The role of retinoids in the prevention and repair of aged and photoaged skin

Retinoids, either naturally occurring or synthetic, are defined by their ability to bind nuclear retinoid receptors of the steroid/thyroid superfamily. Their protean but key function in physiology is control of cellular proliferation and differentiation. Topical retinoids, namely tretinoin, have been proven to prevent and repair clinical features of photoageing; these processes are facilitated by an ability to prevent loss of collagen from, and stimulate new collagen formation in, the papillary dermis of sun-exposed skin. Emerging evidence indicates that intrinsic, chronological ageing of the skin shares several mechanistic features with photoageing. Indeed aged skin is characterized by retinoid sensitivity and is probably reparable by application of topical retinoids.     

Decreased retinoid concentration and retinoid signalling pathways in human atopic dermatitis

Atopic dermatitis (AD) is one of the most common skin diseases. Various features present in AD like inflammation, reduced apoptosis, altered epidermal differentiation and hyperproliferation as well as permeability dysfunction are also regulated by retinoids. The aim of our study is to identify the retinoid signalling pathways and retinoid concentration profiles in AD skin. Human skin biopsies were obtained from healthy volunteers (HS) (n=6) and patients with AD (n=6), with both affected (AS) and non-affected (NAS) skin. The gene expression of retinoid receptors, retinoid-binding proteins and retinoid-metabolizing enzymes was investigated by QRT-PCR. Retinoid concentrations in serum and skin were measured via high performance liquid chromatography mass spectrometry-mass spectrometry. Our results show that the target gene expression of retinoid receptor regulated pathways is significantly decreased in AS and NAS of patients with AD. CYP26A1, transglutaminase 2 and retinoic acid receptor responder 1 decreased in NAS and AS in comparison with HS. The main retinoic acid synthesizing enzyme, retinal dehydrogenase 1, was significantly lower expressed in NAS (0.1%) and AS (1%) in patients with AD. Analysis of retinoid concentration in serum and skin showed comparable all-trans retinoic acid (ATRA) and retinol (ROL) concentrations from AD and healthy serum, but strongly reduced ATRA and ROL concentrations in affected and non-affected skin in comparison with healthy skin. Our data indicate that retinoid transport, synthesis, concentrations and signalling are strongly decreased in the affected but also in non-affected skin of patients with AD suggesting a general intrinsic influence on skin retinoid signalling pathway in patients with AD.     

Qualitative and quantitative analysis of cytosol retinoid binding proteins in human skin

The distribution of the cytosol retinol and retinoic acid binding proteins are known to vary greatly within the different layers of the eye, a retinoid target organ. We have analyzed the cytosol retinoid binding from adult human skin, another retinoid target organ, and examined the relative contribution of the epidermis and dermis to the total retinoid binding. The mean specific activity of [3H]retinol (0.52 +/- 0.06 pmol/mg protein) and [3H]retinoic acid (3.20 +/- 0.45 pmol/mg protein) binding to cytosol preparations from different specimens of adult human skin was determined. On the average these skins bound 7-fold more retinoic acid than retinol. When skin was treated with EDTA and separated into epidermal and dermal fractions, [3H]retinol and [3H]retinoic acid binding was found in the cytosol derived from epidermis (0.36 +/- 0.03 pmol/mg protein, 3.69 +/- 0.13 pmol/mg protein, respectively) but not from dermis. To confirm that the absence of dermal binding was not due to loss during the EDTA separation, we assayed skin keratomed at 0.1, 0.2, and 0.3 mm. The skin obtained at 0.1 mm was upper epidermis and exhibited binding for both retinol and retinoid acid. The 0.2 mm skin, which added lower epidermis but little dermal contamination, had higher specific activities for both retinol and retinoic acid binding. The 0.3 mm skin which added primarily dermis, had lower specific activities for binding both retinoids. This is consistent with the concept that the epidermis is responsible for the majority of retinoid binding in adult human skin obtained from the lower limb.     

Quantitative evaluation of chronological ageing and photoageing in vivo: studies on skin echogenicity and thickness

Skin ageing is divided into chronological ageing and photoageing due to the cumulative effects of solar ultraviolet radiation. It is, however, difficult to measure the degree of photoageing and chronological ageing in humans in vivo . Here, we have evaluated the usefulness of ultrasonography for measurement of chronological ageing and photoageing in vivo . Twenty megahertz ultrasonography was performed in 90 individuals (29 men, 61 women, age 18–94) to describe age-related changes in sun-exposed regions with different levels of sun exposure (dorsal and ventral forearm, forehead, ankle) and non-exposed buttock skin. Skin thickness and skin echogenicity in different layers of the dermis were measured in ultrasound images. Additionally, cutaneous photodamage was scored clinically. Age-related changes were dependent on body site as well as layer of the dermis. A progressive, age-related decrease in echogenicity of the upper dermis was found in sun-exposed regions (dorsal forearm, forehead), but not in moderately exposed regions (ventral forearm, ankle). In the buttock an increase in echogenicity was observed. The echogenicity of the lower dermis increased in all examined sites. Skin thickness increased with age in the forehead and buttock, but decreased in the extremity skin. Our findings show that photoageing causes a decrease in echogenicity in the upper dermis. In contrast, chronological ageing is associated with an increase in echogenicity in the lower dermis. Although both increases and decreases in skin thickness were observed in different anatomical regions, there was no general relationship between skin thickness and age. Dermal echogenicity was deemed valuable for in vivo study of chronological ageing and photoageing.     

The role of specific retinoid receptors in sebocyte growth and differentiation in culture

Retinoic acid derivatives (retinoids) exert their pleiotropic effects on cell development through specific nuclear receptors, the retinoic acid receptors and retinoid X receptors. Despite recent progress in understanding the cellular and molecular mechanisms of retinoid activity, it is unknown which of the retinoid receptor pathways are involved in the specific processes of sebocyte growth and development. In this study, we investigated the roles of specific retinoid receptors in sebocyte growth and differentiation, by testing the effects of selective retinoic acid receptor and retinoid X receptor ligands at concentrations between 10-10 M and 10-6 M in a primary rat preputial cell monolayer culture system. Cell growth was determined by number of cells and colonies, and cell differentiation by analysis of lipid-forming colonies. All-trans retinoic acid and selective retinoic acid receptor agonists (CD271 = adapalene, an RAR-beta,gamma agonist; CD2043 = retinoic acid receptor pan-agonist; and CD336 = Am580, an RAR-alpha agonist) caused significant decreases in numbers of cells, colonies, and lipid-forming colonies, but with an exception at high doses of all-trans retinoic acid (10-6 M), with which only a small number of colonies grew but they became twice as differentiated as controls (42.2 +/- 4.0% vs 22.6 +/- 2.7%, mean +/- SEM, lipid-forming colonies, p < 0.01). Furthermore, the RAR-beta,gamma antagonist CD2665 antagonized the suppressive effects of all-trans retinoic acid, adapalene, and CD2043 on both cell growth and differentiation. In contrast, the retinoid X receptor agonist CD2809 increased cell growth slightly and lipid-forming colonies dramatically in a clear dose-related manner to a maximum of 73.7% +/- 6.7% at 10-6 M (p < 0. 001). Our data suggest that retinoic acid receptors and retinoid X receptors differ in their roles in sebocyte growth and differentiation: (i) retinoic acid receptors, especially the beta and/or gamma subtypes, mediate both the antiproliferative and antidifferentiative effects of retinoids; (ii) retinoid X receptors mediate prominent differentiative and weak proliferative effects; (iii) the antiproliferative and antidifferentiative effects of all-trans retinoic acid are probably mediated by retinoic acid receptors, whereas its differentiative effect at high dose may be mediated by retinoid X receptors via all-trans retinoic acid metabolism to 9-cis retinoic acid, the natural ligand of retinoid X receptors.     

Cellular, immunologic and biochemical characterization of topical retinoic acid-treated human skin.

Histologic and clinical improvement of sun-exposed skin following topical treatment with retinoic acid has been reported. Daily application of retinoic acid typically results within 2-5 d in an erythematous scaling reaction, which lessens with continued usage. The cellular, immunologic, and biochemical basis of this retinoid reaction and its role in the repair of photodamaged skin are not known. To investigate the retinoid reaction in man, we have treated non-sun-exposed skin with 0.1% retinoic acid cream (Retin-A, Ortho Pharmaceutical Corporation, Raritan, NJ) under occlusion for 4 d to induce erythema and then examined changes in 1) histology, 2) expression of cell-surface molecules, 3) the enzymes and second messengers of the phospholipase C/protein kinase C signal-transduction system, 4) levels of eicosanoids, and 5) levels of interleukin-1 protein and mRNA. These parameters were chosen for measurement both because they are indicators of epidermal function and previous studies suggest they may be responsive to retinoic acid treatment. Epidermal cell growth as judged by increased epidermal thickness and mitotic figures was significantly increased in retinoic acid-treated skin compared to vehicle-treated controls. Increased numbers of CD4+ T cells accompanied by prominence of dermal dendrocytes in the papillary dermis and focal keratinocyte expression of intercellular adhesion molecule-1 were observed in retinoic acid-treated biopsies. Phosphoinositide-specific phospholipase C activity and 1,2-diacylglycerol content were also elevated in retinoic acid-treated epidermis. Protein kinase C activity was reduced by one third in both the soluble and membrane fraction, suggesting down-regulation. Surprisingly, in view of the inflammatory nature of the retinoid reaction, no increases were observed in arachidonic acid, its metabolites, interleukin-1 alpha, or interleukin-1 beta. To examine the specificity of the retinoid reaction, subjects were treated with the irritant sodium lauryl sulfate, under conditions that resulted in a reaction clinically similar to that observed with retinoic acid. The histologic alterations induced by sodium lauryl sulfate were found to be indistinguishable from those induced by retinoic acid. These data indicate that, although a wide range of cellular and molecular alterations occur in retinoic acid-treated skin, these changes may not be necessarily specific or unique for retinoic acid.     

Developments in topical retinoid therapy for acne

Topical retinoic acid was introduced for acne treatment three decades ago. Since that time, researchers have discovered thousands of retinoids, originally defined as chemical analogs of vitamin A. After the identification of nuclear retinoid receptors in 1987, the definition of this class expanded to include molecules that bind to and activate such receptors. The receptor-selective retinoid agents, adapalene and tazarotene, were developed in the 1990s. Other innovations of the past decade include retinoid formulations and methods aimed at limiting retinoid absorption. Cutaneous irritation may be reduced without losing retinoid efficacy by inhibiting retinoid penetration into the deep epidermis and dermis. Examples include tretinoin in slow-release vehicles and the short-contact method of tazarotene gel therapy. Only trace amounts of adapalene are absorbed after topical application, perhaps explaining its relatively low irritancy. New formulations of existing agents, such as additional concentrations of tretinoin in microsphere gel and cream formulations of tazarotene, are now under investigation for acne. Current research focused on receptor selectivity holds the promise of yielding new retinoid molecules with improved benefits and safety.     

Chronic sun exposure alters both the content and distribution of dermal glycosaminoglycans

Summary Chronic sun exposure leads to structural and functional alterations in exposed skin. Photoageing is a process distinct from the changes taking place due to chronological ageing. Unique alterations in the dermal extracellular matrix occur as a result of photoageing and are responsible for many of these physiological changes taking place in sun-damaged skin. Accompanying the deposition of abnormal elastic tissue, or solar elastosis, are significant alterations in dermal glycosaminoglycans (GAGs). Accumulation of GAGs as a result of photoageing as demonstrated in both humans and animal models of photoageing seems almost paradoxical in view of the large amounts of GAGs present in the skin of newborns, making their skin well hydrated and supple, in sharp contrast to the weathered appearance of photoaged skin. We investigate the relative GAG content of photoaged skin using immunoperoxidase stains specific for hyaluronic acid and chondroitin sulphate, and determine the location of these GAGs using confocal laser scanning microscopy. Our results demonstrate significant increases in GAG staining in sun-damaged vs. sun-protected skin from the same individuals, as measured by computer-based image analysis. Furthermore, confocal laser scanning microscopy reveals that the increased dermal GAGs in sun-damaged skin are deposited on the elastotic material of the superficial dermis of photodamaged skin, and not between collagen and elastic fibres as in normal skin. The abnormal location of GAGs on these fibres may explain the apparent paradoxical weathered appearance of photodamaged skin despite increased GAGs.     

In vivo assessment of chronological ageing and photoageing in forearm skin using reflectance confocal microscopy

Background Skin ageing is a complex process due to intrinsic chronological factors (chronoageing) and extrinsic environmental factors. The primary extrinsic factor is cumulative ultraviolet (UV) exposure, and is therefore termed photoageing. The current standards for measuring cumulative sun damage are biopsy histology and skin microtopography. However, skin biopsies are too invasive for population studies and skin replicas render only superficial skin architecture data. Reflectance confocal microscopy (RCM) is a noninvasive imaging tool that allows for in vivo imaging of the skin at quasihistological resolution. Objectives To define and identify RCM features associated with chronological ageing and photoageing on the forearm in two age groups with different skin phototypes and to assess whether these results agree with previous findings. Methods We obtained RCM images of dorsal and volar nonlesional skin of the lower forearm of 75 individuals with skin Fitzpatrick phototypes I–III in two age groups (20–30 years and 50–60 years). From each participant and body site, 21 RCM features were assessed and statistically significant differences between the two age groups and different forearm sites determined. Results RCM enabled identification of changes in architecture, cell morphology and extracellular matrix (collagen) at the level of the epidermis, dermoepidermal junction and papillary dermis. Changes that were correlated with chronological ageing and which were aggravated on the UV‐exposed dorsal forearm were: loss of small skin furrows resulting in wider and less intersecting furrows; irregularity of the epidermal honeycomb pattern; irregularly distributed (mottled) pigmented keratinocytes/melanocytes; irregularity of the papillary rings and/or effacement of the rete ridges; and loss of thin collagen fibres and presence of collagen clods. Conclusion We have tested previously reported and new parameters for skin ageing evaluation by RCM, and identified 15 statistically significant RCM features that can be used to quantify ageing and photoageing in forearm skin noninvasively.     

Effects of ageing on dermal echogenicity

Background/aims: Changes in the dermis associated with ageing can be detected by high-frequency skin ultrasonography. In photoaged skin, this technique shows a subepidermal low echogenic band (SLEB) that is probably an ultrasound manifestation of elastosis and oedema in the papillary dermis. Since some authors found an association between age and SLEB thickness or its echogenicity on exposed sites, it has been proposed to use these parameters to quantify skin photoageing.
Methods: To determine whether SLEB can be used as a quantitative marker of ageing, its prevalence was determined on forearm skin in a group of 55 individuals (age 18–57 years). The size of SLEB has been measured by quantifying the number of low echogenic pixels in the subepidermal area, which is an accurate method for assessing SLEB severity.
Results: The prevalence of SLEB increased with age, but SLEB was also present in young subjects. The echogenicity of the subepidermal area did not show any age dependence. However, when a ratio of echogenicity between upper and lower dermis was calculated, a linear dependence on age was found.
Conclusions: This study indicates that skin echogenicity measured as a ratio between the upper and lower dermis may be used to objectively estimate photoageing.     

Extrinsic ageing in the human skin is associated with alterations in the expression of hyaluronic acid and its metabolizing enzymes

Abstract: Extrinsic skin ageing or ‘photoageing’, as opposed to intrinsic skin ageing, is the result of exposure to external factors, mainly ultraviolet irradiation. Glycosaminoglycans (GAG) and particularly hyaluronic acid (HA) are major components of the cutaneous extracellular matrix involved in tissue repair. However, their involvement in extrinsic skin ageing remains elusive. In this study, we investigated the expression of HA and its metabolizing enzymes in photoexposed and photoprotected human skin tissue specimens, obtained from the same patient. Total GAG were isolated, characterized using specific GAG‐degrading enzymes and separated by electrophoresis on cellulose acetate membranes and polyacrylamide gels. Quantitation of HA in total GAG was performed using ELISA. Gene expression of hyaluronan synthases (HAS), hyaluronidases (HYAL) and HA receptors CD44 and receptor for HA‐mediated motility (RHAMM) was assessed by RT‐PCR. We detected a significant increase in the expression of HA, of lower molecular mass, in photoexposed skin as compared with photoprotected skin. This increase was associated with a significant decrease in the expression of HAS1 and an increase in the expression of HYAL1‐3. Furthermore, the expression of HA receptors CD44 and RHAMM was significantly downregulated in photoexposed as compared with photoprotected skin. These findings indicate that extrinsic skin ageing is characterized by distinct homoeostasis of HA. The elucidation of the role of HA homoeostasis in extrinsic skin ageing may offer an additional approach in handling cutaneous ageing.     

Human in vivo pharmacology of topical retinoids

All-trans retinoic acid is used topically for treating a variety of dermatologic conditions ranging from acne to photoaged skin. Although the clinical effects of retinoic acid treatment are often considerable, relatively little is known about the basic mechanisms underlying such effects. With the development of an in vivo human assay we have investigated the pleiotypic effects of topical retinoids from the histologic to the molecular. Histologically, retinoic acid induces epidermal proliferation and differentiation coupled with dermal fibroblast production of collagen. Immunologic effects include stimulation of the antigen-presenting capacity of Langerhans cells and induction of keratinocyte ICAM-1 expression. At the biochemical level, retinoic acid regulates transglutaminase and tyrosinase activities and activates protein kinase C. Both polar metabolites and stereoisomers of all-trans retinoic acid are also biologically active. Molecular biologic techniques have revealed that elevation of mRNA for cellular retinoic acid binding protein II is a retinoid-related event and that nuclear receptors such as retinoic acid receptors and retinoid X-receptors may transduce the retinoid response.     

Are dark‐skinned people really protected from ultraviolet radiation?

Background. Premature ageing of the skin (photoageing) results from the action of ultraviolet radiation (UVR) on skin. One of the histopathological findings of photoageing is the presence of solar elastosis in the dermis. Skin pigmentation is protective against UVR. Aim. To evaluate the presence of solar elastosis in dark‐skinned people. Methods. Normal facial skin biopsies of 147 dark‐skinned and 140 light‐skinned people were examined histopathologically for solar elastosis. The degree of solar elastosis was graded on a five‐point scale by a panel of dermatopathologists blinded to patient demographics. Results. There were 112 of 140 (80%) light‐skinned and 50 of 147 (34%) dark‐skinned patients with high‐grade solar elastosis. In the dark‐skinned patient group, high‐grade solar elastosis was seen in 29 of 61 (47.5%) Hispanic and 5 of 49 (10.2%) African American subjects. Conclusions. Dark‐skinned people are not completely protected from the effects of UVR.     

Topical tretinoin or adapalene in acne vulgaris: an overview

Retinoids target several pathoetiologic events of acne vulgaris. The undisputed efficacy of tretinoin, and yet its underutilization, due to apprehension of retinoid dermatitis, triggered a search for newer, well-tolerated retinoids. The discovery of nuclear retinoic acid receptors has provided clues to a rational design of synthetic, receptor-selective retinoic acid agonists. Adapalene is an addition to the arsenal of topical retinoids. It possesses the biological properties of tretinoin, but has a distinct physiochemical profile, including high lipophilicity and increased chemical and photostability. It exhibits selective affinity for nuclear retinoic acid receptors and does not bind to cytosolic retinoic acid binding proteins. It exemplifies the formulation of a novel retinoid with specific pharmacologic profile and clinical objectives. Accordingly, numerous clinical trials have compared adapalene and tretinoin in the management of acne vulgaris and concluded that tretinoin 0.05% gel exhibits a greater anti-acne efficacy than adapalene 0.1% gel, but has higher skin irritation potential. This article reviews the pharmacology of adapalene, including its retinoid receptor binding profile, antiproliferative effects, cell differentiation modulation, comedolytic and anti-inflammatory activity, and specifically focuses on the comparison of the efficacy and irritation profile of adapalene and tretinoin.     

In vitro metabolism by human skin and fibroblasts of retinol, retinal and retinoic acid

The metabolism of radio-labelled retinol, retinal and retinoic acid by fresh human skin as well as by human dermal fibroblasts have been investigated in vitro . Surgically removed human skin biopsies were placed at the air-liquid interface, and treated topically for 24 h with retinoids. At the end of the treatment period, epidermis and dermis were separated by heat. Epidermis, dermis and medium were subsequently extracted and resulting fractions were analysed by HPLC. Dermal fibroblast cultures were treated and analysed in a comparable manner. Topical application of retinoids resulted in gradient concentrations within the skin. For each fraction, metabolites and unchanged product proportions were determined by HPLC. After treatment with retinol and retinal, low but significant amounts of retinoic acid were detected in the epidermis, as well as in the dermis (30 pmol to 90 pmol). In comparison, treatments with retinoic acid itself, led to higher level of retinoic acid in the epidermis and in the dermis (respectively 2050 and 420 pmol). Cultured human dermal fibroblasts, treated with retinol and retinal, formed retinoic acid as well as several other metabolites (retinol esters, reduction of retinal to retinol…). Taken together, our results are consistent with an action of retinol or retinal on the skin via a retinoic acid formation and a metabolic function of the dermis.     

Therapy with a synthetic retinoid--(Ro 10-1670) etretin--increases the cellular retinoic acid-binding protein in nonlesional psoriatic skin

Cellular retinol (CRBP)-and retinoic acid (CRABP)-binding proteins were determined in samples of lesional and nonlesional skin of psoriatic patients, before and during oral administration of a synthetic retinoid, Etretin (Ro 10-1670). A 200% increase in CRABP levels, measured by the ability of the protein to bind retinoic acid, was observed in the normal skin during treatment. The CRBP levels were not altered during therapy. The results show that CRBP and CRABP are independently regulated in human skin and suggest that synthetic retinoids may exert their pharmacologic effects by interfering with the regulation of natural retinoic acid receptors.