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T Bârzu

Publications and source records attributed to T Bârzu.

At least 19 recordsLinked to original sources
PubMed1997

Synthesis and pharmacological properties of a close analogue of an antithrombotic pentasaccharide (SR 90107A/ORG 31540).

The synthetic pentasaccharide (1) corresponding to the heparin sequence which binds to, and activates, antithrombin III (AT III) is a potent antithrombotic compound in several animal models of venous thrombosis. We describe here the preparation and the pharmacological properties of 34, an analogue of oligosaccharide 1 with the latter's N-sulfates being replaced by sulfate esters and hydroxyl groups being methylated. These structural modifications allow a simpler and more efficient synthesis of such anionic oligosaccharides. Affinity for human AT III, anti-factor Xa activity, ability to inhibit thrombin generation, antithrombotic activity in a rat model of venous thrombosis, and elimination half-life in the rat have been determined for 1 and 34. Surprisingly, introduction of O-sulfates in place of N-sulfates, and methylation of hydroxyl groups, contributes to reinforce the binding to AT III, resulting in an improved pharmacological profile.

Animals↗
PubMed1996

Entry and distribution of fluorescent antiproliferative heparin derivatives into rat vascular smooth muscle cells: comparison between heparin-sensitive and heparin-resistant cultures.

We studied the binding and entry of fluorescein (FITC)-labeled heparin derivatives into rat aortic smooth muscle cells (SMC) by confocal microscopy. FITC-labeled heparin fractions or FITC-labeled SR 80037A, a potent antiproliferative heparin derivative (Bârzu et al., Eur. J. Pharmacol., 219:225-233 1992), were prepared and their antiproliferative activity was confirmed. By incubating SMC with FITC-labeled heparins, a specific cell-associated fluorescence was found. Cellular fluorescence was mostly located around the nucleus and at the level of cell contacts or cell adhesion. The fluorescence was displaced neither by chasing with excess of unlabeled heparins nor by washing with 1 M NaCl, which proved that labeled heparins had been internalized by SMC. Kinetics of internalization of FITC-heparins suggested receptor-mediated endocytosis of heparins by SMC. Double labeling of SMC with biotinylated Concanavalin A and FITC-SR 80037A also indicated that heparin derivative enters the endocytic pathway. The process was accelerated when serum was present in the incubation medium. Treatment of cells with chloroquine (50 microM) induced accumulation of FITC-SR 80037A in the late endosomes, around the nucleus. No fluorescence labeling could be evidenced inside the nucleus. Neither electron microscopy nor cell fractionation experiments performed with SMC previously incubated with [3H]-heparin were able to ascertain nuclear uptake of heparin, as proposed by other workers (Busch et al., Cell Biol., 116:31-42; 1992; Sing et al., Drug Dev. Res., 29:129-136 1993). The cell-associated fluorescence was very weak in SMC resistant to the antiproliferative activity of heparin, selected by long-term heparin treatment (HT-SMC) as previously shown [Bârzu et al., J. Cell. Physiol., 160:239-248, 1994]. The HT-SMC differed from control SMC with regard to expression of extracellular matrix proteins. These cells exhibited very low expression of fibronectin and prevalent expression of laminin and synthesized less cell-associated glycosaminoglycans. From our results, the following conclusions can be drawn: (1) the antiproliferative heparins are bound and internalized by SMC without being taken up into the nucleus; (2) there is a correlation between the binding and/or the internalization process and the sensitivity of SMC to the antiproliferative activity of heparins; and (3) selection of heparin-resistant SMC by long treatment with heparin results in particular growth pattern of SMC (absence of focal overgrowth), associated with changes in the expression of the extracellular matrix components (fibronectin, laminin, and cell-bound glycosaminoglycans).

Animals↗
PubMed1994

Characterization of rat aortic smooth muscle cells resistant to the antiproliferative activity of heparin following long-term heparin treatment.

Vascular smooth muscle cells (SMC) do not represent a homogeneous population (Schwartz et al., 1990, Am. J. Pathol. 136: 1417-1428). Cellular clones resistant to the antiproliferative activity of heparin were isolated from rat aortic SMC cultures (Pukac et al., 1990, Cell Regul., 1:435-443; San Antonio et al., 1993, Arterioscler. Thromb., 13:748-757) and from explant of human arterial restenotic lesions (Chan et al., 1993, Lancet, 341:341-342). We have shown in the present study that long-term treatment (growth medium supplemented with 200 micrograms/ml heparin, from the second to the tenth passage) of rat aortic SMC, without cell cloning, resulted in a significant loss of sensitivity to the growth inhibition by heparin and its derivatives. The heparin resistance was stable after growing cells for two passages in heparin-free medium, suggesting the selection of a particular phenotype. We tried to characterize these cells and to determine the causes of the resistance to the growth inhibition by heparin. Heparin-treated SMC (HT-SMC) were smaller than their control culture at the same passage, expressed less alpha-SM actin, and did not overgrow after reaching confluence. As in the heparin-resistant clones (San Antonio et al., 1993, Cell Regul., 1:435-443) expression of alpha-SM actin could be increased in HT-SMC by heparin addition before Western blotting. Heparin resistance was associated with a tenfold decrease in [3H]-heparin binding capacity (Bmax = 1.9 x 10(6) sites per cell) compared to control cultures (Bmax = 1.7 x 10(7) sites per cell), which was irreversible after growing the cells for two additional passages in heparin-free medium. We also investigated protein kinase C (PKC) in HT-SMC in terms of both enzymatic activity and protein expression (evaluated by [3H]-staurosporine and [3H]-phorbol-12,13-dibutyrate binding). We found that HT-SMC had only half the PKC activity and expression as control SMC. Therefore, long-term treatment of rat aortic SMC with heparin allowed the selection of a less differentiated subpopulation of cells, exhibiting low sensitivity to the growth inhibition by heparin, which could be related to the low capacity of binding heparin and to a lower PKC activity and/or expression.

Animals↗
PubMed1993

Preparation and anti-HIV activity of O-acylated heparin and dermatan sulfate derivatives with low anticoagulant effect.

In order to increase the ratio of anti-HIV activity to anticoagulant activity, glycosaminoglycan derivatives selectively substituted at OH and/or COOH groups were prepared. Standard heparin, heparin fragments, or dermatan sulfate were converted to their tributylammonium or tetrabutylammonium salts. Their selective O-acylation to various (controlled) degrees was carried out in a homogeneous way in N,N-dimethylformamide using carboxylic acid anhydrides and 4-(dimethylamino)pyridine as catalyst. Esterification of the COOH groups was performed by the addition of alkyl halide to an N,N-dimethylformamide solution of glycosaminoglycan tetrabutylammonium salts. The in vitro anticoagulant activity, the activity against HIV-1 and HIV-2 cytopathicity, the cytotoxicity, and the activity on the induction of giant cell formation were determined. O-acylation (O-butyrylation or O-hexanoylation) of the heparin fragments obtained by periodate depolymerization (compounds 2d and 2e), and their esters (compounds 7i and 7j), yielded products with very low anticoagulant effects in vitro, yet potent activity against both HIV-1 and HIV-2 induced cytopathicity, and low, if any, cytotoxicity. As compared to other anionic polysaccharides, these acylated derivatives are more active as inhibitors of HIV-induced giant-cell formation. Their anti-HIV activity is related to the degree of O-acylation and is mainly due to the inhibition of virus adsorption to the target cells.

Acylation↗
PubMed1992

O-acylated heparin derivatives with low anticoagulant activity decrease proliferation and increase alpha-smooth muscle actin expression in cultured arterial smooth muscle cells.

Selectively O-acylated derivatives of various glycosaminoglycans were prepared and tested in vitro for their anticoagulant activity and their antiproliferative effect on rat and rabbit smooth muscle cells. When O-acylation (butyrylation or hexanoylation) had been performed on periodate-depolymerized heparin fragments having very low anticoagulant activity, the antiproliferative potency was markedly increased (IC50 = 2 and 1 micrograms/ml respectively, versus 31 micrograms/ml for starting compound) without an increase in anticoagulant activity. The antiproliferative activity was related to the degree of acylation. The O-acylated derivatives of heparin fragments were also very active in reversing the de-differentiation of smooth muscle cell in culture, as estimated by the increase in the expression of alpha-smooth muscle actin and alpha-smooth muscle actin mRNA.

Actins↗
PubMed1989

Heparin-derived oligosaccharides: affinity for acidic fibroblast growth factor and effect on its growth-promoting activity for human endothelial cells.

The minimal structural requirements for the interaction of heparin with acidic fibroblast growth factor (aFGF) were investigated. Oligosaccharides (tetra- to decasaccharides) obtained by nitrous acid depolymerisation of standard heparin were separated by affinity chromatography on Sepharose-immobilised aFGF. The shortest fragment retained by the affinity column at 0.2 M NaCl and eluted at 1 M NaCl was a "regular" hexasaccharide, a trimer of the most abundant disaccharide sequence in heparin. More complex octa- and decasaccharides were also retained by the column. The oligosaccharides eluted by 1 M NaCl from the affinity column ("high-affinity" oligosaccharides) and those washed from the column at 0.2 M NaCl ("low-affinity" oligosaccharides) were compared for their capacity to protect aFGF from proteolysis and to potentiate its mitogenic activity. At a low ionic strength, all oligosaccharides tested, except the "regular" disaccharide, protected aFGF against trypsin and collagenase digestion. At higher ionic strength (greater than 0.2 M NaCl), only high-affinity oligosaccharides showed a protective effect. The high-affinity oligosaccharides (hexa- to decasaccharides) potentiated the mitogenic activity of aFGF, as measured by [3H]thymidine incorporation into DNA of human fibroblasts. The effect of the oligosaccharides on human endothelial cell proliferation was more complex: inhibition of proliferation was observed in the presence of serum and low concentrations of aFGF (1-5 ng/ml) and potentiation in the presence of higher concentrations of aFGF. The potentiating effect increased as a function of molecular size of the heparin fragments and, for a given size, as a function of the anionic charge of the oligosaccharide. Our results suggest that inhibition of cell proliferation by heparin may result from interference with an autocrine basic FGF-like activity.

Cell Division↗
PubMed1986

Endothelial binding sites for heparin. Specificity and role in heparin neutralization.

The specificity of endothelial binding sites for heparin was investigated with heparin fractions and fragments differing in their Mr, charge density and affinity for antithrombin III, as well as with heparinoids and other anionic polyelectrolytes (polystyrene sulphonates). The affinity for endothelial cells was estimated by determining I50 values in competition experiments with 125I-heparin. We found that affinity for endothelial cells increases as a function of Mr and charge density (degree of sulphation). Binding sites are not specific receptors for heparin. Other anionic polyelectrolytes, such as pentosan polysulphates and polystyrene sulphonates, competed with heparin for binding to endothelial cells. Fractions of standard heparin with high affinity for antithrombin III also had greater affinity for endothelium. However, these two properties of heparin (affinity for antithrombin III and affinity for endothelial cells) could be dissociated. Oversulphated heparins and oversulphated low-Mr heparin fragments had lower anticoagulant activity and higher affinity for endothelial cells than did their parent compounds. Synthetic pentasaccharides, bearing the minimal sequence for binding to antithrombin III, did not bind to endothelial cells. Binding to endothelial cells involved partial neutralization of heparin. Bound heparin exhibited only 5% and 7% of antifactor IIa and antifactor Xa specific activity, respectively. In the presence of 200 nM-antithrombin III, and in the absence of free heparin, a limited fraction (approx. 30%) of bound heparin was displaced from endothelial cells during a 1 h incubation period. These data suggested that a fraction of surface-bound heparin could represent a pool of anticoagulant.

Antithrombin III↗
PubMed1986

Modulation of endothelial cells growth induced by heparin.

We have previously shown that heparin was bound and internalized by cultured human endothelial cells. In this study, we have investigated the effect of heparin on endothelial cells growth. We found that heparin inhibited 3H-thymidine uptake as well as actual cell growth in a dose-dependent manner in the presence of low concentrations of human serum. Inhibition was maximal at 1% serum concentration and was abolished at 10%. Chasing experiments supported the role of membrane-bound heparin in this inhibition. Low molecular weight heparin fractions, or pentosan polysulfate, were equally effective in inhibiting 3H-thymidine uptake. On the other hand, the simultaneous addition of heparin and ECGS was synergic in stimulating 3H-thymidine uptake. These results suggest a modulatory role of heparin in endothelial cells growth.

Cell Division↗
PubMed1985

Binding and endocytosis of heparin by human endothelial cells in culture.

Binding of heparin and low molecular weight heparin fragments (CY 222, Mr range 1500-8000) to human vascular endothelial cells was studied. Primary culture of human umbilical vein endothelial cells and either 125I or 3H-labeled heparin or [125I]CY 222 were used. Slow, saturable and specific binding was found. No other tested glycosaminoglycan, excepting a highly sulfated heparan fraction, was able to compete for heparin binding. Two groups of binding sites for [3H]heparin could be distinguished: one with high affinity (Kd = 0.12 microM) and another with lower affinity (Kd = 1.37 microM) and a relative large capacity of binding (1.16 X 10(7) molecules/cell) was calculated. The Kd for unlabeled heparin, as calculated from competition experiments, was 0.23 microM. Much lower affinity was calculated for unlabeled low molecular weight heparin fragments CY 222 (Kd = 4.3 microM) from competition experiments with [125I]CY 222. The binding reversibility was only partial for unfractionated heparin. Even by chasing with unlabeled compound, a fraction of 25-30% was not dissociable from endothelial cells. This fraction was much lower if incubation was carried out at 4 degrees C. The addition of basic proteins (histones) to the incubation medium greatly enhanced the undissociable binding at 37 degrees C, but not at 4 degrees C. The undissociable fraction of heparin was not available to degradation by purified microbial heparinase. These results suggest that a fraction of bound heparin is internalized by the vascular endothelium.

Binding Sites↗
PubMed1984

Binding of heparin and low molecular weight heparin fragments to human vascular endothelial cells in culture.

The interaction of standard heparin and some low molecular weight heparin fragments (CY 222, mw 1,500-8,000 daltons) with human vascular endothelium in culture was studied using both 125I and 3H labeled ligands. A specific and saturable binding was shown for both labeled standard heparins. Two populations of binding sites for 3H-standard heparin could be distinguished: one of high affinity (KD = 0.12 microM), and another of lower affinity (KD = 1.37 microM). Total binding capacity was in the order of 10(7) molecules per cell. The same high level of affinity was calculated for unlabeled compounds from competition experiments with 125I-standard heparin. No other glycosaminoglycans, except a highly sulfated heparan (fraction IIA) could compete for heparin binding sites. A specific binding was also shown for 125I-CY 222. The affinity of unlabeled CY 222 was approximately ten times lower than that of unfractionated heparin. However, CY 222 could compete for approximatively 30% of standard heparin binding. Binding was not completely reversible. Even in the presence of a large excess of unlabeled compounds, a fraction of 25% of radioactive heparins remained bound to the endothelium. This fraction was three times lower if incubation was carried out at +4 degrees C, suggesting a possible incorporation of heparin into the endothelial cells.

Binding Sites↗
PubMed1977

Regulatory effects of purine nucleotide analogs with liver glutamate dehydrogenase.

A total of 26 different purine nucleotides with specific modifications in the base moiety and/or in the polyphosphate chain as well as various combinations of nucleotides were tested as allosteric effectors of beef liver glutamate dehydrogenase (L-glutamate : NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3). The capacity of these nucleotide analogs to activate or to inhibit the glutamate dehydrogenase activity is expressed quantitatively and scaled between the extreme effects of ADP and GTP, respectively. The significance of distinct structural elements for the enzyme-effector interaction is discussed. While the inhibitory GTP site is less specific, accepting many natural and most modified nucleoside triphosphates as inhibitors, the activating ADP site shows a much higher specificity for nucleotides as activators.

Allosteric Regulation↗