Endothelin-1 mRNA Levels Research Articles

Eicosapentaenoic acid suppresses basal and insulin-stimulated endothelin-1 production in human endothelial cells.

cis-Polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) are the major fatty acids contained in fish oil, and are known to affect the various physiological properties of cell membranes in humans. The present study investigated the effects of polyunsaturated fatty acids on endothelin-1 (ET-1) production in human umbilical vein endothelial cells (HUVECs) and on insulin activity. After addition of various concentrations of EPA, docosahexaenoic acid, arachidonic acid, or linoleic acid to a culture medium, the concentration of ET-1 was measured using ELISA, and that of ET-1 mRNA was determined by RT-PCR. The results showed that EPA had the strongest inhibitory effect (p<0.05) on both basal ET-1 production and ET-1 mRNA levels. In addition, insulin (1 micromol/l) markedly increased ET-1 production, and EPA also significantly decreased the effect induced by insulin. Pretreatment with Ca2+ chelator EGTA (1 mmol/l), NOS inhibitor L-NAME (300 micromol/l), or calmodulin antagonist W-7 (300 micromol/l) inhibited NO production by EPA (100 micromol/l), but these pretreatments had no effect on ET-1 production by EPA. These findings suggest that EPA reduces basal and insulin-enhanced ET-1 production by inhibiting ET-1 mRNA production. These effects of EPA may contribute to its vasorelaxant and anti-atherosclerotic effects.

Erythropoietin-induced excessive erythrocytosis activates the tissue endothelin system in mice.

The endothelium controls blood flow and pressure by releasing several vasoactive factors, among them the vasodilator nitric oxide (NO) and the potent vasoconstrictor endothelin-1 (ET-1). Although increased NO levels have been found in excessive erythrocytosis, little is known concerning ET-1 expression in this condition. Thus, we examined the endothelin system in transgenic mice that due to constitutive overexpression of erythropoietin (Epo) reached hematocrit levels of approximately 80%. Surprisingly, despite generalized vasodilatation, polycythemic mice exhibited a two- to fivefold elevation in ET-1 mRNA levels in aorta, liver, heart, and kidney. In line with this, increased expression of ET-1 protein was detected in the pulmonary artery by immunohistochemical analysis. Compared with their wild-type littermates, aortic rings of Epo transgenic animals exhibited a marked reduction in vascular reactivity to ET-1 and big ET-1, but this effect was abrogated upon preincubation with the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME). Pretreatment of polycythemic mice with the ET(A) receptor antagonist darusentan for 3 wk significantly prolonged their survival upon acute exposure to L-NAME. Taken together, these results demonstrate for the first time that excessive erythrocytosis induces a marked activation of the tissue endothelin system that results in increased mortality upon blockade of NO-mediated vasodilatation. Because ETA antagonism prolonged survival after acute blockade of NO synthesis, endothelin may be regarded as a contributor to the adverse cardiovascular effects of erythrocytosis and may thus represent a new target in the treatment of cardiovascular disease associated with erythrocytosis.

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.

Liver-derived insulin-like growth factor-I is involved in the regulation of blood pressure in mice.

IGF-I has been suggested to be of importance for cardiovascular structure and function, but the relative role of locally produced and liver-derived endocrine IGF-I remains unclear. Using the Cre-LoxP recombination system, we have previously created transgenic mice with a liver-specific, inducible IGF-I knockout (LI-IGF-I-/-). To examine the role of liver-derived IGF-I in cardiovascular physiology, liver-derived IGF-I was inactivated at 4 wk of age, resulting in a 79% reduction of serum IGF-I levels. At 4 months of age, systolic blood pressure (BP) was increased in LI-IGF-I-/- mice. Echocardiography showed increased posterior wall thickness in combination with decreased stroke volume and cardiac output, whereas other systolic variables were unchanged, suggesting that these cardiac effects were secondary to increased peripheral resistance. Acute nitric oxide-synthase inhibition increased systolic BP more in LI-IGF-I-/- mice than in control mice. LI-IGF-I-/- mice showed impaired acetylcholine-induced vasorelaxation in mesenteric resistance vessels and increased levels of endothelin-1 mRNA in aorta. Thus, the increased peripheral resistance in LI-IGF-I-/- mice might be attributable to endothelial dysfunction associated with increased expression of endothelin-1 and impaired vasorelaxation of resistance vessels. In conclusion, our findings suggest that liver-derived IGF-I is involved in the regulation of BP in mice.

Stimulation of peroxisome-proliferator-activated receptor alpha (PPAR alpha) attenuates cardiac fibrosis and endothelin-1 production in pressure-overloaded rat hearts.

Endothelin-1 (ET-1) production is increased in hypertrophied hearts accompanied with fibrosis. ET-1 is a potent mitogen of fibroblasts and ET receptor antagonists are reported to inhibit the proliferation of fibroblasts and cardiac fibrosis. Peroxisome-proliferator-activated receptor alpha (PPAR alpha), one of the nuclear hormone receptors, suppresses activator protein-1 (AP-1), one of the nuclear transcription factors. Activation of PPAR alpha is reported to inhibit thrombin-induced ET-1 production by repressing the AP-1 signalling pathway in vascular endothelial cells. We investigated effects of the PPAR alpha activator fenofibrate (80 mg/kg per day, per os) on mRNA levels of ET-1, collagen type I and type III and histological features of myocardial fibrosis in hypertrophied rat hearts due to pressure-overload by abdominal aortic banding (AB). The treatment with fenofibrate or vehicle was started 7 days before the AB operation. Four days after the AB operation, fenofibrate treatment significantly reduced ET-1 mRNA expression compared with vehicle treatment in AB rat hearts. Collagen type I and type III mRNA expression, and interstitial and perivascular fibrosis were attenuated in the fenofibrate-treated AB rat group. Since the ET-1 gene has AP-1 response elements in the 5'-flanking region, it is suggested that myocardial fibrosis is effectively inhibited by fenofibrate through suppression of AP-1-mediated ET-1 gene augmentation in the pressure-overloaded heart caused by aortic banding in rats.

Expression of endothelin-1 and vasoactive intestinal contractor genes in mouse organs during the perinatal period.

In an attempt to understand the significance of endothelin-1 (ET-1) and vasoactive intestinal contractor (VIC)/ET-2 peptides in organs during perinatal development, we performed quantitative analysis of ET-1 and VIC gene expression in mouse organs obtained from embryos at days 14 and 17 (E-14 and E-17) of pregnancy, neonates at days 0, 1, 3 and 7 after birth (N-0, -1, -3 and -7), and adult mice (10 weeks old). In intestine, VIC gene expression progressively increased between E-14 and N-1 (approximately 10-fold) and then remained constant into adulthood. ET-1 gene expression exhibited a one-step increase between E-17 and N-0, subsequently remaining constant. In lung, a sharp increase in ET-1 mRNA level (approximately 10-fold) was noticed between E-14 and N-0. The gene expression pattern of VIC, with a peak at N-0, was similar to that of ET-1 although the expression level of VIC was two to three orders of magnitudes lower than that of ET-1. Gene expression patterns of ET-1 and VIC remained nearly constant in brain, heart, liver and kidney throughout the period examined. Considering that the intestinal and pulmonary gene expression levels of both genes reached almost the same level as observed in adult soon after birth, we suggest that these peptides may be involved in the emergence and maintenance of intestinal and pulmonary functions vital after birth.

Co-expression of endothelin-1 and vascular endothelial growth factor mediates increased vascular permeability in lung grafts before reperfusion

Endothelin-1 (ET-1) protein levels increase in lung transplant recipients, although the source of ET-1 remains uncertain. Evidence is accumulating that ET-1 coregulates the expression of vascular endothelial growth/permeability factor (VEGF-A). By using real-time polymerase chain reaction and Western blotting, we describe upregulated ET-1 mRNA and VEGF-A protein levels as well as increased fluid content in donor lung-tissue biopsies before reperfusion. Hypoxia and ET-1 co-upregulate VEGF-A overexpression, therefore temporary VEGF-A antagonism during graft preservation might be of benefit in lung transplantation.

Endothelin receptor function in mesenteric veins from deoxycorticosterone acetate salt-hypertensive rats

To identify the receptors by which endothelin-1 (ET-1) increases venomotor tone in hypertension. Vascular reactivity to ET-1 and the selective endothelin receptor subtype B (ET(B)) agonist, sarafotoxin 6c (S6c), was studied in mesenteric blood vessels from deoxycorticosterone acetate (DOCA-salt) hypertensive and normotensive control rats. The diameter of small (< or = 280 microm) mesenteric arteries and veins was monitored in vitro using computer-assisted video microscopy. Contractions of mesenteric arteries (< or= 250 microm diameter) were also studied, using a myograph. ET-1 mRNA levels were measured in mesenteric arteries and veins using real-time RT-PCR techniques. ET-1-induced contractions were reduced in arteries of DOCA-salt hypertensive rats compared with those of normotensive control rats; S6c produced negligible contractions in arteries from both groups. ET-1 concentration-responses curves in arteries measured using video microscopy or a myograph were similar. ET-1 and S6c caused veins to contract, and there were no differences between responses to these agonists in tissues from DOCA-salt hypertensive rats or normotensive control rats. Studies using ET(A) and ET(B) receptor antagonists indicated that ET-1-induced venoconstriction was mediated by ET(A) receptors. Potassium chloride concentration-response curves were similar in arteries and veins from normotensive control rats and DOCA-salt hypertensive rats. ET-1 mRNA levels in DOCA-salt hypertensive rat arteries or veins were not different from those in normotensive control rat arteries and veins. These data indicate that ET-1 reactivity is maintained in mesenteric veins, but not arteries, in DOCA-salt hypertension. Therefore, the sustained increase in venomotor tone mediated by ET(A) receptors that is known to occur in vivo in DOCA-salt hypertensive rats is not caused by direct venoconstriction.

Crosstalk of endothelin-1 and platelet-derived growth factor in cardiac allograft arteriosclerosis

ObjectivesIn this study, we investigated the crosstalk of endothelin-1 (ET-1) and platelet-derived growth factor (PDGF) in coronary artery smooth muscle cell (SMC) proliferation in the rat cardiac allograft model. BackgroundPrevious studies have suggested an independent role of ET-1 and PDGF in the development of cardiac allograft arteriosclerosis (i.e., chronic rejection). MethodsHeterotopic heart transplantations were performed from Dark Agouti to Wistar Furth rats. Grafts were harvested after five days in an acute rejection model and after 60 days in a chronic rejection model. In the in vitro part of the study, SMC proliferation and migration were quantitated, as well as messenger ribonucleic acid (mRNA) levels of ET-1 and PDGF ligands and receptors after growth factor stimulation. ResultsAcute rejection induced both ET-1 receptors in the arterial wall. On linear regression analysis of chronically rejecting cardiac allografts, a strong correlation between intimal thickening and immunoreactivity of ET-1 and ET receptors A and B (ETAand ETB) in the arterial walls was observed. Treatment with Bosentan, a mixed ET-1 receptor antagonist, significantly reduced the incidence and intensity of arteriosclerotic lesions in rat cardiac allografts, as well as total intragraft ETAand ETBmRNA expression and intimal cell ET-1 and receptor immunoreactivity. This was associated with significantly reduced intragraft PDGF beta-receptor (PDGF-Rbeta) mRNA expression. In contrast, CGP 53716, a protein tyrosine kinase inhibitor selective for the PDGF receptor, did not reduce intragraft ET-1, ETAor ETBmRNA expression. In rat coronary artery SMC cultures, ET-1 stimulation significantly upregulated PDGF-Ralpha and -Rbeta mRNA expression and augmented PDGF-BB–mediated SMC proliferation as well as PDGF-AB– and PDGF-BB–mediated SMC migration. ConclusionsOur results suggest that the ET-1/PDGF-Rbeta/PDGF-BB axis may operate in SMC migration and proliferation in cardiac allograft arteriosclerosis, thus explaining the marked beneficial effects of blocking the signaling downstream of ET-1 receptors.

Activated mast cells increase the level of endothelin-1 mRNA in cocultured endothelial cells and degrade the secreted Peptide.

Subendothelial mast cells have been implicated in the pathogenesis of allergic inflammation, in atherosclerosis, and in the regulation of vascular tone. Because endothelin-1 (ET-1) is an important regulator of vascular tone and has also been implicated in the pathogenesis of atherosclerosis, we studied the role of mast cells in the metabolism of endothelial cell-derived ET-1. In mast cell-endothelial cell cocultures, activation of the mast cells with ensuing degranulation was accompanied by the increased expression of ET-1 mRNA in the endothelial cells, yet the immunoreactive ET-1 protein in the coculture medium disappeared almost completely during the 24-hour coculture. Activation of the mast cells with the ensuing degranulation resulted in proteolytic degradation of ET-1 by the 2 neutral proteases, chymase and carboxypeptidase A, of the exocytosed mast cell granules. With synthetic ET-1 and purified mast cell granule enzymes, efficient degradation of ET-1 by chymase and carboxypeptidase A was verified. These in vitro results imply a novel role for mast cell-derived neutral proteases in ET-1 metabolism and suggest that activated subendothelial mast cells are important local regulators of ET-1 metabolism.

The effect of statins on mRNA levels of genes related to inflammation, coagulation, and vascular constriction in HUVEC. Human umbilical vein endothelial cells.

Large-scale clinical trials have demonstrated significant reductions in cardiovascular events following statin therapy. The observed benefit of statin therapy, however, may be greater in these trials than is to be expected from lowering lipid levels alone. In order to clarify the mechanism by which statins prevent cardiovascular events in vascular wall cells, we investigated the changes in gene expression profiles after incubation with atorvastatin or pitavastatin in cultured human umbilical vein endothelial cells using DNA microarrays. Statins affected the expression levels of genes involved in inflammation, coagulation, and vascular constriction. The mRNA levels for interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) decreased after statin treatment. Statins reduced mRNA levels of plasminogen activator inhibitor-1 (PAI-1) and increased the mRNA levels of thrombomodulin. Statins reduced the mRNA levels of endothelin-1 and increased the mRNA levels of nitric oxide synthase-3 (eNOS). These results show that, statins are clinically effective because of their ability to change the gene expression profile of endothelial cells thereby preventing vascular events.

Endothelin-1 in smooth muscle cells and mast cells of mouse uterus after parturition.

To elucidate the physiological importance of endothelin-1 (ET-1) in mouse uterus, we investigated quantitative changes in ET-1 mRNA levels in the uterus during the estrous cycle, pregnancy and post-parturient period by use of the real-time PCR technique and we examined the cellular distribution of the ET-1 peptide by use of immunohistochemical techniques. Low and constant mRNA levels were observed in the uterus from cyclic or pregnant mice. However, a significant increase in mRNA levels was found immediately after parturition (day 0 postpartum) which then decreased gradually to a basal level at day 14 postpartum. Discernible immunopositivity was found in myometrial cells as well as in endometrial epithelial cells in the post-parturient uterus. Myometrial cells showed the strongest staining at day 0 postpartum, and some large cells in the myometrial layers, intensely positive for ET-1, were characterized as mast cells. These findings suggest the possibility that in mouse uterus ET-1 may play a role in recovery from the uterine changes caused by pregnancy and parturition.

C-Abl Is Required for the Development of Hyperoxia-Induced Retinopathy

The requirement for the nonreceptor tyrosine kinase c-abl in the pathogenesis of retinopathy of prematurity (ROP) was examined using the mouse model for ROP and c-abl-deficient mice. Hyperoxia-induced retinal neovascularization was observed in wild-type and heterozygous mice but animals that were homozygous null for c-abl did not develop a vasoproliferative retinopathy in response to hyperoxia. Two gene products, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF), have been implicated in the pathogenesis of ROP. The mRNA expression of ET-1 and VEGF was assessed in mice maintained in normoxia and in hyperoxia-exposed mice. ET-1 mRNA levels were unchanged in wild-type mice throughout the hyperoxia treatment, suggesting that ET-1 mRNA expression is not regulated by the increase in inspired oxygen. In wild-type mice maintained in room air, VEGF mRNA levels rose threefold from postnatal day 6 (P6) to P17. When wild-type mice were treated with the hyperoxia regimen, a fivefold decrease in VEGF mRNA expression was observed from P7 to P16. However, retinal VEGF expression in hyperoxia-treated homozygous null mice did not decrease and remained at control levels. These data suggest that c-abl is required for the hyperoxia-induced retinal neovascularization and hyperoxia-induced decrease in VEGF mRNA levels.

Altered gene expression of endothelin-A and endothelin-B receptors, but not endothelin-1, in myometrium and placenta from pregnancies complicated by preeclampsia.

The aim of the present study was to compare the mRNA expression of endothelin-1 (ET-1), ET-1 receptors ET(A) and ET(B) in myometrium and placenta obtained from biopsies collected from women with preeclampsia (n=10) or normal pregnant (n=12). The mRNA levels of ET-1 ET(A) and ET(B) were determined using RT-PCR and expressed as arbitrary units after correction for control GAPDH gene mRNA levels. The mRNA levels of ET-1 in myometrium and placenta were not altered in women with preeclampsia compared to normal pregnant. The mRNA expression of ET(A) was significantly reduced (p<0.05) in both placenta and myometrium from women with preeclampsia. The mRNA levels of ET(B) were similar in placentas from both preeclamptic and normal pregnant women, but higher in myometrium (p<0.05) from women with preeclampsia. These results indicate that the higher levels of ET-1 seen in preeclamptic women do not depend on an altered mRNA transcription of ET-1. The significantly reduced mRNA expression of ET(A) receptor in myometrium and placenta in women with preeclampsia might represent downregulation of the receptors due to the increased levels of ET-1 in uteroplacental circulation in this disorder. Much more research is needed before the role of ET(B) receptors in preeclampsia can be clarified.

Exposure of endothelial cells to recombinant human erythropoietin induces nitric oxide synthase activity

Anemic patients with chronic renal failure receiving recombinant human erythropoietin (rHuEPO) therapy frequently develop hypertension through an unknown mechanism. We hypothesize that EPO receptors (EPORs) on endothelial cells (ECs) in various sites of vasculature may mediate the activities of nitric oxide synthase (NOS) and/or the release of endothelin-1 (ET-1), contributing to blood pressure changes. We tested this hypothesis using primary cultures of ECs obtained from human coronary artery (HCAEC), pulmonary artery (HPAEC), dermis (HDEC), and umbilical vein (HUVEC). EPORs were measured by 125I-EPO binding. The effect of EPO on EPOR, ET-1, and NOS mRNA levels was assessed by quantitative reverse transcription-polymerase chain reaction. Cellular NOS activity and ET-1 release into the medium was measured by the NOSdetect assay and by radioimmunoassay kits. Short-term (4 h) treatment with EPO (4 U/mL) did not change the number or affinity of EPOR per cell. Neither were there any changes in the amount of EPOR, ET-1, and NOS transcripts (cDNA/microg of mRNA) nor in ET-1 release and NOS activity. In HUVEC only, 24-hour exposure to EPO caused a threefold increase in NOS transcript. In other cells, EPO treatment for six days increased NOS activity by twofold to fourfold. We show that upon extended exposure, EPO induces NOS activity but does not affect ET-1 release. These findings indicate that the hypertensive effect of EPO is not likely to be caused by a direct effect on ECs.

Augmented Expression of Tissue Endothelin-1 Messenger RNA is a Common Feature in Hypertensive Rats

Summary: Recent studies revealed important roles for endothelin-1 (ET-1) in the pathogenesis of hypertension. Whether ET-1 could be a primary cause of hypertension or a secondary factor associated with hypertension, however, remains unknown. In this study, we determined plasma ET-1 levels and the expression of ET-1 mRNA in tissues of rats rendered hypertensive using distinct mechanisms: deoxycorticosterone acetate (DOCA)-salt hypertension; NG-nitro-L-arginine-methyl ester-(L-NAME) induced hypertension; and spontaneously hypertensive rats (SHR-SP). ET-1 mRNA expression level was determined by reverse transcriptase polymerase chain reaction (RT-PCR) followed by Southern blotting. There was no significant difference in plasma ET-1 levels between the hypertensive rats and normotensive rats. By contrast, ET-1 mRNA level was significantly increased in various tissues including the adrenal, lung, kidney and brain of these hypertensive rats compared with control rats. Thus, ET-1 gene expression was ubiquitously augmented in tissues of hypertensive rats irrespective of the cause of the hypertension. The results suggest that the increase in ET-1 expression is not the primary cause of hypertension but a secondary outcome which may further exacerbate the hypertension.

Augmented expression of tissue endothelin-1 messenger RNA is a common feature in hypertensive rats.

Recent studies revealed important roles for endothelin-1 (ET-1) in the pathogenesis of hypertension. Whether ET-1 could be a primary cause of hypertension or a secondary factor associated with hypertension, however, remains unknown. In this study, we determined plasma ET-1 levels and the expression of ET-1 mRNA in tissues of rats rendered hypertensive using distinct mechanisms: deoxycorticosterone acetate (DOCA)-salt hypertension: N(G)-nitro-L-arginine-methyl ester- (L-NAME) induced hypertension; and spontaneously hypertensive rats (SHR-SP). ET-1 mRNA expression level was determined by reverse transcriptase polymerase chain reaction (RT-PCR) followed by Southern blotting. There was no significant difference in plasma ET-1 levels between the hypertensive rats and normotensive rats. By contrast, ET-1 mRNA level was significantly increased in various tissues including the adrenal, lung, kidney and brain of these hypertensive rats compared with control rats. Thus, ET-1 gene expression was ubiquitously augmented in tissues of hypertensive rats irrespective of the cause of the hypertension. The results suggest that the increase in ET-1 expression is not the primary cause of hypertension but a secondary outcome which may further exacerbate the hypertension.

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.

The cardiac endothelin system in established pressure overload left ventricular hypertrophy.

In normal hearts, endothelin-1 (ET-1) has been shown to initiate myocyte growth and to modulate cardiac function. However, regulation of the various components of the system and the functional effects of ET-1 in established left ventricular hypertrophy (LVH) are less clear. We thus studied ET-1, ET(A) receptor, and endothelin converting enzyme (ECE-1) mRNA regulation as well as the effects of ET-1 on coronary resistance, LV contractility and relaxation in hypertrophied rat hearts. Cardiac pressure overload, secondary to banding of the ascending aorta, resulted in a transient increase of cardiac ET-1 and ET(A) receptor mRNAs that reached a maximum at 2 days (+75% and +40%, respectively, P<0.05, each). ET-1 mRNA levels reached a second peak at 84 days of pressure overload (+60%, P<0.05), at the later time point in conjunction with elevated ECE-1 mRNA levels (+20%, P<0.05). The functional implications of ET-1 were examined in a study of isolated perfused hearts. Both hearts with established LVH and sham control hearts responded to ET-1 perfusion (10(-1)] to 10(-9) M) with an increase of coronary perfusion pressure (CPP; +85+/-15 and +75+/-8 mm Hg; P<0.001 each) and a slight decrease of LV systolic pressure (LVP; -12+/-9 and -9+/-7 mm Hg; P = NS). In contrast, ET-1 increased LV end-diastolic pressure (LVEDP) only in LVH hearts (+22+/-7 mm Hg, P<0.05 versus baseline and +20+/-7 mm Hg, P<0.05 versus sham). Direct stimulation of protein kinase C mimicked the effects of ET-1, whereas inhibition of this kinase or the Na+ -H+ exchanger blunted the effects of ET-1 on CPP, LVP, and LVEDP. Interestingly, coadministration of the vasodilator and the nitric oxide (NO) donor nitroglycerin not only prevented the increase of CPP and LVEDP, but also uncovered a slight positive inotropic effect of ET-1 in LVH hearts. Thus, the cardiac expression of ET-1, ET(A), and ECE-1 mRNAs displays a distinct pattern during early and advanced cardiac pressure overload. Furthermore, ET-1 mediates a slight depression of systolic, and a profound depression of diastolic, functional parameters in hearts with established LVH, effects that appear to be secondary to ET-1-related coronary vasoconstriction. The data suggest a functional role of the endothelin system in hearts with established pressure overload hypertrophy.

Endothelin-1 Gene Suppression by Shear Stress: Pharmacological Evaluation of the Role of Tyrosine Kinase, Intracellular Calcium, Cytoskeleton, and Mechanosensitive Channels

Physiological fluid shear stress regulates endothelin-1 (ET-1) gene expression in endothelial cells by inducing an early transient upregulation followed by a sustained suppression, at times greater than 2 h in duration. We evaluated the mechanism of ET-1 mRNA downregulation in confluent monolayers of bovine aortic endothelial (BAE) cells by applying a 6 h steady laminar shear stress of magnitude 20 dyn/cm2. Inhibition of tyrosine kinases using herbimycin A (875 nm) abolished the shear-induced decrease in ET-1 mRNA expression. Similarly, chelation of intracellular calcium ( [Ca2+]i) with quin2-AM (10μm) blocked the suppression of ET-1 mRNA by shear. To examine the role of the endothelial cytoskeleton in the response to flow, cytochalasin D was used to disrupt F-actin microfilaments. This treatment induced cell retraction and detachment under flow, whereas stabilization of F-actin with phalloidin (1μm) did not affect shear-induced ET-1 downregulation. In contrast, disruption of the microtubule network with nocodazole (10μg/ml) completely prevented, while microtubule stabilization with taxol (10μm) did not affect the suppression of ET-1 mRNA by flow. To determine the possible contributions of mechanosensitive channels, barium (1 mmBaCl2), was added to confluent BAE monolayers in a low-sulfate/low-phosphate modified medium and was noted to abrogate the downregulation of ET-1 gene expression and to attenuate the shear-induced increase in cytoplasmic free calcium concentration. Tetraethylammonium (3 mmTEA) partially inhibited the suppression of ET-1 mRNA by shear; in contrast, gadolinium (10μmGdCl3), an inhibitor of the stretch-activated cation channel ISA, had no effect. Membrane depolarization by elevated extracellular potassium ( [K+]o) also attenuated the suppression of ET-1 mRNA by flow at [K+]o=70 mmand completely inhibited it at [K+]o=135 mm. In summary, the steady-state downregulation of ET-1 mRNA by physiological levels of fluid shear stress shares signaling features with the morphological and cytoskeletal response to shear stress. These include requirement for intracellular calcium, tyrosine kinase activity, an intact microtubule network, and independence from a Gd3+-sensitive ISA. Unlike shear-induced changes in cell morphology and the actin cytoskeleton, the shear-induced decrease in ET-1 mRNA level is blocked by cell depolarization and by Ba2+, a blocker of the shear-activated IKSwhich also decreases shear-induced cytoplasmic calcium increase.