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Vasculature plays an essential role in skin physiology and its architecture and function are altered in aged and diseased skin. Quite remarkably, papillary and reticular dermis show very distinct extracellular matrix (ECM) and vascularization. Furthermore, fibroblasts freshly isolated from their native microenvironment have different gene expression patterns, morphology and proliferation rate. Whereas 2D culture or embedding in hydrogels has been used to characterize both fibroblast subpopulations, the lack of relevant models has hindered investigation of their contribution to vascularization. We thus cultured human papillary and reticular fibroblasts as cell sheets over two to three weeks and used RNA-seq differential expression analysis to identify genes involved in microenvironment generation. Bioinformatics analysis revealed that cell sheet culture maintains specific expression of matrisome gene signatures resulting in papillary and reticular ECMs that differ in composition and structure. The transcriptomic also revealed layer-specific expression of angiogenesis-related genes. The impact of secreted and ECM-bound factors was then assessed using two independent 3D angiogenesis assays. The first assay consisted in stimulating endothelial cells embedded in a fibrin gel with conditioned media from each fibroblast subpopulation, while the second assay was based on cell-sheet co-culture of fibroblasts and endothelial cells, thus allowing direct interactions of endothelial cells with the microenvironment generated by fibroblasts. Vascularization was analysed in 3D using in-house developed software (3D-skel; Atlas et al, 2021) with both assays. These analyses revealed that papillary fibroblasts secrete highly angiogenic factors and produce a microenvironment characterised by ECM remodelling capacity and formation of dense small vessels, whereas reticular fibroblasts produced more structural components of the ECM associated with less but larger vessels. These features mimick the characteristics of both the ECM and vasculature of native dermis subcompartments. In addition to showing that skin fibroblast populations differentially regulate angiogenesis via both secreted and ECM factors, our work emphasizes the importance of papillary and reticular fibroblasts for tissue engineering and modelling dermis microenvironment and its vascularization.
Atlas Y, Gorin C, Novais A, Marchand MF, Chatzopoulou E, Lesieur J, Bascetin R, Binet-Moussy C, Sadoine J, Lesage M, Opsal-Vital S, Péault B, Monnot C, Poliard A, Girard P, Germain S, Chaussain C, Muller L. Microvascular maturation by mesenchymal stem cells in vitro improves blood perfusion in implanted tissue constructs. (2021). Biomaterials. 268: 520194
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