Papillary and reticular fibroblasts generate distinct microenvironments that differentially impact angiogenesis.

Papillary and reticular dermis show distinct extracellular matrix (ECM) and vascularization corresponding to their specific functions. These characteristics are associated with gene expression patterns of fibroblasts freshly isolated from their native microenvironment. In order to assess the relevance of these fibroblast subpopulations in a tissue engineering context, we investigated their contribution to matrix production and vascularization using cell sheet culture conditions. We first performed RNA-seq differential expression analysis to determine whether several rounds of cell amplification and high-density culture affected their gene expression profile. Bioinformatics analysis revealed that expression of angiogenesis-related and matrisome gene signatures were maintained, resulting in papillary and reticular ECMs that differ in composition and structure. The impact of secreted or ECM-associated factors was then assessed using two independent 3D angiogenesis assays: -1/ a fibrin hydrogel-based assay allowing investigation of diffusible secreted factors, -2/ a scaffold-free cell-sheet based assay for investigation of fibroblast-produced microenvironment. These analyses revealed that papillary fibroblasts secrete highly angiogenic factors and produce a microenvironment characterised by ECM remodelling capacity and dense and branched microvascular network, whereas reticular fibroblasts produced more structural core components of the ECM associated with less branched and larger vessels. These features mimick the characteristics of both the ECM and the vasculature of 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 engineering and modelling dermis microenvironment and vascularization. STATEMENT OF SIGNIFICANCE: Recent advances have brought to the forefront the central role of microenvironment and vascularization in tissue engineering for regenerative medicine and microtissue modelling. We have investigated the role of papillary and reticular fibroblast subpopulations using scaffold-free cell sheet culture. This approach provides differentiated cells conditions allowing the production of their own microenvironment. Analysis of gene expression profiles and characterisation of the matrix produced revealed strong and specific angiogenic properties that we functionally characterized using 3D angiogenesis models targeting the respective role of either secreted or matrix-bound factors. This study demonstrates the importance of cell-generated extracellular matrix and questions the importance of cell source and the relevance of hydrogels for developing physio-pathologically relevant tissue engineered substitutes.

Angiopoietin-like 4-Induced 3D Capillary Morphogenesis Correlates to Stabilization of Endothelial Adherens Junctions and Restriction of VEGF-Induced Sprouting.

Angiopoietin-like 4 (ANGPTL4) is a target of hypoxia that accumulates in the endothelial extracellular matrix. While ANGPTL4 is known to regulate angiogenesis and vascular permeability, its context-dependent role related to vascular endothelial growth factor (VEGF) has been suggested in capillary morphogenesis. We here thus develop in vitro 3D models coupled to imaging and morphometric analysis of capillaries to decipher ANGPTL4 functions either alone or in the presence of VEGF. ANGPTL4 induces the formation of barely branched and thin endothelial capillaries that display linear adherens junctions. However, ANGPTL4 counteracts VEGF-induced formation of abundant ramified capillaries presenting cell-cell junctions characterized by VE-cadherin containing reticular plaques and serrated structures. We further deciphered the early angiogenesis steps regulated by ANGPTL4. During the initial activation of endothelial cells, ANGPTL4 alone induces cell shape changes but limits the VEGF-induced cell elongation and unjamming. In the growing sprout, ANGPTL4 maintains cohesive VE-cadherin pattern and sustains moderate 3D cell migration but restricts VEGF-induced endothelium remodeling and cell migration. This effect is mediated by differential short- and long-term regulation of P-Y1175-VEGFR2 and ERK1-2 signaling by ANGPTL4. Our in vitro 3D models thus provide the first evidence that ANGPTL4 induces a specific capillary morphogenesis but also overcomes VEGF effect.