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Photodermatol Photoimmunol Photomed 2000; 16: 239244 Printed in Denmark All rights reserved Munksgaard Copenhagen

Copyright C Munksgaard 2000

ISSN 0905-4383

Review article

Photoaging of human skin


M. Berneburg, H. Plettenberg, J. Krutmann
Clinical and Experimental Photodermatology, Dept. of Dermatology, Heinrich-Heine-University, Du sseldorf, Germany

Chronic sun exposure causes photoaging of human skin, a process that is characterized by clinical, histological and biochemical changes which differ from alterations in chronologically aged but sun-protected skin. Within recent years, substantial progress has been made in unraveling the underlying mechanisms of photoaging. Induction of matrix metalloproteinases as a consequence of activator protein (AP)-1 and nuclear factor (NF)-kB activation as well as mutations of mitochondrial DNA have been identied recently.

This has increased our understanding of photoaging signicantly and has led to new prophylactic and therapeutic strategies aimed at the prevention and repair of the detrimental effects of chronic sun-exposure on the skin.

Key words: antioxidants; mitochondrial DNA; photoaging; reactive oxygen species; repetitive sun exposure; retinoic acid; sunscreens; ultraviolet light.

he term photoaging describes distinct clinical, histological and functional features of chronically sunexposed skin. It has evolved from a variety of terms such as heliodermatosis, actinic dermatosis, and accelerated skin aging. Photoaged, chronically sun-exposed skin has characteristics in common with sun-protected, chronologically aged skin. However, there are features which are found exclusively in photoaged skin, making it an independent entity with its own pathophysiology. Extended life-span, more spare time and excessive exposure to ultraviolet (UV) radiation from natural sunlight or tanning devices, especially in the western population, has resulted in an ever increasing demand to protect human skin against the detrimental effects of UV-exposure of the skin to ultraviolet light. Therefore, photoaging will be of increasing concern in the future. The clinical and histological characteristics of photoaged skin have been known for some time (1); however, not until recently have the underlying molecular mechanisms responsible for the specic macro- and micro-

scopic alterations been discovered. The role of selected transcription factors (AP-1, NF-kB) in photoaging has been demonstrated and it has been found that mutations of mitochondrial DNA may also be involved. The elucidation of these pathophysiological mechanisms provides the basis for evaluating the efcacy of photo(aging)protective substances and might help in the development of new strategies which will provide protection and repair of photoaged human skin. Previous reviews on this topic have described the different aspects of photoaging (2 4). Hence, this review will only briey summarize the clinical and histological features of photoaged skin and then focus on recent ndings regarding the photobiological and molecular mechanisms responsible for photoaging of human skin.

Clinical features
Normally aged skin which has not been chronically exposed to sunlight is characterized by generalized wrinkling, dry and thin appearance, and seborrheic keratoses (1). Photoaged skin partly overlaps and superimposes these changes. However, changes induced by chronic sunexposure can occur well before signs of chronic skin aging. While there is wide interindividual variation with regards to clinical features of photoaged skin, depending mostly on factors such as skin type, nature of sun-exposure (occupational vs. recreational), hairstyle, dress and possible individual repair capacity, there are several common characteristics. These features occur strictly on sun-ex-

Abbreviations: EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; GAG, glycosaminoglycans; JNK, c-Jun amino terminal kinase; mt, mitochondrial; MAP, mitogen-activated protein; MMP, matrix metalloproteinase; MED, minimal erythema dose; NF-kB, Nuclear factor kB; nm, nanometer; OXPHOS, oxidative phosphorylation; RA, retinoic acid; ROS, reactive oxygen species; TIMP, tissue specic inhibitor of matrix metalloproteinases.

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, face, posed areas of the body such as the neck, decollete forearms and hands. They comprise leathery appearance, increased wrinkle formation, reduced recoil capacity, increased fragility of the skin with blister formation and impaired wound healing (4). Most of these attributes are caused by dermal changes. The most prominent epidermal changes are pigmentary alterations such as lentigines and diffuse hyperpigmentation (1).

Histological features
Photoaged skin has a variable but characteristic histological appearance, which differs quantitatively and qualitatively from sun-protected skin of the same individual. The stratum corneum of the epidermis may show hyperkeratosis but is usually normal. The epidermis can be hypertrophic, atrophic or unaltered. The thickness of the basal membrane is increased, possibly reecting damage to basal keratinocytes and the distribution of melanocytes along the basal membrane is irregular and these cells vary widely in size, dendricity and pigmentation (5, 6). In the dermis there is a vertical gradient of damage consistent with progressive attenuation of UV exposure. Depth and severity of dermal changes depend on the degree of acquired damage. The most prominent histological feature of photoaging is elastosis (1). Altered elastic bers

can span a varying portion of the dermal compartment. Elastosis generally begins at the junction of papillary and reticular dermis (7) and it is not observed in chronologically aged skin. Another prominent feature of photoaged skin is the replacement of mature collagen bers by collagen with a distinct basophilic appearance. This is called basophilic degeneration. Further changes characterizing photoaged skin include a large increase in deposition of glycosaminoglycans and fragmented elastic bers, (8, 9) as well as dermal extracellular matrix proteins such as elastin (1013), glycosaminoglycans (10, 14) and interstitial collagen (1517).

Photobiology
Which parts of the sunlight cause which feature of photoaging? UV light penetrates into the skin; depending on its wavelength, it interacts with different cells that are located at different depths (Fig. 1). UV light of the shorter wavelengths (UVB, 280320 nm) is mostly absorbed in the epidermis and predominantly affects epidermal cells, i.e. keratinocytes, while longer wavelength UV light (UVA 320400 nm) penetrates deeper and can interact with both epidermal keratinocytes and dermal broblasts. Melaninpigmentation of the skin absorbs UV light and thus protects skin cells from the detrimental effects of UV exposure. This provides a rationale of why individuals with darker skin exhibit clinical signs of photoaging at much later stages than fair-skinned people (1). Induction of skin pigmentation by oligonucleotides containing thymine dinucleotide (pTpT) sequence motifs has been shown to protect from skin cancer- and photoaging-related features (18, 19). Induction of skin pigmentation therefore may be one of the feasible strategies to protect skin from photoaging and will be discussed later in this review. Once UV light has reached the cells of the skin, the different wavelengths exert their specic effects. UVA light mostly acts indirectly through generation of reactive oxygen species (ROS), which subsequently can exert a multitude of effects such as lipid peroxidation, activation of transcription factors and generation of DNA-strand breaks. While UVB light can also generate ROS, its main mechanism of action is the direct interaction with DNA via induction of DNA damage.

Fig. 1. Photoaging of human skin: UVB light is mostly absorbed in the epidermis, primarily comprising keratinocytes. Transcription factors such as AP-1 and NF-kB are induced in the epidermis. These factors in turn then induce the expression of MMPs in a yet uncharacterized fashion. UVA light reaches into the dermis where it is absorbed by broblasts. UVA-induced generation of ROS leads to the expression of MMPs and induction of mutations of mtDNA.

Matrix-metalloproteinases
A wealth of evidence exists indicating that the induction of matrix metalloproteinases (MMP) play a major role in the pathogenesis of photoaging. While it has been demonstrated that UV light affects the post-translational modication of dermal matrix proteins such as collagen (20, 21) it has been known for some years that UV light also induces a wide variety of an ever increasing family of MMPs. These MMPs can be induced by both UVB and

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UVA light (2224). As indicated by their name, MMPs show proteolytic activity to degrade matrix proteins. Each MMP degrades different components of the dermal matrix proteins, for example, MMP-1 cleaves collagen type I, II, III and MMP-9, also called gelatinase, degrades collagen type IV, V and gelatin. The activity of MMPs is tightly regulated not only by transcriptional regulation. It has also been shown that tissue-specic inhibitors of MMPs (TIMP) exist that specically inactivate certain MMPs (4). Work by Fisher et al. (25, 26) indicated that activation of transcription factors might be responsible for MMP induction. Accordingly, UV exposure of human skin not only leads to the induction of MMPs but, within hours of UVB exposure, transcription factors AP-1 and NF-kB, which are known stimulatory factors of MMP genes (27, 28), are induced. It has been shown at the RNA and protein levels that in human skin, exposure to UVB light that was one tenth of the dose necessary for skin reddening (0.1 minimal erythema dose) induced the expression of AP-1 and NF-kB within minutes and the expression of MMPs within hours. Subsequent work by the same group (26) claried the pathway by which UV exposure leads to the degradation of matrix proteins in human skin. Low dose UVB irradiation activated MAP kinase pathways, involving the upregulation of epidermal growth factor (EGF) receptors, the GTP-binding regulatory protein p21Ras, extracellular signal-regulated kinase (ERK), c-jun amino terminal kinase (JNK) and p38. Elevated c-jun together with constitutively expressed c-fos increased activation of transcription factor AP-1. Thus, this elegant work not only unraveled the complex mechanistic pathways underlying the process of photoaging but also provided a rationale for the efcacy of retinoic acid (RA) which has previously been demonstrated in a multitude of trials (for reviews see 2931). In addition to activation of transcription factors, a second pathophysiological pathway leading to photoaging of human skin has recently been identied. This pathway is initiated by alterations at the level of mitochondrial DNA.

Mitochondrial DNA
Mitochondria are cell-organelles whose main function is to generate energy for the cell. This is achieved by a multistep process called oxidative phosphorylation (OXPHOS) or electron-transport-chain. Located at the inner mitochondrial membrane are ve multi-protein complexes which generate an electrochemical proton gradient used in the last step of the process to turn ADP and organophosphate into ATP. This process is not completely error free and ultimately this leads to the generation of ROS, making the mitochondrion the site of the highest ROS

turnover in the cell. In close proximity to this site lies the mitochondrions own genetic material, the mtDNA. The human mtDNA is a 16 559-bp-long, circular and doublestranded molecule of which four to ten copies exist per cell. Mitochondria do not contain any repair mechanism to remove bulky DNA lesions; although they do contain base excision repair mechanisms and repair mechanisms against oxidative damage (32), the mutation frequency of mtDNA is approximately 50-fold higher than nuclear DNA (33). Mutations of mtDNA have been found to play a causative role in degenerative diseases such as Alzheimers disease, chronic progressive external ophthalmoplegia and Kearns-Sayre syndrome. In addition to degenerative diseases, it has been found that mutations of mtDNA may play a causative role in the normal aging process with an accumulation of mtDNA mutations accompanied by a decline of mitochondrial function (34, 35). Recent evidence indicates that mtDNA mutations not only play a role in the normal aging process but that they may also be involved in the process of photoaging. Initial indications for a role of mtDNA in photoaging has come from several groups which have demonstrated that chronically sun-exposed skin showing clinical signs of photoaging has a higher mutation frequency of the mtDNA than sun-protected skin (3639). These studies found several large-scale deletions of mtDNA in photoaged skin. To explain the generation of these largescale deletions in mtDNA, a modied slip-replication mechanism and a central role of ROS have been postulated (3941). Recent work has been able to provide a possible link for the involvement of ROS in the generation of the most frequent mtDNA deletion, the so-called common deletion (42). Employing an in vitro model system, it has been possible to demonstrate that normal human broblasts when repetitively exposed for 3 weeks to sublethal doses of UVA light exhibit a time- and dose-dependent increase of the common deletion. In the same study, it was shown that this UVA-induced mtDNA mutagenesis is mediated by singlet oxygen. This not only provided a link between the proposed generation mechanism of largescale deletions and ROS but also further supported a possible role of mtDNA mutations in the process of photoaging. These in vitro studies have been extended in vivo (Plettenberg, Berneburg, Krutmann, unpublished results) where repetitive irradiation of normal human skin also led to the induction of the common deletion. In vitro, the common deletion disappears after the cells are no longer exposed to UV light, while in vivo, in human skin the common deletion in human skin could still be detected up to 4 months after cessation of the irradiation regimen. An initial indication as to whether these mutations are of functional relevance for photoaging has recently been given. Employing the in vitro test system described before,

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Fig. 2. Proposed pathophysiology of mitochondrial mutations: Exposure to UV light induces the generation of ROS, which in turn generate mutations of mtDNA. These mutations may (i) serve as a memory for damage inicted to cells and (ii) reduce the cells capacity to carry out OXPHOS. This process may in turn lead to the generation of more ROS.

it could be demonstrated that there is a close correlation between the existence of the common deletion and a decrease of mitochondrial function as well as expression of a metalloproteinase that is causally involved in photoaging. The appearance of the common deletion was paralleled by a reduction in cellular oxygen consumption and mitochondrial membrane potential (yD), which are markers for mitochondrial function. Most interestingly, there was also a close association between the induction of the metalloproteinase MMP-1 with the occurrence of the common deletion, while its tissue-specic inhibitor remained unaltered (Berneburg, Plettenberg, Krutmann, unpublished results). These changes of photoaged skin may provide a memory-function for previously inicted UV damage and the reduction of the OXPHOS, which may lead to more ROS (Fig. 2). However, more studies are needed to strengthen the link between mtDNA mutations and the process of photoaging. Assessment of the underlying photobiological mechanisms has revealed that, similar to UVA-induced MMP-induction, the generation of mtDNA mutations is due to production of singlet oxygen. This indicates that substances with ROS-quenching potential may be employed to prevent photoaging of human skin. By inhibiting the translation of transiently damaging ROS effects into genetically imprinted mutations, quenchers may not only protect from short-term damage of UV but also prevent longterm effects of UV exposure.

Photoprotection
Understanding the underlying mechanisms of photoaging may provide strategies of protection and repair of these processes. As discussed above, production of melanin in

the skin is one of the most effective ways to protect against the sun. Work by Eller & Gilchrest indicated that oligonucleotides that contain thymine dinucleotides (pTpT) induce tanning of the skin (18). The presence of these pTpTs not only induced tanning of the skin but also provided protective effects against photocarcinogenesis and photoaging (19), providing a new and possibly powerful tool to improve the bodys own sun-protection. A well established way to protect skin against detrimental effects of sunlight is the application of organic and inorganic UV lters found in conventional sunscreen preparations. Newer formulas provide protection against UVB and UVA light and some even against infrared radiation. The efcacy of these substances has been demonstrated in a wide variety of studies and their protective effect against photocarcinogenesis and photoaging is widely accepted. However, there is controversy in the literature with regards to the effects that these substances have on the immune function of the skin, since protected skin can then be exposed much longer to sunlight without getting sunburned. A new protective strategy has emerged from our understanding that oxidative stress plays a major role in the induction of photoaging. A large number of antioxidants have been found to exhibit protective effects against the different ROS involved in photoaging (4346). The data suggesting these protective effects against ROS-induced photoaging derives mainly from in vitro studies. Although the above-mentioned substances are already commercially available, in order to prove their efcacy, in vivo studies are needed that provide reproducible data in human skin. As described earlier, new model systems are emerging that make these studies feasible and that allow the investigation of the different pathophysiological endpoints such as induction of MMP, transcription factors and mitochondrial DNA. Improvement of the bodys endogenous pigmentation and the application of exogenous sun-protectants are merely prophylactic tactics. Improving the repair of already existing damage would complete a strategy to decrease detrimental effects of sun exposure. A large body of data exists demonstrating that a derivative of vitamin A, all-trans retinoic acid, exibits such properties. In vitro and in vivo studies have recently demonstrated that all-trans retinoic acid, which is a known transrepressor of the photoaging-involved transcription factor AP-1, when applied before UVB irradiation substantially abrogated the induction of AP-1 and MMPs. This abrogation was achieved in a posttranscriptional mechanism in which RA antagonized AP-1 activation by inhibition of c-jun protein induction (26). Subsequent work by the same group indicated that ultraviolet radiation causes a functional vitamin A deciency and that

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this deciency could be overcome by pretreating the skin with RA. Thus, this work not only provided a mechanistical model for the process of photoaging but also a rationale for the efcacy of RA in the repair of photoaged skin. Another strategy for photoprotection is to repair existing photodamage. Progress in this area may come from the eld of photocarcinogenesis. Studies employing a liposome-encapsulated repair enzyme called photolyase, derived from the algae Anacystis nidulans, demonstrated that when applied to human skin, photolyase reached the lower levels of the skin and removed DNA damage in the cells (47). Furthermore, removal of the pre-existing DNA damage led to physiological effects. Previous studies had demonstrated that immunosuppression of UV-irradiated skin is caused by generation of DNA damage in immune cells of the skin. In a recent study, application of the repair enzyme photolyase restored the skins immune responsiveness; this was shown to be due to the removal of DNA damage (48). Since photocarcinogenesis and photoaging have features in common, it is tempting to speculate that removal of DNA damage in skin cells may not only protect against skin cancer but also prevent photoaging. Overall, within recent years several promising strategies have emerged that may allow us, in spite of increasing sun exposure of the population, to protect and repair the alterations associated with photoaging of our skin.

Conclusions
Our understanding of the complex process of photoaging has increased signicantly in recent years. Elucidating the underlying mechanisms involved in photoaging is of paramount importance for the design of specic effective therapeutic and protective strategies for the improvement of public health.

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Acknowledgments This work has been supported in part by the BMBF (07UVB C5/7) and the European Commission (QRCT-199901590). Accepted for publication May 17, 2000 Corresponding author: J. Krutmann Clinical and Experimental Photodermatology Dept. of Dermatology Heinrich-Heine-University Moorenstr.5 40225 Du sseldorf Germany Tel.: 49 211 811 7627 Fax.: 49 211 811 8830 e-mail: krutmann/rz.uni-duesseldorf.de

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