Endothelin-1 Protein Levels Research Articles

Endothelin(A) receptor blockade reduces ischemia/reperfusion injury in pig pancreas transplantation.

The effect of prophylactic administration of a selective endothelin(A) receptor antagonist (ET(A)-RA) on ischemia/reperfusion injury in an experimental model of graft pancreatitis after pancreas transplantation was evaluated. It is well established that endothelin-1 (ET-1), a powerful vasoconstrictor, plays an important role in the development of pancreatitis. Recent studies have shown a beneficial effect of endothelin receptor antagonists in the therapy for experimental pancreatitis. Relevant ischemia/reperfusion injury was induced in pig pancreas transplants after 6 hours hypothermic preservation in University of Wisconsin solution. The recipients were randomized into 2 groups: control pigs received isotonic saline and the treated group received the selective ET(A)-RA BSF 208075 at the beginning of reperfusion. On postoperative days 2 and 5, animals were relaparotomized to obtain tissue specimens. Blood monitoring included lipase, amylase, C-reactive protein, trypsinogen-activation peptide, thiobarbituric acid-reacting substances, and ET-1. Partial oxygen tension (p(ti)O(2)) was measured by a Clarke-type electrode and blood flow by laser doppler. A semiquantitative score index was used for assessment of histologic injury and for immunohistochemical analysis of ET-1 and ET(A) receptor expression. Tissue mRNA levels of prepro ET-1, ET(A) receptor, pro-interleukin (IL)-6, and pro-IL-1beta were quantified using TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR). Prophylactic treatment with ET(A)-RA significantly reduced the severity of graft pancreatitis evidenced by C-reactive protein. The finding of transient capillary perfusion at the beginning of reperfusion supports the application of the ET(A)-RA during this period. The dramatic increase of plasma ET-1 in the therapy group is a clear evidence of effective receptor blockade. Mean trypsinogen-activation peptide levels from the portal venous effluent, but not mean systemic plasma TAP values were significantly lower in the treated group. Analysis of p(ti)O(2) and blood flow revealed a significant improvement of capillary perfusion and blood flow in the treated group and was associated with relevant reduction of tissue injury. Intrapancreatic ET-1 and IL-6 mRNA expression and ET-1 protein levels were significantly lower in the therapy group as compared with the control group. In contrast, ET(A) mRNA showed a marked up-regulation by ET(A) receptor blockade. Application of a ET(A)-RA reduces ischemia/reperfusion induced graft pancreatitis in a pig transplantation model by improving microcirculation and reducing tissue injury.

Antenatal dexamethasone enhances endothelin-1 synthesis and gene expression in the heart in congenital diaphragmatic hernia in rats

Background/Purpose: Although high levels of endothelin-1 (ET-1) in plasma may be relevant in certain pathophysiologic states, such as pulmonary hypertension accompanying congenital diaphragmatic hernia (CDH), experimental evidence favors a local, paracrine, or autocrine role for ET-1 in most tissues. Evidence of ET-1 production has been documented in fetal heart tissue where it exerts growth-enhancing and mitogenic effects. ET-1 also has a potent positive inotrope action on cardiac muscle. ET-1 −/− homozygous mice display a wide variety of cardiac anomalies, which also are features of the human and of the experimental CDH. Autopsy reports have shown that total heart weight is reduced significantly in the presence of CDH, and animal models have documented the presence of cardiac hypoplasia associated with CDH. Experimental and clinical studies have shown that prenatal exposure to corticosteroids improves cardiovascular function in the immediate newborn period. The aim of this study was to determine cardiac gene expression of ET-1 and of its receptor ETA and the cardiac ET-1 content in the heart of nitrofen-induced CDH in rats and to evaluate the effect of antenatal Dexamethasone (Dex) treatment. Methods: A CDH model was induced in pregnant rats after administration of 100 mg of nitrofen on day 9.5 of gestation (term, 22 days). Dex (0.25 mg/kg) was given by intraperitoneal injection on days 18.5 and 19.5 of gestation. Cesarean section was performed on day 21 of gestation. The fetuses were divided into 3 groups: group I, control (n = 8); group II, nitrofen-induced CDH (n = 8); group III, nitrofen-induced CDH with antenatal Dex treatment (n = 8). ET-1 protein was measured using ELISA. RT-PCR was performed to evaluate the relative amount of ET-1 and ETA mRNA expression. Results: There was a reduction in ET-1 mRNA (P <.05) and in ETA mRNA (P <.01) in the heart of CDH group compared with controls. ET-1 protein level also was reduced in heart of CDH compared with controls. Antenatal Dex treatment increased significantly both ET-1 mRNA and protein levels in the heart of CDH animals (P <.05 and P <.01, respectively). Conclusions: The reduced cardiac ET-1 gene expression and ET-1 synthesis may be responsible for the heart hypoplasia associated with CDH. Prenatal corticosteroids increase the cardiac production of ET-1, and this may enhance heart growth and cardiac inotropism at birth. J Pediatr Surg 37:1563-1567. Copyright 2002, Elsevier Science (USA). All rights reserved.

Chronic ET(A) receptor blockade prevents endothelial dysfunction of small arteries in apolipoprotein E-deficient mice.

This study investigated whether endothelial dysfunction occurs in mesenteric arteries of apoE-deficient mice and determined the role of endothelin (ET)-1, which is increased in human atherosclerosis, using an orally active endothelin ET(A) receptor antagonist. ApoE-deficient and C57BL/6J control mice were fed for 30 weeks with normal chow or high-fat Western-type diet alone or in combination with darusentan (LU135252; 50 mg/kg/day). Vasomotor reactivity of isolated small mesenteric arteries (I.D. 200-250 microm) was studied in vitro under perfused and pressurized conditions. In both mouse strains, about one fourth of the endothelium-dependent relaxant response to acetylcholine was insensitive to inhibition by L-NAME and indomethacin. In mesenteric arteries of apoE-deficient mice on Western-type diet, increased intima-media thickness and levels of endothelin-1 protein were observed. In addition, NO-mediated endothelium-dependent relaxation to acetylcholine was reduced without affecting L-NAME/indomethacin insensitive relaxation and contractions to endothelin-1 and serotonin were enhanced. Treatment with darusentan normalized vascular structure, NO-mediated relaxation to acetylcholine and contractions to endothelin-1 and serotonin without affecting blood pressure or plasma cholesterol levels. Severe hypercholesterolemia in apoE-deficient mice is associated with attenuation of NO-mediated relaxation to acetylcholine and increased vascular endothelin-1 content. Chronic ET(A) receptor blockade may provide a new therapeutic approach to improve NO-mediated endothelium-dependent vasomotion in small arteries.

The role of endothelin-1 in strain-related susceptibility to develop hypoxic pulmonary hypertension in rats

The Hilltop (H) strain compared to the Madison (M) strain of Sprague–Dawley rats develops severe pulmonary hypertension in response to chronic hypoxia. We tested the hypothesis that endothelin-1 (ET-1) contributes to these strain-related differences. Plasma ET-1 content was not modified by chronic hypoxia in either strain. The lung ET-1 peptide and preproET-1 mRNA content were significantly increased to the same magnitude in both strains at 2 and 3 weeks of hypoxia. The ET A receptor mRNA increased more at 3 weeks of hypoxia in the lungs of H rats than in M rats, but not at other time points. The ET B receptor mRNA was not modified by hypoxia in either strain. After 3 days of normoxic recovery following 2 weeks of hypoxia, ET-1 protein and mRNA levels decreased to baseline levels in both rat strains. We conclude that ET-1 does not contribute to the development of cardiopulmonary differences between the H and M strains in response to hypoxia.

Expression and activity of pulmonary endothelin converting enzyme in heart failure: Relation to endothelin biosynthesis and receptor distribution

Background: Although reduced pulmonary clearance of endothelin-1 (ET-1) has been suggested to contribute to increased circulating levels in congestive heart failure (CHF), the regulation of the pulmonary ET system with CHF remains to be defined. Accordingly, the aim of the present study is to investigate the expression and activity of the ET system with the development of CHF. Methods and Results: Pulmonary tissue samples were collected from pigs with pacing CHF (240 bpm, 3 wks, n = 10) and controls (n = 10). The pulmonary messenger RNA (mRNA) and protein levels of endothelin converting enzyme-1 (ECE-1) subisoforms, ET-1, and ET receptor profiles were determined. The gene expression of ET-1 precursor, ECE-1a, and ETA was upregulated 4-, 3-, and 2-fold, respectively, with CHF. Pulmonary tissue ET-1 was increased to 13 ± 2 fmol/mg protein from control values of 5 ± 1 fmol/mg protein (P <.05), and ECE-1 activity was augmented from 3,264 ± 665 fmol/mg protein in control animals to 14,073 ± 654 fmol/mg protein per hour in CHF animals (P <.05). The ETB receptor density decreased, whereas ETA receptors were increased in CHF, indicating a shift in the ETA to ETB ratio. Conclusions: Both the increased synthesis and the decreased clearance of ET-1 via ETB receptors may contribute to the increased systemic and pulmonary ET-1 levels in CHF.

Aldosterone Receptor Antagonism Normalizes Vascular Function in 11β-Hydroxysteroid Dehydrogenase Deficient Hypertension

76 Background: The enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD2) provides mineralocorticoid receptor specificity for aldosterone by metabolising glucocorticoids to their receptor inactive 11-dehydro derivatives. The present study investigated the effect of the aldosterone receptor antagonist spironolactone on endothelial function in liquorrhice induced hypertension. Methods: Glycyrrhizic acid (GA), a recognised inhibitor of 11β-HSD2 was supplemented to the drinking water (3g/L) of Wistar Kyoto rats over a period of 21 days. From day 8 to 21 spirolonoactone (5.8±0.6 mg/kg/d) or placebo was added to chow (n=7/group). Endothelium-dependent and -independent vascular function was assessed as relaxation of preconstricted aortic rings to acetylcholine (10 -10 -10 -5 mol/L) or sodium nitroprusside (10 -10 -10 -5 mol/L). Furthermore aortic eNOS protein content, nitrate tissue levels, endothelin-1 (ET-1) protein levels were determined. Results: GA application increased SBP to 185±9 mmHg vs 142±8 mmHg in control animals, p&lt;0.01). In the GA group endothelium-dependent relaxation was impaired as compared to controls (73±6% vs 99±5% of norepinephrine 3x10 -7 mol/L), whereas endothelium independent relaxation remained unchanged. In the aorta of 11β-HSD2 deficient rats, eNOS protein content and nitrate tissue levels (1114±128 vs 518±77μg/g protein, p&lt;0.05) decreased. In contrast, aortic endothelin-1 (ET-1) protein levels were enhanced by GA. (308±38 vs 497±47 pg/mg tissue, p&lt;0.05). Treatment with spironolactone normalized blood pressure in animals on GA (142±9 mmHg vs 189±8 mmHg in the placebo group; p&lt;0.01) and restored endothelium-dependent relaxation (96±3%, p&lt;0.01 vs placebo). Spironolactone furthermore blunted the decrease in vascular eNOS protein content and nitrate tissue levels as well as the elevation of ET-1 protein levels. Conclusion: In 11β-HSD2-deficient hypertension, aldosterone receptor antagonism normalizes blood pressure, prevents up-regulation of vascular ET-1 and restores NO-mediated endothelial dysfunction and therefore may advance as a novel therapeutic approach.

Aldosterone Receptor Antagonism Normalizes Vascular Function in 11β-Hydroxysteroid Dehydrogenase Deficient Hypertension

76 Background: The enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD2) provides mineralocorticoid receptor specificity for aldosterone by metabolising glucocorticoids to their receptor inactive 11-dehydro derivatives. The present study investigated the effect of the aldosterone receptor antagonist spironolactone on endothelial function in liquorrhice induced hypertension. Methods: Glycyrrhizic acid (GA), a recognised inhibitor of 11β-HSD2 was supplemented to the drinking water (3g/L) of Wistar Kyoto rats over a period of 21 days. From day 8 to 21 spirolonoactone (5.8±0.6 mg/kg/d) or placebo was added to chow (n=7/group). Endothelium-dependent and -independent vascular function was assessed as relaxation of preconstricted aortic rings to acetylcholine (10-10-10-5 mol/L) or sodium nitroprusside (10-10-10-5 mol/L). Furthermore aortic eNOS protein content, nitrate tissue levels, endothelin-1 (ET-1) protein levels were determined. Results: GA application increased SBP to 185±9 mmHg vs 142±8 mmHg in control animals, p<0.01). In the GA group endothelium-dependent relaxation was impaired as compared to controls (73±6% vs 99±5% of norepinephrine 3x10-7 mol/L), whereas endothelium independent relaxation remained unchanged. In the aorta of 11β-HSD2 deficient rats, eNOS protein content and nitrate tissue levels (1114±128 vs 518±77μg/g protein, p<0.05) decreased. In contrast, aortic endothelin-1 (ET-1) protein levels were enhanced by GA. (308±38 vs 497±47 pg/mg tissue, p<0.05). Treatment with spironolactone normalized blood pressure in animals on GA (142±9 mmHg vs 189±8 mmHg in the placebo group; p<0.01) and restored endothelium-dependent relaxation (96±3%, p<0.01 vs placebo). Spironolactone furthermore blunted the decrease in vascular eNOS protein content and nitrate tissue levels as well as the elevation of ET-1 protein levels. Conclusion: In 11β-HSD2-deficient hypertension, aldosterone receptor antagonism normalizes blood pressure, prevents up-regulation of vascular ET-1 and restores NO-mediated endothelial dysfunction and therefore may advance as a novel therapeutic approach.

Diagnosis and Rectification of Stuck and Sluggish Fermentations

The presence of the HLA-B35 allele has emerged as an important risk factor for the development of isolated pulmonary hypertension in patients with scleroderma, however the mechanisms underlying this association have not been fully elucidated. The goal of our study was to determine the molecular mechanisms that mediate the biological effects of HLA-B35 in endothelial cells (ECs). Our data demonstrate that HLA-B35 expression at physiological levels via adenoviral vector resulted in significantly increased endothelin-1 (ET-1) and a significantly decreased endothelial NO synthase (eNOS), mRNA, and protein levels. Furthermore, HLA-B35 greatly upregulated expression of chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induces the endoplasmic reticulum (ER) stress and unfolded protein response in ECs. Examination of selected mediators of the unfolded protein response, including H chain binding protein (BiP; GRP78), C/Ebp homologous protein (CHOP; GADD153), endoplasmic reticulum oxidase, and protein disulfide isomerase has revealed a consistent increase of BiP expression levels. Accordingly, thapsigargin, a known ER stress inducer, stimulated ET-1 mRNA and protein levels in ECs. This study suggests that HLA-B35 could contribute to EC dysfunction via ER stress-mediated induction of ET-1 in patients with pulmonary hypertension.

Inverse regulation of endothelin-1 and nitric oxide metabolites in tissue with aging: implications for the age-dependent increase of cardiorenal disease.

Aging is an independent risk factor for cardiovascular and renal disease. The study reported here investigated whether aging affects endothelin-1 (ET-1) and tissue levels of the nitric oxide metabolites nitrite/nitrate in the kidney of rodents. Blood pressure was measured by the tail-cuff method, ET-1 protein was determined by radioimmunoassay/high-performance liquid chromatography (RIA/HPLC) and nitrite/nitrate was measured by ion-pairing chromatography. Compared to young male Wistar Kyoto rats (3 months of age), renal ET-1 protein levels in whole kidneys increased 3.6-fold at 24 months of age (from 70 +/- 9 to 253 +/- 43 pg/g tissue, p < 0.05, n = 6 each group). Similarly, renal ET-1 protein increased 1.7-fold in 18-month-old C57BL/6J mice as compared to 8-month-old adult animals (from 188 +/- 18 to 319 +/- 14 pg/g tissue, p < 0.05, n = 5-7). In female RoRo-Wistar rats (6, 18 and 33 months of age), tissue nitrite/nitrate levels in whole kidneys decreased with increasing age (from 232 +/- 25 to 130 +/- 6 micromol/l/g tissue, p < 0.05). Thus, aging in healthy rodents is associated with a marked upregulation of renal ET-1 protein content and a decrease in tissue nitrite/nitrate levels in whole kidneys, independent of blood pressure. Activation of the ET pathway with aging may promote the development of age-dependent diseases such as glomerulosclerosis, hypertension and atherosclerosis.

Inverse Regulation of Endothelin-1 and Nitric Oxide Metabolites in Tissue with Aging

Summary: Aging is an independent risk factor for cardiovascular and renal disease. The study reported here investigated whether aging affects endothelin-1 (ET-1) and tissue levels of the nitric oxide metabolites nitrite/nitrate in the kidney of rodents. Blood pressure was measured by the tail-cuff method, ET-1 protein was determined by radioimmunoassay/high-performance liquid chromatography (RIA/HPLC) and nitrite/nitrate was measured by ionpairing chromatography. Compared to young male Wistar Kyoto rats (3 months of age), renal ET-1 protein levels in whole kidneys increased 3.6-fold at 24 months of age (from 70 ± 9 to 253 ± 43 pg/g tissue, p < 0.05, n = 6 each group). Similarly, renal ET-1 protein increased 1.7-fold in 18-month-old C57BL/6J mice as compared to 8-month-old adult animals (from 188 ± 18 to 319 ± 14 pg/g tissue, p < 0.05, n = 5-7). In female RoRo-Wistar rats (6, 18 and 33 months of age), tissue nitrite/nitrate levels in whole kidneys decreased with increasing age (from 232 ± 25 to 130 ± 6 μmol/l/g tissue, p < 0.05). Thus, aging in healthy rodents is associated with a marked upregulation of renal ET-1 protein content and a decrease in tissue nitrite/nitrate levels in whole kidneys, independent of blood pressure. Activation of the ET pathway with aging may promote the development of age-dependent diseases such as glomerulosclerosis, hypertension and atherosclerosis.

Endothelin Regulates Angiotensin-Converting Enzyme in the Mouse Kidney

Using the orally active endothelin-A- (ET(A)) receptor antagonist LU135252, we determined whether endothelin-1 (ET-1) and/or dietary fat may be involved in angiotensin-converting enzyme (ACE) regulation in vivo. In C57BL6/J mice, renal and pulmonary tissue ACE activity (nmol/l His-Leu/mg protein) was measured and ACE mRNA expression, tissue ET-1 protein content and nitrite/nitrate level were measured in the kidney. Western-type diet increased renal ACE activity by 70% (55 +/- 4 vs 33 +/- 3 nmol/l His-Leu/mg protein, p < 0.05) and increased renal ET-1 levels (267 +/- 19 pg/g vs 190 +/- 18, p < 0.05). Chronic LU135252 treatment completely prevented activation of renal ACE activity (13.3 +/- 0.3 His-Leu/mg protein nmol/l, p < 0.05) independent of ACE mRNA expression or renal ET-1 protein levels. Thus, dietary fat activates renal ACE activity and ET-1 is involved in regulation of tissue ACE activity in vivo independently of ACE mRNA expression.

Endothelial Dysfunction in Acute Renal Failure

Abstract. The kidney is an important target and source of the potent vasoconstrictor and mitogen endothelin-1 (ET-1). However, its exact role in acute renal failure (ARF) remains to be determined. ARF was induced in male Wistar-Kyoto rats (n= 7) in a 2-kidney, 2-clip model of 30-min clamping. Twentyfour hours after clamp release, contractions to angiotensin I (AngI) and II, ET-1, and big ET-1 were studied in isolated aortic and renal artery rings. Endothelium-dependent and -independent relaxations were assessed by acetylcholine and sodium nitroprusside. ET-1 clearance, tissue uptake, plasma levels, and vascular and kidney content were investigated. In addition, ETAand EtBreceptor mRNA expression was determined. Sham-operated animals served as controls (n= 7). In ARF, ET-1 plasma levels and tissue content of the renal artery, the aorta, and the kidney markedly increased (P&lt; 0.01). Plasma half-life of radiolabeled125I-ET-1 was markedly prolonged, whereas125I-ET-1 tissue uptake decreased in the kidney in ARF. Contractions to AngI and AngII were blunted (P&lt; 0.05) and those to KC1 were unchanged, whereas vascular responses to big ET-1 and ET-1 were enhanced in the renal artery and also in the aorta in ARF (P&lt; 0.05 to 0.001). Correspondingly, ETAand EtBreceptor mRNA expression significantly increased in both vascular beds. In addition, endothelium-dependent relaxation to acetylcholine was diminished and inversely correlated with vascular ET-1 protein levels in the renal artery (r= -0.827,P&lt; 0.001) and the aorta (r= -0.812,P&lt; 0.001). In conclusion, the present study demonstrates that increase of circulating and tissue ET-1 protein levels and ETAand EtBreceptor gene expression occurs, which induces endothelial dysfunction and enhanced vasoconstriction in different vascular beds in ARF.

Quinapril decreases renal endothelin-1 expression and synthesis in a normotensive model of immune-complex nephritis.

Angiotensin-converting enzyme (ACE) inhibitors diminish proteinuria and the progression to renal failure in several experimental models of renal injury. Endothelin-1 (ET-1) possesses potent biological actions on renal vessels and has been considered as a potential mediator of renal damage. Because angiotensin II (Ang II) induces ET-1 synthesis in endothelial and mesangial cells, we hypothesized that some of the beneficial effects of the ACE inhibitors could result from the blockade of ET-1 synthesis. In a normotensive model of immune-complex nephritis, in which there exists an increase in renal ACE activity, the effect of the ACE inhibitor quinapril on preproET-1 and ETA receptor mRNA expression, as well as on ET-1 protein levels, was examined in this study. In relation to controls, nephritic rats showed an increase in preproET-1 and ETA receptor gene expression in renal cortex and medulla, coinciding with the maximal renal ACE activity. PreproET-1 mRNA (in situ hybridization) and ET-1 protein (immunohistochemistry) were localized in glomerular capillary walls, mesangial and glomerular epithelial cells, as well as in the brush border of some proximal tubules, and in small vessels. In nephritic rats, there was an increase in preproET-1 mRNA levels and ET-1 protein in all of these areas, without modification of their distribution. The administration of the ACE inhibitor quinapril decreased proteinuria and morphological lesions, preproET-1 gene transcription, and ET-1 protein levels, as well as the ETA receptor mRNA. The results from this study show that in a normotensive model of immune-complex nephritis, there was an overexpression of ET-1 in several structures of the kidney that was downregulated by quinapril administration. The beneficial effect of ACE inhibitors could be a result of the modulation of local production of Ang II and ET-1.

Effect of reactive oxygen species on endothelin-1 production by human mesangial cells

Reactive oxygen species (ROS) have been implicated in the pathophysiology of renal ischemia/reperfusion injury. Endothelin-1 (ET-1) is generated in abundance in renal ischemia/reperfusion with resultant decreases in renal blood flow and glomerular filtration rate. To determine if ROS regulate ET-1 production, the effect of ROS donors or scavengers on ET-1 protein and mRNA levels in cultured human mesangial cells was examined. Incubation with xanthine/xanthine oxidase, glucose oxidase, or H2O2 caused a dose-dependent rise in ET-1 release. Similarly, xanthine/xanthine oxidase or H2O2 augmented ET-1 mRNA levels. In contrast, the ROS scavengers dimethylthiourea (DMTU), dimethylpyrroline N-oxide, or pyrrolidine dithiocarbamate reduced basal ET-1 release, while DMTU lowered ET-1 mRNA levels. Deferoxamine, an iron chelator, also decreased basal ET-1 release. Superoxide dismutase potentiated the ET-1 stimulatory effect of xanthine/xanthine oxidase, while catalase abrogated the effect of xanthine/xanthine oxidase and H2O2. The effects of ROS were unrelated to changes in nitric oxide production or cytotoxicity. These data indicate that exogenously or endogenously-derived ROS can increase ET-1 production by human mesangial cells. While superoxide anion reduces ET-1 levels, H2O2 leads to enhanced production of the peptide. ROS stimulation of mesangial cell ET-1 production may contribute to impaired glomerular hemodynamics in the setting of renal ischemia/reperfusion injury.

Increased endothelin-1 production in fibroblasts derived from patients with systemic sclerosis.

To determine whether fibroblasts from patients with systemic sclerosis (SSc) produce excessive amounts of endothelin-1 (ET-1), which is recognised as having vasoconstrictive properties and as having a potent mitogenic effect on fibroblasts. Dermal fibroblasts were removed from 11 patients with SSc and from five normal controls (NC). The assay of ET-1 protein was measured by an ELISA that used two anti-ET-1 antibodies. The gene expression of prepro ET-1 mRNA was evaluated by a reverse-transcriptase polymerase chain reaction (RT-PCR) method. Levels of ET-1 protein were significantly higher in SSc fibroblast cultures than in those of normal fibroblasts (p < 0.01). The expression of prepro ET-1 mRNA was also higher in SSc fibroblasts than in normal fibroblasts. The addition of interleukin-1 beta (IL-1 beta) increased the production of ET-1 by fibroblasts. The findings indicate that the overproduction of ET-1 is a novel abnormal function in SSc fibroblasts, and that ET-1 induced by fibroblasts may play a role in the fibrosis and Raynaud's phenomenon of SSc.