Mitochondrial Transplantation from Bone Marrow Mesenchymal Stromal Cells Combined with Sildenafil Attenuated Vascular Remodeling and Improved Right Ventricular Dysfunction in Experimental Pulmonary Arterial Hypertension

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Onofre and colleagues explore a new approach to treating pulmonary arterial hypertension (PAH) associated with experimental mitochondrial transplantation therapy. PAH is a severe, progressive disease associated with pulmonary vascular remodeling, impaired blood flow, and increased vascular resistance. Despite the use of modern vasodilators such as sildenafil, existing treatments are unable to completely halt or reverse structural vascular changes. In this study, the authors test the hypothesis that transplantation of mitochondria isolated from bone marrow mesenchymal stromal cells can improve mitochondrial homeostasis and attenuate vascular remodeling. Particular attention is paid to the combined use of mitochondrial therapy and sildenafil, which may lead to a synergistic effect. The study was conducted in a rat model of monocrotaline-induced PAH. The authors assessed the effects of therapy on hemodynamics, right ventricular remodeling, pulmonary vascular health, and key molecular markers associated with mitochondrial function and cellular plasticity. The results suggest that this combined approach could be a promising avenue for developing metabolically targeted therapy for PAH. Overall, the study is well-designed, however, despite the positive impression, I have several critical comments:

1. Intravenously administered mitochondria accumulate predominantly in the liver and kidneys, with minimal signaling in the lungs and heart. This raises a key question. If the drug targets the pulmonary vessels and right ventricle, and the majority of injected mitochondria are localized in the liver and kidneys, what mechanisms mediate the observed cardiopulmonary effects? The authors suggest indirect or immunomodulatory mechanisms, but do not provide experimental evidence.
2. Although the authors hypothesize that BM-MSC-derived mitochondria are «relatively resistant to oxidative stress», direct measurements of reactive oxygen species production are lacking. Measurements of mitochondrial membrane potential are also absent, and markers of oxidative damage are not provided.
3. Respirometry was performed on frozen lung tissue samples. This is a clear limitation, as freezing negatively impacts mitochondrial function. High-resolution respirometry should be used on fresh perfused lungs or isolated cells for greater physiological relevance.
4. Dysfunction of calcium homeostasis is recognized as a key mechanism in pulmonary vascular remodeling in PAH. It promotes smooth muscle cell proliferation, endothelial-mesenchymal transition, and vascular calcification. Despite this, the authors did not evaluate calcium homeostasis in their model. This is an important gap, given recent studies showing that mitochondrial transplantation specifically reduces tissue calcification in dystrophic muscle of mdx mice by restoring mitochondrial calcium buffering. I recommend assessing calcium deposits in the lung tissue of animal models, or at least discussing this potential mechanism for the protective effect of mitochondrial transplantation. Furthermore, the authors observed a decrease in α-SMA in animals receiving mitochondria, a marker of endothelial-mesenchymal transition that is dependent on Ca²⁺.
5. It is crucial to assess the immune response to mitochondrial transplantation in animals, as mitochondria are a source of DAMPs.

Author Response

Response to Reviewers’ Comments

Reviewer #1

Onofre and colleagues explore a new approach to treating pulmonary arterial hypertension (PAH) associated with experimental mitochondrial transplantation therapy. PAH is a severe, progressive disease associated with pulmonary vascular remodeling, impaired blood flow, and increased vascular resistance. Despite the use of modern vasodilators such as sildenafil, existing treatments are unable to completely halt or reverse structural vascular changes. In this study, the authors test the hypothesis that transplantation of mitochondria isolated from bone marrow mesenchymal stromal cells can improve mitochondrial homeostasis and attenuate vascular remodeling. Particular attention is paid to the combined use of mitochondrial therapy and sildenafil, which may lead to a synergistic effect. The study was conducted in a rat model of monocrotaline-induced PAH. The authors assessed the effects of therapy on hemodynamics, right ventricular remodeling, pulmonary vascular health, and key molecular markers associated with mitochondrial function and cellular plasticity. The results suggest that this combined approach could be a promising avenue for developing metabolically targeted therapy for PAH. Overall, the study is well-designed, however, despite the positive impression, I have several critical comments:
Response: We thank the Reviewer for the careful evaluation and constructive comments.

1. Intravenously administered mitochondria accumulate predominantly in the liver and kidneys, with minimal signaling in the lungs and heart. This raises a key question. If the drug targets the pulmonary vessels and right ventricle, and the majority of injected mitochondria are localized in the liver and kidneys, what mechanisms mediate the observed cardiopulmonary effects? The authors suggest indirect or immunomodulatory mechanisms, but do not provide experimental evidence.
   Response: We thank the Reviewer for this important point. In vivo imaging demonstrated that intravenously administered mitochondria predominantly accumulate in the liver and kidneys, with comparatively low signal in the lungs and heart. Accordingly, we have revised the manuscript to avoid implying direct pulmonary uptake as the primary mechanism. We now explicitly frame the cardiopulmonary effects as potentially mediated through systemic and immunomodulatory pathways rather than local mitochondrial engraftment.
   
   To support this hypothesis, we performed new analyses of circulating inflammatory mediators at day 28. Plasma IL-6 and IL-1β levels were significantly elevated in PAH animals compared with controls and were significantly reduced by combined sildenafil and mitochondrial therapy. These new data (Figure 9) have been added to the Results (page 10) and incorporated into a more cautious mechanistic discussion.
   
   
2. Although the authors hypothesize that BM-MSC-derived mitochondria are «relatively resistant to oxidative stress», direct measurements of reactive oxygen species production are lacking. Measurements of mitochondrial membrane potential are also absent, and markers of oxidative damage are not provided.
   Response: We agree with the Reviewer that direct measurements of oxidative stress and mitochondrial membrane potential would strengthen the mechanistic interpretation. These experiments were not feasible within the scope of the current study and are now explicitly acknowledged as a limitation (page 13).
   
   We have revised the Discussion to clarify that the relative resistance of BM-MSC–derived mitochondria to oxidative stress is inferred from prior literature (page 11) and our recent complementary work (reference #21; https://doi.org/10.1093/stcltm/szaf053), rather than directly demonstrated here. Speculative language has been toned down accordingly.
   
   
3. Respirometry was performed on frozen lung tissue samples. This is a clear limitation, as freezing negatively impacts mitochondrial function. High-resolution respirometry should be used on fresh perfused lungs or isolated cells for greater physiological relevance.
   Response: We fully agree that respirometry on frozen tissue represents a methodological limitation. This has been clearly stated and emphasized in the revised Discussion (pages 12 and 13). While prior studies support partial preservation of respiratory capacity in frozen samples, we now explicitly caution against overinterpretation and restrict conclusions to comparative rather than absolute functional assessments.
   
   
4. Dysfunction of calcium homeostasis is recognized as a key mechanism in pulmonary vascular remodeling in PAH. It promotes smooth muscle cell proliferation, endothelial-mesenchymal transition, and vascular calcification. Despite this, the authors did not evaluate calcium homeostasis in their model. This is an important gap, given recent studies showing that mitochondrial transplantation specifically reduces tissue calcification in dystrophic muscle of mdx mice by restoring mitochondrial calcium buffering. I recommend assessing calcium deposits in the lung tissue of animal models, or at least discussing this potential mechanism for the protective effect of mitochondrial transplantation. Furthermore, the authors observed a decrease in α-SMA in animals receiving mitochondria, a marker of endothelial-mesenchymal transition that is dependent on Ca²⁺.
   Response: We acknowledge this important mechanistic gap. Direct assessment of calcium homeostasis was not performed and is now explicitly listed as a limitation (page 13). We expanded the Discussion to note that reduced α-SMA expression may indirectly reflect altered Ca²⁺-dependent endothelial–mesenchymal transition (page 12), but we clearly state that this interpretation remains speculative and requires direct validation.
   
   
5. It is crucial to assess the immune response to mitochondrial transplantation in animals, as mitochondria are a source of DAMPs
   Response: We agree that immune activation is a critical concern. As noted above, new analyses of systemic cytokines (IL-6, IL-1β) were performed and added (Figure 9 and page 10), demonstrating attenuation rather than exacerbation of inflammation with combined therapy. We now explicitly frame immune modulation as a supported but still partial explanation (page 11, second paragraph) rather than a definitive mechanism.

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigated the intravenous administration of BM-MSC-derived mitochondria in a monocrotaline-induced PAH rat model and compared the effects of sildenafil alone and in combination. This study reports improvements in key readouts, including RVSP, echocardiographic parameters, pulmonary vascular remodeling, and mitochondrial-related measurements. However, several points listed below need to be clarified to support reproducibility and strengthen the main conclusions.

Major

1. The Methods section states that liver-derived mitochondria were used for validation/function/distribution, and that in vivo therapeutic experiments used BM-MSC-derived mitochondria only, but the Results section describes day 14 administration of liver-derived mitochondria in PAH animals with 100% mortality. This is inconsistent and must be reconciled (L361–364 and L120–124). The authors should clearly define whether liver-derived administration was a pilot/safety exploration and report the dosing conditions, group allocation, observations, and exact number of animals.
2. The Discussion section links the fatal outcome after liver-derived mitochondria to possible mechanisms, such as metabolic mismatch, excessive redox signaling, or immune activation; however, no dose-response assessment is provided (L120–124, L258–267). The interpretation should be made more cautious, and the authors should either add dose-finding/safety data or explicitly limit the conclusion to poor tolerability under tested conditions
3. The dose of 100 µg and injection time points of days 14 and 21 were based on a pilot study and published reports; however, the pilot study design is not described (L366–368). The authors should briefly state what was tested in the pilot study and how the final dose/timing was selected, including animal numbers and endpoints, in the main text or supplementary information.
4. The mitochondria preparations are described as a mitochondrial-enriched fraction, and BM-MSC mitochondria are isolated using a commercial kit, but information supporting preparation quality is limited (L369–374, L380–384). To support the claim of “mitochondrial transplantation,” the authors should provide basic quality controls, such as representative mitochondrial markers and evidence minimizing non-mitochondrial contamination and residual cellular components.
5. The authors suggest that the benefit may be systemic or immunomodulatory because organ distribution is liver/kidney-dominant and lung/heart uptake is low; however, the manuscript does not present direct immunologic measurements (L279–286). The authors should present this as a hypothesis and avoid overinterpretation or add supporting data (for example, inflammatory cytokines or immune cell readouts) if they want to emphasize an immunomodulatory mechanism.
6. The disease stage at the time of intervention is not explained in a reader-friendly manner. The schedule is clear, but it is difficult to judge whether the treatment targets a developing or established PAH phenotype (L125–132, L395–404). The authors should add a short description of the time course of the monocrotaline model and explicitly place the day 14/day 21 injections within that context.
7. The distinction between mitochondria and sildenafil alone is not clear across endpoints. For example, RV outflow tract diameter improvement is described mainly in the sildenafil and combination groups, while RVSP is reduced across the intervention groups (L129–132, L142–145). The authors should summarize, for each key endpoint, which effects are seen with mitochondria alone versus sildenafil alone, and ensure that the narrative is consistent with the stated “additive” effect of the combination therapy.

Minor

1. Figure 1 legend states mean ± SD with n = 3. The figure display should match the legend, and error bars or data presentation should be corrected if necessary (L99–105).
2. Group names are not fully consistent across the text and figures (for example, “PAH-saline” vs “PAH-SAL”). Standardizing terminology would reduce confusion (L129–132, L148–152).
3. The manuscript shows functional differences between BM-MSC-derived and liver-derived mitochondria, but broader statements about “organ-specific” rules should be phrased carefully and kept within the scope of the presented comparisons (L106–119).

Author Response

Response to Reviewers’ Comments

Reviewer #2

This study investigated the intravenous administration of BM-MSC-derived mitochondria in a monocrotaline-induced PAH rat model and compared the effects of sildenafil alone and in combination. This study reports improvements in key readouts, including RVSP, echocardiographic parameters, pulmonary vascular remodeling, and mitochondrial-related measurements. However, several points listed below need to be clarified to support reproducibility and strengthen the main conclusions.
Response: We thank the Reviewer for the detailed critique and valuable suggestions to improve rigor and reproducibility.

Major

1. The Methods section states that liver-derived mitochondria were used for validation/function/distribution, and that in vivo therapeutic experiments used BM-MSC-derived mitochondria only, but the Results section describes day 14 administration of liver-derived mitochondria in PAH animals with 100% mortality. This is inconsistent and must be reconciled (L361–364 and L120–124). The authors should clearly define whether liver-derived administration was a pilot/safety exploration and report the dosing conditions, group allocation, observations, and exact number of animals.
   Response: We appreciate this critical observation. We have clarified that liver-derived mitochondria were used in pilot biodistribution and safety experiments (page 14, section 4.3), not as part of the therapeutic arm. All PAH animals receiving liver-derived mitochondria died shortly after administration, and these data are now transparently reported as negative safety findings, with explicit group allocation, dosing, timing, and animal numbers added to the Methods (page 15, section 4.4).
   
   
2. The Discussion section links the fatal outcome after liver-derived mitochondria to possible mechanisms, such as metabolic mismatch, excessive redox signaling, or immune activation; however, no dose-response assessment is provided (L120–124, L258–267). The interpretation should be made more cautious, and the authors should either add dose-finding/safety data or explicitly limit the conclusion to poor tolerability under tested conditions
   Response: We agree and have revised the Discussion to limit conclusions strictly to poor tolerability under the tested conditions. No claims regarding mechanism or general organ incompatibility are made, and the absence of dose–response data is now explicitly acknowledged as a limitation (page 13).
   
   
3. The dose of 100 µg and injection time points of days 14 and 21 were based on a pilot study and published reports; however, the pilot study design is not described (L366–368). The authors should briefly state what was tested in the pilot study and how the final dose/timing was selected, including animal numbers and endpoints, in the main text or supplementary information.
   Response: We have added a concise description of the pilot dosing experiments (liver-derived vs BM-MSC–derived mitochondria, doses tested, survival outcomes, rationale for timing) to the Methods section (page 14, section 4.3), improving transparency and reproducibility.
   
   
4. The mitochondria preparations are described as a mitochondrial-enriched fraction, and BM-MSC mitochondria are isolated using a commercial kit, but information supporting preparation quality is limited (L369–374, L380–384). To support the claim of “mitochondrial transplantation,” the authors should provide basic quality controls, such as representative mitochondrial markers and evidence minimizing non-mitochondrial contamination and residual cellular components.
   Response: We have expanded the Methods (page 14, section 4.3.3.) and Results (page 3) to include basic quality control measures, including respirometry, MitoTracker labeling, and flow cytometry characterization. Claims are now restricted to “mitochondria-enriched fractions,” avoiding overstatement of purity.
   
   
5. The authors suggest that the benefit may be systemic or immunomodulatory because organ distribution is liver/kidney-dominant and lung/heart uptake is low; however, the manuscript does not present direct immunologic measurements (L279–286). The authors should present this as a hypothesis and avoid overinterpretation or add supporting data (for example, inflammatory cytokines or immune cell readouts) if they want to emphasize an immunomodulatory mechanism.
   Response: We thank the Reviewer for this very important question. Reviewer #1 also asked about immunologic measurements. We have revised the language throughout to clearly distinguish observed data from mechanistic hypotheses. Immunomodulatory effects are now presented as supported by cytokine measurements (Figure 9 and page 10) but not fully mechanistically resolved, in line with the presented evidence.
   
   
6. The disease stage at the time of intervention is not explained in a reader-friendly manner. The schedule is clear, but it is difficult to judge whether the treatment targets a developing or established PAH phenotype (L125–132, L395–404). The authors should add a short description of the time course of the monocrotaline model and explicitly place the day 14/day 21 injections within that context.
   Response: We added a concise description of the monocrotaline PAH timeline, clarifying that day-14 intervention corresponds to early but established functional PAH, supported by echocardiographic abnormalities (Supplemental Figure 3 and page 11). This contextualizes the intervention as clinically relevant rather than preventive.
   
   
7. The distinction between mitochondria and sildenafil alone is not clear across endpoints. For example, RV outflow tract diameter improvement is described mainly in the sildenafil and combination groups, while RVSP is reduced across the intervention groups (L129–132, L142–145). The authors should summarize, for each key endpoint, which effects are seen with mitochondria alone versus sildenafil alone, and ensure that the narrative is consistent with the stated “additive” effect of the combination therapy.
   Response: We have added a new summary figure (Figure S5) comparing percentage changes across treatment arms and revised the Discussion to consistently describe additive rather than interchangeable effects.

Minor

1. Figure 1 legend states mean ± SD with n = 3. The figure display should match the legend, and error bars or data presentation should be corrected if necessary (L99–105).
2. Group names are not fully consistent across the text and figures (for example, “PAH-saline” vs “PAH-SAL”). Standardizing terminology would reduce confusion (L129–132, L148–152).
3. The manuscript shows functional differences between BM-MSC-derived and liver-derived mitochondria, but broader statements about “organ-specific” rules should be phrased carefully and kept within the scope of the presented comparisons (L106–119).
   Response: All minor issues raised (Figure 1 and Supplemental Figure 1 presentation, terminology consistency, scope of conclusions) have been fully addressed and corrected in the revised manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The work has been significantly enhanced through the revision process. But there appears to be a slight misalignment or inconsistency in the numerical sequence of the in-text citations and the reference list. This may have occurred during the integration of new references or text adjustments.

Author Response

Comments 1: The work has been significantly enhanced through the revision process. But there appears to be a slight misalignment or inconsistency in the numerical sequence of the in-text citations and the reference list. This may have occurred during the integration of new references or text adjustments.
Response: We thank the Reviewer for noting it. Indeed, there was misalignment or inconsistency in the numerical sequence of the in-text citations and the reference list. The reference list was corrected. We corrected the in-text citations.

Reviewer 2 Report

Comments and Suggestions for Authors

I thank the authors for their thoughtful and appropriate responses to the comments. 

Author Response

Comment 1: I thank the authors for their thoughtful and appropriate responses to the comments. 
Response: We thank the Reviewer for the suggestions which really helped to improve the quality of manuscript.