Matrix Biology Plus

Oncofetal fibronectin (oFN) is a known biomarker of cancer progression and is involved in events such as epithelial mesenchymal transition and multidrug resistance. It is also understood that fibronectin (FN) and its splice variants are differentially arranged by cancer associated fibroblasts (CAFs) to support cancer cell proliferation and migration. Contrastingly, little is understood about the mechanobiology of oFN, a post-translationally modified FN isoform defined by O-glycosylation of a specific threonine residue. To investigate the architectural features of the oFN matrix, CAFs were simulated by culturing human mammary fibroblasts (HMFs) on stiff polyacrylamide gels (20 kPa) in media collected from HCC-1937 (primary site breast adenocarcinoma) and MDA-MB-231 (metastatic breast adenocarcinoma) cells. As a control, HMFs were also cultured on softer gels representing healthy adipose tissue (2 kPa) in media not exposed to cancer cells. After immunocytochemical analysis, tumor conditioned HMFs displayed an expected CAF-like phenotype that was most prominent with MDA-MB-231 conditioned media. Matrices produced by tumor-conditioned HMFs contained more oFN with structural features mostly following that of the overall dense, aligned FN matrix. Cancer cells reseeded on their corresponding decellularized tumor-associated matrices had greater F-actin/oFN colocalization than control matrix. The lack of distinct oFN topographical cues may indicate that oFN O-glycosylation is more directly involved in biochemical signaling than mechanosignaling through matrix architecture. Together, this study established a platform for investigating stromal oFN which revealed that tumor-conditioned media sourced from cancer cells with different metastatic potential influences HMF activation, leading to distinct total FN and thus oFN matrix microarchitecture features.

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.

Seroma is a common complication following abdominoplasty and other procedures involving extensive soft tissue dissection. Although animal seroma models have been reported, the stromal pathology underlying post-abdominoplasty seroma, particularly the role of oxidative stress, remains poorly characterized. Here, we developed a rat abdominal seroma model by surgically separating the subcutaneous tissue from the underlying muscle fascia and investigated the temporal evolution of the associated stromal response. Seroma developed in 9 of 20 operated rats, corresponding to a formation rate of 45.0%. Longitudinal ultrasound demonstrated that fluid accumulation peaked around postoperative day (POD) 7 and largely resolved by POD30. Despite macroscopic fluid resolution, substantial stromal remodeling persisted. Compared with normal abdominal tissue, seroma-associated tissues at POD14 and POD35 exhibited increased collagen deposition, macrophage accumulation, oxidative injury, and vascular remodeling. Additionally, at POD35, macrophage accumulation and 4-hydroxynonenal staining were greater than at POD14, whereas collagen deposition and vascular density remained elevated with no significant differences between postoperative time points. To our knowledge, this study presents the first rat model specifically designed to reproduce post-abdominoplasty seroma and the first temporal characterization of oxidative stress within seroma-associated tissue. These findings reveal that seroma fluid resolution does not coincide with stromal recovery and identify persistent oxidative stress, inflammation, fibrosis, and vascular remodeling as important features of seroma pathology.

Background. Despite the growing demand for transplantation, a shortage of donor organs persists, limiting access to this lifesaving procedure. To meet the increasing demand, organs are frequently transplanted across an age discrepancy between donor and recipients. Methods. We delineated biological age dynamics in heterochronic heart transplants performed in both mice and patients. We carried out multiomic profiling and applied advanced biomarkers of aging to understand the interactions between the transplanted organ and the host. Results. We show that the biological age of experimental heart transplants rapidly assimilates the age of recipients. Interestingly, this effect was limited to the grafted tissue without reciprocal effects on systemic biological age of the recipient. These effects were confirmed at the level of DNA methylation and gene expression. Extending our findings clinically using both omics and functional analyses, we show that the biological age of heterochronically transplanted hearts is strongly associated with the age of the recipient, rather than the donor, confirming the clinical relevance of our experimental findings. Conclusions. These data identify the systemic environment as a driving force on tissue biological age. The rejuvenation of older organs in young recipients suggests novel approaches to organ allocation that may expand the pool of transplantable organs.

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.

Collagen biosynthesis is a complex multistep process, essential for functional tissue formation and repair. It is highly active during embryogenesis and wound healing, leading to physiological fibrogenesis, while its dysregulation induces pathologies affecting connective tissues, namely fibrosis. There are several key cellular microenvironmental factors regulating collagen biosynthesis, including vitamin C, oxygen tension and growth factors. Whilst the function of these distinct factors has previously been studied individually, how they operate together, synergistically or not, is not fully characterized. Using primary human dermal fibroblasts, we demonstrated that ascorbic acid (AA) and hypoxia both induced collagen I deposition when applied individually and had additive effects when combined together. Their effects were associated to the expected increase in collagen prolyl hydroxylation. However, AA induced a much stronger collagen secretion compared to hypoxia, while hypoxia leads to a higher collagen I hydroxylation. This observation challenged the common view of collagen prolyl hydroxylation being the central mechanism of AA-induced collagen secretion. Using proteomic analysis of the secretome, biochemical and cell imaging approaches, we further showed that increased CXCL12 extracellular levels and activation of its cognate CXCR4 receptor are at the core of AA-induced collagen intracellular trafficking and secretion. This mechanism is specific to AA and is not involved under hypoxia. The existence of distinct mechanisms underlying the effects of AA and hypoxia is further demonstrated by the different ultrastructure of collagen fibrils in the two conditions. Together, our results contribute to explain how these two triggers drive collagen biosynthesis and pave the way for potential pro- or anti-fibrotic strategies aimed at improving tissue repair.

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.

Neonatal mammals exhibit a remarkable capacity for cardiac repair that declines rapidly after birth and continues to diminish with age. This loss of regenerative potential is accompanied by age-dependent changes in macrophage behavior, shifting from pro-regenerative to pro-fibrotic responses following injury. We investigated whether extracellular matrix (ECM)-derived matrikines from different developmental ages regulate macrophage polarization. Cardiac ECM was isolated from neonatal (P2), adolescent (6-week), and adult (16-20-week) rat hearts through decellularization and pepsin digestion. ECM peptides were adsorbed onto tissue culture surfaces, and murine RAW 264.7 macrophages were cultured on ECM-coated or uncoated controls. After 24 hours, macrophage phenotype and gene expression were assessed using marker analysis and RNA sequencing. Macrophages cultured on neonatal cardiac ECM displayed reduced inflammatory activation and maintained expression of pro-reparative markers, even in the presence of inflammatory stimuli. Transcriptomic analysis revealed a distinct ECM-driven phenotype that differed from canonical M1 and M2 polarization states. Gene ontology analysis demonstrated significant downregulation of pathways associated with antiviral responses, immune activation, and interferon signaling in macrophages exposed to neonatal ECM. These effects were not observed with adolescent or adult ECM. Collectively, these findings suggest that neonatal ECM matrikines suppress interferon-mediated inflammatory signaling, potentially contributing to the reduced inflammation and enhanced repair observed following myocardial injury in neonatal hearts.

Individuals with fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder caused by mutations in the bone morphogenetic protein receptor ACVR1 (also known as ALK2), experience progressive and severely debilitating endochondral heterotopic ossification (HO). Previous studies have identified fibro-adipogenic progenitors (FAPs) as a major cellular source of HO in FOP mouse models. Here, we used mRNA sequencing of FAPs isolated from injured muscle of FOP mice to capture the earliest gene expression changes associated with the transformation of muscle tissues into bone. Whereas the transcriptomes of mutant and wild-type FAPs were indistinguishable prior to injury and at 1 day post injury, dramatic dysregulation of gene expression was observed at 3 and 5 days after injury. Surprisingly, gene expression changes suggested early specification of osteochondral lineages by 3 days post injury, approximately 3 to 4 days before the appearance of histologically identifiable cartilage. We identified dysregulation of genes encoding components of major signaling pathways, cell cycle regulators, extracellular matrix components, and hypoxia response mediators, and showed that these gene expression changes are temporally associated with abnormal population growth of mutant FAPs. We further demonstrated that treatment of FOP mice with either an anti-activin A monoclonal antibody or the ACVR1 kinase inhibitor BLU-782 (IPN60130; fidrisertib) normalized the expression of the majority of dysregulated FAP genes. However, the relative efficacy of the drugs differed, and distinct sets of overlapping genes were normalized by the treatments. The substantial transcriptional changes caused by these drugs in FAPs from normally regenerating muscle were also enumerated and described. These findings define early transcriptional events associated with pathogenic FAP reprogramming and provide a molecular framework for understanding the shared and distinct effects of activin A and ACVR1 inhibition.

Prostate cancer (PC) bone metastases (PCBM) are a debilitating disease morbidity that primarily affects the axial skeleton. PCBM are predominantly osteosclerotic with mixed osteosclerotic/osteolytic features, including irregular bone deposition, loss of collagen alignment and increased porosity that mimic features of woven bone formed during development and repair. We hypothesize that progression of PCBM is driven by interactions between tumor cells and the bone microenvironment and is associated with changes in the stromal compartment. We performed tandem mass tag mass spectrometry proteomics on lumbar vertebral specimens from 32 cadaveric PCBM specimens and 3 age-matched controls. Unsupervised clustering segregated specimens into three clusters associated with benign, mixed, and osteosclerotic lesion states, characterized by distinct immune-, tumor-, and osteogenic-associated protein signatures. Histological analysis showed tumor infiltration was accompanied by marrow fibrosis and increased collagen III deposition. We identified a marker signature associated with osteosclerotic remodeling and mapped this signature to independent metastatic PC transcriptomic datasets. The proteomics-derived markers reproduced similar molecular groupings at RNA level, and robustly segregated bone PCBMs from soft tissue metastases PC in RNA data. Together, these findings define molecularly distinct states of tumor-driven changes in PCBM that extend beyond histological classification and may inform biomarker development and therapeutic stratification.

Introduction: Endothelial-mesenchymal transition (EndoMT) is a dynamic endothelial cell-state transition increasingly implicated in fibrosis, vascular dysfunction, inflammation and cancer. Despite expansion of EndoMT-focused transcriptomic research, EndoMT representation remains limited to broader epithelial-mesenchymal transition (EMT) frameworks in standard gene set repositories. Methods: To address the absence of a general-purpose EndoMT gene set for transcriptomic enrichment analysis, we developed an open EndoMT gene set resource and accompanying R package. A literature-informed EndoMT gene set was curated and directionally assigned to EndoMT UP and EndoMT DOWN signatures according to reported or hypothesised expression during partial EndoMT, defined here as an active transitional state rather than a complete mesenchymal endpoint, to improve capture of EndoMT-associated transcriptional changes across different stages of the process. Genes were annotated by marker subtype and implemented as Gene Matrix Transposed files distributed through the R package, together with exported R objects and gene-level metadata. Benchmarking was performed using bulk RNA-seq data sets GSE118446, comprising seven-day IL-1l{beta}/TGF-{beta}2-treated human umbilical vein endothelial cells (HUVECs) and human pulmonary arterial endothelial cells, and GSE342578, comprising three-day TGF-{beta}1/2/3-treated HUVECs; single-cell RNA-seq data set GSE237895, comprising seven-day TGF-{beta}1-treated and SNAI1-overexpressing HUVECs; and endothelial cell and cancer-associated fibroblast populations from human metastatic melanoma single-cell RNA-seq data set GSE72056. Results: In GSE118446, EndoMT UP was positively enriched and EndoMT DOWN negatively enriched in chronically IL-1{beta}/TGF-{beta}2-treated endothelial cells, concordant with experimentally validated EndoMT induction, while partial EndoMT-associated enrichment was detected in single ligand-treatment. In contrast, EndoMT UP was negatively enriched following acute TGF-{beta}1/2/3 treatment, suggesting temporal differences between acute TGF-{beta} stimulation and chronic EndoMT induction. In GSE237895 mesenchymal-like endothelial cells, EndoMT UP was positively enriched and EndoMT DOWN negatively enriched in both TGF-{beta}1-treated and SNAI1-overexpressing models. EndoMT gene sets also discriminated endothelial and mesenchymal/stromal transcriptional states in GSE72056 metastatic melanoma. Marker subtype and Gene Ontology Biological Process analyses provided additional exploratory resolution of EndoMT-associated components beyond MSigDB Hallmark EMT. Discussion: The EndoMT R package provides a dedicated, accessible and interoperable resource for EndoMT-focused transcriptomic enrichment analysis. Benchmarking demonstrates utility for detecting established, partial and divergent EndoMT-associated states, supporting broad applicability across EndoMT biology research.

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.

Kidney disease affects over 850 million people and leads to degradation of the kidney cortical compartments and kidney failure. Interstitial fibrosis and tubular atrophy (IFTA) is a universal feature of chronic kidney disease, yet the sequence of tubulointerstitial matrix protein (matrisome) changes that underlie the development and progression of IFTA are not fully understood. In particular, post-translational modifications which may stabilize key matrisome components and result in irreversible and functionally detrimental interstitial fibrosis or tubular basement membrane thickening, have yet to be characterized. Post-translational modifications (e.g., acetylation) have been shown to contribute to kidney diseases, yet they have not been investigated in chronic kidney disease progression. Herein, we established feasibility of matrisome protein quantification in the tubulointerstitium, with concurrent matrisome protein post-translational modification quantification, in archived diagnostic human kidney biopsies, using laser capture microdissection, liquid chromatography tandem mass spectrometry (LC-MS/MS), and bioinformatics analysis. In this pilot study, we focused on two conditions associated with IFTA progression: diabetic nephropathy and kidney transplantation. We examined both healthy and chronically damaged tubulointerstitium in two human kidney biopsies, one representing each condition. We identified signature protein markers of tubulointerstitium (e.g., UMOD, SLC12A1) in abundance, confirming sample composition, and observed uniformity in protein abundance across replicates from the same biopsies. We found increases in certain collagen isoforms (COL1A1, COL6A3) and other ancillary proteins in areas of IFTA. We also successfully quantified post-translational modifications, confirming that hydroxylproline was the most abundant modification in healthy tubulointerstitium from the kidney transplant biopsy, and that hydroxyllysine was also highly abundant and could be confidently quantitated on two collagen isoforms (COL1A1, COL4A2). Lastly, we performed power analysis to determine the required sample size to achieve significant power and false discovery rate. Several proteins exhibited large Hedge effect sizes in the tubulointerstitium (ANPEP, PRODH2), indicating their potential usefulness as fibrosis signatures. Our study is a first step toward comprehensive characterization of tubulointerstitial matrisome changes, including post-translational modifications seen with IFTA progression. Subsequent studies will potentially reveal new molecular markers of key early events in chronic kidney disease progression, and new therapeutic targets for mitigating this process. Data are available via ProteomeXchange identifier PXD079025.

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.

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.

Decellularized extracellular matrix (dECM) scaffolds are increasingly used in regenerative medicine, yet the extent to which processed placental dECM retains properties capable of influencing cellular responses remains unclear. This study combines functional cell assays with deep learning-enabled quantitative imaging to determine how dehydrated placental ECM regulates cellular behavior across multiple human cell lineages. Human dermal fibroblasts, cardiac fibroblasts, and osteoblasts were cultured on dehydrated placental ECM or standard cell culture surfaces and assessed for cell attachment, viability, extracellular matrix production, and nuclear morphology. Placental dECM supported attachment and survival across all three cell types, while Pro-Collagen I Alpha 1 secretion varied by cell lineage relative to negative controls. To identify structural responses associated with scaffold culture, an automated imaging pipeline combining Cellpose-based nuclear segmentation with nuclear morphometric analysis was used to quantify nuclear area, eccentricity, and circularity. Quantitative profiling of hundreds of nuclei revealed scaffold-dependent remodeling of nuclear morphology that was not apparent by conventional microscopy. Cells cultured on placental dECM exhibited reduced nuclear area and increased nuclear eccentricity, while cardiac fibroblasts and osteoblasts showed alterations in nuclear circularity. These lineage-dependent morphological responses demonstrate that placental dECM provides more than a permissive substrate for cell attachment and is associated with measurable changes in cellular architecture following processing. Together, these findings support the biological relevance of processed placental dECM as a regenerative biomaterial and demonstrate the utility of quantitative single-cell morphometric analysis for detecting cell-material interactions that may not be apparent through qualitative imaging alone, guiding the rational design of regenerative therapies.

Alterations in the female urinary microbiome have been linked to several several common urinary conditions that increase in prevalence after menopause. Here we use sequencing and metabolomic data to compare the urinary bladder microenvironments between women who are premenopausal, postmenopausal, and postmenopausal using vaginal estrogen. While the urinary microbiome differs between pre- and postmenopausal women, at least 6 weeks of vaginal estrogen is associated with higher urinary lactobacilli and fewer opportunistic pathogens in the urinary postmenopausal microbiome, which closer approximates what is seen in the premenopausal context. Changes in urinary Lactobacillus species are correlated with vaginal pH and the same Lactobacillus species found in the vaginal microbiome tend to also be identified within the urinary bladder microbiome. Urinary metabolites differ by context (e.g., pre-, post-, and post-menopausal using vaginal estrogen), but the majority of metabolites that drive these differences are unannotated. Of the metabolites with shared associations among different Lactobacillus species, L. iners have consistently inverse relationships compared to L. crispatus, L. gasseri, or L. jensenii.