Korean J Physiol Pharmacol.  2021 Jan;25(1):59-68. 10.4196/kjpp.2021.25.1.59.

Ref-1 protects against FeCl₃ -induced thrombosis and tissue factor expression via the GSK3β–NF-κB pathway

• Lee I
• Nagar H
• Kim S
• Choi Sj
• Piao S
• Ahn M
• Jeon BH
• Oh SH
• Kang SK
• Kim CS

• KMID: 2509655
• DOI: http://doi.org/10.4196/kjpp.2021.25.1.59

Abstract

Arterial thrombosis and its associated diseases are considered to constitute a major healthcare problem. Arterial thrombosis, defined as blood clot formation in an artery that interrupts blood circulation, is associated with many cardiovascular diseases. Oxidative stress is one of many important factors that aggravates the pathophysiological process of arterial thrombosis. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ref-1) has a multifunctional role in cells that includes the regulation of oxidative stress and anti-inflammatory function. The aim of this study was to investigate the therapeutic effect of adenovirus-mediated Ref-1 overexpression on arterial thrombosis induced by 60% FeCl₃ solution in rats. Blood flow was measured to detect the time to occlusion, thrombus formation was detected by hematoxylin and eosin staining, reactive oxygen species (ROS) levels were detected by high-performance liquid chromatography, and the expression of tissue factor and other proteins was detected by Western blot. FeCl₃ aggravated thrombus formation in carotid arteries and reduced the time to artery occlusion. Ref-1 significantly delayed arterial obstruction via the inhibition of thrombus formation, especially by downregulating tissue factor expression through the Akt-GSK3β-NF-κB signaling pathway. Ref-1 also reduced the expression of vascular inflammation markers ICAM-1 and VCAM-1, and reduced the level of ROS that contributed to thrombus formation. The results showed that adenovirus-mediated Ref-1 overexpression reduced thrombus formation in the rat carotid artery. In summary, Ref-1 overexpression had anti-thrombotic effects in a carotid artery thrombosis model and could be a target for the treatment of arterial thrombosis.

Keyword

APE/Ref-1; Carotid artery thrombosis; Endothelium; Tissue factor

Figure

• 

  Fig. 1 Redox factor-1 (Ref-1) suppresses FeCl3-induced arterial thrombosis formation. (A) Timeline of experimental thrombus induction. (B) The arteries were infected with adβ-gal or adRef-1 for 24 h. Thrombosis was induced for 5 min, followed by 4 h incubation. The arteries were then harvested and Western blotting was performed to confirm Ref-1 overexpression. β-actin was used as an internal control. (C) The expression level of Ref-1 was quantified by densitometric analysis using ImageJ software. (D) FeCl3-mediated injury was induced in the adenovirus-infected carotid arteries for 5 min, and arterial blood flow was monitored constantly using a flow module until it had ceased entirely for 5 min. (E) Statistical graph of the TTO data. (F) After adenovirus infection and FeCl3-mediated injury, the arterial segments were isolated and stained with H&E. Scale bar 200 μm. (G) Statistical data are presented as ratios of the thrombus area. All data are presented as means ± standard deviations of three independent experiments. *p < 0.05 vs. Adβ-gal + FeCl3 (n = 3/group).

• 

  Fig. 2 Effect of redox factor-1 (Ref-1) overexpression on the Akt/glycogen synthase kinase 3 beta (GSK3β)/nuclear factor (NF)-κB signaling pathway and the expression of TF pathway inhibitor (TFPI), tissue factor (TF) and thrombomodulin. The arteries were infected with adβ-gal or adRef-1 for 24 h. Thrombosis was induced for 5 min, followed by 4 h incubation. The arteries were harvested and Western blotting was performed to measure the phosphorylation of (A) Akt and GSK3β, (C) p65, and the protein expressions of (C) TF, (E) TFPI and thrombomodulin. The total forms of the proteins and GAPDH were used as internal controls. The expression levels of the proteins were quantified by densitometric analysis using ImageJ software (B, D), and (F). All data are presented as means ± standard deviations of three independent experiments. *p < 0.05 vs. Adβ-gal + FeCl3 (n = 3/group).

• 

  Fig. 3 Redox factor-1 (Ref-1) suppresses the expression of inflammatory molecules in the carotid artery. The arteries were infected with adβ-gal or adRef-1 for 24 h. Thrombosis was induced for 5 min, followed by 4 h incubation. The arteries were harvested and Western blotting was performed to measure the protein expressions of (A) vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). GAPDH was used as an internal control. (B) The expression levels of VCAM-1 and ICAM-1 were quantified by densitometric analysis using ImageJ software. All data are presented as means ± standard deviations of three independent experiments. *p < 0.05 vs. Adβ-gal + FeCl3 (n = 3/group).

• 

  Fig. 4 Redox factor-1 (Ref-1) suppresses reactive oxygen species (ROS) production in the carotid artery. Arterial segments were isolated and stained with dihydroethidium (DHE) after adenovirus infection and FeCl3-mediated injury. (A) The fluorescent signal of 2-hydroxyethidium (2-EOH, the oxidative form of DHE) was measured by high-performance liquid chromatography to detect ROS levels. Arrows indicate the 2-EOH signals. (B) Statistical graph of the 2-EOH signal. All data are presented as means ± standard deviations of three independent experiments. *p < 0.05 vs. Adβ-gal + FeCl3 (n = 3/group).

• 

  Fig. 5 Schematic model of the pathways involved in FeCl3-induced thrombus formation and the possible role of apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) in the inhibition of thrombus formation. GSK3β, glycogen synthase kinase 3 beta; TF, tissue factor; TFPI, TF pathway inhibitor; ROS, reactive oxygen species.

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