Noriko Asuwa

The effects of a myocardial bridge on coronary atherosclerosis and ischaemia

The term myocardial bridge (MB) describes the surprisingly common situation in which part of the left anterior descending coronary artery (LAD), running in epicardial adipose tissue, is covered by a bridge of myocardial tissue. The presence of an MB may influence arterial tissue through the alteration of haemodynamic forces by the myocardial contraction of the bridge itself. Histopathologically and ultrastructurally, any manifestations of atherosclerosis elsewhere in the LAD are suppressed in the intima beneath the MB. By scanning electron microscopy, abrupt changes in endothelial cell morphology indicate that the intima beneath the bridge is protected by haemodynamic factors. Furthermore, the closer the bridge to the left coronary ostium, the greater the extent of proximal intimal thickening. In parallel with this, considering the occurrence of myocardial infarction in cases of proximal MB together with previous reports on relationships between MB and coronary ischaemia, it appears that anatomical characteristics such as the location, length, and thickness of the MB have a bearing on the effects of this abnormality. When the pathologist examines the heart at autopsy, this quite common condition should be borne in mind, in view of its potential but complex relationship to atherosclerosis and ischaemic heart disease.

Eicosapentaenoic acid inhibits tube formation of vascular endothelial cellsin vitro

We previously have described a quantitative angiogenesisin vitro model, in which endothelial cells are cultured between two layers of type I collagen gel and become organized into tube-like structures. Using this model, the effect of eicosapentaenoic acid (20∶5n−3) on tube formation was investigated. When the endothelial cells isolated from bovine carotid artery were treated for 2 days with 5 μg/mL of arachidonic acid (20∶4n−6), eicosapentaenoic acid or docosahexaenoic acid (22∶6n−3), these polyunsaturated fatty acids were extensively incorporated into cellular phospholipids. The content of arachidonic, eicosapentaenoic and docosahexaenoic acid increased from 9.58% to 23.29%, from 0.98% to 11.76% and from 6.88% to 18.40%, respectively. When the eicosapentaenoic acid-treated cells were cultured between collagen gels, the tube-forming ability of the cells was markedly inhibited. The inhibition was dose-dependent between 1.0 and 5.0 μg/mL of eicosapentaenoic acid. At 5.0 μg/mL of eicosapentaenoic acid the inhibition reached 76%. By contrast, arachidonic acid increased tube formation, and docosahexaenoic acid had no effect. To elucidate the mechanism of eicosapentaenoic acid induced inhibition ofin vitro tube formation, we examined the effect of the acid on the proliferation of endothelial cells. Eicosapentaenoic acid at any dose (<5.0 μg/mL) had no effect on the proliferation of endothelial cells cultured on plastic plates without collagen gel. However, when the cells were cultured between collagen gels, eicosapentaenoic acid inhibited cell growth in a dose-dependent manner with maximum inhibition being observed at 2.5 μg/mL. These data suggest that eicosapentaenoic acid suppresses tube formation of endothelial cells, at least in part,via its inhibitory effect on cellular proliferation. Thus eicosapentaenoic acid may act as an endogenous inhibitor of angiogenesis under various pathological conditions, including tumor growth and chronic inflammation.

Absence of atherosclerosis evolution in the coronary arterial segment covered by myocardial tissue in cholesterol-fed rabbits.

The evolution of atherosclerotic lesions is suppressed in the intima of the human coronary artery, beneath myocardial bridges. To elucidate the mechanism of the protective effect, we investigated morphological changes using the rabbit coronary artery as a model. Rabbits fed a 1%-cholesterol diet were killed at intervals up to 20 weeks. Two short segments of the left coronary arteries running in the epicardial adipose tissue (EpiLAD) and subsequently running in the myocardium (MyoLAD) were compared morphologically. The intima of the EpiLAD had flat endothelial cells with a polygonal shape, and demonstrated raised atherosclerotic lesions with increase in serum cholesterol level. In contrast, the intima of the MyoLAD was free of atherosclerotic lesions throughout the study, and the endothelial cells were spindle-shaped and engorged. While ferritin particles reached only the surroundings of the internal elastic lamina in the MyoLAD, they permeated into the media of the EpiLAD. We suggest that myocardial bridges suppress coronary atherosclerosis by an alteration of endothelial permeability, which may be due to changes in haemodynamic force tending towards a higher shear stress. The data provide an insight into the relationship between haemodynamics and the development of coronary atherosclerosis.

Distribution and Synthesis of Apolipoprotein J in the Atherosclerotic Aorta

The distribution of apolipoprotein (apo) J during the development of atherosclerosis in the human aorta was evaluated by immununohistochemical observation, together with the other apolipoprotein A-I, A-II, B, C-III, and E. Although apoJ was never observed in the normal aorta (ie, without any intimal lesions or intimal thickening), it was distributed not only in the intima but also in the media of aortas with diffuse, intimal thickening or atherosclerotic lesions. Double immunostaining with antibodies for apoJ and alpha-smooth muscle actin revealed apoJ deposition in smooth muscle cells (SMCs) or the aortic stroma in the vicinity of SMCs. The extent of apoJ distribution in the aortic wall increased with the degree of atherosclerosis development. In addition, the distribution pattern of apoJ was very similar to that of apoA-I and E. In situ hybridization with human apoJ cDNA demonstrated intense signals in cells scattered within the subendothelial space and medial SMCs of the aorta with advanced atherosclerosis but not in those of the normal aorta without intimal thickening. Furthermore, reverse transcriptase-polymerase chain reaction of the cultured human aortic SMCs revealed apoJ mRNA expression in these cells. The results indicate that apoJ in the aortic wall originates from not only apoJ circulated in the plasma but also apoJ produced by SMCs in the aortic wall. Considering the similarities of the distribution between apoJ and apo-A-I or E, we hypothesize that apoJ possibly has a protective role against human atherosclerosis by its involvement with cholesterol transport from the aortic wall to the liver.

Leukotriene C4 stimulates angiogenesis in bovine carotid artery endothelial cells in vitro

In order to investigate the mechanism of angiogenesis involved in inflammatory processes, the effects of leukotrienes and prostaglandin E2 on in vitro tube formation of cultured vascular endothelial cells were examined. Endothelial cells from bovine carotid artery were cultured for 4 days between two layers of collagen gel and the lengths of organized tubes were quantitatively estimated with an image analyzer. Treatment with 10(-8)-10(-6)M of prostaglandin E2 increased the tubular lengths, and leukotriene C4 stimulated tube formation at far lower concentrations (10(-15)-10(-9)M) but leukotriene B4 and D4 were not effective on the tube formation. It was also found that endothelial cell migration was stimulated by almost the same concentrations of leukotriene C4 as those stimulating tube formation. These data suggest that leukotriene C4 is, at least, one of the important factors involved in angiogenesis during inflammatory processes.

Collagen alteration in vascular remodeling by hemodynamic factors.

Abstract The collagen alterations in the vascular wall remodeled by hemodynamic change were investigated by electron microscopy and immunohistochemistry. The left anterior descending coronary artery (LAD) without a myocardial bridge (MB) showed both lower matrix metalloproteinase-1 (MMP-1) expression and a smaller extent of spiraled collagen (SC) distribution than the LAD wall with MB, in which the intima was influenced by high shear stress. In the wall of the varicose great saphenous vein (GSV) the expression of MMP-1 was lower, while the expression of prolyl 4-hydroxylase was higher, than in the normal GSV. The extent of SC distribution in the intima and media of the varicose GSV was smaller than that in the normal GSV. An analogous difference in results was demonstrated between the portal vein (PV) of patients with liver cirrhosis and normal PV. However, the levels of expression of MMP-2, MMP-9 and tissue inhibitors of MMP (TIMPs) in these pathologic vessels were not different from those in the corresponding normal vessels. The results indicate that hemodynamic forces such as shear stress and increased intravascular blood pressure contribute to the collagen alterations in the vascular wall, which may lead to vascular wall remodeling.

Enhanced Expression of Caspase-3 in Hypertrophic Scars and Keloid: Induction of Caspase-3 and Apoptosis in Keloid Fibroblasts In Vitro

Recent studies have suggested that the regulation of apoptosis during wound healing is important in scar establishment and development of pathological scarring. To examine the phenomenon of apoptosis and its involvement in the process of pathological scarring, we immunohistochemically quantified differential levels of expression of caspase-3 and -2, which are activated during apoptosis in vitro, in surgical resected scar tissues. We divided 33 cases of normally healed flat scars and 18 cases of pathological scars (15 cases of hypertrophic scars and 3 cases of keloid) into three groups (S1 = <10 months’ duration; S2 = 10 to 40 months’ duration; and S3 = >40 months’ duration) according to the duration of scar. In all three groups examined, the semiquantitative scores for caspase-3 staining were significantly higher for the combination of hypertrophic scars and keloid as a group compared with normally healed flat scars, suggesting reduced cell survival and increased apoptotic cell death in hypertrophic scars and keloid. Apoptosis and caspase proteolytic activities were examined in vitro using two flat scar-derived fibroblast lines (FSFB-1 and -2) and two keloid-derived fibroblast lines (KFB-1 and -2). After 24 hours of serum deprivation, apoptotic cells were significantly increased in both KFB lines, whereas serum deprivation of FSFB-1 cells did not result in a significant increase in apoptotic cell number. After serum deprivation, significant increases in caspase-3 proteolytic activities were detected in both KFB lines compared with both FSFB lines. In contrast, no significant differences with caspase-8 activity were observed between similarly treated KFB and FSFB lines. Furthermore, serum deprivation-induced apoptosis of KFB-2 cells was significantly inhibited by the caspase-3 inhibitor Ac-Asp-Glu-Val-Asp-fluoromethyl ketone (DEVD-FMK), indicating that caspase-3 is important for serum deprivation-induced apoptosis in KFB-2 cells. Considering the role of caspase-3 as a key effector molecule in the execution of apoptotic stimuli, our results suggested that enhanced expression of caspase-3 in hypertrophic scars and keloid induces apoptosis of fibroblasts, which may play a role in the process of pathological scarring.

Effects of glucose on migration, proliferation and tube formation by vascular endothelial cells

SummaryIn order to elucidate the association between hyperglycemia and the vascular complications of diabetes, the effects of high glucose concentrations on the migration, proliferation and tube formation of bovine carotid artery endothelial cells were investigated. Cells treated with 16.7 and 33.3 mM glucose for 6 days showed 1.69- and 1.75-fold increase in serum-induced migration compared with cells treated with 5.6 mM glucose (p<0.05). The effect of glucose on cell proliferation was affected by serum concentration. When this was below 0.5%, a high glucose concentration stimulated cell growth to a maximum of 1.73 times that at a serum concentration of 0.05% (p<0.01) whereas at a serum concentration of 10%, growth was inhibited (p<0.05). Tube formation was studied by culturing the cells between two layers of collagen gel. Ultrastructurally, tubular structures were composed of one to several endothelial cells containing pinocytotic vesicles and cytoplasmic projections, and linked by junctional complexes. A basal lamina-like structure surrounded the abluminal surface. Treatment of the cells with 16.7 and 27.8 mM glucose for 4 days stimulated tubular elongation 1.85 and 1.71 times, respectively (p<0.01). Other osmogenic molecules such as mannitol and sucrose did not affect tube formation. These data imply that high glucose concentrations mimicking diabetic hyperglycemia may not inhibit the repair of endothelial injury and could act as a stimulator of neovascularization.

A case of basaloid‐squamous carcinoma of the esophagus: Immunohistochemical and ultrastructural studies

A case of basaloid‐squamous carcinoma of the esophagus in an 83 year old man is reported. The esophageal tumor showed a fungating growth at the junction of the middle and lower esophagus and was composed microscopically of submucosal multiple nests with solid and cribriform‐like patterns accompanied with a small focus of squamous cell carcinoma adjacent to the overlying esophageal epithelium. The structural features closely resembled those of basaloid‐squamous carcinoma. The submucosal tumor cells were immunohistochemicaliy positive for epithelial membrane antigen, wide spectral keratin, alpha actin and S‐100 protein. By electron microscopy, the tumor cells had microvilli, des‐mosomes and bundles of myofilaments, and replicated basement membranes were frequently observed adjacent to the nests. The positive immunoreaction of S‐100 protein and alpha actin and the existence of bundles of myofilaments indicated that the present tumor did not correspond well with basaloid‐squamous carcinoma. In addition, there was no evidence of true glandular lumina in the tumor nests, a finding which was inconsistent with that of adenoid cystic carcinoma. From the immunoreactivity of S‐100 protein and ultrastructural features, it was considered that the present submucosal tumor had originated from undifferentiated pluripotential primitive cells, which differentiated to myoepithelial cells.

AN ACTH-PRODUCING PITUITARY CARCINOMA DEVELOPING CUSHING'S DISEASE

An autopsy case of an ACTH-producing pituitary carcinoma in a 59-year-old man who developed Cushings disease is reported. The surgically removed pituitary tumor was diagnosed as chromophobe adenoma, however, pulmonary metastases appeared 2 years after the operation. Autopsy revealed a residual pituitary tumor in the sella turcica with systemic metastases to the lungs, liver, pulmonary lymph nodes, hypothalamus, dura mater, and the subarachnoid space of the midbrain and spinal cord. Immunohistochemistry revealed ACTH positivity in the tumor cells. Further immunohistochemical study showed positive high expression of Ki-67 in the tumor removed at surgery as well as in the autopsied tumor. Ki-67 labeling index provided valuable information about the invasive and proliferative potential compared to noninvasive benign pituitary adenoma.