Objective To investigate the pathogenetic mechanisms leading to the development of calcific degeneration of the aortic valve in the elderly patients with particular reference to the relationship between apoptosis and calcification in the aortic valve tissue.Methods High resolution scanning and transmission electron microscopic observations of the calcified aortic valves obtained during aortic valve replacement were carried out in 10 patients with senile calcific aortic stenosis.Results　Various degrees of endothelial alterations from focal disruption of individual endothelial cells to extensive denudation of entire endothelium were observed particularly on the aortic side of the valve tissues. The apoptotic changes occurring in the nuclei of endothelial cells and fibroblasts were common findings in the calcified valve tissues. It was noteworthy that the severity of endothelial damage was closely related to apoptotic changes of the fibroblasts. Calcific deposits were frequently observed in association with the cellular fragments mainly derived from the apoptotic fibroblasts.Conclusions　Our results strongly indicate that apoptosis may play an important role in the alterations of endothelial integrity leading to the increased filtration of calcium into the deeper layer of the valve tissues. Then, the cellular degradation products and organelles extruded from the dead cells, mainly resulted from apoptosis provided the substrates for calcium binding with progressive development of calcification in the valve tissue. Although the role of apoptosis in contribution to the pathogenesis of senile calcific aortic stenosis is evident, further studies using modern molecular biotechnology are mandatory in order to clarify the mechanism for the initiation of apoptotic process in the endothelial cells and fibroblasts.

Objective To characterize ultrastructurally and biochemically catecholamine release mechanisms of cultured human pheochromocytoma cells in the basal and stimulated states.Methods The cultured pheochromocytoma cells were prepared from human adrenal pheochromocytoma tumors. Biochemical determinations of catecholamine secretion from the cultured cells were carried out in the basal and stimulated states. Transmission electron microscopy was used to observe the modes of catecholamine release from the cells without and with stimulation by depolarization of the cells with the administration of 50 mmol/L KCl.Results Biochemical determinations consistently showed spontaneous secretion of catecholamines from the cultured cells in the basal state without stimulation. Catecholamine release in a calcium-dependent manner could be enhanced in the cells in response to high extracellular potassium concentration. A series of electron microscopic observations of the cultured cells consistently disclosed the classical exocytotic profiles on the cell surface in the basal state. In addition to abundant increase in the number of classical single exocytosis, compound exocytosis was frequently observed in the stimulated cells. Furthermore, other modes of catecholamine release mechanism involving the formation of pseudopodial and/or tubule-like structures, which were different from classical exocytosis, were often present in the intensely stimulation cells. Conclusions Based on the biochemical and electron microscopic findings, we concluded: (1) classical single exocytosis is considered to be a primary mechanism responsible for spontaneous secretion of catecholamines from the cells in the basal state; (2) compound exocytosis is an essential mechanism for extruding large amounts of catecholamines in the stimulated cells; and (3) other modes of catecholamine release mechanism may operate in the cells in response to intense stimulation. These morphological data may be helpful in explanation of biochemical variability and extreme diversity of clinical manifestatons in patients with pheochromocytoma tumor.

Objective To investigate molecular events of exocytosis in cultured human pheochromocytoma cells with stimulation.Methods The cultured pheochromocytoma cells prepared from human adrenal pheochromocytoma tumor were stimulated for the release of catecholamines by depolarization with the administration of 50 mmol/L KCl. Transmission electron microscopy (TEM) and high resolution scanning electron microscopy (HR-SEM) combined with autoradiography and cytochemistry were used to observe molecular mechanisms of exocytotic release of catecholamines from the stimulated cells labelled with 3H-noradrenaline and the filipin-treated cells.Results TEM and HR-SEM observations of the stimulated cells labelled with 3H-noradrenaline revealed that the initial exocytotic fusion pores even less than 10 nm in diameter in human pheochromocytoma cells can be clearly observed in a single lipid bilayer. Furthermore, HR-SEM examinations of the filipin-treated cells showed that the derangement of the particles of the filipin-sterol complexes (FSCs) in the fused membranes of granule and plasma membranes occurred as the exocytotic fusion pores opened. In addition, the aggreates of the FSCs particles were consistently demonstrated around the openings of the differently sized closing exocytotic pores.Conclusions Based on our results, it is suggested that the rearrangement of the sterol molecules in the fused membranes of granule and plasma membranes plays an important role in the opening and closing mechanisms of exocytotic fusion pores. We hope that morphological data obtained in this study can provide some new insights into the understanding of molecular mechanisms of exocytosis, particularly the opening and closing of exocytotic fusion pores in relation to the distribution of the membrane sterols.

Objective　To investigate the ultrastructural pathological alterations of the microvasculature and nerve fibers in the endomyocardial biopsied specimens of the left ventricular myocardium obtained from patients with idiopathic dilated cardiomyopathy and chronic heart failure.Methods　Transmission electron microscopic observations of endomyocardial biopsied specimens of the left ventricular myocardium were carried out in 10 patients with idiopathic dilated cardiomyopathy and chronic heart failure.Results　Various degrees of ultrastructural pathological alterations in the microvessels and sympathetic nerves in the diseased myocardium were consistently demonstrated in idiopathic dilated cardiomyopathy. In addition, abnormal accumulation of collagen tissue and edematous fluid were often seen in the interspace between myocardial cells and nerve endings and capillaries.Conclusions　Based on the ultrastructural pathological findings in this study, we consider that all the structures forming the muscle cells and the tissues around them, namely the microvessels and nerves may participate in the pathological process in the course of dilated cardiomyopathy. The damage of microvasculature and sympathetic nerves resulting from the underlying disease processes are considered to be an important pathogenetic mechanism responsible for progressive development of myocardial degeneration and dysfunction throughout the course of the disease. It is hoped that our data may provide some insights into the understanding of the role of microcirculation and sympathetic nerves in the etiopathogenesis of dilated cardiomyopathy.

Objective Familial hypertrophic cardiomyopathy (FHCM) is a primary myocardial disease characterized by unexplained ventricular hypertrophy. The application of the techniques of reverse genetics has identified at least five chromosomal loci as the major causes for FHCM in diverse ethnic populations, suggesting substantial genetic heterogeneity for FHCM. Recently, the defective gene loci of two Chinese families with FHCM have been mapped to chromosome 11 and 14q1, respectively. For further understanding of the molecular basis of FHCM in Chinese, we analyzed the linkage between four other Chinese kindreds and DNA markers from chromosome 14q1. Methods Six unrelated Chinese families with FHCM, including two previously reported, were studied. Totally 90 family members were included for analysis. DNA from 80 individuals was extracted and polymerase chain reactions were performed using the primers designed according to the sequences derived from the α and β myosin heavy chain gene. Totally four polymorphisms were studied, including three polymorphic microsatellite sequences and one single strand conformation polymorphism. Genetic linkage analysis were performed using the Linkage program.Results In the six studied families, 39 of the 90 family members were found to be affected diagnosed either by echocardiography or by clinical evaluation. The pattern of inheritance in all six studied families was most consistent with an autosomal dominant trait with a high degree of penetrance. Genetic linkage analysis using polymorphisms on the α and β MHC genes showed a combined maximal lod score of 6.2 for trinucleotide repeat polymorphism AMHC-I 15 at θ=0.00 for three studied families without recombination. Exclusion of linkage to the chromosome 14q1 location was noted in two of three other families with the maximal lod score of -2 or less.Conclusions These results provide further evidence that FHCM in Chinese is genetically heterogeneous. Chromosome 14q1 locus, probably the β myosin heavy chain gene, is important as the molecular basis for FHCM in Chinese.