The Effect of Prostaglandin Analogue Bimatoprost on Thyroid-Associated Orbitopathy

Abstract

Purpose: We characterize the effect of bimatoprost on orbital adipose tissue in thyroid-associated orbitopathy (TAO) with clinicopathologic correlation.

Methods: Orbital adipose-derived stem cells (OASCs) from types 1 and 2 TAO and control patients with and without exposure to 1 μm bimatoprost were examined via immunohistochemistry, RT-PCR, and Western blot for cell viability, migration capacity, lipid content, adipocyte morphology, mitochondrial content, and levels of adipogenic markers. A retrospective chart review was performed for clinicopathologic correlation. In mice, optical coherence tomography and pattern electroretinography were performed at baseline and at 1 month following a retrobulbar injection of bimatoprost, followed by orbital exenteration for histopathologic examination.

Results: Types 1 and 2 TAO-derived cells had a significantly higher migration capacity and lipid content than those of healthy controls. With the addition of bimatoprost, types 1 and 2 TAO and control adipocytes exhibited a significant decrease in lipid content with morphologic transformation into smaller and multilocular lipid droplets, and an increase in mitochondrial load and UCP-1 expression consistent with an increase in brown adipose tissue turnover. Retrobulbar injection of bimatoprost in mice did not alter the gross morphology, retinal thickness, or ganglion cell function in vivo.

Conclusions: Bimatoprost inhibits adipogenesis in OASCs and upregulates pathways involved in the browning of adipocytes. Furthermore, retrobulbar injection of bimatoprost is tolerated without immediate adverse effects in mice. Our results suggest a potential future application of prostaglandin analogues in the treatment of TAO.

Figure 1

Differential expression of PG receptors in TAO and the effect of bimatoprost on cell viability and migration. (A) RNA-sequencing analysis of OASCs from TAO patients compared to control patients demonstrating differential expression of PG receptors identified by EdgeR and DESeq2. (B) Dose-dependent cell viability of OASCs from healthy controls treated with bimatoprost. (C) Cell proliferation of OASCs from healthy controls, and types 1 and 2 TAO patients treated with bimatoprost on days 3 and 7. (D) Scratch assay showing the effect of 1 μM bimatoprost on OASC migration over a 24-hour period. (E) Quantification of 24-hour migration data in control and TAO OASCs. *P < 0.05, **P < 0.01. NS, not significant. Scale bar: 200 μm.

Figure 2

Bimatoprost inhibits OASC adipogenesis. (A) A timeline of the adipogenesis assay. (B) Oil red O stain demonstrating bimatoprost-treated adipocytes with smaller, multilocular lipid droplets compared to vehicle-treated cells containing larger, unilocular lipid droplets. (C) Quantification of oil red O assay showing higher baseline neutral lipid content in type 1 > type 2 TAO adipocytes compared to control, and inhibition of adipogenesis in control and TAO OASCs with bimatoprost. *P < 0.05, **P < 0.01. Scale bar: 200 μm.

Figure 3

Bimatoprost induces mitochondrial biogenesis and triggers white-to-brown adipose trans-differentiation. (A) Mitochondrial content labeled with MitoTracker (green) and nuclear counterstaining with DAPI (blue) showing increased mitochondrial contents in adipocytes treated with bimatoprost (arrows). (B) Western blot analysis and (C) RT-PCR demonstrating increased expression of UCP-1 and PGC-1α with bimatoprost treatment, while PPAR-γ remained unchanged. (D) Immunofluorescence staining demonstrating formation of multiple small multilocular lipid droplets and increased expression of UCP-1 in response to bimatoprost treatment. *P < 0.05. Scale bars: (A) 200 μm; (D) 100 μm.

Figure 4

MAPK and Akt signaling pathways are activated by bimatoprost. (A) Western blot analysis demonstrating upregulation of phosphorylated ERK1/2, Akt, and p38 in response to bimatoprost treatment. (B) Schematic timeline of the adipogenic differentiation protocol. MEK inhibitor U0216, AKT inhibitor MK-2206, or p38 inhibitor SB202190 was added concurrently with bimatoprost after adipocyte lineage induction. (C) Oil red O staining and (D) quantification showing partial rescue from the inhibitory effect of bimatoprost on adipogenesis by all three inhibitors. (E) Fluorescent images and quantification (F) of intracellular transient calcium flux induced by bimatoprost treatment in OASCs. Arrow indicates time point of 1 μM bimatoprost addition. Data depicted as mean ± 95% confidence interval for n = 25 cells. *P < 0.05. Scale bar: 200 μm.

Figure 5

Gross morphology and histology of orbital tissues, retinal layer thickness, and RGC function after retrobulbar injection of bimatoprost. (A) H&E sections showing normal gross morphology of the orbit and surrounding orbital tissue in the bimatoprost-treated eyes (OS) and PBS-treated eyes (OD). (B) Representative images of OCT and fundus auto fluorescence (FAF) overlaid with retinal thickness heat maps generated by Heidelberg retina tomography. (C) RNFL thickness quantification showing no difference between bimatoprost-treated eyes (OS) and PBS-treated eyes (OD) at 1 month. (D) Representative PERG amplitudes waveforms showing no significant differences between bimatoprost- and PBS-treated eyes at baseline, and at 1 month after treatment. (E) Quantitative representation of PERG amplitude recordings in PBS- and bimatoprost treated eyes. NS, not significant. Scale bars: (A) 500 μm; (B) 200 μm.

Figure 6

Working model for the mechanism of action: bimatoprost (via bimatoprost acid) can bind to the prostanoid FP receptor, which is a cell surface G-protein-coupled receptor (GPCR), and initiates its intracellular signaling cascades including MAPK, PI3/Akt, p38 MAPK and calcium signaling pathways. Together, these pathways are thought to lead to mitochondrial biogenesis and a thermogenic switch from white to brown adipose tissue.