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Fibrillin-Rich Microfibrils are Reduced in Photoaged Skin. Distribution at the Dermal–Epidermal Junction

1999; Elsevier BV; Volume: 112; Issue: 5 Linguagem: Inglês

10.1046/j.1523-1747.1999.00562.x

1523-1747

Rachel Watson, C.E.M. Griffiths, N. M. Craven, C. Adrian Shuttleworth, Cay M. Kielty,

Lymphatic System and Diseases

Chronic sun exposure results in photoaged skin with deep coarse wrinkles and loss of elasticity. We have examined the distribution and abundance of fibrillin-rich microfibrils, key structural components of the elastic fiber network, in photoaged and photoprotected skin. Punch biopsies taken from photoaged forearm and from photoprotected hip and upper inner arm of 16 subjects with a clinical range of photoaging were examined for fibrillin-1 and fibrillin-2 expression and microfibril distribution. In situ hybridization revealed decreased fibrillin-1 mRNA but unchanged fibrillin-2 mRNA levels in severely photoaged forearm biopsies relative to photoprotected dermal sites. An immunohistochemical approach demonstrated that microfibrils at the dermal–epidermal junction were significantly reduced in moderate to severely photoaged forearm skin. Confocal microscopy revealed that the papillary dermal microfibrillar network was truncated and depleted in photoaged skin. These studies highlight that the fibrillin-rich microfibrillar network associated with the upper dermis undergoes extensive remodeling following solar irradiation. These changes may contribute to the clinical features of photoaging, such as wrinkle formation and loss of elasticity. Chronic sun exposure results in photoaged skin with deep coarse wrinkles and loss of elasticity. We have examined the distribution and abundance of fibrillin-rich microfibrils, key structural components of the elastic fiber network, in photoaged and photoprotected skin. Punch biopsies taken from photoaged forearm and from photoprotected hip and upper inner arm of 16 subjects with a clinical range of photoaging were examined for fibrillin-1 and fibrillin-2 expression and microfibril distribution. In situ hybridization revealed decreased fibrillin-1 mRNA but unchanged fibrillin-2 mRNA levels in severely photoaged forearm biopsies relative to photoprotected dermal sites. An immunohistochemical approach demonstrated that microfibrils at the dermal–epidermal junction were significantly reduced in moderate to severely photoaged forearm skin. Confocal microscopy revealed that the papillary dermal microfibrillar network was truncated and depleted in photoaged skin. These studies highlight that the fibrillin-rich microfibrillar network associated with the upper dermis undergoes extensive remodeling following solar irradiation. These changes may contribute to the clinical features of photoaging, such as wrinkle formation and loss of elasticity. Skin aging can be divided into intrinsic (or chronologic) and extrinsic due to environmental insults such as chronic sun exposure. Intrinsically aged skin has characteristic fine wrinkling and appears smooth (Montagna et al., 1989Montagna W. Kirchner S. Carlisle K. Histology of sun-damaged skin.J Am Acad Dermatol. 1989; 21: 907-918Abstract Full Text PDF PubMed Scopus (149) Google Scholar). At the histologic level, such skin shows general atrophy of the extracellular matrix (ECM) with reduced elastin and disintegration of elastic fibers (Braverman and Fornferko, 1982Braverman I.M. Fornferko E. Studies on cutaneous ageing. I. the elastic fiber network.J Invest Dermatol. 1982; 78: 434-443Abstract Full Text PDF PubMed Scopus (375) Google Scholar). Photoaged skin, in contrast, appears coarse, roughened, and deeply wrinkled with marked loss of elasticity and recoil (Smith et al., 1962Smith J.G. Davidson E.A. Sams W.M. Clark R.D. Alterations in human dermal connective tissue with age and chronic sun exposure.J Invest Dermatol. 1962; 39: 347-350Abstract Full Text PDF PubMed Scopus (233) Google Scholar;Warren et al., 1991Warren R. Gartstein V. Kligman A.M. Montagna W. Allendorf R.A. Riddler G.M. Age, sunlight, and facial skin: a histologic and quantitative study.J Am Acad Dermatol. 1991; 25: 751-760Abstract Full Text PDF PubMed Scopus (210) Google Scholar). By routine light microscopy, photoaged skin contains abundant dystrophic elastotic material in the reticular dermis, which is immunopositive for tropoelastin and fibrillin (Mitchell, 1967Mitchell R.E. Chronic solar dermatosis: a light and electron microscopic study of the dermis.J Invest Dermatol. 1967; 43: 203-230Google Scholar;Chen et al., 1986Chen V.L. Fleischmajer R. Scheartz E. Palia M. Timpl R. Immunochemistry of elastotic material in sun damaged skin.J Invest Dermatol. 1986; 87: 334-337Crossref PubMed Scopus (151) Google Scholar;Mera et al., 1987Mera S.L. Lovell C.R. Jones R.R. Davies J.D. Elastic fibres in normal and sun-damaged skin: an immunohistological study.Br J Dermatol. 1987; 117: 21-27Crossref PubMed Scopus (85) Google Scholar;Werth et al., 1996Werth V.P. Kalathil S.E. Jaworsky C. Elastic fiber-associated proteins of skin in development and photoaging.Photochem Photobiol. 1996; 63: 308-313Crossref PubMed Scopus (24) Google Scholar). Recently, ultraviolet (UV) B irradiation has been shown directly to upregulate tropoelastin gene expression in vivo and in vitro (Uitto et al., 1997Uitto J. Brown D.B. Gasparro F.P. Bernstein E.F. Molecular aspects of photoaging.Eur J Dermatol. 1997; 7: 210-214Google Scholar), and increased fibrillin expression and deposition have been reported within the reticular dermis of photoaged skin (Bernstein et al., 1994Bernstein E.F. Chen Y.Q. Tamai K. et al.Enhanced elastin and fibrillin gene expression in chronically photodamaged skin.J Invest Dermatol. 1994; 103: 182-186Crossref PubMed Scopus (169) Google Scholar). Elastic fibers are insoluble structural elements of connective tissues that have a central core of amorphous, hydrophobic cross-linked elastin surrounded by fibrillin-rich microfibrils (Mecham and Heusar, 1991Mecham R.P. Heusar J.E. The elastic fibre.in: Hay E.D. 2nd edn. Cell Biology of the Extracellular Matrix. Plenum Press, New York1991Google Scholar). The process of elastic fiber formation is under tight developmental control with tropoelastin deposited upon a preformed microfibrillar template perinatally and during early development. In skin, the elastic fiber network forms a continuum from the dermal–epidermal junction (DEJ) to the deep dermis. It comprises thick elastin-rich fibers within the reticular dermis, a network of finer fibers with reduced elastin in the lower papillary dermis, and cascades of discrete microfibrillar bundles, without measurable elastin, in the upper papillary dermis terminating at the DEJ (Cotta-Periera et al., 1978Cotta-Periera G. Guerro Rodriguez F. Bittencourt-Sampaio S. Oxytalan, elaunin and elastic fibers in the human skin.J Invest Dermatol. 1978; 66: 143-148Crossref Scopus (239) Google Scholar;Dahlbäck et al., 1990Dahlbäck K. Ljungquist A. Löfberg H. Dahlbäck B. Engvall E. Sakai L.Y. Fibrillin immunoreactive fibers constitute a unique network in the human dermis: immunohistochemical comparison of the distributions of fibrillin, vitronectin, amyloid P component, and orcein stainable structures in normal skin and elastosis.J Invest Dermatol. 1990; 94: 284-291Abstract Full Text PDF PubMed Google Scholar). Fibrillin is a product of dermal fibroblasts, but keratinocytes also express fibrillin and assemble microfibrils (Haynes et al., 1997Haynes S.L. Shuttleworth C.A. Kielty C.M. Expression of fibrillin by keratinocytes: implications for dermal microfibril and elastic fibre organisation.Br J Dermatol. 1997; 137: 17-23Crossref PubMed Scopus (42) Google Scholar). Following keratinocyte autografting, a fine cascade of papillary dermal microfibrils is deposited in association with the DEJ (Raghunath et al., 1996Raghunath M. Bächi T. Meuli M. Altermatt S. Gobet R. Bruckner-Tuderman L. Steinmann B. Fibrillin and elastin expression in skin regenerating from cultured keratinocyte autographs: morphogenesis of microfibrils begins at the dermo-epidermal junction and precedes elastic fiber formation.J Invest Dermatol. 1996; 106: 1090-1096Crossref PubMed Scopus (61) Google Scholar). These studies demonstrate that loss of microfibrillar integrity in the upper dermis may contribute to the clinical appearance and reduced elasticity of photoaged skin. To examine this hypothesis, we have studied fibrillin expression and the distribution of fibrillin-rich microfibrils in sun-protected and sun-exposed skin of individuals with a clinical range of photoaging. Sixteen healthy Caucasian volunteers (six male, 10 female; age range 24–80 y) were graded for overall severity of photoaging on the extensor forearm using an established 0–9 scale, where 0 is no clinically detectable photoaging and 9 is severe photoaging (Griffiths et al., 1992Griffiths C.E.M. Wang T.S. Hamilton T.A. Voorhees J.J. Ellis C.N. A photonumeric scale for the assessment of cutaneous photodamage.Arch Dermatol. 1992; 128: 347-351Crossref PubMed Scopus (172) Google Scholar,Griffiths et al., 1993Griffiths C.E. Russman A.N. Majmudar G. Singer R.S. Hamilton T.A. Voorhees J.J. Restoration of collagen formation in photodamaged human skin by tretinoin (retinoic acid).N Engl J Med. 1993; 329: 530-535Crossref PubMed Scopus (378) Google Scholar). Volunteers were separated into clinical groups: no clinical photoaging (grade 0; n = 3); minimal photoaging (grades 1–4; n = 3); and moderate to severe photoaging (grades 5–9; n = 10). Of the 10 individuals with moderate to severe photoaging, six had spent more than 2 y living and working abroad (India, Malaysia, and Australia). Eight of the 10 reported that they had previously been on sunbathing holidays (but not in the preceding 3 mo), although all admit to using sun-block and to wearing protective clothing. Seven patients had previously presented with skin tumors (six basal cell carcinomas; one squamous cell carcinoma) and one individual was a habitual user of a solarium. Of the minimally photoaged group, all reported to have been on sunbathing holidays, but similarly, these had not taken place within the previous 3 mo. None of the remaining individuals worked out of doors. Four millimeter punch biopsies were taken from the extensor aspect of the forearm (photoaged) following 1% lidocaine local anesthesia, and from the photoprotected hip and upper inner arm (as an anatomic site control). Biopsies were snap frozen in liquid nitrogen and stored at –70°C until use. The study was approved by the Salford and Trafford local research ethics committee and all subjects gave written, informed consent. Seven millimeters cryostat sections from biopsies taken from moderate to severely photoaged subjects were pretreated for in situ hybridization using standard protocols (Harrison and Pearson, 1990Harrison P.J. Pearson R.C.A. In situ hybridization histochemistry and the study of gene expression in the human brain.Prog Neurobiol. 1990; 34: 271-312Crossref PubMed Scopus (56) Google Scholar). Once pretreated (fixation in 4% paraformaldehyde, acetylation, serial dehydration and delipidation) sections were stored at –70°C until required. Fibrillin-1 anti-sense probe was 5′-ACA CAG GCC ATT TTT ACA CAC TCC TGG GAA-3′ and fibrillin-1 sense probe was 5′-TTC CCA GGA GTG TGT AAA AAT GGC CTG TGT-3′ (Pereira et al., 1993Pereira L. D'Alessio M. Ramirez F. Lynch J.R. Sykes B. Pangilinan T. Bonadio J. Genomic organization of the sequence coding for fibrillin, the defective gene product in Marfan syndrome.Hum Mol Genet. 1993; 2: 961-968Crossref PubMed Scopus (247) Google Scholar). Fibrillin-2 anti-sense probe was 5′-CAC AGT GAT ACC TAC TCC ACT ACT ACA AAG-3′ and fibrillin-2 sense probe was 5′-CTT TGT AGT AGT GGA GTA GGT ATC ACT GTG-3′ (Lee et al., 1991Lee B. Godfrey M. Vitale E. et al.Linkage of Marfan syndrome and a phenotypically related disorder to two different fibrillin genes.Nature. 1991; 352: 330-334Crossref PubMed Scopus (541) Google Scholar). Specificity of the chosen sequences (5′-untranslated sequences of both molecules) was checked using the MRC gene database and by northern blotting of total human skin RNA (Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62307) Google Scholar;Farrell, 1993Farrell R.E. RNA Methodologies: a Laboratory Guide for Isolation and Characterization. Academic Press, London1993Google Scholar). Oligonucleotides (at a concentration of 2 pmol per ml) were 3′ tail-labeled with [35S]αdATP using a commercially available kit (NEN/DuPont, Stevenage, U.K.). After purification, 106 cpm of labeled probe was applied to each section in hybridization buffer (4 × sodium citrate/chloride buffer; 1 × Denhardt's; 10 mM EDTA; 2 mg per ml denatured herring sperm; 1 mg per ml yeast tRNA; 1 mg per ml poly(A); 50% de-ionized formamide; 10% dextran sulfate; 20 mM phosphate buffer). Three sections per biopsy were hybridized for each anti-sense probe and one section for each control sense strand. Incubation was performed overnight and followed by stringency washes to remove unbound label. Incubation temperature (Ti) was 26°C and wash temperature (Tw) was 48°C. Following washing, sections were air-dried and message visualized by film autoradiography. Films were exposed for 21 d and developed (M35-M X-OMAT processor; Kodak, Hemel Hempstead, U.K.). Following film development, sections were subjected to autoradiographic emulsion (K5; Ilford, Mobberley, U.K.) to visualize cellular localization of mRNA. Sections were exposed for 6 wk and developed following standard protocols (Harrison and Pearson, 1990Harrison P.J. Pearson R.C.A. In situ hybridization histochemistry and the study of gene expression in the human brain.Prog Neurobiol. 1990; 34: 271-312Crossref PubMed Scopus (56) Google Scholar), stained with hematoxylin and eosin, and examined on a Leica Axioskop microscope. Quantitation of the number of cells positive for fibrillin-1 mRNA was done automatically using a Kontron KAT IBAS system with IBAS software (version 2.0) (Imaging Associates of Thame, Oxford, U.K.). Autoradiographic images were coded such that their anatomic origin was unknown and quantitated so as to produce mean optical densities for each section (Molecular Analyst, BioRad, Hemel Hempstead, U.K.). To ensure the clinical assessment, elastin was visualized in 7 μm frozen sections (OFT cryostat, Bright Instruments, Cambridge, U.K.) using standard peroxidase histochemistry with the monoclonal antibody BA4 (Sigma, Poole, U.K.;Wrenn et al., 1986Wrenn D.S. Griffin G.L. Senior R.M. Mecham R.P. Characterization of biologically active domains on elastin: Identification of a monoclonal antibody to a cell recognition site.Biochemistry. 1986; 25: 5172-5176Crossref PubMed Scopus (79) Google Scholar). For visualization of the fibrillin-rich microfibrils at the DEJ, triplicate sections were fixed for 10 min in chilled 100% acetone. Following washes in Tris-buffered saline (TBS; 100 mM Tris, 150 mM sodium chloride), sections were solubilized in 0.5% Triton X-100 for 15 min. Sections were blocked with 3% normal goat serum (Vector Laboratories, Peterborough, U.K.) plus 3% bovine serum albumin (BSA) for 1 h at room temperature before application of a well-characterized primary polyclonal anti-fibrillin-rich microfibril antibody (5507;Shuttleworth et al., 1992Shuttleworth C.A. Berry L. Kielty C.M. Microfibrillar elements in dental pulp: Presence of type VI collagen and fibrillin-containing microfibrils.Arch Oral Biol. 1992; 37: 1079-1084Abstract Full Text PDF PubMed Scopus (15) Google Scholar;Waggett et al., 1993Waggett A.D. Kielty C.M. Shuttleworth C.A. Microfibrillar elements in the synovial joint: Presence of type VI collagen and fibrillin-containing microfibrils.Ann Rheum Dis. 1993; 52: 449-453Crossref PubMed Scopus (25) Google Scholar;Kielty et al., 1994Kielty C.M. Woolley D.E. Whittaker S.P. Shuttleworth C.A. Catabolism of intact fibrillin microfibrils by neutrophil elastase, chymotrypsin and trypsin.FEBS Lett. 1994; 351: 85-89Abstract Full Text PDF PubMed Scopus (49) Google Scholar;Raghunath et al., 1996Raghunath M. Bächi T. Meuli M. Altermatt S. Gobet R. Bruckner-Tuderman L. Steinmann B. Fibrillin and elastin expression in skin regenerating from cultured keratinocyte autographs: morphogenesis of microfibrils begins at the dermo-epidermal junction and precedes elastic fiber formation.J Invest Dermatol. 1996; 106: 1090-1096Crossref PubMed Scopus (61) Google Scholar). Titration experiments ascertained an optimal working dilution of 1:200. Incubation was performed overnight at 4°C. Following rigorous washes in TBS to remove unbound antibody, sections were incubated with an FITC-conjugated goat antirabbit IgG secondary antiserum (45 min at room temperature). Sections were stained with 4,6-diamino-2-phenylindole (DAPI) to identify nuclei and mounted in aqueous mounting media containing p-phenylene diamine (an "antifade" agent). Negative controls were performed for each biopsy by omission of primary antibody. Immunostained sections were coded and randomized such that the single investigator (REBW) reading them was unaware of their anatomic site of origin. The staining intensity and both abundance and continuity of the microfibrillar arrays extending from the DEJ to the deeper dermis were assessed in four high power microscopic fields for each section and were graded using a 0–4 ordinal scale: 0, absent; 1, minimum; 2, moderate; 3, high; and 4, maximum. In all cases, both photoprotected and photodamaged skin from each individual was quantitated. The same sections that were processed for immunofluorescence were also visualized by confocal microscopy (BioRad MRC 600 focal system). Fluorescence was visualized with an argon 25 mW laser (488 nm) to reveal the complete microfibrillar network running throughout the 7 μm specimen. Optical dissections (z series) were taken through the section every 0.7 μm (Kalman series, n = 8) with a mean pinhole diameter of 0.5 mm. Images were converted from BioRad plc format to Targa format and pseudo-colored using Confocal Assistant and Adobe PhotoShop, prior to printing on CL5000 film (Polaroid). These sections per specimen were merged to show extended focus views. Data were tested nonparametrically to compare fibrillin immunostaining among the three anatomic regions from sun-protected and photoaged skin using Friedman's two-way ANOVA. Summary data are expressed as mean ± SEM. Data were analyzed with the SPSS package (SPSS, Chicago, IL). Autoradiographic data were analyzed using Student's t-test to identify differences between photoprotected and photoaged groups of each individual (with significance taken at the 5% level). Localization of elastin in forearm skin sections by immunohistochemistry from non-photoaged and severely photoaged individuals confirmed the accumulation of abundant elastotic material in the upper papillary dermis following chronic sun exposure (data not shown). In situ hybridization of photoprotected and photoaged skin showed that fibrillin-1 mRNA was present in both dermal fibroblasts and epidermal keratinocytes (Figure 1,Figure 2,Figure 3). Film autoradiography highlighted that fibrillin-1 mRNA in skin from hip, upper inner arm and forearm was abundant around the DEJ and within the deeper dermis (Figure 1). In histologic sections of all skin sites, keratinocytes were heavily labeled, but signals were also evident in dermal fibroblasts and around hair follicles (Figure 2). Quantitation of label confirmed that the epidermal layer was more heavily labeled than the papillary dermis (Figure 3). Close examination showed that both hip and upper inner arm skin generally had higher levels of fibrillin-1 mRNA than forearm samples; there were no apparent differences between photoprotected areas. Fibrillin-1 mRNA expression was decreased in photoaged forearm compared with photoprotected hip and upper inner arm in eight of 10 moderate to severely photoaged individuals. This decrease was significant in two of these patients (Figure 3).Figure 2Cellular localization of fibrillin-1 mRNA. The cellular localization of fibrillin-1 mRNA in photoprotected skin and severely photoaged skin showed that keratinocytes were heavily labeled, and signals were also evident in dermal fibroblasts and around hair follicles. Scale bar: ≈25 μm. (a) Sense strand control; (b) photoprotected hip; (c) photoprotected upper inner arm; (d) non-photoaged forearm [parts (a)–(d) are from the same individual]; (e) photoprotected hip; (f) photoprotected upper inner arm; (g) severely photoaged forearm skin [parts (e)–(g) are from a second individual].View Large Image Figure ViewerDownload (PPT)Figure 3Keratinocytes and dermal fibroblasts show a reduction in the number of cells positive for fibrillin-1 mRNA. Histologic assessment of fibrillin-1 positive cells in individuals with moderate to severe photoaging (n = 10). (a) Epidermal keratinocytes; (b) dermal fibroblasts. Sites are photoprotected hip (filled bars), photoprotected upper inner arm (shaded bars), and photoaged forearm (open bars), respectively.View Large Image Figure ViewerDownload (PPT) Low levels of fibrillin-2 mRNA were detected in dermis and epidermis of non-photoaged and minimally or severely photoaged skin biopsies (n = 10). Mean optical densities of 0.04 (hip), 0.035 (upper inner arm), and 0.045 (forearm) were recorded for severely photoaged skin (n = 6), and 0.015 (hip), 0.018 (upper inner arm), and 0.012 (forearm) were recorded for minimally photoaged skin (n = 3). There were no significant differences between photoprotected skin sites, or between minimally or severely photoaged forearm skin. Candelabra-like fibers extending perpendicularly from the DEJ into the papillary dermis were seen in all normal sections including photoprotected hip and upper arm and non-photoaged forearm (Figure 4). Severely photoaged extensor forearm sections also contained microfibrils at the DEJ, but in these sections the microfibrils were reduced in amount and appeared severely truncated (Figure 4f) relative to the long arrays present in photoprotected skin from the same patients (Figure 4d, e) and in non-photoaged forearm skin (Figure 4c). This analysis highlighted intraindividual differences between light-exposed and light-protected skin. Clinically, mild photoaged forearm samples showed similar marked disruption of the microfibrillar network (not shown). Semi-quantitative analysis confirmed that fibrillin-rich microfibril levels were significantly decreased in both severely and minimally photoaged forearm biopsies compared with sun-protected control sites (p < 0.0001 and p < 0.05, respectively). Confocal microscopy of microfibrils subjacent to the DEJ confirmed and extended the light microscopy observations (Figure 4g–l). Intact microfibrillar networks were observed in all sites from clinically non-photoaged skin and all photoprotected sites of patients with minimal or moderate-to-severe photoaging (Figure 4g–k). In contrast, microfibrils were markedly disrupted and reduced in the papillary dermis of both minimally and severely photoaged skin (Figure 4l). The fibrillin-containing microfibrillar network of the papillary dermis, close to the DEJ is greatly re-organized and reduced following chronic sun exposure. Previous studies have examined elastin and fibrillin distribution in the deep dermis of photoaged skin and showed that both of these matrix components are increased following chronic sun exposure (Bernstein et al., 1994Bernstein E.F. Chen Y.Q. Tamai K. et al.Enhanced elastin and fibrillin gene expression in chronically photodamaged skin.J Invest Dermatol. 1994; 103: 182-186Crossref PubMed Scopus (169) Google Scholar;Schwarz et al., 1995Schwarz E. Feinberg E. Lebwohl M. Mariani T.J. Boyd C.D. Ultraviolet radiation increases tropoelastin accumulation by a post-transcriptional mechanism in dermal fibroblasts.J Invest Dermatol. 1995; 105: 65-69Crossref PubMed Scopus (24) Google Scholar). Little attention has been paid to the fibrillin-containing microfibrils (devoid of elastin), however, which are found in close proximity to the DEJ. In photoprotected skin (hip and upper inner arm) we observe a complex, intertwining system of small fibers running from the DEJ into the papillary dermis. This distribution is in agreement with that previously described in normal skin (Cotta-Periera et al., 1978Cotta-Periera G. Guerro Rodriguez F. Bittencourt-Sampaio S. Oxytalan, elaunin and elastic fibers in the human skin.J Invest Dermatol. 1978; 66: 143-148Crossref Scopus (239) Google Scholar;Dahlbäck et al., 1990Dahlbäck K. Ljungquist A. Löfberg H. Dahlbäck B. Engvall E. Sakai L.Y. Fibrillin immunoreactive fibers constitute a unique network in the human dermis: immunohistochemical comparison of the distributions of fibrillin, vitronectin, amyloid P component, and orcein stainable structures in normal skin and elastosis.J Invest Dermatol. 1990; 94: 284-291Abstract Full Text PDF PubMed Google Scholar). The distribution of fibrillin-positive fibers, however, in severely photoaged skin shows that, although immunohistochemically identifiable material is present along the DEJ, discrete microfibrils are markedly reduced in number. Examination of minimally photoaged skin reveals a similar marked loss of fibrillin-positive structures at the DEJ. Such data implies that this molecule is one of the first constituents of the dermal extracellular matrix to be affected or damaged by solar irradiation. Mutations in the genes encoding fibrillin-1 (FBN-1) and fibrillin-2 (FBN-2) have been shown to be causative in a number of inherited diseases such as Marfan syndrome (FBN-1;Lee et al., 1991Lee B. Godfrey M. Vitale E. et al.Linkage of Marfan syndrome and a phenotypically related disorder to two different fibrillin genes.Nature. 1991; 352: 330-334Crossref PubMed Scopus (541) Google Scholar) and congenital contractural arachnodactyly (FBN-2;Lee et al., 1991Lee B. Godfrey M. Vitale E. et al.Linkage of Marfan syndrome and a phenotypically related disorder to two different fibrillin genes.Nature. 1991; 352: 330-334Crossref PubMed Scopus (541) Google Scholar). Of interest in these diseases is the presentation of striae distensae in the skin (Pinkus et al., 1966Pinkus H. Keech M.K. Mehregan A.H. Histopathology of striae distensae, with special reference to striae and wound healing in the Marfan syndrome.J Invest Dermatol. 1966; 46: 283-292Abstract Full Text PDF PubMed Scopus (45) Google Scholar;Aoyama et al., 1995Aoyama T. Francke U. Gasner C. Furthmayr H. Fibrillin abnormalities and prognosis in Marfan syndrome and related disorders.Am J Med Genet. 1995; 58: 169-176Crossref PubMed Scopus (68) Google Scholar). Recent work in this laboratory has identified reductions in the elastic fiber network in striae distensae, where both fibrillin microfibrils and elastin in the papillary dermis are affected equally (Watson et al., 1998Watson R.E.B. Parry E.J. Humphries J.D. Jones C.J.P. Polson D.W. Kielty C.M. Griffiths C.E.M. Fibrillin microfibrils are reduced in skin exhibiting striae distensae.Br J Dermatol. 1998; 138: 931-937Crossref PubMed Scopus (135) Google Scholar). It, therefore, seems unlikely that the observed reduction in fibrillin-rich microfibrils in photoaged skin is due to any spatial confinement brought about by increased abnormal elastin production and deposition in the papillary dermis. Fibrillin-1 expression by keratinocytes and dermal fibroblasts in photoprotected and photoaged skin suggests that both cell types contribute actively to maintaining the integrity of the microfibrillar network that extends from the DEJ into the papillary dermis. The presence of fibrillin-1 mRNA even in photoprotected adult skin further suggests that remodeling of the microfibrillar network occurs throughout adult life. Investigation of the distribution of fibrillin-1 mRNA-positive cells revealed extensive labeling within the epidermis. This labeling was not confined to basal keratinocytes, but extended into the granular cell layer. Such a distribution provides evidence that epidermal keratinocytes have potential to make a major contribution to the production of microfibrillar arrays in normal skin, similar to that observed with type VII collagen and anchoring fibrils in the same region. Examination of fibrillin-1 gene expression in skin derived from sites of severe photoaging indicates that long-term exposure to solar irradiation has major effects at the mRNA level. Decreases in the number of keratinocytes and dermal fibroblasts expressing this mRNA were observed in 80% of the severely affected extensor forearm biopsies examined. This decrease mirrors those observed in other major matrix components of the papillary dermis in photoaging; collagen VII-containing anchoring fibrils of the DEJ (Craven et al., 1997Craven N.M. Watson R.E.B. Jones C.J.P. Shuttleworth C.A. Kielty C.M. Griffiths C.E.M. Clinical features of photodamaged human skin are associated with a reduction of collagen VII.Br J Dermatol. 1997; 137: 344-350Crossref PubMed Scopus (75) Google Scholar) and procollagens I and III (Talwar et al., 1995Talwar H.S. Griffiths C.E.M. Fisher G.J. Hamilton T.A. Voorhees J.J. Reduced type I and type III procollagens in photodamaged adult human skin.J Invest Dermatol. 1995; 105: 285-290Crossref PubMed Scopus (289) Google Scholar). It can be argued that this reduction is a general one, a result of the pathobiology of photoaging itself. It would appear, however, that not all matrix proteins within the papillary dermis are affected by solar irradiation. Studies examining the distribution of the microfibrillar collagen type VI and its mRNA expression in photoaged skin have identified little alteration with sun exposure (Boorsma et al., 1996Boorsma J. Watson R.E.B. Craven N.M. Shuttleworth C.A. Kielty C.M. Griffiths C.E.M. Collagen type VI in photoaged skin.J Invest Dermatol. 1996; 107 (Abstract): 516Google Scholar). The distribution and role of fibrillin-2 in normal human skin has not been examined in detail. It is, however, known to be localized to predominantly elastic tissues such as cartilage and bronchal tissue (Boileau et al., 1997Boileau C. Collod G. Bonnet D. Genetic input into the pathophysiology and the diagnosis of marfan syndrome.Arch Mal Coeur Vaiss. 1997; 90: 1707-1713PubMed Google Scholar).Ashcroft et al., 1997Ashcroft G.S. Kielty C.M. Horan M.A. Ferguson M.W. Age-related changes in temporal and spatial distributions of fibrillin and elastin mRNAs and proteins in acute cutaneous wounds of healthy humans.J Pathol. 1997; 183: 80-89Crossref PubMed Scopus (67) Google Scholar examined the distribution and abundance of fibrillin-1, fibrillin-2, and elastin in wound healing. These studies reveal fibrillin-1 to be the major component of the microfibrillar arrays in skin repair following punch biopsies, with the distribution of fibrillin-2 mainly confined to areas within the dermis proximal to blood vessels. This is very much in accord with data presented here on expression of fibrillin-2 mRNA in photoaged skin. Fibrillin-2 is expressed in much lower amounts than fibrillin-1 and there appears to be little difference in the number of cells expressing it, nor in the level of expression in photoprotected versus photoaged skin. Examination of severely photoaged skin provides data for understanding the end point of the pathologic process due to prolonged exposure to UV irradiation. Conclusions cannot be drawn from these data as to the effect of acute UV irradiation on the expression of either fibrillin-1 or fibrillin-2. It is well known that UV has the ability to modulate quickly the expression of elastin via transcriptional promoters (Schwarz et al., 1995Schwarz E. Feinberg E. Lebwohl M. Mariani T.J. Boyd C.D. Ultraviolet radiation increases tropoelastin accumulation by a post-transcriptional mechanism in dermal fibroblasts.J Invest Dermatol. 1995; 105: 65-69Crossref PubMed Scopus (24) Google Scholar,Schwartz et al., 1998Schwartz E. Gelfand J.M. Mauch J.C. Kligman L.H. Generation of tropoelastin mRNA variant by alternative polyadenylation site selection in sun-damaged and UV-B irradiated fibroblasts.Biochem Biophys Res Commun. 1998; 246: 217-221Crossref PubMed Scopus (10) Google Scholar;Bernstein et al., 1996Bernstein E.F. Gasparro F.P. Brown D.B. Tackeuchi T. Kang S.G. Uitto J. 8-methoxypsoralen and UV-A radiation activate the human elastin promoter in transgenic mice: In vivo and in vitro evidence for gene induction.Photochem Photobiol. 1996; 64: 369-374Crossref PubMed Scopus (18) Google Scholar;Uitto et al., 1997Uitto J. Brown D.B. Gasparro F.P. Bernstein E.F. Molecular aspects of photoaging.Eur J Dermatol. 1997; 7: 210-214Google Scholar). It is, therefore, vital to examine further the effect of acute UV irradiation on the turnover of both these molecules in vivo. Microfibril depletion observed as a result of prolonged, chronic UV exposure could reflect inflammatory cell proteinase activities such as neutrophil elastase, which we have previously shown capable of rapidly degrading intact microfibrils (Kielty et al., 1994Kielty C.M. Woolley D.E. Whittaker S.P. Shuttleworth C.A. Catabolism of intact fibrillin microfibrils by neutrophil elastase, chymotrypsin and trypsin.FEBS Lett. 1994; 351: 85-89Abstract Full Text PDF PubMed Scopus (49) Google Scholar). Fibrillin degradation is also possible by matrix metalloproteinases (Ashworth et al., 1999Ashworth J.L. Murphy M. Rock M.J. Sherratt M.J. Shapiro S.D. Shuttleworth C.A. Kielty C.M. Fibrillin degradation by matrix metalloproteinases: Implications for connective tissue remodelling.Biochem J. 1999Crossref Google Scholar).Fisher et al., 1997Fisher G.J. Wang Z.Q. Datta S.C. Varani J. Kang S. Voorhees J.J. Pathophysiology of premature skin aging induced by ultraviolet light.N Engl J Med. 1997; 337: 1419-1428Crossref PubMed Scopus (1081) Google Scholar have further shown that matrix metalloproteinase expression is enhanced in photoaged skin. It is, therefore, likely that the end process observed in photoaging is due (at least in part) to the effects of these enzymes. It remains to be determined if the microfibril-immunopositive material at the DEJ of photoaged skin is newly deposited fibrillin-rich microfibrils elaborated as a repair attempt similar to that previously shown to follow keratinocyte autografting of burns (Raghunath et al., 1996Raghunath M. Bächi T. Meuli M. Altermatt S. Gobet R. Bruckner-Tuderman L. Steinmann B. Fibrillin and elastin expression in skin regenerating from cultured keratinocyte autographs: morphogenesis of microfibrils begins at the dermo-epidermal junction and precedes elastic fiber formation.J Invest Dermatol. 1996; 106: 1090-1096Crossref PubMed Scopus (61) Google Scholar). Additional data need to be acquired to address this situation. In conclusion, these studies suggest that the fibrillin-containing microfibrillar elements of the DEJ are susceptible to damage caused by solar irradiation, likely to be due to the activity of matrix degrading enzymes, and that such alterations may contribute to some of the clinical characteristics of chronically sun-exposed skin. We are grateful to Steven Bagley for his expertise with confocal microscopy and Fiona Campbell for statistical analyses. This work was supported by a grant from Unilever Research, Colworth, Bedfordshire, U.K.