Transcriptomic Profiling of Control and Thyroid-Associated Orbitopathy (TAO) Orbital Fat and TAO Orbital Fibroblasts Undergoing Adipogenesis.

Purpose: Orbital fat hyperplasia commonly occurs in thyroid-associated orbitopathy (TAO). To understand molecular mechanisms underlying orbital adipogenesis, we used transcriptomics to compare gene expression in controls and patients with TAO, as well as in orbital fibroblasts (OFs) undergoing adipogenic differentiation. Methods: We performed bulk RNA sequencing (RNA-Seq) on intraconal orbital fat from controls and patients with TAO. We treated cultured OFs derived from patients with TAO with adipogenic media to induce adipogenesis. We used single nucleus RNA-Seq (snRNA-Seq) to profile treated and control OFs, identifying genes that are dynamically expressed during orbital adipogenesis in vitro, and compared these results to data from control and TAO orbital fat. Results: Gene expression profiles in control and TAO orbital fat are distinct. Signaling pathways including PI3K-Akt signaling, cAMP signaling, AGE-RAGE signaling, regulation of lipolysis, and thyroid hormone signaling are enriched in orbital fat isolated from patients with TAO. SnRNA-Seq of orbital fibroblasts undergoing adipogenesis reveals differential expression of the adipocyte-specific genes FABP4/5, APOE, PPARG, and ADIPOQ during adipogenic differentiation. The insulin-like growth factor-1 receptor and Wnt signaling pathways appear to be enriched early in adipogenesis. Gene modules that are enriched in TAO orbital fat are upregulated in orbital adipocytes during differentiation in vitro, whereas genes that are enriched in control orbital fat are enriched in undifferentiated OFs. Conclusions: We identified pathways enriched in TAO orbital fat, and dynamic changes in gene expression that occur during adipogenic differentiation of orbital fibroblasts. These findings may help guide functional studies of genes and pathways critical for orbital adipogenesis.