Chronic venous diseases, including varicose veins, are characterized by hemodynamic disturbances due to valve defects, venous insufficiency, and orthostatism. Veins are physiologically low shear stress systems, and how altered hemodynamics drives focal endothelial dysfunction and causes venous remodeling is unknown. Here we demonstrate the occurrence of endothelial to mesenchymal transition (EndMT) in human varicose veins. Moreover, the BMP4-pSMAD5 pathway was robustly upregulated in varicose veins. In vitro flow-based assays using human vein, endothelial cells cultured in microfluidic chambers show that even minimal disturbances in shear stress as may occur in early stages of venous insufficiency induce BMP4-pSMAD5-based phenotype switching. Furthermore, low shear stress at uniform laminar pattern does not induce EndMT in venous endothelial cells. Targeting the BMP4-pSMAD5 pathway with small molecule inhibitor LDN193189 reduced SNAI1/2 expression in venous endothelial cells exposed to disturbed flow. TGFβ inhibitor SB505124 was less efficient in inhibiting EndMT in venous endothelial cells exposed to disturbed flow. We conclude that disturbed shear stress, even in the absence of any oscillatory flow, induces EndMT in varicose veins via activation of BMP4/pSMAD5-SNAI1/2 signaling. The present findings serve as a rationale for the possible use of small molecular mechanotherapeutics in the management of varicose veins.

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 (pT (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.

Given the high prevalence of chronic venous diseases (CVD), defining criteria to screen patients who are in need for intervention is attaining primacy. An important clinical criterion for treating CVD is incompetence of larger veins. We have assessed the association of size of afflicted veins with disease severity in patients with CVD to define an acceptable criterion to identify patients who need intervention. Demographic characteristics and risk factors were recorded from 6350 patients. Based on physical examination and venous duplex ultrasound study, patients were classified into clinical severity, etiology, anatomy, and pathophysiology (CEAP) classes and grouped according to the size of the veins which had varicosities. Patients with reflux in smaller veins (vein size < 4 mm diameter) were considered as type I and those with varicosities in truncal veins (> 4 mm diameter) as type II. Risk ratio was determined by multivariate regression analysis. About 47.67% of patients in this study were found to have CEAP class 3 disease. Compared with varicose veins of large truncal veins, patients with varicosities in smaller superficial veins had 2.85-fold (p < 0.01) more risk of edema and 5.71-fold (p < 0.01) higher prevalence of hyperpigmentation. Varicosities in small superficial veins were associated with higher risk of ulceration (odds ratio 3.93, 95% confidence interval 2.51-6.18) compared with truncal vein reflux. Our study reveals that presence of small varicose veins in patients without truncal saphenous reflux involvement is associated with severe manifestations of venous insufficiency such as edema and skin lesions even in the absence of varicosities in truncal saphenous veins.