Abstract: Exosomes are mediators of intercellular communication. In the present work, test models were developed to explore these activities in vitro and ex vivo to develop natural actives that regulate endogenous cell communication for hair and skin benefits.
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Abstract: Exosomes are mediators of intercellular communication. In the present work, test models were developed to explore these activities in vitro and ex vivo to develop natural actives that regulate endogenous cell communication for hair and skin benefits.
Intercellular communication corresponds to all signals, physical or chemical, that enable cells to mutually interact and effectively adapt themselves to changes in their environment. In a tissue as heterogeneous and complex as the skin, the dialogue between its different cell types is indispensable for guaranteeing renewal and maintaining biological functions.1-3
Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication, with multiple roles in physiological processes.4-6 This heterogeneous group of cell-derived membranous structures includes exosomes, microvesicles and apoptotic bodies (see Figure 1).
EVs transfer proteins, lipids and nucleic acids (mRNA, miRNA, etc.) from a donor cell to a recipient cell at both near and far distances. Indeed, EVs are released into the environment surrounding the cell and can cross biological barriers (vascular system, dermal-epidermal junction, etc.) to enter the integumentary and circulatory systems.7
At the skin level, EVs — and among them, exosomes — have garnered significant attention due to their ability to influence cellular processes including hair loss, and skin pigmentation and aging.8 Nevertheless, it is critical to study exosomal communication in a well-defined context before attempting to modulate it, since exosomes can carry messengers that activate or inhibit biological pathways depending on the transmitting and receiving cells as well as the environment.
In this context, three models were developed to assess the role of exosomes in physiological contexts ex vivo and in vitro. These models were then used to substantiate the capacity of natural yeast and red algae extracts to modulate this endogenous communication system for cosmetic benefits dedicated to hair and skin care. The extracts were chosen based on their promising biological activities as determined by initial investigations.
Fibroblast to Hair Follicle Communication
To explain the effects of the actives in test models, some background on the biology is helpful. Hair follicles (HFs) are dynamic mini organs composed of two principal components. The dermal papilla is the engine of its growth and is located at the base of the follicle. It contains dermal papilla cells (HFDPC) that emit growth signals received by hair matrix cells.
The papilla is surrounded by a structure containing keratinocytes that respond to signals by proliferating to trigger the formation of a new hair shaft.9, 10 HFs may also receive signals from the dermis via exosomal communication, although the impact of EVs in the hair follicle microenvironment or macroenvironment had not previously been well-studied.
In 2019, the authors’ company participated in a study that found exosomes from dermal fibroblasts that are stimulated with growth factors are efficient activators of dermal papilla cells, promoting hair follicle length.11 Thus, the efficacy of a natural yeast extract was evaluated on an ex vivo model mimicking androgenetic alopecia with exosomal communication between dermal fibroblasts and HFDPC.
Materials and Methods: Hair Growth
Fibroblasts: Human primary fibroblasts were seeded in culture medium and incubated at 37°C in an atmosphere containing 5% CO2. After five days of culture, fibroblasts were treated for 48 hr with or without a yeast extract at 0.04% (v/v). On day seven, exosomes secreted by these fibroblasts were isolated from culture supernatants by ultracentrifugation and stored at –80°C before being assayed with a nanoparticle characterization instrumenta. Purification was validated by Western blot analysis of exosome-specific markers.
Hair follicles: Hair follicles were isolated from scalp samples and placed in a culture medium at 37°C in an incubator containing 5% CO2. After one day of culture, follicles were photographed and measured with a microscope coupled to an image analysis systemb.
Follicles in anagen phase were selected and then treated with a solution of dihydrotestosterone (DHT) to mimic androgenetic alopecia, with or without control exosomes at 5 x108 particles/mL, and exosomes from fibroblasts stimulated with the natural active ingredient at 5 x108 particles/mL. On day five, follicles were photographed and measured with the same equipment. Any elongation of the follicles was equal to growth between D1 and D5.
Results: Results demonstrated that control exosomes had no impact on hair follicle growth in the ex vivo model mimicking alopecia. Exosomes isolated from fibroblasts stimulated with the yeast extract, however, showed a significant 29% increase in hair growth, compared with the control model (see Figure 2).
Hence, the natural active demonstrated the ability to stimulate the production of biologically active exosomes by fibroblasts, favoring hair growth. The extract was also found to act directly on the dermal papilla to limit hair loss and increase hair density in Caucasian men with slight to moderate alopecia (data not shown).
Keratinocyte to Melanocyte Communication
Next, the potential to modulate communication between keratinocytes and melanocytes was examined using a red algae extract (INCI: Palmaria palmata extract). For context, pigmentation occurs in the epidermal unit. Melanin pigments are produced by melanocytes – specialized dendritic cells – and then distributed to neighboring keratinocytes. This tandem arrangement thereby ensures skin color at birth but is also responsible for pigmentation induced as an adaptive response to stimuli including prolonged exposures to UV.
Keratinocytes can also stimulate the synthesis of melanin in response to UVB exposure through the secretion and transfer of exosomes to melanocytes. A previous study has shown that UVB exposure increases the miRNA 3196 content of keratinocyte exosomes.12
The transmission of this messenger to melanocytes leads to an increase in tyrosinase activity, pigmentation gene expression, and thereby melanin content. This results in hyperpigmentation of the skin.12 An in vitro model reproducing this exosomal communication thus was developed to explore the efficacy of a natural red algae active (INCI: Palmaria palmata extract) to inhibit this transmission.
Materials and Methods: Skin Pigmentation
Keratinocyte protocol: Keratinocytes were seeded in culture medium and incubated at 37°C in an atmosphere containing 5% CO2. After one day of culture, keratinocytes were subjected to UVB irradiation (30 mJ/cm²). The cells were then treated in culture medium with or without Palmaria palmata extract at 1.0% (v/v) and placed in a 37°C incubator in a moist atmosphere containing 5% CO2 for three days.
Exosomes secreted by the irradiated keratinocytes were isolated using a series of centrifugations and ultracentrifugations. A rigorous validation procedure ensured the purity and integrity of these exosomes.
Melanocyte treatments: Melanocytes were then seeded in culture medium and incubated at 37°C in an atmosphere containing 5% CO2. From days three to five, the melanocytes were treated daily with the exosomes from keratinocytes treated (or not) with the natural active ingredient. The cells were recovered and total RNA was extracted. The expressions of MITF and TYRP1, involved in melanin synthesis, and of Myo5a, involved in melanosome transport, were analyzed by quantitative PCR.
Results: Results revealed that exosomes isolated from UVB-irradiated keratinocytes and applied to melanocytes induced a significant increase in MITF and TYRP1 genes. Moreover, the expression of Myo5a was also significantly increased (Figure 3a). To limit skin pigmentation and recover an even and radiant complexion, the expression of these genes must be limited. Exosomes derived from keratinocytes treated with the natural active ingredient (INCI: Palmaria palmata extract) at 1% showed a significant decrease in the expression of MITF, TYRP1 and Myo5a (see Figure 3b).
Hence, the natural active ingredient regulated intercellular communication mediated by exosomes, inhibiting the expression of genes involved in melanogenesis. Complementing this efficacy pathway, the Palmaria palmata extract also targeted steps in the melanogenesis process to combat the signs of cutaneous photoaging (data not shown). Studies also have indicated the active attenuated pigmentation spots in Caucasian and Asian skin (data not shown).
Fibroblast to Keratinocyte Communication
Finally, EV communication between the dermis and epidermis was explored. The dermis, its aging and the visible signs of age in the skin are closely related. The epidermis and its properties, on the other hand, are not as directly associated with the aging phenomenon. However, the compartments of the dermis and the epidermis cannot be reduced to two separate and autonomous entities. Indeed, in a tissue as heterogeneous and complex as the skin, the dialogue between the different cell types is indispensable for guaranteeing development and maintaining biological functions.1, 2, 13
Among the biological messengers involved in this communication are miRNAs – small RNAs that can control gene expression. miRNAs bind to complementary target mRNAs and are powerful communication agents. Produced at the cellular level, they can repress the synthesis of proteins in a target cell after being transported by extracellular vesicles.
In this context, an in vitro model was established to reproduce EV-mediated communication from the dermis to the epidermis. Nine miRNAs transported by fibroblast-secreted EVs were targeted for their ability to inhibit epidermal cell functions such as proliferation, differentiation and cohesion.14-16 The efficacy of a natural yeast active (INCI: Pichia ferment lysate filtrate) was then tested by this in vitro model.
Materials and Methods: Dermal-epidermal Communication
Fibroblast protocol: Young (< P6) human fibroblasts or those artificially aged by successive replications (> P20) were seeded and incubated at 37°C in an atmosphere containing 5% CO2. After seven days of culture, fibroblasts were treated or not with the Pichia ferment lysate filtrate at 0.4% (v/v) for 48 hr and incubated at 37°C in an atmosphere containing 5% CO2.
EVs secreted by fibroblasts were then isolated from secretomesc, characterizedd and stored at –80°C. The miRNAs in EVs were extracted, reverse-transcribed and the complementary DNAs obtained were analyzed by quantitative PCR.
Keratinocyte treatments: To determine the impact of the age-induced communication shortfall of the dermal compartment on epidermal functions, keratinocytes from old donors (> 60 years old) were submitted to the secretome of aged fibroblasts (SAF). The Ki-67 proliferation marker was then examined by immunocytofluorescence. Markers of epidermal differentiation (filaggrin), cell cohesion (desmoglein-1), hydration (aquaporin-3) and epidermis to dermis anchoring (laminin-332) were investigated by quantitative PCR.
Results: Results demonstrated the nine miRNAs were expressed more significantly in exosomes secreted by aged fibroblasts (see Figure 4a), suggesting a communication shortfall that could induce an alteration in epidermal biological functions. To corroborate this hypothesis, as stated, old keratinocytes were exposed to the SAF. This significantly decreased keratinocyte proliferation and differentiation, cell cohesion and the anchoring of the dermal-epidermal junction, thus accentuating the impact of aging on the biological functions of the epidermis (data not shown).
The capacity of the natural active to regulate exosomal communication between the dermis and the epidermis was also investigated. Tested at 0.4% on aged fibroblasts, the Pichia ferment lysate filtrate significantly limited the expression of miRNAs involved in epidermal aging (see Figure 4a). Moreover, keratinocytes exposed to SAF previously treated with the natural active ingredient showed a significant improvement in epidermal functions (see Figure 4b).
This natural active was therefore able to regulate the fibroblast to keratinocyte exosomal communication. Hence, by reactivating miR-30a-3p, and thereby communication from the dermis to the epidermis, the Pichia ferment lysate filtrate could increase epidermal renewal and thickness for smoother microrelief, boosted hydration and restored radiance.
Discussion and Conclusions
Exosomes have emerged as innovative tools in cosmetics, driven by their ability to deliver biological messengers and cross biological barriers. However, the use of exosomes directly in formulations requires ongoing research to prove their stability and efficacy.
The development of natural active ingredients to act directly on endogenous skin exosomal communication is thus an innovative solution. The use of in vitro or ex vivo biological models is nevertheless a prerequisite to understanding exosomal communication and developing an active ingredient able to stimulate or inhibit this communication pathway for hair and skin benefits.
Footnotes
a NanoSight LM10, Malvern
b IX 70 microscope (Olympus) and NIS-Elements imaging system (Nikon)
c exoEasy Maxi Kit, Qiagen
d NanoSight LM14, Malvern
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