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.

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.

Monocyte chemoattractant protein 1 plasma concentration in blood from varicose veins decreases under venoactive drug treatment.

BACKGROUND: Vein-specific inflammation leads to vascular smooth muscle cells proliferation and extracellular matrix degradation of vein wall. This process is known as remodeling and is promoted by "trapped" leukocytes. Monocyte chemoattractant protein 1 (MCP-1) is a chemokine responsible for trafficking of leukocytes from blood to vein wall. The aim of this study was to measure the MCP-1 concentration in varicose veins blood before and after venoactive drug therapy and to compare it with a concentration of blood from varicose veins of subjects who did not receive drug treatment. METHODS: Non-randomized comparative study was conducted on 30 patients with primary varicose veins. 20 patients of the study group received diosmin 900 mg/hesperidin 100 mg once daily. 10 controls received no treatment. MCP-1 level was measured (pg/mL) in the blood from varicose veins twice, at the day of inclusion and after 60 days. Legs discomfort related to chronic venous disease (CVD) symptoms was measured with 10-cm Visual Analogue Scale (VAS) at inclusion and at completion of the study. RESULTS: Median (interquartile range, IQR) MCP-1 concentrations in treatment and control groups at inclusion were 171.9 (124.4-216.0) and 157.0 (120.1-163.1), resp., P=0.285. After 60 days of treatment MCP-1 level decreased, but non-significantly to 152.3 (124.1-178.3). In patients who did not receive treatment chemokine level slightly increased to 163.0 (134.0-172.9). Median changes over time were -6.6 (-30.9-7.4) and 10.6 (-3.7-19.2) in the study and control groups, resp. (P=0.048). After 60 days in 12 of 19 and 2 of 9 patients of treatments and control groups MCP-1 decreased (P=0.103). Odds ratio for MCP-1 decreasing was 9.5 (95% CI 1.1-81.5, P=0.043) for those who received venoactive drug. Mean (± standard deviation [SD]) legs discomfort significantly dropped in the study group from 5.7 (±2.5) to 1.9 (±2.2) (P=0.0003), while in controls no changes were registered: 3.4 (±1.3) and 3.5 (± 1.4), resp., P=0.28). Mean difference of VAS at baseline and at follow-up was -3.5 (±2.6) and 0.9 (±2.1), resp. (P<0.0001). CONCLUSIONS: Plasma concentration of MCP-1 in varicose veins blood demonstrates a tendency to decrease under two months treatment with a venoactive drug. Future studies are needed to reveal a possible role of MCP-1 as a target considering its role in varicose veins pathogenesis.

Biomechanical stretch-induced CLOCK upregulation in venous smooth muscle cells promotes phenotypic and functional transformation.

BACKGROUND: A chronic change in hemodynamic forces might activate the pathophysiological process of maladaptive venous remodeling. Biomechanical stretching stimulates venous smooth muscle cells (SMCs) in the media, and biomechanical loads exceeding physiological levels affect the intrinsic circadian rhythm and cellular phenotype. This study aimed to investigate the changes in the expression patterns of circadian clock genes under biomechanical stretching and their role in the regulation of the SMC phenotype. METHODS: Circadian genes were detected in venous specimens and venous SMCs from patients with varicose veins (VVs) and patients with autologous vein grafts (normal veins). Molecular mechanism studies of SMC phenotypic switching under biomechanical stretching were performed in human umbilical venous SMCs (HUVSMCs). RESULTS: CLOCK upregulation was observed in VVs. The circadian rhythm was disrupted in venous SMCs derived from VVs. In addition, CLOCK expression and cell proliferation and migration were increased in HUVSMCs exposed to biomechanical stretch. CLOCK overexpression activated NF-κB signaling and phenotypic transformation in HUVSMCs, whereas CLOCK depletion had inhibitory effects on these pathways. Further experiments revealed that the CLOCK protein regulates phenotypic and functional transformation via the RHOA/ROCK1 pathway. CONCLUSIONS: Our results demonstrate that CLOCK is a crucial regulator of the SMC phenotype under mechanical stretch. The CLOCK/RHOA/ROCK1 pathway is important in phenotypic adaptation, and targeting RHOA/ROCK1 could potentially reverse stretch-induced phenotypic switching.

Genome-wide association analysis and replication in 810,625 individuals with varicose veins.

Varicose veins affect one-third of Western society, with a significant subset of patients developing venous ulceration, costing $14.9 billion annually in the USA. Current management consists of either compression stockings, or surgical ablation for more advanced disease. Most varicose veins patients report a positive family history, and heritability is ~17%. We describe the largest two-stage genome-wide association study of varicose veins in 401,656 individuals from UK Biobank, and replication in 408,969 individuals from 23andMe (total 135,514 cases and 675,111 controls). Forty-nine signals at 46 susceptibility loci were discovered. We map 237 genes to these loci, several of which are biologically plausible and tractable to therapeutic targeting. Pathway analysis identified enrichment in extracellular matrix biology, inflammation, (lymph)angiogenesis, vascular smooth muscle cell migration, and apoptosis. Using a polygenic risk score (PRS) derived in an independent cohort, we demonstrate its predictive utility and correlation with varicose veins surgery.

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.

Shear Stress Alterations Activate BMP4/pSMAD5 Signaling and Induce Endothelial Mesenchymal Transition in Varicose Veins.

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. TGFß 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.

Elevated c-fos expression is correlated with phenotypic switching of human vascular smooth muscle cells derived from lower limb venous varicosities.

BACKGROUND: Lower limb venous varicosities (VVs) are clinically common; however, their molecular underpinnings are far from well elucidated. Previous studies have demonstrated that the phenotypic transition of vascular smooth muscle cells (VSMCs) plays a critical role in VV pathogenesis and that c-fos is upregulated in VSMCs from VVs. The present study investigated the histologic and cytologic changes in VVs and the correlation between c-fos upregulation and VSMC phenotypic switching. METHODS: Thirty-four patients with VVs (VV group) and 13 patients undergoing coronary artery bypass using autologous great saphenous vein segments (normal vein [NV] group) were enrolled in the present study. The great saphenous veins of both groups were harvested for subsequent experiments. Hematoxylin and eosin staining was performed for vein morphologic analysis. Real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blot assays were used to assess mRNA and protein expression of c-fos, α-smooth muscle actin (α-SMA), and osteopontin (OPN). Simple linear regression was used to evaluate the correlation between c-fos and OPN/α-SMA. Primary VSMCs were isolated from both groups and cultured in vitro. A cell counting kit-8 assay and scratch-wound assay were used to analyze the proliferation and migration abilities of the cells, respectively. RESULTS: The mean age of the patients in the NV and VV groups was 61.4 ± 3.8 years and 59.5 ± 10.4 years, respectively. The vein cavities of the VV group were dilated, and the arrangement of the cells was disordered. The tunica media of the VV group was thicker than that of the NV group owing to the accumulation and proliferation of VSMCs. Significantly elevated mRNA levels of c-fos and OPN were observed in the VV group compared with the NV group, and a positive correlation was further demonstrated between the mRNA levels of c-fos and OPN/α-SMA (R2, 0.5524; P < .001). The VSMCs derived from the VV group were more numerous (as shown by the cell counting kit-8 assay) and had a significantly greater migration speed (as shown by the scratch-wound assay) than those derived from the NV group. Moreover, the protein expression of c-fos was significantly upregulated in VSMCs derived from the VV group, and this change was accompanied by a decrease in α-SMA and an increase in OPN expression. CONCLUSIONS: Both mRNA and protein expression of c-fos were upregulated in VV specimens, and the phenotypic biomarkers (OPN/α-SMA) were altered concurrently. VSMCs derived from VVs showed increased proliferation and migration abilities. Upregulation of c-fos might play a role in the phenotypic switching of VSMCs and subsequently participate in the pathogenesis of VVs. CLINICAL RELEVANCE: C-fos is an immediate early gene owing to the transient and rapid change in its expression in response to stimuli. It is involved in the regulation of cell proliferation, cell growth, and cell movement. In the present study, varicose vein specimens showed increased mRNA and protein expression of c-fos, accompanied by altered phenotypic biomarkers. The upregulation of the c-fos gene in smooth muscle cells cultured from varicose vein specimens might be associated with phenotypic switching and functional disturbance. These results could contribute to the exploration of the molecular mechanisms underlying the pathogenesis of varicose veins and the development of new therapeutic strategies.

Compression stockings attenuate the expression of proteins associated with vascular damage in human varicose veins.

OBJECTIVE: The objective of this study was to analyze whether compression stocking therapy in the human varicose vein wall may change the levels of biomarkers associated with vein insufficiency. METHODS: Dilated collateral varicose vein samples were obtained from patients showing chronic venous disease (class 2 of the Clinical, Etiology, Anatomy, and Pathophysiology classification). Before elective surgery, 12 patients underwent compression stocking therapy (for 1 month) and 9 patients did not (control group). Expression levels of biomarkers associated with endothelial functionality (nitric oxide synthase 3), inflammation (interleukin-6, interleukin-10), oxidative stress (Gp91phox subunit of NADPH oxidase), and coagulation (factor Xa) were determined. P-selectin, an inflammatory and thrombosis-related biomarker, was also measured. RESULTS: Compression stockings increased the content of nitric oxide synthase 3 (control, 16.48 [16.04-17.40] AU; compression, 83.71 [67.70-91.85] AU; P < .001) in the varicose vein wall that was accompanied by reduction of both interleukin-6 levels (control, 38.72 [33.48-48.52] pg/µg protein; compression, 14.49 [11.05-17.41] pg/µg protein; P = .001) and the expression of Gp91phox subunit of NADPH oxidase (control, 63.24 [53.79-77.03] AU; compression, 36.85 [35.66-52.27] AU; P < .010). P-selectin (control, 77.37 [61.86-85.00] AU; compression, 54.31 [49.60-67.50] AU; P = .017) and factor Xa (control, 90.78 [75.02-100.00] AU; compression, 14.50 [13.77-36.20] AU; P < .001) were also reduced in the varicose vein wall of compression stocking-treated patients. However, P-selectin lost its statistical significance after adjustment by dyslipidemia. CONCLUSIONS: In the varicose vein wall, compression stocking therapy improved the content levels of biomarkers associated with endothelial functionality, inflammation, oxidative stress, and coagulation.

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.

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.

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.

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.

Disequilibrium in MMPs and the tissue inhibitor of metalloproteinases in different segments of the varicose great saphenous vein wall.

BACKGROUND: Varicose great saphenous veins (VGSVs) are a common disorder with a high incidence, but the pathogenesis is unclear. This study was designed to measure the changes in matrix metalloproteinases (MMPs) and the tissue inhibitor of metalloproteinases (TIMPs) in different segments from VGSV walls to determine the relationship between MMPs, TIMPs expression, and expansion of the venous wall. METHODS: Twenty-one VGSV and 12 normal great saphenous vein (GSV) specimens were collected. Venous walls in the two groups, expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 proteins, protein-positive expression ratios, mRNA expression, and protein content were determined by immunohistochemistry, PCR, and western blot. RESULTS: The MMP-2, MMP-9, TIMP-1, and TIMP-2 protein-positive expression ratios, mRNA expression in the upper, middle, and lower segments in the VGSV group were significantly higher than the corresponding regions in the GSV group, respectively. The MMP-2, MMP-9, TIMP-1, and TIMP-2 protein-positive expression ratios, mRNA expression, and protein concentrations in the lower segments in the VGSV group were also significantly higher than the upper and middle segments in the VGSV group and the corresponding regions in the GSV group, respectively. CONCLUSIONS: Under high hemodynamics, disequilibrium of MMPs and TIMPs from VGSVs exists within the upper, middle, and lower segments of VGSVs. These results suggested that MMPs and TIMPs participate in the process of venous wall remodeling and may be one of the mechanisms associated with the formation and development in varicose veins.

Cold-Stored Venous Allografts In Different Preserving Solutions: A Study On Changes In Vein Wall Morphology.

BACKGROUND:: The saphenous vein is the most frequently used bypass conduit for vascular reconstructions, which may need to be stored for a prolonged time. The aim of this study was to compare the effect of different preservation solutions on the morphology of saphenous veins during the long-term cold storage. DESIGN:: An in vitro study. MATERIAL AND METHODS:: Saphenous vein samples, collected from 29 patients undergoing varicose vein surgery, were stored at +4°C in (1) 10% formalin, (2) isotonic saline with heparin and antibiotic, (3) phosphate-buffered saline, (4) 2.5% glutaraldehyde + phosphate-buffered saline, and (5) Custodiol (histidine-tryptophan-ketoglutarate). Changes in the vein wall were histologically investigated up to day 35. Possible retention of the capacity of endothelial function was evaluated by immunohistochemical detection of endothelial nitric oxide synthase. RESULTS:: Formalin as the control medium well preserved the vein wall morphology, but endothelial nitric oxide synthase immunostaining was very weak. Phosphate-buffered saline and isotonic saline with heparin and antibiotic poorly preserved vein wall morphology. Phosphate-buffered saline endothelial nitric oxide synthase staining decreased dramatically throughout the study period. Compared to phosphate-buffered saline, stronger isotonic saline with heparin and antibiotic endothelial nitric oxide synthase staining was noted at day 35 (p < 0.001). Custodiol and glutaraldehyde better preserved vein morphology compared to ISHA and PBS at day 5 and later (p < 0.001), but compared to stronger isotonic saline with heparin and antibiotic their endothelial nitric oxide synthase staining was weaker. CONCLUSION:: In terms of preserving the morphology of saphenous veins, phosphate-buffered saline and isotonic saline with heparin and antibiotic were the poorest, while Custodiol and glutaraldehyde were the best. Demonstrating good retention of endothelial nitric oxide synthase staining throughout the study period, isotonic saline with heparin and antibiotic seems to have the best potential to retain vein wall functionality, despite relatively poor morphological preservation.

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.