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(Circulation. 1997;95:1201-1206.)
© 1997 American Heart Association, Inc.

Articles

AT₁ and AT₂ Angiotensin Receptor Gene Expression in Human Heart Failure

Guy A. Haywood, MD, MRCP; Lars Gullestad, MD, PhD; Tomohiro Katsuya, MD, PhD; Howard G. Hutchinson, MD; Richard E. Pratt, PhD; Masatsugu Horiuchi, MD, PhD; Michael B. Fowler, MB, MRCP

the Division of Cardiovascular Medicine, Stanford (Calif) University.

Correspondence to Guy Haywood, MD, MRCP, Consultant Cardiologist, Derriford Hospital, Plymouth PL6 8DH, UK.

	Abstract

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Background The availability of selective antagonists for angiotensin II receptors has focused interest on the gene expression of angiotensin II–receptor subtypes in the human heart.

Methods and Results We analyzed expression of the AT₁ and AT₂ subtypes of the angiotensin II receptor in ventricular myocardium taken from 9 donor hearts before implantation and from 12 patients with heart failure (6 with dilated cardiomyopathy and 6 with ischemic heart disease). Competitive reverse transcription–polymerase chain reaction with synthetic RNA internal standards was used to detect mRNA for both subtypes and to quantify relative differences in levels between failing and nonfailing ventricular myocardium. AT₁- and AT₂-receptor mRNA could be detected in all samples. AT₁-receptor gene expression was 2.5-fold greater in nonfailing hearts than in patients with failing hearts (P=.015). There was no significant difference in AT₂-receptor mRNA expression in failing and nonfailing hearts.

Conclusions The level of expression of the angiotensin AT₁ receptor appears to decrease in the failing human ventricle whereas the level of AT₂ expression is unaffected. These changes parallel the changes found in human ventricular myocardium at the receptor level, suggesting that the changes in receptor level may result from changes in gene expression or mRNA stability.

Key Words: angiotensin • receptors • heart failure

	Introduction

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The survival benefit conferred by blockade of the renin-angiotensin system in patients with heart failure has led to intense interest in the mechanisms underlying the action of angiotensin II. Recently, a new class of drugs that antagonize angiotensin binding to angiotensin II receptors has been developed¹ ; these drugs are undergoing clinical trials in patients with heart failure. Although beneficial actions have been reported from initial research with these agents, details of their mechanisms of action at the receptor level are limited. Angiotensin II has been shown to bind to receptors present in human myocardium, and the first reports analyzing failing and nonfailing ventricular myocardium showed that there was a nonsignificant trend toward decreased angiotensin II receptor binding in hearts from patients with dilated cardiomyopathy.² Further comparisons of atrial myocardium from failing and nonfailing human hearts found a significant decrease in total angiotensin II–receptor subtype binding.³

In the last 3 years, the angiotensin II receptor population has been subdivided. The human genes encoding the angiotensin II receptor subtypes (AT₁ and AT₂) were cloned in 1992⁴ and 1994,⁵ respectively. Subsequently, the AT₁ subtype has been further subdivided into the AT1a and AT1b genes that have 98% amino acid homology. The AT1b gene seems restricted in its tissue expression to placenta, lung, and liver and is not expressed in human heart.⁶ The AT1a receptor-subtype gene is a single-copy gene located on chromosome 3.⁷ The human AT2 receptor-subtype gene is located on the X chromosome⁸ and shares 92.6% amino acid homology with the rat AT2 subtype gene.⁵ In the rat, there is only 32% amino acid–sequence homology between the AT1a and AT2 receptor subtype genes.⁹

The present study investigated the level of gene expression at the mRNA level for the AT1a and AT2 receptor subtype genes in failing and nonfailing human ventricular myocardium.

	Methods

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Subjects
Right ventricular myocardium from the endocardial surface of the intraventricular septum was obtained at the time of cardiac transplantation from patients with heart failure. Control myocardium was obtained from the endocardial surface of the right side of the intraventricular septum from donor hearts before implantation.

Reverse Transcription–Polymerase Chain Reaction
RNA Extraction
In all cases, the myocardial tissue obtained was immediately frozen in liquid nitrogen and stored at -80°C. Total RNA was isolated from tissue samples by use of the method of Chomozynski and Sacchi¹⁰ using Trizol reagent (Gibco BRL Life Technologies Inc). RNA was extracted with the use of chloroform and precipitated by isopropanol. The RNA was washed in 75% ethanol and then treated with DNAse 1 (Gibco BRL) for 15 minutes at room temperature before reextraction using the same method. After reextraction, the RNA was dried and stored at -70°C until required. RNA was quantified by spectrophotometry using the A_260/280 method. Before use, samples were centrifuged at 4°C and resuspended in RNase free water.

Synthesis of RNA Internal Standards
Synthetic RNA internal standards were manufactured by use of a polymerase chain reaction (PCR)–based technique. For each target mRNA, an oligonucleotide primer pair was synthesized that under the conditions specified in Table 1 gave amplification of a PCR product of the predicted size visible as a single band on ethidium gel. The PCR product was purified and cut with a restriction enzyme to confirm amplification of the correct sequence. A third oligonucleotide primer was then synthesized corresponding to a position in the amplified target mRNA sequence that was inset from either the 5' or 3' end by 200 base pairs (bp). This third primer was then used in combination with the appropriate forward or reverse primer from the original pair and conditions were optimized to result in amplification of a PCR product of the predicted size visible as a single band on ethidium gel (200 bp shorter than the original PCR product) (Fig 1). If the third primer was a forward primer, a new forward primer was then synthesized that linked a T7 recognition sequence to the original forward-primer sequence, which was in turn linked to the third primer (forward-primer) sequence. A new reverse-primer sequence was also synthesized linking the reverse primer to an 18 poly-T tail. These primers were used to synthesize a PCR product that formed a DNA template for transcription of RNA by use of a T7 RNA polymerase (Promega Co). The RNA formed was then extracted, treated with DNAse 1, and reextracted. The quantity of RNA present was determined by A_260/280 spectrophotometry, and reverse transcription–PCR (RT-PCR) amplification from RNA was confirmed with the original primer pair with an RNAse-treated sample used as a negative control to ensure no amplification arose from any template DNA still contaminating the RNA.

View this table: [in this window] [in a new window]   Table 1. Oligonucleotide Primer Sequences and Reaction Conditions for Polymerase Chain Reactions

View larger version (26K): [in this window] [in a new window]   Figure 1. Schema of mimic synthesis. PCR indicates polymerase chain reaction.

The PCR product resulting from amplification of the AT₁ target cDNA was 255 bp in length, and the AT₁ internal standard PCR product was 137 bp. The AT₂ target cDNA product was 293 bp long, and the AT₂ internal standard product was 254 bp. To verify that the AT₁ PCR product arose from the target cDNA, a restriction enzyme cut was performed with the use of Ssp I. This gave a single cut at position 83 on the product sequence. The PCR product amplified by the AT₂ primer pair was subcloned into a vector with the use of the TA cloning kit (Invitrogen Inc) and sequenced to confirm it matched the cDNA sequence.

Competitive RT-PCR
Serial dilutions of the synthetic RNA mimics were made and added to equal quantities of total RNA extracted from failing or nonfailing hearts in a series of RT-PCR reaction mixes. Each tube therefore contained 500 ng of total RNA and a specific concentration of both the AT₁ and AT₂ mimic RNA strands. These were then reverse transcribed and amplified by one of the original primer pairs over 45 cycles. First-strand cDNA was synthesized from total RNA with the use of monkey Moloney leukemia virus reverse transcriptase (Perkin-Elmer Cetus) and random hexamers. Aliquots (5 µL) of the cDNA from each combination of total RNA and mimic RNA were amplified against a single pair of primers. Amplification by the PCR was performed in a total reaction volume of 50 µL. The sequences of the primers and the conditions used are shown in Table 1. Thermal cycling was performed on a PTC 100 thermal cycler (MJ Research). Electrophoresis of the amplified products was performed on 1.5% agarose gel containing Tris acetate/EDTA and ethidium bromide. A Hae III digest of 174 DNA (Gibco BRL) was used as a molecular size standard. Gels were visualized with UV irradiation and photographed. When the synthetic mimic RNA strand exceeded the patient's target mRNA, competition for the primers favored the mimic, and a PCR product resulted that was the size of the synthetic internal standard. When the patient's target mRNA exceeded the mimic RNA, the product was the longer target product, but when the quantity of synthetic mimic RNA in the tube equaled the quantity of the target mRNA present, amplification of both sized bands was equal (Fig 2). This allowed comparison between the failing and nonfailing heart series in relation to the dilution sequence so that relative changes in concentration of the target mRNA between samples could be derived. Ethidium gels were analyzed by blinded observers to determine the point of equivalence between the intensity of the mimic and target product bands. The point of equivalence determined from ethidium gel analysis for an individual total RNA sample was not found to differ between consecutive RT-PCR analyses. To quantify the level of reproducibility, we ran five separate RT-PCR analyses on serial runs of the thermocycler against 1 pg of the synthetic target RNA. The coefficient of variance was .053.

View larger version (25K): [in this window] [in a new window]   Figure 2. Schema of competitive reverse transcription–polymerase chain reaction (RT-PCR).

Noncompetitive amplification of ß-actin was used to demonstrate the presence of intact mRNA in each total RNA sample and to help to demonstrate approximate equivalence of mRNA loading in each patient's RT-PCR reaction series.

RT-PCR Control Experiments
The absence of genomic contamination in the cDNA samples was confirmed by the use of a PCR reaction using primers that amplify the promoter region of apolipoprotein(a)-related gene C, a nontranscribed region of genomic DNA.¹¹ These primers were designed to sensitively and specifically amplify genomic DNA only, and the technique was validated against serial dilutions of genomic DNA and total RNA with or without RNAse pretreatment before RT-PCR (G.A.H., MD, MRCP, and C. Byrne, MD, MRCP, unpublished data, 1994). This method allows screening of cDNA for genomic contamination without the need to use reverse-transcriptase–negative RNA controls, thus saving on the consumption of total RNA. A subgroup of samples were also put through the reverse-transcription stage without the addition of reverse transcriptase to act as supplementary negative controls. The AT₂ primers spanned intron sequences (T.K., MD, and M.H., MD, unpublished data, 1995) to enable identification of amplification of cDNA from any genomic DNA amplification by the size of the product. The AT₁ primers were located within the single intron that contains the entire coding sequence.¹²

Validation of Competitive RT-PCR Measurements
A synthetic RNA standard containing terminal sequences for the forward and reverse primers of both the AT₁-receptor and AT₂-receptor subtypes was synthesized by PCR, purified on an electrophoretic gel, and quantified by spectrophotometry. One picogram was then amplified against a serial dilution of the mimic RNAs for AT₁ and AT₂ using each of the primer pairs in turn to test whether differences in amplification efficiency resulted in different measurements of the quantity of the synthetic RNA standard present.

Variability in absolute quantities of mimic RNA in successive dilutive series was controlled for by comparison with the synthetic RNA standard, and appropriate corrections were applied.

In addition, total RNA from a patient with heart failure was diluted 10-fold and run against a 10-fold serial dilution of the mimic RNA to check that the point of equivalence showed a 10-fold difference between the two concentrations.

Statistical Analysis
All results are expressed as mean±SD. Statistical analysis was performed with the use of a statistical software package (Statview, Abacus) on an Apple Macintosh computer. The two-tailed Mann-Whitney U test was used to compare relative concentrations of the AT₁ and AT₂ mimics in failing and nonfailing hearts and relative concentrations of AT₁ versus AT₂ in each group. Differences between patients with ischemic heart disease, dilated cardiomyopathy, and donor hearts were analyzed by use of ANOVA with appropriate post hoc tests.

	Results

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Patient Characteristics
Subjects (n=9) from whom donor control myocardium was obtained were significantly younger (33.3±12.6 years; range, 19 to 52 years) than patients with heart failure (50.2±8.9 years; range, 33 to 65 years; P<.01). However, there was overlap in the ages of patients from the two groups, and there was no significant correlation between age of the donor hearts and either AT₁ (r=-.51, P=NS) or AT₂ (r=.014, P=NS) mRNA level. None of the subjects from whom donor control myocardium was obtained showed any evidence of cardiac disease. Clinical characteristics of the patients with heart failure are shown in Table 2. None of the patients with ischemic heart disease had clinical evidence to suggest they had suffered acute myocardial infarction within 6 months of cardiac transplantation (range, 6.5 to 30 months).

View this table: [in this window] [in a new window]   Table 2. Clinical Characteristics of Subjects With Heart Failure

PCR Results
Representative gels for the AT₁- and AT₂-receptor subtypes analyzed by competitive RT-PCR amplification to 45 cycles are shown in Fig 3.

View larger version (79K): [in this window] [in a new window]   Figure 3. Representative gels of donor and failing hearts (competitive reverse transcription–polymerase chain reaction analyses for AT1 and AT2). Lanes 1-6: serial dilutions of AT1 mimic RNA internal standard (1=1 pg, 2=600 pg, 3=200 pg, 4=100 fg, 5=50 fg, and 6=0 fg). Lanes 7-12: serial dilutions of AT2 mimic RNA internal standard (7=1 pg, 8=100 fg, 9=10 fg, 10=1 fg, 11=100 attograms, and 12=0 fg). For AT1, the point of equivalence is lower in the failing heart; for AT2, the point of equivalence is the same in both the failing and nonfailing hearts.

Amplification of ß-actin was confirmed in all samples. In all cases, screening for genomic contamination after DNAse treatment of the RNA was negative. The AT₁ mRNA levels were higher in nonfailing than in failing hearts (200±104 versus 79±41 fg; P=.015). There was no significant difference in the levels of AT₂ mRNA between the two groups (113±65 versus 137±150 fg; P=NS) The differences between levels of mRNA for AT₁ and AT₂ in the two groups are shown in Fig 4. AT₁ mRNA levels were similar in patients with ischemic heart disease versus dilated cardiomyopathy (81±45 versus 77±41 fg; P=NS). However, the numbers in each group were small (n=6 in each group) and thus the statistical power for the detection of any significant difference between the two etiologies was low.

View larger version (33K): [in this window] [in a new window]   Figure 4. Changes in the levels of AT1 (A) and AT2 (B) mRNA in failing and nonfailing hearts.

Dilution of a sample of total RNA by 10-fold resulted in a shift in the point of equivalence against the mimic serial dilution of 10-fold (Fig 5). Amplification of AT₁ and AT₂ mimic serial dilution ranging from 20 pg to 10 fg against 1 pg of the synthetic target RNA confirmed that the point of equivalence was at the 1-pg dilution of each mimic (Fig 5). The ratio of mRNA for AT₁ compared with mRNA for AT₂ in nonfailing hearts was 1.78:1 (P=NS); the ratio of mRNA for AT₁ compared with mRNA for AT₂ in failing hearts was 0.58:1 (P=NS).

View larger version (45K): [in this window] [in a new window]   Figure 5. A, An ethidium gel showing a 10-fold shift in the point of equivalence within the mimic RNA serial dilution in response to a 10-fold dilution of a subject's total RNA. Lanes a-f: 10-fold dilutions of mimic RNA internal standard, with lane a representing the highest concentration and lane f the lowest concentration. Lane g is a water control lane. The upper series is a 1:1 dilution of the subject's total RNA; the lower series is a 1:10 dilution of the subject's total RNA. The point of equivalence in each series is denoted by an asterisk. B, Measurement of the point of equivalence against 1 pg of the synthetic RNA target strand using an AT1 dilutive series. Mimic concentrations: lane 1=water control, lane 2=20 pg, lane 3=10 pg, lane 4=1 pg, lane 5=100 fg, lane 6=10 fg, lane 7=0 fg, lane 8=1 pg AT2 mimic with no synthetic target RNA strand, and lane 9=1 pg AT1 mimic with no synthetic target RNA strand. C, Measurement of the point of equivalence against 1 pg of the synthetic RNA target strand using an AT2 serial dilution. Mimic concentrations: lane 10=water control, lane 11=20 pg, lane 12=10 pg, lane 13=1 pg, lane 14=100 fg, lane 15=10 fg, lane 16=0 fg, lane 17=1 pg AT2 mimic with no synthetic target RNA strand, and lane 18=1 pg AT1 mimic with no synthetic target RNA strand.

	Discussion

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The decrease in the concentration of AT₁ mRNA in failing compared with nonfailing myocardium agrees with the findings of a previous report that analyzed atrial myocardium³ and with findings in failing human ventricular myocardium at the receptor level.¹³ The lack of significant change in the level of AT₂ mRNA between failing and nonfailing myocardium also reflects findings at the receptor level in human ventricular myocardium¹³ but differs from the findings reported at the receptor level in human atrial myocardium, in which it appears that the level of AT₂ receptors is also decreased under the conditions of chronic heart failure.³

The similarity of relative changes in receptor density and mRNA level suggest that the principal mechanism regulating the level of angiotensin II receptors may be the concentration of mRNA for the two receptor subtypes present in the myocardium. This is not necessarily the case, because the mRNA concentrations could be just an epiphenomenon associated with another regulatory mechanism, but regulation at the mRNA expression level is thought to be the primary means by which cells regulate the availability of proteins.¹⁴ The cause of the decrease in the level of mRNA for the AT₁ subtype cannot be determined from the present study. The mechanism may be factors resulting in decreased transcription, decreased message stability, or both.

Although the results obtained in the present study give a definitive answer to the question being investigated, it is important to avoid concluding too much. Because others have performed quantification at the receptor level in detailed studies in subjects with heart failure, we did not attempt to obtain receptor density data on the patients we studied. Paired comparisons of mRNA and receptor concentrations in individual patients therefore were not possible.

Because of the low level of message present and the very limited quantities of donor myocardium available, we were unable to localize mRNA expression to individual cell types and could only provide data for ventricular myocardium as a whole. The observations were confined to myocardium taken from the right side of the interventricular septum because this is the area from which the donor biopsy samples were taken, and thus regional differences in the level of RNA expression could not be assessed. Finally, the functional significance of the observation that the level of AT1 gene expression decreases in failing hearts cannot be determined from the present study. Even if the numbers of subjects studied were greatly increased, it would be difficult to draw associations between the level of gene expression and clinical features because the number of uncontrolled variables present in such patients is necessarily high.

The findings in the present study have interesting implications for the predicted actions of angiotensin II receptor inhibitors. The preservation of gene expression for the AT₂ receptor in patients with heart failure indicates that a situation will exist in the presence of AT₁-receptor blockade in which angiotensin II will act unopposed on the AT₂ receptors. The removal of feedback inhibition by the actions of angiotensin II via the AT₁ receptor may also result in an increase in circulating and tissue levels of angiotensin II,¹⁵ thus increasing the occupation of the AT₂-receptor population. Although the functional effects mediated by the AT₂ receptor remain unclear, there is increasing evidence that in some tissues the AT₂ receptor may activate pathways that result in inhibition of cell growth¹⁶ or even stimulation of apoptotic pathways.^{17 18} Although proapoptotic effects acting via the AT₂ receptor have not been investigated in myocardium, there is evidence in PC12W (rat pheochromocytoma) cells and R3T3 (mouse fibroblast) cells that apoptosis may be induced by dephosphorylation of mitogen-activated protein kinase secondary to AT₂-receptor stimulation.¹⁸ Thus, it is possible that a change in the balance of the effects mediated by angiotensin II on the two receptor subtypes may not only decrease the stimulation of hypertrophy through the AT₁ receptor but also potentially increase antihypertrophic, proapoptotic effects on the myocardium.

The decrease in AT₁-receptor subtype expression seen in human heart failure is in marked contrast to the increase observed in the ventricle of animal models of acute myocardial infarction.¹⁹ In the rat infarct model, there appears to be upregulation of AT₁–receptor-subtype gene expression, both acutely and persisting up to 8 months after infarction.²⁰ Despite the fact that patients with ischemic heart disease were included in the heart failure group in our study, the effects of myocardial infarction and ischemic cardiomyopathy appear to have been opposite. This raises intriguing questions about what the major regulators of AT₁-receptor gene expression in the myocardium are. One possibility is that the cell types expressing AT₁ in these two situations differ. There is some evidence that the upregulation of AT₁ after infarction localizes to scar tissue and may be related to fibroblasts²⁰ ; the downregulation observed in failing hearts, however, may be related to cardiac myocytes. It is also possible that species differences are important. In the rat, there is a transcription factor AP1 recognition site present on the AT₁-promoter region,⁹ which might act to stimulate AT₁ transcription under hypoxic conditions. This site is absent on the human AT₁ promoter and may result in oxidative mechanisms predominating and causing inhibition of the binding of oxidation-sensitive transcription factors such as stimulator protein 1 (SP1).²¹ SP1 recognition sites are present just upstream of the human AT₁ transcription start site, and inhibition of the binding of SP1 would be predicted to decrease transcription of the gene.

Summary
In the interventricular septum, myocardial levels of mRNA for the angiotensin II AT₁-receptor subtype are 2.5-fold greater in nonfailing hearts than in patients with heart failure. In contrast, levels of AT₂-receptor subtype mRNA are similar. These results are very similar to the changes observed in the receptor density of the two subtypes in failing and nonfailing hearts, suggesting that regulation of the alteration in expression is at the mRNA level. The alteration in the balance of gene transcription between the two subtypes may alter the actions of angiotensin II on failing hearts and may influence the effects of specific AT₁-receptor antagonists in patients with heart failure.

	Acknowledgments

 
This work was supported by a medical school grant from Merck and Co, West Point, Pa. Dr Gullestad was supported by The Research Council of Norway. We are most grateful to Gail Yee for expert technical assistance with the extraction and preparation of RNA. We would also like to thank the surgical staff at Stanford, and in particular Hermann Reichenspurner and John Stevens, who assisted us in the collection of the myocardial samples.

Received September 21, 1995; revision received March 20, 1996; accepted October 28, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Timmermans P, Wong P, Chiu A, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JM, Smith RD. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev. 1993;45:205-251.[Medline] [Order article via Infotrieve]

2. Urata H, Healy B, Stewart RW, Bumpus FM, Husain A. Angiotensin II receptors in normal and failing human hearts. J Clin Endocrinol Metab. 1989;69:54-66.[Abstract/Free Full Text]

3. Regitz-Zagrosek V, Friedel N, Heymann A, Bauer P, Neuss M, Rolfs A, Steffen C, Hildebrandt A, Hetzer R, Fleck E. Regulation, chamber localization, and subtype distribution of angiotensin II receptors in human hearts. Circulation. 1995;91:1461-1471.[Abstract/Free Full Text]

4. Furuta H, Gou D, Inagami T. Molecular cloning and sequencing of the gene encoding human angiotensin II type 1 receptor. Biochem Biophys Res Commun. 1992;183:8-13.[Medline] [Order article via Infotrieve]

5. Tsuzuki S, Ichiki T, Nakakubo H, Kitami Y, Guo DF, Shirai H, Inagami T. Molecular cloning and expression of the gene encoding human angiotensin II type 2 receptor. Biochem Biophys Res Commun. 1994;200:1449-1454.[Medline] [Order article via Infotrieve]

6. Konishi H, Kuroda S, Inada Y, Fujisawa Y. Novel subtype of human angiotensin II type 1 receptor: cDNA cloning and expression. Biochem Biophys Res Commun. 1994;199:467-474.[Medline] [Order article via Infotrieve]

7. Curnow K, Pascoe L, White P. Genetic analysis of the human type-1 angiotensin II receptor. Mol Endocrinol. 1992;6:1113-1118.[Abstract/Free Full Text]

8. Koike G, Horiuchi M, Yamada T, Szpirier C, Jacob H, Dzau V. Human type 2 angiotensin II receptor gene: cloned, mapped to the X chromosome, and its mRNA is expressed in the human lung. Biochem Biophys Res Commun. 1994;203:1842-1850.[Medline] [Order article via Infotrieve]

9. Inagami T, Guo D, Kitami Y. Molecular biology of angiotensin II receptors: an overview. J Hypertens. 1994;12(suppl 10):S83-S94.

10. Chomozynski P, Sacchi N. Single step method of RNA isolation by acid guanidinium-thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159.[Medline] [Order article via Infotrieve]

11. Byrne C, Schwartz K, Meer K, Cheng J-F, Lawn R. The human apolipoprotein (a)/plasminogen gene cluster contains a novel homologue transcribed in liver. Arterioscler Thromb. 1994;14:534-541.[Abstract/Free Full Text]

12. Guo D, Furuta H, Mizukoshi M, Inagami T. The genomic organization of human angiotensin II type 1 receptor. Biochem Biophys Res Commun. 1994;200:313-319.[Medline] [Order article via Infotrieve]

13. Asano K, Minobe W, Mitchusson K, Dutcher D, Roden RL, Port JD, Bristow MR. Selective down-regulation of angiotensin II AT1 receptors in failing human heart: relationship to ß₁-receptor down regulation. J Am Coll Cardiol. 1995;25:291A. Abstract.

14. Rosenthal N. Molecular medicine: regulation of gene expression. N Engl J Med. 1994;331:931-933.[Free Full Text]

15. Goldberg M, Tanaka W, Barchowsky A, Bradstreet TE, McCrea J, Lo M-W, McWilliams EJ, Bjornsson TD. Effects of losartan on blood pressure, plasma renin activity, and angiotensin II in volunteers. Hypertension. 1993;21:704-713.[Abstract/Free Full Text]

16. Stoll M, Steckelings U, Paul M, Bottari S, Metzger R, Unger T. The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells. J Clin Invest. 1995;95:651-657.

17. Tanaka M, Ohnishi J, Ozawa Y, Sugimoto M, Usuki S, Naruse M, Murakami K, Miyazaki H. Characterization of angiotensin II receptor type 2 during differentiation and apoptosis of rat ovarian cultured granulosa cells. Biochem Biophys Res Commun. 1995;207:593-598.[Medline] [Order article via Infotrieve]

18. Yamada T, Horiuchi M, Dzau V. The novel angiotensin II type 2 receptor mediates programmed cell death. Proc Natl Acad Sci U S A. In press.

19. Meggs L, Coupet J, Huang H, Cheng W, Li P, Capasso JM, Homcy CJ, Anversa P. Regulation of angiotensin II receptors on ventricular myocytes after myocardial infarction in rats. Circ Res. 1993;72:1149-1162.[Abstract/Free Full Text]

20. Lefroy D, Wharton J, Crake T, Knock GA, Rutherford RAD, Polak JM, Poole-Wilson PA. Angiotensin II receptor expression after myocardial infarction. Br Heart J. 1995;73:P32. Abstract.

21. Ammendola R, Mesuraca M, Russo T, Cimino F. The DNA-binding efficiency of Sp1 is affected by redox changes. Eur J Biochem. 1994;225:483-489.[Medline] [Order article via Infotrieve]

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				H. Han, S. Hoffmann, K. Hu, and G. Ertl Angiotensin II subtype 1 (AT1) receptors contribute to ischemic contracture and regulate chemomechanical energy transduction in isolated transgenic rat ({alpha}MHC-hAT1)594-17 hearts Eur J Heart Fail, March 1, 2002; 4(2): 131 - 137. [Abstract] [Full Text] [PDF]

				D. Schultz, X. Su, C.-C. Wei, S. P. Bishop, P. Powell, G. H. Hankes, A. R. Dillon, P. Rynders, F. G. Spinale, G. Walcott, et al. Downregulation of ANG II receptor is associated with compensated pressure-overload hypertrophy in the young dog Am J Physiol Heart Circ Physiol, February 1, 2002; 282(2): H749 - H756. [Abstract] [Full Text] [PDF]

				S.B. GURLEY, T.H. LE, and T.M. COFFMAN Gene-targeting Studies of the Renin-Angiotensin System: Mechanisms of Hypertension and Cardiovascular Disease Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 451 - 458. [Abstract] [PDF]

				C. Berry, R. Touyz, A. F. Dominiczak, R. C. Webb, and D. G. Johns Angiotensin receptors: signaling, vascular pathophysiology, and interactions with ceramide Am J Physiol Heart Circ Physiol, December 1, 2001; 281(6): H2337 - H2365. [Abstract] [Full Text] [PDF]

				M. P. Schuijt, M. Basdew, R. van Veghel, R. de Vries, P. R. Saxena, R. G. Schoemaker, and A. H. Jan Danser AT2 receptor-mediated vasodilation in the heart: effect of myocardial infarction Am J Physiol Heart Circ Physiol, December 1, 2001; 281(6): H2590 - H2596. [Abstract] [Full Text] [PDF]

				H. Nagashima, Y. Sakomura, Y. Aoka, K. Uto, K.-i. Kameyama, M. Ogawa, S. Aomi, H. Koyanagi, N. Ishizuka, M. Naruse, et al. Angiotensin II Type 2 Receptor Mediates Vascular Smooth Muscle Cell Apoptosis in Cystic Medial Degeneration Associated With Marfan's Syndrome Circulation, September 18, 2001; 104 (2009): I-282 - I-287. [Abstract] [Full Text] [PDF]

				S. Lee, C. M. Kramer, S. Mankad, S.-e. Yoo, and K. Sandberg Combined angiotensin converting enzyme inhibition and angiotensin AT1 receptor blockade up-regulates myocardial AT2 receptors in remodeled myocardium post-infarction Cardiovasc Res, July 1, 2001; 51(1): 131 - 139. [Abstract] [Full Text] [PDF]

				S. C. Verduyn, C. Ramakers, G. Snoep, J. D. M. Leunissen, H. J. J. Wellens, and M. A. Vos Time course of structural adaptations in chronic AV block dogs: evidence for differential ventricular remodeling Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2882 - H2890. [Abstract] [Full Text] [PDF]

				H. Matsubara Renin-Angiotensin System in Human Failing Hearts : Message From Nonmyocyte Cells to Myocytes Circ. Res., May 11, 2001; 88(9): 861 - 863. [Full Text] [PDF]

				L. H. Opie and M. N. Sack Enhanced Angiotensin II Activity in Heart Failure : Reevaluation of the Counterregulatory Hypothesis of Receptor Subtypes Circ. Res., April 13, 2001; 88(7): 654 - 658. [Abstract] [Full Text] [PDF]

				T. Hisaoka, M. Yano, T. Ohkusa, M. Suetsugu, K. Ono, M. Kohno, J. Yamada, S. Kobayashi, M. Kohno, and M. Matsuzaki Enhancement of Rho/Rho-kinase system in regulation of vascular smooth muscle contraction in tachycardia-induced heart failure Cardiovasc Res, February 1, 2001; 49(2): 319 - 329. [Abstract] [Full Text] [PDF]

				M. E El-Sabban, K. A Hassan, A. E Birbari, K. M Bitar, and A. B Bikhazi Angiotensin II binding and extracellular matrix remodelling in a rat model of myocardial infarction Journal of Renin-Angiotensin-Aldosterone System, December 1, 2000; 1(4): 369 - 378. [Abstract] [PDF]

				Chiming Wei, M. G Cardarelli, S. W Downing, and J. S McLaughlin The effect of angiotensin II on mitogen-activated protein kinase in human cardiomyocytes Journal of Renin-Angiotensin-Aldosterone System, December 1, 2000; 1(4): 379 - 384. [Abstract] [PDF]

				S. L. Malendowicz, P. V. Ennezat, M. Testa, L. Murray, E. H. Sonnenblick, T. Evans, and T. H. LeJemtel Angiotensin II Receptor Subtypes in the Skeletal Muscle Vasculature of Patients With Severe Congestive Heart Failure Circulation, October 31, 2000; 102(18): 2210 - 2213. [Abstract] [Full Text] [PDF]

				Y. Xu, A. S. Clanachan, and B. I. Jugdutt Enhanced Expression of Angiotensin II Type 2 Receptor, Inositol 1,4,5-Trisphosphate Receptor, and Protein Kinase C{epsilon} During Cardioprotection Induced by Angiotensin II Type 2 Receptor Blockade Hypertension, October 1, 2000; 36(4): 506 - 510. [Abstract] [Full Text] [PDF]

				E. Chabielska, T. Matys, I. Kucharewicz, D. Pawlak, R. Rolkowski, and W. Buczko The involvement of AT2-receptor in the antithrombotic effect of losartan in renal hypertensive rats Journal of Renin-Angiotensin-Aldosterone System, September 1, 2000; 1(3): 263 - 267. [Abstract] [PDF]

				S. Busche, S. Gallinat, R.-M. Bohle, A. Reinecke, J. Seebeck, F. Franke, L. Fink, M. Zhu, C. Sumners, and T. Unger Expression of Angiotensin AT1 and AT2 Receptors in Adult Rat Cardiomyocytes after Myocardial Infarction : A Single-Cell Reverse Transcriptase-Polymerase Chain Reaction Study Am. J. Pathol., August 1, 2000; 157(2): 605 - 611. [Abstract] [Full Text] [PDF]

				K. Yamamoto, T. Masuyama, Y. Sakata, T. Mano, N. Nishikawa, H. Kondo, N. Akehi, T. Kuzuya, T. Miwa, and M. Hori Roles of renin-angiotensin and endothelin systems in development of diastolic heart failure in hypertensive hearts Cardiovasc Res, August 1, 2000; 47(2): 274 - 283. [Abstract] [Full Text] [PDF]

				Yi Xu, V. Menon, and B. I Jugdutt Cardioprotection after angiotensin II type 1 blockade involves angiotensin II type 2 receptor expression and activation of protein kinase C-{varepsilon} in acutely reperfused myocardial infarction in the dog: Effect of UP269-6 and losartan on AT1- and AT2-receptor expression and IP3 receptor and PKC{varepsilon} proteins Journal of Renin-Angiotensin-Aldosterone System, June 1, 2000; 1(2): 184 - 195. [Abstract] [PDF]

				T. Matsumoto, R. Ozono, T. Oshima, H. Matsuura, T. Sueda, G. Kajiyama, and M. Kambe Type 2 angiotensin II receptor is downregulated in cardiomyocytes of patients with heart failure Cardiovasc Res, April 1, 2000; 46(1): 73 - 81. [Abstract] [Full Text] [PDF]

				H. Kawano, Y. S. Do, Y. Kawano, V. Starnes, M. Barr, R. E. Law, and W. A. Hsueh Angiotensin II Has Multiple Profibrotic Effects in Human Cardiac Fibroblasts Circulation, March 14, 2000; 101(10): 1130 - 1137. [Abstract] [Full Text] [PDF]

				S. Gallinat, S. Busche, M. K. Raizada, and C. Sumners The angiotensin II type 2 receptor: an enigma with multiple variations Am J Physiol Endocrinol Metab, March 1, 2000; 278(3): E357 - E374. [Abstract] [Full Text] [PDF]

				M. Horiuchi, W. Hayashida, M. Akishita, S. Yamada, J. Y. A. Lehtonen, K. Tamura, L. Daviet, Y. E. Chen, M. Hamai, T.-X. Cui, et al. Interferon-{gamma} Induces AT2 Receptor Expression in Fibroblasts by Jak/STAT Pathway and Interferon Regulatory Factor-1 Circ. Res., February 4, 2000; 86(2): 233 - 240. [Abstract] [Full Text] [PDF]

				B. I. Jugdutt, Yi Xu, M. Balghith, R. Moudgil, and V. Menon Cardioprotection Induced by AT1R Blockade After Reperfused Myocardial Infarction: Association With Regional Increase in AT2R, IP3R and PKC{varepsilon} Proteins and cGMP Journal of Cardiovascular Pharmacology and Therapeutics, January 1, 2000; 5(4): 301 - 311. [Abstract] [PDF]

				G.A.J. Riegger, H. Bouzo, P. Petr, J. Munz, R. Spacek, H. Pethig, V. von Behren, M. George, and H.-J. Arens Improvement in Exercise Tolerance and Symptoms of Congestive Heart Failure During Treatment With Candesartan Cilexetil Circulation, November 30, 1999; 100(22): 2224 - 2230. [Abstract] [Full Text] [PDF]

				S. Gunasegaram, R. S. Haworth, D. J. Hearse, and M. Avkiran Regulation of Sarcolemmal Na+/H+ Exchanger Activity by Angiotensin II in Adult Rat Ventricular Myocytes : Opposing Actions via AT1 Versus AT2 Receptors Circ. Res., November 12, 1999; 85(10): 919 - 930. [Abstract] [Full Text] [PDF]

				K. C Wollert and H. Drexler The renin-angiotensin system and experimental heart failure Cardiovasc Res, September 1, 1999; 43(4): 838 - 849. [Abstract] [Full Text] [PDF]

				M. Malmsjo, A. Bergdahl, S. Moller, X.-H. Zhao, X.-Y. Sun, T. Hedner, L. Edvinsson, and D. Erlinge Congestive heart failure induces downregulation of P2X1-receptors in resistance arteries Cardiovasc Res, July 1, 1999; 43(1): 219 - 227. [Abstract] [Full Text] [PDF]

				M. J. Mann, G. H. Gibbons, H. Hutchinson, R. S. Poston, E. G. Hoyt, R. C. Robbins, and V. J. Dzau Pressure-mediated oligonucleotide transfection of rat and human cardiovascular tissues PNAS, May 25, 1999; 96(11): 6411 - 6416. [Abstract] [Full Text] [PDF]

				M. Tanimura, V. G. Sharov, H. Shimoyama, T. Mishima, T. B. Levine, S. Goldstein, and H. N. Sabbah Effects of AT1-receptor blockade on progression of left ventricular dysfunction in dogs with heart failure Am J Physiol Heart Circ Physiol, April 1, 1999; 276(4): H1385 - H1392. [Abstract] [Full Text] [PDF]

				M. Horiuchi, M. Akishita, and V. J. Dzau Recent Progress in Angiotensin II Type 2 Receptor Research in the Cardiovascular System Hypertension, February 1, 1999; 33(2): 613 - 621. [Abstract] [Full Text] [PDF]

				J. Bartunek, E. O. Weinberg, M. Tajima, S. Rohrbach, and B. H. Lorell Angiotensin II Type 2 Receptor Blockade Amplifies the Early Signals of Cardiac Growth Response to Angiotensin II in Hypertrophied Hearts Circulation, January 12, 1999; 99(1): 22 - 25. [Abstract] [Full Text] [PDF]

				H. Matsubara Pathophysiological Role of Angiotensin II Type 2 Receptor in Cardiovascular and Renal Diseases Circ. Res., December 14, 1998; 83(12): 1182 - 1191. [Abstract] [Full Text] [PDF]

				Y. Tsutsumi, H. Matsubara, N. Ohkubo, Y. Mori, Y. Nozawa, S. Murasawa, K. Kijima, K. Maruyama, H. Masaki, Y. Moriguchi, et al. Angiotensin II Type 2 Receptor Is Upregulated in Human Heart With Interstitial Fibrosis, and Cardiac Fibroblasts Are the Major Cell Type for Its Expression Circ. Res., November 16, 1998; 83(10): 1035 - 1046. [Abstract] [Full Text] [PDF]

				R. L. Skolnick, S. E. Litwin, W. H. Barry, and K. W. Spitzer Effect of ANG II on pHi, [Ca2+]i, and contraction in rabbit ventricular myocytes from infarcted hearts Am J Physiol Heart Circ Physiol, November 1, 1998; 275(5): H1788 - H1797. [Abstract] [Full Text] [PDF]

				L. S. Zisman, K. Asano, D. L. Dutcher, A. Ferdensi, A. D. Robertson, M. Jenkin, E. W. Bush, T. Bohlmeyer, M. B. Perryman, and M. R. Bristow Differential Regulation of Cardiac Angiotensin Converting Enzyme Binding Sites and AT1 Receptor Density in the Failing Human Heart Circulation, October 27, 1998; 98(17): 1735 - 1741. [Abstract] [Full Text] [PDF]

				O. Zolk, M. Flesch, G. Nickenig, P. Schnabel, and M. Bohm Alteration of intracellular Ca2+-handling and receptor regulation in hypertensive cardiac hypertrophy: insights from Ren2-transgenic rats Cardiovasc Res, July 1, 1998; 39(1): 242 - 256. [Abstract] [Full Text] [PDF]

				R.S. Krombach, M. J Clair, J. W Hendrick, W. V Houck, J. L Zellner, S. B Kribbs, S. Whitebread, R. Mukherjee, M. de Gasparo, and F. G Spinale Angiotensin converting enzyme inhibition, AT1 receptor inhibition, and combination therapy with pacing induced heart failure: effects on left ventricular performance and regional blood flow patterns Cardiovasc Res, June 1, 1998; 38(3): 631 - 645. [Abstract] [Full Text] [PDF]

				L. Gullestad, G. Haywood, H. Aass, H. Ross, G. Yee, T. Ueland, O. Geiran, J. Kjekshus, S. Simonsen, N. Bishopric, et al. Angiotensin II receptor subtype AT1 and AT2 expression after heart transplantation Cardiovasc Res, May 1, 1998; 38(2): 340 - 347. [Abstract] [Full Text] [PDF]

				K. Ponicke, M. Vogelsang, M. Heinroth, K. Becker, O. Zolk, M. Bohm, H.-R. Zerkowski, and O.-E. Brodde Endothelin Receptors in the Failing and Nonfailing Human Heart Circulation, March 3, 1998; 97(8): 744 - 751. [Abstract] [Full Text] [PDF]

				N. Ohkubo, H. Matsubara, Y. Nozawa, Y. Mori, S. Murasawa, K. Kijima, K. Maruyama, H. Masaki, Y. Tsutumi, Y. Shibazaki, et al. Angiotensin Type 2 Receptors Are Reexpressed by Cardiac Fibroblasts From Failing Myopathic Hamster Hearts and Inhibit Cell Growth and Fibrillar Collagen Metabolism Circulation, December 2, 1997; 96(11): 3954 - 3962. [Abstract] [Full Text]

				M. van Bilsen Signal transduction revisited: recent developments in angiotensin II signaling in the cardiovascular system Cardiovasc Res, December 1, 1997; 36(3): 310 - 322. [Full Text] [PDF]

				K. Asano, D. L. Dutcher, J. D. Port, W. A. Minobe, K. D. Tremmel, R. L. Roden, T. J. Bohlmeyer, E. W. Bush, M. J. Jenkin, W. T. Abraham, et al. Selective Downregulation of the Angiotensin II AT1-Receptor Subtype in Failing Human Ventricular Myocardium Circulation, March 4, 1997; 95(5): 1193 - 1200. [Abstract] [Full Text]

				G. G. N. Serneri, M. Boddi, I. Cecioni, S. Vanni, M. Coppo, M. L. Papa, B. Bandinelli, I. Bertolozzi, G. Polidori, T. Toscano, et al. Cardiac Angiotensin II Formation in the Clinical Course of Heart Failure and Its Relationship With Left Ventricular Function Circ. Res., May 11, 2001; 88(9): 961 - 968. [Abstract] [Full Text] [PDF]

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