Reply to the letter to the editor entitled “Bridging the species divide: The limits of rat models in capturing human PVOD mechanisms” by Perros F. et al.

We thank Drs. Perros, Chaveroux, and Montani for their interest in our articles describing the activation of the integrated stress response (ISR) pathway as a mechanism underlying the pathogenesis of pulmonary veno-occlusive disease (PVOD), based on the rat mitomycin C (MMC) model of PVOD.1, 2 Their letter highlights the differences observed between our findings in the rat PVOD model1, 2 and those reported by Dr. Perros's group, which shed light on the potential role of general control nonderepressible 2 (GCN2) in PVOD pathogenesis.3 Their letter raises concerns regarding the relevance of ISR activation in the pathogenesis of human PVOD, as well as the effectiveness of therapeutics developed based on the rat model.4 Specifically, they emphasize two points: (i) the lack of robust ISR-ATF4 activation in human PVOD samples and (ii) the potential role of GCN2 in PVOD pathogenesis. First, we would like to clarify that in our article in Nature Cardiovascular Research, we demonstrated elevated levels of the auto-phosphorylated, active form of protein kinase R (PKR) and activating transcription factor 4 (ATF4) in the pulmonary vasculature of PVOD patients, which indicates robust activation of the PKR-ISR axis in these patients.1 This activation was observed in two lung samples of PVOD patients but not those with idiopathic pulmonary arterial hypertension.1 Notably, Manaud et al. also reported a significant increase in C/EBP homologous protein and heme oxygenase 1 (HO-1), which are ATF4 targets and downstream effectors of the ISR pathway, in five patients with heritable and five patients with sporadic forms of PVOD.3 However, due to the small sample sizes in both studies and the heterogeneity of human pathogenesis, it remains unclear whether PKR-ISR activation is broadly present across different forms of PVOD or at all stages of the disease. Hence, our studies do not suggest PKR-ISR activation is the sole mechanism driving PVOD. Future studies with a larger cohort of PVOD patients are necessary to address this question. Such research could help stratify PVOD patients in whom PKR-ISR axis activation plays a central role in pathogenesis and who may benefit from therapies targeting this pathway, as demonstrated in the rat PVOD model. Second, we reported the depletion of GCN2 in pulmonary vascular cells following MMC treatment in rats,1 consistent with the findings by Manaud et al.,3 and acknowledge that the depletion of GCN2 likely plays a role in PVOD pathogenesis, particularly in cases with impaired bone morphogenetic protein receptor type 2 signaling.4, 5 We further demonstrated that treatment with the ISR inhibitor ISRIB restores GCN2 protein levels to their physiological state, suggesting that GCN2 depletion occurs as a consequence of ISR activation.1 Therefore, inhibiting the PKR-ISR axis with ISRIB or PKR antagonists could mitigate the pathological effects of GCN2 depletion. We appreciate the letter emphasizing the critical issue of potential discrepancies between preclinical models and human disease—a pervasive problem in biomedical research. The MMC-induced rat model of PVOD is based on clinical observations that the administration of alkylating agents like MMC can lead to PVOD in cancer patients,6 making it a relevant approach for studying human PVOD pathogenesis. Nonetheless, we acknowledge the need for improved animal models that accurately replicate human disease to bridge the gap between animal studies and clinical observations. Validating critical findings from preclinical studies using patient samples, as we demonstrated with the PKR–ISR axis,1 is crucial for identifying the limitations of existing animal models and assessing the translational potential of therapeutics tested in those models. Given the rarity of PVOD, we propose that enhancing access to patient specimens from clinical centers worldwide would significantly accelerate discoveries relevant to patient care. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Amit Prabhakar, Rahul Kumar, Meetu Wadhwa, Rubin M. Tuder, Nicholas W. Morrell, Brian B. Graham, Giorgio Lagna, and Akiko Hata wrote the manuscript. National Heart, Lung, and Blood Institute (NHLBI; R01HL132058, R01HL153915, and R01HL164581) to A. H.; American Heart Association 19CDA34730030, Cardiovascular Medical Research Fund (CMREF) and United Therapeutics Jenesis Innovative Research Award to R. K.; by NHLBI (P01HL014985) to R. M. T.; by NHLBI (R01HL135872 and P01HL152961) to B. B. G. The authors declare no conflict of interest. The authors have nothing to report.