Nobuyuki Shirai

Telomere Shortening in Human Coronary Artery Diseases

Background—Increased cell turnover in response to injury is considered to be important in the development of atherosclerotic plaques. Telomere shortening has been shown to be associated with cell turnover. We assessed the telomere length of human coronary endothelial cells to clarify whether there is a relationship between telomere shortening and coronary artery disease (CAD). Methods and Results—Coronary endothelial cells were obtained from 11 patients with CAD who underwent autopsy and 22 patients without CAD who underwent autopsy by scraping off the luminal surface of coronary arteries. DNA extracted from the endothelial cells were blotted and hybridized with telomere-specific oligonucleotide ([TTAGGG]4). The hybridization signal intensity, which represented telomeric DNA content, was standardized with centromeric DNA content (T/C ratio) to estimate telomere length. The T/C ratios were significantly smaller (P <0.0001) in CAD patients than in age-matched non-CAD patients (CAD patients, 0.462±0.135; non-CAD patients, 1.002±0.212). In 6 individual CAD patients, the T/C ratio at the atherosclerotic lesion was significantly smaller (P <0.05) than that at the non-atherosclerotic portion. Conclusions—These findings suggest that focal replicative senescence and telomere shortening of endothelial cells may play a critical role in coronary atherogenesis and CAD.

Persistent High Levels of Plasma Oxidized Low-Density Lipoprotein After Acute Myocardial Infarction Predict Stent Restenosis

Objective—Recently, elevated levels of plasma oxidized low-density lipoprotein (LDL) have been shown to relate to plaque instability in human atherosclerotic lesions. We investigated prospectively patients admitted with acute myocardial infarction (AMI) who underwent primary coronary stenting to evaluate whether the 6-month outcome could be predicted by measuring plasma oxidized LDL (ox-LDL) levels at the time of hospital discharge. Methods and Results—Plasma ox-LDL levels were measured in 102 patients with AMI undergoing primary coronary stenting using a highly sensitive ELISA method. Measurements were taken on admission and at discharge, and the findings related to the clinical outcome. At 6-month follow-up, angiographic stent restenosis occurred in 25 (25%) of the 102 AMI patients. Plasma ox-LDL levels at discharge were significantly (P=0.0074) higher in the restenosis group than those in the no-restenosis group (1.03±0.65 versus 0.61±0.34 ng/5 &mgr;g LDL protein). Multiple regression analysis showed that only plasma ox-LDL levels at discharge were a statistically significant independent predictor for late lumen loss after stenting (&bgr;=0.645; P<0.0001). Conclusions—This prospective study demonstrates that persistence of an increased level of plasma ox-LDL at discharge is a strong independent predictor of stent restenosis at 6-month follow-up in AMI patients.

Immunohistochemical Expression of Multidrug Resistance Proteins as a Predictor of Poor Response to Chemotherapy and Prognosis in Patients with Nodal Diffuse Large B-Cell Lymphoma

Background/Aims: The aim of this study was to determine whether expression of P-glycoprotein (P-gp), multidrug-resistance-related protein 1 (MRP1), and lung resistance protein (LRP) was related to the response to induction chemotherapy and prognosis in untreated diffuse large B-cell lymphoma (DLBCL). Methods: We assessed immunohistochemical expression of P-gp, MRP1 and LRP, using formalin-fixed and paraffin-embedded specimens of lymph node in 41 patients with DLBCL. Association between expression of these three proteins and their impact on clinical outcome and prognosis was statistically evaluated. Results: P-gp was positive in 37% of subjects, MRP1 in 63%, and LRP in 68%. The complete remission rates achieved in the group expressing these multidrug resistance (MDR) proteins was significantly lower than in the group not expressing them (20 versus 58%; p = 0.025 in P-gp, 23 versus 80%; p < 0.001 in MRP1 and 32 versus 69%, p = 0.043 in LRP, respectively). Furthermore, the patients expressing LRP had a shorter overall survival rate than those that did not (median of 26 months versus median not reached; p = 0.013). Conclusions: These findings suggest that the three MDR proteins are important predictive factors for the clinical outcome and prognosis in patients with DLBCL.

Neopterin is associated with plaque inflammation and destabilisation in human coronary atherosclerotic lesions

Background: Previous studies have shown that recent activation of the inflammatory response in coronary atherosclerotic lesions contributes to rapid progressive plaque destabilisation. Neopterin, a by-product of the guanosine triphosphate pathway, is produced by activated macrophages and serves as an activation marker for monocytes/macrophages. Objective: To elucidate the role of neopterin in coronary plaque destabilisation by immunohistochemical study of the presence of neopterin in coronary atherectomy specimens obtained from patients with stable angina pectoris (SAP) and unstable angina pectoris (UAP). Patients and methods: All patients underwent atherectomy of the primary atherosclerotic lesions responsible for SAP (n = 25) and UAP (n = 25). Frozen samples were studied with antibodies against smooth muscle cells, macrophages, T cells, neutrophils and neopterin. Results: In 22/25 patients with UAP, abundant neopterin-positive macrophages were found at the sites of coronary culprit lesions. However, in 25 lesions from patients with SAP, only 11 lesions showed neopterin positivity. Quantitatively, the neopterin-positive macrophage score was significantly higher (p<0.001) in patients with UAP than in patients with SAP. Moreover, the neopterin-positive macrophage score showed a significant positive correlation with the number of neutrophils or T cells, respectively (neutrophils, r = 0.55, p<0.001; T cells, r = 0.70, p<0.001). Conclusions: Neopterin can be considered as one of the significant factors in the process of plaque inflammation and destabilisation in human coronary atherosclerotic lesions. Its exact role in the process needs to be investigated further.

Elevated levels of neopterin are associated with carotid plaques with complex morphology in patients with stable angina pectoris

OBJECTIVE Neopterin is an activation marker for monocytes/macrophages, and circulating levels of neopterin are elevated in patients with coronary complex lesions in unstable angina pectoris. We investigated the possible association between neopterin and complex carotid plaques which may be associated with the risk of ischemic stroke in patients with stable angina pectoris (SAP). METHODS We measured plasma levels of neopterin in 102 patients with SAP and carotid ultrasound was performed for evaluation of the presence of carotid plaques and plaque surface characteristics categorized as complex or noncomplex. In addition, endarterectomy specimens of extracranial high-grade carotid stenosis with complex plaques from five patients with SAP were immunohistochemically examined with antibodies to smooth muscle cells, endothelial cells, platelets, macrophages, and T cells. RESULTS Plasma neopterin levels were significantly higher in patients with complex carotid plaques than in those with noncomplex plaques (median [interquartile range]: 24.2 [19.2-39.3]nmol/L vs. 19.4 [11.9-25.1]nmol/L; P=0.01) or without any plaques (18.8 [14.9-23.6]nmol/L; P=0.001). On multivariate logistic analyses after adjustment for traditional atherosclerotic risk factors, multi-vessel coronary disease and high sensitivity C-reactive protein, neopterin levels were independently associated with the presence of complex carotid plaques (adjusted OR 2.21 per SD increase, 95%CI 1.13-4.33, P=0.02). Immunohistochemical staining revealed abundant neopterin-positive macrophages in carotid complex lesions. CONCLUSION These findings demonstrate that carotid plaques with complex morphology have increased circulating neopterin levels and immunohistochemical localization of neopterin in patients with SAP. Neopterin can be considered an important biomarker of plaque destabilization in carotid artery atherosclerotic lesions in this population.

Close association between activated platelets and neutrophils in the active phase of ulcerative colitis in humans

Background: Neutrophils are considered to play a causative role in inflammatory mucosal injury in ulcerative colitis (UC), and an association between platelets and neutrophils may contribute to the progression of the inflammatory processes. To test this hypothesis, we performed immunohistochemical and flow cytometric analyses on tissue and blood samples from patients with UC. Materials and Methods: Colonic mucosal tissues of patients with active (n = 27) or inactive (n = 16) UC and normal controls (n = 11) were subjected to immunohistochemical staining for markers of activated platelets (glycoprotein IIb/IIIa and P‐selectin) and neutrophils (neutrophil elastase, myeloperoxidase, and CD66b). The amounts of stained cells were evaluated by computer‐aided morphometry. Peripheral blood samples from patients (n = 8) and healthy volunteers (n = 8) were subjected to comparative flow cytometric analysis of activated platelets. Results: P‐selectin‐positive activated platelets were frequently aggregated in the inflamed mucosa, especially in ulcerative lesions, and were close to regions of dense neutrophil infiltration. An increase in the number of activated platelets in the colonic lesions was associated with an increase in infiltrating neutrophils and was related to the severity of the disease. The flow cytometric analysis indicated that circulating platelets of patients with UC were highly activated. Conclusions: The present study demonstrated that a close association between activated platelets and neutrophils is a prominent pathological change in both the affected colonic mucosa and peripheral blood of patients with active‐phase UC. This suggests that platelet‐neutrophil association may play an important role in the progression of inflammatory processes in UC.

Relation of Elevated Levels of Plasma Myeloperoxidase to Impaired Myocardial Microcirculation After Reperfusion in Patients With Acute Myocardial Infarction

Previous studies have shown that oxidative stress and endothelial dysfunction are related to impaired myocardial microcirculation after reperfusion. Moreover, elevated myeloperoxidase (MPO) levels are associated with endothelial dysfunction. Plasma MPO levels were measured in patients with ST-segment elevation acute myocardial infarction (n = 160) who had undergone percutaneous coronary stenting within 12 hours of symptom onset. We investigated whether the plasma MPO level at admission was associated with impaired myocardial microcirculation, as indicated by ST-segment resolution and myocardial blush grade after reperfusion, and left ventricular ejection fraction and remodeling at 6 months. The patients were divided into 2 groups according to the median MPO value for the entire cohort (low-MPO group < or =50 ng/ml, n = 80; high-MPO group >50 ng/ml, n = 80). ST-segment resolution and the myocardial blush grade were significantly lower in the high-MPO than in the low-MPO group (48 +/- 27% vs 61 +/- 24%, p <0.005; and 2.1 +/- 0.8 vs 2.4 +/- 0.7, p <0.01; respectively). Moreover, the percentage of increase in the left ventricular end-diastolic volume index was significantly greater and the left ventricular ejection fraction at 6 months was significantly lower in the high-MPO group than in the low-MPO group (8.2 +/- 24.7% vs -1.9 +/- 23.5%, p <0.05; and 46 +/- 9% vs 54 +/- 9%, p <0.0001, respectively). Multiple regression analysis showed that the plasma MPO level was an independent predictor of incomplete ST-segment resolution (odds ratio 6.94, 95% confidence interval 2.10 to 22.9, p = 0.0015). In conclusion, elevated plasma MPO levels at admission were associated with impaired myocardial microcirculation after reperfusion in patients with acute myocardial infarction.

Expression of cyclooxygenase-2 in Hodgkin's lymphoma:its role in cell proliferation and angiogenesis

Although many studies have revealed the association between cyclooxygenase-2 (COX-2) and carcinogenesis, the association between COX-2 and Hodgkins lymphoma (HL) remains unknown. We examined the immunohistochemical expression of COX-2, p53, bcl-2, and Ki-67 in 33 patients with HL, and counted microvessels stained with CD34. Hodgkin and Reed – Sternberg (HRS) cells with COX-2 expression were scored as 0 = no staining; 1 = <25% of cells staining; 2 = 25 – 49%; 3 = 50 – 75%; and 4 = ≥75%. COX-2 expression was observed in 15 cases of classical HL. Nevertheless, neither accumulation of p53 nor bcl-2 expression was associated with COX-2 expression. The percentage of Ki-67 positive-HRS cells and microvessel density in COX-2 score groups 2 – 4 were significantly higher than those in score group 0, respectively. We show that COX-2 expression is associated with cell proliferation and angiogenesis in HL. These findings suggest that COX-2 may be a target for therapy in HL.

Expression of endothelin-converting enzyme, endothelin-1 and endothelin receptors at the site of percutaneous coronary intervention in humans.

Objective The repair process at the site of injury after percutaneous coronary intervention (PCI) is dominated by neointimal formation composed mainly of smooth muscle cells (SMC). Endothelin-1 (ET-1) is a powerful vasoconstrictor and SMC mitogen. Endothelin-converting enzyme (ECE) is the final key enzyme of endothelin processing. The effects of ET-1 are mediated by binding to endothelin type A (ETA) and endothelin type B (ETB) receptors. The ligand/receptor/ligand-producing system (ET system) could be involved in the pathogenesis of neointimal formation in humans. Methods Fifteen post-PCI sites obtained at autopsy and eight atherectomy specimens obtained from restenotic sites were investigated using immunohistochemical single and double staining techniques. Frozen sections were stained with antibodies against ECE, ET-1, ETA and ETB receptors, SMC, macrophages and endothelial cells. Results At the early stage, less than 3 months after PCI, neointimal SMC were positive for ECE, ET-1, ETA and ETB receptors. The expression of ECE, ET-1, ETA and ETB receptors in these neointimal SMC decreased markedly from 6 months onwards. The ECE, ET-1, ETA and ETB receptor-positive cell areas were significantly (P < 0.005) greater in the first 3 months after PCI compared with 6 months or more after PCI. Atherectomy specimens also showed similar positivity. Conclusions These observations strongly suggest that the expression of ECE, ET-1, ETA and ETB receptors is enhanced in neointimal SMC at early stages after PCI injury in human coronary arteries. The increased expression of the ET system may contribute to SMC proliferation/migration and vasoconstriction in human post-PCI coronary lesions.

Comparison of determinations of left atrial volume by the biplane area-length and Simpson's methods using 64-slice computed tomography

OBJECTIVES There is increasing evidence that left atrial (LA) size is an important predictor of adverse cardiovascular outcomes such as atrial fibrillation, stroke, and congestive heart failure. The aim of this study was to determine whether there is a difference in results of quantification of LA volume by the area-length and Simpsons methods using multislice computed tomography (MSCT). METHODS AND RESULTS The study population consisted of 51 patients with sinus rhythm (sinus group) and 20 patients with atrial fibrillation (af group) clinically indicated for MSCT angiography for evaluation of coronary arteries. Maximum LA volume, obtained at end-systole from the phase immediately preceding mitral valve opening, was measured using the area-length and Simpsons methods. In the sinus group, the mean LA volumes, indexed to body surface area, were 48.4+/-17.9 ml/m(2) with the area-length method and 48.3+/-17.0 ml/m(2) with the Simpsons method. In the af group, the mean indexed LA volumes with the area-length method and the Simposons method were 91.5+/-47.5 ml/m(2) and 90.3+/-45.9 ml/m(2), respectively. LA volumes calculated by the area-length method exhibited a strong linear relationship and agreement with those calculated using Simpsons method in both the groups (sinus group: r=0.99, P<0.0001, af group: r=0.99, P<0.0001). CONCLUSIONS The area-length method is a simple and reproducible means of assessment of LA volume. Standardization of LA volume assessment using MSCT is important for serial follow-up and meaningful communication of results of testing among institutions and physicians.