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Rheological abnormalities and thromboembolic complications in heart disease: spontaneous echo contrast and red cell aggregation.

The role of abnormal rheological changes in the pathogenesis of thromboembolism has received much attention in recent years, especially in the field of cardiology. Such changes are sometimes seen in an echocardiogram as a smokelike haze known as spontaneous echo contrast (SEC). The presence and severity of SEC correlate with dilated cardiac chambers and the incidence of thromboembolic complications. It is caused by increased red cell aggregation and increased fibrinogen levels, both of which are known risk factors for thrombosis. Although not used clinically, measurements of red cell aggregation can be made in research settings. This can provide findings that give insight into factors causing increased red cell aggregation. A small series of patients with angina pectoris was studied with the Myrenne aggregometer for red cell aggregation. The results, which show correlation between the plasma fibrinogen and triglyceride levels, are presented. As yet, there are only a few therapeutic guidelines for the correction of abnormally high fibrinogen levels in patients at risk.

Blood Viscosity↗

Electrical activity in embryonic heart cell aggregates. Developmental aspects.

Action potential parameters were measured in beating heart cell aggregates which were formed from trypsin-dissociated cells of embryonic chick heats aged 2 1/2, 4 or 7 days. 1. In aggregates composed of cells from the whole heart there was an increase in the maximum diastolic potential, overshoot, maximum rate of rise of the action potential (V max), and action potential duration between days 2 1/2 and 7. 2. Action potential parameters from 4- or 7-day aggregates composed exclusively of atrial or ventricular cells were similar to those in whole heart aggregates of the same age with the exception of the action potential duration in which atrial less than whole heart less than ventricular. Between days 4 and 7 the increases in duration were approximately 14% in atrial, 35% in whole heart, and 50% in ventricular aggregates. Differences in action potential duration, within or between ages, were not due solely to differences in the rate of beating. 3. Action potentials in whole heart aggregates aged 2 1/2 days were insensivitive to TTX (10-5 g/ml) but abolished by D600 (1 MUG/ML). Conversely, at 7 days activity was suppressed by TTX (2 X 10-8 G/ML) WHILE D600 (1 mug/ml) shortened the action potential duration and reduced the overshoot without influencing V max. 4. Adrenaline (1 mug/ml) restored the action potential overshoot and duration in 7-day aggregates treated with D600. 5. Action potential development in embryonic heart cells appears to be characterized by the functional appearance of fast inward channels. The slow channel mechanism, previously utilized in action potential generation, may gradually assume its adult role of carrying inward current during the plateau phase. 6. In contrast to monolayer cultures, embryonic heart cells cultured in aggregate form seem to have membrane properties similar to those of intact tissue.

Action Potentials↗

The intracellular domain of cadherin-11 is not required for the induction of cell aggregation, adhesion or gap-junction formation.

The cadherin family of cell adhesion molecules demonstrates calcium-dependent homophilic binding, leading to cellular recognition and adhesion. The adhesion mediated by the classical type I cadherins is strengthened through catenin-mediated coupling of the cytoplasmic domain to the cytoskeleton. This cytoskeletal interaction may not be essential for the adhesion promoted by all cadherins, several of which lack cytosolic catenin-binding sequences. Cadherin-11, a classical cadherin, possesses a cytoplasmic domain that interacts with catenins, but may also occur as a variant form expressing a truncated cytoplasmic domain. To study the role of the cytoplasmic sequence in cadherin-11 mediated adhesion we have constructed and expressed a truncated cadherin-11 protein lacking the cytoplasmic domain and unable to bind beta-catenin. Expression of the truncated cadherin-11 in MDA-MB-435S human mammary carcinoma cells reduced their motility and promoted calcium-dependent cell aggregation, frequent cell contacts, and functional gap-junctions. We conclude that the intracellular catenin-binding domain of cadherin-11, and by inference cytoskeletal interaction, is not required for the initiation and formation of cell adhesion.

Blotting, Western↗

Infection of human neural cell aggregate cultures with a clinical isolate of cytomegalovirus.

Human neural cell aggregate cultures were prepared from dissociated fetal brain tissue and maintained in rotation culture. After 35 days in culture, aggregates had the histologic appearance of dense, immature, neural cells in a tightly packed neuropil. Electron microscopy revealed ultrastructural features suggestive of immature neurons and neuroglia. In addition, neuron-specific enolase and glial fibrillary acidic protein associated with radial glial cells were detected within the aggregates by immunoperoxidase staining. When infected with a laboratory-adapted strain of cytomegalovirus (CMV), [AD169], cells containing large, bizarre, nuclei and CMV-induced intranuclear inclusion bodies were dispersed throughout the aggregates at 16 days postinfection. In situ hybridization using a CMV-specific DNA probe and electron microscopy confirmed the presence of virus sequences as well as virus particles at histologic sites of cytopathology. In sharp contrast, aggregate cultures infected with a CMV strain recovered from the retina of an acquired immune deficiency syndrome (AIDS) patient with CMV retinitis and encephalitis displayed distinct foci of cytopathology at 23 days postinfection, a pattern not observed in CMV [AD169]-infected aggregates. Our findings suggest that human neural cell aggregates represent a a promising multicellular non-neoplastic culture system in which to study the replication of human neurotropic viruses within neural tissue.

Brain Diseases↗

Benign nevus cell aggregates in the thymus: a case report.

Aggregates of benign nevus cells occurring in lymph nodes are a well-described incidental finding. Nevus cell aggregates (NCAs) can mimic foci of metastatic carcinoma or other disease processes, so the surgical pathologist should be familiar with this lesion. The purpose of this report is to describe the potential diagnostic difficulties created by benign NCAs within the thymus of a 32-year-old man with dysplastic nevus syndrome and malignant melanoma involving mediastinal lymph nodes and the right lung. Morphologically, the NCAs in this case elicited the differential diagnoses of metastatic melanoma and thymoma. Immunohistochemical studies helped to establish the correct diagnosis by demonstrating reactivity for S-100 protein and negative staining for keratin and HMB-45. Unlike malignant melanomas, NCAs show no p53 protein immunoreactivity, and low proliferative activity was detected by Ki-67 antigen immunostaining. Although melanocytic cells were rarely reported in thymic neoplasms, we are not aware of any previous reports of NCAs occurring in the normal thymus.

Adult↗

Red blood cell aggregation in experimental sepsis.

Red blood cell (RBC) aggregation was investigated in a rat model of sepsis with special emphasis on RBC-related factors. Sepsis was produced by cecal ligation/puncture, whereas another group had only laparotomy (sham operation); blood samples also were obtained from control, unoperated-on animals. RBC aggregation was measured in autologous plasma and in 3% dextran 70, 18 hours after the operations, by using a Myrenne Aggregometer system and the zeta sedimentation ratio (ZSR) method. RBC aggregation in autologous plasma was found to be enhanced in both sham-operated and septic animals and was consistent with their increased plasma fibrinogen levels. However, RBC aggregation in dextran was significantly higher than control only in the sepsis group. RBCs from septic animals also aggregated more in septic plasma compared with RBCs from control animals. In the sepsis group, RBC deformability was significantly decreased, whereas RBC lipid peroxidation was significantly increased. Our results thus confirm the known increase of RBC aggregation in septicemia and, in addition, demonstrate marked alterations of intrinsic RBC properties that further enhance red cell aggregation.

Animals↗

The topology of phase response curves induced by single and paired stimuli in spontaneously oscillating chick heart cell aggregates.

The topological properties of the phase resetting of biological oscillators by an isolated stimulus delivered at various phases of the cycle depend on whether the stimulus is "weak" or "strong." When multiple stimuli are delivered to the oscillator, the response to stimulation also depends on the time between the stimuli, and the rate at which the oscillator returns to an underlying limit cycle attractor. If the time between two consecutive "weak" stimuli is sufficiently short, the effects produced by the pair of stimuli may be characteristic of a single "strong" stimulus. These results are demonstrated in a model experimental system, spontaneously beating aggregates of cells derived from embryonic chick heart, and are illustrated by consideration of a simple theoretical model of nonlinear oscillators, the Poincaré oscillator.

Animals↗

Effect of cell aggregation on intravenous tumor transplantation.

We have studied how the presence of cell aggregates affects the pattern of intravenously induced "experimental metastases" from two syngeneic murine tumors. Aggregates were produced mechanically by centrifugation, or chemically by a lectin (wheat-germ agglutinin). Compared to well-dissociated suspensions, aggregated suspensions tended to give a greater total metastasis volume in the lungs of recipient mice. Disaggregated suspensions, on the other hand, gave rise to more extrapulmonary metastases. Presumably, aggregates are preferentially retained in lung vessels, while single cells are let through to other sites. Nor are aggregates superior to single cells in producing tumor growth when the total metastasis yield is considered.

Abdominal Neoplasms↗

Transient expression of syndecan in mesenchymal cell aggregates of the embryonic kidney.

Induction of the embryonic kidney mesenchyme is followed by formation of cell aggregates which subsequently transform into epithelial tubules. Syndecan, which binds various matrix components and growth factors, is a candidate molecule to be involved in this process. We have analyzed the changes in the expression of syndecan during tubule morphogenesis by using in situ hybridization and slot-blot analysis. The expression pattern of syndecan was compared with the distribution of cell proliferation analyzed by immunohistochemistry. Furthermore, the expression of syndecan during formation of the pretubular aggregates was studied in hanging-drop cultures of experimentally induced mesenchymal cells. Syndecan mRNA was expressed in the metanephric mesenchyme prior to induction, was intensely present during formation of the pretubular cell aggregates, but was lost during maturation of the nephron. Slot-blot analyses of the kidney mesenchymes (11-day kidney) cultured in a transfilter situation with a heterotypic inductor tissue that triggers a complete tubulogenic program in the nephric mesenchyme during the first 24 hr suggested the presence of syndecan mRNA in the uninduced mesenchymes with no change during induction. Expression of mRNA was stimulated later (13-day kidney) followed by subsequent decrease. Immunoisolation of sulfate-labeled syndecan, however, revealed a marked stimulation in the induced kidney mesenchyme during the first 24-hr inductive period when the DNA level still remained constant. In hanging-drop cultures where either induced or uninduced mesenchymal cells were dissociated and reaggregated, syndecan was detected only in the induced and aggregating mesenchymal cells. Double-immunostaining demonstrated a close correlation between syndecan expression and cell proliferation analyzed by bromodeoxyuridine incorporation. Thus, it appears that syndecan expression in the mesenchyme is initially induced post-transcriptionally and later during differentiation at the mRNA level. Syndecan may have a dual function during early kidney morphogenesis; it may be involved in cell aggregation through its adhesive properties, and it may contribute to proliferation of the induced mesenchymal cells by binding growth factors.

Animals↗

Effect of ionic and non-ionic contrast media on red cell aggregation in vitro.

The effect of solutions of the ionic contrast media diatrizoate, iocarmate, and metrizoate and the non-ionic metrizamide on red cell aggregation in vitro was examined. The aggregation was recorded by both microphotography and photometry in a counter-rotating rheoscope chamber. All the contrast media decreased the formation of red cell aggregates. This desaggregating ability increased with both increasing volume ratio (contrast media/blood) and with increasing osmolality of the contrast media. The desaggregating effect was also obtained with the contrast media solutions isotonic with blood. The iocarmate and diatrizoate solutions induced less reduction in red cell aggregation than the metrizoate and metrizamide solutions.

Contrast Media↗

The receptor tyrosine kinase ARK mediates cell aggregation by homophilic binding.

The ARK (AXL, UFO) receptor is a member of a new family of receptor tyrosine kinases whose extracellular domain contains a combination of fibronectin type III and immunoglobulin motifs similar to those found in many cell adhesion molecules. ARK mRNA is expressed at high levels in the mouse brain, prevalently in the hippocampus and cerebellum, and this pattern of expression resembles that of adhesion molecules that are capable of promoting cell aggregation through homophilic or heterophilic binding. We report here the ability of the murine ARK receptor to mediate homophilic binding. Expression of the ARK protein in Drosophila S2 cells induces formation of cell aggregates consisting of ARK-expressing cells, and aggregation leads to receptor activation, with an increase in receptor phosphorylation. Homophilic binding does not require ARK tyrosine kinase activity, since S2 cells expressing a receptor in which the intracellular domain was deleted were able to undergo aggregation as well as cells expressing the wild-type ARK receptor. Similar results were obtained with NIH 3T3 and CHO cells expressing high levels of ARK, although in this case ARK expression appeared to be accompanied by constitutive activation. The purified recombinant extracellular domain of ARK can induce homotypic aggregation of coated fluorescent beads (Covaspheres), and this protein can also function as a substrate for adhesion by S2 and NIH 3T3 cells expressing ARK. These results suggest that ARK represents a new cell adhesion molecule that through its homophilic interaction may regulate cellular functions during cell recognition.

3T3 Cells↗

The function and distinctive regulation of the integrin VLA-3 in cell adhesion, spreading, and homotypic cell aggregation.

To assess directly the functional role of the integrin VLA-3 (alpha 3 beta 1), we transfected human alpha 3 cDNA into erythroleukemia (K562) cells and rhabdomyosarcoma (RD) cells. The resulting transfectants (KA3 and RA3) expressed alpha 3 beta 1 on the cell surface as confirmed using a panel of nine anti-alpha 3 monoclonal antibodies. Neither of the transfected cells exhibited increased adhesion to the extracellular matrix proteins fibronectin, laminin, and collagen. However, the KA3 transfectants did bind strongly to the extracellular matrix deposited by epidermal and carcinoma cell lines, allowing the cells to attach and spread. Binding to this cell-deposited ligand, probably containing epiligrin/kalinin, was specific to VLA-3 and could be inhibited by anti-alpha 3 antibodies and by EDTA, but not by RGD peptides. In marked contrast to other integrins (VLA-2 and VLA-4), VLA-3 showed high constitutive activity in K562 cells, but was minimally active in RD cells. Also contrasting with other beta 1 integrins, VLA-3 was minimally stimulated by the anti-beta 1 monoclonal antibody TS/216 under normal conditions. VLA-3-mediated adhesive function was well supported by either Mg2+ or Mn2+, but was almost completely abolished by the presence of 1 mM Ca2+. Surprisingly, this negative Ca2+ effect was completely overcome by the addition of the stimulatory anti-beta 1 monoclonal antibody TS2/16. Together, these results point to markedly distinct regulation for VLA-3 function compared to other beta 1 integrins. Also, all anti-VLA-3 antibodies were able to induce temperature-dependent homotypic cell aggregation of KA3 cells, but not K562 cells. However, this aggregation did not appear to be directly mediated by VLA-3 since it was not inhibited by EDTA. In addition, no enhancement of heterotypic cell-cell adhesion was observed in alpha 3-transfected cells.

Animals↗

Study of red cell aggregation in pulsatile flow from ultrasonic Doppler power measurements.

Human red cell aggregability and disaggregability represent important hemorheological parameters of blood. Several techniques have been proposed to evaluate the tendency of red cells to form aggregates and to disrupt in the presence of shear stress. One of the most recent approaches is based on the characterization of the intensity of ultrasonic scattered signals. A pulsatile flow loop model is used in the present study to demonstrate the potential applicability of Doppler ultrasound to detect and characterize the hemodynamic behavior of red cell aggregates. Porcine whole blood specimens collected from 20 different pigs were circulated in the flow model (tube diameter of 0.476 cm) at different mean velocities and pulsation rates. At a pulsation of 70 beats/min for mean velocities of 13 cm/sec and 63 cm/sec, no cyclic variation of the Doppler power was observed, suggesting the absence of rouleaux build-up and rouleaux disruption. At a pulsation of 20 beats/min and mean velocities of 11 cm/sec and 38 cm/sec, statistically significant cyclic variations (p < 0.01) were measured. It is suggested that aggregate size enlargement, rouleaux orientation with the flow field and the effect of shear stress on rouleaux disruption are possible causes for the observed cyclic variation of the Doppler power within the flow cycle at a pulsation of 20 beats/min. A discussion of the potential application of this technique for in vivo study in large vessels is given.

Animals↗

[The red blood cell aggregation in men with coronary risk factors].

1. The in vitro test for red blood cell aggregation has been improved, the coefficient of variation was decreased to 14.2%. 2. By the improved method the earlier findings were confirmed with high statistical significance that heavy smokers have a significantly higher red blood cell aggregation value in comparison with nonsmokers. 3. It could be shown that other coronary risk factors, especially hypercholesterolaemia, increase the aggregation value too. 4. The aggregation of red blood cells increases in the presence of several coronary risk factors in the same patient to higher values than expected from the addition of the aggregations caused by single risk factors.

Erythrocyte Aggregation↗