Abstract
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.
Highlights
Chronic venous disease (CVD) of the lower extremities is one of the most prevalent diseases worldwide though the prevalence estimates differ extensively due to the different disease evaluation methods
Distribution of genetic variants in 59, 39 flanking regions and coding sequence of forkhead box C2 (FoxC2) gene in patients with CVD and healthy controls are presented in tables 3 and 4
Human FoxC2 is a forkhead/winged helix transcription factor coding gene located on chromosome 16q24.1 (NCBI Reference Sequence No: NG_012025.1)
Summary
Chronic venous disease (CVD) of the lower extremities is one of the most prevalent diseases worldwide though the prevalence estimates differ extensively due to the different disease evaluation methods. CVD comprises of visible venous disorders which are not associated with an identifiable mechanism of venous dysfunction [1]. They are manifested by a variety of signs ranging from telangiectasis and varicose veins to venous ulceration. CVD is generally termed as varicose veins, that being the most common form of clinical manifestation. The great saphenous vein and its tributaries are the anatomic sites frequently affected in CVD of lower limbs [2]. Structural failures of vein such as valve weakness or vein wall dilatation may result in venous retrograde flow in limb leading to distal high venous pressure causing CVD. The primary events resulting in valvular incompetence and primary vein wall changes are not yet elucidated
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