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HOW THE EPIDERMIS AND DERMIS INTERACT WITH EACH OTHER JULIAN M MENTER PhD Department of Microbiology, Biochemistry and Immunology Morehouse School of Medicine Atlanta, GA. LIGHT MICROSCOPY OF NORMAL HUMAN SKIN • The epidermis and dermis are commonly considered to be two separate entities… • …However, there is actually a great deal of interaction between the two layers (Some) Examples of Epidermal/Dermal Interactions in the Ground State Blood Vessels in Dermis Enter the Epidermis as Capillaries (1) EXAMPLES OF EPIDERMAL/DERMAL INTERACTIONS (2) • Epidermal Vascular Endothelial Growth Factor Production Is Required for Permeability Barrier Homeostasis, Dermal Angiogenesis, and the Development of Epidermal Hyperplasia • Implications for the Pathogenesis of Psoriasis • Peter M. Elias,* Jack Arbiser,† Barbara E. Brown,* et al • Am J Pathol. 2008 Sep; 173(3): 689–699. • doi: 10.2353/ajpath.2008.080088 • PMCID: PMC2527083 • Interactions between Whisker Dermal Papillae and Epidermis RESPONSES OF ORAL EPITHELIUM TO THE INFLUENCE OF WHISKER DERMAL PAPILLAE IN THE ADULT RAT R. F. OLIVER* Arch oral Bid. Vol. 18p, p.4 134211, (1973) The EXPERIMENTAL INDUCTION OF WHISKER GROWTH IN THE HOODED RAT BY IMPLANTATION OF DERMAL PAPILLAE R. F. OLIVER J Embryol Exp Morphol, 18 (1967), pp. 43–51 “Whisker dermal papillae developed various epidermal modulations, including generalized hyperplasia and the development of spikes of keratinized cells, hair follicles, sebaceous gland cells and stellate reticulum-like configurations reminiscent of the tooth germ.” Nitric Oxide in Skin (1) • NO plays a key role in orchestrating the skin's response to external stimuli such as heat, ultraviolet (UV) light, response to infection, and wound healing, as well as possibly underlying certain pathological conditions • MM Cals-Grierson, OD Ormerod Nitric Oxide 2004 (4) 179 - 193 Nitric Oxide in Skin • Keratinocytes, constitutively express the neuronal isoform of NO synthase (NOS1), • Fibroblasts in the dermis and other cell types in the skin express the endothelial isoform (NOS3). • Under certain conditions, virtually all skin cells appear to be capable of expressing the inducible NOS isoform (NOS2) • MM Cals-Grierson, OD Ormerod Nitric Oxide 2004 (4) 179 - 193 Melanin Couples Reaction of NO with Oxygen Menter JM, Nokkaew C, Eatman D, Sprewell A, Silvestrov N, Patta AM et al., Open J Phys Chem 3:157–162 (2013). (Some) Examples of Epidermal/Dermal Interactions in the Excited State Solar Spectrum (Wehrli, 1985) 2.0 2 Irradiance (W/cm /nm) 2.5 1.5 1.0 0.5 Wavelength (nm) vs W/cm2/nm 0.0 200 300 400 500 600 Wavelength (nm) 700 800 1 2 Irradiance (W/cm /nm) Solar Spectrum (Wehrli, 1985) 0.1 Wavelength (nm) vs W/cm2/nm 200 300 400 500 600 Wavelength (nm) 700 800 THE SOLAR SPECTRUM Long Wave UVA Augments UV Sunburn Damage EFFECTS OF LONG ·ULTRAVIOLET RAYS ON HUMAN SKIN: PHOTOPROTECTIVE OR PHOTOAUGMENTATIVE?* ISAAC WILLIS, M.D., ALBERT KLIGMAN, M.D., Ph.D., AND JOHN EPSTEIN, M.D. T HE JOURNAL OF INVESTIGATI VE DERMATOLOGY Vol. 59, No. 6, 416 – 420 (1972) ABSTRACT: “The effects of long ultraviolet (LUV) rays on human skin are generally believed to be either harmless or protective against sunburn damage. Responses to LUV alone, LUV plus sunburn radiation, and sunburn radiation alone were evaluated both clinically and microscopically by routine histologic and autoradiographic techniques. Observations revealed that contrary to previous accepted hypotheses, LUV rays have a striking augmentative effect on sunburn damage. In addition, although LUV radiation alone causes no discernable adverse clinical or histologic changes in skin , an effect on DNA metabolism does occur…” Long Wave UVA Augments UV Photocarcinogenesis The rapid induction of cancers in the hairless mouse utilizing the principle of photoaugmentation. Willis I, Menter JM, Whyte HJ. J Invest Dermatol. 1981 May;76(5):404-8 • • Abstract Exposure to highly erythemogenic, but otherwise clinically noninjurious, dose of broad spectrum (290-400 nm) ultraviolet light is increased by 20% every 6th day. Clinical and histological observations reveal the presence of squamous cell cancer after as little as 18 days of irradiation. The rate of cancer induction is enhanced by the 320-400 nm component and this enhancement is shown to be a photoaugmentative effect. The results support the idea that stratum corneum and/or malpighian layer thickening produced in early stages of tumor induction tends to protect against the detrimental effects of UV radiation. Strict monitoring of both the spectral distribution and output of the radiation source is imperative for reproducible rates of tumor induction. UVA and UVB Radiation Have Different, Separate, Molecular Targets. UVA – (mainly) dermal targets UVB – (mainly) epidermal targets Important Epidermal Target Chromophores • DNA (Major Significance) • (a) Pyridine 2 + 2 Photodimers • (b) 6 - 4 Photoproducts • KERATIN AND OTHER PROTEINS: • Photoproducts essentially unknown • Melanin: • These absorb strongly at wavelengths • < 300 nm Important Difference Between UVB and UVA – Mediated Reactions: • UVB Reactions generally do not require molecular O2, and are mostly “direct” reactions • UVA Reactions generally do require molecular O2 and are “sensitized” reactions Important Difference Between UVB and UVA – Mediated Reactions: • UVB Reactions are generally in the epidermis, • UVA reactions are mainly in the dermis Action Spectra for CPD Dimers, Skin Cancer, and 8-Oxoguanine Production Rünger TM1, Kappes UP..Mechanisms of mutation formation with long-wave ultraviolet light (UVA) .Photodermatol Photoimmunol. Photomed.2008 Feb;24 Feb;24(1):2-10. doi: 10.1111/j.1600-0781.2008.00319.x SOME IMPORTANT DERMAL TARGETS • COLLAGEN: • (a) Aromatic amino acids on teloptide region and their postranslational products • (b) Peptide linkage (UVC) • ELASTIN • (a) Desmosine • (b) Amino acids (see above) • PROTEINS, DRUGS, ENDOGENOUS, CHROMOPHORES ,(e.g. Riboflavin, porphyrins, flavins and melanin) Some UVA Sensitizers in the Dermis Peter Karran, Reto Brem Protein oxidation, UVA and human DNA repair DNA Repair 2016 Aug 44 178 - 185 “The Situation is Complicated” • • • Summary “Long-wave ultraviolet (UV) A light is able to damage DNA, to cause mutations, and to induce skin cancer, but the exact mechanisms of UVA-induced mutation formation remain a matter of debate. While pyrimidine dimers are well established to mediate mutation formation with shortwave UVB, other types of DNA damage, such as oxidative base damage, have long been thought to be the premutagenic lesions for UVA mutagenesis. However, pyrimidine dimers can also be generated by UVA, and there are several lines of evidence that these are the most important premutagenic lesions not only for UVB- but also for UVAinduced mutation formation. C→T transition mutations, which are generated by pyrimidine dimers, are called UV-signature mutations. They cannot be interpreted to be solely UVB-induced, as they are induced by UVA as well. Furthermore, there is no consistent evidence for a separate UVA-signature mutation that is only generated with UVA*. We hypothesize that a weaker anti-mutagenic cellular response, but not a different type of DNA damage, may be responsible for a higher mutation rate per DNA photoproduct with UVA, as compared with UVB.” • Rünger TM1, Kappes UP..Mechanisms of mutation formation with long-wave ultraviolet light (UVA).Photodermatol, Photoimmunol. Photomed.2008 Feb;24(1):2-10. doi: 10.1111/j.16000781.2008.00319.x. • *This lack of “completely separate” UVA and UVB signature mutations seems to illustrate yet another aspect of dermal/epidermal interactions. JMM Optical Properties of Skin PENETRATION OF LIGHT INTO SKIN Type I Collagen is a Prognosticator of Skin Damage • Fluorescence Spectral and (Photo)chemical Changes are Functions of: • • • • Time Temperature Age of sample Previous history Telopeptides are Non – Helical Portions and the N- and C- Terminal Ends of the Collagen Molecule • (a) Telopeptides have an antiparallel β – pleated sheet* • (b) Telopeptides are high in tyrosine and phenylalanine residues, low in arginine and (hydroxy)lysine residues** • (c) Tyrosine residues in favorable position to form dimers, “excimers” and higher oxidation products** • (d) Telopeptides (Mainly N- telopeptides) are necessary for fibrillogenesis • • * D. Helseth et al, Biopolymers 18 3005 – 3014 (1979) * *A.L. Rubin et al, Science 139 37 – 38 (1963) Tyrosine Can be Degraded by Thermal Oxidation or UV radiation. Degraded Collagen Has Different Fluorescence Properties than Normal Spectral Differences in Calf Skin Collagen Depend on Previous History Lot# 159 “New” (obtained Feb 2012 and used July 2012) 80 Wavelength (nm) vs Icorr#121 Wavelength (nm) vs Icorr#159 60 40 20 0 300 320 340 360 380 Wavelength (nm) 400 420 440 Corrected Fluorescence Intensity (a.u.) Corrected Fluorescence Intensity (a.u.) Lot# 121 (5 years old; kept in refrigerator in dark at 4o C) 80 60 Wavelength (nm) vs Icorr#159 Wavelength (nm) vs Icorr#121 40 20 0 300 320 340 360 380 Wavelength (nm) 400 420 440 Temperature Dependence of Collagen Photochemical Fluorescence Fading • (1) Fluorescent State (radiative) not the same as the photochemical state (radiationless) • (2) In general, fluorescence intensity decreases with temperature with concomitant increase of photochemical or other radiationless transistions (e.g. vibration) back to the ground state. Effect of Temperature on Fading and Fluorescence Emission • Higher Temperatures Favor Photochemical Reaction (“Fading”) and Disfavor Fluorescence Emission • Lower Temperatures Favor Fluorescence Emission and Disfavor Photochemical Fading Tyrosine Dimerization to Dityrosine Decreased in Autoxidized Collagen • Fresh Collagen was irradiated with UVC (254 nm) as a function of time. • Black Dots - “aged” collagen sample • White Dots - fresh collagen sample • See O Shimizu Photochem Photobiol 18(3) 123 – 133, 1973 Photodestruction of DOPA Oxidation Product is Increased in Autoxidized Collagen • Oxidized Sample “aged” in dark at 4 C for ~ 5 years was irradiated with UVC (254 nm) as a function of time • Black Dots – “aged” collagen sample • White Dots - new collagen sample Collagen as a Prognosticator of Skin Damage • Fluorescence spectral and photochemical behavior indicate relative stability. The more the spectra differ from a “tyrosine – like” spectrum, the less stable they tend to be. • Abnormal amounts of certain fluorescent species (e.g. dityrosine, AGE’s, DOPA oxidation products can reflect pathological conditions (e.g. diabetes, skin cancer, collagen vascular diseases) HOW ARE THE VARIOUS COMPONENTS ORGANIZED? The ECM has an organization where ECM structural protein and polysaccharide components are linked to different cells. Cells can then sense changes to the ECM. The main organized components are the adhesive glycoprotein: 1. Fibronectin 2. Laminin PROTEOGLYCAN & GAGS Preliminary Data • Excess Hyaluronate Does Not Seem to Affect Collagen Fading Rate. Possible Reasons: • Tyrosine is buried in hydrophobic region of telopeptide • Photochemical reaction involves free radical mechanism • Collagen/HA molecules are not packed close enough to each other as they are in skin. Acknowledgements • • • • • • • Isaac Willis M.D. Abrienne M Patta Natalya Silvestrov Latoya Freeman Otega Edukuye Gina Chu Many Other Colleagues • Supported in part by • DOD Grant # W911NF-10-1-0445 from the US Army Research Office • RCMI Grant # 8G12 MD007602 to Morehouse School of Medicine