Targeting lysyl oxidase like 2 attenuates OVA-induced airway remodeling partly via the AKT signaling pathway.

BACKGROUND: Airway epithelium is an important component of airway structure and the initiator of airway remodeling in asthma. The changes of extracellular matrix (ECM), such as collagen deposition and structural disturbance, are typical pathological features of airway remodeling. Thus, identifying key mediators that derived from airway epithelium and capable of modulating ECM may provide valuable insights for targeted therapy of asthma. METHODS: The datasets from Gene Expression Omnibus database were analyzed to screen differentially expressed genes in airway epithelium of asthma. We collected bronchoscopic biopsies and serum samples from asthmatic and healthy subjects to assess lysyl oxidase like 2 (LOXL2) expression. RNA sequencing and various experiments were performed to determine the influences of LOXL2 knockdown in ovalbumin (OVA)-induced mouse models. The roles and mechanisms of LOXL2 in bronchial epithelial cells were explored using LOXL2 small interfering RNA, overexpression plasmid and AKT inhibitor. RESULTS: Both bioinformatics analysis and further experiments revealed that LOXL2 is highly expressed in airway epithelium of asthmatics. In vivo, LOXL2 knockdown significantly inhibited OVA-induced ECM deposition and epithelial-mesenchymal transition (EMT) in mice. In vitro, the transfection experiments on 16HBE cells demonstrated that LOXL2 overexpression increases the expression of N-cadherin and fibronectin and reduces the expression of E-cadherin. Conversely, after silencing LOXL2, the expression of E-cadherin is up-regulated. In addition, the remodeling and EMT process that induced by transforming growth factor-ß1 could be enhanced and weakened after LOXL2 overexpression and silencing in 16HBE cells. Combining the RNA sequencing of mouse lung tissues and experiments in vitro, LOXL2 was involved in the regulation of AKT signaling pathway. Moreover, the treatment with AKT inhibitor in vitro partially alleviated the consequences associated with LOXL2 overexpression. CONCLUSIONS: Taken together, the results demonstrated that epithelial LOXL2 plays a role in asthmatic airway remodeling partly via the AKT signaling pathway and highlighted the potential of LOXL2 as a therapeutic target for airway remodeling in asthma.

Selective depletion of hepatic stellate cells-specific LOXL1 alleviates liver fibrosis.

The role of LOXL1 in fibrosis via mediating ECM crosslinking and stabilization is well established; however, the role of hepatic stellate cells (HSCs)-specific LOXL1 in the development of fibrosis remains unknown. We generated HSCs-specific Loxl1-depleted mice (Loxl1Gfap-cre mice) to investigate the HSCs-specific contribution of LOXL1 in the pathogenesis of fibrosis. Loxl1fl/fl mice were used as the control. Furthermore, we used RNA sequencing to explore the underlying changes in the transcriptome. Results of the sirius red staining, type I collagen immunolabeling, and hydroxyproline content analysis, coupled with the reduced expression of profibrogenic genes revealed that Loxl1Gfap-cre mice with CCl4 -induced fibrosis exhibited decreased hepatic fibrosis. In addition, Loxl1Gfap-cre mice exhibited reduced macrophage tissue infiltration by CD68-positive cells and decreased expression of inflammatory genes compared with the controls. RNA sequencing identified integrin α8 (ITGA8) as a key modulator of LOXL1-mediated liver fibrosis. Functional analyses showed that siRNA silencing of Itga8 in cultured fibroblasts led to a decline in the LOXL1 expression and inhibition of fibroblast activation. Mechanistic analyses indicated that LOXL1 activated the FAK/PI3K/AKT/HIF1a signaling pathway, and the addition of inhibitors of FAK or PI3K reversed these results via downregulation of LOXL1. Furthermore, HIF1a directly interacted with LOXL1 and upregulated its expression, indicating that LOXL1 can positively self-regulate by forming a positive feedback loop with the FAK/PI3K/AKT/HIF1a pathway. We demonstrated that HSCs-specific Loxl1 deficiency prevented fibrosis, inflammation and that ITGA8/FAK/PI3K/AKT/HIF1a was essential for the function and expression of LOXL1. Knowledge of this approach can provide novel mechanisms and targets to treat fibrosis in the future.

Deletion of the lysyl oxidase-like 1 gene induces impaired elastin fiber synthesis and inefficient urethral closure in rats.

We investigated the bladder and urethral function in a rat model lacking the protein lysyl oxidase-like 1 (Loxl1). Female nulliparous rats of Loxl1-/- or age-matched wild type (WT) rats had leak-point pressure testing, cystometry, histopathological analyses of lower urinary tract, and contractile response of isolated detrusor strips to carbachol and electric field stimulation. The Loxl1-/- rats showed increased looseness and redundancy of the skin, the decreased intercontraction interval and voided volume in cystometry, the lower leak-point pressure, thinner elastic fibers of the mesentery, bladder, urethra and vagina, and smaller contractile response of detrusor strips to carbachol when compared to the WT rats. Thus, the insufficient hydrostatic mechanism of urethra via submucosal impaired elastin synthesis might reduce the resting urethral closure pressure and the diminished cholinergic contractile response of detrusor smooth muscle might be involved in bladder activity in the Loxl1-/- rats.

Differential Lysyl oxidase like 1 expression in pseudoexfoliation glaucoma is orchestrated via DNA methylation.

Pseudoexfoliation syndrome (PXF) is the most common cause of secondary open angle glaucoma worldwide. Single nucleotide polymorphisms (SNPs) in the gene Lysyl oxidase like 1 (LOXL1) are strongly associated with the development of pseudoexfoliation glaucoma (PXFG). However, these SNPs are also present in 50-80% of the general population, suggestive of other factors being involved in the pathogenesis of PXFG. In this study, we aimed to investigate the influence of epigenetic regulation, specifically DNA methylation, on LOXL1 expression in PXFG using human tenons fibroblasts (HTFs), aqueous humour and serum samples from donors with and without PXFG. LOXL1 expression in HTFs was measured by qPCR and Western Blotting and LOXL1 concentration in aqueous humour was determined by ELISA. Global DNA methylation levels were quantified using an ELISA for 5-methylcytosine. MeDIP assays assessed the methylation status of the LOXL1 promoter region. Expression of methylation-associated enzymes (DNMT1, DNMT3a and MeCP2) were determined by qPCR and inhibited by 0.3 µM 5-azacytidine (5-aza). Results showed that LOXL1 expression was significantly decreased in PXFG HTFs compared with Control HTFs at gene (Fold change 0.37 ± 0.05, P < 0.01) level and showed a decrease, when measured at the protein level (Fold change 0.65 ± 0.42, P = 0.22), however this was not found to be significant. LOXL1 concentration was increased in the aqueous of PXFG patients compared with Controls (2.76 ± 0.78 vs. 1.79 ± 0.33 ng/ml, P < 0.01). Increased global methylation (56.07% ± 4.87% vs. 32.39% ± 4.29%, P < 0.01) was observed in PXFG HTFs compared with Control HTFs, as was expression of methylation-associated enzymes (DNMT1 1.58 ± 0.30, P < 0.05, DNMT3a 1.89 ± 0.24, P < 0.05, MeCP2 1.63 ± 0.30, P < 0.01). Methylation-associated enzymes were also increased when measured at protein level (DNMT1 5.70 ± 2.64, P = 0.04, DNMT3a 1.79 ± 1.55, P = 0.42, MeCP2 1.64 ± 1.33, P = 0.45). LOXL1 promoter methylation was increased in patients with PXFG compared to Control patients in both blood (3.98 ± 2.24, 2.10 ± 1.29, P < 0.05) and HTF cells (37.31 ± 22.0, 8.66 ± 10.40, P < 0.01). Treatment of PXFG HTFs with in 5-azacytidine increased LOXL1 expression when compared with untreated PXFG HTFs (Fold change 2.26 ± 0.67, P < 0.05). These data demonstrate that LOXL1 expression is altered in PXFG via DNA methylation and that reversal of these epigenetic changes may represent future potential therapeutic targets in the management of PXFG.

Elevated exosomal lysyl oxidase like 2 is a potential biomarker for head and neck squamous cell carcinoma.

OBJECTIVES: The secretory enzyme lysyl oxidase like 2 (LOXL2) is speculated to contribute to tumor progression through its functions in the remodeling of extracellular matrix and epithelial-mesenchymal transition. We previously identified elevated expression of LOXL2 in metastatic human head and neck squamous cell carcinoma (HNSCC) cells in a mouse lymph node metastases model. Here we performed a case series study examining LOXL2 expression levels in human serum from HNSCC patients to evaluate whether LOXL2 is worth evaluation in a large cohort study. METHODS: LOXL2 protein levels in three serum samples from HNSCC patients were assessed by immunoblotting and LOXL2 tissue expression was examined in one human tongue squamous cell carcinoma (SCC) tissue by immunohistochemistry as a representative of HNSCC tissue. Serum samples were further fractionated in exosomes and supernatants by ultracentrifugation, which were then subjected to immunoblot and in vitro LOX activity analyses. Exosomal LOXL2 levels of 36 serum samples from HNSCC patients and seven healthy volunteers were measured using polymer sedimentation exosome preparation followed by ELISA measurement and subjected to statistical analyses. RESULTS: Immunoblot analyses revealed that LOXL2 was present in serum exosomal fractions from three HNSCC patients, and we observed approximately threefold higher levels of LOXL2 in HNSCC patients compared with three healthy volunteers. Immunohistochemical LOXL2 staining was detected in HNSCC cells in addition to non-cancerous lipid tissues and some muscles in human tongue HNSCC tissue. Further measurements of exosomal LOXL2 by ELISA showed over ninefold higher mean LOXL2 levels in patients compared with controls. Statistical analysis revealed a correlation between elevated serum exosomal LOXL2 levels and low-grade, but not high-grade, HNSCC. CONCLUSIONS: Our case series study that elevated serum exosomal LOXL2 levels exhibited a correlation with low-grade HNSCCs. A follow-up large cohort clinical study will be required to determine the potential clinical utility of LOXL2 as a new biomarker and/or therapy target for HNSCCs. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:E327-E334, 2020.

Understanding Solar Skin Elastosis-Cause and Treatment.

Photoageing, also called actinic ageing, is the main cause of prematurely aged skin. Our expertise in elastic fibers has led us to discover a process triggered in response to ultraviolet (UV) light and which upsets the balance of elastin fibers: there is too much elastin and insufficient lysyl oxidase (LOXL1) enzyme to form functional elastic fibers. This imbalance then leads to an accumulation of nonfunctional elastin, which forms aggregates. In addition to this imbalance, UV rays also induce elafin synthesis by fibroblasts. Known to be a marker of elastotic aggregates, elafin crystallizes the elastin fibers and stimulates the formation of aggregates that cannot be naturally eliminated by the skin. We developed a Hamamelis virginiana leaf extract that was able to restore both the balance between elastin and LOXL1 and to decrease the elafin synthesis to fight and correct the damage. This specific Hamamelis virginiana extract increased LOXL1 expression by twofold and decreased elafin synthesis. As a consequence, elastic fibers became functional and aggregates of unfunctional fibers decreased. The specific Hamamelis extract activity was confirmed in vivo with decreasing wrinkles and improving skin firmness.

Inhibition of lysyl oxidase-like 1 (LOXL1) expression arrests liver fibrosis progression in cirrhosis by reducing elastin crosslinking.

Mature crosslinked-poly-elastin deposition has been found to be associated with liver fibrosis. However, the regulation of crosslinked/insoluble elastin in liver fibrosis remains largely unknown. Here, we investigated the contribution of lysyl oxidases (LOXs) family, mediated elastin crosslinking, to liver fibrogenesis. We established carbon tetrachloride (CCl4)-induced liver fibrotic and cirrhotic models and found that crosslinked/insoluble elastin levels spiked only in cirrhosis stage during disease progression, in comparison to collagen Ι levels which increased continuously though all stages. Among the LOXs family members, only LOX-like 1 (LOXL1) levels were coincident with the appearance of crosslinked/insoluble elastin. These coincidences included that LOXL1 expression increased (34 fold) in cirrhosis, localized with α-smooth muscle actin (SMA) and was absent in normal and fibrotic livers. In LX-2 cells, LOXL1 silencing arrested expression of α-SMA, elastin and collagen Ι. Our previously characterized adeno-associated vector (AAV) 2/8 shRNA was shown to effectively downregulate LOXL1 expression in CCl4 induced fibrosis mice models. These resulted in delicate and thinner septa and less crosslinked elastin, with a 58% loss of elastin area and 51% decrease of collagen area. Our findings strongly suggested that elastin crosslinking and LOXL1 were co-associated with liver cirrhosis, while selective inhibition of LOXL1 arrested disease progression by reducing crosslinking of elastin.

No Effects of Hyperosmolar Culture Medium on Tissue Regeneration by Human Degenerated Nucleus Pulposus Cells Despite Upregulation Extracellular Matrix Genes.

STUDY DESIGN: An in vitro study using human degenerated nucleus pulposus cells. OBJECTIVE: To determine the effect of osmolality and different osmolytes on the regeneration by human nucleus pulposus cells through gene expression and extracellular matrix production. SUMMARY OF BACKGROUND DATA: Intervertebral disc (IVD) degeneration is a major problem in developed countries. Regeneration of the IVD can prevent pain and costs due to diminished work absence and health care, and improve quality of life. The osmotic value of a disc decreases during degeneration due to loss of proteoglycans and might increase degeneration. It is known that gene expression of matrix genes of nucleus pulposus (NP) cells increases when cultured in hyperosmotic medium. Thus, increasing the osmolality of the disc might be beneficial for disc regeneration. METHODS: In the current study, isolated degenerated human NP cells were used in regeneration culture with medium of different osmolalities, adjusted with different osmolytes. NaCl, urea and sucrose. The cells were cultured for 28 days and expression of matrix genes and production of glycosaminoglycans and collagen II were measured. RESULTS: Gene expression for both collagen II and aggrecan increased with increasing osmolality using NaCl or sucrose, but not urea. Protein production however, was not affected by increasing osmolality and was decreased when using urea and sucrose. Expression of genes for Col1A1, MMP13, and MMP14 decreased with increasing osmolality, whereas expression of LOXL2 and LOXL3 increased. Transient expression of TonEBP was found 6 hours after the start of culture, but not at later time points. CONCLUSION: Although expression of matrix genes is upregulated, hyperosmolality does not enhance matrix production by nucleus pulposus cells. Raising osmolality can potentially increase matrix production, but in itself is not sufficient to accomplish regeneration in the current in vitro culture system. LEVEL OF EVIDENCE: N /A.

Posttranscriptional Regulation of LOXL1 Expression Via Alternative Splicing and Nonsense-Mediated mRNA Decay as an Adaptive Stress Response.

Purpose: Alternative mRNA splicing coupled to nonsense-mediated decay (NMD) is a common mRNA surveillance pathway also known to dynamically modulate gene expression in response to cellular stress. Here, we investigated the involvement of this pathway in the regulation of lysyl oxidase-like 1 (LOXL1) expression in response to pseudoexfoliation (PEX)-associated pathophysiologic factors. Methods: Transcript levels of LOXL1 isoforms were determined in ocular tissues obtained from donor eyes without and with PEX syndrome. Pseudoexfoliation-relevant cell types, including human Tenon's capsule fibroblasts (hTCF) and trabecular meshwork cells (hTMC), were exposed to puromycin, caffeine, TGF-ß1, homocysteine, IL-6, retinoic acid, UV-B radiation, oxidative stress, and mechanical stress for up to 48 hours. Western blot analysis was carried out using antibodies against LOXL1, (phosphorylated-) eukaryotic initiation factor 2-α (eIF2-α), and regulator of nonsense transcripts 2 (UPF2). RNA interference was used to knockdown UPF1-3 and Serine/threonine-protein kinase (SMG1). Results: Constitutive expression of wild-type LOXL1 and alternatively spliced LOXL1-a transcripts was detected in all ocular tissues showing highest levels in trabecular meshwork and differential expression between PEX and control specimens. LOXL1-a transcripts were upregulated in hTCF and hTMC by NMD inhibitors puromycin and caffeine (≥6-fold; P < 0.01) or after knockdown of NMD core factors (≥2-fold; P < 0.05), whereas mRNA and protein levels of LOXL1 were reduced (≤0.8 fold; P < 0.05). Exposure of cells to various PEX-associated (stress) factors, including TGF-ß1, UV-B light, oxidative stress, mechanical stress, and retinoic acid enhanced LOXL1-a transcript levels (≥1.5-fold; P < 0.05), while partially downregulating LOXL1 levels (≤0.7-fold; P < 0.05). Stress-induced inhibition of NMD was dependent on phosphorylation of eIF2α. Conclusions: These findings provide evidence for a functional role of alternative splicing coupled to NMD in the posttranscriptional regulation of LOXL1 gene expression and suggest this mechanism to represent a dynamic mode of adapting LOXL1 expression to PEX-associated environmental and nutritional cues.

Insulin resistance promotes Lysyl Oxidase Like 2 induction and fibrosis accumulation in non-alcoholic fatty liver disease.

In patients with non-alcoholic fatty liver disease (NAFLD), insulin resistance (IR) associates with fibrosis progression independently of the hepatic inflammation, but the mechanisms are still unclear. We modeled the independent contribution of inflammation (non-alcoholic steatohepatitis: NASH) by exploiting the methionine-choline deficient (MCD) diet, and that of IR by insulin receptor (InsR) haploinsufficiency (InsR+/-) in the pathogenesis of liver fibrosis in C57BL/6 mice. We confirmed the study findings in 96 patients with NAFLD. InsR+/- enhanced hepatic fat content and impaired hepatic insulin signaling leading to Forkhead box protein O1 (FoxO1) accumulation in MCD-fed mice. Remarkably, despite reduced inflammation and hampered transdifferentiation of hepatic stellate cells (HSCs), InsR+/- promoted hepatic fibrosis accumulation, which correlated with the induction of the Lysyl Oxidase Like 2 (Loxl2), involved in matrix stabilization. Loxl2 up-regulation was not a cell autonomous property of insulin resistant HSCs, but was dependent on microparticles (MPs) released specifically by insulin resistant hepatocytes (HEPs) exposed to fatty acids. The mechanism entailed FoxO1 up-regulation, as FoxO1 silencing normalized Loxl2 expression reversing fibrosis in InsR+/- MCD-fed mice. Loxl2 up-regulation was similarly detected during IR induced by obesity, but not by lipogenic stimuli (fructose feeding). Most importantly, LOXL2 up-regulation was observed in NAFLD patients with type 2 diabetes (T2D) and LOXL2 hepatic and circulating levels correlated with histological fibrosis progression. IR favors fibrosis deposition independently of the classic 'inflammation - HSC transdifferentiation' pathway. The mechanism entails a cross-talk between enhanced lipotoxicity in insulin resistant HEPs and Loxl2 production by HSCs, which was confirmed in patients with diabetes, thereby facilitating extracellular matrix (ECM) stabilization.

Immunohistochemical Profiles of LOXL-1, FBN1, TGF-ß1, and COX-2 in Pseudoexfoliation Syndrome.

PURPOSES: To (i) determine expression patterns of lysyl oxidase-like 1 (LOXL1), fibrillin-1 (FBN1), transforming growth factor beta-1 (TGF-ß1), and cyclooxygenase-2 (COX-2) in lens epithelium and anterior lens capsule in pseudoexfoliation (PEX) syndrome and (ii) delineate the roles of these proteins in the etiopathogenesis of PEX. MATERIALS AND METHODS: Study participants, all of whom had undergone cataract surgery, comprised 47 patients with and 27 patients without (controls) PEX syndrome. Immunohistochemistry on paraffin sections of lens capsule and lens epithelium was performed. RESULTS: Immunoexpression of LOXL1 and FBN1 on the outer surface of the lens capsule was significantly higher (p < 0.001), and nuclear immunopositivity for LOXL1 was more frequently observed (p = 0.017), in PEX patients compared with control patients. Cytoplasmic expression of LOXL1 and COX-2 was significantly lower (p = 0.015 and p = 0.042, respectively) in PEX patients compared with controls. TGF-ß1 exhibited diffuse immunostaining detected in all cell layers in PEX patients (p <0.001). Significant direct correlations of cytoplasmic LOXL1 with FBN1 and TGF-ß1, and of COX-2 with FBN1, TGF-ß1, and LOXL-1, were observed only in PEX patients. CONCLUSIONS: Results of our study provide valuable information vis-à-vis expression and localization of TGF-ß1, LOXL1, and FBN1, as well as their associations in the lens epithelium and lens capsule. These data not only advance our knowledge of the etiopathogenesis of PEX syndrome, but also include novel findings, for example, immunostaining patterns of TGF-ß1 in PEX syndrome. We suggest that COX-2 plays a role in the pathobiology of PEX syndrome and should be the subject of future investigations.

High-level expression of l-glutamate oxidase in Pichia pastoris using multi-copy expression strains and high cell density cultivation.

l-glutamate oxidase (GLOD), encoded by the gox gene, catalyses the transformation of l-glutamic acid into α-ketoglutaric acid (α-KG). In the present study, Pichia pastoris was used for heterologous production of GLOD following optimization of the gox coding sequence for expression in the yeast host. A series of constructs based on the pHBM905BDM plasmid were engineered and transformed into P. pastoris to increase the gox copy number. The results indicated that GLOD protein levels and enzyme activity increased with increasing gox copy number. Strain PGLOD4, which contained four copies of the target gene, was chosen for subsequent fermentation experiments, and a fermentation strategy involving two exponential feeding phases was developed. During the preinduction phase, glycerol was fed exponentially at µG = 0.15/h. When the cell density reached 300 g/l, methanol was fed exponentially at µM = 0.03/h to induce GLOD production. After 84 h of cultivation, the final cell density and total enzyme activity reached 420 g/L and 247.8 U/mL, respectively. The recombinant enzyme displayed an optimum temperature of 40 °C, which was higher than recombinant enzyme expressed in E. coli. This is important because increasing the temperature could accelerate enzymatic transformation of l-glutamic acid to α-KG. Experiments also demonstrated superior thermo-stability for the enzyme produced in yeast, which further enhances its potential for industrial applications.

Inhibition of Tobacco Ringspot Virus by the Culture Fluid of L-Lysine-α-Oxidase Producing Strain.

A producing strain of an anti-tumor and antiviral enzyme L-lysine-α-oxidase from Trichoderma was cultured using a technological device of G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences (Pushchino). L-lysine-α-oxidase activity in the obtained metabolite concentrate was 5.4 U/ml. We studied the effects of the concentrate of active L-lysine-α-oxidase producer on the highly infectious Tobacco ringspot virus and revealed anti-viral activity of it when enzyme concentration was at least 1.0 U/ml.

All-trans retinoic acid promotes wound healing of primary amniocytes through the induction of LOXL4, a member of the lysyl oxidase family.

Thirty percent of preterm births directly result from preterm premature rupture of fetal membranes (PPROM). Clinical management currently proposes using a collagen plug to mechanically stop loss of amniotic fluid. Vitamin A and its active metabolite (retinoic acid) have well-known pro-healing properties and could thus make good candidates as a proposable adjuvant to this mechanical approach. Here we investigate the molecular mechanisms involved in the pro-healing properties of all-trans retinoic acid (atRA) in fetal membranes via an approach using an in vitro primary amniocyte wound model and transcriptomics. The results demonstrate that atRA promotes migration in primary amniocytes, improving wound healing in vitro by up to 90%. This effect is mediated by the induction of LOXL4, which plays a crucial role in the dynamics of the extracellular matrix by regulating collagen reticulation. This new insight into how atRA exerts its pro-healing properties prompts us to propose using atRA as a candidate strategy to help prevent future PPROM.

Loss of lysyl oxidase-like 3 causes cleft palate and spinal deformity in mice.

In mammals, embryonic development are highly regulated morphogenetic processes that are tightly controlled by genetic elements. Failure of any one of these processes can result in embryonic malformation. The lysyl oxidase (LOX) family genes are closely related to human diseases. In this study, we investigated the essential role of lysyl oxidase-like 3 (LOXL3), a member of the LOX family, in embryonic development. Mice lacking LOXL3 exhibited perinatal lethality, and the deletion of the Loxl3 gene led to impaired development of the palate shelves, abnormalities in the cartilage primordia of the thoracic vertebrae and mild alveolar shrinkage. We found that the obvious decrease of collagen cross-links in palate and spine that was induced by the lack of LOXL3 resulted in cleft palate and spinal deformity. Thus, we provide critical in vivo evidence that LOXL3 is indispensable for mouse palatogenesis and vertebral column development. The Loxl3 gene may be a candidate disease gene resulting in cleft palate and spinal deformity.

The Role of LOX and LOXL2 in the Pathogenesis of an Experimental Model of Choroidal Neovascularization.

PURPOSE: We investigated whether lysyl oxidase (LOX) and lysyl oxidase-like 2 (LOXL2) play a role in an experimental model of choroidal neovascularization (CNV). The therapeutic potential of antibodies against LOX (M64) and LOXL2 (AB0023) was evaluated in a murine laser-induced CNV model. METHODS: Expression of LOX and LOXL2 in the posterior eye cups (including retina, retinal pigment epithelium, choroid, and sclera) was studied by qRT-PCR and immunohistochemistry. In the murine model of CNV, both antibodies were administered intraperitoneally every other day until the day killed. On different time points after laser, treatment outcome was studied by immunohistochemical analysis of inflammation, angiogenesis and fibrosis, and by transcript analysis of different cytokines. RESULTS: Levels of LOX and LOXL2 in the posterior eye cups were increased after CNV-induction at different time points after laser. At day 35, their protein expression patterns appeared to correlate with retinal glial cells and endothelial cells, respectively. Both antibodies significantly inhibited fibrosis, whereas AB0023 also significantly reduced angiogenesis and inflammation. Transcript levels of α-1 type I collagen (COL1A1) in the posterior eye cups were significantly decreased in lasered mice treated with either M64 or AB0023. Vascular endothelial growth factor expression was also reduced only after AB0023 treatment, whereas activated fibroblast marker α-smooth muscle actin (αSMA) levels were not significantly changed. CONCLUSIONS: This study suggests that LOX and LOXL2 may play an important role in the pathogenesis of AMD. Targeting LOXL2 could have a broader efficacy than targeting LOX, by reducing angiogenesis and inflammation, as well as fibrosis.

Characterization of recombinant biosynthetic precursors of the cysteine tryptophylquinone cofactors of l-lysine-epsilon-oxidase and glycine oxidase from Marinomonas mediterranea.

The lysine-ε-oxidase, LodA, and glycine oxidase, GoxA, from Marinomonas mediteranea each possesses a cysteine tryptophylquinone (CTQ) cofactor. This cofactor is derived from posttranslational modifications which are covalent crosslinking of tryptophan and cysteine residues and incorporation of two oxygen atoms into the indole ring of Trp. In this manuscript, it is shown that the recombinant synthesis of LodA and GoxA containing a fully synthesized CTQ cofactor requires coexpression of a partner flavoprotein, LodB for LodA and GoxB for GoxA, which are not interchangeable. An inactive precursor of LodA or GoxA which contained a monohydroxylated Trp residue and no crosslink to the Cys was isolated from the soluble fraction when they were expressed alone. The structure of LodA revealed an Asp residue close to the cofactor which is conserved in quinohemoprotein amine dehydrogenase (QHNDH), containing CTQ, and methylamine dehydrogenase (MADH) containing tryptophan tryptophylquinone (TTQ) as cofactor. To study the role of this residue in the synthesis of the LodA precursor, Asp-512 was mutated to Ala. When the mutant protein was coexpressed with LodB an inactive protein was isolated which was soluble and contained no modifications at all, suggesting a role for this Asp in the initial LodB-independent hydroxylation of Trp. A similar role had been proposed for this conserved Asp residue in MADH. It is noteworthy that the formation of TTQ in MADH from the precursor also requires an accessory enzyme for its biosynthesis but it is a diheme enzyme MauG and not a flavoprotein. The results presented reveal novel mechanisms of post-translational modification involved in the generation of protein-derived cofactors. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Elastin development-associated extracellular matrix constituents of subepithelial connective tissue in human pterygium.

PURPOSE: We evaluated the expression of several extracellular matrix constituents implicated in the synthesis and reticulation of elastin in human pterygium, according to age and sex of the patients. METHODS: Pterygia and normal conjunctiva samples were divided into groups according to age (<50/≥50 years) and sex. Tissue was subjected to immunohistochemical staining with anti-lysyl oxidase (LOX), lyxyl oxidase-like 1 (LOXL-1), fibulin (FBLN)-5 and FBLN4, and fibrillin-1 (FBN1) antibodies. Specific primers for the same constituents were used in a quantitative real-time PCR (qRT-PCR) analysis to determine gene expression. RESULTS: The LOXL-1 (P = 0.0002), FBLN5 (P = 0.0035), and FBN1 (P < 0.0001) mRNAs were significantly higher in pterygium than conjunctiva. No differences were found for LOX and FBLN4 mRNA. The expression of LOXL-1 was not affected by age or sex; however, pterygium from men and patients over 50 years old exhibited significantly higher FBLN5/FBN1 expression than did controls. The LOX gene expression was higher in the pathologic samples from the over 50-year-olds compared to the conjunctiva (P = 0.0396) and in men's versus women's pterygium (P = 0.0173). In general, the levels of LOX, LOXL-1, and FBLN5 decreased with age in healthy samples, while the pathology seemed to have increased expression of the three proteins, and even more so in older patients. The FBLN4 and FBN1 immunostaining was slight in all samples, displaying no differences between groups. CONCLUSIONS: Several extracellular matrix constituents, LOXs, FBN1, and FBLN5, implicated in the development of elastin, are overexpressed in the subepithelial connective tissue extracellular matrix of human pterygium, supporting our hypothesis that elastic synthesis and reticulation is dysregulated in this type of pathology.

Lysyl oxidase-like 2 (LOXL2) from stromal fibroblasts stimulates the progression of gastric cancer.

The aim of this study was to clarify the role of fibroblast-derived Lysyl oxidase-like 2 (LOXL2) in the development of gastric cancer. The correlation between the clinicopathological features of 548 primary gastric carcinomas and LOXL2 expression in stromal cells was examined by immunohistochemistry. Two gastric cancer cell lines, OCUM-12 and NUGC-3, and cancer-associated fibroblasts (CAFs) were used in this in vitro study. The effect of fibroblast-derived LOXL2 on the motility of gastric cancer cells was analyzed by using a wound-healing assay, a double-chamber invasion assay, and western blot. LOXL2 expression in stromal cells was significantly associated with tumor invasion depth, lymph node metastasis, lymphatic invasion, venous invasion, and peritoneal dissemination. Multivariable logistic regression analysis revealed that LOXL2 expression in stromal cells could be an independent predictive parameter for the overall survival of patients. CAFs significantly stimulated the migration and invasion of OCUM-12 and NUGC-3 cells. This motility-stimulating ability of CAFs was inhibited by LOXL2 siRNA. Western blot analysis indicated that phosphorylation of focal adhesion kinase (FAK) in cancer cells was increased by the conditioned medium from CAFs, and was decreased by the conditioned medium from LOXL2 siRNA-treated CAFs. LOXL2 expression in stromal cells may be a useful prognostic factor for patients with gastric cancer. Fibroblast-derived LOXL2 may stimulate the motility of gastric cancer cells.