Matrix Biology Plus

Identification of pathways preventing recovery from acute respiratory viral infection is under-studied but essential for long-term health. Using unbiased proteomics, we reveal an unexpected persistent reduction in lung basement membrane proteins in mice recovered from influenza infection. Basement membrane provides a critical scaffold for heterogeneous cell types and the proteins they secrete/express at the endothelial and epithelial barrier. Further peptide location fingerprinting analysis shows inherent structure-associated changes within core collagen IV and laminin components, particularly within matrikine-producing regions of collagen IV. Our results imply lingering damage to the basement membrane network despite full recovery from viral infection. Surprisingly, these structure-associated changes in laminin and collagen IV components are also observed in non-infected aged mice indicating that inflammation-driven basement membrane degeneration may contribute to tissue ageing. Interestingly, macrophages in regions deficient in basement membrane express collagen IV and laminin chains. Repair of the basement membrane should therefore be targeted to improve overall lung health. Non-technical summaryLung virus infection is a constant global threat, despite developments in vaccination and anti-viral treatments. We have a deep understanding of this inflammatory condition, but less is known about the drivers of persistent problems, including fatigue and breathlessness as illustrated by "long COVID". Here, we reveal a novel finding that a critical structure in the lung (the basement membrane) remains damaged even after the virus and symptoms have cleared. This structure supports a variety of cells that and forms a barrier that lines the airspaces. It also regulates fluid and cell movement into these airspaces. Remarkably, we show that similar persistent changes after virus infection are also evident in aged lungs, which implies that lung complications with age may be due to repeated inflammation. By deciphering the processes causing persistent basement membrane changes, we provide an entirely novel area to target with new medicines to treat complications arising from viral infection.

At least 10% of the global population is impacted by chronic kidney disease (CKD) and ageing is a key risk factor. CKD is characterised by the build-up of extracellular matrix and a loss of functional nephrons. However, the mechanisms that maintain matrix homeostasis across the physiological lifespan remain elusive. Using {superscript 1}3C-lysine metabolic labelling, we quantified kidney matrix protein turnover in healthy mice at four timepoints (8, 22, 52, and 78 weeks). We found that basement membrane components, including collagen IV, laminin-521, nidogens and perlecan, were more long-lived over age, with collagen IV half-lives extending from weeks in young kidneys to years in aged kidneys, suggesting a reduced capacity for basement membrane renewal. The half-lives of fibrillar collagens I and III also increased over age up to forty-fold, which is consistent with minimal degradation. In contrast, collagen XV retained rapid turnover despite increased abundance, indicating a persistent role in tissue remodelling. Using peptide location fingerprinting to predict structural alterations and proteolytic processing we identified age-dependent meprin oligomerisation and altered nidogen-laminin interaction states. We predicted structural alterations within assembly domains of collagen VI and reduced accessibility of integrin-binding regions, suggesting altered microfibril organisation and cell-surface binding. Collagen XV had predicted structural changes across the NC1 domain encoding the matrikine restin, consistent with altered protease accessibility and matrikine release during ageing. These findings indicate that age-related kidney fibrosis is primarily caused by impaired matrix degradation, with protease accessibility and altered matrix interactions likely playing key roles in this remodeling process.

Collagens, the most abundant proteins in mammals, play pivotal roles in the maintenance of tissue structure, functions, cell-to-cell communication, cellular migration, behavior, and growth. Collagens are highly complex in structure due to the dynamic post-translational modifications (PTMs) such as hydroxylations (on prolines and lysine residues) and O-glycosylation (on hydroxylysines) enzymatically catalyzed during biosynthesis. The most prevalent modification in fibrillar collagens is prolyl 4-hydroxylation catalyzed by collagen prolyl 4-hydroxylases (C-P4hs). Prolyl 4-hydroxylation on collagens plays a critical role in collagen biosynthesis, thermostability, and cell-collagen interactions. However, the site-specificity of prolyl 4-hydroxylase 1 (P4ha1) and P4ha2 is not comprehensively studied yet. Further, the effect of P4ha1 and P4ha2 on the plethora of other site-specific collagen PTMs is not known to date. In-depth mass-spectrometry data (PXD008802) analysis of mice skin collagen I extracted from wild-type and different deletion mutants of C-P4hs revealed that partial or full deletion of prolyl 4-hydroxylases (P4ha1 and P4ha2) significantly decreases collagen deposition in ECM hinting towards perturbed biosynthesis. A total of 421 site-specific PTMs on fibrillar collagen chains (Col1a1, Col1a2, and Col3a1) were identified. Further, novel 23 P4ha1 specific, 8 P4ha2 specific, and 18 C-P4hs promiscuous sites on fibrillar collagen chains were identified. Partial deletion of P4ha1 and full deletion of P4ha2 also resulted in altered levels of the site-specific prolyl-3-hydroxylation occupancy in collagen I. Surprisingly, an increased level of site-specific lysyl hydroxylation (Col1a1-K731, Col1a2-K183,315) was documented upon partial deletion of P4ha1 and full deletion of P4ha2. Our findings showcased that the activity of prolyl 4-hydroxylases is not limited to 4-hydroxylation of specific proline sites, but simultaneously can perturb the entire biosynthetic network by modulating prolyl 3-hydroxylation and lysyl hydroxylation occupancy levels in the fibrillar collagen chains in a site-specific manner.

ObjectiveAnterior cruciate ligament (ACL) tears increase the risk for developing posttraumatic osteoarthritis (PTOA). Females have greater risk for both. However, studies defining sex-specific protein responses in human cartilage after ACL injury are lacking. We hypothesize that articular cartilages response to an injurious environment differs depending on sex. DesignWe compared the proteomic profiles of normal cartilage with injured cartilage harvested from the intercondylar area during ACL surgery. Sex-specific injury effects were estimated through contrasts between Injured Male and Normal Male and between Injured Female and Normal Female. Pathway enrichment analysis was done using gene ontology (GO) and compared against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Extracellular matrix (ECM) proteins were further analyzed using the Matrisome AnalyzeR. ResultsFrom the 2,188 proteins identified, males and females shared 1,121 upregulated and 23 downregulated proteins in injured compared to normal cartilage. Analysis of ECM proteins and enriched pathways revealed mostly similar male and female responses to an injurious environment, with evidence of early cartilage remodeling in both sexes. Nevertheless, more than 240 proteins were affected specifically by sex, and significant sex differences were found in inflammation, ECM-related, and metabolic pathways. Males were enriched mostly in "ECM-receptor interaction", while females were enriched in "Citrate cycle (TCA cycle)", "Fatty acid degradation", and "Fatty acid metabolism" pathways. ConclusionArticular cartilage shows signs of remodeling soon after ACL injury, even when only exposed to an injurious environment rather than being physically impacted. Sex differences were observed in inflammation, metabolic pathways, and ECM synthesis.

The human adrenal is one of the pivotal glands of the endocrine system. Recently, the extracellular matrix (ECM) of the adrenal capsule and cortex was explored by dividing them into two fractions: outer (OF) and inner (IF). A significant variation in the levels of ECM proteins, including collagens, was documented. During the biosynthesis of collagen, it undergoes a plethora of PTMs exhibiting crucial roles such as cell-matrix interaction, adhesion, crosslinking, stability, etc. However, the site-specific identification and characterization of collagen PTMs remained challenging and is unknown for the human adrenal gland. We applied our in-house developed proteomics pipeline to identify several PTMS in 25 collagen chains from human adrenal-ECM. In the entire collagenome, we identified a total of 963 4-hydroxyproline (4-HyP), 201 3-hydroxyproline (3-HyP), 105 hydroxylysine (HyK), 17 galactosyl-hydroxylysine (G-HyK), and 35 glucosyl galactosyl-hydroxylysine (GG-HyK) sites. Although the site-specificity of collagen PTMs (3-HyP, HyK, G/GG-HyK) across fractions is conserved, the occupancies were different in a site-specific manner. Classically, a fully 3-hydroxylated site (P1164) of COL1A1 associated with osteogenesis imperfecta was found to be approximately fully hydroxylated ([~]99%) across fractions. Similarly, we also looked at the microheterogeneity of lysine modifications on one lysine residue (K862) of COL1A1. We observed that the hydroxylation level was higher in OF, while glycosylation levels were higher in IF. This may suggest a change in the crosslinking of collagen I across both fractions. Further, our analysis revealed much higher site-specific O-glycosylation in basement membrane collagen-IV, potentially facilitating the secretion of steroids from the adrenal gland. For the first time, we have annotated the collagen PTMs, developed a COL1A1 PTM map, and quantitated site-specific PTMs in the human adrenal gland. Taken together, this work revealed that intra-tissue-specific site-specific PTM collagen heterogeneity and lay the foundation for understanding their role in region-specific functions.

Joint injury is a risk factor for post-traumatic osteoarthritis. However, metabolic and microarchitectural changes within the joint post-injury in both sexes remain unexplored. This study identified tissue-specific and spatially-dependent metabolic signatures in male and female mice using matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) and LC-MS metabolomics. Male and female C57Bl/6J mice were subjected to non-invasive joint injury. Eight days post-injury, serum, synovial fluid, and whole joints were collected for metabolomics. Analyses compared between injured, contralateral, and naive mice, revealing local and systemic responses. Data indicate sex influences metabolic profiles across all tissues, particularly amino acid, purine, and pyrimidine metabolism. MALDI-MSI generated 2D ion images of bone, the joint interface, and bone marrow, highlighting increased lipid species in injured limbs, suggesting physiological changes across injured joints at metabolic and spatial levels. Together, these findings reveal significant metabolic changes after injury, with notable sex differences. Significance statementOsteoarthritis, the leading cause of disability worldwide, disproportionately affects females with sex being one of the strongest predictors of disease. This disparity is partly driven by sex-specific differences in injury susceptibility, increasing the likelihood of traumatic injury to the anterior cruciate ligament (ACL), other ligaments, and menisci. Using a non-invasive injury model, we demonstrate that injury perturbs the local joint environment and has systemic effects in a sex-specific manner. Furthermore, by leveraging matrix-assisted laser desorption ionization-mass spectrometry imaging of the joint, we provide new insight into the composition of osteochondral tissue at the metabolite level. These sexually dimorphic metabolic responses to joint injury advance current understanding of the complex sexual dimorphism in OA pathogenesis providing a foundation for targeted therapeutic strategies and improved patient outcomes for female patients.

BackgroundDysregulated proteolysis is implicated in thoracic (TAA) and abdominal aortic aneurysm (AAA) pathogenesis, but the proteolytic landscapes (degradomes) of aneurysmal and normal aorta, and contributions of individual proteases remain undefined. Here, a proteome-wide approach was used to uncover TAA and AAA degradomes, compare them quantitatively and define the specific role in aortic remodeling of two proteases consistently identified in the aneurysms, mast cell chymase (CMA1) and matrix metalloprotease 9 (MMP9). MethodsThe mass spectrometry-based N-terminomics strategy Terminal Amine Isotopic Labeling of Substrates (TAILS) was applied to Marfan syndrome TAAs (n=5), AAAs (n=16) and corresponding non-diseased aorta (TAs, n=4, and AAs, n=8) as a forward degradomics application, i.e., to define substrate and protease degradomes, and 8-plex iTRAQ-TAILS was used for quantitative comparison. Cleavage sites of CMA1 and MMP9 were sought by reverse degradomics, i.e., digestion of aortic proteins with these proteases, followed by 6-plex iTRAQ-TAILS. CMA1 and MMP9 proteolysis of biglycan was investigated using Amino-Terminal Oriented Mass spectrometry of Substrates (ATOMS). ResultsWe experimentally annotated 16,923 proteolytically derived peptides (substrate degradome) and 90 proteases (protease degradome) in the aorta. Quantitative substrate degradome comparisons identified specific differentially modulated pathways and networks in TAA and AAA. Reverse degradomics elucidated > 300 CMA1 and MMP9 substrate cleavage sites, of which, many, including orthogonally validated biglycan cleavage, occurred in the disease degradomes. ConclusionsUnbiased, proteome-wide forward degradomics of the aortic wall from TAA, AAA and non-diseased tissue generated the first systems biology view of vascular wall breakdown and public resource for the hitherto occult proteolytic landscape, demonstrating widespread extracellular matrix remodeling. The findings provide insights on aortic aneurysm pathways and potential disease biomarkers. Mapping of specific contributions of CMA1 and MMP9 on the aortic forward substrate degradome using reverse degradomics provides a strategy for defining the activities of all proteases involved in aortic disease.

Cartilage is characterized by a highly specialized extracellular matrix (ECM) secreted by chondrocytes and limited self-regenerative capacity. In vivo investigations of chondrogenesis are limited by difficult and traumatic access, especially in humans. While it is known for decades that disturbances of chondrocyte differentiation and changed cartilage ECM composition cause severe skeletal phenotypes in vertebrates, a detailed molecular understanding of chondrogenesis and cartilage ECM formation is still missing, especially in the context of human genetic skeletal diseases. ATDC5 cells, derived from AT805 mouse teratocarcinoma cells, have been used in the past to model chondrogenic differentiation, however, most studies have investigated few major cellular differentiation markers only so that the composition of the secreted ECM as well as effects on the ATDC5 transcriptome upon differentiation are still unclear. Here, we performed time-resolved transcriptomic and ECM proteomic analyses of differentiating ATDC5 cells. Both datasets confirmed the formation of a cartilage-like matrix with increasing expression of key chondrocyte genes over the course of differentiation. ECM proteomics further revealed a number of ECM components not previously reported in ATDC5 cells or the secreted ECM, encompassing collagens, proteoglycans, glycoproteins and other secreted factors. Overall, our findings provide a more detailed molecular characterization of ATDC5 chondrogenesis and highlight the potential of this model system for ECM-focused studies.

Understanding how connective tissue cells respond to mechanical stimulation is important to human health and disease processes in musculoskeletal diseases. Injury to articular cartilage is a key risk factor in predisposition to osteoarthritis. Here we identified a ubiquitin signature that is unique to injured articular cartilage tissue (the "injury ubiquitinome"). A total of 408 ubiquitinated peptides mapped to 114 proteins were identified, with an enrichment of ubiquitinated peptides of proteins involved in protein processing in the endoplasmic reticulum(ER), also known as the ER-associated degradation(ERAD) response, including YOD1, BRCC3, ATXN3 and USP5 as well as the ER stress regulators, RAD23B, VCP/p97 and Ubiquilin 1. Enrichment of these proteins suggested an injury-induced ER stress response and, for instance, ER stress markers DDIT3/CHOP and BIP/GRP78 were upregulated following cartilage injury on the protein and gene expression levels. Similar ER stress induction was also observed in response to tail fin injury in zebrafish larvae, suggesting a generic response to tissue injury. Furthermore, a rapid increase in global DUB activity following injury and significant activity in human osteoarthritic cartilage was observed using DUB specific activity probes. Inhibition of DUBs using a broad-spectrum inhibitor caused a reduction in the injury-induced inflammatory response in a zebrafish tail fin injury model. These results implicate the involvement of ubiquitination events and activation of a set of DUBs and ER stress regulators in cellular responses to cartilage tissue injury and osteoarthritis. This link through the ERAD pathway makes this protein set attractive for further investigation in in vivo models of tissue injury and for targeting in osteoarthritis and related musculoskeletal diseases.

Intervertebral disc (IVD) degeneration is characterized by a loss of cellularity, and changes in cell-mediated activity that drives anatomic changes to IVD structure. In this study, we use single cell RNA-sequencing analysis of cells extracted from the degenerating tissues of the rat IVD following lumbar disc puncture. Two control, uninjured IVDs (L2-3, L3-4) and two degenerated, injured IVDs (L4-5, L5-6) from each animal were examined either at two- and eight-week post-operative time points. The cells from these IVDs were extracted and transcriptionally profiled at a single-cell resolution. Unsupervised cluster analysis revealed the presence of 4 known cell types in both non-degenerative and degenerated IVDs based on previously established gene markers: IVD cells, endothelial cells, myeloid cells, and lymphoid cells. As a majority of cells were associated with the IVD cell cluster, sub-clustering was used to further identify the cell populations of the nucleus pulposus, inner and outer annulus fibrosus. The most notable difference between control and degenerated IVDs was the increase of myeloid and lymphoid cells in degenerated samples at 2- and 8- weeks post-surgery. Differential gene expression analysis revealed multiple distinct cell types from the myeloid and lymphoid lineages, most notably macrophages and B lymphocytes and demonstrated a high degree of immune specificity during degeneration. In addition to the heterogenous infiltrating immune cell populations in the degenerating IVD, the increased number of cells in the AF sub-cluster expressing Ngf and Ngfr, encoding for p75NTR, suggest that NGF signaling may be one of the key mediators of the IVD crosstalk between immune and neuronal cell populations. These findings provide the basis for future work to understand the involvement of select subsets of non-resident cells in IVD degeneration.

Fibroblasts display complex functions associated with distinct gene expression profiles that influence matrix production and cell communications and the autonomy of tissue development and repair. Thrombospondin-2 (TSP-2), produced by fibroblasts, is a potent angiogenesis inhibitor and negatively associated with tissue repair. Single-cell (sc) sequencing analysis on WT and TSP2KO skin fibroblasts demonstrate distinct cell heterogeneity. Specifically, we found an enrichment of Sox10+ multipotent progenitor cells, identified as Schwann precursor cells, in TSP2KO fibroblasts, while fibrosis-related subpopulations decreased. Immunostaining of tissue and cells validated the increase of this Sox10+ population in KO fibroblasts. Furthermore, in silico analysis suggested enhanced pro-survival signaling, including WNT, TGF-{beta}, and PDGF-{beta}, alongside a reduced BMP4 response. Additionally, the creation of two TSP2KO NIH3T3 cell lines using the CRISPR/Cas9 technique allowed functional and signaling validation in a less complex system. Moreover, KO 3T3 cells exhibited enhanced migration and proliferation, with elevated levels of pro-regenerative molecules including TGF-{beta}3 and Wnt4, and enrichment of nuclear {beta}-catenin. These functional and molecular alterations likely contribute to improved healing and increased neurogenesis in TSP2-deficient wounds. Overall, our findings describe the heterogeneity of dermal fibroblasts and identify pro-regenerative features of TSP2KO fibroblasts.

Collagen fibrils constitute the structural scaffold of bone, and their hierarchical organization is central to biomineralization. While hydroxylation and glycosylation of lysine residues are well-known collagen post-translational modifications, their structural consequences remain poorly understood. Here we show that excess of glycosylation of hydroxylysine residues leads to significant alterations in the local packing of collagen molecules within the fibrils. By prolonging the enzymatic modification window during helix folding through the use of cyclosporin A, an increase in double glycosylation was observed at specific sites. These overglycosylated residues were pinpointed by mass spectrometry, while cryogenic TEM revealed fibrils with reduced diameters and distinct displacements of defined sub-bands within the D-period, without altering the overall periodicity. By mapping the modified residues onto the quarter-staggered model, we have been able to correlate site-specific glycosylation with sub-band shifts, linking chemical modification to supramolecular order. These results provide molecular-level evidence that collagen glycosylation is an active determinant of fibril structure. Such insights not only advance fundamental understanding of collagen assembly but could also illuminate mechanisms underlying bone fragility disorders, including osteogenesis imperfecta, that feature altered glycosylation.

The long-term morbidity of tendon disease in an increasingly active and ageing population represents a growing area of unmet clinical need. Tendon disorders commonly affect the lower limb, ranging from isolated tendon rupture to degenerative joint deformity. In the absence of valid animal models of chronic tendinopathy, traditional methods to isolate and identify crucial sub types involved in disease are limited by the heterogeneity of tendon cells, by their relative paucity in tissue and by the density of the surrounding collagen matrix. To overcome this, we have used next generation CITE-sequencing to combine surface proteomics with in-depth, unbiased gene expression analysis of single cells derived ex vivo from healthy and diseased tendon. For the first time we have been able to show that human tendon consists of at least eight sub-populations of cells. In addition to endothelial cells, Tc cells, and macrophages, there are five distinct tenocyte populations expressing COL1A genes. These consist of a population of resident cells expressing microfibril associated genes (FBN1, VCAN, DCN, EMILIN1, MFAP5), a group of SCX+ cells co-expressing high levels of pro-inflammatory markers, a population of APOD+ fibro-adipogenic progenitors (FAPs), TPPP3/PRG4+ chondrogenic cells (COMP, CILP, PRG4) and ITGA7+ Smooth Muscle-Mesenchymal Cells, recently described in mouse muscle but not, as yet, in human tendon. Surface proteomic analysis identified markers by which these sub-classes could be isolated and targeted in future. In comparison to healthy tendon, diseased tendon harboured a greater proportion of SCX+ tendon cells and these expressed high levels of pro-inflammatory markers including CXCL1, CXCL6, CXCL8, PDPN and previously undescribed PTX3. We were also able to show that whereas disease associated genes such as CD248 and PDPN were expressed by COL1+ tenocytes, IL33 was restricted to endothelial cells of chronically diseased tendon.

Animal microbiota have complex interactions with hosts and environment that determines its composition. Yet the ability of hosts to determine their microbiota composition is less well studied. In this study, to investigate the role host genetics in determining skin microbiota, we used 30 different mouse strains from the recombinant inbred panel, the Collaborative Cross. Murine skin microbiota composition was strongly dependent on murine strain with > 50% of the variation explained by murine strain. In particular, a quantitative trait locus on chromosome 4 associates both with Staphylococcus abundance and principal-component multi-trait analyses. Additionally, excisional wound associated changes in microbiota composition were not uniform across mouse strains and were host-specific, the genetic background accounting for about 40% of the variation in microbiota. Genetic background also had the highest effect on the healing speed of wounds accounting for over 50% of the variation while mouse age and microbiota composition change accounted only for 20% and 5% of the healing speed despite reaching statistical significance. In conclusion, host genetics has a significant impact on the skin microbiota composition during both homeostasis and wound healing. These findings have long reaching implications in our understanding of associations between microbiota dysbiosis and disease.

Short abstractOvarian fibrosis is a known pathology of reproductive aging, becoming a growing concern for infertility and complex ovarian diseases. In research, mouse and human ovary samples are utilized, though distinct differences between species warrant validation of architectural phenotypes to accurately define its pathology. Using polarized light microscopy and orientation analysis of collagen fibers in mouse and human ovaries, we define ovarian fibrosis as the accumulation and/or anisotropic organization of fibrillar collagen within the ovarian stroma and/or cortex.

Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde pretreated heterograft tissue, such as bovine pericardium (BP) are the most commonly used heart valve replacements. However, BHV durability is limited by structural valve degeneration (SVD) resulting from both calcification and advanced glycation end product (AGE) deposition together with serum protein infiltration. In the present study we investigated the hypothesis that anti-AGE agents, Aminoguanidine (AG), Pyridoxamine (PYR), and N-Acetylcysteine (NAC) could mitigate AGE-serum protein mechanisms in model studies, both in vitro and in vivo, using rat subdermal implants of BP. In vitro studies demonstrated that each of these agents could significantly inhibit AGE formation in BP. However, in rat 28 days BP subdermal implants, only PYR demonstrated both significant inhibition of AGE and serum albumin accumulation per immunostaining. BHV calcification was not mitigated by PYR. It is concluded that AGE-serum protein pathophysiology contributing to SVD can be ameliorated by PYR.

Pulmonary fibrosis is characterized by extracellular matrix (ECM) deposition driven by fibroblast-to-myofibroblast transition (FMT) and by an altered proteolytic balance. While the roles of several cysteine proteases have been documented, the specific contribution of cathepsin V (CatV) and legumain (LGMN) remains poorly explored. LGMN, CatV and their dual inhibitor cystatin M/E (CysM/E) are significantly increased in lung specimens and bronchoalveolar lavage fluids from patients with idiopathic pulmonary fibrosis. TGF-{beta}1 triggered CysM/E expression and LGMN transcription, intracellular maturation, enzymatic activity, and pro-LGMN secretion via the Smad-3 pathway, whereas CatV was downregulated in human lung fibroblasts (CCD-19Lu and primary HPF cells) undergoing myodifferentiation. Genetic silencing of LGMN or CatV, and pharmacological inhibition of LGMN, led to accumulation of fibronectin and elastin, implying that both proteases contribute to ECM remodeling. LGMN cleaved fibronectin, while CatV predominantly regulated elastin levels. Conversely, broad-spectrum inhibitor cystatin C (hCC) markedly reduced elastin and fibronectin degradation, whereas CysM/E exerted a weaker effect, mainly on elastin turnover. LGMN inhibition transiently delayed fibroblast wound closure, establishing a functional role in tissue repair through fibronectin remodeling. Neither LGMN nor CatV influenced -SMA expression, distinguishing them from CatB, which participates in FMT. Altogether, LGMN was identified as an effector of matrix remodeling rather than myodifferentiation, acting in concert with CatV. Within the proteolytic network governing fibrosis progression, the present findings identify a cystatin-regulated LGMN/CatV partnership, participating in ECM turnover and cell migration. Present results also provide new perspectives on potential therapeutic protease-based strategies targeting ECM turnover underlying lung fibrosis.

Tendinopathy is a debilitating tendon disorder affecting millions of people, characterized by pain, swelling, and diminished biomechanical properties. While the precise mechanisms underlying tendon homeostasis remain unclear, metabolic regulation plays a critical role. In this study, we combine transcriptomic analysis of human tendinopathic samples with a conditional mouse model in which Prkaa1 (encoding AMPK1) is selectively deleted in tendon progenitors to elucidate the role of AMPK signaling in tendon homeostasis. RNA sequencing of diseased human tendons revealed downregulation of key metabolic genes, including several involved in the mitochondrial electron transport chain and AMPK signaling pathways, alongside an increase in markers associated with senescence and a secretory inflammatory profile. In parallel, mice with loss of Prkaa1 function exhibited normal postnatal development; however, by one month of age, tendons demonstrated widespread transcriptional alterations related to cell cycle regulation and ECM organization. By three months, AMPK1-deficient tendons showed significant reductions in mechanical strength and increased expression of senescence markers p21 and p16, progressing to prominent ectopic calcification with age. In vitro studies further confirmed that tendon fibroblasts lacking AMPK1 have altered ECM substrate adhesion profiles. Importantly, voluntary exercise partially rescued these deficits by enhancing ECM organization and reducing senescence marker expression. Collectively, our findings demonstrate that AMPK1 is critical for maintaining energy balance, regulating ECM remodeling, and preventing premature cellular senescence in tendon. These insights highlight AMPK signaling as a promising therapeutic target and underscore the beneficial role of exercise in mitigating tendinopathic changes.

ObjectiveTo validate SERPINB2 and SERPINA9 as chondrogenic biomarker candidates across independent transcriptomic platforms and cell sources, to characterise the complete SERPIN expression landscape during kartogenin (KGN)-induced chondrogenic differentiation of human mesenchymal stem cells (hMSCs), and to identify novel SERPIN biomarker candidates and their signalling context during cartilage lineage commitment. DesignMulti-platform transcriptomic analysis across three independent datasets: (i) Affymetrix HGU133+2 microarray of KGN-induced chondrocytes versus undifferentiated hMSCs (ATCC source); (ii) Affymetrix Clariom D whole-transcriptome array of KGN-treated versus control hMSCs from an independent Mexican source (SINREG Laboratories); and (iii) previously published qPCR validation. Differential expression was computed using limma with Benjamini,Hochberg correction. SERPIN-focused cross-platform correlation and targeted pathway analysis were performed. ResultsThe Clariom D dataset yielded 1,869 differentially expressed genes (925 upregulated, 944 downregulated; FDR < 0.05) from 29,124 transcripts tested. SERPINB2 was concordantly upregulated across all three platforms (Clariom D: fold-change [FC] +3.54, FDR = 0.006; HGU133+2: log2FC = +3.29, nominal P = 0.027; qPCR confirmed), establishing it as one of the most reproducible transcriptomic signals in chondrogenic differentiation. In the direct Bone versus Cart comparison, SERPINB2 showed [~]45-fold chondrogenic enrichment (log2FC = -5.45, adjusted P < 0.0001). Cross-platform SERPIN correlation was significant (Pearson r = 0.54, P = 0.0025; n = 29 shared genes). Four additional SERPINs reached genome-wide significance on Clariom D: SERPINE2 (FC +2.57), SERPING1, SERPIND1, and SERPINE1. SERPINA9 was not replicated in the independent SINREG source, identifying it as a context-dependent marker. ConclusionsSERPINB2 is a robust, cross-platform chondrogenic biomarker with translational potential for osteoarthritis (OA) monitoring. The coordinated SERPIN programme activates a multi-layered proteolytic and signalling network during cartilage lineage commitment, positioning SERPINB2 as a functional regulator of the chondro-osteogenic lineage decision.

Age-related delays in fracture healing are prevalent and contribute to morbidity and mortality in elderly populations. Clinical and preclinical studies demonstrate that aging is associated with slower and less complete fracture repair characterized by delayed cartilage and bone formation, impaired matrix resorption, and an increased risk of delayed union or non-union. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) enables spatially resolved, in situ molecular analysis of proteins directly from murine fracture tissues. We applied collagenase type III (MMP-13) mediated proteolytic digestion to formalin-fixed, paraffin-embedded (FFPE) tibia fracture callus sections harvested 10 days post-tibial fracture from young (3-month-old) and aged (18-month-old) mice to perform spatially resolved proteomic profiling. Spatial MS Imaging revealed pronounced age-dependent differences in extracellular matrix protein composition and remodeling within the fracture callus. We identified upregulation of canonical bone and matrix proteins, including Col1a1 and Col1a2 specifically in the young fracture callus demonstrating advancement into harden callus formation. Conversely, Col2a1 and other soft callus proteins were only seen in the aged callus tissues. Further, protein indicators of tissue state, such as fibronectin (upregulated) and calreticulin (downregulated) were selectively regulated aged tissues, demonstrating a failure for aged tissues to fully progress into harden calluses. Spatial proteomic patterns demonstrated a marked delay in progression from cartilaginous to osseous callus in aged mice, consistent with impaired matrix remodeling during fracture repair. Together, these findings establish spatial MS Imaging based proteomics as a powerful approach to elucidate age-related alterations in fracture healing and to identify molecular regulators of impaired skeletal regeneration. Lay SummaryUsing spatially-resolved proteomics via mass spectrometry imaging on fracture callus tissues from young and aged mice, we observed delayed healing in aged animals based on the composition of the extracellular matrices. Higher levels of bone specific collagens were detected in young animals, whereas cartilage specific collagens were detected in aged animals at higher levels. Further, detection of novel, non-canonical callus proteins revealed critical transitional steps that are delayed in aged-callus tissues, and these may also contribute to the delayed healing aged animals.