Biogerontology (2012) 13:37–48 DOI 10.1007/s10522-011-9332-9 RESEARCH ARTICLE Differential expression of elastic fibre components in intrinsically aged skin Abigail K. Langton • Michael J. Sherratt • Christopher E. M. Griffiths • Rachel E. B. Watson Received: 11 February 2011 / Accepted: 21 March 2011 / Published online: 2 April 2011 Ó Springer Science+Business Media B.V. 2011 Abstract Intrinsic ageing of the skin is a subtle network components were unchanged with age. process resulting in some degree of skin laxity. The However, three differentially expressed genes were dermal elastic fibre network imbues skin with the identified: latent TGFb-binding protein (LTBP)-2 capacity to recoil and loss of this property contributes which was up-regulated with age (fold change ?1.58, to an aged, wrinkled appearance. Whilst elastic fibres P = 0.041); LTBP3 (fold change -1.67, P = 0.025) have a complex, composite structure which allows and the lysyl oxidase-like enzyme (LOXL1, fold them to fulfil multiple roles, the effects of intrinsic change -1.47, P = 0.008) which were both down- ageing on their discrete molecular components has regulated with age. Although the changes in gene not previously been explored. In this study, we have expression for LTBP3 were not confirmed by either used a microarray-based approach to perform a novel qPCR or immunostaining, the expression and tissue survey of the changes in gene expression that occur in deposition of both LTBP2 and LOXL1 were signif- components of cutaneous elastic fibres as a result of icantly enhanced in intrinsically aged skin. Whilst the intrinsic ageing. Age-related changes in gene expres- functional implications of these altered expression sion were validated at the mRNA and protein levels profiles remains to be elucidated, LTBP2 and LOXL1 using quantitative real-time polymerase chain reac- are thought to play important roles in controlling and tion (qPCR) and immunostaining, respectively. The maintaining elastic fibre deposition, assembly and microarray revealed that the majority of elastic fibre structure via binding to fibulin-5. Consequently, any age-related perturbations in the expression of these components may have important consequences on Electronic supplementary material The online version of remodelling of the extracellular matrix and hence on this article (doi:10.1007/s10522-011-9332-9) contains the mechanical properties of intrinsically aged skin. supplementary material, which is available to authorized users. A. K. Langton (&) Á C. E. M. Griffiths Á R. E. B. Watson Keywords Elastic fibre Á Intrinsic ageing Á Dermatological Sciences, The University of Manchester, Fibrillin-rich microfibril Manchester Academic Health Science Centre, 1.222 Stopford Building, Oxford Road, Manchester M13 9PT, UK Abbreviations e-mail: [email protected] AUC Area under the curve bp Base pair M. J. Sherratt DAPI 40-6-Diamidino-2-phenylindole Regenerative Biomedicine, The University of Manchester, Manchester Academic Health Science Centre, DEJ Dermal-epidermal junction Manchester, UK GC-RMA GeneChip-Robust Multiarray Averaging 123 38 Biogerontology (2012) 13:37–48 LOX Lysyl oxidase proteins (Mithieux and Weiss 2005). In the mature LOXL Lysyl oxidase-like elastic fibre, the elastin core comprises over 90% of LTBP Latent transforming growth factor-b the volume whilst the fibrillin-rich microfibrils are binding protein largely confined to an outer mantle (Mecham and qPCR Quantitative real-time polymerase chain Davis 1994). Although elastin and fibrillin-1 are the reaction predominant protein constituents of skin-derived TBS Tris-buffered saline elastic fibres, several proteins, including the micro- fibril associated glycoproteins (MAGPs), latent trans- forming growth factor-b binding proteins (LTBPs) Introduction and fibulins are frequently associated with microfi- brils, whilst decorin, biglycan, versican and the Whilst facial wrinkles occur primarily as a conse- emilins may also be sporadically co-localised (Kielty quence of chronic exposure to environmental insults et al. 2002). such as sunlight (Fisher et al. 1996) and smoking Both the intrinsic and extrinsic ageing processes (Ernster et al. 1995; Kadunce et al. 1991), in the have profound, yet differing effects on the structure, absence of such exogeneous factors, the clinical and hence potentially the function, of the elastic fibre effects of chronological (also known as intrinsic network (Sherratt 2009). The characteristic smooth ageing) may only become apparent from the age of appearance and fine wrinkles of intrinsically aged 70 years (Kafi et al. 2007). This slow ageing process skin are associated with a progressive fragmentation causes subtle changes in tissue structure (Montagna and decline of the elastic fibre network (Braverman and Carlisle 1979) which in turn impairs function. As and Fonferko 1982; Montagna and Carlisle 1979). In a consequence, intrinsically aged skin is relatively contrast, the deep wrinkles that characterise photo- smooth, finely wrinkled and unblemished. Histolog- aged skin are associated with not only loss of the ically, whilst few changes are noted in the epidermis, fibrillin-rich microfibrils at the DEJ (Watson et al. the topology of the dermal-epidermal junction (DEJ) 1999) but crucially the gain of elastotic material is altered in that it exhibits a flattened appearance (disorganised elastic fibre proteins) within the deep with a correspondingly diminished connecting sur- dermis (Watson et al. 2001). These architectural and face area (Giangreco et al. 2010). Intrinsically aged compositional alterations are thought to occur as a skin is also more fragile and less elastic (both result of structural degenerative changes within the resilience and compliance) than young skin (Escoffier elastic fibre which may be mediated by cellular et al. 1989). mechanisms, principally extracellular proteases The dermal elastic fibre network, which is a major (Kielty et al. 1994; Ashworth et al. 1999) and/or by contributor to elastic recoil (Uitto 1979), is composed cellular mechanisms such as exposure to ultraviolet of complex and highly ordered macromolecular radiation (Sherratt et al. 2010). assemblies (Cotta-Pereira et al. 1976). Discrete Although these elastic fibre components are cascades of candelabra-like oxytalan fibres (which required to fulfil important yet disparate mechanical are composed solely of fibrillin-rich microfibrils) and biochemical roles in structurally and functionally connect the DEJ to fibrillin and elastin containing dissimilar ocular, vascular and cutaneous tissues, the elaunin fibres in the lower papillary dermis. Finally, effects of intrinsic ageing on this group of biomol- in the reticular dermis, large diameter elastin-rich ecules has not previously been explored. Therefore, fibres run parallel to the DEJ (for a review see the aim of this study was to perform a novel survey of (Langton et al. 2010). Hence, with increasing depth the changes that occur in commonly-associated from the DEJ, the ratio of elastin to fibrillin increases. elastic fibre components in young and intrinsically The developmental deposition of elastic fibres is also aged human skin. Age-related changes in cutaneous highly regulated: tropoelastin, the soluble precursor gene expression were identified using an AffymetrixÒ of elastin, is deposited onto a pre-formed fibrillin-rich microarray and were validated at the mRNA and microfibrillar scaffold. Mature elastin is then formed protein levels using quantitative real-time polymer- via the enzyme-mediated cross-linking of tropoelastin ase chain reaction (qPCR) and immunostaining, by lysyl oxidase (LOX) and/or LOX-like (LOXL) respectively. 123 Biogerontology (2012) 13:37–48 39 Materials and methods tested statistically with CyberT on logarithmic scale data (Baldi and Long 2001). False-discovery correc- Skin biopsy procurement tion was performed with Q-value software (Storey and Tibshirani 2003). Significantly changed probe Skin biopsies (6 mm diameter) were obtained from sets were selected on P \ 0.05 and expression level the photoprotected buttock of nine young (five men ([50). and four women; age range 18–29 years) and nine aged (five men and four women; age range 65–75 years) healthy Caucasian volunteers. Each Real-time qPCR punch biopsy was obtained under 1% lignocaine local anaesthesia and bisected. One half of the biopsy cDNA was transcribed from total RNA using random was immersed in RNAlater solution (Ambion, Foster primers and the cloned AMV first-strand cDNA City, CA, USA) and stored at -80°C, the remaining synthesis kit (Invitrogen). The resulting cDNA was half was embedded in optimal cutting temperature subjected to qPCR analysis using human TaqManÒ compound (Miles Laboratories, Elkhart, IN, USA), gene expression assays (Applied Biosystems, snap frozen in liquid nitrogen and stored at -80°C. Warrington, UK) and the StepOne PlusTM Real-Time Local ethical committee approval was obtained for PCR system (Applied Biosystems). TaqManÒ gene the study and all subjects gave written, informed expression assays were obtained for LTBP2 consent. (Hs01553358_m1, RefSeq: NM_000428.2, amplicon size = 64 bp), LTBP3 (Hs01105729_m1, RefSeq: RNA extraction NM_001130144.2, amplicon size = 86 bp) and LOXL1 (Hs00935937_m1, RefSeq: NM_005576.2, Total RNA was isolated from frozen biopsy tissue amplicon size = 73 bp). Two types of controls were using reagents from the TRIzolÒ Plus RNA Purifica- included for each gene analysed: (i) reverse tran- tion Kit (Invitrogen, Paisley, UK) according to scription mixture synthesised without