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Long-term safety and efficacy of drug-eluting stents remains controversial. The CREDO-Kyoto registry cohort-2 is a physician-initiated non-company sponsored multi-center registry enrolling consecutive patients undergoing first coronary revascularization in 26 centers in Japan. We compared 3-year outcome between patients treated with sirolimus-eluting stent (SES) only (5092 patients) and bare-metal stent (BMS) only (5405 patients). SES-use as compared with BMS-use was associated with significantly lower adjusted risk for all-cause death [hazard ratio (HR) [95% confidence interval (CI)] 0.72 (0.59–0.87), P = 0.0007], which was mainly driven by the reduction in non-cardiac death [HR (95% CI) 0.64 (0.48–0.85), P = 0.002]. The risk of cardiac death [HR (95% CI) 0.82 (0.63–1.07), P = 0.15], myocardial infarction [HR (95% CI) 0.73 (0.51–1.03), P = 0.07] and definite stent thrombosis [HR (95% CI) 0.62 (0.35–1.09), P = 0.1] was not different between the two groups. Despite longer duration of thienopyridine administration, SES-use was associated with significantly lower risk for bleeding [HR (95% CI) 0.75 (0.6–0.95), P = 0.02] and similar risk for stroke [HR (95% CI) 1.0 (0.75–1.34), P = 1.0]. The risk for target-lesion revascularization (TLR) was markedly lower in the SES group [HR (95% CI) 0.42 (0.36–0.48), P < 0.0001]. The direction and magnitude of the effect of SES relative to BMS in patients presenting acute myocardial infarction (AMI) were similar to those in patients presenting otherwise. In conclusion, SES-use as compared with BMS-use was associated with marked reduction of TLR without any increases in death, myocardial infarction, stent thrombosis, stroke and bleeding in real world clinical practice regardless of clinical presentation including AMI.

Previous studies have shown inconsistent results regarding the effects of concomitant use of clopidogrel and proton pump inhibitors (PPI) on cardiovascular outcomes. We sought to evaluate the clinical impact of PPI-use in patients treated with thienopyridines after percutaneous coronary intervention (PCI) in a large Japanese observational database. Among 12446 patients discharged alive on thienopyridines (ticlopidine 90.4% and clopidogrel 9.6%), 3223 patients were treated with PPIs and 9223 patients without PPI at the time of hospital discharge. The PPI group included more patients with co-morbidities than the non-PPI group. The adjusted hazard ratio (HR) of PPI-use for a composite of cardiovascular death, myocardial infarction, and stroke was 1.26 (95% confidence interval (CI) 1.09-1.47, p = 0.002). The adjusted HR of PPI-use for bleeding was 1.26 (95% CI 1.05-1.52, p = 0.013). Cardiovascular and bleeding outcomes were not different among the three groups receiving three different types of PPI. The negative effect of PPI on cardiovascular outcome was consistently seen in both drug-eluting stent (DES) [HR 1.31 (95% CI 1.07-1.6, p = 0.0097)] and non-DES strata [HR 1.25 (95% CI: 0.99-1.57, p = 0.057)] (Interaction p = 0.79) despite the fact that the duration of thienopyridine administration was significantly longer in patients receiving DES. In conclusion, cardiovascular outcomes after PCI were significantly worse in patients with PPI than in patients without PPI in the Japanese real clinical practice. However, the observed poorer cardiovascular outcome in patients receiving PPI was most likely to be related to residual confounding and seemed not causally related to attenuation of antiplatelet effect of thienopyridine through interaction with PPI.

Background Japanese patients with acute myocardial infarction (MI) have a greater incidence of coronary artery spasm than Caucasians. Some beta-blockers have been reported to aggravate coronary spasm. This study sought to assess the effects of beta-adrenoceptor blockade on coronary vasospasm in Japanese patients with acute MI who had been treated with primary angioplasty. Methods In 69 patients we analyzed the effect of atenolol 50 mg/day initiated the day after emergency primary angioplasty on the results of intracoronary ergonovine provocation test performed 4 weeks after onset. Results Among 35 patients in the atenolol group, the drug was discontinued in 9 (26%) due to hemodynamic compromise. The remaining 26 in the atenolol group and 34 in the control group underwent the spasm provocation test. Atenolol did not significantly increase the incidence of coronary vasospasm (31% vs. 15% in the atenolol and control groups, respectively, p = 0.135). Multivariate analysis revealed that only the pre-provocation diameter of the distal segment of the infarct-related artery predicted coronary spasm whereas atenolol did not. Conclusions This study showed that atenolol 50 mg/day did not increase coronary spasm in Japanese acute MI patients. It is suggested that beta-blockers can be safely used soon after coronary intervention for acute MI without the risk of increasing coronary spasm; however, attention should be paid to hemodynamic change in the acute phase.

Background Adjunctive thrombus aspiration ( TA ) during primary percutaneous coronary intervention ( PCI ) was reported to promote better coronary and myocardial reperfusion. However, long‐term mortality benefit of TA remains controversial. The objective of this study is to investigate the clinical impact of TA on long‐term clinical outcomes in patients with ST ‐segment elevation myocardial infarction (STEMI) undergoing primary PCI . Methods and Results The CREDO ‐Kyoto AMI Registry is a large‐scale cohort study of acute myocardial infarction patients undergoing coronary revascularization in 2005–2007 at 26 hospitals in Japan. Among 5429 patients enrolled in the registry, the current study population consisted of 3536 patients who arrived at the hospital within 12 hours after the symptom onset and underwent primary PCI . Clinical outcomes were compared between the 2 patient groups with or without TA . During primary PCI procedures, 2239 out of 3536 (63%) patients underwent TA ( TA group). The cumulative 5‐year incidence of all‐cause death was significantly lower in the TA group than in the non‐ TA group (18.5% versus 23.9%, log‐rank P TA group was not significantly lower than that in the non‐ TA group (hazard ratio: 0.90, 95% CI : 0.76 to 1.06, P =0.21). The adjusted risks for cardiac death, myocardial infarction, stroke, and target‐lesion revascularization were also not significantly different between the 2 groups. Conclusions Adjunctive TA during primary PCI was not associated with better 5‐year mortality in STEMI patients.

To examine whether we could predict myocardial ischemia when coronary artery calcification is detected by non-gated multidetector CT in patients with suspected ischemic heart disease.Eighty-three patients suspected of having ischemic heart disease (55 men, 28 women; age range 36-83 years; mean age 68 years) underwent multidetector CT and T1-201 single photon emission computed tomography. Prediction of myocardial ischemia by coronary arterial calcification detected on CT was evaluated by comparing the coronary artery territories that showed calcification with the area of myocardial ischemia determined by SPECT. The sensitivity, specificity, positive predictive value, and negative predictive value of multidetector CT for predicting myocardial ischemia were calculated. Coronary angiography was also examined and compared with multidetector CT. Risk factors, including hypertension, smoking, hyperlipidemia, diabetes, and family history, were compared for evidence of coronary artery calcification detected by multidetector CT and myocardial ischemia detected by thallium nuclear scans.For analysis by patients, the sensitivity, specificity, positive predictive value, and negative predictive value of coronary artery calcification for myocardial ischemia detection were 65, 63, 56, and 71%, respectively. Similarly, for analysis by coronary arterial territories, those values were 56, 77, 41 and 86%, respectively. Coronary stenosis on CAG was also related to the ischemia determined by SPECT and calcification on multidetector CT. Ischemia was better influenced by risk factors than was coronary arterial calcification.For analysis by coronary arterial territories, the specificity and negative predictive value of coronary arterial calcification seen by multidetector CT are relatively high.

FTIR spectra of the four isotopically substituted 1:1 complexes of acetonitrile and boron trifluoride were recorded in Ar, N2 and Xe matrices. In Ar, previously unreported three vibrational modes of the complex were clearly observed. Several significant vibrational bands were also observed in N2 and Xe. The observed frequency shifts on complexation, Δν, were qualitatively in good agreement with the computational results, which were calculated at the B3LYP/6-311++G(d,p) level using the optimized geometry of the C3v eclipsed conformation. The observed magnitudes of Δν for most of the complex bands were larger than the calculated values. The BF3 symmetric deformation mode is an exception. The observed frequency shits for this mode were smaller than the calculated values, especially in N2. This suggests that even an inert matrix can significantly affect the vibrational spectrum of the complex.

The FTIR spectra of CH3NC and BF3 as well as their isotopomers co-deposited in low temperature Ar matrices were recorded. We observed several new infrared bands absent in the spectra of each component. These bands were assigned to the 1:1 EDA complex of CH3NC and BF3 based on the DFT calculations at the B3LYP/6-311++G** level. The vibrational assignments and frequencies of the observed bands are the NC stretching at 2319.9 cm−1, the BF3 degenerate stretching at 1203.8 cm−1, the BF3 symmetric stretching at 840.4 cm−1 and the BF3 symmetric deformation at 638.0 cm−1 for the CH3NC−11BF3 isotopomer. Although the BF3 symmetric stretching is infrared inactive for free BF3 of D3h symmetry, complexation with methyl isocyanide would have induced a structural change in the BF3 moiety and the activation of this mode. Apart from some numerical discrepancies, the observed frequency shifts on complexation were in good agreement with the results of DFT calculation. Therefore, the structure of CH3NC-BF3 in solid Ar would be similar to the optimized C3v geometry, which corresponds to the gas phase structure.

Background It has been well-established that aspirin therapy reduces the risk of vascular events in the acute phase of evolving acute myocardial infarction (AMI). However, the adequate dose of aspirin for AMI has not yet been determined. Methods To compare alternate-day aspirin 81 mg and daily aspirin 162 mg on the effects of in-hospital cardiovascular events after AMI, we performed a multicenter, open-label, controlled, randomized clinical trial. The aspirin regimen was started immediately, with 162 mg on the first and second day, and randomly allocated to alternate-day aspirin 81 mg and 162 mg aspirin daily after the third day and continued in the hospital. Three hundred sixty-five patents with AMI were allocated to alternate-day aspirin 81 mg and 386 patients with AMI to daily aspirin 162 mg. Results The observation period was 29.9 ± 0.6 days. There were no statistically significant differences between the 2 treatment groups in age, sex, hours from onset to admission. Killip classification, number of thrombolytic therapies, percutaneous transluminal coronary angioplasty, and other drugs (nitrates, calcium antagonists, β-blockers, and angiotensin-converting enzyme inhibitors). There were also no differences between the 2 groups in the incidence of refractory angina to drug therapy, reinfarction, cerebral vascular accidents, and occurrence of side effects. However, cardiac death occurred in 9 patients in the daily aspir n 162 mg group but in none of those in the alternate-day aspir n 81 mg group (P = .0037). On the other hand, fatal cereb-al infarction occurred in 2 patients in the alternate-day 81 mg aspirin group but in none of those in the daily aspirin 162 mg group. Conclusions We conclude that this study, although underpowered to show meaningful differences in outcome, did not show any difference in in-hospital cardiovascular events with alternate-doy aspirin 81 mg compared with daily aspirin 162 mg.

Background —Enhanced coronary vasomotion may contribute to acute coronary occlusion during the acute phase of myocardial infarction (AMI). Japanese have a higher incidence of variant angina than Caucasian patients, but racial differences in vasomotor reactivity early after AMI are controversial. Methods and Results —The same team studied 15 Japanese and 19 Caucasian patients within 14 days of AMI by acetylcholine injection into non–infarct-related (NIRA) and infarct-related (IRA) coronary arteries followed by nitroglycerin. Incidence of vasodilation, vasoconstriction, spasm, and basal tone were assessed in proximal, middle, and distal segments after each drug bolus by quantitative angiography. Japanese patients had much lower cholesterol levels than Caucasians (183±59 versus 247±53 mg/dL, P P P P Conclusions —Soon after AMI, Japanese patients exhibited a 3-fold-greater incidence of spasm and greater vasoconstriction of nonspastic segments after acetylcholine than Caucasians. The causes of such differences warrant further investigation because they may have relevant pathophysiological and therapeutic implications.

Aspirin therapy confers conclusive net benefits in the acute phase of evolving myocardial infarction, but no clear evidence of benefit from the long-term use of aspirin after acute myocardial infarction (AMI) has been shown in any single study. This multicenter study, the Japanese Antiplatelets Myocardial Infarction Study, was performed to find out whether aspirin or trapidil would improve clinical outcome compared with no antiplatelets in postinfarction patients. The study was a multicenter, open-label, randomized controlled trial of aspirin 81 mg/day, trapidil 300 mg/day, and no antiplatelets in patients with AMI admitted within 1 month from the onset of symptoms. Seven hundred twenty-three patients were enrolled at 70 hospitals in 18 prefectures of Japan; 250 were randomly assigned to treatment with 81 mg aspirin (aspirin group), 243 to that with trapidil (trapidil group), and 230 were not given antiplatelet agents. The mean follow-up period was 475 days. This study demonstrated that long-term use of aspirin at the dose of 81 mg/day reduced the incidence of recurrent AMI compared with the group receiving no antiplatelets after AMI (p = 0.0045) and that trapidil also reduced the occurrence of reinfarction compared with the group receiving no antiplatelets, but the difference was not significant (p = 0.0810). The incidence of cardiovascular events including cardiovascular death, reinfarction, uncontrolled unstable angina requiring admission to hospital, and nonfatal ischemic stroke was reduced in the group receiving 300 mg trapidil daily compared with the group receiving no antiplatelets (p = 0.0039). The use of aspirin 81 mg/day provided almost no benefit over no antiplatelets therapy in the incidence of cardiovascular events. In conclusion, low-dose aspirin (81 mg) effectively prevented recurrent AMI in postinfarction patients after thrombolysis or coronary angioplasty when used over a long term. Furthermore, the long-term use of trapidil resulted in a significant reduction in the incidence of cardiovascular events.

Background— Late adverse events such as very late stent thrombosis (VLST) or late target-lesion revascularization (TLR) after first-generation sirolimus-eluting stents (SES) implantation have not been yet fully characterized at long term in comparison with those after bare-metal stent (BMS) implantation. Methods and Results— Among 13 058 consecutive patients undergoing first percutaneous coronary intervention in the Coronary REvascularization Demonstrating Outcome study-Kyoto registry Cohort-2, 5078 patients were treated with SES only, and 5392 patients were treated with BMS only. During 7-year follow-up, VLST and late TLR beyond 1 year after SES implantation occurred constantly and without attenuation at 0.24% per year and at 2.0% per year, respectively. Cumulative 7-year incidence of VLST was significantly higher in the SES group than that in the BMS group (1.43% versus 0.68%, P P =0.91). Cumulative incidences of late TLR (both overall and clinically driven) were also significantly higher in the SES group than in the BMS group (12.0% versus 4.1%, P P Conclusions— Late catch-up phenomenon regarding stent thrombosis and TLR was significantly more pronounced with SES than that with BMS. This limitation should remain the target for improvements of DES technology.

Background Oxidized LDL and lysophosphatidylcholine (LPC) have been reported to inhibit the endothelium-dependent relaxation (EDR) mediated by nitric oxide. Recently, a new vasorelaxing factor, endothelium-derived hyperpolarizing factor (EDHF), which hyperpolarizes and relaxes the porcine coronary artery in the presence of N ω -nitro- l -arginine (NNA) and indomethacin (IM), has been reported. We examined whether LPC also inhibits both the EDHF-mediated relaxation and membrane hyperpolarization of the porcine coronary artery. Methods and Results EDHF was evaluated as the bradykinin- or A23187-induced relaxation of the porcine coronary artery contracted by prostaglandin F 2α in the presence of NNA and IM. We also directly measured the membrane potential of the porcine coronary artery. The effects of LPC on both relaxation and membrane hyperpolarization were investigated. At concentrations of 0 to 20 μmol/L, LPC dose-dependently inhibited the NNA/IM-resistant EDR induced by bradykinin and A23187, and the relaxation was reversible after the absorption of LPC with albumin. LPC also inhibited the bradykinin- and A23187-induced hyperpolarization of the porcine coronary artery. Conclusions In the present study, LPC was found to inhibit not only nitric oxide–mediated but also EDHF-mediated relaxation of the porcine coronary artery. Our findings suggest a new regulatory mechanism in the atherosclerotic coronary artery.

Nitric oxide accounts for the activity of endothelium-derived relaxing factor, which seems to have an important role in vasodilation and inhibition of platelet aggregation. In endothelial cells, one isoform of nitric-oxide synthase is constitutively expressed. Analysis of the cDNA encoding the human endothelial nitric-oxide synthase revealed that the mRNA is 4.1 kb in size and that the translated protein consists of 1203 amino acids. We have cloned a genomic DNA encoding the human endothelial nitric-oxide synthase and analyzed the entire nucleotide sequence of the gene. The gene consists of 26 exons with a total size of 21 kb. The 5' flanking region of the gene lacks TATA boxes, but it contains putative Sp1-binding sites in (G+C)-rich regions. Of particular interest is the fact that a shear-stress-responsive element is located at position -985, which probably regulates the nitric-oxide-synthase gene in response to fluid mechanical forces at the transcriptional level in the vascular endothelium. Two minisatellite sequences are detectable in introns 2 and 8; a 32-bp consensus sequence repeats 38 times and a 57-bp consensus sequence repeats ten times. We found polymorphisms of the BamHI fragment containing the former minisatellite sequence in genomic DNA from pedigree family members. Furthermore, five tandem repeats of a 27-bp core consensus sequence and 35 repeats of a dinucleotide (CA) are located in introns 4 and 13, respectively. These repeat sequences will probably provide genetic markers for gene mapping and linkage analysis of inherited diseases including cardiovascular diseases.

術後気管支断端瘻は, 肺切除後の合併症のなかで最も重篤なものの一つである. 治療法として胸腔ドレナージ, 洗浄, 再手術などがおこなわれるが, 今回我々は右肺全摘術後に合併した気管支断端瘻に対し, 早期に局麻下にて胸腔鏡による観察を行い, フィブリン糊の塗布により治癒した症例を経験した. この処置は侵襲も軽く, 種々の条件を満たせば, 断端瘻の治療法として有用な方法の一つになると考えられる.

We have cloned a cDNA encoding rat neutrophil inducible nitric oxide synthase (iNOS). The amino acid sequence of the iNOS in the rat neutrophils was 95% identical to that in the mouse macrophages, and the difference between the two sequences was most likely the result of species variability. Moreover, the iNOS in the rat neutrophils was 99% identical in amino acid sequence to that in the rat hepatocytes and vascular smooth muscle cells (VSMC). Northern blot analysis of total RNA from both rat macrophages and neutrophils showed a single band above 4 kilo-nucleotides. These findings suggest that iNOS in rat neutrophils, macrophages, hepatocytes, and VSMC are almost identical in the primary structure.

We have produced 14 monoclonal antibodies to inducible nitric oxide synthase purified from rat peritoneal cytotoxic activated macrophages. None of the antibodies showed neutralizing activity, but some of them enhanced the enzyme activity through stabilization of the enzyme.

It has been known that cytotoxic-activated macrophages release an unstable vasorelaxing substance, nitric oxide. We have found that a more stable relaxing factor is released from those cells. This factor seems to be synthesized from L-arginine. It acts through the activation of soluble guanylate cyclase without affecting membrane potential. It has little charge at nearly neutral pH, and its molecular weight is < 500.

Among patients undergoing surgery for lung cancer during a three-year period from 1988 to 1990, 15 patients who were suspected of having ischemic heart disease (HID) and underwent preoperative coronary angiography (CAG) were subjected. In the present study, preoperative evaluation, preoperative treatment, and perioperative and postoperative management were studied. Ten of the 15 subjects tested with CAG showed significant abnormalities of the coronary arteries and 5 out of the ten revealed no ECG abnormalities in preoperative ECG. Ergometrine maleate tests induced coronary spasms in 5 patients. Two of them had percutaneous coronary angioplasty preoperatively, and another 2 had coronary bypass surgery. All of them had coronary vasodilators during and after surgery, and no serious heart complications were observed during or after surgery. In cases with a slightest suspicion of IHD in preoperative ECG and interviews, operation for lung cancer can be carried out safely, if CAG is carried out and coronary artery lesions are identified.

A cytological examination of pleural lavage with 50 ml saline sterilized was performed before resection in 53 cases of pulmonary lung cancer. The results of pleural lavage were positive in 13 of 53 patients (24.5%). In these 13 patients with positive findings, 9 patients had adenocarcinoma, and 6 patients were p-stage I, one in p-stage II, 4 in p-stage III A, and 2 in p-stage IV. Positive findings significantly correlated to p-factor as compared with negative findings. Nine of 13 patients with positive findings died within 2 years because of distant metastases (5 cases), intrathracic recurrence (2), or other desease (2). When the survival rate was studied according to Kaplan-Meier method, the prognosis in patients with positive findings was poor and no survival for longer than 3 years was recorded. If occult cancer cells present in the thoracic cavity at thoracotomy, even radical operation can allow postoperative recurrence, which might be a possible index for poor prognosis.

Percutaneous transluminal coronary angioplasty (PTCA) was successful in 91% of 76 patients with unstable angina pectoris refractory to pharmacological treatment. However, the rate of acute occlusion and reocclusion was rather high (95). Restenosis developed in 56.5% of successful cases after initial PTCA, and 29 patients underwent 2nd, and nine 3rd PTCA. Most refractory unstable angina can be controlled by PTCA, which may require repeating in some patients.

We recently experienced two cases of diaphragmatic hernia accompanied by no gastrointestinal gas, which could be preoperatively diagnosed by MRI. One was Bochdalek hernia, and the other was Morgagni hernia. In both cases, it was difficult to diagnose, because of homogenous shadows on chest X-ray films. MR imaging showed that each mass was continuous with the subphrenic tissue. As a result of operation, prolapse organs were the retroperitoneal fat tissue and greater omentum. It is important for decision-making of therapeutic methods and proper approach for operation to establish the definitive diagnosis preoperatively. MRI has an advantage over angiography and pneumoperitoneum in having lesser invasion, and its combined use with CT is recommended when this disease is suspected.

The effects of U-61,431F, ciprostene, a stable prostacyclin analogue, were examined on the proliferation of cultured quiescent bovine aortic endothelial cells (EC) and smooth muscle cells (SMC). After stimulation with 5% fetal calf serum, U-61,431F suppressed both the DNA synthesis and proliferation of SMC dose-dependently at the concentration of 3-100 microM, but had no effect on either of them in EC at a concentration of up to 30 microM. The inhibitory effect on DNA synthesis was greater in SMC than in EC at 3-50 microM. When SMC were stimulated with platelet-derived growth factor (PDGF) for 2 hrs followed by a 22-hr incubation with insulin, U-61,431F (1-50 microM) administered at the time of PDGF stimulation did not inhibit DNA synthesis. SMC initiated and terminated DNA synthesis at about 15-18 h and 24 h after stimulation with serum, respectively. Inhibition of DNA synthesis in serum-stimulated SMC as a function of the addition time of U-61,431F reduced at 3-12 h after the stimulation. U-61,431F raised the cyclic AMP (cAMP) content in SMC. Moreover, a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and a more specific cAMP phosphodiesterase inhibitor, Ro 20-1724, augmented the inhibition of DNA synthesis in SMC concomitant with further elevation of cAMP level. These results suggest that U-61,431F inhibits DNA synthesis of SMC acting in the progression stage rather than in the competence stage, with little antiproliferative effect on EC. cAMP may play an important role in its antiproliferative action in SMC.

We compared the effects on the proliferation of bovine vascular smooth muscle cells (VSMC) of serum from 36 patients without restenosis (group A), and 21 patients with restenosis (group B) after percutaneous transluminal coronary angioplasty (PTCA). Baseline characteristics were similar in both groups, except for the greater number of patients with unstable angina at the time of PTCA (52 vs 22%, p = 0.020) and the shorter interval between PTCA and repeat angiography in group B (106 +/- 30 vs 153 +/- 112 days, p = 0.022). Cultured bovine VSMC were stimulated with patient serum (5%) obtained at repeat angiography in either Ca(2+)-containing or Ca(2+)-free culture medium. DNA synthesis was assessed by [3H]thymidine incorporation. The following indices of VSMC proliferation were used: S(+) = [3H]thymidine uptake stimulated by 5% serum in Ca(2+)-containing medium/[3H]thymidine uptake stimulated by 5% fetal calf serum (FCS) in Ca(2+)-containing medium, S(-) = [3H]thymidine uptake stimulated by 5% serum in Ca(2+)-free medium/[3H]thymidine uptake stimulated by 5% FCS in Ca(2+)-free medium, and D = S(-)-S(+). D represented the preserved DNA synthesis in Ca(2+)-deprived medium. S(-) was lower than S(+) in group A (1.35 +/- 0.56 vs 1.57 +/- 0.58, p less than 0.0001), whereas it was higher than S(+) in group B (1.64 +/- 0.66 vs 1.50 +/- 0.58, p = 0.010). D was significantly higher in group B than in group A (0.14 +/- 0.23 vs -0.22 +/- 0.28, p less than 0.0001), and was not associated with any continuous variables including serum calcium level on univariate regression analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

A selective inhibitor of thromboxane A2 synthetase, CV-4151, has the unique property of increasing prostacyclin synthesis in addition to its anti-platelet aggregating effect. Prostacyclin inhibits the growth of smooth muscle cells which is considered to be an underlying mechanism of restenosis occurring after successful coronary angioplasty. A prospective randomized trial was conducted to determine whether CV-4151 could prevent restenosis. Administration was begun greater than or equal to 2 days before angioplasty, and continued until the follow-up study performed between 3 and 6 months after angioplasty. Follow-up angiography was performed in 58 patients (81 segments) taking the active drug and in 27 patients (35 segments) taking the placebo. The incidence of angiographic restenosis was 38.3% in the former group and 31.4% in the latter group. Thus, CV-4151 did not significantly reduce the incidence of restenosis, perhaps due to endothelial denudation after angioplasty preventing an increase of the subendothelial local prostacyclin level.

We hypothesized that oxidative processes in myocardial tissues initiated by ischemia also cause the same kind of changes in blood proteins and lead to oxidation of sulfhydryl groups (SH). To test this hypothesis, the ratio (%MA) of mercaptalbumin to total albumin (= mercaptalbumin + nonmercaptalbumin) and the total SH contents of serum were measured in patients with coronary artery disease. There was a positive correlation between %MA and total SH contents and both values were reduced in proportion to the severity of the coronary artery disease. Furthermore, these values were lower early hours after the onset of acute myocardial infraction than 4 weeks later. These results support our hypothesis, and may provide a basis for the administration of agents which can counteract oxidative stress in order to protect myocardium in patients with coronary artery disease.

Prostacyclin (PGI2) has been reported to stimulate activities of acid cholesteryl ester hydrolase (ACEH; EC 3.1.1.13) and neutral cholesteryl ester hydrolase (NCEH; EC 3.1.1.13) in the smooth muscle cells leading to a decrease in intracellular cholesteryl ester. Recently, we have found that the half-life of PGI2 was prolonged through stabilization by HDL. HDL is known to have anti-atherogenic properties, although its precise mechanism has not been fully clarified. We therefore hypothesized that HDL can exert anti-atherogenic action by augmenting PGI2-stimulated increases in the activities of ACEH and NCEH. After incubation with PGI2 and HDL, a cell homogenate was made from which the activities of ACEH and NCEH were assessed. HDL significantly augmented the PGI2-induced increase in the activities of both enzymes. This effect of HDL was abolished in the absence of PGI2. Elevated intracellular levels of cyclic AMP were maintained for longer periods by HDL. The increase in both intracellular cyclic AMP levels and enzyme activities disappeared in the presence of an inhibitor of adenylate cyclase, 2'5'-dideoxyadenosine. Radiolabeled smooth muscle cells demonstrated a significant loss in total cholesterol and cholesteryl ester after treatment with PGI2 and HDL, due to the increase in cholesteryl ester hydrolytic activities. These data suggest that HDL enhanced the PGI2-stimulated hydrolysis of cholesteryl ester and augmented the PGI2-induced reduction of cellular cholesteryl ester content by stabilizing PGI2.

We found that the cytosol of rat peritoneal polymorphonuclear neutrophils contains factor(s) that can stabilize an unstable enzyme, nitric oxide synthetase, in the cytosol. This enzyme has been purified to a single protein from the cytosol. Its half-life was 3 hours at 4 degrees C and was prolonged to greater than 24 hours by the stabilizing factor in the cytosol. The molecular weight of the stabilizing factor was greater than 100,000. Its activity was lost by the treatment with heating or alkali for 1 min or with acid for 5 min. It did not adhere to the carboxymethyl or diethylaminoethyl column at neutral pH. This stabilizing factor(s) may play a role in the regulation of the nitric oxide synthetase.

The present study was performed to investigate plasma inhibitors of endothelium-dependent relaxation other than hemoglobin and low-density lipoprotein (LDL). We purified an inhibitor that contained a protein of 28,000 Da from human plasma by ammonium sulfate precipitation and serial chromatography. NH2-terminal sequence analysis revealed the protein to be homologous with human apolipoprotein A-I (Apo A-I), a major apolipoprotein of high-density lipoprotein (HDL). Very low-density lipoprotein (VLDL), LDL, and HDL obtained from rabbit plasma reversed endothelium-dependent relaxation of rabbit aorta induced by acetylcholine (ACh) and A23187 but did not inhibit relaxations induced by nitroglycerin or nitric oxide. These inhibitory activities were lost by delipidation of lipoproteins, and there were no differences in the inhibitory activity among these three lipoproteins on the basis of phospholipid concentration. Moreover, phospholipids such as phosphatidylcholine, phosphatidylinositol, and sphingomyelin reversed relaxations by ACh and A23187. Thus all lipoproteins inhibit endothelium-dependent relaxation, and this nonspecific inhibition seems to be due to the inhibition of production or release of endothelium-derived relaxing factor by phospholipids in the lipoprotein complex.

The inhibition of nitric oxide synthase (NOS) by ebselen, 2-phenyl-1,2-benzisoselenazole-3(2H)-one, was reversed by the addition of 10−5 M dithiothreitol, suggesting that ebselen reacts with a critical thiol group of NOS in the inhibitory mechanism. In the presence of 10−4 to 10−3 M dithiothreitol, ebselen dose-dependently enhanced NOS activity, implicating another interaction of ebselen with NOS under these conditions. Thus, the effect of ebselen on the NOS activity is modified by thiols.

We describe a rare case of Becker’s muscular dystrophy (BMD) in a 28-year-old man complicated by rapidly progressing heart failure without apparent clinical signs of neuromuscular disease. He showed rhabdomyolysis, which repeatedly occurred causing acute renal failure as heart failure worsened. His serum creatine kinase (CK) level was generally below 300 IU/l. However, it exceeded more than 10,000 IU/l at the time of myoglobinuria. This suggests that the worsening of heart failure could induce rhabdomyolysis in a BMD patient. Gene analysis for BMD should be considered when the elevation of serum CK is noted in heart failure.

The relationships between different dendritic cell (DC) populations are not clearly established. In particular, it is not known how DC generated in vitro relate to those identified in vivo. Here we have characterized rat bone marrow-derived DC (BMDC) and compared them with DC isolated from spleen (SDC) and pseudo-afferent lymph (LDC). BMDC express typical DC markers and are mostly OX41 positive and CD4 negative. In contrast to ex vivo DC, some BMDC express Fc receptors. FcR+ and FcR- BMDC express similar levels of major histocompatibility complex class II molecules (MHC) and are B7 positive, but some FcR- BMDC express high levels of B7. In contrast to freshly isolated or cultured ex vivo SDC and LDC, both BMDC subpopulations can express inducible nitric oxide synthase (iNOS) and can secrete nitric oxide (NO) in amounts similar to those secreted by peritoneal macrophages. Despite expressing MHC class II and B7, FcR+ BMDC stimulate only a very weak MLR and inhibit stimulation by FcR- BMDC and ex vivo DC. Inhibition is only partially NO dependent. FcR+ BMDC are not macrophages, as judged by adherence and phagocytosis. Both subpopulations are able to present antigen to primed T cells in vitro and are able to prime naive CD4 T cells in vivo. However, unlike SDC, BMDC are unable to stimulate cytotoxic T-lymphocyte (CTL) responses to a minor histocompatibility antigen. Thus, BMDC show marked differences to ex vivo DC and their relationship to those of in vivo DC populations, to date, is unclear.

Nitric oxide synthase [EC 1.14.23] from the particulate fraction of rat cerebella was purified and characterized. The homogenate of rat cerebella was centrifuged to obtain a pellet, which was washed and incubated with Triton X-100 containing buffer. The enzyme activity appeared in the 100,000 x g supernatant after incubation with the detergent. The solubilized enzyme was then purified by sequential affinity chromatography using adenosine 2',5'-diphosphate agarose and calmodulin Sepharose 4B, which gave a product that migrated as a single protein band on SDS/PAGE with a molecular mass of about 150 kDa. The purified enzyme exhibited an absolute requirement for FAD, in addition to NADPH and Ca2+/calmodulin. Thus, there is an insoluble nitric oxide synthase in rat cerebellum that has similar characteristics to the soluble type.

In the present report we investigated the differential expression of three types of nitric oxide synthase (NOS) in the left ventricle after myocardial infarction in rats. One, 3, 7, 14, 28 and 56 days (n=6-12 for each group) after ligation of a coronary artery, tissue samples were obtained from infarcted and non-infarcted tissues. The mRNA and protein levels of neuronal (n) NOS, endothelial (e) NOS and inducible (i) NOS were sequentially determined by semi-quantitative reverse transcription-polymerase chain reaction and Western blotting. Progressive left ventricular dilatation and gradual reduction in fractional shortening were confirmed by echocardiography. The expression levels of nNOS were significantly increased 1, 3 and 7 days post-infarct compared to those of sham-operated rats in both the infarcted (P0.01) and non-infarcted regions (P0.01). Immunohistochemical analysis showed that nNOS was localized in nerve fibers in the left ventricle and that the number of positive fibers after myocardial infarction had increased compared to that in sham-operated rats. With regard to eNOS, no significant changes in expression levels were detected between infarcted hearts and sham-operated controls. The level of iNOS expression peaked three days post-infarct and then decreased in the infarcted tissue, whereas it increased one day post-infarct, peaked at 14 and 28 days post-infarct and was still elevated in the chronic stage in the ventricular septum. iNOS immunoreactivity was detected in spared cardiomyocytes and macrophages in the infarcted region, and in cardiomyocytes in the ventricular septum. The expressions of three types of NOS were differentially regulated and iNOS produced in the non-infarcted region may contribute to the progression of heart failure after myocardial infarction in rats.

Cardiotrophin-1 (CT-1) is a potent cytokine that stimulates the assembly of sarcomeric units in series in cardiomyocytes through gp130 signaling, resulting in myocardial cell hypertrophy. To clarify the role of CT-1 and the gp130-signaling pathway during ventricular remodeling after myocardial infarction, we examined the expression of CT-1 and gp130 in a rat model of myocardial infarction. At 1, 3, 7, 14, 28 and 56 days (n=12 for each group) after ligation of a coronary artery, tissue samples were obtained from infarct tissue, the ventricular septum and the right ventricle. All animals developed large myocardial infarctions, with infarct sizes ranging from 39.8% to 50.3%. Progressive left ventricular dilatation and inadequate hypertrophy of the surviving myocardium were confirmed by echocardiography. CT-1 and gp130 mRNA levels were determined by semiquantitative reverse transcription-polymerase chain reaction using 1 or 5 microg of total RNA followed by Southern blotting. The densitometric analysis of the Southern blots revealed a significant increase in CT-1 and gp130 mRNA levels (P

Recently, the purification of nitric oxide synthase (EC 1.14.23) from rat cerebellum has been reported, and the enzyme is a calmodulin-requiring enzyme (Bredt, D. S., and Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685). In this paper, nitric oxide synthase has been purified to near homogeneity from the cytosol fraction of rat polymorphonuclear neutrophils. The purification procedure involves affinity chromatography with adenosine 2',5'-diphosphate-agarose and an anion exchange column, DEAE-Bio-Gel A. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrated as a single protein band with Mr = 150,000. The molecular weight was estimated to be 150,000 by gel filtration on a Superose 12 HR 10/30. The purified enzyme was unstable with a half-life of 3 h at pH 7.4 and 4 degrees C. The enzyme activity required the presence of Ca2+, NADPH, FAD, and (6R)-5,6,7,8-tetrahydro-L-biopterin. Calmodulin antagonists (W5, W7, W13, and trifluoperazine dihydrochloride) did not inhibit the enzyme activity, and the addition of calmodulin was also ineffective for the increase in the enzyme activity. The neutrophil enzyme appears to be a calmodulin-independent type of nitric oxide synthase.

Nitric oxide synthases (NOS) convert L-arginine to nitric oxide in the presence of tetrahydrobiopterin (BH4). Two constitutive isoforms of NOS exist in normal skeletal muscle fibers, however, the existence of a third, the inducible isoform (iNOS), has never been detected in these fibers in vivo. Therefore, we assessed the influence of in vivo endotoxemia on skeletal muscle expression of constitutive and inducible NOS isoforms and GTP cyclohydrolase I, the rate limiting enzyme of BH4 synthesis. Two groups of rats were infused i.p. either with E. coli endotoxin (20 mg/kg, LPS group) or saline (saline group). Animals were killed 6 h later and the ventilatory and limb muscles were quickly frozen. Endotoxin infusion elicited a significant rise in NOS activity of the diaphragm, intercostal, soleus and gastrocnemius muscles. Reverse transcription-polymerase chain reaction (RT-PCR) on muscle total RNA detected very low expression of iNOS and GTP cyclohydrolase I mRNA in the saline group, but significant upregulation of both enzymes was found in the ventilatory and limb muscles of the LPS group. Immunoblotting detected no iNOS protein in the saline group but a significant iNOS protein expression was found in the diaphragm, intercostal and soleus muscles and to a lesser extent, in the gastrocnemius of the LPS group. Endotoxemia was also associated with increased protein expression of constitutive NOS isoforms mainly in the diaphragm and to lesser extent in the intercostal, gastrocnemius and soleus muscles. We conclude that in vivo exposure to endotoxin leads to differential induction of both iNOS and GTP cyclohydrolase I in the ventilatory and limb muscles.

We evaluated the temporal profile of the number of neurons containing neuronal nitric oxide synthase (nNOS neurons) in the brain of a neonatal hypoxic-ischemic rat model. Hypoxic-ischemic insults were produced in the brains of 7-day-old rat pups using a combination of unilateral carotid artery ligation and hypoxic (8% oxygen) exposure. Sections of brain from rats killed at 0-24 h after the onset of hypoxia were stained immunohistochemically using a polyclonal anti-nNOS antibody. Histological changes of neuronal injury were evaluated in the adjacent Nissl stained sections. The number of nNOS neurons in the hemisphere ipsilateral to the carotid ligation was significantly increased (P < 0.05) at 3 h, when the neuronal injury consisted of clusters of degenerating hyperchromic neurons. Neuronal degeneration and an increased number of nNOS neurons were seen only in the ipsilateral hemisphere and the increase was most prominent in the dorsolateral area of the striatum. The increase in the number of nNOS neurons continued at 6 h, when the area of neuronal injury continued to expand. At 24 h, the neuronal injury was diffuse, and the number of nNOS neurons on the ipsilateral side significantly decreased. The increase of the number of nNOS neurons in the early phase of neonatal neuronal injury suggests its possible involvement in the hypoxic-ischemic injury. The delineation of its role in neuronal injury may lead to an improvement in managing neonatal hypoxic-ischemic brain injury.

In order to clarify the relationship between the patency of the infarcted arteries and subsequent long-term prognosis after thrombolytic therapy, we evaluated 116 patients with acute myocardial infarction treated with intracoronary (112 patients) or intravenous (four patients) urokinase. Patients treated with angioplasty after thrombolysis were excluded. The infarcted vessel was recanalized in 52 patients (patent group) and was not in the remaining 64 patients (occluded group). Five-year and 8-year follow up was conducted in 91% and 81% of the patients, respectively. The 1-, 5- and 8-year survival rate for the patent and occluded group was 91.8 and 80.9%, 80.8 and 79.2%, and 75.9 and 75.6%, respectively. The survival rate in the patent group tended to be higher than that in the occluded group up to 4 years. However, after 5 years, both groups showed similar survival rates. Therefore, reopening of the infarcted arteries with thrombolysis was not an independent predictor for late cardiac death (Cox regression analysis).

An antirat monoclonal antibody (mAb) against inducible nitric oxide synthase (iNOS), ANOS11, was used for immunohistochemistry to examine the expression of iNOS in various organs and tissues of adult rats in experimental endotoxic shock induced by lipopolysaccharide (LPS) injection. The phenotype of iNOS-expressed cells was also examined immunohistochemically using various mAbs. In control rats, very few cells were positive for ANOS11 except in the thymus. After intravenous injection of LPS, the number of iNOS-positive cells increased rapidly in almost all organs, except the thymus and brain, peaked 6 h after the injection, and decreased slowly. Of the numerous inflammatory cells that infiltrated the lungs, liver, and spleen after LPS injection, many were positive for ANOS11. Besides inflammatory cells, hepatocytes and endothelial cells of the aorta were also positive for ANOS11 but only around 6 h after injection. The cellular composition of iNOS-positive infiltrated cells changed along with the progression of endotoxic shock. At 4 to 6 h after injection, most iNOS-positive cells were considered polymorphonuclear leukocytes judging by their positive reactivity to 0X42 and their nuclear morphology. The population of iNOS-positive macrophages positive for EDI or ED2 increased with time. After 24 h, many iNOS-positive macrophages were found around the focal necrosis in the liver and spleen. These results indicate that the expression of iNOS in neutrophils, endothelial cells, and hepatocytes precedes that of macrophages in experimental endotoxic shock. The expression of iNOS in various cells and organs is closely associated with the progress and pathological changes of endotoxic shock. J. Leukoc. Biol. 57: 36–44; 1995.

GABAergic interneurons in the rat frontal cortex were subdivided on the basis of immunoreactivity for calcium binding proteins, neuropeptides and nitric oxide synthase, using double immunofluorescence and mirror image immunohistochemical methods. The results indicate that in this region of the neocortex there are at least three distinct subpopulations of local circuit neurons. The first subgroup consists of parvalbumin-immunoreactive cells. Those do not contain neuropeptide, calretinin or nitric oxide synthase immunoreactivity. A substantial number of parvalbumin-immunoreactive cells in layer II/III were also immunoreactive for calbindin D28k. The second subgroup consists of cells immunoreactive for calretinin. Most were usually immunoreactive for vasoactive intestinal polypeptide as well, but a few cells in layer II/III were immunoreactive for one or the other only. Calretinin-immunoreactive cells do not colocalize parvalbumin, somatostatin or nitric oxide synthase, and only a few colocalize calbindin D28k. The third subgroup consists of cells most of which contain somatostatin, and is entirely separate from the parvalbumin- and calretinin-immunoreactive populations. There was substantial colocalization of somatostatin and calbindin D28k and of somatostatin and neuropeptide Y. Some somatostatin-immunoreactive cells showed nitric oxide synthase immunoreactivity. All of the populations of immunoreactive cells examined in the present study also showed GABA immunoreactivity. About 10% of calbindin D28k-immunoreactive cells and all of those strongly stained for calbindin D28k in layer II/III showed GABA immunoreactivity. Most calbindin D28k-positive cells in deep layers also showed GABA immunoreactivity. These results support that almost all calbindin D28k-immunoreactive non-pyramidal cells are probably GABAergic.

The distributions of reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and nitric oxide synthase (NOS) containing neurons and the extent of NADPH-d and NOS colocalization have been analyzed by histochemical and immunocytochemical techniques in the neocortex of Macaca fuscata monkeys. NADPH-d positive cells were consistently also NOS positive and presented a relatively uniform distribution from area to area, but area-specific differences were observed in the pattern of distribution of fiber plexuses.

The lability of an intracoronary thrombus, which is common in the case of unstable angina. indicates the presence of endogenous fibrinolysis. To prove our hypothesis that suppression of coagulation facilitates the thrombolytic process, 10w molecular weight heparin (LMWH) was given intravenously to dogs with intracoronary thrombi induced by copper coils. LMWH caused reperfusion in this model and suppressed the formation of intracoronary thrombi, thus sup-porting our hypothesis.

THE FACTORS RESPONSIBLE FOR EARLY OCCLUSION OF THE INFARCT VESSEL AFTER emergency percutaneous transluminal coronary angioplasty (PTCA) were retrospectively examined in 191 patients with acute myocardial infarction. During the 24-hour period after the initial balloon inflation, 47 patients (25%) had occlusion of the vessel (occlusion group), whereas 144 did not (nonocclusion group). The former patients immediately underwent repeat PTCA, which was successful in 37. Univariate correlates of early occlusion were a shorter time interval between the onset of symptoms and PTCA (3.5 +/- 2.2 vs 4.5 +/- 2.9 hours, p = 0.025), right coronary artery involvement (53% vs 30%, p = 0.015), prior thrombolytic therapy (49% vs 32%, p = 0.035), and undersized inflation (43% vs 17%, p < 0.001). With multivariate analysis the three independent predictors were undersized inflation (p < 0.001), right coronary artery involvement (p = 0.004), and a shorter time interval until PTCA (p = 0.011). Thus patients undergoing early PTCA and having right coronary artery involvement appear to be at greater risk of having early occlusion. Thrombolytic agents and undersized inflation may also play an important role in its development.