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Chronic liver injury leads to decreased liver function and increased fibrosis. Fibrosis is not only associated with the development of portal hypertension and carcinogenesis, but with the occurrence of events and a poor prognosis, highlighting the importance of non‐invasive fibrosis assessment in patients. In the present study, we searched for markers related to liver fibrosis via proteomic analysis of small extracellular vesicles (sEVs). In the discovery cohort, proteomic analysis was carried out in the sEVs extracted from the sera of 5 patients with decompensated cirrhosis, 5 patients with compensated cirrhosis, and 5 controls without liver disease. Interestingly, in this cohort, fibulin‐4 was significantly associated with cirrhosis while in the validation cohort [formed by 191 patients: 7 patients without disease, 16 patients without liver disease (other diseases), 38 patients with chronic liver disease (CLD), 75 patients with cirrhosis of Child–Pugh class A (36 without hepatocellular carcinoma [HCC], 29 with HCC), and 65 patients with cirrhosis of Child–Pugh class B–C (39 without HCC, 26 with HCC)], fibulin‐4/CD9 levels increased with cirrhosis progression. Furthermore, the fibulin‐4/CD9 ratio was significantly higher in patients with varices. Immunostaining also revealed strong fibulin‐4 expression in cholangiocytes within the fibrous areas and mesothelial cells in liver tissue blood vessels. Taken together, our results suggest that fibulin‐4, essential for lysyl oxidase activation, might be a new liver fibrosis marker found in the sEVs of patients with cirrhosis. [ABSTRACT FROM AUTHOR]

[Display omitted] • Single-cell RNA sequence analysis revealed the heterogeneity of fibroblast of hearts in diabetic mouse for the first time. • The number of cluster 4 fibroblasts were higher and we reveal the up-regulation of Lox in diabetic mouse group. • We validate that the up-regulation of Lox is associated with the decreased cardiac function, increased interstitial fibrosis, higher expression of HYP and the amounts of collagen in the DM group. • Upregulation of Lox might be involved in the development of diabetic myocardial fibrosis and cardiac dysfunction via activation of TGF-β1-Smad2/3 signaling pathway. • Inhibition of Lox could improve cardiac function and fibrosis, which may indicate a potential target for the treatment of diabetic cardiomyopathy. Myocardial fibrosis and cardiac dysfunction are the main characteristics of diabetic heart disease. However, the molecular mechanisms underlying diabetic myocardial fibrosis remain unclear. This study aimed to investigate the heterogeneity of cardiac fibroblasts in diabetic mice and its possible mechanism in the development of diabetic myocardial fibrosis. We established a diabetic mouse model by injecting mice with streptozotocin. The overall cell profiles in diabetic hearts were analyzed using single-cell RNA transcriptomic techniques. Cardiac function was evaluated by echocardiography. Cardiac fibrosis was assessed by Masson's trichrome and Sirius red staining. Protein expression was analyzed using Western blotting and immunofluorescence staining. A total of 11,585 cells were captured in control (Ctrl) and diabetic (DM) hearts. Twelve cell types were identified in this study. The number of fibroblasts was significantly higher in the DM hearts than in the Ctrl group. The fibroblasts were further re-clustered into nine subsets. Interestingly, cluster 4 fibroblasts were significantly increased in diabetic hearts compared with other fibroblast clusters. Lysyl oxidase (Lox) was highly expressed in DM fibroblasts (especially in cluster 4). Beta-aminopropionitrile, a Lox inhibitor, inhibited collagen expression and alleviated cardiac dysfunction in the diabetic group. Lysyl oxidase inhibition also reduced high glucose-induced collagen protein upregulation in primary fibroblasts. Moreover, a TGF-β receptor inhibitor not only prevented an increase in Lox and Col I but also inhibited the phosphorylation of Smad2/3 in fibroblasts. This study revealed the heterogeneity of cardiac fibroblasts in diabetic mice for the first time. Fibroblasts with high expression of Lox (cluster 4 fibroblasts) were identified to play a crucial role in fibrosis in diabetic heart disease. The findings of this study may provide a possible therapeutic target for interstitial fibrosis. [ABSTRACT FROM AUTHOR]

The primary source of strength in menisci, tendons, and ligaments are hierarchical collagen fibers; however, these fibers are not regenerated after injury nor in engineered replacements, resulting in limited repair options. Collagen strength is reliant on fiber alignment, density, diameter, and crosslinking. Recently, we developed a culture system which guides cells in high-density collagen gels to develop native-like hierarchically organized collagen fibers, which match native fiber alignment and diameters by 6 weeks. However, tensile moduli plateau at 1MPa, suggesting crosslinking may be lacking. Collagen crosslinking is regulated by lysyl oxidase (LOX) which forms immature crosslinks that condense into mature trivalent crosslinks. Trivalent crosslinks are thought to be the primarily source of strength in fibers, but it's not well understood how they form. The objective of this study was to evaluate the effect of exogenous LOX in our culture system at different stages of hierarchical fiber formation to produce stronger replacements and to better understand factors affecting crosslink maturation. We found treatment with LOX isoform LOXL2 did not restrict hierarchical fiber formation, with constructs still forming aligned collagen fibrils by 2 weeks, larger fibers by 4 weeks, and early fascicles by 6 weeks. However, LOXL2 treatment did significantly increase mature pyridinium (PYD) crosslink accumulation and tissue mechanics, with timing of LOXL2 supplementation during fiber formation having a significant effect. Overall, we found one week of LOXL2 supplementation at 4 weeks produced constructs with native-like fiber organization, increased PYD accumulation, and increased mechanics, ultimately matching the tensile modulus of immature bovine menisci. Collagen fibers are the primary source of strength and function in connective tissues throughout the body, however it remains a challenge to develop these fibers in engineered replacements, greatly reducing treatment options. Here we demonstrate lysyl oxidase like 2 (LOXL2) can be used to significantly improve the mechanics of tissue engineered constructs, but timing of application is important and will most likely depend on degree of collagen organization or maturation. Currently there is limited understanding of how collagen crosslinking is regulated, and this system is a promising platform to further investigate cellular regulation of LOX crosslinking. Understanding the mechanism that regulates LOX production and activity is needed to ultimately regenerate functional repair or replacements for connective tissues throughout the body. [Display omitted] [ABSTRACT FROM AUTHOR]

Recent insights collectively suggest the important roles of lysyl oxidase (LysOX) in the pathological processes of several acute and chronic neurological diseases, but the molecular regulatory mechanisms remain elusive. Herein, we explore the regulatory role of LysOX in the seizure-induced ferroptotic cell death of neurons. Mechanistically, LysOX promotes ferroptosis-associated lipid peroxidation in neurons via activating extracellular regulated protein kinase (ERK)-dependent 5-lipoxygenase (Alox5) signaling. In addition, overexpression of LysOX via adeno-associated viral vector (AAV)-based gene transfer enhances ferroptosis sensitivity and aggravates seizure-induced hippocampal damage. Our studies show that pharmacological inhibition of LysOX with β -aminopropionitrile (BAPN) significantly blocks seizure-induced ferroptosis and thereby alleviates neuronal damage, while the BAPN-associated cardiotoxicity and neurotoxicity could further be reduced through encapsulation with bioresponsive amorphous calcium carbonate-based nanocarriers. These findings unveil a previously unrecognized LysOX-ERK-Alox5 pathway for ferroptosis regulation during seizure-induced neuronal damage. Suppressing this pathway may yield therapeutic implications for restoring seizure-induced neuronal injury. A previously unrecognized LysOX/ERK/Alox5 signaling axis for ferroptosis regulation during seizure-induced neuronal damage is delineated. Selective inhibition of this pathway provides a therapeutic approach for treating post-seizure neuronal impairment. [Display omitted] [ABSTRACT FROM AUTHOR]

Suitable scaffold structures and mechanical loading are essential for functional tendon engineering. However, the bipolar fibril structure of native tendon collagen is yet to be recaptured in engineered tendons. This study compared the development of Achilles tendons of postnatal rats with and without (via surgical section) mechanical loading to define the mechanism of mechanical stimulation-mediated tendon development. The results demonstrated that the severed tendons weakened mechanically and exhibited disorganization without a bipolar fibril superstructure. Proteomic analysis revealed differentially expressed key regulatory molecules related to the collagen assembly process, including decreased fibromodulin, keratocan, fibroblast growth factor-1, and increased lumican and collagen5a1 in the severed tendons with immunohistochemical verification. Additionally, a complex regulatory network of mechanical stimulation-mediated collagen assembly in a spatiotemporal manner was also revealed using bioinformatics analysis, wherein PI3K–Akt and HDAC4 may be the predominant signaling pathways. A wavy microgrooved surface (Y = 5.47sin(0.015x)) that biomimics tendon topography was observed to enhance the expression of collagen assembly molecules under mechanical loading, and the aforementioned pathways are particularly involved and verified with their respective inhibitors of LY-294002 and LMK-235. Furthermore, an electrospun crimped nanofiber scaffold (approximately 2 μm fiber diameter and 0.12 crimpness) was fabricated to biomimic the tenogenic niche environment; this was observed to be more effective on enhancing collagen production and assembly under mechanical stimulation. In conclusion, the synergistic effect between topographical niche and mechanical stimulation was observed to be essential for collagen assembly and maturation and should be applied to functional tendon engineering in the future. In biomaterial-mediated tendon regeneration, mechanical stimulation is essential for tendon collagen assembly. However, the underlying mechanisms remain not fully defined, leading to the failure of the native-like collagen regeneration. In this study, a mechanical stimulation deprivation model of rat tendon was established to reveal the mechanisms in tendon development and define the key regulatory molecules including small leucine-rich proteoglycans, lysyl oxidase and collagen V. After ensuring the importance of biomimetic structure in tendon remodeling, crimped nanofibers were developed to verify these regulatory molecules, and demonstrated that mechanical stimulation significantly enhanced collagen assembly via PIK3 and HDAC4 pathways in biomaterial-regulated tendon regeneration. This study provides more insightful perspectives in the physiologically remodeling progression of tendon collagen and design of tendon scaffolds. [Display omitted] [ABSTRACT FROM AUTHOR]

Energetic electrons have recently evolved as a powerful tool for crosslinking bio-derived hydrogels without the need for adding potentially hazardous reagents. Application of this approach allows for synthesis of biomimetic collagen-derived networks of highly tunable properties and functionalization. Yet, the underlying reaction kinetics are still not sufficiently established at this point. While hydroxyl radicals are generated by energetic electron-induced hydrolysis of water and play a key role in introducing covalent bonds between network fibers, a detailed mechanistic understanding would significantly increase applicability. We present a comprehensive analysis of central aspects of the reactivity between the hydroxyl radical (•OH) and collagen, elastin, glycine (Gly) and l -lysine (Lys). Pulse radiolysis (PR), solid state nuclear magnetic resonance (NMR), ultraviolet-visible absorption spectroscopy (UV/VIS) and electron spray ionization mass spectrometry (ESI-MS) shine light on distinct features of the crosslinking process. These highlight retained protein backbone integrity in collagen and elastin whilst Lys's ability to form several imine bonded Lys-Lys-species suggests striking similarities to crosslinking via lysyl oxidase catalysis in vivo. Thus, energetic electron based crosslinking opens the venue for customized hybrid gels of outstanding biomimicry and –compatibility. Energetic electron beam treatment constitutes a highly attractive approach to establish chemical bonds between (bio) molecules. Although a convincing number of publications showed the versatility regarding crosslinking of bioderived hydrogels, insights into the underlying chemistry are still unestablished at this point. The present work unravels the mechanistics of energetic electron induced processes in collagen and elastin hydrogels as well as several abundant amino acids in aqueous solution. As key finding we demonstrate, that i) the connection between polymer chains is dominated by amino acid side chain interaction and ii) two single l -lysine molecules form an imine bond between the terminal amino group of one molecule and the delta carbon of the second molecule. We also consider the formation of H-bonds as a second crosslinking pathway. These findings open up for advanced, optionally spatially resolved biomaterials design. [Display omitted] [ABSTRACT FROM AUTHOR]

Liver fibrosis is a common feature of progressive liver disease and is manifested as a dynamic series of alterations in both the biochemical and biophysical properties of the liver. Hepatic stellate cells (HSCs) reside within the perisinusoidal space of the liver sinusoid and are one of the main drivers of liver fibrosis, yet it remains unclear how changes to the sinusoidal microenvironment impact HSC phenotype in the context of liver fibrosis. Cellular microarrays were used to examine and deconstruct the impacts of bio-chemo-mechanical changes on activated HSCs in vitro. Extracellular matrix (ECM) composition and stiffness were found to act individually and in combination to regulate HSC fibrogenic phenotype and proliferation. Hyaluronic acid and collagen III promoted elevated collagen I expression while collagen IV mediated a decrease. Previously activated HSCs exhibited reduced lysyl oxidase (Lox) expression as array substrate stiffness increased, with less dependence on ECM composition. Collagens III and IV increased HSC proliferation, whereas hyaluronic acid had the opposite effect. Meta-analysis performed on these data revealed distinct phenotypic clusters (e.g. low fibrogenesis/high proliferation) as a direct function of their microenvironmental composition. Notably, soft microenvironments mimicking healthy tissue (1 kPa), promoted higher levels of intracellular collagen I and Lox expression in activated HSCs, compared to stiff microenvironments mimicking fibrotic tissue (25 kPa). Collectively, these data suggest potential HSC functional adaptations in response to specific bio-chemo-mechanical changes relevant towards the development of therapeutic interventions. These findings also underscore the importance of the microenvironment when interrogating HSC behavior in healthy, disease, and treatment settings. In this work we utilized high-throughput cellular microarray technology to systematically interrogate the complex interactions between HSCs and their microenvironment in the context of liver fibrosis. We observed that HSC phenotype is regulated by ECM composition and stiffness, and that these phenotypes can be classified into distinct clusters based on their microenvironmental context. Moreover, the range of these phenotypic responses to microenvironmental stimuli is substantial and a direct consequence of the combinatorial pairing of ECM protein and stiffness signals. We also observed a novel role for microenvironmental context in affecting HSC responses to potential fibrosis therapeutics. [Display omitted] [ABSTRACT FROM AUTHOR]

Abdominal aortic aneurysms (AAAs) are characterized histopathologically by compromised elastic fiber integrity, lost smooth muscle cells or their function, and remodeled collagen. We used a recently introduced mouse model of AAAs that combines enzymatic degradation of elastic fibers and blocking of lysyl oxidase, and thus matrix cross-linking, to study progressive dilatation of the infrarenal abdominal aorta, including development of intraluminal thrombus. We quantified changes in biomaterial properties and biomechanical functionality within the aneurysmal segment as a function of time of enlargement and degree of thrombosis. Towards this end, we combined multi-modality imaging with state-of-the art biomechanical testing and histology to quantify regional heterogeneities for the first time and we used a computational model of arterial growth and remodeling to test multiple hypotheses, suggested by the data, regarding the degree of lost elastin, accumulation of glycosaminoglycans, and rates of collagen turnover. We found that standard histopathological findings can be misleading, while combining advanced experimental and computational methods revealed that glycosaminoglycan accumulation is pathologic, not adaptive, and that heightened collagen deposition is ineffective if not cross-linked. In conclusion, loss of elastic fiber integrity can be a strong initiator of aortic aneurysms, but it is the rate and effectiveness of fibrillar collagen remodeling that dictates enlargement. Precise mechanisms by which abdominal aortic aneurysms enlarge remain unclear, but a recent elastase plus β-aminopropionitrile mouse model provides new insight into disease progression. As in the human condition, the aortic degeneration and adverse remodeling are highly heterogeneous in this model. Our multi-modality experiments quantify and contrast the heterogeneities in geometry and biomaterial properties, and our computational modeling shows that standard histopathology can be misleading. Neither accumulating glycosaminoglycans nor frustrated collagen synthesis slow disease progression, thus highlighting the importance of stimulating adaptive collagen remodeling to limit lesion enlargement. [Display omitted] [ABSTRACT FROM AUTHOR]

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). 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. 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. 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. [Display omitted] Lysyl oxidase expression is enhanced in cholangiocarcinoma tumors and predictive of poor survival. Lysyl oxidase is found mainly in cancer-associated fibroblasts, resident stromal cells responsible for shaping the tumor microenvironment. Lysyl oxidase inactivation in vivo shows diminished cholangiocarcinoma burden in murine livers. Therefore, targeting lysyl oxidase may be a promising therapeutic strategy in cholangiocarcinoma. [ABSTRACT FROM AUTHOR]

Lysyl oxidase (LOX) is required for the formation of bone collagen cross-links. Inactivation of the LOX gene in osteoblasts by DNA methylation and JAK signaling has been reported to cause loss of cross-links and an increased risk of fractures. Tocotrienols (T3s) have proven benefits on bone strength, but their potential effects on LOX remain largely unknown. Thus, the present study investigates the in vitro effects of T3s on LOX expression in human osteoblastic MG-63 cells. Results indicated that Tocotrienol-Rich Fraction (TRF), the δ-T3 rich oil extracted from Annatto was the most effective and significantly increased LOX expression. TRF treatment decreased de-novo methyltransferases (DNMTs), DNMT3A and DNMT3B levels. In addition, TRF significantly inhibited JAK2 activation and decreased expression of Fli1, a transcription factor of DNMTs. We conclude that TRF induced an increase in LOX expression via inhibition of de-novo methylation and reduction of Fli1 expression by the inactivation of JAK2. Abbreviations: CpG: cytosine-guanine dinucleotide; DNMT: DNA methyltransferase; Fli1: friend leukemia virus integration 1; JAK: janus kinase; LOX: lysyl oxidase; PCR: polymerase chain reaction; STAT: signal transducers and activators of transcription; T3s: tocotrienols; TPs: tocopherols; TRF: Tocotrienol-Rich Fraction. TRF induced amelioration of LOX expression in MG-63 via inhibition of de-novo methylation through the reduction of Fli1 expression by the inactivation of JAK2. [ABSTRACT FROM AUTHOR]

Background: Lysyl oxidase is an extracellular regulatory enzyme with an imperative role in interlinking of collagen and elastin by oxidizing lysine residues. Lysyl oxidase has been implicated in incidence of mammary tumors in bitches. Therefore, it becomes significant to study the structural and functional features of this enzyme for a better understanding of its molecular mechanisms. Results: The detailed computational investigation of the canine lysyl oxidase protein was analyzed in silico with respect to its physicochemical properties, secondary and tertiary structure predictions and functional analysis using standard bioinformatic tools. Lysyl oxidase is a flexible protein with an average molecular weight of around 46 kDa, unstable, hydrophilic, and extracellular (secretory) in nature. Twelve cysteine residues and a disulfide bridge were also found. Secondary structure analysis shows that most of the protein has predominant coiled configuration. A putative copper-binding region signature was predicted. The phylogenetic relationship of canine lysyl oxidase with a vast range of mammalian species indicates that the protein was very well conserved throughout the course of evolution. Top 10 interacting proteins were identified using STRING v10.0 analysis, elastin being the closest interacting protein. Functional analysis by InterproScan predicted protein’s biological role in oxidation-reduction process. Conclusion: Understanding the structural and functional properties of the protein will facilitate a better understanding of its mechanism of enzyme action. Further, the predicted 3D model will serve as a cornerstone for further understanding towards the tumorigenesis potential of the protein. [ABSTRACT FROM AUTHOR]

Fibrosis and stricture are major comorbidities in patients with eosinophilic esophagitis (EoE). Lysyl oxidase (LOX), a collagen cross-linking enzyme, has not been investigated in the context of EoE. We investigated regulation of epithelial LOX expression as a novel biomarker and functional effector of fibrostenotic disease conditions associated with EoE. LOX expression was analyzed by using RNA-sequencing, PCR assays, and immunostaining in patients with EoE; cytokine-stimulated esophageal 3-dimensional organoids; and fibroblast–epithelial cell coculture, the latter coupled with fluorescence-activated cell sorting. Gene ontology and pathway analyses linked TNF-α and LOX expression in patients with EoE, which was validated in independent sets of patients with fibrostenotic conditions. TNF-α–mediated epithelial LOX upregulation was recapitulated in 3-dimensional organoids and coculture experiments. We find that fibroblast-derived TNF-α stimulates epithelial LOX expression through activation of nuclear factor κB and TGF-β–mediated signaling. In patients receiver operating characteristic analyses suggested that LOX upregulation indicates disease complications and fibrostenotic conditions in patients with EoE. There is a novel positive feedback mechanism in epithelial LOX induction through fibroblast-derived TNF-α secretion. Esophageal epithelial LOX might have a role in the development of fibrosis with substantial translational implications. [ABSTRACT FROM AUTHOR]

Copyright of Revista Española de Cardiología (18855857) is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Although fibrosis and emphysema are in many ways on opposite ends of the pulmonary parenchymal disease spectrum, they seem to share common pathomechanistic steps. This is illustrated by the coexistence of both entities in lungs of individuals with combined pulmonary fibrosis and emphysema. Macroproteins elastin and collagen are major constituents of the pulmonary extracellular matrix. The prevailing paradigm states that emphysema is caused by an imbalance between destructive proteolytic and protective antiproteolytic enzymes leading to accelerated degradation of elastin fibers in the lungs. Rates of elastin breakdown, however, are equally enhanced in patients with idiopathic pulmonary fibrosis (IPF) and emphysema. Excessive accumulation of collagen is a hallmark of IPF. Surprisingly, collagen levels in the lung parenchyma of patients with emphysema are also higher than in controls. The concentration of elastin fibers is elevated in fibrotic lungs, despite accelerated elastinolysis, suggesting that elastin repair is also enhanced in IPF. Since elastin concentrations are reduced in emphysematous lungs, the factor of divergence between emphysema and fibrosis seems to be the degree of elastin repair. Multiple elastin repair steps can be deduced of which tropoelastin synthesis and crosslinking of tropoelastin polymers by the copper dependent enzyme lysyl oxidase seem to be the most important ones. We suspect that the distinction in the pathogeneses of lung fibrosis and emphysema depends on the local availability of copper to activate sufficient lysyl oxidase for elastin crosslinking, and suggest assessing the effects of inhalation therapy with copper plus heparin in emphysema and heparin monotherapy in IPF. [ABSTRACT FROM AUTHOR]

Background and Aims: Crohn's disease is a chronic inflammatory disorder of the intestine and often leads to fibrosis, characterized by excess extracellular matrix [ECM] deposition, increased tissue stiffness, and stricture formation. Here we evaluated the contribution of myofibroblast--ECM interactions to the development of intestinal fibrosis in Crohn's disease. Methods: Matched primary human myofibroblasts were isolated from stenotic, inflamed and normal-appearing small intestine within the same Crohn's disease patient [n = 10]. Cells were analyzed by gene expression profiling, microscopy and functional assays, including matrix metalloproteinase [MMP] production and ECM contraction. Results: We demonstrated that myofibroblasts isolated from stenotic intestine differed both in phenotype and function from those isolated from purely inflammatory or normal-appearing intestine of the same patient. Stenotic myofibroblasts displayed increased expression of genes associated with ECM modulation and collagen deposition. Upon culture in a fibrotic environment, normal myofibroblasts increased expression of MMPs to counteract the mechanical force exerted by the matrix. Interestingly, stenotic myofibroblasts showed a paradoxical response with decreased expression of MMP3. In addition, stenotic myofibroblasts expressed increased levels of the collagen crosslinking enzyme lysyl oxidase [LOX] and induced significantly more ECM contraction than both normal and inflamed myofibroblasts. Importantly, LOX inhibition completely restored MMP3 activity in stenotic myofibroblasts grown in a fibrotic environment, and prevented excessive ECM contraction. Conclusions: Together these data indicate aberrancies in the myofibroblast--ECM interaction in Crohn's disease, and identify LOX inhibition as a potential anti-fibrotic agent in this condition. [ABSTRACT FROM AUTHOR]

Background/objectives Lysyl oxidase (LOX) is an enzyme crucial for collagen fibre crosslinking and thus for fibrosis development. Fibrosis is characterised by a surplus of collagen fibre accumulation and is amongst others also a feature of obesity-associated dysfunctional adipose tissue (AT) which has been linked with type 2 diabetes. We hypothesised that in type 2 diabetes and obesity LOX expression and activity will be increased as a consequence of worsening AT dysfunction. This study aimed to provide a comprehensive characterisation of LOX in human AT. Methods LOX mRNA expression was analysed in omental and abdominal subcutaneous AT obtained during elective surgery from subjects with a wide range of BMI, with and without diabetes. In addition, LOX expression was studied in subcutaneous AT before and 9.5 months after bariatric surgery. To study the mechanism of LOX changes, its expression and activity were assessed after either hypoxia, recombinant human leptin or glucose treatment of AT explants. In addition, LOX response to acute inflammation was tested after stimulation by a single injection of lipopolysaccharide versus saline solution (control) in healthy men, in vivo. Quantity of mRNA was measured by RT-qPCR. Results LOX expression was higher in obesity and correlated with BMI whilst, in vitro, leptin at high concentrations, as a potential feedback mechanism, suppressed its expression. Neither diabetes status, nor hyperglycaemia affected LOX . Hypoxia and lipopolysaccharide-induced acute inflammation increased LOX AT expression, latter was independent of macrophage infiltration. Conclusions Whilst LOX may not be affected by obesity-associated complications such as diabetes, our results confirm that LOX is increased by hypoxia and inflammation as underlying mechanism for its upregulation in adipose tissue with obesity. [ABSTRACT FROM AUTHOR]

Objective To explore the role of microRNA (miR)-30a in the development of aortic dissection. Methods Human aortic specimens of aortic dissections and aneurysms were harvested. Aortic specimens from donors for heart transplantation served as controls. Rat aortic vascular smooth muscle cells (VSMCs) were transfected with agomiR-30a or antagomiR-30a, and control cells were incubated with empty vectors. Rats were pretreated with agomiR-30a or antagomiR-30a (5 × 10 7 transfection units every 3 days for 4 weeks), and empty vectors were infused to controls. Acute aortic dissection was induced by subcutaneous infusion of angiotensin II (1 μg · kg −1 · min −1 for 24 hours). Protein expressions of lysyl oxidase (LOX) and elastin and gene expression of miR-30a were measured in VSMCs and human and rat aortic specimens by Western blot analysis and quantitative real-time polymerase chain reaction. Results Gene expression of miR-30a was much higher, and protein abundance of LOX and elastin was significantly lower, in the aortic dissection specimens ( P < .05 vs controls). Transfection of agomiR-30a markedly decreased the luciferase activity of LOX in VSMCs of wild type, but not of LOX 3′-UTR mutant ( P = .002). In cultured VSMCs, transfection of agomiR-30a dramatically enhanced the gene expression of miR-30a and down-regulated the protein abundance of LOX and elastin ( P < .05 vs controls). Pretreatment with agomiR-30a in vivo enhanced miR-30a expression and down-regulated the protein abundance of LOX and elastin in rat aortas ( P < .05 vs controls). The rate of dissection was significantly higher in rats pretreated with agomiR-30a ( P = .003 vs controls). Conclusions Overexpression of miR-30a contributes to the development of aortic dissection, possibly by targeting LOX. [ABSTRACT FROM AUTHOR]

Abnormalities in the elastic fiber biology are seen in pulmonary emphysema (PE). The copper-dependent lysyl oxidases regulate the production and accumulation of elastic fibers in the connective tissue. This study focused on the relationship between lysyl oxidase (LOX), LOX-like protein 1 (LOXL1), and LOXL2 and PE pathogenesis. Lung samples with or without PE from patients with chronic obstructive lung disease (n = 35) were used. Protein levels of elastin, LOX, LOXL1, LOXL2, hypoxia inducible factor 1-alpha (HIF-1α), copper metabolism domain containing-1 (COMMD1), and phosphatase and tensin homolog (PTEN) were assayed using microscopic and biochemical methods The emphysematous areas were characterized by enlargement of the alveoli, destruction of the alveolar structure, accumulation of macrophages in the alveolar lumens, and showed increased HIF-1α immunoreactivity. Additionally, the emphysematous areas had significantly lower elastin, LOX, LOXL1, LOXL2, HIF-1α, COMMD1, and PTEN protein levels than the non-emphysematous areas. We suppose that the reductions in the HIF-1α levels led to decreases in the protein levels of active LOX, LOXL1, and LOXL2. These decreases might cause abnormalities in the elastic fiber biology. HIF-1α activation induced by decreased COMMD1 and protease activation induced by decreased PTEN might contribute to the development of PE. Finally, methods aimed at increasing the protein levels of LOXs, COMMD1 and PTEN might be effective for treating PE. [ABSTRACT FROM AUTHOR]

Purpose: Lysyl oxidase (LOX) controls the cross-linking and maturation of elastin and collagen fibers. In this study, we investigated the association between LOX gene polymorphisms and intracranial aneurysm (IA) formation in a homogeneous Korean population. Materials and Methods: This cross-sectional study involved 80 age-sex matched patients with IA and controls. Fisher's exact test was performed to analyze allelic associations between ten single nucleotide polymorphisms (SNPs) and IA, including 41 ruptured and 39 unruptured cases. Haplotype-specific associations were analyzed using the omnibus test estimating asymptotic chi-square statistics. Results: Of ten SNPs, three SNPs (rs2303656, rs3900446, and rs763497) were significantly associated with IA (p<0.01). The C allele of rs3900446 was significantly related to increased IA risk with a significant threshold [odds ratio (OR)=20.15, p=4.8×10-5]. Meanwhile, the A allele of rs2303656 showed a preventive effect against IA formation (p=8.2×10-4). Seventeen of 247 haplotype structures showed a suggestive association with IA (asymptotic p<0.001). Of ten SNP haplotype combinations, the CG combination of rs3900446 and rs763497 reached Bonferroni-adjusted significant threshold in IA patients (minor haplotype frequency=0.113, asymptotic p=1.3×10-5). However, there was no association between aneurysm rupture and the LOX gene. Conclusion: This preliminary study indicated that LOX gene polymorphisms, such as rs2303656, rs3900446, and rs763497, may play crucial roles in IA formation in the Korean population. Our novel findings need to be validated in a large-scale independent population. [ABSTRACT FROM AUTHOR]

The formation of cross-linkages between and within collagen is catalyzed by lysyl oxidase, which can be inhibited by β-aminopropionitrile (BAPN), a lathyrogen from sweet pea (Lathyrus odoratus) seeds. The quality and integrity of the collagenous template of skeletal elements depend on an appropriate concentration of collagen cross-links. In this study, chick embryos treated in ovo with BAPN on embryonic days (ED) 4-9 were found to develop multiple skeletal deformities. The most readily discernible and highly reproducible deformity was evident in the tibiotarsus, on which we focused to explore the chronology of the malformation process. Several lines of observation indicated that the bending deformity observable at ED10 in the tibiotarsus was inducible by BAPN administered on ED4-8; in other words, administration of BAPN on ED8 was sufficient to induce the deformity by ED10, whereas administration on ED9 was ineffective. Ultrastructurally, osteoclasts appeared to show enhanced activity in the medullary surface of the bone collar after BAPN administration. In addition, bone hyperplasia associated with the bending deformity was suggested to be correlated with higher osteoblast activity on the concave (or flexor) side of the tibiotarsal skeleton. These findings indicate that the bending deformity due to reduced mechanical integrity of the collagenous template is also associated with aberrant bone remodeling. (J Oral Sci 58, 255-263, 2016). [ABSTRACT FROM AUTHOR]

The lysyl oxidase propeptide (LOX-PP) is derived from pro-lysyl oxidase (Pro-LOX) by extracellular biosynthetic proteolysis. LOX-PP inhibits breast and prostate cancer xenograft tumor growth and has tumor suppressor activity. Although, several intracellular targets and molecular mechanisms of action of LOX-PP have been identified, LOX-PP uptake pathways have not been reported. Here we demonstrate that the major uptake pathway for recombinant LOX-PP (rLOX-PP) is PI3K-dependent macropinocytosis in PWR-1E, PC3, SCC9, MDA-MB-231 cell lines. A secondary pathway appears to be dynamin- and caveola dependent. The ionic properties of highly basic rLOX-PP provide buffering capacity at both high and low pHs. We suggest that the buffering capacity of rLOX-PP, which serves to limit endosomal acidification, sustains PI3K-dependent macropinocytosis in endosomes which in turn is likely to facilitate LOX-PP endosomal escape into the cytoplasm and its observed interactions with cytoplasmic targets and nuclear uptake. [ABSTRACT FROM AUTHOR]

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Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid tumors. A dense desmoplastic and specifically aligned stroma plays integral roles in the progression of PDAC, and inhibits drug penetration to impede treatment. However, direct depletion of stromal components for PDAC therapy interferes with the stromal balance, possibly accelerating tumor progression. Specific fiber alignment of the extracellular matrix, the most significant type of stroma topology in PDAC, is an important feature of PDAC and often overlooked. Here, we found that the alignment of collagen fibers, as determined by second harmonic generation (SHG) imaging, negatively correlated with pancreatic cancer prognosis. We report an integrated, two-step strategy for PDAC treatment, utilizing pretreatment with polymer-lipid-peptide nanoparticles that deliver inhibitors of lysyl oxidase like 2 (LOXL2) and discoidin domain receptor 1 (DDR1), followed by treatment with albumin-bound-paclitaxel (Nab-paclitaxel). The LOXL2 and DDR1 inhibitors intervene with collagen crosslinking and matrix metallopeptidase 1 (MMP1) expression to remodel stromal alignment without decreasing the collagen content, thus facilitating the transport of Nab-paclitaxel through the stromal barrier to enhance drug penetration and accumulation in the PDAC tumor tissue. Through this mechanism, the combined strategy resulted in significant antitumor efficacy in PDAC orthotopic xenograft mice. The current work highlights the key role of stromal alignment in therapeutics transport through stromal barriers. The strategy of remodeling stromal alignment may represent a promising approach for improving drug delivery in other stroma-rich tumors and fibrosis-related diseases. [Display omitted] • Collagen topology was characterized by aligned arrangement in PDAC, but it was disoriented fibers in normal adjacent tissue. • LOXL2-DDR1@MLP facilitated the permeability of nanoparticles through remodeling stromal alignment with stable content. • The antitumor efficacy of Nab-paclitaxel was boosted by the combined strategy. [ABSTRACT FROM AUTHOR]

Abstract: Aims: Lysyl oxidase (LOX) expression is elevated in colorectal cancer (CRC) tissue and associated with disease progression. A blood test may form a more acceptable diagnostic test for CRC although LOX has not previously been measured in the serum. We therefore sought to determine the clinical usefulness of a serum LOX test for CRC in a symptomatic population. Methods: Adult patients referred to a hospital colorectal clinic with bowel symptoms completed a questionnaire and provided a blood sample for serum LOX measurement. Associations between presenting symptoms, serum LOX concentrations and outcomes of investigations were tested by univariate and multivariate analyses to determine if serum LOX was clinically useful in the prediction of CRC. LOX expression in CRC and adjacent colon biopsies was evaluated by ELISA and immunohistochemistry. Results: Thirty-one cases of colorectal cancer and 16 high-risk polyps were identified from a total of 962 participants. There was no association between serum LOX concentration and the presence of CRC, high-risk polyps or cancers at any site. LOX expression was significantly increased in CRC tissue compared to adjacent colon. Conclusion: Despite overexpression of LOX in CRC tissue, elevated serum levels could not be demonstrated. Serum LOX measurement is therefore not a clinically useful test for CRC. [Copyright &y& Elsevier]

Abstract: Mechanical property elaboration of engineered tissues is often assumed on the basis of gene and protein characterizations, rather than mechanical testing. However, we recently demonstrated that mechanical properties are not consistently correlated with matrix content and organization during embryonic tissue development. Based on this, mechanical properties should be assessed independently during natural or engineered tissue formation. Unfortunately, mechanical testing is destructive, and thus alternative means of assessing these properties are desirable. In this study, we examined lysyl oxidase (LOX)-mediated crosslinks as markers for mechanical properties during embryonic tendon formation and the potential to detect them non-destructively. We used tandem mass spectrometry (LC-MS/MS) to quantify changes in hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP) crosslink density in embryonic chick tendon as a function of developmental stage. In addition, we assessed a multiphoton imaging approach that exploits the natural fluorescence of HP and LP. With both techniques, we quantified crosslink density in normal and LOX-inhibited tendons, and correlated measurements with mechanical properties. HP and LP crosslink density varied as a function of developmental stage, with HP-to-dry mass ratio correlating highly to elastic modulus, even when enzymatic crosslink formation was inhibited. Multiphoton optical imaging corroborated LC-MS/MS data, identifying significant reductions in crosslink density from LOX inhibition. Taken together, crosslink density may be useful as a marker of tissue mechanical properties that could be assessed with imaging non-destructively and perhaps non-invasively. These outcomes could have significant scientific and clinical implications, enabling continuous and long-term monitoring of mechanical properties of collagen-crosslinked tissues or engineered constructs. [Copyright &y& Elsevier]

Abstract: Bone morphogenetic protein-2 (BMP2) is among the most popular anabolic agents and substantially increase bone volume related to enhanced osteoblast differentiation. Here we demonstrate a remarkable deterioration in the nanomechanical properties of mineralized tissue induced from osteoblasts solely by the function of BMP2. Mineralized tissue of primary osteoblasts cultured with BMP2 shows molecular features of both bone and cartilage, but depletion of lysyl oxidase family members leads to poor nanomechanical properties of the mineralized tissue. Lysyl oxidase like-2 supplementation reinforces the inferior mineralized tissue induced from osteoblasts by BMP2 through intermolecular cross-linking of type II or type X collagen-rich extracellular matrix. This may also mimic a consolidation of bone fracture gaps, despite the fact that the distribution of the bone properties in such microenvironments has been poorly elucidated. These findings confirm the importance of testing newly induced bone down to the microscale and nanoscale in bone tissue engineering. From the Clinical Editor: Bone morphogenetic protein-2 is known to substantially increase bone volume related to enhanced osteoblast differentiation; however, this team of investigators report a remarkable deterioration in the nanomechanical properties of mineralized tissue induced from osteoblasts solely by the function of BMP2. [Copyright &y& Elsevier]

Abstract: The developmental pathway that gives rise to mature adipocytes involves commitment and terminal differentiation. Our previous findings indicate that BMP4 (bone morphogenetic protein 4) induces nearly complete commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage and knockdown of lysyl oxidase (Lox) disrupts this commitment process. Here, we found that an epithelial–mesenchymal transition (EMT)-like response is required for adipocyte lineage commitment and that Lox is indispensable for this process. When C3H10T1/2 cells were treated with BMP4, Vim and Cdh2 showed up-regulated expression while Cdh1 and Ocln were down-regulated along with enhanced cell migration, which are EMT-like responses. Silencing of Lox in BMP4-treated C3H10T1/2 cells induced a mesenchymal–epithelial transition (MET)-like response associated with the repression of mesenchymal markers, induction of epithelial markers and decreased cell migration. Importantly, blocking the EMT-like response by knocking down Cdh2 or over-expression of Cdh1 impairs adipocyte lineage commitment. EMT is regulated by distinct transcription factors such as Snai1, Snai2 and Twist. In this study, we also found that only Twist was down-regulated after Lox silencing in C3H10T1/2 cells treated with BMP4. This study provides new insights into adipocyte lineage commitment. [Copyright &y& Elsevier]

Summary: Lysyl oxidase family members have various roles in cancer progression. The aim of this study was to investigate their expression and clinical significance in esophageal squamous cell carcinoma. We examined messenger RNA expression of lysyl oxidase family members including lysyl oxidase and lysyl oxidase–like proteins (lysyl oxidase L) in 10 esophageal squamous cell carcinoma cell lines and 83 pairs of tumor samples by quantitative real-time polymerase chain reaction. All except lysyl oxidase L3 were expressed at high levels in esophageal squamous cell carcinoma, but only lysyl oxidase L2 was associated with lymph node metastasis (P = .034). We examined lysyl oxidase L2 protein further by immunohistochemistry staining in 178 surgically resected esophageal squamous cell carcinoma tissue samples. The protein manifested decreased nuclear expression and increased cytoplasmic expression. Moreover, these 2 events both had significant correlation with the presence of lymph node metastasis (P = .001 and P < .001). Overall survival rates of the patients with esophageal squamous cell carcinoma with decreased nuclear expression or increased cytoplasmic expression of lysyl oxidase L2 were significantly lower than those of the patients with esophageal squamous cell carcinoma with the reverse expression pattern (P = .040 or P = .022). Multivariate analyses revealed that nuclear expression of lysyl oxidase L2 was an independent prognostic factor for esophageal squamous cell carcinoma. These results suggest that lysyl oxidase L2 exerts a critical effect on esophageal squamous cell carcinoma progression and can be a predictive marker of lymph node metastasis and outcome. [Copyright &y& Elsevier]

Abstract: Introduction: The lysyl oxidase (LOX) family is an emerging family of amine oxidases responsible for the formation of collagen fibrils in the extracellular matrix. To date, 5 LOX family genes have been identified in humans, encoding LOX and LOX-like proteins (LOXL, LOXL2, LOXL3, and LOXL4). The goal of this study was to evaluate the expression and function of the LOX family genes in odontoblastic differentiation of human dental pulp (HDP) cells. Methods: Expression of the LOX family genes was assessed by reverse transcriptase polymerase chain reaction analysis, and the amine oxidase activity of HDP cells was evaluated by peroxidase-coupled fluorometric assays. Mineral nodule formation and expression of odontoblastic marker genes were assessed in the presence and absence of specific small interfering RNAs (siRNAs) of the LOX family genes. Results: Among the LOX family genes, only LOX and LOXL showed prominent expression during odontoblastic differentiation of HDP cells. Suppression of LOX and LOXL expression by siRNA-induced interference substantially decreased the amine oxidase activity of the differentiating HDP cells. Furthermore, interference of LOX and LOXL expression inhibited mineral nodule formation and expression of odontoblastic marker genes during odontoblastic differentiation of HDP cells. Conclusions: These findings show for the first time that the LOX- and LOXL-mediated organization of collagen fibrils in extracellular matrices of HDP cells might be an important regulator for odontoblastic differentiation of HDP cells. [Copyright &y& Elsevier]

Background/purpose: Oral squamous cell carcinoma (OSCC) is highly prevalent worldwide. Lysyl oxidase (LOX) is an important enzyme that modulates the extracellular. The LOX superfamily contains LOX and LOX-like (LOXL) enzymes. The aim of the present study was to determine the expressions of LOXL genes in OSCC. Materials and methods: RT-PCR was performed to detect messenger (m)RNA expression of the LOXL genes, LOX-Li, -L3, and -L4, in paired OSCC and non-cancerous matched tissues (NCMTs). Results: Increased mRNA expressions of LOX-LI and LOX-L3 in 05CC relative to NCMT5 were identified in 41% and 65% of 05CC samples, respectively. The normalized mRNA expression of LOXL-3 was 1.11 ± 0.15 in OSCC, which was significantly higher than 0.68 ± 0.12 in NCMTs (P = 0.004). The increased expression of LOX-L3 was associated with worse survival of OSCC patients (P = 0.028). Conclusion: The results indicated the frequent increase in LOX-L3 mRNA expression in OSCC, and the value of LOX-L3 mRNA expression might have clinical uses in monitoring OSCC. [ABSTRACT FROM AUTHOR]

Abstract: Excessive exercise may induce osteoarthritis, which is a leading cause of lameness and decreased use in horses. The purpose of this study was to utilize a sheep model to determine the effects of circular and linear exercise on biochemical markers in serum and synovial fluid (SF). Twenty lambs (5 months) were assigned to three groups: circular exercise (C, 8.5 m diameter; n = 8), straight-line exercise (S, treadmill; n = 8) and nonexercised control (CON, n = 4). Lambs (C and S) were exercised up to 8 weeks at 1.3 m/s for up to 30 minutes, 6 days per week. Serum and SF from a metacarpophalangeal (MCP) joint was collected. Serum total protein (P = .66), Collagen Type II cleavage [3/4] fragments concentrations (P = .44) and lysyl oxidase (LOX) (P = .15) activity were not different among treatment groups throughout the study. There was a polynomial response to serum LOX over time (R 2 = 0.7464). There were no differences in SF total protein (P = .94, right MCP; P = .68, left MCP) or SF LOX specific activities (P = .9, right MCP; P = .93, left MCP) among groups throughout the study. However, SF LOX specific activity did increase over time (P = .001) for all treatment groups. These results indicated that the biochemical markers utilized in this study were unable to detect differences between groups with respect to exercise protocols. However, the LOX activity did increase over time possibly as a result of growth. [Copyright &y& Elsevier]

Aortic aneurysms are common among the elderly population. A large majority of aortic aneurysms are located at two distinct aneurysm-prone regions, the abdominal aorta and thoracic aorta involving the ascending aorta. In this study, we combined two factors that are associated with human aortic aneurysms, hypertension and degeneration of elastic lamina, to induce an aortic aneurysm in mice. Roles of hemodynamic conditions in the formation of aortic aneurysms were assessed using two different methods for inducing hypertension and antihypertensive agents. In 9-week-old C57BL/6J male mice, hypertension was induced by angiotensin II or deoxycorticosterone acetate-salt hypertension; degeneration of elastic lamina was induced by infusion of beta-aminopropionitrile, a lysyl oxidase inhibitor. Irrespective of the methods for inducing hypertension, mice developed thoracic and abdominal aortic aneurysms (38% to 50% and 30 to 49%, respectively). Aneurysms were found at the two aneurysm-prone regions with site-specific morphological and histological characteristics. Treatment with an antihypertensive agent, amlodipine, normalized blood pressure and dramatically reduced aneurysm formation in the mice that received angiotensin II and beta-aminopropionitrile. However, treatment with captopril, an angiotensin-converting enzyme inhibitor, did not affect blood pressure or the incidence of aortic aneurysms in the mice that received deoxycorticosterone acetate-salt and beta-aminopropionitrile. In summary, we have shown that a combination of hypertension and pharmacologically induced degeneration of elastic laminas can induce both thoracic and abdominal aortic aneurysms with site-specific characteristics. The aneurysm formation in this model depended on hypertension but not on direct effects of angiotensin II to the vascular wall. [ABSTRACT FROM AUTHOR]

Background/purpose: Lysyl oxidase (LOX) is involved in the initial steps of converting soluble monomers of collagen and elastin into insoluble fibers in the extracellular matrix. LOX was found to be upregulated in some fibrotic diseases. However, little is known about the correlation between LOX and cyclosporin A (CsA)-induced gingival overgrowth. The aim of this study was to compare LOX expression in normal healthy gingival tissues and CsA-induced gingival overgrowth specimens. Materials and methods: Fifteen CsA-induced gingival overgrowth specimens and five normal gingival tissues were examined by immunohistochemistry. Three oral submucous fibrosis specimens were used as positive controls. In addition, one section from each CsA-induced gingival overgrowth specimen was stained with hematoxylin and eosin to evaluate the magnitude of inflammation at the histologic level. Differences in LOX expression between tissues with low and high Levels of inflammation were subsequently analyzed using Fisher's exact test. Results: LOX staining in gingival tissue was stronger in the CsA-induced gingival overgrowth group than in the normal gingival group (P<0.05). LOX staining was detected in the epithelium, connective tissue, inflammatory infiltrates, and endothelium. The LOX signal was mainly expressed in inflammatory cells (100%), followed by endothetial cells (93.3%), fibroblasts (80%) and epithelial cells (60%). In addition, LOX expression was significantly higher in CsA-induced gingival overgrowth specimens with higher levels of inflammatory infiltrates (P=0.017). Conclusion: LOX expression was significantly upregulated in CsA-induced gingival overgrowth specimens. In addition, the expression of LOX increased with the grade of inflammation in CsA-induced gingival overgrowth. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate whether torasemide modifies collagen cross-linking in the failing human heart. We analyzed the degree of cross-linking and the expression of the enzyme lysyl oxidase, which regulates cross-linking, in the myocardium of patients with chronic heart failure at baseline and after 8 months of treatment with either torasemide or furosemide in addition to their standard heart failure therapy. Whereas lysyl oxidase protein expression was very scarce in normal hearts, it was highly expressed in failing hearts. Cross-linking was increased (P<0.001) in heart failure patients compared with normal hearts. These 2 parameters decreased (P=0.021 and P=0.034) in torasemide-treated patients and remained unchanged in furosemide-treated patients. In addition, more (P=0.009) patients showed normalization of left ventricular chamber stiffness in the torasemide subgroup than in the furosemide subgroup after treatment. Lysyl oxidase expression correlated with cross-linking (r=0.661; P<0.001), and cross-linking correlated with left ventricular chamber stiffness (r=0.452; P=0.002) in all patients. These findings show for the first time that lysyl oxidase overexpression is associated with enhanced collagen cross-linking in the failing human heart. In addition, we report that the ability of torasemide to correct both lysyl oxidase overexpression and enhanced collagen cross-linking results in normalization of left ventricular chamber stiffness in patients with heart failure. Lysyl oxidase may thus represent a target for reduction of stiff collagen and improvement of left ventricular mechanical properties in heart failure patients. [ABSTRACT FROM AUTHOR]

The article discusses a study which examines messenger RNA (mRNA) expression of lysyl oxidase (LOX) and matrix metalloproteinase-12 (MMP-12) in mouse skin. The study detected gender-based differences in LOX mRNA expression, with expression lower in females. Higher MMP-12 mRNA expression was found in the light period. The findings suggest that elastic fibers might be degraded in the light rather than the dark period.

Objective: To clarify the influence of occlusal hypofunction on the integrity of gingival tissue and gingival extracellular matrix biosynthesis. Materials and Methods: Thirteen-week-old male Wistar rats were divided into two groups. To eliminate occlusal forces, all the right maxillary molars were extracted in the hypofunctional group. The control group was anesthetized but not subjected to surgery. The rats were killed at 2 and 4 weeks after the procedure, and the lower right second molars were prepared for histological analysis. To investigate the effect of occlusal hypofunction on collagen biosynthesis, the expression of connective tissue growth factor (CTGF) and lysyl oxidase (LOX) was determined by immunohistochemistry as well as histological examination by hematoxylin and eosin staining. Results: Disorientation of the collagen fibers, proliferation of the connective tissue fibroblasts, and enlargement of epithelial intercellular gaps were observed in gingival tissue of rat molars with experimental occlusal hypofunction. Immunohistochemically, the expression of CTGF and LOX was increased significantly (P < .05) in the hypofunctional group. Conclusion: These results suggest that occlusal hypofunction can affect the structural integrity and the expression of CTGF and LOX in gingival tissue. [ABSTRACT FROM AUTHOR]

The role of lysyl oxidase in fibroblasts with drug-induced gingival overgrowth is not well understood. Cyclosponne-A (CsA) may have direct effects on lysyl oxidase produced by gingival fibroblasts which may increase collagen accumulation. Normal human gingival fibroblast cultures were obtained which were either treated with CsA or not; these cultures were respectively defined as the test and control groups. RNA isolation, Northern blot analyses, and lysyl oxidase activity assays were carried out. Some differences in lysyl oxidase mRNA levels between the 4- and 6-hour groups (p=O.03 by ANOVA) and in elastin mRNA levels between the 12- and 48-hour groups (p=O.O47 by ANOVA) were found to be statistically significant. No statistically significant differences were found in lysyl oxidase activities (t-test, p>O.O5) or in a-i-type I collagen, elastin, and type ifi collagen mRNA levels between other groups (ANOVA, p>O.O5). Trends towards increased lysyl oxidase enzyme activity and some upregulation of α-1-type I collagen and lysyl oxidase mRNA levels were observed. However, these effects were inconsistent and not statistically significant. Direct regulation by cyclosporine-A of lysyl oxidase, type I and type ifi collagen, and elastin mRNA levels in gingival fibroblast cultures is unlikely to account for the altered extracellular matrix production in CsA-induced gingival overgrowth. It is proposed that the relationship of CsA-induced gingival overgrowth with lysyl oxidase and collagen may be modified by some other factors, which have yet to be confirmed. [ABSTRACT FROM AUTHOR]

Background: Systemic therapy with cyclosporin A, phenytoin, and nifedipine modulates cytokine levels in human gingival tissues. Functional relationships between altered cytokine levels and gingival extracellular matrix production are partially characterized. The present study investigates in cultured human gingival fibroblasts the regulation of lysyl oxidase, α-1 type I collagen, and elastin by selected cytokines that are elevated in drug-induced gingival overgrowth tissues. Methods: Normal human gingival fibroblasts were cultured and then treated with selected cytokines: interleukin (IL)-1β, IL-6, platelet-derived growth factor (PDGF)-BB, and basic fibroblast growth factor (bFGF or FGF-2). Cells were harvested at intervals, and changes in lysyl oxidase enzyme activity, and in mRNA levels of lysyl oxidase, α-1 type 1 collagen, and elastin were determined. Results: bFGF reproducibly and significantly decreased human gingival fibroblast lysyl oxidasc and α-1 type 1 collagen mRNA levels in a dose- and time-dependent manner; 1 nM bFGF reduced lysyl oxidase and collagen mRNA levels to 53% and to less than 10% of control after 48 hours of treatment. Interestingly, bFGF downregulated lysyl oxidase enzyme activity by 10% to 20%. IL-1, IL-6. and PDGF-BB did not significantly regulate lysyl oxidase enzyme activity, or α-1 type I collagen, elastin, and lysyl oxidase mRNA levels under the conditions tested. Conclusions: Previous studies have shown that modulated levels of bFGF occur in gingiva as a result of certain pharmacology therapies. The present study suggests that, modulated levels of bFGF likely influence gingival connective tissue metabolism. [ABSTRACT FROM AUTHOR]

• This is the first report that determined lysyl oxidase activity in bovine heart of copper deficient animals. • The commercial kit used to measure lysyl oxidase activity for the first time in bovines, was successful. • Cardiac pathology in Cu-deficient cattle may be associated, at least in part, to a decrease in LOX enzymatic activity. Lysyl oxidase (LOX) is a metalloenzyme that requires Cu as a cofactor and it is responsible for the formation of collagen and elastin cross-linking. The objective of this work was to measure the LOX enzyme activity in the heart of bovines with Cu deficiency induced by high molybdenum and sulfur levels in the diet. Eighteen myocardial samples were obtained from Cu-deficient (n = 9) and control (n = 9) Holstein bovines during two similar assays. The samples were frozen in liquid nitrogen and stored at −70 °C to measure enzymatic activity. A commercial kit was used, following producer instructions. The results showed that LOX activity from the hearts of Cu-deficient bovines is 29 % lower than the ones of control bovines, being this difference statistically significant (p = 0.03). To our knowledge, this is the first report that determined LOX enzymatic activity in bovine heart of Cu-deficient animals. The microscopic alterations found in these animals in our previous work, could be explained by a diminished LOX activity. The results are in agreement with other authors, who found a relationship between LOX activity and dietary Cu intake. The information provided by this work could help to clarify the pathogenesis of cardiac lesions in cattle with dietary Cu deficiency. [ABSTRACT FROM AUTHOR]

Lysyl oxidase (LOX) is a cell-secreted amine oxidase that crosslinks collagen and elastin in extracellular microenvironment. LOX-traceable nanoparticles (LOX ab -NPs) consisting of LOX antibodies (LOX ab) and paclitaxel, can accumulate at high concentrations at radiation-treated target sites, as a tumor-targeting drug carrier for chemotherapy. Tumor-targeting and anticancer effects of PLGA based LOX ab -NPs in vitro and in vivo were evaluated at radiation-targeted site. In the in vivo A549 lung carcinoma xenograft model , we showed highly specific tumor targeting (above 7.0 times higher) of LOX ab -NPs on irradiated tumors. Notably, systemically administered NPs delayed tumor growth, reducing tumor volumes by more than 2 times compared with non-irradiated groups (222% vs. >500%) over 2 weeks. Radiotropic LOX ab -NPs can serve as chemotherapeutic vehicles for combined targeted chemo-radiotherapy in clinical oncology. LOX-traceable PLGA based nanoparticles consisting of LOX antibodies and paclitaxel (LOX-NPs(P)), which can accumulate at high concentrations at radiation-treated target sites, as a tumor-targeting drug carrier for chemotherapy. Biodistribution of NPs on extracted organs from sacrificed mice after 7 days. Photograph of organs (Heart, Liver, Spleen, lung, kidney, 10 Gy irradiated A549 tumor and non-irradiated tumor). Ex vivo images of organs administrated nanoparticles after 7 days. Photographs of extracted A549 tumor from animal euthanasia. IR (−) indicated non-irradiated (only drugs series administration) groups. Control group administrated only saline. Unlabelled Image [ABSTRACT FROM AUTHOR]

Hypoxia stimulates angiogenesis during development, as well as in the course of ischemic cardiovascular diseases and tumor growth. In a hypoxic environment, endothelial cells (EC) are key players of the angiogenic response. Their activation leads to remodeling of the extracellular matrix (ECM): a degradation step generates a provisional ECM supporting EC proliferation and migration; assembly of a new basal lamina leads to pericyte recruitment and neovessel maturation. In order to identify endothelial ECM proteins regulated by hypoxia, 2D gel electrophoresis was performed on ECM samples prepared from cultured EC of micro or large vessels (HDMEC or HUVEC respectively). Mass spectrometry identified lysyl oxidase-like protein 2 (LOXL2) as a highly hypoxia-induced protein. Lysyl oxidases are secreted enzymes involved in ECM maturation through covalent cross-linking of its major components, collagens and elastin. The induction of LOXL2 expression was detected both at the mRNA and protein levels. Using siRNA, we demonstrated that LOXL2 is responsible for 65 % of lysyl oxidase total activity in hypoxic EC. In addition, LOXL2 protein was colocalised with type IV collagen in endothelial ECM. We further investigated the expression of LOXL2 in vivo. The enzyme was expressed in rat EC from retina during postnatal vascular development, and from adult skeletal muscle. In a murine model of hindlimb ischemia, LOXL2 was upregulated at the protein and mRNA levels. In situ hybridization revealed its expression in both EC and macrophages. Involvement of LOXL2 at different steps of the angiogenic process was further studied in vitro. We demonstrated that LOXL2 increases EC migration on fibronectin, as well as migration and tube formation in fibrin 3D gels. In addition, LOXL2 knock-down inhibited type IV collagen deposition in the ECM, suggesting a major role for LOXL2 in the organisation of endothelial basal lamina. Finally, whereas the efficiency of endothelial ECM for recruiting VSMC was increased by hypoxia, this effect was reduced upon knocking down endothelial LOXL2 expression. Altogether, these data suggest that LOXL2 plays a major role in EC migration, vascular basal lamina deposition and neovessel stabilisation during hypoxia-induced angiogenesis. [Copyright &y& Elsevier]