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 intervertebral disc (IVD) has a complex and multiscale extracellular matrix structure which provides unique mechanical properties to withstand physiological loading. Low back pain has been linked to degeneration of the disc but reparative treatments are not currently available. Characterising the disc's 3D microstructure and its response in a physiologically relevant loading environment is required to improve understanding of degeneration and to develop new reparative treatments. In this study, techniques for imaging the native IVD, measuring internal deformation and mapping volumetric strain were applied to an in situ compressed ex vivo rat lumbar spine segment. Synchrotron X-ray micro-tomography (synchrotron CT) was used to resolve IVD structures at microscale resolution. These image data enabled 3D quantification of collagen bundle orientation and measurement of local displacement in the annulus fibrosus between sequential scans using digital volume correlation (DVC). The volumetric strain mapped from synchrotron CT provided a detailed insight into the micromechanics of native IVD tissue. The DVC findings showed that there was no slipping at lamella boundaries, and local strain patterns were of a similar distribution to the previously reported elastic network with some heterogeneous areas and maximum strain direction aligned with bundle orientation, suggesting bundle stretching and sliding. This method has the potential to bridge the gap between measures of macro-mechanical properties and the local 3D micro-mechanical environment experienced by cells. This is the first evaluation of strain at the micro scale level in the intact IVD and provides a quantitative framework for future IVD degeneration mechanics studies and testing of tissue engineered IVD replacements. STATEMENT OF SIGNIFICANCE: Synchrotron in-line phase contrast X-ray tomography provided the first visualisation of native intact intervertebral disc microstructural deformation in 3D. For two annulus fibrosus volumes of interest, collagen bundle orientation was quantified and local displacement mapped as strain. Direct evidence of microstructural influence on strain patterns could be seen such as no slipping at lamellae boundaries and maximum strain direction aligned with collagen bundle orientation. Although disc elastic structures were not directly observed, the strain patterns had a similar distribution to the previously reported elastic network. This study presents technical advances and is a basis for future X-ray microscopy, structural quantification and digital volume correlation strain analysis of soft tissue.

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.

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 β1. 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.

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.