LOX Expression Research Articles

Xiaoliu Pingyi Pecipe Inhibits Lung Pre-Metastatic Niche Formation and Prevents Myeloid-Derived Suppressor Cells Recruitment.

Metastasis, a leading cause of cancer-related deaths, involves complex mechanisms. The premetastatic niche (PMN) is a crucial contributor to this process. Myeloid-derived suppressor cells (MDSCs) play an important role in PMN formation and promote tumor progression and metastasis. The Xiaoliu Pingyi recipe (XLPYR), a traditional Chinese medicine, is effective in preventing postoperative recurrence and metastasis in cancer patients. This study investigated the effects of XLPYR on MDSCs recruitment and on the expression of PMN markers and elucidated the mechanisms involved in the prevention of tumor metastasis. C57BL/6 mice were subcutaneously injected with Lewis cells and treated with cisplatin and XLPYR. Tumors were resected after 14 days after the establishment of a model of lung metastasis, and tumor volume and weight were measured. Lung metastases were observed 21 days after resection. MDSCs in the lung, spleen, and peripheral blood were detected by flow cytometry. Western blotting, qRT-PCR and ELISA were used to detect the expression of S100A8, S100A9, MMP9, LOX, and IL-6/STAT3 in premetastatic lung tissue. XLPYR treatment inhibited tumor growth and prevented lung metastasis. Compared to mice without subcutaneous tumor cell transplantation, the model group had an increased proportion of MDSCs, higher expression of S100A8, S100A9, MMP9, and LOX in the premetastatic lung. XLPYR treatment reduced the proportion of MDSCs, S100A8, S100A9, MMP9, and LOX expression, and downregulated the IL-6/STAT3 pathway. XLPYR may prevent MDSCs recruitment and reduce the expression of S100A8, MMP9, LOX, and IL6/STAT3 in premetastatic lung tissue, thus reducing lung metastases.

Abstract 11565: Improvement in Cardiopulmonary Function in a Rat Model of Pulmonary Arterial Hypertension Observed With RKER-012, a Novel Activin Receptor Type II Ligand Trap, was Associated With Reduced Markers of Inflammation and Fibrosis in the Right Ventricle

Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary vascular resistance, impaired cardiac output, and right ventricle (RV) overload. PAH is associated with imbalanced TGF-ß signaling, marked by elevated SMAD2/3 signaling, which is associated with inflammation and fibrosis contributing to heart failure. Initially, the RV can compensate for increased pressure due to its adaptive contractility, but chronic pressure overload leads to maladaptive RV remodeling through inflammatory and fibrotic pathways. Current therapies for PAH are primarily vasodilators which do not treat the underlying pathology. RKER-012 is a research form of KER-012, an investigational modified ActRIIB ligand trap designed to inhibit signaling through SMAD2/3. We investigated RKER-012’s mechanism of prevention of cardiac pathology in a Sugen/Hypoxia (SH) PAH model. Rats received a subcutaneous (SQ) dose of 25 mg/kg Sugen 5416 under hypoxic conditions. For 3 weeks, SH rats received vehicle (SH-VEH) or 10 mg/kg RKER-012 (SH-RKER-012) twice weekly SQ. Control rats received VEH twice weekly in normoxic conditions. Systolic pulmonary arterial pressure (sPAP) and Fulton index (FI) were measured. qPCR of RV tissue was used to evaluate the mechanism of RKER-012 in this model. SH-VEH rats had increased FI and sPAP (both p≤0.0001) compared to NX, consistent with development of cardiac and pulmonary impairment. Relative to SH-VEH, RKER-012 reduced increased FIand prevented increased sPAP (both p≤0.001) in SH rats so that sPAP did not differ from NX. Relative to NX, SH-VEH had increased RV expression of fibrotic markers such as Timp1, Col1a1, Col3a1, Runx2, and LOX (all p≤0.05). In contrast, SH-RKER-012 rats had reduced expression of Timp1, Col1a1, Col3a1, Runx2 and LOX (all p≤0.05) relative to SH-VEH. Inflammation markers CD68 and MCP1 (both p≤0.05) were increased in SH-VEH compared to NX. CD68 expression was reduced in SH-RKER-012 (p≤0.01) compared to NX but MCP1 expression was not affected (p=0.54). These results support that RKER-012 attenuated upregulation of key inflammation and fibrosis markers in a PAH preclinical model and suggest that KER-012 has a distinct mechanism from vasodilation and could be developed as a cardioprotective treatment for PAH.

Delivery of Mir-196c-3p with NIR-II light-triggered gel attenuates cardiomyocyte ferroptosis in cardiac ischemia-reperfusion injury

Ferroptosis plays an important role in ischemia-reperfusion (I/R)-induced cardiac injury and there are many defects in current targeted delivery of miRNAs for the treatment of ferroptosis. We herein report a unique hydrogel (Gel) that can be triggered by a near-infrared-II (NIR-II) light with deep tissue penetration and biocompatible maximum permissible exposure (MPE) value for in situ treatment after I/R. The mir-196c-3p mimic (mimics) and photothermal nanoparticles (BTN) were co-encapsulated in an injectable Gel (mimics + Gel/BTN) with NIR-II light-triggered release. Using 1064 nm light irradiation, local microenvironment photothermal-triggered on-demand noninvasive controllable delivery of miRNA was achieved, aiming to inhibit I/R-induced ferroptosis. Consequently, declined ferroptosis in cardiomyocytes and improved cardiac function, survival rate in rats was achieved through the controlled release of Gel/BTN mimics in I/R model to simultaneously inhibit ferroptosis hub genes NOX4, P53, and LOX expression.

Engineering sigma factors and chaperones for enhanced heterologous lipoxygenase production in Escherichia coli

BackgroundLipoxygenase (EC. 1.13.11.12, LOX) can catalyze the addition of oxygen into polyunsaturated fatty acids to produce hydroperoxides, which are widely used in the food, chemical, and pharmaceutical industries. In recent years, the heterologous production of LOX by Escherichia coli has attracted extensive attention. However, overexpressed recombinant LOX in E. coli aggregates and forms insoluble inclusion bodies owing to protein misfolding.ResultsIn this study, a split green fluorescent protein-based screening method was developed to screen sigma (σ) factors and molecular chaperones for soluble LOX expression. Three mutant libraries of Skp, GroES, and RpoH was analyzed using the high-throughput screening method developed herein, and a series of mutants with significantly higher yield of soluble heterologous LOX were obtained. The soluble expression level of LOX in the isolated mutants increased by 4.2- to 5.3-fold. Further, the highest LOX activity (up to 6240 ± 269 U·g-DCW−1) was observed in E. coli REopt, with the regulatory factor mutants, RpoH and GroES. Based on RNA-Seq analysis of the selected strains, E. coli Eopt, E. coli Sopt, E. coli Ropt, and wild type, amino acid substitutions in σ factors and molecular chaperones regulated the expression level of genes related to gene replication, recombination, and repair. Furthermore, the regulatory factor mutants were identified to be beneficial to the soluble expression of two other heterologous proteins, amylase and bone morphological protein 12.ConclusionIn this study, a high-throughput screening method was developed for improved soluble LOX expression. The obtained positive mutants of the regulatory factor were analyzed and employed for the expression of other heterologous proteins, thus providing a potential solution for the inclusion-body protein.

Deletion of the extracellular matrix glycoprotein ADAMTSL3 increases pro-fibrotic signalling and exacerbates heart failure in mice

Abstract Background/Introduction Cardiac fibrosis is a central pathophysiological process accompanying most cardiac diseases including heart failure, and is a predictor of poor outcomes. The fibrotic process is mediated by activated cardiac fibroblasts (CFBs), so-called myofibroblasts, which produce excessive amounts of type I collagen and other ECM molecules. The ADAMTSL family of glycoproteins is upregulated in the cardiac extracellular matrix (ECM) during heart failure, but their function in the heart is unknown. Some ADAMTSL proteins are suggested to regulate the TGFβ, a major driver of myofibroblast activation and cardiac fibrosis. Purpose The purpose of this study was to examine the role of ADAMTSL3 in TGFβ-regulation and heart failure. Methods ADAMTSL3 expression was analysed in failing hearts of patients and mice. To study the functional role of ADAMTSL3 in the failing heart, an Adamtsl3 knock-out (L3-KO) mouse was generated, and WT and L3-KO littermates were subjected to experimental heart failure by aortic banding (AB), or sham surgery, for a total of six weeks. The mice were followed with echocardiography and MRI, and the left ventricles (LVs) were harvested one and six weeks after AB or sham surgery, with molecular analyses performed. To elucidate molecular functions of ADAMTSL3 in vitro, we overexpressed ADAMTSL3 in CFBs producing an extensive ECM. Results We determined that ADAMTSL3 was produced by CFBs and upregulated in failing hearts of patients and mice. The L3-KO mice had a normal cardiac phenotype at baseline, but upon increased LV afterload developed a dilated cardiomyopathy phenotype with increased LV dilation and reduced systolic function from one week post-AB. Furthermore, high mortality was observed in the L3-KO mice post-AB, with 60% vs. 96% survival of WTs over six weeks. At one week post-AB RNA sequencing of LVs revealed 233 differentially expressed genes in L3-KO vs. WT, with Col1a1 and Postn among the most upregulated, suggesting increased fibrosis and TGFβ signalling. Increased canonical TGFβ signalling was confirmed by increased SMAD2/3 phosphorylation and increased Lox expression in L3-KOs six weeks post-AB. In vitro, ADAMTSL3 overexpression in cultured CFBs resulted in reduced collagen synthesis and reduced expression of COL1A1, LOX, CTGF and POSTN, indicating anti-fibrotic properties. Furthermore, ADAMTSL3 inhibited the expression of ACTA2 and SPP1, reduced α-SMA protein by 25%, and reduced proliferation and CFB contraction, suggesting attenuated TGFβ signalling and inhibited myofibroblast differentiation. Conclusions ADAMTSL3 was upregulated in human and mouse heart failure, and served an anti-fibrotic and cardio-protective role in failing mouse hearts. Mechanistically, ADAMTSL3 was produced by CFBs and inhibited myofibroblast differentiation and collagen synthesis through TGFβ in cultured CFBs. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The Research Council of Norway

LOX and Its Methylation Impact Prognosis of Diseases and Correlate with TAM Infiltration in ESCA.

Background ESCA is one of the digestive tract tumors with a high fatality. It is implicated in an intricate gene regulation process, but the pathogenesis remains ambiguous. Methods The study used the packages of Limma from R software to analyze DEGs of ESCA in the GEO database and TCGA database. We employed the DAVID website for enrichment analysis, and the string database constructed the PPI network. Hub genes were identified from ESCA DEGs with Cytoscape MCODE. We evaluated the clinical relevance of LOX expression and its DNA methylation in the cBioPortal database and explored the roles of LOX in ESCA immunity, especially immune cell infiltration levels and immune checkpoint expression, by immunedeconv package of R software. Conclusions The overexpression of LOX in ESCA is regulated by DNA hypomethylation; LOX overexpression or LOX hypomethylation can predict a worse prognosis in patients with ESCA. Besides, LOX may be involved in TIME regulation, promoting the infiltration levels and function of TAM. Hence, high LOX expression affected by DNA hypomethylation has an essential role in patients with ESCA, which may become an effective prognostic marker and therapeutic target.

Correlation of Matrisome-Associatted Gene Expressions with LOX Family Members in Astrocytomas Stratified by IDH Mutation Status.

Tumor cell infiltrative ability into surrounding brain tissue is a characteristic of diffusely infiltrative astrocytoma and is strongly associated with extracellular matrix (ECM) stiffness. Collagens are the most abundant ECM scaffolding proteins and contribute to matrix organization and stiffness. LOX family members, copper-dependent amine oxidases, participate in the collagen and elastin crosslinking that determine ECM tensile strength. Common IDH mutations in lower-grade gliomas (LGG) impact prognosis and have been associated with ECM stiffness. We analyzed the expression levels of LOX family members and matrisome-associated genes in astrocytoma stratified by malignancy grade and IDH mutation status. A progressive increase in expression of all five LOX family members according to malignancy grade was found. LOX, LOXL1, and LOXL3 expression correlated with matrisome gene expressions. LOXL1 correlations were detected in LGG with IDH mutation (IDHmut), LOXL3 correlations in LGG with IDH wild type (IDHwt) and strong LOX correlations in glioblastoma (GBM) were found. These increasing correlations may explain the increment of ECM stiffness and tumor aggressiveness from LGG-IDHmut and LGG-IDHwt through to GBM. The expression of the mechanosensitive transcription factor, β-catenin, also increased with malignancy grade and was correlated with LOXL1 and LOXL3 expression, suggesting involvement of this factor in the outside–in signaling pathway.

Glucocorticoid Dose Dependency on Gene Expression in Vocal Fold Fibroblasts and Macrophages.

Glucocorticoids (GCs) modulate multiple cellular activities including inflammatory and fibrotic responses. Outcomes of GC treatment for laryngeal disease vary, affording opportunity to optimize treatment. In the current study, three clinically employed GCs were evaluated to identify optimal in vitro concentrations at which GCs mediate favorable anti-inflammatory and fibrotic effects in multiple cell types. We hypothesize a therapeutic window will emerge as a foundation for optimized therapeutic strategies for patients with laryngeal disease. In vitro. Human vocal fold fibroblasts and human macrophages derived from THP-1 monocytes were treated with 0.03-1000 nM dexamethasone, 0.3-10,000 nM methylprednisolone, and 0.3-10,000 nM triamcinolone in combination with interferon-γ, tumor necrosis factor-α, or interleukin-4. Real-time polymerase chain reaction was performed to analyze inflammatory (CXCL10, CXCl11, PTGS2, TNF, IL1B) and fibrotic (CCN2, LOX, TGM2) genes, and TSC22D3, a target gene of GC signaling. EC50 and IC50 to alter inflammatory and fibrotic gene expression was calculated. Interferon-γ and tumor necrosis factor-α increased inflammatory gene expression in both cell types; this response was reduced by GCs. Interleukin-4 increased LOX and TGM2 expression in macrophages; this response was also reduced by GCs. GCs induced TSC22D3 and CCN2 expression independent of cytokine treatment. EC50 for each GC to upregulate CCN2 was higher than the IC50 to downregulate other genes. Lower concentrations of GCs repressed inflammatory gene expression and only moderately induced genes involved in fibrosis. These data warrant consideration as a foundation for optimized clinical care paradigms to reduce inflammation and mitigate fibrosis. NA Laryngoscope, 133:1169-1175, 2023.

Macrophages alter inflammatory and fibrotic gene expression in human vocal fold fibroblasts

Macrophage phenotypes are simplistically classified as pro-inflammatory (M1) or anti-inflammatory/pro-fibrotic (M2). Phenotypically different macrophages are putatively involved in vocal fold (VF) fibrosis. The current study investigated interactions between macrophages and VF fibroblasts. THP-1 monocyte-derived macrophages were treated with interferon-gamma (IFN-γ), lipopolysaccharide (LPS)/IFN-γ, interleukin-10 (IL10), transforming growth factor-β1 (TGF-β), or interleukin-4 (IL4) for 24 h (M(IFN), M(IFN/LPS), M(IL10), M(TGF), and M(IL4), respectively; M(−) denotes untreated macrophages). Differentially activated macrophages and human VF fibroblasts were co-cultured ± direct contact. Expression of CXCL10, CCN2, ACTA2, FN1, TGM2, and LOX was quantified by real-time polymerase chain reaction. Type I collagen and smooth muscle actin (SMA) were observed by immunofluorescence. CXCL10 and PTGS2 were upregulated in fibroblasts indirectly co-cultured with M(IFN) and M(IFN/LPS). M(TGF) stimulated CCN2, ACTA2, and FN1 in fibroblasts. Enzymes involved in extracellular matrix crosslinking (TGM2, LOX) were increased in monocultured M(IL4) compared to M(−). Direct co-culture with all macrophages increased type I collagen and SMA in fibroblasts. Macrophage phenotypic shift was consistent with stimulation and had downstream differential effects on VF fibroblasts. Direct contact with macrophages, regardless of phenotype, stimulated a pro-fibrotic response in VF fibroblasts. Collectively, these data suggest meaningful interactions between macrophages and fibroblasts mediate fibrosis.

Exogenous melatonin improved the growth and development of naked oat seedlings under cadmium stress

Heavy metal pollution has become a global problem, which affect more and more crop yields. Melatonin (MT) is widely used in plant stress resistance to alleviate the toxicity caused by heavy metals and other stresses. In this paper, the effects of exogenous 50μM and 100μM MT on the growth and development of naked oat seedlings under cadmium stress (25mg L-1) were studied. The results showed that different concentrations of MT could promote the growth of naked oat seedlings under 25-mg L-1 cadmium stress. The application of exogenous melatonin could significantly increase the plant height, fresh weight, dry weight, chlorophyll, and proline contents of naked oats. MT could also reduce the contents of hydrogen peroxide, superoxide anion, and malondialdehyde in the cells of naked oat seedlings, and increase the activities of SOD, POD, and CAT. In addition, exogenous melatonin could affect the gene expression of LOX, POX, and Asmap1 in MAPK family and NAC and WRKY1 in TFS family in naked oat seedlings, thus promoting the growth and development of naked oat seedlings. In conclusion, this study is the first to demonstrate that MT is able to alleviate the negative effects to treat naked oat seedlings with cadmium stress. Therefore, melatonin has the potential to be applied in crops threatened by heavy metals.

Immunomodulatory Molecular Mechanisms of Luffa cylindrica for Downy Mildews Resistance Induced by Growth-Promoting Endophytic Fungi.

Downy mildew (DM), caused by P. cubensis, is harmful to cucurbits including luffa, with increased shortcomings associated with its control through cultural practices, chemical fungicides, and resistant cultivars; there is a prompt need for an effective, eco-friendly, economical, and safe biocontrol approach. Current research is therefore dealt with the biocontrol of luffa DM1 through the endophytic fungi (EF) consortium. Results revealed that T. harzianum (ThM9) and T. virens (TvA1) showed pathogen-dependent inducible metabolic production of squalene and gliotoxins by higher gene expression induction of SQS1/ERG9 (squalene synthase) and GliP (non-ribosomal peptide synthetase). Gene expression of lytic enzymes of EF was also induced with subsequently higher enzyme activities upon confrontation with P. cubensis. EF-inoculated luffa seeds showed efficient germination with enhanced growth potential and vigor of seedlings. EF-inoculated plants showed an increased level of growth-promoting hormone GA with higher gene expression of GA2OX8. EF-pre-inoculated seedlings were resistant to DM and showed an increased GSH content and antioxidant enzyme activities (SOD, CAT, POD). The level of MDA, H2O2, REL, and disease severity was reduced by EF. ACC, JA, ABA, and SA were overproduced along with higher gene expression of LOX, ERF, NCED2, and PAL. Expression of defense-marker genes (PPO, CAT2, SOD, APX, PER5, LOX, NBS-LRR, PSY, CAS, Ubi, MLP43) was also modulated in EF-inoculated infected plants. Current research supported the use of EF inoculation to effectively escalate the systemic immunity against DM corresponding to the significant promotion of induced systemic resistance (ISR) and systemic acquired resistance (SAR) responses through initiating the defense mechanism by SA, ABA, ET, and JA biosynthesis and signaling pathways in luffa.

Differential roles for lysyl oxidase (like), family members in chronic obstructive pulmonary disease; from gene and protein expression to function.

Chronic obstructive pulmonary disease (COPD) is characterized by long-term airflow obstruction with cigarette smoke as a key risk factor. Extracellular matrix (ECM) alterations in COPD may lead to small airway wall fibrosis. Altered collagen cross-linking, potentially mediated by the lysyl oxidase (LO) family of enzymes (LOX, LOXL1-4), orchestrates disturbed ECM homeostasis. In this study, we investigated the effects of smoking status and presence and severity of COPD on LOs gene and protein expression in the airways and the impact of LOs inhibition on airway contraction in an ex vivo mouse model. We used gene expression data from bronchial brushings, airway smooth muscle (ASM) cells in vitro and immunohistochemistry in lung tissue to assess smoke- and COPD-associated differences in LOs gene and protein expression in the small airways. We found higher LOX expression in current- compared to ex-smokers and higher LOXL1 expression in COPD compared to non-COPD patients. LOX and LOXL2 expression were upregulated in COPD ASM cells treated with cigarette smoke extract. LOXL1 and LOXL2 protein levels were higher in small airways from current- compared to non-smokers. In COPD patients, higher LOXL1 and lower LOX protein levels were observed, but no differences for LOXL2, LOXL3, and LOXL4 protein were detected in small airways. Inhibiting LOs activity increased airway contraction in murine lung slices. COPD-associated changes in LOs, in particular LOX and LOXL1, may be related to smoking and contribute to impaired airway function, providing potential novel targets for preventing or treating small airways changes in COPD.

Target Mechanism of Iturinic Lipopeptide on Differential Expression Patterns of Defense-Related Genes against Colletotrichum acutatum in Pepper.

Bacillus subtilis KB21 is an isolate with broad spectrum antifungal activity against plant pathogenic fungi. Our aim was to produce and purify antifungal lipopeptides via fermentation using B. subtilis KB21 and verify their antifungal mechanism against pepper anthracnose. When the KB21 strain was cultured in tryptic soy broth medium, the antifungal activity against pepper anthracnose correlated with biosurfactant production. However, there was no antifungal activity when cultured in Luria-Bertani medium. KB21 filtrates showed the highest degree of inhibition of mycelia (91.1%) and spore germination (98.9%) of Colletotrichum acutatum via increases in the biosurfactant levels. Using liquid chromatography-mass spectrometry (LC-MS) and LC-tandem MS (LC-MS/MS) analyses, the component with antifungal activity in the fermentation medium of the KB21 strain was determined to be the cyclic lipopeptide (CLP) antibiotic, iturin A. When the iturin fractions were applied to pepper fruits inoculated with conidia of C. acutatum, the lesion diameter and hyphal growth on the fruit were significantly suppressed. In addition, iturin CLP elevated the gene expression of PAL, LOX, and GLU in the treatments both with and without following fungal pathogens. Overall, the results of this study show that iturin CLPs from B. subtilis KB21 may be potential biological control agents for plant fungal diseases.

Sleep Apnea and Kidney Disease: The Role of Hypoxia and Lysyl Oxidase

Study Objective/HypothesisChronic kidney disease (CKD) or progressive loss of kidney function, is a serious health condition that affects 1 in 7 adults or an estimated 37 million Americans. Epidemiological studies have linked CKD with Sleep Apnea (SA), a breathing disorder characterized by erratic breathing, and episodes of hypoxia. However, the mechanisms that underlie the association between CKD and SA are not well understood. Hypoxia Inducible Factor‐1α (HIF‐1α) is a transcription factor that regulates the body’s response to hypoxia. Lysyl oxidase and lysyl oxidase likes (LOX and LOX‐Ls) are a group of copper enzymes that are hypoxia responsive and pro‐fibrotic. We hypothesized that hypoxia in SA upregulates HIF‐1α to drive expression of LOX or LOX‐L enzymes, fibrosis, and CKD.Materials & MethodsSamples from the Sleep and Nephrology and Outcomes Research Study (SNORE), a prospective observational study of sleep disorders in veterans with CKD, were used for this study. The patients were divided into three main groups: CKD with no SA (n=14), CKD with severe SA (n=12) and healthy controls (n=13). RNA from peripheral blood mononuclear cells (PBMC) was isolated using PAXGene Blood RNA tubes (Qiagen). The RNA was then used as a template for reverse transcription, converting it into cDNA and fold changes in gene expression between groups were examined using real time PCR. Differences between groups were compared using nonparametric Mann Whitney U tests and a p‐value < 0.05 was considered statistically significant.ResultsLOX expression was lower in patients with CKD compared to healthy controls, regardless of SA (CKD with SA: 0.28 ± 0.06 (p=0.04) and CKD without SA: 0.33 ± 0.07 (p=0.09)). There were no significant differences seen in any of the LOX‐L expression (LOX‐L1 (p=0.84), LOX‐L2 (p=0.35), or LOX‐L3 (p=0.93)). Similarly, HIF‐1α was lower in patients with CKD (CKD with SA: 0.69 ± 1.0 (p=0.47) and CKD without SA: 0.46 ± 0.06 (p=0.01)). There was no statistically significant correlation between LOX and HIF‐1α (r=0.04, p=0.83).ConclusionOur results showed that HIF‐1α and LOX expression in PBMC was decreased in patients with CKD regardless of if they had SA or not. The mechanisms underlying these observations, the functional significance, and if these changes are representative of tissue LOX expression needs further studies.

Proteomic Identification of a Gastric Tumor ECM Signature Associated With Cancer Progression.

The extracellular matrix (ECM) plays an undisputable role in tissue homeostasis and its deregulation leads to altered mechanical and biochemical cues that impact cancer development and progression. Herein, we undertook a novel approach to address the role of gastric ECM in tumorigenesis, which remained largely unexplored. By combining decellularization techniques with a high-throughput quantitative proteomics approach, we have performed an extensive characterization of human gastric mucosa, uncovering its composition and distribution among tumor, normal adjacent and normal distant mucosa. Our results revealed a common ECM signature composed of 142 proteins and indicated that gastric carcinogenesis encompasses ECM remodeling through alterations in the abundance of 24 components, mainly basement membrane proteins. Indeed, we could only identify one de novo tumor-specific protein, the collagen alpha-1(X) chain (COL10A1). Functional analysis of the data demonstrated that gastric ECM remodeling favors tumor progression by activating ECM receptors and cellular processes involved in angiogenesis and cell-extrinsic metabolic regulation. By analyzing mRNA expression in an independent GC cohort available at the TGCA, we validated the expression profile of 12 differentially expressed ECM proteins. Importantly, the expression of COL1A2, LOX and LTBP2 significantly correlated with high tumor stage, with LOX and LTBP2 further impacting patient overall survival. These findings contribute for a better understanding of GC biology and highlight the role of core ECM components in gastric carcinogenesis and their clinical relevance as biomarkers of disease prognosis.

Identification of thrombin as a key regulator of chondrocyte catabolic activity through RNA-Seq and experimental verification

The present study was designed to explore the relationship between thrombin and catabolic activity in chondrocytes. Primary rat chondrocytes were cultured for 24 h with rat serum (RS), rat plasma (RP), or rat plasma supplemented with thrombin (RPT). RNA-sequencing was then performed. Cell proliferation was analyzed by EdU uptake, CCK-8 assays and protein–protein interaction (PPI) network of proliferation-related genes. Heatmaps were used to visualize differences in gene expression. Gene Ontology (GO) enrichment analyses of up- and down-regulated differentially expressed genes were conducted. Molecular probes were used to label the endoplasmic reticulum in chondrocytes from three treatment groups. Immunofluorescence and Safranin O staining were used to assess type II collagen (Col2a1) expression and proteoglycan synthesis, whereas Lox expression was assessed by immunocytochemistry. The expression of enzymes involved in the synthesis and maturation of extracellular matrix (ECM) components and chemokines were measured by qPCR while matrix metalloproteinases (MMPs) levels were evaluated by Western blotting. Relevant nodules were selected through further PPI network analyses. A total of 727 and 1162 genes were up- and down-regulated based on the Venn diagrams comparison among groups. Thrombin was thus able to promote chondrocyte proliferation and a shift towards fibrotic morphology, while upregulating MMPs and chemokines linked to ECM degradation. In addition, thrombin decreased the enzyme expression involved in the synthesis and maturation of ECM.

MiR-29a Curbs Hepatocellular Carcinoma Incidence via Targeting of HIF-1α and ANGPT2.

A high-fat diet is responsible for hepatic fat accumulation that sustains chronic liver damage and increases the risks of steatosis and hepatocellular carcinoma (HCC). MicroRNA-29a (miR-29a), a key regulator of cellular behaviors, is present in anti-fibrosis and modulator tumorigenesis. However, the increased transparency of the correlation between miR-29a and the progression of human HCC is still further investigated. In this study, we predicted HIF-1α and ANGPT2 as regulators of HCC by the OncoMir cancer database and showed a strong positive correlation with HIF-1α and ANGPT2 gene expression in HCC patients. Mice fed the western diet (WD) while administered CCl4 for 25 weeks induced chronic liver damage and higher HCC incidence than without fed WD mice. HCC section staining revealed signaling upregulation in ki67, severe fibrosis, and steatosis in WD and CCl4 mice and detected Col3a1 gene expressions. HCC tissues significantly attenuated miR-29a but increased in HIF-1α, ANGPT2, Lox, Loxl2, and VEGFA expression. Luciferase activity analysis confirms that miR-29a specific binding 3′UTR of HIF-1α and ANGPT2 to repress expression. In summary, miR-29a control HIF-1α and ANGPT2 signaling in HCC formation. This study insight into a novel molecular pathway by which miR-29a targeting HIF-1α and ANGPT2 counteracts the incidence of HCC development.

Single-cell transcriptomics reveals the regulative roles of cancer associated fibroblasts in tumor immune microenvironment of recurrent osteosarcoma.

Rationale: Osteosarcoma (OS) is the most common primary bone tumor with a poor prognosis, but the detailed mechanism is still unclear. A comprehensive investigation of tumor microenvironment (TME) of OS might help find effective anti-tumor strategies. Single-cell transcriptomics is a powerful new tool to explore TME. Therefore, this study is designed to investigate the TME and gene expression pattern of primary and recurrent OS at the single-cell level.Methods: The single-cell RNA sequencing and bioinformatic analysis were conducted to investigate the cellular constitution of primary, recurrent, and lung metastatic OS lesions according to the datasets of GSE152048 and GSE162454. TIMER database was used to investigate the role of LOX in the prognosis of sarcoma. The functions of related cells and markers were further confirmed by in vitro and in vivo experiments.Results: Cancer associated fibroblasts (CAFs) were found with a higher infiltrating level in recurrent OS, and were enriched in the epithelial-mesenchymal transition (EMT) pathway. CAFs showed remarkably increased expression of LOX, which might lead to EMT and poor prognosis of OS. Mechanically, LOX regulated the function of CAFs and macrophage polarization to remodel the tumor immune microenvironment. Moreover, LOX inhibitor could inhibit migration and promote apoptosis of OS both in vitro and in vivo.Conclusions: This study revealed the heterogeneity of recurrent OS and highlighted an innovative mechanism that CAFs regulate EMT of OS via LOX. Targeting LOX of CAFs showed promising efficacy in remodeling TME and treating recurrent OS.

The Chaperone Protein GRP78 Released From MPN Cells Increases the Expression of LOX in a Human Stromal Cell Line

The Chaperone Protein GRP78 Released From MPN Cells Increases the Expression of LOX in a Human Stromal Cell Line

The miR-23b/27b/24-1 Cluster Inhibits Hepatic Fibrosis by Inactivating Hepatic Stellate Cells.

Background & AimsHepatic fibrosis is characterized by hepatic stellate cell (HSC) activation and transdifferentiation-mediated extracellular matrix (ECM) deposition, which both contribute to cirrhosis. However, no antifibrotic regimen is available in the clinic. microRNA-23b/27b/24-1 cluster inhibition of transforming growth factor-β (TGF-β) signaling during hepatic development prompted us to explore whether this cluster inhibits HSC activation and hepatic fibrosis.MethodsExperimental fibrosis was studied in carbon tetrachloride (CCl4)-treated C57BL/6 mice. After administration of miR-23b/27b/24-1 lentivirus or vehicle, animals were euthanized for liver histology. In primary rat HSC and HSC-T6, the anti-fibrotic effect of miR-23b/27b/24-1 cluster was furtherly investigated by RNA-sequencing, luciferase reporter assay, western blotting and bioinformatic means.ResultsIn this study, we showed that increasing the miR-23b/27b/24-1 level through intravenous delivery of miR-23b/27b/24-1 lentivirus ameliorated mouse hepatic fibrosis. Mechanistically, the miR-23b/27b/24-1 cluster directly targeted messenger RNAs, which reduced the protein expression of 5 secretory profibrotic genes (TGF-β2, Gremlin1, LOX, Itgα2, and Itgα5) in HSCs. Suppression of the TGF-β signaling pathway by down-regulation of TGF-β2, Itgα2, and Itgα5, and activation of the bone morphogenetic protein signaling pathway by inhibition of Gremlin1, decreased extracellular matrix secretion of HSCs. Furthermore, down-regulation of LOX expression softened the ECM. Moreover, a reduction in tissue inhibitors of metalloproteinase 1 expression owing to weakened TGF-β signaling increased ECM degradation.ConclusionsHepatic overexpression of the miR-23b/27b/24-1 cluster blocked hepatic fibrosis and may be a novel therapeutic regimen for patients with hepatic fibrosis.