Omidenepag, a non-prostanoid EP2 receptor agonist, induces enlargement of the 3D organoid of 3T3-L1 cells
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SUBMITTER: Ida Y
PROVIDER: S-EPMC7524797 | biostudies-literature | 2020 Jan
REPOSITORIES: biostudies-literature
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Project description:PurposeThe purpose of this study was to present the effects of the prostanoid EP2 agonist, omidenepag (OMD) on human orbital fibroblasts (HOFs) using a three-dimension (3D) cell culture.MethodsDuring adipogenesis of 3D HOFs organoids, changes in size, lipids staining, mRNA expression of adipogenesis related genes, PPARγ, AP2, and ADIPOQ, and extracellular matrix, collagen 1 (COL 1), COL 4, COL 6, and fibronectin (FN), and stiffness by a micro-squeezer were examined in the presence and absence of either 100 nM bimatoprost acid (BIM-A) or 10, 100, or 10,000 nM OMD.ResultsThe size of the 3D organoids increased dramatically during adipogenesis, and these were further enhanced in the presence of OMD in contrast to the BIM-A induced suppression effect. The intensity of lipid staining and the mRNA expression of PPARγ were significantly increased upon adipogenesis, and both or latter was markedly inhibited in the presence of OMD or BIM-A, respectively. AP2 expression was also upregulated by adipogenesis, and was further enhanced by BIM-A. The adipogenesis-induced downregulation of COL 1 and FN, or the upregulation of the expression of COL 4 and COL 6 were all suppressed in the presence of BIM-A. In contrast, OMD caused similar effects on COL 4, COL 6, or FN expression, but caused a significant increase in COL 1 expression. Stiffness was significantly increased upon adipogenesis, and was further increased or substantially decreased by BIM-A or OMD, respectively.ConclusionsThe present study indicates that the FP2 agonist, OMD, had different effects on 3D HOFs organoids, as compared to BIM-A.Translational relevanceThe current study suggests that OMD may not induce deepening of upper eyelid sulcus (DUES).
| S-EPMC8039573 | biostudies-literature
Project description:BackgroundTo obtain new insights into the activation of the thyroid-stimulating hormone (TSH) and insulin-like growth factor 1 (IGF-1) receptors in human orbital fibroblasts (n-HOFs), the effects of the prostanoid EP2 agonist, omidenepag (OMD), and a rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) were evaluated using three-dimension (3D) n-HOFs spheroids in the absence and presence of the recombinant human TSH receptor antibodies, M22 and IGF-1.MethodsThe effects of 100 nM OMD or 10 μM Rip on the physical properties, size, stiffness, and mRNA expression of several extracellular matrix (ECM) molecules, their regulator, inflammatory cytokines, and endoplasmic reticulum (ER) stress-related factors were examined and compared among 3D spheroids of n-HOFs, M22-/IGF-1-activated n-HOFs and GO-related human orbital fibroblasts (GHOFs).ResultsThe physical properties and mRNA expressions of several genes of the 3D n-HOFs spheroids were significantly and diversely modulated by the presence of OMD or Rip. The OMD-induced effects on M22-/IGF-1-activated n-HOFs were similar to the effects caused by GHOHs, but quite different from those of n-HOFs.ConclusionsThe findings presented herein indicate that the changes induced by OMD may be useful in distinguishing between n-HOFs and GHOFs.
| S-EPMC8622545 | biostudies-literature
Project description:To elucidate the effects of switching a PGF2α agonist, bimatoprost acid (BIM-A), to an EP2 agonist (Omidenepag-OMD; butaprost-Buta) or reversing the switching on adipose tissue, two-dimensional (2D) and three-dimensional (3D) cultures of 3T3-L1 cells were analyzed by lipid staining and according to the mRNA expression of adipogenesis-related genes (Pparγ, Ap2, and Leptin), components of the extracellular matrix (ECM; collagen1 (Col1), Col4, Col6, and fibronectin (Fn)), and the sizes and stiffness of the 3D spheroids. Switching from BIM-A to EP2 agonists caused (1) suppression of lipid staining and downregulation of most adipogenesis-related genes, (2) smaller and stiffer 3D spheroids, and (3) upregulation of Col1 and Fn, downregulation of Col4 (2D), or up-regulation of all ECM genes (3D, BIM-A to OMD), as well as downregulation of Col6 (3D, BIM-A to Buta). In contrast, reversing the switching resulted in (1) an enhancement in lipid staining (2D) and a significant upregulation of adipogenesis-related genes (2D, 3D Buta to BIM-A), (2) larger and slightly stiffer 3D spheroids, and (3) upregulation of Col1 and Fn (2D). These collective findings indicate that the switching orders of BIM-A and EP2 agonists have a significant effect on lipid metabolism, ECM expression, and the physical stiffness of 3T3-L1 cells.
| S-EPMC8698783 | biostudies-literature
Project description:This study investigated the effects of omidenepag (OMD), a novel selective EP2 receptor agonist, on human trabecular meshwork (HTM) cells, monkey Schlemm's canal endothelial (SCE) cells, and porcine ciliary muscle (CM) to clarify the mechanism of intraocular pressure (IOP) reduction involving conventional outflow pathway. In HTM and SCE cells, the effects of OMD on transforming growth factor-β2 (TGF-β2)-induced changes were examined. The expression of actin cytoskeleton and extracellular matrix (ECM) proteins, myosin light chain (MLC) phosphorylation in HTM cells were evaluated using real-time quantitative PCR, immunocytochemistry, and western blotting. The expression of barrier-related proteins, ZO-1 and β-catenin, and permeability of SCE cells were evaluated using immunocytochemistry and transendothelial electrical resistance. The CM contraction was determined by contractibility assay. OMD significantly inhibited expression of TGF-β2 induced mRNA, protein, and MLC-phosphorylation on cytoskeletal and ECM remodeling in the HTM dose dependently. In SCE cells, OMD suppressed TGF-β2-induced expression of the barrier-related proteins and decreased SCE monolayer permeability. OMD at 3 µM significantly inhibited CM contraction, however, the effect was not significant at lower concentrations. IOP lowering effect of OMD through conventional outflow pathway is exerted by increasing outflow facilities with the modulation of TM cell fibrosis and SCE cell permeability.
| S-EPMC8355290 | biostudies-literature
Project description:To elucidate the additive effects of an EP2 agonist, omidenepag (OMD) or butaprost (Buta) on the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) on adipose tissue, two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells were analyzed by lipid staining, the mRNA expression of adipogenesis-related genes, extracellular matrix (ECM) molecules including collagen (Col) -1, -4 and -6, and fibronectin (Fn), and the sizes and physical properties of 3D organoids, as measured by a micro-squeezer. The results indicate that adipogenesis induced (1) an enlargement of the 3D organoids; (2) a substantial enhancement in lipid staining as well as the expression of the Pparγ, Ap2 and Leptin genes; (3) a significant softening of the 3D organoids, the effects of which were all enhanced by Rip except for Pparγ expression; and (4) a significant downregulation in Col1 and Fn, and a significant upregulation in Col4, Col6, the effects of which were unchanged by Rip. When adding the EP2 agonist to Rip, (1) the sizes of the 3D organoids were reduced substantially; (2) lipid staining was increased (OMD), or decreased (Buta); (3) the stiffness of the 3D organoids was substantially increased in Buta; (4-1) the expression of Pparγ was suppressed (2D, OMD) or increased (2D, Buta), and the expressions of Ap2 were downregulated (2D, 3D) and Leptin was increased (2D) or decreased (3D), (4-2) all the expressions of four ECM molecules were upregulated in 2D (2D), and in 3D, the expression of Col1, Col4 was upregulated. The collective findings reported herein indicate that the addition of an EP2 agonist, OMD or Buta significantly but differently modulate the Rip-induced effects on adipogenesis and the physical properties of 2D and 3D cultured 3T3-L1 cells.
| S-EPMC8125646 | biostudies-literature
Project description:BACKGROUND AND PURPOSE:Prostanoid EP2 receptor agonists exhibit several activities including ocular hypotension, tocolysis and anti-inflammatory activity. This report describes the affinity and selectivity of a structurally novel, non-prostanoid EP2 receptor agonist, PGN-9856, and its therapeutic potential. EXPERIMENTAL APPROACH:The pharmacology of a series of non-prostanoid EP2 receptor agonists was determined according to functional and radioligand binding studies, mostly using human recombinant prostanoid receptor transfectants. The selectivity of PGN-9856, as the preferred compound, was subsequently determined by using a diverse variety of non-prostanoid target proteins. The therapeutic potential of PGN-9856 was addressed by determining its activity in relevant primate cell, tissue and disease models. KEY RESULTS:PGN-9856 was a selective and high affinity (pKi ? 8.3) ligand at human recombinant EP2 receptors. In addition to high affinity binding, it was a potent and full EP2 receptor agonist with a high level of selectivity at EP1 , EP3 , EP4 , DP, FP, IP and TP receptors. In cells overexpressing human recombinant EP2 receptors, PGN-9856 displayed a potency (pEC50 ? 8.5) and a maximal response (increase in cAMP) comparable to that of the endogenous agonist PGE2 . PGN-9856 exhibited no appreciable affinity (up 10 ?M) for a range of 53 other receptors, ion channels and enzymes. Finally, PGN-9856 exhibited tocolytic, anti-inflammatory and long-acting ocular hypotensive properties consistent with its potent EP2 receptor agonist properties. CONCLUSIONS AND IMPLICATIONS:PGN-9856 is a potent, selective and efficacious prostanoid EP2 receptor agonist with diverse potential therapeutic applications: tocolytic, anti-inflammatory and notably anti-glaucoma.
| S-EPMC6365485 | biostudies-literature
Project description:ObjectivesWe evaluated early medication persistence with new topical antiglaucoma eyedrops, omidenepag isopropyl 0.002% (a selective prostaglandin EP2 agonist).Design and settingRetrospective two-institute study in Himeji and Akashi in Japan.ParticipantsWe analysed patients with glaucoma who were prescribed topical omidenepag isopropyl from November 2018 to December 2019. From the last outpatient visit of patients until February 2020, 235 patients were prescribed a new solution of omidenepag isopropyl (129 patients in the initial monotherapy group, 85 in the switching group (switched from another topical antiglaucoma eyedrops), 19 added to another topical antiglaucoma eyedrops group, and 2 were lost to follow-up)). Additionally, we recruited 98 patients (3 were lost to follow-up) who received initial latanoprost 0.005% monotherapy during the same period as a control group.OutcomesMedication persistence failure was defined as drug discontinuation due to any adverse effects or change of therapy. Kaplan-Meier survival analysis was performed with a Cox regression analysis.ResultsAmong 233 patients, 48 (20%) showed failure of treatment; the median persistence time of all patients was 165 days, and the median time until discontinuation of omidenepag isopropyl was 45 days. The total persistence rates were 85%, 80% and 70% at 3, 6 and 12 months, respectively. Risk factors for failure were male gender (HR: 1.45, p=0.023) and monotherapy/switching (HR: 1.715, p=0.002). Comparison between latanoprost and omidenepag isopropyl monotherapy, only male gender (HR: 1.43, p=0.016) was a significant risk factor. Failures associated with omidenepag isopropyl were due to insufficient intraocular pressure-lowering efficiency (n=26, observed during all the period), followed by conjunctival hyperaemia (n=10) and visual acuity disturbance (n=5) in patients who were observed until 3 months.ConclusionMedication persistence with omidenepag isopropyl is mostly positive; however, clinicians should also be cautious of early failure.
| S-EPMC7849894 | biostudies-literature
Project description:Exposure to endocrine disrupting chemicals is an established risk factor for obesity. The most commonly used pesticide active ingredients have never been tested in an adipogenesis assay. We tested for the first time the potential of glyphosate, 2, 4-dichlorophenoxyacetic acid, dicamba, mesotrione, isoxaflutole, and quizalofop-p-ethyl (QpE) to induce lipid accumulation in murine 3T3-L1 adipocytes. Only QpE caused a dose-dependent statistically significant triglyceride accumulation from a concentration of 5 up to 100 µM. The QpE commercial formulation Targa Super was 100 times more cytotoxic than QpE alone. Neither the estrogen receptor antagonist ICI 182, 780 nor the glucocorticoid receptor antagonist RU486 was able to block the QpE-induced lipid accumulation. RNAseq analysis of 3T3-L1 adipocytes exposed to QpE suggests that this compound exerts its lipid accumulation effects via a peroxisome proliferator-activated receptor gamma (PPARγ)-mediated pathway, a nuclear receptor whose modulation influences lipid metabolism. QpE was further shown to be active in a PPARγ reporter gene assay at 100 µM, reaching 4% of the maximal response produced by rosiglitazone, which acts as a positive control. This indicates that lipid accumulation induced by QpE is only in part caused by PPARγ activation. The lipid accumulation capability of QpE we observe suggest that this pesticide, whose use is likely to increase in coming years may have a hitherto unsuspected obesogenic property.
| S-EPMC6657571 | biostudies-literature
Project description:Brown adipose tissue (BAT) activity plays a key role in regulating systemic energy. The activation of BAT results in increased energy expenditure, making this tissue an attractive pharmacological target for therapies against obesity and type 2 diabetes. Sirtuin 5 (SIRT5) affects BAT function by regulating adipogenic transcription factor expression and mitochondrial respiration. We analyzed the expression of SIRT5 in the different adipose depots of mice. We treated 3T3-L1 preadipocytes and mouse primary preadipocyte cultures with the SIRT5 inhibitor MC3482 and investigated the effects of this compound on adipose differentiation and function. The administration of MC3482 during the early stages of differentiation promoted the expression of brown adipocyte and mitochondrial biogenesis markers. Upon treatment with MC3482, 3T3-L1 adipocytes showed an increased activation of the AMP-activated protein kinase (AMPK), which is known to stimulate brown adipocyte differentiation. This effect was paralleled by an increase in autophagic/mitophagic flux and a reduction in lipid droplet size, mediated by a higher lipolytic rate. Of note, MC3482 increased the expression and the activity of adipose triglyceride lipase, without modulating hormone-sensitive lipase. Our findings reveal that SIRT5 inhibition stimulates brown adipogenesis in vitro, supporting this approach as a strategy to stimulate BAT and counteract obesity.
| S-EPMC8148511 | biostudies-literature
Project description:BackgroundAquaporin-8 (AQP8), a member of the aquaporin water channel family, is expressed in various tissue and cells, including liver, testis, and pancreas. AQP8 appears to have functions on the plasma membrane and/or on the mitochondrial inner membrane. Mitochondrial AQP8 with permeability for water, H2O2 and NH3 has been expected to have important role in various cells, but its information is limited to a few tissues and cells including liver and kidney. In the present study, we found that AQP8 was expressed in the mitochondria in mouse adipose tissues and 3T3-L1 preadipocytes, and investigated its role by suppressing its gene expression.MethodsAQP8-knocked down (shAQP8) cells were established using a vector expressing short hairpin RNA. Cellular localization of AQP8 was examined by western blotting and immunocytochemistry. Mitochondrial function was assessed by measuring mitochondrial membrane potential, oxygen consumption and ATP level measurements.ResultsIn 3T3-L1 cells, AQP8 was expressed in the mitochondria. In shAQP8 cells, mRNA and protein levels of AQP8 were decreased by about 75%. The shAQP8 showed reduced activities of complex IV and ATP synthase; it is probable that the impaired mitochondrial water handling in shAQP8 caused suppression of the electron transport and ADP phosphorylation through inhibition of the two steps which yield water. The reduced activities of the last two steps of oxidative phosphorylation in shAQP8 cause low routine and maximum capacity of respiration and mitochondrial hyperpolarization.ConclusionMitochondrial AQP8 contributes to mitochondrial respiratory function probably through maintenance of water homeostasis.General significanceThe AQP8-knocked down cells we established provides a model system for the studies on the relationships between water homeostasis and mitochondrial function.
| S-EPMC5668916 | biostudies-literature