Apoptosis regulates human vascular calcification in vitro: evidence for initiation of vascular calcification by apoptotic bodies.

The mechanisms involved in the initiation of vascular calcification are not known, but matrix vesicles, the nucleation sites for calcium crystal formation in bone, are likely candidates, because similar structures have been found in calcified arteries. The regulation of matrix vesicle production is poorly understood but is thought to be associated with apoptotic cell death. In the present study, we investigated the role of apoptosis in vascular calcification. We report that apoptosis occurs in a human vascular calcification model in which postconfluent vascular smooth muscle cell (VSMC) cultures form nodules spontaneously and calcify after approximately 28 days. Apoptosis occurred before the onset of calcification in VSMC nodules and was detected by several methods, including nuclear morphology, the TUNEL technique, and external display of phosphatidyl serine. Inhibition of apoptosis with the caspase inhibitor ZVAD.fmk reduced calcification in nodules by approximately 40%, as measured by the cresolphthalein method and alizarin red staining. In addition, when apoptosis was stimulated in nodular cultures with anti-Fas IgM, there was a 10-fold increase in calcification. Furthermore, incubation of VSMC-derived apoptotic bodies with (45)Ca demonstrated that, like matrix vesicles, they can concentrate calcium. These observations provide evidence that apoptosis precedes VSMC calcification and that apoptotic bodies derived from VSMCs may act as nucleating structures for calcium crystal formation.

Sensitivity to Fas-mediated apoptosis is determined below receptor level in human vascular smooth muscle cells.

Despite Fas expression, many cells resist Fas-induced apoptosis. Although differences in surface Fas expression can explain Fas resistance, multiple proteins below receptor level also inhibit Fas-induced apoptosis. To examine the mechanism of Fas resistance, we studied Fas-induced apoptosis in human medial vascular smooth muscle cells (VSMCs) from healthy coronary arteries. VSMCs showed marked heterogeneity to Fas-induced apoptosis, exhibiting both Fas-resistant (98.1+/-2.3% viable, n = 4, P = NS) and Fas-sensitive (31.3+/-2.6% viable, n = 3, P<0.01) cells. Fas-resistant VSMCs expressed surface Fas and could recruit RIP, indicating that functional receptor complexes were formed. However, Fas-resistant cells showed reduced expression of FADD, Fas ligand, and caspases 3, 7, and 8 and increased expression of FLIP and c-IAP-1. Fas-induced apoptosis was associated with cleavage of caspase 3 and blocked by inhibitors of caspase 3 or 8 but not caspase 1, 6, or 7. Selective inhibition of caspase 3 or 8 by antisense transfection inhibited Fas-induced apoptosis, but their reexpression could not rescue the Fas-resistant phenotype. In vivo, medial VSMCs showed marked heterogeneity of expression of caspase 3. We conclude that Fas sensitivity is determined not only by expression of surface Fas but by differential expression of Fas-signaling proteins below receptor level. Subpopulations of cells within the same tissue have different sensitivities to apoptosis, determined by expression of specific death-signaling proteins.

Sex-related and age-related differences in platelet function in vitro: influence of hematocrit.

Even minor changes in the sodium citrate concentration of citrated plasma from young probands due to diverse hematocrit values were found to affect platelet functional activity following in vitro stimulation. There were no differences between men and women in the platelet aggregation and TXB2 production of young probands with similar hematocrit values. Platelets from elderly persons virtually failed to respond to such changes in plasma sodium citrate. Mechanisms of increased platelet activity in the elderly are discussed. It is assumed that the age-related differences are caused by a more pronounced platelet response even to lower plasma calcium levels in citrated plasma.

Histochemical investigation into the molecular mechanisms of malignant transformation in a benign glomus tumour.

A glomangiosarcoma arose in a benign glomus tumour. The histological and immunohistochemical characteristics of the tumour were investigated. Apoptotic cells were identified by terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end labelling (TUNEL). The proportion of apoptotic cells was found to be low and TUNEL positive nuclei were present in the benign part of the tumour. Bcl-2 protein, an inhibitor of apoptosis, was strongly expressed in the glomangiosarcoma with only weak staining in the benign area. The proliferation index of the glomangiosarcoma was almost 10-fold higher than that of the benign glomus tumour. Numerous nuclei in the glomangiosarcoma were intensely stained for the tumour suppressor protein p53. The results of the this study may contribute to an understanding of the molecular basis of malignant transformation in benign glomus tumours.

[Antiphagocytic activity of sera treated with organic solvents (chloroform, ether)]. / Antifagocytárna aktivita sér opracovaných organickými rozpúst'adlami (chloroform, éter).

The opsonization of C. albicans by means of sera treated with chloroform and ethyl ether resulted in significantly decreased values of PMN leucocyte phagocytic activity. In contrast to ethyl ether treated sera (decrease of phagocytic activity about 45%), chloroform processed sera possessed increased antiphagocytic activity (decrease of phagocytic activity about 63%). Significant changes were observed in the candidacidal capacity to C. albicans (decrease about 50%) and in the capacity of NBT (INT) reduction measured spectrophotometrically (decrease about 66% in chloroform treated sera and about 42% in ethyl ether treated sera). (Fig. 1, Ref. 22.)

[Changes in the capacity of T-lymphocytes for spontaneous recovery of selected differentiation antigens in relation to age]. / Zmena schopnosti spontánnej znovuobnovy vybraných diferenciacných antigénov T-lymfocytov v závislosti od veku.

BACKGROUND: During physiological ageing changes of the immune system take place at several levels. The objective of the submitted work was to compare the ability of spontaneous restoration of selected differentiation antigens on lymphocytes in the peripheral blood stream after previous trypsin treatment in a group of healthy elderly and adult subjects. METHODS AND RESULTS: Twenty-four adults were examined (19-59 years) and 36 elderly subjects (60-90 years). Isolated lymphocytes from the peripheral blood stream were treated with trypsin and then incubated in a cultivation medium. The authors investigated the capacity of restoration of differentiation antigens CD2, CD4, CD8 and CD45RA. Antigen CD2 was not restored in any of the investigated groups to original levels. However the difference between its expression on lymphocytes before trypsin treatment and on lymphocytes after 16-hour incubation was higher in the elderly subjects 16% (p < 0.001) than in the group of adults 7% (p < 0.01). Restoration of antigen CD4 was in both investigated groups almost equal. The number of CD8+ T-lymphocytes was in elderly people lower (p < 0.05), spontaneous restoration of antigen CD8 did not differ among the investigated groups and reached in both instances the baseline value. Antigen CD45RA was restored more slowly in elderly subjects, the difference between groups was at borderline of statistical significance (p < 0.0595). CONCLUSION: From the results ensues that during physiological ageing the ability of spontaneous restoration of antigens CD2 and CD45RA declines but not of antigens CD4 and CD8. So far there is no unequivocal explanation why this change occurs, it is probably conditioned by several factors. Investigation of these changes and an attempt to influence them can help to understand age-conditioned immunological dysregulation, its consequences and the possibility to influence them by treatment.

[Arrhythmogenic right ventricular dysplasia. Case report and review of the literature]. / Arrhythmogen jobb kamrai dysplasia. Esetismertetés és irodalmi áttekintés.

Two cases of arrhythmogenic right ventricular dysplasia are described. The most important clinical features of the disease are malignant ventricular arrhythmia and sudden death. It is characterized by the loss of right ventricular musculature and by the fatty and connective tissue infiltration of the right ventricular wall. The diagnosis is based on the typical echocardiographic appearance of right ventricular dilatation, on the presence of negative T waves in leads V1-4 on the resting ECG and on ventricular tachycardia of left bundle branch block pattern. Right heart failure develops only in the late phase of the disease. Genetic defect might be an etiologic factor. In conclusion authors suggest that in case of left bundle branch block ventricular tachycardia or Adams-Stokes syndrome in young adults echocardiography and family screening are necessary.

[Geriatric maladaptation syndrome]. / Geriatrický maladaptacný syndróm.

Geriatric maladaptation syndrome is a demonstration of adaptation failure, which is typical for higher age. This failure occurred on the basis of chronic stress caused usually by relevant psychosocial stimulus, with clinical manifestation mainly in cardiovascular or immune systems followed with serious threaten of health and life. A lost of independence, loneliness, change of dwelling-place and living standard, low income, stages of confusion, mental disease, passive life attitude belong to psychosocial risks of the origin of geriatric maladaptation syndrome. Higher age and bad health status belong to biological risks of the origin of geriatric maladaptation syndrome. Clinical image of geriatric maladaptation syndrome has three phases (1. development of stress reaction, 2. full clinical image development of adaptation failure, 3. followed phase of adaptation reaction). It is a set of somatic disorders, which threaten a life of old people. These disorders arose in a consequence of inadequate adaptation to stress life event. Geriatric maladaptation syndrome is significantly different from mentioned adaptation disorders because of its clinical course and frequently unfavourable prognosis. In international classification of diseases it should have an independent item G 43.8.

Malignant myoepithelioma arising in adenomyoepithelioma of the breast and coincident multiple gastrointestinal stromal tumours in a patient with neurofibromatosis type 1.

A 41-year-old female patient with neurofibromatosis type 1 (NF-1) presented with a breast lump and anaemia related to gastrointestinal bleeding. She was found to have malignant myoepithelioma of the breast and simultaneously multiple gastrointestinal stromal tumours (GISTs) of the small bowel. Molecular studies showed a silent germline mutation in exon 9 of the KIT gene of both tumours. The common gene mutations characteristic of sporadic GISTs were not identified in these tumours, consistent with the literature, suggesting that gene mutations in GISTs are either absent or late events in patients with NF-1.

Cell death in perinatal hypoxic-ischaemic brain injury.

Perinatal hypoxic brain injury is a major cause of death and morbidity, in which the onset of injury can be prenatal, and the effects may be delayed. Selective neuronal necrosis, with isolated karyorrhectic nuclei in the pons, is a common pattern of injury in mature perinatal deaths. Other evidence implicates apoptosis in hypoxic brain injury. In this study the mode of cell death in hypoxic injury was investigated in 11 fresh stillbirths and 10 neonatal deaths. Sections of pons were stained using several methods including terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) and immunocytochemistry. Karyorrhectic nuclei were counted on adjacent haemotoxylin and eosin sections. A high percentage of apoptotic cells was significantly associated with the presence of karyorrhexis in the pons, but there were five stillbirths in whom apoptosis in the pons was the sole evidence of hypoxic brain injury. PCNA positive neuronal nuclei were seen in 19 out of 21 cases. The results suggest that both apoptosis and necrosis are occurring following hypoxic injury, so that the pattern of injury in the pons may be better termed 'selective neuronal death'. Variations in severity and duration of the insult might explain the differences between cases. The presence of PCNA-positive neurons may suggest DNA repair in these nuclei, which might be activated at an early stage of apoptosis. However the precise mechanism by which apoptosis is induced in hypoxic brain injury remains to be elucidated.

Complete recovery from paraquat poisoning causing severe unilateral pulmonary lesion.

Poisoning by paraquat, a plant-protecting agent, its clinical manifestations and treatment are discussed. The case of a 5-year-old boy who had ingested an unknown quantity of paraquat is described. Peritoneal dialysis proved to be effective in overcoming renal and hepatic failure. Subsequently, a pulmonary lesion with unilateral preponderance developed; this showed marked radiological regression and in a year nearly complete functional recovery ensued.

Macrophage death and the role of apoptosis in human atherosclerosis.

The arterial disease atherosclerosis is responsible for severe morbidity and is the most common cause of death in the Western population. The complete pathogenesis of the disease is unknown, but multiple risk factors have been identified that correlate with the development of its complications such as heart attack and stroke. Evidence suggests that atherosclerosis is an inflammatory disease and the major cell types involved are smooth muscle cells, macrophages, and T lymphocytes. In this paper, we review the function of macrophages in the context of atherosclerosis and we also discuss the role and significance of macrophage death, including apoptosis. There is much evidence, certainly in vitro, suggesting that low-density lipoprotein becomes atherogenic when it undergoes cell-mediated oxidation within the artery wall. Besides inducing apoptosis in vitro, oxidized low-density lipoprotein may also cause extensive DNA damage in intimal cells, which might presage apoptosis. We review the results of experimental and clinical studies, which may indicate how the complications of atherosclerosis could be prevented by using different therapeutical strategies including bone marrow transplantation and gene therapy.

Foam cell apoptosis and the development of the lipid core of human atherosclerosis.

A characteristic feature of the advanced atherosclerotic lesion is the acellular lipid core, which appears to result at least partly from the death of macrophage foam cells. This study shows that foam cell death at the edge of the lipid core includes both necrosis and apoptosis and that remnants of apoptotic nuclei are present within the lipid core. Apoptotic cells were identified by transmission electron microscopy and by nick end-labelling using terminal deoxynucleotidyl transferase (TUNEL). Some TUNEL-positive cells also expressed proliferating cell nuclear antigen (PCNA). The cause of foam cell death in atherogenesis is unknown, but oxidized low-density lipoprotein (LDL) can cause macrophage apoptosis in vitro and might therefore play a role in the formation and enlargement of the lipid core.

The role of apoptosis in the initiation of vascular calcification.

The initiation sites for calcification in cartilage and bone are cellular products called matrix vesicles. Similar structures have been found in calcified arteries and recent studies suggest that these may be derived from apoptotic cells. It is well established that there is a link between cell death and calcification but the mechanism involved is not known. Since apoptotic cell death is known to occur in the vasculature, we set out to investigate the role of apoptosis in the initiation of vascular calcification. We used a human vascular calcification model in which postconfluent vascular smooth muscle cell (VSMC) cultures form nodules spontaneously and calcify after approximately 28 days. Our studies revealed that apoptosis occurred prior to the onset of calcification and that VSMC "blebs" or apoptotic bodies (ABs) could concentrate calcium in a crystallised form. These observations suggest that apoptosis is involved in the development of VSMC calcification and that VSMC-derived ABs have similarities with matrix vesicles.

Nesprins: a novel family of spectrin-repeat-containing proteins that localize to the nuclear membrane in multiple tissues.

In search of vascular smooth muscle cell differentiation markers, we identified two genes encoding members of a new family of type II integral membrane proteins. Both are ubiquitously expressed, and tissue-specific alternative mRNA initiation and splicing generate at least two major isoforms of each protein, with the smaller isoforms being truncated at the N-terminus. We have named these proteins nesprin-1 and -2 for nuclear envelope spectrin repeat, as they are characterized by the presence of multiple, clustered spectrin repeats, bipartite nuclear localization sequences and a conserved C-terminal, single transmembrane domain. Transient transfection of EGFP-fusion expression constructs demonstrated their localization to the nuclear membrane with a novel C-terminal, TM-domain-containing sequence essential for perinuclear localization. Using antibodies to nesprin-1, we documented its colocalization with LAP1, emerin and lamins at the nuclear envelope, and immunogold labeling confirmed its presence at the nuclear envelope and in the nucleus where it colocalized with heterochromatin. Nesprin-1 is developmentally regulated in both smooth and skeletal muscle and is re-localized from the nuclear envelope to the nucleus and cytoplasm during C2C12 myoblast differentiation. These data and structural analogies with other proteins suggest that nesprins may function as 'dystrophins of the nucleus' to maintain nuclear organization and structural integrity.

Apoptosis in human monocyte-macrophages exposed to oxidized low density lipoprotein.

This study has demonstrated the toxicity to human monocyte-macrophages of low-density lipoprotein (LDL) which had been artificially oxidized using copper sulphate. The assays of cell damage used were tritiated adenine release, neutral red staining, lactate dehydrogenase leakage, and MTT dye reduction. Toxicity was concentration- and time-dependent. Exposure to native LDL under the same conditions did not result in toxicity. Transmission electron microscopy of cells exposed to oxidized LDL showed characteristic changes of apoptosis, including chromatin condensation and a decrease in cell volume. There was extensive loss of cell surface protrusions and evidence of the phagocytosis of apoptotic cells by neighbouring monocyte-macrophages. Apoptotic features preceded the increased membrane permeability revealed by the release of radioactivity from cells preloaded with tritiated adenine and by lactate dehydrogenase leakage. DNA fragmentation was indicated by nick end-labelling using the terminal transferase enzyme (TUNEL). The number of TUNEL-positive cells was markedly greater in cells exposed to oxidized LDL, compared with those incubated as no-additions controls. Inhibition of de novo protein synthesis with cycloheximide and of Ca2+/Mg(2+)-activated endonuclease activity with aurintricarboxylic acid or zinc ion did not inhibit the toxicity produced by oxidized LDL.

Macrophages, lipid oxidation, ceroid accumulation and alpha-tocopherol depletion in human atherosclerotic lesions.

Necropsy samples of atherosclerotic lesions of different histological stages have been analysed. Ceroid was present in all the lesions, within lipid-laden macrophage foam cells and extracellularly in the atheromatous core of advanced lesions. Mean levels of 7 beta-hydroxycholesterol, 26-hydroxycholesterol and hydroxyoctadecadienoic acids were all significantly greater in lesions than in normal intima. Levels of hydroxycholesterols were very low or undetectable in normal intima. Fatty streaks showed the highest ratio of 7 beta-hydroxycholesterol to cholesterol, and the lowest ratio of linoleate to oleate, suggesting that this type of lesion experiences the greatest free radical activity. Levels of 26-hydroxycholesterol, a product of the cytochrome P-450 enzyme sterol 26-hydroxylase, and the ratio of 26-hydroxycholesterol to cholesterol were significantly higher in advanced lesions than in intermediate lesions or fatty streaks. The ratio of alpha-tocopherol to cholesterol levels varied widely in normal intima but was consistently low in lesions, especially those rich in macrophage foam cells, suggesting that oxidative activity in the lesion may lead to significant oxidation of the lesion constituents only after alpha-tocopherol has been depleted. Macrophage death was a characteristic feature of advanced lesions, with apoptotic bodies present, and occasionally, intact apoptotic cells were seen in lesions. These striking correlations between macrophages, lipid oxidation, alpha-tocopherol depletion, ceroid accumulation, and macrophage death in advanced lesions, strongly support a role for oxidative damage in atherosclerosis, and lend credence to the idea that alpha-tocopherol dietary supplementation may slow the progression of atherosclerosis in humans.

Changes in elemental concentrations are associated with early stages of apoptosis in human monocyte-macrophages exposed to oxidized low-density lipoprotein: an X-ray microanalytical study.

This study examines ion homeostasis in monocyte-macrophages committed to death by apoptosis. X-ray microanalysis has been used to demonstrate that intracellular concentrations of potassium decreased whilst those of sodium increased following 3 h of exposure to 100 microg/ml of oxidized low-density lipoprotein (LDL) in vitro. In contrast, the maximal incidence of cell death, as determined by the inability to exclude trypan blue, was not seen until 24 h of exposure. At 12 h, less than 1 per cent of cells were stained using terminal transferase-mediated DNA nick-end labelling, which is generally accepted as a marker of late stages in the apoptotic pathway. This is the first demonstration of early perturbations of ion homeostasis in monocyte-macrophages exposed to concentrations of oxidized LDL known to cause apoptosis.