Adventitial fibroblasts direct smooth muscle cell-state transition in pulmonary vascular disease

Reviewer #1 (Public Review):

Summary:

The authors isolated and cultured pulmonary artery smooth muscle cells (PASMC) and pulmonary artery adventitial fibroblasts (PAAF) of the lung samples derived from the patients with idiopathic pulmonary arterial hypertension (PAH) and the healthy volunteers. They performed RNA-seq and proteomics analyses to detail the cellular communication between PASMC and PAAF, which are the main target cells of pulmonary vascular remodeling during the pathogenesis of PAH. The authors revealed that PASMC and PAAF retained their original cellular identity and acquired different states associated with the pathogenesis of PAH, respectively.

Strengths:

Although previous studies have shown that PASMC and PAAF cells each have an important role in the pathogenesis of PAH, there have been scarce reports focusing on the interactions between PASMC and PAAF. These findings may provide valuable information for elucidating the pathogenesis of pulmonary arterial hypertension.

Weaknesses:

The results of proteome analysis using primary culture cells in this paper seem a bit insufficient to draw conclusions. In particular, the authors described "We elucidated the involvement of cellular crosstalk in regulating cell state dynamics and identified pentraxin-3 and hepatocyte growth factor as modulators of PASMC phenotypic transition orchestrated by PAAF." However, the presented data are considered limited and insufficient.

Reviewer #2 (Public Review):

Summary:

Utilizing a combination of transcriptomic and proteomic profiling as well as cellular phenotyping from source-matched PASMC and PAAFs in IPAH, this study sought to explore a molecular comparison of these cells in order to track distinct cell fate trajectories and acquisition of their IPAH-associated cellular states. The authors also aimed to identify cell-cell communication axes in order to infer mechanisms by which these two cells interact and depend upon external cues. This study will be of interest to the scientific and clinical communities of those interested in pulmonary vascular biology and disease. It also will appeal to those interested in lung and vascular development as well as multi-omic analytic procedures.

Strengths:

(1) This is one of the first studies using orthogonal sequencing and phenotyping for the characterization of source-matched neighboring mesenchymal PASMC and PAAF cells in healthy and diseased IPAH patients. This is a major strength that allows for direct comparison of neighboring cell types and the ability to address an unanswered question regarding the nature of these mesenchymal "mural" cells at a precise molecular level.

(2) Unlike a number of multi-omic sequencing papers that read more as an atlas of findings without structure, the inherent comparative organization of the study and presentation of the data were valuable in aiding the reader in understanding how to discern the distinct IPAH-associated cell states. As a result, the reader not only gleans greater insight into these two interacting cell types in disease but also now can leverage these datasets more easily for future research questions in this space.

(3) There are interesting and surprising findings in the cellular characterizations, including the low proliferative state of IPAH-PASMCs as compared to the hyperproliferative state in IPAH-PAAFs. Furthermore, the cell-cell communication axes involving ECM components and soluble ligands provided by PAAFs that direct cell state dynamics of PASMCs offer some of the first and foundational descriptions of what are likely complex cellular interactions that await discovery.

(4) Technical rigor is quite high in the -omics methodology and in vitro phenotyping tools used.

Weaknesses:

There are some weaknesses in the methodology that should temper the conclusions:

(1) The number of donors sampled for PAAF/PASMCs was small for both healthy controls and IPAH patients. Thus, while the level of detail of -omics profiling was quite deep, the generalizability of their findings to all IPAH patients or Group 1 PAH patients is limited.

(2) While the study utilized early passage cells, these cells nonetheless were still cultured outside the in vivo milieu prior to analysis. Thus, while there is an assumption that these cells do not change fundamental behavior outside the body, that is not entirely proven for all transcriptional and proteomic signatures. As such, the major alterations that are noted would be more compelling if validated from tissue or cells derived directly from in vivo sources. Without such validation, the major limitation of the impact and conclusions of the paper is that the full extent of the relevance of these findings to human disease is not known.

(3) While the presentation of most of the manuscript was quite clear and convincing, the terminology and conclusions regarding "cell fate trajectories" throughout the manuscript did not seem to be fully justified. That is, all of the analyses were derived from cells originating from end-stage IPAH, and otherwise, the authors were not lineage tracing across disease initiation or development (which would be impossible currently in humans). So, while the description of distinct "IPAH-associated states" makes sense, any true cell fate trajectory was not clearly defined.

Author response:

Reviewer #1 (Public Review):

Summary:

The authors isolated and cultured pulmonary artery smooth muscle cells (PASMC) and pulmonary artery adventitial fibroblasts (PAAF) of the lung samples derived from the patients with idiopathic pulmonary arterial hypertension (PAH) and the healthy volunteers. They performed RNA-seq and proteomics analyses to detail the cellular communication between PASMC and PAAF, which are the main target cells of pulmonary vascular remodeling during the pathogenesis of PAH. The authors revealed that PASMC and PAAF retained their original cellular identity and acquired different states associated with the pathogenesis of PAH, respectively.

Strengths:

Although previous studies have shown that PASMC and PAAF cells each have an important role in the pathogenesis of PAH, there have been scarce reports focusing on the interactions between PASMC and PAAF. These findings may provide valuable information for elucidating the pathogenesis of pulmonary arterial hypertension.

We appreciate the reviewer’s positive view of our study.

Weaknesses:

The results of proteome analysis using primary culture cells in this paper seem a bit insufficient to draw conclusions. In particular, the authors described "We elucidated the involvement of cellular crosstalk in regulating cell state dynamics and identified pentraxin-3 and hepatocyte growth factor as modulators of PASMC phenotypic transition orchestrated by PAAF." However, the presented data are considered limited and insufficient.

We thank the reviewer for drawing our attention to this point and we will modify our statements and conclusions accordingly, in order to avoid making too general and broad claims.

Reviewer #2 (Public Review):

Summary:

Utilizing a combination of transcriptomic and proteomic profiling as well as cellular phenotyping from source-matched PASMC and PAAFs in IPAH, this study sought to explore a molecular comparison of these cells in order to track distinct cell fate trajectories and acquisition of their IPAH-associated cellular states. The authors also aimed to identify cell-cell communication axes in order to infer mechanisms by which these two cells interact and depend upon external cues. This study will be of interest to the scientific and clinical communities of those interested in pulmonary vascular biology and disease. It also will appeal to those interested in lung and vascular development as well as multi-omic analytic procedures.

We thank the reviewer forvery positive assessment of our study.

Strengths:

(1) This is one of the first studies using orthogonal sequencing and phenotyping for the characterization of source-matched neighboring mesenchymal PASMC and PAAF cells in healthy and diseased IPAH patients. This is a major strength that allows for direct comparison of neighboring cell types and the ability to address an unanswered question regarding the nature of these mesenchymal "mural" cells at a precise molecular level.

We value the reviewer’s kind and objective summary of our study.

(2) Unlike a number of multi-omic sequencing papers that read more as an atlas of findings without structure, the inherent comparative organization of the study and presentation of the data were valuable in aiding the reader in understanding how to discern the distinct IPAH-associated cell states. As a result, the reader not only gleans greater insight into these two interacting cell types in disease but also now can leverage these datasets more easily for future research questions in this space.

We thank the reviewer for this highly positive comment.

(3) There are interesting and surprising findings in the cellular characterizations, including the low proliferative state of IPAH-PASMCs as compared to the hyperproliferative state in IPAH-PAAFs. Furthermore, the cell-cell communication axes involving ECM components and soluble ligands provided by PAAFs that direct cell state dynamics of PASMCs offer some of the first and foundational descriptions of what are likely complex cellular interactions that await discovery.

We agree with the reviewer’s assessment that some of the novel data in our study helps to formulate testable hypothesis that can be followed up in future research.

(4) Technical rigor is quite high in the -omics methodology and in vitro phenotyping tools used.

We are grateful for reviewer’s recognition and positive assessment of our work.

Weaknesses:

There are some weaknesses in the methodology that should temper the conclusions:

(1) The number of donors sampled for PAAF/PASMCs was small for both healthy controls and IPAH patients. Thus, while the level of detail of -omics profiling was quite deep, the generalizability of their findings to all IPAH patients or Group 1 PAH patients is limited.

We share the reviewers concerns regarding the generalizability of the findings. Indeed, the initial number of samples used for the omics study (n=4 in each group) was limited due to the unique setup of using source-matched cells from the same pulmonary artery. While we included additional samples in our phenotypic assays (n=6) which further confirmed our findings, we will acknowledge the small number of samples in the revised manuscript as a limiting factor in drawing definite conclusions for all PAH patients.

(2) While the study utilized early passage cells, these cells nonetheless were still cultured outside the in vivo milieu prior to analysis. Thus, while there is an assumption that these cells do not change fundamental behavior outside the body, that is not entirely proven for all transcriptional and proteomic signatures. As such, the major alterations that are noted would be more compelling if validated from tissue or cells derived directly from in vivo sources. Without such validation, the major limitation of the impact and conclusions of the paper is that the full extent of the relevance of these findings to human disease is not known.

We thank the reviewer for this constructive and excellent suggestion. Changes induced by ex vivo culturing are a common challenge when working with primary human cells. We agree with the reviewer that the proposed comparison with the publicly available sequencing datasets utilizing fresh samples will provide the readers with sufficient information to more objectively put the findings of our study into perspective.

(3) While the presentation of most of the manuscript was quite clear and convincing, the terminology and conclusions regarding "cell fate trajectories" throughout the manuscript did not seem to be fully justified. That is, all of the analyses were derived from cells originating from end-stage IPAH, and otherwise, the authors were not lineage tracing across disease initiation or development (which would be impossible currently in humans). So, while the description of distinct "IPAH-associated states" makes sense, any true cell fate trajectory was not clearly defined.

In accordance with reviewer’s comment, we will more carefully choose the wording in order to better reflect our findings.