Endothelin-1 Gene Expression Research Articles

Effects of sildenafil on pulmonary hypertension and levels of ET-1;eNOS;and cGMP in aorta-banded rats

Sildenafil, an oral phosphodiesterase Type 5 inhibitor, has vasodilatory effects through a cGMP-dependent mechanism. We previously showed that aortic banding could result in left ventricular overloading and pulmonary hypertension (PH). In this study, we investigated whether early administration of sildenafil, either immediately after or 2 weeks after aortic banding, could ameliorate the development of PH and alter gene expression of endothelin (ET)-1 and endothelial nitric oxide synthase (eNOS), and alter the levels of cGMP in rats undergoing an ascending aortic banding. Rats (n = 32) were divided into sham-operated and banding groups with or without treatment. The banded rats were further divided into three groups: (i) receiving saline on Days 1-28 (AOB28; n = 8), (ii) receiving saline on Days 1-14 followed by treatment with 50 mg/kg/day sildenafil on Days 15-28 (AOB28/Sil(15-28); n = 8), and (iii) receiving 50 mg/kg/day sildenafil on days 1-28 (AOB28/Sil(1-28); n = 8). The sham-operated rats were administrated saline on Days 1-28 (n = 8). Four weeks after banding, there was a significant development of PH with pulmonary vascular remodeling. Although both sildenafil-treatment groups had significant increases in cGMP and had reductions in the thickening in the medial layer of pulmonary arteriole, notable attenuation of PH occurred only in the AOB28/Sil(1-28) group. PreproET-1 and eNOS messenger RNA (mRNA) expressions were measured by competitive reverse transcription polymerase chain reaction, and eNOS protein was determined by Western blotting. Sildenafil did not alter the elevated ET-1 or preproET-1 mRNA in banded rats. Interestingly, pulmonary eNOS increased in the AOB28/Sil(1-28) group. In conclusion, early treatment with sildenafil inhibited the rise in pulmonary arterial pressure and pulmonary vascular remodeling in PH secondary to heart failure, and cGMP, but not ET-1, might be involved. Clinically, early repeated administration of sildenafil may offer an alternative in protecting against PH in heart failure.

Altered laminar gene expression indicative of vascular, inflammatory and metabolic events in horses with acute laminitis

Laminitis is a common disease of the equine digit characterized by cardiovascular alterations (ischemia/reperfusion) associated with other complex pathophysiologic cascades with many unidentified components. This results in varied therapeutics utilized by veterinarians. The objective was to examine potential cardiovascular, metabolic and inflammatory mediators in naturally-acquired and experimentally-induced equine laminitis. Samples were collected from 54 horses from naturally-acquired (acute & chronic), 9-hours post-experimental induction (black walnut extract model), 72-hours post-experimental induction, and controls. TaqMan RT-PCR was completed for endothelin-1 (ET-1), interleukin (IL)-8, cyclooxygenase (COX)-1, COX-2 and Glut-1. When the equine gene sequence was not available, it was cloned, sequenced and validated. Significant differences were found for all genes. ET-1, IL-8, COX-1 and COX-2 gene expression increased in horses with acute disease. IL-8 and COX-2 returned to basal levels, but COX-1 and Glut-1 significantly decreased below basal levels in chronic disease. Horses with naturally-acquired disease had the most alterations, especially those with gross alterations of the distal phalanx. These findings demonstrate the roles of vascular alterations, inflammation and altered metabolism in the onset of this devastating disease. Funding by the Equine Health Studies Program of the LSU, School of Veterinary Medicine.

Suppression of Hyperglycemia-Induced Superoxide Formation and Endothelin-1 Gene Expression by Carvedilol

Carvedilol (CV) is a beta-blocker with favorable effects on cardiovascular disease. To determine whether CV can prevent increases in superoxide (SO) production due to hyperglycemia, human umbilical vein endothelial cells (HUVEC) were treated with either 100 or 500 mg/dL dextrose in the presence or absence of 0.1, 1.0, and 10 micromol/L CV. Superoxide levels were measured using the hydroethidine (HE) fluorescence method. Generation of SO was linear from time 0 to 60 minutes. At 60 minutes, the HE fluorescence in cells treated with 500 mg/dL dextrose (123.3+/-4.9 units) was significantly higher than that in control cells treated with 100 mg/dL dextrose (84.0+/-3.5 units) (P<0.002). Addition of 0.1, 1.0, and 10 micromol/L CV to cells treated with 500 mg/dL dextrose decreased SO generated to 113.3+/-1.8, 98.7+/-8.3, and 70.0+/-1.0 units, respectively (P<0.13, P<0.05, and P<0.004, respectively). Cellular endothelin-1 mRNA and endothelin-1 protein secreted in culture media were significantly increased in the presence of 500 mg/dL dextrose. The addition of 10 micromol/L CV significantly decreased both endothelin-1 (1-21) mRNA and protein levels. Measurements of media lactate dehydrogenase activity indicated that CV inhibited cytotoxicity caused by 500 mg/dL dextrose. These findings suggest that CV not only prevents dextrose-induced SO generation in endothelial cells but may also have favorable effects on gene expression and cell survival.

Tu-P10:508 Leptin induces endothelin-1 gene expression via RAS/RAF/extracellular signal-regulated kinase pathway in rat aortic smooth muscle cells

Tu-P10:508 Leptin induces endothelin-1 gene expression via RAS/RAF/extracellular signal-regulated kinase pathway in rat aortic smooth muscle cells

Salutary effects of androstenediol on hepatic function after trauma-hemorrhage are mediated via peroxisome proliferators–activated receptor gamma

A recent study suggested that administration of androstenediol (Adiol) after trauma-hemorrhage (T-H) improves hepatic functions; however, the mechanism responsible for the salutary effect of Adiol remains unknown. Although studies indicate similarities and association between the anti-inflammatory properties of Adiol and peroxisome proliferator-activated receptor gamma (PPARgamma), whether the salutary effects of Adiol are mediated via upregulation of PPARgamma remains unclear. Male Sprague-Dawley rats underwent laparotomy and approximately 90 minutes of hemorrhagic shock (40 mm Hg), followed by resuscitation with 4 times the shed blood volume in the form of Ringer's lactate. Adiol (1 mg per kilogram of body weight, iv) was administered at the end of resuscitation. An additional group of rats were treated with PPARgamma antagonist (GW9662, 1 mg/kg ip) along with Adiol and the rats were sacrificed 5 hours thereafter. Hepatic functions were markedly depressed and plasma tumor necrosis factor-alpha, C-reactive protein and endothelin-1 were markedly increased after T-H. DNA-binding activity of nuclear factor kappa B and AP-1, and gene expressions of inducible nitric oxide synthase and endothelin-1 in the liver also increased significantly. These parameters were attenuated by Adiol treatment. These effects were accompanied an increased DNA-binding activity of PPARgamma in T-H-Adiol-treated rats. Treatment of rats with GW9662 prevented the salutary effects of Adiol after T-H. Since blockade of PPARgamma prevented the salutary effects of Adiol on hepatic functions and proinflammatory factors, this finding suggests that Adiol mediated its salutary effects after T-H via the PPARgamma-related pathways.

Resveratrol inhibits angiotensin II-induced endothelin-1 gene expression and subsequent proliferation in rat aortic smooth muscle cells

Resveratrol is a phytoestrogen naturally found in grapes and is the major constituent of wine thought to have a cardioprotective effect. The aims of this study were to examine whether resveratrol alters angiotenisn II-induced cell proliferation and endothelin-1 gene expression and to identify the putative underlying signaling pathways in rat aortic smooth muscle cells. Cultured rat aortic smooth muscle cells were preincubated with resveratrol then stimulated with angiotensin II, after which [3H]thymidine incorporation and endothelin-1 gene expression were examined. The intracellular mechanism of resveratrol in cellular proliferation and endothelin-1 gene expression was elucidated by examining the phosphorylation level of angiotensin II-induced extracellular signal-regulated kinase (ERK). The inhibitory effects of resveratrol (1–100 μM) on angiotensin II-induced DNA synthesis and endothelin-1 gene expression were demonstrated with Northern blot and promoter activity assays. Measurements of 2′7′-dichlorofluorescin diacetate, a redox-senstive fluorescent dye, showed a resveratrol-mediated inhibition of intracellular reactive oxygen species generated by the effects of angiotensin II. The inductive properties of angiotensin II and H2O2 on ERK phosphorylation and activator protein-1-mediated reporter activity were found reversed with resveratrol and antioxidants such as N-acetyl-cysteine. In summary, we speculate that resveratrol inhibits angiotensin II-induced cell proliferation and endothelin-1 gene expression, and does so in a manner which involves the disruption of the ERK pathway via attenuation of reactive oxygen species generation. Thus, this study provides important insight into the molecular pathways that may contribute to the proposed beneficial effects of resveratrol on the cardiovascular system.

Additive amelioration of left ventricular remodeling and molecular alterations by combined aldosterone and angiotensin receptor blockade after myocardial infarction

The mechanisms underlying the clinical benefits of mineralocorticoid receptor antagonism in patients with left ventricular (LV) dysfunction and heart failure (CHF) after myocardial infarction (MI) are poorly understood. We investigated whether long-term (9 weeks) aldosterone antagonism with eplerenone (100 mg/kg/day) provides additional benefit to angiotensin II type 1 (AT1) receptor inhibition with irbesartan (50 mg/kg/day) on cardiac remodeling after MI in rats. Eplerenone monotherapy, like AT1 receptor blockade, significantly reduced LV end-diastolic pressure (LVEDP), end-systolic volume (LVESV) and end-diastolic volume (LVEDV) compared to placebo. Improvement of LV dilation by aldosterone antagonism was associated with a significant reduction of increased AT1 receptor, angiotensin-converting enzyme (ACE) and endothelin-1 gene expression in the noninfarcted LV myocardium. Combination therapy with irbesartan led to a substantial further leftward shift of the LV pressure-volume curve and decrease in LVEDP, LVESV and LVEDV. Moreover, combination therapy significantly improved LV systolic and diastolic function and reversed LV alterations of alpha- and beta-myosin heavy-chain isoforms, ANF and SERCA2 ATPase expression more effectively than monotherapies. LV collagen type I and type III expression as well as interstitial fibrosis were substantially increased in placebo CHF rats, similarly decreased by eplerenone and irbesartan, and further reduced by eplerenone/irbesartan. However, no additive effects of eplerenone/irbesartan on myocardial AT1 receptor, ACE and endothelin-1 mRNAs were observed. Aldosterone receptor antagonism provides additional benefit to AT1 receptor blockade on LV function and remodeling associated with improvement of molecular alterations responsible for progressive contractile dysfunction post-MI.

Hemorrhagic Shock-Induced Vascular Hyporeactivity in the Rat: Relationship to Gene Expression of Nitric Oxide Synthase, Endothelin-1, and Select Cytokines in Corresponding Organs

Our previous work observed that vascular hyporeactivity to norepinephrine (NE) developed after hemorrhage and the response was not the same in the 4 arteries examined. To evaluate possible mechanisms involved, the present study investigated the gene expression of iNOS, eNOS, IL-1beta, IL-6, TNF-alpha, and endothelin-1 in the corresponding organs, and the roles of nitric oxide (NO) and endothelin (ET). LAnesthetized rats (n=7/time point/group) were hemorrhaged to a mean arterial pressure of 50 mmHg for 60 min. The vascular reactivity of the superior mesenteric (SMA), celiac (CA), left renal (LRA), and left femoral arteries (LFA) to NE was measured at baseline, at the end of the hypotensive period (E), and at 1, 2, and 4 h later in the three groups (hemorrhage, hemorrhage+NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, or hemorrhage+PD142893, an ET receptor antagonist). Gene expression in ileum, left kidney, liver, and skeletal muscle was determined by quantitative RT-PCR at these times. Vascular reactivity of SMA, CA, LRA, and LFA to NE decreased as much as 98% over 4 h compared with baseline. This loss of responsiveness in CA and LFA was more severe than in SMA and LRA. Gene expression of iNOS, eNOS, IL-1beta, IL-6, TNF-alpha, and endothelin-1 in the corresponding organs of select vasculatures increased markedly over baseline levels and the fold increase in mRNA levels of these enzymes and mediators in liver and skeletal muscle was higher than in ileum and left kidney. For example, at 4 h, iNOS expression was over 16-fold higher than baseline in liver and skeletal muscle, but 5- and 7-fold higher in ileum and kidney, respectively. L-NAME or PD142893 partially attenuated the decreased vascular reactivity induced by hemorrhagic shock and attenuated the changes in gene expression observed. These findings suggest that the differential expression of NOS, cytokines, and endothelin-1 in different organs are associated with the development of vascular hyporeactivity after hemorrhagic shock and may account, at least in part, for the vascular bed diversity observed.

Luteolin Inhibits Endothelin-1 Secretion in Cultured Endothelial Cells

We discovered that luteolin, a typical flavonoid contained in various kinds of plants, inhibits the secretion and gene expression of endothelin-1 (ET-1), a potent vasoconstrictor regulating blood pressure, in porcine aortic endothelial cells. Its ED50 was about 10 microM. In addition, the inhibition of ET-1 by a glycoside compound of luteolin (luteolin-6-C-glucoside) was weak.

Use of a mixed endothelin receptor antagonist in portopulmonary hypertension: A safe and effective therapy?

Portopulmonary hypertension (PPHTN), a severe complication of portal hypertension is observed in 3%-6% of patients evaluated for liver transplantation. Endothelin-1, a potent vasoconstrictor, is likely to play a role in the pathogenesis of primary pulmonary hypertension, and, in 2 recent trials, the dual endothelin receptor antagonist bosentan has shown beneficial effects in this disease. A role for endothelins in the development of both pulmonary hypertension and cirrhosis has been suggested. We therefore hypothesized that endothelin receptor blockade may be beneficial in the treatment of PPHTN. We report a case of a 42-year-old patient with PPHTN and alcoholic cirrhosis treated with the mixed endothelin receptor antagonist bosentan. The patient rapidly improved from NYHA IV to stage II, experienced a remarkable improvement of 6-minute walking distance from 0 to 590 m within 6 months, and resumed working full-time as a locksmith after 7 months of treatment. Improvement of cardiovascular parameters included a reduction of pulmonary vascular resistance by 60%, a decrease of mean pulmonary artery pressure (mPAP) from 55 to 44 mm Hg at 9 months, and a decline of plasma B-type natriuretic peptide (BNP) from 339 pg/mL to 19 pg/mL after 1 year. There were no adverse events except for a transient decrease in systemic blood pressure. To our knowledge, this is the first report of a patient with PPHTN treated with an endothelin receptor antagonist. The marked and sustained improvement supports the undertaking of controlled studies of the safety and efficacy of bosentan in PPHTN.

Xanthoangelol D Isolated from the Roots of Angelica keiskei Inhibits Endothelin-1 Production through the Suppression of Nuclear Factor-.KAPPA.B

Transcription factor nuclear factor-kappa B (NF-kappaB) has been demonstrated to be important in regulating various gene expressions such as cytokines, adhesion molecules, and endothelin-1 (ET-1) in vascular endothelial cells. In the present study, we show the effects of xanthoangelol, xanthoangelol D, E, and F, which isolated from the root of Angelica keiskei KOIDZUMI (Umbelliferae), on NF-kappaB activation and ET-1 gene expression in cultured porcine aortic endothelial cells (PAECs). Treatments of xanthoangelol D but not xanthoangelol, xanthoangelol E and F markedly suppressed both of basal and tumor necrosis factor-alpha (TNF-alpha)-induced NF-kappaB activation in PAECs. To clarify the mechanism of xanthoangelol D-induced suppression on NF-kappaB activation, we evaluated the effects of xanthoangelol D on phosphorylation and degradation of IkappaBalpha, an inhibitory protein bound to NF-kappaB, and obtained evidence that xanthoangelol D selectively suppresses the phosphorylation of IkappaBalpha rather than the degradation of phosphorylated IkappaBalpha. In addition, xanthoangelol D significantly attenuated basal and TNF-alpha-induced prepro ET-1 mRNA expression in PAECs. These results suggest that xanthoangelol D may be useful for the treatment of various vascular diseases involved NF-kappaB activation.

TNF-α induces interleukin-8 and endothelin-1 expression in human endothelial cells with different redox pathways

We investigated the effect of TNF-α on interleukin-8 (IL-8) and endothelin-1 (ET-1) expression, and their different signal transduction pathways. Human dermal microvascular endothelial cells (HMECs) were treated with TNF-α. By Northern blot analysis, TNF-α at 50, 100, 200, and 400 U/ml significantly induced IL-8 mRNA expression by 206%, 252%, 211%, and 158%, respectively, as compared to controls ( p < 0.05). Overexpression of human superoxide dismutase (SOD) by adenovirus-mediated gene transfer or addition of exogenous hydrogen peroxide (H 2O 2) significantly enhanced TNF-α-induced IL-8 mRNA expression. Furthermore, HMECs treated with TNF-α at 50, 100, and 200 U/ml significantly increased ET-1 mRNA expression by 71%, 82%, and 66%, respectively ( p < 0.05). By contrast, SOD gene transfer and exogenous H 2O 2 significantly inhibited TNF-α-induced ET-1 mRNA expression. Thus, TNF-α significantly induces both IL-8 and ET-1 gene expression in HMECs possibly through different redox signaling pathways. H 2O 2 enhances TNF-α-induced IL-8 expression, but inhibits TNF-α-induced ET-1 expression.

Sesamin induces nitric oxide and decreases endothelin-1 production in HUVECs

Sesamin has been proved to be antihypertensive. Nitric oxide (NO) is the most important vascular relaxing factor that is regulated in endothelium. Endothelin-1 (ET-1) is characterized as a potent vasoconstrictor and is also regulated in endothelium. Alterations in the endothelial production of NO and ET-1 are known to correlate with hypertension. This study investigated the effect of sesamin on NO and ET-1 in the human umbilical vein endothelial cells (HUVECs). The concentrations of NO and ET-1 in the medium of HUVECs treated by sesamin were measured. The mRNA and protein expressions of nitric oxide synthase (NOS), endothelin converting enzyme-1 (ECE-1), and endothelin-1 (ET-1) were also investigated. Other than the mRNA and protein expression, NOS activity and cyclic GMP (cGMP) were detected. The NO concentration was detected by colorimetric assay. The cGMP and ET-1 were analyzed by EIA. The eNOS, ECE-1, and ET-1 mRNA expressions were assayed by Northern blot. The eNOS and ECE-1 protein expressions were analyzed by Western blot. The NOS activity was assayed by detecting the level of [H]-1-citrullin transformed from [H]-1-arginine. Sesamin not only increased the NO concentration in the medium of HUVECs in a dose-dependent manner after 24 h, but also induced eNOS mRNA and protein expressions. NOS activity in the HUVECs was also induced by sesamin. The content of cGMP was induced by sesamin through NO signaling. On the other hand, the ET-1 concentration in the medium of HUVECs treated by sesamin was suppressed in a dose-dependent manner after 24 h. The ECE-1 protein and mRNA expressions were also inhibited by sesamin. However, the mRNA expression of prepro ET-1 was not influenced by sesamin. From the above results, it is suggested that sesamin may improve hypertension by its ability to induce NO and inhibit ET-1 production from endothelial cells. The increase of NO by sesamin is through the induction of eNOS gene expression. The decrease of ET-1 by sesamin is through the inhibition of ECE gene expression, but is not through the inhibition of prepro ET-1 gene expression.

PDE-4 Inhibitor Rolipram Prevents Hypoxia Induced Pulmonary Hypertension in Transgenic Sickle Cell Sad Mice.

Pulmonary hypertension (PH) is one of the major causes of morbidity and mortality for young-adult patients with sickle cell disease (SCD). Little is known about the role of vaso-active agents, pro-inflammatory and pro-fibrotic mediators in the genesis of PH in SCD.We developed a model for studying PH in transgenic sickle cell SAD mice. Wild-type (WT) and SAD mice aged between 4 and 6 months were divided in groups of 7 animals each. One group from each strain served as control under normoxia, while a second group from each strain was exposed to 7 days hypoxia (8% oxygen). The following parameters were evaluated in normoxic and hypoxic groups: lung histopathology, complete blood count, reticulocytes, bronchoalveolar lavage (BAL) total leokocyte and neutrophil count, BAL levels of IL-6, IL-10, IL-1β and TNF-α. The expression of nine genes was examined by quantitative RT-PCR. These included endothelin-1 (ET-1), endothelin-1 receptor (ET-1R), cycloxygenase-2 (COX2), NF-kBp65, transforming-growth-factor-1β (TGF-1β), metalloproteinase-2 and 9 (MMP-2, MMP-9), IL-10 and IL-1β.Two of the seven SAD mice exposed to hypoxia died at day fourth. The remaining five mice were sacrified at day seven and showed: a) increased pulmonary arterial wall thickness and α-actin staining, suggesting a vascular remodeling; b) lung injury compatible with PH in SCD; c) increased BAL total leukocyte and neutrophil count; d) increased BAL IL1β, IL10, IL6 and TNF-α; e) increased peripheral neutrophil and reticulocyte counts, hematocrit and hemoglobin levels. In lungs of hypoxic SAD mice, ET-1, ET-1R, TGF-1β, MMP-2, MMP-9, IL-1β and IL-10 gene expression were induced, with no significant changes in NF-Kbp65 gene expression. These data were consistent with a model of PH in SCD.We then assessed the effect of the PDE-4 inhibitor ROLIPRAM on the PH in seven hypoxic SAD mice. ROLIPRAM was administrated at the dosage of 30 mg/Kg/day by gavage. No deaths were observed in this group. ROLIPRAM a) protected SAD mice from hypoxia induced lung injury; b) decreased BAL total leukocyte and neutrophil count; c) decreased BAL IL-1β, IL-6 and IL-10 levels; d) reduced peripheral neutrophil count; d) normalized and/or modulated the expression of the hypoxia-induced genes. These data suggest that the PDE-4 inhibitor ROLIPRAM has anti-inflammatory properties and could have beneficial effects on the hypoxia-induced airway-remodeling and possibly PH in sickle cell disease.

Exercise-induced Tissue-specific Change in Gene Expression of Endothelin-1

Exercise causes an integrated physiological response (e.g., an increase in cardiac output and a significant redistribution of tissue blood flow), which greatly increases blood flow in active muscles but decreases it in the splanchnic circulation. Endothelin-1 (ET-1) is a potent vasoconstrictor peptide produced by vascular endothelial cells. We hypothesized that exercise causes a tissue-specific change in ET-1 production in various organs. We studied whether a bout of exercise alters the expression of preproET-1 mRNA in various tissues of rats. The rats were randomly divided into exercise rats and control rats. Exercise rats performed treadmill running for 30 minutes at a speed of 30 m/minute. Control rats remained at rest during the same period (30 minutes). Immediately after the exercise and rest, the abdominal artery, liver, muscles (tibialis anterior muscle, plantar muscle and soleus) and adipose tissue were quickly removed and the expression of preproET-1 mRNA in these tissues was determined by real-time quantitative polymerase chain reaction analysis. The expression of preproET-1 mRNA in these tissues was changed in a different manner by acute exercise (i.e., increased, decreased, or unchanged). The expression of preproET-1 mRNA in the aorta was significantly decreased by exercise, whereas that in the plantar muscle was significantly increased by exercise. There were no significant differences in the expression of preproET-1 mRNA in the liver, tibialis anterior muscle, soleus and adipose tissue between the exercise rats and the control rats. These results show that the expression of preproET-1 mRNA was changed tissue-specifically by acute exercise. We demonstrate that acute exercise causes tissue-specific changes in the production of ET-1 and that these alterations may participate in the integrated physiological response during exercise.

Endothelin Gene Expression During Ischemia and Reperfusion

Endothelin-1 (ET-1) probably plays an important role in myocardial damage in acute ischemia. Coronary sinus ET-1 and its precursor big endothelin-1 (big ET-1) levels and also tissue levels of preproendothelin-1 mRNA (ET-1 mRNA) were investigated in an in vivo canine ischemia-reperfusion model in nine consecutive mongrel dogs, surviving 30-minute ligation of the left descending coronary artery followed by a 90-minute reperfusion period. Samples were collected before and at the end of ischemia and during reperfusion. ET-1 and big ET-1 were obtained by immunoprecipitation and detected by western blotting. The ET-1 mRNA level was assessed by reverse transcription-polymerase chain reaction. During ischemia the plasma ET-1 levels and big ET-1 levels did not change significantly, while the myocardial ET-1 mRNA level decreased to 57.8%. During reperfusion an increase of the coronary sinus ET-1 and big ET-1 levels was observed (control versus reperfusion, 90 minutes; ET-1, 15.2 +/- 4.18 fmol/mL versus 23.2 +/- 5.23 fmol/mL, P < 0.01; big ET-1, 14.7 +/- 5.9 fmol/mL versus 27.2 +/- 7.1 fmol/mL, P < 0.001). Simultaneously, the ET-1 mRNA level increased by 322% relative to the ischemic and by 214% relative to the baseline level. The decrease of ET-1 mRNA during ischemia may be due to degradation and decreased metabolism in the hypoxic cells locally. The elevation of the ET-1 mRNA level during reperfusion indicates rapid big ET-1 synthesis. This was confirmed by the increases in big ET-1 and ET-1 plasma levels. This latter can be associated with the generation of reperfusion arrhythmias or other complications of acute myocardial infarction.

Differential Changes in Endothelin-1 and Endothelial Nitric Oxide Synthase Gene Expression in Rats with a Chronic High Pulmonary Flow

The expressions of endothelin-1 (ET-1) and endothelial nitric oxide synthase were assessed in the lung of adult Wistar rats (n = 6/group) undergoing an abdominal aortocaval shunt to increase pulmonary blood flow for 4, 8 or 12 weeks. The shunt resulted in significant medial hypertrophy of the pulmonary artery without significant increases in pulmonary and systemic arterial pressure. A competitive reverse transcriptase-polymerase chain reaction demonstrated significant increases in pulmonary preproET-1 mRNA at 12 weeks (mean +/- standard error of the mean; shunt, 1.82 +/- 0.12; sham, 1.00 +/- 0.15; P < 0.05) and in pulmonary endothelial nitric oxide synthase mRNA at both 8 weeks (shunt, 1.57 +/- 0.12; sham, 1.00 +/- 0.18; P < 0.05) and 12 weeks (shunt, 1.89 +/- 0.18; sham, 1.00 +/- 0.13; P < 0.05). In addition, western blot analysis showed increases in pulmonary endothelial nitric oxide synthase protein by 126% and 164% at 8 and 12 weeks, respectively, in the shunt animals. However, the plasma ET-1 concentrations and the lung ET-1 contents were unchanged. These results indicate that endothelial nitric oxide synthase gene expression was upregulated, prior to that of ET-1, at the transcriptional level during pulmonary vascular remodeling in this chronic shunt model.

Time Course Alteration of Endothelin-1 Gene Expression in the Heart during Exercise and Recovery from Post-exercise Periods in Rats

Endothelin-1 (ET-1) is produced by endothelial cells and cardiac myocytes. ET-1 has potent positive inotropic and chronotropic effects in the heart and causes myocardial cell hypertrophy. We investigated the alteration of gene expression of ET-1 in the heart of rats during acute exercise and 24 hour postexercise periods. Sprague-Dawley rats performed treadmill running for 30 minutes at a speed of 30 m/minute. We determined the expression of preproendothelin-1 mRNA in the rat hearts of resting (control) and 0, 0.5, 1, 3, 6, 12, and 24 hour post-exercise time points, respectively. The percent changes in expression of preproendothelin-1 mRNA in the heart from resting control rats were significantly increased at the time point of 1 hour post-exercise (199.0 +/- 33.6%, P < 0.05), and this enhancement returned to the level of resting control rats at the time points of 6, 12, and 24 hours post-exercise. These results suggest that a bout of exercise causes time-related enhancement of gene expression of ET-1 in the rat hearts during acute exercise and 24 hour post-exercise periods. Therefore, an exercise-induced change of ET-1 gene expression in the heart may participate in mechanisms of exercise-induced and/or training-induced adaptive responses of the heart.

Effect of Electrical Modification of Cardiomyocytes on Transcriptional Activity through 5'-AMP-activated Protein Kinase

Endothelin-1 (ET-1) is known as an aggravating factor of the failing cardiomyocytes and, therefore, a therapeutic method is indispensable to decrease cardiac ET-1 expression. To study the mechanisms of how cardiac ET-1 gene expression can be modified, we investigated the effect of electrical stimulation against cardiomyocytes. Considering the physiology of cardiomyocytes, in vitro cultured cardiomyocytes demonstrate distinctive features from in vivo cardiomyocytes (i.e. the absence of a stretch along with electrical stimulation). In this study, we especially focused on the effect of electrical stimulation. The electrical stimulation reduced the gene expression of ET-1 mRNA in rat primary cultured cardiomyocytes. Furthermore, this effect on the transcriptional modification of ET-1 was also identified in H9c2 cells. Luciferase activity using H9c2 cells was decreased by electrical stimulation in the early phase, suggesting that the attenuation of the ET-1 gene transcription by electrical stimulation should be due to a transcriptional repression. To further investigate a trigger signal involved in the transcriptional repression, phosphorylation of 5'-AMP-activated protein kinase (AMPK) was evaluated. It was revealed that AMPK was phosphorylated in the early phase of electrical stimulation of H9c2 cells as well as in rat primary cultured cardiomyocytes, and that AMPK phosphorylation was followed by ET-1 transcriptional repression, suggesting that electrical stimulation directly regulates AMPK. This study suggests that AMPK activation in cardiomyocytes plays a crucial role in the transcriptional repression of ET-1.

Periostin as a novel factor responsible for ventricular dilation.

Periostin is highly expressed in the myocardium in patients with heart failure. However, no report has documented the function of periostin. To identify the function of periostin in the pathophysiology of heart failure, overexpression or loss of function of the periostin gene was examined by direct transfection into the rat heart. Rats transfected with the periostin gene by the HVJ-liposome method showed left ventricular (LV) dilation as assessed by echocardiography, accompanied by an increase in periostin expression. Consistently significant differences were observed in LV pressure, LV end-diastolic pressure, LV dP/dt(max), and LV dP/dt(min) at 6 and 12 weeks after transfection in rats transfected with the periostin gene, accompanied by a decrease in cardiac myocytes and an increase in collagen deposition. Importantly, periostin has the ability to inhibit the spreading of myocytes and the adhesion of cardiac fibroblasts with or without fibronectin. Markers of cardiac dysfunction such as brain natriuretic peptide and endothelin-1 gene expression were significantly increased after transfection in the LV of rats transfected with the periostin gene. These data demonstrate that overexpression of the periostin gene led to cardiac dysfunction. Thus, we examined the inhibition of periostin in Dahl salt-sensitive rats by an antisense strategy because periostin is highly expressed in heart failure. Importantly, inhibition of periostin gene expression resulted in a significant increase in survival rate, accompanied by an improvement of LV function. The present study demonstrates the contribution of the periostin gene to cardiac dilation in animal models. Inhibition of periostin might become a new therapeutic target for the treatment of heart failure.