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Prostacyclin synthesis by proliferative aortic smooth muscle cells A kinetic in vivo and in vitro study
Atherosclerosis, 50 (1984) 63-12 Elsevier Scientific Publishers Ireland, Ltd
63
ATH 03420
Prostacyclin Synthesis by Proliferative Aortic Smooth Muscle Cells A Kinetic In Vivo and In Vitro Study J. Larrue,
D. Daret,
J. Demond-Hem-i,
Unit; 8 de Cardiologie, I.N.S.E.R.M.,
C. All&es
and H. Bricaud
Avenue du Haut Lpv~que, 33600 Pessac (France)
(Received 21 February, 1983) (Revised, received 25 July, 1983) (Accepted 26 July, 1983)
Summary The capacity of arterial SMCs to produce PGI, when stimulated by exogenous AA was studied in proliferative and confluent cultured cells and at different periods following endothelial denudation in vivo. PGI, production per cell was doubled during the exponential growth-phase in culture. By contrast, increased PGI, formation did not correlate with mitotic activity in intimal regeneration tissue but with the presence of SMCs in a synthetic phenotype. The present results suggest a potential role for PGI, in SMC differentiation and proliferation. Key words:
Atherosclerosis - Cell culture - Proliferation - Prostacyclin - Vascular smooth muscle cells
The discovery that vascular cells synthesize PGI, from either endogenous or exogenous AA [1,2] has generated a great interest in the field of platelet-vascular wall interaction in several pathological conditions including atherosclerosis [3]. This work was supported by the Institut National de la Sante et de la Recherche Medicale (CRL 81.5.037) and by the University of Bordeaux II. Abbreviations: AA = arachidonic acid; ASA = acetyl salicylic acid; FCS = fetal calf serum; IT = thickened intima; 6-KF,, = 6-keto prostaglandin Fi,; M = media; PBS = phosphate-bound saline; PGE, = prostaglandin E,; PGF,, = prostaglandin F,,; PGI, = prostacyclin; SMC = smooth muscle cell; TEM = transmission electron microscopy; TLC = thin-layer chromatography; w/w = wet/weight.
0021-9150/84/$03.00
Q 1984 Elsevier Scientific Publishers Ireland, Ltd.
64
Nevertheless, the possible role of prostanoids in vascular cell biology and function has not been well defined as yet. The importance of the vascular SMCs in atherogenesis has been established [4] and their replicative potential may play a key role in the formation of atherosclerotic plaque [5]. In addition to‘the fact that SMCs are able to produce large amounts of PGI, [6] and that this capacity was modulated under different pathophysiological conditions [3,7], eicosanoids have been implicated in cell proliferation and differentiation [8,9]. Therefore, we studied the capacity of cultured and neointimal SMCs from rabbit aorta to produce PGI,. Materials and Methods Animals Adult male ‘Fauve de Bourgogne’ rabbits weighing 2-2.5 kg were maintained a commercial laboratory chow under standardized conditions 2 months prior beginning of experiments.
on the
Cells Aortic smooth muscle cells were obtained from medial explants of thoracic aortas as previously described [7]. Cells were grown using HAM FlO medium (SEROMED), supplemented by 20% FCS, at 37°C in 25cm* plastic flasks in an atmosphere of 5% CO, in air during the first weeks. When confluency was achieved, cells were trypsinized and subcultivated with 1: 4 split ratio in 10% FCS-supplemented HAM FlO medium. All the experiments were carried out using cultures below passage 5. Cell counts were performed with a haemocytometer after trypsinization. De-endothelialization by balloon catheter Endothelium was removed from rabbit aorta by a balloon catheter (Fogarthy 3F) as described by Baumgartner et al. [lo]. Animals were killed 10, 40, 60, 120 and 180 days after intimal injury. Control unballooned sham-operated rabbits were killed at the same time with an overdose of sodium pentobarbital. Aortas were immediately dissected from adjacent tissues, rinsed and maintained in cold (4’C) PBS buffer. Adventitia was removed under a dissecting microscope. Aortas were opened, then the thickened intima (IT) was carefully dissected from underlying media (M). Tissue fragments were processed for PGI,-synthetic capacity. At the end of incubation period, fragments were weighed, then treated for histological examinations. In some animals, adjacent segments of tissue were immediately removed after killing and processed for TEM. PGIz synthesis determination Cultured SMCs (2 X 106) or tissue biopsies (50 mg w/w) were incubated with 10 nmol [14C]AA in 0.05 M Tris-HCI buffer (pH 7.4) containing NaCl (0.15 M) at 37°C for 20 min as previously described [7]. After incubation, the medium was immediately recovered, acidified to pH 3 with HCl(N) and extracted with 3 volumes of ethyl acetate. The organic phase was evaporated to dryness under nitrogen. Dried extracts were redissolved in 0.1 ml ethyl acetate and subjected to TLC in the organic
65
phase of ethyl acetate/iso-octane/acetic acid/water (110 :‘50 : 20 : 100, v/v). The radioactive products were located using a radiochromatogram scanner (Packard) or by autoradiography using Kodak X ray films (X-omat) after 2 days exposure. The position of the standards (6-KF,,, PGF,,, PGE,) on the TLC plates was visualized by iodine vapors. Results Characterization
of cultured aortic smooth muscle cells
Electron-microscope characteristics of cultured aortic smooth muscle cells were previously described [ll]. Briefly, cells appeared as ‘modulated’ SMCs. These cells contained a large number of plasmalemmal vesicles, numerous bundles of filaments (SO-100 A diameter); sometimes a defined but discontinuous basal lamina could be observed. These cells originating from healthy media have higher doubling time and saturation density values than corresponding adventitial fibroblasts as well as a characteristic ratio of type III to type I collagen synthesis. Morphological
observations of de-endothelialized
rabbit aorta
Light microscopy and TEM of aortas of rabbits de-endothelialized 10 days before killing revealed a luminal surface lined by a neointima consisting predominantly of SMCs, without endothelial coverage. Occasionally, scattered platelet aggregates could be observed. The most striking phenomenon observed until day 60 within both the XT and the inner part of the media is the presence of SMCs in a synthetic stage essentially characterized by a large development of rough endoplasmic reticulum and few myofilaments (Fig. 1). At day 120 after de-endothelialization, SMCs appeared predominantly in a contractile state characterized by a large number of myofilaments and a discrete development of cytoplasmic organelles (Fig. 2) either in the intimal thickening or in the media. At this interval, intimal thickening as measured as IT/M ratio began to decrease (Fig. 5). PGI, production by cultured SMC
Previous experiments have shown that total prostaglandin production by cultured arterial SMCs as expressed by percent transformation of [14C]AA was 2.5% for lo6 cells under our experimental conditions. This production increased linearly as a function of cell number between 1 x lo6 to 6 X lo6 cells. The major prostanoid formed was PGI, as determined by its hydrolysis product 6-KF,, followed by PGF,, am. PGE, (Table 1). The 6-KF,, accounts for 86.1 f 2.2% of the total prostaglandin production in confluent cells. This percentage was independent from the cell number between 0.7-6 X lo6 cells. The evaluation of total prostaglandins and 6-KF,, synthetic capacity by dish between day 1 and 7 after planting is shown in Fig. 3. It appears that total prostaglandin synthetic capacity increased significantly during the first 3 days after seeding but PGI, synthesis did not represent more than 50% of the total prostaglandin production within the first 2 days. This percentage increased quickly and reached normal values by day 3. Nevertheless, PGI, production from exogenous AA remains significantly higher during the exponential phase of growth
Fig. 1. Intimal thickening of rabbit thoracic aorta 40 days after de-endothelialization. SMC in a synthetic state. Er = rough endoplasmic reticulum; M = mitochondria, G = Golgi complex. X 13 500.
67
Fig. 2. Intimal thickening of rabbit thoracic aorta 180 days after de-endothelialization. SMCs in a contractile state. Adjacent to the plasma membrane there is an electron-dense basement membrane. Micropinocytotic vesicles (arrow heads). Myofilaments (arrows). M = mitochondria; EF = elastic fiber. x13500.
Fig. 3. Total prostaglandin (0) and 6-KF,, (0) values by cultured cells between 1 and 7 days after seeding. Each value represents the mean of 4 experiments f SEM. Fig. 4. Prostacyclin production by 1 x lo6 cells as a function of cell growth (0) t = P ( 0.01. Mean of 3 experiments f SEM.
than in confluent cells as expressed vs 30.2 f 6.3) (Fig. 4). PGI, production by arterial tissue In arterial tissue derived from
as pM formed by 1 X lo6 cells (60.0 f 14.5 pM
healthy
aortas
incubated
as described above,
.a5
3 ‘...
‘I.
‘2..
.Q)
“.* ....
o...
‘V....
-02
Lr.n r
“0
,:.: :‘\:, ,:::
.a25
:::
1:.
j:
::.
:.: l> :> :::
.> :.: ::: .:.
10
120
.:: 1_:
.Ql.
;i:
:::
:I:
180 days
Fig. 5. Kinetic evolution of 6-KF,, by tissue fragments from: Intimal thickening (0) and media (0) of de-endothelialized rabbit aorta as compared to normal media tissue (day 0): ? Z ?The intimal thickening is expressed as intima/media ratio (0 . . . . . . 0). Mean of 8 determinations f SEM.
69
TABLE
1
[‘4C]ARACHIDONIC Confluent eicosanoid
ACID TRANSFORMATION
BY AORTIC
TISSUE
AND CULTURED
SMCs were on the 5th passage. Results expressed as % transformation measured were the mean of 5 experiments + SEM.
of [r4C]AA
6-KF,,
PGF,,
PGE,
HETEs
Arachidonic
7.ll*1.1
1.0*0.4
0.8jzO.3
1.8kO.6
86.5 f 3.7
1.8*0.5
0.4fO.l
0.4fO.l
1.3*0.3
95.8kO.8
Confluent SMCs control
2.3 f 0.2
0.8*0.3
0.4dzO.l
1.23to.5
94.3 + 2.6
Confluent SMCs + ASA (5.10-4 M)
0.5 f 0.1
0.2*0.1
0.2*0.2
1.250.6
97.1 f 1.3
Aortic media control
SMCs for each
acid
Aortic media + ASA (15 mg/kg)
HETEs = hydroxylated
derivatives.
exogenous AA metabolism led to the formation of 6-KF,,, PGF,, and PGE, as shown in Table 1. Eighteen hours after oral administration of ASA (15 mg/kg), the prostanoid formation remained significantly inhibited in aortas. After 10 days following de-endotheliahzation, PGI, synthetic activity (expressed as pM 6-KF,, formed/mg w/w) remained essentially identical to that of normal vessel (IT: 34 f 12, M: 30 f 6, vs 23 f 7). On the other hand, a significant increase of 6-KF,, formation occurred at day 40 following endothelial removal, both in IT (116 f 28) and underlying M (60 f 8). This enhanced production remained until day‘ 60 and was significant when compared to normal media (IT: 92 f 16); (M: 46 + 2). Later (day 120) 6-KF,, concentration decreased (IT: 46 f 6), (M: 34 f 8) to reach normal values at day 180, either in IT (34 + 7) or underlying media (28 f 6) (Fig. 3). Discussion
The participation of arterial SMCs in the pathogenesis of atherosclerosis is well documented [4,5]. SMCs are significantly involved in the generation of PGI, by arterial wall [a], but the possible effects of endogenously produced prostaglandins on vascular physiology and function remain essentially unknown. Recently, the critical role of PGI, formation by SMCs in early arterial lesions has been suggested both in experimental [12] and human lesions [13]. In order to evaluate PGI,-synthetic capacities of arterial SMCs at several and well characterized stages of differentiation (i.e. synthetic phenotype vs contractile phenotype [14]), the transformation of exogenously added [14C]AA was comparatively studied in proliferative (exponential phase) and confluent SMCs in vitro and at different stages following de-endothelialization in vivo both in IT and underlying media. The results show that (a) under culture conditions, the level of PGI, production per cell is higher during the exponential growth phase of SMCs than at confluency, and (b) after de-endothelialization, an increased PGI, formation occurs in intimal regeneration tissue. This enhanced PGI, synthesis correlates with the presence of
70
SMCs in a synthetic phenotype as defined by ultrastructural features. Prostaglandin synthesis by confluent rabbit aortic SMCs in culture has been described [7,11]. An activation of prostaglandin synthetic capacity during the exponential growth phase in vitro has been recently reported in endothelial and fibroblast-like cells [15]. Accordingly, in our experiment, increased PGI, production in growing SMCs essentially results from an activation of cyclooxygenase rather than prostacyclin synthetase per se. Interestingly, it seems that cyclooxygenase may represent an actual rate-limiting enzyme in the metabolic conversion of AA to PGI, in arterial SMCs. These cells have a relative inability to form endoperoxide precursors of PGI, synthetase due to a low concentration of cyclooxygenase as compared to endothelial cells [16]. An increased PGI, production can result either from a general activation of protein synthesis leading to an increased cyclooxygenase content or from a stimulation of cyclooxygenase activity. Alternatively, explanations regarding enzymatic competition upon AA substrate may be also taken into account, since lipoxygenase activity occurs significantly in cultured arterial SMCs (171 and since hydroperoxide derivatives have been previously implicated both in the regulation of PGI, production [18] and cell growth [19]. PGI, synthesis is studied at different times following endothelial denudation, each corresponding to define stages of arterial repair, respectively: intimal proliferation (day lo), maximal IT (days 40-60) and regression (days 120-180) [20]. Arterial PGI, synthesis remains unchanged 10 days following endothelial denudation, despite a great mitotic activity in the tissue [21]. It is not possible to exclude the occurrence of any peak of stimulation within the first 10 days. However, this observation is in agreement with a previous report showing no alteration of PGI, synthesis in arterial punch biopsies 48 h after balloon injury when compared to uninjured aorta [12]. By contrast, the PGI, production is significantly enhanced in tissue biopsies from IT and, to a lesser extent from underlying media, at days 40 and 60 after injury. This interval corresponds to the maximal development of IT as measured by the IT/M ratio and is highly correlated with the presence of SMCs in a synthetic state as defined by ultrastructural features. At days 120 and 180, SMCs are in a contractile state. Despite no significant alteration in the cell number per unit square as compared to days 40-60 (not shown) PGI, synthesis returns to normal values, so that no differences may be observed in IT, underlying media and control media, respectively. This is certainly not linked to the rate of endothelial repair since identical recovery of PGI, has been obtained at the luminal surface of aorta from de-endothelialized and re-endothelialized areas
WI. These results suggest that activation of PGI, production by SMCs may be associated with their phenotypic expression. This is consistent with the finding that SMCs from fetal arteries, which are in a synthetic state, synthesize higher levels of PGI, than contractile SMCs ,from adult arteries [23]. These observations, when taken together with recent data showing that PGI, may stimulate adenylate cyclase in SMCs producing an increase of CAMP intracellular levels [24,25] and a report showing increased levels of CAMP in intimal thickening of
71
rabbit aorta [26], suggest that PGI, can affect growth and function of SMCs. However, the relationships between PGI, and CAMP regarding SMCs proliferation remain puzzling since elevated concentrations of CAMP inhibit SMCs proliferation in vitro [27] and since several types of anti-inflammatory drugs, which block PGI, synthesis, act as inhibitors of SMC proliferation [28,29]. Whatever the mechanism, the exact physiological relevance of the increasing capacities of secretory SMCs to produce PGI, cannot be precisely established from these experiments. However, the results do offer evidence that PGI,, in addition to its contribution to the thromboregulatory capacity of surface lining cells, may also participate in the biological behaviour of arterial SMCs and influence differentiation and proliferation. Acknowledgement
The authors wish to thank Mrs. N. Grillon for typing the manuscript. References 1 Moncada, S., Gryglewski, R., Bunting, S. and Vane, J.R., An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation, Nature (Lond.), 263 (1976) 663. 2 Moncada, S., Herman, A.G., Higgs, E.A. and Vane, J.R., Differential formation of prostacyclin by layers of the arterial wall - An explanation for the anti-thrombotic properties of vascular endothelium, Thromb. Res., 11 (1977) 323. 3 Dembinska-Kiec, A., Gryglewska, T., Zmuda, A. and Gryglewski, R.J., The generation of prostacyclin by arteries and by coronary vascular bed is reduced in experimental atherosclerosis in rabbits, Prostaglandins, 14 (1977) 1025. 4 Wissler, R., The arterial medial cell, smooth muscle of multifunctional mesenchyme? J. Atheroscler. Res., 8 (1968) 201. 5 Ross, R. and Glomset, J., The pathogenesis of atherosclerosis, N. Engl. J. Med., 295 (1976) 369. 6 Baenziger, J.L., Dillender, M.J. and Majerus, P.W., Cultured human skin fibroblasts and arterial cells produced a labile platelet inhibitory prostaglandin, B&hem. Biophys. Res. Commun., 78 (1977) 294. 7 Larrue, J., Rigaud, M., Daret, D., Demond, J., Durand, J. and Bricaud, H., Prostacyclin production by cultured smooth muscle cells from atherosclerotic rabbit aorta, Nature (Lond.), 285 (1980) 480. 8 Hopkins, N.K. and Gorman, R.R., Regulation of 3T3Ll fibroblast differentiation by prostacyclin, B&him. Biophys. Acta, 663 (1981) 457. 9 Kom, J.H., Halushka, P.V. and Le Roy, E.D., Mononuclear cell modulation of connective tissue function - Suppression of fibroblast growth by stimulation of endogenous prostaglandin production, J. Clin. Invest., 65 (1980) 543. 10 Baumgartner, H.R., Stemerman, M.B. and Spaet, T.H., Adhesion of blood platelets to subendothelial surface - Distinct from adhesion to collagen, Experientia (Basel), 27 (1971) 383. 11 Larrue, J., Leroux, C., Daret, D. and Bricaud, H., Decreased prostaglandin production in cultured smooth muscle cells from atherosclerotic aorta, B&him. Biophys. Acta, 710 (1982) 257. 12 Elder, A., Falcone, D.J., Hajjar, D.P., Minick, C.R. and Weksler, B.B., Recovery of prostacyclin production by the de-endothelialized rabbit aorta - Critical role of neointimal smooth muscle cells, J. Clin. Invest., 67 (1981) 735. 13 Sinzinger, M., Silberbauer, K. and Auerswald, W., Does prostacyclin (PGI,) regulate human intimal smooth muscle cell proliferation in early atherogenesis? Blood Vessels, 17 (1980) 58. What controls smooth muscle phenotype? 14 Charnley-Campbell, J.H. and Campbell, G.R., Atherosclerosis, 40 (1981) 347.
72
15 Ali, A.E., Barrett, J.C. and Eling, T.E., Prostaglandin and thromboxane production by fibroblasts and vascular endothelial cells, Prostaglandins, 20 (1980) 667. 16 Smith, W.L., De Witt, D.L. and Day, J.S., Quantitation and localisation of PGH, and PGI, synthetase activities in smooth muscle and endothelial cells. In: Vth Int. Conf. Prostaglandins, Florence, 18-21 May 1982, p. 229 (Abstr.). I7 Larrue, J., Rigaud, M., Razaka, G., Daret, D., Demond-Hem+, J. and Bricaud, H., Formation of monohydroxyeicosatetraenoic acids from arachidonic acid by cultured rabbit aortic smooth muscle cells, B&hem. Biophys. Res. Commun., 112 (1983) 242. 18 Salmon, J.A., Smith, D.R., Flower, R.J., Moncada, S. and Vane, J.R., Further studies on the enzymatic conversion of prostaglandin endoperoxides into prostacyclin by porcine aorta microsomes, Biochim. Biophys. Acta, 523 (1978) 250. 19 Cornwell, D.G., Huttner, J.J., Mile, G.E., Panganamala, R.V., Sharma, H.M. and Geer, J.C., Polyunsaturated fatty acids, vitamin E and the proliferation of aortic smooth muscle cells, Lipids, 14 (1979) 104. 20 Stemerman, M.B., Spaet, T.H., Pitlick, F.A., Cintron, J.R., Lejnicks, I. and Tiell, M.L., Intimal healing - The pattern of re-endothelialization and intimal thickening, Amer. J. Path., 87 (1977) 125. 21 Goldberg, I.D., Stemerman, M.B., Ransil, B.J. and Fuhro, R.L., In vivo aortic muscle cell growth kinetics, Circ. Res., 47 (1980) 182. 22 Elder, A., Falcone, D.J., Hajjar, D.P., Minick, C.R. and Weksler, B.B., Diet induced hypercholesterolemia inhibits the recovery of prostacyclin production by injured rabbit aorta, Amer. J. Path., 107 (1982) 186. 23 Clyman, R.I., Mauray, F., Koerper, M.A., Wiemer, F., Heyman, M.A. and Rudolph, A.M., Formation of prostacyclin (PGI,) by the ductus arteriosus of fetal lambs at different stages of gestation, Prostaglandins, 16 (1978) 633. 24 Hajjar, D.P., Weksler, B.B., Falcone, D.J., Hefton, J.M., Tack-Goldman, K. and Minick, C.R., Prostacyclin modulates cholesteryl ester hydrolytic activity by its effects on CAMP in rabbit aorta, J. Chn. Invest., 70 (1982) 479. 25 Larrue, J., Dorian, B., Braquet, P. and Bricaud, H., Regulation of smooth muscle cell cyclic nucleotide metabolism by prostacyclin, 5th Int. Conf. Cyclic Nucleotides and Protein Phosphorylation, Milan, 27 June-l July 1983, p. 120 (Abstr.). 26 Numano, F., Cyclic nucleotides and atherosclerosis. In: A.M. Gotto, Jr., L.C. Smith and B. Allen (Eds.), Atherosclerosis V, Springer-Verlag, New York, 1980, p. 537. 27 Stout, R.W., Cyclic AMP - A potent inhibitor of DNA synthesis in cultured arterial endothelial and smooth muscle cells, Diabetologia, 22 (1982) 51. 28 Longenecker, J.P., Kilty, L.A. and Johnson, L.K., Glucocorticoid influence on growth of vascular wall cells in culture, J. Cell Physiol., 113 (1982) 197. 29 Bayer, B.M., Kruth, H.S., Vaughan, M. and Beaven, M.A., Arrest of cultured cells in the G, phase of cell cycle by indomethacin, J. Pharm. Exp. Therap., 210 (1979) 106.
63
ATH 03420
Prostacyclin Synthesis by Proliferative Aortic Smooth Muscle Cells A Kinetic In Vivo and In Vitro Study J. Larrue,
D. Daret,
J. Demond-Hem-i,
Unit; 8 de Cardiologie, I.N.S.E.R.M.,
C. All&es
and H. Bricaud
Avenue du Haut Lpv~que, 33600 Pessac (France)
(Received 21 February, 1983) (Revised, received 25 July, 1983) (Accepted 26 July, 1983)
Summary The capacity of arterial SMCs to produce PGI, when stimulated by exogenous AA was studied in proliferative and confluent cultured cells and at different periods following endothelial denudation in vivo. PGI, production per cell was doubled during the exponential growth-phase in culture. By contrast, increased PGI, formation did not correlate with mitotic activity in intimal regeneration tissue but with the presence of SMCs in a synthetic phenotype. The present results suggest a potential role for PGI, in SMC differentiation and proliferation. Key words:
Atherosclerosis - Cell culture - Proliferation - Prostacyclin - Vascular smooth muscle cells
The discovery that vascular cells synthesize PGI, from either endogenous or exogenous AA [1,2] has generated a great interest in the field of platelet-vascular wall interaction in several pathological conditions including atherosclerosis [3]. This work was supported by the Institut National de la Sante et de la Recherche Medicale (CRL 81.5.037) and by the University of Bordeaux II. Abbreviations: AA = arachidonic acid; ASA = acetyl salicylic acid; FCS = fetal calf serum; IT = thickened intima; 6-KF,, = 6-keto prostaglandin Fi,; M = media; PBS = phosphate-bound saline; PGE, = prostaglandin E,; PGF,, = prostaglandin F,,; PGI, = prostacyclin; SMC = smooth muscle cell; TEM = transmission electron microscopy; TLC = thin-layer chromatography; w/w = wet/weight.
0021-9150/84/$03.00
Q 1984 Elsevier Scientific Publishers Ireland, Ltd.
64
Nevertheless, the possible role of prostanoids in vascular cell biology and function has not been well defined as yet. The importance of the vascular SMCs in atherogenesis has been established [4] and their replicative potential may play a key role in the formation of atherosclerotic plaque [5]. In addition to‘the fact that SMCs are able to produce large amounts of PGI, [6] and that this capacity was modulated under different pathophysiological conditions [3,7], eicosanoids have been implicated in cell proliferation and differentiation [8,9]. Therefore, we studied the capacity of cultured and neointimal SMCs from rabbit aorta to produce PGI,. Materials and Methods Animals Adult male ‘Fauve de Bourgogne’ rabbits weighing 2-2.5 kg were maintained a commercial laboratory chow under standardized conditions 2 months prior beginning of experiments.
on the
Cells Aortic smooth muscle cells were obtained from medial explants of thoracic aortas as previously described [7]. Cells were grown using HAM FlO medium (SEROMED), supplemented by 20% FCS, at 37°C in 25cm* plastic flasks in an atmosphere of 5% CO, in air during the first weeks. When confluency was achieved, cells were trypsinized and subcultivated with 1: 4 split ratio in 10% FCS-supplemented HAM FlO medium. All the experiments were carried out using cultures below passage 5. Cell counts were performed with a haemocytometer after trypsinization. De-endothelialization by balloon catheter Endothelium was removed from rabbit aorta by a balloon catheter (Fogarthy 3F) as described by Baumgartner et al. [lo]. Animals were killed 10, 40, 60, 120 and 180 days after intimal injury. Control unballooned sham-operated rabbits were killed at the same time with an overdose of sodium pentobarbital. Aortas were immediately dissected from adjacent tissues, rinsed and maintained in cold (4’C) PBS buffer. Adventitia was removed under a dissecting microscope. Aortas were opened, then the thickened intima (IT) was carefully dissected from underlying media (M). Tissue fragments were processed for PGI,-synthetic capacity. At the end of incubation period, fragments were weighed, then treated for histological examinations. In some animals, adjacent segments of tissue were immediately removed after killing and processed for TEM. PGIz synthesis determination Cultured SMCs (2 X 106) or tissue biopsies (50 mg w/w) were incubated with 10 nmol [14C]AA in 0.05 M Tris-HCI buffer (pH 7.4) containing NaCl (0.15 M) at 37°C for 20 min as previously described [7]. After incubation, the medium was immediately recovered, acidified to pH 3 with HCl(N) and extracted with 3 volumes of ethyl acetate. The organic phase was evaporated to dryness under nitrogen. Dried extracts were redissolved in 0.1 ml ethyl acetate and subjected to TLC in the organic
65
phase of ethyl acetate/iso-octane/acetic acid/water (110 :‘50 : 20 : 100, v/v). The radioactive products were located using a radiochromatogram scanner (Packard) or by autoradiography using Kodak X ray films (X-omat) after 2 days exposure. The position of the standards (6-KF,,, PGF,,, PGE,) on the TLC plates was visualized by iodine vapors. Results Characterization
of cultured aortic smooth muscle cells
Electron-microscope characteristics of cultured aortic smooth muscle cells were previously described [ll]. Briefly, cells appeared as ‘modulated’ SMCs. These cells contained a large number of plasmalemmal vesicles, numerous bundles of filaments (SO-100 A diameter); sometimes a defined but discontinuous basal lamina could be observed. These cells originating from healthy media have higher doubling time and saturation density values than corresponding adventitial fibroblasts as well as a characteristic ratio of type III to type I collagen synthesis. Morphological
observations of de-endothelialized
rabbit aorta
Light microscopy and TEM of aortas of rabbits de-endothelialized 10 days before killing revealed a luminal surface lined by a neointima consisting predominantly of SMCs, without endothelial coverage. Occasionally, scattered platelet aggregates could be observed. The most striking phenomenon observed until day 60 within both the XT and the inner part of the media is the presence of SMCs in a synthetic stage essentially characterized by a large development of rough endoplasmic reticulum and few myofilaments (Fig. 1). At day 120 after de-endothelialization, SMCs appeared predominantly in a contractile state characterized by a large number of myofilaments and a discrete development of cytoplasmic organelles (Fig. 2) either in the intimal thickening or in the media. At this interval, intimal thickening as measured as IT/M ratio began to decrease (Fig. 5). PGI, production by cultured SMC
Previous experiments have shown that total prostaglandin production by cultured arterial SMCs as expressed by percent transformation of [14C]AA was 2.5% for lo6 cells under our experimental conditions. This production increased linearly as a function of cell number between 1 x lo6 to 6 X lo6 cells. The major prostanoid formed was PGI, as determined by its hydrolysis product 6-KF,, followed by PGF,, am. PGE, (Table 1). The 6-KF,, accounts for 86.1 f 2.2% of the total prostaglandin production in confluent cells. This percentage was independent from the cell number between 0.7-6 X lo6 cells. The evaluation of total prostaglandins and 6-KF,, synthetic capacity by dish between day 1 and 7 after planting is shown in Fig. 3. It appears that total prostaglandin synthetic capacity increased significantly during the first 3 days after seeding but PGI, synthesis did not represent more than 50% of the total prostaglandin production within the first 2 days. This percentage increased quickly and reached normal values by day 3. Nevertheless, PGI, production from exogenous AA remains significantly higher during the exponential phase of growth
Fig. 1. Intimal thickening of rabbit thoracic aorta 40 days after de-endothelialization. SMC in a synthetic state. Er = rough endoplasmic reticulum; M = mitochondria, G = Golgi complex. X 13 500.
67
Fig. 2. Intimal thickening of rabbit thoracic aorta 180 days after de-endothelialization. SMCs in a contractile state. Adjacent to the plasma membrane there is an electron-dense basement membrane. Micropinocytotic vesicles (arrow heads). Myofilaments (arrows). M = mitochondria; EF = elastic fiber. x13500.
Fig. 3. Total prostaglandin (0) and 6-KF,, (0) values by cultured cells between 1 and 7 days after seeding. Each value represents the mean of 4 experiments f SEM. Fig. 4. Prostacyclin production by 1 x lo6 cells as a function of cell growth (0) t = P ( 0.01. Mean of 3 experiments f SEM.
than in confluent cells as expressed vs 30.2 f 6.3) (Fig. 4). PGI, production by arterial tissue In arterial tissue derived from
as pM formed by 1 X lo6 cells (60.0 f 14.5 pM
healthy
aortas
incubated
as described above,
.a5
3 ‘...
‘I.
‘2..
.Q)
“.* ....
o...
‘V....
-02
Lr.n r
“0
,:.: :‘\:, ,:::
.a25
:::
1:.
j:
::.
:.: l> :> :::
.> :.: ::: .:.
10
120
.:: 1_:
.Ql.
;i:
:::
:I:
180 days
Fig. 5. Kinetic evolution of 6-KF,, by tissue fragments from: Intimal thickening (0) and media (0) of de-endothelialized rabbit aorta as compared to normal media tissue (day 0): ? Z ?The intimal thickening is expressed as intima/media ratio (0 . . . . . . 0). Mean of 8 determinations f SEM.
69
TABLE
1
[‘4C]ARACHIDONIC Confluent eicosanoid
ACID TRANSFORMATION
BY AORTIC
TISSUE
AND CULTURED
SMCs were on the 5th passage. Results expressed as % transformation measured were the mean of 5 experiments + SEM.
of [r4C]AA
6-KF,,
PGF,,
PGE,
HETEs
Arachidonic
7.ll*1.1
1.0*0.4
0.8jzO.3
1.8kO.6
86.5 f 3.7
1.8*0.5
0.4fO.l
0.4fO.l
1.3*0.3
95.8kO.8
Confluent SMCs control
2.3 f 0.2
0.8*0.3
0.4dzO.l
1.23to.5
94.3 + 2.6
Confluent SMCs + ASA (5.10-4 M)
0.5 f 0.1
0.2*0.1
0.2*0.2
1.250.6
97.1 f 1.3
Aortic media control
SMCs for each
acid
Aortic media + ASA (15 mg/kg)
HETEs = hydroxylated
derivatives.
exogenous AA metabolism led to the formation of 6-KF,,, PGF,, and PGE, as shown in Table 1. Eighteen hours after oral administration of ASA (15 mg/kg), the prostanoid formation remained significantly inhibited in aortas. After 10 days following de-endotheliahzation, PGI, synthetic activity (expressed as pM 6-KF,, formed/mg w/w) remained essentially identical to that of normal vessel (IT: 34 f 12, M: 30 f 6, vs 23 f 7). On the other hand, a significant increase of 6-KF,, formation occurred at day 40 following endothelial removal, both in IT (116 f 28) and underlying M (60 f 8). This enhanced production remained until day‘ 60 and was significant when compared to normal media (IT: 92 f 16); (M: 46 + 2). Later (day 120) 6-KF,, concentration decreased (IT: 46 f 6), (M: 34 f 8) to reach normal values at day 180, either in IT (34 + 7) or underlying media (28 f 6) (Fig. 3). Discussion
The participation of arterial SMCs in the pathogenesis of atherosclerosis is well documented [4,5]. SMCs are significantly involved in the generation of PGI, by arterial wall [a], but the possible effects of endogenously produced prostaglandins on vascular physiology and function remain essentially unknown. Recently, the critical role of PGI, formation by SMCs in early arterial lesions has been suggested both in experimental [12] and human lesions [13]. In order to evaluate PGI,-synthetic capacities of arterial SMCs at several and well characterized stages of differentiation (i.e. synthetic phenotype vs contractile phenotype [14]), the transformation of exogenously added [14C]AA was comparatively studied in proliferative (exponential phase) and confluent SMCs in vitro and at different stages following de-endothelialization in vivo both in IT and underlying media. The results show that (a) under culture conditions, the level of PGI, production per cell is higher during the exponential growth phase of SMCs than at confluency, and (b) after de-endothelialization, an increased PGI, formation occurs in intimal regeneration tissue. This enhanced PGI, synthesis correlates with the presence of
70
SMCs in a synthetic phenotype as defined by ultrastructural features. Prostaglandin synthesis by confluent rabbit aortic SMCs in culture has been described [7,11]. An activation of prostaglandin synthetic capacity during the exponential growth phase in vitro has been recently reported in endothelial and fibroblast-like cells [15]. Accordingly, in our experiment, increased PGI, production in growing SMCs essentially results from an activation of cyclooxygenase rather than prostacyclin synthetase per se. Interestingly, it seems that cyclooxygenase may represent an actual rate-limiting enzyme in the metabolic conversion of AA to PGI, in arterial SMCs. These cells have a relative inability to form endoperoxide precursors of PGI, synthetase due to a low concentration of cyclooxygenase as compared to endothelial cells [16]. An increased PGI, production can result either from a general activation of protein synthesis leading to an increased cyclooxygenase content or from a stimulation of cyclooxygenase activity. Alternatively, explanations regarding enzymatic competition upon AA substrate may be also taken into account, since lipoxygenase activity occurs significantly in cultured arterial SMCs (171 and since hydroperoxide derivatives have been previously implicated both in the regulation of PGI, production [18] and cell growth [19]. PGI, synthesis is studied at different times following endothelial denudation, each corresponding to define stages of arterial repair, respectively: intimal proliferation (day lo), maximal IT (days 40-60) and regression (days 120-180) [20]. Arterial PGI, synthesis remains unchanged 10 days following endothelial denudation, despite a great mitotic activity in the tissue [21]. It is not possible to exclude the occurrence of any peak of stimulation within the first 10 days. However, this observation is in agreement with a previous report showing no alteration of PGI, synthesis in arterial punch biopsies 48 h after balloon injury when compared to uninjured aorta [12]. By contrast, the PGI, production is significantly enhanced in tissue biopsies from IT and, to a lesser extent from underlying media, at days 40 and 60 after injury. This interval corresponds to the maximal development of IT as measured by the IT/M ratio and is highly correlated with the presence of SMCs in a synthetic state as defined by ultrastructural features. At days 120 and 180, SMCs are in a contractile state. Despite no significant alteration in the cell number per unit square as compared to days 40-60 (not shown) PGI, synthesis returns to normal values, so that no differences may be observed in IT, underlying media and control media, respectively. This is certainly not linked to the rate of endothelial repair since identical recovery of PGI, has been obtained at the luminal surface of aorta from de-endothelialized and re-endothelialized areas
WI. These results suggest that activation of PGI, production by SMCs may be associated with their phenotypic expression. This is consistent with the finding that SMCs from fetal arteries, which are in a synthetic state, synthesize higher levels of PGI, than contractile SMCs ,from adult arteries [23]. These observations, when taken together with recent data showing that PGI, may stimulate adenylate cyclase in SMCs producing an increase of CAMP intracellular levels [24,25] and a report showing increased levels of CAMP in intimal thickening of
71
rabbit aorta [26], suggest that PGI, can affect growth and function of SMCs. However, the relationships between PGI, and CAMP regarding SMCs proliferation remain puzzling since elevated concentrations of CAMP inhibit SMCs proliferation in vitro [27] and since several types of anti-inflammatory drugs, which block PGI, synthesis, act as inhibitors of SMC proliferation [28,29]. Whatever the mechanism, the exact physiological relevance of the increasing capacities of secretory SMCs to produce PGI, cannot be precisely established from these experiments. However, the results do offer evidence that PGI,, in addition to its contribution to the thromboregulatory capacity of surface lining cells, may also participate in the biological behaviour of arterial SMCs and influence differentiation and proliferation. Acknowledgement
The authors wish to thank Mrs. N. Grillon for typing the manuscript. References 1 Moncada, S., Gryglewski, R., Bunting, S. and Vane, J.R., An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation, Nature (Lond.), 263 (1976) 663. 2 Moncada, S., Herman, A.G., Higgs, E.A. and Vane, J.R., Differential formation of prostacyclin by layers of the arterial wall - An explanation for the anti-thrombotic properties of vascular endothelium, Thromb. Res., 11 (1977) 323. 3 Dembinska-Kiec, A., Gryglewska, T., Zmuda, A. and Gryglewski, R.J., The generation of prostacyclin by arteries and by coronary vascular bed is reduced in experimental atherosclerosis in rabbits, Prostaglandins, 14 (1977) 1025. 4 Wissler, R., The arterial medial cell, smooth muscle of multifunctional mesenchyme? J. Atheroscler. Res., 8 (1968) 201. 5 Ross, R. and Glomset, J., The pathogenesis of atherosclerosis, N. Engl. J. Med., 295 (1976) 369. 6 Baenziger, J.L., Dillender, M.J. and Majerus, P.W., Cultured human skin fibroblasts and arterial cells produced a labile platelet inhibitory prostaglandin, B&hem. Biophys. Res. Commun., 78 (1977) 294. 7 Larrue, J., Rigaud, M., Daret, D., Demond, J., Durand, J. and Bricaud, H., Prostacyclin production by cultured smooth muscle cells from atherosclerotic rabbit aorta, Nature (Lond.), 285 (1980) 480. 8 Hopkins, N.K. and Gorman, R.R., Regulation of 3T3Ll fibroblast differentiation by prostacyclin, B&him. Biophys. Acta, 663 (1981) 457. 9 Kom, J.H., Halushka, P.V. and Le Roy, E.D., Mononuclear cell modulation of connective tissue function - Suppression of fibroblast growth by stimulation of endogenous prostaglandin production, J. Clin. Invest., 65 (1980) 543. 10 Baumgartner, H.R., Stemerman, M.B. and Spaet, T.H., Adhesion of blood platelets to subendothelial surface - Distinct from adhesion to collagen, Experientia (Basel), 27 (1971) 383. 11 Larrue, J., Leroux, C., Daret, D. and Bricaud, H., Decreased prostaglandin production in cultured smooth muscle cells from atherosclerotic aorta, B&him. Biophys. Acta, 710 (1982) 257. 12 Elder, A., Falcone, D.J., Hajjar, D.P., Minick, C.R. and Weksler, B.B., Recovery of prostacyclin production by the de-endothelialized rabbit aorta - Critical role of neointimal smooth muscle cells, J. Clin. Invest., 67 (1981) 735. 13 Sinzinger, M., Silberbauer, K. and Auerswald, W., Does prostacyclin (PGI,) regulate human intimal smooth muscle cell proliferation in early atherogenesis? Blood Vessels, 17 (1980) 58. What controls smooth muscle phenotype? 14 Charnley-Campbell, J.H. and Campbell, G.R., Atherosclerosis, 40 (1981) 347.
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15 Ali, A.E., Barrett, J.C. and Eling, T.E., Prostaglandin and thromboxane production by fibroblasts and vascular endothelial cells, Prostaglandins, 20 (1980) 667. 16 Smith, W.L., De Witt, D.L. and Day, J.S., Quantitation and localisation of PGH, and PGI, synthetase activities in smooth muscle and endothelial cells. In: Vth Int. Conf. Prostaglandins, Florence, 18-21 May 1982, p. 229 (Abstr.). I7 Larrue, J., Rigaud, M., Razaka, G., Daret, D., Demond-Hem+, J. and Bricaud, H., Formation of monohydroxyeicosatetraenoic acids from arachidonic acid by cultured rabbit aortic smooth muscle cells, B&hem. Biophys. Res. Commun., 112 (1983) 242. 18 Salmon, J.A., Smith, D.R., Flower, R.J., Moncada, S. and Vane, J.R., Further studies on the enzymatic conversion of prostaglandin endoperoxides into prostacyclin by porcine aorta microsomes, Biochim. Biophys. Acta, 523 (1978) 250. 19 Cornwell, D.G., Huttner, J.J., Mile, G.E., Panganamala, R.V., Sharma, H.M. and Geer, J.C., Polyunsaturated fatty acids, vitamin E and the proliferation of aortic smooth muscle cells, Lipids, 14 (1979) 104. 20 Stemerman, M.B., Spaet, T.H., Pitlick, F.A., Cintron, J.R., Lejnicks, I. and Tiell, M.L., Intimal healing - The pattern of re-endothelialization and intimal thickening, Amer. J. Path., 87 (1977) 125. 21 Goldberg, I.D., Stemerman, M.B., Ransil, B.J. and Fuhro, R.L., In vivo aortic muscle cell growth kinetics, Circ. Res., 47 (1980) 182. 22 Elder, A., Falcone, D.J., Hajjar, D.P., Minick, C.R. and Weksler, B.B., Diet induced hypercholesterolemia inhibits the recovery of prostacyclin production by injured rabbit aorta, Amer. J. Path., 107 (1982) 186. 23 Clyman, R.I., Mauray, F., Koerper, M.A., Wiemer, F., Heyman, M.A. and Rudolph, A.M., Formation of prostacyclin (PGI,) by the ductus arteriosus of fetal lambs at different stages of gestation, Prostaglandins, 16 (1978) 633. 24 Hajjar, D.P., Weksler, B.B., Falcone, D.J., Hefton, J.M., Tack-Goldman, K. and Minick, C.R., Prostacyclin modulates cholesteryl ester hydrolytic activity by its effects on CAMP in rabbit aorta, J. Chn. Invest., 70 (1982) 479. 25 Larrue, J., Dorian, B., Braquet, P. and Bricaud, H., Regulation of smooth muscle cell cyclic nucleotide metabolism by prostacyclin, 5th Int. Conf. Cyclic Nucleotides and Protein Phosphorylation, Milan, 27 June-l July 1983, p. 120 (Abstr.). 26 Numano, F., Cyclic nucleotides and atherosclerosis. In: A.M. Gotto, Jr., L.C. Smith and B. Allen (Eds.), Atherosclerosis V, Springer-Verlag, New York, 1980, p. 537. 27 Stout, R.W., Cyclic AMP - A potent inhibitor of DNA synthesis in cultured arterial endothelial and smooth muscle cells, Diabetologia, 22 (1982) 51. 28 Longenecker, J.P., Kilty, L.A. and Johnson, L.K., Glucocorticoid influence on growth of vascular wall cells in culture, J. Cell Physiol., 113 (1982) 197. 29 Bayer, B.M., Kruth, H.S., Vaughan, M. and Beaven, M.A., Arrest of cultured cells in the G, phase of cell cycle by indomethacin, J. Pharm. Exp. Therap., 210 (1979) 106.