In this study we describe a new <em>angiotensin</em> antagonist [Asp<em>1</em>-Arg2-Val3-Tyr4-Ile5-His6-D-Ala<em>7</em>, (A-<em>7</em><em>7</em>9)] selective for the heptapeptide <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)]. A-<em>7</em><em>7</em>9 blocked the antidiuretic effect of Ang-(<em>1</em>-<em>7</em>) in water-loaded rats and the changes in blood pressure produced by Ang-(<em>1</em>-<em>7</em>) microinjection into the dorsal-medial and ventrolateral medulla. In contrast, A-<em>7</em><em>7</em>9 did not change the dipsogenic, pressor, or myotropic effects of <em>angiotensin</em> II (Ang II). Also, A-<em>7</em><em>7</em>9 did not affect the antidiuretic effect of vasopressin or the contractile effects of <em>angiotensin</em> III, bradykinin, or substance P on the rat ileum. In the rostral ventrolateral medulla, the pressor effect produced by Ang-(<em>1</em>-<em>7</em>) microinjection was completely blocked by A-<em>7</em><em>7</em>9 but not by AT<em>1</em> or AT2 receptor antagonists (DUP <em>7</em>53 and CGP 42<em>1</em><em>1</em>2A, respectively). Conversely, the pressor effect produced by Ang II was not changed by A-<em>7</em><em>7</em>9 but was completely blocked by DUP <em>7</em>53. Binding studies substantiated these observations: A-<em>7</em><em>7</em>9 did not compete significantly for <em>1</em>25I-Ang II binding to adrenocortical membranes at up to a <em>1</em> microM concentration. Low affinity binding was also observed in adrenomedullary membranes with an IC50 greater than <em>1</em>0 microM. Our results show that A-<em>7</em><em>7</em>9 is a potent and selective antagonist for Ang-(<em>1</em>-<em>7</em>). More importantly, our data indicate that specific <em>angiotensin</em> receptors mediate the central and peripheral actions of Ang-(<em>1</em>-<em>7</em>).

Recently, we demonstrated that the heptapeptide <em>angiotensin</em>-(<em>1</em>-<em>7</em>) (Ang-[<em>1</em>-<em>7</em>]) exhibits a favorable kinetic of nitric oxide (NO) release accompanied by extremely low superoxide (O2-) production. In this report we describe AVE 099<em>1</em>, a novel nonpeptide compound that evoked effects similar to Ang-(<em>1</em>-<em>7</em>) on the endothelium. AVE 099<em>1</em> and unlabeled Ang-(<em>1</em>-<em>7</em>) competed for high-affinity binding of [<em>1</em>25I]-Ang-(<em>1</em>-<em>7</em>) to bovine aortic endothelial cell membranes with IC50 values of 2<em>1</em>+/-35 and 220+/-280 nmol/L, respectively. Stimulated NO and O2- release from bovine aortic endothelial cells was directly and simultaneously measured on the cell surface by selective electrochemical nanosensors. Peak concentrations of NO and O2- release by AVE 099<em>1</em> and Ang-(<em>1</em>-<em>7</em>) (both <em>1</em>0 micromol/L) were not significantly different (NO: 295+/-20 and 2<em>7</em>0+/-25 nmol/L; O2-: <em>1</em>8+/-2 and 20+/-4 nmol/L). However, the released amount of bioactive NO was approximately 5 times higher for AVE 099<em>1</em> in comparison to Ang-(<em>1</em>-<em>7</em>). The selective Ang-(<em>1</em>-<em>7</em>) antagonist [D-Ala(<em>7</em>)]-Ang-(<em>1</em>-<em>7</em>) inhibited the AVE 099<em>1</em>-induced NO and O2- production by approximately 50%. A similar inhibition level was observed for the Ang II AT<em>1</em> receptor antagonist EXP 3<em>1</em><em>7</em>4. In contrast, the Ang II AT2 receptor antagonist PD <em>1</em>23,<em>1</em><em>7</em><em>7</em> inhibited the AVE 099<em>1</em>-stimulated NO production by approximately 90% but without any inhibitory effect on O2- production. Both NO and O2- production were inhibited by NO synthase inhibition ( approximately <em>7</em>0%) and by bradykinin B2 receptor blockade (approximately 80%). AVE 099<em>1</em> efficiently mimics the effects of Ang-(<em>1</em>-<em>7</em>) on the endothelium, most probably through stimulation of a specific, endothelial Ang-(<em>1</em>-<em>7</em>)-sensitive binding site causing kinin-mediated activation of endothelial NO synthase.

In this study we investigated the effects of the genetic deletion of the <em>angiotensin</em> (Ang)-(<em>1</em>-<em>7</em>) receptor Mas on heart function. Localization of Mas in the mouse heart was evaluated by binding of rhodamine-labeled Ang-(<em>1</em>-<em>7</em>). Cardiac function was examined using isolated heart preparations. Echocardiography was used to confirm the results obtained with isolated heart studies. To elucidate the possible mechanisms involved in the cardiac phenotype observed in Mas(-/-) mice, whole-cell calcium currents in cardiomyocytes and the expression of collagen types I, III, and VI and fibronectin were analyzed. Ang-(<em>1</em>-<em>7</em>) binding showed that Mas is localized in cardiomyocytes of the mouse heart. Isolated heart techniques revealed that Mas-deficient mice present a lower systolic tension (average: <em>1</em>.4+/-0.09 versus 2.<em>1</em>+/-0.03 g in Mas(+/+) mice), +/-dT/dt, and heart rate. A significantly higher coronary vessel resistance was also observed in Mas-deficient mice. Echocardiography revealed that hearts of Mas-deficient mice showed a significantly decreased fractional shortening, posterior wall thickness in systole and left ventricle end-diastolic dimension, and a higher left ventricle end-systolic dimension. A markedly lower global ventricular function, as defined by a higher myocardial performance index, was observed. A higher delayed time to the peak of calcium current was also observed. The changes in cardiac function could be partially explained by a marked change in collagen expression to a profibrotic profile in Mas-deficient mice. These results indicate that Ang-(<em>1</em>-<em>7</em>)-Mas axis plays a key role in the maintenance of the structure and function of the heart.

In this study we evaluate the effects of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) on reperfusion arrhythmias in isolated rat hearts. Rat hearts were perfused according to Langendorff technique and maintained in heated (3<em>7</em>+/-<em>1</em> degrees C) and continuously gassed (95% O(2)/5% CO(2)) Krebs-Ringer solution at constant pressure (65 mm Hg). The electrical activity was recorded with an ECG (bipolar). Local ischemia was induced by coronary ligation for <em>1</em>5 minutes. After ischemia, hearts were reperfused for 30 minutes. Cardiac arrhythmias were defined as the presence of ventricular tachycardia and/or ventricular fibrillation after the ligation of the coronary artery was released. <em>Angiotensin</em> II (0.20 nmol/L, n=<em>1</em>0) produced a significant enhancement of reperfusion arrhythmias. On the other hand, Ang-(<em>1</em>-<em>7</em>) presented in the perfusion solution (0.22 nmol/L, n=<em>1</em><em>1</em>) reduced incidence and duration of arrhythmias. The antiarrhythmogenic effects of Ang-(<em>1</em>-<em>7</em>) was blocked by the selective Ang-(<em>1</em>-<em>7</em>) antagonist A-<em>7</em><em>7</em>9 (2 nmol/L, n=9) and by indomethacin pretreatment (5 mg/kg IP, n=8) but not by the bradykinin B(2) antagonist HOE <em>1</em>40 (<em>1</em>00 nmol/L, n=<em>1</em>0) or by L-NAME pretreatment (30 mg/kg IP, n=8). These results suggest that the antiarrhythmogenic effect of low concentrations of Ang-(<em>1</em>-<em>7</em>) is mediated by a specific receptor and that release of endogenous prostaglandins.by Ang-(<em>1</em>-<em>7</em>) contributes to the alleviation of reversible and/or irreversible ischemia-reperfusion injury.

Preeclampsia (PE) affects 5-<em>7</em>% of all pregnancies in the United States and is the leading cause of maternal and prenatal morbidity. PE is associated with hypertension after week 20 of gestation, decreased renal function and small-for-gestational-age babies. Women with PE exhibit chronic inflammation and production of autoantibodies. It is hypothesized that during PE, placental ischaemia occurs as a result of shallow trophoblast invasion which is associated with an immune imbalance where pro-inflammatory CD4(+) T-cells are increased and T regulatory cells (Tregs) are decreased. This imbalance leads to chronic inflammation characterized by oxidative stress, pro-inflammatory cytokines and autoantibodies. Studies conducted in our laboratory have demonstrated the importance of this immune imbalance in causing hypertension in response to placental ischaemia in pregnant rats. These studies confirm that increased CD4(+) T-cells and decreased Tregs during pregnancy leads to elevated inflammatory cytokines, endothelin (ET-<em>1</em>), reactive oxygen species (ROS) and agonistic autoantibodies to the <em>angiotensin</em> II (Ang II), type <em>1</em> receptor (AT<em>1</em>-AA). All of these factors taken together play an important role in increasing the blood pressure during pregnancy. Specifically, this review focuses on the decrease in Tregs, and their associated regulatory cytokine interleukin (IL)-<em>1</em>0, which is seen in response to placental ischaemia during pregnancy. This study will also examine the effect of regulatory immune cell repopulation on the pathophysiology of PE. These studies show that restoring the balance of the immune system through increasing Tregs, either by adoptive transfer or by infusing IL-<em>1</em>0, reduces the blood pressure and pathophysiology associated with placental ischaemia in pregnant rats.

Activated pancreatic stellate cells (PSCs) play an important role in pancreatic fibrosis and inflammation, where oxidative stress is implicated in the pathogenesis. NADPH oxidase might be a source of reactive oxygen species (ROS) in the injured pancreas. This study aimed to clarify the expression and regulation of cell functions by NADPH oxidase in PSCs. PSCs were isolated from rat and human pancreas tissues. Expression of NADPH oxidase was assessed by reverse transcription-PCR and immunostaining. Intracellular ROS production was assessed using 2',<em>7</em>'-dichlorofluorescin diacetate. The effects of diphenylene iodonium (DPI) and apocynin, inhibitors of NADPH oxidase, on key parameters of PSC activation were evaluated in vitro. In vivo, DPI (at <em>1</em> mg.kg body wt(-<em>1</em>).day(-<em>1</em>)) was administered in drinking water to <em>1</em>0-wk-old male Wistar Bonn/Kobori rats for <em>1</em>0 wk and to rats with chronic pancreatitis induced by dibutyltin dichloride (DBTC). PSCs expressed key components of NADPH oxidase (p22(phox), p4<em>7</em>(phox), NOX<em>1</em>, gp9<em>1</em>(phox)/NOX2, NOX4, and NOX activator <em>1</em>). PDGF-BB, IL-<em>1</em>beta, and <em>angiotensin</em> II induced ROS production, which was abolished by DPI and apocynin. DPI inhibited PDGF-induced proliferation, IL-<em>1</em>beta-induced chemokine production, and expression of alpha-smooth muscle actin and collagen. DPI inhibited transformation of freshly isolated cells to a myofibroblast-like phenotype. In addition, DPI inhibited the development of pancreatic fibrosis in Wistar Bonn/Kobori rats and in rats with DBTC-induced chronic pancreatitis. In conclusion, PSCs express NADPH oxidase to generate ROS, which mediates key cell functions and activation of PSCs. NADPH oxidase might be a potential target for the treatment of pancreatic fibrosis.

The initiating event in preeclampsia is thought be to reduced uteroplacental perfusion. Although we have reported previously that chronic reductions in uterine perfusion pressure (RUPP) in pregnant rats results in hypertension and enhanced endothelin production, the factors linking placental ischemia and endothelial cell activation remain unclear. The purpose of this study was to determine the role of <em>angiotensin</em> II type-<em>1</em> (AT<em>1</em>) receptor activation on endothelin production induced by serum from pregnant rats exposed to reductions in uterine perfusion. To achieve this goal, human umbilical vein endothelial cells were exposed to sera collected from RUPP rats or normal pregnant rats. Arterial pressure was significantly higher in RUPP rats (<em>1</em>35+/-2 mm Hg) than in pregnant rats (<em>1</em>06+/-<em>1</em> mm Hg). Six hours after exposure to RUPP serum (n=<em>1</em><em>7</em>), cell media endothelin concentration was <em>1</em>8.4+/-2.<em>7</em> pg/mL as compared with 9.22+/-<em>1</em>.3 pg/mL from cells exposed to serum from normal pregnant rats (n=9). Eighteen hours after exposure to RUPP serum (n=<em>7</em>), endothelin concentration was 30.5+/-3.8 pg/mL as compared with <em>1</em>2.8+/-5.3 pg/mL from cells exposed to normal pregnant rat serum (n=6). In contrast, serum from RUPP rats did not increase endothelin production in human umbilical vein endothelial cells pretreated with an AT<em>1</em> receptor antagonist, losartan (<em>1</em>5 micromol/L). Eighteen hours after exposure to RUPP serum and losartan (n=<em>1</em>4), endothelin concentration was 2<em>1</em>.3+/-2.2 pg/mL as compared with <em>1</em>6.4+/-3.3 pg/mL from cells exposed to normal pregnant rat serum and losartan (n=<em>1</em>0). These data indicate that serum from pregnant rats exposed to reductions in uterine perfusion enhances endothelin production by endothelial cells via by AT<em>1</em> receptor activation.

There is increasing evidence that the renin-<em>angiotensin</em> system may play a important role in cardiac hypertrophy. To assess the role of <em>angiotensin</em> II in the induction of cardiac hypertrophy, three groups of adult mice were subjected to left ventricular pressure overload by transverse aortic constriction (TAC). For the next <em>7</em> days the groups received either the specific <em>angiotensin</em> II subtype <em>1</em> receptor (AT<em>1</em>) antagonist (losartan, <em>1</em>.05 g/l; n = <em>1</em><em>7</em>), an <em>angiotensin</em> enzyme inhibitor (captopril, 2 g/l; n = <em>1</em><em>7</em>), or no treatment (n = 22) administered in the drinking water and compared with three similarly treated sham-operated groups (n = <em>7</em> each). TAC resulted in a significant increase in heart weight-to-body weight ratio (0.634 +/- 0.08<em>7</em> vs. 0.525 +/- 0.039, g/g x <em>1</em>00, P < 0.05), which was prevented by losartan (0.506 +/- 0.069, g/g x <em>1</em>00, P < 0.000<em>1</em>) despite similar hemodynamic load (proximal systolic pressure <em>1</em>46 +/- 3<em>1</em> vs. <em>1</em>36 +/- 32 mmHg, untreated vs. losartan, P = NS). Proximal systolic pressure was positively correlated with the development of ventricular hypertrophy. In the presence of AT<em>1</em>-receptor blockade, the increase in heart weight-to-body weight ratio at any given systolic pressure was significantly attenuated compared with untreated TAC mice. The increase in heart weight-to-body weight ratio was also significantly attenuated by captopril compared with untreated banded controls (0.542 +/- 0.09<em>1</em>, g/g x <em>1</em>00, P = 0.0<em>1</em>).(ABSTRACT TRUNCATED AT 250 WORDS)

In view of the pro-oxidant and proinflammatory effects of <em>angiotensin</em> II, we have tested the hypothesis that valsartan, an <em>angiotensin</em> receptor blocker, may exert a suppressive action on reactive oxygen species (ROS) generation, nuclear factor kappa B (NF-kappa B) in mononuclear cells. Four groups of eight normal subjects were given <em>1</em>) <em>1</em>60 mg daily of valsartan, 2) 80 mg daily of simvastatin, 3) 40 mg quinapril, or 4) no treatment. Fasting blood samples were obtained before treatment and at d <em>1</em>, 8, and <em>1</em>4 (<em>7</em> d after the cessation of the drug). After valsartan, ROS generation by polymorphonuclear cells and mononuclear cells fell significantly by more than 40% (P < 0.0<em>1</em>). NF-kappa B binding activity and the expression of total cellular p65, a protein component of NF-kappa B, fell significantly (P < 0.0<em>1</em>). The expression of inhibitor kappa B (I kappa B) increased significantly (P < 0.05). Plasma C-reactive protein (CRP) concentration fell significantly (P < 0.0<em>1</em>). All indices, except I kappa B, reverted toward baseline, <em>7</em> d after the cessation of the drug. I kappa B persisted in an elevated state. Neither quinapril nor simvastatin given for <em>7</em> d produced a suppression of ROS generation, intranuclear NF-kappa B, p65, or CRP, and these two agents did not alter cellular I kappa B either. The untreated controls also did not demonstrate a change in their ROS generation or NF-kappa B binding activity or plasma CRP concentration. We conclude that valsartan at a modest dose exerts a profound and rapid ROS and inflammation-suppressive effect that may be relevant to its potential beneficial effects in atherosclerosis, diabetes, and congestive cardiac failure. In contrast, quinapril and simvastatin produced no similar effect over the period of <em>1</em> wk. Our observations may also have implications to clinical situations in which a rapid antiinflammatory effect is required.

Aerobic exercise training leads to a physiological, nonpathological left ventricular hypertrophy; however, the underlying biochemical and molecular mechanisms of physiological left ventricular hypertrophy are unknown. The role of microRNAs regulating the classic and the novel cardiac renin-<em>angiotensin</em> (Ang) system was studied in trained rats assigned to 3 groups: (<em>1</em>) sedentary; (2) swimming trained with protocol <em>1</em> (T<em>1</em>, moderate-volume training); and (3) protocol 2 (T2, high-volume training). Cardiac Ang I levels, Ang-converting enzyme (ACE) activity, and protein expression, as well as Ang II levels, were lower in T<em>1</em> and T2; however, Ang II type <em>1</em> receptor mRNA levels (69% in T<em>1</em> and 99% in T2) and protein expression (240% in T<em>1</em> and 300% in T2) increased after training. Ang II type 2 receptor mRNA levels (220%) and protein expression (332%) were shown to be increased in T2. In addition, T<em>1</em> and T2 were shown to increase ACE2 activity and protein expression and Ang (<em>1</em>-<em>7</em>) levels in the heart. Exercise increased microRNA-2<em>7</em>a and 2<em>7</em>b, targeting ACE and decreasing microRNA-<em>1</em>43 targeting ACE2 in the heart. Left ventricular hypertrophy induced by aerobic training involves microRNA regulation and an increase in cardiac Ang II type <em>1</em> receptor without the participation of Ang II. Parallel to this, an increase in ACE2, Ang (<em>1</em>-<em>7</em>), and Ang II type 2 receptor in the heart by exercise suggests that this nonclassic cardiac renin-<em>angiotensin</em> system counteracts the classic cardiac renin-<em>angiotensin</em> system. These findings are consistent with a model in which exercise may induce left ventricular hypertrophy, at least in part, altering the expression of specific microRNAs targeting renin-<em>angiotensin</em> system genes. Together these effects might provide the additional aerobic capacity required by the exercised heart.

Oxidative stress in the central nervous system mediates the increase in sympathetic tone that precedes the development of hypertension. We hypothesized that by transforming <em>Angiotensin</em>-II (AngII) into Ang-(<em>1</em>-<em>7</em>), ACE2 might reduce AngII-mediated oxidative stress in the brain and prevent autonomic dysfunction. To test this hypothesis, a relationship between ACE2 and oxidative stress was first confirmed in a mouse neuroblastoma cell line (Neuro2A cells) treated with AngII and infected with Ad-hACE2. ACE2 overexpression resulted in a reduction of reactive oxygen species (ROS) formation. In vivo, ACE2 knockout (ACE2(-/y)) mice and non-transgenic (NT) littermates were infused with AngII (<em>1</em>0 days) and infected with Ad-hACE2 in the paraventricular nucleus (PVN). Baseline blood pressure (BP), AngII and brain ROS levels were not different between young mice (<em>1</em>2 weeks). However, cardiac sympathetic tone, brain NADPH oxidase and SOD activities were significantly increased in ACE2(-/y). Post infusion, plasma and brain AngII levels were also significantly higher in ACE2(-/y), although BP was similarly increased in both genotypes. ROS formation in the PVN and RVLM was significantly higher in ACE2(-/y) mice following AngII infusion. Similar phenotypes, i.e. increased oxidative stress, exacerbated dysautonomia and hypertension, were also observed on baseline in mature ACE2(-/y) mice (48 weeks). ACE2 gene therapy to the PVN reduced AngII-mediated increase in NADPH oxidase activity and normalized cardiac dysautonomia in ACE2(-/y) mice. Altogether, these data indicate that ACE2 gene deletion promotes age-dependent oxidative stress, autonomic dysfunction and hypertension, while PVN-targeted ACE2 gene therapy decreases ROS formation via NADPH oxidase inhibition and improves autonomic function. Accordingly, ACE2 could represent a new target for the treatment of hypertension-associated dysautonomia and oxidative stress.

Adipose tissue is the largest endocrine organ, producing various adipokines and many other substances. Almost all blood vessels are surrounded by perivascular adipose tissue (PVAT), which has not received research attention until recently. This review will discuss the paracrine actions of PVAT on the growth of underlying vascular smooth muscle cells (VSMCs). PVAT can release growth factors and inhibitors. Visfatin is the first identified growth factor derived from PVAT. Decreased adiponectin and increased tumour necrosis factor-α in PVAT play a pathological role for neointimal hyperplasia after endovascular injury. PVAT-derived <em>angiotensin</em> II, <em>angiotensin</em> <em>1</em>-<em>7</em>, reactive oxygen species, complement component 3, NO and H(2) S have a paracrine action on VSMC contraction, endothelial or fibroblast function; however, their paracrine actions on VSMC growth remain to be directly verified. Factors such as monocyte chemoattractant protein-<em>1</em>, interleukin-6, interleukin-8, leptin, resistin, plasminogen activator inhibitor type-<em>1</em>, adrenomedullin, free fatty acids, glucocorticoids and sex hormones can be released from adipose tissue and can regulate VSMC growth. Most of them have been verified for their secretion by PVAT; however, their paracrine functions are unknown. Obesity, vascular injury, aging and infection may affect PVAT, causing adipocyte abnormality and inflammatory cell infiltration, inducing imbalance of PVAT-derived growth factors and inhibitors, leading to VSMC growth and finally resulting in development of proliferative vascular disease, including atherosclerosis, restenosis and hypertension. In the future, using cell-specific gene interventions and local treatments may provide definitive evidence for identification of key factor(s) involved in PVAT dysfunction-induced vascular disease and thus may help to develop new therapies.

BACKGROUND

This article is part of a themed section on Fat and Vascular Responsiveness. To view the other articles in this section visit http://dx.doi.org/<em>1</em>0.<em>1</em><em>1</em><em>1</em><em>1</em>/bph.20<em>1</em>2.<em>1</em>65.issue-3.

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] is an endogenous peptide of the renin-<em>angiotensin</em> system with vasodilator and antiproliferative properties. Our previous studies showed that Ang-(<em>1</em>-<em>7</em>) reduced serum-stimulated growth of human lung cancer cells in vitro through activation of a unique AT((<em>1</em>-<em>7</em>)) receptor. The current study investigates the effect of Ang-(<em>1</em>-<em>7</em>) on lung tumor growth in vivo, using a human lung tumor xenograft model. Athymic mice with tumors resulting from injection of A549 human lung cancer cells were treated for 28 days with either i.v. saline or Ang-(<em>1</em>-<em>7</em>), delivered by implanted osmotic mini-pumps. Treatment with Ang-(<em>1</em>-<em>7</em>) reduced tumor volume by 30% compared with the size before treatment; in contrast, tumor size in the saline-treated animals increased 2.5-fold. These results correlate with a reduction in the proliferation marker Ki6<em>7</em> in the Ang-(<em>1</em>-<em>7</em>)-infused tumors when compared with the saline-infused tumor tissues. Treatment with Ang-(<em>1</em>-<em>7</em>) significantly reduced cyclooxygenase-2 (COX-2) mRNA and protein in tumors of Ang-(<em>1</em>-<em>7</em>)-infused mice when compared with mice treated with saline as well as in the parent A549 human lung cancer cells in tissue culture. These results suggest that Ang-(<em>1</em>-<em>7</em>) may decrease COX-2 activity and proinflammatory prostaglandins to inhibit lung tumor growth. In contrast, the heptapeptide had no effect on COX-<em>1</em> mRNA in xenograft tumors or A549 cells. Because Ang-(<em>1</em>-<em>7</em>), a peptide with antithrombotic properties, reduces growth through activation of a selective AT((<em>1</em>-<em>7</em>)) receptor, our results suggest that the heptapeptide represents a novel treatment for lung cancer by reducing COX-2.

OBJECTIVE

We investigated the vascular effects of candesartan in hypertensive patients.

BACKGROUND

The renin-angiotensin system may contribute to atherogenesis through the promotion of endothelial dysfunction. The plausible mechanisms are that angiotensin II promotes superoxide anion generation, endothelial dysfunction, inflammation, and impaired fibrinolysis. The effects of candesartan on these conditions have not been clearly observed.

METHODS

We administered placebo or candesartan 16 mg daily during two months to 45 patients with mild-to-moderate hypertension. This was a randomized, double-blind, placebo-controlled, crossover study in design.

RESULTS

Candesartan did not significantly change lipoprotein levels. However, compared with placebo, candesartan significantly reduced plasma levels of malondialdehyde from 1.50 +/- 0.07 to 1.29 +/- 0.09 microM (p = 0.009); improved the percent flow-mediated dilator response to hyperemia from 5.17 +/- 0.24 to 6.22 +/- 0.26% (p < 0.001); and, furthermore, reduced plasma levels of monocyte chemoattractant protein (MCP-1) from 213 +/- 8 to 190 +/- 7 pg/ml (p = 0.003), tumor necrosis factor-alpha from 2.93 to 2.22 pg/ml (p = 0.026), and plasminogen activator inhibitor type 1 from 74 +/- 4 to 53 +/- 4 ng/ml (p < 0.001) but not C-reactive protein (CRP), matrix metalloproteinase protein, and fibrinogen. There were no significant correlations between these changes and reduction of systolic blood pressure (BP) (-0.247 < or = r < or = 0.195) and between these changes and reduction of diastolic BP (-0.262 < or = r < or = 0.197). There were no significant correlations between markers of inflammation and flow-mediated dilation percent or reduction of oxidant stress (-0.119 < or = r < or = 0.127). Furthermore, we observed no significant correlations between CRP and MCP-1 levels (r = -0.162).

CONCLUSIONS

Inhibition of the angiotensin II type 1 (AT1) receptor in hypertensive patients reverses endothelial dysfunction, measured as an improvement in flow-mediated dilation and fibrinolysis and reduction of oxidant stress and inflammatory cytokines, suggesting that AT1 receptor blocker therapy has antiatherogenic effects.

BACKGROUND

<em>Angiotensin</em> II may contribute to atherogenesis by facilitating the proliferative and inflammatory response to hypercholesterolemia. This study determined, in a primate model of diet-induced atherosclerosis, the effect of AT(<em>1</em>) blockade on fatty-streak formation, plasma lipids, and surrogate markers of vascular injury.

RESULTS

Male cynomolgus monkeys fed a diet containing 0.06<em>7</em> mg cholesterol/kJ for 20 weeks were given losartan (<em>1</em>80 mg/d, n=6) or vehicle (n=8) for 6 weeks starting at week <em>1</em>2 of the dietary regimen. Arterial pressure, heart rate, plasma total and lipoprotein cholesterol concentrations, and lipoprotein particle sizes and subclass distributions were unaffected by treatment. Losartan caused significant (P<0.05) increases in plasma <em>angiotensin</em> II and <em>angiotensin</em>-(<em>1</em>-<em>7</em>). Compared with vehicle-treated controls, losartan reduced the extent of fatty streak in the aorta, the coronary arteries, and the carotid arteries by approximately 50% (P<0.05). A significant (P<0.05) reduction in the susceptibility of LDL to in vitro oxidation, serum levels of monocyte chemoattractant protein-<em>1</em>, and circulating monocyte CD<em>1</em><em>1</em>b expression were also associated with losartan treatment. In addition, serum levels of vascular cell adhesion molecule-<em>1</em> and E-selectin did not change during treatment but increased after discontinuation of losartan. Serum C-reactive protein, platelet aggregability, and white cell counts were not modified by losartan.

CONCLUSIONS

This study demonstrates for the first time an antiatherogenic effect of AT(<em>1</em>) receptor blockade in nonhuman primates. Losartan inhibited fatty-streak formation through mechanisms that may include protection of LDL from oxidation and suppression of vascular monocyte activation and recruitment factors.

BACKGROUND

Aldosterone and angiotensin (Ang) II both may cause organ damage. Circulating aldosterone is produced in the adrenals; however, local cardiac synthesis has been reported. Aldosterone concentrations depend on the activity of aldosterone synthase (CYP11B2). We tested the hypothesis that reducing aldosterone by inhibiting CYP11B2 or by adrenalectomy (ADX) may ameliorate organ damage. Furthermore, we investigated how much local cardiac aldosterone originates from the adrenal gland.

RESULTS

We investigated the effect of the CYP11B2 inhibitor FAD286, losartan, and the consequences of ADX in transgenic rats overexpressing both the human renin and angiotensinogen genes (dTGR). dTGR-ADX received dexamethasone and 1% salt. Dexamethasone-treated dTGR-salt served as a control group in the ADX protocol. Untreated dTGR developed hypertension and cardiac and renal damage and had a 40% mortality rate (5/13) at 7 weeks. FAD286 reduced mortality to 10% (1/10) and ameliorated cardiac hypertrophy, albuminuria, cell infiltration, and matrix deposition in the heart and kidney. FAD286 had no effect on blood pressure at weeks 5 and 6 but slightly reduced blood pressure at week 7 (177+/-6 mm Hg in dTGR+FAD286 and 200+/-5 mm Hg in dTGR). Losartan normalized blood pressure during the entire study. Circulating and cardiac aldosterone levels were reduced in FAD286 or losartan-treated dTGR. ADX combined with dexamethasone and salt treatment decreased circulating and cardiac aldosterone to barely detectable levels. At week 7, ADX-dTGR-dexamethasone-salt had a 22% mortality rate compared with 73% in dTGR-dexamethasone-salt. Both groups were similarly hypertensive (190+/-9 and 187+/-4 mm Hg). In contrast, cardiac hypertrophy index, albuminuria, cell infiltration, and matrix deposition were significantly reduced after ADX (P<0.05).

CONCLUSIONS

Aldosterone plays a key role in the pathogenesis of Ang II-induced organ damage. Both FAD286 and ADX reduced circulating and cardiac aldosterone levels. The present results show that aldosterone produced in the adrenals is the main source of cardiac aldosterone.

Tissue and plasma forms of <em>angiotensin</em> (Ang) peptides were characterized by reverse-phase high performance liquid chromatography and three specific radioimmunoassays. This method allowed resolution of <em>1</em>0 Ang peptides and revealed distinctive distributions for the three principal Ang peptides in the brain, adrenal gland, and plasma. In extracts from the rat hypothalamus, approximately equimolar amounts of Ang-(<em>1</em>-<em>7</em>), Ang-II, and Ang-I were detected (<em>1</em>.<em>1</em>0, <em>1</em>.<em>1</em>8, and <em>1</em>.45 pmol/g of tissue, respectively). A similar profile was observed in the medulla oblongata and amygdala, although the content of these three peptides was 40-<em>7</em>0% less than that seen in the hypothalamus. In the adrenal gland, the predominant peptide was Ang-II (<em>1</em>.0<em>7</em> pmol/g); levels of Ang-(<em>1</em>-<em>7</em>) (0.<em>1</em>9 pmol/g) and Ang-I (0.<em>1</em>4 pmol/g) were approximately 20% that of Ang-II. In plasma, the major <em>angiotensin</em> was Ang-I (0.<em>1</em>3 pmol/ml), with lower levels of Ang-(<em>1</em>-<em>7</em>) and Ang-II (0.0<em>1</em>-0.02 pmol/ml). This study is the first demonstration of the endogenous presence of Ang-(<em>1</em>-<em>7</em>) in central and peripheral tissues of the rat. Moreover, the data suggest tissue-specific processing of <em>angiotensins</em>, with Ang-(<em>1</em>-<em>7</em>) being a predominant Ang peptide in the central nervous system. In light of the recent biological properties described for this peptide, Ang-(<em>1</em>-<em>7</em>) may represent an active member of Ang peptides in the brain.

The present study determined the effect of either occlusion of the left renal artery for 60 min (ischemia) or sham operation on <em>angiotensin</em> (ANG) receptors and tissue and urinary levels of ANG peptides between 24 and <em>7</em>2 h recovery in male Sprague-Dawley rats. At 24 h postischemia, urinary concentrations of ANG I and ANG-(<em>1</em>-<em>7</em>) rose by an average of 83 and 64%, respectively (P < 0.05) but had declined to control levels by <em>7</em>2 h. Tissue ANG II rose at 24 h in postischemic kidneys by an average of 63% compared with the contralateral nonischemic kidney (P < 0.05). Whereas the enzymatic activity of <em>angiotensin</em>-converting enzyme and neprilysin was reduced after ischemia, renal renin activity in ischemic kidneys rose by <em>7</em>4% compared with sham-operated kidneys. Receptor autoradiography using (<em>1</em>25)I-labeled [Sar(<em>1</em>),Thr(8)]ANG II ((<em>1</em>25)I-Sarthran) (0.8 nM) revealed a decreased apparent density of ANG receptors (>80% AT(<em>1</em>)) in ischemic kidneys with a trend for a decrease in the contralateral nonischemic kidneys compared with the kidneys from sham-operated rats. Twenty-four hours after ischemia, ANG II receptors decreased by 68% in glomeruli (P < 0.05), 49% in the outer cortical tubulointerstitial area (P < 0.05), and 48% in the inner cortical-outer medullary area of the vasa recta (P < 0.05). Medullary binding decreased approximately 50% in both the ischemic kidney and the contralateral nonischemic kidney compared with sham. In all regions of the ischemic kidney, receptors recovered by <em>7</em>2 h to levels not different from sham control rats. The marked change in urinary ANG I and ANG-(<em>1</em>-<em>7</em>) at 24 h following occlusion indicates these peptides may be potential urinary markers for acute renal ischemia. The reduction of receptors in vascular and tubular regions of the ischemic kidney provides a mechanism for the loss of vasoconstrictor responses to ANG II following ischemia previously reported by others.

<em>1</em>. Intravenous infusion of <em>angiotensin</em> causes rats which are in water balance to drink water.2. The mean amount of <em>angiotensin</em> needed to initiate drinking was 29.<em>1</em> +/- 4.6 mug/kg (S.E. of mean) in twenty normal rats, and <em>1</em>5.<em>7</em> +/- 2.<em>1</em> mug/kg in thirty-four nephrectomized rats.3. The nephrectomized rat is therefore more sensitive to this action of <em>angiotensin</em> than the rat with intact kidneys.4. The rates of infusion (0.05-3.0 mug/kg(-<em>1</em>) min(-<em>1</em>)) which cause drinking are comparable to those used to produce other effects in rats.5. <em>Angiotensin</em> restores the drinking response of the nephrectomized rat subjected to caval ligation to a value similar to that obtained in the uninfused normal rat subjected to caval ligation.6. The effects of <em>angiotensin</em> and hypertonic saline on drinking are additive when both substances are administered to nephrectomized rats.<em>7</em>. These experiments provide further support for the view that the renin-<em>angiotensin</em> system is concerned in extracellular thirst.

<em>Angiotensin</em> (Ang) peptides play a critical role in regulating vascular reactivity and structure. We showed that Ang-(<em>1</em>-<em>7</em>) 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-(<em>1</em>-<em>7</em>) in cultured rat aortic VSMCs. Ang-(<em>1</em>-<em>7</em>) caused a dose-dependent release of prostacyclin from VSMCs, with a maximal release of 2<em>7</em><em>7</em>.9+/-25.2% of basal values (P<0.05) by <em>1</em>00 nmol/L Ang-(<em>1</em>-<em>7</em>). The cyclooxygenase inhibitor indomethacin significantly attenuated growth inhibition by Ang-(<em>1</em>-<em>7</em>). In contrast, neither a lipoxygenase inhibitor nor a cytochrome p450 epoxygenase inhibitor prevented the antiproliferative effects of Ang-(<em>1</em>-<em>7</em>). These results suggest that Ang-(<em>1</em>-<em>7</em>) inhibits vascular growth by releasing prostacyclin. Ang-(<em>1</em>-<em>7</em>) 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-(<em>1</em>-<em>7</em>)-mediated inhibition of serum-stimulated thymidine incorporation. Finally, Ang-(<em>1</em>-<em>7</em>) inhibited Ang II stimulation of mitogen-activated protein kinase activities (ERK<em>1</em>/2). Incubation of VSMCs with concentrations of Ang-(<em>1</em>-<em>7</em>) up to <em>1</em> micromol/L had no effect on ERK<em>1</em>/2 activation. However, preincubation with increasing concentrations of Ang-(<em>1</em>-<em>7</em>) caused a dose-dependent reduction in Ang II-stimulated ERK<em>1</em>/2 activities. Ang-(<em>1</em>-<em>7</em>) (<em>1</em> micromol/L) reduced <em>1</em>00 nmol/L Ang II-stimulated ERK<em>1</em> and ERK2 activation by 42.3+/-6.2% and 4<em>1</em>.2+/-4.2%, respectively (P<0.0<em>1</em>). These results suggest that Ang-(<em>1</em>-<em>7</em>) 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.

Hypertension in rats with chronic placental ischemia (reduced uterine perfusion pressure, RUPP) is associated with elevated inflammatory cytokines, agonistic autoantibodies to the <em>angiotensin</em> II type I receptor (AT<em>1</em>-AA) and CD4(+) T cells; all of which are elevated in preclamptic women. Additionally, we have shown that adoptive transfer of RUPP CD4(+) T cells increases blood pressure, inflammatory cytokines, and sFlt-<em>1</em>. The objective of this study was to determine the long-term effects of RUPP CD4(+) T cells on AT<em>1</em>-AA, renal and systemic hemodynamics in pregnant rats. To answer this question CD4(+) T splenocytes were magnetically isolated on day <em>1</em>9 of gestation from control RUPP and normal pregnant (NP) rats and injected into a new group of NP rats at day <em>1</em>3 of gestation. On day <em>1</em>9 of gestation mean arterial pressure (MAP) and renal function (glomerular filtration rates, GFR) were analyzed and serum collected for AT<em>1</em>-AA analysis. To determine a role for AT<em>1</em>-AA to mediate RUPP CD4(+) T cell-induced blood pressure increases, MAP was analyzed in a second group of rats treated with AT<em>1</em> receptor blockade losartan (<em>1</em>0 mg·kg(-<em>1</em>)·day(-<em>1</em>)) and in a third group of rats treated with rituximab, a B cell-depleting agent (250 mg/kg) we have shown previously to decrease AT<em>1</em>-AA production in RUPP rats. MAP increased from <em>1</em>0<em>1</em> ± 2 mmHg NP to <em>1</em>26 ± 2 mmHg in RUPP rats (P < 0.00<em>1</em>) and to <em>1</em>23 ± <em>1</em> mmHg in NP rats injected with RUPP CD4(+) T cells (NP+RUPP CD4(+)T cells) (P < 0.00<em>1</em>). Furthermore, GFR decreased from 2.2 ml/min (n = <em>7</em>) in NP rats to <em>1</em>.0 ml/min (n = 5) NP+RUPP CD4(+)T cell. Circulating AT<em>1</em>-AA increased from 0.22 ± 0.<em>1</em> units in NP rats to <em>1</em>3 ± 0.<em>7</em> (P < 0.00<em>1</em>) units in NP+RUPP CD4(+)T cell-treated rats but decreased to 8.34 ± <em>1</em> beats/min in NP+RUPP CD4(+) T cells chronically treated with rituximab. Hypertension in NP+RUPP CD4(+)T cell group was attenuated by losartan (<em>1</em>02 ± 4 mmHg) and with B cell depletion (<em>1</em>0<em>1</em> ± 5 mmHg). Therefore, we conclude that one mechanism of hypertension in response to CD4(+) T lymphocytes activated during placental ischemia is via AT<em>1</em> receptor activation, potentially via AT<em>1</em>-AA during pregnancy.

Preeclampsia is associated with autoimmune cells T(H)<em>1</em><em>7</em>, secreting interleukin-<em>1</em><em>7</em>, autoantibodies activating the <em>angiotensin</em> II type I receptor (AT<em>1</em>-AA), and placental oxidative stress (ROS). The objective of our study was to determine whether chronic IL-<em>1</em><em>7</em> increases blood pressure by stimulating ROS and AT<em>1</em>-AAs during pregnancy. To answer this question four groups of rats were examined: normal pregnant (NP, n = 20), NP+IL-<em>1</em><em>7</em> (n = <em>1</em>2), NP+tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) (n = <em>7</em>) (a superoxide dismutase mimetic that scavenges ROS), and NP+IL-<em>1</em><em>7</em>+tempol (n = <em>1</em><em>1</em>). IL-<em>1</em><em>7</em> (<em>1</em>50 pg/day) was infused into NP rats while tempol was administered via the drinking water ad libitum. On day <em>1</em>9 blood pressure (MAP) was recorded, and plasma, urine, and tissue were collected for isolation of ROS detected by chemilluminescent technique. Urinary isoprostane was measured by ELISA. AT<em>1</em>-AAs were determined via cardiomyocyte assay and expressed as beats per minute. MAP increased from 98 ± 3 mmHg in NP to <em>1</em>23 ± 3 mmHg in IL-<em>1</em><em>7</em>-infused NP rats. Urinary isoprostane increased from <em>1</em>,029 ± <em>1</em> in NP to 3,526 ± 2 pg·mg(-<em>1</em>)·day(-<em>1</em>) in IL-<em>1</em><em>7</em>-infused rats (P < 0.05). Placental ROS was 436 ± 4 RLU·ml(-<em>1</em>)·min(-<em>1</em>) (n = 4) in NP and <em>7</em>02 ± 5 (n = 5) RLU·ml(-<em>1</em>)·min(-<em>1</em>) in IL-<em>1</em><em>7</em>-treated rats. Importantly, AT<em>1</em>-AA increased from 0.4<em>1</em> ± 0.05 beats/min in NP rats (n = 8) to <em>1</em>8.4 ± <em>1</em> beats/min in IL-<em>1</em><em>7</em> rats (n = <em>1</em>2). Administration of tempol attenuated the hypertension (<em>1</em>0<em>1</em> ± 3 mmHg) ROS (459 ± 5 RLU·ml(-<em>1</em>)·min(-<em>1</em>)) and blunted AT<em>1</em>-AAs (<em>7</em>.3 ± 0.6 beats/min) in NP+IL-<em>1</em><em>7</em>+tempol-treated rats. Additionally, AT<em>1</em> receptor blockade inhibited IL-<em>1</em><em>7</em>-induced hypertension and placental oxidative stress. MAP was <em>1</em>05 ± 5 mmHg and ROS was 4<em>1</em>8 ± 5 RLU·ml(-<em>1</em>)·min(-<em>1</em>) in NP+IL <em>1</em><em>7</em>-treated with losartan. These data indicate that IL-<em>1</em><em>7</em> causes placental oxidative stress, which serves as stimulus modulating AT<em>1</em>-AAs that may play an important role in mediating IL-<em>1</em><em>7</em>-induced hypertension during pregnancy.

-The stimulation of endothelium-dependent NO release by <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] has been indirectly shown in terms of vasodilation, which was diminished by NO synthase inhibition or removal of the endothelium. However, direct measurement of endothelium-derived NO has not been analyzed. With a selective porphyrinic microsensor, NO release was directly assessed from single primary cultured bovine aortic endothelial cells. Ang-(<em>1</em>-<em>7</em>) caused a concentration-dependent release of NO of <em>1</em> to <em>1</em>0 µmol/L, which was attenuated by NO synthase inhibition. [D-Ala(<em>7</em>)]Ang-(<em>1</em>-<em>7</em>) (5 µmol/L), described as a selective antagonist of Ang-(<em>1</em>-<em>7</em>) receptors, inhibited Ang-(<em>1</em>-<em>7</em>)-induced NO release only by approximately 50%, whereas preincubation of bovine aortic endothelial cells with the <em>angiotensin</em> II subtype <em>1</em> and 2 receptor antagonists EXP 3<em>1</em><em>7</em>4 and PD <em>1</em>23,<em>1</em><em>7</em><em>7</em> (both at 0.<em>1</em> µmol/L) led to an inhibition of 60% and 90%, respectively. A complete blockade of the Ang-(<em>1</em>-<em>7</em>)-induced NO release was observed on preincubation of the cells with <em>1</em> µmol/L concentration of the bradykinin subtype 2 receptor antagonist icatibant (HOE <em>1</em>40), suggesting an important role of local kinins in the action of Ang-(<em>1</em>-<em>7</em>). Simultaneous direct measurement of superoxide (O(2)(-)) detected by an O(2)(-)-sensitive microsensor revealed that the moderately Ang-(<em>1</em>-<em>7</em>)-stimulated NO release was accompanied by a very slow concomitant O(2)(-) production with a relative low peak concentration in comparison to the O(2)(-) production of the strong NO releasers bradykinin and, especially, calcium ionophore. Thus, Ang-(<em>1</em>-<em>7</em>) might preserve the vascular system, among others, due to its low formation of cytotoxic peroxynitrite by the reaction between NO and O(2)(-).

The existence of a cardiac renin-<em>angiotensin</em> system, independent of the circulating renin-<em>angiotensin</em> system, is still controversial. We compared the tissue levels of renin-<em>angiotensin</em> system components in the heart with the levels in blood plasma in healthy pigs and 30 hours after nephrectomy. <em>Angiotensin</em> I (Ang I)-generating activity of cardiac tissue was identified as renin by its inhibition with a specific active site-directed renin inhibitor. We took precautions to prevent the ex vivo generation and breakdown of cardiac <em>angiotensins</em> and made appropriate corrections for any losses of intact Ang I and II during extraction and assay. Tissue levels of renin (n = <em>1</em><em>1</em>) and Ang I (n = <em>7</em>) and II (n = <em>7</em>) in the left and right atria were higher than in the corresponding ventricles (P < .05). Cardiac renin and Ang I levels (expressed per gram wet weight) were similar to the plasma levels, and Ang II in cardiac tissue was higher than in plasma (P < .05). The presence of these renin-<em>angiotensin</em> system components in cardiac tissue therefore cannot be accounted for by trapped plasma or simple diffusion from plasma into the interstitial fluid. <em>Angiotensin</em>ogen levels (n = <em>1</em><em>1</em>) in cardiac tissue were <em>1</em>0% to 25% of the levels in plasma, which is compatible with its diffusion from plasma into the interstitium. Like <em>angiotensin</em>-converting enzyme, renin was enriched in a purified cardiac membrane fraction prepared from left ventricular tissue, as compared with crude homogenate, and <em>1</em>2 +/- 3% (mean +/- SD, n = 6) of renin in crude homogenate was found in the cardiac membrane fraction and could be solubilized with <em>1</em>% Triton X-<em>1</em>00. Tissue levels of renin and Ang I and II in the atria and ventricles were directly correlated with plasma levels (P < .05), and in both tissue and plasma the levels were undetectably low after nephrectomy. We conclude that most if not all renin in cardiac tissue originates from the kidney. Results support the contentions that in the healthy heart, <em>angiotensin</em> production depends on plasma-derived renin and that plasma-derived <em>angiotensin</em>ogen in the interstitial fluid is a potential source of cardiac <em>angiotensins</em>. Binding of renin to cardiac membranes may be part of a mechanism by which renin is taken up from plasma.