Background/objective Increased lysyl oxidase (LOX) activity favors pathologic cartilage and vessel calcification. LOX promotes disease through enhanced collagen cross-linking, inflammation, reactive oxygen species (ROS) production, cell trans-differentiation, and fibrosis. This study investigates the therapeutic potential of cystathionine gamma lyase (CSE)-generated hydrogen sulfide (H2S) to inhibit tendon calcification by targeting LOX in human samples and murine models of calcific tendinopathy (CT). Methods Human shoulder supraspinatus tendons with varying degrees of CT were analyzed using Alizarin Red staining and LOX and CSE immunohistochemistry to evaluate the correlation between CSE and LOX/calcification. Mechanistic studies were performed using wild-type (WT) and CSE knockout murine tenocytes cultured in calcification-inducing medium with or without H2S donors or the LOX inhibitor β-aminopropionitrile (BAPN). Achilles tendon CT was induced in WT and CSE knockout mice via surgical intervention or aging. Tendon calcification, LOX expression, biomechanical integrity, and transcriptomic changes were assessed. Persulfidation of total proteins and recombinant human LOX (rhLOX) was measured using the dimedone-switch method. Results An inverse correlation between CSE levels and LOX/calcification was observed in human tendons and in the surgery-induced CT murine model. In murine tenocytes and in the aging murine model, CSE deficiency led to increased LOX expression, enhanced calcification, and reduced tendon biomechanical integrity.Transcriptomic analysis confirmed the negative association between CSE and LOX in murine CT. Mechanistically, H2S increased total cellular protein persulfidation, including rhLOX, resulting in inhibition of its enzymatic activity. Conclusion Dysregulated LOX activity is a key driver of calcific tendinopathy. CSE-generated H2S effectively suppresses LOX activity, highlighting its potential as a therapeutic strategy for CT and other calcification-related disorders. The translational potential of this article This study identifies LOX as a therapeutic target in CT and supports H2S as a promising treatment strategy for this condition.

Many studies analyze tissue-resident or blood-borne leukocytes to monitor disease progression. We hypothesized that the microvasculature serves as a distinct site for immune cell activity. Here, we investigate microvascular leukocyte phenotypes before, during and after acute kidney injury (AKI) in mice, uncovering unique characteristics in the kidney, liver, and lung. Using single-cell sequencing, we identify several immune cells that were up to 100-fold expanded in the kidney vasculature, including macrophages, dendritic cells (DC), and B cells. Regeneration after AKI is characterized by sustained remodeling of the renal microvascular interface. Homeostatic microvascular C1q+ macrophages withdraw from the vascular barrier which is subsequently repopulated by new subsets, including CD11c+F480+ and CD11c+F480- cells. These newly arrived macrophages exhibit enhanced phagocytic activity toward circulating bacteria and secretion of tumor necrosis factor, pointing to maladaptive repair mechanisms after AKI. These data suggest organ- and disease-specific microvascular immune dynamics which are not detectable through conventional blood and tissue analysis.

Objectives Oral enzyme combination (OEC) therapy with bromelain, trypsin and rutoside reduces pain and improves function in patients with knee osteoarthritis (OA). Here, we investigated several potential biological mechanisms underlying the clinical effects of OEC therapy in patients with established knee OA with respect to innate immunity, systemic inflammation and cartilage turnover (EudraCT 2020-003154-80, NCT05038410).Methods Patients (age ≥40 years, body mass index (BMI) ≤35 kg/m2) with symptomatic knee OA were randomised to either placebo or OEC, administered 2×3 tablets/day, for 8 weeks before crossing over after a 4-week washout period. Different markers exploring innate immunity, inflammation and cartilage matrix degradation have been measured in the blood using immunoassays or cytometric methods. Data from the modified intention-to-treat population (mITT) were analysed using a generalised linear mixed model. No correction for multiple comparisons was made due to the exploratory nature of the study.Results Altogether, 45 patients were randomised; 43 completed both treatment sequences (mITT; mean age: 63.3 years; mean BMI: 27.4 kg/m2; mean global Knee injury and Osteoarthritis Outcome Score (KOOS): 48.7). OEC significantly increased levels of α2-macroglobulin (p=0.038) and interleukin-10 (p<0.0001) while decreasing urinary carboxyl-terminal cross-linked telopeptide of type II collagen (p=0.038). Patients administered OEC exhibited significant improvements in KOOS Pain (p=0.0464) and Symptoms (p=0.026) subdomains but not globally. OEC was well tolerated, with no serious related adverse events reported in either group.Conclusions One of the key findings of this proof-of-mechanism study is that OEC modulates IL-10 production, suggesting an anti-inflammatory effect in patients with knee OA. This main finding contributes to explaining the effects of OEC on pain and function in these patients.Trial registration number NCT05038410.

Fibroblast-like synoviocytes (FLS) are key cells promoting cartilage damage and bone loss in rheumatoid arthritis (RA). They are activated to assume an invasive and migratory phenotype. While mechanisms of FLS activation are unknown, evidence suggests that pre-damaged extracellular matrix (ECM) of the cartilage can trigger FLS activation. Integrin α11β1 might be involved in the activation, as it is increased in RA patients and hTNFtg mice, an RA mouse model. We treated murine chondrocytes with TNFα to produce a damaged, RA-like matrix. Comparison to healthy chondrocyte matrix revealed decreased ECM proteins, e.g. collagens and proteoglycans, increased matrix-degrading proteins and elevated levels of inflammatory cytokines. FLS responded to the damaged chondrocyte matrix with a matrix-remodeling and pro-inflammatory phenotype characterized by a gene signature involved in matrix degradation and increased production of CLL11 and CCL19. Damaged chondrocyte matrix stimulated increased Itga11 expression in FLS, correlating with the increased α11β1 amounts in RA patients. FLS deficient in integrin α11β1 released lower amounts of inflammation-associated cytokines. Our results demonstrate differences in healthy and RA-like chondrocyte ECM and distinctly different responses of wt FLS to damaged versus healthy ECM.

OBJECTIVES:In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) acquire an aggressive, tumour-like phenotype characterised by increased adhesion to extracellular matrix, contributing to joint degradation. The collagen-binding integrin alpha11beta1 is involved in similar processes in cancer-associated fibroblasts, but its role in RA and arthritic mice remains unclear. METHODS:Integrin α11 expression was analysed in synovial tissue and FLS from RA and osteoarthritis patients and human tumour necrosis factor transgenic (hTNFtg) and wild-type mice supported by Accelerating Medicines Partnership Rheumatoid Arthritis and Pathobiology of Early Arthritis Cohort data. A novel 3-dimensional (3D) organoid coculture model and electron microscopy were used to analyse FLS invasion into cartilage explants, Itga11-/- were crossed with hTNFtg mice, and disease severity was evaluated using microcomputed tomography (µCT) and histology. Functional assays using FLS included cell morphology, adhesion, degradation, and matrix metalloproteinase expression and were complemented by osteoclast and coculture studies. RESULTS:In the context of RA, strong α11 expression was detected in the synovium, particularly in sublining clusters of FLS within fibroid-type synovial tissue in vivo and at focal adhesions of arthritic FLS and at invasion sites within the 3D coculture model in vitro. Clinical scores, µCT imaging, and histomorphological analyses revealed significantly reduced cartilage degradation, bone erosions, and FLS attachment to cartilage in Itga11-/-hTNFtg compared to hTNFtg mice. In vitro studies revealed that α11 deficiency led to a decreased receptor activator of nuclear factor kappa-B ligand/osteoprotegerin ratio along with reduced TNFα-induced proteolytic degradation activity, and signalling pathway activation. CONCLUSIONS:Integrin α11 levels are increased in RA, and its deficiency notably diminishes joint destruction in hTNFtg mice, emphasising its potential as promising therapeutic target.

Breast cancer (BC)-derived bone metastases colonize bone and drive severe bone degradation through complex interactions with bone-resorbing osteoclasts (OCs). Subsequent bone resorption liberates matrix-stored factors, such as TGF-β and calcium, which further stimulate tumor proliferation and exacerbate bone destruction. Myostatin (Mstn), a member of the TGF-β superfamily, is known to enhance OC differentiation and bone resorption in models of musculoskeletal disease; however, its role in BC-associated bone lesions and metastases remains unknown. Here, we demonstrate that bone metastases from BC patients express Mstn, predominantly localized at the osteoclast-rich bone–tumor interface. In vitro, both direct and indirect interactions between BC cells and OC precursors significantly increased OC formation and resorptive activity. Antibody-mediated blockade of Mstn attenuated these effects by inhibiting SMAD2 phosphorylation. In vivo, targeting Mstn in 4T1 and MDA-MB-231 murine models of BC-induced bone destruction resulted in elevated bone density, increased muscle mass, and reduced OC numbers compared to controls. Furthermore, anti-Mstn treatment decreased the burden of bone metastases in MDA-MB-231-bearing mice. Collectively, these findings identify Mstn as a previously unrecognized driver of BC-induced osteolysis and metastases, highlighting its potential as a therapeutic target in metastatic BC.

Fibronectin 1 (FN1), a general organizer of extracellular matrix (ECM) in various connective tissues, contains disulfide bridges formed by protein disulfide isomerases (PDIs). ERp57 (PDIA3), an ER resident glycoprotein-specific PDI, is also detectable in cartilage extracellular matrix (ECM). Here, we analyzed the extracellular role of ERp57 in FN1 fibrillogenesis in cartilage. ERp57 KO mice exhibited reduced ECM density. Isolated chondrocytes thereof and C28/I2 ERp57 KO chondrocytes formed fewer and shorter FN1 fibrils than WT cells. Significantly, cell membrane-impermeable thiol blockers reduced FN1 assembly in WT cells, while active recombinant ERp57 protein increased it only in the absence of thiol blockers, emphasizing the necessity of ERp57-mediated disulfide bridge formation for FN1 fibrillogenesis. Co-immunofluorescence and proximity ligation assays revealed a direct interaction between ERp57 and FN1. This study highlights a key role for extracellular ERp57 PDI activity in cartilage and further explains phenotypic changes in ERp57 KO animals.

Transient receptor potential channel 1 (TRPC1) is a widely expressed mechanosensitive ion channel located within the endoplasmic reticulum membrane, crucial for refilling depleted internal calcium stores during activation of calcium-dependent signaling pathways. Here, we have demonstrated that TRPC1 activity is protective within cartilage homeostasis in the prevention of cellular senescence-associated cartilage breakdown during mechanical and inflammatory challenge. We revealed that TRPC1 loss is associated with early stages of osteoarthritis (OA) and plays a nonredundant role in calcium signaling in chondrocytes. Trpc1-/- mice subjected to destabilization of the medial meniscus-induced OA developed a more severe OA phenotype than WT controls. During early OA development, Trpc1-/- mice displayed an increased chondrocyte survival rate; however, remaining cells displayed features of senescence including p16INK4a expression and decreased Sox9. RNA-Seq identified differentially expressed genes related to cell number, apoptosis, and extracellular matrix organization. Trpc1-/- chondrocytes exhibited accelerated dedifferentiation, while demonstrating an increased susceptibility to cellular senescence. Targeting the mechanism of TRPC1 activation may be a promising therapeutic strategy in OA prevention.

Purpose (the aim of the study): Osteoarthritis (OA) is a multifactorial degenerative joint disorder that results in cartilage breakdown, subchondral bone remodelling and disability. Our previous work has implicated the chemokine receptor Cxcr2 as a critical regulator of cartilage homeostasis, whereby Cxcr2 knockout mice develop more severe cartilage destruction following destabilization of medial meniscus (DMM). The Cxcr2 ligand Cxcl6 has been identified to limit hypertrophic differentiation of chondrocytes. Integrins are heterodimeric transmembrane receptors linking the actin cytoskeleton to the extracellular matrix. Their activity is regulated via an allosteric switch between low- and high-affinity states, induced by effector binding to the cytoplasmic tail of the α- and β-subunits or by ligand binding to the ectodomain. β1-integrin activity in chondrocytes has been shown to increase during osteoarthritis leading to increased catabolism, however β1-integrin signaling is required during chondrogenic differentiation. This project aims to investigate whether Cxcr2 signaling can control β1-integrin activation within cartilage homeostasis and may be harnessed to limit activation during OA development.

Nature realizes protein and peptide depots by catalyzing covalent bonds with the extracellular matrix (ECM) of tissues. We are translating this natural blueprint for the sustained delivery of a myostatin-inhibiting peptide (Anti-Myo), resulting in an enzyme depot established from injectable solutions. For that, we fused Anti-Myo to the D-domain of insulin-like growth factor I, a transglutaminase (TG) substrate. TG catalyzed the covalent binding of the D-domain to ECM proteins, such as laminin and fibronectin, on bioengineered ECM and in mice. ECM decorated with Anti-Myo suppressed myostatin activity and pathway activation and reduced the differentiation of preconditioned bone marrow-derived macrophages into osteoclasts in vitro.

Nature realizes protein and peptide depots by catalyzing covalent bonds with the extracellular matrix (ECM) of tissues. We are translating this natural blueprint for the sustained delivery of a myostatin-inhibiting peptide (Anti-Myo), resulting in an enzyme depot established from injectable solutions. For that, we fused Anti-Myo to the D-domain of insulin-like growth factor I, a transglutaminase (TG) substrate. TG catalyzed the covalent binding of the D-domain to ECM proteins, such as laminin and fibronectin, on bioengineered ECM and in mice. ECM decorated with Anti-Myo suppressed myostatin activity and pathway activation and reduced the differentiation of preconditioned bone marrow-derived macrophages into osteoclasts in vitro.

Bone resorption is highly dependent on the dynamic rearrangement of the osteoclast actin cytoskeleton to allow formation of actin rings and a functional ruffled border. Hem1 is a hematopoietic-specific subunit of the WAVE-complex which regulates actin polymerization and is crucial for lamellipodia formation in hematopoietic cell types. However, its role in osteoclast differentiation and function is still unknown. Here, we show that although the absence of Hem1 promotes osteoclastogenesis, the ability of Hem1-/- osteoclasts to degrade bone was severely impaired. Global as well as osteoclast-specific deletion of Hem1 in vivo revealed increased femoral trabecular bone mass despite elevated numbers of osteoclasts in vivo. We found that the resorption defect derived from the morphological distortion of the actin-rich sealing zone and ruffled border deformation in Hem1-deficient osteoclasts leading to impaired vesicle transport and increased intracellular acidification. Collectively, our data identify Hem1 as a yet unknown key player in bone remodeling by regulating ruffled border formation and consequently the resorptive capacity of osteoclasts.

Objective Loose bodies are free-floating tissues of cartilage and bone that can cause pain, swelling, the inability to straighten the knee, or intermittent locking of the knee. Loose bodies can arise from degenerative joint disease, flake fractures, osteochondritis dissecans, or chondromatosis. We hypothesized that loose bodies can be classified in stages with tissue characteristics similar to endochondral ossification. Design Loose bodies were harvested from patients undergoing joint replacement. Samples were processed for histology, gene expression analysis, and micro-computed tomography (µCT). Cartilage- and bone-related genes and proteins were selected for immunofluorescence stainings (collagen type I, II, and X, SOX9 [SRY-box transcription factor 9], and MMP13 [matrix metalloproteinase 13]) and gene expression analysis ( FN [fibronectin], COL1A1, COL2A1, COL10A1, SOX9, MMP13, and aggrecan [ ACAN]). Results Loose bodies were grouped in 4 stages: fibrous, (mineralized) cartilaginous, cartilage and bone, and bone. Hyaline-like cartilage tissue with Benninghoff arcades was present in stages 2 and 3. A transition from cartilaginous to mineralized tissue and bone trabecula was defined by an increase in COL1A1 and COL10A1 (stage 3 vs. 4: p = 0.047) positive area. Stage 4 showed typical trabecular bone tissue. The relative volume of calcified tissue (mineralized cartilage and bone tissue) decreased with stages (stages 1-2 vs. 3: p = 0.002; stage 1-2 vs. 4: p = 0.012). COL2A1 expression and stained area decreased from stages 1-2 to 4 ( p = 0.010 and p = 0.004). ACAN expression decreased from stage 1-2 to stage 3 ( p = 0.049) and stage 4 ( p = 0.002). Conclusion Loose bodies show tissue characteristics similar to endochondral ossification. They are probably a relevant substrate for regenerative therapeutic interventions in joint disease.