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Animal models of pulmonary hypertension: role in translational research
Drug Discovery Today: Disease Models
Vol. 7, No. 3–4 2010
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Andrew McCulloch – University of California, SanDiego, USA
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Pulmonary vascular disease
Animal models of pulmonary hypertension: role in translational research Oleg Pak1,3, Wiebke Janssen2,3, Hossein Ardeschir Ghofrani1, Werner Seeger1,2, Friedrich Grimminger1, Ralph Theo Schermuly1,2, Norbert Weissmann1,* 1
Excellence Cluster Cardio-Pulmonary System, University of Giessen Lung Center, Dept. of Internal Medicine II/IV/V, Justus-Liebig-University Giessen, Aulweg 130, 35392 Giessen, Germany 2 Max-Planck-Institute for Heart and Lung Research, Parkstr. 1, 61231 Bad Nauheim, Germany
Pulmonary hypertension (PH) is a severe progressive disorder with an unclear etiology and a poor prognosis. Current treatments can alleviate the symptoms and even revert the characteristic vascular remodeling process, but cannot cure the disease. A variety of
Section editors: Jason Yuan – Department of Medicine and Institute for Personalized Respiratory Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA Amy Firth – Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA
animal models have been suggested to mimic PH and
successful
translation
of
new
therapeutic
approaches from bench to bedside demonstrates the value of such models. Our review highlights the role of different animal models of PH in translational research. Introduction Pulmonary hypertension (PH) is a severe progressive disorder characterized by extensive narrowing of the pulmonary vascular bed, leading to an increase in pulmonary vascular resistance, which ultimately produces a compensatory right ventricular hypertrophy and results in heart failure and premature death [1]. A variety of conditions can lead to the development of PH. The modern classification of PH groups different diseases, which share similarities in pathophysiological mechanisms, clinical manifestation, and therapeutic approaches in five groups. These 5 groups are: 1st – pulmonary arterial hypertension group; 2nd – PH associated with left*Corresponding author.: N. Weissmann ([email protected]) 3 Both these authors contributed equally. 1740-6757/$ ß 2011 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmod.2011.02.002
sided heart diseases; 3rd – PH related with hypoxemia/lung diseases, 4th – thromboembolic PH and 5th – PH with unclear multifactorial mechanisms [1] (Table 1a). The pathogenesis of PH is poorly understood and reliable treatment is still lacking. Current treatments can improve clinical symptoms, but cannot cure PH.
Animal models of pulmonary hypertension Animal models of PH are used expansively in current research. These models have been the source of a plethora of scientific information, such as the role of certain molecular mechanisms and genetic contributions in PH. Additionally, animal models have been utilized in the discovery and testing of possible therapeutic approaches. PH can be induced in the animal either through pharmacologic/toxic agents [2,3], genetic techniques [4–8], environmental factors [9–11] or through surgical interventions [12–15] (Table 1a). The most commonly used animal models of PH are the injection of monocrotaline and the chronic exposure to hypoxia (classical models). There are a variety of other 89
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Table 1. Current classification of PH and animal models (a) Revised Dana Point classification of PH and animal models of PH Group of PH
Animal models
1. Pulmonary arterial hypertension (PAH)
MCT [21]; MCT + pneumonectomy [33]; BMPR2 knockout [4]; Tg+ mice [5]; Fawn-hooded rat [7]; S100A4 overexpressing [6]; SHIV-nef-infected macaques [16]; hypoxia + SU-5416 [28]; Schistosomiasis [17]; left-to-right shunt [12,13], closure of the ductus arteriosus [14]
2. Pulmonary hypertension with left heart disease
Congestive heart failure models [15]
3. Pulmonary hypertension associated with lung diseases and/or hypoxemia
Chronic hypoxia [9], hypoxia + SU-5416 [28]; Intermittent hypoxia [10]; Smoke exposure [11]; Bleomycin [2]; 5-HTT overexpression [8]
4. Pulmonary hypertension owing to chronic thrombotic and/or embolic disease
Repeated microembolization with microspheres [18]
5. Miscellaneous
?
(b) WHO functional class assessment classification WHO functional assessment classification
Animal model
Class I
Patients with PH but without resulting limitation of physical activity. Ordinary physical activity does not cause undue dyspnea or fatigue, chest pain, or near syncope.
Hypoxia
Class II
Patients with PH resulting in slight limitation of physical activity. They are comfortable at rest. Ordinary physical activity causes undue dyspnea or fatigue, chest pain, or near syncope.
Hypoxia/hypoxia + SU-5416
Class III
Patients with PH resulting in marked limitation of physical activity. They are comfortable at rest. Less than ordinary activity causes undue dyspnea or fatigue, chest pain, or near syncope.
MCT
Class IV
Patients with PH with inability to carry out any physical activity without symptoms. These patients manifest signs of right heart failure. Dyspnea and/or fatigue may even be present at rest. Discomfort is increased by any physical activity.
MCT/MCT + pneumectomy
in vivo animal models of PH that mimic features of different PH groups: 1st group – bone morphogenetic protein receptor 2 (BMPR2) knockout mice [4], transgenic mice overexpressing Interleukin 6 [5], S100A4 (Mts1) overexpressing mice [6], SHIV-nef-infected macaques (a chimeric viral construct containing the HIV nef gene in a simian immunodeficiency virus backbone) [16], Fawn-hooded rats [7], schistosomiasisinduction of PH [17], experimental left-to-right shunt [12,13], closure of the ductus arteriosus [14]; 2nd group – congestive heart failure (CHF) models [15]; 3rd group – intermittent hypoxia [10], cigarette smoke exposure [11], bleomycin application [2], 5-hydroxytryptamine transporter protein (5-HTT) overexpression [8]; 4th group – repeated microembolization with microspheres [18] (Table 1a). Our review is focused on in vivo models of PH and especially on the classical models, because they are the most frequently used models in translational research due to their good reproducibility and their well described histopathology (Fig. 1).
Chronic hypoxia Chronic hypoxia is the most commonly used physiological stimulus for PH development in animal models. Since 1946 it has been known that acute hypoxia is a trigger of hypoxic pulmonary vasoconstriction (HPV), which redistributes the pulmonary blood flow from poorly to better ventilated areas 90
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of the lung to maintain the ventilation-perfusion match in adults [19]. By contrast, systemic vessels of adults dilate during hypoxia. Global chronic alveolar hypoxia that occurs in permanent residents of high altitude or in pathological conditions and diseases leading to alveolar hypoxia (group 3 of the Dana Point classification of PH), may result in pulmonary vascular remodeling and PH development. Normobaric hypoxia (reduction of oxygen partial pressure at sea level to approximately 10%) and hypobaric hypoxia (e.g. 400 mm Hg atmospheric pressure, simulating high altitude) exposure for 2 weeks and more induces PH in a wide variety of animal species including rats, mice, guinea pigs, dogs, cows, pigs and sheep. The most commonly used species in laboratory models of PH are rats and mice. This experimental model represents the group 3 of the Dana Point classification of PH (Table 1a). The most common pathological findings are muscularization of previously nonmuscularized vessels (Fig. 1a) and a moderate medial thickening of muscular resistance vessels (Fig. 1b; Table 2). Both pulmonary smooth muscle cells (PASMC) and adventitial fibroblasts proliferate under hypoxia. These features are largely reversible by return to normoxia. The details of the molecular steps of the hypoxic pulmonary vasculature remodeling from the oxygen sensing to cell proliferation are not yet fully understood. The hypoxia-inducible transcription factors (HIF) play a major role in this process [20].
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Drug Discovery Today: Disease Models | Pulmonary vascular disease
Pulmonary artery remodeling in animal models compared to IPAH
63x,vessels <100 µm EE EI N
L M
A
IPAH
Mouse HOX
Rat HOX
Donor control
Mouse NOX
Rat NOX
Rat HOX + SU-5416
Rat NOX
MCT Rat
MCT + pneumonectomy
Rat control
Rat control
MCT Rat
MCT + pneumonectomy
Rat control
Rat control
40x,vessels < 200 µm
PL N
IPAH
Mouse HOX
Rat HOX
Donor control
Mouse NOX
Rat NOX
Rat HOX + SU-5416
Rat NOX
Staining - elastic van Gieson. Elastic fibres – blue/black/brown; nuclei - black/brown; collagen fibres – red; media, epithelia, nerves, erythrocytes – yellow IPAH – idiopathic pulmonary arterial hypertension; HOX – 3 weeks hypoxic exposure, NOX – normoxic control; MCT – monocrotaline injection; EE – elastica externa; EI elastica interna; L – lumen; N – neointima, M – media; A – adventitia; PL – plexiform lesion. Drug Discovery Today: Disease Models
Figure 1. Staining, elastic van Gieson. Elastic fibres, blue/black/brown; nuclei, black/brown; collagen fibres, red; media, epithelia, nerves, erythrocytes, yellow; IPAH, idiopathic pulmonary arterial hypertension; HOX, 3 weeks hypoxic exposure; NOX, normoxic control; MCT, monocrotaline injection; EE, elastica externa; EI, elastica interna; L, lumen; N, neointima, M, media; A adventitia; PL, plexiform lesion.
Chronic hypoxia in mice In 1900, the French biologist Lucien Cuenot used mice to repeat Mendel’s work in plants. Since then mice have become the most widespread model in research culminating with the creation of knockout mice in 1987 and the publication of the mice genome sequence in 2002. Chronic exposure of mice to normobaric or hypobaric hypoxia leads to an elevation in pulmonary artery pressure, vascular remodeling and RV hypertrophy (Fig. 1) [9]. The role of the nitric oxide (NO) pathway, reactive oxygen species (ROS), and cytoskeletal architecture in PH development has been investigated in the hypoxic mouse model [3,9,21]. NO is a vasodilatator and an important signaling molecule. Soluble guanylyl cyclase (sGC) is the known receptor for NO. Upregulation of sGC was detected in the structurally remodeled smooth muscle layer in chronic hypoxic mice lungs. Treatment of wild-type mice with the activator of sGC, or the stimulator of sGC after full establishment of PH significantly reduced pulmonary pressure, RV hypertrophy, and structural remodeling of the lung
vasculature. Additionally, an endogenous inhibitor of the nitric oxide (NO) pathway, PDE1A (phosphodiesterase 1 A) was significantly upregulated in pulmonary arterial smooth muscle cells (PASMC) of chronically hypoxic mice and the PDE1 inhibitor 8MM-IBMX reversed chronic PH [21]. ROS could play a role in hypoxic pulmonary vasoconstriction and PH development [19]. It was shown that NOX4 (NADPH oxidase 4) mRNA is upregulated under hypoxia in homogenized lung tissue, concomitant with increased levels in microdissected pulmonary arterial vessels of mice. Treatment of PASMCs with siRNA directed against NOX4 decreased NOX4 mRNA levels and reduced PASMC proliferation as well as generation of ROS [9]. Growth factors regulate diverse physiological processes and it was suggested that disturbance in their regulation, expression or alterations in downstream effects can contribute to the development of PH. It was reported that the platelet derived growth factor (PDGF) receptor antagonist STI571 (imatinib) reversed advanced pulmonary vascular disease in a mouse model. STI571 prevented phosphorylation of www.drugdiscoverytoday.com
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Table 2. Comparison of different PH animal models Animal model Chronic hypoxia
Chronic hypoxia + SU-5416
Monocrotaline
Monocrotaline + pneumonectomy
Species
Rat and mice
Rat
Rat
Rat
Stimulus
(1) Physiological – hypoxia
(1) Physiological – hypoxia (2) Chemical – VEGF inhibitor
(1) Toxic – MCT
(1) Toxic – MCT (2) Physiological – increased flow
Main histological features
(1) Moderate increased media of muscularized pulmonary arteries (Fig. 1b)
(1) Moderate increased media of muscularized pulmonary arteries (Fig. 1b)
(2) Muscularization of normally non-muscular arteries (Fig. 1a)
(2) Muscularization of normally non-muscular arteries (Fig. 1a)
(1) Prominent increased media of muscularized pulmonary arteries (Fig. 1b) (2) Muscularization of normally non-muscular arteries (Fig. 1a)
(1) Prominent increased media of muscularized pulmonary arteries (Fig. 1b) (2) Muscularization of normally non-muscular arteries (Fig. 1a) (3) Neointima formation
(3) Neointima formation Pro
Physiological stimulus; predictable and reproducible
Physiological stimulus; proliferation of endothelial cells
Severe PH; RV failure model; predictable and reproducible
Severe PH; RV failure model; proliferation of endothelial cells
Con
Moderate PH; using more severe hypoxia than in human; absence of plexiform lesion
Not clear which group mimics
Toxic stimulus; absence of plexiform lesions
Toxic stimulus; difficult in manipulation
Classification group mimicking
3. Pulmonary hypertension owing to lung diseases and/or hypoxia
3. Pulmonary hypertension owing to lung diseases and/or hypoxia; 1. Pulmonary arterial hypertension?
1. Pulmonary arterial hypertension
1. Pulmonary arterial hypertension
the PDGF receptor and suppressed activation of downstream signaling pathways [22]. There are genetic modified strains of mice, which mimic chronic hypoxia. 5-HTT (serotonin transporter) overexpression imitates the hypoxic stimulus and induces PH in mice, whereas mice lacking 5-HTT exhibit attenuated hypoxiainduced PH [8]. Increased serine elastase activity can contribute to hypoxic pulmonary remodeling. Overexpression of a serine elastase inhibitor – elafin – protects transgenic mice from hypoxic PH [23]. Moreover, the genetic modifications that characterize the 1st PH group of the PH classification can modulate the chronic hypoxic response. IL-6-overexpressing mice exhibit increased muscularization of the proximal arterial tree, formation of occlusive neointimal angioproliferative lesions, elevated RV systolic pressure and RV hypertrophy [5]. Chronic hypoxia possibly synergistically acts via the same downstream effectors molecules and worsens the vasculopathy in IL-6 overexpressing mice. Mice overexpressing the S100A4/Mts1 (a member of the S100 family of EF hand calcium-binding proteins) have PH under normoxic conditions and chronic hypoxia even further increases the pulmonary arterial pressure probably because it diminishes the compensatory mechanisms that protect against pulmonary remodeling [6]. Currently, mutations of the type 2 bone 92
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morphogenetic protein receptor (BMPR2) have been associated with familiar and idiopathic pulmonary arterial hypertension. Heterozygous BMPR2-deficient mice basically have a slight increase in the pulmonary arterial pressure. Chronic exposure to hypoxia, however, reduced arterial remodeling, maybe because BMPR2 mutation interferes with the hypoxiainduced proliferation of smooth muscle cells [4].
Chronic hypoxia in rats Hypoxic pulmonary remodeling in rats is more severe than in mice (Fig. 1). Microarray analysis of the lung tissue demonstrates distinct differences in gene expression induced by hypoxia between the species, which can explain a discrepancy in pathological findings [24]. Kv 1.5 (potassium) channel downregulation has been observed in rat pulmonary arterial smooth muscle cells (PASMCs). Kv 1.5 channel downregulation increases PASMC proliferation and reduces apoptosis, which contributes to obstructive vascular remodeling [25]. In addition, there is a significant role of the adventitia in hypoxic pulmonary remodeling in rats. It was described that the thickening and fibrosis of the large proximal pulmonary arteries ultimately results in stiffening vessels and consequently increase of right ventricular (RV) afterload [26]. The fawn-hooded rat discovered in 1947 has a platelet storage
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disease comparable to the Hermansky–Pudlak syndrome in humans. The genetic defect in rab38 gene of the 1st chromosome provokes the increase of plasma serotonin levels and as a consequence the animals spontaneously develop PH, which is markedly aggravated by mild hypoxia [7]. Inflammation appears to play a significant role in the hypoxia-induced remodeling process in rats. It was shown that monocytes and dendritic cells are accumulated in the vessel wall of rats exposed to chronic hypoxia together with upregulation of stromal cell-derived factor-1 (SDF-1), VEGF, growth-related oncogene protein-alpha (GRO-alpha), ICAM1, osteopontin (OPN), and transforming growth factor-beta (TGF-beta) [27].
Chronic hypoxia in combination with the VEGF 2 receptor antagonist SU-5416 Patients with idiopathic pulmonary arterial hypertension and PH associated with left-to right shunt or HIV have a unique vasculopathy with the occurrence of plexiform lesions, which consist of a network of poorly organized vascular channels surfaced by endothelial cells that may completely obliterate the vascular lumen (Fig. 1). Dysfunction of endothelial cells and alterations in VEGF (vascular endothelial growth factor) function are suggested to be responsible for the excessive angiogenesis, as chronic exposure of rats and mice to hypoxia only does not to lead to plexiform lesion formation (Table 2). It was suggested that combination of an alteration of VEGF signaling and exposure to chronic hypoxia can mimic the neointima formation and it has been successfully shown that pharmacologic inhibition of the VEGF receptor 2 with combined chronic hypoxia results in severe pulmonary arterial hypertension, which is similar to human PH vasculopathy – including neointima formation [28] (Fig. 1). The authors have shown that VEGFR-2 blockade with SU5416 initiates the selection of apoptosis resistant endothelial cells with a high proliferative potential and that chronic hypobaric hypoxia causes extensive proliferation of these cells.
The monocrotaline model The monocrotaline model is a, if not the, classical model for pulmonary hypertension (PH) and widely used as such. The preferred species for the study of monocrotalineinduced PH is currently the rat. Almost 50 years ago, Lalich and Merkow first described that oral treatment with monocrotaline induced progressive PH in rats [29]. Monocrotaline (MCT) is a phytotoxin, which is present in the seed and vegetation of the plant Crotalaria spectabilis. Lalich and Merkow discovered that ingestion of the toxic pyrrolizidine alkaloid MCT resulted in pulmonary arteritis [29]. In the liver MCT has to be activated by mixed function oxidases (cytochrome P450) to form the reactive compound MCTpyrrole (MCTP), which is a major metabolite, leading to
Drug Discovery Today: Disease Models | Pulmonary vascular disease
vascular injury in the pulmonary vessels [30]. MCTP exhibits relatively selective pulmonary vascular effects without influencing the systemic blood vessels because the lung is the first vascular bed distal of the liver. Either intravenous or subcutaneous injection of MCT or MCTP leads to an increased muscularization in precapillary pulmonary arterioles in laboratory animals. The MCT model has features of group 1 of the Dana Point classification of PH (Fig. 1; Table 1a).
Monocrotaline in rats In rats after MCT-injection a strong inflammatory reaction develops including endothelial cell death in small arterioles, a rarefaction of peripheral arteries as well as an increase of the alveoli to arteries ratio. Several hours after injection an interstitial edema of the lung including vascular leakage and an accumulation of platelets occur. The increase of mast cells can be found as well as elastolysis of the alveolar wall [30]. MCT exhibits its direct toxic effect on endothelial and interstitial cells initially damaging the pulmonary small arteries and capillaries. In the first two weeks after MCT injection, the animals appear normal and no clinical disorder can be seen. Within 2–4 weeks, their condition deteriorates involving a progressive thickening of the media in muscularized arteries (Fig. 1b) and muscularization of normally non-muscularized arteries (Fig. 1a). Together with an adventitial proliferation these changes finally lead to an increased pulmonary vascular resistance with a right ventricular systolic pressure increasing from 25 to 80 mm Hg [22]. The animals become increasingly sick displaying impaired breathing, cyanotic mucus membranes and a hunched posture. After six weeks the animals develop severe pulmonary hypertension including right ventricular hypertrophy (RVH). After six to eight weeks after MCT injection the animals die due to right heart failure. The early onset of an inflammatory response to MCT seems to be a crucial event for the development of PH in this animal model. Several authors describe the finding of a mononuclear vasculitis with increased numbers of leukocytes, which is accompanied by an increase in their activity [31]. Additionally, the number of abnormally appearing alveolar macrophages is increased. The mononuclear cells, which accumulate in the lungs of treated animals, are randomly distributed from the medial layer of the pulmonary arteries to the external layer, the adventitia. Some authors found largely macrophages with lesser numbers of lymphocytes, plasma cells, eosinophils, and mast cells. All these findings correlate with the human manifestation of the disease; several investigators found an inflammatory response in human PH patients [32]. In the experimental setting, the differential MCT sensitivity between rat strains can be problematic, and even inter-individual differences in time of onset and extent of toxic effects can vary markedly due to the pharmacokinetics of MCT. www.drugdiscoverytoday.com
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Monocrotaline in combination with pneumonectomy In contrast to the large number of vessels with neointimal remodeling observed in PH patients, a neointima does not form in the pulmonary arteries of MCT-treated animals (Fig. 1). Therefore, in 1997 Okada et al. developed an animal model combining pneumectomy with MCTtreatment to aggravate the disease [33]. With the change in hemodynamics by pneumonectomy, the injury by MCT exceeds its normal extent and leads to neointimal formation in the distal pulmonary arteries. The increased shear stress seems to be responsible for the neointimal formation in the presence of MCT [33]. Since then, several different groups have used this model to further characterize the disease mechanisms and the response to treatment.
Monocrotaline in mice As the opportunities to investigate genetic contributions to the pathophysiology of PH are limited in rats, it is desirable to use the (genetically engineered) mouse as a laboratory animal in PH models. Although it appears that mice are more resistant to the effects of MCT compared to rats, multiple groups have attempted to establish a MCT model in the mouse [34– 36]. Only liver damage, moderate pulmonary fibrosis and immunotoxicity are caused by an injection or oral application of MCT to mice [36]. As the liver metabolism of MCT to MCTP by the cytochrome P450 system differs between species, methods have been developed to synthesize MCTP chemically. In 2007, Raoul et al. reported elevated pulmonary pressure and right heart hypertrophy in mice 15 days after MCTP injection into the tail vein [35]. This could not be confirmed in a later study by Dumitrascu et al. [34]. Even with a doubled MCTP dosage compared to the study of Raoul et al., this study did not find significant changes in PH-related parameters. Only a tendency toward an elevation of pulmonary pressure and right heart hypertrophy, which was not significant compared to the control animals, was seen in this study. In the early phase histopathological changes like perivascular and interstitial lung edema and diffuse alveolar damage with alveolar hyaline membranes and inflammatory cell infiltration occur in the lungs of mice injected with MCTP [34]. Therefore it was proposed that administration of MCTP in mice is rather a model for acute lung injury than a model for PH.
Animal models of persistent pulmonary hypertension of the newborn Persistent pulmonary hypertension of the newborn (PPHN) is a cardiopulmonary disorder, which is characterized by systemic arterial hypoxemia secondary to elevated pulmonary vascular resistance. PPHN is the result of elevated pulmonary vascular resistance to the point that venous blood is diverted to some degree through the fetal channels (i.e. the 94
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ductus arteriosus and foramen ovale) into the systemic circulation bypassing the lungs. This leads to proliferation of pulmonary arterial medial smooth muscle cells and extension of muscularization to normally non-muscularized pulmonary arteries. As some infants die within the first 24 h of life, there is great need for intrauterine models of persistent pulmonary hypertension to elucidate the mechanism of this particular form of PH. The prenatal models generally employ fetal lambs. The relatively large size of the fetal lamb makes it suitable for surgical intervention and physiological study as a fetus and newborn [14]. Furthermore, uterine surgical intervention is well tolerated in the ewes. Because of these advantages, many investigations of mechanisms of the normal pulmonary vascular adaptation at birth were performed in fetal and newborn lambs. After constriction or closure of the ductus arteriosus of fetal lambs, pulmonary blood flow acutely increases. Fetal lambs born seven to fourteen days after (surgical) ductal constriction or ligation show persistent pulmonary hypertension. This implicates many pathophysiological hallmarks of PPHN also seen in the human syndrome, including a pulmonary arterial pressure equal to the aortic pressure and hypoxemia unresponsive to ventilation with 100% oxygen. Structural alterations like the spread of PASMCs into the normally non-muscularized distal arteries, as well as adventitial fibrosis surrounding the intra-acinar arteries can be seen in this model. This model has been widely studied and was used for several preclinical studies of different substances for the treatment of PPHN.
Animal models of hypercirculation induced PH Congenital heart defects that are characterized by chronic left-to-right shunting can evoke PH. The histopathological abnormalities seen in patients with PH associated with congenital systemic-to-pulmonary shunts are similar to those observed in idiopathic PH (group 1 of Dana Point classification). Surgical creation of left to right shunts to mimic some congenital heart diseases (e.g. anastomosis of the left subclavian artery to the pulmonary arterial trunk in piglets [12]; aortocaval shunt in rats; shunt between the left upper lobe pulmonary artery and the aorta in sheep [13]) leads to an increase in pulmonary blood flow and pulmonary resistance with resulting development of PH. This model is characterized by medial hypertrophy of the pulmonary arteries, abnormal extension of smooth muscle layer in the walls of more peripheral arteries, and later intimal proliferation and complete obliteration of small arteries [12,13]. Plexiform lesions have been profusely found after one and a half years in sheep with increased flow hypertension [13]. It was revealed that experimental hypercirculation-induced PH evokes increased circulating plasma endothelin-1 levels and is completely prevented by the dual endothelin receptor antagonist bosentan [12].
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Animal models of PH due to left heart disease Left heart diseases are the most frequent cause of secondary PH. Increased left atrial pressure due to the congestive heart failure produces subsequent congestion of the pulmonary venous circulation and increase in pulmonary arterial pressure (Table 1a). Animal models of congestive heart failure can be used to reproduce group 2 of the Dana Point classification of PH. Different approaches include ligation of a coronary artery to initiate the myocardial infarction, fast pacing models and inherited cardiomyopathy (e.g. LIM protein knockout) to induce PH development in different animals [15]. Despite the fact that all of these models are primarily used for investigation of the left ventricular function, they could be utilized as model for the investigation of new therapeutic approaches with regard to a treatment of secondary PH. Pulmonary arterial remodeling owing to chronic heart failure (CHF) is characterized by medial hypertrophy and lung fibrosis. The important role of angiotensin-II in pulmonary structural remodeling after myocardial infarct has been proven by showing that receptor antagonist irbesartan completely reduced the right ventricular hypertrophy [15].
Comparison of models and translation to the human system Although the animal models described above have been proven to be useful for the investigation of signaling pathways, which contribute to pulmonary vascular remodeling in PH, and help us find novel treatments, they lack important features displayed in the human disease. Both classical models do not provoke plexiform lesions, which is an important limitation of these animal models. The main advantages of classical models are the high predictability and the good reproducibility within the selected animal strain. Compared to the MCT model (group 1 of Dana Point Classification), which needs the toxic stimulus, one main advantage of the chronic hypoxia model is the physiological stimulus, which is similar to the human disease (group 3 of Dana Point Classification). The chronic hypoxia model leads to a moderate remodeling of the pulmonary vascular bed as compared to the MCT model, which evokes a more prominent vasculopathy with extensive media hypertrophy and a more than doubled increase in pulmonary artery pressure and signs of right heart failure (Fig. 1; Table 2). Compared to the MCT rat model, which resembles WHO functional classes III and IV, the chronic hypoxia model is less severe and is therefore used as a model, which resembles the symptoms of WHO functional classes I and II (Table 1b). Other in vivo animal models of PH represent special forms of pulmonary hypertension. Both PPHN and hypercirculationinduced PH models have the advantage of reproducing the exact pathophysiological disorder of humans, but carries the same limitations due to the use of very sophisticated surgical approaches. Pathological alterations can appear late – after
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many months or even years. Hypercirculation-induced PH models imitate the formation of plexiform lesions in human severe PH [13]. Animal models of PH owing to left heart diseases are perfect tools to investigate secondary PH. None of these models have been developed especially focused on PH. Additionally, heart failure models are very severe acute disorder models with a short biological period for pathological development and none of these models completely reproduces the slow progression of PH caused by CHF in humans. The knowledge obtained from animal models has been successfully confirmed in human. For example, inflammation plays a significant role in the MCT model and the same is true for various types of human PH, such as idiopathic PAH (IPAH) and PAH associated with connective tissue diseases and HIV infection [29]. Current progress in the treatment of PH illustrates how the scientific achievements based on the animal models (Table 3) ultimately result in dramatic improvement in PH patient care. More than 25 randomized clinical trials have confirmed effectiveness of three groups of drugs: prostacyclin analogues (prostanoids), endothelin receptor antagonists and phosphodiesterase type-5 (PDE-5) inhibitors in PH patients after an enormous number of experiments with different animal models (Table 3). Today, new potential therapeutic agents including inhibitors of Rho kinase, growth factor receptor inhibitors, HMG-CoA (or 3hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitors, soluble guanylate cyclase stimulator and endothelial progenitor cell administration have been tested in animal models and have showed their possible therapeutic potential (Table 3). In addition, future therapeutic agents should target RV failure, which is a main cause of disability and death of PH patients. Animal models, especially the MCT model, are a crucial tool to answer some challenging questions. Scientists should keep in mind that there is no ideal animal model which perfectly mimics all forms of PH. Application of different triggers simulates various forms of PH with a large number of cellular and molecular alterations (Table 1a and b). In spite of the diversity of initial factors/triggers the downstream effects of different stimuli could be analogous. The comparison of different animal models is therefore necessary to understand the differential pathway impact in PH development. Using both classical models simulates different stages of disease progression from a moderate hypertension to the severe RV failure, and allows to try various preventive and, even more important, curative therapeutic approaches. However, not all novel drugs, which effectively reverse PH in the animal models, will work in human PH. Thus, successful therapeutic effects observed in at least two different animal models make success of translation to human PH more likely. For that reason, from our point of view, it is important to use both most common animal models of PH (rat and mouse) in new therapeutic agent trials to decrease the frequency of false positive results. www.drugdiscoverytoday.com
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Table 3. The animal models of PH and translation research Animal model Species
Translation to human Pathway/agent/year
Effect
Trial/agent/year
Effect
Drugs used in clinical practice Neonatal piglets
Prostacyclin and analogues/Prostacylin/1978
Decreases hypoxic pulmonary vasoconstriction by prostacyclin infusion [37].
PPHSG/Epoprostenol/1996
Improves symptoms and survival [38].
Rats
Endothelin receptor antagonist/BQ 123/1996
Attenuates hypoxic PH [39].
BENEFiT/Bosentan/2002
Improves exercise capacity and symptoms [40].
Mice
PDE-5 inhibitors/ Sildenafil/2001
Attenuates hypoxic PH [41].
SUPER/sildenafil/2005
Improves exercise capacity, WHO functional class, and hemodynamics [42].
New potential agents Rats
HMG-CoA reductase inhibitor/Simvastatin/2002
Attenuates pulmonary remodeling in MCT + pneumonectomy PH [43].
SiPHT/Simvastatin/2010
Added to conventional therapy produces a small and transient early reduction in RV mass [44].
Mice Rats
Soluble guanylate cyclase stimulator/BAY 63-252/2008
Reverses hypoxic PH in mice and MCT PH in rats [3].
Phase 2 trial/Riociguat (Bay 63-252)/2010
Improves exercise capacity and symptoms [45].
Mice Rats
PDGF inhibitor/Imatinib/2005
Reverses hypoxic PH in mice and MCT PH in rats [22].
Phase 2/Imatinib/2010
Improves hemodynamic [46].
Rats
Serine-threonine kinase inhibition/Sorafenib/2008
Prevents pulmonary remodeling in MCT PH in rats [47].
Phase Ib/Sorafenib/2010
Well tolerated [48].
Mice Rats
Rho-kinase inhibitor/ Azaindole-1/2010
Reverses hypoxic PH in mice and MCT PH in rats [49].
–
–
Conclusion The complexity of pulmonary hypertension and the lack of a robust model of PH do not allow the use of a single inbred animal model of PH to unravel all possible pathways leading to PH development in the human population. Moreover, no animal model reproduces the full spectrum of pathological changes seen in the human lung obtained from PH patients. In our opinion the different models have different strengths and weaknesses. Therefore, before translating the knowledge gained in animal models to the human system, more than one experimental animal model needs to be used. Nevertheless, animal models can be used as valuable scientific tools to take a major step in the understanding of PH development and effective treatment improvement.
References 1 McLaughlin, V.V. et al. (2009) ACCF/AHA 2009 expert consensus document on pulmonary hypertension a report of the American College of Cardiology Foundation Task Force on Expert Consensus Documents and the American Heart Association developed in collaboration with the American College of Chest Physicians; American Thoracic Society, Inc.; and the Pulmonary Hypertension Association. J. Am. Coll. Cardiol. 53, 1573–1619 2 Schroll, S. et al. (2010) Improvement of bleomycin-induced pulmonary hypertension and pulmonary fibrosis by the endothelin receptor antagonist Bosentan. Respir. Physiol. Neurobiol. 170, 32–36
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3 Schermuly, R.T. et al. (2008) Expression and function of soluble guanylate cyclase in pulmonary arterial hypertension. Eur. Respir. J. 32, 881–891 4 Beppu, H. et al. (2004) BMPR-II heterozygous mice have mild pulmonary hypertension and an impaired pulmonary vascular remodeling response to prolonged hypoxia. Am. J. Physiol. Lung Cell Mol. Physiol. 287, L1241–L1247 5 Steiner, M.K. et al. (2004) Interleukin-6 overexpression induces pulmonary hypertension. Circ. Res. 104, 236–244 28p 6 Greenway, S. et al. (2004) S100A4/Mts1 produces murine pulmonary artery changes resembling plexogenic arteriopathy and is increased in human plexogenic arteriopathy. Am. J. Pathol. 164, 253–262 7 Bonnet, S. et al. (2006) An abnormal mitochondrial-hypoxia inducible factor-1alpha-Kv channel pathway disrupts oxygen sensing and triggers pulmonary arterial hypertension in fawn hooded rats: similarities to human pulmonary arterial hypertension. Circulation 113, 2630–2641 8 Guignabert, C. et al. (2006) Transgenic mice overexpressing the 5hydroxytryptamine transporter gene in smooth muscle develop pulmonary hypertension. Circ. Res. 98, 1323–1330 9 Mittal, M. et al. (2007) Hypoxia-dependent regulation of nonphagocytic NADPH oxidase subunit NOX4 in the pulmonary vasculature. Circ. Res. 101, 258–267 10 McGuire, M. and Bradford, A. (2001) Chronic intermittent hypercapnic hypoxia increases pulmonary arterial pressure and haematocrit in rats. Eur. Respir. J. 18, 279–285 11 Wright, J.L. et al. (2006) Vasoactive mediators and pulmonary hypertension after cigarette smoke exposure in the guinea pig. J. Appl. Physiol. 100, 672–678 12 Rondelet, B. et al. (2003) Bosentan for the prevention of overcirculationinduced experimental pulmonary arterial hypertension. Circulation 107, 1329–1335
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Schnader, J. et al. (1996) Chronic pulmonary hypertension in sheep: temporal progression of lesions. J. Surg. Res. 62, 243–250 Steinhorn, R. et al. (1997) Models of persistent pulmonary hypertension of the newborn (PPHN) and the role of cyclic guanosine monophosphate (GMP) in pulmonary vasorelaxation. Semin. Perinatol. 21, 393–408 Jasmin, J. et al. (2003) Lung structural remodeling and pulmonary hypertension after myocardial infarction: complete reversal with irbesartan. Cardiovasc. Res. 58, 621–631 Sehgal, P.B. et al. (2009) Golgi dysfunction is a common feature in idiopathic human pulmonary hypertension and vascular lesions in SHIVnef-infected macaques. Am. J. Physiol. Lung Cell Mol. Physiol. 297, L729–L737 Crosby, A. et al. (2010) Pulmonary vascular remodeling correlates with lung eggs and cytokines in murine schistosomiasis. Am. J. Respir. Crit. Care Med. 181, 279–288 Zagorski, J. et al. (2003) Chemokines accumulate in the lungs of rats with severe pulmonary embolism induced by polystyrene microspheres. J. Immunol. 171, 5529–5536 Sommer, N. et al. (2008) Regulation of hypoxic pulmonary vasoconstriction: basic mechanisms. Eur. Respir. J. 32, 1639–1651 Weissmann, N. et al. (2006) Hypoxia in lung vascular biology and disease. Cardiovasc. Res. 71, 618–619 Schermuly, R.T. et al. (2007) Phosphodiesterase 1 upregulation in pulmonary arterial hypertension: target for reverse-remodeling therapy. Circulation 115, 2331–2339 Schermuly, R.T. et al. (2005) Reversal of experimental pulmonary hypertension by PDGF inhibition. J. Clin. Invest. 115, 2811–2821 Zaidi, S.H. (2002) Overexpression of the serine elastase inhibitor elafin protects transgenic mice from hypoxic pulmonary hypertension. Circulation 105, 516–521 Bull, T.M. et al. (2007) Gene expression profiling in pulmonary hypertension. Proc. Am. Thorac. Soc. 4, 117–120 Platoshyn, O. et al. (2001) Chronic hypoxia decreases K(V) channel expression and function in pulmonary artery myocytes. Am. J. Physiol. Lung Cell Mol. Physiol. 280, L801–L812 Stenmark, K.R. et al. (2009) Animal models of pulmonary arterial hypertension: the hope for etiological discovery and pharmacological cure. Am. J. Physiol. Lung Cell Mol. Physiol. 297, L1013–L1032 Burke, D.L. et al. (2009) Sustained hypoxia promotes the development of a pulmonary artery-specific chronic inflammatory microenvironment. Am. J. Physiol. Lung Cell Mol. Physiol. 297, L238–L250 Taraseviciene-Stewart, L. et al. (2001) Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension. FASEB J. 15, 427–438 Lalich, J.J. and Merkov, L. (1961) Pulmonary arteritis produced in rat by feeding Crotalaria spectabilis. Lab Invest. 10, 744–750 Marsboom, G. and Janssens, S. (2004) Models for pulmonary hypertension. Drug Discover Today: Disease Models 1, 289–296 Stenmark, K.R. et al. (1985) Alveolar inflammation and arachidonate metabolism in monocrotaline-induced pulmonary hypertension. Am. J. Physiol. 248, H859–H866
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Dorfmuller, P. et al. (2003) Inflammation in pulmonary arterial hypertension. Eur. Respir. J. 22, 358–363 Okada, K. et al. (1997) Pulmonary hemodynamics modify the rat pulmonary artery response to injury. A neointimal model of pulmonary hypertension. Am. J. Pathol. 151, 1019–1025 Dumitrascu, R. et al. (2008) Characterization of a murine model of monocrotaline pyrrole-induced acute lung injury. BMC. Pulm. Med. 8, 25 Raoul, W. et al. (2007) Effects of bone marrow-derived cells on monocrotaline- and hypoxia-induced pulmonary hypertension in mice. Respir. Res. 8, 8 Molteni, A. et al. (1989) Monocrotaline pneumotoxicity in mice. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 57, 149–155 Starling, M.B. et al. (1979) Control of elevated pulmonary vascular resistance in neonatal swine with prostacyclin (PGI2). Prostaglandins Med. 3, 105–117 Barst, R.J. et al. (1996) A comparison of continuous intravenous epoprostenol (prostacyclin) with conventional therapy for primary pulmonary hypertension. The Primary Pulmonary Hypertension Study Group. N. Engl. J. Med. 334, 296–302 Bonvallet, S.T. et al. (1994) BQ123, an ETA-receptor antagonist, attenuates hypoxic pulmonary hypertension in rats. Am. J. Physiol. 266, H1327– H1331 Jais, X. et al. (2008) Bosentan for treatment of inoperable chronic thromboembolic pulmonary hypertension: BENEFiT (Bosentan Effects in iNopErable Forms of chronIc Thromboembolic pulmonary hypertension), a randomized, placebo-controlled trial. J. Am. Coll. Cardiol. 52, 2127–2134 Zhao, L. et al. (2001) Sildenafil inhibits hypoxia-induced pulmonary hypertension. Circulation 104, 424–428 Galie, N. et al. (2005) Sildenafil citrate therapy for pulmonary arterial hypertension. N. Engl. J. Med. 353, 2148–2157 Nishimura, T. et al. (2002) Simvastatin attenuates smooth muscle neointimal proliferation and pulmonary hypertension in rats. Am. J. Respir. Crit. Care Med. 166, 1403–1408 Wilkins, M.R. et al. (2010) Simvastatin as a treatment for pulmonary hypertension trial. Am. J. Respir. Crit. Care Med. 181, 1106–1113 Ghofrani, H.A. et al. (2010) Riociguat for chronic thromboembolic pulmonary hypertension and pulmonary arterial hypertension: a phase II study. Eur. Respir. J. 36, 792–799 Ghofrani, H.A. et al. (2010) Imatinib in Pulmonary Arterial Hypertension Patients with Inadequate Response to Established Therapy. Am. J. Respir. Crit. Care Med. 182, 1171–1177 Klein, M. et al. (2008) Combined tyrosine and serine/threonine kinase inhibition by sorafenib prevents progression of experimental pulmonary hypertension and myocardial remodeling. Circulation 118, 2081–2090 Gomberg-Maitland, M. et al. (2010) A dosing/cross-development study of the multikinase inhibitor sorafenib in patients with pulmonary arterial hypertension. Clin. Pharmacol. Ther. 87, 303–310 Dahal, B.K. et al. (2010) Therapeutic efficacy of azaindole-1 in experimental pulmonary hypertension. Eur Respir J. 36, 808–818
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Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Andrew McCulloch – University of California, SanDiego, USA
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Pulmonary vascular disease
Animal models of pulmonary hypertension: role in translational research Oleg Pak1,3, Wiebke Janssen2,3, Hossein Ardeschir Ghofrani1, Werner Seeger1,2, Friedrich Grimminger1, Ralph Theo Schermuly1,2, Norbert Weissmann1,* 1
Excellence Cluster Cardio-Pulmonary System, University of Giessen Lung Center, Dept. of Internal Medicine II/IV/V, Justus-Liebig-University Giessen, Aulweg 130, 35392 Giessen, Germany 2 Max-Planck-Institute for Heart and Lung Research, Parkstr. 1, 61231 Bad Nauheim, Germany
Pulmonary hypertension (PH) is a severe progressive disorder with an unclear etiology and a poor prognosis. Current treatments can alleviate the symptoms and even revert the characteristic vascular remodeling process, but cannot cure the disease. A variety of
Section editors: Jason Yuan – Department of Medicine and Institute for Personalized Respiratory Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA Amy Firth – Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA
animal models have been suggested to mimic PH and
successful
translation
of
new
therapeutic
approaches from bench to bedside demonstrates the value of such models. Our review highlights the role of different animal models of PH in translational research. Introduction Pulmonary hypertension (PH) is a severe progressive disorder characterized by extensive narrowing of the pulmonary vascular bed, leading to an increase in pulmonary vascular resistance, which ultimately produces a compensatory right ventricular hypertrophy and results in heart failure and premature death [1]. A variety of conditions can lead to the development of PH. The modern classification of PH groups different diseases, which share similarities in pathophysiological mechanisms, clinical manifestation, and therapeutic approaches in five groups. These 5 groups are: 1st – pulmonary arterial hypertension group; 2nd – PH associated with left*Corresponding author.: N. Weissmann ([email protected]) 3 Both these authors contributed equally. 1740-6757/$ ß 2011 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmod.2011.02.002
sided heart diseases; 3rd – PH related with hypoxemia/lung diseases, 4th – thromboembolic PH and 5th – PH with unclear multifactorial mechanisms [1] (Table 1a). The pathogenesis of PH is poorly understood and reliable treatment is still lacking. Current treatments can improve clinical symptoms, but cannot cure PH.
Animal models of pulmonary hypertension Animal models of PH are used expansively in current research. These models have been the source of a plethora of scientific information, such as the role of certain molecular mechanisms and genetic contributions in PH. Additionally, animal models have been utilized in the discovery and testing of possible therapeutic approaches. PH can be induced in the animal either through pharmacologic/toxic agents [2,3], genetic techniques [4–8], environmental factors [9–11] or through surgical interventions [12–15] (Table 1a). The most commonly used animal models of PH are the injection of monocrotaline and the chronic exposure to hypoxia (classical models). There are a variety of other 89
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Table 1. Current classification of PH and animal models (a) Revised Dana Point classification of PH and animal models of PH Group of PH
Animal models
1. Pulmonary arterial hypertension (PAH)
MCT [21]; MCT + pneumonectomy [33]; BMPR2 knockout [4]; Tg+ mice [5]; Fawn-hooded rat [7]; S100A4 overexpressing [6]; SHIV-nef-infected macaques [16]; hypoxia + SU-5416 [28]; Schistosomiasis [17]; left-to-right shunt [12,13], closure of the ductus arteriosus [14]
2. Pulmonary hypertension with left heart disease
Congestive heart failure models [15]
3. Pulmonary hypertension associated with lung diseases and/or hypoxemia
Chronic hypoxia [9], hypoxia + SU-5416 [28]; Intermittent hypoxia [10]; Smoke exposure [11]; Bleomycin [2]; 5-HTT overexpression [8]
4. Pulmonary hypertension owing to chronic thrombotic and/or embolic disease
Repeated microembolization with microspheres [18]
5. Miscellaneous
?
(b) WHO functional class assessment classification WHO functional assessment classification
Animal model
Class I
Patients with PH but without resulting limitation of physical activity. Ordinary physical activity does not cause undue dyspnea or fatigue, chest pain, or near syncope.
Hypoxia
Class II
Patients with PH resulting in slight limitation of physical activity. They are comfortable at rest. Ordinary physical activity causes undue dyspnea or fatigue, chest pain, or near syncope.
Hypoxia/hypoxia + SU-5416
Class III
Patients with PH resulting in marked limitation of physical activity. They are comfortable at rest. Less than ordinary activity causes undue dyspnea or fatigue, chest pain, or near syncope.
MCT
Class IV
Patients with PH with inability to carry out any physical activity without symptoms. These patients manifest signs of right heart failure. Dyspnea and/or fatigue may even be present at rest. Discomfort is increased by any physical activity.
MCT/MCT + pneumectomy
in vivo animal models of PH that mimic features of different PH groups: 1st group – bone morphogenetic protein receptor 2 (BMPR2) knockout mice [4], transgenic mice overexpressing Interleukin 6 [5], S100A4 (Mts1) overexpressing mice [6], SHIV-nef-infected macaques (a chimeric viral construct containing the HIV nef gene in a simian immunodeficiency virus backbone) [16], Fawn-hooded rats [7], schistosomiasisinduction of PH [17], experimental left-to-right shunt [12,13], closure of the ductus arteriosus [14]; 2nd group – congestive heart failure (CHF) models [15]; 3rd group – intermittent hypoxia [10], cigarette smoke exposure [11], bleomycin application [2], 5-hydroxytryptamine transporter protein (5-HTT) overexpression [8]; 4th group – repeated microembolization with microspheres [18] (Table 1a). Our review is focused on in vivo models of PH and especially on the classical models, because they are the most frequently used models in translational research due to their good reproducibility and their well described histopathology (Fig. 1).
Chronic hypoxia Chronic hypoxia is the most commonly used physiological stimulus for PH development in animal models. Since 1946 it has been known that acute hypoxia is a trigger of hypoxic pulmonary vasoconstriction (HPV), which redistributes the pulmonary blood flow from poorly to better ventilated areas 90
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of the lung to maintain the ventilation-perfusion match in adults [19]. By contrast, systemic vessels of adults dilate during hypoxia. Global chronic alveolar hypoxia that occurs in permanent residents of high altitude or in pathological conditions and diseases leading to alveolar hypoxia (group 3 of the Dana Point classification of PH), may result in pulmonary vascular remodeling and PH development. Normobaric hypoxia (reduction of oxygen partial pressure at sea level to approximately 10%) and hypobaric hypoxia (e.g. 400 mm Hg atmospheric pressure, simulating high altitude) exposure for 2 weeks and more induces PH in a wide variety of animal species including rats, mice, guinea pigs, dogs, cows, pigs and sheep. The most commonly used species in laboratory models of PH are rats and mice. This experimental model represents the group 3 of the Dana Point classification of PH (Table 1a). The most common pathological findings are muscularization of previously nonmuscularized vessels (Fig. 1a) and a moderate medial thickening of muscular resistance vessels (Fig. 1b; Table 2). Both pulmonary smooth muscle cells (PASMC) and adventitial fibroblasts proliferate under hypoxia. These features are largely reversible by return to normoxia. The details of the molecular steps of the hypoxic pulmonary vasculature remodeling from the oxygen sensing to cell proliferation are not yet fully understood. The hypoxia-inducible transcription factors (HIF) play a major role in this process [20].
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Drug Discovery Today: Disease Models | Pulmonary vascular disease
Pulmonary artery remodeling in animal models compared to IPAH
63x,vessels <100 µm EE EI N
L M
A
IPAH
Mouse HOX
Rat HOX
Donor control
Mouse NOX
Rat NOX
Rat HOX + SU-5416
Rat NOX
MCT Rat
MCT + pneumonectomy
Rat control
Rat control
MCT Rat
MCT + pneumonectomy
Rat control
Rat control
40x,vessels < 200 µm
PL N
IPAH
Mouse HOX
Rat HOX
Donor control
Mouse NOX
Rat NOX
Rat HOX + SU-5416
Rat NOX
Staining - elastic van Gieson. Elastic fibres – blue/black/brown; nuclei - black/brown; collagen fibres – red; media, epithelia, nerves, erythrocytes – yellow IPAH – idiopathic pulmonary arterial hypertension; HOX – 3 weeks hypoxic exposure, NOX – normoxic control; MCT – monocrotaline injection; EE – elastica externa; EI elastica interna; L – lumen; N – neointima, M – media; A – adventitia; PL – plexiform lesion. Drug Discovery Today: Disease Models
Figure 1. Staining, elastic van Gieson. Elastic fibres, blue/black/brown; nuclei, black/brown; collagen fibres, red; media, epithelia, nerves, erythrocytes, yellow; IPAH, idiopathic pulmonary arterial hypertension; HOX, 3 weeks hypoxic exposure; NOX, normoxic control; MCT, monocrotaline injection; EE, elastica externa; EI, elastica interna; L, lumen; N, neointima, M, media; A adventitia; PL, plexiform lesion.
Chronic hypoxia in mice In 1900, the French biologist Lucien Cuenot used mice to repeat Mendel’s work in plants. Since then mice have become the most widespread model in research culminating with the creation of knockout mice in 1987 and the publication of the mice genome sequence in 2002. Chronic exposure of mice to normobaric or hypobaric hypoxia leads to an elevation in pulmonary artery pressure, vascular remodeling and RV hypertrophy (Fig. 1) [9]. The role of the nitric oxide (NO) pathway, reactive oxygen species (ROS), and cytoskeletal architecture in PH development has been investigated in the hypoxic mouse model [3,9,21]. NO is a vasodilatator and an important signaling molecule. Soluble guanylyl cyclase (sGC) is the known receptor for NO. Upregulation of sGC was detected in the structurally remodeled smooth muscle layer in chronic hypoxic mice lungs. Treatment of wild-type mice with the activator of sGC, or the stimulator of sGC after full establishment of PH significantly reduced pulmonary pressure, RV hypertrophy, and structural remodeling of the lung
vasculature. Additionally, an endogenous inhibitor of the nitric oxide (NO) pathway, PDE1A (phosphodiesterase 1 A) was significantly upregulated in pulmonary arterial smooth muscle cells (PASMC) of chronically hypoxic mice and the PDE1 inhibitor 8MM-IBMX reversed chronic PH [21]. ROS could play a role in hypoxic pulmonary vasoconstriction and PH development [19]. It was shown that NOX4 (NADPH oxidase 4) mRNA is upregulated under hypoxia in homogenized lung tissue, concomitant with increased levels in microdissected pulmonary arterial vessels of mice. Treatment of PASMCs with siRNA directed against NOX4 decreased NOX4 mRNA levels and reduced PASMC proliferation as well as generation of ROS [9]. Growth factors regulate diverse physiological processes and it was suggested that disturbance in their regulation, expression or alterations in downstream effects can contribute to the development of PH. It was reported that the platelet derived growth factor (PDGF) receptor antagonist STI571 (imatinib) reversed advanced pulmonary vascular disease in a mouse model. STI571 prevented phosphorylation of www.drugdiscoverytoday.com
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Table 2. Comparison of different PH animal models Animal model Chronic hypoxia
Chronic hypoxia + SU-5416
Monocrotaline
Monocrotaline + pneumonectomy
Species
Rat and mice
Rat
Rat
Rat
Stimulus
(1) Physiological – hypoxia
(1) Physiological – hypoxia (2) Chemical – VEGF inhibitor
(1) Toxic – MCT
(1) Toxic – MCT (2) Physiological – increased flow
Main histological features
(1) Moderate increased media of muscularized pulmonary arteries (Fig. 1b)
(1) Moderate increased media of muscularized pulmonary arteries (Fig. 1b)
(2) Muscularization of normally non-muscular arteries (Fig. 1a)
(2) Muscularization of normally non-muscular arteries (Fig. 1a)
(1) Prominent increased media of muscularized pulmonary arteries (Fig. 1b) (2) Muscularization of normally non-muscular arteries (Fig. 1a)
(1) Prominent increased media of muscularized pulmonary arteries (Fig. 1b) (2) Muscularization of normally non-muscular arteries (Fig. 1a) (3) Neointima formation
(3) Neointima formation Pro
Physiological stimulus; predictable and reproducible
Physiological stimulus; proliferation of endothelial cells
Severe PH; RV failure model; predictable and reproducible
Severe PH; RV failure model; proliferation of endothelial cells
Con
Moderate PH; using more severe hypoxia than in human; absence of plexiform lesion
Not clear which group mimics
Toxic stimulus; absence of plexiform lesions
Toxic stimulus; difficult in manipulation
Classification group mimicking
3. Pulmonary hypertension owing to lung diseases and/or hypoxia
3. Pulmonary hypertension owing to lung diseases and/or hypoxia; 1. Pulmonary arterial hypertension?
1. Pulmonary arterial hypertension
1. Pulmonary arterial hypertension
the PDGF receptor and suppressed activation of downstream signaling pathways [22]. There are genetic modified strains of mice, which mimic chronic hypoxia. 5-HTT (serotonin transporter) overexpression imitates the hypoxic stimulus and induces PH in mice, whereas mice lacking 5-HTT exhibit attenuated hypoxiainduced PH [8]. Increased serine elastase activity can contribute to hypoxic pulmonary remodeling. Overexpression of a serine elastase inhibitor – elafin – protects transgenic mice from hypoxic PH [23]. Moreover, the genetic modifications that characterize the 1st PH group of the PH classification can modulate the chronic hypoxic response. IL-6-overexpressing mice exhibit increased muscularization of the proximal arterial tree, formation of occlusive neointimal angioproliferative lesions, elevated RV systolic pressure and RV hypertrophy [5]. Chronic hypoxia possibly synergistically acts via the same downstream effectors molecules and worsens the vasculopathy in IL-6 overexpressing mice. Mice overexpressing the S100A4/Mts1 (a member of the S100 family of EF hand calcium-binding proteins) have PH under normoxic conditions and chronic hypoxia even further increases the pulmonary arterial pressure probably because it diminishes the compensatory mechanisms that protect against pulmonary remodeling [6]. Currently, mutations of the type 2 bone 92
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morphogenetic protein receptor (BMPR2) have been associated with familiar and idiopathic pulmonary arterial hypertension. Heterozygous BMPR2-deficient mice basically have a slight increase in the pulmonary arterial pressure. Chronic exposure to hypoxia, however, reduced arterial remodeling, maybe because BMPR2 mutation interferes with the hypoxiainduced proliferation of smooth muscle cells [4].
Chronic hypoxia in rats Hypoxic pulmonary remodeling in rats is more severe than in mice (Fig. 1). Microarray analysis of the lung tissue demonstrates distinct differences in gene expression induced by hypoxia between the species, which can explain a discrepancy in pathological findings [24]. Kv 1.5 (potassium) channel downregulation has been observed in rat pulmonary arterial smooth muscle cells (PASMCs). Kv 1.5 channel downregulation increases PASMC proliferation and reduces apoptosis, which contributes to obstructive vascular remodeling [25]. In addition, there is a significant role of the adventitia in hypoxic pulmonary remodeling in rats. It was described that the thickening and fibrosis of the large proximal pulmonary arteries ultimately results in stiffening vessels and consequently increase of right ventricular (RV) afterload [26]. The fawn-hooded rat discovered in 1947 has a platelet storage
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disease comparable to the Hermansky–Pudlak syndrome in humans. The genetic defect in rab38 gene of the 1st chromosome provokes the increase of plasma serotonin levels and as a consequence the animals spontaneously develop PH, which is markedly aggravated by mild hypoxia [7]. Inflammation appears to play a significant role in the hypoxia-induced remodeling process in rats. It was shown that monocytes and dendritic cells are accumulated in the vessel wall of rats exposed to chronic hypoxia together with upregulation of stromal cell-derived factor-1 (SDF-1), VEGF, growth-related oncogene protein-alpha (GRO-alpha), ICAM1, osteopontin (OPN), and transforming growth factor-beta (TGF-beta) [27].
Chronic hypoxia in combination with the VEGF 2 receptor antagonist SU-5416 Patients with idiopathic pulmonary arterial hypertension and PH associated with left-to right shunt or HIV have a unique vasculopathy with the occurrence of plexiform lesions, which consist of a network of poorly organized vascular channels surfaced by endothelial cells that may completely obliterate the vascular lumen (Fig. 1). Dysfunction of endothelial cells and alterations in VEGF (vascular endothelial growth factor) function are suggested to be responsible for the excessive angiogenesis, as chronic exposure of rats and mice to hypoxia only does not to lead to plexiform lesion formation (Table 2). It was suggested that combination of an alteration of VEGF signaling and exposure to chronic hypoxia can mimic the neointima formation and it has been successfully shown that pharmacologic inhibition of the VEGF receptor 2 with combined chronic hypoxia results in severe pulmonary arterial hypertension, which is similar to human PH vasculopathy – including neointima formation [28] (Fig. 1). The authors have shown that VEGFR-2 blockade with SU5416 initiates the selection of apoptosis resistant endothelial cells with a high proliferative potential and that chronic hypobaric hypoxia causes extensive proliferation of these cells.
The monocrotaline model The monocrotaline model is a, if not the, classical model for pulmonary hypertension (PH) and widely used as such. The preferred species for the study of monocrotalineinduced PH is currently the rat. Almost 50 years ago, Lalich and Merkow first described that oral treatment with monocrotaline induced progressive PH in rats [29]. Monocrotaline (MCT) is a phytotoxin, which is present in the seed and vegetation of the plant Crotalaria spectabilis. Lalich and Merkow discovered that ingestion of the toxic pyrrolizidine alkaloid MCT resulted in pulmonary arteritis [29]. In the liver MCT has to be activated by mixed function oxidases (cytochrome P450) to form the reactive compound MCTpyrrole (MCTP), which is a major metabolite, leading to
Drug Discovery Today: Disease Models | Pulmonary vascular disease
vascular injury in the pulmonary vessels [30]. MCTP exhibits relatively selective pulmonary vascular effects without influencing the systemic blood vessels because the lung is the first vascular bed distal of the liver. Either intravenous or subcutaneous injection of MCT or MCTP leads to an increased muscularization in precapillary pulmonary arterioles in laboratory animals. The MCT model has features of group 1 of the Dana Point classification of PH (Fig. 1; Table 1a).
Monocrotaline in rats In rats after MCT-injection a strong inflammatory reaction develops including endothelial cell death in small arterioles, a rarefaction of peripheral arteries as well as an increase of the alveoli to arteries ratio. Several hours after injection an interstitial edema of the lung including vascular leakage and an accumulation of platelets occur. The increase of mast cells can be found as well as elastolysis of the alveolar wall [30]. MCT exhibits its direct toxic effect on endothelial and interstitial cells initially damaging the pulmonary small arteries and capillaries. In the first two weeks after MCT injection, the animals appear normal and no clinical disorder can be seen. Within 2–4 weeks, their condition deteriorates involving a progressive thickening of the media in muscularized arteries (Fig. 1b) and muscularization of normally non-muscularized arteries (Fig. 1a). Together with an adventitial proliferation these changes finally lead to an increased pulmonary vascular resistance with a right ventricular systolic pressure increasing from 25 to 80 mm Hg [22]. The animals become increasingly sick displaying impaired breathing, cyanotic mucus membranes and a hunched posture. After six weeks the animals develop severe pulmonary hypertension including right ventricular hypertrophy (RVH). After six to eight weeks after MCT injection the animals die due to right heart failure. The early onset of an inflammatory response to MCT seems to be a crucial event for the development of PH in this animal model. Several authors describe the finding of a mononuclear vasculitis with increased numbers of leukocytes, which is accompanied by an increase in their activity [31]. Additionally, the number of abnormally appearing alveolar macrophages is increased. The mononuclear cells, which accumulate in the lungs of treated animals, are randomly distributed from the medial layer of the pulmonary arteries to the external layer, the adventitia. Some authors found largely macrophages with lesser numbers of lymphocytes, plasma cells, eosinophils, and mast cells. All these findings correlate with the human manifestation of the disease; several investigators found an inflammatory response in human PH patients [32]. In the experimental setting, the differential MCT sensitivity between rat strains can be problematic, and even inter-individual differences in time of onset and extent of toxic effects can vary markedly due to the pharmacokinetics of MCT. www.drugdiscoverytoday.com
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Monocrotaline in combination with pneumonectomy In contrast to the large number of vessels with neointimal remodeling observed in PH patients, a neointima does not form in the pulmonary arteries of MCT-treated animals (Fig. 1). Therefore, in 1997 Okada et al. developed an animal model combining pneumectomy with MCTtreatment to aggravate the disease [33]. With the change in hemodynamics by pneumonectomy, the injury by MCT exceeds its normal extent and leads to neointimal formation in the distal pulmonary arteries. The increased shear stress seems to be responsible for the neointimal formation in the presence of MCT [33]. Since then, several different groups have used this model to further characterize the disease mechanisms and the response to treatment.
Monocrotaline in mice As the opportunities to investigate genetic contributions to the pathophysiology of PH are limited in rats, it is desirable to use the (genetically engineered) mouse as a laboratory animal in PH models. Although it appears that mice are more resistant to the effects of MCT compared to rats, multiple groups have attempted to establish a MCT model in the mouse [34– 36]. Only liver damage, moderate pulmonary fibrosis and immunotoxicity are caused by an injection or oral application of MCT to mice [36]. As the liver metabolism of MCT to MCTP by the cytochrome P450 system differs between species, methods have been developed to synthesize MCTP chemically. In 2007, Raoul et al. reported elevated pulmonary pressure and right heart hypertrophy in mice 15 days after MCTP injection into the tail vein [35]. This could not be confirmed in a later study by Dumitrascu et al. [34]. Even with a doubled MCTP dosage compared to the study of Raoul et al., this study did not find significant changes in PH-related parameters. Only a tendency toward an elevation of pulmonary pressure and right heart hypertrophy, which was not significant compared to the control animals, was seen in this study. In the early phase histopathological changes like perivascular and interstitial lung edema and diffuse alveolar damage with alveolar hyaline membranes and inflammatory cell infiltration occur in the lungs of mice injected with MCTP [34]. Therefore it was proposed that administration of MCTP in mice is rather a model for acute lung injury than a model for PH.
Animal models of persistent pulmonary hypertension of the newborn Persistent pulmonary hypertension of the newborn (PPHN) is a cardiopulmonary disorder, which is characterized by systemic arterial hypoxemia secondary to elevated pulmonary vascular resistance. PPHN is the result of elevated pulmonary vascular resistance to the point that venous blood is diverted to some degree through the fetal channels (i.e. the 94
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ductus arteriosus and foramen ovale) into the systemic circulation bypassing the lungs. This leads to proliferation of pulmonary arterial medial smooth muscle cells and extension of muscularization to normally non-muscularized pulmonary arteries. As some infants die within the first 24 h of life, there is great need for intrauterine models of persistent pulmonary hypertension to elucidate the mechanism of this particular form of PH. The prenatal models generally employ fetal lambs. The relatively large size of the fetal lamb makes it suitable for surgical intervention and physiological study as a fetus and newborn [14]. Furthermore, uterine surgical intervention is well tolerated in the ewes. Because of these advantages, many investigations of mechanisms of the normal pulmonary vascular adaptation at birth were performed in fetal and newborn lambs. After constriction or closure of the ductus arteriosus of fetal lambs, pulmonary blood flow acutely increases. Fetal lambs born seven to fourteen days after (surgical) ductal constriction or ligation show persistent pulmonary hypertension. This implicates many pathophysiological hallmarks of PPHN also seen in the human syndrome, including a pulmonary arterial pressure equal to the aortic pressure and hypoxemia unresponsive to ventilation with 100% oxygen. Structural alterations like the spread of PASMCs into the normally non-muscularized distal arteries, as well as adventitial fibrosis surrounding the intra-acinar arteries can be seen in this model. This model has been widely studied and was used for several preclinical studies of different substances for the treatment of PPHN.
Animal models of hypercirculation induced PH Congenital heart defects that are characterized by chronic left-to-right shunting can evoke PH. The histopathological abnormalities seen in patients with PH associated with congenital systemic-to-pulmonary shunts are similar to those observed in idiopathic PH (group 1 of Dana Point classification). Surgical creation of left to right shunts to mimic some congenital heart diseases (e.g. anastomosis of the left subclavian artery to the pulmonary arterial trunk in piglets [12]; aortocaval shunt in rats; shunt between the left upper lobe pulmonary artery and the aorta in sheep [13]) leads to an increase in pulmonary blood flow and pulmonary resistance with resulting development of PH. This model is characterized by medial hypertrophy of the pulmonary arteries, abnormal extension of smooth muscle layer in the walls of more peripheral arteries, and later intimal proliferation and complete obliteration of small arteries [12,13]. Plexiform lesions have been profusely found after one and a half years in sheep with increased flow hypertension [13]. It was revealed that experimental hypercirculation-induced PH evokes increased circulating plasma endothelin-1 levels and is completely prevented by the dual endothelin receptor antagonist bosentan [12].
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Animal models of PH due to left heart disease Left heart diseases are the most frequent cause of secondary PH. Increased left atrial pressure due to the congestive heart failure produces subsequent congestion of the pulmonary venous circulation and increase in pulmonary arterial pressure (Table 1a). Animal models of congestive heart failure can be used to reproduce group 2 of the Dana Point classification of PH. Different approaches include ligation of a coronary artery to initiate the myocardial infarction, fast pacing models and inherited cardiomyopathy (e.g. LIM protein knockout) to induce PH development in different animals [15]. Despite the fact that all of these models are primarily used for investigation of the left ventricular function, they could be utilized as model for the investigation of new therapeutic approaches with regard to a treatment of secondary PH. Pulmonary arterial remodeling owing to chronic heart failure (CHF) is characterized by medial hypertrophy and lung fibrosis. The important role of angiotensin-II in pulmonary structural remodeling after myocardial infarct has been proven by showing that receptor antagonist irbesartan completely reduced the right ventricular hypertrophy [15].
Comparison of models and translation to the human system Although the animal models described above have been proven to be useful for the investigation of signaling pathways, which contribute to pulmonary vascular remodeling in PH, and help us find novel treatments, they lack important features displayed in the human disease. Both classical models do not provoke plexiform lesions, which is an important limitation of these animal models. The main advantages of classical models are the high predictability and the good reproducibility within the selected animal strain. Compared to the MCT model (group 1 of Dana Point Classification), which needs the toxic stimulus, one main advantage of the chronic hypoxia model is the physiological stimulus, which is similar to the human disease (group 3 of Dana Point Classification). The chronic hypoxia model leads to a moderate remodeling of the pulmonary vascular bed as compared to the MCT model, which evokes a more prominent vasculopathy with extensive media hypertrophy and a more than doubled increase in pulmonary artery pressure and signs of right heart failure (Fig. 1; Table 2). Compared to the MCT rat model, which resembles WHO functional classes III and IV, the chronic hypoxia model is less severe and is therefore used as a model, which resembles the symptoms of WHO functional classes I and II (Table 1b). Other in vivo animal models of PH represent special forms of pulmonary hypertension. Both PPHN and hypercirculationinduced PH models have the advantage of reproducing the exact pathophysiological disorder of humans, but carries the same limitations due to the use of very sophisticated surgical approaches. Pathological alterations can appear late – after
Drug Discovery Today: Disease Models | Pulmonary vascular disease
many months or even years. Hypercirculation-induced PH models imitate the formation of plexiform lesions in human severe PH [13]. Animal models of PH owing to left heart diseases are perfect tools to investigate secondary PH. None of these models have been developed especially focused on PH. Additionally, heart failure models are very severe acute disorder models with a short biological period for pathological development and none of these models completely reproduces the slow progression of PH caused by CHF in humans. The knowledge obtained from animal models has been successfully confirmed in human. For example, inflammation plays a significant role in the MCT model and the same is true for various types of human PH, such as idiopathic PAH (IPAH) and PAH associated with connective tissue diseases and HIV infection [29]. Current progress in the treatment of PH illustrates how the scientific achievements based on the animal models (Table 3) ultimately result in dramatic improvement in PH patient care. More than 25 randomized clinical trials have confirmed effectiveness of three groups of drugs: prostacyclin analogues (prostanoids), endothelin receptor antagonists and phosphodiesterase type-5 (PDE-5) inhibitors in PH patients after an enormous number of experiments with different animal models (Table 3). Today, new potential therapeutic agents including inhibitors of Rho kinase, growth factor receptor inhibitors, HMG-CoA (or 3hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitors, soluble guanylate cyclase stimulator and endothelial progenitor cell administration have been tested in animal models and have showed their possible therapeutic potential (Table 3). In addition, future therapeutic agents should target RV failure, which is a main cause of disability and death of PH patients. Animal models, especially the MCT model, are a crucial tool to answer some challenging questions. Scientists should keep in mind that there is no ideal animal model which perfectly mimics all forms of PH. Application of different triggers simulates various forms of PH with a large number of cellular and molecular alterations (Table 1a and b). In spite of the diversity of initial factors/triggers the downstream effects of different stimuli could be analogous. The comparison of different animal models is therefore necessary to understand the differential pathway impact in PH development. Using both classical models simulates different stages of disease progression from a moderate hypertension to the severe RV failure, and allows to try various preventive and, even more important, curative therapeutic approaches. However, not all novel drugs, which effectively reverse PH in the animal models, will work in human PH. Thus, successful therapeutic effects observed in at least two different animal models make success of translation to human PH more likely. For that reason, from our point of view, it is important to use both most common animal models of PH (rat and mouse) in new therapeutic agent trials to decrease the frequency of false positive results. www.drugdiscoverytoday.com
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Table 3. The animal models of PH and translation research Animal model Species
Translation to human Pathway/agent/year
Effect
Trial/agent/year
Effect
Drugs used in clinical practice Neonatal piglets
Prostacyclin and analogues/Prostacylin/1978
Decreases hypoxic pulmonary vasoconstriction by prostacyclin infusion [37].
PPHSG/Epoprostenol/1996
Improves symptoms and survival [38].
Rats
Endothelin receptor antagonist/BQ 123/1996
Attenuates hypoxic PH [39].
BENEFiT/Bosentan/2002
Improves exercise capacity and symptoms [40].
Mice
PDE-5 inhibitors/ Sildenafil/2001
Attenuates hypoxic PH [41].
SUPER/sildenafil/2005
Improves exercise capacity, WHO functional class, and hemodynamics [42].
New potential agents Rats
HMG-CoA reductase inhibitor/Simvastatin/2002
Attenuates pulmonary remodeling in MCT + pneumonectomy PH [43].
SiPHT/Simvastatin/2010
Added to conventional therapy produces a small and transient early reduction in RV mass [44].
Mice Rats
Soluble guanylate cyclase stimulator/BAY 63-252/2008
Reverses hypoxic PH in mice and MCT PH in rats [3].
Phase 2 trial/Riociguat (Bay 63-252)/2010
Improves exercise capacity and symptoms [45].
Mice Rats
PDGF inhibitor/Imatinib/2005
Reverses hypoxic PH in mice and MCT PH in rats [22].
Phase 2/Imatinib/2010
Improves hemodynamic [46].
Rats
Serine-threonine kinase inhibition/Sorafenib/2008
Prevents pulmonary remodeling in MCT PH in rats [47].
Phase Ib/Sorafenib/2010
Well tolerated [48].
Mice Rats
Rho-kinase inhibitor/ Azaindole-1/2010
Reverses hypoxic PH in mice and MCT PH in rats [49].
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Conclusion The complexity of pulmonary hypertension and the lack of a robust model of PH do not allow the use of a single inbred animal model of PH to unravel all possible pathways leading to PH development in the human population. Moreover, no animal model reproduces the full spectrum of pathological changes seen in the human lung obtained from PH patients. In our opinion the different models have different strengths and weaknesses. Therefore, before translating the knowledge gained in animal models to the human system, more than one experimental animal model needs to be used. Nevertheless, animal models can be used as valuable scientific tools to take a major step in the understanding of PH development and effective treatment improvement.
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