HIV-1 infection induces functional alterations in human liver endothelial cells in primary culture.

OBJECTIVES: Since human liver endothelial cells allow HIV-1 multiplication in vitro, we investigated whether HIV induced functional alterations in these cells in primary culture. DESIGN: Direct evidence of the replication of HIV in endothelial cells is sparse, but clotting abnormalities and thrombi, which suggest the existence of an endothelial dysfunction, have been observed in HIV-infected patients. We therefore studied the storage and release of endothelial-specific factors in primary cultures of liver endothelial cells infected with HIV, as well as their cytoskeleton, pinocytic and phagocytic properties. METHODS: Intracellular storage of von Willebrand's factor (vWF) was determined by immunofluorescence and computer image analysis. Excretion of vWF, protein S and endothelin-1 was measured using an enzyme-linked immunosorbent assay and radioimmunoassay. Cytoskeletal constituents were studied by light microscopy. The pinocytosis of acetylated low-density lipoproteins and the phagocytosis of latex beads were analysed under light and electron microscopy. RESULTS: The synthesis of vWF is markedly decreased in HIV-infected liver endothelial cells, as is the excretion of endothelin-1. In contrast, the excretion of protein S remains unaffected and the cytoskeletal network appears to be unaltered. Pinocytosis and phagocytosis are preserved. CONCLUSIONS: HIV infection triggers non-lethal functional alterations in cultured human liver sinusoidal endothelial cells, with a selective impairment in the storage and/or the excretion of endothelial-specific factors such as vWF. This functional modulation could play a role in the pathophysiology of HIV-induced disease.

Increased aggregation and secretion responses of human platelets when loaded with the calcium fluorescent probes quin2 and fura-2.

Incorporation into human platelets of the calcium fluorescent indicators quin2 or fura-2 at low concentrations used to measure intracellular free calcium leads to the potentiation of the effects of agonists on platelets. This was shown by increased aggregatory and secretory responses of quin2 or fura-2 loaded platelets after stimulation with ADP, PAF and with low concentrations of thrombin, collagen, the endoperoxide analog U-46619 and the calcium ionophore A 23187. Quin2 and fura-2 mediated platelet sensitisation could be due to altered arachidonic acid metabolism since it was inhibited by prior treatment with the cyclooxygenase inhibitor acetylsalicylate. In contrast, platelets loaded with higher concentrations of calcium chelators exhibited diminished aggregation responses to all aggregating agents. This latter effect was accompanied by increased fluidity of the platelet plasma membrane bilayer and by the exposure of a new pool of membranes to the outer surface of platelets, as monitored with trimethyl-ammonium-diphenylhexatriene (TMA-DPH) in platelets loaded with the non-fluorescent calcium probe analog MAPT. In contrast, low concentrations of quin2 did not potentiate shape change of platelets activated with ADP. Thus, shape change and aggregation can be influenced separately by intracellular Ca2+ chelators. We conclude that platelet responses are altered by the incorporation of intracellular calcium chelators at concentrations used to monitor intracellular calcium changes.

A simple in vitro model of mechanical injury of confluent cultured endothelial cells to study quantitatively the repair process.

A model of in vitro mechanical injury of confluent human endothelial cells (EC) in culture was developed. Human EC were obtained from umbilical veins and grown to confluence. Application on the EC monolayer of a calibrated disk of cellulose polyacetate paper resulted in removal of the EC, leaving a continuous subendothelial extracellular matrix (ECM) on the culture dish. The regeneration time depended on the original size of the lesion. Regeneration was similar with EC grown on different substrates such as human fibronectin, human subendothelial ECM, bovine collagen type I or surfaces coated with Transglutine, a surgical glue containing adhesive proteins. A human brain extract containing growth factor activity accelerated significantly the repair of the lesion, especially at low serum concentration. This simple in vitro model of mechanical injury allows the quantitative study of the effects of matrices, growth factors and pharmacological agents on the repair process.

Experimental thrombosis on a collagen coated arterioarterial shunt in rats: a pharmacological model to study antithrombotic agents inhibiting thrombin formation and platelet deposition.

A rat thrombosis model was developed to assess the efficacity of antithrombotic drugs. It had the following characteristics: controlled hemodynamic and rheological conditions corresponding to arterial flow, a collagen coated surface as a relevant thrombogenic stimulus, a method of measurement allowing dynamic monitoring of thrombus formation and the possibility to assess the thrombus structure. A shunt composed of polyethylene and silicone catheters, including in the middle of the shunt a collagen coated glass capillary, was inserted between the two primitive carotids of the rat. The duration of patency of the shunt was recorded using a thermic probe fixed on its central part. In this model, the patency of the shunt was 539 +/- 55 s. Platelet and fibrinogen-fibrin accumulation in successive one centimeter segments along the shunt were measured using 111In labeled platelets and 125I labeled fibrinogen. Platelet accumulation occurred on the collagen coated surface and at the junctions between the different components of the shunt, where flow was disturbed. The effects of four antithrombotic agents were measured: aspirin, clopidogrel, heparin and r-hirudin. Clopidogrel, heparin and hirudin significantly prolonged patency duration of the shunt, whereas aspirin was inactive. Aspirin did not reduce platelet or fibrinogen-fibrin accumulation on the collagen coated surface. Platelet accumulation on the collagen surface was significantly lower in the clopidogrel group (50 mg/kg) than in the group treated with heparin (500 U/kg), demonstrating the direct antiplatelet effect of clopidogrel. Hirudin at doses giving similar values of APTT as heparin (500 U/kg) prolonged the occlusion time to over 2 h while the heparin occlusion time was only 20 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies to thrombomodulin induce receptor-mediated endocytosis in human saphenous vein endothelial cells.

The membrane glycoprotein thrombomodulin (TM) is an essential endothelial cell (EC) cofactor, which forms a 1:1 stoichiometric complex with thrombin. Binding of thrombin to the high affinity TM receptor transforms its procoagulant activity into an anticoagulant potential, by activating protein C. The fate of TM in the presence of thrombin is still unclear: some authors claim that the thrombin-TM complex is internalized in EC, while others find this complex to be stable for at least 2 h at 37 degrees C on the EC surface. In the present study, we investigated the interactions of thrombin and Fab-fragments of anti-TM antibodies, coupled to 5 or 15 nm gold particles with saphenous vein endothelial cells. Our results demonstrate that TM can be observed both on the plasma membrane and in coated structures only in the presence of anti-TM antibodies. Addition of thrombin decreased the extent of this labeling, while in double labeling experiments, where cells were incubated with 5 nm gold coupled thrombin and 15 nm gold coupled Fab fragments of anti-TM antibodies, thrombin was cointernalized only when anti-TM antibodies were present. These results show that thrombin-TM complex is not significantly internalized in EC. The internalization of this complex induced by anti-TM antibodies could play an important role in the thrombotic complications induced by anti-EC autoantibodies.

Nitric oxide production and endothelium-dependent vasorelaxation induced by wine polyphenols in rat aorta.

1. The aim of this work was to investigate the mechanism of vasorelaxation induced by red wine polyphenolic compounds (RWPC) and two defined polyphenols contained in wine, leucocyanidol and catechin. The role of the endothelium, especially endothelium-derived nitric oxide (NO), was also investigated. 2. Relaxation produced by polyphenols was studied in rat aortic rings with and without functional endothelium, pre-contracted to the same extent with noradrenaline (0.3 and 0.1 microM, respectively). RWPC and leucocyanidol, but not catechin, produced complete relaxation of vessels with and without endothelium. However, 1000 fold higher concentrations were needed to relax endothelium-denuded rings compared to those with functional endothelium. 3. High concentrations of catechin (in the range of 10(-1) gl-1) only produced partial relaxation (maximum 30%) and had the same potency in rings with and without endothelium. 4. The NO synthase inhibitor, N omega-nitro-L-arginine-methyl-ester (L-NAME, 300 microM) completely abolished the endothelium-dependent but not the endothelium-independent relaxations produced by all of the polyphenolic compounds. 5. In contrast to superoxide dismutase (SOD, 100 u ml-1), neither RWPC nor leucocyanidol affected the concentration-response curve for the NO donor, SIN-1 (3-morpholino-sydnonimine) which also produces superoxide anion (O2-). 6. In aortic rings with endothelium, RWPC (10(-2) gl-1) produced, a 7 fold increase in the basal production of guanosine 3':5'-cyclic monophosphate (cyclic GMP) which was prevented by L-NAME (300 microM). 7. Electron paramagnetic resonance (e.p.r.) spectroscopy studies with Fe(2+)-diethyldithiocarbamate as an NO spin trap demonstrated that RWPC and leucocyanidol increased NO levels in rat thoracic aorta about 2 fold. This NO production was entirely dependent on the presence of the endothelium and was abolished by L-NAME (300 microM). 8. These results show that RWPC and leucocyanidol, but not the structurally closely related polyphenol catechin, induced endothelium-dependent relaxation in the rat aorta. They indicate that this effect results from enhanced synthesis of NO rather than enhanced biological activity of NO or protection against breakdown by O2. It is concluded that some polyphenols, with specific structure, contained in wine possess potent endothelium-dependent vasorelaxing activity.

Role of cyclic AMP in promoting the thromboresistance of human endothelial cells by enhancing thrombomodulin and decreasing tissue factor activities.

1. The effects of forskolin, prostaglandin E1 (PGE1), dibutyryl cyclic AMP (db cyclic AMP), dibutyryl cyclic GMP (db cyclic GMP) and 3-isobutyl-l-methyl-xanthine (IBMX) were investigated on the expression of tissue factor and thrombomodulin activities on the surface of human saphenous vein endothelial cells (HSVEC) in culture. 2. Forskolin (10(-6) to 10(-4) M), PGE1 (10(-7) to 10(-5) M) and db cyclic AMP (10(-4) to 10(-3) M) caused a concentration-dependent decrease of cytokine-induced tissue factor activity. 3. Similar concentrations of forskolin, PGE1 and db cyclic AMP enhanced significantly constitutive thrombomodulin activity and reversed the decrease of this activity caused by interleukin-1 (IL-1). 4. IBMX (10(-4) M) decreased tissue factor activity and enhanced the effect of forskolin on tissue factor and thrombomodulin activities. 5. Forskolin (10(-4) M) decreased the IL-1-induced tissue factor mRNA and increased the thrombomodulin mRNA level. IL-1 did not change the thrombomodulin mRNA level after 2 h of incubation with HSVEC in culture. 6. Dibutyryl cyclic GMP (10(-4) M to 10(-3) M) did not influence tissue factor or thrombomodulin activity. 7. Our data suggest that elevation of intracellular cyclic AMP levels may participate in the regulation of tissue factor and thrombomodulin expression, thus contributing to promote or restore antithrombotic properties of the endothelium.

Role of cyclic AMP in the inhibition of human platelet aggregation by quercetin, a flavonoid that potentiates the effect of prostacyclin.

Quercetin (3.3',4',5,7-pentahydroxyflavone) has previously been shown to inhibit cyclic nucleotide phosphodiesterases prepared from various cell homogenates and the function of intact human platelets. We now report that (1) high concentrations of quercetin raise platelet cAMP levels; and (2) quercetin potentiates the inhibitory effect of prostacyclin (PGI2) on ADP-induced washed human platelet aggregation and the elevation of platelet cAMP levels elicited by PGI2. These results suggest a role for cAMP in the mechanism of action of quercetin on blood platelets.

Inhibition of human platelet cyclic AMP phosphodiesterase and of platelet aggregation by a hemisynthetic flavonoid, amentoflavone hexaacetate.

Amentoflavone hexaacetate (AmAc) was synthesized from natural amentoflavone (Am), a biflavonoid extracted from Viburnum lantana L. Am does not inhibit aggregation of intact platelets up to a concentration of 100 microM but inhibits human platelet cAMP phosphodiesterase (IC50 = 22.0 microM). AmAc is a potent inhibitor of the aggregation of washed human platelets induced by ADP (IC50 = 2.3 microM) or collagen (IC50 = 4.7 microM). AmAc inhibits crude (IC50 = 8.6 microM) or partially purified (IC50 = 42.2 microM) human platelet cAMP phosphodiesterase. In the presence of prostaglandin E1, AmAc (10 microM) induces a 3.7-fold increase in total platelet cAMP. The characteristics of this action suggest a role for cAMP in the mechanism of action of AmAc. The incubation of AmAc with intact platelets for 5 min is necessary for its activity.

Cyclic nucleotide phosphodiesterase inhibitors prevent aggregation of human platelets by raising cyclic AMP and reducing cytoplasmic free calcium mobilization.

Cyclic nucleotide phosphodiesterase inhibitors (HL-725, RO 15-2041, cilostamide, quercetin and MY-5445) potently inhibit human platelet aggregation induced by ADP. In parallel, PDE inhibitors inhibit the increase in cytoplasmic free Ca2+ evoked by ADP, as measured with the fluorescent probe quin 2. The inhibition of ADP-induced aggregation and rise in [Ca2+]i is potentiated by PGE1 which stimulates adenylate cyclase and is inhibited by adrenaline which inhibits adenylate cyclase. PDE inhibitors increase human platelet cAMP levels in the presence of low concentrations of PGE1. It is suggested that PDE inhibitors prevent platelet aggregation by raising cAMP levels and by subsequent inhibition of cytoplasmic free Ca2+ mobilization.

Artifact of blood pressure recording using heparin-filled catheter: effects on blood pressure and coagulation parameters.

A usual complication of catheterization procedures of arteries for blood pressure recording is the clogging of catheters due to activation of coagulation. This is usually avoided by filling the catheters with a heparinized saline solution. We have studied rats implanted with four catheters, one of which was used to monitor blood pressure for 6 h. Catheters were filled with 100 IU/ml heparin in saline. Using this standard protocol, approximately 50-200 IU of heparin were injected into the animals. This induces significant anticoagulation. The activated partial thromboplastin time (APTT) increased from 27 s to more than 240 s. We devised a modified "low heparin" protocol, in which the concentration of heparin in the wash solution of the catheters was lowered from 100 to 0.1 IU/ml, and the pressor transducer was back-perfused with saline solution without heparin. Using this new protocol, no significant modification of the APTT was observed, indicating that only trace amounts of heparin were injected. Subsequent controlled administration of heparin induced a significant decrease in blood pressure. To avoid all effects associated with this unwanted infusion of heparin, mainly a decrease in blood pressure and a major anticoagulant effect, we suggest the use of such "low heparin" catheterization protocol.

Modulation by cytokines of leukocyte-endothelial cell interactions. Implications for thrombosis.

Endothelial cells play an important role in the regulation of thrombosis. Normal resting (i.e. unstimulated) endothelial cells exhibit antithrombotic activity. This property is due to an active participation of endothelial cells in the inhibition of platelet adhesion and aggregation, in the inhibition of thrombin generation, in the direct inactivation of thrombin, and in clot lysis through the fibrinolytic system. When endothelial cells are stimulated by cytokines such as tumor necrosis factor (TNF) or interleukin 1 (IL-1), they may switch to an active procoagulant state. On the one hand, thrombin generation can be regulated on the endothelial cell surface by thrombomodulin, which allows the activation by thrombin of protein C which subsequently acquires and expresses potent anticoagulant properties. On the other hand, after activation, the same endothelial cell can express tissue factor on its surface, which will lead to the triggering of the coagulation cascade resulting in the generation of thrombin. TNF has been shown both to induce tissue factor gene expression and to suppress transcription of the thrombomodulin gene in endothelial cells. Many cytokines induce tissue factor gene expression and procoagulant activity in the monocyte/macrophage lineage; they also stimulate adhesion of leukocytes to endothelial cells. Cytokines such as IL-1 or TNF can thus be characterized as important intercellular messengers during the onset of coagulation. The role of these compounds can be schematized as: 1) agents of the stimulation of endothelial cells by leukocytes, 2) agents of stimulation of leukocytes by endothelial cells, 3) localization of the coagulation response through the initiation of endothelial cell-leukocyte interactions. Pharmacological modulation of these responses is possible along two pathways: 1) inhibition of the activation of endothelial cells or leukocytes responsible for cytokine release, 2) inhibition of the cytokine-induced cellular activation responsible for potentiation of procoagulant activity.

Endothelial cell seeding: coating Dacron and expanded polytetrafluoroethylene vascular grafts with a biological glue allows adhesion and growth of human saphenous vein endothelial cells.

The establishment of an endothelial lining on vascular grafts to obtain a highly thromboresistant surface in a clinical situation requires optimization of cell collection, quality, adhesion and growth. We have studied the conditions for collection, seeding and growth of human saphenous vein endothelial cells (HSVEC), on Dacron or Gore-Tex expanded polytetrafluoroethylene (PTFE) vascular grafts. Carefully handled veins, as opposed to veins obtained using the usual procedures for coronary bypass graft preparation, yielded a higher rate of successful culture (94% vs 43%) and reached confluence in primary culture sooner (9.4 +/- 3 days vs 13.4 +/- 4.5 days). HSVEC were seeded at a density of 6 x 10(3) cells/cm2 on graft fragments coated with fibronectin (FN) or Transglutine (TGL), a biological glue. There was no HSVEC adhesion on Dacron or PTFE without protein pretreatment of the artificial surface. FN improved HSVEC adhesion but there was no cell growth. Adhesion, doubling time and cell density at confluence on PTFE pretreated with TGL were similar to those on conventional tissue culture polystyrene (TCP) pretreated with TGL or FN. HSVEC adhesion on Dacron pretreated with TGL was lower than on TCP pretreated with TGL; the doubling time was similar but the density at confluence was 40% lower. We conclude that pretreatment of vascular grafts with TGL, besides being an alternative to preclotting of the Dacron graft, allows adhesion and growth to confluence of HSVEC on these surfaces.

Culture of vascular cells in tridimensional (3-D) collagen: a methodological review.

Experimental models based on the culture of cells within tridimensional (3-D) gels add a 3-D organization to the classical 2-D culture of vascular cells. They allow the study of cell structure in an environment which is more representative of the in vivo situation and the investigation of cellular functions which cannot be studied using the basic 2-D models. This review shows examples of the use of cultures of vascular cells (endothelial cells, smooth muscle cells as well as fibroblasts) in 3-D collagen matrices for the study of cellular functions as diverse as angiogenesis, extracellular matrix reorganization, migration through 3-D collagen gels or phenotype modulation. It also describes recent advances in the in vitro reconstruction of biological blood vessels by bioengineering. A method for the preparation of 3-D collagen gels is described.

[Methods of studying platelet function. Biological markers of the activation of human platelets in vivo and in vitro]. / Méthodes d'exploration des fonctions plaquettaires. Marqueurs biologiques de l'activation des plaquettes humaines in vivo et in vitro.

Platelets play a key role in hemostasis, thrombosis, atherosclerosis and their pathological consequences. It is possible to follow platelet activation in vivo by measuring bleeding time, platelet count, existence of circulating platelet aggregates or platelet survival and sequestration. In vitro tests include measurement of platelet adhesion, aggregation alpha, and dense granule secretion. It is also possible to follow biochemical events linked to platelet activation such as prostaglandin metabolism, Ca2+ levels or platelet membrane modifications (receptors, glycoproteins, coagulant activities, antigens). Some of these markers of platelet activation are modified in diseases (thrombotic events, hyperlipoproteinemia) and the use of artificial surfaces. It is not always possible to know if the modifications are the cause or the consequence of the pathological event. Unfortunately, some results are questionable because of methodological procedures. Some of these tests have been used to follow the involvement of platelets in a pathological event or to evaluate a prethrombotic state in a patient. It is not yet possible to identify directly, or by the mean of a marker of platelet activation, a patient who is likely to experience a thrombotic episode.

[Platelet aggregation: a tool for clinical investigation and pharmacological study. Methodology]. / L'agrégation plaquettaire: outil d'investigation clinique et d'étude pharmacologique. Méthodologie.

Platelet aggregation can be measured quantitatively by continuous recording of the transmission of a beam of light across a suspension of platelets in constant agitation in an aggregometer. In routine clinical investigation, the study of platelet aggregation is performed on platelet-rich citrated plasma (PRPc). The blood sample has to be excellent to eliminate all traces of thrombin. The blood is collected in 3.8% sodium dihydrate citrate (1 volume for 9 volumes of blood). It is centrifuged at 190 g for 15 minutes at ambiant temperature. The PRPc obtained can be diluted with platelet-poor plasma (PPP) to adjust the concentration of platelets to 3 X 10(5)/microliters. The PRPc is stored at ambiant temperature in stoppered tubes under CO2 to avoid variations in pH. The maximal delay between the collection of the blood and the end of the study of aggregation is 3 hours. In certain cases, in order to define the platelet lesion and to eliminate the influence of plasma proteins, clotting factors and anti-coagulants, a suspension of washed platelets is used. The blood is collected on acid-citrate-dextrose (ACD) and centrifuged at + 37 degrees C to obtain the PRPc. The platelet deposit obtained by centrifugation of the PRPc is washed twice, according to Mustard's method, in Tyrode-albumin buffer at a concentration of 0.35% at + 37 degrees C. It is re-suspended in the same buffer solution at + 37 degrees C in the presence of apyrase and the platelet concentration is adjusted to 3 X 10(5)/microliters. The aggregation or agglutination of the platelets is induced by several agents: ADP, adrenalin, collagen, thrombin, arachidonic acid, ionophor A 23187, PAF-acether and ristocetin. The quantitative study of the aggregation curves of human platelets allows us to study the physiological and biochemical mechanisms control platelet aggregation, to recognize and classify the hereditary or acquired platelet abnormalities which lead to clinical haemorrhagic or thrombotic manifestations and to study the effect of drugs which inhibit platelet aggregation and to understand their mechanism of action.

[Flavonoids: antithrombotic agents or nutrients?]. / Les flavonoïdes: médicaments antithrombotiques ou nutriments?

Flavonoids are a vast group of natural substances, but their pharmacological properties have not all been explored. The term flavonoid is used at large to designate a series of more than 4,000 molecules, which in fact can have very heterogenous molecular structures. We have shown that some flavonoids are good inhibitors of cyclic nucleotide phosphodiesterase (PDE). The most active PDE inhibitors among the flavonoids were also good inhibitors of the aggregation of human platelets in vitro. This suggests that flavonoids could serve as a template for the development of new anti-platelet drugs. However, a direct extrapolation of our experimental results to possible therapeutical use of flavonoid-containing medicinal plant extracts is not possible. The metabolic fate of these plant flavonoids is poorly understood, and their absence of toxicity has not always been clearly demonstrated. Flavonoids are also present in a regular diet in significant amounts. The role of these dietary flavonoids in the prevention of thrombotic diseases or atherosclerosis should also be investigated.