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.

Temperature-dependent basal tone in isolated human saphenous veins: implication of TP-receptors.

Cutaneous blood vessels are very sensitive to changes in environmental temperature. The influence of variations in local temperature on the mechanisms involved in the basal tone, present in isolated human saphenous veins has not yet been studied. In the present study, segments with and without endothelium of human saphenous veins obtained from coronary bypass surgery patients were mounted for isometric tension recording in oxygenated physiological salt solution (PSS). After stabilisation of the basal tone, the local temperature was rapidly either decreased from 37 degrees C to 24 degrees C (cooling) or increased from 37 degrees C to 42 degrees C (warming). When antagonists or inhibitors were used the preparations were incubated for 30 min with the drugs. During basal conditions, cooling caused relaxations of the saphenous vein segments with endothelium and warming caused contractions; the absence of the endothelium did not modify these responses. In veins without endothelium, the warming-induced contractions were significantly inhibited by verapamil (10 microM) and by the antagonist of TP-receptors (receptors for thromboxane A2) Bay u 3405 (1 microM). The warming induced contractions were not affected by cyclooxygenase or lipoxygenase inhibition. At 37 degrees C, the isoprostanes (8-iso-PGE2 and 8-iso-PGF2alpha) induced potent contractions that were significantly inhibited by Bay u 3405 (1 microM). The data show that a basal tone is present in isolated resting human saphenous vein segments at 37 degrees C. This basal tone is decreased by local cooling and enhanced by local warming and is not dependent on the presence of the endothelium. The warming-induced contraction of the veins is mediated by a non-cyclooxygenase, non-lipoxygenase metabolite (isoprostane?) that interacts with TP-receptors and via an extracellular calcium-dependent pathway.

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.

Evidence for induction of nonendothelial NO synthase in aortas of cholesterol-fed rabbits.

The aim of our study was to examine the effects of the inhibitor of nitric oxide (NO)-synthase, nitro-L-arginine (L-NNA), in atherosclerotic aortas obtained from cholesterol-fed rabbits. In the atherosclerotic aortas, L-NNA (100 microM) caused endothelium-independent contractions that were not observed in aortas from control rabbits. L-NNA (100 microM) significantly enhanced the contractile responses to norepinephrine and 5-hydroxytryptamine (5-HT) in atherosclerotic aortas with and without endothelium; in control aortas, L-NNA only augmented the response to 5-HT when the endothelium was present. The concentration-dependent increases in the norepinephrine-induced contractions caused by L-NNA (1 to 100 microM) could be reversed by L-arginine (1 mM) both in atherosclerotic aortas with and without endothelium. L-NMMA also evoked concentration-dependent augmentations of the norepinephrine-induced contraction; the effect of L-NMMA was equipotent to that of L-NNA. Finally, L-NNA (100 microM) prevented the paradoxical endothelium-independent contraction to hypoxia in atherosclerotic aortas. These data strongly suggest that nonendothelial NO synthase has been induced in the aortas of the hyperlipidemic rabbit.

Venous and arterial endothelial cells respond differently to thrombin and its endogenous receptor agonist.

The effects of thrombin and a peptide mimicking the amino terminus of its receptor, Res (42-55), on vascular reactivity were compared in isolated canine blood vessels. In saphenous veins contracted with endothelin-1, both thrombin and Res (42-55) caused relaxation in rings with endothelium and contraction in rings without endothelium. In coronary arteries, thrombin caused similar responses while Res (42-55) only caused contraction. These data suggest that different thrombin receptors are present on venous and arterial endothelial cells.

Daflon 500 mg depresses bradykinin-ischemia-induced microvascular leakage of FITC dextran in rat cremaster muscle.

We studied the in vivo effects of Daflon 500 mg on transvascular movement of macromolecules induced by bradykinin (BK) and ischemia. The experimental preparation involved the rat cremaster muscle. The muscle was fashioned as a single bag (new procedure), placed in a transparent chamber and superfused with a bicarbonate buffer solution equilibrated with a 5% CO2 95% N2 gas mixture in order to obtain pH 7.40, PCO2 = 40 mmHg, PO2 = 20-40 mmHg and thermostated at 35 degrees C. FITC-Dextran 150 (MW 150,000) was injected i.v. as a macromolecular tracer. BK was added to the buffer solution at the concentration of 2 micrograms/ml five minutes after a control period of 60 minutes. Ischemia was performed during 60 min by a clamp positioned on the main artery of the cremaster muscle. Animals treated with Daflon 500 mg (100 mg/kg) 18 and 2 hours before experiments showed a significant reduction in FITC-Dextran 150 leakage in both BK and ischemia protocols. Leakage of FITC-Dextran 150 started 2-3 min after application of BK in the two animal groups but the response was less important (+ 270%) and the preparation returned to control appearance after 40 min in the treated rats in contrast with control rats (+ 450% and 70 min). The amplitude of FITC-Dextran 150 leakage was identical just one hour after ischemia in the two animal groups, but microvascular permeability returned to basal state in treated animals (30 min), a fact non observed in non treated animals. These data demonstrate the protective effect of Daflon 500 mg on the microvascular muscle network in vivo.