Epigallocatechin gallate increase the prostacyclin production of bovine aortic endothelial cells

We describe the effect of (-) epigallocatechin gallate (EGCg), one of catechins known in tea, on the prostacyclin (PGI) production by bovine aortic endothelial cells. The amounts of 6-keto-PGF(1alpha) and Delta(17)-6-keto-PGF(1alpha), stable metabolites of PGI(2) and PGI(3), released in culture medium were measured using gas chromatography/selected ion monitoring (GC/SIM). The prostacyclin production of endothelial cells was increased by EGCg in a dose- and time-dependent manner. The effect by EGCg was stronger than any other catechins (catechin, epicatechin, epigallocatechin, and epicatechin gallate). When endothelial cells incubated with EGCg and arachidonic acid (AA) or eicosapentaenoic acid (EPA), PGI(2), and PGI(3) production were increased greater than those incubated with AA or EPA alone. Furthermore, gallic acid, that also has a pyrogallol structure, increased PGI(2) production. These observations indicate that catechins increase the prostacyclin production and that the pyrogallol structure is significant to this function.     

Recovery of prostacyclin capacity of irradiated endothelial cells and the protective effect of vitamin C

Ionizing irradiation has been reported to affect prostacyclin (PGI2) production by intact blood vessels and cultured endothelial cells (EC) due to damage of enzymes of the arachidonate cascade. In the present study, we investigated whether EC can recover from radiation injury and regain their capacity to produce PGI2. Bovine aortic EC were exposed to radiation doses of 3 and 6 Gy and their capacity to produce PGI2 in response to stimulation with arachidonic acid was tested, at various times after irradiation. The results of these experiments showed clearly that EC exposed to single or fractionated irradiation could recover their capacity to produce PGI2 depending on the radiation dose and the time period following radiation. Radiation damage is associated with oxidant stress and the production of free radicals. We therefore tested the ability of an oxygen radical scavenger, vitamin C, to protect the capacity of irradiated EC to produce PGI2. Pretreatment of EC with low concentrations of vitamin C inhibited the radiation induced release of PGI2 to the culture medium. Vitamin C also enhanced the capacity of irradiated EC to produce PGI2 following short stimulation with arachidonic acid. Treatment with this scavenger however, did not protect the cells against the cytopathic effects of radiation.     

Reorganization of endothelial cells cytoskeleton during formation of functional monolayer in vitro

Endothelium lining the inner surface of vessels regulates permeability of vascular wall by providing exchange between blood circulation in vessels and tissue fluid and therefore performs a barrier function. Endothelial cells (ECs) in culture are able to maintain the barrier function peculiar to cells of vascular endothelium in vivo. The endothelial monolayer in vitro is a unique model system that allows studying interaction of cytoskeletal and adhesive structures of endotheliocytes from the earliest stages of its formation. In the present work, we described and quantitatively characterized the changes of EC cytoskeleton from the moment of spreading of endotheliocytes on glass and the formation of the first contacts between neighbor cells until formation of a functional confluent monolayer. The main type of intermediate filaments of ECs are vimentin filaments. At different stages of endothelial monolayer formation, disposition of vimentin filaments and their amount do not change essentially, they occupy more than 80% of the cell area. Actin filaments system of endotheliocytes is represented by cortical actin at the cell periphery and by bundles of actin stress fibers organized in parallel. With formation of contacts between cells in native endothelial cells, the number of actin filaments rises and thickness of their bundles increases. With formation of endothelial monolayer, there are also changes in the microtubules system—their number increases at the cell edge. At all stages of EC monolayer formation, the number of microtubules in the region of the already formed intercellular contacts exceeds the number of microtubules in the free lamella region of the cell.     

Cultured endothelial cells produce heparinlike inhibitor of smooth muscle cell growth

Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 μg/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.     

Culturing of human vascular endothelial cells strongly affects their endothelin-1 and prostacyclin production

Cultured human umbilical vein endothelial cells (HUVECs) are a widely used model to study the regulation of endothelial production of vasoactive substances such as endothelin-1 (ET-1) and prostacyclin (PGI₂) in human. As even short term culturing is known to affect the function of many cell types, we studied whether there are differences in the production of ET-1 and PGI₂ between freshly isolated HUVECs and HUVECs cultured for two passages, and whether variation in cell density affects the production of ET-1 and PGI₂ by these cells. At confluency, freshly isolated HUVECs produced only from one-tenth to one-fifth of ET-1, but 46-86 times more PGI2 (p < 0.001), when compared to respective productions by similar amounts of cultured HUVECs. When the cell density of freshly isolated HUVECs was lowered either by diluting the cell suspension or by plating the same amount of cells on different size wells, the production of ET-1 increased: lowering cell density to one-tenth led to 18 fold increase in ET-1 production (p < 0.001). PGI₂ production was not affected by cell density. Thus our data imply that the production of both ET-1 and PGI₂ are differently regulated in freshly isolated and cultured HUVECs, and that cell density is an important determinant in the regulation of ET-1 production.     

Aortic endothelial cells synthesize a large chondroitin sulphate proteoglycan capable of binding to hyaluronate.

Confluent cultures of mouse aortic endothelial (END-D) were incubated with either [35S]methionine or 35SO4 2-, and the radiolabelled proteoglycans in media and cell layers were analysed for their hyaluronate-binding activity. The proteoglycan subfraction which bound to hyaluronate accounted for about 18% (media) and 10% (cell layers) of the total 35S radioactivity of each proteoglycan fraction. The bound proteoglycan molecules could be dissociated from the aggregates either by digestion with hyaluronate lyase or by treatment with hyaluronate decasaccharides. Digestion of [methionine-35S]proteoglycans with chondroitinase and/or heparitinase, followed by SDS/polyacrylamide-gel electrophoresis, indicated that the medium and cell layer contain at least three chondroitin sulphate proteoglycans, one dermatan sulphate proteoglycan, and two heparan sulphate proteoglycans which differ from one another in the size of core molecules. Among these, only the hydrodynamically large chondroitin sulphate species with an Mr 550,000 core molecule was shown to bind to hyaluronate. A very similar chondroitin sulphate proteoglycan capable of binding to hyaluronate was also found in cultures of calf pulmonary arterial endothelial cells (A.T.C.C. CCL 209). These observations, together with the known effects of hyaluronate on various cellular activities, suggest the existence of possible specialized functions of this proteoglycan subspecies in cellular processes characteristic of vascular development and diseases.     

Docosatetraenoic acid in endothelial cells: formation, retroconversion to arachidonic acid, and effect on prostacyclin production

Cultured bovine aortic endothelial cells convert arachidonic acid to docosatetraenoic acid and also take up docosatetraenoic acid from the extracellular fluid. After a 24-h incubation with biosynthetically prepared [3H]docosatetraenoic acid, about 20% of the cellular fatty acid radioactivity was converted to arachidonic acid. Furthermore, in pulse-chase experiments, the decrease in phospholipid docosatetraenoic acid content was accompanied by an increase in arachidonic acid, providing additional evidence for retroconversion. These findings suggest that one possible function of docosatetraenoic acid in endothelial cells is to serve as a source of arachidonic acid. The endothelial cells can release docosatetraenoic acid when they are stimulated with ionophore A23187, but they do not form appreciable amounts of eicosanoids from docosatetraenoic acid. Enrichment of the endothelial cells with docosatetraenoic acid reduced their capacity to produce prostacyclin (PGI2) in response to ionophore A23187. This may be related to the fact that docosatetraenoic acid enrichment caused a 40% reduction in the arachidonic acid content of the inositol phosphoglycerides. In addition, less prostacyclin was formed when the enriched cells were incubated with arachidonic acid, suggesting that docosatetraenoic acid also may act as an inhibitor of prostaglandin synthesis in endothelial cells.     

Human chorion cells respond to growth factors but lose steroidogenic capacity in primary monolayer cell culture

Summary This study has defined a method for preparation and monolayer culture of cells fromchorion laeve. Cell number and cell protein content are table over 7 d in culture. The cells will divide in response to epidermal growth factor in the presence of a supplemented, enriched medium and a collagen matrix, but they lose steroidogenic activity over time in culture. This culture system can be used as the starting point for the development of a chemically defined hormone-supplemented, serum-free culture system for studies of chorion cell differentiation and fetal membrane cell interactions. This work was supported by the Medical Research Council of Canada through Grant MT-7867 (to A. K. T.), Grant MA-7731 (to B. F. M.), and a Summer Studentship (to K. A. F.).     

Possible inhibitory function of endogenous 15-hydroperoxyeicosatetraenoic acid on prostacyclin formation in bovine aortic endothelial cells

Arachidonic acid is metabolized via the cyclooxygenase pathway to several potent compounds that regulate important physiological functions in the cardiovascular system. The proaggregatory and vasoconstrictive thromboxane A2 produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of prostacyclin (prostaglandin I2) synthesized by blood vessels. Furthermore, arachidonic acid is metabolized by lipoxygenase enzymes to different isomeric hydroxyeicosatetraenoic acids (HETE's). This metabolic pathway of arachidonic acid was studied in detail in endothelial cells obtained from bovine aortae. It was found that this tissue produced 6-ketoprostaglandin F1 alpha as a major cyclooxygenase metabolite of arachidonic acid, whereas prostaglandins F2 alpha and E2 were synthesized only in small amounts. The monohydroxy fatty acids formed were identified as 15-HETE, 5-HETE, 11-HETE and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The latter two compounds were produced by cyclooxygenase activity. Nordihydroguaiaretic acid (NDGA), a rather selective lipoxygenase inhibitor and antioxidant blocked the synthesis of 15- and 5-HETE. It also strongly stimulated the cyclooxygenase pathway, and particularly the formation of prostacyclin. This could indicate that NDGA might exert its effect on prostacyclin levels by preventing the synthesis of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a potent inhibitor of prostacyclin synthetase. 15-HPETE could therefore act as an endogenous inhibitor of prostacyclin production in the vessel wall.     

Structural and functional alterations in the surface of vascular endothelial cells associated with the formation of a confluent cell monolayer and with the withdrawal of fibroblast growth factor

Vascular endothelial cells cultured in the presence of fibroblast growth factor (FGF) devide actively when seeded at low or clonal cell densities and upon reachin confluence adopt a morphologic appearance and differentiated properties similar to those of the vascular endothelium in vovi. In this review, we present some of our recent observations regarding the characteristics (both structural and functional) of these endothelial cells and the role of FGF in controlling their proliferation and normal differentation. At confluence the endothelial cells from a monolayer of closely apposed and nondividing cell that have a nonthrombogenic apical surface and can no longer internalize bound ligands such as low-density lipoprotein (LDL). The adoption of these properties is correlated and possibly causally related to changes in the cell surface such as the appearance of a 60,000 molecular weight protein (CSP-60); the disappearance of fibronectin from the apical cell surface and its concomitant accumulation in the basal lamina; and a restriction of the lateral mobility of various cell surface receptor sites. In contrast, endothelial cells that are maintained in the absence of FGF undergo within three passages alterations that are incompatible with their in vivo morphologic apperarance and physiologic beharior. They grow at confluence on top of each other and hence can no longer adopt both the structural (CSP-60, cell surface polarity) and functional (barrier function, nonthrombogenicity) attributes of differentiated endothelial cell. Since these characteristics can be reacquired in response to readdition of FGF, in addition to being a mitogen FGF may also be involved in controlling the differentitation and phenotypic expression of the vascular endothelium.     

Volume changes of an endothelial cell monolayer on exposure to anisotonic media

The osmotic process plays an important role in controlling the distribution of water across cell membranes and thus the cell volume. A system was designed to detect the volume changes of an endothelial cell monolayer when cells were exposed to media with altered osmolalities. Electrodes housed in a flow chamber measured the resistance of ionic media flowing over a cultured cell layer. Assuming the cell membrane acts as an electrical insulator, volume changes of the cell layer can be calculated from the corresponding changes in chamber resistance. The media used in the experiments had osmolalities in the range 120-630 mmol/kg. When cells were exposed to hypertonic media, there was rapid shrinkage with an approximate 30% reduction in total cell volume for a twofold increase in osmolality. On exposure to hypotonic media, the cells initially swelled with an approximate 20% volume increase for a decrease in osmolality by half. With sustained exposure to low osmolality media, there was a gradual and partial return of cell volume towards isotonic values that started 10 minutes after and was complete within 30 minutes of the osmolality alteration. This finding suggests regulatory volume decrease (RVD); however, no regulatory volume increase (RVI) was observed with the continued exposure to hypertonic media over 45 minutes.     

Cultured chick-embryo heart cells respond differently to ouabain as measured by the increase in their intracellular Na+ concentration.

Using digital imaging microscopy with the sodium-sensitive fluorescent indicator sodium-binding benzofuran isophtalate (SBFI), we examined the cytosolic free sodium ion concentration ([Na+]i) in single chick-embryo heart cells. The distribution of the [Na+]i was homogeneous within one cell, but we found a wide cell to cell variation in the range of 3 to 18 mM [Na+]i. In contrast to former experiments showing a heterogeneity of chick-embryo heart cells with respect to their [Ca2+]i (Ahlemeyer et al. (1992) Eur. J. Biochem. 205, 269-275), we could not distinguish cell populations with different [Na+]i. We found a lognormal distribution of the resting [Na+]i with a median value of 8.8 mM with a standard deviation of 4.5 mM (n = 90). After the addition of varying concentrations of ouabain, we found a biphasic dose-response curve as measured by the increase in [Na+]i. Ouabain showed its half-maximal effect on the [Na+]i between 10(-9) M and 10(-8) M and at 4.3.10(-6) M under steady-state conditions. The finding of a heterogeneity of chick-embryo heart cells with respect to their ouabain-induced increase in [Na+]i is consistent with our previous observations of cells differing in their [Ca2+]i and in the sensitivity of their sodium pumps to cardiac glycosides.     

Vascular endothelial cells synthesize a plasma membrane protein indistinguishable from the platelet membrane glycoprotein IIa

To define the role of membrane components that function in endothelial cell physiology and to characterize them biochemically, we have attempted to prepare monoclonal antibodies specific for endothelial cells. Several clones were obtained producing antibodies which bound to endothelial cells and also to platelets. The antibody of one of these clones, CLB-HEC 75, was studied in more detail. This antibody is directed against a single protein which is synthesized constitutively by endothelial cells and is expressed on the surface of both endothelial cells and platelets. The CLB-HEC 75 antigen was isolated from Nonidet P-40-solubilized endothelial cells and platelets by immunoprecipitation and exhibited an apparent molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 145,000 in the presence of 2-mercaptoethanol. Two-dimensional polyacrylamide gel electrophoresis and crossed immunoelectrophoresis revealed that the mobility of the CLB-HEC 75 antigen relative to platelet glycoproteins Ib, IIa, IIb, and IIIa fits previously defined criteria for platelet membrane glycoprotein IIa. The CLB-HEC 75 antigen isolated from endothelial cells co-migrated under all conditions tested with the antigen from platelets. These results indicate that endothelial cells share a plasma membrane protein indistinguishable from platelet membrane glycoprotein IIa. This protein may be a component involved in the interaction of endothelial cells with their environment including coagulation factors, platelets, and the subendothelial matrix. CLB-HEC 75 may serve as a useful tool for studying these processes.     

Retraction of cultured endothelial cell monolayer by human breast cancer cells, MCF-7.

A human breast cancer cell line (MCF-7), when sealed on confluent bovine pulmonary aortic endothelial cell (CPAE) monolayers, induced morphological changes (retraction) in CPAE cells. The area of retraction depended on the incubation time and the number of MCF-7 cells, suggesting that MCF-7 cells had the capacity to retract CPAE cells. This capacity was reduced by 60% by pretreatment of MCF-7 cells with 17β-estradiol (E) and progesterone (Pg). The extent of retraction was not affected by the addition of various protease inhibitors. CPAE retraction was induced also by adding conditioned medium (CM) from the culture of MCF-7 cells. Considerably less activity was detected in the CM obtained from MCF-7 cells cultured in the presence of E and Pg. The retraction was reversed in 24 h by culturing the monolayer in fresh medium without CM and was not induced by trypsin treatment of the CM.     

Correlation between trypsin binding to a specific receptor and prostacyclin production in cultured vascular endothelial cells

The correlation between the binding and processing of trypsin and its effect on prostacyclin (PGI2) production in cultured adult bovine aortic endothelial (ABAE) cells was studied. ABAE cells demonstrated an ability to produce PGI2 in a dose-response manner to trypsin at the range of 0.1-2.0 micrograms/ml with a saturation at a concentration of 1 microgram/ml. Likewise, 125I-trypsin binding to the cultured cells increased in a dose-response way and reached saturation at a concentration of about 1 microgram/ml; 125I-trypsin was bound to a specific high-affinity cell-surface receptor with a dissociation constant (Kd) of 1.5 X 10(-8) M and each of the confluent ABAE cells has about 1.2 X 10(5) such receptors sites. The cell-surface receptor for trypsin displayed specific characteristics and an excess amount of unlabeled trypsin successfully abolished 125I-trypsin binding while thrombin in excess failed to compete for 125I-trypsin binding. Only a small fraction of the cell-surface-bound 125I-trypsin was internalized and subsequently degraded by ABAE cells as compared to the process of 125I-trypsin internalization by human skin fibroblasts (HSF). This study demonstrated that the stimulatory effect of trypsin on prostacyclin production and release by ABAE cells might be mediated by a specific cell-surface receptor for trypsin on these cells distinct from the thrombin receptor.     

Cultured endothelial cells do not respond to a platelet-derived growth-factor-like protein in an autocrine manner

Cultured endothelial cells produce a growth factor similar or identical to platelet-derived growth factor (PDGF). Endothelial cells are able to proliferate in plasma-supplemented medium, while most nontransformed cells require serum-supplemented medium. Since PDGF is a major serum mitogen, we have tested the possibility that endothelial cells interact with and respond to the autologously produced PDGF-like (PDGF-c) protein. We have found that bovine aortic and rat heart endothelial cells express little or no cell surface PDGF receptors as determined by binding of pure 125I-PDGF. Treating these cells under acidic conditions, which release receptor-bound PDGF in control cells without affecting receptor function, did not reveal a population of cryptic receptors. In addition, when rat heart endothelial cells were grown in the presence of an antibody to PDGF, proliferation was unimpaired, though no detectable free PDGF was present in the medium. An equivalent amount of antibody completely blocked the mitogenic response of human fibroblasts that had been preincubated for 1 h at 37 degrees C with an equivalent dose of PDGF. Thus, endothelial cells do not respond mitogenically in a manner that would be expected from the interaction of autologously produced PDGF with its cell surface receptor. Endothelial cells were detergent-solubilized and immobilized on nitrocellulose in an attempt to detect the presence of intracellular PDGF receptors. Specific binding of 125I-PDGF to adsorbed, solubilized bovine aortic or rat heart endothelial cells was undetectable, though significant binding to adsorbed, solubilized fibroblasts, used as a positive control, was observed. We conclude that endothelial cells do not have detectable intracellular PDGF receptors.     

Cultured gastrointestinal smooth muscle cells: cell response to contractile agonists depends on their phenotypic state

In the digestive tract, the transit of ingested food induces a local contraction-relaxation reflex of which the smooth muscle cell (SMC) represents the functional unit. Although freshly isolated SMCs have been extensively used for in vitro studies, in specific cases cultured cells appear necessary. Because conventionally cultured SMCs lose their contractile properties, we have developed: (1) differentiated, contractile rabbit gastric SMCs (D-stim cells), cultured in a medium supplemented with insulin, and (2) proliferative, dedifferentiated rabbit gastric SMCs (P-stim cells), cultured in a medium supplemented with insulin, fetal serum, EGF and b-FGF. The proliferative index was 5±4% and 82±10%, respectively, for D-stim and P-stim cells. Expression of SM-myosin heavy chain was observed in 90% of D-stim cells, whereas it was progressively lost in P-stim cells. Carbachol (1–100 nM), glicentin (2 nM) and gastrin-17 (100 nM) induced contraction of D-stim cells cultured for 3 or 6 days, whereas they did not induce the contraction of P-stim cells; in contrast, gastrin-17 (10 nM) was able to stimulate DNA synthesis (1.86±0.09-fold increase) in P-stim cells. The coupling of muscarinic receptors to intracellular transduction pathways was evaluated in D-stim cells: at day 3, carbachol (100 nM) induced a twofold increase in the production of inositol tri-tetra-phosphates; in parallel, a phosphorylation of ERK MAP kinases occurred within 1 min of carbachol stimulation. In conclusion, cultured functional myocytes derived from mature tissue may be used for long-term studies concerning the events coupled either to proliferation or to motility regulation of differentiated SMCs due to the activation of G-protein-coupled receptors.This study was supported in part by grants from the AFM (Association Française contre les Myopathies).     

Stimulation of cell growth and inhibition of prostacyclin production by heparin in human umbilical vein endothelial cells

We characterized human umbilical vein (HUV) endothelial cells as to cell growth and prostacyclin production to get a better understanding of the properties of endothelial cells. Endothelial cell growth supplement (ECGS) and basic fibroblast growth factor (FGF) stimulated HUV endothelial cell growth. Heparin further enhanced the cell growth stimulated by ECGS, but not the cell growth stimulated by FGF or in the absence of these growth factors. In the presence of ECGS, the prostacyclin-producing capacity of the cells was inhibited by heparin. However, in the presence of FGF of in the absence of growth factors, heparin did not inhibit prostacyclin production. Therefore, it is likely that there is a specific correlation between heparin and growth factors for endothelial cells in the blood vessel to maintain nonthrombogenicity properly. Heparin-treated cultures may not be suitable for some examinations of prostacyclin production by vascular endothelial cells.