Atsushi Yamashita

Thrombin generation by intimal tissue factor contributes to thrombus formation on macrophage-rich neointima but not normal intima of hyperlipidemic rabbits

Arterial thrombosis occurs in atherosclerotic, but rarely in non-atherosclerotic arteries. The present study investigates how hyperlipidemic condition affects thrombus formation on macrophage-rich neointima or normal intima in rabbits. Rabbits were fed with a 0.5% cholesterol diet, and then the femoral artery on one side of each rabbit was injured with a balloon catheter. Three weeks later, bilateral femoral arteries were similarly injured with a balloon catheter to produce thrombi on neointima and normal intima. We compared the expression and activity of intimal tissue factor (TF) as well as thrombus size and composition between these femoral arteries. 0.5% cholesterol diet combined with a balloon injury induced macrophage-rich neointima in injured arteries. The whole blood coagulation activity or plasma thrombin generation activity did not differ after consuming the 0.5% cholesterol diet for 4 weeks, and an anti-TF antibody did not affect the measured parameters. TF activities were increased in the neointima/media compared with normal intima/media. Balloon injury induced large platelet-fibrin thrombi on macrophage-rich neointima, whereas small platelet thrombi were produced in normal arteries even under hyperlipidemic conditions. Recombinant human tissue factor pathway inhibitor (25microg/(kgmin)) and argatroban (100microg/(kgmin)), a specific thrombin inhibitor, significantly reduced thrombus formation on induced neointima, but not on normal intima. Thrombin generation mediated by TF in intima contributes to thrombus formation on macrophage-rich neointima, but not on normal intima. The TF content in disrupted atherosclerotic plaques might play a more important role than hyperlipidemia in the development of atherothrombosis.

ADAMTS-13 attenuates thrombus formation on type I collagen surface and disrupted plaques under flow conditions

Plaque disruption with subsequent thrombus formation is a major cause of atherothrombotic diseases and von Willebrand factor (VWF), which is cleaved by ADAMTS-13, plays a critical role in thrombus formation. However, the role of ADAMTS-13 during thrombogenesis on atherosclerotic vessel remains unknown. We examined the localization of ADAMTS-13 in coronary thrombi obtained from patients with acute myocardial infarction. We also investigated the roles of ADAMTS-13 in thrombus formation using type I collagen-coated flow chambers (100S(-1) and 1500S(-1)) and on injured neointima of rabbit femoral arteries. ADAMTS-13 was present in thrombi of human coronary arteries, where it co-localized with VWF. In a flow chamber, both the average of the surface covered by platelet adhesion and the long axes of platelet thrombi were significantly augmented by an antibody to the ADAMTS-13 disintegrin-like domain (WH2-22-1A) at a shear rate of 1500s(-1), but not by an antibody to the ADAMTS-13 thrombospondin 1-3 domain (WH10). WH2-22-1A also reduced the activity of plasma ADAMTS-13 to cleave large VWF multimers during perfusion. Thrombi on injured neointima were induced by repeated balloon injury of rabbit femoral arteries, and were composed of platelet and fibrin, like human coronary thrombi. WH2-22-1A significantly augmented thrombus formation on injured neointima. These results suggest that the disintegrin-like domain of ADAMTS-13 functions in attenuating thrombus growth on diseased arteries exposed to a high shear rate.

Thioredoxin in coronary culprit lesions: possible relationship to oxidative stress and intraplaque hemorrhage.

The present study investigated the expression of thioredoxin (TRX), an important anti-oxidative protein, and its relationship to plaque instability in atherectomy specimens from 43 and 42 patients with stable (SAP) and unstable (UAP) angina pectoris, respectively. We histologically assessed thrombus formation, cellular elements, localization of TRX and of oxidized low density lipoprotein (ox-LDL), intraplaque hemorrhage, and transition metal iron (Fe(2+), Fe(3+)) deposition in these specimens. The clinical characteristics of the two groups did not differ except for aspirin administration. The incidence of thrombus formation was more frequent (P=0.005) and immunopositive areas of macrophage, TRX and ox-LDL were significantly larger in patients with UAP than SAP (P<0.001, each). Macrophages were mainly immunoreactive for TRX and ox-LDL. Intraplaque hemorrhage evaluated by glycophorin A immunoreactivity and Fe(2+)/Fe(3+) deposition was also more obvious in lesions from patients with UAP than SAP (P<0.001, each). Additionally, immunopositive areas of TRX and ox-LDL positively correlated with Fe(2+)/Fe(3+) deposition and were also associated with thrombus formation. Although the underlying mechanisms remain unknown, TRX was up-regulated in response to increased oxidative stress and associated with intraplaque hemorrhage of coronary culprit lesions, and thus might be a potent marker of plaque instability.

Serotonin induces vasoconstriction of smooth muscle cell-rich neointima through 5-hydroxytryptamine2A receptor in rabbit femoral arteries.

Summary.u2002 Background:u2002Smooth muscle cell (SMC)‐rich intima is a morphological feature of atherosclerotic lesions that is observed in eroded plaque and spastic arteries. Arteries with SMC‐rich intima are susceptible to vasoconstriction or vasospasm against some vasoactive agents. Objective:u2002The present study evaluates the contribution of SMC‐rich intima to thrombogenic vasoconstriction. Methods:u2002We established SMC‐rich neointima by damaging rabbit femoral arteries using balloons and then measured the isometric tension of the femoral strips against 5‐hydroxytryptamine (5‐HT), adenosine diphosphate, adenosine triphosphate and thrombin. Results:u2002Among these agents, only 5‐HT induced a hypercontractile response of the injured arteries with SMC‐rich neointima, compared with non‐injured arteries. Smooth muscle cells of both the neointima and media expressed 5‐HT2A receptor, and sarpogrelate, a selective 5‐HT2A receptor antagonist significantly inhibited the hypercontraction. Furthermore, 5‐HT induced contraction of separated neointima and hypercontraction of separated media compared with non‐injured media. Sarpogrelate and fasudil, a specific Rho‐kinase inhibitor, significantly suppressed such contraction of both the neointima and media of injured arteries. Conclusions:u2002These results suggest that 5‐HT plays a crucial role in thrombogenic vasoconstriction, and that SMC‐rich intima as well as media directly contributes to the hypercontractile response of atherosclerotic vessels through the 5‐HT2A receptor and the Rho‐kinase pathway.

BOTH 5-HYDROXYTRYPTAMINE 5-HT2A AND 5-HT1B RECEPTORS ARE INVOLVED IN THE VASOCONSTRICTOR RESPONSE TO 5-HT IN THE HUMAN ISOLATED INTERNAL THORACIC ARTERY

1 The 5‐hydroxytryptamine (5‐HT, serotonin) receptor subtypes that mediate vasoconstriction in the human internal thoracic artery (ITA), which is frequently used as an arterial graft, remain unclear. The aim of the present study was to elucidate the 5‐HT receptor subtypes responsible for 5‐HT‐induced contraction of the human ITA. 2 The contractile responses to 5‐HT of endothelium‐denuded human ITA obtained from patients undergoing coronary bypass surgery were examined. In addition, we investigated the effects of sarpogrelate and SB224289, antagonists of 5‐HT2A and 5‐HT1B receptors, respectively, on the 5‐HT‐induced vasoconstriction. Finally, 5‐HT2A and 5‐HT1B receptors in the human ITA were immunolabelled. 3 5‐Hydroxytryptamine (1 nmol/L–10 mmol/L) caused vasoconstriction in a concentration‐dependent manner. Both sarpogrelate (1 mmol/L) and SB224289 (1 mmol/L) significantly, but not completely, inhibited 5‐HT‐induced vasoconstriction. 4 Conversely, simultaneous pretreatment with supramaximum concentrations (1 mmol/L for both) of sarpogrelate and SB224289 almost completely inhibited the 5‐HT‐induced vasoconstriction. 5 Immunopositive staining for 5‐HT2A and 5‐HT1B receptors was detected in smooth muscle cells of the human ITA. 6 These results demonstrate that, in human ITA, 5‐HT‐induced vasoconstriction is mediated by activation of both 5‐HT2A and 5‐HT1B receptors. Thus, when the human ITA is used as an arterial graft, a combination of 5‐HT2A and 5‐HT1B receptor antagonists would appear to be most useful to prevent 5‐HT‐induced vasospasm.

Interleukin-10 correlates with oxidized low density lipoprotein in coronary culprit plaques

Inflammation plays an important role in the pathogenesis of atherosclerosis and plaque instability [1,2]. We and others have demonstrated that many pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, IL-18 and interferon-γ are expressed in human atherosclerotic plaques, and the expression levels of most of them are increased at sites of plaque rupture [1–3]. However, the roles of antiinflammatory cytokines and their localization in coronary culprit plaque have not been defined. Oxidized low density lipoprotein (oxLDL) is also involved in the disruption of atherosclerotic plaque [2,4]. Vulnerable plaques contain more significant numbers of ox-LDLpositive macrophages [4], which when stimulated by ox-LDL release many pro-inflammatory cytokines that contribute to plaque instability [2], as well as IL-10, which is an important anti-inflammatory cytokine [1,5]. We therefore immunohistochemically investigated the localiza-