Abstract
Purpose :
Multiple cellular pathways have been researched in an attempt to better understand Graves Ophthalmopathy (GO), an inflammatory state of the orbital muscles and fat due to orbital fat hypertrophy. Rho-kinases (ROCK) modulate various signaling pathways affecting cell structure and morphology. In ophthalmology, ROCK inhibitors are being used to treat glaucoma. Rho-kinases (ROCK) inhibitors have been shown to inhibit adipogenesis. We hypothesize that ROCK inhibitors may reduce adipogenesis in patients with GO. In this study, we evaluated the effect of ROCK inhibitor (KD025) on human orbital preadipocytes in vitro.
Methods :
Orbital adipose tissue was obtained during orbital surgery from eight consented patients with GO who were euthyroid, radiation-naïve and off steroids for at least 3 months as well as eight controls. Total mRNA levels of RhoA, Rho-associated kinase 1 (ROCK1), and Rho-associated kinase 2 (ROCK2) were measured in GO and control patients adipose tissue using RT-PCR. Orbital preadipocytes were isolated from orbital tissues and cultured in DMEM/F12 medium. Adipogenesis was initiated in orbital preadipocytes cultured in adipogenic media with a PPARγ agonist to heightened stimulation of adipogenesis. KD025 (1 or 5 µM) were presented in the adipogenic differentiation culture for 14 days. At day 14 of culture cell were stained with Oil Red O staining
Results :
mRNA transcription levels for RhoA, ROCK1 and ROCK2 were more highly expressed in GO tissue than in controls. Orbital preadipocytes exposed to KD025 demonstrated dose-dependent reduction in size and intracytoplasmic lipid droplets in the cells that underwent differentiation than the preadipocytes without KD025.
Conclusions :
RhoA, ROCK1, and ROCK2 levels are all expressed at a higher, statistically significant level in GO orbital adipose tissues than in control tissues. There was a dose dependent reduction in GO adipocyte size and intracytoplasmic lipid droplets in cells that underwent differentiation with the use of the KD025, suggesting that ROCK inhibitors are potential therapeutics for patients with GO. Further research into ROCK inhibitor effects on GO is warranted.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.