Vascular smooth muscle cells preloaded with eicosapentaenoic acid
and docosahexaenoic acid fail to respond to serotonin stimulation
Rajbabu Pakala *, Rajashree Pakala, Wen Lu Sheng, Claude R. Benedict
Department of Internal Medicine,Di6ision of Cardiology,Uni6ersity of Texas Health Science Center-Medical School,6431Fannin,MSB6.039,
Houston,TX77030,USA
Received 14 July 1999; received in revised form 16 November 1999; accepted 19 January 2000
Abstract
Epidemiological, animal and clinical studies indicate that n-3 fatty acids may benefit individuals with known history of cardiovascular disease or at risk of developing it. Though there is indirect evidence to suggest that the beneficial effects of n-3 fatty acids may be because of their ability to inhibit smooth muscle cell (SMC) proliferation, there are no studies that have examined this hypothesis. In this study, the mitogenic effect of serotonin (5HT) and platelet derived growth factor (PDGF), known mitogens for vascular SMC, on aortic SMCs preloaded with eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) is examined. 5HT and PDGF could only partially stimulate proliferation of SMC that were preloaded with EPA or DHA as compared to the control cells.g-Linolenic acid (LA) and oleic acid (OA) did not block the 5HT or PDGF induced3[H]thymidine incorporation
suggesting that the anti-proliferative effect was specific to n-3 fatty acids only. Further, when EPA and DHA were combined in the ratio they are present in fishoils, there was a synergistic interaction in inhibiting the proliferation of SMC. Further, SMC grown in the presence of EPA or DHA, when stimulated with 5HT, failed to show an increase in 5HT2receptor mRNA. One of the potential mechanism by which fish oils may prevent the development of atherosclerosis or restenosis could be inhibition of the mitogen induced SMC proliferation. Combination of EPA with DHA is likely to be more beneficial. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Eicosapentaenoic acid; Docosahexaenoic acid; Vascular smooth muscle cells; Atherosclerosis; Restenosis
www.elsevier.com/locate/atherosclerosis
1. Introduction
Evidence from epidemiological [1 – 4], biochemical [5 – 8], animal [9 – 16] and clinical studies [17 – 19] indi-cate that dietary n-3 fatty acids may benefit individuals with established atherosclerosis or at risk of developing it, including patients undergoing interventional proce-dures for symptomatic coronary artery disease. Popula-tions consuming traditional diets rich in n-3 fatty acids, or western diets supplemented with n-3 fatty acids, primarily C20: 5n-3, eicosapentaenoic acid (EPA) and C22:6 n-3 docosahexaenoic acid (DHA) exhibit charac-teristic biochemical and functional changes including decreased serum triglycerides, variable decrease in total serum cholesterol and an increase in high density lipo-protein cholesterol [5,6], altered cell membrane
compo-sition [8], decreased blood viscosity [7] and impaired platelet Hemostatic functions [7,8]. Although the reduc-tion in thrombotic vascular events has been attributed to the metabolic effects of substituting n-3 fatty acids for arachidonic acid and the generation of eicosanoid products that modify platelet and vascular functions, animal experiments and clinical data provide only indi-rect evidence in this regard. Dietary n-3 fatty acids reduce experimental vascular lesion formation in dogs [10,20], swine [11,12], rabbits [13,14] and non-human primates [15,16], and increased fish consumption has been associated with decreased mortality from coronary artery disease [17,19 – 21]. However, the effects of di-etary n-3 fatty acids on restenosis in patients undergo-ing coronary angioplasty has been inconclusive [22,23]. Restenosis following angioplasty involves migration and proliferation of vascular smooth muscle cells (SMC) probably in response to mitogens released from: (a) aggregating platelets [24,25]; (b) monocyte that are
* Corresponding author. Tel.: +1-713-5006622; fax: + 1-713-5006625.
derived from macrophages accumulating at the injured site [26], and from endothelial cells subjected to injury [27,28]. In vitro cell culture and in vivo animal studies have indicated that the vasoactive compounds like sero-tonin (5HT) and thromboxane A2 released from the
aggregating platelets may play a major role in the development of neointima [29 – 32]. These compounds have been shown not only to act as mitogens by themselves but also act as amplification factors for known peptide growth factors as platelet derived growth factor (PDGF) and fibroblast growth factor (FGF) [24,32]. Recently, it has been reported that 5HT and thromboxane A2can also act synergistically among
themselves in inducing SMC proliferation [31]. The objective of the present study is to find out whether preloading of SMC with EPA and/or docasahexaenoic acid (DHA) (n-3 fatty acids present in fish oils) makes them less responsive to mitogens that are known to play an important role in the development of atherosclerosis and restenosis. Results of this study indicate that SMC grown in the presence of (EPA) and/or (DHA) are non-responsive to 5HT and PDGF stimulation. This suggests that one of the mechanisms by which fishoils may reduce the neointima formation is by inhibiting the SMC responsiveness to mitogenic factors released by aggregating platelets.
2. Materials and methods
2.1. Materials
5HT (as creatine sulphate), ethylene diamine te-traacetic acid (EDTA), pargyline, Hank’s balanced salts (HBSS), g-linolenic acid (6,9,12-octadecatrienoic acid, LA), oleic acid (cis-9 octadecenoic acid, OA) and lyser-gic acid diethylamide (LSD) were obtained from Sigma, St. Louis, MO; Dulbecco’s modified Eagles medium (DMEM) and fetal bovine serum (FBS) were obtained from Whittaker Bioproducts, Walkersville, MD; Hu-man PDGF was obtained from Amersham Life Science, Arlington Heights, IL, 3[H]thymidine (20 Ci
/mol) and
3[H]LSD (
N-methyl-3[H]lysergic acid diethylamide from
New England Nuclear, Boston, MA). Other reagents were purchased from local vendors. EPA and DHA were provided by the United States Department of Commerce, National Oceanic and Atmospheric Admin-istration, Charleston, SC.
2.2. Isolation, culture and characterization of canine primary aortic SMC
Canine primary aortic SMCs were isolated using the explant method described by Pakala et al. [31]. Briefly the intima was first peeled off from the aorta and then the media carefully stripped away from the adventitia
and placed in a pettridish containing warmed DMEM (37°C). The medial layer was cut into approximately 1 mm squares, which were transferred into a 25 cm2
tissue culture flask and barely covered with DMEM supplemented with 20% FBS. The blocks of tissue were cultured in a humidified atmosphere of 95% air and 5% CO2 (vol/vol) at 37°C. After 1 – 2 weeks, the tissue
blocks were removed and the migrated SMCs were cultured. Following isolation, the identity of the SMC was confirmed by morphological examination and by staining for a-actin.
Subcultures of SMC done once they became conflu-ent, media from the plates aspirated and the cells washed with 10 ml of phosphate buffered saline (PBS). Then 2 – 3 ml of trypsin – EDTA (0.05% trypsin, 0.53 mM EDTA in Ca2+, Mg2+ free HBSS) were added to the cells and incubated at room temperature for 2 – 3 min. The action of trypsin was stopped by the addition of 7 – 8 ml of DMEM containing 10% FBS. The cells were collected by centrifugation at 150×g for 10 min. After removing the supernatant, the pelletes cells were dispersed in 10 ml of DMEM containing 10% FBS and fresh cultures were initiated from these cells.
2.3. 3[H]Thymidine incorporation
SMCs from passages 2 or 3 were seeded into 35 mm diameter plates at a density of 65 000 – 75 000 cells/
plate in DMEM containing 10% FBS and allowed to proliferate for 72 h. After 72 h, the growth medium was replaced with 2 ml DMEM containing 0.1% FBS and incubated for an additional 72 h for growth arrest and synchronization. After growth arrest cells were stimu-lated with serum free medium containing 500 mg/ml bovine serum albumin (BSA), 10 mg/ml bovine insulin, 25 ng/ml selenium, 0.2 nM ascorbate and 100 mM pargyline or the same medium with indicated concen-trations of 5HT or PDGF. After 20 h of incubation with 5HT or PDGF, 1mCi of3[H]thymidine was added to each plate and then incubated for an additional 4 h. Twenty-four hours after addition of compounds, the medium was removed and the plates washed three times with ice cold PBS. Then 6% trichloroacetic acid was added to the cells and the acid insoluble thymidine collected on to glass fiber filter. The filters were washed with 100% ethanol, air dried and the amount of
3[H]thymidine incorporated into the DNA was
growth arrest, the cells were stimulated with 1% FBS containing medium and the amount of 3[H]thymidine
incorporated into the DNA determined as described earlier. In a third set of experiment SMC were growth arrested and synchronized in non-cytotoxic concentra-tions of EPA, DHA, OA or LA and then stimulated with mitogenic concentrations of 5HT or PDGF in serum free medium and the amount of 3[H]thymidine
incorporated into the DNA measured as described ear-lier. The cells were counted (Coulter counter) on the day of seeding and before changing to 0.1% FBS con-taining medium to ensure that they were growing nor-mally, and finally on day 6 to determine that the cells were growth arrested. All experiments were performed in triplicate and each experiment was repeated mini-mum 3 times.
2.4. Determination of SMC number
After preloading the SMC with fatty acids as de-scribed in the3[H]thymidine incorporation section, cells
were stimulated with either 5HT or PDGF. Twenty-four hours after stimulation, 0.4 ml of 2% (w/v) crude pancreatic trypsin in Dulbeeco’s PBS containing 152 mM EDTA was added to each dish. The dishes were incubated at room temperature for 2 min before addi-tion of 0.8 ml of horse serum. The contents of each dish were diluted to 20 ml with isotone II (Coulter Electron-ics) and the cell number determined by using a Coulter counter. Triplicate counts were taken for each plate and quadruplicate plates were used for each determination.
2.5. Isolation of poly(A+) RNA and Northern blot
analysis
After preloading SMC with EPA and DHA as de-scribed earlier cells were stimulated with serum free medium alone or with serum free medium containing 100mM 5HT. Twenty-four hours after stimulation cells were rinsed twice with PBS, and mRNA isolated using the ‘on track’ mRNA isolation kit (Biotex Laborato-ries, Houston, TX) and 5HT2 receptor mRNA
quantified Poly(A+) RNA was denatured with formal-dehyde and formamide and size-fractionated on a 0.66 M formaldehyde/1.3% agarose gel for 304 h at 80 AV RNA transferred overnight onto a magnagraph nylon transfer membrane (MSI, Westboro, MA) by electro-blotting in 10× SSC (1× SSC contains 0.15 M Nacl, 0.015 M trisodium citrate). The RNA was cross-linked to the membrane at 120 000 mJ using UV cross linker (Hoeffer Scientific Instruments, San Francisco, CA) and prehybridized in 50% deionized formamide, 4×
SSC, 20 mM Tris Hel (PH 8.0), 1× Denhardt’s solu-tion (0.02% ficoll, 0.02% polyvinal pyrrolidine, 0.02% BSA), 0.1% sodium dodecyl sulfate (SDS), 200 mg/ml denatured salmon sperm DNA and 10% dextran sulfate
for 20 min. 35S-labeled cRNA probe was synthesized
using the full-length 5HT2receptor coding region
(pro-vided by Dr Julies) and the ‘MAXIscript’ kit (Ambion, Austin, TX). Approximately 3 ng/ml of probe was added to the pre-hybridization solution and hybridiza-tion was performed overnight at 42°C. Filters were washed 3 times for 20 min with 0.1× SSC containing 0.1% SDS at room temperature and 3 times for 20 min with 0.1× SSC containing 0.1% SDS at 60°C. The filters were then exposed to X-ray film at −80°C. For semi-quantification filters were stripped of 5HT2 probe
and reprobed with random primed GAPDH. The rela-tive quantity of 5HT2 mRNA in each sample was
analyzed by densitometry using OPTIMIUS program on a Toshiba Image analyzer and corrected for loading conditions by the quantity of mRNA for GAPDH. The concentrations of mRNA are expressed as densitomet-ric units.
2.6. Radioligand binding studies
After preloading the cells with EPA and DHA and stimulating with 5HT as described earlier, cell mem-branes isolated and radioligand binding studies were performed. Briefly the medium was removed and cell layers rinsed twice with PBS and scrapped into ice cold homogenizing buffer (50 mM Tris – Hcl, 0.5 mM Na2
EDTA, 10 mM Mg 504, PH 7.4). Cells were homoge-nized and membranes pelleted at 30 000×gfor 15 min. Pellets were resuspended in homogenizing buffer, incu-bated at 37°C for 15 min. and re-centrifuged at 30 000×g for 15 min, Supernatant decanted, and pel-lets stored at −45°C until use. Saturation analysis for 5HT2 receptors was performed with3[H]LSD (0.3125 –
20 nM). Non-specific binding was determined in the presence of 100-fold excess of unlabeled ligand. Assays were performed with approximately 120 mg of cell membrane protein in a final volume of 1 ml. The assay buffer was identical to the homogenizing buffer de-scribed above except that it contained 10 mM pargyline and 0.1% ascorbic acid. Samples were incubated for 30 min at 25°C and filtered through glass fiber filters which were presoaded in 0.1% poly-ethlemine for 30 min. Filters were washed rapidly with 10 ml of ice cold 50 mM Tris – HCl (PH 7.0) and radioactivity that re-mained bound to the filters was measured using liquid scintillation counter.
2.7. Statistical analyses
Data were analyzed by one way analysis of variance (ANOVA) for each interaction. When a statistically significant difference was obtained further analysis was conducted using Scheffe’s post-hoc tests. For all com-parisons statistical significance was assumed as PB
shown. Radioligand binding results were analyzed by standard linear regression analysis.
3. Results
3.1. Effect of 5HT 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 of3[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 induced3
[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 ng/ml.
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 of3[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 7.5mM 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 7.5mM, 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 – 100mM) 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 100mM pargyline, as described in Section 2. One hundred percent equals the baseline
value of3[H]thymidine uptake. 100%=81309210 CPM/106cells for A, 100%=76429350 CPM/106cells for B. The experiments were performed
Fig. 2. Effect of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), oleic acid (OA) andg-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 of3[H]thymidine incorporated into DNA was
measured as described in Section 2. One hundred percent equals the baseline value of3[H]thymidine uptake. 100%=79609300 CPM/106
cells. The experiments were performed with two different batches of cells and each batch was tested in triplicate. Results are mean9S.D., +PB0.05, *PB0.01.
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 (50mM) could stimulate3[H]thymidine incorpo-ration in SMC grown in 3.33mM 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 0.22mM EPA; or (b) 0.055 or 0.11mM 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 0.22mM EPA and 0.11mM DHA (0.33mM 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 0.33mM of n-3 fatty acids (0.22mM EPA and 0.11mM 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 5009
6750 cells over the control, whereas when 3.2, 3mM EPA or DHA or 0.33mM EPA+DHA preloaded cells were stimulated with the same concentration of 5HT the cell numbers were increased only by 11 25091050, 16 75092100, 21 50093100 cells respectively (Fig. 6A). Similarly with PDGF stimulated cells, the cell numbers were increased by 52 50095670, 56 9009
6070, 56 25095750 cells respectively as compared to an increase of 756 500912 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 5HT 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 – 3.33mM) 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 – 3.33mM) 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 ng/ml 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
3.5. Effect of n-3 fatty acids on5HT2 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 5HT2 receptor and 5HT2 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 5HT2 receptor mRNA level was
determined. Stimulation of SMC with 5HT induced an
:55% increase in the 5HT2 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 5HT2 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 5HT2 receptor in SMC.
3.6. Effect of n-3 fatty acids on 5HT2 receptor number
To determine whether the changes in 5HT2 mRNA
levels induced by EPA or DHA, translates to an alter-ation in 5HT2 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 5HT2 receptor, there was no significant
changes in 5HT2receptor density (data not presented).
4. Discussion
Platelets aggregate in response to arterial injury re-leasing vasoactive compounds and peptide growth fac-tors [33,34]. Some of these facfac-tors have been shown to stimulate the proliferation of SMC [25,31,32,35,36], indicating that factors released from aggregating platelets may play an important role in the develop-ment of restenosis. Antiplatelet agents have been shown to decrease intimal hyperplasia and to improve long
Fig. 3. Effect of serotonin (5HT) (A, B) and platelet derived growth factor (PDGF) (C, D) on proliferation of smooth muscle cell (SMC) preincubated with (A, C) eicosapentaenoic acid (EPA) (B, D) docosahexaenoic acid (DHA). Aortic SMC were grown in the presence of different concentrations of EPA or DHA in 10% fetal bovine serum (FBS) containing medium for 72 h, and then with the same concentrations of EPA or DHA in 0.1% containing medium for 72 h and then stimulated with serum free medium containing 100 mM pargyline (control) or given concentrations of 5HT and 100 mM pargyline or PDGF and the amount of3[H]thymidine incorporated into DNA were measured as described
in Section 2. One hundred percent equals the baseline value of3[H]thymidine uptake. 100%=84209260 CPM/106cells for A, 100%=81909230
CPM/106cells for B, 100%=74509316 CPM/106cells for C, 100%=79809270 CPM/106cells for D. The experiments were performed with two
Fig. 4. Effect of serotonin (5HT) (A, B) and platelet derived growth factor (PDGF) (C, D) on proliferation of smooth muscle cell (SMC) preincubated with (A, C) oleic acid (OA) (B, D)g-linolenic acid (LA). Aortic SMC were grown in the presence of different concentrations of OA
or LA in 10% fetal bovine serum (FBS) containing medium for 72 h, and then with the same concentrations of OA or LA in 0.1% containing medium for 72 h and stimulated with serum free medium containing 100 mM pargyline (control) or given concentrations of 5HT and 100 mM pargyline or PDGF. The amount of3[H]thymidine incorporated into DNA was measured as described in Section 2. One hundred percent equals
the baseline value of3[H]thymidine uptake. 100%=80209220 CPM/106cells for A, 100%=82109320 CPM/106 cell for B 100%=7759260
CPM/106cells for C, 100%=75609220 CPM/106cells for D. The experiments were performed with two different batches of cells and each batch
was tested in triplicate. Results are mean9S.D.
term patency of autologous vein grafts [37,38]. Fish oil feeding has been shown to decrease platelet counts, prolong bleeding time and reduce platelet aggregation [5,7]. These effects of fish oils on platelet metabolism resulted in studies to examine the effect of fish oils on intimal hyperplasia and atherosclerosis [9,11,15,16,20]. Although these studies indicate that fish oils may have an inhibitory effect on SMC proliferation, efforts to understand the mechanisms of action of n-3 fatty acids have focused largely on their effects on eicosanoid production [39,40]. Further, an ever-growing number of other effects, potentially linked to the inhibition of atherogenesis or restenosis appear also to be eicosanoid independent, which include decreased production of tissue factor and cytokines [41,42], inhibition of pro-duction of growth factor like mitogens [43,44] and attenuation of expression of endothelial cell adhesion molecules [45]. In general, polyunsaturated fatty acids (PUFA) have been reported to inhibit proliferation of vascular SMC [46,47], but the difference in efficacy between different PUFAs and the underlying mecha-nism involved have not yet been fully characterized. In
this study, the effects of fish oil derived n-3 PUFAs EPA and DHA are examined. OA (n-9 monounsatu-rated fatty acid) and LA (n-6 PUFA) were used as control fatty acids. In some studies arachidonic acid, has been used as a control, but since arachidonic acid and it is metabolites, like prostaglandins can directly influence SMC proliferation [48], LA might be a better alternative for n-6 PUFA because it is not converted to prostaglandins. In this study at low concentrations none of the fatty acids tested had any effect on SMC proliferation.
DHA at a concentration of 20mM and above inhibited SMC proliferation by 30 and 20%, respectively. They also demonstrated that incubating the cells with free radical Scavenger butylated hydroxytoluene (BHT) re-versed this inhibitory effect, which indicates that the concentrations of EPA and DHA used by Shiina et al. may have produced free radicals that are known to be cytotoxic at higher concentration [50]. In the present study, also when preloaded with 30 mM and higher concentrations of EPA or DHA or LA or 250mM and higher concentration of OA, SMC failed to grow at a normal growth rate when transferred to normal growth medium (data not presented). However cells preloaded with 7.5mm and lower concentrations of EPA or DHA or LA or OA continued to grow at normal rate (data
Fig. 6. Effect of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) preloading on serotonin (5HT) (A) or platelet derived growth factor (PDGF) (B) induced smooth muscle cell (SMC) cell number. Aortic SMC were grown in the presence of different concentrations of EPA and DHA individually or together (EPA+DHA combined) in 10% fetal bovine serum (FBS) containing medium for 72 h, and then with the same concentrations of EPA and DHA in 0.1% FBS containing medium for 72 h and then stimulated with serum free medium containing 100 mM pargyline (control) or given concentra-tions of 5HT or PDGF. Twenty-four hours after stimulation number of cells per plate was counted as described in Section 2, results are expressed as net increase in cell number over control. Control plates have 256 000911 560 cells/plate. Experiments were performed with three different batches of cells, and each batch were tested in tripli-cate, values are mean9S.D. +PB0.05; *PB0.01.
Fig. 5. Effect of serotonin (5HT) (A) and platelet derived growth factor (PDGF) (B) on proliferation of smooth muscle cell (SMC) preincubated with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Aortic SMC were grown in the presence of different concentrations of EPA and DHA in 10% fetal bovine serum (FBS) containing medium for 72 h, and then with the same concentrations of EPA and DHA in 0.1% FBS containing medium for 72 h and then stimulated with serum free medium containing 100 mM pargyline (control) or given concentrations of 5HT with 100 mM pargyline or PDGF. The amount of3[H]thymidine incorporated into DNA was
measured as described in Section 2. One hundred percent equals the baseline value of3[H]thymidine uptake. 100%=83109240 CPM/106
cells for A, 74309250 CPM/106 cells for B. The experiments were
performed with two different batches of cells and each batch was tested in triplicate. Results are mean9SD, +PB0.05, *PB0.01.
not presented) indicating that the concentrations of fatty acids used in this study are not cytotoxic. Addi-tion of BHT to the culture medium did not alter the blocking effect of EPA and DHA (data not presented) indicating that the blocking effects observed in this study were not due to the formation of cytotoxic con-centrations of free radicals.
In vascular SMC 5HT has been shown to excert its proliferative effect via the 5HT2 receptors [51]. In this
study stimulation of SMC with 5HT, resulted in upreg-ulation of the 5HT2 mRNA. Ligand induced
the same level as in the control cells, suggesting that receptor down regulation may be one of the mechanism by which EPA and DHA prevent 5HT induced prolifer-ation of the SMC. Even though there is a decrease in the amount of mRNA for 5HT2 receptors in EPA or
DHA preloaded SMC, the receptor affinity or the number was not significantly different from the control SMC. Fitzgerald et al. have reported that in uterine 5HT and its analogues unregulated and antagonists down regulated the 5HT2 receptor mRNA levels
with-out any significant difference in the affinity between the ligand and receptor number [54]. Changes in the levels of mRNA without analogous changes in corresponding protein levels have been shown for epidermal growth factor and interleukin-2 also [52,53]. In some instances this was found to be the result of specific processes
regulating receptor turnover [55,56]. Moreover the dis-parity between mRNA levels and receptor densities could be the result of mechanisms regulating distinct steps of protein synthesis like transcription and transla-tion [56].
On the basis of the present knowledge growth factors can be divided into two classes based on their transmembrane signaling: (1) the ones act through re-ceptor tyrosine kinases (PDGF, FGF epidermal growth factor insulin like growth factor) [57]; (2) The ones act through G-protein coupled receptors (bomberin, bradykinin, vasopressin, thrombin, 5HT and throm-boxane A2[31,58 – 60]. In the present study, it has been
demonstrated that upregulation of 5HT2 mRNA is
blocked by n-3 fatty acid pretreatment. Kiminski et al. [44] have already shown that n-3 fatty acids down regulates PDGF mRNA. Thus the present results and that of others suggest that n-3 fatty acids block the mitogenic effect of both the classes of growth factors. The partial blocking effect on PDGF induced stimula-tion maybe because of the fact that growth factors which act through tyrosine kinase pathway (‘classical’ growth factors) are very strong mitogens as compared to the mitogens which act through G-protein coupled receptors (hormones).
The effective concentrations of EPA and DHA in the present study are well below the concentrations nor-mally used in in vitro studies Wallace et al. [61] and Tremoli et al. [62] have shown that in healthy humans even low concentration of fish oil supplementation (2 – 3 g/day as compared to 5.4 – 20 g/day) has significantly reduced cytokine and tissue factor production. Results from this study and that of Wallace et al. [61] and Tremoli et al. [62] indicate that the beneficial effects of fishoils could be ascertained even lower concentrations. In fact, using lower concentrations of EPA and DHA may be more beneficial, because presence of high con-centrations of EPA and DHA in the membrane phos-pholipids makes them more prone to lipid peroxidation [63 – 65] and free radicals generated as a result of in-creased lipid peroxidation are known to be cytotoxic [50,66,67].
Although earlier studies have indicated that the de-creased rate of intimal hyperplasia [9,20] and atherosclerosis [15,16] in fish oil fed animals may be due to the decreased production of SMC growth factors by fish oil treated endothelial cells [43] and monocytes [44], a specific effect of EPA and DHA on vascular SMC proliferation was demonstrated that suggests ad-ditional mechanistic beneficial effects of n-3 fatty acid rich diets in reducing cardiovascular events.
Acknowledgements
The authors would like to thank Dr David Julius,
Fig. 7. Northern blot analysis for 5HT2 receptor mRNA in aortic
smooth muscle cell (SMC). Aortic SMC were grown in the presence of 10% fetal bovine serum (FBS) containing medium alone (control) or with either eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) for 72 h and then in 0.1% fetal bovine serum (FBS) contain-ing the medium (for control) or same medium with EPA (EPA group) or DHA (DHA group) for 72 h then cells was stimulated either with serum free medium containing 100 mM pargyline (control, EPA and DHA) or with serum free medium containing 100 mM pargyline with 50 mM serotonin (5HT, EPA-5HT, and DHA-5HT). mRNA was isolated from these cells and used for northern analysis: Panel A shows the expression of mRNA for 5HT2 receptor; and panel B for
the mRNA for GAPDH. Panel C shows the densitometric ratio of the 5HT2 receptor mRNA normalized to loading conditions with
Department of Pharmacology UCSF for providing 5HT2receptor probe, to the United States Department
of Commerce, National Oceanic and Atmosphere Ad-ministration, Charleston, South Carolina for providing EPA and DHA, Takuya Watanabe for his technical help and Shirley McWhorter for typing the manuscript. This study was supported by National Institute of Health/National Heart, Lung and Blood Institute grants, RO1-HL-39916, RO1-HL50653 and American Heart Association Grant-In-Aid (C.R. Benedict).
References
[1] Goodnight SH, Cairns JA, Fisher M, Fitzgerald GA. Assessment of the therapeutic use of n-3 fatty acids in vascular disease and thrombosis. Chest 1992;102:374S – 84S.
[2] Burr ML, Fehily AM, Gilbert JF, Rogers S, Holliday RM, Sweetnam PM, Elwood PC, Deadman NM. Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART). Lancet 1989;2:757 – 61. [3] Von Schacky C. Prophylaxis of atherosclerosis with marinev-3
fatty acids: a comprehensive strategy. Ann Intern Med 1987;107:890 – 9.
[4] Ballard-Barbash R, Callaway CW. Marine fish oils: role in prevention of coronary artery disease. Mayo Clin Proc 1987;62:113 – 8.
[5] Mortensen JZ, Schmidt EB, Nielsen AH, Dyerberg J. The effect of n-6 and n-3 polyunsaturated fatty acids on haemostasis, blood lipids and blood pressure. Thromb Haemost 1983;350:543 – 6. [6] Nestel PJ, Connor WE, Reardon MF, Connor S, Wong S,
Boston R. Suppression by diets rich in fish oil of very low density lipoprotein production in man. J Clin Invest 1984;74:82 – 9.
[7] Lorenz R, Spengler U, Fischer S, Duhm J, Weber PC. Platelet functions, thromboxane formation and blood pressure control during supplementation of the western diet with cod liver oil. Circulation 1983;67:504 – 11.
[8] Bruckner GG, Lokesh B, German B, Kinsella JE. Biosynthesis of prostanoids, tissue fatty acid composition and thrombotic parameters in rats fed diets enriched with docosahexaenoic (22:6n3) or eicosapentaenoic (20:5n3) acids. Thromb Res 1984;34:479 – 97.
[9] Landymore RW, Kinley CE, Cooper JH, MacAulay M, Sheri-dan B, Cameron C. Cod-liver oil in the prevention if intimal hyperplasia in autologous vein grafts used for arterial bypass. J Thorac Cardiovasc Surg 1985;89:351 – 7.
[10] Casali RE, Hale JA, LeNarz L, Faas F, Morris MD. Improved graft patency associated with altered platelet function induced by marine fatty acids in dogs. J Surg Res 1986;40:6 – 12.
[11] Weiner BH, Ockene IS, Levine PH, Cue´noud HF, Fisher M, Johnson BF, Daoud AS, Jarmolych J, Hosmer D, Johnson MH, Natak A, Vauderuil C, Hoogasian JJ. Inhibition of atherosclero-sis by cod-liver oil in a hyperlipidemic swine model. N Engl J Med 1986;315:841 – 6.
[12] Hartag JM, Lamers JM, Essed CE, Schalkwijk WP, Verdouw PD. Does platelet aggregation play a role in the reduction in the localized intimal proliferation in normolipidemic pigs with fixed coronary artery stenosis fed dietary fish oil. Atherosclerosis 1989;76:79 – 88.
[13] Zhu BQ, Smith DL, Sievers RE, Isenberg WM, Parmley WW. Inhibition of atherosclerosis by fish oil in cholesterol fed rabbits. J Am Coll Cardiol 1988;12:1073 – 8.
[14] Thiery J, Seidel D. Fish oil feeding results in an enhancement of
cholesterol-induced atherosclerosis in rabbits. Atherosclerosis 1987;63:53 – 6.
[15] Davis HR, Bridenstine T, Vasselinovitch D, Wissler R. Fish oil inhibits development of atherosclerosis in rhesus monkeys. Arte-riosclerosis 1987;7:441 – 9.
[16] Harker LA, Kelly AB, Hanson SR, Krupski W, Bass A, Osterud B, Fitgerald GA, Goodnight SH, Connor WE. Interruption of vascular thrombus formation and vascular lesion formation by dietary n-3 fatty acids in fish oil in non-human primates. Circula-tion 1993;87:1017 – 29.
[17] De Lorgeril M, Renaud S, Mamelle N, Salen P, Martin J-L, Monjaud I, Guidollet J, Touboul P, Delaye J. Mediterranean alpha-linoleic acid-rich diet in secondary prevention of coronary heart disease. Lancet 1994;343:1454 – 9.
[18] Leaf A, Weber PC. Cardiovascular effects of n-3 fatty acids. N Engl J Med 1988;318:549 – 57.
[19] Mehta J, Lopez LM, Wargovich T. Eicosapentaenoic acid: its relevance in atherosclerosis and coronary artery disease. Am J Cardiol 1987;59:155 – 9.
[20] Landymore RW, Cameron C, Sheridan B, MacAulay MA. Reduction of intimal hyperplasia in canine autologous vein grafts with cod-liver oil and dipyridamole. Can J Surg 1986;29:537 – 58.
[21] Israel DH, Gorlin R. Fish oils in the prevention of atherosclero-sis. J Am Coll Cardiol 1992;19:174 – 9.
[22] Dehmer GJ, Potms JJ, Van Den Berg EK, Eichhorn EJ, Prewitt JB, Campbell WB, Jennings L, Willerson JT, Schmitz JM. Reduction in the rate of early restenosis after coronary angio-plasty by a diet supplemented withv-3 fatty acids. N Engl J
Med 1988;319:733 – 40.
[23] Cairns JA, Gill J, Morton B, Roberts R, Gent M, Hirsh J, Holder D, Finnie K, Marquis JF, Naqvi S, Cohen E. Fish oils and low-molecular-weight heparin for the reduction of restenosis after percutaneous transluminal coronary angioplasty. The EM-PAR Study. Circulation 1996;94:1553 – 60.
[24] Crowley ST, Dempsey, EC, Horwitz KB, Horwitz LD. Platelet induced vascular smooth muscle cell proliferation is modulated by the growth amplification factors and adenosine diphosphate. Circulation 1994;90:1908 – 1918.
[25] Hwang DL, Latus LJ, Lev-Ran A. Effects of platelet-contained growth factors (PDGF, EGF, IGF-1, and TGF-beta) on DNA synthesis in porcine aortic smooth muscle cells in culture. Exp Cell Res 1992;200:358 – 60.
[26] Totani L, Piccoli A, Pellegrini G, Di Santo A, Lorenzet R. Polymorphonuclear Leukocytes enhance release of growth fac-tors by cultured endothelial cells. Arterioscler Thromb 1994;4:125 – 32.
[27] Harlan JM, Thompson PJ, Ross RR, Bowen-Pope DF. Alpha-thrombin induces release of platelet-derived growth factor-like molecule(s) by cultured human endothelial cells. J Cell Biol 1986;103:1129 – 33.
[28] Gajdusek C, Carbon S, Ross R, Nawroth P, Stern D. Activation of coagulation releases endothelial cell mitogens. J Cell Biol 1986;103:419 – 28.
[29] Willerson JT, Yao SK, McNatt J, Benedict CR, Anderson HV, Golino P, Murphree SS, Buja LM. Frequency and severity of cyclic flow alterations and platelet aggregation predict the sever-ity of neointimal proliferation following experimental coronary stenosis and endothelial injury. Proc Natl Acad Sci USA 1991;88:10624 – 8.
[30] Pakala R, Willerson JT, Benedict CR. Mitogenic effect of sero-tonin on vascular endothelial cells. Circulation 1994;90:1919 – 26. [31] Pakala R, Willerson JT, Benedict CR. Effect of serotonin, thromboxane A2 and specific receptor antagonists on vascular
[32] Sachinidis A, Flesch M, Ko Y, Schror K, Bohm M, Dusing R, Vetter H. Thromboxane A2 and vascular smooth muscle cell
proliferation. Hypertension 1995;26:771 – 80.
[33] Ross R. The pathogenesis of atherosclerosis — an update. N Engl J Med 1986;314:488 – 500.
[34] Adams PC, Badimon JJ, Badimon L, Chesebro JH, Fuster V. Role of platelets in atherogenesis: relevance to coronary arterial restenosis after angioplasty. Cardiovasc Clin 1987;18:49 – 71. [35] Nemecek GM, Coughlin SR, Handley DA, Moskowitz MA.
Stimulation of aortic smooth muscle cell mitogenesis by sero-tonin. Proc Natl Acad Sci USA 1986;83:674 – 8.
[36] Dorn GW, Becker MW, Davis M. Dissociation of the contractile and hypertrophic effects of vasoconstrictor prostanoids in vascu-lar smooth muscle cells. J Biol Chem 1992;267:24897 – 905. [37] Landymore R, Karmazyn M, MacAulay MA, Sheridan B,
Cameron C. Correlation between the effects of aspirin and dipyridamole on platelet function and prevention of intimal hyperplasia in autologous vein grafts. Can J Cardiol 1988;4:56 – 9.
[38] Metke MP, Lie J, Fuster V, Jova M, Kaye M. Reduction of intimal thickening in canine coronary bypass vein grafts with dipyridamole and aspirin. Am J Cardiol 1979;43:1144 – 8. [39] Harris WS. Fish oils and plasma lipid and lipoprotein
metabolism in humans: a critical review. J Lipid Res 1989;30:785 – 807.
[40] Appel LJ, Miller ER, Seidler AJ, Whelton PK. Does supplemen-tation of diet with ‘fish oil’ reduce blood pressure? A meta-anal-ysis of controlled trials. Arch Intern Med 1993;153:1429 – 38. [41] Hansen JB, Olsen JO, Wilsgard L, Osterud B. Effects of dietary
supplementation with cod liver oil on monocyte thromboplastin synthesis, coagulation and fibrinolysis. J Intern Med Suppl 1989;225:133 – 9.
[42] Endres S, Ghorbani R, Kelly VE, Georgilis K, Lonneman NG, van der Meer JW, Cannon JG, Rogers TS, Klempner MS, Weber PC, Schaefer EJ, Wolff SM, Dinarello CA. The effect of dietary supplementation withv3 polyunsaturated fatty acids on the synthesis of interleukin-1 and tumor necrosis factor by mononuclear cells. N Engl J Med 1989;320:265 – 71.
[43] Fox PL, DiCorleto PE. Fish oils inhibit endothelial cell produc-tion of platelet-derived growth factor-like protein. Science 1988;241:453 – 6.
[44] Kaminski WE, Jendraschak E, Kiefl R, von Schacky C. Dietary omega-3 fatty acids lower levels of platelet-derived growth factor mRNA in human mononuclear cells. Blood 1993;81:1871 – 9. [45] De Caterina R, Libby P. Control of endothelial leucocyte
adhe-sion molecules by fatty acids. Lipids 1996;31:S57 – 63.
[46] Morisaki N, Sprecher H, Milo GE, Cornwell DG. Fatty acid specificity in the inhibition of cell proliferation and its relation-ship to lipid peroxidation and prostaglandin biosynthesis. Lipids 1982;17:893 – 9.
[47] Huttner JJ, Gwebu ET, Pangnamala RV, Milo GE, Cornwell DC, Sharma HM, Greer JC. Fatty acids and their prostaglandin derivatives. Inhibitors of proliferation in aortic smooth muscle cells. Science 1977;197:289 – 91.
[48] Rao GN, Baas AS, Glasgow WC, Eling TE, Runge MS, Alexan-der RW. Activation of mitogen-activated kinases by arachidonic acid and its metabolites in vascular smooth muscle cells. J Biol Chem 1994;269:32586 – 91.
[49] Shiina T, Terano T, Saito J, Tamura Y, Yoshida S. Eicosapen-taenoic acid and docosahexaenoic acid suppress the proliferation of vascular smooth muscle cells. Atherosclerosis 1993;104:95 – 103.
[50] Rao GN, Berck BC. Active oxygen species stimulate vascular smooth muscle cell growth and proto-oncogene expression. Circ Res 1992;70:593 – 9.
[51] Corson MA, Alexander RW, Berk BC. 5HT2receptor mRNA is
over expressed in cultured rat aortic smooth muscle cells relative to normal aorta. Am J Physiol 1992;262(Cell. Physiol. 31):C309 – 15.
[52] Clark AJ, Ishii S, Richert N, Merlino GT, Pastan I. Epidermal growth factor regulates the expression of its own receptor. Proc Natl Acad Sci USA 1985;82:8374 – 8.
[53] Reem GH, Yeh NH. Interleukin 2 regulates expression of its receptor and synthesis of gamma interon by human T lymphocytes. Science 1984;225:429 – 30.
[54] Rydelck-Fitzgerald L, Wilcox BD, Teitler M, Jeffrey JJ. Sero-tonin-mediated 5HT2receptor gene regulation in rat myometrial
smooth muscle cells. Mol Cell Endocrinol 1993;92:253 – 9. [55] Versnel MA, Bouts MJ, Langerak AW, van der Kwast TH,
Hoogsteden HC, Hegemeijer A, Heldin C-H. Hydrocortisone induced increase of PDGF beta-receptor expression in a human malignant mesothelioma cell line. Exp Cell Res 1992;200:83 – 8. [56] Dinarello CA. Dissociation of transcription from translation of
human 1L-1beta. Induction of steady state mRNA by adherence or recombinant c5a in the absence of translation. Proc Soc Exp Biol Med 1992;200:228 – 32.
[57] Yarden Y, Ullrich A. Growth factor receptor tyrosine kinases. Annu Rev Biochem 1988;57:443 – 78.
[58] Zachary I, Penella JW, Rozengurt E. A role for neuropeptides in the control of cell proliferation. Dev Biol 1987;124:295 – 308.
[59] Vicentini LM, Villereal ML. Serum. bradykinin and vasopressin stimulate release of inositol phosphates from human fibroblasts. Biochem Biophys Res Commum 1984;123:663 – 70.
[60] Chen LB, Buchanan JM. Mitogenic activity of blood compo-nents. I. Thrombin and prothrombin. Proc Natl Acad Sci USA 1975;72:131 – 5.
[61] Wallace JMW, Turley E, Gilmore WS, Strain JJ. Dietary fish oil supplementation alters leukocyte function and cytokine produc-tion in healthy women. Arterioscler Thromb Vasc Biol 1995;15:185 – 9.
[62] Tremoli E, Eligini S, Colli S, Maderna P, Rise P, Pazzucconi F, Marangoni F, Sirtori CR, Galli C. n-3 Fatty acid ethyl ester administration to healthy subjects and to hypertriglyceridemic patients reduces tissue factor activity in adherent monocytes. Arterioscler Thromb 1994;14:1600 – 8.
[63] Gavino VC, Miller JS, Ikharebha SO, Milo GE, Cornwell DG. Effect of polyunsaturated fatty acid and antioxidants on lipid peroxidation in tissue cultures. J Lipid Res 1981;22:763. [64] Morisaki N, Sprecher H, Milo GE, Cornwell DG. Fatty acid
specificity in the inhibition of cell proliferation and its relation-ship to lipid peroxidation and prostaglandin biosynthesis. Lipids 1982;17:893.
[65] Hutter JJ, Gwebu ET, Pangnamala RV, Milo GE, Cornwell DG. Fatty acids and their prostaglandin derivatives: inhibitors of proliferation in aoartic smooth muscle cells. Science 1977;197:289.
[66] Halliwell B. Current status review: free radicals, reactive oxygen species and human disease: a critical evaluation with special reference to atherosclerosis. Br J Exp Pathol 1989;70:737 – 57. [67] Fridovich I. The biology of oxygen radicals. Science
1978;201:875 – 80.
![Fig. 1. Graph showing concentration dependent stimulation of aortic smooth muscle cell (SMC) by serotonin (5HT) (A) and platelet derivedgrowth factor (PDGF) (B).value of5HT and PDGF in serum a free medium in the presence of 100 3[H]Thymidine incorporation](https://thumb-ap.123doks.com/thumbv2/123dok/3148130.1384396/4.612.109.449.411.657/concentration-dependent-stimulation-serotonin-derivedgrowth-presence-thymidine-incorporation.webp)
