Matrix Biology

Collagens, the most abundant proteins in mammals, play pivotal roles in the maintenance of tissue structure, functions, cell-to-cell communication, cellular migration, behavior, and growth. Collagens are highly complex in structure due to the dynamic post-translational modifications (PTMs) such as hydroxylations (on prolines and lysine residues) and O-glycosylation (on hydroxylysines) enzymatically catalyzed during biosynthesis. The most prevalent modification in fibrillar collagens is prolyl 4-hydroxylation catalyzed by collagen prolyl 4-hydroxylases (C-P4hs). Prolyl 4-hydroxylation on collagens plays a critical role in collagen biosynthesis, thermostability, and cell-collagen interactions. However, the site-specificity of prolyl 4-hydroxylase 1 (P4ha1) and P4ha2 is not comprehensively studied yet. Further, the effect of P4ha1 and P4ha2 on the plethora of other site-specific collagen PTMs is not known to date. In-depth mass-spectrometry data (PXD008802) analysis of mice skin collagen I extracted from wild-type and different deletion mutants of C-P4hs revealed that partial or full deletion of prolyl 4-hydroxylases (P4ha1 and P4ha2) significantly decreases collagen deposition in ECM hinting towards perturbed biosynthesis. A total of 421 site-specific PTMs on fibrillar collagen chains (Col1a1, Col1a2, and Col3a1) were identified. Further, novel 23 P4ha1 specific, 8 P4ha2 specific, and 18 C-P4hs promiscuous sites on fibrillar collagen chains were identified. Partial deletion of P4ha1 and full deletion of P4ha2 also resulted in altered levels of the site-specific prolyl-3-hydroxylation occupancy in collagen I. Surprisingly, an increased level of site-specific lysyl hydroxylation (Col1a1-K731, Col1a2-K183,315) was documented upon partial deletion of P4ha1 and full deletion of P4ha2. Our findings showcased that the activity of prolyl 4-hydroxylases is not limited to 4-hydroxylation of specific proline sites, but simultaneously can perturb the entire biosynthetic network by modulating prolyl 3-hydroxylation and lysyl hydroxylation occupancy levels in the fibrillar collagen chains in a site-specific manner.

Asprosin, the C-terminal furin cleavage product of profibrillin-1, was reported to act as a hormone that circulates at nanomolar levels and is recruited to the liver where it induces G protein-coupled activation of the cAMP-PKA pathway and stimulates rapid glucose release into the circulation. Although derived upon C-terminal cleavage of fibrillin-1, a multidomain extracellular matrix glycoprotein with a ubiquitous distribution in connective tissues, little is known about the mechanisms controlling the bioavailability of asprosin in tissues. In the current view, asprosin is mainly produced by white adipose tissue from where it is released into the blood in monomeric form. Here, by employing newly generated specific asprosin antibodies we monitored the distribution pattern of asprosin in human and murine connective tissues such as placenta, and muscle. Thereby we detected the presence of asprosin positive extracellular fibers. Further, by screening established cell lines for asprosin synthesis we found that most cells derived from musculoskeletal tissues render asprosin into an oligomerized form. Our analyses show that asprosin already multimerizes intracellularly, but that stable multimerization via covalent bonds is facilitated by transglutaminase activity. Further, asprosin fiber formation requires an intact fibrillin-1 fiber network for proper linear deposition. Our data suggest a new extracellular storage mechanism of asprosin in an oligomerized form which may regulate its cellular bioavailability in tissues.

Formation of 4-hydroxyproline (4Hyp) in -X-Pro-Gly- collagen sequences is essential for the thermal stability of collagen molecules. 4Hyp formation is catalyzed by collagen prolyl 4-hydroxylases (C- P4H). Here we identify specific roles for the two main C-P4H isoenzymes by 4Hyp analysis of type I and IV collagens. Loss of C-P4H-I mainly affected prolines preceded by an X-position amino acid with a positively charged or a polar uncharged side chain. In contrast, loss of C-P4H-II affected triplets with a negatively charged glutamate or aspartate in the X-position, and their hydroxylation was found to be important as loss of C-P4H-II alone resulted in reduced collagen melting temperature and altered assembly of collagen fibrils and basement membrane. The C-P4H isoenzyme differences in substrate specificity were explained by selective substrate binding to the active site resulting in differences in Km and Vmax values. In conclusion, this study provides a molecular level explanation for the need of multiple C-P4H isoenzymes to generate collagen molecules capable to assemble into intact extracellular matrix structures.

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.

Mucopolysaccharidoses (MPS) are a group of syndromes characterized by the accumulation of sulfated glycosaminoglycans (sGAGs), leading to profound connective tissue alterations, including impaired endochondral ossification. The function of sGAGs involves determining the mechanical properties of the extracellular matrix and regulating growth factor signaling pathways, such as Fgf2. In this study, we investigated the deposition of chondroitin sulfate and dermatan sulfate, two major sGAGs, and their resemblance to wound healing states in human fibroblasts. Our findings indicate that this condition alters cell adhesion, providing a potential explanation for fibrosis-like changes observed in MPS patients. Furthermore, we elucidate the molecular pathway responsible for this effect, wherein increased Cathepsin L activation leads to the processing of the transcription factor Cux1 into a stable form capable of regulating the expression of target genes, including SERPINB2. The presence of similar changes in cell adhesion in human-induced pluripotent stem cell-derived mesenchymal cells further reinforces the significance of sGAGs in cell adhesion and sheds light on possible mechanisms underlying altered endochondral ossification in MPS patients.

Wound healing is a well-orchestrated process that typically recruits the immune and vascular systems to restore the structure and function of the injured tissue. Injuries to the enthesis, a hypocellular and avascular tissue, often result in fibrotic scar formation and loss of mechanical properties, thereby severely affecting musculoskeletal function and life quality. This raises questions about the healing capabilities of the enthesis. Here, we established an injury model to the Achilles entheses of neonatal mice to study the possibility that at an early age, the enthesis can heal more effectively. Histology and immunohistochemistry analyses revealed an atypical process that did not involve inflammation or angiogenesis. Instead, neonatal enthesis healing was mediated by secretion of collagen types I and II by resident cells, which formed a permanent hypocellular and avascular scar. Transmission electron microscopy showed that the cellular response to injury, including ER stress, autophagy and cell death, varied between the tendon and cartilage ends of the enthesis. Single-molecule in situ hybridization, immunostaining, and TUNEL assays verified these differences. Finally, gait analysis showed that these processes effectively restored function of the injured leg. Collectively, these findings reveal a novel healing mechanism in neonatal entheses, whereby local ECM secretion by resident cells forms an acellular ECM deposit in the absence of inflammation markers, allowing gait restoration. These insights into the healing mechanism of a complex transitional tissue may lead to new therapeutic strategies for adult enthesis injuries.

Matrix metalloproteinase-3 (MMP-3 or stromelysin 1) participates in normal extracellular matrix (ECM) turnover during embryonic development, organ morphogenesis and wound healing, and in tissue-destructive diseases, such as aneurysm, cancer, arthritis and heart failure. Despite its ability to hydrolyse numerous proteins in the ECM, MMP-3 fails to cleave the triple helix of interstitial fibrillar collagens. Nonetheless, it can still bind to these collagens although the mechanism, location and role of binding are not known. We used the Collagen Toolkits, libraries of triple-helical peptides that embrace the entire helical domains of collagens II and III, to map MMP-3 interaction sites. The enzyme recognises five sites on collagen II and three sites on collagen III. They share a glycine-phenylalanine-hydroxyproline/alanine (GFO/A) motif that is recognised by the enzyme in a context-dependent manner. Neither MMP-3 zymogen (proMMP-3) nor the individual catalytic (Cat) and hemopexin (Hpx) domains of MMP-3 interact with the peptides, revealing cooperative binding of both domains to the triple helix. The Toolkit binding data combined with molecular modelling enabled us to deduce the putative collagen-binding mode of MMP-3, where all three collagen chains make contacts with the enzyme in the valley running across both Cat and Hpx domains. The observed binding pattern casts light on how MMP-3 could regulate collagen turnover and compete with various collagen-binding proteins regulating cell adhesion and proliferation.

Fibulin-3 (FBLN3), also known as EFEMP1, is a secreted extracellular matrix (ECM) glycoprotein that contains forty cysteine residues. These cysteines, which are distributed across one atypical and five canonical calcium-binding epidermal growth factor (EGF) domains, are important for regulating FBLN3 structure, secretion, and presumably function. As evidence of this importance, a rare homozygous p.C55R mutation in FBLN3 negates its function, alters disulfide bonding, and causes marfanoid syndrome. Additional studies suggest that heterozygous premature stop codon mutations in FBLN3 may also cause similar, albeit less severe, connective tissue disorders. Interestingly, a series of twenty-four cysteine mutations in FBLN3 have been identified in the human population and published in the Clinical Variation (ClinVar) and gnomAD databases. We tested how seven of these cysteine mutants (five loss-of-cysteine variants: C42Y, C190R, C218R, C252F, and C365S, two gain-of-cysteine variants: R358C, Y369C) and two newly developed mutations (G57C and Y397C) altered FBLN3 secretion, disulfide bonding, MMP2 zymography, and stress response activation Surprisingly, we found a wide variety of biochemical behaviors: i) loss-of-cysteine variants correlated with an increased likelihood of disulfide dimer formation, ii) N-terminal mutations were less likely to disrupt secretion, and were less prone to aggregation, iii) in contrast to wild-type FBLN3, multiple, but not all variants failed to induce MMP2 levels in cell culture, and iv) C-terminal mutations (either loss or gain of cysteines) were more prone to significant secretion defects, intracellular accumulation/misfolding, and stress response activation. These results provide molecular and biochemical insight into FBLN3 folding, secretion, and function for many cysteine mutations found in the human population, some of which may increase the likelihood of subclinical connective tissue or other FBLN3-associated haploinsufficiency diseases.

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.

Collagen has been postulated to be the most abundant protein in our body, making up one-third of the total protein content in mammals. However, to the best of our knowledge, a direct assessment of the total collagen levels of an entire mammal to confirm this estimate is missing. Here we measured hydroxyproline levels as a proxy for collagen content together with total protein levels of entire mice or of individual tissues. Collagen content normalized to the total protein is approximately 0.1% in the brain and liver, 1% in the heart and kidney, 4% in the muscle and lung, 6% in the colon, 20-40% in the skin, 25-35% in bones, and 40-50% in tendons of wild-type (CD1 and CB57BL/6) mice, consistent with previous reports. Mice consist of 37 mg of collagen and 265 mg of protein per g of body weight. To our surprise, we find that collagen is approximately 12% in females and 17% in males of the total protein content of entire wild-type (CD1 and CB57BL/6) mice. High-Performance Liquid Chromatography approaches confirmed a 10-12% collagen over total protein estimates for female mice. Collagen staining methods and extracellular matrix-enriched proteomics estimated 5-6% of collagens over the total protein extracted. Although collagen type I is the most abundant collagen, the most abundant proteins are albumin, hemoglobulin, histones, actin, serpina, and then collagen type I. Analyzing amino acid compositions of mice revealed glycine as the most abundant amino acid. Thus, we provide reference points for collagen, matrisome, protein, and amino acid composition of healthy wild-type mice that are important for tissue and biomaterial engineering and for the comparison of these factors in various disease models.

The secretion of extracellular matrix (ECM) proteins is vital to the maintenance of tissue health. One major control point of this process is the Golgi apparatus, whose dysfunction causes numerous connective tissue disorders. Golgi function is tightly linked to its structure, which is maintained by the cytoskeleton and Golgi organising proteins. We sought to investigate the role of two of these organising proteins, the golgins GMAP210 and Golgin-160, in ECM secretion. We found that loss of either protein had distinct impacts on Golgi organisation. GMAP210 loss caused cisternal fragmentation and dilation, alongside the accumulation of tubulovesicular structures. Meanwhile, Golgin-160 knockout lead to Golgi fragmentation and vesicle build-up. Nonetheless, loss of each protein had a similar impact on ECM secretion and glycosaminoglycan synthesis. We therefore propose that golgins are collectively required to create the correct physical-chemical space to support efficient ECM protein secretion and modification. This is the first time that Golgin-160 has been shown to be required for ECM secretion. SummaryIn this study, Thompson et al demonstrate that two cis-Golgi golgins, GMAP210 and Golgin-160, have distinct, non-redundant roles in maintaining Golgi organisation and that both are required to support the efficient secretion, assembly, and modification of extracellular matrix proteins.

Semaphorin 3A (Sema3A) is a secreted protein that signals to cells through binding to neuropilin and plexin receptors and provides neurons with guidance cues key for axon pathfinding, and also controls cell migration in several other biological systems. Sema3A interacts with glycosaminoglycans (GAGs), an interaction that could localize the protein within tissues and involves the C-terminal domain of the protein. This domain comprises several furin cleavage sites that are processed during secretion and in previous works have hampered recombinant production of full-length wild type Sema3A, and the biochemical analysis of Sema3A interaction with GAGs. In this work, we have developed a strategy to purify the full-length protein in high yield and identified two sequences in the C-terminal domain, KRDRKQRRQR and KKGRNRR, which confer to the protein sub nM affinity for chondroitin sulfate and heparan sulfate polysaccharides. Using chemically defined oligosaccharides and solid phase binding assays, we report that Sema3A recognizes a (GlcA-GalNAc4S6S)2 motif but not a (GlcA2S-GalNAc6S)2 motif and is thus highly specific for type E chondroitin sulfate. Functionally, we found that Sema3A rigidified CS-E films that mimic the GAG presentation within extracellular matrices (ECMs), suggesting that Sema3A may have a previously unidentified function to cross-link and thus stabilize GAG-rich ECMs. Finally, we demonstrated that the full-length Sema3A is more potent at inhibiting neurite outgrowth than the truncated or mutant forms that were previously purified and that the GAG binding sites are required to achieve full activity. The results suggest that Sema3A can rigidify and cross-link GAG matrices, implicating Sema3A could function as an extracellular matrix organizer in addition to binding to and signaling through its cognate cell surface receptors.

Nerves in bone play well-established roles in pain and vasoregulation and have been associated with progression of skeletal disorders including osteoporosis, fracture, arthritis and tumor metastasis. However, isolation of the region-specific mechanisms underlying these relationships is limited by our lack of comprehensive maps of skeletal innervation. To overcome this, we mapped sympathetic adrenergic and sensory peptidergic axons within the limb in two strains of mice (B6 and C3H). In the periosteum, these maps were related to the surrounding musculature, including entheses and myotendinous attachments to bone. Locally, three distinct patterns of innervation (Type I, II, III) were defined within established sites that are important for bone pain, bone repair, and skeletal homeostasis. In addition, we mapped the major nerve branches and areas of specialized mechanoreceptors. This work is intended to serve as a guide during the design, implementation, and interpretation of future neuroskeletal studies and was compiled as a resource for the field as part of the NIH SPARC consortium.

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a debilitating genodermatosis caused by pathogenic mutations in the COL7A1 gene, which induce absence or reduction in the number of anchoring fibrils. The severity of RDEB depends on the mutation type and localization, but many aspects of this dependence remain to be elucidated. Here, we report a novel variant of RDEB Intermediate in two unrelated patients. Their disease manifestation includes early skin and oral mucosa blistering and is associated with localized atrophic scarring. According to the exome and Sanger sequencing results, both investigated Probands are the carriers of complex heterozygosity in the COL7A1 gene with the same deletion in intron 19 of the COL7A1 gene. RT-PCR followed by sequence analysis revealed skipping of the part of exon19, as well as the rescue of the open reading frame (ORF) of COL7A1 in both Probands. We hypothesize that the mutation in the acceptor splice site leads to the activation of the cryptic donor splice site, resulting in the truncated but partially functional protein and the milder phenotype of intermediate RDEB. This rare type of mutation expands our understanding of RDEB etiology and invites further investigation.

Extracellular matrix (ECM) is the main component of cartilage, making it an ideal environment to study cell-matrix interactions. Among ECM constituents, heparan sulfate (HS)-carrying proteoglycans (PGs) are of particular interest since they are not only structural components but are also involved in cell matrix adhesion and signalling processes. We previously demonstrated that transgenic mice with a clonal loss of HS synthesis in chondrocytes (Col2-rtTA-Cre;Ext1e2fl/e2fl) develop clusters of enlarged cells in the articular cartilage (AC), which are surrounded by a glycosaminoglycan (GAG)-rich ECM. This led to the questions how HS regulate the molecular composition and mechanical properties of the ECM, how they sense alterations in the HS structure and how they respond to it. We stained tissue sections of Col2-rtTA-Cre;Ext1e2fl/e2f animals and detected increased levels of chondroitin sulfate (CS), Aggrecan (Acan), Perlecan (Pcan), Matrilin (Matn)-3 and-4, Collagen type II (Col2) and Col9, while Col12 was abolished in the HS-deficient clusters. We assessed the stiffness of the mutant matrix by Atomic Force Microscopy (AFM) and found that it was markedly softer than the surrounding, HS-containing tissue. Likely in response to this altered texture, HS-deficient clones showed increased protein levels of Integrin pathway components. To model a loss of HS-function in vitro, we treated murine embryonic fibroblasts (MEFs) with the HS-antagonist Surfen. Treatment during cell adhesion resulted in impaired cell-substrate adhesion, increased formation of filopodia-like membrane protrusions, decreased cell polarisation and migration, reduced formation of FA and SF, and a translocation of YAP into the cytoplasm. Similarly, we observed reduced cell polarisation in HS-deficient CHO pgsD-667 cells, which could not be rescued by external presentation of HS. When MEFs were treated with Surfen after the completion of the initial cell adhesion process, inhibition of HS-function led to an increased formation of FA and SF, in line with the increased levels of Integrin pathway components observed in HS-deficient chondrocytes in vivo. We detected high levels of Yes1-associated protein (YAP) in the HS-deficient clusters, and we investigated the effect of YAP modulation on high density micromass cultures from primary murine chondroprogenitors. YAP activation induced an increased GAG synthesis similar to Surfen, while YAP inactivation partially abolished the effect of Surfen, showing that YAP acts downstream of HS function and controls GAG synthesis. Taken together, we demonstrated that HS-function is essential for Integrin-dependent cell-matrix interactions. Information on the impaired cell matrix adhesion upon loss of HS is conveyed into the nucleus via YAP, which at least partially controls the synthesis of GAGs in chondrocytes.

Immune-stromal crosstalk governs tissue fibrosis, which is marked by dysregulated extracellular matrix (ECM) production and aberrant vasculature. Here, we investigate how {gamma}{delta} T cell interactions with stromal cells shape fibrosis in the foreign body response. During the acute reaction, type-1 ({gamma}{delta}IFN{gamma}) and type-17 ({gamma}{delta}17) effector subsets accumulated at the implant. While {gamma}{delta}IFN{gamma} decreased as fibrosis progressed, activated {gamma}{delta}17 persisted as dominant interleukin-17 producers. The {gamma}{delta}17 increased with aging and high-fat diet, both factors associated with chronic inflammation and fibrosis. Co-culture with {gamma}{delta}17 stimulated fibroblast expression of collagen genes and intercellular communication inference linked {gamma}{delta} T cell ligands to activation of ECM remodeling and vascular development programs in fibroblasts and endothelial cells. Finally, genetic deletion of {gamma}{delta} T cells altered expression of ECM components and increased vessel size within the fibrotic matrix. Altogether, our findings implicate {gamma}{delta} T cells in regulating stromal behavior to modulate composition and vascularity of fibrotic tissues.

Type 2 diabetes affects multiple organ systems, including the skeletal system. Diabetes reduces bones mechanical properties and impacts bone cells, such as osteocytes, which are crucial to preserving bone health. Osteocytes maintain bone health through the lacunar canalicular network (LCN), a highly interconnected system vital for remodeling, mechanotransduction, and nutrient transport. Yet the specific impact diabetes has on this network has remained unclear. Here, we used confocal laser scanning microscopy combined with advanced connectomics modeling to achieve high-resolution, three-dimensional reconstructions of the LCN in Zucker Diabetic Sprague Dawley rats, a polygenic model that closely mimics human type 2 diabetes. Diabetes profoundly disrupted LCN connectivity in the femoral mid-cortex, with canalicular and node density reduced by 21% and 30%, respectively. Additionally, we observed a 30-40% increase in lacunar density and highly connected nodes. These architectural shifts impair bone permeability, diminishing mechanosensitivity and compromising nutrient and oxygen transport. Our findings uncover a previously unrecognized mechanism of skeletal fragility in diabetes and highlight the LCN as a promising therapeutic target.

Marfan syndrome (MFS) is the most prevalent inherited connective tissue disorder, still remains uncurable, and is characterized by high mortality at early age driven by dissection and rupture of thoracic aortic aneurysms. MFS is caused by mutations in the fibrillin-1 gene and aberrant TGF{beta} signaling. Here we addressed whether myeloperoxidase (MPO), a leukocyte derived enzyme with potent matrix modulating properties also influences the aortic phenotype in MFS. MFS patients displayed increased circulating MPO levels compared to controls as well as marked aortic MPO deposition. In an MFS mouse model, MPO induced inflammatory endothelial activation and endothelial to mesenchymal transition which triggered aortic leukocyte recruitment. Moreover, MPO directly contributed to adverse extracellular matrix remodeling by promoting oxidative stress and nitration of proteins within the vascular wall. Genetic MPO deficiency and pharmacological MPO inhibition attenuated MFS-related aneurysm formation. We herein identify MPO as a critical mediator of MFS-related thoracic aortic aneurysm formation and - in the absence of any pharmacological treatment so far in this disease - a first anti-inflammatory target to modulate disease progression.

Osteoarthritis (OA) is characterized by progressive cartilage degeneration, yet the initiating molecular events remain incompletely understood. Transmembrane protein 2 (TMEM2), a cell-surface hyaluronidase that degrades hyaluronan (HA), has been implicated in extracellular matrix homeostasis. Here, we delineate the spatiotemporal expression pattern of TMEM2 in mouse knee joints and assess its functional role during OA development. Single-cell RNA sequencing and histological analyses of normal joints revealed predominant Tmem2 expression in non-calcified articular chondrocytes and synovial cells. Following destabilization of the medial meniscus (DMM), Tmem2 was transiently upregulated during early OA, particularly within the non-calcified cartilage zone, coinciding with a pronounced reduction in HA content indicative of accelerated HA turnover. In contrast, Tmem2 expression declined at later stages, suggesting a temporally restricted activation phase. Functionally, Tmem2 deficiency exacerbated DMM-induced OA, leading to more severe structural deterioration, increased chondrocyte apoptosis, reduced proliferation, and elevated type X collagen, consistent with impaired cartilage homeostasis. Collectively, these findings identify TMEM2 as a key regulator of HA metabolism and a context-dependent modulator of OA progression. We propose that early, transient TMEM2 upregulation represents an adaptive remodeling response, whereas dysregulated or prolonged activity may contribute to HA depletion and cartilage breakdown.