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Endothelin-1 Molecular Ribonucleic Acid Expression in Pulmonary Hypertensive and Nonhypertensive Chickens

A. P. Gomez^*, M. J. Moreno^*, R. M. Baldrich and A. Hernández^*,1

^* Facultad de Medicina Veterinaria y de Zootecnia, and Instituto de Genética, Universidad Nacional de Colombia, Bogotá DC1, Colombia

¹ Corresponding author: ahernandezv{at}unal.edu.co

	ABSTRACT

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Four hundred 1-d-old Cobb broilers were distributed in 3 groups: group A comprised broilers maintained under natural hypobaric hypoxia (Bogotá, Colombia); group B comprised broilers under relative normoxia (Villavicencio, Colombia); and group C comprised broilers maintained at 460 m above sea level (Villavicencio, Colombia) from d 1 to 25 of age, and then moved to 2,638 m above sea level (Bogotá, Colombia). Broilers were designated as nonpulmonary hypertensive (NPHB) and pulmonary hypertensive (PHB), to estimate possible differences between them in the lung expression of endothelin 1 (ET-1) mRNA at 24 and 42 d of age. In group A, 12 NPHB and 12 PHB were used for determination of ET-1 mRNA expression at 42 d. In group B, nonPHB were found, and therefore, ET-1 mRNA expression was detected in 48 NPHB, 24 of them in each age group (24 and 42 d). In group C, only NPHB were encountered at 42 and 53 d, and ET-1 mRNA expression was determined at 42 d in 24 birds. The ET-1 mRNA levels of PHB of group A at 42 d were significantly higher than the correspondent ones in NPHB of groups A (P < 0.001) and C (P < 0.05) at the same age. No differences in ET-1 mRNA expression were encountered between NPHB of groups A and B at 42 d (P > 0.05). However, ET-1 mRNA expression was higher in group C than the correspondent one in NPHB of groups A and B at 42 d (P < 0.001). The present data suggest that ET-1 may play a major role in pulmonary hypertension pathophysiology. It is possible that chickens should be exposed to hypobaric hypoxia before d 24, as a requisite to develop pulmonary hypertension. These results might provide clues for future studies in pulmonary vasoconstriction and vascular remodeling.

Key Words: cardiac index • endothelin-1 • hypobaric hypoxia • normoxia • pulmonary hypertension

	INTRODUCTION

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Cardiac index (CI) is widely recognized as an accepted measure to assess the presence or absence of pulmonary hypertension (PH; Burton and Smith, 1967; Cueva et al., 1974; Hernandez, 1987). High-altitude hypoxia is a known cause of PH in humans and broilers resident at high altitude (Meyrick and Reid, 1978; Currie, 1999; Cogo et al., 2004; Bartsch et al., 2005; Kanazawa et al., 2005; Reeves and Grover, 2005; Remillard and Yuan, 2005; Rhodes, 2005). Previous studies demonstrated that pulmonary vascular resistance is enhanced by constriction of pulmonary vascular smooth muscle and structural remodeling of the vascular bed (Reid, 1979; Stenmark and Mecham, 1997). These events are associated with changes in serum levels and endothelial expression of molecules in the lungs of humans and animals exposed to acute or chronic hypoxia (Yoshibayashi et al., 1991; Elton et al., 1992; Goerre et al., 1995). Nevertheless, plasma concentrations of some molecules such as endothelin 1 (ET-1) and nitric oxide (NO) are poor predictors of their biological activities, as compared with correspondent tissue concentrations and mRNA expressions (Pollock, 1998). Inappropriately elevated pulmonary vascular resistance is an important factor to contribute to the development of PH in susceptible broilers (Kouyoumdjian et al., 1994; Wideman and Kirby, 1995).

Endothelial dysfunction is postulated to contribute to PH pathophysiology through several mechanisms, including the loss of NO-dependent dilatation and increased ET-1 production (Tozzi et al., 1989; Adnot et al., 1991; Inagami et al., 1995; Mawji and Marsden, 2003). The response of the correspondent genes in models of cellular activation reflects a reciprocal pattern of regulation known as transcriptional induction of ET-1 and destabilization of endothelial nitric oxide synthase (eNOS) mRNA, which is responsible for NO generation (Mawji and Marsden, 2003).

Endothelins (ET) are vasoconstrictor peptides that contribute to various functions in different tissues (Inagami et al., 1995). Among ET, ET-1 is the most abundant molecule produced by endothelial cells (Yanagisawa et al., 1988). It plays an important role in vasoconstriction, smooth muscle cells proliferation, and hypertrophy of pulmonary arterioles. Under conditions of acute and chronic hypoxia, endothelial cells release factors influencing vascular tone and remodeling (Faller, 1999). In response to low oxygen tension, cultured endothelial cells increase ET-1 transcription and expression (Kourembanas et al., 1991). The transcription response to hypoxia localizes to a hypoxia inducible factor-1 (HIF-1) element at –124/–118 of the human promoter (Hu et al., 1998; Minchenko and Caro, 2000; Mawji and Marsden, 2003), which is potentiated by p300/CBP-enhanced binding of activator protein-1 and GATA-2 (Bandyopadhyay et al., 1995; Yamashita et al., 2001; Mawji and Marsden, 2003).

It was found that ET-1, connective tissue growth factor (CTGF), and adrenomedullin were upregulated in pulmonary hypertensive broilers by hypobaric hypoxia at 24 d of age (Gomez et al., 2007). In contrast, the activity of NOS enzyme was reduced in endothelial cells of pulmonary hypertensive broilers (Moreno de Sandino and Hernandez, 2003). These data suggested that these molecules can play a major role in PH pathophysiology; however, their role in normal cardiovascular homeostasis and PH is unclear.

There are not previous reports about ET-1 mRNA expression in broilers maintained at high and low altitude. Neither, it is known how the change of altitude (from low to high) of 25-d-old broilers can regulate this expression. The aim of this study was to detect possible differences in ET-1 mRNA expression in lungs of 24- and 42-d-old broilers under hypobaric hypoxia and relative normoxia, and 42-d-old broilers remained at 460 m above sea level (masl) from d 1 to 25 of age, and then moved to 2,638 masl.

	MATERIALS AND METHODS

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Birds and Tissue Samples
Four hundred 1-d-old Cobb male broilers were obtained from a commercial hatchery and reared on floor, using standard nutritional and management procedures for commercial operations. The broilers were distributed in 3 groups: group A: broilers maintained under natural hypobaric hypoxia at 2,638 masl in Bogotá, Colombia (barometric pressure, P_B: 560 mmHg; oxygen partial pressure, PO₂: 117 mmHg; inspired PO₂: 105 mmHg; n = 200); group B: broilers under relative normoxia at 460 masl in Villavicencio, Colombia (P_B: 739 mmHg; PO₂: 155 mmHg; inspired PO₂: 143 mmHg; n = 100); and group C: broilers remaining at 460 masl (Villavicencio, Colombia) from d 1 to 25 of age, and then moved to 2,638 masl (Bogotá, Colombia; n = 100). Feed and water were provided for ad libitum consumption, and lighting was continuous. Temperature was initially 32°C and gradually allowed to drop to a range between 18 and 21°C after the third week of the growing period. At 460 masl (in Villavicencio, Colombia), temperature did not descend below 24°C, due to natural environmental conditions.

From the main groups, 144 broilers were chosen. For groups A and B, 2 age-groups were studied (24- and 42-d-old birds), and for group C, an age-group (42-d-old birds). To choose pulmonary hypertensive (PHB) and nonhypertensive broilers (NPHB), the animals were subjected to clinical examination and anesthetized with an intravenous injection of a mixture of ketamine (Ketalar; 10 mg/kg) and xylasine (Rompun; 0.1 mg/kg of body weight). After anesthesia, the body cavity was opened and the heart and left lungs were removed. The heart was dried and dissected after fixation in 10% buffered formalin to calculate CI. To evaluate PH in each group, the CI values were calculated according to the procedure used by Alexander and Jensen (1959) (CI = right ventricular weight/total ventricular mass weight x 100). Chickens with CI above 30 were designed as PHB and those with CI below 26 as NPHB (Hernandez, 1987; Moreno de Sandino and Hernandez, 2006; Gomez et al., 2007). In groups A and B, CI was calculated at 24 and 42 d of age; for group C, at 42 and 53 d of age. The latter age was included to give the broilers in group C a sufficiently long hypoxic stimulus.

Thus, in groups A and B, 96 broilers were used for determination of ET-1 mRNA expression (48 birds/group). In each group, 24 broilers were studied at 24 d of age and 24 at 42 d of age. In group C, ET-1 mRNA expression was determined in twenty-four 42-d-old broilers.

The apical regions of left lungs were obtained in all samples, frozen in liquid N, and stored at –80°C before the RNA extraction period. All experimental procedures used in this study were reviewed and approved by the Ethics Committee of the National University of Colombia, in accordance to international normative for the care and use of experimental animals.

Total RNA Extraction, Reverse-Transcription, and Real-Time PCR Analysis
Total RNA was extracted from 200 mg of lung tissue using Trizol reagent according to the manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA) and then mRNA was isolated using a Dynabeads mRNA Direct kit (Invitrogen Corp.). The mRNA concentration was quantified using light absorption at 260 and 280 nm (A260/280) with an UV-visible spectrophotometer (Spectronic BioMate 3 UV-Vis Spectrophotometer, Thermo Electron Corp., Waltham, MA). Gene mRNA expression of ET-1 in the lungs was determined by reverse-transcription PCR using random hexamer primers (Invitrogen Corp.), and it was quantified by real-time PCR using a Light Cycler thermocycler (Roche, Mannheim, Germany) as described previously (Steuerwald et al., 1999; Nogueiras et al., 2003; Hazari et al., 2004; Gomez et al., 2007). Reverse-transcription reactions without addition of reverse transcriptase served as negative controls to ensure PCR amplification specificity (RT-). The real-time PCR was performed using SYBR green PCR core reagents (Roche) and specific sense and antisense primers as previously reported (Gomez et al., 2007). The mRNA levels were normalized with respect to chick hypoxanthine phosphoribosyltransferase level in each sample, which was used as a control housekeeping gene. Product purity was confirmed by dissociation curves. The amplified products were resolved in 1.5% agarose gels and visualized with ethidium bromide.

Statistical Analysis
The CI and ET-1 mRNA levels are shown as the means ± SD. Significant differences (P < 0.05) in ET-1 mRNA levels between groups were determined using Student’s 2-tailed unpaired t-test. The CI comparisons among experimental groups were made by ANOVA (GraphPad Instat, GraphPad Software Inc., San Diego, CA).

	RESULTS

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Cardiac Index and ET-1 mRNA Expression
Group A.
The NPHB and PHB under hypobaric hypoxia conditions were found at both ages. PH incidence was 13% at 24 d, and 7.5% at 42 d of age. The PHB had depression, cyanosis, ascites, and generalized congestion. These broilers had enlarged hearts and dilation of the atria and right ventricles. Lungs and livers were congested. The NPHB did not have any gross lesions.

The CI values and statistical differences among NPHB and PHB at 24 d of age were previously reported (Gomez et al., 2007). The CI values in NPHB were between 11 and 21% and between 35 and 59% in PHB at 42 d. Statistical differences in the 2 groups were highly significant (P < 0.001, Table 1). No differences were encountered when comparing the 2 ages between PHB and NPHB (P > 0.05, Table 1).

View larger version (26K): [in this window] [in a new window]   Figure 1. Representative reverse-transcription PCR for hypoxanthine phosphoribosyltransferase (HPRT; 179 bp) and endothelin 1 (ET-1; 141 bp) mRNA in lung samples from broilers. A 100-bp molecular weight marker (M) was used. The PCR products were separated on 1.5% agarose gel, stained with ethidium bromide, examined with UV light, and visualized with a Gel Doc system (Bio-Rad, Hercules, CA). Reverse-transcription reactions without addition of reverse transcriptase (negative controls; RT-) are shown and resulted in no bands after amplification.

View larger version (14K): [in this window] [in a new window]   Figure 2. Paired comparisons of endothelin 1 (ET-1) mRNA levels in the lung samples of experimental groups. Lung ET-1 mRNA expression was evaluated using semiquantitative real-time reverse-transcription PCR. The ET-1 mRNA levels were normalized to those of the internal control hypoxanthine phosphoribosyltransferase (HPRT). Group A: broilers subjected to hypobaric hypoxia (2,638 m above sea level, masl). Group B: broilers maintained under relative normoxia (460 masl). Group C: birds moved from 460 to 2,638 masl. Data are represented as mean ± SD. *P < 0.05, ***P < 0.001. Data of ET-1 mRNA expression levels, at high altitude and at 24 d old, were taken from a previous report (Gomez et al., 2007).

The CI values were between 9 and 18% at 24 d of age. The CI values ranged from 12 to 20% at 42 d. No statistical differences were found when comparing corresponding values at 24 and 42 d (P > 0.05, Table 1).

No differences in ET-1 mRNA levels were encountered between the 2 above-mentioned ages (P > 0.05, Figure 2).

Group C.
Only NPHB were found at 42 and 53 d. CI values were between 13 and 22% at 42 d, and between 14 and 23% at 53 d. Statistical differences between the 2 ages were not significant (P > 0.05, Table 1).

The ET-1 mRNA lung levels were significantly higher in broilers of group C than those detected in lungs of NPHB of group A at 42 d (P < 0.001, Figure 2).

Interactions among Experimental Groups
Statistical differences were not found to compare CI values of group B and NPHB of group A at 24 and 42 d (17.61 and 16.76 respectively, P > 0.05). The CI values were lower in group C at 42 d (17.18) than those detected in PHB of group A at 42 d (P < 0.001). No differences in CI values were encountered between group C and NPHB of groups A and B at 42 d (P > 0.05).

The ET-1 mRNA expression in lung samples of NPHB of group A was higher than the detected one in group B, at 24 d of age (P < 0.001). Also, ET-1 mRNA levels in the lungs of PHB of group A at 42 d of age were significantly higher than the correspondent ones in group C (P < 0.05) at the same age. We did not find differences to compare ET-1 mRNA lung levels in group B and NPHB of group A at 42 d (P > 0.05). However, ET-1 mRNA expression was higher in group C than in NPHB of groups A and B at 42 d of age (P < 0.001).

	DISCUSSION

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The present results are in agreement with previous reports, in which it was demonstrated that CI is a sensitive measurement of pulmonary artery pressure in poultry (Burton and Smith, 1967; Burton et al., 1968; Cueva et al., 1974). The CI is a valid parameter to diagnose PH at necropsy, which allowed us to classify broilers as PHB or NPHB at 2 ages and at different altitudes. In the present work it was corroborated that high altitude hypoxia is a cause of PH in susceptible strains of broilers (Meyrick and Reid, 1978; Currie, 1999; Cogo et al., 2004; Bartsch et al., 2005; Kanazawa et al., 2005; Reeves and Grover, 2005; Remillard and Yuan, 2005; Rhodes, 2005) because PHB were only found at high altitude (group A). Also, it is possible that CI might have a direct correlation with expression of molecules associated with PH pathophysiology, according with a previous report, where it was demonstrated that ET-1, CTGF, and adrenomedullin mRNA expression is higher in the lung of PHB with high CI at 24 d old (Gomez et al., 2007). This result together with previous observations made in humans and various animal models have shown that ET-1 could be involved in the pathogenesis of PH (Mortensen and Fink, 1992; Rabelink et al., 1994; Potter et al., 1997; Giaid, 1998; Cardillo et al., 1999).

It has been found that ET-1 induces vasoconstriction, promotes fibrosis, has mitogenic potential, and is important in the regulation of vascular tone, arterial remodeling, and vascular injury. Although constitutive ET-1 expression is primarily observed in endothelial cells, lower levels of ET-1 mRNA and protein are evident in a variety of nonendothelial cell types, including vascular smooth muscle cells, cardiac myocytes, neurons, and epithelial cells of the gut and kidney (Rubanyi and Polokoff, 1994; Mawji and Marsden, 2003). The ET-1 has been suggested as a marker of endothelial dysfunction because it plays a pathophysiologic role in various forms of cardiovascular disease (Endemann and Schiffrin, 2004). However, its function in normal cardiovascular homeostasis and PH is unclear (Touyz and Schiffrin, 2003).

In the present study, ET-1 mRNA expression levels in PHB were higher than detected in NPHB at 42 d of age. In addition, ET-1 mRNA expression was higher in group A than in group B at 24 d of age. The present findings suggested that at high altitude, the augmented release of the potent pulmonary vasoconstrictor peptide ET-1 may represent one of the mechanisms underlying the pulmonary vasoconstriction observed in susceptible broilers to PH due to hypobaric hypoxia. Cultured endothelial cells increase ET-1 transcription and expression in response to low oxygen tension (Kourembanas et al., 1991; McQuillan et al., 1994; Mawji and Marsden, 2003). Hypoxic induction of the ET-1 promoter was detected in vivo in organs from transgenic mice carrying a luciferase reporter under the transcriptional control of the human ET-1 promoter (Aversa et al., 1997; Mawji and Marsden, 2003).

It has been found that PH susceptibility may be associated with a defect in endothelium-dependent NO synthesis (Scherrer et al., 1996), and ET release can be inhibited by NO (Boulanger and Luscher, 1990; Warner et al., 1992; Sartori et al., 1999). An impairment of this NO-induced inhibition could be another mechanism contributing to the augmented ET-1 mRNA expression levels in susceptible broilers. These results agree with previous observations suggested that under conditions of acute hypoxia, endothelial cells of broilers release factors influencing vascular tone and remodelling, such as ET-1, CTGF, and adrenomedullin (Gomez et al., 2007), and decrease NOS expression (Moreno de Sandino and Hernandez, 2006). However, the exact underlying mechanisms of PH by hypobaric hypoxia are incompletely understood.

We did not find differences when comparing ET-1 mRNA lung levels of group B with the detected ones in NPHB of group A at 42 d old. It is possible that persistent hypoxia acts on the ET-1 promoter, restoring its constitutive expression in adapted broilers at 42 d. However, persistent hypoxia could promote upregulation of ET-1 in susceptible broilers. The response to hypoxia of the ET-1 promoter was confirmed in this study because mRNA expression was higher in group C than in NPHB of groups A and B at 42 d of age.

From the present results it appears that broilers have a period of susceptibility before 24 d of age, which might not be extended beyond that day because animals moved from low to high altitude at 24 d of age (group C) did not develop PH. Further studies are presently carried out as to corroborate this finding.

Physiological responses to hypoxia involve changes in gene expression, mediated by the transcriptional activator HIF-1, which functions as a regulator of oxygen homeostasis. The HIF-1 activity is induced by hypoxia in all nucleated cell types, via a post-translational mechanism and plays important roles in the responses of the cardiovascular and respiratory systems to hypoxia (Semenza et al., 2000). The ET-1 transcription response to hypoxia localizes to HIF-1 at –124/–118 of the human promoter (Hu et al., 1998; Minchenko and Caro, 2000; Mawji and Marsden, 2003). However, a possible interaction between HIF-1 and ET-1 remains unknown in broilers.

Various molecules have been implicated in the regulatory mechanism of pulmonary vascular tone, such as serotonin (Chapman and Wideman, 2002), NO (Moreno de Sandino and Hernandez, 2003), and thromboxane (Wideman et al., 1999), among others. It is evident that any factor contributing to an increase in pulmonary vascular resistance and pressure may amplify the incidence of PH in susceptible broilers (Wideman et al., 1993, 1998; Wideman and Kirby, 1995).

	ACKNOWLEDGMENTS

 
We thank Eduardo Caminos (Facultad de Medicina, Universidad Nacional de Colombia), Víctor Julio Vera (Instituto de Genética, Universidad Nacional de Colombia), and Susana Bravo (Departamento de Fisiología, Universidad de Santiago de Compostela, Spain) for their kind and wise guidance in molecular biology procedures, Martha Pulido (Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia) and Agustín Góngora (Programa de Medicina Veterinaria y Zootecnia, Universidad de los Llanos, Colombia) for their assistance in poultry health management. We are also indebted to Incubacol S.A. for generous donation of chickens. The financial support for the present study was provided by División de Investigación de Bogotá, Universidad Nacional de Colombia.

Received for publication October 3, 2007. Accepted for publication March 13, 2008.

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