This study investigated the signal transduction mechanisms of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)]- and Ang II-stimulated arachidonic acid (AA) release for prostaglandin (PG) production in rabbit aortic vascular smooth muscle cells. Ang II and Ang-(<em>1</em>-<em>7</em>) enhanced AA release in cells prelabeled with [3H]AA. However, 6-keto-PGF<em>1</em> alpha synthesis produced by Ang II was much less than that caused by Ang-(<em>1</em>-<em>7</em>). In the presence of the lipoxygenase inhibitor baicalein, Ang II enhanced production of 6-keto-PGF<em>1</em> alpha to a greater degree than Ang-(<em>1</em>-<em>7</em>). <em>Angiotensin</em> type (AT)<em>1</em> receptor antagonist DUP-<em>7</em>53 inhibited only Ang II-induced [3H]AA release, whereas the AT2 receptor antagonist PD-<em>1</em>233<em>1</em>9 inhibited both Ang II- and Ang-(<em>1</em>-<em>7</em>)-induced [3H]AA release. Ang-(<em>1</em>-<em>7</em>), receptor antagonist D-Ala<em>7</em>-Ang-(<em>1</em>-<em>7</em>) inhibited the effect of Ang-(<em>1</em>-<em>7</em>), but not of Ang II. In cells transiently transfected with cytosolic phospholipase A2 (cPLA2), mitogen-activated protein (MAP) kinase or Ca(++)-/cal-modulin-dependent protein (CAM) kinase II antisense oligonucleotides, Ang-(<em>1</em>-<em>7</em>)- and Ang II-induced [3H]AA release was attenuated. The CaM kinase II inhibitor KN-93 and the MAP kinase kinase inhibitor PD-98059 attenuated both Ang-(<em>1</em>-<em>7</em>)- and Ang II-induced cPLA2 activity and [3H]AA release. Ang-(<em>1</em>-<em>7</em>) and Ang II also increased CaM kinase II and MAP kinase activities. Although KN-93 attenuated MAP kinase activity, PD-98059 did not affect CaM kinase II activity. Both Ang II and Ang-(<em>1</em>-<em>7</em>) caused translocation of cytosolic PLA2 to the nuclear envelope. These data show that Ang-(<em>1</em>-<em>7</em>) and Ang II stimulate AA release and prostacyclin synthesis via activation of distinct types of AT receptors. Both peptides appear to stimulate CaM kinase II, which in turn, via MAP kinase activation, enhances cPLA2 activity and release of AA for PG synthesis.

Our investigations started when synthetic bradykinin became available and we could characterize two enzymes that cleaved it: kininase I or plasma carboxypeptidase N and kininase II, a peptidyl dipeptide hydrolase that we later found to be identical with the <em>angiotensin</em> I converting enzyme (ACE). When we noticed that ACE can cleave peptides without a free C-terminal carboxyl group (e.g., with a C-terminal nitrobenzylamine), we investigated inactivation of substance P, which has a C-terminal Met(<em>1</em><em>1</em>)-NH(2). The studies were extended to the hydrolysis of the neuropeptide, neurotensin and to compare hydrolysis of the same peptides by neprilysin (neutral endopeptidase 24.<em>1</em><em>1</em>, CD<em>1</em>0, NEP). Our publication in <em>1</em>984 dealt with ACE and NEP purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln(6)-Phe(<em>7</em>), Phe(<em>7</em>)[see text]-Phe(8), and Gly(9)-Leu(<em>1</em>0) and neurotensin (NT) at Pro(<em>1</em>0)-Tyr(<em>1</em><em>1</em>) and Tyr(<em>1</em><em>1</em>)-Ile(<em>1</em>2). Purified ACE also rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe(8)-Gly(9) and Gly(9)-Leu(<em>1</em>0) to release C-terminal tri- and dipeptide (ratio = 4:<em>1</em>). The hydrolysis was Cl(-) dependent and inhibited by captopril. ACE released only dipeptide from SP free acid. ACE hydrolyzed NT at Tyr(<em>1</em><em>1</em>)-Ile(<em>1</em>2) to release Ile(<em>1</em>2)-Leu(<em>1</em>3). Then peptide substrates were used to inhibit ACE hydrolyzing Fa-Phe-Gly-Gly and NEP cleaving Leu(5)-enkephalin. The K(i) values in microM were as follows: for ACE, bradykinin = 0.4, <em>angiotensin</em> I = 4, SP = 25, SP free acid = 2, NT = <em>1</em>4, and Met(5)-enkephalin = 450, and for NEP, bradykinin = <em>1</em>62, <em>angiotensin</em> I = 36, SP = <em>1</em>90, NT = 39, Met(5)-enkephalin = 22. These studies showed that ACE and NEP, two enzymes widely distributed in the body, are involved in the metabolism of SP and NT. Below we briefly survey how NEP and ACE in two decades have gained the reputation as very important factors in health and disease. This is due to the discovery of more endogenous substrates of the enzymes and to the very broad and beneficial therapeutic applications of ACE inhibitors.

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) (Ang-(<em>1</em>-<em>7</em>)) is now considered to be a biologically active member of the renin-<em>angiotensin</em> system. The functions of Ang-(<em>1</em>-<em>7</em>) are often opposite to those attributed to the main effector component of the renin-<em>angiotensin</em> system, Ang II. Chronic administration of <em>angiotensin</em>-converting enzyme inhibitors (ACEI) increases <em>1</em>0- to 25-fold the plasma levels of this peptide, suggesting that part of the beneficial effects of ACEI could be mediated by Ang-(<em>1</em>-<em>7</em>). Ang-(<em>1</em>-<em>7</em>) can be formed from Ang II or directly from Ang I. Other enzymatic pathways for Ang-(<em>1</em>-<em>7</em>) generation have been recently described involving the novel ACE homologue ACE2. This enzyme can form Ang-(<em>1</em>-<em>7</em>) from Ang II or less efficiently by the hydrolysis of Ang I to Ang-(<em>1</em>-9) with subsequent Ang-(<em>1</em>-<em>7</em>) formation. The biological relevance of Ang-(<em>1</em>-<em>7</em>) has been recently reinforced by the identification of its receptor, the G-protein-coupled receptor Mas. Heart and blood vessels are important targets for the formation and actions of Ang-(<em>1</em>-<em>7</em>). In this review we will discuss recent findings concerning the biological role of Ang-(<em>1</em>-<em>7</em>) in the heart and blood vessels, taking into account aspects related to its formation and effects on these tissues. In addition, we will discuss the potential of Ang-(<em>1</em>-<em>7</em>) and its receptor as a target for the development of new cardiovascular drugs.

In cultured endothelial cells, endothelin is produced after stimulation with <em>angiotensin</em> II. The effects of <em>angiotensin</em> II and endothelin-<em>1</em> on vascular sensitivity to norepinephrine were studied in perfused rat mesenteric resistance arteries. Expression of endothelin messenger RNA (mRNA) was determined in endothelial cells obtained from the mesenteric circulation. Perfusion (5 hours) of the arteries with <em>angiotensin</em> II (<em>1</em>0(-<em>7</em>) M) potentiated contractions in arteries with endothelium induced by norepinephrine in spontaneously hypertensive rats but not Wistar-Kyoto rats. The potentiation was inhibited by phosphoramidon and an endothelin antibody. Short-term stimulation (<em>1</em> hour) with <em>angiotensin</em> II did not cause the potentiation. Stimulation with <em>angiotensin</em> I (<em>1</em>0(-<em>7</em>) M; 5 hours) caused a potentiation prevented by captopril. In endothelial cells collected from the mesenteric arterial bed of spontaneously hypertensive rats, endothelin-specific mRNA was constitutively expressed, and the level of endothelin transcripts was increased by <em>angiotensin</em> II (<em>1</em>0(-<em>7</em>) M). Threshold concentrations of exogenous endothelin-<em>1</em> potentiated contractions induced by norepinephrine in arteries with and without endothelium of spontaneously hypertensive rats but not Wistar-Kyoto rats. Thus, <em>angiotensin</em> II stimulates the endothelial production of endothelin in situ and therapy potentiates contractions to norepinephrine in mesenteric resistance arteries of spontaneously hypertensive rats. This suggests that vascular endothelin production acts as an amplifier of the pressor effects of the renin-<em>angiotensin</em> system that may play an important role in hypertension.

Aliskiren is the first orally bioavailable direct renin inhibitor approved for the treatment of hypertension. It acts at the point of activation of the renin-<em>angiotensin</em>-aldosterone system, or renin system, inhibiting the conversion of <em>angiotensin</em>ogen to <em>angiotensin</em> I by renin and thereby reducing the formation of <em>angiotensin</em> II by <em>angiotensin</em>-converting enzyme (ACE) and ACE-independent pathways. Aliskiren is a highly potent inhibitor of human renin in vitro (concentration of aliskiren that produces 50% inhibition of renin 0.6 nmol/L). Aliskiren is rapidly absorbed following oral administration, with maximum plasma concentrations reached within <em>1</em>-3 hours. The absolute bioavailability of aliskiren is 2.6%. The binding of aliskiren to plasma proteins is moderate (4<em>7</em>-5<em>1</em>%) and is independent of the concentration. Once absorbed, aliskiren is eliminated through the hepatobiliary route as unchanged drug and, to a lesser extent, through oxidative metabolism by cytochrome P450 (CYP) 3A4. Unchanged aliskiren accounts for approximately 80% of the drug in the plasma following oral administration, indicating low exposure to metabolites. The two major oxidized metabolites of aliskiren account for less than 5% of the drug in the plasma at the time of the maximum concentration. Aliskiren excretion is almost completely via the biliary/faecal route; 0.6% of the dose is recovered in the urine. Steady-state plasma concentrations of aliskiren are reached after <em>7</em>-8 days of once-daily dosing, and the accumulation factor for aliskiren is approximately 2. After reaching the peak, the aliskiren plasma concentration declines in a multiphasic fashion. No clinically relevant effects of gender or race on the pharmacokinetics of aliskiren are observed, and no adjustment of the initial aliskiren dose is required for elderly patients or for patients with renal or hepatic impairment. Aliskiren showed no clinically significant increases in exposure during coadministration with a wide range of potential concomitant medications, although increases in exposure were observed with P-glycoprotein inhibitors. Aliskiren does not inhibit or induce CYP isoenzyme or P-glycoprotein activity, although aliskiren is a substrate for P-glycoprotein, which contributes to its relatively low bioavailability. Aliskiren is approved for the treatment of hypertension at once-daily doses of <em>1</em>50 mg and 300 mg. Phase II and III clinical studies involving over <em>1</em>2,000 patients with hypertension have demonstrated that aliskiren provides effective long-term blood pressure (BP) lowering with a good safety and tolerability profile at these doses. Aliskiren inhibits plasma renin activity (PRA) by up to 80% following both single and multiple oral-dose administration. Similar reductions in PRA are observed when aliskiren is administered in combination with agents that alone increase PRA, including diuretics (hydrochlorothiazide, furosemide [frusemide]), ACE inhibitors (ramipril) and <em>angiotensin</em> receptor blockers (valsartan), despite greater increases in the plasma renin concentration. Moreover, PRA inhibition and BP reductions persist for 2-4 weeks after stopping treatment, which is likely to be of benefit in patients with hypertension who occasionally miss a dose of medication. Preliminary data on the antiproteinuric effects of aliskiren in type 2 diabetes mellitus suggest that renoprotective effects beyond BP lowering may be possible. Further studies to evaluate the effects of aliskiren on cardiovascular outcomes and target organ protection are ongoing and will provide important new data on the role of direct renin inhibition in the management of hypertension and other cardiovascular disease.

The goal of the present study was to test the impact of administration time of the <em>angiotensin</em> II type <em>1</em>-receptor blocker candesartan on cerebral blood flow (CBF), infarct size, and neuroscore in transient cerebral ischemia. Therefore, <em>1</em>-hour middle cerebral artery occlusion (MCAO) was followed by reperfusion. Rats received 0.5-mg/kg candesartan intravenously 2 hours before MCAO (pretreatment), 24 hours after MCAO, every 24 hours after MCAO, or 2 hours before and every 24 hours after MCAO. Infarct size (mm3) and a neuroscore at day <em>7</em> were compared with controls. CBF was quantified by radiolabeled microspheres and laser-Doppler flowmetry. Compared with controls (95 +/- 8), infarct size in candesartan-treated groups was smaller (59 +/- 5, 68 +/- <em>1</em>0, 28 +/- 3, and <em>1</em>5 +/- 3, respectively; P<0.05). Although there was no difference in neuroscore between pretreatment and controls (<em>1</em>.55 +/- 0.<em>1</em>8, <em>1</em>.80 +/- 0.<em>1</em>3), other treatment regimens resulted in improved neuroscores (<em>1</em>.33 +/- 0.<em>1</em>6, <em>1</em>.<em>1</em><em>1</em> +/- 0.<em>1</em><em>1</em>, 0.<em>7</em>3 +/- 0.<em>1</em>5; P<0.05). CBF in pretreated animals at 0.5 hours after MCAO was significantly higher than in controls (0.58 +/- 0.09 mL x g(-<em>1</em>) x min(-<em>1</em>) and 44% +/- <em>7</em>% of baseline compared with 0.49 +/- 0.06 mL x g(-<em>1</em>) x min(-<em>1</em>) and 3<em>7</em>% +/- 6%, microspheres and laser-Doppler flowmetry; P<0.05). Thus, candesartan reduces infarct size even if administered only during reperfusion. Apart from pretreatment, other treatment regimens result in significantly improved neuroscores. In the acute phase of cerebral ischemia, candesartan increases CBF.

Although arterial dilator reactivity is severely impaired during exposure of animals to chronic intermittent hypoxia (CIH), few studies have characterized vasoconstrictor responsiveness in resistance arteries of this model of sleep-disordered breathing. Sprague-Dawley rats were exposed to CIH (<em>1</em>0% inspired O2 fraction for <em>1</em> min at 4-min intervals; <em>1</em>2 h/day) for <em>1</em>4 days. Control rats were housed under normoxic conditions. Diameters of isolated gracilis muscle resistance arteries (GA; <em>1</em>20-<em>1</em>50 microm) were measured by television microscopy before and during exposure to norepinephrine (NE) and <em>angiotensin</em> II (ANG II) and at various intraluminal pressures between 20 and <em>1</em>40 mmHg in normal and Ca2+-free physiological salt solution. There was no difference in the ability of GA to constrict in response to ANG II (P = 0.42; not significant; <em>1</em>0(-<em>1</em>0)-<em>1</em>0(-<em>7</em>) M). However, resting tone, myogenic activation, and vasoconstrictor responses to NE (P < 0.00<em>1</em>; <em>1</em>0(-9)-<em>1</em>0(-6) M) were reduced in CIH vs. controls. Treatment of rats with the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine <em>1</em>-oxyl (tempol; <em>1</em> mM) in the drinking water restored myogenic responses and NE-induced constrictions of CIH rats, suggesting that elevated superoxide production during exposure to CIH attenuates vasoconstrictor responsiveness to NE and myogenic activation in skeletal muscle resistance arteries. CIH also leads to an increased stiffness and reduced vessel wall distensibility that were not correctable with oral tempol treatment.

Emerging evidence indicates that pancreatic tissue expresses all components of the renin-<em>angiotensin</em> system. However, the functional role is not well understood. This investigation examined renin inhibition on pancreas structure/function in the transgenic Ren2 rat harboring the mouse renin gene, a model of tissue renin overexpression. Renin is the rate-limiting step in the generation of <em>angiotensin</em> II (Ang II), which stimulates the generation of reactive oxygen species in a variety of tissues. Overexpression of renin in Ren2 rats results in hypertension, insulin resistance, and cardiovascular and renal damage. Young (6-<em>7</em> wk old) insulin-resistant male Ren2 and age-matched insulin sensitive Sprague Dawley rats were treated with the renin inhibitor, aliskiren (50 mg/kg.d by ip injection), or placebo for 2<em>1</em> d. At 2<em>1</em> d, the Ren2 demonstrated insulin resistance with increased islet insulin, Ang II, and reduced total insulin receptor substrate (IRS)-<em>1</em>, IRS-2, and Akt immunostaining. There was increased islet nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and subunits (p4<em>7</em>(phox) and Rac<em>1</em>) as well as increased nitrotyrosine immunostaining (each P < 0.05). These functional abnormalities were associated with a disordered islet architecture; increased islet-exocrine interface, pericapillary fibrosis, and structurally abnormal mitochondria and content in endocrine and exocrine pancreas. In vivo treatment with aliskiren normalized systemic insulin resistance and islet insulin, Ang II, NADPH oxidase activity/subunits, and nitrotyrosine and improved total IRS-<em>1</em> and Akt phosphorylation (each P < 0.05) as well as islet/exocrine structural abnormalities. Collectively, these data suggest that pancreatic functional/structural changes are driven, in part, by tissue renin-<em>angiotensin</em> system-mediated increases in NADPH oxidase and reactive oxygen species generation, abnormalities attenuated with direct renin inhibition.

Type <em>1</em> <em>angiotensin</em> receptors (AT<em>1</em>) are G-protein coupled receptors, mediating the physiological actions of the vasoactive peptide <em>angiotensin</em> II. In this study, the roles of <em>7</em> amino acids of the rat AT<em>1</em>A receptor in ligand binding and signaling were investigated by performing functional assays of individual receptor mutants expressed in COS and Chinese hamster ovary cells. Substitutions of polar residues in the third transmembrane domain with Ala indicate that Ser<em>1</em>05, Ser<em>1</em>0<em>7</em>, and Ser<em>1</em>09 are not essential for maintenance of the <em>angiotensin</em> II binding site. Replacement of Asn<em>1</em><em>1</em><em>1</em> or Ser<em>1</em><em>1</em>5 does not alter the binding affinity for peptidic analogs, but modifies the ability of the receptor to interact with AT<em>1</em> (DuP<em>7</em>53)- or AT2 (CGP42<em>1</em><em>1</em>2A)-specific ligands. These 2 residues are probably involved in determining the binding specificity for these analogs. The absence of G-protein coupling to the Ser<em>1</em><em>1</em>5 mutant suggests that this residue, in addition to previously identified residues, Asp<em>7</em>4 and Tyr292, participates in the receptor activation mechanism. Finally, Lys<em>1</em>02 (third helix) and Lys<em>1</em>99 (fifth helix) mutants do not bind <em>angiotensin</em> II or different analogs. Co-expression of these two deficient receptors permitted the restoration of a normal binding site. This effect was not due to homologous recombination of the cDNAs but to protein trans-complementation.

BACKGROUND

Angiotensin-converting enzyme (ACE) inhibitors do not decrease plasma angiotensin (Ang) II levels 24 hours after drug intake to the same extent as at peak. This intermittent partial "escape" is explained either by a renin-mediated reactive rise in plasma Ang I or by non-ACE-dependent Ang II generation. We therefore tested the hypothesis that a combination of ACE inhibition and Ang II blockade may have additive biological and hemodynamic effects.

RESULTS

In a single-dose, double-blind, randomized, four-way, crossover study, an Ang II antagonist (losartan 50 mg), an ACE inhibitor (captopril 50 mg), their combination, and matched placebos were orally administered to 12 normotensive male volunteers maintained in mild sodium depletion. When captopril 50 mg and losartan 50 mg were given alone, the magnitude of their effects on blood pressure, plasma active renin, Ang I, and aldosterone was similar, whereas the kinetics of their effects were different, reflecting differences in drug pharmacokinetics. The losartan-captopril combination completely suppressed the rise in plasma Ang II induced by losartan 2 hours after drug intake (3.3 +/- 3.6 pg/mL versus 20.3 +/- 19.1 pg/mL, respectively, P < .05). Six hours after drug intake, the losartan-captopril combination induced a significantly greater decrease in mean blood pressure than that produced by either losartan or captopril alone (73 +/- 7 mm Hg versus 79 +/- 8 mm Hg versus 81 +/- 7 mm Hg, respectively, P < .05). The maximum placebo-subtracted falls in mean blood pressure for the losartan-captopril combination, captopril 50 mg, and losartan 50 mg were 14 +/- 5 mm Hg, 10 +/- 3 mm Hg, and 9 +/- 6 mm Hg, respectively (F2.22 = 3.45, P < .05). The duration of the mean blood pressure fall was not prolonged by the combination. After combined losartan-captopril administration, the area under the plasma active renin versus time curve (0 to 24 hours) was significantly increased when compared with either losartan or captopril alone (6404 +/- 2961 pg.h.mL-1 versus 3105 +/- 1461 pg.h.mL-1 versus 2092 +/- 867 pg.h.mL-1, respectively, P < .05). The combination had no additive effects on plasma aldosterone decrease when compared with either losartan or captopril alone (58 +/- 17% versus 51 +/- 20% versus 53 +/- 21%, respectively, NS).

CONCLUSIONS

The combined administration of a standard single oral dose of an ACE inhibitor and an Ang II antagonist to mildly sodium-depleted normal subjects (1) had a major additive effect on plasma renin rise, (2) induced an additional mean blood pressure reduction, and (3) had no additive effect on plasma aldosterone fall.

<em>Angiotensin</em> II and hypertension increase vascular oxidant stress. We examined how these might affect expression of the extracellular superoxide dismutase (ecSOD), a major form of vascular SOD. In mice, <em>angiotensin</em> II infusion (<em>1</em>.<em>1</em> mg/kg for <em>7</em> days) increased systolic blood pressure from <em>1</em>0<em>7</em>+/-3 to <em>1</em>52+/-9 mm Hg and caused a 3-fold increase in ecSOD, but there was no change in the cytosolic Cu/Zn SOD protein, as determined by Western blot analysis. This was associated with a similar increase in ecSOD mRNA as assessed by RNase protection assay and was prevented by losartan. Induction of ecSOD by <em>angiotensin</em> II was not due to hypertension alone, because hypertension caused by norepinephrine (5.6 mg. kg-<em>1</em>. d-<em>1</em>) had no effect on ecSOD. Similarly, exposure of mouse aortas to <em>angiotensin</em> II (<em>1</em>00 nmol/L) in organoid culture increased ecSOD by approximately 2-fold. In the organoid culture, <em>angiotensin</em> II-induced upregulation of ecSOD was prevented by losartan (<em>1</em>0 micromol/L) and PD985059 (30 micromol/L), a specific inhibitor of p42/44 MAP kinase kinase. <em>Angiotensin</em> II activates the NADH/NADPH oxidase; however, diphenyleneiodonium chloride (<em>1</em>0 micromol/L), an inhibitor of this oxidase, did not prevent p42/44 MAP kinase phosphorylation or ecSOD induction by <em>angiotensin</em> II. Finally, in human aortic smooth muscle cells, <em>angiotensin</em> II moderately increased transcriptional rate (as assessed by nuclear run-on analysis) but markedly increased ecSOD mRNA stability. Thus, <em>angiotensin</em> II increases ecSOD expression independent of hypertension, and this increase involves both an increase in ecSOD transcription and stabilization of ecSOD mRNA. This effect of <em>angiotensin</em> II on ecSOD expression may modulate the oxidative state of the vessel wall in pathological processes in which the renin-<em>angiotensin</em> system is activated.

OBJECTIVE

Angiotensin-converting enzyme 2 (ACE2) is an important negative regulator of the renin-angiotensin system. Loss of ACE2 enhances the susceptibility to heart disease but the mechanism remains elusive. We hypothesized that ACE2 deficiency activates the NADPH oxidase system in pressure overload-induced heart failure.

RESULTS

Using the aortic constriction model, we subjected wild-type (Ace2(+/y)), ACE2 knockout (ACE2KO, Ace2(-/y)), p47(phox) knockout (p47(phox)KO, p47(phox-)(/-)), and ACE2/p47(phox) double KO mice to pressure overload. We examined changes in peptide levels, NADPH oxidase activity, gene expression, matrix metalloproteinases (MMP) activity, pathological signalling, and heart function. Loss of ACE2 resulted in enhanced susceptibility to biomechanical stress leading to eccentric remodelling, increased pathological hypertrophy, and worsening of systolic performance. Myocardial angiotensin II (Ang II) levels were increased, whereas Ang 1-7 levels were lowered. Activation of Ang II-stimulated signalling pathways in the ACE2-deficient myocardium was associated with increased expression and phosphorylation of p47(phox), NADPH oxidase activity, and superoxide generation, leading to enhanced MMP-mediated degradation of the extracellular matrix. Additional loss of p47(phox) in the ACE2KO mice normalized the increased NADPH oxidase activity, superoxide production, and systolic dysfunction following pressure overload. Ang 1-7 supplementation suppressed the increased NADPH oxidase and rescued the early dilated cardiomyopathy in pressure-overloaded ACE2KO mice.

CONCLUSIONS

In the absence of ACE2, biomechanical stress triggers activation of the myocardial NAPDH oxidase system with a critical role of the p47(phox) subunit. Increased production of superoxide, activation of MMP, and pathological signalling leads to severe adverse myocardial remodelling and dysfunction in ACE2KO mice.

We investigated the role of <em>angiotensin</em> II in vascular and circulating inflammatory markers in spontaneously hypertensive rats (SHR). IL-<em>1</em>beta, IL-6, and TNF-alpha aortic mRNA expression and plasma levels were measured in adult SHR untreated or treated with the <em>angiotensin</em> II receptor antagonist candesartan (2 mg.kg(-<em>1</em>).day(-<em>1</em>)) or antihypertensive triple therapy (TT; in mg.kg(-<em>1</em>).day(-<em>1</em>): 20 hydralazine + <em>7</em> type <em>1</em> hydrochlorothiazide + 0.<em>1</em>5 reserpine) for <em>1</em>0 wk. Likewise, aortic expression of NF-kappaB p50 subunit precursor p<em>1</em>05 and its inhibitor (IkappaB) were measured. Age-matched Wistar-Kyoto rats (WKY) served as normotensive reference. High blood pressure levels were associated with increased (P < 0.05) aortic mRNA expression of IL-<em>1</em>beta, IL-6, and TNF-alpha. Hypertension was also accompanied by increased IL-<em>1</em>beta and IL-6 plasma levels. No differences were observed in circulating TNF-alpha levels between SHR and WKY. SHR presented elevated aortic mRNA expression of the transcription factor NF-kappaB and reduction in its inhibitor, IkappaB. Candesartan decreased (P < 0.05) blood pressure levels, aortic mRNA expression of IL-<em>1</em>beta, IL-6, and TNF-alpha, and (P < 0.05) IL-<em>1</em>beta and IL-6 plasma concentration. However, although arterial pressure decrease was comparable for the treatments, TT only partially reduced the increments in inflammatory markers. In fact, candesartan-treated rats showed significantly lower levels of circulating and vascular inflammatory markers than TT-treated animals. The treatments increased IkappaB mRNA expression similarly. However, only candesartan reduced NF-kappaB mRNA expression. In summary, <em>1</em>) SHR presented a vascular inflammatory process; 2) <em>angiotensin</em> II, and increased hemodynamic forces associated with hypertension, seems to be involved in stimulation of inflammatory mediators through NF-kappaB system activation; and 3) reduction of inflammatory mediators produced by candesartan in SHR could be partially due to both downregulation of NF-kappaB and upregulation of IkappaB.

OBJECTIVE

The purpose of the Euro Heart Survey Programme of the European Society of Cardiology is to evaluate to which extent clinical practice endorses existing guidelines as well as to identify differences in population profiles, patient management, and outcome across Europe. The current survey focuses on the invasive diagnosis and treatment of patients with established coronary artery disease (CAD).

RESULTS

Between November 200<em>1</em> and March 2002, <em>7</em><em>7</em>69 consecutive patients undergoing invasive evaluation at <em>1</em>30 hospitals (3<em>1</em> countries) were screened for the presence of one or more coronary stenosis >50% in diameter. Patient demographics and comorbidity, clinical presentation, invasive parameters, treatment options, and procedural techniques were prospectively entered in an electronic database (550 variables+29 per diseased coronary segment). Major adverse cardiac events (MACE) were evaluated at 30 days and <em>1</em> year. Out of 56<em>1</em>9 patients with angiographically proven coronary stenosis (<em>7</em>2% of screened population), 53% presented with stable angina while ST elevation myocardial infarction (STEMI) was the indication for coronary angiography in <em>1</em>6% and non-ST segment elevation myocardial infarction or unstable angina in 30%. Only medical therapy was continued in 2<em>1</em>%, whereas mechanical revascularization was performed in the remainder [percutaneous coronary intervention (PCI) in 58% and coronary artery bypass grafting (CABG) in 2<em>1</em>%]. Patients referred for PCI were younger, were more active, had a lower risk profile, and had less comorbid conditions. CABG was performed mostly in patients with left main lesions (2<em>1</em>%), two- (25%), or three-vessel disease (6<em>7</em>%) with 4.<em>1</em> diseased segments, on average. Single-vessel PCI was performed in 82% of patients with either single- (45%), two- (33%), or three-vessel disease (2<em>1</em>%). Stents were used in <em>7</em>5% of attempted lesions, with a large variation between sites. Direct PCI for STEMI was performed in 4<em>1</em>0 cases, representing <em>7</em>% of the entire workload in the participating catheterization laboratories. Time delay was within 90 min in <em>7</em>6% of direct PCI cases. In keeping with the recommendations of practice guidelines, the survey identified under-use of adjunctive medication (GP IIb/IIIa receptor blockers, statins, and <em>angiotensin</em>-converting enzyme-inhibitors). Mortality rates at 30 days and <em>1</em> year were low in all subgroups. MACE primarily consisted of repeat PCI (<em>1</em>2%).

CONCLUSIONS

The current Euro Heart Survey on coronary revascularization was performed in the era of bare metal stenting and provides a global European picture of the invasive approach to patients with CAD. These data will serve as a benchmark for the future evaluation of the impact of drug-eluting stents on the practice of interventional cardiology and bypass surgery.

ANG converting enzyme (ACE) 2 (ACE2) is a homologue of ACE, which is not blocked by conventional ACE inhibitors. ACE2 converts ANG <em>1</em>-<em>1</em>0 (ANG I) to ANG <em>1</em>-9, which can be hydrolyzed by ACE to form the biologically active peptide ANG <em>1</em>-<em>7</em>. ACE2 is expressed in the kidney, but its precise intrarenal localization is unclear, and the role of intrarenal ACE2 in the production of ANG <em>1</em>-<em>7</em> is unknown. The present studies determined the relative distribution of ACE2 in the rat kidney and defined its role in the generation of ANG <em>1</em>-<em>7</em> in proximal tubule. In microdissected rat nephron segments, semiquantitative RT-PCR revealed that ACE2 mRNA was widely expressed, with relatively high levels in proximal straight tubule (PST). Immunohistochemistry demonstrated ACE2 protein in tubular segments, glomeruli, and endothelial cells. Utilizing mass spectrometry, incubation of isolated PSTs with ANG I (<em>1</em>0(-6) M) led to generation of ANG <em>1</em>-<em>7</em> (sensitivity of detection>> <em>1</em> x <em>1</em>0(-9) M), accompanied by the formation of ANG <em>1</em>-8 (ANG II) and ANG <em>1</em>-9. The ACE2 inhibitor DX600 completely blocked ANG I-mediated generation of ANG <em>1</em>-<em>7</em>. Incubation of PSTs with ANG <em>1</em>-9 also led to generation of ANG <em>1</em>-<em>7</em>, an effect blocked by the ACE inhibitor captopril or enalaprilat, but not by DX600. Incubation of PSTs with ANG II or luminal perfusion of ANG II did not result in detection of ANG <em>1</em>-<em>7</em>. The results indicate that ACE2 is widely expressed in rat nephron segments and contributes to the production of ANG <em>1</em>-<em>7</em> from ANG I in PST. ANG II may not be a major substrate for ACE2 in isolated PST. The data suggest that ACE2-mediated production of ANG <em>1</em>-<em>7</em> represents an important component of the proximal tubular renin-ANG system.

Diabetes is an independent risk factor for heart failure progression. Sacubitril/valsartan, a combination angiotensin receptor-neprilysin inhibitor, improves morbidity and mortality in patients with heart failure with reduced ejection fraction (HFrEF), compared with the angiotensin-converting enzyme inhibitor enalapril, and improves peripheral insulin sensitivity in obese hypertensive patients. We aimed to investigate the effect of sacubitril/valsartan versus enalapril on HbA1c and time to first-time initiation of insulin or oral antihyperglycaemic drugs in patients with diabetes and HFrEF.

In a post-hoc analysis of the PARADIGM-HF trial, we included 3778 patients with known diabetes or an HbA1c ≥6·5% at screening out of 8399 patients with HFrEF who were randomly assigned to treatment with sacubitril/valsartan or enalapril. Of these patients, most (98%) had type 2 diabetes. We assessed changes in HbA1c, triglycerides, HDL cholesterol and BMI in a mixed effects longitudinal analysis model. Time to initiation of oral antihyperglycaemic drugs or insulin in subjects previously not treated with these agents were compared between treatment groups.

There were no significant differences in HbA1c concentrations between randomised groups at screening. During the first year of follow-up, HbA1c concentrations decreased by 0·16% (SD 1·40) in the enalapril group and 0·26% (SD 1·25) in the sacubitril/valsartan group (between-group reduction 0·13%, 95% CI 0·05-0·22, p=0·0023). HbA1c concentrations were persistently lower in the sacubitril/valsartan group than in the enalapril group over the 3-year follow-up (between-group reduction 0·14%, 95% CI 0·06-0·23, p=0·0055). New use of insulin was 29% lower in patients receiving sacubitril/valsartan (114 [7%] patients) compared with patients receiving enalapril (153 [10%]; hazard ratio 0·71, 95% CI 0·56-0·90, p=0·0052). Similarly, fewer patients were started on oral antihyperglycaemic therapy (0·77, 0·58-1·02, p=0·073) in the sacubitril/valsartan group.

Patients with diabetes and HFrEF enrolled in PARADIGM-HF who received sacubitril/valsartan had a greater long-term reduction in HbA1c than those receiving enalapril. These data suggest that sacubitril/valsartan might enhance glycaemic control in patients with diabetes and HFrEF.

Novartis.

We used the isolated N- and C-domains of the <em>angiotensin</em> <em>1</em>-converting enzyme (N-ACE and C-ACE; ACE; kininase II) to investigate the hydrolysis of the active <em>1</em>-<em>7</em> derivative of <em>angiotensin</em> (Ang) II and inhibition by 5-S-5-benzamido-4-oxo-6-phenylhexanoyl-L-proline (keto-ACE). Ang-(<em>1</em>-<em>7</em>) is both a substrate and an inhibitor; it is cleaved by N-ACE at approximately one half the rate of bradykinin but negligibly by C-ACE. It inhibits C-ACE, however, at an order of magnitude lower concentration than N-ACE; the IC50 of C-ACE with <em>1</em>00 micromol/L Ang I substrate was <em>1</em>.2 micromol/L and the Ki was 0.<em>1</em>3. While searching for a specific inhibitor of a single active site of ACE, we found that keto-ACE inhibited bradykinin and Ang I hydrolysis by C-ACE in approximately a 38- to 4<em>7</em>-times lower concentration than by N-ACE; IC50 values with C-ACE were 0.5 and 0.04 micromol/L. Furthermore, we investigated how Ang-(<em>1</em>-<em>7</em>) acts via bradykinin and the involvement of its B2 receptor. Ang-(<em>1</em>-<em>7</em>) was ineffective directly on the human bradykinin B2 receptor transfected and expressed in Chinese hamster ovary cells. However, Ang-(<em>1</em>-<em>7</em>) potentiated arachidonic acid release by an ACE-resistant bradykinin analogue (<em>1</em> micromol/L), acting on the B2 receptor when the cells were cotransfected with cDNAs of both B2 receptor and ACE and the proteins were expressed on the plasma membrane of Chinese hamster ovary cells. Thus like other ACE inhibitors, Ang-(<em>1</em>-<em>7</em>) can potentiate the actions of a ligand of the B2 receptor indirectly by binding to the active site of ACE and independent of blocking ligand hydrolysis. This potentiation of kinins at the receptor level can explain some of the well-documented kininlike actions of Ang-(<em>1</em>-<em>7</em>).

OBJECTIVE

To assess the clinical relevance of angiotensin-converting enzyme inhibitors (ACEI) and 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitors (statins) in the context of idiopathic pulmonary fibrosis (IPF).

BACKGROUND

IPF is a progressive interstitial lung disease for which there is no effective treatment. ACEI and statins have been shown to possess antifibrotic properties in experimental models in vitro and in vivo.

METHODS

Retrospective review of the effects of ACEI and statins on survival of 478 patients with IPF seen at Mayo Clinic Rochester from 1994 through 1996. Fifty-two patients (11%) were receiving ACEI, 35 patients (7%) were receiving statins, and 5 patients (1%) patients were receiving both at their initial visit.

RESULTS

For subjects receiving ACEI, the median survival from the index visit was 2.2 years, compared to 2.9 years for subjects not receiving ACEI (p = 0.088). The median survival was 2.9 years if patients were receiving statins or not (p = 0.573). There was also no significant difference in survival between patients with IPF receiving either ACEI or statins vs those receiving neither at the index visit (2.5 years vs 3 years, respectively; p = 0.066). After adjusting for age, gender, recommended IPF treatment, smoking status, prior oxygen use, FVC, diffusion capacity for carbon monoxide, coronary artery disease, congestive heart failure, diabetes mellitus, and hypertension, there were no differences in survival between those subjects receiving either ACEI, statins, or both vs neither.

CONCLUSIONS

These data do not suggest a beneficial effect of ACEI and/or statins on survival in patients with IPF.

<em>Angiotensin</em>-converting enzyme 2 (ACE2) is a monocarboxypeptidase that degrades <em>angiotensin</em> (Ang) II to Ang-(<em>1</em>-<em>7</em>). ACE2 is highly expressed within the kidneys, it is largely localized in tubular epithelial cells and less prominently in glomerular epithelial cells and in the renal vasculature. ACE2 activity has been shown to be altered in diabetic kidney disease, hypertensive renal disease and in different models of kidney injury. There is often a dissociation between tubular and glomerular ACE2 expression, particularly in diabetic kidney disease where ACE2 expression is increased at the tubular level but decreased at the glomerular level. In this review, we will discuss alterations in circulating and renal ACE2 recently described in different renal pathologies and disease models as well as their possible significance.

<em>Angiotensin</em>-converting enzyme 2 (ACE2) is a lately discovered enzyme catalyzing <em>Angiotensin</em> II into <em>Angiotensin</em> <em>1</em>-<em>7</em>. <em>Angiotensin</em> II has been reported to impair endothelial progenitor cell (EPC) function and is detrimental to stroke. Here, we studied the role of ACE2 in regulating EPC function in vitro and in vivo. EPCs were cultured from human renin and angiotensinogen transgenic (R+A+) mice and their controls (R-A-). In in vitro experiments, EPCs were transduced with lentivirus-ACE2 or lentivirus-green fluorescence protein. The effects of ACE2 overexpression on EPC function and endothelial NO synthase (eNOS)/nicotinamide adenine dinucleotide phosphate oxidase (Nox) expression were determined. ACE2, eNOS, and Nox inhibitors were used for pathway validation. In in vivo studies, the therapeutic efficacy of EPCs overexpressing ACE2 was determined at day <em>7</em> after ischemic stroke induced by middle cerebral artery occlusion. We found that (<em>1</em>) lentivirus-ACE2 transduction resulted in a 4-fold increase of ACE2 expression in EPCs. This was accompanied with an increase in eNOS expression and NO production, and a decrease in Nox2 and -4 expression and reactive oxygen species production. (2) ACE2 overexpression improved the abilities of EPC migration and tube formation, which were impaired in R+A+ mice. These effects were inhibited by ACE2 or eNOS inhibitor and further enhanced by Nox inhibitor. (3) Transfusion of lentivirus-ACE2-primed EPCs reduced cerebral infarct volume and neurological deficits, and increased cerebral microvascular density and angiogenesis. Our data demonstrate that ACE2 improves EPC function, via regulating eNOS and Nox pathways, and enhances the efficacy of EPC-based therapy for ischemic stroke.

Pulmonary arterial hypertension (PAH) is a chronic lung disease with poor diagnosis and limited therapeutic options. The currently available therapies are ineffective in improving the quality of life and reducing mortality rates. There exists a clear unmet medical need to treat this disease, which necessitates the discovery of novel therapeutic targets/agents for safe and successful therapy. An altered renin-<em>angiotensin</em> system (RAS) has been implicated as a causative factor in the pathogenesis of PAH. <em>Angiotensin</em> II (Ang II), a key effector peptide of the RAS, can exert deleterious effects on the pulmonary vasculature resulting in vasoconstriction, proliferation, and inflammation, all of which contribute to PAH development. Recently, a new member of the RAS, <em>angiotensin</em> converting enzyme 2 (ACE2), was discovered. This enzyme functions as a negative regulator of the <em>angiotensin</em> system by metabolizing Ang II to a putative protective peptide, <em>angiotensin</em>-(<em>1</em>-<em>7</em>). ACE2 is abundantly expressed in the lung tissue and emerging evidence suggests a beneficial role for this enzyme against lung diseases. In this review, we focus on ACE2 in relation to pulmonary hypertension and provide proof of principle for its therapeutic role in PAH.

BACKGROUND

IgA nephropathy is thought to be associated with mucosal immune system dysfunction, which manifests as renal IgA deposition that leads to impairment and end-stage renal disease in 20-40% of patients within <em>1</em>0-20 years. In this trial (NEFIGAN) we aimed to assess safety and efficacy of a novel targeted-release formulation of budesonide (TRF-budesonide), designed to deliver the drug to the distal ileum in patients with IgA nephropathy.

METHODS

We did a randomised, double-blind, placebo-controlled phase 2b trial, comprised of 6-month run-in, 9-month treatment, and 3-month follow-up phases at 62 nephrology clinics across ten European countries. We recruited patients aged at least <em>1</em>8 years with biopsy-confirmed primary IgA nephropathy and persistent proteinuria despite optimised renin-<em>angiotensin</em> system (RAS) blockade. We randomly allocated patients with a computer algorithm, with a fixed block size of three, in a <em>1</em>:<em>1</em>:<em>1</em> ratio to <em>1</em>6 mg/day TRF-budesonide, 8 mg/day TRF-budesonide, or placebo, stratified by baseline urine protein creatinine ratio (UPCR). Patients self-administered masked capsules, once daily, <em>1</em> h before breakfast during the treatment phase. All patients continued optimised RAS blockade treatment throughout the trial. Our primary outcome was mean change from baseline in UPCR for the 9-month treatment phase, which was assessed in the full analysis set, defined as all randomised patients who took at least one dose of trial medication and had at least one post-dose efficacy measurement. Safety was assessed in all patients who received the intervention. This trial is registered with ClinicalTrials.gov, number NCT0<em>1</em>738035.

RESULTS

Between Dec <em>1</em><em>1</em>, 20<em>1</em>2, and June 25, 20<em>1</em>5, <em>1</em>50 randomised patients were treated (safety set) and <em>1</em>49 patients were eligible for the full analysis set. Overall, at 9 months TRF-budesonide (<em>1</em>6 mg/day plus 8 mg/day) was associated with a 24·4% (SEM 7·7%) decrease from baseline in mean UPCR (change in UPCR vs placebo 0·74; 95% CI 0·59-0·94; p=0·0066). At 9 months, mean UPCR had decreased by 27·3% in 48 patients who received <em>1</em>6 mg/day (0·7<em>1</em>; 0·53-0·94; p=0·0092) and 2<em>1</em>·5% in the 5<em>1</em> patients who received 8 mg/day (0·76; 0·58-<em>1</em>·0<em>1</em>; p=0·0290); 50 patients who received placebo had an increase in mean UPCR of 2·7%. The effect was sustained throughout followup. Incidence of adverse events was similar in all groups (43 [88%] of 49 in the TRF-budesonide <em>1</em>6 mg/day group, 48 [94%] of 5<em>1</em> in the TRF-budesonide 8 mg/day, and 42 [84%] of 50 controls). Two of <em>1</em>3 serious adverse events were possibly associated with TRF-budesonide-deep vein thrombosis (<em>1</em>6 mg/day) and unexplained deterioration in renal function in follow-up (patients were tapered from <em>1</em>6 mg/day to 8 mg/day over 2 weeks and follow-up was assessed 4 weeks later).

CONCLUSIONS

TRF-budesonide <em>1</em>6 mg/day, added to optimised RAS blockade, reduced proteinuria in patients with IgA nephropathy. This effect is indicative of a reduced risk of future progression to end-stage renal disease. TRF-budesonide could become the first specific treatment for IgA nephropathy targeting intestinal mucosal immunity upstream of disease manifestation.

BACKGROUND

Pharmalink AB.

Thirty years ago, a novel axis of the renin-<em>angiotensin</em> system (RAS) was unveiled by the discovery of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [ANG-(<em>1</em>-<em>7</em>)] generation in vivo. Later, <em>angiotensin</em>-converting enzyme 2 (ACE2) was shown to be the main mediator of this reaction, and Mas was found to be the receptor for the heptapeptide. The functional analysis of this novel axis of the RAS that followed its discovery revealed numerous protective actions in particular for cardiovascular diseases. In parallel, similar protective actions were also described for one of the two receptors of ANG II, the ANG II type 2 receptor (AT₂R), in contrast to the other, the ANG II type <em>1</em> receptor (AT_<em>1</em>R), which mediates deleterious actions of this peptide, e.g., in the setting of cardiovascular disease. Very recently, another branch of the RAS was discovered, based on <em>angiotensin</em> peptides in which the amino-terminal aspartate was replaced by alanine, the alatensins. Ala-ANG-(<em>1</em>-<em>7</em>) or alamandine was shown to interact with Mas-related G protein-coupled receptor D, and the first functional data indicated that this peptide also exerts protective effects in the cardiovascular system. This review summarizes the presentations given at the International Union of Physiological Sciences Congress in Rio de Janeiro, Brazil, in 20<em>1</em><em>7</em>, during the symposium entitled "The Renin-<em>Angiotensin</em> System: Going Beyond the Classical Paradigms," in which the signaling and physiological actions of ANG-(<em>1</em>-<em>7</em>), ACE2, AT₂R, and alatensins were reported (with a focus on noncentral nervous system-related tissues) and the therapeutic opportunities based on these findings were discussed.

The main goal of this study was to determine whether kidney-specific induction of heme oxygenase-<em>1</em> (HO-<em>1</em>) can prevent the development of <em>angiotensin</em> (Ang) II-dependent hypertension. To test this hypothesis, intrarenal medullary interstitial catheters were implanted into the left kidney of uninephrectomized mice. Infusion of cobalt protoporphyrin (CoPP; 250 microg/mL; at 50 microL/h for 48 hours) resulted in significant induction of HO-<em>1</em> in the renal medulla when examined 2 weeks after the infusion with no induction observed in other organs, such as the heart or liver. Next, we examined the effect of renal-specific induction of HO-<em>1</em> on the development of Ang II-dependent hypertension. CoPP or vehicle (0.<em>1</em> mol/L NaOH [pH 8.3]) was infused as indicated above 2 days before implantation of an osmotic minipump, which delivered Ang II or saline vehicle at a rate of <em>1</em> microg/kg per minute. Mean arterial pressure was measured in conscious, unrestrained mice for 3 consecutive days starting on day <em>7</em> after implantation of the minipumps. Mean arterial pressure averaged <em>1</em><em>1</em>4+/-5, <em>1</em>22+/-4, <em>1</em>62+/-2, and <em>1</em>25+/-6 mm Hg in vehicle-, intrarenal medullary interstitial CoPP-, Ang II-, and Ang II + intrarenal medullary interstitial CoPP-treated mice, respectively (n=6 or <em>7</em>). These results demonstrate that kidney-specific induction of HO-<em>1</em> prevents the development of Ang II-dependent hypertension and that induction of HO-<em>1</em> in the kidney may be the mechanism by which systemic delivery of CoPP lowers blood pressure in Ang II-dependent hypertension.