Evidence for a two-step process in prostaglandin secretion. Intracellular accumulation of prostacyclin precedes its release from human endothelial cells in culture

Cultured endothelial cells (EC) from human umbilical vein were incubated with [U-14C]arachidonic acid (AA) followed by a challenge with thrombin (2 units/ml) or calcium ionophore A23187 (5 microM) for 0.5-10 min at 37 degrees C. In both cases, AA was rapidly liberated from phospholipids and converted into prostaglandin I2 (PGI2), as determined by the radioactivity of the stable derivative 6-keto-PGF1 alpha. Maximal liberation of AA and synthesis of PGI2 were achieved within 2 min, but the two compounds first accumulated in EC prior to their release into supernatants. This finding, which was never reported before, raises the question of the mechanism of AA and PG release through the cell membranes and offers a convenient model to investigate this still obscure process.     

Halothane potentiates the alcohol-adduct induced TNF-alpha release in heart endothelial cells

BACKGROUND: The possibility exists for major complications to occur when individuals are intoxicated with alcohol prior to anesthetization. Halothane is an anesthetic that can be metabolized by the liver into a highly reactive product, trifluoroacetyl chloride, which reacts with endogenous proteins to form a trifluoroacetyl-adduct (TFA-adduct). The MAA-adduct which is formed by acetaldehyde (AA) and malondialdehyde reacting with endogenous proteins, has been found in both patients and animals chronically consuming alcohol. These TFA and MAA-adducts have been shown to cause the release of inflammatory products by various cell types. If both adducts share a similar mechanism of cell activation, receiving halothane anesthesia while intoxicated with alcohol could exacerbate the inflammatory response and lead to cardiovascular injury. METHODS: We have recently demonstrated that the MAA-adduct induces tumor necrosis factor-alpha (TNF-alpha) release by heart endothelial cells (HECs). In this study, pair and alcohol-fed rats were randomized to receive halothane pretreatments intra peritoneal. Following the pretreatments, the intact heart was removed, HECs were isolated and stimulated with unmodified bovine serum albumin (Alb), MAA-modified Alb (MAA-Alb), Hexyl-MAA, or lipopolysaccharide (LPS), and supernatant concentrations of TNF-alpha were measured by ELISA. RESULTS: Halothane pre-treated rat HECs released significantly greater TNF-alpha concentration following MAA-adduct and LPS stimulation than the non-halothane pre-treated in both pair and alcohol-fed rats, but was significantly greater in the alcohol-fed rats. CONCLUSION: These results demonstrate that halothane and MAA-adduct pre-treatment increases the inflammatory response (TNF-alpha release). Also, these results suggest that halothane exposure may increase the risk of alcohol-induced heart injury, since halothane pre-treatment potentiates the HEC TNF-alpha release measured following both MAA-Alb and LPS stimulation.     

Histamine-induced phosphoinositide metabolism in cultured human umbilical vein endothelial cells. Association with thromboxane and prostacyclin release

Histamine stimulation of cultured human umbilical vein endothelial cells induced dose- and time-dependent increases in glycerophosphoinositol (GroPIns), inositol-1-phosphate (InsP), inositolbisphosphate (InsP2) and inositoltrisphosphate (InsP3) in addition to release of thromboxane A2 and prostacyclin. Increases in InsP2 and InsP3 were immediate while increases in GroPIns and InsP occurred only after 1 min. Thromboxane A2 and prostacyclin release paralleled GroPIns and InsP production. The data indicate that, in endothelial cells, histamine evokes early hydrolysis of polyphosphoinositides, and that subsequent mobilization of arachidonic acid for thromboxane and prostacyclin synthesis involves both deacylation and phosphodiesteratic cleavage of phosphatidylinositol.     

Synthesis and metabolism of leukotrienes by human endothelial cells: influence on prostacyclin release

The synthesis and metabolism of leukotrienes (LTs) by endothelial cells was investigated using reverse-phase high-performance liquid chromatography. Cells were incubated with [14C]arachidonic acid. LTA4 or [3H]LTA4 and stimulated with ionophore A23187. The cells did not synthesize leukotrienes from [14C]arachidonic acid. LTA4 and [3H]LTA4 were converted to LTC4, LTD4, LTE4 and 5,12-diHETE. Endothelial cells metabolized [3H]LTC4 to [3H]LTD4 and [3H]LTE4. The metabolism of [3H]LTC4 was inhibited by L-serine-borate complex, phenobarbital and acivicin in a concentration-related manner, with maximal inhibition occurring at a concentration of 0.1 M, 0.01 M and 0.01 M, respectively. LTC4, LTB4 and LTD4 stimulated the synthesis of prostacyclin, measured by radioimmunoassays as 6-keto-PGF1 alpha. The stimulation by LTC4 was greater than that by LTD4 or LTB4. LTE4, 14,15-LTC4 and 14,15-LTD4 failed to stimulate the synthesis of prostacyclin. LTD4 and LTB4 also stimulated the release of PGE2, whereas LTC4 did not. Serine-borate and phenobarbital inhibited LTC4-stimulated synthesis of prostacyclin in a concentration-related manner. They also inhibited the release of prostacyclin by histamine, A23187 and arachidonic acid. Acivicin had no effect on the release of prostacyclin by LTC4, histamine or A23187. Furthermore, FPL-55712, an LT receptor antagonist, inhibited LTC4-stimulated prostacyclin synthesis but had no effect on histamine-stimulated release of prostacyclin or PGE2. Indomethacin inhibited both LTC4- and histamine-stimulated release. The results show that (a) endothelial cells metabolize LTA4, LTC4 and LTD4 but do not synthesize LTs from arachidonic acid; (b) LTC4 act directly at the leukotriene receptor to stimulation prostacyclin synthesis; (c) the presence of the glutathione moiety at the C-6 position of the eicosatetraenoic acid skeleton is necessary for leukotriene stimulation of prostacyclin release; and (d) the metabolism of LTC4 to LTD4 and LTE4 does not appear to alter the ability of LTC4 to stimulate the synthesis of PGI2.     

17 alpha-ethinylestradiol decreases production and release of prostacyclin in cultured human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC) were incubated with the estrogen 17 alpha-Ethinylestradiol (30 ng/ml) in order to examine its regulating influence on synthesis and release of prostacyclin (PGI2). ATP (1 mg/ml) was used to stimulate PGI2-production through the purine receptors. We demonstrated that this level of estrogen decreases PGI2-synthesis by 11% and PGI2-release by 32% within 300 sec. Longer incubation times (48 hrs) resulted in the same inhibitory effect. Intracellular ATP content and methyl-3H-thymidine uptake demonstrated that the decrease of prostacyclin-concentration is not caused by reduced viability of the cells but by a direct inhibitory effect on prostacyclin synthesis and release.     

Effect of lipopolysaccharides on cultured human endothelial cells. Relationship between tissue factor activity and prostacyclin release

Exposure to lipopolysaccharides (LPS; 10 micrograms/ml derived from either S. enteritidis or E. coli or to their lipid A moiety alone induced procoagulant activity in cultured human endothelial cells. This exclusively cell-associated activity was identified as tissue factor activity by two criteria: Firstly, the presence of Factor VII was required for its expression and, secondly, clotting was abolished by the addition of the IgG fraction of anti-human tissue factor antibodies. Concomitant analysis of prostacyclin (PGl2) formation by the cells showed a substantial increase in the production of this potent platelet inhibiting substance during exposure to endotoxin. LPS-induced release of PGl2 did not result in refractoriness of the cells to generate new PGl2 as indicated by the retained response to stimulation with 20 microM arachidonic acid. While the release of PGl2 could be inhibited by pretreatment of the cells with 100 microM acetylsalicylic acid (ASA), the induction of tissue factor activity remained unaffected by ASA. In contrast to LPS-free control cultures, ASA did not completely prevent PGl2 formation by human endothelial cells after exposure to LPS suggesting the induction of a cyclooxygenase-independent pathway by LPS.     

人血管内皮细胞中腺苷代谢的定量研究

目的:通过对人脐静脉内皮细胞腺苷分泌进行定性及定量研究,了解人类血管内皮细胞的腺苷代谢及机制.方法:收集并测定不同干预下细胞柱流出液中分离的人脐静脉内皮细胞分泌的腺苷量.结果:在无干预、抑制腺苷激酶及去氨酶、抑制细胞膜腺苷转运情况下,人脐静脉内皮细胞腺苷分泌率分别为13.5±7.1 pmol·min-1·mg-1、32.5±14.2 pmol·min-1·mg-1和20.8±15.7 pmol·min-1·mg-1.结论:人类血管内皮细胞内腺苷合成高于胞外,而细胞膜腺苷转运被抑制后的腺苷分泌率反而高于生理状态下分泌率,则表明腺苷在胞内分解代谢非常迅速,使部分腺苷反由胞外扩散入胞内.     

Selenium deficiency inhibits prostacyclin release and enhances production of platelet activating factor by human endothelial cells

Selenium is an essential component of glutathione peroxidase, an enzyme which protects cells against peroxidation and controls concentrations of intracellular peroxides. Since selenium deficiency is clinically associated with an increased degree of atherosclerosis, the effects of selenium deficiency on prostacyclin (PGI2) and platelet activating factor (PAF) production by cultured human umbilical vein endothelial cells (HUVEC) were investigated. In selenium-deficient HUVEC, histamine-induced PGI2 synthesis was significantly decreased when compared to selenium-supplemented HUVEC; in contrast, histamine-induced PAF production was increased by selenium deficiency. Histamine-induced inositol trisphosphate and [Ca2+]i responses and the conversion of PGG2 and PGH2 to PGI2 were not altered by selenium deficiency. However, selenium deficiency decreased the conversion of exogenous arachidonate to PGI2 and markedly suppressed glutathione peroxidase activity. These results suggest that selenium deficiency, by decreasing glutathione peroxidase activity, makes HUVEC susceptible to peroxide-induced inhibition of the cyclooxygenase activity of PGH2 synthase, resulting in decreased PGI2 production. These changes may alter platelet function in vivo and thus play a role in the increased incidence of atherosclerosis reported in selenium-deficient individuals.     

Insulin potentiates cytokine-induced VCAM-1 expression in human endothelial cells

Hyperinsulinemia is an independent risk factor for cardiovascular events and may contribute to cardiovascular disease. Low-grade chronic inflammation has been implicated in the pathogenesis of atherosclerosis. We aimed at determining the impact of pathophysiologically high insulin concentrations on cytokine-induced endothelial activation in human umbilical vein endothelial cells (HUVEC). HUVEC were incubated with insulin (0-24 h)+/-tumor necrosis factor (TNF)-alpha or lipopolysaccharide (LPS). At pathophysiological/pharmacological concentrations (10(-9)-10(-7) mol/L), insulin selectively induced VCAM-1 expression and potentiated the effects of TNF-alpha andLPS, effects reverted by the proteasome inhibitor lactacystin. Compared with TNF-alpha alone, insulin+TNF-alpha doubled U937 cell adhesion. Insulin markedly increased TNF-alpha-induced NF-kappaB activation and induced phosphorylated IkappaB-alpha accumulation. Therefore, hyperinsulinemia enhances cytokine-induced VCAM-1 expression in endothelial cells, thus potentially contributing to detrimental effects of other inflammatory stimuli on atherogenesis.     

Regulation of histamine-mediated prostacyclin synthesis in cultured human vascular endothelial cells

Histamine stimulates the production of prostacyclin (PGI₂) in cultured human endothelial cells. We have examined the cell specificity of histamine-mediated PGI₂ synthesis in primary and subcultured human cells. Venous and arterial smooth muscle cells and skin fibroblasts synthesized PGI₂ from exogenous arachidonic acid, but they did not synthesize a significant amount of PGI₂ when treated with histamine. Endothelial cells, however, produced similar amounts of PGI₂ in response to histamine and arachidonic acid. Thrombin also stimulates PGI₂ production in endothelial cells. Histamine and thrombin yielded an additive production of PGI₂ when added simultaneously to endothelial cells. When histamine and thrombin were added sequentially, the amount of PGI₂ produced was not additive but equaled the amount characteristic of the first agonist alone. Following an initial treatment with histamine, endothelial cells were unable to respond to histamine for 3 hr, after which the PGI₂ biosynthetic response rapidly returned to normal by $\text{4}\text{1}\text{2}$ hr. When the initial histamine treatment was carried out under mildly alkaline conditions, the complete return of activity was delayed to 8 hr after treatment. The synthesis of PGI₂ from exogenous arachidonic acid was unaffected by prior treatment with histamine. Recovery of histamine-mediated PGI₂ production was not dependent on protein synthesis but required a component of fetal calf serum that is nondialyzable and moderately heat stable. Thus endothelial cell PGI₂ synthesis in response to a physiologic agonist is subject to several levels of regulation, reflecting not only intracellular events but also the extracellular environment.     

Characterization of prostacyclin synthesis in cultured human arterial smooth muscle cells, venous endothelial cells and skin fibroblasts.

The interaction of human platelets with one another and with the blood vessel wall is thought to be regulated in part by a balance between two arachidonic acid metabolites: thromboxane A₂, synthesized by platelets, and prostacyclin (PGI₂), synthesized by the vessel wall. We have studied the ability of cultured human vascular cells to synthesize PGI₂ from arachidonic acid. Four strains of human arterial smooth muscle cells synthesized a mean of 1.36 ng PGI₂ per 10⁵ cells, with a range of 0.2–5.3 ng PGI₂ per 10⁵ cells among the different strains. Human umbilical vein endothelial cells synthesized a mean of 7.16 ng PGI₂ per 10⁵ cells with a range of 2.3–14.0 ng per 10⁵ cells. In contrast, cultured human diploid skin fibroblasts synthesized only 0.27 ng PGI₂ per 10⁵ cells with a range of 0.05–0.6 ng per 10⁵ cells. When cultured cells were mixed with platelets, PGI₂ synthesis from added arachidonate was reduced rather than stimulated. Thus the major precursor cyclic endoperoxides utilized for PGI₂ synthesis are formed within the cells and not from endoperoxides synthesized by platelet cyclooxygenase. Aspirin has been proposed as an anti-thrombotic agent. Aspirin could be ineffective, however, if it inhibited not only platelet cyclooxygenase but that of vessel wall cells as well. Measurement of the rate constant or potency for aspirin inhibition of PGI₂ synthesis in cultured cells indicates that the cyclooxygenase in both cell types of the blood vessel wall is 14–44 fold less sensitive to aspirin inactivation than that in platelets, and appropriate levels of aspirin can selectively block human platelet thromboxane A₂ synthesis without compromising the capacity of the vasculature to produce PGI₂.     

Physiological concentrations of ADP stimulate the release of prostacyclin from bovine aortic endothelial cells

ADP (0.2-200 microM) stimulated the synthesis of prostacyclin (PGI2), as reflected by the release of 6-keto-prostaglandin F1 alpha (6-K-PGF1 alpha), in endothelial cells cultured from bovine aorta. This effect of ADP was mimicked by ATP, whereas AMP and adenosine were completely inactive. The release of 6-K-PGF1 alpha triggered by ADP was rapid and onset (within 5 min), transient (10 min) and followed by a period of refractoriness to a new ADP challenge. Growing and confluent cells were equally responsive to ADP. ADP stimulated the release of free arachidonic acid from the endothelial cells. ADP could thus exert two opposite actions on platelet aggregation in vivo: a direct stimulation and an inhibition mediated by PGI2. This last action might contribute to limit thrombus formation to areas of endothelial cell damage.     

Homologous desensitization of ATP-mediated elevations in cytoplasmic calcium and prostacyclin release in human endothelial cells does not involve protein kinase C.

The interaction of echinomycin with a kinetoplast DNA fragment which contains phased runs of adenine residues has been examined by various footprinting techniques. DNAase I footprinting confirms that all drug-binding sites contain the dinucleotide CpG. However, not all such sequences are protected. Three sites, each of which is located between two adenine tracks in the sequence GCGA, are not protected from DNAase I attack. Enhanced cleavage by DNAase I, DNAase II and micrococcal nuclease is observed in regions surrounding drug-binding sites. The results suggest that echinomycin alters the conformation of the AT tracks, making them more like an average DNA structure. Echinomycin renders adenine residues in the sequence CGA hyper-reactive to diethyl pyrocarbonate.     

Physiological concentrations of ADP stimulate the release of prostacyclin from bovine aortic endothelial cells

ADP (0.2−200 μN) stimulated the synthesis of prostacyclin (PGI₂), as reflected by the release of 6-keto-prostaglandin F_1α (6-K-PGF_1α), in endothelial cells cultured from bovine orta. This effect of ADP was mimicked by ATP, whereas AMP and adenosine were completely inactive. The release of 6-K-PGF_1α triggered by ADP was rapid in onset (within 5 min), transient (10 min) and followed by a period of refractoriness to a new ADP challenge. Growing and confluent cells were equally responsive to ADP. ADP stimulated the release of free arachidonic acid from the endothelial cells. ADP could be thus exert two opposite actions on platelet aggregation in vivo: a direct stimulation and an inhibition mediated by PGI₂. This last action might contribute to limit thrombus formation to areas of endothelial cell damage.     

Histamine stimulates prostacyclin synthesis in cultured human umbilical vein endothelial cells

Human venous endothelial cells synthesize prostacyclin (PGI₂) in response to treatment with histamine. The amount of PGI₂ produced is proportional to the histamine concentration over the range of 10^?7 to 10^?5 M, with a maximal response at 2–5 × 10^?6 M. PGI₂ synthesis occurs as a burst lasting less than 3 minutes after histamine addition. The H1 histamine receptor antagonist pyrilamine causes an 87% inhibition of PGI₂ synthesis, whereas the H2 antagonist cimetidine gives no significant inhibition, suggesting that PGI₂ synthesis in response to histamine is mediated by an H1 receptor.     

Alpha-thrombin induces release of platelet-derived growth factor-like molecule(s) by cultured human endothelial cells

Cultured endothelial cells secrete a platelet-derived growth factor-like molecule (PDGFc). We examined the effects of purified human alpha-thrombin on the production of PDGFc in cultures of human umbilical vein endothelial cells (HUVE) using a specific radioreceptor assay for PDGF. Addition of physiologically relevant concentrations of alpha-thrombin (0.1 to 10 U/ml) induced a time- and dose-dependent increase in the release of PDGFc into the culture medium. Significant stimulation of PDGFc release was observed as early as 1.5 h after addition of alpha-thrombin (10 U/ml) with a 4.9 +/- 1.1 fold increase at 24 h (mean +/- SEM of nine experiments, P less than 0.01). alpha-Thrombin treatment of HUVE did not affect cell viability as assessed by trypan blue dye exclusion. The receptor binding of PDGFc secreted by HUVE in response to alpha-thrombin was inhibited by monospecific antibody to purified human PDGF indicating that the molecule(s) is closely related to PDGF. alpha-Thrombin inactivated with diisopropylfluorophosphate was without stimulatory effect. Lysis of HUVE by repeated cycles of freeze/thaw released minimal PDGFc (less than 0.3 ng per 10(6) cells) compared to levels of PDGFc released into supernatant medium in response to alpha-thrombin (greater than 5.0 ng per 10(6) cells after a 24-h incubation with 10 U/ml alpha-thrombin). Moreover, incubation of freeze/thaw lysates of HUVE with alpha-thrombin failed to release PDGFc. Over a 3-h time course, however, alpha-thrombin-induced secretion of PDGFc was not prevented by cycloheximide. We conclude that alpha-thrombin induces secretion of PDGFc from HUVE by a nonlytic mechanism requiring the serine esterase activity of the enzyme. Although this effect does not initially require de novo protein synthesis, it does require cell-mediated conversion of PDGFc from an inactive to an active form.     

Lipoteichoic acid inhibits lipopolysaccharide-induced adhesion molecule expression and IL-8 release in human lung microvascular endothelial cells

Cell adhesion molecule expression (CAM) and IL-8 release in lung microvascular endothelium facilitate neutrophil accumulation in the lung. This study investigated the effects of lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria, alone and with LPS or TNF-alpha, on CAM expression and IL-8 release in human lung microvascular endothelial cells (HLMVEC). The concentration-dependent effects of Staphylococcus aureus (S. aureus) LTA (0.3-30 microg/ml) on ICAM-1 and E-selectin expression and IL-8 release were bell shaped. Streptococcus pyogenes (S. pyogenes) LTA had no effect on CAM expression, but caused a concentration-dependent increase in IL-8 release. S. aureus and S. pyogenes LTA (30 microg/ml) abolished LPS-induced CAM expression, and S. aureus LTA reduced LPS-induced IL-8 release. In contrast, the effects of S. aureus LTA with TNF-alpha on CAM expression and IL-8 release were additive. Inhibitory effects of LTA were not due to decreased HLMVEC viability, as assessed by ethidium homodimer-1 uptake. Changes in neutrophil adhesion to HLMVEC paralleled changes in CAM expression. Using RT-PCR to assess mRNA levels, S. aureus LTA (3 microg/ml) caused a protein synthesis-dependent reduction (75%) in LPS-induced IL-8 mRNA and decreased the IL-8 mRNA half-life from >6 h with LPS to approximately 2 h. These results suggest that mechanisms exist to prevent excessive endothelial cell activation in the presence of high concentrations of bacterial products. However, inhibition of HLMVEC CAM expression and IL-8 release ultimately may contribute to decreased neutrophil accumulation, persistence of bacteria in the lung, and increased severity of infection.     

Staphylococcus aureus infection of human endothelial cells potentiates Fc receptor expression

Vasculitis, a recognized complication of staphylococcal-endovascular infections, may result in part, from the expression of FcR by Staphylococcus aureus-infected endothelial cells. FcR were measured using [51]Cr labeled SRBC preincubated with rabbit anti-SRBC IgG. FcR were not detected on uninfected endothelial cells, but were demonstrated on S. aureus infected cells using IgG, but not IgM labeled SRBC. FcR expression was dependent on the initial bacterial density (greater than or equal to 8 x 10(7) cfu/ml) and on phagocytosis of the staphylococci, but not on new protein synthesis. IgG labeled SRBC binding was blocked by aggregated IgG but not IgM. SRBC coated with the F(ab')2 portion of IgG did not bind, thus confirming that FcR were specifically involved in this interaction. FcR are expressed after S. aureus invasion of human endothelial cells and may contribute to the vasculitis which often accompanies S. aureus-endovascular infections.