Atheromatous plaque from human carotid artery: Potential involvement of the endothelin-1 and their receptors

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that can modulate the behaviour of vascular smooth muscle cells and thus impact on the development of human atherosclerosis. Circulating plasma levels of ET-1 were measured from 82 patients with ischemic cardiomyopathy (ICM) and 42 healthy controls. A significant increase was found in plasma levels of ET-1 in the patients compared to the controls. These circulating levels of ET-1 were greater in patients with diabetes or involvement of several territories. Gene expression of pre-proET-1 and its receptors ET_A and ET_B was analyzed in the atheromatous plaques from carotid arteries (n = 8) and the internal mammary artery (IMA) (n = 8). Our group observed an increase in pre-proET-1 and ET_A in IMA compared with the atheromatous plaques. Immunohistochemical studies in the atherosclerotic plaque showed that the expression of ET-1 was greater in the areas where the macrophages and lipid nucleus were located.Our findings in this group of patients with symptomatic vascular disease suggest that the endothelin system may play an important role in atherothrombosis.     

The effect of acute ischemia on ET-1 and its receptors in patients with underlying chronic ischemia of the lower limb

Elevated plasma and tissue endothelin (ET)-1 levels in patients with critical limb ischemia (CLI) has been described. Here the effect of a period of acute ischemia and subsequent reperfusion on plasma ET-1 and tissue ET-1/ET receptors in skeletal muscle biopsies from CLI patients undergoing femoro-distal bypass surgery was studied. Peripheral and "local" blood and muscle biopsies were obtained from patients undergoing femoro-distal bypass surgery, at the start of the procedure (control), after a period of vascular clamping (ischemia), and after clamp release (reperfusion). Plasma ET-1 was determined by enzyme-linked immunosorbent assay. Tissue ET-1 was assessed by counting ET-1 immunostaining cells per unit area, and ET(A)/ET(B) receptors were identified on sections by in vitro autoradiography in which binding was quantitatively assessed by densitometry. There was no significant effect of ischemia or reperfusion on plasma ET-1 levels or on ET(A)/ET(B) receptor binding. However, tissue ET-1 increased during both acute ischemia and reperfusion (P < 0.05). A high proportion of positive ET-1 immunostaining was associated with microvessels and also exhibited a similar distribution to macrophages. Previously, it has been shown that both plasma ET-1 and tissue ET-1/ET receptors are increased in CLI patients compared with atherosclerotic controls. Also, increased muscle ET-1 levels have been described in acute ischemia caused by tourniquet application in nonischemic patients undergoing total knee replacement. In CLI patients, in whom ET-1 is already upregulated, this further increase may exacerbate existing pathologic processes and contribute to ischemia-reperfusion injury. ET-1 antagonists may therefore be useful adjuncts in CLI and other surgical procedures in which ischemia-reperfusion damage occurs.     

Stimuli of Sensory-Motor Nerves Terminate Arterial Contractile Effects of Endothelin-1 by CGRP and Dissociation of ET-1/ETA-Receptor Complexes

Background

Endothelin-1 (ET-1), a long-acting paracrine mediator, is implicated in cardiovascular diseases but clinical trials with ET-receptor antagonists were not successful in some areas. We tested whether the quasi-irreversible receptor-binding of ET-1 (i) limits reversing effects of the antagonists and (ii) can be selectively dissociated by an endogenous counterbalancing mechanism.

Methodology/Principal findings

In isolated rat mesenteric resistance arteries, ET_A-antagonists, endothelium-derived relaxing factors and synthetic vasodilators transiently reduced contractile effects of ET-1 but did not prevent persistent effects of the peptide. Stimuli of peri-vascular vasodilator sensory-motor nerves such as capsaicin not only reduced but also terminated long-lasting effects of ET-1. This was prevented by CGRP-receptor antagonists and was mimicked by exogenous calcitonin gene-related peptide (CGRP). Using 2-photon laser scanning microscopy in vital intact arteries, capsaicin and CGRP, but not ET_A-antagonism, were observed to promote dissociation of pre-existing ET-1/ET_A-receptor complexes.

Conclusions

Irreversible binding and activation of ET_A-receptors by ET-1 (i) occur at an antagonist-insensitive site of the receptor and (ii) are selectively terminated by endogenously released CGRP. Hence, natural stimuli of sensory-motor nerves that stimulate release of endogenous CGRP can be considered for therapy of diseases involving ET-1.     

Endothelin-1 and endothelin receptors in the basilar artery of the capybara

Little is known about cerebral vasculature of capybara, which seems may serve as a natural model of studying changes in cerebral circulation due to internal carotid artery atrophy at animal sexual maturation. This is the first study of the light- and electron-immunocytochemical localisation of endothelin-1 (ET-1) and ET_A and ET_B endothelin receptors in the basilar artery of capybaras (6 to 12-month-old females and males) using an ExtrAvidin detection method. All animals examined showed similar patterns of immunoreactivity. Immunoreactivity for ET-1 was detected in the endothelium and adventitial fibroblasts, whilst immunoreactivity for ET_A and ET_B receptors was present in the endothelium, vascular smooth muscle, perivascular nerves and fibroblasts. In endothelial cells immunoreactivity to ET-1 was pronounced in the cytoplasm or on the granular endoplasmic reticulum. Similar patterns of immunolabelling were observed for ET_A and ET_B receptors, though cytoplasmic location of clusters of immunoprecipitate seems dominant. These results suggest that the endothelin system is present throughout the wall of the basilar artery of capybara.     

Large Vessel Adventitial Vasculitis Characterizes Patients with Critical Lower Limb Ischemia with as Compared to without Human Immunodeficiency Virus Infection

Objectives

Whether a human immunodeficiency virus (HIV)-associated vasculitis in-part accounts for occlusive large artery disease remains uncertain. We aimed to identify the histopathological features that characterize large vessel changes in HIV sero-positive as compared to sero-negative patients with critical lower limb ischemia (CLI).

Materials and Methods

Femoral arteries obtained from 10 HIV positive and 10 HIV negative black African male patients admitted to a single vascular unit with CLI requiring above knee amputation were subjected to histopathological assessment. None of the HIV positive patients were receiving antiretroviral therapy.

Results

As compared to HIV negative patients with CLI, HIV positive patients were younger (p<0.01) and had a lower prevalence of hypertension (10 vs 90%, p<0.005) and diabetes mellitus (0 vs 50%, p<0.05), but a similar proportion of patients previously or currently smoked (80 vs 60%). 90% of HIV positive patients, but no HIV negative patient had evidence of adventitial leukocytoclastic vasculitis of the vasa vasorum (p<0.0001). In addition, 70% of HIV positive, but no HIV negative patient had evidence of adventitial slit-like vessels. Whilst T-lymphocytes were noted in the adventitia in 80% of HIV positive patients, T-lymphocytes were noted only in the intima in HIV negative patients. The presence of femoral artery calcified multilayered fibro-atheroma was noted in 40% of HIV positive and 90% of HIV negative patients with CLI.

Conclusions

An adventitial vasculitis which characterizes large artery changes in CLI in HIV-infected as compared to non-infected patients, may contribute toward HIV-associated occlusive large artery disease.     

An endothelin type A receptor-expressing cell to characterize endothelin-1 binding and screen antagonist

Endothelin-1 (ET-1) induces contraction of vascular smooth muscle through binding to endothelin type A receptor (ET_AR). COS-7 cells stably expressing high levels of the ET_AR were established (designated COS-7(ET_AR)). The COS-7(ET_AR) cell bound [¹²⁵I]ET-1 with a K_d of 932 ± 161 pM and a B_max of 74 ± 13 fmol/2 × 10⁵ cells. [¹²⁵I]ET-1 binding was inhibited by ET-1 and the ET_AR antagonist BQ-610, but not by the endothelin type B receptor (ET_BR) antagonist BQ-788. In clones expressing two ET_AR mutants containing D46N or R53Q substitutions in the first extracellular domain of the receptor, [¹²⁵I]ET-1 binding activity was dramatically reduced. This suggests that these single amino acid substitutions alter the three-dimensional structure of the ligand-binding domain of the ET_AR. Using COS-7(ET_AR) cell, we showed that Ca²⁺ or Mg²⁺ was essential for ET-1 binding to the ET_AR and that ET-1 treatment induced postreceptor signaling, that is, intracellular accumulation of cyclic AMP (cAMP) and Ca²⁺ mobilization. The COS-7(ET_AR) established in this study will be a useful tool for screening ET-1 antagonists for treating hypertension.     

Endothelin causes transactivation of the EGFR and HER2 in non-small cell lung cancer cells

Endothelin (ET)-1 is an important peptide in cancer progression stimulating cellular proliferation, tumor angiogenesis and metastasis. ET-1 binds with high affinity to the ET_A receptor (R) and ET_BR on cancer cells. High levels of tumor ET-1 and ET_AR are associated with poor survival of lung cancer patients. Here the effects of ET-1 on epidermal growth factor (EGF)R and HER2 transactivation were investigated using non-small cell lung cancer (NSCLC) cells. ET_AR mRNA was present in all 10 NSCLC cell lines examined. Addition of ET-1 to NCI-H838 or H1975 cells increased EGFR, HER2 and ERK tyrosine phosphorylation within 2 min. The increase in EGFR and HER2 transactivation caused by ET-1 addition to NSCLC cells was inhibited by lapatinib (EGFR and HER2 tyrosine kinase inhibitor (TKI)), gefitinib (EGFR TKI), ZD4054 or BQ-123 (ET_AR antagonist), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor) or Tiron (superoxide scavenger). ET-1 addition to NSCLC cells increased cytosolic Ca²⁺ and reactive oxygen species. ET-1 increased NSCLC clonal growth, whereas BQ123, ZD4054, lapatinib or gefitinib inhibited proliferation. The results indicate that ET-1 may regulate NSCLC cellular proliferation in an EGFR- and HER2-dependent manner.     

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.     

Effects of Cyclic Intermittent Hypoxia on ET-1 Responsiveness and Endothelial Dysfunction of Pulmonary Arteries in Rats

Obstructive sleep apnoea (OSA) is a risk factor for cardiovascular disorders and in some cases is complication of pulmonary hypertension. We simulated OSA by exposing rats to cyclic intermittent hypoxia (CIH) to investigate its effect on pulmonary vascular endothelial dysfunction. Sprague-Dawley Rats were exposed to CIH (FiO₂ 9% for 1 min, repeated every 2 min for 8 h/day, 7 days/wk for 3 wk), and the pulmonary arteries of normoxia and CIH treated rats were analyzed for expression of endothelin-1 (ET-1) and ET receptors by histological, immunohistochemical, RT-PCR and Western Blot analyses, as well as for contractility in response to ET-1. In the pulmonary arteries, ET-1 expression was increased, and ET-1 more potently elicited constriction of the pulmonary artery in CIH rats than in normoxic rats. Exposure to CIH induced marked endothelial cell damage associated with a functional decrease of endothelium-dependent vasodilatation in the pulmonary artery. Compared with normoxic rats, ET_A receptor expression was increased in smooth muscle cells of the CIH rats, while the expression of ET_B receptors was decreased in endothelial cells. These results demonstrated endothelium-dependent vasodilation was impaired and the vasoconstrictor responsiveness increased by CIH. The increased responsiveness to ET-1 induced by intermittent hypoxia in pulmonary arteries of rats was due to increased expression of ET_A receptors predominantly, meanwhile, decreased expression of ET_B receptors in the endothelium may also participate in it.     

Endothelin-1 induces the expression of C-reactive protein in rat vascular smooth muscle cells

Numerous studies have shown that both vasoconstrictive peptide endothelin-1 (ET-1) and inflammatory marker C-reactive protein (CRP) are implicated in the inflammatory process of atherosclerosis. The purpose of the present study was to observe effect of ET-1 on CRP production and the molecular mechanisms in rat vascular smooth muscle cells (VSMCs). The results showed that ET-1 was capable of stimulating VSMCs to produce CRP both in protein and in mRNA levels in vitro and in vivo. ET_A receptor antagonist BQ123, but not ET_B receptor antagonist BQ788, inhibited CRP production in VSMCs. In addition, ET-1 was able to elicit reactive oxygen species (ROS) generation and mitogen-activated protein kinase (MAPK) activation, and antioxidant pyrrolidine dithiocarbamate and p38MAPK inhibitor SB203580 inhibited ET-1-induced CRP expression. The results demonstrate that ET-1 induces CPR production in VSMCs via ET_A receptor followed by ROS and MAPK signal pathway, which may contribute to better understanding of the role of ET-1 in inflammatory activation of the vessel wall during atherogenesis.     

Plasma endothelin-1 levels and albumin excretion rate in normotensive,microalbuminuric type 2 diabetic patients

BACKGROUND: Endothelin-1 (ET-1) is able to determine functional and structural renal alterations and plasma levels of this vasoconstrictor peptide are increased in diabetic patients. In a selected group of type 2 normotensive diabetic patients with microalbuminuria, we investigated circulating ET-1 levels compared to a control group and verified whether there is a relationship between ET-1 levels and albumin excretion rate in diabetics. SUBJECTS AND METHODS: Thirty-two microalbuminuric type 2 diabetic patients (12 males and 20 females; mean age 57 +/- 8 years) without hypertension, renal failure, hypercholesterolemia or atherosclerotic damage were selected. The control group was made up of 28 healthy subjects matched for sex and age. Blood pressure, creatinine clearance, serum cholesterol and plasma ET-1 values were determined in diabetic and control group. In diabetic patients, glycosilated hemoglobin and urinary albumin excretion rate were also assayed. Mean ET-1 values in diabetics and controls were compared using Student's t-test. Linear regression test was done to relate two variables. Statistical significance was set at p<0.05. RESULTS: Mean ET-1 values were significantly higher in the diabetic group than in controls (11.77 +/- 1.16 pg/ml vs 8.9 +/- 2.1 pg/ml; p<0.05). No relationship (p>0.05) was found between circulating ET-1 and blood pressure, creatinine clearance, serum cholesterol and metabolic control in diabetics. There was a significant positive correlation (r=0.403; p=0.03) between plasma ET-1 levels and albumin excretion rate in diabetic patients. CONCLUSIONS: Our results showed that circulating ET-1 values were increased in microalbuminuric, normotensive, type 2 diabetic patients and correlated with albumin excretion rate. These findings confirm that endothelial dysfunction, as expressed by ET-1 levels, occurs early in these patients and support the hypothesis of a potential role for this peptide in development of microalbuminuria in diabetic nephropathy.     

Jab1 regulates levels of endothelin type A and B receptors by promoting ubiquitination and degradation

Endothelin type A receptor (ET_AR) plays an important role in some cardiovascular disorders where ET_AR levels are increased. However, regulatory mechanisms for ET_AR levels are unknown. Here, we identified Jun activation domain-binding protein 1 (Jab1) as an ET_AR-interacting protein by yeast two-hybrid screening of human heart cDNA library using carboxyl terminal tail (C-tail) of ET_AR as a bait. The interaction was confirmed by glutathione S-transferase pull-down assay, co-immunoprecipitation in HEK293T cells expressing ET_AR-myc and FLAG-Jab1, and confocal microscopy. Jab1 knockdown increased whole cell and cell surface levels of ET_AR and ET-1-induced ERK1/2 phosphorylation in HEK293T cells expressing ET_AR, whereas Jab1 overexpression decreased them. Jab1 overexpression accelerated disappearance rate of ET_AR after protein synthesis inhibition as an index of a degradation rate. ET_AR was constitutively ubiquitinated, and the level of ubiquitination was enhanced by Jab1 overexpression. Long-term ET-1 stimulation markedly accelerated the rate of ET_AR degradation and increased the amount of Jab1 bound to ET_AR with a maximal level of 500% at 3 h. In the absence of ET-1 stimulation, the level of ET_BR was lower than that of ET_AR and the degradation rate of ET_BR was markedly faster than that of ET_AR. Notably, the amount of Jab1 bound to ET_BR and ubiquitination level of ET_BR were markedly higher than those for ET_AR. Taken together, these results suggest that the amount of Jab1 bound to ETR regulates the degradation rate of ET_AR and ET_BR by modulating ubiquitination of these receptors, leading to changes in ET_AR and ET_BR levels.     

NLRP3 activation contributes to endothelin-1-induced erectile dysfunction

In the present study, we hypothesized that endothelin (ET) receptors (ET_A and ET_B) stimulation, through increased calcium and ROS formation, leads to Nucleotide Oligomerization Domain-Like Receptor Family, Pyrin Domain Containing 3 (NLRP3) activation. Intracavernosal pressure (ICP/MAP) was measured in C57BL/6 (WT) mice. Functional and immunoblotting assays were performed in corpora cavernosa (CC) strips from WT, NLRP3^−/− and caspase^−/− mice in the presence of ET-1 (100 nM) and vehicle, MCC950, tiron, BAPTA AM, BQ123, or BQ788. ET-1 reduced the ICP/MAP in WT mice, and MCC950 prevented the ET-1 effect. ET-1 decreased CC ACh-, sodium nitroprusside (SNP)-induced relaxation, and increased caspase-1 expression. BQ123 an ET_A receptor antagonist reversed the effect. The ET_B receptor antagonist BQ788 also reversed ET-1 inhibition of ACh and SNP relaxation. Additionally, tiron, BAPTA AM, and NLRP3 genetic deletion prevented the ET-1-induced loss of ACh and SNP relaxation. Moreover, BQ123 diminished CC caspase-1 expression, while BQ788 increased caspase-1 and IL-1β levels in a concentration-dependent manner (100 nM–10 μM). Furthermore, tiron and BAPTA AM prevented ET-1-induced increase in caspase-1. In addition, BAPTA AM blocked ET-1-induced ROS generation. In conclusion, ET-1-induced erectile dysfunction depends on ET_A- and ET_B-mediated activation of NLRP3 in mouse CC via Ca²⁺-dependent ROS generation.     

Pulmonary clearance of circulating endothelin-1 in dogs in vivo: exclusive role of ETB receptors

Dupuis, Jocelyn, Carl A. Goresky, and Alain Fournier.Pulmonary clearance of circulating endothelin-1 in dogs in vivo: exclusive role of ET_B receptors.J. Appl. Physiol. 81(4):1510-1515, 1996.The pulmonary circulation plays an importantrole in the removal of circulating endothelin-1 (ET-1). Plasma ET-1levels are increased in pulmonary hypertensive states of variousetiologies (e.g., idiopathic, heart failure, and congenital anomalies)in proportion to the severity of pulmonary hypertension. It is possible that reduced pulmonary clearance of this peptide contributes to thehyperendothelinemia of those pathologies. TheET_A andET_B receptors are abundant in lungtissues: on the vascular endothelium, theET_B receptor is predominant andmay contribute to ET-1 extraction through receptor-mediatedendocytosis. We designed experiments to determine and quantify theimportance of the ET_A andET_B receptors in the pulmonaryextraction of circulating ET-1 in anesthetized dogs. The single-passcumulative tracer ET-1 extraction by the lung was measured with theindicator-dilution technique before and 5 min after intrapulmonaryinjection of the specific ET_Aantagonist BQ-123 (n = 5, 120-960nmol) and the specific ET_Bantagonist BQ-788 (n = 6, 1,000 nmol).The inhibitors had no significant effect on pulmonary and systemichemodynamics. Mean cumulative pulmonary ET-1 extraction was notmodified by BQ-123 [control (C): 36 ± 4%, antagonist (A): 34 ± 6%] but was completely abolished by BQ-788 (C: 34 ± 6%, A: 0 ± 2%, P < 0.001). Thepulmonary rate constant (K) for ET-1removal was also unaffected by BQ-123 (C: 0.050 ± 0.0085 s¹, A: 0.047 ± 0.012 s¹) but significantlydecreased and became close to zero after BQ-788 (C: 0.058 ± 0.014 s¹, A: 0.009 ± 0.007 s¹,P < 0.001). We conclude that theET_B receptor is completely andexclusively responsible for pulmonary ET-1 removal in vivo. Futurestudies are needed to show whether desensitization or downregulation ofthe ET_B receptor may contribute tothe increase in circulating ET-1 levels in conditions associated withpulmonary hypertension. This novel pulmonary endothelial cell functionmay play a protective role by modulating circulating ET-1 levels in thesystemic circulation.

     

Effects of endothelin family on ANP secretion

The endothelins (ET) peptide family consists of ET-1, ET-2, ET-3, and sarafotoxin (s6C, a snake venom) and their actions appears to be different among isoforms. The aim of this study was to compare the secretagogue effect of ET-1 on atrial natriuretic peptide (ANP) secretion with ET-3 and evaluate its physiological meaning. Isolated nonbeating atria from male Sprague-Dawley rats were used to evaluate stretch-activated ANP secretion in response to ET-1, ET-2, ET-3, and s6C. Changes in mean blood pressure (MAP) were measured during acute injection of ET-1 and ET-3 with and without natriuretic peptide receptor-A antagonist (A71915) in anesthetized rats. Changes in atrial volume induced by increased atrial pressure from o to 1, 2, 4, or 6 cm H₂O caused proportional increases in mechanically-stimulated extracellular fluid (ECF) translocation and stretch-activated ANP secretion. ET-1 (10 nM) augmented basal and stretch-activated ANP secretion in terms of ECF translocation, which was blocked by the pretreatment with ET_A receptor antagonist (BQ123, 1 μM) but not by ET_B receptor antagonist (BQ788, 1 μM). ET_A receptor antagonist itself suppressed stretch-activated ANP secretion. As compared to ET-1- induced ANP secretion (3.2-fold by 10 nM), the secretagogue effects of ANP secretion by ET-2 was similar (2.8-fold by 10 nM) and ET-3 and s6C were less potent (1.7-fold and 1.5-fold by 100 nM, respectively). Acute injection of ET-1 or ET-3 increased mean blood pressure (MAP), which was augmented in the presence of natriuretic peptide receptor-A antagonist. Therefore, we suggest that the order of secretagogue effect of ET family on ANP secretion was ET-1 ≥ ET-2 >> ET-3 > s6C and ET-1-induced ANP secretion negatively regulates the pressor effect of ET-1.     

Angiotensin II enhances endothelin-1-induced vasoconstriction through upregulating endothelin type A receptor

Endothelin-1 (ET-1) is the most potent vasoconstrictor by binding to endothelin receptors (ET_AR) in vascular smooth muscle cells (VSMCs). The complex of angiotensin II (Ang II) and Ang II type one receptor (AT₁R) acts as a transient constrictor of VSMCs. The synergistic effect of ET-1 and Ang II on blood pressure has been observed in rats; however, the underlying mechanism remains unclear. We hypothesize that Ang II leads to enhancing ET-1-mediated vasoconstriction through the activation of endothelin receptor in VSMCs. The ET-1-induced vasoconstriction, ET-1 binding, and endothelin receptor expression were explored in the isolated endothelium-denuded aortae and A-10 VSMCs. Ang II pretreatment enhanced ET-1-induced vasoconstriction and ET-1 binding to the aorta. Ang II enhanced ET_AR expression, but not ET_BR, in aorta and increased ET-1 binding, mainly to ET_AR in A-10 VSMCs. Moreover, Ang II-enhanced ET_AR expression was blunted and ET-1 binding was reduced by AT₁R antagonism or by inhibitors of PKC or ERK individually. In conclusion, Ang II enhances ET-1-induced vasoconstriction by upregulating ET_AR expression and ET-1/ET_AR binding, which may be because of the AngII/Ang II receptor pathways and the activation of PKC or ERK. These findings suggest the synergistic effect of Ang II and ET-1 on the pathogenic development of hypertension.     

Endothelin-1 expression in vascular adventitial fibroblasts

Endothelial cells are a major source of endothelin (ET)-1, but the possibility that vascular adventitial fibroblasts generate ET-1 has not been explored. We hypothesized that aortic adventitial fibroblasts have the ability to produce ET-1, which may contribute to extracellular matrix synthesis. Vascular adventitial fibroblasts were isolated from mouse aorta and incubated with various concentrations of angiotensin II (ANG II). mRNA levels of preproET-1 and type I procollagen were detected with relative RT-PCR. ET-1 levels in culture medium were measured with ELISA. Protein levels of procollagen were detected with Western blotting. ANG II (10 and 100 nM, 1 microM) induced a time- and concentration-dependent increase in preproET-1 mRNA levels (P < 0.05). Induction of preproET-1 mRNA was accompanied by release of immunoreactive peptide ET-1 (P < 0.05). ANG II-evoked increases in preproET-1 mRNA expression and ET-1 release were blocked by losartan (100 microM), an AT1 receptor antagonist, but not PD-123319 (100 microM), an AT2 receptor antagonist. To further confirm our findings, we cloned and then sequenced vascular fibroblast preproET-1 bidirectionally with T7 and M13 reverse sequencing primers. Their nucleotide sequences were identical to preproET-1 cDNA from mouse vascular endothelial cells (accession no. AB081657). Moreover, ANG II-induced type I procollagen mRNA and protein expression were inhibited by BQ-123 (10 microM), an ET(A) receptor inhibitor, but not BQ-788 (10 microM), an ET(B) receptor inhibitor, suggesting a significant role of adventitial ET-1 in regulation of extracellular matrix synthesis. The results demonstrate that vascular adventitial fibroblasts are able to synthesize and release ET-1 in response to ANG II.     

Improvement in cardiac function of diabetic rats by bosentan is not associated with changes in the activation of PKC isoforms

We previously demonstrated that chronic treatment with the mixed endothelin A and B (ET_A and ET_B) receptor blocker bosentan improved isolated working heart function in streptozotocin (STZ) diabetic rats. Endothelin-1 (ET-1) peptide levels, ET-1 mRNA and ET_A and ET_B receptor mRNA were all increased in diabetic hearts, but were unaffected by bosentan treatment, indicating that the beneficial effects of bosentan on heart appear to be on downstream effectors of ET-1 and ET receptors rather than the ET-1 system itself. Stimulation of ET-1 receptors leads to increased activation of protein kinase C (PKC), which is associated with PKC translocation from the cytosol to the membrane. Persistent activation of specific PKC isoforms has been proposed to contribute to diabetic cardiomyopathy. The purpose of this study was to determine whether chronic treatment with bosentan influences the activation of PKC isoforms in hearts from diabetic rats. Male Wistar rats were divided into four groups: control, bosentan-treated control, diabetic, and bosentan-treated diabetic. Diabetes was induced by the intravenous injection of 60 mg/kg streptozotocin. One week later, treatment with bosentan (100 mg/kg/day) by oral gavage was begun and continued for 10 weeks. The heart was then removed, homogenized, separated into soluble (cytosolic) and particulate (membrane) fractions and PKC isoform content in each fraction was determined by Western blotting. PKC α, β2, δ, ε and ζ were all detected in hearts from both control and diabetic rats. However, no change in the levels or distribution between the soluble and particulate fractions of any of these isoforms could be detected in chronic diabetic hearts compared to control, whether untreated or treated with bosentan. These observations indicate that bosentan does not improve cardiac performance in STZ diabetic rats by affecting the activation of PKC isoforms.     

Role of ERK/MAPK in endothelin receptor signaling in human aortic smooth muscle cells

Background  

Endothelin-1 (ET-1) is a potent vasoactive peptide, which induces vasoconstriction and proliferation in vascular smooth muscle cells (VSMCs) through activation of endothelin type A (ET_A) and type B (ET_B) receptors. The extracellular signal-regulated kinase 1 and 2 (ERK1/2) mitogen-activated protein kinases (MAPK) are involved in ET-1-induced VSMC contraction and proliferation. This study was designed to investigate the ET_A and ET_B receptor intracellular signaling in human VSMCs and used phosphorylation (activation) of ERK1/2 as a functional signal molecule for endothelin receptor activity.     

Endothelin-1 stimulates cyclin D1 expression in rat cultured astrocytes via activation of Sp1

Endothelins (ETs), a family of vasoconstrictor peptides, are up-regulated in several pathological conditions in the brain, and induce astrocytic proliferation. We previously observed that ET-1 increased the expression of cyclin D1 protein. Thus, we confirmed the intracellular up-regulation of cyclin D1 by ET-1 in rat cultured astrocytes. Real-time PCR analysis indicated that ET-1 (100 nM) and Ala^1,3,11,15-ET-1 (100 nM), a selective agonist of the ET_B receptor, induced a time-dependent and transient increase in cyclin D1 mRNA. The effect of ET-1 was diminished by an ET_B antagonist (1 μM BQ788) or inhibitors of Sp1 (500 nM mithramycin), ERK (50 μM PD98059), p38 (20 μM SB203580) and JNK (1 μM SP600125), but not inhibitors of NF-κB (10 μM SN50 and 100 μM pyrrolidine dithiocarbamate). The binding assay for Sp1 indicated that ET-1 increased the binding activity of Sp1 to consensus sequences, and two oligonucleotides of the cyclin D1 promoter including the Sp1-binding sites diminished the effect of ET-1. Western blot analysis showed that ET-1 induced time-dependent and transient phosphorylation of Sp1 on Thr453 and Thr739 via the ET_B receptor. ET-1-induced phosphorylation of Sp1 was attenuated by PD98059 and SP600125. Additionally, ET-1 increased the incorporation of bromodeoxyuridine (BrdU) in cultured astrocytes and the number of BrdU-positive cells decreased in the presence of PD98059, SP600125 and mithramycin. These results suggest that ET-1 increases the expression of cyclin D1 via activation of Sp1 and induces astrocytic proliferation.