Eur. J. Biochem. 206, 367-372 (1992) 0FEBS 1992
Effects of angiotensins on cellular hypertrophy and c-fos expression in cultured arterial smooth muscle cells Dominique MILLET ’, Claude DESGRANGES’, Michel CAMPAN ’, Alain-Pierre GADEAU’ and Olivier COSTEROUSSE’ *
Unitit 8 de Cardiologie de 1’Institut National de la Sante et de la Recherche Medicale, Avenue du Haut-LevCque, Pessac, France Unite 36 de Pathologie Vasculaire et Endocrinologie Renale de 1’Institut National de la Santk et de la Recherche Medicale, Paris, France
(Received February 19, 1992) - EJB 92 0234
An increase in cell size and protein content was observed when quiescent arterial smooth muscle cells in culture were incubated with either angiotensin I1 or 111. These effects were inhibited by the specific angiotensin type-1 receptor antagonist losartan (DuP 753) but not by CGP 42112A. In parallel, a transient and dose-dependent induction of czfos was demonstrated not only with angiotensins I1 and I11 but also with angiotensin I. Both angiotensins I1 and 111 exerted their maximal effect at 1 pM, while angiotensin I needed a tenfold-higher concentration to exert an identical effect. As for hypertrophy, losartan also inhibits angiotensin-induced c-fos expression, suggesting that this gene may be involved into the hypertrophic process. Angiotensin-I-mediated c-jos induction is partially inhibited by the angiotensin-converting enzyme inhibitors captopril and trandolaprilate; given that an angiotensin-converting enzyme activity was detected in these smooth muscle cell cultures, these results suggest that angiotensin-I-induced c-fos expression is mediated in part via angiotensin-I conversion to angiotensin 11, but also by other unidentified pathway(s). Angiotensin I could essentially induce smooth muscle cell hypertrophy by indirect mechanisms, while angiotensins I1 and 111 act directly on smooth muscle cells.
The effect of hypertension on the arterial wall is characterized by an increase in smooth muscle mass due to cellular hypertrophy and/or hyperplasia (for review, see [l]). In chronic hypertension models such as spontaneously hypertensive rats (SHR) or two-kidney one-clip Goldblatt hypertensive rats, the increased mass of smooth muscle cells (SMC) in aortas is due principally to SMC hypertrophy [2-41. In contrast, an increase in medial SMC number, resulting from their proliferation, is accounted for by the increase in smooth muscle mass in models of acute severe hypertension such as aortic coarctation [5, 61. Factors inducing in vivo arterial SMC hypertrophy are not yet well defined. However, some reports suggest that the vasoconstrictor peptide angiotensin (Ang) I1 may play a role in this induction. Indeed, administration of angiotensin-converting enzyme (ACE) inhibitors decreases SMC hypertrophy in the aorta of SHR during the development of hypertension. As this effect is not entirely mediated by reduction in blood pressure, it has been postulated that local angiotensin may participate in the development of SMC hypertrophy [7, 81. In vitro data corroborate the role of AngII in SMC hypertrophy, since AngII induces an increase in cell volume and protein content in quiescent aortic SMC in culture [9 - 111. In addition Correspondence to C. Desgranges, Unite 8 de Cardiologie de 1’Institut National de la Santk et de la Recherche Mitdicale, Avenue du Haut-Ltvkque, F-33600 Pessac, France Abbreviations. SHR, spontaneously hypertensive rats; SMC, smooth muscle cells; Ang, angiotensin; ACE, angiotensin-converting enzyme; DMEM, Dulbecco’s modified Eagle’s medium; AT, angiotensin-I1 receptor; Sar, sarcosine. Enzyme. Angiotensin-converting enzyme (EC 3.4.15.1).
to this trophic effect, AngII induces the expression of some protooncogenes such as c-fos, c-myc and c-jun in quiescent SMC, without inducing cell proliferation [12- 151; this suggests that these protooncogenes, which are generally involved in the proliferative process [16], could also accompany the hypertrophic process. Moreover, it has previously been demonstrated that c-myc induction accompanies hypertrophy induction in non-proliferating cardiac myocytes [17]. Several reports suggest that AngII may be locally produced. Indeed, the presence of various components of the renin/angiotensin system in vascular tissue has been reported : Ang is produced at the vascular level [18 -201 ; AngI can result from the action of locally produced renin (for a review, see [20]); ACE is found at different levels in blood vessels, where it may convert inactive AngI to the potent vasoconstrictor AngII. ACE is widespread on endothelial cells [21], but some data suggest that it may also exist in SMC. Indeed, the presence of ACE has been directly or indirectly demonstrated in arterial tissues, even after endothelial removal [22 - 241 or in SMC in culture [25 - 271. Moreover, AngI, AngII and AngIII are generated and excreted by vascular tissue, since the peptides have been detected in perfusates of blood vessels [28]. Of these three angiotensins, AngII is not the only one capable of producing vasoactive effects. AngIII exerts vasopressive or vasoconstrictive effects on the arterial system [29, 301, and several studies have demonstrated that it can bind to Angreceptor (AT) subtypes of cultured arterial SMC [31- 341. AngI is also able to induce contraction of aortic rings, even in the absence of endothelium [22, 24, 271, and to increase intracellular free-calcium concentrations [27], but these effects are essentially dependent on its enzymic conversion to AngII.
368 Since AngI, AngII and AngIII may be generated at the vascular level, and may have various effects on the vascular wall or its SMC, we have studied the role of these peptides on the induction of SMC hypertrophy and the related c-fos expression in a cell-culture system. This study demonstrates that AngI-induced c-fos expression is mostly mediated via its conversion to AngII and that AngIII, the degradation product of AngII, induces c-fos expression and SMC hypertrophy in vitro to the same degree as AngII after linkage to ATI. MATERIALS AND METHODS
dehyde, 1.7% SDS, 0.04 M Mops, pH 7, 0.01 M sodium acetate and 1 mM EDTA were added. This mixture was incubated for 15 min at 65 “C and centrifuged. The supernatant containing total RNA resulting from 106 cells was size-fractionated by electrophoresis on 1YOagarose gel according to Manniatis et al. [38]. RNA were transferred on nylon membrane by pressure and hybridized with 32P-labelled c-fos probe. This probe was the 1.3-kb BglII-PvuII fragment of the v-fos sequence of the murine osteosarcoma virus [39] and was labelled with [ L X - ~ ~ P I ~by C Trandom P oligomer priming. Hybridization was revealed by autoradiography using Kodak X-OMAT AR films.
Cell culture SMC were isolated from thoracic aorta of Wistar-Kyoto rats by enzymic dissociation and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, as previously described [35]. At post-confluence, secondary cultures were obtained after harvesting the cells with 0.5 g/1 trypsin and 0.2 g/1 EDTA (trypsin/EDTA) and reseeding in fresh DMEM containing 10% fetal calf serum. The cells were subcultured regularly and the medium was changed every two days. Cells at passages 5 - 10 were used in this study. These secondary cultures were exclusively constituted with SMC [35]. Unless otherwise stated, cells were plated preconfluence (3-5x lo4 cells/cm2 in a 25-cm2 flask) in DMEM supplemented with 10% fetal calf serum for 3 h then made quiescent by incubation in 5 ml serum-free DMEM for 72 h. Cell volume determination Quiescent SMC were incubated for different times in serum-free medium with or without 1 pM AngII or 1 pM AngIII. Media were changed every day. At each time, SMC were harvested with trypsin/EDTA and cell volume analysis was immediately performed with a cell counter-channelyser ZM (Coultronics) equipped with a 140-pm aperture orifice tube for determination of 200 - 3850 fl. Modal and mean volumes were automatically determined. Determination of protein and DNA Cell monolayers were washed three times with Dulbecco’s phosphate-buffered saline, then lysed by 0.5 M sodium hydroxide for 1 h at 37°C. One part of the lysate was used for protein determination using the Bio-Rad (Richmond, CA) assay with bovine serum albumin as standard protein. The remaining part was treated with 1 M perchloric acid for 30 min at +4”C, then centrifuged for 15 min at 12000 g; the DNA contained in the centrifugation pellet was hydrolysed by a 20-min incubation at 80°C in 1 M perchloric acid and DNA concentration was determined by the colorimetric method of Burton [36].
ACE activity determination ACE catalytic activity was determined by hydrolysis of the synthetic substrate Bz-Gly-His-Leu (Bz, benzoyl) according to Cushman and Cheung [40]. Acellular extracts (corresponding to 1.5 x lo6 cells), prepared by sonication of quiescent SMC membranes in Dulbecco’s phosphate-buffered saline containing 8 mM Chaps, were incubated at 37 “C with 5 mM substrate in 100 mM potassium phosphate, pH 8.3, 300 mM sodium chloride and 10 pM zinc sulfate (250 pl final volume) for 2 - 5 h. The amount of hippuric acid liberated from the substrate was then determined by HPLC [41]. 1 U activity was defined as the amount of enzyme catalyzing the release of 1 pmol hippuric acid/min from the substrate. Determination of total RNA content by flow cytofluorimetry Cells were stained by acridine orange and analysed in an ATC 3000 (ODAM) as previously reported [35]. Products Serum and culture medium were obtained from GibcoBRL Life Technologies (Uxbridge, UK); NA-agarose from Pharmacia (Uppsala, Sweden);cycloheximide, Ang and [Sarl, Ile8lAngII (Sar, sarcosine) from Sigma Chemicals Co. (St Louis, MO); captopril from Squibb Laboratories (Princeton, NJ); trandolaprilate from Roussel-Uclaf (Romainville, France); [w3’P]dCTP (3000 Ci/mmol), nylon membrane Hybond N + and the multiprime DNA-labelling kit from Amersham (Little Chalfont, UK). Losartan (DuP 753) and CGP 42112A were generous gifts from Dr M. de Gasparo from Ciba-Geigy (Basel, Switzerland). Statistics Statistical data are means k SD and were analysed by Student’s t-test; P < 0.05 was considered significant. RESULTS Effect of Ang on arterial SMC hypertrophy
RNA analysis Quiescent SMC were incubated in serum-free DMEM (lo6 cells/5 ml medium in a 25-cm2 flask) with Ang, as described in each figure. The cells were harvested with trypsin/EDTA and washed with cold Dulbecco’s phosphate-buffered saline. Total RNA was prepared according to the method of Hatch and Bonner [37]. A cell pellet containing lo6 cells was resuspended in 10 pl Dulbecco’s phosphate-buffered saline containing 2% bentonite, then 3 vol. 46% formamide, 6.3% formal-
As shown in Fig. 1, a 24-h treatment of quiescent SMC by 1 pM AngII induced a 27% increase in modal cell volume. In the continuous presence of AngII, the modal cell volume continued to increase regularly to 160% of that of unstimulated cells after 4 days. Mean cell volume increased in the same way (Table 1). A similar increase was found when quiescent SMC were submitted to 1 pM AngIII (Fig. 1 and Table 1). This hypertrophic effect was concentration dependent. Modal cell volumes were increased, on the third day of
369 2200
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Table 2. Effect of AngII and AngIII on protein and DNA content of arterial SMC. SMC were seeded at 0.5 x lo6 in 35 mm diameter wells and made quiescent by serum deprivation. SMC were then treated daily for 2 days with 1 pM AngII or 1 pM AngIII, and both cellular protein and DNA content were determined as described in Materials and Methods. Values are means k SD (n = 3). Treatment
Protein
DNA
Protein/DNA
Control AngII AngIII 10% FCS
mg/well 0.10 k 0.02 0.20 0.01 a 0.21 f 0.02" 0.36 0.01 a
pg/welI 3.2 k 0.2 2.9 5 0.2 3.2 5 0.1 8.8 k 0.4"
Pg/N 31 69 a 66a 41 a
1600
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0
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a Statistically significant difference (P< 0.05) between control and fetal calf serum (FCS)-treated or angiotensin-treated cells.
Days
Fig. 1. Effect of AngII and AngIII on SMC volume. SMC were seeded at 0.8 x lo6 cells/25-cmz flask and made quiescent by serum deprivation. Cultures were then treated daily with DMEM only (A),1 pM AngII (0)or 1 pM AngIII (m), and cell volume was determined at the indicated times with a Coulter counter-channelyzer. Each individual point on this figure represents the modal value of cell volume determined with a total number of 5 x lo5 cells. Table 1. Effects on AngII and AngIII on SMC volume. SMC were seeded at 0.8 x lo6 cells/25-cm2flask, made quiescent by serum deprivation then treated daily with DMEM with or without 1 pM AngII or 1 pM AngIII. Values of minimal, modal, mean and maximal cell volumes were determined at days 1 and 4 of experiment from data obtained with a Coulter counter-channelyzer. Incubation time
Sample
SMC volume RNA content
minimal modal day
mean
maximal
fl
1
control AngII AngIII
534 570 606
1212 1548 1594
1361 1618 2994
2567 2594 3065
4
control AngII AngIII
571 749 713
1323 2103 2103
1634 >2345 >2345
3146 $3850 $3850
treatment, by 9%, 26% and 49% for 10 nM, 0.1 pM and 1 pM Ang, respectively; such an effect was not detectable for lower concentrations. This increase in cell volume was probably due to an increase in protein content in cells exposed to either AngII or AngIII. Indeed, as shown in Table 2, the DNA content did not significantly differ between control and AngIIor AngIII-stimulated cells, while the protein content of the stimulated cells increased significantly. The protein/DNA ratio of the AngII- or AngIII-stimulated cells was significantly higher t h a n that of control cells, demonstrating protein accumulation during SMC treatment with the Ang. Cytofluorimetric determinations demonstrated that both AngII and AngIII significantly increased total RNA content (Fig. 2) without modification of DNA content. This increase in cell volume was entirely inhibited when the cells were treated with 10 pM [Sarl, Ile81AngI1, a non-specific AT antagonist, 10 min before AngII or AngIII addition (Table 3). Moreover, this hypertrophic process was dose-dependently inhibited by losartan, a specific AT1 inhibitor [42]; in contrast, CGP 42112A, a specific antagonist of AT2 [34], did not induce any inhibitory effect (Table 4).
(arbitrary
units)
Fig. 2. Effect of AngII and AngIII on total RNA content of quiescent SMC. Quiescent SMC were obtained as described in Fig. 1 and treated daily with 1 pM AngII or 1 pM AngIII. On day 3, total RNA was determined by flow cytofluonmetry, as described in Materials and Methods.
Table 3. Inhibitory effects of [Sarl, Ile8jAngII on AngII- and AngIIImediated increase in cell volume of arterial SMC. Quiescent SMC were obtained under the same conditions as those given in Fig. 1, and modal cell volume was determined after 3 days of a daily treatment with angiotensin and/or [Sarl, Ile81AngII. Each day, cells were fed with fresh culture medium containing the inhibitor and supplemented 10 min later with AngII or AngIII. Treatment
SMC volume control
1 pM AngII
1 pM AngIII
1788 1296
1312
fl
None 1329 [Sarl,Ile8]AngII 1302
1755
Effects of Ang on c-fos induction As previously described [13, 141, AngII induced a rapid and transient expression of c-fos mRNA in quiescent SMC, which peaked around 30 min after stimulation then decreased to basal level after 2 h (Fig. 3A). This induction was observed over the range 0.001 - 10 pM AngII; the maximum response occurring for 1 pM AngII remained lower than that obtained after serum stimulation (Fig. 3B). This AngII-increase in c-fos
3 70
@
Table 4. Inhibitory effects of losartan and CGP 42112A on AngII- and Anglll-mediated increase in cell volume of arterial SMC. Quiescent SMC were obtained in the same conditions as in Fig. 1, and modal cell volume was determined after 3 days of a daily treatment with angiotensin and/or losartan and CGP 42112A. Each day, cells were fed with fresh culture medium containing one of these inhibitors and supplemented 10 min later with AngII or AngIII. n.d., not determined. Treatment
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15
30
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60 90 120
15
45 60 90 120
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fl None 1 pM CGP 42112A 10 pM losartan 1 nM losartan 0.1 pM losartan
1312 1329 1412 n.d. n.d.
1722 1722 1312 1378 1525
Fig. 4. Comparativeeffects of Angl, AngII and AngIII on c-fos induction in quiescent SMC in culture. (A) Dose effect. Quiescent SMC were treated by 1 pM AngI (l), 10 pM AngI (2), 1 pM AngII (3), I pM AngIII (4) or 10 pM AngIII ( 5 ) for 30 min, then processed as described in Fig. 3 for c-fos mRNA detection. (0)Control cells in DMEM; (S) 30-min-I 0%-fetal-calf-serum-stimulated cells. Autoradiography was exposed for 2 days. (B) Kinetics. Quiescent SMC of the same batch ofcells were incubated with 10 pM AngI, 1 pM AngII or 1 pM AngIII for different times. c-fos mRNA detection was determined after total RNA extraction. With this batch of cells, maximal c-fos induction occurred 15 min after cell stimulation. The autoradiograph was exposed for 3 days.
1722 1575 1411 1296 1443
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Fig. 3. c-fos induction by AngII in quiescent SMC in culture. (A) Time course of c-fos mRNA induction. Quiescent SMC were treated for the indicated times with 1 pM AngII in DMEM. Total RNA from lo6 cells was analyzed by Northern blotting and hybridized with a 32P-labelledc-jos probe, as described in Materials and Methods. The autoradiograph was exposed for 2 days. (B) Dose/response of the effect of AngII on c-fos expression. Quiescent SMC were exposed for 30 min to various concentrations of AngIl: (1) 1 nM, (2) 10 nM, (3) 0.1 pM, (4) 1 pM, (5) 10 pM or (S) 10% fetal calf serum in DMEM; (C) DMEM alone. The autoradiograph was exposed for 2 days. (C) Effect of protein synthesis inhibition on c-fos mRNA induction by AngII. Quiescent SMC were exposed for the indicated times with 1 pM AngII in the absence or presence of 1.5 pg/ml cycloheximide. At the end of each incubation, cells were harvested and processed as previously described for clfos mRNA evaluation. The autoradiograph was exposed for 2 days.
mRNA was not dependent on protein synthesis, since cycloheximide did not produce any inhibition of c-fos expression, but, in contrast, induced an overproduction and probably a stabilization of the c-fos mRNA, which remained detectable 2 h after stimulation (Fig. 3C). In the same conditions, cycloheximide alone did not induce any e-fos expression in quiescent SMC (not shown).
As shown in Fig. 4, c-fos expression was also induced by AngIII and AngI. Like AngII, 1 pM AngIII induced maximal expression of c-fos mRNA. In contrast, 10 pM AngI was needed to induce a c-fos expression equivalent to that produced by 1 pM AngII (Fig. 4A). c--0s induction occurred with the same kinetics after stimulation by AngI, AngIT or AngIII (Fig. 4B). The AngII induction of c-fos mRNA was blocked by [Sarl , IleSIAngII, a non-specific AT antagonist (Fig. 5). Induction of c-jos expression by AngI and AngIII was also inhibited by this inhibitor (Fig. 5B and A). In contrast, [Sarl, lleS]AngII did not inhibit serum-induced expression of c-fos mRNA (not shown). Losartan, a selective antagonist of AT1, dose-dependently inhibited c-fos expression induced by either AngIl or AngIII. In contrast, these inductions were not inhibited by CGP 42112A, a specific inhibitor of AT2 (Fig. 5C and D). Addition of 10 pM amastatin and 10 pM bestatin, two inhibitors of aminopeptidases, to the culture medium before AngIl addition, did not inhibit or increase c-fos induction by AngII (not shown), demonstrating that this induction was not due to AngII conversion. 1 pM captopril, an inhibitor of ACE, inhibits AngI-induced expression by 75% (Fig. 6A). Higher captopril concentrations (10 pM and 100 pM) did not significantly increase this inhibition (Fig. 6B). AngI-induced c-fos expression was also partially inhibited by trandolaprilate (Fig. 6B), another ACE inhibitor, suggesting that the AngI effect is, in part, mediated via its conversion to AngII by ACE. Under the same conditions, captopril ( 3 pM and 10 pM) and trandolaprilate (10 pM) did not induce significant modification of AngIImediated c-fos induction (Fig. 6A and B). Moreover, an ACElike activity (5.7 pU/cell) was demonstrated in the membrane fraction of quiescent SMC cultures maintained in serum-free medium. DISCUSSION The present study clearly demonstrates that AngIII induces both cellular hypertrophy and c:fos expression when
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Fig. 6. Influence of captopril and trandolaprilate on c-fbs induction by Angl in quiescent SMC in culture. After a 10-min incubation with captopril or trandokaprilate, 10 pM AngI or 1 pM AngII was added for 30 min to the culture medium of quiescent SMC. c-fos mRNA expression was compared to that of cells stimulated in the absence of captopril or trandolaprilate. (A) Lane 0, quiescent cells; 1, AngI; 2, AngII; 3, 10% fetal calf serum; 4, 1 pM captopril; 5, 1 phII captopril and AngI; 6, 1 pM captopril and AngII. (B) Lane 0, quiescent cells; I , 1 pM trandolaprilate and AngI; 2, 10 pM trandolaprilate and AngI; 3,O. 1 mM trandolaprilate and Angl; 4, Angl; 5 , l pM captopril and AngI; 6, 10 pM captopril and AngI; 7, 0.1 mM captopril and AngI; 8, 10 pM trandolaprilate and AngII; 9, AngII; 10, 10 pM captopril and AngII. Autoradiographs were exposed for 2 days.
6
7
8
Fig. 5. Inhibition of Angl, AngII or AngIII c-fos induction in quiescent SMC in culture by [Sarl, IleSjAngII (Sar', Ile'-AngII), losartan and CGP 42112A. Quiescent SMC were incubated for 10 min with 10 pM [Sarl, Ile8lAngII (A and B), 10 pM losartan or 10 pM CGP 42112A (C) before addition of 10 pM AngI, 1 pM AngII or 1 pM AngIII to the culture medium for 30 min. c-fos mRNA content was compared with cells incubated 30 min with AngI, AngI1, AngIII or serum ( S ) in the absence of any inhibitor. (D) SMC were incubated with various concentrations of losartan (0.1 pM, 1 pM, 10 pM, 0.1 mM; 1-4, respectively) or CGP 42112A (10 nM, 0.1 pM, 1 pM, 10 pM; 5-8, respectively), before addition of 1 pM AngII for 30 min. (0)Control cells; ,411, 30-min-1 pM-AngII-stimulated cells in the absence of any inhibitor. Autoradiographs were exposed for 3 days.
added to the culture medium of quiescent rat aortic SMC. The vasopressive effects of AngIII in vivo [29, 30, 431 and the induction of arterial-strip contraction by AngIII in vitro [44, 451 have previously been reported. More recently, Dostal et al. [46] described AngIII induction of a transient increase in cytosolic calcium identical to that obtained with AngII in cultured arterial SMC. All these effects are probably due to AngIII binding to a subtype of AT, since AngIII displaces AngII from its membrane receptors in cultured arterial SMC [31- 341. In our study, both hypertrophy and c-jos induction by AngIII were inhibited not only by [Sarl, Ile8lAngII but also by the specific AT1 antagonist losartan, suggesting that AngIII binds to the same subclass of AT as AngII. These results are in agreement with those describing the inhibition of vasopressive effects of AngIII by [Sarl, IleSIAnglI [43], although other studies suggest that AngIII causes vasoconstriction in peripheral arteries by acting on a subclass of AT different from those activated by AngII [29, 301. The parallel-
ism in induction of both hypertrophy and c-fos mRNA expression obtained after stimulation of quiescent SMC by AngIII or AngII, suggests that AngIII, which may be produced at the level of the arterial wall [25,28], could participate with AngII in SMC hypertrophy during the development of hypertension in vivo. Moreover, this study suggests that cTfos induction, which is inhibited at the same time as angiotensininduced hypertrophy, could be involved in this hypertrophic process. The present study also demonstrates a dose-dependent increase in c-jos expression after AngI stimulation of quiescent arterial SMC in culture. However, a tenfold-higher concentration is necessary to induce the same effect as that of Angll. This increase in c-fos mRNA by AngI is inhibited by [Sarl, Ile81AngI1, suggesting that AngI could bind to the same receptor as AngII, since it has previously been demonstrated that AngI can bind with a lower affinity to the same site as AngII on SMC from rat mesenteric arteries [31]. However, in the present study, partial inhibition of AngI-mediated c-jos induction when SMC were incubated beforehand with the ACE inhibitors captopril and trandolaprilate, and the presence of an ACE-like activity in these cells, suggest that the AngI effect is, in part, mediated via its conversion to AngII. The same kind of observation has recently been described in primary cultures of rat aortic SMC: AngI induction of transient cytosolic calcium increase is inhibited by captopril[27]. However, in this study, inhibition of AngI effects by ACE inhibitors was complete, while c-jos induction was only partially inhibited in our study, suggesting that AngI exerts its effects mostly via its degradation by an ACE-like enzyme (inhibited by ACE inhibitors) but also by other undefined mechanisms. Indeed, residual c--0s induction observed at a high concentration of ACE inhibitors suggests that AngI could be either converted to AngII by another enzyme not inhibited by ACE inhibitors [23, 471, converted to other angiotensin-derived biologically active peptides, such as Angl - 7 [48], which may be produced in SMC culture [49], or be active by itself. These different hypotheses must be verified by the use of specific protease inhibitors. Nevertheless, conversion of AngI to AngI1, which
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Mizuno, K., Nakamura, M., Higashimori, K. & Inagami, T. (1988) Hypertension (Dallas) 11, 223-229. 29. Carey, R. M., Peach, M. J., Vaughan, E. D. & Ayers, C. R. (1978) Circ. Res. 43, 63 - 68. 30. Tabrizchi, R. & Pang, C. C. Y. (1987) Eur. J. Pharmacol. 142, 359-366. 31. Gunther, S., Alexander, R. W., Atkinson, W. J. & Gimbrone, M. A. (1982) J. Cell Biol. 92, 289-298. 32. McQueen, J., Murray, G. D. & Semple, P. F. (1984) Biochem. J . This work was supported by INSERM grants, Squibb Laborato223,659-671. ries grants, EPR grants and fellowships from Squibb Laboratories 33. Chiu, A. T., McCall, D. E., Price, W. A., Wong, P. C.. Carini, D. (A.-P.G.) and from GRRC (M.C.). We thank Dr F. Alhenc-Gelas for J., Duncia, J. V., Wexler, R. R., Johnson, A. L., Taber, R. I. & helpful discussion in the preparation of manuscript. We wish to thank Timmermans, P. B. M. W. M. (1989) Hypertension (Dallas) F. Dupuch and I. Belloc for their excellent technical assistance and 14, 358. M. Faucher for her expert editorial work. 34. Whitebread, S., Mele, M., Kamber, B. & De Gasparo, M. (1989) Biochem. Biophys. Res. Commun. 163,284 - 291. REFERENCES 35. Desgranges, C., Campan, M., Gadeau, A. P., Guerineau, N., 1. Owens, G. K. (1989) Am. J . Physiol. 257, H1755-H1765. Mollard, P. & Razaka, G. (1991) Biochem. Pharmacol. 41, 2. Owens, G. K. & Schwartz, S. M. (1982) Circ. Res. 51, 280-289. 1045- 1054. 3. Owens, G. K. & Schwartz, S. M. (1983) Circ. Res. 53,491 -501. 36 Burton, K. (1956) Biochem. J . 62, 315-323. 4. Black, M. J., Adams, M. A., Bobik, A., Campbell, J. H. & 37. Hatch, C. L. & Bonner, W. M. (1987) Anal. Biochem. 162, 283Campbell, G. R. (1988) Clin. Exp. Pharmacol. Physiol. 15, 290. 345 - 348. 38. Manniatis, T., Fritsch, E. F. & Sambrook, J. (1982) in Molecular 5. Bevan, R. D., Van Marthens, E. V. & Bevan, J. A. (1976) Circ. cloning: a laboratory manual, Cold Spring Harbor Laboratory, Res. 38, 58-62. Cold Spring Harbor, NY. 6. Owens, G. K. & Reidy, M. A. (1985) Circ. Res. 57, 695-705. 39. Curran, T., Peters, G., Van Beveren, C., Teich, N. M. & Verma, 7. Freslon, J. L. & Giudicelli, J. F. (1983) Br. J. Pharmacol. 80, I. M. (1982) J . Virol. 44, 674-682. 533 - 543. 40. Cushman, D. W. & Cheung, H. S. (1971) Biochem. Pharmacol. 8. Owens, G. K. (1987) Hypertension (Dallas) 9, 178-187. 20, 1637-1648. 9. Berck, B. C., Vekshtein, V., Cordon, H. M. & Tsuda, T. (1989) 41. Wei, L., Alhenc-Gelas, F., Soubrier, F., Michaud, A., Corvol, Hypertension (Dallas) 13, 305 - 314. P. & Clauser, E. (1991) J. Biol. Chem. 266, 5540-5546. 10. Geisterfer, A. A. T., Peach, M. J. & Owens, G. K. (1988) Circ. 42. Chiu, A. T., McCall, D. E., Aldrich, P. E. & Timmermans, P. B. Res. 62, 749 -756. M. W. M. (1990) Biochem. Biophys. Res. Commun. 172,119511. Kohane, D. S., Sarzani, R., Schwartz, J. H., Chobanian, A. V. & 1202. Brecher, P. (1990) Am. J. Physiol. 258, H1699-H1705. 43. Turker, R. K. & Ercan, 2. S. (1978) Res. Commun. Chem. Pathol. 12. Naftilan, A. J., Pratt, R. E. & Dzau, V. J. (1989) J. Clin. Invest. Pharmacol. 21,15 -24. 83, 1419-1424. 44. Ackerly, J. A., Moore, A. F. & Peach, M. J. (1977) Proc. Nut1 13. Naftilan, A. J., Pratt, R. E., Eldridge, C. S., Lin, H. L. & Dzau, Acad. Sci. USA 74,5725 - 5728. V. J. (1989) Hypertension (Dallas) 13, 706-711. 14. Taubman, M. B., Berk, B. C., Izumo, S., Tsuda, T., Alexander, 45. Moore, G. J. & Scanlon, M. N. (1989) Gen. Pharmacol. 20,193 198. R. W. & Nadal-Ginard, B. (1989) J. Biol. Chem. 264, 52646. Dostal, D. E., Murahashi, T. & Peach, M. J. (1990) Hypertension 530. (Dallas) 15, 815-8822, 15 Naftilan, A. J., Gilliland, G. K., Eldridge, C. S. & Kraft, A. S. 47. Lanzillo, J. J., Dasarathy, Y., Stevens, J. & Fanburg, B. L. (1986) (1990) Mol. Cell. Biol. 10, 5536- 5540. Biochem. Biophys. Res. Comrnun. 134, 770 - 716. 16 Gadeau, A. P., Campan, M. & Desgranges, C. (1991) J. Cell. 48. Kohara, K., Brosnihan, K. B., Chappell, M. C., Khosla, M. C. & Physiol. 146, 356-361. Ferrario, C. M. (1991) Hypertension (Dallas) 17, 131 -138. 17 Starksen, N. F., Simpson, P. C., Bishopric, N., Coughlin, S. R., Lee, W. M. F., Escobedo, J. A. & Williams, L. T. (1986) Proc. 49. Chappell, M. C., Jaiswal, R., Brosnihan, K. B. & Ferrario, C. M. (1991) Hypertension (Dallas) 18, 385-386. Natl Acad. Sci. USA 83,8348 - 8350.
seems to be the major route for AngI c-fos induction in cultured aortic SMC, is confirmed by the demonstration of an ACE-like activity which, in spite of its low level in SMC cultures, may be sufficient to explain the local formation of an AngII level sufficient for maximal c-fos induction. The presence of binding sites for ACE inhibitors has also been described in human vascular SMC in culture [26].ACE-like activities have also been described both in dog aorta homogenates, after endothelium and adventitia removal 1231, and in cultured arterial SMC [25].Complementary studies are needed to verify if the ACE-like enzyme found in secondary SMC in culture is really produced by these cells or if it originates from the serum of the culture medium and remains adsorbed onto the SMC surface, despite several washes in serum-free medium. In conclusion, this s t d y strongly suggests that AngI may have an indirect hypertrophic effect on SMC, essentially after its local transformation into AngII or other peptides, although a direct effect cannot be totally excluded. This study also demonstrates that c-fos induction accompanies hypertrophy induced by either AngII or AngIII, and confirms the previous hypothesis that the expression of this oncogene is involved in the hypertrophic process. Moreover, the finding that AngIII induces both c-fos and hypertrophy at a level similar to AngII suggests that AngIII in vivo could have an action similar to AngII on medial thickening during the hypertensive process.
Effects of angiotensins on cellular hypertrophy and c-fos expression in cultured arterial smooth muscle cells Dominique MILLET ’, Claude DESGRANGES’, Michel CAMPAN ’, Alain-Pierre GADEAU’ and Olivier COSTEROUSSE’ *
Unitit 8 de Cardiologie de 1’Institut National de la Sante et de la Recherche Medicale, Avenue du Haut-LevCque, Pessac, France Unite 36 de Pathologie Vasculaire et Endocrinologie Renale de 1’Institut National de la Santk et de la Recherche Medicale, Paris, France
(Received February 19, 1992) - EJB 92 0234
An increase in cell size and protein content was observed when quiescent arterial smooth muscle cells in culture were incubated with either angiotensin I1 or 111. These effects were inhibited by the specific angiotensin type-1 receptor antagonist losartan (DuP 753) but not by CGP 42112A. In parallel, a transient and dose-dependent induction of czfos was demonstrated not only with angiotensins I1 and I11 but also with angiotensin I. Both angiotensins I1 and 111 exerted their maximal effect at 1 pM, while angiotensin I needed a tenfold-higher concentration to exert an identical effect. As for hypertrophy, losartan also inhibits angiotensin-induced c-fos expression, suggesting that this gene may be involved into the hypertrophic process. Angiotensin-I-mediated c-jos induction is partially inhibited by the angiotensin-converting enzyme inhibitors captopril and trandolaprilate; given that an angiotensin-converting enzyme activity was detected in these smooth muscle cell cultures, these results suggest that angiotensin-I-induced c-fos expression is mediated in part via angiotensin-I conversion to angiotensin 11, but also by other unidentified pathway(s). Angiotensin I could essentially induce smooth muscle cell hypertrophy by indirect mechanisms, while angiotensins I1 and 111 act directly on smooth muscle cells.
The effect of hypertension on the arterial wall is characterized by an increase in smooth muscle mass due to cellular hypertrophy and/or hyperplasia (for review, see [l]). In chronic hypertension models such as spontaneously hypertensive rats (SHR) or two-kidney one-clip Goldblatt hypertensive rats, the increased mass of smooth muscle cells (SMC) in aortas is due principally to SMC hypertrophy [2-41. In contrast, an increase in medial SMC number, resulting from their proliferation, is accounted for by the increase in smooth muscle mass in models of acute severe hypertension such as aortic coarctation [5, 61. Factors inducing in vivo arterial SMC hypertrophy are not yet well defined. However, some reports suggest that the vasoconstrictor peptide angiotensin (Ang) I1 may play a role in this induction. Indeed, administration of angiotensin-converting enzyme (ACE) inhibitors decreases SMC hypertrophy in the aorta of SHR during the development of hypertension. As this effect is not entirely mediated by reduction in blood pressure, it has been postulated that local angiotensin may participate in the development of SMC hypertrophy [7, 81. In vitro data corroborate the role of AngII in SMC hypertrophy, since AngII induces an increase in cell volume and protein content in quiescent aortic SMC in culture [9 - 111. In addition Correspondence to C. Desgranges, Unite 8 de Cardiologie de 1’Institut National de la Santk et de la Recherche Mitdicale, Avenue du Haut-Ltvkque, F-33600 Pessac, France Abbreviations. SHR, spontaneously hypertensive rats; SMC, smooth muscle cells; Ang, angiotensin; ACE, angiotensin-converting enzyme; DMEM, Dulbecco’s modified Eagle’s medium; AT, angiotensin-I1 receptor; Sar, sarcosine. Enzyme. Angiotensin-converting enzyme (EC 3.4.15.1).
to this trophic effect, AngII induces the expression of some protooncogenes such as c-fos, c-myc and c-jun in quiescent SMC, without inducing cell proliferation [12- 151; this suggests that these protooncogenes, which are generally involved in the proliferative process [16], could also accompany the hypertrophic process. Moreover, it has previously been demonstrated that c-myc induction accompanies hypertrophy induction in non-proliferating cardiac myocytes [17]. Several reports suggest that AngII may be locally produced. Indeed, the presence of various components of the renin/angiotensin system in vascular tissue has been reported : Ang is produced at the vascular level [18 -201 ; AngI can result from the action of locally produced renin (for a review, see [20]); ACE is found at different levels in blood vessels, where it may convert inactive AngI to the potent vasoconstrictor AngII. ACE is widespread on endothelial cells [21], but some data suggest that it may also exist in SMC. Indeed, the presence of ACE has been directly or indirectly demonstrated in arterial tissues, even after endothelial removal [22 - 241 or in SMC in culture [25 - 271. Moreover, AngI, AngII and AngIII are generated and excreted by vascular tissue, since the peptides have been detected in perfusates of blood vessels [28]. Of these three angiotensins, AngII is not the only one capable of producing vasoactive effects. AngIII exerts vasopressive or vasoconstrictive effects on the arterial system [29, 301, and several studies have demonstrated that it can bind to Angreceptor (AT) subtypes of cultured arterial SMC [31- 341. AngI is also able to induce contraction of aortic rings, even in the absence of endothelium [22, 24, 271, and to increase intracellular free-calcium concentrations [27], but these effects are essentially dependent on its enzymic conversion to AngII.
368 Since AngI, AngII and AngIII may be generated at the vascular level, and may have various effects on the vascular wall or its SMC, we have studied the role of these peptides on the induction of SMC hypertrophy and the related c-fos expression in a cell-culture system. This study demonstrates that AngI-induced c-fos expression is mostly mediated via its conversion to AngII and that AngIII, the degradation product of AngII, induces c-fos expression and SMC hypertrophy in vitro to the same degree as AngII after linkage to ATI. MATERIALS AND METHODS
dehyde, 1.7% SDS, 0.04 M Mops, pH 7, 0.01 M sodium acetate and 1 mM EDTA were added. This mixture was incubated for 15 min at 65 “C and centrifuged. The supernatant containing total RNA resulting from 106 cells was size-fractionated by electrophoresis on 1YOagarose gel according to Manniatis et al. [38]. RNA were transferred on nylon membrane by pressure and hybridized with 32P-labelled c-fos probe. This probe was the 1.3-kb BglII-PvuII fragment of the v-fos sequence of the murine osteosarcoma virus [39] and was labelled with [ L X - ~ ~ P I ~by C Trandom P oligomer priming. Hybridization was revealed by autoradiography using Kodak X-OMAT AR films.
Cell culture SMC were isolated from thoracic aorta of Wistar-Kyoto rats by enzymic dissociation and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, as previously described [35]. At post-confluence, secondary cultures were obtained after harvesting the cells with 0.5 g/1 trypsin and 0.2 g/1 EDTA (trypsin/EDTA) and reseeding in fresh DMEM containing 10% fetal calf serum. The cells were subcultured regularly and the medium was changed every two days. Cells at passages 5 - 10 were used in this study. These secondary cultures were exclusively constituted with SMC [35]. Unless otherwise stated, cells were plated preconfluence (3-5x lo4 cells/cm2 in a 25-cm2 flask) in DMEM supplemented with 10% fetal calf serum for 3 h then made quiescent by incubation in 5 ml serum-free DMEM for 72 h. Cell volume determination Quiescent SMC were incubated for different times in serum-free medium with or without 1 pM AngII or 1 pM AngIII. Media were changed every day. At each time, SMC were harvested with trypsin/EDTA and cell volume analysis was immediately performed with a cell counter-channelyser ZM (Coultronics) equipped with a 140-pm aperture orifice tube for determination of 200 - 3850 fl. Modal and mean volumes were automatically determined. Determination of protein and DNA Cell monolayers were washed three times with Dulbecco’s phosphate-buffered saline, then lysed by 0.5 M sodium hydroxide for 1 h at 37°C. One part of the lysate was used for protein determination using the Bio-Rad (Richmond, CA) assay with bovine serum albumin as standard protein. The remaining part was treated with 1 M perchloric acid for 30 min at +4”C, then centrifuged for 15 min at 12000 g; the DNA contained in the centrifugation pellet was hydrolysed by a 20-min incubation at 80°C in 1 M perchloric acid and DNA concentration was determined by the colorimetric method of Burton [36].
ACE activity determination ACE catalytic activity was determined by hydrolysis of the synthetic substrate Bz-Gly-His-Leu (Bz, benzoyl) according to Cushman and Cheung [40]. Acellular extracts (corresponding to 1.5 x lo6 cells), prepared by sonication of quiescent SMC membranes in Dulbecco’s phosphate-buffered saline containing 8 mM Chaps, were incubated at 37 “C with 5 mM substrate in 100 mM potassium phosphate, pH 8.3, 300 mM sodium chloride and 10 pM zinc sulfate (250 pl final volume) for 2 - 5 h. The amount of hippuric acid liberated from the substrate was then determined by HPLC [41]. 1 U activity was defined as the amount of enzyme catalyzing the release of 1 pmol hippuric acid/min from the substrate. Determination of total RNA content by flow cytofluorimetry Cells were stained by acridine orange and analysed in an ATC 3000 (ODAM) as previously reported [35]. Products Serum and culture medium were obtained from GibcoBRL Life Technologies (Uxbridge, UK); NA-agarose from Pharmacia (Uppsala, Sweden);cycloheximide, Ang and [Sarl, Ile8lAngII (Sar, sarcosine) from Sigma Chemicals Co. (St Louis, MO); captopril from Squibb Laboratories (Princeton, NJ); trandolaprilate from Roussel-Uclaf (Romainville, France); [w3’P]dCTP (3000 Ci/mmol), nylon membrane Hybond N + and the multiprime DNA-labelling kit from Amersham (Little Chalfont, UK). Losartan (DuP 753) and CGP 42112A were generous gifts from Dr M. de Gasparo from Ciba-Geigy (Basel, Switzerland). Statistics Statistical data are means k SD and were analysed by Student’s t-test; P < 0.05 was considered significant. RESULTS Effect of Ang on arterial SMC hypertrophy
RNA analysis Quiescent SMC were incubated in serum-free DMEM (lo6 cells/5 ml medium in a 25-cm2 flask) with Ang, as described in each figure. The cells were harvested with trypsin/EDTA and washed with cold Dulbecco’s phosphate-buffered saline. Total RNA was prepared according to the method of Hatch and Bonner [37]. A cell pellet containing lo6 cells was resuspended in 10 pl Dulbecco’s phosphate-buffered saline containing 2% bentonite, then 3 vol. 46% formamide, 6.3% formal-
As shown in Fig. 1, a 24-h treatment of quiescent SMC by 1 pM AngII induced a 27% increase in modal cell volume. In the continuous presence of AngII, the modal cell volume continued to increase regularly to 160% of that of unstimulated cells after 4 days. Mean cell volume increased in the same way (Table 1). A similar increase was found when quiescent SMC were submitted to 1 pM AngIII (Fig. 1 and Table 1). This hypertrophic effect was concentration dependent. Modal cell volumes were increased, on the third day of
369 2200
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Table 2. Effect of AngII and AngIII on protein and DNA content of arterial SMC. SMC were seeded at 0.5 x lo6 in 35 mm diameter wells and made quiescent by serum deprivation. SMC were then treated daily for 2 days with 1 pM AngII or 1 pM AngIII, and both cellular protein and DNA content were determined as described in Materials and Methods. Values are means k SD (n = 3). Treatment
Protein
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Control AngII AngIII 10% FCS
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Fig. 1. Effect of AngII and AngIII on SMC volume. SMC were seeded at 0.8 x lo6 cells/25-cmz flask and made quiescent by serum deprivation. Cultures were then treated daily with DMEM only (A),1 pM AngII (0)or 1 pM AngIII (m), and cell volume was determined at the indicated times with a Coulter counter-channelyzer. Each individual point on this figure represents the modal value of cell volume determined with a total number of 5 x lo5 cells. Table 1. Effects on AngII and AngIII on SMC volume. SMC were seeded at 0.8 x lo6 cells/25-cm2flask, made quiescent by serum deprivation then treated daily with DMEM with or without 1 pM AngII or 1 pM AngIII. Values of minimal, modal, mean and maximal cell volumes were determined at days 1 and 4 of experiment from data obtained with a Coulter counter-channelyzer. Incubation time
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treatment, by 9%, 26% and 49% for 10 nM, 0.1 pM and 1 pM Ang, respectively; such an effect was not detectable for lower concentrations. This increase in cell volume was probably due to an increase in protein content in cells exposed to either AngII or AngIII. Indeed, as shown in Table 2, the DNA content did not significantly differ between control and AngIIor AngIII-stimulated cells, while the protein content of the stimulated cells increased significantly. The protein/DNA ratio of the AngII- or AngIII-stimulated cells was significantly higher t h a n that of control cells, demonstrating protein accumulation during SMC treatment with the Ang. Cytofluorimetric determinations demonstrated that both AngII and AngIII significantly increased total RNA content (Fig. 2) without modification of DNA content. This increase in cell volume was entirely inhibited when the cells were treated with 10 pM [Sarl, Ile81AngI1, a non-specific AT antagonist, 10 min before AngII or AngIII addition (Table 3). Moreover, this hypertrophic process was dose-dependently inhibited by losartan, a specific AT1 inhibitor [42]; in contrast, CGP 42112A, a specific antagonist of AT2 [34], did not induce any inhibitory effect (Table 4).
(arbitrary
units)
Fig. 2. Effect of AngII and AngIII on total RNA content of quiescent SMC. Quiescent SMC were obtained as described in Fig. 1 and treated daily with 1 pM AngII or 1 pM AngIII. On day 3, total RNA was determined by flow cytofluonmetry, as described in Materials and Methods.
Table 3. Inhibitory effects of [Sarl, Ile8jAngII on AngII- and AngIIImediated increase in cell volume of arterial SMC. Quiescent SMC were obtained under the same conditions as those given in Fig. 1, and modal cell volume was determined after 3 days of a daily treatment with angiotensin and/or [Sarl, Ile81AngII. Each day, cells were fed with fresh culture medium containing the inhibitor and supplemented 10 min later with AngII or AngIII. Treatment
SMC volume control
1 pM AngII
1 pM AngIII
1788 1296
1312
fl
None 1329 [Sarl,Ile8]AngII 1302
1755
Effects of Ang on c-fos induction As previously described [13, 141, AngII induced a rapid and transient expression of c-fos mRNA in quiescent SMC, which peaked around 30 min after stimulation then decreased to basal level after 2 h (Fig. 3A). This induction was observed over the range 0.001 - 10 pM AngII; the maximum response occurring for 1 pM AngII remained lower than that obtained after serum stimulation (Fig. 3B). This AngII-increase in c-fos
3 70
@
Table 4. Inhibitory effects of losartan and CGP 42112A on AngII- and Anglll-mediated increase in cell volume of arterial SMC. Quiescent SMC were obtained in the same conditions as in Fig. 1, and modal cell volume was determined after 3 days of a daily treatment with angiotensin and/or losartan and CGP 42112A. Each day, cells were fed with fresh culture medium containing one of these inhibitors and supplemented 10 min later with AngII or AngIII. n.d., not determined. Treatment
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1312 1329 1412 n.d. n.d.
1722 1722 1312 1378 1525
Fig. 4. Comparativeeffects of Angl, AngII and AngIII on c-fos induction in quiescent SMC in culture. (A) Dose effect. Quiescent SMC were treated by 1 pM AngI (l), 10 pM AngI (2), 1 pM AngII (3), I pM AngIII (4) or 10 pM AngIII ( 5 ) for 30 min, then processed as described in Fig. 3 for c-fos mRNA detection. (0)Control cells in DMEM; (S) 30-min-I 0%-fetal-calf-serum-stimulated cells. Autoradiography was exposed for 2 days. (B) Kinetics. Quiescent SMC of the same batch ofcells were incubated with 10 pM AngI, 1 pM AngII or 1 pM AngIII for different times. c-fos mRNA detection was determined after total RNA extraction. With this batch of cells, maximal c-fos induction occurred 15 min after cell stimulation. The autoradiograph was exposed for 3 days.
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Fig. 3. c-fos induction by AngII in quiescent SMC in culture. (A) Time course of c-fos mRNA induction. Quiescent SMC were treated for the indicated times with 1 pM AngII in DMEM. Total RNA from lo6 cells was analyzed by Northern blotting and hybridized with a 32P-labelledc-jos probe, as described in Materials and Methods. The autoradiograph was exposed for 2 days. (B) Dose/response of the effect of AngII on c-fos expression. Quiescent SMC were exposed for 30 min to various concentrations of AngIl: (1) 1 nM, (2) 10 nM, (3) 0.1 pM, (4) 1 pM, (5) 10 pM or (S) 10% fetal calf serum in DMEM; (C) DMEM alone. The autoradiograph was exposed for 2 days. (C) Effect of protein synthesis inhibition on c-fos mRNA induction by AngII. Quiescent SMC were exposed for the indicated times with 1 pM AngII in the absence or presence of 1.5 pg/ml cycloheximide. At the end of each incubation, cells were harvested and processed as previously described for clfos mRNA evaluation. The autoradiograph was exposed for 2 days.
mRNA was not dependent on protein synthesis, since cycloheximide did not produce any inhibition of c-fos expression, but, in contrast, induced an overproduction and probably a stabilization of the c-fos mRNA, which remained detectable 2 h after stimulation (Fig. 3C). In the same conditions, cycloheximide alone did not induce any e-fos expression in quiescent SMC (not shown).
As shown in Fig. 4, c-fos expression was also induced by AngIII and AngI. Like AngII, 1 pM AngIII induced maximal expression of c-fos mRNA. In contrast, 10 pM AngI was needed to induce a c-fos expression equivalent to that produced by 1 pM AngII (Fig. 4A). c--0s induction occurred with the same kinetics after stimulation by AngI, AngIT or AngIII (Fig. 4B). The AngII induction of c-fos mRNA was blocked by [Sarl , IleSIAngII, a non-specific AT antagonist (Fig. 5). Induction of c-jos expression by AngI and AngIII was also inhibited by this inhibitor (Fig. 5B and A). In contrast, [Sarl, lleS]AngII did not inhibit serum-induced expression of c-fos mRNA (not shown). Losartan, a selective antagonist of AT1, dose-dependently inhibited c-fos expression induced by either AngIl or AngIII. In contrast, these inductions were not inhibited by CGP 42112A, a specific inhibitor of AT2 (Fig. 5C and D). Addition of 10 pM amastatin and 10 pM bestatin, two inhibitors of aminopeptidases, to the culture medium before AngIl addition, did not inhibit or increase c-fos induction by AngII (not shown), demonstrating that this induction was not due to AngII conversion. 1 pM captopril, an inhibitor of ACE, inhibits AngI-induced expression by 75% (Fig. 6A). Higher captopril concentrations (10 pM and 100 pM) did not significantly increase this inhibition (Fig. 6B). AngI-induced c-fos expression was also partially inhibited by trandolaprilate (Fig. 6B), another ACE inhibitor, suggesting that the AngI effect is, in part, mediated via its conversion to AngII by ACE. Under the same conditions, captopril ( 3 pM and 10 pM) and trandolaprilate (10 pM) did not induce significant modification of AngIImediated c-fos induction (Fig. 6A and B). Moreover, an ACElike activity (5.7 pU/cell) was demonstrated in the membrane fraction of quiescent SMC cultures maintained in serum-free medium. DISCUSSION The present study clearly demonstrates that AngIII induces both cellular hypertrophy and c:fos expression when
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Fig. 6. Influence of captopril and trandolaprilate on c-fbs induction by Angl in quiescent SMC in culture. After a 10-min incubation with captopril or trandokaprilate, 10 pM AngI or 1 pM AngII was added for 30 min to the culture medium of quiescent SMC. c-fos mRNA expression was compared to that of cells stimulated in the absence of captopril or trandolaprilate. (A) Lane 0, quiescent cells; 1, AngI; 2, AngII; 3, 10% fetal calf serum; 4, 1 pM captopril; 5, 1 phII captopril and AngI; 6, 1 pM captopril and AngII. (B) Lane 0, quiescent cells; I , 1 pM trandolaprilate and AngI; 2, 10 pM trandolaprilate and AngI; 3,O. 1 mM trandolaprilate and Angl; 4, Angl; 5 , l pM captopril and AngI; 6, 10 pM captopril and AngI; 7, 0.1 mM captopril and AngI; 8, 10 pM trandolaprilate and AngII; 9, AngII; 10, 10 pM captopril and AngII. Autoradiographs were exposed for 2 days.
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7
8
Fig. 5. Inhibition of Angl, AngII or AngIII c-fos induction in quiescent SMC in culture by [Sarl, IleSjAngII (Sar', Ile'-AngII), losartan and CGP 42112A. Quiescent SMC were incubated for 10 min with 10 pM [Sarl, Ile8lAngII (A and B), 10 pM losartan or 10 pM CGP 42112A (C) before addition of 10 pM AngI, 1 pM AngII or 1 pM AngIII to the culture medium for 30 min. c-fos mRNA content was compared with cells incubated 30 min with AngI, AngI1, AngIII or serum ( S ) in the absence of any inhibitor. (D) SMC were incubated with various concentrations of losartan (0.1 pM, 1 pM, 10 pM, 0.1 mM; 1-4, respectively) or CGP 42112A (10 nM, 0.1 pM, 1 pM, 10 pM; 5-8, respectively), before addition of 1 pM AngII for 30 min. (0)Control cells; ,411, 30-min-1 pM-AngII-stimulated cells in the absence of any inhibitor. Autoradiographs were exposed for 3 days.
added to the culture medium of quiescent rat aortic SMC. The vasopressive effects of AngIII in vivo [29, 30, 431 and the induction of arterial-strip contraction by AngIII in vitro [44, 451 have previously been reported. More recently, Dostal et al. [46] described AngIII induction of a transient increase in cytosolic calcium identical to that obtained with AngII in cultured arterial SMC. All these effects are probably due to AngIII binding to a subtype of AT, since AngIII displaces AngII from its membrane receptors in cultured arterial SMC [31- 341. In our study, both hypertrophy and c-jos induction by AngIII were inhibited not only by [Sarl, Ile8lAngII but also by the specific AT1 antagonist losartan, suggesting that AngIII binds to the same subclass of AT as AngII. These results are in agreement with those describing the inhibition of vasopressive effects of AngIII by [Sarl, IleSIAnglI [43], although other studies suggest that AngIII causes vasoconstriction in peripheral arteries by acting on a subclass of AT different from those activated by AngII [29, 301. The parallel-
ism in induction of both hypertrophy and c-fos mRNA expression obtained after stimulation of quiescent SMC by AngIII or AngII, suggests that AngIII, which may be produced at the level of the arterial wall [25,28], could participate with AngII in SMC hypertrophy during the development of hypertension in vivo. Moreover, this study suggests that cTfos induction, which is inhibited at the same time as angiotensininduced hypertrophy, could be involved in this hypertrophic process. The present study also demonstrates a dose-dependent increase in c-jos expression after AngI stimulation of quiescent arterial SMC in culture. However, a tenfold-higher concentration is necessary to induce the same effect as that of Angll. This increase in c-fos mRNA by AngI is inhibited by [Sarl, Ile81AngI1, suggesting that AngI could bind to the same receptor as AngII, since it has previously been demonstrated that AngI can bind with a lower affinity to the same site as AngII on SMC from rat mesenteric arteries [31]. However, in the present study, partial inhibition of AngI-mediated c-jos induction when SMC were incubated beforehand with the ACE inhibitors captopril and trandolaprilate, and the presence of an ACE-like activity in these cells, suggest that the AngI effect is, in part, mediated via its conversion to AngII. The same kind of observation has recently been described in primary cultures of rat aortic SMC: AngI induction of transient cytosolic calcium increase is inhibited by captopril[27]. However, in this study, inhibition of AngI effects by ACE inhibitors was complete, while c-jos induction was only partially inhibited in our study, suggesting that AngI exerts its effects mostly via its degradation by an ACE-like enzyme (inhibited by ACE inhibitors) but also by other undefined mechanisms. Indeed, residual c--0s induction observed at a high concentration of ACE inhibitors suggests that AngI could be either converted to AngII by another enzyme not inhibited by ACE inhibitors [23, 471, converted to other angiotensin-derived biologically active peptides, such as Angl - 7 [48], which may be produced in SMC culture [49], or be active by itself. These different hypotheses must be verified by the use of specific protease inhibitors. Nevertheless, conversion of AngI to AngI1, which
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seems to be the major route for AngI c-fos induction in cultured aortic SMC, is confirmed by the demonstration of an ACE-like activity which, in spite of its low level in SMC cultures, may be sufficient to explain the local formation of an AngII level sufficient for maximal c-fos induction. The presence of binding sites for ACE inhibitors has also been described in human vascular SMC in culture [26].ACE-like activities have also been described both in dog aorta homogenates, after endothelium and adventitia removal 1231, and in cultured arterial SMC [25].Complementary studies are needed to verify if the ACE-like enzyme found in secondary SMC in culture is really produced by these cells or if it originates from the serum of the culture medium and remains adsorbed onto the SMC surface, despite several washes in serum-free medium. In conclusion, this s t d y strongly suggests that AngI may have an indirect hypertrophic effect on SMC, essentially after its local transformation into AngII or other peptides, although a direct effect cannot be totally excluded. This study also demonstrates that c-fos induction accompanies hypertrophy induced by either AngII or AngIII, and confirms the previous hypothesis that the expression of this oncogene is involved in the hypertrophic process. Moreover, the finding that AngIII induces both c-fos and hypertrophy at a level similar to AngII suggests that AngIII in vivo could have an action similar to AngII on medial thickening during the hypertensive process.
