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Pulmonary Arteriole Remodeling in Hypoxic Broilers Expressing Different Amounts of Endothelial Nitric Oxide Synthase
Research Note Pulmonary Arteriole Remodeling in Hypoxic Broilers Expressing Different Amounts of Endothelial Nitric Oxide Synthase M. Moreno de Sandino and A. Herna´ndez1 Departamento de Salud Animal Facultad de Medicina Veterinaria y de Zootecnia Universidad Nacional de Colombia, Bogota´, Colombia
Key words: hypoxia, pulmonary hypertension, vascular remodeling, pulmonary arteriole, nitric oxide, broiler 2006 Poultry Science 85:899–901
sive (H) broilers expressing low levels of NOS were compared with pulmonary arterioles from 30 NPH broilers expressing higher levels of NOS at each of the following ages: 17, 30, and 42 d. Lung samples were taken from animals used in a previous study (Moreno de Sandino and Herna´ndez, 2003). Briefly, the cardiac index (right ventricular weight ÷ total ventricular weight × 100) was used to identify H and NPH broilers, and NOS expression was calculated using a histochemical procedure (Moreno de Sandino and Herna´ndez, 2003). The vascular smooth muscle thickness for each animal was calculated in 20 interparabronchial Masson trichromic-stained arterioles per age: 10 measuring between 50- and 100-m external diameter and 10 measuring between 100- and 200-m external diameter. The external vessel diameter was taken as the mean of 2 measurements made at right angles to each other, and medial thickness was estimated as the mean of 4 measurements around the circumference of each vessel (Tucker et al., 1975). For each arteriole, %T was expressed as the mean of the external diameter (medial wall width ÷ external vessel diameter × 100). A total mean value of %T for each broiler was obtained by summing all %T results calculated for individual arterioles and then dividing the corresponding value by the number of arterioles. All measurements were made with an image analysis system (LECO 2001, LECO Instruments Ltd., St. Joseph, MI). Statistical analysis was accomplished using the GLM procedure (SAS Institute, 1985).
INTRODUCTION High altitude hypoxia is a well-known cause of pulmonary hypertension (PH) in broilers. 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). Nitric oxide (NO) may be responsible for vasodilatation in pulmonary circulation. In rats, a likely mechanism of defective release of NO in chronic hypoxic PH is a decrease of NO synthase (NOS) gene expression (Adnot et al., 1991). In a previous study, it was established that broilers with PH expressed greater amounts of NOS than did nonpulmonary hypertensive (NPH) broilers (Moreno de Sandino and Herna´ndez, 2003). However, the previous report in broilers does not comment on the relationship between NOS expression and vascular remodeling. Moreover, it is not clear as yet if there is a difference in the remodeling process between arterioles of various sizes. The present study was aimed to establish possible differences in the remodeling process in 2 types of arterioles between PH and NPH broilers.
MATERIALS AND METHODS Arbor Acres broilers, resident at 2,638 m above sea level, were used. Pulmonary arterioles from 30 hyperten-
RESULTS
2006 Poultry Science Association, Inc. Received August 21, 2005. Accepted January 18, 2006. 1 Corresponding author: [email protected]
The %T was greater in H broilers than in NPH broilers (Table 1). The differences were significant (P < 0.01). In 899
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
from 17-, 30-, and 42-d-old broilers to calculate %T (medial wall width ÷ external vessel diameter × 100). The %T was higher in hypertensive chickens than in nonhypertensive (P < 0.01) chickens and was inversely related to NO synthase enzyme (P < 0.01). It can be inferred from this study that NO is involved in the remodeling process in broilers with hypoxic pulmonary hypertension syndrome.
ABSTRACT Lung samples from 30 pulmonary hypertensive chickens expressing low nitric oxide (NO) synthase activity in endothelial arteriolar cells were compared with samples taken from 30 nonhypertensive animals expressing high activity to investigate a possible relationship between the just-mentioned expressions and pulmonary vascular remodeling. The external diameter and media muscular thickness in 20 arterioles of 50- to 200-m external diameter were measured in lung samples
´ NDEZ MORENO DE SANDINO AND HERNA
900
Table 1. Mean (±SD) values of cardiac index (CI) and medial thickness (%T; medial wall width ÷ external vessel diameter × 100) of pulmonary arterioles in hypertensive [H; expressing low levels of nitric oxide synthase (NOS)] and nonhypertensive (NH; expressing high levels of NOS) broilers at 17, 30, and 42 d of age Age 17 d
CI %T (50- to 100-m external diameter) %T (100- to 200-m external diameter)
30 d
42 d
NH
H
NH
H
NH
H
22.27 ± 2.09 6.78 ± 0.52a 5.89 ± 039a
43.97 ± 4.99 12.11 ± 0.65a 9.72 ± 0.69b
23.72 ± 3.22 7.11 ± 1.07a 6.30 ± 0.81a
44.05 ± 3.24 11.79 ± 1.29b 10.15 ± 1.03b
24.14 ± 3.23 7.52 ± 1.43a 5.77 ± 0.84a
44.54 ± 4.58 13.37 ± 1.02b 11.93 ± 0.87b
Means lacking a common superscript are significantly different (P < 0.01).
a,b
DISCUSSION Present results for arteriole remodeling are in agreement with those reported by other investigators (Montalvo et al., 1979; Sillau and Montalvo, 1982). According to Enkvetchakul et al. (1995) and Xiang et al. (2004) arterioles in PH broilers that were induced by exposure to low temperatures increased their middle muscular layer in a similar manner as presently found, although at 6 wk of age, the increment found in those studies was greater in control broilers. A cold environment and a high-energy diet offered to the broilers ad libitum were sufficient to provoke PH in broilers at low altitude, which caused an increment in pulmonary arteriole remodeling, the latter being related to high cardiac index and ascites development (Pan et al., 2005). Vitamin C supplementation markedly reduced the incidence of PH and the associated muscularization of pulmonary arterioles induced by a cool environmental temperature, a diet supplemented with triiodothyronine, or both (Xiang et al., 2002). Intravenous L-arginine, the NO precursor, was shown to acutely reduce pulmonary arterial pressure in patients with PH by increasing endogenous NO production (Mehta et al., 1995). Mitani et al. (1997) found that L-arginine ameliorated chronic PH and pulmonary vascular remodeling in rats by modifying endogenous NO production. Supplementary dietary L-arginine has been shown to reduce pulmonary arterial pressure as well as the incidence of PH in broilers exposed to low environmental temperature (Wideman et al., 1995). In addition, inhalation of NO by rats exposed to chronic hypoxia has been demonstrated to inhibit the medial thickening of pulmonary arteries and to attenuate muscularization of distal pulmonary arteries (Roberts et al., 1995; Horstman et al., 1998). The inhibition effect might be associated with NO-induced apoptosis in
pulmonary arteriole smooth muscle cells (Tan et al., 2005). The vascular remodeling processes in PH, at least in part, could be mediated by NO released by endothelial cells (Stenmark and Mecham, 1997). In summary, hypoxia-induced PH in broiler chickens appears to be associated with remodeling pulmonary arterioles and decreased endothelial NO production.
ACKNOWLEDGMENTS This research was supported by Colciencias and Universidad Nacional de Colombia.
REFERENCES Adnot, S., B. Rafestin, S. Eddahibi, P. Braquet, and P. Chabrier. 1991. Loss of endothelium-dependent relaxant activity in the pulmonary circulation of rats exposed to chronic hypoxia. J. Clin. Invest. 87:155–162. Envetchakul, B., J. Beasley, and W. Bottje. 1995. Pulmonary arteriole hypertrophy in broilers with pulmonary hypertension syndrome (ascites). Poult. Sci. 74:1676–1682. Horstman, D. J., D. U. Frank, and G. F. Rich. 1998. Prolonged inhaled NO attenuates hypoxic, but not monocrotaline-induced, pulmonary vascular remodelling in rats. Anesth. Analg. 86:74–81. Mehta, S., D. J. Stewart, D. Langleben, and R. D. Levy. 1995. Short-term pulmonary vasodilation with L-arginine in pulmonary hypertension. Circulation 92:1539–1545. Mitani, Y., K. Maruyama, and M. Sakurai. 1997. Prolonged administration of L-arginine ameliorates chronic pulmonary hypertension and pulmonary vascular remodeling in rats. Circulation 96:689–697. Montalvo, C., M. Ayon, and A. H. Sillau. 1979. Pages 16–22 in Histologı´a de las Arteriolas Pulmonares en Aves a Nivel del Mar y en la Altura (3.300 Metros sobre el Nivel del Mar). Anales del Congreso Latinoamericano de Avicultura, Lima, Peru´. Moreno de Sandino, M., and A. Herna´ndez. 2003. Nitric oxide synthase expression in the endothelium of pulmonary arterioles in normal and pulmonary hypertensive chickens subjected to chronic hypobaric hypoxia. Avian Dis. 47:1291–1297. Pan, J. Q., J. C. Li, W. D. Sun, and X. L. Wang. 2005. Effects of early feed restriction and cold temperature on lipid peroxidation, pulmonary vascular remodelling and ascites morbidity in broilers under normal and cold temperatures. Br. Poult. Sci. 46:374–381. Reid, L. M. 1979. The pulmonary circulation: Remodelling in growth and disease. Am. Rev. Respir. Dis. 56:531–546. Roberts, J. D., C. T. Roberts, R. C. Jones, W. M. Zapol, and K. D. Bloch. 1995. Continuous nitric oxide inhalation reduces pulmonary arterial structural changes, right ventricular hy-
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
both groups, %T of the 100- to 200-m diameter arterioles was less than that of the 50- to 100-m diameter arterioles, although there were not statistical differences (Table 1). The %T was 90% dependent on health condition and arteriolar diameter. Interaction analysis showed that %T in the NPH broilers did not change with age. In H broilers, %T changed with age. The %T and cardiac index were highly significantly correlated in a direct manner (P < 0.01) in both 50- to 100-m diameter arterioles (r = 88.77) and in 100- to 200-m diameter arterioles (r = 88.27).
RESEARCH NOTE pertrophy, and growth retardation in the hypoxic newborn rat. Circ. Res. 76:215–222. SAS Institute. 1985. SAS威 User’s Guide. Version 5 Edition. SAS Inst., Inc., Cary, NC. Sillau, A. H., and C. Montalvo. 1982. Pulmonary hypertension and the smooth muscle of the pulmonary arterioles in chickens at high altitude. Comp. Biochem. Physiol. Part A Physiol. 71:125–130. Stenmark, K. R., and R. P. Mecham. 1997. Cellular and molecular mechanisms of pulmonary vascular remodeling. Annu. Rev. Physiol. 59:89–144. Tan, X., J. Q. Pan, J. C. Li, Y. J. Liu, W. D. Sun, and X. L. Wang. 2005. L-arginine inhibiting pulmonary vascular remodelling is associated with promotion of apoptosis in pulmonary arterioles smooth muscle cells in broilers. Res. Vet. Sci. 79:203– 209.
901
Tucker, A., I. F. Mcmurtry, A. F. Alexander, J. T. Reeves, R. F. Grover, and D. H. Alexander. 1975. Lung vascular smooth muscle as a determinant of pulmonary hypertension at high altitude. Am. J. Physiol. 228:762–767. Wideman, R. F., Y. K. Kirby, M. Ismail, W. G. Bottje, R. W. Moore, and R. C. Vardeman. 1995. Supplemental L-arginine attenuates pulmonary hypertension syndrome in broilers. Poult. Sci. 74:323–330. Xiang, R. P., W. D. Sun, J. Y. Wang, and X. L. Wang. 2002. Effect of vitamin C on pulmonary hypertension and muscularisation of pulmonary arterioles in broilers. Br. Poult. Sci. 43:705–712. Xiang, R. P., W. D. Sun, K. C. Zhang, J. C. Li, J. Y. Wang, and X. L. Wang. 2004. Sodium chloride-induced acute and chronic pulmonary hypertension syndrome in chickens. Poult. Sci. 83:732–736. Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
Key words: hypoxia, pulmonary hypertension, vascular remodeling, pulmonary arteriole, nitric oxide, broiler 2006 Poultry Science 85:899–901
sive (H) broilers expressing low levels of NOS were compared with pulmonary arterioles from 30 NPH broilers expressing higher levels of NOS at each of the following ages: 17, 30, and 42 d. Lung samples were taken from animals used in a previous study (Moreno de Sandino and Herna´ndez, 2003). Briefly, the cardiac index (right ventricular weight ÷ total ventricular weight × 100) was used to identify H and NPH broilers, and NOS expression was calculated using a histochemical procedure (Moreno de Sandino and Herna´ndez, 2003). The vascular smooth muscle thickness for each animal was calculated in 20 interparabronchial Masson trichromic-stained arterioles per age: 10 measuring between 50- and 100-m external diameter and 10 measuring between 100- and 200-m external diameter. The external vessel diameter was taken as the mean of 2 measurements made at right angles to each other, and medial thickness was estimated as the mean of 4 measurements around the circumference of each vessel (Tucker et al., 1975). For each arteriole, %T was expressed as the mean of the external diameter (medial wall width ÷ external vessel diameter × 100). A total mean value of %T for each broiler was obtained by summing all %T results calculated for individual arterioles and then dividing the corresponding value by the number of arterioles. All measurements were made with an image analysis system (LECO 2001, LECO Instruments Ltd., St. Joseph, MI). Statistical analysis was accomplished using the GLM procedure (SAS Institute, 1985).
INTRODUCTION High altitude hypoxia is a well-known cause of pulmonary hypertension (PH) in broilers. 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). Nitric oxide (NO) may be responsible for vasodilatation in pulmonary circulation. In rats, a likely mechanism of defective release of NO in chronic hypoxic PH is a decrease of NO synthase (NOS) gene expression (Adnot et al., 1991). In a previous study, it was established that broilers with PH expressed greater amounts of NOS than did nonpulmonary hypertensive (NPH) broilers (Moreno de Sandino and Herna´ndez, 2003). However, the previous report in broilers does not comment on the relationship between NOS expression and vascular remodeling. Moreover, it is not clear as yet if there is a difference in the remodeling process between arterioles of various sizes. The present study was aimed to establish possible differences in the remodeling process in 2 types of arterioles between PH and NPH broilers.
MATERIALS AND METHODS Arbor Acres broilers, resident at 2,638 m above sea level, were used. Pulmonary arterioles from 30 hyperten-
RESULTS
2006 Poultry Science Association, Inc. Received August 21, 2005. Accepted January 18, 2006. 1 Corresponding author: [email protected]
The %T was greater in H broilers than in NPH broilers (Table 1). The differences were significant (P < 0.01). In 899
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
from 17-, 30-, and 42-d-old broilers to calculate %T (medial wall width ÷ external vessel diameter × 100). The %T was higher in hypertensive chickens than in nonhypertensive (P < 0.01) chickens and was inversely related to NO synthase enzyme (P < 0.01). It can be inferred from this study that NO is involved in the remodeling process in broilers with hypoxic pulmonary hypertension syndrome.
ABSTRACT Lung samples from 30 pulmonary hypertensive chickens expressing low nitric oxide (NO) synthase activity in endothelial arteriolar cells were compared with samples taken from 30 nonhypertensive animals expressing high activity to investigate a possible relationship between the just-mentioned expressions and pulmonary vascular remodeling. The external diameter and media muscular thickness in 20 arterioles of 50- to 200-m external diameter were measured in lung samples
´ NDEZ MORENO DE SANDINO AND HERNA
900
Table 1. Mean (±SD) values of cardiac index (CI) and medial thickness (%T; medial wall width ÷ external vessel diameter × 100) of pulmonary arterioles in hypertensive [H; expressing low levels of nitric oxide synthase (NOS)] and nonhypertensive (NH; expressing high levels of NOS) broilers at 17, 30, and 42 d of age Age 17 d
CI %T (50- to 100-m external diameter) %T (100- to 200-m external diameter)
30 d
42 d
NH
H
NH
H
NH
H
22.27 ± 2.09 6.78 ± 0.52a 5.89 ± 039a
43.97 ± 4.99 12.11 ± 0.65a 9.72 ± 0.69b
23.72 ± 3.22 7.11 ± 1.07a 6.30 ± 0.81a
44.05 ± 3.24 11.79 ± 1.29b 10.15 ± 1.03b
24.14 ± 3.23 7.52 ± 1.43a 5.77 ± 0.84a
44.54 ± 4.58 13.37 ± 1.02b 11.93 ± 0.87b
Means lacking a common superscript are significantly different (P < 0.01).
a,b
DISCUSSION Present results for arteriole remodeling are in agreement with those reported by other investigators (Montalvo et al., 1979; Sillau and Montalvo, 1982). According to Enkvetchakul et al. (1995) and Xiang et al. (2004) arterioles in PH broilers that were induced by exposure to low temperatures increased their middle muscular layer in a similar manner as presently found, although at 6 wk of age, the increment found in those studies was greater in control broilers. A cold environment and a high-energy diet offered to the broilers ad libitum were sufficient to provoke PH in broilers at low altitude, which caused an increment in pulmonary arteriole remodeling, the latter being related to high cardiac index and ascites development (Pan et al., 2005). Vitamin C supplementation markedly reduced the incidence of PH and the associated muscularization of pulmonary arterioles induced by a cool environmental temperature, a diet supplemented with triiodothyronine, or both (Xiang et al., 2002). Intravenous L-arginine, the NO precursor, was shown to acutely reduce pulmonary arterial pressure in patients with PH by increasing endogenous NO production (Mehta et al., 1995). Mitani et al. (1997) found that L-arginine ameliorated chronic PH and pulmonary vascular remodeling in rats by modifying endogenous NO production. Supplementary dietary L-arginine has been shown to reduce pulmonary arterial pressure as well as the incidence of PH in broilers exposed to low environmental temperature (Wideman et al., 1995). In addition, inhalation of NO by rats exposed to chronic hypoxia has been demonstrated to inhibit the medial thickening of pulmonary arteries and to attenuate muscularization of distal pulmonary arteries (Roberts et al., 1995; Horstman et al., 1998). The inhibition effect might be associated with NO-induced apoptosis in
pulmonary arteriole smooth muscle cells (Tan et al., 2005). The vascular remodeling processes in PH, at least in part, could be mediated by NO released by endothelial cells (Stenmark and Mecham, 1997). In summary, hypoxia-induced PH in broiler chickens appears to be associated with remodeling pulmonary arterioles and decreased endothelial NO production.
ACKNOWLEDGMENTS This research was supported by Colciencias and Universidad Nacional de Colombia.
REFERENCES Adnot, S., B. Rafestin, S. Eddahibi, P. Braquet, and P. Chabrier. 1991. Loss of endothelium-dependent relaxant activity in the pulmonary circulation of rats exposed to chronic hypoxia. J. Clin. Invest. 87:155–162. Envetchakul, B., J. Beasley, and W. Bottje. 1995. Pulmonary arteriole hypertrophy in broilers with pulmonary hypertension syndrome (ascites). Poult. Sci. 74:1676–1682. Horstman, D. J., D. U. Frank, and G. F. Rich. 1998. Prolonged inhaled NO attenuates hypoxic, but not monocrotaline-induced, pulmonary vascular remodelling in rats. Anesth. Analg. 86:74–81. Mehta, S., D. J. Stewart, D. Langleben, and R. D. Levy. 1995. Short-term pulmonary vasodilation with L-arginine in pulmonary hypertension. Circulation 92:1539–1545. Mitani, Y., K. Maruyama, and M. Sakurai. 1997. Prolonged administration of L-arginine ameliorates chronic pulmonary hypertension and pulmonary vascular remodeling in rats. Circulation 96:689–697. Montalvo, C., M. Ayon, and A. H. Sillau. 1979. Pages 16–22 in Histologı´a de las Arteriolas Pulmonares en Aves a Nivel del Mar y en la Altura (3.300 Metros sobre el Nivel del Mar). Anales del Congreso Latinoamericano de Avicultura, Lima, Peru´. Moreno de Sandino, M., and A. Herna´ndez. 2003. Nitric oxide synthase expression in the endothelium of pulmonary arterioles in normal and pulmonary hypertensive chickens subjected to chronic hypobaric hypoxia. Avian Dis. 47:1291–1297. Pan, J. Q., J. C. Li, W. D. Sun, and X. L. Wang. 2005. Effects of early feed restriction and cold temperature on lipid peroxidation, pulmonary vascular remodelling and ascites morbidity in broilers under normal and cold temperatures. Br. Poult. Sci. 46:374–381. Reid, L. M. 1979. The pulmonary circulation: Remodelling in growth and disease. Am. Rev. Respir. Dis. 56:531–546. Roberts, J. D., C. T. Roberts, R. C. Jones, W. M. Zapol, and K. D. Bloch. 1995. Continuous nitric oxide inhalation reduces pulmonary arterial structural changes, right ventricular hy-
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014
both groups, %T of the 100- to 200-m diameter arterioles was less than that of the 50- to 100-m diameter arterioles, although there were not statistical differences (Table 1). The %T was 90% dependent on health condition and arteriolar diameter. Interaction analysis showed that %T in the NPH broilers did not change with age. In H broilers, %T changed with age. The %T and cardiac index were highly significantly correlated in a direct manner (P < 0.01) in both 50- to 100-m diameter arterioles (r = 88.77) and in 100- to 200-m diameter arterioles (r = 88.27).
RESEARCH NOTE pertrophy, and growth retardation in the hypoxic newborn rat. Circ. Res. 76:215–222. SAS Institute. 1985. SAS威 User’s Guide. Version 5 Edition. SAS Inst., Inc., Cary, NC. Sillau, A. H., and C. Montalvo. 1982. Pulmonary hypertension and the smooth muscle of the pulmonary arterioles in chickens at high altitude. Comp. Biochem. Physiol. Part A Physiol. 71:125–130. Stenmark, K. R., and R. P. Mecham. 1997. Cellular and molecular mechanisms of pulmonary vascular remodeling. Annu. Rev. Physiol. 59:89–144. Tan, X., J. Q. Pan, J. C. Li, Y. J. Liu, W. D. Sun, and X. L. Wang. 2005. L-arginine inhibiting pulmonary vascular remodelling is associated with promotion of apoptosis in pulmonary arterioles smooth muscle cells in broilers. Res. Vet. Sci. 79:203– 209.
901
Tucker, A., I. F. Mcmurtry, A. F. Alexander, J. T. Reeves, R. F. Grover, and D. H. Alexander. 1975. Lung vascular smooth muscle as a determinant of pulmonary hypertension at high altitude. Am. J. Physiol. 228:762–767. Wideman, R. F., Y. K. Kirby, M. Ismail, W. G. Bottje, R. W. Moore, and R. C. Vardeman. 1995. Supplemental L-arginine attenuates pulmonary hypertension syndrome in broilers. Poult. Sci. 74:323–330. Xiang, R. P., W. D. Sun, J. Y. Wang, and X. L. Wang. 2002. Effect of vitamin C on pulmonary hypertension and muscularisation of pulmonary arterioles in broilers. Br. Poult. Sci. 43:705–712. Xiang, R. P., W. D. Sun, K. C. Zhang, J. C. Li, J. Y. Wang, and X. L. Wang. 2004. Sodium chloride-induced acute and chronic pulmonary hypertension syndrome in chickens. Poult. Sci. 83:732–736. Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014