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.

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.

Production of microparticles (MP) by myofibroblasts (Wmyo) during wound healing process is a new finding in the field of cellular communication. To determine the proteome of MP, the 2D‐DIGE method was performed over MP produced by 6 different populations of Wmyo. Each gel allowed us to analysed MP extravesicular proteins and total protein content, due to the use of different Cydye labelling methods. Fluorescent labelling of each gel were acquired using Typhoon Variable Mode Imager system and all images were analysed by Delta2D software. We were looking for highly expressed and extremely conserved proteins. With these parameters and Saffold Viewer software, 133 spots were sent to mass spectrometry analysis for further characterisation. 292 different proteins were identified with a 95% probability of being peptides unique to one protein. This wide variety of proteins included several enzymes such as fructose‐biphosphate aldolase A, L‐lactate dehydrogenase and ATP synthase. Lactadherin, Annexin A2, filamin‐A serpin H1 and elongation factors were also revealed in all samples studied. A large proportion of all the protein listed corresponded to cytoskeleton‐related protein. Knowledge of the protein content of MP used by Wmyo to communicate between each other will enable a better comprehension of the mechanism of wound healing process. Research Support: Thecell network‐FRQS.

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