- Email: [email protected]
Reduced PKCα expression in pulmonary arterioles of broiler chickens is associated with early feed restriction
Available online at www.sciencedirect.com
Research in Veterinary Science 84 (2008) 434–439 www.elsevier.com/locate/rvsc
Reduced PKCa expression in pulmonary arterioles of broiler chickens is associated with early feed restriction q Jia-qiang Pan a
a,b
, Xun Tan a, Jin-chun Li a, Wei-dong Sun a, Guo-qing Huang a, Xiao-long Wang a,*
Institute of Nutritional and Metabolic Disorders in Domestic Animals and Fowls, Nanjing Agricultural University, Nanjing 210095, China b College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China Accepted 21 June 2007
Abstract Objective: The present study was conducted to investigate the effect of early feed restriction on protein kinase Ca (PKCa) expression in pulmonary arterioles, which has been revealed to promote pulmonary vascular remodeling in pulmonary hypertensive broilers. Methods: A total of 270 day-old mixed sex commercial broilers were randomly distributed to a normal temperature control group (NT), a low temperature control group (LT) and a low temperature plus feed restriction group (LR). The PHS incidence, the right/total ventricular weight ratio (RV/TV), the vessel wall area/vessel total area ratio (WA/TA), the mean media thickness in pulmonary arterioles (mMTPA) and the expression of PKCa in the pulmonary arterioles were measured weekly. Results: Low temperature treatment significantly increased the PHS mortality. The RV/TV, WA/TA and mMTPA values of group LT were significantly elevated compared with those of group NT on d 35 and 42. The LT chickens had increased PKCa expression compared with their NT counterparts on d 28 and afterwards. Feed restriction reduced the PHS mortality, RV/TV, WA/TA and mMTPA in cold-exposed broilers. The LR chickens had much lower PKCa expression in pulmonary arterioles than the LT chickens. Conclusion: Early time feed restriction inhibited pulmonary vascular remodeling in broilers, which might be partly attributed to reduced PKCa expression in pulmonary arterioles. 2007 Elsevier Ltd. All rights reserved. Keywords: Early feed restriction; Pulmonary hypertension syndrome; PKCa; Pulmonary vascular remodeling; Broiler
1. Introduction Pulmonary hypertension syndrome (PHS), or ascites syndrome, is a metabolic disease mainly occurring in fast growing broilers. PHS accounts for over 25% of overall mortality in the broiler industry (De Smit et al., 2005) and has been reported to represent a cost to the interna-
q
The present work was supported by the National Natural Science Foundation of China (Project No. 30571366, No. 30371061 and No. 30600440). * Corresponding author. Address: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, China. Tel.: +86 25 84395586; fax: +86 25 84398669. E-mail address: [email protected] (X.-l. Wang). 0034-5288/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2007.06.009
tional broiler industry of about US $1 billion annually (Maxwell and Robertson, 1997). PHS is characterized by accumulation of large amount of serous fluid in the abdominal cavity, pulmonary hypertension, right ventricular hypertrophy and pulmonary vascular remodeling (Xiang et al., 2002). PHS is thought to be a response to oxygen insufficiency that develops in response to rapid growth, low ambient temperature, increased oxygen demand, hypoxemia, or inappropriately elevated vascular resistance to blood flow resulting in pulmonary hypertension (Julian, 1993; Wideman and Kirby, 1995). Pulmonary vascular remodeling, characterized by hypertrophy and proliferation of smooth muscle cells in the pulmonary vascular bed, is an important pathological feature of pulmonary hypertension both in mammals and fowls.
J.-q. Pan et al. / Research in Veterinary Science 84 (2008) 434–439
Protein kinase C (PKC), a family of ubiquitous phospholipid dependent serine/threonine protein kinases, has been demonstrated to play an integral role in intracellular signal transduction. At least 12 distinct PKC isoforms have been identified by molecular and biochemical analysis, which are divided into three major groups according to their mode of activation (Gopalakrishna and Jaken, 2000). Although PKC isoforms have diverse cellular functions, it is clear that activation of PKCa promotes the proliferation and differentiation of vascular smooth muscle cells (Wang et al., 1997). In mammals, PKC signal transduction pathway plays important roles in the process of hypoxic pulmonary hypertension by modulating the hypoxic proliferation of pulmonary artery smooth muscle cells and the pulmonary vascular remodeling (Dempsey et al., 1991; Zhou et al., 2002). Previous experiment has revealed that early time feed restriction inhibited pulmonary vascular remodeling (Pan et al., 2005); however, the underlying mechanisms are not fully understood. It is known that over expression of PKCa in pulmonary arterioles is involved in the development of pulmonary vascular remodeling in hypertensive birds (Tan et al., 2005a). Therefore, the present study was conducted to examine the expression of PKCa in feed restricted birds, so as to further investigate the molecular mechanisms of feed restriction on pulmonary vascular remodeling. 2. Materials and methods 2.1. Groups and treatment A total of two hundred and seventy day-old mixed sex commercial broiler chickens (Avian) were randomly distributed to three groups, which were normal temperature treated control group (NT), low temperature treated control group (LT) and low temperature plus feed restriction treated group (LR). All birds were brooded under about 35 C from 1 to 7 d of age, then 30 C from 8 to 14 d. Thereafter, birds in group NT continued to be reared at approximately 24 C, whereas birds in the LT and LR were subjected to a step-down temperature program of 1–2 C per day down to 12–14 C where it remained constant until the end of the experiment. From d 7 to d 14, the LR chickens were fed 8 h per day (from 09:00 to 17:00 h), then fed ad libitum until the end of the experiment, whereas broilers in the other two groups were given ad libitum access to feed throughout. Water was freely available to all birds, and the lighting schedule was 24 h of light per day. The experiment ended on d 49. 2.2. Diets The chicks were fed commercial maize-soybean pelleted broiler starter (d 1–21), grower (d 22–40) and finisher (d 41–49) diets that were formulated to meet or exceed minimum NRC standards for all ingredients. The metabolic
435
energy of starter, grower and finisher diets were 12.09, 13.00 and 13.02 MJ/kg. The crude protein contents were 210, 195 and 175 g/kg. 2.3. Variables measured 2.3.1. Body weights, right/total ventricle ratio and PHS mortality The incidences of PHS were recorded daily after d 7 of age. Birds were included in PHS mortality when ascitic fluid accumulation was evident or when a plasma clot to adhere to the surface of the liver (Wideman and French, 2000). Body weights were recorded per week. Nine of clinically healthy birds from each group were selected at random and euthanized on d 14, 21, 28, 35 and 42. Hearts were removed and dissected; right ventricle and total ventricle were weighed for calculation of right /total ventricle ratio (RV/TV) as an index of pulmonary hypertension (Julian et al., 1989). 2.3.2. Morphometric analysis of pulmonary arterioles distension The lungs were fixed immediately at the time of necropsy in situ by instilling buffered 10% formalin through a tracheal cannula for 5 min, and then removed and further fixed in formal/saline. Complete transverse sections of the left lungs were cut across the hilum, then gradually dehydrated in alcohol and embedded in paraffin. The tissues were serially sectioned and stained with Wergert’s and Van-Gieson’s stain for elastin (Li et al., 2000). Similar populations of vessels at corresponding anatomical sites were examined in each broiler. Vessel wall area (WA), total vessel area (TA), external and inner diameter of 6–10 muscular arterioles 6100 lm in outer diameter in each slide were measured using Image-Pro Plus 4.0 microscopic image analytic software (Media Cybernetics Inc., 1998), and the average of WA/TA ratios as well as mean media thickness of pulmonary arterioles (mMTPA) were calculated, both of which served as an accurate index of pulmonary vascular remodeling (Li et al., 2000). 2.3.3. Immunohistochemical staining of PKCa and quantitative measurement of PKCa expression Immunohistochemical assay was used to examine the expression of PKCa in pulmonary arterioles as described by Tan et al. (2005b). Briefly, the lung tissue was cut into 5 lm -thick sections. Sections were incubated with mouse monoclonal anti-human PKCa (BD Biosciences, San Diego, CA) at 1:120 dilution for 2 h at 37 C followed by visualization of streptavidin–biotin system (Zymed Laboratories, Inc., San Francisco, CA). Diaminobenzidine (DAB) was used as the final chromogen. The sections were then counterstained with hematoxylin for microscopic examination. Negative controls for each tissue section were prepared by omitting the primary antibody. The expression of PKCa in the wall of pulmonary arterioles was quantified by measuring mean optical density (OD)
436
J.-q. Pan et al. / Research in Veterinary Science 84 (2008) 434–439
using Image-Pro Plus 4.0 image analytic software (Media Cybernetics Inc., 1998). In each slide, 6–10 randomly selected muscular arterioles (u 6 100 lm) were measured and the average was calculated as the representative value for each bird. 2.4. Statistical analysis Percentage data of PHS mortality were compared by chi square test between groups. Other values were expressed as means ± SE and were analyzed by one-way analysis of variance (ANOVA). Tukey’s multiple range test was used to determine significance between means. A P value less than 0.05 is considered statistically significant.
3.4. Morphometric analysis of pulmonary arterioles distension The mean values of WA/TA and mMTPA of muscular arterioles are presented in Table 3. No significant difference was found between LT and NT group on d 21 and d 28. However, from d 35 on, both the WA/TA and mMTPA of LT birds were significantly elevated compared to their NT counterparts. Feed restriction decreased the mMTPA values of cold-exposed birds on d 14, 21 and 42. The WA/TA ratios in group LR were significantly lower than those in group LT on d 14, 35 and 42. There were no differences of WA/TA and mMTPA between NT and LR group except for on d 14. 3.5. PKCa expression in pulmonary arterioles
3. Results 3.1. PHS mortality Cases of PHS in the groups NT, LT and LR are 4, 12 and 3, respectively. The cumulative mortality was 13.3% in the group LT, which was much higher than the 4.44% in the group NT and 3.33% in the group LR. There was no statistically difference of PHS mortality between group NT and LR. 3.2. Body weight The results are given in Table 1. The body weights of birds in group LT were significantly lower than those of group NT from d 28 on. The feed restricted birds had lower body weights than birds in group LT during the experiment. However, the body weight of LR chickens did not differ from that of LT chickens on d 49. LR chickens had lower body weights than NT chickens throughout the experiment. 3.3. RV/TV value Birds in the LT group had much higher RV/TV ratios than those in the NT group on d 35 and d 42, while birds in LR group had lower RV/TV ratios than LT group. There were no differences of RV/TV ratio between NT and LR group on all time points. (See Table 2.)
The pulmonary arterioles in the NT birds were weakly positive for anti-PKCa labeling, and PKCa was mainly distributed within the adventitial and medial layer of vessel (Fig. 1A). There was a much stronger anti-PKCa labeling reaction in the pulmonary arterioles of LT chickens compared with NT chickens from d 28 on. The adventitial and medial smooth muscle layers of pulmonary arterioles in LT chickens were strongly positive for PKCa stain (Fig. 1B). The anti-PKCa labeling reaction in LR chickens was weakly positive (Fig. 1C). Image analysis showed that the mean OD values in vessel wall of pulmonary arterioles of LT broilers were significantly higher than those of their NT counterparts on d 28 and afterwards, indicating an increased PKCa protein expression. However, early time feed restriction led to reduced PKCa expression in LR group compared with LT group, and LR chickens had reduced PKCa expression than NT chickens on d 14, 21 and 28 (See Table 4). 4. Discussion Cumulative evidence showed that the most important cause of PHS in broiler was metabolic hypoxia (Julian et al., 1989), which was primarily triggered by low ambient temperature and inherited fast growth rate. In the present study, feed restriction decreased the body weight of broiler compared with LT controls during the period of restriction and thereafter. Reduced body weight in the early period led to a decreasing of basal metabolic rate, and contributed to
Table 1 The body weight of broiler in different groups n = 9 Groups
NT LT LR
Day-old 14
21
28
35
42
49
400 ± 10a 400 ± 10a 350 ± 10b
721 ± 21a 727 ± 15a 695 ± 30b
1210 ± 40a 1150 ± 30b 1057 ± 25c
1700 ± 46a 1607 ± 29b 1483 ± 47c
2213 ± 21a 2050 ± 20b 1979 ± 52c
2647 ± 15a 2440 ± 53b 2433 ± 21b
Note: Values within the same column with non common superscripts indicate significant differences (P < 0.05). NT – normal temperature; LT – low temperature; LR – low temperature plus feed restriction.
J.-q. Pan et al. / Research in Veterinary Science 84 (2008) 434–439
437
Table 2 The ratio of right to total ventricular weight (RV/TV) of broiler in different groups n = 9 Groups
Day-old 14
21
28
35
42
NT LT LR
0.20 ± 0.026a 0.20 ± 0.026a 0.18 ± 0.023a
0.19 ± 0.022a 0.20 ± 0.019a 0.21 ± 0.033a
0.21 ± 0.023a 0.23 ± 0.032a 0.20 ± 0.026a
0.21 ± 0.013b 0.25 ± 0.03a 0.22 ± 0.024b
0.23 ± 0.033b 0.29 ± 0.044a 0.25 ± 0.045b
Note: Values within the same column with non common superscripts indicate significant differences (P < 0.05). NT – normal temperature; LT – low temperature; LR – low temperature plus feed restriction.
Table 3 The mean medial thickness in pulmonary arterioles (mMTPA) and the average ratio of wall area to total area (WA/TA) of broiler in different groups n = 9 Item
Groups
Day-old 21
28 a
35 ab
42 a
49 b
mMTPA (lm/lm)
NT LT LR
0.324 ± 0.027 0.324 ± 0.027a 0.275 ± 0.034b
0.337 ± 0.027 0.352 ± 0.032a 0.305 ± 0.035b
0.372 ± 0.031 0.384 ± 0.048a 0.369 ± 0.026a
0.379 ± 0.03 0.433 ± 0.038a 0.408 ± 0.036ab
0.392 ± 0.034b 0.434 ± 0.034a 0.408 ± 0.009b
WA/TA (lm2/lm2)
NT LT LR
0.539 ± 0.040a 0.539 ± 0.040a 0.463 ± 0.054b
0.550 ± 0.032a 0.576 ± 0.030a 0.512 ± 0.052a
0.591 ± 0.041a 0.603 ± 0.053a 0.590 ± 0.031a
0.608 ± 0.038b 0.669 ± 0.037a 0.626 ± 0.05b
0.604 ± 0.039b 0.644 ± 0.022a 0.600 ± 0.040b
Note: Values within the same column with non common superscripts indicate significant differences (P < 0.05). NT – normal temperature; LT – low temperature; LR – low temperature plus feed restriction.
Fig. 1. PKCa expression in the pulmonary arteriole from a single bird of different groups on d 42 (immunohistochemical stain, 400·): (A) Section of pulmonary arteriole from a bird in group NT. PKCa is mainly distributed within the adventitial (a) and medial (m) layer of vessel. The vessel wall is weakly positively stained. (B) Section of pulmonary arteriole from a bird in group LT. Adventitial (a) and medial (m) smooth muscle layer of vessel wall is strongly positive for PKCa stain. (C) Section of pulmonary arteriole from a bird in group LR. The vessel wall is weakly positively stained for PKCa (arrow).
Table 4 Value of mean optical density (OD, represents PKCa expression) in the vessel wall of pulmonary arterioles in different groups n = 9 Groups
NT LT LR
Day-old 14
21
28
35
42
0.224 ± 0.019a 0.224 ± 0.019a 0.154 ± 0.03b
0.186 ± 0.022a 0.209 ± 0.023a 0.166 ± 0.01b
0.203 ± 0.005b 0.264 ± 0.038a 0.143 ± 0.047c
0.180 ± 0.02b 0.239 ± 0.033a 0.148 ± 0.043b
0.148 ± 0.030b 0.241 ± 0.053a 0.135 ± 0.038b
Note: Values within the same column with non common superscripts indicate significant differences (P < 0.05). NT – normal temperature; LT – low temperature; LR – low temperature plus feed restriction.
an alleviated systemic hypoxia. As a result, PHS mortality was reduced in feed restricted groups. However, the body weight of LR chickens did not differ from their LT controls on d 49, and this might attribute to the compensatory growth induced by feed restriction, which was obviously showed in the last week (from 42 d to 49 d) in LR group. In this study, cold exposure increased pulmonary pressure as indicated by elevated RV/TV ratio, and led to pul-
monary vascular remodeling as indicated by increased WA/TA and mMTPA, resulting in elevated PHS mortality. However, early feed restriction reduced the cumulative PHS mortality in cold-exposed birds, decreased the pulmonary pressure as reflected by decreased RV/TV ratio, and inhibited pulmonary vascular remodeling as reflected by decreased WA/TA and mMTPA. The inhibition of pulmonary vascular remodeling might be a result of alleviated
438
J.-q. Pan et al. / Research in Veterinary Science 84 (2008) 434–439
systemic hypoxia, but the underlying mechanisms are not well understood. PKC is one of the important intracellular signal transduction systems and a key enzyme implicated in the control of cellular proliferation, differentiation and cytokine secretion. The classical PKCa is Ca2+ and diacylglycerol dependent PKC isoforms. PKCa seems ubiquitously distributed in all tissues so far examined (Pokorski et al., 2000), including pulmonary epithelial cell, vascular smooth muscle cell, vascular endothelial cell and fibroblast. In mammals, chronic hypoxia stimulates an increased expression of PKC protein in pulmonary artery smooth muscle cells (Zhong et al., 1999). In addition, hypoxia stimulates enhanced PKC isozyme activity in pulmonary arterial vascular endothelial cells (Yang et al., 2000). In vitro experiment also confirmed that the PKC protein levels and PKC isozyme activity were higher in pulmonary artery adventitial fibroblasts in response to hypoxia (Das et al., 2000). In current study, the PKCa expression was detected by measuring OD values in the wall of pulmonary arterioles. The reported optical density is a mean optical density rather than an integrated optical density (IOD) to preclude the influence of vessel thickness, because of the fact that there is a large increase in medial/adventitial layer thickness in the LT group versus NT and LR group. The PKCa protein expression in pulmonary arterioles of low temperature treated birds was significantly higher than those of normal temperature treated birds. The present results provide further evidence that the PKCa signal transduction pathway has an important role in the pathogenesis of hypoxic pulmonary hypertension in broilers, similar to mammals. In addition, feed restriction led to a reduced PKCa expression in pulmonary arterioles. Feed restriction improves the systemic hypoxemia in cold-exposed broiler by slowing growth rate. It is also possible that the reduced pulmonary pressure due to depression of growth rate inhibits PKCa expression. In this context, the reduced PHS mortality and inhibited pulmonary vascular remodeling caused by feed restriction may be partially due to reduction of PKCa expression. Pulmonary vascular remodeling, characterized by proliferation of smooth muscle cells and adventitial fibroblasts in pulmonary arteries, is an important contributor to the sustained pulmonary hypertension induced by hypoxia (Xiang et al., 2002). Many factors have been revealed to contribute to this event, among which the PKC signal transduction pathway has been extensively studied. It has been revealed that hypoxia-induced proliferation of pulmonary artery smooth muscle cells and pulmonary artery adventitial fibroblasts is dependent on PKC activation (Dempsey et al., 1991; Stenmark et al., 2002), and the activation of PKC is a requisite step for pulmonary artery smooth muscle cells to proliferate in response to hypoxia in vitro (Dempsey et al., 1997). In addition, PKC activation results in a slow, sustained pulmonary artery contraction (Tsai et al., 2004), which may also lead to pulmonary remodeling. Moreover, further studies concentrated on PKC iso-
forms revealed that the differentiation and proliferation of vascular smooth muscle cells in response to hypoxia were at least partially dependent on PKCa activation (Dempsey et al., 1997). In this study, the over expressed PKCa in the LT group might contribute to the vascular remodeling. In conclusion, the present study confirmed over expression of PKCa in remodeled pulmonary arterioles of hypertensive broilers. Early time feed restriction inhibited pulmonary vascular remodeling, which is associated with reduction of PKCa expression. Acknowledgements The research work was supported by the National Natural Science Foundation of China (Project No. 30571366, No. 30371061 and No. 30600440). References Das, M., Dempsey, E.C., Bouchey, D., Reyland, M.E., Stenmark, K.R., 2000. Chronic hypoxia induces exaggerated growth response in pulmonary artery adventitial fibroblasts: potential contribution of specific protein kinase C isozymes. American Journal of Respiratory Cell and Molecular Biology 22, 15–25. Dempsey, E.C., McMurtry, I.F., O’Brien, R.F., 1991. Protein kinase C activation allows pulmonary artery smooth muscle cells to proliferation to hypoxia. The American Journal of Physiology 260, L136–L145. Dempsey, E.C., Frid, M.G., Aldashev, A.A., Das, M., Stenmark, K.R., 1997. Heterogeneity in the proliferative response of bovine pulmonary artery smooth muscle cells to mitogens and hypoxia: importance of protein kinase C. Canadian Journal of Physiology and Pharmacology 75, 936–944. De Smit, L., Tona, K., Bruggeman, V., Onagbesan, O., Hassanzadeh, M., Arckens, L., Decuypere, E., 2005. Comparison of three lines of broilers differing in ascites susceptibility or growth rate. 2. Egg weight loss, gas pressures, embryonic heat production, and physiological hormone levels. Poultry Science 84, 1446–1452. Gopalakrishna, R., Jaken, S., 2000. Protein kinase C signaling and oxidative stress. Free Radical Biology and Medicine 28, 1349–1361. Julian, R.J., 1993. Ascites in poultry. Avian Pathology 22, 419–454. Julian, R.J., McMillan, I., Quinton, M., 1989. The effect of cold and dietary energy on right ventricle hypertrophy, right ventricle failure and ascites in meat-type chickens. Avian Pathology 18, 675–684. Li, J.C., Wang, X.L., Sun, W.D., Zhang, K.C., 2000. Study on histopathology of pulmonary arterioles of broilers with Na+ induced pulmonary hypertension syndrome by using the microscopic image analysis program. Acta Veterinaria et Zootechnica Sinaca 31, 441–447. Maxwell, M.H., Robertson, G.W., 1997. World broiler ascites survey 1996. Poultry International 36, 16–30. Media Cybernetics INC., 1998. Image-Pro Plus User’s Guide (Silver Spring, MD, Media Cybernetics Inc). National Research Council, 1994. Nutrient Requirements of Poultry, 9th rev. National Academy Press, Washington, DC. Pan, J.Q., Tan, X., Li, J.C., Sun, W.D., Wang, X.L., 2005. Effects of early feed restriction and cold temperature on lipid peroxidation, pulmonary vascular remodeling and ascites morbidity in broilers under normal and cold temperature. British Poultry Science 46, 374–381. Pokorski, M., Sakagami, H., Kondo, H., 2000. Classical protein kinase C and its hypoxic stimulus-induced translocation in the cat and rat carotid body. The European Respiratory Journal 16, 459–463. Stenmark, K.R., Gerasimovskaya, E., Nemenoff, R.A., Das, M., 2002. Hypoxic activation of adventitial fibroblasts: role in vascular remodeling. Chest 122, 326S–334S.
J.-q. Pan et al. / Research in Veterinary Science 84 (2008) 434–439 Tan, X., Liu, Y.J., Li, J.C., Pan, J.Q., Sun, W.D., Wang, X.L., 2005a. Activation of PKCa and pulmonary vascular remodeling in broilers. Research in Veterinary Science 79, 131–137. Tan, X., Liu, Y.J., Pan, J.Q., Li, J.C., Sun, W.D., Wang, X.L., 2005b. Expression of PKCa in pulmonary arterioles and its association with pulmonary vascular remodeling in broilers with pulmonary hypertension induced by cold temperature exposure. Scientia Agricultura Sinica 38, 1917–1922. Tsai, B.M., Wang, M., Pitcher, J.M., Meldrum, K.K., Meldrum, D.R., 2004. Hypoxic pulmonary vasoconstriction and pulmonary artery tissue cytokine expression are mediated by protein kinase C. American Journal of Physiology. Lung Cellular and Molecular Physiology 287, L1215–L1219. Wang, S., Desai, D., Wright, G., Niles, R.M., Wright, G.L., 1997. Effects of protein kinase Ca over-expression on A7r5 smooth muscle cell proliferation and differentiation. Experimental Cell Research 236, 117–126. Wideman, R.F., French, H., 2000. Ascites resistance of progeny from broiler breeders selected for two generations using chronic unilateral pulmonary artery occlusion. Poultry Science 79, 396–401.
439
Wideman, R.F., Kirby, Y.K., 1995. Evidence of ventilation-perfusion mismatch during acute unilateral pulmonary artery occlusion in broilers. Poultry Science 74, 1209–1217. Xiang, R.P., Sun, W.D., Wang, J.Y., Wang, X.L., 2002. Effect of Vitamin C on pulmonary hypertension and muscularisation of pulmonary arterioles in broilers. British Poultry Science 43, 705–712. Yang, X.M., Xie, Y.Z., Nie, H.J., Yin, Z.Y., 2000. The relationship between VEGF level and PKC activity in pulmonary arterial vascular endothelial cells of rat during hypoxia. Chinese Journal of Applied Physiology 16, 250–252. Zhong, S., Xu, Y.J., Zhang, Z.X., 1999. Immunohistochemical study on protein kinase C expression changes of pulmonary artery in chronic hypoxia rats. Acta Universitatis Medicinae Tongji 28, 475– 477. Zhou, H., Chen, S.X., Wang, L.X., Chen, Y.F., Xie, Y.P., Wang, Q.J., Fan, X.F., 2002. An experimental study on the regulation on pulmonary arterial remodeling by protein kinase C in chronic hypoxic rats. Chinese Journal of Applied Physiology 18, 38–42.
Research in Veterinary Science 84 (2008) 434–439 www.elsevier.com/locate/rvsc
Reduced PKCa expression in pulmonary arterioles of broiler chickens is associated with early feed restriction q Jia-qiang Pan a
a,b
, Xun Tan a, Jin-chun Li a, Wei-dong Sun a, Guo-qing Huang a, Xiao-long Wang a,*
Institute of Nutritional and Metabolic Disorders in Domestic Animals and Fowls, Nanjing Agricultural University, Nanjing 210095, China b College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China Accepted 21 June 2007
Abstract Objective: The present study was conducted to investigate the effect of early feed restriction on protein kinase Ca (PKCa) expression in pulmonary arterioles, which has been revealed to promote pulmonary vascular remodeling in pulmonary hypertensive broilers. Methods: A total of 270 day-old mixed sex commercial broilers were randomly distributed to a normal temperature control group (NT), a low temperature control group (LT) and a low temperature plus feed restriction group (LR). The PHS incidence, the right/total ventricular weight ratio (RV/TV), the vessel wall area/vessel total area ratio (WA/TA), the mean media thickness in pulmonary arterioles (mMTPA) and the expression of PKCa in the pulmonary arterioles were measured weekly. Results: Low temperature treatment significantly increased the PHS mortality. The RV/TV, WA/TA and mMTPA values of group LT were significantly elevated compared with those of group NT on d 35 and 42. The LT chickens had increased PKCa expression compared with their NT counterparts on d 28 and afterwards. Feed restriction reduced the PHS mortality, RV/TV, WA/TA and mMTPA in cold-exposed broilers. The LR chickens had much lower PKCa expression in pulmonary arterioles than the LT chickens. Conclusion: Early time feed restriction inhibited pulmonary vascular remodeling in broilers, which might be partly attributed to reduced PKCa expression in pulmonary arterioles. 2007 Elsevier Ltd. All rights reserved. Keywords: Early feed restriction; Pulmonary hypertension syndrome; PKCa; Pulmonary vascular remodeling; Broiler
1. Introduction Pulmonary hypertension syndrome (PHS), or ascites syndrome, is a metabolic disease mainly occurring in fast growing broilers. PHS accounts for over 25% of overall mortality in the broiler industry (De Smit et al., 2005) and has been reported to represent a cost to the interna-
q
The present work was supported by the National Natural Science Foundation of China (Project No. 30571366, No. 30371061 and No. 30600440). * Corresponding author. Address: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, China. Tel.: +86 25 84395586; fax: +86 25 84398669. E-mail address: [email protected] (X.-l. Wang). 0034-5288/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2007.06.009
tional broiler industry of about US $1 billion annually (Maxwell and Robertson, 1997). PHS is characterized by accumulation of large amount of serous fluid in the abdominal cavity, pulmonary hypertension, right ventricular hypertrophy and pulmonary vascular remodeling (Xiang et al., 2002). PHS is thought to be a response to oxygen insufficiency that develops in response to rapid growth, low ambient temperature, increased oxygen demand, hypoxemia, or inappropriately elevated vascular resistance to blood flow resulting in pulmonary hypertension (Julian, 1993; Wideman and Kirby, 1995). Pulmonary vascular remodeling, characterized by hypertrophy and proliferation of smooth muscle cells in the pulmonary vascular bed, is an important pathological feature of pulmonary hypertension both in mammals and fowls.
J.-q. Pan et al. / Research in Veterinary Science 84 (2008) 434–439
Protein kinase C (PKC), a family of ubiquitous phospholipid dependent serine/threonine protein kinases, has been demonstrated to play an integral role in intracellular signal transduction. At least 12 distinct PKC isoforms have been identified by molecular and biochemical analysis, which are divided into three major groups according to their mode of activation (Gopalakrishna and Jaken, 2000). Although PKC isoforms have diverse cellular functions, it is clear that activation of PKCa promotes the proliferation and differentiation of vascular smooth muscle cells (Wang et al., 1997). In mammals, PKC signal transduction pathway plays important roles in the process of hypoxic pulmonary hypertension by modulating the hypoxic proliferation of pulmonary artery smooth muscle cells and the pulmonary vascular remodeling (Dempsey et al., 1991; Zhou et al., 2002). Previous experiment has revealed that early time feed restriction inhibited pulmonary vascular remodeling (Pan et al., 2005); however, the underlying mechanisms are not fully understood. It is known that over expression of PKCa in pulmonary arterioles is involved in the development of pulmonary vascular remodeling in hypertensive birds (Tan et al., 2005a). Therefore, the present study was conducted to examine the expression of PKCa in feed restricted birds, so as to further investigate the molecular mechanisms of feed restriction on pulmonary vascular remodeling. 2. Materials and methods 2.1. Groups and treatment A total of two hundred and seventy day-old mixed sex commercial broiler chickens (Avian) were randomly distributed to three groups, which were normal temperature treated control group (NT), low temperature treated control group (LT) and low temperature plus feed restriction treated group (LR). All birds were brooded under about 35 C from 1 to 7 d of age, then 30 C from 8 to 14 d. Thereafter, birds in group NT continued to be reared at approximately 24 C, whereas birds in the LT and LR were subjected to a step-down temperature program of 1–2 C per day down to 12–14 C where it remained constant until the end of the experiment. From d 7 to d 14, the LR chickens were fed 8 h per day (from 09:00 to 17:00 h), then fed ad libitum until the end of the experiment, whereas broilers in the other two groups were given ad libitum access to feed throughout. Water was freely available to all birds, and the lighting schedule was 24 h of light per day. The experiment ended on d 49. 2.2. Diets The chicks were fed commercial maize-soybean pelleted broiler starter (d 1–21), grower (d 22–40) and finisher (d 41–49) diets that were formulated to meet or exceed minimum NRC standards for all ingredients. The metabolic
435
energy of starter, grower and finisher diets were 12.09, 13.00 and 13.02 MJ/kg. The crude protein contents were 210, 195 and 175 g/kg. 2.3. Variables measured 2.3.1. Body weights, right/total ventricle ratio and PHS mortality The incidences of PHS were recorded daily after d 7 of age. Birds were included in PHS mortality when ascitic fluid accumulation was evident or when a plasma clot to adhere to the surface of the liver (Wideman and French, 2000). Body weights were recorded per week. Nine of clinically healthy birds from each group were selected at random and euthanized on d 14, 21, 28, 35 and 42. Hearts were removed and dissected; right ventricle and total ventricle were weighed for calculation of right /total ventricle ratio (RV/TV) as an index of pulmonary hypertension (Julian et al., 1989). 2.3.2. Morphometric analysis of pulmonary arterioles distension The lungs were fixed immediately at the time of necropsy in situ by instilling buffered 10% formalin through a tracheal cannula for 5 min, and then removed and further fixed in formal/saline. Complete transverse sections of the left lungs were cut across the hilum, then gradually dehydrated in alcohol and embedded in paraffin. The tissues were serially sectioned and stained with Wergert’s and Van-Gieson’s stain for elastin (Li et al., 2000). Similar populations of vessels at corresponding anatomical sites were examined in each broiler. Vessel wall area (WA), total vessel area (TA), external and inner diameter of 6–10 muscular arterioles 6100 lm in outer diameter in each slide were measured using Image-Pro Plus 4.0 microscopic image analytic software (Media Cybernetics Inc., 1998), and the average of WA/TA ratios as well as mean media thickness of pulmonary arterioles (mMTPA) were calculated, both of which served as an accurate index of pulmonary vascular remodeling (Li et al., 2000). 2.3.3. Immunohistochemical staining of PKCa and quantitative measurement of PKCa expression Immunohistochemical assay was used to examine the expression of PKCa in pulmonary arterioles as described by Tan et al. (2005b). Briefly, the lung tissue was cut into 5 lm -thick sections. Sections were incubated with mouse monoclonal anti-human PKCa (BD Biosciences, San Diego, CA) at 1:120 dilution for 2 h at 37 C followed by visualization of streptavidin–biotin system (Zymed Laboratories, Inc., San Francisco, CA). Diaminobenzidine (DAB) was used as the final chromogen. The sections were then counterstained with hematoxylin for microscopic examination. Negative controls for each tissue section were prepared by omitting the primary antibody. The expression of PKCa in the wall of pulmonary arterioles was quantified by measuring mean optical density (OD)
436
J.-q. Pan et al. / Research in Veterinary Science 84 (2008) 434–439
using Image-Pro Plus 4.0 image analytic software (Media Cybernetics Inc., 1998). In each slide, 6–10 randomly selected muscular arterioles (u 6 100 lm) were measured and the average was calculated as the representative value for each bird. 2.4. Statistical analysis Percentage data of PHS mortality were compared by chi square test between groups. Other values were expressed as means ± SE and were analyzed by one-way analysis of variance (ANOVA). Tukey’s multiple range test was used to determine significance between means. A P value less than 0.05 is considered statistically significant.
3.4. Morphometric analysis of pulmonary arterioles distension The mean values of WA/TA and mMTPA of muscular arterioles are presented in Table 3. No significant difference was found between LT and NT group on d 21 and d 28. However, from d 35 on, both the WA/TA and mMTPA of LT birds were significantly elevated compared to their NT counterparts. Feed restriction decreased the mMTPA values of cold-exposed birds on d 14, 21 and 42. The WA/TA ratios in group LR were significantly lower than those in group LT on d 14, 35 and 42. There were no differences of WA/TA and mMTPA between NT and LR group except for on d 14. 3.5. PKCa expression in pulmonary arterioles
3. Results 3.1. PHS mortality Cases of PHS in the groups NT, LT and LR are 4, 12 and 3, respectively. The cumulative mortality was 13.3% in the group LT, which was much higher than the 4.44% in the group NT and 3.33% in the group LR. There was no statistically difference of PHS mortality between group NT and LR. 3.2. Body weight The results are given in Table 1. The body weights of birds in group LT were significantly lower than those of group NT from d 28 on. The feed restricted birds had lower body weights than birds in group LT during the experiment. However, the body weight of LR chickens did not differ from that of LT chickens on d 49. LR chickens had lower body weights than NT chickens throughout the experiment. 3.3. RV/TV value Birds in the LT group had much higher RV/TV ratios than those in the NT group on d 35 and d 42, while birds in LR group had lower RV/TV ratios than LT group. There were no differences of RV/TV ratio between NT and LR group on all time points. (See Table 2.)
The pulmonary arterioles in the NT birds were weakly positive for anti-PKCa labeling, and PKCa was mainly distributed within the adventitial and medial layer of vessel (Fig. 1A). There was a much stronger anti-PKCa labeling reaction in the pulmonary arterioles of LT chickens compared with NT chickens from d 28 on. The adventitial and medial smooth muscle layers of pulmonary arterioles in LT chickens were strongly positive for PKCa stain (Fig. 1B). The anti-PKCa labeling reaction in LR chickens was weakly positive (Fig. 1C). Image analysis showed that the mean OD values in vessel wall of pulmonary arterioles of LT broilers were significantly higher than those of their NT counterparts on d 28 and afterwards, indicating an increased PKCa protein expression. However, early time feed restriction led to reduced PKCa expression in LR group compared with LT group, and LR chickens had reduced PKCa expression than NT chickens on d 14, 21 and 28 (See Table 4). 4. Discussion Cumulative evidence showed that the most important cause of PHS in broiler was metabolic hypoxia (Julian et al., 1989), which was primarily triggered by low ambient temperature and inherited fast growth rate. In the present study, feed restriction decreased the body weight of broiler compared with LT controls during the period of restriction and thereafter. Reduced body weight in the early period led to a decreasing of basal metabolic rate, and contributed to
Table 1 The body weight of broiler in different groups n = 9 Groups
NT LT LR
Day-old 14
21
28
35
42
49
400 ± 10a 400 ± 10a 350 ± 10b
721 ± 21a 727 ± 15a 695 ± 30b
1210 ± 40a 1150 ± 30b 1057 ± 25c
1700 ± 46a 1607 ± 29b 1483 ± 47c
2213 ± 21a 2050 ± 20b 1979 ± 52c
2647 ± 15a 2440 ± 53b 2433 ± 21b
Note: Values within the same column with non common superscripts indicate significant differences (P < 0.05). NT – normal temperature; LT – low temperature; LR – low temperature plus feed restriction.
J.-q. Pan et al. / Research in Veterinary Science 84 (2008) 434–439
437
Table 2 The ratio of right to total ventricular weight (RV/TV) of broiler in different groups n = 9 Groups
Day-old 14
21
28
35
42
NT LT LR
0.20 ± 0.026a 0.20 ± 0.026a 0.18 ± 0.023a
0.19 ± 0.022a 0.20 ± 0.019a 0.21 ± 0.033a
0.21 ± 0.023a 0.23 ± 0.032a 0.20 ± 0.026a
0.21 ± 0.013b 0.25 ± 0.03a 0.22 ± 0.024b
0.23 ± 0.033b 0.29 ± 0.044a 0.25 ± 0.045b
Note: Values within the same column with non common superscripts indicate significant differences (P < 0.05). NT – normal temperature; LT – low temperature; LR – low temperature plus feed restriction.
Table 3 The mean medial thickness in pulmonary arterioles (mMTPA) and the average ratio of wall area to total area (WA/TA) of broiler in different groups n = 9 Item
Groups
Day-old 21
28 a
35 ab
42 a
49 b
mMTPA (lm/lm)
NT LT LR
0.324 ± 0.027 0.324 ± 0.027a 0.275 ± 0.034b
0.337 ± 0.027 0.352 ± 0.032a 0.305 ± 0.035b
0.372 ± 0.031 0.384 ± 0.048a 0.369 ± 0.026a
0.379 ± 0.03 0.433 ± 0.038a 0.408 ± 0.036ab
0.392 ± 0.034b 0.434 ± 0.034a 0.408 ± 0.009b
WA/TA (lm2/lm2)
NT LT LR
0.539 ± 0.040a 0.539 ± 0.040a 0.463 ± 0.054b
0.550 ± 0.032a 0.576 ± 0.030a 0.512 ± 0.052a
0.591 ± 0.041a 0.603 ± 0.053a 0.590 ± 0.031a
0.608 ± 0.038b 0.669 ± 0.037a 0.626 ± 0.05b
0.604 ± 0.039b 0.644 ± 0.022a 0.600 ± 0.040b
Note: Values within the same column with non common superscripts indicate significant differences (P < 0.05). NT – normal temperature; LT – low temperature; LR – low temperature plus feed restriction.
Fig. 1. PKCa expression in the pulmonary arteriole from a single bird of different groups on d 42 (immunohistochemical stain, 400·): (A) Section of pulmonary arteriole from a bird in group NT. PKCa is mainly distributed within the adventitial (a) and medial (m) layer of vessel. The vessel wall is weakly positively stained. (B) Section of pulmonary arteriole from a bird in group LT. Adventitial (a) and medial (m) smooth muscle layer of vessel wall is strongly positive for PKCa stain. (C) Section of pulmonary arteriole from a bird in group LR. The vessel wall is weakly positively stained for PKCa (arrow).
Table 4 Value of mean optical density (OD, represents PKCa expression) in the vessel wall of pulmonary arterioles in different groups n = 9 Groups
NT LT LR
Day-old 14
21
28
35
42
0.224 ± 0.019a 0.224 ± 0.019a 0.154 ± 0.03b
0.186 ± 0.022a 0.209 ± 0.023a 0.166 ± 0.01b
0.203 ± 0.005b 0.264 ± 0.038a 0.143 ± 0.047c
0.180 ± 0.02b 0.239 ± 0.033a 0.148 ± 0.043b
0.148 ± 0.030b 0.241 ± 0.053a 0.135 ± 0.038b
Note: Values within the same column with non common superscripts indicate significant differences (P < 0.05). NT – normal temperature; LT – low temperature; LR – low temperature plus feed restriction.
an alleviated systemic hypoxia. As a result, PHS mortality was reduced in feed restricted groups. However, the body weight of LR chickens did not differ from their LT controls on d 49, and this might attribute to the compensatory growth induced by feed restriction, which was obviously showed in the last week (from 42 d to 49 d) in LR group. In this study, cold exposure increased pulmonary pressure as indicated by elevated RV/TV ratio, and led to pul-
monary vascular remodeling as indicated by increased WA/TA and mMTPA, resulting in elevated PHS mortality. However, early feed restriction reduced the cumulative PHS mortality in cold-exposed birds, decreased the pulmonary pressure as reflected by decreased RV/TV ratio, and inhibited pulmonary vascular remodeling as reflected by decreased WA/TA and mMTPA. The inhibition of pulmonary vascular remodeling might be a result of alleviated
438
J.-q. Pan et al. / Research in Veterinary Science 84 (2008) 434–439
systemic hypoxia, but the underlying mechanisms are not well understood. PKC is one of the important intracellular signal transduction systems and a key enzyme implicated in the control of cellular proliferation, differentiation and cytokine secretion. The classical PKCa is Ca2+ and diacylglycerol dependent PKC isoforms. PKCa seems ubiquitously distributed in all tissues so far examined (Pokorski et al., 2000), including pulmonary epithelial cell, vascular smooth muscle cell, vascular endothelial cell and fibroblast. In mammals, chronic hypoxia stimulates an increased expression of PKC protein in pulmonary artery smooth muscle cells (Zhong et al., 1999). In addition, hypoxia stimulates enhanced PKC isozyme activity in pulmonary arterial vascular endothelial cells (Yang et al., 2000). In vitro experiment also confirmed that the PKC protein levels and PKC isozyme activity were higher in pulmonary artery adventitial fibroblasts in response to hypoxia (Das et al., 2000). In current study, the PKCa expression was detected by measuring OD values in the wall of pulmonary arterioles. The reported optical density is a mean optical density rather than an integrated optical density (IOD) to preclude the influence of vessel thickness, because of the fact that there is a large increase in medial/adventitial layer thickness in the LT group versus NT and LR group. The PKCa protein expression in pulmonary arterioles of low temperature treated birds was significantly higher than those of normal temperature treated birds. The present results provide further evidence that the PKCa signal transduction pathway has an important role in the pathogenesis of hypoxic pulmonary hypertension in broilers, similar to mammals. In addition, feed restriction led to a reduced PKCa expression in pulmonary arterioles. Feed restriction improves the systemic hypoxemia in cold-exposed broiler by slowing growth rate. It is also possible that the reduced pulmonary pressure due to depression of growth rate inhibits PKCa expression. In this context, the reduced PHS mortality and inhibited pulmonary vascular remodeling caused by feed restriction may be partially due to reduction of PKCa expression. Pulmonary vascular remodeling, characterized by proliferation of smooth muscle cells and adventitial fibroblasts in pulmonary arteries, is an important contributor to the sustained pulmonary hypertension induced by hypoxia (Xiang et al., 2002). Many factors have been revealed to contribute to this event, among which the PKC signal transduction pathway has been extensively studied. It has been revealed that hypoxia-induced proliferation of pulmonary artery smooth muscle cells and pulmonary artery adventitial fibroblasts is dependent on PKC activation (Dempsey et al., 1991; Stenmark et al., 2002), and the activation of PKC is a requisite step for pulmonary artery smooth muscle cells to proliferate in response to hypoxia in vitro (Dempsey et al., 1997). In addition, PKC activation results in a slow, sustained pulmonary artery contraction (Tsai et al., 2004), which may also lead to pulmonary remodeling. Moreover, further studies concentrated on PKC iso-
forms revealed that the differentiation and proliferation of vascular smooth muscle cells in response to hypoxia were at least partially dependent on PKCa activation (Dempsey et al., 1997). In this study, the over expressed PKCa in the LT group might contribute to the vascular remodeling. In conclusion, the present study confirmed over expression of PKCa in remodeled pulmonary arterioles of hypertensive broilers. Early time feed restriction inhibited pulmonary vascular remodeling, which is associated with reduction of PKCa expression. Acknowledgements The research work was supported by the National Natural Science Foundation of China (Project No. 30571366, No. 30371061 and No. 30600440). References Das, M., Dempsey, E.C., Bouchey, D., Reyland, M.E., Stenmark, K.R., 2000. Chronic hypoxia induces exaggerated growth response in pulmonary artery adventitial fibroblasts: potential contribution of specific protein kinase C isozymes. American Journal of Respiratory Cell and Molecular Biology 22, 15–25. Dempsey, E.C., McMurtry, I.F., O’Brien, R.F., 1991. Protein kinase C activation allows pulmonary artery smooth muscle cells to proliferation to hypoxia. The American Journal of Physiology 260, L136–L145. Dempsey, E.C., Frid, M.G., Aldashev, A.A., Das, M., Stenmark, K.R., 1997. Heterogeneity in the proliferative response of bovine pulmonary artery smooth muscle cells to mitogens and hypoxia: importance of protein kinase C. Canadian Journal of Physiology and Pharmacology 75, 936–944. De Smit, L., Tona, K., Bruggeman, V., Onagbesan, O., Hassanzadeh, M., Arckens, L., Decuypere, E., 2005. Comparison of three lines of broilers differing in ascites susceptibility or growth rate. 2. Egg weight loss, gas pressures, embryonic heat production, and physiological hormone levels. Poultry Science 84, 1446–1452. Gopalakrishna, R., Jaken, S., 2000. Protein kinase C signaling and oxidative stress. Free Radical Biology and Medicine 28, 1349–1361. Julian, R.J., 1993. Ascites in poultry. Avian Pathology 22, 419–454. Julian, R.J., McMillan, I., Quinton, M., 1989. The effect of cold and dietary energy on right ventricle hypertrophy, right ventricle failure and ascites in meat-type chickens. Avian Pathology 18, 675–684. Li, J.C., Wang, X.L., Sun, W.D., Zhang, K.C., 2000. Study on histopathology of pulmonary arterioles of broilers with Na+ induced pulmonary hypertension syndrome by using the microscopic image analysis program. Acta Veterinaria et Zootechnica Sinaca 31, 441–447. Maxwell, M.H., Robertson, G.W., 1997. World broiler ascites survey 1996. Poultry International 36, 16–30. Media Cybernetics INC., 1998. Image-Pro Plus User’s Guide (Silver Spring, MD, Media Cybernetics Inc). National Research Council, 1994. Nutrient Requirements of Poultry, 9th rev. National Academy Press, Washington, DC. Pan, J.Q., Tan, X., Li, J.C., Sun, W.D., Wang, X.L., 2005. Effects of early feed restriction and cold temperature on lipid peroxidation, pulmonary vascular remodeling and ascites morbidity in broilers under normal and cold temperature. British Poultry Science 46, 374–381. Pokorski, M., Sakagami, H., Kondo, H., 2000. Classical protein kinase C and its hypoxic stimulus-induced translocation in the cat and rat carotid body. The European Respiratory Journal 16, 459–463. Stenmark, K.R., Gerasimovskaya, E., Nemenoff, R.A., Das, M., 2002. Hypoxic activation of adventitial fibroblasts: role in vascular remodeling. Chest 122, 326S–334S.
J.-q. Pan et al. / Research in Veterinary Science 84 (2008) 434–439 Tan, X., Liu, Y.J., Li, J.C., Pan, J.Q., Sun, W.D., Wang, X.L., 2005a. Activation of PKCa and pulmonary vascular remodeling in broilers. Research in Veterinary Science 79, 131–137. Tan, X., Liu, Y.J., Pan, J.Q., Li, J.C., Sun, W.D., Wang, X.L., 2005b. Expression of PKCa in pulmonary arterioles and its association with pulmonary vascular remodeling in broilers with pulmonary hypertension induced by cold temperature exposure. Scientia Agricultura Sinica 38, 1917–1922. Tsai, B.M., Wang, M., Pitcher, J.M., Meldrum, K.K., Meldrum, D.R., 2004. Hypoxic pulmonary vasoconstriction and pulmonary artery tissue cytokine expression are mediated by protein kinase C. American Journal of Physiology. Lung Cellular and Molecular Physiology 287, L1215–L1219. Wang, S., Desai, D., Wright, G., Niles, R.M., Wright, G.L., 1997. Effects of protein kinase Ca over-expression on A7r5 smooth muscle cell proliferation and differentiation. Experimental Cell Research 236, 117–126. Wideman, R.F., French, H., 2000. Ascites resistance of progeny from broiler breeders selected for two generations using chronic unilateral pulmonary artery occlusion. Poultry Science 79, 396–401.
439
Wideman, R.F., Kirby, Y.K., 1995. Evidence of ventilation-perfusion mismatch during acute unilateral pulmonary artery occlusion in broilers. Poultry Science 74, 1209–1217. Xiang, R.P., Sun, W.D., Wang, J.Y., Wang, X.L., 2002. Effect of Vitamin C on pulmonary hypertension and muscularisation of pulmonary arterioles in broilers. British Poultry Science 43, 705–712. Yang, X.M., Xie, Y.Z., Nie, H.J., Yin, Z.Y., 2000. The relationship between VEGF level and PKC activity in pulmonary arterial vascular endothelial cells of rat during hypoxia. Chinese Journal of Applied Physiology 16, 250–252. Zhong, S., Xu, Y.J., Zhang, Z.X., 1999. Immunohistochemical study on protein kinase C expression changes of pulmonary artery in chronic hypoxia rats. Acta Universitatis Medicinae Tongji 28, 475– 477. Zhou, H., Chen, S.X., Wang, L.X., Chen, Y.F., Xie, Y.P., Wang, Q.J., Fan, X.F., 2002. An experimental study on the regulation on pulmonary arterial remodeling by protein kinase C in chronic hypoxic rats. Chinese Journal of Applied Physiology 18, 38–42.