j eur acad dermatol venereol 2011

REVIEW ARTICLE

Skin ageing

E. Kohl,†,* J. Steinbauer,† M. Landthaler,† R.-M. Szeimies‡

†Department of Dermatology, Regensburg University Hospital, Regensburg, and ‡Department of Dermatology and Allergology,

Hospital Vest, Recklinghausen, Germany

*Correspondence: E. Kohl. E-mail: [email protected]

AbstractSimilar to the entire organism, skin is subject to an unpreventable intrinsic ageing process. Additionally, skin ageing

is also influenced by exogenous factors. Ultraviolet radiation in particular results in premature skin ageing, also

referred to as extrinsic skin ageing or photoageing, which is the main cause of the changes associated with the

ageing process in sun-exposed areas. Despite their morphological and pathophysiological differences, intrinsic and

extrinsic ageing share several molecular similarities. The formation of reactive oxygen species and the induction of

matrix metalloproteinases reflect the central aspects of skin ageing. Accumulation of fragmented collagen fibrils

prevents neocollagenesis and accounts for the further degradation of the extracellular matrix by means of positive

feedback regulation. The importance of extrinsic factors in skin ageing and the detection of its mechanisms have

furthered the development of various therapeutic and preventive strategies.

Received: 13 May 2010; Accepted: 14 December 2010

Keywordsextrinsic skin ageing, intrinsic skin ageing, matrix metalloproteinases, photoageing, retinoids, UV radiation

Conflict of interestNone declared.

IntroductionSkin ageing, a highly complex but not yet fully understood pro-

cess, is particularly interesting because of the continuously increas-

ing life expectancy in many countries. Several theories have been

developed to comprehend this progressive process. Ageing may be

considered as the accumulation of different deleterious changes in

cells and tissues. These changes may progressively impair biologi-

cal functions, increase the risk of developing diseases and ulti-

mately lead to death.1 Up to now, no integrative concept exists

connecting the ageing models evolved so far.

Intrinsic and extrinsic skin ageing

Pathomechanisms of intrinsic skin ageing

Intrinsically aged skin is usually found in sun-protected areas.

Photoageing may be considered a superposition of chronological

skin ageing by UV-radiation. Skin may serve as a model organ for

investigating both endogenous and exogenous ageing models.

Extrinsic and intrinsic skin ageing show similarities in molecular

mechanisms.

The following aspects are discussed in several theories on intrin-

sic skin ageing: Cellular ageing (Hayflick-Limit) and shortening of

telomeres, mutations of mitochondrial DNA, oxidative stress,

genetic mutations and decrease of several hormone levels.2

According to the free radical theory of ageing, reactive oxygen spe-

cies (ROS), primarily arising from oxidative cell metabolism, play

a major role in both chronological ageing and photoageing.3

Despite several antioxidative mechanisms, which deteriorate with

increasing age, abound ROS damage cellular components. This

damage leads to increasing ROS and decreasing antioxidative

capacities and finally to cellular ageing.2,4 ROS in extrinsic and

intrinsic skin ageing may be assumed to induce the transcription

factor c-Jun via MAPK (mitogen-activated protein kinases). This

induction activates the decisive transcription factor AP-1 (activa-

tor protein 1), leads to the expression of matrix metalloproteinases

MMP-1 (interstitial collagenase), MMP-3 (stromelysin 1), and

MMP-9 (gelatinase b) and prevents the expression of procollagen-

1.5 In accordance with these results, elevated levels of partially

degraded collagen are present in intrinsically aged skin similar to

photoaged skin. Recently, an in vivo study has indicated that

reduced expression of the connective tissue growth factor (CTGF)

and reduced transforming growth factor (TGF)-b ⁄ Smad signalling

are probably responsible for the loss of type I procollagen expres-

sion in intrinsically aged skin.6

Intrinsic skin ageing is strongly influenced by hormonal

changes.7 Production of sex hormones in the gonads, the pituitary,

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JEADV 2011, 25, 873–884 Journal of the European Academy of Dermatology and Venereology ª 2011 European Academy of Dermatology and Venereology

DOI: 10.1111/j.1468-3083.2010.03963.x JEADV

and adrenal glands already gradually declines in the mid-twenties.

Oestrogens and progesterone decay in line with the menopause. In

particular, the deficiency in oestrogens and androgens cause dry-

ness, wrinkling, epidermal atrophy, collagen breakdown and loss

of elasticity.8,9

Pathomechanisms of photoageing

Extrinsic skin ageing primarily arises from UV-light exposure.

Approximately 80% of facial skin ageing is attributed to UV-expo-

sure.10 Further relevant exogenous factors are exposure to tobacco

smoke, airborne particulate matter, infrared radiation, ozone and

malnutrition. Premature skin ageing or photoageing are synonyms

for extrinsic skin ageing.

The biological effects of UV-radiation are based on light absorp-

tion in chromophores and the subsequent conversion of energy in

chemical reactions. UVA ⁄ UVB-radiation contribute to biological

effects, resulting in skin ageing and photocarcinogenesis. The exact

action spectrum, i.e., the biological effects depending on wave-

lengths, remains unclear. Short wave UVB is mainly absorbed in

the epidermis, generating DNA-damage by forming photocarcino-

genic cyclobutane pyrimidine dimers (CPDs) and 6,4-photoprod-

ucts. UVA-light is absorbed by cellular chromophores, such as

urocanic acid, melanin precursors and riboflavin. These light-

exposed chromophores generate ROS, which damage lipids, pro-

teins and DNA. UVA-light is exceptionally relevant in photoageing

because of its high penetration depth.11

Mutations of mitochondrial DNA The main endogenous source

of ROS is mitochondria, and ROS are built from approximately

1% of the oxygen consumed in the mitochondria. ROS, in addi-

tion to their physiological role as signalling molecules, lead to oxi-

dative stress after exhaustion of cellular defence mechanisms.

Because of their proximity, ROS generated in the respiratory chain

are particularly important for mitochondrial (mt) DNA. The

human mtDNA consists of up to 10 copies of a double-stranded

circular DNA-molecule comprising 16 559 base pairs, which

encodes proteins of the respiratory chain. mtDNA shows consider-

ably higher mutation rates than nuclear DNA because of its ineffi-

cient recognition and repair mechanisms.12

Mutations of mitochondrial DNA are important for ageing

processes, photoageing and various neurological diseases. Photo-

aged skin shows more mutations in mtDNA than sun-protected

skin.13 The most frequent mitochondrial mutation is a large-scale

4977 base-pair deletion termed ‘common deletion’, whose induc-

tion is directly related to chronic UVA-exposure. In vivo repetitive

exposure of previously unirradiated skin to physiological doses of

UVA-light increased the level of common deletion in human skin

by 40%. The 4977 bp-deletions persisted and were detectable even

months after cessation of irradiation. The levels of common dele-

tion continued to increase and partly showed an up to 32-fold

accumulation 16 months after irradiation.14 These observations

are in accordance with the theory proposing that ROS-induced

mutations of mitochondrial DNA lead to dysfunctional oxidative

phosphorylation. Thus, more ROS are generated, causing even

more mutations.15 A causative relationship must be assumed for

the depletion of mitochondrial DNA, resulting in oxidative stress

and increased expression of MMP-1.16

Role of telomeres Telomeres consisting of 1000-fold tandem

repeats of TTAGGG in humans form the chromosome ends. Telo-

meres do not encode any gene products and constitute the last

7000–12 000 base pairs. Telomere length varies with species and

decreases with increasing age.17

Telomeres consist of a double-strand region composed of

TTAGGG repeats and a much shorter single-strand 3¢ overhang at

the distal end. Telomeres form a 3-dimensional structure (t-loop)

that is required for telomere capping. The t-loop is secured by

insertion of the 3¢ overhang into the proximal double-stranded

DNA. T-loops prevent the chromosome ends to be recognized as

double-strand breaks, defending them from instability as a fusion

of chromosomes. A complete replication of the final bases by

DNA-polymerase is not possible. For this reason, telomeres

shorten with each cell division by approximately 100 base pairs.18

Therefore, the number of cell divisions is confined to 50–70

because of critical telomere shortening.19

Critical shortening of telomeres induces disruption of the t-loop

configuration, exposing the 3¢ overhang. This process initiates

DNA-damage response, entailing to apoptosis, senescence or cell

cycle arrest.20

Besides shortening telomeres with each round of cell division,

UV-radiation or other DNA-damage may lead to loop disruption,

exposing the TTAGGG tandem repeat sequence. This process elic-

its the above-mentioned DNA-damage response, indicating the

possible existence of a common mechanism for intrinsic ageing

and photoageing.21

UV-exposure particularly damages telomeres because of their

higher number of TT- and G-bases compared with the other part

of the chromosome. UV-radiation particularly targets TT- and

G-bases, possibly destabilizing the t-loop configuration and acti-

vating the common signalling pathway.22

The impact of telomeres on the aetiopathology of photoageing

was also put into question.23 Telomere lengths in photoaged and

photoprotected skin did not differ; altogether, telomere length was

shorter in the epidermis than in the dermis.24 Expression of telo-

merase is maintained in early embryonic, malignant and germ

cells. Telomerase acts as a maintenance mechanism of telomere

length, synthesizing TTAGGG sequences.23 Contrary to most

somatic cells, expression of telomerase is maintained in the hema-

topoietic system and in the gastrointestinal tract. Evidence suggests

expression of telomerase in the keratinocytes of rapidly regenerat-

ing epidermis.25 The epidermis in situ showed a mean telomere

loss of 25 bp per year. A telomerase-based mechanism to maintain

telomere length in keratinocytes must be assumed because of

the high proliferation rate of keratinocytes in contrast to slowly

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874 Kohl et al.

proliferating fibroblasts with a reported telomere loss of 25 bp per

year. The evidence for functionally active telomerase in human

epidermis and the lack of considerable telomere loss of keratino-

cytes and fibroblasts suggest that telomere loss does not play a key

role in intrinsic skin ageing.23

Matrix metalloproteinases and signal transduction pathways As

a major structural protein of the extracellular matrix (ECM), type

I collagen is secreted by fibroblasts, forming more than 90% of the

dry weight of the dermis. Besides, MMPs are secreted, hydrolyzing

ECM proteins, such as collagen fibres. After exposure to subery-

thematous UVB-doses, sustained elevations of MMPs were

assessed.26 MMP-1 initiates cleavage of type I collagen and is thus

particularly important for degrading the ECM.

Additionally, UV-exposure involves considerably decreased col-

lagen production. Photoaged skin is designated with a collagenous

matrix fragmented by MMPs. UV-induced oxidative stress initi-

ates signalling cascades via the activation of several cell surface

receptors. This activation results in the degradation of the ECM

and in the down-regulation of neocollagenesis. Receptor activation

effects stimulation of MAP-kinases p38, JNK (c-Jun amino-termi-

nal kinase), and ERK (extracellular signal-regulated kinases), con-

sequently inducing the transcription factor AP-1. AP-1 induces

collagen degradation by up-regulation of MMP-1, MMP-3 and

MMP-9.26 In addition, procollagen synthesis is inhibited by the

UV-induced transcription factor AP-1. Increased transcription of

AP-1 inhibits the effects of TGF-b (transforming growth factor b),

a cytokine promoting collagen production.27 Extracellular matrix

degradation is further enhanced by UV-induced activation of the

transcription factor NF-jB. This factor stimulates the expression

of MMPs and several cytokines and enforces UV-response by

activating respective cell surface receptors.

UV-induced elevated levels of MMPs are not accompanied by

the compensatory augmented production of their physiological

inhibitors, the TIMPs (tissue inhibitor of matrix metalloproteinas-

es). Induction of MMPs may be effected by UVA and UVB.28

Expression of MMP-1, MMP-3 and MMP-9 occurs in both kerati-

nocytes and fibroblasts. In vivo studies have shown that MMPs in

UV-exposed skin (solar simulated UV irradiation) primarily derive

from epidermal keratinocytes and diffuse into the dermis; here,

they bind directly to ECM.29

Mechanical tension and ECM Loss of mechanical tension con-

tributes to molecular changes detectable in aged skin. Beyond the

cellular ageing process of fibroblasts, reduced neocollagenesis may

be ascribed to the reduced mechanical stimulation of fibroblasts.

In aged skin, fibroblasts collapse because of the loss of mechanical

tension caused by the accumulation of fragmented collagen and

concomitant loss of binding sites between intact collagen and

fibroblasts. This process concurs with an increase in MMPs, the

intracellular oxidative level, protein oxidation, the transcription

factor AP-1 and with a reduction of collagen production, resulting

in a self-perpetuating cycle (see Fig. 1).30 Degradation of elastic

fibres is also relevant in skin ageing processes and particularly in

wrinkle formation. The findings obtained in animal experiments

suggest repetitive UVB-exposure as a cause of wrinkle formation

through loss of skin elasticity. The expression of fibroblast elastase

is stimulated by UVB-induced cytokine secretion of keratinocytes.

Up-regulated activity of fibroblast elastase damages elastic fibres

facilitating wrinkle formation.31

Within the framework of UV-exposure (solar simulated UV

irradiation), infiltrating neutrophil granulocytes release MMP-1,

MMP-8 (neutrophil collagenase), MMP-9 and neutrophil elastase

and participate in the degradation of the ECM. Neutrophil granu-

locytes are assumed to play a critical role in the pathogenesis of

photoageing.32

Vascular alterations Skin ageing also involves alterations of

dermal vascularization. In the papillary dermis of photoaged skin,

both vessel size and density are significantly decreased. However,

intrinsically aged skin only shows a decrease in vessel size because

the density of dermal blood vessels is age-independent.33

Imbalance of the angiogenesis inhibitor thrombospondin-1

(TSP-1) and the vascular endothelial growth factor (VEGF) is

of particular importance in UV-induced vascular changes. Acute

Figure 1 This model schematically depicts factors of pathogenic

relevance for skin ageing. The induction of matrix metalloprotein-

ases is of particular importance as they degrade collagen and

other components of the extracellular matrix (ECM). Mainly UV-induced reactive oxygen species (ROS) and DNA damage lead to

increased induction of matrix metalloproteinases in keratinocytes

and fibroblasts. Proteolytic enzymes such as elastase and matrix

metalloproteinases derived from neutrophil granulocytes contrib-ute to the degradation of the ECM. Besides, UV-exposure

directly stimulates the production of elastase in fibroblasts. As a

result, partially degraded collagen and reduced mechanical ten-sion of fibroblasts inhibit neocollagenesis. Reduced mechanical

tension leads to further production of ROS, which again results in

increased expression of matrix metalloproteinases.

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Skin ageing 875

UVB-irradiation results in pronounced generation of immature

vessels, possibly arising from the down-regulation of TSP-1 and

the up-regulation of VEGF.34 UVB-irradiated transgenic mice with

overexpression of TSP-1 showed decreased skin vascularization as

well as reduced wrinkle formation compared with the control

group.35

The causes of the aforementioned reduction of dermal vessels in

photoaged skin in contrast to the proangiogenic effects of acute

UV-exposure require further investigations. Altered ECM in photo-

aged skin is assumed to allow the regression of dermal vessels.33

Protein oxidation Ageing cells show accession of oxidized pro-

teins and consequently declining proteasome activities.36 Activity

of the proteasome, a multicatalytic protease that degrades oxi-

dized proteins, declines with age.37 Protein oxidation results

from UV-induced depletion of antioxidant enzyme expression.

The epidermis of photodamaged skin shows significantly lower

levels of antioxidant enzymes than photoprotected skin. As the

levels of antioxidant enzymes in the dermis are considerably

lower than those in the epidermis, photoaged skin shows

accumulation of oxidized proteins in the upper dermis. In

combination with the lower proliferative activity of fibroblasts

compared with that of keratinocytes, the low enzyme levels may

explain the pattern of distribution of oxidized proteins.37 Accu-

mulation of oxidized and cross-linked proteins results in the

aggregation of proteins and lipofuscin, progressively inhibiting

all proteasome activities. The quantity of dermal oxidized pro-

teins correlates well with the severity of the clinical features of

photoageing.37

Clinical and histological changes in intrinsically and

extrinsically aged skin

Intrinsically aged skin is uniformly pigmented, showing loss of

elasticity, cigarette paper-like wrinkling, and rarefied hair follicles,

sweat glands and sebaceous glands.

The cumulative UV-dose and the Fitzpatrick skin type assign

the degree of sun-induced cutaneous changes. People with skin

types I and II show atrophic skin changes with focal depigmenta-

tion, epidermal atrophy, ephelides and pseudoscars and may

develop malignant or non-malignant skin cancer. By contrast,

people with skin types III and IV show diffuse irreversible hyper-

pigmentation, leathery appearance, deep wrinkles and lentigines

(Figs 2–4).

Because of the distinct extension of cell cycles, the epidermis is

renewed much slower in elderly people. The epidermal turnover

rate is up to 50% lower in the eighth decade of life. Moreover,

intrinsically aged skin shows epidermal atrophy, which particularly

affects the stratum spinosum, ranging from 10% to 50%.38

From the age of 30 onwards, the number of melanocytes abates

by 8% to 20% per decade.39 The notably fewer Langerhans cells

present show morphological alterations and are functionally

impaired. The dermis of photoprotected aged skin shows fewer

mast cells and fibroblasts than photoprotected young skin, and

collagen fibres and elastic fibres are rarefied.40 Collagen synthesis

declines by 30% in the first 4 years of menopause, then by 2%

annually.41

The epidermis in sun-exposed areas is thicker than in intrinsi-

cally aged skin, whereas severe photodamage elicits epidermal

Figure 2 Deep furrows, solar elastosis, focal hypopigmentationand solar lentigines in the face of a 66-year-old patient with

Fitzpatrick’s skin type II.

Figure 3 Deep perioral wrinkles of the same patient with a

10-year history of smoking cigarettes.

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876 Kohl et al.

atrophy.42 Sun-induced molecular effects are primarily located in

the dermis and the dermoepidermal junction. Histopathologically,

photoaged skin is distinguished by solar elastosis, i.e., the accumu-

lation of degraded elastic tissue in the dermis. Elastotic material

consists of elastin, fibrillin, glycosaminoglycans, particularly hyal-

uronic acid, and versican, a large chondroitin sulphate proteogly-

can. The pathogenesis of solar elastosis is not yet fully understood

but is assumed to derive from both degradation and de novo syn-

thesis.43

Skin ageing induced by other exogenous factors

Tobacco smoking Besides UV-exposure, alcohol consumption,

skin type and gender, exposure to tobacco smoke is a major factor

contributing to premature skin ageing.44 Several studies have indi-

cated that cigarette smoking furthers wrinkle formation. Analysis

of skin surface structure showed that people with a smoking his-

tory of at least 35 pack years showed significantly deeper wrinkles

than non-smokers, yet line density was decreased compared with

non-smokers.45 Similar to UV-exposure, nicotine abuse induces

expression of MMPs degrading ECM in human skin.46

Infrared radiation Infrared (IR) radiation (k = 760 nm to

1 mm) comprises 54.3% of total incident solar energy and may be

subdivided according to wavelength into IRA (k = 760 nm

to 1440 nm), IRB (k = 1440 nm to 3000 nm), and IRC

(k = 3000 nm to 1 mm). The depth of penetration into the skin

declines with increasing wavelength in the IR region.

Similar to UV-radiation, IRA is capable of inducing ECM deg-

radation, thus accelerating the skin ageing process. Already in

1982 was exposure to IRA found to result in dermal damage,

resembling solar elastosis.47 IRA radiation causes ROS formation

in the mitochondria and increases expression of MMP-1 and

MMP-9. Antioxidants that specifically target the mitochondria

[MitoQ (mitoquinone)] block the IRA-induced expression of

MMP-1 in human fibroblasts, whereas the UVA ⁄ UVB-induced

increased expression of MMP-1 remains unaffected.48

Besides the increased expression of MMP-1, IRA-exposure

decreases the production of procollagen 1.49 Similar to UV-expo-

sure, acute exposure to IRA elicits neoangiogenesis and accumula-

tion of infiltrating inflammatory cells.50

Prevention and therapy

Inorganic and organic sunscreens

Photoprotection is essential for preventing UV-induced premature

skin ageing. In vivo studies have indicated that the regular applica-

tion of sunscreen may avoid or at least diminish UV-induced

epidermal and dermal changes.51 Topical sunscreens are divided

into inorganic (formerly referred to as physical) and organic sun

protection (formerly referred to as chemical).

Inorganic sunscreens as titanium dioxide and zinc oxide reflect,

scatter, or absorb UV-radiation depending on the particle size and

the wavelength of the light. Zinc oxide and titanium dioxide nei-

ther have any skin-irritating or skin-sensitizing properties nor pen-

etrate into the layers below the stratum corneum. Modern

micronized forms show a decreased particle size of 10–50 nm,

offering more transparent and cosmetically appealing formula-

tions. Organic sunscreens absorb UV-radiation, whereby the

agent’s electrons reach an excited state. For a more detailed

description of this topic, we refer to several excellent reviews.52,53

Antioxidants

The formation of free radicals and ROS is of particular importance

for photocarcinogenesis and skin ageing. Because the topical applica-

tion of sunscreens does not offer complete protection against UV-

damage, antioxidants play a major role in the prevention and ther-

apy of UV-induced skin ageing. The enzymatic and non-enzymatic

antioxidants of the skin are depleted by UV-induced oxidative

stress.54 In the skin, important non-enzymatic antioxidants are asc-

orbic acid, coenzyme Q 10, vitamin E, niacinamide and b-carotene.

Besides the topical application of antioxidants, endogenous

photoprotection through dietary micronutrients is becoming more

important, because the biggest part of a cumulative UV-dose is

obtained in everyday life without topically applied sunscreens.

Contrary to previous assumptions, at least 75% of a lifetime

UV-dose is attained after the age of 18.55 Topical application of

antioxidants and supplementation with micronutrients, such as

polyphenols and b-carotene, should be complementarily effected

with topical sunscreen.56

Figure 4 The back of the hands of a 23-year-old woman (top left

corner) without any signs of photoageing and of a 46-year-oldwoman (top right corner) with lentigines and fine wrinkles. On the

bottom right, the back of the hand of a 61-year-old woman with

lentigines and wrinkles and, on the bottom left, the back of thehand of an 83-year-old woman showing loss of subcutaneous

fat, lentigines, hypopigmented spots, deep wrinkles and atrophy.

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Skin ageing 877

However, no significant long-term studies are yet available on

topical antioxidants and supplementation of antioxidants proving

photoprotective properties.

Vitamins As other antioxidants, vitamin C is very instable when

exposed to air because of oxidation. Therefore, esterified deriva-

tives are used for topical application. Besides its antioxidative

properties, vitamin C is essential for collagen biosynthesis because

it serves as a cofactor for two enzymes.57 In an animal model, vita-

min C has shown to be photoprotective by decreasing UVB-

induced erythema and the number of sunburn cells.58

The photoprotective effects of vitamin E have been shown in

numerous studies.59 Vitamin C and coenzyme Q10 synergistically

interact with vitamin E by reducing oxidized vitamin E, thus

enhancing the antioxidative properties of vitamin E.60

Niacinamide, also known as nicotinamide, is the amide of nia-

cin (vitamin B3). The application of creams containing 4% and

5% niacinamide significantly reduced wrinkles and improved skin

elasticity (see Table 1).61,62 Although less potent than retinoids,

niacinamide is particularly suited for the periocular region because

of its non-irritating properties.

Carotenoids protect plants against oxidative stress and excess

light and exhibit an extended system of conjugated double bonds

essential for antioxidative properties.63 A meta-analysis showed

that oral supplementation with b-carotene protects against UVB-

induced erythema. For significant protection against sunburn, a

minimum supplementation period of 10 weeks was noted.64 Car-

otenoids at high doses may have prooxidative effects. Endogenous

photoprotection attainable with b-carotene complies with sun

protection factor 4 at the most.64

Coenzyme Q10 Coenzyme Q10 (CoQ10, ubiquinone) is a fat-

soluble antioxidant. In vitro CoQ10 reduced the UVA-induced

production of MMP in human fibroblasts.65 So far, only one study

showed wrinkle improvement using 1% CoQ10 cream for

5 months.66

Table 1 Overview of studies on topical antioxidants for the treatment of skin ageing

Authors No.patients

Study design Study medication Application Duration Clinical results

Bissett et al.(2005)

50 Double-blind,left-rightrandomized

5% niacinamide vs.vehicle

Twice daily to half ofthe face and itsvehicle control tothe other half

12 weeks After 12 weeks significantreductions in- fine lines and wrinkles

(P = 0.0005)- hyperpigmented spots

(P = 0.006)- red blotchiness (P = 0.03)- skin sallowness (P = 0.0004)

(yellowing) compared withvehicle control

- improvement of skinelasticity compared withvehicle control (P < 0.05)

Kawada et al.(2008)

30 Randomized,placebo-controlled,split face study

4% niacinamide vs.vehicle

Once daily to half ofthe face and itsvehicle control tothe other half

8 weeks After 8 weeks- marked resp. moderate

improvement of wrinkles in64% of the subjects with asignificant differencecompared with the control site(P < 0.001)

- wrinkle grades weresignificantly reducedcompared with control(P < 0.001)

Humbert et al.(2003)

20 Randomizedplacebo-controlled,double-blind study

Cream containing5% vitamin C vs.vehicle

Once daily onlow-neck and arms

6 months - significant improvement, interms of the ‘global score’(hydration, roughness,suppleness, wrinkles andlaxity) compared with control

- highly significant increase inthe density of skin microrelief

- ultrastructural evidence of theelastic tissue repair due toreappearance of ‘composite’elastic fibres in the papillarydermis of vitamin c-treatedsides

- no changes of dermal collagen

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878 Kohl et al.

Polyphenols

Polyphenols are secondary plant substances consisting of polycy-

clic aromatic compounds bonded with hydroxyl groups. Polyphe-

nols dispose of both anti-inflammatory and antioxidant effects

and are capable of modulating several signalling cascades.

The photoprotective properties of green tea have been exten-

sively investigated. Green tea is obtained from the leaves of the

plant camellia sinensis that mainly contain the polyphenols (-)-

epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin-3-

gallate (ECG) and (-)-epigallocatechin-3-gallate (EGCG), which

have antioxidant properties. Green tea offers potent anti-inflam-

matory, antioxidant and photoprotective qualities67,68 that may

prevent UV-induced skin changes.

In animal models, topical application or oral supplementation

with green tea polyphenols decreased UVA-induced roughness

and sagginess,69 attenuated UVB-induced erythema,70 and inhib-

ited UVB-induced protein oxidation in the dermis. Besides, green

tea polyphenols were shown to diminish expression of MMPs

in vivo and in vitro.71

Topically administered polyphenols from green tea dose-

dependently inhibited UVB-induced CPD formation in the

human epidermis and dermis.72 Moreover, topically applied

green tea extracts prior to exposure to solar simulated radiation

reduced the number of histologically verifiable sunburn cells,

inhibited UV-induced erythema, maintained the depletion of

Langerhans cells,68 and inhibited UV-induced increase of epider-

mal thickness (Table 2).9 The UV-protective effects of green tea

polyphenols are presumably due to enhanced IL-12 mediated

DNA-repair.73

Silymarin, a flavonoid from milk thistle (Silybum marianum),

also reduced UVA ⁄ B-induced oxidative stress in animal models74

and attenuated photocarcinogenesis.75

Extracts of the fern polypodium leucotomos is endued with

strong antioxidant, anti-inflammatory and photoprotective prop-

erties.76–78 In humans, UV-induced skin changes were reduced by

oral supplementation with Polypodium leucotomos, which – prior

to exposure to solar simulated radiation – decreased UV-induced

erythema, the formation of sunburn cells, DNA-damage and

Table 2 Overview of clinical studies on photoprotective effects of green tea polyphenols and Polypodium leucotomos

Authors No.subjects

Study design Study medication Results

Elmets et al. (2001) 6 Areas of skin were treatedwith an extract of greentea or one of itsconstituents. 30 min later,the treated sites wereexposed to a 2 MED solarsimulated UVR

5% solution of green teapolyphenols (GTPs)

Green tea polyphenols resulted ina dose-dependent inhibition ofUV-induced erythema.The (-)-epigallocatechin-3-gallate(EGCG) and (-)-epicatechin-3-gallate (ECG) polyphenolicfractions were most efficient atinhibiting erythema.Histologically, application ofgreen tea polyphenols resulted ina reduction of sunburn cells by66% (P < 0.01) and reduceddepletion of Langerhans cells.Green tea polyphenols alsoreduced significantly the DNAdamage that formed after UVradiation.

Middelkamp-Hup et al.(2004)

9 Subjects were exposed tovarying doses of UVradiation without and afteroral administration ofPolypodium leucotomos(7.5 mg ⁄ kg)

Oral administration ofPolypodium leucotomos(7.5 mg ⁄ kg)

A significant decrease in erythemawas found in PL-treated skin(P < 0.01).Histologically, PL-treated biopsyspecimens showed less sunburncells (P < 0.05), cyclobutanepyrimidine dimers (P < 0.001),proliferating epidermal cells(P < 0.001), and dermal mast cellinfiltration (P < 0.05).A trend towards Langerhans cellpreservation was seen.

Villa et al. (2010) 10 Randomized, investigator-blinded, controlled study.Subjects were exposed toUVA without and after oraladministration ofPolypodium leucotomos

Oral administration ofPolypodium leucotomos240 mg, 8 h and 2 hbefore UVA exposure

A trend towards prevention of theincrease of the common deletionin the Polypodium leucotomos-group was seen.

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Skin ageing 879

dermal mast cell infiltration.76 Polypodium leucotomos may be

administered orally and topically.

Eicosapentaenoic acid

In human skin, topical application of eicosapentaenoic acid (EPA)

before UVA ⁄ B-exposure reduced epidermal thickening and inhib-

ited UV-induced decrease of procollagen expression in vivo. In

intrinsically aged human skin, topical application of EPA increased

expression of ECM proteins (Table 3).79

Retinoids

The efficacy of topically applied tretinoin (all-trans-retinoic acid)

for the treatment of photoageing has been convincingly docu-

mented in several trials (Table 4). Significant effects were induced

by concentrations of 0.02% or higher, whereas a dose–response

relationship exists for both effectiveness and skin irritation. Signifi-

cant improvements were found for fine wrinkling, skin roughness,

mottled hyperpigmentation, sallowness and the overall severity of

photodamage.80,81 Clinical improvement occurred after several

months of application; in addition, skin conditions continued to

improve with an application duration of at least 10–12 months.

Clinical results are reversible after cessation of therapy; thus, long-

term treatment three to four times a week is recommended to

maintain clinical benefits. In the US, Tretinoin 0.05% is approved

by the FDA for the mitigation of fine wrinkles, mottled hyperpig-

mentation and tactile roughness of facial skin. Tazarotene, another

retinoid approved by the FDA to ameliorate some of the signs of

photoageing, is assumed to be as effective as tretinoin.82

Topical application of tretinoin prevents the UVB-induced

expression of MMPs through inhibition of the nuclear transcrip-

tion factor AP-1 and obviates collagen degradation, which is

already initiated by low-dose UVB-irradiation.26

Histological effects differ depending on use duration. Initially,

increase of epidermal thickness and anchoring fibrils are observed.

Dermal effects in terms of neocollagenesis are not evident before

12 month application.83 According to the clinical effects described

above, histological examination shows an increase of collagen in

the papillary dermis accompanied by a decrease of solar elastosis.84

The melanin content also continues to decrease with the increasing

duration of therapy, correlating with improved mottled hyperpig-

mentation and solar lentigines.83 Evidence suggests that intrinsi-

cally aged skin may also benefit from the application of topical

retinoids.85

Hormones and growth factors

Study results on the cutaneous effects of hormone replacement

therapy are inconsistent.86 However, several studies have indicated

that hormone replacement therapy or the topical application of

oestrogen may ameliorate the effects of hormone deficiency. In

comparison to control groups, prevention of and improvements

in wrinkles have been documented87 as well as enhanced hydra-

tion88 and elasticity,88,89 accession of skin thickness88 and

increased collagen.8 Because of the well-documented side-effects,

risks and benefits should be diligently analysed before the incep-

tion of oestrogen supplementation.90

Table 3 Photoprotective effects and effects on extracellular matrix of eicosapentaenoic acid in human skin

Author No.subjects

Study design Study medication Results

Kim et al. (2006) 7 resp. 4 (A) Topical application of EPA2% under occlusion vs.vehicle once daily for 2 daysbefore UV-exposure (2 MED)in volunteers (average age:28 years)(B) Topical application of EPA2% vs. vehicle three times aweek for 2 weeks underocclusion in volunteers(average age: 76 years)

2% EPA in ethanol-polyethylene glycol (70 : 30)vs. vehicle

(A) EPA inhibited UV-inducedepidermal thickening by72 ± 12.6% (P < 0.05) comparedwith vehicle.EPA prevented UV-induceddecrease of procollagenexpression compared with vehicle.Inhibition of UV-induced MMP-1expression by 55 ± 13%(P < 0.05) and MMP-9 expressionby 75 ± 7% (P < 0.05) comparedwith vehicle.Inhibition of UV-induced c-Junphosphorylation by 79 ± 11%(P < 0.05) compared to vehicle.Inhibition of UV-induced COX-2expression by 76 ± 4% (P < 0.05)compared with vehicle.(B) EPA increased the expressionof procollagen by 218 ± 39%(P < 0.05) compared with vehicle.EPA increased the level oftropoelastin and fibrillin-1 by420 ± 53% (P < 0.05) comparedwith vehicle.

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880 Kohl et al.

Enhancing collagen production with topical oestradiol seems to

be restricted to intrinsically aged skin.91 Evidence suggests that

topical application of growth factors derived from human fibro-

blasts improves several features of photoaged skin.92

DNA repair enzymes

DNA-damage, such as CPDs, plays an essential role in photocar-

cinogenesis but also contributes to skin ageing. UVB-irradiated

human keratinocytes are involved in the induction of MMP-1

in dermal fibroblasts via paracrine mechanisms. MMP-1 induc-

tion was reduced when keratinocytes were treated with the

DNA-repair enzyme T4 endonuclease V (T4N5).93 The bacterial

DNA-incision repair enzyme T4N5 is encapsulated in liposomes

for delivery into the living cells of the skin; here, the enzyme

recognizes CPDs and enhances dimer removal. The topical

application of a lotion containing T4N5 in patients with xero-

derma pigmentosum lowered the incidence of non-melanoma

skin cancer.94

Photolyase is another DNA repair enzyme, which can be found

in numerous plants and animals but not in humans. Enzymatic

removal of CPDs requires exposure of the dimer-photolyase com-

plex to photoreactivating light. The application of photolyase con-

taining liposomes considerably reduces the amount of CPDs in

human skin. Besides, evidence suggests that topical application of

photolyase prevents UVB-induced immunosuppression, formation

of sunburn cells, erythema and tanning reactions.95 A sunscreen

Table 4 Overview of studies on topical retinoids for treatment of skin ageing

Authors No.patients

Study design Retinoid Frequency ofapplication

Duration Clinical results

Weiss et al.(2006)

45 Randomized,double-blind,placebo-controlledphase and anadditional 6-monthopen-label phase

Tretinoinmicrosphere gel0.1%

Once daily onfacial skin

6 months resp.12 months

At 6 months statistically significantimprovement relative to placebo in- overall severity of photodamage

(P = 0.0003)- investigator’s global assessment

of clinical response (P < 0.0001)- fine wrinkling (P < 0.0001),- mottled hyperpigmentation

(P = 0.0002)- yellowing ⁄ sallowness

(P < 0.0001)- lentigines (P = 0.0054)

Kang et al.(2005)

204 Randomized placebo-controlled

Tretinoinemollient cream0.05%


24 months Significantly greater improvementrelative to placebo in- fine wrinkling- coarse wrinkling- mottled hyperpigmentation- lentigines- sallowness- overall photodamage severity- investigator’s global assessment

of clinical response (P < 0.05)

Phillips et al.(2002)

563 24-week multicentre,double-blind,randomized, vehicle-controlled interventionstudy followed by a28-week open-labelextension

Tazarotenecream 0.1%


12 months Double-blind period:Tazarotene resulted in asignificantly greater incidence ofpatients achieving treatmentsuccess (‡50% globalimprovement) and at least a1-grade improvement in finewrinkling, mottledhyperpigmentation, lentigines,elastosis, pore size, irregulardepigmentation, tactile roughness,coarse wrinkling and the overallintegrated assessment ofphotodamage (P < 0.01)

Kafi et al.2007)

36 Randomized, double-blind, vehicle-controlled, left andright arm comparisonstudy

0.4% retinol(vitamin A) lotion

Up to threetimes perweek on theupper innerportions ofthe arms

24 weeks After 24 weeks significantdifferences between retinol-treatedand vehicle-treated skin forchanges in fine wrinkling scores[)1.64 (95% CI: )2.06 to )1.22)vs. )0.08 (95% CI: )0.17 to 0.01);P < 0.001]

ª 2011 The Authors


Skin ageing 881

containing the DNA-repair enzyme photolyase is commercially

available.

Innovative approaches for photoprotection

DNA-oligonucleotides DNA-oligonucleotides (T-oligos) show

another possibility to enhance DNA-repair. DNA-oligonucleotides

homologous to the telomere 3-prime overhang sequence mimic a

telomere loop disruption and overhang exposure and initiate pro-

tective DNA-damage response. Treatment with t-oligos stimulates

melanogenesis and enhances DNA repair capacity. This displays

an innovative option for photoprotection, as both tanning and

increased DNA-repair rates are physiologically induced by UV-

exposure and DNA-damage to protect from further damage.96

Forskolin Tanning of the skin may also be induced by the topi-

cal application of forskolin, a cell permeable diterpene that stimu-

lates adenylate cyclase activity. This enzyme is physiologically

activated after the binding of a-MSH to the melanocortin 1 recep-

tor (MC1R), thus up-regulating cAMP-levels in melanocytes. As

effects of a-MSH are cAMP-mediated, the binding of a-MSH to

the MC1R may be obviated, and melanin production takes place

without any previous sun-exposure.

In animal models, pigmentation was compassed despite the

absence of a functional MC1R. Application of the cyclic AMP-ago-

nist forskolin induced pigmentation in fair-skinned individuals

with defective MC1R who show sequence variants of the MC1R-

gene.97 Besides, in vitro induction of pigmentation with forskolin

also showed enhanced DNA-repair, removing CPDs and 6,4-

photoproducts more efficiently.98

a-MSH analogues a-MSH, mediating UV-induced tanning, is

regarded as a cytoprotective agent. Synthesis of melanin may also

be stimulated by analogues of a-MSH that bind to melanocortin-

1-receptor on melanocytes. [Nle4-D-Phe7]-a-MSH (MT-1 or

Melanotan-1), a derivative of a-MSH with higher potency and

prolonged chemical stability than a-MSH, entails an explicit

increase of the melanin content in human skin in vivo when

injected subcutaneously. This effect was most pronounced in fair

subjects with a low minimal erythema dose threshold.99 However,

new findings indicate that a-MSH may actually increase mtDNA

damage because of increased oxidative stress within the frame of

elevated melanin synthesis.100

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ª 2011 The Authors


884 Kohl et al.

j eur acad dermatol venereol 2011

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