Characterization of AMSC-EV and HUMSC-EV and their intense uptake in the skin
MSCs could be isolated from a variety of tissues. In this study, we used two types of MSCs, human adipose-derived MSCs (hAMSCs) and umbilical cord-derived MSCs (hHUMSCs), and EV were isolated from the supernatants by ultracentrifugation. To characterize hAMSCs-derived EV (AMSC-EV) and hHUMSCs-derived EV (HUMSC-EV), we performed transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), western blot, and ExoView analysis. Both EV had enrichment of marker proteins, including CD63, CD9, TSG101, and Alix, and the absence of endoplasmic reticulum membrane marker calnexin (Fig. 1a). They presented a classic cup-shaped appearance under TEM (Fig. 1b). According to NTA, EV had a size distribution peaking at about 150 nm in diameter (Fig. 1c). Furthermore, ExoView analysis demonstrated high expression levels of EV transmembrane proteins CD63, CD81 and CD9 (Fig. 1d, e). No significant differences were observed between AMSC-EV and HUMSC-EV regarding morphology, size distribution, and marker expression patterns. We then investigated the distribution of EV in the skin by subcutaneous injection. Over time, in vivo imaging revealed the intense uptake of EV in the skin (Fig. 1f). Histological analysis of skin biopsies confirmed the dispersed presence of EV in the dermis (Fig. 1g). Additionally, no adverse events were detected in mice after MSC-EV injection as evidenced by histological examination of vital organs (Supplementary Fig. 1a), and blood biochemistry analysis of liver, renal and cardiac function indices (Supplementary Fig. 1b). Together, these results demonstrated a high sample purity of AMSC-EV and HUMSC-EV and showed EV intense uptake in the skin.
Characterization of AMSC-EV and HUMSC-EV and their intense uptake in the skin. a Western blot analysis for exosomal marker proteins CD63, CD9, TSG101, and Alix and cell-specific marker Calnexin. b Morphology of AMSC-EV and HUMSC-EV was examined by TEM (scale bar, 200 nm). c Size distribution profile of EV detected by NTA. d ExoView analysis detected CD63, CD81, and CD9. Mouse IgG was used as a negative control. e Quantitation of the expression of CD63, CD81, CD9, and Mouse IgG in EV. f Representative in vivo fluorescent imaging of PKH26-labeled EV in mice at 1 h and 24 h after injection. g Biodistribution of PKH26-labeled EV in mice skin at 24 h, 48 h and 72 h after EV treatment
AMSC-EV and HUMSC-EV mitigated photoaging of keratinocytes and fibroblasts in vitro
Exposure to UVB radiation instigates a cascade of skin aging processes, oxidative stress manifested as a dramatic increase in ROS levels,27 and extensive DNA damage that threatens genome stability and cell viability. To investigate whether AMSC-EV and HUMSC-EV have a protective effect on photoaging, we used human keratinocytes (HaCaTs), human primary keratinocytes (HKCs), and human dermal fibroblasts (HDFs) as in vitro cellular models.
HaCaTs are a spontaneously immortalized human keratinocyte line that can mimic the characteristics of normal epidermal cells, a major cell component in the epidermis, which is the outermost skin layer directly affected by UVB.28 We first investigated whether UVB irradiation affects the uptake of AMSC-EV and HUMSC-EV by HaCaTs. EV were labeled with PKH26 and the fluorescence intensity in both control and photoaged HaCaTs were compared. No significant differences were observed (Fig. 2a and Supplementary Fig. 2a), indicating that UVB irradiation did not affect the uptake of AMSC-EV and HUMSC-EV by HaCaTs. We then evaluated the effects of EV by analyzing various biomarkers related to photoaging. After irradiation, there was a notable increase in ROS levels, whereas treatment with EV reduced ROS levels (Fig. 2b, c). UVB exposure induced the accumulation of SA-β-gal positive cells in HaCaTs, which was attenuated by EV treatment (Fig. 2d, e), while senescence markers P16 and LMNB1 in HaCaTs were also rescued after EV stimulation (Supplementary Fig. 2b, c). The level of γ-H2Ax increased significantly after irradiation and notably decreased in the AMSC-EV and HUMSC-EV groups (Fig. 2f, g). Under physiological conditions, the proliferation and migration of epidermal cells play an essential role.29 CCK8 assay showed that proliferation was inhibited after irradiation, and AMSC-EV and HUMSC-EV had the effect of promoting proliferation (Fig. 2h). Both transwell and migration experiments showed that migration was affected by irradiation, and AMSC-EV and HUMSC-EV promoted migration (Fig. 2i–l). Additionally, there was a significant increase in inflammatory factors after UVB irradiation, significantly ameliorated by EV (Fig. 2m, n). Moreover, we utilized human primary keratinocytes (HKCs) to replicate key experiments involving senescence, yielding results consistent with those obtained from HaCaT cells (Supplementary Fig. 3a–f). The above results indicated that UVB can increase epidermal ROS, aging, DNA damage, proliferation, migration, and inflammatory response, while AMSC-EV and HUMSC-EV can alleviate these effects.
AMSC-EV and HUMSC-EV mitigated photoaging of HaCaTs in vitro. a Representative image of MSC-EV uptake by HaCaTs pretreated with or without UVB irradiation (scale bar, 20 μm). b Representative immunofluorescence staining images of positive cells of ROS (green) and DAPI (scale bar, 20 μm). c Fluorescence intensity of ROS levels. n = 3, ****P < 0.0001. d Representative images of SA-β-gal staining in HaCaTs (scale bar, 100 μm). e Quantitation of SA-β-gal positive cells in HaCaTs. f Representative immunofluorescence staining images of positive cells of γ-H2Ax (red) and DAPI (scale bar, 20 μm). g Quantitation of the mean number of γ-H2Ax foci/cell. n = 3, ****P < 0.0001. h Quantitation of HaCaTs proliferation detected by CCK8 assay with the OD value on Day 1, Day 2, Day 3, Day 4, and Day 5. n = 3, ***P < 0.001, ****P < 0.0001. i Representative images of transwell assays of HaCaTs (scale bar, 100 μm). j Quantitation of transwell assays of HaCaTs. n = 3, ****P < 0.0001. k Representative images of migration assay and the image was taken at the indicated times (scale bar, 100 μm). l Quantitation of migration assays of HaCaTs. n = 3, *P < 0.05, **P < 0.01. m Quantitation of IL-1β, IL-6, and TNF-α released by HaCaTs was detected through qRT-PCR. n = 3, ****P < 0.0001. n Quantitation of IL-1β, IL-6, and TNF-α released by HaCaTs was detected through ELISA. n = 3, **P < 0.01, ****P < 0.0001
The dermis, the second layer of the skin beneath the epidermis, is also affected by UVB. Fibroblasts are one of the important cells in the dermis with functions such as secretion of collagen and MMPs that affect the ECM and ultimately the appearance of the skin such as wrinkles.30 To study the effects of UVB on the dermis, we used HDFs, which produce multiple ECM proteins that are essential for skin structure and function. There was no significant difference in PKH26-labeled EV that were up-taken by HDFs whether in the control group or under UVB irradiation (Fig. 3a and Supplementary Fig. 2d). We found that UVB irradiation caused similar changes in HDFs as in HaCaTs, such as increased ROS levels (Fig. 3b, c), senescence (Fig. 3d, e and Supplementary Fig. 2i, j), DNA damage (Fig. 3f, g), inflammation (Fig. 3m, n), migration (Supplementary Fig. 2e–h) and reduced proliferation (Fig. 3h), which were all alleviated by EV treatment.
AMSC-EV and HUMSC-EV mitigated photoaging of HDFs in vitro. a Representative images of MSC-EV uptake by HDFs pretreated with or without UVB irradiation (scale bar, 20 μm). b Representative immunofluorescence staining images ROS (green) and DAPI (scale bar, 20 μm). c Fluorescence intensity of ROS levels. n = 3, ***P < 0.001, ****P < 0.0001. d Representative images of SA-β-gal staining in HDFs (scale bar, 100 μm). e Quantitation of SA-β-gal positive cells in HDFs. n = 3, ****P < 0.0001. f Representative immunofluorescence staining images of positive cells of γ-H2Ax (red) and DAPI (scale bar, 20 μm). g Quantitation of a mean number of γ-H2Ax foci/cell. h Quantitation of HDFs proliferation detected by CCK8 assay with the OD value on Day 1, Day 2, Day 3, Day 4, and Day 5. n = 3, ***P < 0.001, ****P < 0.0001. i Quantitative of COL31A in HDFs by qRT-PCR. n = 3, *P < 0.05, **P < 0.01. j Quantitation of TIMP1, MMP1, and MMP9 released by HDFs by qRT-PCR. n = 3, *P < 0.05, ***P < 0.001, ****P < 0.0001. k Western blot analysis showing the change of COL3, TIMP1, MMP1 and MMP9 in HDFs. l Quantitative of COL3, TIMP1, MMP1, and MMP9 in HDFs by western blot. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. m Quantitation of IL-1β, IL-6, and TNF-α released by HDFs by qRT-PCR. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n Quantitation of IL-1β, IL-6, and TNF-α released by HDFs was detected through ELISA. n = 3, **P < 0.01, ****P < 0.0001
Moreover, we investigated the impact of UVB and EV on collagen synthesis, which is a key process in maintaining skin elasticity and integrity. Matrix metalloproteinase (MMP) and the tissue inhibitor of metalloproteinase (TIMP) play crucial roles in preserving extracellular matrix (ECM) homeostasis that is directly related to wrinkles and sagging in skin appearance.31 We found that UVB irradiation decreased TIMP1 and COL3 levels while increasing MMP1 and MMP3 levels (Fig. 3i–l), indicating a disruption of collagen homeostasis and degradation of collagen fibers. In contrast, EV treatment restored the balance of TIMP1, MMP1, MMP3, and COL3 levels (Fig. 3i–l), suggesting a protective role of EV against UVB-induced collagen damage. These results indicate that UVB irradiation induces oxidative stress, DNA damage, senescence, inflammation, and impaired collagen synthesis in HDFs, the main features of photoaging in the dermis. Remarkably, treatment with EV demonstrates a capacity to mitigate these effects, thereby preserving the function and structure of the dermal layer.
Collectively, our in vitro study demonstrates that AMSC-EV and HUMSC-EV can protect both epidermal and dermal cells from UVB-induced photoaging by modulating various cellular processes.
AMSC-EV and HUMSC-EV mitigated photoaging in a reconstructed full-thickness skin model
The protective effects of AMSC-EV and HUMSC-EV in keratinocyte and fibroblast cell models prompted us to investigate further their role in an in vitro T-Skin photoaging model (Fig. 4a). T-Skin is a commercialized reconstructed full-thickness skin model, which shares some similarities with normal human skin in structure and biomarkers (Fig. 4b). Following various doses of UVB irradiation, we observed that 180mJ/cm2 every day for 3 days of irradiation induced a senescence phenotype, as evidenced by the increased presence of senescence (Fig. 4c, d), enhanced oxidative stress (Fig. 4e, f) and DNA damage (Fig. 4g, h). We added PKH26-stained EV and found that partial uptake occurred after 48 h, and EV uptake was completed in the whole model after 72 h (Fig. 4i). The addition of EV to the photoaging T-Skin model significantly alleviated SA-β-gal levels, ROS levels, and γ-H2Ax levels (Fig. 4c–h). Moreover, senescence markers P16 and LMNB1 were also rescued after EV stimulation (Supplementary Fig. 4a, b). UVB is a pivotal trigger of skin inflammation, which leads to the causation and exacerbation of numerous skin diseases.32 We then measured the expression levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α using ELISA and found that UVB irradiation increased their secretion in the T-Skin model, and treatment with EV decreased their secretion, indicating an anti-inflammatory effect of AMSC-EV and HUMSC-EV (Fig. 4j).
AMSC-EV and HUMSC-EV mitigated photoaging in constructed full-thickness skin model. a Schematic representation of establishment and treatment of T-Skin model (Created with BioRender.com). b Representative images of histological analysis and biomarker staining of the T-Skin model (scale bar, 15 μm). c Representative images of SA-β-gal staining (scale bar, 20 μm). d Quantitation of SA-β-gal positive cells. n = 3, **P < 0.01, ***P < 0.001, ****P < 0.0001. e Representative images of ROS staining (scale bar, 20 μm). f Quantitation of ROS-positive cells. n = 3, ****P < 0.0001. g Representative images of γ-H2Ax staining (scale bar, 20 μm). h Quantitation of γ-H2Ax positive cells. n = 3, **P < 0.01, ****P < 0.0001. i Representative images of MSC-EV uptake by T-Skin model after 48 and 72 h (left, scale bar, 150 μm; right, scale bar, 25 μm). j Quantitation of IL-1β, IL-6, and TNF-α released by T-Skin model was detected through ELISA. n = 3, ****P < 0.0001
Taken together, these results suggested that AMSC-EV and HUMSC-EV significantly ameliorated their photoaging oxidative stress and DNA damage levels while also alleviating their secretion of inflammatory factors in skin organoids.
The therapeutic effect of AMSC-EV and HUMSC-EV in nude mice model of photoaging
The promising outcomes of AMSC-EV and HUMSC-EV in the in vitro cellular and T-Skin model motivated us to conduct additional investigation into their effect on skin photoaging in vivo. We established a photoaging model as previously reported30 in which UVB was administered every other day for 8 weeks in nude mice. Mice were randomly divided into four groups: control group and UVB exposure groups, which were separately treated with PBS, AMSC-EV, or HUMSC-EV (Fig. 5a). After establishing the UVB irradiation model, we captured images and investigated the wrinkle formation of the back skin of mice at week 0, 2, 3, and 4. At week 0, there were almost no wrinkles in the control group. In contrast, the UVB-treated groups exhibited deep and wide wrinkles, indicating the successful establishment of the photoaging model (Fig. 5b). Remarkably, from the second week, there were significantly fewer and thinner wrinkles in the EV-treated group (Fig. 5c–e). We investigated transepidermal water loss (TEWL) to characterize skin barrier function. Excess moisture content evaporated from the skin after UVB irradiation, and moisture loss was significantly reduced in EV-treated groups (Fig. 5f).
Histological analysis of the dorsal skin in nude mice after UVB irradiation and EV treatment. a Schematic description of the establishment and treatment of the photoaging model (Created with BioRender.com). b–e Representative images of mouse dorsal skin. From left to right: control group, UVB-exposed groups treated with PBS, AMSC-EV, and HUMSC-EV. f Transepidermal water loss content. n = 5, *P < 0.05, ***P < 0.001. g Representative images of HE staining (top, scale bar, 400 μm; bottom, scale bar, 200 μm). h Epidermal and dermal thickness analysis. n = 6, *P < 0.05, **P < 0.01. i Representative images of SA-β-gal staining. j Quantitation of SA-β-gal positive cells. n = 6, **P < 0.01, ***P < 0.001
Further, histological analysis was performed to evaluate the skin condition and treatment effect. Hematoxylin and eosin (HE) staining was utilized to show the alterations in skin structure. Compared to the control group, UVB irradiation caused thicker epidermis and thinner dermis, and EV treatment significantly recovered thickness changes (Fig. 5g, h). Additionally, the number of SA-β-gal positive cells significantly increased after UVB irradiation and decreased significantly after EV-treated (Fig. 5i, j). Senescence markers P16 and LMNB1 were also restored after EV stimulation (Supplementary Fig. 5a, b). The above results indicate that EV can effectively mitigate the detrimental effects of UVB radiation on skin structure and aging.
Skin histology analysis could elucidate the amount of collagen deposition. Both Masson’s staining and picrosirius red staining were carried out to visualize alterations in dermal collagen levels. UVB exposure led to a reduction in the total amount of collagen. Compared with the control group, there were abundant and dense collagen fibers in the AMSC-EV and HUMSC-EV groups (Fig. 6a, c). Similarly, picrosirius red staining confirmed these findings (Fig. 6b, d), showing enhanced collagen restoration following EV treatment. Additionally, we performed Verhoeff’s Van Gieson staining to evaluate changes in elastin and found EV treatment restored elastin levels (Supplementary Fig. 5c, d).
Histological analysis and inflammation antibody array of the dorsal skin in nude mice after UVB irradiation and EV treatment. a Representative images of Masson’s staining (scale bar, 50 μm). b Representative images of picrosirius red staining (scale bar, 50 μm). c Quantitation of collage area from Masson’s staining statistics. n = 6, **P < 0.01, ***P < 0.001. d Quantitation of collage area from picrosirius red staining statistics. n = 6, ***P < 0.001. e Western blot analysis for COL1, COL3, TIMP1, MMP1 and MMP3. f The top inflammatory factors examined by Inflammation Antibody Array. g Heatmap highlighting the representative inflammatory factors examined by the Inflammation Antibody Array. h Western blot analysis for inflammatory factors IL-1β, IL-6, TNF-α, and GM-CSF
We then analyzed collagen proteins and factors associated with dermal matrix remodeling. We found a significant decrease in the protein expression levels of COL1 and COL3, a significant increase in the levels of MMP1 and MMP3, and a significant reduction in the level of TIMP1 after UVB irradiation. These changes were restored after the addition of EV. (Fig. 6e). Additionally, we evaluated the expression of inflammatory factors by Inflammation Antibody Array (Fig. 6f). We show the results of representative inflammatory factor changes, including GCSF, IFN-γ, IL-6, and others (Fig. 6g). UVB exposure increased the expression of inflammatory factor, while treatment effectively restores these factor levels. After treatment, representative Western blot results confirmed the reduction trend in inflammatory factors, including IL-1β, IL-6, TNF-α, and GM-CSF (Fig. 6h).
Collectively, these findings demonstrate that UVB exposure induces skin changes in appearance, function, and histology, accompanied by inflammatory responses in vivo, and MSC-EV reversed these changes in a positive direction.
In addition, the observed therapeutic effect of HUMSC-EV seems better than that of AMSC-EV, which warrants further investigation.
MSC-EV rescues HDFs and HaCaTs photoaging by transferring TIMP1
Since both AMSC-EV and HUMSC-EV exhibited protective effects against photoaging, we speculate that there should be shared components within their cargo responsible for this outcome. As a crucial component of EV cargo, proteins are known to exert significant influence on EV functions. We first performed a protein analysis of MSC-EV. Overall, we detected 1,370 proteins, of which 1023 proteins were common to AMSC-EV and HUMSC-EV (Fig. 7a).
MSC-EV rescues HDFs and HaCaTs photoaging by upregulating TIMP1. a Venn diagram of the protein contents of AMSC-EV and HUMSC-EV. b Heatmap of the protein contents of AMSC-EV and HUMSC-EV. c Heatmap of extracellular matrix organization. d Western blot analysis showing COL1, COL3, COL6, and TIMP1 expression in AMSC-EV and HUMSC-EV. e Quantitation of TIMP1 in AMSC-EV and HUMSC-EV was detected by Western blot. n = 3. f Representative immunofluorescence staining images of positive cells of ROS (green) and DAPI (scale bar, 35 μm). g Quantitation of ROS-positive cells. n = 3, ****P < 0.0001. h Representative immunofluorescence staining images of positive cells of γ-H2Ax (red) and DAPI (scale bar, 20 μm). i Quantitation of a mean number of γ-H2Ax foci/cell. n = 3, ***P < 0.001, ****P < 0.0001. j Representative images of SA-β-gal staining in HDFs and HaCaTs (scale bar, 100 μm). k Quantitation of SA-β-gal positive cells in HDFs and HaCaTs. n = 3, ***P < 0.001, ****P < 0.0001. l Representative images of transwell assays of HaCaTs (scale bar, 100 μm). m epresentative images of migration assay of HaCaTs (scale bar, 100 μm). n Western blot analysis showing the change of COL3, TIMP1, MMP1 and MMP9 in HDFs
Gene ontology (GO) analysis showed that proteins that are highly expressed in both AMSC-EV and HUMSC-EV are mainly enriched in extracellular matrix organization (Fig. 7b, c), suggesting their potential impact on extracellular matrix remodeling mediated by the shared highly expressed proteins. We performed Western blot for the highly expressed TOP10 proteins (COL1A2, DCN, COL3A1, COL6A1, TIMP1, FSTL1, LUM, NID1, PTX3, and ENO1). The levels of TIMP1, COL1A2, COL3A1, and COL6A1, closely associated with extracellular matrix homeostasis, are all high in AMSC-EV and HUMSC-EV (Fig. 7d, e and Supplementary Fig. 6a). NID1 and FSTL1, which may be related to extracellular matrix homeostasis, are also highly expressed (Supplementary Fig. 6b, c).
We further analyzed TIMP1 due to its well-established role in ECM remodeling27,33 and potential involvement in the photoaging process. In aged mice skin, we observed a decrease in TIMP1 protein expression compared to young mice (Supplementary Fig. 6d, e). Silence of TIMP1 by siRNAs in HaCaTs and HDFs resulted in a significant increase in SA-β-gal positive cells (Supplementary Fig. 6f, g) and expression of senescence-associated markers (Supplementary Fig. 6h–k), indicating a crucial role of TIMP1 in the aging process. To investigate whether EV exert protective effects against photoaging by transferring TIMP1, we first added TIMP1 into the cell culture medium according to the concentration gradient. We found that 5 ng/mL of TIMP1 was able to reduce UVB-induced oxidative stress (Fig. 7f, g) and DNA damage (Fig. 7h, i). Assays for ROS and γ-H2Ax were also performed on days 1, 2, and 3 after the addition of 5 ng/mL of TIMP1, and remarkable reductions were observed on day 3 (Supplementary Fig. 7a–d). Other hallmarks of photoaging, including elevated SA-β-gal positive cells in the HDFs and HaCaTs (Fig. 7j, k), reduction of HaCaTs migration ability (Fig. 7l, m and Supplementary Fig. 7e, f), as well as extracellular matrix degradation (Fig. 7n and Supplementary Fig. 7g), were all inhibited after TIMP1 treatment. In addition, a significant decrease in the levels of MMP1, MMP3, and MMP9 after TIMP1 treatment was observed (Supplementary Fig. 8a–f).
Taken together, we demonstrated that TIMP1 is a pivotal protein in EV and plays a crucial role in preventing photoaging, particularly in oxidative stress, DNA damage, cell migration, and extracellular matrix remodeling.
The therapeutic effect of MSC-EV is mediated through the downregulation of Notch signaling pathway
To investigate the impact of MSC-EV on signaling pathways, we performed RNA sequencing (RNA-seq) analysis on HDFs control, UVB exposure, and treatment with AMSC-EV and HUMSC-EV groups, and analyzed different expression genes (DEGs) overlapped in both AMSC-EV and HUMSC-EV treatment groups. DEGs up-regulated by UVB but repressed by EV and DEGs downregulated by UVB but restored by EV were identified, resulting in a set of Rev-photoaging DEGs, while genes that changed in the same direction were categorized as Pro-photoaging DEGs (Fig. 8a). We observed a significantly higher count of Rev-photoaging DEGs compared to Pro-photoaging DEGs. GO analysis indicated that the Rev-photoaging DEGs were enriched in ECM degradation and immune responses (Fig. 8b). Further investigation into altered signaling pathways unveiled that EV downregulated the UVB-induced genes in Notch signaling pathway (Fig. 8c). Subsequent qPCR analysis confirmed the changes in related molecules Hes1, Tle1, Lfng, Dll1 and HeyL (Fig. 8d).
MSC-EV rescues HDFs photoaging by downregulating NOTCH signaling pathway. a Venn diagram showing the overlapped genes between photoaging (UVB/Control) and Treatment (EV/UVB) based on data from RNA-seq. Rev-photoaging-DEGs were defined as subsets of overlapped DEGs that were changed in the opposite direction in photoaging and treatment. b Representative GO terms of Rev-photoaging DEGs for Biological Process (green), Cellular Component (orange), and Molecular Function (blue) were shown. c Heatmaps showing the expression profile of genes in response to UVB and EV treatment in Notch signaling. d Quantitation of different mRNA levels of Hes1, Tle1, Lfng, Dll1, and HeyL. e Western blot analysis showing expression of NOTCH1, NICD1, HES1, SIRT1, P16, P21, P53 and GAPDH after EV treatment. f Western blot analysis showing expression of NOTCH1, NICD1, HES1, SIRT1, P16, P21, P53 and GAPDH after TIMP1 treatment. g Representative immunofluorescence staining images of positive cells of HES1 in nude mice dorsal skin injected PBS, AMSC-EV, or HUMSC-EV (top, scale bar, 30 μm; bottom, scale bar, 10 μm). h Representative immunofluorescence staining images of SA-β-gal (scale bar, 100 μm), ROS (green), and γ-H2Ax (red) (scale bar, 30 μm). i Luciferase-reporter assays of HES1 transcriptional activity in HDFs after UVB, FM (culture medium), EV, TIMP1, GI254023X, and VPA treatment. n = 3, **P < 0.01, ***P < 0.001, ****P < 0.0001. j Schematic of the role of EV in mediating UVB-induced photoaging of Notch signaling and cellular senescence. (Created with BioRender.com)
The Notch signaling pathway is an important signaling pathway vital to cell fate, proliferation, and tissue homeostasis. The NOTCH receptor is first cleaved in the Golgi and translocated to the cell membrane. The Notch receptor is activated by binding to ligands presented by neighboring cells, such as Delta-like 1 (DLL1), which promotes a conformational change that exposes Notch to cleavage by A disintegrin and metalloproteinases (ADAMs) at the S2 site. The remainder of the NOTCH receptor is further cleaved to form the Notch intracellular domain (NICD) by γ-secretase at the cell membrane. NICD can translocate to the nucleus, and transcriptional activation of Notch target genes is initiated, such as those of the Hes and Hey family.34 Transducin-like Enhancer of Split 1 (TLE1) is a multitasked transcriptional corepressor that acts through the Notch signaling pathway.35 Lunatic Fringe (Lfng), the Notch modulator gene, is mainly involved in intercellular coupling and delays intercellular Notch signaling transmission.36 Hes1, a crucial downstream molecule of the Notch signaling pathway, prompted us to explore its role in EV-mediated alleviation of photoaging. Literature reports suggest that p16, p21, and p53 are downstream molecules of HES1, which further influence the release of SASP.37,38 In UVB-induced HDFs, western blot analysis demonstrated that changes in Notch1 and its downstream genes HES1, SIRT1, P16, P21, and P53 were all recovered by AMSC-EV and HUMSC-EV (Fig. 8e and Supplementary Fig. 9a). Additionally, we found that TIMP1 exerted a similar role in mitigating the activation of the NOTCH signaling pathway (Fig. 8f and Supplementary Fig. 9b). Furthermore, immunofluorescence staining showed that AMSC-EV and HUMSC-EV exhibited much less HES1 in the photoaged skin in nude mice (Fig. 8g and Supplementary Fig. 9c).
To determine whether EV and TIMP1 modulate downstream pathways by inhibiting Notch signaling, we introduced Valproic Acid Sodium (VPA), a Notch pathway activator. Following VPA addition in HDFs, we observed a reduction in the efficacy of EV and TIMP1 in mitigating senescence, clearing ROS, and repairing DNA damage (Fig. 8h and Supplementary Fig. 9d–f). ADAM10, an essential substrate for Notch activation, is a proposed target of TIMP1. We then employed GI254023X, an ADAM10 inhibitor, and found that it replicated the photoaging alleviation effects observed with TIMP1 (Fig. 8h and Supplementary Fig. 9d–f). Similar findings were also observed in the T-skin model. As demonstrated in Supplementary Fig. 10a–d, the addition of Notch activator VPA decreased the efficacy of EV and TIMP1, while ADAM10 inhibitor GI254023X partially recapitulated the effects of EV and TIMP1. Additionally, it inhibited the UVB-induced upregulation of HES1 transcriptional activity and protein expression (Fig. 8i and Supplementary Fig. 10e, f).
In summary, these results indicate that EV and its content TIMP1 may exert their effects by inhibiting the Notch signaling pathway through TIMP1-mediated suppression of ADAM10 (Fig. 8j).
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- Source: https://www.nature.com/articles/s41392-024-01993-z