LOX Expression Research Articles

Unveiling Novel Regulatory Mechanisms of miR-5195-3p in Pelvic Organ Prolapse Pathogenesis†.

Pelvic organ prolapse (POP) is a condition that significantly affects women's quality of life. The pathological mechanism of POP is not yet fully understood, and its pathogenesis is often caused by multiple factors, including the metabolic imbalance of the extracellular matrix (ECM). This study aims to investigate the role of miR-5195-3p, a microRNA, in the pathology of POP and its regulatory mechanism. Using various molecular biology techniques such as qRT-PCR, FISH, immunohistochemistry, and western blot, miR-5195-3p expression was examined in vaginal wall tissues obtained from POP patients. Results revealed an up-regulation of miR-5195-3p expression in these tissues, showing a negative correlation with the expression of ECM-related proteins. Further analysis using bioinformatics tools identified LOX as a potential target in POP. Dual luciferase reporter gene experiments confirmed LOX as a direct target of miR-5195-3p. Interestingly, regulating the expression of LOX also influenced the TGF-β1 signaling pathway and had an impact on ECM metabolism. This finding suggests that miR-5195-3p controls ECM metabolism by targeting LOX and modulating the TGF-β1 signaling pathway. In conclusion, this study unveils the involvement of miR-5195-3p in the pathological mechanism of POP by regulating ECM metabolism through the LOX/TGF-β1 axis. These findings reveal new mechanisms in the pathogenesis of pelvic POP, providing a theoretical foundation and therapeutic targets for further research on POP treatment.

Integrative microbiomics, proteomics and lipidomics studies unraveled the preventive mechanism of Shouhui Tongbian Capsules on cerebral ischemic stroke injury

Ethnopharmacological relevanceCerebral ischemic stroke (CIS) is one of the most important factors leading to death and disability, which seriously threaten the survival and health of patients. The intentional flora and its derived metabolites are demonstrated to play vital roles in the physiology and onset of CIS. Shouhui Tongbian Capsules (SHTB), a Traditional Chinese Medicine, could regulate gut microbiota and metabolites. Study has found that SHTB has protective effect on CIS, but the mechanism is still unclear. Aim of studyThis study was designed to evaluate the preventive effects and the mechanism of SHTB on CIS injury. Materials and methodsThe rats were pretreated with SHTB for 5 days, then the middle cerebral artery occlusion/reperfusion (MCAO/R) was established. Neurological deficit score, TTC staining, brain water content, H&E and Nissl staining were preformed to evaluate the preventive effects of SHTB on CIS. The Occludin and ZO-1 were analyzed to evaluate the blood-brain barrier (BBB). 16S rDNA sequencing and LC-ESI-MS/MS-based metabolomics profiling were performed to analyze the gut microbiota composition and short chain fatty acids (SCFAs) profile in gut. Serum lipopolysaccharide specific IgA antibody (LPS-SIgA) and diamine oxidase (DAO), as well as colon Claudin 5 and ZO-1 were analyzed to evaluate the intestinal barrier. Proteomics was used to evaluated the proteins profile in brain. Lipidomics were used to evaluate the brain SCFAs as well as medium and long chain fatty acids (MCFAs and LCFAs). Malondialdehyde (MDA), Total Superoxide dismutase (T-SOD), Glutathione (GSH), Glutathione peroxidase (GSH-Px), Catalase (CAT) and reactive oxygen species (ROS) were assayed to evaluate the oxidative stress in brain. Western blot was performed to evaluate the expression of PPARγ, Nrf2, SLC3A2, SCL7A11, GPX4, ACSL4 and LOX. ResultsSHTB prevented rats from MCAO/R injury, which was confirmed by lower cerebral infarct rate, brain water content, neurological deficit score and nissl body loss, and improved brain pathology. Meanwhile, SHTB upregulated the expression of ZO-1 and Occludin to maintain the integrity of BBB. 16S rDNA sequencing and LC-ESI-MS/MS-based targeted metabolomics found that SHTB increased the abundance of gut microbiota, regulated the numbers of intestinal bacteria to increase the production of Acetic acid, Propionic acid, and Butyric acid, as well as decrease the production of Valeric acid and Hexanoic acid in the gut. Meanwhile, SHTB improved the intestinal barrier by upregulating the protein levels of Claudin 5 and ZO-1, which was confirmed by low concentrations of LPS-SIgA and DAO in serum. Multi omics and spearman correlation analysis indicated that SHTB regulated the abundance of Escherichia-Shigella and Lactobacillus to increase Acetic acid, Propionic acid, and Butyric acid to induce the expression of PPARγ, thereby regulating fatty acid metabolism and degradation, improving lipid metabolism disorders, downregulating lipid oxidative stress, inhibiting ferroptosis, and alleviating brain injury. ConclusionThis study confirmed that SHTB improved the disturbance of fatty acid metabolism in brain tissue by regulating gut microbiota and the production of fecal SCFAs to inhibit ferroptosis caused by lipid oxidative stress and prevent CIS injury, which provided a potential candidate drug for the prevention of CIS.

Storage temperature affects metabolism of sweet corn

Temperature is one of the critical factors affecting the postharvest storage quality of sweet corn, but effects of storage temperatures have not been elucidated at the molecular level. In this study, the effects of storage at 0 °C, 4 °C and 20 °C, on quality and associated effects on the transcriptome and metabolome were investigated. Compared with storage at 20 °C, cobs stored at 0 °C and 4 °C had up-regulated expression of genes associated with cell wall disassembly (PG, PE, BMY, β-Gal) and down-regulated expression of genes involved in lignin synthesis, the up-regulation of SPP, SPS and SUS involved in sucrose synthesis, and the down-regulation of IN, FK, HXK and PFK involved in sucrose catabolism. Most of the genes involved in ethylene biosynthesis and signaling were inhibited at 0 °C, except for ERFs, while abscisic acid synthesis and signal transduction were most active at this temperature. Near-freezing temperature also maintains the integrity of the cell membrane by inhibiting the expression of PLD, PLC and LOX, which encode membrane lipid-degrading enzymes. Metabolomics and combined analysis showed that the contents of lysophospholipid, glutathione and L-ascorbic acid in sweet corn increased at low temperatures. These results provide insight into the effects of low temperatures on sweet corn quality.

Matrine protects against experimental autoimmune encephalomyelitis through modulating microglial ferroptosis

Multiple sclerosis (MS) is an inflammatory demyelination neurodegenerative disease of the central nervous system (CNS). Ferroptosis has been implicated in a range of brain disorders, and iron-loaded microglia are frequently found in affected brain regions. However, the molecular mechanisms linking ferroptosis with MS have not been well-defined. The present study seeks to bridge this gap and investigate the impact of matrine (MAT), a herbal medicine with immunomodulatory capacities, on the regulation of oxidative stress and ferroptosis in the CNS of mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. CNS of EAE mice contained elevated levels of ferroptosis-related molecules, e.g., MDA, LPCAT3 and PTGS2, but decreased expression of antioxidant molecules, including GSH and SOD, GPX4 and SLC7A11. This pathogenic process was reversed by MAT treatment, together with significant reduction of disease severity and CNS inflammatory demyelination. Furthermore, the expression of PTGS2 and LOX was largely increased in microglia of EAE mice, accompanied with increased production of IL-6 and TNF-α, indicating a proinflammatory phenotype of microglia that undergo oxidative stress/ferroptosis, and their expression was significantly reduced after MAT treatment. Together, our results indicate that ferroptosis/inflammation plays an important role in the pathogenesis of CNS autoimmunity, and inhibiting ferroptosis-induced microglial activation/inflammation could be a novel mechanism underlying the therapeutic effects of MAT on CNS inflammatory demyelination in EAE.

Zishen Qingre Lishi Huayu Recipe May Ameliorate the Symptoms of PCOS Model Rats via Alleviating Systemic and Ovarian Inflammation.

Zishen Qingre Lishi Huayu recipe (ZQLHR) has shown significant therapeutic effects in treating sex hormone levels and follicular developmental disorders in patients with polycystic ovary syndrome (PCOS). However, little is known about the potential mechanisms of its treatment. Dehydroepiandrosterone and a high-fat diet induced the PCOS model rat. The serum of rats was collected to detect the levels of sex hormones and inflammatory cytokines by enzyme-linked immunosorbent assay, and the ovaries were collected for ovarian histopathology and qPCR assay to detect the levels of inflammatory cytokines in ovarian tissues. Granulosa cells (GCs) were collected for western blot assay to detect of IL-1β, IL-6R, and LOX protein expression levels. ZQLHR could reduce body weight, regulate estrous cycles, and improve serum sex hormone levels, follicular development, and insulin resistance (IR) in PCOS model rats. In addition, ZQLHR treatment improved the levels of inflammatory cytokines in serum and ovary, and regulated the protein expression of IL-6R, IL-1β, and LOX in GCs of PCOS model rats. The results showed that the HOMA-IR index increased with the increasing levels of IL-6, IL-1β, and CRP, and decreased with the increased IL-10. This study reveals that the treatment of endocrine disorders and ovulation disorders in PCOS with ZQLHR may be closely related to the improvement of systemic and ovarian inflammation in PCOS patients, as well as the inhibition of IL-6R, IL-1β, and LOX expression in GCs, which reemphasizes the role of reducing chronic inflammatory states in the treatment of PCOS. Moreover, this study reemphasizes the correlation between multiple inflammatory mediators and IR.

Long-Term In Vitro Culture Alters Gene Expression Pattern of Genes Involved in Ontological Groups Representing Cellular Processes.

The oviduct provides an optimal environment for the final preparation, transport, and survival of gametes, the fertilization process, and early embryonic development. Most of the studies on reproduction are based on in vitro cell culture models because of the cell's accessibility. It creates opportunities to explore the complexity of directly linked processes between cells. Previous studies showed a significant expression of genes responsible for cell differentiation, maturation, and development during long-term porcine oviduct epithelial cells (POECs) in vitro culture. This study aimed at establishing the transcriptomic profile and comprehensive characteristics of porcine oviduct epithelial cell in vitro cultures, to compare changes in gene expression over time and deliver information about the expression pattern of genes highlighted in specific GO groups. The oviduct cells were collected after 7, 15, and 30 days of in vitro cultivation. The transcriptomic profile of gene expression was compared to the control group (cells collected after the first day). The expression of COL1A2 and LOX was enhanced, while FGFBP1, SERPINB2, and OVGP1 were downregulated at all selected intervals of cell culture in comparison to the 24-h control (p-value < 0.05). Adding new detailed information to the reproductive biology field about the diversified transcriptome profile in POECs may create new future possibilities in infertility treatments, including assisted reproductive technique (ART) programmes, and may be a valuable tool to investigate the potential role of oviduct cells in post-ovulation events.

Comparison Of Transient ACE Inhibitor Vs. Angiotensin Receptor Blocker Treatment On Angiotensin II-Induced Oxidative Stress In The Heart Of Spontaneously Hypertensive Rats

Angiotensin II (Ang II) is a primary mediator of the renin angiotensin system (RAS) with strong profibrotic effects, mediated in part through increased levels of reactive oxygen species (ROS). We have previously shown that transient treatment with an Ang II converting enzyme inhibitor (ACEi) produces long-term protection against Ang II-induced fibrosis. Our goal was to compare the impact of prior transient AT1 receptor antagonist (ARB) vs. ACEi treatment on the oxidative stress response and fibrotic signaling profile in the left ventricle (LV) in response to Ang II. Male and female spontaneously hypertensive rats were initially treated with vehicle, ARB (losartan, 30mg/kg per day) or ACEi (enalapril, 30mg/kg per day) for two weeks. To assess long term protection of ARB and ACEi treatment, after two-weeks of washout, the rats then received Ang II (400ng/kg/day) for 2 additional weeks. Protein levels of pro-oxidant (NOX-2) and antioxidant (SOD1, SOD2) enzymes, fibrosis marker (LOX) in the left ventricle (LV) were measured by western blotting. Cardiac hypertrophy was evaluated by the LV mass/tibia Length (LV/TL) ratio. Data were analyzed by 2-way ANOVA. After a transient ARB treatment, Ang II promoted an increase in LV/TL in both male (+7%) and female (+9%) rats when compared to vehicle+Ang II (treatment effect, p&lt;0.009). Ang II had no effect on LV/TL in rats previously treated with ACEi or vehicle. In response to Ang II, male and female rats that were previously treated with ACEi showed a decrease in LOX expression (treatment effect, p=0.02) in both male (-21%) and female (-22%) rats. No changes were observed in LOX in response to Ang II after ARB treatment. In rats transiently treated with ARB or ACEi, the expression of NOX2 was reduced after Ang II infusion. In the ARB group the NOX2 was reduced 25% in male and 28% in female (treatment effect, P=0.003). Similarly, in the ACEi group, NOX2 was reduced 27% in male and 26% in female rats (treatment effect, p&lt;0.0001). Prior ACEi and ARB produced sex-selective changes in SOD1 expression following Ang II infusion. Specifically, following transient ARB treatment, Ang II increased SOD1 in females (24%), but not males (sex x ARB treatment interaction: p=0.0033). Similarly, prior treatment with ACEi produced sex-dependent effects, whereby AngII increased SOD1 in females (10%) and decreased expression in males (-8%, ACEi x sex interaction: p=0.043). Neither ARB, ACEi nor Ang II affected the expression of SOD2 Our data indicate a sex and treatment-specific response to Ang II following transient ARB or ACEi treatment. Both ARB and ACEi showed a long-term beneficial effect on oxidative stress. However, ARB appeared to sensitize males and females to a hypertrophy response to AngII. Future studies will evaluate this differential response observed in cardiac hypertrophy and the mechanisms leading to cardiac fibrosis. NHBL R01HL153112. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Comprehensive phytohormone metabolomic and transcriptomic analysis of tobacco (Nicotiana tabacum) infected by tomato spotted wilt virus (TSWV)

Tomato spotted wilt virus (TSWV) is ranked among the top 10 most destructive viruses globally. It results in abnormal leaf growth, stunting, and even death, significantly affecting crop yield and quality. Phytohormones play a crucial role in regulating plant-virus interactions. However, there is still limited research on the effect of TSWV on phytohormone levels, particularly growth hormones and genes involved in the phytohormone pathway. In our study, we combined phytohormone metabolomics and transcriptomics to examine the impact of TSWV infection on phytohormone content and gene expression profile. Metabolomic results showed that 41 metabolites, including major phytohormones and their precursors and derivatives were significantly altered after 14 days of TSWV inoculation tobacco plants cvK326, with 31 being significantly increased and 10 significantly reduced. Specifically, the levels of abscisic acid (ABA) and jasmonoyl-isoleucine (JA-Ile) were significantly reduced. The levels of indole-3-acetic acid (IAA) have remained unchanged. However, the levels of cytokinin isopentenyladenine (iP) and salicylic acid (SA) significantly increased. The transcriptome analysis revealed 2,746 genes with significant changes in expression. Out of these, 1,072 genes were significantly downregulated, while 1,674 genes were significantly upregulated. Among them, genes involved in ABA synthesis and signaling pathways, such as 9-cis-epoxycarotenoid dioxygenase (NCED), protein phosphatase 2C (PP2C), serine/threonine-protein kinase (SnRK2), and abscisic acid responsive element binding factor (ABF), exhibited significant downregulation. Additionally, expression of the lipoxygenase gene LOX, Jasmonate ZIM domain-containing protein gene JAZ, and transcription factor gene MYC were significantly down-regulated. In the cytokinin pathway, while there were no significant changes in the expression of the cytokinin synthesis genes, a significant downregulation of transcriptionally active factor type-B response regulators (type-B RRs) was observed. In terms of SA synthesis and signaling pathways, the isochorismate synthase gene ICS1 and the pathogenesis-related gene PR1 were significantly upregulated. These results can strengthen the theoretical foundation for understanding the interaction between TSWV and tobacco and provide new insights for the future prevention and control of TSWV.

Ferrous Ion Alleviates Lipid Deposition and Inflammatory Responses Caused by a High Cottonseed Meal Diet by Modulating Hepatic Iron Transport Homeostasis and Controlling Ferroptosis in Juvenile Ctenopharyngodon idellus.

To investigate the mechanisms through which ferrous ion (Fe2+) addition improves the utilization of a cottonseed meal (CSM) diet, two experimental diets with equal nitrogen and energy content (low-cottonseed meal (LCM) and high-cottonseed meal (HCM) diets, respectively) containing 16.31% and 38.46% CSM were prepared. Additionally, the HCM diet was supplemented with graded levels of FeSO4·7H2O to establish two different Fe2+ supplementation groups (HCM + 0.2%Fe2+ and HCM + 0.4%Fe2+). Juvenile Ctenopharyngodon idellus (grass carps) (5.0 ± 0.5 g) were fed one of these four diets (HCM, LCM, HCM + 0.2%Fe2+ and HCM + 0.4%Fe2+ diets) for eight weeks. Our findings revealed that the HCM diet significantly increased lipid peroxide (LPO) concentration and the expression of lipogenic genes, e.g., sterol regulatory element binding transcription factor 1 (srebp1) and stearoyl-CoA desaturase (scd), leading to excessive lipid droplet deposition in the liver (p < 0.05). However, these effects were significantly reduced in the HCM + 0.2%Fe2+ and HCM + 0.4%Fe2+ groups (p < 0.05). Plasma high-density lipoprotein (HDL) concentration was also significantly lower in the HCM and HCM + 0.2%Fe2+ groups compared to the LCM group (p < 0.05), whereas low-density lipoprotein (LDL) concentration was significantly higher in the HCM + 0.2%Fe2+ and HCM + 0.4%Fe2+ groups than in the LCM group (p < 0.05). Furthermore, the plasma levels of liver functional indices, including alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glucose (GLU), were significantly lower in the HCM + 0.4%Fe2+ group (p < 0.05). Regarding the expression of genes related to iron transport regulation, transferrin 2 (tfr2) expression in the HCM group and Fe2+ supplementation groups were significantly suppressed compared to the LCM group (p < 0.05). The addition of 0.4% Fe2+ in the HCM diet activated hepcidin expression and suppressed ferroportin-1 (fpn1) expression (p < 0.05). Compared to the LCM group, the expression of genes associated with ferroptosis and inflammation, including acyl-CoA synthetase long-chain family member 4b (acsl4b), lysophosphatidylcholine acyltransferase 3 (lpcat3), cyclooxygenase (cox), interleukin 1β (il-1β), and nuclear factor kappa b (nfκb), were significantly increased in the HCM group (p < 0.05), whereas Fe2+ supplementation in the HCM diet significantly inhibited their expression (p < 0.05) and significantly suppressed lipoxygenase (lox) expression (p < 0.05). Compared with the HCM group without Fe2+ supplementation, Fe2+ supplementation in the HCM diet significantly upregulated the expression of genes associated with ferroptosis, such as heat shock protein beta-associated protein1 (hspbap1), glutamate cysteine ligase (gcl), and glutathione peroxidase 4a (gpx4a) (p < 0.05), and significantly decreased the expression of the inflammation-related genes interleukin 15/10 (il-15/il-10) (p < 0.05). In conclusion, FeSO4·7H2O supplementation in the HCM diet maintained iron transport and homeostasis in the liver of juvenile grass carps, thus reducing the occurrence of ferroptosis and alleviating hepatic lipid deposition and inflammatory responses caused by high dietary CSM contents.

Biocontrol efficacy of Burkholderia pyrrocinia S17-377 in controlling rice sheath blight

Rice sheath blight, caused by Rhizoctonia solani, is a major disease of rice that significantly affects rice production. To find sustainable and effective methods to control this disease, biocontrol strains have emerged as a promising solution. In this study, a total of 28 strains were screened from the rhizosphere of rice plants in order to identify strains that are effective against rice sheath blight. Among these strains, strain S17-377 showed remarkable antifungal activity, resulting in a 10 mm diameter inhibitory band against R. solani. Furthermore, it exhibited a wide range of antimicrobial properties in vitro. Through genetic analysis using the 16S rDNA and recA genes, strain S17-377 was identified as Burkholderia pyrrocinia. Importantly, it was found to be non-pathogenic to onions and did not possess the virulence genes BCESM and cblA. Therefore, this strain is safe for both people and plants. The strain S17-377 showed an efficacy of 64.81% and 55.78% in controlling rice sheath blight in pot experiments and field trials, respectively. Strain S17-377 displayed persistent colonization ability in rice plants for up to 30 days after inoculation. It also induced the expression of several rice plant genes, including NH1, PR1a, PR10, PAL/ZB8, LOX, and AOS2, as well as enzymes such as POD, PPO, and PAL. Additionally, S17-377 produced proteases, chitinases, non-volatile metabolites, and biofilms. In terms of disease control, the aseptic fermentation solution of S17-377, when diluted with PDA medium at ratios of 1:20, 1:10, and 1:5, exhibited inhibition rates of 77.14%, 86.92%, and 98.26%, respectively, against R. solani. S17-377 also had noticeable effects on the expansion and collapse of fungal mycelium. Moreover, the bacterial culture filtrates(BCF) of S17-377 demonstrated high thermal stability with an inhibition rate of 96.16%, even after being subjected to a temperature of 121 °C for 30 min. The BCF of strain S17-377 exhibited significant inhibition of R. solani, with inhibition rates of 95.23%, 94.43%, and 75.11% for the n-butanol, crude protein, and crude lipopeptide extracts, respectively. Further analysis of the n-butanol extract using liquid chromatography-mass spectrometry (LC-MS) identified eight compounds with potent antibacterial activity. These findings highlight the potential of strain S17-377 as a biocontrol agent for the management of rice sheath blight.

LOX and AAT Genes Affect the Aroma of “Xiaobai” Apricot during Postharvest Storage

“Xiaobai” apricot is one of the most popular fruits in China. However, during postharvest storage, fruit aroma loss occurs easily. In this study, factors affecting the aroma changes in different ripening stages of “Xiaobai” apricot during postharvest storage were searched. Immature and mature “Xiaobai” apricot samples were collected and monitored for sensory changes during postharvest storage. After 25 days of storage, the aromatic ester and alcohol abundance in mature showed a decrease, related to the loss of characteristic aroma. Immature fruit still tasted sour with an indistinct characteristic aroma, as a result of decreased alcohol and increased acid abundance. The ATT, ADH, PDC, and LOX genes were identified, and their expression levels were detected at different ripening stages during storage. The correlation analysis showed that the expression of LOX and ATT was positively correlated with the variation of aromatic ester ( P &lt; 0.05 ), a key factor affecting the apricot aroma during postharvest storage.

Identification of LOX as a candidate prognostic biomarker in Glioblastoma multiforme

BackgroundGlioblastoma multiforme (GBM) is the most malignant type of glioma. GBM tumors grow rapidly, have a high degree of malignancy, and are characterized by a fast disease progression. Unfortunately, there is a lack of effective treatments. An effective strategy for the treatment of GBM would be to identify key biomarkers correlating with the occurrence and progression of GBM and developing these biomarkers into therapeutic targets. Method and ResultsIn this study, using integrated bioinformatics analysis, we identified differentially expressed genes (DEGs), including 130 genes that were upregulated in GBM compared to normal brain tissue, and 128 genes that were downregulated in GBM. Based on Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis, these genes were associated with regulation of tumor cell adhesion, differentiation, morphology in GBM and were mainly enriched in Complement and coagulation cascades pathway. The Search Tool for the Retrieval of Interacting Genes (STRING) database was used to construct a Protein-Protein Interaction network. Ten hub genes were identified, including FN1, CD44, MYC, CDK1, SERPINE1, COL3A1, COL1A2, LOX, POSTN and EZH2, all of which were significantly upregulated in GBM, these results were confirmed by oncomine database exploration. Alteration analysis of hub genes found that patients with alteration in at least one of the hub genes showed shorter median survival times (p = 0.013) and shorter median disease-free survival times (p = 2.488E-3) than patients without alterations in any of the hub genes. Multiple tests for survival analysis showed that among individual hub genes only expression of LOX was correlated with patient survival (P < 0.05).GDS4467 data set was used to analyze the expression of LOX in gliomas with different degrees of malignancy, and it was found that the expression level of LOX was positively correlated with the malignant degree of gliomas.By analyzing GDS 4535 data set showed that the expression level of LOX was positively correlated with the differentiation degree of GBM cells ConclusionThis research suggests that FN1, CD44, MYC, CDK1, SERPINE1, COL3A1, COL1A2, LOX, POSTN and EZH2 are key genes in GBM. However, only LOX is correlated with patient survival and promotes glioblastoma cell differentiation and tumor recurrence. LOX may be a candidate prognostic biomarker and potential therapeutic target for GBM.

Validation of Fourier Transform Infrared Microspectroscopy for the Evaluation of Enzymatic Cross-Linking of Bone Collagen.

Enzymatic cross-linking of the bone collagen is important to resist to crack growth and to increased flexural strength. In the present study, we proposed a new method for assessment of enzymatic cross-link based on Fourier transform infrared (FTIR) microspectroscopy that takes into account secondary structure of type I collagen. Briefly, femurs were collected from sham or ovariectomized mice and subjected either to high-performance liquid chromatography-mass spectrometry or embedded in polymethylmethacrylate, cut and analyzed by FTIR microspectroscopy. FTIR acquisition was recorded before and after ultraviolet (UV) exposure or acid treatment. In addition, femurs from a second animal study were used to compare gene expression of Plod2 and Lox enzymes and enzymatic cross-links determined by FTIR microspectroscopy. We evidenced here that intensities and areas of subbands located at ~1660, ~1680, and ~1690cm-1 were positively and significantly associated with the concentration of pyridinoline (PYD), deoxypyridinoline, or immature dihydroxylysinonorleucine/hydroxylysinonorleucine cross-links. Seventy-two hours exposure to UV light significantly reduced by ~86% and ~89% the intensity and area of the ~1660cm-1 subband. Similarly, 24h of acid treatment significantly reduced by 78% and 76% the intensity and area of the ~1690cm-1 subband. Plod2 and Lox expression were also positively associated to the signal of the ~1660 and ~1690cm-1 subbands. In conclusion, our study provided a new method for decomposing the amide I envelope of bone section that positively correlates with PYD and immature collagen cross-links. This method allows for investigation of tissue distribution of enzymatic cross-links in bone section.

Fatty acid binding protein 1 and fatty acid synthetase over-expression have differential effects on collagen III synthesis and cross-linking in Zongdihua pig primary adipocytes.

The purpose of this study was to determine whether FABP1 and FAS regulate expression of collagen and its crosslinking via lysyl oxidase in isolated adipocytes from Zongdihua pigs. We aimed to identify biochemical processes affecting meat quality using molecular tools to provide a basis for breeding improvement of these animals. We measured expression levels of FABP1 and related genes using qRT-PCR in longissimus dorsi muscle and subcutaneous adipose tissues. Primary adipocytes from fat tissues were isolated and FABP1 and FAS were over-expressed from recombinant plasmids. Sequence analysis of the cloned genes indicated that FABP1 encodes a hydrophobic protein of 128 amino acids and contained 12 predicted phosphorylation sites and no transmembrane regions. The basal levels of FABP1 and FAS expression in pig tissues were 3-3.5-fold higher in subcutaneous fat compared with muscle (P < 0.01). Recombinant expression plasmids were successfully transfected into the cloned preadipocytes and (a) over-expression of FAS resulted in significantly increased expression of COL3A1 (P < 0.05) and significantly inhibited lysyl oxidase LOX expression (P < 0.01); (b) over-expression of FABP1 significantly increased COL3A1 expression (P < 0.01) and significantly inhibited LOX expression (P< 0.05) and significantly reduced lysyl oxidase activity (P < 0.01). Therefore, FAS enhanced FABP1 expression resulting in increased collagen accumulation and this preliminarily suggested that FAS and FABP1 can serve as fat-related candidate genes and provide a theoretical basis for the study of fat deposition in Zongdihua pigs.

Intermittent hypoxia induces fibroblast activation: An in vitro approach in NIH/3T3 cells

Obstructive sleep apnea (OSA) is recognized as an independent cardiovascular (CV) risk factor. Intermittent hypoxia (IH), the landmark feature of OSA, represents the major detrimental factor triggering CV complications. In rodent models, IH contributes to cardiac remodeling, mainly characterized by early interstitial fibrosis. However, the direct effect of IH on cardiac fibrosis, as well as involved mechanisms are unclear. The aim of this study is to investigate in vitro whether IH induces fibroblast activation. NIH/3T3 murine fibroblasts were exposed to normoxic condition (N) or fast IH cycles, including 5-min normoxia (16% O2) followed by 5-min hypoxia (2% O2), in the presence or not of 10 ng/ml transforming growth factor (Tgf-∝). Cell proliferation was assessed after 48 h N or IH exposure by MTT assay; cell migration was evaluated during 12 h epxosure by scratch assay; mRNA expression of several markers of fibrosis was evaluated after 6 h exposure. Fibroblast differentiation was evaluated through α Smooth Muscle Actin (Acta2) mRNA expression and fibrosis through expression of Collagen 1 (Col1a1), lysyl oxidase (Lox), Tgf∝ and Tgf∝ receptor (Tgf-∝R). Data were analyzed by 2-ways Anova (Impact of IH and Tgf-β), followed by a Tukey post-hoc test. Our results confirm the well-known role of Tgf-∝ on fibroblast activation, characterized by increased proliferation (P = 0.004) and mRNA expression of Acta2 (P = 0.002), Col1a1 (P = 0.008) and Lox (P = 0.04). Independently of Tgf-β, IH does not increase cell proliferation, tends to increase fibroblasts migration (P = 0.1) and significantly increases mRNA expression of Acta2 (P = 0.002) and Col1a1 (P = 0.05), whereas increases in Lox and Tgf-β expression do not reach significance (P = 0.06 and P = 0.08). Interestingly, concomitant stimulation with Tgf-β and IH does not potentiate cell proliferation and migration, but synergistically increases Acta2, Col1a1 and Lox mRNA expression. Our in vitro results shows that IH induces fibroblast activation, promoting migration, differentiation into myofibroblast and expression of fibrotic markers. Furthermore, IH and Tgf-∝ seem to exert synergistic effects on fibrosis. Further studies are needed to understand specific mechanisms involved in IH-induced fibrosis.

Long term in vivo Dlk1 gene transfer protects against myocardial fibrosis-induced cardiac dysfunction

Introduction: Cardiac fibrosis can independently contribute to the impairment of cardiac function and development of heart failure. Early activation of pro-fibrotic genetic pathways has been suggested to occur well before adverse ventricular remodeling occurs in fibrosis as it progresses to heart failure. Delta-like homolog 1 (Dlk1), a paternally imprinted gene that encodes a transmembrane protein that contains multiple epidermal growth factor repeats, has been shown to play a critical role as a regulator of cell differentiation, neuronal differentiation, bone differentiation, and hematopoiesis. We hypothesize that Dlk-1 could regulate myocardial fibrosis via inhibition of fibroblast-myofibroblast conversion and therefore restrain cardiac fibrosis progression. Methods: Wildtype (WT) and Dlk1 knockout (Dlk1-/-) mice were subjected to myocardial infarction (MI) by permanent ligation of the left anterior descending artery to induce heart failure. A recombinant adeno-associated virus 9 (AAV9) was used to deliver Dlk1 into the myocardium in vivo. Cardiac function and hemodynamic changes were measured by echocardiography and left ventricular catheterization. Immunohistochemistry and confocal microscopy were used to determine the expression of pro-fibrotic markers. Total RNA was prepared from left ventricular (LV) tissue or cardiomyocytes and fibroblasts from Dlk1-/- and WT hearts. Masson trichrome staining was used to determine abnormal collagen deposition in the heart. Result: Myocardial fibrosis was demonstrated by increased expression of alpha-smooth muscle actin (αSMA), collagen, fibronectin, connective tissue growth factor, along with a substantial increase in the deposition of collagen as evident from Masson's trichrome staining. We observed downregulated Dlk-1 mRNA and protein expression in ischemic tissues from human patients and infarcted pigs’ hearts. The reduced levels of Dlk1 were associated with increased mRNA expression of pro-fibrotic molecules, collagen1a, LOX and αSMA in the scar area. Echocardiographic analysis revealed substantial LV chamber dilatation and reduced fractional shortening (FS %) in Dlk1 null mice as compared to WT, whereas the mice treated with AAV9-Dlk1 for 8-10 weeks showed a significant improvement in cardiac function. Consistently, Dlk1-deficient mice exhibited impairment in heart function, cardiac fibrosis and myocyte hypertrophy and increased expression of atrial natriuretic peptide, and B-type natriuretic peptide, while AAV9-Dlk1-treated mice significantly reduced cardiac remodeling and myocardial fibrosis. Conclusion: Restoration of Dlk1 in the heart following MI-induced heart failure improved cardiac function and attenuated myocardial fibrosis. The findings from the current study suggest a critical role for Dlk1 in myocardial fibrosis-induced cardiac remodeling, indicating that Dlk1 gene therapy may be a viable therapeutic approach for heart failure. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 5957: Chemotherapy-induced extracellular matrix remodeling in HGSOC

Abstract In this study we explored the effects of chemotherapy on the extracellular matrix of high-grade serous ovarian cancer metastasis (HGSOC) at structural-textural protein level and transcriptionally, in order to identify the microenvironmental features that accompany a successful response. We studied two murine orthotopic, transplantable syngeneic models of HGSOC, one that displays excellent response to carboplatin/paclitaxel (60577) and one that is resistant to treatment (HGS2)1. Using immunohistochemistry, we stained omental tumors from both models for the ECM components fibronectin (FN1), collagen 1A1 (COL1A1) and versican (VCAN), also using Masson’s Trichrome to stain for collagen. We analyzed their abundance, textural features (Haralick features) and structure with the image analysis softwares, QuPath and TWOMBLI respectively. A compilation of 44 ECM metrics, which we named the “structure index”, correlated with response to chemotherapy, as measured by reduction in omental tumor weight. In parallel, we performed RNAseq on the omental tumors, identifying clusters of genes that change over time with chemotherapy treatment of the responsive model 60577, while the chemoresistant model HGS2 displayed limited alteration of gene expression. We integrated RNAseq and ECM structure and texture data, and showed the structure index was correlated with 144 core matrisome genes. To identify potential therapeutic targets, we then focused our study on genes that remained highly expressed in resistant tumors post chemotherapy, but were originally low or decreased with treatment in the sensitive tumors. Members of the LOX and P4HA family were found amongst these genes. We confirmed by immunohistochemical analysis that expression of LOX, LOXL2, P4HA1 and P4HA2 was downregulated by chemotherapy in the 60577 chemo-sensitive tumors while their levels remained relatively unaffected in the resistant HGS2 tumors. This study refines our understanding of the mechanisms of microenvironmental chemoresistance in HGSOC, highlighting putative therapeutic targets to increase the efficacy of platinum-based chemotherapy in ovarian cancer. 1 Maniati, E. et al. Mouse Ovarian Cancer Models Recapitulate the Human Tumor Microenvironment and Patient Response to Treatment. Cell Rep 30, 525-540.e527, doi:10.1016/j.celrep.2019.12.034 (2020). Citation Format: Panoraia Kotantaki, Florian Laforêts, Eleni Maniati, Chiara Berlato, Anna Malliouri, Michael John Devlin, Beatrice Malacrida, Samar Elorbany, Ranjit Manchanda, Frances R. Balkwill. Chemotherapy-induced extracellular matrix remodeling in HGSOC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5957.

Exposure to oxLDL impairs TGF-β activity in human tendon cells

BackgroundPrevious studies have shown that patients with hypercholesterolemia experience elevated levels of oxidized LDL (oxLDL), a molecule which triggers inflammation and collagenase activity. In this study we discovered novel mechanistic effects of oxLDL on tendon cells and the mediators regulating matrix remodeling by analyzing the expression and activity of related proteins and enzymes. These effects may contribute to tendon damage in patients with high cholesterol.MethodsIsolated human tendon cells (male and female donors age 28 ± 1.4 age 37 ± 5.7, respectively) were incubated in the presence or absence of oxLDL. The influence of oxLDL on the expression level of key mRNA and proteins was examined using real time quantitative PCR, ELISA and Western blots. The activities of enzymes relevant to collagen synthesis and breakdown (lysyl oxidase and matrix metalloproteinases) were quantified using fluorometry. Finally, the isolated human tendon cells in a 3D construct were exposed to combinations of oxLDL and TGF-β to examine their interacting effects on collagen matrix remodeling.ResultsThe one-way ANOVA of gene expression indicates that key mRNAs including TGFB, COL1A1, DCN, and LOX were significantly reduced in human tendon cells by oxLDL while MMPs were increased. The oxLDL reduced the activity of LOX at 50 µg/ml, whereas conversely MMP activities were induced at 25 µg/ml (P ≤ 0.01). COL1A1 synthesis and TGF-β secretion were also inhibited (P ≤ 0.05). Adding recombinant TGF-β reversed the effects of oxLDL on the expression of collagens and LOX. OxLDL also impaired collagen matrix remodeling (P ≤ 0.01), and adding TGF-β restored the native phenotype.ConclusionExposure to oxLDL in patients with hypercholesterolemia may adversely affect the mechanical and structural properties of tendon tissue through a direct action of oxLDL on tendon cells, including impairment of TGF-β expression. This impairment leads to disturbed matrix remodeling and synthesis, thereby potentially leading to increased risk of acute or chronic tendon injury. Our discovery may provide an opportunity for developing effective treatments for tendon injury in hypercholesterolemia patients by targeting the TGF-β pathway.

Identification of Prognosis-Related Oxidative Stress Model with Immunosuppression in HCC

For hepatocellular carcinoma (HCC) patients, we attempted to establish a new oxidative stress (OS)-related prognostic model for predicting prognosis, exploring immune microenvironment, and predicting the immunotherapy response. Significantly differently expressed oxidative stress-related genes (DEOSGs) between normal and HCC samples from the Cancer Genome Atlas (TCGA) were screened, and then based on weighted gene coexpression network analysis (WGCNA), HCC-related hub genes were discovered. Based on the least absolute shrinkage and selection operator (LASSO) and cox regression analysis, a prognostic model was developed. We validated the prognostic model's predictive power using an external validation cohort: the International Cancer Genome Consortium (ICGC).Then a nomogram was determined. Furthermore, we also examined the relationship of the risk model and clinical characteristics as well as immune microenvironment. 434 DEOSGs, comprising 62 downregulated and 372 upregulated genes (p < 0.05 and |log2FC| ≥ 1), and 257 HCC-related hub genes were recognized in HCC. Afterward, we built a five-DEOSG (LOX, CYP2C9, EIF2B4, EZH2, and SRXN1) prognostic risk model. Using the nomogram, the risk model was shown to have good prognostic value. Compared to the low risk group, HCC patients with high risk had poorer outcomes, worse pathological grades, and advanced tumor stages (p < 0.05). There were significant increases in LOX, EIF2B4, EZH2, and SRXN1 expression in HCC samples, while CYP2C9 expression was decreased. Finally, Real-time PCR (RT-qPCR) confirmed the mRNA expressions of five genes (CYP2C9, EIF2B4, EZH2, SRXN1, LOX) in HCC cell lines. Our study constructed a prognostic OS-related model with strong predictive power and potential as an immunosuppressive biomarker for HCC leading to improving prediction and providing new insights for HCC immunotherapy.

Single-cell analysis reveals lysyl oxidase (Lox)+ fibroblast subset involved in cardiac fibrosis of diabetic mice

IntroductionMyocardial fibrosis and cardiac dysfunction are the main characteristics of diabetic heart disease. However, the molecular mechanisms underlying diabetic myocardial fibrosis remain unclear. ObjectivesThis study aimed to investigate the heterogeneity of cardiac fibroblasts in diabetic mice and its possible mechanism in the development of diabetic myocardial fibrosis. MethodsWe established a diabetic mouse model by injecting mice with streptozotocin. The overall cell profiles in diabetic hearts were analyzed using single-cell RNA transcriptomic techniques. Cardiac function was evaluated by echocardiography. Cardiac fibrosis was assessed by Masson’s trichrome and Sirius red staining. Protein expression was analyzed using Western blotting and immunofluorescence staining. ResultsA total of 11,585 cells were captured in control (Ctrl) and diabetic (DM) hearts. Twelve cell types were identified in this study. The number of fibroblasts was significantly higher in the DM hearts than in the Ctrl group. The fibroblasts were further re-clustered into nine subsets. Interestingly, cluster 4 fibroblasts were significantly increased in diabetic hearts compared with other fibroblast clusters. Lysyl oxidase (Lox) was highly expressed in DM fibroblasts (especially in cluster 4). Beta-aminopropionitrile, a Lox inhibitor, inhibited collagen expression and alleviated cardiac dysfunction in the diabetic group. Lysyl oxidase inhibition also reduced high glucose-induced collagen protein upregulation in primary fibroblasts. Moreover, a TGF-β receptor inhibitor not only prevented an increase in Lox and Col I but also inhibited the phosphorylation of Smad2/3 in fibroblasts. ConclusionsThis study revealed the heterogeneity of cardiac fibroblasts in diabetic mice for the first time. Fibroblasts with high expression of Lox (cluster 4 fibroblasts) were identified to play a crucial role in fibrosis in diabetic heart disease. The findings of this study may provide a possible therapeutic target for interstitial fibrosis.