Kazuhira Maehara

Attenuation of Ischemia/Reperfusion Injury in Rats by a Caspase Inhibitor

BACKGROUND Z-Val-Ala-Asp(OMe)-CH2F (ZVAD-fmk), a tripeptide inhibitor of the caspase interleukin-1beta-converting enzyme family of cysteine proteases, may reduce myocardial reperfusion injury in vivo by attenuating cardiomyocyte apoptosis within the ischemic area at risk. METHODS AND RESULTS Sprague-Dawley rats were subjected to a 30-minute coronary occlusion followed by a 24-hour reperfusion. An inert vehicle (dimethylsulfoxide; group 1, n=8) or ZVAD-fmk, at a total dose of 3.3 mg/kg (group 2, n=8), was administered intravenously every 6 hours starting at 30 minutes before coronary occlusion until 24 hours of reperfusion. At this 24-hour point, hemodynamics were assessed by means of cardiac catheterization; then, the rats were killed, and the left ventricle was excised and sliced. The myocardial infarct size/ischemic area at risk and the count of presumed apoptotic cardiomyocytes (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling [TUNEL]-positive cells) within the ischemic area at risk were assessed through triphenyltetrazolium chloride staining and TUNEL methods, respectively. Peak positive left ventricular dP/dt was higher (P=.02) and left ventricular end-diastolic pressure was lower (P=.04) in group 2 than in group 1. The infarct size/ischemic area at risk of group 2 (52.4+/-4.0%) was smaller (P=.02) than that of group 1 (66.6+/-3.7%), and TUNEL-positive cells were fewer (P=.0002) (group 2, 3.1+/-0.9%; group 1, 11.1+/-1.0%). Agarose gel electrophoresis revealed DNA laddering in the border zone myocardium of group 1, but DNA ladder formation was attenuated in group 2. CONCLUSIONS ZVAD-fmk was effective in reducing myocardial reperfusion injury, which could at least be partially attributed to the attenuation of cardiomyocyte apoptosis.

Apoptosis in relevant clinical situations: contribution of apoptosis in myocardial infarction.

Myocardial infarction is associated with increased TUNEL-positivity in cardiac resident and infiltrated cells. Apoptosis of proliferated interstitial myofibroblasts and infiltrated inflammatory cells may have a role in terminating tissue repair processes after infarction. Lateral and endocardial border zones of infarction within the risk area have frequent appearance of TUNEL-positive cardiomyocytes. Although the typical ultrastructural morphology of apoptosis has rarely been detected in ischaemic cardiomyocytes, there are many reports in which the TUNEL method was used for assessment of cardiomyocyte apoptosis. It has become evident that TUNEL-positivity reflects a wide range of cellular conditions; viable cells undergoing DNA repair, apoptosis, and necrosis. Therefore, it is controversial whether TUNEL-positive cardiomyocytes in infarcted myocardium are all apoptotic. Methods which will be more specific for identifying apoptosis are required for future study. TUNEL-positivity can be attenuated by anti-apoptotic interventions such as inhibition of caspases, mitochondrial protection, free radical scavenging, and some conventional pharmacotherapies. However, it remains to be determined whether anti-apoptotic interventions result in satisfactory reduction of infarct size. The injurious impact of myocardial ischaemia comes from a mixture of pro-apoptotic and necrosis-promoting signals, and the target of both signals is mitochondria. Through a common pathway they may cause permeability transition. Interventions which act only at the post-mitochondrial stage of apoptosis may fail to reduce infarct size, whereas those acting at pre-mitochondrial and mitochondrial stages may reduce infarct size. Progress in investigating the basic mechanisms of apoptosis and recognition of the modes of cardiomyocytes death will contribute to advances in cardioprotective therapy in myocardial infarction.

A comparison of ultrastructural changes on endomyocardial biopsy specimens obtained from patients with diabetes mellitus with and without hypertension

SummaryThe pathogenesis of diabetic cardiomyopathy is unknown. The synergistic, or enhanced, effect of hypertension on pathological changes in the heart of diabetic patients has been highly suspected. The purpose of this study was to evaluate the myocardial changes related to diabetes mellitus with and without hypertension, using biopsy specimens. We examined the ultrastructural changes in biopsy specimens of the endomyocardium obtained from 25 patients. They were divided into four groups: controls without hypertension or diabetes mellitus (n=6), and patient with hypertension (n=3), diabetes mellitus (n=8), and diabetes with hypertension (n=8). The diabetic patients showed nearly normal or mildly depressed systolic left ventricular function. Ultrastructural pictures were analyzed for thickening of the capillary basement membrane, presence of toluidine blue-positive materials (i.e., materials showing metachromasia) in the myocytes, size of myocytes, and interstitial fibrosis. The thickening of the capillary basement membrane, the accumulation of toluidine blue-positive materials, and interstitial fibrosis were all significantly greater in the patients with diabetes mellitus compared to the control subjects. The myocytes tended to be small (cell atrophy) in the diabetes group. Although these pathological changes in the heart were characteristic of diabetic patients, irrespective of the presence or absence of hypertension, the presence of hypertension increased the pathological changes of myocardial cells as well as abnormality in the capillary vessels in patients with diabetes mellitus. Alterations in the myocardial cells and capillaries, caused by diabetes mellitus, may lead to myocardial cell injury and interstitial fibrosis and, ultimately, to ventricular systolic and diastolic dysfunction, especially when the diabetes is accompanied by hypertension.

Increased plasminogen activator inhibitor-1 and apolipoprotein (a) in coronary atherectomy specimens in acute coronary syndromes.

BACKGROUND Although increased tissue factor expression is known in vulnerable plaques, there is no reported study to compare plaque fibrinolysis in stable and unstable plaques. This study investigates the extent of plasminogen activator inhibitor-1 (PAI-1) and apolipoprotein (a) [apo(a)] in the plaques of different types of coronary artery disease as well as the correlation between these molecules and infiltration of macrophages to plaques. METHODS Using immunohistochemical staining, we examined PAI-1 expression and apo(a) deposition in coronary atherosclerotic specimens obtained by directional coronary atherectomy from 19 patients with acute myocardial infarction (AMI), 12 with unstable angina pectoris (UAP), and 13 with stable angina pectoris (SAP). The percentages of the total areas of specimens stained with PAI-1 or apo(a) were estimated by an NIH image program. The proportion of macrophages as a percentage of all cells in plaques was calculated as the macrophage density. RESULTS We found significantly higher percentages of total areas of specimens stained with PAI-1 in AMI (25.5 +/- 8.6%, P < 0.001) and UAP (22.2 +/- 10.4%, P < 0.005) than in SAP (9.5 +/- 5.0%), as well as with apo(a) (AMI; 11.7 +/- 7.1%, P < 0.005, UAP; 11.1 +/- 5.5%, P < 0.01 versus SAP; 3.9 +/- 1.5%). Linear regression analysis of all the samples showed a correlation between PAI-1 or apo(a) and macrophage density (PAI-1: r = 0.75, P < 0.001 and apo(a): r = 0.56, P < 0.001). CONCLUSIONS Our results suggest a possible contribution of increased PAI-1 and apo(a) in plaques to the pathogenesis of acute coronary syndromes including impaired fibrinolysis.

Multiple endothelial injury in epicardial coronary artery induces downstream microvascular spasm as well as remodeling partly via thromboxane A2.

OBJECTIVES The study was undertaken to develop a coronary microvascular spasm model in pigs by repeated epicardial coronary artery endothelial injury. BACKGROUND The pathophysiologic mechanisms responsible for coronary microvascular spasm remain unclear, in large part because a suitable animal model has yet to be found. METHODS Balloon endothelial denudation was done just distal to the site of an implanted Doppler flowmeter in the left anterior descending coronary artery (LAD) every two weeks for a total of four times. Changes in LAD blood flow by intracoronary administration of vasoactive agents were assessed before each denudation. RESULTS In the epicardial LAD endothelial denudation pigs, decreases in LAD blood flow caused by acetylcholine were augmented. Before denudation, it was - 15 +/- 4%, and at week 8 (i.e., two weeks after the fourth denudation) it was -100% (i.e., zero flow [p < 0.01]). The LAD flow changes in response to 5-hydroxytryptamine (5-HT) changed from an increase to a decrease, accompanied by medial thickening of microvessels in the LAD perfusion area. These flow responses were observed without significant changes in LAD diameter. In contrast, the LAD blood flow responses to acetylcholine and 5-HT did not change throughout the experiment in pigs given aspirin and a thromboxane A2 (TXA2) synthase inhibitor orally. CONCLUSIONS This microvascular spasm model indicates that hypersensitivity to vasoactive substances in the microvascular beds as well as microvascular remodeling are brought about partly through TXA2. This model should be useful for examining the pathophysiology and treatment of microvascular angina.

Morphological and functional changes in coronary vessel evoked by repeated endothelial injury in pigs

OBJECTIVE We examined the morphological changes induced by repeated endothelial denudation in coronary artery (CA), as well as functional changes in the endothelium-dependent and smooth muscle responses to various vasoactive agents during the process of intimal thickening. METHODS We observed vascular responses in denuded and non-denuded portions of pig CA while being fed a normal diet (n = 11, N group) or 2% cholesterol diet (n = 25, C group) to intracoronary acetylcholine (ACh), 5-hydroxytryptamine (5-HT), substance P (SP), and isosorbide dinitrate (ISDN) with and without the nitric oxide synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg i.v.) over a period of 8 weeks. Balloon endothelial denudation of the left anterior descending CA was carried out every 2 weeks. RESULTS In N group, maximum vasoconstriction was obtained with ACh 2 weeks after the first denudation [26 +/- 5% vs. 1 +/- 1% pre-denudation, p < 0.05]. L-NAME did not affect ACh-induced CA diameter changes. Thereafter, the response to ACh was attenuated by repeated denudation in N groups. However, the degree of 5-HT-induced CA narrowing at the denuded portion increased from 7 +/- 4% (0 week) to 88 +/- 8% (8 weeks) (p < 0.05). The changes resulted in severe myocardial ischaemia, and suggested that endothelium-dependent vasodilation was progressively attenuated while hyperreactivity of vascular smooth muscle simultaneously increased. Vasodilation induced by SP was attenuated somewhat, but ISDN-induced vasodilation was preserved. Although mild hypercholesterolaemia was induced in C group, the vascular responses to these vasoactive agents did not differ from those of N group. CONCLUSIONS Repeated CA endothelial injury and regeneration induce the change of morphology and vascular reactivity in the denuded portion regardless of atherogenic diet. This study strongly suggests that intimal thickening caused by repeated endothelial injury and regeneration induces specific vascular responses to vasoactive agents. Moreover, it is also suggested that during the progression of intimal thickening, increased vascular smooth muscle contraction and decreased endothelium-dependent dilation appear in a stimulus-dependent manner, often leading to severe coronary vasoconstriction accompanied with definitive ECG ST change.

Management Strategy of Isolated Spontaneous Dissection of the Superior Mesenteric Artery

OBJECTIVE Isolated spontaneous dissection of the superior mesenteric artery (SMA) is very rare among of the visceral artery dissection and its treatment is not established. In this paper we present our experiences and consider the treatment of isolated SMA dissection. METHODS A retrospective review of our cases from 2005 was performed. Clinical symptoms, radiologic findings and results were evaluated. There were 14 cases of visceral artery dissection, in which all cases were with SMA dissection. There were 12 males and 2 females with a mean age of 57 years (range 41-78 years). RESULTS We categorized SMA dissection into the six types according to the Sakamotos and Zerbibs classification. One patient with type VI underwent emergent endovascular surgery with stent. One patient with type VI received thrombectomy and intimectomy with open surgery. One patient with type II underwent aneurysmectomy due to enlarged dissected SMA 3 months later from onset. The other eleven patients were managed conservatively. At follow-up, the diameter of SMA did not enlarged and the length of the dissection significantly decreased to 20.7 ± 15.7 mm from 38.0 ± 15.1 mm at onset (p <0.01). After treatment, imaging indicated the following changes in classification: type I, one patient; type II, 4 patients; type IV, 4 patients; complete remodeling, one patient, all without any event during the follow-up period of 5-82 months. CONCLUSION Most patients with isolated visceral artery dissection occurred in superior mesenteric artery and can be treated conservatively; however, endovascular or surgical procedures including laparotomy are indicated when there is suspicion of severe mesenteric ischemia. Because the dissection configuration will change, long term follow-up is necessary. (English translation of Jpn J Vasc Surg 2013; 22: 695-701).

Effects of antioxidants on coronary microvascular spasm induced by epicardial coronary artery endothelial injury in pigs.

ObjectivesThe effect of oxidative stress on coronary microvascular disease is unknown. We investigated whether chronic administration of ascorbic acid (ASC) or glutathione (GSH) prevents microvascular dysfunction and remodeling induced by upstream repeated coronary artery endothelial injury. MethodsBalloon endothelial injury was repeated at the left anterior descending coronary artery (LAD), just distal to an implanted flow meter, every 2 weeks for 6 weeks in pigs. Changes in LAD blood flow induced by acetylcholine (ACh) and 5-hydroxytryptamine were assessed before each endothelial injury and at 8 weeks after the first endothelial injury in pigs without treatment (endothelial injury group, n=12) and in pigs treated with oral ASC (3 g/day) (ASC group, n=12) and ASC (3 g/day) plus GSH (1 g/day) (ASC+GSH group, n=12). ResultsIn the endothelial injury group, reduced blood flow in response to ACh was augmented from a decrease of 18±17% to a decrease of 100% (that is, zero flow, 8 weeks, P<0.01), accompanied by an increase of ascorbyl free radicals (AFRs) in coronary sinus blood. In contrast, in the ASC+GSH group, blood flow response to ACh was altered to a decrease of 45±17% (8 weeks, P<0.01 compared with the endothelial injury group), coronary sinus blood AFRs did not change (8 weeks, 21.4±12.5 signal intensities, P<0.01 compared with the endothelial injury group) and the rate of platelet aggregation induced by adenosine diphosphate was small (8 weeks, 56±17%, P<0.01 compared with the endothelial injury group). ConclusionsChronic administration of antioxidants suppressed microvascular hypercontraction, suggesting that it may be a promising therapeutic strategy for treating coronary microvessel disorders, including microvascular angina.

Characteristics of death of neonatal rat cardiomyocytes following hypoxia or hypoxia–reoxygenation: the association of apoptosis and cell membrane disintegrity

Abstract. Irreversibly injured cardiomyocytes are positive for terminal deoxynucleotidyl transferase nick end-labeling (TUNEL), making it controversial as to whether TUNEL-positive cardiomyocytes induced by hypoxia–reoxygenation are apoptotic (secondarily necrotic) or primarily necrotic. We investigated the relationship between plasma membrane integrity and DNA fragmentation in hypoxic-reoxygenated cardiomyocytes. Cardiomyocytes were prepared from neonatal rat heart and exposed to hypoxia. The plasma membrane integrity was assessed by propidium iodide (PI) staining. The mode of DNA fragmentation was assessed by TUNEL and in situ polymerase chain reaction ligation assay. Furthermore, caspase-3 activity was measured in hypoxic-reoxygenated cardiomyocytes. Reoxygenation for 24 h after 3–8 h of hypoxia increased TUNEL positivity. However, the appearance of PI-positivity preceded that of TUNEL at various time points following reoxygenation. In contrast, TUNEL-positive but PI-negative cells were rarely found. In the hypoxic-reoxygenated cells, caspase-3 activity was increased, and PI- and TUNEL-positive cardiomyocytes possessed a sufficient number of double-strand DNA breaks with single-base 3′-OH terminals. In cardiomyocytes subjected to hypoxia–reoxygenation, the appearance of TUNEL positivity was delayed in comparison to membrane disintegrity, but in these cells caspase-3 has been activated and the mode of DNA fragmentation was apoptosis-specific. Thus, hypoxia–reoxygenation induces apoptosis associated with cell membrane disintegrity in cardiomyocytes.

Repeated epicardial coronary artery endothelial injuries lead to a global spontaneous coronary artery spasm.

ObjectivesThis study was conducted to develop a spontaneous coronary spasm model. Materials and methodsBalloon endothelial denudation was carried out in the epicardial left anterior descending coronary artery (LAD) every 2 weeks, for a total of four times, in 12 pigs. Changes in the denuded site diameter and LAD blood flow caused by acetylcholine or serotonin were assessed before each denudation and at week 8. Blood pressure, electrocardiogram (ECG) from the LAD area and LAD blood flow were monitored continuously in conscious and unrestrained pigs. ResultsSpontaneous ECG ST depression with a decrease in LAD blood flow appeared at around 2 weeks. In accordance with this, 0.5 μg/kg acetylcholine induced similar ECG and LAD blood flow changes without denuded site narrowing, suggesting microvascular spasm. Thereafter, ECG ST depression or elevation by serotonin via a denuded site spasm was found after 6 weeks and spontaneous ECG ST changes due to epicardial coronary artery spasm were observed. ConclusionEpicardial coronary artery endothelial injury may induce spontaneous vasospasticity in the downstream coronary microvessels as well as in the denuded portion, suggesting functional abnormality through the entire coronary arterial tree.