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Conducting efficacy evaluations for pulmonary arterial hypertension (PAH): how should one choose among the three classic animal models?

 

In the development of new drugs for pulmonary arterial hypertension (PAH), the appropriateness of model selection directly affects whether drug efficacy can be reliably observed, quantified, and validated. Among the three classic PAH models—hypoxia‑induced, monocrotaline‑induced, and Sugen‑5416 plus chronic hypoxia‑induced—which key phenotypes do they each recapitulate, and how do they respond to pharmacological interventions? In this study, we systematically elucidate the characteristics and practical utility of these models by comparing them side by side and integrating cardiac function assessments, Doppler echocardiography, and histopathological analyses.

01 Unified Baseline: Ensuring the Validity of Multi-Model Comparisons

To minimize the effects of variations in animal batch, age, and testing conditions, a total of 35 male C57BL/6 mice aged 6–8 weeks were selected, with a standardized 28-day modeling and intervention period. The experiment employed three modeling strategies and added a positive-drug intervention group on top of the H+SU regimen.

G1｜Control group: Rats were kept under normoxic conditions at atmospheric pressure for 28 days and received vehicle treatment.

G2｜Hypoxia group: Continuous exposure to a 10% O₂ hypoxic chamber for 28 days.

G3｜H+SU group: SU5416 was administered by subcutaneous injection once weekly for 3 weeks, concurrently with exposure to 10% O₂ hypoxia, for a total duration of 28 days.

G4｜MCT group: Subcutaneous injection of MCT Once a week for 3 weeks, under standard housing conditions for 28 days.

G5｜H+SU + Sild group: Sildenafil was administered concurrently on the basis of H+SU model induction (gavage, twice daily for 28 days).

The entire experiment was conducted according to the following protocol: animal randomization into groups → model induction and drug administration → dynamic ultrasound monitoring → harvesting of heart and lung tissues on day 28 → histopathological analysis. On the endpoint day 28, high-frequency ultrasound was used for noninvasive dynamic monitoring, assessing 14 parameters including pulmonary hemodynamics, and the structure and function of the right and left hearts; at day 28, heart and lung tissues were collected uniformly, and HE staining, Masson’s trichrome staining, and α‑SMA immunohistochemical analysis were performed.

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Using animals from the same batch, with a uniform experimental period and standardized testing procedures, enhances the comparability of cross‑strategies comparisons.

02 Pulmonary vascular remodeling exhibits marked model-specific differences.

One of the key pathological changes in PAH is medial thickening of pulmonary arterioles, enhanced smooth muscle–related phenotypes, and progressive luminal narrowing. Therefore, this study first assessed the degree of pulmonary vascular remodeling in different models at two levels: tissue architecture and vascular muscularization.

HE: By how much exactly has the vessel wall thickened?

In the control group, pulmonary small vessels exhibited a clear structure with relatively thin walls and no obvious inflammatory cell infiltration. In the hypoxia group, mild medial thickening, smooth muscle cell proliferation, and slight luminal narrowing were observed, suggesting chronic hypoxia-induced vascular remodeling. In the MCT group, the vascular media was thickened and the vessel wall was irregular; muscularization of distal pulmonary arteries was present, accompanied by scattered inflammatory cells in the surrounding tissue. The H+SU group showed the most pronounced changes: marked medial hypertrophy of the pulmonary arteries, significant thickening of the smooth muscle layer, and severe luminal stenosis, with a 192% increase compared to the control group (P < 0.001).

H+SU is the most severe phenotype of pulmonary vascular disease; after Sild treatment, vascular wall thickness decreased to a level comparable to that of the Control group, indicating that sildenafil exerts a significant inhibitory effect on pulmonary vascular remodeling in PAH.

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α-SMA: Is there a more pronounced myofibroblastic phenotype in the vessels?

If HE staining indicates the degree of pulmonary artery wall thickening, α‑SMA staining further assesses smooth muscle–related phenotypes and muscularization of small vessels. The results showed that in the Control group, only sparse α‑SMA positivity was observed in medial smooth muscle cells and perivascular pericytes of the pulmonary arteries, with a relatively thin medial smooth muscle layer. In the Hypoxia group, α‑SMA positivity was enhanced around both pulmonary arteries and small airways, indicating smooth muscle cell proliferation and phenotypic transformation. In the MCT group, strong positive staining revealed marked medial hypertrophy, though the severity of vascular lesions was slightly milder than in H+SU. The H+SU group exhibited the strongest positive staining, with a thick, brownish‑yellow signal throughout the vessel wall; non‑muscular vessels displayed evidence of muscularization, reflecting the most severe pathology. When HE and α‑SMA findings are considered together, the trends across both parameters are broadly consistent: H+SU not only shows greater vessel wall thickness but also more pronounced smooth muscle–related phenotypes, with the highest positive area (↑71%). Sild treatment reduced this compared with H+SU (↓37%), thereby inhibiting smooth muscle phenotypic transformation. If the candidate drug primarily targets pulmonary vascular remodeling, abnormal smooth muscle proliferation, or mechanisms related to muscularization, H+SU can provide a more pronounced histological phenotypic window.

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03 When the pulmonary vessels change, how does the heart adapt?

With increased pulmonary vascular resistance, the right heart is subjected to greater afterload. Therefore, the evaluation of PAH models should not be limited to pulmonary vascular histology; it must also assess myocardial structural changes and fibrosis.

HE staining of myocardial tissue showed that in the Control group, myocardial fibers were regularly arranged with nuclei of normal morphology; in the Hypoxia group, cardiomyocytes exhibited mild hypertrophy. Both the H+SU and MCT groups displayed varying degrees of cardiomyocyte hypertrophy, enlarged and deeply stained nuclei, and disorganized arrangement, with these morphological features partially ameliorated following sildenafil intervention. However, Masson’s trichrome staining yielded different results: at day 28, the right ventricular collagen volume fraction (CVF) across all groups generally ranged from 3.7% to 4.1%, with no statistically significant intergroup differences overall, reflecting predominantly focal collagen deposition. These findings suggest that, under the conditions of this study and at the 28-day observation time point, pulmonary vascular remodeling and myocardial collagen reorganization are not synchronized. The emergence of distinct pathological changes in pulmonary vessels does not necessarily imply that myocardial fibrosis will concurrently attain a comparable phenotypic severity. Should the research focus extend further to right ventricular fibrosis, evaluation would require longer observation periods and incorporation of molecular markers related to collagen metabolism.

Different pathological endpoints exhibit temporal asynchrony, so model evaluation should not rely on a single endpoint to represent the entire disease course.

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04 Functional Evaluation Further Differentiates Model Features

Pathological sections reveal structural lesions in tissues, while echocardiography is used to confirm whether these changes have led to functional abnormalities in vivo. This batch of ultrasound examinations assessed pulmonary artery blood flow as well as the structure and function of both the right and left heart. The results showed that the three models exhibited distinct patterns of functional impairment:

 

Pulmonary circulation and the right heart: H+SU shows the most pronounced changes and exhibits a clear response to the positive control drug.

The core indicator reflecting pulmonary vascular resistance, PAT/PET, decreased significantly in all three modeling groups. The H+SU group showed the largest reduction (down 27% from baseline to 0.249, P < 0.001 ), Consistent with severe medial thickening of the pulmonary vasculature. Regarding right heart remodeling, the right ventricular free wall thickness (RVFW) was increased by 20%–27% in all three groups, reflecting right ventricular systolic function… TAPSE decreased by 27%–35% Following sildenafil intervention, PAT/PET increased significantly by 16% (0.249 → 0.290), and TAPSE rose by 9.6% (0.68 → 0.745 mm), demonstrating the drug’s effective improvement of pulmonary circulation and right ventricular function.

 

Left ventricular structure and function: The MCT group showed the most severe involvement, while hypoxia was relatively mild.

The end-systolic volume (ESV) increased 2.1–3.4-fold in all model groups. Among them, The MCT group exhibited the most pronounced impairment in left cardiac structure and function: The end-systolic internal diameter (LVIDs) increased by 62%, and the end-systolic volume increased to 3.4 times that of the control group (10.2→35.0 μL), while the left ventricular ejection fraction EF decreased by 39% (73.5%→45.0%) , characterized primarily by marked left ventricular remodeling and systolic dysfunction. In contrast, Hypoxia group EF decreased by 25%, overall cardiovascular function changed relatively mildly, and pulmonary arterial hemodynamics showed moderate changes.

The ultrasound findings were corroborated by the histopathological results: The H+SU group showed more pronounced pulmonary vascular resistance and pulmonary vascular remodeling, the MCT group exhibited more marked left ventricular remodeling and impaired cardiac function, while the Hypoxia group displayed a relatively mild pathological process. This provides in vivo functional data to support the selection of evaluation models based on the mechanisms of action of candidate drugs.

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Creating the 05 model is only the first step—where exactly lies the true therapeutic window?

Previous results have shown that the three models produce distinct phenotypes: Hypoxia is characterized primarily by relatively mild pulmonary vascular remodeling, MCT exhibits more pronounced cardiac dysfunction, while H+SU displays prominent pulmonary vascular remodeling, muscularization, and abnormalities in pulmonary circulation. Crucially, in the H+SU group, treatment with sildenafil led to varying degrees of improvement across multiple endpoints, including pulmonary vascular wall thickness, α‑SMA‑positive area, as well as PAT/PET and TAPSE. This means that, for evaluating drug efficacy, the value of a model lies not only in whether a model can be successfully established, but also in whether it can generate a clear, stable, and quantifiable window of drug response. From this perspective, in the present study, H+SU not only induces distinct PAH-related pathological and functional phenotypes but also allows detection of post-treatment changes across multiple levels, including histology and ultrasound; therefore, it is better suited as A pharmacological efficacy evaluation model for candidate drugs related to pulmonary vascular remodeling and pulmonary circulatory function.

06 Back to R&D Decision-Making: How Should We Choose a PAH Animal Model?

Model selection should not be based on a simple comparison of which model exhibits the most severe phenotype, but rather on which best addresses the specific R&D question at hand. Based on the results from this batch of animals, the three classic models do not represent a straightforward progressive replacement relationship; instead, each corresponds to distinct R&D scenarios and phenotypic windows:

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Conclusion

A robust PAH efficacy evaluation system goes beyond merely establishing models and conducting assays; it establishes a correspondence between the model’s phenotype, evaluation metrics, and the mechanism of action of candidate drugs. In new drug development, models are not definitive solutions but tools for addressing specific R&D challenges. By defining target phenotypes, selecting appropriate models, and setting quantifiable endpoints, animal studies can truly inform efficacy assessments.

 

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