Matrix Biology

Collagen biosynthesis within the extracellular matrix (ECM) relies on finely regulated enzymatic steps to ensure proper collagen maturation and fibrillar assembly. Among these, bone morphogenetic protein-1 (BMP1) and the canonical lysyl oxidase (LOX) act on the collagen telopeptide to promote procollagen processing and oxidative cross-linking, respectively. However, the mechanisms that ensure precise coordination of their activities remain poorly understood. Using NanoBiT assays, we identified and characterized a stable LOX/BMP1 protein complex that assembles intracellularly during trafficking through the ER/Golgi pathway and persists after secretion. Analysis of BMP1 and LOX domains involved in the interaction showed that BMP1 binding requires its CUB2/3 domains, while LOX recognition depends on a conserved, positively charged segment of LOX (residues 259-285) located immediately upstream of its catalytic domain. Formation of the LOX/BMP1 complex did not substantially alter LOX enzymatic activity but markedly enhanced LOX association with collagen type I through the carboxy-telopeptide region, facilitating the assembly of a ternary LOX/BMP1/collagen complex. This pre-assembled complex promoted efficient targeting of LOX to nascent collagen fibrils. Our findings reveal a previously unrecognized layer of regulation in collagen biosynthesis, in which LOX and BMP1 act as a functional unit to ensure precise localization and proper processing of collagen. This mechanism offers new insights into ECM assembly and identifies the LOX/BMP1 interface as a potentially druggable node for anti-fibrotic strategies.

Anchoring fibrils formed by collagen VII play a critical role in stabilizing the dermal-epidermal junction. The N-terminal non-collagenous (NC1) domain of collagen VII binds firmly to basement membrane components including collagen IV and has also been reported to interact with mesenchymal fibrillar collagens via its von Willebrand factor A-like domain 2 (A2 domain). To elucidate how collagen VII recognizes fibrillar collagen, we performed yeast two-hybrid screening using a triple-helical random peptide library, which resulted in the identification of a Met-Gly-{Phi} ({Phi}; aromatic amino acid residue) motif. Biochemical analysis with synthetic triple-helical peptides revealed a binding preference of Trp > Phe as the {Phi} residue by the A2 domain despite Trp being absent in native collagens. The crystal structure of the A2 domain in complex with the Nle (Met surrogate)-Gly-Trp-containing peptide revealed a unique mechanism by which two distinct hydrophobic pockets of the A2 domain accommodate the Nle and Trp residues corresponding to the Met-Gly-{Phi} motif, engaging all three chains of the triple helix. Subsequent molecular dynamics simulations demonstrated that the A2 domain recognizes the corresponding native Met-Gly-Phe motif in a similar manner, but with lower affinity, implying a transient interaction with mesenchymal collagens. The findings obtained in this work suggest models in which transient A2-triple helix interaction promotes the recruitment of collagen I and III fibrils into the arc-shaped structure of anchoring fibrils. This also provides a foundation for linking structural understanding to skin fragility diseases caused by collagen VII dysfunction.

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

The myotendinous junction (MTJ) is a critical interface between muscle fibers and tendons, essential for force transmission between muscle and bone. Laminin-2, a key extracellular matrix (ECM) component, is strongly enriched at this interface. Mutations in the LAMA2 gene cause LAMA2-related muscular dystrophy (LAMA2 MD), an early-onset severe congenital muscular dystrophy. Here, we examined the MTJ in dyW/dyWmice, a mouse model for LAMA2 MD. We find a strong disruption of MTJ morphology, including altered muscle fiber tips, collagen XXII mislocalization, and reduced muscle tendon interface. As MTJ loading is altered in dyW/dyW mice and MTJ maintenance requires loading and unloading, we also examined MTJ structures upon denervation-induced unloading. While muscle fiber tip morphology resembled that of dyW/dyW mice, collagen XXII distribution was not affected and the muscle-tendon interface was preserved. Finally, proteomic profiling via laser capture microdissection and mass spectrometry revealed significant regional and global shifts in MTJ protein composition in dyW/dyW and denervated mice. Across both models, we identified integrin-associated remodeling as a shared response linked to the perturbed muscle fiber tip morphology. These findings demonstrate that laminin-2 is required for MTJ stability, and that mechanical unloading contributes to the observed phenotype. Importantly, our results suggest that disruptions in MTJ structure and protein composition may contribute to the pathology observed in LAMA2 MD.

Despite the growing prevalence of non-healing diabetic wounds, no current treatment options overcome multifactorial deficits in repair. To this end, a mesenchymal stromal cell-derived regenerative extracellular matrix (rECM) was evaluated for the ability to accelerate cutaneous wound repair in leptin receptor-deficient (db/db) diabetic mice with paired full-thickness dorsal skin defects. A single dose of rECM significantly accelerated wound closure compared with vehicle controls. Also, rECM dose-dependently improved overall histological healing scores and modulated granulation tissue dynamics, with the highest dose promoting rapid resolution of granulation tissue relative to wound area. Spatial transcriptomics and immunofluorescence revealed that rECM drove robust formation of de novo peripheral nerve clusters characterized by the Schwann cell marker, p75. The rECM also enhanced vascular maturation in healed wounds, increasing average blood vessel size, smooth muscle actin-positive vessels, and vessel density within myofibroblast-rich regions. In a complementary 3D angiogenic sprouting model, rECM accelerated endothelial invasion and filopodia extension, and at higher concentrations induced contraction of collagen matrices consistent with accelerated resolution of granulation tissue. These data demonstrate that rECM accelerates closure of diabetic skin defects by coordinating faster granulation tissue remodeling with enhanced peripheral nerve formation and vascular maturation.

Apical extracellular matrices (aECMs) act as barriers against pathogens and shape tissue organization throughout animals. In nematodes, the cuticle is the aECM performing this function, representing a defining feature of this largest animal phylum. Morphological and molecular evidence supports that worm cuticles extend from the head into the mouth, the latter contributing to the large diversity of nematode feeding structures. However, the biochemical understanding of the compositions of nematode cuticles and head structures is largely limited to collagens. Here, we characterized a recently identified mucin-type protein DPY-6 as a constituent of the cuticle and mouth in the model organisms Pristionchus pacificus and Caenorhabditis elegans. Utilizing bioinformatic tools, we discover a unique cysteine-consisting motif at the N-terminus of DPY-6 with the amino acid sequence CxCxCxC. We demonstrate through in vitro biophysical experiments that this cysteine motif facilitates the intermolecular dimerization of DPY-6 proteins. In vivo studies in both species reveal that this motif is involved in the proper localization of the protein in the cuticle, but functions synergistically with other protein domains in a species-specific manner. Given that dpy-6 transcriptomic expression precedes other cuticle components, we speculate that DPY-6 acts as scaffold molecule for nematode cuticular aECM formation.

BackgroundBone formation during development and repair is divergently modulated by osteoblast (OB)-derived vascular endothelial growth factor (VEGF) which drives the skeletal sexual dimorphism of the bone vasculature. While the extracellular matrix (ECM) provides both structural and instructive cues to developing vasculature, the contributions of the bone matrix to this skeletal vascular dimorphism in bone remains undefined at the cellular level. MethodsPrimary OBs were isolated from neonatal female and male C57BL/6 long bones and cultured under basal or osteogenic conditions. ECM composition was quantified by Raman spectroscopy. Primary murine bone marrow endothelial cells (BMECs) were seeded directly onto established OB layers and maintained in heterotypic cocultures to assess contact-mediated effects of OB ECM on BMEC survival and expansion. OB-conditioned media (CM) was used to evaluate soluble-factor contributions, with VEGF-A concentration quantified by ELISA. ResultsRaman spectroscopy, on individual OBs from monotypic cultures, revealed sexually dimorphic ECM signatures that were independent of cellular growth profiles. Female OB matrices were enriched with type I collagen-specific proline and hydroxyproline and octacalcium phosphate with enhanced collagen intra-strand stability consistent with a matrix-dominant signature. Male OB matrices exhibited relatively lower type I collagen content and higher carbonated apatite resulting in an elevated mineral-to-matrix ratio indicative of advanced mineral maturation. After 24-hours of heterotypic culture of BMECs with OBs, BMEC numbers were 1.39-fold higher when in contact with male OBs. CM treatment of BMECs did not recapitulate these effects despite higher VEGF-A release from male OBs. ConclusionsSex differences in OB ECM are linked to divergent, contact-dependent regulation of BMEC behaviour. These findings suggest that differences in matrix maturation stat contribute to the sex-specific regulation of the skeletal vascular niche. Elucidating the mechanisms that regulate sex-specific OB-ECM production may reveal new therapeutic targets for selectively modulating pathological skeletal angiogenesis in men and women. SummaryBone is a sexually dimorphic organ, with men and women differing in bone size, strength and risk of fracture. The skeletal vasculature is essential for bone growth and repair, with bone forming osteoblast (OB) cells influencing blood vessel development through the skeletal extracellular matrix (ECM). Although the interactions between OB and vascular cells are crucial for lifelong skeletal health, it is not yet known whether sex differences in bone structure between men and women arise from differences in OB activity, or whether this divergence is driven by sex differences in blood vessel growth. Here, we show that male and female mouse OBs deposit distinct ECMs that differentially influence vascular endothelial cell behaviour. Female OBs produce a collagen-rich matrix with low mineral content. In contrast, male OB matrices contain less collagen and more mineral while releasing elevated levels of blood vessel promoting VEGF-A than females. When placed directly onto these OBs, vascular cell growth was greater when in contact with male than female OBs. Notably, this sex-dependent effect requires direct contact between both cell types and was not reproduced by exposure to OB-derived substances alone. These findings identify a cellular mechanism by which sex differences in OB matrix composition influences vascular cell behaviour in bone. Understanding how OB-vascular interactions differ by sex may help explain variation in bone health, healing capacity and disease risk between men and women. Further, our approach may support the discovery of new therapeutic targets that support bone growth and repair while targeting abnormal blood vessel growth in a sex-specific manner. HighlightsO_LIPrimary OBs from male and female C57BL/6 mouse long bones synthesise compositionally distinct ECMs. C_LIO_LIFemale OB matrices are type I collagen-rich and enriched with octacalcium phosphate, whereas male OB matrices contain less type I collagen and higher levels of carbonated apatite. C_LIO_LIBone marrow-derived endothelial cell (BMEC) growth is enhanced in heterotypic cocultures with male, but not female, OBs after 24 hours. C_LIO_LIMale OBs release higher levels of the pro-angiogenic factor VEGF-A than female OBs. C_LIO_LIThe sex-specific effects of the OB ECM on BMECs is contact-dependent and are not reproduced by treatment with OB-derived conditioned media. C_LI

The Hippo pathway effectors YAP and TAZ are key regulators of cell proliferation, apoptosis, and differentiation, thereby maintaining tissue homeostasis and controlling organ size. While their roles in epithelial tissues and cancer are well established, their role in dermal fibroblast extracellular matrix (ECM) regulation is less understood. Here, we investigated the role of Yap/Taz during postnatal skin dermis development. During postnatal growth, mouse skin steadily grows and undergoes significant surface expansion. Postnatal deletion of Yap/Taz in dermal fibroblasts, the primary cells responsible for dermal ECM homeostasis, significantly impaired dermal maturation, as evidenced by marked deficiencies in collagen synthesis and deposition. Isolated fibroblasts from Yap/Taz knockout mice showed reduced proliferation and diminished expression of Yap/Taz target genes (Ccn2, Col1a1), which were rescued by reintroduction of active Yap/Taz. RNA-seq, and spatial transcriptomics and proteomics of Yap/Taz knockout skin revealed substantial downregulation of matrisome genes, including type I (Col1a1, Col1a2) and type III (Col1a3) collagens, which together constitute more than 90% of the skins collagen content. These findings demonstrate that Yap/Taz are essential for dermal ECM homeostasis, highlighting their therapeutic potential in skin regeneration, fibrosis, and aging-related ECM decline.

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

The extracellular matrix (ECM) is a complex scaffold of proteins that supports multicellular structures. Interactions between cells and the ECM via receptors, like integrins, govern cellular phenotypes (e.g., proliferation, adhesion), but also contribute to ECM assembly. Understanding how ECM-receptor interactions regulate matrix assembly is critical to uncover how alterations of the ECM cause or accompany congenital diseases, cancer, or fibrosis. SNED1 is a novel ECM protein with roles in development and metastasis. However, the mechanisms governing its assembly and signaling functions remain largely unknown. SNED1 contains two integrin-binding motifs, RGD and LDV, and we recently showed that its interaction with RGD-integrins mediates cell adhesion. Here, we investigated the role of SNED1/integrin interactions in SNED1 ECM assembly. While SNED1/integrin interactions were not necessary for its initial incorporation in the ECM, interaction with LDV-, but not RGD-, integrins, was required for ECM build-up and the patterning of SNED1 and the fibrillar proteins fibronectin and collagen I. Moreover, SNED1/LDV-integrin interaction promoted ECM alignment, cell alignment, and cell proliferation, processes essential to SNED1-driven neural crest cell migration during craniofacial development and breast cancer invasion. SUMMARY STATEMENTInteraction of SNED1 with LDV-binding integrins, but not RGD-binding integrins, mediates ECM remodeling and controls cytoskeletal rearrangement and cell proliferation.

BackgroundEffective skin wound healing is essential for restoring tissue integrity following injury. Repair proceeds through phases of hemostasis, inflammation, proliferation, and remodeling, but molecular mechanisms governing these stages remain poorly defined. Vascular niche cells (VNCs)-including endothelial cells, vascular smooth muscle cells (SMCs), and fibroblasts-are central regulators of healing, but the lack of longitudinal in vivo human data has limited identification of VNC-derived signals that distinguish effective repair from pathological healing such as ulcers. Thus, defining the regulation of VNCs in wound healing addresses a critical knowledge gap. MethodsWe developed a protocol for wound healing using dermal forearm punch biopsies to track longitudinal repair in healthy volunteers. Single-cell and spatial transcriptomics were performed to identify and validate signaling activities within VNCs. ResultsWe spatiotemporally defined the inflammation, proliferation, and remodeling phases of human skin wound healing with a focus on VNCs. Spatial analysis localized this activity for VNCs and immune cells within a heterogenous granulation zone that later led to re-epithelializion. Angiogenesis was dominated by Vegf, Egf and Hif1 signaling. Extracellular matrix (ECM) remodeling occurred through Collagen, Laminin, Thrombospondin, and Fibronectin. SMCs emerged as dominant drivers of injury-induced remodeling including basement membrane and interstitial ECM components compared to fibroblasts. This SMC-led program was further defined by robust induction of TIMP1, an inhibitor of matrix degradation, which localized to granulation tissue and correlated with re-epithelialization and wound resolution. Lastly, we compared remodeling factors between healing and non-healing human diabetic foot ulcers (DFUs). SMCs in non-healing DFUs had deficient expression for core remodeling factors, including TIMP1, indicating SMC activity is needed for effective healing. ConclusionWe identified an SMC-driven model of wound repair in which TIMP1-dependent activity underpins granulation zone formation. Failure of this program defined a mechanistic basis for impaired healing in ulcers, identifying SMCs and TIMP1 as therapeutic targets.

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

Aging impairs the regenerative capacity of bone and is associated with poor healing outcomes. The mechanical environment is of fundamental importance to the regenerative response in bone - yet the effect of aging on the mechano-responsive capacity of bone regeneration remains largely unresolved. To investigate age-dependent mechanobiological responses in bone regeneration, we established an experimental framework consisting of: (i) an established femur defect mouse model, (ii) the use of PolgAD257A/D257A (PolgA) mice - a mouse model of premature aging, and (iii) our recently established spatial transcriptomics-based "mechanomics" platform which permits gene expression to be analyzed as a function of the local in vivo mechanical environment. Aging impaired the regenerative response in PolgA mice, resulting in an increased occurrence of delayed and non-unions, delayed bone formation / resorption responses, impaired osteogenesis and delayed mineralization of new bone. Cyclic mechanical loading significantly enhanced the regenerative response in young PolgA mice inducing sustained bone formation, suppressing bone resorption, and enhancing mineralization, with the strongest effects observed in peripheral regions of the fracture site. In aged PolgA mice, the mechanosensitivity of the regenerative response was retained with an anabolic response localized to the defect center. Cyclic mechanical loading applied during the reparative and remodelling phases of fracture healing thus represents a potential translational strategy to harness the mechanosensitivity of aged bone.

The extracellular matrix (ECM) is a meshwork of proteins that orchestrates a broad range of cellular phenotypes, including proliferation, adhesion, migration, and differentiation. SNED1 is a newly characterized ECM glycoprotein that promotes cell adhesion and is essential for embryonic development. Its upregulation is also associated with breast cancer metastasis and poor prognosis for breast cancer patients. We recently showed that SNED1 assembles into fibrillar structures, but the mechanisms guiding its incorporation into the ECM scaffold remain unknown. Combining biochemical assays and confocal immunofluorescence imaging, we found that SNED1 assembly in the ECM occurs early in the process of ECM building and is concomitant and overlaps with the deposition of fibronectin and collagen I, two major ECM proteins. By knocking down fibronectin or destabilizing collagen I fibers, we further demonstrate that SNED1 requires the presence of these proteins for its assembly. Last, using biolayer interferometry, we identify collagen I as the first direct binding partner of SNED1. Altogether, our results lay the foundation for future studies aimed at determining the mechanisms by which SNED1 fibers contribute to SNED1 pathophysiological functions. SUMMARY STATEMENTThe novel protein SNED1 requires the presence of fibronectin and collagen I to assemble into fibrillar structures in the extracellular matrix scaffold.

Aberrant bone remodeling is a hallmark of osteoarthritis, the most common arthritis affecting over 27 million US adults. Subchondral bone sclerosis, one sign of aberrant bone remodeling observable by routine x-rays, occurs as the trabeculae thicken, leading to increased bone volume. Toll-like receptors, pattern-recognition receptors of the innate immune system, have been implicated in OA pathogenesis, with TLR ligands, receptors, and co-receptors shown to mediate the severity and progression of OA. We have previously shown that CD14-deficiency protects mice against post-traumatic OA, and specifically reduces subchondral sclerosis post-injury. We hypothesized that depletion of CD14 protects against TLR4-dependent inhibition of osteoclastogenesis and therefore increases OC density in the SCB after injury, mitigating aberrant bone deposition in a murine model of OA. To determine how cellular changes correlate with bone structure derangements post-DMM, we performed MicroCT, Tartrate-resistant acid phosphatase staining, and alkaline phosphatase staining. To establish mechanistic changes in cellular signaling, we isolated WT and CD14-deficient osteoclast precursors and subjected them to LPS, an osteoarthritis-relevant TLR ligand, during differentiation. CD14-deficient mice, as well as WT mice treated with an anti-CD14 monoclonal antibody, show protection from post-injury increases in both bone volume fraction and bone mineral density. CD14-deficient mice had an increased osteoclast presence in the SCB two weeks post-injury, potentially protecting them from increases in bone volume and density. In vitro, CD14-deficient OCPs differentiated faster than WT OCPs, due to reduced Type I Interferon (IFN-I) signaling. In the presence of an LPS challenge, CD14-deficient OCPs were protected against LPS and TLR4-mediated inhibition, likely due to decreased MyD88-dependent TLR4 signaling. This work opens up new potential pathways to therapeutically target aberrant bone remodeling in the setting of joint injury and PTOA. Lay SummaryOsteoarthritis is one of the leading causes of disability worldwide. One of the hallmarks is subchondral sclerosis, or thickening of the bone in and around the joint. In this work, we used a mouse model of osteoarthritis to show that decreasing inflammatory signaling, through removal of CD14, protects against subchondral sclerosis, due to an increased presence of osteoclasts, cells that combat bone thickening. Osteoclasts without CD14 differentiate faster than osteoclasts with CD14, due to decreased Type I Interferon, an inflammatory cytokine. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/705094v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@176bdd5org.highwire.dtl.DTLVardef@a914bborg.highwire.dtl.DTLVardef@902748org.highwire.dtl.DTLVardef@2f9b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pregnancy is a unique period regarding immune cell regulation. Within the placenta, maternal immune cells play a central role in immune surveillance and tissue remodeling. However, regulatory mechanisms of systemic immunity during pregnancy are less clear. Here, we show that neutrophil function is altered in pregnant mice (E13.5), indicated by increased slow rolling velocity and reduced adhesion. Mechanistically, PreImplantation factor (PIF), a 15 amino acid peptide which is produced by human and murine trophoblast cells of the placenta, is continuously secreted into the maternal circulation and plays a key role in modulating neutrophil function via blocking the voltage-gated potassium channel KV1.3. This resulted in impaired intracellular Ca2+ signaling and subsequently disturbance of neutrophil post-arrest modifications and a higher susceptibility to physiological shear forces in vivo and in vitro. Furthermore, PIF-mediated KV1.3 blockade impaired E-selectin-mediated release of S100A8/A9 and phagocytosis. Taken together, we have identified PIF as an important modulator of neutrophil function during pregnancy suggesting a critical role in regulating innate immune responses throughout gestation.

Exposure to stress in adulthood alters the manifestation of persistent pain, yet its role in chronic pain vulnerability remains unclear. Here, we report that sub-chronic restraint stress experienced two weeks before unilateral osteoarthritis (OA) induction via intra-articular mono-iodoacetate (MIA) promoted the emergence of a resilient-like phenotype in adult male mice. This was evidenced by decreased MIA-induced mechanical hypersensitivity, improved gait dynamics and lower anxiety-like behaviour. In contrast, stress exposed females exhibited augmented pain-associated symptoms. In males, sub-chronic stress mitigated several MIA-induced molecular changes, including reduced adult hippocampal neurogenesis, increased glucocorticoid receptor levels in the hypothalamic paraventricular nucleus (PVN) and elevated c-Fos expression in deep spinal laminae, periaqueductal gray (PAG) and PVN. RNA sequencing suggested that restraint stress in males primed the spinal cord for an exacerbated GABAergic response post-MIA and pre-empted some pro-nociceptive MIA-induced transcriptional changes. However, the combination of stress and injury disrupted longevity-associated programs and shifted neurons toward a stress-vulnerable state. These findings reveal that prior stress exposure can modulate the long-term pain experience in osteoarthritis, with sexually dimorphic outcomes. Importantly, these results challenge the notion of stress as inherently maladaptive and underscore its potential to foster resilience to chronic pain.

In chronic inflammatory diseases such as obesity, tissue and cellular homeostasis are disrupted by persistent low-grade inflammation, where one of the most prominent pro-inflammatory cytokines that correlates with age and body mass index (BMI) is IL-6. All members of the IL-6 family of cytokines signal through their obligate co-receptor gp130, in whichsignaling tyrosines on the intracellular portion of gp130 activate multiple downstream pathways such as the canonical STAT and MAPK pathways. However, non-canonical gp130 pathways such as SRC family of kinases (SFKs) signaling have emerged as drivers of cellular stress response. Our recently published mutant mouse model carrying a constitutive inactivation of gp130-Y814 (F814 mice), which impairs SFK activation, exhibited an enhanced resolution of inflammatory responses and improved regenerative outcomes in both acute skin wound healing and post-traumatic osteoarthritis models. The current study was designed to explore whether the gp130-Y814 mutation reduces systemic chronic inflammation and multimorbidity in a high-fat diet (HFD)-induced model and to interrogate the possible downstream cellular mechanisms that are affected. In response to HFD, F814 mice showed significantly reduced systemic and tissue-specific inflammatory responses and protection from obesity-induced bone loss and osteoarthritis compared to wild type (WT) mice. After extensive characterization, the role of gp130-Y814 in monocytes/macrophages appeared to be dispensable, but we discovered that the F814 mutation blunts gp130 receptor internalization, p38/MAPK activation and the release of matrix metalloproteinases (MMPs) in chondrocytes. Finally, we showed that F814 chondrocytes had markedly reduced activation of the SFK-dependent GTPase dynamin 2 (Dyn2) in response to catabolic IL-6 cytokines. Introduction of a dominant-negative Dyn2 mutant into WT chondrocytes phenocopied the effects of the F814 mutation, resulting in attenuated p38/MAPK signaling and reduced MMP activation following stimulation with IL-6 cytokines. This study demonstrates, for the first time, that the Y814 residue is directly implicated in Dyn2-mediated internalization of the gp130 receptor, thereby modulating downstream signaling and contributing to pathological outcomes in chronic inflammatory and degenerative diseases in a cell type-specific manner.

Fibrotic scarring is a common response to tissue injury. Repeated or severe insults can cause fibrosis, leading to excessive extracellular matrix deposition and a substantial clinical risk of organ dysfunction. Despite its high prevalence, few therapeutic options exist, and fibrotic diseases collectively represent a major global health burden. Fibrotic diseases affect virtually all organs, yet they have been explored mainly in isolation for each organ. As a result, proposed shared fibrotic mechanisms are often based on indirect comparisons between independent datasets rather than on a unified, systematic, cross-organ meta-analysis. To overcome this gap, we conducted a large-scale meta-analysis of single-cell transcriptomic data from healthy and fibrotic human tissues to identify both shared and organ-specific transcriptomic profiles. We constructed a single-cell fibrosis atlas of over five million cells from 20 studies, covering more than 25 disease etiologies affecting the heart, liver, kidney, and lung. Through systematic comparison of these datasets, we identified organ-specific as well as cross-organ fibrosis-associated gene expression profiles in major cell types and defined disease fibroblast subpopulations with excessive extracellular matrix production. These analyses revealed a conserved fibrotic response shared across tissues. Our analysis spans global comparisons of fibrosis-associated changes in cellular composition and predictive disease signatures to detailed examinations of individual genes, transcription factors, and intercellular communication patterns observed in fibrotic diseases across organs. We provide our cross-organ integration as a user-friendly open resource for investigating fibrotic diseases across organs. This resource will enable an accelerated discovery of disease mechanisms and faster development of broadly effective antifibrotic strategies in the future. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/709232v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@37e26borg.highwire.dtl.DTLVardef@f1ef53org.highwire.dtl.DTLVardef@1974d4corg.highwire.dtl.DTLVardef@53f5ea_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

Pathological cardiac remodeling is driven by the proliferation and differentiation of resident fibroblasts into active myofibroblasts and results in excessive extracellular matrix (ECM) deposition and tissue stiffening. Expression of the matricellular protein WISP1 has previously been shown to be increased with cardiac fibrosis and promote myofibroblast activity, but the mechanisms by which this occurs remain unknown. Primary cardiac fibroblasts were isolated from adult mouse hearts and treated with recombinant WISP1 or TGF{beta}1 both alone and in combination to determine the functional role of the matricellular protein WISP1 in driving cardiac myofibroblast activity. WISP1 significantly increased alpha-smooth muscle actin and collagen type I expression, total collagen secretion, collagen gel contractility, and wound healing equally in fibroblasts from both male and female mice. However, WISP1 alone failed to induce expression of periostin, a hallmark myofibroblast marker, suggesting the resulting WISP1-dependent cell phenotype is unique and/or acting through non-canonical pathways. Indeed, inhibition of P38 MAPK completely ablated the WISP1-dependent increase in SMA and collagen expression, while having little to no impact on TGF{beta}1-dependent expression of myofibroblast marker genes. We next employed a multi-omics approach to define the functional impact of WISP1 on fibroblast cell-state within the transcriptome, cytosolic, and secreted ECM proteome. RNA-seq results show that WISP1 broadly promotes the expression of proliferative and immune modulatory genes at the transcriptomic level, while having very little impact on traditional myofibroblast and ECM modifying gene expression programs. At the proteome level, WISP1 was again a much weaker mediator of traditional myofibroblast and ECM proteins. However, in agreement with RNA-seq data, we observed a strong WISP1-dependent enrichment for proliferation-associated proteins in the cytosolic proteome and inflammation-associated proteins in the ECM proteome. Interestingly, WISP1 also showed a context-dependent response with TGF{beta}1, suggesting a more complex and yet to be elucidated signaling interaction between these independent mediators of myofibroblast activity. In conclusion, our data suggests that WISP1 promotes a unique proliferative and immune-modulatory myofibroblast phenotype. HighlightsO_LIWISP1 is sufficient to drive myofibroblast SMA and collagen expression and ECM deposition C_LIO_LIWISP1 promotes canonical myofibroblast contractility and wound healing activity C_LIO_LIWISP1 mediates myofibroblast activity via a non-canonical, P38 MAPK-dependent signaling pathway C_LIO_LIMulti-omics analysis of WISP1-dependent RNA and protein expression show that WISP promotes a proliferative and immune modulatory myofibroblast phenotype C_LI