Matrix Biology Plus

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.

Background and aimsAtherosclerotic plaques form preferentially at vascular sites exposed to disturbed blood flow, yet the protein changes underlying this site-specific plaque development remain unclear. Mouse models are widely used to study atherosclerosis but yield only limited amounts of tissue, previously restricting proteomic studies. However, recent advances in mass spectrometry now enable proteomic profiling of very small tissue samples. We aimed to utilise this to uncover site-specific protein changes in aortic regions prone or resistant to plaque formation. MethodsAortic arches from apolipoprotein E-deficient (ApoE-/-) mice fed a Western diet (WD) for 16 weeks were dissected into plaques from the major branches and inner curvature and visibly healthy regions. Proteins were extracted, enzymatically digested, and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). ResultsMore than 4000 proteins were identified per sample despite their small size (< 1 mg tissue). Principal component analysis showed clustering by both disease status and anatomical location within the aortic arch, indicating distinct proteomes. Proteins known to drive atherosclerosis - including vascular cell adhesion molecule 1 (Vcam1), apolipoprotein B (Apob), lipoprotein lipase (Lpl), and galectin 3 (Lgals3) - were most abundant in advanced plaques and decreased progressively across anatomical regions, reaching their lowest levels in healthy regions furthest from the plaques. Enrichment analysis highlighted pathways related to the extracellular matrix, immune system, hemostasis, and lipoprotein transport as central to disease progression. ConclusionsThis study demonstrates the feasibility of region-resolved proteomics in individual murine aortas and provide new molecular insights into the site-specific nature of atherosclerotic plaque development.

Sensory neuropeptides, particularly Substance P (SP) and -calcitonin gene-related peptide (CGRP), are implicated in osteoarthritis (OA) pathogenesis. This study elucidates their specific roles in spontaneous, age-related OA. Male and female mice deficient in SP (Tac1-/-), CGRP (CGRP-/-), or both (DKO) were evaluated at 6, 12, and 18 months of age. Assessments included histological OARSI scoring for articular cartilage matrix structure, Luminex arrays for systemic serum cytokines, and flow cytometry for local synovial immune cell profiling. Wild type (WT) mice developed early-stage, age-related cartilage degradation, predominantly in the lateral compartment. Conversely, all neuropeptide-deficient strains exhibited significant structural protection against this process. Systemically, SP deficiency distinctly altered cytokine profiles (e.g., decreased IL-23, increased IP-10), whereas CGRP deficiency caused minimal systemic shifts, highlighting a disconnect between circulating markers and local joint preservation. Locally, flow cytometry revealed profound, sexually dimorphic, and age-dependent neuroimmune alterations. In young males, neuropeptide deficiency significantly reduced synovial macrophage counts to levels comparable to those of aged WT mice. Furthermore, male CGRP-/- mice exhibited an age-related accumulation of CD8+ cytotoxic T cells. In contrast to males, young WT females demonstrated higher baseline CD8+ T cell counts that declined with age, whereas these subpopulations remained persistently low in KO mice. SP and CGRP act as critical modulators of age-related cartilage degradation. Their absence provides robust structural protection mediated through highly localized, sexually dimorphic neuroimmune pathways. These findings emphasize the necessity of targeting the local joint microenvironment for future personalized, sex-specific OA therapies.

The lung extracellular matrix (ECM) governs tissue architecture, mechanics, and function, yet how it remodels with age across sex and anatomical regions remains poorly understood. Here, we performed a systematic multi-factor proteomic analysis of rat lungs to define age-, sex-, and region-dependent remodeling across the tissue landscape. Age emerged as the dominant source of variation, with a conserved aging signature modified by region- and sex-specific effects. Young lungs showed coordinated ECM assembly, balanced proteolysis, and active biosynthetic programs consistent with structural adaptability and mechanical resilience. In contrast, aged lungs exhibited accumulation of mature collagen crosslinks and a more stabilized matrix architecture, indicating progressive matrix maturation and reduced structural plasticity. These changes were accompanied by proteomic signatures of metabolic stress and immune activation, suggesting coordinated remodeling across ECM, metabolic, and immune pathways during lung aging. Aging effects varied across anatomical regions and were more pronounced in females, highlighting context-dependent trajectories within the broader aging program. Age also partially reshaped spatial proteomic heterogeneity across lung compartments. Together, these findings identify matrix stabilization as a central feature of lung aging that links structural remodeling to metabolic-inflammatory imbalance and increased pulmonary vulnerability.

The axolotl salamander can fully regenerate amputated limbs, yet the systemic consequences underlying this process remain largely understudied. Cerebrospinal fluid is an emerging signaling medium capable of communicating with both the central and peripheral nervous systems, but its composition and potential role in salamander limb regeneration have not yet been examined using modern multi-omics techniques. Here, we developed a protocol for extracting mixed perilymph-cerebrospinal fluid (P-CSF) from axolotl and provided the first proteomic and metabolomic characterization of this biological fluid. We identified 2,626 unique proteins and 173 high-confidence metabolites and quantified them across four time points of early limb regeneration. We demonstrated that limb amputation drives progressive shifts in P-CSF proteins, including an elevation of sarcomeric muscle proteins, regeneration-associated factors, and protease/extracellular matrix proteins. We observed shifts in metabolites involved in oxidative stress, polyunsaturated fatty acid oxidation, and histamine metabolism. Injury-comparison experiments revealed that the observed proteomic changes as a result of limb amputation are different than crush injury, denervation, or tail amputation. This study proposes axolotl P-CSF as a reservoir for limb amputation-associated systemic signaling and as a potential conduit of signals involved in limb regeneration.

The sperm surface glycocalyx undergoes extensive remodeling during epididymal maturation, required for sperm to reach and bind the oviductal epithelium. N-acetylglucosamine (GlcNAc)-containing glycans are candidate mediators of these events, however, how these residues are regulated across the reproductive tract, and whether their changes depend on specific epididymal enzymes or functionally contribute to sperm-oviduct epithelial adhesion, remains poorly defined. Here, we addressed this gap by examining how surface GlcNAc changes as sperm mature and become functionally competent, from epididymal maturation through capacitation and the acrosome reaction. We further asked whether these changes relate to the ability of spermatozoa to bind the oviductal epithelium. Surface GlcNAc, assessed by WGA reactivity, increased progressively from caput to cauda epididymal spermatozoa, with a corresponding shift in GlcNAc-bearing protein profiles, while remaining predominantly localized to the acrosomal region throughout maturation. Incubation of caput spermatozoa with cauda epididymal fluid reduced WGA labeling, an effect blocked by the selective {beta}-N-acetylglucosaminidase ({beta}-NAG) thiourea derived hydroxy pyrrolidine inhibitor VP150, identifying luminal {beta}-NAG as an active contributor to GlcNAc remodeling in the epididymis. In ejaculated spermatozoa, capacitation induced minor changes in surface GlcNAc, whereas the calcium ionophore-induced acrosome reaction produced a marked reduction in WGA reactivity and acrosomal labeling, consistent with glycoprotein loss during acrosomal exocytosis. Functionally, spermatozoa that bound to BOEC monolayers were preferentially WGA-positive, and pre-incubation of BOECs with WGA significantly reduced sperm adhesion, implicating surface GlcNAc in sperm-oviduct epithelial recognition. Together, these findings define surface GlcNAc as a dynamically regulated glycan that is progressively established during epididymal transit, partly through luminal {beta}-NAG activity, redistributed during capacitation and acrosomal exocytosis, and functionally engaged during sperm-BOEC adhesion, providing a mechanistic framework for glycocalyx-mediated sperm selection in cattle.

Species identification in palaeoproteomics relies on genome-derived protein sequences which are often poor-quality, and lacks tools to cope with multi-species samples. Here, we address both challenges through the analysis of physical and genetic mixtures. Species that are absent from our database are considered a genetic mixture, i.e. a patchwork of peptides from closely related species. Inversely, various overlapping peptide stretches allow us to resolve complex physical mixtures. This is benchmarked by analysing physical mixtures of modern bone fragments, including genetic mixtures. We illustrate the impact of our approach via a rapid and high-throughput analysis of >2500 bone fragments, revealing the Eemian-era faunal environment around Scladina Cave, including the first Palaeoloxodon antiquus identified at this site. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/732552v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@75c1d3org.highwire.dtl.DTLVardef@1084481org.highwire.dtl.DTLVardef@1c9a4f9org.highwire.dtl.DTLVardef@16dd859_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundDuctal carcinoma in situ (DCIS) is a noninvasive breast lesion with variable risk of progression to invasive breast cancer (IBC). Current transcription and cell marker investigations suggest ECM decreases in later events but are limited in details of ECM proteomic composition, including post-translational modifications. We investigated whether the extracellular matrix (ECM) proteome alters with later breast events of DCIS or IBC. MethodsECM-targeted mass spectrometry imaging and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were applied to ten tissue microarrays from the Resource of Archival Human Breast Tissue cohort (RAHBT). Primary DCIS specimens (n=136) were analyzed in relation to later events of DCIS (n=40) or IBC(n=30), with a mean follow-up of 192.1 months 95% CI [179.1,205.1]. Statistical modeling, survival analyses, and exploratory machine learning approaches were used to identify ECM peptide signatures associated with later events. ResultsDistinct ECM peptide profiles were associated with later events of DCIS or IBC. Fifteen peptides derived from fibrillar collagens (COL1A1, COL1A2, COL3A1) and elastin, showed significantly reduced abundance in patients who developed IBC. Lower expression of specific collagen peptides associated with overall 19.9% 95% CI [17.92, 21.81] decreased disease-free survival for IBC. Lower expression of these peptides was significantly associated with reduced disease-free survival (age-adjusted hazard ratio [HR] = 2.45, 95% CI: 2.33-2.57; P < 0.05). Patient-matched samples of primary DCIS, later DCIS, and later invasive breast cancer further demonstrated reduction in ECM peptide detection. Exploratory predictive modeling from patient-matched samples achieved high performance (AUROC >0.98, accuracy >93%) in distinguishing primary from later events. Following prior work in the RAHBT cohort, reduction of certain collagen peptides was also observed in primary DCIS samples from higher risk patient groups. ConclusionsECM proteomic remodeling, particularly decreases of specific collagen domains, is strongly associated with later events of DCIS and IBC. These findings highlight ECM proteome as a critical regulator of breast cancer emergence with potential as a prognosticator of risk stratification to guide clinical management of DCIS.

Members of the transforming growth factor-{beta} (TGF-{beta}) superfamily, including inhibins and activins, are structurally related glycoprotein dimers that regulate reproductive and endocrine signaling. Their high degree of molecular similarity presents challenges for label-free analytical discrimination. To evaluate the ability of Raman spectroscopy to distinguish closely related TGF-{beta} superfamily proteins based on intrinsic vibrational fingerprints. Raman spectra of recombinant human Inhibin -subunit, Inhibin B ({beta}B homodimer), and Activin A ({beta}A--{beta}A) were acquired using confocal Raman microscopy with 532 nm excitation. Spectra were baseline-corrected, area-normalized, and analysed using principal component analysis (PCA). Distinct spectral signatures were observed across the 500--1800 cm-1 region. Differences within the S--S stretching region (500--550 cm-1) were consistent with variations in disulfide-bond environments, with the Inhibin -subunit exhibiting the highest relative intensity in this region. Variations in the amide I band (1600--1700 cm-1) suggested differences in protein secondary structure, while aromatic amino acid vibrations provided additional discriminatory features. PCA revealed clear clustering and separation of all three protein classes based on their Raman fingerprints. Raman spectroscopy enables label-free differentiation of structurally related endocrine glycoproteins and demonstrates potential for the structural characterization and classification of inhibin and activin proteins within the TGF-{beta} superfamily.

Hepatic fibrosis progression involves a well-characterized but computationally unmodeled feedback loop: Advanced Glycation End-products (AGEs) accumulate on permanent collagen via Maillard chemistry, activate the Receptor for Advanced Glycation End-products (RAGE) on hepatic stellate cells (HSCs) and Kupffer cells, drive NF-{kappa}B-mediated HSC activation and anti-apoptotic signaling, and deplete soluble RAGE (sRAGE) through hepatocyte loss -- creating a closed positive feedback loop. To our knowledge, we present the first spatial agent-based model incorporating the complete AGE-RAGE-sRAGE axis in a three-dimensional GPU-accelerated liver tissue simulation. The model produces three key findings: (i) stage-dependent irreversibility thresholds emerge without explicit stage-gating, with resolution declining from [~]68% at F2 to <10% at F4; (ii) sRAGE trajectories diverge at F2-F3: recovering during abstinence from F2 (0.68 [-&gt;] 0.87) but remaining depleted from F3 (0.54), predicting a clinically testable biomarker transition; and (iii) RAGE-driven HSC activation becomes self-sustaining at F3+ independent of exogenous injury, explaining why late-stage fibrosis resists resolution despite removal of the primary insult. No prior computational model -- ODE, PDE, or agent-based -- has formalized the complete RAGE-AGE-sRAGE feedback loop in hepatic fibrosis. The sRAGE divergence prediction is independently testable in clinical cohorts.

Fibroblasts mediate tissue repair after damage, but aberrant fibroblast behavior in response to injury can result in impaired wound healing. Severe burn injury often results in tissue scarring, but the underlying mechanisms by which fibroblasts respond to burn injury and the role of inflammation in fibrosis are not well understood. Here we developed fluorescent reporters of collagen expressing mesenchymal cells enabling real time imaging of fibroblasts during homeostatic development and in response to burn injury using larval zebrafish. We find that fibroblasts derived from the mesenchyme respond to burn injury by engaging in a maturation process, characterized by the expression of vimentin, which is reminiscent of larval development. In burned tissue, fibroblast maturation is perturbed by prolonged neutrophil infiltration, resulting in disorganized extracellular matrix (ECM) and delayed ECM remodeling, which can be rescued by neutrophil depletion. This work adds to our understanding of fibroblast development in zebrafish and shows that collagen expressing mesenchymal cells regulate ECM remodeling in coordination with immune cells during burn wound healing. Summary StatementVimentin-positive fibroblasts are required for normal wound healing in larval zebrafish, but the presence of inflammatory neutrophils after burn injury impairs fibroblast maturation and collagen remodeling.

Endometriosis, a common inflammatory gynecological disorder affecting up to 10% of women worldwide, is characterized by the presence of endometrium-like tissue outside the uterus. Current diagnostic methods, such as ultrasound and MRI, effectively detect ovarian and deep endometriosis but fail to detect more common peritoneal type. Diagnosing peritoneal endometriosis currently necessitates invasive laparoscopy and histological confirmation. Despite numerous efforts, no new reliable biomarkers have successfully transitioned into routine clinical use. This study aimed to investigate the use of targeted metabolomics to discover metabolite ratios capable of identifying endometriosis in plasma samples. We analyzed a discovery population of 235 patients and a validation population of 278 patients. All cases and controls in both populations were diagnosed by laparoscopy. Control subjects included individuals presenting with symptoms such as pain, dysmenorrhea, infertility, or other benign conditions, but who had no laparoscopic evidence of endometriosis. Using generalized linear models (GLMs) and machine learning, the study identified specific metabolite ratios as potential biomarkers that can distinguish different types of endometriosis and enable mass spectrometry-based diagnostics for peritoneal endometriosis. The best-validated GLM, derived from the concentration ratios of amino acids, acylcarnitines, sphingomyelins, and phosphatidylcholines, consisted of Thr/SM(OH) C22:2 + PC aa C40:5/SFA_PC + lysoPC a C16:0/SM(OH) C16:1. This model yielded an AUC of 0.82 (95% CI 0.619-0.891, with 76% sensitivity and 81% specificity) for peritoneal endometriosis. This innovative approach offers a robust diagnostic model, addressing an unmet medical need by facilitating earlier detection of peritoneal endometriosis and improving overall clinical management.

BackgroundThe etiology and molecular mechanisms of Moyamoya disease (MMD) remain unclear. Exosomes, as carriers of bioactive molecules, may reflect disease-specific alterations and serve as potential biomarkers. This study aimed to investigate disease mechanisms using proteomic profiling of serum-derived exosomes (SDEs) in MMD. Materials and MethodsPeripheral blood each from 15 MMD patients and 15 healthy-controls were used to isolate SDEs via ultracentrifugation. Proteins from pooled SDEs were extracted, digested, and analyzed by LC-MS/MS. Differentially expressed proteins were examined using MetaboAnalyst, DAVID, Enrichr, STRING, and Cytoscape. Key targets were validated at transcript and protein levels using RT-qPCR and ELISA in independent cohorts. ResultsA total of 2,554 proteins were identified, with 213 showing differential expression (118 upregulated, 95 downregulated; p [&le;] 0.05). Functional and pathway analyses revealed enrichment in angiogenesis, cytoskeletal remodeling, and endothelial signaling. PRKG2 and MYC were upregulated, while RHOA was downregulated, highlighting their involvement in focal adhesion and PI3K-AKT pathways. Validation confirmed these findings. ConclusionDysregulated proteins were linked to RHOA-ROCK and PI3K-Akt signaling, suggesting their role in driving VSMC phenotypic switching, contributing to vascular occlusion. These findings indicate that altered exosomal-proteins may participate in maladaptive vascular remodeling, although the initial trigger for VSMC transition remains unknown.

BackgroundVein graft failure due to intimal hyperplasia and maladaptive remodeling remains a major limitation of peripheral bypass surgery. Although vascular remodeling is recognized as a multilayered process, layer-specific molecular mechanisms that distinguish adaptive from negative remodeling remain incompletely understood. We aimed to investigate the vascular microenvironment of patent and stenotic grafts using spatial transcriptomics. MethodsVein specimens were obtained from three patients undergoing revision surgery. For each patient, a matched set of three samples was collected: unused saphenous vein (Denovo), normally healed vein graft (Non-stenosed), and stenosed vein graft (Stenosed) (n = 3 patients). GeoMx Digital Spatial Profiling with the Human Whole Transcriptome Atlas was used to map gene expression across intima, medial, and adventitial layers. Differential expression, gene ontology, spatial deconvolution, and immunohistochemistry were integrated for analysis. ResultsNon-stenosed and Stenosed grafts shared transcriptional features distinct from Denovo veins, particularly in pathways related to cell proliferation. Non-stenosed grafts showed increased expression of CDKN1A across all vascular layers, whereas Stenosed grafts exhibited enhanced mitogen-activated protein kinase (MAPK) pathway activity, reduced DUSP1-mediated regulation, and increased macrophage accumulation. ECM remodeling showed layer-specific organization, with VCAN and ACAN enriched in the intima and DCN in the adventitia, while Stenosed grafts demonstrated a trend toward collagen-dominant remodeling. Cell deconvolution suggested a shift toward vascular smooth muscle cell (VSMC)-dominant architecture after arterialization, with modest enrichment of synthetic VSMC signatures in stenotic regions. ConclusionsVein graft stenosis appears to be associated with layer-specific alterations in cell cycle regulation, inflammatory signaling, extracellular matrix remodeling, and VSMC phenotype. Spatial transcriptomic analysis reveals molecular heterogeneity not captured by bulk approaches and provides preliminary insight into graft remodeling. These findings may inform future studies to improve long-term graft patency.

Chondrosarcoma is a hyper-vascularised, chemoresistant cartilage malignancy driven by VEGF-centred angiogenesis, and local adipose depots are increasingly recognised as paracrine drivers of tumour angiogenesis via adipokines and extracellular vesicles (EVs). The infrapatellar fat pad (IFP), an inflammatory adipose depot within the articular joint in direct cartilage contact, is a key local source of adipose-derived EVs, and thus a candidate driver of angiogenesis in chondrosarcoma. The aim of this study was to determine whether the IFP is a productive source of EVs, and whether IFP-derived EVs induce angiogenesis in articular chondrocytes. The IFP released significantly more EVs than subcutaneous fat (n = 8 per depot; p = 0.027). Treating primary human articular chondrocytes with IFP EVs for 24 h upregulated VEGFA (+1.6-fold, p = 0.036) and downregulated BMP4 (-2.4-fold, p = 0.011), engaging the VEGF/eNOS/ERK axis that drives chondrosarcoma angiogenesis. Re-analysis of a previously published phospho-kinase dataset from the same donor EVs, corroborated by a pooled donor-group analysis (n = 3), supported activation of eNOS, ERK1/2, PLC-{gamma}1 and HSP27. These findings identify the IFP as a dominant source of EVs within the articular joint, which can induce a pro-angiogenic, VEGF-axis switch in articular cartilage cells, supporting a signalling model relevant to chondrosarcoma angiogenesis.

The surface layer of articular cartilage provides for low-friction joint movement and protects the tissue from mechanical wear. The superficial zone chondrocytes (SZCs) of the surface layer produce proteoglycan-4 (PRG4), which is a lubricant that is necessary to reduce friction. Articular cartilage has limited capacity for self-repair and cell-based therapies, such as autologous chondrocyte implantation (ACI), is used to stimulate repair. However, in ACI, cells are expanded on tissue culture polystyrene where SZC poorly attach, proliferate slowly and dedifferentiate. Consequently, expanded SZC produce fibrocartilage tissue with insufficient PRG4. We previously demonstrated that culturing SZC on chondrocyte-derived decellularized extracellular matrix (CM) enhances SZC attachment and preserves phenotype. Since fibronectin (FN) was identified as the most abundant matrix protein within CM, here we tested the hypothesis that FN-coated culture surfaces would partially reproduce the beneficial effects of CM. We found that, similar to CM, SZC on FN-coated polystyrene increased SZC attachment and proliferation. However, unlike CM, SZCs expanded on FN-coated polystyrene remained more dedifferentiated as indicated by spread cells, elevated fibroblastic and contractile mRNA levels, and increased formation of SMA positive stress fibers. Consistent with the dedifferentiated phenotype, SZC on FN-coated polystyrene displayed extensive stress fibers, and higher nuclear myocardin-related-transcription-factor-a (MRTF-A). In contrast, CM reduced stress fiber formation and diminished nuclear MRTF-A in SZC. CM provides matrix cues beyond FN that suppress dedifferentiation and preserve the SZC phenotype. Identifying the matrix cues necessary to improve SZC expansion could lead to the generation of a superior surface in ACI repair tissue.

Pathological collagen degradation is a central feature of impaired wound healing, dermal aging, periodontal breakdown, intestinal barrier injury and connective tissue degeneration. Current strategies often focus on direct inhibition of matrix metalloproteinases or collagenases; however, complete blockade of collagen remodeling may interfere with normal repair. UT-018, a bioactive formulation that acts as a tissue-protective and regenerative agent, was evaluated as a collagenous extracellular matrix modulator. Across in vitro kinetic assays, endpoint signal analysis, integrated area-under-curve (AUC) analysis and substrate preincubation studies, UT-018 produced concentration-dependent preservation of collagen against collagenase challenge. Importantly, collagen protection persisted after substrate preincubation with UT-018, with approximately 33%, 60% and 65% protection at 5, 10 and 25 mM UT-018 concentrations, respectively. Exploratory kinetic transformations did not support a simple competitive collagenase inhibitor model. Instead, the collective pattern supports a substrate-directed mechanism involving collagen shielding, reduced cleavage susceptibility and indirect modulation of collagenase activity. These findings position UT-018 as a potential first-in-class collagen resilience modulator for wound healing, gastrointestinal barrier protection, oral care, dermal preservation and regenerative medicine applications. Highlights- UT-018 preserves collagen content in in vitro collagenase challenge assays. - The Protection is UT-018 concentration-dependent across kinetic, endpoint and AUC readouts. - Preincubation of substrate with UT-018 retains protection after collagenase challenge. - The data support matrix-directed stabilization by UT-018 rather than classical active-site collagenase inhibition.

At the onset of intervertebral disc degeneration, within the disc core, the pH and osmolarity decrease, and the residing notochordal cells (NCs) gradually transition towards nucleus pulposus cells (NPCs). How these microenvironmental cues shape the NCs extracellular vesicles (EV)-enriched secretome, and thus EV-mediated communication with NPCs during this transition, remains poorly understood. To study this, we collected the secretome from pig NC-rich tissue cultured for 4 days in either healthy or degenerate disc media to mimic these changes. In both conditions, NC-rich tissues were largely comparable at the histological and biochemical levels. Despite, tissues released glycosaminoglycans (GAGs), depleting the extracellular matrix. Surprisingly, degenerative media did not differentially release inflammatory regulators, though it reduced PGE2 release. We asked whether this extended to EV-enriched secretome media (SM_EV+), and found that the degenerative media reduced the number of EVs without altering their morphology or size. We then determined NC-EV association of inflammatory and matrix regulators. NC-EV isolation enriched MMP1, IL6 and IL10 and depleted soluble GAGs. Conversely, EV-depletion (SM_EV-) removed most GAGs without affecting MMP1, IL6, and IL10, suggesting that they contribute to the NC-EV soft corona. Functionally, healthy SM_EV+ improved GAG production by NPCs, but attenuated TBXT expression. Degenerate SM_EV+ did not elicit detectable EV-specific effects. These findings suggest that, in health, secretome-mediated communication from NCs to NPCs is only partially EV-mediated. At the onset of IVD degeneration, low pH and osmolarity impair the release of NC-EVs and negate the EV-specific beneficial matrix-anabolic effects on NPCs, contributing to the NC-to-NPC transition.

Pressure ulcers arise from sustained mechanical loading that impairs perfusion and damages skin tissues, yet the coupled mechanical and biological mechanisms of their formation and healing remain poorly characterized. We addressed this gap using a mouse model in which dorsal skin underwent 72 hours of magnet-induced ischemia followed by reperfusion, with tissue collected at 0, 3, 6, and 9 days and compared with baseline controls. From each mouse, we obtained paired samples from pressure ulcer and remote control (non-loaded) sites, mapped thickness by tissue profilometry, and performed equibiaxial testing with full-field digital image correlation and inverse finite element analysis to estimate regional material parameters. In parallel, we quantified CD31+ vasculature, F4/80+ macrophages, collagen content, and key cytokines. Pressure ulcer sites were compressed and thinner at Day 0, developed ulcers by Day 3, and continued to remodel through Day 9. Mechanical tests revealed heterogeneous strain fields with elevated deformation along ulcer borders, while remote control tissue deformed more homogeneously. These mechanical changes evolved alongside dynamic vessel and macrophage repopulation, increased collagen content at early time points, and cytokine upregulation within pressure ulcer tissue. Collectively, our data define the spatiotemporal co-evolution of tissue geometry, mechanics, collagen remodeling, and inflammation in pressure ulcers and provide a quantitative foundation for predictive mechanobiological models.

Tendons are commonly injured, not only in athletes, but also during normal everyday activities, particularly in older age. Tendons are rich in extracellular matrix proteins, many of which are turned over extremely slowly during an individuals lifetime. As a result, tight regulation of the extracellular matrix is essential for tendons to remain resilient to the mechanical load placed on them. However, tendons are prone to age-associated functional decline, characterised by chronic inflammation, accumulation of damaged collagen, and matrix remodelling. These degenerative changes often precede injury, wherein additional inflammation and fibrotic tissue deposition make treatment difficult and reinjury highly likely. Understanding and preventing the causes of tendon functional decline is therefore vital to improving quality of life in the ageing population. Using an equine model, here we show that tendon fibroblasts, or tenocytes, isolated from aged tendons exhibit markers of senescence when cultured in vitro. Further, we describe how senescence in tenocytes drives inflammation, hypercontractility, and dysregulation of tendon matrix components. By replicatively senescing tenocytes isolated from young tendons, we observed that senescent tenocytes instigated proinflammatory signalling, had impaired capacity in wound healing assays, increased contractility, and secreted factors that induced senescence in healthy tenocytes. Further, tenocyte senescence dysregulated matrix turnover both at the gene and protein level. Finally, we demonstrate how senotherapeutic treatment of senescent tenocytes can reduce expression of senescence markers and restore proliferative capacity. Our results uncover systemic links between tendon ageing and cellular senescence, and identify mechanisms and therapeutic strategies for age-associated tendon degeneration.