Identification of miR‐126 as a new regulator of skin ageing (2016)

Skin ageing is commonly associated with wrinkle formation, reduced wound healing capacity, loss of elasticity and pale complexion.1, 2 In addition, capillary density decreases with age, resulting in a decreased oxygen and nutrient supply.1, 2 Although several factors that are involved in skin ageing have been already identified, only few molecular mechanisms regulating these processes are understood. MicroRNAs (miRs) are short endogenous RNA molecules negatively regulating gene expression by binding to the 3′-untranslated region or coding region of target mRNAs and thus initiate translational repression or mRNA degradation 3 and might play a role in the development of ageing phenotypes. A deregulation of miR expression in skin-associated diseases such as psoriasis and atopic dermatitis has been reported previously.4, 5 However, to our knowledge, no study has been published yet, showing that specific secretory miRs become deregulated in the course of skin ageing and are involved in human skin cell crosstalk. Thus, we analysed, if secreted miRs might play a role in age-related dysfunctions. We generated miR profiles of interstitial fluid collected from younger and older volunteers after suction blistering and identified age-dependent differences in miR levels (eg miR-126). In vitro, we analysed whether miR-126 is age-dependently secreted by dermal endothelial cells and incorporated by other skin cells. To investigate the levels of secreted miRs during skin ageing, human interstitial fluid of the skin was analysed.6 Quantitative PCR-based microRNA profiling was performed, and 175 miRs were detected with a Ct-value <40 which was sufficient to be considered in the following analysis (Figure S1 and Table S1). Setting a threshold of 1.5-fold regulation (up/down), we identified 61 miRs that were regulated in an age-related manner in suction blister fluids collected from young and old volunteers. The results could be confirmed in an independent experiment for three selected miRs (miR-100, miR-126 and miR-223) (Figure 1A-C). In vitro analysis was performed to delineate whether the age-related miR expression changes are triggered during cellular ageing. We cultivated dermal endothelial cells isolated from volunteers aged between 22 and 73 years and determined miR-100, miR-126 and miR-223 abundance in the supernatant 48 hours after cell seeding. All three miRs were significantly decreased in the supernatant of endothelial cell populations isolated from aged volunteers compared to the younger cell population (Figure S2A-C). In contrast, intra-cellular miR levels detected for miR-100 and miR-126 (miR-223 was not analysed) in the intra-cellular compartment remain stable throughout age (Figure S3A-B). Previous studies suggested that miRs detected in cell-free serum/plasma may function as cell–cell communicators and play key roles in many cellular processes associated with cell signalling, for example cardiomyocyte hypertrophy.7 Thus, there is evidence that secretory miRs may be incorporated by “acceptor cells” to regulate their intra-cellular gene expression. To analyse whether miR-100 and endothelial-enriched miR-126 secreted by dermal endothelial cells can be internalized by other skin cell types, keratinocytes, fibroblasts and preadipocytes were incubated in the presence of the supernatant collected from endothelial cells (Figure 2A). Interestingly, it was evident that miR-100 was not significantly incorporated by these different cell types (Figure 2B). However, a significant increase of miR-126 concentration was observed in fibroblasts and preadipocytes and a clear trend towards miR-126 uptake was detected in keratinocytes (Figure 2C). The molecular pathways contributing to the vascular changes during ageing are not fully understood yet. In this study, we could highlight that miR-126 expression levels are reduced in suction blister fluids of aged volunteers and this age-related decrease could be confirmed in vitro. MiR-126 is known to protect blood vessels, induces neovascularization and is the most abundant miR in endothelial cells.8 We postulate that the observed age-related decrease of miR-126 is in part driven by the age-dependent secretion changes of dermal endothelial cells. This is in good agreement with the ageing phenotype, because it is known that the number and the functionality of capillary structures decrease with age.1 The exact molecular mechanisms were not addressed in the present study; however, our data indicate that the secretion of miR-126 is affected rather than its basal endogenous expression. In the literature, the participation of active secretory mechanisms for miRs has been reported via (A) exosome-mediated delivery of miRNAs via NSmase as suggested by Bang et al.7 in a model of crosstalk between cardiac cells or (B) mediated by Rab proteins emphasized in a recent study in endothelial cells.9 Interestingly, uptake mechanisms in recipient cells seem to differ as we did not observe a significant uptake of miR-100, but incorporation of miR-126 into fibroblasts. The identification of detailed miRNA uptake mechanisms will be an interesting goal for future research. Our findings suggest the following working model (Figure S4): the crosstalk between endothelial cells and fibroblasts in human skin is among others mediated by miR-126 and might be especially relevant for the ratio between pro- and anti-angiogenic factors. In fact, preliminary results indicate that pro- and anti-angiogenic factors are deregulated in dermal fibroblasts after miR-126 transfection (data not shown). We speculate that enhanced secretion of endothelial miR-126 in young skin stimulates fibroblasts to secret angiogenic-relevant factors to support vascularity and may furthermore trigger biological processes that improve skin parameters not directly linked to endothelial cells. At old age, the amount of circulatory miR-126 decreases consistently and might result ultimately in an impaired dermal vasculature and further dysfunctions within the skin. Following this, we suggest directed therapeutic strategies (eg via small chemical compounds) to stimulate endothelial miR-126 expression and/or secretion to circumvent age-related dysfunctional human skin. EG, SB, KS, LT, HW and MW were employees of Beiersdorf AG at the time the study was performed. TT received consultation fees from Beiersdorf AG. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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