shown. Radioligand binding results were analyzed by standard linear regression analysis.
3. Results
3
.
1
. Effect of
5
HT and PDGF on
3
[
H
]
thymidine incorporation by SMC
To find out the mitogenic concentration of 5HT and PDGF growth arrested and synchronized SMC were
stimulated with 5HT or PDGF in serum free medium and the amount of
3
[H]thymidine incorporated into the DNA determined as described in Section 2. The effect
of 5HT and PDGF on SMC proliferation was deter- mined on growth arrested SMC. 5HT at an added
concentration of 100 nM induced
3
[H]thymidine incor- poration into SMC and the effect was maximal at a
concentration of 100 mM of 5HT. At concentrations greater then 200 mM of 5HT, there was a decrease in
3
[H]thymidine incorporation, suggesting that higher concentrations of 5HT may be cytotoxic to SMC Fig.
1. Exposure of SMC to PDGF was similarly associated with enhanced DNA synthesis Fig. 1B. SMCs re-
sponded to PDGF with concentration dependent in- crease in
3
[H]thymidine incorporation into DNA. Stimulation of
3
[H]thymidine incorporation was maxi- mal at PDGF concentrations above 20 ngml.
3
.
2
. Effect of n-
3
fatty acid pretreatment on
3
[
H
]
thymidine incorporation by SMC To find out the effect of preloading of SMC with
EPA, DHA, OA or LA on SMC proliferation, SMC were preloaded and synchronized in the presence of
different concentrations of fatty acids. After synchro- nization SMC were stimulated with 1 FBS containing
DMEM and the amount of
3
[H]thymidine incorporated into the DNA determined as described in Section 2.
When SMC were preloaded with different concentra- tions of EPA or DHA, up to a concentration of 7.5 mM
of EPA or DHA there was no significant effect on
3
[H]thymidine incorporation by the SMC Fig. 2. However, when SMC were grown in concentrations of
EPA or DHA higher than 7.5 mM, there was a gradual decrease in the amount of
3
[H]thymidine incorporation upto a concentration of 30 mM of EPA or DHA Fig.
2. Concentrations higher than 30 mM EPA or DHA completely inhibited
3
[H]thymidine incorporation by SMC. These results indicate that higher concentrations
of EPA and DHA may be cytotoxic to SMC. To determine whether this response was specific to n-3
fatty acids or is it common to all classes of fatty acids, SMC were preincubated with either LA n-6 or OA
n-9 in the same concentration range 0 – 100 mM and the amount of
3
[H]thymidine incorporated into the DNA measured. The results indicate that when SMCs
were incubated with LA the
3
[H]thymidine incorpora-
Fig. 1. Graph showing concentration dependent stimulation of aortic smooth muscle cell SMC by serotonin 5HT A and platelet derived growth factor PDGF B.
3
[H]Thymidine incorporation into DNA was determined in synchronized cells stimulated by various concentrations of 5HT and PDGF in serum a free medium in the presence of 100 mM pargyline, as described in Section 2. One hundred percent equals the baseline
value of
3
[H]thymidine uptake. 100 = 8130 9 210 CPM10
6
cells for A, 100 = 7642 9 350 CPM10
6
cells for B. The experiments were performed with two different batches of cells and each batch was tested in triplicate. Results are mean 9 S.D.,
+
P B 0.05, P B 0.01.
Fig. 2. Effect of eicosapentaenoic acid EPA, docosahexaenoic acid DHA, oleic acid OA and g-linolenic acid LA on smooth muscle
cell SMC proliferation. Aortic SMC were grown in the presence of different concentrations of EPA, DHA, LA or OA in 10 fetal
bovine serum FBS containing medium for 72 h, and then with the same concentrations of fatty acids in 0.1 containing medium for 72
h and then stimulated with serum free medium containing 100 mM pargyline. The amount of
3
[H]thymidine incorporated into DNA was measured as described in Section 2. One hundred percent equals the
baseline value of
3
[H]thymidine uptake. 100 = 7960 9 300 CPM10
6
cells. The experiments were performed with two different batches of cells and each batch was tested in triplicate. Results are mean 9 S.D.,
+
P B 0.05, P B 0.01.
3
[H]thymidine incorporated into the DNA of EPA treated cell was decreased by 40 Fig. 3C and DHA
treated cells by 30 Fig. 3D as compared to the control cells. In contrast, preloading of SMC with LA or OA did
not inhibit their proliferative response either to 5HT or to PDGF Fig. 4. In fact, even modest concentrations
of 5HT 50 mM could stimulate
3
[H]thymidine incorpo- ration in SMC grown in 3.33 mM of LA or OA Fig. 4B.
These results suggest that preloading of SMC with either EPA or DHA makes them non-responsive to 5HT and
partially responsive to PDGF and this effect was specific for only n-3 fatty acids not shared by n-6 or n-9 fatty
acids.
Next the combined effect of EPA and DHA on SMC in preventing 5HT or PDGF induced SMC proliferation
is determined. Since EPA and DHA are present approx- imately in 2.1 ratio in fishoils, SMC were preloaded with:
a 0.11 or 0.22 mM EPA; or b 0.055 or 0.11 mM DHA alone; or c with 0.11 mM EPA and 0.055 mM DHA
0.165 nM of total n-3 fatty acids; or d with 0.22 mM EPA and 0.11 mM DHA 0.33 mM of total n-3 fatty acids
and then stimulated with 5HT or PDGF. The results indicate that EPA or DHA at the concentrations tested
did not prevent 5HT or PDGF induced SMC prolifera- tion Fig. 5. Further, EPA or DHA by themselves, at
a concentration of 0.33 mM alone produced only a modest inhibition of SMC proliferation Fig. 3A for EPA
and Fig. 3B for DHA. However, when EPA and DHA were added together at a total concentration of 0.33 mM
of n-3 fatty acids 0.22 mM EPA and 0.11 mM DHA 5HT induced SMC proliferation was completely inhibited
Fig. 5A and PDGF induced SMC proliferation was partially inhibited Fig. 5B. These results indicate that
EPA and DHA may act synergistically in inhibiting the SMC proliferative response to 5HT and PDGF.
3
.
4
. Effect of n-
3
fatty acids on SMC cell number To determine whether the inhibition of DNA synthesis
results in decreased cell number, SMCs were preloaded with EPA or DHA or EPA and DHAs together were
stimulated with 5HT or PDGF and the number of cells counted. The results show that the inhibition of DNA
synthesis by EPA and DHA preloading results in de- creased cell number Fig. 6. Stimulation of control cells
with 100 mM 5HT resulted in an increase of 103 500 9 6750 cells over the control, whereas when 3.2, 3 mM EPA
or DHA or 0.33 mM EPA + DHA preloaded cells were stimulated with the same concentration of 5HT the cell
numbers
were increased
only by
11 250 9 1050, 16 750 9 2100, 21 500 9 3100 cells respectively Fig.
6A. Similarly with PDGF stimulated cells, the cell numbers were increased by 52 500 9 5670, 56 900 9
6070, 56 250 9 5750 cells respectively as compared to an increase of 756 500 9 12 500 cells in the control Fig.
6B. tion response was similar to that of EPA or DHA. In
contrast, with OA there was only a minimal inhibitory effect on SMC growth Fig. 2.
3
.
3
. Effect of
5
HT and PDGF on
3
[
H
]
thymidine incorporation by SMC grown in the presence of n-
3
fatty acids To determine the effect of EPA and DHA on 5HT and
PDGF induced SMC proliferation, primary SMC were grown in the presence of different concentrations of EPA
or DHA 0 – 3.33 mM in 10 FBS containing medium for 72 h. Only the non-inhibitory and non-cytotoxic
concentrations of EPA and DHA were selected to make sure that the observed effects of EPA and DHA on 5HT
and PDGF induced SMC proliferation were not due to the inhibitory or cytotoxic effect of EPA and DHA. As
controls, another group of cells was grown in the presence of same concentrations 0 – 3.33 mM of LA or
OA. Following this, the cells were growth arrested for 72 h in the same concentrations of fatty acids. Then SMC
were stimulated with 50, 100 or 200 mM of 5HT or 10, 20 or 30 ngml PDGF. The results indicated that when
SMC preloaded with 3.33 mM EPA or DHA was stimulated with 5HT, even the highest concentration of
5HT tested failed to induce significant
3
[H]thymidine incorporation Fig. 3A,B. However, when the same cells
were stimulated
with PDGF,
the amount
of
3
.
5
. Effect of n-
3
fatty acids on
5
HT
2
receptor mRNA le6els
Next the mechanism by which EPA and DHA may inhibit the proliferative response of SMC to 5HT is
determined. In vascular cells 5HT mediate its prolifera- tive effect via the 5HT
2
receptor and 5HT
2
receptor antagonists can block the 5HT induced
3
[H]thymidine incorporation [30,31]. Therefore, the effect of 5HT
alone, or n-3 fatty acids alone, or the effect of n-3 fatty acids with 5HT on 5HT
2
receptor mRNA level was determined. Stimulation of SMC with 5HT induced an
: 55 increase in the 5HT
2
receptor mRNA levels when compared to the unstimulated cells control. When
SMC preloaded with EPA or DHA were stimulated with 5HT, there was an 11 or a 22 increase respec-
tively of the 5HT
2
receptor mRNA levels as compared to controls : 55 increase Fig. 7. These results
suggest that EPA or DHA may attenuate the SMC proliferative response to 5HT by down regulating the
mRNA levels for 5HT
2
receptor in SMC.
3
.
6
. Effect of n-
3
fatty acids on
5
HT
2
receptor number To determine whether the changes in 5HT
2
mRNA levels induced by EPA or DHA, translates to an alter-
ation in 5HT
2
receptor number, Scatchard analyses were performed using
3
[H]LSD as a 5HT
2
receptor ligand. The results suggest, that although EPA and
DHA attenuate the 5HT induce increase in mRNA levels for 5HT
2
receptor, there was no significant changes in 5HT
2
receptor density data not presented.
4. Discussion
