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Introduction Lysyl oxidase-like 4 is an amine oxidase from the lysyl oxidase family that was previously shown to be overexpressed in head and neck cancer and upregulated in response to hypoxia. The possible role of lysyl oxidase-like 4 as a tumor marker in advanced stage larynx cancer was investigated. Objective To investigate the expression of lysyl Oxidase-Like 4 protein in advanced stage laryngeal cancer and elucidate its possible role as a tumor marker, predictor of treatment response and prognosticator. Methods Diagnostic specimens of 72 patients treated for stage III–IV laryngeal squamous cell carcinoma were evaluated for lysyl oxidase-like 4 expression by immunohistochemistry. Results Lysyl oxidase-like 4 expression was correlated with advanced tumor stage (p = 0.041) and better differentiation (p = 0.025) but was independent of tumor diameter (p = 0.456). Response to induction chemotherapy or the need for salvage laryngectomy were not affected by lysyl oxidase-like 4 expression (p = 0.999, p = 0.070 respectively). Increased lysyl oxidase-like 4 expression was associated with better 2 year overall survival in both univariate (p = 0.036) and multivariate analyses (p = 0.014). Conclusion Lysyl oxidase-like 4 expression emerges with advancing stages, is lost with worsening differentiation, and may have tumor suppressive properties in larynx cancer.

International audience; Lysyl oxidases are major actors of microenvironment and extracellular matrix (ECM) remodeling. These cross-linking enzymes are thus involved in many aspects of physiopathology, including tumor progression, fibrosis and cardiovascular diseases. We have already shown that Lysyl Oxidase-Like 2 (LOXL2) regulates collagen IV deposition by endothelial cells and angiogenesis. We here provide evidence that LOXL2 also affects deposition of other ECM components, including fibronectin, thus altering structural and mechanical properties of the matrix generated by endothelial cells. LOXL2 interacts intracellularly and directly with collagen IV and fibronectin before incorporation into ECM fibrillar structures upon exocytosis, as demonstrated by TIRF time-lapse microscopy. Furthermore, surface plasmon resonance experiments using recombinant scavenger receptor cysteine-rich (SRCR) domains truncated for the catalytic domain demonstrated their direct binding to collagen IV. We thus used directed mutagenesis to investigate the role of LOXL2 catalytic domain. Neither enzyme activity nor catalytic domain were necessary for collagen IV deposition and angiogenesis, whereas the SRCR domains were effective for these processes. Finally, surface coating with recombinant SRCR domains restored deposition of collagen IV by LOXL2-depleted cells. We thus propose that LOXL2 SRCR domains orchestrate scaffolding of the vascular basement membrane and angiogenesis through interactions with collagen IV and fibronectin, independently of the enzymatic crosslinking activity.

Organ fibrosis is characterized by epithelial injury and aberrant tissue repair, where activated effector cells, mostly fibroblasts and myofibroblasts, excessively deposit collagen into the extracellular matrix. Fibrosis frequently results in organ failure and has been estimated to contribute to at least one third of all global deaths. Also lung fibrosis, in particular idiopathic pulmonary fibrosis (IPF), is a fatal disease with rising incidence worldwide. As current treatment options targeting fibrogenesis are insufficient, there is an urgent need for novel therapeutic strategies. During the last decade, several studies have proposed to target intra- and extracellular components of the collagen biosynthesis, maturation, and degradation machinery. This includes intra- and extracellular targets directly acting on collagen gene products, but also such that anabolize essential building blocks of collagen, in particular glycine and proline biosynthetic enzymes. Collagen, however, is a ubiquitous molecule in the body and fulfils essential functions as a macromolecular scaffold, growth factor reservoir, and receptor binding site in virtually every tissue. This review summarizes recent advances and future directions in this field. Evidence for the proposed therapeutic targets and where they currently stand in terms of clinical drug development for treatment of fibrotic disease is provided. The drug targets are furthermore discussed in light of (1) specificity for collagen biosynthesis, maturation and degradation, and (2) specificity for disease-associated collagen. As therapeutic success and safety of these drugs may largely depend on targeted delivery, different strategies for specific delivery to the main effector cells and to the extracellular matrix are discussed.

Objective Aortic aneurysm and dissection are major life-threatening complications of Marfan syndrome. Avoiding factors that promote aortic damage is critical in managing the care of these patients. Findings from clinical and animal studies raise concerns regarding fluoroquinolone use in patients at risk for aortic aneurysm and dissection. Therefore, we examined the effects of ciprofloxacin on aortic aneurysm and dissection development in Marfan mice. Methods Eight-week-old Marfan mice (Fbn1C1041G/+) were given ciprofloxacin (100 mg/kg/d; n = 51) or vehicle (n = 59) for 4 weeks. Mice were monitored for 16 weeks. Aortic diameters were measured by using ultrasonography, and aortic structure was examined by using histopathologic and immunostaining analyses. Results Vehicle-treated Fbn1C1041G/+ mice showed progressive aortic enlargement, with aortic rupture occurring in 5% of these mice. Compared with vehicle-treated Fbn1C1041G/+ mice, ciprofloxacin-treated Fbn1C1041G/+ mice showed accelerated aortic enlargement (P = .01) and increased incidences of aortic dissection (25% vs 47%, P = .03) and rupture (5% vs 25%, P = .005). Furthermore, ciprofloxacin-treated Fbn1C1041G/+ mice had higher levels of elastic fiber fragmentation, matrix metalloproteinase expression, and apoptosis than did vehicle-treated Fbn1C1041G/+ mice. Conclusions Ciprofloxacin accelerates aortic root enlargement and increases the incidence of aortic dissection and rupture in Marfan mice, partially by suppressing lysyl oxidase expression and further compromising the inherited defect in aortic elastic fibers. Our findings substantiate that ciprofloxacin should be avoided in patients with Marfan syndrome.

Accumulating evidence from both animal and human studies suggests that activation of beige fat increases cellular energy expenditure, ultimately reducing adiposity. Here, we report the central role of adipocyte-derived lysyl oxidase (Lox) in the formation of thermogenic beige fat. Mice exposed to cold or a β3 agonist showed drastically lower Lox expression in thermogenically activated beige fat. Importantly, inhibition of Lox activity with BAPN stimulated biogenesis of beige fat in inguinal white adipose tissue (iWAT) under housing conditions and potentiated cold-induced adaptive thermogenesis and beiging in both iWAT and epididymal white adipose tissue (eWAT). Notably, white adipocytes with Lox repression undergo transdifferentiation into beige adipocytes which can be suppressed by tumor necrosis factor-α (TNFα) via ERK activation. This work provides new insight into the molecular control to expand beige fat by Lox inhibition and suggest the potential for utilizing inhibitor of Lox to treat the emerging epidemics of obesity and diabetes.

In obese adipose tissue (AT), hypertrophic expansion of adipocytes is not matched by new vessel formation, leading to AT hypoxia. As a result, hypoxia inducible factor-1⍺ (HIF-1⍺) accumulates in adipocytes inducing a transcriptional program that upregulates profibrotic genes and biosynthetic enzymes such as lysyl oxidase (LOX) synthesis. This excess synthesis and crosslinking of extracellular matrix (ECM) components cause AT fibrosis. Although fibrosis is a hallmark of obese AT, the role of fibroblasts, cells known to regulate fibrosis in other fibrosis-prone tissues, is not well studied. Here we have developed an in vitro model of AT to study adipocyte-fibroblast crosstalk in a hypoxic environment. Further, this in vitro model was used to investigate the effect of hypoxia on adipocyte mechanical properties via ras homolog gene family member A (RhoA)/Rho-associated coiled-coil kinases (ROCK) signaling pathways. We confirmed that hypoxia creates a diseased phenotype by inhibiting adipocyte maturation and inducing actin stress fiber formation facilitated by myocardin-related transcription factor A (MRTF-A/MKL1) nuclear translocation. This work presents new potential therapeutic targets for obesity by improving adipocyte maturation and limiting mechanical stress in obese AT.

Background: The effects of Nerve growth factor (NGF) in chondrosarcoma are not confirmed, although NGF is capable of promoting the progression and metastasis of several different types of tumors. Here we aim to explore the role of NGF in chondrosarcoma and elucidate how NGF acts.Methods: Immunohistochemistry (IHC)-stained tissue samples from chondrosarcoma patients were stained with NGF and LOX antibodies. Cell migration was examined by Transwell migration and invasion assays. The expression levels of LOX, microRNA-149-5p (miR-149-5p) were measured by quantitative real-time polymerase chain reaction. LOX, PI3K, Akt, and mTOR protein expression were examined by Western blot assays. The interaction between LOX 3’-UTRs and miR-149-5p binding site was explored by luciferase assay. We established the orthotopic in vivo model of chondrosarcoma lung metastasis to further investigate the promoting effects of NGF in metastatic chondrosarcoma. Results: Here, we found that the levels of NGF and lysyl oxidase (LOX) correlated with tumor stage in patients with chondrosarcoma. NGF facilitated LOX-dependent cellular migration in human chondrosarcoma JJ012 cells, while overexpression of NGF enhanced lung metastasis in a mouse model of chondrosarcoma. NGF promoted LOX synthesis and cell migration by inhibiting miR-149-5p expression through the PI3K, Akt and mTOR signaling cascades. NGF appears to be a worthwhile therapeutic target in the treatment of metastatic chondrosarcoma.Conclusions: Our study has identified that NGF promotes LOX-dependent cell migration in human chondrosarcoma tissue by inhibiting miR-149-5p synthesis via the PI3K, Akt and mTOR signaling cascades

Metastasis is the main cause of cancer-related mortality. Despite intense efforts to understand the mechanisms underlying the metastatic process, treatment of metastatic cancer is still challenging. Here we describe a chemotherapy-induced, host-mediated mechanism that promotes remodeling of the extracellular matrix (ECM), ultimately facilitating cancer cell seeding and metastasis. Paclitaxel (PTX) chemotherapy enhanced rapid ECM remodeling and mechanostructural changes in the lungs of tumor-free mice, and the protein expression and activity of the ECM remodeling enzyme lysyl oxidase (LOX) increased in response to PTX. A chimeric mouse model harboring genetic LOX depletion revealed chemotherapy-induced ECM remodeling was mediated by CD8+ T cells expressing LOX. Consistently, adoptive transfer of CD8+ T cells, but not CD4+ T cells or B cells, from PTX-treated mice to naïve immunodeprived mice induced pulmonary ECM remodeling. Lastly, in a clinically relevant metastatic breast carcinoma model, LOX inhibition counteracted the metastasis-promoting, ECM-related effects of PTX. This study highlights the role of immune cells in regulating ECM and metastasis following chemotherapy, suggesting that inhibiting chemotherapy-induced ECM remodeling represents a potential therapeutic strategy for metastatic cancer. Significance: Chemotherapy induces prometastatic pulmonary ECM remodeling by upregulating LOX in T cells, which can be targeted with LOX inhibitors to suppress metastasis. See related commentary by Kolonin and Woodward, p. 197

Introduction Photoaging is the process by which ultraviolet rays gradually induce clinical and histological changes in the skin through the production and organization of biological molecules, such as elastin, which is critical to skin strength and elasticity. After exposure to radiation, elastin may undergo alternative mRNA splicing, resulting in modified proteins that contribute to the formation of aging characteristics, such as solar elastosis. The present work aimed to study two different forms of elastin under these conditions: normal elastin and elastin that had been altered in exon 26A. Methods These different forms of elastin were characterized for gene expression by quantitative real-time polymerase chain reaction (qPCR) and for protein expression by immunohistochemistry of ex vivo skins (from photoexposed and non-photoexposed areas) and in vitro reconstituted skin. In addition, up- and downstream molecules in the elastin signaling cascade were evaluated. Results As a result, a significant increase in the gene expression of elastin 26A was observed in both ex vivo photoexposed skin tissues and the in vitro photoexposed reconstituted skins. Additionally, significant increases in the gene expression levels of matrix metalloproteinase-12 (MMP12) and lysyl oxidase (LOX) were observed in the ex vivo skin model. The evaluation of protein expression levels of some photoaging markers on the reconstituted skin revealed increased tropoelastin and fibrillin-1 expression after photoexposure. Conclusion This work contributes to a better understanding of the biological mechanisms involved in photoaging, making it possible to obtain new strategies for the development of dermocosmetic active ingredients to prevent and treat skin aging.

Objective Abdominal aortic aneurysm (AAA) is a significant medical problem with a high mortality rate. Nevertheless, the underlying mechanism for the progression and regression of AAA is unknown. Methods Experimental model of AAA was first created by porcine pancreatic elastase incubation around the infrarenal aorta of C57BL/6 mice. Then, AAA progression and regression were evaluated based on the diameter and volume of AAA. The aortas were harvested for hematoxylin-eosin staining (HE), orcein staining, sirius red staining, immunofluorescence analysis and perls' prussian blue staining at the indicated time point. Finally, β-aminopropionitrile monofumarate (BAPN) was used to explore the underlying mechanism of the regression of AAA. Results When we extended the observation period to 100 days, we not only observed an increase in the AAA diameter and volume in the early stage, but also a decrease in the late stage. Consistent with AAA diameter and volume, the aortic thickness showed the same tendency based on HE staining. The elastin and collagen content first degraded and then regenerated, which corresponds to the early deterioration and late regression of AAA. Then, endogenous up-regulation of lysyl oxidase (LOX) was detected, accompanying the regression of AAA, as detected by an immunofluorescent assay. BAPN and LOX inhibitor considerably inhibited the regression of AAA, paralleling the degradation of elastin lamella and collagen. Conclusion Taken together, we tentatively conclude that endogenous re-generation of LOX played an influential role in the regression of AAA. Therefore, regulatory factors on the generation of LOX exhibit promising therapeutic potential against AAA.

Inflammatory factors have been considered to contribute to keratoconus (KC) pathogenesis. This study aims to determine the immune cells subsets and soluble inflammatory factor profile on the ocular surface of KC patients. 32 KC subjects (51 eyes) across different grades of severity and 15 healthy controls (23 eyes) were included in the study. Keratometry and pachymetry measurements were recorded. Ocular surface immune cells (collected by ocular surface wash) immunophenotyped using flow cytometry include leukocytes, neutrophils, macrophages, natural killer (NK) cells, pan-T cells, gamma delta T (gamma delta T) cells and NKT cells. Tear fluid collected using Schirmer's strip was used to measure 50 soluble factors by multiplex ELISA. Proportions of activated neutrophils, NK cells and gamma delta T cells were significantly increased in KC patients. Significantly higher levels of tear fluid IL-1 beta, IL-6, LIF, IL-17A, TNF alpha, IFN alpha/beta/gamma, EPO, TGF beta 1, PDGF-BB, sVCAM, sL-selectin, granzyme-B, perforin, MMP2, sFasL and IgE, along with significantly lower levels of IL-1 alpha and IL-9 were observed in KC patients. Alterations observed in few of the immuno-inflammatory parameters correlated with grades of disease, allergy, eye rubbing and keratometry or pachymetry measurements. The observation implies a distinct immuno-inflammatory component in KC pathogenesis and its potential as an additional therapeutic target in KC management.

In the three-dimensional (3D) tumor microenvironment, matrix stiffness is associated with the regulation of tumor cells behaviors. In vitro tumor models with appropriate matrix stiffness are urgently desired. Herein, we prepare 3D decellularized extracellular matrix (DECM) scaffolds with different stiffness to mimic the microenvironment of human breast tumor tissue, especially the matrix stiffness, components and structure of ECM. Furthermore, the effects of matrix stiffness on the drug resistance of human breast cancer cells are explored with these developed scaffolds as case studies. Our results confirm that DECM scaffolds with diverse stiffness can be generated by tumor cells with different lysyl oxidase (LOX) expression levels, while the barely intact structure and major components of the ECM are maintained without cells. This versatile 3D tumor model with suitable stiffness can be used as a bioengineered tumor scaffold to investigate the role of the microenvironment in tumor progression and to screen drugs prior to clinical use to a certain extent., Graphical abstract Image 1, Highlights • Novel 3D bioengineered tumor scaffolds with different stiffness were developed. • Cells with different LOX expression levels were used to generate tumor tissue. • DECM scaffold has good cytocompatibility. • DECM with high stiffness promotes the resistance of MDA-MB-231 cells to cisplatin. • DECM with high stiffness increases the expression of Bcl-2 and ABCB1.

Small extracellular vesicles (sEVs) operate as a signaling platform due to their ability to carry functional molecular cargos. However, the role of sEVs in hypoxic tumor microenvironment-mediated premetastatic niche formation remains poorly understood. Methods: Protein expression profile of sEVs derived from normoxic and hypoxic head and neck squamous cell carcinoma (HNSCC) cells were determined by Isobaric Tagging Technology for Relative Quantitation. In vitro invasion assay and in vivo colonization were performed to evaluate the role of sEV-delivering proteins. Results: We identified lysyl oxidase like 2 (LOXL2) which had the highest fold increase in hypoxic sEVs compared with normoxic sEVs. Hypoxic cell-derived sEVs delivered high amounts of LOXL2 to non-hypoxic HNSCC cells to elicit epithelial-to-mesenchymal transition (EMT) and induce the invasion of the recipient cancer cells. Moreover, LOXL2-enriched sEVs were incorporated by distant fibroblasts and activate FAK/Src signaling in recipient fibroblasts. Increased production of fibronectin mediated by FAK/Src signaling recruited myeloid-derived suppressor cells to form a premetastatic niche. Serum sEV LOXL2 can reflect a hypoxic and aggressive tumor type and can serve as an alternative to tissue LOXL2 as an independent prognostic factor of overall survival for patients with HNSCC. Conclusion: sEVs derived from the hypoxic tumor microenvironment of HNSCC can drive local invasion of non-hypoxic HNSCC cells and stimulate premetastatic niche formation by delivering LOXL2 to non-hypoxic HNSCC cells and fibroblasts to induce EMT and fibronectin production, respectively.

In recent years, breakthroughs in the field of tumor immunotherapy with immune checkpoint inhibitors (ICIs) have made a therapeutic revolution, which has been shown to improve the prognosis of patients with hepatocellular carcinoma (HCC). Immune infiltrates represent a major component of tumor microenvironment (TME), and play an essential role in both tumor progression and therapeutic response. The major unmet challenge in tumor immunotherapy is exploring the intrinsic and extrinsic mechanisms of TME promoting the management of HCC. Lysyl oxidase like 3 (LOXL3) participates in the remodeling of extracellular matrix (ECM) and the cross-linking of collagen and elastic fibers. It has been reported that LOXL3 is associated with the development and tumorigenesis of multiple types of cancer. RNA sequencing data and corresponding clinical information were extracted from The Cancer Genome Atlas (TCGA) databases, then subjected to gene expression, tumor microenvironment, survival, enrichment analyses utilizing R packages. In this study, we first found that LOXL3 gene was upregulated in tumor tissues compared with the normal tissues. Furthermore, LOXL3 expression is positively correlated with the infiltration of multiple immune cells and the expression of immune checkpoint genes in HCC. Meanwhile, high LOXL3 expression predicted poor outcomes of the patients with HCC. Functional enrichment analysis suggested that LOXL3 was mainly linked to extracellular structure and matrix organization, cell-cell adhesion, and T cell activation. This is the first comprehensive study to indicate that LOXL3 is correlated with immune infiltrates and may serve as a novel biomarker predicting prognosis and immunotherapy in HCC.

Introduction: Enzymatic cross-linking of the collagens within the extracellular matrix (ECM) catalyzed by enzymes such as lysyl oxidase (LOX) and lysyl oxidase like-enzymes 1–4 (LOXL), transglutaminase 2 (TG2), and peroxidasin (PXDN) contribute to fibrosis progression through extensive collagen cross-linking. Studies in recent years have begun elucidating the important role of collagen cross-linking in perpetuating progression of organ fibrosis independently of inflammation through an increasingly stiff and noncompliant ECM. Therefore, collagen cross-linking and the cross-linking enzymes have become new targets in anti-fibrotic therapy as well as targets of novel biomarkers to properly assess resolution of the fibrotic ECM. Areas covered: The enzymatic actions of enzymes catalyzing collagen cross-linking and their relevance in organ fibrosis. Potential biomarkers specifically quantifying proteolytic fragments of collagen cross-linking is discussed based on Pubmed search done in November 2020 as well as the authors knowledge. Expert opinion: Current methods for the assessment of fibrosis involve the use of invasive and/or cumbersome and expensive methods such as tissue biopsies. Thus, an unmet need exists for the development and validation of minimally invasive biomarkers of proteolytic fragments of cross-linked collagens. These biomarkers may aid in the development and proper assessment of fibrosis resolution in coming years.

Muscle texture, which plays an important role in the acceptability and commercialization of seafood products, can be affected by collagen, the principal component of connective tissue in animals. I...

According to clinical studies, the use of fluoroquinolone antibacterial agents is associated with such rare, but serious adverse reactions as aortic injuries. The aim of the study was to analyse scientific literature data on the risk of aortic injury during fluoroquinolone treatment. The analytical review showed that the risk factors for fluoroquinolone-induced aortic injury are male gender, age over 45 years, underlying aortic disease, as well as smoking and associated atherosclerosis. Clinical and morphological forms of fluoroquinolone-associated aortic injuries include dilatation (aneurysm development), dissection, and rupture. The analysis of data on the association between aortic injuries and the use of most common fluoroquinolones (ciprofloxacin, levofloxacin, and moxifloxacin) showed that development of aneurysm and dissection was most often observed for levofloxacin, and least often for ciprofloxacin. The mechanism of aortic injury is due to fluoroquinolone-mediated activation of matrix metalloproteinases which damage elastic components of vascular walls, as well as reduction in lysyl oxidase expression and collagen synthesis. The ability of fluoroquinolones to form complexes with magnesium ions reduces the availability of magnesium to the cell enzyme systems, which delays synthesis of extracellular matrix structural proteins, leads to metalloproteinase activation and calcification of the vascular walls. Prevention, early detection, and timely management of the above-mentioned issues depend on the awareness of different medical specialists about the risks of aortic injury associated with the use of fluoroquinolone antibiotics.

Canine mammary tumors (CMT) are the second most common form of neoplasia accounting for 40% of tumors in dogs. Today, as we know cancer is a disease of multiple genes and molecules. Research across the last three decades has enhanced our understanding of these molecules which is making possible new strategies of predicting, diagnosing and clinical treatment of this malady. Biomarkers are the modern day “neoplastic signatures” which enhances the specificity of tumor diagnosis allowing the researchers to differentiate tumors at the molecular level. This review enlists a concise summary of novel serum biomarkers counting matrix metalloproteinases, lysyl oxidase, heat shock proteins, mammaglobin, kynurenine 3-monooxygenase, WT1, nectin 4, and ERBB2. These new-fangled markers of canine malignancy could might help in early and accurate diagnosis resulting in more effective treatment and better prognosis. Nevertheless, more detailed research of these biomarkers along with their role in other cancers should be further validated in future.

Background: The aim of this study was to investigate associations between polymorphisms in the Lysyl oxidase (LOX) gene with susceptibility to cancer. The role of LOX in carcinogenesis has prompted...

Background and aims Lysyl oxidase-like 4 (LOXL4) is an amine oxidase that is primarily involved in extracellular matrix remodeling and is highly expressed in HCC tissues, but its functional role in mediating liver carcinogenesis is poorly understood. Therefore, we aimed to investigate the role of LOXL4 in hepatocarcinogenesis. Approach and results Here, we demonstrate that hepatic LOXL4 expression was increased during the liver carcinogenesis in mice concomitantly fed a choline-deficient, l-amino acid-defined diet. LOXL4 was secreted by the neoplastic cells and primarily localized within hepatic macrophages through exosome internalization. Supplementation of LOXL4 had minimal effect on neoplastic cells. In vitro exposure of macrophages to LOXL4 invoked an immunosuppressive phenotype and activated programmed death ligand 1 (PD-L1) expression, which further suppressed the function of CD8+ T cells. Injection of LOXL4 promoted macrophages infiltration into the liver and accelerated tumor growth, which was further abolished by adoptive T-cell transfer or PD-L1 neutralization. Label-free proteomics analysis revealed that the immunosuppressive function of LOXL4 on macrophages primarily relied on interferon (IFN)-mediated signal transducer and activator of transcription-dependent PD-L1 activation. Hydrogen peroxide scavenger or copper chelation on macrophages abolished the IFN-mediated PD-L1 presentation by LOXL4. In human HCC tissue, expression of LOXL4 in CD68+ cells was positively correlated with PD-L1 level. High expression of LOXL4 in CD68+ cells and low expression of CD8A in tumor tissue cooperatively predict poor survival of patients with HCC. Conclusions LOXL4 facilitates immune evasion by tumor cells and leads to hepatocarcinogenesis. Our study unveils the role of LOXL4 in fostering an immunosuppressive microenvironment during hepatocarcinogenesis.

Background Skin aging is marked by progressive loss in elastin and collagen that causes wrinkling and sagging of skin. Tropoelastin (TE) is the precursor monomer of elastin secreted by cells that cross-links extracellularly to create functional elastic fibers. Cells maintain the capacity to make TE during the aging process. However, the process of extracellular tropoelastin cross-linking diminishes with age. Others have shown that TE production by cells increases with UV exposure. Objective We hypothesize that polyphenols may help coacervate cell secreted TE due to its elastin binding property and increase insoluble elastin in human dermal fibroblasts (HDFs). Increase in TE production by short term UV exposure may further improve elastin deposition by polyphenols. Methods We treated HDFs with polyphenols viz epigallocatechin gallate (EGCG) and pentagalloyl glucose (PGG) either with or without intermittent (UVA, 12 min three times a week) exposure for 3, 7, and 14 days. Results Polyphenols increased insoluble elastin deposition several folds as compared to control untreated cells. Furthermore, short UVA light exposure led to several-fold increased TE production in HDFs. Still, UVA exposure alone was unable to increase insoluble elastic fibers. When polyphenols were introduced with UVA exposure, insoluble elastin deposition was further enhanced in HDFs (30−45-fold increase). Polyphenol treatments with UVA exposure also led to increased collagen deposition in cell cultures. Polyphenols also prevented cell oxidation during UVA exposure. Conclusions Polyphenols in combination with short exposure to UVA light increase extracellular matrix deposition of elastin and collagen and may improve skin properties.

Elastic fibers are essential components of the arterial extracellular matrix. They consist of the protein elastin and an array of microfibrils that support the protein and connect it to the surrounding matrix. The elastin gene encodes tropoelastin, a protein that requires extensive cross-linking to become elastin. Tropoelastin is expressed throughout human life, but its expression levels decrease with age, suggesting that the potential to synthesize elastin persists during lifetime although declines with aging. The initial abnormality documented in human atherosclerosis is fragmentation and loss of the elastic network in the medial layer of the arterial wall, suggesting an imbalance between elastic fiber injury and restoration. Damaged elastic structures are not adequately repaired by synthesis of new elastic elements. Progressive collagen accumulation follows medial elastic fiber disruption and fibrous plaques are formed, but advanced atherosclerosis lesions do not develop in the absence of prior elastic injury. Aging is associated with arterial extracellular matrix anomalies that evoke those present in early atherosclerosis. The reduction of elastic fibers with subsequent collagen accumulation leads to arterial stiffening and intima-media thickening, which are independent predictors of incident hypertension in prospective community-based studies. Arterial stiffening precedes the development of hypertension. The fundamental role of the vascular elastic network to arterial structure and function is emphasized by congenital disorders caused by mutations that disrupt normal elastic fiber production. Molecular changes in the genes coding tropoelastin, lysyl oxidase (tropoelastin cross-linking), and elastin-associated microfibrils, including fibrillin-1, fibulin-4, and fibulin-5 produce severe vascular injury due to absence of functional elastin.

IntroductionNeonatal lung injury as a consequence of hyperoxia (HO) therapy and ventilator care contribute to the development of bronchopulmonary dysplasia (BPD). Increased expression and activity of lysyl oxidase (LOX), a key enzyme that cross-links collagen, was associated with increased sphingosine kinase 1 (SPHK1) in human BPD. We, therefore, examined closely the link between LOX and SPHK1 in BPD.MethodThe enzyme expression of SPHK1 and LOX were assessed in lung tissues of human BPD using immunohistochemistry and quantified (Halo). In vivo studies were based on Sphk1−/− and matched wild type (WT) neonatal mice exposed to HO while treated with PF543, an inhibitor of SPHK1. In vitro mechanistic studies used human lung microvascular endothelial cells (HLMVECs).ResultsBoth SPHK1 and LOX expressions were increased in lungs of patients with BPD. Tracheal aspirates from patients with BPD had increased LOX, correlating with sphingosine-1-phosphate (S1P) levels. HO-induced increase of LOX in lungs were attenuated in both Sphk1−/− and PF543-treated WT mice, accompanied by reduced collagen staining (sirius red). PF543 reduced LOX activity in both bronchoalveolar lavage fluid and supernatant of HLMVECs following HO. In silico analysis revealed STAT3 as a potential transcriptional regulator of LOX. In HLMVECs, following HO, ChIP assay confirmed increased STAT3 binding to LOX promoter. SPHK1 inhibition reduced phosphorylation of STAT3. Antibody to S1P and siRNA against SPNS2, S1P receptor 1 (S1P1) and STAT3 reduced LOX expression.ConclusionHO-induced SPHK1/S1P signalling axis plays a critical role in transcriptional regulation of LOX expression via SPNS2, S1P1 and STAT3 in lung endothelium.

Objective Preeclampsia is a pregnancy-specific disorder that is a major cause of maternal and foetal morbidity and mortality, with a prevalence of 6-8% of pregnancies. Although the downregulation of lysyl oxidase (LOX) and LOX-like protein 2 (LOXL2), which leads to reduced trophoblast cell migration and invasion through activation of the TGF-β1/Smad3/collagen pathway, is relevant to preeclampsia, the mechanisms regulating differences in the gene expression of LOX and LOXL2 in placentas are not yet understood. This study aimed to investigate the mechanisms regulating differences in the gene expression of LOX and LOXL2 in placentas. Methods The expression of miRNAs, LOX and LOXL2 in preeclamptic placentas and control placentas was analysed by qPCR. Localisation of miR29a and LOXL2 in preeclamptic placentas was performed by RNA-Fluorescence in-situ hybridization assay. The direct regulation of LOXL2 by miR-29a was assessed by dual-luciferase reporter assays in human extravillous trophoblast cells (HTR8/SVneo). Cell migration and invasion were evaluated by Transwell assays in HTR8/SVneo cells. Results miR-29a expression was upregulated in preeclamptic placentas and negatively correlated with LOXL2 mRNA expression levels. RNA-Fluorescence in-situ hybridization assay revealed a clear overlap between miR-29a and LOXL2 in the placentas of preeclampic women. LOXL2 was a direct target gene of miR-29a, as confirmed by a dual-luciferase reporter assay in HTR8/SVneo trophoblast cells. miR-29a suppressed HTR8/SVneo trophoblast cell migration and invasion. LOXL2 overexpression reversed the inhibitory effects of miR-29a on HTR8/SVneo trophoblast cell migration and invasion. Conclusion Our results suggest that the upregulation of miR-29a suppresses the migration and invasion of HTR8/SVneo trophoblast cells by directly targeting LOXL2 in preeclampsia.

Non-alcoholic steatohepatitis (NASH) is an increasing cause of chronic liver disease characterized by steatosis, inflammation, and fibrosis which can lead to cirrhosis, hepatocellular carcinoma, and mortality. Quantitative, noninvasive methods for characterizing the pathophysiology of NASH at both the preclinical and clinical level are sorely needed. We report here a multiparametric magnetic resonance imaging (MRI) protocol with the fibrogenesis probe Gd-Hyd to characterize fibrotic disease activity and steatosis in a common mouse model of NASH. Mice were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) to induce NASH with advanced fibrosis. Mice fed normal chow and CDAHFD underwent MRI after 2, 6, 10 and 14 weeks to measure liver T1, T2*, fat fraction, and dynamic T1-weighted Gd-Hyd enhanced imaging of the liver. Steatosis, inflammation, and fibrosis were then quantified by histology. NASH and fibrosis developed quickly in CDAHFD fed mice with strong correlation between morphometric steatosis quantification and liver fat estimated by MRI (r = 0.90). Sirius red histology and collagen quantification confirmed increasing fibrosis over time (r = 0.82). Though baseline T1 and T2* measurements did not correlate with fibrosis, Gd-Hyd signal enhancement provided a measure of the extent of active fibrotic disease progression and correlated strongly with lysyl oxidase expression. Gd-Hyd MRI accurately detects fibrogenesis in a mouse model of NASH with advanced fibrosis and can be combined with other MR measures, like fat imaging, to more accurately assess disease burden.

Background and aims The mechanism of vascular calcification (VC) resembles that of bone metabolism, and a correlation has frequently been reported between calcification and vascular extracellular matrix (ECM) regulating its integrity; however, the detailed mechanisms remain unclear. In this study, we examined how the vascular ECM, especially collagen metabolism, is involved in the process of VC. Methods VC was modeled using 5-week-old male Sprague–Dawley rats fed a diet containing warfarin and vitamin K1 (WVK). Additionally, β-aminopropionitrile (BAPN) was administered to inhibit lysyl oxidase (LOX), which is an enzyme that mediates collagen cross-linking. Harvested aortic samples were analyzed by staining with alizarin red (AR), immunohistochemistry (IHC), transmission electron microscopy (TEM), and ex vivo microcomputed tomography (μCT). Results Rats fed WVK developed increasing numbers of aortic medial calcifications (AMCs) over time. TEM images indicated punctate calcification within collagen fibers in the early phase of AMC. AR staining of translucent samples revealed the distribution and severity of calcification, and these lesions were significantly decreased in the BAPN group. Three-dimensional reconstructed μCT images that allowed the quantification of calcified volumes revealed that BAPN significantly reduced the bulk of calcification. Moreover, IHC showed that both LOX and collagen I were present around the sites of AMC, and thus the IHC-positive area was reduced in the BAPN group compared to the WVK group. Conclusions The results indicated that inhibition of LOX by BAPN attenuated AMC, and that collagen metabolism plays a significant role in the early pathogenesis of VC.

The role of lysyl oxidase (LOX) in prostate cancer remains controversial. Studies have shown that LOX may inhibit the progression of prostate cancer (PCa), whereas other studies demonstrate that LOX may act as a tumor activator in PCa. Here, we report that low LOX expression contributes to CRPC progression through upregulation of IGFBP3. We showed that LOX expression decreased in the more advanced and aggressive castration-resistant prostate cancer (CRPC), compared to castration-sensitive prostate cancer (CSPC). We demonstrated that LOX was negatively correlated with IGFBP3 and may directly bind to the promoter of IGFBP3 and thus decrease the expression of IGFBP3. Inhibition of IGFBP3 by siRNA suppressed the growth and migration of CRPC cells, suggesting a critical role for IGFBP3 in CRPC. The preclinical study in a mouse model suggested that introducing back LOX inhibited the progression of CRPC. In summary, we identified a new function of LOX in PCa and discovered that LOX downregulation contributed to progression via IGFBP3, and that the restoration of LOX may be a promising therapeutic strategy for PCa.

Background: Oral submucous fibrosis (OSF) is a debilitating collagen-metabolic disorder leading to submucosal fibrosis and trismus. Lysyl oxidase (LOX), a critical collagen biosynthetic enzyme, is up-regulated in OSF. Polymorphisms in the Lysyl oxidase gene have been associated with increased risk of OSF and might affect normal collagen synthesis, accumulation, or degradation, crucial in determining fibrosis severity. Methods: One hundred OSF cases and 100 controls were genotyped for LOX G473A(Arg158Gln) polymorphism by polymerase chain reaction-restriction fragment length polymorphism. The expression of LOX was estimated both by quantitative mRNA analysis and western blot. Total soluble collagen was evaluated from mucosal tissue obtained from OSF cases. Immunohistochemical (IHC) localization of type 1 collagen was performed in mucosal tissue obtained from patients carrying various genotypes. Results: Heterozygous G473A genotype was significantly higher in OSF cases [2.063(95% CI =1.059-4.016)], among 26-40 years age-group [4.375(95% CI=1.323-14.267),p=0.029] and in male patients [2.38 (95% CI= 1.107-5.121), p= 0.042]. LOX expression was significantly higher in cases of the heterozygous or homozygous carrier (p

Surgical resection is a common therapeutic option for primary solid tumors. However, high cancer recurrence and metastatic rates after resection are the main cause of cancer related mortalities. This implies the existence of a “fertile soil” following surgery that facilitates colonization by circulating cancer cells. Myeloid-derived suppressor cells (MDSCs) are essential for premetastatic niche formation, and may persist in distant organs for up to 2 weeks after surgery. These postsurgical persistent lung MDSCs exhibit stronger immunosuppression compared with presurgical MDSCs, suggesting that surgery enhances MDSC function. Surgical stress and trauma trigger the secretion of systemic inflammatory cytokines, which enhance MDSC mobilization and proliferation. Additionally, damage associated molecular patterns (DAMPs) directly activate MDSCs through pattern recognition receptor-mediated signals. Surgery also increases vascular permeability, induces an increase in lysyl oxidase and extracellular matrix remodeling in lungs, that enhances MDSC mobilization. Postsurgical therapies that inhibit the induction of premetastatic niches by MDSCs promote the long-term survival of patients. Cyclooxygenase-2 inhibitors and β-blockade, or their combination, may minimize the impact of surgical stress on MDSCs. Anti-DAMPs and associated inflammatory signaling inhibitors also are potential therapies. Existing therapies under tumor-bearing conditions, such as MDSCs depletion with low-dose chemotherapy or tyrosine kinase inhibitors, MDSCs differentiation using all-trans retinoic acid, and STAT3 inhibition merit clinical evaluation during the perioperative period. In addition, combining low-dose epigenetic drugs with chemokine receptors, reversing immunosuppression through the Enhanced Recovery After Surgery protocol, repairing vascular leakage, or inhibiting extracellular matrix remodeling also may enhance the long-term survival of curative resection patients.

Stromal stiffening accompanies malignancy, compromises treatment and promotes tumour aggression. Clarifying the molecular nature and the factors that regulate stromal stiffening in tumours should identify biomarkers to stratify patients for therapy and interventions to improve outcome. We profiled lysyl hydroxylase-mediated and lysyl oxidase-mediated collagen crosslinks and quantified the greatest abundance of total and complex collagen crosslinks in aggressive human breast cancer subtypes with the stiffest stroma. These tissues harbour the highest number of tumour-associated macrophages, whose therapeutic ablation in experimental models reduced metastasis, and decreased collagen crosslinks and stromal stiffening. Epithelial-targeted expression of the crosslinking enzyme, lysyl oxidase, had no impact on collagen crosslinking in PyMT mammary tumours, whereas stromal cell targeting did. Stromal cells in microdissected human tumours expressed the highest level of collagen crosslinking enzymes. Immunohistochemical analysis of biopsies from a cohort of patients with breast cancer revealed that stromal expression of lysyl hydroxylase 2, an enzyme that induces hydroxylysine aldehyde-derived collagen crosslinks and stromal stiffening, correlated significantly with disease specific mortality. The findings link tissue inflammation, stromal cell-mediated collagen crosslinking and stiffening to tumour aggression and identify lysyl hydroxylase 2 as a stromal biomarker.

Objective Increased gingival elasticity has been implicated as the cause of relapse following orthodontic rotational tooth movement and approaches to reduce relapse are limited. This study aimed to investigate the effects of sulforaphane (SFN), an inhibitor of osteoclastogenesis, on gene expression in gingival fibroblasts and relapse after rotational tooth movement in beagle dogs. Methods The lower lateral incisors of five beagle dogs were rotated. SFN or dimethylsulfoxide (DMSO) were injected into the supra-alveolar gingiva of the experimental and control group, respectively, and the effect of SFN on relapse tendency was evaluated. Changes in mRNA expression of extracellular matrix components associated with gingival elasticity in beagles were investigated by real-time polymerase chain reaction. Morphology and arrangement of collagen fibers were observed on Masson’s trichrome staining of buccal gingival tissues of experimental and control teeth. Results SFN reduced the amount and percentage of relapse of orthodontic rotation. It also decreased the gene expression of lysyl oxidase and increased the gene expression of matrix metalloproteinase (MMP) 1 and MMP 12, compared with DMSO control subjects. Histologically, collagen fiber bundles were arranged irregularly and were not well connected in the SFN-treated group, whereas the fibers extended in parallel and perpendicular directions toward the gingiva and alveolar bone in a more regular and well-ordered arrangement in the DMSO-treated group. Conclusions Our findings demonstrated that SFN treatment may be a promising pharmacologic approach to prevent orthodontic rotational relapse caused by increased gingival elasticity of rotated teeth in beagle dogs.

Background: Malignancy is a complex process resulting from different changes such as extracellular matrix (ECM) remodeling and stiffness. One of the important enzymes that contribute to ECM remodeling is lysyl oxidase (Lox) that is overexpressed in different types of human cancers. Because of the high prevalence and poor survival of gastrointestinal (GI) malignancies in this review, we discuss the association between Lox activity and the progression of GI cancers. Lox proteins are a group of extracellular enzymes that catalyzed the cross-linking of collagen and elastin, so they have important roles in the control of structure and homeostasis of ECM. Abnormal activation and expression of the Lox family of proteins lead to changes in the ECM toward increased rigidity and fibrosis. Stiffness of ECM can contribute to the pathogenesis of cancers. Summary: Dysregulation of Lox expression is a factor in both fibrotic diseases and cancer. ECM stiffness by Lox overactivity creates a physical barrier against intratumoral concentration of chemotherapeutic drugs and facilitates cancer inflammation, angiogenesis, and metastasis. Key Message: Because of the roles of Lox in GI cancers, development targeting Lox protein isotypes may be an appropriate strategy for treatment of GI cancers and improvement in survival of patients.

Backgrounds Kidney renal clear cell carcinoma (KIRC) is a major pathological type of renal cell carcinoma (RCC), and the prognosis of advanced KIRC patients is often unsatisfactory. Some lysine oxidase (LOX) family genes have been proven to be upregulated in some malignancies and play pivotal roles in the carcinogenesis. However, their roles in KIRC remain unclear. Materials and Methods Here, we used some online databases (eg, ONCOMINE, GEPIA, UALCAN, c‐BioPortal, Human Protein Altas) to comprehensively explored the expression levels and the prognostic values of LOX family genes in KIRC using bioinformatic methods. Results The results revealed that lysyl oxidase (LOX) and lysyl oxidase‐like 2 (LOXL2) were significantly overexpressed in KIRC at the level of mRNA expression, protein expression, and RCC cell lines. Further analysis demonstrated that higher mRNA expression of LOX and LOXL2 were significantly correlated with poor survival, tumor grade, individual cancer stages, and nodal metastasis status. DNA copy number amplifications and mRNA upregulation, DNA deep deletion, and mRNA upregulation were the main genetic mutations of LOX and LOXL2, respectively. Prognostic analysis showed that the altered group had significantly poorer overall survival (OS) compared to the unaltered group (p = .0387). Co‐expression analysis showed CP, PLOD2, and COL5A1 were significantly correlated with LOX, and COL1A2 was positively correlated with LOXL2. Further analysis confirmed that these co‐expressed genes were significantly upregulated and predicted unfavorable prognosis in KIRC. Conclusion Multi‐level analysis demonstrated that LOX and LOXL2 were significantly upregulated and predicted poor survival in KIRC, which may apply as promising biomarkers for diagnosis and therapy of KIRC in the future., We firstly comprehensively explored the role of LOX family genes in kidney renal clear cell carcinoma, and found that LOX and LOXL2 may be used as new therapeutic targets for kidney renal clear cell carcinoma.

The effects of human umbilical cord blood mesenchymal stem cells conditioned medium (hMSC-CM) on Lysyl oxidase (LOX) family gene expression and proteins were evaluated. Fifty-four male Wistar rats were divided into nine groups. The control groups received olive oil and phosphate-buffered saline (PBS) while untreated groups received CCl4. The treated groups received hMSC-CM after the 4th week of CCl4 injection. LOX family expression and proteins were evaluated by RT-PCR and western blot. CCl4 enhanced significantly the expression of LOX genes and proteins. hMSC-CM significantly decreased the expression of the LOX genes and proteins. hMSC-CM decreased cross-linked collagen in the treated groups. hMSC-CM improved liver fibrosis through decreased LOX family gene expression.