Effects of thyroid-stimulating hormone on adhesion molecules and pro-inflammatory cytokines secretion in human umbilical vein endothelial cells

Abstract

Atherosclerosis is a multifactorial disorder, which affects the arterial wall. It has been reported that, hypothyroidism and thyroid hormone deficiency are related to cardiovascular disorders. Also, endothelial dysfunction plays an essential role in the development of atherosclerosis. We aimed to evaluate the effects of thyroid-stimulating hormone (TSH) on pro-inflammatory tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), angiogenic vascular endothelial growth factor (VEGF) and leukocyte adhesion, intercellular adhesion molecule 1 (ICAM-1) and E-selectin in human umbilical vein endothelial cells (HUVECs). In this study, HUVEC cells were treated with 1 and 2 μM of TSH in different treatment times. The gene and protein expression of ICAM-1, VEGF, and E-selectin were performed by real-time polymerase chain reaction and western blotting, respectively. Likewise, TNF-α and IL-6 protein levels were determined by the ELISA method. VEGF, ICAM-1, and E-selectin as endothelial dysfunction markers and also, TNF-α and IL-6 as pro-inflammatory cytokines were detectable in HUVEC. Besides, the results of this study revealed that TSH treatment down-regulates TNF-α and IL-6. Evaluating the gene and protein expression data revealed the upregulation of ICAM-1, E-selectin, and VEGF in TSH treated cases in different periods of exposure. Considering the multiple actions of TSH, it could be concluded that TSH plays a controversial role in atherogenesis by anti-inflammatory effects and on the other side, angiogenesis and leukocyte adhesion induction which is related to vascular cell proliferation.

Keywords: E-selectin; HUVEC; ICAM-1; TNF-α; Thyrotropin; VEGF.

Fig. 1

Down regulation of tumor necrosis factor-α (TNF-α) level in HUVECs by thyroid-stimulating hormone (TSH) exposure. After treatments with TSH (1 and 2 μM) for 12, 24, and 48 h, protein expression evaluated by ELISA method. * Indicates significant differences (P< 0.05) compared to control group. Data are presented as mean ± SD values derived from three independent experiments.

Fig. 2

Down regulation of interleukin-6 (IL-6) levels in HUVECs by thyroid-stimulating hormone (TSH) exposure. After treatments with TSH (1 and 2 μM) for 12, 24, and 48 h, protein expression evaluated by ELISA method. * Indicates significant differences (P< 0.05) compared to control group. Data presented as mean ± SD values derived from three independent experiments.

Fig. 3

mRNA levels of E-selectin in HUVECs cells treated by thyroid-stimulating hormone (TSH) (1 and 2 μM) for (A) 24 h and (B) 48 h exposure times. Data presented as mean fold change ± SD values derived from three independent experiments. *P< 0.01.

Fig. 4

mRNA levels of intercellular adhesion molecule 1 (ICAM-1) in HUVECs cells treated by thyroid-stimulating hormone (TSH) (1 and 2 μM) for (A) 24 h and (B) 48 h exposure times. Data presented as mean fold change ± SD values derived from three independent experiments.*P< 0.01.

Fig. 5

mRNA levels of vascular endothelial growth factor (VEGF) in HUVECs cells treated by thyroid-stimulating hormone (TSH) (1 and 2 μM) for (A) 24 h and (B) 48 h exposure times. Data presented as mean fold change ± SD values derived from three independent experiments.*P< 0.05, **P< 0.01.

Fig. 6

Prortein detection for evaluation of E-selectin expression performed using western blotting technique. (A) Protein expression levels of E-selectin in HUVECs cells treated by thyroid-stimulating hormone (TSH) (1 and 2 μM) for 6 and 24 h and (B) TSH treatment significantly increased relative expression of E-selectin. For analysis protein's bands densities, Image J software was used to analyze the densities. Each protein (in one repeat) bands normalized to β-actin bands.

Fig. 7

Prortein detection for evaluation of E-selectin expression performed using western blotting technique. (A) Protein expression levels of E-selectin in HUVECs cells treated by thyroid-stimulating hormone (TSH) (1 and 2 μM) for 6 and 24 hand (B) TSH treatment significantly increased relative expression of E-selectin. For analysis protein's bands densities, Image J software was used to analyze the densities. Each protein (in one repeat) bands normalized to β-actin bands.