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.

Polymorphisms in inflammation-related genes and the risk of primary varicose veins in ethnic Russians.

Inflammation was shown to be activated in varicose veins, although its role in the development of vein wall transformation remains inconclusive. We aimed to investigate the influence of 13 inflammation-related single nucleotide polymorphisms (SNPs) TNF rs1800629 and rs3093661, IL1A rs1800587, IL1RN rs4251961, IL6 rs1800795 and rs1800796, IFNG rs2430561, IL10 rs1800896, TGFB1 rs1800469, HIF1A rs11549465, NFKB1 rs28362491, and rs4648068 on the risk of primary varicose veins (PVVs) in ethnic Russians. We genotyped 709 patients with PVVs and 278 individuals without a history of chronic venous disease and performed a single SNP and a haplotype analysis. Several associations with P < 0.05 were revealed in our study. Variant allele HIF1A rs11549465 T, TNF rs3093661 A, and NFKB1 rs28362491 ATTG deletion showed the reverse association with PVV risk, and allele IL6 rs1800795 C was associated with the increased risk of the studied pathology. Haplotype analysis revealed associations of TNF haplotypes rs3093661 A-rs1800629 G and IL6 rs1800795 C-rs1800796 G with the decreased and the increased risk of PVVs, correspondingly. However, all the observed associations failed to reach statistical significance after the correction for multiple testing, which was set at a level of 10-3 due to many tests performed. Our study therefore provides evidence that investigated polymorphisms do not play a major role in susceptibility to PVVs.

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.

Glycosaminoglycan sulodexide modulates inflammatory pathways in chronic venous disease.

Inflammation represents an important epiphenomenon in the etiopathogenesis of chronic venous disease, a worldwide debilitating condition affecting millions of subjects. The pathophysiology of chronic venous disease (CVD) is based on the hemodynamic abnormalities in conjunction to alterations in cellular and extracellular matrix biocompounds. The endothelial dysfunction results from early perturbation in the endothelium linked to glycocalyx injury and promoted by inflammatory cells and mediators (such as matrix metalloproteinases and interleukins), which lead to progressive dilation of the vein resulting in chronic venous insufficiency. Activated leukocytes during the inflammatory process release enzymes, free radicals, chemokines and inflammatory cytokines in the vessel microenvironment, which are responsible for the changes of the venous wall and venous valve, reflux and venous hypertension, and the development/progression of tissue destruction and skin changes. Sulodexide, a highly purified mixture of glycosaminoglycans composed by 80% fast moving heparin and 20% of dermatan sulphate, exhibits anti-thrombotic and profibrinolytic properties, restoring also the essential endothelial glycocalyx. Glycosaminoglycan sulodexide has been also characterized to reduce the release of inflammatory cytokines/chemokines and to inhibit the matrix metalloproteinases-related proteolytic cascades, counteracting endothelial dysfunctions. The pleiotropic effects of sulodexide set the basis for a very promising agent in treating the spectrum of CVD.

Sulodexide down-regulates the release of cytokines, chemokines, and leukocyte colony stimulating factors from human macrophages: role of glycosaminoglycans in inflammatory pathways of chronic venous disease.

Chronic venous disease (CVeD) is a debilitating condition that affects millions of individuals worldwide. The condition can result in varicose veins, or advance to severe skin changes and venous ulceration. The fundamental basis for CVeD is inflammation within the venous circulation and that it is subjected to increased hydrostatic pressure resulting in increased ambulatory venous pressure. The inflammation involves leukocytes, in particular macrophages and monocytes, inflammatory modulators and chemokines, cytokine expression, growth factors, metalloproteinase (MMP) activity, and many regulatory pathways that perpetuate inflammation. Sulodexide (SDX) is a glycosaminoglycan with pro-fibrinolytic and anti-thrombotic properties. We have previously demonstrated that SDX inhibits the secretion of pro-zymogen MMP-9 from human leukocytes without displacing high molecular complexes of MMP-9. The anti-inflammatory properties of SDX on activated leukocytes have not been well established. We hypothesized that SDX will reduce the secretion of inflammatory mediators from lipopolysaccharide (LPS)-stimulated macrophages. Therefore, we evaluated the effects of SDX on LPS-stimulated macrophage secretion of various inflammatory and anti-inflammatory cytokines, chemokines, and colony stimulating factors. We used microplatebased multiplex immunoassays. LPS-stimulated macrophages in vitro caused a substantial increase of interleukins, tumor necrosis factor, interferon, chemokines and colony stimulating factors. The addition of SDX caused both a dose-dependent and dose-independent decrease in nearly all of the inflammatory cytokines, chemokines and colony stimulating factors. These findings suggest that SDX has a significant effect on the release of inflammatory mediators from macrophages, and may be useful in the treatment of early and advanced CVeD.

Increased levels of lysosomal cysteinyl cathepsins in human varicose veins: a histology study.

Varicose veins are a major chronic venous disease characterised by extensive remodelling of the extracellular matrix architecture in the vascular wall. Although matrix metalloproteinases have been implicated in these pathologic events, little is known about the functional relevance of other protease family members. Here, we studied the distribution of lysosomal cysteine proteases, cathepsins B, L, K, and S, and their endogenous inhibitor, cystatin C, in long saphenous vein specimens from nine normal donors and 18 patients with varicose veins (VVs). Immunohistochemical analysis demonstrated increased levels of cathepsins L, K, B, and S and reduced levels of cystatin C in VVs. This imbalance between cysteinyl cathepsins and cystatin C may favour VV remodelling. To investigate the inflammatory mechanism of their expression, we examined a detailed inflammatory cell profile in VVs, including macrophages, T lymphocytes, and mast cells. Increased numbers of CD3-positive T cells and tryptase-positive mast cells were found in VVs, and enhanced levels of cysteinyl cathepsins were detected from lesion CD3-positive T cells, chymase-positive mast cells, endothelial cells, and smooth-muscle cells. Elevated cathepsins, and their co-localisation to infiltrated inflammatory cells and to vascular cells, suggest that these proteases participate in extracellular matrix degradation in response to inflammation during VV pathogenesis.

Varicose veins show enhanced chemokine expression.

OBJECTIVES: Leucocyte infiltration in the wall of varicose veins has been reported previously. This study was designed to investigate the expression of pro-inflammatory cytokines and chemokines in control and in patients with varicose veins and to test the effect of treating varicose vein patients with acetylsalicylic acid (ASA) on cytokine expression prior to removal of varices. MATERIAL AND METHODS: Sections of vein were removed during operation from both patient groups, and ribonuclease protection assays (RPAs) were performed to assess the expression of chemokines. Group I included non-varicose saphenous veins from healthy patients undergoing amputation for trauma. Varicose veins were obtained from patients with primary varicose undergoing surgical treatment who received no drug (group II) or treatment with 300 mg day(-1) of ASA for 15 days before surgery (group III). RESULTS: Non-varicose veins constitutively expressed low levels of monocyte-chemoattractant protein (MCP-1) and interleukin (IL)-8 mRNA. Varicose veins had a distinct chemokine expression pattern, since significant up-regulation of MCP-1 and IL-8 and a marked expression of IP-10, RANTES, MIP-1alpha and MIP-1beta mRNA were detected. Removal of the endothelium did not alter this pattern. Varicose veins obtained from patients treated with ASA showed a consistent decrease in chemokine expression, although it did not reach statistical significance. CONCLUSIONS: Varicose veins showed increased expression of several chemokines compared to control veins. A non-significant reduction of activation was observed following treatment with ASA for 15 days.

Evaluation of the memory CD4+ and CD8+ T cells homeostasis during chronic venous disease of lower limbs.

More and more is known about the role of venous wall abnormalities and valvular incompetence in the development of chronic venous disorders (CVD). Unfortunately detailed mechanisms of CVD pathophysiology are not well understood. Recent studies focus on involvement of the inflammatory process in the structural remodeling of venous valves and venous wall. The aim of this study is to investigate and to document the memory T cells homeostasis in CVD patients. In this study we present lymphocytic changes in blood from varicose veins in terms of total CD4+ and CD8+ T cells and their particular subsets of memory T cells: TN, TCM and TEM. Results suggest that immunological memory may be involved in the CVD development.

The impact of gastrocnemius muscle cell changes in chronic venous insufficiency.

OBJECTIVE: To investigate the pathological and metabolic changes in the gastrocnemius muscle in patients with chronic vein insufficiency (CVI). METHOD: Thirty-six patients with varicose veins were investigated by ambulatory venous pressure (AVP) and duplex ultrasonography. Twelve age and height-matched controls were used for comparison. Patients and controls consented to participate in this study. Twenty-one patients with primary vein varicose (group AI) and 15 patients (group AII) with primary deep venous valve incompetence (DVI) underwent biopsies of the gastrocnemius muscle during operation. Adductor biopsies obtained from the same limbs served as a control group (group B) and specimens from controls subjects without venous disease served as the second control group (group C). All the specimens were investigated by superoxide dismutase (SOD), nitric oxide (NO), Na+-K+-ATPase, Ca2+-ATPase and lactic acid (LD) determinations. Samples were subjected to light and electron microscopy following H & E staining, special ATPase, cytochrome oxidase/succinate dehydrogenase (COX/SDH) stains. RESULTS: Normal muscle architecture was seen following H & E, ATPase and COX/SDH staining and normal cell metabolism was observed in specimens of groups B and C. In group A, pathological changes were encountered in the gastrocnemius muscle including disseminated myofibril atrophy, cell denaturation and necrosis, inflammatory cell infiltration, proliferation and dilation of interfascicular veins. ATPase staining (pH 9.4) demonstrated grouping of atrophic fibres, especially type I myofibril grouping, accompanied by moderate to severe atrophy of type II muscle fibres. However, no patient had selective type I fibre atrophy. Enhanced enzymatic activity in single or multiple myofibrils was demonstrated by COX/SDH staining in approximately half of the specimens in group AII. In group AII, electron microscopy showed swelling, myelin figure denaturation of mitochondria, disruption of the myofibrils and increased lipid droplets in the gastrocnemius muscle. Increased concentration of LD was found in most specimens from group A patients. There were also reductions of SOD, NO, biochemical activity of Na+-K+-ATPase, Ca2+-ATPase with increasing concentration of LD in these patients, most prominently in group AII. We found correlation between AVP assessments and the biochemical measurements as well as morphological appearances of the gastrocnemius muscle. CONCLUSION: Venous hypertension results in pathophysiological changes in the gastrocnemius muscles of patients with DVI, associated with decreased calf pump function.

Immunocytochemical characterisation of the inflammatory cell infiltrate of varicose veins.

OBJECTIVES: To characterise the inflammatory cell infiltrate in varicose vein wall, and its relationship to the valve complex. DESIGN: A comparative study of the distribution of inflammatory cells in normal and varicose vein. MATERIALS: Specimens of proximal human long saphenous vein were obtained from patients with duplex Doppler confirmed long saphenous vein reflux (n=14). Control vein was obtained from patients undergoing coronary artery bypass without clinical evidence of venous insufficiency (n=6). Longitudinal 7 microm frozen sections of vein, displaying valve, were prepared. METHODS: Using immunohistochemistry, T-lymphocytes (CD3), macrophage/monocytes (CD68), neutrophils (CD15s) and mast cells (anti-mast cell tryptase) were identified. The number of cells per unit length vein were counted using light microscopy. RESULTS: There were significantly more mast cells and macrophage/monocytes in varicose vein as compared to control. There was a non-significant trend towards more T-lymphocytes in varicose vein. Few neutrophils were present in varicose or normal vein. The distribution of inflammatory cells with respect to the valve was not found to be significant. CONCLUSIONS: Varicose veins display a greater inflammatory cell infiltrate than normal vein. The key role of macrophage/monocytes and mast cells in tissue damage and remodelling should stimulate further research into whether they play a significant role in the development of chronic venous insufficiency.

Matrix metalloproteinase-9 and urokinase-type plasminogen activator in varicose veins.

The purpose of this study was to evaluate the roles of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) with regard to varicose veins (VVs). Immunohistochemical staining and ELISA were performed on samples from 73 patients with the following leg VVs: 82 greater saphenous veins (GSV) from the groin (GSV groin), 28 GSV from the ankle (GSV ankle), 85 VVs, and 13 normal GSV groin (control [CR]) obtained during coronary artery bypass surgery. Immunohistochemically, MMP-9 was localized in the smooth muscle cells (SMCs) in the tunica media. The ratio of immunopositive cells of MMP-9 in the GSV groin, VVs, and GSV ankle were significantly higher than that of CR. The ratios of immunopositive cells of uPA and uPA receptor (uPAR) were not significantly different among the groups. uPA and uPAR were found to be positive in a different set of SMCs of the MMP-9-positive cells. An ELISA showed that the amount of uPA in the culture of the GSV groin was significantly higher than that in CR. For the remodeling process, MMP-9 may be produced in the VV wall and degrade elastic lamellae and other extracellular components of the venous wall. uPA may be produced by groin tissue of the GSV and flow downward because of valvular incompetence, activating MMP-9 at VV tissues.

Treatment of varicose veins: an assessment of intraoperative and postoperative compression sclerotherapy.

Since thrombotic complications, such as superficial thrombophlebitis and subsequent skin pigmentation, are common after sclerotherapy, we conducted a study to evaluate whether combining sclerotherapy with ligation of varicose veins minimizes complications and what timing for sclerotherapy would be most beneficial-accompanying surgery or several weeks postsurgery. Surgical intervention and compression sclerotherapy were performed consecutively on 111 limbs (group A), and sclerotherapy was performed 28 days after surgical intervention on 87 limbs (group B). The volume of sclerosant used and the frequency of complications (thrombus formation and pigmentation) were analyzed. Plasma levels of thrombin-antithrombin III complex (TAT) and D-dimer (DD), as markers for activation of coagulation, were compared. In group A, the total volume of sclerosant used in patients with complications was significantly higher than that in patients without complications. The frequency of thrombus formation and of pigmentation was significantly lower (p <0.01) in group B (10% and 18%, respectively) than in group A (21% and 37%, respectively). The plasma levels of TAT 7 days after treatment were significantly lower in group B (3.4 +/- 1.2 mg/L) than in group A (4.9 +/- 1.1 mg/L). Performing compression sclerotherapy 28 days after surgical intervention is effective for reducing complications and a good alternative for patients with an underlying hypercoagulable state.

Circulating leucocyte adhesion molecules in chronic venous insufficiency.

BACKGROUND: Enzyme-linked immunosorbent assay (ELISA) techniques have detected the existence of circulating forms of intercellular adhesion molecule-1 (ICAM-1), vascular endothelial adhesion molecule-1 (VCAM-1) and E-selectin, all of which mediate leucocyte-endothelial adhesion. This study determined whether circulating cell adhesion molecules were increased in patients with chronic venous insufficiency (CVI) which causes venous stasis. PATIENTS AND METHODS: Before and after walking and upon recovery blood samples were drawn from the saphenous vein in 20 CVI patients: 10 with varicose veins (group 1), 10 with deep venous insufficiency (group 2). 10 healthy controls were enrolled. The total leucocyte count and the soluble levels of ICAM-1, VCAM-1 and E-selectin were determined. RESULTS: After walking, the total leucocyte count decreased significantly (p < 0.01) only in group 2 and sICAM-1 and sVCAM-1 increased significantly (p < 0.01). Upon recovery, these significant differences remained in group 2. No significant modification was observed at any stage of the study in group 1 or in the control group. CONCLUSIONS: These results suggest persistently high levels of circulating adhesion molecules may contribute to worsen microvascular perfusion, which leads to the onset of trophic damage in CVI.

Endothelial activation response to oral micronised flavonoid therapy in patients with chronic venous disease--a prospective study.

BACKGROUND: Endothelial activation is important in the pathogenesis of skin changes due to chronic venous disease (CVD). Purified micronised flavonoid fraction has been used for symptomatic treatment of CVD for a considerable period of time. The exact mode of action of these compounds remains unknown. AIM: To study the effects of micronised purified flavonoidic fraction (Daflon 500 mg, Servier, France) treatment on plasma markers of endothelial activation. MATERIALS AND METHODS: Twenty patients with chronic venous disease were treated for 60 days with DAFLON 500 mg twice daily. Duplex ultrasonography and PPG was used to assess the venous disease. Blood was collected from a foot vein immediately before starting treatment and within 1 week of stopping treatment. Plasma markers of endothelial activation were measured using commercial ELISA kits. RESULTS: Reduction in the level of ICAM-1, 32% (141 ng/ml: 73 ng/ml) and VCAM 29% (1292 ng/ml: 717 ng/ml) was seen. Reduction in plasma lactoferrin (36% decrease, 760 ng/ml: 560 ng/ml) and VW factor occurred in the C4 group only. CONCLUSIONS: Micronised purified flavonoidic fraction treatment for 60 days seems to decrease the levels of some plasma markers of endothelial activation. This could ameliorate the dermatological effects of (CVD). This could also explain some of the pharmacological actions of these compounds. Our study demonstrates the feasibility of using soluble endothelial adhesion molecules as markers for treatment.

Fibrin- and fibrinogen-related antigens in patients with venous disease and venous ulceration.

Abnormalities in systemic fibrinolysis have been implicated in the pathogenesis of venous ulceration. Patients with venous disease have a prolonged euglobulin lysis time and elevated plasma fibrinogen levels, yet little is known about the metabolism of fibrinogen and fibrin in such patients. In this study, we have used a technique that involves electrophoresis and densitometric analysis of captured fibrin-related antigens to measure the concentration and proportions of the individual fibrin and fibrinogen degradation products in patients with venous disease of the lower extremity. As a group, patients with venous disease had markedly elevated levels of total fibrin-related antigen and D-dimer, the terminal degradation product of cross-linked fibrin. Levels of D-monomer, the breakdown product of fibrinogen and non-cross-linked fibrin monomer, and a measure of fibrinogenolysis were normal in all patients. In patients with ulcers, the levels of D-dimer were disproportionately higher than expected from fibrin monomer levels. On an individual basis, significant elevations of D-dimer were present in six (55%) of the 11 patients with venous disease with ulcers and in three (43%) of the seven patients with venous disease without ulcers. We conclude that patients with venous disease have uniform evidence for enhanced fibrin formation, as evidenced by elevated levels of total fibrin-related antigen and D-dimer. This is regardless of whether the venous disease is accompanied by ulceration.

Primary varicose veins and HLA.

138 patients with primary varices of the lower extremities were typed in two independent studies for 41 HLA antigens A, B, C, and their frequencies were compared to those in controls. In both studies, the patients showed higher frequency of HLA-B7 (36.2 vs. 23.4%, relative risk 1.86, p = 0.0013) and lower frequencies of HLA-Aw19 (9.4 vs. 25.0%, relative risk 0.312, p = 0.00006), Cw5 (0.7 vs. 9.8%, relative risk 0.067, p = 0.0001) and Cw6 (7.3 vs. 27.0%, relative risk 0.212, p = 0.00003). These differences were most pronounced in patients whose fathers also suffered from primary varices. No relationship was found between HLA antigens and the sex of the patients or the age at the onset of varices.