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. 2015 Mar 31;94(4):628–638. doi: 10.3382/ps/pev018

Plexogenic arteriopathy in broiler lungs: Evaluation of line, age, and sex influences1

R F Wideman Jr 1,2, J G Mason 1, N B Anthony 1, D Cross 1
PMCID: PMC4990984  PMID: 25681478

Abstract

Plexiform lesions form in the terminal pulmonary arterioles of human patients suffering from prolonged pulmonary arterial hypertension. Plexiform lesions also develop in broiler lungs, but lesion incidences are not strongly correlated with sustained pulmonary hypertension as reflected by right to total ventricular weight (RVTV) ratios. The present study was conducted to assess plexiform lesion incidences in broiler lines that have been divergently selected for susceptibility or resistance to pulmonary hypertension. Broilers from susceptible (SUS) and resistant (RES) lines were reared together and only clinically healthy (nonascitic, noncyanotic) individuals were evaluated to minimize potential line differences in cardiopulmonary hemodynamics. The objective was to determine if an innate genetic predisposition for plexogenic arteriopathy would be exposed in SUS broilers when compared with RES broilers in the absence of extreme differences in cardiopulmonary hemodynamics. Broilers up to 12 wk age from the SUS and RES lines had essentially equivalent BW, indices of cardiopulmonary function (left ventricle + septum weight, total ventricle weight, and RVTV ratios), and lung volumes within a sex. Average RVTV ratios for broilers from both lines were indicative of normal pulmonary arterial pressures at all ages sampled. Nevertheless, plexiform lesions were detected in SUS and RES broiler lungs immediately posthatch and thereafter at all ages sampled. Lesion incidences were consistently low and did not differ between the lines within any of the sampling ages. This evidence demonstrates that plexiform lesions develop extremely rapidly in broiler chicks, apparently without the prerequisite for vascular stress caused by severe, prolonged pulmonary arterial hypertension. No innate genetic predisposition for complex vascular lesion development appeared to exist in the SUS line when compared with the RES line.

Keywords: broilers, lungs, plexiform lesions, pulmonary hypertension

Abbreviations

IPAH

= idiopathic pulmonary arterial hypertension;

PAP

= pulmonary arterial pressure;

RES

= resistant line;

RVTV

= right to total ventricular weight ratio;

SUS

= susceptible line

INTRODUCTION

Idiopathic pulmonary arterial hypertension (IPAH) is characterized by elevated pulmonary arterial pressure (PAP) and pulmonary vascular resistance attributable to vasoconstriction, hypertrophy of the medial muscle layer in pulmonary arterioles, and pruning of the arterial tree by complex vascular lesions. Early in the pathogenesis of IPAH the right ventricle undergoes work hypertrophy as it develops a progressively higher PAP to propel the cardiac output through the pulmonary vasculature. Indeed, an increase in the right-to-total ventricular weight (RVTV) ratio is widely accepted as an index of the specific work hypertrophy that is characteristic of sustained pulmonary hypertension (Cueva et al., 1974; Hernandez, 1987; Huchzermeyer and DeRuyck, 1986; Julian, 1988; Peacock et al., 1989, 1990; Wideman, 1999, 2000, 2001). The terminal pathogenesis of IPAH includes right ventricular decompensation (weakening and dilation), regurgitation by the right atrioventricular valve, right-sided congestive heart failure, central venous hypertension, hepatic cirrhosis, and ultimately ascites (Julian, 1993, 2000; Wideman, 2000, 2001; Wideman et al., 2007, 2010, 2013). The pathophysiology of IPAH is remarkably similar in humans and fast-growing broiler chickens, and frequently includes the formation of pathognomonic complex vascular lesions known as plexiform lesions (Eddahibi et al., 2002; Humbert et al., 2004; Cool et al., 2005; Wideman et al., 2011; 2013; Wideman and Hamal, 2011; Hamal et al., 2012). High pressures and flow rates are believed to create turbulent blood flow and excessive shear stress in terminal pulmonary arterioles, causing localized endothelial damage that initiates lesion development. Plexiform lesions have been described as dynamic angiogenic lesions driven by disordered or neoplastic-like endothelial proliferation, accompanied by fibrosis and myofibroblast and inflammatory cell infiltration. Lesion morphology usually is complicated by the presence of micro-thrombi, platelet aggregates, and inflammatory cells (Dorfmüller et al., 2003; Nicolls et al., 2005; Hoeper et al., 2006; Wideman et al., 2011, 2013).

Plexogenic arteriopathy in human IPAH patients progressively obliterates pulmonary arterioles and thus proportionally elevates the pulmonary vascular resistance and PAP, thereby further amplifying the pressure and shear stress impinging upon the vessels that remain unobstructed (Tuder et al., 1994, 2001; Cool et al., 1997, 1999; Lee et al., 1998; Eddahibi et al., 2002). The pathogenesis of plexogenic arteriopathy appears to be considerably less aggressive in broilers. In a survey of broilers from an IPAH-susceptible line, plexiform lesions were observed in approximately 40% of the lung sections examined, but lesion densities (lesion numbers per section area) were low relative to the much higher densities typically observed in human IPAH patients. Correlations between RVTV ratios and plexiform lesion incidences also were low, and individual broilers having the highest lesion densities were not necessarily those having the highest RVTV ratios (Wideman et al., 2011). A subsequent experiment also did not demonstrate a consistent association between increases in PAP and increased plexiform lesion incidences in broilers (Kluess et al., 2012). Sparsely distributed plexiform lesions clearly would only modestly increase the pulmonary vascular resistance, indicating that plexogenic arteriopathy likely is a consequence rather than the proximate cause of the elevated pulmonary vascular resistance and pulmonary hypertension in broilers developing IPAH (Wideman et al., 2011, Kluess et al., 2012). Alternatively, the rapid three-dimensional expansion of the pulmonary vascular tree in fast-growing broilers may occasionally lead to developmental misalignments or distortions of the arterial branches, resulting in localized turbulent blood flow rather than laminar blood flow. Plexiform lesions have been identified in broiler lungs during the first 5 wk posthatch, coincident with a period of very rapid lung development and expansion accompanying the exponential phase of growth (Wideman et al., 2013). Within this context of normal lung maturation, plexogenic arteriopathy may constitute a naturally occurring remedial process that serves to prune inappropriately aligned arterioles independent of the onset of IPAH (Wideman and Hamal, 2011; Wideman et al., 2011; 2013).

The present study was conducted to assess plexiform lesion incidences in 2 broiler lines that were divergently selected for over 14 generations for susceptibility or resistance to IPAH when exposed to low atmospheric partial pressures of oxygen (hypobaric hypoxia) at simulated high altitudes (Anthony et al., 2001; Balog, 2003; Pavlidis et al., 2007). Progeny from the 14th generation of the susceptible (SUS) line exhibited 98% IPAH mortality during 6 wk exposure to hypobaric hypoxia at a simulated altitude of 8,000 ft or 2,438 m, whereas only 7% of the broilers from the resistant (RES) line succumbed to IPAH under a more rigorous challenge at a more extreme simulated altitude of 12,000 ft or 3,658 m (Wideman et al., 2013). Physiological evaluations of broilers reared under normoxic conditions near sea level demonstrated higher pulmonary arterial resistances and pressures, but similar cardiac outputs in the SUS line when compared with the RES line (Chapman and Wideman, 2006; Lorenzoni et al., 2008; Wideman et al., 2010). If the formation of plexiform lesions innately precedes or coincides with the onset of pulmonary hypertension in broilers that are genetically predisposed to develop IPAH, then broilers from the SUS line should exhibit higher plexiform lesion incidences than broilers from the RES line when both groups are reared together under normoxic and thermoneutral (nonchallenging) conditions. Alternatively, if plexiform lesions are indicative of a naturally occurring pruning of misaligned arterioles that cause turbulent (i.e., high resistance) rather than laminar (i.e., low resistance) blood flow, then it was our hypothesis that comparably low incidences of plexogenic arteriopathy should develop in the lungs of rapidly growing broilers from both lines.

MATERIALS AND METHODS

Experiment 1

Animal procedures were approved by the University of Arkansas Institutional Animal Care and Use Committee (Protocol No. 06067). Straight run chicks (n = 200 SUS, n = 170 RES) were wing banded on the day of hatch (d 1; 09/02/2009), and randomly assigned to 4 environmental chambers (8 m2 floor space/chamber). Clean pine shavings were used for litter. Single-pass ventilation was maintained at a constant rate of 6 m3/min in all chambers. Birds were brooded at 32°C from d 1 to 3, and then the temperature was reduced to 31°C during d 4 through 6, 25°C on d 7 through 11, 25°C on d 11 through 14, and 24°C on d 15 through 86. The photoperiod was 23L:1D for d 1 to 4, and 16L:8D thereafter. Chambers were equipped with 2 rows of nipple waterers, and 2 tube feeders. During the study birds were fed a corn/soybean meal-based feed, designed to conform to or exceed the minimum National Research Council (1994) standards for all ingredients including but not limited to 22.7% CP, 3,059 kcal ME/kg, 1.5% arginine, and 1.43% lysine. Feed and water were provided ad libitum for the duration of the study. A minimum of 10 clinically healthy (noncyanotic, nonascitic) birds per line (5 males, 5 females) were sampled at 2, 4, 6, 8, 10, and 12 wk age. Procedures for fixing the lungs in situ were the same as previously described (Wideman et al., 2011; Kluess et al., 2012). Birds were weighed and anesthetized to a surgical plane with intramuscular injections of allobarbitol (5, 5-diallylbarbituric acid, 3.0 mL/kg BW, 25 mg/mL; Sigma–Aldrich, St. Louis, MO) and ketamine HCl (1.0 to 2.5 mL of 100 mg/mL; Henry Schein, Melville, NY). Heparinized saline with papaverine (2 mL/bird, 200 units/mL ammonium heparin in 0.9% NaCl containing 0.4 mg/mL papaverine; Sigma–Aldrich, St. Louis, MO) was injected intravenously to prevent clotting (heparin) and relax (dilate) the vascular smooth muscle (papaverine). Birds then were euthanized by exsanguination. The sternum was retracted to expose the heart, the right atrium was clamped with a hemostat, the left atrium was cut open for drainage, and polyethylene tubing (2.5 mm inner diameter) was inserted via a slit in the right ventricle to flush the pulmonary vasculature with 200 mL 0.9% NaCl containing 50 units/mL ammonium heparin and 0.1 mg/mL papaverine at room temperature. The lungs were fixed by transcardiac perfusion with 200 to 400 mL 4% phosphate-buffered paraformaldehyde at room temperature. Carboys containing the saline and the paraformaldehyde were positioned at an elevation sufficient to obtain a gravimetric perfusion pressure of 46 cm H2O (34 mm Hg) at the level of the heart. The thoracic cavity was flooded with 4% paraformaldehyde and the lungs remained fixing in situ for a minimum 3 additional h. Hearts were dissected and weighed to calculate the RVTV ratios. Lungs were harvested intact from the thoracic cavity and the volume (in cubic centimeters, i.e., milliliters) was measured by gravimetric displacement of water (Owen et al., 1995). The lungs were sliced in the transverse plane at the 4 major rib indentations (costal sulci), and were stored overnight in 4% paraformaldehyde. The tissues were rinsed in tap water and dehydrated in 25, 50, and 70% ethyl alcohol for 30 min each, and stored in 70% ethyl alcohol. One interrib division from the middle of each lung was embedded in paraffin, sectioned in the transverse plane at 5 to 7 μm thickness, mounted at one section per slide, and stained with hematoxylin and eosin. One slide per lung division was searched for plexiform lesions using overlapping fields of view and sequential horizontal traverses at 40× total magnification. Suspected lesions were noted on a slide template, photographed at 400×, and their coordinates recorded using a Micro-Slide Field Finder (Gurley Precision Instruments, Troy NY). Two independent researchers reviewed each lung section to confirm lesion identifications and locations.

Experiment 2

A second independent study was conducted to comprehensively evaluate plexiform lesion incidences during the early posthatch period. Straight run chicks (n = 90 SUS, n = 85 RES) wing banded and randomly assigned to 4 environmental chambers. Bird rearing and management were the same as described for Experiment 1. Lung tissue samples were collected for histological evaluation on the d of hatch (d 1; 05/05/2010) and every wk thereafter through 5 wk age. Chicks (n = 15) were sampled per line on d 1, and 10 birds (5 males and 5 females) were sampled per line during subsequent weeks. Clinically healthy (noncyanotic, nonascitic) chicks from the SUS and RES lines were anesthetized to a surgical plane with intramuscular injections of allobarbitol and ketamine HCl, and they were injected intravenously with heparinized saline and papaverine (1 to 2 mL/bird, 200 units/mL ammonium heparin in 0.9% NaCl containing 0.4 mg/mL papaverine). On d 1 and 7, the chicks were euthanized and the lungs were fixed in situ by flooding the thoracic cavity with 4% phosphate-buffered paraformaldehyde at room temperature for 24 h. On subsequent sampling days the lungs were fixed in situ by transcardiac perfusion as described for Experiment 1. After the initial fixation, all lungs were processed for histology as described for Experiment 1. BW, sex, ventricle weights, and lung volumes were not recorded in Experiment 2.

Data Analysis

Plexiform lesion incidences were calculated in percentages as follows: [(number of lung sections or slides with ≥1 plexiform lesion) / (number of lung sections or slides examined) × 100], as described previously (Wideman et al., 2011; Kluess et al., 2012). Plexiform lesion incidences were compared between sexes or lines within a sampling age using the SigmaStat Z-test (Jandel Scientific, 1994). Plexiform lesion densities were calculated from the total number of lesions present per lung section or slide examined. Within a sampling age, the SigmaStat ANOVA package was used to compare plexiform lesion densities, BW, ventricle weights, RVTV ratios, and lung volumes between sexes and lines. Values were considered to differ at P ≤ 0.05.

RESULTS

Experiment 1

One bird from the SUS line and no birds from the RES line developed ascites during the course of Experiment 1. Values for BW, lung volume, and relative lung weight are shown by age, line, and sex in Table 1. Sex and BW were not recorded on d 14. Within each of the ages sampled BW did not differ between lines within a sex, with the solitary exception of wk 10 when SUS males were heavier than RES males. For both lines the males were consistently heavier than females at each of the ages sampled. Differences between lines were sparse and inconsistent with regard to left, right, and total lung volumes. Instead, these absolute lung volumes usually were higher in males than in females at all ages sampled. Many of the sex-related differences in absolute lung volume were normalized by dividing total lung volume by BW to calculate the relative lung volume. The remaining differences in relative lung volume were sporadic and inconsistent. For example when compared with females from the SUS line, females from the RES line had lower relative values on wk 4 and 10, but higher relative values on wk 8 and 12. Across all ages, lines and sexes the relative lung volume consistently averaged approximately 0.01 cm3/g BW (Table 1). Right ventricle weights, left ventricle + septum weights, total ventricle weights, relative ventricle weights, and RVTV ratios are provided by age, line, and sex in Table 2. Within an age and regardless of line, left ventricle + septum and total ventricle weights tended to be higher in males than in females, and these sex-related differences generally were normalized by dividing the total ventricle weight by BW to calculate the relative ventricle weight. The RVTV ratios did not differ by line or sex within any of the sampling ages (Table 2). Plexiform lesions were present in the lungs of SUS and RES broilers, of both sexes and at all ages (Figures 1 to 7; vide infra). As shown in Table 3, plexiform lesion densities tended to average less than one lesion per histological section, and plexiform incidences ranged from a low of 20% of the sections examined from SUS males at 4 wk to a high of 70% of the sections examined from SUS females at 4 wk. The lesion densities and incidences did not differ by line, sex, or lung (right vs. left) within any of the sampling ages. No consistent age-related trends were evident for lesion densities calculated in terms of lesions observed per section evaluated (Table 3).

Table 1.

BW, lung volumes, and relative total lung volume in Experiment 1 for male and female broilers from lines selected for susceptibility or resistance to pulmonary arterial hypertension syndrome.

Age Number BW Left Lung Right Lung Total Lung Relative Lung Volume
(wk) Line Sex (n) (g) (cm3) (cm3) (cm3) (cm3/g BW)
2 S M+F 10 – 1.88 ± 0.08 1.66 ± 0.07b 3.54 ± 0.14 –
R M+F 10 – 1.94 ± 0.07 2.01 ± 0.12a 3.95 ± 0.17 –
4 S M 5 1,356 ± 27a 7.51 ± 0.39a 7.46 ± 0.27a 14.97 ± 0.62a 0.0110 ± 0.0004a
R M 6 1,294 ± 21a 6.85 ± 0.32ab 6.97 ± 0.38ab 13.82 ± 0.62ab 0.0107 ± 0.0004a
S F 5 1,118 ± 40b 6.27 ± 0.23b 6.21 ± 0.43b 12.48 ± 0.61b 0.0112 ± 0.0004a
R F 5 1,195 ± 31b 5.00 ± 0.36c 4.99 ± 0.40c 9.99 ± 0.75c 0.0083 ± 0.0006b
6 S M 6 2,223 ± 75a 10.92 ± 0.31ab 11.06 ± 0.36b 21.98 ± 0.66a 0.0099 ± 0.0001
R M 5 2,445 ± 49a 11.65 ± 0.50a 12.39 ± 0.25a 24.04 ± 0.71a 0.0098 ± 0.0002
S F 6 1,962 ± 55b 9.65 ± 0.34b 9.48 ± 0.23c 19.13 ± 0.56b 0.0098 ± 0.0004
R F 5 1,953 ± 43b 9.74 ± 0.16b 9.62 ± 0.37c 19.36 ± 0.39b 0.0099 ± 0.0002
8 S M 5 3,452 ± 76a 16.63 ± 1.00a 16.84 ± 1.30a 33.46 ± 2.26a 0.0097 ± 0.0006ab
R M 5 3,365 ± 115a 17.92 ± 0.50a 17.58 ± 0.64a 35.50 ± 1.03a 0.0106 ± 0.0003a
S F 5 2,710 ± 58b 11.52 ± 0.30c 11.39 ± 0.46b 22.91 ± 0.62b 0.0085 ± 0.0002b
R F 5 2,586 ± 51b 13.46 ± 0.53b 13.45 ± 0.55b 26.91 ± 1.07b 0.0104 ± 0.0005a
10 S M 5 4,467 ± 144a 22.89 ± 0.48a 22.53 ± 0.58a 45.42 ± 0.65a 0.0102 ± 0.0003b
R M 5 3,866 ± 141b 23.84 ± 1.72a 23.54 ± 1.21a 47.38 ± 2.86a 0.0093 ± 0.0007b
S F 5 3,507 ± 60b 16.98 ± 1.08b 15.76 ± 1.17b 32.74 ± 1.83b 0.0123 ± 0.0005a
R F 5 3,537 ± 113b 17.46 ± 0.63b 17.01 ± 0.93b 34.47 ± 1.52b 0.0104 ± 0.0002b
12 S M 5 4,948 ± 161a 26.19 ± 0.83a 25.36 ± 1.32a 51.54 ± 1.88a 0.0105 ± 0.0005a
R M 5 4,780 ± 187a 27.13 ± 1.55a 28.22 ± 1.58a 55.35 ± 3.11a 0.0116 ± 0.0002a
S F 5 3,813 ± 120b 17.06 ± 0.63b 17.97 ± 0.61b 35.03 ± 0.94b 0.0092 ± 0.0004b
R F 5 3,669 ± 72b 19.44 ± 1.29b 19.53 ± 0.89b 38.97 ± 2.17b 0.0106 ± 0.0006a
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a,bValues with different superscripts within an age group differed significantly between sexes or lines at P ≤ 0.05.

Table 2.

Heart weights (right ventricle, left ventricle + septum, total ventricle), relative total ventricle weights, and right/total ventricular weight ratios in Experiment 1 for male and female broilers from lines selected for susceptibility or resistance to pulmonary arterial hypertension syndrome.

Age Number Right Ventricle Left Ventricle Total Ventricle Relative Ventricle
(wk) Line Sex (n) (g) (g) (g) (g/g BW) Right/Total Ventricle
2 S M+F 10 0.37 ± 0.02 1.15 ± 0.08 1.51 ± 0.09 – 0.24 ± 0.01
R M+F 10 0.38 ± 0.04 1.27 ± 0.07 1.65 ± 0.09 – 0.23 ± 0.01
4 S M 5 1.68 ± 0.38 5.05 ± 0.05a 6.74 ± 0.39a 0.0050 ± 0.0003 0.24 ± 0.04
R M 6 1.23 ± 0.22 4.83 ± 0.35a 6.06 ± 0.40a 0.0047 ± 0.0003 0.20 ± 0.03
S F 5 0.95 ± 0.07 3.71 ± 0.12b 4.66 ± 0.15b 0.0042 ± 0.0001 0.20 ± 0.01
R F 5 1.17 ± 0.10 4.22 ± 0.13b 5.40 ± 0.21b 0.0045 ± 0.0002 0.22 ± 0.01
6 S M 6 1.96 ± 0.14 6.58 ± 0.31b 8.54 ± 0.39a 0.0039 ± 0.0002b 0.23 ± 0.01
R M 5 2.05 ± 0.25 7.82 ± 0.29a 9.88 ± 0.46a 0.0040 ± 0.0001ab 0.20 ± 0.02
S F 6 1.32 ± 0.10 4.82 ± 0.19c 6.13 ± 0.28b 0.0031 ± 0.0001b 0.21 ± 0.01
R F 5 2.06 ± 0.33 6.83 ± 0.23ab 8.89 ± 0.48a 0.0046 ± 0.0003a 0.22 ± 0.02
8 S M 5 2.96 ± 0.17a 9.80 ± 0.78a 12.75 ± 0.79a 0.0037 ± 0.0003 0.24 ± 0.02
R M 5 2.17 ± 0.22b 8.92 ± 0.69a 11.08 ± 0.89a 0.0033 ± 0.0002 0.19 ± 0.01
S F 5 1.78 ± 0.29b 6.59 ± 0.64b 8.37 ± 0.86b 0.0031 ± 0.0003 0.21 ± 0.02
R F 5 1.61 ± 0.10b 6.56 ± 0.25b 8.17 ± 0.32b 0.0032 ± 0.0002 0.20 ± 0.01
10 S M 5 2.64 ± 0.21 11.77 ± 0.78a 14.41 ± 0.97a 0.0032 ± 0.0002 0.18 ± 0.01
R M 5 2.09 ± 0.11 9.37 ± 0.71ab 11.46 ± 0.61a 0.0030 ± 0.0001 0.24 ± 0.03
S F 5 2.46 ± 0.43 7.75 ± 0.52b 10.21 ± 0.73b 0.0029 ± 0.0002 0.19 ± 0.02
R F 5 1.84 ± 0.15 8.24 ± 1.45b 10.08 ± 1.53b 0.0028 ± 0.0004 0.19 ± 0.02
12 S M 5 3.51 ± 0.19a 12.64 ± 0.10a 16.15 ± 0.23a 0.0033 ± 0.0001 0.22 ± 0.01
R M 5 3.60 ± 0.60a 12.37 ± 1.19a 15.98 ± 1.75a 0.0033 ± 0.0003 0.22 ± 0.02
S F 5 2.19 ± 0.11b 8.38 ± 0.53b 10.57 ± 0.59b 0.0028 ± 0.0002 0.21 ± 0.01
R F 5 1.94 ± 0.23b 7.59 ± 0.24b 9.54 ± 0.34b 0.0026 ± 0.0001 0.20 ± 0.02
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a–cValues with different superscripts within an age group differed significantly between sexes or lines at P ≤ 0.05.

Figure 1.

Figure 1.

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Plexiform lesion incidences (upper panel: average percentage of lung sections with ≥1 plexiform lesion/number of lung sections examined × 100) and plexiform lesion densities (lower panel: average number of lesions per lung section examined) on the d of hatch (wk 0) and during subsequent sampling intervals, for broilers from the IPAH-susceptible and IPAH-resistant lines. Data from Experiments 1 and 2 and for both sexes are combined. P values are shown for comparisons between lines within an age.

Figure 7.

Figure 7.

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Photomicrographs of a plexiform lesion in the lung of a 12-week-old broiler from the IPAH-susceptible line, photographed in the same section at original magnifications of 100× (7A, upper panel) and 400× (7B, lower panel). 7A: This plexiform lesion (PL) occupies a midsized interparabronchial artery that may be a branch of the nearby large muscular artery (ar). Perivascular mononuclear cell infiltrates (PMCI) permeate the interparabronchial septum separating adjacent parabronchi (PB = parabronchial lumen). 7B: The plexiform lesion presents a glomeruloid appearance and contains peripherally located foam-type macrophages (MΦ). Arrows indicate the remnants of the wall of the larger artery in which the lesion developed. Within the matrix of the lesion proliferating intimal cells (IP) are clustered into laminated elongated bundles sheathed by encircling layers of connective tissue and smooth muscle (indicated with an asterisk). The appearance and structure of this lesion is consistent with the interpretation that the parent artery and its minor branches were occluded by a progressive retrograde intimal cell proliferation that extended from an initial focal lesion within a smaller (downstream) interparabronchial arteriole. Hematoxylin and eosin staining.

Table 3.

Plexiform lesion densities and incidences in Experiment 1 for male and female broilers from lines selected for susceptibility or resistance to pulmonary arterial hypertension syndrome.

Age Number Left Lung Right Lung Lung Average Plexiform Incidence
(wk) Line Sex (n) (lesions/section) (lesions/section) (lesions/section) (lungs with lesions/lungs evaluated)
2 S M+F 10 0.70 ± 0.26 0.30 ± 0.21 0.50 ± 0.20 35% (7/20)
R M+F 10 0.20 ± 0.13 0.60 ± 0.31 0.40 ± 0.16 30% (6/20)
4 S M 5 0.20 ± 0.20 0.20 ± 0.20 0.20 ± 0.12 20% (2/10)
R M 6 0.50 ± 0.37 0.88 ± 0.44 0.67 ± 0.34 42% (5/12)
S F 5 0.20 ± 0.20 0.40 ± 0.40 0.30 ± 0.20 70% (7/10)
R F 5 0.60 ± 0.24 1.40 ± 0.87 1.00 ± 0.52 30% (3/10)
6 S M 6 0.33 ± 0.16 0.33 ± 0.26 0.33 ± 0.19 25% (3/12)
R M 5 0.40 ± 0.17 0.60 ± 0.17 0.50 ± 0.11 50% (5/10)
S F 6 0.67 ± 0.16 0.50 ± 0.17 0.58 ± 0.12 58% (7/12)
R F 5 0.60 ± 0.28 0.20 ± 0.14 0.40 ± 0.13 30% (3/10)
8 S M 5 1.20 ± 0.73 1.00 ± 0.45 1.10 ± 0.33 50% (5/10)
R M 5 0.20 ± 0.20 0.40 ± 0.24 0.30 ± 0.12 30% (3/10)
S F 5 1.00 ± 0.32 0.20 ± 0.20 0.60 ± 0.19 50% (5/10)
R F 5 0.60 ± 0.40 0.80 ± 0.80 0.70 ± 0.25 60% (6/10)
10 S M 5 1.00 ± 0.63 0.20 ± 0.20 0.60 ± 0.40 30% (3/10)
R M 5 1.40 ± 0.75 0.60 ± 0.40 1.00 ± 0.35 50% (5/10)
S F 5 0.40 ± 0.24 0.40 ± 0.24 0.40 ± 0.19 50% (5/10)
R F 5 0.40 ± 0.24 0.40 ± 0.24 0.40 ± 0.19 40% (4/10)
12 S M 5 0.80 ± 0.37 0.40 ± 0.24 0.60 ± 0.19 50% (5/10)
R M 5 1.00 ± 0.55 0.80 ± 0.58 0.90 ± 0.56 50% (5/10)
S F 5 0.20 ± 0.20 0.60 ± 0.24 0.40 ± 0.19 40% (4/10)
R F 5 0.80 ± 0.37 0.40 ± 0.40 0.60 ± 0.29 40% (4/10)
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Figure 3.

Figure 3.

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Photomicrograph of a section from the lung of a newly hatched (d 1) broiler chick from the IPAH-susceptible line showing a plexiform lesion in an interparabronchial arteriole coursing between three adjacent parabronchi (PB), including multiple foam-type macrophages (MΦ) and proliferating intimal cells (IP). The parabronchial parenchyma is congested with nucleated avian red blood cells. Original magnification 400×; hematoxylin and eosin staining.

Experiment 2

No birds from either line developed ascites during the course of Experiment 2. Plexiform lesion densities tended to average less than one lesion/slide evaluated, ranging from an average of 0.05 lesions/section for SUS broilers at 4 wk to 0.85 lesions/section for SUS broilers at 8 wk (Table 4). Plexiform lesion incidences ranged from 5% of the sections evaluated in SUS broilers at 4 wk, to 50% of the sections evaluated in SUS broilers at 3 wk (Table 4). The lesion densities and incidences did not differ significantly between lines or right or left lungs for any of the sampling ages. For example, comparing the 1.10 vs. 0.30 average lesions/section for right lungs from 3-week-old broilers yielded a probability of P = 0.130 (ANOVA), and comparing the 50% vs. 25% lesion incidences at 3 wk yielded a probability of P = 0.191 (Z-test).

Table 4.

Plexiform lesion densities and incidences in Experiment 2 for male and female broilers from lines selected for susceptibility or resistance to pulmonary arterial hypertension syndrome1.

Age Number Left Lung Right Lung Lung Average Plexiform Incidence
(wk) Line Sex (n) (lesions/section) (lesions/section) (lesions/section) (lungs with lesions/lungs evaluated)
0 S M+F 15 0.27 ± 0.11 0.53 ± 0.19 0.40 ± 0.12 33% (10/30)
R M+F 15 0.27 ± 0.15 0.20 ± 0.10 0.23 ± 0.10 20% (6/30)
1 S M+F 10 0.40 ± 0.16 0.30 ± 0.21 0.35 ± 0.13 30% (6/20)
R M+F 10 0.30 ± 0.15 0.30 ± 0.15 0.30 ± 0.10 30% (6/20)
2 S M+F 10 0.50 ± 0.17 0.40 ± 0.16 0.45 ± 0.17 35% (7/20)
R M+F 10 0.40 ± 0.16 0.20 ± 0.20 0.30 ± 0.13 25% (5/20)
3 S M+F 10 0.60 ± 0.34 1.10 ± 0.31 0.85 ± 0.23 50% (10/20)
R M+F 10 0.30 ± 0.15 0.30 ± 0.21 0.30 ± 0.13 25% (5/20)
4 S M+F 10 0.00 ± 0.00 0.10 ± 0.10 0.05 ± 0.05 5% (1/20)
R M+F 10 0.10 ± 0.10 0.10 ± 0.10 0.10 ± 0.07 10% (2/20)
5 S M+F 10 0.00 ± 0.00 0.60 ± 0.31 0.30 ± 0.16 20% (4/20)
R M+F 12 0.25 ± 0.11 0.42 ± 0.20 0.33 ± 0.13 25% (6/24)
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1None of the values within an age group differed significantly between sexes or lines (P ≥ 0.05).

Metaanalysis of Lesion Incidences and Densities

The data from both Experiments 1 and 2, for plexiform lesion incidences or densities, are pooled by line in the upper and lower panels, respectively, of Figure 1. Lesion incidences did not differ between the SUS and RES broilers at any of the ages evaluated. Differences in lesion densities between the lines were sporadic and presumably reflect the relative scarcity and chance occurrence of these lesions in both lines. For example, the SUS line had a higher lesion density at 3 wk age, but a lower lesion density at 4 wk age when compared with the RES line (Figure 1).

Histological Observations for Experiments 1 and 2

Representative photomicrographs of plexiform lesions in broiler lungs from the d of hatch through 12 wk age are provided in Figures 2 to 7. Plexiform lesions did not appear to differ histologically between SUS and RES broilers at any age. Lesions in the lungs from 1-day-old and 1-week-old chicks were relatively small in size, and contained a tightly packed matrix of proliferating intimal cells surrounding dispersed mononuclear cells, and large macrophages that had not yet developed a characteristic foam-type appearance. These “early” plexiform lesions did not present a recanalized glomeruloid appearance, nor were they consistently associated with obvious perivascular mononuclear cell infiltrates (Figures 2 to 4). The earliest lesions clearly developed near sites where muscularized interparabronchial arteries were branching from their parent arteries (Figure 4). Plexiform lesions in the lungs of 4- to 6-week-old old broilers typically were found within interparabronchial septa adjacent to large muscular parent arteries supplying smaller interparabronchial arterioles. These maturing and evidently expanding lesions typically contained obvious foam-type macrophages aligned along the dilated and often indistinct remnants of the arteriolar wall. The matrix tended to be less compact and more glomeruloid in structure, with the proliferating intimal cells forming laminated swaths or clusters that appeared to extend lengthwise along the remnants of the arteriolar lumen. Perivascular mononuclear cell infiltrates typically were observed along the extramural aspects of the lesions (Figures 5 and 6). Well-developed plexiform lesions in 10- to 12-week-old broiler lungs contain multiple large foam-type macrophages encased in a glomeruloid-like matrix of proliferating intimal cells. The intimal cells are grouped together in elongated laminated bundles sheathed by encircling layers of connective tissue and smooth muscle. The appearance and structure of this lesion is consistent with the interpretation that the parent artery, and its minor branches, were occluded by a progressive retrograde intimal cell proliferation that extended from an initial focal lesion within a smaller (downstream) interparabronchial arteriole. Substantial extramural aggregations of perivascular mononuclear cell infiltrates routinely were observed adjacent to the lesion and nearby blood vessels (Figure 7).

Figure 2.

Figure 2.

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Photomicrograph of a section from the lung of a newly hatched (d 1) broiler chick from the IPAH-susceptible line showing an immature plexiform lesion forming in an interparabronchial arteriole, including macrophages (MΦ) that do not yet exhibit foam-type characteristics, proliferating intimal cells (IP), and blood capillaries (c). The adjacent parabronchial parenchyma (PB) is congested with nucleated avian red blood cells. Atria (at) open into the lumen of the parabronchi. Original magnification 400×; hematoxylin and eosin staining.

Figure 4.

Figure 4.

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Photomicrographs of a plexiform lesion in the lung of a 1-week-old broiler chick from the IPAH-susceptible line, photographed in adjacent sections at original magnifications of 100× (4A, upper panel) and 400× (4B, lower panel). 4A: This plexiform lesion (PL) has formed in an interparabronchial arteriole coursing between three adjacent parabronchi (PB = parabronchial lumen). Additional muscular interparabronchial arterioles are indicated by arrows. 4B: The plexiform lesion contains macrophages (MΦ) and a matrix of proliferating intimal cells (IP). Dark-staining mononuclear cells appear to be dispersed within the IP matrix. The parabronchial parenchyma (PB) is congested with nucleated avian red blood cells. Arrows indicate small arterioles. Hematoxylin and eosin staining.

Figure 5.

Figure 5.

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Photomicrographs of a plexiform lesion in the lung of a 4-week-old broiler from the IPAH-susceptible line, photographed in the same section at original magnifications of 100× (5A, upper panel) and 400× (5B, lower panel). 5A: This plexiform lesion (PL) formed in a small arteriole that appears to have branched from the adjacent large muscular artery (ar). Another branch point from the same artery is indicated by the arrow. Abundant expanded and uncongested air capillaries are preserved within the parabronchial parenchyma (PB) that has been effectively cleared and fixed by trans-cardiac perfusion. 5B: The plexiform lesion contains foam-type macrophages (MΦ) and a laminated bundle of proliferating intimal cells (IP). Scattered nucleated red blood cells and an aggregate of perivascular mononuclear cell infiltrates (PMCI) are evident adjacent to the lesion and a small muscular arteriole branch (ar). Arrow indicates muscular wall of parent artery adjacent to the plexiform lesion. Hematoxylin and eosin staining.

Figure 6.

Figure 6.

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Photomicrographs of a plexiform lesion in the lung of an 8-wk-old broiler from the IPAH-resistant line, photographed in the same section at original magnifications of 100× (6A, upper panel) and 400× (6B, lower panel). 6A: This mature plexiform lesion (PL) appears to occupy an arteriole branching from the adjacent large muscular artery (ar). Another emerging branch point from the same artery is indicated by the arrow. Abundant air capillaries are preserved within the parabronchial parenchyma (PB). 6B: The plexiform lesion contains several foam-type macrophages (MΦ) and proliferating intimal cells (IP). The mature lesion presents a glomeruloid appearance, having been recanalized by a small arteriole (ar) and vascular capillaries (c). Arrow indicates muscular wall of parent artery adjacent to the plexiform lesion. Hematoxylin and eosin staining.

DISCUSSION

The histopathogenesis of plexiform lesion development in broiler lungs has been documented previously (Wideman et al., 2011; Wideman and Hamal, 2011; Wideman et al., 2013). In the present study the lesions observed in both sexes from both lines closely resembled plexiform lesions described previously for 8- to 52-week-old broilers from the SUS line (Wideman et al., 2011; Hamal et al., 2012). One novel observation in the present study was the occurrence of plexogenic arteriopathy in newly hatched chicks. This benchmark of very rapid and early lesion development is remarkable because the air capillaries and blood capillaries in embryonic avian lungs are undeveloped and sparsely distributed until approximately 2 d prior to hatch. Up to that stage of embryonic development the majority of blood returning to the heart bypasses the lungs via right-to-left cardiac shunts (atrial foramina). However, after the embryo penetrates the air cell to initiate gaseous breathing 2 d prehatch, an explosive phase of air- and blood-capillary proliferation is triggered, the pulmonary vascular architecture expands dramatically, and the right-to-left cardiac shunts close (Duncker, 1978; Timmwood et al., 1987). Accordingly, appreciable levels of cardiac output only are propelled through avian lungs by the right ventricle beginning shortly before hatch, thus it is only during this brief interval that pulmonary arterial hypertension potentially could occur in ovo. Clearly the acute development of plexiform lesions in newly hatched broiler chicks is incompatible with the apparent prerequisite for prolonged, severe pulmonary hypertension over the course of several months or years before plexiform lesions develop in human IPAH patients (Naeye and Vennart, 1960; Wagenvoort and Wagenvoort, 1970; Heath et al., 1987; Loyd et al., 1988; Wagenvoort, 1989) and in an experimental rat model (Abe et al., 2010). In future studies it clearly will be important to determine when plexiform lesions first appear in the lungs of broiler embryos.

Broilers from the SUS line are consistently more susceptible to developing IPAH than broilers from the RES line when both groups are challenged with hypobaric hypoxia, cool temperature exposure, or microvascular occlusion (Anthony et al., 2001; Wideman et al., 2002, 2007, 2013; Chapman and Wideman, 2006; Pavlidis et al., 2007; Lorenzoni et al., 2008). When challenged by microvascular occlusion, these lines also exhibited markedly divergent pulmonary gene expression patterns for vasoactive mediators and their receptors, and for inflammatory chemokines and cytokines (Hamal et al., 2008, 2010a,b). However, the present study was designed to compare clinically healthy (nonascitic, noncyanotic) broilers from the SUS and RES lines, that were reared together under ideal (nonchallenging) environment and management conditions. The objective was to determine if an innate genetic predisposition for plexogenic arteriopathy would be exposed in SUS broilers when compared with RES broilers in the absence of extreme differences in cardiopulmonary hemodynamics. In Experiment 1 the SUS and RES lines exhibited, within a sex, minor and inconsistent differences in BW, lung volumes, and ventricular weights. No differences in RVTV ratios were detected between the lines. Cardiac output is positively correlated with BW, left ventricle + septum weight, and total ventricle weight, and PAP is positively correlated with RVTV ratios (Wideman, 1999; Chapman and Wideman, 2001; Lorenzoni et al., 2008; Wideman et al., 2010). Therefore the conditions of the present study appear to have minimized cardiopulmonary hemodynamic differences between the lines. Moreover, the numerically highest RVTV ratios of 0.24 (wk 2, 4, and 8 for the SUS line, and wk 10 for the RES line) are not indicative of sustained pulmonary hypertension in broilers (Huchzermeyer and DeRuyck, 1986; Julian, 1988; Peacock et al., 1989, 1990; Wideman, 1999, 2000, 2001). Within this context, plexiform lesions were detected in SUS and RES broilers in both Experiments 1 and 2, and at all ages sampled, and the lesion densities and incidences did not differ between the lines within any of the sampling ages in either Experiment 1 or 2 (Tables 3 and 4). The overall average lesion incidence of approximately 34% for both lines across all ages in the present study (Figure 1) was similar to the average lesion incidence of approximately 40% for the SUS line in a previous study (Wideman et al., 2011). Lesion densities appeared to gradually trend upward with age for both lines (Figure 1), however age-related increases in absolute lung volume occurred concurrently, ranging from approximately 2 cm3/lung at 2 wk age to approximately 20 cm3/lung by 10 wk age (Table 1). The age-related increase in lung volume translated into obvious age-related increases in the overall size (square centimeters) of the transverse histological sections from the lungs (not shown). Therefore the lesion densities per square centimeter of histological section evaluated must have declined with age, as was demonstrated in the previous evaluation of the SUS line (Wideman et al., 2011). In all cases the lesion incidences and densities were so low that the associated vascular obstruction must be considered to only modestly increase the resistance to pulmonary blood flow.

In conclusion, plexiform lesions were detected in SUS and RES broilers immediately posthatch and thereafter in both lines at all ages sampled. Furthermore the lesion densities and incidences did not differ between the lines within any of the sampling ages in either Experiment 1 or 2. The available evidence demonstrates that plexiform lesions can develop extremely rapidly in broiler chicks, apparently without the prerequisite for vascular stress caused by severe, prolonged pulmonary arterial hypertension. After minimizing potential differences in cardiopulmonary hemodynamics, no innate genetic predisposition for complex vascular lesion development appeared to exist in the SUS line when compared with the RES line. The absence of consistent differences in lesion incidences and densities between the lines within all sampling ages in 2 independent experiments is consistent with the hypothesis that plexiform lesions in broiler lungs may be symptomatic of a naturally occurring and ongoing pruning of misaligned arterioles that are creating turbulent blood flow, and thus localized damage to the endothelium. This conclusion does not preclude the possibility that broilers experiencing sustained IPAH may exhibit elevated plexiform lesion incidences if the increased pressure transforms laminar blood flow into turbulent flow in increasing numbers of terminal inter- and intraparabronchial arterioles (Kluess et al., 2012). In that case plexogenic arteriopathy may serve to prune arterioles in which step-wise increases in arterial pressure sequentially elicit turbulent blood flow in marginally aligned elements of the maturing pulmonary vascular tree (Wideman and Hamal, 2011; Wideman et al., 2011).

Footnotes

1

J. G. Mason conducted a portion of this study in partial fulfillment for the Masters of Science degree. This study was supported by National Institutes of Health (NIH)/National Heart Lung Blood Institute Grant 1R15HL092517-01.

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