(Hypertension. 1999;34:617-624.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
In Vivo Study of AT1 and AT2 Angiotensin Receptors in Apoptosis in Rat Blood Vessels
From the Medical Research Council of Canada Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal, Quebec, Canada.
Correspondence to Ernesto L. Schiffrin, MD, PhD, FRCPC, Clinical Research Institute of Montreal, 110 Pine Ave W, Montreal, Quebec, Canada H2W 1R7. E-mail schiffe{at}IRCM.qc.ca
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Abstract |
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Abstract—In vitro experiments suggest that angiotensin II (Ang II) may cause growth via angiotensin type 1 (AT1) receptors and apoptosis via angiotensin type 2 (AT2) receptors. To answer the question of whether AT1 or AT2 receptor activation could induce apoptosis in the vasculature in vivo, Wistar rats were infused for 7 days with Ang II (120 ng · kg-1 · min-1 subcutaneously) and treated with the AT2 receptor antagonist PD 123319 (30 mg · kg-1 · d-1 subcutaneously) or the AT1 receptor antagonist losartan (10 mg · kg-1 · d-1 orally). Apoptosis in thoracic aorta was quantified by radiolabeled DNA laddering and by terminal deoxynucleotide transferase–mediated dUTP nick end-labeling. The expression of p53, bax, bcl-2, and caspase-3, which play critical roles in apoptotic signaling, was examined by Western blot analysis. The mRNA expression of AT1 and AT2 receptors was determined by reverse transcription–polymerase chain reaction. The increase in systolic blood pressure and aortic growth induced by Ang II infusion was completely prevented by losartan alone or losartan given with PD 123319, whereas PD 123319 resulted in a greater increase in systolic blood pressure and aortic growth than Ang II alone. Radiolabeled DNA laddering showed that Ang II infusion±losartan or PD 123319 significantly increased apoptosis (147±8%, 178±20%, and 238±41%, respectively, P<0.05 compared with control). Expression of bax and active forms of caspase-3 was increased in the Ang II+PD 123319 group, whereas the expression of p53 and bcl-2 was not significantly different in all groups. The expression of AT1 and AT2 receptor mRNA was downregulated by losartan and PD 123319, respectively. Thus, when AT1 or AT2 receptors are stimulated in vivo, apoptosis is enhanced in the media of blood vessels. In the case of AT1 receptor stimulation, this may occur secondary to vascular growth and modulate the latter. Both bax and caspase-3 participate in the pathways of apoptosis triggered by in vivo AT1 receptor stimulation.
Key Words: muscle, smooth, vascular • apoptosis • angiotensin II • p53 • cysteine proteases
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Introduction |
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Angiotensin (Ang) II, the most important peptide that mediates the effects of the renin-angiotensin system, may play an important role in hypertension via its effects on vascular smooth muscle cell (VSMC) growth.1 Interestingly, Ang II exerts positive or negative effects on cell growth depending on which subtype of its seven-transmembrane domain receptors the peptide binds. There are 2 main subtypes of Ang II receptors, and these may be blocked by specific antagonists; losartan for angiotensin type 1 (AT1) receptors and PD 123319 for angiotensin type 2 (AT2) receptors.2 3 4 Most of the known functional effects of Ang II on VSMCs are mediated via AT1 receptors.5 Although it has been hypothesized that AT2 receptor activation may be involved in the control of cell differentiation, proliferation, and apoptosis,6 7 8 the roles of AT2 receptors in vivo are poorly understood.
Apoptosis is an active, gene-directed process that is distinguished from necrosis by a number of morphological and biochemical criteria.9 10 11 12 13 Morphologically, apoptosis is defined by cell shrinkage, membrane blebbing, condensation and fragmentation of the nuclei, and chromatin condensation.9 10 11 The key biochemical feature of apoptosis is DNA fragmentation at internucleosomal units induced by an endogenous endonuclease.12 13 DNA fragmentation can be detected by agarose gel electrophoresis (DNA laddering) and by terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL).
Apoptosis plays a critical role in both the normal development and the pathology of a variety of tissues.14 Apoptosis is recognized as a contributing cause of cardiac myocyte loss with ischemia/reperfusion injury, myocardial infarction, and vascular wall remodeling. In VSMCs, apoptosis has been detected after serum and growth factor removal15 16 and after cells have been exposed to reactive oxygen species.17 Apoptosis has been detected in cultured VSMCs after exposure to nitric oxide donors,18 whereas Ang II may offer a protective effect. However, Ang II has been shown to induce apoptosis in human endothelial cells, whereas nitric oxide provides a protective effect.19 It has been proposed that apoptosis may represent an important process that contributes to vascular remodeling in hypertension. However, the molecular mechanisms responsible for VSMC apoptosis remain largely unknown. Because some of the genes involved mechanistically in growth and apoptosis (such as immediate early genes like c-myc) are activated in both processes, increased apoptosis may accompany enhanced growth as a controlling and fine-tuning mechanism.20
The present study was designed to examine the hypothesis that AT1 receptor–induced VSMC growth in vivo could be associated with increased apoptosis. We also asked whether AT2 receptor–mediated apoptosis may occur and be demonstrated in an in vivo experimental paradigm. The association of administration of Ang II with a selective Ang receptor subtype antagonist was used to enhance cellular responses to Ang II at the subtype receptor that was not blocked. Because there may be non-AT1, non-AT2 Ang receptors (AT4, ATx), Ang II was also infused into rats treated simultaneously with both AT1 and AT2 antagonists. Furthermore, to study the pathways of apoptosis induced by Ang II without and in the presence of blockade of AT1 and AT2 receptors, we measured gene expression of different proapoptotic and antiapoptotic proteins in the vasculature.
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Methods |
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Animal Experiments
The study was approved by the Animal Care Committee of the Clinical Research Institute of Montreal and was performed according to the guidelines of the Canadian Council for Animal Care. As previously described,21 male Wistar rats 7 weeks of age (200 g, n=7 to 8) were infused subcutaneously with the use of osmotic minipumps (Alzet, Alza Corp) with Ile-Ang II 5 (Peninsula) at a dose of 120 ng · kg-1 · min-1 or PD 123319 (AT2 receptor antagonist was a gift from Dr Joan Keiser, Parke-Davis, Ann Arbor, Mich) at the dose of 30 mg · kg-1 · d-1. Losartan (AT1 receptor antagonist) was given in the drinking water at a dose of 10 mg · kg-1 · d-1. The dose of Ang II used in the present study is the pharmacological dose used previously by others22 and us.21 After 7 days of treatment, systolic blood pressure (SBP) was taken by the tail-cuff method. Rats were killed by decapitation. A segment of thoracic aorta was dissected out and immediately frozen in dry ice and kept at -70°C until use.
Morphometry of Aorta
The cross-sectional area (CSA) of the media was evaluated as
previously described.21 The growth index was calculated as
(CSAtr-
CSActrl)/CSActrl where
CSActrl and CSAtr were the
CSA of aorta from normotensive and treated rats, respectively.
Measurement of Apoptosis in Aorta
Fat, adventitia, and endothelium were removed
from the aorta, and DNA was extracted from the media and nick end
radiolabeled as previously described.23 Radiolabeled DNA
was loaded in 1.5% agarose gel. After electrophoresis, DNA was
transferred onto a nylon membrane and the radioactivity associated with
100- to 1500-bp DNA fragments was quantified with a phosphorimager
(Molecular Dynamics). Apoptosis was also evaluated
histologically by the TUNEL technique as previously
described.23
Western Blot Analysis of p53, bax, bcl-2, and
Caspase-3
Protein was extracted from frozen tissue as previously
described.23 Protein concentration was determined with a
protein assay (BioRad Laboratories, Inc). Thirty micrograms of total
protein was separated by electrophoresis on a 15%
polyacrylamide gel at 100 V for 1 hour and transferred onto a
PVDF membrane in the cooling system at 100 V for 1 hour.
Membranes were incubated with specific antibody to p53
(Calbiochem-Novabiochem International), bax, and bcl-2 (both from Santa
Cruz Biotechnology, Inc) at a dilution 1:1000, 1:2000, and 1:1600,
respectively, for 1 hour at room temperature. To evaluate the
expression of inactive (Mr=32 kDa) and active
caspase-3 (20 kDa and 17 kDa), membranes were incubated overnight with
rabbit antiserum to human CPP32 (antibody against caspase-3 was a gift
from Dr Rafick Pierre Sekaly, Clinical Research Institute of Montreal,
Canada) at a dilution 1:1000.24 Signals were revealed with
chemiluminescence and visualized by
autoradiography.
Reverse Transcription–Polymerase Chain Reaction Analysis
of AT1 and AT2 Receptors
mRNA expression of AT1 and
AT2 receptors was measured with reverse
transcription–polymerase chain reaction (RT-PCR) as previously
described25 with some modifications. Total RNA samples
were treated with RNAse-free DNAse (Gibco Life Technologies) and
extracted with phenol-chloroform to eliminate possible residual DNA
before RT-PCR.
Statistical Analysis
Results are presented as mean±SEM. Data were
analyzed by 1-way ANOVA followed by a Newman-Keuls test or by a
Kruskal-Wallis nonparametric test followed by a
t test for Gaussian populations with different SDs where
applicable. P<0.05 was considered statistically
significant.
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Results |
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Body Weight, SBP, and Growth Index
Body weight was greater (P<0.001) in Ang II–infused rats treated simultaneously with losartan and PD 123319 compared with normotensive rats, whereas body weight was similar in all other groups (Table). The increase in SBP and aortic growth induced by Ang II–infusion (P<0.001 and P<0.05 versus control, respectively) was completely prevented by treatment with losartan alone or losartan given with PD 123319 (Table 1, Figure 1). Interestingly, PD 123319, when given to Ang II–infused rats, resulted in a greater increase in SBP and aortic growth than Ang II alone. Treatment of normotensive rats with either losartan or PD 123319 separately or together had no effect on SBP and aortic growth.
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P<0.05 vs Ang II group, and
P<0.05 vs Ang+PD group. Bottom, Bar graph shows
growth index [(CSAtr -
CSActrl)/CSActrl] of aortic media. The control
group has a growth index of zero and is not shown. Error bars indicate
SEM, n=7 to 8. *P<0.05 vs control,
DNA Laddering and TUNEL
Because we used the 3'-end DNA labeling method, which is more
sensitive than ethidium bromide DNA staining, to detect DNA laddering
typical of apoptosis, we could detect basal levels of
apoptosis in untreated rats. Figure 2 shows a significant increase in
apoptosis by increased DNA laddering in the Ang II–infused
group (147±8%) and groups infused with Ang II that received
losartan (178±20%) or PD 123319 (238±41%) in comparison to
control rats (P<0.05). Losartan and PD 123319 alone
did not have a detectable effect on apoptosis. In situ 3'-end
labeling with an in situ apoptosis detection kit (ApopTag,
Intergan Company) in aorta (Figure 3) demonstrated
histologically sparse apoptotic smooth muscle
cells in the media of aorta in all treated groups but not in the
control group.
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Expression of Proapoptotic and Antiapoptotic
Proteins
Western blot analysis was performed to quantify the
expression of the proapoptotic proteins p53 and bax, the
antiapoptotic protein bcl-2, and the inactive and the active
fragments of a cysteine protease (caspase-3) critically situated
in the proteolytic pathway of apoptosis
(Mr=32 kDa for the inactive form and 20 kDa and
17 kDa for the active forms). Figure 4
shows that the expression of bax was increased in Ang II–infused rats
treated with PD 123319, whereas expression of p53 and bcl-2 remained
unchanged in all groups. Expression of the active forms of caspase-3
(particularly the 20-kDa peptide) was increased relative to the
inactive 32-kDa protein only in Ang II–infused rats that received PD
123319 (Figure 5). In
losartan-treated rats (infused or not infused with Ang II),
absolute amounts of caspase-3, both the 32-kDa inactive peptide and the
17-kDa active form, were increased, but relative amounts of the active
form (active/inactive caspase-3) were not.
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AT1 and AT2 Receptor mRNA by
RT-PCR
Expression of AT1 receptor mRNA was reduced
in rats that received losartan with or without Ang II (Figure 6), whereas AT2
receptor mRNA expression was significantly diminished in the Ang II+PD
123319 group.
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Discussion |
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To evaluate the hypothesis that AT1 receptor–induced smooth muscle cell growth is associated with secondarily increased apoptosis and that AT2 receptor–mediated apoptosis of smooth muscle cells demonstrated previously in vitro could be shown to occur in vivo, we examined the following paradigm. Ang II–infused rats treated with the AT2 receptor antagonist PD 123319 were used as a model of AT1 receptor stimulation, and Ang II–infused rats treated with the AT1 receptor antagonist losartan were used as a model of AT2 receptor stimulation. Ang II–infused rats treated simultaneously with the AT1 and AT2 antagonists were used as a model of ATx- (non-AT1, non-AT2) mediated effects. Our results show that both AT1 and AT2 receptor stimulation were associated with enhanced apoptosis of smooth muscle cells in aorta of rats. AT1 receptor activation was accompanied by apoptosis, which may modulate the proliferation of smooth muscle cells, whereas AT2 receptors in the present paradigm only induced apoptosis and thus may exert an antigrowth effect in vivo. No effects except for a greater body weight could be attributed to non-AT1, non-AT2 receptors in this experimental paradigm. However, this need not be attributed to a non-AT1, non-AT2 (AT4, ATx) effect. Blood pressure elevation is often associated with lower body weight in some rat models. Blockade of blood pressure elevation and of AT1- and AT2-mediated apoptosis could presumably, particularly in combination, be associated with greater body weight gain. However, this remains speculative. Furthermore, we also show that AT1 receptor activation (Ang II–infused rats receiving PD 123319) induces apoptosis in blood vessels via a bax-dependent pathway and via activation of the caspase cascade, as evidenced by proteolytical cleavage of caspase-3 into its active 20-kDa and 17-kDa subunits. These findings extend our understanding of the role of Ang II and its receptor subtypes, particularly AT1 receptors as important contributors and regulators of remodeling of blood vessels in hypertension, which is shown by the increase in DNA fragmentation, bax expression, and caspase-3 activation in the Ang II+PD 123319 group ( AT1 stimulation).
Cell hypertrophy, proliferation, or both are mediated by AT1 receptors, whereas stimulation of AT2 receptors has been proposed to lead to inhibition of cell proliferation or to apoptosis.5 6 7 8 The distribution of AT1 and AT2 receptors in tissues varies depending on the cell type or tissue considered. In rat aorta, 60% of Ang receptors are AT1, whereas AT2 receptors are abundantly and widely expressed in aorta of fetal and young rats but present only to a limited extent in adult tissues.26 Several in vitro studies have demonstrated different effects of Ang II on different cell types.6 7 8 18 19 27 28 29 In addition, Ang II may induce growth in VSMCs.27 However, recent studies in non–smooth muscle cells have demonstrated that Ang II is also capable of inducing apoptosis via stimulation of AT2 receptors.7 8 It has been suggested that the antigrowth effect of Ang II on endothelial cells is mediated by AT2 receptors,6 although the underlying mechanisms have not been elucidated. Pollman et al18 failed to induce apoptosis by Ang II in VSMCs, which may be explained by the fact that the expression of AT2 receptors in VSMCs disappears in passaged cells during cell culture. Interestingly, these authors showed that Ang II directly antagonized nitric oxide donor-induced apoptosis via activation of AT1 receptors. In contrast, Dimmeler et al19 demonstrated that activation of programmed cell death by Ang II clearly involves the stimulation of AT2 receptors in human endothelial cells. In contrast to the antiproliferative effect of AT2 that has been shown in endothelial cells and in PC12W cells that overexpress AT2 receptors, AT1 receptors play a more important proapoptotic role in cardiomyocytes of both neonatal and adult rats.28 29 In these studies, the AT1 receptor antagonist losartan completely blocked Ang II–induced apoptosis, whereas the AT2 receptor antagonist PD 123319 failed to reduce Ang II–induced apoptosis. Moreover, Leri et al30 showed that the increase in Ang II–induced apoptosis triggered by stretch was abolished by losartan, which indicated that apoptosis in cardiomyocytes is mediated by AT1 receptors. Taken together, these data suggest that Ang II induces apoptosis via different receptor subtypes depending on the cell type and the ratio between the expression of AT1 and AT2 receptors. When PD 123319 was given to Ang II–infused rats, AT2 receptor mRNA expression was reduced as shown by RT-PCR (Figure 6), and any expressed AT2 receptors were furthermore presumably blocked by the AT2 receptor antagonist. Growth in this experimental condition was stimulated via unblocked AT1 receptors, and secondary apoptosis may have occurred as evidenced by a 2.5-fold increase in DNA laddering (Figure 2b), indicating a compensatory balance between cell replication and deletion in the maintenance of tissue homeostasis. When both losartan and PD 123319 were given to Ang II–infused rats, there were no demonstrable additional effects except for a greater body weight gain. In this group, both AT1 and AT2 receptors are blocked, and Ang II could be acting on non-AT1, non-AT2 Ang receptors or degraded to Ang IV and act on AT4 receptors. However, the present results do not provide definitive evidence of non-AT1, non-AT2–mediated effects, because as mentioned earlier, this could result from a combined effect of blockade of blood pressure increase and of inhibition of AT1- and AT2-mediated apoptosis. AT1 and AT2 receptor mRNA was evaluated by RT-PCR, which is only semiquantitative, and thus the reported levels of mRNA can only be considered as an approximation. However, previous work from our laboratory has already demonstrated a correlation between levels of AT1 and AT2 receptor protein and mRNA.25
In this study, we investigated molecular steps involved in the mechanisms that trigger apoptosis in aorta. Cell death is controlled in part by a complex interplay between regulatory proteins. The bax protein, a member of the Bcl family, is one of these regulatory proteins and is found in various tissues.31 The present study shows bax expression even in tissues from normotensive rats. Overexpression of bax was found in aorta of Ang II–infused rats after treatment with PD 123319 despite the absence of changes in the expression of p53. Thus, p53 is not necessarily the main regulator of bax expression. Bcl-2 is a suppressor of apoptosis that homodimerizes or forms heterodimers with the homologous protein bax. A previous study from our laboratory has shown that both bax and bcl-2, another member of the Bcl family, are involved in pathways of apoptosis in aorta.23 In the present study, we extended those observations and demonstrated that caspase-3 also participates in the mechanisms that trigger apoptosis of VSMCs in rat aorta. Recent work has supported a central role for the caspase family of cysteine proteases, especially caspase-3, as effectors of apoptosis. Among the caspases, caspase-3 (CPP32, YAMA, or apopain) has been considered a central component of the proteolytic cascade during apoptosis.32 We report here on the expression of both the caspase-3 proenzyme (Mr=32 kDa) and 2 active forms of caspase-3 (20 kDa and 17 kDa) present in SMCs in aorta of rats. After activation, caspase-3 is also cleaved to a 12-kDa subunit, which is not detectable by Western blotting. We have also shown that activation of caspase-3 is one of the events that occur in VSMCs that undergo apoptosis, which results from the simultaneous treatment with Ang II and PD 123319 (blockade of AT2 receptors and AT1 receptor stimulation). To the best of our knowledge, the present study is the first to show the involvement of caspase-3 in VSMCs that undergo apoptosis induced by Ang II via the AT1 receptor in an in vivo study. The main finding in this study is that when AT1 receptor activation occurs in the presence of AT2 receptor blockade, inactive caspase-3, as a percent of total caspase-3, is reduced, whereas the active caspase-3 fragments are increased.
In conclusion, the balance between growth and apoptosis in blood vessels, which may play an important role in vascular remodeling in hypertension, may be regulated in vivo by Ang receptors, particularly by AT1 receptors. Activation of AT1 receptors in vivo in rats results in SBP increase, blood vessel growth, and associated VSMC apoptosis, which may modulate the degree of growth. Whether apoptosis that is induced by AT1 receptors in VSMCs is primary, or secondary as we believe, awaits clarification. Thus, the present results extend our knowledge on the essential role of AT1 receptors in blood pressure control, VSMC growth, and apoptosis, as shown by increases in blood pressure, aortic growth, DNA fragmentation, bax expression, and caspase-3 activation.
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Acknowledgments |
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This work was supported by a group grant of the Medical Research Council of Canada (MRC) to the Multidisciplinary Research Group on Hypertension and by a Medical School Grant from Merck and Co, Inc, Whitehouse Station, NJ. Q.N. Diep was supported by a postdoctoral fellowship from MRC. The authors are grateful to André Turgeon for his excellent technical assistance.
Received April 20, 1999; first decision May 3, 1999; accepted June 10, 1999.
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E. L. Schiffrin and R. M. Touyz From bedside to bench to bedside: role of renin-angiotensin-aldosterone system in remodeling of resistance arteries in hypertension Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H435 - H446. [Full Text] [PDF]
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 B. I. Levy Can Angiotensin II Type 2 Receptors Have Deleterious Effects in Cardiovascular Disease?: Implications for Therapeutic Blockade of the Renin-Angiotensin System Circulation, January 6, 2004; 109(1): 8 - 13. [Full Text] [PDF]
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V. Grishko, V. Pastukh, V. Solodushko, M. Gillespie, J. Azuma, and S. Schaffer Apoptotic cascade initiated by angiotensin II in neonatal cardiomyocytes: role of DNA damage Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2364 - H2372. [Abstract] [Full Text] [PDF]
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M. Ruperez, O. Lorenzo, L. M. Blanco-Colio, V. Esteban, J. Egido, and M. Ruiz-Ortega Connective Tissue Growth Factor Is a Mediator of Angiotensin II-Induced Fibrosis Circulation, September 23, 2003; 108(12): 1499 - 1505. [Abstract] [Full Text] [PDF]
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 N. Werner and G. Nickenig AT1 receptors in atherosclerosis: biological effects including growth, angiogenesis, and apoptosis Eur. Heart J. Suppl., January 1, 2003; 5(suppl_A): A9 - A13. [Abstract] [PDF]
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A. Virdis, M. F. Neves, F. Amiri, E. Viel, R. M. Touyz, and E. L. Schiffrin Spironolactone Improves Angiotensin-Induced Vascular Changes and Oxidative Stress Hypertension, October 1, 2002; 40(4): 504 - 510. [Abstract] [Full Text] [PDF]
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 T. WALTHER, L. OLAH, C. HARMS, B. MAUL, M. BADER, H. HORTNAGL, H.-P. SCHULTHEISS, and G. MIES Ischemic injury in experimental stroke depends on angiotensin II FASEB J, February 1, 2002; 16(2): 169 - 176. [Abstract] [Full Text] [PDF]
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M. Stoll, A. W.A. Hahn, U. Jonas, Y. Zhao, B. Schieffer, J. W. Fischer, and T. Unger Identification of a Zinc Finger Homoeodomain Enhancer Protein After AT2 Receptor Stimulation by Differential mRNA Display Arterioscler. Thromb. Vasc. Biol., February 1, 2002; 22(2): 231 - 237. [Abstract] [Full Text] [PDF]
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J.-L. Bascands, J.-P. Girolami, M. Troly, I. Escargueil-Blanc, D. Nazzal, R. Salvayre, and N. Blaes Angiotensin II Induces Phenotype-Dependent Apoptosis in Vascular Smooth Muscle Cells Hypertension, December 1, 2001; 38(6): 1294 - 1299. [Abstract] [Full Text] [PDF]
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H. Ono, Y. Ono, A. Takanohashi, H. Matsuoka, and E. D. Frohlich Apoptosis and Glomerular Injury After Prolonged Nitric Oxide Synthase Inhibition in Spontaneously Hypertensive Rats Hypertension, December 1, 2001; 38(6): 1300 - 1306. [Abstract] [Full Text] [PDF]
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H. D. Intengan and E. L. Schiffrin Vascular Remodeling in Hypertension: Roles of Apoptosis, Inflammation, and Fibrosis Hypertension, September 1, 2001; 38(3): 581 - 587. [Abstract] [Full Text] [PDF]
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S. A. Mezzano, M. Ruiz-Ortega, and J. Egido Angiotensin II and Renal Fibrosis Hypertension, September 1, 2001; 38(3): 635 - 638. [Abstract] [Full Text] [PDF]
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Q. N. Diep and E. L. Schiffrin Increased Expression of Peroxisome Proliferator-Activated Receptor-{alpha} and -{gamma} in Blood Vessels of Spontaneously Hypertensive Rats Hypertension, August 1, 2001; 38(2): 249 - 254. [Abstract] [Full Text] [PDF]
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Q. N. Diep, M. El Mabrouk, R. M. Touyz, and E. L. Schiffrin Expression of Cell Cycle Proteins in Blood Vessels of Angiotensin II-Infused Rats : Role of AT1 Receptors Hypertension, February 1, 2001; 37(2): 604 - 608. [Abstract] [Full Text] [PDF]
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 R. M. Touyz and E. L. Schiffrin Signal Transduction Mechanisms Mediating the Physiological and Pathophysiological Actions of Angiotensin II in Vascular Smooth Muscle Cells Pharmacol. Rev., December 1, 2000; 52(4): 639 - 672. [Abstract] [Full Text] [PDF]
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Q. N. Diep, R. M. Touyz, and E. L. Schiffrin Docosahexaenoic Acid, a Peroxisome Proliferator-Activated Receptor-{alpha} Ligand, Induces Apoptosis in Vascular Smooth Muscle Cells by Stimulation of p38 Mitogen-Activated Protein Kinase Hypertension, November 1, 2000; 36(5): 851 - 855. [Abstract] [Full Text] [PDF]
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H. D. Intengan and E. L. Schiffrin Structure and Mechanical Properties of Resistance Arteries in Hypertension : Role of Adhesion Molecules and Extracellular Matrix Determinants Hypertension, September 1, 2000; 36(3): 312 - 318. [Abstract] [Full Text] [PDF]
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 M. Ruiz-Ortega, O. Lorenzo, M. Ruperez, S. Konig, B. Wittig, and J. Egido Angiotensin II Activates Nuclear Transcription Factor {kappa}B Through AT1 and AT2 in Vascular Smooth Muscle Cells : Molecular Mechanisms Circ. Res., June 23, 2000; 86(12): 1266 - 1272. [Abstract] [Full Text] [PDF]
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B.-S. Tea, S. Der Sarkissian, R. M. Touyz, P. Hamet, and D. deBlois Proapoptotic and Growth-Inhibitory Role of Angiotensin II Type 2 Receptor in Vascular Smooth Muscle Cells of Spontaneously Hypertensive Rats In Vivo Hypertension, May 1, 2000; 35(5): 1069 - 1073. [Abstract] [Full Text] [PDF]
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E. L. Schiffrin, J. B. Park, H. D. Intengan, and R. M. Touyz Correction of Arterial Structure and Endothelial Dysfunction in Human Essential Hypertension by the Angiotensin Receptor Antagonist Losartan Circulation, April 11, 2000; 101(14): 1653 - 1659. [Abstract] [Full Text] [PDF]
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 Jeong Bae Park, H. D Intengan, and E. L Schiffrin Reduction of resistance artery stiffness by treatment with the AT1-receptor antagonist losartan in essential hypertension Journal of Renin-Angiotensin-Aldosterone System, March 1, 2000; 1(1): 40 - 45. [Abstract] [PDF]
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Q. N. Diep, M. El Mabrouk, P. Yue, and E. L. Schiffrin Effect of AT1 receptor blockade on cardiac apoptosis in angiotensin II-induced hypertension Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1635 - H1641. [Abstract] [Full Text] [PDF]
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