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Biomedical subjects

J M Herbert

Publications and source records attributed to J M Herbert.

At least 19 recordsLinked to original sources
PubMed2004

Gi-dependent and -independent mechanisms downstream of the P2Y12 ADP-receptor.

The P2Y12 ADP receptor is one of the major regulators of platelet activation and the target of antithrombotic thienopyridines (ticlopidine and clopidogrel). It has been recently cloned but the signaling pathways triggered by this receptor are still poorly documented. Here, we show that stimulation of the human P2Y12 receptor stably expressed in Chinese hamster ovary cells activates two major intracellular signaling mechanisms leading either to cell proliferation or to actin cytoskeleton reorganization. Both effects were blocked by the active metabolite of clopidogrel, a specific antagonist of P2Y12. The P2Y12-mediated stimulation of proliferation required the pertussis toxin-sensitive activation of PI3-kinase/Akt upstream of MAP-kinases. A partial contribution of a transactivation mechanism, through the tyrosine kinase receptor platelet-derived growth factor (PDGF)-R-beta, was also observed. Conversely, the P2Y12-mediated reorganization of the actin cytoskeleton was Gi-independent, requiring activation of RhoA and Rho-kinase. Our results provide new insights into the molecular basis of P2Y12-mediated intracellular signaling. These data may prove to be useful for a better understanding of the physiological role of P2Y12, particularly in platelets and glial cells which express this important therapeutic target.

Actins↗
PubMed2004

Activity of a synthetic hexadecasaccharide (SanOrg123781A) in a pig model of arterial thrombosis.

The activity of SanOrg123781A, a new synthetic antithrombotic drug inhibiting both factor Xa and thrombin through antithrombin (AT), was compared to that of unfractionated heparin (UFH) and of the synthetic pentasaccharide (fondaparinux, SP) in an ex vivo arterial thrombosis model in the pig. Six groups of four pigs were administered intravenously with SanOrg123781A (1, 3, 10 and 30 nmol kg(-1)), UFH (30 nmol kg(-1)) or SP (30 nmol kg(-1)). In this arterial model in which platelet thrombus was formed on a thrombogenic surface under a constant high shear rate, UFH and SP had moderate antithrombotic effects while SanOrg123781A exhibited a strong, dose-dependent inhibitory activity on platelet adhesion and platelet thrombus formation. In contrast to UFH, SanOrg123781A did not modify the activated partial thromboplastin time (aPTT) even at 30 nmol kg(-1), but strongly inhibited thrombin generation. At the same dose, despite a lower antithrombotic activity than SanOrg123781A, UFH significantly affected all the coagulation parameters. Taken together, these results show that SanOrg123781A, due to its potent and selective antifactor Xa and antifactor IIa activities is a promising new antithrombotic agent even in arterial setting.

Animals↗
PubMed2003

Binding characteristics of SSR180575, a potent and selective peripheral benzodiazepine ligand.

The peripheral benzodiazepine receptor (PBR), has been recently shown to play a key role in the regulation of the mitochondrial process leading to apoptosis, which occurs during cardiac ischemia. The present work shows that SSR180575, a novel PBR ligand of potential interest in pathological cardiovascular indications, irreversibly and specifically binds with high affinity on both rat heart mitochondria and on a cell line transfected with the human PBR (K(d)=1.95+/-0.22 and 4.58+/-0.83nM, respectively). In conclusion, SSR180575 is a specific and potent PBR ligand which irreversible binding to PBR appears of high interest in various therapeutic indications where apoptosis occurs.

Acetamides↗
PubMed2003

Efficacy of pentasaccharide on a prethrombosis model based on a calibrated stasis by the increase in up-stream venous pressure.

On a previous model using Wessler's principle in the rat, we have demonstrated that a partial ligature of the inferior vena cava leading to a 40% increase in up-stream venous pressure was thrombogenic only in association with the infusion of low dose of thromboplastin (90 microg/kg). In these thrombogenic conditions, the infusion of pentasaccharide (Arixtra, fondaparinux) should lead to a strong inhibition of thrombus formation. Therefore, we performed on five groups of 10 rats: stasis alone (group S) with a 40% increase in up-stream venous pressure; stasis and thromboplastin (group ST90); and stasis, thromboplastin and pentasaccharide (groups SPT50, SPT100 and SPT250) at three different dosages (50, 100 and 250 microg/kg). The efficacy of pentasaccharide was measured according to the variations in up-stream venous pressure, thrombus weight and thrombin-antithrombin complexes levels. Only 250 microl/kg pentasaccharide significantly reduced the thrombus weight in comparison with group ST90 (5 mg versus 23.8 mg, P = 0.01) but it was not sufficient to induce a return to the basic state (5 mg versus 0.2 mg in group S, P = 0.049). Thrombin-antithrombin complex levels measured at the end of the experiment were significantly reduced in comparison with group ST90 (16.7 versus 57.8 mg, P = 0.01) and were not statistically different from group S (16.1 versus 16.6 mg, P = 0.65). In conclusion, in a very borderline model toward thrombogenesis, pentasaccharide was able to reduce thrombus weight and abolished biological hypercoagulability.

Animals↗
PubMed2002

Clopidogrel-induced qualitative changes in thrombus formation correlate with stent patency in injured pig cervical arteries.

Thienopyridines (ticlopidine or clopidogrel) alone or in combination with aspirin are now the reference antiplatelet therapy after stent implantation. To better understand the high efficacy and low risk of bleeding with these agents, we tested clopidogrel alone or with aspirin in an acute ex vivo flow chamber model and in a subacute in vivo arterial thrombosis model. Clopidogrel induced a dose-dependent increase in bleeding time (BT), inhibited ADP-induced platelet aggregation and in the flow chamber reduced thrombus size, and changed thrombus structure to broad-based structure composed of nondegranulated loosely attached platelets contrasting with the tight clumps of degranulated platelets seen without clopidogrel. The in vivo model involved angioplasty and stenting at the site of a preinduced arterial lesion and thrombosis in pig carotid arteries. Clopidogrel alone or with aspirin (but not aspirin alone) decreased the number of stented vessels occluded for more than 24 h and conversely reduced the number of occluding thrombus. At 96 h after stenting, 100% and 90% of the arteries were patent with clopidogrel/aspirin and clopidogrel alone, respectively (vs. 67% and 44% with aspirin and saline, respectively). Clopidogrel destabilizes thrombus without complete abolishment of platelet reactivity.

Animals↗
PubMed2002

Pharmacokinetics of new synthetic heparin mimetics.

Pharmacokinetics of oligosaccharides displaying various affinities for antithrombin (AT) allowed us to show that there was a close relationship between the plasma half-life of these antithrombotic oligosaccharides and their affinity for AT. Recently, we have described new heparin mimetics comprising an AT binding domain and a thrombin binding domain separated by a neutral methylated saccharide sequence. These compounds displayed strong anti-Xa and antii-IIA activities and, in contrast to heparin, escaped neutralisation by platelet factor 4. The aim of this work was to compare the pharmacokinetics of several of these heparin mimetics in rats. These compounds differed by their length, charge and affinity for AT (AT-binding domain). The results obtained indicate that the prolongation of the AT binding domain did not modify significantly their affinities for AT. However, an increase in the number of charges leads to a decrease in the half-life. When a methylated spacer was added in order to minimise the non-specific interactions to other proteins, half-lives of the heparin mimetic were in the same range than that of the pentasaccharide used as an AT binding domain. In conclusion, this study shows the influence of the charge of the oligosaccharides on their pharmacokinetics and underlinesthe importance of minimising their non-specific binding to plasma proteins in order to obtain compounds with predictive pharmacokinetics.

Animals↗
PubMed2002

The structure of synthetic oligosaccharides in relation to factor IXa inhibition.

We investigated the effect of various oligosaccharides (OS) on the inhibition of factor IXa by antithrombin (AT) in a purified system. The OS comprised the AT-binding pentasaccharide sequence prolonged by saccharide chains with various lengths and charges. We show that factor IXa inhibition depended on the molecular weight of the OS. Factor IXa was not inhibited by the AT-binding pentasaccharide alone, but was inhibited if it was prolonged by a sulphated dodecasaccharide at the non-reducing end. The overall charge was also important since factor IXa inhibition was negligible if the pentasaccharide was prolonged by a non-sulphated dodecasaccharide. Using compounds containing a non-sulphated spacer, we showed that the central part of the OS was not critical. This study therefore demonstrates that the minimal OS structure necessary for catalysing factor IXa inhibition by AT is close to that required for catalysing thrombin inhibition.

Antithrombin III↗
PubMed2001

Synthesis of conformationally locked L-iduronic acid derivatives: direct evidence for a critical role of the skew-boat 2S0 conformer in the activation of antithrombin by heparin.

We have used organic synthesis to understand the role of L-iduronic acid conformational flexibility in the activation of antithrombin by heparin. Among known synthetic analogues of the genuine pentasaccharidic sequence representing the antithrombin binding site of heparin, we have selected as a reference compound the methylated anti-factor Xa pentasaccharide 1. As in the genuine original fragment, the single L-iduronic acid moiety of this molecule exists in water solution as an equilibrium between three conformers 1C4, 4C1 and 2S0. We have thus synthesized three analogues of 1, in which the L-iduronic acid unit is locked in one of these three fixed conformations. A covalent two atom bridge between carbon atoms two and five of L-iduronic acid was first introduced to lock the pseudorotational itinerary of the pyranoid ring around the 2S0 form. A key compound to achieve this connection was the D-glucose derivative 5 in which the H-5 hydrogen atom has been replaced by a vinyl group, which is a progenitor of the carboxylic acid. Selective manipulations of this molecule resulted in the 2S0-type pentasaccharide 23. Starting from the D-glucose derivative 28, a covalent two atom bridge was now built up between carbon atoms three and five to lock the L-iduronic acid moiety around the 1C4 chair form conformation, and the 1C4-type pentasaccharide 43 was synthesized. Finally the L-iduronic acid containing disaccharide 58 which, due to the presence of the methoxymethyl substituent at position five adopts a 4C1 conformation, was directly used to synthesize the 4C1-type pentasaccharide 61. The locked pentasaccharide 23 showed about the same activity as the reference compound 1 in an antithrombin-mediated anti-Xa assay, whereas the two pentasaccharides 43 and 61 displayed very low activity. These results clearly establish the critical importance of the 2S0 conformation of L-iduronic acid in the activation of antithrombin by heparin.

Antithrombins↗
PubMed2001

Targeted deletion of the cytosolic domain of tissue factor in mice does not affect development.

The role of the cytosolic domain of tissue factor (TF) in signal transduction and gene regulation was studied in mice with a targeted deletion of the 18 carboxy-terminal intracellular amino acids. This deletion was introduced in exon 6 along with a floxed neo(R) selection cassette in intron 5 using homologous recombination in embryonic stem cells. Removal of the floxed neo(R) cassette by in vivo Cre-mediated loxP recombination yielded TF(+/deltaCT) and TF(deltaCT/deltaCT) mice. In contrast to TF(-/-) mice, TF(+/deltaCT) and TF(deltaCT/deltaCT) mice displayed normal embryonic development, survival, fertility, and blood coagulation. Factor VIIa or factor Xa stimulation produced similar p44/42 MAPK activation in TF(+/+) and TF(deltaCT/deltaCT) fibroblasts. These data, based on expression of a TF(deltaCT) molecule from the endogenous TF locus, provide conclusive proof that the cytosolic domain of TF is not essential for signal transduction in embryogenesis and in physiological postnatal processes.

Animals↗
PubMed2001

Hypoxia-inducible factor-2alpha (HIF-2alpha) is involved in the apoptotic response to hypoglycemia but not to hypoxia.

Deprivation of oxygen (hypoxia) and/or glucose (hypoglycemia) represents a serious stress that affects cellular survival. The hypoxia-inducible transcription factor-1alpha (HIF-1alpha), which has been implicated in the cellular response to hypoxia (Semenza, G. L. (1999) Annu. Rev. Cell Dev. Biol. 15, 551-578), mediates apoptosis during hypoxia (Halterman, M. W., Miller, C. C., and Federoff, H. J. (1999) J. Neurosci. 19, 6818-6824 and Carmeliet, P., Dor, Y., Herbert, J. M., Fukumura, D., Brusselmans, K., Dewerchin, M., Neeman, M., Bono, F., Abramovitch, R., Maxwell, P., Koch, C. J., Ratcliffe, P., Moons, L., Jain, R. K., Collen, D., and Keshet, E. (1998) Nature 394, 485-490), but the function of its homologue HIF-2alpha remains unknown. Therefore, the role of HIF-2alpha in cellular survival was studied by targeted inactivation of the HIF-2alpha gene (HIF-2alpha(-/-)) in murine embryonic stem (ES) cells. In contrast to HIF-1alpha deficiency, loss of HIF-2alpha did not protect ES cells against apoptosis during hypoxia. Both HIF-1alpha(-/-) and HIF-2alpha(-/-) ES cells were, however, resistant to apoptosis in response to hypoglycemia. When co-cultured with wild type ES cells, HIF-2alpha(-/-) ES cells became rapidly and progressively enriched in hypoglycemia but not in hypoxia. Thus, HIF-1alpha and HIF-2alpha may have distinct roles in responses to environmental stress, and despite its name, HIF-2alpha may be more important in the survival response to environmental variables other than the level of oxygen.

Animals↗
PubMed2001

P2y(12), a new platelet ADP receptor, target of clopidogrel.

The binding characteristics of (33)P-2MeS-ADP, a stable analogue of ADP, were determined on CHO cells transfected with the human P2Y(12) receptor, a novel purinergic receptor. These transfected CHO cells displayed a strong affinity for (33)P-2MeS-ADP, the binding characteristics of which corresponded in all points to those observed on platelets. In particular, this receptor recognised purines with the following order of potency: 2MeS-ADP = 2MeS-ATP > ADP = ATPgammaS = ATP >> UTP, a binding profile which is similar to that obtained in platelets. The binding of (33)P-2MeS-ADP was antagonised by pCMPS but not by MRS2179 and FSBA, antagonists of P2Y(1) and aggregin, respectively. Moreover, the binding of (33)P-2MeS-ADP to these cells was strongly and irreversibly inhibited by the active metabolite of clopidogrel with a potency which was consistent with that observed for this compound on platelets. Like in platelets, 2MeS-ADP induced adenylyl cyclase down-regulation in these P2Y(12) transfected CHO cells, an effect which was absent in the corresponding non-transfected cells. As already shown in platelets, the active metabolite of clopidogrel antagonised 2MeS-ADP-induced inhibition of adenylyl cyclase on transfected cells. Our results confirm that P2Y(12) is the previously called "platelet P2t(AC)" receptor and show that this receptor is antagonised by the active metabolite of clopidogrel.

Adenosine Diphosphate↗
PubMed2001

Detection of bradykinin B1 receptors in rat aortic smooth muscle cells.

The tritiated bradykinin B1 receptor agonist [3H]des-Arg(10)-kallidin bound to a single class of high-affinity binding sites (K(d) = 0.5 +/- 0.16 nM; B(max) = 15,000 +/- 8,000 sites/cell) on cultured rat aortic smooth muscle cells. [3H]Des-Arg(10)-kallidin association and dissociation kinetics were monoexponential, making it possible to determine the association and dissociation rate constants (k(+1) = 1.5 10(5) M(-1) sec(-1); k(-1) = 4.2 10(-5) sec(-1)). [3H]Des-Arg(10)-kallidin binding was inhibited by specific ligands of bradykinin B1 and B2 receptors with a rank order of potency consistent with that known for bradykinin B1 receptors in other species (des-Arg(9)-[Leu(8)]bradykinin = des-Arg(10)-kallidin = des-Arg(9)-bradykinin = des-Arg(10)-[Leu(9)]kallidin > des-Arg(10)-HOE-140 >> bradykinin >> HOE-140). Bradykinin B1 receptor mRNA was also detected in these cells. Des-Arg(10)-kallidin increased cytosolic free Ca2+ levels, phosphoinositide turnover, and arachidonic acid release at nanomolar concentrations (respective EC(50) values: 16 +/- 2, 4 +/- 2.7, 6 +/- 2 nM). These functional effects of des-Arg(10)-kallidin could be blocked by the bradykinin B1 receptor antagonist des-Arg(9)-[Leu(8)]bradykinin, but were not sensitive to bradykinin B2 receptor antagonists. These results therefore show that rat aortic smooth muscle cells in culture express functional bradykinin B1 receptors.

Animals↗
PubMed2001

The calcium inhibitor SR33805 reduces intimal formation following injury of the porcine carotid artery.

We studied the effect of SR33805, a calcium channel blocker, in vitro on the proliferation of vascular smooth muscle cells (SMC) stimulated by foetal calf serum, basic fibroblast growth factor and platelet derived growth factor, and in vivo with regard to SMC migration and proliferation which occurred following injury of the porcine carotid artery. The intimal lesion was induced by a silasten collar surgically positioned around the carotid artery and by a stenosis reducing blood flow by 50% for 30 days. Animals received SR33805 (5 mg/kg/day) 8 days before the induction of the lesion and up to 30 days after. In vitro, SR33805 inhibited in a dose-dependent manner growth factor-induced proliferation of SMC (0.20<IC(50)<0.46 microM). In vivo, SR33805 reduced the intima/media ratio of the cross sectional surface area (decrease of 60%, P<0.05) without affecting neointimal SMC density. The medial SMC density was 40% lower in treated than in control animals (upstream, P<0.05 and downstream to the stenosis, P<0.01). Thus, it appears that SR33805 significantly reduced intimal hyperplasia, which occurred after perivascular manipulation of the artery, an effect consistent with its in vitro proliferation inhibitory activity, suggesting that long-term treatment with SR33805 may reduce or delay SMC proliferation.

Animals↗
PubMed2001

Experimental proof for the structure of a thrombin-inhibiting heparin molecule.

Kinetic studies of thrombin inhibition by antithrombin in the presence of heparin have shown that thrombin binds to heparin in a preformed heparin-antithrombin complex. To study the relative position of the thrombin binding domain and the antithrombin binding domain on a heparin molecule we have designed and synthesized heparin mimetics, which structurally are very similar to the genuine polysaccharide. Their inhibitory properties with respect to factor Xa and thrombin provide experimental evidence that in heparin the thrombin binding domain must be located at the nonreducing end of the antithrombin binding domain to observe thrombin inhibition. As expected, factor Xa inhibition is not affected by elongation of the antithrombin binding pentasaccharide sequence, regardless of the position in which this elongation takes place.

Antithrombins↗
PubMed2001

Tissue factor is not involved in the mitogenic activity of factor VIIa.

The present study was undertaken to evaluate in vitro the importance of tissue factor in the mitogenic effect of factor VIIa for embryonic fibroblasts. For that purpose, embryonic fibroblasts were isolated from either wild-type or transgenic mice showing a single inactivation of the tissue factor gene or expressing a truncated form (lacking the cytosolic domain) of this protein. Factor VIIa stimulated in a dose-dependent manner the growth of the 3 types of fibroblasts, thus showing that TF is not involved in the mitogenic activity of factor VIIa. The mitogenic activity of factor VIIa disappeared in serum immunopurified in factor X and was almost totally inhibited by DX9065, a selective factor Xa inhibitor, showing that this effect of factor VIIa occurred via factor Xa generated during the incubation period. Hirudin did not show any significant effect on factor VIIa-induced fibroblast proliferation, thus showing that the effect observed for factor VIIa was selectively mediated by factor Xa and was not due to thrombin formation. Our results therefore represent the first evidence for the possible importance of factor Xa in the mitogenic effect of factor VIIa and show the negligible role of tissue factor in this process.

Animals↗
PubMed2001

Isolation of vascular smooth muscle cells from a single murine aorta.

The vascular smooth muscle cell plays a significant role in many important cardiovascular disorders, and smooth muscle biology is therefore important to cardiovascular research. The mouse is critical to basic cardiovascular research, largely because techniques for genetic manipulation are more fully developed in the mouse than in any other mammalian species. We describe here a technique for isolating smooth muscle cells from a single mouse aorta. This technique is particularly useful when material is limiting, as is frequently the case when genetically modified animals are being characterized.

Actins↗
PubMed2001

Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by activating VEGF receptor-2 (VEGFR-2). The role of its homolog, placental growth factor (PlGF), remains unknown. Both VEGF and PlGF bind to VEGF receptor-1 (VEGFR-1), but it is unknown whether VEGFR-1, which exists as a soluble or a membrane-bound type, is an inert decoy or a signaling receptor for PlGF during angiogenesis. Here, we report that embryonic angiogenesis in mice was not affected by deficiency of PlGF (Pgf-/-). VEGF-B, another ligand of VEGFR-1, did not rescue development in Pgf-/- mice. However, loss of PlGF impaired angiogenesis, plasma extravasation and collateral growth during ischemia, inflammation, wound healing and cancer. Transplantation of wild-type bone marrow rescued the impaired angiogenesis and collateral growth in Pgf-/- mice, indicating that PlGF might have contributed to vessel growth in the adult by mobilizing bone-marrow-derived cells. The synergism between PlGF and VEGF was specific, as PlGF deficiency impaired the response to VEGF, but not to bFGF or histamine. VEGFR-1 was activated by PlGF, given that anti-VEGFR-1 antibodies and a Src-kinase inhibitor blocked the endothelial response to PlGF or VEGF/PlGF. By upregulating PlGF and the signaling subtype of VEGFR-1, endothelial cells amplify their responsiveness to VEGF during the 'angiogenic switch' in many pathological disorders.

Animals↗