Pulmonary hypertension (PH) is a life-threatening pulmonary vascular disorder characterized by pathological pulmonary vascular remodeling and lumen obstruction, leading to a persistent increase in pulmonary vascular resistance and ultimately resulting in right-sided heart failure [1, 2, 3]. Although current therapies (e.g., endothelin receptor antagonists and prostacyclin analogues) can partially alleviate vasoconstriction, they generally fail to effectively halt or reverse structural vascular remodeling [4, 5, 6]. Therefore, exploring novel targets capable of directly reversing vascular remodeling is of great importance.

Excessive proliferation, migration, and phenotypic switching of pulmonary arterial smooth muscle cells (PASMCs) from a contractile to a synthetic secretory phenotype are considered central cellular mechanisms driving pulmonary vascular remodeling and lumen occlusion [7].

N-myc downstream-regulated gene 2 (NDRG2) is a member of the N-myc downstream-regulated gene family, which possesses an $\alpha$/$\beta$ hydrolase domain and has been demonstrated to be widely involved in regulating key processes such as stress responses, cell proliferation, and migration [8, 9]. NDRG2 expression is upregulated under various stress conditions, including hypoxia and cerebral ischemia, yet its expression pattern exhibits significant cell-type specificity. For instance, hypoxia promotes NDRG2 expression via direct binding of HIF-1$\alpha$ to its promoter in A549 cells [10], whereas hypoxia-induced miR-346 exerts an inhibitory effect in cells such as OS-RC-2 [11]. Our previous study established a crucial role for NDRG1 in promoting pulmonary vascular remodeling in PH [12]. Given the high homology between NDRG2 and NDRG1, we hypothesize that NDRG2 may play a similar role. Furthermore, supporting this notion, NDRG2 has been reported to participate in the progression of various cardiovascular diseases, such as cardiac remodeling in models of myocardial ischemia–reperfusion injury [13, 14] and heart failure [15]. However, despite these suggestive associations, the specific role and mechanism of NDRG2 in PH remain unknown.

On the contrary, aberrant mitochondrial fragmentation has been identified as a significant contributor to PH, exacerbating vascular remodeling by promoting the proliferation, migration, and phenotypic switching of PASMCs, whereas the inhibition of this process confers therapeutic benefits [16, 17, 18]. This pathological mitochondrial fission event is primarily regulated by the post-translational modifications of dynamin-related protein 1 (DRP1): phosphorylation of DRP1 at serine 616 (Ser616) promotes its translocation to mitochondria and initiates fission, whereas the phosphorylation at serine 637 (Ser637) exerts an inhibitory effect [16, 19]. Given the central role of NDRG2 in regulating cellular signaling pathways, we hypothesize that NDRG2 may influence mitochondrial dynamics, and thereby contribute to PH progression, by perturbing the balance of DRP1 phosphorylation.

Based on this background, this study was conducted to systematically address three core questions: (1) to determine whether NDRG2 is involved in the pathogenesis and progression of PH; (2) to investigate the molecular mechanism by which NDRG2 promotes vascular remodeling, specifically examining whether it acts by regulating DRP1 phosphorylation-mediated mitochondrial fragmentation; and (3) to evaluate the potential therapeutic value of targeting NDRG2 for ameliorating pathological vascular remodeling.

Our study identified NDRG2 as an additional member of the NDRG family involved in pulmonary vascular remodeling in PH, demonstrating its crucial role in regulating mitochondrial dynamics. We established that NDRG2 acted as a molecular switch in pathological PASMCs, linking hypoxic stress to mitochondrial fission and metabolic dysregulation through direct interaction with DRP1 and precise modulation of its phosphorylation status. This pathway ultimately drove disease progression. Our findings not only provide novel insights into PH pathogenesis but also reveal a potential therapeutic target.

The biological functions of NDRG2 are highly context dependent, varying by cell type and pathological setting, with reported roles spanning from cytoprotection to disease promotion [24, 25]. This functional diversity is reflected in its variable response to hypoxia across various cell lineages, thereby underscoring the complexity of its regulation [26]. Our data definitively showed that NDRG2 is upregulated and exerts a net pro-pathogenic effect in hypoxic PASMCs, aligning with its characterization as a stress-responsive gene. Notably, our previous work identified a pro-remodeling function for its homolog NDRG1 in PH [12]. The congruent pathogenic role of NDRG2 highlighted in this study points to a conserved importance of the NDRG protein family in pulmonary vascular pathology. A comparative analysis suggests that both NDRG1 and NDRG2 may converge on disrupting mitochondrial homeostasis; however, NDRG1 has also been implicated in modulating additional pathways such as PI3K–Akt signaling. This indicates a shared nodal role in metabolic dysregulation, alongside isoform-specific mechanisms that may collectively drive vascular remodeling. Furthermore, our in vitro studies employed a sustained 1% O₂ exposure to robustly elicit cellular responses. In human PH, vascular cells may experience more moderate or intermittent hypoxia. Future studies investigating the threshold and dynamics of NDRG2 induction and DRP1 activation across a range of oxygen tensions may be valuable to better understand how this pathway engages across the spectrum of hypoxic stress relevant to clinical disease.

The central mechanistic advance of our work is the delineation of the NDRG2–DRP1–mitochondrial fission axis. Mitochondrial fragmentation is a well-established driver of PH pathogenesis [27, 28], facilitating vascular remodeling by promoting PASMC proliferation and conferring resistance to apoptosis [17, 29]. DRP1 serves as the master regulator of mitochondrial fission, with its activity predominantly controlled by opposing post-translational modifications: activating phosphorylation at Ser616 and inhibitory phosphorylation at Ser637 [16, 30]. We found that NDRG2 specifically enhanced DRP1 phosphorylation at the Ser616 activation site via a direct molecular interaction, without altering total DRP1 levels or the phosphorylation status at Ser637. This selective effect suggests that NDRG2 interfaces with upstream regulators specific to the Ser616 pathway (e.g., ERK1/2 or CDK1), thereby shifting the phosphorylation balance decisively toward activation to drive pathological fission. This modification is crucial for DRP1 activation and its subsequent translocation to mitochondria. Consequently, we placed NDRG2 at a pivotal regulatory node, directly governing the DRP1 activation switch to transduce extracellular hypoxic cues into intracellular metabolic pathology, evidenced here by bioenergetic failure (reduced ATP levels) and oxidative stress (ROS accumulation). Based on this observed dysfunction, we predicted that NDRG2 overexpression also impaired mitochondrial respiratory function, leading to reduced oxygen consumption rates and compromised membrane potential. Direct measurement of these parameters in future studies can provide a more detailed resolution of the metabolic alterations downstream of the NDRG2-DRP1 axis. These pathological changes collectively propel the proliferation and migration of PASMCs.

Despite these insights, our study had certain limitations. First, our mechanistic conclusions were derived primarily from studies on PASMCs. Although this focused approach allowed detailed dissection of the NDRG2–DRP1 axis, we acknowledge that PH is a multicellular disease involving endothelial dysfunction, fibroblast activation, and immune cell infiltration. Our in vivo AAV9-mediated knockdown was not cell specific, and thus the therapeutic effects observed might have resulted from NDRG2 inhibition in multiple cell types. The contribution of NDRG2 within nonsmooth muscle vascular cells to PH pathogenesis remains an important area for future investigation. Second, the precise upstream mechanism whereby NDRG2 facilitates DRP1 Ser616 phosphorylation remains to be fully elucidated. Although our study established the functional consequence of this modification, identifying the responsible kinase(s) is a key remaining question. NDRG2 may act as a scaffold to recruit specific kinases known to phosphorylate DRP1 at Ser616 (e.g., ERK1/2, CDK1, or PKC$\delta$) or may modulate the activity of counteracting phosphatases. Definitively identifying these upstream effectors using approaches such as phospho-proteomic screening or targeted genetic/pharmacologic intervention represents an essential and immediate future direction arising from this study. Third, validating NDRG2 upregulation and its correlation with disease severity in lung tissues from patients with PH may be essential to underscore the translational potential of our findings. Fourth, our in vivo studies were conducted exclusively in male rats. Given the established sexual dimorphism in PH incidence and progression, future investigations should include both sexes to determine the generalizability and potential sex-specific nuances of the NDRG2–DRP1 pathway. Furthermore, although our localized intratracheal delivery of AAV9-shNDRG2 aimed to minimize systemic exposure, a comprehensive assessment of potential off-target effects and the consequences of long-term NDRG2 inhibition in other organs may be crucial considerations for future therapeutic development. Finally, from a translational perspective, our in vivo intervention demonstrated the preventive potential of NDRG2 inhibition when administered prior to disease induction. It is important to note that this paradigm models a preventive strategy. While the central role of the NDRG2-DRP1 axis in sustaining mitochondrial fission and PASMC dysfunction suggests that its inhibition could also ameliorate established pathology, future studies explicitly evaluating the efficacy of NDRG2-targeted therapies initiated after the full establishment of PH are needed to define their potential for disease reversal.

In summary, our study identified NDRG2 as a key upstream regulator driving pathological mitochondrial fission and vascular remodeling in PH. We demonstrated that NDRG2 was upregulated in hypoxic PASMCs and experimental PH models, where it promoted a synthetic secretory phenotype, proliferation, and migration. Mechanistically, NDRG2 directly interacted with DRP1 to specifically enhance its phosphorylation at Ser616, leading to excessive mitochondrial fission, bioenergetic dysfunction, and oxidative stress. Crucially, the genetic inhibition of NDRG2 in vivo ameliorated hemodynamic and structural pathologies in rats with SuHx-induced PH. These findings delineate a novel NDRG2–DRP1 axis central to PH pathogenesis in preclinical models, thereby nominating this pathway for further mechanistic and translational research (Graphical Abstract).

The datasets used and analyzed in this study are available from the corresponding author upon reasonable request.

JYL, JYC, YL, YSS, YYZ, YRZ, EFX, YQX, SST, HXL and WMH significantly contributed to the study, including its conception, design, execution, data acquisition, analysis, and interpretation. JYL, JYC, YL, YSS, YYZ, YRZ, EFX, YQX, SST, HXL and WMH were also involved in drafting, revising, or critically reviewing the manuscript; All authors approved the final version for publication; agreed on the journal for manuscript submission; and accepted accountability for all aspects of the study. JYL designed the study, conducted the experiments, interpreted the results, and wrote the manuscript. YSS and JYC conducted the experiments and interpreted the results. YL, YRZ and EFX contributed to the study design, conducted the experiments, and participated in manuscript revision. YYZ, YQX, SST and HXL drew icons for statistical analysis. WMH provided financial support.

The Research Ethics Committee at Ningbo University granted approval for all animal experiments and methodologies (AEWC-NBU20250371). All procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The Laboratory Animal Science Department facilitated the procurement of Sprague–Dawley rats. Strict compliance with the established protocols for the care and use of laboratory animals was maintained throughout all experimental processes.

The authors would like to express their gratitude to all those who helped them in writing this manuscript. They thank all the peer reviewers for their opinions and suggestions.

This work was financially supported by the National Natural Science Foundation of China (Grant No. 82500082). Additional support was provided by the One Health Interdisciplinary Research Project at the Institute of One Health Science, Ningbo University, the project “Establishment of the Coronary Heart Disease Interventional Diagnosis and Treatment Center at the Cixi Integrated Traditional Chinese and Western Medicine Medical Health Group” (Grant No. 812410050), and the project “The Role of Leukocyte-Derived Chemokine 2 in Regulating Macrophage Polarization” (Grant No. 882500370).

The authors declare no conflict of interest.

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