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Cardiovascular Research 2001 49(2):330-339; doi:10.1016/S0008-6363(00)00264-9
© 2001 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Pulmonary endothelinergic system in experimental congestive heart failure

Delphine Lepailleur-Enoufa, Giorgia Egidyb, Monique Philippea, Liliane Louedeca, Jean-Paul Henryc, Paul Mulderc and Jean-Baptiste Michela,*

aINSERM U460, UFR X. Bichat, 16 rue Henri Huchard, 75018 Paris, France
bINSERM U36, Collège de France, 75005 Paris, France
cE 9920 INSERM VACOMED, Department of Pharmacology, Rouen, France

* Corresponding author. Tel.: +33-14-485-6152; fax: 33-14-485-6157 u460{at}bichat.inserm.fr

Received 31 January 2000; accepted 24 October 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Endothelin-1 (ET-1), plays an important role in the pathophysiology of CHF and the pulmonary endothelium is an early hemodynamic target in diastolic left ventricular dysfunction. Therefore we hypothesized that the lung is a main source of humoral endothelin in CHF and that its secretion is proportional to the degree of heart failure. Methods and results: We used rats with coronary artery ligation as an experimental model of either compensated or decompensated heart failure, depending on infarct size. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that in the lung, the expression of preproET-1 mRNA was higher in decompensated HF than in control and compensated HF rats (P<0.001). Run-on assay demonstrated that ET-1 overexpression is regulated at a transcriptional level (P<0.01). In contrast, there was no change in ET-1 mRNA expression in aortae, left ventricular myocardium and skeletal muscle. The expression of endothelin-converting enzyme (ECE)-1 mRNA was not modified and the expression of ETB receptor mRNA in the congestive lung was significantly lower than in control and compensated HF rats (P<0.0001), while the expression of ETA receptor mRNA did not differ between groups. The lung and plasma ET-1 peptide levels were respectively 4.2 and 9 fold higher in the rats with decompensated HF than in control rats (P<0.05; P<0.0001). Organoculture experiments showed that the lung ET-1 peptide secretion level in rats with decompensated HF was higher than that in control rats (P<0.01). In contrast, there was no change in ET-1 peptide secretion by the left ventricular myocardium and skeletal muscle. In plasma of rats with decompensated HF, the rate of bigET-1 conversion to ET-1 was 22%. ET-1 peptide was also present in the pleural effusion of decompensated heart failure. Plasma ET-1 concentration was significantly correlated with upstream markers of left ventricular diastolic dysfunction, with the expression of preproET-1 mRNA in the lung, with lung and pleural ET-1 concentration and with the expression ratio of ET-1/ETB receptor mRNA. Conclusion: Taken together, these data suggest that overexpression of ET-1 and down-regulation of ETB receptors in the lung are determinants of circulating endothelin in CHF. As a corollary, increased plasma endothelin may provide evidence of pulmonary endothelial dysfunction in CHF.

KEYWORDS Endothelins; Gene expression; Heart failure; Receptors


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Left ventricular dysfunction has upstream hemodynamic consequences on pulmonary endothelial function in early diastole [1,2]. For example we have reported a downregulation of the expression of angiotensin converting enzyme which correlates with the degree of heart failure in the model of experimental myocardial infarction in rats [3]. Similarly, constitutive eNOS expression also decreased early in the pulmonary circulation upstream to the dysfunctional left ventricle [2].

The lung is an endothelium-rich organ in which it is possible to evaluate endothelial secretion in plasma and the endothelial response to circulating agents. For example, we have already shown that the pulmonary endothelium responds to plasma atrial natriuretic peptide by secreting plasma cyclic GMP in experimental congestive heart failure [4].

Endothelin-1 (ET-1) is a peptide constitutively secreted by endothelial cells [5]. ET-1 is initially synthesized as a large inactive precursor protein called preproET-1, which is first cleaved at two pairs of basic amino acids to generate bigET-1. It is then cleaved by endothelin-converting enzyme (ECE) to produce the active peptide ET-1. Nevertheless the relative importance of the intracellular versus the extracellular conversion of bigET-1 remains unclear. Circulating ET-1 levels are elevated in an experimental model of heart failure [6] as well as in humans [7]. Moreover, blockade of the endothelin system has a beneficial effect on survival rate and hemodynamic parameters in heart failure [8–10]. Nevertheless, the hemodynamic stimulus for the elevation of plasma ET-1 concentration in congestive heart failure remains to be defined. The pulmonary vascular bed is an important site for plasma production of ET-1 in congestive heart failure [11]. Therefore we hypothesized that the lung is a main source of endothelial ET-1 expression and secretion in response to left ventricular diastolic dysfunction.

To demonstrate this point, we have evaluated the expression of the different components of the endothelin system, bigET-1, ET-1, ECE-1, ETA and ETB receptors in lungs in comparison with aorta, left ventricular myocardium and skeletal muscle, of rats with experimentally-induced congestive heart failure. We observed a transcriptional upregulation of the ET-1 gene, a downregulation of ETB endothelial receptor expression and an increase in ET-1 secretion in the lungs, the presence of ET-1 in the pleural effusion, and a correlation between all these different parameters and the degree of heart failure. In contrast, these parameters were not influenced by the degree of heart failure in other tissues.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Experimental design
Normotensive male Wistar rats (Charles River, France) weighing 300 g were used. Left ventricular infarction was produced by ligation of the left coronary artery under ether anesthesia as described by Fishbein et al. [12,13]. The left descending anterior coronary artery was ligated proximally to obtain moderate or large infarcts leading to compensated or decompensated heart failure [14]. Control rats were sham-operated using a similar procedure without coronary artery ligation. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).

The experimental period was 3 months. The animals were examined twice weekly. At the end of the experimental protocol, rats were sacrificed. Blood and pleural effusion samples were collected in prechilled 10 ml tubes containing EDTA or heparin, leupeptin and PMSF, giving approximate final concentrations of 2 mM, 100 U/ml, 20 µM and 200 µM, respectively. These samples were centrifuged for 10 min at 3000 g, the supernatants stored at –20°C. Pleural effusion volume was measured. The heart was excised and weighed. The left ventricle was then separated from the rest of the heart (i.e. right ventricle and the two atria) and weighed. Lungs, thoracic aortae, left ventricular myocardium and tibialis anterior skeletal muscle were rapidly excised in 37 rats, rinsed in cold phosphate-buffered saline, frozen in liquid nitrogen, and stored at –80°C.

2.2 Infarct size determination
Myocardial infarction (MI) size was measured in 34 rats by use of techniques previously described by Chien et al. [3,15]. The left ventricle was opened via an incision along the septum from base to apex so that the left ventricular tissue could be pressed flat. The circumference of the entire flat left ventricle and the observable infarcted area, as judged from both epicardial and endocardial sides, were outlined on a transparent plastic sheet. The difference in weight between the two marked areas on the sheet was used to determine the size of MI and was expressed as a percentage of left ventricular surface area. The degree of congestive heart failure in the MI group was defined according to one major criterion, the presence of pleural effusion corresponding to an increased heart weight–left ventricle weight index.

2.3 Run-on assay
The run-on assay permits the study of the transcription rate of a given gene in vitro. This transcriptional activity is dependent on proteic transcription factors present in the nucleus. For this purpose, lung nuclei were isolated on ice at 4°C as described by Boggaram et al. [16]. In vitro transcriptions were carried out in the presence of 4x107 isolated nuclei and ({alpha}-32P)CTP, as described by Mak et al. [17] with slight modifications. On the other hand, a large amount (10 µg) of cDNA encoding for preproET-1 and GAPDH amplified in Bluescript KS plasmids and pGEM plasmids respectively, were denatured and immobilized on a nylon filter (Hybond-N, Amersham). Empty Bluescript KS and pGEM plasmids were used as negative controls. Slot blots were then hybridized with equivalent amounts of radioactive RNA (7x106 cpm) obtained from lungs of myocardial infarction or control rats. After hybridization, the blots were washed and exposed to Kodak Biomax MS film with an intensifying screen at –80°C for 7 days. Therefore the intensity of the band was proportional to the in vitro transcription rate giving evidence of the activity of the preproET-1 gene.

2.4 RT-PCR to evaluate levels of mRNA expressions
Total RNA were extracted from lung, left ventricle, skeletal muscle and aortae using the method described by Chomczynski and Sacchi [18]. The quality of isolated mRNA was verified by gel electrophoresis. PreproET-1, ECE-1, ETA receptor, ETB receptor and GAPDH mRNA expression were evaluated in lung by semi-quantitative RT-PCR. ET-1 mRNA expression was evaluated in aortae and skeletal muscle. For the RT stage, 100 ng of total lung RNA, 1 µg of total skeletal muscle and 600 ng of total aorta RNA were primed with 1 µg oligo(dT) (Pharmacia Biotech) and reverse transcribed in the presence of Moloney murine leukemia virus reverse transcriptase (M-MLV reverse transcriptase) (GIBCO). The cDNA was amplified by PCR with the use of specific oligonucleotide primers for rat preproET-1 [5] ECE-1 [19], ETA receptor, ETB receptor and GAPDH [20]. The sequences of the oligonucleotides are summarized in Table 1. Double-stranded cDNAs were synthesized and amplified using 1.25 U Taq polymerase (GIBCO), 20 mM TrisHCl (pH 8.0), 50 mM KCl, 0.2 mM of dNTP, 10 pmol of each of the primers except for GAPDH where primers were used at 50 pmol, 1.5 mM MgCl2, and 4x105 counts/min (cpm) of 33P-labeled primer (Isotopchim) in a 25 µl reaction final volume. The amplification was carried out in a DNA thermal cycler (Techne). The annealing temperature and the number of reaction cycles of PCR were set as follows: preproET-1 62°C, 32 cycles (in aortae and in lung), 35 cycles (in left ventricle) and 34 cycles (in skeletal muscle); ECE-1 55°C, 32 cycles; ETA receptor 55°C, 29 cycles; ETB receptor 55°C, 25 cycles and GAPDH 55°C, 28 cycles (in lung), 24 cycles (in aortae), 22 cycles (in left ventricle) and 21 cycles (in skeletal muscle).


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  		Table 1 Primer sequences used in this study (bp=base pairs)

 
PCR fragments were analyzed by 8% PAGE and were visualized by ethidium bromide staining. Bands were cut out, dissolved in periodic acid (25 mM), and counted with the use of a RackBeta liquid scintillation counter. PCR amplification was verified to be exponential, and the amplification products were proportional to sample input. PreproET-1,ECE-1, ETA and ETB receptors mRNA expression were calculated by normalizing preproET-1, ECE-1, ETA and ETB receptors mRNA to GAPDH mRNA.

2.5 Determination of plasma, pleural and tissue ET-1 levels
Plasma, pleural and lung ET-1 levels were measured using a commercial ELISA kit (Parameter, R & D Systems, Minneapolis, USA).

The anti-ET-1 antibody showed 100% specificity for ET-1, <1% cross-reactivity to bigET-1. However, this assay is not suitable for the direct assay of plasma and pleural effusion samples. An extraction procedure is required. To 2ml of plasma or pleural effusion was added 3 ml of extraction solvent (acetone: 1 N HCl:Water (40:1:5). After mixing by inversion, these samples were centrifuged for 20 min at 2000 g at 4°C. The supernatants were dried down in a centrifugal evaporator. The pellets were resuspended in 0.25 ml of sample diluent. Then 100 µl of this sample extract were used to measure ET-1 levels. Pleural effusion ET-1 levels were only measured in 8 rats since the immunoassay used required a minimal volume of 2 ml. The range of collected pleural effusion volumes was heterogeneous (0.4 to 15.9 ml) of which 10 samples contained less than 2 ml.

Lung ET-1 tissue was extracted as previously described [21]. Briefly, 100 mg of lung were homogenized in 4 M guanidium thiocyanate/0.1% trifluoroacetic acid on ice. An aliquot was taken for protein measurement (Bio-Rad, Hercules, CA, USA). The homogenate was then centrifuged at 4000 g for 20 min at 4°C. The supernatant was then loaded on a Sep Pak C18 cartridge. The cartridge was washed with H2O and methanol. The absorbed peptides were then eluted with 90% ethanol. ET-1 tissue levels were determined by EIA kit (Parameter, R&D Systems, Minneapolis, USA). Results were expressed as picograms per milligram of total protein.

2.6 Endothelin-1 peptide secretion in organocultures
As ET-1 is secreted, approximatively 1 g of lung, left ventricle and skeletal muscle were incubated in 1 ml of medium consisting of DMEM supplemented with 20 mM HEPES (Boerhinger Mannheim), 2 mM L-glutamine, 50 UI/ml penicillin, 50 µg/ml streptomycin, and 0.125 µg amphotericin B (Sigma, St Louis, MO) for 48 h. ET-1 levels in the culture medium were measured using a commercial kit (R & D Systems) as previously mentioned. ET-1 secretion was expressed in pg per g of total secreted proteins in 1 ml of culture medium.

2.7 BigET-1 conversion
To measure bigET-1 levels, a stably transfected CHO cell line expressing a soluble form of human ECE (sECE) [22] was used as source of endothelin converting enzyme activity. Partial purification of sECE-1 from the conditioned medium of CHO/sECE cells was performed as described [22]. The integrity of the enzymatic preparation was monitored by analyzing the enzymatic conversion of bigET-1 into ET-1 peptide carried out in 100 µl 50 mM HEPES buffer (pH 6.8), 150 mM NaCl, 0.1 µM ZnCl2 and 0.1% BSA at different enzyme and bigET-1 concentrations. Plasma and pleural samples were fractionated in 1 ml aliquots. One ml of heparinized plasma was supplemented with 0.1 µM ZnCl2, 20 mM 2-[N-Morpholino]-ethanesulfonic acid (pH 5.5) and 200 µM PMSF and incubated at 37°C in the presence of soluble ECE for 30 min. The reaction was stopped by adding 5 µl of 0,5 M EDTA, the sample concentrated as describe above and then subjected to a commercial ELISA kit to quantify ET-1 produced by the enzymatic conversion of bigET-1 into ET-1 peptide. Specificity of the conversion was monitored by preincubation of the enzyme with 100 µM phosphoramidon, an ECE inhibitor prior to the incubation with plasma samples. Endogenous ET-1 was quantified from plasma or pleural effusion not submitted to ECE incubation. All determinations were performed in duplicate.

2.8 Statistical analysis
Results are expressed as mean±S.E.M. The difference in each biological parameter (in lung, aorta or plasma) was evaluated by a one-way ANOVA for the increase of each variable measured. ANOVA was followed by Scheffe's F test to compare the effect of different pathophysiological conditions on these parameters. Regression curves and correlation coefficients were obtained by the least-squares method (Statview software). Statistical significance was accepted for P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Heart parameters
A total of 18 rats with decompensated HF (severe myocardial infarction) and nine with compensated HF (moderate myocardial infarction) were studied for determination of heart failure graduation. There was no significant difference in body weight between rats with myocardial infarction and control rats (Table 2). Both the total heart weight and the ratio of total heart weight to body weight (heart weight index) were significantly increased in the decompensated as compared compensated and control rats (F = 30, P<0.0001) (Table 2). The absolute weight of the part of the myocardium hemodynamically upstream to the infarcted left ventricle (i.e. heart weight–left ventricle weight) and its ratio to body weight (heart weight–left ventricle weight index) were also significantly increased in decompensated HF rats (F = 25, P<0.0001) (Table 2). Infarct size averaged 24±1.6% of the left ventricle area in rats with compensated HF and 41±1.6% in rats with decompensated HF (F = 179, P<0.0001). The heart weight–left ventricle weight was related to infarct size (r = 0.88, F = 114.2, P<0.0001). All rats assigned to the decompensated stage presented pleural effusion.


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  		Table 2 Rat body weight, heart weight, and oedemaa

 
3.2 Expression of endothelin system mRNAs
The expression of preproET-1 mRNA, ECE-1 mRNA, ETA and ETB receptor mRNAs in lung from rats with infarct and from controls was evaluated by comparative RT-PCR. The expression of preproET-1 mRNA was significantly increased in the lungs of the rats with decompensated HF (Fig. 1A). The ratio of the levels of preproET-1 mRNA to GAPDH mRNA was significantly increased from 1.17±0.07 in the sham-operated group and 1.19±0.04 in the compensated HF group to 1.62±0.09 in the decompensated HF group (P<0.0004). The lung preproET-1 mRNA expression was related to the degree of heart failure and was therefore positively correlated with the markers of severity: the heart weight–left ventricle weight (r = 0.48, F = 10, P<0.01) and the infarct size (r = 0.61, F = 21.1, P<0.0001). In contrast, aortic, left ventricle and skeletal muscle expression of preproET-1 mRNA did not differ between decompensated and compensated HF and control rats (Fig. 1B).


Figure 1
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  		Fig. 1 Column graph showing the expression of preproET-1 mRNA (semi-quantitative RT-PCR) in lung (A) and in aorta (a), skeletal muscle (m) and left ventricle (lv) (B) and the expression of ECE-1 mRNA (C) in congestive heart failure (CHF) versus control rats. Comp indicates compensated HF; and Decomp, decompensated HF. Each column and bar represents the mean±S.E.M.

 
Semi-quantitative RT-PCR showed a similar ECE-1 expression in the lung of HF and control rats (1.28±0.18 versus 1.02±0.08) (Fig. 1C). Similarly the expression of ETA receptor mRNA did not differ between control and HF rats (1.4±0.1 versus 1.32±0.6) (Fig. 2). In contrast, the expression of ETB receptor mRNA in the lung was significantly lower in rats with decompensated HF than in compensated HF and control rats (F = 48, P<0.0001). The expression of ETB receptor mRNA was decreased four fold in decompensated HF rats compared with control rats (Fig. 2). The ETB receptor mRNA expression was negatively correlated with the markers of HF severity: the heart–left ventricle weight index (r = –0.85, F = 84.3, P<0.0001) and the infarct size (r = –0.76, F = 44.9, P<0.0001).


Figure 2
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  		Fig. 2 RT-PCR semi-quantitative analysis of ETB and ETA receptor mRNA in lung in rats with heart failure versus controls. Comp indicates compensated HF; and D, decompensated HF. Values are mean±S.E.M.

 
3.3 Endothelin gene transcription
To determine whether the increase in preproET-1 mRNA expression in decompensated HF rat lungs occurs via transcriptional or post-transcriptional mechanisms, we performed a nuclear run-on transcriptional assay (Fig. 3). The rate of preproET-1 gene transcription was increased 3.8 fold in decompensated HF rats compared with control rats (F = 9.1, P<0.01).


Figure 3
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  		Fig. 3 Nuclear run-on experiment. Nuclei were isolated from lungs of control rats (n = 4), rats with compensated heart failure (Comp) (n = 4) and rats with decompensated heart failure (Decomp) (n = 3). Values are mean±S.E.M.

 
3.4 Biochemical parameters
The plasma ET-1 concentration in rats with decompensated HF was markedly (nine fold) higher than that of control rats: 3.6±0.39 pg/ml for decompensated versus 0.37±0.035 pg/ml for control and 0.84±0.28 pg/ml for compensated HF rats. Plasma ET-1 level was significantly correlated with the heart–left ventricle weight index (r = 0.81, F = 79.4, P<0.0001) (Fig. 4B), with the infarct size (r = 0.76, F = 48.5, P<0.0001), with the expression of preproET-1 mRNA in the lung (r = 0.72, F = 8 P<0.001), with the pleural effusion ET-1 concentration (r = 0.77, F = 10, P<0.05) and negatively with the expression of ETB receptor mRNA (r = 0.79, F = 55, P<0.0001). Plasma endothelin was also correlated with the expression ratio of ET-1/ETB receptor mRNA (r = 0.73, F = 38, P<0.0001) (Fig. 4C).


Figure 4
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  		Fig. 4 Plasma ET-1 and lung ET-1 concentrations in controls and heart failure (HF) rats (compensated HF, Comp; decompensated HF, Decomp). (A): the relation between the expression ratio of ET-1/ETB receptor mRNA and plasma ET-1 concentration. (B): the relation between Heart–LV weight index and plasma ET-1 concentration. Curvilinear relationship is fitted with a natural exponential function (r2=0.81). (C): the relation between lung ET-1 concentration and plasma ET-1 concentration. (D): Linear regression of the relationship between ET-1 level in pleural effusion and lung ET-1 level (r2=0.53).

 
The lung ET-1 peptide level was similar between control and compensated HF rats (2.28±0.76 versus 2.68±0.65 pg/ml). The lung ET-1 peptide level of rats with decompensated HF (10.5±2.73 pg/ml) was significantly (4.2 fold) higher than the two other groups (control and compensated HF) (F = 4.53, P<0.05). The changes in lung ET-1 concentration were closely correlated with the ET-1 concentration in the pleural effusion (r = 0.76, F = 9.44, P<0.02) (Fig. 4D) and with plasma ET-1 concentration (r = 0.7, F = 31, P<0.001) (Fig. 4A).

The lung ET-1 peptide secretion level in rats with decompensated HF was significantly higher than that of control rats (Table 3). In contrast, left ventricle and skeletal muscle ET-1 peptide secretion levels did not differ between groups. The lung ET-1 peptide secretion was correlated with heart weight (r = 0.63, F = 9.8, P<0.01).


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  		Table 3 Endothelin-1 peptide secretion in organoculturesa

 
The conversion rate of bigET-1 into ET-1 peptide was 22±2.39% in plasma of decompensated HF rats. In view of the low level of circulating bigET-1, significant conversion could not be detected in the plasma of control and compensated HF rats. Similarly, no detectable conversion could be measured in the pleural effusions.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Endothelin is a peptide constitutively secreted by the endothelial cells [5], circulating at a low level because of its high affinity for its endothelial receptor which functions as one of the main clearance mechanisms for the circulating peptide [23].

The lung is an organ particularly rich in endothelium [24] and pulmonary endothelium is an early hemodynamic target upstream to the diastolic left ventricular dysfunction [2] due to myocardial infarction in rats [14]. Pulmonary endothelial dysfunction is mainly due to a chronic decrease in shear stress as a consequence of the diastolic pressure-dependent increase in pulmonary vascular dimensions associated with a decrease in blood flow [2]. Therefore we tested the hypothesis that endothelin expression is upregulated in the lung in experimental heart failure and could be a main source of humoral endothelin in congestive heart failure.

The present study shows that the ET-1 gene is overexpressed in the lungs of rats in HF, and this expression correlates with the degree of heart failure, suggesting an interdependency between hemodynamic disturbance and endothelin expression in the lung. Run-on assay demonstrated that this overexpression is transcriptionally regulated. The presence of ET-1 in the pleural effusion and the correlation of its concentration with that in the lung, also suggested the pulmonary source of the peptide. Pleural liquid is a transudate of pulmonary interstitial fluid in heart failure. Therefore the secreted peptide diffused from the interstitial compartment to the pleural liquid. These data have been confirmed by the assessment of in vitro secretion of ET-1 by lung tissues. Previously published data have already shown an overexpression of ET-1 in pulmonary endothelium of HF in rats [25,26] and the absence of its upregulation in the kidney. The present data, as in a dog model of heart failure [27] extend them to show: first, that this overexpression is transcriptionally regulated probably by hemodynamic forces in a tissue submitted to low shear stress in vivo [2] and second, that this overexpression is probably a main source of the peptide present in the pleural effusion and the plasma. Therefore all these parameters, lung, plasma and pleural ET-1 concentration, and lung ET-1 mRNA are all correlated with each other and also with the degree of heart failure.

The regulation of endothelial endothelin expression by shear stress has been modeled in vitro by several groups [28,29] leading to conflicting results. Morita et al. showed that shear-stress induced endothelin-1 gene expression in cultured endothelial cells [30] whereas Malek et al. [28,31] reported a down-regulation of endothelin expression in endothelial cells submitted to shear stress. Our in vivo data confirm the latter in vitro results, showing that left ventricular dysfunction induced a decrease in shear stress in the pulmonary circulation [2] which was associated with a decrease in endothelial NO synthase function [1] and expression [2] and an increase in endothelin expression in the lung of animals with congestive heart failure.

Because endothelin is a peptide secreted by the endothelium into the tissue interstitium and the plasma, the quantity of extracellular peptide in plasma and pleural effusion correlates better with the degree of heart failure than with the tissue concentration in the lung. The down-regulation of ETB receptor mRNA also suggested that the endothelium is a main source of plasma endothelin and that circulating endothelin interacted mainly with its endothelial receptor. It has been shown that ETB receptors are mainly expressed in the endothelium [32]. Endothelial ETB receptors play a predominant role in the physiological clearance of extracellular endothelin-1 in vitro [33] and of plasma circulating endothelin-1 in vivo [34,35]. Moreover, the level of ETB receptor mRNA was down-regulated by endothelins and this phenomenon appears to be related to an endothelin-induced decrease in intracellular stability of mRNA molecules [36]. It has been shown that reduced pulmonary clearance of endothelin-1, via the down-regulation of ETB receptors [37] contributed to the increase in circulating levels of the peptide in heart failure [38]. Our data confirm the results of these previous studies, showing a clear negative correlation between plasma endothelin and lung ETB receptor mRNA expression. This down-regulation was restricted to the ETB receptor isoform, suggesting that the phenomenon was limited to the endothelium.

Nevertheless, an extra-endothelial and extra-pulmonary source of endothelin cannot be completely excluded. Sakai and Oie et al. [6,39] showed an overexpression of the ET-1 gene and an upregulation of ET-1 binding sites in the non-scarred myocardium of HF rats. Similar data have been reported by Picard et al. [40]. But, in contrast with our observations in the lung, ETA and ETB receptor mRNA expression and ET-1 binding sites increased in parallel [6,40]. Moreover in the study of Oie et al. [39] the overexpression of ET-1 mRNA was transient and predominant in the inflammatory scar of the infarcted area. These data of myocardial overexpression of ET-1 and receptors suggested a local action of endothelin within the myocardial tissue rather than a systemic one. In the present study, we did not observe any overexpression and oversecretion of ET-1 from cardiac muscle in decompensated heart failure as compared to compensated and controls. These results are supported by human data [7] showing a correlation between plasma endothelin concentrations and hemodynamic perturbations and an absence of relation to myocardial peptide secretion in coronary plasma effluent in human congestive heart failure. Serneri et al. [41] recently showed a complete dissociation between plasma level of ET-1 and myocardial overexpression of the endothelin system in different forms of congestive heart failure. Similarly, Stangl et al. [42] recently demonstrated that the lung acted as producer and the heart and the periphery acted as consumers of elevated circulating endothelin in human congestive heart failure.

In order to evaluate whether humoral bigET-1 concentration could be a more valuable marker in congestive heart failure than ET-1 itself, we have measured the conversion rate of bigET-1 to ET-1 in plasma and pleural effusion using a soluble form of human ECE. In view of the very low level of circulating endothelin in control and compensated HF rats and the lack of sensitivity of the assay on heparinized plasma, we could not detect any conversion in these groups. In contrast, in decompensated HF rats, in which the plasma endothelin concentration was higher, we could detect a conversion rate around 20%. These data suggest that ECE activity is not a rate limiting step in ET-1 production in the plasma endothelium compartment and confirm our observations that endothelial cell cultures are able to convert all the bigET-1 [43] and those reported after bigET-1 intravenous infusion in vivo [44]. Similarly, we could not detect any bigET-1 conversion in pleural effusion, suggesting that ECE activity is not a rate limiting step in ET-1 production in the lung [44]. Therefore, it appears that the measurement of plasma bigET-1 is not more relevant than the measurement of ET-1 itself in experimental congestive heart failure. Nevertheless, to provide a definitive answer to the question, the development of a specific direct immunoassay of bigET-1 in rat is required as has been already developed for the human form [45].

In conclusion, these data suggest that dysfunction of the pulmonary endothelium, including overexpression of the endothelin gene and down-regulation of the ETB receptor, is probably a main source of plasma endothelin in experimental congestive heart failure. As a corollary, an increase in circulating endothelin may provide evidence of endothelial dysfunction in congestive heart failure.

Time for primary review 25 days.


   Acknowledgements
 
This study was supported by INSERM and by grants from the «Fondation de France» and from the «Fondation pour la Recherche Médicale».


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Ontkean M., Gay R., Greenberg B. Diminished endothelium-derived relaxing factor activity in an experimental model of chronic heart failure. Circ Res (1991) 69:1088–1096.[Abstract/Free Full Text]
  2. Ben Driss A.B., Devaux C., Henrion D., Duriez M., Thuillez C., Levy B.I., Michel J.B. Hemodynamic stresses induce endothelial dysfunction and remodeling of pulmonary artery in experimental compensated heart failure. Circulation (2000) 101:2764–2770.[Abstract/Free Full Text]
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