Idiopathic Pulmonary Arterial Hypertension: An Avian Model for Plexogenic Arteriopathy and Serotonergic Vasoconstriction

Abstract

Idiopathic pulmonary arterial hypertension (IPAH) is a disease of unknown cause that is characterized by elevated pulmonary arterial pressure and pulmonary vascular resistance attributable to vasoconstriction and vascular remodeling of small pulmonary arteries. Vascular remodeling includes hypertrophy and hyperplasia of smooth muscle (medial hypertrophy) accompanied in up to 80% of the cases by the formation of occlusive plexiform lesions (plexogenic arteriopathy). Patients tend to be unresponsive to vasodilator therapy and have a poor prognosis for survival when plexogenic arteriopathy progressively obstructs their pulmonary arteries. Research is needed to understand and treat plexogenic arteriopathy, but advances have been hindered by the absence of spontaneously developing lesions in existing laboratory animal models. Young domestic fowl bred for meat production (broiler chickens, broilers) spontaneously develop IPAH accompanied by semi-occlusive endothelial proliferation that progresses into fully developed plexiform lesions. Plexiform lesions develop in both females and male broilers, and lesion incidences (lung sections with lesions/lung sections examined) averaged approximately 40% in 8 to 52 week old birds. Plexiform lesions formed distal to branch points in muscular interparabronchial pulmonary arteries, and were associated with perivascular mononuclear cell infiltrates. Serotonin (5-hydroxytryptamine, 5-HT) is a potent vasoconstrictor and mitogen known to stimulate vascular endothelial and smooth muscle cell proliferation. Serotonin has been directly linked to the pathogenesis of IPAH in humans, including IPAH linked to serotonergic anorexigens that trigger the formation of plexiform lesions indistinguishable from those observed in primary IPAH triggered by other causes. Serotonin also plays a major role in the susceptibility of broilers to IPAH. This avian model of spontaneous IPAH constitutes a new animal model for biomedical research focused on the pathogenesis of IPAH and plexogenic arteriopathy.

4.1 Idiopathic Pulmonary Arterial Hypertension (IPAH) in Humans

IPAH, formerly known as primary pulmonary hypertension (PPH), is a disease of unknown cause characterized by elevated pulmonary vascular resistance (PVR) and a mean pulmonary arterial pressure (PAP) ≥ 25 mm Hg at rest in the absence of other chronic lung or heart disease. The increased PVR seen in IPAH has been attributed to vasoconstriction and remodeling of the pulmonary vasculature, diminished vascular compliance, and vascular occlusion by thrombi. The pathogenesis of IPAH is not clearly understood, and the likelihood exists that IPAH encompasses a variety of etiologies exhibiting common end stage lung pathology. Patients with IPAH are partitioned into ‘familial’ and ‘sporadic’ categories. Assignment to the ‘familial’ category is based on genealogical evidence and confirmation of the presence of a key mutation in the bone morphogenetic protein receptor type II (BMPR2) gene. Familial PAH (FPAH) encompasses approximately 12% of all PAH patients, although as many as 25% of the patients assigned to the sporadic IPAH category also test positive for mutation of the BMPR2 gene (Wagenvoort and Wagenvoort, 1970; Loyd and Newman, 1997; Pietra, 1997; Rubin, 1997; Voelkel et al., 1997; Lee et al., 1998; Nauser and Stites, 2001; McLaughlin, 2002; Newman et al., 2004; Simonneau et al., 2004; Morse, 2005; Stenmark et al., 2009).

4.2 Pulmonary Vascular Pathology in IPAH

The early pathogenesis of IPAH is relatively poorly understood because most patients seek medical evaluation only after their disease has progressed sufficiently to provoke overt clinical symptoms. Pathological evaluations traditionally indicated that early pulmonary vascular remodeling progressively evolves over a number of years into the severe obstructive vascular pathology observed in patients confirmed to have advanced IPAH (Wagenvoort and Wagenvoort, 1970; Voelkel et al., 1997). In very young patients increases in PVR and PAP appear to be correlated with vasoconstriction and medial hypertrophy of small (<400 µ diameter) pre-acinar to intra-acinar pulmonary arteries. Medial hypertrophy encompasses hypertrophy and hyperplasia of the smooth muscle fibers in the tunica media of muscular arteries, distal extension (neomuscularization) of smooth muscle into non-muscularized intra-acinar arteries, and increased densities of elastin and collagen fibers within the tunica media (Wagenvoort and Wagenvoort, 1970; Pietra, 1997; Voelkel et al., 1997; Yi et al., 2000; Pietra et al., 2004). Medial hypertrophy often is accompanied in smaller arterioles by intimal thickening attributable to the accumulation of one or more layers of myofibroblasts and fibrous matrix proteins within the neointimal space between the endothelium (tunica intima) and the internal elastic lamina (Palevsky et al., 1989; Yi et al., 2000; Pietra et al., 2004). In more severe or advanced stages, small pre- and intra-acinar arterioles typically exhibit complex lesions that functionally occlude the vessel’s lumen, including concentric laminar intimal proliferation (”onionskin” or concentric-obliterative lesions), and glomeruloid-like plexiform lesions. Plexiform lesions traditionally have been regarded as characteristic of sustained, rapidly progressing or severe vasoconstrictive pulmonary hypertension, and are present in the lungs of up to 80% of the patients with severe IPAH. Survival times are reported to be shortest when PAH patients’ biopsies reveal the presence of plexiform lesions (Wagenvoort and Wagenvoort, 1970; Palevsky et al., 1989; Tuder et al., 1994; Voelkel et al., 1997; Pietra et al., 2004). Plexiform lesions have been described in several diseases that evoke PAH and are not considered pathognomonic for the idiopathic or familial PAH categories (Voelkel et al., 1997; Pietra, 1997; Yi et al., 2000; Pietra et al., 2004).

Plexiform lesions form immediately downstream from branching points in small muscular pre- and intra-acinar arterioles. Arteriole bifurcation sites are thought to promote the development of complex lesions as a consequence of localized turbulent blood flow and shear stress that damage the normally quiescent endothelium (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). 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). 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 tend to be monoclonal rather than polyclonal in origin, supporting the concept that somatic gene mutations promote proliferation of the altered cells. Endothelial cells from plexiform lesions in anorexigen-triggered pulmonary arterial hypertension (PAH) also tend to be monoclonal in origin, whereas those from patients with secondary PAH are polyclonal in origin (Lee et al., 1998; Tuder et al., 1998b). Dysregulated endothelial cells proliferate until the lumen of an arteriole is functionally 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; Cool et al., 1997, 1999; Pietra, 1997; Pietra et al., 2002; Stevens, 2005). Thrombi and platelet aggregates commonly are found within established plexiform lesions, an anticipated response to endothelial changes that create thrombogenic surfaces. In the absence of anticoagulant therapy, ongoing in situ thrombotic occlusion further complicates the vascular pathology and increases the PVR (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 tend to infiltrate the perivascular zone of plexiform lesions, and fibrosis can develop in the intimal and adventitial layers of the lesion (Tuder et al., 1994; Voelkel et al., 1997; Dorfmüller et al., 2003; Nicolls et al., 2005).

Endothelial cells within plexiform lesions over-express several key markers of angiogenesis, including vascular endothelial growth factor (VEGF) and its receptor (VEGFR-2). At the central core of plexiform lesions the endothelial cells are actively proliferating and do not express transforming growth factor-β (TGF-β) signaling molecules, whereas at the periphery of the plexiform lesion the endothelial cells appear to be involved in de novo angiogenesis and actively express TGF-β signaling molecules (Cool et al., 1999; Geiger et al., 2000; Tuder et al., 2001; Richter et al., 2004; Abe et al., 2010). Endothelial cells within plexiform lesions also tend to over-express components of pathways leading to endothelium-derived vasoconstrictor production (e.g., leukotrienes, thromboxane, and endothelin) (Giaid et al., 1993; Wright et al., 1998; Budhiraja et al., 2004), and variably express components of pathways leading to endothelium-derived vasodilator production (e.g., nitric oxide and prostacyclin) (Giaid and Saleh, 1995; Mason et al., 1998; Tuder et al., 1999; Berger et al., 2001). Conflicting observations of both enhanced (Mason et al., 1998; Berger et al., 2001) and suppressed (Giaid and Saleh, 1995) nitric oxide synthase (NOS) expression within plexiform lesions may reflect variable leukocyte infiltration as well as the distribution of affected arterioles within a transitional zone between macro-vascular (high NOS expressing) and micro-vascular (low NOS expressing) endothelial cell phenotypes (Stevens et al., 2001; Stevens, 2005). Nitric oxide (NO) inhibits platelet aggregation, reduces pulmonary artery contractile responses to serotonin (5-hydroxytryptamine, 5-HT) and endothelin-1 (ET-1), and modulates (attenuates) mitogenic responses to ET-1 and 5-HT (Martinez-Lemus et al., 1999; Tyler et al., 1999; Villamor et al., 2002; Jones et al., 2003; Wideman et al., 2004, 2007).

The precise stimuli responsible for initiating vasoconstriction, medial hypertrophy, and plexiform lesion formation remain unknown. The sustained vasoconstriction and medial hypertrophy of small pulmonary arterioles may reflect enhanced production of or sensitivity to endothelium- or leukocyte-derived pro-mitogenic vasoconstrictors (5-HT, ET-1), attenuated production of or sensitivity to endothelium-derived anti-mitogenic vasodilators (NO, PGI₂), altered function of K⁺ ion channels, and epigenetically triggered apoptosis followed by proliferation of apoptosis-resistant smooth muscle and endothelial cells (Giaid et al., 1993; Giaid and Saleh, 1995; Voelkel et al., 1997; Wright et al., 1998, Tuder et al., 1999; Archer and Rich, 2000; Eddahibi et al., 2001a, 2002; Taraseviciene-Stewart et al., 2001; Archer et al., 2004; Budhiraja et al., 2004; Humbert et al., 2004; Cogolludo et al., 2006). Current concepts suggest the pathology of pulmonary hypertension can be divided into vessel wall abnormalities attributable primarily to vasoconstriction (e.g., medial hypertrophy and adventitial thickening) and concurrently or independently evolving endothelial damage and clonal proliferation attributable to elevated shear stress (e.g., concentric intimal thickening and plexiform lesion formation) (Tuder et al., 2001; Richter et al., 2004; Cool et al., 2005; Stevens, 2005; Stenmark et al., 2009). To the extent that medial hypertrophy and plexiform lesion formation are independent rather than sequential processes, then pharmacologic therapies designed to reverse smooth muscle vasoconstriction are unlikely to impede the deteriorating pathology attributable to progressive plexogenic pulmonary arteriopathy. Increases in PVR and PAP appear to be most amenable to pharmacologic reversal through vasodilator administration or vasoconstrictor receptor blockade when the vascular pathology is limited primarily to medial hypertrophy. Patients confirmed by cardiac catheterization to have severe PAH are more likely to exhibit complex obstructive pulmonary vascular pathology and are less likely to benefit from pharmacologic interventions designed to reduce their PVR (Wagenvoort and Wagenvoort, 1970; Barst, 1986; Palevsky et al., 1989; Voelkel et al., 1997). New approaches are needed to understand and treat advanced cases of PAH involving plexogenic arteriopathy (Nishimura et al., 2002, 2003; Taraseviciene-Stewart et al., 2006; Stenmark et al., 2009; Abe et al., 2010; Firth et al., 2010).

4.3 Pulmonary Vascular Pathology in Mammalian Models of PAH

Attempts to understand the pathogenesis of complex vascular lesion development have been hampered by the fact that plexiform lesions do not develop spontaneously in current animal models of PAH (Stelzner et al., 1992; Rabinovitch, 1997; Rich, 1998; Zabka et al., 2006; Stenmark et al., 2009; Abe et al., 2010; Firth et al., 2010). The Fawn-hooded rat (FHR) spontaneously develops PAH and extensive medial hypertrophy, but spontaneous intimal proliferation and plexogenic arteriopathy have not been observed in the FHR (Kentera et al., 1988; Sato et al., 1992; Le Cras et al., 1999; Nagaoka et al., 2006). Chronic Hypoxia, monocrotaline toxin, pulmonary emboli, pulmonary arterial banding, and hyperdynamic systemic-to-pulmonary artery shunts have been used to induce PAH and medial hypertrophy in various mammalian models. However, the pathogenic mechanisms attributable to these initiating stimuli likely differ from those in spontaneous human IPAH, and none of these models have been found to recapitulate the progressive development of severe, obstructive plexiform lesions similar to those observed in human IPAH (Zamora et al., 1996; Rabinovitch, 1997; Botney, 1999; Tyler et al., 1999; Gust and Schuster, 2001; Taraseviciene-Stewart et al., 2001; Medhora et al., 2002; Stenmark et al., 2009; Firth et al., 2010). Neointimal development can be induced in laboratory animals by imposing multiple experimental challenges to create very severe PAH. For example, occlusive neointimal lesions formed in the pulmonary arterioles of rats when monocrotaline toxin administration was combined with severe hemodynamic stress caused by unilateral pneumonectomy or the creation of an anastomosis between systemic and pulmonary arteries (Tanaka et al., 1996; Botney, 1999; Vaszar et al., 2004; Albada et al., 2005). Neointimal lesions also formed when rats deficient in endothelin type B receptors (ET_B) were injected with monocrotaline toxin (Ivy et al., 2005). The active metabolite of monocrotaline toxin directly damages the pulmonary endothelium (Huxtable, 1990; Lame et al., 2000). Endothelial cell proliferation clearly contributes to neointimal obstruction of pulmonary arterioles in combined-challenge animal models (Taraseviciene-Stewart et al., 2001, 2006; Nishimura et al., 2002, 2003). Chronic hypoxia combined with blockade of VEGF receptors triggered the formation of occlusive neointimal lesions in rats (Taraseviciene-Stewart et al., 2001, 2006). Recently the same combined-challenge model (VEGF receptor blockade plus chronic hypoxia) was used to initiate pulmonary hypertension, after which the rats were maintained under normoxic conditions. Severe, progressive pulmonary hypertension ensued, accompanied by complex plexiform-like lesions closely resembling human plexiform lesions (Abe et al., 2010). Plexiform lesions also were detected in a retrospective study of dogs whose tissues had been archived between 1983 and 2003. These dogs had well documented IPAH and the lungs of four individuals contained plexiform lesions that were regionally clustered downstream from branching points of pulmonary arterioles (Zabka et al., 2006). Plexiform lesions also developed in dogs and sheep two to four 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). This evidence supports the hypothesis that complex obstructive lesions may fail to develop in most laboratory mammals because the PAP and shear stress are insufficiently elevated, the hyperdynamic forces are not sustained long enough, or the animals succumb too rapidly to severe PAH (Rabinovitch, 1997; Albada et al., 2005; Abe et al., 2010). Recent comprehensive reviews emphasize that many of the current mammalian models of PAH greatly facilitated pioneering advances in understanding and treating human IPAH. However none of these animal models fully replicate the pathogenesis of human IPAH nor, absent multiple challenges, does plexogenic arteriopathy arise spontaneously in the current mammalian models of PAH (Stenmark et al., 2009; Firth et al., 2010).

4.4 Avian Model of Spontaneous IPAH

Anatomical and functional descriptions of the lungs, airways and pulmonary vasculature of domestic fowl have been provided previously by Duncker (1972), Abdalla and King (1975), Maina (1988), and Brown et al. (1997). Chickens bred for rapid growth and meat production (broiler chickens, broilers) provide an excellent model of spontaneous IPAH, encompassing many of the classic symptoms and a genetically tractable system for elucidating the underlying causes. Broiler chicks typically hatch at a weight of 40 g and can grow to 4 kg within 8 weeks. Thus in two months a broiler’s body weight doubles and redoubles almost 7 times. If human infants grew at the same rate, their body weight would increase from 3 kg (6.6 lb) at birth to 310 kg (690 lb) at 2 months of age. The extremely rapid early growth performance of broilers imposes proportional challenges to their developmentally immature pulmonary and cardiovascular systems, thereby triggering a suite of “metabolic diseases” that are attributable primarily to rapid growth rather than to infectious pathogens. Approximately 3% of all broiler chickens spontaneously develop IPAH when reared under conditions that promote maximal growth. Rapid growth incurs corresponding increments in cardiac output that must be propelled through lungs that remain essentially isovolumetric throughout the respiratory cycle, and that are constrained in volume by the dimensions of the dorsal thoracic rib cage (Wideman, 1999, 2000, 2001). The pulmonary vasculature of broilers exhibits low compliance and is 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 (Peacock et al., 1989; Wideman and Kirby, 1995a,b; Wideman et al., 1996a,b; Wideman, 2000). Total lung volume also is poorly correlated with body mass in broilers, creating an incipient pulmonary hemodynamic insufficiency that continues to be further exacerbated by ongoing genetic selection for rapid muscle accretion and thus increased metabolic demand for O₂ (Julian, 1989; Peacock et al., 1989, 1990; Owen et al., 1995a,b; Silversides et al., 1997; Wideman, 1999). These observations serve as the basis for the hypothesis that the pulmonary vascular capacity of broiler chickens is marginally adequate to accommodate the cardiac output required to support fast growth. The pulmonary vascular capacity can be broadly defined to encompass metabolic limitations related to the tone (degree of contraction) maintained by the primary resistance vessels (arterioles), as well as anatomical constraints related to the compliance and effective volume of the blood vessels (Wideman 2000, 2001; Wideman et al., 2007).

Abundant evidence demonstrates that broilers with the most limited pulmonary vascular capacity spontaneously develop IPAH when the right ventricle is forced to develop an excessively elevated PAP to propel the cardiac output through their lungs. 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; 2010a). 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; Wideman et al., 2010a). Sustained pulmonary hypertension leads to right ventricular work hypertrophy, as demonstrated by increases in the right-to-total ventricular weight ratios. Following the onset of PAH a distinctive pathophysiological progression includes the development of systemic arterial hypoxemia (cyanosis), polycythemia, systemic arterial hypotension attributable to reduced total peripheral resistance, regurgitation by the monocuspid right atrio-ventricular valve, cardiac decompensation, right-sided congestive heart failure, central venous hypertension, hepatic cirrhosis, transudation of plasma from the liver into the abdominal cavity (ascites), and death. The systemic arterial hypoxemia is attributable to a diffusion limitation initiated by erythrocytes flowing too rapidly past the pulmonary gas exchange surfaces to permit full equilibration with oxygen (Peacock et al., 1990; Wideman et al., 1996a, 2007; Wideman, 2000, 2001). Minimally invasive methods such as electrocardiography and pulse oximetry can be used to identify subclinical/intermediate stages of PAH, including the onset of right ventricular hypertrophy (electrocardiography) and systemic arterial hypoxemia (pulse oximetry and hematocrit) (Wideman, 2000, 2001). Mortality attributable to IPAH can exceed 20% of the broilers subjected to experimental conditions designed to excessively increase the cardiac output or pulmonary vascular resistance, including exposure to sustained sub- or supra-thermoneutral environmental temperatures, chronic hypoxia, respiratory disease, poor air quality, hyperthyroidism, or partial occlusion of the pulmonary vasculature (Cueva et al., 1974; Sillau and Montalvo, 1982; Huchzermeyer and DeRuyck, 1986; Hernandez, 1987; Julian, 1988, 1993; Peacock et al., 1989, 1990; Wideman and Kirby, 1995a,b; Wideman, 1999, 2000, 2001; Wideman et al., 1996a,b, 1997, 2000, 2007).

Procedures used to reveal sub-clinical genetic susceptibility to IPAH within breeding populations of broilers include exposure to hypobaric hypoxia (hypoxic pulmonary vasoconstriction) (Ploog, 1973; Owen et al., 1990, 1995a,b,c; Anthony et al., 2001; Balog, 2003), surgical occlusion of one pulmonary artery (50% reduction in pulmonary vascular capacity) (Wideman and Kirby, 1995a,b; Wideman and French, 1999; 2000), and intravenous microparticle injections (proportional occlusion of pulmonary arterioles and initiation of focal inflammatory responses) (Wideman and Erf, 2002; Wideman et al., 2002; Wang et al., 2003; Hamal et al., 2008, 2010a,b). Exposure to sustained hypobaric hypoxia has been used to select IPAH-susceptible and IPAH-resistant broiler lines for multiple generations (Anthony et al., 2001; Balog, 2003). Broilers from the IPAH-susceptible line succumb to IPAH during exposure to hypobaric hypoxia or cool temperatures, or when microparticles are injected i.v. to partially occlude pulmonary arterioles (vide infra), whereas broilers from the IPAH-resistant line remain markedly unperturbed by these challenges (Wideman et al., 2002; Chapman and Wideman, 2006b). Clinically healthy broilers from the IPAH-susceptible line spontaneously develop higher PAP and PVR when compared with clinically healthy individuals from the IPAH-resistant line (Wideman et al., 2002, 2007; Bowen et al., 2006a,b; Chapman and Wideman, 2006b; Lorenzoni et al., 2008). Lung volume as a percentage of BW does not differ between broilers from these lines (Wideman, unpublished observations). The cumulative evidence therefore demonstrates that selection pressures rigorously focused to challenge the pulmonary vascular capacity readily expose the genetic basis for spontaneous IPAH in broilers. The genes responsible for avian IPAH have a high heritability, estimated at 0.4 to 0.6 (Huchzermeyer et al., 1988; Peacock et al., 1989; Lubritz et al., 1995; Wideman and French, 1999, 2000; Moghadam et al., 2001; Deeb et al., 2002; Pakdel et al., 2002). Attempts to correlate mutations in the chicken BMPR2 with IPAH susceptibility were unsuccessful. Fourteen single nucleotide polymorphisms (SNPs) were identified in broiler BMPR2 mRNA, but 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).

4.5 Pulmonary Vascular Pathology in the Avian Model of IPAH

Broilers with IPAH consistently exhibit medial hypertrophy within 25 to 100µ diameter interparabronchial pulmonary arterioles that are homologous to the pre-acinar 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). Medial hypertrophy has been attributed, in part, to reduced apoptosis of smooth muscle cells in the pulmonary arterioles (Tan et al., 2005b). Intimal proliferation and activation of endothelial protein kinase C (PKCα) also have been observed within the muscularized pulmonary arterioles of broilers developing PAH (Xiang et al., 2002; Tan et al., 2005a,b). However, until recently there were no reports of plexiform lesions in avian lungs. Accordingly, we surveyed the lungs of male and female broilers from an IPAH-susceptible line to estimate the relative age- and gender-specific incidences of plexiform lesion development (Wideman et al., 2010b). We focused on the interparabronchial arterioles (Abdalla and King, 1975) because these key branch points within the avian pulmonary vascular bed appear to be homologous to the arteriole branch sites that promote plexiform lesion development in humans and laboratory mammals (Cool et al., 1999; Zabka et al., 2006; Stenmark et al., 2009; Abe et al., 2010). Plexiform lesions developed in both females and males, and lesion incidences (lung sections with lesions/lung sections examined) averaged approximately 40% in 8 to 52 week old birds. Plexiform lesion densities averaged 0.20 lesions per cm² which is the lower range of lesion densities (0.1 to 11.7 pe r cm²) reported for human patients confirmed to have plexogenic pulmonary arteriopathy (Wagenvoort et al., 1970; 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 elevate the right ventricular afterload. The existing hemodynamic challenge is further aggravated when the hypertrophied right ventricle is forced to elevate the PAP to propel the cardiac output through the decreasing numbers of pulmonary blood vessels that remain unobstructed (Naeye and Vennart, 1959; Dammann et al., 1961; Voelkel et al., 1998; Stenmark et al., 2009). The limited extent of plexogenic vascular obstruction in broilers does not seem likely to significantly increase the PVR, and we have not detected age-related increases in plexiform lesion incidences or densities. Accordingly, plexiform lesion development appears to be a consequence rather than the proximate cause of pulmonary hypertension in IPAH-susceptible broilers (Wideman et al., 2010b).

Our observations suggest a maturational process occurs through which compact cellular lesions having a relatively homogeneous matrix and sparse vascular channels transition into larger ‘mature’ plexiform lesions exhibiting numerous vascular channels and multiple cell types including connective tissue, inflammatory cells and proliferating intimal cells. Plexiform lesions are most likely to develop in regions of broiler lungs where perivascular mononuclear cell infiltrates surround interparabronchial arteries at their branch points from parent arteries. Perivascular mononuclear cell infiltrates often occur in the absence of notable inflammation within the adjacent gas exchange parenchyma or airways. Plexiform lesions also are likely to develop in portions of the lung where muscularized pulmonary arteries exhibit cellular intimal proliferation. Well developed medial hypertrophy and intimal proliferation are readily detected but unevenly distributed within broiler lungs. Small, apparently early-stage, plexiform lesions forming within interparabronchial arteries typically consisted of an intimal cellular matrix and embedded macrophages, an apparently un-distended smooth muscle layer, and extramural inflammatory cell infiltrates (Figures 1 and 2). Plexiform lesions have been detected in broiler lungs as early as 7 days post-hatch, clearly forming at branch points of interparabronchial arterioles (Figure 3). Medium sized plexiform lesions typically occur shortly downstream from the point where an interparabronchial artery branches from a larger parent artery, or in supernumerary branches from large conducting arteries. Mononuclear cell infiltrates typically are evident around the perimeter of the lesions and nearby arterial branches. Macrophages having a foam-type appearance are arrayed adjacent to the remnants of the dilated arterial wall, surrounding a compact, fairly homogeneous core of intimal cells (Figure 4). In ‘maturing’ plexiform lesions the glomeruloid-like main body (120 to 200 µm in diameter) undergoes aneurysmal expansion well beyond the circumference of a typical interparabronchial artery, foam-type macrophages reside at the periphery of the main body, and remnants of smooth muscle in the arterial wall are scarce or absent (Figures 5 and 6). Perivascular mononuclear cell infiltrates persist in surrounding the parent arteriole, and inflammatory cell infiltrates also may surround the dilated margins of mature plexiform lesions in broiler lungs (Figures 6 and 7). Similar inflammatory processes have been associated with IPAH and plexogenic arteriopathy in humans and laboratory mammals (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; Stenmark et al., 2009). It remains to be determined if vascular injury induced by shear stress attracts 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).

Figure 1.

Section through an interparabronchial arteriole reveals intimal proliferation (IP) within the arteriole lumen. Closed arrow indicates perivascular mononuclear cells outside the smooth muscle wall of the arteriole (open arrow). Hematoxylin and eosin staining in all figures.

Figure 2.

An arteriole with the lumen occluded by proliferating intimal cells (IP) and macrophages (MΦ). Sparse perivascular mononuclear cells surround the arteriole wall (open arrow).

Figure 3.

A small plexiform lesion in a branch of an interparabronchial arteriole exhibits macrophages (MΦ) arrayed inside the remnants of the smooth muscle wall (open arrow), with proliferating intimal cells (IP) occluding the lumen. Adjacent branches of the same interparabronchial arteriole contain nucleated avian erythrocytes within the smooth muscle wall (open arrow). This lung from a 7 day old broiler chick was fixed by immersion in phosphate buffered formalin.

Figure 4.

A. This intermediate sized plexiform lesion developed in an interparabronchial arteriole branch coursing within the septum between adjacent parabronchi. The lesion contains foam-type macrophages (MΦ) adjacent to the remnant of the arteriole wall on the bottom right and a channel filled intimal proliferating cells (IP) adjacent to the arteriole smooth muscle (open arrow) on the upper left. Filled arrow indicates perivascular mononuclear cells outside the wall of the arteriole. Figure 4B. Higher magnification shows the intimal proliferating cells (IP) and a foam-type macrophage (MΦ).

Figure 5.

Maturing plexiform lesions contain foam-like macrophages (MΦ) at the periphery near the remnants of the arteriole wall, with the core of the lesion becoming glomeruloid in form due to penetration by multiple vascular channels. A swath of intimal proliferating cells (IP) courses within the lumen of an adjacent segment of the arteriole.

Figure 6.

This plexiform lesion exhibits a central matrix of intimal proliferating cells penetrated by vascular channels, with multiple foam-type macrophages (MΦ) arrayed adjacent to the indistinct remnants of the arteriole’s wall. Filled arrow indicates mononuclear cells.

Figure 7.

A. Mature plexiform lesion in a 24 week old male broiler evidently developed in an interparabronchial arteriole coursing between three parabronchi (PB). The central matrix of the lesion contains epithelioid cells and myofibroblasts, and is penetrated by vascular channels that were preserved in an open configuration by in situ trans-cardiac perfusion fixation. Figure 7B. Higher magnification shows vascular channels penetrating the central core of the lesion, which is surrounded by perivascular mononuclear cell infiltrates.

4.6 Role of Serotonin (5-HT) in IPAH and Vascular Remodeling

Serotonin (5-HT) has been implicated in the pathogenesis of IPAH (Hervé et al., 1995; MacLean et al., 2000; Eddahibi et al., 2001a, 2002; Marcos et al., 2003, 2004; Naeije and Eddahibi, 2004; Eddahibi and Adnot, 2005; Lawrie et al., 2005), PAH triggered by serotonergic appetite suppressant drugs (Seiler et al., 1974; Douglas et al., 1981; Brenot et al., 1993a,b; Abenhaim et al., 1996; Louis, 1999; Eddahibi et al., 2001b; Eddahibi and Adnot, 2002; Naeije and Eddahibi, 2004), PAH initiated by hypoxia and monocrotaline toxin (Demiryurek et al., 1991; Uzun et al., 1998; Eddahibi et al., 1997, 1999, 2000; Marcos et al., 2003; Guignabert et al., 2005), and PAH associated with Gram-negative sepsis, ARDS, COPD, and other inducers of secondary PAH in humans (Sibbald et al., 1980; Heffner and Repine, 1997; Egermayer et al., 1999; MacLean et al., 2000; Eddahibi et al., 2003). Serotonin initiates rapid and sustained increases in PVR, reflecting acute vasoconstriction followed by structural remodeling associated with smooth muscle cell proliferation. Acute pulmonary vasoconstriction is mediated by 5-HT_2A, 5-HT_2B, and 5-HT_1B/1D receptors expressed on vascular smooth muscle cells (McGoon and Vanhoutte, 1984; Choi and Maroteaux, 1996; MacLean et al., 1996, 2000; Morecroft et al., 1999; Keegan et al., 2001; Lawrie et al., 2005; Cogolludo et al., 2006; Rodat-Despoix et al., 2008). Serotonin receptor expression did not appear to be altered in one evaluation of patients with IPAH (Marcos et al., 2004), but increased 5-HT_1B and 5-HT_2B receptor expressions have been implicated in other studies of human IPAH (Morecroft et al., 1999; Launay et al., 2002). Structural remodeling is mediated by the high affinity serotonin transporter (5-HTT, SERT) that primarily is expressed by pulmonary vascular smooth muscle but also is expressed by the endothelium. Serotonin translocated into the smooth muscle cells by 5-HTT is mitogenic, causing hypertrophy and proliferation of the medial muscle layer in pulmonary arterioles (Lee et al., 1991, 1994; Ramamoorthy et al., 1993; Pitt et al., 1994; Zamora et al., 1996; Fanburg and Lee, 1997; Eddahibi et al., 1999; Marcos et al., 2003, 2004). 5-HTT expression and smooth muscle cell proliferation mediated by serotonin are markedly amplified in lung tissues, pulmonary arteries, and platelets from human IPAH patients (Eddahibi et al., 2001a, 2002). Multi-fold increases in 5-HTT gene transcription and 5-HTT expression in IPAH patients have been attributed to the L allele of the 5-HTT gene promoter. The homozygous (LL) genotype appears to confer susceptibility to IPAH when combined with other predisposing factors, including the BMPR2 gene mutation (Heils et al., 1996; Lesch et al., 1996; Eddahibi et al., 2001a, 2002; Du et al., 2003; Sullivan et al., 2003; Eddahibi and Adnot, 2005). Thus, mice with targeted disruption (knockout) of the BMPR2 gene and their wild-type littermates did not exhibit differences in pulmonary hemodynamics or vascular morphometry under normoxic or hypoxic conditions until serotonin was infused chronically, after which the BMPR2 deficient mice developed markedly amplified PAH and vascular remodeling (Long et al., 2006). The LL genotype of the 5-HTT gene promoter also predisposes human COPD patients to more severe PAH and vascular remodeling (Eddahibi et al., 2003).

Competitive inhibitors of 5-HTT but not 5-HT_1B/1D receptor antagonists eliminate the ability of serotonin and its agonists to stimulate human pulmonary artery smooth muscle cell proliferation in vitro (Eddahibi et al., 2001a; Marcos et al., 2003). Monocrotaline toxin increases 5-HTT expression, vascular remodeling and PAH, whereas subsequent inhibition of 5-HTT completely reverses the PAH and vascular remodeling induced by monocrotaline toxin (Guignabert et al., 2005). Direct evidence of the role played by 5-HTT in pulmonary vascular remodeling has been demonstrated in 5-HTT gene knockout mice, and in mice treated with 5-HTT competitive inhibitors. Indices of PAH (right ventricular hypertrophy, elevated right ventricular systolic pressure) and arteriole muscularization were attenuated in mice lacking functional 5-HTT when compared with the PAH responses of wild-type or vehicle-injected control mice during chronic (2 week) exposure to normobaric hypoxia or following monocrotaline toxin injection (Eddahibi et al., 2000; Marcos et al., 2003). Conversely, transgenic mice overexpressing 5-HTT predominantly in vascular smooth muscle cells developed marked PAH, right ventricular hypertrophy and muscularization of distal pulmonary arterioles without changes in systemic arterial pressure (Guignabert et al., 2006). Chronic hypoxia triggers increased 5-HTT expression leading to enhanced smooth muscle cell proliferation and PAH in response to serotonin supplementation (Eddahibi et al., 1999, 2001b). Paradoxically, competitive inhibition of 5-HTT potentiated the PAH response of mice exposed to acute (5 minute) normobaric hypoxia when compared with vehicle-treated controls. The latter observations are consistent with the hypothesis that inhibiting serotonin uptake by platelets increased the plasma pool available to bind to serotonin receptors on smooth muscle cells, resulting in amplified pulmonary vasoconstriction during the acute PAH response to hypoxia. Co-treatment with competitive 5-HTT inhibitors plus 5-HT_1B receptor antagonists blunted the PAH response to acute hypoxia (Marcos et al., 2003), and 5-HT_1B receptor knockout mice were less susceptible to the onset of hypoxic PAH and vascular remodeling than were wild-type controls (Keegan et al., 2001; Launay et al., 2002). The 5-HT_1B receptor and 5-HTT interact to regulate Rho kinase-mediated pulmonary vascular remodeling (Liu et al., 2004; Hyvelin et al., 2005), and interactions between 5-HT_1B receptors and 5-HTT regulate the S100A4/Mts1 gene that is induced in clinical IPAH (Lawrie et al., 2005). These studies reemphasize the importance of 5-HTT expression and co-dependent interactions with serotonin receptors in the chronic pathogenesis of PAH (Marcos et al., 2003; Guignabert et al., 2005; Lawrie et al., 2005).

An important linkage between plexiform lesions and serotonin can be deduced from the pathogenesis of IPAH triggered by anorexigens (appetite suppressants) such as aminorex, dexfenfluramine and the combination of fenfluramine and phentermine (fen/phen). In a small percentage of anorexigen users severe PAH evolved with a latency of up to 23 years. The histopathological, morphological and clinical features were considered indistinguishable from those in familial IPAH, including medial hypertrophy and the formation of plexiform lesions in pulmonary arterioles (Tuder et al., 1998a,b). Endothelial cells from plexiform lesions 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., 1998a,b). Anorexigens are serotonergic and are thought to trigger IPAH by: stimulating serotonin release from cellular stores such as nerve terminals and platelets; inhibiting 5-HTT and thus serotonin uptake by platelets and neurons; increasing plasma serotonin levels; and, directly stimulating serotonin receptors (Seiler et al., 1974; Celada et al., 1994; Abenhaim et al., 1996; Weir et al., 1996; DeJonghe and Swinkels, 1997; Rothman et al., 1999; Louis, 1999; Russell and Laverty, 2000; Eddahibi et al., 2001a, 2001b; Eddahibi and Adnot, 2002; Launay et al., 2002). Individuals may be particularly susceptible to anorexigen-related IPAH when the presence of the LL genotype of the 5-HTT promoter imparts high basal level of serotonin uptake by pulmonary vascular smooth muscle cells (Eddahibi et al., 2001a).

Mechanisms by which serotonin and anorexigens contribute to endothelial proliferation and plexiform lesion formation remain to be clarified. Serotonin is actively transported into endothelial cells and endothelial serotonin uptake is stimulated by hypoxia (Lee and Fanberg, 1986a,b, 1987). Human pulmonary endothelial cells exhibit 5-HTT and 5-HT_2B-receptor immunoreactivity, and 5-HTT mRNA expression was observed in cultured endothelial cells from patients with non-familial IPAH but not in endothelial cells from control subjects (Eddahibi et al., 2001a, 2006; Marcos et al., 2004). Active serotonin uptake by the pulmonary endothelium may primarily serve to clear serotonin from the circulation (Fanberg and Lee, 1997). However serotonin also is mitogenic for isolated endothelial cells, leading to the proposal that platelets aggregating at sites of endothelial damage may release serotonin in quantities sufficient to induce endothelial proliferation (Pakala et al., 1994). The development of thrombogenic surfaces in response to elevated shear stress likely promotes in situ thrombosis and platelet aggregation within the pulmonary arterioles of patients with IPAH. Platelets and the endothelium to which they adhere can release of a variety of mitogens (5-HT, ET-1, PAF, PDGF, VEGF, FGFa, TGF- β) that potentially can stimulate endothelial and smooth muscle cell proliferation (Farber and Loscalzo, 1999; Humbert et al., 2004; Hoeper et al., 2006). Serotonin is the most potent mitogen of all the endothelial- and platelet-derived growth factors evaluated in isolated pulmonary arterial smooth muscle cell cultures (Eddahibi et al., 2001a; 2006). Evaluations of pulmonary vascular remodeling have focused on paracrine (cross-talk) pathways involving serotonin, smooth muscle cells and the endothelium. Vascular smooth muscle cells produce the protein angiopoietin 1 (Ang-1) which promotes angiogenesis by binding to TIE2 receptors that are localized exclusively on endothelial cells. Ang-1 activation of TIE2 receptors stimulates pulmonary endothelial cells to synthesize and secrete serotonin, which in turn triggers vascular remodeling. Extremely strong positive correlations (r = 0.97) have been demonstrated between pulmonary Ang-1 expression, TIE2 activation, and PVR in patients with non-familial IPAH (Du et al., 2003; Sullivan et al., 2003). Quiescent pulmonary endothelial cells from patients with non-familial IPAH also synthesize and secrete more serotonin and exhibit markedly upregulated expression of tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin biosynthesis, when compared with control endothelial cells. TPH immunoreactivity was confined to the intima and was not observed in smooth muscle cells in tissues from IPAH patients with marked medial hypertrophy (Eddahibi et al., 2006). These studies provide a plausible linkage between dysregulated Ang-1 or TPH gene expression and the involvement of serotonin in the increased PVR and associated vascular remodeling that are characteristic of IPAH.

4.7 Serotonin is a Key Vasoconstrictor in Avian IPAH

A primary role for serotonin in avian IPAH was revealed through the use of the i.v. microparticle injection technique. Microparticles having a size suitable for occluding arterioles are injected into a systemic vein to be carried to the lungs by the returning venous blood. Pulmonary arterioles become occluded in proportion to the number and size of the microparticles injected, thereby increasing the PVR and triggering acute physiological responses that mirror those previously observed following acute unilateral pulmonary artery occlusion and that are characteristic of broilers that spontaneously develop IPAH (Wideman and Erf, 2002; Wideman et al., 2005, 2006). As shown in Figure 8, entrapped microparticles also trigger a marked focal pulmonary inflammatory response that includes the perivascular infiltration of mononuclear cells in combination with luminal accumulations of thrombocytes and macrophages (Wideman and Erf, 2002, Wideman et al., 2002, 2003, 2005, 2006, 2007; Wang et al., 2003; Hamal et al., 2008; 2010a,b). Microparticle entrapment elicits higher chemokine and cytokine expression in the lungs of IPAH-resistant than in IPAH-susceptible broilers (Hamal et al., 2010a). Thrombocytes rapidly aggregate around microparticles lodged in the lumen of inter-parabronchial arterioles (Wideman et al., 2002, 2004, 2007; Wang et al., 2003). Thrombocytes are the most numerous leukocytes in avian blood and are functional homologs of mammalian platelets. Thrombocytes and platelets accumulate serotonin within intracellular storage granules that are released upon activation (Kuruma et al., 1970; Simoneit et al., 1970; Sorimachi et al., 1970, 1974; Lacoste-Eleaume et al., 1994). Microparticle injections more than double plasma serotonin levels in broilers (Chapman et al., 2008). Serotonin increases the PVR and PAP in broilers, and is singularly the most potent pulmonary vasoconstrictor we have evaluated. Serotonin is capable of triggering an essentially instantaneous and fully obstructive vasoconstriction leading to an immediate >90% reduction in cardiac output and terminal suffocation within 30 seconds in clinically healthy broilers unless i.v. infusion rates are carefully titrated to 100-fold lower than levels typically used to elicit PAH in mammals (Chapman and Wideman, 2002). The pulmonary hemodynamic responses to serotonin were evaluated in broilers pretreated with the selective 5-HT_2A receptor antagonist ketanserin or with the nonselective 5-HT_1/2 receptor antagonist methiothepin (Engel et al., 1986; Barnes and Sharp, 1999). Pre-treating broilers with ketanserin failed to alter the PAH response to subsequent serotonin infusion, whereas pretreatment with methiothepin reduced PAP below baseline values and virtually eliminated increases in PVR and PAP elicited by i.v. infusions of serotonin. Methiothepin clearly blocks serotonin-mediated increases in PVR and PAP in broilers, although the specific receptor subtype involved remains to be determined (Chapman and Wideman, 2006a). In a subsequent study methiothepin was used to evaluate the role of serotonin in the onset of PAH triggered by i.v. microparticle injections (Chapman and Wideman, 2006b). Pretreatment with methiothepin reduced PAP below baseline values, demonstrating serotonin likely exerts tonic control of PVR. Microparticle injections increased PAP by 90% in control broilers, but the same dose of microparticles failed to significantly elevate PAP in broilers that had been pretreated with methiothepin.

Figure 8.

A. Intrapulmonary inflammatory response 20 minutes after cellulose microparticles were injected i.v. Arrows show microparticles entrapped in interparabronchial arterioles coursing between three parabronchi (PB). Figure 8B. Entrapped microparticles (MP) attract numerous macrophages (thin arrow) and a swarm of nucleated thrombocytes (thick arrow).

Subsequent to injecting a suitable dose of microparticles, broilers with the most limited pulmonary vascular capacity rapidly succumb to respiratory insufficiency whereas those having a sufficiently robust pulmonary vascular capacity thrive as clinically healthy/resistant survivors. Broiler lines selected for IPAH-resistance are substantially more resistant to microparticle injections when compared with their respective unselected (Base) populations or IPAH-susceptible lines (Wideman et al., 2002, 2007). Injecting a high dose of microparticles into broilers from the IPAH-susceptible line elicited 78% mortality in controls as compared with only 20% in those pretreated with methiothepin. Injecting the same microparticle dose into the IPAH-resistant line elicited 12% mortality in controls as compared with zero mortality in those pretreated with methiothepin (Chapman and Wideman, 2006b).

We also evaluated the impact of i.v. microparticle injections on the incidence of plexiform lesions in the lungs of susceptible broilers. Three months after an LD₅₀ injection, some microparticles remained entrapped in the lungs of the survivors, however no plexiform lesions were observed in the vicinity of persisting microparticles. Instead these survivors exhibited a significant overall reduction in their plexiform lesion incidence when compared with the incidence for age-matched controls. Evidently the broilers that succumbed to the microparticle injection were more susceptible than the survivors to plexogenic arteriopathy (Wideman et al., 2010b). Microparticle injections likely serve to eliminate individuals having excessive serotonin biosynthesis, impaired thrombocyte uptake or enhanced release or leakage of serotonin, enhanced receptor-mediated vasoconstrictive responsiveness to serotonin, or altered internalization of serotonin by 5-HTT. Microparticle entrapment evokes patterns of gene expression consistent with our hypothesis that susceptibility to IPAH in broilers is a consequence an anatomically inadequate pulmonary vascular capacity combined with functional predominance of vasoconstriction attributable to serotonin over vasodilation and immune modulation attributable to NO (Wideman et al., 2002, 2004, 2007; Hamal et al., 2008, 2010a,b)

4.8 Summary and Conclusions

Plexiform lesions elevate the pulmonary vascular resistance PVR) by progressively occluding pulmonary arterioles in up to 80% of the human patients with idiopathic pulmonary arterial hypertension (IPAH). Patients with plexogenic arteriopathy tend to be unresponsive to vasodilator therapy and have a poor prognosis for survival. Research is needed to understand and treat plexogenic arteriopathy, but advances have been hindered by the absence of spontaneously developing plexiform lesions in most mammalian models of PAH. Chickens bred for meat production (broiler chickens, broilers) provide an excellent model of spontaneous, genetically based IPAH triggered by increases in PVR attributable in part to serotonin. Serotonin is a potent vasoconstrictor and mitogen known to stimulate the proliferation of vascular endothelial and smooth muscle cells in mammals. Serotonin has been directly linked to the pathogenesis of IPAH in humans, including IPAH linked to serotonergic anorexigens that trigger the formation of plexiform lesions indistinguishable from those observed in other forms of primary IPAH. The mechanisms by which serotonin and anorexigens contribute to endothelial proliferation and plexiform lesion formation remain to be clarified. The onset of IPAH in broiler chickens clearly involves perturbations of the pre-capillary vasculature (e.g., elevated arteriole resistance and vascular remodeling). In this regard, although extreme differences exist between mammals and birds with regard to the anatomy of air passageways, gas exchange compartments, and the processes involved in ventilation, nevertheless the pre-capillary pulmonary vasculature of mammals and birds exhibits striking structural and functional similarities. Moreover, key similarities clearly exist when the avian and mammalian vascular beds and hemodynamic profiles are compared during the pathogenesis of IPAH. The broiler chicken model of IPAH constitutes the only animal model that develops plexogenic arteriopathy spontaneously. Caveats regarding the use of this model include the relatively low plexiform lesion incidences and densities in most birds, as well as the apparent absence of progressively increasing lesion densities (and associated increases in PVR) as the birds age. Progressive, irreversible pruning of the pulmonary vasculature is a critical problem associated with plexogenic arteriopathy in humans suffering from severe IPAH. Our studies have focused on the spontaneous development of plexogenic arteriopathy in rapidly growing broilers reared under optimal (non-challenging) environmental conditions. Under these fairly benign circumstances perhaps relatively few plexiform lesions develop because the PAP and shear stresses are insufficiently elevated and/or are not sustained long enough. It also is possible that the most susceptible broilers succumb too rapidly to terminal right-sided congestive heart failure. The development of plexiform lesions shortly post-hatch (Figure 3) coincides with a period of rapid lung development in concert with equally challenging increases in body mass and cardiac output. Younger birds are easier to maintain and may present an opportunity to obtain escalated lesion incidences and densities in response to appropriate environmental challenges designed to trigger sustained increases in PAP. Higher lesion incidences are desirable if an animal model is to be useful for assessing therapeutic treatments. Alternatively, it is evident that the immune system plays an important role in the pathogenesis of plexiform lesion development. Plexiform lesions may prove to be triggered primarily by vascular damage and the initiating or accompanying immunological responses, rather than by elevated pressure or shear stress per se. Identifying the processes involved in the pathogenesis of IPAH and plexogenic arteriopathy in the broiler model likely will provide key insights that we hope will prove to be relevant to the pathogenesis of plexogenic arteriopathy and IPAH in humans