Plexiform Lesions in the Lungs of Domestic Fowl Selected for Susceptibility to Pulmonary Arterial Hypertension: Incidence and Histology

Authors


Abstract

Plexiform lesions develop in the pulmonary arteries of humans suffering from idiopathic pulmonary arterial hypertension (IPAH). Plexogenic arteriopathy rarely develops in existing animal models of IPAH. In this study, plexiform lesions developed in the lungs of rapidly growing meat-type chickens (broiler chickens) that had been genetically selected for susceptibility to IPAH. Plexiform lesions developed spontaneously in: 42% of females and 40% of males; 35% of right lungs, and 45% of left lungs; and, at 8, 12, 16, 20, 24, and 52 weeks of age the plexiform lesion incidences averaged 52%, 50%, 51%, 40%, 36%, and 22%, respectively. Plexiform lesions formed distal to branch points in muscular interparabronchial pulmonary arteries exhibiting intimal proliferation. Perivascular mononuclear cell infiltrates consistently surrounded the affected arteries. Proliferating intimal cells fully or partially occluded the arterial lumen adjacent to plexiform lesions. Broilers reared in clean stainless steel cages exhibited a 50% lesion incidence that did not differ from the 64% incidence in flock mates grown on dusty floor litter. Microparticles (30 μm diameter) were injected to determine if physical occlusion and focal inflammation within distal pulmonary arteries might initiate plexiform lesion development. Three months postinjection no plexiform lesions were observed in the vicinity of persisting microparticles. Broiler chickens selected for innate susceptibility to IPAH represent a new animal model for investigating the mechanisms responsible for spontaneous plexogenic arteriopathy. Anat Rec, 2011. © 2011 Wiley-Liss, Inc.

INTRODUCTION

Idiopathic pulmonary arterial hypertension (IPAH) is a disease of unknown cause characterized by elevated pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) in the absence of other apparent disease. Human patients with IPAH are partitioned into “sporadic” or “familial” categories based on genealogical evidence and the presence of key mutations in the bone morphogenetic protein receptor Type II (BMPR2) gene (Wagenvoort and Wagenvoort,1970; Loyd and Newman,1997; Pietra,1997; Rubin,1997; Voelkel et al.,1997; Lee et al.,1998; McLaughlin,2002; Newman et al.,2004; Simonneau et al.,2004; Morse,2005). In very young patients, increases in PVR and PAP appear to be correlated with vasoconstriction and medial hypertrophy of small pre- and intra-acinar pulmonary arteries. Medial hypertrophy encompasses hypertrophy and hyperplasia of the smooth muscle fibers of muscular arteries, distal extension of smooth muscle into nonmuscularized intra-acinar arteries, and increased densities of elastin and collagen fibers within the tunica media (Heath and Edwards,1958; Wagenvoort and Wagenvoort,1970; Pietra,1997; Voelkel et al.,1997; Yi et al.,2000; Pietra et al.,2004). Medial hypertrophy often is accompanied by intimal thickening attributable to cellular intimal proliferation (endothelial proliferation) followed by the accumulation of one or more layers of myofibroblasts and fibrous matrix proteins within the neointimal space between the endothelium and the internal elastic lamina (Palevsky et al.,1989; Yi et al.,2000; Pietra et al.,2004). Pathological evaluations traditionally indicated that early pulmonary vascular remodeling progressively evolves into the obstructive vascular pathology observed in patients confirmed to have advanced IPAH. Complex obstructive vascular lesions include concentric laminar intimal proliferation and glomeruloid-like plexiform lesions that develop within the lumen and physically occlude multiple arterial branches, elevating the PVR and rendering the lungs hypo-responsive to pharmacologic interventions designed to alleviate inappropriate vasoconstriction (Naeye and Vennart,1959; Wagenvoort and Wagenvoort,1970; Yamaki and Wagenvoort,1985; Barst,1986; Palevsky et al.,1989; Tuder et al.,1994; Voelkel et al.,1997; Archer and Rich,2000; Pietra et al.,2004). Plexiform lesions form immediately downstream from branching points in small muscular pre- and intra-acinar arteries. The arterial wall near a plexiform lesion can exhibit medial hypertrophy, aneurismal dilatation, or deterioration with extension of the lesion into the perivascular tissue, but upstream from the branch point the obstructed artery may appear to be normal (Heath and Edwards,1958; Naeye and Vennart,1959; Cool et al.,1999; Pietra et al.,2004). In patients with large left to right shunts caused by congenital heart disease, plexiform lesions occur most frequently in supernumerary arterioles branching laterally from large conducting arteries in the lungs (Yaginuma et al.,1990). Arteriole bifurcation sites are thought to promote the development of complex vascular lesions as a consequence of localized turbulent blood flow and shear stress or as a result of a zonal transition from distinctive macro-vascular to micro-vascular cell phenotypes (Mecham et al.,1987; Frid et al.,1997; Kelly et al.,1998; Stevens et al.,2001; Aird,2003; Archer et al.,2004; Cool et al.,2005; Stevens,2005). When present, concentric laminar lesions tend to occur immediately upstream from a plexiform lesion and may represent a more advanced, consolidated stage of plexogenic arteriopathy (Cool et al.,1997,1999; Tuder et al.,2001).

Plexiform lesions have been described as dynamic angiogenic lesions driven by disordered or neoplastic-like endothelial proliferation and myofibroblast infiltration (Lee et al.,1998; Cool et al.,1999; Yi et al.,2000; Berger et al.,2001; Tuder et al.,2001). Endothelial cells in most but not all of the plexiform lesions from patients with IPAH and anorexigen-triggered PAH tend to be monoclonal rather than polyclonal in origin, supporting the concept that somatic gene mutations promote proliferation of the altered cells (Lee et al.,1998; Tuder et al.,1998b). Dysregulated, apoptosis-resistant endothelial cells are thought to proliferate until the lumen is obstructed. Slit-like anastomosing endothelial channels supported by connective tissue and myofibroblasts canalize the plexiform obstruction. Newly formed endothelial channels circumvent the obstruction by penetrating through the arterial wall into the perivascular connective tissue (Tuder et al.,1994,2001; Cool et al.,1997,1999; Pietra,1997; Pietra et al.,2004; Stevens,2005). Thrombi and platelet aggregates commonly are found within established plexiform lesions, an anticipated response to endothelial changes that create thrombogenic surfaces (Heath and Edwards,1958; Voelkel et al.,1997; Farber and Loscalzo,1999; Archer and Rich,2000; Yi et al.,2000; Humbert et al.,2004; Hoeper et al.,2006). Inflammatory cells consisting of macrophages, T and B cells (mononuclear cells) infiltrate the perivascular region of affected pulmonary arterioles and plexiform lesions. Identification of these perivascular mononuclear cell infiltrates supported the concept that inflammatory responses may play an important role in the etiology of PAH and plexogenic arteriopathy (Tuder et al.,1994; Voelkel et al.,1997; Nicolls et al.,2005). Dorfmüller et al. (2002) confirmed the involvement of mononuclear cells in the vascular inflammation associated with PAH attributable to a variety of etiologies and subsequently concluded that inflammatory mechanisms can play an important role in the pathogenesis or progression of PAH as well as the development of complex vascular lesions (Dorfmüller et al.,2003).

Attempts to better understand the pathogenesis of obstructive vascular pathology in humans with severe IPAH have been hampered by a dearth of animal models in which plexiform lesions develop spontaneously. Chickens bred for rapid growth and meat production (broiler chickens, broilers) provide an excellent model of spontaneous IPAH. The pulmonary vasculature of broilers is minimally compliant and fully engorged with blood at a normal (resting) cardiac output, unlike the situation in mammals in which the pulmonary vasculature is compliant and recruitable when cardiac output increases (Powell et al.,1985; Peacock et al.,1989; Wideman and Kirby,1995; Wideman et al.,1996a,b; Wideman,2000). Rapid growth in broilers incurs corresponding increases in cardiac output (Wideman,1999), which must be propelled through lungs that remain essentially isovolumetric throughout the respiratory cycle (Jones et al.,1985), and that are constrained in volume by the dimensions of the dorsal thoracic rib cage. Accordingly lung volume and pulmonary vascular capacity are poorly correlated with body mass, creating an incipient pulmonary hemodynamic insufficiency that continues to be further exacerbated by ongoing genetic selection for very rapid muscle accretion and thus increased metabolic oxygen demand (Julian,1989; Peacock et al.,1989,1990; Wideman,1999; Owen et al.,1995a,b; Silversides et al.,1997). Susceptible individuals having the most limited pulmonary vascular capacity spontaneously develop IPAH leading to terminal right-sided congestive heart failure and ascites when the right ventricle is forced to develop an excessively elevated PAP to propel the cardiac output through the lungs. Approximately 3% of all broiler chickens develop IPAH when reared under conditions that promote rapid growth. Incidences of IPAH exceeding 20% have been exposed in broilers subjected to conditions that further increase the cardiac output or PVR, including exposure to sub-thermoneutral temperatures, hypobaric hypoxia, poor air quality, respiratory disease, or partial occlusion of the pulmonary vasculature (Wideman,2000,2001; Wideman et al.,2007). Broiler chickens that otherwise appear to be clinically healthy can be demonstrated, by pulmonary arterial catheterization, to have an elevated PAP that precedes characteristic work hypertrophy of the right ventricle (Wideman et al.,2006). Wedge pressure measurements confirm the precapillary vasculature as the primary source of excessive resistance to blood flow (Chapman and Wideman,2001; Lorenzoni et al.,2008). Broilers with IPAH consistently exhibit medial hypertrophy within the 25 to 100 μm diameter inter-parabronchial pulmonary arteries that are homologous to the preacinar arteries of mammals (Cueva et al.,1974; Hernandez,1987; Peacock et al.,1989; Sillau and Montalvo,1982; Maxwell,1991; Enkvetchakul et al.,1995; Xiang et al.,2002,2004; Moreno de Sandino and Hernandez,2003,2006; Pan et al.,2005; Tan et al.,2005a,b). Intimal proliferation has been observed within the muscularized pulmonary arteries of broilers developing IPAH (Xiang et al.,2002; Tan et al.,2005a,b), but complex concentric laminar lesions and plexiform lesions have not been reported previously.

In this study, we evaluated the spontaneous development of complex vascular lesions in the lungs of broilers from a line selected for enhanced susceptibility to IPAH (Anthony et al.,2001; Balog,2003; Pavlidis et al.,2007). After the 8th generation of selection, 98.6% of the progeny from this susceptible line succumbed to IPAH when exposed to sustained hypobaric hypoxia (Pavlidis et al.,2003). Progeny from the susceptible line also exhibited enhanced susceptibility to IPAH during chronic exposure to cool temperatures or after microparticles were injected i.v. to occlude the pulmonary arterioles (Wideman et al.,2002; Chapman and Wideman,2006b). The PVR and PAP in clinically healthy broilers from the susceptible line are significantly higher than the respective values in clinically healthy resistant individuals, whereas wedge pressures and cardiac output do not differ (Wideman et al.,2002,2007; Bowen et al.,2006a,b; Chapman and Wideman,2006b; Lorenzoni et al.,2008). Attempts to correlate susceptibility with mutations in BMPR2 were unsuccessful. Fourteen single nucleotide polymorphisms were identified in broiler BMPR2 mRNA, however no mutations unique to IPAH susceptibility were present nor were differences in BMPR2 mRNA expression levels detected between susceptible and resistant individuals (Cisar et al.,2003a,b). It was our hypothesis that the sustained pulmonary hypertension and elevated hemodynamic shear stress should, over time, initiate the development of plexiform lesions in the pulmonary arteries of fast growing IPAH-susceptible broilers. Our primary objective was to survey the lungs of 8- to 52-week-old males and females to estimate the relative age- and gender-specific incidences of plexiform lesion development. We also evaluated the potential impact of environmental conditions by comparing lesion formation in the lungs of broilers grown relatively slowly in clean stainless steel cages versus in broilers grown more rapidly on the floor in a dusty environment created by wood shavings litter. Previous studies provided evidence that pulmonary function deteriorated in broilers reared on floor litter when compared with hatch mates reared in stainless steel cages (Bottje et al.,1998; Wang et al.,2002). Finally, we evaluated the impact of microparticle injections on the incidence of plexiform lesions in the lungs of susceptible broilers. Microparticles entrapped in the pulmonary arteries increase the PVR and PAP, and they also trigger a marked focal pulmonary inflammatory response that includes the perivascular infiltration of mononuclear cells in combination with luminal accumulations of thrombocytes (the nucleated avian homolog of platelets) and macrophages (Wideman and Erf,2002, Wideman et al.,2002,2003,2005,2006; Wang et al.,2003; Hamal et al.,2008). Anatomical descriptions and nomenclature for the lungs, airways and pulmonary vasculature of domestic fowl have been provided previously by Duncker (1972), Abdalla and King (1975), King et al. (1978), King (1979), and Maina (1988).

MATERIALS AND METHODS

Animal Source and Management

Animal procedures were approved by the University of Arkansas Institutional Animal Care and Use Committee (Protocol #08036). Male and female chicks from three separate hatches of the IPAH-susceptible broiler line were reared in environmental chambers (dimensions: 3.7 m long × 2.5 m wide × 2.5 m high) within the Poultry Environmental Research Laboratory. Single-pass ventilation was maintained at a constant rate of 6 m3 per minute per chamber. The photoperiod was set for 23 hr light:1 hr dark for the first 4 days, and 16 hr light:8 hr dark thereafter. Thermoneutral temperatures were maintained throughout: 32°C for days 1 to 3, 31°C for days 4 to 6, 29°C for days 5 to10, 26°C for days 11 to 14, and 24°C thereafter. Chicks initially were placed on the floor on wood shavings litter at a density of 930 cm2/bird, and by 6 weeks of age the bird density had been reduced to >2,000 cm2/bird. A corn and soybean meal-based feed formulated to meet or exceed the minimum National Research Council (1994) standards for all ingredients (22.7% crude protein, 3,059 kcal ME/kg, 1.5% arginine, and 1.43% lysine) was provided ad libitum until the birds reached 15 weeks of age, after which all birds were subjected to complete feed withdrawal on 1 out of every 3 days to attenuate the development of lameness attributable to obesity. Water was available ad libitum via nipple watering systems throughout the study.

Age and Gender Comparisons of Complex Vascular Lesion Incidences

At 8, 12, 16, 20. and 24 weeks of age, at least 12 randomly selected birds of each gender were anesthetized to a surgical plane with intra-muscular injections of allobarbitol (5,5-diallylbarbituric acid, 3.0 mL, 25 mg/mL) and ketamine HCl (1.0 to 2.5 mL of 100 mg/mL). Heparinized saline (1 mL per bird of 200 units/mL ammonium heparin in 0.9% NaCl) was injected i.v. after which the birds were euthanized by exsanguination. The sternum was retracted to expose the heart, the right atrium was clamped with a hemostat, the left atrium was opened for drainage, and polyethylene tubing (2.5 mm I.D.) inserted via a slit in the right ventricle was used to flush the pulmonary vasculature with 200 mL of 0.9% NaCl at room temperature. The lungs then were fixed in situ by trans-cardiac perfusion with 200 to 400 mL of freshly prepared 4% phosphate buffered paraformaldehyde at room temperature. Carboys containing the 0.9% NaCl and the 4% paraformaldehyde were positioned at an elevation sufficient to obtain a gravimetric perfusion pressure of 46 cm H2O (34 mmHg) at the level of the heart. Susceptible broilers developing IPAH exhibit PAP values averaging 30 to 35 mmHg (Chapman and Wideman,2001; Wideman et al.,2006; Lorenzoni et al.,2008). The thoracic cavity was flooded with 4% paraformaldehyde and the lungs remained fixing in situ for a minimum of three hours, after which the lungs were removed and cut in the transverse plane at the major rib indentations (costal sulci) labeled 1 through 4 by Abdalla and King (1975). Each lung thus yielded four separate inter-rib divisions that we labeled A, B, C. and D along the anterior to posterior axis. These divisions were immersed overnight in 4% paraformaldehyde, after which they were rinsed briefly in tap water, dehydrated in 25% and 50% ETOH for 30 min each, and stored in 70% ETOH. Body weights were recorded and heart ventricles were dissected and weighed to calculate right-to-total ventricular weight ratios (RV:TV ratios) as an index of the right ventricular work hypertrophy that is characteristic of sustained IPAH (Cueva et al.,1974; Sillau and Montalvo,1982; Hernandez,1987; Huchzermeyer and DeRuyck,1986; Julian,1988; Peacock et al.,1989; Wideman,2000). At the end of the 2007 and 2008 breeding seasons, the lungs of >52 week-old hens and roosters from the susceptible line also were fixed in situ by trans-cardiac perfusion. The RV:TV ratios for these breeders were recorded but their body weights were not.

Lung Histology

Lungs from five individuals known to have IPAH were surveyed to assess the distributions of complex vascular lesions within the four inter-rib divisions. These birds included two males and three females between 16 and 23 weeks of age, with RV:TV ratios averaging 0.29 (indicative of significant right ventricular hypertrophy), and whose lungs exhibited perivascular mononuclear cell infiltrates, arterial muscularization, cellular intimal proliferation, and plexiform lesions. Inter-rib divisions A through D from both lungs were embedded in paraffin, sectioned at 5 to 7 μm thickness, and stained with hematoxylin and eosin (H&E). Sections from each division were searched using overlapping fields of view and sequential horizontal traverses at 40× total magnification. The locations of lesions were recorded on a template representative of a typical lung section, the lesions were photographed, and their coordinates were recorded using a Micro-Slide Field Finder (Gurley Precision Instruments, Troy, NY). Multiple plexiform lesions were observed in several of the lung sections, with a maximum of four observed in a single section from Division C. Plexiform lesions were equally likely to occur in left lungs (53% or 10/19) and right lungs (50% or 10/20) when compared using “plexiform incidences” calculated as the: (number of lung sections with at least 1 plexiform lesion)/(number of lung sections examined). Along the anterior to posterior axis, plexiform lesions were observed in 30%, 60%, 50%, and 67% of the sections from inter-rib divisions A through D, respectively. These observations indicated the lesions were equally likely to develop in the medial and posterior inter-rib divisions. Within the transverse plane of section regardless of inter-rib division most lesions were located in the ventral-medial portion of lung sections near the Facies septalis. Thereafter, slides stained with H&E were prepared from the medial inter-rib divisions (Divisions B or C) of both lungs. In a number of cases ineffective vascular perfusion or fixation prevented one lung from being evaluated. One slide per lung division was searched for plexiform lesions using overlapping fields of view and sequential horizontal traverses. The coordinates were recorded and lesion incidences were compared within and between age categories using a z-test (Jandel Scientific,1994). Lung sections were projected onto graph paper at 10× magnification, traced, and the tracings were cut out and weighed to the nearest 0.001 g to determine the area in cm2 of each section. Lung section areas were compared by gender and age using ANOVA with means separated at P< 0.05 using the Tukey test (Jandel Scientific,1994). “Plexiform densities” were estimated for each gender and age category as: (number of lesions per section)/(cm2 per section).

Microparticle Injections

In this study, 24 female broilers from the IPAH susceptible line were injected with microparticles at 5 weeks of age. Micro-granular CM-32 ion exchange cellulose (Fisher Scientific, St. Louis, MO) was suspended at 0.02 g/mL in heparinized saline (150 units/mL of ammonium heparin in 0.9% NaCl). This suspension was vortexed continuously on a magnetic stirring plate to keep the microparticles evenly distributed and was injected via a wing vein at the LD50 dose of 0.4 to 0.5 mL/bird using a 1-mL tuberculin syringe attached to a 22-gauge needle (Becton Dickinson, Franklin Lake, NJ). When the survivors of this procedure reached 17 weeks of age (12 weeks postinjection) their lungs were fixed in situ by trans-cardiac perfusion.

Cage vs. Floor Evaluations

Newly hatched female broiler chicks were housed in environmental chambers, either on wood shavings litter in floor pens (Floor group) or in stainless steel cages (Cage group). All chicks were brooded at thermoneutral temperatures for the first 3 weeks. Thereafter for the Floor group the temperature was reduced to 16°C (sub-thermoneutral), lights were on for 18 hr/day, and feed was continuously available. For the Cage group the temperatures were maintained within the age-appropriate thermoneutral range, lights were on for 8 hr/day, feed was withdrawn for 1 out of every 3 days, and cage pans were cleaned every 3 to 4 days. The intent was to cause birds in the Floor group to grow as rapidly as possible (full feed, long photoperiod) under an increased metabolic challenge (cool temperatures) and comparatively dirty environmental conditions (floor litter and associated aerosolized dust and accumulated droppings). In contrast, the birds in cages were induced to grow more slowly (short photoperiod, periodic feed withdrawal) under clean environmental conditions. Broilers grown in cages also exercise substantially less than those grown on the floor. When these birds reached 6 to 7 weeks of age the lungs of 14 birds from each group were fixed in situ by trans-cardiac perfusion.

RESULTS

Lung Histology

Plexiform lesions developed in regions of the lungs where perivascular mononuclear cell infiltrates surrounded interparabronchial arteries at their branch points from parent arteries (Fig. 1). Perivascular mononuclear cell infiltrates often occurred in the absence of notable inflammation within the adjacent gas exchange parenchyma or airways (Figs. 1, 2). Affected arteries were ringed by accumulations of macrophages and lymphocytes, frequently accompanied by dispersed heterophils, the avian homolog of neutrophils (Fig. 2b). Vasculitis occasionally was evident, with macrophages and heterophils penetrating the adventitial and medial layers of terminal arteries (not shown). Plexiform lesions also were likely to develop in portions of the lung where muscularized pulmonary arteries exhibited cellular intimal proliferation (Fig. 2). A tangential section through an artery with mild perivascular inflammation accompanied by a localized region of cellular intimal proliferation is shown in Fig. 2a. The affected segment includes mononuclear cells and heterophils within the lumen as well as in the adventitial and parenchymal tissues (Fig. 2b). Longitudinal sections demonstrated that the proliferating intimal cells, presumably endothelial cells, were elongated in parallel with the direction of blood flow (Fig. 2), whereas in cross sections the proliferating intimal cells appeared to project centripetally into the lumen. The vacuolated cytoplasm of proliferating intimal cells appeared distinctly blue in color when stained with H&E (Fig. 2).

Figure 1.

An interparabronchial arteriole (A) coursing between three parabronchi (P) branches into a terminal intraparabronchial arteriole (IA) that penetrates through the gas exchange parenchyma toward the lumen (L) of a parabronchus. Complex vascular lesions typically develop in lungs exhibiting perivascular mononuclear cell infiltrates at arteriole branch points (arrows). Note the absence of inflammation within the surrounding parenchyma.

Figure 2.

a: Section through an interparabronchial arteriole (A) coursing between two parabronchi (P) reveals a localized region of intimal proliferation (IP) within the arteriole lumen. Cellular intimal proliferation (IP) and medial hypertrophy are characteristic of lungs in which plexiform lesions develop. Arrows indicate perivascular mononuclear cell infiltrates. b: At higher magnification the zone of cellular intimal proliferation (IP) contains mononuclear cells (arrows) and heterophils (arrowheads) in the lumen and perivascular spaces. The elongated, vacuolated (presumably endothelial) cells within the IP segment are oriented parallel to the direction of blood flow.

Small, apparently early-stage, plexiform lesions forming within interparabronchial arteries typically consisted of an intimal cellular matrix and embedded macrophages, an indistinct smooth muscle layer, and extramural inflammatory cell infiltrates (Fig. 3). At this stage, affected arteries did not appear to be excessively dilated, the lumen appeared to be partially or fully occluded, and the cells occluding the lumen had a distinctively pink cytoplasm when stained with H&E. Medium sized plexiform lesions typically developed shortly downstream from the point where an interparabronchial artery branched from a larger parent artery (Figs. 4, 5). Mononuclear cell infiltrates typically were evident around the perimeter of the lesions and nearby arterial branches. Macrophages having a foam-type appearance tended to be arrayed adjacent to the remnants of the dilated arterial wall, surrounding a compact, fairly homogeneous core of intimal cells (Figs. 4, 5). The vascular wall frequently was indistinct and appeared to be comprised primarily of a single connective tissue lamina, although remnants of the vascular smooth muscle sometimes were evident. Sparse, slit-like vascular channels within the lesions generally appeared to be free of erythrocytes in well perfused lungs (Figs. 4, 5). In “maturing” plexiform lesions the main body had expanded beyond the circumference of a typical longitudinal interparabronchial artery, foam-type macrophages were established around the periphery of the main body, and remnants of smooth muscle in the arterial wall were scarce or absent (Figs. 5, 6). Mature plexiform lesions also were observed adjacent to large conducting arteries, presumably arising within supernumerary arterioles branching at right angles from the parent artery (not shown). Vasculitis with mural aggregates of heterophils occasionally was observed in vessels within or adjacent to plexiform lesions (not shown). Serial sections indicated the lesions were spherical or ovoid in shape and ∼120 to 200 μm in diameter. These lesions often were associated with a swath of intimal cells that engorged the lumen of the host artery both upstream (Fig. 5) and occasionally downstream (not shown) from the site of lesion formation. The plexiform lesion and its swath of intimal cells characteristically were associated with lymphoid aggregates and heterophils (Figs. 5, 7, 8). The glomeruloid-like microvascular penetration of mature lesions was best demonstrated when the blood was not fully cleared during perfusion fixation, revealing nucleated avian erythrocytes filling numerous vascular channels coursing among a mixture of cell types (Fig. 7). Foam-type macrophages appeared to reside within or adjacent to the microvascular vascular channels of mature plexiform lesions (Figs. 3, 4, 5, 6, 7, 8, 3–8).

Figure 3.

An arteriole with an occluded lumen penetrates the connective tissue septum separating three adjacent parabronchi. The affected arteriole is occluded by proliferating intimal cells (IP) and macrophages (Mϕ). The perivascular connective tissue exhibits mononuclear cell infiltration (arrow).

Figure 4.

This plexiform lesion developed in one of four arteriole branches coursing within the septum between three adjacent parabronchi. The lesion contains foam-type macrophages (Mϕ) adjacent to the remnant of the arteriole wall on the left and intimal proliferating cells (IP) adjacent to the arteriole smooth muscle (SM) on the right.

Figure 5.

a,b,c: Serial sections through a plexiform lesion (PL). The lesion contains foam-like macrophages within a matrix of intimal proliferating cells. A swath of intimal proliferating cells (IP) courses between a terminal intraparabronchial arteriole (arrowhead) and the dilated body of the PL. The intraparabronchial arteriole arises from an interparabronchial arteriole (A) that parallels a venule (V) coursing between two parabronchi. Perivascular mononuclear cell infiltrates (arrows) surround arteriole branch points as well as the body of the lesion. d: Higher magnification from section shown in 5b showing the junction of the IP swath with the intraparabronchial arteriole. A cuff of perivascular mononuclear cells rings the terminal arteriole branches, and heterophils (arrowheads) typically are observed in the vicinity of the lesion and affected arterioles.

Figure 6.

This plexiform lesion exhibits a central matrix of intimal proliferating cells with multiple foam-type macrophages arrayed next to the indistinct remnants of the arteriole's smooth muscle wall (SM).

Figure 7.

The glomeruloid-like structure of this “mature” plexiform lesion is indicated by the multiple vascular channels (c) containing nucleated avian erythrocytes. The matrix is composed of intimal proliferating cells (IP) and foam-type macrophages (Mϕ) that appear to reside within vascular channels.

Figure 8.

This foam-type macrophage is located adjacent to the wall of a blood vessel (SM) at the periphery of a plexiform lesion. At high magnification foam-type macrophages can appear to contain multiple nuclei and exhibit a vacuolated appearance. Heterophils (arrowheads) typically are observed in the vicinity of lesions. Nucleated avian erythrocytes also are evident.

Age and Gender Comparisons

A preliminary survey of inter-rib divisions A through D in five broilers with IPAH indicated that plexiform lesions are equally likely to develop in left and right lungs, and in divisions B, C, or D of either lung. In all subsequent studies, sections were evaluated from inter-rib divisions B or C of both lungs. Table 1 shows the numbers of female and male broilers evaluated within each age category. Sufficient numbers of birds were available from hatches two and three to obtain duplicate groups at 24 weeks of age. These duplicate groups were obtained from the same breeder parents but were hatched 25 days apart and were raised to 24 weeks of age in separate environmental chambers. Also shown by gender and age are the plexiform incidences (number of lung sections with at least 1 plexiform lesion/number of lung sections examined), lung section area, plexiform densities (number of lesions per section)/(cm2 per section), body weights, RV:TV ratios, and the relative ventricle weights (Table 1). Figure 9 illustrates the plexiform incidences pooled within each age group by right or left lung independent of gender; by gender independent of lung; and, regardless of gender or lung (All). Within each age category the plexiform incidences did not differ for the lung or gender comparisons (P> 0.10, z-test). Across age categories the incidences averaged approximately 50% at 8, 12, and 16 weeks, declined numerically to an average of 38% at 20 and 24 weeks, and declined significantly to 22% by 52 weeks of age (Table 1, Fig. 9). The lesions in 8-week-old birds generally appeared to be smaller and less extensively developed (e.g., Figs. 3, 4) than those in >16-week-old birds (e.g., Figs. 5, 6, 7, 5–7). Two or more plexiform lesions (up to a maximum of four) were observed per lung section from 28 females and 30 males through 24 weeks of age. Thereafter, none of the lung sections from the 52 week old breeders had more than one plexiform lesion, and few regions of the lungs of these older birds exhibited perivascular mononuclear cell infiltrates, arterial muscularization or cellular intimal proliferation. Age-related decreases in plexiform incidences occurred in spite of the contemporaneous trend for lung section areas to increase as the birds matured. Consequently, when compared with 8 and 12 week old birds within a gender, 52-week-old breeders had lower plexiform incidences combined with larger lung section areas thereby yielding lower plexiform densities (Table 1). Within the transverse plane of section most complex vascular lesions in birds of all ages were distributed in the ventral-medial portion of the lung within approximately 5 mm of the Facies septalis.

Figure 9.

The plexiform lesion incidence (number of lung sections with at least 1 lesion/number of lung sections examined) observed in 8- to 52-week-old broilers from an IPAH-susceptible genetic line, comparing incidences pooled by lung independent of gender (R vs. L lung), by gender independent of lung (Female vs. Male), or by both gender and lung (All). Sufficient numbers of birds were available from independent hatches 2 and 3 to obtain duplicate groups at 24 weeks of age (H2 and H3, respectively). None of the incidences differed within an age group (P > 0.10). Across age groups values with different superscripts (a,b) differed significantly (P < 0.05, z-test).

Table 1. Plexiform lesion incidences and densities in the lungs of male and female broilers between 8 and 52 weeks of age
VariableSex8 Weeks12 Weeks16 Weeks20 Weeks24 Weeks (hatch 2)24 Weeks (hatch 3)52 Weeks (old breeders)
  1. Across all age groups values with different superscripts (a-e) differed significantly (P < 0.05).

Number of birdsF16121718152115
M16121916162216
Plexiform incidence (Lungs w lesions/Lungs evaluated)F0.55 (16/29)0.52 (11/21)0.59 (17/29)0.32 (7/22)0.37 (10/27)0.40 (17/43)0.20 (5/25)
M0.48 (13/27)0.48 (10/21)0.44 (14/32)0.48 (11/23)0.34 (10/29)0.36 (14/39)0.24 (6/25)
Lung section area (cm2)F3.19 ± 0.13d3.58 ± 0.15d3.88 ± 0.16c,d3.64 ± 0.11d3.88 ± 0.15c,d3.86 ± 0.11c,d4.39 ± 0.09c
M3.62 ± 0.13d4.34 ± 0.14b,c4.59 ± 0.12b,c4.73 ± 0.11b,c5.30 ± 0.14a,b4.98 ± 0.13b5.61 ± 0.09a
Plexiform density (number of lesions/cm2)F0.210.170.310.090.150.180.05
M0.220.160.160.130.100.110.04
Body weight (g)F2,500 ± 78e3,442 ± 105d4,352 ± 117c4,565 ± 90c5,009 ± 141b,c4,803 ± 156b,cn/a
M2,755 ± 179d,e4,205 ± 236c,d4,711 ± 188b,c5,288 ± 158b6,163 ± 168a6,140 ± 188an/a
Right:total ventricle Wt. (RV:TV ratio)F0.245 ± 0.01a0.221 ± 0.01a,b0.228 ± 0.01a,b0.226 ± 0.01a,b0.230 ± 0.01a,b0.246 ± 0.01a,b0.206 ± 0.01b
M0.271 ± 0.02a0.238 ± 0.02a,b0.227 ± 0.01a,b0.238 ± 0.01a,b0.249 ± 0.01a,b0.253 ± 0.01a0.200 ± 0.01b
Relative right ventricle (RV, g/BW, g)F0.0008 ± 0.00010.0006 ± 0.00010.0006 ± 0.00010.0006 ± 0.00010.0005 ± 0.00010.0006 ± 0.0001n/a
M0.0011 ± 0.00010.0008 ± 0.00010.0007 ± 0.00010.0008 ± 0.00010.0008 ± 0.00010.0008 ± 0.0001n/a
Relative left ventricle (LV ± S, g/BW, g)F0.0025 ± 0.00010.0020 ± 0.00010.0020 ± 0.00010.0020 ± 0.00010.0018 ± 0.00010.0018 ± 0.0001n/a
M0.0028 ± 0.00010.0026 ± 0.00010.0025 ± 0.00010.0025 ± 0.00010.0024 ± 0.00010.0023 ± 0.0001n/a
Relative total ventricle (TV, g/BW, g)F0.0033 ± 000010.0026 ± 0.00010.0026 ± 0.00010.0025 ± 0.00010.0023 ± 0.00010.0024 ± 0.0001n/a
M0.0040 ± 0.00020.0034 ± 0.00020.0032 ± 0.00010.0033 ± 0.00010.0032 ± 0.00010.0031 ± 0.0001n/a

The feed restriction imposed on all birds by 15 weeks of age prevented the females from gaining significantly more body weight after week 16, whereas the highest body weights for males were recorded at 24 weeks of age (Table 1). The mean RV:TV ratios did not differ when compared between age and gender categories through 24 weeks of age (P = 0.132), but the RV:TV ratios in 52 week old males and females were significantly lower than the ratios at 8 weeks of age (P = 0.002). Average RV:TV values consistently were below the ratio of 0.28, which is the acknowledged threshold for sustained IPAH in broiler chickens. Relative right, left and total ventricle weights were similarly consistent within each gender across the age categories (Table 1). However the RV:TV ratios for 37 individuals did reach or exceed the 0.28 threshold (Fig. 10). By 52 weeks of age the downward trend in RV:TV ratios (Fig. 10) paralleled the downward trend in plexiform incidences and densities in both genders (Fig. 9, Table 1). Correlations between RV:TV ratios and plexiform lesion incidences or densities were not obtained for each of the age- and gender-groups because in the majority of individuals the lesion incidence was zero or one per lung. In addition, those rare individuals having 3 or 4 lesions per lung section did not necessarily also have the highest RV:TV ratios of the group.

Figure 10.

Right to total ventricular weight ratios (RV:TV) for 8- to 52-week-old female (F) and male (M) broilers from an IPAH-susceptible genetic line. Sufficient numbers of birds were available from independent hatches 2 and 3 to obtain duplicate groups at 24 weeks of age (H2 and H3, respectively). RV:TV ratios > 0.28 (dashed line) are indicative of sustained pulmonary arterial hypertension.

Microparticle Injections

Twenty-four female broilers from the PAH-Susceptible line were injected with an LD50 dose of microparticles at 5 weeks of age. Eleven of the 24 injected broilers survived to 17 weeks of age, when their lungs were fixed for histological examination. Plexiform lesions were found in 27% of the lung sections from microparticle-injected survivors, which is lower (P = 0.047) than the 59% lesion incidence in lung sections from the noninjected 16-week-old female flock mates (Table 1). There was no evidence of complex vascular lesion development at sites where microparticles remained entrapped for 3 months. Instead, the persisting microparticles appeared to have been marginalized and continued to serve as a focus for mononuclear cells and heterophils (Fig. 11).

Figure 11.

Three months postinjection microparticles (mp) that initially were trapped in a branch of an interparabronchial arteriole (A) continue to serve as a focus for perivascular inflammatory cell infiltrates (arrows) including heterophils (arrowheads) although plexiform lesions did not develop at these foci of arteriole obstruction and inflammation.

Cage vs. Floor Evaluations

This study was conducted to assess the potential impact of environmental conditions on the incidence of plexiform lesions in broiler lungs. Between 6 and 7 weeks of age the lungs of 14 birds from each group were fixed for histology. Birds in the Cage group had a significantly lower body weight than those in the Floor group (2,095 + 56 g vs. 2,890 + 54g, respectively). In the Cage group, 14 out of 28 (50%) of the lungs examined had one or more plexiform lesion and in 4 individuals neither of the lungs exhibited lesions. In the Floor group, 18 out of 28 (64%) of the lungs examined had one or more plexiform lesion, and in one individual neither of the lungs exhibited lesions. In both groups, seven of the lungs exhibited two to four lesions per section. The lesion incidences for the Cage and Floor groups at 6 to 7 weeks of age (50% and 64%, respectively) did not differ significantly from one another, nor did they differ from the 55% incidence for 8-week-old female broilers in the primary study (Table 1, Fig. 9).

DISCUSSION

Our understanding of the pathogenesis of complex vascular lesion development is incomplete due to a dearth of animal models of spontaneous plexogenic pulmonary arteriopathy (Stelzner et al.,1992; Rabinovitch,1997; Rich,1998; Zabka et al.,2006). Laboratory mammals exposed to chronic hypoxia, monocrotaline toxin, pulmonary emboli, pulmonary arterial banding, or serotonergic appetite suppressant drugs can develop PAH accompanied by medial hypertrophy and intimal proliferation in their pulmonary arteries, however these experimental challenges do not induce the formation of plexiform lesions (Saldana et al.,1968; Gurtner,1985; Tanaka et al.,1996; Zamora et al.,1996; Rabinovitch,1997; Botney,1999; Gust and Schuster,2001; Taraseviciene-Stewart et al.,2001,2006; Medhora et al.,2002; Nishimura et al.,2002,2003; Vaszar et al.,2004; Albada et al.,2005; Ivy et al.,2005). The Fawn-hooded rat (FHR) spontaneously develops PAH and extensive medial hypertrophy, but intimal proliferation and complex vascular lesions have not been reported in the FHR (Kentera et al.,1988; Sato et al.,1992; Le Cras et al.,1999; Nagaoka et al.,2006). Plexiform lesions were detected in a retrospective study of tissues archived from six dogs confirmed to have severe IPAH. The lungs of four dogs contained plexiform lesions that were regionally clustered downstream from branching points of pulmonary arterioles. Concentric laminar lesions were situated immediately upstream from the plexiform lesions and no dog had a plexiform lesion independent of medial hypertrophy and neointimal thickening (Zabka et al.,2006). Plexiform lesions also developed in dogs and sheep within 2 to 4 months after severe PAH was initiated by creating hyperdynamic systemic-to-pulmonary artery shunts in the superior lobe of one lung (Saldana et al.,1968; Schnader et al.,1996). Human plexiform lesions and concentric laminar lesions generally are believed to arise from foci of proliferating endothelial cells. Blood propelled at elevated pressure through branch points in pulmonary arteries presumably creates turbulent flow and shear stress that can damage the normally quiescent endothelium. The resulting apoptosis of normal endothelial cells is thought to permit clones of apoptosis-resistant endothelial cells to proliferate, leading to the progressive formation of plexiform lesions (Tuder et al.,1994; Lee et al.,1998; Botney,1999; Taraseviciene-Stewart et al.,2001; Budhiraja et al.,2004; Humbert et al.,2004; Cool et al.,2005; Sakao et al.,2005). The available evidence suggests that plexiform lesions fail to develop in laboratory mammals because the PAP and hemodynamic shear stress are insufficiently elevated or the animals succumb too rapidly to severe PAH (Rabinovitch,1997; Albada et al.,2005).

Rapid growth forces the right ventricle of IPAH-susceptible broilers to propel the cardiac output at excessively high pressures and flow rates through a functionally noncompliant pulmonary vascular bed that maintains an elevated precapillary resistance. Blood flows so rapidly through the gas exchange capillaries that the ensuing diffusion limitation triggers systemic arterial hypoxemia (cyanosis), a consistent symptom in broilers developing IPAH (Peacock et al.,1990; Wideman et al.,1996a,2007; Wideman,2000,2001). It was our hypothesis that sustained pulmonary hypertension and elevated hemodynamic shear stress should, over time, initiate the development of plexiform lesions in the lungs of birds from the IPAH-susceptible line. Indeed, small presumably “early” plexiform lesions developed in regions of the lungs where many of the arterioles exhibited perivascular mononuclear cell infiltration, medial hypertrophy, and cellular intimal proliferation. Medial hypertrophy and cellular intimal proliferation are considered early histological features of hypertensive pulmonary vascular disease in humans (Heath and Edwards,1958; Wagenvoort et al.,1984; Heath et al.,1988). Our observations suggest a maturational process occurred through which compact cellular lesions having a relatively homogeneous matrix and sparse vascular channels (Figs. 3, 4) transitioned into larger “mature” plexiform lesions exhibiting numerous slit-like vascular channels and multiple cell types including connective tissue, inflammatory cells, and proliferating intimal cells (Figs. 5, 6, 7, 5–7). Proliferating intimal cells extended into the lumen of the host artery, but neither cellular nor acellular concentric laminar fibrosis was observed in broilers (Fig. 5). Human and canine plexiform lesions appear to follow a similar dynamic pattern of maturation, with cellular intimal proliferation and compact cellular lesions preceding the formation of concentric laminar intimal proliferation and mature, increasingly fibrotic plexiform lesions (Heath and Edwards,1958; Heath et al.,1988; Caslin et al.,1990; Tuder et al.,1994,1998a,b; Fishman,2000; Yi et al.,2000; Zabka et al.,2006). It remains to be determined if the proliferating intimal cells of broilers are endothelial or myofibroblast in origin. Ultrastructural and immunohistochemical evidence initially indicated that human plexiform lesions develop from myofibroblasts migrating from the media into the intima and lumen of pulmonary arteries (Heath et al.,1987,1988; Yi et al.,2000), whereas subsequent studies implicated hyper-proliferating endothelial cells as the principal cellular component at the core of the lesion (Tuder et al.,1994; Lee et al.,1998; Cool et al.,1999; Richter et al.,2004). The arterial wall surrounding larger lesions in broilers was intermittent or indistinct in structure, which likely can be attributed to dilation and deterioration associated with the activity of foam-type macrophages arrayed along the inner surfaces of the arterial wall. The parent vessel wall surrounding mature plexiform lesions in canine lungs has been described as being dilated, degenerative and undergoing remnant fibrinoid necrosis, with the lesion extending into the perivascular tissue (Zabka et al.,2006). Plexiform lesions in humans were described as having dilated, fragile walls consisting of a single elastic lamina or an exceedingly thin layer of muscle between elastic lamina (Heath and Edwards,1958). In other cases, the hypertrophied arterial smooth muscle layer was described as forming an uncomplicated “sleeve” surrounding the distended lesion (Tuder et al.,1998a).

The frequency with which lesions are detected can vary widely among human patients known to have plexogenic pulmonary arteriopathy. In some cases, multiple biopsy specimens or blocks of lung tissue must be sectioned to find a solitary plexiform lesion, whereas in other cases multiple lesions are observed in every lung section (Wagenvoort,1980; Tuder et al.,1994; Jamison and Michel,1995; Mark et al.,1997). Lesion densities ranging from 0.1 to 11.7 per cm2 were reported for 77 cases of human plexogenic arteriopathy (Wagenvoort et al.,1970), and from 0 to 4.2 per cm2 in children and adults with primary plexogenic arteriopathy (Yamaki and Wagenvoort,1985). In humans with severe IPAH ongoing lesion development irreversibly obliterates small pulmonary arteries, pruning the pulmonary vasculature and fueling a positive feedback cycle in which the accumulating vascular obstruction is thought to progressively increase the PVR and further aggravate the existing pulmonary hemodynamic challenge (Naeye and Vennart,1959; Dammann et al.,1961; Voelkel et al.,1998). We anticipated a similar pathophysiological progression in IPAH-susceptible broilers, if they survived long enough for aggressive plexogenic pulmonary arteriopathy to evolve. Plexiform lesions were observed in approximately half of the lung sections from 6- to 7-week-old female broilers in the cage vs. floor experiment, as well as in 8-week-old males and females in the age- and gender-experiment. The average plexiform incidence of ∼50% persisted up to 16 weeks of age, with affected lung sections rarely exhibiting more than one lesion. Indeed, plexiform lesion densities averaged 0.20 per cm2 which is the lower range of lesion densities reported for humans (vide supra). This limited extent of vascular obstruction does not seem likely to have significantly increased the PVR. For example, when microparticles are injected in numbers sufficient to increase the PVR and PAP, an estimated 10% of the interparabronchial pulmonary arteries contain entrapped microparticles that are readily visible within virtually any field of view at 40× total magnification (Wideman and Erf,2002; Wideman et al.,2002; Wang et al.,2003). Accordingly, plexiform lesion development appears to be a consequence rather than the proximate cause of pulmonary hypertension in IPAH-susceptible broilers. Alternatively, factors other than pulmonary hypertension per se may trigger the development of plexiform lesions. Clearly plexiform lesions developed rapidly in relatively immature birds, during the exponential phase of growth when broilers are most likely to succumb to spontaneous IPAH (Wideman,2000,2001). Plexogenic arteriopathy also developed rapidly in susceptible humans who developed IPAH after consuming serotonergic appetite suppressant drugs for relatively brief intervals (Gurtner,1985; Mark et al.,1997).

After 16 weeks of age the plexiform lesion incidences and densities trended downward whereas lung section areas increased. By 52 weeks of age, only 22% of the lung sections exhibited plexiform lesions and lesion densities had declined to 0.05 per cm2. Age-related reductions in lesion densities may indicate that the most susceptible individuals succumbed preferentially during the course of the experiment. Undefined maturational changes may have attenuated the pulmonary vascular susceptibility to complex vascular lesion formation. It also is possible that lesion formation began to subside and existing lesions began to regress when feed restriction was imposed to prevent obesity, immobility and lameness. The reduction in the lesion incidence coincided with reductions in RV:TV ratios, with only one bird having an RV:TV ratio exceeding 0.28 by 52 weeks of age. Virtually any strategy that slows the growth rate of broilers also reduces the incidence of IPAH, presumably by moderating the mismatch between cardiac output and pulmonary vascular capacity (Fedde et al.,1998; Wideman,2000). Conclusive examples of “regressing” plexiform lesions were not identified in lung sections from 24- or 52-week-old broilers, although such examples may be difficult to detect in view of the relatively gradual age-dependent decline in the lesion incidence. Reversibility and regression of pulmonary vascular pathology has been documented in human patients subsequent to alleviating the hemodynamic challenges to their lungs. However, lesion regression typically applies to reversal of medial hypertrophy, cellular intimal proliferation and mild concentric laminar intimal fibrosis, usually after palliative treatment is administered to very young patients. Advanced concentric laminar fibrosis and mature plexiform lesions are rarely thought to regress and may even progress in severity despite treatment (Dammann et al.,1961; Rabinovitch et al.,1984; Wagenvoort et al.,1984; Palevsky,1989). One notable exception pertains to human patients who rapidly developed severe IPAH and plexogenic pulmonary arteriopathy while consuming serotonergic appetite suppressant drugs. Subsequently after cessation of drug use many of the same patients exhibited marked long term reversal of their IPAH that was presumed to be accompanied by reversal of their pulmonary vascular obstruction (Gurtner,1985).

Most of the plexiform lesions in broilers were distributed along the ventral-medial portion of the lung within ∼5 mm of the Facies septalis (King,1979). This region of the lung is gravitationally dependent but less than 4 cm lower than the dorsal aspect of the lung. Lesion distributions in humans tend to be uniform throughout both lungs except in cases of pulmonary venous hypertension or gross anatomical abnormalities of the vasculature (Wagenvoort,1980). Increased postcapillary resistance and pulmonary venous hypertension have been dismissed as factors that potentially might contribute to IPAH in susceptible broilers (Lorenzoni et al.,2008). The ventral-medial zone of the chicken lung is supplied by a primary branch of the pulmonary artery coursing medial to the intrapulmonary primary bronchus (Abdalla and King,1975). Plexiform lesions in humans are not found in arteries supplying the conducting airways, nor are plexiform lesions preferentially distributed near the conducting airways (Jamison and Michel,1995). The ventral-medial surface is tightly constrained by a thin membrane, the pulmonary aponeurosis, anchored by costoseptal (costopulmonary) skeletal muscles to the internal thoracic wall. These muscles contract during expiration to tighten the pulmonary aponeurosis, thereby maintaining patency of the ostea to the caudal air sacs and minimizing the tendency of these air sacs to compress the lung parenchyma during expiration (Jones et al.,1985). The ventral portions of domestic fowl lungs also contain neopulmonic parabronchi that accommodate bidirectional air flow originating from the intrapulmonary primary bronchus, whereas the dorsal portion contains paleopulmonic parabronchi that receive unidirectional air flow primarily from the caudal air sacs (Duncker,1972). It is possible that within the ventral-medial region of the lung parenchyma the shear stress exerted within terminal arteries may be cyclically affected by forces transmitted to the lung parenchyma during respiration. The factors responsible for the regional clustering of plexiform lesions near the ventral-medial surfaces of broiler lungs remain to be identified. Understanding the relevant contributing factors likely will provide valuable insight into the pathogenesis of lesion formation.

Two preliminary experiments were conducted in an attempt to experimentally influence the incidence of plexiform lesions. Comparisons of the lesion incidence for broilers reared in clean cages (50%) versus the incidence for flock mates grown on dusty floor litter (64%) failed to support a major influence of environmental quality on the development of plexiform lesions in young birds. Microparticles were injected at an LD50 dose to determine if physical occlusion in combination with the initiation of focal inflammation might trigger the development of plexiform lesions. Three months postinjection some microparticles remained entrapped in the lungs of the survivors, with lymphocytes and heterophils continuing to surround the outer boundary of the marginalized microparticles. However no plexiform lesions were observed in the vicinity of persisting microparticles. Instead these survivors exhibited a significant overall reduction in their lesion incidence (27%) when compared with incidence for age-matched noninjected flock mates (59%). Microparticles injections are used to eliminate IPAH-susceptible individuals from commercial genetic populations, based on evidence that the survivors of microparticles injections produce progeny that are innately resistant to IPAH (Wideman et al.,2007). Entrapped microparticles stimulate responding thrombocytes to release serotonin (5-hydroxytryptamine, 5-HT) (Chapman et al.,2008). Serotonin is an exceptionally potent pulmonary vasoconstrictor in broilers (Chapman and Wideman,2002). Pretreatment with the nonselective 5-HT1/2 receptor antagonist methiothepin virtually eliminated increases in PVR and PAP elicited by serotonin and by microparticle injections, and reduced by two thirds the mortality caused by injecting microparticles into IPAH-susceptible broilers (Chapman and Wideman,2006a,b). The results of the present study suggest the most susceptible individuals succumbed to the initial microparticle injection, with fewer plexiform lesions developing in the survivors. The possibility therefore exists that serotonin is involved in plexiform lesion development in IPAH-susceptible broilers. Serotonin clearly is involved in the pathogenesis of IPAH and plexogenic arteriopathy in humans (Hervé et al.,1995; Eddahibi et al.,2002; Marcos et al.,2004; Naeije and Eddahibi,2004; Eddahibi and Adnot,2005; Lawrie et al.,2005) as well as PAH and plexogenic arteriopathy triggered by serotonergic appetite suppressant drugs (Douglas et al.,1981; Gurtner,1985; Brenot et al.,1993a,b; Abenhaim et al.,1996; Louis,1999; Eddahibi and Adnot,2002; Naeije and Eddahibi,2004). For example, some appetite suppressant drug users developed PAH with histopathological, morphological, and clinical features considered indistinguishable from those in patients with familial IPAH, including medial hypertrophy, concentric laminar proliferation and the formation of plexiform lesions (Tuder et al., 1998). Endothelial cells from plexiform lesions also tend to be monoclonal in origin when obtained from patients with either anorexigen-triggered PAH or spontaneous IPAH, but polyclonal in origin when obtained from patients with secondary PAH (Lee et al.,1998; Tuder et al., 1998).

Plexiform lesions were most likely to be found in regions of broiler lungs where perivascular mononuclear cell infiltrates surrounded branch points of muscularized interparabronchial arteries. Abundant perivascular inflammation was readily apparent at low magnification, and was not categorized as vasculitis or arteritis because the adjacent vessel wall and its branches did not appear to be deteriorating or necrotic. Vasculitis was sporadically identified based on the presence of macrophages and heterophils in the adventitial and medial layers of smaller blood vessels within or adjacent to mature plexiform lesions. Large foam-type macrophages were consistently present around the periphery of maturing plexiform lesions, evidently participating in the degenerative changes affecting the wall of the host arteriole. Dense lymphocytic foci also routinely surrounded the extramural margins of plexiform lesions. Similar inflammatory processes have been associated with IPAH and plexogenic arteriopathy in humans and dogs. Elevated pulmonary arterial systolic pressures, shear stress and turbulent blood flow may cause pulmonary arterioles to release cytokines and chemokines that attract perivascular inflammatory cell infiltrates (Dorfmüller et al.,2002,2003; Nicolls et al.,2005). Perivascular inflammatory infiltrates including T cells, B cells and macrophages have been observed surrounding distal pulmonary arterioles in a variety of conditions associated with plexogenic arteriopathy (Caslin et al.,1990; Tuder et al.,1994; Chazova et al.,1995; Cool et al.,1997; Dorfmüller et al.,2002; Nicolls et al.,2005; Zabka et al.,2006). Lymphocytes and macrophages also have been detected as isolated cells or small clusters of cells within plexiform lesions (Tuder et al.,1994; Cool et al.,1997; Wright et al.,1998; Dorfmüller et al.,2002; Zabka et al.,2006). The immune response to distal arterial damage is believed to promote remodeling and repair of injured vascular channels. In contrast, necrotizing fibrosis (arteritis, vasculitis) destroys affected blood vessels, cumulatively leading to the structural pruning of the pulmonary arterial tree observed in severe terminal IPAH (Heath and Edwards,1958; Saldana et al.,1968; Heath et al.,1987; Mark et al.,1997). Fishman (2000) summarized evidence indicating necrotizing arteritis may precede and initiate plexogenic arteriopathy under conditions that promote vascular inflammation. Evidence supporting a connection between IPAH, vascular inflammation and immune dysregulation has accumulated from animal models of PAH as well as from observations that human patients with autoimmune disorders, collagen vascular diseases, HIV infection and other viral infections are predisposed to the onset of PAH and plexogenic arteriopathy (Wagenvoort and Wagenvoort,1970; Meyrick et al.,1987; Tuder et al.,1994; Voelkel et al.,1997; Barst and Loyd,1998; Dorfmüller et al.,2003; Nicolls et al.,2005). Severe IPAH may be both preceded and accompanied by pulmonary arterial inflammation (Voelkel et al.,1997), and it is our belief that the immune system plays an important role in the pathogenesis of plexiform lesion development in broilers. It remains to be determined if pressure- and shear stress-induced vascular injury and remodeling attract lymphocytes to areas of lesion formation, or if lymphocytes responding to injury contribute to changes in intimal and medial cells that promote the development of plexiform lesions (Dorfmüller et al.,2002,2003; Nicolls et al.,2005). The broiler chicken model of susceptibility to IPAH provides an important experimental model in which pulmonary vascular inflammation can be evaluated in conjunction with complex vascular lesion development.

Acknowledgements

MTB conducted a portion of this research for his undergraduate Honor's Thesis in Biological Sciences. The authors are grateful to Mallory Eanes and Amanda Wideman for assistance in data analysis and determining the area of the lung sections.

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