The renin-<em>angiotensin</em> system (RAS) is critically involved in cardiovascular and renal function and in disease conditions, and has been shown to be a far more complex system than initially thought. A recently discovered homologue of <em>angiotensin</em>-converting enzyme (ACE)--ACE2--appears to negatively regulate the RAS. ACE2 cleaves Ang I and Ang II into the inactive Ang <em>1</em>-9 and Ang <em>1</em>-<em>7</em>, respectively. ACE2 is highly expressed in kidney and heart and is especially confined to the endothelium. With quantitative trait locus (QTL) mapping, ACE2 was defined as a QTL on the X chromosome in rat models of hypertension. In these animal models, kidney ACE2 messenger RNA and protein expression were markedly reduced, making ACE2 a candidate gene for this QTL. Targeted disruption of ACE2 in mice failed to elicit hypertension, but resulted in severe impairment in myocardial contractility with increased <em>angiotensin</em> II levels. Genetic ablation of ACE in the ACE2 null mice rescued the cardiac phenotype. These genetic data show that ACE2 is an essential regulator of heart function in vivo. Basal renal morphology and function were not altered by the inactivation of ACE2. The novel role of ACE2 in hydrolyzing several other peptides-such as the apelin peptides, opioids, and kinin metabolites-raises the possibility that peptide systems other than <em>angiotensin</em> and its derivatives also may have an important role in regulating cardiovascular and renal function.

Estrogen replacement therapy is cardioprotective in postmenopausal women; however, the precise molecular mechanisms for this modulation are not fully elucidated. We previously showed that chronic estrogen replacement therapy reduced <em>angiotensin</em>-converting enzyme (ACE) activity in tissue extracts and serum with an associated reduction in plasma <em>angiotensin</em> II. A reverse transcriptase-polymerase chain reaction assay was developed to determine whether estrogen treatment regulates tissue ACE mRNA concentration. Total RNA was isolated from kidney cortex, kidney medulla, lung, and aorta of ovariectomized Sprague-Dawley rats after 2<em>1</em> days of chronic <em>1</em><em>7</em>beta-estradiol replacement therapy (5 mg pellet per rat SC) or placebo. A marked decrease in densitometric intensity ratios of amplified ACE cDNA to elongation factor-<em>1</em>alpha control cDNA was observed in all tissues from placebo-treated rats compared with the estradiol-treated rats (renal cortex: 0.29+/-0.04 versus 0.<em>1</em>4+/-0.02; renal medulla: 0. 3<em>7</em>+/-0.04 versus 0.24+/-0.03; lung: 4.49+/-0.3<em>7</em> versus 2.49+/-0.59; and aorta: 0.4<em>1</em>+/-0.04 versus 0.29+/-0.02; all P<0.05). A comparable reduction in ACE activity was detected in tissue extracts from kidney cortex, kidney medulla, and lung of hormone-treated animals. Incubation of purified rat lung ACE with <em>1</em> or <em>1</em>0 micromol/L <em>1</em><em>7</em>beta-estradiol had no effect on enzyme activity. These results suggest that estrogen treatment regulates tissue ACE activity by reducing ACE mRNA concentrations. Thus, the beneficial cardiovascular effects of estrogen may be mediated in part by downregulation of ACE with a consequent reduction in the circulating levels of the vasoconstrictor <em>angiotensin</em> II, a decrease in the metabolism of the vasodilator bradykinin, and an increase in the production of the vasorelaxant <em>angiotensin</em>-(<em>1</em>-<em>7</em>).

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] was recently recognized to have novel biological functions that are distinct from those of Ang II. In these studies, we determined the vasoactive effects of Ang-(<em>1</em>-<em>7</em>) together with the endothelium-dependent mediator(s) of these responses in canine coronary arteries. Isometric tension was measured in intact canine coronary artery rings suspended in organ chambers perfused with 95% O2/5% CO2 at 3<em>7</em> degrees C. Ang-(<em>1</em>-<em>7</em>) caused significant concentration-dependent vascular relaxation (2.<em>7</em>3 +/- 0.58 micromol/L, EC50) of rings precontracted with the thromboxane A2 analogue U46,6<em>1</em>9. Pretreatment with the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (<em>1</em> mol/L) abolished the vasodilator response to Ang-(<em>1</em>-<em>7</em>), whereas treatment with the cyclooxygenase inhibitor indomethacin (<em>1</em>0 micromol/L) was without effect. The vasodilator response produced by Ang-(<em>1</em>-<em>7</em>) was blocked by <em>7</em>5% with the bradykinin B2 receptor antagonist Hoe <em>1</em>40 (<em>1</em> micromol/L) or by 80% with the nonselective Ang II antagonist [Sar<em>1</em>,Thr8]-Ang II (<em>1</em> micromol/L). In contrast, the selective AT<em>1</em> or AT2 Ang II antagonists CV <em>1</em><em>1</em>9<em>7</em>4 (<em>1</em> micromol/L), and PD <em>1</em>233<em>1</em>9 (<em>1</em> micromol/L), respectively, were ineffective in inhibiting the Ang-(<em>1</em>-<em>7</em>)-elicited vasodilation. Furthermore, pretreatment of the coronary rings with 2 micromol/L Ang-(<em>1</em>-<em>7</em>) markedly potentiated the bradykinin response. These results suggest that Ang-(<em>1</em>-<em>7</em>) elicits coronary vasodilation that is specifically mediated by the endothelium-dependent release of nitric oxide. These responses involve a B2 bradykinin receptor and a non-AT<em>1</em>, non-AT2, <em>angiotensin</em> receptor. These data suggest that increases in circulating levels of Ang-(<em>1</em>-<em>7</em>) accompanying long-term administration of converting enzyme inhibitors or Ang II receptor blockers may contribute to the cardioprotective actions of these drugs.

Adipose tissue expresses components of the renin-<em>angiotensin</em> system (RAS). <em>Angiotensin</em> converting enzyme (ACE2), a new component of the RAS, catabolizes the vasoconstrictor peptide ANG II to form the vasodilator <em>angiotensin</em> <em>1</em>-<em>7</em> [ANG-(<em>1</em>-<em>7</em>)]. We examined whether adipocytes express ACE2 and its regulation by manipulation of the RAS and by high-fat (HF) feeding. ACE2 mRNA expression increased (threefold) during differentiation of 3T3-L<em>1</em> adipocytes and was not regulated by manipulation of the RAS. Male C5<em>7</em>BL/6 mice were fed low- (LF) or high-fat (HF) diets for <em>1</em> wk or 4 mo. At <em>1</em> wk of HF feeding, adipose expression of <em>angiotensin</em>ogen (twofold) and ACE2 (threefold) increased, but systemic <em>angiotensin</em> peptide concentrations and blood pressure were not altered. At 4 mo of HF feeding, adipose mRNA expression of <em>angiotensin</em>ogen (twofold) and ACE2 (threefold) continued to be elevated, and liver <em>angiotensin</em>ogen expression increased (twofold). However, adipose tissue from HF mice did not exhibit elevated ACE2 protein or activity. Increased expression of ADAM<em>1</em><em>7</em>, a protease responsible for ACE2 shedding, coincided with reductions in ACE2 activity in 3T3-L<em>1</em> adipocytes, and an ADAM<em>1</em><em>7</em> inhibitor decreased media ACE2 activity. Moreover, ADAM<em>1</em><em>7</em> mRNA expression was increased in adipose tissue from 4-mo HF-fed mice, and plasma ACE2 activity increased. However, HF mice exhibited marked increases in plasma <em>angiotensin</em> peptide concentrations (LF: 2,<em>1</em>4<em>1</em> +/- 253; HF: 6,829 +/- <em>1</em>,0<em>7</em>5 pg/ml) and elevated blood pressure. These results demonstrate that adipocytes express ACE2 that is dysregulated in HF-fed mice with elevated blood pressure compared with LF controls.

BACKGROUND

<em>Angiotensin</em> II type <em>1</em> (AT<em>1</em>) receptor activation is potentially involved in the multifactorial pathogenesis of atherosclerosis.

RESULTS

Apolipoprotein E-deficient (ApoE-/-) mice were crossed with AT<em>1</em>A receptor-deficient (AT<em>1</em>-/-) mice to obtain homozygous double-knockout animals (ApoE-/--AT<em>1</em>-/- mice). Wild-type (C57BL/6J), ApoE-/-, AT<em>1</em>-/-, and ApoE-/--AT<em>1</em>-/- mice were fed a high-cholesterol diet for 7 weeks. In contrast to wild-type and AT<em>1</em>-/- mice, this treatment led to severe atherosclerotic lesion formation in the aortic sinus and the aorta (oil red O staining) and to an impaired endothelium-dependent vasodilation (organ chamber experiments with isolated aortic segments) in ApoE-/- mice. In the age-matched ApoE-/--AT<em>1</em>-/- littermates, development of diet-induced endothelial dysfunction and atherosclerotic lesion formation was profoundly inhibited. Concomitantly, aortic release of superoxide radicals was increased 2-fold in ApoE-/- mice compared with wild-type animals, whereas aortic superoxide production was normalized in ApoE-/--AT<em>1</em>-/- mice (L-0<em>1</em>2 chemiluminescence). There were no significant differences in plasma cholesterol levels between ApoE-/- and ApoE-/--AT<em>1</em>-/- animals. Systolic blood pressure was significantly lower in ApoE-/--AT<em>1</em>-/- animals than in ApoE-/- mice (tail-cuff measurements). Oral treatment of ApoE-/- mice with either hydralazine or irbesartan reduced systolic blood pressure to the same level; however, only AT<em>1</em> receptor antagonist treatment reduced atherosclerotic lesion formation and improved endothelial function.

CONCLUSIONS

Genetic disruption of the AT<em>1</em>A receptor leads to inhibition of vascular oxidative stress, endothelial dysfunction, and atherosclerotic lesion formation in ApoE-/- mice irrespective of blood pressure and plasma cholesterol levels. These results indicate a fundamental role of AT<em>1</em> receptor activation in atherogenesis.

Vascular injury, characterized by endothelial dysfunction, structural remodelling, inflammation and fibrosis, plays an important role in cardiovascular diseases. Cellular processes underlying this include altered vascular smooth muscle cell (VSMC) growth/apoptosis, fibrosis, increased contractility and vascular calcification. Associated with these events is VSMC differentiation and phenotypic switching from a contractile to a proliferative/secretory phenotype. Inflammation, associated with macrophage infiltration and increased expression of redox-sensitive pro-inflammatory genes, also contributes to vascular remodelling. Among the many factors involved in vascular injury is Ang II. Ang II, previously thought to be the sole biologically active downstream peptide of the renin-<em>angiotensin</em> system (RAS), is converted to smaller peptides, [Ang III, Ang IV, Ang-(<em>1</em>-<em>7</em>)], that are functional and that modulate vascular tone and structure. The actions of Ang II are mediated via signalling pathways activated upon binding to AT<em>1</em>R and AT2R. AT<em>1</em>R activation induces effects through PLC-IP3-DAG, MAP kinases, tyrosine kinases, tyrosine phosphatases and RhoA/Rho kinase. Ang II elicits many of its (patho)physiological actions by stimulating reactive oxygen species (ROS) generation through activation of vascular NAD(P)H oxidase (Nox). ROS in turn influence redox-sensitive signalling molecules. Here we discuss the role of Ang II in vascular injury, focusing on molecular mechanisms and cellular processes. Implications in vascular remodelling, inflammation, calcification and atherosclerosis are highlighted.

BACKGROUND

<em>Angiotensin</em>-converting enzyme (ACE)2 opposes the actions of <em>angiotensin</em> (Ang) II by degrading it to Ang <em>1</em>-<em>7</em>.

OBJECTIVE

Given the important role of Ang II/Ang <em>1</em>-<em>7</em> in atherogenesis, we investigated the impact of ACE2 deficiency on the development of atherosclerosis.

RESULTS

C5<em>7</em>Bl6, Ace2 knockout (KO), apolipoprotein E (ApoE) KO and ApoE/Ace2 double KO mice were followed until 30 weeks of age. Plaque accumulation was increased in ApoE/Ace2 double KO mice when compared to ApoE KO mice. This was associated with increased expression of adhesion molecules and inflammatory cytokines, including interleukin-6, monocyte chemoattractant protein-<em>1</em>, and vascular cell adhesion molecule-<em>1</em>, and an early increase in white cell adhesion across the whole aortae on dynamic flow assay. In the absence of a proatherosclerotic (ApoE KO) genotype, ACE2 deficiency was also associated with increased expression of these markers, suggesting that these differences were not an epiphenomenon. ACE inhibition prevented increases of these markers and atherogenesis in ApoE/ACE2 double KO mice. Bone marrow macrophages isolated from Ace2 KO mice showed increased proinflammatory responsiveness to lipopolysaccharide and Ang II when compared to macrophages isolated from C5<em>7</em>Bl6 mice. Endothelial cells isolated from Ace2 KO mice also showed increased basal activation and elevated inflammatory responsiveness to TNF-α. Similarly, selective inhibition of ACE2 with MLN-4<em>7</em>60 also resulted in a proinflammatory phenotype with a physiological response similar to that observed with exogenous Ang II (<em>1</em>0(-<em>7</em>) mol/L).

CONCLUSIONS

Genetic Ace2 deficiency is associated with upregulation of putative mediators of atherogenesis and enhances responsiveness to proinflammatory stimuli. In atherosclerosis-prone ApoE KO mice, these changes potentially contribute to increased plaque accumulation. These findings emphasize the potential utility of ACE2 repletion as a strategy to reduce atherosclerosis.

OBJECTIVE

To compare the effects on proteinuria and blood pressure of addition of dietary sodium restriction or angiotensin receptor blockade at maximum dose, or their combination, in patients with non-diabetic nephropathy receiving background treatment with angiotensin converting enzyme (ACE) inhibition at maximum dose.

METHODS

Multicentre crossover randomised controlled trial.

METHODS

Outpatient clinics in the Netherlands.

METHODS

52 patients with non-diabetic nephropathy.

METHODS

All patients were treated during four 6 week periods, in random order, with angiotensin receptor blockade (valsartan 320 mg/day) or placebo, each combined with, consecutively, a low sodium diet (target 50 mmol Na(+)/day) and a regular sodium diet (target 200 mmol Na(+)/day), with a background of ACE inhibition (lisinopril 40 mg/day) during the entire study. The drug interventions were double blind; the dietary interventions were open label.

METHODS

The primary outcome measure was proteinuria; the secondary outcome measure was blood pressure.

RESULTS

Mean urinary sodium excretion, a measure of dietary sodium intake, was 106 (SE 5) mmol Na(+)/day during a low sodium diet and 184 (6) mmol Na(+)/day during a regular sodium diet (P<0.001). Geometric mean residual proteinuria was 1.68 (95% confidence interval 1.31 to 2.14) g/day during ACE inhibition plus a regular sodium diet. Addition of angiotensin receptor blockade to ACE inhibition reduced proteinuria to 1.44 (1.07 to 1.93) g/day (P=0.003), addition of a low sodium diet reduced it to 0.85 (0.66 to 1.10) g/day (P<0.001), and addition of angiotensin receptor blockade plus a low sodium diet reduced it to 0.67 (0.50 to 0.91) g/day (P<0.001). The reduction of proteinuria by the addition of a low sodium diet to ACE inhibition (51%, 95% confidence interval 43% to 58%) was significantly larger (P<0.001) than the reduction of proteinuria by the addition of angiotensin receptor blockade to ACE inhibition (21%, (8% to 32%) and was comparable (P=0.009, not significant after Bonferroni correction) to the reduction of proteinuria by the addition of both angiotensin receptor blockade and a low sodium diet to ACE inhibition (62%, 53% to 70%). Mean systolic blood pressure was 134 (3) mm Hg during ACE inhibition plus a regular sodium diet. Mean systolic blood pressure was not significantly altered by the addition of angiotensin receptor blockade (131 (3) mm Hg; P=0.12) but was reduced by the addition of a low sodium diet (123 (2) mm Hg; P<0.001) and angiotensin receptor blockade plus a low sodium diet (121 (3) mm Hg; P<0.001) to ACE inhibition. The reduction of systolic blood pressure by the addition of a low sodium diet (7% (SE 1%)) was significantly larger (P=0.003) than the reduction of systolic blood pressure by the addition of angiotensin receptor blockade (2% (1)) and was similar (P=0.14) to the reduction of systolic blood pressure by the addition of both angiotensin receptor blockade and low sodium diet (9% (1)), to ACE inhibition.

CONCLUSIONS

Dietary sodium restriction to a level recommended in guidelines was more effective than dual blockade for reduction of proteinuria and blood pressure in non-diabetic nephropathy. The findings support the combined endeavours of patients and health professionals to reduce sodium intake. Trial registration Netherlands Trial Register NTR675.

<em>Angiotensin</em>-converting enzyme type 2 (ACE2) is a pivotal component of the renin-<em>angiotensin</em> system, promoting the conversion of <em>angiotensin</em> II (Ang-II) to Ang-(<em>1</em>-<em>7</em>). We previously reported that decreased ACE2 expression and activity contributes to the development of Ang-II-mediated hypertension in mice. The present study aimed to investigate the mechanisms involved in ACE2 downregulation during neurogenic hypertension. In ACE2-transfected Neuro-2A cells, Ang-II treatment resulted in a significant attenuation of ACE2 enzymatic activity. Examination of the subcellular localization of ACE2 revealed that Ang-II treatment leads to ACE2 internalization and degradation into lysosomes. These effects were prevented by both the Ang-II type <em>1</em> receptor (AT<em>1</em>R) blocker losartan and the lysosomal inhibitor leupeptin. In contrast, in HEK293T cells, which lack endogenous AT<em>1</em>R, Ang-II failed to promote ACE2 internalization. Moreover, this effect could be induced after AT<em>1</em>R transfection. Furthermore, coimmunoprecipitation experiments demonstrated that AT<em>1</em>R and ACE2 form complexes, and these interactions were decreased by Ang-II treatment, which also enhanced ACE2 ubiquitination. In contrast, ACE2 activity was not changed by transfection of AT2 or Mas receptors. In vivo, Ang-II-mediated hypertension was blunted by chronic infusion of leupeptin in wildtype C5<em>7</em>Bl/6, but not in ACE2 knockout mice. Overall, this is the first demonstration that elevated Ang-II levels reduce ACE2 expression and activity by stimulation of lysosomal degradation through an AT<em>1</em>R-dependent mechanism.

OBJECTIVE

The increasing number of podocyte-expressed genes implicated in steroid-resistant nephrotic syndrome (SRNS), the phenotypic variability, and the uncharacterized relative frequency of mutations in these genes in pediatric and adult patients with SRNS complicate their routine genetic analysis. Our aim was to compile the clinical and genetic data of eight podocyte genes analyzed in <em>1</em><em>1</em>0 cases (<em>1</em>25 patients) with SRNS (ranging from congenital to adult onset) to provide a genetic testing approach.

METHODS

Mutation analysis was performed by sequencing the NPHS<em>1</em>, NPHS2, TRPC6, CD2AP, PLCE<em>1</em>, INF2, WT<em>1</em> (exons 8 and 9), and ACTN4 (exons <em>1</em> to <em>1</em>0) genes.

RESULTS

We identified causing mutations in 34% (37/<em>1</em><em>1</em>0) of SRNS patients, representing 67% (<em>1</em>6/24) familial and 25% (2<em>1</em>/86) sporadic cases. Mutations were detected in <em>1</em>00% of congenital-onset, 57% of infantile-onset, 24 and 36% of early and late childhood-onset, 25% of adolescent-onset, and <em>1</em>4% of adult-onset patients. The most frequently mutated gene was NPHS<em>1</em> in congenital onset and NPHS2 in the other groups. A partial remission was observed in 7 of 26 mutation carriers treated with immunosuppressive agents and/or angiotensin-converting enzyme inhibitors. Patients with NPHS<em>1</em> mutations showed a faster progression to ESRD than patients with NPHS2 mutations. None of these mutation carriers relapsed after kidney transplantation.

CONCLUSIONS

We propose a genetic testing algorithm for SRNS based on the age at onset and the familial/sporadic status. Mutation analysis of specific podocyte-genes has a clinical value in all age groups, especially in children.

OBJECTIVE

Obesity promotes hypertension, but it is unclear if sex differences exist in obesity-related hypertension. <em>Angiotensin</em> converting enzyme 2 (ACE2) converts <em>angiotensin</em> II (AngII) to <em>angiotensin</em>-(<em>1</em>-<em>7</em>) (Ang-[<em>1</em>-<em>7</em>]), controlling peptide balance. We hypothesized that tissue-specific regulation of ACE2 by high-fat (HF) feeding and sex hormones contributes to sex differences in obesity-hypertension.

RESULTS

HF-fed females gained more body weight and fat mass than males. HF-fed males exhibiting reduced kidney ACE2 activity had increased plasma <em>angiotensin</em> II levels and decreased plasma Ang-(<em>1</em>-<em>7</em>) levels. In contrast, HF-fed females exhibiting elevated adipose ACE2 activity had increased plasma Ang-(<em>1</em>-<em>7</em>) levels. HF-fed males had elevated systolic and diastolic blood pressure that were abolished by losartan. In contrast, HF-fed females did not exhibit increased systolic blood pressure until females were administered the Ang-(<em>1</em>-<em>7</em>) receptor antagonist, D-Ala-Ang-(<em>1</em>-<em>7</em>). Deficiency of ACE2 increased systolic blood pressure in HF-fed males and females, which was abolished by losartan. Ovariectomy of HF-fed female mice reduced adipose ACE2 activity and plasma Ang-(<em>1</em>-<em>7</em>) levels, and promoted obesity-hypertension. Finally, estrogen, but not other sex hormones, increased adipocyte ACE2 mRNA abundance.

CONCLUSIONS

These results demonstrate that tissue-specific regulation of ACE2 by diet and sex hormones contributes to sex differences in obesity-hypertension.

Cardiac fibrosis is a key component of heart disease and involves the proliferation and differentiation of matrix-producing fibroblasts. The effects of an antifibrotic peptide hormone, relaxin, in inhibiting this process were investigated. We used rat atrial and ventricular fibroblasts, which respond to profibrotic stimuli and express the relaxin receptor (LGR<em>7</em>), in addition to two in vivo models of cardiac fibrosis. Cardiac fibroblasts, when plated at low density or stimulated with TGF-beta or <em>angiotensin</em> II (Ang II), accelerated fibroblast differentiation into myofibroblasts, as demonstrated by significantly increased alpha-smooth muscle actin expression, collagen synthesis, and collagen deposition (by up to 95% with TGF-beta and 40% with Ang II; all P < 0.05). Fibroblast proliferation was significantly increased by <em>1</em>0(-8) m and <em>1</em>0(-<em>7</em>) m Ang II (63-<em>7</em>5%; P < 0.0<em>1</em>) or 0.<em>1</em>-<em>1</em> microg/ml IGF-I (2<em>7</em>-40%; P < 0.05). Relaxin alone had no marked effect on these parameters, but it significantly inhibited Ang II- and IGF-I-mediated fibroblast proliferation (by <em>1</em>5-50%) and Ang II- and TGF-beta-mediated fibroblast differentiation, as detected by decreased expression of alpha-smooth muscle actin (by 65-88%) and collagen (by 60-80%). Relaxin also increased matrix metalloproteinase-2 expression in the presence of TGF-beta (P < 0.0<em>1</em>) and Ang II (P < 0.05). Furthermore, relaxin decreased collagen overexpression when administered to two models of established fibrotic cardiomyopathy, one due to relaxin deficiency (by 40%; P < 0.05) and the other to cardiac-restricted overexpression of beta2-adrenergic receptors (by 58%; P < 0.0<em>1</em>). These coherent findings indicate that relaxin regulates fibroblast proliferation, differentiation, and collagen deposition and may have therapeutic potential in diseased states characterized by cardiac fibrosis.

<em>Angiotensin</em>-converting enzyme 2 (ACE2) is a homolog of ACE, which is not blocked by ACE inhibitors. High amounts of ACE2 are present in the proximal tubule, and ACE2 catalyzes generation of <em>angiotensin</em> <em>1</em>-<em>7</em> (Ang-(<em>1</em>-<em>7</em>)) by this segment. Ang-(<em>1</em>-<em>7</em>) binds to a receptor distinct from the AT<em>1</em> or AT2 Ang II receptor, identified as the mas receptor. We studied the effects of Ang-(<em>1</em>-<em>7</em>) on Ang II-mediated cell signaling pathways in proximal tubule. In primary cultures of rat proximal tubular cells, activation of mitogen-activated protein kinases (MAPK) was detected by immunoblotting, in the presence or absence of agonists/antagonists. Transforming growth factor-beta<em>1</em> (TGF-beta<em>1</em>) was measured by enzyme-linked immunosorbent assay. Ang II (5 min, <em>1</em>0(-<em>7</em>) M) stimulated phosphorylation of the three MAPK (p38, extracellular signal-related kinase (ERK <em>1</em>/2), and c-Jun N-terminal kinase (JNK)). While incubation of proximal tubular cells with Ang-(<em>1</em>-<em>7</em>) alone did not significantly affect MAPK phosphorylation, Ang-(<em>1</em>-<em>7</em>) (<em>1</em>0(-<em>7</em>) M) completely inhibited Ang II-stimulated phosphorylation of p38, ERK <em>1</em>/2, and JNK. This inhibitory effect was reversed by the Ang-(<em>1</em>-<em>7</em>) receptor antagonist, D-Ala<em>7</em>-Ang-(<em>1</em>-<em>7</em>). Ang II significantly increased production of TGF-beta<em>1</em> in proximal tubular cells, an effect that was partly inhibited by Ang-(<em>1</em>-<em>7</em>). Ang-(<em>1</em>-<em>7</em>) had no significant effect on cyclic 3',5'-adenosine monophosphate production in these cells. In summary, Ang-(<em>1</em>-<em>7</em>) inhibits Ang II-stimulated MAPK phosphorylation in proximal tubular cells. Generation of Ang-(<em>1</em>-<em>7</em>) by proximal tubular ACE2 could thereby serve a protective role by counteracting the effects of locally generated Ang II.

BACKGROUND

Preeclampsia is considered a disease of immunological origin associated with abnormalities in inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), and activated lymphocytes secreting autoantibodies to the <em>angiotensin</em> II receptor (AT<em>1</em>-AA). Recent studies have also demonstrated that an imbalance of angiogenic factors, soluble fms-like tyrosine kinase (sFlt-<em>1</em>), and sEndoglin, exists in preeclampsia; however, the mechanisms that initiate their overproduction are unclear.

METHODS

To determine the role of immune regulation of these factors, circulating and placental sFlt-<em>1</em> and/or sEndoglin was examined from pregnant rats chronically treated with TNF-alpha or AT<em>1</em>-AA. On day <em>1</em>9 of gestation blood pressure was analyzed and serum and tissues were collected. Placental villous explants were excised and cultured on matrigel coated inserts for 24 h and sFlt-<em>1</em> and sEndoglin was measured from media.

RESULTS

In response to TNF-alpha-induced hypertension, sFlt-<em>1</em> increased from <em>1</em>80 +/- 5 to 2,907 +/- 4<em>1</em>2 pg/ml. sFlt-<em>1</em> was also increased from cultured placental explants of TNF-alpha induced hypertensive pregnant rats (n = <em>1</em>2) (2,544 +/- <em>1</em>,<em>1</em>32 pg/ml) vs. explants from normal pregnant (NP) rats (n = <em>1</em>2) (2,<em>1</em>89 +/- 586 pg/ml) where as sEng was undetectable. Circulating sFlt-<em>1</em> increased from 245 +/- 38 to 3,920 +/- 798 pg/ml in response to AT<em>1</em>-AA induced hypertension. sFlt-<em>1</em> levels were higher (3,400 +/- 350 vs. 2,480 +/- 900 pg/ml) in placental explants from AT<em>1</em>-AA infused rats (n = <em>1</em>2) than NP rats (n = 7). In addition, sEndoglin increased from 30 +/- 2.7 to 44 +/- 3.3 pg/ml (P < 0.047) in AT<em>1</em>-AA infused rats but was undetectable in the media of the placental explants.

CONCLUSIONS

These data suggest that immune factors may serve as an important stimulus for both sFlt-<em>1</em> and sEndoglin production in response to placental ischemia.

Advanced glycation end-product (AGE) is important in the pathogenesis of diabetic nephropathy (DN), and captopril (an <em>angiotensin</em> converting enzyme inhibitor) is effective in treating this disorder. We have shown that the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) cascade is responsible for AGE-induced mitogenesis in NRK-49F (normal rat kidney fibroblast) cells, but its role in renal fibrosis in DN remains unknown. Therefore, we have sought to determine whether JAK/STAT is involved in AGE-regulated collagen production in NRK-49F cells. We found that AGE time (<em>1</em>-<em>7</em> days) and dose (<em>1</em>0-200 microg/ml)-dependently increased collagen production in these cells. Additionally, AGE increased RAGE (receptor for AGE) protein expression. AGE-induced RAGE expression was dose-dependently inhibited by antisense RAGE oligodeoxynucleotide (ODN) and captopril. AGE-induced type I collagen production and JAK2-STAT<em>1</em>/STAT3 activation were decreased by AG-490 (a specific JAK2 inhibitor), antisense RAGE ODN and captopril. Meanwhile, STAT<em>1</em> and STAT3 decoy ODNs also suppressed the induction of collagen by AGE. We concluded that RAGE and the JAK2-STAT<em>1</em>/STAT3 pathway were involved in AGE-induced collagen production in NRK-49F cells. Furthermore, captopril was found to reverse AGE-induced collagen production, probably by attenuating RAGE expression and JAK2-STAT<em>1</em>/STAT3 activities.

OBJECTIVE

To quantify and evaluate the distribution of angiotensin II (Ang II) and its receptors in the human retina.

METHODS

Donor eyes were obtained within 12 hours postmortem and classified as hypertensive or normotensive and diabetic or nondiabetic, based on the donors' medical histories. Ang II in retina and vitreous was quantified by RIA. Ang II receptors were characterized and quantified by competitive membrane-binding assays. Ang II, its heptapeptide metabolite Ang-(1-7), and AT1 and AT2 receptors were localized by immunohistochemistry and confocal imaging.

RESULTS

Levels of Ang II in the retina were significantly higher than in vitreous (P < 0.05). Ang II in the diabetic retina had a higher median compared with that in the nondiabetic retina. Ang II and Ang-(1-7) colocalized in retinal Müller cells. The retina had the highest levels of Ang II receptors that were significantly higher than the optic nerve, retinal pigment epithelium-choroid complex, and ciliary body-iris complex (P < 0.05). AT1 receptors were more abundant than AT2 receptors in the retina. Immunoreactivity for AT1 was detected in Müller cells and on blood vessels. AT2 receptors were localized throughout the Müller cells and nuclei of ganglion cells and neurons in the inner nuclear layer.

CONCLUSIONS

In the human retina, identification of Ang II and its bioactive metabolite Ang-(1-7) in Müller cells suggests that these glial cells are able to produce and process Ang II. Ang receptors were localized in the blood vessels and neural cells. Local Ang II signaling may thus allow for autoregulation of neurovascular activity. Such an autonomous system could modulate the onset and severity of retinovascular disease.

OBJECTIVE

This study prospectively evaluated the relationship between cardiovascular risk factors, selected biomarkers (high-sensitivity C-reactive protein [hs-CRP], interleukin [IL]-6, and osteoprotegerin [OPG]), and the progression of coronary artery calcification (CAC) in type 2 diabetic subjects.

BACKGROUND

Coronary artery calcification is pathognomonic of coronary atherosclerosis. Osteoprotegerin is a signaling molecule involved in bone remodeling that has been implicated in the regulation of vascular calcification and atherogenesis.

METHODS

Three hundred ninety-eight type 2 diabetic subjects without prior coronary disease or symptoms (age 52 +/- 8 years, 6<em>1</em>% male, glycated hemoglobin [HbA(<em>1</em>)c] 8 +/- <em>1</em>.5) were evaluated serially by CAC imaging (mean follow-up 2.5 +/- 0.4 years). Progression/regression of CAC was defined as a change>> or =2.5 between the square root transformed values of baseline and follow-up volumetric CAC scores. Demographic data, risk factors, glycemic control, medication use, serum hs-CRP, IL-6, and plasma OPG levels were measured at baseline and follow-up.

RESULTS

Two hundred eleven patients (53%) had CAC at baseline. One hundred eighteen patients (29.6%) had CAC progression, whereas 3 patients (0.8%) had regression. Age, male gender, hypertension, baseline CAC, HbA(<em>1</em>)c >7, waist-hip ratio, IL-6, OPG, use of beta-blockers, calcium channel antagonists, angiotensin-converting enzyme (ACE) inhibitors, statins, and Framingham/UKPDS (United Kingdom Prospective Diabetes Study) risk scores were univariable predictors of CAC progression. In the multivariate model, baseline CAC (odds ratio [OR] for CAC >400 = 6.38, 95% confidence interval [CI] 2.63 to <em>1</em>5.5, p < 0.00<em>1</em>), HbA(<em>1</em>)c >7 (OR <em>1</em>.95, CI <em>1</em>.08 to 3.52, p = 0.03), and statin use (OR 2.27, CI <em>1</em>.38 to 3.73, p = 0.00<em>1</em>) were independent predictors of CAC progression.

CONCLUSIONS

Baseline CAC severity and suboptimal glycemic control are strong risk factors for CAC progression in type 2 diabetic subjects.

BACKGROUND

Angiotensin-converting-enzyme (ACE) inhibitors not only decrease the production of angiotensin II but also decrease the degradation of bradykinin. In this study, a specific bradykinin-receptor antagonist, icatibant acetate (HOE 140), was used to determine the contribution of bradykinin to the short-term effects of ACE inhibition on blood pressure and plasma renin activity in both normotensive and hypertensive subjects.

METHODS

We compared the hemodynamic, renal, and endocrine effects of captopril alone (25 mg), captopril plus icatibant (100 microg per kilogram of body weight), the angiotensin II subtype 1-receptor antagonist losartan (75 mg), and placebo in 20 subjects with normal blood pressure and 7 subjects with hypertension. The subjects were studied while they were salt depleted (i.e., in balance on a diet in which they were allowed 10 mmol of sodium per day). The drugs were administered on four separate study days in a single-blind, randomized fashion.

RESULTS

The coadministration of icatibant significantly attenuated the hypotensive effect of captopril (maximal decrease in mean arterial pressure for all subjects combined, 10.5+/-1.0 mm Hg, as compared with 14.0+/-1.0 mm Hg for captopril alone; P=0.001), in such a way that the decrease in blood pressure after the administration of captopril plus icatibant was similar to that after the administration of losartan (maximal decrease in mean arterial pressure, 11.0+/-1.7 mm Hg). Icatibant did not alter the renal hemodynamic response to captopril, but it significantly altered the change in plasma renin activity in response to ACE inhibition (-0.4+/-0.4 ng of angiotensin I per milliliter per hour, as compared with 2.0+/-0.7 ng per milliliter per hour for captopril alone; P=0.007). The magnitude of these effects was similar in both the normotensive and the hypertensive subjects, as well as in both the black subjects and the white subjects.

CONCLUSIONS

These data confirm that bradykinin contributes to the short-term effects of ACE inhibition on blood pressure in normotensive and hypertensive persons and suggest that bradykinin also contributes to the short-term effects of ACE inhibition on the renin-angiotensin system.

OBJECTIVE

The degradation of <em>angiotensin</em> (Ang) II by ACE2, leading to the formation of Ang <em>1</em>-<em>7</em>, is an important step in the renin-<em>angiotensin</em> system (RAS) and one that is significantly altered in the diabetic kidney. This study examines the role of ACE2 in early renal changes associated with diabetes and the influence of ACE2 deficiency on ACE inhibitor-mediated renoprotection.

METHODS

Diabetes was induced by streptozotocin in male c5<em>7</em>bl6 mice and ACE2 knockout (KO) mice. After 5 weeks of study, animals were randomized to receive the ACE inhibitor perindopril (2 mg x kg(-<em>1</em>) x day(-<em>1</em>)). Wild-type mice were further randomized to receive the selective ACE2 inhibitor MLN-4<em>7</em>60 (<em>1</em>0 mg x kg(-<em>1</em>) x day(-<em>1</em>)) and followed for an additional 5 weeks. Markers of renal function and injury were then assessed.

RESULTS

Induction of diabetes in wild-type mice was associated with a reduction in renal ACE2 expression and decreased Ang <em>1</em>-<em>7</em>. In diabetic mice receiving MLN-4<em>7</em>60 and in ACE2 KO mice, diabetes-associated albuminuria was enhanced, associated with an increase in blood pressure. However, renal hypertrophy and fibrogenesis were reduced in diabetic mice with ACE2 deficiency, and hyperfiltration was attenuated. Diabetic wild-type mice treated with an ACE inhibitor experienced a reduction in albuminuria and blood pressure. These responses were attenuated in both diabetic ACE2 KO mice and diabetic mice receiving MLN-4<em>7</em>60. However, other renoprotective and antifibrotic actions of ACE inhibition in diabetes were preserved in ACE2-deficient mice.

CONCLUSIONS

The expression of ACE2 is significantly modified by diabetes, which impacts both pathogenesis of kidney disease and responsiveness to RAS blockade. These data indicate that ACE2 is a complex and site-specific modulator of diabetic kidney disease.

OBJECTIVE

Angiotensin II (Ang II) has been shown to have both central and peripheral effects in mediating hypertension, for which the hypothalamic paraventricular nucleus (PVN) is an important brain cardio-regulatory centre. Angiotensin-converting enzyme 2 (ACE2) has been identified as a negative regulator of the pro-hypertensive actions of Ang II. Recent findings from our laboratory suggest that Ang II infusion decreases ACE2 expression in the PVN. In the present study, we hypothesized that ACE2 overexpression in the PVN will have beneficial effects in counteracting Ang II-induced hypertension.

RESULTS

Male Sprague-Dawley rats were used in this study. Bilateral microinjection of an adenovirus encoding hACE2 (Ad-ACE2) into the PVN was used to overexpress ACE2 within this region. Mean arterial pressure measured by radiotelemetry was significantly increased after 14 days in Ang II-infused (200 ng/kg/min) rats vs. saline-infused controls (162.9 ± 3.6 vs. 102.3 ± 1.5 mmHg). Bilateral PVN microinjection of Ad-ACE2 attenuated this Ang II-induced hypertension (130.2 ± 5.7 vs. 162.9 ± 3.6 mmHg). ACE2 overexpression also significantly decreased AT(1)R and ACE expression and increased AT(2)R and Mas expression in the PVN. Additionally, ACE2 overexpression in the PVN attenuated the Ang II-induced increase in the expression of the pro-inflammatory cytokines tumour necrosis factor-α, interleukin (IL)-1β and IL-6 in the PVN.

CONCLUSIONS

Our findings suggest that attenuation of pro-inflammatory cytokines in the PVN in combination with the shift of the renin-angiotensin system towards the anti-hypertensive axis (ACE2/Ang-(1-7)/Mas) may be responsible for the overall beneficial effects of ACE2 overexpression in the PVN on the Ang II-induced hypertensive response.

AT<em>1</em>R (<em>angiotensin</em> type <em>1</em> receptor) and AT2R (<em>angiotensin</em> type 2 receptor) are well known to be involved in the complex cardiovascular actions of AngII (<em>angiotensin</em> II). However, shorter peptide fragments of AngII are thought to have biological activity in their own right and elicit effects that oppose those mediated by AngII. In the present study, we have used HEK (human embryonic kidney)-293 cells stably transfected with either AT<em>1</em>R or AT2R to perform a systematic analysis of binding affinities of all the major <em>angiotensin</em> peptides. Additionally, we tested the novel AT2R agonist Compound 2<em>1</em>, as well as the MasR (Mas receptor) agonist and antagonist AVE099<em>1</em> and A-<em>7</em><em>7</em>9 respectively, for their ability to bind to AT<em>1</em>R or AT2R. Candesartan, CGP422<em>1</em>4 and PD<em>1</em>233<em>1</em>9 were used as reference compounds. Binding studies using <em>1</em>25I-[Sar<em>1</em>Ile8]AngII on the AT<em>1</em>R-transfected HEK-293 cells revealed only AngII, AngIII [<em>angiotensin</em> III; <em>angiotensin</em>-(2-8)] and candesartan to have high affinity for AT<em>1</em>R. In the AT2R-transfected HEK-293 cells, competition for <em>1</em>25I-[Sar<em>1</em>Ile8]AngII binding was observed for all ligands except candesartan, AVE099<em>1</em> and A-<em>7</em><em>7</em>9, the latter two compounds having negligible affinity at either AT<em>1</em>R or AT2R. The rank order of affinity of ligands at AT2R was CGP42<em>1</em><em>1</em>2>AngII≥AngIII>Compound 2<em>1</em>≥PD<em>1</em>233<em>1</em>9≫AngIV [<em>angiotensin</em> IV; <em>angiotensin</em>-(3-8)>>Ang-(<em>1</em>-<em>7</em>) [<em>angiotensin</em>-(<em>1</em>-<em>7</em>)]. Of note, although AngIV and Ang-(<em>1</em>-<em>7</em>) exhibited only modest affinity at AT2R compared with AngII, these two <em>angiotensin</em> peptides, together with AngIII, had substantial AT2R selectivity over AT<em>1</em>R. Collectively, our results suggest that shorter <em>angiotensin</em> peptides can act as endogenous ligands at AT2R.

The early and long-term effects of coronary artery ligation on the plasma and left ventricular <em>angiotensin</em>-converting enzyme (ACE and ACE2) activities, ACE and ACE2 mRNA levels, circulating <em>angiotensin</em> (Ang) levels [Ang I, Ang-(<em>1</em>-<em>7</em>), Ang-(<em>1</em>-9), and Ang II], and cardiac function were evaluated <em>1</em> and 8 weeks after experimental myocardial infarction in adult Sprague Dawley rats. Sham-operated rats were used as controls. Coronary artery ligation caused myocardial infarction, hypertrophy, and dysfunction 8 weeks after surgery. At week <em>1</em>, circulating Ang II and Ang-(<em>1</em>-9) levels as well as left ventricular and plasma ACE and ACE2 activities increased in myocardial-infarcted rats as compared with controls. At 8 weeks post-myocardial infarction, circulating ACE activity, ACE mRNA levels, and Ang II levels remained higher, but plasma and left ventricular ACE2 activities and mRNA levels and circulating levels of Ang-(<em>1</em>-9) were lower than in controls. No changes in plasma Ang-(<em>1</em>-<em>7</em>) levels were observed at any time. Enalapril prevented cardiac hypertrophy and dysfunction as well as the changes in left ventricular ACE, left ventricular and plasmatic ACE2, and circulating levels of Ang II and Ang-(<em>1</em>-9) after 8 weeks postinfarction. Thus, the decrease in ACE2 expression and activity and circulating Ang-(<em>1</em>-9) levels in late ventricular dysfunction post-myocardial infarction were prevented with enalapril. These findings suggest that in this second arm of the renin-<em>angiotensin</em> system, ACE2 may act through Ang-(<em>1</em>-9), rather than Ang-(<em>1</em>-<em>7</em>), as a counterregulator of the first arm, where ACE catalyzes the formation of Ang II.

The renin-<em>angiotensin</em> system is an important component of the cardiovascular system. Mounting evidence suggests that the metabolic products of <em>angiotensin</em> I and II - initially thought to be biologically inactive - have key roles in cardiovascular physiology and pathophysiology. This non-canonical axis of the renin-<em>angiotensin</em> system consists of <em>angiotensin</em> <em>1</em>-<em>7</em>, <em>angiotensin</em> <em>1</em>-9, <em>angiotensin</em>-converting enzyme 2, the type 2 <em>angiotensin</em> II receptor (AT₂R), the proto-oncogene Mas receptor and the Mas-related G protein-coupled receptor member D. Each of these components has been shown to counteract the effects of the classical renin-<em>angiotensin</em> system. This counter-regulatory renin-<em>angiotensin</em> system has a central role in the pathogenesis and development of various cardiovascular diseases and, therefore, represents a potential therapeutic target. In this Review, we provide the latest insights into the complexity and interplay of the components of the non-canonical renin-<em>angiotensin</em> system, and discuss the function and therapeutic potential of targeting this system to treat cardiovascular disease.

We have recently shown that mechanical stress induces cardiomyocyte hypertrophy partly through the enhanced secretion of <em>angiotensin</em> II (ATII). Endothelin-<em>1</em> (ET-<em>1</em>) has been reported to be a potent growth factor for a variety of cells, including cardiomyocytes. In this study, we examined the role of ET-<em>1</em> in mechanical stress-induced cardiac hypertrophy by using cultured cardiomyocytes of neonatal rats. ET-<em>1</em> (<em>1</em>0(-8) approximately <em>1</em>0(-<em>7</em>) M) maximally induced the activation of both Raf-<em>1</em> kinase and mitogen-activated protein (MAP) kinases at 4 and 8 min, respectively, followed by an increase in protein synthesis at 24 h. All of these hypertrophic responses were completely blocked by pretreatment with BQ<em>1</em>23, an antagonist selective for the ET-<em>1</em> type A receptor subtype, but not by BQ<em>7</em>88, an ET-<em>1</em> type B receptor-specific antagonist. BQ<em>1</em>23 also suppressed stretch-induced activation of MAP kinases and an increase in phenylalanine uptake by approximately 60 and 50%, respectively, but BQ<em>7</em>88 did not. ET-<em>1</em> was constitutively secreted from cultured cardiomyocytes, and a significant increase in ET-<em>1</em> concentration was observed in the culture medium of cardiomyocytes after stretching for <em>1</em>0 min. After 24 h, an approximately 3-fold increase in ET-<em>1</em> concentration was observed in the conditioned medium of stretched cardiomyocytes compared with that of unstretched cardiomyocytes. ET-<em>1</em> mRNA levels were also increased at 30 min after stretching. Moreover, ET-<em>1</em> and ATII synergistically activated Raf-<em>1</em> kinase and MAP kinases in cultured cardiomyocytes. In conclusion, mechanical stretching stimulates secretion and production of ET-<em>1</em> in cultured cardiomyocytes, and vasoconstrictive peptides such as ATII and ET-<em>1</em> may play an important role in mechanical stress-induced cardiac hypertrophy.