(D Macmillan Press Ltd, 1991
Br. J. Pharmacol. (1991), 102, 101-106
Effects of cyclic nucleotides and phorbol myristate acetate on proliferation of pig aortic endothelial cells Shonna Alexandra Moodie & 'William Martin Department of Pharmacology, University of Glasgow, Glasgow, G12 8QQ 1 The role of cyclic nucleotides and protein kinase C in controlling proliferation of pig aortic endothelial cells (PAEC) in culture was investigated. 2 Dibutyryl cyclic AMP (30.uM), added twice daily, inhibited proliferation but 8 bromo cyclic GMP (30pM) had no effect. Two other stimuli known to increase PAEC cyclic GMP content by stimulating particulate and soluble guanylate cyclase respectively, atriopeptin II (10nM) and sodium nitroprusside (1 pM), were also without effect on proliferation. 3 Two agents known to inhibit soluble guanylate cyclase and lower intercellular cyclic GMP content, haemoglobin (10pM) and methylene blue (1pOM), each inhibited proliferation of PAEC. 4 The inhibitory effect of haemoglobin (10pM) was mediated by inhibition of soluble guanylate cyclase since it was reversed by agents known to increase cyclic GMP content, i.e. atriopeptin 1I(10 nM), 8 bromo cyclic GMP (30,uM) or sodium nitroprusside (1 uM). The inhibitory effect of methylene blue (10pM) was not reversed by these agents. 5 Phorbol 12-myristate 13-acetate (PMA, 0.1 nM-l UM), which activates protein kinase C, inhibited proliferation in a concentration-dependent manner. No early stimulation of proliferation was seen wtih PMA. The inactive isomer, 4a-phorbol 12,13-didecanoate (0.3,pM), lacked the ability of PMA to inhibit proliferation of PAEC. 6 PMA-induced inhibition of proliferation appeared not to be due to stimulated production of destructive oxygen-derived free radicals since it was unaffected by the radical scavengers, vitamin E (30,UM) or butylated hydroxytoluene (30puM). The antiproliferative actions of paraquat (10pM), an agent which generates free radicals intracellularly, was, in contrast, inhibited by vitamin E or butylated hydroxytoluene. Furthermore, neither dibutyryl cyclic AMP (30,UM) nor 8 bromo cyclic GMP (30pM) had any effect on the ability of PMA to inhibit proliferation. 7 This study suggests that cyclic AMP, cyclic GMP and protein kinase C play a role in controlling the proliferation of PAEC.
Introduction Identification of the hormonal factors and intracellular pathways that control migration and proliferation of vascular endothelial and smooth muscle cells is important since dysfunction of both cell types is observed in several vascular diseases (Hoshi et al., 1988a; Klagsbrun & Edelman, 1989). With the exception of cells from the microvasculature (Bar et al., 1989), endothelial cells exhibit a growth independence from platelet-derived mitogens by being able to grow equally well in serum- or plasm-supplemented medium (Kazlauskas & DiCorleto, 1985). Several endothelial cell mitogens have been identified, including, basic fibroblast growth factor, epidermal growth factor and a family of acidic polypeptide mitogens, including acidic fibroblast growth factor, endothelial cell growth factor and eye-derived growth factor (Gospodarowicz et al., 1978; 1986; Schreiber et al., 1985; Sato & Rifkin, 1988). The mitogenic actions of growth factors are mediated by interactions with cell surface receptors, and protein phosphorylation is known to be an early biochemical signal initiated upon activation of these receptors. Furthermore, differences in protein phosphorylation patterns occur in proliferating and density-inhibited endothelial cells (Kazlauskas & DiCorletto, 1987). Several studies indicate that cyclic nucleotides acting via specific protein kinases may regulate proliferation of cells (Friedman, 1981). Subsequent reports have shown that cyclic AMP has differing actions on proliferation of endothelial cells from different sources in culture: inhibition of proliferation was observed with bovine aortic and rat brain microvascular endothelial cells (Leitman et al., 1986; Kempski et al., 1987), ' Author for
correspondence.
but stimulation was observed in a foetal bovine aortic endothelial cell line (Presta et al., 1989). In contrast to cyclic AMP, the role of cyclic GMP in endothelial cell proliferation has not been reported. The role of the phospholipid/calcium-dependent kinase, protein kinase C, in signal transduction is well documented (Nishizuka, 1984; Blackshear, 1988). The activation of protein kinase C by tumour promoting phorbol esters has been shown to have differing actions on proliferation of endothelial cells from different sources in culture: inhibition of proliferation was observed with human aortic and bovine capillary endothelial cells (Doctrow & Folkman, 1987; Hoshi et al., 1988b); no effect was seen on bovine aortic endothelial cells (Doctrow & Folkman, 1987); and stimulation was observed on foetal bovine aortic and bovine cerebral cortex capillary endothelial cells (Daviet et al., 1989; Presta et al., 1989). In pig aortic endothelial cells, phorbol esters have been reported to produce an initial stimulation of proliferation which is followed within hours by inhibition (Uratsuji & DiCorleto, 1988). In this study we have attempted to evaluate more fully the influence of cyclic nucleotides and of protein kinase C stimulation upon proliferation of pig aortic endothelial cells in culture.
Methods
Endothelial cell culture Pig aortae were obtained from a local abattoir and the endothelial cells isolated by collagenase (0.2%) treatment as previously described (Gordon & Martin, 1983). Pig aortic endothelial cells (PAEC) were grown in Dulbecco's modified
102
SA. MOODIE & W. MARTIN
Eagle's medium (DMEM) supplemented with foetal calf serum (10%), newborn calf serum (10%), penicillin (100unitsml-'), streptomycin (lOOpgml-1), kanamycin (lOOpgml-') and glutamine (4mM): this is subsequently referred to in the text as normal serum-supplemented DMEM. Cells were grown at 370C under an atmosphere of 5% CO2 in air. Cells were seeded initially into 80cm2 tissue culture flasks and grown to confluence, attained in 6-8 days. The serumsupplemented DMEM was aspirated off and replaced every 2 or 3 days. Cells were characterized as endothelial cells by their growth in a strict monolayer and typical cobblestone appearance. Furthermore, we have previously reported their ability to secrete prostacyclin and endothelium-derived relaxing factor (Martin et al., 1988), and to fluoresce when incubated with the selective marker, acetylated low-density lipoprotein labelled with 1, 1'dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchorate (CMD, UK Ltd) (Voyta et al., 1984).
(Poole, Dorset). [Methyl-3H]-thymidine was purchased from Amersham (Bucks).
Statistical analysis Results are expressed as the mean + s.e.mean and comparisons were made by use of Student's t test or the MannWhitney test when there was unequal variance between samples. A probability of 0.05 or less was considered significant. In the Results, n represents the number of wells of cells.
Results Effect of phorbol 12-myristate 13-acetate on endothelial cell proliferation Pig aortic endothelial cells (PAEC) seeded at a density of 104
cells/cm' in normal serum-supplemented DMEM grew to Proliferation studies For proliferation studies, PAEC were seeded at a density of approximately 104 cells/cm2 in six-well plates (9.6cm2). The effects of various treatments on proliferation were then examined over the following 6-10 days. PMA, vitamin E, butylated hydroxytoluene and 4a-phorbol 12,13-didecanoate (4aPDD) were dissolved in 100% ethanol. At the dilutions used, the maximum concentration of ethanol (0.1% v/v) had no effect by itself on proliferation. All other drugs were dissolved in distilled water and sterilised by filtration through a Millipore filter (0.2 yM pore size). Solutions of haemoglobin were reduced to the ferrous form before use with sodium dithionite as previously described (Martin et al., 1985). All drugs were added twice daily with the exception of methylene blue which was added once daily. At the various time points indicated in the Results, cells were detached with a solution of trypsin (0.05%) and EDTA (0.02%) and counted by haemocytometry.
[3H]-thymidine incorporation experiments PAEC were plated at a density of approximately 1.5 x 105 cells/cm2 in six-well plates. The cells were grown in normal serum-supplemented DMEM for 24h. After a further 24h incubation in serum-free medium, the cells were challenged with drugs in low serum (2% foetal calf and 2% newborn calf serum) containing DMEM and pulsed with a mixture of [3H]-thymidine (2juCi/well) and unlabelled thymidine (1 pM) for various times as indicated in the Results. At the end of the incubation period, the cells were washed 3 times with 2 ml of 5% trichloroacetic acid and solubilised in 1 ml of 0.25 M NaOH. The radioactivity in the solubilised cells was determined by liquid scintillation counting and expressed in d.p.m.
confluence within 6-8 days (Figure 1). The activator of protein kinase C, PMA (0.3pM), when added twice daily, produced a marked inhibition of proliferation: 86 + 2% (n = 6) inhibition was observed at day 8 (Figure 1). The ability of PMA to inhibit proliferation was observed over the concentration range 0.1 nM-i /IM (Figure 2), but the inactive phorbol ester, 4a-PDD (0.3 pM), lacked the ability of PMA to inhibit proliferation (Figure 1). PMA did not stimulate trypan blue uptake by cells (data not shown). Phorbol esters are know to stimulate production of oxygenderived free radicals in endothelial cells (Matsubara & Ziff, 1986). To determine if these radicals contribute to the ability of PMA to inhibit proliferation of PAEC, we examined the effects of vitamin E (30pM) and butylated hydroxytoluene (BHT, 30,UM). Vitamin E and BHT, when added twice daily, each had no effect on proliferation by themselves and had no effect on the ability of PMA (0.3 pM) to inhibit proliferation (Figure 3a). Paraquat (10pM), an agent which generates oxygen-derived free radicals intracellularly (Minakami et al., 1990), when added twice daily, also inhibited proliferation of PAEC (Figure 3b). This inhibition was associated with accumulation of trypan blue, and was prevented by the presence of vitamin E (30pM) or BHT (30pM) (Figure 3b).
Effect of PMA on [3H]-thymidine incorporation by endothelial cells Phorbol esters have been reported initially to stimulate then inhibit proliferation of pig aortic endothelial cells (Uratsuji & 10-
Ln
8-
*;-G
0
Materials Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, glutamine, foetal calf serum, newborn calf serum and trypan blue were purchased from Gibco Ltd. (Paisley, Scotland). Trypsin - EDTA and kanamycin were purchased from Flow Laboratories (Irvine, Scotland). Tissue culture flasks (80 cm2) and six-well multidishes were supplied by Nunc (Denmark). Atriopeptin II, 8 bromo guanosine 3': 5'-cyclic monophosphate (8 bromo cyclic GMP), butylated hydroxytoluene, collagenase (type II), dibutyryl adenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP), haemoglobin (bovine erythrocytes), methylene blue, paraquat (,_1'dimethyl-4-4'bipyridinium dichloride), 4a-phorbol 12,13-didecanoate (4aPDD), phorbol 12-myristate 13-acetate (PMA), sodium nitroprusside, vitamin E (D,L-a-tocopherol acetate) and thymidine were purchased from the Sigma Chemical Company Ltd. (Poole, Dorset). Sodium dithionite was purchased from BDH
6-
S
4-
0~~~
-
CD .0
E:
a)
2-
a~~~ 2
4 T Time (days)
6
8
10
Figure 1 The effect of phorbol myristate acetate (PMA) and 4aphorbol 12,13-didecanoate (4ca-PDD) on the proliferation of pig aortic endothelial cells. Cells were seeded at a density of 104 cells/cm2 in normal serum-supplemented DMEM and received either no drug (O), PMA (0.3 pM, A), or 4a-PDD (0.3 gM, *) twice daily. At the time points indicated, cells were counted by haemocytometry. Points shown mean cell numbers (n = 6); all s.e.means are contained within the symbols. * P < 0.05; ** P < 0.0005; denotes a significant difference from untreated cells on that day.
ENDOTHELIAL CELL PROLIFERATION 70-
a, 60-
Table 1 Effect of phorbol myristate acetate (PMA) on [3H]thymidine incorporation by pig aortic endothelial cells (PAEC)
9
-0
E
** ~
T/
0~
:350-
103
~
*
[3H]-thymidine incorporation (d.p.m.)
8 40-
Treatment
4h
12h
6454 + 553** 12430 + 1060 8530 + 550**
14259 + 2865** 31380 + 2520 17730 + 1890**
C
c
0
30-
No serum Low serum Low serum and PMA (0.3 gM)
0
C.,
r 20-
*
a)
O-
g 10-
-9
-10
-8
-7
-6
log PMA(M)
Figure 2 Concentration-effect curve showing the ability of phorbol myristate acetate (PMA) to inhibit proliferation of pig aortic endotheHal cells (PAEC) in normal serum-supplemented DMEM. Cells were seeded at a density of 104 cells/cm2. PMA (0.1 nM-l pM) was added twice daily. The cells were allowed to grow for 4 days and then counted by haemocytometry. The results are expressed as the mean (%) of cell number (s.e.mean reduction shown by vertical bars) when compared with untreated cells (n = 6); where error bars are not seen they are contained within the symbols. * P < 0.005; ** P
Br. J. Pharmacol. (1991), 102, 101-106
Effects of cyclic nucleotides and phorbol myristate acetate on proliferation of pig aortic endothelial cells Shonna Alexandra Moodie & 'William Martin Department of Pharmacology, University of Glasgow, Glasgow, G12 8QQ 1 The role of cyclic nucleotides and protein kinase C in controlling proliferation of pig aortic endothelial cells (PAEC) in culture was investigated. 2 Dibutyryl cyclic AMP (30.uM), added twice daily, inhibited proliferation but 8 bromo cyclic GMP (30pM) had no effect. Two other stimuli known to increase PAEC cyclic GMP content by stimulating particulate and soluble guanylate cyclase respectively, atriopeptin II (10nM) and sodium nitroprusside (1 pM), were also without effect on proliferation. 3 Two agents known to inhibit soluble guanylate cyclase and lower intercellular cyclic GMP content, haemoglobin (10pM) and methylene blue (1pOM), each inhibited proliferation of PAEC. 4 The inhibitory effect of haemoglobin (10pM) was mediated by inhibition of soluble guanylate cyclase since it was reversed by agents known to increase cyclic GMP content, i.e. atriopeptin 1I(10 nM), 8 bromo cyclic GMP (30,uM) or sodium nitroprusside (1 uM). The inhibitory effect of methylene blue (10pM) was not reversed by these agents. 5 Phorbol 12-myristate 13-acetate (PMA, 0.1 nM-l UM), which activates protein kinase C, inhibited proliferation in a concentration-dependent manner. No early stimulation of proliferation was seen wtih PMA. The inactive isomer, 4a-phorbol 12,13-didecanoate (0.3,pM), lacked the ability of PMA to inhibit proliferation of PAEC. 6 PMA-induced inhibition of proliferation appeared not to be due to stimulated production of destructive oxygen-derived free radicals since it was unaffected by the radical scavengers, vitamin E (30,UM) or butylated hydroxytoluene (30puM). The antiproliferative actions of paraquat (10pM), an agent which generates free radicals intracellularly, was, in contrast, inhibited by vitamin E or butylated hydroxytoluene. Furthermore, neither dibutyryl cyclic AMP (30,UM) nor 8 bromo cyclic GMP (30pM) had any effect on the ability of PMA to inhibit proliferation. 7 This study suggests that cyclic AMP, cyclic GMP and protein kinase C play a role in controlling the proliferation of PAEC.
Introduction Identification of the hormonal factors and intracellular pathways that control migration and proliferation of vascular endothelial and smooth muscle cells is important since dysfunction of both cell types is observed in several vascular diseases (Hoshi et al., 1988a; Klagsbrun & Edelman, 1989). With the exception of cells from the microvasculature (Bar et al., 1989), endothelial cells exhibit a growth independence from platelet-derived mitogens by being able to grow equally well in serum- or plasm-supplemented medium (Kazlauskas & DiCorleto, 1985). Several endothelial cell mitogens have been identified, including, basic fibroblast growth factor, epidermal growth factor and a family of acidic polypeptide mitogens, including acidic fibroblast growth factor, endothelial cell growth factor and eye-derived growth factor (Gospodarowicz et al., 1978; 1986; Schreiber et al., 1985; Sato & Rifkin, 1988). The mitogenic actions of growth factors are mediated by interactions with cell surface receptors, and protein phosphorylation is known to be an early biochemical signal initiated upon activation of these receptors. Furthermore, differences in protein phosphorylation patterns occur in proliferating and density-inhibited endothelial cells (Kazlauskas & DiCorletto, 1987). Several studies indicate that cyclic nucleotides acting via specific protein kinases may regulate proliferation of cells (Friedman, 1981). Subsequent reports have shown that cyclic AMP has differing actions on proliferation of endothelial cells from different sources in culture: inhibition of proliferation was observed with bovine aortic and rat brain microvascular endothelial cells (Leitman et al., 1986; Kempski et al., 1987), ' Author for
correspondence.
but stimulation was observed in a foetal bovine aortic endothelial cell line (Presta et al., 1989). In contrast to cyclic AMP, the role of cyclic GMP in endothelial cell proliferation has not been reported. The role of the phospholipid/calcium-dependent kinase, protein kinase C, in signal transduction is well documented (Nishizuka, 1984; Blackshear, 1988). The activation of protein kinase C by tumour promoting phorbol esters has been shown to have differing actions on proliferation of endothelial cells from different sources in culture: inhibition of proliferation was observed with human aortic and bovine capillary endothelial cells (Doctrow & Folkman, 1987; Hoshi et al., 1988b); no effect was seen on bovine aortic endothelial cells (Doctrow & Folkman, 1987); and stimulation was observed on foetal bovine aortic and bovine cerebral cortex capillary endothelial cells (Daviet et al., 1989; Presta et al., 1989). In pig aortic endothelial cells, phorbol esters have been reported to produce an initial stimulation of proliferation which is followed within hours by inhibition (Uratsuji & DiCorleto, 1988). In this study we have attempted to evaluate more fully the influence of cyclic nucleotides and of protein kinase C stimulation upon proliferation of pig aortic endothelial cells in culture.
Methods
Endothelial cell culture Pig aortae were obtained from a local abattoir and the endothelial cells isolated by collagenase (0.2%) treatment as previously described (Gordon & Martin, 1983). Pig aortic endothelial cells (PAEC) were grown in Dulbecco's modified
102
SA. MOODIE & W. MARTIN
Eagle's medium (DMEM) supplemented with foetal calf serum (10%), newborn calf serum (10%), penicillin (100unitsml-'), streptomycin (lOOpgml-1), kanamycin (lOOpgml-') and glutamine (4mM): this is subsequently referred to in the text as normal serum-supplemented DMEM. Cells were grown at 370C under an atmosphere of 5% CO2 in air. Cells were seeded initially into 80cm2 tissue culture flasks and grown to confluence, attained in 6-8 days. The serumsupplemented DMEM was aspirated off and replaced every 2 or 3 days. Cells were characterized as endothelial cells by their growth in a strict monolayer and typical cobblestone appearance. Furthermore, we have previously reported their ability to secrete prostacyclin and endothelium-derived relaxing factor (Martin et al., 1988), and to fluoresce when incubated with the selective marker, acetylated low-density lipoprotein labelled with 1, 1'dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchorate (CMD, UK Ltd) (Voyta et al., 1984).
(Poole, Dorset). [Methyl-3H]-thymidine was purchased from Amersham (Bucks).
Statistical analysis Results are expressed as the mean + s.e.mean and comparisons were made by use of Student's t test or the MannWhitney test when there was unequal variance between samples. A probability of 0.05 or less was considered significant. In the Results, n represents the number of wells of cells.
Results Effect of phorbol 12-myristate 13-acetate on endothelial cell proliferation Pig aortic endothelial cells (PAEC) seeded at a density of 104
cells/cm' in normal serum-supplemented DMEM grew to Proliferation studies For proliferation studies, PAEC were seeded at a density of approximately 104 cells/cm2 in six-well plates (9.6cm2). The effects of various treatments on proliferation were then examined over the following 6-10 days. PMA, vitamin E, butylated hydroxytoluene and 4a-phorbol 12,13-didecanoate (4aPDD) were dissolved in 100% ethanol. At the dilutions used, the maximum concentration of ethanol (0.1% v/v) had no effect by itself on proliferation. All other drugs were dissolved in distilled water and sterilised by filtration through a Millipore filter (0.2 yM pore size). Solutions of haemoglobin were reduced to the ferrous form before use with sodium dithionite as previously described (Martin et al., 1985). All drugs were added twice daily with the exception of methylene blue which was added once daily. At the various time points indicated in the Results, cells were detached with a solution of trypsin (0.05%) and EDTA (0.02%) and counted by haemocytometry.
[3H]-thymidine incorporation experiments PAEC were plated at a density of approximately 1.5 x 105 cells/cm2 in six-well plates. The cells were grown in normal serum-supplemented DMEM for 24h. After a further 24h incubation in serum-free medium, the cells were challenged with drugs in low serum (2% foetal calf and 2% newborn calf serum) containing DMEM and pulsed with a mixture of [3H]-thymidine (2juCi/well) and unlabelled thymidine (1 pM) for various times as indicated in the Results. At the end of the incubation period, the cells were washed 3 times with 2 ml of 5% trichloroacetic acid and solubilised in 1 ml of 0.25 M NaOH. The radioactivity in the solubilised cells was determined by liquid scintillation counting and expressed in d.p.m.
confluence within 6-8 days (Figure 1). The activator of protein kinase C, PMA (0.3pM), when added twice daily, produced a marked inhibition of proliferation: 86 + 2% (n = 6) inhibition was observed at day 8 (Figure 1). The ability of PMA to inhibit proliferation was observed over the concentration range 0.1 nM-i /IM (Figure 2), but the inactive phorbol ester, 4a-PDD (0.3 pM), lacked the ability of PMA to inhibit proliferation (Figure 1). PMA did not stimulate trypan blue uptake by cells (data not shown). Phorbol esters are know to stimulate production of oxygenderived free radicals in endothelial cells (Matsubara & Ziff, 1986). To determine if these radicals contribute to the ability of PMA to inhibit proliferation of PAEC, we examined the effects of vitamin E (30pM) and butylated hydroxytoluene (BHT, 30,UM). Vitamin E and BHT, when added twice daily, each had no effect on proliferation by themselves and had no effect on the ability of PMA (0.3 pM) to inhibit proliferation (Figure 3a). Paraquat (10pM), an agent which generates oxygen-derived free radicals intracellularly (Minakami et al., 1990), when added twice daily, also inhibited proliferation of PAEC (Figure 3b). This inhibition was associated with accumulation of trypan blue, and was prevented by the presence of vitamin E (30pM) or BHT (30pM) (Figure 3b).
Effect of PMA on [3H]-thymidine incorporation by endothelial cells Phorbol esters have been reported initially to stimulate then inhibit proliferation of pig aortic endothelial cells (Uratsuji & 10-
Ln
8-
*;-G
0
Materials Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, glutamine, foetal calf serum, newborn calf serum and trypan blue were purchased from Gibco Ltd. (Paisley, Scotland). Trypsin - EDTA and kanamycin were purchased from Flow Laboratories (Irvine, Scotland). Tissue culture flasks (80 cm2) and six-well multidishes were supplied by Nunc (Denmark). Atriopeptin II, 8 bromo guanosine 3': 5'-cyclic monophosphate (8 bromo cyclic GMP), butylated hydroxytoluene, collagenase (type II), dibutyryl adenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP), haemoglobin (bovine erythrocytes), methylene blue, paraquat (,_1'dimethyl-4-4'bipyridinium dichloride), 4a-phorbol 12,13-didecanoate (4aPDD), phorbol 12-myristate 13-acetate (PMA), sodium nitroprusside, vitamin E (D,L-a-tocopherol acetate) and thymidine were purchased from the Sigma Chemical Company Ltd. (Poole, Dorset). Sodium dithionite was purchased from BDH
6-
S
4-
0~~~
-
CD .0
E:
a)
2-
a~~~ 2
4 T Time (days)
6
8
10
Figure 1 The effect of phorbol myristate acetate (PMA) and 4aphorbol 12,13-didecanoate (4ca-PDD) on the proliferation of pig aortic endothelial cells. Cells were seeded at a density of 104 cells/cm2 in normal serum-supplemented DMEM and received either no drug (O), PMA (0.3 pM, A), or 4a-PDD (0.3 gM, *) twice daily. At the time points indicated, cells were counted by haemocytometry. Points shown mean cell numbers (n = 6); all s.e.means are contained within the symbols. * P < 0.05; ** P < 0.0005; denotes a significant difference from untreated cells on that day.
ENDOTHELIAL CELL PROLIFERATION 70-
a, 60-
Table 1 Effect of phorbol myristate acetate (PMA) on [3H]thymidine incorporation by pig aortic endothelial cells (PAEC)
9
-0
E
** ~
T/
0~
:350-
103
~
*
[3H]-thymidine incorporation (d.p.m.)
8 40-
Treatment
4h
12h
6454 + 553** 12430 + 1060 8530 + 550**
14259 + 2865** 31380 + 2520 17730 + 1890**
C
c
0
30-
No serum Low serum Low serum and PMA (0.3 gM)
0
C.,
r 20-
*
a)
O-
g 10-
-9
-10
-8
-7
-6
log PMA(M)
Figure 2 Concentration-effect curve showing the ability of phorbol myristate acetate (PMA) to inhibit proliferation of pig aortic endotheHal cells (PAEC) in normal serum-supplemented DMEM. Cells were seeded at a density of 104 cells/cm2. PMA (0.1 nM-l pM) was added twice daily. The cells were allowed to grow for 4 days and then counted by haemocytometry. The results are expressed as the mean (%) of cell number (s.e.mean reduction shown by vertical bars) when compared with untreated cells (n = 6); where error bars are not seen they are contained within the symbols. * P < 0.005; ** P
