Endocrinol Metab.  2024 Oct;39(5):767-776. 10.3803/EnM.2024.1952.

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

• Lee J
• Lee J
• Baek H
• Lim DJ
• Lee SB
• Lee JM
• Jang SA
• Kang MI
• Yang SW
• Kim MH

• KMID: 2560285
• DOI: http://doi.org/10.3803/EnM.2024.1952

Abstract

Background
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.

Keyword

Thyroid; Graves ophthalmopathy; Hypoxia; Hypoxia-inducible factor 1; Adipogenesis

Figure

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  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.

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  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×).

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  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.

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  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.

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  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).

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  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.

Reference

1. Bahn RS. Graves’ ophthalmopathy. N Engl J Med. 2010; 362:726–38.

2. Smith TJ, Hegedus L. Graves’ disease. N Engl J Med. 2016; 375:1552–65.

3. Lacheta D, Miskiewicz P, Gluszko A, Nowicka G, Struga M, Kantor I, et al. Immunological aspects of Graves’ ophthalmopathy. Biomed Res Int. 2019; 2019:7453260.

4. Smith TJ, Janssen JA. Insulin-like growth factor-I receptor and thyroid-associated ophthalmopathy. Endocr Rev. 2019; 40:236–67.

5. Metcalfe RA, Weetman AP. Stimulation of extraocular muscle fibroblasts by cytokines and hypoxia: possible role in thyroid-associated ophthalmopathy. Clin Endocrinol (Oxf). 1994; 40:67–72.

6. Chng CL, Lai OF, Chew CS, Peh YP, Fook-Chong SM, Seah LL, et al. Hypoxia increases adipogenesis and affects adipocytokine production in orbital fibroblasts: a possible explanation of the link between smoking and Graves’ ophthalmopathy. Int J Ophthalmol. 2014; 7:403–7.

7. Gortz GE, Horstmann M, Aniol B, Reyes BD, Fandrey J, Eckstein A, et al. Hypoxia-dependent HIF-1 activation impacts on tissue remodeling in Graves’ ophthalmopathy: implications for smoking. J Clin Endocrinol Metab. 2016; 101:4834–42.

8. Eltzschig HK, Carmeliet P. Hypoxia and inflammation. N Engl J Med. 2011; 364:656–65.

9. Trayhurn P. Hypoxia and adipose tissue function and dysfunction in obesity. Physiol Rev. 2013; 93:1–21.

10. Dutton JJ. Anatomic considerations in thyroid eye disease. Ophthalmic Plast Reconstr Surg. 2018; 34(4S Suppl 1):S7–12.

11. Walasik-Szemplinska D, Pauk-Domanska M, Sanocka U, Sudol-Szopinska I. Doppler imaging of orbital vessels in the assessment of the activity and severity of thyroid-associated orbitopathy. J Ultrason. 2015; 15:388–97.

12. Lee K, Kang JE, Park SK, Jin Y, Chung KS, Kim HM, et al. LW6, a novel HIF-1 inhibitor, promotes proteasomal degradation of HIF-1alpha via upregulation of VHL in a colon cancer cell line. Biochem Pharmacol. 2010; 80:982–9.

13. Lee K, Ban HS, Naik R, Hong YS, Son S, Kim BK, et al. Identification of malate dehydrogenase 2 as a target protein of the HIF-1 inhibitor LW6 using chemical probes. Angew Chem Int Ed Engl. 2013; 52:10286–9.

14. Kim MH, Lee TH, Lee JS, Lim DJ, Lee PC. Hif-1α inhibitors could successfully inhibit the progression of differentiated thyroid cancer in vitro. Pharmaceuticals (Basel). 2020; 13:208.

15. Ke Q, Costa M. Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol. 2006; 70:1469–80.

16. Semenza GL. Hypoxia-inducible factors in physiology and medicine. Cell. 2012; 148:399–408.

17. Liu ZZ, Tian YF, Wu H, Ouyang SY, Kuang WL. LncRNA H19 promotes glioma angiogenesis through miR-138/HIF-1α/VEGF axis. Neoplasma. 2020; 67:111–8.

18. Befani C, Liakos P. The role of hypoxia-inducible factor-2 alpha in angiogenesis. J Cell Physiol. 2018; 233:9087–98.

19. Luo L, Luo G, Fang Q, Sun Z. Stable expression of hypoxiainducible factor-1α in human renal proximal tubular epithelial cells promotes epithelial to mesenchymal transition. Transplant Proc. 2014; 46:130–4.

20. Palsson-McDermott EM, O’Neill LA. The Warburg effect then and now: from cancer to inflammatory diseases. Bioessays. 2013; 35:965–73.

21. Xie Y, Shi X, Sheng K, Han G, Li W, Zhao Q, et al. PI3K/ Akt signaling transduction pathway, erythropoiesis and glycolysis in hypoxia (review). Mol Med Rep. 2019; 19:783–91.

22. Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer. 2003; 3:721–32.

23. Hammond EM, Giaccia AJ. The role of p53 in hypoxia-induced apoptosis. Biochem Biophys Res Commun. 2005; 331:718–25.

24. Dong S, Liang S, Cheng Z, Zhang X, Luo L, Li L, et al. ROS/PI3K/Akt and Wnt/β-catenin signalings activate HIF-1αinduced metabolic reprogramming to impart 5-fluorouracil resistance in colorectal cancer. J Exp Clin Cancer Res. 2022; 41:15.

25. Sharma A, Sinha S, Shrivastava N. Therapeutic targeting hypoxia-inducible factor (HIF-1) in cancer: cutting gordian knot of cancer cell metabolism. Front Genet. 2022; 13:849040.

26. Abdi Sarabi M, Shiri A, Aghapour M, Reichardt C, Brandt S, Mertens PR, et al. Normoxic HIF-1α stabilization caused by local inflammatory factors and its consequences in human coronary artery endothelial cells. Cells. 2022; 11:3878.

27. Ruan H, Zhang Q, Zhang YP, Li SS, Ran X. Unraveling the role of HIF-1α in sepsis: from pathophysiology to potential therapeutics-a narrative review. Crit Care. 2024; 28:100.

28. Smith TJ, Janssen JA. Building the case for insulin-like growth factor receptor-I involvement in thyroid-associated ophthalmopathy. Front Endocrinol (Lausanne). 2017; 7:167.

29. Gortz GE, Philipp S, Bruderek K, Jesenek C, Horstmann M, Henning Y, et al. Macrophage-orbital fibroblast interaction and hypoxia promote inflammation and adipogenesis in Graves’ orbitopathy. Endocrinology. 2022; 164:bqac203.

30. Van Regemorter E, Joris V, Van Regemorter V, Marique L, Behets C, Lengele B, et al. Downregulation of caveolin-1 and upregulation of deiodinase 3, associated with hypoxia-inducible factor-1α increase, are involved in the oxidative stress of Graves’ orbital adipocytes. Thyroid. 2021; 31:627–37.

31. Gao J, Zhu J, Zhao Y, Gan X, Yu H. Leptin attenuates hypoxia-induced apoptosis in human periodontal ligament cells via the reactive oxygen species-hypoxia-inducible factor-1α pathway. Exp Physiol. 2021; 106:1752–61.

32. Tang QQ, Lane MD. Adipogenesis: from stem cell to adipocyte. Annu Rev Biochem. 2012; 81:715–36.

33. Valyasevi RW, Harteneck DA, Dutton CM, Bahn RS. Stimulation of adipogenesis, peroxisome proliferator-activated receptor-gamma (PPARgamma), and thyrotropin receptor by PPARgamma agonist in human orbital preadipocyte fibroblasts. J Clin Endocrinol Metab. 2002; 87:2352–8.

34. Westra J, Brouwer E, Bos R, Posthumus MD, Doornbos-van der Meer B, Kallenberg CG, et al. Regulation of cytokine-induced HIF-1alpha expression in rheumatoid synovial fibroblasts. Ann N Y Acad Sci. 2007; 1108:340–8.