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(Hypertension. 2003;42:574.)
© 2003 American Heart Association, Inc.

Scientific Contributions

Molecular Mechanisms of Inhibition of Vascular Growth by Angiotensin-(1-7)

From the Hypertension and Vascular Disease Center, Wake Forest University School of Medicine, Winston-Salem, NC.

Correspondence to E. Ann Tallant, PhD, Hypertension and Vascular Disease Center, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1032. E-mail atallant{at}wfubmc.edu

	Abstract

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Angiotensin (Ang) peptides play a critical role in regulating vascular reactivity and structure. We showed that Ang-(1-7) reduced smooth muscle growth after vascular injury and attenuated the proliferation of vascular smooth muscle cells (VSMCs). This study investigated the molecular mechanisms of the antiproliferative effects of Ang-(1-7) in cultured rat aortic VSMCs. Ang-(1-7) caused a dose-dependent release of prostacyclin from VSMCs, with a maximal release of 277.9±25.2% of basal values (P<0.05) by 100 nmol/L Ang-(1-7). The cyclooxygenase inhibitor indomethacin significantly attenuated growth inhibition by Ang-(1-7). In contrast, neither a lipoxygenase inhibitor nor a cytochrome p450 epoxygenase inhibitor prevented the antiproliferative effects of Ang-(1-7). These results suggest that Ang-(1-7) inhibits vascular growth by releasing prostacyclin. Ang-(1-7) caused a dose-dependent release of cAMP, which might result from prostacyclin-mediated activation of adenylate cyclase. The cAMP-dependent protein kinase inhibitor Rp-adenosine-3',5'-cyclic monophosphorothioate attenuated the Ang-(1-7)–mediated inhibition of serum-stimulated thymidine incorporation. Finally, Ang-(1-7) inhibited Ang II stimulation of mitogen-activated protein kinase activities (ERK1/2). Incubation of VSMCs with concentrations of Ang-(1-7) up to 1 µmol/L had no effect on ERK1/2 activation. However, preincubation with increasing concentrations of Ang-(1-7) caused a dose-dependent reduction in Ang II–stimulated ERK1/2 activities. Ang-(1-7) (1 µmol/L) reduced 100 nmol/L Ang II–stimulated ERK1 and ERK2 activation by 42.3±6.2% and 41.2±4.2%, respectively (P<0.01). These results suggest that Ang-(1-7) inhibits vascular growth through the release of prostacyclin, through the prostacyclin-mediated production of cAMP and activation of cAMP-dependent protein kinase, and by attenuation of mitogen-activated protein kinase activation.

Key Words: angiotensin II • muscle, smooth, vascular • vasculature • peptides • prostacyclin • cyclic AMP • protein kinases

	Introduction

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Regulation of vascular smooth muscle cell (VSMC) growth is critical in the maintenance of vascular perfusion pressure and blood flow. VSMC growth is regulated by proliferative and antiproliferative factors produced and released by vascular endothelial cells and fibroblasts; by circulating platelets, neutrophils, and monocytes; or by VSMCs in an autocrine and paracrine fashion. Classic growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor, and angiotensin II (Ang II) stimulate VSMC growth in vitro and in vivo.^1,2 For example, Ang II infusion into rats markedly stimulates DNA synthesis in both neointimal and medial SMCs,³ an effect that is independent of the Ang II–mediated increase in blood pressure.⁴ Furthermore, neointimal formation after vascular injury is attenuated by reducing Ang II formation with angiotensin-converting enzyme (ACE) inhibitors or by inhibiting Ang II activity with angiotensin type 1 (AT₁) receptor antagonists.^5,6

In contrast, atrial natriuretic factor, prostacyclin (PGI₂), and nitric oxide inhibit the growth of cultured VSMCs.^7–10 We recently showed that the heptapeptide fragment of Ang II, Ang-(1-7), also attenuated mitogen-stimulated VSMC growth.¹¹ Ang-(1-7) caused a dose-dependent inhibition of Ang II-, PDGF-, or serum-stimulated VSMC growth. The antiproliferative effects of Ang-(1-7) were blocked by sarcosine¹-threonine⁸-Ang II ([Sar¹-Thr⁸]-Ang II) or by D-alanine⁷-angiotensin-(1-7) ([D-Ala⁷]-Ang-[1-7]) but were not prevented by subtype-selective AT₁ or AT₂ receptor antagonists.^11,12 Furthermore, we showed that a 2-fold elevation in circulating Ang-(1-7), produced by an intravenous infusion of the heptapeptide, significantly inhibited neointimal formation after vascular injury but had no effect on blood pressure, heart rate, or medial cross-sectional area.¹³ The associated discovery that plasma Ang-(1-7) is increased to a similar level after ACE inhibitor therapy or long-term administration of AT₁ receptor antagonists^14–16 suggests that Ang-(1-7) might either limit the proliferative response of VSMCs in hypertension or contribute to the mechanisms that account for the reversal of vascular hypertrophy or hyperplasia due to inhibition of Ang II formation or activity.

Ang-(1-7) increases prostaglandin formation in various types of cells,^17–19 and prostaglandins inhibit vascular growth.^8,9 Infusions of Ang-(1-7) into rats at the concentration that inhibited neointimal formation in balloon-injured rats increased urinary prostaglandin excretion.²⁰ Thus, Ang-(1-7) might inhibit vascular growth through the production of prostaglandins and the activation of prostaglandin-mediated cellular events. In this study, we investigated the molecular mechanisms that participate in inhibition of vascular growth by Ang-(1-7).

	Materials

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Ang-(1-7) was obtained from Bachem California. Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Gibco BRL. Indomethacin, cinnamyl-3,4-dihydroxy--cyanocinnamate, 17-octadecynoic acid, carbacyclin, PGI₂, and Rp-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPS) were obtained from Biomol. Phospho-specific antibodies against extracellular signal–regulated kinase (ERK) 1/2 and an ERK2 antibody were obtained from Cell Signaling.

VSMC Isolation
VSMCs were isolated by explant culture from the thoracic aortas of 12- to 14 week-old Hannover Sprague-Dawley rats bred and raised at Wake Forest University School of Medicine, as described previously.¹¹ Cells were used between passages 4 and 10 and were made quiescent by a 48-hour treatment with defined serum-free medium containing Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium, penicillin, streptomycin, 3 µg/mL insulin, 5 µg/mL transferrin, and 0.2 mmol/L ascorbic acid.

Measurement of Thymidine Incorporation
Quiescent cells in 24-well plates were treated for 48 hours in the presence or absence of angiotensin peptides, 1% FBS, and metabolic inhibitors, as indicated. During the last 24 hours of treatment, 0.5 µCi [³H]thymidine per milliliter was added to each well, and [³H]thymidine incorporation was determined as previously described.¹¹

Radioimmunoassays
PGI₂ release from cells incubated for 15 minutes with Ang-(1-7) was measured in Krebs-Ringer solution (125 mmol/L NaCl, 5 mmol/L KCl, 1.2 mmol/L MgSO₄, 6 mmol/L glucose, 1 mmol/L CaCl₂, and 25 mmol/L HEPES, pH 7.4). PGI₂ was measured by a radioimmunoassay for its stable metabolite, 6-keto-PGF₁, by using a kit from PerCeptive Diagnostics.

The production of cAMP was measured in confluent monolayers of cells preincubated with 1 mmol/L isobutylmethylxanthine and incubated for 15 minutes at 37°C with Ang-(1-7). The medium was extracted twice in ice-cold 95% ethanol and evaporated to dryness under N₂. cAMP was measured by a specific radioimmunoassay (Amersham Pharmacia).

Measurement of ERK1/ERK2 Activities
Quiescent cells were preincubated with increasing concentrations of Ang-(1-7) for 30 minutes, followed by a 10-minute treatment with 100 nmol/L Ang II. Cell lysates were prepared in lysis buffer (100 mmol/L NaCl, 50 mmol/L NaF, 5 mmol/L EDTA, 1% Triton X-100, and 50 mmol/L Tris-HCl, pH 7.4) containing 0.01 mmol/L NaVO₄, 0.1 mmol/L phenylmethylsulfonylfluoride, and 0.6 µmol/L leupeptin, and protein concentrations were measured by the method of Lowry et al.²¹ Solubilized proteins were separated by electrophoresis and transferred to polyvinyl membranes. Nonspecific binding was blocked by incubation in 5% evaporated milk and 4% Triton X-100 in Tris-buffered saline (Blotto). Membranes were subsequently probed with a specific antibody to the activated phosphorylated form of the p44/p42 mitogen-activated protein (MAP) kinases (ERK1/2; 1:5000 dilution), followed by incubation with a goat anti-rabbit antibody coupled to horseradish peroxidase. Immunoreactive bands were visualized by enhanced chemiluminescence reagents and quantified by densitometry. Multiple exposures were used to ensure that densitometry was performed in the linear range of the film. Protein loading was visualized by probing the stripped membranes with an antibody against ERK2.

Statistics
All data are presented as mean±SEM. Statistical differences were evaluated by repeated-measures, 1-way ANOVA followed by the Dunnett post hoc test or by Student t test. The criterion for statistical significance was set at P<0.05.

	Results

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Role of Prostaglandins in Growth Inhibition by Ang-(1-7)
PGI₂ release from VSMCs stimulated with Ang-(1-7) was measured to determine whether Ang-(1-7) stimulated the production of PGI₂ in VSMCs isolated from Sprague-Dawley rat aortas. VSMCs were incubated with increasing concentrations of Ang-(1-7) for 15 minutes, and PGI₂ release into the medium was measured by a radioimmunoassay specific for the stable metabolite of prostacyclin, 6-keto-PGF₁. Ang-(1-7) caused a dose-dependent release of PGI₂, with a maximal release of 177.9±25.2% above basal value at 100 nmol/L Ang-(1-7), as shown in Figure 1.

View larger version (14K): [in this window] [in a new window]   Figure 1. Effect of Ang-(1-7) on PGI2 release from VSMCs. Confluent monolayers of VSMCs were incubated with increasing concentrations of Ang-(1-7) for 15 minutes at 37°C. PGI2 released into the medium was measured by a radioimmunoassay for its stable analogue, 6-keto-PGF1. Results are presented as the percentage of basal release, which averaged 360±120 pg/mL of incubation medium. Data shown are mean±SEM of duplicate samples of VSMCs isolated from 3 different rat aortas. *P<0.05 vs vehicle.

PGI₂ is produced by the cyclooxygenase-mediated conversion of arachidonic acid to PGG₂/prostaglandin H₂, which is subsequently processed by PGI₂ synthase to PGI₂. The cyclooxygenase inhibitor indomethacin was used to determine whether an increase in prostaglandin production contributes to the Ang-(1-7)–mediated inhibition of VSMC growth. Ang-(1-7) caused a significant inhibition of serum-stimulated [³H]thymidine incorporation (79.1±5.1% of total; n=5, P<0.05). Pretreatment with the cyclooxygenase inhibitor indomethacin (10 µmol/L) effectively blocked the growth inhibition mediated by Ang-(1-7), as shown in Figure 2. In contrast, neither the lipoxygenase inhibitor cinnamyl-3,4-dihydroxy--cyanocinnamate (1 µmol/L) nor the cytochrome P450 inhibitor 17-octadecynoic acid (10 µmol/L) had any effect on growth inhibition by the heptapeptide.

View larger version (16K): [in this window] [in a new window]   Figure 2. Effect of inhibitors of arachidonic acid metabolism on Ang-(1-7)–mediated inhibition of serum-stimulated VSMC growth. Confluent monolayers of VSMCs were treated with 1 µmol/L Ang-(1-7)/1% FBS (vehicle, VEH) in the presence or absence of the cyclooxygenase inhibitor indomethacin (IND, 10 µmol/L), the lipoxygenase inhibitor cinnamyl-3,4-dihydroxy--cyanocinnamate (CDC, 1 µmol/L), or the cytochrome P450 inhibitor 17-octadecynoic acid (ODYA, 10 µmol/L) for 48 hours at 37°C. [3H]thymidine was added during the final 24 hours, and thymidine incorporation was determined. Inhibition of [3H]thymidine incorporation by Ang-(1-7) was calculated as the percentage of serum-stimulated VSMCs incubated with the same concentration of each inhibitor. Results are presented as the percentage of inhibition of serum-stimulated growth by Ang-(1-7), which averaged 20.9±5.1%. Data are mean±SEM of triplicate samples from VSMCs isolated from 5 different rat aortas. *P<0.05 vs growth inhibition by 1 µmol/L Ang-(1-7)/1% FBS.

Role of cAMP in the Antiproliferative Response to Ang-(1-7)
The addition of PGI₂ (or stable analogues of PGI₂) to VSMCs activates adenylate cyclase, resulting in an elevation in the cellular levels of cAMP.¹⁰ Because Ang-(1-7) stimulates VSMCs to release PGI₂, VSMCs were treated with Ang-(1-7), and cellular cAMP production was measured to determine whether the PGI₂ released in response to Ang-(1-7) stimulation caused a subsequent release of cAMP. VSMCs were pretreated with 1 mmol/L isobutylmethylxanthine, a cyclic nucleotide phosphodiesterase inhibitor, to prevent breakdown of cellular cAMP. VSMCs were then treated with Ang-(1-7) for 15 minutes. Cellular cyclic nucleotides were extracted with ethanol and measured by radioimmunoassay. Ang-(1-7), at a concentration of 1 µmol/L, caused a significant increase in the cellular levels of cAMP, to 131.9±9.7% of basal value (n=3, P<0.05).

We hypothesize that Ang-(1-7) inhibits vascular growth by the PGI₂-mediated increase in cAMP and its activation of the cAMP-dependent protein kinase. To test this hypothesis, quiescent VSMCs were treated with 1% FBS and 1 µmol/L Ang-(1-7) in the presence or absence of the protein kinase A inhibitor Rp-cAMPS. Ang-(1-7) significantly reduced serum-stimulated [³H]thymidine incorporation to 85.9±1.8% of total (n=3, P<0.05), as shown in Figure 3. The Ang-(1-7)–dependent inhibition of [³H]thymidine incorporation was completely blocked by pretreatment with the protein kinase A inhibitor (98.7±1.8% of serum-stimulated incorporation; n=3, P<0.05). PGI₂ or its stable analogue carbacyclin, at a concentration of 5 µmol/L, also caused a significant inhibition of serum-stimulated thymidine incorporation (82.9±6.0% and 62.6±2.6% of serum-stimulated incorporation, respectively). The PGI₂-mediated inhibition of thymidine incorporation was also significantly blocked by pretreatment with the protein kinase A inhibitor.

View larger version (19K): [in this window] [in a new window]   Figure 3. Blockade of the antitrophic effects of Ang-(1-7) by a cAMP-dependent protein kinase A inhibitor. Quiescent VSMCs were pretreated for 48 hours with 1% FBS, 1 µmol/L Ang-(1-7), 5 µmol/L carbacyclin, or 5 µmol/L PGI2 in the presence or absence of the protein kinase A inhibitor Rp-cAMPS (10 µmol/L). [3H]thymidine was added during the last 24 hours, and thymidine incorporation was determined. Inhibition of [3H]thymidine incorporation by Ang-(1-7) was calculated as the percentage of thymidine incorporation into serum-stimulated VSMCs incubated in the presence or absence of Rp-cAMPS. Results are mean±SEM of triplicate samples from VSMCs isolated from 3 different rat aortas. *P<0.05 vs in the presence and absence of Rp-cAMPS.

Reduction in Ang II–Stimulated ERK1/ERK2 Activity by Ang-(1-7)
Ang II stimulates the MAP kinases ERK1 and ERK2 in VSMCs. Ang II caused a dose-dependent increase in both ERK1 and ERK2 activity (37- and 166-fold increase over basal), with maximal stimulation by 100 nmol/L Ang II (data not shown). Incubation of VSMCs with concentrations of Ang-(1-7) up to 1 µmol/L had no effect on ERK1 or ERK2 phosphorylation. However, preincubation with increasing concentrations of Ang-(1-7) caused a dose-dependent reduction in 100 nmol/L Ang II–stimulated ERK1/2 activities, with maximal inhibition at 1 µmol/L Ang-(1-7), as shown in Figure 4. Ang-(1-7) at 1 µmol/L reduced 100 nmol/L Ang II–stimulated ERK1 and ERK2 activation by 42.3±6.2% and 41.2±4.2%, respectively (P<0.01).

View larger version (44K): [in this window] [in a new window]   Figure 4. Regulation of ERK1/ERK2 activities by Ang-(1-7). Quiescent VSMCs were treated for 30 minutes with increasing amounts of Ang-(1-7), followed by a 10-minute incubation with 100 nmol/L Ang II. Cellular proteins were harvested and analyzed by Western blot hybridization with an antibody against phospho-specific ERK1/ERK2. Protein loading was visualized by incubation of the stripped gel with an ERK2 antibody. Immunoreactive bands of a representative experiment are visualized in A. Different exposures of the same gel are shown for ERK1 and ERK2. Statistical analysis of ERK1 and ERK2 activities in quiescent VSMCs treated with 100 nmol/L Ang II and increasing concentrations of Ang-(1-7) is shown in B. Data were quantified by densitometry. Results are mean±SEM of samples from VSMCs isolated from 3 different rat aortas. *P<0.05 vs Ang II alone.

	Discussion

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In previous studies, we showed that Ang-(1-7) inhibited mitogen-stimulated VSMC growth and reduced neointimal formation after vascular injury.^11,13 Ang-(1-7) stimulated PGI₂ release from VSMCs, and the Ang-(1-7)–mediated inhibition of serum-stimulated [³H]thymidine incorporation into VSMCs was blocked by indomethacin, a cyclooxygenase inhibitor that blocks prostaglandin production. Ang-(1-7) also increased the vascular content of cAMP in VSMCs, and Rp-cAMPS, an inhibitor of the cAMP-dependent protein kinase, blocked the Ang-(1-7)–mediated inhibition of serum-stimulated [³H]thymidine incorporation. Finally, Ang-(1-7) reduced the Ang II–stimulated MAP kinase activities of ERK1 and ERK2. These results suggest that Ang-(1-7) inhibits VSMC growth by a cyclooxygenase-mediated increase in PGI₂ through an elevation in cellular levels of cAMP and activation of the cAMP-dependent protein kinase and by a reduction in MAP kinase activity.

Ang-(1-7) caused a dose-dependent increase in PGI₂ production in VSMCs, in agreement with previous studies.^17–19 PGI₂ inhibited the growth of cultured VSMCs,^8,9 and overexpression of PGI₂ synthase in rat VSMCs increased PGI₂ production and decreased DNA synthesis in response to serum stimulation.²² Because PGI₂ is generated from arachidonic acid by the cyclooxygenase-mediated increase in PGG₂/prostaglandin H₂ and its subsequent conversion to PGI₂ by PGI₂ synthase, we blocked PGI₂ production in response to Ang-(1-7) by using a cyclooxygenase inhibitor. The Ang-(1-7)–mediated increase in serum-stimulated [³H]thymidine conversion was prevented by the cyclooxygenase inhibitor indomethacin, suggesting that a metabolite of the cyclooxygenase pathway, such as PGI₂, mediates the response to Ang-(1-7). In agreement with these studies, Muthalif et al¹⁹ showed that indomethacin blocked the Ang-(1-7)–mediated increase in PGI₂ production in rabbit VSMCs.

Alternatively, other prostaglandins generated by the cyclooxygenase pathway in VSMCs might mediate the response to Ang-(1-7). Bayorh et al²³ showed that Ang-(1-7) reduced the release of thromboxane B₂ from rat aortic rings, which was blocked by the AT_(1-7) receptor antagonist [D-Ala⁷]-Ang-(1-7). Because thromboxanes are cyclooxygenase products that stimulate VSMC growth,^24,25 an Ang-(1-7)–mediated reduction in thromboxane formation could also contribute to vascular growth inhibition. Inhibition of vascular lipoxygenase activity by cinnamyl-3,4-dihydroxy--cyanocinnamate or blockade of cytochrome P450 epoxygenase activity by octadecynoic acid had no effect on growth inhibition by Ang-(1-7). In contrast, blockade of the lipoxygenase pathway inhibited the proliferative response to Ang II in Chinese hamster ovary–AT_1A cells.²⁶ Furthermore, a lipoxygenase inhibitor potentiated PGI₂ release by Ang-(1-7) in rabbit VSMCs, suggesting that Ang-(1-7) also stimulated the production of lipoxygenase metabolites, which attenuated PGI₂ production in rabbit VSMCs.¹⁹ Indomethacin prevented the vasodepressor response to Ang-(1-7) in the pithed rat²⁷ as well as the dilation of piglet pial arteries.²⁸ In spontaneously hypertensive rats treated with lisinopril, losartan, or both, an antibody against Ang-(1-7), an inhibitor of Ang-(1-7) formation, or [D-Ala⁷]Ang-(1-7) caused an increase in blood pressure.²⁹ Indomethacin caused a similar increase in blood pressure, and the increase in blood pressure by [D-Ala⁷]Ang-(1-7) was prevented by pretreatment with the cyclooxygenase inhibitor. These results suggest that the production of prostaglandins represents a common signaling pathway in both the vasodepressor and antiproliferative effects of Ang-(1-7).

PGI₂ receptors on VSMCs activate adenylate cyclase to increase cellular levels of cAMP.¹⁰ Treatment of VSMCs with Ang-(1-7) also resulted in a significant increase in cAMP in conjunction with an increase in PGI₂ production. These results suggest that Ang-(1-7) might stimulate the release of PGI₂ to activate vascular PGI₂ receptors and produce cAMP. Alternatively, Ang-(1-7) might directly activate adenylate cyclase to generate cAMP. Inhibition of vascular growth by prostaglandins is correlated with an increase in cAMP,³⁰ and pharmacologic agents that increase the intracellular concentration of cAMP (membrane-permeant cAMP analogues, forskolin or phosphodiesterase inhibitors) reduced serum-stimulated growth of rabbit and rat VSMCs.^31,32

Many of the known effects of cAMP are mediated through the activation of the cAMP-dependent protein kinase. The Ang-(1-7)- or prostacyclin-mediated inhibition of serum-stimulated [³H]thymidine incorporation was blocked by Rp-cAMPS, an inhibitor of the cAMP-dependent protein kinase. The growth-inhibitory response to phosphodiesterase inhibition was also completely reversed by pretreatment with a peptide inhibitor of the cAMP-dependent protein kinase inhibitor.³² These results suggest that Ang-(1-7) inhibits vascular growth through activation of the cAMP-dependent protein kinase and subsequent stimulation of downstream signaling pathways by cAMP-mediated protein phosphorylation.

VSMC growth stimulated by PDGF and Ang II was mediated, at least in part, by activation of MAP kinases,³³ and inhibition of the MAP kinase pathway prevented VSMC proliferation in response to PDGF.³⁴ Ang-(1-7) significantly reduced Ang II–mediated ERK1/ERK2 activity in VSMCs, which might attenuate vascular growth. Vascular MAP kinase activated by PDGF was reduced by an increase in the cellular content of cAMP,³⁵ which caused cell cycle arrest by stimulating an inhibitor of cyclin-dependent kinase 4, p27^Kip1.³⁶ These results suggest that Ang-(1-7) might inhibit vascular growth by a cAMP-mediated inhibition of MAP kinase. A reduction in MAP kinase activity by Ang-(1-7) might result from the inhibition or downregulation of the ERK1/2 kinases by the heptapeptide, by a reduction or inhibition of MEK, the enzyme that activates ERK1/2, or by an increase in MAP kinase phosphatase activity. Takeda-Matsubara et al³⁷ showed that estrogen activates both the serine/threonine MAP kinase phosphatase (MKP-1) and a tyrosine phosphatase (SHP-1) to inhibit VSMC growth.

In previous studies, we showed that Ang-(1-7) infusion reduced neointimal formation in rat carotid arteries injured with a balloon catheter.¹³ Neointimal formation in the injured rabbit aorta was reduced by the stable PGI₂ analogue TFC-132,³⁸ whereas treatment with ciprostene, a chemically stable PGI₂ analogue, reduced restenosis of human coronary arteries after angioplasty.³⁹ Beraprost, another stable PGI₂ analogue, had a protective effect on cardiac allograft vasculopathy in rats⁴⁰ through an increase in cAMP and downregulation of the cyclin-dependent protein kinase inhibitor p27^Kip1.⁴¹ Local administration of 8-bromo-cAMP or phosphodiesterase inhibitors reduced neointimal formation in the balloon-injured rat carotid artery,³² and systemic infusion of 8-chloro-cAMP significantly reduced neointimal formation after balloon injury to the rat carotid artery.⁴² The reduction in intimal hyperplasia by analogues of PGI₂ and cAMP is in agreement with our results, showing that Ang-(1-7) stimulates the production of PGI₂ and cAMP and blockade of cyclooxygenase or the cAMP-dependent protein kinase attenuates the response to Ang-(1-7). We also demonstrated a reduction in Ang II–stimulated ERK1/ERK2 activity by Ang-(1-7). The MAP kinases ERK1 and ERK2 are rapidly activated in balloon-injured rat carotid arteries.⁴³ Gene transfer of a dominant-negative mutant of ERK1/ERK2⁴⁴ or the MAP kinase kinase MEK⁴⁵ or downregulation of ERK1 and ERK2 by antisense oligonucleotides⁴⁶ prevented neointimal formation in balloon-injured arteries. Because Ang-(1-7) reduced Ang II–stimulated ERK1/ERK2 activity in vitro, a similar reduction in MAP kinase activity by Ang-(1-7) might account for the reduction in neointimal formation after Ang-(1-7) infusion into carotid artery–injured rats.

The results of the present study show that Ang-(1-7) releases PGI₂ and stimulates cAMP production in rat VSMCs. Furthermore, inhibition of PGI₂ production by indomethacin and blockade of cAMP-dependent protein kinase activity by Rp-cAMPS attenuated the antiproliferative effects of Ang-(1-7). Ang-(1-7) reduced Ang II–stimulated ERK1/ERK2 activation, the signaling pathway that plays a predominant role in the proliferative response to Ang II. These results suggest that Ang-(1-7) releases PGI₂, which has autocrine and paracrine effects on vascular PGI₂ receptors to increase cAMP, activate the cAMP-dependent protein kinase, and reduce MAP kinase activities to inhibit vascular growth. These mechanisms might account for the Ang-(1-7)–mediated reduction in neointimal formation after vascular injury.

Perspectives
Ang-(1-7) inhibited the growth of cultured VSMCs and reduced neointimal formation after endothelial denudation of the rat carotid artery. We now report that Ang-(1-7) increased PGI₂ and cAMP production and attenuated Ang II–stimulated ERK1/ERK2 activities in cultured VSMCs. In addition, blockade of cyclooxygenase or the cAMP-dependent protein kinase attenuated the Ang-(1-7)–mediated inhibition of VSMC growth, suggesting a role for PGI₂ and cAMP production and MAP kinase inhibition in the antiproliferative response to Ang-(1-7). Treatment with stable analogues of PGI₂ and cAMP or downregulation of ERK1/ERK2 by gene transfer or antisense oligonucleotides reduced neointimal hyperplasia after vascular injury. These data suggest that the beneficial effects of Ang-(1-7) infusion after vascular injury might be mediated through similar mechanisms. Furthermore, because plasma Ang-(1-7) is elevated after treatment with ACE inhibitors or AT₁ receptor antagonists, Ang-(1-7) stimulation of PGI₂ and cAMP production, as well as inhibition of MAP kinase activity, might account for the reversal of vascular hypertrophy or hyperplasia due to inhibition of Ang II formation or activity. In addition, Ang-(1-7) is the major product of a newly discovered enzyme of the renin-angiotensin system, ACE2.⁴⁷ Reduced ACE2 was associated with hypertension quantitative trait loci as detected by linkage analysis, and ACE2 ablation results in mice with severe cardiac dysfunction. Reduced ACE2 expression or activity might result in a shift of the balance between Ang II and Ang-(1-7), resulting in loss of the counteracting vasodepressor and antiproliferative effects of Ang-(1-7).

	Acknowledgments

 
This work was supported in part by grants HL-51952 and NS-31664 from the National Institutes of Health and a grant-in-aid from the North Carolina Affiliate of the American Heart Association.

	Footnotes

 
M.A.C. is currently affiliated with the College of Pharmacy, Department of Pharmaceutical Sciences, Nova Southeastern University, Fort Lauderdale, Fla.

Received March 3, 2003; first decision April 3, 2003; accepted July 29, 2003.

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