S35972, a direct-acting thrombin inhibitor with high oral bioavailability and antithrombotic efficacy.

OBJECTIVES: Dabigatran etexilate is the first oral thrombin inhibitor to demonstrate superior efficacy to warfarin for stroke prevention in patients with atrial fibrillation. This study describes the in vitro, ex vivo anticoagulant and in vivo antithrombotic effects of an oral thrombin inhibitor, S35972, in comparison with dabigatran etexilate. METHODS: Enzyme assays with thrombin and related serine proteases were performed. Clotting times, including activated partial thromboplastin time (APTT) and thrombin time (TT), were measured in vitro in different species and ex vivo in dogs and rats to determine pharmacologic bioavailabilities. The formation of occlusive venous and arterial thrombi in the rat vena cava and aorta was induced with stasis plus thromboplastin or ferrous chloride, respectively. RESULTS: S35972 inhibited human thrombin with an IC(50) of 3.7 nm, and did not inhibit other serine proteases. The anticoagulant activities of S35972 in vitro were comparable in dog and human plasmas, and the sensitivity of the clotting times to S35972 was TT > APTT > prothrombin time. In the fasted dog, oral administration of 3 mg kg(-1) S35972 increased TT rapidly and for at least 8 h, and its pharmacologic bioavailability was 75.4% ± 0.1%. In the rat venous thrombosis model, 3 mg kg(-1) oral S35972 or dabigatran etexilate significantly decreased the thrombus weight. In the rat aortic thrombosis model, oral S35972 at 10mg kg(-1) significantly decreased thrombus weight, by approximately 50%, whereas, at this dose, no effect was obtained with dabigatran etexilate. CONCLUSIONS: S35972 is a non-prodrug thrombin inhibitor with high selectivity, oral bioavailability, and antithrombotic efficacy.

Openers of calcium-activated potassium channels and endothelium-dependent hyperpolarizations in the guinea pig carotid artery.

This study was designed to determine whether putative openers of calcium-activated potassium channels of small and/or intermediate conductance (SK(Ca) and IK(Ca)) induce vascular smooth muscle hyperpolarizations and to identify the underlying mechanisms. The membrane potential of guinea pig carotid artery smooth muscle cells was recorded with intracellular microelectrodes in the presence of N(omega)-nitro-L-arginine and indomethacin. Acetylcholine and NS-309 produced endothelium-dependent hyperpolarizations. The effects of acetylcholine were partially and significantly inhibited by apamin. The combinations of charybdotoxin plus apamin and TRAM-34 plus apamin markedly and significantly reduced these hyperpolarizations. 1-ethyl-2-benzimidazolinone (1-EBIO) induced hyperpolarizations that were unaffected by TRAM-34 but partially inhibited by charybdotoxin, apamin, TRAM-34 plus apamin, and charybdotoxin plus apamin. Riluzole produced only marginal hyperpolarizations. Therefore, in the guinea pig carotid artery, endothelium-dependent hyperpolarization to acetylcholine involves the activation of both SK(Ca) and IK(Ca), with a predominant role for the former channel. 1-EBIO is a non-selective and weak opener of SK(Ca), while riluzole is virtually ineffective. By contrast, NS-309 is a reasonably potent and selective opener of both SK(Ca) and IK(Ca), and this compound mimics the endothelium-dependent hyperpolarizations to acetylcholine.

Different flavonoids present in the micronized purified flavonoid fraction (Daflon 500 mg) contribute to its anti-hyperpermeability effect in the hamster cheek pouch microcirculation.

AIM: This study evaluated microcirculatory effects of the flavonoid substances that constitute the micronized purified flavonoid fraction (MPFF) (Daflon 500 mg) in comparison to diosmin. METHODS AND RESULTS: In groups of 3 male hamsters, oral treatment with MPFF or diosmin (15 min before anesthesia) did not alter blood pressure. At 10 or 30 mg/kg, both MPFF and diosmin significantly decreased the leaky sites caused by ischemia/reperfusion (I/R) (30 min) in the hamster cheek pouch; the effect was significantly higher with MPFF (39+/-1% and 52+/-1%, respectively) than diosmin (18+/-1% and 37+/-3%, respectively). Eight groups of 3 hamsters each were treated with the components of MPFF. Diosmetin only decreased the number leaky sites at 30 mg/kg (decrease: 15+/-2%). The decrement at 10 and 30 mg/kg averaged at: 17+/-3% and 44+/-1%, respectively, for hesperidin; 19+/-1% and 46+/-2%, respectively, for linarin; and 30+/-1% and 44+/-1%, respectively, for isorhoifolin. Hesperidin, linarin, and isorhoifolin each displayed an anti-leakage effect comparable to or greater than diosmin. MPFF decreases permeability more than any of its single constituents, suggesting that the flavonoids present in its formulation have a synergistic action. CONCLUSION: These results illustrate that MPFF is more potent than single diosmin in this model of hyperpermeability and that each of the flavonoid substances present in MPFF contribute to its action.

Assessment of anti-migraine potential of a novel alpha-adrenoceptor agonist S19014: effects on porcine carotid and regional haemodynamics and human coronary artery.

Taking into account the drawbacks associated with the use of triptans, attempts are being made to explore other avenues for the treatment of migraine. Recently, it has been shown that both alpha1- and alpha2-adrenoceptors mediate the constriction of porcine carotid arteriovenous anastomoses, which has effectively served as an experimental model predictive of anti-migraine activity. The present study investigated the effects of a novel alpha-adrenoceptor agonist S19014 (spiro[(1,3-diazacyclopent-1-ene)-5 : 2'-(4',5'-dimethylindane)] fumarate) on carotid and systemic haemodynamics in anaesthetized pigs, and on human isolated coronary arteries. Increasing doses of S19014 (1-30 micro g/kg, i.v.) produced a dose-dependent initial short-lasting vasopressor response and a decrease of total carotid blood flow and conductance. The carotid blood flow and conductance changes were exclusively due to constriction of carotid arteriovenous anastomoses (capillary blood flow increased) and were accompanied by an increase in arterio-jugular venous oxygen saturation difference. Whereas prazosin (100 micro g/kg, i.v.) was ineffective, rauwolscine (300 micro g/kg, i.v.) attenuated the responses to S19014. The compound did not much affect the distribution of cardiac output to peripheral organs when compared with the vehicle group. Furthermore, S19014 only slightly contracted the human isolated coronary artery and its contractions, contrary to those of sumatriptan, were not increased in blood vessels pre-contracted with U46619. These results suggest that (i) the systemic and carotid vascular effects of S19014 are mainly mediated by alpha2-adrenoceptors, and (ii) S19014 could be effective in the treatment of migraine with an improved cardiovascular tolerance.

Thrombin-induced platelet PAR4 activation: role of glycoprotein Ib and ADP.

Thrombin activates human platelets via the cleavage of two protease-activated G-protein coupled receptors (PARs), PAR1 and PAR4 that respond to low and high concentrations of thrombin, respectively. The aim of the present study was to examine the relative contributions of GPIbalpha and ADP receptors in response to thrombin-induced PAR1 and PAR4 stimulation. Platelet responses (aggregation, secretion and calcium mobilization) elicited by low thrombin concentrations were impaired when thrombin interaction with GPIbalpha was blocked. In contrast, blockade of thrombin interaction with GPIbalpha had no effect when PAR4-coupled responses were specifically elicited by high thrombin concentrations in the presence of PAR1 antagonists or after PAR1 desensitization. These results confirmed that unlike PAR1, PAR4 does not require GPIbalpha as a cofactor for thrombin-mediated activation. Both apyrase and selective antagonists of P2Y1 and P2Y12 inhibited PAR1-coupled responses but did not modify PAR4-coupled responses, indicating that in contrast to PAR1, PAR4 signals are not reinforced by ADP secretion and binding to the platelets. These results provide the direct evidence that, in human platelets, GPIbalpha and ADP act in synergy to amplify PAR1 coupled responses while PAR4 is activated independently of GPIbalpha and ADP.

Imbalance in the synthesis of collagen type I and collagen type III in smooth muscle cells derived from human varicose veins.

Varicose veins have a thickening wall. Their smooth muscle cells are disorganized as regards proliferation and production of extracellular matrix protein. An imbalance between the synthesis of collagen type I protein (collagen I) and collagen type III protein (collagen III) could explain the lack of elasticity of varicose veins. Therefore, collagen synthesis was compared in the media and in cultured smooth muscle cells derived from human control and varicose saphenous veins. An increase in total collagen synthesis was observed in the media and in smooth muscle cells derived from varicose veins. This augmentation was due to an overproduction of collagen I in cultured cells from varicose veins consistent with an increase in the release of collagen I metabolites in the media. A concomitant decrease in collagen III was observed in cultures of smooth muscle cells from varicose veins. The increase in the synthesis of collagen I in cells from varicose veins was correlated with an overexpression of the gene since mRNAs for collagen I were augmented without change in mRNA-half-life. This augmentation in the synthesis of collagen I was reduced by the addition of exogenous collagen III in cultures from varicose veins. These findings suggest a dysregulation of the synthesis of collagen I and III in smooth muscle cells derived from varicose veins.

S17834, a new inhibitor of cell adhesion and atherosclerosis that targets nadph oxidase.

microdant stress is involved in the events that accompany endothelial cell expression of adhesion molecules and leukocyte adherence in many disease states, including atherosclerosis. A recently discovered benzo(b)pyran-4-one derivative, S17834 (10 to 50 micromol/L), reduced tumor necrosis factor-stimulated vascular cell adhesion molecule-1 (VCAM) mRNA accumulation and protein expression in human umbilical vein endothelial cells. Intercellular cell adhesion molecule-1 and E-selectin were also inhibited by S17834, but platelet endothelial cell adhesion molecule-1 was not. Adherence of U937 monocytic cells to the endothelial cells as well as to plastic plates coated with soluble VCAM, intercellular cell adhesion molecule-1, P-selectin, and E-selectin was also decreased. Consistent with an antioxidant mechanism of action, S17834 (10 to 50 micromol/L) inhibited tumor necrosis factor-stimulated release of superoxide from endothelial cells measured by cytochrome c reduction. S17834 had no effect on superoxide produced by xanthine oxidase, indicating that rather than by acting as a scavenger of superoxide anion, the drug acts by inhibiting the production of free radicals. Indeed, S17834 inhibited NADPH oxidase activity of endothelial cell membranes. The ability to inhibit superoxide anion production appears to be key in the effect of S17834 on superoxide anion production and VCAM expression, because these actions were mimicked by adenovirus-mediated overexpression of superoxide dismutase. Furthermore, these actions may be relevant in vivo, because S17834 reduced aortic superoxide anion levels by 40% and aortic atherosclerotic lesions by 60% in apolipoprotein E-deficient mice. These results indicate that S17834 inhibits adhesion molecule expression and adherence of leukocytes to endothelial cells as well as aortic atherogenesis and that perhaps these effects can be explained by its ability to inhibit endogenous superoxide anion production.

Comparison of the contractile responses of human coronary bypass grafts and monkey arteries to human urotensin-II.

Human urotensin-II (hU-II) is a cyclic peptide recently cloned in humans and present in human cardiac tissue and human arteries. The effects of hU-II were studied on human coronary bypass grafts in vitro. In three out of eight human mammary arteries, and two out of three human radial arteries, hU-II caused contraction; human saphenous veins did not respond to hU-II. When it exists, the contraction slowly develops and has a low-to-moderate intensity. All radial arteries obtained from young healthy non-human primates contracted strongly to hU-II.

Inactivation of plasminogen activator inhibitor-1 accelerates thrombolysis of a platelet-rich thrombus in rat mesenteric arterioles.

To investigate the role of active plasminogen activator inhibitor 1 (PAI-1) in the evolution of a microthrombus generated in the arteriolar microcirculation, the monoclonal antibody, 33H1F7, which transforms active PAI-1 to a tissue type plasminogen activator (t-PA) substrate, was evaluated in an arteriolar thrombosis model in the rat mesentery. Arterioles (200-300 um) were stimulated electrically to create an endothelial lesion; ADP was then perfused for 2 min to induce the formation of a platelet-rich thrombus which lysed spontaneously in 140 +/- 24 s. Two successive ADP superfusions produced comparable thrombi which lysed in comparable times. Different doses of 33H1F7 were infused to rats for 30 min and the dose which inactivates rapidly and totally active rat PAI-1 (300 microg/kg/min) was selected to be tested on the thrombosis model. Infusion of 33H I F7 beginning 10 min before the ADP application significantly reduced the lysis time in comparison to the control (123 +/- 30 s versus 169 +/- 33 s, P < 0.05, paired Student's t-test) and the cumulative thrombus area during the lysis period was decreased by 56 +/- 7%. These results demonstrate that inactivation of PAI-1 is able to accelerate lysis of a platelet-rich clot in a mesenteric arteriole of the rat. Thus active PAI-1 most likely participates to the resistance to thrombolysis in the arteriolar microcirculation and its inactivation may shorten ischemic periods after microvascular obstruction such as e.g. during cerebral stroke.

Long-lasting cyclic guanosine-3',5'-monophosphate accumulation in the medium of cultured smooth muscle cells from atherosclerotic rabbit aortas in response to exogenous or endogenous nitric oxide.

Although atherosclerosis causes a marked inhibition of the endothelium-dependent vasorelaxation it also leads to expression of inducible nitric oxide synthase (iNOS), accompanied by an increase in cyclic GMP content, in the arterial wall. The aim of our present study as to evaluate the influence of atherosclerosis on the soluble guanylyl cyclase pathway in viable cultured smooth muscle cells (SMC) from rabbit atherosclerotic rabbit aortas (atherosclerotic SMC) and from control rabbit aortas (control SMC). In response to 100 microM sodium nitroprusside (SNP), the intracellular production of cyclic GMP was similar in both types of cells, reaching a maximum after 5 min of incubation. In the culture medium, SNP evokes an increased cyclic GMP concentration lasting 6 h in control SMC and 24 h in atherosclerotic SMC. Interleukin-1beta (100 IU/mL), which induces iNOS in SMC from both control and atherosclerotic aortas causes accumulation of cyclic GMIP in the extracellular medium between 3 and 6 h for control SMC and between 3 and 24 h with atherosclerotic SMC. These results demonstrate a long-lasting egression of cyclic GMP in the extracellular medium of cultured SMC from rabbit aortas in response to endogenous or exogenous NO. Since this egression of cyclic GMP lasts longer in atherosclerotic than in control SMC, we suggest that atherosclerosis dysregulates the long-term soluble guanylyl cyclase response to NO in SMC.

In vivo analysis of adrenergic and serotoninergic constrictions of the rabbit saphenous vein.

We aimed to develop a model to study in vivo the rabbit saphenous vein pharmacology and to investigate constrictions mediated by adrenoceptor and 5-HT receptor subtypes. We used the technique of high precision ultrasonic echo-tracking for direct measurement of saphenous vein diameters in pentobarbital anesthetized rabbits. Saphenous vein constrictions induced in rabbits by the alpha(1)-adrenoceptor agonist L-phenylephrine and the 5-HT(1B) receptor agonist sumatriptan were comparable with those induced in dogs but those induced by the 5-HT(1B) and 5-HT(7) receptor agonist 5-carboxamidotryptamine failed to appear in dogs. Dose-related constrictions of rabbit veins were obtained with L-phenylephrine and the alpha(2)-adrenoceptor agonist dexmedetomidine. Frequency-related constrictions of rabbit veins induced by nerve stimulation were partially inhibited by an alpha(1)-adrenoceptor or a postsynaptic alpha(2)-adrenoceptor antagonist (prazosin and SKF 104,078) but not affected by the pre- and post-synaptic alpha(2)-adrenoceptor antagonists BRL 44408 or rauwolscine. Constrictions of rabbit veins to sumatriptan and 5-CT were inhibited by GR 127935 and those induced by quipazine, a 5-HT(2) receptor agonist were prevented by ritanserin. The initial constrictions induced by 5-CT were followed by dilatations which were inhibited by the 5-HT(7) receptor antagonist mesulergine. These data indicate that rabbit saphenous veins, in vivo and at rest, respond to activation of 5-HT(1B) and 5-HT(2) receptors, alpha(1)- and alpha(2)-adrenoceptors and nerve stimulation; the dilator effect mediated by 5-HT(7) receptor activation was also detected. The data validate a new animal model to study superficial vein reactivity and its pharmacological sensitivity.

Effect of chronic treatment with the inducible nitric oxide synthase inhibitor N-iminoethyl-L-lysine or with L-arginine on progression of coronary and aortic atherosclerosis in hypercholesterolemic rabbits.

BACKGROUND: We examined the implications of iNOS in atherosclerosis progression using the selective inducible NO synthase (iNOS) inhibitor N:-iminoethyl-L-lysine (L-NIL) in hypercholesterolemic rabbits. METHODS AND RESULTS: Nine rabbits were fed a 0.3% cholesterol diet for 24 weeks (Baseline group); 25 animals were maintained on the diet and treated for 12 extra weeks with L-NIL (5 mg x kg(-1) x d(-1), L-NIL group, n=8), vehicle (Saline group, n=9), or L-arginine (2.25%, L-Arg group, n=8). In abdominal aortas of Saline rabbits, the lesions (53.7+/-5.7%, Baseline) increased to 75.0+/-5.0% (P:<0.05) but remained unaltered in the L-NIL group (63. 4+/-6.6%). Similar results were obtained for the intima/media ratio in thoracic aortas. In coronary arteries, the intima/media ratio was comparable in Baseline (0.68+/-0.18) and Saline (0.96+/-0.19) rabbits but decreased to 0.34+/-0.19 (P:<0.05) in L-NIL rabbits. L-Arginine had beneficial effects only in abdominal aortas. An increased thoracic aorta collagen content was found in Saline and L-Arg but not in L-NIL rabbits. In thoracic aortas of the Saline group, acetylcholine caused modest relaxations that slightly increased by L-arginine but not by L-NIL. Relaxations to nitroglycerin were ameliorated by L-NIL. CONCLUSIONS: This is the first study showing that chronic treatment with an iNOS inhibitor, L-NIL, limits progression of preexisting atherosclerosis in hypercholesterolemic rabbits. Increased intimal collagen accumulation may participate in iNOS-induced atherosclerosis progression.

The thromboxane receptor antagonist S18886 but not aspirin inhibits atherogenesis in apo E-deficient mice: evidence that eicosanoids other than thromboxane contribute to atherosclerosis.

Atherosclerosis involves a complex array of factors, including leukocyte adhesion and platelet vasoactive factors. Aspirin, which is used to prevent secondary complications of atherosclerosis, inhibits platelet production of thromboxane (Tx) A(2). The actions of TxA(2) as well as of other arachidonic acid products, such as prostaglandin (PG) H(2), PGF(2alpha), hydroxyeicosatetraenoic acids, and isoprostanes, can be effectively antagonized by blocking thromboxane (TP) receptors. The purpose of this study was to determine the role of platelet-derived TxA(2) in atherosclerotic lesion development by comparing the effects of aspirin and the TP receptor antagonist S18886. The effect of 11 weeks of treatment with aspirin (30 mg. kg(-1). d(-1)) or S18886 (5 mg. kg(-1). d(-1)) on aortic root atherosclerotic lesions, serum levels of intercellular adhesion molecule-1 (ICAM-1), and the TxA(2) metabolite TxB(2) was determined in apolipoprotein E-deficient mice at 21 weeks of age. Both treatments did not affect body or heart weight or serum cholesterol levels. Aspirin, to a greater extent than S18886, significantly decreased serum TxB(2) levels, indicating the greater efficacy of aspirin in preventing platelet synthesis of TxA(2). S18886, but not aspirin, significantly decreased aortic root lesions as well as serum ICAM-1 levels. S18886 also prevented the increased expression of ICAM-1 in cultured human endothelial cells stimulated by the TP receptor agonist U46619. These results indicate that inhibition of platelet TxA(2) synthesis with aspirin has no significant effect on atherogenesis or adhesion molecule levels. The effects of S18886 suggest that blockade of TP receptors inhibits atherosclerosis by a mechanism independent of platelet-derived TxA(2), perhaps by preventing the expression of adhesion molecules whose expression is stimulated by eicosanoids other than TxA(2).

Endothelial dysfunction in diabetes.

Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease. The endothelium controls the tone of the underlying vascular smooth muscle through the production of vasodilator mediators. The endothelium-derived relaxing factors (EDRF) comprise nitric oxide (NO), prostacyclin, and a still elusive endothelium-derived hyperpolarizing factor (EDHF). Impaired endothelium-dependent vasodilation has been demonstrated in various vascular beds of different animal models of diabetes and in humans with type 1 and 2 diabetes. Several mechanisms of endothelial dysfunction have been reported, including impaired signal transduction or substrate availibility, impaired release of EDRF, increased destruction of EDRF, enhanced release of endothelium-derived constricting factors and decreased sensitivity of the vascular smooth muscle to EDRF. The principal mediators of hyperglycaemia-induced endothelial dysfunction may be activation of protein kinase C, increased activity of the polyol pathway, non-enzymatic glycation and oxidative stress. Correction of these pathways, as well as administration of ACE inhibitors and folate, has been shown to improve endothelium-dependent vasodilation in diabetes. Since the mechanisms of endothelial dysfunction appear to differ according to the diabetic model and the vascular bed under study, it is important to select clinically relevant models for future research of endothelial dysfunction.

Histochemical evidence for inducible nitric oxide synthase in advanced but non-ruptured human atherosclerotic carotid arteries.

In response to cytokine stimulation, the inducible isoform of the nitric oxide synthase (iNOS) produces large amounts of nitric oxide with potential consequences in the pathophysiology of atherosclerosis. Previous investigations have demonstrated the presence of iNOS in human atherosclerotic lesions. The goal of this study was to evaluate the occurrence of the expression of iNOS in ruptured versus non-ruptured human carotid atherosclerotic plaques. Using plastic-embedded sections, we performed in situ hybridization and immunohistochemistry on very advanced atherosclerotic lesions type V (non-ruptured) and type VI (ruptured) from 12 atheromatous carotid arteries from endarterectomy and six non-atherosclerotic internal mammary arteries from aorto-coronary bypass. Only one internal mammary artery expressed iNOS in the endothelium. In contrast, iNOS mRNA and protein were repeatedly expressed in advanced lesions type V in 5/7 cases, particularly in inflammatory regions. Specific cell markers identified iNOS-positive cells as macrophages and T-lymphocytes but also as smooth muscle cells and endothelial cells adjacent to these inflammatory regions. Nitration of protein tyrosines was not always associated to iNOS expression but more likely to the presence of inflammatory cells. In complicated lesions type VI, the occurrence of iNOS mRNA and protein expression diminished drastically (1/5 cases). Combined expression of iNOS mRNA and protein is frequently found in advanced but non-ruptured human atherosclerotic carotid lesions while it becomes rare after the plaque has ruptured. These findings suggest that iNOS could be an active participant in the plaque rupture event.

Platelets and prostacyclin in arterial bypasses: implications for coronary artery surgery.

BACKGROUND: We investigated effects of platelets and prostacyclin formation in human internal mammary (IMA) and radial (RA) arteries. METHODS: IMA and RA segments were suspended in organ bath with increasing concentrations of platelets. Experiments were applied with and without ketanserin, a 5HT2 receptor antagonist, or U3405, a TXA2 receptor antagonist. The release of prostacyclin (PGI2) was assessed by enzyme immunoassay in vessels without endothelium, before and after contraction with angiotensin (AT) I-II. RESULTS: In IMA and RA with endothelium, platelets caused contractions, significantly enhanced in arteries without endothelium. Contractions to platelets were higher in RA than in IMA. U3405 reduced the platelet induced contractions in RA but not in IMA. Ketanserin inhibited the platelet induced contractions in IMA and RA. The basal release of PGI2 was more important in IMA than in RA. Addition of AT/I-II significantly reduced the release of PGI2 in IMA but not in RA. CONCLUSIONS: The RA responds more powerfully to platelets than IMA. Protective system with PGI2 seems to be more powerless in RA than in IMA. This accentuates the importance of antispastic and antiplatelet drugs when arteries are used for coronary artery bypass surgery.

Off-line analysis of red blood cell velocity in renal arterioles.

Videomicroscopic methods with off-line analysis of microcirculatory parameters by multifunctional computer-assisted image analysis systems have significant advantages for in vivo microvascular research. A limitation of these methods is, however, that red blood cell velocities (V(RBC)) exceeding 2 mm/s cannot be measured using standard video framing rates. In the present study, a high-speed video camera, recording up to 600 frames per second, was incorporated in the set-up, and V(RBC) was measured off-line with the line-shift-diagram method. The aim of this study was to test the reproducibility and validity of the method using a high-speed video camera and to evaluate its applicability in vivo. V(RBC) were measured in arterioles of the split hydronephrotic kidney. The intra- and interindividual variability was small for V(RBC) below 40 mm/s. The validity of the method was tested using the mass conservation principle and found to be at least as good as that of the dual-slit photometric technique. The present approach extends the application of videomicroscopy coupled to image analysis systems to the analysis of high V(RBC).

Urinary nitrate excretion in cholesterol-fed rabbits: effect of a chronic treatment by N-iminoethyl-L-lysine, a selective inhibitor of inducible nitric oxide synthase.

To evaluate the influence of atherosclerosis on the global production of NO, we studied the effect of a 0.3% cholesterol-enriched diet on urinary nitrate excretion in rabbits during 69 weeks. To examine whether the inducible nitric oxide synthase (iNOS) present in atherosclerotic lesions could participate in NO excretion, hypercholesterolemic rabbits were treated chronically with the selective iNOS inhibitor, N-iminoethyl-L-lysine (L-NIL; 5 mg/kg/day). Urinary nitrate excretion was higher in hypercholesterolemic than in control rabbits throughout the study period and decreased progressively with time in both groups; L-NIL had no significant effect on urinary nitrate excretion. These data illustrate that systemic NO production is enhanced in hypercholesterolemia and that iNOS, present within the plaque, might not participate in this enhanced NO production.

Regulation of adhesion molecules: a new target for the treatment of chronic venous insufficiency.

As more insight into the mechanisms leading to chronic venous insufficiency (CVI) is gained, novel targets for drug treatment of the disease, or of its complications, become available. Studies using chemical entities capable of inhibiting leukocyte adhesion in postcapillary venules have led to the discovery of selective inhibitors of cell adhesion mechanisms. The aim of the current review is to describe the pharmacology, biochemistry, and molecular biology studies performed with some new inhibitors of adhesion molecule expression. Compounds such as hydroxy triallyl farnisine (S 17834) may offer new and efficient treatment of the microcirculatory complications that accompany chronic venous disease.

Distribution and prevalence of inducible nitric oxide synthase in atherosclerotic vessels of long-term cholesterol-fed rabbits.

The expression of inducible nitric oxide synthase (iNOS) as well as its functional activity has recently been reported in atherosclerotic lesions. The aim of the present study was to evaluate the expression of iNOS in various arteries of rabbits fed a long-term but low-level cholesterol-enriched diet which promotes different types of atherosclerotic lesions resembling human diseased vessels. No iNOS expression was revealed in arteries from control rabbits and in fatty streaks found in carotid and femoral arteries from hypercholesterolemic rabbits. In transitional lesions from the thoracic and abdominal aortas, the coronary and pulmonary arteries, a punctiform iNOS staining was detected in the intima. When lesions were more advanced, iNOS expression was found more intense and diffuse and localized in the subendothelial layer as well as in the media. Smooth muscle cell accumulation in intimal layers of the arteries is a marker of the degree of evolution of the atherosclerotic lesion; since we found a correlation between the smooth muscle cell infiltration in the intima and the iNOS expression in the intima and the subendothelial layer, our results suggest a link between the severity of the lesion and the iNOS expression.