Oxidative DNA damage promotes vascular aging associated with changes in extracellular matrix-regulating proteins.

AIMS: Vascular aging is characterized by vessel stiffening, with increased deposition of extracellular matrix (ECM) proteins including collagens. Oxidative DNA damage occurs in vascular aging, but how it regulates ECM proteins and vascular stiffening is unknown. We sought to determine the relationship between oxidative DNA damage and ECM regulatory proteins in vascular aging. METHODS AND RESULTS: We examined oxidative DNA damage, the major base excision repair (BER) enzyme 8-Oxoguanine DNA Glycosylase (Ogg1) and its regulators, multiple physiological markers of aging, and ECM proteomics in mice from 22-72w. Vascular aging was associated with increased oxidative DNA damage, and decreased expression of Ogg1, its active acetylated form, its acetylation regulatory proteins P300 and CBP, and the transcription factor Foxo3a. Vascular stiffness was examined in vivo in control, Ogg1-/-, or mice with vascular smooth muscle cell-specific expression of Ogg1+ (Ogg1) or an inactive mutation (Ogg1KR). Ogg1-/- and Ogg1KR mice showed reduced arterial compliance and distensibility, and increased stiffness and pulse pressure, whereas Ogg1 expression normalised all parameters to 72w. ECM proteomics identified major changes in collagens with aging, and downregulation of the ECM regulatory proteins Protein 6-lysyl oxidase (LOX) and WNT1-inducible-signaling pathway protein 2 (WISP2). Ogg1 overexpression upregulated LOX and WISP2 both in vitro and in vivo, and downregulated Transforming growth factor ß1 (TGFb1) and Collagen 4α1 in vivo compared with Ogg1KR. Foxo3a activation induced Lox, while Wnt3 induction of Wisp2 also upregulated LOX and Foxo3a, and downregulated TGFß1 and fibronectin 1. In humans, 8-oxo-G increased with vascular stiffness, while active OGG1 reduced with both age and stiffness. CONCLUSIONS: Vascular aging is associated with oxidative DNA damage, downregulation of major BER proteins, and changes in multiple ECM structural and regulatory proteins. Ogg1 protects against vascular aging, associated with changes in ECM regulatory proteins including LOX and WISP2.

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.

Matrikines in kidney ageing and age-related disease.

PURPOSE OF REVIEW: Matrikines are cell-signalling extracellular matrix fragments and they have attracted recent attention from basic and translational scientists, due to their diverse roles in age-related disease and their potential as therapeutic agents. In kidney, the matrix undergoes remodelling by proteolytic fragmentation, so matrikines are likely to play a substantial, yet understudied, role in ageing and pathogenesis of age-related diseases. RECENT FINDINGS: This review presents an up-to-date description of known matrikines with either a confirmed or highly anticipated role in kidney ageing and disease, including their point of origin, mechanism of cleavage, a summary of known biological actions and the current knowledge which links them to kidney health. We also highlight areas of interest, such as the prospect of matrikine cross-tissue communication, and gaps in knowledge, such as the unexplored signalling potential of many kidney disease-specific matrix fragments. SUMMARY: We anticipate that knowledge of specific matrikines, and their roles in controlling processes of kidney pathology, could be leveraged for the development of exciting new future therapies through inhibition or even with their supplementation.

Peptide location fingerprinting identifies structural alterations within basement membrane components in ageing kidney.

During ageing, the glomerular and tubular basement membranes (BM) of the kidney undergo a progressive decline in function that is underpinned by histological changes, including glomerulosclerosis and tubular interstitial fibrosis and atrophy. This BM-specific ageing is thought to result from damage accumulation to long-lived extracellular matrix (ECM) protein structures. Determining which BM proteins are susceptible to these structure-associated changes, and the possible mechanisms and downstream consequences, is critical to understand age-related kidney degeneration and to identify markers for therapeutic intervention. Peptide location fingerprinting (PLF) is an emerging proteomic mass spectrometry analysis technique capable of identifying ECM proteins with structure-associated differences that may occur by damage modifications in ageing. Here, we apply PLF as a bioinformatic screening tool to identify BM proteins with structure-associated differences between young and aged human glomerular and tubulointerstitial compartments. Several functional regions within key BM components displayed alterations in tryptic peptide yield, reflecting potential age-dependent shifts in molecular (e.g. laminin-binding regions in agrin) and cellular (e.g. integrin-binding regions in laminins 521 and 511) interactions, oxidation (e.g. collagen IV) and the fragmentation and release of matrikines (e.g. canstatin and endostatin from collagens IV and XVIII). Furthermore, we found that periostin and the collagen IV α2 chain exhibited structure-associated differences in ageing that were conserved between human kidney and previously analysed mouse lung, revealing BM components that harbour shared susceptibilities across species and organs.

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.

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.

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.

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.

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.

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.

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.