Involvement of the TP receptor in TNF-α-induced endothelial tissue factor expression.

BACKGROUND: Thromboxane (TX) A2, prostaglandin endoperoxides and F2-isoprostanes exert their effects through a TX-prostanoid (TP) receptor, also expressed in endothelial cells. We investigated a role of the TP receptor in the endothelial expression of tissue factor (TF), a key trigger to thrombosis. METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVEC) exposed to the TP receptor agonist U46619 featured a concentration-dependent increase in TF surface exposure and procoagulant activity. HUVEC pre-incubation with the TP receptor antagonist S18886, followed by stimulation with either U46619 or tumor necrosis factor-α (TNF-α), attenuated TF surface exposure and activity compared with stimulated control. Aspirin or indomethacin, while inhibiting cyclooxygenase (COX)-1 and -2 activities, did not mimic this effect. Probing of underlying mechanisms by selective pharmacological and gene silencing experiments showed that S18886 reduced U46619- or TNF-α-induced TF expression inhibiting ROS production, NAD(P)H oxidase and PKC activation. In addition, S18886 also inhibited ERK activation in the presence of both U46619 and TNF-α alone, while inhibition of JNK activation only occurred in the presence of U46619. CONCLUSION: The endothelial TP receptor contributes to TF surface exposure and activity induced not only by known TP receptor agonists, but also by TNF-α. Such findings expand the therapeutic potential of TP receptor inhibition.

Terutroban, a thromboxane/prostaglandin endoperoxide receptor antagonist, prevents hypertensive vascular hypertrophy and fibrosis.

Thromboxane A(2) and other eicosanoids such as isoprostanes contribute to vascular proliferation and atherosclerosis by binding to the thromboxane/prostaglandin endoperoxide receptors. The effects of terutroban, a thromboxane/prostaglandin endoperoxide receptor antagonist, on aorta remodeling were evaluated in spontaneously hypertensive stroke-prone rats (SHRSPs), a model of severe hypertension, endothelial dysfunction, vascular inflammation, and cerebrovascular diseases. Male SHRSPs were allocated to three groups receiving a standard diet (n = 5) or a high-sodium permissive diet plus vehicle (n = 6) or plus terutroban (30 mg · kg(-1) · day(-1); n = 6). After 6 wk of dietary treatment, all of the animals were injected with bromodeoxyuridine and simultaneously euthanized for aorta collection. The aortic media thickness-to-lumen ratio significantly (P < 0.0001) increased in the salt-loaded rats compared with the rats fed a standard diet, whereas terutroban treatment completely prevented media thickening (P < 0.001). When compared with vehicle, terutroban was also effective in preventing cell proliferation in the media, as indicated by the reduced number of bromodeoxyuridine-positive (P < 0.0001) and proliferating cell nuclear antigen-positive cells (P < 0.0001). Severe fibrosis characterized by a significant accumulation of collagen and fibronectin in the vascular wall was observed in the vehicle-treated rats (P < 0.01) but was completely prevented by terutroban (P < 0.001). The latter also inhibited heat shock protein-47 (P < 0.01) and TGF-1ß expression (P < 0.001), which were significantly increased by the high-salt diet. In conclusion, terutroban prevents the development of aorta hyperplasia and has beneficial effects on fibrotic processes by affecting TGF-ß and heat shock protein-47 expression in SHRSPs. These findings provide mechanistic data supporting the beneficial effects of terutroban in preventing or retarding atherogenesis.

Terutroban, a thromboxane/prostaglandin endoperoxide receptor antagonist, increases survival in stroke-prone rats by preventing systemic inflammation and endothelial dysfunction: comparison with aspirin and rosuvastatin.

This study investigated the efficacy of terutroban, a specific thromboxane/prostaglandin endoperoxide receptor antagonist, on stroke incidence in spontaneously hypertensive stroke-prone rats (SHRSP). The effects of terutroban were compared with those of aspirin, another antiplatelet agent, and rosuvastatin, known to exert end-organ protection in SHRSP. Salt-loaded male SHRSP were treated orally once a day with vehicle, terutroban (30 mg/kg/day), aspirin (60 mg/kg/day), or rosuvastatin (10 mg/kg/day). Compared with vehicle, and regardless of any effect on blood pressure or serum thromboxane B(2) levels, terutroban significantly increased survival (p < 0.001) as a consequence of a delayed brain lesion occurrence monitored by magnetic resonance imaging (p < 0.001), and a delayed increase of proteinuria (p < 0.001). Terutroban decreased cerebral mRNA transcription of interleukin-1beta, transforming growth factor-beta, and monocyte chemoattractant protein-1 after 6 weeks of dietary treatment. Terutroban also prevented the accumulation of urinary acute-phase proteins at high molecular weight, identified as markers of systemic inflammation, and assessed longitudinally by one-dimensional electrophoresis. Terutroban also has protective effects on the vasculature as suggested by the preservation of endothelial function and endothelial nitric-oxide synthase expression in isolated carotid arteries. These effects are similar to those obtained with rosuvastatin, and superior to those of aspirin. Terutroban increases survival in SHRSP by reducing systemic inflammation as well as preserving endothelial function. These data support clinical development of terutroban in the prevention of cerebrovascular and cardiovascular complications of atherothrombosis.

Micronization enhances the protective effect of purified flavonoid fraction against postischaemic microvascular injury in the hamster cheek pouch.

1. The present study was designed to evaluate the effect of micronization on the protective effect of the purified flavonoid fraction (MPFF) on increases in macromolecular permeability induced by ischaemia-reperfusion in the hamster cheek pouch microcirculation. 2. Male hamsters (Mesocricetus auratus) were treated orally, twice a day, with vehicle (lactose), MPFF and non-micronized purified flavonoid fraction (PFF) at 5, 20, 80 and 320 mg/kg per day for 10 consecutive days. On the 11th day, cheek pouches of anaesthetized animals were prepared for intravital microscopy. 3. Local ischaemia was obtained by clamping the neck of the everted pouch and the increase in microvascular permeability was quantified as leakage (leaks) of intravenously injected fluorescein isothiocyanate-labelled dextran (FITC-dextran 150; MW = 150 000). 4. Reperfusion, after 30 min ischaemia, resulted in an immediate but reversible increase in post-capillary leakage. The MPFF induced a significant dose-related reduction in the increased permeability, with 83.4% inhibition compared with control at 320 mg/kg per day (19.2 +/- 1.9 vs 115.7 +/- 4.1 leaks/cm2; P < 0.0001). Non-micronized PFF was significantly less effective: only 47.9% inhibition compared with control was observed at 320 mg/kg per day (60.3 +/- 1.0 vs 115.7 +/- 4.1 leaks/cm2; P < 0.0001) and there was no dose-effect relationship. 5. In conclusion, micronization significantly enhances the protective effects of the purified flavonoid fraction on reperfusion injury in the hamster cheek pouch. This improvement is likely to be related to the better absorption of the micronized formulation, which could explain the superior clinical efficacy shown in previous studies.

Enhancement of reperfusion injury by elevation of microvascular pressures.

Elevated venous pressure can be associated with severe tissue injury. Few links, however, between venous hypertension and tissue damage have been established. We examined here the effects of micropressure elevation on the outcome of venular occlusion/reperfusion in the mesenteric microvasculature of male Wistar rats. One hour of venular occlusion (diameter approximately 50 microm) by micropipette occlusion followed by reperfusion were carried out with sham surgery without occlusion as control. Leukocyte rolling, adhesion, and migration, oxygen radicals detected by dichlorofluorescein (DCF), and parenchymal cell death detected by propidium iodide (PI) were recorded simultaneously in the same vessel at a location upstream of the occlusion site with elevated micropressure and at a downstream location with low micropressure. The number of rolling, adhering, and migrating leukocytes increased on the upstream side of the occlusion to a higher level than downstream of the occlusion site. During occlusion, DCF intensity on the venular endothelium was greater on the upstream side than in the downstream side, but there were no differences during reperfusion. The number of PI-positive cells adjacent to the venules increased significantly compared with controls, and it remained greater on the upstream higher-pressure side than the downstream side. Leukocyte adhesion and transvascular migration in postcapillary venules as well as parenchymal cell death could be significantly reduced by the hydroxyl radical scavenger dimethylthiourea. Microhemorrhages of blood cells into the mesentery interstitium were observed only on the upstream side of the occlusion. These results indicate that an elevation of the venular blood pressure during occlusion/reperfusion exacerbates the inflammatory cascade and tissue injury. Venous occlusion may constitute an important mechanism for tissue injury.