Biochemical analysis revealed that <em>angiotensin</em>-converting enzyme related carboxy-peptidase (ACE2) cleaves <em>angiotensin</em> (Ang) II to Ang-(<em>1</em>-<em>7</em>), a heptapeptide identified as an endogenous ligand for the G protein-coupled receptor Mas. No data are currently available that systematically describe ACE2 distribution and activity in rodents. Therefore, we analyzed the ACE2 expression in different tissues of mice and rats on mRNA (RNase protection assay) and protein levels (immunohistochemistry, ACE2 activity, western blot). Although ACE2 mRNA in both investigated species showed the highest expression in the ileum, the mouse organ exceeded rat ACE2, as also demonstrated in the kidney and colon. Corresponding to mRNA, ACE2 activity was highest in the ileum and mouse kidney but weak in the rat kidney, which was also confirmed by immunohistochemistry. Contrary to mRNA, we found weak activity in the lung of both species. Our data demonstrate a tissue- and species-specific pattern for ACE2 under physiological conditions.

The current study was undertaken to determine whether Ang-(<em>1</em>-<em>7</em>) is effective in improving metabolic parameters in fructose-fed rats (FFR), a model of metabolic syndrome. Six-week-old male Sprague-Dawley rats were fed either normal rat chow (control) or the same diet plus <em>1</em>0% fructose in drinking water. For the last 2 wk of a 6-wk period of either diet, control and FFR were implanted with subcutaneous osmotic pumps that delivered Ang-(<em>1</em>-<em>7</em>) (<em>1</em>00 ng.kg(-<em>1</em>).min(-<em>1</em>)). A subgroup of each group of animals (control or FFR) underwent a sham surgery. We measured systolic blood pressure (SBP) together with plasma levels of insulin, triglycerides, and glucose. A glucose tolerance test (GTT) was performed, with plasma insulin levels determined before and <em>1</em>5 and <em>1</em>20 min after glucose administration. In addition, we evaluated insulin signaling through the IR/IRS-<em>1</em>/PI3K/Akt pathway as well as the phosphorylation levels of IRS-<em>1</em> at inhibitory site Ser(30<em>7</em>) in skeletal muscle and adipose tissue. FFR displayed hypertriglyceridemia, hyperinsulinemia, increased SBP, and an exaggerated release of insulin during a GTT, together with decreased activation of insulin signaling through the IR/IRS-<em>1</em>/PI3K/Akt pathway in skeletal muscle, liver, and adipose tissue, as well as increased levels of IRS-<em>1</em> phospho-Ser(30<em>7</em>) in skeletal muscle and adipose tissue, alterations that correlated with increased activation of the kinases mTOR and JNK. Chronic Ang-(<em>1</em>-<em>7</em>) treatment resulted in normalization of all alterations. These results show that Ang-(<em>1</em>-<em>7</em>) ameliorates insulin resistance in a model of metabolic syndrome via a mechanism that could involve the modulation of insulin signaling.

It has been described recently that the nonpeptide AVE 099<em>1</em> (AVE) mimics the effects of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] in bovine endothelial cells. In this study, we tested the possibility that AVE is an agonist of the Ang-(<em>1</em>-<em>7</em>) receptor Mas, in vitro and in vivo. In water-loaded C5<em>7</em>BL/6 mice, AVE (0.58 nmol/g body weight) produced a significant reduction in urinary volume (0.06+/-0.03 mL/60 min [n=9] versus 0.2<em>7</em>+/-0.05 [n=9]; P<0.0<em>1</em>), associated with an increase in urinary osmolality. The Ang-(<em>1</em>-<em>7</em>) antagonist A-<em>7</em><em>7</em>9 completely blocked the antidiuretic effect of AVE. As observed previously for Ang-(<em>1</em>-<em>7</em>), the antidiuretic effect of AVE after water load was blunted in Mas-knockout mice (0.3<em>7</em>+/-0.<em>1</em>0 mL/60 min [n=9] versus 0.2<em>7</em>+/-0.03 mL/60 min [n=<em>1</em><em>1</em>] AVE-treated mice). In vitro receptor autoradiography in C5<em>7</em>BL/6 mice showed that the specific binding of <em>1</em>25I-Ang-(<em>1</em>-<em>7</em>) to mouse kidney slices was displaced by AVE, whereas no effects were observed in the binding of <em>1</em>25I-<em>angiotensin</em> II or <em>1</em>25I-<em>angiotensin</em> IV. Furthermore, AVE displaced the binding of <em>1</em>25I-Ang-(<em>1</em>-<em>7</em>) in Mas-transfected monkey kidney cells (COS) cells (IC50=4.<em>7</em>5x<em>1</em>0(-8) mol/L) and of rhodamine-Ang-(<em>1</em>-<em>7</em>) in Mas-transfected Chinese hamster ovary (CHO) cells. It also produced NO release in Mas-transfected CHO cells blocked by A-<em>7</em><em>7</em>9 but not by <em>angiotensin</em> II type-<em>1</em> (AT<em>1</em>) and AT2 antagonists. Contrasting with these data, the antidiuretic effect of AVE was totally blocked by AT2 antagonists and partially blocked (approximately 60%) by AT<em>1</em> antagonists. The binding data, the results obtained in Mas-knockout mice and in Mas-transfected cells, show that AVE is a Mas receptor agonist. Our data also suggest the involvement of AT2/AT<em>1</em>-related mechanisms, including functional antagonism, oligomerization or cross-talk, in the renal responses to AVE.

Activation of the renin-<em>angiotensin</em> (Ang) system induces inflammation via interaction between Ang II and type <em>1</em> receptor on leukocytes. The relevance of the new arm of the renin-Ang system, namely Ang-converting enzyme-2/Ang-(<em>1</em>-<em>7</em>)/Mas receptor, for inflammatory responses is not known and was investigated in this study. For this purpose, two experimental models were used: Ag-induced arthritis (AIA) in mice and adjuvant-induced arthritis (AdIA) in rats. Male C5<em>7</em>BL/6 wild-type or Mas(-/-) mice were subjected to AIA and treated with Ang-(<em>1</em>-<em>7</em>), the Mas agonist AVE 099<em>1</em>, or vehicle. AdIA was performed in female rats that were given AVE 099<em>1</em> or vehicle. In wild-type mice, Mas protein is expressed in arthritic joints. Administration of AVE 099<em>1</em> or Ang-(<em>1</em>-<em>7</em>) decreased AIA-induced neutrophil accumulation, hypernociception, and production of TNF-α, IL-<em>1</em>β, and CXCL<em>1</em>. Histopathological analysis showed significant reduction of inflammation. Mechanistically, AVE 099<em>1</em> reduced leukocyte rolling and adhesion, even when given after Ag challenge. Mas(-/-) mice subjected to AIA developed slightly more pronounced inflammation, as observed by greater neutrophil accumulation and cytokine release. Administration of AVE 099<em>1</em> was without effect in Mas(-/-) mice subjected to AIA. In rats, administration of AVE 099<em>1</em> decreased edema, neutrophil accumulation, histopathological score, and production of IL-<em>1</em>β and CXCL<em>1</em> induced by AdIA. Therefore, activation of Mas receptors decreases neutrophil influx and cytokine production and causes significant amelioration of arthritis in experimental models of arthritis in rats and mice. This approach might represent a novel therapeutic opportunity for arthritis.

The distribution of hepatic binding sites for the calcium-mobilizing second messenger, inositol <em>1</em>,4,5-trisphosphate (IP3), was analyzed in subcellular fractions of the rat liver by binding studies with [32P]IP3 and compared with the Ca2+ release elicited by IP3 in each fraction. Three major subcellular fractions enriched in plasma membrane, mitochondria, and endoplasmic reticulum were characterized for their 5'-nucleotidase, glucose-6-phosphatase, succinate reductase, and <em>angiotensin</em> II binding activities. The fraction enriched in plasma membrane showed <em>7</em>- and 20-fold increases in IP3 binding capacity over those enriched in endoplasmic reticulum and mitochondria, respectively, and contained a single class of high-affinity binding sites with Kd of <em>1</em>.<em>7</em> +/- <em>1</em>.0 nM and concentration of 239 +/- 9<em>1</em> fmol/mg protein. IP3 binding reached equilibrium in 30 min at 0 degrees C, and the half-time of dissociation was about <em>1</em>5 min. The specificity of the IP3 binding sites was indicated by their markedly lower affinities for inositol <em>1</em>-phosphate, phytic acid, fructose <em>1</em>,6-bisphosphate, 2,3-bisphosphoglycerate, and inositol <em>1</em>,3,4,5-tetrakisphosphate. The Ca2+-releasing activity of IP3 in the subcellular fractions was monitored with the fluorescent indicator, Fura-2. All three fractions showed ATP-dependent Ca2+ uptake and rapidly released Ca2+ in response in IP3. The fraction enriched in plasma membrane was the most active in this regard, releasing <em>1</em><em>7</em>4 +/- 6<em>7</em> pmol Ca2+/mg of protein compared to 45 +/- <em>1</em>0 and 48 +/- <em>7</em> pmol/mg protein for the fractions enriched in endoplasmic reticulum and mitochondria, respectively. These data suggest that the [32P]IP3 binding sites represent specific intracellular receptors through which IP3 mobilizes Ca2+ from a storage site associated (or co-purifying) with the plasma membrane of the rat liver. It is likely that a specialized vesicular system (to which IP3 can bind and trigger the release of Ca2+) is located in close proximity with the plasma membrane and is thus adjacent to the site at which IP3 is produced during stimulation of the hepatocyte by Ca2+-mobilizing hormones.

<em>Angiotensin</em> II (AII) binds to specific G protein-coupled receptors and is mitogenic in adrenal, liver epithelial, and vascular smooth muscle cells. Since the cyclin D<em>1</em> gene encodes the regulatory subunit of the cyclin D<em>1</em>-dependent kinase (CD<em>1</em>K) required for phosphorylation of the retinoblastoma protein (pRB), an essential and rate-limiting step in G<em>1</em> phase progression of the cell cycle, we examined the effect of AII on cyclin D<em>1</em> expression and CD<em>1</em>K activity in the human adrenal cell line H295R. AII (<em>1</em>0(-6) M) stimulated G<em>1</em> phase progression within <em>1</em>2 h, with a maximal effect after <em>7</em>2 h. This action was antedated by the induction of cyclin D<em>1</em> mRNA (3-fold), cyclin D<em>1</em> nuclear protein abundance (4-fold), and CD<em>1</em>K activity (4-fold). AII induced cyclin D<em>1</em> promoter activity 4-fold, via the AT<em>1</em> receptor through an enhancer sequence at -954 base pairs. c-Fos and c-Jun bound the cyclin D<em>1</em> -954 enhancer sequence, and the abundance of c-Fos within this complex was increased by AII treatment. AII induced extracellular signal-regulated kinase (ERK) activity <em>7</em>-fold, and dominant-negative mutants of either p2<em>1</em>(ras) or ERK reduced AII-stimulated cyclin D<em>1</em> promoter activity. These findings suggest that AII may stimulate mitogenesis by increasing CD<em>1</em>K activity through a p2<em>1</em>(ras)/ERK/activator protein <em>1</em> pathway.

Intraglomerular ANG II has been linked to glomerular injury. However, little is known about the contribution of podocytes (POD) to intraglomerular ANG II homeostasis. The aim of the present study was to examine the processing of <em>angiotensin</em> substrates by cultured POD. Our approach was to use matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for peptide determination from conditioned cell media and customized AQUA peptides for quantification. Immortalized mouse POD were incubated with <em>1</em>-2 microM ANG I, ANG II, or the renin substrate ANG-(<em>1</em>-<em>1</em>4) for different time intervals and coincubated in parallel with various inhibitors. Human mesangial cells (MES) were used as controls. POD incubated with <em>1</em> microM ANG I primarily formed ANG-(<em>1</em>-9) and ANG-(<em>1</em>-<em>7</em>). In contrast, MES incubated with ANG I primarily generated ANG II. In POD, ANG-(<em>1</em>-<em>7</em>) was the predominant product, and its formation was inhibited by a neprilysin inhibitor. Modest <em>angiotensin</em>-converting enzyme (ACE) activity was also detected in POD, although only after cells were incubated with 2 microM ANG I. In addition, we observed that POD degraded ANG II into ANG III and ANG-(<em>1</em>-<em>7</em>). An aminopeptidase A inhibitor inhibited ANG III formation, and an ACE2 inhibitor led to ANG II accumulation. Furthermore, we found that POD converted ANG-(<em>1</em>-<em>1</em>4) to ANG I and ANG-(<em>1</em>-<em>7</em>). This conversion was inhibited by a renin inhibitor. These findings demonstrate that POD express a functional intrinsic renin-<em>angiotensin</em> system characterized by neprilysin, aminopeptidase A, ACE2, and renin activities, which predominantly lead to ANG-(<em>1</em>-<em>7</em>) and ANG-(<em>1</em>-9) formation, as well as ANG II degradation. These findings may reflect a specific role of POD in maintenance of intraglomerular renin-<em>angiotensin</em> system balance.

It is widely accepted that proteinuria reduction is an appropriate therapeutic goal in chronic proteinuric kidney disease. Based on large randomized controlled clinical trials (RCT), ACE inhibitor (ACEI) and <em>angiotensin</em> receptor blocker (ARB) therapy have emerged as the most important antiproteinuric and renal protective interventions. However, there are numerous other interventions that have been shown to be antiproteinuric and, therefore, likely to be renoprotective. Unfortunately testing each of these antiproteinuric therapies in RCT is not feasible. The nephrologist has two choices: restrict antiproteinuric therapies to those shown to be effective in RCT or expand the use of antiproteinuric therapies to include those that, although unproven, are plausibly effective and prudent to use. The goal of this work is to provide the documentation needed for the nephrologist to choose between these strategies. This work describes 25 separate interventions that are either antiproteinuric or may block injurious mechanisms of proteinuria. Each intervention is assigned a level of recommendation (Level <em>1</em> is the highest; Level 3 is the lowest) according to the strength of the evidence supporting its antiproteinuric and renoprotective efficacy. Pathophysiologic mechanisms possibly involved are also discussed. The number of interventions at each level of recommendation are: Level <em>1</em>, n = <em>7</em>; Level 2, n = 9; Level 3, n = 9. Our experience indicates that we can achieve in most patients the majority of Level <em>1</em> and many of the Level 2 and 3 recommendations. We suggest that, until better information becomes available, a broad-based, multiple-risk factor intervention to reduce proteinuria can be justified in those with progressive nephropathies. This work is intended primarily for clinical nephrologists; therefore, each antiproteinuria intervention is described in practical detail.

Our previous studies have indicated that chronic treatment with <em>1</em>-[(2-dimethylamino) ethylamino]-4-(hydroxymethyl)-<em>7</em>-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one (XNT), an <em>angiotensin</em>-converting enzyme 2 (ACE2) activator, reverses hypertension-induced cardiac and renal fibrosis in spontaneously hypertensive rats (SHRs). Furthermore, XNT prevented pulmonary vascular remodelling and right ventricular hypertrophy and fibrosis in a rat model of monocrotaline-induced pulmonary hypertension. The aim of this study was to determine the mechanisms underlying the protective effects of XNT against cardiac fibrosis. Hydroxyproline assay was used to measure cardiac collagen content in control and XNT-treated (200 ng kg(-<em>1</em>) min(-<em>1</em>) for 28 days) SHRs. Cardiac ACE2 activity and protein levels were determined using the fluorogenic peptide assay and Western blot analysis, respectively. Extracellular signal-regulated kinases (ERKs; p44 and p42) and <em>angiotensin</em> II type <em>1</em> (AT(<em>1</em>)) receptor levels were quantified by Western blotting. Cardiac ACE2 protein levels were ∼<em>1</em>5% lower in SHRs compared with Wistar-Kyoto control animals (ACE2/glyceraldehyde 3-phosphate dehydrogenase ratio: Wistar-Kyoto, <em>1</em>.00 ± 0.02 versus SHR, 0.8<em>7</em> ± 0.0<em>1</em>). However, treatment of SHRs with XNT completely restored the decreased cardiac ACE2 levels. Also, chronic infusion of XNT significantly increased cardiac ACE2 activity in SHRs. This increase in ACE2 activity was associated with decreased cardiac collagen content. Furthermore, the antifibrotic effect of XNT correlated with increased cardiac <em>angiotensin</em>-(<em>1</em>-<em>7</em>) immunostaining, though no change in cardiac AT(<em>1</em>) protein levels was observed. The beneficial effects of XNT were also accompanied by a reduction in ERK phosphorylation (phospho-ERK/total ERK ratio: Wistar-Kyoto, <em>1</em>.00 ± 0.04; control SHR, <em>1</em>.46 ± 0.25; treated SHR, 0.86 ± 0.02). Our observations demonstrate that XNT activates cardiac ACE2 and inhibits fibrosis. These effects are associated with increases in <em>angiotensin</em>-(<em>1</em>-<em>7</em>) and inhibition of cardiac ERK signalling.

BACKGROUND

Diabetes mellitus is a predictor of morbidity and mortality in patients with heart failure. The effect of angiotensin-converting enzyme (ACE) inhibitors on the prevention of diabetes in patients with left ventricular dysfunction is unknown. The aim of this retrospective study was to assess the effect of the ACE inhibitor enalapril on the incidence of diabetes in the group of patients from the Montreal Heart Institute enrolled in the Studies of Left Ventricular Dysfunction (SOLVD).

RESULTS

Clinical charts were evaluated for fasting plasma glucose (FPG) levels by blinded reviewers. A diagnosis of diabetes was made when a FPG>> or =126 mg/dL (7 mmol/L) was found at 2 visits (follow-up, 2.9+/-1.0 years). Of the 391 patients enrolled at the Montreal Heart Institute, 291 were not diabetic (FPG <126 mg/dL without a history of diabetes), 153 of these were on enalapril and 138 were on placebo. Baseline characteristics were similar in the 2 groups. Forty patients developed diabetes during follow-up, 9 (5.9%) in the enalapril group and 31 (22.4%) in the placebo group (P<0.0001). By multivariate analysis, enalapril remained the most powerful predictor for risk reduction of developing diabetes (hazard ratio, 0.22; 95% confidence intervals, 0.10 to 0.46; P<0.0001). The effect of enalapril was striking in the subgroup of patients with impaired FPG (110 mg/dL [6.1 mmol/L] < or =FPG <126 mg/dL) at baseline: 1 patient (3.3%) in the enalapril group versus 12 (48.0%) in the placebo group developed diabetes (P<0.0001).

CONCLUSIONS

Enalapril significantly reduces the incidence of diabetes in patients with left ventricular dysfunction, especially those with impaired FPG.

OBJECTIVE

Recent studies have demonstrated that perivascular adipose tissue (PVAT) releases vascular relaxation factor(s), but the identity of this relaxation factor remains unknown. Here, we examined if <em>angiotensin</em> <em>1</em>-<em>7</em> [Ang-(<em>1</em>-<em>7</em>)] is one of the relaxation factors released by PVAT.

METHODS

Morphological and functional methods were used to study aorta from adult Wistar rats.

RESULTS

Immunohistochemical staining showed abundant presence of Ang-(<em>1</em>-<em>7</em>) in aortic PVAT. In vessels with PVAT removed but intact endothelium (PVAT - E+), contraction induced by phenylephrine was attenuated by preincubation with Ang-(<em>1</em>-<em>7</em>). PVAT - E+ vessels precontracted with phenylephrine showed a concentration-dependent relaxation response to Ang-(<em>1</em>-<em>7</em>), and this response was abolished by the removal of endothelium. Relaxation response induced by Ang-(<em>1</em>-<em>7</em>) was also prevented by Ang-(<em>1</em>-<em>7</em>) receptor (Mas) antagonist (A<em>7</em><em>7</em>9), nitric oxide synthase inhibitor, and nitric oxide scavenger. Ang-(<em>1</em>-<em>7</em>) did not cause a relaxation response in aorta precontracted with KCl, and the relaxation response to Ang-(<em>1</em>-<em>7</em>) was also blocked by calcium-dependent potassium (K(Ca)) channel blockers. Incubation of PVAT + E+ vessels with A<em>7</em><em>7</em>9 or <em>angiotensin</em>-converting enzyme 2 inhibitor DX600 or <em>angiotensin</em>-converting enzyme inhibitor enalaprilat increased the contraction induced by phenylephrine. Transfer of donor solution incubated with PVAT + E+ vessel to recipient PVAT - E+ vessel caused a relaxation response. This relaxation response was abolished when donor vessels were incubated with DX600 or enalaprilat or when recipient vessels were incubated with A<em>7</em><em>7</em>9.

CONCLUSIONS

Ang-(<em>1</em>-<em>7</em>) released by PVAT acts on the endothelium to cause the release of nitric oxide, and nitric oxide acts as a hyperpolarizing factor through K(Ca) channels to cause relaxation of the blood vessel.

BACKGROUND

We have demonstrated that accumulated macrophages in human coronary arteries strongly express angiotensin converting enzyme in accordance with the development of atheromatous plaques. However, there are few reports on the regulation of the renin-angiotensin system in macrophages and in monocytes as their source.

OBJECTIVE

To examine whether the renin-angiotensin system is upregulated during the differentiation of monocytes to macrophages, and whether it is further regulated by angiotensin II and cytokines.

METHODS

We used a human leukemia cell line, THP-1, for monocytes. Differentiated THP-1, induced by adding phorbol 12-myristate 13-acetate for 24 h, were used as macrophages. Expression of messenger RNA of the renin-angiotensin system components was measured by quantitative reverse-transcriptase polymerase chain reaction. Angiotensin converting enzyme activity and subtype-specific angiotensin-binding sites of cultured cells, and angiotensin II production in the culture medium were measured.

RESULTS

Macrophages expressed all components of the renin-angiotensin system except chymase. Cellular angiotensin converting enzyme activity and angiotensin II in the medium were increased 3.2- and 4.5-fold during differentiation, respectively. Expression of angiotensin II type 1 (AT1) and type 2 (AT2) receptors was increased 6.2-and 6.4-fold during differentiation, and was sustained for 7 days. Incubation with angiotensin II for 24 h caused downregulation of both AT1 and AT2 receptor messenger RNA, but the expression levels were still more than threefold higher compared with monocytes. The density of binding sites of AT1 and AT2 receptors in macrophages was 0.26 +/- 0.02 and 0.15 +/- 0.01 fmol/10(6) cells, respectively.

CONCLUSIONS

The renin-angiotensin system is markedly activated during monocyte/macrophage differentiation, and may participate in the development of atherosclerosis.

Increased plasma renin activity (PRA) has been associated with an increased risk of myocardial infarction (MI), whereas <em>angiotensin</em>-converting enzyme (ACE) inhibition appears to reduce the risk of recurrent MI in patients with left ventricular dysfunction. These observations may be partially explained by an interaction between the renin-<em>angiotensin</em> system (RAS) and fibrinolytic system. To test this hypothesis, we examined the effect of salt depletion on tissue-type plasminogen activator (tPA) antigen and plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>) activity and antigen in normotensive subjects in the presence and absence of quinapril (40 mg BID). Under low (<em>1</em>0 mmol/d) and high (200 mmol/d) salt conditions there was significant diurnal variation in PAI-<em>1</em> antigen and activity and tPA antigen. Morning (8 AM through 2 PM) PAI-<em>1</em> antigen levels were significantly higher during low salt intake compared with high salt intake conditions (ANOVA, F=5.8, P=0.048). PAI-<em>1</em> antigen correlated with aldosterone (r=0.56, P(<em>1</em>0(-<em>7</em>)) during low salt intake. ACE inhibition significantly decreased 24-hour (ANOVA for 24 hours, F=6. <em>7</em>, P=0.04) and morning (F=24, P=0.002) PAI-<em>1</em> antigen and PAI-<em>1</em> activity (F=6.48, P=0.038) but did not alter tPA antigen. Thus, the mean morning PAI-<em>1</em> antigen concentration was significantly higher during low salt intake than during either high salt intake or low salt intake and concomitant ACE inhibition (22.<em>7</em>+/-4.6 versus <em>1</em>6. <em>1</em>+/-3.3 and <em>1</em>6.3+/-3.<em>7</em> ng/mL, respectively; P<0.05). This study provides evidence of a direct functional link between the RAS and fibrinolytic system in humans. The data suggest that ACE inhibition has the potential to reduce the incidence of thrombotic cardiovascular events by blunting the morning peak in PAI-<em>1</em>.

The renin-<em>angiotensin</em> system (RAS) is a complex network that regulates blood pressure, electrolyte and fluid homeostasis, as well as the function of several organs. <em>Angiotensin</em>-converting enzyme 2 (ACE2) was identified as an enzyme that negatively regulates the RAS by converting Ang II, the main bioactive molecule of the RAS, to Ang <em>1</em>-<em>7</em>. Thus, ACE2 counteracts the role of <em>angiotensin</em>-converting enzyme (ACE) which generates Ang II from Ang I. ACE and ACE2 have been implicated in several pathologies such as cardiovascular and renal disease or acute lung injury. In addition, ACE2 has functions independent of the RAS: ACE2 is the receptor for the SARS coronavirus and ACE2 is essential for expression of neutral amino acid transporters in the gut. In this context, ACE2 modulates innate immunity and influences the composition of the gut microbiota, which can explain diarrhea and intestinal inflammation observed in Hartnup disorder, Pellagra, or under conditions of severe malnutrition. Here we review and discuss the diverse functions of ACE2 and its relevance to human pathologies.

OBJECTIVE

<em>Angiotensin</em>-converting enzyme 2 (ACE2), its product, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) and its receptor, Mas, may moderate the adverse effects of <em>angiotensin</em> II in liver disease. We examined the expression of these novel components of the renin <em>angiotensin</em> system (RAS) and the production and vasoactive effects of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) in the bile duct ligated (BDL) rat.

METHODS

BDL or sham-operated rats were sacrificed at <em>1</em>, 2, 3 and 4 weeks. Tissue and blood were collected for gene expression, enzyme activity and peptide measurements. In situ perfused livers were used to assess <em>angiotensin</em> peptide production and their effects on portal resistance.

RESULTS

Hepatic ACE2 gene and activity (P<0.0005), plasma <em>angiotensin</em>-(<em>1</em>-<em>7</em>) (P<0.0005) and Mas receptor expression (P<0.0<em>1</em>) were increased following BDL compared to shams. Perfusion experiments confirmed that BDL livers produced increased <em>angiotensin</em>-(<em>1</em>-<em>7</em>) (P<0.05) from <em>angiotensin</em> II and this was augmented (P<0.0<em>1</em>) by ACE inhibition. Whilst <em>angiotensin</em> II increased vasoconstriction in cirrhotic livers, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) had no effect on portal resistance.

CONCLUSIONS

RAS activation in chronic liver injury is associated with upregulation of ACE2, Mas and hepatic conversion of <em>angiotensin</em> II to <em>angiotensin</em>-(<em>1</em>-<em>7</em>) leading to increased circulating <em>angiotensin</em>-(<em>1</em>-<em>7</em>). These results support the presence of an ACE2-<em>angiotensin</em>-(<em>1</em>-<em>7</em>)-Mas axis in liver injury which may counteract the effects of <em>angiotensin</em> II.

Cigarette smoking is the single most important risk factor for the development of cardiovascular and pulmonary diseases (CVPD). Although cigarette smoking has been in constant decline since the <em>1</em>950's, the introduction of e-cigarettes or electronic nicotine delivery systems <em>1</em>0 years ago has attracted former smokers as well as a new generation of consumers. Nicotine is a highly addictive substance, and it is currently unclear whether e-cigarettes are "safer" than regular cigarettes or whether they have the potential to reverse the health benefits, notably on the cardiopulmonary system, acquired with the decline of tobacco smoking. Of great concern, nicotine inhalation devices are becoming popular among young adults and youths, emphasizing the need for awareness and further study of the potential cardiopulmonary risks of nicotine and associated products. This review focuses on the interaction between nicotine and the renin-<em>angiotensin</em> system (RAS), one of the most important regulatory systems on autonomic, cardiovascular and pulmonary functions in both health and disease. The literature presented in this review strongly suggests that nicotine alters the homeostasis of the RAS by up-regulating the detrimental <em>angiotensin</em> converting enzyme (ACE)/<em>Angiotensin</em> (Ang)-II/Ang-II type <em>1</em> receptor (AT<em>1</em>R) axis and down-regulating the compensatory ACE2/Ang-(<em>1</em>-<em>7</em>)/Mas receptor axis, contributing to the development of CVPD.

BACKGROUND

Many antihyperglycaemic drugs, including insulin, are primarily cleared by the kidneys, restricting treatment options for patients with kidney disease. Dulaglutide is a long-acting glucagon-like peptide-<em>1</em> receptor agonist that is not cleared by the kidneys, and confers a lower risk of hypoglycaemia than does insulin. We assessed the efficacy and safety of dulaglutide in patients with type 2 diabetes and moderate-to-severe chronic kidney disease.

METHODS

AWARD-7 was a multicentre, open-label trial done at 99 sites in nine countries. Eligible patients were adults with type 2 diabetes and moderate-to-severe chronic kidney disease (stages 3-4), with an HbA<em>1</em>c of 7·5-<em>1</em>0·5%, and who were being treated with insulin or insulin plus an oral antihyperglycaemic drug and were taking a maximum tolerated dose of an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker. Participants were randomly assigned (<em>1</em>:<em>1</em>:<em>1</em>) by use of a computer-generated random sequence with an interactive response system to once-weekly injectable dulaglutide <em>1</em>·5 mg, once-weekly dulaglutide 0·75 mg, or daily insulin glargine as basal therapy, all in combination with insulin lispro, for 52 weeks. Insulin glargine and lispro doses were titrated as per an adjustment algorithm; dulaglutide doses were masked to participants and investigators. The primary outcome was HbA<em>1</em>c at 26 weeks, with a 0·4% non-inferiority margin. Secondary outcomes included estimated glomerular filtration rate (eGFR) and urine albumin-to-creatinine ratio (UACR). The primary analysis population was all randomly assigned patients who received at least one dose of study treatment and had at least one post-randomisation HbA<em>1</em>c measurement. The safety population was all patients who received at least one dose of study treatment and had any post-dose data. This study is registered with ClinicalTrials.gov, number NCT0<em>1</em>62<em>1</em><em>1</em>78.

RESULTS

Between Aug <em>1</em>5, 20<em>1</em>2, and Nov 30, 20<em>1</em>5, 577 patients were randomly assigned, <em>1</em>93 to dulaglutide <em>1</em>·5 mg, <em>1</em>90 to dulaglutide 0·75 mg, and <em>1</em>94 to insulin glargine. The effects on HbA<em>1</em>c change at 26 weeks of dulaglutide <em>1</em>·5 mg and 0·75 mg were non-inferior to those of insulin glargine (least squares mean [LSM] -<em>1</em>·2% [SE 0·<em>1</em>] with dulaglutide <em>1</em>·5 mg [<em>1</em>83 patients]; -<em>1</em>·<em>1</em>% [0·<em>1</em>] with dulaglutide 0·75 mg [<em>1</em>80 patients]; -<em>1</em>·<em>1</em>% [0·<em>1</em>] with insulin glargine [<em>1</em>86 patients]; one-sided p≤0·000<em>1</em> for both dulaglutide doses vs insulin glargine). The differences in HbA<em>1</em>c concentration at 26 weeks between dulaglutide and insulin glargine treatments were LSM difference -0·05% (95% CI -0·26 to 0·<em>1</em>5, p<0·000<em>1</em>) with dulaglutide <em>1</em>·5 mg and 0·02% (-0·<em>1</em>8 to -0·22, p=0·000<em>1</em>) with dulaglutide 0·75 mg. HbA<em>1</em>c-lowering effects persisted to 52 weeks (LSM -<em>1</em>·<em>1</em>% [SE 0·<em>1</em>] with dulaglutide <em>1</em>·5 mg; -<em>1</em>·<em>1</em>% [0·<em>1</em>] with dulaglutide 0·75 mg; -<em>1</em>·0% [0·<em>1</em>] with insulin glargine). At 52 weeks, eGFR was higher with dulaglutide <em>1</em>·5 mg (Chronic Kidney Disease Epidemiology Collaboration equation by cystatin C geometric LSM 34·0 mL/min per <em>1</em>·73 m2 [SE 0·7]; p=0·005 vs insulin glargine) and dulaglutide 0·75 mg (33·8 mL/min per <em>1</em>·73 m2 [0·7]; p=0·009 vs insulin glargine) than with insulin glargine (3<em>1</em>·3 mL/min per <em>1</em>·73 m2 [0·7]). At 52 weeks, the effects of dulaglutide <em>1</em>·5 mg and 0·75 mg on UACR reduction were not significantly different from that of insulin glargine (LSM -22·5% [95% CI -35·<em>1</em> to -7·5] with dulaglutide <em>1</em>·5 mg; -20·<em>1</em>% [-33·<em>1</em> to -4·6] with dulaglutide 0·75 mg; -<em>1</em>3·0% [-27·<em>1</em> to 3·9] with insulin glargine). Proportions of patients with any serious adverse events were similar across groups (20% [38 of <em>1</em>92] with dulaglutide <em>1</em>·5 mg, 24% [45 of <em>1</em>90] with dulaglutide 0·75 mg, and 27% [52 of <em>1</em>94] with insulin glargine). Dulaglutide was associated with higher rates of nausea (20% [38 of <em>1</em>92] with dulaglutide <em>1</em>·5 mg and <em>1</em>4% [27 of <em>1</em>90] with 0·75 mg, vs 5% [nine of <em>1</em>94] with insulin glargine) and diarrhoea (<em>1</em>7% [33 of <em>1</em>92] with dulaglutide <em>1</em>·5 mg and <em>1</em>6% [30 of <em>1</em>90] with 0·75 mg, vs 7% [<em>1</em>4 of <em>1</em>94] with insulin glargine) and lower rates of symptomatic hypoglycaemia (4·4 events per patient per year with dulaglutide <em>1</em>·5 mg and 4·3 with dulaglutide 0·75 mg, vs 9·6 with insulin glargine). End-stage renal disease occurred in 38 participants: eight (4%) of <em>1</em>92 with dulaglutide <em>1</em>·5 mg, <em>1</em>4 (7%) of <em>1</em>90 with dulaglutide 0·75 mg, and <em>1</em>6 (8%) of <em>1</em>94 with insulin glargine.

CONCLUSIONS

In patients with type 2 diabetes and moderate-to-severe chronic kidney disease, once-weekly dulaglutide produced glycaemic control similar to that achieved with insulin glargine, with reduced decline in eGFR. Dulaglutide seems to be safe to use to achieve glycaemic control in patients with moderate-to-severe chronic kidney disease.

BACKGROUND

Eli Lilly and Company.

Many cells (including <em>angiotensin</em> II target cells) respond to external stimuli with accelerated hydrolysis of phosphatidylinositol 4,5-bisphosphate, generating <em>1</em>,2-diacylglycerol and inositol <em>1</em>,4,5-trisphosphate, a rapidly diffusible and potent Ca2+-mobilizing factor. Following its production at the plasma membrane level, inositol <em>1</em>,4,5-trisphosphate is believed to interact with specific sites in the endoplasmic reticulum and triggers the release of stored Ca2+. Specific receptor sites for inositol <em>1</em>,4,5-trisphosphate were recently identified in the bovine adrenal cortex (Baukal, A. J., Guillemette, G., Rubin, R., Spät, A., and Catt, K. J. (<em>1</em>985) Biochem. Biophys. Res. Commun. <em>1</em>33, 532-538) and have been further characterized in the adrenal cortex and other target tissues. The inositol <em>1</em>,4,5-trisphosphate-binding sites are saturable and present in low concentration (<em>1</em>04 +/- 48 fmol/mg protein) and exhibit high affinity for inositol <em>1</em>,4,5-trisphosphate (Kd <em>1</em>.<em>7</em> +/- 0.6 nM). Their ligand specificity is illustrated by their low affinity for inositol <em>1</em>,4-bisphosphate (Kd approximately <em>1</em>0(-<em>7</em>) M), inositol <em>1</em>-phosphate and phytic acid (Kd approximately <em>1</em>0(-4) M), fructose <em>1</em>,6-bisphosphate and 2,3-bisphosphoglycerate (Kd approximately <em>1</em>0(-3) M), with no detectable affinity for inositol <em>1</em>-phosphate and myo-inositol. These binding sites are distinct from the degradative enzyme, inositol trisphosphate phosphatase, which has a much lower affinity for inositol trisphosphate (Km = <em>1</em><em>7</em> microM). Furthermore, submicromolar concentrations of inositol <em>1</em>,4,5-trisphosphate evoked a rapid release of Ca2+ from nonmitochondrial ATP-dependent storage sites in the adrenal cortex. Specific and saturable binding sites for inositol <em>1</em>,4,5-trisphosphate were also observed in the anterior pituitary (Kd = 0.8<em>7</em> +/- 0.3<em>1</em> nM, Bmax = <em>1</em>4.8 +/- 9.0 fmol/mg protein) and in the liver (Kd = <em>1</em>.66 +/- 0.<em>7</em> nM, Bmax = <em>1</em>4<em>7</em> +/- 24 fmol/mg protein). These data suggest that the binding sites described in this study are specific receptors through which inositol <em>1</em>,4,5-trisphosphate mobilizes Ca2+ in target tissues for <em>angiotensin</em> II and other calcium-dependent hormones.

Identification of <em>angiotensin</em>-(<em>1</em>-<em>1</em>2) as an intermediate precursor derived directly from <em>angiotensin</em>ogen led us to explore whether the heart has the capacity to process <em>angiotensin</em>-(<em>1</em>-<em>1</em>2) into biologically active <em>angiotensin</em> peptides. The generation of <em>angiotensin</em> I, <em>angiotensin</em> II, and <em>angiotensin</em>-(<em>1</em>-<em>7</em>) from exogenous <em>angiotensin</em>-(<em>1</em>-<em>1</em>2) was evaluated in the effluent of isolated perfused hearts mounted on a Langendorff apparatus in three normotensive and two hypertensive strains: Sprague-Dawley, Lewis, congenic mRen2.Lewis, Wistar-Kyoto, and spontaneously hypertensive rats. Hearts were perfused with Krebs solution for 60 min before and after the addition of <em>angiotensin</em>-(<em>1</em>-<em>1</em>2) (<em>1</em>0 nmol/l). <em>Angiotensin</em>-(<em>1</em>-<em>1</em>2) caused the rapid appearance of both <em>angiotensin</em> I and <em>angiotensin</em> II in the perfusate that peaked between 30 and 60 min of recirculation. Production of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) from exogenous <em>angiotensin</em>-(<em>1</em>-<em>1</em>2) rose steadily over the course of the 60-min experiment. These data directly demonstrate that <em>angiotensin</em>-(<em>1</em>-<em>1</em>2) is a substrate for the formation of <em>angiotensin</em> peptides in cardiac tissue. This finding further suggests that this <em>angiotensin</em>ogen-derived product is a previously unrecognized important precursor peptide to the renin-<em>angiotensin</em> system cascade.

BACKGROUND

Food allergy (FA) is an important health problem for which epidemiologic studies are needed.

OBJECTIVE

We performed an epidemiologic survey in France to determine the prevalence, clinical pictures, allergens, and risk factors of FA.

METHODS

This study was conducted on 33,<em>1</em><em>1</em>0 persons who answered a questionnaire addressed to a representative sample of the French population on a scale of <em>1</em>:<em>1</em>000 (44,000 subjects aged </=60 years). One thousand one hundred twenty-nine persons with FA selected during phase <em>1</em> received a second questionnaire.

RESULTS

The reported prevalence of FA is 3.52%: 3.24% evolutionary FA; 0.<em>1</em>2% asymptomatic cases thanks to eviction diets; and 0.<em>1</em>7% cured FA. The subjects were characterized by overrepresentation of city dwellers (80% vs 76%), women (63% vs 50%), and health care personnel (<em>1</em><em>1</em>% vs 4%). Fifty-seven percent (vs <em>1</em>7%) presented with atopic diseases (P <.0<em>1</em>). FA was often persistent, lasting more than 7 years in 9<em>1</em>% of the adults. The most frequent allergens were <em>1</em>4% Rosaceae, 9% vegetables, 8% milk, 8% crustaceans, 5% fruit cross-reacting with latex, 4% egg, 3% tree nuts, and <em>1</em>% peanut. Sensitization to pollen was significantly correlated with angioedema, asthma, rhinitis, and fruit allergy (P <.0<em>1</em>). FA was 4 times more frequent in patients with latex allergy. The main manifestations of FA were atopic dermatitis for subjects under 6 years of age, asthma for subjects between 4 and 6 years of age, and anaphylactic shock in adults over 30 years of age (P <.007). Shocks were correlated with alcohol or nonsteroidal anti-inflammatory drug intake (P <.0<em>1</em> and P <.04, respectively).

CONCLUSIONS

The prevalence of FA is estimated at 3.24% (range, 3.04% to 3.44%) in France. This study emphasizes the increasing risk of FA in well-developed countries and draws attention to certain FA risk factors, such as the intake of drugs (nonsteroidal anti-inflammatory drugs, beta-blockers, and angiotensin-converting enzyme inhibitors) or alcohol, intolerance of latex gloves, and socioprofessional status.

<em>Angiotensin</em>-converting enzyme 2 (ACE2) is expressed at high levels in the kidney and converts <em>angiotensin</em> II (ANG II) to ANG-(<em>1</em>-<em>7</em>). We studied the effects of ACE2 inhibition and ANG-(<em>1</em>-<em>7</em>) in the (5/6) nephrectomy ((5/6) Nx) mouse model of chronic kidney disease (CKD). Male FVB mice underwent sham surgery (Sham) or (5/6) Nx and were administered either vehicle, the ACE2 inhibitor MLN-4<em>7</em>60 (MLN), the AT(<em>1</em>) receptor antagonist losartan, MLN plus losartan, or ANG-(<em>1</em>-<em>7</em>) for 4 wk. In (5/6) Nx mice with or without MLN, kidney cortical ACE2 protein expression was significantly decreased at 4 wk, compared with Sham. Inhibition of ACE2 caused a decrease in renal cortical ACE2 activity. Kidney cortical ACE expression and activity did not differ between groups of mice. In (5/6) Nx mice treated with MLN, kidney levels of ANG II were significantly increased, compared with Sham. (5/6) Nx induced a mild but insignificant increase in blood pressure (BP), a 50% reduction in FITC-inulin clearance, and a significant increase in urinary albumin excretion. ACE2 inhibition in (5/6) Nx mice did not affect BP or FITC-inulin clearance but significantly increased albuminuria compared with (5/6) Nx alone, an effect reversed by losartan. Treatment of (5/6) Nx mice with ANG-(<em>1</em>-<em>7</em>) increased kidney and plasma levels of ANG-(<em>1</em>-<em>7</em>) but did not alter BP, FITC-inulin clearance, or urinary albumin excretion, and it increased relative mesangial area. These data indicate that kidney ACE2 is downregulated in the early period after (5/6) Nx. Inhibition of ACE2 in (5/6) Nx mice increases albuminuria via an AT(<em>1</em>) receptor-dependent mechanism, independent of BP. In contrast, ANG-(<em>1</em>-<em>7</em>) does not affect albuminuria after (5/6) Nx. We propose that endogenous ACE2 is renoprotective in CKD.

Exercise training (EX) normalizes sympathetic outflow and plasma ANG II in chronic heart failure (CHF). The central mechanisms by which EX reduces this sympathoexcitatory state are unclear, but EX may alter components of the brain renin-<em>angiotensin</em> system (RAS). <em>Angiotensin</em>-converting enzyme (ACE) may mediate an increase in sympathetic nerve activity (SNA). ACE2 metabolizes ANG II to ANG-(<em>1</em>-<em>7</em>), which may have antagonistic effects to ANG II. Little is known concerning the regulation of ACE and ACE2 in the brain and the effect of EX on these enzymes, especially in the CHF state. This study aimed to investigate the effects of EX on the regulation of ACE and ACE2 in the brain in an animal model of CHF. We hypothesized that the ratio of ACE to ACE2 would increase in CHF and would be reduced by EX. Experiments were performed on New Zealand White rabbits divided into the following groups: sham, sham + EX, CHF, and CHF + EX (n = 5 rabbits/group). The cortex, cerebellum, medulla, hypothalamus, paraventricular nucleus (PVN), nucleus tractus solitarii (NTS), and rostral ventrolateral medulla (RVLM) were analyzed. ACE protein and mRNA expression in the cerebellum, medulla, hypothalamus, PVN, NTS, and RVLM were significantly upregulated in CHF rabbits (ratio of ACE to GAPDH: 0.3 +/- 0.03 to 0.8 +/- 0.<em>1</em>0 in the RVLM, P < 0.05). EX normalized this upregulation compared with CHF (0.8 +/- 0.<em>1</em> to 0.4 +/- 0.<em>1</em> in the RVLM). ACE2 protein and mRNA expression decreased in CHF (ratio of ACE2 to GAPDH: 0.3 +/- 0.02 to 0.<em>1</em> +/- 0.0<em>1</em> in the RVLM). EX increased ACE2 expression compared with CHF (0.<em>1</em> +/- 0.0<em>1</em> to 0.8 +/- 0.<em>1</em> in the RVLM). ACE2 was present in the cytoplasm of neurons and ACE in endothelial cells. These data suggest that the activation of the central RAS in animals with CHF involves an imbalance of ACE and ACE2 in regions of the brain that regulate autonomic function and that EX can reverse this imbalance.

Age-related baroreflex reductions in function may originate from central neural dysregulation as well as vascular structural/functional changes. We determined the role of 2 <em>angiotensin</em> (Ang) peptides at the nucleus tractus solitarii in age-related baroreflex impairment. Baroreflex sensitivity control of heart rate in response to increases in blood pressure was tested in younger (3 to 5 months) and older (<em>1</em>6 to 20 months) anesthetized male Sprague-Dawley rats before and after bilateral solitary tract injections of the Ang II type <em>1</em> (AT<em>1</em>) receptor antagonist candesartan (24 pmol) or the Ang-(<em>1</em>-<em>7</em>) antagonist (D-Ala<em>7</em>)-Ang-(<em>1</em>-<em>7</em>) (<em>1</em>44 fmol or 24 pmol). Basal reflex sensitivity of older rats was significantly lower than younger rats. In younger rats, the reflex was facilitated by bilateral candesartan injections and attenuated by bilateral (D-Ala<em>7</em>)-Ang-(<em>1</em>-<em>7</em>) injections. In older rats, the reflex was facilitated by AT<em>1</em> blockade; however, (D-Ala<em>7</em>)-Ang-(<em>1</em>-<em>7</em>) injected into the solitary tract nucleus had no effect. Neprilysin mRNA in the medulla was lower in older rats compared with younger rats, whereas <em>angiotensin</em>-converting enzyme (ACE), ACE2, and mas receptor mRNA levels of older rats did not differ from values of younger rats. Thus, opposing actions of endogenous Ang II and Ang-(<em>1</em>-<em>7</em>) in the solitary tract nucleus contribute to baroreflex function in response to increases in mean arterial pressure of younger rats. The attenuated counterbalancing effect of Ang-(<em>1</em>-<em>7</em>) on baroreflex function is lost in older rats, which may be attributable to diminished production of the peptide from neprilysin.

In <em>7</em>6 patients with heart failure (HF) (New York Heart Association [NYHA] classes I through IV) and in <em>1</em>5 control subjects, cardiac <em>angiotensin</em> II (Ang II) generation and its relationship with left ventricular function were investigated by measuring aorta-coronary sinus concentration gradients of endogenous <em>angiotensins</em> and in a part of patients by studying (<em>1</em>25)I-labeled Ang I kinetics. Gene expression and cellular localization of the cardiac renin-<em>angiotensin</em> system components, the density of AT(<em>1</em>) and AT(2) on membranes and isolated myocytes, and the capacity of isolated myocytes for synthesizing the hypertrophying growth factors insulin-like growth factor-I (IGF-I) and endothelin (ET)-<em>1</em> were also investigated on 22 HF explanted hearts (NYHA classes III and IV) and <em>7</em> nonfailing (NF) donor hearts. Ang II generation increased with progression of HF, and end-systolic wall stress was the only independent predictor of Ang II formation. Angiotensinogen and <em>angiotensin</em>-converting enzyme mRNA levels were elevated in HF hearts, whereas chymase levels were not, and mRNAs were almost exclusively expressed on nonmyocyte cells. Ang II was immunohistochemically detectable both on myocytes and interstitial cells. Binding studies showed that AT(<em>1</em>) density on failing myocytes did not differ from that of NF myocytes, with preserved AT(<em>1</em>)/AT(2) ratio. Conversely, AT(<em>1</em>) density was lower in failing membranes than in NF ones. Ang II induced IGF-I and ET-<em>1</em> synthesis by isolated NF myocytes, whereas failing myocytes were unable to respond to Ang II stimulation. This study demonstrates that (<em>1</em>) the clinical course of HF is associated with progressive increase in cardiac Ang II formation, (2) AT(<em>1</em>) density does not change on failing myocytes, and (3) failing myocytes are unable to synthesize IGF-I and ET-<em>1</em> in response to Ang II stimulation.