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Montreal Heart Institute and University of Montreal, Montreal; Institut National de la Recherche .... bars represent control group; solid bars represent ET group.
Chronically Elevated Endothelin Levels Reduce Pulmonary Vascular Reactivity to Nitric Oxide Annik Migneault, Ste´phanie Sauvageau, Louis Villeneuve, Eric Thorin, Alain Fournier, Normand Leblanc, and Jocelyn Dupuis Montreal Heart Institute and University of Montreal, Montreal; Institut National de la Recherche Scientifique–Institut Armand Frappier, Universite´ du Que´bec, Pointe-Claire, Quebec, Canada

Although local tissue activation of the endothelin (ET) system contributes to the development of pulmonary hypertension, the impact of isolated chronic plasma hyperendothelinemia on the pulmonary circulation is unknown. Methods: Mini-osmotic pumps were implanted in rats to deliver ET-1 during 7 or 28 days. After in vivo hemodynamics, the lungs were isolated to derive pressure–flow relations. Small pulmonary arteries (ⵑ 250 ␮m) were mounted on an isometric myograph to study their reactivity. Results: Plasma ET-1 approximately doubled (p ⬍ 0.05) after 7 and 28 days. Lung tissue ET-1 level increased fourfold after 7 days (p ⬍ 0.001) but was no longer significantly elevated after 28 days. Right ventricular systolic pressure was unaffected. The pulmonary pressure–flow relation shifted upward with a steeper slope (p ⬍ 0.05) at 7 days, but not after 28 days. Maximum dilatations to both acetylcholine (p ⬍ 0.01) and sodium nitroprusside (p ⬍ 0.001) were greatly reduced by approximately 50% after 28 days and were normalized by the addition of the nitric oxide synthase inhibitor L-NNA and the antioxidant N-acetyl-L-cysteine, respectively. Conclusion: Chronic hyperendothelinemia reduces the pulmonary vasodilator reserve in response to nitric oxide. Correction by an antioxidant and L-NNA suggests that this relates to increased production of reactive oxygen species, which may have clinical relevance for conditions associated with chronic increase of ET. Further studies are required to determine if, in the long term, this could contribute to the development of pulmonary hypertension. Keywords: acetylcholine; endothelium; pulmonary circulation; pulmonary hypertension; smooth muscle

Circulating endothelin (ET) levels are elevated in animal models of pulmonary hypertension (PH) as well as in human PH of various causes (1). In many studies, the increase in ET levels correlates well with the severity of the underlying disease process (2–7). Whether this increase in ET levels is merely a marker of the PH that reflects an increased pulmonary ET production, with or without reduced plasma clearance of this peptide, or actually contributes to the development and maintenance of PH is still a subject of debate (8). Chronic elevation of circulating ET levels could modulate vascular reactivity by direct action on ET receptors, and indirectly by modifying receptor expression and/or sensitivity. It could also alter smooth muscle cell properties to vasoactive agents as previously demonstrated in small rat cerebral arteries (9).

(Received in original form March 15, 2004; accepted in final form November 29, 2004) Supported by a grant from the Canadian Institutes for Health Research. J.D. is a senior scholar from the Fonds de la Recherche en Sante´ du Que´bec. Correspondence and requests for reprints should be addressed to Jocelyn Dupuis, M.D., Ph.D., Research Center, Montreal Heart Institute, 5000 Belanger Street, Montreal, PQ, H1T 1C8, Canada. E-mail: [email protected] Am J Respir Crit Care Med Vol 171. pp 506–513, 2005 Originally Published in Press as DOI: 10.1164/rccm.200403-340OC on December 3, 2004 Internet address: www.atsjournals.org

Knowledge of the impact of chronically elevated ET levels on the pulmonary circulation is important not only to broaden our understanding of the physiopathology of PH but also to appreciate the potential impact on pulmonary vascular reactivity in other conditions associated with increased ET levels. Cirrhosis of the liver, for example, is associated with increased plasma ET levels (10) caused in part to a reduction in liver clearance of this peptide (11). Whether this increase in ET levels in hepatic outflow could modify pulmonary vascular reactivity and contribute to the rare occurrence of porto-PH is unknown. The present study was therefore designed to evaluate the impact of isolated acute and chronic hyperendothelinemia, mimicking usual physiopathologic levels, on pulmonary vascular reactivity and right ventricular function. We determined the effect of increased levels of circulating ET-1 in rats for a duration of 7 and 28 days on in vivo hemodynamics, on isolated lung vascular resistive properties, on isolated small pulmonary artery reactivity (ⵑ 250 ␮m), and on their expression of ET-1, ETA, and ETB receptors measured by confocal microscopy. Some of the results of these studies have been previously reported in the form of an abstract (12).

METHODS The animal ethics committee of our institution approved the study, which was performed on male Wistar rats (Charles River, St. Constant, Quebec) weighing 250 to 300 g. Rats were anesthetized with halothane; the left jugular vein was exposed, and a subcutaneous pouch was formed. A polyethylene catheter connected to a mini-osmotic pump (Alzet, Cupertino, CA) was inserted into the jugular vein and securely tied in place. The control animals were subjected to the same procedure except for the insertion of the pumps. The mini-osmotic pump was filled with an aqueous solution of ET-1 to be delivered at a rate of 10 ng/kg/ minute for a period of 7 or 28 days. Four experimental groups were thus created: (1 ) control, 7 days; (2 ) ET, 7 days; (3 ) control, 28 days; and (4 ) ET, 28 days.

Determination of ET-1 Stability and Biological Activity ET-1 was incubated at 37⬚C for durations of 7 and 28 days. Stability was evaluated with analytic reverse-phase HPLC using a Phenomenex ˚ , 5 mm, 250 ⫻ 4.6 mm) column (Torrance, CA) Jupiter C18 (300 A connected to a Beckman 128 solvent module and a Beckman 168 PDA detector (Mississauga, ON, Canada), with a flow rate maintained at 1 ml/minute. Peptide elution was performed with a linear gradient from 20 to 60% acetonitrile in aqueous TFA (Sigma-Aldrich, Oakville, ON, Canada) 0.06%, and peaks were resolved at 230 nm. Biological activity was evaluated by cumulative concentration–response curves of isolated small pulmonary vessels to the incubated ET-1 according to the isometric recording of tension methods described later.

In Vivo Hemodynamic Measurements and Pressure–Flow Relationship in Isolated Lungs The rats were anesthetized with a mixture of ketamine and xylazine followed by intraperitoneal injection of 2,000 U of heparin. The right jugular vein and right carotid artery were isolated, incised, and cannulated with polyethylene catheters to measure central venous, right ventricular, systemic arterial, and left ventricular pressures. All measurements were recorded and monitored on a Gould TA 400 polygraph

Migneault, Sauvageau, Villeneuve, et al.: ET and Lung Vascular Reactivity

(Gould Electronics, Valley View, OH). Finally, 3 ml of blood were sampled for plasma ET-1 measurement. The trachea was then isolated, cannulated, and connected to a rodent ventilator. The lungs were ventilated with a tidal volume of 1 ml at 60 cycles/minute with 2 cm H2O positive end-expiratory pressure. After a midline sternotomy, the pulmonary artery was rapidly cannulated through an incision in the right ventricle. The lung perfusion was initiated by infusing Krebs solution with heparin (100 U/ml) at 2 ml/minute. The Krebs solution had the following composition (in mmol/L): NaCl, 120; NaHCO3, 25; KCl, 4.7; KH2PO4, 1.18; MgSO4, 1.17; CaCl2, 2.5, and glucose, 5.5. Before each experiment, the Krebs solution was passed through a 0.22-␮m filter (Sarstedt, Newton, NC) and adjusted to a pH of 7.4. The lungs were then rapidly removed and suspended in a waterjacketed chamber at 37⬚C to be perfused at a constant flow rate (5 ml/ minute) using a Masterflex roller pump (Cole-Palmer Instruments Co., Vernon Hills, IL) with Krebs solution supplemented with 3% albumin. The pulmonary flow rate was constantly monitored with a flow probe (Transonic, Ithaca, NY). After 10 minutes of equilibration, the relationship between perfusion pressure and flow rate was first obtained during baseline conditions and after treatment with the nonselective nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (L-NNA; 100 ␮mol/L). The flow rate was increased in the range of 5 to 25 ml/minute, and the corresponding perfusion pressure was measured. At the end of each experiment, the right ventricle and the left ventricle plus septum weight were determined to calculate the right ventricle/(left ventricle plus septum) ratio. The wet and dry weights of the pulmonary right lower lobe were determined. The remaining lung tissues were rapidly frozen at ⫺80⬚C to measure tissue ET-1 levels.

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by ELISA according to the manufacturer’s instructions (Biomedica, distributed by Medicorp, Montreal, PQ, Canada). Tissue protein content was determined by a Lowry assay.

Confocal Imaging, Deconvolution, and Fluorescence Quantification of ET-1, ETA, and ETB Receptors Rat lungs were removed, snap-frozen in liquid nitrogen, and immersed in 2-methyl-butane (Sigma-Aldrich). The lungs were oriented to crosssection the arteries of interest and 14-␮m cryocuts were performed. Tissues were fixed with fresh 4% paraformadehyde, pH 7.2, and blocked in 2% normal donkey serum (Jackson, West Grove, PA) and 0.5% Triton X-100 (Sigma-Aldrich). Anti-ETA receptor antibody (rabbit; Alomone, Jerusalem, Israel) and anti-ETB receptor antibody (rabbit; Alomone) were incubated respectively with anti-␣ smooth muscle actin antibody (mouse; Sigma-Aldrich). They were diluted in 1% normal donkey serum and 0.1% Triton X-100. For the determination of ET-1 expression, slides were blocked with 2% bovine serum albumin (SigmaAldrich) and 0.5% Triton X-100 and then were incubated with anti–ET-1 antibody (rabbit; Phoenix Pharmaceuticals, Belmont, CA) diluted in 1% bovine serum albumin and 0.1% Triton X-100. Primary antibody incubation was performed overnight at 4⬚C. Antirabbit Alexa 555 (donkey; Molecular Probes, Eugene, Oregon) and antimouse Alexa 647 (donkey; Molecular Probes) in their respective antibody diluents

Isometric Recording of Tension of Isolated Microvessels Rat pulmonary arteries (270 ⫾ 10 ␮m) were gently dissected and placed in Krebs solution with ethylenediaminetetraacetic acid (0.026 mmol/L) and aerated with 12% O2, 5% CO2, and 83% N2 (pH 7.4). Segments 2 mm in length were mounted on 20-␮m tungsten wires in microvessel myographs (IMF; University of Vermont, Burlington, VT). Vessels were equilibrated for 30 minutes at their optimal tension and then challenged twice with 40 mmol/L KCl followed by the addition of 100 ␮mol/L of the endothelium-dependent vasodilator acetylcholine (Ach) to test the endothelium integrity. The arteries’ reactivity was then evaluated with two different protocols, one for vasoconstriction and one for vasodilatation. Dose–response constricting curves were performed using U-46619 (0.1 nmol/L to 1 ␮mol/L) and ET-1 (1 pmol/L to 3 ␮mol/L). To evaluate vasodilatation, arteries were preconstricted with U-46619 (100 nmol/L) before each dose–response curve with Ach (1 nmol/L to 100 ␮mol/L) and the endothelium-independent vasodilator sodium nitroprusside (SNP; 1 nmol/L to 10 ␮mol/L). For the ET 28-day group, responses to Ach were also assessed in the presence of 100 mM of L-NNA, and responses to SNP were obtained in the presence of 10 mM of the antioxydant N-acetyl-l-cysteine; both solutions were preincubated with the arteries for 30 minutes before the stimulation protocols.

ET-1 Levels Plasma and lung tissue homogenate samples were passed on Sep-Pak C18 columns (Waters, Milford, MA) before determination of ET-1 levels

Figure 1. Reactivity of small pulmonary arteries to endothelin-1 (ET-1) that has been freshly prepared (n ⫽ 8) or incubated at 37⬚C for 7 (n ⫽ 6) or 28 (n ⫽ 4) days.

Figure 2. (A ) Venous plasma levels of ET-1. Control 7 days (n ⫽ 12); ET 7 days (n ⫽ 11); control 28 days (n ⫽ 11); ET 28 days (n ⫽ 13). (B ) Pulmonary tissue concentration of ET-1. Control 7 days (n ⫽ 11); ET 7 days (n ⫽ 12); control 28 days (n ⫽ 12); ET 28 days (n ⫽ 11). White bars represent control group; solid bars represent ET group.

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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 171 2005 TABLE 1. HEMODYNAMIC AND GRAVIMETRIC PARAMETERS 7 Days Groups HR, beats/min MAP, mm Hg LV ⫹ dP/dT, mm Hg/s LV ⫺ dP/dT, mm Hg/s RVSP, mm Hg RV ⫹dP/dT, mm Hg/s RV ⫺ dP/dT, mm Hg/s Body weight, g RV weight, mg/g (LV ⫹ septum) weight, mg/g RV/(LV ⫹ septum) weight Lung dry/wet weight

28 Days

Control (n ⫽ 21)

ET (n ⫽ 22)

Control (n ⫽ 22)

ET (n ⫽ 23)

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫽

267 ⫾ 4 91.6 ⫾ 5.4 6348 ⫾ 245 6000 ⫾ 270* 26.8 ⫾ 0.5 1488 ⫾ 42 974 ⫾ 32 317 ⫾ 5* 0.498 ⫾ 0.012 1.871 ⫾ 0.016 0.265 ⫾ 0.006 0.213 ⫾ 0.003 (n ⫽ 9)

273 ⫾ 5 98.3 ⫾ 2.8 6997 ⫾ 277 5122 ⫾ 285 26.6 ⫾ 0.3 1611 ⫾ 49 997 ⫾ 31 442 ⫾ 6 0.455 ⫾ 0.008 1.781 ⫾ 0.023 0.256 ⫾ 0.005 0.217 ⫾ 0.003 (n ⫽ 10)

270 ⫾ 4 105.5 ⫾ 3.1 7581 ⫾ 266 6073 ⫾ 266* 27.2 ⫾ 0.4 1592 ⫾ 53 1002 ⫾ 26 422 ⫾ 6* 0.464 ⫾ 0.010 1.831 ⫾ 0.026 0.254 ⫾ 0.005 0.225 ⫾ 0.003 (n ⫽ 12)

277 89.9 6414 5049 27.3 1522 1015 336 0.517 1.882 0.276 0.216 (n

4 3.0 243 320 0.4 76 43 7 0.008 0.031 0.005 0.003 8)

Definition of abbreviations: ET ⫽ endothelin; HR ⫽ heart rate; MAP ⫽ mean arterial pressure; LV ⫽ left ventricle; RV⫽ right ventricle; RVSP ⫽ right ventricular systolic pressure. Gravimetric parameters are indexed according to body weight. * p ⬍ 0.05 versus control.

were then applied. Slides were mounted using 0.2% 1.4-diazabicyclo(2.2.2)-octane (Sigma-Aldrich) diluted with glycerol (1:5). We used phosphate-buffered saline (NaCl, 137 mM; KCl, 2.7 mM; Na2HPO4, 4.3 mM; KH2PO4, 1.4 mM) for all the washes that followed fixation and primary and secondary antibody incubations.

Slides were analyzed using a Zeiss LSM 510 confocal microscope (North York, ON, Canada). Images were collected with a Plan-Apochromat 63⫻/1.4 oil differential interference contrast (DIC) objective. HeNe1 (543 nm) and HeNe2 (633 nm) lasers were used for excitation of the antirabbit Alexa 555 and antimouse Alexa 647 antibody, respec-

Figure 3. Relationship between perfusion pressure and the flow rate in isolated lungs from (A ) 7-day groups (n ⫽ 13) and (B ) 28-day groups (n ⫽ 12).

Migneault, Sauvageau, Villeneuve, et al.: ET and Lung Vascular Reactivity

tively. Internal elastic and external elastic lamina autofluorescence was obtained with the argon laser line (488 nm) and collected between 505 and 530 nm. Z stacks of each tissue were performed, and images were taken at every 0.16 ␮m (top to bottom) to respect the Nyquist criteria in Z-sampling. The Z stacks were then deconvolved using the maximum likelihood estimation algorithm of the Huygens Pro software (version 2.4.1; Scientific Volume Imaging). Transparent projections (in face view) were applied to each Z stack using the projection tool of the LSM 510 software. Images were saved in TIFF file format. To quantify fluorescence intensity of ETA, ETB, and ET-1, we used internal and external elastic lamina autofluorescence to identify the limits of the media and the endothelium. Using the “close free shape curve” tool of the LSM image software, the endothelium or the media was isolated by masking the remaining image. Mean fluorescence intensity was calculated over the nonmasked region by the LSM 510 software. This operation was executed at every five images of each Z stack. The mean fluorescence intensity of all the images in a Z stack was then averaged.

Study Drugs The thromboxane A2 mimetic U-46619, L-NNA, Ach, SNP, and N-acetyll-cysteine were purchased from Sigma Chemicals, and ET-1 was purchased from American Peptide (Sunnyvale, CA). All drugs were dissolved in nanopure water except for U-46619, which was dissolved in 95% ethanol. All solutions were kept at ⫺20⬚C at different concentrations of 1 pmol/L to 100 ␮mol/L, except for L-NNA, which was freshly prepared.

Statistical Analysis Results are expressed as mean ⫾ SEM. Hemodynamics, gravimetric parameters, and ET-1 levels compared by analysis of variance followed by multiple groups comparisons using the Bonferroni correction. Differences in the isolated lungs’ pressure–flow relations were evaluated by repeated-measures analysis of variance followed by multiple groups comparisons with Bonferroni correction. The individual pressure–flow relationships for each group were fitted by linear regression to determine their slope and intercept. Vasoconstriction of pulmonary vessels was expressed as a percentage of the maximal response (Emax) obtained in the presence of 127 mmol/L KCl; vasodilatations are expressed as the percentage of inhibition of the preconstricting tone induced by 100 nmol/L U46619. The EC50 and the Emax were measured from each individual dose–response curve using a five-parameter logistic function with SigmaPlot curve-fitting software. The pD2 value reported is the negative log of the EC50. For these parameters, differences between the ET 7-day or ET 28-day groups and their respective control groups were evaluated with a two-tailed unpaired Student’s t test. Statistical significance was assumed at p ⬍ 0.05.

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RESULTS Stability and Biological Activity of ET-1 after 7 and 28 Days

After in vitro incubation for 7 and 28 days, there was no degradation of ET-1 as measured by HPLC (data not shown). The biological activity of ET-1, as assessed from its vasoconstrictive action on isolated small pulmonary vessels, was also preserved (Figure 1). The maximal constriction and pD2 values for nonincubated ET-1 were 90.8 ⫾ 3.4% and 8.06 ⫾ 0.12%. The same parameters for ET-1 incubated for 7 days were 90.8 ⫾ 6.6% and 8.14 ⫾ 0.22%, respectively, and 90.2 ⫾ 8.0% and 7.50 ⫾ 0.29%, respectively, for the ET-1 incubated for 28 days. Effect of ET-1 Infusion on Hemodynamic and Gravimetric Parameters

Infusion of ET-1 almost doubled plasma levels after both 7 and 28 days (Figure 2A). This finding was accompanied by an increase in lung tissue ET-1 levels after 7 days, and levels remained increased, although not significantly, after 28 days (Figure 2B). The mean arterial pressure was nonsignificantly higher in the ET 28-day group (105.5 ⫾ 3.1 mm Hg) compared with the control group (98.3 ⫾ 2.8 mm Hg; Table 1). The ET-1–perfused rats did not develop PH as demonstrated by similar right ventricular systolic pressure values. The only significant hemodynamic change was a higher rate of relaxation of the left ventricle (LVdP/dT) in both the ET 7- and 28-day groups compared with the control group. At Days 7 and 28, the body weight of the ETinfused rats was significantly lower (p ⬍ 0.05) compared with control rats (Table 1). There were no differences in the right ventricle/(left ventricle plus septum) weight and the lung dry/ wet weight ratios between the four experimental groups. Pulmonary Pressure–Flow Relationships

The pressure–flow relationship was shifted upward (p ⬍ 0.05) in the ET 7-day group compared with the control group (Figure 3). Furthermore, the slope of this relationship was higher in the ET 7-day group (0.53 ⫾ 0.02 mm Hg/ml/second) compared with the 7-day control group (0.47 ⫾ 0.01 mm Hg/ml/second; p ⬍ 0.05), but there was no difference for the intercept. On the other hand, the 28-day groups did not display any significant difference. The administration of L-NNA did not modify the pressure–flow relationships in any group (Figure 4).

Figure 4. Pressure–flow relationship for each group under baseline conditions and after pretreatment with L-NNA (100 ␮mol/L). (A ) Control 7 days; (B ) ET 7 days; (C ) control 28 days; (D ) ET 28 days.

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Pulmonary Vascular Reactivity

The maximal vasodilator response to acetylcholine (Ach) was greatly reduced in the ET 28-day group (23.7 ⫾ 4.0%) compared with 28-day control group (53.6 ⫾ 6.1%, p ⬍ 0.01) but without any difference in the pD2 values (Figure 5A; Table 2). ET infusion for 7 days did not modify the maximal relaxation to Ach, but mildly increased the sensitivity of the small pulmonary arteries as evidenced by a higher pD2 value when compared with control animals (7.24 ⫾ 0.27 vs. 6.52 ⫾ 0.28; p ⬍ 0.05). The dose–response curves to SNP were nearly identical for the 7-day groups (Figure 5B; Table 2). The maximal endothelium-independent vasodilator response of small arteries from the ET 28-day group, however, was greatly reduced from 64.8 ⫾ 4.2% in control animals to 35.1 ⫾ 4.0% (p ⬍ 0.001). All four experimental groups demonstrated similar vasoconstriction when challenged with the thromboxane A2 mimetic U-46619 (Figure 6A; Table 3), except for a greater maximal vasoconstriction in the ET 28-day group (77.5 ⫾ 6.3% vs. 60.4 ⫾ 2.0%; p ⬍ 0.05). The vasoconstrictor response to ET-1 was also unaffected in the experimental groups (Figure 6B; Table 3), except for a reduced sensitivity in the ET 7-day groups as evidenced by a slightly lower pD2 value (8.00 ⫾ 0.03) compared with the control group (8.38 ⫾ 0.08; p ⬍ 0.001).

To further evaluate the mechanisms responsible for the reduced vasodilator response to Ach and SNP in the ET 28-day group, experiments were performed with the addition of L-NNA and N-acetyl-l-cysteine (Figure 7). The addition of L-NNA normalized the response to Ach and increased the maximum vasodilator response from 23.7 ⫾ 4.0% to 54.6 ⫾ 8.8% (p ⫽ 0.013). The addition of N-acetyl-l-cysteine also normalized the response to SNP from 35.1 ⫾ 4% to 69.5 ⫾ 13.9% (p ⫽ 0.029). ET-1, ETA, and ETB Receptors in Small Pulmonary Arteries

Examples of composite Z-stacked images obtained with antibodies to ET-1, the ETA, and ETB receptors and smooth muscle actin are shown in Figure 8. Autofluorescence of the internal and external elastic lamina enables easy demarcation of the endothelium from the media. As expected, there was no detectable ETA receptor on the endothelium. ET-1 and the ETB receptor were present on both the endothelium and in the media in similar proportion. Fluorescence intensity revealed an approximate threefold increase in ETB receptor in both the intima and media of small pulmonary arteries in the ET 7-day group (p ⬍ 0.01; Figure 8), which returned to control levels after 28 days. There was no significant modification of the ETA receptor and of ET-1.

DISCUSSION This study was designed to evaluate the impact of chronically elevated plasma ET-1 levels on pulmonary vascular reactivity. We found that increased ET levels for a period of up to 4 weeks did not cause PH or right ventricular hypertrophy (RVH) but was associated with modifications of pulmonary vascular reactivity characterized by a markedly reduced response to NO. Effects of ET-1 Infusion on Plasma and Tissue Levels

Chronic ET-1 perfusion almost doubled plasma levels after 7 and 28 days. The levels attained are similar to those reported in many animal models of PH as well as in human subjects with PH. This finding was accompanied by an increase in lung tissue ET-1 levels after 7 days, which was less marked and no longer significant after 28 days. The pulmonary circulation is an important site for ET-1 removal from the circulation that is mediated by the endothelial ETB receptor (13). In isolated rat lungs, close

TABLE 2. EFFECT OF ENDOTHELIN-1 PERFUSION ON pD2 VALUES AS MAXIMAL AND CALCULATED MAXIMAL RELAXATION TO ACETYLCHOLINE AND SODIUM NITROPRUSSIDE IN SMALL PULMONARY ARTERIES PRECONTRACTED WITH U-46619 (100 nmol/L) n Ach Control 7 days ET 7 days Control 28 days ET 28 days SNP Control 7 days ET 7 days Control 28 days ET 28 days

Figure 5. Reactivity of small pulmonary artery segments to (A ) acetylcholine (Ach) and (B ) sodium nitroprusside (SNP). Relaxations are expressed as mean ⫾ SEM of six to nine experiments. White squares represent control 7-day group; solid squares represent ET 7-day group; white circles represent control 28-day group; solid circles represent ET 28-day group.

pD2

Maximum Relaxation (%)

9 9 8 7

6.52 7.24 6.89 6.51

⫾ ⫾ ⫾ ⫾

0.13 0.27* 0.12 0.24

50.2 52.1 53.6 23.7

⫾ ⫾ ⫾ ⫾

4.4 7.6 6.1 4.0†

9 6 6 7

7.66 7.87 7.73 7.27

⫾ ⫾ ⫾ ⫾

0.17 0.11 0.12 0.20

50.5 50.8 64.8 35.1

⫾ ⫾ ⫾ ⫾

7.3 8.1 4.2 4.0‡

Definition of abbreviations: Ach ⫽ acetylcholine; ET ⫽ endothelin; SNP ⫽ sodium nitroprusside. Results are expressed as mean ⫾ SEM. * p ⬍ 0.05 versus control 7 days. † p ⬍ 0.01 versus control 28 days. ‡ p ⬍ 0.001 versus control 28 days.

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Figure 7. Reactivity of small pulmonary artery segments in the ET 28-day group to Ach in the absence or presence of the nitric oxide synthase inhibitor L-NNA (upper panel), and to SNP in the presence or absence of the antioxidant N-acetylL-cysteine (NAC; lower panel). Relaxations are expressed as mean ⫾ SEM of six to eight experiments. Upper panel: circles represent ET 28-day group; triangles represent ET 28-day ⫹ L-NNA group. Lower panel: squares represent ET 28day group; inverted triangles represent ET 28-day ⫹ NAC group.

Figure 6. Reactivity of small pulmonary artery segments to (A ) U-46619 and (B ) ET-1. Constrictions are expressed as mean ⫾ SEM of 7 to 10 experiments. White squares represent control 7-day group; solid squares represent ET 7-day group; white circles represent control 28-day group; solid circles represent ET 28-day group.

to 60% of injected ET is removed within a single transit time (14). It is thus possible that the increased tissue levels were a direct consequence of ET-1 uptake and internalization. This may have been facilitated by the measured increase in endothelium

TABLE 3. EFFECT OF ENDOTHELIN-1 PERFUSION ON pD2 VALUES AS MAXIMAL AND CALCULATED MAXIMAL CONSTRICTION (%Emax) TO U-46619 AND ENDOTHELIN-1 IN SMALL PULMONARY ARTERIES n U-46619 Control 7 days ET 7 days Control 28 days ET 28 days ET-1 Control 7 days ET 7 days Control 28 days ET 28 days

pD2

Maximal Constriction (%)

8 7 9 10

7.05 7.07 7.06 6.87

⫾ ⫾ ⫾ ⫾

0.06 0.05 0.04 0.08

67.2 64.0 60.4 77.5

⫾ ⫾ ⫾ ⫾

2.3 3.4 2.0 6.3†

7 7 8 10

8.38 8.00 8.39 8.36

⫾ ⫾ ⫾ ⫾

0.08 0.03* 0.07 0.16

100.7 104.4 100.4 110.5

⫾ ⫾ ⫾ ⫾

1.8 2.3 1.8 5.5

Definition of abbreviation: ET ⫽ endothelin. Results are expressed as mean ⫾ SEM. * p ⬍ 0.001 versus control 7 days. † p ⬍ 0.05 versus control 28 days.

ETB that we found after 7 days of ET-1 infusion. In isolated porcine endothelial cells, binding of exogenous ET-1 to the ETB receptor has also been shown to modulate ET-1 synthesis through a negative feedback loop (15, 16). This second mechanism together with the reduction of endothelium ETB receptor may explain the lowering of lung tissue ET-1 levels after 28 days of ET-1 infusion. It has also been demonstrated that cultured peripheral lung microvascular smooth muscle cells can synthesize ET-1, and that ET-1 itself can stimulate prepro-ET-1 expression in these cells through stimulation of both the ETA and ETB receptors (17). The increased expression of ET-1 in cultured sheep endothelial cells after ET-1 stimulation is transient, however (17). Our findings of normal total lung tissue ET-1 levels after 28 days, and of unchanged small pulmonary artery ET-1 expression in the media after both 7 and 28 days, suggest a lack of in vivo effect of increased plasma ET-1 levels on vascular smooth muscle cell ET-1 production. Effects of ET-1 Infusion on Hemodynamics

Except for an improvement in the rate of relaxation of the left ventricle, chronic ET-1 infusion did not significantly modify in vivo hemodynamics in the systemic and pulmonary circulations. ET-1 is a potent inotropic agent and a negative lusitropic agent when used at pharmacologic doses in vitro. ET-1 also has a tonic positive chronotropic effect in normal subjects, which can be unmasked by selective ETA receptor blockade, but opposite negative inotropic effects in subjects with dilated cardiomyopathy (18). It is therefore difficult to determine if the positive lusitropic effects observed in the present study represent a compensatory mechanism or a direct effect of the ET infusion on the heart. Overall, the lack of important effects of isolated higher plasma ET levels on hemodynamics suggests a greater importance of local tissue production of ET-1 in pathologic conditions because ET-1 is principally a paracrine substance. The secondary increase in circulating ET levels may, however, contribute to alterations in vascular reactivity of more distant organs, such as the lung. Effects of ET Infusion on Pulmonary Vascular Reactivity

The isolated lung pressure–flow relationship was mildly but significantly shifted with a steeper slope after 7 days of ET infusion, suggesting an increase in pulmonary vascular resistance. This

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Figure 8. Immunofluorescence quantification of the distribution of ETA and ETB receptors and of ET-1 in transverse 14-␮m-thick sections of small pulmonary arteries. The images represent examples of composite Z stacks that were deconvolved and projected with the LSM 510 software (Zeiss). The first row of figures displays the fluorescence for the ETA and the ETB receptors and for ET-1, from left to right (in red). The second row displays the same components, but with the addition of the internal elastic lamina (IEL) and external elastic lamina (EEL; in green), which enables easy demarcation of the endothelium (E) from the media (M). The third row displays the fluorescence for smooth muscle actin (in blue), which is limited to the media (middle image) and colocalizes with the ETA receptor (on left). A phase contrast image is shown in the bottom right corner. Bars represent a scale of 10 ␮m. The computed mean fluorescence intensities are presented in the bar graphs. *p ⬍ 0.01 versus control 7 days.

finding was no longer apparent after 28 days of infusion, possibly because of compensatory mechanisms. The more than threefold increase in medial ETB receptor that we found after 7 days of ET infusion, with the subsequent reduction after 28 days, may represent such a mechanism. Among the possible mediators that could be modified by ET infusion, we evaluated the role of basal NO production and found that it did not modulate isolated pulmonary vascular tone in either control or ET-infused animals. We further explored the impact of chronically elevated plasma ET levels by studying isolated small pulmonary arteries. The most striking finding of these experiments was an important reduction of both endothelium-dependent and -independent vasodilatation to Ach and SNP, respectively, supporting either reduced smooth muscle sensitivity to NO or reduced biovavailability of produced NO. Although increased exposure of pulmonary artery smooth muscle cells to NO can reduce soluble guanylate cyclase mRNA and enzyme activity (19), this mechanism is unlikely in the present study. Furthermore, the lack of effect of NO

synthase inhibition on pulmonary pressure–flow relationships does not support a compensatory increase in basal NO production after ET infusion. Another possible explanation for our findings could stem from demonstrated effects of ET-1 on smooth muscle cell properties. It has been demonstrated in cerebral vessels that ET-1 can desensitize smooth muscle cell responsiveness to NO by a protein kinase C–independent pathway (20). We believe, however, that the most likely explanation for our findings resides in ET-1–induced augmentation of reactive oxygen species production in endothelial cells and smooth muscle cells, which would contribute to a reduction in the bioavailability of NO (21–23). This hypothesis is strongly supported by the normalization of SNP-induced vasodilatation by the addition of the antioxidant N-acetyl-l-cysteine. The apparently paradoxic normalization of the vasodilator response to Ach by the addition of the nonselective NO synthase inhibitor L-NNA may also be explained by increased reactive oxygen species production. Although highly speculative at this point, a possible explanation

Migneault, Sauvageau, Villeneuve, et al.: ET and Lung Vascular Reactivity

could reside in an increased superoxide formation through a modification of NO synthase isoforms and/or their substrates (24, 25), whereas maintained vasodilatation could occur by the release of endothelium-derived hyperpolarizing factor (26). The previous demonstrations that chronic ET receptor blockade could improve endothelium-dependent vasodilation to Ach in isolated rat lungs from animals with monocrotaline-induced PH as well as in lungs from rats with venous PH after myocardial infarction also support a deleterious effect of ET on endothelium-dependent relaxation (27, 28). Implications of These Findings

Chronic elevations of circulating ET levels are found in numerous pathologic conditions, such as congestive heart failure, liver disease, hypertension, and atherosclerosis. Because PH from all causes is associated with elevated ET levels and because some conditions cited previously (e.g., congestive heart failure and liver disease) may be associated with secondary PH, it becomes relevant to determine if elevated ET levels could contribute to altered pulmonary vascular reactivity. The impact of elevated levels on lung circulation may be particularly important because this organ is a primary site for circulating ET-1 clearance (29). Our major finding of an important reduction in the pulmonary vasodilator reserve in response to NO may therefore have significant pathologic implications in that context. Conclusions

One month of chronic hyperendothelinemia failed to induce PH or right ventricular hypertrophy. However, this condition causes an important modification of pulmonary vascular reactivity characterized by a reduced response of smooth muscle to NO. These findings suggest that conditions associated with increased circulating ET levels may reduce the pulmonary vasodilatory reserve in relation to an increased production of reactive oxygen species. Further studies are needed to determine if, in the long term, this situation could contribute to the development of PH. Conflict of Interest Statement : A.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; L.V. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; E.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; N.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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