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.

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.

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.

Fibrillin microfibrils and elastic fibre proteins: Functional interactions and extracellular regulation of growth factors.

Fibrillin microfibrils are extensible polymers that endow connective tissues with long-range elasticity and have widespread distributions in both elastic and non-elastic tissues. They act as a template for elastin deposition during elastic fibre formation and are essential for maintaining the integrity of tissues such as blood vessels, lung, skin and ocular ligaments. A reduction in fibrillin is seen in tissues in vascular ageing, chronic obstructive pulmonary disease, skin ageing and UV induced skin damage, and age-related vision deterioration. Most mutations in fibrillin cause Marfan syndrome, a genetic disease characterised by overgrowth of the long bones and other skeletal abnormalities with cardiovascular and eye defects. However, mutations in fibrillin and fibrillin-binding proteins can also cause short-stature pathologies. All of these diseases have been linked to dysregulated growth factor signalling which forms a major functional role for fibrillin.

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.

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.

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.

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.

Lysyl oxidase-like 2 (LOXL2)-mediated cross-linking of tropoelastin.

Lysyl oxidases (LOXs) play a central role in extracellular matrix remodeling during development and tumor growth and fibrosis through cross-linking of collagens and elastin. We have limited knowledge of the structure and substrate specificity of these secreted enzymes. LOXs share a conserved C-terminal catalytic domain but differ in their N-terminal region, which is composed of 4 repeats of scavenger receptor cysteine-rich (SRCR) domains in LOX-like (LOXL) 2. We investigated by X-ray scattering and electron microscopy the low-resolution structure of the full-length enzyme and the structure of a shorter form lacking the catalytic domain. Our data demonstrate that LOXL2 has a rod-like structure with a stalk composed of the SRCR domains and the catalytic domain at its tip. We detected direct interaction between LOXL2 and tropoelastin (TE) and also LOXL2-mediated deamination of TE. Using proteomics, we identified several allysines together with cross-linked TE peptides. The elastin-like material generated was resistant to trypsin proteolysis and displayed mechanical properties similar to mature elastin. Finally, we detected the codistribution of LOXL2 and elastin in the vascular wall. Altogether, these data suggest that LOXL2 could participate in elastogenesis in vivo and could be used as a means of cross-linking TE in vitro for biomimetic and cell-compatible tissue engineering purposes.-Schmelzer, C. E. H., Heinz, A., Troilo, H., Lockhart-Cairns, M.-P., Jowitt, T. A., Marchand, M. F., Bidault, L., Bignon, M., Hedtke, T., Barret, A., McConnell, J. C., Sherratt, M. J., Germain, S., Hulmes, D. J. S., Baldock, C., Muller, L. Lysyl oxidase-like 2 (LOXL2)-mediated cross-linking of tropoelastin.

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.

Structural and compositional diversity of fibrillin microfibrils in human tissues.

Elastic fibers comprising fibrillin microfibrils and elastin are present in many tissues, including the skin, lungs, and arteries, where they confer elasticity and resilience. Although fibrillin microfibrils play distinct and tissue-specific functional roles, it is unclear whether their ultrastructure and composition differ between elastin-rich (skin) and elastin-poor (ciliary body and zonule) organs or after in vitro synthesis by cultured cells. Here, we used atomic force microscopy, which revealed that the bead morphology of fibrillin microfibrils isolated from the human eye differs from those isolated from the skin. Using newly developed pre-MS preparation methods and LC-MS/MS, we detected tissue-specific regions of the fibrillin-1 primary structure that were differentially susceptible to proteolytic extraction. Comparing tissue- and culture-derived microfibrils, we found that dermis- and dermal fibroblast-derived fibrillin microfibrils differ in both bead morphology and periodicity and also exhibit regional differences in fibrillin-1 proteolytic susceptibility. In contrast, collagen VI microfibrils from the same dermal or fibroblast samples were invariant in ultrastructure (periodicity) and protease susceptibility. Finally, we observed that skin- and eye-derived microfibril suspensions were enriched in elastic fiber- and basement membrane-associated proteins, respectively. LC-MS/MS also identified proteins (such as calreticulin and protein-disulfide isomerase) that are potentially fundamental to fibrillin microfibril biology, regardless of their tissue source. Fibrillin microfibrils synthesized in cell culture lacked some of these key proteins (MFAP2 and -4 and fibrillin-2). These results showcase the structural diversity of these key extracellular matrix assemblies, which may relate to their distinct roles in the tissues where they reside.

How stiff is skin?

The measurement of the mechanical properties of skin (such as stiffness, extensibility and strength) is a key step in characterisation of both dermal ageing and disease mechanisms and in the assessment of tissue-engineered skin replacements. However, the biomechanical terminology and plethora of mathematical analysis approaches can be daunting to those outside the field. As a consequence, mechanical studies are often inaccessible to a significant proportion of the intended audience. Furthermore, devices for the measurement of skin function in vivo generate relative values rather than formal mechanical measures, therefore limiting the ability to compare studies. In this viewpoint essay, we discuss key biomechanical concepts and the influence of technical and biological factors (including the nature of the testing apparatus, length scale, donor age and anatomical site) on measured mechanical properties such as stiffness. Having discussed the current state-of-the-art in macro-mechanical and micromechanical measuring techniques and in mathematical and computational modelling methods, we then make suggestions as to how these approaches, in combination with 3D X-ray imaging and mechanics methods, may be adopted into a single strategy to characterise the mechanical behaviour of skin.

Macro- and micromechanical remodelling in the fish atrium is associated with regulation of collagen 1 alpha 3 chain expression.

Numerous pathologies lead to remodelling of the mammalian ventricle, often associated with fibrosis. Recent work in fish has shown that fibrotic remodelling of the ventricle is 'reversible', changing seasonally as temperature-induced changes in blood viscosity alter haemodynamic load on the heart. The atrial response to varying haemodynamic load is less understood in mammals and completely unexplored in non-mammalian vertebrates. To investigate atrial remodelling, rainbow trout were chronically cooled (from 10 ± 1 to 5 ± 1 °C) and chronically warmed (from 10 ± 1 to 18 ± 1 °C) for a minimum of 8 weeks. We assessed the functional effects on compliance using ex vivo heart preparations and atomic force microscopy nano-indentation and found chronic cold increased passive stiffness of the whole atrium and micromechanical stiffness of tissue sections. We then performed histological, biochemical and molecular assays to probe the mechanisms underlying functional remodelling of the atrial tissue. We found cooling resulted in collagen deposition which was associated with an upregulation of collagen-promoting genes, including the fish-specific collagen I alpha 3 chain, and a reduction in gelatinase activity of collagen-degrading matrix metalloproteinases (MMPs). Finally, we found that cooling reduced mRNA expression of cardiac growth factors and hypertrophic markers. Following long-term warming, there was an opposing response to that seen with cooling; however, these changes were more moderate. Our findings suggest that chronic cooling causes atrial dilation and increased myocardial stiffness in trout atria analogous to pathological states defined by changes in preload or afterload of the mammalian atria. The reversal of this phenotype following chronic warming is particularly interesting as it suggests that typically pathological features of mammalian atrial remodelling may oscillate seasonally in the fish, revealing a more dynamic and plastic atrial remodelling response.

Three-dimensional visualisation of soft biological structures by X-ray computed micro-tomography.

Whereas the two-dimensional (2D) visualisation of biological samples is routine, three-dimensional (3D) imaging remains a time-consuming and relatively specialised pursuit. Current commonly adopted techniques for characterising the 3D structure of non-calcified tissues and biomaterials include optical and electron microscopy of serial sections and sectioned block faces, and the visualisation of intact samples by confocal microscopy or electron tomography. As an alternative to these approaches, X-ray computed micro-tomography (microCT) can both rapidly image the internal 3D structure of macroscopic volumes at sub-micron resolutions and visualise dynamic changes in living tissues at a microsecond scale. In this Commentary, we discuss the history and current capabilities of microCT. To that end, we present four case studies to illustrate the ability of microCT to visualise and quantify: (1) pressure-induced changes in the internal structure of unstained rat arteries, (2) the differential morphology of stained collagen fascicles in tendon and ligament, (3) the development of Vanessa cardui chrysalises, and (4) the distribution of cells within a tissue-engineering construct. Future developments in detector design and the use of synchrotron X-ray sources might enable real-time 3D imaging of dynamically remodelling biological samples.

Raised mammographic density: causative mechanisms and biological consequences.

High mammographic density is the most important risk factor for breast cancer, after ageing. However, the composition, architecture, and mechanical properties of high X-ray density soft tissues, and the causative mechanisms resulting in different mammographic densities, are not well described. Moreover, it is not known how high breast density leads to increased susceptibility for cancer, or the extent to which it causes the genomic changes that characterise the disease. An understanding of these principals may lead to new diagnostic tools and therapeutic interventions.

Increased peri-ductal collagen micro-organization may contribute to raised mammographic density.

BACKGROUND: High mammographic density is a therapeutically modifiable risk factor for breast cancer. Although mammographic density is correlated with the relative abundance of collagen-rich fibroglandular tissue, the causative mechanisms, associated structural remodelling and mechanical consequences remain poorly defined. In this study we have developed a new collaborative bedside-to-bench workflow to determine the relationship between mammographic density, collagen abundance and alignment, tissue stiffness and the expression of extracellular matrix organising proteins. METHODS: Mammographic density was assessed in 22 post-menopausal women (aged 54-66 y). A radiologist and a pathologist identified and excised regions of elevated non-cancerous X-ray density prior to laboratory characterization. Collagen abundance was determined by both Masson's trichrome and Picrosirius red staining (which enhances collagen birefringence when viewed under polarised light). The structural specificity of these collagen visualisation methods was determined by comparing the relative birefringence and ultrastructure (visualised by atomic force microscopy) of unaligned collagen I fibrils in reconstituted gels with the highly aligned collagen fibrils in rat tail tendon. Localised collagen fibril organisation and stiffness was also evaluated in tissue sections by atomic force microscopy/spectroscopy and the abundance of key extracellular proteins was assessed using mass spectrometry. RESULTS: Mammographic density was positively correlated with the abundance of aligned periductal fibrils rather than with the abundance of amorphous collagen. Compared with matched tissue resected from the breasts of low mammographic density patients, the highly birefringent tissue in mammographically dense breasts was both significantly stiffer and characterised by large (>80 µm long) fibrillar collagen bundles. Subsequent proteomic analyses not only confirmed the absence of collagen fibrosis in high mammographic density tissue, but additionally identified the up-regulation of periostin and collagen XVI (regulators of collagen fibril structure and architecture) as potential mediators of localised mechanical stiffness. CONCLUSIONS: These preliminary data suggest that remodelling, and hence stiffening, of the existing stromal collagen microarchitecture promotes high mammographic density within the breast. In turn, this aberrant mechanical environment may trigger neoplasia-associated mechanotransduction pathways within the epithelial cell population.

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.

Cross-linking of structural proteins in ageing skin: an in situ assay for the detection of amine oxidase activity.

With increasing age, dynamic tissues such as lungs, blood vessels and skin lose their ability to both deform and recoil, culminating in tissue stiffening. This loss of tissue elasticity, which profoundly impacts tissue function and thus morbidity, may be due not only to changes in the relative abundance of key extracellular matrix proteins within tissues but also to their accumulation of post-translational modifications. Whilst to date attention has focussed primarily on the age-related non-enzymatic formation of advanced glycation end products, the accumulation of pathological enzyme-mediated cross-links may also lead to age-related tissue stiffening. The lysyl oxidase (LOX) family of enzymes are constitutively expressed in adult tissues and are known to drive the catalysis of cross-links in both fibrillar collagens and elastin. Although immunochemical approaches are commonly used to localise the inactive pro-enzyme of LOX, and biochemical methods are employed to quantify activity in homogenised tissue, they do not allow for the in situ localisation of the enzyme. Thus, we have developed a novel assay to both detect and localise LOX enzyme activity in situ. LOX family members are amine oxidases and this assay uses the principle that an amine substrate in the presence of this class of enzyme will be oxidised to an aldehyde and hydrogen peroxide (H2O2). In turn, H2O2, when combined with luminol and horseradish peroxidase, will produce a light-emitting reaction that can be detected by film autoradiography. The development of a technique to localise specific amine oxidase activity in tissue sections may provide crucial additional information on the exact role played by this class of enzymes in mediating age-related tissue stiffening.

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.