PLGF-1 contained in normal wound myofibroblast-derived microvesicles stimulated collagen production by dermal fibroblasts

Abstract

During the last stages of wound healing, myofibroblasts differentiate mainly from fibroblasts. Myofibroblasts from normal skin wounds (Wmyo) can communicate with its surrounding using secreted factors. They also have the capacity to produce microvesicles (MVs), a type of extracellular vesicles, as mediators of intercellular communication. MVs cargo are potentially capable of regulating the behavior of targeted cells and tissues. The aim of this study is to evaluate the effect of Wmyo-derived MVs on dermal fibroblasts and to determine the responsible signaling molecule. Microvesicles were obtained from culture media of myofibroblasts and characterized using protein quantification, dynamic light scattering and transmission electron microscopy. Uptake of fluorescent MVs in fibroblasts was assessed by flow cytometry. Cytokines concentrations were quantified in MV samples by a multiplex ELISA. Different concentration of MVs or a selected cytokine were used as treatments over fibroblasts culture for 5 days. Following the treatments, parameters linked to the extracellular matrix were studied. Lastly, the selected cytokine was neutralized within MVs before evaluating collagen production. We showed that Wmyo derived-MVs were internalized by dermal fibroblasts. Cytokine array analysis revealed that a large amount of placental growth factor 1 (PLGF-1) (0.88 ± 0.63 pg/μg proteins in MVs) could be detected in MVs samples. Cutaneous fibroblasts treated with MVs or PLGF-1 showed significantly stimulated procollagen I level production (Fold change of 1.80 ± 0.18 and 2.07 ± 0.18, respectively). Finally, the neutralization of PLGF-1 in MVs significantly inhibited the production of procollagen I by fibroblasts. Our study shows that Wmyo derived-MVs are involved in intercellular communication by stimulating collagen production by fibroblasts during wound healing. This effect is possibly attained through PLGF-1 signalling. These findings represent a promising opportunity to gain insight into how MVs and Wmyo may mediate the healing of a skin wound.

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Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Code Availability

Not applicable.

Abbreviations

Wmyo:

Myofibroblasts from normal skin wounds

MVs:

microvesicles

PLGF-1:

Placental growth factor 1

ECM:

Extracellular matrix

TGFß1:

Transforming growth factor-ß

α-SMA:

Alpha-Smooth Muscle Actin

A2M:

Alpha-2-macroglobulin

MMPs:

Matrix metalloproteinases

DME:

Dulbecco’s Modified Eagle’s medium

FBS:

Fetal bovine serum

RFP-670:

Red Fluorescent Protein

PBS:

Phosphate-buffered saline

DLS:

Dynamic light scattering

PLGF-1-NAb:

PLGF-1 neutralizing antibodies

LTA:

Lymphotoxin alpha/ tumor necrosis beta

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Acknowledgements

The authors thank Caroline Gilbert from the CHU of the Université Laval for the use of the Nanosizer® apparatus, Richard Janvier from IBIS of the Université Laval for the use of their TEM microscope and, Annie Karakeussian-Rimbaud from the CHUM for the handling of the multiplex ELISA device.

Funding

This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) (RGPIN2014–04404); les Fonds de recherche du Québec-Santé (FRQS) (Research Centre funding grant); the Quebec Cell, Tissue and Gene Therapy Network–ThéCell (a thematic network supported by FRQS).

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S.A. performed the experiments and wrote the manuscript, S.L conceived the transduction of Wmyo with a fluorescent protein, V.J.M conducted the project and wrote the manuscript. All authors reviewed the manuscript.

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Correspondence to Véronique J. Moulin.

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All procedures involving patients were reviewed and approved by the Research Ethical Committee of the Centre Hospitalier Universitaire (CHU) de Québec-Université Laval, 2014–04404.

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Arif, S., Larochelle, S. & Moulin, V.J. PLGF-1 contained in normal wound myofibroblast-derived microvesicles stimulated collagen production by dermal fibroblasts. J. Cell Commun. Signal. (2020). https://doi.org/10.1007/s12079-020-00572-5

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Keywords

  • Collagen
  • Fibroblasts
  • Microvesicles
  • Myofibroblasts
  • PLGF-1
  • Wound healing