Is Netrin-1 Associated with the Development of Fibrosis in Systemic Sclerosis?

  1. Yüksel Maraş
  2. Ahmet Kor
  3. Esra Fırat Oğuz
  4. Alper Sarı
  5. Kevser Gök
  6. Ali Akdoğan

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Abstract

Background

Netrin-1 is a laminin class protein that guides the axonal during the first embryonic development, has pushing and pulling properties, and has axonal chemoattractant activity. Netrin-1 has been shown to increase the effect of fibrosis in mouse lung and human SSc lung cell cultures. In this study, we aimed to investigate the relationship between Netrin-1 and Systemic sclerosis (SSc) and to emphasize the role of Netrin-1 in the pathophysiology of SSc by increasing the known VEGF and M2 macrophage expression, which supports the fibrotic process.

Methods

The study included 56 SSc patients with a mean age of 48·.08±13.59 years and 58 healthy volunteers with a mean age of 48.01±11.59 years. SSc organ involvements were scanned retrospectively from patient files, and patients were grouped according to SSc complications. Calculation of Netrin-1 levels was performed using a quantitative sandwich enzyme immunoassay method with an ELISA kit (Elabscience, Texas, USA; catalog number: E-EL-H2328; lot number: GZWTKZ5SWK). The modified Rodnan skin score (mRSS) was used for skin thickness scoring in SSc patients.

Results

The median value of Netrin-1 was found to be significantly higher in SSc (268.8 [82.75-1006.64]) than in controls (108.63 [21.02-351.49]) (p<0.0001). In ROC analysis, a cut-off value of 354.24 for Netrin in SSc was found to provide a sensitive confidence interval with 32.8% sensitivity and 98.3% specificity (AUC[95% CI]: 0.746-0.895, p<0.0001). There was no significant correlation between Netrin-1 level, organ involvement in SSc, and mRSS (p>0.05).

Conclusion

We found a significant relationship between Netrin-1 levels and SSc disease in this study. Our study is the first clinical study in which Netrin-1 elevation was demonstrated in SSc patients.

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  1. PeerRef

    Peer review report

    Reviewer: Suleyman Sedar Koca

    ORCID: 0000-0003-4995-430X

    email: kocassk@yahoo.com

    General assessment

    The latest version (bioRxiv V3) of the manuscript is acceptable/publishable as it stands.

    Essential revisions that are required to verify the manuscript

    There are no suggestions for revisions

    Decision

    Verified manuscript: The content is scientifically sound, only minor amendments (if any) are suggested.

  2. Version published to 10.1101/2022.02.05.22270510v3 on medRxiv
  3. PeerRef

    Peer review report

    Reviewer: Barbara Ruaro Institution: University of Trieste email: barbara.ruaro@yahoo.it

    Section 1 – Serious concerns

    • Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No
    • Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? Yes

    Section 2 – Language quality

    • How would you rate the English language quality?

    Low to medium quality, but I understand the content (e.g. Our study is the first clinical study in which Netrin-1 elevation was demonstrated in SSc patients. It is better “studied” than “demonstrated”

    Section 3 – validity and reproducibility

    • Does the work cite relevant and sufficient literature? No
    • Is the study design appropriate and are the methods used valid? Yes
    • Are the methods documented and analysis provided so that the study can be replicated? Yes
    • Is the source data that underlies the result available so that the study can be replicated? Yes
    • Is the statistical analysis and its interpretation appropriate? No
    • Is quality of the figures and tables satisfactory? No
    • Are the conclusions adequately supported by the results? No
    • Are there any objective errors or fundamental flaws that make the research invalid? No

    Section 4 – Suggestions

    • In your opinion how could the author improve the study?

    1. Abstract. Results There was no significant correlation between netrin-1 level, organ involvement in SSc, and MRS (p>0.05). Please clarify MRS acronym. Is it mRSS (modified Rodnan Skin Score)?

    2. Abstract. Conclusion: In this study, we found that there is a significant relationship between Netrin-1 levels and SSc disease. Our study is the first clinical study in which Netrin-1 elevation was demonstrated in SSc patients. Please in the last sentence use “elevation was shown”

    3. Systemic sclerosis (SSc), often called scleroderma, is an autoimmune, destructive systemic connective tissue disease characterized by organ fibrosis and vasculopathy. Pathophysiological mechanisms that may play a role in disease development include platelet activation, fibroblast proliferation, endothelial disruption, fetal microchimerism, and increased transforming growth factor-β. In addition, VEGF is an important signaling factor contributing to the pathogenesis of SSc, even in the earliest clinically detectable stages of the disease.[1] Please add some references.

    4. Introduction. In this study, we aimed to evaluate the levels of Netrin1 between SSc and healthy controls and to emphasize the role of the known effects of Netrin-1 in the pathophysiology of SSc, which increases VEGF and supports the fibrotic process. Please change “to evaluate the levels of Netrin1 between SSc and healthy controls” and write” to compare the levels of Netrin1 in SSc patients and healthy controls”

    5. Methods. Modified Rodnan scoring (MRS) was used for skin thickness scoring in SSc patients. For MRS, 17 body areas were evaluated and scored in the range of 0-51 points. Please use the acronym mRSS.

    6)Results. Please underline the statistical values to support the conclusions.

    1. DISCUSSION SSc is a progressive disease that pathophysiologically starts with microvascular damage and then develops widespread fibrosis due to increased autoimmune response and inflammation.[18]. Please summarise and underlie here the most important data of the study.

    Section 5 – Decision

    Verified with reservations: The content is scientifically sound but has shortcomings that could be improved by further studies and/or minor revisions.

  4. PeerRef

    SciScore rigor report

    Sciscore is an AI platform that assesses the rigor of the methods used in the manuscript. SciScore assists expert referees by finding and presenting information scattered throughout a manuscript in a simple format.

    Not required = Field is not applicable to this study

    Not detected = Field is applicable to this study, but not included.

    Ethics

    IRB: The ethics committee approval of the research protocol was made by the Ankara City Hospital Consent: Informed consent was obtained from the patients to participate in the study.

    Inclusion and Exclusion Criteria

    not detected.

    Attrition

    Two publications are evaluating the association with Netrin-1 in bleomycin-induced lung fibrosis in mice and SSc lung cell culture in humans.

    Sex as a biological variable

    A total of 56 SSc patients (mean age: 48.08±13.59) consisting of 53 females and 3 males, who were followed up in the rheumatology department of Ankara city hospital, diagnosed according to the 2013 ACR (American College of Rheumatology)/EULAR (European League Against Rheumatism) SSc classification criteria were included in the study.

    Subject Demographics

    Age: For the control group, 58 healthy volunteers (mean age: 48.01±11.59 years) consisting of 54 females and 4 males were included in the study.

    Randomization

    not detected.

    Blinding

    not detected.

    Power Analysis

    not detected.

    Replication

    not required.

    Data Information

    Availability: It is made available under a CC-BY-NC-ND 4.0 International license .

    Identifiers: preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in medRxiv preprint doi: https:// doi.org/10.1101/2022.02.05.22270510; this version posted February 10, 2022. https://doi.org/10.1101/2022.02.05.22270510

  5. Version published to 10.1101/2022.02.05.22270510v2 on medRxiv
  6. Version published to 10.1101/2022.02.05.22270510v1 on medRxiv