The lupus anticoagulant stimulates the release of prostacyclin from human endothelial cells

Decreased endothelial cell production of prostacyclin (PGI₂) in response to the lupus anticoagulant has been previously demonstrated and postulated to have a causal relationship to the thrombotic events associated with the lupus anticoagulant. Five patients who exhibited the anticoagulant were studied in an effort to determine if a relationship exists between exposure of endothelial cells to the lupus anticoagulant and decreased production of PGI₂. Human endothelial cells derived from human umbilical vein grown in culture were exposed to IgG fractions of patient plasmas containing the lupus anticoagublant. PGI₂ released per 10 cells was determined by radioimmunoassay for 6-keto-PGF-1-alpha. The6overall means for the patient and control groups are given by 47 pM/10⁶ cells and 12 pM/10 cells respectively. This is a statistically significant difference (F-10.65, p-0.017) when the effects of different batches of endothelial cells and thrombin stimulation are adjusted for in the analysis of variance model. These results demonstrate that in this homologous human system exposure of endothelial cells to the lupus anticoagulant leads to stimulation rather than inhibition of PGI₂ release.     

Hirudin stimulates prostacyclin but not endothelin-1 production in cultured human vascular endothelial cells

To study the effect of hirudin on endothelial cell prostacyclin (PGI₂) and endothelin-1 (ET-1) production, we cultured human umbilical vein endothelial cells (HUVECs), stimulated them with 0.00001–10 kU/l of hirudin for 12–24 hours, and measured by radioimmunoassays the concentrations of 6-ketoprostaglandinF_{1 alfa} (6-keto, a metabolite of PGI₂) and ET-1 in the incubation medium. In incubation medium containing 10% serum hirudin stimulated PGI₂-production dose-dependently. The lowest stimulatory hirudin concentration was 0.001 kU/l, which increased the concentration of 6-keto by 10.8±4.4% (mean±S.E) (p<0.01). The greatest stimulation rate (28.6±6.2 %, p<0.001) was obtained with the highest hirudin concentration (10 kU/l), when the culture medium contained 10% human serum. The PGI₂-stimulating activity was exaggerated in the absence of serum, when 1 kU/l of hirudin increased PGI₂-production by 59.7±6.2% (p<0.001, N=14). Stimulation of PGI₂ appeared after 12 hour incubation. Hirudin had no effect on the conversion of exogenous arachidonic acid to 6-keto or on the production of ET-1. We thus conclude that hirudin stimulates PGI₂-production through de novo protein synthesis. Stimulation of PGI₂-production by hirudin may contribute to its antithrombotic activity, since PGI₂ favours vasodilatation and attenuates platelet aggregation.     

Prostaglandin synthesis in bovine coronary endothelial cells: comparison with other commonly studied endothelial cells

Comparison of arachidonic acid metabolism by bovine coronary artery endothelial cells, bovine aortic endothelial cells and human umbilical endothelial cells indicated potentially important differences in relative amounts of the different prostaglandins produced. Bovine coronary endothelial cells converted ¹⁴C-arachidonic acid to radioactive 6-keto PGF₁ (the stable metabolite of PGI₂) and to a lesser extent PGE₂. Bovine aortic cells synthesized 6-keto PGF₁ and 6,15-diketo PGF₁ as the major products. PGE₂, 6-keto PGE₁, PGE₂, and PGD₂ were minor metabolites. By comparison, endothelia cells isolated from human umbilical artery or vein formed mainly 6-keto PGF₁ and substantial amounts of PGF₂, PGE₂ and PGD₂. Basal concentrations of 6-keto PGF₁ were two-fold higher in bovine coronary cells than in bovine aortic endothelial cells, but seven-fold less than in endothelial cells cultured from human umbilical vessels. Histamine, bradykinin and thrombin stimulated PGI₂ synthesis in both coronary endothelial cells and human umbilical cells, but only bradykinin stimulated PGI₂ synthesis in bovine aortic cells. This comparative study indicates that endothelial cells vary in the metabolites of arachidonic acid that they produce depending upon the vascular origin of the cells. Also, endothelial cells from different vascular beds respond differently to specific vasoactive agents.     

Epidermal growth factor stimulates prostacyclin production by cultured human vascular endothelial cells

Epidermal growth factor (EGF) stimulated prostacyclin (PGI2) production by cultured human umbilical vein endothelial cells, as measured by radioimmunoassay of its stable metabolite 6-keto-prostaglandin F1 alpha. This effect of EGF was dose-dependent, the lowest stimulatory concentration of EGF was 1.0 ng/ml and 100 ng/ml caused a 2.7 +/- 0.3 (mean +/- SEM) fold increase in the PGI2 synthesis. The stimulation appeared at 3-6 h of incubation and lasted at least 24 h. It was suppressed by EGF antibodies and blocked by protein synthesis inhibitor cycloheximide. Cells preincubated 12 h with EGF released also higher amounts of PGI2 when incubated with thrombin for 5 min. It is concluded that EGF liberated from platelets during aggregation may prevent local thrombogenesis and atherogenesis by stimulating the release of the antiaggregatory, vasodilatory PGI2 from vascular endothelial cells.     

Ethanol stimulates prostacyclin biosynthesis by human neutrophils and potentiates anti-platelet aggregatory effects of prostacyclin

Previous reports on the direct effects of ethanol on human platelet aggregation function have been inconsistent. Ethanol ingestion produces vasodilation and raises intracellular cyclic AMP concentrations, effects similar to those of prostacyclin. We, therefore, hypothesized that ethanol may influence biosynthesis and/or bioactivity of prostacyclin. In our experiments, ethanol in concentrations up to 400 mg% had no consistent inhibitory effect on platelet aggregation in response to epinephrine, ADP, or combination of subthreshold concentrations of epinephrine plus ADP. However, ethanol in concentrations as low as 10 mg% potentiated the platelet aggregation inhibitory effects of prostacyclin. In addition, ethanol (20 mg%) decreased formation of thromboxane A₂ in whole blood by 41% and stimulated formation of prostacyclin by 160% (both P c 0.01). Additional studies using isolated human cells demonstrated synthesis of prostacyclin by neutrophils in the presence of platelets, and this neutrophil prostacyclin formation was enhanced in the presence of ethanol. These effects of alcohol in concentrations achieved after moderate intake may relate to the hemodynamic, biochemical, and cardioprotective effects of ethanol.     

全反式维甲酸对胶质瘤干细胞VEGF和bFGF表达的影响

目的 研究全反式维甲酸(ATRA)对胶质瘤干细胞(GSCs)血管内皮细胞生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)表达的影响. 方法 从人胶质母细胞瘤细胞系U87中分离培养GSCs,免疫荧光染色检测CD133、巢蛋白(nestin)的表达进行鉴定.将取GSCs分成3组分别培养:(1)ATRA组:培养基中含10 nmol/L ATRA;(2)空载体组:培养基中含与ATRA组等量的二甲基亚砜(DMSO);(3)对照组:单纯培养基,培养10 d后免疫荧光染色检测胶质纤维酸性蛋白(GFAP)、β-微管蛋白Ⅲ(β-tubulinⅢ)、半乳糖脑苷脂(GalC)的表达;CCK8法检测各组细胞的增殖;ELISA和RT-PCR分别检测各组GSCs VEGF、bFGF的分泌水平和mRNA的表达. 结果 二代细胞球表达神经干细胞(NSCs)的标记抗体CD133和nestin.免疫荧光染色检测显示分化后GSCs能够分化为多种同源子代细胞(分别表达星形胶质细胞、神经元、少突胶质细胞标志物GFAP、β-tubulinⅢ、Galc);培养10d后ATRA组细胞GFAP的阳性表达率高于对照组及空载体组,差异有统计学意义(P＜0.05).第3.～7天ATRA组细胞增殖速度较对照组和空载体组明显变缓,差异有统计学意义(P＜0.05);分化24 h后ATRA组GSCs VEGF、bFGF的分泌水平和mRNA的表达均少于对照组和空载体组,差异有统计学意义(P＜0.05). 结论 ATRA能诱导GSCs分化并抑制其增殖,其可能通过抑制VEGF和bFGF的表达发挥抗胶质母细胞瘤的作用.     

Minimal effect of estrogens on endothelial cell growth and production of prostacyclin

The effects of natural and synthetic sex hormones (10(-8)M) have been studied on human umbilical endothelial cell proliferation and prostacyclin production. 17 B estradiol and progesterone had no effect on cell multiplication. Ethinyl-estradiol increased proliferation when the initial plating density was 40,000 cells/cm2. However, the production of 6-keto PGF1 alpha induced by the Ca++ ionophore A 23187 was not different between control, 17 B estradiol-or ethinyl estradiol-treated cultures. These results demonstrate an in vitro effect of synthetic estrogen, ethinyl estradiol on endothelial cell proliferation. At the present time it is however difficult to correlate these results with the clinical observation of an increase in thromboembolic complications in women under oral contraceptives.     

In vitro effects of hyperlipoproteinemic sera on human endothelium: Inhibition of endothelial cell migration by familial hypercholesterolemic sera

Since hyperlipoproteinemia is associated with an increased risk of atherosclerosis we have evaluated the effects of sera of different hyperlipoproteinemic clinical patterns on human endothelial cells in vitro. Cultured human umbilical vein endothelial cells were treated with sera from 2 patients homozygous for familial hypercholesterolemia and 4 patients heterozygous for that disorder. Familial hypercholesterolemic sera inhibited endothelial cell migration by 50% during a 72 hour incubation (p <0.0001) compared to normal pooled human serum or single donor AB serum when measured by an agarose gel technique. The inhibition of migration was not observed when cells were treated with familial combined hyperlipidemic sera (4 patients) or familial hypertriglyceridemic sera (5 patients). Endothelial cell detachment in vitro was not induced by any of the classical patterns of hyperlipoproteinemic sera tested. The development of atherosclerosis in familial hypercholesterolemia may be in part related to an impairment of endothelial repair.     

Heparin-like glycosaminoglycan is a receptor for Antithrombin III-dependent but not for thrombin-dependent prostacyclin production in human endothelial cells

Antithrombin III (ATIII) induced a marked increase in prostacyclin (PGI₂) release from cultured human umbilical vein endothelial cells (HUVEC) after incubation for more than 2 hr and the induction continued for 8 hr, while thrombin induced the increase within 10 min. ATIII-dependent production of PGI₂ was abolished by addition of heparin, but pretreatment of HUVEC with polyclonal antibody against thrombomodulin could not prevent the PGI₂ productions by ATIII and thrombin. ATIII-dependent PGI₂ production was significantly inhibited by pretreatment of HUVEC with β- -xylosides or heparitinase, though neither pretreatment affected thrombin-induced PGI₂ production. After treatment of HUVEC with 1 μg/ml cycloheximide, ATIII-dependent PGI₂ production was completely abolished. These results indicate that the mechanism of the induction of PGI₂ production by ATIII involves heparin-like glycosaminoglycans on HUVEC and the stimulation of synthesis of a protein related to PGI₂ production. The ATIII-induced PGI₂ production is very different from that induced by thrombin.     

PPACK attenuates plasmin-induced changes in endothelial integrity

In order to determine whether plasmin affects endothelial cell integrity directly, confluent bovine aortic endothelial cells were treated with plasminogen and a plasminogen activator. The permeability of the monolayer to [¹²⁵I]-albumin was shown to be increased significantly (P < 0.01) with a concomitant decrease in viability. Plasmin activity correlated significantly with endothelial cell permeability (p < 0.004; R = 0.82). Coincubation with D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone, a tripeptide inhibitor of plasmin, reduced the increase in endothelial permeability induced by plasmin by 59% (p = 0.033). Monolayers studied in parallel were stained with rhodamine-phalloidin to visualize F-actin. There were significant morphologic changes in the endothelial monolayers exposed to plasmin compared to control monolayers, and these changes could be attenuated by coincubation with D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone. These studies show that: 1) plasmin induces significant increases in endothelial cell permeability with accompanying morphologic changes; and 2) these deleterious functional and morphologic effects can be attenuated by coincubation with the plasmin inhibitor, D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone.     

Characterization of cytosolic phospholipases C from porcine aortic endothelial cells

Phospholipases C (PLCs) are ubiquitous enzymes which play key roles in the response of cells to extracellular agonists. Endothelial cells are involved in myriad normal and pathophysiologic functions. Although it is known that agonists activate PLCs in endothelial cells, second messengers form, and cellular responses ensue, more knowledge is needed about the specific types of PLCs in these cells. To this end, cytosolic PLCs from porcine aortic endothelial cells were partially purified by ammonium sulfate fractionation and column chromatography on DEAE-Sepharose CL-6B and heparin-agarose. Three PLC isozymes immunologically similar to bovine brain PLC-β, PLC-γ, and PLC-δ were identified. The relative levels of PLC activities in the cytosol were: PLC-β, 50%; PLC-γ, 44%; PLC-δ, 6%. The level of PLC-β activity in porcine endothelial cells appeared higher than the levels reported for several established cell lines, suggesting that this enzyme may play a specific role in endothelial cell function. Elution profiles of PLC activity with phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P₂) as substrate were similar to those with phosphatidylinositol (Ptdlns) as substrate, indicating that cytosolic PLCs hydrolyze both Ptdlns and Ptdlns(4,5)P₂ and no Ptdlns(4,5)P₂-specific PLC was present in the cytosol. The catalytic properties of the partially purified PLC isozymes from porcine endothelial cells were similar to their counterparts from bovine brain. These include the dependence of hydrolysis of Ptdlns on Ca²⁺, the optimal Ca²⁺ concentrations for the hydrolysis of Ptdlns and Ptdlns(4,5)P₂, the pH optima, and the stimulatory effects of deoxycholate.     

Effect of bradykinin and thrombin on prostacyclin synthesis in endothelial cells from calf and pig aorta and human umbilical cord vein

We have discovered that bradykinin is effective in causing the synthesis of prostacyclin in endothelial cells cultured from calf and pig aorta and human umbilical cord vein. Maximal stimulation is attained at 10 ng bradykinin per ml with a 10 fold increase of PGI₂ as assayed by radioimmunoassay for 6-k-PGF_1α. Ionophore A23187, trypsin, and the precursor arachidonic acid are also stimulatory. We have confirmed the study of Weksler $\text{et al}$ ($\text{J. Clin. Invest., 62}$, 923, 1978) and Czervionke $\text{et al}$ ($\text{Thrombosis Research, 14}$, 781, 1979) that thrombin is effective in stimulating PGI₂ synthesis in endothelial cells from human umbilical cord vein. However, we found that cells from calf or pig aorta are not stimulated as well by thrombin. Thus, there appears to be a difference in the response to thrombin between these cells. When calf cells were grown in the presence of [³H]arachidonic acid, the radioactivity is incorporated mainly into the phospholipids. Bradykinin, ionophore A23187, and trypsin stimulated the release of radioactive materials into medium from [³H]arachidonic acid labeled calf cells. Arachidonic acid is the major radioactive substance released and PGI₂ is the major known arachidonic acid metabolite formed.     

Phosphatidylcholine is the major phospholipid providing arachidonic acid for prostacyclin synthesis in thrombin-stimulated human endothelial cells

Upon incubation for 24 hours with [3H]arachidonic acid (AA, 1 mu Ci/ml), cultured endothelial cells from human umbilical vein incorporated one half of the added radioactivity, mostly into phospholipids (83% of the total cell radioactivity). Distribution of the label between the various phospholipid classes was found to reflect the distribution of endogenous AA. Stimulation with human thrombin (2 U/ml) promoted a rapid release of radioactive material into supernatants, which contained essentially 6-keto-prostaglandin F1 alpha and non-converted AA. This process levelled off at 10 min, at which time phosphatidylcholine displayed a decrease accounting for 3.7% of the total cell radioactivity. Phosphatidylinositol also appeared significantly diminished, but this decrease was almost 2.5 fold less than that observed in phosphatidylcholine. It is concluded that AA availability for prostacyclin biosynthesis is mostly regulated by a phospholipase A2.     

Deficiency of a plasma factor stimulating vascular prostacyclin generation in patients with lupus nephritis and glomerular thrombi and its correction by ancrod: In-vivo and in-vitro observations

Glomerular thrombi occur frequently in active lupus nephritis. Their presence has been correlated with low platelet counts and with subsequent development of glomerular sclerosis. We have examined the plasma PGI₂ generating capacity of 8 patients with active lupus nephritis with thrombi that were to undergo defibrination therapy with ancrod. PGI₂ generation by these plasma samples was significantly decreased as compared both to normals and to 6 individuals with lupus nephritis and no glomerular thrombi. Significant improvement in the capacity to generate PGI₂ was seen in the post-ancrod treatment plasma samples. The pathogenesis of this defect is discussed.     

The culture of vascular endothelial cells to confluence on microporous membranes

We have designed a two compartment system in which upper and lower compartments are separated by a layer of vascular endothelial cells grown on a porous membrane. The culture of pig aortic endothelial cells and their growth to confluence on porous PTFE membranes is described and the principal characteristics of membrane-cultured endothelium are compared with those of similar cells cultured on solid surfaces. While growth of endothelium to visual confluence was slower on the PTFE membranes than on solid surfaces, gross morphology and ratio of 6 keto prostaglandin F₁ to prostaglandin E₂ production by the cells was similar. A decrease in fluid flow across the membrane-cultured cells was associated with their growth to visual confluence: from low levels, fluid flow could be increased markedly by altering the environmental temperature of the cells. The possible uses and limitations of such a system in the interpretation of the role of endothelial cells in selective transport and compartmentation of fluid and cellular components of the blood are discussed.     

Factor V in human vascular endothelium and in endothelial cells in culture

Human blood vessels and human umbilical vein endothelial cells in culture were examined for the presence and synthesis of factor V. Factor V antigen was detected on the luminal surface of blood vessels washed with buffers containing calcium but was absent from segments of the same vessels perfused with buffers containing EDTA. Very low levels of endogenous factor V antigen were found in endothelial cells in culture but these cells did not synthesise factor V in sufficient quantities to be detected by the present methods. Factor V activity was not detected in any of the present cell preparations.