Oxidative stress is a cardinal feature of the inflammatory process and is involved in various pathologies including atherosclerosis. One of the important mechanisms in which oxidative stress may play a role is activation of matrix metalloproteinases such as MMP-2, which are involved in plaque destabilization. We investigated the mechanisms by which oxidative stress induces MMP-2 activation in cultured human coronary artery smooth muscle cells. Using zymography and Western blot analysis, we showed that oxidized low-density lipoproteins activate MMP-2 through up-regulation of the expression and activation of a membrane-type 1 matrix metalloproteinase (MT1-MMP). A second mechanism of MMP-2 activation involves oxidative radicals generated by the xanthine/xanthine oxidase complex (X/Xo). Research on these two mechanisms of MMP activation could lead to the elaboration of new vascular therapies for the treatment of atheroma based on interruption of a specific oxidative stress pathway.

The 8-iso-prostaglandin F 2␣ , a prostanoid produced in vivo by cyclooxygenase-independent free-radical-catalyzed lipid peroxidation, acts as a partial agonist on the thromboxane receptor (TXA 2 R) and is a potent vasoconstrictor in the oxidatively stressed isolated perfused rat heart. We hypothesized that the response in the isolated heart may be due to augmentation of TXA 2 R density, which may be initiated by the presence of oxidative radicals. Previous studies have shown that TXA 2 R density is increased during atherosclerosis on both the medial and intimal smooth muscle layers in human coronary arteries. Here we describe the effect of oxidative stress on TXA 2 R. The thromboxane A 2 receptor ␤ isoform (TXA 2 R␤) was transiently expressed in COS-7 cells. (TXA 2 ) 1 is an unstable arachidonate metabolite, implicated as a mediator in diseases such as myocardial infarction, stroke, and bronchial asthma (1). Binding of TXA 2 to its receptor, a polytopic membrane-spanning protein, induces vasoconstriction and platelet aggregation, as well as mitogenesis and hypertrophy of vascular smooth muscle cells (2). Two TXA 2 receptor (TXA 2 R) isoforms have been identified, TXA 2 R␣ (343 amino acids), which is mainly located in the placenta, and TXA 2 R␤ (407 amino acids), located in the endothelium; these isoforms are generated by the alternative splicing of a single gene (3, 4). The TXA 2 R is part of the G proteincoupled receptor superfamily, and evidence suggests that TXA 2 -induced production of second messenger inositol polyphosphates results from the activation of the G q11 family of heterotrimeric G proteins (5).Isoprostanes are formed by free radical attack on membrane phospholipids during oxidative stress (6). They are found in increased concentration in patients with coronary heart disease and are potent vasoconstrictors (7). We have shown that one of these, the 8-iso-prostaglandin F 2␣ , is a potent coronary vasoconstrictor, and its effect is exerted via partial agonist action on the TXA 2 R (8). This mechanism of action on TXA 2 R, in vascular smooth muscle and in platelets, has been confirmed in a TXA 2 R knock-out mouse (9). Our data suggested that a critical determinant of the intrinsic activity of the isoprostane is the TXA 2 R reserve, and this has subsequently been supported by another study (10). We have shown that, in the normal rat heart perfused at constant pressure in the Langendoff mode, 8-iso-prostaglandin F 2␣ had no effect, even though U46619, a TXA 2 R agonist, produced a pronounced vasoconstriction. However, after an oxidative stress induced by 30 min of low flow and reperfusion or by a superoxide-generating system (i.e. xanthine and xanthine oxidase), 8-iso-prostaglandin F 2␣ became a potent vasoconstrictor, whereas the response to U46619 was unchanged (11). Responses to both agonists were inhibited by the TXA 2 R antagonist SQ29548, suggesting that they act upon the same receptor.The rapidity of the change in response suggests that this is unlikely to be due to alterations in gene expressio...

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Thromboxane A 2 (TXA 2 ) is a key mediator of platelet aggregation and smooth muscle contraction. Its action is mediated by its G protein-coupled receptor of which two isoforms, termed TP␣ and TP␤, occur in humans. TXA 2 has been implicated in pathologies such as cardiovascular diseases, pulmonary embolism, atherosclerosis, and asthma. This study describes the pharmacological characterization of BM-613 [N-n-pentyl-NЈ-[2-(4Ј-methylphenylamino)-5-nitrobenzenesulfonyl]urea], a new combined TXA 2 receptor antagonist and TXA 2 synthase inhibitor. It exhibits a strong affinity for human platelet TP receptors (IC 50 ϭ 1.4 nM), TP␣ and TP␤ expressed in COS-7 cells (IC 50 ϭ 2.1 and 3.1 nM, respectively), and TPs expressed in human coronary artery smooth muscle cells (IC 50 ϭ 29 M). BM-613 shows a weak ability to prevent contraction of isolated rat aorta (ED 50 ϭ 1.52 M) and guinea pig trachea (ED 50 ϭ 2.5 M) induced by TXA 2 agonist U-46619 (9.11-dideoxy-9.11-methanoepoxy-prostaglandin F 2 ). Besides, BM-613 antagonizes TP␣ (IC 50 ϭ 0.11 M) and TP␤ (IC 50 ϭ 0.17 M) calcium mobilization induced by U-46619 and inhibits human platelet aggregation induced by U-46619 (ED 50 ϭ 0.278 M), arachidonic acid (ED 50 ϭ 0.375 M), and the second wave of ADP. BM-613 also dose dependently prevents TXA 2 production by human platelets (IC 50 ϭ 0.15 M). In a rat model of ferric chloride-induced thrombosis, BM-613 significantly reduces weight of formed thrombus by 79, 49, and 28% at 5, 2, and 1 mg/kg i.v., respectively. In conclusion, BM-613 is a dual and potent TP receptor antagonist and TXA 2 synthase inhibitor characterized by a strong antiplatelet and antithrombotic potency. These results suggest that BM-613 could be a potential therapeutic drug for thrombotic disorders.Thromboxane A 2 (TXA 2 ) is a key lipid mediator characterized by several implications in physiological homeostasis, including platelet aggregation and vascular and bronchial smooth muscle constriction (Hamberg et al., 1975;Moncada and Vane, 1978). An overproduction of TXA 2 has been associated with many pathological states such as myocardial infarction, thrombosis and thrombotic disorders, unstable angina, pulmonary embolism, shock, atherosclerosis, preeclampsia, and asthma (Dogné et al., 2004a).TXA 2 is a metabolite of arachidonic acid (AA), a 20-carbon