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Montagna Symposium 2013—Light and Skin: How Light Sustains, Damages, Treats, Images and Modifies Skin Biology

      The 62nd Annual Montagna Symposium on the Biology of Skin, “Light and Skin: How Light Sustains, Damages, Treats, Images and Modifies Skin Biology,” was held from October 10 to 14, 2013, in Stevenson, Washington. Life and skin evolved under sunlight; dermatology will forever be entwined with photomedicine. We routinely use microscopy, optical diagnostics, phototherapy, photodynamic therapy, and lasers while dealing with melanoma, non-melanoma skin cancer, photosensitivity disorders, and photoaging. Moreover, there are misunderstandings, recent surprises, mysteries, and challenges. How do the protean cutaneous and systemic effects of light really happen? Exactly what is healthy and unhealthy about light? Can these effects be separated? What is the ideal sunscreen? What can we “see” inside live skin? Which technologies are pushing the limits for therapy and diagnosis? How to “translate” promising research all the way to practical impact? The Symposium was organized by Molly Kulesz-Martin and chaired by Rox Anderson with Session Chairs David Fisher, Barbara A. Gilchrest, and Steven Jacques.
      The meeting started (and ended) with provocative talks by British dermatologist-scientists. Antony Young delivered the keynote address, “Impact of Climate Change on Skin,” describing the first large prospective database regarding human behavior, sun exposure, sunscreen use, and biomarkers for UV exposure. He reported large prospective, collaborative studies measuring solar UV exposure, hydroxy-vitamin D3 levels, and biomarkers of DNA damage and repair in “free-ranging” Europeans at home and on holiday. The subjects kept detailed logs of their activities and sunscreen use, wore a watch-like monitor of solar UV exposure, and provided blood and urine samples. Sun exposure was highly correlated with thymidine dimer products in the urine and significantly correlated with circulating 25-hydroxy-vitamin D3 levels, and both were decreased with sunscreen use. Surprisingly, the slope of the solar exposure–vitamin D3 dose-response curve was independent of skin pigmentation, suggesting that substantial vitamin D3 photochemistry occurs in the upper epidermis above the heavily pigmented basal cell layer.
      Session Chair David Fisher presented the molecular, cell, and skin biology underlying pigmentation and melanoma. The UV-induced melanogenesis cascade includes keratinocyte synthesis of pro-opiomelanocortin macropeptide, which is cleaved to produce both MSH (melanocyte-stimulating hormones) and endorphins. Mice (much like Young’s Europeans) become addicted to UV exposure and actively choose it, a preference that was suppressed when endorphin receptors were blocked by naloxone. Why has a systemic system for sun-seeking behavior evolved, with skin at its center? Melanocortin receptor (MC1R) sequence polymorphisms that ablate function lead to red hair and increased susceptibility to UV-induced skin cancers. In mice, forskolin, a cAMP agonist, bypasses the MC1R defect and leads to the production of eumelanin. Potentially, phosphodiesterase PDE4D3 inhibitors that increase cAMP levels in humans could reduce melanoma risk in redheads. But how does MC1R deficiency increase skin cancer risk? Mice lacking pheomelanin (deficient in both MC1R and tyrosinase) are less susceptible, strongly suggesting that pheomelanin per se enhances UV-independent and UV-induced skin cancers. Furthermore, increased DNA oxidation products were found without any UV exposure in the red-haired mice. These results from the Fisher lab suggest that pheomelanin elevates production of reactive oxygen species, which can drive UV-induced mutations and skin cancer, and that antioxidant protection strategies may work best in redheads.
      Emi Nishimura reported that targeting c-kit produces temporary graying of hair in mice, without loss of follicular melanocytes. In contrast, aging decreases and eventually depletes the melanocyte stem cells (MSCs) in the bulge region of hair follicles, after which the hair remains permanently white. Mutation and/or genetic instability appears to have a central role in this. Aging and X-ray exposure are associated with increased expression of γH2AX, a DNA damage marker, in MSCs of the hair follicle bulge. Nishimura hypothesized that follicular MSCs may have unusual susceptibility to ionizing radiation damage, and subsequently found that activated MSCs are less sensitive to X-ray than nonactivated MSCs. The mechanisms involved may shed light on hair graying, including its prevention and reversal.
      Andrew Borkowski reported mechanisms by which damage-associated molecular pathways (DAMPs), in particular small noncoding RNAs, are important for skin-barrier maintenance. Toll-like receptor 3 (TRL3) is a target for DAMPs. Inhibition of TLR3 caused transient profound decrease of skin-barrier response genes. In particular, UVB exposure damages the skin barrier and stimulates a robust epidermal barrier repair response. Richard Gallo further discussed the role of DAMPs in mediating UV-induced immunosuppression and apoptosis. Keratinocytes not exposed to UV but to media from UV-exposed cells exhibit a bystander injury response, which can be blocked by addition of RNAse or TLR3 inhibitors. Gallo noted that the skin-commensal bacterium S. epidermidis partially inhibits some TLR3-mediated responses, presumably conferring tolerance and perhaps some abrogation of UV responses. Could TLR3 inhibitors treat certain photosensitivity diseases—such as lupus erythematosus—with DAMP-mediated pathophysiology?
      UV- and light-induced genotoxicity occurs from multiple mechanisms, including primary DNA photochemistry, oxidative photoproducts, delayed damage from endogenous response pathways, and faulty repair. Sergiy Kyryachenko introduced the topic of keratinocyte-derived factors, such as alpha-melanocyte-stimulating hormonesand endothelin, in promoting melanoma, and Arup Indra discussed RXR retinoid receptors in modulating innate immunity and cell survival after UV exposure. Sancy Leachman discussed the complex mechanisms and action spectrum for UV-induced genotoxicity, with emphasis on oxidative damage. Nrf-2 is part of a MC1R-mediated pathway that regulates antioxidant genes. In MC1R-deficient mice, Nrf-2 is inactive and antioxidant gene expression is reduced. Leachman reported a series of studies showing that sulforaphane, an antioxidant contained in broccoli sprouts, restores Nrf-2 activity through interaction with a protein called Keap-1.
      Session Chair Barbara Gilchrest spoke about the presumptive role of telomeres in UV responses. She noted that the disruption of the telomere loop experimentally can initiate DNA damage–like signaling, resulting in cell senescence (permanent inability to divide) or apoptosis depending on the cell type. Because telomeres in all mammalian species are tandem repeats of TTAGGG and its complement, the telomere is an excellent target for DNA damage: thymidine dinucleotides, the substrate for perhaps 70% of UVB damage; and guanine, the target for oxidative DNA damage and major target for UVA irradiation. The extensive damage introduced during UV irradiation or the influx of repair proteins might therefore disrupt the telomere loop, leading to DNA damage signaling that, if sufficiently severe, would result in cell senescence and apoptosis in the skin, consistent with photoaging, or if less severe to p53-mediated protective responses such as the well-documented enhanced melanogenesis (tanning) and the upregulation of DNA repair capacity. As further support for a central role of telomeres in cellular response to UV, identical responses can be induced by treatment of cells or intact skin with telomere homolog oligonucleotides that interact with telomeric DNA to activate the identical signaling pathway.
      Craig Elmets discussed the role of cyclooxygenase (COX) genes in mediating UV-induced skin cancer. UV induces COX-2 and downstream prostaglandin E2, a major inflammatory cytokine. In mice, knocking out COX-2 or administering the selective inhibitor celecoxib significantly reduces UV-induced skin cancer. In a randomized controlled clinical trial of celecoxib in >200 skin cancer–prone subjects, there was no significant reduction in actinic keratosis, but non-melanoma skin cancers were significantly reduced by more than 50%. Topical COX-2 inhibitors could possibly be developed for skin cancer reduction in organ transplant recipients.
      Which proteases have a role in photoaging? Thomas Ruenger reported that solar elastosis involves cathepsin K, a lysosomal enzyme that is debatably the most potent of human collagenases. Cathepsin K knockout mice exhibit hypertrophic scarring. Cathepsin K also processes elastin, which accumulates in photoaged dermis. In fibroblasts from young individuals, but not old individuals, cathepsin K was induced by UVA (not UVB). Other disorders exist in which abnormal extracellular proteins accumulate during aging due to the failure of lysosome functioning as chaperone protein-mediated autophagy decreases. In the Hutchinson Gilford progeria syndrome, a protein called progerin accumulates. Runger reported that UVA (again, not UVB) induced accumulation of progerin in normal skin fibroblasts, along with cell nuclear changes similar to Hutchinson Gilford progeria syndrome. Preventive strategies may emerge from this research.
      A promising new tool for evaluating the pathogenesis of various types of skin inflammation was presented by Phillip Tong. He described a cutaneous “immune atlas” created by using intravital multiphoton microscopy (MPM) to visualize fluorescently tagged immune populations in reporter mice. He showed three-dimensional imaging of immune cells infiltrating living mouse skin, and quantified macrophages, dermal dendritic cells, mast cells, and T cells in defined locations in the skin at multiple body sites.
      Session Chair Steven Jacques led a tour through imaging technologies introducing a superb session on optical imaging, microscopy, and spectroscopy. He explained the energy source and wavelength-dependent ability to use light in order to probe tissue structure on the nano-, micro-, and meso-scales. In particular, second-harmonic-generation imaging nanoscale sensing (1–10 nm) and its dependence on asymmetrical structure was explained. Light scattering as a contrast mechanism, offering macroscale sensing (50–10 μm), was discussed with examples of monitoring collagen fibrils transition to larger collagen fiber bundles.
      The intrinsic optical absorption and scattering properties of skin can be determined from external measurements. Anthony Durkin is using spatial light modulation over a range of wavelengths to noninvasively map the oxygen saturation of cutaneous blood. He found that the detection of cutaneous burn injury depth was successful in a swine model, even for burns deeper than the eye could see.
      In vivo microscopy is painless and harmless, and yields images immediately without artifacts, but lacks the advantage of specific tissue staining. Haishan Zeng presented impressive images of skin by coregistering reflectance confocal microscopy (RCM), MPM, and Raman spectroscopy (RS). RCM has been in steady use for live human skin imaging for more than a decade. RS interrogates molecular structure, e.g., of small metabolites, lipids, nucleic acids, and proteins. Zeng discussed microscopes that combine RCM for structural imaging with MPM for identification of elastin, NADH, collagen, and other molecules, and with RS for point determination of chemical structures.
      Impressive progress of RCM toward clinical applications was presented by Milind Rajadhyaksha. Tumor cell nuclei are easily seen when aluminum chloride or acetic acid is applied as contrast agent for about 1 minute. For basal cell carcinoma, RCM has sensitivity of 92–100% and specificity of 88–97%. Rajadhyaksha proposed a “real-time” analog of Mohs surgery, using RCM to guide an erbium (Er) laser for tissue removal. Imaging was possible through the thin layer of thermal injury left by Er laser, and a false color rendition of RCM images was able to mimic the familiar appearance of hematoxylin and eosin (H&E). Daniel Gareau presented computer vision algorithms to differentiate histologically identified melanomas from benign nevi. Using a novel polar representation of dermoscopy image metrics, neural network training was performed on a set of 88 lesions, which yielded 10 metrics significantly correlated with melanoma. Next, in a prospective set of 60 melanomas and 60 nevi, the neural network achieved 100% sensitivity and 30% specificity, which is much better than that expected for a commercially available mole-imaging device.
      Optical coherence tomography (OCT) is an optical analog to ultrasound imaging, detecting back-scattered light as a function of depth in tissue. Blood flow modulates the back-scattered light, and Ruikang Wang showed remarkable images of skin microvasculature obtained by Doppler optical coherence tomography. Another strategy for label-free imaging of perfusion is speckle modulation. Speckle is an interference pattern of back-scattered light from coherent (laser) sources, which moves when the light-scattering objects move, e.g., with blood flow. Overall, this session showed that huge strides have been made in the imaging field, and that these techniques are heading toward clinical applications.
      At the Saturday banquet, John Parrish presented via video (now available at www.MontagnaSymposium.org) an entertaining and substantial personal history of the birth of sunscreens, PUVA, narrow-band UVB, and dermatological lasers, including rich stories that sparked (literally) reminiscences of early lasers in the clinic.
      The final day focused on emerging optical therapies. Rox Anderson discussed target-selective optical treatments, including the use of 1720 nm laser pulses preferentially absorbed by sebum lipids as a potential treatment for acne. Another strategy uses light-absorbing gold nanoshells forced into the glands by a topical route, followed by laser exposure. In collaborative studies, the nanoshells were driven into sebaceous glands by mechanical vibration then exposed to a near infrared laser resulting in local thermal damage to sebaceous glands, with clinical improvement. Conor Evans presented outstanding 3D images using coherent antistokes Raman scattering of lipids in live-mouse sebaceous glands obtained in situ without stains. After exposing mouse ears to cold temperatures, Evans and colleagues showed that sebocytes in vivo suffer selective injury as a result of cytoplasmic lipid crystallization.
      The potential therapeutic role of “ultrashort” lasers was considered. Although there is but one picosecond laser now in dermatological use (for tattoo removal), there are no femtosecond (fs) lasers in dermatology practice (for anything). Focused fs lasers are capable of very precise dermal cutting without thermal damage. Tracy (Hequn) Wang discussed the potential of fs laser pulses for skin treatment, showing local tissue effects near a laser focus within intact skin. A system that simultaneously provides skin imaging by MPM and precise cutting of the imaged tissue appears to be feasible.
      Highly effective treatment of microvascular skin malformations, especially portwine stains (PWS), remains an elusive goal. Evidence is growing that hypoxia after pulsed dye laser (PDL) treatment may induce unwanted angiogenesis. PDL treatment of neonates less than about 3 months old is much more effective than treatment of older children or adults with PWS. A novel suggestion was made during discussion that fetal hemoglobin, which confers greater tolerance to hypoxia, may be responsible. Studies at the University of California, Irvine by Kristen Kelly, Wesley Moy, and others have shown better response when angiogenesis inhibitors (rapamycin, imiquimod) are administered before and after PDL treatment. The addition of photosensitizing drugs also enhances efficacy. Wesley Moy reported that intravenous administration of the photodynamic photosensitizer NPe6 produces significantly greater vessel clearance after PDL in animal studies, whereas Dr Kelly discussed new clinical evidence. Several photodynamic therapy drugs have been shown to be effective for PWS after intravenous administration and light exposure. Laser speckle modulation imaging was also used to monitor local microvascular blood flow during PDL treatment of PWS. A laser fluence-dependent immediate decrease in perfusion was observed representing microvascular injury. However, loss of perfusion was patchy, with reperfusion noted in some areas that could be reduced by multiple “passes” of laser treatment. At present, it remains unknown whether local perfusion is a reliable guide during PWS treatment.
      Fractional laser treatments use a narrow laser beam to create an array of small zones of thermal damage that heal rapidly without scarring, stimulating a tissue remodeling process. Ablative fractional CO2 or Er lasers make an array of vertical microchannels, extending to controllable depths in the skin. Merete Haedersdal is using these microchannels as conduits for topical drug delivery. In ex vivo human skin, there was a very large increase in the transport of small and large hydrophilic molecules. A study was performed comparing clinical cure rates of actinic keratosis lesions by PDT using topical methyl-aminolevulinic acid and red light-emitting diode light sources with vs. those without fractional Er laser to assist the drug uptake. There was a significantly greater clearance, with significantly reduced recurrence, after fractional laser-assisted drug delivery. Considering that there are little data available about fractional laser-assisted uptake for the host of topical medications available in dermatology, caution was advised.
      A critical update on fractional laser treatments was presented by Dieter Manstein. Photoaging, scars, tattoos, drug-induced pigmentation, and some pigmentary disorders respond. Manstein suggested a new strategy in which the microchannels created by laser ablation are used to deliver a different laser wavelength that would otherwise not penetrate well into skin. He reported that a large volume of deep dermal or even subcutaneous tissue can be affected this way. Dr Manstein is exploring fractional laser treatment in other organs, i.e., fibrosis in heart and liver.
      The meeting ended with something controversial and something hopeful. Properly revealed, changes in appearance motivate people to actually use sunscreen. Trevor Jones summarized a pilot study in which 112 adults attending a SnowSports convention were briefly shown UV camera images of themselves that highlighted photodamage. More than three-fourth of the 41% who replied 2 months later reported greater sun avoidance and sunscreen use.
      Richard Weller delivered a surprising and controversial talk. High blood pressure, the leading Disability Adjusted Life Years risk factor for death and disability, is correlated with latitude (r2=0.26) and season. Evidence is mounting that UVA exposure of skin at solar levels decreases blood pressure, probably through nitric oxide (NO) pathways. Weller and colleagues previously reported in this journal that UV could release NO stores in skin, independent of NO synthetases (
      • Mowbray M.
      • McLintock S.
      • Weerakoon R.
      • et al.
      Enzyme-independent NO stores in human skin: quantification and influence of UV radiation.
      ). An NO-activated fluorescent imaging probe revealed that UVA releases a dose- and time-dependent pool of NO, primarily in the upper epidermis. UVA exposure to subjects’ forearms produces a local increase in cutaneous blood flow, but a decrease in the peripheral vascular resistance may not be the dominant mechanism for UVA-induced decrease in blood pressure. In 24 adults exposed to UVA, blood pressure was lowered, along with concomitant decreases in circulating nitrate and increases in circulating nitrite.
      A final brainstorming session was stimulated by William Ju’s presentation on how to foster and support innovative, impactful treatments for patients with skin conditions. There is a jagged continuum of steps including problem definition, scientific discovery, invention, investment, product development, regulatory approval, commercialization, access (including affordability), and finally patient use. Our skin research community has outstanding diversity, scientific substrate, creativity, and resourcefulness but is challenged to navigate this jagged pathway.
      All told, the 2013 Montagna Symposium brought together a group of skin scientists as diverse, interactive, and provocative as photobiology itself. This field is poised to make major contributions and fits John Parrish’s definition of dermatology—“the skin and everything in it.” Unprecedented optical diagnostics are on clinical dermatology’s threshold, and so are novel light therapies. I sincerely thank the organizers, cochairs, speakers, poster authors, sponsoring organizations, and all who attended, especially students and residents.

      Society for Investigative Dermatology Eugene M. Farber Travel Awards For Young Investigators

      Andrew W. Borkowski, University of California, San Diego; Marcus Calkins, PhD, Oregon Health & Science University; Daniel Gareau, PhD, The Rockefeller University; Jodi L. Johnson, PhD, Northwestern University; Kivanc Kose, PhD, Memorial Sloan-Kettering Cancer Center; Rajan Kulkarni, MD, PhD, University of California, Los Angeles; Wesley Moy, MS, University of California, Irvine; Philip L. Tong, MD, Centenary Institute, Royal Prince Albert Hospital/University of Sydney; Tracy (Hequn) Wang, PhD, University of Washington.
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      ACKNOWLEDGMENTS

      The Montagna Symposium on the Biology of Skin is supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases and the National Institute of Aging (R13 AR009431). Other 2013 supporters included: Advancing Innovation in Dermatology; Cynosure; the Epidermolysis Bullosa Medical Research Foundation; The Procter & Gamble Company; The Company of Biologists on behalf of the journals Development, Journal of Cell Science, The Journal of Experimental Biology, Disease Models & Mechanisms, and Biology Open; Curtis T. Thompson, MD and Associates; the National Alopecia Areata Foundation; Carl Zeiss Microscopy; the OHSU Knight Cancer Institute; the Orentreich Family Foundation; and John A. Parrish, MD. The Montagna Symposium on the Biology of Skin, directed by Molly F. Kulesz-Martin, PhD, is an annual nonprofit scientific meeting that was inaugurated in 1950 by William Montagna, PhD, and gathers leading cutaneous biologists and dermatologists to discuss new findings, techniques, and goals in skin biology. Initiated in 2013 were two new travel awards for young investigators sponsored by the Japanese Society for Investigative Dermatology (details at MontagnaSymposium.org), podcast interviews, and blogs available at http://www.scilogs.com/jid/category/meeting-notes/.

      REFERENCES

        • Mowbray M.
        • McLintock S.
        • Weerakoon R.
        • et al.
        Enzyme-independent NO stores in human skin: quantification and influence of UV radiation.
        J Invest Dermatol. 2009; 129: 834-842