Acute exposure to ultraviolet radiation targets proteins involved in collagen fibrillogenesis.

Introduction: Exposure to chronic, low-dose UV irradiation (UVR) can lead to premature ageing of the skin. Understanding which proteins are affected by acute UVR and photo-dynamically produced reactive oxygen species (ROS) could help to inform strategies to delay photoageing. Conventional biochemical analyses can be used to characterize UVR/ROS-induced damage on a protein-by-protein basis and we have previously shown using SDS-PAGE that collagen I and plasma fibronectin are respectively resistant and susceptible to physiological doses of UVR. The aim of this study was to screen a complex proteome for UVR-affected proteins. Methods: This study employed a sensitive mass spectrometry technique (peptide location fingerprinting: PLF) which can identify structure associated differences following trypsin digestion to characterize the impact of UVR exposure on purified collagen I and tissue fibronectin and to identify UVR-susceptible proteins in an ECM-enriched proteome. Results: Using LC/MS-MS and PLF we show that purified mature type-I collagen is resistant to UVR, whereas purified tissue fibronectin is susceptible. UV irradiation of a human dermal fibroblast-deposited ECM-enriched proteome in vitro, followed by LC/MS-MS and PLF analysis revealed two protein cluster groups of UV susceptible proteins involved in i) matrix collagen fibril assembly and ii) protein translation and motor activity. Furthermore, PLF highlighted UV susceptible domains within targeted matrix proteins, suggesting that UV damage of matrix proteins is localized. Discussion: Here we show that PLF can be used to identify protein targets of UVR and that collagen accessory proteins may be key targets in UVR exposed tissues.

Prediction, screening and characterization of novel bioactive tetrapeptide matrikines for skin rejuvenation.

BACKGROUND: Extracellular matrices play a critical role in tissue structure and function and aberrant remodelling of these matrices is a hallmark of many age-related diseases. In skin, loss of dermal collagens and disorganization of elastic fibre components are key features of photoageing. Although the application of some small matrix-derived peptides to aged skin has been shown to beneficially affect in vitro cell behaviour and, in vivo, molecular architecture and clinical appearance, the discovery of new peptides has lacked a guiding hypothesis. OBJECTIVES: To identify, using protease cleavage site prediction, novel putative matrikines with beneficial activities for skin composition and structure. METHODS: Here, we present an in silico (peptide cleavage prediction) to in vitro (proteomic and transcriptomic activity testing in cultured human dermal fibroblasts) to in vivo (short-term patch test and longer-term split-face clinical study) discovery pipeline, which enables the identification and characterization of peptides with differential activities. RESULTS: Using this pipeline we showed that cultured fibroblasts were responsive to all applied peptides, but their associated bioactivity was sequence-dependent. Based on bioactivity, toxicity and protein source, we further characterized a combination of two novel peptides, GPKG (glycine-proline-lysine-glycine) and LSVD (leucine-serine-valine-aspartate), that acted in vitro to enhance the transcription of matrix -organization and cell proliferation genes and in vivo (in a short-term patch test) to promote processes associated with epithelial and dermal maintenance and remodelling. Prolonged use of a formulation containing these peptides in a split-face clinical study led to significantly improved measures of crow's feet and firmness in a mixed population. CONCLUSIONS: This approach to peptide discovery and testing can identify new synthetic matrikines, providing insights into biological mechanisms of tissue homeostasis and repair and new pathways to clinical intervention.

Like other organs and tissues, the skin is composed of both cells and a complex network of molecules and proteins called an extracellular matrix. This matrix contains proteins such as collagen and elastin and undergoes many changes when the skin is damaged by the sun. We know from previous studies that small parts of matrix proteins (called peptide 'matrikines') can help to treat the signs of sun-related skin ageing. In this UK study, we show that new beneficial peptides (with matrikine activity) can be identified using machine learning (artificial intelligence) techniques that predict where common matrix proteins might be 'cut' by skin enzymes. Candidate peptides were first made in the laboratory and then applied to skin cells in culture. These cell culture screens demonstrated that, while all the peptides showed some matrikine activity, two were particularly promising. These two peptides were then tested in a short-term study on the forearm skin of volunteers and, in a longer-term study, on the face. We found that the combination of these two peptides can prompt forearm skin cells to express genes that are involved in many different aspect of skin health and, over the longer 6-month period, produce visible benefits in the appearance of fine lines and wrinkles and firmness on the face. Our findings suggest that this approach may be able to identify beneficial peptide treatments for not only skin ageing and diseases, but also unwanted changes in the extracellular matrix of other tissues and organs.

Fibrillin microfibril structure identifies long-range effects of inherited pathogenic mutations affecting a key regulatory latent TGFß-binding site.

Genetic mutations in fibrillin microfibrils cause serious inherited diseases, such as Marfan syndrome and Weill-Marchesani syndrome (WMS). These diseases typically show major dysregulation of tissue development and growth, particularly in skeletal long bones, but links between the mutations and the diseases are unknown. Here we describe a detailed structural analysis of native fibrillin microfibrils from mammalian tissue by cryogenic electron microscopy. The major bead region showed pseudo eightfold symmetry where the amino and carboxy termini reside. On the basis of this structure, we show that a WMS deletion mutation leads to the induction of a structural rearrangement that blocks interaction with latent TGFß-binding protein-1 at a remote site. Separate deletion of this binding site resulted in the assembly of shorter fibrillin microfibrils with structural alterations. The integrin αvß3-binding site was also mapped onto the microfibril structure. These results establish that in complex extracellular assemblies, such as fibrillin microfibrils, mutations may have long-range structural consequences leading to the disruption of growth factor signaling and the development of disease.

Peptide location fingerprinting identifies species- and tissue-conserved structural remodelling of proteins as a consequence of ageing and disease.

Extracellular matrices (ECMs) in the intervertebral disc (IVD), lung and artery are thought to undergo age-dependant accumulation of damage by chronic exposure to mechanisms such as reactive oxygen species, proteases and glycation. It is unknown whether this damage accumulation is species-dependant (via differing lifespans and hence cumulative exposures) or whether it can influence the progression of age-related diseases such as atherosclerosis. Peptide location fingerprinting (PLF) is a new proteomic analysis method, capable of the non-targeted identification of structure-associated changes within proteins. Here we applied PLF to publicly available ageing human IVD (outer annulus fibrosus), ageing mouse lung and human arterial atherosclerosis datasets and bioinformatically identified novel target proteins alongside common age-associated differences within protein structures which were conserved between three ECM-rich organs, two species, three IVD tissue regions, sexes and in an age-related disease. We identify peptide yield differences across protein structures which coincide with biological regions, potentially reflecting the functional consequences of ageing or atherosclerosis for macromolecular assemblies (collagen VI), enzyme/inhibitor activity (alpha-2 macroglobulin), activation states (complement C3) and interaction states (laminins, perlecan, fibronectin, filamin-A, collagen XIV and apolipoprotein-B). Furthermore, we show that alpha-2 macroglobulin and collagen XIV exhibit possible shared structural consequences in IVD ageing and arterial atherosclerosis, providing novel links between an age-related disease and intrinsic ageing. Crucially, we also demonstrate that fibronectin, laminin beta chains and filamin-A all exhibit conserved age-associated structural differences between mouse lung and human IVD, providing evidence that ECM, and their associating proteins, may be subjected to potentially similar mechanisms or consequences of ageing across both species, irrespective of differences in lifespan and tissue function.

Matrikines as mediators of tissue remodelling.

Extracellular matrix (ECM) proteins confer biomechanical properties, maintain cell phenotype and mediate tissue repair (via release of sequestered cytokines and proteases). In contrast to intracellular proteomes, where proteins are monitored and replaced over short time periods, many ECM proteins function for years (decades in humans) without replacement. The longevity of abundant ECM proteins, such as collagen I and elastin, leaves them vulnerable to damage accumulation and their host organs prone to chronic, age-related diseases. However, ECM protein fragmentation can potentially produce peptide cytokines (matrikines) which may exacerbate and/or ameliorate age- and disease-related ECM remodelling. In this review, we discuss ECM composition, function and degradation and highlight examples of endogenous matrikines. We then critically and comprehensively analyse published studies of matrix-derived peptides used as topical skin treatments, before considering the potential for improvements in the discovery and delivery of novel matrix-derived peptides to skin and internal organs. From this, we conclude that while the translational impact of matrix-derived peptide therapeutics is evident, the mechanisms of action of these peptides are poorly defined. Further, well-designed, multimodal studies are required.

Early Alterations of Intra-Mural Elastic Lamellae Revealed by Synchrotron X-ray Micro-CT Exploration of Diabetic Aortas.

Diabetes is a major concern of our society as it affects one person out of 11 around the world. Elastic fiber alterations due to diabetes increase the stiffness of large arteries, but the structural effects of these alterations are poorly known. To address this issue, we used synchrotron X-ray microcomputed tomography with in-line phase contrast to image in three dimensions C57Bl6J (control) and db/db (diabetic) mice with a resolution of 650 nm/voxel and a field size of 1.3 mm3. Having previously shown in younger WT and db/db mouse cohorts that elastic lamellae contain an internal supporting lattice, here we show that in older db/db mice the elastic lamellae lose this scaffold. We coupled this label-free method with automated image analysis to demonstrate that the elastic lamellae from the arterial wall are structurally altered and become 11% smoother (286,665 measurements). This alteration suggests a link between the loss of the 3D lattice-like network and the waviness of the elastic lamellae. Therefore, waviness measurement appears to be a measurable elasticity indicator and the 3D lattice-like network appears to be at the origin of the existence of this waviness. Both could be suitable indicators of the overall elasticity of the aorta.

Defining the Protease and Protease Inhibitor (P/PI) Proteomes of Healthy and Diseased Human Skin by Modified Systematic Review.

Proteases and protease inhibitors (P/PIs) are involved in many biological processes in human skin, yet often only specific families or related groups of P/PIs are investigated. Proteomics approaches, such as mass spectrometry, can define proteome signatures (including P/PIs) in tissues; however, they struggle to detect low-abundance proteins. To overcome these issues, we aimed to produce a comprehensive proteome of all P/PIs present in normal and diseased human skin, in vivo, by carrying out a modified systematic review using a list of P/PIs from MEROPS and combining this with key search terms in Web of Science. Resulting articles were manually reviewed against inclusion/exclusion criteria and a dataset constructed. This study identified 111 proteases and 77 protease inhibitors in human skin, comprising the serine, metallo-, cysteine and aspartic acid catalytic families of proteases. P/PIs showing no evidence of catalytic activity or protease inhibition, were designated non-peptidase homologs (NPH), and no reported protease inhibitory activity (NRPIA), respectively. MMP9 and TIMP1 were the most frequently published P/PIs and were reported in normal skin and most skin disease groups. Normal skin and diseased skin showed significant overlap with respect to P/PI profile; however, MMP23 was identified in several skin disease groups, but was absent in normal skin. The catalytic profile of P/PIs in wounds, scars and solar elastosis was distinct from normal skin, suggesting that a different group of P/PIs is responsible for disease progression. In conclusion, this study uses a novel approach to provide a comprehensive inventory of P/PIs in normal and diseased human skin reported in our database. The database may be used to determine either which P/PIs are present in specific diseases or which diseases individual P/PIs may influence.

The Effects of Ionising and Non-Ionising Electromagnetic Radiation on Extracellular Matrix Proteins.

Exposure to sub-lethal doses of ionising and non-ionising electromagnetic radiation can impact human health and well-being as a consequence of, for example, the side effects of radiotherapy (therapeutic X-ray exposure) and accelerated skin ageing (chronic exposure to ultraviolet radiation: UVR). Whilst attention has focused primarily on the interaction of electromagnetic radiation with cells and cellular components, radiation-induced damage to long-lived extracellular matrix (ECM) proteins has the potential to profoundly affect tissue structure, composition and function. This review focuses on the current understanding of the biological effects of ionising and non-ionising radiation on the ECM of breast stroma and skin dermis, respectively. Although there is some experimental evidence for radiation-induced damage to ECM proteins, compared with the well-characterised impact of radiation exposure on cell biology, the structural, functional, and ultimately clinical consequences of ECM irradiation remain poorly defined.

Peptide Location Fingerprinting Reveals Tissue Region-Specific Differences in Protein Structures in an Ageing Human Organ.

In ageing tissues, long-lived extracellular matrix (ECM) proteins are susceptible to the accumulation of structural damage due to diverse mechanisms including glycation, oxidation and protease cleavage. Peptide location fingerprinting (PLF) is a new mass spectrometry (MS) analysis technique capable of identifying proteins exhibiting structural differences in complex proteomes. PLF applied to published young and aged intervertebral disc (IVD) MS datasets (posterior, lateral and anterior regions of the annulus fibrosus) identified 268 proteins with age-associated structural differences. For several ECM assemblies (collagens I, II and V and aggrecan), these differences were markedly conserved between degeneration-prone (posterior and lateral) and -resistant (anterior) regions. Significant differences in peptide yields, observed within collagen I α2, collagen II α1 and collagen V α1, were located within their triple-helical regions and/or cleaved C-terminal propeptides, indicating potential accumulation of damage and impaired maintenance. Several proteins (collagen V α1, collagen II α1 and aggrecan) also exhibited tissue region (lateral)-specific differences in structure between aged and young samples, suggesting that some ageing mechanisms may act locally within tissues. This study not only reveals possible age-associated differences in ECM protein structures which are tissue-region specific, but also highlights the ability of PLF as a proteomic tool to aid in biomarker discovery.

X-ray microtomography reveals a lattice-like network within aortic elastic lamellae.

The arterial wall consists of three concentric layers: intima, media, and adventitia. Beyond their resident cells, these layers are characterized by an extracellular matrix (ECM), which provides both biochemical and mechanical support. Elastin, the major component of arterial ECM, is present in the medial layer and organized in concentric elastic lamellae that confer resilience to the wall. We explored the arterial wall structures from C57Bl6 (control), db/db (diabetic), and ApoE-/- (atherogenic) mice aged 3 months using synchrotron X-ray computed microtomography on fixed and unstained tissues with a large image field (8 mm3 ). This approach combined a good resolution (0.83 µm/voxel), large 3D imaging field. and an excellent signal to noise ratio conferred by phase-contrast imaging. We determined from 2D virtual slices that the thickness of intramural ECM structures was comparable between strains but automated image analysis of the 3D arterial volumes revealed a lattice-like network within concentric elastic lamellae. We hypothesize that this network could play a role in arterial mechanics. This work demonstrates that phase-contrast synchrotron X-ray computed microtomography is a powerful technique which to characterize unstained soft tissues.

Matrix-Bound Growth Factors are Released upon Cartilage Compression by an Aggrecan-Dependent Sodium Flux that is Lost in Osteoarthritis.

Articular cartilage is a dense extracellular matrix-rich tissue that degrades following chronic mechanical stress, resulting in osteoarthritis (OA). The tissue has low intrinsic repair especially in aged and osteoarthritic joints. Here, we describe three pro-regenerative factors; fibroblast growth factor 2 (FGF2), connective tissue growth factor, bound to transforming growth factor-beta (CTGF-TGFß), and hepatoma-derived growth factor (HDGF), that are rapidly released from the pericellular matrix (PCM) of articular cartilage upon mechanical injury. All three growth factors bound heparan sulfate, and were displaced by exogenous NaCl. We hypothesised that sodium, sequestered within the aggrecan-rich matrix, was freed by injurious compression, thereby enhancing the bioavailability of pericellular growth factors. Indeed, growth factor release was abrogated when cartilage aggrecan was depleted by IL-1 treatment, and in severely damaged human osteoarthritic cartilage. A flux in free matrix sodium upon mechanical compression of cartilage was visualised by 23Na -MRI just below the articular surface. This corresponded to a region of reduced tissue stiffness, measured by scanning acoustic microscopy and second harmonic generation microscopy, and where Smad2/3 was phosphorylated upon cyclic compression. Our results describe a novel intrinsic repair mechanism, controlled by matrix stiffness and mediated by the free sodium concentration, in which heparan sulfate-bound growth factors are released from cartilage upon injurious load. They identify aggrecan as a depot for sequestered sodium, explaining why osteoarthritic tissue loses its ability to repair. Treatments that restore matrix sodium to allow appropriate release of growth factors upon load are predicted to enable intrinsic cartilage repair in OA. SIGNIFICANCE STATEMENT: Osteoarthritis is the most prevalent musculoskeletal disease, affecting 250 million people worldwide.1 We identify a novel intrinsic repair response in cartilage, mediated by aggrecan-dependent sodium flux, and dependent upon matrix stiffness, which results in the release of a cocktail of pro-regenerative growth factors after injury. Loss of aggrecan in late-stage osteoarthritis prevents growth factor release and likely contributes to disease progression. Treatments that restore matrix sodium in osteoarthritis may recover the intrinsic repair response to improve disease outcome.

Predicting Proteolysis in Complex Proteomes Using Deep Learning.

Both protease- and reactive oxygen species (ROS)-mediated proteolysis are thought to be key effectors of tissue remodeling. We have previously shown that comparison of amino acid composition can predict the differential susceptibilities of proteins to photo-oxidation. However, predicting protein susceptibility to endogenous proteases remains challenging. Here, we aim to develop bioinformatics tools to (i) predict cleavage site locations (and hence putative protein susceptibilities) and (ii) compare the predicted vulnerabilities of skin proteins to protease- and ROS-mediated proteolysis. The first goal of this study was to experimentally evaluate the ability of existing protease cleavage site prediction models (PROSPER and DeepCleave) to identify experimentally determined MMP9 cleavage sites in two purified proteins and in a complex human dermal fibroblast-derived extracellular matrix (ECM) proteome. We subsequently developed deep bidirectional recurrent neural network (BRNN) models to predict cleavage sites for 14 tissue proteases. The predictions of the new models were tested against experimental datasets and combined with amino acid composition analysis (to predict ultraviolet radiation (UVR)/ROS susceptibility) in a new web app: the Manchester proteome susceptibility calculator (MPSC). The BRNN models performed better in predicting cleavage sites in native dermal ECM proteins than existing models (DeepCleave and PROSPER), and application of MPSC to the skin proteome suggests that: compared with the elastic fiber network, fibrillar collagens may be susceptible primarily to protease-mediated proteolysis. We also identify additional putative targets of oxidative damage (dermatopontin, fibulins and defensins) and protease action (laminins and nidogen). MPSC has the potential to identify potential targets of proteolysis in disparate tissues and disease states.

Peptide location fingerprinting reveals modification-associated biomarker candidates of ageing in human tissue proteomes.

Although dysfunctional protein homeostasis (proteostasis) is a key factor in many age-related diseases, the untargeted identification of structurally modified proteins remains challenging. Peptide location fingerprinting is a proteomic analysis technique capable of identifying structural modification-associated differences in mass spectrometry (MS) data sets of complex biological samples. A new webtool (Manchester Peptide Location Fingerprinter), applied to photoaged and intrinsically aged skin proteomes, can relatively quantify peptides and map statistically significant differences to regions within protein structures. New photoageing biomarker candidates were identified in multiple pathways including extracellular matrix organisation (collagens and proteoglycans), protein synthesis and folding (ribosomal proteins and TRiC complex subunits), cornification (keratins) and hemidesmosome assembly (plectin and integrin α6ß4). Crucially, peptide location fingerprinting uniquely identified 120 protein biomarker candidates in the dermis and 71 in the epidermis which were modified as a consequence of photoageing but did not differ significantly in relative abundance (measured by MS1 ion intensity). By applying peptide location fingerprinting to published MS data sets, (identifying biomarker candidates including collagen V and versican in ageing tendon) we demonstrate the potential of the MPLF webtool for biomarker discovery.

Remodelling of fibrillin-rich microfibrils by solar-simulated radiation: impact of skin ethnicity.

Fibrillin-rich microfibrils (FRMs) constitute integral components of the dermal elastic fibre network with a distinctive ultrastructural 'beads-on-a-string' appearance that can be visualised using atomic force microscopy and characterised by measurement of their length and inter-bead periodicity. Their deposition within the dermis in photoprotected skin appears to be contingent on skin ethnicity, and influences the ultrastructure of papillary - but not reticular - dermal FRMs. Truncation and depletion of FRMs at the dermal-epidermal junction of skin occurs early in photoageing in people with lightly pigmented skin; a process of accelerated skin ageing that arises due to chronic sun exposure. Accumulation of ultraviolet radiation (UVR)-induced damage, either by the action of enzymes, oxidation or direct photon absorption, results in FRM remodelling and changes to ultrastructure. In the current study, the direct effect of UVR exposure on FRM ultrastructure was assayed by isolating FRMs from the papillary and reticular dermis of photoprotected buttock skin of individuals of either black African or white Northern European ancestry and exposing them to solar-simulated radiation (SSR). Exposure to SSR resulted in significant reduction in inter-bead periodicity for reticular dermis-derived FRMs across both cohorts. In contrast, papillary dermal FRMs exhibited significantly increased inter-bead periodicity, with the magnitude of damage greater for African FRMs, as compared to Northern European FRMs. Our data suggest that FRMs of the dermis should be considered as two distinct populations that differentially accrue damage in response to SSR. Furthermore, papillary dermal FRMs derived from black African subjects show greater change following UVR challenge, when extracted from skin. Future studies should focus on understanding the consequences of UVR exposure in vivo, regardless of skin ethnicity, on the molecular composition of FRMs and how this UVR-induced remodelling may affect the role FRMs play in skin homeostasis.

X-ray Micro-Computed Tomography: An Emerging Technology to Analyze Vascular Calcification in Animal Models.

Vascular calcification describes the formation of mineralized tissue within the blood vessel wall, and it is highly associated with increased cardiovascular morbidity and mortality in patients with chronic kidney disease, diabetes, and atherosclerosis. In this article, we briefly review different rodent models used to study vascular calcification in vivo, and critically assess the strengths and weaknesses of the current techniques used to analyze and quantify calcification in these models, namely 2-D histology and the o-cresolphthalein assay. In light of this, we examine X-ray micro-computed tomography (µCT) as an emerging complementary tool for the analysis of vascular calcification in animal models. We demonstrate that this non-destructive technique allows us to simultaneously quantify and localize calcification in an intact vessel in 3-D, and we consider recent advances in µCT sample preparation techniques. This review also discusses the potential to combine 3-D µCT analyses with subsequent 2-D histological, immunohistochemical, and proteomic approaches in correlative microscopy workflows to obtain rich, multifaceted information on calcification volume, calcification load, and signaling mechanisms from within the same arterial segment. In conclusion we briefly discuss the potential use of µCT to visualize and measure vascular calcification in vivo in real-time.

Heterogeneity of fibrillin-rich microfibrils extracted from human skin of diverse ethnicity.

The dermal elastic fibre network is the primary effector of skin elasticity, enabling it to extend and recoil many times over the lifetime of the individual. Fibrillin-rich microfibrils (FRMs) constitute integral components of the elastic fibre network, with their distribution showing differential deposition in the papillary dermis across individuals of diverse skin ethnicity. Despite these differential findings in histological presentation, it is not known if skin ethnicity influences FRM ultrastructure. FRMs are evolutionarily highly conserved from jellyfish to man and, regardless of tissue type or species, isolated FRMs have a characteristic 'beads-on-a-string' ultrastructural appearance, with an average inter-bead distance (or periodicity) of 56 nm. Here, skin biopsies were obtained from the photoprotected buttock of healthy volunteers (18-27 years; African: n = 5; European: n = 5), and FRMs were isolated from the superficial papillary dermis and deeper reticular dermis and imaged by atomic force microscopy. In the reticular dermis, there was no significant difference in FRM ultrastructure between European and African participants. In contrast, in the more superficial papillary dermis, inter-bead periodicity was significantly larger for FRMs extracted from European participants than from African participants by 2.20 nm (p < .001). We next assessed whether these differences in FRM ultrastructure were present during early postnatal development by characterizing FRMs from full-thickness neonatal foreskin. Analysis of FRM periodicity identified no significant difference between neonatal cohorts (p = .865). These data suggest that at birth, FRMs are developmentally invariant. However, in adults of diverse skin ethnicity, there is a deviation in ultrastructure for the papillary dermal FRMs that may be acquired during the passage of time from child to adulthood. Understanding the mechanism by which this difference in papillary dermal FRMs arises warrants further study.

The systemic influence of chronic smoking on skin structure and mechanical function.

One of the major functions of human skin is to provide protection from the environment. Although we cannot entirely avoid, for example, sun exposure, it is likely that exposure to other environmental factors could affect cutaneous function. A number of studies have identified smoking as one such factor that leads to both facial wrinkle formation and a decline in skin function. In addition to the direct physical effects of tobacco smoke on skin, its inhalation has additional profound systemic effects for the smoker. The adverse effects on the respiratory and cardiovascular systems from smoking are well known. Central to the pathological changes associated with smoking is the elastic fibre, a key component of the extracellular matrices of lungs. In this study we examined the systemic effect of chronic smoking (>40 cigarettes/day; >5 years) on the histology of the cutaneous elastic fibre system, the nanostructure and mechanics of one of its key components, the fibrillin-rich microfibril, and the micromechanical stiffness of the dermis and epidermis. We show that photoprotected skin of chronic smokers exhibits significant remodelling of the elastic fibre network (both elastin and fibrillin-rich microfibrils) as compared to the skin of age- and sex-matched non-smokers. This remodelling is not associated with increased gelatinase activity (as identified by in situ zymography). Histological remodelling is accompanied by significant ultrastructural changes to extracted fibrillin-rich microfibrils. Finally, using scanning acoustic microscopy, we demonstrated that chronic smoking significantly increases the stiffness of both the dermis and the epidermis. Taken together, these data suggest an unappreciated systemic effect of chronic inhalation of tobacco smoke on the cutaneous elastic fibre network. Such changes may in part underlie the skin wrinkling and loss of skin elasticity associated with smoking. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Proteomic fingerprints of damage in extracellular matrix assemblies.

In contrast to the dynamic intracellular environment, structural extracellular matrix (ECM) proteins with half-lives measured in decades, are susceptible to accumulating damage. Whilst conventional approaches such as histology, immunohistochemistry and mass spectrometry are able to identify age- and disease-related changes in protein abundance or distribution, these techniques are poorly suited to characterising molecular damage. We have previously shown that mass spectrometry can detect tissue-specific differences in the proteolytic susceptibility of protein regions within fibrillin-1 and collagen VI alpha-3. Here, we present a novel proteomic approach to detect damage-induced "peptide fingerprints" within complex multi-component ECM assemblies (fibrillin and collagen VI microfibrils) following their exposure to ultraviolet radiation (UVR) by broadband UVB or solar simulated radiation (SSR). These assemblies were chosen because, in chronically photoaged skin, fibrillin and collagen VI microfibril architectures are differentially susceptible to UVR. In this study, atomic force microscopy revealed that fibrillin microfibril ultrastructure was significantly altered by UVR exposure whereas the ultrastructure of collagen VI microfibrils was resistant. UVR-induced molecular damage was further characterised by proteolytic peptide generation with elastase followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Peptide mapping revealed that UVR exposure increased regional proteolytic susceptibility within the protein structures of fibrillin-1 and collagen VI alpha-3. This allowed the identification of UVR-induced molecular changes within these two key ECM assemblies. Additionally, similar changes were observed within protein regions of co-purifying, microfibril-associated receptors integrins αv and ß1. This study demonstrates that LC-MS/MS mapping of peptides enables the characterisation of molecular post-translational damage (via direct irradiation and radiation-induced oxidative mechanisms) within a complex in vitro model system. This peptide fingerprinting approach reliably allows both the identification of UVR-induced molecular damage in and between proteins and the identification of specific protein domains with increased proteolytic susceptibility as a result of photo-denaturation. This has the potential to serve as a sensitive method of identifying accumulated molecular damage in vivo using conventional mass spectrometry data-sets.

Circadian rhythms in skin and other elastic tissues.

Circadian rhythms are daily oscillations that, in mammals, are driven by both a master clock, located in the brain, and peripheral clocks in cells and tissues. Approximately 10% of the transcriptome, including extracellular matrix components, is estimated to be under circadian control. Whilst it has been established that certain collagens and extracellular matrix proteases are diurnally regulated (for example in tendon, cartilage and intervertebral disc) the role played by circadian rhythms in mediating elastic fiber homeostasis is poorly understood. Skin, arteries and lungs are dynamic, resilient, elastic fiber-rich organs and tissues. In skin, circadian rhythms influence cell migration and proliferation, wound healing and susceptibility of the tissues to damage (from protease activity, oxidative stress and ultraviolet radiation). In the cardiovascular system, blood pressure and heart rate also follow age-dependent circadian rhythms whilst the lungs exhibit diurnal variations in immune response. In order to better understand these processes it will be necessary to characterise diurnal changes in extracellular matrix biology. In particular, given the sensitivity of peripheral clocks to external factors, the timed delivery of interventions (chronotherapy) has the potential to significantly improve the efficacy of treatments designed to repair and regenerate damaged cutaneous, vascular and pulmonary tissues.