Metabolic Activation of Flavin Monooxygenase-mediated Trimethylamine-N-Oxide Formation in Experimental Kidney Disease

Abstract Cardiovascular disease (CVD) remains the leading cause of death in chronic kidney disease (CKD) patients despite treatment of traditional risk factors, suggesting that non-traditional CVD risk factors are involved. Trimethylamine-N-oxide (TMAO) correlates with atherosclerosis burden in CKD patients and may be a non-traditional CVD risk factor. Serum TMAO concentrations are significantly increased in CKD patients, which may be due in part to increased hepatic flavin monooxygenase (FMO)-mediated TMAO formation. The objective of this work was to elucidate the mechanism of increased FMO activity in CKD. In this study, FMO enzyme activity experiments were conducted in vitro with liver microsomes isolated from experimental CKD and control rats. Trimethylamine was used as a probe substrate to assess FMO activity. The FMO activator octylamine and human uremic serum were evaluated. FMO gene and protein expression were also determined. FMO-mediated TMAO formation was increased in CKD versus control. Although gene and protein expression of FMO were not changed, metabolic activation elicited by octylamine and human uremic serum increased FMO-mediated TMAO formation. The findings suggest that metabolic activation of FMO-mediated TMAO formation is a novel mechanism that contributes to increased TMAO formation in CKD and represents a therapeutic target to reduce TMAO exposure and CVD.

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Uremia Impacts VE-Cadherin and ZO-1 Expression in Human Endothelial Cell-to-Cell Junctions

Toxins ◽

10.3390/toxins10100404 ◽

2018 ◽

Vol 10 (10) ◽

pp. 404 ◽

Cited By ~ 8

Author(s):

Rayana Maciel ◽

Regiane Cunha ◽

Valentina Busato ◽

Célia Franco ◽

Paulo Gregório ◽

...

Keyword(s):

Protein Expression ◽

Intercellular Junctions ◽

Serum Levels ◽

Endothelial Damage ◽

Cell Junctions ◽

Cell Junction ◽

Uremic Serum ◽

Gene And Protein Expression ◽

The Impact

Endothelial dysfunction in uremia can result in cell-to-cell junction loss and increased permeability, contributing to cardiovascular diseases (CVD) development. This study evaluated the impact of the uremic milieu on endothelial morphology and cell junction’s proteins. We evaluated (i) serum levels of inflammatory biomarkers in a cohort of chronic kidney disease (CKD) patients and the expression of VE-cadherin and Zonula Occludens-1 (ZO-1) junction proteins on endothelial cells (ECs) of arteries removed from CKD patients during renal transplant; (ii) ECs morphology in vitro under different uremic conditions, and (iii) the impact of uremic toxins p-cresyl sulfate (PCS), indoxyl sulfate (IS), and inorganic phosphate (Pi) as well as of total uremic serum on VE-cadherin and ZO-1 gene and protein expression in cultured ECs. We found that the uremic arteries had lost their intact and continuous endothelial morphology, with a reduction in VE-cadherin and ZO-1 expression. In cultured ECs, both VE-cadherin and ZO-1 protein expression decreased, mainly after exposure to Pi and uremic serum groups. VE-cadherin mRNA expression was reduced while ZO-1 was increased after exposure to PCS, IS, Pi, and uremic serum. Our findings show that uremia alters cell-to-cell junctions leading to an increased endothelial damage. This gives a new perspective regarding the pathophysiological role of uremia in intercellular junctions and opens new avenues to improve cardiovascular outcomes in CKD patients.

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Identification of pluripotent cells in bovine uterus: in situ and in vitro studies

Reproduction ◽

10.1530/rep-14-0348 ◽

2015 ◽

Vol 149 (4) ◽

pp. 317-327 ◽

Cited By ~ 14

Author(s):

Martyna Łupicka ◽

Gabriel Bodek ◽

Nahum Shpigel ◽

Ehud Elnekave ◽

Anna J Korzekwa

Keyword(s):

Flow Cytometry ◽

Protein Expression ◽

Mrna Expression ◽

Uterine Horn ◽

Uterine Tissue ◽

Pluripotent Cells ◽

Flow Cytometry Assay ◽

Myometrial Cells ◽

Gene And Protein Expression

The aim of this study was to identify uterine pluripotent cells both in bovine uterine tissues as well in epithelial, stromal, and myometrial uterine cell populations. Moreover, the relationship of pluripotent markers expression with age and the uterine horn side was considered. Uterine tissue was collected from ipsilateral and contralateral horns (days 8–10 of the estrous cycle). Immunohistostaining for C-KIT, OCT3/4, NANOG, and SOX2 in uterine tissue was determined. mRNA expression of C-KIT, OCT3/4, NANOG and SOX2 was evaluated in uterine tissue relative to the age of the cow and uterine horn side. Gene and protein expression of these markers in the uterine luminal epithelial, stromal, and myometrial cells was evaluated by real-time PCR and western blotting respectively. The expression of pluripotent cell markers OCT3/4, NANOG, and SOX2 was identified by flow cytometry assay in epithelial, stromal, and myometrial cells. Multilineage differentiation of the bovine uterine cells was performed. mRNA expression of OCT3/4, NANOG, and SOX2 in uterine tissue was higher in the ipsilateral horn than in the contralateral horn. Flow cytometry assay revealed positive fluorescence for OCT3/4, NANOG, and SOX2 in all uterine cell types. Results showed the age-dependent expression of pluripotent markers in uterine tissue. Beside, the different expression of pluripotent cells in each horn of uterus suggests the influence of ovarian hormones on these characteristics. The highest mRNA and protein expression for pluripotent markers was observed in stromal cells among uterine cells, which indicates this population of cells as the main site of pluripotent cells in the cow uterus.

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Evidence of Autophagy Gene and Protein Expression Response to Heat and Oxidative Stress in Avian Muscle Cells: In Vivo and in Vitro Studies.

Free Radical Biology and Medicine ◽

10.1016/j.freeradbiomed.2014.10.352 ◽

2014 ◽

Vol 76 ◽

pp. S72

Author(s):

Sami Dridi ◽

Alissa Piekarski ◽

Kentu Lassiter ◽

Elizabeth Greene ◽

Nick Anthony ◽

...

Keyword(s):

Oxidative Stress ◽

Protein Expression ◽

Muscle Cells ◽

Autophagy Gene ◽

Avian Muscle ◽

Gene And Protein Expression ◽

Expression Response ◽

And Oxidative Stress

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Cell signaling pathways in human mutant PAX6 corneal cells: an in vitro model for aniridia-related keratopathy

10.1101/2021.03.19.436143 ◽

2021 ◽

Author(s):

Marta Słoniecka ◽

André Vicente ◽

Berit Byström ◽

Fátima Pedrosa Domellöf

Keyword(s):

Protein Expression ◽

Signaling Pathways ◽

Cell Fate ◽

Expression Patterns ◽

Pax6 Gene ◽

Cas9 Nuclease ◽

Extracellular Matrix Components ◽

Gene And Protein Expression ◽

Human Keratocytes

ABSTRACTPURPOSETo establish an in vitro model of aniridia-related keratopathy (ARK) using CRISPR/Cas9 engineered human keratocytes with mutations in the PAX6 gene, and to study the Notch Homolog 1, Translocation-Associated (Notch1), sonic hedgehog (SHH), mammalian target of rapamycin (mTOR), and Wnt/β-catenin signaling pathways in the PAX6 mutant keratocytes.METHODSPrimary human keratocytes were isolated from healthy corneas. Keratocytes were transduced with Cas9 lentiviral particles in order to create cells stably expressing Cas9 nuclease. Lentiviral particles carrying PAX6 sgRNA were transduced into the Cas9 keratocytes creating mutants. Analysis of signaling pathways was assessed by RT-qPCR for gene expression and western blot for protein expression.RESULTSHuman keratocytes stably expressing Cas9 nuclease were created. Keratocytes carrying PAX6 gene mutation were successfully generated. PAX6 mutant keratocytes showed modified expression patterns of extracellular matrix components such as collagens and fibrotic markers. Analysis of the Notch1, SHH, mTOR, and Wnt/β-catenin signaling pathways in the PAX6 mutant keratocytes revealed altered gene and protein expression of the key players involved in these pathways.CONCLUSIONSA properly functioning PAX6 gene in keratocytes is crucial for the regulation of signaling pathways important for cell fate determination, proliferation, and inflammation. Pax6 mutation in the in vitro settings leads to changes in these pathways which resemble those found in corneas of patients with ARK.

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Effect of Hesperidin on Cardiovascular Disease Risk Factors: The Role of Intestinal Microbiota on Hesperidin Bioavailability

Nutrients ◽

10.3390/nu12051488 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1488 ◽

Cited By ~ 3

Author(s):

Anna Mas-Capdevila ◽

Joan Teichenne ◽

Cristina Domenech-Coca ◽

Antoni Caimari ◽

Josep M Del Bas ◽

...

Keyword(s):

Risk Factors ◽

Gut Microbiota ◽

Disease Risk ◽

Cardiovascular Disease Risk ◽

Current Evidence ◽

Cvd Risk Factors ◽

Cvd Risk ◽

Beneficial Effects ◽

High Interindividual Variability

Recently, hesperidin, a flavonone mainly present in citrus fruits, has emerged as a new potential therapeutic agent able to modulate several cardiovascular diseases (CVDs) risk factors. Animal and in vitro studies demonstrate beneficial effects of hesperidin and its derived compounds on CVD risk factors. Thus, hesperidin has shown glucose-lowering and anti-inflammatory properties in diabetic models, dyslipidemia-, atherosclerosis-, and obesity-preventing effects in CVDs and obese models, and antihypertensive and antioxidant effects in hypertensive models. However, there is still controversy about whether hesperidin could contribute to ameliorate glucose homeostasis, lipid profile, adiposity, and blood pressure in humans, as evidenced by several clinical trials reporting no effects of treatments with this flavanone or with orange juice on these cardiovascular parameters. In this review, we focus on hesperidin’s beneficial effects on CVD risk factors, paying special attention to the high interindividual variability in response to hesperidin-based acute and chronic interventions, which can be partly attributed to differences in gut microbiota. Based on the current evidence, we suggest that some of hesperidin’s contradictory effects in human trials are partly due to the interindividual hesperidin variability in its bioavailability, which in turn is highly dependent on the α-rhamnosidase activity and gut microbiota composition.

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Calciprotein particle inhibition explains magnesium-mediated protection against vascular calcification

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfz190 ◽

2019 ◽

Vol 35 (5) ◽

pp. 765-773 ◽

Cited By ~ 6

Author(s):

Anique D ter Braake ◽

Coby Eelderink ◽

Lara W Zeper ◽

Andreas Pasch ◽

Stephan J L Bakker ◽

...

Keyword(s):

Electron Microscopy ◽

Chronic Kidney Disease ◽

Smooth Muscle ◽

Cardiovascular Risk ◽

Kidney Disease ◽

Vascular Calcification ◽

Healthy Controls ◽

Gene And Protein Expression ◽

Calciprotein Particles

Abstract Background Phosphate (Pi) toxicity is a strong determinant of vascular calcification development in chronic kidney disease (CKD). Magnesium (Mg2+) may improve cardiovascular risk via vascular calcification. The mechanism by which Mg2+ counteracts vascular calcification remains incompletely described. Here we investigated the effects of Mg2+ on Pi and secondary crystalline calciprotein particles (CPP2)-induced calcification and crystal maturation. Methods Vascular smooth muscle cells (VSMCs) were treated with high Pi or CPP2 and supplemented with Mg2+ to study cellular calcification. The effect of Mg2+ on CPP maturation, morphology and composition was studied by medium absorbance, electron microscopy and energy dispersive spectroscopy. To translate our findings to CKD patients, the effects of Mg2+ on calcification propensity (T50) were measured in sera from CKD patients and healthy controls. Results Mg2+ supplementation prevented Pi-induced calcification in VSMCs. Mg2+ dose-dependently delayed the maturation of primary CPP1 to CPP2 in vitro. Mg2+ did not prevent calcification and associated gene and protein expression when added to already formed CPP2. Confirmatory experiments in human serum demonstrated that the addition of 0.2 mmol/L Mg2+ increased T50 from healthy controls by 51 ± 15 min (P < 0.05) and CKD patients by 44 ± 13 min (P < 0.05). Each further 0.2 mmol/L addition of Mg2+ led to further increases in both groups. Conclusions Our results demonstrate that crystalline CPP2 mediates Pi-induced calcification in VSMCs. In vitro, Mg2+ delays crystalline CPP2 formation and thereby prevents Pi-induced calcification.

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Adiponectin Expression in the Porcine Ovary during the Oestrous Cycle and Its Effect on Ovarian Steroidogenesis

International Journal of Endocrinology ◽

10.1155/2014/957076 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 28

Author(s):

Anna Maleszka ◽

Nina Smolinska ◽

Anna Nitkiewicz ◽

Marta Kiezun ◽

Katarzyna Chojnowska ◽

...

Keyword(s):

Protein Expression ◽

Energy Homeostasis ◽

Oestrous Cycle ◽

Adiponectin Receptors ◽

Basal Secretion ◽

Ovarian Steroidogenesis ◽

Gene And Protein Expression ◽

Adiponectin Mrna ◽

Theca Interna

Adiponectin is an adipose-secreted hormone that regulates energy homeostasis and is also involved in the control of the reproductive system. The goal of the present study was to investigate changes in adiponectin gene and protein expression in porcine ovarian structures during the oestrous cycle and to examine the effects ofin vitroadministration of adiponectin on basal and gonadotrophin- and/or insulin-induced secretion of ovarian steroid hormones. Both gene and protein expression of adiponectin were enhanced during the luteal phase of the cycle. Adiponectin affected basal secretion of progesterone by luteal cells, oestradiol by granulosa cells, and testosterone by theca interna cells. The gonadotrophin/insulin-induced release of progesterone from granulosa and theca interna cells and the release of oestradiol and androstenedione from theca cells was also modified by adiponectin. In conclusion, the presence of adiponectin mRNA and protein in the porcine ovary coupled with our previous results indicating adiponectin receptors expression suggest that adiponectin may locally affect ovarian functions. The changes in adiponectin expression throughout the oestrous cycle seem to be dependent on the hormonal status of pigs related to the stage of the oestrous cycle. The effect of adiponectin on ovarian steroidogenesis suggests that this adipokine influences reproductive functions in pigs.

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Wastewaters of meat-processing enterprise: assessment of genotoxic potential

MATEC Web of Conferences ◽

10.1051/matecconf/201824518002 ◽

2018 ◽

Vol 245 ◽

pp. 18002 ◽

Cited By ~ 4

Author(s):

Olga Ivanchenko ◽

Rustem Khabibullin ◽

Rahat Bhat

Keyword(s):

Waste Water ◽

Ames Test ◽

Excision Repair ◽

Mutagenic Activity ◽

Liver Microsomes ◽

Metabolic Activation ◽

Rat Liver Microsomes ◽

Meat Processing ◽

Processing Enterprise

Environmental pollution and ecosystem dysfunction are one of the most important problems of the today’s world. Assessment of toxigenic properties of effluents from the meat-processing enterprise was carried out using the short-term microorganisms biotests in vitro. Both native waste water and its ether and water fractions were investigated. The probes’ sterilization was carried out by filtration through the sterile membrane filters Synpor with pores diameter of 0.45 m. Mutagenic activity of wastewaters was determined using the Salmonella/microsomes plate with in vitro metabolic activation and without metabolic activation (Ames test). As a metobolic activation the rat liver microsomes were used. Studying of the DNA-damaging activity was carried out using the suspension method modification on the mutant Escherichia coli strains, in which the functioning of one reparation systems is suppressed: uvrA-, recAand рol A-. Native waste water doesn’t have an influence on the mutant strains recAand рol A-, its survivability degree is in the range 96-100%. However, DNA-damaging action was registered for the strain with the damaged excision repair (uvrA-), survivability of which was 81.31%. Ames test of wastewater and its fractions didn’t reveal any mutagenic activity. The tests used in this work allow one to comprehensively estimate the genetic danger of the enterprise wastewaters within a short time and are recommended as test-systems for monitoring the ecological safety of wastewaters.

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The Histone Deacetylase (HDAC) Inhibitor Entinostat (SNDX-275) Targets Hodgkin Lymphoma through a Dual Mechanism of Immune Modulation and Apoptosis Induction.

Blood ◽

10.1182/blood.v114.22.1562.1562 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1562-1562 ◽

Cited By ~ 3

Author(s):

Noor M Khaskhely ◽

Daniela Buglio ◽

Jessica Shafer ◽

Catherine M. Bollard ◽

Anas Younes

Keyword(s):

Immune Response ◽

Cell Death ◽

Protein Expression ◽

Cell Lines ◽

Dependent Manner ◽

Chemokine Production ◽

Apoptosis Pathway ◽

Gene And Protein Expression ◽

Combination Studies

Abstract Abstract 1562 Poster Board I-585 Purpose SNDX-275 is an oral, class 1 isoform selective HDACi. Phase 1 studies in leukemia demonstrated the agent has a long half-life and that weekly or every other week dosing is sufficient for antitumor activity. Based on recent favorable in vitro and in vivo activity of several HDAC inhibitors in HL, we investigated the in vitro activity of SNDX275 in HL-derived cell lines. Methods For apoptosis and gene expression analysis 05 × 106 cells were incubated with 0.1-2 μM of SNDX-275 for 24-72 hours before they were examined for proliferation and cell death by the MTS assay and the annexin-PI and FACS analysis. For combination studies, cells were incubated with 0.1-2 uM of SNDX-275 and 1-20 nM of either gemcitabine or bortezomib for 48-72 hours. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. SNDX-275 effects on a panel of 30 cytokines and chemokines was assayed on 05 × 106 cells after incubation of 48 hrs using a multiplex assay. Results SNDX-275 induced cell death in a dose and time dependent manner with an IC50 of 0.4 μM. At the molecular level, SNDX-275 increased H3 acetylation, up-regulated p21 protein expression, and activated the intrinsic apoptosis pathway by down-regulating the anti-apoptotic X-linked inhibitor or apoptosis (XIAP) protein, which was associated with activation of caspase 9 and 3. Combination studies demonstrated that SNDX-275 had synergistic effects when combined with gemcitabine and bortezomib. To further investigate the potential for SNDX-275 activity in HL we measured the effect of SNDX-275 on pathways that may contribute to an anti-tumor immune response. Dysregulated cytokine/chemokine production has been shown to contribute to HL pathology, including immune tolerance of the cancer cells. SNDX-275 increased IL12 p40-70, IP10, and RANTES, and decreased the level of IL13 and IL4, thus favoring Th1-type cytokines/chemokines. In addition, recent data has demonstrated that a variety of epigenetic-modulating drugs may up-regulate the expression of cancer testis tumor associated antigens, leading to a favorable immune response. None of the lines expressed the CTAs without induction. SNDX275 was able to induce CTA expression of SSX2 in L428 but not HDLM2 whereas MAGE-A was induced in both HL cell lines. NY-ESO expression was not induced. Conclusions Our studies demonstrate that SNDS-275 has dual effect on apoptotic and immunomodulatory pathways in HL. Furthermore, this data demonstrates that SNDX-275 may upregulate CTAs suggesting that this treatment may render the tumor more immunogeneic and susceptible to immune mediated killing with tumor-specific cytotoxic T lymphocytes. The selectivity profile of SNDX-275 also suggests that HDAC1 and 2 are the primary targets for HDAC inhibition in these cells. Phase 2 studies with SNDX-275 in HL are ongoing. Disclosures Younes: MethylGene: Honoraria, Research Funding.

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