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Biomedical subjects

A Beretz

Publications and source records attributed to A Beretz.

At least 37 records · Page 2Linked to original sources
PubMed1992

Opposing effects of heparin with TGF-beta or aFGF during repair of a mechanical wound of human endothelium. Influence of cAMP on cell migration.

The effects on vascular wound repair in vitro of aFGF and TGF-beta, growth factors having opposite influences on endothelial cell growth and angiogenesis, were studied using as a model a mechanical lesion of confluent endothelium. Modulation by heparin of the activities of these growth factors during the repair process was also examined. Whereas heparin alone inhibited repair by lowering both cell proliferation and cell migration, TGF-beta alone mainly inhibited cell proliferation. When added together, TGF-beta and heparin exerted a combined inhibitory effect resulting in a residual lesion 50% larger than in controls. aFGF alone accelerated lesion coverage and this effect was enhanced by 40% over control values when heparin was added with aFGF. This acceleration was slightly (less than 10%) but consistently diminished by TGF-beta. Cell density in confluent unwounded areas was increased by 40% in the presence of aFGF, but TGF-beta diminished cell density by 20%. A small (30%) increase in intracellular cAMP was measured whenever aFGF was present during the repair process. In comparison, intracellular cAMP inducing agents (forskolin, dbcAMP) accelerated cell migration by 20% during lesion recovery without affecting cell proliferation or density. The present results show that the inhibitory effects of TGF-beta during vascular wound repair are opposed by aFGF. Furthermore, heparin (or heparan sulfates in vivo) modulates growth factors having activating or inhibiting functions and thus plays a regulatory role during the repair process. cAMP-inducing substances other than growth factors are able to accelerate cell migration.

Cell Count↗
PubMed1991

Heterogeneous regulation of constitutive thrombomodulin or inducible tissue-factor activities on the surface of human saphenous-vein endothelial cells in culture following stimulation by interleukin-1, tumour necrosis factor, thrombin or phorbol ester.

Thrombomodulin and tissue-factor activities were measured on the surface of confluent human saphenous-vein endothelial cells (HSVEC) cultivated in 96-multiwell plates. Thrombomodulin activity was measured in the presence of purified human thrombin (2.2 nM) and protein C (65 nM). Tissue-factor activity was measured with purified human Factor VII (5 nM) and Factor X (400 nM). Generated activated protein C and Factor Xa released in the supernatant were assayed with chromogenic substrates. Resting cells exhibited significant thrombomodulin activity, but no detectable tissue-factor activity. After 4 h of preincubation with tumour necrosis factor (TNF, 22-2200 pM), interleukin-1 (IL-1, 5.7-570 nM) or phorbol myristate acetate (PMA, 1.61-161 nM) there was an increase in tissue-factor activity and a concomitant decrease in thrombomodulin activity. However, the extent of both responses varied according to the nature of the stimulus. Thrombin (0.44-44 nM) also induced an increase in tissue-factor activity, but had no effect on thrombomodulin activity. Kinetic studies showed that for all stimuli the increase in tissue factor was transient, reaching a maximum after 4-8 h of preincubation with the stimulating agent and returning to normal values after 24 h. IL-1 and TNF induced a time-dependent decrease in thrombomodulin, by respectively 47% and 67% of control values after 24 h. However, PMA induced only a transient down-regulation of thrombomodulin, full activity being recovered after 18 h. Hence this simultaneous assay system, using intact HSVEC and purified human coagulation factors, enabled us to observe that the regulation of thrombin generation could be diversely affected by various substances known to stimulate the endothelium. This suggests that the simultaneous and opposite modulation of these proteins does not represent an unified response of the endothelial cells to procoagulant stimuli. These results also confirm the absence of effect of thrombin on the expression of thrombomodulin on the cell surface.

Cell Membrane↗
PubMed1991

Old and new natural products as the source of modern antithrombotic drugs.

Natural compounds have been the first historical source of antithrombotic compounds (heparin, vitamin K antagonists, streptokinase, urokinase); molecules extracted from plants or animals still provide some of the most original and promising approaches for the discovery of new drugs in this class. In this review, we will briefly describe three examples of current research trends that could lead to the development of new antithrombotic drugs of natural origin. Flavonoids have been shown to be inhibitors of cyclic nucleotide phosphodiesterase; this enzymatic activity is one of the main mechanisms of inhibition of aggregation of blood platelets by flavonoids. Some of these compounds could represent templates for the development of new inhibitors of platelet activation. Garlic ( ALLIUM SATIVUM) has been shown to inhibit platelet aggregation IN VIVO and IN VITRO; a number of active principles has now been identified and their mechanisms of action are currently being explored. An ancient remedy, the medicinal leech ( HIRUDO MEDICINALIS), has been found to contain several potent anticoagulant proteins. Among them, hirudin, a polypeptide of 65 amino acids, has been identified as one of the most potent inhibitors of thrombin. The production of sufficient amounts of hirudin through molecular biology techniques has now allowed the performance of clinical trials. These three examples show that careful consideration of biochemical, ethnopharmacological, or toxicological properties of natural products can still constitute a valuable basis for the development of new drugs.

Journal Article↗
PubMed1991

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.

Blood Vessel Prosthesis↗
PubMed1991

Old and new natural products as the source of modern antithrombotic drugs.

Natural compounds have been the first historical source of antithrombotic compounds (heparin, vitamin K antagonists, streptokinase, urokinase); molecules extracted from plants or animals still provide some of the most original and promising approaches for the discovery of new drugs in this class. In this review, we will briefly describe three examples of current research trends that could lead to the development of new antithrombotic drugs of natural origin. Flavonoids have been shown to be inhibitors of cyclic nucleotide phosphodiesterase; this enzymatic activity is one of the main mechanisms of inhibition of aggregation of blood platelets by flavonoids. Some of these compounds could represent templates for the development of new inhibitors of platelet activation. Garlic (Allium sativum) has been shown to inhibit platelet aggregation in vivo and in vitro; a number of active principles has now been identified and their mechanisms of action are currently being explored. An ancient remedy, the medicinal leech (Hirudo medicinalis), has been found to contain several potent anticoagulant proteins. Among them, hirudin, a polypeptide of 65 amino acids, has been identified as one of the most potent inhibitors of thrombin. The production of sufficient amounts of hirudin through molecular biology techniques has now allowed the performance of clinical trials. These three examples show that careful consideration of biochemical, ethnopharmacological, or toxicological properties of natural products can still constitute a valuable basis for the development of new drugs.

Animals↗
PubMed1990

Behavior of confluent endothelial cells after irradiation. Modulation of wound repair by heparin and acidic fibroblast growth factor.

Image analysis was used to study the repair process of a circular mechanical lesion of confluent human endothelial cells in culture after irradiation (10 Gy) prior to wounding. Coverage of denuded areas 48 and 96 h after injury of endothelial cells was identical in control and irradiated cultures, although the labeling index was lowered by 80 to 95% in irradiated cultures. The cell density of non damaged irradiated areas was decreased by 50%. When cultures were submitted to increasing doses of radiation (5.0-30 Gy), the labeling index of the cells diminished rapidly between 0 and 5.0 Gy and reached a plateau at 10 Gy. The decrease in cell density (50% of control at 96 h) was identical at each dose of radiation. Thus cell migration alone could be sufficient for the repair of the lesion, while cell proliferation would mainly maintain the original cell density. The addition of heparin to the culture medium slowed down cell migration and proliferation, but the speed of repair was identical in irradiated and non-irradiated cultures. Acidic fibroblast growth factor plus heparin accelerated equally the repair process whether the cultures were irradiated or not. In irradiated cultures the presence of acidic fibroblast growth factor and heparin maintained cell density in confluent areas at a level similar to that in non-irradiated damaged control cultures without addition of mitogens. Thus heparin and acidic fibroblast growth factor play a role in cell proliferation, in the maintenance of the cell monolayer integrity and in restoring a continuous layer by rapid cell migration and elongation after irradiation.

Autoradiography↗
PubMed1990

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.

Cytokines↗
PubMed1989

Stability of the thrombin-thrombomodulin complex on the surface of endothelial cells from human saphenous vein or from the cell line EA.hy 926.

Protein C activation by alpha-thrombin on the surface of endothelial cells depends on an essential membrane-glycoprotein cofactor, thrombomodulin. In the present study we have monitored the activity of thrombin-thrombomodulin complexes on human saphenous-vein endothelial cells (HSVEC) or on the endothelial cell line EA.hy 926. Cell monolayers were exposed for 5 min to 8.5 nM human alpha-thrombin and then washed to remove unbound thrombin. The cells were then incubated at 37 degrees C for 5-180 min. At the end of the respective incubation periods, purified human protein C (120 nM) was added in order to assay the activity of the thrombin-thrombomodulin complexes present on the cell surface. HSVEC pre-exposed to thrombin retained their full capacity to promote protein C activation up to 90 min after free thrombin was removed. This capacity then decreased slowly to reach 56% of control value after 180 min of incubation. Original activity was 3.8 +/- 0.9 pmol of activated protein C formed/min per ml per 10(6) cells (mean +/- S.E.M., n = 5). The capacity of protein C activation of EA.hy 926 cells remained constant for 120 min after free thrombin was removed, then decreased to 76% of control after 180 min. Original activity was 2.0 +/- 0.4 pmol of activated protein C formed/min per ml per 10(6) cells (mean +/- S.E.M., n = 3). Similar results were obtained with cells fixed with 3% paraformaldehyde. However, during the 5-180 min incubation period, non-fixed cells of both types were capable of significantly internalizing fluorescent acetylated low-density lipoprotein. In the experimental protocol used here, an eventual inhibition of thrombin internalization by protein C can be excluded, as protein C is only added at the end of the incubation period. We conclude that there is no evidence of rapid internalization of thrombin-thrombomodulin complexes on HSVEC or the EA.hy 926 cell line, as assessed by the ability of membrane-bound thrombin to activate protein C.

Cell Line↗
PubMed1989

Mitogenic activity on human arterial smooth muscle cells is increased in the plasma of patients undergoing hemodialysis with cuprophane membranes.

During hemodialysis on cuprophane membranes, platelets are activated and release in plasma alpha-granule-specific substances such as PF4 or platelet-derived growth factor (PDGF). PDGF is the main source of mitogenic activity found in serum. In vitro, it induces the proliferation of smooth muscle cells (SMC) which is known to be involved in the development of atherosclerotic lesions. Atherosclerosis is one of the major complications of uremic patients undergoing chronic hemodialysis. To investigate whether this complication could be due to the dialysis itself, we measured the mitogenic activity in plasma of 10 patients undergoing hemodialysis on cuprophane membrane, using human arterial SMC in culture. Mitogenic activity in plasma increased about 3-fold during dialysis. These results may provide an argument in favor of a contribution of platelet activation and release of mitogenic activity to atherosclerosis in patients dialysed with cuprophane membranes.

Adult↗
PubMed1988

Interference of blood-coagulation vitamin K-dependent proteins in the activation of human protein C. Involvement of the 4-carboxyglutamic acid domain in two distinct interactions with the thrombin-thrombomodulin complex and with phospholipids.

Human protein C is the precursor of a serine proteinase in plasma which contains nine 4-carboxyglutamic acid residues and functions as a potent anticoagulant. It is activated by thrombin in the presence of an essential endothelial-cell-membrane glycoprotein cofactor, thrombomodulin. In a purified human system, vitamin K-dependent proteins such as factor X, prothrombin and prothrombin fragment 1 were able to inhibit protein C activation by the thrombin-thrombomodulin complex, using either detergent-solubilized thrombomodulin or thrombomodulin reconstituted into vesicles consisting of phosphatidylcholine and phosphatidylserine (1:1, w/w). Factors VII and IX and protein S were much less efficient. Prothrombin fragment 1 behaved as a non-competitive inhibitor with apparent Ki values of 4 microM in the absence, and of 2-2.5 microM in the presence, of phospholipids. Heat decarboxylation of fragment 1 abolished its ability to interfere in protein C activation, and high phospholipid concentrations could attenuate its inhibitory effect and were responsible for a gradual loss of the non-competitive character. Fragment 1 also inhibited the activation of 4-carboxyglutamic acid-domainless protein C, a proteolytic derivative of protein C lacking the 4-carboxyglutamic acid residues, without any influence from phospholipids. At high thrombin concentrations, with respect to thrombomodulin, the inhibitory effect of fragment 1 was diminished. Fragment 1, at 3.8 microM, inhibited by 50% the activation of protein C (0.1 or 0.3 microM) by thrombin. These results suggest that the 4-carboxyglutamic acid domain of vitamin K-dependent proteins can act as a modulator of the protein C anticoagulant pathway through two distinct types of interaction. The functional 4-carboxyglutamic acid domain would be necessary to allow the enhancement of protein C activation in the presence of anionic phospholipids and it could recognize a phospholipid-independent binding site on the thrombin-thrombomodulin complex.

Depression, Chemical↗
PubMed1988

Epinephrine potentiates human platelet activation but is not an aggregating agent.

Epinephrine can in certain in vitro conditions induce the aggregation of human platelets and could play an important role in vivo in the appearance of thrombotic disorders when catecholamine levels are increased. This study examines some functional and biochemical responses to epinephrine. Epinephrine induces the aggregation and serotonin secretion of human platelets in citrated plasma. This is not due to a direct effect of citrate itself, such as the lowering of plasma free Ca2+ but more likely to the generation of traces of thrombin during blood collection, as suggested by abrogation of these platelet responses when hirudin was added before citrate. When washed human platelets suspended in Tyrode buffer containing 2 mM Ca2+, 0.35% albumin and apyrase, and 0.1-100 microM epinephrine were used, no shape change, aggregation, or secretion of serotonin was observed, nor was the platelet ultrastructure modified. Epinephrine does not modify platelet membrane fluidity, as studied with the lipophilic fluorescent probe trimethylammonium-diphenylhexatriene. It has no direct effect on fibrinogen binding to intact platelets, intracellular Ca2+ levels measured by quin2, or protein phosphorylation. Epinephrine potentiates the action of all types of aggregating agents on aggregation, secretion, intracellular Ca2+ levels, membrane fluidity, fibrinogen binding, or protein phosphorylation. These effects are mediated by alpha 2-adrenergic agonists and inhibited by alpha 2-adrenergic antagonists. This study shows that epinephrine alone does not induce modifications of morphology, metabolism, or function of intact and functional washed human platelets and that it cannot be considered per se as an aggregating agent. However, epinephrine interacts with alpha 2-adrenergic receptors on human platelets and potentiates biochemical and aggregatory responses induced by other platelet agonists.

Adenosine Diphosphate↗
PubMed1987

Adhesion of human breast cancer cell line MCF-7 to human vascular endothelial cells in culture. Enhancement by activated platelets.

The interactions of MCF-7 tumor cells with human vascular endothelial cells (EC) and subendothelial extracellular matrices (ECM) were morphologically observed by electron microscopy and quantitatively evaluated by labelling tumor cells with 111Indium-oxine. MCF-7 tumor cells adhered more rapidly to ECM than to the apical surface of a confluent monolayer of EC. The affinity of MCF-7 cells for type-IV collagen was greater than for fibronectin, suggesting that type-IV collagen contributes to the higher rate of adhesion of MCF-7 cells to the subendothelial ECM. Otherwise, the attachment of tumor cells to EC was increased in the presence of both washed platelets and 0.1% citrated platelet-poor plasma (cPPP), a condition accelerating platelet aggregation by tumor cells. The enhancement of MCF-7 adhesion to EC in the presence of platelets and cPPP was completely blocked by the addition of prostacyclin, or hirudin, a specific thrombin inhibitor. In ultrastructural studies, MCF-7 initiated EC retraction, and firm attachment and flattening occurred on exposed ECM. When MCF-7 cells were incubated with platelets and cPPP, most of the tumor cells adhering to the EC and inducing disruption of endothelial monolayer were closely packed and associated with platelet aggregates. MCF-7 cells appeared to adhere more efficiently to exposed subendothelial ECM when they were associated into multicellular aggregates containing platelets and trapped in a fibrin thrombus. Thus, this homologous human system of cultured vascular EC and breast carcinoma line MCF-7 cells may be used to assess anti-aggregant compounds for their ability to alter tumor-cell implantation on EC-lined surfaces.

Adenosine Diphosphate↗
PubMed1987

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.

Aminoquinolines↗
PubMed1987

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.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1986

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.

Cells, Cultured↗