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.

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.

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.

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.

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.

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.

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.

The Impact of Ultraviolet Radiation on Barrier Function in Human Skin: Molecular Mechanisms and Topical Therapeutics.

A competent epidermal barrier is crucial for terrestrial mammals. This barrier must keep in water and prevent entry of noxious stimuli. Most importantly, the epidermis must also be a barrier to ultraviolet radiation (UVR) from the sunlight. Currently, the effects of ultraviolet radiation on epidermal barrier function are poorly understood. However, studies in mice and more limited work in humans suggest that the epidermal barrier becomes more permeable, as measured by increased transepidermal water loss, in response UVR, at doses sufficiently high to induce erythema. The mechanisms may include disturbance in the organisation of lipids in the stratum corneum (the outermost layer of the epidermis) and reduction in tight junction function in the granular layer (the first living layer of the skin). By contrast, suberythemal doses of UVR appear to have positive effects on epidermal barrier function. Topical sunscreens have direct and indirect protective effects on the barrier through their ability to block UV and also due to their moisturising or occlusive effects, which trap water in the skin, respectively. Some topical agents such as specific botanical extracts have been shown to prevent the loss of water associated with high doses of UVR. In this review, we discuss the current literature and suggest that the biology of UVR-induced barrier dysfunction, and the use of topical products to protect the barrier, are areas worthy of further investigation.

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.

Skin health in older age.

As people age, their skin undergoes changes which result in reduced elasticity, increased fragility and an altered immune response; in essence it becomes frail. As life expectancy is increasing the health of older skin is becoming a progressively more important facet of overall care. In addition to the consequences of ageing for otherwise healthy skin, the relative incidence of some dermatological conditions is age-dependent. In particular, xerosis (dry skin), cutaneous malignancies and skin injuries are more common in older people. In this review we describe the functional consequences of skin ageing and discuss the current evidence on how skin health may be maintained and dermatological conditions prevented in an ageing population. The future of dermatological health-care provision in the older population relies on the development of coordinated pathways of care, which start from a young age. Better quality research coordinated by the establishment of institutions dealing with skin health and ageing would be a method of addressing these needs.

Growth differentiation factor 6 and transforming growth factor-beta differentially mediate mesenchymal stem cell differentiation, composition, and micromechanical properties of nucleus pulposus constructs.

INTRODUCTION: Currently, there is huge research focus on the development of novel cell-based regeneration and tissue-engineering therapies for the treatment of intervertebral disc degeneration and the associated back pain. Both bone marrow-derived (BM) mesenchymal stem cells (MSCs) and adipose-derived MSCs (AD-MSCs) are proposed as suitable cells for such therapies. However, currently no consensus exists as to the optimum growth factor needed to drive differentiation to a nucleus pulposus (NP)-like phenotype. The aim of this study was to investigate the effect of growth differentiation factor-6 (GDF6), compared with other transforming growth factor (TGF) superfamily members, on discogenic differentiation of MSCs, the matrix composition, and micromechanics of engineered NP tissue constructs. METHODS: Patient-matched human AD-MSCs and BM-MSCs were seeded into type I collagen hydrogels and cultured in differentiating media supplemented with TGF-ß3, GDF5, or GDF6. After 14 days, quantitative polymerase chain reaction analysis of chondrogenic and novel NP marker genes and sulfated glycosaminoglycan (sGAG) content of the construct and media components were measured. Additionally, construct micromechanics were analyzed by using scanning acoustic microscopy (SAM). RESULTS: GDF6 stimulation of BM-MSCs and AD-MSCs resulted in a significant increase in expression of novel NP marker genes, a higher aggrecan-to-type II collagen gene expression ratio, and higher sGAG production compared with TGF-ß or GDF5 stimulation. These effects were greater in AD-MSCs than in BM-MSCs. Furthermore, the acoustic-wave speed measured by using SAM, and therefore tissue stiffness, was lowest in GDF6-stiumlated AD-MSC constructs. CONCLUSIONS: The data suggest that GDF6 stimulation of AD-MSCs induces differentiation to an NP-like phenotype and results in a more proteoglycan-rich matrix. Micromechanical analysis shows that the GDF6-treated AD-MSCs have a less-stiff matrix composition, suggesting that the growth factor is inducing a matrix that is more akin to the native NP-like tissue. Thus, this cell and growth-factor combination may be the ideal choice for cell-based intervertebral disc (IVD)-regeneration therapies.

Age-Related Tissue Stiffening: Cause and Effect.

SIGNIFICANCE: Tissue elasticity is severely compromised in aging skin, lungs, and blood vessels. In the vascular and pulmonary systems, respectively, loss of mechanical function is linked to hypertension, which in turn is a risk factor for heart and renal failure, stroke, and aortic aneurysms, and to an increased risk of mortality as a result of acute lung infections. RECENT ADVANCES: Although cellular mechanisms were thought to play an important role in mediating tissue aging, the reason for the apparent sensitivity of elastic fibers to age-related degradation remained unclear. We have recently demonstrated that compared with type I collagen, a key component of the elastic fiber system, the cysteine-rich fibrillin microfibril is highly susceptible to direct UV exposure in a cell-free environment. We hypothesized therefore that, as a consequence of both their remarkable longevity and cysteine-rich composition, many elastic fiber-associated components will be susceptible to the accumulation of damage by both direct UV radiation and reactive oxygen species-mediated oxidation. CRITICAL ISSUES: Although elastic fiber remodeling is a common feature of aging dynamic tissues, the inaccessibility of most human tissues has hampered attempts to define the molecular causes. CLINICAL CARE RELEVANCE: Although, currently, the localized repair of damaged elastic fibers may be effected by the topical application of retinoids and some cosmetic products, future studies may extend the application of systemic transforming growth factor ß antagonists, which can prevent cardiovascular remodeling in murine Marfan syndrome, to aging humans. Acellular mechanisms may be key mediators of elastic fiber remodeling and hence age-related tissue stiffening.

Low-dose ultraviolet radiation selectively degrades chromophore-rich extracellular matrix components.

Photoageing of human skin due to chronic exposure to ultraviolet radiation (UVR) is characterized histologically by extensive remodelling of the dermal elastic fibre system. Whilst enzymatic pathways are thought to play a major role in mediating extracellular matrix (ECM) degeneration in UV-exposed skin, the substrate specificity of UVR-up-regulated and activated matrix metalloproteinases (MMPs) is low. It is unclear, therefore, how such cell-mediated mechanisms alone could be responsible for the reported selective degradation of elastic fibre components such as fibrillin-1 and fibulin-5 during the early stages of photoageing. Here we use atomic force microscopy (AFM) and scanning transmission electron microscopy (STEM) to demonstrate that physiologically attainable doses (20-100 mJ/cm(2)) of direct UV-B radiation can induce profound, dose-dependent, changes in the structure of, and mass distribution within, isolated fibrillin microfibrils. Furthermore, using reducing and native PAGE in combination with AFM, we show that, whilst exposure to low-dose UV-B radiation significantly alters the macromolecular and quaternary structures of both UV chromophore (Cys, His, Phe, Trp and Tyr)-rich fibrillin microfibrils (fibrillin-1, 21.0%) and fibronectin dimers (fibronectin, 12.9%), similar doses have no detectable effect on UV chromophore-poor type I collagen monomers (2.2%). Analysis of the published primary amino acid sequences of 49 dermal ECM components demonstrates that most elastic fibre-associated proteins, but crucially neither elastin nor members of the collagen family, are rich in UV chromophores. We suggest, therefore, that the amino acid composition of elastic fibre-associated proteins [including the fibrillins, fibulins, latent TGFbeta binding proteins (LTBPs) and the lysyl oxidase family of enzymes (LOK/LOXLs)] may predispose them to direct degradation by UVR. As a consequence, this selective acellular photochemical pathway may play an important role in initiating and/or exacerbating cell-mediated ECM remodelling in UVR-exposed skin.

Liver X receptor ß: maintenance of epidermal expression in intrinsic and extrinsic skin aging.

Aging in human skin is the composite of time-dependent intrinsic aging plus photoaging induced by chronic exposure to ultraviolet radiation. Nuclear hormone receptors coordinate diverse processes including metabolic homeostasis. Liver X receptor ß (LXRß) is a close human homologue of daf-12, a regulator of nematode longevity. LXRß is positively regulated by sirtuin-1 and resveratrol, while LXRß-null mice show transcriptional profiles similar to those seen in aged human skin. In these studies, we examined LXRß expression in aged and photoaged human skin. Volunteers were recruited to assess intrinsic aging and photoaging. Epidermal LXRß mRNA was examined by in situ hybridization while protein was identified by immunofluorescence. No significant changes were observed in either LXRß mRNA or protein expression between young and aged volunteers (mRNA p = 0.90; protein p = 0.26). Similarly, LXRß protein expression was unaltered in photoaged skin (p = 0.75). Our data therefore suggest that, while not playing a major role in skin aging, robust cutaneous expression implies a fundamental role for LXRß in epidermal biology.

Mass-mapping of ECM macromolecules by scanning transmission electron microscopy.

In the scanning transmission electron microscope, the degree of electron scattering induced by biological specimens, such as ECM macromolecules, is dependent on the molecular mass. By calibrating the ratio of scattered to non-scattered electrons against a known mass standard, such as tobacco mosaic virus, it is possible to quantify absolute changes in both mass and mass distribution. These mass mapping approaches can provide important information on ECM assembly, organisation, and interactions which is not obtainable by other means.

Expression and supramolecular assembly of recombinant alpha1(viii) and alpha2(viii) collagen homotrimers.

Collagen VIII is an extracellular matrix macromolecule comprising two polypeptide chains, alpha1(VIII) and alpha2(VIII), that can form homotrimers in vitro and in vivo. Here, recombinant collagen VIII was expressed to study its supramolecular assembly following secretion. Cells transfected with alpha1(VIII) or alpha2(VIII) assembled and secreted homotrimers that were stable in denaturing conditions and had a molecular mass of approximately 180 kDa on SDS-PAGE gels. Co-transfection with prolyl 4-hydroxylase generated homotrimers with stable pepsin-resistant triple-helical domains. Size fractionation of native recombinant collagen VIII molecules expressed with or without prolyl 4-hydroxylase identified urea-sensitive high molecular mass assemblies eluting in the void volume of a Superose 6HR 10/30 column and urea-resistant assemblies of approximately 700 kDa, all of which were composed of homotrimers. Immunofluorescence analysis highlighted the extracellular deposition of recombinant alpha1(VIII)(3), alpha2(VIII)(3), and co-expressed alpha1(VIII)(3)/alpha2(VIII)(3). Microscopy analysis of recombinant collagen VIII identified rod-like molecules of 134 nm in length that assembled into angular arrays with branching angles of approximately 114 degrees and extensive networks. Based on these data, we propose a model of collagen VIII assembly in which four homotrimers form a tetrahedron stabilized by central interacting C-terminal NC1 trimers. Tetrahedrons may then act as building blocks of three-dimensional hexagonal lattices generated by secondary interactions involving terminal and helical sequences.