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Although it is well established that 5 to 15% of radiotherapy patients exhibit severe side-effects in non-cancerous tissues, the molecular mechanisms involved are still poorly known, and the links between cellular and tissue radiosensitivity are still debated. We here studied fibroblasts from non-irradiated skin of patients with severe sequelae of radiotherapy, to determine whether specific basal cell activities might be involved in susceptibility to side-effects in normal tissues. Compared to control cells, patient fibroblasts exhibited higher radiosensitivity together with defects in DNA repair. Transcriptome profiling of dermal fibroblasts from 16 radiotherapy patients with severe side-effects and 8 healthy individuals identified 540 genes specifically deregulated in the patients. Nuclear factor of activated T cells 2 (NFATC2) was the most differentially expressed gene, poorly expressed at both transcript and protein level, whereas the NFATC2 gene region was hypermethylated. Furthermore, NFATC2 expression correlated with cell survival after irradiation. Finally, silencing NFATC2 in normal cells by RNA interference led to increased cellular radiosensitivity and defects in DNA repair. This study demonstrates that patients with clinical hypersensitivity also exhibit intrinsic cellular radiosensitivity in their normal skin cells. It further reveals a new role for NFATC2 as a potential regulator of cellular sensitivity to ionizing radiation.

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.