In Vivo Target Engagement Assessment of Nintedanib in a Double-Hit Bleomycin Lung Fibrosis Rat Model

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors present an in vivo study evaluating the anti-fibrotic effects and target engagement of nintedanib in a double-hit bleomycin-induced lung fibrosis rat model. The topic is relevant, and the dataset is extensive. However, several substantial issues should be addressed before the manuscript can be considered for publication.

Major comments

1. The central objective of the study is not clearly defined. Although the authors mention pharmacodynamic biomarkers and target engagement, the specific hypothesis and the precise knowledge gap being addressed remain unknown. The manuscript should explicitly state whether the primary aim is to evaluate VEGF as a biomarker, clarify nintedanib’s mechanism of action, or characterize PK/PD relationships.

2. The rationale for VEGF as an indicator of target engagement is insufficiently supported. The manuscript does not clearly explain the mechanistic basis for VEGF elevation, nor does it establish how the phosphoproteomic findings corroborate this interpretation. The linkage between results, mechanistic reasoning, and final conclusions requires a more coherent and evidence-based explanation.

3. The translational implications of the findings are not adequately discussed. While an increase in VEGF after nintedanib treatment is showned in the rat model, its relevance to human IPF remains unclear. The authors should integrate existing clinical evidence, clarify whether similar patterns are seen in patients, and address how these findings may inform biomarker development or therapeutic monitoring.

 

Minor comments

1. Some figure legends are insufficiently detailed. For example, Figure C lacks an explanation of the statistical methods used and does not indicate which comparisons reached statistical significance.

2. The term “double-hit model” should be defined upon first mention. It is unclear whether this modeling approach is intended to represent acute exacerbation of IPF or chronic fibrosis resulting from repeated bleomycin exposure.

3. Please clarify whether VEGF measurements represent total VEGF or specific isoforms.

Author Response

Comment 1. The central objective of the study is not clearly defined. Although the authors mention pharmacodynamic biomarkers and target engagement, the specific hypothesis and the precise knowledge gap being addressed remain unknown. The manuscript should explicitly state whether the primary aim is to evaluate VEGF as a biomarker, clarify nintedanib’s mechanism of action, or characterize PK/PD relationships.

Response 1. Thanks for this comment. We understand the central objective was not clearly stated. The primary objective of this study was to identify potential tissue and circulating biomarkers to elucidate Nintedanib’s target engagement and to robustly support its pharmacodynamic activity in a preclinical model of lung fibrosis. Our results support that modulation of specific biomarkers, particularly VEGF, could serve as an indicator of Nintedanib’s engagement, thereby providing translationally relevant evidence, which, to our knowledge, had not been previously established in preclinical models. This central aim has been now clearly articulated and consistently addressed throughout the entire manuscript. All changes are highlighted in red in the document (Abstract page 1, lines 24-27 & 38-41; Introduction page 2, lines 76-82 and Discussion page 7, lines 227-231).   

Comment 2. The rationale for VEGF as an indicator of target engagement is insufficiently supported. The manuscript does not clearly explain the mechanistic basis for VEGF elevation, nor does it establish how the phosphoproteomic findings corroborate this interpretation. The linkage between results, mechanistic reasoning, and final conclusions requires a more coherent and evidence-based explanation.

Response 2. We thank Reviewer 1 for raising this important point. While the mechanism behind increased plasma VEGF after VEGFR inhibitor treatment remains unclear, several clinical studies provide insight. For example, sunitinib, a multitargeted TKI inhibiting VEGFRs and other kinases, has been shown to markedly elevate plasma VEGF in metastatic renal cell carcinoma patients (J Transl Med. 2007;5:32). After the first treatment cycle, VEGF rose over threefold in 44% of cases, returning to near baseline during off-treatment periods, indicating a drug-dependent effect. Similar findings were reported by Liu et al. (Clin Cancer Res. 2011;17:7634-44), where VEGF increased significantly during sunitinib exposure across different dosing schedules and normalized upon withdrawal. Drevs et al. (Ann Oncol. 2005;16:558-65) observed a dose-dependent VEGF-A rise with PTK/ZK, another VEGFR inhibitor. Collectively, these studies support a class effect of VEGFR blockade on circulating VEGF, and our findings in rodents suggest a comparable feedback mechanism. In summary, clinical use of VEGFR inhibitors is consistently associated with increased circulating VEGF levels, supporting the concept of a class effect for agents targeting VEGFRs. Our current findings align closely with these clinical observations, suggesting that a comparable feedback mechanism may also operate in rodent models.  Main reasons suggested by the literature for this on-target effect, could be explained mainly throughout two hypothesis: 1) the Compensatory Upregulation of VEGF, as a Feedback mechanism, to maintain VEGF system deeply regulated, as proposed from the above mentioned clinical studies or 2) Loss of VEGF Clearance by a reduced VEGFR-VEGF complex internalisation, as already discosed (Nat Rev Mol Cell Biol. 2016 Oct;17(10):611-25 & Am J Physiol Heart Circ Physiol. 2004 Jan;286(1):H153-64). In the last 2 references, it has been postulated that internalization of VEGFR2 after VEGF binding regulates how much VEGF is available at the cell surface. When VEGFR2 is internalized and degraded or sequestered inside the cell, less receptor is present on the membrane to bind VEGF, which can reduce VEGF signalling. Thus, the internalization of VEGF receptors (VEGFR1 and VEGFR2) and their ligand-bound complexes plays a central role in regulating VEGF availability at the cell surface. To better understand the sequence of events leading to the observed increase in VEGF levels in rats following Nintedanib treatment, a more extensive analysis will be required and may constitute the focus of future studies. A potential mechanism underlying the observed increased VEGF levels may involve the activation, or removal of inhibitory regulators on VEGF promoter activity. To explore this, we revisited our previous research on nintedanib’s transcriptomic profile in a rat model of bleomycin-induced lung fibrosis and we observed that RNA sequencing data from lungs of Bleomycin + Nintedanib treated rats revealed the upregulation of genes such as Kruppel-like factor 4 (KLF4) and ETS transcription factor 6 (Etv6), both of which are mechanistically linked to VEGF regulation and expression,  in comparison to BLM + Vehicle animals. However, phosphoproteomic data were not able to corroborate this interpretation. This limitation is primarily attributable to the insufficient abundance of some proteins of interest in the analyzed samples, which precluded their reliable detection and quantification. As a result, direct evidence for the modulation of specific low-abundance proteins could not be obtained, and our conclusions are therefore based on the available data from more abundant targets and indirect pathway analysis. The above piece ok knowledge has been now integrated into the manuscript. (Discussion section, page 9-10, lines 302-331 & 360-369). Supporting references have been added.

Comment 3. The translational implications of the findings are not adequately discussed. While an increase in VEGF after nintedanib treatment is showned in the rat model, its relevance to human IPF remains unclear. The authors should integrate existing clinical evidence, clarify whether similar patterns are seen in patients, and address how these findings may inform biomarker development or therapeutic monitoring.

Response 3. Clinical evidence from Sunitinib and PTK787/ZK 222584, together with our data on elevated VEGF levels in rats, suggests that circulating VEGF may serve as a dynamic biomarker indicative of effective target engagement of VEGFR modulators. In clinical settings, this parameter could help establish optimal dosing and correlate with therapeutic outcomes. In non-clinical models, monitoring VEGF and related proteins in plasma not only may supports efficacy evaluation and dose selection but also deepens mechanistic understanding by revealing biological pathways influenced by tyrosine kinase inhibitors. Therefore, plasma VEGF measurement represents a versatile tool bridging mechanistic research and translational applications. This concept has been integrated into the manuscript (Discussion section, page 9-10, lines 302-314 & 360-373). Supporting references have been added. 

Minor comment 1. Some figure legends are insufficiently detailed. For example, Figure C lacks an explanation of the statistical methods used and does not indicate which comparisons reached statistical significance.

Response 1. We thank the reviewer for this helpful observation. In the revised manuscript, we have updated all figure legends to specify more details, including statistical methods. 

Minor comment 2. The term “double-hit model” should be defined upon first mention. It is unclear whether this modeling approach is intended to represent acute exacerbation of IPF or chronic fibrosis resulting from repeated bleomycin exposure.

Response 2. We thank for the comment. In our study, the “double-hit model” refers to the administration of two intratracheal doses of bleomycin (1 U/kg) on day 0 and day 4, as described in the Methods section and illustrated in Figure 1A. 
The double-hit BLM model was chosen based on accumulating evidence that it more closely recapitulates key features of human IPF compared to the traditional single-dose model. While the single-dose BLM model is widely used, it often results in transient inflammation and partial resolution of fibrosis, which may not fully reflect the chronic and progressive nature of IPF. Specifically, the double administration of BLM (on days 0 and 4) induces a more persistent and severe fibrotic response, compared to single instillation, with a stronger induction of lung biomarkers (TIMP-1, PAI-1, HYP). This approach is supported by a Poster presented at ERS International Congress 2017 comparing lung fibrosis induced by single or double bleomycin administrationin rats (Pittelli et al., https://publications.ersnet.org/highwire_display/entity_view/node/545459/full) and by our own previous work (Bonatti et al., BMJ Open Respir Res 2023), demonstrating that the double-hit protocol yields a robust and reproducible fibrotic phenotype, including sustained upregulation of translationally relevant ECM-related genes. Moreover, the work presented at ERS 2017, demonstrated a more evident and significant effect of Nintedanib in the double BLM administration group. As already demonstrated for murine models (Wang et al., Biochem. Cell Biol. 103: 1–7; 2025), we also have previously confirmed, in a double intratracheal instillation of BLM in rats,  the presence of a cell population defined KRT8+ alveolar differentiation intermediate (ADI) cells with a transcriptional profile similar to the KRT5-/KRT17+ cells identified in the IPF lung (Bocchi et al., Front. Biosci.  2024; 29(8): 305). Growing evidence indicates that KRT8+ cells are progenitor cells derived from AT2 that could be responsible for the establishment of an aberrant tissue repair process and ECM deposition in the Bleomycin rodent models. In conclusion, our findings enforces previous evidence and further support the use of a double-hit Bleomycin rat model as translational preclinical platform for investigating therapeutic targets for IPF. The intention of this modeling approach is not to mimic acute exacerbation of IPF, but rather to establish a stable and severe fibrotic response. The definition of  “double-hit” is nowstated at the first mention in the main text of revised manuscript (Results session, Paragraph 2.2, page3, line 97) and the rational of the use of a double-hit Bleomycin is clarified in the Discussion session, adding recent studies that validate the double-hit model’s translational relevance (page 7-8, lines 232-239). Supporting references have been added (24-26).  

Minor comment 3. Please clarify whether VEGF measurements represent total VEGF or specific isoforms.

Response 3. Thank you for pointing it out. In our study, VEGF levels were measured using a commercially available ELISA kit (Rat VEGF-A ELISA Kit, ab100787, Abcam, Cambridge, UK), as described in the Materials and Methods section. This assay is designed to detect VEGF-A, without distinguishing between specific VEGF-A isoforms. Therefore, all VEGF measurements reported in our manuscript represent total VEGF-A levels. We clarified this information in the revised version of the manuscript, in the Methods section, Par. 4.4. page 12, line 447.

 

 

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors need to address the following comments for better clarity.

1. In the manuscript “In Vivo Anti-Fibrotic Effect and Target Engagement Assessment of Nintedanib in a Double-Hit Bleomycin Lung Fibrosis Rat Model,” the authors claim that VEGF elevation is a reliable marker of Nintedanib target engagement; however, the current data do not sufficiently support this conclusion. The mechanistic link between VEGFR inhibition and VEGF upregulation remains largely inferential, with no direct demonstration of VEGFR phosphorylation status, receptor occupancy, or downstream signalling blockade beyond broad proteomic trends. Alternative explanations such as BLM-induced epithelial injury, hypoxia-related VEGF induction, or off-target pathway activation have not been ruled out. Stronger mechanistic evidence (e.g., Western blot validation, time-course receptor phosphorylation, or cell-type-specific expression analysis) is required before VEGF can be confidently designated as a biomarker of Nintedanib target engagement.
2. The use of a double-hit bleomycin model is not sufficiently justified, despite its central role in driving all subsequent conclusions. The authors neither explain why this model was chosen over the more widely used single-dose BLM model nor provide adequate information on its reproducibility, severity, or relevance to clinical IPF pathology. Histological and molecular distinctions between the two approaches particularly whether the double-hit system better mimics progressive fibrotic remodeling remain unaddressed. A deeper methodological rationale and comparative context are necessary to validate the choice of model and enhance the translational strength of the study.
3. The authors state that PK/PD modelling supports their conclusions, yet no actual PK/PD modelling outputs are presented. The pharmacokinetic results are purely descriptive, without any connect-the-dots quantitative linking of Nintedanib plasma or lung concentrations to target inhibition, biomarker modulation, or anti-fibrotic outcomes. Critical modelling parameters such as EC50/EC80, exposure response curves, predicted receptor occupancy, or PK/PD relationships are missing. As written, the PK/PD interpretation is overstated, and the manuscript either needs true model-based PK/PD analysis or a substantial revision of the claims regarding PK/PD integration.
4. The absence of key control groups significantly weakens the study’s conclusions. The authors include only acutely dosed naïve animals (1–24 h) for mechanistic evaluation, but do not assess chronic Nintedanib exposure in healthy rats, which is necessary to distinguish drug-specific effects from injury-induced changes. Additionally, no comparator such as pirfenidone despite extensive discussion in the introduction is included to contextualize antifibrotic or biomarker effects. The lack of BALF-based biomarker evaluation also limits interpretation of lung-specific versus systemic effects. The study requires more comprehensive controls to support the claims of specificity and mechanistic insight.

Author Response

Comment 1. In the manuscript “In Vivo Anti-Fibrotic Effect and Target Engagement Assessment of Nintedanib in a Double-Hit Bleomycin Lung Fibrosis Rat Model,” the authors claim that VEGF elevation is a reliable marker of Nintedanib target engagement; however, the current data do not sufficiently support this conclusion. The mechanistic link between VEGFR inhibition and VEGF upregulation remains largely inferential, with no direct demonstration of VEGFR phosphorylation status, receptor occupancy, or downstream signalling blockade beyond broad proteomic trends. Alternative explanations such as BLM-induced epithelial injury, hypoxia-related VEGF induction, or off-target pathway activation have not been ruled out. Stronger mechanistic evidence (e.g., Western blot validation, time-course receptor phosphorylation, or cell-type-specific expression analysis) is required before VEGF can be confidently designated as a biomarker of Nintedanib target engagement.

Response 1. Thank you to Reviewer 2 for highlighting this important issue. While the mechanisms underlying the increase in plasma VEGF levels following VEGFR inhibitor therapy remain to be fully elucidated, several clinical studies have already provided valuable evidence. For example, sunitinib—a multitargeted oral tyrosine kinase inhibitor used in various cancers—has been shown to cause a marked, statistically significant rise in plasma VEGF in patients with metastatic renal cell carcinoma, with levels increasing more than threefold from baseline in 44% of cases by the end of the first treatment cycle (J Transl Med. 2007 Jul 2;5:32). This effect was consistently observed across multiple cycles and reversed during off-treatment periods, indicating a drug-dependent phenomenon. Notably, patients with lower baseline VEGF experienced greater increases. These results suggest that elevated circulating VEGF is a robust pharmacodynamic marker of sunitinib activity and likely reflects VEGF pathway inhibition in vivo. Further supporting this, Glenn Liu et al. (Clin Cancer Res. 2011 Dec 15;17(24):7634-44) demonstrated significant increases in plasma VEGF during sunitinib exposure, with levels returning to baseline after drug withdrawal, regardless of dosing schedule. This underscores the utility of plasma VEGF as a dynamic biomarker for monitoring the biological effects of sunitinib in renal cell carcinoma and other solid tumors. Similarly, J. Drevs et al. (Ann Oncol. 2005 Apr;16(4):558-65) reported that treatment with the VEGFR inhibitor PTK787/ZK 222584 led to a dose-dependent rise in plasma VEGF-A, particularly at higher doses. The early increase in VEGF-A is interpreted as a biological response to VEGFR inhibition.
In summary, clinical use of VEGFR inhibitors is consistently associated with increased circulating VEGF levels, supporting the concept of a class effect for agents targeting VEGFRs. Our current findings align closely with these clinical observations, suggesting that a comparable feedback mechanism may also operate in rodent models.   Main reasons for this on-target effect, could be explained mainly throughout two hypothesis: 1) the Compensatory Upregulation of VEGF, as a Feedback mechanism, to maintain VEGF system deeply regulated, as proposed from the above mentioned clinical studies or 2) Loss of VEGF Clearance by a reduced VEGFR-VEGF complex internalisation, as already discosed (Nat Rev Mol Cell Biol. 2016 Oct;17(10):611-25 & Am J Physiol Heart Circ Physiol. 2004 Jan;286(1):H153-64. In the last 2 references, it has been postulated that internalization of VEGFR2 after VEGF binding regulates how much VEGF is available at the cell surface. When VEGFR2 is internalized and degraded or sequestered inside the cell, less receptor is present on the membrane to bind VEGF, which can reduce VEGF signalling. Thus, the internalization of VEGF receptors (VEGFR1 and VEGFR2) and their ligand-bound complexes plays a central role in regulating VEGF availability at the cell surface. To better understand the sequence of events leading to the observed increase in VEGF levels in rats following Nintedanib treatment, a more extensive analysis will be required and may constitute the focus of future studies. A potential mechanism underlying the observed increased VEGF levels may involve the activation, or removal of inhibitory regulators on VEGF promoter activity. To explore this, we revisited our previous research on nintedanib’s transcriptomic profile in a rat model of bleomycin-induced lung fibrosis and we observed that RNA sequencing data from lungs of Bleomycin + Nintedanib treated rats revealed the upregulation of genes such as Kruppel-like factor 4 (KLF4) and ETS transcription factor 6 (Etv6), both of which are mechanistically linked to VEGF regulation and expression,  in comparison to BLM + Vehicle animals. However, phosphoproteomic data were not able to corroborate this interpretation. This limitation is primarily attributable to the insufficient abundance of some proteins of interest in the analyzed samples, which precluded their reliable detection and quantification. As a result, direct evidence for the modulation of specific low-abundance proteins could not be obtained, and our conclusions are therefore based on the available data from more abundant targets and indirect pathway analysis. The above piece ok knowledge has been now integrated into the manuscript. (Discussion section, page 9-10, lines 302-331 & 360-369). Supporting references have been added.

Comment 2. The use of a double-hit bleomycin model is not sufficiently justified, despite its central role in driving all subsequent conclusions. The authors neither explain why this model was chosen over the more widely used single-dose BLM model nor provide adequate information on its reproducibility, severity, or relevance to clinical IPF pathology. Histological and molecular distinctions between the two approaches particularly whether the double-hit system better mimics progressive fibrotic remodeling remain unaddressed. A deeper methodological rationale and comparative context are necessary to validate the choice of model and enhance the translational strength of the study.

Response 2. We thank the reviewer for this observation and the suggestion to clarify our rationale for selecting the double-hit BLM model. The double-hit BLM model was chosen based on accumulating evidence that it more closely recapitulates key features of human IPF compared to the traditional single-dose model. While the single-dose BLM model is widely used, it often results in transient inflammation and partial resolution of fibrosis, which may not fully reflect the chronic and progressive nature of IPF. Specifically, the double administration of BLM (on days 0 and 4) induces a more persistent and severe fibrotic response, compared to single instillation, with a stronger induction of lung biomarkers (TIMP-1, PAI-1, HYP). This approach is supported by a Poster presented at ERS International Congress 2017 comparing lung fibrosis induced by single or double bleomycin administrationin rats (Pittelli et al., https://publications.ersnet.org/highwire_display/entity_view/node/545459/full) and by our own previous work (Bonatti et al., BMJ Open Respir Res 2023), demonstrating that the double-hit protocol yields a robust and reproducible fibrotic phenotype, including sustained upregulation of translationally relevant ECM-related genes. Moreover, the work presented at ERS 2017, demonstrated a more evident and significant effect of Nintedanib in the double BLM administration group. As already demonstrated for murine models (Wang et al., Biochem. Cell Biol. 103: 1–7; 2025), we also have previously confirmed, in a double intratracheal instillation of BLM in rats,  the presence of a cell population defined KRT8+ alveolar differentiation intermediate (ADI) cells with a transcriptional profile similar to the KRT5-/KRT17+ cells identified in the IPF lung (Bocchi et al., Front. Biosci.  2024; 29(8): 305). Growing evidence indicates that KRT8+ cells are progenitor cells derived from AT2 that could be responsible for the establishment of an aberrant tissue repair process and ECM deposition in the Bleomycin rodent models. In conclusion, our findings enforces previous evidence and further support the use of a double-hit Bleomycin rat model as translational preclinical platform for investigating therapeutic targets for IPF. The experimental design performed in this work included four independent studies, all yielding consistent results in terms of fibrotic marker induction, histological severity (Ashcroft scores), and response to Nintedanib treatment. No mortality was observed, and the model was well tolerated.  We hope these clarifications and manuscript revisions address the reviewer’s concerns and strengthen the translational validity of our findings. We have now expanded the Discussion to clarify rational for using double BLM administration instead of single dose, adding recent studies that validate the double-hit model’s translational relevance (see Bonatti et al., BMJ Open Respir Res 2023 etc, Bocchi et al., Front. Biosci.  2024; 29(8): 305; Wang et al., Biochem. Cell Biol. 103: 1–7; 2025). Manuscript Revisions:  The Discussion section has been revised to include a more detailed justification for the double-hit model and its relevance to human IPF (page 7-8, lines 232-239) and supporting references have been added (24-26). We hope these clarifications and manuscript revisions address the reviewer’s concerns and strengthen the translational validity of our findings.

Comment 3. The authors state that PK/PD modelling supports their conclusions, yet no actual PK/PD modelling outputs are presented. The pharmacokinetic results are purely descriptive, without any connect-the-dots quantitative linking of Nintedanib plasma or lung concentrations to target inhibition, biomarker modulation, or anti-fibrotic outcomes. Critical modelling parameters such as EC50/EC80, exposure response curves, predicted receptor occupancy, or PK/PD relationships are missing. As written, the PK/PD interpretation is overstated, and the manuscript either needs true model-based PK/PD analysis or a substantial revision of the claims regarding PK/PD integration.

Response 3. We thank the reviewer for this observation. The statement regarding the PK/PD modelling is reported only in the abstract but it is definitely a refuse which doesn't find any correspondence in the manuscript; such refuse is probably due to a preliminary idea of performing such activity and that was successively abandoned. As you have correctly highlighted, the pharmacokinetic results are here only descriptive, with the aim to compare the observed exposure with that reported in literature in the clinical setting, as a rough evidence of the efficacy oberved in the rat model and as clearly stated in the  discussion section. We apologise for this discrepancy and thank again the reviewer for noticing it; we have changed the abstract, accordingly.

Comment 4. The absence of key control groups significantly weakens the study’s conclusions. The authors include only acutely dosed naïve animals (1–24 h) for mechanistic evaluation, but do not assess chronic Nintedanib exposure in healthy rats, which is necessary to distinguish drug-specific effects from injury-induced changes. Additionally, no comparator such as pirfenidone despite extensive discussion in the introduction is included to contextualize antifibrotic or biomarker effects. The lack of BALF-based biomarker evaluation also limits interpretation of lung-specific versus systemic effects. The study requires more comprehensive controls to support the claims of specificity and mechanistic insight.

Response 4. We thank the reviewer for his feedback.  The inclusion of an additional animal group to asses chronic Nintedanib exposure in healthy rats, was carefully considered. We observed that VEGF levels increased at 24 hours in healthy animals after acute Nintedanib administration, and a similar VEGF increase was seen in the bleomycin model after chronic Nintedanib treatment. These findings support that the effects of Nintedanib on VEGF increase is consistent across both healthy and diseased animals. Therefore, the addition of a chronic Nintedanib group in healthy animals would not provide further mechanistic insight, while increasing animal use results contrary to ethical principles and the ARRIVE guidelines. Our study was designed in strict accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) and the European Directive 2010/63/EU, Italian D. Lgs 26/2014, which emphasize the 3Rs principle (Replacement, Reduction, and Refinement) in animal research. Our approach was to minimize animal numbers and interventions without compromising the scientific validity of our findings. Although pirfenidone is discussed in the introduction to provide clinical context, direct comparison with pirfenidone was not within the scope of this study. Our primary objective was to demonstrate the anti-fibrotic effects and target engagement of Nintedanib in the double-hit bleomycin model. Finally, the decision to limit comparison between lung and plasma VEGF levels and omit BALF analysis was dictated by consideration that plasma biomarker analysis offers greater translational relevance, whereas BALF collection is invasive and not routinely performed in IPF patients. Moreover, in previous not published experiments we demonstrated that biomarkers measured in BALF (including VEGF), primarily reflect the acute inflammatory phase and show greater temporal variability, while those assessed in lung tissue are more stable and representative of the fibrotic process.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed all of the reviewers' comments, and the manuscript has been improved satisfactorily.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have now addressed all the comments.