Protecting and rejuvenating ageing skin by regulating endogenous hyaluronan metabolism using adipose-derived stem cell-secreted siRNAs

Abstract

Background: Loss of moisture is the primary cause of skin ageing and dysfunction. The skin's hydration largely depends on hyaluronan (HA) and its ability to retain water. Ultraviolet (UV) irradiation, which accounts for 80% of skin ageing (commonly referred to as photoaging), gradually disrupts the balance of HA metabolism, leading to a reduction in HA levels, dehydration, and, ultimately, the formation of wrinkles.

Methods: In this study, we develop an RNAi-based strategy to treat aged skin by modulating endogenous HA metabolism. Hyaluronidase 2 (HYAL2), an enzyme responsible for HA degradation, is selected as the therapeutic target, given its significant upregulation in photoaged skin. To deliver the siRNA targeting HYAL2 to the skin, human adipose-derived stem cells (ADSCs) are engineered to stably express and secrete HYAL2-targeting siRNAs (ADSC/siR^H) via small extracellular vesicles (sEVs).

Results: In vitro experiments demonstrate that ADSC-delivered siRNAs are successfully internalised by recipient cells, where they restore UV-induced HA reduction by inhibiting HYAL2 expression. In vivo experiments revealed that subcutaneous implantation of engineered ADSCs prior to UV exposure significantly protects mouse skin from accelerated HA degradation, helping to retain water content and prevent UV-induced dryness. Furthermore, the application of engineered ADSCs to aged mouse skin can markedly restore HA and water content, effectively smoothing deep wrinkles and improving skin appearance.

Conclusion: We developed an effective biological strategy to combat skin ageing and damage by preserving endogenous HA levels, which could be applied for facial rejuvenation in the future.

Keywords: adipose-derived stem cells (ADSCs); extracellular vesicles (EVs); hyaluronan (HA); siRNA therapy; skin aging.

Figure 1

(A) qPCR analysis of HYAL2 mRNA levels in untreated NIH/3T3 cells or NIH/3T3 cells transfected with scrambled RNA, si-hyal2-1, si-hyal2-2, and si-hyal2-3 (n = 6). (B,C) A western blotting analysis of HYAL2 protein levels in untreated NIH/3T3 cells or NIH/3T3 cells transfected with scrambled RNA, si-hyal2-2, and si-hyal2-3 (n = 3). (D) Schematic of the generation of engineered ADSCs. (E) The infection efficiency was expressed by lenti-GFP in NIH/3T3 cells. (F) qPCR analysis of si-hyal2 concentration in ADSCs or ADSC/siR^H (n = 3). (G) Nanoparticle tracking analysis (NTA) of sEVs isolated from the medium of ADSC/siR^H. (H) Representative western blot analysis of the sEV membrane markers CD63, CD9, CD81, and proteins that mediate the budding and abscission processes during sEV exocytosis in ADSC/siR^H and their sEVs. (I) qPCR analysis of si-hyal2 concentrations in sEVs isolated from the medium of ADSCs and ADSC/siR^H (n = 3). The results represent the mean ± SD; p-values are calculated using one-way ANOVA; ^**p < 0.001.

Figure 2

(A) Schematic of NIH/3T3 cells treated with irradiation and sEVs. (B) qPCR analysis of HYAL2 mRNA levels in unirradiated NIH/3T3 cells and irradiated NIH/3T3 cells treated with PBS, sEVs derived from ADSCs, and sEVs derived from ADSC/siR^H (n = 3). (C) A western blotting analysis of HYAL2 protein levels in unirradiated NIH/3T3 cells and irradiated NIH/3T3 cells treated with PBS, sEVs derived from ADSCs, and sEVs derived from ADSC/siR^H (n = 3). (D) qPCR analysis of si-hyal2 concentrations in unirradiated NIH/3T3 cells and irradiated NIH/3T3 cells treated with PBS, sEVs derived from ADSCs, and sEVs derived from ADSC/siR^H (n = 3). (E) The representative immunofluorescence images and fluorescence intensity analysis of HA (red) in unirradiated NIH/3T3 cells and irradiated NIH/3T3 cells treated with PBS, sEVs derived from ADSCs, and sEVs derived from ADSC/siR^H, and blue staining (DAPI) represents nuclei. The results represent the mean ± SD; p-values are calculated using a one-way ANOVA; ^*p < 0.05 and ^**p < 0.01.

Figure 3

(A) A schematic design of the generation of the photodamage mouse model and ADSC/siR^H treatment. (B) The representative dorsal skin photos of the unirradiated mice and the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H. (C) The wrinkle analysis of the unirradiated mice and the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H by clinical wrinkle score (n = 3). (D–F) The representative dorsal skin photos (D), water content (E), and elasticity analysis (F) by Skin Analyzer of the unirradiated mice and the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H (n = 3). (G,I) The representative histochemistry staining images (G) and epidermal thickness analysis (I) of the dorsal skin tissue of the unirradiated mice and the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H (n = 3). (H,J) The representative Masson trichrome staining images (H) and dermal thickness analysis (J) of the dorsal skin tissue of the unirradiated mice and the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H (n = 3). (K,L) The representative immunofluorescence images (K) and fluorescence intensity analysis of HA (red) (L) of the dorsal skin tissue of the unirradiated mice and the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H, and blue staining (DAPI) represents nuclei (n = 3). (M) qPCR analysis of si-hyal2 concentrations in the dorsal skin tissue of the unirradiated mice and the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H (n = 3). (N) qPCR analysis of HYAL2 mRNA levels in the dorsal skin tissue of the unirradiated mice and the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H (n = 3). (O) A western blotting analysis of HYAL2 protein levels in the dorsal skin tissue of the unirradiated mice and the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H (n = 3). The results represent the mean ± SD; p-values are calculated using a one-way ANOVA; ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.

Figure 4

(A) Schematic of the generation of the skin ageing mouse model and ADSC/siR^H treatment. (B) The representative dorsal skin photos of the unirradiated mice and the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H. (C) The wrinkle analysis of the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H before and after treatment by clinical wrinkle score (n = 3). (D–F) The representative dorsal skin photos (D), water content (E), and elasticity analysis (F) by Skin Analyzer of the unirradiated mice and the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H (n = 3). (G,I) The representative histochemistry staining images (G) and epidermal thickness analysis (I) of the dorsal skin tissue of the unirradiated mice and the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H (n = 3). (H,J) The representative Masson’s trichrome staining images (H) and dermal thickness analysis (J) of the dorsal skin tissue of the unirradiated mice and the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H (n = 3). (K,L) The representative immunofluorescence images (K) and fluorescence intensity analysis of HA (red) (L) of the dorsal skin tissue of the unirradiated mice and the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H, and blue staining (DAPI) represents nuclei (n = 3). (M) qPCR analysis of si-hyal2 concentrations in the dorsal skin tissue of the unirradiated mice and the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H (n = 3). (N) qPCR analysis of HYAL2 mRNA levels in the dorsal skin tissue of the unirradiated mice and the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H (n = 3). (O) A western blotting analysis of HYAL2 protein levels in the dorsal skin tissue of the unirradiated mice and the irradiated mice treated with PBS, ADSCs, and ADSC/siR^H (n = 3). The results represent the mean ± SD, and p-values are calculated using a one-way ANOVA; ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.