Cultures Of Aortic Endothelial Cells Research Articles

Comparative toxicity of the cardiovacular toxin allylamine to porcine aortic smooth muscle and endothelial cells

This study supports a recent hypothesis that the cardiovascular toxin, allylamine, is toxic to smooth muscle cells of large elastic arteries (aorta). Cultures of the porcine aortic smooth muscle, endothelial, and fibroblastic cells were exposed to varying concentrations of allylamine ranging from 5 μM to 340 μM. Monitored cytotoxic and cytolytic activities demonstrated that final concentrations of 60 μM allylamine decreased cell population viability of smooth muscle cells as much as 50%. Viability decreased approximately linearly with increasing concentrations of allylamine including spontaneous lysing of smooth muscle cells at 90 μM. Endothelial cells were more resistant to lower concentrations of allylamine requiring 90 μM to decrease cell population viability by 50%. In contrast, fibroblastic cells were very resistant to lower concentrations of allylamine. The specific lytic response of these cells in culture, measured by release of [ 3H]thymidine, gave findings parallel to the viability studies, i.e. at 100 μM allylamine smooth muscle cells demonstrated 75% specific lysis while endothelial cells showed 29%. Growth studies of cells surviving an 8-h exposure to allylamine indicate that surviving endothelial cells have better growth characteristics than surviving smooth muscle cells; both cell lines are also apparently injured at concentrations of allylamine much lower than the CT 50. These studies show that of the cellular components of the vascular wall, smooth muscle cells appear to be the most sensitive to the toxic effects of allylamine.

Bovine coronary artery endothelium: In vitro culture and production of plasminogen activator

A new method for isolation and culture of endothelial cells from bovine coronary artery (BCoAEC) is presented. This method involves in situ perfusion and digestion of main coronary arteries with a collagenase solution. The isolated cells were cultured and maintained through many cell passages in Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum derived from either whole blood or plasma. Confirmation of these cells' endothelial origin was obtained by demonstration of typical morphologic and growth characteristics of endothelium, immunofluorescent staining with antibodies to von Willebrand factor (Factor VIII: vWF), and measurement of plasminogen activator (PA). In addition, production of PA was inhibited by enzymatically active thrombin as has been previously described with bovine aortic endothelial cells in culture.

Cellular requirements for tissue factor generation by bovine aortic endothelial cells in culture

Cultured bovine aortic endothelial cells acquired the ability to initiate coagulation after treatment with endotoxin or phorbol ester. The acquired procoagulant activity was identified as tissue factor since cells treated with endotoxin or phorbol ester activated factor X only in the presence of factor VIIa, and factor X activation could be completely blocked by a specific antibody to bovine tissue factor apoprotein. The generation of tissue factor activity was evident after 6 hours of incubation and was dependent on RNA and protein synthesis, as indicated by the inhibitory effects of actinomycin D and cycloheximide. Endotoxin and phorbol ester are toxic to cultured endothelial cells as evidenced by release of LDH and detachment from the culture dish. Surviving endothelial cells lose their stress fibers and assume a cytoskeletal organization characteristic of mobility or radial extension. Because these changes in cell shape occurred parallel with the acquisition of procoagulant activity, the effects of drugs interfering with organization of the cytoskeleton were tested. Cytochalasins B and D, vinblastine, and cotchicine, each decreased the generation of tissue factor activity when cells were exposed to endotoxin or phorbol ester. Trifluoperazine, a calmodulin antagonist, also prevented the generation of tissue factor activity in a dose-dependent fashion. Thus, perturbation of endothelial cells by treatment with phorbol ester or endotoxin induces potent tissue factor procoagulant activity. This cellular response appears to require protein and RNA synthesis, normal cytoskeletal functions, and the Ca ++-calmodulin system.

A new protective factor in coronary artery disease Very low density lipoprotein toxicity-preventing activity

A newly discovered activity in human serum protects porcine aortic endothelial cells in culture from injury by very low density lipoproteins (VLDL). This factor, toxicity-preventing activity (TxPA), was measured in 29 relatively young men (43 ± 8 years) who had undergone coronary angiography. The level of TxPA was found to be significantly reduced ( P < 0.001) in men who demonstrated more than 15% narrowing of their coronary arteries. Men (n = 18) who had 15% or less narrowing were found to have 104 ± 48 units of TxPA while men (n = 11) with coronary artery disease had 48 ± 24 units of TxPA. A value derived from the product of TxPA and the high density lipoprotein cholesterol (HDL-C) level divided by the non-HDL-C (total cholesterol-HDL-C) accurately separated 97% of the men into 2 groups. TxPA thus appears to be a new protective factor in coronary artery disease, which, when combined with total cholesterol and high density lipoprotein cholesterol values, provides an accurate classification of established coronary artery disease in these subjects.

Effects of hyperoxia on DNA synthesis in cultured porcine aortic endothelial cells.

The mode of action of hyperoxia on the inhibition of DNA synthesis from thymidine (dThd) was studied in primary cultures of porcine aortic endothelial cells (EC) at confluence. A significant effect of hyperoxia on dThd uptake was detected only after a 48-h exposure to 95% O2. On the other hand, decrease in dThd kinase activity was already observed after a 12-h exposure, and the time course of its reduction followed closely that of the inhibition of dThd incorporation into DNA. The incorporation of dThd triphosphate into DNA in permeabilized EC was unaffected by hyperoxia. Determination of DNA alpha- and beta-polymerase activities showed that hyperoxia reduced the activity of the alpha-polymerase and increased that of the beta-polymerase. We conclude that most of the O2 effects on DNA synthesis from dThd can be attributed to dThd kinase inhibition. The increased activity of DNA beta-polymerase, an enzyme involved in DNA repair, also supports the view that hyperoxia could damage DNA.

Effect of variable glutathione peroxidase activity on H2O2-related cytotoxicity in cultured aortic endothelial cells.

Primary cultures of porcine aortic endothelial cells were used to assess the effects of O2 intermediates produced by 10-40 mU/ml xanthine oxidase (XO; +2 mM hypoxanthine) or 25-100 mU/ml glucose oxidase (GO; +5 mM glucose). A 60-min incubation in the presence of the enzyme systems resulted in a dose-dependent toxic effect with evidence of cytolysis (increased LDH release) and cell loss (decrease in DNA and protein content), when these indexes were measured 24 hr after completion of the enzyme reaction. Decreased [3H]thymidine incorporation into DNA was the most sensitive index of cell dysfunction for both enzyme systems. The effects of various scavengers and enzymes indicated that H2O2 was the main O2 intermediate involved in the cytotoxicity resulting from the XO-hypoxanthine reaction. Increased glutathione peroxidase activity associated with the addition of 2 X 10(-7) M selenomethionine to culture medium had a partial protective effect which could be related to an increased rate of H2O2 degradation. Evidence for increased DNA synthesis after injury was found in cells previously exposed to XO-hypoxanthine, the degree of increase in [3H]thymidine incorporation being dependent on the intensity of the initial cytotoxicity. Cultured endothelial cells provide a useful tool to evaluate the role of O2 intermediates in endothelial cell injury, to test the effects of protective agents, and to study the repair process.

Processing and transport of insulin by vascular endothelial cells: Effects of sulfonylureas on insulin receptors

Polypeptide hormones such as insulin must cross the vascular barrier to mediate their biologic actions. A substantial vascular barrier may be encountered in muscle and fat tissues, which are supplied by continuous capillaries lined with tightly joined endothelial cells. Endothelial cells have previously been shown to bind and release insulin with minimal degradation. Because 125I-labeled insulin transport was demonstrated to be receptor-mediated, factors regulating insulin-receptor binding may also affect the insulin transport rate across the vascular barrier. Since sulfonylureas may have a glucose-lowering action by altering insulin receptors, the effects of tolazamide and glyburide on vascular endothelial cell insulin receptors were evaluated. Exposure of aortic endothelial cells in culture to insulin at 37 degrees C resulted in a 75 percent loss of receptors. Five to seven days of tolazamide exposure led to a 35 percent time-dependent increase in insulin binding. More strikingly, tolazamide altered the dose-receptor to insulin-induced down-regulation. Cells down-regulated with insulin (10 ng/ml) showed a 100 percent increase in binding in the presence of tolazamide; a dose-dependent effect occurred at the 75 to 200 micrograms/ml dosage level. Scatchard analysis indicated that the increase in 125I-labeled insulin binding was due to an increase in receptor number. When insulin receptors were identified with 125I-labeled insulin, tolazamide-treated cells clearly showed an increase of a band at Mr = 145 K, the alpha subunit of the receptor. Tolazamide may thus help normalize glucose in diabetes by preventing receptor down-regulation in endothelial cells.

Enhancement of endothelial plasminogen activator synthesis by lymphokines.

Long-term cultures of pig aortic endothelial (E) cells secrete plasminogen activator (PA). When these cells were cultured with murine lymphokine(s) produced in response to antigen, mitogen, or allogeneic stimuli, there was consistently increased PA secretion by E cells. The evidence suggests that initiator of plasminogen activator (IPA) is produced by a nylon-wool--nonadherent cell, probably a T cell. The kinetics of this activation of PA suggest that the effect of IPA on E cells is considerably delayed compared with that on phagocytic cells. The significance of these findings is discussed in terms of their possible relationship to several immunopathological states.

Renin expression by vascular endothelial cells in culture.

Cultured bovine aortic endothelial cells were examined for renin activity by biochemical, immunological, and immunohistochemical techniques. When cell sonicates were incubated with renin substrate, linear generation of angiotensin I was observed (1.12 +/- 0.2 ng angiotensin I/10(6) cells per hr). The effect of pH on this activity was similar to that of bovine renal renin, and renin antibodies inhibited a large portion of the enzymatic activity. Furthermore, immunofluorescence microscopy with antirenin antisera confirmed the presence of renin within these cells. Biosynthetic radiolabeling, followed by immunoprecipitation, demonstrated de novo synthesis of a renin precursor in the endothelial cells, which was processed to a more mature protein. Thus, bovine aortic endothelial cells in culture contain and biosynthesize renin, a key component of the renin-angiotensin system. The expression of renin activity by endothelium may contribute to the local regulation of vascular tone.

Extracellular matrix interactions with the apical surface of vascular endothelial cells.

Cultured aortic endothelial cells, like their in vivo counterparts, form highly organized, confluent monolayers of polarized epithelioid cells that secrete, exclusively at their basal surface, an extracellular matrix to which they then attach. The influence of isolated subendothelial matrix preparations on cell polarity and monolayer organization was studied by presenting fragments of the matrix to confluent bovine aortic endothelial cell cultures. The matrix particles were immediately bound to the apical aspect of the cell monolayer and induced rapid reorganization of the monolayer into cells with a fibroblastoid morphology. To determine if fibronectin, the major glycoprotein of the subendothelial matrix, could be involved in the observed apical cell surface-matrix interactions, latex beads or small discs of Nucleopore filters were coated with the glycoprotein and presented to confluent monolayers. In a fashion similar to that observed with matrix fragments, materials coated with fibronectin caused focal reorganization of the cell layer. After contact with the coated beads, the underlying endothelial cells flowed upward and spread over the entire bead, forming a canopy of confluent cells that draped the particle. Contact of confluent monolayers with the coated filters induced similar behaviour, except that monolayer reorganization into the fibroblastoid phenotype was followed by emigration of the majority of underlying cells through the pores to the upper filter surface, where they formed a new organized cell monolayer with the typical endothelial cell morphology. Thus contact of the apical surface of endothelial cells with structures to which they adhere initiates a rapid disruption of the organized cell monolayer, followed immediately by a concerted effort of the local population to re-establish both cell polarity and monolayer contiguity. The expression of this behaviour may be important during tissue remodeling that occurs in neovascularization and during interactions with thromboemboli.

Antibodies to nuclear thyroid hormone-binding proteins: Antibody characterization and immunofluorescent localization

To further investigate the mechanism by which thyroid hormones regulate target cell function, we have prepared and partially characterized antibodies to highly purified nuclear thyroid hormone-binding proteins (NTBP). NTBPs were prepared from bovine liver nuclear extracts by bio-specific elution from an affinity gel containing immobilized 3,5,3′-triiodo- l-thyronine (T 3). Antibodies (Ab) raised to NTBP in BALB/c mice were assayed for Ab-NTBP complex formation on HPLC TSK SW3000 molecular exclusion gels and found to be species-specific and non-cross-reactive with serum thyroid hormone-binding proteins. Most of the antibody activity was directed against two fractions of molecular weight (MW) 89000 and 53000, which were associated with thyroxine (T 4)-binding activity. The 89000 D T 4-binding activity was shifted to a higher MW complex when incubated with specific antibody. Indirect immunofluorescence showed antibody activity against discrete, clumped chromatin sites, nuclear envelope and plasma membrane in hepatocytes. Intense fluorescence was also observed in the cells lining the hepatic sinusoids and in the cytoskeleton of bovine aortic endothelial cells in culture. The data suggest that thyroid hormone target cells contain extranuclear loci that share antigenic sites with NTBP and may also represent specific NTBP-like sites of thyroid hormone binding.

Effect of oxygen and endotoxin on lactate dehydrogenase release, 5-hydroxytryptamine uptake, and antioxidant enzyme activities in endothelial cells.

We compared the effects of 95% O2 (hyperoxia) alone, endotoxin (20 ng/ml) alone, and 95% O2 plus endotoxin on the release of lactate dehydrogenase (LDH), uptake of 5-hydroxytryptamine (5-HT), and antioxidant enzyme activities in porcine pulmonary arterial and aortic endothelial cells in monolayer culture. Hyperoxia increased LDH release and decreased 5-HT in both endothelial cell types. Hyperoxia also caused a decrease in catalase (CAT) activity and an increase in total superoxide dismutase (SOD) and glutathione reductase (GSH-Red) activities in both cell types. Endotoxin alone had no effect on LDH release, 5-HT uptake, or antioxidant enzyme activities. However, endotoxin prevented the hyperoxic increase in LDH release and the hyperoxic decrease in 5-HT uptake. Endotoxin plus 95% O2 had no consistent effect on the antioxidant enzyme profile in pulmonary artery or aortic endothelial cells. These results indicate that (1) hyperoxia injures both pulmonary artery and aortic endothelial cells in culture and causes changes in the antioxidant enzyme profile that are similar in the two cell types; (2) hyperoxia-induced decreases in CAT activity and increases in SOD activity may be responsible for increased sensitivity of endothelial cells to O2 toxicity; and (3) endotoxin protects against hyperoxic injury to endothelial cells in vitro, but increases in antioxidant enzyme activities are not the mechanism for this protection.

Thromboxane production by perturbed bovine aortic endothelial cells in culture.

Bovine aortic endothelial cells in culture were incubated with endotoxin. The amount of thromboxane A2 synthesized was then determined by a specific radioimmunoassay for thromboxane B2. After a lag of several hours the cells changed their shape and parallel to the change in cell shape release of thromboxane B2 occurred. At 24 h the amount of thromboxane B2 generated in response to endotoxin was 200-fold above baseline. Thromboxane B2 generation could be blocked by aspirin and the specific thromboxane synthetase inhibitor UK 37248. The endotoxin effect was dependent on protein and RNA synthesis as evidenced by the inhibitory action of cycloheximide (1.5 microM) and actinomycin D (2 micron).

Regulation of angiotensin I‐converting enzyme activity in serially cultivated bovine endothelial cells

Angiotensin I-converting enzyme (ACE) activity was measured in lysates of cloned and uncloned cultures of bovine fetal aortic endothelial cells. The expression of ACE activity in these cells was complex, and influenced by subcultivation, cell density, serum, cumulative population doublings, and clonal heterogeneity. The ACE specific activity at any point in the in vitro lifespan was determined, at least in part, by interaction of these culture variables. After subcultivation to subconfluent densities, cellular ACE specific activity decreased markedly and did not reach detectable levels until cells attained confluent densities. The use of different suppliers' lots of serum in the growth medium resulted in different cellular ACE specific activities. The ACE specific activity decreased as cultures were serially subcultivated, but remained detectable throughout the lifespan, suggesting a linkage between the proliferative history of an endothelial cell and its remaining capacity to express ACE. Increased ACE activity was observed when cells at the end of their lifespan were cultured at high densities. Cloned strains behaved similarly to the uncloned parent culture, except that they exhibited a wide range of ACE specific activities.

Prostacyclin production by perturbed bovine aortic endothelial cells in culture

This study reports that endotoxin (Escherichia coli serotype 026:B6) and 12-O-tetradecanoyl-phorbol-13-acetate stimulate cultured bovine aortic endothelial cells to generate prostacyclin. The prostacyclin concentration of the culture medium was measured indirectly by radioimmunoassay for 6-keto-PGF1 alpha. The amount of prostacyclin generated depended on the concentration of endotoxin or phorbol diester. Prostacyclin generation was not immediate, but occurred slowly after a six-hour lag period. The perturbed cells contracted and showed marked shape changes that correlated temporally with the start of enhanced prostacyclin production. Cytochalasins B and D, vinblastine, and colchicine inhibited prostacyclin production, indicating involvement of the cytoskeleton in the cellular response to endotoxin and phorbol diester. The increase in prostacyclin production was prevented by trifluoperazine, an inhibitor of the Ca++-calmodulin system, which is known to be involved in cytoskeletal function. Generation of prostacyclin was inhibited by cycloheximide and actinomycin D, indicating dependence on protein and ribonucleic acid synthesis. It is postulated that exposure to endotoxin or phorbol diester leads, via a series of reactions that involve RNA and protein synthesis and require intact cytoskeletal function, to the generation of toxic active intermediate(s) that stimulate the enzymes necessary for prostacyclin production.

Prostacyclin Production by Perturbed Bovine Aortic Endothelial Cells in Culture

This study reports that endotoxin (Escherichia coli serotype 026:B6) and 12-O-tetradecanoyl-phorbol-13-acetate stimulate cultured bovine aortic endothelial cells to generate prostacyclin. The prostacyclin concentration of the culture medium was measured indirectly by radioimmunoassay for 6-keto-PGF1 alpha. The amount of prostacyclin generated depended on the concentration of endotoxin or phorbol diester. Prostacyclin generation was not immediate, but occurred slowly after a six-hour lag period. The perturbed cells contracted and showed marked shape changes that correlated temporally with the start of enhanced prostacyclin production. Cytochalasins B and D, vinblastine, and colchicine inhibited prostacyclin production, indicating involvement of the cytoskeleton in the cellular response to endotoxin and phorbol diester. The increase in prostacyclin production was prevented by trifluoperazine, an inhibitor of the Ca++-calmodulin system, which is known to be involved in cytoskeletal function. Generation of prostacyclin was inhibited by cycloheximide and actinomycin D, indicating dependence on protein and ribonucleic acid synthesis. It is postulated that exposure to endotoxin or phorbol diester leads, via a series of reactions that involve RNA and protein synthesis and require intact cytoskeletal function, to the generation of toxic active intermediate(s) that stimulate the enzymes necessary for prostacyclin production.

Leukotriene induction of TxB2 in cultured bovine aortic endothelial cells.

The leukotrienes (LT) are potent mediators of inflammation, capable of inducing plasma leakage from postcapillary venules and vasoconstriction of terminal arterioles. Some microvascular effects may be attributable to LT stimulation of thromboxane (Tx) synthesis. Incubation of primary cultures of bovine aortic endothelial cells with buffer, LTB4 (10(-8) M) or LTD4 (10(-8)M), resulted in TxA2 production in pg/10(5) cells to the extent of: 67 +/- 20, 571 +/- 180, and 333 +/- 60 respectively, as measured by radioimmunoassay of the stable metabolite TxB2. Endothelial pretreatment with the LTD4 receptor antagonist FPL55712 (10(-5)M) significantly blocked any LTB4- or LTD4-stimulated TxA2 synthesis. Pretreatment with the TxA2 synthetase inhibitor ketoconazole (10(-6)M) also prevented LTB4 of LTD4 stimulation of TxA2. Preincubation with DMTU (10(-5)M), an hydroxyl radical scavenger, decreased LTB4-induced release of TxA2 (405 +/- 40 and 366 +/- 20, respectively). These findings suggest that LT may mediate their inflammatory actions in vascular beds by stimulation of Tx release from endothelial cells.

Aortic endothelial cells in culture secrete glycoproteins reacting with blood platelets

The culture medium of bovine aortic endothelial cells contains proteins which inhibit the aggregation of platelets induced by aortic microfibrils but not by type III collagen. From this medium, fibronectin, thrombospondin and a glycoprotein with MW of 128 Kd (GP 128), similar to a glycoprotein described in a microfibrillar extract from bovine aorta were separated by affinity and ion exchange chromatography. GP 128 was further purified by molecular sieve chromatography on SW 3000 column. GP 128 inhibited the aggregation of platelets by microfibrils. This suggests a role of GP 128 in the platelet/subendothelium interaction.

Processing of insulin by bovine endothelial cells in culture. Internalization without degradation.

Insulin binding and processing was studied in monolayer cultures of bovine aortic endothelial cells. Specific 125I-insulin binding was both time and temperature dependent. Maximum binding at 37 degrees C occurred at 90 min, and was 3.8%/mg protein and, at 15 degrees C, 7%/mg protein at 4 h. 125I-insulin was crosslinked to its receptor using disuccinimidyl suberate (DSS), and the structure of the receptor complex was identified by SDS-polyacrylamide gel electrophoresis and autoradiography; a major band with Mr = 145,000 was identified, which corresponds to the alpha-subunit of the insulin receptor reported in other tissues. Receptor-bound insulin was internalized, and both the rate and the amount of internalization were temperature dependent. The rate of internalization was slowest at 4 degrees C, and fastest at 37 degrees C, and the maximum amount of 125I-insulin internalized in 120 min was 16% at 4 degrees C, 45% at 15 degrees C, and 81% at 37 degrees C. Despite the high rate of internalization, endothelial cells do not appear to degrade insulin significantly, as determined by gel chromatography and TCA solubility (7% at 4 h) of media-associated radioactivity. In addition, the majority of internalized insulin (75%) was released by 60 min, largely as intact insulin. Chloroquine treatment at high concentration did not exert any major effect on insulin binding or degradation within the first 60 min, but thereafter produced a marked increase in cell-associated radioactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Bovine aortic endothelial cells elaborate an inhibitor of the generation of lipopolysaccharide-stimulated human blood monocyte procoagulant activity.

We examined the effect of bovine aortic endothelial cell culture supernatants upon the generation of procoagulant activity by human blood monocytes. Confluent endothelial monolayers were cultured for up to 96 h. At timed intervals, culture supernatants were collected and incubated for 5 h with lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The procoagulant activity of mononuclear cell lysates was determined in a one-stage clotting assay. In five experiments, procoagulant activity with culture supernatant (time 0) was 2,294 +/- 761 U/ml (mean +/- SEM). Culture supernatants from endothelial cells incubated for 24-96 h strongly inhibited mononuclear cell generation of procoagulant activity. Indomethacin (10 microM) added to endothelial cells delayed the appearance of procoagulant inhibitor for 72 h. Bovine aortic smooth muscle cell culture supernatants did not inhibit procoagulant activity. The inhibitor was heat stable, effective at 1:50 dilution, soluble, and acid sensitive, with a molecular weight of less than 1,500. Further studies on subpopulations of mononuclear cells demonstrated that endothelial inhibitor selectively decreased the generation of monocyte procoagulant activity and interfered with T lymphocyte amplification of monocyte production of procoagulant activity. Thus, we have demonstrated that endothelial cells elaborate a potent inhibitor of monocyte procoagulant activity.