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.

Granulation tissue myofibroblasts during normal and pathological skin healing: The interaction between their secretome and the microenvironment

“…These results concur with other studies that have explored the angiogenesis triggered by myofibroblasts 32,33 or the beneficial effect of the myofibroblast secretome on the wound healing process 34 (Figure 3). Additionally, myofibroblasts isolated from normal human skin wounds release vascular endothelial growth factor (VEGF)-A, a proangiogenic cytokine and FGF2, a profibrotic cytokine, but in the same quantities as skin fibroblasts.…”

“…Culture supernatants were collected and quantified using a multiplex assay as described in our previous research paper. 34 These are the extended unpublished results representing the concentrations of cytokines produced per cell. N = 6.…”

“…Additionally, myofibroblasts isolated from normal human skin wounds release vascular endothelial growth factor (VEGF)-A, a proangiogenic cytokine and FGF2, a profibrotic cytokine, but in the same quantities as skin fibroblasts. 32 These cutaneous myofibroblasts also secrete placental growth factor (PlGF)-1, IL-23 and TGFß1 34 and numerous other cytokines such as IL-6, monocyte chemoattractant protein-1 (MCP-1; also known as CCL2), stromal cell-derived factor 1 (SDF-1α; also known as CXCL12) and growth-regulated oncogenealpha (Gro-α) (also known as CXCL1) (Figure 4).…”

“…It has been shown that myofibroblasts isolated from human skin wounds produce microvesicles that contain cytokines. 33,34,39 F I G U R E 4 (a) Cytokine multiplex analysis of myofibroblast-conditioned media. Myofibroblasts isolated from human skin wounds were cultured until confluence at 37 C + 5% CO 2 .…”

“…(b) Result is taken from our previous research paper. 34 TGF-b1 has been analysed by an aside ELISA (since it was absent in the batch of 45 cytokines). TGF-b1 has not been detected (ND) in either myofibroblast cells or microvesicle samples (N = 3 samples of MV derived from three different Wmyo populations).…”