© 1999 American Heart Association, Inc.
Vascular Biology |
Eicosapentaenoic Acid and Docosahexaenoic Acid Block Serotonin-Induced Smooth Muscle Cell Proliferation
From the Department of Internal Medicine, Division of Cardiology, University of Texas Houston Medical School.
Correspondence to Rajbabu Pakala, The University of Texas Health Science Center, Dept of Cardiology, 6431 Fannin, MSB 6.039, Houston, TX 77030.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract—Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) present in fish oils have been ascribed as having significant antithrombotic and antiatherosclerotic effects. Vascular smooth muscle cell (SMC) proliferation plays an important role in the pathogenesis of atherosclerosis and restenosis. Recent studies have indicated that serotonin at concentrations present at sites of vascular injury stimulates SMC proliferation and may contribute to the restenotic process. In the present study we demonstrate that among the fatty acids tested, only EPA and DHA could block the mitogenic effect of serotonin on vascular SMC. Further, when added together these fatty acids act synergistically in blocking the mitogenic effect of serotonin. EPA and DHA blocked the 5HT-induced increase in the 5-HT2 receptor mRNA. This antimitogenic effect of EPA and DHA may partially explain some of the beneficial effects of fish oils.
Key Words: smooth muscle cell • eicosapentaenoic acid • docosahexaenoic acid • serotonin • restenosis
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Coronary events appear to be less frequent among populations consuming large amounts of
3 polyunsaturated fatty acids
(fish oils).1 2 Clinical trials involving survivors of
acute myocardial infarction have demonstrated a reduction in subsequent
coronary events through an increase in the consumption of
fish3 or 
-linoleic acid, a precursor of 3 fatty
acids derived from vegetable sources.4 However, the exact
mechanism(s) of action of these complex heterogeneous
compounds remain incompletely characterized. Among the diverse
biological effects of 3 fatty acids, favorable alteration of
lipoprotein levels, changes in eicosanoid metabolism, and
inhibition of platelet aggregation have been implicated in the
prevention of atherosclerosis.5 6 7 8 Dietary
3 fatty acids have also been shown to reduce experimental vascular
lesion formation in dogs,9 10 swine,11 12
rabbits,13 14 and nonhuman primates.15 16 Yet
the results of dietary 3 fatty acids on restenosis in
patients undergoing coronary angioplasty have been
inconclusive.17 18 Restenosis after coronary angioplasty involves intimal proliferation of vascular smooth muscle cell (SMC), probably in response to mitogens released from aggregating platelets as well as from monocyte-derived macrophages that accumulate at the site of vascular injury. Platelets contain peptide growth factors like platelet derived growth factor (PDGF), epidermal growth factor (EGF), and transforming growth factor-ß,19 20 21 and nonpeptide vasoactive compounds like serotonin (5HT), thromboxane A2, (TXA2), norepinephrine, histamine, bradykinin, and platelet activating factor.22 23 Recent studies from our laboratory and those of others indicate that some of these vasoactive compounds like 5HT and TXA2 are also mitogens to vascular SMC in culture,22 24 25 26 which suggests that vasoactive compounds may play an important role in the development of neointima. In the present study we investigated the putative mechanism by which eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), the active ingredients of fish oils, may inhibit the 5HT-induced vascular SMC proliferation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Serotonin (as creatine sulfate), EDTA, pargyline, Hanks' balanced salts (HBSS) Arachidonic acid ethyl ester (5, 8, 11, 14-eicosatetroenoic acid, AA, 20:4n-6),
-linolenic acid ethyl ester (9, 12, 15-octadecatrienoic
acid, -LA; 18:3n-3), -linolenic acid ethyl ester (6, 9,
12-octadecatrienoic acid, -LA, 18:3n-6), oleic acid ethyl ester
(cis-9-octadecenoic acid, OA, 18:1n-9), and lysergic acid diethylamide
(LSD) were obtained from Sigma Chemical Co; DMEM and fetal bovine serum
(FBS) were obtained from Whittaker Bioproducts, Walkersville, Md;
3[H]-thymidine (20 Ci/mole) and
3[H]-LSD
(N-methyl-3[H]-lysergic acid diethylamide),
from New England Nuclear Corp, Boston, Mass. Other reagents were
purchased from local vendors. EPA ethyl ester and DHA ethyl ester were
provided by the United States Department of Commerce, National Oceanic
and Atmospheric Administration, Charleston, SC.
Isolation, Culture, and Characterization of Primary Aortic
SMCs
Primary canine aortic SMC were isolated using the explant method
as described by Pakala et al.24 Briefly, the intima was
first peeled off from the aorta and then the media carefully stripped
away from the adventitia and placed in a petri dish containing warmed
DMEM (37°C). The medial layer was cut into 
1-mm squares, which
were transferred into a 25 cm2 tissue culture
flask and barely covered with DMEM supplemented with 10% FBS. The
blocks of tissue were cultured in a humidified atmosphere of 95% air
and 5% CO2 (vol/vol) at 37°C. After 1 to 2
weeks, the tissue blocks were removed and the migrated SMC were
cultured. Following isolation, the identity of the SMC was confirmed by
morphological examination and by staining for -actin.
Subcultures of SMC were done once they became confluent, media from the plates was aspirated and the cells washed with 10 mL of phosphate buffered saline. Then, 2 to 3 mL of trypsin EDTA (0.05% trypsin, 0.53 mmol/L EDTA in Ca2+, Mg2+-free HBSS) was added to the cells and incubated at room temperature for 2 to 3 minutes. The action of trypsin was stopped by the addition of 7 to 8 mL of DMEM containing 10% FBS. The cells were collected by centrifugation at 150g for 10 minutes. After removing the supernatant, the pelleted cells were dispersed in 10 mL of DMEM containing 10% FBS and fresh cultures were initiated from these cells.
3[H]-Thymidine Incorporation
SMCs from passages 2 or 3 were seeded into 35-mm plates at a
density of 65 000 to 75 000 cells/plate in DMEM containing 10% FBS,
and allowed to proliferate for 72 hours. After 72 hours, the growth
medium was replaced with 2 mL DMEM containing 0.1% FBS and incubated
for 72 hours for arresting cell growth and synchronization. After
growth arrest, the medium was replaced with 2 mL DMEM containing 1%
FBS and 100 mmol/L pargyline. Where indicated,
serotonin with pargyline and with or without the respective
fatty acid (EPA, DHA, OA, AA, -LA, -LA) were added to this
medium. After 20 hours of incubation, 1 µCi of
3[H]-thymidine was added to each plate and then
incubated for an additional 4 hours (24 hours after addition of
compounds); the medium was removed and the plates washed 3 times with
ice-cold phosphate-buffered saline. Then, 6% trichloroacetic acid was
added to the cells and the acid insoluble thymidine collected on a
glass fiber filter. The filters were washed with 100% ethanol, air
dried, and 3[H]-thymidine was quantified using
a liquid scintillation counter. The cells were counted (Coulter
counter) on the day of seeding, before changing to 0.1% FBS containing
medium to ensure that they were growing, and finally on day 6 to
determine that the cells were growth-arrested. All experiments were
performed in triplicate.
Isolation of poly(A+) RNA and Northern Blot
Analysis
SMCs from 2 or 3 passage were seeded into 150-mm tissue culture
plates in DMEM containing 10% FBS and allowed to proliferate for 72
hours. The growth medium was replaced with DMEM containing 0.1% FBS
and cultured for another 72 hours for arresting cell growth and
synchronization. After growth arrest, cells were stimulated with DMEM
containing 1% FBS and 100 µmol/L pargyline (control), or 1
µmol/L EPA or 1 µmol/L DHA with or without 5HT. After 24 hours
of incubation, cells were rinsed twice with phosphate buffered saline
(PBS, 160 mmol/L NaCl, 1.5 mmol/L KH2
PO4, 8 mmol/L Na2
HPO4). Poly(A+) RNA was
isolated from SMC using the On Track mRNA isolation kit (Biotecx
laboratories). Poly(A+) RNA was denatured with
formaldehyde and formamide and size-fractionated on a 0.66 mol/L
formaldehyde/1.3% agarose gel for 3 to 4 hours at 80 V. RNA was
transferred overnight onto a magnagraph nylon transfer membrane (MSI)
by electro-blotting in 10xSSC (1xSSC contains 0.15 mol/L NaCl, 0.015
mol/L trisodium citrate). The RNA was cross-linked to the membrane at
120 000 µJ using UV cross linker (Hoeffer Scientific Instruments)
and prehybridized in 50% deionized formamide, 4xSSC, 20 mmol/L
Tris HCl (pH 8.0), 1xDenhardt's solution (0.02% ficoll, 0.02%
polyvinyl pyrrolidine, 0.02% bovine serum albumin), 0.1%
sodium dodecyl sulfate, 200 mg/mL denatured salmon sperm DNA,
and 10% dextran sulfate for 30 minutes.
35S-labeled cRNA probe was synthesized using the
full-length 5-HT2 receptor coding (providing by
Dr Julies) region by the MAXIscript kit (Ambion). Approximately 3 ng/mL
of probe was added to the prehybridization solution, and hybridization
was performed overnight at 42°C. Filters were washed 3 times for 20
minutes with 2xSSC containing 0.1% SDS at room temperature and 3
times for 20 minutes with 0.1xSSC 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 GAPDH. The relative
quantity of 5HT2 mRNA in each sample was
analyzed by densitometry using OPTIMUS program on a Toshiba
Image analyzer and corrected for loading conditions by the
quantity of mRNA for GAPDH. Results were expressed as densitometric
units.
Scatchard Analysis for 5-HT2 Receptor
Binding Studies
Primary SMC from 2 or 3 passage were seeded into 150-mm tissue
culture plates in DMEM containing 10% FBS and allowed to proliferate
for 72 hours. The growth medium was replaced with DMEM containing
0.1% FBS and cultured for another 72 hours for arresting cell growth
and synchronization. After growth arrest, cells were stimulated with
DMEM containing 1% FBS and 100 µmol/L pargyline (control), or
1 µmol/L EPA or 1 µmol/L DHA with or without 5HT. After
24 hours of incubation, SMC were rinsed twice with phosphate-buffered
saline (PBS, 160 mmol/L NaCl, 1.5 mmol/L
K2HPO4, 8 mmol/L
Na2HPO4) and scraped into
ice-cold homogenizing buffer (50 mmol/L Tris-HCl,
0.5 mmol/L Na2 EDTA, 10 mmol/L
MgSO4, pH7.4). Cells were homogenized
and membranes pelleted at 30 000g for 15 minutes. Pellets
were resuspended in homogenizing buffer, incubated at
37°C for 15 minutes, and recentrifuged at 30 000g
for 15 minutes; the supernatant was decanted and pellets stored at
-45°C until use. Saturation analysis for
5-HT2 receptors was performed with 0.3 to 20
nmol/L 3[H]- LSD. Nonspecific binding was
determined in the presence of 100-fold excess concentration of
unlabeled ligand. Assays were performed with 120 mg of cell membrane
protein/assay (determined by Bradford method) in a final volume of 1
mL. The assay buffer was identical to homogenizing
buffer described above, except that it contained 10 mmol/L
pargyline and 0.1% ascorbic acid. Samples were incubated for 30
minutes at 25°C and filtered through glass fiber filters which had
been presoaked in 0.1% polyethylenimine for 30 minutes. Filters were
washed rapidly with 10 mL of ice-cold 50 mmol/L Tris-HCl (pH.7.0)
and radioactivity that remained bound to the filters was measured using
a liquid scintillation counter
Statistical Analyses
Data were analyzed by 1-way ANOVA for each interaction.
When a statistically significant difference was obtained, further
analysis were conducted using Scheffe's post-hoc tests. For
all comparisons, statistical significance was assumed as
P>0.05. In each figure, mean values ±SD are shown.
Radioligand binding results were analyzed by
standard linear regression methods.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Effect of 5HT on 3[H]-Thymidine Incorporation by SMCs
The effect of increasing concentrations of 5HT on thymidine incorporation into the DNA of quiescent SMC was assessed. Figure 1
illustrates the increase in SMC
proliferation as measured by 3[H]-thymidine
incorporation in the presence of 5HT. 5HT at an added concentration
>10 µmol/L induced an increase in
3[H]-thymidine incorporation. At 150
µmol/L of added 5HT, there was 4-fold increase in the amount of
3[H]-thymidine incorporated into the DNA of SMC
when compared with controls incubated with media alone and without
fatty acids (P<0.01). However, at higher concentrations of
added 5HT, there was a decrease in the amount of
3[H]-thymidine incorporated.
|
Effect of Fatty Acids on 3[H]-Thymidine Incorporation
by SMCs
Next, the effect of 3 fatty acids EPA and DHA or the effect of
non-3 fatty acids AA, -LA, -LA, and OA (as control fatty
acids) on 3[H]-thymidine incorporation into the
DNA of growth-arrested SMC was measured. Incubation of SMC with -LA,
-LA, or OA at concentrations tested did not have any significant
effect on 3[H]-thymidine incorporation (Figure 2). In contrast, when growth-arrested SMC
were incubated with AA, up to an added concentration of 1.67
µmol/L, AA did not have a significant effect on
3[H]-thymidine incorporation. However, at
higher concentrations, AA significantly induced
3[H]-thymidine incorporation, resulting in
approximately 3-fold increase to 16.7 µmol/L (Figure 2). When growth-arrested SMC were incubated with EPA or DHA, EPA
up to a concentration of 3.3 µmol/L and DHA up to a
concentration of 7.5 µmol/L, did not have any significant effect
on the 3[H]-thymidine incorporation (Figure 2). With concentrations higher than these values, there was a
gradual decrease in the amount of
3[H]-thymidine incorporated into the DNA of SMC
(Figure 2). At concentrations >75 µmol/L, both EPA and
DHA appeared to be cytotoxic to SMC (data not shown).
|
Effect of 3 Fatty Acids on Serotonin-Induced
3[H]-Thymidine Incorporation by SMCs
Growth-arrested SMC were incubated with different concentrations
of EPA or DHA (0.1665 to 1.665 µmol/L along with
mitogenic concentrations of 5HT, 50 to 200 µmol/L).
EPA at a concentration of 0.33 µmol/L or greater (Figure 3A) and DHA at a concentration of
1.67 µmol/L or greater (Figure 3B), completely blocked
the 5HT induced 3 [H]-thymidine incorporation.
These results suggest that EPA and DHA can block the proliferative
effect of 5HT. In contrast, when SMC were incubated with the same
concentrations of AA, -LA, -LA, or OA along with
mitogenic concentrations of 5HT, AA, -LA, -LA, or OA
did not inhibit 5HT-induced 3[H]-thymidine
incorporation (Figure 4). These results
suggest that the growth inhibitory effects of fatty acids
on SMC appears to be specific for the 3 class of fatty acids only.
Because both EPA and DHA are present in 2:1 ratio in fish oils,
we also examined the combined effect of EPA and DHA (at a ratio
of 2:1) on 5HT-induced 3[H]-thymidine
incorporation. Growth-arrested SMC were incubated with 0.11 or
0.22 µmol/L EPA or 0.055 or 0.11 µmol/L DHA along with
mitogenic concentrations of 5HT (100 µmol/L to
200 µmol/L). The above concentrations of EPA or DHA did not
inhibit the mitogenic effect of 5HT. However, when SMC were
incubated with both EPA (0.11 µmol/L) and DHA (0.055
µmol/L) (total 3 fatty acid concentration 0.165 µmol/L) and
mitogenic concentrations of 5HT, the combination
(0.165 µmol/L of EPA+DHA) could significantly inhibit the
proliferative effects of 5HT (P<0.01) (Figure 5). With higher concentrations of 3
fatty acids (0.22 µmol/L EPA+0.11 µmol/L DHA),
mitogenic effect of 5HT was completely abolished (Figure 5). These results suggest that when EPA and DHA are present
together they can act synergistically in reversing the proliferative
effect of 5HT on SMC.
|
|
|
Effect of 3 Fatty Acids on 5-HT2 Receptor mRNA
Levels
It has been shown that in vascular SMC, 5HT mediates its effects
via the 5HT2 receptors. Therefore, we examined
whether the 5HT-induced mitogenic effect and its reversal
by 3 fatty acids was due to an alteration in the regulation of mRNA
levels for 5HT2 receptors. Growth-arrested SMC
were incubated with medium alone (control) or with the same medium
containing 5HT (50 µmol/L), EPA (1 µmol/L), or DHA
(1 µmol/L) alone or EPA or DHA (1 µmol/L) with 5HT
(50 µmol/L) for 24 hours. mRNA levels for
5HT2 were determined as described in Methods.
Incubation of SMC with 5HT resulted in 100% increase in the
5HT2 receptor mRNA levels as compared with the
controls (Figure 6). Although EPA and DHA
did not have a significant effect on 5HT2
receptor mRNA levels, when SMC were stimulated with 5HT in the presence
of EPA, the 5HT-induced increase in mRNA for 5HT2
receptor was significantly blunted. Similarly, in the presence of DHA,
5HT-induced increase in 5HT2 mRNA levels was
limited to 35% (Figure 6). These results indicate that one
of the mechanism for the effect of EPA and DHA could be to produce a
decrease in mRNA for 5HT2 receptors in vascular
SMC.
|
Effect of 3 Fatty Acids on 5HT2 Receptor
Number
We also examined the effect of 3 fatty on
5HT2 receptor numbers. Growth-arrested SMC were
incubated with medium alone (control) or the same medium containing 5HT
(50 µmol/L), EPA (1 µmol/L), or DHA (1 µmol/L)
alone or EPA or DHA with 5HT for 24 hours. Scatchard analysis
revealed that despite the alteration in regulation of
5HT2 mRNA levels by 5HT and EPA or DHA, there
were no significant differences in the steady state levels of
5HT2 receptors (Figure 7).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Platelets aggregate in areas of endothelial dysfunction (atherosclerosis) or at sites of vascular injury such as those following percutaneous transluminal angioplasty. These aggregating platelets release peptide growth factors like PDGF from alpha granules and nonpeptide growth factors like 5HT and adenosine diphosphate (ADP) from dense granules.22 23 Recent studies from our group and others have indicated that nonpeptide growth factors like 5HT, TXA2, and ADP can stimulate vascular SMC to proliferate.22 24 25 26 These nonpeptide growth factors have been shown to act as amplification factors to known peptide growth factors like PDGF and EGF24 25 26 and also act synergistically among themselves to stimulate SMC proliferation.24 SMC migration and proliferation contributes to the intimal hyperplasia of the arterial wall, which is the main pathological feature underlying the development of the fibrofatty atherosclerotic lesions and neointima that develops following percutaneous transluminal angioplasty. Fish oils exert antiatherosclerotic effects as demonstrated in number of epidemiological and clinical studies.27 28 Diets rich in fish oils have been shown to reduce vascular lesion formation following endarterectomy16 or intimal hyperplasia in autologous vein grafts.9 10 Depending on the regimen used, fish oil supplementation has also been shown to reduce restenosis following angioplasty.17 18 All these beneficial effects of fish oils have been ascribed to the
3
fatty acids EPA and DHA present in the fish oils. Despite the
epidemiological and clinical evidence to suggest that EPA and DHA may
reduce SMC proliferation, there are very few studies to indicate this
at the cellular level. This is the first study to demonstrate that EPA
and DHA inhibit 5HT-induced SMC proliferation at concentrations that
are not cytotoxic to SMC. Shiina et al29 have shown that
20 µmol/L of EPA and DHA in 10% FBS-containing medium,
inhibited SMC proliferation by 30% and 20%, respectively. They have
also demonstrated that this inhibitory effect of EPA and
DHA was reversed by incubating the cells with antioxidants, which
indicates that the concentrations of EPA and DHA used by Shiina et
al29 may have produced cytotoxic concentrations of
oxidized species, thus resulting in inhibition of vascular SMC
proliferation.30 31 Several investigators have reported
that polyunsaturated fatty acids like EPA and DHA generally inhibit the
proliferation of cells in culture mainly through the formation of free
radicals.32 33 Although free radicals are known to be
cytotoxic to different types of cells, recent studies have indicated
that these molecules at low concentrations can also promote SMC
proliferation.34 In this study, we demonstrate that
5HT-induced 3[H]-thymidine incorporation was
dose-dependently inhibited by both EPA and DHA at concentrations that
did not induce free radical formation. Even at very low concentrations
(0.3 µmol/L), both EPA and DHA inhibited 5HT-induced
3[H]-thymidine incorporation by 60% to 70%
and completely abolished the effect at a concentration of 1.6
µmol/L EPA or DHA. At lower concentrations, EPA was more potent then
DHA in inhibiting the 5HT-induced
3[H]-thymidine incorporation. The
inhibitory effect on 5HT-induced
3[H]-thymidine incorporation was specific for
3 fatty acids EPA and DHA only, because -LA (n-3) AA, -LA
(n-6), or OA (n-9) failed to block the 5HT-induced proliferative
effect. The inhibitory effect of EPA and DHA was not due to
cytotoxicity, as cells were morphologically intact and resumed normal
growth after transferring to serum containing medium without 3 fatty
acids (data not shown). Further, because of the synergistic interaction
between EPA and DHA when used in combination, very low concentration of
EPA and DHA are necessary to demonstrate the same beneficial
effects. In vascular SMC, 5HT mediates its effects via the 5HT2 receptor.35 Incubation of SMC with 5HT resulted in an upregulation of 5HT2 receptor mRNA levels. Ligand-induced upregulation of 5HT2 receptor mRNA has been demonstrated in myometrial SMC.36 Ligand-mediated upregulation of receptor mRNA levels has also been reported for EGF and inerleukin-2.37 38 Incubation of SMC with EPA or DHA downregulated the 5HT2 receptor mRNA. Similarly, when SMC were incubated with EPA or DHA along with 5HT, there was a partial decrease in 5HT-induced 5HT2 receptor mRNA levels, indicating that EPA and DHA may inhibit the 5HT-induced SMC proliferation by downregulating the 5HT2 receptor mRNA levels. Surprisingly, the changes in the 5HT2 receptor mRNA levels brought about by 5HT and EPA or DHA were not followed by a corresponding change in the 5HT2 receptor affinity or numbers. Fitzgerald et al36 have reported that in uterine SMC, 5HT and its analogues upregulated the 5HT2 receptor mRNA levels and antagonists downregulated the 5HT2 receptor mRNA levels. However, in both instances they did not observe a significant difference in the affinity for the ligand or 5HT2 receptor numbers.36 Changes in the levels of mRNA without analogous changes in the corresponding protein has also been shown for other receptors, like EGF and interleukin-2,37 38 and in some instances this was found to be the result of specific processes regulating receptor turnover.39 40 Moreover, the disparity between mRNA levels and receptor densities could be the result of mechanisms regulating distinct steps of protein synthesis like transcription and translation.40 Several studies, including the classic experiments of Davis et al15 and Weiner et al11 have shown that fish oil feeding inhibits the development of atherosclerosis. The present study may explain the mechanism by which fish oils exert their beneficial effects.
|
Acknowledgments |
|---|
The authors would like to thank Dr David Julius, Department of Pharmacology, University of California at San Francisco, for providing 5HT2 receptor probe; the US Department of Commerce, National Oceanic and Atmosphere Administration, Charleston, SC, for providing EPA and DHA; 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 an American Heart Association Grant-In-Aid (C.R. Benedict).
Received August 24, 1998; accepted March 5, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Ryan TJ, Bauman WB, Kennedy JW, Kereiakes DJ, King SB, McCallister BD, Smith SC, Ullyot DJ, ACC/AHA Task Force Report. Guidelines for percutaneous transluminal coronary angioplasty: a report of the American College of Cardiology/American Heart Association Task Force on Assessment of Diagnostic and Therapeutic cardiovascular Procedures (Committee on Percutaneous Transluminal Coronary Angioplasty). Circulation. 1993;88:2987–3007.
2. 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–384S.
3. 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;2:757–761.[Medline] [Order article via Infotrieve]
4. 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;343:1454–1459.[Medline] [Order article via Infotrieve]
5. Leaf A, Weber PC. Cardiovascular effects of n-3 fatty acids. N Engl J Med. 1988;318:549–557.[Medline] [Order article via Infotrieve]
6.
Von Schacky C. Prophylaxis of
atherosclerosis with marine -3 fatty acids. A
comprehensive strategy. Ann Intern Med. 1987;107:890–899.
7. Mehta J, Lopez LM, Wargovich T. Eicosapentaenoic acid. Its relevance in atherosclerosis and coronary artery disease. Am J Cardiol. 1987;59:155–159.[Medline] [Order article via Infotrieve]
8. Ballard-Barbash R, Callaway CW. Marine fish oils: role in prevention of coronary artery disease. Mayo Clin Proc. 1987;62:113–118.[Medline] [Order article via Infotrieve]
9. Landymore RW, Kinley CE, Cooper JH, MacAulay M, Sheridan B, Cameron C. Cod-liver oil in the prevention of intimal hyperplasia in autologous vein grafts used for arterial bypass. J Thorac Cardiovasc Surg. 1985;89:351–357.[Abstract]
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.[Medline] [Order article via Infotrieve]
11. Weiner BH, Ockene IS, Levine PH, Cuénoud HF, Fisher M, Johnson BF, Daoud AS, Jarmolych J, Hosmer D, Johnson MH, Natak A, Vauderuil C, Hoogasian JJ. Inhibition of atherosclerosis by cod-liver oil in a hyperlipidemic swine model. N Engl J Med. 1986;315:841–846.[Abstract]
12. Hartag JM, Lamers JMJ, 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.[Medline] [Order article via Infotrieve]
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;12:1073–1078.[Abstract]
14. Thiery J, Seidel D. Fish oil feeding results in an enhancement of cholesterol-induced atherosclerosis in rabbits. Atherosclerosis. 1987;63:53–56.[Medline] [Order article via Infotrieve]
15.
Davis HR, Bridenstine T, Vasselinovitch D, Wissler R.
Fish oil inhibits development of atherosclerosis in
rhesus monkeys. Arteriosclerosis. 1987;7:441–449.
16.
Harker LA, Kelly AB, Hanson SR, Krupski W, Bass A,
Osterud B, Fitzgerald GA, Goodnight SH, Connor WE. Interruption of
vascular thrombus formation and vascular lesion formation by dietary
n-3 fatty acids in fish oil in nonhuman primates.
Circulation. 1993;87:1017–1029.
17.
Dehmer GA, 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 angioplasty by a diet supplemented with -3
fatty acids. N Engl J Med. 1988;319:733–740.[Abstract]
18.
Cairns JH, 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 EMPAR Study. Circulation. 1996;94:1553–1560.
19. Oka Y, Orth DN. Human plasma epidermal growth factor/beta-urogastrone is associated with blood platelets. J Clin Invest. 1983;72:249–259.
20.
Ferns GA, Raines EW, Sprugel KH, Motani AS, Reidy MA,
Ross R. Inhibition of neointimal smooth muscle accumulation
after angioplasty by an antibody to PDGF. Science. 1991;253:1129–1132.
21. 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;200:358–360.[Medline] [Order article via Infotrieve]
22. 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;1908–1918.
23.
Bell L, Madri JA. Effect of platelet factors on
migration of cultured bovine aortic endothelial and
smooth muscle cells. Circ Res. 1989;65:1057–1065.
24.
Pakala R, Willerson JT, Benedict CR. Effect of
serotonin, thromboxane
A2, and specific receptor antagonists
on vascular smooth muscle cell proliferation. Circulation.. 1997;96:2280–2286.
25.
Nemecek GM, Coughlin SR, Handley DA, Moskowitz MA.
Stimulation of aortic smooth muscle cell mitogenesis by
serotonin. Proc Natl Acad Sci U S A. 1986;83:674–678.
26.
Sachinidis A, Flesch M, Ko Y, Schror K, Bohm M, Dusing
R, Vetter H. Thromboxane A2 and vascular
smooth muscle cell proliferation. Hypertension. 1995;26:771–780.
27. Kromhout D, Bosschieter EB, De Lezenne-Coulander C. The inverse relation between fish consumption and 20-year mortality from coronary heart disease. N Engl J Med. 1985;312:1205–1209.[Abstract]
28. Israel DH, Gorlin R. Fish oils in the prevention of atherosclerosis. J Am Coll Cardiol. 1992;19:174–179.[Abstract]
29. Shiina T, Terano T, Saito J, Tamura Y, Yoshida S. Eicosapentaenoic acid and docosahexaenoic acid suppress the proliferation of vascular smooth muscle cells. Atherosclerosis. 1993;104:95–103.[Medline] [Order article via Infotrieve]
30. Morisaki N, Sprecher H, Milo GE, Cornwell DG. Fatty acid specificity in the inhibition of cell proliferation and its relationship to lipid peroxidation and prostaglandin biosynthesis. Lipids. 1982;17:893–899.[Medline] [Order article via Infotrieve]
31.
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;197:289–291.
32. 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;70:737–757.
33.
Fridovich I. The biology of oxygen radicals.
Science. 1978;201:875–880.
34. Rao GN, Berck BC. Active oxygen species stimulate vascular smooth muscle cell growth and proto-oncogene expression Circ Res. 1992;70:593–599.
35. Corson MA, Alexander RW, Berk BC. 5-HT2 receptor mRNA is overexpressed in cultured rat aortic smooth muscle cells relative to normal aorta. Am J Physiol. 1992;262 (suppl2, pt 1):C309–315.
36. Rydelex-Fitzgerald L, Wilcox BD, Teitler M, Jeffrey JJ. Serotonin-mediated 5-HT2 receptor gene regulation in rat myometrial smooth muscle cells. Mol Cell Endocrinol. 1993;92:253–259.[Medline] [Order article via Infotrieve]
37.
Clark AJ, Ishii S, Richert N, Merlino GT, Pastan I.
Epidermal growth factor regulates the expression of it's own receptor.
Proc Natl Acad Sci. U S A. 1985;82:8374–8378.
38.
Reem G H, Yeh NH. Interleukin 2 regulates expression of
it's receptor and synthesis of gamma interon by human T lymphocytes.
Science. 1984;225:429–430.
39. Versnel MA, Bouts MJ, Langerok AW, van der Kwast TH, Hoogsteden HC, Hegemeijer A, Heldin CH. Hydrocortisone induced increase of PDGF beta-receptor expression in a human malignant mesothelioma cell line. Exp Cell Res. 1992;200:83–88.[Medline] [Order article via Infotrieve]
40. 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;200:228–232.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  C. M. C. Dupasquier, E. Dibrov, A. L. Kneesh, P. K. M. Cheung, K. G. Y. Lee, H. K. Alexander, B. K. Yeganeh, M. H. Moghadasian, and G. N. Pierce Dietary flaxseed inhibits atherosclerosis in the LDL receptor-deficient mouse in part through antiproliferative and anti-inflammatory actions Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2394 - H2402. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Suzuki, K. Fukuo, T. Suhara, O. Yasuda, N. Sato, Y. Takemura, M. Tsubakimoto, and T. Ogihara Eicosapentaenoic Acid Protects Endothelial Cells Against Anoikis Through Restoration of cFLIP Hypertension, September 1, 2003; 42(3): 342 - 348. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||