2Professor & Chairman Dept of Dermatology TREATMENT OF AGING SKIN: MOLECULAR CONSIDERATIONS AND HOW THEY INFLUENCE CLINICAL PRACTICEErin Boh M.D.Ph.D.Professor & Chairman Dept of DermatologyTulane University Health Sciences CenterNew Orleans, LA, USAIACD; Lisbon,Portugal 2008
3Cutaneous Aging Chronologic Aging Extrinsic Aging Genetics Metabolic processesOxidationReductionGlycosylationExtrinsic AgingEnvironmental exposuresUV irradiationToxinsXenobioticsMechanical stressorsIntrinsic AgingHormonal changesSo, WHY do we age? Cutaneous aging was historically classified into three separate entities; chronologic aging which describes our inherent genetic program, and the insults accrued over the course of ones lifetime just to keep them alive, namely processes involved in metabolism.There is extrinsic aging, which includes the environmental exposures which damage us. And, intrinsic aging, which has mainly been studied in the context of hormonal status.
5Dermal Matrix Homeostasis DegradationAmiphregulin,betacellulin,epiregulin, TGFαEGFRAP MMPssequestration/degradationSynthesisTGF/TGFRFibroblastCollagen, elastin, proteoglycans, glycosaminoglycansEGFRTGFIn order to best understand how environmental insults modify dermal homeostasis, its important that we are familiar with the normal processes.Dermal matrix homeostasis can be divided into two categories: synthesis and degradationTGGbeta, binding to its receptor, the TGFR, stimulates synthesis of proteins and glycoproteins in the dermis.In contrast, EGFR ligands (of which there are many) bind to the EGFR and stimulate synthesis of proteins which both interfere with synthetic functions and promote degradation.Without interference, both processes peacefully coexist with activity levels that create an equilibrium between synthesis and degradation of dermal tissues.
6TGF- increase in fibroblast proliferation History1980’s:Murine studies with subcutaneous injection of TGF- dose and time dependentincrease in fibroblast proliferationincrease in collagen synthesisincrease in vasculogenesisAdministration of antibodies to TGF- markedly reduced collagen synthesisIt was in the 1980’s that investigators first discovered the importance of TGFbeta in dermal homeostasis. Murine studies were performed using subcutaneous injection of TGFbeta, finding a dose and time dependent increase in fibroblast number, collagen content, and vasculogenesis. Co-administration of antibodies to TGFbeta blunted the effect, confirming the relationship between TGFbeta and dermal collagen synthesis.
7TGF- Signaling SMAD-P Elastin CollagenGAGs TGFR This graphic is important as it reveals properties about TGF beta signalling that we’ll return to later. Intracellular signaling ensues when TGF beta becomes unassociated with its latency protein (depicted here as the red curved bar). This association between Latency associated protein and TGFbeta is a competitive one, the greater the quantity of latency associated protein, the less available the active TGF molecule. TGF once released, binds to the TGFbeta receptor, which then dimerizes. Dimerization activates the phosphorylation of SMAD2,3 protein, which enters the nucleus and activates transcription of genes for elastin, collagen, glycosaminoglycans, and additional TGFbeta receptors.Elastin CollagenGAGs TGFR
8Epidermal growth factor receptor As I stated before, the phosphorylation of the EGFR can occur spontaneously, upon exposure to oxidative pressure generated through UV radiation, which alter intrinsic tyrosine kinase activity. The dimerized receptor, now activated, initiates an intracellular cascade of events culminating in transcription of “activator-protein-1” (AP-1). AP-1s effects are which stimulates reduced function of TGF and increased synthesis of products responsible for matrix degradation, the matrix metalloproteinases.↓TGF↑MMPsAP-1
9Cutaneous Aging Chronologic Aging Extrinsic Aging Genetics Metabolic processesOxidationReductionGlycosylationExtrinsic AgingEnvironmental exposuresToxinsXenobioticsMechanical stressorsUV irradiationIntrinsic AgingHormonal changesSo, WHY do we age? Cutaneous aging was historically classified into three separate entities; chronologic aging which describes our inherent genetic program, and the insults accrued over the course of ones lifetime just to keep them alive, namely processes involved in metabolism.There is extrinsic aging, which includes the environmental exposures which damage us. And, intrinsic aging, which has mainly been studied in the context of hormonal status.
10Aging ProcessIntrinsic aging: dryness, laxity, fine wrinkles, atrophy
12Histologic changes of intrinsic aging Changes in epidermal and dermal cellularityThinning of rete ridgesDegeneration of dermal matrix/decreased elasticityLoss of glycoaminoglycansDisorganization of microvasculare
13Histologic changes of extrinsic Aging Clinical features of photoaged skinWrinklesFurrowsDrynessLoss of elasticityDyspigmentationHistologic features of aged skinReduced numbers of fibroblastsReduced amount of extracellular matrix (ECM)Abnormal organization of ECMAtrophy of subcutisMost of us are well versed in both the clinical and histological features of aged skin listed here. Typically, the description of aged skin assumes that environmental contributions have caused insult overtime. Without additional insults insults that disrupt the cellular repair capacity, many of these features never become not present. Rather, in isolated chronologic aging, more frequently there is loss of subcutaneous fat, mild thinning of the epidermis, and scant alteration in elastic tissue. It is when additional environmental factors are superimposed, that change cellular function specifically as it relates to the balance in synthesis and degradation of dermal tissue, that we witness what we call “aged” skin. So, beyond each of these features is a mechanism of action…a molecular pathway that is driven away from homeostasis.
14Dermal Matrix Homeostasis DegradationAmiphregulin,betacellulin,epiregulin, TGFαEGFRAP MMPssequestration/degradationSynthesisTGFß/TGFRFibroblastCollagen, elastin, proteoglycans, glycosaminoglycansEGFRTGFIn order to best understand how environmental insults modify dermal homeostasis, its important that we are familiar with the normal processes.Dermal matrix homeostasis can be divided into two categories: synthesis and degradationTGGbeta, binding to its receptor, the TGFR, stimulates synthesis of proteins and glycoproteins in the dermis.In contrast, EGFR ligands (of which there are many) bind to the EGFR and stimulate synthesis of proteins which both interfere with synthetic functions and promote degradation.Without interference, both processes peacefully coexist with activity levels that create an equilibrium between synthesis and degradation of dermal tissues.
15Extrinsic Aging Ultraviolet Radiation UV irradiation disrupts the collagen matrix by two independent pathwaysAnabolic pathwayReduces procollagen synthesisIncrease in non-functional latent TGFß proteinDownregulate expression of TGFRCatabolic pathwayIncreases matrix metalloproteinasesAuto-dimerization (activation) of EGFRIncrease in AP-1Inhibitors of TGFßA Study by Quan et al in 2004 showed that UVA radiation reduces procollagen synthesis by 75% within only 8 hours of UV exposure. Quan identified that this disruption in the anabolic pathway was due to UV induced increases in synthesis of latent TGFbeta protein, and downregulation of the TGFR.Quan also identified that UV irradiation upregulates the transcription of genes coding for matrix metalloproteinases, and it does so by inducing oxidative pressures which drive autodimerization (activation) of the EGFR, which drives synthesis of AP-1 and inhibitors of TGFbeta.Quan T, et al. American Journal of Pathology, vol 165, 9/ Xu Y , et al. Journal of Biological Chemistry, Vol 281, 9/ Wan Y, et al. Cellular Signalling, Vol 13, 2/2001
16SMAD inhibition TGF inhibition So, if we were to combine the intracellular events occurring with the oxidative pressures caused by either UV radiation or tobacco smoke we’d observe1) Autodimerization of the EGFR (or spontaneous activation of the degradative pathway)2) Phosphatases remain unable to dephosphorylate the activated EGFR3) Inhibitors of Smad and TGF are synthesized4) And AP-1 is upregulatedIn addition, the excess latent TGF binding protein decreases the active TGF available to bind TGF receptors, and there is a decrease in the presence of TGF receptors.The sum of these events is an increase in MMP synthesis, a decrease in collagen synthesis, and a dwindling of the dermal matrix.
17Epidermal growth factor receptor As I stated before, the phosphorylation of the EGFR can occur spontaneously, upon exposure to oxidative pressure generated through UV radiation, which alter intrinsic tyrosine kinase activity. The dimerized receptor, now activated, initiates an intracellular cascade of events culminating in transcription of “activator-protein-1” (AP-1). AP-1s effects are which stimulates reduced function of TGF and increased synthesis of products responsible for matrix degradation, the matrix metalloproteinases.↓TGF↑MMPsAP-1
21PhotoagingFree radical theory of aging & photoaging UVR ROS cellular damage Repair mechanisms: Redox enzymes Antioxidants
22External Environmental Aging Ultraviolet Radiation Photo-Redox ReactionsUV chromophore → 1O2 or •O2—NADH-/NADPHTryptophanRiboflavinTrans-urocanic acid2 •O H+ → H2O2 + O2H2O2 + Fe++ → OH• + OH-- + Fe+++The generation of UV-induced reactive oxygen species has been well described.In the first reaction,UV absorbing chromophores present in the dermis, such as the NADH system, amino acids like tryptophan, B vitamins, and transurocanic acid to name a few, absorb energy and transfer it to molecular oxygen, generating a superoxide anion. The electrons are shuttled and the reaction self-propagates until, in the end, a highly reactive hydroxyl radical is generated.
27External Environmental Aging Ultraviolet Radiation UVR ROSdirectly interact with the phosphorylation status of growth factor signal transduction cascadesSpecific growth factor functions altered↓ cellular antioxidantsInduces apoptosisAlters cross linking of collagen↑ collagen degradation↑ DNA damageThe generation of the hydroxyl radical is important because it represents the first domino taking the plunge. The preferred path of least resistance for energy transfer alters the phosphorylation state of growth factors that are crucial to dermal matrix homeostasis, namely TGFbeta and EGFR.
30Treatment of Photoaged Skin Reversal of photodamage-Replacement of antioxidants-Absorption of UVR by chromophores- Replacement of growth factors-Stimulation of new dermalmatrixUpregulation of new collagen synthesisInhibition of collagen breakdown
31Treatment of Photoaged Skin Replacement of antioxidantsTopical formulationsVitamin A derivativesVitamin C,B,ECo-enzyme QAHAAlpha lipoic acidPeptides: Cu; NH2EndogenousGlutathioneVitamins C,E,AUbiquinolOxidative enzymes: catalase,superoxide dismutase,GSH peroxidases
32Treatment of Photoaged Skin Effective Topical AntioxidantsAgent must be in stable formulationMust penetrate/be absorbed in sufficient concentrationsMust release reducing equivalents
33Treatment of Photoaged Skin: Replacement of antioxidants VITAMIN A DERIVATIVESVitamin A ( retinoic acid; retinol; retinaldehyde)TretinoinAdapaleneTazarotene
34Treatment of Photoaged Skin VITAMIN A DERIVATIVESAntioxidantAntiinflammatoryUpregulation of new collagen: Histologic restoration of photodamaged skinNormalization of cellular atypiaNormalization of cellular architectureStimulates new collagen/elastinStimulates angiogenesisNormalizes gag synthesisBr J Dermatol 2007Br J Dermatol 2007;157:
35MOLECULAR MECHANISMS OF PHOTOAGING Tretinoin effectsUVR changesInduces c-Jun ÞMMPInduces TIMPInduces elastin mRNAIncreases gag ( -SO4)Inhibits c-Jun induction: no ↑ MMPNo effect on TIMPInhibits elastin mRNANormalizes gag
36TRETINOIN: Net Effects Thin atrophic epidermis with atypical architecture & cellularityAltered dermisNet Effects: Molecular Inhibition of collagen breakdownNew collagen synthesisIncreased dermal ECM
37TRETINOIN: Net effects Net Effects: ClinicalSmooth skinLess pigmentaryalterationsLess wrinkling/laxityof skin
38Botanicals: Soy isoflavones Pycnogenol Green tea extracts Vitamin C TREATMENT OF PHOTOAGED SKIN:Replacement of antioxidantsANTIOXIDANTSBotanicals: Soy isoflavonesPycnogenolGreen tea extractsVitamin CVitamin ECoQ-10Alpha lipoic acid
39Antioxidants VITAMIN C Cofactor for collagen synthesis VITAMIN ENaturally occurringInhibits UVR induced lipid peroxidationReplenishes Vitamin C, GSHVITAMIN CCofactor for collagen synthesisReduces UVR induced changesPhotoprotectionSome formulations unstableDermatologic Therapy 2007;20:
40Treatment of Photoaged skin AntioxidantsAlpha Lipoic AcidInvolved in oxidative metabolismROS scavenger ( inhibits lipid peroxidation)ß NF-BChelates metalsRegenerates vitamins E & CBr J Dermatol 2003;149: 841-9
42Replacement of Growth Factors/Stimulate cytokines Treatment of Photoaged skinReplacement of Growth Factors/Stimulate cytokinesTransforming growth factor Epidermal growth factor (receptor)Keratinocyte growth factorFibroblast growth factorPlatelet-derived growth factorVascular endothelial growth factorDermatologic Therapy 2007;20:Dermatologic Surgery 2006;32:
43Topical TGF- Products Well toleratedMinimizes appearance of rhytides when compared to Vitamin CFew studies at molecular level of reversal of photodamageTNS Recovery ComplexBio-restoratiive Skin CreamCell rejuvenation serum ( CRS)Dermatolog Surg 2006;32:Dermotologic Therapy 2007;20:
45CLINICAL STUDIES WITH MULTIPLE GROWTH FACTOR COSMECEUTICALS Study Design:18 Female subjects2 Months twice daily,followed by 2 week wash-outFacial wrinkle scoring(5-point score)J Drugs Dermatol 2007, 6: BIO-RESTORATIVE SKIN CREAM WITH PSP® (NEOCUTIS)45
46Proposed Mechanism of Action of Growth Factor Cosmeceuticals Large hydrophilic molecules unlikely to penetrate skin barrier but may penetrate through follicles, sweat glands and damaged skinProduce epidermal signaling cytokines which affect dermal fibroblastsMechanism is not elucidated
47CONCLUSIONSHuman growth factor cosmeceuticals are effective for skin rejuvenationMultiple growth factor cosmeceuticals are particularly beneficial as demonstrated in several clinical studiesGrowth factor cosmeceuticals may be also helpfulto improve outcome after cosmetic and dermatologic proceduresAdditional studies warranted to further elucidate promising technology: double blinded placebo controlled studies -both clinical and at molecular level47
48Topical treatment of Photoaged Skin Sunscreens as protection against ongoing damage and combination ofTretinoin Alpha hydroxy acidsVitamin CVitamin EBotanical antioxidantsGrowth factor cosmeceuticals
49QUESTIONS TO BE ASKED OF COSMECEUTICALS Can agent penetrate?Does agent have MOA in target tissue?Is there peer-reviewed data to support claim?
50PERSPECTIVES FOR PHOTOAGING Provide patient with sound reasonable advise about cosmeceuticalsProspective objective studies are needed
51CONCLUSIONSPopularity of noninvasive treatments of photoaging ( cosmeceuticals) useful when care is taken to construct a treatment regimen based on understanding of the molecular mechanisms in placePatient needs to have realistic expectations and know limitations of products
52CONCLUSIONS Assess the patient relative to what bothers patient Recognize that each of these therapies offers mild to modest effects but may prove more efficacious when used in combination
54Environemental Affects Upon Cutaneous Aging Internal Environmental FactorEstrogen statusChronologic AgingOxidative metabolismChronologic aging, a bit of a redundant term, is the umbrella agent, the background upon which all additional contributions to aging occur. Chronologic aging describes the inherent unique genetic program carried by mammalian cells. There is an expected lifetime output of each cell that is tied to the pre-programmed cellular repair capacity. How cells handle free radical formation during oxidative metabolism provides a good example. These free-radical byproducts, which are a necessary consequence of generating energy for life, are, although not perfectly, in large part quenched by antioxidant enzymatic processes built-in to the cell.I will focus most of the discussion on a few factors encountered over a lifetime that accelerate the aging process by either exhausting cell-repair capacity, as we’ll see with external environmental factors, namely UV radiation and tobacco smoke; or by directly altering cell signaling capacity, which occurs with the internal environmental alteration that is secondary to the menopausal state, estrogen deprivation.My goal is to make clear that each of these independent environmental factors, whether external or internal, overlap pathophysiologically as they converge upon this. A final common pathway involved in maintaining dermal homeostasis.External Environmental FactorsUVRTobacco