Sewon Kang

Pathophysiology of premature skin aging induced by ultraviolet light

BACKGROUND Long-term exposure to ultraviolet irradiation from sunlight causes premature skin aging (photoaging), characterized in part by wrinkles, altered pigmentation, and loss of skin tone. Photoaged skin displays prominent alterations in the collagenous extracellular matrix of connective tissue. We investigated the role of matrix-degrading metalloproteinases, a family of proteolytic enzymes, as mediators of collagen damage in photoaging. METHODS We studied 59 whites (33 men and 26 women, ranging in age from 21 to 58 years) with light-to-moderate skin pigmentation, none of whom had current or prior skin disease. Only some of the participants were included in each of the studies. We irradiated their buttock skin with fluorescent ultraviolet lights under standard conditions and obtained skin samples from irradiated and nonirradiated areas by keratome or punch biopsy. In some studies, tretinoin and its vehicle were applied to skin under occlusion 48 hours before ultraviolet irradiation. The expression of matrix metalloproteinases was determined by in situ hybridization, immunohistology, and in situ zymography. Irradiation-induced degradation of skin collagen was measured by radioimmunoassay of soluble cross-linked telopeptides. The protein level of tissue inhibitor of matrix metalloproteinases type 1 was determined by Western blot analysis. RESULTS A single exposure to ultraviolet irradiation increased the expression of three matrix metalloproteinases -- collagenase, a 92-kd gelatinase, and stromelysin -- in skin connective tissue and outer skin layers, as compared with nonirradiated skin. The degradation of endogenous type I collagen fibrils was increased by 58 percent in irradiated skin, as compared with nonirradiated skin. Collagenase and gelatinase activity remained maximally elevated (4.4 and 2.3 times, respectively) for seven days with four exposures to ultraviolet irradiation, delivered at two-day intervals, as compared with base-line levels. Pretreatment of skin with tretinoin (all-trans-retinoic acid) inhibited the induction of matrix metalloproteinase proteins and activity (by 70 to 80 percent) in both connective tissue and outer layers of irradiated skin. Ultraviolet irradiation also induced tissue inhibitor of matrix metalloproteinases-1, which regulates the enzyme. Induction of the inhibitor was not affected by tretinoin. CONCLUSIONS Multiple exposures to ultraviolet irradiation lead to sustained elevations of matrix metalloproteinases that degrade skin collagen and may contribute to photoaging. Treatment with topical tretinoin inhibits irradiation-induced matrix metalloproteinases but not their endogenous inhibitor.

CTLA4Ig-mediated blockade of T-cell costimulation in patients with psoriasis vulgaris

Engagement of the B7 family of molecules on antigen-presenting cells with their T cell-associated ligands, CD28 and CD152 (cytotoxic T lymphocyte-associated antigen-4 [CTLA-4]), provides a pivotal costimulatory signal in T-cell activation. We investigated the role of the CD28/CD152 pathway in psoriasis in a 26-week, phase I, open-label dose-escalation study. The importance of this pathway in the generation of humoral immune responses to T cell-dependent neoantigens, bacteriophage phiX174 and keyhole limpet hemocyanin, was also evaluated. Forty-three patients with stable psoriasis vulgaris received 4 infusions of the soluble chimeric protein CTLA4Ig (BMS-188667). Forty-six percent of all study patients achieved a 50% or greater sustained improvement in clinical disease activity, with progressively greater effects observed in the highest-dosing cohorts. Improvement in these patients was associated with quantitative reduction in epidermal hyperplasia, which correlated with quantitative reduction in skin-infiltrating T cells. No markedly increased rate of intralesional T-cell apoptosis was identified, suggesting that the decreased number of lesional T cells was probably likely attributable to an inhibition of T-cell proliferation, T-cell recruitment, and/or apoptosis of antigen-specific T cells at extralesional sites. Altered antibody responses to T cell-dependent neoantigens were observed, but immunologic tolerance to these antigens was not demonstrated. This study illustrates the importance of the CD28/CD152 pathway in the pathogenesis of psoriasis and suggests a potential therapeutic use for this novel immunomodulatory approach in an array of T cell-mediated diseases.

Injury enhances TLR2 function and antimicrobial peptide expression through a vitamin D–dependent mechanism

An essential element of the innate immune response to injury is the capacity to recognize microbial invasion and stimulate production of antimicrobial peptides. We investigated how this process is controlled in the epidermis. Keratinocytes surrounding a wound increased expression of the genes coding for the microbial pattern recognition receptors CD14 and TLR2, complementing an increase in cathelicidin antimicrobial peptide expression. These genes were induced by 1,25(OH)2 vitamin D3 (1,25D3; its active form), suggesting a role for vitamin D3 in this process. How 1,25D3 could participate in the injury response was explained by findings that the levels of CYP27B1, which converts 25OH vitamin D3 (25D3) to active 1,25D3, were increased in wounds and induced in keratinocytes in response to TGF-beta1. Blocking the vitamin D receptor, inhibiting CYP27B1, or limiting 25D3 availability prevented TGF-beta1 from inducing cathelicidin, CD14, or TLR2 in human keratinocytes, while CYP27B1-deficient mice failed to increase CD14 expression following wounding. The functional consequence of these observations was confirmed by demonstrating that 1,25D3 enabled keratinocytes to recognize microbial components through TLR2 and respond by cathelicidin production. Thus, we demonstrate what we believe to be a previously unexpected role for vitamin D3 in innate immunity, enabling keratinocytes to recognize and respond to microbes and to protect wounds against infection.

New insights into the management of acne: an update from the Global Alliance to Improve Outcomes in Acne group.

The Global Alliance to Improve Outcomes in Acne published recommendations for the management of acne as a supplement to the Journal of the American Academy of Dermatology in 2003. The recommendations incorporated evidence-based strategies when possible and the collective clinical experience of the group when evidence was lacking. This update reviews new information about acne pathophysiology and treatment-such as lasers and light therapy-and relevant topics where published data were sparse in 2003 but are now available including combination therapy, revision of acne scarring, and maintenance therapy. The update also includes a new way of looking at acne as a chronic disease, a discussion of the changing role of antibiotics in acne management as a result of concerns about microbial resistance, and factors that affect adherence to acne treatments. Summary statements and recommendations are provided throughout the update along with an indication of the level of evidence that currently supports each finding. As in the original supplement, the authors have based recommendations on published evidence as much as possible.

Retinoic acid inhibits induction of c-Jun protein by ultraviolet radiation that occurs subsequent to activation of mitogen-activated protein kinase pathways in human skin in vivo.

Human skin is exposed daily to solar ultraviolet (UV) radiation. UV induces the matrix metalloproteinases collagenase, 92-kD gelatinase, and stromelysin, which degrade skin connective tissue and may contribute to premature skin aging (photoaging). Pretreatment of skin with all-trans retinoic acid (tRA) inhibits UV induction of matrix metalloproteinases. We investigated upstream signal transduction pathways and the mechanism of tRA inhibition of UV induction of matrix metalloproteinases in human skin in vivo. Exposure of human skin in vivo to low doses of UV activated EGF receptors, the GTP-binding regulatory protein p21Ras, and stimulated mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38. Both JNK and p38 phosphorylated, and thereby activated transcription factors c-Jun and activating transcription factor 2 (ATF-2), which bound to the c-Jun promoter and upregulated c-Jun gene expression. Elevated c-Jun, in association with constitutively expressed c-Fos, formed increased levels of transcription factor activator protein (AP) 1, which is required for transcription of matrix metalloproteinases. Pretreatment of human skin with tRA inhibited UV induction of c-Jun protein and, consequently, AP-1. c-Jun protein inhibition occurred via a posttranscriptional mechanism, since tRA did not inhibit UV induction of c-Jun mRNA. These data demonstrate, for the first time, activation of MAP kinase pathways in humans in vivo, and reveal a novel posttranscriptional mechanism by which tRA antagonizes UV activation of AP-1 by inhibiting c-Jun protein induction. Inhibition of c-Jun induction likely contributes to the previously reported prevention by tRA of UV induction of AP-1-regulated matrix-degrading metalloproteinases in human skin.

c-Jun–dependent inhibition of cutaneous procollagen transcription following ultraviolet irradiation is reversed by all-trans retinoic acid

The aged appearance of skin following repeated exposure to solar ultraviolet (UV) irradiation stems largely from damage to cutaneous connective tissue, which is composed primarily of type I and type III collagens. We report here that a single exposure to UV irradiation causes significant loss of procollagen synthesis in human skin. Expression of type I and type III procollagens is substantially reduced within 24 hours after a single UV exposure, even at UV doses that cause only minimal skin reddening. Daily UV exposures over 4 days result in sustained reductions of both type I and type III procollagen protein levels for at least 24 hours after the final UV exposure. UV inhibition of type I procollagen synthesis is mediated in part by c-Jun, which is induced by UV irradiation and interferes with procollagen transcription. Pretreatment of human skin in vivo with all-trans retinoic acid inhibits UV induction of c-Jun and protects skin against loss of procollagen synthesis. We have reported previously that UV irradiation induces matrix-degrading metalloproteinases in human skin and that pretreatment of skin with all-trans retinoic acid inhibits this induction. UV irradiation, therefore, damages human skin connective tissue by simultaneously inhibiting procollagen synthesis and stimulating collagen breakdown. All-trans retinoic acid protects against both of these deleterious effects and may thereby retard premature skin aging.

Solar Ultraviolet Irradiation Reduces Collagen in Photoaged Human Skin by Blocking Transforming Growth Factor-β Type II Receptor/Smad Signaling

Ultraviolet (UV) irradiation from the sun reduces production of type I procollagen (COLI), the major structural protein in human skin. This reduction is a key feature of the pathophysiology of premature skin aging (photoaging). Photoaging is the most common form of skin damage and is associated with skin carcinoma. TGF-beta/Smad pathway is the major regulator of type I procollagen synthesis in human skin. We have previously reported that UV irradiation impairs transforming growth factor-beta (TGF-beta)/Smad signaling in mink lung epithelial cells. We have investigated the mechanism of UV irradiation impairment of the TGF-beta/Smad pathway and the impact of this impairment on type I procollagen production in human skin fibroblasts, the major collagen-producing cells in skin. We report here that UV irradiation impairs TGF-beta/Smad pathway in human skin by down-regulation of TGF-beta type II receptor (TbetaRII). This loss of TbetaRII occurs within 8 hours after UV irradiation and precedes down-regulation of type I procollagen expression in human skin in vivo. In human skin fibroblasts, UV-induced TbetaRII down-regulation is mediated by transcriptional repression and results in 90% reduction of specific, cell-surface binding of TGF-beta. This loss of TbetaRII prevents downstream activation of Smad2/3 by TGF-beta, thereby reducing expression of type I procollagen. Preventing loss of TbetaRII by overexpression protects against UV inhibition of type I procollagen gene expression in human skin fibroblasts. UV-induced down-regulation of TbetaRII, with attendant reduction of type I procollagen production, is a critical molecular mechanism in the pathophysiology of photoaging.

Inhibition of Type I Procollagen Synthesis by Damaged Collagen in Photoaged Skin and by Collagenase-Degraded Collagen in Vitro

Type I and type III procollagen are reduced in photodamaged human skin. This reduction could result from increased degradation by metalloproteinases and/or from reduced procollagen synthesis. In the present study, we investigated type I procollagen production in photodamaged and sun-protected human skin. Skin samples from severely sun-damaged forearm skin and matched sun-protected hip skin from the same individuals were assessed for type I procollagen gene expression by in situ hybridization and for type I procollagen protein by immunostaining. Both mRNA and protein were reduced ( approximately 65 and 57%, respectively) in photodamaged forearm skin compared to sun-protected hip skin. We next investigated whether reduced type I procollagen production was because of inherently reduced capacity of skin fibroblasts in severely photodamaged forearm skin to synthesize procollagen, or whether contextual influences within photodamaged skin act to down-regulate type I procollagen synthesis. For these studies, fibroblasts from photodamaged skin and matched sun-protected skin were established in culture. Equivalent numbers of fibroblasts were isolated from the two skin sites. Fibroblasts from the two sites had similar growth capacities and produced virtually identical amounts of type I procollagen protein. These findings indicate that the lack of type I procollagen synthesis in sun-damaged skin is not because of irreversible damage to fibroblast collagen-synthetic capacity. It follows, therefore, that factors within the severely photodamaged skin may act in some manner to inhibit procollagen production by cells that are inherently capable of doing so. Interactions between fibroblasts and the collagenous extracellular matrix regulate type I procollagen synthesis. In sun-protected skin, collagen fibrils exist as a highly organized matrix. Fibroblasts are found within the matrix, in close apposition with collagen fibers. In photodamaged skin, collagen fibrils are shortened, thinned, and disorganized. The level of partially degraded collagen is approximately 3.6-fold greater in photodamaged skin than in sun-protected skin, and some fibroblasts are surrounded by debris. To model this situation, skin fibroblasts were cultured in vitro on intact collagen or on collagen that had been partially degraded by exposure to collagenolytic enzymes. Collagen that had been partially degraded by exposure to collagenolytic enzymes from either bacteria or human skin underwent contraction in the presence of dermal fibroblasts, whereas intact collagen did not. Fibroblasts cultured on collagen that had been exposed to either source of collagenolytic enzyme demonstrated reduced proliferative capacity (22 and 17% reduction on collagen degraded by bacterial collagenase or human skin collagenase, respectively) and synthesized less type I procollagen (36 and 88% reduction, respectively, on a per cell basis). Taken together, these findings indicate that 1) fibroblasts from photoaged and sun-protected skin are similar in their capacities for growth and type I procollagen production; and 2) the accumulation of partially degraded collagen observed in photodamaged skin may inhibit, by an as yet unidentified mechanism, type I procollagen synthesis.

Collagen Fragmentation Promotes Oxidative Stress and Elevates Matrix Metalloproteinase-1 in Fibroblasts in Aged Human Skin

Aged human skin is fragile because of fragmentation and loss of type I collagen fibrils, which confer strength and resiliency. We report here that dermal fibroblasts express increased levels of collagen-degrading matrix metalloproteinases-1 (MMP-1) in aged (>80 years old) compared with young (21 to 30 years old) human skin in vivo. Transcription factor AP-1 and alpha2beta1 integrin, which are key regulators of MMP-1 expression, are also elevated in fibroblasts in aged human skin in vivo. MMP-1 treatment of young skin in organ culture causes fragmentation of collagen fibrils and reduces fibroblast stretch, consistent with reduced mechanical tension, as observed in aged human skin. Limited fragmentation of three-dimensional collagen lattices with exogenous MMP-1 also reduces fibroblast stretch and mechanical tension. Furthermore, fibroblasts cultured in fragmented collagen lattices express elevated levels of MMP-1, AP-1, and alpha2beta1 integrin. Importantly, culture in fragmented collagen raises intracellular oxidant levels and treatment with antioxidant MitoQ(10) significantly reduces MMP-1 expression. These data identify positive feedback regulation that couples age-dependent MMP-1-catalyzed collagen fragmentation and oxidative stress. We propose that this self perpetuating cycle promotes human skin aging. These data extend the current understanding of the oxidative theory of aging beyond a cellular-centric view to include extracellular matrix and the critical role that connective tissue microenvironment plays in the biology of aging.

Effects of administration of a single dose of a humanized monoclonal antibody to CD11a on the immunobiology and clinical activity of psoriasis

BACKGROUND CD11a/CD18 comprise subunits of leukocyte function associated antigen (LFA-1), a T-cell surface molecule important in T-cell activation, T-cell emigration into skin, and cytotoxic T-cell function. OBJECTIVE We explored the immunobiologic and clinical effects of treating moderate to severe psoriasis vulgaris with a single dose of humanized monoclonal antibody against CD11a (hu1124). METHODS This was an open label study with a single dose of hu1124 at doses of 0.03 to 10 mg/kg. Clinical (Psoriasis Area and Severity Index [PASI]) and immunohistologic parameters (epidermal thickness, epidermal and dermal T-cell numbers, and keratinocyte intercellular adhesion molecule 1 [ICAM-1] expression) were followed. RESULTS Treatment with hu1124, at doses higher than 1.0 mg/kg (group III), completely blocks CD11a staining for at least 14 days in both blood and psoriatic plaques. At 0.3 to 1.0 mg/kg, T-cell CD11a staining was completely blocked; however, blockade lasted less than 2 weeks (group II). Only partial saturation of either blood or plaque cellular CD11a was observed at doses of hu1124 between 0.01 and 0.1 mg/kg (group I). This pharmacodynamic response was accompanied by decreased numbers of epidermal and dermal CD3(+) T cells, decreased keratinocyte and blood vessel expression of ICAM-1, and epidermal thinning. Statistically significant drops in PASI compared with baseline were observed in group II patients at weeks 3 and 4 and in group III patients at weeks 2 through 10. No significant drop in PASI score was observed in group 1. Adverse events were mild at doses of 0.3 mg/kg or less and included mild chills, abdominal discomfort, headache, and fever. At a single dose of 0.6 mg/kg or higher, headache was the most common dose-limiting toxicity observed. CONCLUSION Targeting CD11a may improve psoriasis by inhibiting T-cell activation, T-cell emigration into the skin, and cytotoxic T-cell function.