Lysyl Oxidase Regulates Epithelial Differentiation and Barrier Integrity in Eosinophilic Esophagitis.

BACKGROUND & AIMS: Epithelial disruption in eosinophilic esophagitis (EoE) encompasses both impaired differentiation and diminished barrier integrity. We have shown that lysyl oxidase (LOX), a collagen cross-linking enzyme, is up-regulated in the esophageal epithelium in EoE. However, the functional roles of LOX in the esophageal epithelium remains unknown. METHODS: We investigated roles for LOX in the human esophageal epithelium using 3-dimensional organoid and air-liquid interface cultures stimulated with interleukin (IL)13 to recapitulate the EoE inflammatory milieu, followed by single-cell RNA sequencing, quantitative reverse-transcription polymerase chain reaction, Western blot, histology, and functional analyses of barrier integrity. RESULTS: Single-cell RNA sequencing analysis on patient-derived organoids revealed that LOX was induced by IL13 in differentiated cells. LOX-overexpressing organoids showed suppressed basal and up-regulated differentiation markers. In addition, LOX overexpression enhanced junctional protein genes and transepithelial electrical resistance. LOX overexpression restored the impaired differentiation and barrier function, including in the setting of IL13 stimulation. Transcriptome analyses on LOX-overexpressing organoids identified an enriched bone morphogenetic protein (BMP) signaling pathway compared with wild-type organoids. In particular, LOX overexpression increased BMP2 and decreased the BMP antagonist follistatin. Finally, we found that BMP2 treatment restored the balance of basal and differentiated cells. CONCLUSIONS: Our data support a model whereby LOX exhibits noncanonical roles as a signaling molecule important for epithelial homeostasis in the setting of inflammation via activation of the BMP pathway in the esophagus. The LOX/BMP axis may be integral in esophageal epithelial differentiation and a promising target for future therapies.

A lysyl oxidase-responsive collagen peptide illuminates collagen remodeling in wound healing.

Tissue repair and fibrosis involve the dynamic remodeling of collagen, and accurate detection of these sites is of utmost importance. Here, we use a collagen peptide sensor (1) to visualize collagen formation and remodeling during wound healing in mice and humans. We show that the probe binds selectively to sites of collagen formation and remodeling at different stages of healing. Compared to conventional methods, the peptide sensor localizes preferentially to areas of collagen synthesis and remodeling at the wound edge and not in matured fibrillar collagen. We also demonstrate its applicability for in vivo wound imaging and for discerning differential remodeling in wounds of transgenic mice with altered collagen dynamics. Our findings show the value of 1 as a diagnostic tool to rapidly identify the sites of matrix remodeling in tissue sections, which will aid in the conception of new therapeutic strategies for fibrotic disorders and defective tissue repair.

Lysine Deacetylation Is a Key Function of the Lysyl Oxidase Family of Proteins in Cancer.

Mammalian members of the lysyl oxidase (LOX) family of proteins carry a copper-dependent monoamine oxidase domain exclusively within the C-terminal region, which catalyzes ε-amine oxidation of lysine residues of various proteins. However, recent studies have demonstrated that in LOX-like (LOXL) 2-4 the C-terminal canonical catalytic domain and N-terminal scavenger receptor cysteine-rich (SRCR) repeats domain exhibit lysine deacetylation and deacetylimination catalytic activities. Moreover, the N-terminal SRCR repeats domain is more catalytically active than the C-terminal oxidase domain. Thus, LOX is the third family of lysine deacetylases in addition to histone deacetylase and sirtuin families. In this review, we discuss how the LOX family targets different cellular proteins for deacetylation and deacetylimination to control the development and metastasis of cancer.

Inhibiting Lysyl Oxidases prevents pathologic cartilage calcification.

Lysyl oxidases (LOX(L)) are enzymes that catalyze the formation of cross-links in collagen and elastin fibers during physiologic calcification of bone. However, it remains unknown whether they may promote pathologic calcification of articular cartilage, an important hallmark of debilitating arthropathies. Here, we have studied the possible roles of LOX(L) in cartilage calcification, related and not related to their cross-linking activity. We first demonstrated that inhibition of LOX(L) by ß-aminoproprionitrile (BAPN) significantly reduced calcification in murine and human chondrocytes, and in joint of meniscectomized mice. These BAPN's effects on calcification were accounted for by different LOX(L) roles. Firstly, reduced LOX(L)-mediated extracellular matrix cross-links downregulated Anx5, Pit1 and Pit2 calcification genes. Secondly, BAPN reduced collagen fibrotic markers Col1 and Col3. Additionally, LOX(L) inhibition blocked chondrocytes hypertrophic differentiation (Runx2 and COL10), pro-inflammatory IL-6 release and reactive oxygen species (ROS) production, all triggers of chondrocyte calcification. Through unbiased transcriptomic analysis we confirmed a positive correlation between LOX(L) genes and genes for calcification, hypertrophy and extracellular matrix catabolism. This association was conserved throughout species (mouse, human) and tissues that can undergo pathologic calcification (kidney, arteries, skin). Overall, LOX(L) play a critical role in the process of chondrocyte calcification and may be therapeutic targets to treat cartilage calcification in arthropathies.

LOXL4 Shuttled by Tumor Cells-derived Extracellular Vesicles Promotes Immune Escape in Hepatocellular Carcinoma by Activating the STAT1/PD-L1 Axis.

Emerging evidence has validated that extracellular vesicles (EVs) regulate hepatocellular carcinoma (HCC) progression, while its role in HCC immune escape remains to be elucidated. This study investigates the role of EVs-encapsulated lysyl oxidase like-4 (LOXL4) derived from tumor cells in HCC immune escape. HCC-related microarray data sets GSE36376 and GSE87630 were obtained for differential analysis, followed by identifying the essential genes related to the prognosis of HCC patients. Bone marrow-derived macrophages were treated with EVs derived from mouse Hepa 1-6 cells and cocultured with CD8 + T cells to observe the CD8 + T-cell activity. At last, a mouse HCC orthotopic xenograft model was constructed to verify the effects of HCC cell-derived EVs on the immune escape of HCC cells and tumorigenicity in vivo by delivering LOXL4. It was found that ACAT1, C4BPA, EHHADH, and LOXL4 may be the essential genes related to the prognosis of HCC patients. On the basis of the TIMER database, there was a close correlation between LOXL4 and macrophage infiltration in HCC. Besides, STAT1 was closely related to LOXL4. In vitro experiments demonstrated that LOXL4 could induce programmed death-ligand 1 expression in macrophages and immunosuppression by activating STAT1. In vivo experiments also verified that HCC cell-derived EVs promoted the immune escape of HCC cells and tumorigenicity by delivering LOXL4. LOXL4 was delivered into macrophages via EVs to induce programmed death-ligand 1 by activating STAT1 and inhibiting the killing ability of CD8 + T cells to HCC cells, thus promoting immune escape in HCC.

Quercetin antagonized advanced glycated end products induced apoptosis and functional inhibition of fibroblasts from the prolapsed uterosacral ligament.

The altered behaviors and functions of pelvic floor fibroblasts are pathophysiological changes of pelvic organ prolapse (POP). Our previous study showed that advanced glycated end products (AGEs) accumulated in the pelvic tissues of POP and induced fibroblast apoptosis. The study was designed to investigate whether quercetin antagonize AGEs-induced apoptosis and functional inhibition of fibroblasts. The uptake of 5-ethynyl-2'-deoxyuridine (EdU) was evaluated for cell proliferation. Flow cytometric analysis was applied for cell apoptosis. Intracellular reactive oxygen species (ROS) content was determined by the fluorescence of dichlorofluorescein (DCF). The contractility of fibroblasts was measured by collagen gel contraction assay. The expressions of extracellular matrix (ECM) related genes and the expression of miR-4429 and caspase-3 were quantified by qPCR. The expressions of phosphatase and tensin homolog (PTEN), phosphoinositide 3-kinase (PI3K), serine-threonine kinase (Akt), and phosphorylated Akt (p-Akt) were analyzed by Western Blot. The down-regulation of miR-4429 was achieved by cell transfection. Quercetin antagonized AGEs-induced apoptosis, proliferation inhibition, and ROS increase in fibroblasts. Quercetin did not alleviate AGEs-induced contractile impairment of fibroblasts. Quercetin reduced the gene expressions of lysyl oxidase like protein 1 (LOXL1)and matrix metallopeptidase 1 (MMP1), and increased the gene expressions of lysyl oxidase (LOX) and fibrillin 2 (FBN2) in fibroblasts. Quercetin reversed AGEs-induced upregulation of PTEN and downregulation of PI3K, P-Akt, and miR-4429 in fibroblasts. The inhibitory effect of quercetin on AGEs-induced fibroblast apoptosis was inhibited by downregulating the expression of miR-4429. In conclusion, quercetin antagonized AGEs-induced apoptosis and functional inhibition of fibroblasts from the prolapsed uterosacral ligament. And inhibiting AGEs-induced down-regulation of miR-4429/PTEN/PI3K/Akt pathway was the mechanism underlying the antagonistic effect of quercetin on AGEs-induced apoptosis.

Short-Term Effect of Rose Bengal Photodynamic Therapy (RB-PDT) on Collagen I, Collagen V, NF-κB, LOX, TGF-ß and IL-6 Expression of Human Corneal Fibroblasts, In Vitro.

PURPOSE: To investigate collagen I, collagen V, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), lysyl oxidase (LOX), transforming growth factor ß1 (TGF-ß1) and interleukin-6 (IL-6) expression in healthy and keratoconus human corneal fibroblasts (HCFs and KC-HCFs), 24 h after Rose Bengal photodynamic therapy (RB-PDT). METHODS: HCFs were isolated from healthy human corneal donors (n = 5) and KC-HCFs from elective penetrating keratoplasties (n = 5). Both cell cultures underwent RB-PDT (0.001% RB concentration, 0.17 J/cm2 fluence) and 24 h later collagen I, collagen V, NF-κB, LOX, TGF-ß1 and IL-6 mRNA and protein expression have been determined using qPCR and Western blot, IL-6 concentration in the cell culture supernatant by ELISA. RESULTS: TGF-ß1 mRNA expression was significantly lower (p = 0.02) and IL-6 mRNA expression was significantly higher in RB-PDT treated HCFs (p = 0.01), than in HCF controls. COL1A1, COL5A1 and TGF-ß1 mRNA expression was significantly lower (p = 0.04; p = 0.02 and p = 0.003) and IL-6 mRNA expression was significantly higher (p = 0.02) in treated KC-HCFs, than in KC-HCF controls. TGF-ß1 protein expression in treated HCFs was significantly higher than in HCF controls (p = 0.04). IL-6 protein concentration in the HCF and KC-HCF culture supernatant after RB-PDT was significantly higher than in controls (p = 0.02; p = 0.01). No other analyzed mRNA and protein expression differed significantly between the RB-PDT treated and untreated groups. CONCLUSIONS: Our study demonstrates that RB-PDT reduces collagen I, collagen V and TGF-ß1 mRNA expression, while increasing IL-6 mRNA and protein expression in KC-HCFs. In HCFs, RB-PDT increases TGF-ß1 and IL-6 protein level after 24 h.

Fibroblast-Derived Lysyl Oxidase Increases Oxidative Phosphorylation and Stemness in Cholangiocarcinoma.

BACKGROUND & AIMS: Metabolic and transcriptional programs respond to extracellular matrix-derived cues in complex environments, such as the tumor microenvironment. Here, we demonstrate how lysyl oxidase (LOX), a known factor in collagen crosslinking, contributes to the development and progression of cholangiocarcinoma (CCA). METHODS: Transcriptomes of 209 human CCA tumors, 143 surrounding tissues, and single-cell data from 30 patients were analyzed. The recombinant protein and a small molecule inhibitor of the LOX activity were used on primary patient-derived CCA cultures to establish the role of LOX in migration, proliferation, colony formation, metabolic fitness, and the LOX interactome. The oncogenic role of LOX was further investigated by RNAscope and in vivo using the AKT/NICD genetically engineered murine CCA model. RESULTS: We traced LOX expression to hepatic stellate cells and specifically hepatic stellate cell-derived inflammatory cancer-associated fibroblasts and found that cancer-associated fibroblast-driven LOX increases oxidative phosphorylation and metabolic fitness of CCA, and regulates mitochondrial function through transcription factor A, mitochondrial. Inhibiting LOX activity in vivo impedes CCA development and progression. Our work highlights that LOX alters tumor microenvironment-directed transcriptional reprogramming of CCA cells by facilitating the expression of the oxidative phosphorylation pathway and by increasing stemness and mobility. CONCLUSIONS: Increased LOX is driven by stromal inflammatory cancer-associated fibroblasts and correlates with diminished survival of patients with CCA. Modulating the LOX activity can serve as a novel tumor microenvironment-directed therapeutic strategy in bile duct pathologies.

Pan-Cancer Analysis of the LOX Family Reveals that LOX Affects Tumor Prognosis by Affecting Immune Infiltration.

The lysyl oxidase (LOX) gene family encodes for a group of copper-dependent enzymes that play a crucial role in the cross-linking of collagen and elastin fibers in the extracellular matrix (ECM). Dysregulation of LOX gene expression has been implicated in various pathological conditions, including cancer. Several studies have shown that the LOX gene family is involved in cancer progression and metastasis. The goal of this article is to conduct a comprehensive analysis of the LOX family's role in pan-cancer multiplexes. We utilized pan-cancer multi-omics sequencing data from TCGA to investigate the relationship between LOX family genes and tumors at four different levels: mutation, copy number variation, methylation, and gene expression. In addition, we also examined the relationship between LOX family genes and tumors at the cell line level using tumor cell line sequencing data from CCLE. Taking into account the impact of LOX family genes on lung cancer, we developed a LOX family lung cancer prognostic model to forecast the disease's prognosis. Our findings revealed that LOXL2 had the highest mutation frequency in tumors, while all four LOX family genes experienced some degree of copy number variation in diverse tumors. We observed that LOX, LOXL1 to LOXL3 were predominantly highly expressed in tumors including LUAD. The expression trends of LOX and LOXL1 to LOXL3 were consistent across tumor cell lines, but differed somewhat from LOXL4. Utilizing 25 LOX family-related genes, we constructed a LOX family prognostic model that performed well in predicting the prognosis of lung cancer. Through pan-cancer analysis, we gain further knowledge of the role of LOX family genes in different tumors, offering a novel pathway for future research into the relationship between LOX family genes and tumors.

Lysyl oxidase inhibits BMP9-induced osteoblastic differentiation through reducing Wnt/ß-catenin via HIF-1a repression in 3T3-L1 cells.

BACKGROUND: Bone morphogenetic protein 9 (BMP9) is a promising growth factor in bone tissue engineering, while the detailed molecular mechanism underlying BMP9-oriented osteogenesis remains unclear. In this study, we investigated the effect of lysyl oxidase (Lox) on the BMP9 osteogenic potential via in vivo and in vitro experiments, as well as the underlying mechanism. METHODS: PCR assay, western blot analysis, histochemical staining, and immunofluorescence assay were used to quantify the osteogenic markers level, as well as the possible mechanism. The mouse ectopic osteogenesis assay was used to assess the impact of Lox on BMP9-induced bone formation. RESULTS: Our findings suggested that Lox was obviously upregulated by BMP9 in 3T3-L1 cells. BMP9-induced Runx2, OPN, and mineralization were all enhanced by Lox inhibition or knockdown, while Lox overexpression reduced their expression. Additionally, the BMP9-induced adipogenic makers were repressed by Lox inhibition. Inhibition of Lox resulted in an increase in c-Myc mRNA and ß-catenin protein levels. However, the increase in BMP9-induced osteoblastic biomarkers caused by Lox inhibition was obviously reduced when ß-catenin knockdown. BMP9 upregulated HIF-1α expression, which was further enhanced by Lox inhibition or knockdown, but reversed by Lox overexpression. Lox knockdown or HIF-1α overexpression increased BMP9-induced bone formation, although the enhancement caused by Lox knockdown was largely diminished when HIF-1α was knocked down. Lox inhibition increased ß-catenin levels and decreased SOST levels, which were almost reversed by HIF-1α knockdown. CONCLUSION: Lox may reduce the BMP9 osteoblastic potential by inhibiting Wnt/ß-catenin signaling via repressing the expression HIF-1α partially.

Targeting Lysyl Oxidase as a Potential Therapeutic Approach to Reducing Fibrotic Scars Post-operatively: Its Biological Role in Post-Surgical Scar Development.

Abdominal and pelvic surgery, or any surgical injury of the peritoneum, often leads to chronic abdominal adhesions that may lead to bowel obstruction, infertility, and pain. Current therapeutic strategies are usually ineffective, and the pathological mechanisms of the disease are unclear. Excess collagen cross-linking is a key mediator for extra-cellular matrix deposition and fibrogenesis. Lysyl oxidase is a key enzyme that catalyzes the formation of stabilizing cross-links in collagen. Dysregulation of Lysyl oxidase (Lox) expressing upregulates collagen cross-linking, leading ECM deposition. Tissue hypoxia during surgery induces molecular mechanisms and active transcription factors to promote the expression of several genes related to inflammation, oxidative stress, and fibrosis, such as transforming growth factor beta, and Lox. Studies have shown that targeting Lox improves clinical outcomes and fibrotic parameters in liver, lung, and myocardial fibrosis, therefore, Lox may be a potential drug target in the prevention of postsurgical adhesion.

Sox6 impairs the adipogenic commitment of mesenchymal stem cells by targeting lysyl oxidase and preadipocyte factor 1.

The commitment of mesenchymal stem cells (MSCs) to preadipocytes and the termination of differentiation to adipocytes are critical for maintaining systemic energy homeostasis. However, our knowledge of the molecular mechanisms governing the commitment of MSCs to preadipocytes and the subsequent termination of their differentiation into adipocytes remain limited. Additionally, the role of Sox6 sex-determining region Y (SRY)-box6 (Sox6), a transcription factor that regulates gene transcription, is reportedly involved in various cellular processes, including adipogenesis; however, its function in regulating preadipocyte development and the factors involved in the termination of adipogenic differentiation remain unexplored. Therefore, we investigated the role of Sox6 in regulating the differentiation of adipocytes by monitoring the effects of its overexpression in C3H10T1/2 cells (in vitro) and C57BL/6J mouse (in vivo) models of adipogenesis. We observed lower Sox6 expression in the adipose tissue of obese mice than that in control mice. Sox6 overexpression inhibited the differentiation of MSC by directly binding to the lysyl oxidase (Lox) and preadipocyte factor 1 (Pref1) promoters, which was potentiated by histone deacetylase-1(HDAC1). Our findings suggest that Sox6 is a key regulator of MSC commitment to adipocytes; therefore, targeting the Sox6-mediated regulation of this process could offer potential therapeutic avenues for addressing obesity and related metabolic disorders.

The possibilities of LOXL4 as a prognostic marker for carcinomas.

Lysyl oxidase-like 4 (LOXL4), a member of lysyl oxidase family, is a copper and lysine tyrosylquinone-dependent amine oxidase that serves the role of catalyzing the cross-linking of elastin and collagen in the extracellular matrix. Numerous studies have shown a significant association between LOXL4 expression levels and tumor proliferation, migration, invasion and patients' prognosis and overall survival in different types of tumors. Here we review their relationship and the molecular pathogenesis behind them, aiming to explore the possibilities of LOXL4 as a prognostic marker for diverse carcinomas and provide some indications for further research in this field.

Carcinoma-associated fibroblast-derived lysyl oxidase-rich extracellular vesicles mediate collagen crosslinking and promote epithelial-mesenchymal transition via p-FAK/p-paxillin/YAP signaling.

Carcinoma-associated fibroblasts (CAFs) are the main cellular components of the tumor microenvironment and promote cancer progression by modifying the extracellular matrix (ECM). The tumor-associated ECM is characterized by collagen crosslinking catalyzed by lysyl oxidase (LOX). Small extracellular vesicles (sEVs) mediate cell-cell communication. However, the interactions between sEVs and the ECM remain unclear. Here, we demonstrated that sEVs released from oral squamous cell carcinoma (OSCC)-derived CAFs induce collagen crosslinking, thereby promoting epithelial-mesenchymal transition (EMT). CAF sEVs preferably bound to the ECM rather than being taken up by fibroblasts and induced collagen crosslinking, and a LOX inhibitor or blocking antibody suppressed this effect. Active LOX (αLOX), but not the LOX precursor, was enriched in CAF sEVs and interacted with periostin, fibronectin, and bone morphogenetic protein-1 on the surface of sEVs. CAF sEV-associated integrin α2ß1 mediated the binding of CAF sEVs to collagen I, and blocking integrin α2ß1 inhibited collagen crosslinking by interfering with CAF sEV binding to collagen I. CAF sEV-induced collagen crosslinking promoted the EMT of OSCC through FAK/paxillin/YAP pathway. Taken together, these findings reveal a novel role of CAF sEVs in tumor ECM remodeling, suggesting a critical mechanism for CAF-induced EMT of cancer cells.

The Expression Pattern and Clinical Significance of Lysyl Oxidase Family in Gliomas.

LOX (Lysyl oxidase) family participates in the catalysis of collagen and elastin to maintain ECM homeostasis. Glioma is the most common primary brain tumor and LOX family has not been systemic studied in glioma. In this study, we found LOX family members are upregulated expressed in gliomas samples. A protein-protein interaction network (PPIN) was construct to visualize and understand the differential expression pattern, as well as functional annotation, for LOX family and their interacting proteins, which involved in collagen fibril organization and MAPK signaling pathway. Through subcellular localization distribution, the LOX family members distribute both intracellular and extracellular. All five LOX members are consistently significantly correlate with dendritic cell both in immune infiltrate of GBM and LGG. Survival analysis showed that high expression of LOX family is associated with a poor prognosis of gliomas patients. These analyses provide important clues to identify the potential biological roles for LOX family in gliomas, which might serve as diagnosis markers.

Exploring the Interplay between Polyphenols and Lysyl Oxidase Enzymes for Maintaining Extracellular Matrix Homeostasis.

Collagen, the most abundant structural protein found in mammals, plays a vital role as a constituent of the extracellular matrix (ECM) that surrounds cells. Collagen fibrils are strengthened through the formation of covalent cross-links, which involve complex enzymatic and non-enzymatic reactions. Lysyl oxidase (LOX) is responsible for catalyzing the oxidative deamination of lysine and hydroxylysine residues, resulting in the production of aldehydes, allysine, and hydroxyallysine. These intermediates undergo spontaneous condensation reactions, leading to the formation of immature cross-links, which are the initial step in the development of mature covalent cross-links. Additionally, non-enzymatic glycation contributes to the formation of abnormal cross-linking in collagen fibrils. During glycation, specific lysine and arginine residues in collagen are modified by reducing sugars, leading to the creation of Advanced Glycation End-products (AGEs). These AGEs have been associated with changes in the mechanical properties of collagen fibers. Interestingly, various studies have reported that plant polyphenols possess amine oxidase-like activity and can act as potent inhibitors of protein glycation. This review article focuses on compiling the literature describing polyphenols with amine oxidase-like activity and antiglycation properties. Specifically, we explore the molecular mechanisms by which specific flavonoids impact or protect the normal collagen cross-linking process. Furthermore, we discuss how these dual activities can be harnessed to generate properly cross-linked collagen molecules, thereby promoting the stabilization of highly organized collagen fibrils.

The Lysyl Oxidase G473A Polymorphism Exacerbates Oral Cancer Development in Humans and Mice.

Oral cancer is primarily squamous-cell carcinoma with a 5-year survival rate of approximately 50%. Lysyl oxidase (LOX) participates in collagen and elastin maturation. The propeptide of LOX is released as an 18 kDa protein (LOX-PP) in the extracellular environment by procollagen C-proteinases and has tumor-inhibitory properties. A polymorphism in the propeptide region of LOX (rs1800449, G473A) results in a single amino acid substitution of Gln for Arg. Here we investigated the frequency of rs1800449 in OSCC employing TCGA database resources and determined the kinetics and severity of precancerous oral lesion development in wildtype and corresponding knockin mice after exposure to 4-nitroquinoline oxide (4 NQO) in drinking water. Data show that the OSCC is more common in humans carrying the variant compared to the wildtype. Knockin mice are more susceptible to lesion development. The immunohistochemistry of LOX in mouse tissues and in vitro studies point to a negative feedback pathway of wildtype LOX-PP on LOX expression that is deficient in knockin mice. Data further demonstrate modulations of T cell phenotype in knockin mice toward a more tumor-permissive condition. Data provide initial evidence for rs1800449 as an oral cancer susceptibility biomarker and point to opportunities to better understand the functional mechanism of LOX-PP cancer inhibitory activity.

CD11b Deficiency Favors Cartilage Calcification via Increased Matrix Vesicles, Apoptosis, and Lysyl Oxidase Activity.

Pathological cartilage calcification is a hallmark feature of osteoarthritis, a common degenerative joint disease, characterized by cartilage damage, progressively causing pain and loss of movement. The integrin subunit CD11b was shown to play a protective role against cartilage calcification in a mouse model of surgery-induced OA. Here, we investigated the possible mechanism by which CD11b deficiency could favor cartilage calcification by using naïve mice. First, we found by transmission electron microscopy (TEM) that CD11b KO cartilage from young mice presented early calcification spots compared with WT. CD11b KO cartilage from old mice showed progression of calcification areas. Mechanistically, we found more calcification-competent matrix vesicles and more apoptosis in both cartilage and chondrocytes isolated from CD11b-deficient mice. Additionally, the extracellular matrix from cartilage lacking the integrin was dysregulated with increased collagen fibrils with smaller diameters. Moreover, we revealed by TEM that CD11b KO cartilage had increased expression of lysyl oxidase (LOX), the enzyme that catalyzes matrix crosslinks. We confirmed this in murine primary CD11b KO chondrocytes, where Lox gene expression and crosslinking activity were increased. Overall, our results suggest that CD11b integrin regulates cartilage calcification through reduced MV release, apoptosis, LOX activity, and matrix crosslinking. As such, CD11b activation might be a key pathway for maintaining cartilage integrity.

SPECT Imaging of Lysyl Oxidase-like 2 in a Model of Idiopathic Pulmonary Fibrosis.

Noninvasive imaging of idiopathic pulmonary fibrosis (IPF) remains a challenge. The aim of this study was to develop an antibody-based radiotracer targeting Lysyl Oxidase-like 2 (LOXL2), an enzyme involved in the fibrogenesis process, for SPECT/CT imaging of pulmonary fibrosis. The bifunctional chelator DOTAGA-PEG4-NH2 was chemoenzymatically conjugated to the murine antibody AB0023 using microbial transglutaminase, resulting in a degree of labeling (number of chelators per antibody) of 2.3. Biolayer interferometry confirmed that the binding affinity of DOTAGA-AB0023 to LOXL2 was preserved with a dissociation constant of 2.45 ± 0.04 nM. DOTAGA-AB0023 was then labeled with 111In and in vivo experiments were carried out in a mice model of progressive pulmonary fibrosis induced by intratracheal administration of bleomycin. [111In]In-DOTAGA-AB0023 was injected in three groups of mice (control, fibrotic, and treated with nintedanib). SPECT/CT images were recorded over 4 days p.i. and an ex vivo biodistribution study was performed by gamma counting. A significant accumulation of the tracer in the lungs of the fibrotic mice was observed at D18 post-bleomycin. Interestingly, the tracer uptake was found selectively upregulated in fibrotic lesions observed on CT scans. Images of mice that received the antifibrotic drug nintedanib from D8 up to D18 showed a decrease in [111In]In-DOTAGA-AB0023 lung uptake associated with a decrease in pulmonary fibrosis measured by CT scan. In conclusion, we report the first radioimmunotracer targeting the protein LOXL2 for nuclear imaging of IPF. The tracer showed promising results in a preclinical model of bleomycin-induced pulmonary fibrosis, with high lung uptake in fibrotic areas, and accounted for the antifibrotic activity of nintedanib.

LOXL4, but not LOXL2, is the critical determinant of pathological collagen cross-linking and fibrosis in the lung.

Idiopathic pulmonary fibrosis is a progressive fibrotic disease characterized by excessive deposition of (myo)fibroblast produced collagen fibrils in alveolar areas of the lung. Lysyl oxidases (LOXs) have been proposed to be the central enzymes that catalyze the cross-linking of collagen fibers. Here, we report that, while its expression is increased in fibrotic lungs, genetic ablation of LOXL2 only leads to a modest reduction of pathological collagen cross-linking but not fibrosis in the lung. On the other hand, loss of another LOX family member, LOXL4, markedly disrupts pathological collagen cross-linking and fibrosis in the lung. Furthermore, knockout of both Loxl2 and Loxl4 does not offer any additive antifibrotic effects when compared to Loxl4 deletion only, as LOXL4 deficiency decreases the expression of other LOX family members including Loxl2. On the basis of these results, we propose that LOXL4 is the main LOX activity underlying pathological collagen cross-linking and lung fibrosis.