Charbel Bouez

The Lysyl Oxidase LOX Is Absent in Basal and Squamous Cell Carcinomas and Its Knockdown Induces an Invading Phenotype in a Skin Equivalent Model

Lysyl oxidase initiates the enzymatic stage of collagen and elastin cross-linking. Among five isoforms comprising the lysyl oxidase family, LOX is the better studied. LOX is associated to an antitumor activity in ras-transformed fibroblasts, and its expression is down-regulated in many carcinomas. The aim of this work was to shed light on LOX functions within the epidermis by studying its expression in human basal and squamous cell carcinomas and analyzing the effect of its enzymatic activity inhibition and protein absence on human keratinocytes behavior in a skin equivalent. In both carcinomas, LOX expression by epidermal tumor cells was lacking, while it was up-regulated around invading tumor cells in association with the stromal reaction. Lysyl oxidase activity inhibition using β-aminoproprionitrile in a skin equivalent model prepared with both primary human keratinocytes and HaCaT cell line affected keratin 10 and filaggrin expression and disorganized the collagen network and the basement membrane. In spite of all these changes, no invasion phenotype was observed. Modelization of the invasive phenotype was only noticed in the skin equivalent developed with LOX antisense HaCaT cell line, where the protein LOX is specifically absent. Our results clearly indicate that lysyl oxidase enzymatic activity is essential not only for the integrity maintenance of the dermis but also for the homeostasis of the epidermis. Moreover, LOX protein plays a role in the skin carcinomas and invasion but not through its enzymatic activity.

Supplementation with a complex of active nutrients improved dermal and epidermal characteristics in skin equivalents generated from fibroblasts from young or aged donors

Cultured skin equivalent (SE, Mimeskin®) was generated by co-culturing skin fibroblasts and keratinocytes on a collagen-glycosaminoglycan-chitosan dermal substrate. In order to examine donor age effect, fibroblasts from 19- (young) or 49- (aged) year-old females were used. Culture medium was supplemented with nutrients complex containing soy extract, tomato extract, grape seed extract, white tea extract, sodium ascorbate, tocopherol acetate, zinc gluconate and BioMarine™ complex. Epidermal and dermal structure and composition were examined after 42 and 60xa0days of culture. In untreated samples, SE generated from young fibroblasts was superior to SE from aged fibroblasts in all characteristics. Those include number and regularity of keratinocyte layers, number of keratinocytes expressing proliferation marker Ki67, content of collagen type I, fibrillin-1, elastin, and SE lifespan. Effects of nutritional supplementation were observed in SE from both young and aged fibroblasts, however, those effects were more pronounced in SE from aged fibroblasts. In epidermis, the treatment increased number of keratinocyte layers and delayed epidermal senescence. The number of cells expressing Ki67 was nine folds higher than those of controls, and was similar to that of young cell SE. In dermis, the treatment increased mRNA synthesis of collagen I, fibrillin-1 and elastin. In conclusion, skin cell donor age had major important effect on formation of reconstructed SE. Imperfections in epidermal and dermal structure and composition as well as life span in SE from aged cells can be improved by supplementation with active nutrients.

Epidermal and Dermal Characteristics in Skin Equivalent after Systemic and Topical Application of Skin Care Ingredients

Abstract:u2002 Effects of active ingredients from topical and systemic skincare products on structure and organization of epidermis, dermal–epidermal junction (DEJ), and dermis were examined using an in vitro reconstructed skin equivalent (SE). Imedeen Time Perfection (ITP) ingredients (a mixture of BioMarine Complex, grape seed extract, tomato extract, vitamin C) were supplemented systemically into culture medium. Kinetin, an active ingredient from Imedeen Expression Line Control Serum, was applied topically. Both treatments were tested separately or combined. In epidermis, all treatments stimulated keratinocyte proliferation, showing a significant increase of Ki67‐positive keratinocytes (P < 0.05). Kinetin showed a twofold increase of Ki67‐positive cells, ITP resulted in a fivefold, and ITP+kinetin showed a nine‐fold increase. Differentiation of keratinocytes was influenced only by kinetin since filaggrin was found only in kinetin and kinetin+ITP samples. At the DEJ, laminin 5 was slightly increased by all treatments. In dermis, only ITP increased the amount of collagen type I. Both kinetin and ITP stimulated formation of fibrillin‐1 and elastin deposition. The effect of kinetin was seen in upper dermis. It stimulated not only the amount of deposited fibrillin‐1 and elastin fibers but also their organization perpendicularly to the DEJ. ITP stimulated formation of fibrillin‐1 in deeper dermis. In summary, the combination of topical treatment with kinetin and systemic treatment with ITP had complementary beneficial effects in the formation and development of epidermis and dermis.