNA synthesis stimulated by ET-1 were studied in cultured rat vascular smooth muscle cells (VSMC). BQ-123 competitively inhibited the binding of [125I]ET-1 to VSMC with the apparent Ki of 4 x 10(-9) M. BQ-123 dose-dependently inhibited formation of inositol-1,4,5-trisphosphate and [3H]thymidine uptake stimulated by ET-1. These data suggest that the ET-1-induced DNA synthesis in VSMC is mainly mediated by ETA receptor subtype.     

Characterization and localization of Ac-SDKP receptor binding sites using 125I-labeled Hpp-Aca-SDKP in rat cardiac fibroblasts

We have shown that the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibited endothelin-1 (ET-1)-induced cell proliferation and collagen synthesis in cultured rat cardiac fibroblasts (CFs) and reduced left ventricle collagen deposition in rats with aldosterone (salt)- and ANG II-induced hypertension. However, it is not known whether these effects are mediated by receptor binding sites specific for Ac-SDKP. We hypothesized that Ac-SDKP exerts antifibrotic effects by binding to specific receptor sites in cultured rat CFs, which mediate the inhibitory effects of Ac-SDKP on ET-1-stimulated collagen synthesis. Ac-SDKP binding sites in rat CFs and hearts were characterized by a specific radioligand, (125)I-labeled 3-(p-hydroxyphenyl)-propionic acid (or desaminotyrosine) (Hpp)-Aca-SDKP, a biologically active analog of Ac-SDKP. (125)I-labeled Hpp-Aca-SDKP bound to rat CFs and fractionated membranes with similar affinities and specificity in a concentration- and time-dependent fashion. Scatchard plot analyses revealed a single class of high-affinity Hpp-Aca-SDKP binding sites (maximal binding: 1,704 +/- 198 fmol/mg protein; dissociation constant: 3.3 +/- 0.6 nM). (125)I-labeled Hpp-Aca-SDKP binding in CFs was displaced by unlabeled native peptide Ac-SDKP (inhibition constant: 0.69 +/- 0.15 nM) and the analog Hpp-Aca-SDKP (inhibition constant: 10.4 +/- 0.2 nM) but not the unrelated peptide ANG II or ET-1 (10 microM). In vitro, both Ac-SDKP and Hpp-Aca-SDKP inhibited ET-1-stimulated collagen synthesis in CFs in a dose-dependent fashion, reaching a maximal effect at 1 nM (control: 7.5 +/- 0.4, ET-1: 19.9 +/- 1.2, ET-1+SDKP: 7.7 +/- 0.4, ET-1+Hpp-Aca-SDKP: 9.7 +/- 0.1 microg/mg protein; P < 0.001). Ac-SDKP also significantly attenuated ET-1-induced increases in intracellular calcium and MAPK ERK1/2 phosphorylation in CFs. In the rat heart, in vitro autoradiography revealed specific (125)I-labeled Hpp-Aca-SDKP binding throughout the myocardium, primarily interstitially. We believe that these results demonstrate for the first time that Hpp-Aca-SDKP is a functional ligand specific for Ac-SDKP receptor binding sites and that both Ac-SDKP and Hpp-Aca-SDKP exert antifibrotic effects by binding to Ac-SDKP receptors in rat CFs.     

Effects of human peptide endothelin-1 and two of its sterically unrestrained C-terminal fragments on coronaryvascular smooth muscle

Clearance of human peptide endothelin-1 (ET-1) has been proposed to follow a receptor pathway involving a cascade of ET-1 receptor endocytosis and lysosomal degradation by a family of proteinases expressed constitutively by most cells. Genetically distinct endopeptidases produce ET-1 and degrade mature peptide. The ET-1 degradation products were considered to be inactive, however, recent evidence suggests that ET-1 fragments sustain most of the homeostatic response produced by parent peptides. The purpose of this study was to establish whether the overall structure of human ET-1 or the structure of its C-terminus is responsible for the subtype-selectivity, down-regulation and clearance of endothelin, and whether D-aminoacid substitution in the moiety of synthetic peptide is involved in effective ET-1 antagonism in coronary vascular smooth muscle. To characterize specific mechanism(s) leading to subtype-selective ET-receptor down-regulation and/or to ET-1 antagonism, ligand binding studies were accomplished with radioactive human (1-21)ET-1 and with C-terminal ET-1 fragments, both peptide agonists and antagonists, in adult male porcine coronary artery vascular smooth muscle (CVSM). The subcellular membranes of CVSM were isolated by isopycnic gradient centrifugation. Exposure of porcine coronary artery to exogenous ET-1 induced endothelin-ETB selective down-regulation. ETA-mediated subtype-ETB down-regulation was observed with distribution of ligand-ETB receptor complexes in light, endosomal, membranes. The ETA selective PD151242 significantly attenuated [3H]-thymidine incorporation, and the ETB selective antagonist BQ788 blocked down-regulation observed in porcine vascular fibroblasts (PF). Preincubation of coronary arteries with ETB selective BQ3020 was accompanied with a more intense down-regulation. CONCLUSION: our data are indicative of short-term ETB selective down-regulation of endothelin receptors in coronary vascular smooth muscle after exposure to ET-1. The presence in the carboxy-terminus of (Ala11,15) substitution in peptide fragments IRL1620 and BQ3020 determined the differential specificity of ETB-receptor coupling and was important for subtype-ETB-receptor down-regulation. The activation of the dominating ETA-receptor by ET-1 facilitated mitogenic responses to ET-1 in porcine vascular fibroblasts.     

Both endothelin-A and endothelin-B receptors are present on adult rat cardiac ventricular myocytes

Endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors have been demonstrated in intact heart and cardiac membranes. ET(A) receptors have been demonstrated on adult ventricular myocytes. The aim of the present study was to determine the presence of ET(B) and the relative contribution of this receptor subtype to total endothelin-1 (ET-1) binding on adult ventricular myocytes. Saturation binding experiments indicated that ET-1 bound to a single population of receptors (Kd = 0.52 +/- 0.13 nM, n = 4) with an apparent maximum binding (Bmax) of 2.10 +/- 0.25 sites (x 10(5))/cell (n = 4). Competition experiments using 40 pM [125I]ET-1 and nonradioactive ET-1 revealed a Ki of 660 +/- 71 pM (n = 10) and a Hill coefficient (nH) of 0.99 +/- 0.10 (n = 10). A selective ET(A) antagonist, BQ610, displaced 80% of the bound [125I]ET-1. No displacement was observed by concentrations of an ET(B)-selective antagonist, BQ788, up to 1.0 microM. However, in the presence of 1.0 microM BQ610, BQ788 inhibited the remaining [125I]ET-1 binding. Similarly, in the presence of 1.0 microM BQ788, BQ610 inhibited the remaining specific [125I]ET-1 binding. Binding of an ET(B1)-selective agonist, [125I]IRL-1620, confirmed the presence of ET(B). ET(B) bound to ET-1 irreversibly, whereas binding to ET(A) demonstrated both reversible and irreversible components, and BQ610 and BQ788 bound reversibly. Reducing the incubation temperature to 0 degrees C did not alter the irreversible component of ET-1 binding. Hence, both ET(A) and ET(B) receptors are present on intact adult rat ventricular myocytes, and the ratio of ET(A):ET(B) binding sites is 4:1. Both receptor subtypes bind to ET-1 by a two-step association involving the formation of a tight receptor-ligand complex; however, the kinetics of ET-1 binding to ET(A) versus ET(B) differ.     

ETA and ETB receptors are expressed in vascular adventitial fibroblasts

The adventitia has been recognized to play important roles in vascular oxidative stress, remodeling, and contraction. We recently demonstrated that adventitial fibroblasts are able to express endothelin (ET)-1 in response to ANG II. However, it is unclear whether ET-1 receptors are expressed in the adventitia. We therefore investigated the expression and roles of both ET(A) and ET(B) receptors in collagen synthesis and ET-1 clearance in adventitial fibroblasts. Adventitial fibroblasts were isolated and cultured from the mouse thoracic aorta by the explant method. Cultured cells were treated with ANG II (100 nmol/l) or ET-1 (10 pM) in the presence or absence of the ANG II type 1 receptor antagonist losartan (100 μM), the ET-1 receptor antagonists BQ-123 (ET(A) receptor, 1 μM) and BQ-788 (ET(B) receptor, 1 μM), and the ET(B) receptor agonist sarafotoxin 6C (100 nM). ET-1 peptide levels were determined by ELISA, whereas ET(A), ET(B), and collagen levels were determined by Western blot analysis. ANG II increased ET-1 peptide levels in a time-dependent manner. ANG II increased ET(A) and ET(B) receptor protein levels as well as collagen in a similar fashion. ANG II-induced collagen was reduced while in the presence of BQ-123, suggesting a role for the ET(A) receptor in the regulation of the extracellular matrix. ANG II treatment in the presence of BQ-788 significantly increased ET-1 peptide levels. Conversely, the ET(B) receptor agonist sarafotoxin 6C significantly decreased ET-1 peptide levels. These data implicate a role for the ET(B) receptor in the clearance of the ET-1 peptide. In conclusion, both ET(A) and ET(B) receptors are expressed in adventitial fibroblasts, which paves the ground for the biological significance of adventitial ET-1. The ET(A) receptor subtype mediates collagen I expression, whereas the ET(B) receptor subtype may play a protective role through increasing the clearance of the ET-1 peptide.     

Effects of endothelin-1 1-31 on cell viabililty and [Ca2+]i in cultured neonatal rat cardiomyocytes

We previously found that Endothelin-1(1-31) (ET-1(1-31)) exhibited a pro-arrhythmogenic effect in isolated rat hearts. In this study, we further investigated the effects of ET-1(1-31) on a cell viability and observed [Ca(2+)](i) in cultured cardiomyocytes. Cultured neonatal rat cardiomyocytes were treated with 0.1, 1, and 10 nM ET-1(1-31) for 24h in the presence or absence of ET(A) receptor antagonist (BQ(123)) or phosphoramidon, a NEP/ECE inhibitor. Cell injury was evaluated by supernatant lactate dehydrogenase (LDH) assay, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content. Cell viability was assessed by MTT assay. [Ca(2+)](i) was measured with Fluo-3/AM under a laser confocal microscope. 1) ET-1(1-31) dose-dependently increased LDH release and decreased cell viability. 2) LDH and MDA levels were significantly elevated and SOD activity decreased after administration of 1 nM ET-1(1-31) for 24h, and these changes were markedly attenuated by 1 uM BQ(123). 3) Exposure to 10 nM ET 1(1-31) caused a continuous increase in [Ca(2+)](i) to cultured beating cardiomyocytes and termination of [Ca(2+)](i) transient within 6 min, and this change was reversed by 1 uM BQ(123) and attenuated by 0.5 mM phosphoramidon. These results suggest that ET-1(1-31) could cause cell injury, and that the effect of ET-1(1-31) on [Ca(2+)](i) transients is mainly mediated by ET(A) receptor and partially attributed to the conversion of ET-1(1-31) to ET-1(1-21).     

Dual role of endothelin-1 via ETA and ETB receptors in regulation of cardiac contractile function in mice

An increase in coronary perfusion pressure leads to increased cardiac contractility, a phenomenon known as the Gregg effect. Exogenous endothelin (ET)-1 exerts a positive inotropic effect; however, the role of endogenous ET-1 in the contractile response to elevated load is unknown. We characterized here the role of ETA and ETB receptors in regulation of contractility in isolated, perfused mouse hearts subjected to increased coronary flow. Elevation of coronary flow from 2 to 5 ml/min resulted in 80 +/- 10% increase in contractile force (P < 0.001). BQ-788 (ETB receptor antagonist) augmented the load-induced contractile response by 35% (P < 0.05), whereas bosentan (ETA/B receptor antagonist) and BQ-123 (ETA receptor antagonist) attenuated it by 34% and 56%, respectively (P < 0.05). CV-11974 (ANG II type 1 receptor antagonist) did not modify the increase in contractility. These results show that endogenous ET-1 is a key mediator of the Gregg effect in mouse hearts. Moreover, ET-1 has a dual role in the regulation of cardiac contractility: ETA receptor-mediated increase in contractile force is suppressed by ETB receptors.     

Proliferative effects of angiotensin II and endothelin-1 on guinea pig gingival fibroblast cells in culture

We investigated whether phenytoin (PHT) and nifedipine (NIF) induce angiotensin II (Ang II) and endothelin-1 (ET-1) generation by cultured gingival fibroblasts derived from guinea pigs and whether Ang II and ET-1 induce proliferation of these cells. Immunohistochemical experiments showed that PHT (250 nM) and NIF (250 nM) increased the immunostaining intensities of immunoreactive Ang II and ET-1 (IRET-1) in these cells. Captopril (3 microM), an angiotensin-converting enzyme inhibitor, reduced these enhanced intensities to control levels. Ang II (100 nM) enhanced the immunostaining intensity of IRET-1. PHT (250 nM) and NIF (250 nM)-induced cell proliferation. Both PHT- and NIF-induced proliferation was inhibited by captopril (3 microM). Ang II (100 nM) and ET-1 (100 nM) also induced cell proliferation. Ang II-induced proliferation was inhibited by CV11974 (1 microM), an AT(1) receptor antagonist and saralasin (1 microM), an AT(1)/AT(2) receptor antagonist, but not by PD123,319 (1 microM), an AT(2) receptor antagonist. ET-1-induced proliferation was inhibited by BQ123 (10 microM), an ET(A) receptor antagonist, but not by BQ788 (1 microM), an ET(B) receptor antagonist. These findings suggest that PHT- and NIF-induced gingival fibroblast proliferation is mediated indirectly through the induction of Ang II and ET-1 and probably mediated through AT(1) and ET(A) receptors present in or on gingival fibroblasts.     

Signalling and regulation of collagen I synthesis by ET-1 and TGF-beta1

Endothelin-1 (ET-1) plays an important role in tissue remodelling and fibrogenesis by inducing synthesis of collagen I via protein kinase C (PKC). ET-1 signals are transduced by two receptor subtypes, the ETA- and ETB-receptors which activate different Galpha proteins. Here, we investigated the expression of both ET-receptor subtypes in human primary dermal fibroblasts and demonstrated that the ETA-receptor is the major ET-receptor subtype expressed. To determine further signalling intermediates, we inhibited Galphai and three phospholipases. Pharmacologic inhibition of Galphai, phosphatidylcholine-phospholipase C (PC-PLC) and phospholipase D (PLD), but not of phospholipase Cbeta, abolished the increase in collagen I by ET-1. Inhibition of all phospholipases revealed similar effects on TGF-beta1 induced collagen I synthesis, demonstrating involvement of PC-PLC and PLD in the signalling pathways elicited by ET-1 and TGF-beta1. ET-1 and TGF-beta1 each stimulated collagen I production and in an additive manner. ET-1 further induced connective tissue growth factor (CTGF), as did TGF-beta1, however, to lower levels. While rapid and sustained CTGF induction was seen following TGF-beta1 treatment, ET-1 increased CTGF in a biphasic manner with lower induction at 3 h and a delayed and higher induction after 5 days of permanent ET-1 treatment. Coincidentally at 5 days of permanent ET-1 stimulation, a switch in ET-receptor subtype expression to the ETB-receptor was observed. We conclude that the signalling pathways induced by ET-1 and TGF-beta1 leading to augmented collagen I production by fibroblasts converge on a similar signalling pathway. Thereby, long-time stimulation by ET-1 resulted in a changed ET-receptor subtype ratio and in a biphasic CTGF induction.     

内皮素-1对大鼠血管平滑肌细胞产生MCP-1的影响及其机制

目的：观察内皮素-1（ET-1）对大鼠血管平滑肌细胞（VSMCs）产生单核细胞趋化蛋白-1（MCP-1）的影响及其机制。方法：培养大鼠血管平滑肌细胞（VSMCs）。细胞分为2组：ET-1刺激组：以不同浓度ET-1刺激VSMCs不同时间;阻断剂干预组：VSMCs分别与不同阻断剂[ET_AR、ET_BR阻断剂BQ123、BQ788,抗氧化剂N-乙酰半胱氨酸（NAC）,ERK、p38MAPK、JNK及NF-κB抑制剂PD98059、SB203580、SP600125及PDTC]预先孵育30 min,再加入ET-1刺激24 h。在预定时间,以酶联免疫吸附（ELISA）法、逆转录聚合酶链反应（RT-PCR）法分别测定不同因素下VSMCs MCP-1蛋白质及mRNA表达量。VSMCs分别与不同阻断剂（BQ123、BQ788、NAC、PD98059、SB203580及SP600125预先孵育20 min,再加入ET-1刺激5 min,免疫印迹（WB）法测定VSMCs胞浆中细胞外调节蛋白激酶（ERK）、p38丝裂原活化蛋白激酶（p38MAPK）、c-Jun氨基末端激酶（JNK）及其各自磷酸化蛋白质的水平。各项检测均重复3次。结果：ET-1能刺激VSMCs MCP-1蛋白质及mRNA表达,其表达量随ET-1浓度及刺激时间的增加呈升高趋势（P<0.05,P<0.01）;BQ123、NAC、PD98059、SB203580及PDTC能显著抑制ET-1诱导的大鼠VSMCs MCP-1蛋白质及mRNA表达（P<0.01）,而BQ788及SP600125对此作用无明显影响。BQ123、NAC与PD98059或SB203580能分别抑制ET-1刺激后VSMCs胞浆内ERK及p38MAPK的磷酸化（P<0.05,P<0.01）,而ET-1对JNK的磷酸化无明显激活作用。结论：ET-1通过ET_AR、ROS、ERK、p38MAPK及NF-κB诱导大鼠VSMCs产生MCP-1。     

脑室注射ET－1拮抗剂BQ123和ET抗血清使主动脉狭窄──高盐型高血压大鼠血压下降

侧脑室注射ＥＴ－１受体阻断剂ＢＱ１２３可明显降低腹主动脉狭窄──高盐摄入型实验性高血压大鼠的血压,并呈剂量效应关系。大剂量的ＢＱ１２３还可以使该型高血压大鼠心率减慢。侧脑室注射血管内皮素抗血清也可使该型高血压大鼠血压下降。以上结果表明中枢内皮素在高血压发生的病理生理中可能起一定作用。另外ＥＴ－１的拮抗剂ＢＱ１２３与ＥＴ－抗血清的抗高血压作用可能具有一定的临床意义。     

The involvement of transforming growth factor-beta1 secretion in urotensin II-induced collagen synthesis in neonatal cardiac fibroblasts

As the most potent vasoconstrictor in mammals, urotensin II (U II) has recently been demonstrated to play an important role in adverse cardiac remodeling and fibrosis. However, the mechanisms of U II-induced myocardial fibrosis remain to be clarified. We postulated that U II alters transforming growth factor-beta1 (TGF-beta1) expression, and thereby modulates cardiac fibroblast collagen metabolism. Experiments were conducted using cardiac fibroblast from neonatal Wistar rats to determine the expression of TGF-beta1, and the role of U II receptor UT in this process. The functional role of TGF-beta1 and UT in modulating U II effects on type I, III collagen mRNA expression and 3H-proline incorporation was also analyzed. TGF-beta1 gene and protein expression were consistently identified in quiescent cardiac fibroblasts. U II increased the expression of TGF-beta1 mRNA and protein in a time-dependent manner. This effect was UT mediated, because UT antagonist urantide abolished U II-induced TGF-beta1 expression. U II-induced increase in type I, III collagen mRNA expression and 3H-proline incorporation were both inhibited by a specific TGF-beta1 neutralizing antibody and UT receptor antagonist urantide. Hence, our results indicate that TGF-beta1 is upregulated in cardiac fibroblasts by U II via UT and modulates profibrotic effects of U II. These findings provide novel insights into U II-induced cardiac remodeling.