Visualization of internal carotid artery atherosclerotic plaques in symptomatic and asymptomatic patients: a comparison of optical coherence tomography and intravascular ultrasound.

BACKGROUND AND PURPOSE: OCT has been reported as a high-resolution imaging tool for characterizing plaque in the coronary arteries. The present study aimed to evaluate the ability of OCT to visualize carotid artery plaques compared with that of IVUS in asymptomatic and symptomatic patients. MATERIALS AND METHODS: OCT was performed for 34 plaques (17 symptomatic, 17 asymptomatic) in 30 patients during CAS under a proximal cerebral protection method. OCT was performed before balloon angioplasty and after stent placement. IVUS was also performed just after OCT. RESULTS: No technical or neurologic complications were encountered by using OCT. An inner catheter was used in 12 of 34 procedures (35.3%) for advancing the OCT image wire beyond the site of stenosis. OCT clearly visualized intraluminal thrombus in 15 of 34 plaques (44.1%), whereas IVUS detected a thrombus in 1 plaque (2.9%, P < .001). Neovascularization was demonstrated in 13 of 34 plaques (38.2%) by OCT, but not by IVUS (0%, P < .001). Intraluminal thrombus was more frequently observed in symptomatic plaques (13 of 17, 76.5%) than in asymptomatic plaques (2 of 17, 11.8%; P < .001). Interobserver and intraobserver variability with OCT diagnosis was excellent for thrombus, ulceration, neovascularization, and lipid pool. CONCLUSIONS: The present findings suggest that OCT can safely and precisely visualize human carotid plaques during CAS and that intraluminal thrombus and neovascularization are more frequently detected in symptomatic plaques.

Anti-tumor effect of gallic acid on LL-2 lung cancer cells transplanted in mice.

We previously reported that gallic acid (3,4,5-trihydroxybenzoic acid), a naturally occurring plant phenol, can induce apoptosis in four kinds of human lung cancer cell lines in vitro. The present study further investigated the in vivo anti-tumor effects of orally administered gallic acid. Gallic acid reduced cell viability of LL-2 mouse lung cancer cells in vitro dose dependently, with a 50% inhibitory concentration (IC50) value of around 200 microM. C57Black mice were transplanted with LL-2 cells, and administered gallic acid (1 mg/ml in drinking water, ad libitum) and/or cisplatin (4 mg/kg i.p. injection, once a week). The average weight of the transplanted tumors, obtained at 29 days after transplantation, in the mice of control, gallic acid-treated cisplatin-treated and cisplatin plus gallic acid-treated groups was 4.02, 3.65, 3.19 and 1.72 g, respectively. The average tumor weight of the mice treated with cisplatin combined with gallic acid was significantly smaller than that of the control group (p<0.05). The amount of apoptotic cells in the tumor tissues of mice treated with gallic acid and/or cisplatin was significantly higher than those of the control mice. Combination of gallic acid and cisplatin increased the tumor cell apoptosis compared with the treatment with cisplatin alone. The present findings suggest that the combination of gallic acid with an anti-cancer drug, including cisplatin, may be an effective protocol for lung cancer therapy.

Caspase-dependent and serine protease-dependent DNA fragmentation of myocytes in the ischemia-reperfused rabbit heart: these inhibitors do not reduce infarct size.

Some infarcted myocytes undergo caspase-dependent DNA fragmentation, but serine protease-dependent DNA fragmentation may also be involved. There is controversy regarding whether caspase inhibitors can reduce infarct size, so the present study investigated whether serine protease inhibitor can reduce the DNA fragmentation of infarcted myocytes and whether serine protease or caspase inhibitors attenuates myocardial infarct size in Japanese white rabbits without collateral circulation. Rabbits were subjected to 30-min coronary occlusion followed by 48-h reperfusion. A vehicle (dimethylsulfoxide, control group, n=8) or Z-Val-Ala-Asp(Ome)-CH2F (ZVAD-fmk, a caspase inhibitor, ZVAD group, 0.8 mg/kg iv at 20 min before coronary occlusion and 0.8 mg/kg at 90 min after reperfusion, n=8) or 3,4-dichloroisocoumarin (DCI, a serine protease inhibitor, 2 mg/kg iv at 20 min before coronary occlusion, DCI group, n=8) was administered. Animals were killed at 48h after reperfusion for the detection of myocardial infarct size and at 4h after reperfusion for the detection of dUTP nick end-labeling (TUNEL)-positive myocytes, the electrophoretic pattern of DNA fragmentation and ultrastructural analysis. The left ventricle (LV) was excised and sliced. The myocardial infarct size as a percentage of the area at risk was assessed by triphenyltetrazolium chloride staining. DNA fragmentation was assessed by in situ TUNEL at the light microscopic level. ZVAD and DCI significantly reduced the mean blood pressure during reperfusion without affecting heart rate. There was no significant difference in the % area at risk (AAR) of LV among the 3 groups (control: 26.3+/-3.0%; ZVAD: 25.6+/-2.6%; DCI: 25.6+/-2.0%). The % infarct size as a percentage of the AAR in the ZVAD group (41.3+/-4.5%) and the DCI group (50.4+/-3.8%) was not significantly different from the control group (43.5+/-4.5%). However, the percent DNA fragmentation in the infarcted area in the ZVAD (3.5+/-0.8%) and DCI groups (4.2+/-0.9%) was significantly reduced compared with the control group (10.7+/-1.9%). The DNA ladder pattern observed in the control group was attenuated in both the ZVAD and DCI groups. There was no difference in electron microscopic changes among the 3 groups. Serine protease-dependent DNA fragmentation is present in infarcted myocytes, in addition to caspase-dependent DNA fragmentation, but an infarct-size reducing effect was not observed with either of these inhibitors.

Apoptosis, rather than oncosis, is the predominant mode of spontaneous death of isolated adult rat cardiac myocytes in culture.

A number of isolated adult cardiomyocytes dies within a few days of culture and the mode of death has recently been suggested to be apoptosis, based on its association with the appearance of DNA fragmentation. However, morphological evidence is still lacking and precise analysis, including quantification, has not been performed. Adult rat ventricular cardiomyocytes isolated by enzymatic dissociation were incubated for 7 days in a serum-free medium (the rapid attachment model) and after various incubation periods, both attached and floating cells were counted and classified based on combined criteria of morphology and membrane permeability (dye exclusion): type 1, rod cells with intact membranes; type 2, non-rod cells with intact membranes; and type 3, non-rod cells with ruptured membranes. The number of both rod-shaped and dye-excluding cells decreased with the incubation period. After 7 days culture, the number of residual cells decreased to 12% of the initial value. Electron microscopy identified type 1 cells as viable, type 2 cells as viable or apoptotic, and type 3 as undergoing oncosis (primary necrosis) or secondary post-apoptotic necrosis. Ultrastructural morphometry revealed that oncotic cell death occurred predominantly during the early phase of culture whereas the more abundant apoptotic cell death occurred throughout the culture period. In conclusion, although both apoptotic and oncotic death occur in the natural course of adult rat cardiomyocytes in short-term culture, apoptosis is more predominant. Because of the high incidence of spontaneous cell death predominantly via apoptosis, this information is important for the interpretation of studies using this cell type in culture.

Dynamic process of apoptosis in adult rat cardiomyocytes analyzed using 48-hour videomicroscopy and electron microscopy: beating and rate are associated with the apoptotic process.

Dynamic process of apoptosis has not been elucidated in adult rat cardiomyocytes. Soluble Fas ligand (0.1 microg/ml) in the presence of actinomycin D (0.05 microg/ml) induced apoptosis in cultured adult rat cardiomyocytes, as documented by activated caspase-3, DNA fragmentation, and apoptotic ultrastructure. In the present model, we observed 60 adult cardiomyocytes with a normal rod shape under a real-time videomicroscope continuously for 48 hours. Seventeen cells (28%) were unchanged and 7 cells (12%) showed oncosis (so-called necrosis) in which no beating was evident. In the remaining 36 cells (apoptosis, 60%), a slow beating (17 +/- 3/min) was initiated 16 +/- 1 hours later. Approximately 1 hour later, the rod cells showed long-axial shortening as bone- or club-like, or square-shaped, accompanied with faster beating rates (35 +/- 7/min). In 29 cells (type A1 and A2), marked shrinkage occurred; the cellular shape became almost completely round with a smooth surface and the beating ceased 3.0 +/- 0.4 hours later. Then, smooth budding appeared 0.6 +/- 0.2 hours later. Apoptotic bodies were found in 8 cells 10 +/- 4 hours later (type A1, 13%) but not in 21 cells (type A2, 35%). In the other 7 cells (type A3, 12%), the cell surface became rough 8 +/- 3 hours later and the beating ceased. Maximal beating rate was greatest in type A1 (72 +/- 26/min) and greater in type A2 (29 +/- 5/min) than in type A3 (10 +/- 2/min). Electron microscopy confirmed apoptotic ultrastructure even in the cardiomyocytes with bone-, club-like, or square shapes, suggesting that type A3 as well as A1 and A2 is also under apoptotic process. A caspase inhibitor, zVAD.fmk, blocked beating, apoptotic morphology, and DNA fragmentation, indicating these depended on caspase activation. In the caspase-dependent apoptotic process of cultured adult cardiomyocytes, beating and the following deformity of the cellular edges were the initial signs and the rate of beating was related with the subsequent three different processes of apoptosis.

Combination of miglitol, an anti-diabetic drug, and nicorandil markedly reduces myocardial infarct size through opening the mitochondrial K(ATP) channels in rabbits.

The anti-diabetic drug miglitol, an alpha-glucosidase inhibitor, which is currently used clinically, reduces myocardial infarct size by reducing the glycogenolytic rate through inhibition of the alpha-1,6-glucosidase of glycogen-debranching enzyme in the heart. Nicorandil, a K(ATP) channel opener with a nitrate-like effect, which is also currently used clinically, also reduces the infarct size. Therefore, we hypothesized that combination of nicorandil and submaximal dose of miglitol could markedly reduce myocardial infarct size more than miglitol or nicorandil alone, and investigated the mechanism for the infarct size-reducing effect. Japanese white rabbits without collateral circulation were subjected to 30 min coronary occlusion followed by 48 h reperfusion. Pre-ischaemic treatment with submaximal dose of miglitol (5 mg kg(-1), i.v.) and nicorandil alone (100 microg kg(-1) min(-1) 5 min) moderately reduced the infarct size as a percentage of area at risk (24+/-4 and 25+/-4%, respectively), and 10 mg kg(-1) of miglitol markedly reduced the infarct size (15+/-2%) compared with the controls (42+/-2%). Combination of 5 mg kg(-1) of miglitol and nicorandil (100 microg kg(-1) min(-1) 5 min), and 10 mg kg(-1) of miglitol and nicorandil (100 microg kg(-1) min(-1) 5 min) significantly reduced the infarct size (13+/-4 and 12+/-3%, respectively) more than miglitol or nicorandil alone. Pretreatment with 5HD completely abolished the infarct size-reducing effect of 10 mg kg(-1) of miglitol alone (36+/-7%) and that of combination of 5 mg kg(-1) of miglitol and nicorandil (46+/-2%). Combination of nicorandil and submaximal dose of miglitol markedly reduced the myocardial infarct size more than miglitol or nicorandil alone. This effect was suggested to be related to the opening of mitochondrial K(ATP) channels.

Noninvasive quantitative tissue characterization and two-dimensional color-coded map of human atherosclerotic lesions using ultrasound integrated backscatter: comparison between histology and integrated backscatter images.

OBJECTIVES: The purpose of the present study was to define clinicopathologically whether integrated backscatter (IB) combined with conventional two-dimensional echo (2DE) can differentiate the tissue characteristics of calcification (CL), fibrosis (FI), lipid pool (LP) with fibrous cap, intimal hyperplasia (IH) and thrombus (TH) and can construct two-dimensional tissue plaque structure in vivo. BACKGROUND: It is difficult to characterize the components of plaque using conventional 2DE techniques. METHODS: Integrated backscatter values of plaques were measured in the right common carotid and femoral arteries (total 24 segments) both during life and after autopsy in 12 patients (age 68 to 84 years, 10 men and two women). Integrated backscatter values were determined using a 5-12 MHz multifrequency transducer, setting the region of interests (ROIs) (11 x 11 pixels) on the echo tomography of the entire arterial wall (55 +/- 10 ROI/segment) and comparing it with histologic features in the autopsied arterial specimens. RESULTS: Corrected IB values obtained before death and at autopsy were significantly correlated (r = 0.93, p < 0.01). Corresponding to the histologic features, corrected IB values on the rectangle ROIs obtained during life were divided into five categories: category 1 (TH) 4 < IB < or = 6; category 2 (media and IH or LP in the intima) 7 < IB < or = 13; category 3 (FI) 13 < IB < or = 18, category 4 (mixed lesion) 18 < IB < or = 27 and category 5 (CL) 28 < IB < or = 33. In category 2, media and intima were differentiated using conventional 2DE. Under the above procedures, color-coded maps constructed with IB-2DE obtained during life precisely reflected the histologic features of media and intima. CONCLUSIONS: Integrated backscatter with 2DE represents a useful noninvasive tool for evaluating the tissue structure of human plaque.

Treatment with sheng-mai-san reduces myocardial infarct size through activation of protein kinase C and opening of mitochondrial KATP channel.

Sheng-mei-san (SMS), a traditional Chinese formulation comprising Radix Ginseng, Radix Ophiopogonis and Fructus Schisandrae, has long been used for more than 700 years for patients with coronary heart disease. We attempted to clarify 1) whether SMS reduces myocardial infarct size, and 2) whether the infarct size-reducing effect of SMS is related to activation of protein kinase C and the opening of the mitochondrial KATP channels in Japanese white rabbits without collateral circulation. The results indicate that three days treatment but not acute treatment with SMS reduces myocardial infarct size through activation of protein kinase C and opening of the mitochondrial KATP channels.

Combination of N-methyl-1-deoxynojirimycin and ischemic preconditioning markedly reduces the size of myocardial infarcts in rabbits.

N-methyl-1-deoxynojirimycin (NMDN), an a-glucosidase inhibitor, reduces myocardial infarct size by reducing the glycogenolytic rate through inhibition of the alpha-1,6-glucosidase of glycogen-debranching enzyme in the heart, in addition to possessing an antihyperglycemic action by blocking alpha-1,4-glucosidase in the intestine. Ischemic preconditioning (PC), which markedly reduces the size of the myocardial infarct, is known to reduce the activity of phosphorylase and reduce the glycogenolytic rate. Therefore, it was hypothesized that a combination of pharmacological inhibition of glycogenolysis by an alpha-1,6-glucosidase inhibitor, NMDN, and PC could markedly reduce myocardial infarct size more than NMDN or PC alone. Japanese white rabbits without collateral circulation were subjected to a 30-min coronary occlusion followed by 48-h reperfusion. The infarct sizes as a percentage of area at risk were significantly reduced by pre-ischemic treatment with either 100mg/kg of NMDN or PC of 5 min ischemia and 5 min reperfusion alone (15.9+/-2.0%, n=8, and 10.3+/-1.2%, n=8, respectively) as compared with the control (43.9+/-2.2%, n=8). However, the combination of 100mg/kg of NMDN and PC significantly reduced the infarct size (4.9+/-1.2, n=8) compared with NMDN or PC alone. Another 40 rabbits, also given 100mg of NMDN, PC, NMDN+PC or saline before ischemia (n=10 in each group), were killed for biochemical analysis after 30 min of ischemia. NMDN and PC preserved the glycogen content and attenuated the lactate accumulation, respectively, as compared with the control. However, the combination of NMDN and PC preserved significantly more glycogen and significantly reduced lactate accumulation than either NMDN or PC alone. The combination of NMDN and PC markedly reduced the myocardial infarct size more than either process alone. The marked preservation of glycogen and marked attenuation of lactate accumulation by the combination of NMDN and PC suggest that the mechanism for this effect of NMDN+PC is related to the inhibition of glycogenolysis.

Role of protein kinase C in the reduction of infarct size by N-methyl-1-deoxynojirimycin, an alpha-1,6-glucosidase inhibitor.

Preischaemic treatment with N-methyl-1-deoxynojirimycin (MOR-14), an alpha-1,6-glucosidase inhibitor, attenuates glycogenolysis and lactate accumulation during ischaemia and markedly reduces infarct size in rabbit hearts. In the present study, we have investigated whether protein kinase C (PKC), a principal mediator of ischaemic preconditioning, is also involved in the cardioprotective effect of MOR-14. To assess the effect of PKC inhibition on infarct size in MOR-14-treated hearts, 38 rabbits were subjected to 30 min of ischaemia followed by 48 h of reperfusion. Infarct size, as a per cent of area at risk, was significantly smaller in rabbits administered 100 mg kg(-1) of MOR-14 10 min before ischaemia (17+/-2%, n=10), than in a control group (46+/-5%, n=10). This beneficial effect of MOR-14 was abolished when 5 mg kg(-1) of chelerythrine, a PKC inhibitor, was given 10 min prior to MOR-14 injection (39+/-4%, n=10), although chelerythrine alone did not alter infarct size (43+/-4%, n=8). Further, chelerythrine had no effect on MOR-14-induced attenuation of glycogen breakdown and lactate accumulation in hearts excised at 30 min of ischaemia. Immunoblot analysis of PKC in homogenates of Langendorff-perfused rabbit hearts revealed that MOR-14 significantly increased levels of PKC-epsilon in the particulate fraction at 20 and 30 min of ischaemia and in the cytosolic fraction at 30 min of ischaemia. Taken as a whole, our data suggest that PKC acts downstream of the inhibition of glycogenolysis by MOR-14 to reduce infarct size. Thus, activation of PKC is a more direct mediator of the cardioprotection afforded by MOR-14 than is inhibition of glycogenolysis.

Relationship between thallium-201 myocardial SPECT and findings of endomyocardial biopsy specimens in dilated cardiomyopathy.

The purpose of this study was to clarify which myocardial histological findings associated with dilated cardiomyopathy (DCM) are reflected in quantitative 201Tl myocardial SPECT. We obtained studied SPECT images from 21 patients with DCM 10 minutes and 2 hours after they received an injection of 111 MBq 201Tl at rest. We calculated the percent coefficient of variation of myocardial 201Tl counts [%CV(TI)], the washout rate (WR), standard deviation of WR [SD(WR)], extent score (ES) and severity score (SS). We used image analysis to measure % fibrosis, % myocytes, the ratio of fibrous tissue to myocyte tissue (F/My), myocyte size and standard deviation of myocyte size [SD(My)] in left ventricular endomyocardial biopsy specimens. The %CV(Tl) was correlated with % fibrosis and F/My. The ES and SS also correlated with F/My. The correlation between SD(WR) and SD(My) was significant. The present findings suggest that %CV(Tl), ES and SS of rest 201Tl SPECT reflect myocardial fibrosis and that the standard deviation of washout reflects the distribution of myocyte size.

Characterization of ultrastructure and its relation with DNA fragmentation in Fas-induced apoptosis of cultured cardiac myocytes.

The purposes of the present study were to define precisely the ultrastructural features of apoptosis in cultured cardiomyocytes and to determine whether DNA fragmentation is essential for the apoptotic morphology. When cultured neonatal murine cardiomyocytes were incubated with an agonistic anti-Fas antibody in the presence of a non-toxic amount of actinomycin D or cycloheximide, approximately 70% of them had lost their viability after 24 h. The dead cardiomyocytes showed the typical ultrastructural changes of apoptosis on transmission and scanning electron microscopy, as well as by positive in situ nick end-labelling (TUNEL), positive Taq polymerase-based in situ ligation, a DNA ladder pattern on gel electrophoresis, and an increase in the active fragment of caspase-3. According to TUNEL at the electron microscopic level, apoptotic nuclear change, cytoplasmic shrinkage, and DNA fragmentation always occurred simultaneously in apoptotic cardiomyocytes. Other ultrastructural features of apoptosis were the appearance of abundant lipid-like structures in the cytoplasm of cardiomyocytes at the early phase, and a high incidence of plasma membrane rupture and formation of apoptotic bodies at the later phase. When zinc, an inhibitor of Ca2+/Mg2+-dependent endonuclease, was added to the present model, activation of caspase-3 and an apoptotic ultrastructure were still observed in spite of the lack of DNA fragmentation, indicating that this type of myocyte death is also apoptosis. In conclusion, the typical apoptotic ultrastructure and DNA fragmentation occur simultaneously in association with caspase-3 activation in Fas-stimulated cultured cardiomyocytes. Apoptotic morphology can, however, be observed even without DNA fragmentation.

Role of protein kinase C, K(ATP) channels and DNA fragmentation in the infarct size-reducing effects of the free radical scavenger T-0970.

1. In the present study, we investigated the effect of 1-(3-tert-butyl-2-hydroxy-5-methoxyphenyl)-3-(3-pyridylmethyl) urea hydrocloride (T-0970), a novel water-soluble low-molecular weight free radical scavenger, on the generation of hydroxyl radicals in vivo and on myocardial infarct size in an in vivo model of myocardial infarction in rabbits. 2. T-0970 scavenged hydroxyl radicals generated in the myocardium during reperfusion, as assessed by using a microdialysis technique and HPLC in an in vivo model with 30 min coronary occlusion and 30 min reperfusion in rabbits. 3. Another group of rabbits was subjected to 30 min coronary occlusion and 48 h reperfusion. The control group (n = 10) was infused with saline for 190 min from 10 min before occlusion to 180 min after reperfusion. The treatment group (T-0970 group; n = 10) was injected with a bolus 2.5 mg/kg T-0970 and then infused with T-0970 for 190 min from 10 min before reperfusion to 180 min after reperfusion at a rate of 100 microg/kg per min. The T-0970 + CHE group (n = 5) was given chelerythrine (CHE; a selective inhibitor of protein kinase C (PKC); 5 mg/kg, i.v.) 10 min before the administration of T-0970. The T-0970 + 5-HD group (n = 5) was given 5-hydroxydecanoate (5-HD; an inhibitor of mitochondrial K(ATP) channels; 5 mg/kg, i.v.) 10 min before the administration of T-0970. The CHE and 5-HD groups were given CHE (5 mg/kg, i.v.) and 5-HD (5 mg/kg, i.v.) 20 min before reperfusion, respectively. After 48 h reperfusion, infarct size was measured histologically and expressed as a percentage of the area at risk (AAR). In another series of experiments, the control (n = 5) and T-0970 (n = 5) groups were killed 4 h after reperfusion following 30 min coronary occlusion and DNA fragmentation in myocytes was assessed using in situ dUTP nick end-labelling (TUNEL) at the light microscopic level. 4. Infarct size, as a percentage of AAR, in the T-0970 group was significantly reduced compared with the control group (21+/-4 vs 41+/-4%, respectively; P<0.05). This reduction of infarct size by T-0970 was abolished by pretreatment with CHE and 5-HD. Neither CHE nor 5-HD alone had any effect on infarct size. The percentage of infarcted myocytes with DNA fragmentation by TUNEL in the T-0970 group was significantly reduced compared with the number in the control group (4.0+/-1.5 vs 10.7+/-1.9%, respectively; P<0.05). 5. T-0970, a free radical scavenger, improved reperfusion injury. This effect seemed to be mediated by activation of PKC, the opening of mitochondrial K(ATP) channels and inhibition of DNA fragmentation.

Considerable time from the onset of plaque rupture and/or thrombi until the onset of acute myocardial infarction in humans: coronary angiographic findings within 1 week before the onset of infarction.

BACKGROUND: It has been thought that the thrombi and bleeding in plaques that occur after plaque rupture or endothelial damage from vessels with mild stenosis suddenly occlude the lumen and cause acute myocardial infarction (AMI). However, our hypothesis is that thrombi and bleeding may not suddenly occlude the lumen. METHODS AND RESULTS: The study group consisted of 20 patients who had coronary angiograms performed within 1 week (3+/-3 days) before AMI and 20 control patients who had coronary angiograms performed 6 to 18 months (282+/-49 days) before AMI. The features of infarct-related coronary segments (IRCS) at 3 days before AMI were the presence of a significant stenosis of >50% (95% in incidence and 71+/-12% diameter stenosis) and Ambrose's type II eccentric lesions (plus multiple irregularities), an indicator of plaque rupture and/or thrombi (60% [70%]), and the features at 1 year before AMI were mild stenosis of <50% (95% incidence and 30+/-18% diameter stenosis) with rare Ambrose's type II eccentric lesions (plus multiple irregularities) (10% [10%]). The same relation was observed in each of the 4 subgroups with Q-wave infarction, non-Q-wave infarction, preceding effort angina within 1 month before AMI, and no preceding effort angina. CONCLUSIONS: The appearance of marked progression and Ambrose's type II eccentric lesion on coronary angiograms 3 days before AMI suggests the presence of a considerable time from the onset of plaque rupture and/or thrombi until the onset of AMI. These features may be predictors of AMI. The concept provides new insight into the mechanism and prevention of human AMIs.

T-0162, a novel free radical scavenger, reduces myocardial infarct size in rabbits.

1. We investigated the effects of 1-(3-tert-butyl-2-hydroxy-5-methoxyphenyl)-3-(3-pyridylmethyl)urea hydrochloride (T-0162), a novel low-molecular weight free radical scavenger, on the generation of superoxide anions and hydroxyl radicals in vitro and in vivo and on myocardial infarct (MI) size in an in vivo model of MI in rabbits. 2. It was found that T-0162 scavenged both superoxide anions and hydroxyl radicals in a concentration-dependent manner in vitro. 3. In an in vivo rabbit model with 30 min coronary occlusion and 30min reperfusion, T-0162 scavenged hydroxyl radicals generated in the myocardium during reperfusion. 4. Anaesthetized open-chest Japanese white male rabbits were subjected to 30 min coronary occlusion and 48 h reperfusion. The control group (n = 10) was infused with 10% lecithin solution for 220 min from 10 min before occlusion to 180 min after reperfusion. The pretreatment group (n = 10) was infused with T-0162 dissolved in 10% lecithin solution for 220 min from 10 min before occlusion to 180 min after reperfusion at a rate of 400 microg/kg per min. The post-treatment group (n = 10) was injected with an i.v. bolus of 10 mg/kg T-0162 and was then infused with 400 microg/kg per min T-0162 for 190 min from 10 min before reperfusion to 180 min after reperfusion. After 48 h reperfusion, infarct size was measured histologically and expressed as a percentage of area at risk (AAR). 5. There was no significant difference in haemodynamic parameters among the three groups throughout the experimental period. The per cent infarct size of the AAR in the T-0162 groups (24.8+/-4.3 and 30.5+/-3.9% for pre- and posttreatment groups, respectively) was significantly reduced compared with control (44.7+/-4.1%; P<0.05). There was no significant difference in the AAR among the three groups. 6. In conclusion, T-0162 reduces MI size through the inhibition of reperfusion injury.

Three minute, but not one minute, ischemia and nicorandil have a preconditioning effect in patients with coronary artery disease.

OBJECTIVES: This study focused on 1) the determination of the optimal preconditioning (PC) duration, and 2) the protective effect of nicorandil (NC), a hybrid nitrate with a KATP channel opening effect, during a percutaneous transluminal coronary angioplasty (PTCA) model in humans. BACKGROUND: The ischemic PC effect is induced in 180 s ischemia, but not in 120 s ischemia in rabbit hearts. However, the duration of ischemia that induces PC effect and the role of the KATP channel in the PC effect in humans are still unclear. METHODS: Forty-six patients with stable angina were randomly allocated to four groups: the duration of the first inflation as PC ischemia was 60 s in the PC60 group (n = 12), and 180 s in the PC180 group (n = 12). In the other groups, NC (80 microg/kg) was intravenously given for 1 min in the NC group (n = 12), and isosorbide dinitrate (ISDN) (40 microg/kg) was given in the ISDN group (n = 10). Five minutes after first inflation or drug administration, a second inflation was conducted for 120 s in each group. In the ECG, the lead with the largest shift in ST segment (deltaST max), and the sum of elevated ST levels in all leads (sigmaST) were determined. RESULTS: In the PC60 group, no significant difference was observed in either deltaST max or sigmaST between the first and second inflation. However, the second inflation in the PC180 group showed significantly lower levels of deltaST max and sigmaST compared with those of the first inflation. In the NC group, both deltaST max and sigmaST measured at 30 s and 60 s after balloon inflation were significantly lower than those of the first inflation in the PC60 and PC180 control groups. In the ISDN group, no significant difference was observed in deltaST max or sigmaST. CONCLUSION: In human PTCA models, a PC effect is observed in 180 s ischemia, but not in 60 s ischemia. A pharmacological PC effect is induced by NC, a KATP channel opener with a nitrate-like effect but not ISDN. This suggests that the opening of KATP channels plays an important role in the protecting effect of NC.

Gallic acid induces vascular smooth muscle cell death via hydroxyl radical production.

In the present study, we investigated whether gallic acid (GA) can induce death in cultured vascular smooth muscle cells (VSMCs), and whether production of the hydroxyl radical (.OH) is involved in the process of GA action. GA killed cultured VSMCs from rat aorta, in a dosc- and time-dependent manner. Cytoplasmic shrinkage and nuclear condensation were observed light microscopically in GA-treated VSMCs, which appeared apoptotic. However, the ultrastructure of the VSMC was not typical of apoptosis: nuclear condensation was not glossy, and the plasma membrane and subccellular organelles were disrupted. Although the VSMC were positive for in situ nick end-labeling (TUNEL). they did not show a DNA ladder pattern on gel electrophoresis and were negative for T aq polymerase-based in situ ligation, which is more specific for apoptosis than TUNEL. Moreover. GA-induced cell death was not prevented by Boc-Asp-fmk (a pan-caspase inhibitor). Production of OH was detected in GA-treated VSMCs using high-performance liquid chromatography with salicylic acid as a trapping agent. Lipid peroxidation was also observed. The production of .OH was inhibited by catalase (CAT) and deferoxamine (DFX), and these treatments completely rescued VSMCs from cell death. In a cell-free system, GA produced .OH in the presence of Fe2+-EDTA, which was quenched by CAT and DFX, suggesting involvement of the Haber-Weiss reaction. Oxidative stress by reactive oxygen species, .OH in particular, is one of the mechanisms of GA-induced death of VSMCs, the mode of which was different from typical apoptosis.

N-methyl-1-deoxynojirimycin (MOR-14), an alpha-glucosidase inhibitor, markedly improves postischemic left ventricular dysfunction.

We examined whether pharmacological inhibition of glycogenolysis by N-methyl-1-deoxynojirimycin (MOR-14), a new compound which reduces the glycogenolytic rate by inhibiting the alpha-1,6-glucosidase activity of the glycogen-debranching enzyme, can protect the heart against postischemic left ventricular dysfunction. The hearts of male Sprague-Dawley rats were excised, and perfused on a Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O2 and 5% CO2. The hearts were paced at 320 beats/min except during the ischemia. Left ventricular developed pressure (LVDP, mmHg), +/-dP/dt (mmHg/s), and coronary flow (ml/min) were continuously monitored. All hearts were perfused for a total of 120 min including a 30-min preischemic period followed by a 30-min episode of global ischemia and 60 min reperfusion. with or without 0.5 or 2 mM of MOR-14 during the 30-min preischemic period or the first 30 min of reperfusion. In another series of experiments, the myocardial content of glycogen and lactate was measured during the 30-min episode of ischemia in groups treated with and without 2mM of MOR-14. Preischemic but not postischemic treatment with MOR-14 significantly improved LVDP and +/-dP/dt without altering coronary flow during reperfusion in a dose-dependent manner. MOR-14 significantly preserved the glycogen content and significantly attenuated the lactate accumulation during the 30-min episode of ischemia. Preischemic treatment with MOR-14 is protective against postischemic left ventricular dysfunction through the inhibition of glycogenolysis in the isolated rat heart.

A novel anti-diabetic drug, miglitol, markedly reduces myocardial infarct size in rabbits.

1. We examined whether N-hydroxyethyl-1-deoxynojirimycin (miglitol), a new human anti-diabetic drug with effects to inhibit alpha-1, 6-glucosidase glycogen debranching enzyme and reduce the glycogenolytic rate as well as to inhibit alpha-1,4-glucosidase, could reduce infarct size in the rabbit heart. Rabbits were subjected to 30-min coronary occlusion followed by 48-h reperfusion. 2. The infarct size as a percentage of area at risk was not reduced by pre-ischaemic treatment with 1 mg kg(-1) miglitol (42.7+/-4.0%, n=10) compared with the saline control group (41.7+/-2.3%, n=10). However, it was significantly and dose-dependently reduced by pre-ischaemic treatment with 5 or 10 mg kg(-1) of miglitol (25.7+/-4. 5%, n=10, and 14.6+/-2.4%, n=10, respectively) without altering the blood pressure, heart rate or blood glucose level. However, there was no evidence of an infarct-size reducing effect after pre-reperfusion treatment with 10 mg kg(-1) of miglitol (35.0+/-3.0%, n=10). 3. Another 40 rabbits given 1, 5 and 10 mg kg(-1) of miglitol or saline before ischaemia (n=10 in each) were sacrificed at 30 min of ischaemia for biochemical analysis. Miglitol preserved significantly the glycogen content, and attenuated significantly the lactate accumulation in a dose dependent manner in the ischaemic region at 30 min of ischaemia. 4. Pre-ischaemic treatment, but not pre-reperfusion treatment, with miglitol markedly reduced the myocardial infarct size, independently of blood pressure and heart rate. A dose-dependent effect of miglitol on infarct size, glycogenolysis and lactate formation suggests that the mechanism may be related to the inhibition of glycogenolysis. Thus, miglitol may be beneficial for coronary heart disease as well as diabetes mellitus.

Evidence for the delayed effect in human ischemic preconditioning: prospective multicenter study for preconditioning in acute myocardial infarction.

OBJECTIVES: This study aimed to investigate prospectively the protective effect of a first preinfarction angina attack against acute myocardial infarction (AMI) in human hearts without significant collaterals. BACKGROUND: Several retrospective studies and the prospective studies have demonstrated the existence of the preconditioning (PC) effect in humans. However, collaterals were not examined in the prospective studies. In animal models, the PC effect on myocardial infarct size appears soon after PC reperfusion (classic) but disappears within 1 to 2 h. It then reappears 24 to 48 h after reperfusion (the delayed PC effect). Meanwhile, the PC effect on stunning appears 12 h after PC reperfusion (the delayed PC effect). The concept of the classic and delayed PC effects has not been investigated in human AMI studies. If the above concept is also correct in humans, the infarct size and/or impairment of the left ventricular function should be inversely correlated with the time interval between the first preinfarction angina attack and the onset of AMI when that time interval is limited to between 2 and 48 h. METHODS: The subjects were 25 patients with first AMI of the proximal left anterior descending artery who underwent successful direct percutaneous transluminal coronary angioplasty (PTCA) 2 to 6 h after the onset and with no (or poor) collateral circulation (grade 0 or 1). They were divided into two groups: preinfarction angina (PA)(+) group: 11 patients with new onset preinfarction angina from 2 to 48 h before the onset, PA(-) group: 14 patients without angina before infarction. Peak creatine kinase (CK) and cumulative CK were examined, and the left ventricular ejection fraction (LVEF) and the regional wall motion (RWM) were determined from the left ventriculograms during the acute (immediately after the coronary reperfusion) and chronic (four weeks after the onset of AMI) phases. The RWM index (RWMI) was then calculated as the mean motion of chords (standard deviation [SD]/chord) lying in the area of chords of RWM < or = -2 SD in the acute phase (ischemic risk area). RESULTS The increase in the RWMI between the acute and chronic phases was significantly larger in the PA(+) group than in the PA(-) group (1.55 +/- 1.32 and 0.69 +/- 0.75, p < 0.05, respectively) although no significant difference in the enzymatic infarct size was seen between the two groups. The increases in the LVEF and the RWMI were significantly correlated with the time interval from the first preinfarction angina attack to the onset of AMI (r = 0.622, p < 0.05 and r = 0.646, p < 0.05, respectively), but the enzymatic infarct size was not. CONCLUSIONS: The beneficial effect of preinfarction angina on left ventricular wall motion, independently of collateral flows, indicates the existence of the PC effect in humans. The greater protective effect of a longer time interval between angina pectoris and AMI suggests that the protection is due to a delayed PC effect.