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. Author manuscript; available in PMC: 2018 Feb 1.
Published in final edited form as: Int J Cosmet Sci. 2016 Jul 4;39(1):56–65. doi: 10.1111/ics.12348

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

Yuan Shao 1, Tianyuan He 1, Gary J Fisher 1, John J Voorhees 1, Taihao Quan 1,*
PMCID: PMC5136519  NIHMSID: NIHMS793378  PMID: 27261203

Synopsis

OBJECTIVE

Retinoic acid has been shown to improve the aged-appearing skin. However, less is known about the anti-aging 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 retinol anti-aging 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 seven 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-aging 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-aging 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 naturally aged human skin.

Keywords: Retinoid, Skin Aging, TGF-β, ECM, Vascularity

Introduction

Human skin, like all other organs, undergoes natural aging process as a consequence of the passage of time [1]. In addition, human skin, unlike other organs, continuously experience harmful stress and damage from environmental sources such as solar UV irradiation [2]. As skin changes are among the most visible signs of aging, skin is central in the social and visual experience. As such, skin appearance has a significant emotional and psychological impact on our life quality. In clinically, aged skin has a significant pathological impact on many age-related skin diseases, notably impaired wound healing [3, 4] and promotion of cancer development in elderly [5, 6].

Histological and ultrastructural studies have revealed skin undergoes remarkable morphologic changes with aging, which are primarily characterized by thinner epidermis and dermis with reduced numbers of epidermal keratinocytes and dermal stromal cells, respectively [7, 8]. This age-related thinning of epidermis and dermis is the major driving force for the aged-appearing skin in elderly. It is well documented that age-related thinning of the epidermis significantly impairs skin function such as impaired skin barrier function that forms a barrier to prevent water loss and protect against environmental insults. In addition to epidermis, aged skin dermis became thin largely due to loss of collagen, the major structural protein in skin. Age-related thinning of the dermis increases fragility [9, 10], impaired vasculature support [11, 12], poor wound healing [3, 4], and altered tissue microenvironment that promotes cancer development [5, 6].

Anti-aging preventative approaches have been proposed for the purpose of rejuvenating aged human skin and preventing age-related skin diseases. However, safe and effective therapies to reverse the atrophy of aged skin do not exist currently. Topical retinoic acid has been shown to improve clinical features of aged-appearance by reduction in fine wrinkling, increased smoothness, and diminished hyperpigmentation [13]. However, the underlying cellular and molecular mechanisms are remaining to be fully elucidated. Moreover, in contrast to retinoic acid, there are limited studies describing the molecular basis of retinol (ROL, vitamin A), a precursor of retinoic acid, anti-aging effects in human skin in vivo. As human skin has the capacity to convert retinol to its biologically active metabolite retinoic acid, we investigated retinol anti-aging effects in aged human skin in vivo. Our results demonstrate that topical application of 0.4% ROL shows remarkable anti-aging effects in aged human skin through improvement of both epidermis and dermis and enhanced TGF-β/CTGF pathway. These data reveal molecular basis of retinol anti-aging effects and further suggest that topical retinol is a promising and safe treatment to improve naturally aged human skin.

Materials and methods

Procurement of human tissue samples and retinol treatment in human skin in vivo

All procedures involving human volunteers were approved by the University of Michigan Institutional Review Board, and all volunteers provided written informed consent. Full thickness punch biopsies (4 mm) were obtained from aged (76±6 years) healthy sun-protected buttock skin after topical treatment of vehicle (70% ethanal, 30% polyethylene glycol, and 0.05% butylated hydroxytoluene) and retinol (ROL, vitamin A, 0.4%) [14]. Vehicle and retinol were applied topically to sun-protected buttock skin for 7days under occlusion to prevent loss and exposure to light.

Immunohistology

Immunohistology was performed as described previously [15]. Briefly, skin OCT-embedded cryo-sections (7μm) were fixed in paraformaldehyde. Subsequently, the slides were incubated for 1 hour at room temperature with normal control serum followed by incubation of primary antibodies against Ki 67, CD31, type I procollagen, fibronectin, TGF-b1, and CTGF/CCN2 (Santa Cruz Biotechnology, CA, USA). To verify antibody specificity, antibodies were incubated with antigenic peptides (Santa Cruz Biotechnology, CA, USA) for 30 minutes prior to addition to skin sections. All sections were lightly counterstained with hematoxylin, and were mounted with mounting media (Vector, Laboratories, CA, USA). The intensity of positive staining was quantified by computerized image analysis (Image-pro Plus software, version 4.1, Media Cybernetics, MD, USA).

RNA isolation and Northern blot analysis

Total RNA from human skin was extracted with a commercial kit (RNeasy Midi Kit, Qiagen, Chatsworth, CA) according to the manufacturer’s protocol. Samples of total RNA (30–50 μg) were resolved by 1.2% agarose electrophoresis, transferred to nylon membranes, and hybridized with TGF-β1 [16] and CCN2 CTGF/CCN2 [17] cDNA probes labeled with [32P]dCTP by random priming. Each blot was stripped and re-hybridized with 36B4 internal control gene transcript to monitor the sample load in each lane. The intensities of each band were quantified by STORM PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA) and normalized to the 36B4 gene transcript.

In situ hybridization

pCDNA3.1 (Invitrogen, Carlsbad, CA) plasmids containing human TGF-β1 and CTGF/CCN2 were linearized with NotI and BamHI. Digoxigenin-containing sense and antisense riboprobes were synthesized using SP6 and T7 ribonucleic polymerase. Frozen skin sections (5 μm) were mounted, fixed, treated, and hybridized as previously described [17]. The hybridization signals were detected immunohistochemically by alkaline phosphatase-conjugated antidigoxigenin antibody.

RNA isolation and quantitative real-time RT-PCR

Total RNA was extracted from human skin by using a commercial kit (RNeasy mini kit, Qiagen, Chatsworth, CA, USA), as previously described [18]. 200ng total RNA was reverse transcribed using Taqman Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Real-time RT-PCR was performed using a Taqman Universal PCR Master Mix kit (Applied Biosystems, Foster City, CA, USA) and 7700 Sequence Detector (Applied Biosystems, Foster City, CA, USA). All PCR primers and probes were purchased from Applied Biosystems (Assays-on-Demand™ Gene Expression Products, Applied Biosystems, Foster City, CA, USA). Target gene mRNA levels were normalized to the housekeeping gene 36B4 (a ribosomal protein used as an internal control for quantitation) levels as an internal control.

Western blot analysis

Whole cell proteins were extracted from human skin dermis. The epidermis was removed by cutting off epidermis at a depth of 1 mm using cryostat. Equal amounts of protein (~50μg/lane) were resolved by 6–12% gradient sodium dodecyl sulfate-polyacrylamide (SDS) gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were then blocked with PBST (0.1% Tween 20 in PBS) containing 5% nonfat milk for one hour at room temperature. Smad7 primary antibody was generously provided by Dr. Ten Dijke (Leiden University Medical Centrum, Leiden, Netherlands). Primary antibody was incubated for one hour at room temperature. The membranes were washed three times with PBST solution and incubated with rabbit secondary antibody for one hour at room temperature. After washing three times with PBST, the membranes were developed with ECF (Vistra ECF Western blotting system, GE Health Care, Piscataway, NJ, USA) following the manufacturer’s protocol. The membranes were scanned with a STORM PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA), the intensities of each band were quantified using ImageQuant software (GE Health Care, Piscataway, NJ, USA) and normalized to β-actin (control).

Atomic force microscopy (AFM) imaging

Human skin biopsies were embedded in OCT, and cryo-sections (10 μm) were attached onto the microscope cover glass (1.2 mm diameter, Fisher Scientific Co., Pittsburgh, PA). These AFM samples were allowed to air dry for at least 24 hours before imaging them to AFM analysis. Images were taken by Dimension Icon AFM system (Bruker-AXS, Santa Barbara, CA, USA) using a silicon AFM probe (PPP-BSI, force constant 0.01–0.5N/m, resonant frequency 12–45kHz, NANOSENSORS™, Switzerland). AFM was conducted at the Electron Microbeam Analysis Laboratory (EMAL), University of Michigan College of Engineering, and analyzed using Nanoscope Analysis software (Nanoscope Analysis v120R1sr3, Bruker-AXS, Santa Barbara, CA, USA).

Statistical analysis

Comparisons between groups were determined with the Student’s t-test. All p values are two-tailed, and considered significant when p<0.05.

Results

Topical ROL increases epidermal thickness and dermal vascularity by proliferation of epidermal keratinocytes and dermal endothelial cells, respectively, in aged human skin in vivo

As thin epidermis is a characteristic feature of aged human skin, we found that epidermal thickness was greatly improved following ROL treatment (Fig. 1A). Quantitative morphometric analyses revealed that epidermal thickness was increased 2.1-fold by ROL treatment (Fig. 1B). Consistent with thickened epidermis, keratinocyte proliferation, assessed by Ki67 immunostaining (Fig. 1C upper right panel), was increased 12-fold by ROL treatment (Fig. 1D). Interestingly, in addition to epidermal changes, we noticed topical ROL markedly increased the proliferation of dermal stromal cells (Fig. 1C lower right panel, Fig. 1E). No Ki67 positive cell was observed in vehicle-treated aged skin dermis (Fig. 1C lower left panel, Fig. 1E). Ki67 positive staining cells were particularly strong in papillary dermis. To confirm the identity of proliferating dermal stromal cells, we performed double-label immunofluorescence staining. As papillary dermis contains rich vascular networks, we found the majority of Ki67 positive cells were stained with CD31, a marker of endothelial cells (Fig. 1F). This result was further supported by increased prominence of blood vessels, confirmed by CD31 immunostaining (Fig. 1G). Quantitative analyses revealed that blood vessel formation was increased 3.8-fold in ROL-treated aged human skin (Fig. 1H). Together, these data indicate that topical ROL increases epidermal thickness and dermal angiogenesis by stimulating the proliferation of epidermal keratinocytes and dermal endothelial cells, respectively, in aged human skin in vivo.

Figure 1.

Figure 1

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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.

ROL improves dermal ECM microenvironment by stimulating the expression of type I collagen, fibronectin, and tropoelastin in aged human skin in vivo

As thin dermis is a prominent feature of aged skin dermis, we analyzed the expression of major ECM proteins by immunohistochemistry. Type I collagen is the precursor of mature type I collagen fibrils. Compared with vehicle treated aged skin, ROL treatment increased type I collagen staining, the major structural protein in skin (Fig. 2A, right panels). Type I procollagen positive staining was particularly strong in epidermal and dermal junction (Fig. 1A lower right panel). Quantitative analysis indicated that the amount of staining was increased 3-fold by topical ROL treatment (Fig. 2B). In addition to type I collagen, topical ROL markedly increased the expression of fibronectin (FN) (Fig. 2C, right panels) and tropoelastin (Fig. 2E, right panels). Quantification indicated that fibronectin (Fig. 2D) and elastin (Fig. 2F) staining were increased 2.2-fold and 4-fold, respectively. These data indicate that topical ROL improves dermal ECM microenvironment by stimulating the major ECM proteins in aged human skin in vivo.

Figure 2.

Figure 2

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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.

Elevated epidermal-specific c-Jun transcription factor by topical ROL in aged human skin in vivo

Next, we explored the potential mechanisms by which topical ROL stimulates keratinocytes proliferation in aged human skin. It has been reported that AP-1 transcription factor plays a major role in keratinocytes proliferation in response to growth factors, cytokines, and various stimuli [2]. As AP-1 complex largely comprised of c-Jun/c-fos, we examined the expression c-Jun and c-Fos in aged human skin after topical ROL treatment. We found that topical ROL markedly increased epidermal-specific c-Jun protein positive staining (Fig. 3A right panel) along with significant epidermal thickness. No c-Jun positive cell was observed in the dermis. Quantitative analyses revealed that c-Jun positive staining was increased 2.3-fold in ROL-treated aged human skin (Fig. 3A right panel). In contrast, no change of c-Fos protein expression was observed by topical ROL treatment (Fig. 3B). Western analysis further confirmed that while c-fos was constitutively expressed (Fig. 3D), c-Jun protein was elevated 3-fold (Fig. 3C) by topical ROL treatment. These data suggest that topical ROL elevates epidermal-specific c-Jun transcription factor, which in turn drives epidermal keratinocytes proliferation in aged human skin in vivo.

Figure 3.

Figure 3

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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.

Elevated TGF-β/CTGF pathway by topical ROL in aged human skin in vivo

Next, we explored the potential mechanisms by which ROL stimulates dermal ECM production in aged human skin. We first examined whether topical ROL is able to activate dermal fibroblasts, the major ECM producing cells in skin, by staining with HSP47, the marker of fibroblast activation [19]. We observed that topical ROL markedly increased HSP47 positive staining, suggesting the activation of dermal fibroblasts in aged human skin in vivo. (Fig 4A, right panel). Quantitative analyses revealed that HSP47 positive staining was increased 3.8-fold in ROL-treated aged human skin (Fig. 4A right panel).

Figure 4.

Figure 4

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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.

It has been well-documented that activated dermal fibroblasts increase ECM production through TGF-β pathway, the major regulator of ECM production [20]. Therefore, we investigated the possible involvement of TGF-β pathway in increased ECM production by topical ROL in aged human skin. To test this possibility, we examined the effect of topical ROL on TGF-β pathway components such as TGF-β receptors, ligands, and Smads proteins. Interestingly, topical ROL significantly upregulated TGF-β1 mRNA and down-regulated the inhibitory Smad7, while other TGF-β pathway components remained unchanged (Fig. 4B). To further confirm these results, we performed immunostaining of TGF-β1 and its downstream mediator connective tissue growth factor (CTGF). In situ hybridization indicated that intense staining of TGF-β1 (Fig. 4C) and CTGF/CCN2 (Fig. 4D). Quantification indicated that TGF-β1 (Fig. 4C right panel) and CTGF (Fig. 4D right panel) dermal staining were increased 3.1-fold and 2.8-fold, respectively, following topical ROL treatment. Northern analysis further confirmed that TGF-β1 (Fig. 4E) and CTGF/CCN2 (Fig 4F) transcription were increased 2.6-fold and 3.8-fold, respectively, after topical ROL treatment. Consistent with mRNA expression, immunostaining of TGF-β1 (Fig. 4G) and CTGF/CCN2 (Fig. 4H) were increased 2.7-fold and 2.8-fold, respectively, after topical ROL treatment. We further confirmed that ROL treatment significantly reduced Smad7 mRNA (Fig. 4I) and protein (Fig. 4J), a potent inhibitor of TGF-β signaling. These data suggest that topical ROL stimulates dermal fibroblasts ECM production through upregulation of TGF-β/CTGF pathway in aged human skin.

Topical ROL increases the deposition of mature collagen in aged human skin in vivo

Having found that ROL is able to stimulate collagen production, we next assessed the collagen fibrils nanoscale structure and surface topology by atomic force microscopy (AFM). In vehicle-treated aged skin, collagen fibrils appeared disorganized and fragmented (Fig. 5A, left panel). However, topical ROL-treated aged skin revealed highly organized, dense bundles of collagen fibrils, with characteristic banded structure (D-spacing) representing the staggered alignment of individual collagen molecules within fibrils (Fig. 5A, right panel). Three-dimensional topographical AFM images further indicated that vehicle-treated aged skin dermis was much rougher and uneven (Fig. 5B, left panel). In contract, ROL-treated aged skin dermis was smooth and flattened (Fig. 5B, right panel). Quantitative analysis of AFM data indicated that the average roughness (a measure of fibril organization) of dermal collagen fibrils in ROL-treated aged skin is 48% less than in vehicle-treated aged skin dermis (Fig. 5B, 16 nm vs. 31 nm). These findings indicate that ROL treatment stimulates synthesis of procollagen, which is processed into mature collagen in aged human skin.

Figure 5.

Figure 5

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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).

Discussion

Retinoids is the term used for the group of vitamin A derivatives [21]. Retinoic acid is the biologically active form of vitamin A which is also known as retinol (ROL), a precursor to retinoic acid. Human skin has the capacity to convert ROL to its biologically active metabolite retinoic acid. When topically applied to human skin, it penetrates and is sequentially converted to retinaldehyde and then to retinoic acid. One important benefit of topical ROL is that compared to retinoic acid, ROL induces fewer signs of side effects, which is characterized by erythema, scaling, dryness, and pruritis [22, 23]. Our results indicate that although a much higher concentration of ROL (0.4%) is needed to achieve similar results as seen with topical retinoic acid, ROL induced the same histological changes (epidermal hyperplasia and dermal ECM production) as retinoic acid without causing retinoids irritation, the most common side effect of retinoids. Mechanistically, it appears that topical ROL increases epidermal thickness by stimulating keratinocytes proliferation, which involves epidermal-specific upregulation of c-Jun transcription factor. Topical ROL also significantly elevates ECM production, which involves enhanced TGF-β/CTGF pathway in the dermis. Additionally, topical ROL significantly increased dermal vascularity which may have a significant impact on the homeostasis of epidermis and dermis.

Although topical retinoids have been widely used in the treatment of skin aging, the precise mechanism(s) by which retinoids lead to visible improvement in aged skin is not fully understood. One significant finding of our study is that topical ROL improves aged dermal ECM microenvironment through enhancement of TGF-β/CTGF pathway in the dermis, the principle regulator of ECM homeostasis [20]. ROL is able to enhance TGF-β/CTGF pathway through two independent pathways: increases of the expression of TGF-β1/CTGF and suppression of inhibitory Smad7 of TGF-β signaling (Fig. 3). In primary human skin fibroblasts, the major ECM-producing cells in skin, the expression of major ECM proteins is regulated by TGF-β/CTGF pathway [7, 20]. For example, neutralization of endogenous TGF-β or knockdown of CTGF substantially reduced the expression of type I procollagen and fibronectin mRNA and protein. In contrast, overexpression of inhibitory Smad7, which is significantly reduced by topical ROL (Fig. 3B, I, J), abolished TGF-β or CTGF stimulation of ECM production. Importantly, we have previously reported that TGF-β/CTGF pathway is significantly reduced in dermal fibroblasts, in aged (80+ years) human skin in vivo [7, 20, 24, 25]. This impaired TGF-β/CTGF pathway in aged human skin is largely caused by reduced expression of TGF-β1 and CTGF. As impaired TGF-β/CTGF pathway significantly contributes to thinned dermis, our data suggest that the ability of topical ROL to improve aged skin dermis involves enhanced TGF-β/CTGF pathway.

Reduced vasculature and epidermal thinning contribute significantly to skin fragility and impaired wound healing in aged skin [26, 27]. AP-1 transcription factor plays a major role in keratinocytes proliferation [13]. We found that while c-Fos is constitutively expressed in human skin, topical ROL elevates epidermal-specific c-Jun transcription factor. These data suggest that topical ROL may stimulate keratinocytes proliferation by epidermal-specific upregulation c-Jun transcription factor in aged human skin. Our study added another new piece of information that topical ROL improves dermal microenvironment through not only stimulation of ECM production, but also expansion of vasculature by proliferation of endothelial cells in aged human skin (Fig. 1C, F, G). A reduction of the cutaneous vasculature has been reported in aged human skin [27]. Increased vascularity of the dermis by topical ROL could increase skin blood flow and provide a more suitable microenvironment for the homeostasis of skin epidermal and dermal homeostasis. In contrast, it is also conceivable that proliferation of epidermal keratinocytes and restoration of ECM production by topical ROL may provide a more favorable microenvironment for the proliferation of endothelial cells and dermal vasculature. Indeed epidermal keratinocytes are important source of vascular endothelial growth factor (VGEF), a potent angiogenic factor [27]. Additionally, it has been reported that enhanced dermal ECM production resulted in stimulation of endothelial cell proliferation [19]. Clearly, additional studies are warranted to uncover the precise molecular mechanism(s) by which topical ROL stimulates the proliferation of epidermal keratinocytes and dermal endothelial cells in aged human skin. The molecular mechanisms by which retinoids improve aged human skin have been difficult to investigate largely due to lack of appropriate in vitro models. For example, cells in both the epidermis and dermis contain all the proteins and receptors that mediate the biological effects of vitamin A metabolites in the skin. However, primary keratinocytes or dermal fibroblasts are minimally responsive to RA treatment in monolayer culture [14, 28]. This lack of responsiveness is due, at least in part, to low levels of nuclear retinoid receptors, which mediate expression of retinoid-regulated genes [29, 30]. Skin equivalent cultures could be a useful model to investigate retinoids regulation of collagen homeostasis [14]. Skin equivalent cultures are composed of stratified, differentiated keratinocytes (model epidermis) on top of a type I collagen lattice, in which dermal fibroblasts are embedded (model dermis). Treatment of skin equivalent cultures with retinoic acid significantly increased the number of keratinocyte layers and dermal response, similar to the effect of topical application of RA to human skin in vivo. These data suggest that skin equivalent cultures could be a useful model for investigating mechanisms by which retinoids improve aged-appearing skin in human.

Together, our findings extend current knowledge of retinoids anti-aging mechanisms in aged human skin. We propose a working model for the molecular basis of retinol anti-aging properties in aged human skin (Fig. 5C). Topical application of 0.4% ROL to aged human skin leads to remarkable skin changes in both epidermis and dermis through affecting three major types of skin cells, epidermal keratinocytes, dermal endothelial cells and fibroblasts. Topical ROL significantly increases epidermal thickness by stimulating epidermal keratinocytes proliferation, which involves c-Jun transcription factor, a major deriving force for keratinocyte proliferation. In addition to epidermal changes, topical ROL significantly improves dermal ECM microenvironment; increasing dermal blood vessel formation by stimulating endothelial cells proliferation and ECM production by activating fibroblasts. Topical ROL also stimulates TGF-β/CTGF pathway, the major regulator of ECM homeostasis, and thus increased the deposition of mature collagen in aged human skin in vivo.

Increased dermal vasculatures could increase skin blood flow and provide a more active microenvironment for both epidermal keratinocyte proliferation and dermal fibroblasts activation. On the other hand, the proliferation of epidermal keratinocytes may also promote dermal blood vessel growth through stimulation of VEGF expression [27]. Additionally, the restoration of dermal ECM may provide a better, more permissive environment for the proliferation of dermal endothelial cells and epidermal keratinocytes, and activation of dermal fibroblasts (TGF-β/CTGF pathway) [19]. We propose that coupling of the proliferation of keratinocytes and endothelial cells, and dermal fibroblasts activation forms a self-enforcing environment, which might explain the remarkable anti-aging effects of ROL in aged human skin. Proposed model extends current understanding of the retinol anti-aging mechanisms in human skin.

Acknowledgments

We thank Suzan Rehbine for the procurement of tissue specimens and Diane Fiolek for graphic and administrative assistance. We thank Electron Microbeam Analysis Laboratory (EMAL), University of Michigan College of Engineering for AFM. This work was supported by the National Institute of Health (AG019364 to GJ Fisher and T Quan; ES014697 to T Quan), Dermatology Foundation Research grant (to T Quan), and a grant from the Johnson &Johnson Corporation.

Abbreviations

ROL

retinol

ECM

extracellular matrix

AFM

atomic force microscopy

FN

fibronectin

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