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• W2949846784 abstract "Extracellular vesicles (EVs) and their miRNA cargo are intercellular communicators transmitting their pleiotropic messages between different cell types, tissues, and body fluids. Recently, they have been reported to contribute to skin homeostasis and were identified as members of the senescence-associated secretory phenotype of human dermal fibroblasts. However, the role of EV-miRNAs in paracrine signaling during skin aging is yet unclear. Here we provide evidence for the existence of small EVs in the human skin and dermal interstitial fluid using dermal open flow microperfusion and show that EVs and miRNAs are transferred from dermal fibroblasts to epidermal keratinocytes in 2D cell culture and in human skin equivalents. We further show that the transient presence of senescent fibroblast derived small EVs accelerates scratch closure of epidermal keratinocytes, whereas long-term incubation impairs keratinocyte differentiation in vitro. Finally, we identify vesicular miR-23a-3p, highly secreted by senescent fibroblasts, as one contributor of the EV-mediated effect on keratinocytes in in vitro wound healing assays. To summarize, our findings support the current view that EVs and their miRNA cargo are members of the senescence-associated secretory phenotype and, thus, regulators of human skin homeostasis during aging. Extracellular vesicles (EVs) and their miRNA cargo are intercellular communicators transmitting their pleiotropic messages between different cell types, tissues, and body fluids. Recently, they have been reported to contribute to skin homeostasis and were identified as members of the senescence-associated secretory phenotype of human dermal fibroblasts. However, the role of EV-miRNAs in paracrine signaling during skin aging is yet unclear. Here we provide evidence for the existence of small EVs in the human skin and dermal interstitial fluid using dermal open flow microperfusion and show that EVs and miRNAs are transferred from dermal fibroblasts to epidermal keratinocytes in 2D cell culture and in human skin equivalents. We further show that the transient presence of senescent fibroblast derived small EVs accelerates scratch closure of epidermal keratinocytes, whereas long-term incubation impairs keratinocyte differentiation in vitro. Finally, we identify vesicular miR-23a-3p, highly secreted by senescent fibroblasts, as one contributor of the EV-mediated effect on keratinocytes in in vitro wound healing assays. To summarize, our findings support the current view that EVs and their miRNA cargo are members of the senescence-associated secretory phenotype and, thus, regulators of human skin homeostasis during aging. Extracellular vesicles (EVs) are versatile and ubiquitously present membranous particles that participate in intercellular communication by shuttling their functional cargo, such as proteins, RNA, or DNA, to recipient cells (Iraci et al., 2016Iraci N. Leonardi T. Gessler F. Vega B. Pluchino S. Focus on extracellular vesicles: physiological role and signalling properties of extracellular membrane vesicles.Int J Mol Sci. 2016; 17: 171Crossref PubMed Scopus (136) Google Scholar). In the context of the skin, they have been found in ex vivo sections of the human papillary dermis (Cretoiu et al., 2015Cretoiu D. Gherghiceanu M. Hummel E. Zimmermann H. Simionescu O. Popescu L.M. FIB-SEM tomography of human skin telocytes and their extracellular vesicles.J Cell Mol Med. 2015; 19: 714-722Crossref PubMed Scopus (49) Google Scholar), at sites of age-related cutaneous disorders (Nakamura et al., 2016Nakamura K. Jinnin M. Harada M. Kudo H. Nakayama W. Inoue K. et al.Altered expression of CD63 and exosomes in scleroderma dermal fibroblasts.J Dermatol Sci. 2016; 84: 30-39Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), at wounds (Huang et al., 2015Huang P. Bi J. Owen G.R. Chen W. Rokka A. Koivisto L. et al.Keratinocyte microvesicles regulate the expression of multiple genes in dermal fibroblasts.J Invest Dermatol. 2015; 135: 3051-3059Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), and in the stroma of human skin tumors (Jang et al., 2017Jang S.C. Crescitelli R. Cvjetkovic A. Belgrano V. Bagge R.O. Hoog J.L. et al.A subgroup of mitochondrial extracellular vesicles discovered in human melanoma tissues are detectable in patient blood [preprint].bioRxiv. 2017; (accessed 27 June 2019)Google Scholar). In addition, in vitro vesicular cross-talk has been observed between several types of skin cells, including keratinocytes, melanocytes, human dermal fibroblasts (HDF), dermal papilla cells, outer root sheath cells of the hair follicle, and microvascular endothelial cells (Lo Cicero et al., 2015Lo Cicero A. Delevoye C. Gilles-Marsens F. Loew D. Dingli F. Guéré C. et al.Exosomes released by keratinocytes modulate melanocyte pigmentation.Nat Commun. 2015; 6: 7506Crossref PubMed Scopus (101) Google Scholar, Huang et al., 2015Huang P. Bi J. Owen G.R. Chen W. Rokka A. Koivisto L. et al.Keratinocyte microvesicles regulate the expression of multiple genes in dermal fibroblasts.J Invest Dermatol. 2015; 135: 3051-3059Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, Merjaneh et al., 2017Merjaneh M. Langlois A. Larochelle S. Cloutier C.B. Ricard-Blum S. Moulin V.J. Pro-angiogenic capacities of microvesicles produced by skin wound myofibroblasts.Angiogenesis. 2017; 20: 385-398Crossref PubMed Scopus (34) Google Scholar, Wäster et al., 2016Wäster P. Eriksson I. Vainikka L. Rosdahl I. Öllinger K. Extracellular vesicles are transferred from melanocytes to keratinocytes after UVA irradiation.Sci Rep. 2016; 6: 27890Crossref PubMed Scopus (23) Google Scholar, Zhou et al., 2018Zhou L. Wang H. Jing J. Yu L. Wu X. Lu Z. Regulation of hair follicle development by exosomes derived from dermal papilla cells.Biochem Biophys Res Commun. 2018; 500: 325-332Crossref PubMed Scopus (28) Google Scholar). However, nothing is known about EV-mediated cross-talk between skin fibroblasts and keratinocytes during cellular aging. In the elderly, senescent cells have been observed in the dermis and in the epidermis (Ressler et al., 2006Ressler S. Bartkova J. Niederegger H. Bartek J. Scharffetter-Kochanek K. Jansen-Dürr P. et al.p16INK4A is a robust in vivo biomarker of cellular aging in human skin.Aging Cell. 2006; 5: 379-389Crossref PubMed Scopus (329) Google Scholar). Their accumulation with age and at sites of age-associated diseases contributes to cellular, molecular, and structural changes of the dermal and epidermal compartments, where they impair skin homeostasis, causing increased susceptibility for dermatological disorders (Velarde and Demaria, 2016Velarde M.C. Demaria M. Targeting senescent cells: possible implications for delaying skin aging: A mini-review.Gerontology. 2016; 62: 513-518Crossref PubMed Scopus (30) Google Scholar, Waaijer et al., 2016Waaijer M.E.C. Gunn D.A. Adams P.D. Pawlikowski J.S. Griffiths C.E.M. van Heemst D. et al.P16INK4a positive cells in human skin are indicative of local elastic fiber morphology, facial wrinkling, and perceived age.J Gerontol A. Biol Sci Med Sci. 2016; 71: 1022-1028Crossref PubMed Scopus (23) Google Scholar). Senescent cells are irreversibly growth arrested, partially de- or trans-differentiated, and the acquisition of the senescence-associated secretory phenotype (SASP) is discussed as the most potent contributor of senescent cells to organismal aging. The SASP consists of growth factors, cytokines, chemokines, matrix remodeling enzymes (Coppé et al., 2010Coppé J.P. Desprez P.Y. Krtolica A. Campisi J. The senescence-associated secretory phenotype: the dark side of tumor suppression.Annu Rev Pathol. 2010; 5: 99-118Crossref PubMed Scopus (1943) Google Scholar), as well as lipids (Ni et al., 2016Ni C. Narzt M.S. Nagelreiter I.M. Zhang C.F. Larue L. Rossiter H. et al.Autophagy deficient melanocytes display a senescence associated secretory phenotype that includes oxidized lipid mediators.Int J Biochem Cell Biol. 2016; 81: 375-382Crossref PubMed Scopus (29) Google Scholar), and thereby creates a chronically inflamed and pro-tumorigenic microenvironment (Schosserer et al., 2017Schosserer M. Grillari J. Breitenbach M. The dual role of cellular senescence in developing tumors and their response to cancer therapy.Front Oncol. 2017; 7: 278Crossref PubMed Scopus (88) Google Scholar). The selective removal of senescent cells improves tissue homeostasis and repopulation of the hair bulge niche (Yosef et al., 2016Yosef R. Pilpel N. Tokarsky-Amiel R. Biran A. Ovadya Y. Cohen S. et al.Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL.Nat Commun. 2016; 7: 11190Crossref PubMed Scopus (339) Google Scholar), postpones the onset and severity of age-associated diseases, and thereby extends life- and health span of mice (Baker et al., 2016Baker D.J. Childs B.G. Durik M. Wijers M.E. Sieben C.J. Zhong J. et al.Naturally occurring p16 Ink4a -positive cells shorten healthy lifespan.Nature. 2016; 530: 184-189Crossref PubMed Scopus (1125) Google Scholar). However, their elimination in acute wounds delays the healing process, leading to fibrosis and impaired granulation tissue formation (Demaria et al., 2014Demaria M. Ohtani N. Youssef S.A. Rodier F. Toussaint W. Mitchell J.R. et al.An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA.Dev Cell. 2014; 31: 722-733Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar, JIl and Lau, 2010Jun JIl Lau L.F. Cellular senescence controls fibrosis in wound healing.Aging (Albany NY). 2010; 2: 627-631Crossref PubMed Scopus (157) Google Scholar). Recently, EVs and their miRNA cargo emerged as communicators of the SASP of human dermal fibroblasts (EV-SASP; Terlecki-Zaniewicz et al., 2018Terlecki-Zaniewicz L. Lämmermann I. Latreille J. Bobbili M.R. Pils V. Schosserer M. et al.Small extracellular vesicles and their miRNA cargo are anti-apoptotic members of the senescence-associated secretory phenotype. Aging, Albany NY2018Crossref Scopus (76) Google Scholar, Urbanelli et al., 2016Urbanelli L. Buratta S. Sagini K. Tancini B. Emiliani C. Extracellular vesicles as new players in cellular senescence.Int J Mol Sci. 2016; 17Crossref PubMed Scopus (54) Google Scholar). In the skin, the presence of specific miRNAs within different layers and cell types regulates the balanced mRNA to miRNA ratio to maintain functional homeostasis (Botchkareva, 2012Botchkareva N.V. MicroRNA/mRNA regulatory networks in the control of skin development and regeneration.Cell Cycle. 2012; 11: 468-474Crossref PubMed Scopus (47) Google Scholar). Therefore, it is not surprising that the fine tuning of overlapping wound healing phases and skin aging-associated changes are regulated by the transient or constitutive presence of specific miRNAs (Sonkoly et al., 2010Sonkoly E. Wei T. Pavez Loriè E.P. Suzuki H. Kato M. Törmä H. et al.Protein kinase C-dependent upregulation of miR-203 induces the differentiation of human keratinocytes.J Invest Dermatol. 2010; 130: 124-134Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Here we shed light on the existence of EVs in human skin ex vivo and investigate an EV-miRNA cross-talk from fibroblasts to keratinocytes in monolayers and in 3D skin models. Finally, we evaluate how small EVs (sEVs) derived from senescent fibroblasts influence keratinocyte differentiation and their scratch closure capacity in vitro. To address if EVs are present in human skin in vivo, skin sections were studied by transmission electron microscopy (TEM) and images confirmed the presence of EV-like structures within dermal cells, adjacent to dermal cells in extracellular collagen structures (Figure 1a and b), and within intracellular multivesicular bodies (Figure 1c). These structures also stained positive for the EV marker CD63 by immunogold labeling of resin-free ultrathin cryo-cut skin sections (Figure 1d). In order to isolate sEVs from human skin, tissue biopsies from two independent donors were disintegrated using dispase, and sEVs contained in accessible material were purified (see scheme in Supplementary Figure S1a). Particles from this crude extract were enriched by using tangential flow filtration with a cut-off of 300 kDa (Supplementary Figure S1b). Median size was approximately 110 nm as determined by nanoparticle tracking analysis (Figure 1e). These particles were positive for EV-markers TSG101 and syntenin, as shown by western blot analysis. However, calnexin, which is expected to be absent in sEVs, was also detectable (Figure 1f and Supplementary Figure S1c and d). Therefore, we further purified the EV enriched, skin derived preparations using size exclusion chromatography (SEC). Thereby, the majority of particles was eluted in the first six fractions and pools of fractions 1 to 3 and 4 to 6 (SEC 1–3, SEC 4–6) were prepared, and particle number was analyzed by nanoparticle tracking analysis, showing enrichment of particles in SEC 1–3, whereas lower numbers were recovered in fractions SEC 4–6 (Figure 1g); however, particle size of the fractions did not differ significantly (Figure 1h). These were then analyzed by western blotting analysis (Figure 1h). The SEC 4–6 fraction, however, showed strong enrichment of syntenin, whereas calnexin staining was close to the detection limit in western blot analysis (Figure 1i). TEM analysis showed particles below 200 nm, which portrayed a cup-shape characteristic for EVs (Figure 1j). This indicates that with the sequence of purification methods used we were able to isolate sEVs from human skin. As an additional approach to test the existence of EVs in human skin, dermal interstitial fluid (dISF) was collected by dermal open flow microperfusion (Bodenlenz et al., 2013Bodenlenz M. Aigner B. Dragatin C. Liebenberger L. Zahiragic S. Höfferer C. et al.Clinical applicability of dOFM devices for dermal sampling.Skin Res Technol. 2013; 19: 474-483PubMed Google Scholar) and EVs were enriched by two approaches (Supplementary Figure S1e). After removal of cell debris by centrifugation at 500g and 14,000g, respectively, irregularly shaped, double lipid membrane containing, cup-shaped EVs were visible, similar to those isolated from human skin biopsies (Figure 1k). Nanoparticle tracking analysis of these fractions confirmed a particle median size of around 100 nm (Figure 1l). To increase the purity of the EVs isolated from dISF, we performed SEC and analyzed isolated fractions by TEM. As particle counts were very low in the pooled SEC fractions (Supplementary Figure S1f), we were not able to perform western blot analysis or to capture EVs in fractions 4 to 6. However, in pooled SEC fractions 1 to 3, under omission of the 0.22 μm filtration step, cup-shaped particles with sizes between 50–500 nm were detected, albeit too dilute to capture multiple EVs on single frames because of limited dISF sample material (Figure 1m and Supplementary Figure S1g and h). Still, using immunogold labeling, we confirmed the presence of EV marker protein CD81 on the vesicular membrane of these skin derived EVs (Figure 1m, right image, and Supplementary Figure S1h). Taken together these data strongly suggest the existence of EVs in the interstitium of the skin, which we were able to visualize and enrich for by using independent sample materials and enrichment strategies. To confirm an EV-mediated miRNA transfer from HDF to primary normal human epidermal keratinocytes, HDF were transfected with Caenorhabditis elegans–specific cel-miR-39, which is packaged into EVs (Hergenreider et al., 2012Hergenreider E. Heydt S. Tréguer K. Boettger T. Horrevoets A.J.G. Zeiher A.M. et al.Atheroprotective communication between endothelial cells and smooth muscle cells through miRNAs.Nat Cell Biol. 2012; 14: 249-256Crossref PubMed Scopus (871) Google Scholar). The sEVs were isolated from fibroblast supernatants and supplemented to keratinocyte culture media (Figure 2a). Cel-miR-39 was detected in fibroblasts (Figure 2b) and in 2D cultured keratinocytes exposed to the purified sEVs after 48 hours (Figure 2c). In addition, to further test the transfer of miRNAs from fibroblasts to keratinocytes resembling the epidermis of 3D human skin equivalents, cel-miR-39 transfected fibroblasts were embedded into a collagen matrix (“dermis”; Figure 2a). After full maturation over 10 days, dermis and epidermis were separated, RNA isolated, and cel-miR-39 was confirmed to be still present in the dermis (Figure 2d) and in the epidermis (Figure 2e). These findings suggest that miRNA cross-talk between fibroblasts and keratinocytes in skin equivalents is not limited by the collagen matrix. Since we recently identified sEVs and their miRNA cargo as, to our knowledge, previously unreported members of the senescence-associated secretory phenotype (EV-SASP) of fibroblasts (Terlecki-Zaniewicz et al., 2018Terlecki-Zaniewicz L. Lämmermann I. Latreille J. Bobbili M.R. Pils V. Schosserer M. et al.Small extracellular vesicles and their miRNA cargo are anti-apoptotic members of the senescence-associated secretory phenotype. Aging, Albany NY2018Crossref Scopus (76) Google Scholar), we aimed to test how these sEVs might alter the normal homeostasis of primary keratinocytes. Therefore, HDF were driven into stress-induced premature senescence (SIPS) by repetitive exposure to H2O2 (Lämmermann et al., 2018Lämmermann I. Terlecki-Zaniewicz L. Weinmüllner R. Schosserer M. Dellago H. de Matos Branco A.D. et al.Blocking negative effects of senescence in human skin fibroblasts with a plant extract.NPJ Aging Mech Dis. 2018; 4: 4Crossref PubMed Scopus (19) Google Scholar, Terlecki-Zaniewicz et al., 2018Terlecki-Zaniewicz L. Lämmermann I. Latreille J. Bobbili M.R. Pils V. Schosserer M. et al.Small extracellular vesicles and their miRNA cargo are anti-apoptotic members of the senescence-associated secretory phenotype. Aging, Albany NY2018Crossref Scopus (76) Google Scholar). Induction of SIPS was confirmed by increased p21 levels (Supplementary Figure S2a), irreversible growth arrest (not shown), by a flattened and enlarged morphology (Supplementary Figure S2b), and by an increase in SA-ß-Gal activity (Supplementary Figure S2c and d). sEVs of senescent (SIPS) and quiescent control HDF were purified from conditioned media using differential centrifugation and analyzed by TEM (Figure 3a), nanoparticle tracking analysis (Figure 3b), and immunoblotting (Figure 3c). Membranous particles of around 110 nm in size were revealed, which stained positive for the EV-specific marker syntenin and TSG101, but negative for non-EV marker calnexin in western blots. To monitor how the transient presence of senescent fibroblast derived sEVs modulates wound closure of keratinocytes in vitro, we used a 2D culture model to follow the dynamics of wound closure in terms of repopulation of the cell-free area (gap), as well as using scratch assays after a single addition of sEVs. Keratinocytes of three different donors were exposed for 48 hours to sEVs from quiescent or senescent fibroblasts. Exposure to the senescent cell derived sEVs doubled the number of cells in the cell-free area (Figure 3d and e, and Supplementary Figure S3a) and accelerated the closure dynamics in both assay setups compared with cells exposed to sEVs from quiescent fibroblasts (Figure 3f and Supplementary Figure S3b). Although we cannot differentiate between cell migration and proliferation in our experimental setup, appearance of filopodia and lamellipodia-like protrusions (Figure 3d and Supplementary Figure S3a) and an increase in vimentin expression upon exposure to senescent cell derived sEVs (Figure 3g) point toward an at least partial contribution by migration, for which a more mesenchymal-like phenotype is a prerequisite (Yan et al., 2010Yan C. Grimm W.A. Garner W.L. Qin L. Travis T. Tan N. et al.Epithelial to mesenchymal transition in human skin wound healing is induced by tumor necrosis factor-alpha through bone morphogenic protein-2.Am J Pathol. 2010; 176: 2247-2258Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). In order to test the impact of chronic presence of the EV-SASP on keratinocyte differentiation in vitro, we exposed keratinocytes to sEVs from quiescent or senescent fibroblasts for one week. The presence of senescent cell derived sEVs changed the morphology of the confluent keratinocyte layer (Figure 3h) and reduced the expression levels of the late differentiation marker involucrin (Figure 3i), which is reported to be a main initiator of the cornification process in vivo (Robinson et al., 1996Robinson N.A. LaCelle P.T. Eckert R.L. Involucrin is a covalently crosslinked constituent of highly purified epidermal corneocytes: evidence for a common pattern of involucrin crosslinking in vivo and in vitro.J Invest Dermatol. 1996; 107: 101-107Abstract Full Text PDF PubMed Scopus (54) Google Scholar, Watt and Green, 1981Watt F.M. Green H. Involucrin synthesis is correlated with cell size in human epidermal cultures.J Cell Biol. 1981; 90: 738-742Crossref PubMed Scopus (212) Google Scholar). To summarize, we observed an enhanced scratch/gap (wound) closure with a concomitant rise in vimentin expression after the short term presence of senescent derived sEVs, whereas their chronic presence affected terminal differentiation of keratinocytes in vitro. In order to test if imbalanced keratinocyte homeostasis might be attributable to specific miRNAs, we selected miR-23a-3p as a prominent candidate because it was highly secreted in sEVs of senescent fibroblasts (Terlecki-Zaniewicz et al., 2018Terlecki-Zaniewicz L. Lämmermann I. Latreille J. Bobbili M.R. Pils V. Schosserer M. et al.Small extracellular vesicles and their miRNA cargo are anti-apoptotic members of the senescence-associated secretory phenotype. Aging, Albany NY2018Crossref Scopus (76) Google Scholar) and repeatedly connected with cellular senescence and skin aging (Röck et al., 2015Röck K. Tigges J. Sass S. Schütze A. Florea A.M. Fender A.C. et al.miR-23a–3p causes cellular senescence by targeting hyaluronan synthase 2: possible implication for skin aging.J Invest Dermatol. 2015; 135: 369-377Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Indeed, we confirmed secreted miR-23a-3p to be more abundantly secreted by senescent cells (Supplementary Figure S3c). RNAse digestion in the absence of Triton X-100 was then used to determine miR-23a-3p levels presumably protected by lipid membrane structures (Figure 4a). The fraction of miR-23a-3p that is not accessible to RNAse is still elevated in the sEV preparation of senescent cell supernatants, whereas almost all miR-23a-3p is digested by RNAse treatment in the presence of Triton X-100. This suggests that the vast majority of miR-23a-3p is either freely accessible to RNAse or protected by lipid membranes. The remaining small fraction, which is below 7% of the total quantitative reverse transcriptase in real time signal, might be protected by other structures, such as proteinaceous particles. In order to visualize the influence of senescence on freely accessible (Δfree) versus lipid membrane protected miR-23a-3p (ΔEV), we calculated the respective differences (Figure 4a). Indeed, ΔEV increases significantly in senescent versus control cells, while Δfree miR-23a-3p does not. This indicates that the increase of total miR-23a-3p in the sEV preparations of senescent cells is indeed because of an increased EV-based secretion of this miRNA. Then, keratinocytes were exposed to sEVs derived from senescent HDF for 48 hours, which resulted in a significant increase of intracellular miR-23a-3p levels (Figure 4b). This, in combination with the transfer of cel-miR-39 from fibroblasts to keratinocytes shown above, suggests an uptake of this miRNA via sEVs by keratinocytes in vitro. However, we have not excluded whether miR-23a-3p might be induced endogenously after sEV exposure within keratinocytes. To investigate if miR-23a-3p might contribute to the accelerated scratch and gap (wound) closure seen by senescent cell derived sEVs, we transfected keratinocytes with pre-miR-23a-3p and a non-targeting control miRNA. Overexpression was confirmed (Figure 4c) and enhanced gap closure, in terms of cells present in the cell-free area (Figure 4d and e, and Supplementary Figure S3d), as well as closure dynamics (Figure 4f) were observed. In addition, miR-23a-3p transfected cells showed mesenchymal cell-like protrusions (Figure 4e and Supplementary Figure S3d), as it was similarly seen after exposure to senescent cell derived sEVs. In addition, a slight increase in vimentin expression (Figure 4g) and a concomitant decrease of miR-23a-3p’s direct target E-cadherin (Cao et al., 2012Cao M. Seike M. Soeno C. Mizutani H. Kitamura K. Minegishi Y. et al.MiR-23a regulates TGF-β-induced epithelial-mesenchymal transition by targeting E-cadherin in lung cancer cells.Int J Oncol. 2012; 41: 869-875Crossref PubMed Scopus (137) Google Scholar) were observed (Figure 4h), suggesting again an at least partial epithelial-to-mesenchymal transition. Plakophilin 4 (PKP4/p0071) is a predicted putative target of miR-23a-3p in keratinocytes (Agarwal et al., 2015Agarwal V. Bell G.W. Nam J.W. Bartel D.P. Predicting effective microRNA target sites in mammalian mRNAs.Elife. 2015; 4: e05005Crossref Scopus (3100) Google Scholar), which was significantly reduced by miR-23a-3p (Figure 4i). Plakophilin 4A interacts with the desmosomal plaques and the adherens junctions to regulate mechanical strength of keratinocyte monolayers (Calkins et al., 2003Calkins C.C. Hoepner B.L. Law C.M. Novak M.R. Setzer S.V. Hatzfeld M. et al.The armadillo family protein p0071 is a VE-cadherin- and desmoplakin-binding protein.J Biol Chem. 2003; 278: 1774-1783Crossref PubMed Scopus (52) Google Scholar). However, miR-23a-3p did not modulate keratinocyte differentiation as assessed by involucrin levels one week after transfection (data not shown). Cross-talk of fibroblasts to keratinocytes by soluble factors affect skin homeostasis, as knockout of AP-1 components in fibroblasts has been shown to impair epidermal differentiation (Szabowski et al., 2000Szabowski A. Maas-Szabowski N. Andrecht S. Kolbus A. Schorpp-Kistner M. Fusenig N.E. et al.C-Jun and JunB antagonistically control cytokine-regulated mesenchymal-epidermal interaction in skin.Cell. 2000; 103: 745-755Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar) and wound healing in mouse models in vivo (Florin et al., 2006Florin L. Knebel J. Zigrino P. Vonderstrass B. Mauch C. Schorpp-Kistner M. et al.Delayed wound healing and epidermal hyperproliferation in mice lacking JunB in the skin.J Invest Dermatol. 2006; 126: 902-911Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). However, little is known as to whether EVs and their miRNA cargo exist in different layers of the human skin and if they are involved in the regulation of skin homeostasis during aging. Here we report the presence of multivesicular bodies as one source of secreted sEVs in fibroblasts. This presence of EVs in vivo is supported by indications of EVs at sides of wounds from human skin biopsies (Huang et al., 2015Huang P. Bi J. Owen G.R. Chen W. Rokka A. Koivisto L. et al.Keratinocyte microvesicles regulate the expression of multiple genes in dermal fibroblasts.J Invest Dermatol. 2015; 135: 3051-3059Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) and their visualization adjacent to interstitial dermal cells by 3D electron microscopy (Cretoiu et al., 2015Cretoiu D. Gherghiceanu M. Hummel E. Zimmermann H. Simionescu O. Popescu L.M. FIB-SEM tomography of human skin telocytes and their extracellular vesicles.J Cell Mol Med. 2015; 19: 714-722Crossref PubMed Scopus (49) Google Scholar). In the context of fibroblast aging, little is known about EVs (Lehmann et al., 2008Lehmann B.D. Paine M.S. Brooks A.M. McCubrey J.A. Renegar R.H. Wang R. et al.Senescence-associated exosome release from human prostate cancer cells.Cancer Res. 2008; 68: 7864-7871Crossref PubMed Scopus (254) Google Scholar, Terlecki-Zaniewicz et al., 2018Terlecki-Zaniewicz L. Lämmermann I. Latreille J. Bobbili M.R. Pils V. Schosserer M. et al.Small extracellular vesicles and their miRNA cargo are anti-apoptotic members of the senescence-associated secretory phenotype. Aging, Albany NY2018Crossref Scopus (76) Google Scholar). Cellular senescence is a key driver of the aging process, and the SASP has been shown to promote tumorigenesis of epidermal cells (Krtolica et al., 2001Krtolica A. Parrinello S. Lockett S. Desprez P.Y. Campisi J. Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging.Proc Natl Acad Sci U S A. 2001; 98: 12072-12077Crossref PubMed Scopus (1056) Google Scholar) and to increase the number of senescent keratinocytes re-entering the cell cycle, concomitantly with a partial epithelial-to-mesenchymal transition of these escape-keratinocytes (Malaquin et al., 2013Malaquin N. Vercamer C. Bouali F. Martien S. Deruy E. Wernert N. et al.Senescent fibroblasts enhance early skin carcinogenic events via a paracrine MMP-PAR-1 axis.PLoS ONE. 2013; 8Crossref PubMed Scopus (59) Google Scholar). Senescent cell derived EVs have been reported to confer part of this pro-tumorigenic activity (Takasugi et al., 2017Takasugi M. Okada R. Takahashi A. Virya Chen D. Watanabe S. Hara E. Small extracellular vesicles secreted from senescent cells promote cancer cell proliferation through EphA2.Nat Commun. 2017; 8: 15729Crossref PubMed Scopus (142) Google Scholar), which might be partly attributable to increased secretion of senescent fibroblasts derived EVs (Lehmann et al., 2008Lehmann B.D. Paine M.S. Brooks A.M. McCubrey J.A. Renegar R.H. Wang R. et al.Senescence-associated exosome release from human prosta" @default.
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• W2949846784 date "2019-12-01" @default.
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• W2949846784 title "Extracellular Vesicles in Human Skin: Cross-Talk from Senescent Fibroblasts to Keratinocytes by miRNAs" @default.
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