Dill Extract Induces Elastic Fiber Neosynthesis and Functional Improvement in the Ascending Aorta of Aged Mice with Reversal of Age-Dependent Cardiac Hypertrophy and Involvement of Lysyl Oxidase-Like-1.

Elastic fibers (90% elastin, 10% fibrillin-rich microfibrils) are synthesized only in early life and adolescence mainly by the vascular smooth muscle cells through the cross-linking of its soluble precursor, tropoelastin. Elastic fibers endow the large elastic arteries with resilience and elasticity. Normal vascular aging is associated with arterial remodeling and stiffening, especially due to the end of production and degradation of elastic fibers, leading to altered cardiovascular function. Several pharmacological treatments stimulate the production of elastin and elastic fibers. In particular, dill extract (DE) has been demonstrated to stimulate elastin production in vitro in dermal equivalent models and in skin fibroblasts to increase lysyl oxidase-like-1 (LOXL-1) gene expression, an enzyme contributing to tropoelastin crosslinking and elastin formation. Here, we have investigated the effects of a chronic treatment (three months) of aged male mice with DE (5% or 10% v/v, in drinking water) on the structure and function of the ascending aorta. DE treatment, especially at 10%, of aged mice protected pre-existing elastic lamellae, reactivated tropoelastin and LOXL-1 expressions, induced elastic fiber neo-synthesis, and decreased the stiffness of the aging aortic wall, probably explaining the reversal of the age-related cardiac hypertrophy also observed following the treatment. DE could thus be considered as an anti-aging product for the cardiovascular system.

Methylation of LOXL1 Promoter by DNMT3A in Aged Human Skin Fibroblasts.

Lysyl oxidase-like 1 (LOXL1) is an amino-oxidase involved in maturation of elastic fibers. Its downregulation has been associated with elastic fibers repair loss in aging aorta, lung, ligament, and skin. Several evidences of LOXL1 epigenetic silencing by promoter methylation were reported in cancer and cutis laxa syndrome. We hypothesized that this mechanism could be implicated in skin aging process, as far as elastic fibers are also concerned. Anti-DNMT3A chromatin immunoprecipitation was conducted with nuclear extracts from skin fibroblasts isolated from young and elderly individuals, and showed a higher level of DNMT3A protein binding to the LOXL1 promoter in older cells concomitantly to the decrease of LOXL1 mRNA expression and the increase of LOXL1 promoter methylation. Using luciferase reporter assay driven by LOXL1 promoter in HEK293 cells, we demonstrated that LOXL1 transcriptional activity was dramatically reduced when a recombinant DNMT3A was concomitantly overexpressed. LOXL1 promoter transcriptional activity was restored in the presence of a broad-spectrum inhibitor of DNMT activity, 5-aza-2'-deoxycytidine. Finally, to assess whether the interplay between DNMT3A and LOXL1 promoter could be targeted to increase LOXL1 mRNA expression level, an Origanum majorana extract was selected among 43 plant extracts as a new inhibitor of human DNMT3A activity to restore LOXL1 secretion without cytotoxicity in aged skin fibroblasts.

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 60 days 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.

LOXL as a target to increase the elastin content in adult skin: a dill extract induces the LOXL gene expression.

The lysyl oxidases lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) are responsible for elastin cross-linking. It was shown recently that LOXL is essential for the elastic fibres homeostasis and for their maintenance at adult age. We first determined whether or not elastin, LOX and LOXL are less expressed during adulthood. The LOX and LOXL mRNA level, quantified by real-time reverse transcriptase-polymerase chain reaction decreased in adult skin fibroblasts compared with fibroblasts from children. In contrast, the elastin mRNA level remains stable at all ages. The goal of this study was to induce elastogenesis at the adult age. Therefore, both enzymes, and in particular LOXL, of which expression is the most affected by age, could be targeted to induce elastogenesis in adult skin. We screened a library of about 1000 active ingredients to find activators capable to stimulate specifically the LOXL gene expression in adult dermal fibroblasts. The positive effect of selected active ingredients was confirmed on fibroblasts grown on monolayers and on dermal and skin equivalent cultures. One extract, obtained from dill (LYS'LASTINE V, Engelhard, Lyon, France), stimulates the LOXL gene expression in dermal equivalents (+64% increase in the LOXL mRNA level when compared with control). At the same time, the elastin detection is increased in dermal equivalents and under the dermal-epidermal junction of skin equivalents, without increase of the elastin mRNA. In conclusion, LOXL can be considered as a new target to reinduce elastogenesis. Its stimulation by a dill extract is correlated with increased elastin detection, suggesting an increase in elastogenesis efficiency.