JOURNAL OF CELLULAR PHYSIOLOGY 142514-522 (19901
Heparin and Acidic Fibroblast Growth Factor Interact to Decrease Prostacyclin Synthesis in Human Endothelial Cells by Affecting Both Prostaglandin H Synthase and Prostacyclin Synthase BABETTE B. WEKSLER
Division of HernatologyiOncology, Department ot Medicine, Cornell University Medical College, New York, New York 70027 Prostaglandin production by cultured h u m a n endothelial cells varies with growth conditions.We observed a marked diminution in both spontaneous and inducible production of prostacyclin (PGI,) by h u m a n umbilical vein and saphenous vein endothelial cells when they were cultured in the presence of the heparin-binding growth factor, acidic fibroblast growth factor (aFGF) and heparin, compared with PCI, production during culture in medium lacking these tactors. Decreased PGI, production was related to duration of exposure of the cells to aFGF and heparin and depended on the concentration of both substances. Heparin (1-100 Fgiml) strongly potentiated the effects of aFGF but had a limited and variable effect alone. The decrease in PGI, production correlated with a reduction in the cellular content of immunoreactive prostaglandin H synthase and prostacyclin synthase. Arachidonate deacylation was not decreased. In addition, the eicosanoid profile of endothelial cells was changed by exposure to aFGF and heparin. These studies indicate that heparin acts as a modulator of prostaglandin synthesi5 in endothelial cells through its interaction with aFCF, mediated by alterations in two key enzymes in the arachidonate metabolic pathway.
Growth factors for vascular cells may modulate the expression of differentiated cellular characteristics (Saksela et al., 1987; Habenicht et al., 1985; Jaye et al., 1985). When human endothelial cells are cultured in the presence of acidic fibroblast growth factor (aFGF, also called endothelial cell growth factor or heparinbinding growth factor-1) and heparin, we have observed that prostacyclin (PGI,) production is markedly decreased in comparison with PGI, production by the same cultures grown without these supplements. We postulated that aFGF might modulate prostaglandin synthesis in endothelial cells by regulating the level of enzymes involved in arachidonic acid metabolism. This report presents evidence that aFGF and heparin in combination (aFGF/heparin)induce a time- and dosedependent decrease in both prostaglandin H synthase (PGH synthase, cyclooxygenase) and PGI, synthase function in human vascular endothelial cells, without altering enzymatic release of arachidonate. Heparin strongly potentiates the effects of aFGF. Consequently, the capacity of the cells to synthesize prostaglandins declines progressively during exposure of the cells to aFGF and heparin. These results indicate that heparin acts as a modulator of prostaglandin synthesis in endothelial cells through its interaction with aFGF. c, 1990 WILEY-LISS, INC
MATERIALS AND METHODS Materials Tissue culture medium 199 was from Gibco (Grand Island, NY), and disposable plastic flasks and 24-well plastic culture dishes were from Falcon (VWR Scientific, Piscataway, NJ). Culture dishes (96-well) were from Nunc. Crude aFGF (endothelial cell growth supplement) was from Collaborative Research (Waltham, MA), and purified aFGF was a generous gift from Dr. Thomas Maciag, American Red Cross, Rockville, MD. Pooled normal human serum was prepared in our laboratory. I4C-arachidonicacid, 3H-6-keto PGF,,, and 3HTXB, were from New England Nuclear (Boston, MA); unlabeled prostaglandin standards were from Nuchek (Elysian, MN). Ibuprofen was a gift from the Upjohn Co. (Kalamazoo, MI). TLC silica G plates were from Merck, Inc. (St. Louis, MO). Solvents were purchased from Fisher Scientific (Pittsburgh, P A ) and were of nanograde quality. Heparin was from Sigma (St.Louis, MO). Monoclonal anti-PGH synthase and anti-PGI, synthase antibodies were kindly provided by Drs. W. Smith and D. DeWitt, Michigan State University, or were purReceived July 5, 1989; accepted October 30, 1989.
HEPARINIACIDIC FGF ALTER ENDOTHELIAL CELL PGI,
chased from Oxford Biochemicals (Oxford, MI). These antibodies have been well characterized (DeWitt et al., 1982; Smith et al., 1983). Biotinylated anti-mouse IgG and normal horse serum were from Vector (Burlingame, CA), and avidin-alkaline phosphatase conjugates from Enzo Diagnostics (New York, NY).
Culture of endothelial cells Human umbilical vein endothelial cells (EC) were harvested from individual umbilical cords by collagenase treatment and were cultured in gelatin-coated plastic tissue culture dishes in medium 199, containing 20% pooled human serum, glutamine, penicillin, and streptomycin. Cells from different umbilical cords were not pooled. For experiments utilizing growth factors, primary EC from a given culture were divided into two aliquots: one aliquot was subsequently cultured for 1272 hours in the above medium to which aFGF and heparin were added; the other aliquot was cultured in the above medium in the absence of added aFGF and heparin. In some experiments plating densities were adjusted so that cell counts were similar in both types of culture a t the time of testing. The standard concentration of crude aFGF was 40 pgiml and that of heparin 90 pgiml, concentrations widely utilized in published reports to support EC proliferation (for example, D'Amore and Klagsbrun, 1984; Ingerman-Wojenski et al., 1988; Wu et al., 1988; Konkle and Ginsburg, 1988). Crude aFGF was used for all experiments except those specifically designated as using purified aFGF. These concentrations consistently produced doubling of cell numbers in 24-36 hours. Cell viabilities did not differ in the two types of culture conditions. In certain experiments aFGF was varied between 0 and 40 pgiml in the absence or presence of a fixed concentration of heparin, or heparin was varied between 0 and 90 pgiml in the presence of a fixed concentration of aFGF. Purified aFGF was used at 0-500 ng/ml. Human adult venous EC were harvested from normal saphenous veins removed a t the time of coronary artery bypass grafting procedures but not utilized during the surgery, under a protocol approved by the institutional Human Rights Committee. A similar harvesting procedure was used as for umbilical vein EC except that all primary cultures were expanded with aFGFiheparin because of the smaller numbers of cells initially available. Subsequent cultures were carried out with or without aFGFiheparin as detailed above. For experiments in which both prostaglandin production and enzyme measurements were performed, cells were seeded into the 60 central wells of 96-well microtiter plates. For other experiments, 24-well plates were used. For studies of arachidonate release and for analysis of the prostaglandin profile by thin layer radiochromatography, cells were grown in T-75 flasks. Cells were usually used in experiments just prior to confluence, or were cultured for time periods detailed below. Medium was changed the day before the cells were used in a n experiment. Cell counting Endothelial cells were detached from washed monolayer cultures by incubation with 0.02% collagenase0.02% EDTA and were counted in a Coulter Counter model ZBI using a n aperture of 100 pl diameter.
Prostaglandin measurement Prostacyclin and PGE, released into the medium during culture, or elicited by stimulation of washed EC monolayers for 5-15 minutes a t 37°C with sodium arachidonate (25 pM), thrombin (0.5 Uiml), or the calcium ionophore A23187 (1 pM) were measured in unextracted incubation fluids (medium or buffer) by specific radioimmunoassay of the hydrolytic product 6keto PGF,, as previously described (Weksler et al. 1983) for PGI, and by RIA using a commercial kit for PGE,. Fresh incubation fluids were centrifuged to remove any residual cells and were frozen a t -70°C until assay. Arachidonate release Endothelial cells were radiolabeled a t confluence by overnight incubation with 1 pCi 'H-arachidonate in the presence of ibuprofen (100 pM) in tissue culture medium. After removal of the medium, the EC were washed with a t least three changes of HEPES-buffered saline containing 2% fatty acid-free bovine serum albumin to remove unbound 'H-arachidonate, a s ascertained by liquid scintillation counting of the washing fluids. The EC monolayers were then covered with the same buffer-albumin mixture plus 100 pM ibuprofen and were stimulated with 1 Uiml thrombin or 1 pM calcium ionophore, A23187, a t 37°C for 15 minutes. Released 'H-arachidonate, trapped by albumin, was measured by liquid scintillation counting and expressed as a percent of cell-associated arachidonate.
Enzyme-linked immunoassay for PGH synthase and PGI, synthase The enzymes PGH synthase and PGI, synthase were assayed directly using a n enzyme-linked immunoassay (ELISA) on permeabilized fixed monolayers of EC that had been grown in 96-well microtiter plates. The monolayers were washed, the cells were fixed with 0.04% buffered glutaraldehyde and permeabilized with 0.02% Tween in PBS, and nonspecific binding sites were blocked by incubation with 1%horse serum. The cells were then exposed overnight to monoclonal murine antibodies to PGH synthase or PGI, synthase, or to nonspecific mouse immunoglobulin, and amplification of binding was achieved with biotinylated horse-antimouse IgG as second antibody, followed by avidin-alkaline phosphatase. Color was developed by addition of 0.2 M paranitrophenylphosphate and was read at 405 nm in a Titertek Multiscan plate reader. Development was allowed to proceed up to 4 hours, and readings were made along the linear phase of color development. Specific binding was calculated by subtracting the value obtained in the absence of specific primary antibody from that obtained in the presence of primary antibody; triplicate wells were tested for each data point or control. In preliminary studies, specificity of the reaction was established using purified PGH synthase to block binding of the monolconal antibody in a dose-related fashion. Thin layer radiochromatography EC were cultured for 48 hours in the presence or absence of aFGFiheparin in T-75 flasks and were then incubated overnight with 1 pCi 14C-arachidonic acid,
TABLE 1. Spontaneous release of PGI, from endothelial cells cultured for 24 hours in the presence or absence of aFGFiheparin' Umbilical vein EC Saphenous vein EC
17.4 i 2.8 15.8 i 3.6
7.8 2 1.4 8.4 -t 0.2
'Mean i SE in ngiml (n = 6). aFGF
40 pgiml, heparin 90 pgiml
as described above. The medium was then removed, a n aliquot being saved for counting. Each EC monolayer was washed several times with Hepes-buffered saline containing 2% fatty acid-free bovine serum albumin until counts in the wash fluid reached a stable minimum. The monolayer was then stimulated in the culture flask with l Ulml human thrombin in 5 ml buffer for 15 minutes, and the fluid was removed and saved. The cells were harvested with collagenase and counted; a n aliquot was then lysed and subjected to liquid scintillation counting to determine total cell-associated counts. To the fluid removed following incubation of the EC monolayer with thrombin was added 12,000 dpm of 3HTXB, as a n internal standard for recovery plus a mixture of standard 6-keto PGF,,, TXB,, PGF,,, PGE,, and PGD,. The sample was acidified to pH 2.5 and extracted twice with ethyl acetate. The pooled extracts were reconstituted into a small volume of ethyl acetate, applied to a silica gel G thin layer chromatography plate, and developed in a pre-equilibrated tank using a modified A-9 system (ethyl acetate:isooctane:acetic acid:water = 110:50:20:100). After development, the plate was air-dried, and the standards were visualized with 10% phosphomolybdic acid in methanol and heated to 110°C. After obtaining a suitable autoradiogram, the individual bands identified by cold standards were scraped and subjected to liquid scintillation counting. Statistical evaluation Differences between means of paired comparisons were evaluated by Student's t test. For multiple treatments, significance was evaluated by ANOVA with multiple measures. A P value of < 0.05 was considered significant.
RESULTS Both spontaneous and stimulated production of PGI, by cultures of human EC decreased in a time-dependent manner when the cells were exposed to the combination of aFGF plus heparin at concentrations of these factors previously shown to stimulate cell proliferation. Immediate and short-term release of PGI, was not altered by addition of aFGFIheparin or either factor alone to cell cultures for 10-30 minutes (data not shown). For the first 12 hours after changing culture medium, spontaneous PGI, release remained low and was similar in the presence or absence of aFGFiheparin. In contrast, by 24 hours of exposure to these growth factors, spontaneous release of PGIB was consistently less from EC grown in the presence of aFGF and heparin than from cells grown without these factors (Table 1).These results were similar for EC derived from umbilical veins and for EC from saphenous veins.
Synthesis of PGI,, by EC in response to brief stimulation by arachidonic acid or thrombin, a reflection of the maximal capacity of these cells to synthesize PGI, from exogenous or from endogenous substrate, respectively, also fell progressively during exposure of the cells to aFGF and heparin (Fig. 1). To measure PGI, production from exogenous arachidonate, monolayers of EC cultured with or without aFGFiheparin for 24, 48, and 72 hours were washed free of culture medium, covered with fresh HBS, and incubated for 5 minutes with sodium arachidonate. In control EC monolayers that had been cultured without addition of aFGFiheparin, total PGI, production induced by arachidonate did not change significantly over 72 hours (Fig. 1A). In contrast, PGI, produced by parallel monolayers from the same culture to which aFGFiheparin had been added decreased at each time point, differing significantly from controls a t 48 and 72 hours (P < 0.01). When additional monolayers of the same EC cultures grown similarly were stimulated with thrombin (0.5 Uiml) to elicit PGI, production from endogenous cellular arachidonate, PGI, formation decreased steadily over time (Fig. lB, P < 0.01), both in control and in aFGFiheparin-treated monolayers. This finding most probably reflects a decrease in thrombin receptor density as cultures reach confluence. However, thrombininducible PGI, production was consistently less a t each time point in cultures exposed to aFGF/heparin than in control cultures (P < 0.01). Ionophore A23187-induced PGI, production also declined in a manner similar to the decline in thrombin-induced PGI, production in EC exposed to aFGFiheparin (data not shown). To exclude the possibility that residual heparin remaining on the cell monolayer after washing away culture medium might block the capacity of thrombin to induce PGI, production, thrombin-stimulated PGI, production by control EC was compared with that of EC grown in the presence of either heparin alone or both aFGF and heparin. Thrombin-stimulated PGI, production was significantly less in cultures grown with but was aFGFheparin than in control cells (P < 0.01), equivalent to control values in cultures grown with heparin alone (Fig. 2). The effect of heparin alone on arachidonate-induced PGI, production varied with the heparin concentration. Thus exposure of EC to 90 kgiml of heparin re30% decrease in arachidonate-induced sulted in a PGI, release, whereas thrombin-induced PGI, fell by 0-1096. Lower concentrations of heparin in the absence of aFGF did not decrease PGI,; indeed, slight increases were often seen. However, under all conditions tested, the combination of heparin and aFGF was associated with decreased spontaneous and stimulated PGI, production. The extent to which heparin augmented the effect of aFGF on PGI, production was next evaluated. In the presence of a constant concentration of aFGF, increasing the heparin concentration led to progressively lower PGI, production (Fig. 3; P < 0.005 for effect of log heparin dose on PGI,). The ED,, was approximately 2 kgiml, with as little a s 0.5 kgiml heparin having a detectable effect (data not shown). Conversely, a t a constant heparin concentration, the production of PGI, was inversely proportional to the concentration of aFGF (Fig. 5A, see below). The extent of the decrease
HEPARINiACIDIC FGF ALTER ENDOTHELIAL CELL PGI,
48 h Time
Fig. 1. Time-dependent decrease in stimulated PGI, production by EC exposed to aFGF and heparin. Results represent PGI, produced by washed monolayers of human umbilical vein EC a t 24, 48, and 72 hours in controls (no aFGF or heparin added [black bars]) or after addition of 40 pgiml crude aFGF plus 90 pgiml heparin (hatched bars) to cells cultured in HEPES-buffered medium 199 containing 20% pooled human serum. PGI, was measured by RIA of its hydrolytic product 6-keto PGF,,. Bars indicate mean ? SE. In both panels, for each time point n = 9. A: PGI, produced in response to 25 p M sodium
arachidonate during incubation a t 37°C for 5 minutes. In the presence of aFGFheparin, PGI, significantly decreases a t 48 and 72 hours ( P < 0.01); in the absence of growth factors, PGI, does not change significantly ( P = 0.609). B: PGI, produced in response to 0.2 Uiml human thrombin during a 5 minute incubation a t 37°C. In the presence of aFGFiheparin, PGI, significantly decreases at all time points ( P < 0.01)compared with controls. Note that units on ordinate are different in panels A and B.
11.3 2 2 . 5
Fig. 2. Comparison of effect of heparin alone and hepariniaFGF com bined on stimulated PGI, production by EC. Human umbilical vein EC were cultured for 48 hours with either aFGF (40 pg/ml) and heparin (90 pgiml), heparin alone (90 pgiml), or neither additive; cells were then washed and stimulated in buffer with 25 p M sodium arachidonate (dark bars) or 0.5 Uiml thrombin (light bars) for 5 minutes at 37°C. Supernatant fluid was removed and assayed for PGI, content by RIA. Bars represent mean ? SE for five separate experiments. P < 0.01 for the difference between PGI, from EC cultured with aFGFiheparin and control EC cultured without these substances; P = NS for difference between EC cultured in the presence of heparin alone and control EC.
Fig. 3. Heparin promotes aFGF effect to decrease PGI, production by EC. Replicate monolayers of EC were exposed to increasing concentrations of heparin and a constant concentration of crude aFGF (40 pgiml) for 48 hours and then washed and stimulated with arachidonate or thrombin. Dark bars = PGI, produced upon arachidonate stimulation; light bars = PGI, produced upon thrombin stimulation. PGI, falls with increasing heparin concentration, with maximal effect reached at about 20 pgiml under these conditions (P < 0.005 for effect of increasing heparin concentrations with either stimulus). Each bar represents the mean ( * SD) of duplicate samples from a single experiment.
in PGI, depended on the concentrations of both aFGF and heparin, especially a t low concentrations. For example, while stimulated PGI, production fell a s the aFGF concentration increased in combination with either 1or 10 pg/ml heparin, maximal inhibition of PGI, was already achieved a t 10 pg/ml of aFGF when 10 pgiml of heparin was present, but required > 50 pgiml of aFGF when 1 pgiml heparin was present. These changes in PGI,.production by EC exposed to aFGFiheparin were consistently observed not only in primary cell cultures but also in EC cultured for up to
three passages; later passages were not evaluated. The decreases in PGI, followed a similar time pattern and were similar in degree in sequential passages of the same cell isolate, although the absolute amount of PGI, produced in later passages by controls a s well as by growth factor-treated cell cultures tended to be lower than that produced in early passages. Since cells were routinely cultured without aFGF or heparin until the test period, marked differences in population doublings were minimized (population doublings after 72
750 . , -
aFGF ng/ml B
Fig. 4. Increase in PGI, production following removal of aFGF from EC cultures. Monolayers of EC were cultured with aFGFiheparin for 48 hours, washed, and recultured in fresh medium either containing aFGFiheparin (black bars) or neither substance (hatched bars). At the end of an additional 48 hours, the monolayers were washed and PGI, production was stimulated by addition of arachidonate or thrombin as described above. Values are expressed as mean f SE (n = 6);P < 0.05 for differences in PGI, elicited by either stimulus between cultures maintained in aFGFiheparin and cultures from which aFGFiheparin was removed.
hours in culture: 1.68 t 0.13 without aFGFiheparin; 2.72 2 0.19 with aFGFiheparin, mean 2 SE, n = 12). Both the absolute amount of PGI, produced per culture well and the amount produced per cell were decreased by exposure to aFGFiheparin. Cell density did not account for the differences observed in PGI,. Results were similar in EC plated a t equal cell numbers and grown with or without aFGFiheparin and in EC for which initial cell plating densities were adjusted so that the final cell counts a t the time of stimulation were equal under both types of growth conditions. In addition, results obtained with umbilical vein endothelial cells were quantitatively and qualitatively comparable to those obtained with saphenous vein endothelial cells derived from adult human donors. The inhibitory effect of the aFGFiheparin combination on EC production of PGI, was reversible. Removal of aFGF/heparin from the culture medium led to a n increase in PGI, production when EC previously exposed to aFGF/heparin were placed in fresh medium lacking both aFGF and heparin for 48 hours (Fig. 4). The above results obtained using a partially purified aFGF were confirmed using two other preparations of heparin-binding endothelial cell growth factors. PGI, production fell in a dose-dependent fashion in the presence of nanogram per milliliter concentrations of a highly purified aFGF (at a constant amount of heparin), that is, a t concentrations three orders of magnitude lower than those required for a similar effect when partially purified aFGF (endothelial cell growth supplement) was used (Fig. 5A). In addition, crude bovine retinal growth factor, prepared in our laboratory, plus heparin produced a similar diminution of PGI, production (data not shown). Other growth factors tested, such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), did not alter PGI, production by endothelial cells (data not shown). In order to evaluate how exposure of aFGFiheparin
Fig. 5. Effect of highly purified aFGF and heparin on PGI, production and on immunoreactive PGH synthase content in EC. EC were cultured for 48 hours without or in the presence of increasing concentrations of purified aFGF and a constant concentration of heparin (50 pgiml). A Arachidonate-induced (dark bars) and thrombin-induced (light bars) PGI, production by washed EC monolayers, mean (t- SD) of duplicate values in a single experiment. P < 0.001 for effect of aFGF concentration on stimulated PGI,. B: Immunoreactive PGH synthase content of the same EC culture in the same experiment (see Materials and Methods). Each bar represents net absorbance at 405 nm (mean of triplicate determinations); the effect of aFGF concentration on PGH synthase is significant, P < 0.001.Both PG1,production and immunoreactive PGH synthase decrease in parallel with increasing concentrations of aFGF. Purified aFGF is approximately 1,000fold (by weight) more active than crude aFGF.
produced a fall in synthesis of PGI, by EC, we combined studies on conversion of enzyme substrates with a n examination of the cellular content of enzymes involved in prostaglandin synthesis, using a method that permitted evaluation of PGIB production and enzyme content in the same culture plate. Immunoreactive PGH synthase, measured by specific enzyme-linked immunoassay (ELISA) in fixed, permeabilized EC monolayers, decreased in a pattern similar to the decrease in PGI, production following culture of the EC with aFGFiheparin (Fig. 5B). The fall in immunoreactive PGH synthase in cultures treated with aFGFiheparin was both time- and dose-dependent. Only concentrations of aFGF that stimulated EC proliferation were associated with a decreased PGI, production and with a decrease in PGH synthase content. Figure 5 illustrates the relationship between change in enzyme content and fall in PGI, synthesis for EC exposed to highly purified aFGF. Parallel results were obtained with mi-
HEPARINIACIDIC FGF ALTER ENDOTHELIAL CELL PGI,
3000 . n
0 No aFGF/heparin
Fig. 6. Parallel decrease in immunoreactive PGH synthase and PGI, synthase in EC cultured with or without aFGFiheparin. Cell conditions were as described for Fig. 5. Results represent three separate experiments, n = 12 for each variation. Black bars = PGH synthase, striped bars = PGI, synthase. P < 0.001 between the two growth conditions for both enzymes and for PGI, measured in the same experiments (latter not showni.
crogram per milliliter concentrations of crude aFGF, in the presence of a constant amount of heparin. The PGI, synthase activity in these EC cultures was evaluated by measuring the conversion of PGH, to prostacyclin in aspirin-treated EC following culture with or without aFGF/heparin. Additional replicates of the same EC, not treated with aspirin, were simultaneously tested for PGH synthase activity measured as conversion of exogenous or endogenous arachidonate to PGI,. PGI, synthase immunoreactivity was independently measured and was compared with immunoreactive PGH synthase in the same EC cultures. Both PGH synthase and PGI, synthase contents decreased in cells cultured with aFGF/heparin (Fig. 6, P < 0.01 for each enzyme). Under the conditions used, the maximal decrease detected in immunologic PGI, synthase was about 50%. In addition to the change in immunoreactive enzyme content, both PGH synthase and PGI, synthase activities decreased in EC exposed to aFGFIheparin during culture, as shown by parallel declines in PGI, production from arachidonate and in PGI, production from PGH, (Fig. 7). Release of radiolabeled arachidonate from EC preincubated with 14C-sodium arachidonate was measured to determine if net arachidonate deacylatioa, reflecting phospholipase and glycerol lipase activities, also were altered when EC were exposed to growth factors. Incorporation of 14C-arachidonate was equivalent in EC cultured for 48 hours with and without aFGF/heparin. Prelabeled, ibuprofen-treated EC were stimulated with thrombin to activate phospholipases A, and C, resulting in arachidonate release. Cells cultured in the presence of aFGF/heparin, when stimulated with thrombin, released a similar percentage of I4C-arachidonate, as did control cells cultured without aFGF/heparin (6.02 ? 1.15% vs. 7.60 2 2.3%, mean SD of two separate experiments performed in triplicate, p = NS). Comparison of PGI, and PGE, synthesis by EC showed that PGEz production decreased in parallel
Fig. 7. Conversion of PGH, into PGI, is decreased in EC cultured with aFGF and heparin. EC grown for 48 hours in the presence or absence of aFGFiheparin were tested for arachidonate-induced (striped bars) and PGH,-induced (stippled bars) production of PGI,. For measurement of PGH, conversion to PGI, the EC were pretreated with aspirin as described in Materials and Methods. Culture medium in aspirin-free EC was also assayed to determine spontaneous production of PGI, (open bars). Mean i SE, n = 6. In each case, EC exposed to aFGFiheparin produced less PGI, than control cells (all P < 0.0001).
with the fall of PGI, production in cultures exposed to the growth factor (Table 2). Arachidonate-induced, thrombin-induced, and spontaneous (latter not shown) PGE, production were all affected, although the percentage decrease of PGI, (92-95%) was greater than the decrease in PGE, (62-65%) in the same experiments. To evaluate further if aFGF/heparin affected release of other eicosanoid products, additional EC monolayers were cultured with or without aFGF/heparin for 48 hours, prelabeled with 14C-arachidonate, and stimulated with thrombin. Radiolabeled PGI, and PGE, released from cells grown with aFGF and heparin fell (P < 0.01) compared with controls (Fig. 8); small decreases in PGD, and HETEs (minor products) and a trend to increase in PGF,, were also observed ( P = NS). The sum of these eicosanoids was decreased in growth factor-treated cells compared with controls.
DISCUSSION The enzymatic activity of PGH synthase serves as a major rate-limiting factor in prostaglandin production by endothelial cells, since the substrate for this enzyme (arachidonate) is abundant in these cells, and the release of cellular arachidonate from its esterified forms is usually not limiting. Moreover, PGH synthase is rapidly and irreversibly auto-inactivated by free radicals and peroxides produced during formation of its oxygenated products (Egan et al., 1976; Bailey et al., 1985). PGH synthase itself is produced rapidly and continuously by endothelial cells, as estimated from studies of the rate of return of EC capacity to convert arachidonate to endoperoxides after total inhibition of PGH synthase by aspirin, a n irreversible inhibitor of its cyclooxygenating function (Jaffe and Weksler, 1979). In fibroblasts and muscle cells, rapid inhibition of prostaglandin synthesis in the presence of cycloheximide or after stimulation with a n agonist has been observed, similarly suggesting that there is continuous resynthesis of PGH synthase (Fagan and Goldberg, 1986; Bailey
TABLE 2. Comparison of PGE, and PGI, production by human endothelial cells cultured in the presence or absence of aFGFlheDarin'
3.86 i 1.00 4.75 2 0.31
1.33 i 0.22 0.21 i 0.11