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Prostaglandin production in phenotypically distinct cultured bovine pulmonary artery endothelium
Atherosclerosis, 51 (1984) 143-150 Elsevier Scientific Publishers Ireland.
143 Ltd.
ATH 03465
Prostaglandin Production in Phenotypically Distinct Cultured Bovine Pulmonary Artery Endothelium G.L. Hahn and Peter. R. Polgar Department
of Biochemisty,
Boston Uniuersi
Bovine pulmonary artery endothelial cells, maintained in culture for prolonged periods, were observed to enter into a secondary growth pattern commonly referred to as sprouting. Our investigations, as well as those of other workers, indicate that these cells represent a phenotypic variant of the original endothelial cell population, rather than the proliferation of a contaminating cell type. A significant increase in prostaglandin production, particularly prostacyclin, could be correlated with the appearance of these phenotypically distinct endothelial cells in culture. We suggest that the elevation in prostacyclin synthesis is a result of endothelial cell sprouting and hypothesize that increased prostacyclin levels may account for the inhibition of platelet deposition reported in post-confluent endothelial cultures. Key words:
Cell culture - Endothelium
- Prostacyclin
- Prostaglandin
- Sprouting
Introduction Endothelial cells comprise the inner lining of the mammalian circulatory system. Due to their intimate contact with the circulating blood and its contents (factors, hormones, substrates and cells) the endothelium forms a vital segment of vascular physiology [l]. One of the important regulatory features of these cells is their ability to produce relatively large amounts of prostaglandin (PG), particularly prostacyclin (PGI,) [2.3]. PGI, is important in the control of such events as platelet aggregation, This work was supported
0021-9150/84/$03.00
by grant HL-25776
8 1984 Elsevier Scientific
from the National
Publishers
Ireland.
Institute
Ltd.
of Health.
144
the relaxation of vascular smooth muscle, the regulation of cell growth and the maintenance of the differentiated state [4-71. Circumstances which alter the production of PGI, could significantly affect the function of the blood vessel and therefore the physiology of the vasculature in general. Recent work in this laboratory has indicated that one condition which dramatically alters prostaglandin production in cultured endothelium is associated with a change in their morphology. Endothelial cells in culture exist as a homogeneous monolayer of cobblestoneshaped cells [8,9], a morphology quite similar to that which exists in uklo [9.10]. Under certain conditions endothelial cells, both within the organism and in culture. exhibit a secondary growth pattern. Zn uiuo, the endothelium of regenerating or newly formed vessels appears as strands of cells branching off the existing vessel, a phenomenon referred to as sprouting [ll]. In culture, endothelial cells at post-confluent densities begin to undergrow the original monolayer in a branched mycelial pattern which, by analogy, is also referred to as sprouting [12-151 although it has not been demonstrated that these deviations from normal growth patterns represent the same physiological state. Sprouted cells in culture were originally thought to represent contamination by another cell type, probably smooth muscle. More recent evidence, however, has indicated that these cells are indeed of endothelial origin [13]. They are positive for the factor VIII antigen peculiar to endothelial cells and contain little of the actin and 60-A filaments characteristic of smooth muscle [14]. Clones derived from small patches of pure endothelial cells continue to display the dual growth pattern of monolayers followed by sprouting [12-141. These observations suggest that sprouted cells represent a phenotypically altered endothelial cell. In the present study we examined prostaglandin synthesis in cultured bovine pulmonary artery endothelium as a function of their apparent morphology (cobblestone vs. sprouted). We report that as endothelial cultures progress through the sprouted stage their ability to synthesize prostacyclin increases dramatically, particularly in response to the vasoactive, inflammatory agent bradykinin (BK). It has recently been reported that endothelial cultures at post-confluent densities do not bind platelets whereas subconfluent endothelial cultures do [16]. This lack of platelet binding could be a result of the observed increase in prostacyclin production. Materials
and Methods
Cell culture Endothelial cells were isolated from the intima of the calf pulmonary artery by treatment with collagenase and maintained at 37°C 95% air, 5% CO, in a watersaturated incubator in McCoy’s medium supplemented with 20% fetal bovine serum. 50 ;ug/ml streptomycin and 50 units/ml penicillin [16]. Cultures to be used for experimental purposes were seeded into 24 multiwell plates at a density of 200 cells/mm*. The medium of cultures destined for use as monolayers was changed every other day. Sprouted cultures were obtained by prolonged maintenance in culture medium after the onset of confluency. Medium changes were reduced to every 4th day in sprouting cultures. Endothelial cultures used in this report were in the 3rd subcultivation from the primary culture.
145
Incubation procedure
Confluent cultures to be used for analysis were last fed 2 days before the experiment. Sprouted cultures were last fed 4 days before incubation. All incubations were done at 37°C at saturation humidity in 95% air and 5% CO,. Prior to use, the cells were preincubated for 3-5 h in fresh medium supplemented with 1% fetal bovine serum. We have previously demonstrated that this treatment induces a quiescent state which results in maximal PG production [18]. After preincubation, the cultures were washed twice with Hank’s balanced salt solution and resuspended in 0.5 ml serum-free McCoy’s plus any additions. At the end of the 30-min incubation period, the culture medium was removed and was assayed for prostaglandin content by RIA. Assay for prostaglandins
Prostaglandin content was determined by radioimmunoassay (RIA) of incubation medium as previously described [17]. Cross reactivities of the antisera against non-targeted PGs were less than 5%. PGE, was measured directly. Prostacyclin (PGI,) is an unstable compound which breaks down rapidly into its degradative product, 6-keto-PGF,,. Measurement of PGI, production was therefore performed by RIA of 6-keto-PGF,,. 6-Keto-PGF,, was always measured at l-10 dilutions of the original medium. No extraction of prostaglandins was necessary. Results Endothelial cells, inoculated at densities of approximately 200 cell/mm2, readily attached and began growth. Within 5-7 days these cells presented a confluent monolayer with a cobblestoned appearance typical of endothelial cells (Fig. 1A). Densities of approximately 2000 cells/mm2 were common at this stage. Cultures allowed to continue past this point evidenced a second growth pattern at about lo-12 days post-inoculation. Initially cells became tightly packed, reaching densities of 3000-4000 cells/mm2. Foci of elongated and branched cells began to appear beneath the original monolayer usually beginning at the center of the dish (Fig. 1B). By 21 days the entire culture had developed the secondary growth pattern (Fig. 1C) reaching densities in excess of 6 000 cells/mm2. While the time frame presented here was typical for the strain of endothelial cells used in this study not all cell isolates responded in the same fashion. Some isolates entered the secondary growth pattern earlier or later, while a few failed to sprout even after prolonged maintenance in culture. This variation in the ability to form sprouts has been previously reported in bovine aortic endothelium [14]. Numerous subcultivations of sprouted cultures were performed in an attempt to isolate a cell type other than the original endothelial cell. Regardless of the extent of sprouting present when the culture was subcultivated or the cell density employed on reinoculation, passage of these cells always gave rise to a typical cobblestoned endothelial monolayer (Fig. lD), which in turn gave rise to the secondary growth pattern of sprouting. These results support the contention that sprouted endothelial cells represent a phenotypic variant of the original cell type and are not the proliferation of contaminating smooth muscle cells [12-141.
146
Fig. 1. Endothelial cells were isolated and maintained as described in the text. A: Typical cobblestoned confluent monolayer at 7 days post inoculation. B: Initial sprouting beneath the monolayer at 13 days. C: Extensive sprouting at 20 days. Focus is on the sprouted layer beneath the monolayer. D: Typical cobblestoned monolayer derived from sparsely plating a highly sprouted culture.
147
148 TABLE
1
PROSTACYCLIN
SYNTHESIS
IN CULTURED
BOVINE
ENDOTHELIUM
Cultures were inoculated and maintained as described in Methods. At the indicated stage of growth. cultures were twice washed and preincubated for 3 h in McCoy’s medium supplemented with 1.0% fetal bovine serum. Cultures were then incubated for 30 min in serum-free McCoy’s medium with the indicated additions. Bradykinin concentration was 5 Pg/ml, arachidonate 10 PM. Each value presented represents the mean +SEM of measurements made on at least 3 separate cultures. Cell densities were: confluent monolayer 2000 cells/mm2, moderate sprouting 4170 cells/mm2, highly sprouted 5 600 cells/mm2. Cell morphology Confluent monolayer
PGI,
(as 6-keto-PGF,,)(ng/ml)
Control
Bradikinin
Arachidonate
1.2kO.l
12.4* 1.6
15.9+2.4
4.5 f 0.7
85.7+4.8
59.9 * 3.4
Moderate Sprouting Highly sprouted
65.7 + 9.3
502.2 k 46.9
18.7 f 2.8
Once cells had entered the secondary growth state they exhibited considerably elevated synthesis of prostaglandin. The effect of sprouting on PGI, and PGE, synthesis is presented in Tables 1 and 2. Highly sprouted endothelial cultures produced significantly more prostaglandin compared to confluent monolayers. Cultures which were only moderately sprouted showed a response midway between the highly sprouted and monolayer cultures, indicating that the increased prostaglandin synthesis was dependent on the extent of sprouting. This enhanced synthetic capacity was observed both at the basal, unstimulated level or in response to the prostaglandin precursor arachidonate. However, the greatest overall increase in
TABLE PGE,
2 IN CULTURED
BOVINE
Conditions of growth, experimental described in the legend to Table 1.
SYNTHESIS
incubation,
Cell morphology
ENDOTHELIUM cell densities
and concentrations
PGE,(ng/ml) Control
Bradykinin
> 0.02
0.16 + 0.01
Confluent monolayer Moderate sprouting
0.20 f 0.02
1.41 + 0.06
0.63 f 0.23
2.78 + 0.22
Highly sprouted
of additions
were as
149
prostaglandin production in sprouted cultures occurred in response to the vasoactive peptide bradykinin. Bradykinin stimulation of highly sprouted cultures results in an accumulation of PGI, in excess of 500 ng/ml. an almost 30-fold increase over the unstimulated control value. In comparison, confluent monolayers produced only 12 ng/ml as a consequence of BK stimulation. While the overall production of PG in the sprouted cultures increased in response to arachidonate, it did not reach the levels achieved with BK. The principal prostaglandin produced by endothelial cells both in uiuo and in culture is PGI, [3,6]. This was also true of sprouted endothelial cultures. While sprouted endothelial cells produced significantly higher levels of PGEz than monolayers, the amount of PGE, released was only a fraction of the PGI, production. Maximal PGE, production in the highly sprouted cultures also occurred in response to BK. However, the amount of PGE, released in response to BK was only l/200 the amount of PGI, produced.
Discussion The results of this study support the hypothesis that sprouted endothelial cells represent a phenotypic variant of the original cell type and demonstrate that not only has the cellular morphology changed but prostaglandin synthesis has also been significantly affected. We demonstrate an increased capacity for prostaglandin synthesis in cultures which have undergone sprouting. The increases in PG synthesis observed in sprouted endothelial cultures could not be accounted for by the increase in cell number alone. While the cell number increased by a factor of 2.5 as cultures progressed from monolayers to the highly sprouted state, PG levels were elevated up to 20-fold. It appears than that the increased PGI, production represents an activation of PG synthesis which is associated with the altered cellular morphology. It is not clear at this time which segment of the regulatory mechanisms in PG synthesis has been altered or why in sprouted cells the increased response to bradykinin is relatively greater than that to added, free arachidonate. It has recently been reported [16] that aortic endothelial cell cultures bind bovine platelets at subconfluent densities but do not bind platelets at confluence and post-confluence. The elevation in PGI, synthesis observed in sprouted cultures might be the cause of this loss of binding. The nature of the activation of prostaglandin synthesis associated with endothelial cell sprouting is as yet unclear but if in vitro sprouting is analogous to the similar process in vivo then this may represent a physiological mechanism which lends a degree of protection from platelet aggregation. In vivo, endothelial cells which are in the process of regenerating damaged vessels or forming new ones would probably be more susceptible to platelet deposition and thrombus formation. Increased PGIz production could compensate for this vulnerability. However, it must be established whether in vitro and in vivo are truly analogous. The observed increase in prostaglandin synthesis resulting from in vitro sprouting should give us another means of investigating this possibility.
150
References 1 Thorgeirsson, G. and Robertson, A.L., The vascular endothelium - Pathobiologic significance. Amer. J. Path.. 93 (1978) 804. 2 Hong. S.L., The effect of bradykinin and thrombin on prostacyclin synthesis in endothelial cells from calf and pig aorta and human umbilical cord vein, Thromb. Res.. 18 (1980) 787. 3 Hahn, G.L., Menconi,M. and Polgar. P.. The effect of gamma radiation on prostacyclin production in cultured pulmonary artery endothelium. In: T.J. Powles. R.S. Bockman, K.V. Honn and P. Ramwell (Eds.), Prostaglandins and Cancer, Alan R. Liss, New York 1982. p. 381. 4 Moncada, S. and Vane, J.R., Arachidonic acid metabolites and the interaction between platelets and blood vessel walls, N. Engl. J. Med., 300 (1977) 1142. 5 Moncada, S.. Gryg1ewski.R.J.. Bunting, S. and Vane, J.R., An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation, Nature (Lond.). 63 (1976) 663. 6 Armstrong, J.M., Lattimer, N.. Moncada. S. and Vane, J.R.. Comparison of the vasodepressor effects of prostacyclin and 6-oxo-prostaglandin F,, with those of prostaglandin E, in rats and rabbits, Brit. J. Pharmacol., 62 (1978) 125. 7 Hopkins, N.K. and German. R.R.. Regulation of 3T3-Ll fibroblast differentiation by prostacyclin Biochim. Biophys. Acta, 663 (1981) 457. 8 Faris, B.. Mozzicato, P., Mogayzel, P.J.. Ferrera. R., Gerstenfeld, L.C., Glembourtt, M.. Makarski, J.S., Haudenschild, C.C. and Franzblau. C., The effect of protein-hydroxyethylmethacrylate hydrogels on cultured endothelial cells, Exp. Cell Res.. 143 (1983) 15. 9 Gimbrone, M.A., Culture of vascular endothelium. In: T.H. Speat (Ed.), Progress in Hemostasis and Thrombosis, Vol. 3, Grune and Stratton, New York, 1976, p. 1. 10 Macarak. E.J.. Howard, B.V. and Kefalides, N.A., Properties of calf endothelial cells in culture, Lab. Invest., 36 (1977) 62. 11 Ausprunk. D.H. and Folkman. J.G.. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc. Res.. 14 (1977) 53. 12 Gospodarowicz, D. and Mecher, A.L., The control of cellular proliferation by fibroblast and epidermal growth factors, Nat. Cancer Inst. Monogr.. 48 (1978) 109. 13 Cotta-Pereira, G., Sage, H., Bornstein. P.. Ross. R. and Schwartz, S., Studies of morphologically atypical (‘sprouting’) cultures of bovine aortic endothelial cells Growth characteristics and connective tissue protein synthesis, J. Cell Physiol., 102 (1980) 183. 14 Schwartz, S. Selection and characterization of bovine aortic endothelial cells. In Vitro, 14 (1978) 966. 15 Mueller. S.N., Rosen. E.M. and Levine, E.M., Cellular senescence in a cloned strain of bovine fetal aortic endothelial cells, Science, 207 (1980) 889. 16 Feder. J., Marsa, J.C. and Olander. J.V., The formation of capillary-like tubes by calf aortic endothelial cells grown in vitro. J. Cell Physiol., 116 (1983) 1. 17 Polgar. P., Douglas, W.H.J., Terracio. L. and Taylor, L.. Release of prostaglandin and its conversion to prostaglandins in various diploid cell types in culture. In: B. Samuelsson. P.W. Ramwell and R. Paoletti (Eds.). Advances in Prostaglandin and Thromboxane Research, Vol. VI, Raven Press. New York, 1980, p. 225. 18 Hahn, G.L., Menconi. M.. Cahill. M. and Polgar. P.. The influence of gamma radiation on arachidonic acid release and prostacyclin synthesis, Prostaglandins, 25 (1983) 783.
143 Ltd.
ATH 03465
Prostaglandin Production in Phenotypically Distinct Cultured Bovine Pulmonary Artery Endothelium G.L. Hahn and Peter. R. Polgar Department
of Biochemisty,
Boston Uniuersi
Bovine pulmonary artery endothelial cells, maintained in culture for prolonged periods, were observed to enter into a secondary growth pattern commonly referred to as sprouting. Our investigations, as well as those of other workers, indicate that these cells represent a phenotypic variant of the original endothelial cell population, rather than the proliferation of a contaminating cell type. A significant increase in prostaglandin production, particularly prostacyclin, could be correlated with the appearance of these phenotypically distinct endothelial cells in culture. We suggest that the elevation in prostacyclin synthesis is a result of endothelial cell sprouting and hypothesize that increased prostacyclin levels may account for the inhibition of platelet deposition reported in post-confluent endothelial cultures. Key words:
Cell culture - Endothelium
- Prostacyclin
- Prostaglandin
- Sprouting
Introduction Endothelial cells comprise the inner lining of the mammalian circulatory system. Due to their intimate contact with the circulating blood and its contents (factors, hormones, substrates and cells) the endothelium forms a vital segment of vascular physiology [l]. One of the important regulatory features of these cells is their ability to produce relatively large amounts of prostaglandin (PG), particularly prostacyclin (PGI,) [2.3]. PGI, is important in the control of such events as platelet aggregation, This work was supported
0021-9150/84/$03.00
by grant HL-25776
8 1984 Elsevier Scientific
from the National
Publishers
Ireland.
Institute
Ltd.
of Health.
144
the relaxation of vascular smooth muscle, the regulation of cell growth and the maintenance of the differentiated state [4-71. Circumstances which alter the production of PGI, could significantly affect the function of the blood vessel and therefore the physiology of the vasculature in general. Recent work in this laboratory has indicated that one condition which dramatically alters prostaglandin production in cultured endothelium is associated with a change in their morphology. Endothelial cells in culture exist as a homogeneous monolayer of cobblestoneshaped cells [8,9], a morphology quite similar to that which exists in uklo [9.10]. Under certain conditions endothelial cells, both within the organism and in culture. exhibit a secondary growth pattern. Zn uiuo, the endothelium of regenerating or newly formed vessels appears as strands of cells branching off the existing vessel, a phenomenon referred to as sprouting [ll]. In culture, endothelial cells at post-confluent densities begin to undergrow the original monolayer in a branched mycelial pattern which, by analogy, is also referred to as sprouting [12-151 although it has not been demonstrated that these deviations from normal growth patterns represent the same physiological state. Sprouted cells in culture were originally thought to represent contamination by another cell type, probably smooth muscle. More recent evidence, however, has indicated that these cells are indeed of endothelial origin [13]. They are positive for the factor VIII antigen peculiar to endothelial cells and contain little of the actin and 60-A filaments characteristic of smooth muscle [14]. Clones derived from small patches of pure endothelial cells continue to display the dual growth pattern of monolayers followed by sprouting [12-141. These observations suggest that sprouted cells represent a phenotypically altered endothelial cell. In the present study we examined prostaglandin synthesis in cultured bovine pulmonary artery endothelium as a function of their apparent morphology (cobblestone vs. sprouted). We report that as endothelial cultures progress through the sprouted stage their ability to synthesize prostacyclin increases dramatically, particularly in response to the vasoactive, inflammatory agent bradykinin (BK). It has recently been reported that endothelial cultures at post-confluent densities do not bind platelets whereas subconfluent endothelial cultures do [16]. This lack of platelet binding could be a result of the observed increase in prostacyclin production. Materials
and Methods
Cell culture Endothelial cells were isolated from the intima of the calf pulmonary artery by treatment with collagenase and maintained at 37°C 95% air, 5% CO, in a watersaturated incubator in McCoy’s medium supplemented with 20% fetal bovine serum. 50 ;ug/ml streptomycin and 50 units/ml penicillin [16]. Cultures to be used for experimental purposes were seeded into 24 multiwell plates at a density of 200 cells/mm*. The medium of cultures destined for use as monolayers was changed every other day. Sprouted cultures were obtained by prolonged maintenance in culture medium after the onset of confluency. Medium changes were reduced to every 4th day in sprouting cultures. Endothelial cultures used in this report were in the 3rd subcultivation from the primary culture.
145
Incubation procedure
Confluent cultures to be used for analysis were last fed 2 days before the experiment. Sprouted cultures were last fed 4 days before incubation. All incubations were done at 37°C at saturation humidity in 95% air and 5% CO,. Prior to use, the cells were preincubated for 3-5 h in fresh medium supplemented with 1% fetal bovine serum. We have previously demonstrated that this treatment induces a quiescent state which results in maximal PG production [18]. After preincubation, the cultures were washed twice with Hank’s balanced salt solution and resuspended in 0.5 ml serum-free McCoy’s plus any additions. At the end of the 30-min incubation period, the culture medium was removed and was assayed for prostaglandin content by RIA. Assay for prostaglandins
Prostaglandin content was determined by radioimmunoassay (RIA) of incubation medium as previously described [17]. Cross reactivities of the antisera against non-targeted PGs were less than 5%. PGE, was measured directly. Prostacyclin (PGI,) is an unstable compound which breaks down rapidly into its degradative product, 6-keto-PGF,,. Measurement of PGI, production was therefore performed by RIA of 6-keto-PGF,,. 6-Keto-PGF,, was always measured at l-10 dilutions of the original medium. No extraction of prostaglandins was necessary. Results Endothelial cells, inoculated at densities of approximately 200 cell/mm2, readily attached and began growth. Within 5-7 days these cells presented a confluent monolayer with a cobblestoned appearance typical of endothelial cells (Fig. 1A). Densities of approximately 2000 cells/mm2 were common at this stage. Cultures allowed to continue past this point evidenced a second growth pattern at about lo-12 days post-inoculation. Initially cells became tightly packed, reaching densities of 3000-4000 cells/mm2. Foci of elongated and branched cells began to appear beneath the original monolayer usually beginning at the center of the dish (Fig. 1B). By 21 days the entire culture had developed the secondary growth pattern (Fig. 1C) reaching densities in excess of 6 000 cells/mm2. While the time frame presented here was typical for the strain of endothelial cells used in this study not all cell isolates responded in the same fashion. Some isolates entered the secondary growth pattern earlier or later, while a few failed to sprout even after prolonged maintenance in culture. This variation in the ability to form sprouts has been previously reported in bovine aortic endothelium [14]. Numerous subcultivations of sprouted cultures were performed in an attempt to isolate a cell type other than the original endothelial cell. Regardless of the extent of sprouting present when the culture was subcultivated or the cell density employed on reinoculation, passage of these cells always gave rise to a typical cobblestoned endothelial monolayer (Fig. lD), which in turn gave rise to the secondary growth pattern of sprouting. These results support the contention that sprouted endothelial cells represent a phenotypic variant of the original cell type and are not the proliferation of contaminating smooth muscle cells [12-141.
146
Fig. 1. Endothelial cells were isolated and maintained as described in the text. A: Typical cobblestoned confluent monolayer at 7 days post inoculation. B: Initial sprouting beneath the monolayer at 13 days. C: Extensive sprouting at 20 days. Focus is on the sprouted layer beneath the monolayer. D: Typical cobblestoned monolayer derived from sparsely plating a highly sprouted culture.
147
148 TABLE
1
PROSTACYCLIN
SYNTHESIS
IN CULTURED
BOVINE
ENDOTHELIUM
Cultures were inoculated and maintained as described in Methods. At the indicated stage of growth. cultures were twice washed and preincubated for 3 h in McCoy’s medium supplemented with 1.0% fetal bovine serum. Cultures were then incubated for 30 min in serum-free McCoy’s medium with the indicated additions. Bradykinin concentration was 5 Pg/ml, arachidonate 10 PM. Each value presented represents the mean +SEM of measurements made on at least 3 separate cultures. Cell densities were: confluent monolayer 2000 cells/mm2, moderate sprouting 4170 cells/mm2, highly sprouted 5 600 cells/mm2. Cell morphology Confluent monolayer
PGI,
(as 6-keto-PGF,,)(ng/ml)
Control
Bradikinin
Arachidonate
1.2kO.l
12.4* 1.6
15.9+2.4
4.5 f 0.7
85.7+4.8
59.9 * 3.4
Moderate Sprouting Highly sprouted
65.7 + 9.3
502.2 k 46.9
18.7 f 2.8
Once cells had entered the secondary growth state they exhibited considerably elevated synthesis of prostaglandin. The effect of sprouting on PGI, and PGE, synthesis is presented in Tables 1 and 2. Highly sprouted endothelial cultures produced significantly more prostaglandin compared to confluent monolayers. Cultures which were only moderately sprouted showed a response midway between the highly sprouted and monolayer cultures, indicating that the increased prostaglandin synthesis was dependent on the extent of sprouting. This enhanced synthetic capacity was observed both at the basal, unstimulated level or in response to the prostaglandin precursor arachidonate. However, the greatest overall increase in
TABLE PGE,
2 IN CULTURED
BOVINE
Conditions of growth, experimental described in the legend to Table 1.
SYNTHESIS
incubation,
Cell morphology
ENDOTHELIUM cell densities
and concentrations
PGE,(ng/ml) Control
Bradykinin
> 0.02
0.16 + 0.01
Confluent monolayer Moderate sprouting
0.20 f 0.02
1.41 + 0.06
0.63 f 0.23
2.78 + 0.22
Highly sprouted
of additions
were as
149
prostaglandin production in sprouted cultures occurred in response to the vasoactive peptide bradykinin. Bradykinin stimulation of highly sprouted cultures results in an accumulation of PGI, in excess of 500 ng/ml. an almost 30-fold increase over the unstimulated control value. In comparison, confluent monolayers produced only 12 ng/ml as a consequence of BK stimulation. While the overall production of PG in the sprouted cultures increased in response to arachidonate, it did not reach the levels achieved with BK. The principal prostaglandin produced by endothelial cells both in uiuo and in culture is PGI, [3,6]. This was also true of sprouted endothelial cultures. While sprouted endothelial cells produced significantly higher levels of PGEz than monolayers, the amount of PGE, released was only a fraction of the PGI, production. Maximal PGE, production in the highly sprouted cultures also occurred in response to BK. However, the amount of PGE, released in response to BK was only l/200 the amount of PGI, produced.
Discussion The results of this study support the hypothesis that sprouted endothelial cells represent a phenotypic variant of the original cell type and demonstrate that not only has the cellular morphology changed but prostaglandin synthesis has also been significantly affected. We demonstrate an increased capacity for prostaglandin synthesis in cultures which have undergone sprouting. The increases in PG synthesis observed in sprouted endothelial cultures could not be accounted for by the increase in cell number alone. While the cell number increased by a factor of 2.5 as cultures progressed from monolayers to the highly sprouted state, PG levels were elevated up to 20-fold. It appears than that the increased PGI, production represents an activation of PG synthesis which is associated with the altered cellular morphology. It is not clear at this time which segment of the regulatory mechanisms in PG synthesis has been altered or why in sprouted cells the increased response to bradykinin is relatively greater than that to added, free arachidonate. It has recently been reported [16] that aortic endothelial cell cultures bind bovine platelets at subconfluent densities but do not bind platelets at confluence and post-confluence. The elevation in PGI, synthesis observed in sprouted cultures might be the cause of this loss of binding. The nature of the activation of prostaglandin synthesis associated with endothelial cell sprouting is as yet unclear but if in vitro sprouting is analogous to the similar process in vivo then this may represent a physiological mechanism which lends a degree of protection from platelet aggregation. In vivo, endothelial cells which are in the process of regenerating damaged vessels or forming new ones would probably be more susceptible to platelet deposition and thrombus formation. Increased PGIz production could compensate for this vulnerability. However, it must be established whether in vitro and in vivo are truly analogous. The observed increase in prostaglandin synthesis resulting from in vitro sprouting should give us another means of investigating this possibility.
150
References 1 Thorgeirsson, G. and Robertson, A.L., The vascular endothelium - Pathobiologic significance. Amer. J. Path.. 93 (1978) 804. 2 Hong. S.L., The effect of bradykinin and thrombin on prostacyclin synthesis in endothelial cells from calf and pig aorta and human umbilical cord vein, Thromb. Res.. 18 (1980) 787. 3 Hahn, G.L., Menconi,M. and Polgar. P.. The effect of gamma radiation on prostacyclin production in cultured pulmonary artery endothelium. In: T.J. Powles. R.S. Bockman, K.V. Honn and P. Ramwell (Eds.), Prostaglandins and Cancer, Alan R. Liss, New York 1982. p. 381. 4 Moncada, S. and Vane, J.R., Arachidonic acid metabolites and the interaction between platelets and blood vessel walls, N. Engl. J. Med., 300 (1977) 1142. 5 Moncada, S.. Gryg1ewski.R.J.. Bunting, S. and Vane, J.R., An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation, Nature (Lond.). 63 (1976) 663. 6 Armstrong, J.M., Lattimer, N.. Moncada. S. and Vane, J.R.. Comparison of the vasodepressor effects of prostacyclin and 6-oxo-prostaglandin F,, with those of prostaglandin E, in rats and rabbits, Brit. J. Pharmacol., 62 (1978) 125. 7 Hopkins, N.K. and German. R.R.. Regulation of 3T3-Ll fibroblast differentiation by prostacyclin Biochim. Biophys. Acta, 663 (1981) 457. 8 Faris, B.. Mozzicato, P., Mogayzel, P.J.. Ferrera. R., Gerstenfeld, L.C., Glembourtt, M.. Makarski, J.S., Haudenschild, C.C. and Franzblau. C., The effect of protein-hydroxyethylmethacrylate hydrogels on cultured endothelial cells, Exp. Cell Res.. 143 (1983) 15. 9 Gimbrone, M.A., Culture of vascular endothelium. In: T.H. Speat (Ed.), Progress in Hemostasis and Thrombosis, Vol. 3, Grune and Stratton, New York, 1976, p. 1. 10 Macarak. E.J.. Howard, B.V. and Kefalides, N.A., Properties of calf endothelial cells in culture, Lab. Invest., 36 (1977) 62. 11 Ausprunk. D.H. and Folkman. J.G.. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc. Res.. 14 (1977) 53. 12 Gospodarowicz, D. and Mecher, A.L., The control of cellular proliferation by fibroblast and epidermal growth factors, Nat. Cancer Inst. Monogr.. 48 (1978) 109. 13 Cotta-Pereira, G., Sage, H., Bornstein. P.. Ross. R. and Schwartz, S., Studies of morphologically atypical (‘sprouting’) cultures of bovine aortic endothelial cells Growth characteristics and connective tissue protein synthesis, J. Cell Physiol., 102 (1980) 183. 14 Schwartz, S. Selection and characterization of bovine aortic endothelial cells. In Vitro, 14 (1978) 966. 15 Mueller. S.N., Rosen. E.M. and Levine, E.M., Cellular senescence in a cloned strain of bovine fetal aortic endothelial cells, Science, 207 (1980) 889. 16 Feder. J., Marsa, J.C. and Olander. J.V., The formation of capillary-like tubes by calf aortic endothelial cells grown in vitro. J. Cell Physiol., 116 (1983) 1. 17 Polgar. P., Douglas, W.H.J., Terracio. L. and Taylor, L.. Release of prostaglandin and its conversion to prostaglandins in various diploid cell types in culture. In: B. Samuelsson. P.W. Ramwell and R. Paoletti (Eds.). Advances in Prostaglandin and Thromboxane Research, Vol. VI, Raven Press. New York, 1980, p. 225. 18 Hahn, G.L., Menconi. M.. Cahill. M. and Polgar. P.. The influence of gamma radiation on arachidonic acid release and prostacyclin synthesis, Prostaglandins, 25 (1983) 783.