Key Regulators of Angiogenesis and Inflammation Are Dysregulated in Patients with Varicose Veins.

Varicose veins (VVs) are the most common manifestation of chronic venous disease (CVD) and appear as abnormally enlarged and tortuous superficial veins. VVs result from functional abnormalities in the venous circulation of the lower extremities, such as venous hypertension, venous valve incompetence, and venous reflux. Previous studies indicate that enhanced angiogenesis and inflammation contribute to the progression and onset of VVs; however, dysregulations in signaling pathways associated with these processes in VVs patients are poorly understood. Therefore, in our study, we aimed to identify key regulators of angiogenesis and inflammation that are dysregulated in patients with VVs. Expression levels of 18 genes were analyzed in peripheral blood mononuclear cells (PBMC) using real-time PCR, as well as plasma levels of 6 proteins were investigated using ELISA. Higher levels of CCL5, PDGFA, VEGFC, TGF-alpha, TGF-beta 1, and VEGF-A, as well as lower levels of VEGFB and VEGF-C, were found to be statistically significant in the VV group compared to the control subjects without VVs. None of the analyzed factors was associated with the venous localization of the varicosities. The presented study identified dysregulations in key angiogenesis- and inflammation-related factors in PBMC and plasma from VVs patients, providing new insight into molecular mechanisms that could contribute to the development of VVs and point out promising candidates for circulatory biomarkers of this disease.

Overexpression of glycolysis markers in placental tissue of pregnant women with chronic venous disease: a histological study.

Chronic Venous Disease (CVD) refers to a wide variety of venous disorders being the varicose veins its most common manifestation. It is well-established the link between pregnancy and the risk of suffering CVD, due to hormonal or haematological factors, especially during the third trimester. In the same manner, previous studies have demonstrated the detrimental effect of this condition in the placental tissue of pregnant women, including in the normal physiology and the metabolomic profile of this organ. In this context, the aim of this study was to evaluate the glucose homeostasis in the placental tissue of women presenting CVD. Through immunohistochemistry, we studied the protein expression of the glucose transporter 1 (GLUT-1), Phosphoglycerate kinase 1 (PGK1), aldolase (ALD), Glyceraldehyde-3-phosphate dehydrogenase (GA3PDH) and lactate dehydrogenase (LDH). Our results have reported a significative increase in the expression of GLUT-1, PGK1, ALD, GA3PDH and the isoenzyme LDHA in placentas of women with CVD. This work has proven for the first-time an altered glucose metabolism in the placental tissue of women affected by CVD, what may aid to understand the pathophysiological mechanisms of this condition in more distant organs such as placenta. Furthermore, our research also supports the basis for further studies in the metabolic phenotyping of the human placenta due to CVD, which may be considered during the late pregnancy in these women.

Phenotypic and Functional Transformation in Smooth Muscle Cells Derived from a Superficial Thrombophlebitis-affected Vein Wall.

BACKGROUND: Superficial thrombophlebitis (ST) is a frequent pathology, but its exact incidence remains to be determined. This study tested the hypothesis whether relationships exist among smooth muscle cells (SMCs) derived from ST, varicose great saphenous veins (VGSVs), and normal great saphenous veins (GSVs). METHODS: Forty-one samples of ST, VGSVs, and GSVs were collected. SMCs were isolated and cultured. Proliferation, migration, adhesion, and senescence in SMCs from the three vein walls were compared by various methods. Bax, Bcl-2, caspase-3, matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 messenger RNA (mRNA) and protein expressions were detected by fluorescence quantitative PCR and Western blot. RESULTS: An obvious decrease in cytoskeletal filaments was observed in thrombophlebitic vascular smooth muscle cells (TVSMCs). The quantity of proliferation, migration, adhesion, and senescence in TVSMCs was significantly higher than in varicose vascular smooth muscle cells and normal vascular smooth muscle cells (NVSMCs) (all P < 0.05). Bax and caspase-3 mRNA and protein expression were decreased, while Bcl-2 mRNA and protein expression were increased in the TVSMCs compared with the varicose vascular smooth muscle cells and the NVSMCs (all P < 0.05). MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA and protein expression were significantly increased in the TVSMCs compared with the VVGSVs and the NVSMCs (all P < 0.05). CONCLUSION: SMCs derived from ST are more dedifferentiated and demonstrate increased cell proliferation, migration, adhesion, and senescence, as well as obviously decreased cytoskeletal filaments. These results suggest that the phenotypic and functional differences could be related to the presence of atrophic and hypertrophic vein segments during the disease course among SMCs derived from ST, VGSVs, and GSVs.

IGFBP6 regulates vascular smooth muscle cell proliferation and morphology via cyclin E-CDK2.

Despite the high prevalence of varicose veins, the underlying pathogenesis of this disease remains unclear. The present study aims to explore the role of insulin-like growth factor binding protein 6 (IGFBP6) in vascular smooth muscle cells (VSMCs). Using a protein array approach, we identified several differentially expressed proteins between varicose great saphenous veins and normal great saphenous veins. Bioinformatic analysis showed that IGFBP6 was closely related to cell proliferation. Further validation confirmed that IGFBP6 was one of the most highly expressed proteins in varicose vein tissue. Knocking down IGFBP6 in VSMCs significantly attenuated cell proliferation and induced the S phase arrest during the cell cycle. Further experiments demonstrated that IGFBP6 knockdown increased cyclin E ubiquitination, which reduced expression of cyclin E and phosphorylation of CDK2. Furthermore, IGFBP6 knockdown arrested centrosome replication, which subsequently influenced VSMC morphology. Ultimately, IGFBP6 was validated to be involved in VSMC proliferation in varicose vein tissues. The present study reveals that IGFBP6 is closely correlated with VSMC biological function and provides unprecedented insights into the underlying pathogenesis of varicose veins.

Role of fibronectin in chronic venous diseases: A review.

Fibronectin (FN) circulating in the blood and produced by cells provides the basis of the extracellular matrix (ECM) formed in healing acute wounds. The time-dependent deposition of FN by macrophages, its synthesis by fibroblasts and myofibroblasts, and later degradation in the remodeled granulation tissue are a prerequisite for successful healing of wounds. However, the pattern of FN expression and deposition in skin lesions is disturbed. The degradation of the ECM components including FN in varicose veins prevails over ECM synthesis and deposition. FN is inconspicuous in the fibrotic lesions in lipodermatosclerosis, while tenascin-C containing FN-like peptide sequences are prominent. FN is produced in large amounts by fibroblasts at the edge of venous ulcers but FN deposition at the wound bed is impaired. Both the proteolytic environment in the wounds and the changed function of the ulcer fibroblasts may be responsible for the poor healing of venous ulcers. The aim of this review is to describe the current knowledge of FN pathophysiology in chronic venous diseases. In view of the fact that FN plays a crucial role in organizing the ECM, further research focused on FN metabolism in venous diseases may bring results applicable to the treatment of the diseases.

Flow cytometric characterization of the saphenous veins endothelial cells in patients with chronic venous disease and in patients undergoing bypass surgery: an exploratory study.

Recent findings have suggested that the primary factors for development of chronic venous disease (CVD), which commonly manifests as varicose veins (VV), are due to structural and biochemical modifications of the vessel wall. The aim of this exploratory study was to characterize by flow cytometry the endothelial cells (EC) mechanically extracted from the varicose saphenous veins (VSV) segments of patients submitted to VV surgery, and to compare the expression of cell surface molecules in these EC with that observed in the EC from the graft SV (GSV) of patients undergoing bypass surgery. EC were isolated from distal- (varicose trunk) and from proximal- (nearly normal) VSV segments of 30 patients submitted to VV surgery, and from proximal GSV segments of 20 patients submitted to bypass surgery (control group), using a mechanical method, and their immunophenotype was characterized by flow cytometry. EC were identified as being CD45negCD146brightCD31bright, and analyzed for expression of activation-related (CD54, CD62E, CD106), procoagulant (CD142), and cell junction (CD31, CD146) molecules, and for the scavenger receptor, CD36. The EC harvested from the SV segments of CVD patients had lower expression of all the molecules evaluated, in comparison to controls; these differences were more evident for the EC isolated from the distal-VSV. The EC extracted from the proximal- and distal-VSV segments of the CVD patients also differ from each other, the first having lower levels of CD62E, CD106, CD142 and CD36. Groups did not match for gender and controls were heterogeneous concerning the underlying pathologies, which may have a confounding effect. Our study revealed that the EC isolated from varicose (distal) and nearly normal (proximal) VSV segments of the CVD patients differ phenotypically from each other, and from the EC of the control group. The VSV segments more affected by the CVD have the lowest expression of the studied markers. We hypothesize that CVD is associated with a decrease on the EC surface molecules, causing EC dysfunctionality. Further studies with a large number of gender-matched participants are needed, to confirm the results obtained in this exploratory study.

Increased Angiogenesis and Lymphangiogenesis in the Placental Villi of Women with Chronic Venous Disease during Pregnancy.

Pregnancy is a period in a woman's life associated with an increased risk of developing lower extremity chronic venous disease (CVD). Pregnancy-associated CVD is associated with changes in placental villi. We investigated angiogenesis and lymphangiogenesis in the placental villi of women with CVD during pregnancy compared with healthy controls with no history of CVD (HC). An observational, analytical, and prospective cohort study was conducted on 114 women in their third trimester of pregnancy (32 weeks). Sixty-two participants were clinically diagnosed with CVD. In parallel, 52 controls with no history of CVD (HC) were studied. Gene and protein expression of CD31, podoplanin (D2-40), Flt-1, and placental growth factor (PIGF) was analysed by real-time polymerase chain reaction (RT-qPCR) and immunohistochemistry. CD31 and D2-40 gene expression was significantly greater in the placental villi of women with CVD, as were the numbers of vessels positive for CD31 and D2-40. Significantly higher gene and protein expression of Flt-1 and PIGF was observed in the placental villi of women with CVD. Histological analysis showed more placental villi with periodic acid of Schiff (PAS)-positive material in women with CVD. Our results show a connection between pregnancy-associated CVD and leading to higher proangiogenic and lymphangiogenic activity in placental villi.

Augmentation of miR-202 in varicose veins modulates phenotypic transition of vascular smooth muscle cells by targeting proliferator-activated receptor-γ coactivator-1α.

In varicose veins, vascular smooth muscle cells (VSMCs) often show abnormal proliferative and migratory rates and phenotypic transition. This study aimed to investigate whether microRNA (miR)-202 and its potential target, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), were involved in VSMC phenotypic transition. miR-202 expression was analyzed in varicose veins and in VSMCs conditioned with platelet-derived growth factor. The effect of miR-202 on cell proliferation and migration was assessed. Furthermore, contractile marker SM-22α, synthetic markers vimentin and collagen I, and PGC-1α were analyzed by Western blot analysis. The modulation of PGC-1α expression by miR-202 was also evaluated. In varicose veins and proliferative VSMCs, miR-202 expression was upregulated, with decreased SM-22α expression and increased vimentin and collagen I expression. Transfection with a miR-202 mimic induced VSMC proliferation and migration, whereas a miR-202 inhibitor reduced cell proliferation and migration. miR-202 mimic constrained luciferase activity in HEK293 cells that were cotransfected with the PGC-1α 3'-untranslated region (3'-UTR) but not those with mutated 3'-UTR. miR-202 suppressed PGC-1α protein expression, with no influence on its messenger RNA expression. PGC-1α mediated VSMC phenotypic transition and was correlated with reactive oxygen species production. In conclusion, miR-202 affects VSMC phenotypic transition by targeting PGC-1α expression, providing a novel target for varicose vein therapy.

Upregulation of epidermal gap junctional proteins in patients with venous disease.

BACKGROUND: Leg ulceration is a feared complication of venous insufficiency. It is not known whether varicose veins predispose skin to poor wound healing. The expression pattern of gap junctional protein connexin, a known marker of poor wound healing, was investigated across various stages of venous disease. METHODS: Patients undergoing intervention for varicose veins were assessed according to the Clinical Etiologic Anatomic Pathophysiologic (CEAP) classification of varicose veins. Paired 4-mm punch biopsies were taken from above the ankle (pathological) and above the knee (control). Tissues were stained with haematoxylin and eosin, and for connexin 43, connexin 30 and connexin 26. RESULTS: Forty-eight paired biopsies were taken (12 each for CEAP class C0, C2, C4 and C6). The pathological skin showed progressive epithelial hyperthickening, an increase in the number and depth of rete ridges, increased inflammation and loss of dermal architecture with disease progression from C4 onwards. The overall absolute connexin expression and mean connexin expression per cell in the pathological skin similarly increased across the CEAP classes from as early as C2. Increasing levels of connexin in control skin were also noted, indicating progression of the disease proximally. Connexin 43 expression showed the strongest positive correlation between pathological and control skin. CONCLUSION: Connexins were overexpressed in patients with simple varicose veins, with a stepwise increased expression through venous eczema to ulceration. Connexin 43 is a potential biomarker for venous disease. This finding suggests that varicose veins predispose skin to poor wound healing. Surgical relevance The overexpression of connexins, a family of gap junctional proteins, is known to cause poor healing in venous leg ulceration. It is not known whether there is any association with superficial venous disease. Here, connexin proteins were overexpressed in patients with uncomplicated varicose veins, before histological skin changes. Connexin could be a biomarker of venous disease progression.

Innate Effector-Memory T-Cell Activation Regulates Post-Thrombotic Vein Wall Inflammation and Thrombus Resolution.

RATIONALE: Immune cells play an important role during the generation and resolution of thrombosis. T cells are powerful regulators of immune and nonimmune cell function, however, their role in sterile inflammation in venous thrombosis has not been systematically examined. OBJECTIVE: This study investigated the recruitment, activation, and inflammatory activity of T cells in deep vein thrombosis and its consequences for venous thrombus resolution. METHODS AND RESULTS: CD4+ and CD8+ T cells infiltrate the thrombus and vein wall rapidly on deep vein thrombosis induction and remain in the tissue throughout the thrombus resolution. In the vein wall, recruited T cells largely consist of effector-memory T (TEM) cells. Using T-cell receptor transgenic reporter mice, we demonstrate that deep vein thrombosis-recruited TEM receive an immediate antigen-independent activation and produce IFN-γ (interferon) in situ. Mapping inflammatory conditions in the thrombotic vein, we identify a set of deep vein thrombosis upregulated cytokines and chemokines that synergize to induce antigen-independent IFN-γ production in CD4+ and CD8+ TEM cells. Reducing the number of TEM cells through a depletion recovery procedure, we show that intravenous TEM activation determines neutrophil and monocyte recruitment and delays thrombus neovascularization and resolution. Examining T-cell recruitment in human venous stasis, we show that superficial varicose veins preferentially contain activated memory T cells. CONCLUSIONS: TEM orchestrate the inflammatory response in venous thrombosis affecting thrombus resolution.

The Role of Endothelial Dysfunction and Inflammation in Chronic Venous Disease.

Chronic venous disease is a potentially prevalent and debilitating condition affecting millions of individuals, mostly in Western world. Predisposing genetic and environmental factors contribute to its development. However, the main etiology remains to be elucidated. An extensive literature search was conducted in Medline using the following key words algorithm: ("Chronic venous disease" OR "Chronic venous insufficiency" OR "varicose veins") AND ("endothelial dysfunction" OR "inflammation"). Besides being a multifactorial disease, it is now recognized that the hallmark of chronic venous disease pathophysiology likely remains in inflammation, possibly triggered by sustained venous hypertension and valvular incompetence. Shear stress changes are directly sensed by endothelial cells, leading to its activation and subsequent recruitment of leukocytes and release of proinflammatory agents. Dysfunctional endothelium has a pivotal role perpetuating the inflammatory cascade, with consequent pathological venous changes and chronic venous disease worsening. Endothelial dysfunction may be the central player in the link between varicose veins and deep vein thrombosis. In this article, we aim to analyze the crucial role of endothelial activation in the persistent inflammatory cycle that characterizes chronic venous disease.

The Morphofunctional Changes in the Wall of Varicose Veins.

BACKGROUND: Varicose vein (VV) disease is a frequently occurring pathology of the lower extremities. Although the pathogenesis of varicosity development is not clearly defined, the final common pathway leading to chronic venous insufficiency is the development of venous hypertension, which is associated with severe changes in the venous wall. The aim of this study was to clarify the histological and immunohistochemical changes in great saphenous veins (GSVs) in chronic venous insufficiency. METHODS: A histopathological study was conducted on 20 patients with VVs (4 males, 16 females) and 4 (1 male, 3 females) patients undergoing distal bypass surgery. Tissues were processed for histological routine straining and immunohistochemical studies of vascular endothelial growth factor (VEGF), intercellular adhesion molecule (ICAM)-1, vascular adhesion molecule (VCAM)-1, protein gene product 9.5 (PGP 9.5), and collagen type IV, laminin, and fibronectin. A semiquantitative evaluation method was used. RESULTS: Compared with the normal SV, VV sections showed the damaged endothelium areas, significant disorganization of the smooth muscle bundles, and highest density of the vasa vasorum in the tunica media and tunica adventitia, as well as sclerotic blood vessels and neoangiogenesis in almost all specimens. Immunohistochemistry study showed statistically significant difference between the VVs and the control group of several parameters, such as PGP 9.5 positive structures (P < 0.05; 1-tailed significance) and laminin positive structures in subendothelial layer of VVs (P < 0.05; 1-tailed significance). There is also the tendency in increasing of VEGF expression and decreasing of collagen IV structures. Our study did not show statistically significant difference in VEGF, ICAM-1, and VCAM-1 positive structures between varicose and normal veins; however, it could be explained by the limitations of the study. CONCLUSIONS: Varicose GSVs represent nonhomogeneous integrity of the basement membrane, smooth muscle disorganization, and active neoangiogenesis, suggesting remodulation of blood vessels. Changes in the appearance of PGP 9.5-containing innervation, laminin, and collagen IV in tunica intima confirm the remodulation of damaged blood vessels.

TGF-ß1 in Vascular Wall Pathology: Unraveling Chronic Venous Insufficiency Pathophysiology.

Chronic venous insufficiency and varicose veins occur commonly in affluent countries and are a socioeconomic burden. However, there remains a relative lack of knowledge about venous pathophysiology. Various theories have been suggested, yet the molecular sequence of events is poorly understood. Transforming growth factor-beta one (TGF-ß1) is a highly complex polypeptide with multifunctional properties that has an active role during embryonic development, in adult organ physiology and in the pathophysiology of major diseases, including cancer and various autoimmune, fibrotic and cardiovascular diseases. Therefore, an emphasis on understanding its signaling pathways (and possible disruptions) will be an essential requirement for a better comprehension and management of specific diseases. This review aims at shedding more light on venous pathophysiology by describing the TGF-ß1 structure, function, activation and signaling, and providing an overview of how this growth factor and disturbances in its signaling pathway may contribute to specific pathological processes concerning the vessel wall which, in turn, may have a role in chronic venous insufficiency.

Variability of MMP/TIMP and TGF-ß1 Receptors throughout the Clinical Progression of Chronic Venous Disease.

Chronic venous disease (CVeD) is a prevalent condition with a significant socioeconomic burden, yet the pathophysiology is only just beginning to be understood. Previous studies concerning the dysregulation of matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases (TIMPs)) within the varicose vein wall are inconsistent and disregard clinical progression. Moreover, it is highly plausible that MMP and TIMP expression/activity is affected by transforming growth factor (TGF)-ß1 and its signaling receptors (TGFßRs) expression/activity in the vein wall. A case-control study was undertaken to analyze genetic and immunohistochemical differences between healthy (n = 13) and CVeD (early stages: n = 19; advanced stages: n = 12) great saphenous vein samples. Samples were grouped based on anatomic harvest site and subjected to quantitative polymerase chain reaction for MMP1, MMP2, MMP8, MMP9, MMP12, MMP13, TIMP1, TIMP2, TIMP3, TIMP4, TGFßR1, TGFßR2, and TGFßR3 gene expression analysis, and then to immunohistochemistry for immunolocalization of MMP2, TIMP2, and TGFßR2. Decreased gene expression of MMP12, TIMP2, TIMP3, TIMP4, and TGFßR2 was found in varicose veins when compared to controls. Regarding CVeD clinical progression, two facts arose: results across anatomical regions were uneven; decreased gene expression of MMP9 and TGFßR3 and increased gene expression of MMP2 and TIMP3 were found in advanced clinical stages. Most immunohistochemistry results for tunica intima were coherent with qPCR results. In conclusion, decreased expression of TGFßRs might suggest a reduction in TGF-ß1 participation in the MMP/TIMP imbalance throughout CVeD progression. Further studies about molecular events in the varicose vein wall are required and should take into consideration the venous anatomical region and CVeD clinical progression.

Arterialization and anomalous vein wall remodeling in varicose veins is associated with upregulated FoxC2-Dll4 pathway.

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.-512C>T 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, α-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.

Estrogen Receptors and Chronic Venous Disease.

OBJECTIVE/BACKGROUND: Chronic venous disease (CVD) is a common and relevant problem affecting Western people. The role of estrogens and their receptors in the venous wall seems to support the major prevalence of CVD in women. The effects of the estrogens are mediated by three estrogen receptors (ERs): ERα, ERß, and G protein-coupled ER (GPER). The expression of ERs in the vessel walls of varicose veins is evaluated. METHODS: In this prospective study, patients of both sexes, with CVD and varicose veins undergoing open venous surgery procedures, were enrolled in order to obtain vein samples. To obtain control samples of healthy veins, patients of both sexes without CVD undergoing coronary artery bypass grafting with autologous saphenous vein were recruited (control group). Samples were processed in order to evaluate gene expression. RESULTS: Forty patients with CVD (10 men [25%], 30 women [75%], mean age 54.3 years [median 52 years, range 33-74 years]) were enrolled. Five patients without CVD (three men, two women [aged 61-73 years]) were enrolled as the control group. A significant increase of tissue expression of ERα, ERß and GPER in patients with CVD was recorded (p < .01), which was also related to the severity of venous disease. CONCLUSION: ERs seem to play a role in CVD; in this study, the expression of ERs correlated with the severity of the disease, and their expression was correlated with the clinical stage.

Evaluation of apoptosis in varicose vein disease complicated by superficial vein thrombosis.

BACKGROUND: The factors contributing to superficial vein thrombosis (SVT) in patients with varicose vein disease are unclear. Differences in vein wall apoptotic activity could be associated with the pathogenesis of SVT. The aim of the study is to address the role of the programmed cell death in the vein wall by comparing varicose veins with history of SVT to uncomplicated varicose veins. PATIENTS AND METHODS: Vein segments from the proximal part of the great saphenous vein (GSV), the distal part of the vein and from a varicose tributary, from 16 patients with varicose vein disease and one episode of SVT, were evaluated for the immunohistochemical expression of pro-apoptotic (Bax, p53, Caspase 3, BCL-6, BCL-xs), anti-apoptotic (BCL-xl and BCL-2) and proliferation (Ki-67) markers. The results of this study were compared to the results from the evaluation of 19 patients suffering from uncomplicated varicose vein disease and 10 healthy GSVs as controls. RESULTS: Overall, there was increased apoptosis in the distal part of GSV compared to the proximal part documented by increased expression of Bax (p < 0.01), Caspase 3 (p = 0.01), BCL-xs (p < 0.01). The comparisons of the markers' expression between patients with varicose veins and patients with a history of SVT showed significant differences among the three different anatomic locations. In the proximal GSV, only BCL-xs was higher in patients with SVT (p = 0.029). In the tributaries, Bax, BCL-xl and Ki-67 were higher in patients with SVT (p < 0.01). In the distal GSV, increased Bax, BCL-xs, BCL-xl and Ki-67 staining was observed in the thrombosis group compared to uncomplicated veins (p < 0.01). CONCLUSIONS: The vein wall in SVT shows increased pro-apoptotic activity compared to uncomplicated disease and normal veins. Whether increased vein wall cell apoptosis is a causative factor for SVT in varicose veins disease or a repairing mechanism of the thrombosis itself needs further research.

Loci cg06256735 and cg15815843 in the MFAP5 gene regulatory regions are hypomethylated in varicose veins apparently due to active demethylation.

Varicose vein disease (VVD) is a common health problem worldwide. Microfibril-associated protein 5 (MFAP5) is one of the potential key players in its pathogenesis. Our previous microarray analysis revealed the cg06256735 and cg15815843 loci in the regulatory regions of the MFAP5 gene as hypomethylated in varicose veins which correlated with its up-regulation. The aim of this work was to validate preliminary microarray data, estimate the level of 5-hydroxymethylcytosine (5hmC) at these loci, and determine the methylation status of one of them in different layers of the venous wall. For this, methyl- and hydroxymethyl-sensitive restriction techniques were used followed by real-time PCR and droplet digital PCR, correspondingly, as well as bisulfite pyrosequencing of +/- oxidized DNA. Our microarray data on hypomethylation at the cg06256735 and cg15815843 loci in whole varicose vein segments were confirmed and it was also demonstrated that the level of 5hmC at these loci is increased in VVD. Specifically, among other layers of the venous wall, tunica (t.) intima is the main contributor to hypomethylation at the cg06256735 locus in varicose veins. Thus, it was shown that hypomethylation at the cg06256735 and cg15815843 loci takes place in VVD, with evidence to suggest that it happens through their active demethylation leading to up-regulation of the MFAP5 gene, and t. intima is most involved in this biochemical process.

Mechanoresponsive ETS1 causes endothelial dysfunction and arterialization in varicose veins via NOTCH4/DLL4 signaling.

Varicose veins are the most common venous disorder in humans and are characterized by hemodynamic instability due to valvular insufficiency and orthostatic lifestyle factors. It is unclear how changes in biomechanical signals cause aberrant remodeling of the vein wall. Our previous studies suggest that Notch signaling is implicated in varicose vein arterialization. In the arterial system, mechanoresponsive ETS1 is a transcriptional activator of the endothelial Notch, but its involvement in sensing disrupted venous flow and varicose vein formation has not been investigated. Here, we use human varicose veins and cultured human venous endothelial cells to show that disturbed venous shear stress activates ETS1-NOTCH4/DLL4 signaling. Notch components were highly expressed in the neointima, whereas ETS1 was upregulated in all histological layers of varicose veins. In vitro microfluidic flow-based studies demonstrate that even minute changes in venous flow patterns enhance ETS1-NOTCH4/DLL4 signaling. Uniform venous shear stress, albeit an inherently low-flow system, does not induce ETS1 and Notch proteins. ETS1 activation under altered flow was mediated primarily by MEK1/2 and, to a lesser extent, by MEK5 but was independent of p38 MAP kinase. Endothelial cell-specific ETS1 knockdown prevented disturbed flow-induced NOTCH4/DLL4 expression. TK216, an inhibitor of ETS-family, prevented the acquisition of arterial molecular identity and loss of endothelial integrity in cells exposed to the ensuing altered shear stress. We conclude that ETS1 senses blood flow disturbances and may promote venous remodeling by inducing endothelial dysfunction. Targeting ETS1 rather than downstream Notch proteins could be an effective and safe strategy to develop varicose vein therapies.

Morphological study of incompetent saphenous veins: apoptosis and ultrastructural changes of smooth muscle cells.

BACKGROUND: Varicose veins affect approximately 25% of people in industrialized countries. METHODS: The study aimed at detecting apoptotic cells and histopathological changes in varicose vein walls. Patients (N.=41) with varicose veins and 30 control group patients were divided into two groups according to their age (younger and older than 50 years). Apoptosis was determined by the TUNEL assay, elastin and collagen IV expression by immunohistochemistry and ultrastructural changes by transmission electron microscopy. RESULTS: The results show that the number of apoptotic cells in the layers of varicose veins increased, in particular in a group of patients aged over 50 years. In the varicose veins as compared to control veins the elastic fibers were found to be thinner, more fragmented and disorderly arranged. Elastin and collagen IV expression was found to decline in the intima and the media of varicose veins in both age groups. Electron microscopy demonstrated hypertrophy and degeneration of smooth muscle cells. Furthermore, cells with ultrastructural feature of apoptosis were noted. In the disorganized and expanded extracellular matrix membrane-bound vesicles, ghost bodies with different size and electron density were observed. Ghost bodies seem to bud off from smooth muscle cells and are likely to be involved in extracellular matrix remodeling as they are seen in close contact with collagen fibers. CONCLUSIONS: The study demonstrates increase of apoptotic cells in the wall of varicose veins along with vein wall structural abnormalities including alterations of smooth muscle cells and decline of elastin and collagen IV expression.