Upregulation of the gene expression of CLOCK is correlated with hypoxia-inducible factor 1a in advanced varicose lesions

Abstract

According to previous literature, venous hypoxia and the hypoxia-inducible factor (HIF) pathway may contribute to the pathogenesis of varicose veins (VVs). It is widely accepted that the circadian locomotor output cycles kaput (CLOCK) gene affects nucleotide excision repair, DNA damage checkpoints and apoptosis in mammalian organisms; however, the expression levels of CLOCK in varicose veins remain to be elucidated. The aim of the present study was to detect the expression of the circadian clock gene in initial and advanced varicose lesions and analyze the correlation between the CLOCK gene, HIF-1a, and its target gene, vascular endothelial growth factor (VEGF), in VVs. Sections of the great saphenous veins (GSVs) were obtained from patients undergoing ligation and stripping for VVs (n=70) and a control group undergoing coronary artery bypass grafting with GSV harvest (n=11). All VV patients had incompetent GSVs, according to color flow duplex scanning. C-class VVs were determined according to the clinical-etiology-anatomy-pathophysiology classification for venous diseases following physical examination of the patients with VV. Reverse transcription-quantitative polymerase chain reaction was used to determine the expression levels of the CLOCK gene, HIF-1a and VEGF. Immunohistochemical analysis was also performed. The patients with VVs were divided into those with initial varicose lesions (C3 and C4) and advanced varicose lesions (C5 and C6). In total, 21 of the patients had C3 lesions, 23 had C4 lesions, 14 had C5 lesions and 12 had C6 chronic venous disease. The expression of the CLOCK gene was significantly higher in the VV lesions of the GSV, compared with the normal GSVs (P<0.0001). The same trend was found in the expression levels of HIF-1a and its target gene, VEGF, in the VV lesions (P=0.003 and P<0.0001, respectively). Subgroup analysis revealed that the expression levels of the CLOCK gene, HIF-1a and VEGF were significantly higher in the advanced stage varicose lesions, compared with the initial varicose lesions (P<0.0001, P=0.0014 and P<0.0001, respectively). However, no statistically significant difference was identified in the expression levels of the aforementioned genes in the C3 and C4 lesions. The results demonstrated that the expression gene levels of CLOCK, HIF-1a and its target gene, VEGF, increased significantly in advanced stage varicose lesions. Therefore, upregulation of the CLOCK gene in the vessel walls of veins may be involved in the pathogenesis of VVs and the progression of venous disease.