Chronic venous disease (CVD) is one of the most prevalent yet underrated disorders worldwide. High heritability estimates of CVD indicate prominent genetic components in its etiology and pathology. Mutations in human forkhead box C2 (FoxC2) gene are strongly associated with valve failure in saphenous and deep veins of lower extremities. We explored the association of genetic variants of FoxC2 as well as FoxC2 mRNA and protein expression levels with CVD of lower limbs. We systematically sequenced the single coding exon, 5′ and 3′ flanking regions of FoxC2 gene in 754 study subjects which includes 382 patients with CVD and 372 healthy subjects. Four novel and three reported polymorphisms were identified in our cohort. Three variants in 5′ flanking region and one in 3′ flanking region of FoxC2 gene were significantly associated with CVD risk. FoxC2 mRNA in vein tissues from 22 patients was 4±1.42 fold increased compared to saphenous veins from 20 normal subjects (p<0.01). FoxC2 protein was also significantly upregulated in varicose veins compared to control samples. The c.-512C>T (rs34221221: C>T) variant which is located in the FoxC2 putative promoter region was further analyzed. Functional analysis of c.-512C>T revealed increased mRNA and protein expression in patients with homozygous TT genotype compared to heterozygous CT and wild CC genotypes. Luciferase assay indicated higher transcriptional activity of mutant compared to wild genotype of this variant. These findings suggested that c.-512C>T variant of FoxC2 was strongly associated with susceptibility to CVD and also that this variant resulted in FoxC2 overexpression. To obtain a mechanistic insight into the role of upregulated FoxC2 in varicosities, we overexpressed FoxC2 in venous endothelial cells and observed elevated expression of arterial markers Dll4 and Hey2 and downregulation of venous marker COUP-TFII. Our study indicates altered FoxC2-Notch signaling in saphenous vein wall remodeling in patients with varicose veins.
Varicose veins of lower extremities are a heritable common disorder. Mechanisms underlying its pathogenesis are still vague. Structural failures such as valve weakness and wall dilatation in saphenous vein result in venous retrograde flow in lower extremities of body. Reflux of blood leads to distal high venous pressure resulting in distended veins. In an earlier study, we observed a positive association between c.-512C4T FoxC2 gene polymorphism and upregulated FoxC2 expression in varicose vein specimens. FoxC2 overexpression in vitro in venous endothelial cells resulted in the elevated mRNA expression of arterial endothelial markers such as Delta-like ligand 4 (Dll4) and Hairy/enhancer-of-split related with YRPW motif protein 2 (Hey2). We hypothesized that an altered FoxC2-Dll4 signaling underlies saphenous vein wall remodeling in patients with varicose veins. Saphenous veins specimens were collected from 22 patients with varicose veins and 20 control subjects who underwent coronary artery bypass grafting. Tissues were processed for paraffin embedding and sections were immunostained for Dll4, Hey2, EphrinB2, a-SMA, Vimentin, and CD31 antigens and examined under microscope. These observations were confirmed by quantitative real-time PCR and western blot analysis. An examination of varicose vein tissue specimens by immunohistochemistry indicated an elevated expression of Notch pathway components, such as Dll4, Hey2, and EphrinB2, and smooth muscle markers, which was further confirmed by gene and protein expression analyses. We conclude that the molecular alterations in Dll4-Hey2 signaling are associated with smooth muscle cell hypertrophy and hyperplasia in varicose veins. Our observations substantiate a significant role for altered FoxC2-Dll4 signaling in structural alterations of saphenous veins in patients with varicose veins. In conjunction with the presence of genes conferring disease susceptibility, various risk factors, such as prolonged standing, an increased body mass index, and preganancy, increases the mean venous pressure, in the lower extremities. 3,4 Pfisterer et al in 2014 found in an experimental mouse model that an escalated venous filling pressure induces varicose-like venous remodeling. This process to a greater extent mimics detrimental remodeling processes observed in human varicose veins. 5 The precise molecular mechanisms underlying the pathogenesis and progression of varicose veins are unclear. We have earlier observed a significant association of FoxC2 c.-512C4T polymorphism with the presence of varicose veins in patients. FoxC2 was also upregulated at both transcript and protein levels in varicose vein tissues of patients with varicose veins. 6 In venous endothelial cells transfected with FoxC2-overexpressing mammalian vectors, the presence of putative arterial endothelial markers Delta-like ligand 4 (Dll4) and Hairy/enhancer-of-split related with YRPW motif protein 2 (Hey2) were found.
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