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.

Elastin is the extracellular matrix (ECM) protein responsible for the elastic recoil property of certain tissues including the skin, arteries, and lung. Elastic fibre assembly begins with the coacervation of soluble monomeric tropoelastin and is driven by interactions with other ECM components such as glycosaminoglycans (GAGs). Previous research shows that GAG interactions can promote tropoelastin coacervation but lack structural and mechanistic details of this interaction. In this study, we describe the key interactions between tropoelastin and heparin using NMR spectroscopy and coacervation experiments. We propose a mechanism in which substoichiometric GAGs can act as a nucleating scaffold, primarily through transient multivalent interactions with domain 36 of tropoelastin, reducing the energetic barrier for coacervation. Our results provide the first detailed molecular view of tropoelastin-GAG interactions and support a role for negatively charged GAGs in modulating tropoelastin coacervation and thus initiating elastic fibre assembly.

The gut-joint axis describes how impaired intestinal epithelial function and increased gut permeability allow luminal factors to enter circulation. This can drive inflammation in Rheumatoid Arthritis, a chronic condition affecting the joint with systemic features. What mechanisms contribute to disease persistence are, as yet, incompletely understood. In health, extensively Oglycosylated intestinal mucins are central to epithelial protection and immune homeostasis; however, whether mucin glycosylation is altered during arthritis has not been addressed. Here, we investigated whether arthritisassociated inflammation alters mucin Oglycosylation, potentially compromising intestinal barrier function. Using a collageninduced arthritis mouse model, we combined epithelial transcriptomics, mass spectrometry-based glycomics, and imaging approaches to profile intestinal glycosylation. We identified distinct glycan remodeling in the colon, characterized by reduced fucosylation, while the ileum remained largely unaffected. In vitro studies using 3D human epithelial cultures further demonstrated that inflammatory cues, particularly from TNFactivated stromal cells, are sufficient to reduce epithelial fucosylation. Together, these findings identify a stromal-inflammatory mechanism that disrupts mucin glycosylation during arthritis. Loss of colonic fucosylation emerges as a novel element of inflammatory arthritis, providing an additional mechanistic link between intestinal inflammation and fibroblast-dependent modulation of the tissue microenvironment.

Type II diabetes mellitus (T2DM) is one of the most prevalent diseases in the United States and is associated with diabetic foot ulcers (DFU) and their impaired, often chronic, wound healing. The T2DM mouse model with dysfunctional leptin receptor (db/db) has been used in basic and translational studies of wound healing due to its systemic phenotypes (hyperphagia, hypometabolism, obesity, T2DM) and its notable delayed skin wound healing. However, a characterization of the temporal cellular dynamics of the db/db wound healing model has not been performed, nor has the model been systematically compared to human DFUs. We performed the first comprehensive single-cell, multi-omic analysis of dermal cells in diabetic (db/db) compared to non-diabetic (ND) mice across three time points ranging from the inflammatory to the delayed proliferative and resolution phases of healing. Single-cell transcriptomics were uniquely linked to their corresponding cells surface protein expressions of cell-specific receptors, including immune cells (CD45) such as neutrophils (CD11b, Ly6G), monocytes/macrophages (CD11b, F4/80, CD11c, Ly6C) and T lymphocytes (CD3, CD4), and dermal cells such as endothelial cells (CD31) and fibroblasts (CD26, CD140a), and showed high concordance between protein cell markers and their gene expressions in major cell types. Differential multi-omic analyses characterized two neutrophil (Tnfaip3+Sod2+Ly6G+, Csf3r+Fos+Ly6G+), three monocyte/macrophage (F4/80highCD11bhigh, Ly6chighCD11bhigh, CD11chighCD11blow) and three fibroblast (Pi16+Dpp4+CD26high, Lrrc15+Tnc+CD140ahigh, Cilp+Mgp+CD26low) subtypes showing dysregulated dynamics across the time course of healing in db/db vs ND mice. Notably, NETotic Tnfaip3+Sod2+Ly6G+ neutrophils and phagocytic F4/80highCD11bhigh macrophage subtypes were drastically up-regulated in diabetic wounds. Differential cell-cell communication analyses revealed striking differences in crosstalk dynamics between fibroblast, macrophage and neutrophil subtypes in the early phase of healing, and ligand-receptor interactome analyses identified CD44 as the hub of dysregulated immune cell interactions in diabetic wounds, implicating cell adhesion, migration and inflammatory pathways, especially those mediated by ICAM1. Inhibition of CD44 using blocking antibodies in primary macrophages from db/db mice and via intradermal injections in db/db mice significantly normalized the early wound immune dysfunction, in part by inhibiting ICAM1 and reversing the excessive neutrophil influx into diabetic wounds. A new integrated dataset of single-cell human chronic wound studies revealed similar CD44-mediated immune cell dysfunctions in diabetic vs non-diabetic foot ulcers, pointing to CD44 as a promising therapeutic target for T2DM-associated chronic wounds.

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

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.

Osteoarthritis is increasingly recognised as a disease of failed integration across the whole joint unit; however, the mechanisms that co-ordinate tissue integrity from development through to adult homeostasis remain largely unresolved. The LIM-homeodomain transcription factor LMX1B is a key determinant of embryonic skeletal patterning, but how it functions to regulate skeletal integrity in the mature skeleton is unknown. Recently, LMX1B was identified as a key driver of osteoarthritis. Here we show that loss of lmx1ba in zebrafish causes premature and progressive severe osteoarthritic pathology in adult spines and jaws, despite largely normal early skeletal patterning, revealing a conserved and continuous requirement for lmx1ba in joint maintenance beyond development. At a cellular level, loss of lmx1ba decouples osteoblast and osteoclast-mediated remodelling leading to bone overgrowth, heterogeneity of bone properties causing increased incidence of spontaneous fractures, and progressive abnormalities in spine morphology. In parallel, we observe degeneration of the intervertebral disc and dysregulation of the proteome and glycosaminoglycans indicative of disrupted extracellular matrix and a breakdown of the coordinated regulation of hard and soft tissue interfaces, which at the organismal level leads to altered joint performance. Notably, degeneration is restricted to mobile joints, and is not observed in cranial sutures, demonstrating a selective requirement for lmx1ba in mechanically active tissues. These changes are consistent with a model of spatially disrupted matrix properties that, under cyclic loading, promote progressive tissue damage. Our findings support a model in which continued expression of LMX1B in adulthood is required to maintain joint structures throughout life.

The terminal differentiation of osteoblasts into osteocytes, the most abundant cell type in cortical bone, is critical for skeletal homeostasis. Osteocyte loss is a hallmark of bone aging and fragility, yet the mechanisms regulating osteocyte formation and survival are poorly understood. We show that inactivation of fibroblast growth factor receptor 1 (Fgfr1) in the mature osteoblast lineage results in extensive osteocyte death, identifying FGFR1 signaling as essential for osteocyte viability and bone integrity. Lineage tracing and analysis of endogenous and induced appositional bone formation revealed that newly embedded osteocytes fail to survive without FGFR1. These osteocytes exhibited ectopic expression of osteocalcin and podoplanin within sclerostin-positive, TUNEL-reactive lacunae, along with defective dendrite formation and disruption of the local lacunocanalicular network. RNA sequencing of cortical bone demonstrated reduced expression of extracellular matrix (ECM) genes and neuronal regulatory genes, while histological and ultrastructural analyses showed disorganized collagen fibrils, diminished osteoid, and abnormal mineralization. In vitro, FGF signaling in Ocy454 cells regulated gene programs involved in development, axon guidance, and bone ECM organization, highlighting a dual function for FGF signaling in which it controls both matrix-dependent and intrinsic cell differentiation mechanisms during the osteoblast-to-osteocyte transition. We propose that FGFR1 deficiency causes ECM disorganization and impaired dendrite formation, disrupting osteocyte communication with neighboring bone and vascular cells, ultimately leading to cell death. These findings establish FGFR signaling as a critical regulator of osteocyte differentiation, viability of bone-embedded osteocytes, and bone homeostasis. Summary StatementFGFR signaling has a profound effect on adult bone extracellular matrix that is vital to maintaining the viability and morphology of newly formed osteocytes, their lacunocanalicular network and the maintenance of bone homeostasis.

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.

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.

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

BackgroundPhysical loading mediates postnatal growth, homeostasis, and healing of the tendon and its attachment to bone, which is critical for rotator cuff functional integrity. Our prior studies have highlighted the mechano-sensing role of primary cilia; However, the mechanisms through which cilia convert mechanical stimuli into structural functional adaptation under altered loading conditions remain unanswered. MethodsPublicly available scRNA-seq datasets of mechanically loaded human patellar tendon cells were re-analyzed to identify cilia-related transcriptional changes. Tendon-specific cilia knockout mice (ScxCre;Ift88fl/fl) and wild-type controls (Ift88fl/fl) underwent mechanical unloading induced by botulinum toxin A injection, followed by micro-computed tomography, biomechanical testing, histology, qPCR, and immunohistochemistry to evaluate structural, mechanical, and Hedgehog (Hh) signaling responses. Primary tendon fibroblasts from wild-type and cilia-deletion mice were treated with Hh agonist or antagonist to assess Hh signaling responsiveness in vitro. Students t-test for two groups and two-way ANOVA for two groups with two treatments were performed for our statistical analysis. ResultsHere, we find that mechanical force causes changes in cilia- and hedgehog (Hh)-related gene expression in human tendon fibroblasts. Cilia ablation in the enthesis blunts force-driven remodeling of tissue structure and mechanical strength. Cilia deletion also leads to impaired Hh signaling in tendon cells and decreased responsiveness to activation and inactivation of hedgehog signaling. ConclusionsOur results demonstrate loading-regulated ciliary Hh signaling during postnatal growth of the tendon and enthesis and provide proof-of-concept for developing new cilia-targeted mechanical and biological therapies for enthesis repair.

Systemic sclerosis (SSc) is an autoimmune connective tissue disease with pronounced sex differences: females are more frequently affected and males develop more severe skin fibrosis. The cellular mechanisms of this disparity remain unclear. Here we use single-cell transcriptomics of lesional, non-lesional, and healthy skin to define fibroblast states and sex-biased transcriptional programs during lesion development. We identify a sex-dependent divergence in SSc fibrotic regulation. Female fibroblasts exhibit heightened inflammatory signaling and canonical TGF-{beta}-driven extracellular matrix production, whereas male fibroblasts preferentially engage non-canonical TGF-{beta} pathways, mechanotransduction, and MYC-associated stress programs. We further reveal that the fibrotic lesional environment shows sex differences: SFRP2DPP4 fibroblasts predominate in females and COL11A1/COCH in males. Our findings uncover cellular mechanisms underlying sex differences in SSc fibrosis, highlight opportunities for sex-informed therapeutic strategies and underscore the necessity of integrating biological sex into precision medicine frameworks to identify divergent molecular drivers of fibrotic disease.

O-fucosylation plays an essential role in controlling protein folding, secretion and protein-protein interactions within the extracellular space. Recently, we identified a new form of protein O-fucosylation occurring on the N-terminal Elastin Microfibril Interaction (EMI) domain of several secreted proteins, mediated by two previously uncharacterized protein O-fucosyltransferases, POFUT3 (FUT10) and POFUT4 (FUT11). As all POFUT enzymes (POFUT1-4) are highly specific for the three-dimensional (3D) structure of their substrate protein domains, we postulated that structural homologues of these domains in other proteins may also be O-fucosylated. Here, we employed iterative protein structural homology searches as a novel strategy for identifying EMI-like domains that may serve as potential substrates for POFUT3/4. We discovered that microfibrillar-associated protein 2 and 5 (MFAP2/MFAP5) contain EMI-like domains and are O-fucosylated at high stoichiometry in human tissues. Unexpectedly, we showed that only POFUT3 is both necessary and sufficient for MFAP2/MFAP5 O-fucosylation, despite POFUT4 also having strong protein-protein interactions with MFAP2/MFAP5. Finally, we determined that O-fucosylation of MFAP2/MFAP5 is required for their efficient secretion, similar to other EMI domain-containing proteins. Together, these data demonstrate the power of sensitive structural homology analysis in identifying new enzyme-substrate relationships and protein-protein interactions.

TRPV1 (transient receptor potential vanilloid 1) is a non-selective cation channel with high permeability to Ca2+ and is best known for its roles in sensory signaling. However, its function in immune cell biology, particularly in macrophage fusion, remains unknown. Cell fusion is a critical process in both physiological and pathological contexts, including development, tissue remodeling, and the foreign body response (FBR) to implanted biomaterials. During FBR, macrophages undergo fusion to form multinucleated foreign body giant cells (FBGCs), which contribute to implant degradation and fibrotic encapsulation. Here, we identify TRPV1 as a key regulator of macrophage multinucleation and FBGC formation. We demonstrate that TRPV1 is endogenously expressed in bone marrow-derived macrophages (BMDMs) and is upregulated in response to fusogenic cytokines and inflammatory stimuli. Functionally, TRPV1 promotes matrix stiffness-dependent macrophage adhesion and spreading, indicating a role in mechanosensitive signaling. We show that TRPV1 is required for efficient macrophage fusion under both cytokine-driven and matrix stiffness-mediated conditions. Mechanistically, TRPV1 links extracellular mechanical cues and cytokine signaling to cytoskeletal remodeling, facilitating the actin reorganization necessary for cell fusion. Importantly, TRPV1 deficiency does not alter TRPV4-mediated Ca2+ signaling, demonstrating that TRPV1 operates independently of TRPV4, a known mechanosensitive channel implicated in FBR and FBGC formation. Collectively, these findings suggest TRPV1 as a previously unrecognized mechanosensitive regulator of macrophage fusion and FBGC formation. This work provides new insight into the molecular mechanisms governing FBR and identifies TRPV1 as a potential therapeutic target for improving biomaterial biocompatibility and mitigating fibrosis.

Saul-Wilson syndrome (SWS) is a skeletal dysplasia characterized by primordial dwarfism and progeroid features caused by a recurrent dominant COG4 variant (p.G516R). We previously showed that this mutation accelerates Golgi retrograde trafficking and disrupts glycosylation of the proteoglycan decorin, while zebrafish models revealed defects in chondrocyte elongation and intercalation. We have also shown that the SW1353 chondrosarcoma cells carrying the SWS variant exhibit reduced secretion of extracellular matrix (ECM) components. While these results indicate a critical function of COG4 in Golgi processing, the developmental process leading to skeletal dysplasia in SWS patients remains unknown. Here, we generated patient-derived iPSC cartilage organoids (SWS organoids), modeling early human chondrogenesis. SWS organoids failed to produce cartilage structures and displayed poor expression of chondrogenic markers. Time-course RNA-seq analysis of the chondrogenic process revealed reduced activation of gene networks involved in skeletal development, ECM organization, ossification, and glycosaminoglycan metabolism. Spatial multiomic analysis of protein and glycosylation by CODEX and GLYPH imaging revealed an altered chondrogenic trajectory, persistence of mesenchymal states, global glycosylation changes, and reduced deposition of chondroitin sulfate proteoglycans. These results indicate that the COG4 mutation disrupts ECM glycosylation and chondrogenic commitment, and that SWS organoids model early defects in cartilage formation underlies impaired skeletal growth in SWS. HighlightsO_LIPatient iPSC-derived cartilage organoids model development defects in Saul-Wilson syndrome C_LIO_LISWS organoids show defective extracellular matrix deposition and attenuated chondrogenic gene expression C_LIO_LIGlycan profiling reveals global glycosylation defects and deficient proteoglycan GAG chains C_LIO_LIAn early developmental impairment in chondrogenesis alters skeletal formation in Saul-Wilson syndrome C_LI

Congenital heart defects frequently arise from alterations in the elongation of the cardiac outflow tract (OFT). Proper elongation of the OFT depends on the coordinated deployment of progenitor cells from the second heart field (SHF) and on dynamic interactions with the extracellular matrix (ECM). Among ECM components, fibronectin (Fn1) and tenascin-C (TnC) have emerged as key regulators of cardiac morphogenesis. Studies in mouse embryos have shown that mesodermal Fn1 is required to maintain proper TnC localization within SHF cells. To study heart development, mammalian models are challenging to use because of their in utero development. This limitation highlights the need for alternative models with external development, where direct observation is possible; however, in these systems, the cellular organization of the SHF and the dynamics of its ECM environment remain poorly characterized Here, we investigated the cellular and extracellular architecture of SHF cells localized to the dorsal pericardial wall (DPW) during heart development in Xenopus laevis. We show that SHF cells undergo a stage-dependent transition from a predominantly monolayered organization at NF35 to a multilayered structure at NF42. This transition is accompanied by dynamic remodeling of the ECM, characterized by increased expression of Fn1, TnC, and Collagen I (ColI) and by redistribution of ECM components within the DPW. Functional experiments revealed that depletion of Fn1 disrupts cardiac morphogenesis, leading to shortening of the OFT and reduced ventricular size. Moreover, loss of Fn1 decreases TnC and ColI levels and alters the spatial organization of TnC within the DPW, indicating that Fn1 is required for proper ECM assembly within the SHF cells. These findings identify Fn1 as a key regulator of ECM assembly within the DPW and highlight how ECM remodeling contributes to the organization of SHF progenitor cells during OFT elongation. Altogether, we demonstrated that Xenopus laevis is a powerful model for studying ECM-driven mechanisms of cardiac morphogenesis.