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Angiotensin II and noradrenaline increase PDGF-BB receptors and potentiate PDGF-BB stimulated DNA synthesis in vascular smooth muscle
Vol. 166, No. 2, 1990 January 30, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 580-588
ANGIOTENSIN II AND NORADRENALINE INCREASE PDGF-BB RECEPTORS AND POTENTIATE PDGF-BB STIMULATED DNA SYNTHESIS IN VASCULAR SMOOTH MUSCLE A. Bobik,*
S. Grinpukel,
P.J. Little, A. Grooms and G. Jackman
The Clinical Research Unit and Baker Medical Research Institute, Alfred Hospital, Prahran, Victoria 3181, Australia Received
November 17, 1989
The effects of angiotensin II and noradrenaline were examined on PDGF-BB and PDGF-AB induced mitogenesis in primary cultures of rat aortic smooth muscle. Incubation of the smooth muscle with either angiotensin II or noradrenaline potentiated the submaximal but not maximal mitogenic effects of PDGF-BB but not PDGF-AB. These effects on PDGF-BB stimulated mitogenesis correlated with an increase in receptor number specific for this homodimer when the smooth muscle was incubated with either angiotensin II or noradrenaline. Mitogenic concentrations of PDGF-AB did not interact with this PDGF receptor subtype. These results indicate that the mitogenic effects of PDGF-AB and -BB are elicited via different PDGF receptor subtypes. Angiotensin II and noradrenaline potentiate the mitogenic effects of PDGF-BB by increasing the steady state concentrations of membrane receptors for this homodimer. 611990Academic Press, Inc. Excessive growth (proliferation) of vascular smooth muscle is an important feature of vascular hypertrophy in some forms of hypertension (1,2). Vasoconstrictor hormones such as angiotensin II (ANG II) and noradrenaline (NA) appear to play important roles in this early development of vascular hypertrophy (3). The mechanisms by which these agents influence vascular smooth muscle proliferation are not known. It is possible that these vasoconstrictors interact with growth factors to increase the proliferative responsiveness of smooth muscle. ANG II and NA share a number of cellular responses with growth factors, including the activation of phospholipase C (4) the mobilisation of calcium (5,6) and the activation of Na+/H+ exchange (7,8). Recent studies indicate that platelet derived growth factor (PDGF)-like peptides may account for at least some of the mitogenic substances with which ANG II and NA could interact to increase the mitogenic responsiveness of vascular smooth muscle. PDGFlike substances have been shown to be produced by vascular smooth muscle from immature Baker Medical *Present address: Prahran,Vic 3181, Australia.
ABBREVIATIONS ANG II, angiotensin
II; NA, noradrenaline;
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rats (9) and also by endothelial cells (10). The recent findings that transcription for PDGF -A and -B chain precursors is independently controlled in rat aortic smooth muscle raises the possibility that distinct combinations of PDGF-A and -B chains may be produced by cells within the vasculature (9,lO). In the present study our aim was to examine the effects of ANG II and NA on smooth muscle mitogenesis induced by two isoforms of PDGF. We demonstrate that ANG II and NA potentiate the ability of sub- maximal concentrations of PDGF-BB, but not PDGF-AB, to increase DNA synthesis in vascular smooth muscle. Differences in the effects of ANG II and NA on PDGF induced mitogenesis could be attributed to the two isoforms binding to separate receptor systems. The increases in sensitivity to PDGF-BB when smooth muscle cells were exposed to either ANG II or NA could be attributed to an increase in the number of membrane receptors for binding this isoform.
METHODS Human PDGF-AB was either purchased from R and D Systems, Minneapolis, MN or prepared from platelet rich plasma as previously described (11). Recombinant PDGF-BB and [nsI]PDGF-BB, labeled via the Bolton-Hunter reagent were purchased from New England Nuclear, Boston, MA. The latter was radiolabeled to a specific activity of 46,000 cpm/ng. Biochemicals were purchased from the Sigma Chemical Company, St. Louis, MO. Tissue culture products were obtained from CSL, Melbourne, Australia. Multi-well plates were obtained from Flow Laboratories, McLean, Virginia. Primary cultures of aortic smooth muscle were prepared from adult Sprague Dawley rats as previously described (12). Briefly, the procedure involved the digestion of medial layers of smooth muscle in Ml99 containing elastase (0.25 mg/ml) and collagenase (1.5 mg/ml) for several hours at 37OC in a shaking water bath. The cells were pelleted by centrifugation (9OOxg, 3 min), resuspended in Ml99 containing 10% fetal calf serum to a concentration of 5.0-7.5 x 104 cells/ml and plated into 24 well plates (1 ml/well). Fresh medium (DMEM containing 10% fetal calf serum) was applied two days after plating and then every alternate day until confluency was reached (N 5 days). Quiescence was induced b exposing the cultures for 24-36 h to serum free medium composed of Ml99 containing 4 +o v/v Monomed (CSL). The effects of various concentrations of PDGF-BB and PDGF-AB either alone or in combination with ANG II or NA on methyl-[sH]-thymidine incorporation into DNA were assessed as described by Di Corleto and Bowen-Pope (13). Briefly, after an 18 h re-incubation of the cells with growth factors and/or appropriate hormones, methyl(1 &i/ml) was added to the Ml99 containing 4% v/v Monomed and its PsH]-thymidine incorporation into DNA assessed 1 h later after washing the cultures with ice cold Dulbecco’s phosphate-buffered-saline and precipitating the DNA with ice cold 10% trichloroacetic acid. The trichloroacetic acid precipitated material was solubilised at 37OC with 1 M sodium hydroxide and the radioactivity determined in a liquid scintillation counter. [fisI]PDGF-BB competition binding assays with the smooth muscle cells were performed essentially as described by Bowen-Pope and Ross (14). Briefly, cultures made quiescent by exposure to Ml99 containing 4% Monomed and then incubated for 24 h either in the absence or presence of ANG II (10 PM) or NA (10 PM), were washed with ice cold phosphate-buffered saline (PBS). The cultures were then incubated at 4OC for 4 h in 0.50 ml of binding medium (Ham’s F12 containing 25 mM HEPES plus 0.1% bovine serum albumin, pH 7.4), 0.5 nCi [aH]PDGF-BB and various concentrations of PDGF-BB or PDGF-AB, both with and without 100 PM suramin (Bayer, Sydney, Australia). The cells were subsequently washed at 4OC (3 x 0.5 ml PBS) and cell bound radioactivity determined 581
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after solubilising albumin.
BIOCHEMICAL
the cells with 1% Triton
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
X-100 solution containing
0.1% bovine serum
Statistical analyses were performed using the paired Students’ t-test or two way analysis of variance. Results are the mean f s.e.m. or s.e.d. The s.e.d. (standard error of the difference) was calculated as (PEMS/ n ) 1/ 2 w h ere EMS is the error mean square from the two way analysis of variance and n the number of individual experiments. RESULTS Effects of ANG II and NA on PDGF induced mitopenesis The addition of either PDGF-BB or PDGF-AB to quiescent cultures of smooth muscle increased methyl-[sH]-thymidine incorporation into DNA in a concentration stimulation of methyl-[aH]dependent manner (Figure 1, upper panels). Maximum thymidine incorporation was similar for the two PDGF isoforms, averaging 17.3 f 1.9 fold above basal with PDGF-BB and 16.3 f 1.3 fold with PDGF-AB (P for difference > 0.05). Half maximal effects were observed with approximately 7 rig/ml of PDGF-BB and 10 Neither ANG II nor NA (10 PM) ‘alone, affected methyl-[sH]rig/ml PDGF-AB. thymidine incorporation into the DNA of the primary quiescent smooth muscle cultures (Table 1). In contrast to this lack of effect of ANG II and NA on basal methyl-[aH]thymidine incorporation, both agents potentiated the sub-maximal mitogenic effect of PDGF-BB (7 rig/ml). Under these conditions methyl-[sH]-thymidine incorporation averaged 125 f 8% and 121 & 7% of the control response upon incubation with ANG II and NA respectively (P < 0.01 in both instances) (Figure 1, middle panels). Neither ANG II nor NA had any effect on the maximal mitogenic response to PDGF-BB. This effect of ANG II and NA was specific for PDGF-BB since it was not observed when mitogenesis was stimulated with submaximal (10 rig/ml) mitogenic concentrations of PDGF-AB (Figure 1, middle panel). Similarly the maximum mitogenic activity of PDGF-AB was not affected by either ANG II or NA. Differential
binding of PDGF-BB and PDGF-AB to smooth muscle Because multiple receptor sub-types for PDGF have been identified on fibroblasts, we investigated the possibility that differences in the abilities of ANG II and NA to potentiate the mitogenic effects of PDGF-BB but not PDGF-AB might be due to the two PDGF-BB readily isoforms interacting with different PDGF receptor sub-types. competed with [Q5I]PDGF-BB specific binding to smooth muscle (Figure 2). In contrast, concentrations of PDGF-AB approximately five times greater than those required to induce maximal effects on DNA synthesis, had no effect on [W]PDGF-BB specific binding specific binding to rat aortic smooth muscle was (P > 0.05) (F i gure 2). fW]PDGF-BB saturable and of high affinity (Figure 2, lower panel). Fifty percent specific binding occurred at approximately 6 “g/ml, a value similar to the concentration which induced 50% of its maximal mitogenic effect (see Figure 1). Scatchard plots of the isotherm of 582
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PDGF-AB
PDGF-BB
c
0
I
10
20
PDGF-A6
10
140
COMMUNICATIONS
30
40
0
10
(nglml)
20
30
PDGF-BB
nglml
f
40
(rag/ml)
7ngiml *
1 120
@j g$$. .:.:.:$. ::::::.:: :::::::: g$$ -----:::::::. :::::::. .,.
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nglml
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NA
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fi
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&
-------
ANG
II
Fieure 1: Concentration-response curves and the effects of angiotensinII (ANG II and noradrenallne(NA) on the mitogenicactivity of PDGF-AB (left panels)and PDGd-BB (right panels). Upper panels: Aortic smooth musclecells were exposedto increasing concentrationsof PDGF-AB or PDGF-BB and the rate of methyl-[sH]-th midine incorporationwasdetermined24 h later. Middle panels: Effectsof ANG II and Nx on the sub-maximalmltogenicactivity of PDGF isoforms. ANG II (10 @) (dotted panels)was addedwith either PDGF-AB rig/ml) for 24 h and the rate of methyl+H]-thymidine incorporationcomparedto cellsnot exposedto ANG II or NA. Lower panels:Effects of ANG II and NA on the maximal mitogenicresponseto PDGF-AB and PDGF-BB. ANG II 10@) (open panels)or NA (10 PM) (dotted panels)wasaddedwith either PDGF-AB rig/ml) or PDGF-BB (40 ng/mI for 24 h and the mitogenicactivit comparedto control cellsas above. Resultsare typic 1 of 3 experiments(upper panels3 and the mean f s.e.m. (n = 7 experiments) (middleand lower panels). * P < 0.05from controls.
[lasI]PDGF-BB
specific binding to the smooth muscle cells are typically
linear at high
radioligand concentrations. At lower concentrations the deviation from linearity is similar to that observed with [W]PDGF specific binding to Swiss 3T3 fibroblasts or monkey smooth muscle cells, possibly reflecting a degreeof positive co-operativity in the binding of [W]PDGF-BB to its receptor on the rat aortic smooth muscle cells (14).
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TABLE 1: Effects of PDGF isoforms,ANG II and NA on methyl-@]-thymidine incorporationinto DNA of rat aortic smoothmuscle AGENT
Methyl-[sH -thymidine incorporation (fo/d stimulation)t
PDGF-BB PDGF-AB ANG II NA
17.3f 16.3f 1.06f 0.8 f
1.9* 1.3* 0.03 0.04
i Meansf s.e.m.of at least 7 ex eriments. * P < 0.05comparedto methyl- PsH]-thymidine incorporationinto quiescentcultures.
c--
1
10
100
PDGF-BB(AB)
g' =E20 p" F z&
1000
nglml
fmole/106cells
011
I 0
IO
30
20
PDGF-BB
40
50
60
nglml
Lower panel: Typical saturation bindingisothermof [W]PDGF-BB muscle. Insert: Scatchardanalysisof the bindingisotherm. 584
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to rat aortic smooth
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50
0
10
PDGF-BE
20
30
rig/ml
Figure 3: ANG II and NA effects on PDGF-BB subtype receptor number and affinity. Quiescent confluent cultures of smooth muscle were exposed to either no drugs (open circles), 10 /,&I ANG II closed circles or 10 $l NA (closed squares) for 24 h and the amount of lW]PDGF-B B specifically bound to intact cells determined as in Figure 2 and Methods. I P < 0.05 from respective controls.
Effects of ANG II and NA on PDGF-BB suecifm binding The possibility that ANG II and NA potentiated the sub-maximal mitogenic responses of PDGF-BB by increasing either the number or affinity of PDGF receptors for the BB isoform was investigated by comparing [fisI]PDGF-BB specific binding to untreated control smooth muscle cells with those exposed for 24 h to either ANG II or NA. Neither ANG II nor NA significantly affected the affinity of PDGF-BB specific binding to smooth muscle (P for differences > 0.05) (Figure 3). Twenty four hour exposure to ANG II and NA however did increase the number of PDGF-BB specific binding sites. The increases averaged 22.5% and 20% after incubation with ANG II and NA respectively (P < 0.05, both instances). As in the previous experiments PDGF-AB did not compete with [BSIJPDGF-BB specific binding to either the ANG II or NA exposed smooth muscle (data not shown).
DISCUSSION Although PDGF-BB and PDGF-AB stimulate DNA biosynthesis in vascular smooth muscle to a similar extent, two lines of evidence indicate that the two isoforms may be interacting with different PDGF receptor sub-types. Firstly, in competition binding experiments, the concentrations of PDGF-AB which caused maximal stimulation of methyl+HJ-thymidine into DNA, exhibited no binding to the receptor system binding PDGF-BB. Secondly, ANG II and NA potentiated the sub-maximal mitogenic effect of PDGF-BB but not that of the -AB isoform. Our findings that ANG II and NA increase
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the number of membrane receptor sites for PDGF-BB on smooth muscle is in accord with the effects of these two agents on PDGF-BB stimulated methyl-[3H]-thymidine incorporation and may represent the mechanism by which ANG II and NA influence the growth of vascular smooth muscle (3). Two receptor sub-types which bind the three PDGF isoforms (-AA, -AB and -BB) have recently been proposed to exist in human dermal fibroblasts (16,17). In these cells one receptor sub-type binds all three isoforms, whilst the other binds the -BB and -AB with high affinity but not PDGF-AA (16). These findings have led to the suggestion that the latter sub-type of receptor may be principally responsible for initiating mitogenesis, chemotaxis and the re-organisation of actin leading to membrane ruffling (18). The -AB and -BB isoforms of PDGF are also potent stimulators of mitogenesis in vascular smooth muscle. However, this effect is not the consequence of the two isoforms binding to the same PDGF receptor sub-type. Mitogenic concentrations of PDGF-AB did not compete with [fiH]PDGF specific binding to vascular smooth muscle. The findings by Reilly and Broski (19) that mitogenic concentrations of PDGF-BB but not PDGF-AB stimulated tyrosine phosphorylation of a number of low molecular weight polypeptides is also suggestive that these two isoforms are interacting with different PDGF receptor sub-types on bovine aortic smooth muscle. Our observation that both ANG II and NA are capable of potentiating the sub-maximal mitogenic effects of PDGF-BB but not PDGF-AB also support this hypothesis. Recently Gronwald et al. (15) have proposed a new model for PDGF receptor sub-types which appears more compatible with our observations in smooth muscle. They suggest that functional PDGF receptors are dimeric structures consisting of two subunits, (Y and p which can associate to form three receptor sub-types. The @-subunit is proposed to bind the B-chain of PDGF whereas the o-subunit is proposed to bind either PDGF-A or the B chain. Assuming that this model is also applicable to smooth muscle, our observations suggest that two separate receptor sub-types composed of ~$3and &3 subunits could be responsible for the mitogenic effects of PDGF-AB and PDGF-BB respectively. The increases in [aH]PDGF-BB specific binding sites following exposure of the smooth muscle to ANG II or NA represent a novel mechanism by which agents which themselves do not stimulate mitogenesis, can increase the sensitivity of smooth muscle to PDGF-BB. Our findings that these two agents had no additional effect on methyl-[aH]thymidine incorporation when the smooth muscle was exposed to sub-maximal mitogenic concentrations of PDGF-AB (Figure 1) or EGF (not shown) indicate that this effect is specific to the PDGF receptor sub-type binding PDGF-BB. Recently [WIPDGF-BB specific binding sites on 3T3 fibroblasts have been shown to increase in number during TGF-/? exposure (15). As in the present study the increase in receptor number during exposure to TGF-@ was modest, between 20 and 40%. The mechanisms underlying these increases are however still unclear. Protein kinase C activation has been shown to stimulate EGF receptor synthesis and receptor mRNA accumulation in MDA468 cells (20). 586
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It is possible that the PDGF receptor sub-type binding PDGF-BB to smooth muscle is under a similar transcriptional control mechanism. ANG II and NA are known to stimulate the breakdown of phosphatidylinositol diphosphate and elevate protein kinase C activity (4). Thickening of the walls of blood vessels due to small (25 to 35%) increases in the number of smooth muscle cells is frequently observed in hypertension (1,2). It has been suggested that unknown trophic mechanisms involving ANG II and NA may be responsible for this vascular hypertrophy, particularly in genetic hypertension (3,21). Indeed vascular hypertrophy in genetic hypertension is not a consequence of an abnormal elevation in blood pressure since it occurs early in development before there is any abnormal elevation in blood pressure and may involve an interaction of ANG II or NA with growth factors (21). Studies on the expression and developmental control of PDGF in rat aortic smooth muscle in culture indicate the presence of high levels of mRNA for PDGF-B chain in smooth muscle of immature rats with genetic hypertension (9). Our observations in culture suggest that the unknown trophic mechanisms by which ANG II and NA increase the growth of smooth muscle in genetically hypertensive animals may involve an increase in the sensitivity of the smooth muscle to PDGF-BB via an increase in the number of receptors for this growth factor. An understanding of the mechanism by which ANG II and NA increase receptors on smooth muscle for PDGF-BB will give greater insights as to how these agents are involved in the growth of smooth muscle in the vasculature. ACKNOWLEDGMENTS This work was supported by grants from the National Heart Foundation of Australia and the Alfred Hospital Research Scholarship Fund. We thank Miss Barbara Smith for assistance with the preparation of the manuscript. REFERENCES 1. i. 4: 5. 6. 7. 8. 9.
10. 11. 12.
M&any, M.J., Hansen, O.K., and Aalkjaer, C. (1978) Circ. Res. 43, 854-864. Owens, G.K., and Reidy, M.A. (1985) Circ. Res. 57, 695-705. Lever, A.F. (1986) J. Hypertens. 4, 515-524. Williamson, J.R., Cooper, R.H., Joseph, S.K., and Thomas, A.P. (1985) Am. J. Physiol. 248, C203-C216. Smith, J.B., Smith, L., Brown, E.R., Barnes, D., Sabir, M.A., Davis, J.S., and Farese, R.V. (1984) Proc. Natl. Acad. Sci. USA 81, 7812-7816. Kanaide, H., Kobayashi, S., Nishimura, J., Hasegawa, M., Sogakiuchi, Y., Matsumoto, T., and Nakamura, M. (1988) Circ. Res. 63, 16-26. Owen, N.E. (1986) J. Cell. Biol. 103, 2053-2060. Berk, B.C., Aronow, M.S., Brock, T.A., Cragoe, E., Gimbrone, M.A., and Alexander, R.W. (1987) J. Biol. Chem. 262, 5057-5064. Majewsky, M.W., Benditt, E.P., and Schwartz, S.M. (1988) Proc. Natl. Acad. Sci. USA 85, 1524- 1528. Kavanaugh, W.M., Harsh, IV G.R., Starkson, N.F., ROCCO,C.M., and Williams, L.T. (1988) J. Biol. Chem. 263, 8470-8472. Raines, E.W., and Ross, R. (1982) J. Biol. Chem. 257, 5154-5160. Weissberg, P.L., Little, P.J., and Bobik, A. (1989) Am. J. Physiol. 256, c951-c957.
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19. 20. 21.
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Di Corleto, P.E., Bowen-Pope, D.F.(1983) Proc. Natl. Acad. Sci. USA 80, 1919-1923. Bowen-Pope, D.F., and Ross, R. (1982 J. Biol. Chem. 257,5161-5171. GronwaId, R.G.K., Seifert, R.A., and B Owen-Pope, D.F. (1989) J. Biol. Chem. 264, 8120-8125. Heldin, C-H., Backstrgm, G., Ostman, A., Hammacher, A., Ronnstrand, L., Rubin, K., Nister, M., and Westermack, B. (1988) EMBO J. 7, 1387-1393. Hart, C.E., Forstrom, J.W., Kelly, J.D., Seifert, R.A., Smith, R.A., Ross, R., Murray, M.J., and Bowen-Pope, D.F. (,!988) Science 240, 1529-1531. Nister, M., Hammacher, A., Mellstrom, K., Siegbahn, A., Ronnstrand, L., Westermack, B., and Heldin, C-H. (1988) Cell 52, 791-799. Reilly, C.F., and Broski, J.E. (1989) Biochem. Biophys. Res. Commun. 160, 1047-1054. Bjorge, J.D., Paterson, A.J., and Kudlow, J.E. (1989) J. Biol. Chem. 264, 40214027. Adams, M.A., Bobik, A., and Korner, P.I. (1989) Hypertension 14, 191-202.
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ANGIOTENSIN II AND NORADRENALINE INCREASE PDGF-BB RECEPTORS AND POTENTIATE PDGF-BB STIMULATED DNA SYNTHESIS IN VASCULAR SMOOTH MUSCLE A. Bobik,*
S. Grinpukel,
P.J. Little, A. Grooms and G. Jackman
The Clinical Research Unit and Baker Medical Research Institute, Alfred Hospital, Prahran, Victoria 3181, Australia Received
November 17, 1989
The effects of angiotensin II and noradrenaline were examined on PDGF-BB and PDGF-AB induced mitogenesis in primary cultures of rat aortic smooth muscle. Incubation of the smooth muscle with either angiotensin II or noradrenaline potentiated the submaximal but not maximal mitogenic effects of PDGF-BB but not PDGF-AB. These effects on PDGF-BB stimulated mitogenesis correlated with an increase in receptor number specific for this homodimer when the smooth muscle was incubated with either angiotensin II or noradrenaline. Mitogenic concentrations of PDGF-AB did not interact with this PDGF receptor subtype. These results indicate that the mitogenic effects of PDGF-AB and -BB are elicited via different PDGF receptor subtypes. Angiotensin II and noradrenaline potentiate the mitogenic effects of PDGF-BB by increasing the steady state concentrations of membrane receptors for this homodimer. 611990Academic Press, Inc. Excessive growth (proliferation) of vascular smooth muscle is an important feature of vascular hypertrophy in some forms of hypertension (1,2). Vasoconstrictor hormones such as angiotensin II (ANG II) and noradrenaline (NA) appear to play important roles in this early development of vascular hypertrophy (3). The mechanisms by which these agents influence vascular smooth muscle proliferation are not known. It is possible that these vasoconstrictors interact with growth factors to increase the proliferative responsiveness of smooth muscle. ANG II and NA share a number of cellular responses with growth factors, including the activation of phospholipase C (4) the mobilisation of calcium (5,6) and the activation of Na+/H+ exchange (7,8). Recent studies indicate that platelet derived growth factor (PDGF)-like peptides may account for at least some of the mitogenic substances with which ANG II and NA could interact to increase the mitogenic responsiveness of vascular smooth muscle. PDGFlike substances have been shown to be produced by vascular smooth muscle from immature Baker Medical *Present address: Prahran,Vic 3181, Australia.
ABBREVIATIONS ANG II, angiotensin
II; NA, noradrenaline;
0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Research
Institute,
PDGF, platelet-derived
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growth factor.
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rats (9) and also by endothelial cells (10). The recent findings that transcription for PDGF -A and -B chain precursors is independently controlled in rat aortic smooth muscle raises the possibility that distinct combinations of PDGF-A and -B chains may be produced by cells within the vasculature (9,lO). In the present study our aim was to examine the effects of ANG II and NA on smooth muscle mitogenesis induced by two isoforms of PDGF. We demonstrate that ANG II and NA potentiate the ability of sub- maximal concentrations of PDGF-BB, but not PDGF-AB, to increase DNA synthesis in vascular smooth muscle. Differences in the effects of ANG II and NA on PDGF induced mitogenesis could be attributed to the two isoforms binding to separate receptor systems. The increases in sensitivity to PDGF-BB when smooth muscle cells were exposed to either ANG II or NA could be attributed to an increase in the number of membrane receptors for binding this isoform.
METHODS Human PDGF-AB was either purchased from R and D Systems, Minneapolis, MN or prepared from platelet rich plasma as previously described (11). Recombinant PDGF-BB and [nsI]PDGF-BB, labeled via the Bolton-Hunter reagent were purchased from New England Nuclear, Boston, MA. The latter was radiolabeled to a specific activity of 46,000 cpm/ng. Biochemicals were purchased from the Sigma Chemical Company, St. Louis, MO. Tissue culture products were obtained from CSL, Melbourne, Australia. Multi-well plates were obtained from Flow Laboratories, McLean, Virginia. Primary cultures of aortic smooth muscle were prepared from adult Sprague Dawley rats as previously described (12). Briefly, the procedure involved the digestion of medial layers of smooth muscle in Ml99 containing elastase (0.25 mg/ml) and collagenase (1.5 mg/ml) for several hours at 37OC in a shaking water bath. The cells were pelleted by centrifugation (9OOxg, 3 min), resuspended in Ml99 containing 10% fetal calf serum to a concentration of 5.0-7.5 x 104 cells/ml and plated into 24 well plates (1 ml/well). Fresh medium (DMEM containing 10% fetal calf serum) was applied two days after plating and then every alternate day until confluency was reached (N 5 days). Quiescence was induced b exposing the cultures for 24-36 h to serum free medium composed of Ml99 containing 4 +o v/v Monomed (CSL). The effects of various concentrations of PDGF-BB and PDGF-AB either alone or in combination with ANG II or NA on methyl-[sH]-thymidine incorporation into DNA were assessed as described by Di Corleto and Bowen-Pope (13). Briefly, after an 18 h re-incubation of the cells with growth factors and/or appropriate hormones, methyl(1 &i/ml) was added to the Ml99 containing 4% v/v Monomed and its PsH]-thymidine incorporation into DNA assessed 1 h later after washing the cultures with ice cold Dulbecco’s phosphate-buffered-saline and precipitating the DNA with ice cold 10% trichloroacetic acid. The trichloroacetic acid precipitated material was solubilised at 37OC with 1 M sodium hydroxide and the radioactivity determined in a liquid scintillation counter. [fisI]PDGF-BB competition binding assays with the smooth muscle cells were performed essentially as described by Bowen-Pope and Ross (14). Briefly, cultures made quiescent by exposure to Ml99 containing 4% Monomed and then incubated for 24 h either in the absence or presence of ANG II (10 PM) or NA (10 PM), were washed with ice cold phosphate-buffered saline (PBS). The cultures were then incubated at 4OC for 4 h in 0.50 ml of binding medium (Ham’s F12 containing 25 mM HEPES plus 0.1% bovine serum albumin, pH 7.4), 0.5 nCi [aH]PDGF-BB and various concentrations of PDGF-BB or PDGF-AB, both with and without 100 PM suramin (Bayer, Sydney, Australia). The cells were subsequently washed at 4OC (3 x 0.5 ml PBS) and cell bound radioactivity determined 581
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after solubilising albumin.
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the cells with 1% Triton
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X-100 solution containing
0.1% bovine serum
Statistical analyses were performed using the paired Students’ t-test or two way analysis of variance. Results are the mean f s.e.m. or s.e.d. The s.e.d. (standard error of the difference) was calculated as (PEMS/ n ) 1/ 2 w h ere EMS is the error mean square from the two way analysis of variance and n the number of individual experiments. RESULTS Effects of ANG II and NA on PDGF induced mitopenesis The addition of either PDGF-BB or PDGF-AB to quiescent cultures of smooth muscle increased methyl-[sH]-thymidine incorporation into DNA in a concentration stimulation of methyl-[aH]dependent manner (Figure 1, upper panels). Maximum thymidine incorporation was similar for the two PDGF isoforms, averaging 17.3 f 1.9 fold above basal with PDGF-BB and 16.3 f 1.3 fold with PDGF-AB (P for difference > 0.05). Half maximal effects were observed with approximately 7 rig/ml of PDGF-BB and 10 Neither ANG II nor NA (10 PM) ‘alone, affected methyl-[sH]rig/ml PDGF-AB. thymidine incorporation into the DNA of the primary quiescent smooth muscle cultures (Table 1). In contrast to this lack of effect of ANG II and NA on basal methyl-[aH]thymidine incorporation, both agents potentiated the sub-maximal mitogenic effect of PDGF-BB (7 rig/ml). Under these conditions methyl-[sH]-thymidine incorporation averaged 125 f 8% and 121 & 7% of the control response upon incubation with ANG II and NA respectively (P < 0.01 in both instances) (Figure 1, middle panels). Neither ANG II nor NA had any effect on the maximal mitogenic response to PDGF-BB. This effect of ANG II and NA was specific for PDGF-BB since it was not observed when mitogenesis was stimulated with submaximal (10 rig/ml) mitogenic concentrations of PDGF-AB (Figure 1, middle panel). Similarly the maximum mitogenic activity of PDGF-AB was not affected by either ANG II or NA. Differential
binding of PDGF-BB and PDGF-AB to smooth muscle Because multiple receptor sub-types for PDGF have been identified on fibroblasts, we investigated the possibility that differences in the abilities of ANG II and NA to potentiate the mitogenic effects of PDGF-BB but not PDGF-AB might be due to the two PDGF-BB readily isoforms interacting with different PDGF receptor sub-types. competed with [Q5I]PDGF-BB specific binding to smooth muscle (Figure 2). In contrast, concentrations of PDGF-AB approximately five times greater than those required to induce maximal effects on DNA synthesis, had no effect on [W]PDGF-BB specific binding specific binding to rat aortic smooth muscle was (P > 0.05) (F i gure 2). fW]PDGF-BB saturable and of high affinity (Figure 2, lower panel). Fifty percent specific binding occurred at approximately 6 “g/ml, a value similar to the concentration which induced 50% of its maximal mitogenic effect (see Figure 1). Scatchard plots of the isotherm of 582
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PDGF-AB
PDGF-BB
c
0
I
10
20
PDGF-A6
10
140
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30
40
0
10
(nglml)
20
30
PDGF-BB
nglml
f
40
(rag/ml)
7ngiml *
1 120
@j g$$. .:.:.:$. ::::::.:: :::::::: g$$ -----:::::::. :::::::. .,.
t _____
em-a-IL
140
40
nglml
r 120
-
loo
-----n-----s----
I ----
NA
ANG
II
40
fi
-----
NA
rig/ml
&
-------
ANG
II
Fieure 1: Concentration-response curves and the effects of angiotensinII (ANG II and noradrenallne(NA) on the mitogenicactivity of PDGF-AB (left panels)and PDGd-BB (right panels). Upper panels: Aortic smooth musclecells were exposedto increasing concentrationsof PDGF-AB or PDGF-BB and the rate of methyl-[sH]-th midine incorporationwasdetermined24 h later. Middle panels: Effectsof ANG II and Nx on the sub-maximalmltogenicactivity of PDGF isoforms. ANG II (10 @) (dotted panels)was addedwith either PDGF-AB rig/ml) for 24 h and the rate of methyl+H]-thymidine incorporationcomparedto cellsnot exposedto ANG II or NA. Lower panels:Effects of ANG II and NA on the maximal mitogenicresponseto PDGF-AB and PDGF-BB. ANG II 10@) (open panels)or NA (10 PM) (dotted panels)wasaddedwith either PDGF-AB rig/ml) or PDGF-BB (40 ng/mI for 24 h and the mitogenicactivit comparedto control cellsas above. Resultsare typic 1 of 3 experiments(upper panels3 and the mean f s.e.m. (n = 7 experiments) (middleand lower panels). * P < 0.05from controls.
[lasI]PDGF-BB
specific binding to the smooth muscle cells are typically
linear at high
radioligand concentrations. At lower concentrations the deviation from linearity is similar to that observed with [W]PDGF specific binding to Swiss 3T3 fibroblasts or monkey smooth muscle cells, possibly reflecting a degreeof positive co-operativity in the binding of [W]PDGF-BB to its receptor on the rat aortic smooth muscle cells (14).
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TABLE 1: Effects of PDGF isoforms,ANG II and NA on methyl-@]-thymidine incorporationinto DNA of rat aortic smoothmuscle AGENT
Methyl-[sH -thymidine incorporation (fo/d stimulation)t
PDGF-BB PDGF-AB ANG II NA
17.3f 16.3f 1.06f 0.8 f
1.9* 1.3* 0.03 0.04
i Meansf s.e.m.of at least 7 ex eriments. * P < 0.05comparedto methyl- PsH]-thymidine incorporationinto quiescentcultures.
c--
1
10
100
PDGF-BB(AB)
g' =E20 p" F z&
1000
nglml
fmole/106cells
011
I 0
IO
30
20
PDGF-BB
40
50
60
nglml
Lower panel: Typical saturation bindingisothermof [W]PDGF-BB muscle. Insert: Scatchardanalysisof the bindingisotherm. 584
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PDGF-BE
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Figure 3: ANG II and NA effects on PDGF-BB subtype receptor number and affinity. Quiescent confluent cultures of smooth muscle were exposed to either no drugs (open circles), 10 /,&I ANG II closed circles or 10 $l NA (closed squares) for 24 h and the amount of lW]PDGF-B B specifically bound to intact cells determined as in Figure 2 and Methods. I P < 0.05 from respective controls.
Effects of ANG II and NA on PDGF-BB suecifm binding The possibility that ANG II and NA potentiated the sub-maximal mitogenic responses of PDGF-BB by increasing either the number or affinity of PDGF receptors for the BB isoform was investigated by comparing [fisI]PDGF-BB specific binding to untreated control smooth muscle cells with those exposed for 24 h to either ANG II or NA. Neither ANG II nor NA significantly affected the affinity of PDGF-BB specific binding to smooth muscle (P for differences > 0.05) (Figure 3). Twenty four hour exposure to ANG II and NA however did increase the number of PDGF-BB specific binding sites. The increases averaged 22.5% and 20% after incubation with ANG II and NA respectively (P < 0.05, both instances). As in the previous experiments PDGF-AB did not compete with [BSIJPDGF-BB specific binding to either the ANG II or NA exposed smooth muscle (data not shown).
DISCUSSION Although PDGF-BB and PDGF-AB stimulate DNA biosynthesis in vascular smooth muscle to a similar extent, two lines of evidence indicate that the two isoforms may be interacting with different PDGF receptor sub-types. Firstly, in competition binding experiments, the concentrations of PDGF-AB which caused maximal stimulation of methyl+HJ-thymidine into DNA, exhibited no binding to the receptor system binding PDGF-BB. Secondly, ANG II and NA potentiated the sub-maximal mitogenic effect of PDGF-BB but not that of the -AB isoform. Our findings that ANG II and NA increase
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the number of membrane receptor sites for PDGF-BB on smooth muscle is in accord with the effects of these two agents on PDGF-BB stimulated methyl-[3H]-thymidine incorporation and may represent the mechanism by which ANG II and NA influence the growth of vascular smooth muscle (3). Two receptor sub-types which bind the three PDGF isoforms (-AA, -AB and -BB) have recently been proposed to exist in human dermal fibroblasts (16,17). In these cells one receptor sub-type binds all three isoforms, whilst the other binds the -BB and -AB with high affinity but not PDGF-AA (16). These findings have led to the suggestion that the latter sub-type of receptor may be principally responsible for initiating mitogenesis, chemotaxis and the re-organisation of actin leading to membrane ruffling (18). The -AB and -BB isoforms of PDGF are also potent stimulators of mitogenesis in vascular smooth muscle. However, this effect is not the consequence of the two isoforms binding to the same PDGF receptor sub-type. Mitogenic concentrations of PDGF-AB did not compete with [fiH]PDGF specific binding to vascular smooth muscle. The findings by Reilly and Broski (19) that mitogenic concentrations of PDGF-BB but not PDGF-AB stimulated tyrosine phosphorylation of a number of low molecular weight polypeptides is also suggestive that these two isoforms are interacting with different PDGF receptor sub-types on bovine aortic smooth muscle. Our observation that both ANG II and NA are capable of potentiating the sub-maximal mitogenic effects of PDGF-BB but not PDGF-AB also support this hypothesis. Recently Gronwald et al. (15) have proposed a new model for PDGF receptor sub-types which appears more compatible with our observations in smooth muscle. They suggest that functional PDGF receptors are dimeric structures consisting of two subunits, (Y and p which can associate to form three receptor sub-types. The @-subunit is proposed to bind the B-chain of PDGF whereas the o-subunit is proposed to bind either PDGF-A or the B chain. Assuming that this model is also applicable to smooth muscle, our observations suggest that two separate receptor sub-types composed of ~$3and &3 subunits could be responsible for the mitogenic effects of PDGF-AB and PDGF-BB respectively. The increases in [aH]PDGF-BB specific binding sites following exposure of the smooth muscle to ANG II or NA represent a novel mechanism by which agents which themselves do not stimulate mitogenesis, can increase the sensitivity of smooth muscle to PDGF-BB. Our findings that these two agents had no additional effect on methyl-[aH]thymidine incorporation when the smooth muscle was exposed to sub-maximal mitogenic concentrations of PDGF-AB (Figure 1) or EGF (not shown) indicate that this effect is specific to the PDGF receptor sub-type binding PDGF-BB. Recently [WIPDGF-BB specific binding sites on 3T3 fibroblasts have been shown to increase in number during TGF-/? exposure (15). As in the present study the increase in receptor number during exposure to TGF-@ was modest, between 20 and 40%. The mechanisms underlying these increases are however still unclear. Protein kinase C activation has been shown to stimulate EGF receptor synthesis and receptor mRNA accumulation in MDA468 cells (20). 586
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It is possible that the PDGF receptor sub-type binding PDGF-BB to smooth muscle is under a similar transcriptional control mechanism. ANG II and NA are known to stimulate the breakdown of phosphatidylinositol diphosphate and elevate protein kinase C activity (4). Thickening of the walls of blood vessels due to small (25 to 35%) increases in the number of smooth muscle cells is frequently observed in hypertension (1,2). It has been suggested that unknown trophic mechanisms involving ANG II and NA may be responsible for this vascular hypertrophy, particularly in genetic hypertension (3,21). Indeed vascular hypertrophy in genetic hypertension is not a consequence of an abnormal elevation in blood pressure since it occurs early in development before there is any abnormal elevation in blood pressure and may involve an interaction of ANG II or NA with growth factors (21). Studies on the expression and developmental control of PDGF in rat aortic smooth muscle in culture indicate the presence of high levels of mRNA for PDGF-B chain in smooth muscle of immature rats with genetic hypertension (9). Our observations in culture suggest that the unknown trophic mechanisms by which ANG II and NA increase the growth of smooth muscle in genetically hypertensive animals may involve an increase in the sensitivity of the smooth muscle to PDGF-BB via an increase in the number of receptors for this growth factor. An understanding of the mechanism by which ANG II and NA increase receptors on smooth muscle for PDGF-BB will give greater insights as to how these agents are involved in the growth of smooth muscle in the vasculature. ACKNOWLEDGMENTS This work was supported by grants from the National Heart Foundation of Australia and the Alfred Hospital Research Scholarship Fund. We thank Miss Barbara Smith for assistance with the preparation of the manuscript. REFERENCES 1. i. 4: 5. 6. 7. 8. 9.
10. 11. 12.
M&any, M.J., Hansen, O.K., and Aalkjaer, C. (1978) Circ. Res. 43, 854-864. Owens, G.K., and Reidy, M.A. (1985) Circ. Res. 57, 695-705. Lever, A.F. (1986) J. Hypertens. 4, 515-524. Williamson, J.R., Cooper, R.H., Joseph, S.K., and Thomas, A.P. (1985) Am. J. Physiol. 248, C203-C216. Smith, J.B., Smith, L., Brown, E.R., Barnes, D., Sabir, M.A., Davis, J.S., and Farese, R.V. (1984) Proc. Natl. Acad. Sci. USA 81, 7812-7816. Kanaide, H., Kobayashi, S., Nishimura, J., Hasegawa, M., Sogakiuchi, Y., Matsumoto, T., and Nakamura, M. (1988) Circ. Res. 63, 16-26. Owen, N.E. (1986) J. Cell. Biol. 103, 2053-2060. Berk, B.C., Aronow, M.S., Brock, T.A., Cragoe, E., Gimbrone, M.A., and Alexander, R.W. (1987) J. Biol. Chem. 262, 5057-5064. Majewsky, M.W., Benditt, E.P., and Schwartz, S.M. (1988) Proc. Natl. Acad. Sci. USA 85, 1524- 1528. Kavanaugh, W.M., Harsh, IV G.R., Starkson, N.F., ROCCO,C.M., and Williams, L.T. (1988) J. Biol. Chem. 263, 8470-8472. Raines, E.W., and Ross, R. (1982) J. Biol. Chem. 257, 5154-5160. Weissberg, P.L., Little, P.J., and Bobik, A. (1989) Am. J. Physiol. 256, c951-c957.
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19. 20. 21.
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Di Corleto, P.E., Bowen-Pope, D.F.(1983) Proc. Natl. Acad. Sci. USA 80, 1919-1923. Bowen-Pope, D.F., and Ross, R. (1982 J. Biol. Chem. 257,5161-5171. GronwaId, R.G.K., Seifert, R.A., and B Owen-Pope, D.F. (1989) J. Biol. Chem. 264, 8120-8125. Heldin, C-H., Backstrgm, G., Ostman, A., Hammacher, A., Ronnstrand, L., Rubin, K., Nister, M., and Westermack, B. (1988) EMBO J. 7, 1387-1393. Hart, C.E., Forstrom, J.W., Kelly, J.D., Seifert, R.A., Smith, R.A., Ross, R., Murray, M.J., and Bowen-Pope, D.F. (,!988) Science 240, 1529-1531. Nister, M., Hammacher, A., Mellstrom, K., Siegbahn, A., Ronnstrand, L., Westermack, B., and Heldin, C-H. (1988) Cell 52, 791-799. Reilly, C.F., and Broski, J.E. (1989) Biochem. Biophys. Res. Commun. 160, 1047-1054. Bjorge, J.D., Paterson, A.J., and Kudlow, J.E. (1989) J. Biol. Chem. 264, 40214027. Adams, M.A., Bobik, A., and Korner, P.I. (1989) Hypertension 14, 191-202.
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