Photo-Enhanced Modification of Human Skin Elastin in Actinic Elastosis by NE-(Carboxymethyl)Lysine, One of the Glycoxidation Products of the Maillard Reaction

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Photo-Enhanced Modification of Human Skin Elastin in Actinic Elastosis by NE-(Carboxymethyl)Lysine, One of the Glycoxidation Products of the Maillard Reaction Photo-Enhanced Modification of Human Skin Elastin in Actinic Elastosis by NE-(Carboxymethyl)lysine, One of the Glycoxidation Products of the Maillard Reaction Kurniko M.izutari, ';' Tomomichi 011.0, * Kazuyoshi Ikeda, t Ken-ichi Kayashima, * and Seikoh Horiuchit ' Department of Dermatology, Kumamoto University School of Medicin c, and i"Departmcnt of Biochcmistry. Kumamoto Uni\'crsity School of Medici ne, HOllj O, Kumamoto. Japan Long- term incubation of proteins with glucose leads were modified by CML. Further immunohistochemi­ to the formation of advanced glycation end products cal and immunoelectron microscopic examination (AGEs), which are characterized by fluorescence, with 6D12 demonstrated CML accumulates pre­ brown color, and cross-linking. Formation of AGEs ill dominantly in elastic fibers especially in the amor­ vitro requires oxygen and is dependent on transition phous electron-dense materials corresponding to metal- catalyzed oxidation of glucose or Amadori photo-induced degenerated area rather than the products. AGEs are thought to be involved in aging electron-lucent region. Imnlunochelnical analyses and age-enhanced diseases such as diabetic compli­ with enzyme-linked il11munosorbent assay (ELISA) of cations, atherosclerosis, dialysis- related amyloidosis, elastase-soluble fractions delnonstrated that the CML and Alzheimer's disease. Chronic exposure of the levels of the sun-exposed area were significantly skin to sunlight induces hyperplasia of the elastic higher than those of the sun-unexposed area. We tissue in the upper dermis known as actinic elastosis. conclude that ultraviolet-induced oxidation l11ay ac­ Herein we used a monoclonal anti- AGE antibody celerate CML formation in actinic elastosis of photo­ (6D12) whose epitope is NE-(carboxymethyl)lysine aged skin. Key 1UD/'ds: plrotoagillgladval/.ced giycatioll e/ld (CML) , one of the glycoxidation products of AGEs, p,'odllctS. ] I,west Del'l/Iatoi 108:797-802, 1997 and demonstrated that the lesions of actinic elastosis ong-term incubation of proteins with glucose ill III'lro Several AGE Structures have been identified including pyrraline leads, through early-stage products such as a Schiff base (Hayase et ai, 1989), pentosidine (Sell el al 1989), NE-(carboxy_ and Amadori rearr~ngem e nt products, to the formation m ethyl)lysine (CML) (Ahmed et ai, 1986). and crosslines (Naka­ of advanced glycatlon end products (AGEs), wluch are mura et ai, 1992). It is still not known, however, whetber one of characterized by fluorescence, brown color, and cross­ these compounds contributes, as a major AGE structure. to the Llinking (Maillard, 1912). We prepared an anti-AGE monoclonal pathogenesis of these diseases or whetber otber structure(s) may antibody (6D12) in mice (Horiuchi et ai, 1991). Immunologic be involved in this process. Although the presence of AGEs in studies using 6D12 not only demonstrated the presence of AGEs in several human tissues was successfully demonstrated by our several human tissues but also suggested a potential link of AGEs to anti-AGE monoclonal antibody (6D12) as described above. no aging (Araki et ai, 1992; Kimura el ai, 1995, 1996) and age­ info rmatio n was available until quite recently about its AGE enhanced diseases such as diabetic complica tions (Yamada cl ai, structure. Our recent study revealed that 6D12 is able to 1994; Makino 1995), atherosclerosis (Kume 1995; et ai, ct ai, recognize a CML-protein adduct as an epitope (Ikeda et ai, Horiu chi, 1996; Meng et ai, 1996), and dialysis-related amyloidosis 1996), stro ngly suggesting an ill lIillO importan ce of CML among (Miyata et 1993, 1996). Immunologic studies using anti-AGE ai, AGE structures. polyclonal antibodies were also performed in other laboratories, In the skin after chronic exposure to sunlight, especially in demonstrating the presence of AGEs in h uman plasma (Makita et nl, persons with a fair complexion, hyperplasia of the ela sti c tissue is 1992a; Bucala et ai, 1993), hemoglobin (Makita et ai, 1992b), usually evident in the upper dermis by the age of30. These changes atherosclerotic coronal'Y artery (Nakamura ef ai, 1993), and lesions found in the photoaged skin are ca ll ed actinic elastosis (Kligman. of Alzheimer's disease (Smith ct ai, 1994; Vitek el al . 1994) . 1969). Accumulated elastotic materials are thought to be derived from elastic fibers because of their reacti vity WitIl the anti-elastin I, 8, Manuscript received October 1996; revised Jan uar)' 1997; accepted antibody and sellSitivi ry to elastase digestion (Pieraggi, 1988). for publication Jalluary 13. 1997. Although it has no t been clear how chronic solar exposure can Reprint requests to: Dr. TOlllomichi OliO, Departlllent of Dermatology. Kumamoto University School of Medicine, Honj o, 1-1-1 . Kumamoto 860. induce accumulation of elastic fibers, deleterious efFects of sunlight japaJl. could often be attributed to oxidation by fi'ee radical intermediates Abbreviations: AGE(s). advanced glycation clld prodllct(s); CML, N <­ (Sugiyama et ai, 1984; Buettner el al. 1987; Miyachi, 1993; Pent­ (carboxymethyl)l ysi ne . land, 1994). It is therefore possible that ultravio let-induced o xida- 0022-202X/ 97 IS1 0.50 • Copyright © 1997 by The Society for In vestiga ti ve Dermarology, Inc. 797 798 MIZUTARI ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY tive stress may have some role in the pathogenesis of actinic One of thesc scctions was used to observe under a confocal laser scanning elastosis through the formation of oxidation products. microscope (MRC~1024; BioRad. Heme! Hempstead, U.K.). A yellow CML is formed ill IJilrO by oxidative cleavage of Amadori addu cts fluorescence occurs when botlt green alld !"ed staills bind to the same location. Nonirnl11unc rabbit alld mOllse serum (Cappel. West Chester, PAl (Ahmed et al. 1986; Dunn et ai, 1991) OJ" Scbitr bases (Glomb and were used as negative control. No il11l1lulJoreactiOll was observed in Monnier, 1995) or by modification with glyoxal gen erated directly formalin-fixed and paraffin-embedded specimens. through autoxidation of g lucose (Wells-Knecht ct ai, 1995) and h as been proposed as a potential biomarker of oxidative damage of Immunoelectron Microscopy T he specimens fi'oll1 the face of patien ~ tissu e proteins ill Pi/)O (Baynes, 199t; Fu et ai, 1994). CML is 48, 49, and 51 were s ul~jccted to il11l11unoelectron microscopic study by a therefore termed a "glycoxidation product" among AGE structures. post-embedding method Ll sing anti-elastin antibody and 6D12. The tis ue There is currently no evidence that AGEs result from UV-induced sa mples were fixed with Karnovsky fixati ve lor 24 h at 4°C, washed with PBS. dehydrated ill a graded scries of ethanol, and embedded in LR White oxidation. To test this hypothesis, we h ave attempted to determine resin (London Resin, Berkshire, U.K.). Ultrathin sections were floated ou whether CML modification could occur in the lesions of actinic drops of buffer A (0.5% BSA. 0.1·y" gelatin, and 20 111M NaN., in PBS) for elastosis. Immuno logic studies using 6D12, a monoclonal antibody 15 min and incubated for 1 h at room temperature with either the specific for CML structure, demonstrated CML accumulation anti-ela sci1l antibody (1 :500 dilution) or 6012 (5 !Lg per ml). The sections predominantly in elastic Etbers especially in the amorphous elec­ were washed with bllffer and further incubated for 3(l min with 15-.I1Ill tron-dense material corresponding to photo-induced degenerated colloidal gold-conjugated goat anti-mouse IgG antibody (Aurioll, area. T h ese results indicate tbat ultraviolet-induced oxidation may Wageningen, Netherlands) ill bulrer A. These sections were washed with accelerate CML formation in actinic elastosis of t he photoaged skin. buffer A, PBS, and distilled water, stained with lIranyl acetate, and observed tinder a Hitachi H300 electron microscope. For double labeling with MATERIALS AND METHODS different size of gold particles. 6- and 15-nm coll oidal gold-conjugated goat Tissue Preparation Fifry-seven specimens were obtained fi'om S'I Japanese anti-mouse IgG antibodies wel'c uscd to locali ze the bound anti-elastin patients undergoing del111atologi c surgery for the treatment of benign and antibody and 6D12, respectively. matignallt epith eli al tllmors at Kumamoto University School of Medicine Biochenlical Analysis We studied skin tissues obtain ed fj'om skin Hospital (Table 1). Specimens were obtai ned ti'om various sires including lace surgery of sun-exposed and sun-unexposed area in 18 individuals. Elastin (cheek, forehead, and nose), neck, back of the hand, fo rearm, shoulder, chest, was solubilized with clastose (Fleischl11,~jer mId Lara. 1966), a1ld its immu­ abdomen, back, groin , and sale of botb male and female patients. Their "ges noreactivity to 6D 12 wa s derermined by noncompetitive enzyme-linked ranged frOI11 9 to 100 y. The peripheral tissues sllrrounding the tumor were il11l'l1unosorbcnt assay (ELISA) using the fo ll owing procedure (Horiu chi II procUl'cd at the time of surgery. Prior informed conse nt. WllS obtained in all (/1 . 1991). Ea ch skin specimen was rapidly fj'ozen and sli ced in to 10-I.t.rn cases. W hen the subjects were juveniles, informed consent was obtained fi'ol11 sections. Skin sections wcrc washed t.hree times with PBS and incubated their parents. Specimens utilized in the srudy were all hi stologica ll y tumor-free overnight with elastase (W,lko Chemica l, Osaka. JapHlI) in 0.1 M Tris(hr­ and contained epidemlis and underl ying dennis. Tire samples were sectioned droxymethyl)arninomethane hydrochloride bulter (pH 8.8) at 3 rc. The and processed in two ways: one W,IS fOt11ralitl-f,xed and paraffin-embedded and elastase-soluble fraction was collected as "peptide fraction. " Protein COll­ the other WaS rapidly frozen lind stored at - BO °C. Paraffll1-embedded samples centrations were dcrcrnlincd by bicincboninic acid protein assay reClgent ill 6-I.UTI sections were staiJ1Cd with henlatoxylin and eosi.n for diagnosis and the (Pierce.
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