Effects of Prostaglandin F_2αon Adipocyte Biology Relevant to Graves' Orbitopathy

Abstract

Background: In Graves' orbitopathy (GO), increased proliferation, excess adipogenesis, and hyaluronan overproduction produce GO exophthalmos. Enophthalmos occurs in some glaucoma patients treated with Bimatoprost (prostaglandin F 2a , PGF 2a ) 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 PGF 2a 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). Methods: 3T3-L1 cells and orbital OFs were cultured alone or with PGF 2a (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 PGF 2a ) 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 vitrodifferentiated 3T3-L1 or mature orbital adipocytes were incubated with norepinephrine and PGF 2a and free glycerol was assayed. Appropriate statistical tests were applied. Results: 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 PGF 2a 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 PGF 2a , again with prolongation of G2/M. Adipogenesis in 3T3-L1 cells was minimally affected by PGF 2a 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 PGF 2a . There was no effect of PGF 2a on basal or norepinephrine-induced lipolysis, in 3T3-L1 or human OFs, either GO or non-GO.Conclusions: The results demonstrate that PGF 2a significantly reduces proliferation and adipogenesis and that human OFs are more sensitive to its effects than 3T3-L1. Consequently, PGF 2a could be effective in the treatment of GO.

Prostaglandin F2-Alpha Eye Drops (Bimatoprost) in Graves' Orbitopathy: A Randomized Controlled Double-Masked Crossover Trial (BIMA Trial)

“…The standard deviation was consistent with power calculations suggesting that we were not underpowered and unlikely that the effect was missed. This is in contrast with the in vitro findings (20,21), anecdotal case reports in people without GO (6)(7)(8) suggesting adipocyte differentiation inhibition with…”

“…Adipogenesis starts early in the disease and it has been shown that it may continue even in the inactive disease stage (23). PGF2α inhibits adipogenesis per se but does not affect lipolysis and hence has no impact on an already fully mature adipocyte (20). Not all glaucoma patients treated with PGF2α develop periorbital fat atrophy with an estimated incidence of 24.1% (24).…”

“…The trial was conducted according to the protocol and in compliance with the principles of the Declaration of Helsinki (1996) proptosis is considered to be clinically relevant (18,19). Assuming a standard deviation of 2.5 mm in proptosis measurements in patients with GO as previously reported [19,20], we calculated that 26 participants would be needed to be able to identify a treatment effect of 2.0 mm as statistically significant (p=0.05, two-sided paired t-test, power 0.88). Allowing for a 15% dropout rate/incomplete datasets, we Secondary and other outcomes were summarized with descriptive statistics.…”

In Vivo Effects of Retrobulbar Bimatoprost Injection on Orbital Fat

“…3 In addition, 1 group of investigators has shown that in a mouse model of thyroid eye disease (TED), the doubling time of orbital fibroblasts was increased when the cells were exposed in vitro to PGF2α analogues. 4 This suggests that the PGF 2α analogues may decrease adipogenesis and thus induce fat atrophy in vivo.…”

“…3 Other reports have shown that exposing orbital fibroblasts to prostaglandin F2α—the common base structure of the 3 major PGA bimatoprost, travoprost, and latanoprost—slowed adipocyte population growth in cell culture by decreasing adipogenesis when compared with cells not exposed to PGA. 4 …”