Matrix Biology Plus

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 African spiny mouse (Acomys cahirinus) exhibits a unique capacity among mammals for scarless tissue regeneration, making it a compelling model for investigating the cellular mechanisms underlying regenerative healing. To determine how cellular heterogeneity and specific phenotypes influence fibroblast behavior, we established an immortalized Acomys fibroblast line along with a CRISPR/Cas9-mediated Col3A1 knockout variant and a DNA damage-induced senescent population. Compared with Mus musculus, NIH 3T3 fibroblasts, Acomys cells displayed distinct morphology, similar migration speeds, reduced directional persistence, and greater biophysical heterogeneity. While previous studies have linked regenerative wound healing to the elevated expression of collagen type III (Col3A1), CRISPR-mediated knockout of Col3A1 in Acomys fibroblasts yielded comparable biophysical profiles to wild-type cells in 2D culture. To examine additional contributors to the enhanced wound-like matrix environment, we established a senescence model in which Acomys fibroblasts exhibited elevated resistance to DNA-damaging agents, complete loss of proliferation, and altered single-cell morphology. In 3D collagen gel contraction assays, Col3A1 knockout attenuated matrix remodeling capacity, whereas the introduction of a small fraction of senescent cells enhanced gel contraction and remodeling dynamics, suggesting that senescent fibroblasts can modulate collective matrix behaviors. Together, these findings demonstrate that both Col3A1 expression and senescence-associated cell states contribute to fibroblast-driven matrix remodeling, highlighting Acomys fibroblasts as a valuable model for investigating how cellular heterogeneity and senescence-associated cell phenotypes could influence regenerative wound healing.

Structural AbstractO_ST_ABSBackgroundC_ST_ABSTriple-negative breast cancer (TNBC) represents a major clinical challenge due to its aggressiveness, heterogeneity and limited availability of effective targeted therapy. We investigated whether LRP-1, a multifunctional cell-surface endocytic and signaling receptor, contributes to TNBC progression. MethodsUsing CRISPR-Cas9, LRP-1-deficient murine 4T1 and human HS578-T TNBC cells were used. Functional consequences were assessed through migration, invasion, and 3D spheroid assays, imaging of focal adhesions and actin organization, atomic force microscopy, and plasmin activity assays. Global molecular reprogramming was analyzed by label-free quantitative proteomics and secretomics. LRP-1-deficient or proficient 4T1 cells were implanted orthotopically in immunocompetent mice; tumor progression was monitored longitudinally while peritumoral collagen architecture and immune microenvironment composition were characterized by second harmonic generation imaging and immunohistochemistry. ResultsWe show that LRP-1 loss reduces TNBC aggressiveness, as reflected by decreased migration and invasive capacity, reduced spheroid evasion, and significant morphological changes in focal adhesion and actin structure. LRP-1-deficient cells became stiffer and showed lower LOXL-4 levels, while pericellular proteolytic activity remained unchanged, suggesting other proteases mechanism. Multi-omic analysis revealed alterations in extracellular matrix (ECM), epithelial-mesenchymal transition, and inflammatory pathways. In vivo, LRP-1-deficiency reduced tumor progression and peritumoral collagen deposition, while increasing CD8+ T and Natural Killer cell infiltration, together with a cytokine profiling compatible with a more immune-permissive microenvironment. ConclusionsLRP-1 act as a key contributor in TNBC progression through matrix remodeling, mechano-adaptation, and immune exclusion. Positioning it as a candidate biomarker for TNBC patients who are likely to benefit from stroma-targeting therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/732906v2_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@1b595c2org.highwire.dtl.DTLVardef@7b208aorg.highwire.dtl.DTLVardef@1956e54org.highwire.dtl.DTLVardef@17e55d0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: Single cell RNA sequencing provides a wealth of information to explore the complexities of the tumour microenvironment, but crucially the spatial topology of the tumour is lost and studying cellular interactions is limited. Spatial transcriptomics aims to address this however the technique remains cost prohibitive for the generation of data from meaningfully-sized clinical cohorts. In contrast, spatial proteomic profiling with multiplex immunofluorescence, preserves spatial interactions, is relatively cost accessible, and is scalable for large clinical cohorts to address powerful translational questions. Whilst multiplex approaches have advanced in recent years, we note that cancer-associated fibroblasts (CAFs) have been explored in less detail, potentially due to difficulties associated with CAF heterogeneity and the diversity of markers used to define them. Methods: We designed, optimised, and validated a multiplex immunofluorescence panel that combines four frequently used CAF markers; alpha smooth muscle actin (aSMA), fibroblast activation protein (FAP), podoplanin (PDPN) and platelet-derived growth factor receptor alpha (PDGFRa) with CD8 and pan-cytokeratin. Here we share our methodology and the practical considerations taken to inform the final panel design. We also highlight the benefits of robust optimisation experiments.

Introduction Stark disparities in endometrial cancer (EC) risk and mortality exist between non-Hispanic Black and White women, with Black women experiencing higher incidence and worse survival. This disparity has been attributed to biological and socioeconomic factors, though how these factors interact to influence EC disparities remains unclear. This study modeled EC outcomes using race, area-level socioeconomic deprivation, clinical phenotypes, genetic ancestry, and molecular alterations. Methods We identified 281 cases of EC diagnosed from 2013-2023 in women who underwent clinical genomic sequencing as part of routine care across multiple Northwell Health sites. We estimated genetic ancestry, oncogenic alterations in 324 genes, microsatellite instability, and molecular classification. Geocoded patient addresses were used to derive the state-level Area Deprivation Index to estimate socioeconomic deprivation. Results African ancestry patients were enriched for high-grade disease (89% vs 64%), serous histology (57% vs 26%), and the TP53-mutant molecular classification (71% vs 51%) compared to European ancestry patients (p-value<0.05). Socioeconomic deprivation quintiles were associated with race, with more deprived quintiles enriched for Black patients (p-value<0.001). Both race and genetic ancestry, but not area-level deprivation, were independently associated with differences in progression-free survival. TP53 mutations were enriched in African ancestry patients, while KRAS, PTEN, and ARID1A mutations were enriched in European ancestry patients (q<0.10). Cox proportional hazards modeling, adjusting for these factors, showed that African ancestry patients had worse progression-free survival (HR 1.91, p-value<0.05). Conclusion Our findings indicate that EC disparities persist after adjusting for socioeconomic, clinical, and molecular factors, highlighting the need to further investigate additional drivers of disparity.

Spatial proteomic imaging technologies enable the simultaneous assessment of immune cell abundance and spatial organization within the tumor microenvironment. Spatial clustering is commonly summarized using measures such as Ripleys K or nearest-neighbor G-functions at a fixed radius. However, these approaches depend on scale selection and may obscure biologically relevant patterns occurring across spatial ranges. We propose a functional data analysis (FDA) framework to model spatial clustering trajectories derived across a continuum of radii. Functional principal component analysis (FPCA) was used to summarize dominant modes of spatial variation, and resulting scores were incorporated into Cox proportional hazards models as both main effects and interaction with immune cell abundance. The approach was applied to multiplex immunofluorescence data from five ovarian cancer studies, comprising 773 high-grade ovarian serous tumors. Analyses focused on CD3+ and CD8+ T cell populations within the tumor compartment of the tissue, adjusting for age at diagnosis and cancer stage, with study-specific estimates combined using random-effects meta-analysis. Higher abundance of both T cells and CD8+ T cells was consistently associated with improved overall survival. Beyond abundance, spatial features captured by the leading functional principal component were independently associated with survival, particularly for CD8+ T cells. Interaction models further showed that the prognostic effect of immune infiltration depended on spatial clustering, with tumors characterized by high abundance and low spatial clustering exhibiting the most favorable outcomes. These findings indicate that spatial organization provides complementary prognostic information beyond abundance alone and suggests that more diffuse immune infiltration may reflect more effective anti-tumor activity in ovarian cancer. Overall, FDA offers a flexible and interpretable framework for modeling spatial clustering across scales and identifying prognostic spatial features not captured by fixed-radius or distance analyses.

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.

Background: Human induced pluripotent stem cells (hiPSCs) hold promise for vascular regeneration, but preliminary research often relies on neonatal donors, whereas clinical applications will use cells derived from aged individuals. Although the impact of donor age on reprogramming efficiency has been studied, its effect on the functionality of hiPSC-derived endothelial progenitors (hiPSC-EPs) remains unclear. This question is the focus of the current study. Methods and Results: We derived EPs from iPSCs sourced from three neonatal donors (ND) and three mature donors (MD) matched 1:1 for sex and somatic cell origin. We assessed their functional, epigenetic, and transcriptomic characteristics. Despite higher CD34? yields from MD-iPSCs, MD-hiPSC-EPs formed poorly interconnected and non-lumenized vascular structures in 3D hydrogels, compared to neonatal donor (ND) lines. In 2D culture, MD-hiPSC-EPs exhibited reduced cell density and aberrant VE-Cadherin localization. DNA methylation analysis revealed that somatic cell origin was the dominant driver of variance, but consistent differences in methylation of mesoderm commitment, angiogenesis, ECM remodeling, and cytoskeleton-related genes were observed between age groups. Epigenetic age prediction showed MD-hiPSC-EPs had more developmentally advanced signatures, potentially explaining their shift away from vasculogenic competence. Our RNA-sequencing findings confirm trends seen in the DNA methylation data and show differential expression of pathways linked to mitochondrial regulation and nitric oxide signaling. Conclusions: Donor age significantly alters the vasculogenic function of hiPSC-EPs. These findings underscore the necessity of donor-specific considerations in hiPSC-based vascular engineering and highlight potential barriers to translating hiPSC-derived therapeutics into aged patient populations.

Muscle satellite cells (SCs) regenerate skeletal muscle, but their regenerative capacity declines with age, in part due to extracellular matrix (ECM) remodeling and aberrant fibroblast activation within the SC niche. In regenerating young mouse muscle, fibronectin remodeling is transient, whereas in aged mouse muscle, fibronectin remodeling is prolonged and disorganized. Fibroblasts in aged mice are activated, increasing fibronectin deposition and expressing elevated -smooth muscle actin (SMA), which negatively influence SC fate. We develop a viscoelastic hydrogel co-encapsulation system, enabling three-dimensional co-culture of intact myofibers with primary fibroblasts. Using this 3D co-culture system, we show that fibroblasts from young mice support SC quiescence and self-renewal, whereas fibroblasts from aged mice aberrantly activate SCs and promote their differentiation on myofibers isolated from either young or aged mice. Knocking down fibronectin (Fn1) in fibroblasts from aged mice partially restores SC function, promoting quiescence and limiting differentiation. Using a novel 3D hydrogel co-culture system, we demonstrate that fibroblast-deposited fibronectin is a key age-associated regulator negatively affecting SC fate within the SC niche of aged mice.

Evolutionary success in mammals requires coordinated regulation of cognitive functions and reproductive capacity. Such coordination must involve shared genes and molecular pathways between the brain and germ cells, yet direct evidence linking cognition to reproduction across species remains limited. Here, proteomic and transcriptomic analyses were performed experimentally in Ovis aries and Rattus norvegicus, while transcriptomic datasets from Mus musculus, Macaca mulatta, and Homo sapiens were analyzed in silico. We identified 8,464 protein-coding genes shared between the brain and sperm/testis and conserved across five species. In rats, 8,444 of these genes were also shared between the brain and the ovary. Functional annotation classified 3,890 genes as associated with both neurological and reproductive functions, and 1,752 as uncharacterized in these contexts, highlighting candidates for future studies on reproductive and neurological disorders. These findings reveal a deeply conserved genetic network linking neurological and reproductive systems, underscoring the evolutionary interplay that supports mammalian fitness.

Cellular senescence is characterized by stable cell-cycle arrest, cytoskeletal remodeling, and altered secretion of senescence-associated secretory phenotype (SASP) factors, including SERPINE1/plasminogen activator inhibitor-1 (PAI-1). Although extracellular matrix (ECM) stiffening has been linked to fibroblast mechanotransduction and SERPINE1-associated remodeling, the molecular pathway connecting substrate stiffness to SERPINE1 regulation in senescent fibroblasts remains incompletely understood. Here, we investigated how defined substrate stiffness affects fibroblast morphology, mechanical phenotype, and SERPINE1 expression, and examined whether the clathrin adaptor AP2A1 participates in this response in replicative senescent human fibroblasts. Using tunable polyacrylamide hydrogels, we found that increasing substrate stiffness enhanced fibroblast spreading, stress fiber thickening, focal adhesion maturation, cellular stiffness, and senescence-associated marker expression. Stiff substrates also increased SERPINE1 expression and its colocalization with actin fibers, with stronger responses observed in senescent than in young fibroblasts. Functional perturbation experiments further suggested that SERPINE1 contributes to stress fiber organization in senescent cells. In addition, AP2A1 colocalized with SERPINE1, and modulation of AP2A1 under knockdown and overexpression conditions altered SERPINE1 signal intensity. Conversely, perturbation of SERPINE1 also affected AP2A1, supporting a potential bidirectional relationship between these two components. Together, these findings identify SERPINE1 as a stiffness-responsive factor associated with senescence-linked cytoskeletal remodeling and support a functional relationship between AP2A1 and SERPINE1 in senescent fibroblasts. These results suggest that the AP2A1-SERPINE1 axis may contribute to the link between extracellular mechanical cues and senescence-associated fibroblast remodeling.

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

Background: Uncontrolled bleeding complicates trauma, surgery and many medical conditions. While currently available procoagulant therapies (e.g., plasma-derived factors, recombinant proteins, antifibrinolytics) have crucial limitations. Methods: N389 (CQQTVTLLPAADLDDFSC) was synthesized by Fmoc solid-phase chemistry, characterized by HPLC and LC-MS, then tested in normal human pooled plasma in microplate mechanical clot-formation assays using incubated and immediate addition formats. Kinetic parameters (plasma recalcification, PRT; maximum absorbance, Amax) were obtained from absorbance curves fit to four-parameter logistic models. Mixing studies with modified (i.e., aged, adsorbed) plasma probed factor dependence. Results: In plasma coagulation assays activated with 25 mM CaCl2, baseline clotting showed a PRT of 23.74 +/- 0.27 min and Amax of 0.1813 +/- 0.0043 (n = 3), whereas N389 significantly reduced PRT to 8.442 +/- 6.0395 min without incubation (p = 0.0012), further decreased PRT after incubation (p < 0.0001), increased Amax to 0.2523, and retained comparable activity across normal, adsorbed, and aged plasma, in contrast to S1255 which showed a faster but incubation-labile effect with PRT 2.353 +/- 1.3685 min (p = 0.0007) and marked attenuation in factor-depleted and aged plasma. Mixing studies showed N389 activity persisted across normal, aged and adsorbed plasma, consistent with a mechanism that does not require intact plasma coagulation factor profiles (specifically factors II, V, VIII, VII, IX, X). Discussion: Collectively with prior evidence on anionic surfaces, Ca2+-binding Gla domains, and peptide-modulated fibrin polymerization, these results support a model in which N389 functions as a stable, charge-based scaffold that coordinates divalent cations and/or directly nucleates fibrin(ogen), while highlighting limitations of bulk clotting assays and the need for targeted thrombin generation, binding, aggregation, and contact-activation studies. Conclusions: The aspartate-rich peptide N389 is a sustained, factor-independent procoagulant at least in vitro. N389 thus merits further mechanistic and translational evaluation as a synthetic hemostatic agent.

BackgroundThe development of clinically viable tissue-engineered heart valves (TEHVs) remains limited by inconsistent host cell infiltration. The dynamic hemodynamic environment may play a central role in driving or inhibiting cell invasion, yet the effects of cyclic stretch on cell migration and proliferation remain largely unexplored in 3D tissues and scaffolds. Given evidence that uniaxial constraint promotes directional invasion in 3D matrices, we hypothesized that uniaxial cyclic stretch would enhance cell invasion, particularly along the stretch direction. MethodsWe embedded multicellular spheroids into collagen hydrogels and subjected them to uniaxial cyclic stretch (3-10%, 1 Hz) for two days and quantified invasion into the surrounding extracellular matrix using a custom image-processing program. Smooth muscle cells, valvular interstitial cells, and dermal fibroblasts were examined to represent cell populations relevant to TEHVs and for comparison across cell types with different contractility. To determine the mechanisms underlying changes in invasion with stretch, effects of cell tension were evaluated using gel compaction assays and inhibition of myosin IIA, and proliferation was assessed by Ki67 immunostaining. ResultsContrary to our hypothesis, cyclic stretch profoundly inhibited cell invasion into the matrix across all cell types and magnitudes of stretch. Invasion decreased by >50% in smooth muscle cells and fibroblasts and by up to 99% in valvular interstitial cells. Invasion suppression was inversely correlated with cell contractility, implicating a role for cell-generated tension. Inhibition of myosin IIA partially rescued invasion with stretch, though not to static levels. Stretched spheroids also exhibited reduced cell proliferation relative to static controls. ConclusionsThese findings implicate actomyosin-mediated mechanotransduction in stretch-induced suppression of cell invasion and suggest that the dynamic valve environment may limit host-cell repopulation of TEHVs. More broadly, this work provides insight into how cyclic stretch regulates 3D cell invasion in mechanically active tissues with implications for wound healing and cancer metastasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/732094v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@2a21b1org.highwire.dtl.DTLVardef@9fbf6org.highwire.dtl.DTLVardef@17ceb17org.highwire.dtl.DTLVardef@2e3bf9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Generating bioengineered cartilage that recapitulates the depth-dependent phenotype, structure, and function of native articular cartilage remains a challenge. While cartilage is rich in aggrecan and type II collagen, proper function depends on depth-dependent protein expression. Superficial zone chondrocytes (SZCs) secrete proteoglycan-4 (PRG4) to lubricate the cartilage surface. Deep zone chondrocytes produce type X collagen (COLX) to support compressive loading and load transfer to subchondral bone. We previously demonstrated that passaged full-thickness chondrocytes (FTCs) and zonal chondrocytes can re-express cartilage and zone-specific markers following scaffold-free three-dimensional (3D) culture in redifferentiation media. However, in the absence of an instructive matrix, cells expressed low levels of zone-specific proteins and exhibited limited depth-dependent organization. We hypothesize that synthetic extracellular matrix with zone-specific microenvironmental cues will guide zonal differentiation. To this end, passaged primary bovine chondrocytes were encapsulated in a soft, hyaluronan (HA)-based, cell-adhesive, and protease-degradable hydrogel established via bioorthogonal tetrazine (Tz) ligation with norbornene (Nb). When supplemented with TGF{beta}3, FTCs deposited aggrecan and type II collagen with minimal type I collagen. Application of interfacial tetrazine ligation with trans-cyclooctene (TCO) during cell culture resulted in matrix stiffening, leading to upregulation of COLX expression. Conversely, SZCs cultured in soft hydrogels exhibited the greatest PRG4 expression. Establishment of a trilayered construct with region-specific stiffness via the diffusion-controlled reaction promoted PRG4 and COLX expression in defined zones. Together, these findings demonstrate that tunable HA-based hydrogels can enhance zone-specific chondrocyte phenotypes and promote the formation of zonally organized cartilage.

BackgroundOsteoarthritis cartilage contains heterogeneous chondrocyte states, but molecular programs linked to state transitions within human cartilage remain incompletely resolved using public single-cell data. MethodsA retrospective reanalysis was conducted of a public human knee cartilage single-cell RNA sequencing dataset (GSE255460) including 8 osteoarthritis donors and 3 non-osteoarthritis donors (19 samples). Cells underwent sample-wise quality control and doublet removal, followed by batch-corrected clustering, chondrocyte subclustering with marker-based annotation, and trajectory inference using Slingshot. Regulatory chondrocytes were prioritized for osteoarthritis versus control differential expression, with downstream Gene Ontology/KEGG enrichment (Benjamini-Hochberg false discovery rate <0.05) and protein-protein interaction network hub screening. ResultsAfter quality control, 27,036 cells were retained. Chondrocytes formed multiple transcriptional states with branching-like continuous relationships, and regulatory chondrocytes localized near the main manifold and adjacent to multiple inferred branches, consistent with a transition-adjacent state. In regulatory chondrocytes, osteoarthritis versus control differential expression was enriched for collagen-containing extracellular matrix and extracellular matrix organization, endoplasmic reticulum lumen-associated secretory/proteostasis processes, cell-matrix adhesion (including focal adhesion), and transforming growth factor beta/SMAD-related signaling. Protein-protein interaction analysis of regulatory-chondrocyte differential genes identified five high-connectivity hub genes: COL5A1, COL5A2, COL6A1, COL1A2, and COL3A1. ConclusionThis public-dataset reanalysis supports a transition-adjacent regulatory chondrocyte program in osteoarthritis characterized by coordinated extracellular matrix remodeling with concurrent secretory/proteostasis and adhesion-transforming growth factor beta signatures, nominating collagen-network hubs as candidates for downstream validation.

AimsPlasminogen activator inhibitor-1 (PAI-1) regulates plasmin-mediated proteolysis, thereby influencing vascular stability and tissue remodeling. Angiotensin II (AngII) induces an increase in PAI-1 during the development of ascending thoracic aortic aneurysm (ATAA). The initial purpose of this study was to determine whether deletion of PAI-1 influenced development of ATAA. Subsequently, this study aimed to define the early pathological events preceding cardiac fibrosis in PAI-1 deficiency and the structural domain responsible for its protective effect. Methods and resultsAngII was infused for 4 weeks in whole-body PAI-1 deficient (PAI-1-/-) mice and their wild-type littermates (PAI-1+/+) to examine the role of PAI-1 in ATAA. PAI-1 deficiency did not alter AngII-induced aortopathy but revealed a striking cardiac phenotype characterized by replacement fibrosis predominantly within the epicardium and posterior septum. Ferric iron, indicative of prior hemorrhage, was coincident with fibrosis. Similar phenotypes were observed in PAI-1-/- mice infused with norepinephrine for 4 weeks. To define the pathological events preceding cardiac fibrosis, either AngII or norepinephrine was infused for 1 week in PAI-1+/+ or -/- mice. Both infusions induced extensive epicardial hemorrhage and posterior septal fibrosis in PAI-1-/- mice. To explore the initiation of cardiac pathology, AngII was infused for approximately 1 day. PAI-1-/- mice developed diffuse hemorrhage and cardiomyocyte injury localized to the posterior septum, pathologic changes that preceded overt fibrosis. Finally, to determine the protective domain of PAI-1, saline or AngII was administered to mice harboring loss-of-function point mutations in the protease inhibitory (PAI-1Ala/Ala) or somatomedin B-binding domains (PAI-1AK/AK). Compared to saline infusion, 1 week of AngII induced hemorrhage and heterogeneous fibrosis in PAI-1Ala/Ala, but not PAI-1AK/AK mice. ConclusionsThese findings support that, under hemodynamic stress, PAI-1 deficiency promotes early cardiac hemorrhage and cardiomyocyte injury that lead to fibrosis. Mutational studies implicate dysregulated plasmin generation as an initiator of cardiac injury and fibrosis. TRANSLATIONAL PERSPECTIVECardiac fibrosis has been reported in a human population with PAI-1 deficiency and currently lacks targeted therapy. Our findings demonstrate that in animal models, PAI-1 deficiency confers susceptibility to cardiac injury in response to hemodynamic stress, which may accelerate fibrotic remodeling. Mutational disruption of the protease-inhibitory domain of PAI-1 induced similar pathology, supporting a protective role for this function. These observations suggest that interventions aimed at controlling hypertension, promoting endothelial integrity, or regulating plasmin activation could reduce fibrotic remodeling in this population.

The neuromuscular junction (NMJ) is a specialised synapse between motor neurons and skeletal muscle, and its progressive deterioration contributes to age-related and metabolic disease-associated declines in muscle function. Advanced glycation end-products (AGEs) accumulate in tissues during ageing, diabetes, and chronic metabolic dysfunction and have been implicated in neuromuscular degeneration, yet their effects on the intact NMJ have not previously been examined in a human model system. This study employed a fully human, serum-free, and neural growth factor-free NMJ co-culture system, combining neural progenitor cells with immortalised human myoblasts derived from an 83-year-old donor, to investigate the effects of AGE exposure on neuromuscular integrity across structural, metabolic, functional, and secretory outcomes. AGE exposure induced significant reductions in motor neuron axonal length, myotube remodelling with centralised nuclear positioning, mitochondrial membrane depolarisation, elevated mitochondrial superoxide production, mitochondrial uncoupling, and reductions in spontaneous contraction intensity and frequency. Neurotrophic and myogenic growth factor signalling was significantly downregulated in AGE-treated co-cultures. These findings identify the NMJ as a sensitive target of glycation stress and establish this fully human co-culture platform as a physiologically relevant model for investigating glycation-related neuromuscular pathology and evaluating candidate therapeutic interventions.

Fibroproliferative diseases such as systemic sclerosis are complex conditions characterized by chronic skin inflammation and progressive fibrosis, with fibroblast activation as a central feature. While Transforming Growth Factor Beta (TGF-{beta}) signaling is a well-established driver of fibrosis in SSc, inflammatory pathways such as Nuclear Factor Kappa B (NF-{kappa}B) also contribute substantially to disease morbidity. We previously identified CD109 as a TGF-{beta} co-receptor and negative regulator of fibrotic signaling; however, its role in inflammatory signaling remains unknown. Here, we investigate the function of CD109 in regulating inflammatory signaling in skin fibroblasts. We show that, CD109 co-localizes and associates with Toll-like receptors (TLR2, TLR4) and tumor necrosis factor receptors (TNFRI, TNFRII), and that loss of CD109 enhances TNF--induced NF-{kappa}B activation and reprograms cytokine production in human dermal fibroblasts. Furthermore, both global and fibroblast-specific CD109 knockout mice exhibit increased immune cell infiltration and skin inflammation. In parallel, single-cell transcriptomic analyses across a pan-disease fibroblast atlas show that CD109 expression is preferentially maintained in structural and homeostatic fibroblast subtypes, whereas immune-interacting fibroblast subsets consistently display decreased CD109 levels. Pathway-level analyses of fibroblast pseudobulk samples reveal altered activity of canonical inflammatory pathways in SSc compared to healthy skin. Together, these findings identify CD109 as a fibroblast-intrinsic negative regulator of inflammatory signaling and suggest a broader role for CD109 in modulating inflammatory responses in systemic sclerosis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/736423v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@be9e08org.highwire.dtl.DTLVardef@794173org.highwire.dtl.DTLVardef@b81eb5org.highwire.dtl.DTLVardef@1e811f5_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract: CD109 Restrains Fibroblast-Driven Inflammation by Modulating NF-{kappa}B Signaling. Generated using FigureLabs.ai and edited using Adobe Photoshop. C_FIG

An autocrine pro-adhesive/pro-contractile signaling loop, through the mechanosensitive transcriptional cofactor YAP, promotes fibrosis. The CCN family of matricellular proteins modify adhesive signaling. Of these, CCN3 is antifibrotic. We show that BLR-200, a CCN3-derived peptide, has anti-fibrotic properties in the bleomycin-induced model of scleroderma skin fibrosis. In vitro, BLR-200 delayed, but did not abolish, fibroblast adhesion to collagen and nuclear YAP localization. In vivo, BLR-200 prevented/treated bleomycin-induced skin fibrosis, and reduced bleomycin-induced expression of profibrotic genes including alpha-smooth muscle actin, CCN1 and CCN2. Lineage tracing and scRNA-seq analyses revealed that the myofibroblasts in this model were quantitatively derived from collagen-lineage Pi16+/Col15+ve fibroblasts. BLR-200 prevented myofibroblast differentiation in this model and trajectory of fibroblasts toward a Sfrp2-positive subset, a cell type associated with poor clinical outcome. BLR-200 impairs YAP activation in vitro and appearance of translationally-relevant fibroblast subtypes in vivo and is a novel anti-fibrotic agent for SSc skin fibrosis.