The value of selected in vitro and in silico methods to predict acute oral toxicity in a regulatory context: results from the European Project ACuteTox.

ACuteTox is a project within the 6th European Framework Programme which had as one of its goals to develop, optimise and prevalidate a non-animal testing strategy for predicting human acute oral toxicity. In its last 6 months, a challenging exercise was conducted to assess the predictive capacity of the developed testing strategies and final identification of the most promising ones. Thirty-two chemicals were tested blind in the battery of in vitro and in silico methods selected during the first phase of the project. This paper describes the classification approaches studied: single step procedures and two step tiered testing strategies. In summary, four in vitro testing strategies were proposed as best performing in terms of predictive capacity with respect to the European acute oral toxicity classification. In addition, a heuristic testing strategy is suggested that combines the prediction results gained from the neutral red uptake assay performed in 3T3 cells, with information on neurotoxicity alerts identified by the primary rat brain aggregates test method. Octanol-water partition coefficients and in silico prediction of intestinal absorption and blood-brain barrier passage are also considered. This approach allows to reduce the number of chemicals wrongly predicted as not classified (LD50>2000 mg/kg b.w.).

Differences and similarities in tyrosine phosphorylation of proteins in platelets from human and pig species.

BACKGROUND: Pigs have been widely used as animal models to study hemostasis. However, there are significant differences when comparing the hemostatic behavior of pig and human platelets. OBJECTIVE: To investigate signaling through tyrosine-phosphorylation of proteins in pig platelets after activation in suspension or by adhesion under flow conditions, in comparison with human platelets. METHODS: Activation of platelet suspensions was performed with thrombin (T; 0.1 and 1 U mL(-1)) and type I collagen (Col-I; 20 microg mL(-1)), at two different time points (30 and 90 s). Activation by adhesion was carried out on Col-I-coated coverslips, using citrated whole blood samples perfused through a parallel-plate chamber. RESULTS AND CONCLUSIONS: Significant differences between pig and human platelets were detected before and after activation. Activation of pig platelets required higher concentrations of thrombin, as well as increased activation times, to achieve similar levels of tyrosine phosphorylation. Proteins p160, p140, p85 and pp62, present in human platelets, were not detected in profiles corresponding to activated pig platelets. A protein of 70 kDa appeared only in pig platelet profiles, p55 was highly phosphorylated, and the phosphorylation levels of some proteins were significantly different from those found in human platelet profiles. In profiles corresponding to adhered pig platelets, p85 and p62 were absent, and p115 appeared highly phosphorylated. As observed in suspension studies, p70 and p55 appeared specifically in adhered pig platelets. Our study shows that the phosphotyrosine proteins involved in the activation of pig platelets are significantly different from those observed in activated human platelets. These findings may help to explain the differing adhesive and cohesive properties of platelets from both species, which should be considered when extrapolating results.

[Uremic media affects hemostatic properties of human endothelial cells in culture and increases the production of von Willebrand factor]. / El medio urémico altera las propiedades hemostáticas de células endoteliales humanas en cultivo y aumenta la producción de factor von Willebrand.

We have investigated the ability of serum from uremic patients to modify the thrombogenic properties of the endothelium. The effect of the uremic media on the morphology of ECs, and their resistance to flow was analyzed. The reactivity of the extracellular matrix (ECM) generated by ECs towards normal platelets was evaluated in a parallel-plate perfusion chamber. Exposure of ECs to uremic media resulted in abnormal morphology and signs of accelerated growth. Detachment of ECs exposed to circulating blood was increased when cells had been grown with media supplemented with uremic serum (22% vs 13%). Platelet deposition and formation of aggregates were significantly elevated on ECMs generated in the presence of uremic media (40.23 +/- 6.43% vs 25.42 +/- 2.69%, p < 0.05, n = 5). Immunocytochemical methods detected an enhanced expression of von Willebrand factor antigen on uremic ECMs (uremic 17.1 +/- 4.2% vs control 13.57 +/- 3.98%, p < 0.05) and its mRNA expression in endothelial cells (uremic 213.24 +/- 6.13 vs control 200.77 +/- 7.52, p < 0.05). These results suggest that uremic medium alters endothelial function and impairs the antithrombotic functions of cultured endothelial cells. This effect may contribute to the increased cardiovascular and thrombotic risk reported in ESRD patients.

Uremic medium disturbs the hemostatic balance of cultured human endothelial cells.

We have investigated the ability of serum from uremic patients to modify the thrombogenic properties of the endothelium. The effects of uremic medium on the morphology of endothelial cells (ECs), and their resistance to flow was analyzed. The influence of uremic media on the reactivity of the extracellular matrix (ECM) generated by ECs towards normal platelets was evaluated in a parallel-plate perfusion chamber. Exposure of ECs to uremic medium resulted in abnormal cell morphology and signs of an accelerated growth. Detachment of ECs exposed to circulating blood was increased when cells had been grown with media supplemented with uremic serum (21% vs. 14% non exposed). Platelet deposition was significantly elevated on ECMs generated in the presence of uremic media (uremicECMs) (p<0.01 vs. control studies). Effects of uremic serum were not observed at short incubation periods (5 h) but were evident after 24 or 72 h of incubation. Northern blot analysis revealed increased expression of tissue factor (TF) mRNA in ECs exposed to uremic conditions. Immunocytochemical methods detected an augmented expression of TF antigen on uremic ECMs. Incubation of ECMs with an antibody to human tissue factor prevented the increase in platelet deposition observed in uremic ECMs, suggesting that the presence of TF in ECM could be responsible for the enhanced platelet deposition. Results from our study indicate that uremic medium impairs the antithrombotic functions of cultured endothelial cells.

Acute and mid-term combined hormone replacement therapy improves endothelial function in post-menopausal women with angina and angiographically normal coronary arteries.

AIMS AND BACKGROUND: Coronary endothelial dysfunction improves after acute oestradiol treatment in women with angina and normal coronary angiograms. We sought to analyse whether this effect is also seen in the peripheral circulation and whether it is sustained after a mid-term period of treatment. METHODS: We studied 20 women with angina, signs suggestive of myocardial ischaemia and normal coronary angiograms. In five of them, coronary and peripheral endothelial functions were studied at baseline. Brachial artery flow-mediated dilation was reanalysed after 24 h of transdermal oestradiol treatment. In the other 15 women, brachial artery vasoreactivity was studied at baseline and after a 6-week period of treatment with transdermal oestradiol and medroxyprogesterone (HRT) or placebo in a double-blinded crossover fashion. RESULTS: An abnormal coronary artery response to acetylcholine was observed in all women as well as impaired brachial flow-mediated dilation. Brachial flow-mediated dilation significantly increased after 24 h of oestradiol treatment (4.8+/-0.8% vs 0.06+/-0.6%, P<0.001). Peripheral flow-mediated dilation also increased after a 6-week period of HRT compared with baseline (4.1+/-3% vs 0.4+/-1%, P<0.01) and placebo treatment (4.1+/-3% vs 0.6+/-1.7%, P<0.01). CONCLUSION: Impaired endothelium-dependent vasodilation exists both at the coronary and peripheral circulation in post-menopausal women with angina and normal coronary angiograms. Flow-mediated dilation improves in these women after short and mid-term therapy with transdermal oestradiol irrespective of concomitant progesterone use.

Differential aspects of the glycoprotein Ib-von Willebrand factor axis in human and pig species.

BACKGROUND AND OBJECTIVE: The role of glycoprotein Ib (GPIb) in platelet adhesion to subendothelium is well established in human species. However, the interaction of GPIb and von Willebrand factor (VWF) in a widely used experimental model in thrombosis research, that of the pig, has not been clearly elucidated. We investigated the differences between human and pig species in the GPIb/VWF axis in several ways. DESIGN AND METHODS: Standard aggregometry and perfusion studies with circulating blood were applied to isolated platelets or to blood reconstituted with isolated platelets, VWF and red blood cells from the different species. Platelet aggregation to VWF in the presence of either ristocetin or botrocetin was tested. RESULTS: Human VWF and ristocetin did not agglutinate pig platelets. However, botrocetin was capable of agglutinating pig platelets. In perfusion studies (800 s(-1), 10 min), washed platelets from both species were suspended in albumin solutions containing human VWF (hVWF) or porcine VWF (pVWF) and red blood cells from the corresponding species. Reconstituted blood with high concentrations of pVWF (> or =0.25 U/mL) caused severe thrombocytopenia during the perfusion procedure when added to human platelets. Nevertheless, lower concentrations (< or =0.1 U/mL) promoted the formation of large aggregates. Under our experimental conditions, hVWF poorly supported pig platelet adhesion. INTERPRETATION AND CONCLUSIONS: In conclusion, pVWF may support human platelet adhesion and even promote aggregation, while hVWF can only partially facilitate pig platelet adhesion. Minimal concentrations of pVWF could facilitate the interaction of human platelets with subendothelium, increasing their adhesive and aggregating capabilities. Understanding the molecular differences of the GPIb-VWF axis in different species may prove useful for developing therapeutic strategies aimed at preventing excessive platelet deposition on damaged vascular surfaces.

Changes in the cooling rate and medium improve the vascular function in cryopreserved porcine femoral arteries.

PURPOSE: The purpose of this study was to design an adequate technique with which to cryopreserve pig femoral arteries and to assess the influence of storage times in vascular function. METHODS: Fifty-two femoral arteries were distributed in seven groups. In group A (control), 10 arteries were studied after harvest; in groups B1 and B2, 19 arteries were suspended in RPMI 1640 plus fetal calf serum plus dimethylsulfoxide and were cryopreserved at 1 degrees C per minute or 0.3 degrees C per minute, respectively. In groups C1 to C4, 23 arteries were suspended in modified Krebs-Henseleit plus dimethylsulfoxide plus sucrose, cryopreserved at 0.7 degrees C per minute, and kept frozen for 1, 15, 60, or 180 days, respectively. After being thawed, arteries were examined for contraction and endothelial-dependent vasodilation (organ bath studies), antithrombotic properties of the endothelial layer(perfusion studies), and vessel structure (electron microscopy). RESULTS: Endothelial cells were present in both cryopreserved and control arteries. The control vessels showed a mean contraction to norepinephrine (10(-7) mol/L) of 13010 +/- 3181 mg. Arteries in groups B1 and B2 did not respond to norepinephrine. Contraction in groups C1 to C4 was as follows: C1, 5354 +/- 1222 mg; C2, 5187 +/- 2672 mg; C3, 6867 +/- 2292 mg; C4, 7000 +/- 2858 mg, which represent 50% of the control values (P <.001). Vasodilation was similar in control (99% +/- 3%) and cryopreserved arteries (C1, 90% +/- 13%; C2, 93% +/- 12%; C3, 89% +/- 15%; C4, 88% +/- 22%). Storage time did not influence vascular function. Platelet interaction was almost absent and similar in all groups. CONCLUSION: A modified cryopreservation technique preserves endothelial function independently of the storage time up to 6 months.

[Effect of cyclooxygenase inhibition on the adaptation of coronary blood flow to tachycardia]. / Efecto de la inhibición de la ciclooxigenasa en la adaptación del flujo coronario a la taquicardia.

BACKGROUND: The role of different endothelium-derived vasoactive substances in the regulation of coronary circulation during tachycardia is not well defined. In order to elucidate the contribution of prostacyclin to the adaptation of coronary blood flow to tachycardia, the effect of meclofenamate, a cyclooxygenase inhibitor on the coronary blood flow response to rapid atrial pacing was analyzed in a porcine model. METHODS: A group of seventeen pigs were instrumented for coronary blood flow, aortic pressure and atrial pacing. Heart rate was increased by 20 beats every 5 minutes. Coronary blood flow and aortic pressure were measured, and coronary resistance calculated, basally and at each pacing interval, before and after saline serum (n = 6), meclofenamate 5 mg/kg, i.v. (n = 7) or meclofenamate 35 mg/kg, i.v. (n = 4). RESULTS: Neither saline nor meclofenamate modified the normal increase of coronary blood flow provoked by rapid atrial pacing (163 +/- 28% increase before versus 172 +/- 29% after saline; 159 +/- 21% increase before versus 161 +/- 22% after meclofenamate low doses and 201 +/- 39% before vs 172 +/- 36 after meclofenamate high doses). There were no differences in the response of coronary vascular resistance to tachycardia before and after meclofenamate (44% reduction vs 40% respectively). CONCLUSION: Cyclooxygenase blockade does not modify the response of coronary circulation to rapid atrial pacing, suggesting that prostacyclin does not play a limiting role in the regulation of coronary blood flow during tachycardia in this model.

Captopril administration reduces thrombus formation and surface expression of platelet glycoprotein IIb/IIa in early postmyocardial infarction stage.

Long-term administration of the angiotensin-converting enzyme inhibitor captopril in survivors of myocardial infarction (MI) reduces the risk of cardiovascular death, recurrence of MI, and unstable angina, suggesting that captopril may possess antithrombotic properties that have not been clearly elucidated. We assessed the short-term antithrombotic effects of captopril on platelet aggregation, platelet-subendothelium interaction, and the expression of major glycoproteins on platelet surface. A double-blind study was carried out in 25 patients with MI. Blood samples were taken before (baseline) and 12 days after treatment in both the control and captopril groups. Platelet aggregation was tested by conventional aggregometry using common activating agents. Platelet interaction with deeply damaged subendothelial surface was evaluated in a perfusion model, with blood maintained under flow conditions. Deposition of platelets was quantified by using computer-assisted morphometric techniques on histological sections, and it was expressed as a percentage of total vessel surface covered by platelets (CS) and as a ratio between large aggregates (T) and surface covered by platelets (100XT/CS). Glycoprotein expression was measured using flow cytometric techniques. Aggregometric responses showed no significant variations; however, in the captopril group, 100XT/CS decreased after 12 days of treatment (100XT/CS: 36+/-12.1% captopril versus 64+/-8.0% baseline; P=0.005). This parameter was also significantly decreased from that found in control group patients (100XT/CS:67=/-4.5%, P=0.008). Flow cytometry showed a 30% reduction in glycoprotein IIb/IIIa expression (P=0.02). Captopril reduced the formation of large aggregates in a perfusion system, which might be related to a down-regulation of glycoprotein IIb/IIIa complex on the platelet surface. These results suggest that captopril exerts an antiplatelet effect that may contribute to its beneficial action in MI.

Characterization of adhesive and aggregating functions of pig platelets: comparative studies with human platelets and differential effects of calcium chelation.

We have characterized functional responses of pig platelets in citrate-anticoagulated blood and compared results with those of human platelets. Platelet aggregation was induced with known activating agents using citrated platelet-rich plasma. Adhesive and cohesive functions of platelets were evaluated using a perfusion flow system with whole anticoagulated blood (10 min, 800/s). To test the differential sensitivity to free calcium levels, two citrate concentrations (19 mM [standard] vs 13 mM) were used as anticoagulants. The ability of platelets to produce thromboxane A2 was also quantified, using radioimmunoassay. In the presence of 19 mM citrate concentration, pig platelets responded poorly to ADP and collagen, and did not respond to arachidonic acid and U46619. Pig platelets adhered well onto subendothelium, but failed to form aggregates on previously spreaded platelets. At 13 mM citrate concentration, the aggregating responses of pig platelets improved, but some of the aggregation patterns were still reversible. The lower citrate concentration had a dramatic impact on perfusion studies, where the formation of platelet aggregates was restored up to levels found in humans. Thromboxane levels in pig serum were only 20 to 30% of that found in humans. So, pig platelets possess functional properties that clearly differ from human platelets: they are more sensitive to the calcium chelating effects of citrate. Arachidonic acid metabolism and thromboxane amplification of platelet responses seem less preponderant in the pig species.

Desmopressin (DDAVP) enhances platelet adhesion to the extracellular matrix of cultured human endothelial cells through increased expression of tissue factor.

The effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DDAVP-treated ECMS were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin, 20 U/ml). Perfusions with run for 5 min at a shear rate of 800 s-1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p < 0.05 and p. < 0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p < 0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

Cocaine administration enhances platelet reactivity to subendothelial components: studies in a pig model.

Myocardial infarction in cocaine abusers may be related to a direct platelet-activating effect. We analysed this possibility in an experimental model. Studies were carried out in eight normal, anaesthetized pigs with a weight of 30.7 +/- 3.7 kg. Blood samples were withdrawn before and 20 min after i.v. administration of cocaine (10 mg kg-1; at 1 mg kg-1 every 2 min). Modifications in platelet responses to arachidonic acid (AA; 1.4 mmol L-1), ADP (1-4 microM), synthetic thromboxane endoperoxide analogue (U46619; 1 microM), collagen (2.5-5 micrograms mL-1), adrenaline (10 microM) and ristocetin (0.8-1 mg mL-1) were tested by conventional aggregometry. Changes in the capacity of platelets to form aggregates on damaged subendothelium were assessed by means of an ex vivo perfusion system in which blood was circulated for 10 min at 800 s-1, a shear rate similar to that found in normal coronary arteries. The interaction of platelets with perfused denuded arterial segments was morphometrically quantified and expressed as a percentage of damaged vessel surface covered by platelets (%CS). Cocaine administration did not influence platelet aggregation patterns in pigs. However, there was a significant increase in the interaction of pig platelets with subendothelial structures after cocaine infusion (%CS = 40 +/- 17% vs. 27 +/- 16% baseline; mean +/- SD; P < 0.01). Cocaine administration in this animal model increases the reactivity of platelets exposed to subendothelium. These results support the concept that the administration of cocaine to pigs has a prothrombotic effect by facilitating the interaction of platelets with damaged arteries.