During wound healing, myofibroblasts play a central role in matrix formation and wound contraction. At the end of healing, there is evidence that myofibroblasts disappear via apoptotic pathways. Hypertrophic scars are a fibroproliferative disorder that leads to considerable morbidity. It has been postulated that a defect in myofibroblast apoptosis could be responsible for the pathological scar formation, but no evidence exists. We have isolated and cultured human normal wound (Wmyo) and hypertrophic scar (Hmyo) myofibroblasts and compared their basal apoptotic rates and their sensitivity to serum starvation and Fas antibody-induced apoptosis to that obtained for dermal fibroblasts (Fb). A higher rate of apoptosis as evidenced by morphological criteria and a propidium iodide assay was observed for Wmyo in comparison to Fb and Hmyo. These results came along with a low level of the anti-apoptotic proteins Bcl-2 and Bclx(L) in Wmyo, whereas there was an increase in the level of the pro-apoptotic molecule Bax when compared to the results obtained for Fb and Hmyo. Hmyo showed a higher level of Bcl-2 compared to Fb but no difference in the Bax or Bclx(L) level. After serum starvation, Wmyo revealed an increased apoptotic rate, whereas Hmyo and Fb did not show any difference. Anti-Fas treatment did not modify the levels of apoptosis but strongly increased the cell growth of Hmyo as compared to Wmyo. This is the first study presenting a broad vision of the apoptotic sensitivity of normal and pathological myofibroblasts. These results confirmed the hypothesis of defects in apoptosis and growth during pathological scar formation impeding myofibroblast disappearance at the end of healing.

Wound healing is a very highly organized process where numerous cell types are tightly regulated to restore injured tissue. Myofibroblasts are cells that produce new extracellular matrix and contract wound edges. We previously reported that the human myofibroblasts isolated from normal wound (WMyos) produced microvesicles (MVs) in the presence of the serum. In this study, MVs were further characterized using a proteomic strategy and potential functions of the MVs were determined. MV proteins isolated from six WMyo populations were separated using two-dimensional differential gel electrophoresis. Highly conserved spots were selected and analyzed using mass spectrometry resulting in the identification of 381 different human proteins. Using the DAVID database, clusters of proteins involved in cell motion, apoptosis and adhesion, but also in extracellular matrix production (21 proteins, enrichment score: 3.32) and in blood vessel development/angiogenesis (19 proteins, enrichment score: 2.66) were identified. Another analysis using the functional enrichment analysis tool FunRich was consistent with these results. While the action of the myofibroblasts on extracellular matrix formation is well known, their angiogenic potential is less studied. To further characterize the angiogenic activity of the MVs, they were added to cultured microvascular endothelial cells to evaluate their influence on cell growth and migration using scratch test and capillary-like structure formation in Matrigel. The addition of a MV-enriched preparation significantly increased endothelial cell growth, migration and capillary formation compared with controls. The release of microvesicles by the wound myofibroblasts brings new perspectives to the field of communication between cells during the normal healing process.

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During wound healing, angiogenesis plays a crucial role in inducing adequate perfusion of the new tissue, thereby allowing its survival. This angiogenic process contributes to the formation of granulation tissue, alongside myofibroblasts. Myofibroblasts are cells specialized in wound contraction and synthesis of new extracellular matrix. Fibroblasts, considered by some to be at the origin of myofibroblasts, have already been shown to promote neovascularization. Thus, we hypothesized that myofibroblasts play a key role during angiogenic development in wound healing. We isolated myofibroblasts from normal human skin wounds and dermal microvascular endothelial cells (HDMVEC) and fibroblasts from skin. Using an in vitro fibrin-based model, we compared the proangiogenic activity of wound myofibroblasts to that of fibroblasts in the presence of HDMVEC. By immunostaining with collagen IV antibodies, we observed the formation of a capillary network significantly more developed when HDMVEC were cultured with myofibroblasts compared to the network formed in the presence of fibroblasts. The differences between these cell types did not result from a differential secretion of Vascular Endothelial Growth Factor or basic Fibroblast Growth Factor. However, in the presence of myofibroblasts, a significant decrease in matrix metalloproteinase activity was observed. This finding was correlated with a significant increase in Tissue Inhibitor of MetalloProteinase (TIMP)-1 and TIMP-3. Furthermore, inhibition of TIMP-1 secretion using shRNA significantly decreased myofibroblasts induced angiogenesis. These results led to the hypothesis that normal wound myofibroblasts contribute to the vascular network development during wound healing. Our data emphasize the critical role of wound myofibroblasts during healing.

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.