Introduction

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Increasing evidence indicates that the renin-angiotensin system is important in the physiology and pathology of the heart and that Ang II acts as a growth factor, influencing myocardial hypertrophy and fibrosis (Blaufarb and Sonnenblick, 1996; Pfeffer et al., 1995; Weber et al., 1993; Weber and Brilla, 1991). Expression of angiotensinogen, renin, ACE and Ang II receptors has been demonstrated in human cardiac tissues (Morgan et al., 1994; Paul et al., 1993; Schütz et al., 1996) and changes in the expression of these genes associated with cardiac hypertrophy, myocardial infarction and impaired cardiac function (Blaufarb and Sonnenblick, 1996; Pfeffer et al., 1995; Weber et al., 1993; Weber and Brilla, 1991). At the cellular level, Ang II induces expression of both immediate-early genes and late markers of cardiac hypertrophy in neonatal rat myocytes (Sadoshima and Izumo, 1993) and stimulates mitogenesis, collagen synthesis and expression of transcription factors in cultured rat cardiac fibroblasts (Crabos et al., 1994; Sadoshima and Izumo, 1993; Schorb et al., 1993; Villarreal et al., 1993). Although relatively little is known about the growth-related actions of Ang II in the human heart, it has been reported to influence collagen (Brilla et al., 1995) and DNA synthesis in isolated human cardiac fibroblasts (Neu et al., 1994, 1996). The importance of the renin angiotensin system in humans has also been underlined by the effectiveness of ACE inhibitors in the treatment of patients with various forms of heart disease (Blaufarb and Sonnenblick, 1996; Pfeffer et al., 1995; Weber et al., 1993; Weber and Brilla, 1991).

The actions of Ang II are mediated by specific membrane bound receptors, of which two main subtypes have been identified, AT₁ and AT₂ receptors, by their pharmacological and molecular characteristics (Timmermans et al., 1993). Although both receptors have a similar affinity for Ang II and the peptide antagonist (Sar¹,Ile⁸)Ang II, they may be distinguished by their distinct affinity for receptor selective peptide analogs and nonpeptide compounds. AT₁ receptors display a high affinity for nonpeptide antagonists such as losartan (Chiu et al., 1990; Whitebread et al., 1989) and eprosartan (Weinstock et al., 1991), whereas AT₂ receptors have high affinity for the nonpeptide antagonist PD 123319 (Dudley et al., 1990), and peptide analogs such as CGP 42112A (Whitebread et al., 1991) and [p-amino-Phe⁶]Ang II (Speth and Kim, 1990). The two receptors may also be identified on the basis of their sensitivity to the disulfide reducing agent DTT, binding to AT₁ receptors being inhibited by DTT, whereas that to AT₂ receptors is unaffected or enhanced in the presence of DTT (Chiu et al., 1989; Whitebread et al., 1989). Agonist binding to the two receptors also exhibits a differential sensitivity to guanine nucleotides (Dudley et al., 1990). Using these criteria, AT₁ and AT₂ receptors have been identified on isolated rat cardiac myocytes (Meggs et al., 1993; Reiss et al., 1993) and fibroblasts (Crabos et al., 1994; Sadoshima and Izumo, 1993; Schorb et al., 1993; Villarreal et al., 1993) and in the myocardium of several mammals (Rogg et al., 1990; Chang and Lotti, 1991; Nozawa et al., 1994), including humans (Nozawa et al., 1994; Regitz-Zagrosek et al., 1995; Rogg et al., 1996). The nucleotide sequences and genomic organization of the human AT₂ (Martin and Elton, 1995; Martin et al., 1994) and AT₁ receptor (Bergsma et al., 1992; Guo et al., 1994; Konishi et al., 1994) genes have been determined, and the expression of both receptors has been demonstrated in human heart. At the receptor level, however, there are significant species differences in the distribution of Ang II receptor subtypes, with most studies indicating that AT₁ sites represent the majority of cardiac receptors in experimental animals (Nozawa et al., 1994), whereas the AT₂ subtype predominates in membrane preparations of the human heart (Nozawa et al., 1994; Regitz-Zagrosek et al., 1995; Rogg et al., 1996).

Expression of the AT₂ receptor subtype in the cardiovascular system changes during development (Hunt et al., 1995; Matsubara et al., 1994; Sechi et al., 1992; Shanmugam et al., 1996) is growth dependent and modulated by vasoactive factors such as Ang II (Kijima et al., 1996). Cardiac expression of the AT₂ receptor is also increased, together with the AT₁ receptor, in the spontaneously hypertensive rat (Suzuki et al., 1993) and after myocardial infarction (Nio et al., 1995). Furthermore, a switch from AT₁ to AT₂ receptor subtype expression has been demonstrated in the hypertrophic rat heart after pressure and volume overload (Lopez et al., 1994; Poole et al., 194) and in a canine model of right ventricular hypertrophy (Lee et al., 1996). Although the functional significance of the cardiac AT₂ receptor has yet to be established, it has been shown to mediate a number of Ang II-induced responses in the cardiovascular system. These include modulation of collagen metabolism in isolated rat cardiac fibroblasts (Brilla et al., 1994), stimulation of arachidonic acid release from rat cardiac myocytes (Lokuta et al., 1994) and inhibition of cell proliferation and growth by antagonizing the growth-promoting effects of the AT₁ receptor (Booz and Baker, 1996; Levy et al., 1996; Nakajima et al., 1995; Stoll et al., 1995).

The proportion of Ang II receptors in human atrial tissues is influenced by cardiac function, with the number of AT₂ binding sites increasing and AT₁ sites declining with the degree of impairment of cardiac function (Rogg et al., 1996). Establishing the localization of these receptors, in ventricular as well as atrial tissues, and determining disease-related differences in receptor distribution represent important steps toward further understanding the cellular targets for Ang II and the pathophysiological significance of Ang II receptor subtypes in the human heart. Two studies to date have attempted to localize these receptors in human cardiac tissues but were either unable to distinguish receptor subtypes due to the lack of selective ligands (Urata et al., 1989) or because only the presence of receptors in the right atrial appendage was examined (Brink et al., 1996). Furthermore, although ACE expression is reported to be increased in the failing human heart (Studer et al., 1994; Zisman et al., 1995) the cellular distribution of ACE expression was not determined and has not been compared with that of Ang II receptors in human cardiac tissues.

The purpose of this study was therefore to determine the localization and binding characteristics of Ang II receptors in the explanted failing and normal human heart, using selective peptide analogs and nonpeptide antagonists to distinguish AT₁ and AT₂ receptor subtypes, and to compare by autoradiography the distribution of specific Ang II and ACE binding sites.

	Methods

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Materials. ¹²⁵I-(Sar¹,Ile⁸)Ang II and ¹²⁵I-CGP 42112A [¹²⁵I-(N--nicotinoyl-Tyr-(N--CBZ-Arg)Lys-His-Pro-Ile-OH); specific activity, 2200 Ci/mmol] were purchased from DuPont-New England Nuclear (Stevenage, UK). ¹²⁵I-Ang IV (specific activity, 2000 Ci/mmol) was obtained from Amersham International (Amersham, UK). Losartan [DuP 753: 2-n-butyl-4-chloro-5-(hydroxymethyl)-l-[(2(1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidazole] was obtained from DuPont Merck (Wilmington, DE). Eprosartan (SKF-108566: E--2-[2-butyl-1-[(carboxyphenyl)methyl]1H-imidazol-5-yl)methylene]-2-thiophenepropanoic acid)was from SmithKline Beecham (Collegeville, PA). PD123319 [1(4-amino-3-methylphenyl)methyl-5-diphenylacetyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid-2HCl] was from Parke-Davis (Ann Arbor, MI). Unlabeled CGP 42112A and [p-amino-Phe⁶]Ang II were purchased from Neosytem Laboratorie (Strasbourg, France). Unlabeled (Sar¹,Ile⁸)Ang II, Ang II, Ang IV (Ang II 3-8 fragment) and other chemicals were purchased from Sigma Chemical (Poole, UK). Primary antisera (JC/70A, A082) were obtained from DAKO (High Wycombe, UK). Biotinylated horse anti-mouse IgG and goat anti-rabbit IgG and avidin-biotin complex were from Vector Laboratories (Peterborough, UK).

Patients and tissues. Heart tissues were obtained from patients undergoing cardiac transplantation and donor tissues not suitable for transplantation (table 1) and comprised transmural samples from the lateral walls of the ventricles and the interventricular septum. Atrial tissues were also obtained in a proportion of cases. Tissue samples were either snap-frozen, for mRNA extraction, or mounted on cork mats, embedded in mounting medium (Tissue Tek; Miles, Elkhart, IN) and frozen in melting dichlorodifluoromethane (Arcton-12; I.C.I., Runcorn, Cheshire, UK) or isopentane, for autoradiography and staining. Frozen tissues were stored either in liquid nitrogen or at 40°C.

In vitro autoradiographic analysis of Ang II binding sites. Cryostat sections (10 µm thick) were cut, and thaw-mounted onto chrome-alum-gelatin-coated microscope slides and stored at 20°C. Sections were preincubated in 10 mmol/liter sodium phosphate buffer (pH 7.4) containing 150 mmol/liter NaCl, 10 mmol/liter MgCl₂ and 28 µmol/liter bacitracin for 10 min at 20° to 22°C, followed by incubation in buffer containing 250 pmol/liter ¹²⁵I-(Sar¹,Ile⁸)Ang II and 1% bovine serum albumin, for 5 to 180 min at 20° to 22°C, as previously described in studies on other human tissues (Knock et al., 1994; Walsh et al., 1994). Sections were washed in ice-cold buffer twice for 5 min at 4°C, quickly rinsed in ice-cold distilled water and dried under a stream of cold air. Nonspecific binding was determined by coincubating adjacent sections with 10⁶ mol/liter unlabeled (Sar¹,Ile⁸)Ang II; the proportion of AT₁ and AT₂ binding sites was assessed by coincubating adjacent sections with 10⁵ mol/liter AT₁-selective (losartan) and AT₂-selective (PD123319) antagonists. Association time course experiments were undertaken at 20° to 22°C, and ligand stability was assessed by reapplying the incubation medium to fresh adjacent sections, under identical conditions. Ang II binding sites were further characterized by incubating consecutive sections with 250 pmol/liter ¹²⁵I-(Sar¹,Ile⁸)Ang II in the presence of increasing concentrations (10¹⁰ to 10⁵ mol/liter) of unlabeled (Sar¹,Ile⁸)Ang II, Ang II, losartan, eprosartan, PD123319, CGP 42112A or [p-amino-Phe⁶]Ang II. Saturation studies were performed by incubating consecutive sections with increasing concentrations (50-2000 pmol/liter) of ¹²⁵I-(Sar¹,Ile⁸)Ang II for 150 min at 20° to 22°C. The sensitivity of ligand binding to the sulfhydryl reducing agent DTT was investigated by incubating sections in the absence and presence of 10 mmol/liter DTT. The distribution of ¹²⁵I-(Sar¹,Ile⁸)Ang II binding sites was also compared with that of 250 pmol/liter ¹²⁵I-CGP 42112A and ¹²⁵I-Ang IV.

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Calculations and statistical analysis. The equilibrium dissociation constant (K_d) and maximum binding capacity (B_max) were derived from saturation experiments. The concentrations of unlabeled ligands producing 50% inhibition of binding (IC₅₀) of 250 pmol/liter ¹²⁵I-(Sar¹,Ile⁸)Ang II were calculated for each case from binding inhibition experiments. Inhibition constant (K_i) values were derived from IC₅₀ values according to the equation of Cheng and Prusoff (1973): K_i = IC₅₀/(1 + [L]/K_d), where [L] is the ligand concentration and K_d is the equilibrium dissociation constant derived from saturation experiments. Data from inhibition experiments were fitted to one- and two-site models, and the "best fit" was compared by F tests. Curve fitting and experimental derivation of binding constants were performed by iterative nonlinear regression using GraphPAD Inplot version 4 (GraphPAD Software, San Diego, CA). Binding data are expressed as arithmetic or geometric means as appropriate, with 95% CI or S.E.M. values. Comparisons between groups were made by Student's t test (two-tailed) or one-way analysis of variance followed by Bonferroni's correction, as appropriate. Values of P < .05 were taken as significant.

In vitro autoradiographic localization of ACE. Autoradiographic techniques were also used to localize ACE in unfixed cryostat sections of human heart, using a ¹²⁵I-labeled tyrosyl-derivative of lisinopril, ¹²⁵I-351A (N-[(s)-1-carboxy-3-phenylpropyl]-L-lysyl-tyrosyl-L-proline), essentially as described previously (Allen et al., 1988). ¹²⁵I-351A (specific activity, 2000 Ci/mmol) was iodinated, as described previously (Fyhrquist et al., 1984), and high-performance liquid chromatography purification was performed with a Waters C18 µBondapak analytical column using a mobile phase consisting of 88% 0.04 mol/liter phosphoric acid with triethylamine, pH 3 (A), and 12% acetonitrile (B) for 10 min, followed by a linear gradient from 12% to 25% B over the next 40 min. The retention time for the monoiodinated product was 24 min. The stability of ¹²⁵I-351A has been determined previously (Jackson et al., 1986). Sections were processed as described above for Ang II receptor autoradiography and incubated in phosphate buffer containing 0.3 nmol/liter ¹²⁵I-351A for 60 min at 20° to 22°C, and labeled sections were exposed to Hyperfilm-³H for 3 to 7 days. Nonspecific binding was defined as that remaining in the presence of either 1 mmol/liter EDTA or 10⁵ mol/liter lisinopril.

Microautoradiography. Further anatomic resolution of ¹²⁵I-(Sar¹,Ile⁸)Ang II and ¹²⁵I-351A binding sites was achieved by microautoradiography. In comparison with other peptide binding sites, ligand binding to angiotensin II receptors is more readily dissociated when labeled sections are immersed directly in emulsion. Dry-apposition techniques were therefore used (Hudson, 1993), with labeled sections being apposed to emulsion (Ilford K5; Ilford, Cheshire, UK) -coated coverslips for 2 to 3 weeks at 4°C. After exposure, microautoradiographs were developed in Kodak D-19 developer for 3 min at 20°C and fixed, and the sections were stained with hematoxylin and eosin and mounted in dibutylpthalate polystyrene xylene (DPX; BDH Laboratory Supplies, Poole, UK).

Picrosirius red staining. Collagen deposition was demonstrated by picrosirius red staining, essentially as described (Dobler and Spach, 1987) and validated (Pickering and Boughner, 1990) previously. This is a collagen-specific stain that enhances the natural birefringence of collagen fibers when viewed with polarized light. Briefly, sections were immersed in 0.2% (w/v) aqueous phosphomolybdic acid for up to 5 min, stained for 60 to 90 min in a 0.1% solution of sirius red F3B (BDH Laboratory Supplies) in saturated picric acid, rinsed in 0.01 M HCl, dehydrated, mounted in DBX and examined with an Olympus BX-60 microscope using polarized light.

Immunohistochemistry. To examine further the localization of specific ligand binding, consecutive sections to those used for autoradiography were processed for immunostaining using primary antisera raised to endothelial cell marker PECAM CD31 (clone JC/70A) (Parums et al., 1990). Cryostat sections were fixed in 10% formal saline for 10 min, rinsed and incubated with antiserum (diluted 1:1000) overnight at 4°C. Immunoreactivity was visualized by the avidin-biotin method of Hsu et al. (1981), with glucose oxidase/nickel enhancement (Shu et al., 1988). Controls included omission of the primary antiserum and replacement with nonimmune serum.

Angiotensin AT₂ receptor expression. Detection of AT₂ receptor expression in human cardiac tissues was confirmed by RT-PCR and Northern blot analysis. Total RNA was extracted from tissue samples using the single-step acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987). Yield (300 µg/g wet weight of tissue) and purity were assessed spectrophotometrically (optical density, 260 and 280 nm). Then, 5-µg amounts of total RNA were reverse transcribed into cDNA with random primers using a cDNA Cycle kit (InVitrogen, San Diego, CA). Oligonucleotide primers were synthesized (R&D Systems Europe, Abingdon, UK) according to the nucleotide sequences and genomic organization of the human AT₂ receptor (Martin and Elton, 1995). AT₂ primer sequences were 5-CGTCCCAGCGTCTGAGAG-3, which starts at position 74 in exon 1 (sense), and 5-TCACAGGTCCAAAGAGCCA-3, which starts at position 1941 in exon 3 (antisense). Aliquots of cDNA (50 ng) were amplified using gene-specific pairs of primers in 50-µl reactions containing 1× NH₄ buffer, 0.5 mmol/liter MgCl₂, 0.1 µmol/liter concentration of each primer, 0.5 mmol/liter concentration of dNTPs, 1 µCi of [-³²P]dCTP and 2.5 units BioTaq DNA polymerase (Bioline, London, UK). After 5-min denaturation at 95°C, hot-start reactions were run into plateau phase (up to 50 cycles, denaturation at 93°C for 30 sec; annealing 60°C for 30 sec; extension at 72°C for 30 sec) and finally incubated at 72°C for 10 min, using a PHC-3 thermocycler (Techne, Cambridge, UK). Appropriate negative controls included omission of the RT step and inclusion of water blanks. PCR products were analyzed by nondenaturing 6% polyacrylamide gel electrophoresis and autoradiography. Individual amplification products were excised from the gel, and the identity of each band was confirmed by direct cycle sequencing using a Circumvent kit (New England Biolabs, Letchworth, Hertfordshire, UK).

	Results

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Localization and characterization of angiotensin binding sites. ¹²⁵I-(Sar¹,Ile⁸)Ang II binding sites were demonstrated in sections of microscopically normal atrium and ventricle obtained from patients with cystic fibrosis, congenital heart defects, craniocerebral trauma or ruptured aorta (table 1, patients 1-8). Specific binding sites were localized to the myocardium, throughout both atria and ventricles; the medial layer in coronary arteries and to nerves running with coronary vessels in the epicardium and myocardium (fig. 1). ¹²⁵I-(Sar¹,Ile⁸)Ang II binding to all these structures was totally inhibited in the presence of an excess (10⁶ mol/liter) of unlabeled (Sar¹,Ile⁸)Ang II or Ang II and was differentially inhibited by AT₁ and AT₂ antagonists (figs. 1, 2, and 3). The relative density of myocardial binding sites was 2- to 3-fold greater (P < .001) in the atria (9.6 amol·mm²; 95% CI, 6.6-12.6; n = 7) compared with the ventricles (3.9 amol·mm²; 95% CI, 2.9-4.9; n = 9) and comprised both AT₁ and AT₂ subtypes. AT₂ sites predominated, representing 77% (95% CI, 70-80) and 71% (95% CI, 58-84) of the total binding in the atrial and ventricular myocardium, respectively (fig. 2). The density of ¹²⁵I-(Sar¹,Ile⁸)Ang II binding to the wall of coronary arteries (4.1 amol·mm²; 95% CI, 2.4-5.8; n = 6) was similar to that observed in ventricular myocardium, but AT₁ sites represented 85% (95% CI, 76-94) of all the binding sites identified (figs. 2 and 3). The density of binding sites localized to nerves (30.2 amol·mm²; 95% CI, 19.6-40.8; n = 10) was 3- to 7-fold greater than that measured in the myocardium and coronary arteries, and AT₁ sites comprised 96% (95% CI, 94-98) of the total binding (figs. 2 and 3). The same relative proportion of AT₁ and AT₂ sites was demonstrated in all the regions examined after inhibition of ¹²⁵I-(Sar¹,Ile⁸)Ang II binding by either losartan or PD123319 (fig. 3). No binding was detected that lacked affinity for either AT₁ or AT₂ antagonists, and no specific ¹²⁵I-Ang IV binding sites were observed. A similar distribution and density of binding sites were observed in cardiac tissues obtained from patients undergoing retransplantation due to acute or chronic allograft failure (table 1, patients 9-14), and no association was found between the distribution of specific ¹²⁵I-(Sar¹,Ile⁸)Ang II binding sites and inflammatory cell infiltration. It was not possible to distinguish between binding to myocardial and interstitial cells due to the homogeneous distribution of ¹²⁵I-(Sar¹,Ile⁸)Ang II binding sites in normal myocardium and the limited resolution of the autoradiographic technique.

View larger version (124K): [in this window] [in a new window]   Fig. 1.   Regional distribution of 125I-(Sar1,Ile8)Ang II binding in left anterior descending coronary artery, epicardial nerves and myocardium in adjacent tissue sections from patients with craniocerebral trauma (A and B) or cystic fibrosis (C-F and G-I). Film autoradiographic images show total (A) and nonspecific [B; binding in the presence of 106 mol/liter (Sar1,Ile8)Ang II] binding to the left anterior descending coronary artery (open arrow), epicardial nerves (arrows) and adjacent myocardium (M). Photomicrographs show that the high density of 125I-(Sar1,Ile8)Ang II binding sites on cardiac nerves (C) comprises mainly AT1 sites (D; binding in the presence of 105 mol/liter PD123319) and relatively few AT2 sites (E; binding in the presence of 105 mol/liter losartan). AT2 binding sites predominate in the myocardium (M), 125I-(Sar1,Ile8)Ang II binding (C) being inhibited to a greater extent in the presence of 105 mol/liter PD123319 (D) than by 105 mol/liter losartan (E). The wall of the coronary artery exhibits relatively low density 125I-(Sar1,Ile8)Ang II binding (G) and comprises mainly AT1 sites, with few binding sites being detected in the presence of 105 mol/liter losartan (H). N, nerve; i, intima; m, media; a, adventitia. F and I, hematoxylin and eosin-stained sections. Bars = 2 mm (A and B) and 100 µm (C-F and G-I).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Relative density of specific 125I-(Sar1,Ile8)Ang II myocardial binding in atrium (n = 7) and ventricle (n = 9), medial layer of epicardial coronary arteries (n = 6) and nerves (n = 10) in tissue sections from hearts of patients without ischemic heart disease, cardiomyopathy or mitral valve disease. Data represent the mean and 95% CI of 125I-(Sar1,Ile8)Ang II binding (250 pmol/liter; 150 min, at 20-22°C) to both AT1 and AT2 sites (Total), binding in the presence of 10 mmol/liter DTT and binding to either AT2 or AT1 sites in the presence of 105 mol/liter losartan (LOS) or PD123319 (PD), respectively. 125I-(Sar1,Ile8)Ang II binding to the myocardium was significantly reduced in the presence of 105 mol/liter PD123319, whereas binding to coronary arteries and nerves was significantly reduced in the presence of either DTT or losartan but not PD123319. *, P < .01; **, P < .001.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Proportion of specific 125I-(Sar1,Ile8)Ang I. I. binding to myocardium in atrium and ventricle (VENT), medial layer of coronary arteries (CA) and epicardial nerves, displaying either AT1 and AT2 binding characteristics as determined by the inhibition of binding in the presence of 105 mol/liter losartan (hatched columns) or PD123319 (open columns). Data represents the mean and 95% CI obtained from autoradiographic analysis of tissue sections from the hearts of patients (n = 10) without ischemic heart disease, cardiomyopathy or mitral valve disease.

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View larger version (105K): [in this window] [in a new window]   Fig. 4.   Film autoradiographic images showing the heterogeneous distribution of 125I-(Sar1,Ile8)Ang II binding sites (A-E) in serial sections of left ventricle (A-D) and interventricular septum (E and F) from patients with ischemic heart disease. Sections were incubated with 125I-(Sar1,Ile8)Ang II (250 pmol/liter; 150 min, at 20-22°C) alone (A; AT1 and AT2 sites) or with the addition of either 105 mol/liter PD123319 (B; AT1 sites), 105 mol/liter losartan (C; AT2 sites) or 10 mmol/liter DTT (D; AT2 sites). Except for nerves (A and B; arrows), areas displaying a relatively high density of binding sites corresponded to endocardial, perivascular, interstitial and infarcted regions and exhibited AT2 receptor binding characteristics. Film autoradiographic images also show the similar distribution of 125I-(Sar1,Ile8)Ang II binding sites (E) and the AT2 receptor-selective ligand 125I-CGP 42112A (F). Bars = 2 mm.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Quantitative analysis of equilibrium 125I-(Sar1,Ile8)Ang II binding (250 pmol/liter; 150 min, at 20-22°C) to sections of ventricle from patients with either ischemic heart disease or dilated cardiomyopathy. The density of binding sites in endocardial, perivascular, interstitial and infarcted regions (A) was significantly greater (P < .001) than that detected in the rest of the myocardium (B). The density of ligand binding in these regions was significantly enhanced in the presence of DTT (10 mmol/liter), inhibited in the presence of PD123319 (105 mol/liter) and unchanged by losartan (105 mol/liter)., indicating that it comprised mainly AT2 sites. Data points represent the mean and 95% CI of binding to sections from 11 distinct patients. ***, P < .001; **, P < .01; *, P < .05.

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View larger version (21K): [in this window] [in a new window]   Fig. 6.   Association (A) and saturation (B) analysis of 125I-(Sar1,Ile8)Ang II binding to endocardial, perivascular, interstitial and infarcted regions in sections of ventricle from patients with ischemic heart disease or dilated cardiomyopathy. Each point represents the mean and 95% CI of binding to sections from six distinct hearts, expressed either as the percentage of maximum specific binding (A) or (B) total (), nonspecific () and specific () ligand binding (amol·mm2).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Competitive inhibition of binding to endocardial, perivascular, interstitial and infarcted regions in sections of ventricle from patients with ischemic heart disease or dilated cardiomyopathy. Binding studies were conducted in the presence of 250 pmol/liter. 125I-(Sar1,Ile8)Ang II (150 min, at 20-22°C) and increasing concentrations of nonselective (sar1,Ile8)Ang II, Ang II), AT2-selective (CGP 42112A, [p-amino-Phe6]Ang II, PD123319) and AT1-selective competitors (losartan, eprosartan). Each point represents the mean of binding to sections from six distinct hearts, expressed as the percentage of maximum specific binding.

View larger version (107K): [in this window] [in a new window]   Fig. 8.   Photomicrographs of serial sections of interventricular septum from a patient with ischemic heart disease, showing the localization of AT2 binding sites (A; 250 pmol/liter. 125I-(Sar1,Ile8)Ang II binding in the presence of 10 mmol/liter DTT). Compared with the myocardium (m), perivascular (*, arteriole) and infarcted regions (open arrows) contain a high density of binding sites (A), prominent picrosirius red staining (B) and a moderate number of fibroblasts (C; hematoxylin and eosin). Bar = 100 µm.

Comparative distribution of AT₂ and ACE binding sites. ¹²⁵I-351A bound specifically to the coronary vascular endothelium in arteries, arterioles and microvessels and was completely inhibited in the presence of either 1 mmol/liter EDTA or 10⁵ mol/liter lisinopril. ¹²⁵I-351A binding was observed in both noninfarcted myocardium and infarcted areas, with the latter corresponding to the regions exhibiting a high density of AT₂ binding sites (fig. 9). A differential distribution of vascular ACE and AT₂ binding sites was demonstrated using microautoradiography, with ¹²⁵I-351A and ¹²⁵I-(Sar¹,Ile⁸)Ang II binding being mainly localized to the endothelial and adventitial layers of the vessel wall, respectively (fig. 9). In contrast to the high density of silver grains overlying the vascular endothelium, relatively few specific ¹²⁵I-351A binding sites were detected either on the endocardium or the other regions that displayed ¹²⁵I-(Sar¹,Ile⁸)Ang II binding characteristic of AT₂ sites (fig. 9). The distribution of ¹²⁵I-351A binding corresponded with that of CD31-immunoreactive endothelium in small arteries, arterioles and microvessels, and the border zone between infarcted areas and noninfarcted myocardium characteristically contained numerous microvessels, exhibiting ¹²⁵I-351A binding to the vessel wall and perivascular AT₂ binding sites (fig. 10).

View larger version (180K): [in this window] [in a new window]   Fig. 9.   Regional distribution of AT2 binding sites (A, C, and F; 250 pmol/liter. 125I-(Sar1,Ile8)Ang II binding in the presence of 10 mmol/liter DTT) and ACE (B, D, and G; 125I-351A binding) in serial sections of left ventricle from a patient with ischemic heart disease (A and B), right ventricle from a patient with dilated cardiomyopathy (C-E) and interventricular septum from a patient with ischemic heart disease (F-H). Film autoradiograms demonstrate that infarcted regions, surrounding islands of noninfarcted myocardium (m), exhibit both AT2 (A) and ACE binding sites (B). ACE binding was also present in noninfarcted myocardium but was not detected in avascular scar tissue (s). At the microscopic level, AT2 binding sites were localized to endocardium (C; arrowheads), perivascular and infarcted regions (F; open arrows). In contrast, 125I-351A binding was localized mainly to vascular endothelium in small arteries, arterioles (*) and microvessels (D and F; arrows), rather than the endocardium (F; arrowheads). E and H, hematoxylin and eosin-stained sections. Bars = 2 mm (A and B) and 100 µm (C-E and F-H).

View larger version (93K): [in this window] [in a new window]   Fig. 10.   Photomicrographs of serial sections of left ventricle from a patient with mitral valve disease, showing microvessels in the border zone between scar tissue and noninfarcted myocardium. Vessels (arrows) display both immunostaining for the endothelial marker PECAM (A) and 125I-351A binding to endothelial ACE (B) and are surrounded by perivascular AT2 binding sites [C; 125I-(Sar1,Ile8)Ang II binding in the presence of 10 mmol/liter DTT; open arrows]. Bar = 100 µm.

AT₂ receptor expression. Specific products corresponding to human AT₂ receptor cDNA sequences were generated by RT-PCR from both atrial and ventricular tissue samples and the expression of the gene displayed alternative splicing of 5 untranslated exons (fig. 11). Two AT₂ receptor transcripts were found, with the identity of each being confirmed by direct cycle sequencing. A 507-bp fragment encoding exons 1, 2 and 3 predominated, and a 448-bp fragment encoding exons 1 and 3 was also detected. Expression of the AT₂ receptor gene was confirmed by Northern blot analysis of mRNA isolated from ventricle tissues of patients with dilated cardiomyopathy or ischemic heart disease, with a single 3-kb band being demonstrated (fig. 11).

View larger version (72K): [in this window] [in a new window]   Fig. 11.   AT2 receptor gene expression in human cardiac tissues. Specific products, corresponding to AT2 receptor cDNA sequences (507 and 448 bp), were detected autoradiographically, after RT-PCR amplification, in samples of atrium (A; lanes 3-5) and ventricle (A; lanes 6 and 7). Lane 1, DNA size markers; lane 2, control amplification in absence of cDNA. The presence of AT2 transcripts (3 kb) was confirmed by Northern blot analysis of poly(A)+ RNA, as illustrated in samples of left ventricle from patients with ischemic heart disease (B).

	Discussion

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The present data indicate that the human heart contains two populations of ¹²⁵I-(Sar¹,Ile⁸)Ang II binding sites, displaying characteristics of AT₁ and AT₂ receptors, and the relative density and proportion of these two binding sites varies according to the specific structures that bear them. We have shown that ¹²⁵I-(Sar¹,Ile⁸)Ang II binding sites are differentially distributed in the human heart and that the AT₂ subtype predominates in the myocardium of both the normal and failing heart, representing 70% to 77% of the specific binding sites identified. This subtype occurred throughout the myocardium and was relatively more numerous in the atria than the ventricles. A selective increase in AT₂ binding sites was observed in the ventricular myocardium of explanted failing hearts from patients with ischemic heart disease or dilated cardiomyopathy and localized to endocardial, interstitial, perivascular and infarcted regions, corresponding to sites of collagen deposition and fibroblast proliferation. In contrast, a homogeneous population of AT₁ binding sites was localized to nerves and, albeit at relatively low density, in coronary vessels, as well as in the myocardium.

The two ¹²⁵I-(Sar¹,Ile⁸)Ang II binding sites were distinguished on the basis of differences in their respective affinity for the AT₁ and AT₂ nonpeptide antagonists losartan and PD123319 and their sensitivity to DTT. Specific high affinity AT₂ sites displayed a similar distribution pattern to binding sites for the AT₂ receptor-selective ligand ¹²⁵I- CGP 42112A and were further characterized by competitive inhibition experiments. The rank order of inhibitory potency of peptide analogs and nonpeptide antagonists for ¹²⁵I-(Sar¹,Ile⁸)Ang II binding sites in the failing human heart (CGP 42112A > (Sar¹,Ile⁸)Ang II > Ang II > [p-amino-Phe⁶]Ang II > PD123319 losartan = eprosartan) corresponded to that exhibited by the AT₂ receptor in other mammalian tissues (Iimmermans, et al., 1993) and by the cloned human AT₂ receptor expressed in vitro (Martin et al., 1994; Tsuzuki et al., 1994). The identification of cardiac ¹²⁵I-(Sar¹,Ile⁸)Ang II binding sites as either AT₁ or AT₂ receptors was verified by demonstrating well established differences in the sensitivity of the two receptors to DTT (Chui et al., 1989; Nozawa et al., 1994; Whitbread et al., 1989), with ligand binding to AT₁ sites being inhibited by DTT, whereas binding to AT₂ sites was increased. Finally, expression of the AT₂ receptor gene in human cardiac tissues was confirmed at the molecular level by the demonstration of a specific 3-kb AT₂ mRNA transcript in both atrial and ventricular tissues, corresponding in size to that demonstrated in other human tissues (Koike et al., 1994).

The proportion of AT₂ binding sites determined autoradiographically in both normal and noninfarcted myocardium in failing explanted hearts corresponds with that found in radioligand binding studies using atrial and ventricular myocardial membrane preparations from normal individuals and patients with impaired cardiac function or end-stage heart failure due to ischemic heart disease or dilated cardiomyopathy (Nozawa et al., 1994; Regitz-Zagrosek et al., 1995; Rogg et al., 1996). Rogg et al. (1996) also demonstrated that the proportion of AT₁ and AT₂ receptors in the right atrial myocardium, from patients with normal or moderately impaired cardiac function, correlates with atrial pressure and left ventricular ejection fraction, but the tissue distribution of the receptor subtypes was not determined. Our data, together with the recent observations of Brink et al. (1996) on atrial appendage tissues, indicate that changes in the proportion of cardiac Ang II receptor subtypes are associated with disease-related differences in cardiac structure, with a selective increase in AT₂ receptor binding occurring in regions of fibroblast proliferation and collagen deposition in the hearts of patients with ischemic heart disease, idiopathic dilated cardiomyopathy, mitral valve disease and adriamycin-induced cardiomyopathy. These observations are in contrast to those obtained in experimental models of heart disease, for example, after myocardial infarction in the rat, where we (Lefroy et al., 1996) and others (Sun and Weber, 1994) have demonstrated a marked increase in specific AT₁ binding sites in regions of fibrosis and collagen deposition. The present observations therefore serve to further emphasize the fact that there are species differences in both the regulation and proportion of cardiac AT₁ and AT₂ receptors.

Although the functional significance of the human cardiac AT₂ receptor has yet to be established, the receptor has been shown to mediate distinct Ang II-induced effects in a variety of cell types, influencing, for example, collagen metabolism in cultured rat cardiac fibroblasts (Brilla et al., 1994). Several studies have also demonstrated an antigrowth role for the AT₂ receptor in the cardiovascular system, inhibiting the proliferation of rat coronary endothelial cells (Stoll et al., 1995) and transfected vascular smooth muscle cells (Nakajima et al., 1995), inhibiting arterial hypertrophy and fibrosis in Ang II-induced hypertensive rats (Levy et al., 1996) and opposing Ang II-induced growth of cultured neonatal rat myocytes (Booz and Baker, 1996). The cellular expression of the AT₂ receptor is growth dependent (Kijima et al., 1996; Yamada et al., 1996), and apoptosis represents one mechanism by which it may induce an antigrowth effect (Yamada et al., 1996). Indeed, apoptotic myocytes have been found to occur relatively frequently in the border zone of infarcted human heart tissues (Saraste et al., 1997), and it has been speculated that the AT₂ receptor may influence changes in myocardial structure by mediating apoptosis (Dzau and Horiuchi, 1996). Our observations indicate that the tissue-selective distribution pattern of AT₂ receptor expression in the failing human heart is common to a number of cardiac diseases in which there is myocardial infarction and fibrosis, suggesting that it is not a primary event specific to a particular disease but rather that it represents part of a general mechanism contributing to disease-related changes in myocardial structure.

The distribution of ¹²⁵I-351A binding sites corresponds with immunohistochemical localization of ACE immunoreactivity to the vascular endothelium in the human heart (Danilov et al., 1987; Falkenhahn et al., 1995; Hokimoto et al., 1996). Increased ACE activity has been reported to occur in human ventricular myocardium after myocardial infarction (Hokimoto et al., 1995, 1996) and increased ACE mRNA levels detected in the failing explanted hearts of patients with ischemic heart disease or dilated cardiomyopathy (Studer et al., 1994; Zisman et al., 1995). The distribution of ¹²⁵I-351A binding demonstrated in the present study, together with the immunohistochemical observations of Falkenhahn et al. (1995), indicate that the vascular endothelium is also the main site of cardiac ACE expression in the failing human heart, with ¹²⁵I-351A binding and ACE immunoreactivity being prominent in the vessels that proliferate in the border zone between infarcted and noninfarcted myocardium. Although cardiac myocytes and interstitial cells in the failing heart displayed relatively little ¹²⁵I-351A binding, the extent to which nonendothelial cells may exhibit ACE activity requires further investigation, particularly as recent immunohistochemical studies have provided conflicting information concerning the localization of ACE immunoreactivity after myocardial infarction (Falkenham et al., 1995; Hokimoto et al., 1996) and its distribution has not been determined by immunohistochemistry in cardiac tissues from patients with dilated cardiomyopathy or other forms of heart disease.

Clinical trials have provided unequivocal evidence that ACE inhibitor therapy has a beneficial effect on patients with heart failure due to ischemic or nonischemic forms of heart disease, notably dilated cardiomyopathy, relieving symptoms and prolonging survival (Blaufarb and Sonnenblick, 1996; Pfeffer et al., 1995). ACE inhibitor therapy also appears to have a beneficial effect on patients with epirubicin-induced cardiomyopathy (Jensen et al., 1996); however, the mechanisms underlying the beneficial actions of ACE inhibitors remain incompletely understood. The inhibition of ACE is not specific for Ang II production because it influences the metabolism of other peptides, notably bradykinin, which may contribute to its vasodilator and growth-inhibiting properties (Blaufarb and Sonnenblick, 1996; Pfeffer et al., 1995). In addition, an alternative ACE-independent pathway for the generation of Ang II, by chymase, exists in the human heart (Urata et al., 1990), with chymase-like immunoreactivity being localized to interstitial cells, as well as endothelial and mast cells, in the ventricular myocardium of the failing human heart (Urata et al., 1993). Nevertheless, recent studies have indicated that ACE rather than chymase is the predominant pathway responsible for the local generation of Ang II in the human (Studer et al., 1994; Zisman et al., 1995). Regardless of the pathways involved in the generation of Ang II in the heart, Ang II receptor subtypes represent a direct means of influencing Ang II-induced changes in the growth and structure of the myocardium and thereby its contribution to the progression of heart failure. Experimentally, the AT₁ receptor appears to mediate many of the known growth-related changes induced by Ang II in isolated rat cardiac fibroblasts and myocytes (Crabos et al., 1994; Sadoshima and Izumo, 1993; Schorb et al., 1993; Villarreal et al., 1993), and treatment of animals with an AT₁ receptor antagonist attenuates most of the growth-related changes identified in the hypertrophic heart, including a reduction in cardiac size, DNA proliferation in interstitial cells and collagen deposition (Kojima et al., 1994; Makino et al., 1996; Nakamura et al., 1994; Schiefer et al., 1994; Smits et al., 1992; Suzuki et al., 1993). Clinically, AT₁ receptor antagonists have also been shown to block the pressor response to Ang II, reduce elevated blood pressure and exert beneficial hemodynamic effects in heart failure (Awan and Mason, 1996; Goodfriend et al., 1996; Johnston, 1995). However, it remains to be seen whether AT₁ receptor antagonists will influence human ventricular hypertrophy, reproducing the results obtained in experimental animals, and display the same reduction in mortality and morbidity associated with ACE inhibitors. Furthermore, AT₁ receptor blockade increases Ang II levels in humans and might therefore indirectly induce AT₂ receptor mediated responses (Awan and Mason, 1996; Goodfriend et al., 1996; Johnston, 1995).

Study limitations. Due to limited resolution of the autoradiographic technique and the homogeneous distribution of relatively low density myocardial ¹²⁵I-(Sar¹,Ile⁸)Ang II binding sites, the cellular resolution of AT₁ and AT₂ sites in normal and noninfarcted human myocardium remains unclear. Ultrastructural examination of ligand binding sites and immunohistochemical studies, using antibodies selective for AT₁ or AT₂ receptors, represent possible alternative strategies for the cellular resolution of Ang II receptor subtypes in human myocardium in situ. Several studies have used isolated rat myocytes and cardiac fibroblasts to investigate the cellular localization of Ang II receptor subtypes (Crabos et al., 1994; Sadoshima and Izumo, 1993; Schorb et al., 1993; Villarreal et al., 1993), but the relevance of their findings to humans is uncertain in view of species differences in the cardiac expression of Ang II receptors. In addition, cultured cardiac myocytes and fibroblasts exhibit differential expression and regulation of Ang II receptor genes (Matsubara et al., 1994), and results obtained using isolated cardiac cells may be confounded by the apparent plasticity of AT₂ receptor gene expression. The proportion of cardiac AT₁ and AT₂ receptors expressed in situ in the rat heart has, for example, been found to vary from that detected in cultured cells (Feolde et al., 1994), possibly reflecting down-regulation of AT₂ receptor expression after the isolation of mammalian cells (Johnson and Aguilera, 1991; Villarreal et al., 1993) and the growth-dependent regulation of AT₂ receptor expression in cultured cells (Kijima et al., 1996; Yamada et al., 1996). Similarly, the results of ligand binding studies on human heart tissues appear to differ from those obtained with isolated cardiac cells. Although AT₂ binding sites predominate in both tissue sections and myocardial membrane preparations of the human heart, cultured human cardiac fibroblasts are reported to display AT₁ rather than AT₂ receptor-mediated effects on collagen synthesis (Brilla et al., 1995). Atypical Ang II binding sites have also been detected, albeit transiently, on cultured human cardiac fibroblasts, which internalize Ang1-7 and Ang II, but not Ang3-8, and influence DNA synthesis (Neu et al., 1994, 1996). Further atypical Ang II binding sites, displaying high affinity for Ang3-8 (Ang IV) and low affinity for losartan and PD123319, have been identified on rabbit cardiac fibroblasts (Wang et al., 1995) and myocardial membranes of guinea pig and rabbit heart (Hanesworth et al., 1993), but they have not been described in the human heart in situ, and in the present study no specific ¹²⁵I-Ang IV binding sites were detected in the cardiac tissues examined.