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

Abstract

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.

Transcriptomic Profiling of Thyroid Eye Disease Orbital Fat Demonstrates Differences in Adipogenicity and IGF-1R Pathway

“…Matrix files generated from the Cellranger run were used for subsequent analysis as described previously (18, 23). Briefly, Seurat V3 (24) was used to perform downstream analysis, only including cells with more than 500 genes, and 1000 UMI, to process control and treated samples separately.…”

“…Moreover, teprotumumab blocks the induction of inflammatory cytokines interleukin 6 (IL-6), and IL-8 in orbital fibroblasts (17). Single nucleus RNA sequencing (snRNA-Seq) has identified that during the early stages of adipogenesis, orbital fibroblasts upregulate the expression of both IGF-1 and IGF-1R in vitro (18).…”

A review of TSHR- and IGF-1R-related pathogenesis and treatment of Graves’ orbitopathy

“…Signalling pathways, including those for cAMP, PI3K-AKT, AGE-RAGE, lipolysis regulation, and thyroid hormone, are enriched in orbital fat isolated from patients with GO. The IGF-1R and Wnt signalling pathways appear to be enriched early in adipogenesis ( 36 ), and Wnt signalling inhibits adipogenesis in GO-OFs ( 37 ). PI3K can be activated by TSH, IGF-1, or multiple cytokine receptors, resulting in AKT activation.…”

Changes of mRNA expression underlying orbital adipogenesis in thyroid-associated orbitopathy

“…Previous studies investigated transcriptomic profiling of TAO orbit fat from orbital fibroblasts undergoing adipogenesis or adipose-derived stem cells [ 4 , 14 ]. By using next generation RNA sequencing technology, transcriptome analysis of orbital adipose tissue in active TAO was conducted [ 15 ]; however, the study did not involve the inactive ones.…”

“…Even after eliminating the influence of inflammatory factors in the vitro experiment, the expression of Wnt signaling pathway components was significantly suppressed in OASCs [ 4 ]. During adipogenesis, WNT5A was overexpressed only during the early stage and the expression gradually decreases during the differentiation process [ 14 ]. Consistent with the results of previous studies, downregulation of WNT and WNT4 expression was observed in TAO, even in patients who had received anti-inflammatory therapies.…”