Kensaku Nishihira

Increased endoplasmic reticulum stress in atherosclerotic plaques associated with acute coronary syndrome

Background— The endoplasmic reticulum (ER) responds to various stresses by upregulation of ER chaperones, but prolonged ER stress eventually causes apoptosis. Although apoptosis is considered to be essential for the progression and rupture of atherosclerotic plaques, the influence of ER stress and apoptosis on rupture of unstable coronary plaques remains unclear. Methods and Results— Coronary artery segments were obtained at autopsy from 71 patients, and atherectomy specimens were obtained from 40 patients. Smooth muscle cells and macrophages in the fibrous caps of thin-cap atheroma and ruptured plaques, but not in the fibrous caps of thick-cap atheroma and fibrous plaques, showed a marked increase of ER chaperone expression and apoptotic cells. ER chaperones also showed higher expression in atherectomy specimens from patients with unstable angina pectoris than in specimens from those with stable angina. Expression of 7-ketocholesterol was increased in the fibrous caps of thin-cap atheroma compared with thick-cap atheroma. Treatment of cultured coronary artery smooth muscle cells or THP-1 cells with 7-ketocholesterol induced upregulation of ER chaperones and apoptosis, whereas these changes were prevented by antioxidants. We also investigated possible signaling pathways for ER-initiated apoptosis and found that the CHOP (a transcription factor induced by ER stress)-dependent pathway was activated in unstable plaques. In addition, knockdown of CHOP expression by small interfering RNA decreased ER stress-dependent death of cultured coronary artery smooth muscle cells and THP-1 cells. Conclusions— Increased ER stress occurs in unstable plaques. Our findings suggest that ER stress-induced apoptosis of smooth muscle cells and macrophages may contribute to plaque vulnerability.

Factor XI contributes to thrombus propagation on injured neointima of the rabbit iliac artery

Summary.  Background: Thrombus formation through the activation of tissue factor (TF) and factor (F) XI is a critical event in the onset of cardiovascular disease. TF expressed in atherosclerotic plaques and circulating blood is an important determinant of thrombogenicity that contributes to fibrin‐rich thrombus formation after plaque disruption. However, the contribution of FXI to thrombus formation on disrupted plaques remains unclear. Methods: A mouse monoclonal antibody against FXI and activated FXI (FXIa) (XI‐5108) was generated by immunization with activated human FXI. Prothrombin time (PT), activated partial thromboplastin time (APTT), bleeding time, and ex vivo platelet aggregation in rabbits were measured before and after an intravenous bolus injection of XI‐5108. We investigated the role of FXI upon arterial thrombus growth in the rabbit iliac artery in the presence of repeated balloon injury. Results: The XI‐5108 antibody reacted to the light chain of human and rabbit FXI/FXIa, and inhibited FXIa‐initiated FXa and FXIa generation. Fibrin‐rich thrombi developed on the injured neointima that was obviously immunopositive for glycoprotein IIb‐IIIa, fibrin, TF, and FXI. Intravenous administration of XI‐5108 (3.0 mg kg−1) remarkably reduced thrombus growth, and the APTT was significantly prolonged. However, PT, bleeding time and platelet aggregation were not affected. Conclusions: These results indicate that plasma FXI plays a potent role in thrombus growth on the injured neointima. Inhibition of plasma FXI activity might help to reduce thrombus growth on ruptured plaques without prolonging bleeding time.

Inhibition of 5-hydroxytryptamine2A receptor prevents occlusive thrombus formation on neointima of the rabbit femoral artery

Summary.  Background: Thrombus propagation on disrupted plaque is a major cause of acute coronary events and serious complication after coronary intervention. 5‐hydroxytryptamine (5‐HT) is a potent vasoactive and platelet‐aggregating substance that is predominantly mediated by 5‐HT2A receptor. However, the roles of 5‐HT2A receptor in occlusive thrombus formation on disrupted plaque remain obscure. Objective: We investigated the role of 5‐HT2A receptor in thrombus formation using a rabbit model of repeated balloon‐injury. Methods: Three weeks after a first balloon‐injury of the femoral arteries, luminal diameter, neointimal growth, and vasoconstriction by 5‐HT in vitro were examined. Thrombus propagation and the role of 5‐HT2A receptor after a second balloon‐injury were evaluated using sarpogrelate, a selective 5‐HT2A receptor antagonist. Results: Three weeks after the first balloon‐injury, luminal stenosis was evident in the femoral arteries, where the neointima expressed tissue factor and 5‐HT2A receptor. The hypercontractile response of the stenotic arteries to 5‐HT was significantly reduced by sarpogrelate. Balloon‐injury of the neointima with substantially reduced blood flow promoted the formation of occlusive thrombus that was immunoreactive against glycoprotein IIb‐IIIa, 5‐HT2A receptor and fibrin. Intravenous injection of sarpogrelate significantly inhibited ex vivo platelet aggregation induced by adenosine 5′‐diphosphate, thrombin and collagen alone as well as with 5‐HT, and significantly prevented occlusive thrombus formation in vivo. Conclusions: The 5‐HT2A receptor appears to play a crucial role in occlusive thrombus formation in diseased arteries via platelet aggregation and vasoconstriction. Inhibition of 5‐HT2A receptor might help reduce the onset of acute coronary events and of acute coronary occlusion after the intervention.

Disturbed blood flow induces erosive injury to smooth muscle cell‐rich neointima and promotes thrombus formation in rabbit femoral arteries

Summary.  Background: Plaque erosion is a cause of atherothrombosis that preferentially occurs on smooth muscle cell (SMC)‐ and proteoglycan‐rich rather than lipid‐rich plaques. However, its underlying mechanisms remain unknown. Objective: To determine whether disturbed blood flow induces erosive injury and thrombus formation on SMC‐rich neointima. Methods: Three weeks after balloon injury, SMC‐rich neointima with increased tissue factor (TF) activity developed in rabbit femoral arteries that were narrowed with a vascular occluder to disturb blood flow after stenosis. Neointimal injury and thrombus formation were assessed at 15, 30, and 180 min after the vascular narrowing. Results: Endothelial detachment, platelet adhesion and neointimal cell apoptosis became evident at the post‐stenotic regions of all femoral arteries (n = 5) within 15 min of narrowing. Mural thrombi composed of platelet and fibrin developed after 30 min, and then occlusive thrombi were generated in three out of five vessels after 180 min. The identical vascular narrowing of normal femoral arteries also induced endothelial detachment with small platelet thrombi at post‐stenotic regions, but fibrin and occlusive thrombi did not develop. Computational simulation analysis indicated that oscillatory shear stress contributes to the development of erosive damage to the neointima. Conclusions: These results suggest that disturbed post‐stenotic blood flow can induce erosive injury in SMC‐rich plaques and promote thrombus formation that results in vascular events.

Late Restenosis Following Sirolimus-Eluting Stent Implantation

OBJECTIVES This serial angiographic study evaluated the incidence and predictors of late restenosis after sirolimus-eluting stent (SES) implantation. BACKGROUND Previous studies showed late restenosis (i.e., late catch-up phenomenon) after implantation of 7-hexanoyltaxol-eluting stents and nonpolymeric, paclitaxel-eluting stents. METHODS Between August 2004 and December 2006, SES implantation was performed in 1,393 patients with 2,008 lesions, in whom 8-month and 2-year follow-up coronary angiography were planned. RESULTS Of 2,008 lesions, 1,659 (83%) underwent 8-month follow-up angiography (8.3 ± 2.2 months). Restenosis was observed in 122 lesions (7.4%). Coronary angiography 2 years (1.9 ± 0.4 years) after SES deployment was performed in 1,168 lesions (74% of lesions without restenosis at 8-month follow-up angiography). Late restenosis was observed in 83 lesions (7.1%). There was significant decrease in minimum luminal diameter (MLD) between 8-month and 2-year follow-up (2.56 ± 0.56 mm vs. 2.35 ± 0.71 mm, p < 0.001). Multivariate analysis showed in-stent restenosis before SES implantation and MLD at 8-month follow-up as independent predictors of late restenosis. CONCLUSIONS Between 8-month and 2-year follow-up after SES implantation, MLD decreases, which results in late restenosis in some lesions. In-stent restenosis before SES implantation and MLD at 8-month follow-up are independent predictors of late restenosis.

Podoplanin expression in advanced atherosclerotic lesions of human aortas

Thrombus formation on disrupted atherosclerotic lesion is a key mechanism of cardiovascular events. Podoplanin (Aggrus), expressed on the surface of several tumor cells, is an endogenous ligand for C-type lectin-like receptor 2 (CLEC-2), and is involved in tumor cell-induced platelet aggregation and its malignant potency. Podoplanin, which is also expressed in lymphatic endothelial cells, facilitates blood/lymphatic vessel separation. However, podoplanin expression in atherosclerotic lesion has not been investigated. To clarify podoplanin expression in atherosclerotic lesion and to assess its importance for the onset of cardiovascular events, we examined podoplanin expression in abdominal aortas obtained from 31 autopsy cases. Immunohistochemical analysis indicated that podoplanin was localized to smooth muscle cells and macrophages. Moreover, podoplanin immunoreactivity was increased in advanced atherosclerotic lesions containing necrotic core, many macrophages and smooth muscle cells, compared with early lesions composed of smooth muscle cells and small numbers of macrophages. Furthermore, Western-blot and real time-PCR analyses showed that podoplanin expression was significantly enhanced in advanced atherosclerotic lesions, compared with early lesions. These results suggest that podoplanin contributes to thrombotic property of advanced stages of atherosclerosis and that it might be a novel molecular target for an anti-thrombus drug.

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.  Background: Smooth 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: The present study evaluates the contribution of SMC‐rich intima to thrombogenic vasoconstriction. Methods: We 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: Among 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: These 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.

Paucity of CD34-positive cells and increased expression of high-mobility group box 1 in coronary thrombus with type 2 diabetes mellitus

To examine the presence of CD34-positive cells and intranuclear factors in acute coronary thrombi, we compared thrombi in patients with type 2 diabetes mellitus (DM, n = 21) and without DM (n = 29). Immunohistochemical staining revealed the constitutive presence of platelets, fibrin, erythrocytes, neutrophils, extracellular high-mobility group box 1 (HMGB-1), and histone H3 in all thrombi. There were significantly more oval or flat CD34-positive cells and significantly larger HMGB-1-positive areas in the thrombi from patients with DM. The flat CD34-positive cells expressed ecto-nucleoside triphosphate diphosphohydrolase (a platelet aggregation inhibitor). The number of CD34-positive cells was negatively correlated with the serum levels of glucose and hemoglobin A1c, whereas the HMGB-1-positive area was positively correlated with the levels of serum glucose. The paucity of CD34-positive cells and the high levels of HMGB-1 expression in acute coronary thrombi from patients with type 2 DM could facilitate thrombus formation.

Presence of older thrombus in patients with late and very late drug-eluting stent thrombosis

BACKGROUND Although drug-eluting stents (DES) have considerably reduced the incidence of in-stent restenosis, late and very late stent thrombosis (ST) after DES implantation have emerged as major safety concerns. We morphologically investigated the age of DES thrombi aspirated during percutaneous coronary intervention (PCI) from patients with either late or very late ST that resulted in acute myocardial infarction (AMI). METHODS AND RESULTS We obtained DES thrombi during PCI from 16 consecutive patients with ST (late and very late ST, n=4 and n=12, respectively), who presented with AMI within 24 h of the onset of anginal symptoms. Thrombi were morphologically classified as fresh, lytic, and organized. Fresh thrombus was identified in 5 (31%) of the 16 patients and lytic thrombus was found in 3 (19%). Organized thrombus was notably found in 8 (50%) patients, of whom 5 (31%) had only the organized type and 3 (19%) had both fresh and organized thrombi. The frequency of fresh thrombus tended to be higher in patients with stent failure such as stent malapposition and fracture, but the difference did not reach significance (p=0.06). CONCLUSIONS Although the study group is small, about two-thirds of DES thrombi in late and very late ST were days or weeks old. These findings suggest an important discrepancy between the time of onset of the intra-stent thrombotic process and the occurrence of acute clinical symptoms, and provide further information about another potential mechanism of DES thrombosis.