Effects Of Prostaglandin F-2 Alpha On Adipocyte Biology Relevant To Graves' Orbitopathy

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-2, PGF(2)) 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(2) 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(2) (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(2)) 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 PGF(2) and free glycerol was assayed. Appropriate statistical tests were applied. Results: The population doubling time of 3T3-L1 was 27.31.4 hourssignificantly 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(2) 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(2), again with prolongation of G2/M. Adipogenesis in 3T3-L1 cells was minimally affected by PGF(2) 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(2). There was no effect of PGF(2) on basal or norepinephrine-induced lipolysis, in 3T3-L1 or human OFs, either GO or non-GO. Conclusions: The results demonstrate that PGF(2) significantly reduces proliferation and adipogenesis and that human OFs are more sensitive to its effects than 3T3-L1. Consequently, PGF(2) could be effective in the treatment of GO.