Molecular basis of retinol anti-ageing properties in naturally aged human skin in vivo

Abstract

Objective: Retinoic acid has been shown to improve the aged-appearing skin. However, less is known about the anti-ageing effects of retinol (ROL, vitamin A), a precursor of retinoic acid, in aged human skin in vivo. This study aimed to investigate the molecular basis of ROL anti-ageing properties in naturally aged human skin in vivo.

Methods: Sun-protected buttock skin (76 ± 6 years old, n = 12) was topically treated with 0.4% ROL and its vehicle for 7 days. The effects of topical ROL on skin epidermis and dermis were evaluated by immunohistochemistry, in situ hybridization, Northern analysis, real-time RT-PCR and Western analysis. Collagen fibrils nanoscale structure and surface topology were analysed by atomic force microscopy.

Results: Topical ROL shows remarkable anti-ageing effects through three major types of skin cells: epidermal keratinocytes, dermal endothelial cells and fibroblasts. Topical ROL significantly increased epidermal thickness by stimulating keratinocytes proliferation and upregulation of c-Jun transcription factor. In addition to epidermal changes, topical ROL significantly improved dermal extracellular matrix (ECM) microenvironment; increasing dermal vascularity by stimulating endothelial cells proliferation and ECM production (type I collagen, fibronectin and elastin) by activating dermal fibroblasts. Topical ROL also stimulates TGF-β/CTGF pathway, the major regulator of ECM homeostasis, and thus enriched the deposition of ECM in aged human skin in vivo. 0.4% topical ROL achieved similar results as seen with topical retinoic acid, the biologically active form of ROL, without causing noticeable signs of retinoid side effects.

Conclusion: 0.4% topical ROL shows remarkable anti-ageing effects through improvement of the homeostasis of epidermis and dermis by stimulating the proliferation of keratinocytes and endothelial cells, and activating dermal fibroblasts. These data provide evidence that 0.4% topical ROL is a promising and safe treatment to improve the naturally aged human skin.

Keywords: TGF-β; extracellular matrix; retinoid; skin ageing; vascularity.

Figure 1

Topical ROL increases epidermal thickness and dermal vascularity by proliferation of epidermal keratinocytes and dermal endothelial cells, respectively, in aged human skin in vivo. OCT-embedded skin sections (7μm) were obtained from aged (76±6 years) healthy sun-protected buttock skin after topical treatment of vehicle and 0.4% retinol for seven days. (A) H&E staining. Representative images of twelve individuals (N=12). White arrows indicate epidermal thickness. Bars=100μm. (B) Quantification of epidermal thickness (μm). (C) Ki67 immunostaining. 3.0× enlargement of the boxed region is shown to lower panels. Bars= 100μm. Representative images of twelve individuals (N=12). Quantification of epidermal (D) and dermal (E) Ki 67 immunostaining. (F) Ki67 and CD31 co-immunofluoresce staining. Skin sections were co-immunofluoresce stained with Ki67 and CD31, a marker of endothelial cells. Arrows indicate double stained cells. Representative of five individuals. Bar=50μm. (G) CD31 immunostaining. Representative images of twelve individuals (N=12). Bar=100μm. (H) Quantification of CD31 immunostaining. All immunostainings were quantified by computerized image analysis (Image-pro Plus software, version 4.1, Media Cybernetics, MD) and data are expressed as mean±SEM, *p<0.05. N=12.

Figure 2

ROL improves dermal ECM microenvironment in aged human skin in vivo. OCT-embedded skin sections (7μm) were obtained from aged (76±6 years) healthy sun-protected buttock skin after topical treatment of vehicle and 0.4% retinol for seven days. (A) Type I procollagen immunostaining. 3× enlargement of the boxed region is shown to lower panels. Representative images of twelve individuals (N=12). Arrows indicate positive cells. Bars=100μm. (B) Quantification of type I procollagen. (C) Fibronectin immunostaining. 2.5× enlargement of the boxed region is shown to lower panels. Representative images of twelve individuals (N=12). Arrows indicate positive cells. Bars= 100μm. (D) Quantification of fibronectin. (E) Tropoelastin immunostaining. 3.0× enlargement of the boxed region is shown to lower panels. Representative images of twelve individuals (N=12). Bars=100μm. (F) Quantification of tropoelastin. All immunostainings were quantified by computerized image analysis (Image-pro Plus software, version 4.1, Media Cybernetics, MD) and data are expressed as mean±SEM, *p<0.05. N=12.

Figure 3

Elevated epidermal-specific c-Jun transcription factor by topical ROL in aged human skin in vivo. OCT-embedded skin sections (7μm) were obtained from aged (76±6 years) healthy sun-protected buttock skin after topical treatment of vehicle and 0.4% retinol for seven days. (A) c-Jun immunostaining. Representative images of twelve individuals (N=12). Bars=100μm. (B) c-Fos immunostaining. Representative images of twelve individuals (N=12). Bars=100μm. All immunostainings were quantified by computerized image analysis (Image-pro Plus software, version 4.1, Media Cybernetics, MD) and data are expressed as mean±SEM, *p<0.05. N=12. (C) c-Jun protein. (D) c-Fos protein. Protein levels were determined by Western analysis. Protein levels were normalized to β-actin (loading control). Insets show representative Western blots. Mean ± SEM, N=6, *p < 0.05.

Figure 4

Topical ROL stimulates TGF-β/CTGF pathway, the major regulator of ECM homeostasis, in aged human skin in vivo. OCT-embedded skin sections (7μm) were obtained from aged (76±6 years) healthy sun-protected buttock skin after topical treatment of vehicle and 0.4% retinol for seven days. (A) HSP47 immunostaining. Representative images of twelve individuals (N=12). Bars=100μm. (B) TGF-β pathway components mRNA levels. Total RNA was prepared from human skin samples. mRNA levels were determined by real-time RT-PCR. mRNA levels were normalized to 36B4 (internal housekeeping gene control). Mean ± SEM, N=12, *p<0.05. (C) TGF-β1 (D) CTGF/CCN2 in situ hybridization. Representative images of twelve individuals (N=12). Bars= 100μm. (E) TGF-β1 (F) CTGF/CCN2 Northern analysis. The intensities were quantified and normalized using 36B4 as loading control. Insets show representative Northern blots. Data are expressed as mean±SEM, *p<0.05. N=12. (G) TGF-β1 (H) CTGF/CCN2 immunostaining. Representative images of twelve individuals (N=12). Bars= 100μm. (I) Smad7 Northern analysis. The intensities were quantified and normalized using 36B4 as loading control. Inset shows representative Northern blots. Data are expressed as mean±SEM, *p<0.05. N=12. (J) Smad7 protein. Smad7 protein levels were determined by Western analysis. Protein levels were normalized to β-actin (loading control). Insets show representative Western blots. Mean ± SEM, N =6, *p < 0.05. All positive staining was quantified by computerized image analysis (Image-pro Plus software, version 4.1, Media Cybernetics, MD). Data are expressed as mean±SEM, *p<0.05. N=12.

Figure 5

Deposition of mature collagen is increased by topical ROL in aged human skin in vivo. OCT-embedded skin sections (7μm) were obtained from aged (76±6 years) healthy sun-protected buttock skin after topical treatment of vehicle and 0.4% retinol for seven days. (A) Nanoscale collagen fibrils were imaged by AFM. Representative AFM images were shown. N=12. The blue arrows indicate intact collagen fibrils and red arrows heads indicate damaged collagen fibrils. (B) Three dimensional collagen fibrils. Dermal roughness was analyzed using Nanoscope Analysis software (Nanoscope_Analysis_v120R1sr3, Bruker-AXS, Santa Barbara, CA). All results are expressed as the mean ± SEM, N=12, *p < 0.05. Bars=100 nm. (C) Proposed model for topical ROL exerts anti-aging effects in aged human skin by proliferation of epidermal keratinocytes and dermal endothelial cells and activation of dermal fibroblasts (see Discussion for details).