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Key regulatory role of dermal fibroblasts in pigmentation as demonstrated using a reconstructed skin model: impact of photo-aging.

Duval C, Cohen C, Chagnoleau C, Flouret V, Bourreau E, Bernerd F - PLoS ONE (2014)

Bottom Line: Pigmentation of skin reconstructed using young unexposed fibroblasts (n = 3) was compared to that of tissues containing natural photo-aged fibroblasts (n = 3) which express a senescent phenotype.A stimulation of pigmentation in the presence of the natural photo-aged fibroblasts was revealed by a significant increase in the skin color (decrease in Luminance) and an increase in both epidermal melanin content and melanogenic gene expression, thus confirming our hypothesis.Altogether, these data demonstrate that the level of pigmentation of the skin model is influenced by dermal fibroblasts and that natural photo-aged fibroblasts can contribute to the hyperpigmentation that is associated with photo-aging.

View Article: PubMed Central - PubMed

Affiliation: L'Oréal Research & Innovation, Aulnay-sous-Bois, France.

ABSTRACT
To study cutaneous pigmentation in a physiological context, we have previously developed a functional pigmented reconstructed skin model composed of a melanocyte-containing epidermis grown on a dermal equivalent comprising living fibroblasts. The present studies, using the same model, aimed to demonstrate that dermal fibroblasts influence skin pigmentation up to the macroscopic level. The proof of principle was performed with pigmented skins differing only in the fibroblast component. First, the in vitro system was reconstructed with or without fibroblasts in order to test the global influence of the presence of this cell type. We then assessed the impact of the origin of the fibroblast strain on the degree of pigmentation using fetal versus adult fibroblasts. In both experiments, impressive variation in skin pigmentation at the macroscopic level was observed and confirmed by quantitative parameters related to skin color, melanin content and melanocyte numbers. These data confirmed the responsiveness of the model and demonstrated that dermal fibroblasts do indeed impact the degree of skin pigmentation. We then hypothesized that a physiological state associated with pigmentary alterations such as photo-aging could be linked to dermal fibroblasts modifications that accumulate over time. Pigmentation of skin reconstructed using young unexposed fibroblasts (n = 3) was compared to that of tissues containing natural photo-aged fibroblasts (n = 3) which express a senescent phenotype. A stimulation of pigmentation in the presence of the natural photo-aged fibroblasts was revealed by a significant increase in the skin color (decrease in Luminance) and an increase in both epidermal melanin content and melanogenic gene expression, thus confirming our hypothesis. Altogether, these data demonstrate that the level of pigmentation of the skin model is influenced by dermal fibroblasts and that natural photo-aged fibroblasts can contribute to the hyperpigmentation that is associated with photo-aging.

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Effect of fibroblasts from photo-aged versus young skin on the expression level of melanogenic proteins in reconstructed skin.Analysis of melanocyte location and activity in the reconstructed skin samples containing either photo-aged fibroblasts (PAF) or young fibroblasts (YF) was analyzed by immunostaining on histological sections using TRP-1 and Tyrosinase antibodies (A) and by measuring expression levels of melanogenic genes by RT-qPCR (B). Immunostaining of tissue sections showed the correct location of melanocytes in the basal layer and increased labeling of Tyrosinase in PAF conditions as compared to YF samples. The analysis of expression levels of genes involved in melanogenesis in the epidermis revealed the increase in mRNA levels of Silver, Tyrosinase, TRP-1 and DCT (TRP-2) in the PAF-containing model as compared to the YF condition confirming that melanocytes were stimulated when photo-aged fibroblasts were present in dermal equivalent. Values are expressed as a mean +/- SD calculated for 3 samples in one experiment and analyzed using the two-tailed unpaired Student's t-test; ** p<0.01, *** p<0.001. Similar results were obtained in three independent experiments. Magnification: A =  x400.
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pone-0114182-g006: Effect of fibroblasts from photo-aged versus young skin on the expression level of melanogenic proteins in reconstructed skin.Analysis of melanocyte location and activity in the reconstructed skin samples containing either photo-aged fibroblasts (PAF) or young fibroblasts (YF) was analyzed by immunostaining on histological sections using TRP-1 and Tyrosinase antibodies (A) and by measuring expression levels of melanogenic genes by RT-qPCR (B). Immunostaining of tissue sections showed the correct location of melanocytes in the basal layer and increased labeling of Tyrosinase in PAF conditions as compared to YF samples. The analysis of expression levels of genes involved in melanogenesis in the epidermis revealed the increase in mRNA levels of Silver, Tyrosinase, TRP-1 and DCT (TRP-2) in the PAF-containing model as compared to the YF condition confirming that melanocytes were stimulated when photo-aged fibroblasts were present in dermal equivalent. Values are expressed as a mean +/- SD calculated for 3 samples in one experiment and analyzed using the two-tailed unpaired Student's t-test; ** p<0.01, *** p<0.001. Similar results were obtained in three independent experiments. Magnification: A =  x400.

Mentions: Interestingly, a higher level of pigmentation was observed in the tissues with natural photo-aged versus young fibroblasts. In the presence of the three PAF strains versus the YF conditions, a significant increase in pigmentation of the skin model was induced as demonstrated by i) a higher amount of melanin granules as seen on histological sections and measured by image analysis, and ii) the darkening of the skin samples as quantified by a decrease in luminance (Fig. 5). Additionally, TRP1 and TYR immunostainings of skin sections revealed that melanocytes were correctly located at the basal layer and activated as shown by intense enzyme staining (Fig. 6A). These results were strengthened by the analysis of expression of melanogenesis genes in the epidermis revealing an increase in mRNA levels of Silver, Tyrosinase, TRP-1 and DCT (TRP-2) in the presence of PAF versus YF (Fig. 6B).


Key regulatory role of dermal fibroblasts in pigmentation as demonstrated using a reconstructed skin model: impact of photo-aging.

Duval C, Cohen C, Chagnoleau C, Flouret V, Bourreau E, Bernerd F - PLoS ONE (2014)

Effect of fibroblasts from photo-aged versus young skin on the expression level of melanogenic proteins in reconstructed skin.Analysis of melanocyte location and activity in the reconstructed skin samples containing either photo-aged fibroblasts (PAF) or young fibroblasts (YF) was analyzed by immunostaining on histological sections using TRP-1 and Tyrosinase antibodies (A) and by measuring expression levels of melanogenic genes by RT-qPCR (B). Immunostaining of tissue sections showed the correct location of melanocytes in the basal layer and increased labeling of Tyrosinase in PAF conditions as compared to YF samples. The analysis of expression levels of genes involved in melanogenesis in the epidermis revealed the increase in mRNA levels of Silver, Tyrosinase, TRP-1 and DCT (TRP-2) in the PAF-containing model as compared to the YF condition confirming that melanocytes were stimulated when photo-aged fibroblasts were present in dermal equivalent. Values are expressed as a mean +/- SD calculated for 3 samples in one experiment and analyzed using the two-tailed unpaired Student's t-test; ** p<0.01, *** p<0.001. Similar results were obtained in three independent experiments. Magnification: A =  x400.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4260844&req=5

pone-0114182-g006: Effect of fibroblasts from photo-aged versus young skin on the expression level of melanogenic proteins in reconstructed skin.Analysis of melanocyte location and activity in the reconstructed skin samples containing either photo-aged fibroblasts (PAF) or young fibroblasts (YF) was analyzed by immunostaining on histological sections using TRP-1 and Tyrosinase antibodies (A) and by measuring expression levels of melanogenic genes by RT-qPCR (B). Immunostaining of tissue sections showed the correct location of melanocytes in the basal layer and increased labeling of Tyrosinase in PAF conditions as compared to YF samples. The analysis of expression levels of genes involved in melanogenesis in the epidermis revealed the increase in mRNA levels of Silver, Tyrosinase, TRP-1 and DCT (TRP-2) in the PAF-containing model as compared to the YF condition confirming that melanocytes were stimulated when photo-aged fibroblasts were present in dermal equivalent. Values are expressed as a mean +/- SD calculated for 3 samples in one experiment and analyzed using the two-tailed unpaired Student's t-test; ** p<0.01, *** p<0.001. Similar results were obtained in three independent experiments. Magnification: A =  x400.
Mentions: Interestingly, a higher level of pigmentation was observed in the tissues with natural photo-aged versus young fibroblasts. In the presence of the three PAF strains versus the YF conditions, a significant increase in pigmentation of the skin model was induced as demonstrated by i) a higher amount of melanin granules as seen on histological sections and measured by image analysis, and ii) the darkening of the skin samples as quantified by a decrease in luminance (Fig. 5). Additionally, TRP1 and TYR immunostainings of skin sections revealed that melanocytes were correctly located at the basal layer and activated as shown by intense enzyme staining (Fig. 6A). These results were strengthened by the analysis of expression of melanogenesis genes in the epidermis revealing an increase in mRNA levels of Silver, Tyrosinase, TRP-1 and DCT (TRP-2) in the presence of PAF versus YF (Fig. 6B).

Bottom Line: Pigmentation of skin reconstructed using young unexposed fibroblasts (n = 3) was compared to that of tissues containing natural photo-aged fibroblasts (n = 3) which express a senescent phenotype.A stimulation of pigmentation in the presence of the natural photo-aged fibroblasts was revealed by a significant increase in the skin color (decrease in Luminance) and an increase in both epidermal melanin content and melanogenic gene expression, thus confirming our hypothesis.Altogether, these data demonstrate that the level of pigmentation of the skin model is influenced by dermal fibroblasts and that natural photo-aged fibroblasts can contribute to the hyperpigmentation that is associated with photo-aging.

View Article: PubMed Central - PubMed

Affiliation: L'Oréal Research & Innovation, Aulnay-sous-Bois, France.

ABSTRACT
To study cutaneous pigmentation in a physiological context, we have previously developed a functional pigmented reconstructed skin model composed of a melanocyte-containing epidermis grown on a dermal equivalent comprising living fibroblasts. The present studies, using the same model, aimed to demonstrate that dermal fibroblasts influence skin pigmentation up to the macroscopic level. The proof of principle was performed with pigmented skins differing only in the fibroblast component. First, the in vitro system was reconstructed with or without fibroblasts in order to test the global influence of the presence of this cell type. We then assessed the impact of the origin of the fibroblast strain on the degree of pigmentation using fetal versus adult fibroblasts. In both experiments, impressive variation in skin pigmentation at the macroscopic level was observed and confirmed by quantitative parameters related to skin color, melanin content and melanocyte numbers. These data confirmed the responsiveness of the model and demonstrated that dermal fibroblasts do indeed impact the degree of skin pigmentation. We then hypothesized that a physiological state associated with pigmentary alterations such as photo-aging could be linked to dermal fibroblasts modifications that accumulate over time. Pigmentation of skin reconstructed using young unexposed fibroblasts (n = 3) was compared to that of tissues containing natural photo-aged fibroblasts (n = 3) which express a senescent phenotype. A stimulation of pigmentation in the presence of the natural photo-aged fibroblasts was revealed by a significant increase in the skin color (decrease in Luminance) and an increase in both epidermal melanin content and melanogenic gene expression, thus confirming our hypothesis. Altogether, these data demonstrate that the level of pigmentation of the skin model is influenced by dermal fibroblasts and that natural photo-aged fibroblasts can contribute to the hyperpigmentation that is associated with photo-aging.

Show MeSH
Related in: MedlinePlus