Transforming growth factor-beta1 and ultraviolet A1 radiation increase production of vascular endothelial growth factor but not endothelin-1 in human dermal fibroblasts.

BACKGROUND: Normal and dysregulated wound healing involves fibroblast activation and angiogenesis, in which polypeptide factors such as transforming growth factor (TGF)-beta, vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) play an important part. Ultraviolet (UV) A1 (365 nm) has recently received attention as a possible treatment for some dermal fibrotic disorders. OBJECTIVES: The aim of this study was to evaluate the effects of TGF-beta1 and UVA1 radiation, as well as that of cobalt chloride, reported to mimic hypoxia both in vivo and in vitro, on the expression of VEGF and ET-1 by cultured human dermal fibroblasts. METHODS: Levels of VEGF and ET-1 were measured by enzyme-linked immunosorbent assay and expression of neutral endopeptidase (NEP, CD10), known to degrade ET-1, was quantified by flow cytometric analysis after cell trypsinization. RESULTS: Our results showed that the cells released minor amounts of VEGF and ET-1. Both TGF-beta1 and UVA1 strongly increased VEGF secretion in a dose- and time-dependent manner, without significantly affecting ET-1 release. Irradiation of TGF-beta1-stimulated fibroblasts resulted in a synergistic effect on increasing levels of VEGF but not ET-1 after 48 h. Cobalt chloride stimulated the secretion of VEGF by fibroblasts; the effects of TGF-beta1 and cobalt were additive. However, no significant effect of cobalt chloride on ET-1 secretion was observed, suggesting that ET-1 production in fibroblasts is not oxygen-sensitive. The expression of NEP was not modified by TGF-beta1 or UVA1 radiation. Addition of a neutralizing anti-CD10 antibody to fibroblast cultures downregulated CD10 expression at the cell surface without changing ET-1 levels in cell supernatants after 24 or 48 h. This suggests that membrane-bound NEP has minimal or no activity against secreted ET-1. CONCLUSIONS: Taken together, these results underline the major role played by TGF-beta1 in increasing VEGF secretion by fibroblasts. This, as well as the documented effect of UVA1 on increasing VEGF production, may have implications for wound healing in vivo.

Neprilysin, a novel target for ultraviolet B regulation of melanogenesis via melanocortins.

Compelling evidence suggest a role for melanocortins in the regulation of melanogenesis by ultraviolet radiation. Within the epidermis, melanocytes and keratinocytes produce alpha-melanocyte-stimulating hormone and adrenocorticotropic hormone. The persistence and the strength of the biologic signal delivered by these peptides depend on their local concentration, which is controlled by the rate of peptide production and by the rate of its degradation. In this study, we investigated the mechanism of melanocortin degradation by melanocytes and the effect of ultraviolet on this process. We have focused our attention on a neutral endopeptidase, neprilysin, which has been implicated in the ending of numerous peptidergic signals. We have shown that this enzyme is expressed at the surface of human melanocytes. Interestingly, its activity and its expression are dramatically downregulated by ultraviolet B treatment. Moreover, in the presence of phosphoramidon, a stable inhibitor of neprilysin, we observed an increased efficiency of alpha-melanocyte-stimulating hormone and adrenocorticotropic hormone to stimulate both tyrosinase activity and microphthalmia expression. Taken together, these data indicate that neprilysin expressed by melanocytes has a physiologic role in the regulation of melanogenesis by proopiomelanocortin peptide. Further, its downregulation by ultraviolet B irradiation shed light on a new and appealing mechanism of ultraviolet B induced melanogenesis via the control of melanocortins degradation.