E. Ann Tallant

Counterregulatory Actions of Angiotensin-(1-7)

Angiotensin (Ang)-(1-7) is a bioactive component of the renin-angiotensin system that is formed endogenously from either Ang I or Ang II. The first actions described for Ang-(1-7) indicated that the peptide mimicked some of the effects of Ang II, including the release of prostanoids and vasopressin. However, Ang-(1-7) is devoid of vasoconstrictor, central pressor, or thirst-stimulating actions. In fact, new findings reveal depressor, vasodilator, and antihypertensive actions that may be more apparent in hypertensive animals or humans. Thus, the accumulating evidence suggests that Ang-(1-7) may oppose the actions of Ang II either directly or by stimulation of prostaglandins and nitric oxide. These observations are significant because they may explain the effective antihypertensive action of converting enzyme inhibitors in a variety of non-renin-dependent models of experimental and genetic hypertension as well as most forms of human hypertension. In this context, studies in humans and animals showed that the antihypertensive action of converting enzyme inhibitors correlated with increases in plasma levels of Ang-(1-7). In this review, we summarize our knowledge of the mechanisms accounting for the counterregulatory actions of Ang-(1-7) and elaborate on the emerging concept that Ang-(1-7) functions as an antihypertensive peptide within the cascade of the renin-angiotensin system.

Angiotensin-(1-7) Inhibits Vascular Smooth Muscle Cell Growth

Although angiotensin II (Ang II) and the heptapeptide Ang-(1-7) differ by only one amino acid, the two peptides produce different responses in vascular smooth muscle cells. We previously showed that Ang II stimulated phosphoinositide hydrolysis, whereas Ang II and Ang-(1-7) released prostaglandins. We now report that Ang II and Ang-(1-7) differentially modulate rat aortic vascular smooth muscle cell growth. Ang-(1-7) inhibited [3H]thymidine incorporation in response to stimulation by fetal bovine serum, platelet-derived growth factor, or Ang II. The reduction in serum-stimulated thymidine incorporation by Ang-(1-7) depended on the concentration of the heptapeptide over the range of 1 nmol/L to 1 mumol/L, with a maximal inhibition of 60% by 1 mumol/L Ang-(1-7). Ang-(1-7) also inhibited the serum-stimulated increase in cell number to a maximum of 77% by 1 mumol/L Ang-(1-7). The attenuation of serum-stimulated thymidine incorporation by Ang-(1-7) was unaffected by antagonists selective for angiotensin type 1 (AT1) or type 2 (AT2) receptors; however, [Sar1,Ile1]Ang II and [Sar1,Thr2]Ang II were effective antagonists, indicating that growth inhibition by Ang-(1-7) was a result of angiotensin receptor activation. In contrast, Ang II stimulated [3H]thymidine incorporation in cultured vascular smooth muscle cells over the same concentration range, with a maximal stimulation of 314% at 1 mumol/L Ang II. Ang II also increased the total number of cells (to 145% of control), suggesting that enhanced thymidine incorporation was associated with vascular smooth muscle cell proliferation. The AT1 antagonist losartan or L-158,809 but not AT2 antagonists blocked [3H]thymidine incorporation by Ang II. These results suggest that Ang-(1-7) and Ang II exhibit opposite effects on the regulation of vascular smooth muscle cell growth. The inhibition of proliferation by Ang-(1-7) appears to be mediated by a novel angiotensin receptor that is not inhibited by AT1 or AT2 receptor antagonists.

Effect of Angiotensin-Converting Enzyme Inhibition and Angiotensin II Receptor Blockers on Cardiac Angiotensin-Converting Enzyme 2

Background—Angiotensin-converting enzyme 2 (ACE2) has emerged as a novel regulator of cardiac function and arterial pressure by converting angiotensin II (Ang II) into the vasodilator and antitrophic heptapeptide, angiotensin-(1–7) [Ang-(1–7)]. As the only known human homolog of ACE, the demonstration that ACE2 is insensitive to blockade by ACE inhibitors prompted us to define the effect of ACE inhibition on the ACE2 gene. Methods and Results—Blood pressure, cardiac rate, and plasma and cardiac tissue levels of Ang II and Ang-(1–7), together with cardiac ACE2, neprilysin, Ang II type 1 receptor (AT1), and mas receptor mRNAs, were measured in Lewis rats 12 days after continuous administration of vehicle, lisinopril, losartan, or both drugs combined in their drinking water. Equivalent decreases in blood pressure were obtained in rats given lisinopril or losartan alone or in combination. ACE inhibitor therapy caused a 1.8-fold increase in plasma Ang-(1–7), decreased plasma Ang II, and increased cardiac ACE2 mRNA but not cardiac ACE2 activity. Losartan increased plasma levels of both Ang II and Ang-(1–7), as well as cardiac ACE2 mRNA and cardiac ACE2 activity. Combination therapy duplicated the effects found in rats medicated with lisinopril, except that cardiac ACE2 mRNA fell to values found in vehicle-treated rats. Losartan treatment but not lisinopril increased cardiac tissue levels of Ang II and Ang-(1–7), whereas none of the treatments had an effect on cardiac neprilysin mRNA. Conclusions—Selective blockade of either Ang II synthesis or activity induced increases in cardiac ACE2 gene expression and cardiac ACE2 activity, whereas the combination of losartan and lisinopril was associated with elevated cardiac ACE2 activity but not cardiac ACE2 mRNA. Although the predominant effect of ACE inhibition may result from the combined effect of reduced Ang II formation and Ang-(1–7) metabolism, the antihypertensive action of AT1 antagonists may in part be due to increased Ang II metabolism by ACE2.

Upregulation of Angiotensin-Converting Enzyme 2 After Myocardial Infarction by Blockade of Angiotensin II Receptors

Abstract—We investigated in Lewis normotensive rats the effect of coronary artery ligation on the expression of cardiac angiotensin-converting enzymes (ACE and ACE 2) and angiotensin II type-1 receptors (AT1a-R) 28 days after myocardial infarction. Losartan, olmesartan, or the vehicle (isotonic saline) was administered via osmotic minipumps for 28 days after coronary artery ligation or sham operation. Coronary artery ligation caused left ventricular dysfunction and cardiac hypertrophy. These changes were associated with increased plasma concentrations of angiotensin I, angiotensin II, angiotensin-(1–7), and serum aldosterone, and reduced AT1a-R mRNA. Cardiac ACE and ACE 2 mRNAs did not change. Both angiotensin II antagonists attenuated cardiac hypertrophy; olmesartan improved ventricular contractility. Blockade of the AT1a-R was accompanied by a further increase in plasma concentrations of the angiotensins and reduced serum aldosterone levels. Both losartan and olmesartan completely reversed the reduction in cardiac AT1a-R mRNA observed after coronary artery ligation while augmenting ACE 2 mRNA by approximately 3-fold. Coadministration of PD123319 did not abate the increase in ACE 2 mRNA induced by losartan. ACE 2 mRNA correlated significantly with angiotensin II, angiotensin-(1–7), and angiotensin I levels. These results provide evidence for an effect of angiotensin II blockade on cardiac ACE 2 mRNA that may be due to direct blockade of AT1a receptors or a modulatory effect of increased angiotensin-(1–7).

Angiotensin-(1–7) Reduces Smooth Muscle Growth After Vascular Injury

Regulation of vascular smooth muscle cell growth is critical to the maintenance of normal blood flow and vessel patency. Angiotensin-(1-7) [Ang-(1-7)] inhibits proliferation of vascular smooth muscle cells in vitro and opposes the mitogenic effects of angiotensin II. The present study investigated whether Ang-(1-7) inhibits vascular smooth muscle cell growth in vivo by determining its effect on neointimal formation and medial remodeling in balloon-injured carotid arteries. The carotid arteries of adult male Sprague-Dawley rats were injured with a balloon embolectomy catheter. Ang-(1-7) in saline (24 microg/kg per hour) or saline alone was infused intravenously for 12 days after injury. Pumps containing bromodeoxyuridine were implanted at the same time to determine DNA synthesis. Intravenous infusion increased plasma Ang-(1-7) to 166. 0+/-41.2 fmol/mL (n=6) compared with 46.9+/-4.2 fmol/mL (n=8) in saline-infused rats. Plasma concentrations of Ang II were not changed by Ang-(1-7) infusion. Elevation in circulating Ang-(1-7) had no effect on either blood pressure or heart rate compared with saline controls. Histomorphometric analysis of carotid arteries indicated that Ang-(1-7) infusion significantly reduced neointimal area compared with rats infused with saline (0.063+/-0.011 versus 0. 100+/-0.009 mm2; P<0.05). In contrast, Ang-(1-7) infusion had no effect on medial area of the injured or the contralateral uninjured artery compared with saline controls. Ang-(1-7) infusion also reduced the rate of DNA synthesis in both the neointima and the media of the injured vessels. Therefore, exogenous Ang-(1-7) inhibited vascular smooth muscle cell proliferation associated with balloon-catheter injury. Similar increases in endogenous plasma Ang-(1-7) and inhibition of neointimal growth were observed in rats after angiotensin-converting enzyme inhibitor or angiotensin type 1 receptor antagonist administration, suggesting that Ang-(1-7) may contribute to the in vivo antiproliferative effects of these agents on vascular smooth muscle.

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

Abstract—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 &mgr;mol/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 &mgr;mol/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.

Antiproliferative Actions of Angiotensin-(1-7) in Vascular Smooth Muscle

Abstract —Hemodynamic factors, circulating hormones, paracrine factors, and intracrine factors influence vascular smooth muscle growth and plasticity. The well-characterized role of angiotensin II in the modulation of vascular tone and cell function may be critically involved in the mechanisms by which vascular smooth muscle responds to signals associated with vascular endothelial dysfunction and increases in oxidative stress. Studies from this laboratory suggest that the trophic actions of angiotensin II may be intrinsically regulated by angiotensin-(1-7), a separate product of the angiotensin system derived from the common substrate, angiotensin I. Exposure of cultured vascular smooth muscle cells to angiotensin-(1-7) inhibited the trophic actions of angiotensin II and reduced the expression of the mitogenic effects of both normal serum and platelet-derived growth factor. The growth-inhibitory actions of angiotensin-(1-7) were blocked by the selective d-alanine 7 -angiotensin-(1-7) antagonist and the nonselective angiotensin receptor blocker sarcosine 1 -threonine 8 -angiotensin II. In contrast, subtype-selective antagonists for the AT 1 and AT 2 receptors had no effect on the inhibitory actions of angiotensin-(1-7), a finding that is consistent with the pharmacological characterization of a high-affinity 125 I-labeled angiotensin-(1-7) binding site in the vasculature by use of selective and nonselective angiotensin II receptor antagonists. The relevance of these findings to the proliferative response of vascular smooth muscle cells after endothelial injury was confirmed by assessment of the effect of a 12-day infusion of angiotensin-(1-7) on neointimal formation. In these experiments, the proliferative response produced by injuring the carotid artery was inhibited by angiotensin-(1-7) through a mechanism that could not be explained by changes in arterial pressure. Because plasma angiotensin-(1-7) increased to levels comparable to those found in animals and human subjects given therapeutic doses of angiotensin-converting enzyme inhibitors, angiotensin-(1-7) may be one factor participating in the reversal of vascular proliferation during inhibition of angiotensin II formation or activity.

Bovine Aortic Endothelial Cells Contain an Angiotensin-(1–7) Receptor

Angiotensin-(1-7) is a novel peptide of the renin-angiotensin system that counteracts the pressor and proliferative responses to angiotensin II. We now report that cultured bovine aortic endothelial cells contain a saturable, high-affinity [125I]angiotensin-(1-7) binding site with an affinity of 19.3 +/- 10.7 nmol/L and a density of 1351 +/- 710 fmol/mg protein. Angiotensin-(1-7) competed at a second lower-affinity site, with an IC50 of 2.9 mumol/L. The high-affinity angiotensin II receptor antagonist sarcosine1-isoleucine8-angiotensin II blocked [125I]angiotensin-(1-7) binding to bovine aortic endothelial cells at both a high- (IC50 = 1.3 nmol/L) and a low-affinity (IC50 = 6.2 mumol/L) binding site. In contrast, D-alanine7-angiotensin-(1-7) completely blocked [125I]angiotensin-(1-7) binding, with an IC50 of 19.8 nmol/L, suggesting that D-alanine7-angiotensin-(1-7) may selectively block responses to angiotensin-(1-7) in endothelial cells. Neither the AT1 antagonist losartan nor the AT2 antagonist PD 123319 exhibited significant competition for [125I]angiotensin-(1-7) binding to endothelial cells isolated from bovine aorta, in agreement with the absence of detectable mRNAs encoding typical angiotensin receptor subtypes 1 or 2 (AT1 or AT2). Angiotensin II also competed for [125I]angiotensin-(1-7) binding to bovine aortic endothelial cells; however, the relative affinity was 13-fold lower than angiotensin-(1-7), suggesting a preference for angiotensin-(1-7) over angiotensin II. These results demonstrate that bovine aortic endothelial cells contain a unique non-AT1, non-AT2 angiotensin receptor that preferentially binds angiotensin-(1-7).

Renin–angiotensin system expression in rat bone marrow haematopoietic and stromal cells

The existence of a bone marrow renin–angiotensin system (RAS) is evidenced by the association of renin, angiotensin converting enzyme (ACE), and angiotensin (Ang) II and its AT1 and AT2 receptors with both normal and disturbed haematopoiesis. The expression of RAS components by rat unfractionated bone marrow cells (BMC), haematopoietic‐lineage BMC and cultured marrow stromal cells (MSC) was investigated to determine which specific cell types may contribute to a local bone marrow RAS. The mRNAs for angiotensinogen, renin, ACE, and AT1a and AT2 receptors were present in BMC and in cultured MSC; ACE2 mRNA was detected only in BMC. Two‐colour flow fluorocytometry analysis showed immunodetectable angiotensinogen, ACE, AT1 and AT2 receptors, and Ang II, as well as binding of Ang II to AT1 and AT2 receptors, in CD4+, CD11b/c+, CD45R+ and CD90+ BMC and cultured MSC; renin was found in all cell types with the exception of CD4+ BMC. Furthermore, Ang II was detected by radioimmunoassay in MSC homogenates as well as conditioned culture medium. The presence of Ang II receptors in both haematopoietic‐lineage BMC and MSC, and the de novo synthesis of Ang II by MSC suggest a potential autocrine–paracrine mechanism for local RAS‐mediated regulation of haematopoiesis.

Angiotensin-(1-7) Downregulates the Angiotensin II Type 1 Receptor in Vascular Smooth Muscle Cells

Abstract—Angiotensin (Ang)-(1-7) is a biologically active peptide of the renin-angiotensin system that has both vasodilatory and antiproliferative activities that are opposite the constrictive and proliferative effects of angiotensin II (Ang II). We studied the actions of Ang-(1-7) on the Ang II type 1 (AT1) receptor in cultured rat aortic vascular smooth muscle cells to determine whether the effects of Ang-(1-7) are due to its regulation of the AT1 receptor. Ang-(1-7) competed poorly for [125I]Ang II binding to the AT1 receptor on vascular smooth muscle cells, with an IC50 of 2.0 &mgr;mol/L compared with 1.9 nmol/L for Ang II. The pretreatment of vascular smooth muscle cells with Ang-(1-7) followed by treatment with acidic glycine to remove surface-bound peptide resulted in a significant decrease in [125I]Ang II binding; however, reduced Ang II binding was observed only at micromolar concentrations of Ang-(1-7). Scatchard analysis of vascular smooth muscle cells pretreated with 1 &mgr;mol/L Ang-(1-7) showed that the reduction in Ang II binding resulted from a loss of the total number of binding sites [Bmax 437.7±261.5 fmol/mg protein in Ang-(1-7)–pretreated cells compared with 607.5±301.2 fmol/mg protein in untreated cells, n=5, P <0.05] with no significant effect on the affinity of Ang II for the AT1 receptor. Pretreatment with the AT1 receptor antagonist L-158,809 blocked the reduction in [125I]Ang II binding by Ang-(1-7) or Ang II. Pretreatment of vascular smooth muscle cells with increasing concentrations of Ang-(1-7) reduced Ang II–stimulated phospholipase C activity; however, the decrease was significant (81.2±6.4%, P <0.01, n=5) only at 1 &mgr;mol/L Ang-(1-7). These results demonstrate that pharmacological concentrations of Ang-(1-7) in the micromolar range cause a modest downregulation of the AT1 receptor on vascular cells and a reduction in Ang II–stimulated phospholipase C activity. Because the antiproliferative and vasodilatory effects of Ang-(1-7) are observed at nanomolar concentrations of the heptapeptide, these responses to Ang-(1-7) cannot be explained by competition of Ang-(1-7) at the AT1 receptor or Ang-(1-7)–mediated downregulation of the vascular AT1 receptor.