LOX Expression Research Articles

Abstract 1493: Lysyl oxidase expression is associated with early recurrence and poor survival in hepatocellular carcinoma

Abstract Background: Hepatectomy is one of the an established one of a curative treatment for hepatocellular carcinoma (HCC). However, HCC has is a disease that high recurrence rates even after curative hepatectomy. We searched the gene for the prediction of the HCC recurrence from intrahepatic metastasis. Methods: For the gene selection, we used public data base (GSE10141) and searched genes with hHigh early recurrence rate and higher expression genes in tumor area compared with background liver. We detected Lysyl oxidase. As a validation cohort, From 2004 to 2012, 149 patients underwent hepatectomy at Kumamoto University Hospital from 2004 to 2012 were enrolled. The expression of target gene was evaluated by real time PCR. Upper 25% patients were considered as the high expression group, and the other 75% patients were considered as the low expression group. The immunehistochemical (IHC) staining was evaluated with the intensity and area of immunoreactivity of LOX and were scored each factors from 0 to 3. Low expression of LOX group was for scores from 0 to 5. The ones with scores from 6 to 9 were defined as high expression of LOX. Result: As a first step toward discovering the targets genes, we investigated using GSE 10141, which is a microarray database of cancerous part and background liver tissue of hepatocellular carcinoma cases to select genes useful as drug therapy targets. We extracted LOX as a genes with high hazard ratio (HR > 3) forof early recurrence to select genes useful as prevention of early recurrence. In real time PCR, LOX high expression group had a significantly high recurrence rate (2 years recurrence rate was 64.3% VS 42.7%, p = 0.024) and poor survival rate (3 years rate was 62.7% VS 82.0%, p = 0.0441) than LOX low expression group. Moreover, in the comparisons of clinicopathological factors between LOX high and low expression, there was significantly more Vp positive patients in the LOX high expression group (p = 0.004). Multivariate Analysis demonstrated that Tumor size larger than 50 mm (HR, 2.20; p = 0.002), LOX high expression (HR, 1.73; p = 0.04) were independent risk factors for early recurrence. In immunohistochemistry, LOX high expression group had a significantly high recurrence rate (2 years recurrence rate was 58.5% VS 42.3%, p = 0.004) and poor survival rate (3 years rate was 65.1% VS 88.8%, p = 0.004) than LOX low expression. In addition the comparisons of clinicopathological factors between LOX high and low expression, there was significantly more Ig positive patients in the LOX high expression group (p = 0.04). Multivariate Analysis demonstrated that PIVKA-II score higher than 100 (HR, 1.84; p = 0.03), multiple tumors tumor more than 2 pieces (HR, 2.11; p = 0.009), LOX high expression (HR, 1.90; p = 0.014) were independent risk factors for early recurrence. Conclusion: LOX high expression is associated with early recurrence and poor survival in HCC. Citation Format: Naoki Umezaki, Shigeki Nakagawa, Takanobu Yamao, Masayo Tsukamoto, Kota Arima, Tatsunori Miyata, Hirohisa Okabe, Katsunori Imai, Yo-ichi Yamashita, Hidetoshi Nitta, Akira Chikamoto, Hideo Baba. Lysyl oxidase expression is associated with early recurrence and poor survival in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1493.

PO-515 Transcriptome analysis identified alcam expression as potential biomarker to LSCC patient’s outcome

IntroductionLaryngeal squamous cell carcinoma (LSCC) is one of the most incidence tumours in the world, especially in developing countries, such as Brazil. The main risk factors for LSCC are tobacco and alcohol consumption and it usually occurs in patients older than 60 years. Similarly to other head and neck tumours, LSCC is a major health problem because of poor prognosis and slight improvement in the five-year survival during the past four decades. Therefore, our objective was to better understand the LSCC transcriptome, identifying possible prognosis biomarkers and novel therapy targets.Material and methodsWas carried out a gene expression profile analysis using the Human Exon 1.0 ST microarray chip. To this end, the expression profile of 14 tumour tissues were compared with 12­matched non­malignant mucosa.Results and discussionTranscriptome analysis pointed out 817 differentially expressed genes (DEG), 315 of which with overexpression in tumour and 502 underexpressed when compared with matched non­malignant mucosa. Performing a survival analysis across all overexpressed DEG, 24 genes were related to patients survival (ACOX1, ACVR1, ADH7, AGFG2, ALCAM, BTBD11, C12orf75, CDK14, CYP2C19, GBP6, GLTO, GNG4, LOX, LYPD6B, ME1, NPEPPS, ODC1, PMM1, PTGR1, SERPINA3, ST3GAL4, TDP52L1, ZDHHC13 and ZNF750). The prognostic values of these 24 genes were, then, validated in an independent LSCC data set of The Cancer Genome Atlas (TCGA) and the expression data, patient’s clinic and pathological features were analysed in Log-rank Test. Perineural invasion, commitment of surgical margins and expression of LOX, ALCAM, BTBD11 and LYPD68 showed p<0.05 and were selected to multivariate analysis applying Cox regression model. After age and tumour stage-adjustment, perineural invasion, commitment of surgical margins and ALCAM expression were classified as independent prognostic factors to LSCC patients. LSCC-patients displaying ALCAM high expression presented 4.5-fold increased death risk. ALCAM gene encodes the activated leukocyte cell adhesion molecule protein, also known as CD166, which is a member of a subfamily of immunoglobulin receptors. This protein binds to T-cell differentiation antigen CD6, and is implicated in the processes of cell adhesion and migration.ConclusionALCAM expression might be an independent prognosis biomarker to LSCC patients. Furthermore, in vitro, in vivo and pre-clinical analyses must be performed to reinforce this hypothesis.

Abstract 5166: Lysyl oxidase regulates cell surface EGFR, and directionality of cancer cell invasion

Abstract Lysyl oxidase (LOX) remodels the tumour microenvironment by cross-linking the extracellular matrix. LOX overexpression is associated with poor cancer outcomes. We find that LOX regulates the epidermal growth factor receptor (EGFR) to drive tumour progression. LOX regulates EGFR by suppressing TGFβ1 signalling through the secreted protease HTRA1. This increases the expression of Matrilin2 (MATN2), an EGF-like domain containing protein that traps EGFR at the cell surface to facilitate its activation by EGF. A novel pharmacological inhibitor of LOX, CCT365623, disrupts EGFR cell surface retention in vitro and delays the growth of primary tumour cells in vivo. In addition to its role in controlling primary tumour growth, LOX expression is also implicated in the processes of cancer cell invasion, and thereby the process of metastasis. Notably, we find that when LOX is depleted or inhibited, cancer cells are no longer able to sence a chemo-attractive gradient, and invade in a directional manner. Importantly, LOX produced by both cancer cells and fibroblasts can mediate this behaviour. Together, we show that LOX regulates EGFR cell surface retention to drive tumour progression. We also find that LOX does not regulate cancer cell invasion per se, but rather that it regulates the directionality of cancer cell migration, and both the cancer and stromal cells appear to contribute to this behaviour. Citation Format: Haoran Tang, Caroline Springer, Richard Marais. Lysyl oxidase regulates cell surface EGFR, and directionality of cancer cell invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5166.

Abstract 82: Epigenetic reader ZMYND8 bridges BRD4 and hypoxia-inducible factors to mediate breast cancer progression and metastasis

Abstract Metastatic breast cancer has high rates of relapse and mortality. The absence of definitive prognostic biomarker and effective therapeutic target is the major obstacle to end this aggressive disease. Epigenetic dysregulation plays a crucial role in breast cancer metastasis. However, the mechanism by which epigenetic dysregulation stimulates the hypoxia-mediated breast cancer progression and metastasis remains unknown. Based on luciferase reporter assays and analysis of expression changes of 720 epigenetic genes in three microarray datasets, we identified epigenetic reader ZMYND8 as a novel hypoxia-inducible factor (HIF) target gene. Human breast cancer tissue microarray data indicated that ZMYND8 is highly expressed in invasive breast tumors and this overexpression is significantly correlated with poor clinical outcomes in patients with breast cancer. ZMYND8 depletion dramatically attenuates the tumorigenic potential of breast cancer cells in colony formation, migration and invasion in vitro, and suppresses breast tumor growth and metastasis in mice. To elucidate the underlying mechanism, we performed co-immunoprecipitation, chromatin immunoprecipitation and quantitative PCR assays and found that ZMYND8 interacts with HIF complex and acetyl lysine 16 of histone H3 at the hypoxia inducible elements (HREs) to provoke the expression of HIF target genes LOX, AGR2, AQP1 and VEGFA. ZMYND8 controls breast cancer growth and metastasis through HIF in mice. We further found that p300 binds and acetylates ZMYND8 in breast cancer cells. Acetylated ZMYND8 interacts with and recruits BRD4 to the HREs to stimulate RNA polymerase II phosphorylation, thereby promoting transcriptional elongation of HIF target genes. ZMYND8 acetylation is necessary and sufficient for breast tumor growth and metastasis in vitro and in mice. Together, p300-ZMYND8-BRD4-HIF axis is critical for breast cancer progression and metastasis. In summary, ZMYND8 represents a positive feedback mechanism that amplifies HIF-mediated breast cancer progression and metastasis, and provides a potential epigenetic target for the prognosis and treatment of breast cancer. Citation Format: Yan Chen, Bo Zhang, Lei Bao, Lai Jin, Mingming Yang, Yan Peng, Jennifer Wang, Chenliang Wang, Xuan Zou, Yingfei Wang, Weibo Luo. Epigenetic reader ZMYND8 bridges BRD4 and hypoxia-inducible factors to mediate breast cancer progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 82.

S-Enantiomer of 19-Hydroxyeicosatetraenoic Acid Preferentially Protects Against Angiotensin II-Induced Cardiac Hypertrophy.

We had recently demonstrated that the racemic mixture of 19-hydroxyeicosatetraenoic acid (19-HETE) protects against angiotensin II (Ang II)-induced cardiac hypertrophy. Therefore, the purpose of this study was to investigate whether the R- or S-enantiomer of 19-HETE confers cardioprotection against Ang II-induced cellular hypertrophy in RL-14 and H9c2 cells. Both cell lines were treated with vehicle or 10 μM Ang II in the absence and presence of 20 μM 19(R)-HETE or 19(S)-HETE for 24 hours. Thereafter, the level of midchain HETEs was determined using liquid chromatography-mass spectrometry. Gene- and protein-expression levels were measured using real-time polymerase chain reaction and Western blot analysis, respectively. The results showed that both 19(R)-HETE and 19(S)-HETE significantly decreased the metabolite formation rate of midchain HETEs, namely 8-, 9-, 12-, and 15-HETE, compared with control group, whereas the level of 5-HETE was selectively decreased by S-enantiomer. Moreover, both 19(R)-HETE and 19(S)-HETE significantly inhibited the catalytic activity of CYP1B1 and decreased the protein expression of 5- and 12-lipoxygenase (LOX) as well as cyclo-oxygenase-2 (COX-2). Notably, the decrease in 15-LOX protein expression was only mediated by 19(S)-HETE. Interestingly, both enantiomers protected against Ang II-induced cellular hypertrophy, as evidenced by a significant decrease in mRNA expression of β/α-myosin heavy chain ratio, atrial natriuretic peptide, and interleukins 6 and 8. Our data demonstrated that S-enantiomer of 19-HETE preferentially protected against Ang II-induced cellular hypertrophy by decreasing the level of midchain HETEs, inhibiting catalytic activity of CYP1B1, decreasing protein expression of LOX and COX-2 enzymes, and decreasing mRNA expression of IL-6 and IL-8.

Lysyl oxidase: A colorectal cancer biomarker of lung and hepatic metastasis.

BackgroundColorectal cancer (CRC) is a common and lethal disease in which distant metastasis remains the primary cause of death. Paradoxical roles of LOX have been reported in CRC, and the intracellular function of LOX has also recently been determined. Correlations of LOX expression and its intracellular localization with clinicopathological features in CRC patients remain largely unknown. The aim of the present study was to explore the potential roles of LOX in CRC.MethodsLOX messenger RNA expression was assayed by quantitative PCR in eight paired normal mucosa and tumor tissues. Immunohistochemistry was conducted using tissue arrays to investigate LOX expression in 201 CRC patients. Regulation of LOX by YAP and TEAD4 was explored by YAP or TEAD4 short hairpin RNA interference in a LoVo cell line.ResultsLOX messenger RNA expression was elevated in some CRC specimens, and LOX nuclear localization was detected in CRC tumor tissues. LOX nuclear localization was found to correlate with lung/hepatic metastasis, elevated serum carcinoembryonic antigen concentration, and mucinous tumor type (P < 0.05). Nuclear LOX expression was found to be associated with poor overall and disease‐free survival (P < 0.05), and postoperative lung/hepatic metastasis (P < 0.05). Knockdown of YAP or TEAD4 induced downregulation of LOX expression.ConclusionsLOX nuclear localization was significantly associated with poor survival in patients with CRC. Nuclear LOX expression was correlated with synchronous or postoperative lung/hepatic metastasis. LOX may prove to be a potential target gene of YAP and TEAD4.

L-Cysteine hydrochloride delays senescence of harvested longan fruit in relation to modification of redox status

Longan fruit has a limited postharvest life due to aril breakdown, a typical senescence characteristics. In this study, effects of l-cysteine hydrochloride (LCH) on senescence of harvested longan fruit associated with modification of redox status were investigated. Application of LCH delayed aril breakdown and maintained fruit quality, accompanied by reduced H2O2 accumulation and alleviated protein oxidation. Furthermore, expression of several genes related to elimination of H2O2 and peroxide, APX and GST, regeneration of glutathione and ascorbic acid, GR and DHAR, were up-regulated by LCH. Moreover, LCH treatment well maintained the expression level of three oxidized-protein repair-related genes, Msr, MsrA5 and MsrB2. Additionally. LCH treatment inhibited expression of EGase, PLD and LOX. EGase was associated with degradation of cell wall, while PLD and LOX were associated with degradation of cell membrane. Overall, these findings suggested that LCH treatment enhanced anti-oxidant and oxidized-protein repair capacities, and maintained higher reducing state, which contributed to reducing oxidative damage and, thereby, delayed senescence of longan fruit.

Overexpression of Lox in triple-negative breast cancer.

Triple negative breast cancer (TNBC) accounts for approximately 15% of breast cancers. It is associated with a poor prognosis and typically earlier onset of metastasis in comparison with other breast cancer subtypes. Since TNBC lacks the expression of estrogen and progesterone receptors and Her2 status is also negative, there is currently no target that can be used for systemic therapy. Epithelial-mesenchymal transition (EMT) plays an important role in tumor progression and metastasis. In this study, we examined a subset of EMT markers consisting of Snail, Twist-1 and Lox in TNBC and non-TNBC breast cancer subtypes and analyzed their expression pattern in regard to subtype, clinico-pathological parameters and prognosis. We analyzed 659 breast cancer samples from two tissue microarrays. Breast cancer samples were categorized into two groups according to hormone receptor expression and Her2 status (n = 146 were triple negative, n = 513 were non triple-negative). Immunohistochemical expression of Snail, Twist-1 and Lox was semi-quantitatively analyzed using a three-tiered (weak-moderate-strong) scoring system. Results were statistically analyzed and correlated to clinico-pathological parameters and overall survival. Strong overexpression of Lox was significantly higher in triple negative breast cancers when compared to non triple-negative breast cancers (p < 0.001). No difference was seen between the groups regarding Snail and Twist expression (p > 0.05). In addition, Lox expression was significantly stronger in poorly differentiated (G3) breast cancers (p < 0.001 for Lox). The EMT marker Lox has a differential expression pattern in breast cancer, being significantly overexpressed in triple negative breast cancers. We could not link this expression to prognosis, however, this marker might be explored in future studies as possible target for systemic therapy of TNBC.

Higher Gene Expression of Healing Factors in Anterior Cruciate Ligament Remnant in Acute Anterior Cruciate Ligament Tear

Background: Anterior cruciate ligament (ACL) reconstruction with remnant preservation has been described and related to potential advantages. Literature is lacking regarding gene expression of potential factors related to ligament healing in the ACL remnant and its relation to time from injury. Hypothesis: The mRNA expression of ligament healing factors in the ACL remnant would be higher in acute tears (<3 months from injury) than in intermediate (3-12 months) and chronic (>12 months) injuries. Study Design: Controlled laboratory study. Methods: Gene expression of 21 genes related to ligament healing factors was analyzed in 46 ACL remnants biopsied during surgical reconstruction with quantitative real-time polymerase chain reaction technique. Specimens were divided into 3 groups according to time from injury: acute (<3 months from injury; n = 19), intermediate (3-12 months; n = 12), and chronic (>12 months; n = 15). Histological and immunohistochemical evaluation was performed by analysis of hematoxylin and eosin, CD-34, and S-100 staining. Results: Expression of COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, COL12A1, LOX, PLOD1, and TNC genes in ACL remnant was greater in acute compared with chronic injuries. COL1A1, COL5A1, COL12A1, and TNC genes were also expressed more in the acute group compared with the intermediate group. Furthermore, expression of the genes COL1A1 and COL5A2 was significantly higher in female than in male patients. No difference in the number of blood vessels and mechanoreceptors among groups was observed in the microscopic evaluation. Conclusion: The present study demonstrates that expression of COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, COL12A1, LOX, PLOD1, and TNC genes in ACL remnant is greater in acute (<3 months from injury) compared with chronic (>12 months) injuries. Furthermore, COL1A1, COL5A1, COL12A1, and TNC genes were expressed more in the acute group compared with the intermediate group (3-12 months from injury). Clinical Relevance: ACL reconstructions with remnant preservation should be performed in patients with acute injuries, as in these cases the ACL remnant may present the greatest healing potential.

Colonization of the cervicovaginal space with Gardnerella vaginalis leads to local inflammation and cervical remodeling in pregnant mice.

The role of the cervicovaginal (CV) microbiome in regulating cervical function during pregnancy is poorly understood. Gardnerella vaginalis (G. vaginalis) is the most common bacteria associated with the diagnosis of bacterial vaginosis (BV). While BV has been associated with preterm birth (PTB), clinical trials targeting BV do not decrease PTB rates. It remains unknown if G. vaginalis is capable of triggering molecular, biomechanical and cellular events that could lead to PTB. The objective of this study was to determine if cervicovaginal colonization with G. vaginalis, in pregnant mice, induced cervical remodeling and modified cervical function. CD-1 timed-pregnant mice received a 5X108 CFU/mL intravaginal inoculation of G. vaginalis or control on embryonic day 12 (E12) and E13. On E15, the mice were sacrificed and cervicovaginal fluid (CVF), amniotic fluid (AF), cervix, uterus, placentas and fetal membranes (FM) were collected. Genomic DNA was isolated from the CVF, placenta, uterus and FM and QPCR was performed to confirm colonization. IL-6 was measured in the CVF and AF and soluble e-cadherin (seCAD) was assessed in the CVF by ELISA. RNA was extracted from the cervices to evaluate IL-10, IL-8, IL-1β, TNF-α, Tff-1, SPINK-5, HAS-1 and LOX expression via QPCR. Mucicarmine and trichrome staining was used to assess cervical mucin and collagen. Biomechanical properties of the cervix were studied using quasi-static tensile load-to-failure biomechanical tests. G. vaginalis successfully colonized the CV space. This colonization induced immune responses (increased IL-6 levels in CVF and AF, increased mRNA expression of cervical cytokines), altered the epithelial barrier (increased seCAD in the CVF), induced cervical remodeling (increased mucin production, altered collagen) and altered cervical biomechanical properties (a decrease in biomechanical modulus and an increase in maximum strain). The ability of G. vaginalis to induce these molecular, immune, cellular and biomechanical changes suggests that this bacterium may play a pathogenic role in premature cervical remodeling leading to PTB.

The Role of Berberine in the Prevention of HIF-1α Activation to Alleviate Adipose Tissue Fibrosis in High-Fat-Diet-Induced Obese Mice.

Berberine (BBR) is the main active ingredient of a traditional Chinese herb Coptis chinensis. It has been reported to exhibit beneficial effects in treating diabetes and obesity. However, the underlying mechanism has not been fully elucidated. Adipose tissue fibrosis is a hallmark of obesity-associated adipose tissue dysfunction. HIF-1α plays a key role in adipose tissue fibrosis, which closely linked to metabolic dysfunction in obese state. We hypothesized that BBR may alleviate obesity-induced adipose tissue fibrosis and associated metabolic dysfunction through inhibition of HIF-1α. To test this hypothesis, we treated high fat diet (HFD) feeding mice with different dose of BBR (100 mg/kg, 200 mg/kg, and 300 mg/kg) for 8 weeks. We found that BBR treatment greatly decreased the body weight gain and reduced insulin resistance induced by HFD. Data also revealed that BBR improved histologic fibrous of epididymal white adipose tissue (eWAT) and was accompanied with inhibition of the abnormal synthesis and deposition of extracellular matrix (ECM) proteins, such as collagen and fibronectin. We also found that BBR treatment suppressed the expression of HIF-1α and decreased the mRNA expression of LOX in epididymal adipose tissue, which plays a key role in fibrosis development. Taken together, these results suggest that BBR can regulate metabolic homeostasis and suppress adipose tissue fibrosis through inhibiting the expression of HIF-1α.

Herbal compound Teng-Long-Bu-Zhong-Tang inhibits metastasis in human RKO colon carcinoma.

Metastasis is one of the primary obstacles to the successful treatment of colorectal cancer. Teng-Long-Bu-Zhong-Tang (TLBZT) is a modern Chinese herbal formula that may be useful in the treatment of metastatic colorectal cancer. The present study evaluated the effects of TLBZT on lung metastasis in human RKO colon carcinoma cells injected into mice via the tail vein. The results demonstrated that TLBZT inhibited the metastasis of human RKO colon carcinoma cells to the lungs. TLBZT downregulated the expression of LOX and hypoxia-inducible factor 1α. TLBZT also inhibited the expression of integrins αV and β3 and the phosphorylation of focal adhesion kinase. These results indicate that TLBZT inhibits the lung metastasis of RKO colon carcinoma by regulating the expression of multiple genes. The results of the present study provide a new basis for the management of colorectal cancer metastasis using treatments derived from Chinese herbs.

Lysyl oxidase and adipose tissue dysfunction

Background/objectivesLysyl 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. MethodsLOX 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 9.5months 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. ResultsLOX 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. ConclusionsWhilst 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.

Targeting lysyl oxidase reduces peritoneal fibrosis.

BackgroundAbdominal surgery and disease cause persistent abdominal adhesions, pelvic pain, infertility and occasionally, bowel obstruction. Current treatments are ineffective and the aetiology is unclear, although excessive collagen deposition is a consistent feature. Lysyl oxidase (Lox) is a key enzyme required for crosslinking and deposition of insoluble collagen, so we investigated whether targeting Lox might be an approach to reduce abdominal adhesions.MethodsFemale C57Bl/6 mice were treated intraperitoneally with multiwalled carbon nanotubes (NT) to induce fibrosis, together with chemical (ß-aminoproprionitrile–BAPN) or miRNA Lox inhibitors, progesterone or dexamethasone. Fibrotic lesions on the diaphragm, and expression of fibrosis-related genes in abdominal wall peritoneal mesothelial cells (PMC) were measured. Effects of BAPN and dexamethasone on collagen fibre alignment were observed by TEM. Isolated PMC were cultured with interleukin-1 alpha (IL-1α) and progesterone to determine effects on Lox mRNA in vitro.ResultsNT-induced fibrosis and collagen deposition on the diaphragm was ameliorated by BAPN, Lox miRNA, or steroids. BAPN and dexamethasone disrupted collagen fibres. NT increased PMC Lox, Col1a1, Col3a1 and Bmp1 mRNA, which was inhibited by steroids. Progesterone significantly inhibited IL-1α induced Lox expression by PMC in vitro.ConclusionOur results provide proof-of-concept that targeting peritoneal Lox could be an effective approach in ameliorating fibrosis and adhesion development.

Perturbations to lysyl oxidase expression broadly influence the transcriptome of lung fibroblasts.

Lysyl oxidases are credited with pathogenic roles in lung diseases, including cancer, fibrosis, pulmonary hypertension, congenital diaphragmatic hernia, and bronchopulmonary dysplasia (BPD). Lysyl oxidases facilitate the covalent intra- and intermolecular cross-linking of collagen and elastin fibers, thereby imparting tensile strength to the extracellular matrix (ECM). Alternative ECM-independent roles have recently been proposed for lysyl oxidases, including regulation of growth factor signaling, chromatin remodeling, and transcriptional regulation, all of which impact cell phenotype. We demonstrate here that three of the five lysyl oxidase family members, Lox, Loxl1, and Loxl2, are highly expressed in primary mouse lung fibroblasts compared with other constituent cell types of the lung. Microarray analyses revealed that small interfering RNA knockdown of Lox, Loxl1, and Loxl2 was associated with apparent changes in the expression of 134, 3,761, and 3,554 genes, respectively, in primary mouse lung fibroblasts. The impact of lysyl oxidase expression on steady-state Mmp3, Mmp9, Eln, Rarres1, Gdf10, Ifnb1, Csf2, and Cxcl9 mRNA levels was validated, which is interesting, since the corresponding gene products are relevant to lung development and BPD, where lysyl oxidases play a functional role. In vivo, the expression of these genes broadly correlated with Lox, Loxl1, and Loxl2 expression in a mouse model of BPD. Furthermore, β-aminopropionitrile (BAPN), a selective lysyl oxidase inhibitor, did not affect the steady-state mRNA levels of lysyl oxidase target genes, in vitro in lung fibroblasts or in vivo in BAPN-treated mice. This study is the first to report that lysyl oxidases broadly influence the cell transcriptome.

E Eosinophils have an essential role in cardiac repair following myocardial infarction

BackgroundLow peripheral blood eosinophil count is associated with increased risk of mortality in ischaemic heart disease patients. Eosinophils contain preformed IL-4 within their cytoplasmic granules, which promotes an anti-inflammatory response...

Molecular characterization of lysyl oxidase‐mediated extracellular matrix remodeling during mouse decidualization

The establishment of decidualization is a prerequisite of successful pregnancy. Lysyl oxidase (Lox) is a copper-containing amine oxidase which catalyzes cross-linking of collagen and elastin in the ECM. Lox is expressed in the subluminal stroma surrounding the implanting blastocyst on day 5 of pregnancy. From days 6 to 8, the signals for Lox mRNA and protein are strongly detected in the decidual cells. The expression of Lox is under the control of estrogen via the GSK-3β/β-catenin/c-myc pathway. Dtprp is decreased by the inhibition of Lox activity. Furthermore, the inhibition of Lox activity decreases stromal cell migration and embryo adhesion. Our findings highlight the crucial role of Lox in endometrial stromal cells and deepen our understanding of decidualization.

Digital gene expression profiling of the pathogen-resistance mechanism of Oryza sativa 9311 in response to Bacillus amyloliquefaciens FZB42 induction

Plant growth-promoting rhizobacteria (PGPR) are beneficial soil microorganisms that colonize plant roots for nutritional purposes and benefit plants by increasing plant growth or reducing plant disease. To clarify the complex mechanisms underlying plant-PGPR interactions, transcriptomic analyses of the response of rice (Oryza sativa) 9311 to Bacillus amyloliquefaciens FZB42 were carried out using RNA-Seq technology. This resulted in the identification of 379 differentially expressed genes in rice roots and 719 in rice leaves following FZB42 interaction that are likely to be related to plant-PGPR interactions, plant stress-resistance, pathogen-resistance, growth and development and basic and energy metabolism. We focused on plant disease-resistance mechanisms induced by FZB42. Variations in the expression of crucial genes COI1, MEKK1, WRKY, and PR that are involved in signal transduction following plant-pathogen interaction at different interaction time points were examined. Significantly up-regulated expression of PR, LOX, and ERF genes involved in the salicylic acid, jasmonic acid, and ethylene signal transduction pathways, respectively, were detected, suggest that defense reactions in rice were induced following interaction with FZB42. Phytoalexin changes in rice induced by FZB42 were examined and momilactone A, phytocassane A, phytocassane D, phytocassane E levels were low at 12h, rose between 24 and 48h, suggesting a PGPR-mediated mechanism of plant pathogen resistance. The study indicated that plant responses to PGPR in multi-metabolism pathways, which includes improving plant pathogen-resistance by mediating multi-signal transduction that regulates the expression of PRs and other defense genes, as well as influencing fundamental metabolic pathways to enhance the plant’s physical condition.

Lysyl Oxidase Is a Strong Determinant of Tumor Cell Colonization in Bone.

Lysyl oxidase (LOX) is a secreted copper-dependent amine oxidase whose primary function is to drive collagen crosslinking and extracellular matrix stiffness. LOX in colorectal cancer synergizes with hypoxia-inducible factor-1 (HIF-1) to promote tumor progression. Here we investigated whether LOX/HIF1 endows colorectal cancer cells with full competence for aggressive colonization in bone. We show that a high LOX expression in primary tumors from patients with colorectal cancer was associated with poor clinical outcome, irrespective of HIF-1 In addition, LOX was expressed by tumor cells in the bone marrow from colorectal cancer patients with bone metastases. In vivo experimental studies show that LOX overexpression in colorectal cancer cells or systemic delivery of the conditioned medium from LOX-overexpressing colorectal cancer cells promoted tumor cell dissemination in the bone marrow and enhanced osteolytic lesion formation, irrespective of HIF-1 Conversely, silencing or pharmacologic inhibition of LOX activity blocked dissemination of colorectal cancer cells in the bone marrow and tumor-driven osteolytic lesion formation. In vitro, tumor-secreted LOX supported the attachment and survival of colorectal cancer cells to and in the bone matrix, and inhibited osteoblast differentiation. LOX overexpression in colorectal cancer cells also induced a robust production of IL6. In turn, both LOX and IL6 were acting in concert to promote RANKL-dependent osteoclast differentiation, thereby creating an imbalance between bone resorption and bone formation. Collectively, our findings show that LOX supports colorectal cancer cell dissemination in the bone marrow and they reveal a novel mechanism through which LOX-driven IL6 production by colorectal cancer cells impairs bone homeostasis. Cancer Res; 77(2); 268-78. ©2016 AACR.

MicroRNA-214 Affects Fibroblast Differentiation of Adipose-Derived Mesenchymal Stem Cells by Targeting Mitofusin-2 during Pelvic Floor Dysfunction in SD Rats with Birth Trauma

This study investigated whether microRNA-214 (miR-214) targets mitofusin-2 (Mfn2) in the process of fibroblast differentiation of adipose-derived mesenchymal stem cells (ADMSCs) during pelvic floor dysfunction (PFD) in Sprague Dawley (SD) rats with birth trauma.The ADMSCs were isolated from 4-6 week male SD rats (n = 20) and were cultured and divided into the blank, miR-214 mimic negative control (NC), miR-214 mimic, miR-214 inhibitor NC, miR-214 inhibitor, empty vector, Mfn2 over-expression and miR-214 + Mfn2 over-expression groups. Fibroblast differentiation of ADMSCs was measured with immunocytochemistry and immunofluorescence methods. The expression of miR-214 and the mRNA and protein expression of Mfn2, FSP1, Collagen I, Collagen III, Elastin, LOX, Fibulin-5, PPAR-γ and Runx2 were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively. A dual-luciferase reporter assay was performed to confirm whether Mfn2 was the target gene of miR-214.During ADMSC differentiation into fibroblasts, miR-214 expression was up-regulated, but the expression of Mfn2 was down-regulated. Fibroblast differentiation of ADMSCs was promoted in the miR-214 mimic group but was inhibited in the miR-214 inhibitor and Mfn2 over-expression groups. The expression of Mfn2 was decreased, but the expression of FSP1, Collagen I, Collagen III, Elastin, LOX, Fibulin-5, PPAR-γ or Runx2 was increased in the miR-214 mimic group; the miR-214 inhibitor group and Mfn2 over-expression group exhibited the opposite results. Mfn2 was the target gene of miR-214.The study provided strong evidence that miR-214 could promote fibroblast differentiation of ADMSCs by down-regulating Mfn2 to improve PFD in SD rats with birth trauma.