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Ann Thorac Surg 1999;68:1714-1721
© 1999 The Society of Thoracic Surgeons

Original Articles

Bosentan prevents hypoxia-reoxygenation–induced pulmonary hypertension and improves pulmonary function

Jeffrey M. Pearl, MDa, Scott A. Wellmann, MDa, Jerri L. McNamara, CCPa, John P. Lombardi, CCPa, Connie J. Wagner, BSa, Jenni L. Raake, RRTa, David P. Nelson, MDa

a Division of Pediatric Cardiothoracic Surgery, Children’s Hospital Medical Center, Cincinnati, Ohio, USA

Address reprint requests to Dr Pearl, Division of Cardiothoracic Surgery, Children’s Hospital Medical Center, 3333 Burnet Ave, OSB-3, Cincinnati, OH 45229
e-mail: pearj0{at}chmcc.org

Presented at the Thirty-fifth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 25–27, 1999.


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 References
 
Background. Acute hypoxia results in increased pulmonary vascular resistance. Despite reoxygenation, pulmonary vascular resistance remains elevated and pulmonary function is altered. Endothelin-1 might contribute to hypoxia-reoxygenation–induced pulmonary hypertension and to reoxygenation injury by stimulating leukocytes. This study was carried out using an established model of hypoxia and reoxygenation to determine whether endothelin-1 blockade with Bosentan could prevent hypoxia-reoxygenation–induced pulmonary hypertension and reoxygenation injury.

Methods. Twenty neonatal piglets underwent 90 minutes of hypoxia, 60 minutes of reoxygenation on cardiopulmonary bypass, and 2 hours of recovery. Control animals (n = 12) received no drug treatment, whereas the treatment group (n = 8) received the endothelin-1 receptor antagonist, Bosentan, throughout hypoxia.

Results. In controls, pulmonary vascular resistance increased during hypoxia to 491% of baseline and remained elevated after reoxygenation; however in the Bosentan group, it increased to only 160% of baseline by end-hypoxia, then decreased to 76% at end-recovery. Arterial endothelin-1 levels in controls increased to 591% of baseline after reoxygenation. Arterial nitrite levels decreased during hypoxia in controls but were maintained in the Bosentan group. Consequently, animals in the Bosentan group had better postreoxygenation pulmonary vascular resistance, A-a gradient, and airway resistance along with lower myeloperoxidase levels than controls.

Conclusions. Acute hypoxia and postreoxygenation pulmonary hypertension was attenuated by Bosentan, which maintained nitric oxide levels during hypoxia, decreased leukocyte-mediated injury, and improved pulmonary function.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 References
 
Acute hypoxia results in rapid and significant increases in pulmonary artery pressures as a result of pulmonary vasoconstriction. Pulmonary mechanics are also affected by hypoxia, resulting in increased airway resistance and decreased compliance. These acute changes often result in significant hemodynamic and respiratory compromise. Despite a return to normoxic conditions after reoxygenation, those perturbations can persist. This phenomenon is commonly seen after repair of cyanotic congenital heart disease on cardiopulmonary bypass.

Reoxygenation has been shown to result in a form of pulmonary reperfusion injury [1]. Pulmonary reoxygenation injury is manifested by elevated pulmonary vascular resistance, altered pulmonary mechanics [2], increased lung water, and impaired gas exchange. These changes have been associated with elevations in endothelin-1 (ET-1), decreased vascular nitric oxide (NO) production [1, 3–6], and decreased exhaled NO [1, 6]. Adhesion and activation of leukocytes is an important component of this inflammatory response.

The increase in leukocyte activity after reoxygenation is not surprising because both NO and ET-1 are believed to have important regulatory roles on leukocyte adhesion and activation [7]. Nitric oxide is thought to be an important inhibitor of reoxygenation-induced intercellular adhesion molecule (ICAM-1) upregulation by acting as a free-radical scavenger and as a direct inhibitor of the intracellular messenger nuclear factor-kappa ß (NF{kappa}B.) Decreased levels of NO at reoxygenation favor the upregulation of ICAM-1 and increase leukocyte adhesion and subsequent activation [7].

Endothelin-1 has potentially conflicting actions with regard to both NO and the predominance of ET-1 receptors. ETa receptor activation results in the adhesion, migration, and activation of neutrophils [8–10]. In contrast, ETb receptor stimulation results in local production of NO and, hence, could decrease neutrophil adherence and blunt vasoconstriction. However, it appears that ETa receptors predominate in the lung, favoring the accumulation of neutrophils and net vasoconstriction.

Although Bosentan is considered both an ETa and ETb receptor blocker, it has higher affinity for ETa receptors than ETb and is likely to block pulmonary vasoconstriction and to prevent ET-1-mediated leukocyte adhesion and activation. Preservation of NO levels might also be expected, further limiting leukocyte adhesion and maintaining vasodilatation. This study was done using an established animal model of hypoxia and reoxygenation to investigate whether ET-1 blockade could block the late phase of pulmonary hypertension after hypoxia-reoxygenation, preserve NO levels, and blunt leukocyte adhesion and activation.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 References
 
Experimental groups
An established model of acute hypoxia and reoxygenation previously described by us [1] and others [11], involved 20 neonatal, 2-week-old piglets (Hampshire-Yorkshire) weighing 4 to 6 kg. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animals Resources and published by the National Institutes of Health (NIH Publication no. 86-23, revised 1985). This protocol was also approved by the animal care and use committee at Children’s Hospital Research Foundation.

The animals were anesthetized with ketamine, intubated with a cuffed endotracheal tube, and mechanically ventilated using a volume-control ventilator (Siemens 900B; Siemens-Elema, Solna, Sweden). A constant inhaled tidal volume of 18 mL/kg was maintained throughout the experiment, and the rate was adjusted to maintain the partial pressure of carbon dioxide between 36 and 44 mm Hg. The initial fraction of inspired oxygen was 40%. Femoral arterial and venous catheters were placed. Deep sedation and paralysis were maintained by an infusion of nembutal (20 mg/kg per hour), intermittent fentanyl (10 µg/kg per hour) and pancuronium (0.2 mg/kg per hour). Arterial blood gasses were analyzed and co-oximetry was done every 15 minutes (Chiron Diagnostics System 865; Chiron Diagnostics Co, East Walpole, MA).

A median sternotomy was done and a catheter was placed in the pulmonary artery to monitor its pressure. Transducer-tipped catheters (Millar Instruments Inc, Houston, TX) were placed in the right (3-F) and left ventricles (2-F) for measuring right and left ventricular end-diastolic pressures. Cardiac output was recorded with a Doppler flow probe placed around the pulmonary arterial trunk (T206 small animal blood flow meter; Transonics Systems, Inc, Ithaca, NY).

After stabilization, baseline measurements were made of pulmonary arterial pressures, left ventricular end-diastolic pressure, and cardiac output from which pulmonary vascular resistance was calculated. Alveolar-arterial (A-a) gradient was determined on the basis of a fraction of inspired oxygen of 40%. Measurements of airway resistance were made using the CO2SMO plus Respiratory Profile Monitor (Novametrix Medical Systems, Inc, Wallingford, CT).

After an equilibration period of 30 minutes, baseline hemodynamic measurements and arterial and pulmonary arterial samples were obtained. Hypoxia was then induced by decreasing the inhaled fraction of inspired oxygen to 12%. This resulted in a systemic saturation of 65% to 70%. Hemodynamic measurements and arterial samples were taken at 5, 15, 30, 45, 60, 75, and 90 minutes of hypoxia. In controls (n = 12) no drug therapy was initiated. Animals in the Bosentan-treated group (BOS, n = 8) received a 10 mg/kg bolus of Bosentan followed by a continuous infusion of 5 mg/kg per hour starting 20 minutes before induction of hypoxia. This is considered a high dose of Bosentan. Bosentan infusion was continued throughout hypoxia and for the first 30 minutes of reoxygenation (140 minutes total infusion time).

After 90 minutes of hypoxia, animals were placed on cardiopulmonary bypass (CPB) with a minimum PO2 in the bypass circuit of 450 mm Hg. The flow rate on bypass was maintained at the estimated cardiac output (100 cc/kg) with a minimum mean arterial pressure of 30 mm Hg. The ventilator FIO2 was increased to 100% during CPB and the rate decreased to 4 breaths per minute. Animals were cooled to 32°C rectal temperature. Blood samples were obtained at 5, 15, 30, and 60 minutes of reoxygenation. Cardiopulmonary bypass was used to replicate the clinical scenario of a cyanotic infant undergoing repair on CPB. Prior data from our laboratory have shown only slight effects of bypass in our model [1].

After 1 hour of CPB, animals were weaned from bypass and returned to normoxic conditions (fraction of inspired oxygen = 40%). They were maintained in a similar fashion as baseline for a recovery period of 2 hours. Blood samples were obtained for arterial NO and ET-1. Hemodynamics were recorded at 15, 30, 60, and 120 minutes of recovery. After 2 hours of recovery, animals were sacrificed by cardiectomy. Lung biopsies were obtained at baseline, end-hypoxia, and end-recovery. Lung samples were immediately sectioned, frozen in liquid nitrogen, and stored at -80°C.

Endothelin-1 assay
Blood samples were collected in ethylenediaminetetra-acetic acid tubes containing indomethacin (10 µg/mL), immediately centrifuged at 4°C, and frozen at -80°C for later analysis. Frozen lung tissue collected at baseline, end-hypoxia, and end-recovery were homogenized in 10 volumes (1 mL/100 mg) of 10% (volume/volume) acetic acid. Homogenates were centrifuged at 21,000 g for 20 minutes to remove precipitates. A commercial ET-1 immunoassay kit (R&D Systems, Minneapolis, MN) was used to measure ET-1 protein concentration, reported in pg/mL of plasma or pg/100 mg wet tissue.

Nitrite assay
Blood levels of NO were measured as nitrate and nitrite using a two-part assay with nitrate being converted to nitrite in a Griess reaction [12]. The optical density of each well was measured at 550 nm, with correction at 650 nm, by using a Labsystems Multiscan MCC/340 (Franklin, MA). Data were analyzed using GENESIS, a Windows-based microplate software (Fisher Scientific Co, Pittsburgh, PA). Nitric oxide level was reported as µmol/L nitrite.

Tissue myeloperoxidase assay
Frozen lung samples (50 mg) were homogenized in 1 mL of a solution containing 0.5% hexadecyltrimethylammonium bromide dissolved in 10 mmol/L 3-[N-morpholino]propanesulfonic acids, then centrifuged at 21,000 g for 20 minutes at 4°C. The supernatant (100 µL) was then mixed with 700 µL of sodium phosphate (80 mmol/L, pH 5.5) and 100 µL of tetramethyl benimide (16 mmol/L) and incubated at 25°C for 5 minutes. Then 100 µL of hydrogen peroxide (1 mmol/L) was added and incubated exactly 3 minutes at 25°C. A blank without hydrogen peroxide was also analyzed for each tissue. The reaction was stopped by the addition of 1 mL of cold acetic acid (2 mol/L). The optical density was read at 650 nm on a spectrophotometer. Myeloperoxidase (MPO) activity is expressed as U/50 mg tissue per 3 minutes.

Tissue lipid peroxidase assay
Frozen lung tissue was homogenized in 10 volumes (1 mL/100 mg) of 10% (volume/volume) 1.15% potassium chloride on ice. A modified thiobarbituric acid reaction technique was used [13]. The optical density at 532 nm was measured, and concentration was determined by using a Spectronic Genesys spectrophotometer (Spectronic Instruments, Rochester, NY). Concentrations were calculated using standard solutions of malondialdehyde-dimethylacetate (0.5 to 16 nmol) and are expressed in nmol/100 mg wet tissue.

Statistical analysis
Analysis of variance and the two-tailed, paired Student t test were used to compared values at different time periods. Data are presented as the mean ± standard deviation. A p value of 0.05 or less was considered significant (Statview 4.01 software; Abacus Concepts Inc, Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 References
 
Pulmonary vascular resistance rapidly increased in control animals during hypoxia reaching 491% of baseline (393 dynes/cm5 per second) and remained elevated after reoxygenation (221% of baseline, 177 dynes/cm5 per second). In contrast, after an initial increase in the Bosentan group, PVR decreased to 160% (176 dynes/cm5 per second) by end-hypoxia, and to 76% of baseline (84 dynes/cm5 per second) at end-recovery (Fig 1). Mean pulmonary arterial pressures also increased in a similar manner in controls but were significantly lower in Bosentan-treated animals throughout hypoxia and recovery (Fig 2).



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  		Fig 1. Pulmonary vascular resistance rapidly increased in the onset of hypoxia in both groups. However, pulmonary vascular resistance began to decrease by 30 minutes of hypoxia in the Bosentan-treated animals and remained lower than controls at all time points thereafter. *p < 0.05 Bosentan versus controls.

 


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  		Fig 2. Mean pulmonary arterial pressures doubled by 30 minutes of hypoxia in the controls. Animals treated with Bosentan throughout hypoxia and recovery had lower mean pulmonary arterial pressure than controls at 30 minutes and later. *p < 0.05 Bosentan versus controls.

 
In controls, arterial ET-1 increased during hypoxia (0.55 pg/mL baseline and 0.90 pg/mL end-hypoxia) and more markedly after reoxygenation (3.1 pg/mL, 591% of baseline). In the Bosentan group arterial ET-1 levels increased markedly after infusion of Bosentan, reflecting the displacement of bound ET-1 by Bosentan (Fig 3). Pulmonary tissue ET-1 levels tended to increase during hypoxia and reoxygenation in control animals but decreased significantly both at end-hypoxia and end-recovery in the Bosentan group, indicating displacement of pulmonary receptor-bound ET-1 by Bosentan (Fig 4). At end-recovery, pulmonary ET-1 levels were significantly lower in the Bosentan group than in controls (95 versus 170 pg/100 mg wet tissue, p = 0.005).



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  		Fig 3. (Top) Endothelin-1 levels in the treated group rapidly increased after the administration of Bosentan because of the release of receptor-bound ET-1. (Bottom) Arterial endothelin-1 increased only slightly in control animals during hypoxia but increased more dramatically after reoxygenation. *p < 0.05 Bosentan versus controls; {ddagger}p < 0.05 time point versus baseline.

 


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  		Fig 4. Pulmonary tissue endothelin-1 levels did not change during hypoxia and reoxygenation in controls. These levels correlated with increased pulmonary vascular resistance and lung injury. In contrast, endothelin-1 levels decreased in the Bosentan-treated animals during hypoxia and reoxygenation because of the release of receptor-bound endothelin-1 by Bosentan. *p < 0.05 Bosentan versus controls; **p < 0.005 Bosentan versus controls; {ddagger}p < 0.005 time point versus baseline.

 
Arterial NO decreased by 50% by end-hypoxia in controls but was preserved at or above baseline at both end-hypoxia and end-recovery in the Bosentan group (Fig 5). Despite a higher baseline value for MPO in the Bosentan-treated animals, levels did not increase significantly after hypoxia-reoxygenation as they did in controls (Fig 6).



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  		Fig 5. Arterial nitric oxide levels, measured as nitrite, decreased 50% in control animals during hypoxia and slowly returned to baseline by end-recovery. Animals treated with Bosentan maintained nitric oxide levels throughout hypoxia and reoxygenation. *p < 0.005 Bosentan versus controls; {ddagger}p < 0.005 time point versus baseline.

 


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  		Fig 6. Pulmonary myeloperoxidase activity increased after reoxygenation in controls but not in Bosentan-treated animals, despite higher baseline values in the Bosentan group. End-recovery myeloperoxidase activity was higher in the controls than in the Bosentan group, suggesting increased leukocyte activity in the control group. *p < 0.05 Bosentan versus controls; {ddagger}p < 0.005 time point versus baseline.

 
End-recovery MPO levels were significantly higher in controls than in animals administered Bosentan (0.41 versus 0.31 U/50 mg tissue per 3 minutes, p = 0.05). Lipid peroxidase levels in lung tissue actually decreased during hypoxia-reoxygenation in the Bosentan group but were significantly increased at end-recovery in controls (8.17 versus 6.34 nmol/100 g wet tissue weight, p = 0.004, Fig 7).



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  		Fig 7. Pulmonary tissue lipid peroxidase activity was higher than baseline and end-hypoxia levels after reoxygenation in control animals. In contrast, there was a trend for lipid peroxidase levels to decrease in the Bosentan group at end-hypoxia and end-recovery. At end-recovery lipid peroxidase levels were greater in controls than in Bosentan-treated animals. *p < 0.05 Bosentan versus controls; {ddagger}p < 0.005 time point versus baseline.

 
Bosentan treatment resulted in lower postreoxygenation wet/dry weight ratios (Fig 8) and lower A-a gradient after reoxygenation than in controls (Fig 9). Inspiratory airway resistance increased during hypoxia in both groups; however, it remained elevated after reoxygenation in controls, whereas inspiratory airway resistance in the Bosentan group decreased after hypoxia and was not different from baseline values at end-recovery (Fig 10).



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  		Fig 8. Total lung water, determined by the wet:dry tissue ratio, was increased over baseline at end-recovery in control animals. The lung wet:dry ratio of the Bosentan-treated animals was lower than that in controls at end-recovery and did not differ from baseline. *p < 0.05 Bosentan versus controls; {ddagger}p < 0.05 time point versus baseline.

 


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  		Fig 9. Alveolar-arterial (A-a) gradient was increased above baseline at end-recovery in both groups. In addition, the A-a gradient of the Bosentan-treated animals was lower than control animals at end-recovery. *p < 0.05 Bosentan versus controls; {ddagger}p < 0.005 time point versus baseline.

 


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  		Fig 10. Inspiratory airway resistance increased during hypoxia in both treatment groups, although less dramatically in the animals treated with Bosentan. The airway resistance returned to baseline levels by end-recovery in the Bosentan-treated group but remained elevated in the controls. *p < 0.05 Bosentan versus controls; {ddagger}p < 0.05 time point versus baseline.

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
References
 
Prior studies suggested that hypoxic pulmonary hypertension is associated with increased systemic and pulmonary tissue ET-1 [14, 15] along with decreased NO level [3, 6]. Decreased exhaled NO, presumably a result of decreased bronchial epithelial nitric oxide synthase activity, has also been demonstrated and correlated with altered pulmonary mechanics [1].

After reoxygenation, PVR fails to return to baseline normoxic levels. Furthermore, pulmonary function remains altered, which compromises ventilation and gas exchange. Persistent elevation of ET-1 and failure of endothelial-dependent vasodilatation have been implicated as causative factors in postreoxygenation pulmonary hypertension [1, 2]. Furthermore, leukocyte-mediated reoxygenation injury has been described with resultant increased pulmonary water content and altered histology [16]. Reoxygenation injury manifests clinically as increased A-a gradient, decreased pulmonary compliance, and increased airway resistance, and occasionally as pulmonary hemorrhage. In combination with the increase in PVR and resultant right ventricular strain, significant cardiopulmonary dysfunction and shock can result.

The increase in pulmonary leukocyte activity after reoxygenation is not surprising because both NO and ET-1 have important regulatory roles in leukocyte adhesion and activation [16, 17]. In addition, ET-1 and NO have a complex counter-regulatory feedback mechanism [18, 19].

Endothelin-1
Endothelin-1 is a 21-amino acid polypeptide with a half-life of 1.5 seconds. Produced by the lungs, as well as other tissues, it is thought to act in a paracrine fashion. It has numerous effects on cardiovascular physiology; most notably, it is a potent pulmonary and coronary vasoconstrictor [20]. Endothelin-1 has also been shown to decrease pulmonary compliance by direct action on bronchial epithelium and smooth muscle. A sustained increase in ET-1 has been implicated in chronic hypoxic pulmonary hypertension, as well as pulmonary hypertension associated with acute lung injury such as from ischemia-reperfusion.

Previous studies have shown that both arterial and pulmonary tissue ET-1 increases during acute hypoxia. Shirakami and associates [21] demonstrated a 46% to 85% increase in ET-1 levels in the lung and a 139% increase in plasma ET-1 after 60 minutes of hypoxia. In the current study, we found that arterial ET-1 nearly doubled by 90 minutes of hypoxia and increased by more than 550% after reoxygenation. These data support a role for ET-1 in the second phase of acute pulmonary hypertension. However, ET-1 increased only minimally in the control animals early in hypoxia (0.55 versus 0.61 pg/mL at 15 minutes) despite significantly increased PVR. This temporal delay in ET-1 increase suggests that other factors might be responsible for this first phase of acute hypoxic pulmonary hypertension [22].

In the current study, Bosentan administration did not significantly lower PVR at 5 and 15 minutes of hypoxia but resulted in significantly lower PVR at 30 minutes and beyond. Others have also shown that ET-1 blockade with either Bosentan or BQ123 failed to prevent the initial acute pulmonary vasoconstriction seen early in hypoxia [23]. It is likely that decreased NO levels, increased thromboxane B2, or direct changes in calcium transients are responsible for the early first phase of hypoxic pulmonary vasoconstriction that occurs as soon as alveolar oxygen tension decreases to hypoxic values. However, the second phase of acute pulmonary hypertension and postreoxygenation pulmonary hypertension appears to correlate with a significant increase in ET-1.

Differential effects of endothelin-1 receptor activation
Two main ET-1 receptors have been identified, ETa and ETb. There is wide variation among tissues within a species and between species regarding the distribution of these receptors. Previous investigators showed that ETa receptors are primarily responsible for the pulmonary vasoconstrictive effects of ET-1. In adult pigs, hypoxic vasoconstriction has been blocked by ETa receptor antagonists as well as by Bosentan [24]. In contrast, it has been suggested that ETb receptors cause vasodilatation by means of a NO pathway [25].

Porcine blood vessels and bronchial epithelium are rich in ETa receptors, and activation can contract isolated airway tissue [26] and increase airway resistance [27]. In our model, increased airway resistance was found during hypoxia and after reoxygenation, in conjunction with the increase in ET-1. Of note, animals treated with Bosentan had significantly less increase in airway resistance. Lower airway resistance could indicate decreased injury or ETa receptor blockade in bronchial epithelium.

Nitric oxide in hypoxia-reoxygenation and its interaction with endothelin-1
In conjunction with increased ET-1 levels in chronic hypoxia, arterial NO and pulmonary endothelial nitric oxide synthase levels are decreased [28]. ET-1 suppresses nitric oxide synthase activity, suggesting a possible mechanism behind the decreased NO levels observed in this study as ET-1 levels increased. It is interesting that in the current study, NO levels decreased to 50% of baseline with acute hypoxia in controls, but were preserved in animals treated with Bosentan.

Bosentan
Bosentan (Ro 74-0203; Hoffmann-LaRoche, Switzerland) is a nonpeptide, nonselective ET-1 receptor antagonist that can be administered orally or intravenously. Bosentan has 20 to 30 times higher affinity for ETa than ETb. Consequently, low doses of Bosentan can block ETa, increasing ET-1 levels in blood, yet leave ETb receptors available. The minimal blockade of ETb and the increase in ET-1 levels could result in a shift in balance from predominately ETa vasoconstriction to ETb vasodilation [29].

Oral Bosentan therapy prevented acute hypoxic pulmonary hypertension, prevented chronic pulmonary hypertension, and reversed established chronic hypoxic pulmonary hypertension in rats [30]. Bosentan also decreased basal vascular resistance and increased plasma levels of ET-1 in pigs [31].

In a model of endotoxin-induced pulmonary hypertension, investigators blocked the late phase in pigs by administering Bosentan. A biphasic pattern was demonstrated with the first phase blocked by cyclooxygenase inhibitors [32] or TXA2 inhibitors [33, 34]. Bosentan did not abolish the first phase but did block the second phase. In the present study, Bosentan attenuated the second phase of acute hypoxic pulmonary hypertension, similar to its effect in acute lung injury. This effect appears to occur by at least two mechanisms, blockade of ETa receptors and preservation of NO levels.

Endothelin-1 and leukocyte adhesion and activation
Endothelin-1 increases CD18 and CD11b expression on neutrophils [35] and ICAM-1 on endothelium [36]. Endothelin-1 also stimulates leukocyte rolling and adherence through ETa receptor activation [9] and increases neutrophil migration [37]. In vivo studies showed that ET-1 results in the adhesion, migration, and activation of neutrophils [8–10]. In a study on the effect of ET-1 on pulmonary neutrophil accumulation, Helset and colleagues [8] found that infusion of ET-1 resulted in a tenfold increase in the volume density of neutrophils in the lung 120 minutes after infusion. They also found that ET-1 infusion into isolated rat lungs resulted in adhesion of leukocytes to the pulmonary vascular endothelium and sequestration of leukocytes in the pulmonary capillaries [8].

In a rat model of ischemia-reperfusion, Boros and colleagues [9] demonstrated that ET-1 blockade prevents the accumulation of neutrophils after reperfusion and subsequently decreases pulmonary damage compared with nontreated animals. Similarly, in the current study we found increased MPO activity in the lungs after hypoxia-reoxygenation in association with increased ET-1. Bosentan administration prevented the increased MPO activity.

Endothelin-1 also primes neutrophils for enhanced superoxide production [10], stimulates platelet-activating factor release by neutrophils, and stimulates cytokine production by mononuclear cells (monocytes) [38]. Macrophages are prevalent in alveoli and throughout the lung. Interestingly, pretreatment of neutrophils with L-arginine blunted the ET-1-induced calcium transient, which is related to their activation [35]. This finding alludes to the complex, and as of yet not fully understood, interaction between the NO and ET-1 pathways. Endothelin-1 blockade prevented reoxygenation-induced increases in pulmonary MPO and lipid peroxidase in the present study.

Nitric oxide and regulation of leukocyte adhesion and activation
Nitric oxide is thought to be an important regulator of reoxygenation-induced ICAM-1 upregulation by acting as a free-radical scavenger and as a direct inhibitor of the intracellular messenger NF{kappa}B [17]. Nitric oxide decreases and prevents leukocyte-mediated adhesion and leukocyte-mediated injury by interfering with ICAM-1 expression and NF{kappa}B and by maintaining local vasodilatation of postcapillary venules. Nitric oxide could also have direct effects on neutrophil adhesion and activation [7, 39].

Endogenous NO is also an inhibitory modulator of ET-1 production, via a cyclic guanosine monophosphate–dependent mechanism [40]. The decreased NO levels found during hypoxia in our model resulted in loss of this modulation and could have contributed to the increased ET-1, with subsequent increased ET-1-stimulated leukocyte adhesion and activation [3]. In conjunction with the decreased levels of NO seen in hypoxia, leukocyte adhesion and activation might proceed unchecked. Preservation of NO levels might be one of the mechanisms by which ET-1 blockade with Bosentan decreased leukocyte-mediated injury.

The second phase of acute hypoxic pulmonary hypertension is mediated by ET-1. Despite normalization of NO levels, PVR remained elevated and gas exchange remained impaired after hypoxia-reoxygenation. Endothelin-1 blockade with Bosentan prevented hypoxia-reoxygenation–induced pulmonary hypertension, maintained NO levels during hypoxia, decreased leukocyte-mediated injury, and thus, resulted in improved pulmonary function.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 References
 

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