The RAS (renin-<em>angiotensin</em> system) is integral to cardiovascular physiology; however, dysregulation of this system largely contributes to the pathophysiology of CVD (cardiovascular disease). It is well established that AngII (<em>angiotensin</em> II), the main effector of the RAS, engages the AT<em>1</em>R (<em>angiotensin</em> type <em>1</em> receptor) and promotes cell growth, proliferation, migration and oxidative stress, all processes which contribute to remodelling of the heart and vasculature, ultimately leading to the development and progression of various CVDs, including heart failure and atherosclerosis. The counter-regulatory axis of the RAS, which is centred on the actions of ACE2 (<em>angiotensin</em>-converting enzyme 2) and the resultant production of Ang-(<em>1</em>-<em>7</em>) [<em>angiotensin</em>-(<em>1</em>-<em>7</em>)] from AngII, antagonizes the actions of AngII via the receptor Mas, thereby providing a protective role in CVD. More recently, another ACE2 metabolite, Ang-(<em>1</em>-9) [<em>angiotensin</em>-(<em>1</em>-9)], has been reported to be a biologically active peptide within the counter-regulatory axis of the RAS. The present review will discuss the role of the counter-regulatory RAS peptides Ang-(<em>1</em>-<em>7</em>) and Ang-(<em>1</em>-9) in the cardiovascular system, with a focus on their effects in remodelling of the heart and vasculature.

OBJECTIVE

Standard drugs post-myocardial infarction (MI) such as angiotensin converting enzyme (ACE) and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) increase levels of endothelial progenitor cells (EPC). However, potential underlying mechanisms have not yet been investigated.

RESULTS

We studied the effects of ACE inhibition or statin treatment on EPC levels and on bone marrow molecular pathways involved in EPC mobilization after MI in rats. Three days post-infarction, acetylated LDL (acLDL)+/Ulex europeus-1 (UEA-1)+/VEGF receptor-2+/eNOS+ EPC levels and formation of endothelial colony forming units (CFU) were reduced to 60+/-12% (p < 0.05) and 68+/-7% (p < 0.05). In bone marrow, extracellular signal-regulated kinase (ERK) phosphorylation and matrix metalloproteinase (MMP)-9 activity were repressed. Endothelial nitric oxide synthase (eNOS) activity was unchanged, whereas reactive oxygen species (ROS) were increased two-fold in bone marrow. ACE or HMG-CoA reductase inhibition resulted in significant increases in EPC levels. ACE inhibition increased bone marrow ERK phosphorylation and MMP-9 activity. Statin therapy enhanced bone marrow VEGF protein levels, Akt phosphorylation, eNOS activity and normalized increased ROS levels. Augmented EPC levels in the early post-infarction phase by ACE inhibition or statin treatment were associated with improved cardiac function and increased capillary density in the peri-infarct area 7 days after MI. Moreover, increased EPC levels in response to ACE inhibition or statin treatment were sustained 10 weeks post-infarction.

CONCLUSIONS

Increased ROS and impaired MMP-9 activity in bone marrow likely contribute to reduced EPC mobilization in the early post-infarction phase. ACE inhibition or statin treatment increased EPC levels with distinct drug-specific effects on bone marrow molecular alterations.

Central administration of <em>angiotensin</em> IV (Ang IV) or its analogues enhance performance of rats in passive avoidance and spatial memory paradigms. The purpose of this study was to examine the effect of a single bolus injection of two distinct AT4 ligands, Nle<em>1</em>-Ang IV or LVV-haemorphin-<em>7</em>, on spatial learning in the Barnes circular maze. Mean number of days for rats treated with either Nle<em>1</em>-Ang IV or LVV-haemorphin-<em>7</em> to achieve learner criterion is significantly reduced compared with controls (P < 0.00<em>1</em> and P < 0.05 respectively). This is due to enhanced ability of the peptide-treated rats to adopt a spatial strategy for finding the escape hatch. In all three measures of learning performance, (<em>1</em>) the number of errors made, (2) the distance travelled and (3) the latency in finding the escape hatch, rats treated with either <em>1</em>00 pmol or <em>1</em> nmol of Nle<em>1</em>-Ang IV or <em>1</em>00 pmol LVV-haemorphin-<em>7</em> performed significantly better than the control groups. As early as the first day of testing, the rats treated with the lower dose of Nle<em>1</em>-Ang IV or LVV-haemorphin-<em>7</em> made fewer errors (P < 0.0<em>1</em> and P < 0.05 respectively) and travelled shorter distances (P < 0.05 for both groups) than the control animals. The enhanced spatial learning induced by Nle<em>1</em>-Ang IV (<em>1</em>00 pmol) was attenuated by the co-administration of the AT4 receptor antagonist, divalinal-Ang IV (<em>1</em>0 nmol). Thus, administration of AT4 ligands results in an immediate potentiation of learning, which may be associated with facilitation of synaptic transmission and/or enhancement of acetylcholine release.

<em>Angiotensin</em> II (Ang II) is implicated in the proinflammatory process in various disease situations. Thus, we sought to determine the role of Ang II in early inflammation-induced fibrosis of pressure-overloaded (PO) hearts. PO was induced by suprarenal aortic constriction (AC) at day 0 in male Wistar rats, and they were orally administered 0.<em>1</em> mg/kg per day candesartan every day from day -<em>7</em>. This was the maximum dose of candesartan that did not change arterial pressure in hypertensive rats with AC (AC rats). In AC rats, cardiac <em>angiotensin</em>-converting enzyme (ACE) activity was transiently enhanced after day <em>1</em> and peaked at day 3, declining to lower levels by day <em>1</em>4, whereas serum ACE activity was not changed. In AC rats, PO induced early fibroinflammatory changes (monocyte chemoattractant factor [MCP]-<em>1</em> and transforming growth factor [TGF]-beta expression, perivascular macrophage accumulation, and fibroblast proliferation), and thereafter, left ventricular hypertrophy developed, featuring myocyte hypertrophy, intramyocardial arterial wall thickening, and perivascular and interstitial fibroses. Candesartan suppressed the induction of MCP-<em>1</em> and TGF-beta and reduced macrophage accumulation and fibroblast proliferation in PO hearts. Candesartan significantly prevented perivascular and interstitial fibrosis. However, candesartan did not affect myocyte hypertrophy and arterial wall thickening. In conclusion, a subdepressor dose of candesartan prevented the MCP-<em>1</em>-mediated inflammatory process and reactive myocardial fibrosis in PO hearts. Ang II might play a key role in reactive fibrosis in hypertensive hearts, independent of arterial pressure changes.

The discovery of <em>angiotensin</em>-I-converting enzyme 2 (ACE2) and a (pro)renin receptor has renewed interest in the physiology of the renin-<em>angiotensin</em> system (RAS). Through the ACE2/<em>angiotensin</em>-(<em>1</em>-<em>7</em>)/Mas counter-regulatory axis, ACE2 balances the vasoconstrictive, proliferative, fibrotic and proinflammatory effects of the ACE/<em>angiotensin</em> II/AT<em>1</em> axis. The (pro)renin receptor system shows an <em>angiotensin</em>-dependent function related to increased generation of <em>angiotensin</em> I, and an <em>angiotensin</em>-independent aspect related to intracellular signalling. Activation of ACE2 and inhibition of ACE and renin have been at the core of the RAS regulation. The aim of this review is to discuss the biochemistry and biological functions of ACE, ACE2 and renin within and beyond the RAS, and thus provide a perspective for future bioactives from natural plant and/or food resources related to the three proteases.

Over-activation of the brain renin-<em>angiotensin</em> system (RAS) has been implicated in the etiology of anxiety disorders. <em>Angiotensin</em> converting enzyme 2 (ACE2) inhibits RAS activity by converting <em>angiotensin</em>-II, the effector peptide of RAS, to <em>angiotensin</em>-(<em>1</em>-<em>7</em>), which activates the Mas receptor (MasR). Whether increasing brain ACE2 activity reduces anxiety by stimulating central MasR is unknown. To test the hypothesis that increasing brain ACE2 activity reduces anxiety-like behavior via central MasR stimulation, we generated male mice overexpressing ACE2 (ACE2 KI mice) and wild type littermate controls (WT). ACE2 KI mice explored the open arms of the elevated plus maze (EPM) significantly more than WT, suggesting increasing ACE2 activity is anxiolytic. Central delivery of diminazene aceturate, an ACE2 activator, to C5<em>7</em>BL/6 mice also reduced anxiety-like behavior in the EPM, but centrally administering ACE2 KI mice A-<em>7</em><em>7</em>9, a MasR antagonist, abolished their anxiolytic phenotype, suggesting that ACE2 reduces anxiety-like behavior by activating central MasR. To identify the brain circuits mediating these effects, we measured Fos, a marker of neuronal activation, subsequent to EPM exposure and found that ACE2 KI mice had decreased Fos in the bed nucleus of stria terminalis but had increased Fos in the basolateral amygdala (BLA). Within the BLA, we determined that ∼62% of GABAergic neurons contained MasR mRNA and expression of MasR mRNA was upregulated by ACE2 overexpression, suggesting that ACE2 may influence GABA neurotransmission within the BLA via MasR activation. Indeed, ACE2 overexpression was associated with increased frequency of spontaneous inhibitory postsynaptic currents (indicative of presynaptic release of GABA) onto BLA pyramidal neurons and central infusion of A-<em>7</em><em>7</em>9 eliminated this effect. Collectively, these results suggest that ACE2 may reduce anxiety-like behavior by activating central MasR that facilitate GABA release onto pyramidal neurons within the BLA.

CD40 ligand (CD40L) is involved in the vascular infiltration of immune cells and pathogenesis of atherosclerosis. Additionally, T cell CD40L release causes platelet, dendritic cell and monocyte activation in thrombosis. However, the role of CD40L in <em>angiotensin</em> II (ATII)-driven vascular dysfunction and hypertension remains incompletely understood. We tested the hypothesis that CD40L contributes to ATII-driven vascular inflammation by promoting platelet-leukocyte activation, vascular infiltration of immune cells and by amplifying oxidative stress. C5<em>7</em>BL/6 and CD40L-/- mice were infused with ATII (<em>1</em> mg/kg/day for <em>7</em> days) using osmotic minipumps. Vascular function was recorded by isometric tension studies, and reactive oxygen species (ROS) were monitored in blood and heart by optical methods. Western blot, immunohistochemistry, FACS analysis and real-time RT-PCR were used to analyze immune cell distribution, pro-inflammatory cytokines, NAPDH oxidase subunits, T cell transcription factors and other genes of interest. ATII-treated CD40L-/- mice showed improved endothelial function, suppression of blood platelet-monocyte interaction (FACS), platelet thrombin generation (calibrated automated thrombography) and coagulation (bleeding time), as well as decreased oxidative stress in the aorta, heart and blood compared to wild-type mice. Moreover, ATII-treated CD40L-/- mice displayed decreased levels of TH<em>1</em> cytokines released by splenic CD4⁺ T cells (ELISA) and lower expression levels of NOX-2, T-bet and P-selectin as well as diminished immune cell infiltration in aortic tissue compared to controls. Our results demonstrate that many ATII-induced effects on vascular dysfunction, such as vascular inflammation, oxidative stress and a pro-thrombotic state, are mediated at least in part via CD40L.

The renin-<em>angiotensin</em> system (RAS), that is known for its role in the regulation of blood pressure as well as in fluid and electrolyte homeostasis, comprises dozens of <em>angiotensin</em> peptides and peptidases and at least six receptors. Six central components constitute the two main axes of the RAS cascade. <em>Angiotensin</em> (<em>1</em>-<em>7</em>), an <em>angiotensin</em> converting enzyme 2 and Mas receptor axis (ACE2-Ang(<em>1</em>-<em>7</em>)-MasR) counterbalances the harmful effects of the <em>angiotensin</em> II, <em>angiotensin</em> converting enzyme <em>1</em> and <em>angiotensin</em> II type <em>1</em> receptor axis (ACE<em>1</em>-AngII-AT<em>1</em>R) Whereas systemic RAS is an important factor in blood pressure regulation, tissue-specific regulatory system, responsible for long term regional changes, that has been found in various organs. In other words, RAS is not only endocrine but also complicated autocrine system. The human eye has its own intraocular RAS that is present e.g. in the structures involved in aqueous humor dynamics. Local RAS may thus be a target in the development of new anti-glaucomatous drugs. In this review, we first describe the systemic RAS cascade and then the local ocular RAS especially in the anterior part of the eye.

The RAS (renin-<em>angiotensin</em> system) plays a role not only in the cardiovascular system, including blood pressure regulation, but also in the central nervous system. AngII (<em>angiotensin</em> II) binds two major receptors: the AT(<em>1</em>) receptor (AngII type <em>1</em> receptor) and AT(2) receptor (AngII type 2 receptor). It has been recognized that AT(2) receptor activation not only opposes AT(<em>1</em>) receptor actions, but also has unique effects beyond inhibitory cross-talk with AT(<em>1</em>) receptor signalling. Novel pathways beyond the classical actions of RAS, the ACE (<em>angiotensin</em>-converting enzyme)/AngII/AT(<em>1</em>) receptor axis, have been highlighted: the ACE2/Ang-(<em>1</em>-<em>7</em>) [<em>angiotensin</em>-(<em>1</em>-<em>7</em>)]/Mas receptor axis as a new opposing axis against the ACE/AngII/AT(<em>1</em>) receptor axis, novel AngII-receptor-interacting proteins and various AngII-receptor-activation mechanisms including dimer formation. ATRAP (AT(<em>1</em>)-receptor-associated protein) and ATIP (AT(2)-receptor-interacting protein) are well-characterized AngII-receptor-associated proteins. These proteins could regulate the functions of AngII receptors and thereby influence various pathophysiological states. Moreover, the possible cross-talk between PPAR (peroxisome-proliferator-activated receptor)-γ and AngII receptor subtypes is an intriguing issue to be addressed in order to understand the roles of RAS in the metabolic syndrome, and interestingly some ARBs (AT(<em>1</em>)-receptor blockers) have been reported to have an AT(<em>1</em>)-receptor-blocking action with a partial PPAR-γ agonistic effect. These emerging concepts concerning the regulation of AngII receptors are discussed in the present review.

To explore the potential therapeutic effects of <em>angiotensin</em>(<em>1</em>-<em>7</em>) (Ang(<em>1</em>-<em>7</em>)), an endogenous ligand of the Mas receptor, on streptozotocin-induced diabetic nephropathy, male Wistar rats were randomly divided into two groups: a control group and a diabetic model group. After <em>1</em>2 weeks, the diabetic rats were divided into subgroups for 4-week treatments consisting of no-treatment group, small-, moderate-, and large-dose Ang(<em>1</em>-<em>7</em>) groups, a valsartan group, a large-dose Ang(<em>1</em>-<em>7</em>) plus valsartan group, and an A<em>7</em><em>7</em>9 (antagonist of the Mas receptor) group, each with <em>1</em>5 rats. Ang(<em>1</em>-<em>7</em>) improved renal function, attenuated glomeruli sclerosis, oxidative stress, and cell proliferation, decreased the expression of collagen IV, TGF-β<em>1</em>, VEGF, NOX4, p4<em>7</em>phox, PKCα, and PKCβ<em>1</em>, and the phosphorylation of Smad3. In the rat mesangial HBZY-<em>1</em> cell line, Ang(<em>1</em>-<em>7</em>) decreased high-glucose-induced oxidative stress, the proliferation and expression of NOX4, p4<em>7</em>phox, and TGF-β<em>1</em>, the phosphorylation of Smad3, collagen IV, and VEGF, and the membrane translocation of PKCα and PKCβ<em>1</em>. A<em>7</em><em>7</em>9 blocked the effects of Ang(<em>1</em>-<em>7</em>) both in vivo and in vitro. The effects of large-dose Ang(<em>1</em>-<em>7</em>) alone and in combination with valsartan were superior to valsartan alone, but the combination had no significant synergistic effect compared with Ang(<em>1</em>-<em>7</em>) alone. Thus, Ang(<em>1</em>-<em>7</em>) ameliorated streptozotocin-induced diabetic renal injury. Large-dose treatment was superior to valsartan in reducing oxidative stress and inhibiting TGFβ<em>1</em>/Smad3- and VEGF-mediated pathways.

Elephant seals are naturally adapted to survive up to three months of absolute food and water deprivation (fasting). Prolonged food deprivation in terrestrial mammals increases reactive oxygen species (ROS) production, oxidative damage and inflammation that can be induced by an increase in the renin-<em>angiotensin</em> system (RAS). To test the hypothesis that prolonged fasting in elephant seals is not associated with increased oxidative stress or inflammation, blood samples and muscle biopsies were collected from early (2-3 weeks post-weaning) and late (<em>7</em>-8 weeks post-weaning) fasted seals. Plasma levels of oxidative damage, inflammatory markers and plasma renin activity (PRA), along with muscle levels of lipid and protein oxidation, were compared between early and late fasting periods. Protein expression of <em>angiotensin</em> receptor <em>1</em> (AT(<em>1</em>)), pro-oxidant (Nox4) and antioxidant enzymes (CuZn- and Mn-superoxide dismutases, glutathione peroxidase and catalase) was analyzed in muscle. Fasting induced a 2.5-fold increase in PRA, a 50% increase in AT(<em>1</em>), a twofold increase in Nox4 and a <em>7</em>0% increase in NADPH oxidase activity. By contrast, neither tissue nor systemic indices of oxidative damage or inflammation increased with fasting. Furthermore, muscle antioxidant enzymes increased 40-60% with fasting in parallel with an increase in muscle and red blood cell antioxidant enzyme activities. These data suggest that, despite the observed increases in RAS and Nox4, an increase in antioxidant enzymes appears to be sufficient to suppress systemic and tissue indices of oxidative damage and inflammation in seals that have fasted for a prolonged period. The present study highlights the importance of antioxidant capacity in mammals during chronic periods of stress to help avoid deleterious systemic consequences.

<em>Angiotensin</em> (ANG)-(<em>1</em>-<em>7</em>) is known to attenuate diabetic nephropathy; however, its role in the modulation of renal inflammation and oxidative stress in type 2 diabetes is poorly understood. Thus in the present study we evaluated the renal effects of a chronic ANG-(<em>1</em>-<em>7</em>) treatment in Zucker diabetic fatty rats (ZDF), an animal model of type 2 diabetes and nephropathy. Sixteen-week-old male ZDF and their respective controls [lean Zucker rats (LZR)] were used for this study. The protocol involved three groups: <em>1</em>) LZR + saline, 2) ZDF + saline, and 3) ZDF + ANG-(<em>1</em>-<em>7</em>). For 2 wk, animals were implanted with subcutaneous osmotic pumps that delivered either saline or ANG-(<em>1</em>-<em>7</em>) (<em>1</em>00 ng·kg(-<em>1</em>)·min(-<em>1</em>)) (n = 4). Renal fibrosis and tissue parameters of oxidative stress were determined. Also, renal levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), ED-<em>1</em>, hypoxia-inducible factor-<em>1</em>α (HIF-<em>1</em>α), and neutrophil gelatinase-associated lipocalin (NGAL) were determined by immunohistochemistry and immunoblotting. ANG-(<em>1</em>-<em>7</em>) induced a reduction in triglyceridemia, proteinuria, and systolic blood pressure (SBP) together with a restoration of creatinine clearance in ZDF. Additionally, ANG-(<em>1</em>-<em>7</em>) reduced renal fibrosis, decreased thiobarbituric acid-reactive substances, and restored the activity of both renal superoxide dismutase and catalase in ZDF. This attenuation of renal oxidative stress proceeded with decreased renal immunostaining of IL-6, TNF-α, ED-<em>1</em>, HIF-<em>1</em>α, and NGAL to values similar to those displayed by LZR. <em>Angiotensin</em>-converting enzyme type 2 (ACE2) and ANG II levels remained unchanged after treatment with ANG-(<em>1</em>-<em>7</em>). Chronic ANG-(<em>1</em>-<em>7</em>) treatment exerts a renoprotective effect in ZDF associated with a reduction of SBP, oxidative stress, and inflammatory markers. Thus ANG-(<em>1</em>-<em>7</em>) emerges as a novel target for treatment of diabetic nephropathy.

OBJECTIVE

Uveitis is a common cause of vision loss. The renin <em>angiotensin</em> system (RAS), which plays a vital role in cardiovascular system, is a potent mediator of inflammation and has been implicated in the pathogenesis of uveitis. A newly identified axis of RAS, ACE2/Ang-(<em>1</em>-<em>7</em>)/Mas, has emerged as a novel target because it counteracts the deleterious effect of <em>angiotensin</em> II. The purpose of this study was to investigate the effect of endogenous ACE2 activation in preventing endotoxin-induced uveitis (EIU) in mice.

METHODS

ACE2 activator diminazene aceturate (DIZE) was administered both systemically and locally. For systemic administration, female BALB/c mice received intraperitoneal injection of DIZE (60 mg/kg body weight [BW]) for 2 days prior to lipopolysaccharide (LPS) intravitreal injection (<em>1</em>25 ng) to induce uveitis. For local study, DIZE was given at 0.5, 0.<em>1</em>, and 0 mg/mL as eyedrops six times per day for 2 days before LPS injection. The anterior segment of the mice was examined at <em>1</em>2, 24, 48, and <em>7</em>2 hours after LPS injection, and clinical scores were determined at the same time. Morphology and infiltrating inflammatory cells were evaluated after 24 hours. The mRNA levels of inflammatory cytokines were analyzed by real-time RT-PCR. ACE2 activity was determined using a self-quenching fluorescent substrate.

RESULTS

At 24 hours, the clinical score of mice treated with DIZE systemically was significantly lower (mean, ∼<em>1</em>.<em>7</em>5) than the saline vehicle group (mean, ∼4) (P < 0.00<em>1</em>). Histological examination showed 63.4% reduction of infiltrating inflammatory cells in the anterior segment and 5<em>7</em>.4% reduction in the posterior segment of DIZE-treated eyes. The number of CD45(+) inflammatory cells in the vitreous of the DIZE-treated group was decreased (43.3%) compared to the vehicle group (P < 0.0<em>1</em>). The mRNA levels of inflammatory cytokines were significantly reduced in the DIZE-treated group (P < 0.0<em>1</em>, P < 0.00<em>1</em>). The number of infiltrating inflammatory cells was also significantly reduced in eyes that received topical administration of DIZE: <em>7</em>3.8% reduction in the 0.5 mg/mL group and 5<em>1</em>.<em>7</em>% reduction in the 0.<em>1</em>mg/mL group compared to the control group. DIZE treatment resulted in significantly increased ACE2 activity in the retina (P < 0.00<em>1</em>).

CONCLUSIONS

Endogenous ACE2 activation by DIZE has a preventive effect on LPS-induced ocular inflammation in the EIU mouse model. These results support the notions that RAS plays a role in modulating ocular immune response and that enhancing ACE2 provides a novel therapeutic strategy for uveitis.

ACE2 and Ang-(<em>1</em>-<em>7</em>) have important roles in preventing acute lung injury. However, it is not clear whether upregulation of the ACE2/Ang-(<em>1</em>-<em>7</em>)/Mas axis prevents LPS-induced injury in pulmonary microvascular endothelial cells (PMVECs) by inhibiting the MAPKs/NF-κB pathways. Primary cultured rat PMVECs were transduced with lentiviral-borne Ace2 or shRNA-Ace2, and then treated or not with Mas receptor blocker (A<em>7</em><em>7</em>9) before exposure to LPS. LPS stimulation resulted in the higher levels of AngII, Ang-(<em>1</em>-<em>7</em>), cytokine secretion, and apoptosis rates, and the lower ACE2/ACE ratio. Ace2 reversed the ACE2/ACE imbalance and increased Ang-(<em>1</em>-<em>7</em>) levels, thus reducing LPS-induced apoptosis and inflammation, while inhibition of Ace2 reversed all these effects. A<em>7</em><em>7</em>9 abolished these protective effects of Ace2. LPS treatment was associated with activation of the ERK, p38, JNK, and NF-κB pathways, which were aggravated by A<em>7</em><em>7</em>9. Pretreatment with A<em>7</em><em>7</em>9 prevented the Ace2-induced blockade of p38, JNK, and NF-κB phosphorylation. However, only JNK inhibitor markedly reduced apoptosis and cytokine secretion in PMVECs with Ace2 deletion and A<em>7</em><em>7</em>9 pretreatment. These results suggest that the ACE2/Ang-(<em>1</em>-<em>7</em>)/Mas axis has a crucial role in preventing LPS-induced apoptosis and inflammation of PMVECs, by inhibiting the JNK/NF-κB pathways.

OBJECTIVE

Multilineage cytopenias occur following myelosuppressive chemotherapy. Most hematopoietic agents differentiate along a single lineage and fail to prevent progressive cytopenias. <em>Angiotensin</em> <em>1</em>-<em>7</em> [A(<em>1</em>-<em>7</em>)] is a hematopoietic agent that stimulates the proliferation of multipotential and differentiated progenitor cells in cultured bone marrow and human cord blood. The purpose of this study was to determine the optimal biologic dose and the maximum tolerated dose of A(<em>1</em>-<em>7</em>).

METHODS

This study determined the safety and activity of A(<em>1</em>-<em>7</em>) following chemotherapy in patients with breast cancer. Toxicity was assessed by administering A(<em>1</em>-<em>7</em>) daily for <em>7</em> days followed by a <em>7</em>-day washout prior to the first cycle of chemotherapy. Beginning 2 days after chemotherapy and continuing daily for at least <em>1</em>0 days, fifteen patients received five different A(<em>1</em>-<em>7</em>) doses and five patients received filgrastim as a comparator group over three cycles of chemotherapy.

RESULTS

No dose-limiting toxicity was observed following A(<em>1</em>-<em>7</em>). The frequency of adverse events was slightly lower in A(<em>1</em>-<em>7</em>) than in filgrastim patients. No patient required a chemotherapy modification due to hematologic toxicity. There was an apparent differential dose-response sensitivity of the various lineages to A(<em>1</em>-<em>7</em>). At a dose of <em>1</em>00 microg/kg, A(<em>1</em>-<em>7</em>) reduced the frequency of grade 2-4 thrombocytopenia, anemia, and grade 3-4 lymphopenia as compared to filgrastim.

CONCLUSIONS

These data suggest that A(<em>1</em>-<em>7</em>) may be beneficial in attenuating multilineage cytopenias following chemotherapy at a dose of <em>1</em>00 mug/kg per day.

OBJECTIVE

As a newer component of the renin-<em>angiotensin</em> system, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>) ] has been shown to facilitate angiogenesis and protect against ischaemic damage in peripheral tissues. However, the role of Ang-(<em>1</em>-<em>7</em>) in brain angiogenesis remains unclear. The aim of this study was to investigate whether Ang-(<em>1</em>-<em>7</em>) could promote angiogenesis in brain, thus inducing tolerance against focal cerebral ischaemia.

METHODS

Male Sprague-Dawley rats were i.c.v. infused with Ang-(<em>1</em>-<em>7</em>), A-<em>7</em><em>7</em>9 (a Mas receptor antagonist), L-NIO, a specific endothelial NOS (eNOS) inhibitor, endostatin (an anti-angiogenic compound) or vehicle, alone or simultaneously, for <em>1</em>-4 weeks. Capillary density, endothelial cell proliferation and key components of eNOS pathway in the brain were evaluated. Afterwards, rats were subjected to permanent middle cerebral artery occlusion (pMCAO), and regional cerebral blood flow (rCBF), infarct volume and neurological deficits were measured 24 h later.

RESULTS

Infusion of Ang-(<em>1</em>-<em>7</em>) for 4 weeks significantly increased brain capillary density via promoting endothelial cell proliferation, which was accompanied by eNOS activation and up-regulation of NO and VEGF in brain. These effects were abolished by A-<em>7</em><em>7</em>9 or L-NIO. More importantly, Ang-(<em>1</em>-<em>7</em>) improved rCBF and decreased infarct volume and neurological deficits after pMCAO, which could be reversed by A-<em>7</em><em>7</em>9, L-NIO or endostatin.

CONCLUSIONS

This is the first evidence that Ang-(<em>1</em>-<em>7</em>) promotes brain angiogenesis via a Mas/eNOS-dependent pathway, which enhances tolerance against subsequent cerebral ischaemia. These findings highlight brain Ang-(<em>1</em>-<em>7</em>)/Mas signalling as a potential target in stroke prevention.

Hypertension is a major risk factor for stroke, coronary events, heart and renal failure, and the renin-<em>angiotensin</em> system (RAS) plays a major role in its pathogenesis. Within the RAS, <em>angiotensin</em> converting enzyme (ACE) converts <em>angiotensin</em> (Ang) I into the vasoconstrictor Ang II. An "alternate" arm of the RAS now exists in which ACE2 counterbalances the effects of the classic RAS through degradation of Ang II, and generation of the vasodilator Ang <em>1</em>-<em>7</em>. ACE2 is highly expressed in the heart, blood vessels, and kidney. The catalytically active ectodomain of ACE2 undergoes shedding, resulting in ACE2 in the circulation. The ACE2 gene maps to a quantitative trait locus on the X chromosome in three strains of genetically hypertensive rats, suggesting that ACE2 may be a candidate gene for hypertension. It is hypothesized that disruption of tissue ACE/ACE2 balance results in changes in blood pressure, with increased ACE2 expression protecting against increased blood pressure, and ACE2 deficiency contributing to hypertension. Experimental hypertension studies have measured ACE2 in either the heart or kidney and/or plasma, and have reported that deletion or inhibition of ACE2 leads to hypertension, whilst enhancing ACE2 protects against the development of hypertension, hence increasing ACE2 may be a therapeutic option for the management of high blood pressure in man. There have been relatively few studies of ACE2, either at the gene or the circulating level in patients with hypertension. Plasma ACE2 activity is low in healthy subjects, but elevated in patients with cardiovascular risk factors or cardiovascular disease. Genetic studies have investigated ACE2 gene polymorphisms with either hypertension or blood pressure, and have produced largely inconsistent findings. This review discusses the evidence regarding ACE2 in experimental hypertension models and the association between circulating ACE2 activity and ACE2 polymorphisms with blood pressure and arterial hypertension in man.

The octapeptide <em>angiotensin</em> II (Ang II) exerts a wide range of effects on the cardiovascular system but has also been implicated in the regulation of cell proliferation, fibrosis, and apoptosis. Ang II is formed by cleavage of Ang I by <em>angiotensin</em>-converting enzyme, but there is also evidence for non-<em>angiotensin</em>-converting enzyme-dependent conversion of Ang I to Ang II. Here we address the role of mast cell proteases in Ang II production by using two different mouse strains lacking mast cell heparin or mouse mast cell protease 4 (mMCP-4), the chymase that may be the functional homologue to human chymase. Ang I was added to ex vivo cultures of peritoneal cells, and the generation of Ang II and other metabolites was analyzed. Activation of mast cells resulted in marked increases in both the formation and subsequent degradation of Ang II, and both of these processes were strongly reduced in heparin-deficient peritoneal cells. In the mMCP-4(-/-) cell cultures no reduction in the rate of Ang II generation was seen, but the formation of Ang-(5-<em>1</em>0) was completely abrogated. Addition of a carboxypeptidase A (CPA) inhibitor to wild type cells caused complete inhibition of the formation of Ang-(<em>1</em>-9) and Ang-(<em>1</em>-<em>7</em>) but did not inhibit Ang II formation. However, when the CPA inhibitor was added to the mMCP-4(-/-) cultures, essentially complete inhibition of Ang II formation was obtained. Taken together, the results of this study indicate that mast cell chymase and CPA have key roles in both the generation and degradation of Ang II.

<em>Angiotensin</em>-converting enzyme 2 (ACE2) is a protein consisting of two domains, the N-terminus is a carboxypeptidase homologous to ACE and the C-terminus is homologous to collectrin and responsible for the trafficking of the neutral amino acid transporter B(0)AT<em>1</em> to the plasma membrane of gut epithelial cells. The carboxypeptidase domain not only metabolizes <em>angiotensin</em> II to <em>angiotensin</em>-(<em>1</em>-<em>7</em>), but also other peptide substrates, such as apelin, kinins and morphins. In addition, the collectrin domain regulates the levels of some amino acids in the blood, in particular of tryptophan. Therefore it is of no surprise that animals with genetic alterations in the expression of ACE2 develop a diverse pattern of phenotypes ranging from hypertension, metabolic and behavioural dysfunctions, to impairments in serotonin synthesis and neurogenesis. This review summarizes the phenotypes of such animals with a particular focus on the central nervous system.

ANG II plays a major role in renal water and sodium regulation. In the immortalized mouse renal collecting duct principal cells (mpkCCD(cl4)) cell line, we treated cells with ANG II and examined aquaporin-2 (AQP2) protein expression, trafficking, and mRNA levels, by immunoblotting, immunofluorescence, and RT-PCR. After 24-h incubation, ANG II-induced AQP2 protein expression was observed at the concentration of <em>1</em>0(-<em>1</em>0) M and increased in a dose-dependent manner. ANG II (<em>1</em>0(-<em>7</em>) M) increased AQP2 protein expression and mRNA levels at 0.5, <em>1</em>, 2, 6, and 24 h. Immunofluorescence studies showed that ANG II increased the apical membrane targeting of AQP2 from 30 min to 6 h. Next, the signaling pathways underlying the ANG II-induced AQP2 expression were investigated. The PKC inhibitor Ro 3<em>1</em>-8220 (5 × <em>1</em>0(-6) M) and the PKA inhibitor H89 (<em>1</em>0(-5) M) blocked ANG II-induced AQP2 expression, respectively. Calmodulin inhibitor W-<em>7</em> markedly reduced ANG II- and/or dDAVP-stimulated AQP2 expression. ANG II (<em>1</em>0(-9) M) and/or dDAVP (<em>1</em>0(-<em>1</em>0) M) stimulated AQP2 protein levels and cAMP accumulation, which was completely blocked by pretreatment with the vasopressin V2 receptor (V2R) antagonist SR<em>1</em>2<em>1</em>463B (<em>1</em>0(-8) M). Pretreatment with the <em>angiotensin</em> AT(<em>1</em>) receptor (AT<em>1</em>R) antagonist losartan (3 × <em>1</em>0(-6) M) blocked ANG II (<em>1</em>0(-9) M)-stimulated AQP2 protein expression and cAMP accumulation, and partially blocked dDAVP (<em>1</em>0(-<em>1</em>0) M)- and dDAVP+ANG II-induced AQP2 protein expression and cAMP accumulation. In conclusion, ANG II regulates AQP2 protein, trafficking, and gene expression in renal collecting duct principal cells. ANG II-induced AQP2 expression involves cAMP, PKC, PKA, and calmodulin signaling pathways via V2 and AT(<em>1</em>) receptors.

Characterization of C- and N-terminal forms of <em>angiotensin</em> (Ang) peptides mandated assessment of methods to determine plasma levels. <em>1</em>25I-Ang I, <em>1</em>25I-Ang II, and <em>1</em>25I-Ang(<em>1</em>-<em>7</em>) were added to blood samples in the presence of protease inhibitors. Ethylenediaminetetraacetic acid (EDTA) inhibited the conversion of <em>1</em>25I-Ang I to <em>1</em>25I-Ang II. o-Phenanthroline and EDTA (EDTA + o-Ph) did not eliminate [des-Asp<em>1</em>] fragments or <em>1</em>25I-Ang(<em>1</em>-<em>7</em>). The combination of EDTA + o-Ph and pepstatin A or 4-(chloromercuri) benzoic acid (PCMB) significantly reduced <em>1</em>25I-Ang(<em>1</em>-<em>7</em>) generation. Only PCMB plus EDTA + o-Ph eliminated [des-Asp<em>1</em>] fragments. Authentic plasma values of Ang peptides require the correct choice of protease inhibitors.

We evaluated the development of arterial hypertension, cardiac function, and collagen deposition, as well as the level of components of the renin-<em>angiotensin</em> system in the heart of transgenic rats that overexpress an <em>angiotensin</em> (Ang)-(<em>1</em>-<em>7</em>)-producing fusion protein, TGR(A<em>1</em>-<em>7</em>)3292 (TG), which induces a lifetime increase in circulating levels of this peptide. After 30 days of the induction of the deoxycorticosterone acetate (DOCA)-salt hypertension model, DOCA-TG rats were hypertensive but presented a lower systolic arterial pressure in comparison with DOCA-Sprague-Dawley (SD) rats. In contrast to DOCA-SD rats that presented left ventricle (LV) hypertrophy and diastolic dysfunction, DOCA-TG rats did not develop cardiac hypertrophy or changes in ventricular function. In addition, DOCA-TG rats showed attenuation in mRNA expression for collagen type I and III compared with the increased levels of DOCA-SD rats. Ang II plasma and LV levels were reduced in SD and TG hypertensive rats in comparison with normotensive animals. DOCA-TG rats presented a reduction in plasma Ang-(<em>1</em>-<em>7</em>) levels; however, there was a great increase in Ang-(<em>1</em>-<em>7</em>) ( approximately 3-fold) accompanied by a decrease in mRNA expression of both <em>angiotensin</em>-converting enzyme and <em>angiotensin</em>-converting enzyme 2 in the LV. The mRNA expression of Mas and Ang II type <em>1</em> receptors in the LV was not significantly changed in DOCA-SD or DOCA-TG rats. This study showed that TG rats with increased circulating levels of Ang-(<em>1</em>-<em>7</em>) are protected against cardiac dysfunction and fibrosis and also present an attenuated increase in blood pressure after DOCA-salt hypertension. In addition, DOCA-TG rats showed an important local increase in Ang-(<em>1</em>-<em>7</em>) levels in the LV, which might have contributed to the attenuation of cardiac dysfunction and prefibrotic lesions.

It is known that the endothelial function is compromised in atherosclerosis and arterial hypertension and that <em>angiotensin</em> is an important factor contributing to both pathophysiologies. The aim of this study was to evaluate the vascular function in a hypercholesterolemia/atherosclerosis model, in the <em>angiotensin</em> II-dependent 2-kidney <em>1</em>-clip (2K<em>1</em>C) hypertension model and when both conditions coexist. Eight-week-old apolipoprotein E knockout (apoE; n=20) and C5<em>7</em>BL/6 (C5<em>7</em>; n=20) mice underwent a 2K<em>1</em>C or sham operation and were studied 28 days later. Mean arterial pressure was higher in apoE-2K<em>1</em>C and C5<em>7</em>-2K<em>1</em>C (<em>1</em>26+/-3 and <em>1</em>28+/-3 mm Hg) when compared with the apoE-Sham and C5<em>7</em>-Sham (<em>1</em>03+/-2 and <em>1</em>04+/-2 mm Hg, respectively; P<0.05). The vascular reactivity to norepinephrine (NE; <em>1</em>0(-9) to 2 x <em>1</em>0(-3) mol/L), acetylcholine (ACh), and sodium nitroprusside (SNP; <em>1</em>0(-<em>1</em>0) to <em>1</em>0(-3) mol/L) was evaluated in the mesenteric arteriolar bed through concentration-effect curves. NE caused vascular hyper-reactivity in apoE-Sham, apoE-2K<em>1</em>C, and C5<em>7</em>-2K<em>1</em>C (maximal response <em>1</em>46+/-5, <em>1</em>44+/-5, and <em>1</em>59+/-4 mm Hg, respectively) compared with C5<em>7</em>-Sham (<em>1</em>22+/-<em>7</em> mm Hg; P<0.05). The ACh-induced relaxation was smaller (P<0.05) in apoE-2K<em>1</em>C and C5<em>7</em>-2K<em>1</em>C (maximal response 53+/-3% and 46+/-3%) than in apoE-Sham and C5<em>7</em>-Sham mice (<em>7</em>8+/-5% and <em>7</em>3+/-4%). SNP-induced vascular relaxation showed similar concentration-effect curves in all groups. We conclude that in C5<em>7</em>-2K<em>1</em>C mice, the increased reactivity to NE and the decreased endothelium-dependent relaxation contribute to the maintenance of hypertension. The apoE mouse, at early stages of atherosclerosis, shows hyper-reactivity to NE but does not have endothelium dysfunction yet. However, the concurrence of both pathophysiologies does not result in additive effects on the vascular function.

<em>Angiotensin</em> (Ang)-(<em>1</em>-<em>7</em>) is now recognized as a biologically active component of the renin-<em>angiotensin</em> system (RAS). The discovery of the <em>angiotensin</em>-converting enzyme homologue ACE2 revealed important metabolic pathways involved in the Ang-(<em>1</em>-<em>7</em>) synthesis. This enzyme can form Ang-(<em>1</em>-<em>7</em>) from Ang II or less efficiently through hydrolysis of Ang I to Ang-(<em>1</em>-9) with subsequent Ang-(<em>1</em>-<em>7</em>) formation. Additionally, it is well established that the G protein-coupled receptor Mas is a functional ligand site for Ang-(<em>1</em>-<em>7</em>). The axis formed by ACE2/Ang-(<em>1</em>-<em>7</em>)/Mas represents an endogenous counter regulatory pathway within the RAS whose actions are opposite to the vasoconstrictor/proliferative arm of the RAS constituted by ACE/Ang II/AT(<em>1</em>) receptor. In this review we will discuss recent findings concerning the biological role of the ACE2/Ang-(<em>1</em>-<em>7</em>)/Mas arm in the cardiovascular and pulmonary system. Also, we will highlight the initiatives to develop potential therapeutic strategies based on this axis.