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Increased non-angiotensin II [125I] CGP 42112 binding in rat carotid artery after balloon injury
Peptides, Vol. 17, No. 4, pp. 695-699, 1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All tights reserved 0196.9781/96 $15.00 + .OO
PI1SO196-9781(96)00064-Z
ELSEVIER
Increased Non-Angiotensin II [ 125I]CGP 42112 Binding in Rat Carotid Artery After Balloon Injury MOHAN
VISWANATHAN,”
OLAF
JijHREN,
ANA
M. DE OLIVEIRA
AND
JUAN
M. SAAVEDRA
Section on Pharmacology, Laboratory of Clinical Science, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892-1514, USA Received VISWANATHAN, M., 0. JijHREN, A. M.
DE
14 December
1995
OLIVEIRA AND J. M. SAAVEDRA. Increased
42112 binding in rat carotid artery after balloon injury. PEF’TIDES 17(4) 695-699,
1996.-In
non-angiotensin II [“‘l]CGP this study, [‘251]CGP 42112, a
ligand of high affimty and selectivity for the angiotensin II AT2 receptor, was used to detect and quantify a non-angiotensin II binding site in the balloon-injured carotid artery of the rat. The amount of [ ‘Z51]CGP42112 binding was significantly enhanced in the adventitia of the injured arteries. Localization of the binding site using emulsion autoradiography and immunocytochemistry suggests that the binding sites may be expressed by macrophages in the inflamed tissue surrounding the injured artery. Injury
Arteries
Macrophages
CGP42112
Binding
DEVELOPMENT of nonpeptidic ligands like losartan (2-nbutyl - 4 - chloro - 5 - hydrox;fmethyl - 1 - [ 2 ’ - ( 1H - tetrazol - 5 - yl ) biphenyl-4-yl) methyl] imldazole; DuPont Merck ) and PD 123177 ( 1-(4-amino-3-methylphenyl)methyl-5-diphenylacetyl4,5,6,7_tetrahydro1H-imidazo [4,5-c] pyridine-6-carboxylic acid-2HC1; Parke Davis ) , and a modified pentapeptide analogue, CGP 42 112 [ nicotinic acid-Tyr- (N’-benzyloxycarbonyl-Arg ) LysHis-Pro-Ile-OH; Ciba-Geigy] have led to the identification of angiotensin II (ANG II) receptor subtypes, ATI and AT2 (3,4). Losartan and CGP 42112 have high selectivity for AT, and AT* receptors, respectively. Although most of the physiological functions of ANG II are mediated by ATI receptors, the physiological role of AT2 receptors is still not understood. We have demonstrated that AT2 receptors are highly expressed in the vasculature of fetal and young rats, suggesting a role for this receptor subtype in vascular growth ( 18). These findings led us to examine whether injury-induced vascular smooth muscle proliferation and remodeling of the vascular wall involved AT2 receptors. Surprisingly, our results from those studies indicated that neointima formed as a result of vascular injury only expressed significantly high levels of AT, receptors (21,22). The role of AT, receptors in neointima formation is supported by the finding that losartan, a specific AT, receptor antagonist, significantly inhibits neointima formation after balloon injury ( 10). AT2 receptors were not expressed either in the neointima or in the media of injured arteries (21,22). However, Janiak et al. (8) reported that continuous perivascular administration of CGP 42112, a selective AT1 receptor ligand, after in-
sites
Inflammation
jury of the carotid artery, significantly inhibited the formation of neointima. The high affinity and selectivity of CGP 42112 to AT1 receptors has been exploited in developing [ lz51]CGP 42112 as a ligand to study this receptor subtype (5,6,23). Our recent studies using the AT2 ligand [ lz51]CGP 42112 unexpectedly revealed a novel binding site that is recognized by CGP 42 112 and not by ANG II in various models of brain injury (9,19,20), and in the rat spleen ( 13 ) The expression of these binding sites appears to be associated with activated microglia in brain lesions (9,19,20) and in macrophages in rat spleen ( 13). This binding site is not recognized by AT, antagonist losartan, AT, selective ligand PD 123177, ANG II fragments, or various cytokines and growth factors known to affect macrophage function ( 13,19). The major aim of the present study was to examine whether inflammation resulting from balloon catheter injury and subsequent accumulation of macrophages would result in the expression of [ ?] CGP 42112 binding. We have now examined balloon catheter-injured rat carotid artery, for the presence of [ “51]CGP 42112 binding sites, and report that the amount of [ ‘251]CGP 42112 binding in the adventitia of the injured arteries is enhanced when compared to intact arteries. METHOD
Male Sprague-Dawley rats weighing 300-350 g (ZivicMiller, Zelienople, PA) were used in the study. Balloon catheter injury of the left common carotid artery was performed after rats
’ Requests for reprints should be addressed to Mohan Viswanathan, Ph.D., Section on Pharmacology, Laboratory Room 2D-45, National Institute of Mental Health, 10 Center Dr. MSC 15 14, Bethesda, MD 20892-15 14.
695
of Clinical
Science,
Bldg.
10;
VISWANATHAN
696
.
.
.
.
.
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ET AI
[‘*‘I] CGP 42112 BINDING
697
SITES
IA
ISpecific
Right
binding
Left
1
( B
*
Right
Left
FIG. 2.Quantification of [‘ZSI]CGP42112 binding to intact (right) and balloon catheterinjured (left) carotid arteries 5 days (A) or 8 days (B) after balloon injury. Binding was quantified individually in arteries obtained from eight rats, at each time. Data are means + SEM. The area quantified, in the adventitia, is indicated in Fig. 1. Specific binding: binding obtained in the presence of 1 ,uM CGP 42112. +ANG II: specific binding not inhibited by 1 pM ANG II. Specific binding was significantly higher in the left carotid artery at both 5 days (**p < 0.0001) and 8 days (*p < 0.05) when compared to that
in thk right carotid-artery.
were anesthetized with halothane. Arterial embolectomy (balloon) catheters (2F-Fogarty, Baxter, Santa Ana, CA) introduced via the left external carotid artery were used to injure the common carotid artery (21). The surgery was performed at the ZivicMiller Laboratories. At 5 and 8 days after surgery, groups of animals (n = 8) were anesthetized with sodium pentobarbital, and segments of injured left common carotid artery and intact right common carotid artery were removed along with the surrounding connective tissue. The tissues were immediately frozen in isopentane at -3O”C, and kept frozen at -70°C. Cross sections (16 pm) of the vessels were cut in a cryostat at -17”C, thaw mounted on gelatin-coated glass slides, and dried overnight in a desiccator at 4°C. Care was taken to mount sections from injured and intact arteries on the same slide, adjacent to each other. [‘*‘I]CGP 42112 binding was measured using quantitative autoradiography. The methodologies used for binding and to obtain autoradiograms were identical to the ones that we have described previously ( 13,19.20). CGP 42112 was iodinated (specific activity 2200 Ci/mmol) by New England Nuclear (Boston, MA). Autoradiograms were obtained from sections after incubation using Hyperfilm-3H ( Amersham) and optical densities and disintegrations per minute per milligram of plastic standard were used to generate standard curves by nonlinear fitting using NIH Image 1.4 computerized analysis system (NIMH, Bethesda, MD) (13,19,20). Because specific [ ‘251]CGP 42112 binding was consistently present in the adventitia of the injured arteries, quantification was restricted to the adventitia in both injured and intact
FIG. 1. Autoradiograms of [“‘I]CGP 42112 binding (E-H) 8 days after injury. (A, E) Histology, sections 42112. (C, G) Sections incubated as in (B) and (F) and (F) in the presence of 1 pM ANG II. Area used Solid arrowheads in (A) indicate neointima. Arrows was seen in some (three out cf eight arteries) of the
vessels, as shown in Fig. 1 (B, F). Results were expressed as mean 5 SEM. Student’s t-test was used for comparisons between intact and injured arteries. To recognize the cell types that express the CGP 42112 binding site, emulsion autoradiography for [ “‘I] CGP 42112 binding and immunocytochemistry for macrophages were performed on consecutive sections. Emulsion autoradiography was performed using Kodak NTB-2 emulsion-coated coverslips apposed to sections on slides ( 14). Sections were exposed at 4”C, coverslips developed, stained with hematoxylin and eosin, and examined by bright- and darkfield microscopy. Cryostat sections adjacent to the ones used for binding assay were processed for immunocytochemistry. Sections were fixed in cold acetone (-20°C) for 10 min. Anti-rat macrophage monoclonal antibody ED-l (Serotec, Harlan, IN) was used at a dilution of 1:50. Primary antibody was replaced with normal goat serum as control. Affinity-purified, rat adsorbed, biotinylated anti-mouse IgG (Vector, CA) and peroxidase-conjugated streptavidin-biotin complex (Dako LSAB kit) were used for the detection of immunopositive cells. 3,3’Diaminobenzidine-tetrahydrochloride (Sigma) was used as the substrate. RESULTS In intact arteries, the amount of specific [‘*‘I] CGP 42112 binding was not significantly different (p > 0.05) from the amount of nonspecific binding, and therefore was considered to
in sections of balloon catheter injured left carotid artery (A-D) and intact right carotid artery stained with hematoxylin-eosin. (B, F) Total binding, sections incubated with 0.2 nM [‘Z51]CGP in the presence of 1 M CGP 42112 (nonspecific binding). (D, H) Sections incubated as in (B) for the measurement of [‘251]CGP 42112 binding is represented by the dotted line in (B) and (F). indicate media. Open arrowheads in (B) and (D) point to the binding beneath the neointima that carotid arteries. M, skeletal muscle.
VISWANATHAN
698
ET AL.
contained significantly higher (p < 0.0001 and p < 0.05, respectively) amounts of [ ‘251]CGP 42 112 binding than intact arteries (Fig. 2). The binding was found in the adventitial layer of the injured arteries, and had a heterogeneous distribution (Fig. 1). In three out of eight animals at 8 days after balloon injury, binding was seen in a layer of cells along the border between the internal elastic lamina and neointima [Fig. 1 (A-D)], in addition to that in the adventitia. Although macrophages (ED-l positive cells) were not noticed in the intact right carotid artery, they were present in the injured left carotid artery (Fig. 3). Macrophages were detected in the adventitia. In three out of eight animals at 8 days after balloon injury, macrophages were also detected along the internal elastic lamina underneath the neointimal cells. Using emulsion autoradiography, aggregation of silver grains could be localized to the same areas where macrophages were detected (Fig. 3). The distribution of macrophages in the adventitia of the injured artery correlated with that of the cells expressing [ ‘251]CGP 42112 binding, indicating that a significant amount of binding was associated with macrophages. DISCUSSION
c
‘s(tm’
FIG. 3. Emulsion autoradiography of [‘*‘I]CGP 42112 binding and immunocytochemical detection of ED-l-positive cells in the left carotid artery, 8 days after balloon injury. (A) Histology of section used for [?]CGP 42112 binding, stained with hematoxylin-eosin under brightfield illumination. Solid arrowhead indicates neointima. (B) [‘251]CGP 42112 binding under darkfield illumination showing localization of silver grains predominantly in clusters (open arrowheads) in the adventitia. Immunocytochemical staining of ED-l-positive cells in an adjacent section to that in B. Note that the distribution of ED-l positive cells (open arrowheads) correlates well with that of the density of distribution of silver grains in (B). Arrows are in the lumen of the artery pointing to the internal elastic lamina.
be at the limit of detection of our method. There was significantly increased expression of specific [ ‘251]CGP 42112 binding in the injured carotid arteries (Figs. 1 and 2). Angiotensin II ( 1 PM) did not inhibit [ iZ51]CGP 42112 binding, indicating that the identified binding sites were not angiotensin receptors (Figs. 1 and 2). At both 5 and 8 days after balloon injury, the injured arteries
We recently reported that in the rat, ANG II AT, receptor expression is elevated in the neointima resulting from balloon catheter injury (21,22). In this study, we describe for the first time that one of the ANG II receptor ligands CGP 42112, which selectively binds to ANG II ATI receptors, recognizes a non-angiotensin binding site in balloon-injured carotid arteries. We have demonstrated previously that in the balloon-injured rat aorta and carotid arteries, it is the neointimal smooth muscle cells that express enhanced levels of ANG II AT, receptors (21,22). In contrast, the present results suggest that the appearance of the CGP 42112 binding sites is correlated with the accumulation of macrophages in the adventitia of ballooninjured carotid artery. These results augment our recent findings that CGP 42112 binding sites are expressed by activated microglia and spleen macrophages, and support our proposal that the macrophage may be the major cell type in the body that expresses CGP 42112 binding sites (9,13,19,20). A few studies in the past have suggested that perturbations in the vasa vasorum located in the adventitia may lead to hyperplastic changes in the intima ( 1,2,11) . In the present study, macrophage accumulation was mainly seen in the inflammation tissue around the adventitia of balloon-injured carotid arteries. It is possible that growth factors and cytokines expressed by macrophages may cause smooth muscle migration and proliferation ( 15- 17)) which constitute neointima formation. However, the involvement of macrophages has not yet been perceived as an important mechanism in the process of myointimal hyperplasia and neointima formation after balloon catheter injury. Inflammation and macrophage accumulation are nevertheless considered to be involved in the formation of atherosclerotic lesions ( 15 17 ) Therefore, it is of importance to study ligands like CGP 42112 that selectively recognize binding sites in macrophages and possibly affect their metabolism. Continuous perivascular administration of CGP 42 112 for 14 days after injury of the carotid artery has indeed been shown to significantly inhibit the formation of neointima (8). The conclusion drawn from this study was that CGP 42112 acted as an antagonist on AT2 receptors in neointima. However, there are several reasons to suspect that CGP 42112 did not cause inhibition of neointima formation through its action on AT2 receptors. First, there are no AT2 receptors in the neointima (21,22). Second, it is possible that perivascular administration of very high concentration (mM) of CGP 42112 may have affected the neoin-
[ ‘*?I CGP 42 112 BIND tNG SITES
timal AT, receptors. Third, it is possible that the effect of CGP 42112 was mediated through its action on the non-ANG II CGP 42112 binding sites described here in the inflamed adventitia. Finally, CGP 42112 has never been shown to be an antagonist because the function of A.T2 receptors or that of the non-ANG II receptors has not yet been fully clarified. Rather, CGP 42112 was recently found to act as an agonist of AT2 receptors (7,12).
In summary, we have identified enhanced expression of a nonANG II binding site in balloon-injured carotid arteries of the rat using [ lz51]CGP 42112, a ligand originally developed for the study of ANG II AT2 receptors. Localization of this binding site using immunocytochemistry suggests that macrophages which infiltrate into the injured vessel may be the cell type that expresses these binding sites.
REFERENCES 1. Barker, S. G. E.; Talben, A.; Cottam, S.; Baskerville, P. A.; Martin, .I. F. Arterial intimal hyperplasia follows occlusion of the adventitial vasa vasorum in the pig. Arterioscl. Thromb. 13:70-77; 1993. 2. Barker, S. G. E.; Tilling, L. C.; Miller, G. C.; et al. The adventitia and atherogenesis: Removal initiates intimal proliferation in the rabbit which regresses on generation of a ‘neoadventitia.’ Atherosclerosis 105:131-144; 1994. 3. Bottari, S. P.; de Gasparo, M.; Steckelings, U. M.; Levens, N. R. Angiotensin II receptor :subtypes: Characterization, signaling mechanisms, and possible physiological implications. Front. Neuroendocrinol. 14:123-171; 1993. 4. Bumpus, F. M.; Catt, K. J.; Chiu, A. T.; et al. Nomenclature for angiotensin receptors: P, report of the nomenclature committee of the council for high blood pressure research. Hypertension 17:720721; 1991. 5. Heemskerk, F. M. J.; Zorad, S.; Seltzer, A.; Saavedra, J. M. Characterization of brain angiotensin II ATI receptor subtype using [‘251]CGP42112A. Neuroreport4:103-105; 1993. 6. Heemskerk, F. M. J.; Saavedra, J. M. Quantitative autoradiography of angiotensin II AT2 rs?ceptors with [‘*51]CGP42112. Brain Res. 677:29-38; 1995. 7. Huang, X. C.; Richards, E. M.; Sumners, C. Angiotensin II type 2 receptor mediated stimulation of protein phosphatase 2A in rat hypothalamic/brainstem ncuronal cocultures. J. Neurochem. 65:21312137; 1995. 8. Janiak, P.; Pillon, A.; Prost, J. F.; Vilane, J. P. Role of angiotensin subtype 2 receptor in neointima formation after vascular injury. Hypertension 201737-745; 1992. 9. JGhren, 0.; Viswanathan, M.; Saavedra, J. M. Expression of nonangiotensin II [lz51] CGP 42112 binding sites on activated microglia after kainic acid induced neurodegeneration. Brain Res. 702: 153161; 1995. 10. Kauffman, R. F.; Bean, J. S.; Zimmerman, K. M.; Brown, R. F.; Steinberg, M. I. Losartan, a nonpeptide angiotensin II (Ang II) receptor antagonist, inhibits neointima formation following balloon injury to rat carotid arteries. Life Sci. 49:PL223-PL228; 1991. 11. Martin, J. F.; Booth, R. F. G.; Moncada, S. Arterial wall hypoxia following hypoperfusion through the vasa vasorum is an initial lesion in atherosclerosis. E:ur. J. Clin. Invest. 21:355-359; 1991. 12. NLveri, L.; Strijmberg, c’.; Saavedra, J. M. Angiotensin II AT* stimulation extends the upper limit of cerebral blood flow autoregulation:
13.
14.
15. 16. 17.
18.
19.
20.
21.
22.
23.
Agonist effects of CGP 42 112 and PD 1233 19. J. Cereb. Blood Flow Metab. 14:38-44; 1994. Oliveira, A. M.; Viswanathan, M.; Heemskerk, F. M. J.; Correa, F. M. A.; Saavedra, J. M. Specific, nonangiotensin, [?I CGP 42112 binding sites in rat spleen macrophages. Biochem. Biophys. Res. Commun. 200:1049-1058; 1994. Rhodes, K. J.; Meiners, K. A.; Rosene, D. L. A novel hinge system and incubation chamber for emulsion-coated coverslip autoradiography. J. Histochem. Cytochem. 41:1419- 1427; 1993. Ross, R. The pathogenesis of atherosclerosis: A perspective for the 1990s. Nature 362:801-809; 1993. Ross, R. Cell biology of atherosclerosis. Annu. Rev. Physiol. 57:791-804: 1995. Ross, R.; Masuda, J.; Raines, E. W.; et al. Localization of PDGF-B protein in macrophages in all phases of atherogenesis. Science 248:1009-1012; 1990. Viswanathan, M.; Tsutsumi, K.; Correa, F. M. A.; Saavedra, J. M. Changes in the expression of angiotensin receptor subtypes in the rat aorta during development. Biochem. Biophys. Res. Commun. 179:1361-1367; 1991. Viswanathan, M.; Oliveira, A. M.; Correa, F. M. A.; Saavedra, J. M. Expression of a novel nonangiotensin II [lz51]CGP 42112 binding site in healing wounds of the rat brain. Brain Res. 658:265-270: 1994. Viswanathan, M.; Oliveira, A. M.; Wu, R.-M.; Chiueh, C. C.; Saavedra, J. M. [‘*‘I] CGP 42112 reveals a non-angiotensin II binding site in 1-methyl-4-phenylpyridine (MPP+)-induced brain injury. Cell. Mol. Neurobiol. 14:97-102; 1994. Viswanathan, M.; Seltzer, A.; Saavedra, J. M. Heterogenous expression of angiotensin II receptors in neointima of rat carotid artery and aorta after balloon catheter injury. Peptides 1.5:1205- 1212; 1994. Viswanathan, M.; StrGmberg, C.; Seltzer, A.; Saavedra, J. M. Balloon angioplasty enhances the expression of angiotensin II ATI receptors in neointima of rat aorta. J. Clin. Invest. 90:1707- 1712; 1992. Whitebread, S. E.; Taylor, V.; Bottari, S. P.; Kamber, B.; de Gasparo, M. Radioiodinated CGP 42112A: A novel high affinity and highly selective ligand for the characterization of angiotensin AT2 receptors. Biochem. Biophys. Res. Commun. 181:1365-1371; 1991.
PI1SO196-9781(96)00064-Z
ELSEVIER
Increased Non-Angiotensin II [ 125I]CGP 42112 Binding in Rat Carotid Artery After Balloon Injury MOHAN
VISWANATHAN,”
OLAF
JijHREN,
ANA
M. DE OLIVEIRA
AND
JUAN
M. SAAVEDRA
Section on Pharmacology, Laboratory of Clinical Science, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892-1514, USA Received VISWANATHAN, M., 0. JijHREN, A. M.
DE
14 December
1995
OLIVEIRA AND J. M. SAAVEDRA. Increased
42112 binding in rat carotid artery after balloon injury. PEF’TIDES 17(4) 695-699,
1996.-In
non-angiotensin II [“‘l]CGP this study, [‘251]CGP 42112, a
ligand of high affimty and selectivity for the angiotensin II AT2 receptor, was used to detect and quantify a non-angiotensin II binding site in the balloon-injured carotid artery of the rat. The amount of [ ‘Z51]CGP42112 binding was significantly enhanced in the adventitia of the injured arteries. Localization of the binding site using emulsion autoradiography and immunocytochemistry suggests that the binding sites may be expressed by macrophages in the inflamed tissue surrounding the injured artery. Injury
Arteries
Macrophages
CGP42112
Binding
DEVELOPMENT of nonpeptidic ligands like losartan (2-nbutyl - 4 - chloro - 5 - hydrox;fmethyl - 1 - [ 2 ’ - ( 1H - tetrazol - 5 - yl ) biphenyl-4-yl) methyl] imldazole; DuPont Merck ) and PD 123177 ( 1-(4-amino-3-methylphenyl)methyl-5-diphenylacetyl4,5,6,7_tetrahydro1H-imidazo [4,5-c] pyridine-6-carboxylic acid-2HC1; Parke Davis ) , and a modified pentapeptide analogue, CGP 42 112 [ nicotinic acid-Tyr- (N’-benzyloxycarbonyl-Arg ) LysHis-Pro-Ile-OH; Ciba-Geigy] have led to the identification of angiotensin II (ANG II) receptor subtypes, ATI and AT2 (3,4). Losartan and CGP 42112 have high selectivity for AT, and AT* receptors, respectively. Although most of the physiological functions of ANG II are mediated by ATI receptors, the physiological role of AT2 receptors is still not understood. We have demonstrated that AT2 receptors are highly expressed in the vasculature of fetal and young rats, suggesting a role for this receptor subtype in vascular growth ( 18). These findings led us to examine whether injury-induced vascular smooth muscle proliferation and remodeling of the vascular wall involved AT2 receptors. Surprisingly, our results from those studies indicated that neointima formed as a result of vascular injury only expressed significantly high levels of AT, receptors (21,22). The role of AT, receptors in neointima formation is supported by the finding that losartan, a specific AT, receptor antagonist, significantly inhibits neointima formation after balloon injury ( 10). AT2 receptors were not expressed either in the neointima or in the media of injured arteries (21,22). However, Janiak et al. (8) reported that continuous perivascular administration of CGP 42112, a selective AT1 receptor ligand, after in-
sites
Inflammation
jury of the carotid artery, significantly inhibited the formation of neointima. The high affinity and selectivity of CGP 42112 to AT1 receptors has been exploited in developing [ lz51]CGP 42112 as a ligand to study this receptor subtype (5,6,23). Our recent studies using the AT2 ligand [ lz51]CGP 42112 unexpectedly revealed a novel binding site that is recognized by CGP 42 112 and not by ANG II in various models of brain injury (9,19,20), and in the rat spleen ( 13 ) The expression of these binding sites appears to be associated with activated microglia in brain lesions (9,19,20) and in macrophages in rat spleen ( 13). This binding site is not recognized by AT, antagonist losartan, AT, selective ligand PD 123177, ANG II fragments, or various cytokines and growth factors known to affect macrophage function ( 13,19). The major aim of the present study was to examine whether inflammation resulting from balloon catheter injury and subsequent accumulation of macrophages would result in the expression of [ ?] CGP 42112 binding. We have now examined balloon catheter-injured rat carotid artery, for the presence of [ “51]CGP 42112 binding sites, and report that the amount of [ ‘251]CGP 42112 binding in the adventitia of the injured arteries is enhanced when compared to intact arteries. METHOD
Male Sprague-Dawley rats weighing 300-350 g (ZivicMiller, Zelienople, PA) were used in the study. Balloon catheter injury of the left common carotid artery was performed after rats
’ Requests for reprints should be addressed to Mohan Viswanathan, Ph.D., Section on Pharmacology, Laboratory Room 2D-45, National Institute of Mental Health, 10 Center Dr. MSC 15 14, Bethesda, MD 20892-15 14.
695
of Clinical
Science,
Bldg.
10;
VISWANATHAN
696
.
.
.
.
.
_.,.~
_.
. .
,...-
_.
_.
.-
ET AI
[‘*‘I] CGP 42112 BINDING
697
SITES
IA
ISpecific
Right
binding
Left
1
( B
*
Right
Left
FIG. 2.Quantification of [‘ZSI]CGP42112 binding to intact (right) and balloon catheterinjured (left) carotid arteries 5 days (A) or 8 days (B) after balloon injury. Binding was quantified individually in arteries obtained from eight rats, at each time. Data are means + SEM. The area quantified, in the adventitia, is indicated in Fig. 1. Specific binding: binding obtained in the presence of 1 ,uM CGP 42112. +ANG II: specific binding not inhibited by 1 pM ANG II. Specific binding was significantly higher in the left carotid artery at both 5 days (**p < 0.0001) and 8 days (*p < 0.05) when compared to that
in thk right carotid-artery.
were anesthetized with halothane. Arterial embolectomy (balloon) catheters (2F-Fogarty, Baxter, Santa Ana, CA) introduced via the left external carotid artery were used to injure the common carotid artery (21). The surgery was performed at the ZivicMiller Laboratories. At 5 and 8 days after surgery, groups of animals (n = 8) were anesthetized with sodium pentobarbital, and segments of injured left common carotid artery and intact right common carotid artery were removed along with the surrounding connective tissue. The tissues were immediately frozen in isopentane at -3O”C, and kept frozen at -70°C. Cross sections (16 pm) of the vessels were cut in a cryostat at -17”C, thaw mounted on gelatin-coated glass slides, and dried overnight in a desiccator at 4°C. Care was taken to mount sections from injured and intact arteries on the same slide, adjacent to each other. [‘*‘I]CGP 42112 binding was measured using quantitative autoradiography. The methodologies used for binding and to obtain autoradiograms were identical to the ones that we have described previously ( 13,19.20). CGP 42112 was iodinated (specific activity 2200 Ci/mmol) by New England Nuclear (Boston, MA). Autoradiograms were obtained from sections after incubation using Hyperfilm-3H ( Amersham) and optical densities and disintegrations per minute per milligram of plastic standard were used to generate standard curves by nonlinear fitting using NIH Image 1.4 computerized analysis system (NIMH, Bethesda, MD) (13,19,20). Because specific [ ‘251]CGP 42112 binding was consistently present in the adventitia of the injured arteries, quantification was restricted to the adventitia in both injured and intact
FIG. 1. Autoradiograms of [“‘I]CGP 42112 binding (E-H) 8 days after injury. (A, E) Histology, sections 42112. (C, G) Sections incubated as in (B) and (F) and (F) in the presence of 1 pM ANG II. Area used Solid arrowheads in (A) indicate neointima. Arrows was seen in some (three out cf eight arteries) of the
vessels, as shown in Fig. 1 (B, F). Results were expressed as mean 5 SEM. Student’s t-test was used for comparisons between intact and injured arteries. To recognize the cell types that express the CGP 42112 binding site, emulsion autoradiography for [ “‘I] CGP 42112 binding and immunocytochemistry for macrophages were performed on consecutive sections. Emulsion autoradiography was performed using Kodak NTB-2 emulsion-coated coverslips apposed to sections on slides ( 14). Sections were exposed at 4”C, coverslips developed, stained with hematoxylin and eosin, and examined by bright- and darkfield microscopy. Cryostat sections adjacent to the ones used for binding assay were processed for immunocytochemistry. Sections were fixed in cold acetone (-20°C) for 10 min. Anti-rat macrophage monoclonal antibody ED-l (Serotec, Harlan, IN) was used at a dilution of 1:50. Primary antibody was replaced with normal goat serum as control. Affinity-purified, rat adsorbed, biotinylated anti-mouse IgG (Vector, CA) and peroxidase-conjugated streptavidin-biotin complex (Dako LSAB kit) were used for the detection of immunopositive cells. 3,3’Diaminobenzidine-tetrahydrochloride (Sigma) was used as the substrate. RESULTS In intact arteries, the amount of specific [‘*‘I] CGP 42112 binding was not significantly different (p > 0.05) from the amount of nonspecific binding, and therefore was considered to
in sections of balloon catheter injured left carotid artery (A-D) and intact right carotid artery stained with hematoxylin-eosin. (B, F) Total binding, sections incubated with 0.2 nM [‘Z51]CGP in the presence of 1 M CGP 42112 (nonspecific binding). (D, H) Sections incubated as in (B) for the measurement of [‘251]CGP 42112 binding is represented by the dotted line in (B) and (F). indicate media. Open arrowheads in (B) and (D) point to the binding beneath the neointima that carotid arteries. M, skeletal muscle.
VISWANATHAN
698
ET AL.
contained significantly higher (p < 0.0001 and p < 0.05, respectively) amounts of [ ‘251]CGP 42 112 binding than intact arteries (Fig. 2). The binding was found in the adventitial layer of the injured arteries, and had a heterogeneous distribution (Fig. 1). In three out of eight animals at 8 days after balloon injury, binding was seen in a layer of cells along the border between the internal elastic lamina and neointima [Fig. 1 (A-D)], in addition to that in the adventitia. Although macrophages (ED-l positive cells) were not noticed in the intact right carotid artery, they were present in the injured left carotid artery (Fig. 3). Macrophages were detected in the adventitia. In three out of eight animals at 8 days after balloon injury, macrophages were also detected along the internal elastic lamina underneath the neointimal cells. Using emulsion autoradiography, aggregation of silver grains could be localized to the same areas where macrophages were detected (Fig. 3). The distribution of macrophages in the adventitia of the injured artery correlated with that of the cells expressing [ ‘251]CGP 42112 binding, indicating that a significant amount of binding was associated with macrophages. DISCUSSION
c
‘s(tm’
FIG. 3. Emulsion autoradiography of [‘*‘I]CGP 42112 binding and immunocytochemical detection of ED-l-positive cells in the left carotid artery, 8 days after balloon injury. (A) Histology of section used for [?]CGP 42112 binding, stained with hematoxylin-eosin under brightfield illumination. Solid arrowhead indicates neointima. (B) [‘251]CGP 42112 binding under darkfield illumination showing localization of silver grains predominantly in clusters (open arrowheads) in the adventitia. Immunocytochemical staining of ED-l-positive cells in an adjacent section to that in B. Note that the distribution of ED-l positive cells (open arrowheads) correlates well with that of the density of distribution of silver grains in (B). Arrows are in the lumen of the artery pointing to the internal elastic lamina.
be at the limit of detection of our method. There was significantly increased expression of specific [ ‘251]CGP 42112 binding in the injured carotid arteries (Figs. 1 and 2). Angiotensin II ( 1 PM) did not inhibit [ iZ51]CGP 42112 binding, indicating that the identified binding sites were not angiotensin receptors (Figs. 1 and 2). At both 5 and 8 days after balloon injury, the injured arteries
We recently reported that in the rat, ANG II AT, receptor expression is elevated in the neointima resulting from balloon catheter injury (21,22). In this study, we describe for the first time that one of the ANG II receptor ligands CGP 42112, which selectively binds to ANG II ATI receptors, recognizes a non-angiotensin binding site in balloon-injured carotid arteries. We have demonstrated previously that in the balloon-injured rat aorta and carotid arteries, it is the neointimal smooth muscle cells that express enhanced levels of ANG II AT, receptors (21,22). In contrast, the present results suggest that the appearance of the CGP 42112 binding sites is correlated with the accumulation of macrophages in the adventitia of ballooninjured carotid artery. These results augment our recent findings that CGP 42112 binding sites are expressed by activated microglia and spleen macrophages, and support our proposal that the macrophage may be the major cell type in the body that expresses CGP 42112 binding sites (9,13,19,20). A few studies in the past have suggested that perturbations in the vasa vasorum located in the adventitia may lead to hyperplastic changes in the intima ( 1,2,11) . In the present study, macrophage accumulation was mainly seen in the inflammation tissue around the adventitia of balloon-injured carotid arteries. It is possible that growth factors and cytokines expressed by macrophages may cause smooth muscle migration and proliferation ( 15- 17)) which constitute neointima formation. However, the involvement of macrophages has not yet been perceived as an important mechanism in the process of myointimal hyperplasia and neointima formation after balloon catheter injury. Inflammation and macrophage accumulation are nevertheless considered to be involved in the formation of atherosclerotic lesions ( 15 17 ) Therefore, it is of importance to study ligands like CGP 42112 that selectively recognize binding sites in macrophages and possibly affect their metabolism. Continuous perivascular administration of CGP 42 112 for 14 days after injury of the carotid artery has indeed been shown to significantly inhibit the formation of neointima (8). The conclusion drawn from this study was that CGP 42112 acted as an antagonist on AT2 receptors in neointima. However, there are several reasons to suspect that CGP 42112 did not cause inhibition of neointima formation through its action on AT2 receptors. First, there are no AT2 receptors in the neointima (21,22). Second, it is possible that perivascular administration of very high concentration (mM) of CGP 42112 may have affected the neoin-
[ ‘*?I CGP 42 112 BIND tNG SITES
timal AT, receptors. Third, it is possible that the effect of CGP 42112 was mediated through its action on the non-ANG II CGP 42112 binding sites described here in the inflamed adventitia. Finally, CGP 42112 has never been shown to be an antagonist because the function of A.T2 receptors or that of the non-ANG II receptors has not yet been fully clarified. Rather, CGP 42112 was recently found to act as an agonist of AT2 receptors (7,12).
In summary, we have identified enhanced expression of a nonANG II binding site in balloon-injured carotid arteries of the rat using [ lz51]CGP 42112, a ligand originally developed for the study of ANG II AT2 receptors. Localization of this binding site using immunocytochemistry suggests that macrophages which infiltrate into the injured vessel may be the cell type that expresses these binding sites.
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