Veno-active drugs in the management of chronic venous disease. An international consensus statement: current medical position, prospective views and final resolution.

BACKGROUND: Veno-active drugs (VAD) have effects on edema and symptoms related to chronic venous disease (CVD), especially so-called venous pain. VAD's effectiveness, although well established, is regularly debated. OBJECTIVE: Our purpose was to select all randomized controlled trials (RCTs) and meta-analyses devoted to VAD and symptoms in CVD, to submit them to a group of international experts in CVD and to vote with secrete ballot to determine the level of efficacy of each drug, according to EBM (Evidence-Based Medicine) rules and critical analysis. METHODS: Publications in any language devoted to VAD and venous symptoms were searched for in different databanks and submitted to the experts prior to the meeting. RESULTS: 83 papers were analyzed, including 72 RCTs or meta-analyses. Experts determined the level of EBM of each drug, according to the literature and personal experience, using 3 levels of recommendation, A, B and C (from large RCTs to non-randomized trials). CONCLUSIONS: VAD are effective and may be applied in CVD when symptomatic, from C0s to C6s. However, etiological treatment of venous reflux and venous hypertension has always priority. In some cases VAD may replace compression and/or complement its effects. If respecting these prerequisites, VAD are safe and effective.

Chronic venous diseases: roles of various pathophysiological factors.

Disturbances in haemodynamic, biochemical and enzymatic factors have been observed in chronic venous diseases (CVD). These changes lead to the development of varices, telangiectasies and skin disorders. They affect vessels, blood, skin tissues and cells. It is now possible to describe their time course and interdependance of these changes. Orthostatism pressure on vein wall may lead to fluid leakage and oedema, these resulting in vein enlargement. These processes may be further influenced by genetic or acquired risk factors. Skin microvessels suffer more from hypoxia than from hypertension. Indeed, hypoxia affects not only endothelial cells, but also red and white blood cells and modifies particularly, but not exclusively, TGF-beta1 production. This substance is, an important modulator of zinc dependent-metallo-proteinases and their tissue inhibitor of metallo-proteinases (TIMP) in the skin. Imbalance in this enzymatic system seems to lead either to sclerosis or ulcer. Of course, other biochemical events (also in this review) play a role in vessel wall and skin deterioration in CVD. The aim of the present review is to assess the role of pathophysiological factors in CVD and the influence of different therapies, including the venotropic agent calcium dobesilate, on some of these haemodynamic or biochemical aspects.

Blood rheology as a marker of venous hypertension in patients with venous disease.

During chronic venous insufficiency (CVI), several microvascular functional abnormalities, due to venous hypertension, develop. To look for blood rheological consequences of venous hypertension "VH", whole blood viscosity and its main determinants were measured in 11 normal controls and 36 patients with CVI exposed to a short-term experimental VH. Patients were subdivided into 2 groups according to the severity of their disease. Blood was taken from a foot vein before and after VH, which was induced by appling a pneumatic tourniquet to 100 mmHg for 15 minutes. Whole blood viscosity at low and high shear rates, red blood cell (RBC) aggregation, RBC rigidity, plasma viscosity and proteins as well as red and white blood cell (WBC) counts were recorded. Patients at baseline, i.e., before application of the tourniquet, showed several hemorheological abnormalities such as an increased RBC aggregation, increased low shear rate viscosity, and a significant elevation in plasma fibrinogen level. Patients with more severe CVI had more marked hemorheological changes. The short term VH in patients led to further aggravation of these changes. There were also at baseline lower values, however not significantly, of hematocrit and RBC count, suggesting that hemoconcentration is not a feature of CVI. These same parameters were slightly, however not significantly, increased after VH, indicating a fluid escape into the extravascular space. A significant fall in WBC count was also observed after VH, in keeping with the white cell trapping hypothesis. In conclusion, even a short-term VH is able to induce several hemorheological impairments, which are probably involved in the failure of the microcirculation and hence the initiation of tissue damage in patients with CVI.

Induced changes of leukocyte slow rolling in an in flow pharmacological model of adhesion to endothelial cells.

BACKGROUND: Rolling of leukocytes at the surface of the vascular endothelium is a prerequisite for a subsequent firm adhesion, particularly the slow rolling appearing on ELAM CD62E. Therefore, it may be considered that increasing the rolling velocities should be a precise therapeutic target in clinical situations where leukocytes accumulate, mainly venous and arterial ischaemia. METHODS: Human neutrophils were allowed to flow on endothelial HUVECs, with and without 4 hours interleukin-1alpha activation, the cells having or not been incubated with INO5042 anti-inflammatory drug. Under a mean shear-stress of 2 dyn/cm(2), rollers and stickers were identified and quantified, using a video-camera and picture analysing software. RESULTS: When the drug had been added to endothelial cells a shift of velocities was observed towards fast speeds (from 3-5 to 7-11 microm/sec). The same results was significantly found when neutrophils, alone or along with endothelium, had been submitted to the drug, the number of stickers and rollers beeing reduced as well. Finally, such a precise pharmacological method proved efficient to detect the exact mechanism of INO5042 on white cell adhesion.

Pharmacological targets of drugs employed in chronic venous and lymphatic insufficiency.

Numerous compounds acting on the venous system have been developed over the last 50 years, including and often associating flavonoids, pycnogenols, saponosides, nucleosides and bilobatides. Some products have proved highly successful such as Cyclo 3 Fort (Fabroven), Phlebodril, made of ruscus extract (flavonoids, saponins), hesperidin-methyl-chalone and ascorbic acid. This presentation is an attempt to classify the scientifically proven mechanisms of such substances. All selected substances have been tested in clinical trials for their action on the symptoms of chronic venous insufficiency (CVI), leg pain and edema. As the distensibility of the leg veins is the main pathological factor determined by plethysmogaphy, it was at first thought that leg pain would be related to it. But observations have shown that this factor is often unrelated to the enlargement of the veins but rather to a whole chain of factors. The effects of different drugs on venous distensibility, rheological disorders, the prevention of wall dystrophy and action on the microcirculation are examined.

[Are rheological markers of poor prognosis present in diabetic arteriopathies?]. / Existe-t-il des marqueurs rhéologiques de mauvais pronostic au cours de l'artériopathie diabétique?

One of the traits of type I and II diabetes lies in the presence of extensive rheological disorders. Rheological changes appear during infancy, mainly in type I diabetes: decreased red cell deformability, leukocyte rigidity, monocyte activation, alteration in microvessel flux (sludge) and functions. Such disorders are however sensitive to insulin and metabolic correction for a long period. Macrorheological disorders develop at the time of puberty and when lipid changes and vascular complications appear (hypertension, visceral obesity, atherosclerosis). Such changes have potent effects on diabetic arteriopathy, as shown by altered TcPO(2). Numerous medical teams are taking into account red cell aggregation measurements reflecting post-capillary flux behavior. In addition, a proposed score may be used based on fibrinogen, hematocrit, triglycerides as viscosity acting factors, and endothelial markers, Willebrand factor and VCAM-1. An increased score is an indication of suspected distal functional alteration of microvessels.

Comparative methodologic study of NFkappaB activation in cultured endothelial cells.

The transcriptional regulatory protein nuclear factor kappaB (NFkappaB) participates in the control of gene expression of many modulators of the inflammatory and immune responses. Various activators trigger NFkappaB release and nuclear translocation after phosphorylation and proteolytic degradation of IkappaB. This study evaluated the abilities of fluorescence and confocal microscopies, laser scanning cytometry (LSC), electrophoretic mobility-shift assay (EMSA), and Western blotting to detect NFkappaB activation in endothelial cells (ECs) and to investigate the role of homocysteine (Hcy) in NFkappaB activation. ECs were treated with interleukin-1B (10 ng/mL) or Hcy thiolactone (1 and 5 mmol/L) as NFkappaB activators. Hcy, a thiol-containing amino acid, has been shown to directly damage ECs in vitro. Experimental evidence suggests that the atherogenic propensity associated with hyperhomocysteinemia results from EC dysfunction. When ECs were pretreated with an inhibitor (pyrrolidine dithiocarbamate, 100 micromol/L) or with staurosporine (5 microL/mL), no NFkappaB activation was observed. NFkappaB activation in ECs could be detected with all five techniques, clearly showing NFkappaB translocation from the cytoplasm to the nuclei. Confocal microscopy was more sensitive and less subjective than immunofluorescence microscopy. LSC was even more sensitive, specific, and reproducible. EMSA, the reference method, has the disadvantages of being radioactive, expensive, and time consuming. Western blot analysis detected the NFkappaB p50 subunit implicated in NFkappaB activation. The techniques usually used to detect NFkappaB activation in ECs are immunofluorescence microscopy and confocal microscopy, LSC, EMSA, and Western blot analysis, but none of them is ready for routine use.

[Interest in variations in soluble ICAM-1 plasma levels. From physiology to clinical applications]. / Intérêt des variations plasmatiques d'ICAM-1 soluble. De la physiologie à l'application clinique.

Intracellular adhesion molecule 1 (ICAM-1) is an adhesion-related molecule belonging to the immunoglobulin superfamily. This molecule is found on the cell membrane of endothelial cells. When activated ICAM-1 allows stable leukocyte adhesion to the endothelial surface. ICAM-1 is found not only in the membrane form but also circulating in serum. ICAM-1 (extracellular part of ICAM-1). This enables ICAM-1s to bind leukocyte integrin receptors such as LFA-1 (CDI1a/CD18) and Mac-1 (CDI1b/CD18) and therefore provide adaptive changes in the adhesion process between circulating cells and the endothelium.

[Drugs for veno-lymphatic insufficiency]. / Médicaments de l'insuffisance veinolymphatique.

Treatment of venous and lymphatic insufficiency of the lower limbs is based on 3 components: elastic support, venotonic drugs and radical treatments (surgery or sclerotherapy) of insufficient veins. Venotonic drugs have specific indications limited to functional impairment: heavy feeling in the legs, pain and impatience in the evening. There are different categories of venolymphatic drugs. Flavonoids have various pharmacological actions, most notably an increase in venous tone, reduction of capillary permeability and increase of capillary resistance. Choice of a venotonic drug is funded on knowledge of pharmacodynamics and pharmacokinetics of the molecule, critical evaluation of clinical studies, physician's personal experience and drug cost. Venotonic drugs are useful when venous insufficiency leads to functional manifestations. They are especially the treatment of heavy leg syndromes during warm seasons when elastic support is uncomfortable.

[Erythrocyte adhesion to vascular endothelium: clinical applications]. / Adhésion des hématies à l'endothélium vasculaire: applications en pathologie.

INTRODUCTION: The objectives of this paper are to review the environmental factors, the different erythrocyte ligands and the corresponding endothelial receptors involved in adhesion. CURRENT KNOWLEDGE AND KEY POINTS: Leukocyte adhesion to vascular endothelium is related to inflammation and has been widely studied. The adhesion of erythrocytes to vascular endothelium has been investigated more recently, mainly in the physiopathology of three diseases: diabetes mellitus, sickle cell disease and malaria. The three diseases are characterized by microvascular complications and are deleterious for the red blood cell membrane. They lead to abnormal erythrocyte adhesion to vascular endothelium. Thus better understanding of the mechanisms involved in red blood cell adhesion to the endothelium is important since it might lead to the development of new therapeutic targets. Progress in this field might contribute to therapeutic improvement in sickle cell disease and to the development of an antimalarial vaccine. FUTURE PROSPECTS AND PROJECTS: However, additional studies focusing on in vivo endothelium heterogeneity, the different subpopulations of red blood cells and the diversity of Plasmodium falciparum strains are required. The consequences of such erythrocytes/endothelium interactions on the endothelial functions remain to be established.

Phosphatidylserine-related adhesion of human erythrocytes to vascular endothelium.

In pathological conditions such as sickle cell disease, falciparum malaria and diabetes, an abnormal adherence of erythrocytes to endothelium is concomitant with loss of phospholipid asymmetry resulting in phosphatidylserine (PS) exposure. We have investigated the involvement of PS in this interaction by studying adhesion of human erythrocytes, treated with Ca2+-ionophore A23187 in combination with N-ethylmaleimide, to human umbilical vein endothelial cells in a flow-based assay. Results showed that erythrocytes which exposed PS, massively adhered to HUVEC in a Ca2+-dependent manner. This adhesion was inhibited by PS liposomes and by annexin V, giving clear evidence of the PS dependence of these interactions.

Automated reticulocyte counting and immature reticulocyte fraction measurement. Comparison of ABX PENTRA 120 Retic, Sysmex R-2000, flow cytometry, and manual counts.

We evaluated reticulocyte counting and measurement of immature reticulocyte fraction (IRF) with the ABX PENTRA 120 Retic blood analyzer on 300 blood samples. Reticulocyte counts were compared with those obtained by visual counting of 2,000 RBCs, by the TOA (Kobe, Japan) Sysmex R-2000 and a flow cytometry method. The parameters analyzed were the percentages of reticulocytes on all analyzers and the IRF with different modalities. The Retic Count kit (Becton Dickinson, San Jose, CA) was used with the Coulter (Hialech, FL) XL, and a mean channel of fluorescence (MCF) was calculated to fit the reticulocyte maturation. Reticulocyte counting with the ABX (Montpellier, France) PENTRA 120 Retic showed excellent precision and linearity with no significant carryover. Reticulocyte counts were stable after blood storage for 72 hours at 4 degrees C but not at room temperature (RT). IRF parameters values were stable for only 8 hours at 4 degrees C and 6 hours at RT. Comparisons of the methods showed good intraclass correlation (RI) for reticulocyte percentages between ABX PENTRA 120 Retic and Sysmex R-2000, ABX PENTRA 120 Retic and flow cytometry, Sysmex R-2000 and flow cytometry, and ABX PENTRA 120 Retic and manual counting. IRF values were correlated between fluorescence rates and RNA content, but in each case, low RI values were found, showing that Sysmex and ABX IRF values were not concordant. We obtained a significant correlation between mean fluorescence index and the MCF measured by flow cytometry, but the 2 methods were not concordant using the RI. The ABX PENTRA 120 Retic is a good instrument for analyzing reticulocyte count and percentage and allows a good analysis of IRF with several modalities.

Synthesis of Bcl-2 in response to anthracycline treatment may contribute to an apoptosis-resistant phenotype in leukemic cell lines.

BACKGROUND: Some forms of chemoresistance in leukemia may start from failure of tumour cells to successfully undergo apoptosis and Bcl-2 may play a role in this defect. Therefore, we evaluated the Bcl-2 content and synthesis in relation with the apoptotic potential in leukemic cell lines after anthracycline treatment. METHODS: U937, HL60, and K562 cells and their drug resistant (DR) variants were treated with varying concentrations of Idarubicin (IDA). Apoptosis was evaluated by fluorescence microscopy after acridine orange staining. Bcl-2 and Bax content were evaluated either by flow cytometry after indirect immunolabelling or by Western blot. RESULTS: High Bcl-2 contents were not related to a poor ability to undergo apoptosis in U937, HL60, K562 and their DR variants. IDA induced a concentration-dependent increase in Bcl-2 content in all cell lines as long as they do not perform apoptosis. Enhanced Bcl-2 expression was inhibited by cycloheximide, actinomycin D, or antisense oligonucleotide directed against bcl-2 mRNA. Bcl-2 expression was also increased in the resistant U937 variant after serum deprivation or C2-ceramide treatment. The synthesis of Bcl-2 led to an increased Bcl-2/Bax ratio solely in the cells with an apoptosis-resistance phenotype. CONCLUSIONS: These data suggest that exposure to IDA induces Bcl-2 expression in leukemic cell lines, and that this mechanism could contribute to apoptosis resistance and participate in the acquisition of chemoresistance. They also confirm that the evolution of the Bcl-2/Bax ratio reflects apoptotic ability better than the steady state level of Bcl-2 expression.