1. This lecture is exclusive for my web site

2. Professor & Chairman Dept of Dermatology
TREATMENT OF AGING SKIN: MOLECULAR CONSIDERATIONS AND HOW THEY INFLUENCE CLINICAL PRACTICE
Erin Boh M.D.Ph.D.
Professor & Chairman Dept of Dermatology
Tulane University Health Sciences Center
New Orleans, LA, USA
IACD; Lisbon,Portugal 2008

3. Cutaneous Aging Chronologic Aging Extrinsic Aging Genetics
Metabolic processes
Oxidation
Reduction
Glycosylation
Extrinsic Aging
Environmental exposures
UV irradiation
Toxins
Xenobiotics
Mechanical stressors
Intrinsic Aging
Hormonal changes
So, 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.

5. Dermal Matrix Homeostasis
Degradation
Amiphregulin,
betacellulin,
epiregulin, TGFα
EGFR
AP MMPs
sequestration/degradation
Synthesis
TGF/TGFR
Fibroblast
Collagen, elastin, proteoglycans, glycosaminoglycans
EGFR
TGF
In 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 degradation
TGGbeta, 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.

6. TGF- increase in fibroblast proliferation
History
1980’s:
Murine studies with subcutaneous injection of TGF-  dose and time dependent
increase in fibroblast proliferation
increase in collagen synthesis
increase in vasculogenesis
Administration of antibodies to TGF-  markedly reduced collagen synthesis
It 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.

7. TGF- 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

8. Epidermal 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
↑MMPs
AP-1

9. Cutaneous Aging Chronologic Aging Extrinsic Aging Genetics
Metabolic processes
Oxidation
Reduction
Glycosylation
Extrinsic Aging
Environmental exposures
Toxins
Xenobiotics
Mechanical stressors
UV irradiation
Intrinsic Aging
Hormonal changes
So, 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.

10. Aging Process
Intrinsic aging: dryness, laxity, fine wrinkles, atrophy

11. Aging Effects Extrinsic aging:
fine/coarse wrinkling.pigmen-tary changes
UVR
Smoking
Chemicals

12. Histologic changes of intrinsic aging
Changes in epidermal and dermal cellularity
Thinning of rete ridges
Degeneration of dermal matrix/decreased elasticity
Loss of glycoaminoglycans
Disorganization of microvasculare

13. Histologic changes of extrinsic Aging
Clinical features of photoaged skin
Wrinkles
Furrows
Dryness
Loss of elasticity
Dyspigmentation
Histologic features of aged skin
Reduced numbers of fibroblasts
Reduced amount of extracellular matrix (ECM)
Abnormal organization of ECM
Atrophy of subcutis
Most 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.

14. Dermal Matrix Homeostasis
Degradation
Amiphregulin,
betacellulin,
epiregulin, TGFα
EGFR
AP MMPs
sequestration/degradation
Synthesis
TGFß/TGFR
Fibroblast
Collagen, elastin, proteoglycans, glycosaminoglycans
EGFR
TGF
In 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 degradation
TGGbeta, 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.

15. Extrinsic Aging Ultraviolet Radiation
UV irradiation disrupts the collagen matrix by two independent pathways
Anabolic pathway
Reduces procollagen synthesis
Increase in non-functional latent TGFß protein
Downregulate expression of TGFR
Catabolic pathway
Increases matrix metalloproteinases
Auto-dimerization (activation) of EGFR
Increase in AP-1
Inhibitors of TGFß
A 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

16. SMAD 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 observe
1) Autodimerization of the EGFR (or spontaneous activation of the degradative pathway)
2) Phosphatases remain unable to dephosphorylate the activated EGFR
3) Inhibitors of Smad and TGF are synthesized
4) And AP-1 is upregulated
In 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.

17. Epidermal 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
↑MMPs
AP-1

18. SMAD inhibition

19. Molecular Consequences of Photoaging
UVR
ROS  TbRII receptor mRNA   TGF ß
ROS   SMAD 7 mRNA
SMAD 7   TGF 
ROS   AP-1(via c-Jun)
ROS   EGFR
Downregulate TGF 
Upregulate MMPs
Decreased collagen, elastin, proteoglycan synthesis
Increased matrix metalloproteinase synthesis

20. Molecular Consequences of Photoaging
UVR  ROS  ↑c-Jun
↑Activator protein 1 (AP-1) & NF-κB  ↑ MMP
( AP-1 (c-Fos & c-Jun)
MMPs ( collagenase, gelatinase,
Stromelysin-1)
Induces TIMP
Increases elastin
Increases polar gag
Decreased collagen, elastin, proteoglycan synthesis

21. Photoaging
Free radical theory of aging & photoaging UVR ROS  cellular damage Repair mechanisms: Redox enzymes Antioxidants

22. External Environmental Aging Ultraviolet Radiation
Photo-Redox Reactions
UV chromophore → 1O2 or •O2—
NADH-/NADPH
Tryptophan
Riboflavin
Trans-urocanic acid
2 •O H+ → H2O2 + O2
H2O2 + 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.

23. External Environmental Aging Ultraviolet Radiation
Photo-Redox Reactions
UV chromophore → 1O2 or •O2—
NADH-/NADPH
Tryptophan
Riboflavin
Trans-urocanic acid
2 •O H+ → H2O2 + O2
H2O2 + Fe++ → OH• + OH-- + Fe+++

24. External Environmental Aging Ultraviolet Radiation
Photo-Redox Reactions
UV chromophore → 1O2 or •O2—
NADH-/NADPH
Tryptophan
Riboflavin
Trans-urocanic acid
2 •O H+ → H2O2 + O2
H2O2 + Fe++ → OH• + OH-- + Fe+++

25. External Environmental Aging Ultraviolet Radiation
Photo-Redox Reactions
UV chromophore → 1O2 or •O2—
NADH-/NADPH
Tryptophan
Riboflavin
Trans-urocanic acid
2 •O H+ → H2O2 + O2
H2O2 + Fe++ → OH• + OH-- + Fe+++

26. External Environmental Aging Ultraviolet Radiation
Photo-Redox Reactions
UV chromophore → 1O2 or •O2—
NADH-/NADPH
Tryptophan
Riboflavin
Trans-urocanic acid
2 •O H+ → H2O2 + O2
H2O2 + Fe++ → OH• + OH-- + Fe+++
Lipid peroxidation
DNA damage

27. External Environmental Aging Ultraviolet Radiation
UVR ROS
directly interact with the phosphorylation status of growth factor signal transduction cascades
Specific growth factor functions altered
↓ cellular antioxidants
Induces apoptosis
Alters cross linking of collagen
↑ collagen degradation
↑ DNA damage
The 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.

28. Repair mechanisms: Redox enzymes -Superoxide dismutase -Catalase
-Glutathione reductase
-GSH peroxidase
Endogenous antioxidants

29. ANTIOXIDANTS Exogenous Vitamin A derivatives Vitamin C,B,E Co-enzyme Q
Endogenous
Glutathione
Vitamins C,E,A
Ubiquinol
Oxidative enzymes: catalase,superoxide dismutase,GSH peroxidases
Exogenous
Vitamin A derivatives
Vitamin C,B,E
Co-enzyme Q
AHA
Alpha lipoic acid
Peptides: Cu; NH2

30. Treatment of Photoaged Skin
Reversal of photodamage
-Replacement of antioxidants
-Absorption of UVR by chromophores
- Replacement of growth factors
-Stimulation of new dermal
matrix
Upregulation of new collagen synthesis
Inhibition of collagen breakdown

31. Treatment of Photoaged Skin
Replacement of antioxidants
Topical formulations
Vitamin A derivatives
Vitamin C,B,E
Co-enzyme Q
AHA
Alpha lipoic acid
Peptides: Cu; NH2
Endogenous
Glutathione
Vitamins C,E,A
Ubiquinol
Oxidative enzymes: catalase,superoxide dismutase,GSH peroxidases

32. Treatment of Photoaged Skin
Effective Topical Antioxidants
Agent must be in stable formulation
Must penetrate/be absorbed in sufficient concentrations
Must release reducing equivalents

33. Treatment of Photoaged Skin: Replacement of antioxidants
VITAMIN A DERIVATIVES
Vitamin A ( retinoic acid; retinol; retinaldehyde)
Tretinoin
Adapalene
Tazarotene

34. Treatment of Photoaged Skin
VITAMIN A DERIVATIVES
Antioxidant
Antiinflammatory
Upregulation of new collagen: Histologic restoration of photodamaged skin
Normalization of cellular atypia
Normalization of cellular architecture
Stimulates new collagen/elastin
Stimulates angiogenesis
Normalizes gag synthesis
Br J Dermatol 2007
Br J Dermatol 2007;157:

35. MOLECULAR MECHANISMS OF PHOTOAGING
Tretinoin effects
UVR changes
Induces c-Jun ÞMMP
Induces TIMP
Induces elastin mRNA
Increases gag ( -SO4)
Inhibits c-Jun induction: no ↑ MMP
No effect on TIMP
Inhibits elastin mRNA
Normalizes gag

36. TRETINOIN: Net Effects
Thin atrophic epidermis with atypical architecture & cellularity
Altered dermis
Net Effects: Molecular Inhibition of collagen breakdown
New collagen synthesis
Increased dermal ECM

37. TRETINOIN: Net effects
Net Effects: Clinical
Smooth skin
Less pigmentary
alterations
Less wrinkling/laxity
of skin

38. Botanicals: Soy isoflavones Pycnogenol Green tea extracts Vitamin C
TREATMENT OF PHOTOAGED SKIN:
Replacement of antioxidants
ANTIOXIDANTS
Botanicals: Soy isoflavones
Pycnogenol
Green tea extracts
Vitamin C
Vitamin E
CoQ-10
Alpha lipoic acid

39. Antioxidants VITAMIN C Cofactor for collagen synthesis
VITAMIN E
Naturally occurring
Inhibits UVR induced lipid peroxidation
Replenishes Vitamin C, GSH
VITAMIN C
Cofactor for collagen synthesis
Reduces UVR induced changes
Photoprotection
Some formulations unstable
Dermatologic Therapy 2007;20:

40. Treatment of Photoaged skin
Antioxidants
Alpha Lipoic Acid
Involved in oxidative metabolism
ROS scavenger ( inhibits lipid peroxidation)
ß NF-B
Chelates metals
Regenerates vitamins E & C
Br J Dermatol 2003;149: 841-9

41. BOTANICALS Green tea extracts: (polyphenols) Isoflavones: Antioxidant
Anti-inflammatory; inhibits UVR damage
Allantoin: Antiinflammatory
promotes repair
Isoflavones: Antioxidant
Increases skin thickness
May inhibit melanosome phago cytosis
Pycnogenol: Reduces Vit. C
Dermatologic Therapy 2007;20:
Dermatplogic Therapy 2007;20:
PhotodermatolPhotoimmunol Photomed 2007;23:

42. Replacement of Growth Factors/Stimulate cytokines
Treatment of Photoaged skin
Replacement of Growth Factors/Stimulate cytokines
Transforming growth factor 
Epidermal growth factor (receptor)
Keratinocyte growth factor
Fibroblast growth factor
Platelet-derived growth factor
Vascular endothelial growth factor
Dermatologic Therapy 2007;20:
Dermatologic Surgery 2006;32:

43. Topical TGF- Products
Well tolerated
Minimizes appearance of rhytides when compared to Vitamin C
Few studies at molecular level of reversal of photodamage
TNS Recovery Complex
Bio-restoratiive Skin Cream
Cell rejuvenation serum ( CRS)
Dermatolog Surg 2006;32:
Dermotologic Therapy 2007;20:

44. CLINICAL STUDIES WITHMULTIPLE GROWTH FACTOR COSMECEUTICALS
Study Design:
14 Female subjects
2 Months twice daily
Facial wrinkle scoring
(9-point score)
Silicon replica
Histology
J Cosmetic & Laser Ther 2003, 5: 25-34
 TNS Recovery Complex® (SkinMedica) 44

45. CLINICAL STUDIES WITH MULTIPLE GROWTH FACTOR COSMECEUTICALS
Study Design:
18 Female subjects
2 Months twice daily,
followed by 2 week wash-out
Facial wrinkle scoring
(5-point score)
J Drugs Dermatol 2007, 6:
 BIO-RESTORATIVE SKIN CREAM WITH PSP® (NEOCUTIS) 45

46. Proposed Mechanism of Action of Growth Factor Cosmeceuticals
Large hydrophilic molecules unlikely to penetrate skin barrier but may penetrate through follicles, sweat glands and damaged skin
Produce epidermal signaling cytokines which affect dermal fibroblasts
Mechanism is not elucidated

47. CONCLUSIONS
Human growth factor cosmeceuticals are effective for skin rejuvenation
Multiple growth factor cosmeceuticals are particularly beneficial as demonstrated in several clinical studies
Growth factor cosmeceuticals may be also helpful
to improve outcome after cosmetic and dermatologic procedures
Additional studies warranted to further elucidate promising technology: double blinded placebo controlled studies -both clinical and at molecular level 47

48. Topical treatment of Photoaged Skin
Sunscreens as protection against ongoing damage and combination of
Tretinoin Alpha hydroxy acids
Vitamin C
Vitamin E
Botanical antioxidants
Growth factor cosmeceuticals

49. QUESTIONS TO BE ASKED OF COSMECEUTICALS
Can agent penetrate?
Does agent have MOA in target tissue?
Is there peer-reviewed data to support claim?

50. PERSPECTIVES FOR PHOTOAGING
Provide patient with sound reasonable advise about cosmeceuticals
Prospective objective studies are needed

51. CONCLUSIONS
Popularity of noninvasive treatments of photoaging ( cosmeceuticals) useful when care is taken to construct a treatment regimen based on understanding of the molecular mechanisms in place
Patient needs to have realistic expectations and know limitations of products

52. CONCLUSIONS 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

53. Pentapeptide( KTTKS) Lys-Thr-Thr-Lys-Ser ↑collagen synthesis
Copper Peptides
Wound healing
↑collagen synthesis

54. Environemental Affects Upon Cutaneous Aging
Internal Environmental Factor
Estrogen status
Chronologic Aging
Oxidative metabolism
Chronologic 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 Factors
UVR
Tobacco