Reactivities of a Prostanoid EP2 Agonist, Omidenepag, Are Useful for Distinguishing between 3D Spheroids of Human Orbital Fibroblasts without or with Graves’ Orbitopathy

Background. To 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. Methods. The 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). Results.The 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. Conclusions. The findings presented herein indicate that the changes induced by OMD may be useful in distinguishing between n-HOFs and GHOFs.

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Simultaneous Use of ROCK Inhibitors and EP2 Agonists Induces Unexpected Effects on Adipogenesis and the Physical Properties of 3T3-L1 Preadipocytes

International Journal of Molecular Sciences ◽

10.3390/ijms22094648 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4648

Author(s):

Yosuke Ida ◽

Megumi Watanabe ◽

Hiroshi Ohguro ◽

Fumihito Hikage

Keyword(s):

Extracellular Matrix ◽

Adipose Tissue ◽

Protein Kinase ◽

Physical Properties ◽

Coiled Coil ◽

Three Dimension ◽

Additive Effects ◽

Rock Inhibitor ◽

Pparγ Expression ◽

2D And 3D

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.

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ROCK inhibitors modulate the physical properties and adipogenesis of 3D spheroids of human orbital fibroblasts in different manners

FASEB BioAdvances ◽

10.1096/fba.2021-00037 ◽

2021 ◽

Author(s):

Fumihito Hikage ◽

Hanae Ichioka ◽

Megumi Watanabe ◽

Araya Umetsu ◽

Hiroshi Ohguro ◽

...

Keyword(s):

Physical Properties ◽

Orbital Fibroblasts ◽

3D Spheroids

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Targeting TSH and IGF-1 Receptors to Treat Thyroid Eye Disease

European Thyroid Journal ◽

10.1159/000511538 ◽

2020 ◽

Vol 9 (Suppl. 1) ◽

pp. 59-65

Author(s):

Susanne Neumann ◽

Christine C. Krieger ◽

Marvin C. Gershengorn

Keyword(s):

Thyroid Stimulating Hormone ◽

Thyroid Eye Disease ◽

Therapeutic Interventions ◽

Eye Disease ◽

Thyroid Cells ◽

Hormone Synthesis ◽

Dependent Component ◽

Graves Orbitopathy ◽

Blocking Antibodies ◽

Orbital Fibroblasts

Graves’ disease (GD) is an autoimmune disease caused in part by thyroid-stimulating antibodies (TSAbs) that activate the thyroid-stimulating hormone receptor (TSHR). In Graves’ hyperthyroidism (GH), TSAbs cause persistent stimulation of thyroid cells leading to continuous thyroid hormone synthesis and secretion. Thyroid eye disease (TED), also called Graves’ orbitopathy, is an orbital manifestation of GD. We review the important roles of the TSHR and the insulin-like growth factor 1 receptor (IGF-1R) in the pathogenesis of TED and discuss a model of TSHR/IGF-1R crosstalk that considers two pathways initiated by TSAb activation of TSHR in the eye, an IGF-1R-independent and an IGF-1R-dependent signaling pathway leading to hyaluronan (HA) secretion in orbital fibroblasts. We discuss current and future therapeutic approaches targeting the IGF-1R and TSHR. Teprotumumab, a human monoclonal anti-IGF-1R-blocking antibody, has been approved as an effective treatment in patients with TED. However, as the TSHR seems to be the primary target for TSAbs in patients with GD, future therapeutic interventions directly targeting the TSHR, e.g. blocking antibodies and small molecule antagonists, are being developed and have the advantage to inhibit the IGF-1R-independent as well as the IGF-1R-dependent component of TSAb-induced HA secretion. Antigen-specific immunotherapies using TSHR peptides to reduce serum TSHR antibodies are being developed also. These TSHR-targeted strategies also have the potential to treat both GH and TED with the same drug. We propose that combination therapy targeting TSHR and IGF-1R may be an effective and better tolerated treatment strategy for TED.

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Selenium rescues orbital fibroblasts from cell death induced by hydrogen peroxide: another molecular basis for the effects of selenium in graves’ orbitopathy

Endocrine ◽

10.1007/s12020-016-1226-9 ◽

2017 ◽

Vol 58 (2) ◽

pp. 386-389 ◽

Cited By ~ 3

Author(s):

Giovanna Rotondo Dottore ◽

Riccardo Chiarini ◽

Maria De Gregorio ◽

Marenza Leo ◽

Giamberto Casini ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Cell Death ◽

Molecular Basis ◽

Graves Orbitopathy ◽

Orbital Fibroblasts

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Erratum to: Selenium rescues orbital fibroblasts from cell death induced by hydrogen peroxide: another molecular basis for the effects of selenium in graves’ orbitopathy

Endocrine ◽

10.1007/s12020-017-1255-z ◽

2017 ◽

Vol 58 (2) ◽

pp. 390-390 ◽

Cited By ~ 1

Author(s):

Giovanna Rotondo Dottore ◽

Riccardo Chiarini ◽

Maria De Gregorio ◽

Marenza Leo ◽

Giamberto Casini ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Cell Death ◽

Molecular Basis ◽

Graves Orbitopathy ◽

Orbital Fibroblasts

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PERK mediates oxidative stress and adipogenesis in Graves’ orbitopathy pathogenesis

Journal of Molecular Endocrinology ◽

10.1530/jme-21-0057 ◽

2021 ◽

Author(s):

JaeSang Ko ◽

Ji-Young Kim ◽

Min Kyung Chae ◽

Eun Jig Lee ◽

Jin Sook Yoon

Keyword(s):

Oxidative Stress ◽

Hydrogen Peroxide ◽

Endoplasmic Reticulum ◽

Transcription Factor ◽

Er Stress ◽

Adipogenic Differentiation ◽

Ros Generation ◽

Mrna Levels ◽

Graves Orbitopathy ◽

Orbital Fibroblasts

We examined endoplasmic reticulum (ER) stress-related gene expression in orbital tissues from patients with Graves’ orbitopathy (GO) and the effects of silencing protein kinase RNA-like endoplasmic reticulum kinase (PERK) in primary orbital fibroblast cultures to demonstrate the therapeutic potential of PERK-modulating agents in GO management. The expression of ER stress related genes in orbital tissue harvested from individuals with or without GO was studied using real-time polymerase chain reaction. The role of PERK in GO pathogenesis was examined through small-interfering RNA (siRNA)-mediated silencing in cultured primary orbital fibroblasts. Intracellular reactive oxygen species (ROS) levels induced in response to cigarette smoke extract (CSE) or hydrogen peroxide were measured using 5-(and 6)-carboxy-20,70-dichlorodihydrofluorescein diacetate staining and flow cytometry. Cells were stained with Oil Red O, and adipogenesis-related transcription factor expression was evaluated through western blotting after adipogenic differentiation. PERK, activating transcription factor 4 (ATF4), and CCAAT-enhancer-binding protein (C/EBP)-homologous protein(CHOP)mRNA levels were significantly higher in GO orbital tissues than in non-GO orbital tissues. PERK silencing inhibited CSE- or hydrogen peroxide-induced ROS generation. After adipogenic differentiation, GO orbital fibroblasts revealed decreased lipid droplets and downregulation of C/EBPα, C/EBPβ, and peroxisome proliferator-activator gamma (PPARγ) in PERK siRNA-transfected cells. The orbital tissues of patients with GO were exposed to chronic ER stress and subsequently exhibited enhanced unfolded protein response (especially through the PERK pathway). PERK silencing reduced oxidative stress and adipogenesis in GO orbital fibroblasts in vitro. Our results imply that PERK-modulating agents can potentially be used to manage GO.

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Persistent disruption of lateral junctional complexes and actin cytoskeleton in parotid salivary glands following radiation treatment

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00388.2017 ◽

2018 ◽

Vol 315 (4) ◽

pp. R656-R667 ◽

Cited By ~ 4

Author(s):

Wen Yu Wong ◽

Maricela Pier ◽

Kirsten H. Limesand

Keyword(s):

Salivary Glands ◽

Structural Integrity ◽

Radiation Treatment ◽

Healing Process ◽

Coiled Coil ◽

Adaptor Proteins ◽

Wound Healing Process ◽

Rock Inhibitor ◽

Actin Organization ◽

E Cadherin

Xerostomia and hyposalivation are debilitating side effects for patients treated with ionizing radiation for head and neck cancer. Despite technological advances, collateral damage to the salivary glands remains a significant problem for patients and severely diminishes their quality of life. During the wound healing process, restoration of junctional contacts is necessary to maintain polarity, structural integrity, and orientation cues for secretion. However, little is known about whether these structural molecules are impacted following radiation damage and more importantly, during tissue restoration. We evaluated changes in adherens junctions and cytoskeletal regulators in an injury model where mice were irradiated with 5 Gy and a restoration model where mice injected postradiation with insulin-like growth factor 1 (IGF1) are capable of restoring salivary function. Using coimmunoprecipitation, there is a decrease in epithelial (E)-cadherin bound to β-catenin following damage that is restored to untreated levels with IGF1. Via its adaptor proteins, β-catenin links the cadherins to the cytoskeleton and part of this regulation is mediated through Rho-associated coiled-coil containing kinase (ROCK) signaling. In our radiation model, filamentous (F)-actin organization is fragmented, and there is an induction of ROCK activity. However, a ROCK inhibitor, Y-27632, prevents E-cadherin/β-catenin dissociation following radiation treatment. These findings illustrate that radiation induces a ROCK-dependent disruption of the cadherin-catenin complex and alters F-actin organization at stages of damage when hyposalivation is observed. Understanding the regulation of these components will be critical in the discovery of therapeutics that have the potential to restore function in polarized epithelium.

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Recent Progress in Cryopreservation of Bovine Oocytes

BioMed Research International ◽

10.1155/2014/570647 ◽

2014 ◽

Vol 2014 ◽

pp. 1-11 ◽

Cited By ~ 13

Author(s):

In-Sul Hwang ◽

Shinichi Hochi

Keyword(s):

Lipid Droplets ◽

Coiled Coil ◽

High Sensitivity ◽

Short Term ◽

Rock Inhibitor ◽

Developmental Potential ◽

Domestic Species ◽

Cytoplasmic Lipid Droplets ◽

Recovery Culture ◽

Bovine Oocytes

Principle of oocyte cryoinjury is first overviewed and then research history of cryopreservation using bovine oocytes is summarized for the last two decades with a few special references to recent progresses. Various types of cryodevices have been developed to accelerate the cooling rate and applied to the oocytes from large domestic species enriched with cytoplasmic lipid droplets. Two recent approaches include the qualitative improvement of IVM oocytes prior to the vitrification and the short-term recovery culture of vitrified-warmed oocytes prior to the subsequent IVF. Supplementation of L-carnitine to IVM medium of bovine oocytes has been reported to reduce the amount of cytoplasmic lipid droplets and improve the cryotolerance of the oocytes, but it is still controversial whether the positive effect of L-carnitine is reproducible. Incidence of multiple aster formation, a possible cause for low developmental potential of vitrified-warmed bovine oocytes, was inhibited by a short-term culture of the postwarm oocytes in the presence of Rho-associated coiled-coil kinase (ROCK) inhibitor. Use of an antioxidantα-tocopherol, instead of the ROCK inhibitor, also supported the revivability of the postwarm bovine oocytes. Further improvements of the vitrification procedure, combined with pre- and postvitrification chemical treatment, would overcome the high sensitivity of bovine oocytes to cryopreservation.

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A visible light-controllable Rho kinase inhibitor based on a photochromic phenylazothiazole

Chemical Communications ◽

10.1039/d1cc04905d ◽

2021 ◽

Author(s):

Kazuya Matsuo ◽

Sampreeth Thayyil ◽

Mitsuyasu Kawaguchi ◽

Hidehiko Nakagawa ◽

Nobuyuki Tamaoki

Keyword(s):

Protein Kinase ◽

Visible Light ◽

Kinase Inhibitor ◽

Coiled Coil ◽

Rho Kinase ◽

Rock Inhibitor ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Rho Kinase Inhibitor

Rho-associated coiled-coil-containing protein kinase (ROCK) is a serine-threonine kinase, whose inhibitors are useful for the regulation of actomyosin system. Here, we developed a photoswitchable ROCK inhibitor based on a phenylazothiazole...

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Wnt signalling inhibits adipogenesis in orbital fibroblasts from patients with Graves’ orbitopathy

British Journal of Ophthalmology ◽

10.1136/bjophthalmol-2020-316898 ◽

2021 ◽

pp. bjophthalmol-2020-316898

Author(s):

Sang Joon Jung ◽

Yeon Jeong Choi ◽

Tae Kwann Park ◽

Sang Earn Woo ◽

Bo-Yeon Kim ◽

...

Keyword(s):

Cyclin D1 ◽

Adipogenic Differentiation ◽

Wnt Signalling ◽

Dependent Manner ◽

Expression Levels ◽

Graves Orbitopathy ◽

Orbital Fibroblast ◽

Vitro Model ◽

Orbital Fibroblasts

Background/AimsTo investigate the role of Wnt signalling in adipogenesis using an in vitro model of Graves’ orbitopathy (GO).MethodsOrbital fat was obtained from patients with GO and non-GO participants for primary orbital fibroblast (OF) culture. Expression levels of Wnt5a, Wnt10b, β-catenin, phospho-β-catenin and cyclin D1 were compared between GO and non-GO OFs. These expression levels were also determined during adipogenesis of GO and non-GO OFs. The effects of a stimulator and inhibitor of Wnt signalling on adipogenesis of GO and non-GO OFs were investigated.ResultsWestern blotting analysis showed significant reductions in β-catenin and cyclin D1 and significant enhancement of phospho-β-catenin in OFs from patients with GO, compared with OFs from non-GO participants (p<0.05). Expression of Wnt5a, Wnt10b, β-catenin and cyclin D1 in OFs was highest on day 0, and then gradually declined after induction of adipogenic differentiation. The expression levels of PPARγ, C/EBPα and C/EBPβ were reduced in Wnt stimulator-treated OFs in a dose-dependent manner. Oil red O staining confirmed that a stimulator of Wnt inhibited adipogenesis in GO OFs.ConclusionThese results indicate that Wnt signalling inhibits adipogenesis in OFs from patients with GO and non-GO participants. Further studies are required to examine the potential of Wnt signalling as a target for therapeutic strategies.

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