Lipid peroxidation--antioxidant activity system in erythrocytes of patients with chronic bronchitis inhaling and not inhaling ozone.

Development of chronic bronchitis in individuals chronically exposed to ozone is associated with complex changes in the lipid peroxidation--antioxidant activity system in erythrocytes characterized by intensification of lipid peroxidation and parallel attenuation of antioxidant activity.

Functional insufficiency of the system responsible for reactive oxygen species generation by blood neutrophils.

We studied functional activity of the system responsible for generation of reactive oxygen species by blood neutrophils and involved in pathophysiological mechanisms of bronchopulmonary diseases. Insufficiency of this system can be classified as relative, latent (type I and II), and severe.

Effects of antibiotics on reactive oxygen species generation by neutrophils.

Antibiotic therapy of patients with exacerbation of chronic obstructive bronchitis exposed and not exposed to ozone did not improve oxidative metabolism in neutrophils. Cefazolin, ceftizoxime, and gentamicin normalized functional biocidal reserves of neutrophils, which correlated with pronounced therapeutic effects.

Expression of cell adhesion molecules and lymphocyte-endothelium interaction under simulated hypogravity in vitro.

Using histochemical staining and FACS-analysis we have studied the basal and TNF-alpha induced expression of E-selectin, ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (ECs) exposed to simulated hypogravity. Control ECs did not contain detectable amounts of E-selectin or VCAM-1 but were ICAM-1 positive. As soon as after 6-8 hrs of clinorotation at 5 RPM the cellular content of ICAM- 1 increased. Moreover, hypogravity potentiated the effect of inflammatory cytokines (TNF-alpha and IL-1) on ICAM-1 expression. No increase in E-selectin or VCAM-1 expression was observed in ECs exposed to hypogravity itself. However, hypogravity reduced E-selectin and VCAM-1 expression in cell cultures activated by cytokines, more visible at their low (5-10 U/ml) concentrations. Both, control and clinorotated ECs poorly supported spontaneous lymphocyte adhesion; the adhesion of PMA-activated leukocytes was 15-20-fold higher. The interaction of unstimulated lymphocytes with cytokine-activated endothelium was more noticeable but significantly lower in cultures exposed to hypogravity. Activated blood cells interacted with endothelium more effectively, particularly, under hypogravity. Obtained results suggest that EC adhesion molecule expression and endothelium-lymphocyte interaction are altered under simulated hypogravity conditions in direction of increase of endotlielial adhesiveness for activated blood cells.

The role of cytoskeleton in cell changes under condition of simulated microgravity.

Single cells and cell culture are very good model for estimation of primary effects of gravitational changes. It is suggested that cell cytoskeleton plays a key role in mechanisms of adaptation to mechanical influences including gravitational ones. Our results demonstrated that cultured cells of human vascular endothelium (correction of endotheliun) are highly sensitive to hypogravity (clinorotation) and respond by significant decrease of cell proliferative activity. Simultaneously it was noted that the formation of confluent monolayer appeared early in cultures exposed to simulated microgravity due to accelerated cells spreading. Long-term hypogravity (several hours or days) leads to significant changes of cell cytoskeleton revealed as microfilament thinning and their redistribution within cell. Such changes were observed only in monolayer cells and not in cell suspensions. Gravitational forces as known to be modificators of cell adhesive ability and determine their mobility. Hypogravity environment stimulated endothelial cell migration in culture: 24-48 hrs pre-exposition to hypogravity significantly increased endothelial cell migration resulting in 2-3-fold acceleration of mechanically injured monolayer repair. Obtained results suggest that the effects of hypogravity on cultured human endothelial cells are, possibly, associated with protein kinase C and/or adenylate cyclase activity and are accompanied by noticeable functional cell changes.

Effect of long-term exposure to ozone on functional activity of human phagocytes.

Functional activity (biocidal properties) of whole blood phagocytes from humans exposed to ozone was studied by measuring spontaneous and zymosan-induced luminol-dependent chemiluminescence. Generation of reactive oxygen species and biocidal properties of phagocytes depended on the time of ozone exposure and development of bronchopulmonary diseases. The data suggest that generation of reactive oxygen species must be assayed for elaboration of individual patient management programs.

Production of soluble P-selectin by platelets and endothelial cells.

The distribution of a soluble form of a cell adhesion molecule, P-selectin, in human platelets and cultivated endothelial cells has been studied by enzyme-linked immunosorbent assay (ELISA). The concentration of soluble P-selectin in the blood plasma of healthy donors and patients with abnormal platelet count has also been determined. P-selectin was measured in the Triton X-100 lysate of platelets and endothelial cells (total P-selectin), in the 100,000g supernatant obtained after sedimentation of the membrane fraction from the homogenate of sonicated platelets and endothelial cells (intracellular soluble P-selectin), in the supernatant of activated and nonactivated platelets, and in the culture medium of endothelial cells. A soluble form of P-selectin which did not coprecipitate with the membrane fraction was detected in platelets and accounted for approximately 10% of the total P-selectin. Platelet activation by thrombin, ADP, or a thromboxane A2 analog resulted in the secretion of 30-50% of the intracellular soluble P-selectin. Measurements of P-selectin in endothelial cell culture revealed that endothelium from aorta contained about twofold more P-selectin than endothelium from umbilical vein. Intracellular soluble P-selectin was identified in both types of endothelial cells. In endothelial cells from the umbilical vein this form made up approximately 10% of the total P-selectin. Soluble P-selectin was also detected in the medium of cultivated endothelial cells, where its content correlated with the total cellular P-selectin. Concentration of P-selectin in blood plasma strongly correlated with the platelet count in the blood of healthy donors and patients with thrombocytosis and thrombocytopenia. These data indicate that platelets serve as one of the main source of plasma P-selectin. However, the presence of P-selectin in the plasma of patients with severe thrombocytopenia suggests that endothelium can also be involved in plasma P-selectin production. Thus, in vitro experiments as well as measurements of plasma P-selectin have shown that both platelets and endothelial cells can produce a soluble form of the protein. Platelet-derived soluble P-selectin and plasma P-selectin were shown to react with antibodies against the cytoplasmic domain of P-selectin. These data prove that at least part of soluble P-selectin is produced by synthesis employing special mRNA which lacks the sequence encoding the transmembrane domain, but not by the proteolytic shedding of the extracellular portion of membrane P-selectin.

Secretion of a novel T-lymphocyte cytokine possessing both chemotactic and growth factor activity by serotonin-stimulated human aortic endothelial cells.

The development of an extralymphatic T-lymphocyte focus of inflammation requires chemoattractant-induced cell migration and growth factor-induced cell proliferation. In a previous study, we identified a novel 13- to 15-kDa T-lymphocyte-specific chemotactic cytokine, endothelial cell-derived lymphocyte chemoattractant activity (ED-LCA), secreted by serotonin-stimulated human aortic endothelial cells. Based on its physicochemical and functional characteristics and antibody inhibition studies, ED-LCA is distinct from previously identified endothelial cell-derived IL-1, IL-6, and IL-8. Because of the association between T-lymphocyte chemotactic and growth factor activity, in the current study, we investigated the effect of ED-LCA on T cell growth by assessing its capacity to induce markers of the passage of T cells from the resting (G0) state into the G1 phase of the cell cycle, such as receptors for IL-2 (IL-2R) and transferrin (TFR), and class II major histocompatibility complex antigens (HLA-DR). Incubation of G0 freshly isolated human T lymphocytes for 48 h with chromatographically resolved, partially purified ED-LCA resulted in a threefold increase in expression of IL-2R, a threefold increase in TFR, and a twofold increase in HLA-DR. Double antibody labeling demonstrated that IL-2R was induced in both CD4+ and CD8+ T cell subsets. Although incubation of human T cells with ED-LCA alone did not induce DNA synthesis, addition of exogenous IL-2 to T cells pulsed with ED-LCA for 24 h caused an increase in DNA synthesis with a stimulation index of 3.5. By up-regulating functional cell surface receptors for IL-2 on T lymphocytes and priming them to respond to exogenous IL-2, ED-LCA is a competence growth factor. By virtue of its effect on T cells, as a chemotactic and competence factor, this endothelial cell-derived mitoattractant could participate with other T-cell growth factors like IL-2 in the generation of an extralymphatic T-lymphocyte inflammatory response.

Interaction of avidin-carrying red blood cells with nucleated cells.

In vivo application of red blood cells (RBC) modified with avidin-biotin complex has been suggested recently for various purposes. However, avidin attachment to RBC alters their biocompatibility. Thus, it has been described that avidin-carrying biotinylated RBC were lysed by the complement. In the present work interaction between avidin-carrying RBC and nucleated cells has been examined. It was found that attachment of avidin, but not streptavidin, to RBC led to binding of avidin-carrying RBC to nucleated cells. Adhesiveness of nucleated cells for avidin-carrying RBC varied for different types of nucleated cells. The strongest adhesion was observed with human fibroblasts and rat Kupffer cells, while rat liver endothelial cells were practically non-adhesive for avidin-carrying RBC of corresponding species. In contrast with avidin (streptavidin)-induced lysis by the complement, avidin-induced adhesion was independent of temperature, the presence of divalent ions and mode of avidin attachment. Polyanions (dextran sulphate and heparin) efficiently inhibited the adhesion presumably due to interaction with the membrane-bound avidin. Polyanions to a much lesser extent inhibited lysis of avidin-carrying RBC, which might be a result of their interaction with the complement components. Polycations also blocked adhesion of avidin-carrying RBC to nucleated cells, presumably due to interaction with negatively charged cell-surface components. Therefore, attachment of avidin to RBC alters their biocompatibility, due to both high positive charge of avidin and the cross-linking of biotinylated membrane proteins.

Cytokine secretion by human aortic endothelial cells is related to degree of atherosclerosis.

We have previously described a 13- to 15-kDa T-lymphocyte-specific chemotactic protein (endothelial cell-derived lymphocyte chemoattractant activity, ED-LCA) secreted by serotonin-stimulated bovine aortic endothelial cells. In the current study, we have identified a similar serotonin-induced chemotaxin secreted by human aortic endothelial cells (HAEC). Like the bovine ED-LCA, secretion of this human T-cell chemotaxin peaked at 10(-5) M serotonin, was blocked by 5-HT2-receptor antagonists, and was not induced by other vasoactive amines, such as histamine or angiotensin II. In addition, human ED-LCA had no effect on neutrophil or monocyte migration. Using HAEC and human pulmonary arterial endothelial cells (HPAEC) from the same individual, we found that serotonin-stimulated HAEC, but not HPAEC, secreted ED-LCA. Because human vascular endothelium affected by atherosclerosis is morphologically, ultrastructurally, and phenotypically distinct from unaffected areas, we evaluated the secretion of this cytokine from cultured HAEC derived from areas of aorta differentially affected by atherosclerosis. We found that the degree of atherosclerotic involvement of an individual vessel was associated with a decrease in the uptake of serotonin and a reduction in serotonin-induced ED-LCA secretion. In response to serotonin, HAEC derived from atherosclerotic plaques did not secrete ED-LCA, whereas HAEC derived from fatty streaks secreted lesser amounts of ED-LCA than HAEC derived from normal areas. These studies demonstrate that in vivo morphological heterogeneity of HAEC is maintained in vitro and is associated with alterations in function, as measured by cytokine secretion.

A monoclonal antibody, VM64, reacts with a 130 kDa glycoprotein common to platelets and endothelial cells: heterogeneity in antibody binding to human aortic endothelial cells.

A new monoclonal antibody (mAb), VM64, reacts with a common antigen on the surface of human platelets and vascular endothelial cells (EC). Under nonreduced conditions it recognized in immunoblotting a protein of 130 kDa both in platelets and EC. VM64 precipitated the same 130 kDa protein from the lysate of surface radioiodinated platelets. Electrophoretic mobility of this protein was not altered by reduction and differed from the bands precipitated by reference mAb against platelet glycoproteins (GP) Ia-IIa, Ib, IIb-IIIa and GMP130. VM64 binding to platelets and EC was specific and saturable. The number of binding sites on platelets was 9.9 +/- 3.5 x 10(3) per platelet and on the surface of EC monolayer -2.40 +/- 0.32 x 10(6) per cell. VM64 also binds to platelets from Glanzmann's thrombasthenia patients which lack GPIIb-IIIa. VM64 did not affect platelet aggregation induced by ADP, collagen, thrombin and ristocetin. In the monolayers of EC from umbilical vein and human aorta, VM64 stained the area at the periphery of the cells adjacent to the cell-cell boundaries. In preconfluent cultures preferential staining was observed at the active leading margins of the cells. Unlike EC cultures from umbilical vein, where all cells were positively stained, in aortic EC cultures some unstained or poorly stained cells were constantly present, indicating a heterogeneity of EC population related to the expression of VM64 antigen. The biochemical characteristics of VM64 antigen, its presence both on platelets and EC and typical distribution on the surface of EC suggested that this antigen is identical to PECAM (CD31) protein.

Mechano-chemical control of human endothelium orientation and size.

Human umbilical vein endothelial cells (EC) were grown on elastic silicone membranes subjected to cyclic stretch, simulating arterial wall motion. Stretching conditions (20% amplitude, 52 cycle/min) stimulated stress fiber formation and their orientation transversely to the strain direction. Cell bodies aligned along the same axis after the actin cytoskeleton. EC orientation response was inhibited by the adenylate cyclase activator, forskolin (10(-5) M), which caused stress fiber disassembly and the redistribution of F-actin to the cortical cytoplasm. Preoriented EC depleted of stress fibers by forskolin treatment retained their aligned state. Thus, stress fibers are essential for the process of EC orientation induced by repeated strain, but not for the maintenance of EC orientation. The monolayer formed by EC grown to confluence in conditions of intermittent strain consisted of uniform elongated cells and was resistant to deformation. In contrast, the monolayer assembled in stationary conditions was less compliant and exposed local denudations on initiation of stretching. When stretched in the presence of 10(-5) M forskolin it rapidly (3-4 h) reestablished integrity but gained a heterogeneous appearance since denuded areas were covered by giant cells. The protective effect of forskolin was because of the stimulation of EC spreading. This feature of forskolin was demonstrated while studying its action on EC spreading and repair of a scratched EC monolayer in conventional culture. Thus mechanical deformation and adenylate cyclase activity may be important factors in the control of endothelium morphology in human arteries.

Effects of forskolin and phorbol-myristate-acetate on cytoskeleton, extracellular matrix and protein phosphorylation in human endothelial cells.

The role that the intracellular mediators, cAMP and Ca2+/phosphatidylserine-dependent protein kinase C, play in the regulation of endothelial cell (EC) motility was investigated. The adenylate cyclase activator, forskolin, at 10 microM induced rapid and reversible alterations in the shape of cultured human EC, disappearance of actin bundles and the concentration of F-actin at cell borders. Actin reorganization provoked by forskolin coincide with redistribution of vinculin to the cell periphery and rapid elimination of surface-associated fibronectin. A protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) at 10-100 microM induced no visible alterations of cell shape, but enhanced the effect of forskolin. PMA stimulated formation of "stress fibers" and increased the number of vinculin plaques in central areas of the cell. A decrease in the amount of the surface-associated fibronectin in PMA-treated cells has also been observed, but, this effect was considerably slower than that produced by forskolin. Forskolin, but not PMA stimulated phosphorylation of the major intermediate filament protein, vimentin.

Identification of intimal subendothelial cells from human aorta in primary culture.

Subendothelial cells (SEC) were obtained from the inner intimal layer of adult human aorta by collagenase treatment. SEC were identified in primary culture either as smooth muscle cells by staining with FITC-labeled antisera against human smooth muscle myosin or as macrophages, foam cells and contaminating endothelial cells by their uptake of malondialdehyde treated low density lipoproteins labeled with fluorescent dye 3,3'-dioctadecylindocarbocyanine. Between 1 and 5 days in culture, along with smooth muscle cells (SMC, 38-82%), endothelial cells (0-9%), macrophages and foam cells (2-32%), one more type of cell was found. This cell type resembled SMC in size and shape, but was not stained by antisera to SMC myosin. By ultrastructural criteria these cells were characterized as modulated SMC for they contained prominent rough endoplastic reticulum and Golgi complex together with basement membrane and a large number of plasmalemmal vesicles. Like SMC they reacted with phalloidin and were stained by anti-vimentin but not by anti-desmin monoclonal antibodies. The proportion of such cells varied from 5 to 33% of total cell number and increased in parallel to macrophages and foam cells in vessels with well developed atherosclerotic lesions. We conclude that the applied technique may be used for identification of cultured vascular cells including modulated SMC.

Morphological alterations in endothelial cells from human aorta and umbilical vein induced by forskolin and phorbol 12-myristate 13-acetate: a synergistic action of adenylate cyclase and protein kinase C activators.

The morphological effects on human endothelial cells of phorbol 12-myristate 13-acetate (PMA) and of agents that increase intracellular cAMP concentration were studied. The adenylate cyclase activator forskolin (10 microM), the cyclic nucleotide phosphodiesterase inhibitor methylisobutylxanthine (100 microM), dibutyryl-cAMP (10 microM), histamine (10 microM), and PMA (0.1 microM) significantly altered the morphology of human aortic and umbilical vein endothelial cells in primary cultures. These effects reached a maximum 40-80 min after the effector addition and became negligible 30-60 min after its removal. PMA and forskolin were strongly synergistic in altering endothelial cell morphology. All the effects of cAMP-elevating compounds and of PMA were abolished completely by 1 microM colchicine. In explants taken from human adult or child aortas, forskolin and PMA produced alterations in endothelial morphology qualitatively identical to those observed in endothelial cell cultures. Endothelium in these preparations closely resembled that found in zones of expected altered hemodynamic stresses of human aorta. Our data suggest that the morphology of endothelium in vivo may be regulated by separate or synergistic action of hormone-dependent adenylate cyclase and of inositol phospholipid turnover systems and might be important for maintenance of endothelial monolayer integrity under normal physiological and pathological conditions.

Diurnal rhythms of cell reproduction and of some morphometric indices in the thyroid gland of young rats.

Monophasic diurnal rhythms of mitosis and DNA synthesis are characteristic of the thyroid gland epithelium of young rats (unlike adults). It is postulated that the diurnal rhythm of mitotic activity is under the influence of a mechanism synchronizing the cell population before mitosis. With an increase in thyrocyte differentiation the number of mitoses and of DNA-synthesizing cells falls. Diurnal changes in the duration of mitosis affect the dynamics of the diurnal rhythm of mitotic activity. Changes in the height of the follicular cells during the 24-hour period are evidence of a diurnal rhythm of thyroid function in young rats.