Hyaluronic acid (HA), used in a variety of medical applications, is associated in rare instances to long-term adverse effects. Although the aetiology of these events is unknown, a number of hypotheses have been proposed, including low molecular weight of HA (LMW-HA) in the filler products. We hypothesized that cross-linked HA and its degradation products, in a low-grade inflammatory microenvironment, could impact immune responses that could affect cell behaviours in the dermis. Using two different cross-linking technologies VYC-15L and HYC-24L+, and their hyaluronidase-induced degradation products, we observed for nondegraded HA, VYC-15L and HYC-24L+, a moderate and transient increase in IL-1β, TNF-α in M1 macrophages under low-grade inflammatory conditions. Endothelial cells and fibroblasts were preconditioned using inflammatory medium produced by M1 macrophages. 24 h after LMW-HA fragments and HA stimulation, no cytokine was released in these preconditioned cells. To further characterize HA responses, we used a novel in vivo murine model exhibiting a systemic low-grade inflammatory phenotype. The intradermal injection of VYC-15L and its degradation products induced an inflammation and cell infiltration into the skin that was more pronounced than those by HYC-24L+. This acute cutaneous inflammation was likely due to mechanical effects due to filler injection and tissue integration rather than its biological effects on inflammation. VYC-15L and its degradation product potentiated microvascular response to acetylcholine in the presence of a low-grade inflammation. The different responses with 2D cell models and mouse model using the two tested cross-linking HA technologies showed the importance to use integrative complex model to better understand the effects of HA products according to inflammatory state.

The aim of the present study was to evaluate the impact of the microenvironment produced by dermal microvascular endothelial cells, secondary to a pro-inflammatory challenge, on 2D culture models using dermal fibroblasts and in 3D reconstructed skin model using dermal fibroblasts and keratinocytes from healthy donors. We hypothesized that specific microvascular endothelial low grade inflammation could change fibroblasts phenotype and be involved in extracellular matrix (ECM) modification and skin alteration. Following IFNγ, TNFα, IL-1β pro-inflammatory stress on Human Dermal Endothelial Cells (HDMEC) we observed the increased release of Chemokine ligand 2 (CCL2), IL-6 and IL-8 but not VEGF-A in the conditioned medium (CM). The subsequent addition of this endothelial pro-inflammatory CM in dermal fibroblasts revealed an upregulation of IL6, IL8 and CCL2 but no NF-κB gene expression. The resulting ECM formation was impaired with a reduction of the collagen 1 network and a decrease in COL1A1 gene expression in 2D and 3D models. Collagen 1 and pro-LOX protein expression were significantly reduced confirming an impairment of the collagen network related to endothelial inflammation secretion. To conclude, this work showed that, without any immune cells, the endothelial secretion in response to a pro-inflammatory stress is able to activate the fibroblasts that will maintain the pro-inflammatory environment and exacerbate ECM degradation.