In Vitro Investigation of HIF-1α as a Therapeutic Target for Thyroid-Associated Ophthalmopathy

Abstract

Backgruound: Thyroid-associated ophthalmopathy (TAO) involves tissue expansion and inflammation, potentially causing a hypoxic microenvironment. Hypoxia-inducible factor (HIF)-1α is crucial in fibrosis and adipogenesis, which are observed in TAO progression. We investigated the effects of hypoxia on orbital fibroblasts (OFs) in TAO, focusing on the role of HIF-1α in TAO progression.

Methods: OFs were isolated from TAO and non-TAO patients (as controls). In addition to HIF-1α, adipogenic differentiation markers including peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein (CEBP) were measured by Western blot, and phenotype changes were evaluated by Oil Red O staining under both normoxia and hypoxia. To elucidate the effect of HIF-1α inhibition, protein expression changes after HIF-1α inhibitor treatment were evaluated under normoxia and hypoxia.

Results: TAO OFs exhibited significantly higher HIF-1α expression than non-TAO OFs, and the difference was more distinct under hypoxia than under normoxia. Oil Red O staining showed that adipogenic differentiation of TAO OFs was prominent under hypoxia. Hypoxic conditions increased the expression of adipogenic markers, namely PPARγ and CEBP, as well as HIF-1α in TAO OFs. Interleukin 6 levels also increased in response to hypoxia. The effect of hypoxia on adipogenesis was reduced at the protein level after HIF-1α inhibitor treatment, and this inhibitory effect was sustained even with IGF-1 stimulation in addition to hypoxia.

Conclusion: Hypoxia induces tissue remodeling in TAO by stimulating adipogenesis through HIF-1α activation. These data could provide insights into new treatment strategies and the mechanisms of adipose tissue remodeling in TAO.

Keywords: Adipogenesis; Graves ophthalmopathy; Hypoxia; Hypoxia-inducible factor 1; Thyroid.

Fig. 1.

Hypoxia-inducible factor (HIF)-1α expression in orbital fibroblasts. (A) HIF-1α levels in primary cells from thyroid-associated ophthalmopathy (TAO) and non-TAO individuals were assessed under normoxic and hypoxic conditions through Western blot analysis. (B) HIF-1α expression, normalized to β-actin, was quantified in both TAO and non-TAO tissues under normoxic (left) and hypoxic (right) conditions. For each condition, cells under hypoxia were incubated for 48 hours in an environment containing 1% O₂, 5% CO₂, and 94% N₂ at 37°C. For normoxia, cells were incubated for 48 hours in 5% CO₂ at 37°C. The samples were taken from TAO patients 112, 144, and 172, and from non-TAO individuals 87, 140, and 143. Data are presented as mean±standard deviation, with n=3 for each group.

Fig. 2.

Adipogenic changes in orbital fibroblasts derived from patients with thyroid-associated ophthalmopathy (TAO) under hypoxic stimuli. The adipogenic changes in orbital fibroblasts obtained from patients with TAO were prominent under hypoxic conditions. A marked increase in the accumulation of lipid droplets was observed under hypoxia (B) compared to normoxia (A) (upper: 40×; lower: 400×).

Fig. 3.

Differential expression of adipogenic proteins in thyroid-associated ophthalmopathy (TAO) orbital fibroblasts (OFs) under normoxic and hypoxic conditions. Higher hypoxia-inducible factor (HIF)-1α expression was universally observed in all TAO OFs exposed to hypoxic conditions. (A) The expression of CCAAT/enhancer binding protein (CEBP), and peroxisome proliferator-activated receptor γ (PPARγ), key adipogenic markers, exhibited elevations under hypoxic conditions compared to normoxic conditions. Boxplots display the relative expression levels of HIF-1α, CEBP, and PPARγ across three independent TAO fibroblast samples (TAO 112, TAO 144, and TAO 172). (B) Light gray boxes indicate normoxia, and dark gray boxes indicate hypoxia. The sample size for each condition is n=7. ^aStatistically significant differences between normoxic and hypoxic conditions within each sample, as determined by the Wilcoxon signed-rank test.

Fig. 4.

Effect of hypoxia-inducible factor (HIF)-1α inhibition on adipogenic protein expression. (A) Inhibition of HIF-1α under hypoxic conditions leads to the modulation of HIF-1α, CCAAT/enhancer binding protein (CEBP), and peroxisome proliferator-activated receptor γ (PPARγ) expression. (B) Boxplots of protein expression levels for HIF-1α, CEBP, and PPARγ under various conditions. The conditions include a control group (hypoxia) and treatments with IDF-11774, a HIF-1α inhibitor, at concentrations of 15 and 30 μM. Statistical analysis was performed using analysis of variance (ANOVA) followed by the Tukey post hoc test to determine significant differences between groups, with each group comprising seven samples. P values are indicated above the brackets connecting the groups being compared. P values less than 0.05 are considered to indicate statistically significant differences between the expression levels of the proteins.

Fig. 5.

Impact of hypoxia-inducible factor (HIF)-1α inhibition on adipogenic protein expression in thyroid-associated ophthalmopathy (TAO) orbital fibroblasts under hypoxia with insulin-like growth factor (IGF)-1 stimulation. (A) The figure shows the expression levels of HIF-1α, CCAAT/enhancer binding protein (CEBP), and peroxisome proliferator-activated receptor γ (PPARγ) in TAO orbital fibroblasts (TAO 144) under hypoxia conditions with IGF-1 (20 ng/mL) stimulation, assessing the impact of HIF-1α inhibition using IDF-11774 at concentrations of 15 and 30 μM. (B) Box plots represent expression values for each protein (HIF-1α, CEBP and PPARγ) across the three conditions: hypoxia alone (black boxes), hypoxia+IDF-11774 15 μM (dark gray boxes), and hypoxia+IDF-11774 30 μM (light gray boxes). The y-axis denotes expression values, and the x-axis indicates treatment conditions. Statistical analysis, including analysis of variance (ANOVA) and post hoc tests, are provided, with P values indicating the significance of differences between groups. Notably, HIF-1α expression decreased in response to IDF-11774 treatment (P=0.062). For CEBP, significant differences were observed between hypoxia and hypoxia+IDF-11774 30 μM (P=0.007), and between IDF-11774 15 and 30 μM (P=0.046). The changes in PPARγ expression were not statistically significant (P=0.553).

Fig. 6.

Effect of hypoxia-inducible factor (HIF)-1α inhibition on interleukin 6 (IL-6) expression levels in thyroid-associated ophthalmopathy (TAO) orbital fibroblasts under hypoxia with insulin-like growth factor (IGF)-1 stimulation. The box plot presents the effect of HIF-1α inhibition on IL-6 expression levels in TAO orbital fibroblasts (TAO 144) under hypoxic conditions with IGF-1 stimulation (20 ng/mL). The three conditions compared are hypoxia alone, hypoxia with IDF-11774, a HIF-1α inhibitor, at 15 μM, and hypoxia with IDF-11774 at 30 μM. The data indicate that hypoxia significantly increased IL-6 expression. However, the addition of IDF-11774 at both concentrations of 15 and 30 μM resulted in a notable reduction in IL-6 levels. This suggests that HIF-1α plays a critical role in IL-6 expression in TAO orbital fibroblasts under hypoxic conditions, and its inhibition can effectively downregulate IL-6 levels.