Increased Plasma Endothelin-1 Levels Research Articles

Genetic associations of cardiovascular risk genes in European patients with coronary artery spasm.

Coronary artery spasm (CAS) is a frequent finding in patients presenting with angina pectoris. Although the pathogenesis of CAS is incompletely understood, previous studies suggested a genetic contribution. Our study aimed to elucidate genetic variants in a cohort of European patients with angina and unobstructed coronary arteries who underwent acetylcholine (ACh) provocation testing. A candidate association analysis of 208 genes previously associated with cardiovascular conditions was performed using genotyped and imputed variants in patients grouped in epicardial (focal, diffuse) CAS (n = 119) and microvascular CAS (n = 87). Patients with a negative ACh test result (n = 45) served as controls. We found no association below the genome-wide significance threshold of p < 5 × 10-8, thus not confirming variants in ALDH2, NOS3, and ROCK2 previously reported in CAS patients of Asian ancestry. However, the analysis identified suggestive associations (p < 10-05) for the groups of focal epicardial CAS (CDH13) and diffuse epicardial CAS (HDAC9, EDN1). Downstream analysis of the potential EDN1 risk locus showed that CAS patients have significantly increased plasma endothelin-1 levels (ET-1) compared to controls. An EDN1 haplotype comprising rs9349379 and rs2070698 was significantly associated to ET-1 levels (p = 0.01). In summary, we suggest EDN1 as potential genetic risk loci for patients with diffuse epicardial CAS, and European ancestry. Plasma ET-1 levels may serve as a potential cardiac marker.

Loss of CEACAM1 in endothelial cells causes hepatic fibrosis

ObjectivesHepatocytic CEACAM1 plays a critical role in NASH pathogenesis, as bolstered by the development of insulin resistance, visceral obesity, steatohepatitis and fibrosis in mice with global Ceacam1 (Cc1) deletion. In contrast, VECadCre+Cc1fl/fl mice with endothelial loss of Cc1 manifested insulin sensitivity with no visceral obesity despite elevated NF-κB signaling and increased systemic inflammation. We herein investigated whether VECadCre+Cc1fl/fl male mice develop hepatic fibrosis and whether this is mediated by increased production of endothelin1 (ET1), a transcriptional NF-κB target. MethodsVECadCre+Et1.Cc1fl/fl mice with combined endothelial loss of Cc1/Et1 genes were generated. Histological and immunohistochemical analyses were conducted on their livers and on liver tissue biopsies from adult patients undergoing bariatric surgery or from patients with NASH diagnosis receiving liver transplant. ResultsHepatic fibrosis and inflammatory infiltration developed in VECadCre+Cc1fl/fl liver parenchyma. This was preceded by increased ET1 production and reversed with combined endothelial loss of Et1. Conditioned media from VECadCre+Cc1fl/fl, but not VECadCre+Et1.Cc1fl/fl primary liver endothelial cells activated wild-type hepatic stellate cells; a process inhibited by bosentan, an ETAR/ETBR dual antagonist. Consistently, immunohistochemical analysis of liver biopsies from patients with NASH showed a decline in endothelial CEACAM1 in parallel with increased plasma endothelin1 levels and progression of hepatic fibrosis stage. ConclusionsThe data demonstrated that endothelial CEACAM1 plays a key role in preventing hepatic fibrogenesis by reducing autocrine endothelin1 production.

Intravenous Endothelin-1 Infusion Does Not Induce Aura or Headache in Migraine Patients With Aura.

To investigate whether intravenously infused provokes migraine aura and migraine headache in migraine patients with aura. Migraine with aura has been associated with endothelial dysfunction and increased stroke risk. The initiating mechanism of migraine aura symptoms is not known. Experimental provocation of migraine headache using vasoactive peptides has provided tremendous advances in the understanding of migraine pathophysiology but substances that can induce migraine aura have not been identified. Endothelin-1 (ET-1), an endogenous, potent vasoconstrictor peptide released from the vascular endothelium, has been proposed to trigger migraine aura. This hypothesis is based on reports of increased plasma ET-1 levels early during the migraine attacks and the observation that ET-1 applied to the cortical surface potently induces the cortical spreading depolarization, the underlying electrophysiological phenomenon of migraine aura, in animals. Further, endothelial damage due to, for example, carotid puncture and vascular pathology is known to trigger aura episodes. We investigated whether intravascular ET-1 would provoke migraine aura in patients. Using a two-way crossover, randomized, placebo-controlled, double-blind design, we infused high-dose (8ng/kg/minutes for 20minutes) intravenous ET-1 in patients with migraine with typical aura. The primary end-point was the difference in incidence of migraine aura between ET-1 and placebo. Experiments were carried out at a public tertiary headache center (Danish Headache Center, Rigshospitalet Glostrup, Denmark). Fourteen patients received intravenous ET-1. No patients reported migraine aura symptoms or migraine headache during or up to 24hours following the ET-1 infusion. Four patients reported mild to moderate headache only on the ET-1 day, 3 patients reported moderate headache on the placebo day, and 1 patient reported mild headache on both days. No serious adverse events occurred during or after infusion. Provocation of migraine aura by procedures or conditions involving vascular irritation is unlikely to be mediated by ET-1.

Genetic susceptibility of five tagSNPs in the endothelin-1 (EDN1) gene to coronary artery disease in a Chinese Han population.

Endothelin-1 (ET-1) plays important roles in endothelial dysfunction, vascular physiology, inflammation, and atherosclerosis. Nonetheless, the role of ET-1 (EDN1) gene variants on coronary artery disease (CAD) risk remains poorly understood. The aim of the present study was to evaluate the role of EDN1 gene polymorphisms on individual susceptibility to CAD. We genotyped five tagSNPs (single-nucleotide polymorphisms) (rs6458155, rs4145451, rs9369217, rs3087459, and rs2070699) within EDN1 gene in 525 CAD patients and 675 control subjects. In a multivariate logistic regression analysis, we detected an association of rs6458155 in EDN1 gene with the CAD risk; compared with the TT homozygotes, the CT heterozygotes (odds ratio (OR) = 1.53, 95% confidence interval (CI) = 1.02–2.29, P=0.040) and the CC homozygotes (OR = 1.55, 95% CI = 1.01–2.36, P=0.043) were statistically significantly associated with the increased risk for CAD. A similar trend of the association was found in dominant model (OR = 1.53, 95% CI = 1.05–2.25, P=0.029). Consistently, the haplotype rs6458155C-rs4145451C containing rs6458155 C allele exhibited the increased CAD risk (OR = 1.22, 95% CI = 1.03–1.43, and P=0.018). In addition, CT genotype of rs6458155 conferred the increased plasma ET-1 levels compared with TT genotype (P<0.05). No association of the other four tagSNPs in EDN1 gene with CAD risk was observed. In conclusion, our study provides the first evidence that EDN1 tagSNP rs6458155 is associated with CAD risk in the Chinese Han population, which is probably due to the influence of the circulating ET-1 levels.

Transition from post-capillary pulmonary hypertension to combined pre- and post-capillary pulmonary hypertension in swine: a key role for endothelin.

Key points Passive, isolated post‐capillary pulmonary hypertension (PH) secondary to left heart disease may progress to combined pre‐ and post‐capillary or ‘active’ PHThis ‘activation’ of post‐capillary PH significantly increases morbidity and mortality, and is still incompletely understood.In this study, pulmonary vein banding gradually produced post‐capillary PH with structural and functional microvascular remodelling in swine.Ten weeks after banding, the pulmonary endothelin pathway was upregulated, likely contributing to pre‐capillary aspects in the initially isolated post‐capillary PH.Inhibition of the endothelin pathway could potentially stop the progression of early stage post‐capillary PH. Passive, isolated post‐capillary pulmonary hypertension (IpcPH) secondary to left heart disease may progress to combined pre‐ and post‐capillary or ‘active’ PH (CpcPH) characterized by chronic pulmonary vascular constriction and remodelling. The mechanisms underlying this ‘activation’ of passive pulmonary hypertension (PH) remain incompletely understood. Here we investigated the role of the vasoconstrictor endothelin‐1 (ET) in the progression from IpcPH to CpcPH in a swine model for post‐capillary PH. Swine underwent pulmonary vein banding (PVB; n = 7) or sham‐surgery (Sham; n = 6) and were chronically instrumented 4 weeks later. Haemodynamics were assessed for 8 weeks, at rest and during exercise, before and after administration of the ET receptor antagonist tezosentan. After sacrifice, the pulmonary vasculature was investigated by histology, RT‐qPCR and myograph experiments. Pulmonary arterial pressure and resistance increased significantly over time. mRNA expression of prepro‐endothelin‐1 and endothelin converting enzyme‐1 in the lung was increased, while ETA expression was unchanged and ETB expression was downregulated. This was associated with increased plasma ET levels from week 10 onward and a more pronounced vasodilatation to in vivo administration of tezosentan at rest and during exercise. Myograph experiments showed decreased endothelium‐dependent vasodilatation to Substance P and increased vasoconstriction to KCl in PVB swine consistent with increased muscularization observed with histology. Moreover, maximal vasoconstriction to ET was increased whereas ET sensitivity was decreased. In conclusion, PVB swine gradually developed PH with structural and functional vascular remodelling. From week 10 onward, the pulmonary ET pathway was upregulated, likely contributing to pre‐capillary activation of the initially isolated post‐capillary PH. Inhibition of the ET pathway could thus potentially provide a pharmacotherapeutic target for early stage post‐capillary PH.

ET-1 Plasma Levels, Aqueous Flare, and Choroidal Thickness in Patients with Retinitis Pigmentosa.

Purpose. To assess endothelin-1 (ET-1) plasma levels, choroidal thickness, and aqueous flare in patients with early stage retinitis pigmentosa (RP) and to search for possible correlations. Methods. We compared 24 RP patients with 24 healthy controls. Choroidal thickness and aqueous flare were measured, respectively, by using a spectral domain optical coherence tomography and a laser flare-cell meter, whereas plasma samples were obtained from each patient to evaluate ET-1 plasma levels. Results. Notably, RP subjects showed significantly increased ET-1 plasma levels and reduced choroidal thickness compared with controls: 2.143 ± 0.258 versus 1.219 ± 0.236 pg/mL, P < 0.002, and 226.75 ± 76.37 versus 303.9 ± 39.87 μm, P < 0.03, respectively. Higher aqueous flare values were also demonstrated in RP compared to controls: in detail, 10.51 ± 3.97 versus 5.66 ± 1.29 photon counts/ms, P < 0.0001. Spearman's correlation test highlighted that the increase of ET-1 plasma levels was related with the decrease of choroidal thickness (r = −0.702; P < 0.023) and the increase of aqueous flare (r = 0.580; P < 0.007). Conclusions. Early stage RP patients show a breakdown of blood-ocular barrier and increased ET-1 plasma levels and these findings may contribute to the reduction of choroidal thickness.

ET-1 plasma levels, choroidal thickness and multifocal electroretinogram in retinitis pigmentosa

AimTo assess the relationship between both photoreceptor function and choroidal thickness and endothelin-1 (ET-1) plasma levels in patients with early stage retinitis pigmentosa (RP). Main methodsWe compared 24 RP patients (14 males and 10 females), 25 to 42years of age (mean age: 34±7years) with 24 healthy controls (12 males and 12 females) aged between 28 and 45years (mean 36±6.8years). All patients underwent visual field test, electroretinogram and multifocal-electroretinogram and choroidal thickness measurement by using spectral domain optical coherence tomography. Key findingsRP patients had a visual acuity of 0.95, a mean defect of the visual field of −7.90±1.75dB, a pattern standard deviation index of 6.09±4.22dB and a b-wave ERG amplitude of 45.08±8.24μV. Notably RP subjects showed significantly increased ET-1 plasma levels and reduced choroidal thickness compared with controls: respectively, 2.143±0.258pg/ml vs. 1.219±0.236pg/ml; p<0.002 and 226.75±76.37μm vs. 303.9±39.87μm; p<0.03. Spearman's correlation test highlighted that the increase of ET-1 plasma levels was related with the decrease of choroidal thickness (r=−0.702; p<0.023) and the increase of implicit time in both ring 2 (r=−0.669; p<0.034) and ring 3 (r=−0.883; p<0.007) of mfERG. SignificanceIncreased ET-1 plasma levels may play a key role in the impairment of retinal and choroidal blood flow due to the vasoconstriction induced by ET-1. This could lead to worsening of the abiotrophic process of the macular photoreceptors.

Circulating Endothelin-1 Alters Critical Mechanisms Regulating Cerebral Microcirculation

Endothelin-1 (ET1) is a potent vasoconstrictor peptide implicated in the cerebrovascular alterations occurring in stroke, subarachnoid hemorrhage, and brain trauma. Brain or circulating levels of ET1 are elevated in these conditions and in risk factors for cerebrovascular diseases. Most studies on the cerebrovascular effects of ET1 have focused on vascular smooth muscle constriction, and little is known about the effect of the peptide on cerebrovascular regulation. We tested the hypothesis that ET1 increases cerebrovascular risk by disrupting critical mechanisms regulating cerebral blood flow. Male C57Bl6/J mice equipped with a cranial window were infused intravenously with vehicle or ET1, and somatosensory cortex blood flow was assessed by laser Doppler flowmetry. ET1 infusion increased mean arterial pressure and attenuated the blood flow increase produced by neural activity (whisker stimulation) or neocortical application of the endothelium-dependent vasodilator acetylcholine but not A23187. The cerebrovascular effects of ET1 were abrogated by the ET(A) receptor antagonist BQ123 and were not related to vascular oxidative stress. Rather, the dysfunction was dependent on Rho-associated protein kinase activity. Furthermore, in vitro studies demonstrated that ET1 suppresses endothelial nitric oxide (NO) production, assessed by its metabolite nitrite, an effect associated with Rho-associated protein kinase-dependent changes in the phosphorylation state of endothelial NO synthase. Collectively, these novel observations demonstrate that increased ET1 plasma levels alter key regulatory mechanisms of the cerebral circulation by modulating endothelial NO synthase phosphorylation and NO production through Rho-associated protein kinase. The ET1-induced cerebrovascular dysfunction may increase cerebrovascular risk by lowering cerebrovascular reserves and increasing the vulnerability of the brain to cerebral ischemia.

Ethanol Consumption Increases Endothelin-1 Expression and Reactivity in the Rat Cavernosal Smooth Muscle

We investigated the effects of chronic ethanol consumption on the cavernosal smooth muscle (CSM) reactivity to endothelin-1 (ET-1) and the expression of ET system components in this tissue. Male Wistar rats were treated with heavy dose of ethanol (20% v/v) for 6 weeks. Reactivity experiments were performed in the isolated rat CSM. Plasma and CSM nitrate generation and also superoxide anion generation in rat CSM were measured by chemiluminescence. Protein and mRNA levels of pre-pro-ET-1, endothelin-converting enzyme-1 (ECE-1), ETA and ETB receptors, eNOS, nNOS and iNOS were assessed by western immunoblotting and quantitative real-time polymerase chain reaction, respectively. Chronic ethanol consumption increased plasma ET-1 levels and the contractile response induced by this peptide in the isolated CSM. The relaxation induced by acetylcholine, but not IRL1620, a selective ETB receptor agonist, was reduced in CSM from ethanol-treated rats. BQ123, a selective ETA receptor antagonist, produced a rightward displacement of the ET-1 concentration-response curves in CSM from control, but not ethanol-treated rats. Reduced levels of nitrate were found in the plasma and CSM from ethanol-treated rats. Ethanol consumption increased superoxide anion generation in the rat CSM. The mRNA levels of pre-pro-ET-1, ECE-1, ETA and ETB receptors, eNOS, nNOS and iNOS were not altered by ethanol consumption. Protein levels of ET-1, ETA receptor and iNOS were higher in the CSM from rats chronically treated with ethanol. The major findings of the present study are that heavy ethanol consumption increases plasma ET-1 levels and the contraction induced by the peptide in the CSM. Increased CSM reactivity to ET-1 and altered protein levels of ET-1 and ETA receptors could play a role in the pathogenesis of erectile dysfunction associated with chronic ethanol consumption.

The activation of endothelin-1 pathway during methionine-induced homocysteinemia mediates endothelial dysfunction in hypertensive individuals

Endothelin-1 (ET-1) is a key regulator of arterial blood pressure in humans, and homocysteinemia is associated with increased oxidative stress. It is still unclear whether homocysteine-induced oxidative stress is implicated in the regulation of ET-1 expression. We examined the impact of acute homocysteinemia on endothelial function in hypertensive patients and healthy individuals, and the potential role of ET-1. In this double-blind, placebo-controlled study, 39 hypertensive and 49 healthy individuals were randomized to receive high-dose vitamins (2 g vitamin C and 800IU vitamin E) or placebo followed by methionine loading 100 mg/kg body weight. Endothelium-dependent dilation (EDD) and endothelium-independent dilation (EID) of the brachial artery were evaluated by plethysmography, at baseline and 4 h postloading (4 h PML). ET-1 was measured by ELISA, whereas total lipid hydroperoxides (per-ox) levels were measured by a commercially available photometric technique. Acute, methionine-induced homocysteinemia decreased EDD in all study groups (P < 0.001 for all), whereas vitamins pretreatment failed to prevent this effect, despite the vitamins-induced reduction of peroxidation in the hypertensives group (P < 0.05). On the contrary, methionine loading significantly increased plasma ET-1 levels only in hypertensives (P < 0.05), an effect which was not prevented by antioxidant vitamins (P < 0.05). EID remained unchanged after methionine loading, in all study groups (P = NS for all groups). Experimental homocysteinemia rapidly blunts endothelial function in both hypertensive individuals and healthy individuals. The rapid elevation of ET-1 levels observed only in hypertensives, suggests that ET-1 may be the key mediator of homocysteine-induced endothelial dysfunction, independently of oxidative stress.

Increased plasma endothelin level as an endothelial marker of cardiovascular risk in patients with active acromegaly: A comparison with plasma homocysteine

The aim of the present study was to evaluate the plasma endothelin-1 (ET-1) and total homocysteine (tHcy) levels as biochemical markers of endothelial dysfunction and atherosclerosis in patients with active and cured acromegaly in order to assess the relationship between the secretory status of growth hormone (GH)/insulin-like growth factor I (IGF-I) and ET-1/tHcy levels. The patients were divided in two subgroups: 1) patients with active disease (n = 30); and 2) patients with nonactive cured acromegaly (n = 21). Plasma ET-1 levels were directly determined by a highly sensitive enzyme immunoassay and plasma tHcy concentrations were measured by a fluorescence polarization immunoassay. In active acromegaly subjects, plasma ET-1 levels were 1.24 +/- 0.2 pmol/L, significantly higher than in both nonactive acromegalics (0.39 +/- 0.1 pmol/L) and age-matched healthy controls (0.49 +/- 0.2 pmol/L) (P < 0.001). Plasma tHcy concentrations, however, did not differ significantly in all studied groups: nonactive acromegalics: 9.54 +/- 4.42 micromol/L; active acromegalics: 9.0 +/- 3.14 micromol/L; and control subjects: 9.96 +/- 2.95 micromol/L (P > 0.05). In conclusion, our study demonstrated that elevated ET-1 levels probably contributed to premature atherosclerosis and cardiovascular disease and represent a new risk factor for endothelial dysfunction and early vascular complications in acromegaly. We propose that GH and IGF-I secretory status are important determinants of plasma ET-1 but not tHcy levels.

Increased plasma endothelin level as an endothelial marker of cardiovascular risk in patients with active acromegaly: A comparison with plasma homocysteine

Increased plasma endothelin level as an endothelial marker of cardiovascular risk in patients with active acromegaly: A comparison with plasma homocysteine

The Endothelin Receptor Blocker Bosentan Inhibits Doxorubicin-Induced Cardiomyopathy

Doxorubicin is a frequently used anticancer drug, but its therapeutic benefit is limited by acute and chronic cardiotoxicity, often leading to heart failure. The mechanisms underlying doxorubicin-induced cardiotoxicity remain unclear. It was previously shown in men that doxorubicin leads to increased endothelin-1 plasma levels. In addition, cardiac-specific overexpression of endothelin-1 in mice resulted in a cardiomyopathy resembling the phenotype following doxorubicin administration. We therefore hypothesized that endothelin-1 is involved in the pathogenesis of doxorubicin cardiotoxicity. In mice (C57Bl/10), we found that doxorubicin (20 mg/kg body weight, i.p.) impaired cardiac function with decreased ejection fraction, diminished cardiac output, and decreased end-systolic pressure points recorded by a microconductance catheter. This impaired function was accompanied by the up-regulation of endothelin-1 expression on mRNA and protein level. In vitro investigations confirmed the regulation of endothelin-1 by doxorubicin and indicated that the doxorubicin-mediated increase of endothelin-1 expression involves epidermal growth factor receptor signaling via the MEK1/2-ERK1/2 cascade, which was further confirmed by immunoblotting studies in the left ventricle of treated animals. Pretreatment of mice with the endothelin receptor antagonist bosentan (100 mg/kg body weight, p.o.) strikingly inhibited doxorubicin-induced cardiotoxicity with preserved indices of contractility. Moreover, bosentan pretreatment resulted in reduced tumor necrosis factor-alpha content, lipid peroxidation, and Bax expression, as well as increased GATA-4 expression. Thus, endothelin-1 plays a key role in mediating the cardiotoxic effects of doxorubicin and its inhibition may be of therapeutic benefit for patients receiving doxorubicin.

Effects of big endothelin-1 in comparison with endothelin-1 on the microvascular blood flow velocity and diameter of rat mesentery in vivo

Synthetic big endothelin-1 (ET-1), a 39-residue precursor of ET-1, has been reported to elicit potent contractile action on helical strip specimens obtained from the porcine coronary artery, but its molar potency was found to be 140-fold lower than that of ET-1 [Saito, Y., Nakao, K., Mukoyama, M., Imura, H., 1990. Increased plasma endothelin level in patients with essential hypertension. N. Engl. J. Med. 322, 205]. It has been hypothesized that the increased rate of production and/or release of ET-1 from the vascular endothelium may contribute to the pathogenesis of hypertension. However, the effects of big ET-1 in comparison with ET-1 on the macrocirculation and microcirculation of the rat mesentery have not been well documented. Thus, our main purpose for this study was to examine the effects of both big ET-1 and ET-1 to clarify the role of phosphoramidon in inhibiting the conversion of big ET-1 to ET-1, by investigating the systemic blood pressure, microvascular blood flow velocity, and diameters of arterioles and venules of the rat mesentery. For this purpose, two groups of experiments were performed. In these experiments, the mesentery was arranged for in situ intravital microscopic observation under transillumination. In the first group of experiments, intravenous cumulative injections of big ET-1 or ET-1 were infused through a catheter inserted into the right jugular vein. Infusion of big ET-1 (1–8 nmol/kg) elicited a long-lasting significant pressor effect. Infusion of big ET-1 (1–2 nmol/kg) elicited a significant dose-dependent increase in the microvascular blood flow velocity both in arterioles (20–30 μm) and venules (30–40 μm). Microvascular diameters exhibited a slight but significant vasodilator effect. However, the infusion of big ET-1 (4–8 nmol/kg) elicited a dose-dependent significant decrease in the blood flow velocities, and diameters returned to control measurements. The administration of ET-1 (0.25–2 nmol/kg) induced a dose-dependent significant decrease in the blood flow velocity of arterioles and venules, and their diameters exhibited a vasoconstrictive effect more prominent in arterioles than in venules. In the second group of experiments, cumulative injections of phosphoramidon (30 mg/kg/10 min) were administered 10 min prior to the infusion of big ET-1. Phosphoramidon significantly suppressed the long-lasting significant pressor effect and significantly inhibited the dose-dependent increase and dose-dependent decrease in the microvascular blood flow velocity produced by big ET-1 in the rat mesenteric microcirculation. This study observed differences in the effects big ET-1 and ET-1 have on the rat mesenteric microcirculation and proposes a possible mechanism explaining these differences. Moreover, phosphoramidon markedly inhibited the conversion of big ET-1 to ET-1 in the rat mesenteric microcirculation, which may suggest an inhibition of the enzyme which converts big ET-1 to ET-1.

Role of endothelin receptor activation in secondary pulmonary hypertension in awake swine after myocardial infarction

We previously observed that pulmonary hypertension secondary to myocardial infarction (MI) in swine is characterized by elevated plasma endothelin (ET) levels and pulmonary vascular resistance (PVR). Consequently, we tested the hypothesis that an increased ET-mediated vasoconstrictor influence contributes to secondary pulmonary hypertension after MI and investigated the involvement of ET(A) and ET(B) receptor subtypes. Chronically instrumented swine with (MI swine; n = 25) or without (normal swine; n = 19) MI were studied at rest and during treadmill exercise (up to 4 km h(-1)), in the absence and presence of the ET(A) antagonist EMD 122946 or the mixed ET(A)/ET(B) antagonist tezosentan. In normal swine, exercise caused a small decrease in PVR. ET(A) blockade had no effect on PVR at rest or during exercise. Conversely, ET(A)/ET(B) blockade decreased PVR but only during exercise (at 4 km h(-1), from 3.0 +/- 0.1 to 2.3 +/- 0.1 mmHg min l(-1); P <or= 0.05). MI increased pulmonary arterial pressure and PVR both at rest and during exercise (both P <or= 0.05). The increased pulmonary arterial pressure correlated with the increased plasma ET levels in resting MI swine (r = 0.71; P <or= 0.01). Furthermore, the pulmonary vasoconstrictor response to ET-1 infusion was enhanced after MI (P <or= 0.05). ET(A)/ET(B) blockade decreased PVR in MI swine from 3.6 +/- 0.3 to 3.1 +/- 0.5 mmHg min l(-1) at rest and from 3.4 +/- 0.3 to 2.4 +/- 0.2 mmHg min l(-1) during exercise at 4 km h(-1) (both P <or= 0.05). This increased response to mixed ET(A)/ET(B) blockade in MI compared to normal swine appeared to be the result of an increased ET(A)-mediated vasoconstriction, as ET(A) blockade decreased PVR in MI swine from 3.4 +/- 0.4 to 2.8 +/- 0.2 mmHg min l(-1) at rest and from 3.1 +/- 0.3 to 2.6 +/- 0.2 mmHg min l(-1) at 4 km h(-1) (both P <or= 0.05). In conclusion, increased plasma ET levels together with increased pulmonary resistance vessel responsiveness to ET result in an exaggerated pulmonary vasoconstrictor influence of ET in swine with a recent MI. This vasoconstrictor influence is the result of an emergent tonic ET(A)-mediated vasoconstriction in addition to the exercise-induced ET(B)-mediated vasoconstriction that is already present in normal swine.

Dynamic and differential changes in myocardial and plasma endothelin in patients undergoing cardiopulmonary bypass

The bioactive peptide endothelin modulates left ventricular function by changing afterload, coronary vascular tone, and myocardial contractility. However, whether increased plasma endothelin levels observed in patients during and after coronary revascularization and cardiopulmonary bypass reflect actual myocardial interstitial levels are unknown. A microdialysis probe (outer diameter: 0.77 mm; length: 4 mm) was placed in the left ventricular apical midmyocardium in 20 patients and myocardial interstitial fluid was collected (2.5 microL/min) at baseline and up to 30 minutes after cardiopulmonary bypass. Myocardial interstitial and systemic arterial endothelin were measured by radioimmunoassay. Baseline myocardial interstitial endothelin was over 6-fold higher than plasma (20.11 +/- 2.07 vs 3.19 +/- 0.25 fmol/mL, P < .05). Plasma endothelin increased by 23% +/- 12% at 60 minutes of cardiopulmonary bypass whereas myocardial interstitial endothelin increased by 105% +/- 24%, P < .05), and this change was higher than in the plasma ( P < .05). Although no further change in plasma endothelin occurred during cardiopulmonary bypass, myocardial interstitial levels increased further after crossclamp removal (400% +/- 75%) and remained significantly higher than plasma at separation from cardiopulmonary bypass. The unique findings of this study were 2-fold: First, significant compartmentalization of endothelin exists within the human myocardium. Second, a significantly higher and temporally disparate change in myocardial interstitial endothelin occurs during and after cardiopulmonary bypass when compared with systemic levels. These dynamic changes in myocardial endothelin likely influence coronary vascular tone and contractility.

Elevated Endothelin-1 Expression in Dogs with Heartworm Disease

We explored the involvement of endothelin-1 (ET-1) in the pathophysiology of dog dirofilariasis (heartworm disease caused by Dirofilaria immitis) by analyzing mRNA levels of preproendothelin-1 (PPET-1), the precursor form of ET-1, in cardiopulmonary organs as well as ET-1 peptide levels in plasma. To determine the cDNA sequence and primary protein structure of dog PPET-1, we performed molecular cloning of the full-length cDNA. Based on the determined sequence information, comparative expression analysis of PPET-1 mRNA was carried out by real-time polymerase chain reaction on cardiopulmonary organs from healthy (n=5) and filarial (n=5) dogs. Filarial dogs showed a significantly (p<0.05) higher mRNA expression level in the heart (about one hundred times) and lung (about ten times) than healthy dogs. Analysis of plasma ET-1 levels in healthy (n=10) and filarial (n=10) dogs showed that filarial dogs (6.9+/-2.7 pg/ml) have significantly (p<0.01) increased plasma ET-1 levels compared with healthy dogs (1.4+/-0.3 pg/ml). To assess the pathophysiological significance of ET-1 in dirofilariasis relative to other cardiopulmonary disorders, plasma ET-1 levels determined in dogs diagnosed with mitral regurgitation (n=10), tricuspid regurgitation (n=5), ventricular septal defect (n=5), and patent ductus arteriosus (n=5) were compared to plasma ET-1 levels in filarial dogs. Filarial dogs, which commonly develop serious pulmonary hypertension, exhibited by far the highest ET-1 levels of the disease states examined. Based on the fact that ET-1 is a potent bioactive mediator that induces vasoconstriction and promotes vascular remodeling, these findings suggest that ET-1 plays an important role in the pathophysiology of dog dirofilariasis as an aggravating factor by inducing pulmonary hypertension.

The hemodynamic and neurohormonal effects of low doses of tezosentan (an endothelin A/B receptor antagonist) in patients with acute heart failure

In previous studies (the RITZ project), tezosentan, an intravenous (i.v.)-balanced dual endothelin (ET-A/B) antagonist, in doses of 50 and 100 mg/h, improved the hemodynamics but not the clinical outcome of patients with acute heart failure (AHF). To evaluate the effect of lower doses of tezosentan in patients with AHF. Included were 130 patients hospitalized due to AHF with dyspnea at rest, despite initial treatment, and were in need of hemodynamic monitoring with cardiac index (CI)<2.5 l/min/m(2) and wedge pressure > or = 20 mm Hg. Patients were randomized in a double-blind fashion to receive placebo or tezosentan: 0.2, 1, 5, or 25 mg/h for 24 h. The primary endpoint of the study, CI increase at 6 h of treatment, was significant in the 5 and 25 mg/h groups. Tezosentan induced a dose-dependent increase in CI and a decrease in wedge pressure, peaking after 3 h in the 5 and 25 mg/h groups. In the 1 mg/h group, this effect was smaller during the first 6 h and increased gradually, becoming significant at 24 h and beyond treatment discontinuation. There was no hemodynamic effect in the 0.2 mg/h arm. Type-B natriuretic peptide (BNP) decreased in the 1, 5, and 25 mg/h groups but not on placebo. Endothelin levels were significantly increased by the 5 and 25 mg/h groups but not in the lower (< or = 1 mg/h) tezosentan doses. Urine output decreased on the 25 mg/h dose. There was a trend towards improvement in patients' subjective dyspnea score and worsening heart failure events, mainly in the 1 mg/h group. In patients admitted with AHF, tezosentan doses of 1-25 mg/h are efficacious in improving the hemodynamics and reducing BNP. Tezosentan doses beyond 1 mg/h increased plasma endothelin levels and reduced urine output, probably limiting their clinical efficacy, as compared to tezosentan 1 mg/h.

Atrasentan (ABT-627, ATN) pharmacokinetics (PK) and endothelin-1 (ET-1) pharmacodynamics (PD) in a phase 2 study of patients with asymptomatic hormone-refractory prostate cancer (HRPC)

4721 Background: ATN, an oral ET antagonist with high selectivity for ETA receptor, is in phase 3 clinical development for the treatment of HRPC. ETA receptors are primarily responsible for ET-1 mediated vasoconstriction and cell proliferation. ET-1 is the main ET isoform in mammalian tissues and fluids. Methods: ATN PK and ET-1 PD were assessed in a phase 2, randomized, double-blind, placebo-controlled, multicenter study (M96–594). 288 HRPC patients (mean±SD 71±8 yrs, 81±13 kg) were assigned to receive PO QD: ATN 2.5 mg, ATN 10 mg, or placebo, along with continued hormone ablation therapy. Predose blood samples were collected starting Day 14, every 2 weeks for 6 months, and monthly thereafter. For a PK subset (N=9), blood samples for ATN and ET-1 assay were collected over 24 h on Day 28. ATN and ET-1 plasma concentrations were determined using validated methods. Mixed model statistical analysis of ATN and ET-1 predose data included effects for day, dose, age, race, body weight and day*dose. Results: Predose ATN (N=1214 from 173 subjects) and ET-1 (N=1876 from 257 subjects) concentrations did not vary with time, body weight or race, but were positively correlated with age. Only 3% of variability in ATN concentration was explained by age. ATN modestly increased average predose ET-1 levels: 2.8, 3.8 and 4.5 pg/mL for 0, 2.5 and 10 mg dose groups, respectively. ATN and ET-1 results for the PK subset (Day 28): Conclusions: ATN PK were approximately linear and time-independent. No dose adjustment for age or body weight is warranted. ATN modestly increased plasma ET-1 levels. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Abbott Laboratories Abbott Laboratories Abbott Laboratories

Atrasentan (ABT-627, ATN) pharmacokinetics (PK) and endothelin-1 (ET-1) pharmacodynamics (PD) in a phase 2 study of patients with asymptomatic hormone-refractory prostate cancer (HRPC)

4721 Background: ATN, an oral ET antagonist with high selectivity for ETA receptor, is in phase 3 clinical development for the treatment of HRPC. ETA receptors are primarily responsible for ET-1 mediated vasoconstriction and cell proliferation. ET-1 is the main ET isoform in mammalian tissues and fluids. Methods: ATN PK and ET-1 PD were assessed in a phase 2, randomized, double-blind, placebo-controlled, multicenter study (M96–594). 288 HRPC patients (mean±SD 71±8 yrs, 81±13 kg) were assigned to receive PO QD: ATN 2.5 mg, ATN 10 mg, or placebo, along with continued hormone ablation therapy. Predose blood samples were collected starting Day 14, every 2 weeks for 6 months, and monthly thereafter. For a PK subset (N=9), blood samples for ATN and ET-1 assay were collected over 24 h on Day 28. ATN and ET-1 plasma concentrations were determined using validated methods. Mixed model statistical analysis of ATN and ET-1 predose data included effects for day, dose, age, race, body weight and day*dose. Results: Predose ATN (N=1214 from 173 subjects) and ET-1 (N=1876 from 257 subjects) concentrations did not vary with time, body weight or race, but were positively correlated with age. Only 3% of variability in ATN concentration was explained by age. ATN modestly increased average predose ET-1 levels: 2.8, 3.8 and 4.5 pg/mL for 0, 2.5 and 10 mg dose groups, respectively. ATN and ET-1 results for the PK subset (Day 28): Conclusions: ATN PK were approximately linear and time-independent. No dose adjustment for age or body weight is warranted. ATN modestly increased plasma ET-1 levels. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Abbott Laboratories Abbott Laboratories Abbott Laboratories