Chronic thromboembolic pulmonary hypertension: animal models

ANIMAL MODELS OF PERSISTENT INTRAVASCULAR THROMBOSIS

The mechanisms by which the pulmonary emboli or thrombi fail to undergo lysis and instead organise into occluding fibrotic material remain unknown. Hypotheses include predisposing pulmonary endothelial cell abnormalities [42] and impairments in the vascular repair process [43]. A role for in situ thrombosis related to endothelial cell dysfunction may explain why up to 63% of patients with CTEPH have no documented history of acute pulmonary embolism [3]. This hypothesis has not been studied in animals, as no endothelial cell abnormalities have been identified to date in humans with CTEPH. In contrast, the process of thrombus organisation has been studied in reliable animal models of low flow-induced inferior vena cava thrombosis (table 2). Most of these animal models were developed in rodents, with the goal of investigating venous thrombus resolution [5–22]. Thrombosis can be induced by venous stasis alone or combined with induced blood hypercoagulability or mechanical endothelial damage. Kang et al. [13] studied a piglet model of jugular vein thrombosis after stenosis and mechanical endothelial damage [15]. Inferior vena cava occlusion has been also studied in monkeys [15]. However, mice and rats remain the most widely used animals due their cost-effectiveness and efficiency [7]. Low-flow rodent models are characterised by a laminar thrombus that resolves within 3–4 weeks via a process of recanalisation that requires inflammatory cell recruitment and angiogenic signals [5–11, 17–22].

In studies of clinical risk factors, CTEPH was associated neither with the classical plasma-factor abnormalities associated with venous thromboembolism nor with impairments in fibrinolysis. However, having a ventriculo-atrial shunt or history of pacemaker infection increased the risk of CTEPH [44, 45]. This finding prompted studies in animal models. Thus, Bonderman et al.[23] used a mouse model of low-flow venous thrombosis induced by inferior vena cava stenosis and endothelial damage to study the role for staphylococcal infection in delaying thrombus resolution and promoting the expression of profibrotic molecules.

DUAL VASCULAR COMPARTMENT THEORY

Moser et al. [4] were the first to describe two compartments in the pulmonary vascular bed of CTEPH patients: an obstructed compartment subjected to chronic ischemia and an unobstructed compartment subjected to increased flow and shear stress (fig. 1). Thus, in addition to the obstruction of large pulmonary arteries, patients with CTEPH have vascular lesions in distal unobstructed territories. These distal lesions are similar to those found in patients with other forms of PH.

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Figure 1–

Anteroposterior pulmonary angiogram showing the two vascular territories in a patient with chronic thromboembolic pulmonary hypertension. The obstructed territory is subjected to chronic ischaemia and the unobstructed territory to an increase in blood flow.

Many observations are consistent with a role for both territories in increasing pulmonary-vascular resistance. First, for the same degree of pulmonary artery obstruction as measured by lung scanning, CTEPH is associated with higher pulmonary resistance values compared to acute pulmonary embolism [46]. Secondly, the onset of CTEPH disease is characterised by an asymptomatic honeymoon period during which pulmonary vascular resistance increases gradually without evidence of recurrent embolism. These two vascular compartments have been reproduced in animals separately and, more recently, simultaneously in a piglet model.

MODELS REPLICATING PULMONARY ARTERY OBSTRUCTION

The first attempt to replicate chronic pulmonary artery obstruction in an animal model was conducted in piglets by Fadel et al. [24] who used proximal embolisation of coils and tissue adhesive into the left pulmonary artery. Chronic obstruction (for 5 weeks) of the left pulmonary artery resulted in chronic lung ischemia without PH. The result was an increase in the systemic blood supply to the lung via bronchial, mediastinal, and intercostal arteries, as well as distal post-obstructive pulmonary vasculopathy. These lesions were similar to those seen after chronic pulmonary artery ligation [25].

The left pulmonary artery was chosen for ligation, because it could be easily re-implanted into the main pulmonary artery to replicate reperfusion injuries after PEA. The systemic vascular response to chronic-pulmonary vascular obstruction differs across species, with proliferation of bronchial arteries into the intraparenchymal airways in large animals (dogs and piglets) and rats or of intercostal arteries into the pleural space in mice [26]. Although CTEPH studies have been conducted chiefly in piglets, a few studies of bronchial circulation have been performed in a rat model of pulmonary artery ligation [27]. Occlusion of one of the main pulmonary arteries stimulates angiogenesis in the bronchial vessels of the ipsilateral lung [28]. Bronchial arteries begin to enlarge as soon as 2–3 days after pulmonary artery ligation and supply the pulmonary circulation via precapillary anastomoses. These anastomoses may maintain airway epithelium oxygenation, thus explaining the lesser degree of reperfusion injury after chronic lung ischemia than after acute lung ischemia [29].

In addition to bronchial circulation hypertrophy, post-obstructive vasculopathy is characterised by pulmonary artery abnormalities including increased media thickness [30], impaired vasoreactivity [31], impaired endothelial nitric-oxide-synthase function [30], and increased reactivity to endothelin (ET)-1 [32] leading to increased resistance after reperfusion [33].

Lung reperfusion after chronic ischemia was followed by gradual reversal of the post-obstructive vasculopathy [25, 34], after an early phase of ischemia-reperfusion injury with endothelial cell damage [35].

Closure of the aorto-pulmonary shunt replicates the hemodynamic conditions in unobstructed territories after PEA. Shunt closure induced normalisation of ET-1 and ETA expression, followed by media hypertrophy reversal in the distal pulmonary arteries. These findings were consistent with the gradual improvement in pulmonary vascular resistance observed 3–6 months after PEA.

MODELS REPLICATING LESIONS OF UNOBSTRUCTED PULMONARY ARTERIES

During the early phase of CTEPH, the unobstructed territories are subjected to a chronic blood flow increase due to cardiac output redistribution. Therefore, systemic-to-pulmonary shunts have been used in animals to replicate the lesions seen in unobstructed territories in patients with CETPH [36]. In an animal model initially developed by Rendas et al. [37] to replicate congenital heart disease, an aorto-pulmonary shunt is induced by implanting a short prosthesis between the ascending aorta and main pulmonary artery. This model has been used to induce high-flow pulmonary vascular lesions similar to those seen in unobstructed territories in CTEPH (fig. 2). As with the left pulmonary artery ligation models, the aorto-pulmonary shunt was achieved through a median sternotomy to avoid pleural opening and a subsequent inflammatory response. High-flow pulmonary vasculopathy induced by 5 weeks of aorto-pulmonary shunting was characterised by increased media thickness of the distal pulmonary arteries (fig. 3) related to smooth muscle cell proliferation (as shown by proliferating cell nuclear antigen labelling) and by elevated levels of ET-1 and its receptor endothelin receptor A (ETA) in lung tissue. ET-1 is a potent vasoconstrictor and mitotic peptide for vascular smooth muscle cells. ET-1 overexpression has been found in other animal models of high pulmonary flow [38–40]. ET-1 overproduction is probably a response to stimuli such as shear stress resulting from arterial pressure elevation [47].

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Figure 2–

Aortography showing in a sham-group piglet. a) The aortic arch with the two supra-aortic arteries. b) Visualisation of the pulmonary vascular bed (white arrow) at the same time as the aortic arch after creation of an aorto-pulmonary shunt. c) No opacification of the pulmonary vascular bed after shunt closure.

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Figure 3–

Box plot of media thickness as a percentage of small pulmonary arteries (<150 μm) calculated in: the sham (35.9±0.8%), shunt-open (55.6±1.2%), 1-week shunt-closed (48.7±1%), and 5-weeks shunt-closed (40.9±1%) groups [36]. Media thickness percentage was calculated as follows: external diameter minus internal diameter and divided by the external diameter. Aorto-pulmonary shunting was created by a graft interposition between the ascending aorta and the pulmonary trunk in piglets. Closure of the shunt was obtained by dividing the graft.

ANIMAL MODEL REPLICATING ALL FEATURES OF CTEPH

Although the animal models described earlier provided useful information on impaired thrombus resolution and on the pathophysiology of lesions in obstructed and unobstructed territories, they failed to replicate important features of human CTEPH including pulmonary hypertension, interactions between the two pulmonary vascular compartments and, above all, RV remodelling and dysfunction.

Since the 1990s, several attempts to develop animal models of CTEPH [48–52] failed because of clot lysis by the very efficient endogenous fibrinolytic system [53] and of the remarkable adaptive capabilities of the pulmonary circulation. Thus, 3 h after acute pulmonary embolism in dogs [49], only 30% of the initial injected thrombus volume remained inside the pulmonary artery. Adding tranexamic acid [49] or plasminogen activator inhibitor-1 [50] to delay thrombus resorption between injections failed to solve this problem. The second difficulty was the large pulmonary circulation reserve, which required obstruction of more than half the pulmonary vasculature to achieve an increase in pulmonary vascular resistance. The adaptive capabilities of the pulmonary vasculature explain why repeated injections of small, inert, non-absorbable materials failed to replicate CTEPH. Thus, in a dog model of chronic pulmonary-artery injections of ceramic beads, 3 mm in diameter, pulmonary pressures and resistances returned to normal within 1 week after each injection [51]. With 100–300 μm microspheres, 60 days of repeated embolisation were required to increase the pulmonary-artery pressure [52]. After 60 days, signs of PH started to develop, but there was no bronchial artery hypertrophy, post-obstructive or high-flow vasculopathy, or proximal vascular obstruction [52]. A third challenge lies in replicating RV remodelling. Extensive acute obstruction of the pulmonary arterial tree is usually lethal by heart failure in the absence of previous RV training and hypertrophy. To tolerate a persistent increase in pulmonary arterial pressure, the RV must undergo remodelling, which consists in gradual ventricular wall hypertrophy followed by right-heart-chamber enlargement with a paradoxical septal motion. In the event of persistent pulmonary hypertension, RV failure develops.

We recently developed a CTEPH piglet model [41], consisting of a primary left pulmonary artery ligation via a sternotomy that was followed by weekly transcatheter embolisations, under fluoroscopic control, of the tissue adhesive enbucrilate (Histoacryl) into the right lower lobe for 5 weeks. Pulmonary artery ligation overwhelmed the pulmonary circulation reserve and led to PH within a few weeks, with progressive obstruction of the remaining lung vasculature. The progressive nature of the obstruction achieved via weekly embolisation allowed the RV to adapt to the pressure increase, thus preventing death by acute RV failure. The tissue adhesive enbucrilate solidifies immediately after contact with blood and adheres to the arterial wall. The result was proximal obstruction by unresolved material in the right lower-lobe artery. Thus, the right upper-lobe arteries remained patent and exhibited lesions replicating those seen in unobstructed territories in CTEPH. After 5 weeks, this piglet model replicated all the features of human CTEPH: increased pulmonary vascular resistance, increased mean pulmonary artery pressure, increased media thickness of distal pulmonary arteries in both obstructed and unobstructed territories, increased systemic blood supply through the bronchial arteries in the obstructed territories, RV hypertrophy, RV enlargement, and paradoxical septal motion (fig. 4). Interestingly, although the embolisations were stopped after 5 weeks, the increase in pulmonary vascular resistance persisted for up to 1 month later after the last embolisation (unpublished data). Overexpression of ET-1 and its receptors ETA and ETB was documented in the remodelled distal arteries of the unobstructed territories, in keeping with previous findings in piglets with high-flow vasculopathy [36, 38]. In addition, ETA overexpression in the obstructed territories was consistent with the results of previous studies of post-obstructive vasculopathy [33]. Further experiments are needed to monitor pulmonary resistance at a distance from the last pulmonary embolisation, to investigate RV remodelling and failure, and to assess interactions between the obstructed and unobstructed territories. However, one should bear in mind that this model does not replicate the impaired thrombus resolution seen in human CTEPH.

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Figure 4–

Chronic thromboembolic pulmonary hypertension piglet model consisting of left pulmonary artery ligation and repeated embolisation in the right lower lobe: bronchial artery hypertrophy in the ischaemic lung territory (b), apparently normal right upper lobe (a), unresolved intravascular material made of tissue adhesive and fibrin moulding the arterial tree, and right ventricle remodelling.

CONCLUSIONS

Animal models developed over several decades have provided valuable information on the pathophysiology of CTEPH. Thrombus resolution and obstructed and unobstructed territories have been studied separately in several animal models. Recently, a model replicating all the major aspects of human CTEPH was developed. This model should prove useful for investigating RV dysfunction and distal lung vessel abnormalities. However, additional models are still needed to elucidate the pathobiology of thrombus persistence.