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Fumihito Hikage, Stephen Atkins, Alon Kahana, Terry Smith, Tae-Hwa Chun; Three-dimensional ex vivo modeling of Graves’ orbitopathy reveals a novel pathogenic role for the HIF2A-LOX pathway. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1142. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Pathophysiology of Graves’ orbitopathy (GO) is elusive, owning in part to insufficient preclinical and translational disease models. We developed a three-dimensional (3D) organoid culture system to recapitulate Graves’ orbital fibroblast (OF) behavior ex vivo, and employed it to mechanistically probe the adipose tissue fibrosis and inflammation underlying GO.
We obtained human OFs from surgical waste samples of de-identified GO and non-GO (N-GO) patients (n=5 per group). We generated 3D OF organoids in hanging-droplet culture, and employed micro-indentation to measure their stiffness, and gene/protein expression analysis to assess their fibrotic phenotype. We evaluated fibrocyte invasion into organoids through co-culture, and defined HIF2A as a mediator of fibrotic and inflammatory responses through shRNA-based knockdown in human OFs, and overexpression of mutant HIF2A in mouse OFs.
Tissue stiffness was significantly greater in GO versus N-GO organoids (GO 1.34±0.05µN/µm, n=43; N-GO 1.06±0.18, n=32; p<0.001), and corresponded with elevated deposition of collagen (COL) 3, 4, 6, and fibronectin (FN). Thyrotropin receptor activation further enhanced COL6 and FN accumulation in GO organoids. GO organoids were more susceptible than N-GO organoids to fibrocyte invasion, and corresponding induction of IL-6 mRNA. Additionally, the transcript encoding lysyl oxidase (LOX), a mediator of collagen fibrillogenesis, was elevated 2-fold (p=0.03) in GO organoids, while that encoding hypoxia-inducible factor 2α (HIF2A), an upstream regulator of LOX, increased by 2.3-fold (p=0.002). The LOX antagonist BAPN ameliorated COL1 and 4 accumulation, while HIF2A knockdown inhibited LOX expression. Both maneuvers reduced GO organoid stiffness. Conversely, a constitutively active HIF2A mutant induced LOX expression and augmented tissue stiffness in mouse OF organoids. In keeping with these findings, expression of HIF2A-LOX pathway components was greater in orbital tissues from GO versus N-GO patients. Importantly, HIF2A and LOX were unchanged between GO and N-GO cells in standard 2D culture, highlighting the importance of 3D cell-cell interaction for study of this clinically relevant pathway.
HIF2A-LOX may serve as a novel therapeutic target of GO and can be mechanistically studied in a novel 3D organoid culture system.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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