Chronic hypoxia aggravates monocrotaline-induced pulmonary arterial hypertension: a rodent relevant model to the human severe form of the disease
- Florence Coste1, 2, 3Email authorView ORCID ID profile,
- Christelle Guibert1, 2,
- Julie Magat1, 2, 4,
- Emma Abell1, 2, 4,
- Fanny Vaillant1, 2, 4,
- Mathilde Dubois1, 2,
- Arnaud Courtois1, 2,
- Philippe Diolez1, 2, 4,
- Bruno Quesson1, 2, 4,
- Roger Marthan1, 2, 3,
- Jean-Pierre Savineau1, 2,
- Bernard Muller1, 2 and
- Véronique Freund-Michel1, 2
© The Author(s). 2017
Received: 1 July 2016
Accepted: 8 March 2017
Published: 14 March 2017
Pulmonary arterial hypertension (PAH) is a severe form of pulmonary hypertension that combines multiple alterations of pulmonary arteries, including, in particular, thrombotic and plexiform lesions. Multiple-pathological-insult animal models, developed to more closely mimic this human severe PAH form, often require complex and/or long experimental procedures while not displaying the entire panel of characteristic lesions observed in the human disease. In this study, we further characterized a rat model of severe PAH generated by combining a single injection of monocrotaline with 4 weeks exposure to chronic hypoxia. This model displays increased pulmonary arterial pressure, right heart altered function and remodeling, pulmonary arterial inflammation, hyperresponsiveness and remodeling. In particular, severe pulmonary arteriopathy was observed, with thrombotic, neointimal and plexiform-like lesions similar to those observed in human severe PAH. This model, based on the combination of two conventional procedures, may therefore be valuable to further understand the pathophysiology of severe PAH and identify new potential therapeutic targets in this disease.
KeywordsAnimal model Plexiform-like lesions Pulmonary arterial hypertension
Pulmonary hypertension (PH) is a severe disease characterized by sustained elevated mean pulmonary arterial pressure (mPAP) over 25 mmHg, development of right heart hypertrophy, leading to cardiac failure and finally death . Pathobiology of PH includes pulmonary arterial inflammation, remodeling and altered reactivity, all contributing to increased pulmonary vascular resistances .
In the current PH classification, five groups have been identified based on pathophysiological and clinical considerations . The pulmonary arterial hypertension (PAH) group (Group 1) includes idiopathic or familial forms of PH, as well as forms associated to other diseases such as connective tissue diseases or HIV infection [1, 3]. PAH is usually a severe form of PH that combines multiple alterations of pulmonary arteries, including thrombotic lesions, and/or complex and disorganized lesions characterized by a network of proliferated channels separated by core cells, the so-called plexiform lesions . Current pharmacological treatments of PAH manage to slow the progression of the disease but do not afford a cure . Since human samples are difficult to obtain, pertinent animal models are therefore needed to better understand PAH pathobiology and identify new therapeutic targets. However, classical PAH animal models do not recapitulate the severe pathology of human disease , and multiple-pathological-insult models have therefore been developed to mimic more closely the human PAH pathophysiology. For instance, pneumonectomy has been associated to MCT injections, leading to neointimal  or plexiform-like lesions . In other severe PAH models, SUGEN (SU5416, a tyrosine-kinase inhibitor of the vascular endothelial growth factor receptor VEGFR-2) has been associated to hypoxia [9, 10] or pneumonectomy . However, these models are time consuming and/or require experimented manipulation for surgical procedure.
Our group has developed an alternative rat model of severe PAH, combining MCT injection to 3 weeks of chronic hypoxia (Hx) . Additional preliminary experiments conducted in the same study on 4 rats suggested the development of plexiform-like lesions when the duration of Hx combined to MCT was increased to 4 weeks . Herein, we aimed to confirm the interest of this latter model based on a combination of two conventional procedures: a single MCT intraperitoneal injection associated to 4 weeks of Hx (MCT + Hx rats).
Description of the model
All animal studies were made according to European and French directives about vertebrate animals protection use for animal experiments. Agreement was obtained from French authorities (number A33-063-907) and all the protocols used were approved by the local ethics committee (Comité d’éthique regional d’Aquitaine, protocol number: 50110016 A).
Male Wistar rats (250-350 g) were randomly assigned into 5 groups: chronic hypoxia (Hx), monocrotaline (MCT), severe PAH (MCT + Hx) for 3 or 4 weeks, and controls (CTRL). Rats exposed to Hx were placed in a hypoxic hypobaric chamber (380 mmHg) for 28 days (4 weeks). In the MCT group, a single intraperitoneal MCT injection (60 mg/kg, Sigma-Aldrich) was performed at Day 1 and rats were maintained in a normobaric/normoxic environment (room air) for 28 days. Severe PAH was induced by combining a single MCT injection (60 mg/kg) at Day 1 with exposure to Hx from Day 2 to Day 28, as previously described [6, 12]. This combined model was also studied at 1, 2 or 3 weeks of chronic hypoxia. Control rats were injected with MCT vehicle and maintained in a normobaric/normoxic environment (room air) for 28 days. Hypobaric chambers were opened three times a week for animal care and cleaning, and all animals had free access to food and water.
For details about other methods and statistical analysis, see the online supplemental methods section.
Complementary to hemodynamic disturbances, structural and functional alterations of pulmonary arteries in human PAH include inflammation, altered reactivity and intense remodeling with a characteristic arteriopathy including thrombotic, neointimal and plexiform lesions [4, 17]. In the previous model combining MCT administration with 3 weeks of Hx, perivascular inflammatory infiltrates were previously evidenced . We further characterized pulmonary arterial inflammation in the present model by showing increased levels of the pro-inflammatory cytokines interleukin-1β and tumor necrosis factor-α released by pulmonary arteries in the MCT + Hx group compared to control animals (Fig. 1d and e). Increased secretion of such pro-inflammatory cytokines has been previously shown in PAH patients , with cytokine levels being predictive of outcome in these patients [19, 20]. Reproducing such increase in this experimental model may therefore be of valuable importance in terms of evaluating PAH severity and outcomes.
We also confirmed the presence of thrombotic lesions (Fig. 3d 6). Thrombotic occlusions have also been observed in other PH models induced by Hx and/or Sugen , and are often observed in several forms of human PAH. Although it may be difficult to distinguish between thrombotic lesions and post-mortem coagulation, thrombotic lesions observed in our experiments were observed in PAH rats but not in control animals, suggesting that such thrombosis may rather be caused by the disease itself.
Finally, for the first time in this MCT + Hx model, we also showed the presence of other characteristic human-like lesions. In particular, eccentric lesions were observed (Fig. 3d 7). Although being often difficult to distinguish, such eccentric lesions have also been reported in human PAH and in other PH animal models .
In addition, concentric non-laminar intimal thickening lesions were also observed (Fig. 3e 8-11), similar to those observed in human severe PAH . A further characterization of these lesions showed positive staining for α-smooth muscle actin (Fig. 3d 9) but negative staining for the von Willebrand factor (Fig. 3d 10-11). This suggests predominance of smooth muscle cells and/or myofibroblasts rather than endothelial cells in these lesions, in accordance with observations of such lesions in human PAH [4, 27].
If classical characteristics of pulmonary arterial remodeling, i.e. medial thickening and luminal occlusion, are easily reproduced in classical models of PAH such as MCT treatment, other severe PAH specific lesions are not observed in these models. This explains the need for developing alternative models more closely related to human PAH pathophysiology. Models such as pneumonectomy associated to MCT injections [7, 8], or the Sugen model , lead to lesions very close to the pulmonary arteriopathy observed in human severe PAH. The present model combining MCT and 4 weeks of Hx may be a valuable alternative model, with a protocol realized in a reasonable time and without requiring surgical skills. Nevertheless, this model involves hypoxia chambers that are not available in all laboratories.
In conclusion, we show here that combining MCT injection with 4 weeks of exposure to chronic hypoxia in rats generates a relevant model to the pathogenesis of human severe PAH. In particular, it reproduces multiple structural and functional alterations of pulmonary arteries, including inflammation, altered reactivity and intense remodeling. Moreover, this model displays a pulmonary arteriopathy with thrombotic, severe intimal lesions and some plexiform-like lesions, similar to those observed in human severe PAH. As human samples of PAH are difficult to obtain, the present model, using classical protocols performed in one month, may therefore be valuable to further understand the pathophysiology of severe PAH. According to the current recommendations on PAH translational research suggesting the use of more than one rodent model , using this model together with other models of severe PAH may also be of interest to identify new potential therapeutic targets in this disease.
Mean pulmonary arterial pressure
Pulmonary arterial hypertension
- PGF2 α:
Prostaglandin F2 α
Standard error of the mean
Tumor necrosis factor α
The authors thank Mrs M. Lepiez and Mrs E. Poinama for excellent animal care, and Dr C. Toussaint for his technical assistance with mPAP measurements. The microscopy was done in the Bordeaux Imaging Center, a service unit of the CNRS-INSERM and Bordeaux University, member of the national infrastructure France BioImaging.
This work was supported by INSERM and University of Bordeaux.
Availability of data and materials
The dataset supporting the conclusions of this article is included within the article, Pressure-volume loop analysis are available from the corresponding author on request.
Conception and design: FC, VFM; Analysis and interpretation: FC, VFM, JM, EA; Drafting the manuscript for important intellectual content: FC, VFM; Revising the manuscript for important intellectual content: FC, RM, BM, VFM; final approval of the manuscript: FC, JM, EA, FV, CG, MD, AC, PD, BQ, RM, JPS, BM, VFM. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
All animal studies were made according to European and French directives about vertebrate animals protection use for animal experiments. Agreement was obtained from French authorities (number A33-063-907) and all the protocols used were approved by the local ethics committee (Comité d’éthique regional d’Aquitaine, protocol number: 50110016-A).
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