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© 1998 American Heart Association, Inc.
Original Contributions |
Angiotensin II Type 2 Receptor Is Upregulated in Human Heart With Interstitial Fibrosis, and Cardiac Fibroblasts Are the Major Cell Type for Its Expression
From the Department of Medicine II, Kansai Medical University (Y.T., H. Matsubara, N.O., Y. Mori, S.M., K.K., K.M., H. Masaki, Y. Moriguchi, Y.S., H.K., M.I., T.I.), Moriguchi, Osaka, Japan, and Pharmacological Laboratory, Taiho Pharmaceutical Co, Ltd (Y.N.), Tokushima, Japan.
Correspondence to Hiroaki Matsubara, MD, Department of Medicine II, Endocrine Hypertension, Metabolism and Renal Division, Kansai Medical University, Fumizonocho 10-15, Moriguchi, Osaka 570-8507, Japan. E-mail matsubah{at}takii.kmu.ac.jp
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Abstract |
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Abstract—The expression pattern of angiotensin (Ang) II type 2 receptor (AT2-R) in the remodeling process of human left ventricles (LVs) remains poorly defined. We analyzed its expression at protein, mRNA, and cellular levels using autopsy, biopsy, or operation LV samples from patients with failing hearts caused by acute (AMI) or old (OMI) myocardial infarction and idiopathic dilated cardiomyopathy (DCM) and also examined functional biochemical responses of failing hearts to Ang II. In autopsy samples from the nonfailing heart group, the ratio of AT1-R and AT2-R was 59% and 41%, respectively. The expression of AT2-R was markedly increased in DCM hearts at protein (3.5-fold) and mRNA (3.1-fold) levels compared with AMI or OMI. AT1-R protein and mRNA levels in AMI hearts showed 1.5- and 2.1-fold increases, respectively, whereas in OMI and DCM hearts, AT1-R expression was significantly downregulated. AT1-R–mediated response in inositol phosphate production was significantly attenuated in LV homogenate from failing hearts compared with nonfailing hearts. AT2-R sites were highly localized in the interstitial region in either nonfailing or failing heart, whereas AT1-R was evenly distributed over myocardium at lower densities. Mitogen-activated protein kinase (MAPK) activation by Ang II was significantly decreased in fibroblast compartment from the failing hearts, and pretreatment with AT2-R antagonist caused an additional significant increase in Ang II–induced MAPK activity (36%). Cardiac hypertrophy suggested by atrial and brain natriuretic peptide levels was comparably increased in OMI and DCM, whereas accumulation of matrix proteins such as collagen type 1 and fibronectin was much more prominent in DCM than in OMI. These findings demonstrate that (1) AT2-R expression is upregulated in failing hearts, and fibroblasts present in the interstitial regions are the major cell type responsible for its expression, (2) AT2-R present in the fibroblasts exerts an inhibitory effect on Ang II–induced mitogen signals, and (3) AT1-R in atrial and LV tissues was downregulated during chronic heart failure, and AT1-R–mediated functional biochemical responsiveness was decreased in the failing hearts. Thus, the expression level of AT2-R is likely determined by the extent of interstitial fibrosis associated with heart failure, and the expression and function of AT1-R and AT2-R are differentially regulated in failing human hearts.
Key Words: angiotensin II type 2 receptor • AT2 receptor • angiotensin II type 1 receptor • AT1 receptor, angiotensin II
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Introduction |
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The presence of 2 isoforms of angiotensin (Ang) II receptor was originally proposed on the basis of differences in sensitivity of receptor-ligand binding to dithiothreitol. Ang type 2 receptor (AT2-R), which is insensitive to dithiothreitol and has a high affinity for PD123319 and CGP42112A, was isolated, and this receptor was shown to have the same seven-transmembrane domain of AT1-R but only minimal homology (see Review in References 1 and 21 2 ). Most of Ang II functions in the cardiovascular system are mediated by AT1-R, whereas there is little information regarding the physiological roles of AT2-R. Mice lacking AT2-R suggested an antipressor action of AT2-R,3 4 and cardiac-specific overexpression mice of AT2-R showed attenuated response to Ang II–induced chronotropic action.5 The AT2-R also has an antigrowth effect upon neointimal formation after vascular injury6 and on coronary endothelial cells.7 Recent evidence has indicated that AT2-R associates with Gi proteins8 9 and mediates inhibitory effects of mitogen-activated protein kinase (MAPK) activation.8 9 10 11 Considering that AT2-R is abundantly and transiently expressed in fetal tissues,12 this receptor may have an anti–AT1-R effect on cellular growth and may also play a role in development and/or differentiation.
The first study that examined human myocardium showed that there were no significant changes in total Ang II receptor numbers between failing and nonfailing hearts.13 Recent studies using human failing hearts14 15 16 17 18 indicated selective downregulation of AT1-R but not AT2-R, leading to an increase in the distribution ratio of AT2-R relative to AT1-R. Clinical treatment with AT1-R antagonists causes elevation of plasma Ang II, which selectively binds to AT2-R, and may exert as-yet undefined cardiac effects.19 In the present study, we report using left ventricle (LV) samples from failing human hearts that AT2-R expression is upregulated in the fibroblasts present in fibrous regions, and the increased AT2-R has an ability to inhibit Ang II–induced mitogen signals, whereas the expression of AT1-R was downregulated and AT1-R–mediated functional biochemical response was significantly attenuated.
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Materials and Methods |
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Patients
LV tissues were obtained at autopsy from 17 subjects, 13 of whom had heart failure due to ischemic heart disease (n=8) or idiopathic dilated cardiomyopathy (DCM, n=9). Four normally functioning hearts (nonfailing group) were obtained from patients with brain death who had no history of cardiac disease or pulmonary disease (age, 52±7.5 years, range, 29 to 61, n=4). LVs obtained during operation for anuerysmectomy from 4 old myocardial infarction (OMI) patients and biopsy LV specimens from 5 DCM patients were also included in the present study. Diagnosis of ischemic heart disease or DCM had been performed by coronary angiography and histological examinations of biopsy samples. Patients with ischemic heart disease were further divided into 2 groups: those who died of acute myocardial infarction (AMI, n=3) within 5 days after onset of first heart attack, and those who died of ischemic cardiomyopathy (n=5) mainly as a result of OMI (n=5). Two subjects in the OMI group (patient No. 4 and No. 5 in the Table
) had received coronary bypass surgery. There were no
significant differences in mean age between AMI, OMI, and DCM groups
and the nonfailing control group (Table). Although we examined
gender-dependent difference in the AT1-R and
AT2-R densities, there appeared to be no specific change
between men and women (Table). Pulmonary artery
diastolic pressure (PAd) was measured by right heart
catheterization (Swan-Ganz), and the worst values
recorded within 24 hours before death were selected. Samples were
obtained from the base of LV including ventricular septum,
and in AMI and OMI patients, noninfarcted portions of LV were used for
preparation of membrane fractions. Biopsy specimens were obtained from
the subendocardium of LV free wall using biotorm.
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Nine of the patients were pretreated with the
angiotensin-converting enzyme (ACE) inhibitors
(25 to 50 mg/d of captopril) (Table
). This low-dose treatment resulted
in no significant difference in receptor numbers (Bmax) or
in dissociation constant (Kd) in patients with
and without ACE inhibitor therapy (Bmax: ACE
inhibitors, 8.4±1.3 fmol/mg protein; no ACE
inhibitors, 9.3±1.7 fmol/mg protein,
Kd, 0.90±0.2 and 0.89±0.2 nmol/L,
respectively). Complications of hypertension, diabetic mellitus, or
hyperlipidemia also did not affect Bmax
values. The study was approved by the Ethical Committee of Kansai
Medical University.
Preparation of Plasma Membrane Fractions
Autopsy samples (10 to 30 g) excised within 1 hour after
death, and tissue samples obtained during operation were immediately
placed in iced, oxygenated Tyrode's solution and then
homogenized by a Polytron at 4°C in 2 volumes of buffer A
composed of ice-cold 0.32 mol/L sucrose, 0.5 mmol/L EDTA, and
25 mmol/L Tris (pH 7.5), including protease inhibitors
(0.5 mmol/L phenylmethylsulfonyl fluoride, 10 mg/L
bacitracin, 4 µg/mL leupeptin, 4 µg/mL pepstatin, 40 U/mL trasylol,
and antipain, phosphoramidon, amastatin, and bestatin,
each at 1 µg/mL).20 21 22 The homogenates were
centrifuged at 500g for 10 minutes at 4°C, and the
supernatant fractions were recentrifuged at 48 000g
for 30 minutes at 4°C. The pellets were suspended in a solution
containing 0.6 mol/L KCl and 30 mmol/L histidine at pH 7.0
(including protease inhibitors mentioned above) to
solubilize actin and myosin filaments and were recentrifuged at
48 000g for 30 minutes at 4°C. The pellets obtained after
the final centrifugation were resuspended in binding
buffer containing protease inhibitors mentioned above and
immediately used for the binding assay. Thus, in the present study,
the tissue samples and membrane preparations were not subject to the
frozen storage process, because we had found that during the freezing
process of tissue samples, AT1-R and AT2-R
numbers were decreased by 30±2.7% and 8.2±1.4% (n=4), respectively.
Ligand binding assay was performed whenever each membrane fraction was
prepared, and interassay variations were very low (4.8±0.7, n=10).
Binding Assay and Analysis of Data
The binding assay of cardiac AT1-R and
AT2-R was performed as previously
described.20 21 22 Briefly, membrane fractions (
500 µg
of protein) were incubated with
125I-[Sar1,Ile8] Ang II for the
saturation experiment (0.2 nmol/L
125I-[Sar1,Ile8] Ang II for the
competition experiment) in a total assay volume of 300 µL for 90
minutes at 21°C. The assay buffer contained 50 mmol/L Tris (pH
7.6), 100 mmol/L NaCl, 10 mmol/L MgCl2, 1
mmol/L EGTA, 0.25 mg/mL BSA, and a variety of protease
inhibitors used for membrane preparation. The degradation
rate of [Sar1Ile8] Ang II after 90 minutes of
incubation at 21°C was determined by reverse-phase HPLC. The results
showed that under the incubation conditions, as much as 97.1±2.4%
(n=3) of the radioligand remained intact. Specific
125I-[Sar1,Ile8] Ang II binding
was determined from the difference between counts in the absence and
presence of 3 µmol/L unlabeled Ang II. Ki
was calculated from the equation
Ki=IC50/(1+L/Kd),
where L is the concentration of
125I-[Sar1,Ile8] Ang II. Total
Ang II receptor numbers were determined from Scatchard
analysis, and AT1-R and AT2-R were
separately calculated from total Ang II receptor numbers on the basis
of distribution ratio determined with nonlinear least-squares
regression analysis by inhibition of CGP42112A using the
GraphPad InPlot computer program (Graph Pad).21
Na+,K+-ATPase Activity
The assay was performed as previously reported in rat
hearts.20 Briefly, membranes were preincubated with or
without 5 mmol/L ouabain at 37°C for 5 minutes. The reaction was
initiated by the addition of 2 mmol/L ATP, and released
Pi was measured according to the modification of the method
of Parvin and Smith.
RNase Protection Analyses and Northern Blotting
RNase protection assays were performed with 40 µg of total RNA
as previously described.23 24 RNA was digested with DNase
in the presence of RNase inhibitor to remove contaminating
genomic DNA. cDNAs encoding human AT1-R and
AT2-R were subcloned into pBS vector. An RNA probe for the
AT1-R (270 bp) was produced from DraI-digested
EcoRI and PstI cDNA fragment of human
AT1-R (nucleotides -280 to +1140). An
AT2-R RNA probe (100 bp) was generated from
AvaII-digested EcoRI and KpnI fragment
of human AT2-R cDNA (nucleotides -189 to
+425). Antisense cRNA was transcribed using RNA polymerase and
hybridized with total RNA at 45°C and digested with RNase T1 and A,
resulting in 210- and 90-bp protected signals for the AT1-R
and AT2-R mRNAs. The difference between the size of the
protected signal and the RNA probe used was due to the digestion of an
additional part of RNA probe transcribed from DNA sequence present
in the linker site of pBS vector. For quantitative analyses,
the signal densities were measured by laser densitometry and normalized
relative to GAPDH mRNA levels quantified by Northern
blotting.25 26 When AT1-R mRNA levels in
nonfailing LV tissues were quantified by this analysis, the
inter- and intraassay variations were less than 10% (7.6%, n=5;
6.4%, n=7, respectively), suggesting that the validity of the data in
mRNA quantification was very high. Human atrial and brain
natriuretic peptides (ANPs and BNPs) cDNA probes were
kindly provided by Dr Y. Saitoh (Kyoto University), and rat
1 (I) collagen cDNA and fibronectin cDNA were kind gifts
from Dr D. Rowe (Connecticut Health Center) and Dr R.O. Hynes
(Massachusetts Institute of Technology).
Inositol Phosphate Assays
Inositol phosphate measurement using cardiac
homogenate from LV tissues was performed according to the
method described by Berridge,27 with a slight
modification. LV tissues kept in oxygenated Tyrode's
solution were minced finely with scissors and homogenized
to small pieces in the buffer A with the use of a
homogenizer. Cardiac homogenate was
centrifuged at 500g for 10 minutes at 4°C, and the
pellet was suspended in 10-volume of DMEM containing 200 µCi
[3H]-myoinositol and incubated for 3 hours at 22°C.
At the end of incubation, cardiac homogenate was rinsed 3
times with the buffer containing 20 mmol/L HEPES (pH 7.4) and
10 mmol/L LiCl and subsequently incubated with various
concentrations of Ang II in the same medium for 30 minutes at
22°C. The reactions were stopped by addition of 5-volume of
ice-cold chloroform/methanol (1:2, vol/vol), and the
chloroform/methanol extract was transferred to a test tube. Cardiac
homogenate was further homogenized with a
Polytron in 0.5 mol/L HCl, which was added to the chloroform/methanol
extract, shaken, and then centrifuged. The upper phase was
aspirated, dried, and stored at -30°C. The water-soluble inositol
phosphates were eluted with formic acid and ammonium formate by using
Dowex columns, according to the method established by
Berridge.27
Preparation of Cell Fraction, Measurement for MAPK Activity,
and Immunoblotting
Isolation of cells from human LV tissue was performed as
previously reported with a slight modification.15 Briefly,
pieces of LV free walls kept in oxygenated Tyrode's
solution were minced finely with scissors, incubated with Joklik medium
containing 0.3% collagenase for 10 minutes at 37°C, then
mechanically pipetted; the obtained supernatant was collected into
ice-cold DMEM with 20% FCS. This procedure was repeated 5 times. Cell
fraction was collected by a brief centrifugation,
incubated with the buffer containing 50 mmol/L Tris (pH 7.6),
100 mmol/L NaCl, 10 mmol/L MgCl2, 0.3 mmol/L
bacitracin, and 0.2% BSA in the presence or absence of Ang II receptor
antagonists for 20 minutes at 37°C, and then exposed to
Ang II (1 µmol/L) for 10 minutes at 37°C. This sample fraction
was considered to contain the dissociated intact cells, mostly cardiac
fibroblasts as shown by the previous study.15 The reaction
sample was lysed using a pestle in ice-cold buffer containing 10
mmol/L Tris-HCl (pH 7.4), 20 mmol/L NaCl, 1 mmol/L sodium
orthovanadate, 10 mmol/L sodium pyrophosphate, 10 nmol/L okadaic
acid, 2 mmol/L EGTA, 2 mmol/L dithiothreitol, and protease
inhibitors used in the buffer A. After brief sonication,
the homogenate was centrifuged for 5 minutes at
14 000g, and the cell lysate (50 µg of protein) in
supernatant was assayed for MAPK activity with an assay kit (Amersham)
that measured incorporation of [
-33P]ATP into a
synthetic peptide as a specific p44/42 MAPK substrate.5
The resultant solution was applied to a phosphocellulose membrane,
washed, and the radioactivity was measured by liquid scintillation. For
immunoblotting, the reaction sample was lysed with
SDS-PAGE buffer (pH 6.8), containing 62.5 mmol/L Tris-HCl, 2%
SDS, 10% glycerol, 50 mmol/L dithiothreitol, and 0.1% bromphenol
blue. After brief sonication, samples were boiled, subjected to
SDS-PAGE, transferred onto Hybound-ECL and blotted with
phospho-specific MAPK antibodies (New England Biolabs Inc) that detect
p42MAPK and p44MAPK as described.5
The protein on the filter was stripped and reprobed with MAPK antibody
(New England Biolabs Inc).
Emulsion Autoradiography and
Immunocytochemistry
Tissue sections (10 to 20 µm) were cut on a
cryostat at -20°C, thaw-mounted onto
poly-L-lysine–coated slides, dried in vacuo at 4°C over
silica gel, and stored at -80°C. The assay procedure was performed
the same as previously described in myopathic hamster
hearts.22 Briefly, endogenous Ang II bound to
Ang II receptors was removed by preincubating the sections for 15
minutes at room temperature in buffer containing 10 mmol/L sodium
phosphate (pH 7.4), 150 mmol/L NaCl, 1 mmol/L disodium EDTA,
0.3 mmol/L bacitracin, and 0.2% BSA. This preincubation was shown
sufficient to remove endogenous Ang II bound by comparing
Ang II binding preincubated with acidic buffer known to remove ligand
binding.22 The buffer was replaced with fresh buffer
containing 125I-[Sar1,Ile8] Ang
II (0.25 nmol/L, Amersham), and the sections were incubated at
21°C for 90 minutes. Preliminary experiments indicated that the
specific binding of
125I-[Sar1,Ile8] Ang II under
these incubation conditions reached more than 90% saturation. The
slides were then rinsed, dried, and used for emulsion
autoradiography. The slides were dipped in photographic
emulsion (Kodak NTB3), exposed for 14 days at 4°C, developed using
Kodak D-19, and stained with Kernechtrot.
Cells present in fibrous regions were characterized by
immunocytochemistry. Tissue sections were cut on a cryostat at -20°C
and fixed in acetone. The following primary antibodies (Sigma
Immunochemicals) were tested: monoclonal antibodies against vimentin
for the detection of fibroblasts, smooth muscle -actin for vascular
smooth muscle cells, desmin for cardiomyocytes, and a
polyclonal antibody against von Willebrand factor for detection
of endothelial cells. Immunocytochemistry was performed
by Biotin/Avidin system (Elite ABC kit, Vector Lab), using
diaminobenzidine tetrahydrochloride as a substrate.
Reagents and Statistical Methods
All reagents were purchased from Sigma Chemical Co, unless
otherwise indicated. Losartan was provided by DuPont Merck
Pharmaceutical (Wilmington, Del). PD123177 and PD123319 were provided
by Parke-Davis and Warner-Lambert Co (Ann Arbor, Mich), and CGP42112A
was purchased from NEOSYSTEM (Strasbourg, France). Results are
expressed as mean±SE. Data analyses were performed using a
commercially available statistical program (SAS, Statistical
Analysis System, SAS Institute Inc) on an IBM PC. Statistical
analyses in Figures 2 through 7 were performed with 1-way
ANOVA, and actual P values from the ANOVAs are shown in the
respective figure legends. We proceeded to pairwise contrasts (control
versus conditions) using Dunnett's multiple-comparison test only if
the outcome of 1-way ANOVA was significant in Figures 2 through 5 and
Figure 7. In Figure 6B and 6C, the pairwise comparisons (untreated
control versus Ang II treatment, Ang II versus Ang II+losartan,
or Ang II+PD) were performed with Holm's stepdown procedure. Data were
considered statistically significant when P was <0.05.
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Both AT1-R and AT2-R Were Expressed in Human LV
Membrane fractions from all autopsy samples had high-affinity binding sites, with a Ki of 0.89±0.15 nmol/L (Figure 1A
and 1B).
Competition experiments suggested the presence of 2 classes of binding
sites (Figure 1C), and nonlinear least-squares regression
analysis indicated the existence of 2 populations of Ang II
binding sites, one with high (Ki=7.2±0.2
nmol/L) and the other with low
(Ki=0.14±0.03 µmol/L) affinity for
losartan. When CGP42112A was used for inhibition,
Ki values of high- and low-affinity sites were
0.15±0.03 nmol/L and 0.64±0.28 µmol/L, respectively. The
relative ratio of high- and low-affinity sites for CGP42112A was
calculated as 59% and 41%, respectively, in the nonfailing
heart group (n=4, Table). The ratio of
AT1-R/AT2-R by losartan inhibition was
calculated as 57/43%.
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To further confirm the accuracy of the assay method, we
determined AT2-R numbers in LVs from 5 DCM patients (Table)
using AT2-R selective ligand [125I]-CGP42112A
and compared them with AT2-R numbers determined using
[125I]-Sar1,Ile8 Ang II.
AT2-R numbers determined using
[125I]-CGP42112A were 9.7±1.4 fmol/mg protein (patient
No. 9: 7.9, No. 10: 7.1, No. 11: 8.0, No. 12: 14.9, and No. 13: 10.7),
similar to AT2-R numbers calculated by CGP42112A inhibition
(Table), and the interassay variation in AT2-R numbers
determined with the 2 diff`erent methods was <10% (8.6±1.4%).
Next we examined the purity of membrane fractions. The activity of the membrane marker ouabain-sensitive Na+,K+-ATPase was 15±0.86, 16±1.2, and 16±1.3 µmol/mg protein per hour in autopsy samples from the nonfailing, OMI, and DCM hearts (patient No. 9 to No. 13), respectively. The activity in autopsy OMI samples was comparable with that in operation samples (17±0.64). These values were similar to those reported in animal hearts,28 suggesting that the membranes contained an enriched cardiac membrane fraction.
Ventricular AT1-R Numbers Were Decreased in
Both OMI and DCM, Whereas Ventricular AT2-R
Numbers Were Upregulated in DCM
As shown in Figure 2 (top) and the
Table, AT1-R numbers were significantly decreased 63%
in the OMI and DCM groups, respectively, compared with those in the
nonfailing heart group (4.1±0.2 fmol/mg protein), whereas
AT1-R numbers in the AMI group were significantly increased
about 54% relative to those in the nonfailing heart group. On the
other hand, AT2-R numbers were markedly (293%) increased
in the DCM group (11.4±1.3 fmol/mg protein) relative to those in the
nonfailing heart group (2.9±0.2). There were no significant
differences in AT2-R numbers between AMI (3.0±0.3), OMI
(3.9±0.5), and nonfailing heart groups.
AT1-R and AT2-R Were Detected in Atrial
Tissues, and Atrial AT1-R but not AT2-R Was
Downregulated in Failing Hearts
We also measured AT1-R and AT2-R numbers
in membrane fractions from right atria. As shown in Figure 3 (top), AT1-R numbers were
significantly decreased about 43% and 51% in the OMI and DCM groups,
respectively, compared with those in the nonfailing heart group
(6.9±0.4 fmol/mg protein), whereas AT1-R numbers in the
AMI group were not significantly different from those in the nonfailing
group. On the other hand, AT2-R numbers in the AMI, OMI,
and DCM groups did not significantly differ from those in the
nonfailing heart group (Figure 3, bottom).
Change in AT1-R Numbers Was due to Regulation in
AT1-R mRNA Level
AT1-R mRNA levels in the AMI group were significantly
increased by 113% compared with those in the control group (Figure 4). In the OMI and DCM groups,
AT1-R mRNA levels were downregulated by 46% and 57%
(P<0.01), respectively, compatible with changes at protein
levels. Similar regulation of AT1-R was also observed in
operation and biopsy samples. GAPDH mRNA levels were similar between
all study groups including nonfailing and ischemic hearts
(Figure 4A).
Upregulation in AT2-R Numbers in DCM Hearts Was due to
Increased AT2-R mRNA Accumulation
AT2-R mRNA levels in both autopsy and biopsy samples
from DCM were increased by 213% and 177%, respectively, compared with
the nonfailing heart group (Figure 5).
This increase agreed with the change at the protein level (Figure 2, top). There was no significant difference in AT2-R mRNA
levels between AMI, OMI, and nonfailing heart groups.
AT1-R–Mediated Inositol Phosphate Production
Was Decreased in Cardiac Homogenate From Failing Heart
Samples
AT1-R–mediated response of inositol phosphate
production was measured using cardiac homogenates
from LV tissue samples including both myocyte and nonmyocyte
compartments in the presence of AT2-R
antagonist PD123319 (Figure 6A). AT1-R–mediated response
was markedly inhibited in OMI and DCM hearts, and this inhibition
(P<0.01) at 1 µmol/L Ang II was about 54% and 72%
in autopsy samples from OMI and DCM, respectively, and 59% in
operation samples from OMI. Although these experiments were performed
in the presence of PD123319, the reduced response was observed to a
similar extent even in the absence of PD123319, and pretreatment with
losartan (10 µmol/L) completely blocked the inositol
phosphate production by 1 µmol/L Ang II (data not
shown). Thus, AT1-R–mediated inositol phosphate response
of failing hearts was decreased in the tissue homogenate,
including both myocyte and nonmyocyte compartments, in good
agreement with the result of AT1-R downregulation in mRNA
levels and membrane fractions prepared from tissue samples of failing
hearts.
Ang II–induced MAPK Activity Was Attenuated in Cell Fraction
Isolated From DCM Heart
Exposure of cell fraction isolated from nonfailing hearts to Ang
II (1 µmol/L) induced a significant increase (94%,
P<0.001) in MAPK activities compared with those in the
untreated samples (Figure 6B). Next we tested the effects of
pretreatment with losartan and PD123319 on the increase in MAPK
activities induced by Ang II. Ang II–induced MAPK activities were
almost completely abolished by pretreatment with 5 µmol/L of
losartan (P<0.001), whereas pretreatment with
PD123319 did not cause a significant change (Figure 6B). On the other
hand, exposure of cell fraction from DCM hearts to Ang II (1
µmol/L) did not result in a significant increase in MAPK activities
relative to those in the untreated samples (Figure 6C). We also tested
the effects of pretreatment with losartan and PD123319 on the
increase in MAPK activities induced by Ang II. Ang II–induced MAPK
activities (274±10 pmol · min-1 ·
mg-1) were further (36%, P<0.001) increased
by PD123319 pretreatment (371±27 pmol · min-1
· mg-1) whereas pretreatment with losartan
significantly (24%, P<0.05) inhibited Ang II–induced MAPK
activities (Figure 6C).
AT1-R and AT2-R Numbers and mRNA Levels in
Autopsy Samples Were Similar to Those in Samples Obtained at Biopsy
or Operation
The above-mentioned studies were mainly examined using autopsy
samples within 1 hour after death. To test whether the length of time
transpired before hearts were removed affected Ang II receptor mRNA and
numbers, we measured its expression level using LV samples obtained
during operation of aneurysmectomy from OMI patients (n=4) and
biopsy specimens from DCM patients (n=5). As shown in Figure 2, total
Ang II receptor numbers and the relative ratio of
AT1-R/AT2-R in operation samples from OMI
hearts were very similar to those in autopsy samples. AT1-R
mRNA levels in operation and biopsy samples from OMI or DCM were
comparably downregulated compared with those in the nonfailing heart
group (Figure 4B), whereas AT2-R mRNA levels were markedly
increased in biopsy samples from DCM (Figure 5B).
AT1-R–mediated inositol phosphate production
response was reduced to a similar extent between operation and autopsy
OMI samples (Figure 6A). Taken together with the findings that GAPDH
mRNA levels were comparable between all study groups (Figure 4) and
that RNA quality, judging from 28S/18S ratio of rRNA, was high in
autopsy samples (data not shown), it was likely that the length of time
transpired before autopsy hearts were removed had no significant
influence on the distribution pattern of Ang II receptor subtypes.
Expression of Hypertrophy Marker and Extracellular
Matrix Genes Was Markedly Increased in DCM Hearts
To examine the development of cardiac
hypertrophy and interstitial fibrosis, ANP and
BNP mRNA levels, as hypertrophy markers,29 30
and collagen type 1 and fibronectin mRNA levels, known as major
extracellular matrix components,31 32 were quantified. ANP
and BNP mRNA levels in OMI hearts were markedly increased compared with
those in nonfailing hearts, and this level was comparable with the
levels in DCM hearts (Figure 7). Collagen
type 1 and fibronectin mRNA levels were also significantly increased in
both OMI and DCM hearts, whereas the increase in DCM was more prominent
than in OMI (Figure 7). Although in the AMI hearts ANP and BNP mRNA
levels were moderately augmented compared with those in OMI and DCM,
collagen type 1 and fibronectin mRNA levels did not significantly
differ from those in nonfailing hearts. These findings suggest that the
development of ventricular hypertrophy was
similar between OMI and DCM hearts, whereas the degree of cardiac
fibrosis estimated by deposition of extracellular matrix components was
more remarkable in DCM hearts than in OMI hearts.
AT2-R Was Highly Localized in Fibrous Regions in
DCM Hearts
To identify the cells expressing AT2-R in DCM
hearts, cryostat LV sections were incubated with
125I-[Sar1,Ile8] Ang II, followed
by exposure to photographic emulsion. Regions with
interstitial fibrosis were distinguished from the
surrounding myocardium (indicated by arrows in Figure 8 No. 1) by staining with hematoxylin and
eosin. Tissue distributions of Ang II receptors were evaluated by the
numbers of silver grains overlying the myocardium. As shown
in Figure 8 No. 2, Ang II binding sites were abundant in the regions
with interstitial fibrosis with fewer binding sites in the
surrounding myocardium. Competition with PD123319 strongly
inhibited Ang II binding in fibrous regions (Figure 8 No. 3), whereas
losartan did not appreciably affect these binding sites (Figure 8 No. 4), indicating that fibrous regions are the major site for
AT2-R expression. Nonspecific bindings in both fibrous
regions and surrounding myocardium (Figure 8 No. 5) were
fewer than the binding sites in the presence of PD123319 (Figure 8 No.
3), suggesting that AT1-R is present in both
fibroblasts and myocytes at a very low expression level. In contrast,
there was no obvious inhibition in Ang II binding by pretreatment with
losartan (Figure 8 No. 4) compared with the binding in the
absence of competitors (Figure 8 No. 2). As the binding affinity
suggested by Ki value is about 10-fold higher in
PD123319 than in losartan,21 such difference in
Ang II binding might be involved in a lack of obvious inhibition by
losartan. Although AT2-R sites were highly
localized in fibrous regions and its expression level in
myocardium was much lower (Figure 8 No. 4), nonspecific
binding sites in myocardium (Figure 8 No. 5) were obviously
fewer than those in the presence of losartan (Figure 8 No. 4),
suggesting that AT2-R is also expressed in cardiac myocytes
at a much lower level.
|
We also examined the distribution of Ang II receptor subtypes in the LV
tissue from the nonfailing heart (Figure 9 No. 1). Ang II binding sites were
present more numerously in the interstitial region than
in the myocardium (Figure 9 No. 2). Interestingly, Ang II
binding in the interstitial region was nearly abolished by
pretreatment with PD123319 (Figure 9 No. 3), and losartan
pretreatment inhibited the Ang II binding to a lesser extent (Figure 9
No. 4), suggesting that AT2-R is the predominant subtype
present in fibroblasts in the normal human heart. On the other
hand, Ang II binding in the myocardium was predominantly
inhibited by losartan pretreatment (Figure 9 No. 4) rather than
by PD123319 pretreatment (Figure 9 No. 3). Considering that the Ang II
binding in the myocardium in the failing state was less
affected by pretreatment with losartan (Figure 8 No. 4), these
findings suggest that the AT1-R is chiefly expressed in
myocardium rather than in interstitial regions,
and in the heart failure state, its expression level is
downregulated.
|
The presence of fibrous regions in myocardium was clearly
shown by hematoxylin-eosin staining (indicated by arrows in Figure 8
No. 1). To further identify the cell types, we stained LV sections
using antibodies against vimentin (for fibroblasts), desmin (for
myocytes), smooth muscle -actin (for vascular smooth muscle cells),
or von Willebrand factor (for endothelial
cells). Cells present in fibrous regions were positive for vimentin
but were negative for other cell markers, whereas cells surrounding
this region were positive for desmin (data not shown), demonstrating
that the majority of the cells present in fibrous regions are
fibroblasts.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The major new findings of the present human study were that (1) AT2-R expression was increased in human hearts with prominent deposition of extracellular matrix components, and fibroblasts present in interstitial regions were the major cell type responsible for AT2-R expression, (2) AT2-R reexpressed in the fibroblasts exerted an inhibitory effect on Ang II–induced mitogen signals, (3) the expression of AT1-R in atrial and LV tissues was downregulated during chronic heart failure, and biochemical responsiveness assessed by Ang II–induced inositol phosphate production was significantly attenuated in the failing hearts. We also showed upregulation of ventricular AT1-R in hearts with AMI, consistent with the data previously demonstrated in animal studies.33 34 35
The present human study was mainly carried out using autopsy
samples obtained immediately after death. Ang II receptor levels and
Na+,K+-ATPase activities in samples obtained at
operation or biopsy were very similar to those in autopsy samples, and
also RNA quality, judging from 28S/18S ratio of rRNA and GAPDH mRNA,
was high in autopsy samples. Ang II–induced inositol phosphate
production was decreased to a similar extent between autopsy
and operation samples. This evidence established that the length of
time transpired before hearts were removed from autopsy samples had no
significant influence on the purity of plasma membrane or
AT1-R and AT2-R numbers. Measurement of the
plasma membrane marker ouabain-sensitive
Na+,K+-ATPase activity indicated that the
membrane was an enriched cardiac membrane fraction. The expression
pattern of AT1-R and AT2-R observed using
plasma membranes was compatible with that in combined membrane/light
vesicle fractions, suggesting that the change in the expression of Ang
II receptors cannot be explained by internalization of receptors. The
result of autoradiography using samples that did not
undergo the process of membrane preparation was also consistent
with the data of binding assays. These findings demonstrate that the
low level of AT1-R and upregulation of AT2-R in
DCM hearts did not result from an inappropriate method for membrane
preparation or the process of receptor internalization.
AT1-R and AT2-R numbers did not differ between
the patients treated or untreated with ACE inhibitors
(Table). Given that production of Ang II is inhibited in the
patients treated with ACE inhibitors and that there is no
relationship between plasma Ang II levels and ventricular
Ang II receptor numbers in cardiomyopathic
hamsters,36 it is unlikely that plasma Ang II levels
influence the expression pattern of cardiac Ang II receptors.
Total Ang II receptor numbers in LV were in the range of 3.8 to 17.3
fmol/mg protein (Table), similar to the results reported by the
previous studies,13 14 15 16 but were lower than the receptor
numbers (118, n=5) determined by de Gasparo et al.17 37 38
de Gasparo et al used combined fractions including plasma membranes and
internalized receptors, whereas we and other studies13 14 15 16
measured Ang II receptor numbers using only membrane fractions. In
fact, Regitz-Zagrosek et al14 found that the
Bmax values determined with combined fractions were
increased compared with those using only membranes. Urata et
al13 reported that Ang II receptor numbers were not
significantly different between normal and DCM hearts. They prepared
membranes using frozen tissue samples from the middle portion of LVs.
In contrast, we used the base of LVs, in which Ang II receptor numbers
were higher than in the middle portion13 and prepared
membranes without the freezing process of tissue samples that caused
decreases in AT1-R and AT2-R numbers (see
Materials and Methods). Thus, the discrepancy may have been due to
methodological differences, such as dissimilarities in the purity of
the membrane fractions, the portion of ventricles examined, or membrane
preparation methods.
We found that cardiac fibroblasts present in the interstitial region are the major cell type expressing AT2-R in LV tissues from either normal or failing heart, in good agreement with localization of AT2-R in human atrial tissues38 or in LV of cardiomyopathic hamsters.22 Because of limited resolution of the autoradiographic technique and the homogeneous distribution of relatively low-density myocardial Ang II binding sites, the cellular resolution of AT1-R and AT2-R in the normal or failing myocardium remained unclear. Although the presence of the interstitial region might suggest that the control nonfailing heart examined in the present study is not perfectly the normal heart, the interstitial region was observed in all control hearts with normal heart function. Thus, it remains to be determined whether the dominant expression of AT2-R in the interstitial region in the control heart reflects the normal distribution pattern of Ang II receptor subtypes in fibroblasts present in the normal human myocardium or is partially modified by pathological changes associated with interstitial fibrosis. However, these findings imply that the expression level of cardiac AT2-R is likely determined by the extent of the region with interstitial fibrosis and also have the interesting pharmacological implications for the predicted actions of AT1-R antagonists. As circulating Ang II levels are increased by administration of AT1-R antagonists19 and Ang II preferentially binds to the cardiac AT2-R, AT2-R–mediated actions are expected to be activated in failing hearts, especially on cardiac fibroblasts. Although the effects mediated by AT2-R remain unclear, there is increasing evidence that AT2-R activates pathways leading to inhibition of cell growth6 7 and that AT2-R overexpression in vascular smooth muscle cells inhibits cell proliferation by decreasing MAPK activity.6 We reported using cardiac fibroblasts from myopathic hamster hearts that AT2-R had an inhibitory effect on AT1-R–mediated DNA and collagenous protein synthesis.22 In the present study, we also found that AT2-R significantly inhibited Ang II–induced MAPK activation in fibroblast compartment from the failing hearts. However, considering that pretreatment with PD123319 does not completely normalize the Ang II–induced MAPK activity, the downregulation of AT1-R in the fibroblast compartment from the failing hearts might be also partially involved in the decrease in Ang II–induced MAPK activity. Taken together with our previous data that AT2-R has an inhibitory effect on AT1-R–mediated DNA and collagenous protein synthesis in fibroblasts from myopathic hamster hearts,22 it is likely that AT2-R in cardiac fibroblasts at least partially has the ability to inhibit the progression of interstitial fibrosis in failing hearts by decreasing Ang II–induced proliferation of fibroblasts as well as extracellular matrix protein accumulation.
We reported that in rats after AMI, AT1-R expression is increased,33 in good agreement with the result in the present human study. Related studies using rats after AMI showed that upregulation of AT1-R is mainly due to myocyte hypertrophy.34 39 In the present study, ANP and BNP mRNA levels were increased in AMI hearts, supporting a positive relationship between hypertrophy and AT1-R expression. However, ANP and BNP mRNA levels were increased more in OMI and DCM hearts, which contrasted with downregulation of AT1-R in failing hearts. Thus, it appears that the expression of AT1-R is differentially regulated between acute and chronic heart failure, and additional studies will be required to identify this differential mechanism.
It was not defined in the present study whether
downregulation of AT1-R in failing hearts occurred at the
myocyte level or was due to relative dilution of the myocyte
compartment by nonmuscle cells. Using hematoxylin and eosin staining,
we found that in nonfailing hearts, interstitial fibrosis
in myocardium was reduced and in OMI hearts, its extent
tended to be slightly increased. However, in DCM hearts (although the
presence of replacement fibrosis was already confirmed by biopsy),
greater fibrous regions were scattered in myocardium.
Collagen type 1 and fibronectin mRNA levels, known as major
extracellular matrix components and as a specific marker for fibroblast
proliferation,31 32 were more markedly increased in DCM
hearts than in OMI hearts (Figure 7). Although these data indicated
that the extent of interstitial fibrosis was much greater
in DCM hearts, AT1-R numbers were similarly downregulated
in both OMI and DCM hearts (Figure 2). We used ANP and BNP as a
specific marker for myocyte hypertrophy and found that both
mRNA levels were markedly increased in OMI and DCM groups to a similar
extent compared with those in nonfailing hearts, suggesting comparable
hypertrophy of remaining viable myocytes in both groups.
Thus, although in the present study we could not quantitatively
define the ratio between myocytes and nonmyocytes by the
histological data, these data using specific markers
for fibroblast and myocyte strongly suggested that AT1-R
downregulation was unlikely due to relative dilution of myocyte
compartment by nonmuscle cells. Although previous studies reported
downregulation of AT1-R in human failing
hearts,15 16 17 18 the exact mechanism responsible for its
regulation has not been clarified yet. Additional studies will be
needed to determine whether AT1-R downregulation reflects a
decrease in numbers of viable myocytes in failing hearts or is due to
specific regulation of gene transcription, transcript stability, or a
currently unidentified mechanism at myocyte level.
The present study demonstrated that AT1-R expression was decreased in failing hearts as observed in ß-adrenergic receptor,40 and that Ang II–induced inositol phosphate response was also decreased in the compartment including both myocytes and nonmyocytes. These findings suggest that AT1-R–mediated effects leading to elevation of intracellular free calcium might conceivably result in sluggish calcium transients and hence reduced contractile force in patients with heart failure. Recently, we have reported that cardiac myocyte–specific overexpression mice of AT2-R shows attenuated response to Ang II–induced chronotropic action.5 Taken together with the inhibitory effect of AT2-R reexpressed in cardiac fibroblasts on cell proliferation and production of collagenous matrix proteins,22 an induction of AT2-R in failing human hearts likely has desirable effects on cardiac function or the remodeling process, and hence selective stimulation of AT2-R by treatment with AT1-R antagonists might result in cardiac protection as explained by an unexpected lower risk of mortality than the ACE inhibitor in older patients with heart failure.41
|
Acknowledgments |
|---|
This study was supported in part by research grants from the Ministry of Education, Science and Culture, Japan, the Study Group of Molecular Cardiology, the Naito Foundation, the Clinical Pharmacology Foundation, Japan, the Japanese Medical Association, and the Japan Smoking Foundation. We thank Dr N. Yamazaki, Pharmaceutical Research Division (Takeda Chemical Industries), and Y. Okayama (Taiho Pharmaceutical Co, Ltd) for excellent assistance and helpful discussion in the statistical analyses of the data.
Received December 3, 1997; accepted September 8, 1998.
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Y. Oishi, R. Ozono, Y. Yano, Y. Teranishi, M. Akishita, M. Horiuchi, T. Oshima, and M. Kambe Cardioprotective Role of AT2 Receptor in Postinfarction Left Ventricular Remodeling Hypertension, March 1, 2003; 41(3): 814 - 818. [Abstract] [Full Text] [PDF]
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S. Kurisu, R. Ozono, T. Oshima, M. Kambe, T. Ishida, H. Sugino, H. Matsuura, K. Chayama, Y. Teranishi, O. Iba, et al. Cardiac Angiotensin II Type 2 Receptor Activates the Kinin/NO System and Inhibits Fibrosis Hypertension, January 1, 2003; 41(1): 99 - 107. [Abstract] [Full Text] [PDF]
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 I. Manabe, T. Shindo, and R. Nagai Gene Expression in Fibroblasts and Fibrosis: Involvement in Cardiac Hypertrophy Circ. Res., December 13, 2002; 91(12): 1103 - 1113. [Abstract] [Full Text] [PDF]
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 E. Pantev, E. Stenman, A. Wackenfors, L. Edvinsson, and M. Malmsjo Comparison of the antagonistic effects of different angiotensin II receptor blockers in human coronary arteries Eur J Heart Fail, December 1, 2002; 4(6): 699 - 705. [Abstract] [Full Text] [PDF]
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S.-M. Herrmann, V. Nicaud, K. Schmidt-Petersen, J. Pfeifer, J. Erdmann, T. McDonagh, H. J. Dargie, M. Paul, and V. Regitz-Zagrosek Angiotensin II type 2 receptor gene polymorphism and cardiovascular phenotypes: the GLAECO and GLAOLD studies Eur J Heart Fail, December 1, 2002; 4(6): 707 - 712. [Abstract] [Full Text] [PDF]
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 A. Goette, U. Lendeckel, and H. U Klein Signal transduction systems and atrial fibrillation Cardiovasc Res, May 1, 2002; 54(2): 247 - 258. [Abstract] [Full Text] [PDF]
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 H. Okada, T. Inoue, Y. Kanno, T. Kobayashi, Y. Watanabe, J. B. Kopp, R. M. Carey, and H. Suzuki Interstitial Fibroblast-Like Cells Express Renin-Angiotensin System Components in a Fibrosing Murine Kidney Am. J. Pathol., March 1, 2002; 160(3): 765 - 772. [Abstract] [Full Text] [PDF]
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 K. Hirasawa, Y. Sato, Y. Hosoda, T. Yamamoto, and H. Hanai Immunohistochemical Localization of Angiotensin II Receptor and Local Renin-Angiotensin System in Human Colonic Mucosa J. Histochem. Cytochem., February 1, 2002; 50(2): 275 - 282. [Abstract] [Full Text] [PDF]
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 L. Wu, M. Iwai, H. Nakagami, R. Chen, J. Suzuki, M. Akishita, M. de Gasparo, and M. Horiuchi Effect of Angiotensin II Type 1 Receptor Blockade on Cardiac Remodeling in Angiotensin II Type 2 Receptor Null Mice Arterioscler Thromb Vasc Biol, January 1, 2002; 22(1): 49 - 54. [Abstract] [Full Text] [PDF]
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A. Gonzalez, B. Lopez, S. Ravassa, R. Querejeta, M. Larman, J. Diez, and M. A. Fortuno Stimulation of Cardiac Apoptosis in Essential Hypertension: Potential Role of Angiotensin II Hypertension, January 1, 2002; 39(1): 75 - 80. [Abstract] [Full Text] [PDF]
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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]
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J. Deinum, J. M.G. van Gool, M. J.M. Kofflard, F. J. ten Cate, and A.H. J. Danser Angiotensin II Type 2 Receptors and Cardiac Hypertrophy in Women With Hypertrophic Cardiomyopathy Hypertension, December 1, 2001; 38(6): 1278 - 1281. [Abstract] [Full Text] [PDF]
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Y. Shibasaki, H. Matsubara, Y. Nozawa, Y. Mori, H. Masaki, A. Kosaki, Y. Tsutsumi, Y. Uchiyama, S. Fujiyama, A. Nose, et al. Angiotensin II Type 2 Receptor Inhibits Epidermal Growth Factor Receptor Transactivation by Increasing Association of SHP-1 Tyrosine Phosphatase Hypertension, September 1, 2001; 38(3): 367 - 372. [Abstract] [Full Text] [PDF]
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M. D. Schneider and B. H. Lorell AT2, Judgment Day: Which Angiotensin Receptor Is the Culprit in Cardiac Hypertrophy? Circulation, July 17, 2001; 104(3): 247 - 248. [Full Text] [PDF]
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S. Ichihara, T. Senbonmatsu, E. Price Jr, T. Ichiki, F. A. Gaffney, and T. Inagami Angiotensin II Type 2 Receptor Is Essential for Left Ventricular Hypertrophy and Cardiac Fibrosis in Chronic Angiotensin II-Induced Hypertension Circulation, July 17, 2001; 104(3): 346 - 351. [Abstract] [Full Text] [PDF]
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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]
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J. Varagic, D. Susic, and E. D. Frohlich Coronary Hemodynamic and Ventricular Responses to Angiotensin Type 1 Receptor Inhibition in SHR : Interaction With Angiotensin Type 2 Receptors Hypertension, June 1, 2001; 37(6): 1399 - 1403. [Abstract] [Full Text] [PDF]
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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]
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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]
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D. T. Dinh, A. G. Frauman, M. Sourial, D. J. Casley, C. I. Johnston, and M. E. Fabiani Identification, Distribution, and Expression of Angiotensin II Receptors in the Normal Human Prostate and Benign Prostatic Hyperplasia Endocrinology, March 1, 2001; 142(3): 1349 - 1356. [Abstract] [Full Text] [PDF]
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D. E. Dostal Regulation of Cardiac Collagen : Angiotensin and Cross-Talk With Local Growth Factors Hypertension, March 1, 2001; 37(3): 841 - 844. [Full Text] [PDF]
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L. Barlucchi, A. Leri, D. E. Dostal, F. Fiordaliso, H. Tada, T. H. Hintze, J. Kajstura, B. Nadal-Ginard, and P. Anversa Canine Ventricular Myocytes Possess a Renin-Angiotensin System That Is Upregulated With Heart Failure Circ. Res., February 16, 2001; 88(3): 298 - 304. [Abstract] [Full Text] [PDF]
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R. E. Schmieder, J. Erdmann, C. Delles, J. Jacobi, E. Fleck, K. Hilgers, and V. Regitz-Zagrosek Effect of the angiotensin II type 2-receptor gene (+1675 G/A) on left ventricular structure in humans J. Am. Coll. Cardiol., January 1, 2001; 37(1): 175 - 182. [Abstract] [Full Text] [PDF]
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M. Akishita, M. Iwai, L. Wu, L. Zhang, Y. Ouchi, V. J. Dzau, and M. Horiuchi Inhibitory Effect of Angiotensin II Type 2 Receptor on Coronary Arterial Remodeling After Aortic Banding in Mice Circulation, October 3, 2000; 102(14): 1684 - 1689. [Abstract] [Full Text] [PDF]
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J. P. van Kats, D. J. Duncker, D. B. Haitsma, M. P. Schuijt, R. Niebuur, R. Stubenitsky, F. Boomsma, M. A. D. H. Schalekamp, P. D. Verdouw, and A. H. J. Danser Angiotensin-Converting Enzyme Inhibition and Angiotensin II Type 1 Receptor Blockade Prevent Cardiac Remodeling in Pigs After Myocardial Infarction : Role of Tissue Angiotensin II Circulation, September 26, 2000; 102(13): 1556 - 1563. [Abstract] [Full Text] [PDF]
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 M. de Gasparo, K. J. Catt, T. Inagami, J. W. Wright, and Th. Unger International Union of Pharmacology. XXIII. The Angiotensin II Receptors Pharmacol. Rev., September 1, 2000; 52(3): 415 - 472. [Abstract] [Full Text] [PDF]
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A. Leri, F. Fiordaliso, M. Setoguchi, F. Limana, N. H. Bishopric, J. Kajstura, K. Webster, and P. Anversa Inhibition of p53 Function Prevents Renin-Angiotensin System Activation and Stretch-Mediated Myocyte Apoptosis Am. J. Pathol., September 1, 2000; 157(3): 843 - 857. [Abstract] [Full Text] [PDF]
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 R. P. MARSHALL, R. J. MCANULTY, and G. J. LAURENT Angiotensin II Is Mitogenic for Human Lung Fibroblasts via Activation of the Type 1 Receptor Am. J. Respir. Crit. Care Med., June 1, 2000; 161(6): 1999 - 2004. [Abstract] [Full Text]
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C. Kupfahl, D. Pink, K. Friedrich, H. R. Zurbrugg, M. Neuss, C. Warnecke, J. Fielitz, K. Graf, E. Fleck, and V. Regitz-Zagrosek Angiotensin II directly increases transforming growth factor {beta}1 and osteopontin and indirectly affects collagen mRNA expression in the human heart Cardiovasc Res, June 1, 2000; 46(3): 463 - 475. [Abstract] [Full Text] [PDF]
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A. Goette, T. Staack, C. Rocken, M. Arndt, J. C. Geller, C. Huth, S. Ansorge, H. U. Klein, and U. Lendeckel Increased expression of extracellular signal-regulated kinase and angiotensin-converting enzyme in human atria during atrial fibrillation J. Am. Coll. Cardiol., May 1, 2000; 35(6): 1669 - 1677. [Abstract] [Full Text] [PDF]
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A. Leri, Y. Liu, B. Li, F. Fiordaliso, A. Malhotra, R. Latini, J. Kajstura, and P. Anversa Up-Regulation of AT1 and AT2 Receptors in Postinfarcted Hypertrophied Myocytes and Stretch-Mediated Apoptotic Cell Death Am. J. Pathol., May 1, 2000; 156(5): 1663 - 1672. [Abstract] [Full Text] [PDF]
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Y. Sun and K. T. Weber Infarct scar: a dynamic tissue Cardiovasc Res, May 1, 2000; 46(2): 250 - 256. [Abstract] [Full Text] [PDF]
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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]
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T. Tsutamoto, A. Wada, K. Maeda, N. Mabuchi, M. Hayashi, T. Tsutsui, M. Ohnishi, M. Sawaki, M. Fujii, T. Matsumoto, et al. Angiotensin II type 1 receptor antagonist decreases plasma levels of tumor necrosis factor alpha, interleukin-6 and soluble adhesion molecules in patients with chronic heart failure J. Am. Coll. Cardiol., March 1, 2000; 35(3): 714 - 721. [Abstract] [Full Text] [PDF]
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 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]
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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]
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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]
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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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X.-P. Yang, Y.-H. Liu, D. Mehta, M. A. Cavasin, E. Shesely, J. Xu, F. Liu, and O. A. Carretero Diminished Cardioprotective Response to Inhibition of Angiotensin-Converting Enzyme and Angiotensin II Type 1 Receptor in B2 Kinin Receptor Gene Knockout Mice Circ. Res., May 25, 2001; 88(10): 1072 - 1079. [Abstract] [Full Text] [PDF]
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