Shinya Minatoguchi

In Vivo Quantitative Tissue Characterization of Human Coronary Arterial Plaques by Use of Integrated Backscatter Intravascular Ultrasound and Comparison With Angioscopic Findings

Background—The purpose of the present study was to define whether integrated backscatter (IB) combined with conventional intravascular ultrasound (IVUS) makes tissue characterization of coronary arterial plaques possible. Methods and Results—IB-IVUS was performed in coronary arteries (total 18 segments) of 9 patients at autopsy, and the findings were compared with the histology. RF signals, which were digitized at 2 GHz in 8-bit resolution, were obtained with an IVUS system with a 40-MHz catheter. IB values of the RF signal from the region of interest (ROI) (100-&mgr;m depth, 1.4° per line) were calculated by use of a personal computer. IB values on the ROIs were divided into 5 categories, compared with each of the plaque histologies: category 1 (thrombus), −88 < IB ≤ −80; category 2 (intimal hyperplasia or lipid core), −73 < IB ≤ −63; category 3 (fibrous tissue), −63 < IB ≤ −55; category 4 (mixed lesions), −55 < IB ≤ −30; and category 5 (calcification), −30 < IB ≤ −23. On the basis of these categories, we analyzed 5120 ROIs per segment in each ring-like arterial specimen. Color-coded maps of plaques were constructed by use of these IB data and conventional IVUS data, which reflected the plaque histology of autopsied coronary arteries well. Then, the same method was undertaken in 24 segments with plaque from 12 patients in vivo with angina pectoris. Comparisons between coronary angioscopy and IB-IVUS revealed that the surface color of plaques in angioscopy reflected the thickness of the fibrous cap rather than the size of the lipid core. Conclusions—IB-IVUS represents a new and useful tool for evaluating the tissue structure of human coronary arterial plaques.

“Apoptotic” Myocytes in Infarct Area in Rabbit Hearts May Be Oncotic Myocytes With DNA Fragmentation Analysis by Immunogold Electron Microscopy Combined With In Situ Nick End-Labeling

BACKGROUNDnModes of cell death have been defined morphologically as apoptosis and oncosis. Infarcted myocytes have been reported to show apoptosis, as revealed by DNA fragmentation by DNA ladder and by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) at the light microscopic level. We investigated whether TUNEL-positive infarcted myocytes have apoptotic or oncotic ultrastructures by using electron microscopic TUNEL, which can simultaneously observe the ultrastructure and DNA fragmentation of the same myocytes.nnnMETHODS AND RESULTSnThirty rabbits were divided into 5 groups (n=6 each) that were subjected to a sham operation or to 30-minute ischemia followed by 0-minute, 30-minute, 2-hour, or 4-hour reperfusion of a coronary artery. In the 2- and 4-hour reperfusion groups only, DNA electrophoresis showed a ladder pattern, and the light microscopic TUNEL finding was positive in the nuclei of myocytes localized in the infarcted area (6+/-2% and 11+/-3%, respectively). Electron microscopic TUNEL showed that nuclei with a significant accumulation of immunogold particles (indicating an electronic microscopic TUNEL-positive result) were observed only in the infarcted myocytes with irreversibly oncotic ultrastructures that were found in the hearts of the 2- and 4-hour reperfusion groups (41+/-3% and 83+/-4%, respectively). Irreversibly oncotic myocytes (indicated by swelling, inhomogeneously clumped chromatin in nuclei, dense bodies in mitochondria, and/or ruptured plasma membranes) were also seen in the 0- and 30-minute reperfusion groups, which did not exhibit TUNEL-positive myocytes. There was no evidence of apoptotic ultrastructures in the myocytes.nnnCONCLUSIONSnDNA fragmentation occurs in the myocytes that had already shown irreversibly oncotic, but not apoptotic, ultrastructures with ischemia and/or reperfusion. Therefore, DNA fragmentation itself does not always mean apoptosis, and so-called apoptotic infarcted myocytes may belong to a category of cell death other than apoptosis.

Role of Apoptosis in the Disappearance of Infiltrated and Proliferated Interstitial Cells After Myocardial Infarction

Myocardial infarction (MI) progresses from the acute death of myocytes and the infiltration of inflammatory cells into granulation, followed by scars. During the healing process, the myocardial interstitial cell population in the infarcted tissues increases markedly and then decreases. We postulated that apoptosis is responsible for this process. Twenty-four male Japanese white rabbits underwent a 30-minute occlusion of the left coronary artery followed by reperfusion for 2 days, 2 weeks, or 4 weeks (n=8 each). The histological features consisted of dead cardiomyocytes and marked leukocyte infiltration at 2 days after MI and granulation consisting of numerous alpha-smooth muscle actin-positive myofibroblasts, macrophage antigen-positive macrophages, and neovascularization at 2 weeks. At 4 weeks, the cellularity decreased markedly, and scars were evident. Interstitial cells with positive nick end labeling were significantly more frequent at the light microscopic level in the 2-day MI samples (5.3+/-3.6% in the center and 6.9+/-3.3% in the periphery of the infarct region) than in the 2-week (2.5+/-1.0%) and 4-week (0.5+/-0.5%) samples. DNA electrophoresis showed a clear ladder in tissues from the ischemic areas at 2 days after MI but not at 2 and 4 weeks after MI. Ultrastructurally, typical apoptotic figures, including apoptotic bodies and condensed nuclei without ruptured plasma membranes, were detected in leukocytes from all hearts with 2-day MI and in myofibroblasts, endothelial cells, and macrophages from all hearts with 2-week MI. In the electron microscopic in situ nick end labeling, immunogold particles intensely labeled the condensed chromatin of the typical apoptotic nuclei. These particles were also accumulated on nuclei of the interstitial cells showing homogeneous density but not definite condensation as typical apoptotic nuclei, suggesting an early stage of apoptosis. Thus, apoptosis plays an important role in the disappearance of both the infiltrated leukocytes and the proliferated interstitial cells after MI. This finding may have therapeutic implications for postinfarct ventricular remodeling through apoptosis handling during the healing stage of MI.

Preventive Effect of Erythropoietin on Cardiac Dysfunction in Doxorubicin-Induced Cardiomyopathy

Background— Doxorubicin is a highly effective antineoplastic drug, but its clinical use is limited by its adverse side effects on the heart. We investigated possible protective effects of erythropoietin against doxorubicin-induced cardiomyopathy. Methods and Results— Cardiomyopathy was induced in mice by a single intraperitoneal injection of doxorubicin (15 mg/kg). In some cases, human recombinant erythropoietin (5000 U/kg) was started simultaneously. Two weeks later, left ventricular dilatation and dysfunction were apparent in mice given doxorubicin but were significantly attenuated by erythropoietin treatment. Erythropoietin also protected hearts against doxorubicin-induced cardiomyocyte atrophy and degeneration, myocardial fibrosis, inflammatory cell infiltration, and downregulation of expression of GATA-4 and 3 sarcomeric proteins, myosin heavy chain, troponin I, and desmin. Cyclooxygenase-2 expression was upregulated in doxorubicin-treated hearts, and that, too, was attenuated by erythropoietin. No doxorubicin-induced apoptotic effects were seen, nor were any changes seen in the expression of tumor necrosis factor-&agr; or transforming growth factor-&bgr;1. Antiatrophic and GATA-4 restoring effects of erythropoietin were demonstrated in the in vitro experiments with cultured cardiomyocytes exposed to doxorubicin, which indicated the direct cardioprotective effects of erythropoietin beyond erythropoiesis. Cardiac erythropoietin receptor expression was downregulated in doxorubicin-induced cardiomyopathy but was restored by erythropoietin. Among the downstream mediators of erythropoietin receptor signaling, activation of extracellular signal-regulated kinase was reduced by doxorubicin but restored by erythropoietin. By contrast, erythropoietin was ineffective when administered after cardiac dysfunction was established in the chronic stage. Conclusions— The present study indicates a protective effect of erythropoietin against doxorubicin-induced cardiomyopathy.

Sensitivity to Apoptosis Signal, Clearance Rate, and Ultrastructure of Fas Ligand-Induced Apoptosis in In Vivo Adult Cardiac Cells

Background—Sensitivity to apoptotic signals, the clearance rate of apoptosis, and the apoptotic ultrastructure have not been studied in cells of the in vivo adult heart. Methods and Results—To minimize the systemic influence, soluble Fas ligand was injected directly into in vivo rat hearts and livers (as the control) at concentrations of 0, 0.5, 2, and 5 &mgr;g/mL (groups C, F0.5, F2, and F5). Apoptotic cardiomyocytes and apoptotic noncardiomyocytes of the heart were identified with similar incidences only in F5. Their incidences peaked at 12 hours after injection (2.0±0.09% in cardiomyocytes) and diminished markedly 24 hours later. Caspase-3 was activated only in F5. Boc-Asp-fmk, a pancaspase inhibitor, inhibited apoptosis, suggesting that the apoptosis sensitivity was regulated upstream of caspase-3. Apoptotic noncardiomyocytes showed typical ultrastructure. In addition to the typical ultrastructure, such as cellular shrinkage, chromatin condensation, and apoptotic bodies, however, apoptotic cardiomyocytes showed unique features: doughnut-like, but not half-moon– or crescent-like, chromatin condensation; frequent plasma membrane rupture even during the early stage; condensed mitochondria with wrinkled cristae inside; the appearance of cytoplasmic lipid-like droplets; and myofibrillar derangement. In the livers, typical apoptosis was induced in hepatocytes and nonhepatocytes of the liver even in the F0.5 group, which were cleared 24 hours later. Conclusions—Compared with liver cells, cardiomyocytes as well as noncardiomyocytes of the heart are more resistant against the apoptotic signal, but the clearance is similarly rapid (within 24 hours). The ultrastructure of apoptotic cardiomyocytes is unique. These findings provide new insights into the dynamics of cell death in the heart.

Role Of Protein Kinase C, KATP 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.

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.

Baroreflex Modifies the Effect of Vasodilators on Systemic Capacitance Vessel in Dogs

We examined whether the vasodilating effect of vasodilators on systemic capacitance vessels is modified by the baroreflex when a fall in arterial blood pressure was induced by the vasodilators in dogs. We estimated the dilation of the systemic capacitance vessels from the fall in mean circulatory pressure (MCP), proposed by Guyton et al. and the dilation of the systemic capacitance vessels from the fall in the total peripheral resistance (TPR). The dose-response curve for percent change in TPR to graded doses of nitroglycerin (NG, 0.8–200 µg/kg) in the untreated group was not different from that in the total spinal anesthesia (TSA) group in which the baroreflex was eliminated. The TSA group gave a dose-response curve for percent change in MCP to NG on the low dose side of NG as compared with the untreated group. NG increased both plasma levels of nor-epinephrine (NA) and epinephrine (A) in the untreated group. However, NG hardly affected plasma levels of NA or A in the TSA group. Milrinone significantly decreased the TPR and there was no significant difference in the percent change in TPR between the untreated and TSA groups. Milrinone did not change the MCP in the untreated group, but decreased it in both TSA group and the group pretreated with α-blocker, suggesting that baroreflex-mediated vasoconstriction is mediated through α-adrenoceptors in the capacitance vessels. Milrinone also increased plasma levels of NA and A in the untreated group but not in the TSA group.