dinoprost and Graves-Ophthalmopathy

Previous in vitro experiments have demonstrated that prostaglandin F2-alpha (PF. A randomized controlled double-masked crossover trial was conducted in a single tertiary care academic medical center. Patients with long-standing, inactive GO but persistent proptosis (>20 mm in at least one eye) were recruited. Allowing for a 15% dropout rate, 31 patients (26 females) were randomized in order to identify a treatment effect of 2.0 mm (p = 0.05; power 0.88). Following informed consent, participants were randomized to receive bimatoprost or placebo for three months, after which they underwent a two-month washout before switching to the opposite treatment. The primary outcome was the change in exophthalmometry readings over the two three-month treatment periods.. The mean exophthalmometer at baseline was 23.6 mm (range 20.0-30.5 mm), and the mean age of the patients was 55 years (range 28-74 years). The median duration of GO was 7.6 years (interquartile range 3.6-12.3 years). The majority were still suffering from diplopia (61.3%) with bilateral involvement (61.3%). Using multi-level modeling adjusted for baseline, period, and carry-over, bimatoprost resulted in a -0.17 mm (reduction) exophthalmometry change ([confidence interval -0.67 to +0.32]; p = 0.490). There was a mean change in intraocular pressure of -2.7 mmHg ([confidence interval -4.0 to -1.4]; p = 0.0070). One patient showed periorbital fat atrophy on treatment, which resolved on stopping treatment. Independent analysis of proptosis by photographic images (all subjects) and subgroup analysis on monocular disease (n = 12) did not show any apparent benefit.. In inactive GO, bimatoprost treatment over a three-month period does not result in an improvement in proptosis.

Topics: Administration, Ophthalmic; Adult; Aged; Cross-Over Studies; Dinoprost; Double-Blind Method; Eye; Female; Graves Ophthalmopathy; Humans; Male; Middle Aged; Ophthalmic Solutions; Time Factors; Treatment Outcome; Wales

Prostaglandin F2α (PGF2α), the first-line anti-glaucoma medication, can cause the deepening of the upper eyelid sulcus due to orbital lipoatrophy. However, the pathogenesis of Graves' ophthalmopathy (GO) involves the excessive adipogenesis of the orbital tissues. The present study aimed to determine the therapeutic effects and underlying mechanisms of PGF2α on adipocyte differentiation. In this study primary cultures of orbital fibroblasts (OFs) from six patients with GO were established. Immunohistochemistry, immunofluorescence, and Western blotting (WB) were used to evaluated the expression of the F-prostanoid receptor (FPR) in the orbital adipose tissues and the OFs of GO patients. The OFs were induced to differentiate into adipocytes and treated with different incubation times and concentrations of PGF2α. The results of Oil red O staining showed that the number and size of the lipid droplets decreased with increasing concentrations of PGF2α and the reverse transcription-polymerase chain reaction (RT-PCR) and WB of the peroxisome proliferator-activated receptor γ (PPARγ) and fatty-acid-binding protein 4 (FABP4), both adipogenic markers, were significantly downregulated via PGF2α treatment. Additionally, we found the adipogenesis induction of OFs promoted ERK phosphorylation, whereas PGF2α further induced ERK phosphorylation. We used Ebopiprant (FPR antagonist) to interfere with PGF2α binding to the FPR and U0126, an Extracellular Signal-Regulated Kinase (ERK) inhibitor, to inhibit ERK phosphorylation. The results of Oil red O staining and expression of adipogenic markers showed that blocking the receptor binding or decreasing the phosphorylation state of the ERK both alleviate the inhibitory effect of PGF2a on the OFs adipogenesis. Overall, PGF2α mediated the inhibitory effect of the OFs adipogenesis through the hyperactivation of ERK phosphorylation via coupling with the FPR. Our study provides a further theoretical reference for the potential application of PGF2α in patients with GO.

Topics: Adipogenesis; Cells, Cultured; Dinoprost; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Graves Ophthalmopathy; Humans

3D organoid cultures were used to elucidate the periocular effects of several anti-glaucoma drugs including a prostaglandin F2α analogue (bimatoprost acid; BIM-A), EP2 agonist (omidenepag; OMD) or a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor (ripasudil; Rip) on Grave's orbitopathy (GO) related orbital fatty tissue. 3D organoids were prepared from GO related human orbital fibroblasts (GHOFs) obtained from patients with GO. The effects of either 100 nM BIM-A, 100 nM OMD or 10 μM Rip on the 3D GHOFs organoids were examined with respect to organoid size, physical properties by a micro-squeezer, and the mRNA expression of extracellular matrix (ECM) proteins including collagen (COL) 1, COL 4, COL 6, and fibronectin (FN), ECM regulatory genes including lysyl oxidase (LOX), Connective Tissue Growth Factor (CTGF) and inflammatory cytokines including interleukin-1β (IL1β) and interleukin-6 (IL6). The size of the 3D GHOFs organoids decreased substantially in the presence of BIM-A, but also increased substantially in the presence of the others (OMD or Rip). The physical stiffness of the 3D GHOFs organoids was significantly decreased by Rip. BIM-A caused significantly the down-regulation of three ECM genes, Col 1, Col 6 and Fn, and two ECM regulatory genes and the up-regulation of IL6. In the presence of OMD, two ECM genes, Col 1 and Fn, and LOX were significantly down-regulated but IL1β and IL6 were significantly up-regulated. In the case of Rip, Col 1, FN and CTGF were significant down-regulated. Our present findings indicate that anti-glaucoma drugs modulate the structures and physical properties 3D GHOFs organoids in different manners by modifying the gene expressions of ECM, ECM regulatory factors and inflammatory cytokines. The results indicate that the benefits and demerits of anti-glaucoma medications need to be scrutinized carefully, in cases of patients with GO.

Topics: Bimatoprost; Cell Culture Techniques; Dinoprost; Extracellular Matrix Proteins; Fibroblasts; Gene Expression Regulation; Glycine; Graves Ophthalmopathy; Humans; Isoquinolines; Molecular Conformation; Orbit; Organoids; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Real-Time Polymerase Chain Reaction; Receptors, Prostaglandin E, EP2 Subtype; rho-Associated Kinases; RNA, Messenger; Sulfonamides

In Graves' orbitopathy (GO), increased proliferation, excess adipogenesis, and hyaluronan overproduction produce GO exophthalmos. Enophthalmos occurs in some glaucoma patients treated with Bimatoprost (prostaglandin F2α, PGF2α) eye drops. We hypothesized that enophthalmos is secondary to reductions in orbital tissue proliferation, adipogenesis, and/or increased lipolysis. We aimed to determine which of these is affected by PGF2α by using the 3T3-L1 murine preadipocyte cell line and primary human orbital fibroblasts (OFs) from GO patients (n=5) and non-GO (n=5).. 3T3-L1 cells and orbital OFs were cultured alone or with PGF2α (all experiments used 10(-8) to 10(-6) M) and counted on days 1/2/3 or 5, respectively; cell cycle analysis (flow cytometry) was applied. Adipogenesis (in the presence/absence of PGF2α) was evaluated (day 7 or 15 for 3T3-L1 and primary cells, respectively) morphologically by Oil Red O staining and quantitative polymerase chain reaction measurement of adipogenesis markers (glycerol-3-phosphate dehydrogenase and lipoprotein lipase, respectively). For lipolysis, in vitro-differentiated 3T3-L1 or mature orbital adipocytes were incubated with norepinephrine and PGF2α and free glycerol was assayed. Appropriate statistical tests were applied.. The population doubling time of 3T3-L1 was 27.3±1.4 hours-significantly increased by dimethyl sulfoxide 0.02% to 44.6±4.8 hours (p=0.007) and further significantly increased (p=0.049 compared with dimethyl sulfoxide) by 10(-8) M PGF2α to 93.6±19.0 hours, indicating reduced proliferation, which was caused by prolongation of G2/M. GO OFs proliferated significantly more rapidly than non-GO (population doubling time 5.36±0.34 or 6.63±0.35 days, respectively, p=0.035), but the proliferation of both was significantly reduced (dose dependent from 10(-8) M) by PGF2α, again with prolongation of G2/M. Adipogenesis in 3T3-L1 cells was minimally affected by PGF2α when assessed morphologically, but the drug significantly reduced transcripts of the glycerol-3-phosphate dehydrogenase differentiation marker. GO OFs displayed significantly higher adipogenic potential than non-GO, but in both populations, adipogenesis, evaluated by all 3 methods, was significantly reduced (dose dependent from 10(-8) M) by PGF2α. There was no effect of PGF2α on basal or norepinephrine-induced lipolysis, in 3T3-L1 or human OFs, either GO or non-GO.. The results demonstrate that PGF2α significantly reduces proliferation and adipogenesis and that human OFs are more sensitive to its effects than 3T3-L1. Consequently, PGF2α could be effective in the treatment of GO.

Topics: 3T3-L1 Cells; Adipocytes; Adipogenesis; Animals; Cell Proliferation; Dinoprost; Fibroblasts; Graves Ophthalmopathy; Humans; Lipolysis; Mice