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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 ., (©2021 The Authors; Published by the American Association for Cancer Research.)

Pulmonary fibrosis is one of the important causes of morbidity and mortality in fibroproliferative disorders such as systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF). Lysyl oxidase (LOX) is a copper-dependent amine oxidase whose primary function is the covalent crosslinking of collagens in the extracellular matrix (ECM). We investigated the role of LOX in the pathophysiology of SSc. LOX mRNA and protein levels were increased in lung fibroblasts of SSc patients compared with healthy controls and IPF patients. In vivo, bleomycin induced LOX mRNA expression in lung tissues, and LOX activity increased in the circulation of mice with pulmonary fibrosis, suggesting that circulating LOX parallels levels in lung tissues. Circulating levels of LOX were reduced upon amelioration of fibrosis with an antifibrotic peptide. LOX induced ECM production at the transcriptional level in lung fibroblasts, human lungs, and human skin maintained in organ culture. In vivo, LOX synergistically exacerbated fibrosis in bleomycin-treated mice. Further, LOX increased the production of interleukin (IL)-6, and the increase was mediated by LOX-induced c-Fos expression, the nuclear localization of c-Fos, and its engagement with the IL-6 promoter region. Our findings demonstrate that LOX expression and activity correlate with fibrosis in vitro, ex vivo, and in vivo. LOX induced ECM production via upregulation of IL-6 and nuclear localization of c-Fos. Thus, LOX has a direct pathogenic role in SSc-associated fibrosis that is independent of its crosslinking function. Our findings also suggest that measuring circulating LOX levels and activity can be used for monitoring response to antifibrotic therapy.

Purpose: To determine whether high glucose (HG) compromises internalization of lysyl oxidase (LOX) through excess binding of LOX with extracellular matrix (ECM) proteins., Methods: To determine whether HG promotes binding of LOX with ECM proteins, fibronectin (FN) and collagen IV (Coll IV), total or ECM-only proteins from rat retinal endothelial cells grown in normal (N; 5 mM) or HG (30 mM) medium were analyzed by coimmunoprecipitation and Western blot (WB). In parallel, coimmunostaining was performed to determine changes in LOX binding to FN or Coll IV. To determine the effect of HG on extracellular LOX levels, medium in which cells were grown for 1, 3, 5, and 7 days were assessed for LOX levels., Results: WB analysis using total protein showed LOX overexpression and elevated levels of LOX bound to Coll IV or FN in HG condition. Similarly, a significant increase in LOX bound to FN or Coll IV was observed in ECM-only protein. These data were supported by Z-stack confocal microscopy images from coimmunostaining. Furthermore, immunostaining performed on ECM layer revealed increased presence of LOX bound to Coll IV or FN. Additionally, when media from cells grown in HG was monitored, a maximal increase in LOX level was observed by day 3, which declined by day 7., Conclusions: Findings indicate that HG promotes binding of LOX to FN and Coll IV extracellularly that results in reduced LOX internalization, attenuation of negative feedback, and upregulation of LOX expression associated with diabetic retinopathy.

As the primary indication for corneal transplantation, the pathogenesis of keratoconus remains elusive. Aiming to identify whether any mutation from extracellular-matrix (ECM)-related genes contributes to the patients with sporadic cases of keratoconus (KC) from Chinese Han population, one hundred and fifty-three participants in total were enrolled in our study, including fifty-three KC patients and one hundred healthy controls. Mutational analysis of three ECM-related genes (LOX, COL5A1 and TIMP3) with next-generation sequencing and Sanger sequencing was performed. To further confirm the function of three ECM-related genes in the pathogenesis of keratoconus, we performed Real-time Quantitative PCR in vitro. Results showed that three new sequence variants (c.95 G > A in LOX, c.1372 C > T in COL5A1 and c.476 C > T in TIMP3) were identified in aforementioned ECM-related genes in KC patients without being detected among the healthy controls. According to the results of QPCR, we found that the expression levels of LOX and TIMP3 were decreased in the KC patients, while COL5A1 showed no significant difference of expression. This is the first time to screen so many ECM-related genes in Chinese keratoconus patients using next-generation sequencing. We find numerous underlying causal variants, enlarging lots of mutation spectrums and thus providing new sites for other investigators to replicate and for further research.

Heart diseases are a major cause of morbidity and mortality world-wide. Lysyl oxidase (LOX) and related LOX-like (LOXL) isoforms play a vital role in remodelling the extracellular matrix (ECM). The LOX family controls ECM formation by cross-linking collagen and elastin chains. LOX/LOXL proteins are copper-dependent amine oxidases that catalyse the oxidation of lysine, causing cross-linking between the lysine moieties of lysine-rich proteins. Dynamic changes in LOX and LOXL protein-expression occur in a variety of cardiac pathologies; these changes are believed to be central to the associated tissue-fibrosis. An awareness of the potential pathophysiological importance of LOX has led to the evaluation of interventions that target LOX/LOXL proteins for heart-disease therapy. The purposes of this review article are: (i) to summarize the basic biochemistry and enzyme function of LOX and LOXL proteins; (ii) to consider their tissue and species distribution; and (iii) to review the results of experimental studies of the roles of LOX and LOXL proteins in heart disease, addressing involvement in the mechanisms, pathophysiology and therapeutic responses based on observations in patient samples and relevant animal models. Therapeutic targeting of LOX family enzymes has shown promising results in animal models, but small-molecule approaches have been limited by non-specificity and off-target effects. Biological approaches show potential promise but are in their infancy. While there is strong evidence for LOX-family protein participation in heart failure, myocardial infarction, cardiac hypertrophy, dilated cardiomyopathy, atrial fibrillation and hypertension, as well as potential interest as therapeutic targets, the precise involvement of LOX-family proteins in heart disease requires further investigation., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Fetal alcohol syndrome (FAS) is the most severe condition of fetal alcohol spectrum disorders (FASD) and is associated with congenital heart defects. However, more subtle defects such as ventricular wall thinning and cardiac compliance may be overlooked in FASD. Our studies focus on the role of cardiac fibroblasts in the neonatal heart, and how they are affected by prenatal alcohol exposure (PAE). We hypothesize that PAE affects fibroblast function contributing to dysregulated collagen synthesis, which leads to cardiac dysfunction. To investigate these effects, pregnant C57/BL6 mice were intraperitoneally injected with 2.9 g EtOH/kg dose to achieve a blood alcohol content of approximately 0.35 on gestation days 6.75 and 7.25. Pups were sacrificed on neonatal day 5 following echocardiography measurements of left ventricular (LV) chamber dimension and function. Hearts were used for primary cardiac fibroblast isolation or protein expression analysis. PAE animals had thinner ventricular walls than saline exposed animals, which was associated with increased LV wall stress and decreased ejection fraction. In isolated fibroblasts, PAE decreased collagen I/III ratio and increased gene expression of profibrotic markers, including α-smooth muscle actin and lysyl oxidase. Notch1 signaling was assessed as a possible mechanism for fibroblast activation, and indicated that gene expression of Notch1 receptor and downstream Hey1 transcription factor were increased. Cardiac tissue analysis revealed decreased collagen I/III ratio and increased protein expression of α-smooth muscle actin and lysyl oxidase. However, Notch1 signaling components decreased in whole heart tissue. Our study demonstrates that PAE caused adverse changes in the cardiac collagen profile and a decline in cardiac function in the neonatal heart.

Peritoneal metastases are the leading cause of morbidity and mortality in high-grade serous ovarian cancer (HGSOC). Accumulating evidence suggests that mesothelial cells are an important component of the metastatic microenvironment in HGSOC. However, the mechanisms by which mesothelial cells promote metastasis are unclear. Here, we report that the HGSOC tumor-mesothelial niche was hypoxic, and hypoxic signaling enhanced collagen I deposition by mesothelial cells. Specifically, hypoxic signaling increased expression of lysyl oxidase (LOX) in mesothelial and ovarian cancer cells to promote collagen crosslinking and tumor cell invasion. The mesothelial niche was enriched with fibrillar collagen in human and murine omental metastases. Pharmacologic inhibition of LOX reduced tumor burden and collagen remodeling in murine omental metastases. These findings highlight an important role for hypoxia and mesothelial cells in the modification of the extracellular matrix and tumor invasion in HGSOC. SIGNIFICANCE: This study identifies HIF/LOX signaling as a potential therapeutic target to inhibit collagen remodeling and tumor progression in HGSOC. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/9/2271/F1.large.jpg., (©2019 American Association for Cancer Research.)

The extracellular matrix (ECM) regulates numerous cellular events in addition to providing structural integrity. Among several protein and enzyme families implicated in functions of the ECM, the lysyl oxidases and ADAMTS proteins are known to participate in microfibril and elastic fiber formation as well as ECM-associated signaling. A yeast two-hybrid screen to identify lysyl oxidase (LOX) binding proteins identified ADAMTSL4 as a potential interactor. We demonstrate here that several members of the LOX and ADAMTS families interact with one another. Upon investigating the interaction between LOX and ADAMTSL2 we found that the absence or inhibition of Lox affected ADAMTSL2 molecular forms and reduced its tissue levels. Thus, ADAMTSL2 stability and inter-molecular complexes may depend on the activity of lysyl oxidases., (Copyright © 2018 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Stretching is a common method used to prevent muscle shortening and improve limited mobility. However, the effect of different time periods on stretching-induced adaptation of the extracellular matrix and its regulatory elements have yet to be investigated. We aimed to evaluate the expression of fibrillar collagens, sarcomerogenesis, metalloproteinase (MMP) activity and gene expression of the extracellular matrix (ECM) regulators in the soleus (SOL) muscle of rats submitted to different stretching periods. The soleus muscles were submitted to 10 sets of passive stretching over 10 (St 10d) or 15 days (St 15d) (1 min per set, with 30 seconds' rest between sets). Sarcomerogenesis, muscle cross-sectional area (CSA), and MMP activity and mRNA levels in collagen (type I, III and IV), connective tissue growth factor (CTGF), growth factor-beta (TGF-β), and lysyl oxidase (LOX) were analyzed. Passive stretching over both time periods mitigated COL-I deposition in the SOL muscle of rats. Paradoxically, 10 days of passive stretching induced COL-I and COL-III synthesis, with concomitant upregulation of TGF-β1 and CTGF at a transcriptional level. These responses may be associated with lower LOX mRNA levels in SOL muscles submitted to 10 passive stretching sessions. Moreover, sarcomerogenesis was observed after 15 days of stretching, suggesting that stretching-induced muscle adaptations are time-dependent responses.

Some solid tumors are characterized by extracellular matrix (ECM) remodeling and stiffening, which is related to solid tumor progression and aggression. However, the relationship between ECM stiffness and colorectal cancer (CRC) remains unclear. In this study, we investigated the relevance of ECM stiffness to clinicopathologic features using human CRC tissue microarrays. The results demonstrate that the expression of ECM components in CRC tissues is closely correlated with CRC progression and poor prognosis, which indicates that ECM stiffness may be associated with CRC development. We further studied lysyl oxidase (LOX) expression in CRC tissue and demonstrated that LOX expression is closely correlated with CRC progression. Previous studies showed that P-selectin-mediated platelet accumulation in CRC tissue may up-regulate LOX expression. Our findings indicate that P-selectin-mediated platelet aggregation may up-regulate LOX expression and enhance the remodeling and stiffening of the tumor ECM, which may promote the progression of colorectal cancer. Therefore, LOX may be a potential effective therapeutic target to treat colorectal cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Introduction: Lysyl oxidase (LOX) participates in the assembly of collagen and elastin fibres. The impact of vascular LOX over-expression on extracellular matrix (ECM) structure and its contribution to oxidative stress has been analysed., Methods: Studies were conducted on mice over-expressing LOX (Tg), specifically in smooth muscle cells (VSMC). Gene expression was assessed by real-time PCR analysis. Sirius Red staining, H 2 O 2 production and NADPH oxidase activity were analysed in different vascular beds. The size and number of fenestra of the internal elastic lamina were determined by confocal microscopy., Results: LOX activity was up-regulated in VSMC of transgenic mice compared with cells from control animals. At the same time, transgenic cells deposited more organised elastin fibres and their supernatants induced a stronger collagen assembly in in vitro assays. Vascular collagen cross-linking was also higher in Tg mice, which showed a decrease in the size of fenestrae and an enhanced expression of Fibulin-5. Interestingly, higher H 2 O 2 production and NADPH oxidase activity was detected in the vascular wall from transgenic mice. The H 2 O 2 scavenger catalase attenuated the stronger deposition of mature elastin fibres induced by LOX transgenesis., Conclusions: LOX over-expression in VSMC was associated with a change in the structure of collagen and elastin fibres. LOX could constitute a novel source of oxidative stress that might participate in elastin changes and contribute to vascular remodelling., (Copyright © 2017 Sociedad Española de Arteriosclerosis. Publicado por Elsevier España, S.L.U. All rights reserved.)

Lysyl oxidase (LOX) family genes catalyze collagen cross-link formation. To determine the effects of lysyl oxidase-like 4 (LOXL4) expression on breast tumor formation and metastasis, we evaluated primary tumor growth and lung metastasis in mice injected with LOXL4-knockdown MDA-MB-231 triple-negative human breast cancer cells. In addition, we analyzed overall survival in breast cancer patients based on LOXL4 expression using a public online database. In the mouse xenograft model, LOXL4 knockdown increased primary tumor growth and lung colonization as well as collagen I and IV, lysine hydroxylase 1 and 2, and prolyl 4-hydroxylase subunit alpha 1 and 2 levels. Second harmonic generation imaging revealed that LOXL4 knockdown resulted in the thickening of collagen bundles within tumors. In addition, weak LOXL4 expression was associated with poor overall survival in breast cancer patients from the BreastMark dataset, and this association was strongest in triple-negative breast cancer patients. These results demonstrate that weak LOXL4 expression leads to remodeling of the extracellular matrix through induction of collagen synthesis, deposition, and structural changes. These alterations in turn promote tumor growth and metastasis and are associated with poor clinical outcomes in triple-negative breast cancer.

The extracellular matrix (ECM) from perilesional and colorectal carcinoma (CRC), but not healthy colon, sustains proliferation and invasion of tumor cells. We investigated the biochemical and physical diversity of ECM in pair-wised comparisons of healthy, perilesional and CRC specimens. Progressive linearization and degree of organization of fibrils was observed from healthy to perilesional and CRC ECM, and was associated with a steady increase of stiffness and collagen crosslinking. In the perilesional ECM these modifications coincided with increased vascularization, whereas in the neoplastic ECM they were associated with altered modulation of matrisome proteins, increased content of hydroxylated lysine and lysyl oxidase. This study identifies the increased stiffness and crosslinking of the perilesional ECM predisposing an environment suitable for CRC invasion as a phenomenon associated with vascularization. The increased stiffness of colon areas may represent a new predictive marker of desmoplastic region predisposing to invasion, thus offering new potential application for monitoring adenoma with invasive potential.

Aims: After myocardial infarction (MI), extensive remodelling of the extracellular matrix contributes to scar formation. While aiming to preserve tissue integrity, this fibrotic response is also associated with adverse events, including a markedly increased risk of heart failure, ventricular arrhythmias, and sudden cardiac death. Cardiac fibrosis is characterized by extensive deposition of collagen and also by increased stiffness as a consequence of enhanced collagen cross-linking. Members of the lysyl oxidase (LOX) family of enzymes are responsible for the formation of collagen cross-links. This study investigates the contribution of LOX family members to the heart response to MI., Methods and Results: Experimental MI was induced in C57BL/6 mice by permanent ligation of the left anterior descending coronary artery. The expression of LOX isoforms (LOX and LOXL1-4) was strongly increased upon MI, and this response was accompanied by a significant accumulation of mature collagen fibres in the infarcted area. LOX expression was observed in areas of extensive remodelling, partially overlapping with α-smooth muscle actin-expressing myofibroblasts. Tumour growth factor-β as well as hypoxia-activated pathways contributed to the induction of LOX expression in cardiac fibroblasts. Finally, in vivo post-infarction treatment with the broadband LOX inhibitor β-aminopropionitrile or, selectively, with a neutralizing antibody against the canonical LOX isoform attenuated collagen accumulation and maturation and also resulted in reduced ventricular dilatation and improved cardiac function., Conclusion: LOX family members contribute significantly to the detrimental effects of cardiac remodelling, highlighting LOX inhibition as a potential therapeutic strategy for post-infarction recovery., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Cell migration is an important feature of glial cells. Here, we used the Drosophila eye disc to decipher the molecular network controlling glial migration. We stimulated glial motility by pan-glial PDGF receptor (PVR) activation and identified several genes acting downstream of PVR. Drosophila lox is a non-essential gene encoding a secreted protein that stiffens the extracellular matrix (ECM). Glial-specific knockdown of Integrin results in ECM softening. Moreover, we show that lox expression is regulated by Integrin signaling and vice versa, suggesting that a positive-feedback loop ensures a rigid ECM in the vicinity of migrating cells. The general implication of this model was tested in a mammalian glioma model, where a Lox-specific inhibitor unraveled a clear impact of ECM rigidity in glioma cell migration., (© 2014. Published by The Company of Biologists Ltd.)

Objectives: To analyze the expression of genes involved in extracellular matrix (ECM) biogenesis and remodeling in vaginal tissue of women with clinically normal pelvic floor support (defined as controls) according to the phase of menstrual cycle and postmenopausal women with and without pelvic organ prolapse (POP)., Materials and Methods: This study examined the expression of matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and the Lysyl oxidase (LOX) family genes in the anterior vaginal wall of Caucasian women by real-time RT-PCR. Initially, mRNA expression was assessed in premenopausal controls in the secretory (group 1, n = 10) vs. proliferative (group 2, n = 8) phase of menstrual cycle. In addition, we compared premenopausal controls in the proliferative phase (group 2) vs. postmenopausal controls (group 3, n = 5). Finally, we analyzed postmenopausal controls (group 3) vs. postmenopausal women with advanced POP (group 4, n = 13)., Results: According to the phase of menstrual cycle, MMP1 was significantly reduced (p = 0.003), whereas the expression of TIMP1 and LOXL4 was significantly up-regulated during proliferative phase (both p < 0.01) when compared to the secretory phase in premenopausal control women. Regarding menopausal status/ageing, all MMPs were down-regulated, while TIMP3, TIMP4 and LOXL2 were significantly up-regulated in postmenopausal control women when compared to premenopausal controls (p = 0.005, p = 0.01 and p < 0.001, correspondingly). TIMP4 and LOXL2 mRNA levels were significantly decreased in postmenopausal POP patients compared to asymptomatic postmenopausal controls (p < 0.01 for both)., Conclusions: Our results indicate that ovarian cycle and age-related changes influence the expression of genes encoding proteins responsible for ECM metabolism in human vagina. Moreover, POP is associated with alteration in vaginal ECM components after menopause.

Arterial stiffening is a risk factor for cardiovascular disease, but how arteries stay supple is unknown. Here, we show that apolipoprotein E (apoE) and apoE-containing high-density lipoprotein (apoE-HDL) maintain arterial elasticity by suppressing the expression of extracellular matrix genes. ApoE interrupts a mechanically driven feed-forward loop that increases the expression of collagen-I, fibronectin, and lysyl oxidase in response to substratum stiffening. These effects are independent of the apoE lipid-binding domain and transduced by Cox2 and miR-145. Arterial stiffness is increased in apoE null mice. This stiffening can be reduced by administration of the lysyl oxidase inhibitor BAPN, and BAPN treatment attenuates atherosclerosis despite highly elevated cholesterol. Macrophage abundance in lesions is reduced by BAPN in vivo, and monocyte/macrophage adhesion is reduced by substratum softening in vitro. We conclude that apoE and apoE-containing HDL promote healthy arterial biomechanics and that this confers protection from cardiovascular disease independent of the established apoE-HDL effect on cholesterol., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

Erosion and rupture of surface layers in atherosclerotic plaque can cause heart attack and stroke; however, changes in luminal surface composition are incompletely defined. Laser-induced fluorescence spectroscopy (LIFS), with limited tissue penetration, was used to investigate the surface of unstable carotid plaque and correlated with microscopy, birefringence and gene expression. Arterial matrix collagens I, III and elastin were assessed in unstable plaques (n = 25) and reference left internal mammary arteries (LIMA, n = 10). LIFS in addition to selective histological staining with picrosirius red, Movat pentachrome and immunostaining revealed decreased elastin and increased collagen I and III (P < 0.05) in carotid plaque when compared with LIMA. Within plaque, collagen I was elevated in the internal carotid region versus the common carotid region. Polarized light microscopy detected layers of aligned collagen and associated mechanical rigidity of the fibrous cap. Microarray analysis of three carotid and three LIMA specimens confirmed up-regulation of collagen I, III and IV, lysyl oxidase and MMP-12. In conclusion, LIFS analysis coupled with microscopy revealed marked regional differences in collagen I, III and elastin in surface layers of carotid plaque; indicative of plaque instability. Birefringence measurements demonstrated mechanical rigidity and weakening of the fibrous cap with complementary changes in ECM gene expression., (© 2011 The Authors. Photochemistry and Photobiology © 2011 The American Society of Photobiology.)

Objective: In diabetes, retinal vascular basement membrane (BM) undergoes significant thickening and compromises vessel function including increased vascular permeability, a prominent lesion of early diabetic retinopathy. In this study we determined whether altered expression and activity of lysyl oxidase (LOX), a cross-linking enzyme, may compromise vascular basement membrane functional integrity under high-glucose (HG) conditions., Research Design and Methods: Rat retinal endothelial cells (RRECs) grown in normal (5 mmol/l) or HG (30 mmol/l glucose) medium for 7 days were assessed for expression of LOX and proLOX by Western blot analysis and LOX enzyme activity. To determine whether HG alters cellular distribution patterns of LOX and proLOX, immunostaining with respective antibodies was performed. Similarly, cells grown in normal or HG medium were subjected to both LOX inhibition with β-aminopropionitrile (BAPN) and by small interfering RNA knockdown, and respectively examined for cell monolayer permeability. Additionally, retinas of streptozotocin (STZ)-induced diabetic rats were analyzed to determine if diabetes altered LOX expression., Results: Western blot analysis revealed significantly increased LOX and proLOX expression in cells grown in HG medium compared with those grown in normal medium. The increased LOX level was strikingly similar to LOX upegulation in the diabetic retinas. In cells grown in HG medium, LOX activity and cell monolayer permeability was significantly increased, as were LOX and proLOX immunostaining. Small interfering RNA- or BAPN-induced-specific blockage of LOX expression or activity, respectively, reduced cell monolayer permeability., Conclusions: HG-induced increased LOX expression and activity compromises barrier functional integrity, a prominent lesion of diabetic retinopathy.

LKB1 loss-of-function mutations, observed in ∼30% of human lung adenocarcinomas, contribute significantly to lung cancer malignancy progression. We show that lysyl oxidase (LOX), negatively regulated by LKB1 through mTOR-HIF-1α signaling axis, mediates lung cancer progression. Inhibition of LOX activity dramatically alleviates lung cancer malignancy progression. Up-regulated LOX expression triggers excess collagen deposition in Lkb1-deficient lung tumors, and thereafter results in enhanced cancer cell proliferation and invasiveness through activation of β1 integrin signaling. High LOX level and activity correlate with poor prognosis and metastasis. Our findings provide evidence of how LKB1 loss of function promotes lung cancer malignancy through remodeling of extracellular matrix microenvironment, and identify LOX as a potential target for disease treatment in lung cancer patients.

Aberrant concentrations of cardiac extracellular matrix (ECM) fibrillar collagen cross-linking have been proposed to be an underlying cause of cardiac diastolic dysfunction however the role of the adaptive immune system in this process has yet to be investigated. Fibrillar collagen cross-linking is a product of the enzymatic activities of lysyl oxidase (LOX and LOXL-3) released by the cardiac fibroblast and possibly cardiac myocytes. Our hypothesis is that stimulation of the TH1 lymphocytes activates lysyl oxidase mediated ECM cross-linking and thereby alters left ventricular function. Three-month old C57BL/J female mice were treated with selective TH1 lymphocyte inducers - T-cell receptor Vβ peptides (TCR). After 6 weeks, candidate gene expression, tissue enzymatic activity, ECM composition, and left ventricular mechanics were quantified. Lymphocyte gene expression and cytokine assay revealed TH1 immune polarization with TCR administration which was associated with a 2.6-fold and 3.1-fold increase of LOX and LOXL3 gene expression, respectively, and a 55% increase in cardiac LOX enzymatic activity. The ECM cross-linked fibrillar collagen increased by 95% when compared with the control. Concurrently, there was a 33% increased ventricular stiffness, decreased cardiac output, and normal ejection fraction. These data implicate the TH1 lymphocyte in the pathogenesis of diastolic dysfunction which has potential clinical application in the pathogenesis of diastolic heart failure., (Copyright © 2010 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Cadmium (Cd) inhalation can result in emphysema. Cd exposure of rat lung fibroblasts (RFL6) enhanced levels of metal scavenging thiols, e.g., metallothionein (MT) and glutathione (GSH), and the heavy chain of gamma-glutamylcysteine synthetase (gamma-GCS), a key enzyme for GSH biosynthesis, concomitant with downregulation of lysyl oxidase (LO), a copper-dependent enzyme for crosslinking collagen and elastin in the extracellular matrix (ECM). Cd downregulation of LO in treated cells was closely accompanied by suppression of synthesis of collagen, a major structure component of the lung ECM. Using rats intratracheally instilled with cadmium chloride (30 microg, once a week) as an animal model, we further demonstrated that although 2-week Cd instillation induced a non-significant change in the lung LO activity and collagen synthesis, 4- and 6-week Cd instillation resulted in a steady decrease in the lung LO and collagen expression. The lung MT and total GSH levels were both upregulated upon the long-term Cd exposure. Emphysematous lesions were generated in lungs of 6-week Cd-dosed rats. Increases of cellular thiols by transfection of cells with MT-II expression vectors or treatment of cells with GSH monoethyl ester, a GSH delivery system, markedly inhibited LO mRNA levels and catalytic activities in the cell model. Thus, Cd upregulation of cellular thiols may be a critical cellular event facilitating downregulation of LO, a potential mechanism for Cd-induced emphysema., (2009 Elsevier Ireland Ltd. All rights reserved.)

Osteoblasts synthesize collagen matrix, which itself regulates the differentiation of precursor cells into mature osteoblasts. They express lysyl oxidase (LOX), which is involved in the collagen cross-linking process. Lathyrogens, like ss-aminopropionitrile (ssAPN), inhibit the formation of a stable matrix. The aim of the present study was to investigate the influence of cross-linking on osteoblastic differentiation. MC3T3-E1 cells were seeded and treated with or without 400 muM ssAPN for 1 week. Thereafter, living cells were removed and, on this extracellular matrix, new MC3T3-E1 cells were seeded and cultured for 1 week without ssAPN. RNA was isolated, and expression of specific marker genes was determined by quantitative reverse transcription-polymerase chain reaction. Changes in specific cross-links after ssAPN treatment were measured with Fourier-transform infrared spectroscopy. The collagen matrix that formed showed a significant reduction of two major cross-links of bone collagen, deH-DHLNL and pyr, compared to control cultures. Gene expression studies showed an increase of collagen alpha1 (I) (COL1A1) to 150%. Expression of LOX and osteocalcin (OCN) mRNA was significantly downregulated to about 75%. When fresh MC3T3-E1 cells were seeded on this altered matrix without ssAPN, COL1A1 mRNA expression was upregulated (140%), OCN was downregulated (60%), and LOX mRNA expression remained unaffected. These results indicate that ssAPN treatment not only disrupts collagen cross-link formation but also affects osteoblastic activity and expression. In conclusion, the disrupted matrix produced in the presence of lathyrogen influences, even in its absence, the expression of osteoblastic genes.

Previous data from spaceflight studies indicate that injured muscle and bone heal slowly and abnormally compared with ground controls, strongly suggesting that ligaments or tendons may not repair optimally as well. Thus the objective of this study was to investigate the biochemical and molecular gene expression of the collagen extracellular matrix in response to medial collateral ligament (MCL) injury repair in hindlimb unloaded (HLU) rodents. Male rats were assigned to 3- and 7-wk treatment groups with three subgroups each: sham control, ambulatory healing (Amb-healing), and HLU-healing groups. Amb- and HLU-healing animals underwent bilateral surgical transection of their MCLs, whereas control animals were subjected to sham surgeries. All surgeries were performed under isoflurane anesthesia. After 3 wk or 7 wk of HLU, rats were euthanized and MCLs were surgically isolated and prepared for molecular or biochemical analyses. Hydroxyproline concentration and hydroxylysylpyridinoline collagen cross-link contents were measured by HPLC and showed a substantial decrement in surgical groups. MCL tissue cellularity, quantified by DNA content, remained significantly elevated in all HLU-healing groups vs. Amb-healing groups. MCL gene expression of collagen type I, collagen type III, collagen type V, fibronectin, decorin, biglycan, lysyl oxidase, matrix metalloproteinase-2, and tissue inhibitor of matrix metalloproteinase-1, measured by real-time quantitative PCR, demonstrated differential expression in the HLU-healing groups compared with Amb-healing groups at both the 3- and 7-wk time points. Together, these data suggest that HLU affects dense fibrous connective tissue wound healing and confirms previous morphological and biomechanical data that HLU inhibits the ligament repair processes.

In this study, we aimed to increase crosslinking in collagen and elastin in the extracellular matrix through overexpression of lysyl oxidase (LO) in order to improve mechanical strength in dermal wounds during healing. We had used a gene activated matrix (GAM) approach to locally deliver plasmid DNA (pDNA) complexed with polyethylenimine (PEI) in collagen gels at the wound site for localized and sustained transfection of cells involved in the healing process. We first demonstrated in vitro that PEI-pDNA complexes in collagen gels could be taken up and expressed by cultured fibroblasts for at least 20 days. In vitro studies showed that fibroblast-seeded GAMs with the LO transgene exhibited over a 3-fold increase in mechanical strength as compared with a green fluorescent protein (GFP)-transgene control. Addition of an inhibitor of LO abolished this increase. We applied this system in a rat dermal wound healing model and showed that treatment with LO-producing GAMs led to significantly enhanced mechanical strength of the wound site.

The study is based on the premise that the enzyme lysyl oxidase (LO), which catalyzes the crosslinking of extracellular matrix (ECM) proteins, participates in ECM modulation and senile plaque formation in Alzheimer's disease (AD). Experiments on hippocampal samples indicate that LO activity is increased (about 30%) in AD, but also in non-Alzheimer's dementia, as compared to controls with unrelated diseases. Immunohistochemistry with specific LO antibody indicates localization in blood vessel walls and in plaque-like structures. The number of LO-positive plaque-like structures in AD was over two-fold higher as compared to both non-Alzheimer's dementia and control groups. The findings lead us to suggest that active LO molecules in the ECM may be associated with plaque formation.