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Photoageing of human skin by cumulative exposure to ultraviolet radiation (UVR) is characterised by extensive remodelling of the long-lived dermal elastic fibre network. Fibrillin microfibrils are key components of this network and are a useful biomarker of photoageing and its repair with topical all-trans retinoic acid (t-RA) treatment. Investigations have shown that fibrillin microfibrils are highly susceptible to UVR in vitro, yet it is unknown how the full contributing effects of photoageing in vivo will affect their complex ultrastructure. Furthermore, whilst t-RA-treatment of photoaged skin leads to an increase in immunohistochemical staining for fibrillin microfibrils, still unanswered is whether treatment leads to new synthesis or repair of structurally and functionally competent fibrillin microfibrils, comparable to those found in photoprotected young skin. Healthy, but severely photoaged, volunteers (mean age 71.3 years) were recruited to the study (n=10). Samples (3mm biopsies) were obtained from photoprotected (intrinsically aged) upper inner arm and photoaged forearm untreated or treated with 0.025% t-RA for four-days. Biopsies were sectioned and stained for fibrillin microfibrils using immunohistochemistry (performed on nine volunteers) or used for extraction of fibrillin microfibrils by bacterial collagenase and size exclusion chromatography (performed on five volunteers). The following parameters were measured on extracted fibrillin microfibrils both by scanning transmission electron microscopy (STEM) and atomic force microscopy (AFM): (i) Abundance (n=10) (ii) length (n=50) (iii) mass per repeat (n=250, STEM only) (iv) flexion angle (n=250) and (v) periodicity (n=250).In eight of the nine volunteers there was a reduction in fibrillin microfibril staining grade of photoaged skin. Fibrillin microfibrils extracted from photoaged skin had a lower mass per repeat than those extracted from photoprotected skin of the same volunteer; which was localised to the centre of the bead. All volunteers displayed a significant increase or decrease in periodicity, but no change in flexibility or abundance. Fibrillin microfibrils extracted from t-RA-treated photoaged skin showed alterations in periodicity in four of the five volunteers and an increase in mass in two of four volunteers, but no change in length, flexibility or abundance. Fibrillin microfibrils extracted from t-RA-treated photoprotected (intrinsically aged) skin, were apparently normalised in terms of length and periodicity, with decreases in mass in three of five volunteers, but no alterations in flexibility or abundance. There was wide individual variation across all fibrillin microfibril populations. Results showed for the first time that fibrillin microfibrils are susceptible to photoageing-related remodelling and damage in vivo, which is likely mediated by a combination of direct UVR absorption by chromophores, ROS-mediated damage and enzyme-mediated remodelling. t-RA was found not to significantly alter the ultrastructure of fibrillin microfibrils, perhaps due to the short length of the patch-test study, the complex macromolecular nature of fibrillin microfibrils, or individual variation in retinoid response.

Exposure of the skin to ultraviolet radiation (UVR) results clinically in the formation of deep wrinkles and mottled pigmentation and histologically, in a vast remodelling of the dermal extracellular matrix (ECM), in particular the elastic fibre network. Fibrillin microfibrils and fibulin-5 are early biomarkers of photoageing, where a loss of these fibres from the dermal epidermal junction is apparent. A study by our group showed that isolated fibrillin microfibrils and fibronectin which are rich in amino acids which absorb energy from UVR (UV-chromophores) are susceptible to UVR-induced damage, whilst UV-chromophore poor collagen type I is not. This research, with other earlier studies, indicates that acellular mechanism may work in tandem with cell-mediated up-regulation of matrix metalloproteinases (MMPs) in the progression of photoageing. This thesis aims to: i) test whether acellular mechanisms of photoageing are a result of direct photon absorption and/or the photodynamic production of reactive oxygen species (ROS); ii) assess the functional consequences of UVB degradation on the susceptibility of fibrillin microfibrils to MMPs and; iii) assay whether ECM proteins are differentially susceptible to solar simulated radiation (SSR) or UVA (315-400nm) alone using physiologically relevant doses of irradiation. Isolated proteins were exposed to UVB (280-315nm) in depleted-O2 conditions and in the presence of deuterium oxide. Depleted-O2 conditions decreased and deuterium oxide conditions increased UVR-induced degradation. Isolated proteins also show a similar pattern of degradation when exposed to H2O2 as an exogenous source of ROS. These results indicate that ROS play an important role in the differential degradation of dermal proteins. MMPs-3 and -9 are both upregulated in the skin after exposure to UVR and have the ability to degrade elastic fibre components. After exposure to UVB, damaged fibrillin microfibrils become more susceptible to degradation by both MMPs-3 and -9. Chromophore-rich fibrillin microfibrils and fibronectin are susceptible to degradation by both SSR and UVA alone, whereas chromophore-poor collagens type I and VI and tropoelastin are not. These results support our previous findings that amino acid composition of proteins is a good indicator of their relative susceptibility to UV-induced damage with a physiologically relevant irradiation system. In conclusion this work shows that ROS are an important mediator of acellular mechanisms of photoageing and that amino acid composition is a good indication of relative susceptibility of proteins to both ROS and UVR. The ability to predict ROS-susceptible proteins also has wider implications for human ageing as a whole.