Coronary bifurcation lesions.

Coronary bifurcations are prone to develop atherosclerotic plaque due to turbulent blood flow and high shear stress. These lesions amount to 15-20% of the total number of interventions. The true bifurcation lesion consist of >50% diameter obstruction of the main vessel (MV) and of the side branch (SB) in an inverted "Y" fashion. Treatment of coronary bifurcation lesions represents a challenging area in interventional cardiology but recent advances in percutaneous coronary interventions (PCI) have led to the dramatic increase in the number of patients successfully treated percutaneously. When compared with non-bifurcation interventions, bifurcation interventions have a lower rate of procedural success, higher procedural costs, longer hospitalization and a higher clinical and angiographic restenosis. Introduction of drug-eluting stents (DES) has resulted in a lower event rate and reduction of main vessel (MV) restenosis in comparison with historical controls. However, side branch (SB) ostial residual stenosis and long-term restenosis remains a problem. Although stenting the MV with provisional SB stenting seems to be the prevailing approach, in the era of DES various two-stent techniques have emerged to allow stenting of the large side branch.

Angiotensin AT1 receptor antagonist irbesartan decreases lesion size, chemokine expression, and macrophage accumulation in apolipoprotein E-deficient mice.

Recent data suggest that angiotensin II AT1 receptor antagonists may be beneficial in the treatment of atherosclerosis. To clarify how AT1 receptor antagonists reduce atherosclerosis, the effect of irbesartan on atherosclerotic lesion development was determined in low-fat, chow-fed apolipoprotein (Apo) E-deficient mice. Irbesartan (50 mg/kg per day) strongly decreased lesion development after a 12-week treatment period (lesion size: irbesartan treated, 20,524 +/- 4,200 microm(2) vs. control, 99,600 +/- 14,500; 79.4% inhibition, p < 0.001). This effect was not due to an effect of irbesartan on lipoprotein levels because irbesartan slightly increased total cholesterol levels and decreased the ratio of Apo A-I relative to Apo B levels. Immunochemical analysis of the atherosclerotic lesions using the mac3 monoclonal antibody showed the presence of macrophages in the lesions of control mice, whereas sections from irbesartan-treated animals only showed occasional labeling in the lesion area. These data suggest that irbesartan inhibits monocyte/macrophage influx into the vessel wall. Therefore, expression levels of monocyte chemoattractant protein-1 (MCP-1), as well as other chemokines involved in macrophage infiltration into the lesion area, were measured in the aortic sinus of control and irbesartan-treated animals. Irbesartan treatment strongly decreased MCP-1 mRNA levels as well as MCP-1 immunostaining in the lesion area. This effect of irbesartan on MCP-1 occurred without an effect on CCR2, the receptor of MCP-1. Expression of macrophage inflammatory protein (MIP)-1alpha, another CC chemokine expressed in atherosclerotic lesions, was also reduced after irbesartan treatment, without effect on CCR3 and CCR5, the receptors of MIP-1alpha. Concomitantly, the expression of the angiogenic chemokines KC and MIP-2, which are functionally related to interleukin-8, were downregulated, whereas their shared receptor CXCR2 was upregulated. These data suggest that inhibition of the inflammatory component of lesion progression plays an important role in the inhibitory effect of AT1 receptor antagonists on atherosclerotic lesion formation.

Relaxant effect of 2-methyl-thio-adenosine diphosphate on rat thoracic aorta: effect of clopidogrel.

The main aim of this study was to determine the functional effect of 2-methyl-thio-adenosine diphosphate (2MeS-ADP) on vascular purinoceptors, in comparison with that of a characterised agonist of the P2Y1 receptor, 2-methyl-thio-adenosine triphosphate (2MeS-ATP), and of the P2Y2 receptor, uridine triphosphate (UTP). On phenylephrine-precontracted rat aortic rings, mounted isometrically in organ baths, we found that 2MeS-ADP (10(-9) to 10(-6) M) induced concentration-dependent relaxation of rings with a functional endothelium. Mechanical removal of the endothelium abolished the relaxant effect of 2MeS-ADP. The 2MeS-ADP-induced relaxation of phenylephrine-precontracted rings was inhibited by Nomega-nitro-L-arginine methyl ester (L-NAME) (100 microM) but not by indomethacin (100 microM) or aspirin (1 mM), indicating that the 2MeS-ADP-induced relaxation was nitric oxide (NO) synthase-mediated but not cyclooxygenase-dependent. Repeated stimulation with 2MeS-ADP resulted in desensitisation of the receptor. Under these conditions, the relaxant effect of 2MeS-ATP was abolished. On the contrary, UTP-induced relaxation was not affected, showing that 2MeS-ADP and 2MeS-ATP but not UTP shared the same receptor. Suramin (100 microM), a non-specific P2 inhibitor, abolished the effect of 2MeS-ADP, 2MeS-ATP and UTP. In contrast, pyridoxal-phosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS) and adenosine-3'-phosphate-5'-phosphosulphate (A3P5PS) abolished only the vasodilator responses to 2MeS-ADP and 2MeS-ATP and did not affect the relaxant effect of UTP, showing that 2MeS-ADP acted through the P2Y1 receptor. Clopidogrel, a potent platelet ADP receptor antagonist, at a dose that strongly inhibited ADP-induced platelet aggregation ex vivo, did not modify the relaxant responses to 2MeS-ADP or 2MeS-ATP. In conclusion, these results showed that 2MeS-ADP induces endothelium-dependent, NO-mediated relaxation of rat aortic rings. This effect, resistant to clopidogrel treatment, occurred through activation of the P2Y1 receptor.

Coagulation factor Xa induces endothelium-dependent relaxations in rat aorta.

The relaxing effect of coagulation factor Xa on phenylephrine-contracted rat aortic rings was compared with the effect of thrombin and trypsin. All three proteases induced a dose-dependent relaxation in the presence of an intact endothelium. EC50 values were 3 +/- 1, 24 +/- 9, and 16 +/- 1 nmol/L for thrombin, trypsin, and factor Xa, respectively. Whereas thrombin induced rapid relaxations followed by partial recontraction, trypsin and factor Xa induced slower sustained effects. Factor Xa-induced relaxations were not affected by hirudin at high concentrations (1 mumol/L) but were abolished by DX9065A, a specific inhibitor of the catalytic activity of factor Xa. Furthermore, no relaxations to factor Xa could be elicited in the presence of the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (100 mumol/L), whereas relaxations were not altered in the presence of the inactive enantiomer N omega-nitro-D-arginine methyl ester (100 mumol/L). Addition of factor Xa together with thrombin induced relaxations that were larger than those induced by thrombin alone, whereas factor Xa had no additional effects on trypsin-induced relaxations. Further-more, factor Xa relaxed thrombin-desensitized aortic rings but was ineffective in trypsin-desensitized tissues. These data suggest that factor Xa acts on a cleavable endothelial receptor that induces NO release, resulting in the relaxation of precontracted rat aortic rings. Factor Xa does not act through endothelial thrombin receptors but may activate another cleavable trypsin-sensitive receptor.

Intimal hyperplasia following vascular injury is not inhibited by an antisense thrombin receptor oligodeoxynucleotide.

Thrombin is a multifunctional serine protease with central functions in hemostasis, but demonstration of its role in the initiation and maintenance of cell proliferation which occurs following vascular injury is still lacking. To determine the role played by thrombin and its receptor in neointimal accumulation of smooth muscle cells in a rabbit carotid artery model, we have used an 18 mer antisense phosphorothioate oligonucleotide (ODN) directed against the translation initiation region of the human thrombin receptor gene. The antisense ODN inhibited in a dose-dependent manner thrombin- or thrombin receptor activating peptide-induced human aortic smooth muscle cell proliferation. The growth-inhibitory effect of thrombin receptor antisense ODN was preventable by an excess of sense oligomer and specific for thrombin. The suppression of growth was accompanied by a marked decrease of the level of thrombin receptor expression as evidenced by [125I]-thrombin binding to smooth muscle cells. Under the same experimental conditions, the corresponding sense ODN was inactive. The effect of the antisense ODN on intimal smooth muscle hyperplasia in rabbit carotid arteries subjected to endothelial injury was then investigated. The topical application of the antisense (500 microg/artery) but not the sense ODN dissolved in F127 pluronic gel around the injured artery resulted, 2 weeks after the application, in a dramatic reduction of the expression of the thrombin receptor mRNA and protein levels as determined by in situ hybridization and immunohistochemistry. However, intimal smooth muscle cell accumulation as estimated by an intimal to medial cross-sectional area ratio was reduced only by 2.7% (vs. 10.3% for the sense ODN), whereas r-hirudin (200 microg/kg/day, s.c.), a potent direct thrombin inhibitor significantly reduced the formation of neointima in denuded carotid arteries (35.4% inhibition, P = 0.03).

Platelet-activating factor (PAF) is not an essential component of the cascade leading to smooth muscle cell proliferation following vascular injury.

In order to evaluate the relative importance platelet-activating factor (PAF) in the proliferative process leading to restenosis, the effect of SR 27417, a novel highly potent PAF receptor antagonist, on PAF-induced rabbit aortic smooth muscle cell (SMC) proliferation and intimal hyperplasia in rabbit carotid arteries subjected to air-drying endothelial injury was investigated. When added to low concentrations of foetal calf serum, PAF showed a dose-dependent mitogenic effect with regard to rabbit arterial SMC. SR 27417 inhibited PAF-induced SMC growth (IC50 = 2.4 +/- 0.4 nM) but remained without effect on the mitogenic effect of foetal calf serum. A 16 day treatment of SR 27417 (10 mg/kg per day, p.o.) abrogated PAF-induced platelet aggregation ex vivo but did not affect the development of intimal thickening, therefore showing that PAF is not an essential component of the cascade leading to restenosis following vascular injury.

Effect of SR 33805 on arterial smooth muscle cell proliferation and neointima formation following vascular injury.

The possible activity of SR 33805 ([[N-[dimethoxy-3,4-phenethyl]-N- methylamino-propoxyl]-4-benzenesulfonyl]-2-isopropyl-3-methyl-1-in dole), a novel Ca2+ channel blocker, in early atherogenesis was investigated. In vitro, SR 33805 strongly inhibited fetal calf serum-induced proliferation of cultured human aortic smooth muscle cells with an IC50 value of 0.3 +/- 0.1 microM (n = 3). In this respect, SR 33805 was several fold more active than the reference compounds: diltiazem, verapamil, nifedipine and fantofarone. SR 33805 was also a potent inhibitor of platelet-derived growth factor- or basic fibroblast growth factor-induced proliferation of human smooth muscle cells. SR 33805 inhibited serum-stimulated 45Ca2+ uptake in these cells, with an IC50 value of 47 +/- 18 nM. The effect of SR 33805 on intimal smooth muscle hyperplasia in rabbit carotid arteries subjected to air-drying endothelial injury was then investigated. After a 16-day treatment, SR 33805 (6.0 mg/kg/day p.o.) inhibited the development of intimal thickening. Under the same experimental conditions, nifedipine, verapamil, diltiazem (2 x 6 mg/kg/day p.o.--16 days) and fantofarone (12 mg/kg/day p.o.--16 days) were inactive. These results show that SR 33805, a novel and potent Ca2+ channel blocker, can reduce myointimal thickening following endothelial injury.

Effect of various antiplatelet agents on acute arterial thrombosis in the rat.

Electrical stimulation of the rat carotid artery causes a deep medial injury and the formation of a platelet-rich thrombus. Occlusive thrombosis at sites of vessel wall injury was significantly reduced after the oral administration of clopidogrel, a potent analogue of ticlopidine, which showed dose-dependent inhibition of the thrombus formation (ED50 = 1.0 +/- 0.2 mg/kg, p.o.). Accumulation of thrombotic material was also considerably reduced after the i.v. administration of SR 27417, a highly potent and selective platelet activating factor receptor antagonist (ED50 = 10 micrograms/kg, i.v.), nafagrel, a thromboxane A2 synthetase inhibitor (ED50 = 1.3 mg/kg, i.v.) and hirudin (ED50 = 140 micrograms/kg, i.v.). A high dose (20 mg/kg, i.v.) of the anti-adhesive tetrapeptide Arg-Gly-Asp-Ser (RGDS) showed only a slight effect on thrombus formation whereas aspirin was ineffective. These results confirm that ADP and thromboxane A2 play key roles in the initiation and progression of arterial thrombus formation and suggest that platelet activating factor may also modulate thrombosis in this experimental model.