Impact of Human Dermal Microvascular Endothelial Cells on Primary Dermal Fibroblasts in Response to Inflammatory Stress

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

Lysyl Oxidase silencing impairs keratinocyte differentiation in a reconstructed-epidermis model

International audienceLysyl Oxidase (LOX) is an extracellular enzyme involved in the maturation of connective tissues. It also acts in many cell types as a regulator of cell behaviour and phenotype through intracellular signalling pathways. Recently, LOX was shown to be present in human epidermis where its precise functions remain unclear. We showed here that in confluent monolayer cultures of normal human keratinocytes (KCs) and N/TERT-1-immortalized KCs, LOX expression was induced during the first differentiation steps. Moreover, the silencing of LOX by stable RNA interference disrupted the expression of early differentiation markers. In a reconstructed-epidermis model, LOX silencing did not impair the stratification process nor the formation of the first differentiated layers. However, terminal differentiation was strongly impaired, as shown by a decreased expression of late differentiation proteins and by the absence of stratum corneum. Nonetheless, inhibition of LOX enzymatic activity by β-aminopropionitrile did not affect the differentiation process. Therefore, LOX protein acts during the first steps of KC differentiation and is important for subsequent commitment into terminal differentiation. Taken together, these results suggest that a finely regulated expression of LOX is necessary for normal KC differentiation and thus for maintenance of epidermal homeostasis

Epigenetic Silencing of Lysyl Oxidase-Like-1 through DNA Hypermethylation in an Autosomal Recessive Cutis Laxa Case

International audienceWe have recently reported a case of cutis laxa caused by a fibulin-5 missense mutation (p.C217R). Skin fibroblasts from this individual showed an abnormal pattern of expression of several genes coding for elastic fiber-related proteins, including lysyl oxidase-like-1 (LOXL1). In this study we intended to elucidate the mechanism responsible for LOXL1 downregulation in these fibulin-5-mutant cells. We identified a proximal region (-442/-342) of the human LOXL1 promoter in which two binding sites for the transcription factor specific protein 1 (Sp-1) are required for gene activity in normal fibroblasts. Binding of Sp-1 to these sequences was dramatically reduced within cutis laxa cells, although Sp-1 expression was normal. Further analysis of the promoter sequence found increased methylation levels in cutis laxa cells compared with cells from unaffected individuals. When DNA methyltransferase activity was transiently inhibited in cutis laxa cells using the 5-aza-2'-deoxycytidine, we found a significant increase in LOXL1 expression. In conclusion, besides changes caused by the fibulin-5 mutation, LOXL1 gene regulation is affected by an epigenetic mechanism that can be reversed by an inhibitor of DNA methyltransferase activity. It is not yet known whether LOXL1 gene expression is affected in all cases of cutis laxa arising from fibulin-5 mutation

Dill extract preserves dermal elastic fibers network and functionality. Implication of elafin.

Elastic skin fibers lose their mechanical properties during aging due to enzymatic degradation, lack of maturation or post-translational modifications. Dill extract has been observed to increase elastin protein expression and maturation in a 3D skin model, to improve mechanical properties of skin, to increase elastin protein expression in vascular smooth muscle cells, to preserve aortic elastic lamella and to prevent glycation

Lysyl oxidase is a strong determinant of tumor cell colonization in bone. Short Title: LOX triggers tumor cell colonization in bone

International audienceLysyl oxidase (LOX) is a secreted copper-dependent amine oxidase whose primary function is to drive collagen crosslinking and extracellular matrix stiffness. LOX in colorectal cancer (CRC) synergizes with hypoxia-inducible factor-1 (HIF-1 to promote tumor progression. Here we investigated whether LOX/HIF1 endows CRC cells with full competence for aggressive colonization in bone. We show that a high LOX expression in primary tumors from CRC patients was associated with poor clinical outcome, irrespective of HIF-1. Additionally, LOX was expressed by tumor cells in the bone marrow from CRC patients with bone metastases. In vivo experimental studies show that LOX overexpression in CRC cells or systemic delivery of the conditioned medium from LOX-overexpressing CRC cells promoted tumor cell dissemination in the bone marrow and enhanced osteolytic lesion formation, irrespective of HIF-1. Conversely, silencing or pharmacological inhibition of LOX activity blocked dissemination of CRC cells in the bone marrow and tumor-driven osteolytic lesion formation. In vitro, tumor-secreted LOX supported the attachment and survival of CRC cells to and in the bone matrix, and inhibited osteoblast differentiation. LOX overexpression in CRC cells also induced a robust production of IL-6. In turn, both LOX and IL-6 were acting in concert to promote RANKL-dependent osteoclast differentiation, thereby creating an imbalance between bone resorption and bone formation. Collectively, our findings show that LOX supports CRC cell dissemination in the bone marrow and they reveal a novel mechanism through which LOX-driven IL-6 production by CRC cells impairs bone homeostasis

Lysyl oxidase is a strong determinant of tumor cell colonization in bone. Short Title: LOX triggers tumor cell colonization in bone

International audienceLysyl oxidase (LOX) is a secreted copper-dependent amine oxidase whose primary function is to drive collagen crosslinking and extracellular matrix stiffness. LOX in colorectal cancer (CRC) synergizes with hypoxia-inducible factor-1 (HIF-1 to promote tumor progression. Here we investigated whether LOX/HIF1 endows CRC cells with full competence for aggressive colonization in bone. We show that a high LOX expression in primary tumors from CRC patients was associated with poor clinical outcome, irrespective of HIF-1. Additionally, LOX was expressed by tumor cells in the bone marrow from CRC patients with bone metastases. In vivo experimental studies show that LOX overexpression in CRC cells or systemic delivery of the conditioned medium from LOX-overexpressing CRC cells promoted tumor cell dissemination in the bone marrow and enhanced osteolytic lesion formation, irrespective of HIF-1. Conversely, silencing or pharmacological inhibition of LOX activity blocked dissemination of CRC cells in the bone marrow and tumor-driven osteolytic lesion formation. In vitro, tumor-secreted LOX supported the attachment and survival of CRC cells to and in the bone matrix, and inhibited osteoblast differentiation. LOX overexpression in CRC cells also induced a robust production of IL-6. In turn, both LOX and IL-6 were acting in concert to promote RANKL-dependent osteoclast differentiation, thereby creating an imbalance between bone resorption and bone formation. Collectively, our findings show that LOX supports CRC cell dissemination in the bone marrow and they reveal a novel mechanism through which LOX-driven IL-6 production by CRC cells impairs bone homeostasis