- Email: [email protected]
Effect of angiotensin II receptor blocker on angiotensin II stimulated dna synthesis of cultured human aortic smooth muscle cells
Life Sciences,
Vol. 56, No. 20 Pp. PL 383388,1995 Cqyright Q 1995ElsevierScienceLtd Printed in the USA. AUrightsreserved 00263205/95$950 + .oo
Pergamon 0024-3205(95)00144-1
II
Shinji Makita, Motoyuki Nakamura *, Hiroaki Yoshida, and Katsuhiko Hiramori Department of Internal Medicine II, Iwate Medical University, Morioka, Japan (Submitted December 12, 1994; accepted January 5, 1995; received in final form February 27, 1995)
Abstract
: To examine the role of the renin-angiotensin system on human vascular smooth muscle cell (VSMC) replication, we studied the effect of DUP 753, an angiotensin II (ANG II) type 1 receptor antagonist, on ANG II stimulated tritiated-thymidine (3H-Tdr) incorporation into cultured human aortic VSMC. ANG II stimulated DNA synthesis of VSMC in a dose-dependent manner as estimated by 3H-Tdr incorporation (control; 2993 + 486 cpm, lO_*M;3360 _+ 350 cpm, 10.‘M; 3474 & 516 cpm, 10v6M; 4889 + 320 cpm, P c 0.01). The effects of ANG II were clearly inhibited by l@‘M DUP 753 (ANG II 10m8M;3360 &350 vs 509 &39 cpm, IO-‘M; 3474 t 516 vs 661 t 36 cpm, 10e6M; 4889 2 320 vs 806 -+ 76 cpm, each P < 0.01). This receptor antagonist decreased the basal 3H-Tdr incorporation of VSMC from 2933 t 486 to 411 t 78 cpm (P < 0.01). Furthermore, DUP 753 decreased 10.‘M ANG II-stimulated 3H-Tdr incorporation of VSMC in a dose-dependent manner (control; 2627 -t 256 cpm, lO_‘M; 2145 -+ 143 cpm, 10F8M;1047 _+ 543 cpm, lO_‘M; 639 -+ 169 cpm, 10e6M;642 + 59 cpm, P < 0.01). These observations suggest that, in human VSMC, ANG II type 1 receptors are important for the regulation of both stimulated and basal cell proliferation. It may therefore be worth while to examine the clinical usefulness of DUP 753 for preventing abnormal VSMC growth. Key Words: aortic smooth muscle cells, angiotensin II, DUP 753
Introduction
Angiotensin (ANG) II is generally recognized as a circulating hormone with potent vasoconstrictory properties, and is suggested as stimulating hypertrophy and hyperplasia of vascular smooth muscle cells (VSMC). It has also been reported as enhancing intimal thickening of rat carotid arteries after balloon injury (1). Angiotensin converting enzyme (ACE) inhibitors which inhibit the conversion of ANG I to ANG II reduce this intimal thickening (2). These observations may indicate that overproduction %To whom all correspondence should be addressed.
g A 2 8
PHARM4COLOGY LEVERS Accelerated Communication
EFFECT OF ANGIOTENSIN II RECEPTOR BLOCKER ON ANGIOTENSIN STIMULATED DNA SYNTHESIS OF CULTURED HUMAN AORTIC SMOOTH MUSCLE CELLS
P
PL-384
ANG II Receptor Blocker and Human VSMC
Vol. 56, No. 20,lWS
of ANG II accelerates the development of atherosclerotic lesions in the vessel, resulting in abnormal vascular wall thickening. It is now apparent that renin, angiotensinogen and ACE are present within the vascular wall, and particularly that these components are markedly expressed in the neointima (3, 4, 5). It is therefore probable that ANG II is produced locally in the vascular wall, and has a direct effect on VSMC in an autocrine or paracrine fashion. In view of this, it has been thought that activation of the local renin-angiotensin system (PAS) in the vascular wall may play an important role in the development of atherosclerosis. Although numerous investigators have reported the effects of ANG II on cellular hypertrophy or hyperplasia of VSMC in animals, experimental studies on human VSMC are scant at present. Furthermore, the biological character of animal vascular tissue may differs from those of human vessels (6). The present experiment attempts to clarify the role of RAS on progression of atherosclerosis in humans by investigating the proliferating effect of ANG II on cultured human aortic VSMC, and to determine whether this effect is inhibited by the concomitant use of the AI1 receptor blocker DUP753 (7) (2-n-butyl-4chloro-5-hydroxymethyl-l-[2’-( lH-tetrazol-9yl) biphenyl4~1) methyl] imidazole, potassium salt) on VSMC proliferation. Materials and Methods Materials : ANG II was purchased from Peptide Laboratory (Osaka, Japan), and the ANG II receptor blocker DUP 753 was gifted by Merck Banyu Co Ltd (Tokyo, Japan). The culture media Ml99 and SGM were obtained from GIBCO (Grand Island, NY, USA) and Kurabou Co Ltd (Osaka, Japan), respectively. The fetal calf serum (FCS) added to these media was purchased from GIBCO (Grand Island, NY, USA), and tritiated thymidine was purchased from Amersham Co Ltd (Buckinghamshire, UK). Cell Culture : The VSMC isolated from normal human aortic medial layer was purchased from Kurabou Co Ltd (Osaka, Japan), seeded in cultured flasks (Be&on Co Ltd, Lincoln Park, NJ, USA), and subcultured by a 1:4 split in SGM under a humidified atmosphere of 5% CO, + 95% air at 37°C. The VSMC were reached confluent after approximately 7 days, with a change of culture medium every 3 days (Fig 1). For experimental purposes, the VSMC were detached by trypsin-EDTA, seeded onto 24well multiplates (Becton Co Ltd, Lincoln Park, NJ, USA) at approximately 2 x 104cells per well, and subcultured by the same procedures. When the VSMC reached confluence (approximately one week after seeding), they were quiescent after 48 hours of serum deprivation in culture medium (M199). After that the medium was replaced by fresh medium containing 2% FCS, tritiated thymidine (3H-Tdr), and ANG II with or without ANG II receptor blocker (final volume of culture medium was 1000 ~1 per well). The VSMC were subsequently incubated for 24 hours, and cells at the 8th to 14th passage were used for the experiment. Quantification of 3H-Tdr incorporation : 3H-Tdr was added to the culture medium at a concentration of 1 MCi per well. In the quantification of 3H-Tdr incorporation, VSMC were trypsinized and harvested on filter paper (labomash filter, N D S Co Ltd, Tokyo,
Vol. 56, No. 20, 1995
PL-385
ANG II Receptor Blocker and Human VSMC
Japan) using a cell-harvester (Labo Science Co Ltd, Tokyo, Japan) after washing in phosphate buffered saline. 3H-Tdr incorporation into these cells was counted as cpm by a liquid gamma scintillation counter to evaluate VSMC proliferation. Statistics: Values were expressed as mean f SD. Statistical analysis of the data was performed using a one-way ANOVA test or an unpaired t test, and significance was assumed at P < 0.05.
Fig. 1 Phase-contrast microscopic view of confluent cultured human aortic smooth muscle cells one week after the subseeding. Original magnification X400
Results 1) DUP 753 (10e7M) caused a decrease in basal ‘PI-Tdr incorporation (2933 t486vs411 f78cpm,P
into VSMC
2) ANG II ( 10e8, 10e7, 10-6M) caused a concentration-dependent increase in ‘H-Tdr incorporation to a maximum of 40% above the control value (control; 2993 t 486 cpm, 10m8M;3360 -+ 350 cpm, lO_‘M;3474 2 516 cpm, 10e6M;4889 -I_320 cpm, P < 0.001; n = 6) (Fig 2). 3) Under ANG II stimulation (lo*‘, 10m7,10e6M),DUP 753 ( 10m7M)caused a decrease in 3H-Tdr incorporation at each ANG II concentration (10e8M; 3360 t 350 vs 509 t39 cpm, lO_‘M; 3474 + 516 vs 661 & 36 cpm, 10‘6M; 4889 t 320 vs 806 -+ 76 cpm, each P < 0.001 ; n = 6) (Fig 2). 4) In the presence of ANG II (10e7M) stimulation, DUP 753 (10m9,10m8,10e7, 10m6M) caused a decrease in 3H-Tdr incorporation in a concentration-dependent manner (control; 2627 t 256 cpm, 10-w; 2145 4 143 cpm, 10e8M;1047 z!z543 cpm, 10’M; 639 &169 cpm, 10-M; 642 -t- 59 cpm, P < 0.001; n = 6) (Fig 3).
PL-386
ANG II Receptor Blocker and Human VSMC
Vol. 56, No. 20, 1995
6000 5000 4000 3000 2000
1000 0
-8
control
-7
-6
log [ANG II] (M)
Fig. 2 Effect of angiotensin II (ANG II ; 10-6-10‘8M) with (I) or without (m ANG II receptor blocker (DUP 753, 1O“M) on DNA synthesis of cultured human aortic smooth muscle cells. *P < 0.05, **P < 0.01 vs control. Bars indicate mean 2 SD ( n = 6 )
control
-9
-8
-7 log [DUP753]
-6 (M)
Fig. 3 Inhibitory effect of angiotensin II receptor blocker (DUP 753 ; lo-‘106M) on angiotensin II (1o“M) -stimulated DNA synthesis of cultured human aortic smooth muscle cells. *P < 0.05, **P < 0.01 vs control. Bars indicate Mean + SD ( n = 6 )
Vol. 56, No. 20,lWS
ANG II Receptor Blocker and Human VSMC
PL-387
Discussion This experiment showed that, in the presence of FCS, ANG II stimulated VSMC proliferation in a concentration-dependent manner. This was inhibited by ANG II type 1 receptor blocker DUP 753, with the inhibitory effect was dependent on the concentration of the receptor blocker. While ACE inhibitors inhibit conversion of ANG I to ANG II, DUP 753 competitively blocks the ANG II type 1 receptor on the cell membrane (8). The inhibitory effect of the ANG II receptor blocker on VSMC proliferation observed here suggests that ANG II has a potent capacity to stimulate VSMC proliferation, and that this effect may he mediated by the ANG II type 1 receptor. Bunkenburg et al reported that ANG II stimulated VSMC proliferation in spontaneously hypertensive rats (9). The proliferative effect of ANG II observed in our experiment was in accord with this previous report. However, Itoh et al (10) and Geisterfer et al (11) showed in rat VSMC that although ANG II stimulated protein synthesis and enlarged cell size, DNA synthesis and cell numbers were not affected. These reports suggest that ANG II induces cell hypertrophy only, and not hyperplasia. In the present experiment, however, ANG II clearly increased cell proliferation. This difference may be due to use of human VSMC, as opposed to that of other species. Alternatively, the cells studied in this experiment may have been liable to express mitogenesis because they were used after frequent subseeding steps. Several previous reports have indicated that ANG II synergistically augments the proliferative effect of platelet derived growth factor (PDGF) due to enhancement of PDGF mRNA expression in VSMC (12, 13, 14, 15), and that this proliferative effect is inhibited by anti PDGF antibody (14). Thus, these observations suggest that ANG II induces a proliferative effect via augmentation of PDGF production and/or sensitivity of vascular growth to PDGF. Recentry, Herbert et al have shown that an ANG II type 1 receptor antagonist, SR47436 and losartan, block the ANG II-stimurated increase in the number of human aortic smoth muscle cells (16). Furthermore, Varty et al have reported that losartan inhibits ANG II-induced growth of human saphenous vein in culture (17). These observations may be consistent with our results. On the other hand, our observations showed that DUP 753 inhibited basal VSMC proliferation. This mechanism may be explained by the following possibilities. First, the proliferative autocrine or paracrine effect of ANG II produced by the local RAS may have been blocked by DUP 753. Indeed, in our independent experiments, ACE inhibitor captopril has been shown to inhibit the basal 3H-Tdr incorporation into these cells to a maximum of 68% below the control value (data not shown). Second, the synergistic proliferative effect of ANG II with other growth factors in FCS may be inhibited by the blocking of the ANG II type 1 receptor. Third, the phenotype of VSMC may be changed from a contractile to a synthetic form by cell culture (18). The synthetic phenotype of VSMC has been reported to produce PDGF (19), smooth musclecell derived growth factor(20), and insulin like growth factor (21). The synergistic effect of ANG II and these growth factors on VSMC proliferation may thus be blocked
PL-388
ANG II Receptor Blocker and Human VSMC
Vol. 56, No. 20,19!25
by the ANG II type 1 receptor blocker. In conclusion,
the present experiment has shown that in human cultured VSMC and that the ANG II receptor blocker DUP 753 significantly inhibits these effects as well as basal DNA synthesis. ANG II has a potent stimulatory effect on cell proliferation,
References 1.
2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12.
13. 14. 15. 16. 17. 18. 19.
M. J. A. P. DAEMEN, D. M. LOMBARDI, F. T. BOSMAN, and S. M. SCHWARTZ. Circ. Res. 68 450-456 (1991). J. S. POWELL, J. P. CLOTEL, R. K. M. MILLER, H. KUHN, F. HEFTI, M. HOSANG, and H. R. BAUMGARTNER. Science 245 186-188 (1989). J. D. SWALES, N. J. SAMANI, and J. HUM. Hypezns. 3_71-77 (1989). H. RAKUGI, H. J. JACOB, J. E. KRIEGER, J. R. INGERFINGER, and R. E. PRATT. Circulation 87 283-290 (1993). H. RAKUGI, J. KRIEER, D. S. WANG, V. J. DZAU, and R. E. PRATT. Circulation 84 (suppl-II) 113 (1991). THE MERCmOR STUDY GROUP, Circulation 86 100-l 10 (1992). P. B. M. W. M. TIMMERMANS, D. J. CARINI, A.T. CHIU, J. V. DUNCIA, W. A. PRICE, G. J. WELLS, P. C. WONG, A. L. JOHNSON, and R. R. WEXLER. Am. J. Hypertens. i 275S- 281s (1991). H. YAZAWA, E. IIDA-KUBOTA, and K. HONDA, Jpn. J. Pharmacol. -62 339-343 (1993). B. BUNKENBURG, T. V. AMELSVOORT, H. ROGG, and J. M. WOOD. Hypertension 20 746-754 (1992). H. ITOH, R. EPRATT, and V. J. DZAU, J. Clin. Invest. 86 1690-1697 (1990). A. T. GEISTERFER, M. J. PEACH, and G. K. OWENS. Czc. Res. -62 749-756 (1988). S. ARAKI, Y. KAWAHARA, K. KARIYA, M. SUNAKO, T. TSUDA, H. FUKUZAKI, and T. YOSHIMI. B&hem. Biophys. Res. Comm. 168 350-357 (1990). Y. KO, H. STIEBLER, G. NICKENIG, A. J. WIECZOREK, H. VETTER, and A. SACHINIDIS. Am.J. Hypertens. 5 4%-499 (1993). K. SUDHIR, E. WILSON, K. CHATTERJEE, and H. E. IVES. J. Clin. Invest. -92 3003-3007 (1993). A. J. NAFIILAN. J. Cardiovasc. Pharm. 20 (suppl-I) S37-S4O (1992). J. M. HERBERT, c. DELISEE, F. DOL, P.SCHAEFFER, c. CAZAUBON, D. NISATO, and P. CHATELAIN. Eur. J. Pharmacol. 251 143-150 (1994). K. VARTY, K. E. ALLEN, L. JONES, R. D. SAYER, P. R. F. BELL, and N. J. M. LONDON. Br. J. Surg. 81819-822 (1994). s. M. SCHWARTZ, G. R. CAMPBELL, and J. H. CAMPBELL, Circ. Res. -58 427-444 (1986). P. LIBBY, S. WARNER, R. SALOMON, and L. K. BIRINYI. N. Engl. J. Med. 318 1493-1498
(1988).
20. N.MORISAKI, T. KANZAKI, T. KOSHIKAWA, Y. SAITO, and s. YOSHIDA. FEBS L&t. 230 186-190 (1988). J. S. FORRESTER, R. H. HERFANT, and J. A. 21. B. CERCEK, M. C. FISmIN, FAGIN. Circ. Res. -66 17551760 (1990).
Vol. 56, No. 20 Pp. PL 383388,1995 Cqyright Q 1995ElsevierScienceLtd Printed in the USA. AUrightsreserved 00263205/95$950 + .oo
Pergamon 0024-3205(95)00144-1
II
Shinji Makita, Motoyuki Nakamura *, Hiroaki Yoshida, and Katsuhiko Hiramori Department of Internal Medicine II, Iwate Medical University, Morioka, Japan (Submitted December 12, 1994; accepted January 5, 1995; received in final form February 27, 1995)
Abstract
: To examine the role of the renin-angiotensin system on human vascular smooth muscle cell (VSMC) replication, we studied the effect of DUP 753, an angiotensin II (ANG II) type 1 receptor antagonist, on ANG II stimulated tritiated-thymidine (3H-Tdr) incorporation into cultured human aortic VSMC. ANG II stimulated DNA synthesis of VSMC in a dose-dependent manner as estimated by 3H-Tdr incorporation (control; 2993 + 486 cpm, lO_*M;3360 _+ 350 cpm, 10.‘M; 3474 & 516 cpm, 10v6M; 4889 + 320 cpm, P c 0.01). The effects of ANG II were clearly inhibited by l@‘M DUP 753 (ANG II 10m8M;3360 &350 vs 509 &39 cpm, IO-‘M; 3474 t 516 vs 661 t 36 cpm, 10e6M; 4889 2 320 vs 806 -+ 76 cpm, each P < 0.01). This receptor antagonist decreased the basal 3H-Tdr incorporation of VSMC from 2933 t 486 to 411 t 78 cpm (P < 0.01). Furthermore, DUP 753 decreased 10.‘M ANG II-stimulated 3H-Tdr incorporation of VSMC in a dose-dependent manner (control; 2627 -t 256 cpm, lO_‘M; 2145 -+ 143 cpm, 10F8M;1047 _+ 543 cpm, lO_‘M; 639 -+ 169 cpm, 10e6M;642 + 59 cpm, P < 0.01). These observations suggest that, in human VSMC, ANG II type 1 receptors are important for the regulation of both stimulated and basal cell proliferation. It may therefore be worth while to examine the clinical usefulness of DUP 753 for preventing abnormal VSMC growth. Key Words: aortic smooth muscle cells, angiotensin II, DUP 753
Introduction
Angiotensin (ANG) II is generally recognized as a circulating hormone with potent vasoconstrictory properties, and is suggested as stimulating hypertrophy and hyperplasia of vascular smooth muscle cells (VSMC). It has also been reported as enhancing intimal thickening of rat carotid arteries after balloon injury (1). Angiotensin converting enzyme (ACE) inhibitors which inhibit the conversion of ANG I to ANG II reduce this intimal thickening (2). These observations may indicate that overproduction %To whom all correspondence should be addressed.
g A 2 8
PHARM4COLOGY LEVERS Accelerated Communication
EFFECT OF ANGIOTENSIN II RECEPTOR BLOCKER ON ANGIOTENSIN STIMULATED DNA SYNTHESIS OF CULTURED HUMAN AORTIC SMOOTH MUSCLE CELLS
P
PL-384
ANG II Receptor Blocker and Human VSMC
Vol. 56, No. 20,lWS
of ANG II accelerates the development of atherosclerotic lesions in the vessel, resulting in abnormal vascular wall thickening. It is now apparent that renin, angiotensinogen and ACE are present within the vascular wall, and particularly that these components are markedly expressed in the neointima (3, 4, 5). It is therefore probable that ANG II is produced locally in the vascular wall, and has a direct effect on VSMC in an autocrine or paracrine fashion. In view of this, it has been thought that activation of the local renin-angiotensin system (PAS) in the vascular wall may play an important role in the development of atherosclerosis. Although numerous investigators have reported the effects of ANG II on cellular hypertrophy or hyperplasia of VSMC in animals, experimental studies on human VSMC are scant at present. Furthermore, the biological character of animal vascular tissue may differs from those of human vessels (6). The present experiment attempts to clarify the role of RAS on progression of atherosclerosis in humans by investigating the proliferating effect of ANG II on cultured human aortic VSMC, and to determine whether this effect is inhibited by the concomitant use of the AI1 receptor blocker DUP753 (7) (2-n-butyl-4chloro-5-hydroxymethyl-l-[2’-( lH-tetrazol-9yl) biphenyl4~1) methyl] imidazole, potassium salt) on VSMC proliferation. Materials and Methods Materials : ANG II was purchased from Peptide Laboratory (Osaka, Japan), and the ANG II receptor blocker DUP 753 was gifted by Merck Banyu Co Ltd (Tokyo, Japan). The culture media Ml99 and SGM were obtained from GIBCO (Grand Island, NY, USA) and Kurabou Co Ltd (Osaka, Japan), respectively. The fetal calf serum (FCS) added to these media was purchased from GIBCO (Grand Island, NY, USA), and tritiated thymidine was purchased from Amersham Co Ltd (Buckinghamshire, UK). Cell Culture : The VSMC isolated from normal human aortic medial layer was purchased from Kurabou Co Ltd (Osaka, Japan), seeded in cultured flasks (Be&on Co Ltd, Lincoln Park, NJ, USA), and subcultured by a 1:4 split in SGM under a humidified atmosphere of 5% CO, + 95% air at 37°C. The VSMC were reached confluent after approximately 7 days, with a change of culture medium every 3 days (Fig 1). For experimental purposes, the VSMC were detached by trypsin-EDTA, seeded onto 24well multiplates (Becton Co Ltd, Lincoln Park, NJ, USA) at approximately 2 x 104cells per well, and subcultured by the same procedures. When the VSMC reached confluence (approximately one week after seeding), they were quiescent after 48 hours of serum deprivation in culture medium (M199). After that the medium was replaced by fresh medium containing 2% FCS, tritiated thymidine (3H-Tdr), and ANG II with or without ANG II receptor blocker (final volume of culture medium was 1000 ~1 per well). The VSMC were subsequently incubated for 24 hours, and cells at the 8th to 14th passage were used for the experiment. Quantification of 3H-Tdr incorporation : 3H-Tdr was added to the culture medium at a concentration of 1 MCi per well. In the quantification of 3H-Tdr incorporation, VSMC were trypsinized and harvested on filter paper (labomash filter, N D S Co Ltd, Tokyo,
Vol. 56, No. 20, 1995
PL-385
ANG II Receptor Blocker and Human VSMC
Japan) using a cell-harvester (Labo Science Co Ltd, Tokyo, Japan) after washing in phosphate buffered saline. 3H-Tdr incorporation into these cells was counted as cpm by a liquid gamma scintillation counter to evaluate VSMC proliferation. Statistics: Values were expressed as mean f SD. Statistical analysis of the data was performed using a one-way ANOVA test or an unpaired t test, and significance was assumed at P < 0.05.
Fig. 1 Phase-contrast microscopic view of confluent cultured human aortic smooth muscle cells one week after the subseeding. Original magnification X400
Results 1) DUP 753 (10e7M) caused a decrease in basal ‘PI-Tdr incorporation (2933 t486vs411 f78cpm,P
into VSMC
2) ANG II ( 10e8, 10e7, 10-6M) caused a concentration-dependent increase in ‘H-Tdr incorporation to a maximum of 40% above the control value (control; 2993 t 486 cpm, 10m8M;3360 -+ 350 cpm, lO_‘M;3474 2 516 cpm, 10e6M;4889 -I_320 cpm, P < 0.001; n = 6) (Fig 2). 3) Under ANG II stimulation (lo*‘, 10m7,10e6M),DUP 753 ( 10m7M)caused a decrease in 3H-Tdr incorporation at each ANG II concentration (10e8M; 3360 t 350 vs 509 t39 cpm, lO_‘M; 3474 + 516 vs 661 & 36 cpm, 10‘6M; 4889 t 320 vs 806 -+ 76 cpm, each P < 0.001 ; n = 6) (Fig 2). 4) In the presence of ANG II (10e7M) stimulation, DUP 753 (10m9,10m8,10e7, 10m6M) caused a decrease in 3H-Tdr incorporation in a concentration-dependent manner (control; 2627 t 256 cpm, 10-w; 2145 4 143 cpm, 10e8M;1047 z!z543 cpm, 10’M; 639 &169 cpm, 10-M; 642 -t- 59 cpm, P < 0.001; n = 6) (Fig 3).
PL-386
ANG II Receptor Blocker and Human VSMC
Vol. 56, No. 20, 1995
6000 5000 4000 3000 2000
1000 0
-8
control
-7
-6
log [ANG II] (M)
Fig. 2 Effect of angiotensin II (ANG II ; 10-6-10‘8M) with (I) or without (m ANG II receptor blocker (DUP 753, 1O“M) on DNA synthesis of cultured human aortic smooth muscle cells. *P < 0.05, **P < 0.01 vs control. Bars indicate mean 2 SD ( n = 6 )
control
-9
-8
-7 log [DUP753]
-6 (M)
Fig. 3 Inhibitory effect of angiotensin II receptor blocker (DUP 753 ; lo-‘106M) on angiotensin II (1o“M) -stimulated DNA synthesis of cultured human aortic smooth muscle cells. *P < 0.05, **P < 0.01 vs control. Bars indicate Mean + SD ( n = 6 )
Vol. 56, No. 20,lWS
ANG II Receptor Blocker and Human VSMC
PL-387
Discussion This experiment showed that, in the presence of FCS, ANG II stimulated VSMC proliferation in a concentration-dependent manner. This was inhibited by ANG II type 1 receptor blocker DUP 753, with the inhibitory effect was dependent on the concentration of the receptor blocker. While ACE inhibitors inhibit conversion of ANG I to ANG II, DUP 753 competitively blocks the ANG II type 1 receptor on the cell membrane (8). The inhibitory effect of the ANG II receptor blocker on VSMC proliferation observed here suggests that ANG II has a potent capacity to stimulate VSMC proliferation, and that this effect may he mediated by the ANG II type 1 receptor. Bunkenburg et al reported that ANG II stimulated VSMC proliferation in spontaneously hypertensive rats (9). The proliferative effect of ANG II observed in our experiment was in accord with this previous report. However, Itoh et al (10) and Geisterfer et al (11) showed in rat VSMC that although ANG II stimulated protein synthesis and enlarged cell size, DNA synthesis and cell numbers were not affected. These reports suggest that ANG II induces cell hypertrophy only, and not hyperplasia. In the present experiment, however, ANG II clearly increased cell proliferation. This difference may be due to use of human VSMC, as opposed to that of other species. Alternatively, the cells studied in this experiment may have been liable to express mitogenesis because they were used after frequent subseeding steps. Several previous reports have indicated that ANG II synergistically augments the proliferative effect of platelet derived growth factor (PDGF) due to enhancement of PDGF mRNA expression in VSMC (12, 13, 14, 15), and that this proliferative effect is inhibited by anti PDGF antibody (14). Thus, these observations suggest that ANG II induces a proliferative effect via augmentation of PDGF production and/or sensitivity of vascular growth to PDGF. Recentry, Herbert et al have shown that an ANG II type 1 receptor antagonist, SR47436 and losartan, block the ANG II-stimurated increase in the number of human aortic smoth muscle cells (16). Furthermore, Varty et al have reported that losartan inhibits ANG II-induced growth of human saphenous vein in culture (17). These observations may be consistent with our results. On the other hand, our observations showed that DUP 753 inhibited basal VSMC proliferation. This mechanism may be explained by the following possibilities. First, the proliferative autocrine or paracrine effect of ANG II produced by the local RAS may have been blocked by DUP 753. Indeed, in our independent experiments, ACE inhibitor captopril has been shown to inhibit the basal 3H-Tdr incorporation into these cells to a maximum of 68% below the control value (data not shown). Second, the synergistic proliferative effect of ANG II with other growth factors in FCS may be inhibited by the blocking of the ANG II type 1 receptor. Third, the phenotype of VSMC may be changed from a contractile to a synthetic form by cell culture (18). The synthetic phenotype of VSMC has been reported to produce PDGF (19), smooth musclecell derived growth factor(20), and insulin like growth factor (21). The synergistic effect of ANG II and these growth factors on VSMC proliferation may thus be blocked
PL-388
ANG II Receptor Blocker and Human VSMC
Vol. 56, No. 20,19!25
by the ANG II type 1 receptor blocker. In conclusion,
the present experiment has shown that in human cultured VSMC and that the ANG II receptor blocker DUP 753 significantly inhibits these effects as well as basal DNA synthesis. ANG II has a potent stimulatory effect on cell proliferation,
References 1.
2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12.
13. 14. 15. 16. 17. 18. 19.
M. J. A. P. DAEMEN, D. M. LOMBARDI, F. T. BOSMAN, and S. M. SCHWARTZ. Circ. Res. 68 450-456 (1991). J. S. POWELL, J. P. CLOTEL, R. K. M. MILLER, H. KUHN, F. HEFTI, M. HOSANG, and H. R. BAUMGARTNER. Science 245 186-188 (1989). J. D. SWALES, N. J. SAMANI, and J. HUM. Hypezns. 3_71-77 (1989). H. RAKUGI, H. J. JACOB, J. E. KRIEGER, J. R. INGERFINGER, and R. E. PRATT. Circulation 87 283-290 (1993). H. RAKUGI, J. KRIEER, D. S. WANG, V. J. DZAU, and R. E. PRATT. Circulation 84 (suppl-II) 113 (1991). THE MERCmOR STUDY GROUP, Circulation 86 100-l 10 (1992). P. B. M. W. M. TIMMERMANS, D. J. CARINI, A.T. CHIU, J. V. DUNCIA, W. A. PRICE, G. J. WELLS, P. C. WONG, A. L. JOHNSON, and R. R. WEXLER. Am. J. Hypertens. i 275S- 281s (1991). H. YAZAWA, E. IIDA-KUBOTA, and K. HONDA, Jpn. J. Pharmacol. -62 339-343 (1993). B. BUNKENBURG, T. V. AMELSVOORT, H. ROGG, and J. M. WOOD. Hypertension 20 746-754 (1992). H. ITOH, R. EPRATT, and V. J. DZAU, J. Clin. Invest. 86 1690-1697 (1990). A. T. GEISTERFER, M. J. PEACH, and G. K. OWENS. Czc. Res. -62 749-756 (1988). S. ARAKI, Y. KAWAHARA, K. KARIYA, M. SUNAKO, T. TSUDA, H. FUKUZAKI, and T. YOSHIMI. B&hem. Biophys. Res. Comm. 168 350-357 (1990). Y. KO, H. STIEBLER, G. NICKENIG, A. J. WIECZOREK, H. VETTER, and A. SACHINIDIS. Am.J. Hypertens. 5 4%-499 (1993). K. SUDHIR, E. WILSON, K. CHATTERJEE, and H. E. IVES. J. Clin. Invest. -92 3003-3007 (1993). A. J. NAFIILAN. J. Cardiovasc. Pharm. 20 (suppl-I) S37-S4O (1992). J. M. HERBERT, c. DELISEE, F. DOL, P.SCHAEFFER, c. CAZAUBON, D. NISATO, and P. CHATELAIN. Eur. J. Pharmacol. 251 143-150 (1994). K. VARTY, K. E. ALLEN, L. JONES, R. D. SAYER, P. R. F. BELL, and N. J. M. LONDON. Br. J. Surg. 81819-822 (1994). s. M. SCHWARTZ, G. R. CAMPBELL, and J. H. CAMPBELL, Circ. Res. -58 427-444 (1986). P. LIBBY, S. WARNER, R. SALOMON, and L. K. BIRINYI. N. Engl. J. Med. 318 1493-1498
(1988).
20. N.MORISAKI, T. KANZAKI, T. KOSHIKAWA, Y. SAITO, and s. YOSHIDA. FEBS L&t. 230 186-190 (1988). J. S. FORRESTER, R. H. HERFANT, and J. A. 21. B. CERCEK, M. C. FISmIN, FAGIN. Circ. Res. -66 17551760 (1990).