Ultrastructural characterisation of fibrillin microfibrils from photoaged and all-trans retinoic acid-treated skin

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.