Chronological aging impacts abundance, function and microRNA content of extracellular vesicles produced by human epidermal keratinocytes.

The disturbance of intercellular communication is one of the hallmarks of aging. The goal of this study is to clarify the impact of chronological aging on extracellular vesicles (EVs), a key mode of communication in mammalian tissues. We focused on epidermal keratinocytes, the main cells of the outer protective layer of the skin which is strongly impaired in the skin of elderly. EVs were purified from conditioned medium of primary keratinocytes isolated from infant or aged adult skin. A significant increase of the relative number of EVs released from aged keratinocytes was observed whereas their size distribution was not modified. By small RNA sequencing, we described a specific microRNA (miRNA) signature of aged EVs with an increase abundance of miR-30a, a key regulator of barrier function in human epidermis. EVs from aged keratinocytes were found to be able to reduce the proliferation of young keratinocytes, to impact their organogenesis properties in a reconstructed epidermis model and to slow down the early steps of skin wound healing in mice, three features observed in aged epidermis. This work reveals that intercellular communication mediated by EVs is modulated during aging process in keratinocytes and might be involved in the functional defects observed in aged skin.

MiR-30a-5p Alters Epidermal Terminal Differentiation during Aging by Regulating BNIP3L/NIX-Dependent Mitophagy.

Chronological aging is characterized by an alteration in the genes' regulatory network. In human skin, epidermal keratinocytes fail to differentiate properly with aging, leading to the weakening of the epidermal function. MiR-30a is particularly overexpressed with epidermal aging, but the downstream molecular mechanisms are still uncovered. The aim of this study was to decipher the effects of miR-30a overexpression in the human epidermis, with a focus on keratinocyte differentiation. We formally identified the mitophagy receptor BNIP3L as a direct target of miR-30a. Using a 3D organotypic model of reconstructed human epidermis overexpressing miR-30a, we observed a strong reduction in BNIP3L expression in the granular layer. In human epidermal sections of skin biopsies from donors of different ages, we observed a similar pattern of BNIP3L decreasing with aging. Moreover, human primary keratinocytes undergoing differentiation in vitro also showed a decreased expression of BNIP3L with age, together with a retention of mitochondria. Moreover, aging is associated with altered mitochondrial metabolism in primary keratinocytes, including decreased ATP-linked respiration. Thus, miR-30a is a negative regulator of programmed mitophagy during keratinocytes terminal differentiation, impairing epidermal homeostasis with aging.

Maintenance of Chronological Aging Features in Culture of Normal Human Dermal Fibroblasts from Old Donors.

Chronological aging is defined as a time-dependent decline of tissue homeostasis which severely impacts skin. Understanding the mechanisms of skin aging is an active research area limited by the lack of relevant in vitro models. Being a component of aging, replicative or stress-induced senescence is repeatedly used to mimic skin aging in vitro, thus presenting only a partial view of the complexity of aging. Herein, we aimed to clarify whether primary normal human dermal fibroblasts retained age-related characteristics when cultured in 2D monolayer, and could be used as a relevant model for aging research. We compared three groups of fibroblasts isolated from different aged donors. We observed strongly decreased population doubling capacities, a reduced clonogenic ability, an impairment in extracellular matrix production together with modifications of respiratory metabolism with an increase in age. These disruptions were particularly marked when comparing fibroblasts isolated from old individuals (over 70 years old) to those isolated from young individuals (18-37 years old), while cells from middle-aged donors exhibited an intermediate profile. These alterations of cell features can be related to the signs of dermis aging, thus showing that cultured primary cells indeed retain some characteristics of the original tissue from which they were extracted.

[Functional integrity of aging skin, from cutaneous biology to anti-aging strategies]. / Vieillissement et intégrité de la peau - De la biologie cutanée aux stratégies anti-âge.

The skin is a sentinel organ making easily visible the passing of time. Chronological and environmental aging weakens skin structure and functions. The skin barrier, the elastic and mechanical properties of the cutaneous tissue as well as its vascular reactivity are impacted by aging. The barrier dysfunction in aged skin is caused by defects in epidermal keratinocytes renewal and differentiation notably linked to abnormal expression of microRNAs regulating cell death and autophagy. An abnormal balance between synthesis and degradation of matrix proteins modifies the mechanical properties of the dermis in aged skin. Finally, a reduction of the vascular reactivity linked to endothelial dysfunctions is observed in elderly people. These biological processes can be targeted by therapeutic approaches either topical or systemic, especially using anti-oxydants or senolytics. These anti-aging strategies might contribute to restore, at least in part, the functional integrity of aged skin.

TITLE: Vieillissement et intégrité de la peau - De la biologie cutanée aux stratégies anti-âge. ABSTRACT: La peau est un organe sentinelle, soumis au vieillissement chronologique et environnemental qui fragilise sa structure et ses fonctions. La fonction barrière de la peau, ses propriétés élastiques et de résistance, ainsi que sa réactivité vasculaire sont atteintes par le vieillissement dans les compartiments épidermiques, dermiques et vasculaires. Les progrès de la recherche ont permis de révéler des processus biologiques sous-jacents, qui peuvent être ciblés par des approches médicamenteuses topiques ou globales à base notamment d'anti-oxydants ou de sénolytiques. Ces stratégies anti-âge pourront contribuer à restaurer, au moins en partie, l'intégrité fonctionnelle de la peau âgée.

NFATC2 Modulates Radiation Sensitivity in Dermal Fibroblasts From Patients With Severe Side Effects of Radiotherapy.

Although it is well established that 5 to 15% of radiotherapy patients exhibit severe side-effects in non-cancerous tissues, the molecular mechanisms involved are still poorly known, and the links between cellular and tissue radiosensitivity are still debated. We here studied fibroblasts from non-irradiated skin of patients with severe sequelae of radiotherapy, to determine whether specific basal cell activities might be involved in susceptibility to side-effects in normal tissues. Compared to control cells, patient fibroblasts exhibited higher radiosensitivity together with defects in DNA repair. Transcriptome profiling of dermal fibroblasts from 16 radiotherapy patients with severe side-effects and 8 healthy individuals identified 540 genes specifically deregulated in the patients. Nuclear factor of activated T cells 2 (NFATC2) was the most differentially expressed gene, poorly expressed at both transcript and protein level, whereas the NFATC2 gene region was hypermethylated. Furthermore, NFATC2 expression correlated with cell survival after irradiation. Finally, silencing NFATC2 in normal cells by RNA interference led to increased cellular radiosensitivity and defects in DNA repair. This study demonstrates that patients with clinical hypersensitivity also exhibit intrinsic cellular radiosensitivity in their normal skin cells. It further reveals a new role for NFATC2 as a potential regulator of cellular sensitivity to ionizing radiation.