A multiparametric, stepwise in vitro approach to identify anti-dark circle and anti-puffiness ingredients
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Presented by: Boris Vogelgesang
Introduction
Dark circles under or around the eyes are a cosmetic concern worldwide, often associated with tiredness or aging. The physiopathology of dark circles and puffiness is multifactorial, and nowadays, no detailed description is available in the literature. Skin under eye is very thin, about 0.5 mm thick (3 times thinner than on the rest of the face), and so extremely sensitive to environmental stress: UV, pollution, sleep deprivation. Owing to its thinness, and subsequent translucency, the highly vascularized and innervated eye contour area easily shows blood and lymphatic circulation disorders. Dark circles and puffiness often co-exist and are aggravated when skin slackens. Environmental stresses trigger a signaling cascade with inflammation upstream, which stimulates dermal microvascular endothelial cells and alters skin microcirculation. The endothelial barrier function is weakened, and the opening of intercellular junctions leads to extravasation of leukocytes followed by their accumulation in skin tissue, responsible for under-eye puffiness formation. At the same time, leakage of red blood cells is the primary cause of hyperpigmentation, characteristic of dark circles. An increase in oxidation is also observed in the subocular area, due to hemoglobin breakdown, the main component of red blood cells, which releases pigmented and toxic degradation products.
The multifactorial nature of dark circles and puffiness represents a real challenge for in vitro efficacy testing of active ingredients because it is necessary to act at several levels of the signaling cascade to improve the clinical appearance of dark circles and puffiness.
Therefore, to select an active ingredient with anti-puffiness and anti-dark circle potential, we have implemented a screening strategy that combined different biological models addressing relevant targets of skin microcirculation and endothelial barrier function. Using this screening model, 22 plant extracts were compared, and results obtained allowed us to identify a particularly interesting plant extract. This ability to improve skin microcirculation was then confirmed on an 3D innovative dermis model including a microcapillary network, while expression of hemoglobin oxygenase 1 (HMOX-1) was measured in cultured fibroblasts obtained from eyelids.
Methods
At first, we used dermal microvascular endothelial cell cultures, challenged with TNF-α, and we used in situ immunostaining coupled with image analysis to analyze VCAM-1 firm adhesion protein synthesis. Then, we used fluorescent labeling coupled with image analysis to measure leukocyte adhesion to dermal microvascular endothelial cell membranes, challenged with TNF-α. In a third step, we measured in vitro membrane permeability of monolayer cultures of dermal microvascular endothelial cells, stimulated with TNF-α, using the Trans-Endothelial Electrical Resistance technique (TEER).
We used a reconstructed 3D dermis model composed of human dermal fibroblasts and human umbilical vein endothelial cells stimulated with TNF-α and combined with in situ immunostaining of laminin and CD31 to confirm the efficacy of the selected plant extract on endothelial cell organization.
To investigate efficacy of the plant extract at degrading hemoglobin, we grew human dermal fibroblasts from eyelids in monolayer cultures and used qRT-PCR technique to assess HMOX-1 transcriptomic expression, which encodes a hemoglobin degrading enzyme. Besides, chelation of ferrous ions was assessed using in tubo assay.
Results
Out of the 22 ingredients tested originally selected, one particular plant extract significantly reduced firm adhesion protein VCAM-1 synthesis in endothelial cell cultures. It also significantly decreased leukocyte adhesion to endothelial cell membranes, while significantly increasing trans-endothelial electrical resistance in monolayer cultures.
The improvement of endothelial barrier function was illustrated in a vascularized 3D dermis model stimulated with TNF-α, in which the plant extract was able to restore basal membrane of capillary-like tubular structures as observed by the stimulation of laminin synthesis.
In addition to skin microcirculation, the extract favored hemoglobin degradation through stimulation of HMOX-1 gene expression in dermal fibroblast cultures. It also increased chelation of ferrous ions, which are hemoglobin by-products that increase skin oxidation.
Conclusions
The stepwise selection model we used allowed us to identify a unique plant extract with promising anti-puffiness and anti-dark circle potential, based on combined proteomic, genomic and biochemical methods in acellular assays and 2D and 3D cell models. Dedicated clinical study should be used in the near future to demonstrate the in vivo benefits of the ingredient.
Dark circles under or around the eyes are a cosmetic concern worldwide, often associated with tiredness or aging. The physiopathology of dark circles and puffiness is multifactorial, and nowadays, no detailed description is available in the literature. Skin under eye is very thin, about 0.5 mm thick (3 times thinner than on the rest of the face), and so extremely sensitive to environmental stress: UV, pollution, sleep deprivation. Owing to its thinness, and subsequent translucency, the highly vascularized and innervated eye contour area easily shows blood and lymphatic circulation disorders. Dark circles and puffiness often co-exist and are aggravated when skin slackens. Environmental stresses trigger a signaling cascade with inflammation upstream, which stimulates dermal microvascular endothelial cells and alters skin microcirculation. The endothelial barrier function is weakened, and the opening of intercellular junctions leads to extravasation of leukocytes followed by their accumulation in skin tissue, responsible for under-eye puffiness formation. At the same time, leakage of red blood cells is the primary cause of hyperpigmentation, characteristic of dark circles. An increase in oxidation is also observed in the subocular area, due to hemoglobin breakdown, the main component of red blood cells, which releases pigmented and toxic degradation products.
The multifactorial nature of dark circles and puffiness represents a real challenge for in vitro efficacy testing of active ingredients because it is necessary to act at several levels of the signaling cascade to improve the clinical appearance of dark circles and puffiness.
Therefore, to select an active ingredient with anti-puffiness and anti-dark circle potential, we have implemented a screening strategy that combined different biological models addressing relevant targets of skin microcirculation and endothelial barrier function. Using this screening model, 22 plant extracts were compared, and results obtained allowed us to identify a particularly interesting plant extract. This ability to improve skin microcirculation was then confirmed on an 3D innovative dermis model including a microcapillary network, while expression of hemoglobin oxygenase 1 (HMOX-1) was measured in cultured fibroblasts obtained from eyelids.
Methods
At first, we used dermal microvascular endothelial cell cultures, challenged with TNF-α, and we used in situ immunostaining coupled with image analysis to analyze VCAM-1 firm adhesion protein synthesis. Then, we used fluorescent labeling coupled with image analysis to measure leukocyte adhesion to dermal microvascular endothelial cell membranes, challenged with TNF-α. In a third step, we measured in vitro membrane permeability of monolayer cultures of dermal microvascular endothelial cells, stimulated with TNF-α, using the Trans-Endothelial Electrical Resistance technique (TEER).
We used a reconstructed 3D dermis model composed of human dermal fibroblasts and human umbilical vein endothelial cells stimulated with TNF-α and combined with in situ immunostaining of laminin and CD31 to confirm the efficacy of the selected plant extract on endothelial cell organization.
To investigate efficacy of the plant extract at degrading hemoglobin, we grew human dermal fibroblasts from eyelids in monolayer cultures and used qRT-PCR technique to assess HMOX-1 transcriptomic expression, which encodes a hemoglobin degrading enzyme. Besides, chelation of ferrous ions was assessed using in tubo assay.
Results
Out of the 22 ingredients tested originally selected, one particular plant extract significantly reduced firm adhesion protein VCAM-1 synthesis in endothelial cell cultures. It also significantly decreased leukocyte adhesion to endothelial cell membranes, while significantly increasing trans-endothelial electrical resistance in monolayer cultures.
The improvement of endothelial barrier function was illustrated in a vascularized 3D dermis model stimulated with TNF-α, in which the plant extract was able to restore basal membrane of capillary-like tubular structures as observed by the stimulation of laminin synthesis.
In addition to skin microcirculation, the extract favored hemoglobin degradation through stimulation of HMOX-1 gene expression in dermal fibroblast cultures. It also increased chelation of ferrous ions, which are hemoglobin by-products that increase skin oxidation.
Conclusions
The stepwise selection model we used allowed us to identify a unique plant extract with promising anti-puffiness and anti-dark circle potential, based on combined proteomic, genomic and biochemical methods in acellular assays and 2D and 3D cell models. Dedicated clinical study should be used in the near future to demonstrate the in vivo benefits of the ingredient.