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Cell cycle related inhibition of mouse vascular smooth muscle cell proliferation by prostaglandin E1: relationship between prostaglandin E1 and intracellular cAMP levels
Prostaglandins, Leukotrienes and Essential FattyAcids (1996) 54(2), 101-107
© PearsonProfessionalLtd 1996
Cell c y c l e related inhibition of m o u s e v a s c u l a r s m o o t h m u s c l e cell proliferation by p r o s t a g l a n d i n El: relationship b e t w e e n p r o s t a g l a n d i n E 1 and i n t r a c e l l u l a r c A M P levels Y.-Yi Fan 1, K. S. Ramos z, R. S. Chapkin ~ 1Faculty of Nutrition and Molecular and Cell Biology Group, 442 Kleberg Center, Texas A&M University, College Station, TX 77843, USA. 2Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843, USA.
Summary Elucidation of the cellular and molecular mechanisms which regulate vascular smooth muscle cell proliferation is critical to the understanding of atherogenesis. The present studies were conducted to evaluate the relationship between prostaglandin E1 (PGG) and cAMP in the regulation of DNA synthesis in mouse vascular smooth muscle cells (SMCs). Quiescent cultures of SMCs were challenged with 10% fetal bovine serum to initiate cell cycle transit and PGE1 (10 pM) or dibutyryl cAMP (1, 10, 100 pM) added at 0, 8, 16, 24, and 32 h. DNA synthesis as measured by [3H] thymidine incorporation and intracellular cAMP levels were measured 24 h following individual treatments. PGE~ modulated DNA synthesis in a cell cycle related fashion, with inhibition only observed in cells challenged 16 h or longer following initiation of cell cycle transit. The decrease in DNA synthesis induced by PGG was associated with increased intracellular cAMP levels at 16 and 24 h, but not 32 h. Exposure of SMCs to dibutyryl-cAMP also inhibited DNA synthesis in a cell cycle related fashion, with the most pronounced effect seen at 16 h. These results demonstrate that the effects of PGE1 are restricted to a defined period within the cell cycle following S phase entry and implicate modulation of intracellular cAMP levels in the inhibitory response.
INTRODUCTION Vascular smooth muscle cell (SMC) proliferation plays a principal role in the fibroproliferative component of atherosclerotic disease. 1-3 The proliferation of SMCs is a complex process regulated by the interaction of growth factors with their respective receptors. Prostaglandins, 20Received 19 June 1995 Accepted 24 July 1995 Correspondence to: Robert S. Chapkin, Tel. 409 845 0419; Fax. 409 845 6433; E-mail: [email protected]
carbon polyunsaturated fatty acid derived eicosanoids, 4 are potent mediators of cell proliferation. 5-7 Prostaglandin E 1 (PGE1) and prostaglandin E2 (PGE2) share the ability to inhibit PDGF-induced DNA synthesis in rat arterial SMCs, but the biological effects of PGE~ are approximately 20 times stronger than PGE2.s PGE1 appears to exert opposite effects on quiescent relative to randomly cycling SMCs suggesting that the biological activity of this PG is influenced by cell cycle related events? Addition of PGE~ to quiescent A-10 cells, a rat vascular SMC line, enhances DNA synthesis, while addition to cycling cells elicits an anti-proliferative 101
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response. These data suggest a potential benefit of PGE~ in wound healing (quiescent state) and prevention of atheromatous plaque formation (proliferating state), and implicate a cell cycle related component in the ability of PGE1 to modulate SMC proliferation. Previous in vitro studies in this laboratory have demonstrated that PGE~ biosynthesis in macrophages can be enhanced by feeding gamma-linolenic acid enriched dietary oils. ~°-~2It is therefore possible that manipulation of macrophage PGE1 synthesis by dietary gamma-linolenic acid may also play an important role in the regulation of SMC proliferation. Although the mouse is naturally resistant to atherosclerosis, 13 the recent development of transgenic mouse models have provided researchers with the unique opportunity to study the complex environmental and genetic interactions underlying atherosclerosis. 14 Techniques for gene manipulation in vivo are more advanced in the mouse than in any other mammal; therefore this model system is currently being aggressively utilized by numerous investigators in the atherosclerosis fieldY,~6 Since alteration of SMC growth programs is a contributing factor in the process of atherogenesis/,2 we have evaluated the effect of select lipid mediators on SMC growth using the highly relevant murine model system. Several studies indicate that the inhibitory response of SMC proliferation to PGE~ is cell cycle dependent. However, published findings are contradictory and final interpretation awaits further studies. For example, the inhibitory effect of PGE~ on human SMC proliferation occurred primarily in G1 phase, with no effect in S phaseY In contrast, PGE1 increased quiescent SMC (majority of cells were in Go/G~phase) DNA synthesis, and reduced cycling (growing) SMC DNA synthesis in rats? It has also been shown that the biological effect of PGE1 in various cells are mediated by activation of adenylate cyclases and subsequent elevation in intracellular cAMP levels? The increase of cAMP levels may contribute to down-regulation of SMC proliferation.8,~z~8Previous studies in this laboratory have shown that dibutyryl cAMP (rib-cAMP), a stable analogue of cAMP, modulates rat SMC growth, differentiation, 19 and growth factor related gene expression. 2° Although addition of PGEj quickly raises intracellular cAMP levels, 8,~z~8whether changes in intracellular cAMP levels by PGE~ correlate with SMC proliferation as a function of cell cycle transit has not been elucidated. The present study was conducted to examine the cell cycle related effect of PGE~ and db-cAMP on SMC DNA synthesis and the changes of intracellular cAMP levels throughout the cell cycle. Our results show that the inhibitory effect of PGE~ on mouse SMC DNA synthesis is cell cycle specific, having a pronounced effect following entry of the cells into the S phase. The inhibitory effect may involve a change in intracellular cAMP levels.
MATERIALS AND METHODS Materials
Tissue culture Media 199 was purchased from Gibco BRL (Grand Island, NY). Heat inactivated fetal bovine serum (FBS) was obtained from Intergen (Purchase, NY). CoUagenase was from Worthington (Freehold, NJ). Prostaglandin standards were obtained from Cayman Chemicals (Ann Arbor, MI). [3H] thymidine was from ICN Radiochemicals (Irvine, CA). The cAMP radioimmunoassay (RIA) kit was purchased from Nuclear Magnetics (Cambridge, MA). Trypsin-EDTA solution, antibiotic/antimycotic solution, dibutyryl cAMP, and glutamine were obtained from Sigma Chemical (St Louis, MO). All solvents were Optima grade (Fisher Scientific, Fair Lawn, NJ). C57BL/6 female mice were from Charles River (Frederick Research Facility, Frederick, MD). Cell culture
SMCs were isolated from aortas of pathogen-free C57BL/6 female mice by a series of enzymatic digestions with collagenase and trypsin as described previously. 21,22Cells were pooled from 3 mice and grown in Medium 199 supplemented with 10% FBS, 2 mM glutamine, 10 000 units/ ml penicillin, lOmg/ml streptomycin, and 25gg/ml amphotericin B. Secondary cultures were prepared by trypsinization of primary cultures and seeded in 35-mm culture dishes at a density of 1 x 104 cells/dish. Passage 3-12 was used in all experiments. Quiescent SMCs were obtained by serum deprivation in medium containing 0.1% FBS for 72 h. a3 Prostaglandin treatment
Quiescent or cycling cultures of SMCs were incubated with [3H] thymidine at 0.5 gCi/ml and treated with 0, 5, 10, 20, 40, or 80 gM PGE1 in the presence of 2 or 10% FBS, respectively, for 24 h. Prostaglandins were diluted in ethanol and the final concentration of ethanol in the cukure medium was less than 0.1%. Control cells were exposed to the same amount of ethanol as treated cells. Cells were harvested at the end of the incubation period and DNA synthesis ([3H]thymidine incorporation) and protein concentration measured as described previouslyY,24 Effects of PGE 1 or db-cAMP on DNA synthesis
Quiescent SMCs were released from Go by incubation in 10% FBS medium (time 0). pH] thymidine (0.5 gCi/ml) and PGE1 (10 gM) or db-cAMP (1, 10, 100 gM) were added to cultures at 0, 8, 16, 24, and 32 h following serum stimulation. Cultures were incubated for an additional 24 h to ensure identical conditions and [3HIthymidine incorpora-
Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 54(2), 101-107
© Pearson Professional Ltd 1996
PGE~ modulation of SMC growth
tion and protein content were subsequently measured at the end of each incubation period. Effect of PGE~ on cAMP levels
Growth-arrested SMCs were stimulated by 10% FBS as described above. PGE1 (10 gM) was added to the cultures at 0, 8, 16, 24, and 32 h after serum stimulation and incubated for an additional 24 h. Cells were harvested at the end of each incubation and intracellular cAMP was measured by radioimmunoassayP Flow cytometry
Quiescent SMC were stimulated by 10% FBS as previously described. Cells were trypsinized and collected at 0, 8, 16, 24, and 32 h after serum stimulation. Mouse lymphocytes were isolated immediately prior to SMC cell cycle analysis and used as a G0/G ~phase controlY Nuclei were stained with propidium iodide and fluorescence measured by flow c y t o m e t r y 7 Effect of PGE~ on cell proliferation
Growth-arrested SMCs were released from Go by 10% FBS as described above. Cells were dosed with PGE~ (10 ~tM) at 0 and 16 h following serum stimulation and grown for 4 days. Fresh medium containing 10 ~zM of PGE1 was applied every other day. At the end of the incubation period, cells were trypsinized and counted using a hemacytometer.
Statistics
Data were analyzed using one-way analysis of variance and Fisher's least significant difference procedure for comparing treatment means. 2s A difference of p < 0.05 was considered statistically significant.
RESULTS
The effect of PGE] on serum-induced DNA synthesis in cycling versus quiescent SMCs is shown in Figures la and lb, respectively. The inhibitory effect was concentration dependent and most pronounced when cells were maintained in 10% serum to preserve continuous cycling. The inhibitory effect of PGE2 was less pronounced than that of PGE~ (data not shown). To determine whether the inhibitory effect of PGE~ on SMC DNA synthesis was cell cycle-related, PGE~ (10 ~tM) was added to SMC cultures at various time points following initiation of cell cycle transit, and subsequently incubated for an additional 24 h. The action of PGE~ on SMC DNA synthesis is shown in Figure 2. The inhibitory effect of PGE~ was only observed at 16 h and beyond, with the most significant effect seen at 16 h. As determined by flow cytometry (Fig. 2 panel), the percentage of control cells in S phase increased as cells progressed through the cycle, reaching the highest proportion at 16 h. These data indicate that the inhibitory action of PGE~ on DNA synthesis was confined to a critical period following S phase entry. We next assessed the inhibitory effect of PGE~ on SMC proliferation at 16 h. For this purpose, cells were dosed
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Fig. 1 Dose-dependent effect of PGE1 on (a) cycling versus (b) quiescent DNA synthesis in SMCs. Quiescent and cycling SMCs were treated with various concentrations of PGE1 (0-80 #M) and pulsed with 0.5 gCi/ml of pH] thymidine for 24 h in the presence of 2% or 10% serum-containing medium, respectively. Values represent the means + SEM of 4 replicate cultures per group (*p < 0.05).
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Fig. 2 Effect of PGE1 on SMC DNA synthesis. Growth-arrested SMCs were stimulated with 10% serum• PGE~ (10 gM) and [3H] thymidine (0.5 gCi/ml) were added at different times following serum stimulation and [aH] thymidine incorporation was measured after 24 h incubation. Results are expressed as percentage of pH] thymidine incorporated in cells treated with PGE1 compared with incubations without PGE, Values represent the means _+SEM of 4 replicate cultures per group (*p< 0.05). Panel represents the percent of SMCs in S phase of the cell cycle at various times. SMCs were serum depleted for 72 h and stimulated with 10% serum for 0, 8, 16, 24, and 32 h. Cells were then harvested and nuclei fluorescence measured by flow cytometry.
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with PGE 1 (10 gM) at 0 and 16 h following serum stimulation and grown for 4 days. A significant anti-proliferative effect was seen at 16 h (Fig. 3), confirming the cell cycle related inhibition of DNA synthesis b y PGE1 Since the biologic effects of PGE1 m a y be linked to its ability to
Fig. 4 Effect of PGE1 on SMC intracellular cAMP concentration. Growth-arrested SMCs were stimulated with 10% serum. PGE1 (10 gM) was added at different times following serum stimulation and the intracellular cAMP level was measured after 24 h incubation. Values represent the means _+SEM of 4 replicate cultures per group. (,op= 0.058)
modulate cAMP levels, the effects of PGEI on the regulation of intracellular cAMP levels in s y n c h r o n o u s l y cycling cells was m e a s u r e d (Fig. 4). Intracellular cAMP levels increased as cells progressed t h r o u g h the cell cycle in b o t h groups. C o m p a r e d to the control group, PGE1 treated cells h a d an increased cAMP level at 16 h ( p = 0.058). If the inhibitory effect of PGE~ is mediated in part by cAMP, t h e n exogenous cAMP s h o u l d also inhibit DNA
Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 54(2), 101-107
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Fig. 5 Effect of Ob-cAMP on SMC DNA synthesis. Growth-arrested SMCs were stimulated with 10% serum. Dibutyryl cAMP (1, 10, 100 #M) and [3H] thymidine (0.5 #Ci/ml) were added at different times following serum stimulation and [3H] thymidine incorporation was measured after a 24 h incubation. Values represent the means + SEM of 4 replicate cultures per group (*p < 0.05).
synthesis in a manner comparable to that of the PGE~. Figure 5 demonstrates the effects of exogenous db-cAMP on DNA synthesis during different stages of the cell cycle. Similar to exogenous PGE~, the strongest concentrationdependent inhibitory effect by db-cAMP was noted at 16 h, suggesting that a PGE~-sensitive point is expressed at this time during cell cycle transit. DISCUSSION
SMC proliferation is one of the main events implicated in the pathogenesis of atherosclerotic vessel diseases. 3 An understanding of anti-proliferative factors may therefore aid in controlling the pathogenesis of vascular diseases. PGE1 is capable of inhibiting SMC DNA synthesis, s,~z~sbut the mechanism of PGE~ interference with SMC proliferation has not been well defined. Because mouse vascular SMCs can produce eicosanoids at gM levels upon stimulation by thrombin, 29 the PGE~ concentration (10 gM) used in our studies likely approximates the physiological conclifton. The concentration of PGE~ required to inhibit SMC proliferation in vitro appears to vary depending on the species and culture conditions. For example, 25 nM of PGG can inhibit h u m a n aortic SMC proliferation induced by 1% serum + PDGF (50 ng/ml) or 200/0 serum by up to 600/0,17 while the ICs0 for PGE~ in cycling -4-10 cells maintained in 10% FBS was 20 gM? Our data demonstrate that PGE~ down-regulates SMC DNA synthesis in a concentration-dependent manner, and that this inhibitory response is influenced by cell cycle kinetics. As shown in Figure 1, PGE 1had a diminished inhibitory effect on quiescent SMC DNA synthesis cul© Pearson Professional Ltd 1996
105
tured in low serum (Fig. lb) relative to cycling SMCs maintained in a higher serum concentration (Fig. la). These data indicate that the ultimate inhibitory effect of PGE~ on vascular SMC DNA synthesis is dependent on culture conditions and cell cycle progression. PGE 2 also exerts an anti-proliferative effect on SMCs, albeit with reduced potency relative to PGE~? The relative difference in the anti-proliferative potential of PGE1 and PGE2 is noteworthy, because in many cell systems these autacoids possess opposing functions. These include effects on the immune response, fibrinolytic system, and vascular contraction. 3°,3~ The stronger anti-proliferative capacity of PGE 1emphasizes the potential benefit of PGE~ as an anti-atherogenic agent. The extent to which PGE1 exerted a down-regulatory effect on SMC DNA synthesis varied throughout the cell cycle. The strongest inhibitory effect was observed when PGE 1 was added 16h after serum stimulation (Fig. 2), which corresponded to S phase as determined by flow cytometry (Fig. 2 panel). This interpretation is also supported by cell proliferation data (Fig. 3). However, these results are contrary to observations reported by Loesberg et allz where the inhibitory effect of PGE~ on DNA synthesis was observed during the G 1 phase of the cell cycle. PGE 1 was less effective at periods following the critical 16 h sensitive point. This discrepancy may be due to the different cell types and/or differences in the culture conditions utilized. For instance, Loesberg et al used h u m a n cells in 20% serum-containing medium as compared to murine cells in lower serum concentration. It is possible that higher growth factor concentrations influenced the anti-proliferative response to PGE~. Since 35% of cells reached S phase by 8 h after serum stimulation (Fig. 2 panel) and addition of PGE1 did not inhibit SMC DNA synthesis at this time, it is likely that specific molecular targets for the anti-proliferative function of PGE~ are expressed at the peak of S phase (16 h). It has been suggested that the biologic effects of PGE 1 are linked to its ability to modulate intracellular cAMP levels.8,~z~s We have previously shown that exogenous db-cAMP (200 gM) and the phosphodiesterase inhibitor theophylline (100 gM) significantly inhibited DNA synthesis in rat SMCs? 9 In order to determine if the cell cycle related anti-proliferative effects of PGE1 correlated with modulation of cAMP levels, we examined the intracellular cAMP levels in response to the addition of PGE1 at various times. The increase in intracellular cAMP levels (Fig. 4) was associated with a decrease in SMC DNA synthesis (Fig. 2), with the most notable effect observed at 16 h. These data indicate that the cell cycle related effect of PGE1 on SMC DNA synthesis involves cAMP. Although studies have indicated that the addition of PGE~ to SMCs rapidly elevates intracellular cAMP levels followed by a gradual decrease to basal levels within several hours, 8,~7
Prostaglandins, Leukotrienes and Essential FattyAcids (1996) 54(2), 101-107
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t h e l o n g - t e r m effect of PGE1 o n i n t r a c e l l u l a r c A M P production has not been previously reported. Our data d e m o n s t r a t e t h a t cAMP a c c u m u l a t e s o v e r t i m e as a f u n c t i o n of cell cycle t r a n s i t (Fig. 4). A p o s s i b l e e x p l a n a t i o n is t h a t t h e a d d i t i o n of PGE1 at 16 h m a y s t r o n g l y activate a d e n y l a t e cyclases i n c o m b i n a t i o n w i t h o t h e r intracellutar mediators which inhibit phosphodiesterase activity, t h e r e b y , r e s u l t i n g i n t h e l o n g - t e r m e l e v a t i o n of i n t r a c e l l u l a r c A M P levels. F u r t h e r s t u d i e s are n e e d e d to clarify t h i s p o i n t . To f u r t h e r d e m o n s t r a t e t h e i n v o l v e m e n t of c A M P as a d o w n s t r e a m m o d u l a t o r of t h e SMC r e s p o n s e to P G E , we t r e a t e d cells w i t h d b - c A M P at different cell cycle stages a n d m e a s u r e d t h e effect o n DNA s y n t h e s i s (Fig. 5). C o n sistently, t h e s t r o n g e s t i n h i b i t i o n was s e e n w h e n d b c A M P was a d d e d at 16 h. T h e DNA s y n t h e s i s i n h i b i t i o n profile of d b - c A M P (Fig. 5) is c o m p a r a b l e w i t h PGE1 (Fig. 2), a l t h o u g h d b - c A M P h a d a l o w e r i n h i b i t o r y effect c o m p a r e d to PGE~. This d i s c r e p a n c y is p r o b a b l y t h e r e s u l t of r e d u c e d p e r m e a b i l i t y of db-cAMP. I n this regard, h i g h c o n c e n t r a t i o n s (100 ~M) of cAMP a n a l o g u e were n e e d e d to m a i n t a i n e l e v a t e d cAMP levels i n r a b b i t aortic SMCs i n o r d e r to o b t a i n a n a n t i - p r o l i f e r a t i v e effect. 32 I n c o n c l u s i o n , o u r d a t a d e m o n s t r a t e t h a t (i) PGE1 i n h i b i t s DNA s y n t h e s i s i n m o u s e SMCs, (ii) t h e antiproliferative effect of PGE~ is cell cycle-related, w i t h t h e m o s t p r o n o u n c e d effect at 16 h f o l l o w i n g i n i t i a t i o n of cell cycle transit, a n d (iii) t h e cell cycle r e l a t e d a c t i o n of PGE~ o n SMC DNA s y n t h e s i s i n v o l v e s m o d u l a t i o n of i n t r a c e l l u l a r cAMP.
ACKNOWLEDGEMENTS The authors wish to thank Ms Cindy H. Thurlow for her excellent technical assistance, and Ms Betty Rosenbaum and Mr Chris Jolly for flow cytometry analyses. This research was supported in part by Scotia Pharmaceutical Ltd to RSC, the Texas A&M interdisciplinary Research Enhancemere Program to KSR and RSC, and NIH grant (DK 41693) to RSC and (ES 00213) to KSR.
REFERENCES 1. Ip J. H., Fuster V., Badimon L., Badimon J., Taubman M. B., Chesebro J. H. Syndromes of accelerated atherosclerosis: role of vascular injury and smooth muscle cell proliferation. JAm Coil Cardio11990; 15: 1667-1687. 2. Querol-Ferrer V., Hnltagardh-Nilsson A., Ringertz N. S., Nilsson J., Jonzon B. Adenosine receptors, cyclic AMP accumulation, and DNA synthesis in aortic smooth cell cultures of adult and neonatal rats. J Cell Physiol 1992; 151: 555-560. 3. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990's. Nature 1990; 362: 801-809. 4. Bergstrom S., Danielsson H., Klenberg D., Samuelsson B. The enzymatic conversion of essential fatty acids into prostaglandins. J Biol Chem 1964; 239: 4006-4008.
5. Thomas D. R., Phflpott G. W., Jaffe B. M. The relationship between concentration of prostaglandin E and rates of cell replication. Exp Cell Res 1974; 84: 40-46. 6. Uehara Y., Ishimitsu T., Kimura K., Ishii M., Ikeda T., Sugimoto T. Regulatory effects of eicosanoids on thymidine uptake by vascular smooth muscle cells of rats. Prostaglandins 1988; 36: 847-85Z Z Pasricha P. J., Hassoun P. M., Teufel E., Landman M. J., Fanbury B. L Prostaglandin E1and E2 stimulate the proliferation of pulmonary artery smooth muscle ceils. Prostaglandins 1992; 43: 5-19. 8. Nilsson J., Olsson A. G. Prostaglandin E1 inhibits DNA synthesis in arterial smooth muscle cells stimulated with platelet-derived growth factor. Atherosclerosis 1984; ~3: 77-82. 9. Owen N. E. Effect of prostaglandin E1 on DNA synthesis in vascular smooth muscle cells. Am J Physiol 1986; 250: C584-C588. 10. Chapkin R. S., Coble K. J. utilization of gammalinolenic acid by mouse peritoneal macrophages. Biochim Biophys Acta 1991; 108~: 365-370. 11. Fan Y-Y,Chapkin R. S. Mouse peritoneal macrophage prostaglandin E1 synthesis is altered by dietary gamma-linolerdc acid. J Nutr 1992; 122: 1600-1606. 12. Fan Y.-Y.,Chapkin R. S. Phospholipid sources of metabolically elongated gammalinolenic acid: Conversion to prostaglandin E1 in stimulated mouse macrophages. J Nutr Biochem 1993; 4: 602-60Z 13. Breslow J. L. Insights into lipoprotein metabolism from studies in transgenic mice. Annu Rev Physio11994; 56: 797-810. 14. Lusis A. J. The mouse model for atherosclerosis. Trends Cardiovasc Med 1993; 3: 135-143. 15. Nakashima Y., Plump A. S., Raines E. W., Brestow J. L., Ross R. Apo E-deficient mice develop lesions during phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb 1994; 14: 133-139. 16. Reddick R. L., Zhang S. H., Maeda N. Atherosclerosis in mice lacking apo E. Evaluation of lesional development and progress. Arterioscler Thromb 1994; 14: 141-14Z 1Z Loesberg C., Van Wijk R., Zandbergen J., Van Aken W. G., Van Mourik J. A., De Groot Ph G. Cell cycle-dependent inhibition of human vascular smooth muscle proliferation by prostaglandin E~. Exp Cell Res 1985; 160:117-125. 18, Hnttner J. J., Gwebu E. T., Panganamala R. V. et al. Fatty acids and their prostaglandin derivatives: Inhibitors of proliferation in aortic smooth muscle cells. Science 1977; 197: 289-291. 19. Cox L. R., Murphy S. K., Ramos K. S. Modulation of phosphoinositide metabolism in rat aortic smooth muscle cells by allylamine. Exp Mol Patho11990; 53: 52-63. 20. Sadhu D. N., Ramos K. S. Cyclic AMP inhibits c-Ha-ras protooncogene expression and DNA synthesis in rat aortic smooth muscle cells. Expem:entia 1993; 49: 567-570. 21. Ramos K. S., Cox L. R. Primary cultures of rat aortic endothelial and smooth muscle cells: An in vitro model to study xenobioticinduced vascular toxicity. In Vitro Cell Dev Bio11987; 23: 288-296. 22. Ramos K. S., Cox L. R. Aortic endothelial and smooth muscle cell cultures. In: Tyson A., Frazier J. M. eds. Methods in Toxicology. New York: Academic Press, 1993: 159-168. 23. Ou X., Ramos K. S. Proliferative responses of quail aortic smooth muscle cells to benzo(a)pyrene: implications in PAH-induced atherogenesis. Toxicology 1992; 74: 243-258, 24. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254. 25. Ramos K. S., Lin H., McGrath J. J. Modulation of cycling
Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 54(2), 101-107
© Pearson Professional Ltd 1996
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26.
2Z
28. 29.
guanosine monophosphate levels in cultured aortic smooth cells by carbon monoxide. Bfachem Pharmaca11989; 38: 1368-1370. Fowler K. H., Chapkin R. S., McMurray D. N. Effects of purified dietary n-3 ethyl esters on murine T-lymphocyte function. J Immuno11993; 151: 5186-519Z Tare E. H., Wilder M. E., Cram L. S., Wharton W. A method for staining 3T3 cell nuclei with propidium iodine in hypotonic solution. Cytometry 1983; 4:211-215. Ott L. An Introduction to Statistical Methods and Analysis, 3rd edn. Boston, MA: PWS-KENT Publishing, 1988. Low C-E, Pupillo M. B., Bryan R. W., Bailey J. M. Inhibffion of phytohemagglutinin-induced lymphocyte mitogenesis by
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lipoxygenase metabolites of arachidonic acid: structure-activity relationships. JLipidRes 1984; 25: 1090-1095. 30. Simmer T. H., Peskar B. A. Prostaglandin E~ and arterial occlusive disease: pharmacological consideration. EurJ Clin Invest 1988; 18: 549-554. 31. Brouard C., Pascaud M. Effects of moderate dietary supplementations with n-3 fatty acids on macrophage eicosanoid synthesis in the rat. Bfochim BiaphysActa 1990; 1047: 19-28. 32. Assender J. W., Southgate K. M., Hallett M. B., Newby A. C. Inhibition of proliferation, but not of Ca 2+mobilization, by cyclic AMP and GMP in rabbit aortic smooth-muscle cells. BiachemJ 1992; 288: 527-532.
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Cell c y c l e related inhibition of m o u s e v a s c u l a r s m o o t h m u s c l e cell proliferation by p r o s t a g l a n d i n El: relationship b e t w e e n p r o s t a g l a n d i n E 1 and i n t r a c e l l u l a r c A M P levels Y.-Yi Fan 1, K. S. Ramos z, R. S. Chapkin ~ 1Faculty of Nutrition and Molecular and Cell Biology Group, 442 Kleberg Center, Texas A&M University, College Station, TX 77843, USA. 2Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843, USA.
Summary Elucidation of the cellular and molecular mechanisms which regulate vascular smooth muscle cell proliferation is critical to the understanding of atherogenesis. The present studies were conducted to evaluate the relationship between prostaglandin E1 (PGG) and cAMP in the regulation of DNA synthesis in mouse vascular smooth muscle cells (SMCs). Quiescent cultures of SMCs were challenged with 10% fetal bovine serum to initiate cell cycle transit and PGE1 (10 pM) or dibutyryl cAMP (1, 10, 100 pM) added at 0, 8, 16, 24, and 32 h. DNA synthesis as measured by [3H] thymidine incorporation and intracellular cAMP levels were measured 24 h following individual treatments. PGE~ modulated DNA synthesis in a cell cycle related fashion, with inhibition only observed in cells challenged 16 h or longer following initiation of cell cycle transit. The decrease in DNA synthesis induced by PGG was associated with increased intracellular cAMP levels at 16 and 24 h, but not 32 h. Exposure of SMCs to dibutyryl-cAMP also inhibited DNA synthesis in a cell cycle related fashion, with the most pronounced effect seen at 16 h. These results demonstrate that the effects of PGE1 are restricted to a defined period within the cell cycle following S phase entry and implicate modulation of intracellular cAMP levels in the inhibitory response.
INTRODUCTION Vascular smooth muscle cell (SMC) proliferation plays a principal role in the fibroproliferative component of atherosclerotic disease. 1-3 The proliferation of SMCs is a complex process regulated by the interaction of growth factors with their respective receptors. Prostaglandins, 20Received 19 June 1995 Accepted 24 July 1995 Correspondence to: Robert S. Chapkin, Tel. 409 845 0419; Fax. 409 845 6433; E-mail: [email protected]
carbon polyunsaturated fatty acid derived eicosanoids, 4 are potent mediators of cell proliferation. 5-7 Prostaglandin E 1 (PGE1) and prostaglandin E2 (PGE2) share the ability to inhibit PDGF-induced DNA synthesis in rat arterial SMCs, but the biological effects of PGE~ are approximately 20 times stronger than PGE2.s PGE1 appears to exert opposite effects on quiescent relative to randomly cycling SMCs suggesting that the biological activity of this PG is influenced by cell cycle related events? Addition of PGE~ to quiescent A-10 cells, a rat vascular SMC line, enhances DNA synthesis, while addition to cycling cells elicits an anti-proliferative 101
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response. These data suggest a potential benefit of PGE~ in wound healing (quiescent state) and prevention of atheromatous plaque formation (proliferating state), and implicate a cell cycle related component in the ability of PGE1 to modulate SMC proliferation. Previous in vitro studies in this laboratory have demonstrated that PGE~ biosynthesis in macrophages can be enhanced by feeding gamma-linolenic acid enriched dietary oils. ~°-~2It is therefore possible that manipulation of macrophage PGE1 synthesis by dietary gamma-linolenic acid may also play an important role in the regulation of SMC proliferation. Although the mouse is naturally resistant to atherosclerosis, 13 the recent development of transgenic mouse models have provided researchers with the unique opportunity to study the complex environmental and genetic interactions underlying atherosclerosis. 14 Techniques for gene manipulation in vivo are more advanced in the mouse than in any other mammal; therefore this model system is currently being aggressively utilized by numerous investigators in the atherosclerosis fieldY,~6 Since alteration of SMC growth programs is a contributing factor in the process of atherogenesis/,2 we have evaluated the effect of select lipid mediators on SMC growth using the highly relevant murine model system. Several studies indicate that the inhibitory response of SMC proliferation to PGE~ is cell cycle dependent. However, published findings are contradictory and final interpretation awaits further studies. For example, the inhibitory effect of PGE~ on human SMC proliferation occurred primarily in G1 phase, with no effect in S phaseY In contrast, PGE1 increased quiescent SMC (majority of cells were in Go/G~phase) DNA synthesis, and reduced cycling (growing) SMC DNA synthesis in rats? It has also been shown that the biological effect of PGE1 in various cells are mediated by activation of adenylate cyclases and subsequent elevation in intracellular cAMP levels? The increase of cAMP levels may contribute to down-regulation of SMC proliferation.8,~z~8Previous studies in this laboratory have shown that dibutyryl cAMP (rib-cAMP), a stable analogue of cAMP, modulates rat SMC growth, differentiation, 19 and growth factor related gene expression. 2° Although addition of PGEj quickly raises intracellular cAMP levels, 8,~z~8whether changes in intracellular cAMP levels by PGE~ correlate with SMC proliferation as a function of cell cycle transit has not been elucidated. The present study was conducted to examine the cell cycle related effect of PGE~ and db-cAMP on SMC DNA synthesis and the changes of intracellular cAMP levels throughout the cell cycle. Our results show that the inhibitory effect of PGE~ on mouse SMC DNA synthesis is cell cycle specific, having a pronounced effect following entry of the cells into the S phase. The inhibitory effect may involve a change in intracellular cAMP levels.
MATERIALS AND METHODS Materials
Tissue culture Media 199 was purchased from Gibco BRL (Grand Island, NY). Heat inactivated fetal bovine serum (FBS) was obtained from Intergen (Purchase, NY). CoUagenase was from Worthington (Freehold, NJ). Prostaglandin standards were obtained from Cayman Chemicals (Ann Arbor, MI). [3H] thymidine was from ICN Radiochemicals (Irvine, CA). The cAMP radioimmunoassay (RIA) kit was purchased from Nuclear Magnetics (Cambridge, MA). Trypsin-EDTA solution, antibiotic/antimycotic solution, dibutyryl cAMP, and glutamine were obtained from Sigma Chemical (St Louis, MO). All solvents were Optima grade (Fisher Scientific, Fair Lawn, NJ). C57BL/6 female mice were from Charles River (Frederick Research Facility, Frederick, MD). Cell culture
SMCs were isolated from aortas of pathogen-free C57BL/6 female mice by a series of enzymatic digestions with collagenase and trypsin as described previously. 21,22Cells were pooled from 3 mice and grown in Medium 199 supplemented with 10% FBS, 2 mM glutamine, 10 000 units/ ml penicillin, lOmg/ml streptomycin, and 25gg/ml amphotericin B. Secondary cultures were prepared by trypsinization of primary cultures and seeded in 35-mm culture dishes at a density of 1 x 104 cells/dish. Passage 3-12 was used in all experiments. Quiescent SMCs were obtained by serum deprivation in medium containing 0.1% FBS for 72 h. a3 Prostaglandin treatment
Quiescent or cycling cultures of SMCs were incubated with [3H] thymidine at 0.5 gCi/ml and treated with 0, 5, 10, 20, 40, or 80 gM PGE1 in the presence of 2 or 10% FBS, respectively, for 24 h. Prostaglandins were diluted in ethanol and the final concentration of ethanol in the cukure medium was less than 0.1%. Control cells were exposed to the same amount of ethanol as treated cells. Cells were harvested at the end of the incubation period and DNA synthesis ([3H]thymidine incorporation) and protein concentration measured as described previouslyY,24 Effects of PGE 1 or db-cAMP on DNA synthesis
Quiescent SMCs were released from Go by incubation in 10% FBS medium (time 0). pH] thymidine (0.5 gCi/ml) and PGE1 (10 gM) or db-cAMP (1, 10, 100 gM) were added to cultures at 0, 8, 16, 24, and 32 h following serum stimulation. Cultures were incubated for an additional 24 h to ensure identical conditions and [3HIthymidine incorpora-
Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 54(2), 101-107
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PGE~ modulation of SMC growth
tion and protein content were subsequently measured at the end of each incubation period. Effect of PGE~ on cAMP levels
Growth-arrested SMCs were stimulated by 10% FBS as described above. PGE1 (10 gM) was added to the cultures at 0, 8, 16, 24, and 32 h after serum stimulation and incubated for an additional 24 h. Cells were harvested at the end of each incubation and intracellular cAMP was measured by radioimmunoassayP Flow cytometry
Quiescent SMC were stimulated by 10% FBS as previously described. Cells were trypsinized and collected at 0, 8, 16, 24, and 32 h after serum stimulation. Mouse lymphocytes were isolated immediately prior to SMC cell cycle analysis and used as a G0/G ~phase controlY Nuclei were stained with propidium iodide and fluorescence measured by flow c y t o m e t r y 7 Effect of PGE~ on cell proliferation
Growth-arrested SMCs were released from Go by 10% FBS as described above. Cells were dosed with PGE~ (10 ~tM) at 0 and 16 h following serum stimulation and grown for 4 days. Fresh medium containing 10 ~zM of PGE1 was applied every other day. At the end of the incubation period, cells were trypsinized and counted using a hemacytometer.
Statistics
Data were analyzed using one-way analysis of variance and Fisher's least significant difference procedure for comparing treatment means. 2s A difference of p < 0.05 was considered statistically significant.
RESULTS
The effect of PGE] on serum-induced DNA synthesis in cycling versus quiescent SMCs is shown in Figures la and lb, respectively. The inhibitory effect was concentration dependent and most pronounced when cells were maintained in 10% serum to preserve continuous cycling. The inhibitory effect of PGE2 was less pronounced than that of PGE~ (data not shown). To determine whether the inhibitory effect of PGE~ on SMC DNA synthesis was cell cycle-related, PGE~ (10 ~tM) was added to SMC cultures at various time points following initiation of cell cycle transit, and subsequently incubated for an additional 24 h. The action of PGE~ on SMC DNA synthesis is shown in Figure 2. The inhibitory effect of PGE~ was only observed at 16 h and beyond, with the most significant effect seen at 16 h. As determined by flow cytometry (Fig. 2 panel), the percentage of control cells in S phase increased as cells progressed through the cycle, reaching the highest proportion at 16 h. These data indicate that the inhibitory action of PGE~ on DNA synthesis was confined to a critical period following S phase entry. We next assessed the inhibitory effect of PGE~ on SMC proliferation at 16 h. For this purpose, cells were dosed
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Fig. 1 Dose-dependent effect of PGE1 on (a) cycling versus (b) quiescent DNA synthesis in SMCs. Quiescent and cycling SMCs were treated with various concentrations of PGE1 (0-80 #M) and pulsed with 0.5 gCi/ml of pH] thymidine for 24 h in the presence of 2% or 10% serum-containing medium, respectively. Values represent the means + SEM of 4 replicate cultures per group (*p < 0.05).
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Fig. 2 Effect of PGE1 on SMC DNA synthesis. Growth-arrested SMCs were stimulated with 10% serum• PGE~ (10 gM) and [3H] thymidine (0.5 gCi/ml) were added at different times following serum stimulation and [aH] thymidine incorporation was measured after 24 h incubation. Results are expressed as percentage of pH] thymidine incorporated in cells treated with PGE1 compared with incubations without PGE, Values represent the means _+SEM of 4 replicate cultures per group (*p< 0.05). Panel represents the percent of SMCs in S phase of the cell cycle at various times. SMCs were serum depleted for 72 h and stimulated with 10% serum for 0, 8, 16, 24, and 32 h. Cells were then harvested and nuclei fluorescence measured by flow cytometry.
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with PGE 1 (10 gM) at 0 and 16 h following serum stimulation and grown for 4 days. A significant anti-proliferative effect was seen at 16 h (Fig. 3), confirming the cell cycle related inhibition of DNA synthesis b y PGE1 Since the biologic effects of PGE1 m a y be linked to its ability to
Fig. 4 Effect of PGE1 on SMC intracellular cAMP concentration. Growth-arrested SMCs were stimulated with 10% serum. PGE1 (10 gM) was added at different times following serum stimulation and the intracellular cAMP level was measured after 24 h incubation. Values represent the means _+SEM of 4 replicate cultures per group. (,op= 0.058)
modulate cAMP levels, the effects of PGEI on the regulation of intracellular cAMP levels in s y n c h r o n o u s l y cycling cells was m e a s u r e d (Fig. 4). Intracellular cAMP levels increased as cells progressed t h r o u g h the cell cycle in b o t h groups. C o m p a r e d to the control group, PGE1 treated cells h a d an increased cAMP level at 16 h ( p = 0.058). If the inhibitory effect of PGE~ is mediated in part by cAMP, t h e n exogenous cAMP s h o u l d also inhibit DNA
Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 54(2), 101-107
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Fig. 5 Effect of Ob-cAMP on SMC DNA synthesis. Growth-arrested SMCs were stimulated with 10% serum. Dibutyryl cAMP (1, 10, 100 #M) and [3H] thymidine (0.5 #Ci/ml) were added at different times following serum stimulation and [3H] thymidine incorporation was measured after a 24 h incubation. Values represent the means + SEM of 4 replicate cultures per group (*p < 0.05).
synthesis in a manner comparable to that of the PGE~. Figure 5 demonstrates the effects of exogenous db-cAMP on DNA synthesis during different stages of the cell cycle. Similar to exogenous PGE~, the strongest concentrationdependent inhibitory effect by db-cAMP was noted at 16 h, suggesting that a PGE~-sensitive point is expressed at this time during cell cycle transit. DISCUSSION
SMC proliferation is one of the main events implicated in the pathogenesis of atherosclerotic vessel diseases. 3 An understanding of anti-proliferative factors may therefore aid in controlling the pathogenesis of vascular diseases. PGE1 is capable of inhibiting SMC DNA synthesis, s,~z~sbut the mechanism of PGE~ interference with SMC proliferation has not been well defined. Because mouse vascular SMCs can produce eicosanoids at gM levels upon stimulation by thrombin, 29 the PGE~ concentration (10 gM) used in our studies likely approximates the physiological conclifton. The concentration of PGE~ required to inhibit SMC proliferation in vitro appears to vary depending on the species and culture conditions. For example, 25 nM of PGG can inhibit h u m a n aortic SMC proliferation induced by 1% serum + PDGF (50 ng/ml) or 200/0 serum by up to 600/0,17 while the ICs0 for PGE~ in cycling -4-10 cells maintained in 10% FBS was 20 gM? Our data demonstrate that PGE~ down-regulates SMC DNA synthesis in a concentration-dependent manner, and that this inhibitory response is influenced by cell cycle kinetics. As shown in Figure 1, PGE 1had a diminished inhibitory effect on quiescent SMC DNA synthesis cul© Pearson Professional Ltd 1996
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tured in low serum (Fig. lb) relative to cycling SMCs maintained in a higher serum concentration (Fig. la). These data indicate that the ultimate inhibitory effect of PGE~ on vascular SMC DNA synthesis is dependent on culture conditions and cell cycle progression. PGE 2 also exerts an anti-proliferative effect on SMCs, albeit with reduced potency relative to PGE~? The relative difference in the anti-proliferative potential of PGE1 and PGE2 is noteworthy, because in many cell systems these autacoids possess opposing functions. These include effects on the immune response, fibrinolytic system, and vascular contraction. 3°,3~ The stronger anti-proliferative capacity of PGE 1emphasizes the potential benefit of PGE~ as an anti-atherogenic agent. The extent to which PGE1 exerted a down-regulatory effect on SMC DNA synthesis varied throughout the cell cycle. The strongest inhibitory effect was observed when PGE 1 was added 16h after serum stimulation (Fig. 2), which corresponded to S phase as determined by flow cytometry (Fig. 2 panel). This interpretation is also supported by cell proliferation data (Fig. 3). However, these results are contrary to observations reported by Loesberg et allz where the inhibitory effect of PGE~ on DNA synthesis was observed during the G 1 phase of the cell cycle. PGE 1 was less effective at periods following the critical 16 h sensitive point. This discrepancy may be due to the different cell types and/or differences in the culture conditions utilized. For instance, Loesberg et al used h u m a n cells in 20% serum-containing medium as compared to murine cells in lower serum concentration. It is possible that higher growth factor concentrations influenced the anti-proliferative response to PGE~. Since 35% of cells reached S phase by 8 h after serum stimulation (Fig. 2 panel) and addition of PGE1 did not inhibit SMC DNA synthesis at this time, it is likely that specific molecular targets for the anti-proliferative function of PGE~ are expressed at the peak of S phase (16 h). It has been suggested that the biologic effects of PGE 1 are linked to its ability to modulate intracellular cAMP levels.8,~z~s We have previously shown that exogenous db-cAMP (200 gM) and the phosphodiesterase inhibitor theophylline (100 gM) significantly inhibited DNA synthesis in rat SMCs? 9 In order to determine if the cell cycle related anti-proliferative effects of PGE1 correlated with modulation of cAMP levels, we examined the intracellular cAMP levels in response to the addition of PGE1 at various times. The increase in intracellular cAMP levels (Fig. 4) was associated with a decrease in SMC DNA synthesis (Fig. 2), with the most notable effect observed at 16 h. These data indicate that the cell cycle related effect of PGE1 on SMC DNA synthesis involves cAMP. Although studies have indicated that the addition of PGE~ to SMCs rapidly elevates intracellular cAMP levels followed by a gradual decrease to basal levels within several hours, 8,~7
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t h e l o n g - t e r m effect of PGE1 o n i n t r a c e l l u l a r c A M P production has not been previously reported. Our data d e m o n s t r a t e t h a t cAMP a c c u m u l a t e s o v e r t i m e as a f u n c t i o n of cell cycle t r a n s i t (Fig. 4). A p o s s i b l e e x p l a n a t i o n is t h a t t h e a d d i t i o n of PGE1 at 16 h m a y s t r o n g l y activate a d e n y l a t e cyclases i n c o m b i n a t i o n w i t h o t h e r intracellutar mediators which inhibit phosphodiesterase activity, t h e r e b y , r e s u l t i n g i n t h e l o n g - t e r m e l e v a t i o n of i n t r a c e l l u l a r c A M P levels. F u r t h e r s t u d i e s are n e e d e d to clarify t h i s p o i n t . To f u r t h e r d e m o n s t r a t e t h e i n v o l v e m e n t of c A M P as a d o w n s t r e a m m o d u l a t o r of t h e SMC r e s p o n s e to P G E , we t r e a t e d cells w i t h d b - c A M P at different cell cycle stages a n d m e a s u r e d t h e effect o n DNA s y n t h e s i s (Fig. 5). C o n sistently, t h e s t r o n g e s t i n h i b i t i o n was s e e n w h e n d b c A M P was a d d e d at 16 h. T h e DNA s y n t h e s i s i n h i b i t i o n profile of d b - c A M P (Fig. 5) is c o m p a r a b l e w i t h PGE1 (Fig. 2), a l t h o u g h d b - c A M P h a d a l o w e r i n h i b i t o r y effect c o m p a r e d to PGE~. This d i s c r e p a n c y is p r o b a b l y t h e r e s u l t of r e d u c e d p e r m e a b i l i t y of db-cAMP. I n this regard, h i g h c o n c e n t r a t i o n s (100 ~M) of cAMP a n a l o g u e were n e e d e d to m a i n t a i n e l e v a t e d cAMP levels i n r a b b i t aortic SMCs i n o r d e r to o b t a i n a n a n t i - p r o l i f e r a t i v e effect. 32 I n c o n c l u s i o n , o u r d a t a d e m o n s t r a t e t h a t (i) PGE1 i n h i b i t s DNA s y n t h e s i s i n m o u s e SMCs, (ii) t h e antiproliferative effect of PGE~ is cell cycle-related, w i t h t h e m o s t p r o n o u n c e d effect at 16 h f o l l o w i n g i n i t i a t i o n of cell cycle transit, a n d (iii) t h e cell cycle r e l a t e d a c t i o n of PGE~ o n SMC DNA s y n t h e s i s i n v o l v e s m o d u l a t i o n of i n t r a c e l l u l a r cAMP.
ACKNOWLEDGEMENTS The authors wish to thank Ms Cindy H. Thurlow for her excellent technical assistance, and Ms Betty Rosenbaum and Mr Chris Jolly for flow cytometry analyses. This research was supported in part by Scotia Pharmaceutical Ltd to RSC, the Texas A&M interdisciplinary Research Enhancemere Program to KSR and RSC, and NIH grant (DK 41693) to RSC and (ES 00213) to KSR.
REFERENCES 1. Ip J. H., Fuster V., Badimon L., Badimon J., Taubman M. B., Chesebro J. H. Syndromes of accelerated atherosclerosis: role of vascular injury and smooth muscle cell proliferation. JAm Coil Cardio11990; 15: 1667-1687. 2. Querol-Ferrer V., Hnltagardh-Nilsson A., Ringertz N. S., Nilsson J., Jonzon B. Adenosine receptors, cyclic AMP accumulation, and DNA synthesis in aortic smooth cell cultures of adult and neonatal rats. J Cell Physiol 1992; 151: 555-560. 3. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990's. Nature 1990; 362: 801-809. 4. Bergstrom S., Danielsson H., Klenberg D., Samuelsson B. The enzymatic conversion of essential fatty acids into prostaglandins. J Biol Chem 1964; 239: 4006-4008.
5. Thomas D. R., Phflpott G. W., Jaffe B. M. The relationship between concentration of prostaglandin E and rates of cell replication. Exp Cell Res 1974; 84: 40-46. 6. Uehara Y., Ishimitsu T., Kimura K., Ishii M., Ikeda T., Sugimoto T. Regulatory effects of eicosanoids on thymidine uptake by vascular smooth muscle cells of rats. Prostaglandins 1988; 36: 847-85Z Z Pasricha P. J., Hassoun P. M., Teufel E., Landman M. J., Fanbury B. L Prostaglandin E1and E2 stimulate the proliferation of pulmonary artery smooth muscle ceils. Prostaglandins 1992; 43: 5-19. 8. Nilsson J., Olsson A. G. Prostaglandin E1 inhibits DNA synthesis in arterial smooth muscle cells stimulated with platelet-derived growth factor. Atherosclerosis 1984; ~3: 77-82. 9. Owen N. E. Effect of prostaglandin E1 on DNA synthesis in vascular smooth muscle cells. Am J Physiol 1986; 250: C584-C588. 10. Chapkin R. S., Coble K. J. utilization of gammalinolenic acid by mouse peritoneal macrophages. Biochim Biophys Acta 1991; 108~: 365-370. 11. Fan Y-Y,Chapkin R. S. Mouse peritoneal macrophage prostaglandin E1 synthesis is altered by dietary gamma-linolerdc acid. J Nutr 1992; 122: 1600-1606. 12. Fan Y.-Y.,Chapkin R. S. Phospholipid sources of metabolically elongated gammalinolenic acid: Conversion to prostaglandin E1 in stimulated mouse macrophages. J Nutr Biochem 1993; 4: 602-60Z 13. Breslow J. L. Insights into lipoprotein metabolism from studies in transgenic mice. Annu Rev Physio11994; 56: 797-810. 14. Lusis A. J. The mouse model for atherosclerosis. Trends Cardiovasc Med 1993; 3: 135-143. 15. Nakashima Y., Plump A. S., Raines E. W., Brestow J. L., Ross R. Apo E-deficient mice develop lesions during phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb 1994; 14: 133-139. 16. Reddick R. L., Zhang S. H., Maeda N. Atherosclerosis in mice lacking apo E. Evaluation of lesional development and progress. Arterioscler Thromb 1994; 14: 141-14Z 1Z Loesberg C., Van Wijk R., Zandbergen J., Van Aken W. G., Van Mourik J. A., De Groot Ph G. Cell cycle-dependent inhibition of human vascular smooth muscle proliferation by prostaglandin E~. Exp Cell Res 1985; 160:117-125. 18, Hnttner J. J., Gwebu E. T., Panganamala R. V. et al. Fatty acids and their prostaglandin derivatives: Inhibitors of proliferation in aortic smooth muscle cells. Science 1977; 197: 289-291. 19. Cox L. R., Murphy S. K., Ramos K. S. Modulation of phosphoinositide metabolism in rat aortic smooth muscle cells by allylamine. Exp Mol Patho11990; 53: 52-63. 20. Sadhu D. N., Ramos K. S. Cyclic AMP inhibits c-Ha-ras protooncogene expression and DNA synthesis in rat aortic smooth muscle cells. Expem:entia 1993; 49: 567-570. 21. Ramos K. S., Cox L. R. Primary cultures of rat aortic endothelial and smooth muscle cells: An in vitro model to study xenobioticinduced vascular toxicity. In Vitro Cell Dev Bio11987; 23: 288-296. 22. Ramos K. S., Cox L. R. Aortic endothelial and smooth muscle cell cultures. In: Tyson A., Frazier J. M. eds. Methods in Toxicology. New York: Academic Press, 1993: 159-168. 23. Ou X., Ramos K. S. Proliferative responses of quail aortic smooth muscle cells to benzo(a)pyrene: implications in PAH-induced atherogenesis. Toxicology 1992; 74: 243-258, 24. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254. 25. Ramos K. S., Lin H., McGrath J. J. Modulation of cycling
Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 54(2), 101-107
© Pearson Professional Ltd 1996
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26.
2Z
28. 29.
guanosine monophosphate levels in cultured aortic smooth cells by carbon monoxide. Bfachem Pharmaca11989; 38: 1368-1370. Fowler K. H., Chapkin R. S., McMurray D. N. Effects of purified dietary n-3 ethyl esters on murine T-lymphocyte function. J Immuno11993; 151: 5186-519Z Tare E. H., Wilder M. E., Cram L. S., Wharton W. A method for staining 3T3 cell nuclei with propidium iodine in hypotonic solution. Cytometry 1983; 4:211-215. Ott L. An Introduction to Statistical Methods and Analysis, 3rd edn. Boston, MA: PWS-KENT Publishing, 1988. Low C-E, Pupillo M. B., Bryan R. W., Bailey J. M. Inhibffion of phytohemagglutinin-induced lymphocyte mitogenesis by
© Pearson Professional Ltd 1996
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lipoxygenase metabolites of arachidonic acid: structure-activity relationships. JLipidRes 1984; 25: 1090-1095. 30. Simmer T. H., Peskar B. A. Prostaglandin E~ and arterial occlusive disease: pharmacological consideration. EurJ Clin Invest 1988; 18: 549-554. 31. Brouard C., Pascaud M. Effects of moderate dietary supplementations with n-3 fatty acids on macrophage eicosanoid synthesis in the rat. Bfochim BiaphysActa 1990; 1047: 19-28. 32. Assender J. W., Southgate K. M., Hallett M. B., Newby A. C. Inhibition of proliferation, but not of Ca 2+mobilization, by cyclic AMP and GMP in rabbit aortic smooth-muscle cells. BiachemJ 1992; 288: 527-532.
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