Review Skin ageing Molecu/a,r aspects q/' skin ageing: recentdam C. Rocquet and F. Bonte ABSTRACT As our body's envelope, the skin acts as a biosensor with the environment and reflects our personality. Skin ageing is therefore an important and interesting topic of study. It results from the combination of intrinsic ageing and photoageing, which is due to the environmental influence, such as reactive oxygen species (ROS). The more recent data are gathered here to remind current knowledge about skin ageing, from a molecular level to the clinical signs, wrinkles and spots mainly. Because knowledge of the prefer- ential biological targets of ageing has recently been making progress, it is possible to delay the manifes- tation of ageing, by acting on key biological processes. 1. Introduction Humanity is ageing. The average life expectancy of people living in industrialized nations has doubled since 1900. This will result in social, economic, and health- care changes that will, in turn, drive pub lic policy worlcl- wide. The cosmetic industry is moving to cater for the ageing population by cleveloping more innovative procl- ucts. The skin provides a large interface with the envi- ronment, and is thus of prime importance. The major changes in the skin that occur with age are a loss of elasticity and a reduction in its protective function. These changes do not just affect the elderly, as they begin when people are younger than thirty. The extent of change depends to a large degree on how much the skin is exposecl to sunlight ancl how stressful is a person's lifestyle. Por years, people have attempted to hicle, reverse or control changes in their skin, such as wrinkling, roughness, mottling, blotching and dryness. Skin of eld- erly people is thin ancl fragile, clue to complex changes very often summarized to reduced dermal collagen ancl clecreasecl cell proliferation. Skin ageing is thought to result from two processes, intrinsic ageing and extrin- sic, or photoageing (1, 2). Intrinsic ageing is believecl to be genetically programmed ancl is thus presentecl as independent of all external and envifonmental influ- ences. Extrinsic ageing is due to UV racliation and other environmental insults; that accelerate the skin changes (3). Photoageing leacls to a rough/dry/leathery skin, a yellow/clull complexion, lentigines or actinic spots ancl wrinkles. The study of the biology of ageing has made Acta Dermatoven APA Vol 11, 2002, No 3 71 Skin ageing R e v i e w rapid progress recently, after decades of stagnation. This Table 1 . Reactive Oxygen species. paper reviews the most recent data on skin ageing, from the cell nucleus to tissue, and discusses their conse- guences for the skin. 2. Damage due to Reactive Oxygen Species (ROS) Most people agree that free radicals are most im- portant agents of ageing. Organic molecules absorb light or UV irradiation and become excited because an elec- tron is transferred to a higher orbita!. The excited mol- ecule is a free radical which may cause secondary reac- tions and damage to various constitutive molecule (4). The energy storage molecule, adenosine triphosphate (ATP) is produced by oxidative phosphorylation in the mitochondria. The energy is produced by the oxida- tion of reducing eguivalents of nutrients via the respi- rato1y chain. However, mitochondria also regulate the intracellular calcium concentration and apoptosis. Abnormaly increased membrane potential is linked to ROS (Reactive Oxygen Species) augmentation anc! mi- tochonc!rial DNA (mtDNA) mutations, constitutive to skin ageing. ROS are normally proc!uced by mitochondria, but they can also be producec! following an external stress . Normal metabolism gives rise to most ROS, primarily via the mitochondrial respirato1y chain , in which ex- cess electrons are donated to molecular oxygen (O) to generate a superoxide anion (02• -) . The superoxide anion is reduced by the enzyme superoxide dismutases (SOD) to hydrogen peroxide (H2O), which, in turn, is rec!uced to water by catalase, located in the primary peroxisomes, and by glutathione peroxidase (GPx) , located in the mitochondria and cytosol. There are three isoforms of SOD (SOD 1 in the cytosol, SOD 2 in the mitochondria anc! SOD 3 in the extracellular space). Hydrogen peroxide can be converted to the highly toxic hydroxyl radical (OH•) in the presence of transition metals, and all three of the ROS (OH•, O, • , H,O,) can damage macromolecules directly or indirectly -(5). ROS are responsible for structural anc! functional alterations of cellular membranes, polyunsaturated fatty acic!s, proteins and DNA (6). For example, recent stud- ies have shown that mitochondrial aconitase, an enzyme of the citric acid cycle that is critical for controlling the rate of ageing, is a target of oxidative damage (7). Cells also contain antioxidant enzymes such as superoxide c!ismutase, glutathione peroxidase, anc! catalase, and reducing agents like vitamin E and glutathione (8-10). Ageing is associated with a decrease in the plasma con- centration of antioxidants, such as glutathione, and with increases in markers of oxidation c!amage, such as lipid peroxidation products (11,12);. The total glutathione concentration in cultured skin fibroblasts decreases with Hydroxyl OH• 0.3 ns Superoxide o.-2 0.4 ns Nitric oxicle NO• Seconds Peroxynitrite ONOO(-) 50 ms - second Singlet oxygen 102 ]-1S Hydrogen peroxic!e H2O2 Stable Peroxicle LOOH Stable age, while glutathione rec!uctase activity is unaffected. The antioxic!ant defenses are less well developec! in the early stages of life than in postnatal life. (13). The first changes that occurs in the skin after chronic or acute UV irracliation is the generation of reactive oxygen species, leadiHg to the peroxic!ation ofunsatur- ated lipids in the celi membrane. Low phototype indi- viduals are more likely to produce large amounts ofROS after exposure to UVA, in particular singlet oxygen that can diffuse across the cell membrane. For example, the oxidation of catalase by singlet oxygen gives rise to more acidic conformers. The integrity of catalase may be a marker ofthe stress clue to exposure to UVA (14). Sin- glet oxygen, due to its reactivity, appears more anc! more as a powerful free rac!ical able to c!amage numerous skin components. Mitochondria: an important target The mitochonclrial theory of ageing states that mi- tochonc!ria are the main site for generating free radicals and reactive oxygen in the cel!. Thus, mitochondria are vulnerable to oxidative stress and damaged mitochon- dria can cause an energy crisis in the cell, leading to senescence and tissue ageing. An accumulation of dam- age decreases the cell 's ability to generate ATP, so that cells, tissues, and individuals function less well. There is now considerable evidence that mitochondria are al- tered in the tissues of ageing individuals and that the clamage to mtDNA increases 1000-fold with age. The phagocytic lysosome system for removing mitochon- dria is also considerably altered in the cells of ageing organisms. Thus, damaged mitochonclria play an ii.n- portant role in apoptosis. The survival ofthe whole celi may depend on the release of caspase activators such as cytochrome C from the mitochonclria (15). The ex- tent of this alteration during senescence is stili debated (16, 17). Oxidative stress increases the production of free radicals and the inner membrane ofthe mitochondrion is chemically and physically altered. ROS are normally produced in mitochondria because ofubiguinones and the cytochrome b family (Complex III). Complexes I and IV are less vulnerable, while complexes II, III and V are affected by oxidative stress, via damage to lipids 72 -----------------------------------Acta Dermatoven APA Vo l 11, 2002, No 3 Review and proteins (18). The electron transport cbain is thus compromised, leading to energy supply failure and celi death (19). The mutation rate of mitochondrial DNA is ten-times higher than that of nuclear DNA. Indirect ob- servations suggest there is transport of nucleic acids between mitochondria, which helps to repah- the dam- aged mitochondrial genome (20). Damage to mtDNA will block mitochondrial turnover and replication, lead- ing to decline in ATP production and protein synthesis. The accumulation of 8-hydroxydeoxy gi.ianine (8-oxo- dG) in mitochonclria also inclicates great oxiclative dam- age (21-23). Exposure ofthe human skin to solar raclia- tion leacls to an accumulation of mtDNA mutations, caused partly by oxidative damage, and these mutations play an important role in photoageing (24). Genes and cellular pathways Alterations in oxiclative metabolism ancl the celi re- dox state can affect many genes and cellular pathways. The influence of oxidation on mitogenic responses and signal transduction pathways, such as MAP kinase and NF-eB, are well documented (25). Human fibroblasts exposed to severa! oxidative stresses also develop mark- ers of replicative senescence. Some genes, such as those encoding fibronectin , osteonectin, a.l(I)-procollagen, apolipoprotein, SM22 (putative Ca binding protein over- expressecl in senescent fibroblasts), and GTP-a. bind- ing protein are overexpressed. The mitogenic response to severa! growth stimuli (serum, PDGF, basic FGF and EGF) is !ost (26). The reduction in connective tissue growth factor by UV radiation may contribute to the reclucecl procollagen synthesis observecl in UV-irracli- ated normal human skin (27). Peroxynitrite (ONOO·) is generated from the trans- ducer molecule nitric oxide (NO) ancl superoxicle an- ions (0 2 •·) under pathological conditions. MMPs ancl pro MMPs have been shown recently to be activatecl by peroxynitrite in vitro (28). The activation of poly(ADP- ribose) polymerase by peroxynitrite is also implicatecl in the pathogenesis of various inflammatory conclitions ancl injuries. Tyrosine nitration is mostly mecliatecl by peroxynitrite, a cytotoxic oxidant clerived from nitric oxide that can cause DNA breaks. Peroxynitrite inhibits celi proliferation ancl high concentrations are also cyto- toxic. Peroxynitrite and poly(ADP-ribose) polymerase also seem to be involvecl in the regulation of keratino- cyte function ancl cleath in contact hypersensivity (29). Nitrogen dioxide and carbonate radical anion must also be taken in consicleration. Nitrogen dioxide can be pro- duced from excessive nitric oxicle autoxidation in hy- drophobic environments such as celi membranes (ni- tratecl lipids) and the interior of proteins (nitratecl pro- teins); carbonate raclical anions are proclucecl in strong oxiclative conditions and can oxiclize nucleic acicl gua- nine resiclues, GSH and proteins (30). Skin ageing J. Proteins O.xidation and its consequences Thin, wrinklecl skin is ve1y often attributecl to a Jack of collagen. The dermis and overlying epiclermis of ageing skin are profoundly altered (31). Slower protein synthesis is one of the most common events observecl cluring ageing. The synthesis of both structural proteins, such as collagen, ancl enzymes that repair and maintain the normal metabolic functions of the celi, is slowed down. This leacls to the inefficient removal of clamagecl molecules ancl clecreased intra-and intercellular signal- ing pathways. The age-relatecl increase m oxidized pro- tems may also be linkecl to modifications of proteins causecl by lipid peroxiclation proclucts (32- 35). Age- related changes in fibroblasts due to the metal-catalyzecl oxiclation of proteins lead to an exponential increase in the concentration of protein carbonyl groups in tissue samples taken from people agecl 10 to 80 years. Oxidative clamage to proteins may be most impor- tant in ageing, because oxiclized proteins become inac- tive and can accumulate in the celi, thereby triggering programmecl cell cleath. ROS increase the carbonyl con- tent of proteins by forming alclehydes and ketones from certain aminoacicl resiclues (36,37). The concentration of adenine nucleotide translocase, a protein in the in- ner mitochondrial membrane that is tightly bouncl to six molecules of carcliolipin, which contains highly un- saturated fatty acids, also clecreases witl1 age. This pro- tein is the prima1y intracellular site for the generation of superoxicle anion and exhibits adducts of the lipid peroxidation product, 4-hyclroxynonenal, a powerful oxidative aldehycle . The concentration of carcliolipin may be a mark er of the real age of the celi, basecl on its energetic capacity (38). Proteasome involvement Protem oxiclation in vivo is a natura! consequence of aerobic life and the proteasome complex is respon- sible for the selective clegradation of oxidizecl proteins. The age-related increase in the concentration of oxi- dized proteins is partly clue to the cell 's decreasecl ca- pacity to degrade them. Lysosomal proteases ancl the proteasome complex normally c;legrade oxidized pro- teins . The 26S proteasome units selectively recognize and degrade oxidized proteins in tl1e cytoplasm, the nucleus and the encloplasmic reticulum. The proteasome activity (multicatalytic proteinase MCP) in- volved in clegracling oxidized protems may be reduced (39). One ofthe lipid peroxiclation products, 4-hydroxy- 2-nonenal (HNE), can cross-lmk proteins via their lysine resiclues. The accumulation of oxiclizecl protein, lipo- fuscin ancl/or ceroicl pigments during ageing may be due to the changes producecl in proteins by HNE and their subsequent inhibition of the proteasome unit Acta Dermatoven APA Vol 11, 2002, No 3 ------------------ --- 7.J Review (proteasome 20S). This woulcl leacl to a vicious circle of cytotoxic protein oxiclation proclucts. (34). Oxygen stress (especially 4-hyclroxy-2-nonenal) may attack pro- teins clirectly or through lipid peroxidation, to inhibit enzymatic activity. Oxiclative clamage to membrane transport proteins leacls to alteration ofthe intercellular concentrations of calcium and potassium. The activity of the cytosolic proteasomal system also cleclines dur- ing the proliferative senescence of human fibroblasts (40). UVA and UVB irradiation both alter proteasome function in human keratinocytes (41, 42). While UV- incluced skin clamage is amelioratecl by retino! and ali- trans retinoic acid, UV irradiation blocks retinoid sig- naling in human skin through the ubiquitin/proteasome- mediated degradation of nuclear retinoid receptors ( 43, 44) . In the future, it would be interesting to focus on the existence of preferential protein targets. Glycation Cross-links can also form between proteins by cou- pling glucose carbonyl group to aminoacids such as lysine. These compounds called Advancecl Glycation End proclucl~ (AGEs) bincl covalently to other proteins, and can cause extensive clamage. The collagen lattice formecl by cross-linkecl type I collagen is uncleformable (unglycated collagen is fully compactible). Cross-link- ing collagen fibrils also alters the physical ancl mechani- cal properties of the extracellular matrix ancl changes the organization of the intracellular actin cytoskeleton (45). Glycatecl collagen may moclify normal celi aclhe- sion (46). As aclhesion is a funclamental celi function, each alteration can damage celi behaviour (apoptosis, etc) ancl then change tissue homeostasis . The clermis ancl elastic fibre network become glycatecl in people over 35 years of age and solar irradiation appeared to enhance it (47). The fluorescence of epidermal tryp- tophan moieties ancl collagen cross-links in the dermal matrix can also be considered to be good in vivo mark- ers of photoageing ( 48). While there are ve1y high con- centrations of antioxidant enzymes (catalase, SOD) in the epiclermis, the concentrations are much lower in the dermis. Protein glycation ancl advanced glycation may be inhibitecl by antioxidant components (49). Photoaged skin has significantly reclucecl concentrations of antioxidant enzymes in the stratum corneum and tl1e epidermis, while the concentration of oxidized proteins in the upper dermis is increasecl. ACllte exposure to UV irradiation depletes the catalase activity in the skin and increases protein oxidation (50). 4. Dermal Matrix alteration Fibroblasts The importance of cytokines, ancl immune celi ho- Acta Dermatoven APA Vol 11, 2002, No 3 Skin ageing meostasis for ageing and its clinical signs is stili not clear. Normal concentrations of cytokines seem to be required for skin celi homeostasis. A clisturbance leacls to de- fects or wrinkle formation. Histological stuclies of chronically sun-exposed skin show that the clermis con- tains inflammatory infiltrates (51), mostly perivascular ancl perifollicular. Mast cells are more abunclant in photodamagecl skin than in normal skin. They synthe- size and release mediators that moclulate clirectly or in- clirectly extracellular matrix production and degrada- tion (including TNFcx, TGF~ ancl prostaglandin 2). They release proteases that can degrade the ECM or activate the proenzyme forms of metalloproteinases. Ultrastrnc- tural studies have also shown infiltration of the epider- mis by macrophage/denclritic-like cells. These studies have recently been confirmed, showing more epider- mal dendritic cells, but fewer Langehans cells in sun- exposecl skin (52). The balance of cytokines in the skin is alterecl dur- ing ageing and fibroblasts become less responsive to growth factors or cytokines. Physiological ageing in human fibroblasts seems to be particularly associated with an altered response to interleukin-1- ~, a cytokine proclucecl by monocytes, macrophages ancl other tran- sit01y cells involvecl in inflammation (53). Transforming growth factor (TGF)-~1 is a cytokine involvecl in the clifferentiation of fibroblasts to myo- fibroblasts. These myofibroblasts are very important for clermal strength ancl may be responsible for the con- traction of the clermis (54). It acts on fibroblast collagen, fibronectin, glycosaminoglycans, elastin procluction, all Exogenous / Endogenous STRESS Protein turnover Decrease of proteasome activity l Proteins Glycation Oxidatively modified proteins Protein synthesis Hydroxynonenal protein adducts Figure 1 . Cellular consequences of stress on proteins. 7.f Skin ageing of which are important for the mechanical properties of the skin. The expression of the TGF-~l gene in epi- dermal keratinocytes does not decline with increasing celi age. Hence, TGF-~l does not appear such as the message from epidermis to dermis affected by age (55). Altered cytoskeleton function may play a key role in the age-related changes in severa! cell types, because it is involved in a variety of functions that are altered with ageing (immunological , endocrine and neurologi- cal changes) (56). The age-related changes in the cy- toskeleton, due to its involvement in metabolic pro- cesses and celi surface receptors expression, can indi- cate defective signal transcluction. Aged fibroblasts, which very often contract collagen gels poorly and do not migrate well , have a clisorclerecl actin microfilament cytoskeleton ancl a reclucecl a.-2-~-integrin. (57). Postmitotic ancl mitotic cells age clifferently. Post- mitotic cells never divicle , such as nerve, muscle and fat cells. Mitotic cells , such as keratinocytes ancl fibro- blasts, clivide. Replicative senescence is the process that limits the number of celi clivisions. As senescent fibro- blasts ancl keratinocytes accumulate w ith age in human skin, this could explain the cleterioration of the appear- ance and properties ofthe skin. Senescent cells secrete degrading enzymes that modify the cytokine / inter- leukin balance, causing the loss of functional integrity (58). Lipofuscin, or age pigment, accumulates in cells within the lysosomal vacuoles, especially in fibroblasts . Lipofuscin can also accelerate ageing ancl senescence under mild hyperoxia (59). The accumulation of lipo- fuscin may also be involvecl in spot formation. A lack of glucose-6-phosphate dehyclrogenase (G6PD), an enzyme involvecl in the celi reclox balance, may also accelerate fibroblast senescence (60). It has been pro- posecl that phospholipid hydroperoxicle glutathione peroxiclase (PHGP) helps to protect fibroblasts against UVA-induced lipid peroxiclation ancl the activation of metalloproteinase 1 (MMP l ) by UVA. Receptors The number, affinity ancl rate of internalization of epidermal growth factor (EGF) receptors are clifferent in young and olcl fibroblasts, explaining the loss of re- sponsiveness to EGF with age and the impairecl wouncl healing in the elderly (61). Extracellular matrix is also climinishecl cluring ageing and the amount of collage- nase in the skin increases with age. Collagenase pro- cluction is controlled by protein kinase C via the mem- bers of the APl transcription factor family and can be inhibited by a.-tocopherol (62). Down-regulation of ligand-activated receptors is important for normal celi functioning. Receptors bearing their ligancl move to specialized regions in the plasma membrane. The re- sulting vesicles are transported through the cytoplasm by microtubules ancl fuse with enclosomes and lysos- omes, where they are clegradecl. ROS can also alter re- ceptor function. For example, oxiclase stress causecl by hyclrogen peroxicle rapidly inhibits the internalization of receptor-bouncl EGF in human fibroblasts, so that the breakclown of the EGF-receptor complex is inhib- itecl. Hydrogen peroxide also alters negative feed-back within the celi, and attenuates growth factor-induced signal transduction, leading to altered celi metabolism (63). Photo-damage increases the tenascin in cells, which might cause competition for the c.<2~1-integrin receptor, reducing cell-collagen binding. a2~1-integrin is the major collagen receptor, but is also a receptor for tenascin (64) . Tenascin C is a large extracellular matrix glycoprotein whose production by keratinocytes is in- creased in wound repair; it is also founcl in normal adult skin. It is often distributed discontinuously in the up- per papilla1y dermis adjacent to the EDJ, close to capil- lary basement membranes. The concentration of tena- scin is increased in photoclamagecl skin and its distri- bution is different from that of skin protected from the sun. Tenascin is found along the dermio-epidermal junc- tion in a continuous pattern and extends further into the p apilla1y dermis. The pattern of gene expression in senescent fibroblasts is clifferent from that of their still- clividing counterparts. Presenescent fibroblasts have low concentrations of metalloproteinases (MMPs) and high concentrations o MMP inhibitors (TIMP-1 and TIMP-3) . The concentration of MMPs increases as cells become senescent, while that ofTIMP clecreases. The MMP pro- duction is stimulatecl by activation of the redox-regu- latecl transcription factor NFKB and protein kinase C via activator protein 1 transcription factor (APl) (65). Exogenous / Endogenous STRESS l Altered receptors Functionality or expression i Loss of growth factors Hormone responsiveness Figure 2. Cellular consequences of stress on receptors. R eview 76 - - - - --------- ----------------------Acta Dermatoven APA Vol 11, 2002, No 3 Review MMP Some of the MMP, a family of at least 16 enzymes that digest matrix macromolecules, are activated by UV irradiation (66). Thus, metalloproteinases 1, 3 and 9 (MMP-1, 3 and 9) in the epidermis are activated by UVB, while UVA stimulates MMP-1 in vivo and MMP-2 and 3 in vitro. The MMPs in the skin are responsible for break- ing down macromolecules of the skin ECM, which en- sures the skin's three-dimensional integrity. The balance between MMPs and MMP inhibitors is perturbed by en- vironmental factors, such as light. This leads to collapse ofthe EMC and the visible effects ofUV damage: wrin- kling, loss of elasticity. Besides chronological ageing, actinic ageing, also called photodamage, causes prema- ture skin ageing: thinning of the dermis, a loss of colla- gen content and protein organization and a breakdown ofthe ECM (67, 68). Type 1 MMPs (interstitial collagenase) and type 9 MMP (gelatinase) break down skin collagen fibers, par- ticularly during photodamage (69, 70). MMP-2 (gela- tinase) acts on collagen types I, IV, and VII. Gelatin, elastin and fibronectin are all substrates for MMP-2, whose activity increases with age (71 , 72). MMP-1 de- grades collagen, which accounts for at least 70% of the dry weight of the dermis. Smoking increases the activ- ity of MMP-1 in the skin in vivo. It leads to an imbal- ance between MMP-1 and the tissue inhibitor of metallo- proteinase 1 (TIMP-1), which could be important for ageing (73). The MMP-1 produced by epidermal keratinocytes and clermal fibroblasts in response to vari- ous stimuli (7 4-78) appears to play a key role in clermal remodeling (79-81). Skin fibroblasts produce MMP-1 in response to UVB irradiation and keratinocytes play a major role through an indirect paracrine mechanism involving the release of epidermal cytokine after UVB- irradiation (82) . MMP are produced in response to UVB irradiation in vivo, ancl are considered to be involved in the changes in connective tissue that occur in photoageing (83). They are associatecl with a variety of normal and pathological conditions that involve degra- dation and remodeling of the matrix (84-87). Severa! MMPs are produced cluring wound healing, such as MMP-3 in epidermis repait· (88, 89). UV rays ancl ageing lead to excess proteolytic activ- ity that clisturbs the skin's three-dimensional integrity. These proteinases are important for breaking down the extracellular matrix during chronic wound repair, in which there is re-epithelialization by keratinocyte mi- gration (90). Thus, MMPs are continuously involved in the remodeling of the skin aft:er chronic aggression. Thrombospondin 2 (TSP2) , a secreted extracellular matrix glycoprotein, is an aclaptator and modulator of cell matrix interactions (91). It binds to heparan sulfate proteoglycan, low-density lipoprotein receptor-related protein (LRP), and the integrin av~3 (92, 93). Increased MMP-2 activity (gelatinase A) leacls to recluced fibro- Acta Dermatoven APA Vol 11, 2002, No 3 Skin ageing blast adhesion which could contribute to abnormal col- lagen fibril structure in the skin and the release of an- giogenic factors. New data show that TSP2 binds to and inhibits MMP-2 indirectly and therefore plays a role in cell-matrix interactions (94). The age modulated hypoxia response causes an imbalance between MMP-1 and MMP-9 and TIMP. Hence, there may well be altered MMP and TIMP gene expression at wrinkle sites (95, 96). Photodamage also results in the accumulation of abnormal elastin in the superficial dermis, and severa! MMPs have been impli- cated in this process. The quantities of matrilysin (MMP- 7) and human macrophage metalloelastase (MMP-12), which have broad substrate specificities, are two key parameters that can be used to evaluate long term antiageing treatments (97). They are increased in the abnormal elastic fibers of chronically photoaged skin and contribute to the remodeling of elastic areas in sun- damaged skin. Human metalloelastase also aids mac- rophage migration, in addition to degrading elastic tis- sue, so amplifying the disturbance of the inflammatory homeostasis of the tissue. Ultrastructural and histopatho- logically studies have demonstrated that sun-exposed skin contains accumulated insoluble material and the normal elastic fiber architecture is lost, resulting in a loss of skin resilience and elasticity and probably wrinkle formation. Fibrillin and Elastin Elastic fibers in the clermis form an amorphous ma- trix of elastin and intertwining bundles of microfibrils, which measure 10-14 nm in diameter. The oxytalan fi- bers are rich in microfibrils and are orientated perpen- dicularly to the basal lamina of the epidermis. A study on photoaged skin has shown that UV irradiation in- creases the tropoelastin mRNA in keratinocytes and fi- broblasts (98). Selective inhibition of skin fibroblast elastase could be one way to fight wrinkle formation following cumulative ultraviolet B irradiation (99). Lysozyme may alter the elastic fibers in the surface, pre- venting further degradation and the accumulation of altered elastic fibers . Photoaged skin contains elastic material in the re- ticular dermis, and the fibrillin deposits in the reticular dermis are enlarged. Elastic fibe_rs have a central core of hydrophobic cross-linked elastin surrounded by fibrillin-rich microfibrils. The papillary dermal micro- fibrillin-rich microfibril network is truncated and de- pleted in photoaged skin. There are fewer fibrillin-rich microfibrils in wrinkled photoaged skin, probably due to inflammato1y cell proteinases (neutrophil elastase), or activation of matrix metalloproteinase (100). Cross- linking causing decreasecl elasticity could also be in- volvecl in wrinkle formation (101). The fluorescence of tryptophan ancl collagen cross- links in the dermal matrix may serve as in vivo markers 77 Review Skin ageing Table 2. Main Matrix Metalloproteinases and their substrates. MMP-1 Matrix collagenase (fibroblast collagenase) Neutrophil collagenase Collagenase 3 Collagenase 4 Gelatinase A Collagens I, II, III, VII and X MMP-8 MMP-13 MMP-18 MMP-2 Collagens I, II , III , Link protein, Aggrecan Collagens I, II , III Collagens I Gelatins, Collagens I, IV, VII and XI, Fibronectin, Laminin, Elastin MMP-9 MMP-3 Gelatinase B Stromelysin 1 Gelatins, Collagens IV, Vand XIV Aggrecan, Elastin Aggrecan, Gelatin, Fibronectin, Laminin, Collagens III, IV, IX and X MMP-10 MMP-14 MMP-7 MMP-11 MMP-12 Stromelysin 2 (membrane type) Matrilysin Stromelysin 3 Aggrecan Collagens I, II ancl III, Laminin Aggrecan, Fibronectin Fibronectin Metalloelastase (Macrophage) Elastin of skin aging, pbotoaging, ancl as a way of assessing exposure to UVA radiation (48) . The morphology of elastic fibers changes significantly during life. The num- ber of elastin microfibrils (mainly composed of fibrillin) graclually clecreases during ageing, ancl the clegenera- tive process is accelerated by exposure to sunlight. Amyloicl P and lysozyme are cleposited in thickened fibers, while amyloid P alone is deposited in oxytalan vertically orientecl fibers in the papilla1y dermis. Deep wrinkles are linkecl to the clegeneration of collagen ancl the deposition of abnormal elastic material (102). Wrinkles are formed by major changes in the dermis matrix and at the dermio-epiclermal junction. The con- tent of fibrillin, a component of the elastic fiber net- work, is increased by prolonged clinical doses of topi- ca! retinoic acid. This is why fibrillin-1 has been pro- posecl as a "reporter" molecule for the efficacy of photoageing. Water and GAG The alterecl skin texture ancl structure of elderly people is caused by changes in proteins , lipicls and water, leading to alterecl mechanical properties , such as wrinkling, sagging, loss of elasticity and apparent d1yness. Water structure is important because water can bind to various proteins and is important for maintain- ing the structural and mechanical properties of proteins. Their natura! interaction is climinishecl in photoaged skin, leacling to decreased collagen stability ancl the frag- mentation of collagen fibrils (103). The clistribution of glycosaminoglycans (GAG) in the dermis seems to be moclified in sun-damagecl skin ancl coulcl be linkecl to alterations of deep protein. Studies using immuno- peroxiclase staining of hyaluronic acicl ancl chonclroitin sulfate and confocal laser scanning microscopy have shown increased dermal GAGs in sun-clamaged skin. The GAGs are deposited on the elastic material of the superficial dermis and not between collagen ancl elas- tic fibers, as in normal skin (104). Hyaluronan is a ma- jor constituent of the skin extracellular matrix. Hya- luronan polymers become more tissue-associated with aclvancing age (105). Together with changes in proteins, this contributes to the pronounced alteration of the skin mechanical properties in the elderly. Lipids and cell membrane The skin barrier is linkecl to the lipicls of the inter- corneocyte space. Intercellular lipicls consist of an or- ganizecl mixture of ceramicles, sterols and fatty acicls (106, 107). The lipids in intercellular membranes form short- ancl long-periodicity lamellar phases (108). Are- cent X-ray cliffraction ancl electron macroscopy study showecl no correlation between clifferences in the or- ganization of stratum corneum lipids ancl ageing, de- spite the changes in skin properties often observecl in the elderly (109). Vitamin A (retino! ancl retinyl esters) is present in the epidermis as free ancl esterifiecl retino!. Acute ex- posure to UVA completely depletes the epidermis of vitamin A and causes lipid peroxidation. In contrast, exposure to UVB results only in the loss of vitamin A (110). The human sebaceous gland unclergoes both ex- trinsic ancl intrinsic ageing (morphological changes in the sebaceous glancl activity). The highly anclrogen-cle- penclent sebum secretion in neonates reaches its maxi- mum in young aclults. The number of sebaceous glands remains unchangecl throughout life, but sebum produc- tion tends to decrease after menopause in women and after the eighth clecacle in men. The age-clepenclent Acta Dermatoven APA Vol 11, 2002, No 3 79 Skin ageing clecrease in anclrogen leacls to a slower celi turnover in the sebaceous glancls, resulting in hyperplasia of the facial sebaceous glands. UV may contribute to this pro- cess. Molecular st:uclies have shown that overexpression of the ageing-associatecl gene Smad7 and parathor- mone-relatecl protein are linked to hyperplasia of the sebaceous glancl, but overexpression of the c-myc gene is associated with enhanced sebum procluction (111). Decreased sebum production is also responsible for skin dryness in the elderly. 5. DNA Damages and consequences The DNA of the skin is constantly submittecl to en- vironmental damage and has developed mechanisms to repail· this darnage. The balance between damage and repair has a major impact on ageing. The most com- mon cause of premature skin ageing is UV irradiation, which damages DNA through photoproducts. DNA re- pair is essential for maintaining the functional integrity of DNA. Selective repair, such as the removal of pyrimi- dine dimers, occurs in the transcribed strand. DNA is repaired by a variety of mechanisms, such as direct re- versal DNA damage for thymin dimers, base excision and mismatch repair. Nucleotide excision repair is very important for damage causecl by UV. A repair complex binds the damaged DNA, then an enclonuclease cuts it on either side of the damaged nucleotide. The original DNA sequence is resynthesized by a DNA polymerase and a ligase. The most reactive oxygen free radical, OH•, reacts with DNA bases to give altered bases, such as 8- hydrmrydeoxy guanine. These are eliminated by the DNA repait· enzyme complex, but some accumulate with ageing (112). Ageing may also result from the injury to mitochondrial DNA and peroxidation of the inner mi- tochondrial membrane lipid. Many studies have sug- gested that mtDNA suffers more from oxidative DNA damage than does nuclear DNA. Genomic and mitochondrial DNA are both intimately involved in the process of ageing. There is some de- cline in DNA repafr capacity with age. Increased DNA fragility or DNA strand breaks, chromosomal aberra- tions based on cytogenetic examination, decreased DNA methylation and changes in ploicly ali increase with age. Rearrangements, translocations, and sequence alter- ations also increase with age (113). The main type of damage generated by apparently ali types of reactive oxygen species (ROS) is oxidative changes to guanine (114) . Free radicals produce a number of lesions in DNA, damaging bases, sugar lesions, DNA-protein cross-links, causing single-strand breaks, double-strand breaks, and abasic sites by different mechanisms (115). A recent 80 review relates the contributions of stress-induced dam- age to cellular DNA: by damage to nuclear DNA and its repafr mediated by poly(ADP-ribose) polymerasel (PARP 1) damage to telomeric DNA ancl its contribution to telomere-driven celi senescence the accumulation of mutations in mitochondrial DNA (116). The effects of oxiclative stress can be dfrect or indi- rect. For example, some celi constituents (flavins, phorphyrin) , many dyes (acridines, methylene blue, neutral red) and drugs can act as photosensitizers in- side cells. The excited state of a photosensitizer can be thought of as genotoxic species similar to other free radicals, because they can directly or indirectly cause DNA modifications (4). Highly selective changes to guanine are also caused by photosensitizers that modify DNA via singlet oxygen (type II photoreaction), or one- electron oxidation (type I photoreaction) (117-120). Endogenous and exogenous oxidative stress can cause serious damages to mitochondrial DNA. Deletions of mitochondrial DNA may be used as markers of skin ageing and exposure to UV irradiation (121-124). Di- rect evidence for the increased presence of UV-induced damage in mt DNA was obtained recently. Ray et al (2000) used a PCR method to show that the number of mtDNA deletions in the epidermis is significantly asso- ciated with increased exposure to UV radiation. UV ra- diation may directly or indirectly act via free radicals to cause mutations at la bile sites in mtDNA, enhancing intra genome recombination, and increasing deletions. Mu- tations of mt DNA accumulate during ageing and in photoaged skin; the most common mutation is a 4977 base pair deletion (called common deletion). Chronic exposure of human skin to sunlight results in more mtDNA mutations than in un-exposecl skin. UVA-irra- cliation procluces singlet oxygen that generates the com- mon mutations of mitochondrial DNA that occur in photoageing (125). It is clifficult to precisely measure oxiclative DNA clamage, because extraction ancl sample treatment may cause oxidation. Various analytical techniques can be used to measure oxiclative clamage to DNA: gas chro- matography (GC) ancl liquicl chromatography (LC) with mass spectromet1y (MS) provicle positive iclentification and accurate quantification. Modified nucleosicles have been measurecl recently by methods using LC/tanc!e'i·n MS (LC/MS/MS) ancl LC/MS (112). 6. Telomerase involvement The closest thing to a cellular clock resides at tl1e tips of chromosomes. The chromosome ends, the te- lomeres , do not contain genes that program hereclitary traits, but are functional complexes. The telomeres at the encls of eukaryote chromosomes protect them from Review Acta Dermatoven APA Vol 11, 2002, No 3 Revi e w degradation or fusion. The telomeres shorten each tirne human cells divide. The celi may finally fall into a se- nescent state . Telomerase is a ribonucleoprotein that synthesizes the repeated sequences at chromosome ends and helps DNA polymerases to complete the rep- lication. New data indica te a new way to link telomeres to senescence. The telomere is a dynamic nucleopro- tein complex that can switch stochastically between an uncapped sta te and a capped state, which preserves the physical integrity of the telomere and allows celi divi- sion to proceed (126-130). It bas been suggested that celi senescence may be good because it is a defense against cancer, which is marked by uncontrolled celi division. Cells unable to regrow their telomeres stop dividing before they can cause too many mistakes. As there is no telomerase in many somatic tissues, telomere erosion may well be a major factor in celi ageing (131). Telomerase therapy might one day help generate a new supply of cells to treat age-related diseases. It bas been showed that there is a telomerase activity in the skin. It is possible to distinguish between ageing and lon- gevity. Telomere shortening is probably more involved in regulating cell longevity than in ageing stricto sensu. Ageing is due to accumulation of molecular disorders and a lack of celi energy. This loss of energy is involved in the deterioration and a gradual loss of the functional integrity ofthe tissues. Nevertheless , telomere shorten- ing (or more precisely telomerase dysfu nction) , oxida- tive damage and hormones could all be signals involved in ageing. Skin ageing The regulation of telomerase in mammalian cells is multifactorial, involving telomerase gene expression, post-translational protein-protein interactions, and pro- tein phosphorylation. Severa! proto-oncogenes and tu- mor suppressor genes are involved in the regulation of telomerase activity (132). Severa! physiological factors, like EGF and/or amphiregulin, and growth factors , can also influence telomerase (133). Telomere length and telomerase activity may determine cell senescence. Hyperoxia accelerates telomere shortening by causing oxidative stress. A reduction of stress, for example by the action of free radical scavengers , delays replicative senescence. Telomere act as a "sentinel" for oxidative damage to the genome and replicative senescence may be triggered by telomeres as a consequence of DNA damage. It is thus ve1y important to ensure that cells have sufficient telomerase (134). Guanine of the telo- merase 3' overhang (TTAGGG) can be considered asa target for reactive oxygen species or UV irradiation (135). Estrogen activates telomerase via direct and in- clirect effects . There may be hormona! control of telomerase activity. Sex steroicls may thus influence cell senescence ancl ageing (136). The activity of telomerase may be also regulatecl by the tumor suppressor protein p53; a lack of this protein may lead to increase cl telomerase activity in cancer celi development (137). Mutations of the p53 gene and telomerase activity are linkecl, ancl these mutations are consiclerecl to be UV specific (138) . The transcription factor NF-KB may act at a specific site to influence the activity of the telomerase ca talytic subunit (hTERT) T (139). More recently, Exogenous / Endogenous STRESS Telomerase interactions Mitochondrial DNA common deletion 8 oxoguanine ~ Accumulation in mitochondrial DNA ~ Mitochondrial dysfuncti on Energetic Crisis Immune system Figure 3. Consequences of external stress on the DNA. DNA-Protein crosslinks Decrease of DNA repair mechanisms - base excis_ion repair - nucleotide excision repair Thymine derivatives i DNA replication Altered Transcription l Genome instability Acta Dermatoven APA Vol 11, 2002, No 3 ------------- - - --- 91 Skin ageing telomerase and its catalytic subunit hTERT have been shown to be involved in oxidative stress, and lN irra- diation disturbs the telomerase activity in human keratinocytes (140). The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP 1) participates in the regulation of both DNA repah- and transcription. Moderation of PARP following DNA damage has also been proposed to protect skin cells from lN induced acute and chronic photodamage (141). The ageing and survival of endothelial cells are linked to molecular mechanisms that control celi pro- liferation, quiescence, apoptosis and senescence. The activation of telomerase in human dermal microvascu- lar endothelial cells also seems to affect their durability both in vitro and in vivo (142). Apoptosis Celi senescence could be, like apoptosis, a part of the body's defenses, a natura! mechanism to prevent cells from accumulating mutations with their physiologi- cal consequence, malignancy (143). The differentiation, apoptosis and senescence of keratinocytes share some molecular pathways. Epidermal differentiation and apoptosis lead to cel! death and the removal of cells by transglutaminase activation and proteolysis. Regulators of apoptosis, such as Bcl-2 (suppressor) or Bax (pro- motor), are also produced during differentiation. Se- nescent cells are cells which can no longer replicate, but can still respond to growth factors. They have !ost their ability to perform correctly tissue homeostasis. Cells lose their ability to divide early in differentiation. Figure 4. Consequences of ageing on main biological targets. 82 Some authors think that senescence is not directly linked to epidermal differentiation, due to differences in the responses to celi cycle inhibitors (144). Apoptosis is a cellular end point of the stress re- sponse. Apoptosis removes damaged cells from lN-ir- radiated tissues. The genes that control apoptosis in the epidermis, such as the bcl-2 gene, are disregulated dur- ing ageing. The decreased efficiency of apoptosis may contribute to chronological ageing and extrinsic skin ageing. Only epidermal stem cells escape cellular se- nescence. It appears that epidermal terminal differen- tiation, apoptosis and celi senescence are ali triggered by stimuli. Nevertheless, keratinocytes will undergo clas- sical epidermal differentiation or will irreversibly enter into senescence or apoptosis, depending on their state and on the nature and strength of the stimuli. Oxidative damage is a cellular stress that can cause senescence like growth arrest, or even apoptosis. Ras-induced se- nescence is also mediated by ROS, but is not clearly associated with telomere shortening. The tumor sup- pressor P16 accumulates as fibroblasts approach senes- cence, and by inhibiting its degradation, P19 indirectly mediates the growth arrest or apoptosis. P (145). 7. Morphological Variations The skin becomes thicker until maturity and then becomes thinner in women over 50-60 years old. Mea- surements of skin physical properties show that it be- comes thinner, stiffer, less tense and elastic with ageing (146). Mechanical Properties Young's modules (elasticity modules) of the skin, a ratio between stress and deformation, increases linearly with age. This is in agreement with data indicating that skin becomes more rigid and less able to stretch in re- sponse to stress with age. This has to be correlated with the increased crosslinking of collagen, the disorganiza- tion of the fibril network and the large amount of free water in the dermis. Ageing decreases skin function and causes clinical changes such as wrinkling, color changes (yellowish, patches, pigmentation), and a loss of elas- ticity (146). Sagging is one of the major age-related morphological changes in the face. While wrinkles and general changes in the face have been well studied, a recent method using photostandards and 3 D analysis of replicas shows that women's cheeks begin to sag- ging when they reach 40 (147). Recently measurements of site-related and age-dependent variations in facial skin show that there is an overall increase of skin echogenicity and thickness with age. The skin on the upper and lower lips, on the chin and infraorbital re- gions is thicker than that on the central forehead, lat- eral forehead and cheeks. The facial skin thickness be- Review Acta Dermatoven APA Vol 11, 2002, No 3 R eview Figure 5. Clinical signs of ageing on a Caucasian woman of 75 years old: presence of wrinkles, spots and sagging. comes greater over the lateral regions of the forehead, lips and nose in elderly subjects, and becomes thinner over the infraorbital regions (148). Fine lines are due to the gradual breakdown of collagen and elastin fibers, and they are exacerbated by sun damage. Very deep wrinkles are associated with the muscle below the skin surface. Muscles contract more with age to compen- sate for the loss of volume. Excessive exposure to sun- light and smoking can cause major changes in the skin (73) . The skin may darken; develop very fine wrinkles, spots, and sag , all of w hich are symptoms of photoageing. This is a very serious concern for middle- aged women, especially women in Asia. Studies using the two point gap discrimination method plus microneurographic recording in response to mechani- cal stimuli have also revealed changes in tactile spatial discrimination in the elderly (149) . The subcutis is also concerned in ageing. Ageing results in larger fat cells in the subcutaneous tissue. Hor- mone changes linked to ageing may also cause differ- ence in body fat distribution (150). Energy metabolism is also regulated via leptin, a fat cells-derived hormone, in adults. The concentration of leptin in the blood var- ies during the menstrual cycle. Leptin binding activity Skin ageing is low at birth and high in the pre-pubertal years, but it is stable during adult life and does not vary with ageing (151-153). The adipocytes also act as estradiol stores. The circulating concentration of this hormone varies with age, and is most important in mature skin. Meno- pause, the physiological cessation of menstruation caused by decreased function of the ovaries, leads to thinning of the dermis, mainly due to a decrease in the collagen content, atrophy of subcutaneous tissues and increased skin dryness (154, 155). Wrinkles Wrinkles are modifications of the skin associated with cutaneous ageing and develop preferentially on sun-exposed skin. Clinicopathological features of wrinkles were studied among the different types of skin relief modifications. Four types of skin depressions can be defined according to their depth: folds, permanent wrinkles, reducible wrinkles and skin micro-relief. De- velopment of wrinkles may be seconda1y to actinic elas- tosis and to the disappearance of microfibrils and col- lagen fibers at the dermoepidermal junction. Epider- mis is involved with a decrease of the cel! renewal, an increase of involucrin, a decrease of integrin ~ 1, type VII collagen and fibrillin 1 (156). Wrinkles are one of the major concerns for women along with spots and freckles. Principal causes of wrinkling are ageing and excessive exposure to UV rays. Wrinkles are the expres- sion of the accumulation of modifications at different levels of the skin . Development of so-called fine wrinkles begins to take place in the thilties, reaching a peak in the fifties, while deep wrinkles increase in the fifties. Little is known about the exact histological changes underlying wrinkle formation. Changes in col- lagen type I, III, type IV and VII at the DEJ have been recorded (157-160). Collagen VI, concentrated in the y= -0,051x+ 5,431 avec r = 0,571et p = 0,05 15 :š [ 4 C, :i. ::, 3 o E .!:, ,, 2 ·.:; "' " :;; o " o "' "' 30 40 50 60 70 80 Age (years) Figure 6. 14C Ascorbic acid intracellular transport in Human Normal Fibroblasts {lifting), n=12 (M. Dumas courtesy). Acta Dermatoven APA Vol 11, 2002, No 3 - -------- ------------ 83 Skin ageing papillary dermis immediately below the dermal-epider- mal junction is similar in photoprotected and photoaged skin (159). Some fibroblasts which are accumulating damages are less stimulated by surrounding ascorbic acid, resulting in a decrease of collagen and a loss of dermis density. Very recently, in aged fibroblasts from photoexposed zones, a decrease of the intracellular transport of pericellular ascorbic acid has been proposed in wrinkle formation and ellagic acid derivatives have been proposed to overturn the phenomenon (161; Marc Dumas, personal communication). The balance between the stress activated (SAPK) and mitogen activated (ERK) MAP kinase signaling pathways regulates celi growth and extracellular matrix produc- tion (ECM). SAPK activity, measured by c-jun phospho- rylation, is increased in the elderly and inhibits ECM production by activating collagenase and inhibiting collagen synthesis (162). Oxidative damage is central to skin ageing, and is particularly involved in wrinkle formation. The extracellular signal-regulated MAP Ki- nase pathway (ERK), which mediates celi responses to growth factors, is less active in old human skin in vivo. At the same tirne, the activity of the stress-activated MAP Kinase pathway (c-jun, p38 MAP Kinase) increases in old human skin in vivo. The amounts of c-Jun mRNA and protein are increased in old skin, but the amounts of c-Fos mRNA and protein are not. It has also been demonstrated that retino! activates the ERK pathway in old skin but does not alter the stress-activated c-jun ki- nase. (163). TGF ~-1 in the extracellular matrix and in keratinocyte may also be an important marker for mea- suring the efficiency of an "anti-wrinkle" treatment (164). Spots / Freckles Melanocytes are specialized cells that are located in the basal layer of the epidermis. They synthesize and transfer melanin pigments to surrounding keratinocytes, thus protecting from UV carcinogenic effects (160, 165, 166). Molecular mechanisms and celi cycle regulatory gene expression leading to melanocyte senescence and transformation differ significantly from fibroblasts. As found in other celi types , progressive telomere short- ening appears to trigger replicative senescence in nor- mal melanocytes. In melanocytes and not fibroblasts, there is a loss of p21 wat"1 and cyclin E expression. Mel- anocytes and fibroblasts present common events as an increase in p16 INK4a levels and down-regulation ofE2Fl , also shared by senescent keratinocytes. In fibroblasts, the senescent phenotype is linked to the repression of the gene c-fos, upregulation of p21 war- 1, and down-regu- lation ofE2F transcription factors. p21 wa1' 1 may be a major regulator of fibroblast senescence. (167). Adult melanocytes are able to stop to proliferate and terminally differentiated melanocytes are stili metaboli- cally active but postmitotic. The altered differentiated functions of senescent melanocyte are not well known. Alterations of melanosomes, melanin synthesizing en- zyme in mitogen activated protein kinase (MAPK), and in celi cycle progression have been reported. Ali these altered functions may have a real impact in tissue (168). The knowledge of the mechanisms of human skin color is of prime importance to develop skin care increasing the skin radiance and fighting spot formation. The change of the absorbance spectrum from reflectance including the scattering effect has been found not to correspond to the molar absorption spectrum of mela- nin and blood (169, 170). Disturbed keratinocytes-melanocytes interactions during melanosome transfer and skin melanosome dis- tribution patterns could be related to spot formation. Melanocytes located in the basal layer of epidermis pro- duce melanin-loaded melanosomes, which are distrib- uted to neighboring keratinocytes. UV radiation lead to an accumulation of melanosomes in melanocytes and treatment with MSH induces exocytosis of melano- somes. UV and MSH increase the phagocytose of mel- anosomes by keratinocytes (171). Kinesin and kinectin are motor proteins that are in- volved in some stage of melanosome transport (166; 172-17 4). Melanosome transport along the dendrite is mediated in part by microtubule proteins and myosin V, which traps melanosomes at the actin-rich periphery of the dendrite SNARE protein (soluble N- ethyl- maleimide-sensitive factor attachment protein receptors) and Rab-3a. Proteins involved in vesicle transport, dock- ing (rab 3) and membrane fusion (SNARE) seem to be involved in the changed melanosome dynamics caused by UV irradiation. This can be thought of as an exocy- tosis towards the keratinocytes Cl 75). Serine protease inhibitors that interfere w ith PAR 2 (protease-activated receptor 2) may cause depigmentation by affecting melanosome transfer and distribution (176). The for- mation of skin spots on photoexposed areas is a ve1y complex problem, but may be viewed asa double prob- lem. The direct and indirect processes stimulated by UV irradiation (endothelin-1 , thymin dimers, NO) in- crease the melanin production Cl 77-179). Their local aggregation can also generate new reactive oxygen species (180). A disturbance of celi-celi signaling (cad- herin E, P, etc) can cause melanin to be transferred to basal keratinocytes instead to suprabasal cells, result- ing in pigment accumulation (181) . Accumulation of pigment in such cells coulcl also be due to local defect of keratinization and to oxidation products, like lipo- fuscin. Changes due to UV irradiation and ageing could make these cells, pigment-loaded by error, less easy to eliminate. Furthermore, melanocytes getting the infor- mation that their protective pigment distribution pat- tem is abnormal will continue to oversynthesize pig- ment, leading to more, uncontrolled accumulation. Other biochemical factors may be responsible for spot formation. Melanogenic stimulatory factors deri ved from epidermal cells in senile freckles include endo- Review 84 --- - -------- ---- ---- ---- -------- ---Acta Dermatoven APA Vol 11, 2002, No 3 Review thelin and stem celi factor (182). Abnormal sphingo- myelin deacylase production leads to high concentra- tions of sphingosylphosphorylcholine insteacl of ceramide in the epidermis of patients w ith atopic der- matitis. The sphingosylphosphorylcholine is associated with the pigmentation defects frequently observed in atopic dermatitis (183). The question of a specific way of spot formation in mature skin linked to the hormona! balance modification is also stili open. UV-induced melanogenesis is mediated by nitric oxicle radicals. There is a clecrease in tyrosinase activity stimulated prior to NO-stimulation (184). Human melanocytes contain the mammalian melanin concentrating hormone (MCH), but human keratinocytes and fibroblasts do not. The MCH is coupled to a G protein receptor, SLCl; the inac- tivatecl complex blocks the cyclic AMP second messen- ger pathway and increases intracellular calcium. Dis- turbance of this pathway by UV irradiation or other el- ements can also favor spot formation (185). The hor- mona! control of skin pigmentation via interaction of POMC peptides (eg a-MSH, ACTH) with other local and circulating hormones may influence melanocyte func- tion, and thus coordinate the pigmenta1y response of the skin, especially after major changes in hormone con centra ti on (186). Metallothioneine, an intracellular free radical scavenger, could be induced in human mela- nocytes. Suppression of melanogenesis is partly due to the induction of metallothioneine. Cutaneous microvasculature The ageing ancl survival of endothelial cells are linked to molecular mechanisms controlling celi prolif- eration, quiescence, apoptosis and cellular senescence. The activation of telomerase in human dermal microvas- cular enclotbelial cell s is linkecl to their clurability both in vitro and in vivo. Knowledge of telomerase activity and other markers of amplifying dermal perivascular cells may reveal more about the regenerative capacity of the skin microvasculature. Telomerase activity / length seems to be directly linkecl to the angiogenic potential (130; 142; 187-190). 8. Spedjicity oj Asian Skin The formation of facial wrinkles is a sign of photoageing. A unique study of over 3000 people from five ethnic groups (Africa, American, East Asian, Cau- casian, Indian Asian ancl Latina) of different ages bas revealecl age-clependent changes of the skin (wrinkles, hyperpigmentation and pores) . The mean fraction of the face area covered with wrinkles is significantly smaller in African Americans than in Caucasians, but East Asians have the smallest wrinkled area at any given age. The authors suggest that racial differences in other genetic factors besides skin pigmentation, such as DNA Acta Dermatoven APA Vol 11, 2002, No 3 Skin ageing repair, are important in determining the development of skin wrinkles. African Americans have more hyper- pigmented spots ancl facial pores than other racial groups. Caucasians have significantly les well hyclratecl skin than African Americans, East Asians ancl Latinos (191). The relationship between skin phototype ancl deep and fine wrinkle scores on the faces of 230 Japa- nese subjects shows that sunlight-sensitive subjects have deeper wrinkles.(192) . A recent study has compared the action of sun pro- tection factor according to COLIPA recommenclations on Asian and Caucasian volunteers. The obse1vecl clif- ference in SPF is not only due to skin color but also to interna! factors affecting response of the skin to UV (193). Asian skins are not ali the same. Differences in the minimal dose causing erythema in Chinese ancl Korean subjects were also linkecl to differences in skin chromopohores (194). A stucly of 230 Japanese incli- vicluals classifiecl according to their skin phototype (type IV preclominant) suggests that cleep wrinkles are more severe in phototype I, and that there is no link between phototype ancl fine wrinkles. Skin phototype cloes not seem to be relatecl to hair or eye color. Some Japanese with dark hair ancl black eyes have sun sensitive low phototype (192). A stucly of the factors causing clark circles around the eyes in 60 healthy Japanese women indicates that the clark circles become darker as the bloocl mass in- creases, showing the importance of hemoclynamics in the area (195). A comparison of the cheek skin color of Caucasian andJapanese women shows an increase in the yellow axis with age in Japanese women, whereas there was an increase in the reci axis in Caucasians, fol- lowed by a decrease around 50 years (196) . East Asians living in Los Angeles andJapaneses living in Akita (Ja- pan) hacl similar clegrees of skin wrinkling. East Asians have less facial hyperpigmentation than Latinos, Afri- can Americans or Caucasians (191). The facial hyper- pigmentation of 56 women (aged 22 to 67 years) and melanin spot distribution showed more melanin gran- ules arouncl the nuclei in chloasma and senile pigmen- tation (197). Age-relatecl alterations in the echogenicity ofthe skin inJapanese women (130 women aged 18-83 years) were studiecl, with measurements on the fore- head, eye corners, ancl cheeks. An increase in the lower layer of the dermis probably clue to the accumulation of degraded or disarranged collagen and a decrease in echogenicity in the upper dermal layer indicate a ten- dency similar to that seen in Caucasians (198). Mea- surements of the sebum excretion rate and skin surface contours of 662 healthy Korean volunteers revealed age- related changes. The forehead/ cheek sebum excretion rates increased with age while there were changes in the skin pores and skin surface texture . The disappear- ance of prima1y lines, seconda1y lines and increased pore size with age is probably linked more to exposure to sunlight (199). As demonstrated for the Japanese Skin ageing volunteers, sunlight also modifies lipid peroxidation, and cholesterol 7-hydroperoxide is a good marker (200). According to JH Chung, the patterns of wrinkles in Asian people differ from those of Caucasians. Por ex- ample, Korean people have cleeply w rinklecl foreheacl ancl perioral areas . Wrinkles ancl pigmentary moclifica- tions are the two main characteristics of photoageing in Koreans (201). The skin of groups of 100 clifferent Asian women was compared . In Japanese the skin was in a better conclition compared to six other populations. Age-relatecl increase of sallowness was more promi- nent in Chinese ancl Korean skin, while . in Philippines the highest tenclency of combinecl skin lesions pre- vailecl (202). There are severa! inclepenclent risk factors of wrin- kles, such as age, exposure to sunlight, menopause, skin color ancl smoking. Pregnancy is another factor of risk for facial wrinkling clue to the high levels of sex hor- mone bouncl to globulin ancl low concentrations of free estradiol. Progesterone also acts as an antagonist to es- trogen. Lactation delays the return of ovulation ancl clecreases the estracliol concentration. Hormone Re- placement Therapy (HRT) clecreases the risks of wrin- kling. The clecrease in skin collagen due to estrogen cleficiency in post-menopausal women may aggravate the severity of wrinkles. Men are also subject to pig- mentation clamage, leacling to seborrheic keratosis ancl solar lentigo, ancl, as in women, it leads to depigmenta- tion (lentigo, freckles, mottled pigmentation). In 189 Korean women, a significant increase in the risk of wrinkles was found associatecl w ith an increasing num- ber of full-term pregnancies ancl menopausal age (203). A multicentric stucly carried out on 3 000 Chinese women showecl no crow's feet area prevalence in North- ern cities and a higher peri-oral ancl glabella wrinkle score in Southern cities. Only 203 women were con- cernecl at 21-25 years of wrinkles in crow's feet area, butat 36 years 75 % ofwomen were concernecl (204). Comparing 160 French and Chinese women (20-65 years) the onset of wrinkles delayecl by arouncl 10 years in Chinese women (205). From 26 to 60 years, 60% of the Chinese women exhibit pigmented spots on their face. Small facial pigmentecl spots characterize young population (18-40 years), spots of more than 6 mm di- ameter increase after 30. Whatever the age group, pig- mentecl spots are always more pronounced on the face than on the hancls. Climatic factors and chronic expo- sure of UV are suggestecl asa main cause of pigmentecl spots (206). B. E J?E.RE' 1-..r ,.-, "C,' 0 ;,/ . -~ . .J ~· "f ;z.,, ~.'J ;.,. J Conclusion The skin acts as a biosensor because it forms a large interface with our environment. It is a dynamic living barrier which is of prime importance in our social life. The recent studies describecl in this paper clearly show the complex interconnections at all levels of the tissue and provide a more predse picture of the importance of the events involved in ageing and photo-damage. It may become possible to clelay the appearance of signs of skin ageing by acting on key mechanisms revealed by research into this fascinating topic Abbreviations AGE ADP ATP DNA ECM ED] EGF FGF G6PD GAG CC GPx GTP HME HNE HRT h1ERT LC LRP MAPK MCP MMP MSH mtDNA PARP PDGF ROS SAPK SNARE TSP2 uv Advanced Glycation products Adenosine di Phosphate Adenosine tri Phosphate Desoxyribonucleic acid Extracellular Matrix Epidermal-Dermal junction Epidermal Growth Factor Fibroblast Growth Factor Glucose-6-Phosphate Deshydrogenase Glycosaminoglycans Gas Chromatography Glutathion Peroxydase Guanosine Triphosphate Human Metalloelastase 4-hydroxy-2-nonenal Hormone Replacemnt Therapy telomerase catalytic subunit Liquid Chromatography Lipoprotein receptor-related protein Mitogen Activated Protein Kinase Multicatalytic Proteinase Matrix Metalloproteinase Melanocyte stimulating Hormon Mitochondrial DNA Poly(ADP-ribose) polymerase 1 Platelet-derived growth factor Reactive Oxygen Species Stress Activated MAP K}nase Soluble N- ethylmaleimide-sensitivefactor attachment protein receptors Thrombospondin 2 Ultraviolet 1. 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A U T H O R S ' Celina Rocquet, LVMH R&D - Parjums et Cosmetiques, 185 Avenue de A D DRE S SE S Verdun,45804SaintleandeBraye,France Frederic Bonte, PhD, biochemist, same address,jbonte @diormail.com Review 94 - - --------------------------------Acta Dermatoven APA Vol 11, 2002, No 3