Eliglustat tartrate (Genz-<em>1</em><em>1</em>2638), a specific inhibitor of glucosylceramide synthase, is under development as an oral substrate reduction therapy for Gaucher disease type <em>1</em> (GD<em>1</em>). A multinational, open-label, single-arm phase 2 study of 26 GD<em>1</em> patients (<em>1</em>6 female, <em>1</em>0 male; mean age, 34 years) evaluated the efficacy, safety, and pharmacokinetics of eliglustat tartrate administered twice daily by mouth at 50- or <em>1</em>00-mg doses based on plasma drug concentrations. Entry criteria required splenomegaly with thrombocytopenia and/or anemia. The composite primary efficacy end point required improvement after 52 weeks in at least 2 of these 3 disease manifestations and was met by <em>7</em><em>7</em>% (95% confidence interval [CI] = 58%-89%) of all patients and 9<em>1</em>% (95% CI = <em>7</em>2%-98%) of the 22 patients completing 52 weeks. Statistically significant improvements occurred in mean hemoglobin level (<em>1</em>.62 g/dL; 95% CI =<em>1</em>.05-2.<em>1</em>8 g/dL), platelet count (40.3%; 95% CI = 23.<em>7</em>-5<em>7</em>.0 g/dL), spleen volume (-38.5%; 95% CI = -43.5%--33.5%), liver volume (-<em>1</em><em>7</em>.0%; 95% CI = -2<em>1</em>.6%-<em>1</em>2.3%), and lumbar spine bone mineral density (0.3<em>1</em> Z-score; 95% CI = 0.09-0.53). Elevated biomarkers (chitotriosidase; chemokine CCL<em>1</em>8; <em>angiotensin</em>-converting enzyme; tartrate-resistant acid phosphatase) decreased by 35% to 50%. Plasma glucosylceramide and ganglioside GM3 normalized. Eliglustat tartrate was well tolerated: <em>7</em> mild, transient adverse events in 6 patients were considered treatment-related. Individual pharmacokinetics varied; mean time to maximal observed concentration was 2.3 hours and mean half-life was 6.8 hours. Eliglustat tartrate appears to be a promising oral treatment for GD<em>1</em>.

It has been shown that the female sex hormones have a protective role in the development of <em>angiotensin</em> II (ANG II)-induced hypertension. The present study tested the hypotheses that <em>1</em>) the estrogen receptor-alpha (ERalpha) is involved in the protective effects of estrogen against ANG II-induced hypertension and 2) central ERs are involved. Blood pressure (BP) was measured in female mice with the use of telemetry implants. ANG II (800 ng.kg(-<em>1</em>).min(-<em>1</em>)) was administered subcutaneously via an osmotic pump. Baseline BP in the intact, ovariectomized (OVX) wild-type (WT) and ERalpha knockout (ERalphaKO) mice was similar; however, the increase in BP induced by ANG II was greater in OVX WT (23.0 +/- <em>1</em>.0 mmHg) and ERalphaKO mice (23.8 +/- 2.5 mmHg) than in intact WT mice (<em>1</em>0.<em>1</em> +/- 4.5 mmHg). In OVX WT mice, central infusion of <em>1</em><em>7</em>beta-estradiol (E(2); 30 microg.kg(-<em>1</em>).day(-<em>1</em>)) attenuated the pressor effect of ANG II (<em>7</em>.0 +/- 0.4 mmHg), and this protective effect of E(2) was prevented by coadministration of ICI-<em>1</em>82,<em>7</em>80 (ICI; <em>1</em>.5 microg.kg(-<em>1</em>).day(-<em>1</em>), <em>1</em>8.8 +/- <em>1</em>.5 mmHg), a nonselective ER antagonist. Furthermore, central, but not peripheral, infusions of ICI augmented the pressor effects of ANG II in intact WT mice (<em>1</em><em>7</em>.8 +/- 4.2 mmHg). In contrast, the pressor effect of ANG II was unchanged in either central E(2)-treated OVX ERalphaKO mice (<em>1</em>9.0 +/- <em>1</em>.<em>1</em> mmHg) or central ICI-treated intact ERalphaKO mice (<em>1</em>9.6 +/- <em>1</em>.6 mmHg). Lastly, ganglionic blockade on day <em>7</em> after ANG II infusions resulted in a greater reduction in BP in OVX WT, central ER antagonist-treated intact WT, central E(2) + ICI-treated OVX WT, ERalphaKO, and central E(2)- or ICI-treated ERalphaKO mice compared with that in intact WT mice given just ANG II. Together, these data indicate that ERalpha, especially central expression of the ER, mediates the protective effects of estrogen against ANG II-induced hypertension.

Chronic low-dose <em>angiotensin</em> II (Ang II) infusion for <em>1</em>3 days mimics two-kidney, one clip Goldblatt hypertension and increase intrarenal Ang II levels. We performed studies to determine the time course for the enhancement of intrarenal Ang II levels and whether the increased intrarenal Ang II is a tissue-specific event and requires a receptor-mediated step. Male Sprague-Dawley rats were uninephrectomized, and either vehicle or Ang II (40 ng/min) was infused via a subcutaneous osmotic minipump. Plasma and renal Ang II levels were measured 3, <em>7</em>, <em>1</em>0, and <em>1</em>3 days after minipump implantation. Compared with controls (<em>1</em>26 +/- 2 mm Hg), systolic pressure in Ang II-infused rats exhibited a detectable increase by day 6 (<em>1</em>46 +/- 2 mm Hg) and continued to increase to <em>1</em>89 +/- 5 mm Hg by day <em>1</em>2. Plasma Ang II levels were elevated by day 3, whereas intrarenal Ang II levels were not significantly elevated until <em>1</em>0 days of Ang II infusion. Renal injury characterized by focal and segmental glomerulosclerosis was evident after <em>1</em>3 days of Ang II infusion. Losartan (30 mg/kg per day) prevented the development of hypertension in the Ang II-infused rats for the duration of the infusion period (<em>1</em>25 +/- <em>1</em> mm Hg) and reduced the degree of glomerular injury. Plasma renin activity was suppressed in the Ang II-infused group but was elevated markedly in both losartan-treated groups. Plasma Ang II levels were elevated in the Ang II-infused rats and were even higher during losartan treatment. Intrarenal Ang II levels were enhanced significantly (354 +/- 60 versus <em>1</em>64 +/- 23 fmol/g) in the Ang II-infused rats. However, losartan treatment prevented the augmentation of intrarenal Ang II caused by Ang II infusion. Heart and adrenal Ang II levels were not significantly increased in the Ang II-infused rats but were significantly elevated during losartan treatment. These results suggest that the tissue-specific elevations of intrarenal Ang II levels caused by chronic Ang II infusion are mediated by <em>angiotensin</em> type <em>1</em> receptor activation, which leads to either receptor-mediated internalization of Ang II, enhancement of intrarenal Ang II formation, or both.

The <em>angiotensin</em> II type <em>1</em> (AT(<em>1</em>)) receptor is a G protein-coupled receptor that has a crucial role in the development of load-induced cardiac hypertrophy. Here, we show that cell stretch leads to activation of the AT(<em>1</em>) receptor, which undergoes an anticlockwise rotation and a shift of transmembrane (TM) <em>7</em> into the ligand-binding pocket. As an inverse agonist, candesartan suppressed the stretch-induced helical movement of TM<em>7</em> through the bindings of the carboxyl group of candesartan to the specific residues of the receptor. A molecular model proposes that the tight binding of candesartan to the AT(<em>1</em>) receptor stabilizes the receptor in the inactive conformation, preventing its shift to the active conformation. Our results show that the AT(<em>1</em>) receptor undergoes a conformational switch that couples mechanical stress-induced activation and inverse agonist-induced inactivation.

BACKGROUND

Nephropathy may develop in patients with sickle cell disease. We determined the prevalence of proteinuria and renal insufficiency in a group of patients with sickle cell disease and investigated the renal pathologic changes and the effects of an angiotensin-converting-enzyme inhibitor (enalapril) on protein excretion in patients found to have nephropathy.

METHODS

We prospectively screened 381 patients with sickle cell disease for the presence of proteinuria and renal insufficiency. Renal biopsy and measurements of glomerular filtration rate, effective renal plasma flow, and urinary protein excretion were performed in 10 patients with mild nephropathy before and after the administration of enalapril, and again two to three weeks after its discontinuation.

RESULTS

Of the 381 patients with sickle cell disease, 26 (7 percent) had serum creatinine concentrations above the normal range and 101 (26 percent) had proteinuria of at least 1+. The renal lesions in the 10 patients who had biopsies consisted of glomerular enlargement and perihilar focal segmental glomerulosclerosis. The mean (+/- SD) glomerular area in these patients was 28.7 +/- 4.1 x 10(3) micron 2, as compared with 15.8 +/- 4.3 x 10(3) micron 2 in 10 control patients without renal disease who had died of trauma (P less than 0.0001). During the administration of enalapril, the mean 24-hour urinary protein excretion decreased 57 percent (range, 23 to 79 percent) below the base-line value (P less than 0.001), and it increased to 25 percent below the base-line value after enalapril was discontinued. The glomerular filtration rate and effective renal plasma flow did not change significantly.

CONCLUSIONS

Approximately 25 percent of patients with sickle cell disease have proteinuria. Treatment with enalapril reduces the degree of proteinuria in these patients, suggesting that glomerular capillary hypertension may be a pathogenic factor in sickle cell nephropathy.

We established a new congenic model of hypertension, the mRen(2). Lewis rat and assessed the intracellular expression of <em>angiotensin</em> peptides and receptors in the kidney. The congenic strain was established from the backcross of the (mRen2)2<em>7</em> transgenic rat that expresses the mouse renin 2 gene onto the Lewis strain. The 20-wk-old male congenic rats were markedly hypertensive compared with the Lewis controls (systolic blood pressure: <em>1</em>95 +/- 2 vs. <em>1</em>0<em>7</em> +/- 2 mmHg, P < 0.0<em>1</em>). Although plasma ANG II levels were not different between strains, circulating levels of ANG-(<em>1</em>-<em>7</em>) were 2<em>7</em>0% higher and ANG I concentrations were 40% lower in the mRen2. Lewis rats. In contrast, both cortical (CORT) and medullary (MED) ANG II concentrations were 60% higher in the mRen2. Lewis rats, whereas tissue ANG I was 66 and 84% lower in CORT and MED. For both strains, MED ANG II, ANG I, and ANG-(<em>1</em>-<em>7</em>) were significantly higher than CORT levels. Intracellular ANG II binding distinguished nuclear (NUC) and plasma membrane (PM) receptor using the ANG II radioligand <em>1</em>25I-sarthran. Isolated CORT nuclei exhibited a high density (Bmax >200 fmol/mg protein) and affinity for the sarthran ligand (KD<0.5 nM); the majority of these sites (>95%) were the AT<em>1</em> receptor subtype. CORT ANG II receptor Bmax and KD values in nuclei were <em>7</em>5 and 50% lower, respectively, for the mRen2. Lewis vs. the Lewis rats. In the MED, the PM receptor density (Lewis: 50 +/- 4 vs. mRen2. Lewis: 2<em>1</em> +/- 5 fmol/mg protein) and affinity (Lewis: 0.3<em>1</em> +/- 0.<em>1</em> vs. 0.69 +/- 0.<em>1</em> nM) were lower in the mRen2. Lewis rats. In summary, the hypertensive mRen2. Lewis rats exhibit higher ANG II in both CORT and MED regions of the kidney. Evaluation of intracellular ANG II receptors revealed lower CORT NUC and MED PM AT<em>1</em> sites in the mRen2. Lewis. The downregulation of AT<em>1</em> sites in the mRen2. Lewis rats may reflect a compensatory response to dampen the elevated levels of intrarenal ANG II.

BACKGROUND

Angiotensin II (ANG II) mediated hypertension accelerates atherosclerosis (AS) and thereby increases the incidence of myocardial infarction (MI). On the other hand, superoxide anion (O2-) is involved in the modification of low density lipoproteins, inhibition of prostacyclin (PGI2) formation and breakdown of nitric oxide. These events finally lead to rapid progression of AS and MI. In the present study, we investigate whether ANG II can induce O2- release from human vascular endothelial cells (HVECs) and the possible mechanisms involved.

RESULTS

The expression of ANG receptors subtype-1 (AT-1) and subtype-2 (AT-2) were identified by using reverse transcription polymerase chain reaction and sequence analysis. The O2- production was dose-dependently increased in HVECs treated with ANG II (10(-7)-10(-9) M) and with a maximum rate after 1 h of incubation. This event was significantly inhibited by pretreatment of cells with the specific AT-1 blocker losartan (10(-7) M) and to a lesser extent by the specific AT-2 receptor blocker PD123319 (10(-7) M). The combined incubation of both receptor blockers was even more effective. In addition, our lucigenin-enhanced chemiluminescence assay showed that the activity of plasma membrane-bound NADH-/NADPH-oxidases derived from ANG II-treated cells was also significantly increased, this effect was reduced in cells pretreated with losartan or to lesser extent by PD123319. However, the activity of xanthine oxidase remained unchanged in response to ANG II. Furthermore, the basal O2- release from HVECs was inhibited in cells treated with angiotensin-converting enzyme (ACE) inhibitor, Lisinopril (10(-6) M), and this event could be reversed by ANG II.

CONCLUSIONS

ANG II induces O2- release in HVECs via activation of membrane-bound NADH-/NADPH-oxidase, an effect, that is mediated by both AT-1 and AT-2 receptors. This suggests that acceleration of AS and MI in ANG II-mediated hypertension may at least be due to ANG II-induced O2- generation from vascular endothelial cells. In this case, the ACE inhibitors and the ANG receptor antagonists may act as causative "antioxidants".

The dorsomedial portion of the nucleus tractus solitarius (dmNTS) is the site of termination of baroreceptor and cardiorespiratory vagal afferents and plays a critical role in cardiovascular regulation. <em>Angiotensin</em> II (Ang II) is a powerful signaling molecule in dmNTS neurons and exerts some of its biological effects by modulating Ca(2+) currents via reactive oxygen species (ROS) derived from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. We investigated whether a Nox2-containing NADPH oxidase is the source of the Ang II-induced ROS production and whether the signaling mechanisms of its activation require intracellular Ca(2+) or protein kinase C (PKC). Second-order dmNTS neurons were anterogradely labeled with 4-(4-[didecylamino]styryl)-N-methylpyridinium iodide transported from the vagus and isolated from the brain stem. ROS production was assessed in 4-(4-[didecylamino]styryl)-N-methylpyridinium iodide-positive dmNTS neurons using the fluorescent dye 6-carboxy-2',<em>7</em>'-dichlorodihydro-fluorescein di(acetoxymethyl ester). Ang II (3 to 2000 nmol/L) increased ROS production in dmNTS neurons (EC(50)=38.3 nmol/L). The effect was abolished by the ROS scavenger Mn (III) porphyrin 5,<em>1</em>0,20-tetrakis (benzoic acid) porphyrin manganese (III), the Ang II type <em>1</em> receptor antagonist losartan, or the NADPH oxidase inhibitors apocynin or gp9<em>1</em>ds-tat. Ang II failed to increase ROS production or to potentiate L-type Ca(2+) currents in dmNTS neurons of mice lacking Nox2. The PKC inhibitor GF<em>1</em>09203X or depletion of intracellular Ca(2+) attenuated Ang II-elicited ROS production. We conclude that the powerful effects of Ang II on Ca(2+) currents in dmNTS neurons are mediated by PKC activation leading to ROS production via Nox2. Thus, a Nox2-containing NADPH oxidase is the critical link between Ang II and the enhancement of Ca(2+) currents that underlie the actions of Ang II on central autonomic regulation.

It is well known that the RAS (renin-<em>angiotensin</em> system) plays a key role in the modulation of many functions in the body. AngII (<em>angiotensin</em> II) acting on AT<em>1</em>R (type <em>1</em> AngII receptor) has a central role in mediating most of the actions of the RAS. However, over the past <em>1</em>0 years, several studies have presented evidence for the existence of a new arm of the RAS, namely the ACE (<em>angiotensin</em>-converting enzyme) 2/Ang-(<em>1</em>-<em>7</em>) [<em>angiotensin</em>-(<em>1</em>-<em>7</em>)]/Mas axis. Ang-(<em>1</em>-<em>7</em>) can be produced from AngI or AngII via endo- or carboxy-peptidases respectively. ACE2 appears to play a central role in Ang-(<em>1</em>-<em>7</em>) formation. As described for AngII, Ang-(<em>1</em>-<em>7</em>) also has a broad range of effects in different organs and tissues which goes beyond its initially described cardiovascular and renal actions. Those effects are mediated by Mas and can counter-regulate most of the deleterious effects of AngII. The interaction Ang-(<em>1</em>-<em>7</em>)/Mas regulates different signalling pathways, such as PI3K (phosphoinositide 3-kinase)/AKT and ERK (extracellularsignal-regulated kinase) pathways and involves downstream effectors such as NO, FOXO<em>1</em> (forkhead box O<em>1</em>) and COX-2 (cyclo-oxygenase-2). Through these mechanisms, Ang-(<em>1</em>-<em>7</em>) is able to improve pathological conditions including fibrosis and inflammation in organs such as lungs, liver and kidney. In addition, this heptapeptide has positive effects on metabolism, increasing the glucose uptake and lipolysis while decreasing insulin resistance and dyslipidaemia. Ang-(<em>1</em>-<em>7</em>) is also able to improve cerebroprotection against ischaemic stroke, besides its effects on learning and memory. The reproductive system can also be affected by Ang-(<em>1</em>-<em>7</em>) treatment, with enhanced ovulation, spermatogenesis and sexual steroids synthesis. Finally, Ang-(<em>1</em>-<em>7</em>) is considered a potential anti-cancer treatment since it is able to inhibit cell proliferation and angiogenesis. Thus the ACE2/Ang-(<em>1</em>-<em>7</em>)/Mas pathway seems to be involved in many physiological and pathophysiological processes in several systems and organs especially by opposing the detrimental effects of inappropriate overactivation of the ACE/AngII/AT<em>1</em>R axis.

<em>Angiotensin</em>-converting enzyme 2 (ACE2) is a homolog of ACE that preferentially forms <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [ANG-(<em>1</em>-<em>7</em>)] from <em>angiotensin</em> II (ANG II). Incubation of neonatal rat cerebellar or medullary astrocytes with ANG II reduced ACE2 mRNA by approximately 60%, suggesting transcriptional regulation of the enzyme. In contrast, ANG II had no effect on ACE mRNA in astrocytes isolated from either brain region, demonstrating a differential regulation of the two enzymes by ANG II. The ANG II-mediated reduction in ACE2 mRNA was blocked by the <em>angiotensin</em> type <em>1</em> (AT(<em>1</em>)) receptor antagonists losartan or valsartan; the <em>angiotensin</em> type 2 (AT(2)) antagonist PD<em>1</em>233<em>1</em>9 was ineffective. The reduction in ACE2 mRNA by ANG II also was associated with a 50% decrease in cerebellar and medullary ACE2 protein, which was blocked by losartan. Treatment of medullary astrocytes with ANG-(<em>1</em>-<em>7</em>), the product of ACE2 hydrolysis of ANG II, did not affect ACE2 mRNA; however, ANG-(<em>1</em>-<em>7</em>) prevented the ANG II-mediated reduction in ACE2 mRNA. The addition of [d-Ala(<em>7</em>)]-ANG-(<em>1</em>-<em>7</em>), a selective AT((<em>1</em>-<em>7</em>)) receptor antagonist, blocked the inhibitory actions of ANG-(<em>1</em>-<em>7</em>). These data are the first to demonstrate transcriptional regulation of ACE2 by ANG II and ANG-(<em>1</em>-<em>7</em>). Because ACE2 preferentially converts ANG II to ANG-(<em>1</em>-<em>7</em>), downregulation of the enzyme by ANG II constitutes a novel positive feed-forward system within the brain that may favor ANG II-mediated neural responses. Furthermore, the modulatory role of ANG-(<em>1</em>-<em>7</em>) in the transcriptional regulation of ACE2 by ANG II suggests a complex interplay between these peptides that is mediated by distinct receptor systems.

Unlike the ubiquitous <em>angiotensin</em>-converting enzyme (ACE), the ACE-related carboxypeptidase 2 (ACE 2) is predominantly expressed in the heart, kidney, and testis. ACE 2 degrades <em>angiotensin</em> (Ang) II to Ang (<em>1</em>-<em>7</em>) and Ang I to Ang (<em>1</em>-9). We investigated the expression of ACE and ACE 2 in a rodent model of type 2 diabetes. ACE and ACE 2 were measured in kidney and heart from 8-week-old no diabetic control (db/m) mice and diabetic (db/db) mice, which at this young age have obesity and hyperglycemia without nephropathy. In renal cortical tissue, ACE mRNA was reduced (db/db 0.3<em>1</em>+/-0.06 versus db/m 0.99+/-0.05; P<0.005), whereas ACE 2 mRNA was not (db/db 0.94+/-0.05 versus db/m <em>1</em>.03+/-0.<em>1</em><em>1</em>, NS). ACE protein was markedly reduced in kidney cortex of db/db mice (db/db 0.24+/-0.<em>1</em>3 versus db/m <em>1</em>.02+/-0.<em>1</em>2; P<0.005), and this was associated with a corresponding decrease in renal ACE activity (db/db <em>1</em>2.<em>7</em>+/-3.<em>7</em> versus db/m 6<em>1</em>.6+/-4.4 mIU/mg protein; P<0.00<em>1</em>). ACE 2 protein, by contrast, was increased in kidneys from diabetic mice (db/db <em>1</em>.39+/-0.<em>1</em>4 versus db/m 0.53+/-0.04; P<0.005). An increase in ACE 2 protein and a decrease in ACE protein, respectively, were also seen by immunostaining of renal cortical tubules from the db/db mice. In heart tissue, there were no significant differences between db/db and db/m mice in either ACE mRNA and protein or ACE 2 mRNA and protein. We conclude that in young db/db mice, ACE 2 protein in renal cortical tubules is increased, whereas ACE protein is decreased. We propose that the pattern of low ACE protein coupled with increased ACE 2 protein expression may be renoprotective in early stages of diabetes.

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] is an endogenous peptide hormone of the renin-<em>angiotensin</em> system with vasodilator and anti-proliferative properties. Human adenocarcinoma SK-LU-<em>1</em> and A549 cells as well as non-small lung cancer SK-MES-<em>1</em> cells were treated with serum in the presence and absence of Ang-(<em>1</em>-<em>7</em>), to determine whether Ang-(<em>1</em>-<em>7</em>) inhibits the growth of lung cancer cells. Ang-(<em>1</em>-<em>7</em>) caused a significant reduction in serum-stimulated growth in all three lung cancer cell lines. Treatment with Ang-(<em>1</em>-<em>7</em>) resulted in both a dose- and time-dependent reduction in serum-stimulated DNA synthesis in all three cell lines, with IC(50)'s in the sub-nanomolar range. The Ang-(<em>1</em>-<em>7</em>) receptor antagonist [D-Ala(<em>7</em>)]-Ang-(<em>1</em>-<em>7</em>) blocked the attenuation of the serum-stimulated DNA synthesis of SK-LU-<em>1</em> cells by Ang-(<em>1</em>-<em>7</em>), while neither AT(<em>1</em>) nor AT(2) <em>angiotensin</em> receptor subtype antagonists prevented the response to the heptapeptide. MAS mRNA and protein, a receptor for Ang-(<em>1</em>-<em>7</em>), was detected in the three lung cancer cell lines, suggesting that the anti-proliferative effect of Ang-(<em>1</em>-<em>7</em>) in the cancer cells may be mediated by the non-AT(<em>1</em>), non-AT(2), AT((<em>1</em>-<em>7</em>)) receptor MAS. Other <em>angiotensin</em> peptides [Ang I, Ang II, Ang-(2-8), Ang-(3-8) and Ang-(3-<em>7</em>)] did not attenuate mitogen-stimulated DNA synthesis of SK-LU-<em>1</em> cells, demonstrating that Ang-(<em>1</em>-<em>7</em>) selectively inhibits SK-LU-<em>1</em> cancer cell growth. Pre-treatment of SK-LU-<em>1</em> cells with <em>1</em>0 nM Ang-(<em>1</em>-<em>7</em>) reduced serum-stimulated phosphorylation of extracellular signal-regulated kinase (ERK)<em>1</em> and ERK2, indicating that the anti-proliferative effects may occur, at least in part, through inhibition of the ERK signal transduction pathway. The results of this study suggest that Ang-(<em>1</em>-<em>7</em>) inhibits lung cancer cell growth through the activation of an <em>angiotensin</em> peptide receptor and may represent a novel chemotherapeutic and chemopreventive treatment for lung cancer.

In the past few years the classical concept of the renin-<em>angiotensin</em> system (RAS) has experienced substantial conceptual changes. The identification of the renin/prorenin receptor, the <em>angiotensin</em>-converting enzyme homologue ACE2 as an <em>angiotensin</em> peptide processing enzyme, Mas as a receptor for Ang-(<em>1</em>-<em>7</em>) and the possibility of signaling through ACE, have contributed to switch our understanding of the RAS from the classical limited-proteolysis linear cascade to a cascade with multiple mediators, multiple receptors, and multi-functional enzymes. In this review we will focus on the recent findings related to RAS and, in particular, on its role in diabetes by discussing possible interactions between RAS mediators, endothelium function, and insulin signaling transduction pathways as well as the putative role of ACE2-Ang-(<em>1</em>-<em>7</em>)-Mas axis in disease pathogenesis.

BACKGROUND

The renin-<em>angiotensin</em> system (RAS) is a key regulator of the cardiovascular system, electrolyte, and water balance. Here, we report identification and characterization of alamandine, a new heptapeptide generated by catalytic action of <em>angiotensin</em>-converting enzyme-2 <em>angiotensin</em> A or directly from <em>angiotensin</em>-(<em>1</em>-<em>7</em>).

OBJECTIVE

To characterize a novel component of the RAS, alamandine.

RESULTS

Using mass spectrometry we observed that alamandine circulates in human blood and can be formed from <em>angiotensin</em>-(<em>1</em>-<em>7</em>) in the heart. Alamandine produces several physiological actions that resemble those produced by <em>angiotensin</em>-(<em>1</em>-<em>7</em>), including vasodilation, antifibrosis, antihypertensive, and central effects. Interestingly, our data reveal that its actions are independent of the known vasodilator receptors of the RAS, Mas, and <em>angiotensin</em> II type 2 receptor. Rather, we demonstrate that alamandine acts through the Mas-related G-protein-coupled receptor, member D. Binding of alamandine to Mas-related G-protein-coupled receptor, member D is blocked by D-Pro(<em>7</em>)-<em>angiotensin</em>-(<em>1</em>-<em>7</em>), the Mas-related G-protein-coupled receptor, member D ligand β-alanine and PD<em>1</em>233<em>1</em>9, but not by the Mas antagonist A-<em>7</em><em>7</em>9. In addition, oral administration of an inclusion compound of alamandine/β-hydroxypropyl cyclodextrin produced a long-term antihypertensive effect in spontaneously hypertensive rats and antifibrotic effects in isoproterenol-treated rats. Alamandine had no noticeable proliferative or antiproliferative effect in human tumoral cell lines.

CONCLUSIONS

The identification of these 2 novel components of the RAS, alamandine and its receptor, provides new insights for the understanding of the physiological and pathophysiological role of the RAS and may help to develop new therapeutic strategies for treating human cardiovascular diseases and other related disorders.

Sex differences in blood pressure are evident in experimental models and human subjects, yet the mechanisms underlying this disparity remain equivocal. The current study sought to define the extent of male-female differences in the circulating and tissue renin-<em>angiotensin</em> aldosterone systems (RAASs) of congenic mRen(2). Lewis and control Lewis rats. Male congenics exhibited higher systolic blood pressure than females [200 +/- 4 vs. <em>1</em>46 +/- <em>7</em> mmHg, P < 0.0<em>1</em>] or Lewis males and females [<em>1</em><em>1</em>3 +/- 2 vs. <em>1</em><em>1</em>2 +/- 2 mmHg, P>> 0.05]. Plasma ANG II levels were twofold higher in male congenics [4<em>7</em> +/- 3 vs. <em>1</em>9 +/- 3 pM, P < 0.0<em>1</em>] and fivefold higher than in male or female Lewis rats [6 +/- <em>1</em> vs. 6 +/- <em>1</em> pM]. ANG I levels were also highest in the males; however, plasma ANG-(<em>1</em>-<em>7</em>) was higher in female congenics. Male congenics exhibited greater circulating renin and <em>angiotensin</em>-converting enzyme (ACE) activities, as well as <em>angiotensin</em>ogen, than female littermates. Renal cortical and medullary ANG II levels were also higher in the male congenics versus all the other groups; ANG I was lower in the males. Cortical ACE2 activity was higher in male congenics, yet neprilysin activity and protein were greater in the females, which may contribute to reduced renal levels of ANG II. These data reveal that sex differences in both the circulating and renal RAAS are apparent primarily in the hypertensive group. The enhanced activity of the RAAS in male congenics may contribute to the higher pressure and tissue injury evident in the strain.

<em>Angiotensin</em> (A) II plays a critical role in vascular remodeling, and its action is mediated by type <em>1</em> AII receptor (AT<em>1</em>R). Recently, <em>1</em>5-deoxy-(Delta)(<em>1</em>2,<em>1</em>4)-prostaglandin J(2) and thiazolidinediones have been shown to be ligands for peroxisome proliferator-activated receptor (PPAR)-gamma and activate PPAR-gamma. In the present work, we have studied the effect of PPAR-gamma on AT<em>1</em>R expression in rat vascular smooth muscle cells (VSMCs). We observed that: <em>1</em>) endogenous AT<em>1</em>R expression was significantly decreased by PPAR-gamma ligands both at messenger RNA and protein levels, whereas AT<em>1</em>R messenger RNA stability was not affected; 2) AII-induced increase of (3)H-thymidine incorporation into VSMCs was inhibited by PPAR-gamma ligands; 3) rat AT<em>1</em>R gene promoter activity was significantly suppressed by PPAR-gamma ligands, and PPAR-gamma overexpression further suppressed the promoter activity; 4) transcriptional analyses using AT<em>1</em>R gene promoter mutants revealed that a GC-box-related sequence within the -58/-34 region of the AT<em>1</em>R gene promoter was responsible for the suppression; 5) Sp<em>1</em> overexpression stimulated AT<em>1</em>R gene transcription via the GC-box-related sequence, which was inhibited by additional PPAR-gamma overexpression; 6) electrophoretic mobility shift assay suggested that Sp<em>1</em> could bind to the GC-box-related sequence whereas PPAR-gamma could not; <em>7</em>) antibody supershift experiments using VSMC nuclear extracts revealed that protein-DNA complexes formed on the GC-box-related sequence, which were decreased by PPAR-gamma coincubation, were mostly composed of Sp<em>1</em>; and 8) glutathione S-transferase pull-down assay revealed a direct interaction between PPAR-gamma and Sp<em>1</em>. Taken together, it is suggested that activated PPAR-gamma suppresses AT<em>1</em>R gene at a transcriptional level by inhibiting Sp<em>1</em> via a protein-protein interaction. PPAR-gamma ligands, thus, may inhibit AII-induced cell growth and hypertrophy in VSMCs by AT<em>1</em>R expression suppression and possibly be beneficial for treatment of diabetic patients with hypertension and atherosclerosis.

BACKGROUND

<em>Angiotensin</em>-converting enzyme 2 (ACE2) is highly expressed in the kidney and converts <em>angiotensin</em> (Ang) II to Ang-(<em>1</em>-<em>7</em>), a renoprotective peptide. Urinary ACE2 has been shown to be elevated in patients with chronic kidney disease. However, the effects of antihypertensive agents on urinary ACE2 remain unclear.

METHODS

Of participants in the Tanno-Sobetsu cohort study in 20<em>1</em><em>1</em> (n = 6<em>1</em><em>7</em>), subjects on no medication (n = <em>1</em>0<em>1</em>) and hypertensive patients treated with antihypertensive agents, including the calcium channel blockers amlodipine and long-acting nifedipine; the ACE inhibitor enalapril; and the Ang II receptor blockers losartan, candesartan, valsartan, telmisartan, and olmesartan, for more than <em>1</em> year (n = <em>1</em>00) were enrolled, and urinary ACE2 level was measured.

RESULTS

Glucose and hemoglobin A<em>1</em>c were significantly higher in patients treated with enalapril, telmisartan or olmesartan than in the control subjects. Urinary albumin-to-creatinine ratio (UACR) was significantly higher in patients treated with enalapril than in the control subjects. Urinary ACE2 level was higher in the olmesartan-treated group, but not the other treatment groups, than in the control group. Urinary ACE2 level was positively correlated with systolic blood pressure (r = 0.2<em>1</em><em>1</em>; P = 0.003), UACR (r = 0.36<em>7</em>; P < 0.00<em>1</em>), and estimated salt intake (r = 0.260; P < 0.00<em>1</em>). Multivariable regression analysis after adjustment of age, sex, and the correlated indices showed that the use of olmesartan was an independent predictor of urinary ACE2 level.

CONCLUSIONS

In contrast with other antihypertensive drugs, olmesartan may uniquely increase urinary ACE2 level, which could potentially offer additional renoprotective effects.

<em>Angiotensin</em> II (Ang II), a bioactive peptide of the renin-<em>angiotensin</em> system (RAAS), plays an important role in the development of cardiovascular diseases (CVD). Pharmacological inhibition of <em>angiotensin</em>-converting enzyme (ACE), the Ang II forming enzyme, or specific blockade of Ang II binding to <em>angiotensin</em> type <em>1</em> receptor (AT<em>1</em>R) through which it exerts its deleterious effects, were shown to provide some protection against progression of CVD. The ACE-Ang II-AT<em>1</em>R axis has been challenged over the last few years with RAAS components able to counterbalance the effects of the main axis. The ACE homologue ACE2 efficiently hydrolyses Ang II to form Ang (<em>1</em>-<em>7</em>), a peptide that exerts actions opposite to those of Ang II. In contrast to the Ang II axis, the role of the ACE2-Ang (<em>1</em>-<em>7</em>) axis in cardiac function is largely obscure. Ang (<em>1</em>-<em>7</em>) is present in the viable myocardium, and its formation depends on Ang II as a substrate. The expression of this peptide is associated with cardiac remodeling: it is lost in the infarcted area and significantly increased in the border area. Low doses of Ang (<em>1</em>-<em>7</em>) improve cardiac output and antagonize Ang II-induced vasoconstriction. The type of Ang (<em>1</em>-<em>7</em>) biological activity is tissue specific and dose dependent. These findings point to a possible protective role for Ang (<em>1</em>-<em>7</em>) in abating the Ang II-induced actions. The elevated expression of Ang (<em>1</em>-<em>7</em>) in failing heart tissue paralleled the expression of its forming enzyme, ACE2. Several observations and experimental evidence suggest a beneficial role for ACE2 in cardiovascular function. Elevated ACE2 expression at the initial stage of several pathologies which decline with progression of disease might indicate a protective role for ACE2. Genetic manipulation of ACE2 expression, either targeted disruption or overexpression, point to the possible significance of this enzyme in cardiac function. Based on the above, a therapeutic approach that will amplify the ACE2-Ang (<em>1</em>-<em>7</em>) axis could provide further protection against the development of CVD. It turns out that the merits of currently used drugs--ACE inhibitors, AT<em>1</em>R blockers and mineralocorticoid receptor blockers (MRB) - lay beyond their direct effects on suppression of the ACE-Ang II-AT<em>1</em>R axis as they also increase cardiac ACE2 and Ang (<em>1</em>-<em>7</em>) significantly.

The aim of this study was to test the hypothesis that treatment with <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [ANG-(<em>1</em>-<em>7</em>)] or ANG-(<em>1</em>-<em>7</em>) nonpeptide analog AVE-099<em>1</em> can produce protection against diabetes-induced cardiovascular dysfunction. We examined the influence of chronic treatment (4 wk) with ANG-(<em>1</em>-<em>7</em>) (5<em>7</em>6 microg.kg(-<em>1</em>).day(-<em>1</em>) ip) or AVE-099<em>1</em> (5<em>7</em>6 microg.kg(-<em>1</em>).day(-<em>1</em>) ip) on proteinuria, vascular responsiveness of isolated carotid and renal artery ring segments and mesenteric bed to vasoactive agonists, and cardiac recovery from ischemia-reperfusion in streptozotocin-treated rats (diabetes). Animals were killed 4 wk after induction of diabetes and/or treatment with ANG-(<em>1</em>-<em>7</em>) or AVE-099<em>1</em>. There was a significant increase in urine protein (23<em>1</em> +/- 2 mg/24 h) in diabetic animals compared with controls (88 +/- 6 mg/24 h). Treatment of diabetic animals with ANG-(<em>1</em>-<em>7</em>) or AVE-099<em>1</em> resulted in a significant reduction in urine protein compared with vehicle-treated diabetic animals (<em>1</em>83 +/- <em>1</em>6 and <em>1</em>49 +/- <em>1</em>5 mg/24 h, respectively). Treatment with ANG-(<em>1</em>-<em>7</em>) or AVE-099<em>1</em> also prevented the diabetes-induced abnormal vascular responsiveness to norepinephrine, endothelin-<em>1</em>, <em>angiotensin</em> II, carbachol, and histamine in the perfused mesenteric bed and isolated carotid and renal arteries. In isolated perfused hearts, recovery of left ventricular function from 40 min of global ischemia was significantly better in ANG-(<em>1</em>-<em>7</em>)- or AVE-099<em>1</em>-treated animals. These results suggest that activation of ANG-(<em>1</em>-<em>7</em>)-mediated signal transduction could be an important therapeutic strategy to reduce cardiovascular events in diabetic patients.

Rat models of hypertension, eg, spontaneously hypertensive stroke-prone rats (SHRSP), display reduced <em>angiotensin</em>-converting enzyme 2 (ACE2) mRNA and protein expression compared with control animals. The aim of this study was to investigate the role of ACE2 in the pathogenesis of hypertension in these models. Therefore, we generated transgenic rats on a SHRSP genetic background expressing the human ACE2 in vascular smooth muscle cells by the use of the SM22 promoter, called SHRSP-ACE2. In these transgenic rats vascular smooth muscle expression of human ACE2 was confirmed by RNase protection, real-time RT-PCR, and ACE2 activity assays. Transgene expression leads to significantly increased circulating levels of <em>angiotensin</em>-(<em>1</em>-<em>7</em>), a prominent product of ACE2. Mean arterial blood pressure was reduced in SHRSP-ACE2 compared to SHRSP rats, and the vasoconstrictive response to intraarterial administration of <em>angiotensin</em> II was attenuated. The latter effect was abolished by previous administration of an ACE2 inhibitor. To evaluate the endothelial function in vivo, endothelium-dependent and endothelium-independent agents such as acetylcholine and sodium nitroprusside, respectively, were applied to the descending thoracic aorta and blood pressure was monitored. Endothelial function turned out to be significantly improved in SHRSP-ACE2 rats compared to SHRSP. These data demonstrate that vascular ACE2 overexpression in SHRSP reduces hypertension probably by locally degrading <em>angiotensin</em> II and improving endothelial function. Thus, activation of the ACE2/<em>angiotensin</em>-(<em>1</em>-<em>7</em>) axis may be a novel therapeutic strategy in hypertension.

We have previously demonstrated that urokinase-type plasminogen activator (uPA) is highly expressed in the aneurysmal segment of the abdominal aorta (AAA) in apolipoprotein E-deficient (apoE-/-) mice treated with <em>angiotensin</em> II (Ang II). In the present study, we tested the hypothesis that uPA is essential for AAA formation in this model. An osmotic minipump containing Ang II (<em>1</em>.44 mg/kg per day) was implanted subcutaneously into <em>7</em>- to <em>1</em><em>1</em>-month-old male mice for <em>1</em> month. Ang II induced AAA in 9 (90%) of <em>1</em>0 hyperlipidemic mice deficient in apoE (apoE-/-/uPA+/+ mice) but in only 2 (22%) of 9 mice deficient in both apoE and uPA (apoE-/-/uPA-/- mice) (P<0.05). Although the expansion of the suprarenal aorta was significantly less in apoE-/-/uPA-/- mice than in apoE-/-/uPA+/+ mice, the aortic diameters of the aorta immediately above or below the suprarenal aorta were similar between the 2 groups. Ang II induced AAA in <em>7</em> (39%) of <em>1</em>8 strain-matched wild-type C5<em>7</em> black/6J control mice. The incidence was significantly higher in atherosclerotic apoE-deficient (apoE-/-) mice, in which 8 (<em>1</em>00%) of 8 mice developed AAA. Only <em>1</em> (4%) of 2<em>7</em> uPA-/- mice developed AAA after Ang II treatment. We conclude the following: (<em>1</em>) uPA plays an essential role in Ang II-induced AAA in mice with or without preexisting hyperlipidemia and atherosclerosis; (2) uPA deficiency does not affect the diameter of the nonaneurysmal portion of the aorta; and (3) atherosclerosis and/or hyperlipidemia promotes but is not essential for Ang II-induced AAA formation in this model.

<em>1</em>. In the rat, hypertension is induced by fetal exposure to maternal low-protein diets. The effect on blood pressure of undernutrition before conception and during discrete periods in early, mid or late pregnancy was assessed using an <em>1</em>8% casein (control) diet and a 9% casein to apply mild protein restriction. 2. The offspring of rats fed 9% casein developed raised blood pressure by weaning age. Feeding a low-protein diet before conception was not a prerequisite for programming of hypertension. 3. Hypertension was observed in rats exposed to low protein during the following gestational periods: days 0-<em>7</em>, days 8-<em>1</em>4 and days <em>1</em>5-22. Blood pressure increases elicited by these discrete periods of undernutrition were lower than those induced by feeding a low-protein diet throughout pregnancy. The effect in early gestation was significant only in male animals. Post-natal growth of male rats exposed to low-protein diets was accelerated, but kidneys were small in relation to body weight. 4. Biochemical indices of glucocorticoid action in liver, hippocampus, hypothalamus and lung were elevated in rats exposed to low-protein diets in utero. The apparent hypersensitivity to glucocorticoids was primarily associated with undernutrition in mid to late gestation. 5. Plasma renin activity was elevated in rats exposed to 9% casein over days <em>1</em>5-55 of gestation. Animals undernourished over days 0-<em>7</em> and 8-<em>1</em>4 produced pups with lower plasma <em>angiotensin</em> II concentrations at weaning. 6. Fetal exposure to maternal low-protein diets for any period in gestation may programme hypertension in the rat. Alterations to renal structure, renal hormone action or the hypothalamic-pituitary-adrenal axis may all play a role in the programming phenomenon, either independently or in concert.

Recent studies have shown that <em>angiotensin</em>-(<em>1</em>-<em>7</em>) (Ang-[<em>1</em>-<em>7</em>]), which is generated endogenously from both Ang I and II, is a bioactive component of the renin-<em>angiotensin</em> system and may play an important role in the regulation of blood pressure. However, little is known about its role in regulating the reactivity of the afferent arteriole or the mechanism(s) involved. We hypothesized that Ang-(<em>1</em>-<em>7</em>), acting on specific receptors, participates in the control of afferent arteriole tone. We first examined the direct effect of Ang-(<em>1</em>-<em>7</em>) on rabbit afferent arterioles microperfused in vitro, and we tested whether endothelium-derived relaxing factor/NO and cyclooxygenase products are involved in its actions. To assess the vasodilator effect of Ang-(<em>1</em>-<em>7</em>), afferent arterioles were preconstricted with norepinephrine, and increasing concentrations of Ang-(<em>1</em>-<em>7</em>) were added to the lumen. We found that <em>1</em>0(-<em>1</em>0) to <em>1</em>0(-6) mol/L Ang-(<em>1</em>-<em>7</em>) produced dose-dependent vasodilatation, increasing luminal diameter from 8.9+/-<em>1</em>.0 to <em>1</em>6.3+/-<em>1</em>.<em>1</em> microm (P<0.006). Indomethacin had no effect on Ang-(<em>1</em>-<em>7</em>)-induced dilatation. N(G)-nitro-L-arginine methyl ester, a NO synthesis inhibitor, abolished the dilatation induced by Ang-(<em>1</em>-<em>7</em>). We attempted to determine which <em>angiotensin</em> receptor subtype is involved in this process. We found that <em>1</em>0(-6) mol/L [d-Ala<em>7</em>]-Ang-(<em>1</em>-<em>7</em>), a potent and selective Ang-(<em>1</em>-<em>7</em>) antagonist, abolished the dilatation induced by Ang-(<em>1</em>-<em>7</em>). An <em>angiotensin</em> II type <em>1</em> receptor antagonist (L<em>1</em>58809) and an <em>angiotensin</em> II type 2 receptor antagonist (PD <em>1</em>233<em>1</em>9) at <em>1</em>0(-6) mol/L had no effect on Ang-(<em>1</em>-<em>7</em>)-induced dilatation. Our results show that Ang-(<em>1</em>-<em>7</em>) causes afferent arteriole dilatation. This effect may be due to production of NO, but not the action of cyclooxygenase products. Ang-(<em>1</em>-<em>7</em>) has a receptor-mediated vasodilator effect on the rabbit afferent arteriole. This effect may be mediated by Ang-(<em>1</em>-<em>7</em>) receptors, because <em>angiotensin</em> type <em>1</em> and type 2 receptor antagonists could not block Ang-(<em>1</em>-<em>7</em>)-induced dilatation. Thus, our data suggest that Ang-(<em>1</em>-<em>7</em>)opposes the action of Ang II and plays an important role in the regulation of renal hemodynamics.

<em>Angiotensin</em> II (AngII) has been shown to play a critical role in diabetic nephropathy and vasculopathy. Although it is well recognized that an <em>angiotensin</em>-converting enzyme (ACE)-dependent AngII-generating system is a major source of intrarenal AngII production, it is here reported that the chymase-dependent AngII-generating system is upregulated in the human diabetic kidney. This becomes particularly strong in those with hypertension. In the normal kidney, while ACE was constitutively expressed by most kidney cells, chymase was weakly expressed by mesangial cells (MC) and vascular smooth muscle cells (VSMC) only. In the diabetic kidney, while ACE expression was significantly upregulated (<em>1</em> to 3-fold) by tubular epithelial cells (TEC) and infiltrating mononuclear cells, there was also markedly increased chymase expression (<em>1</em>0 to <em>1</em>5-fold) by both MC and VSMC, with strong deposition in the collagen-rich extracellular matrix including both diffuse and nodular glomerulosclerosis, tubulointerstitial fibrosis, and vascular sclerosis. Interestingly, while ACE expression showed no difference in patients with or without hypertension, upregulation of chymase in hypertensive patients was much stronger than that seen in those without hypertension (4 to <em>7</em>-fold, P < 0.00<em>1</em>). Correlation analysis showed that, in contrast to the ACE expression, upregulation of chymase correlated significantly with the increase in BP and the severity of collagen matrix deposition within the glomerulus, tubulointerstitium, and arterial walls (all with P < 0.00<em>1</em>). In conclusion, the present study demonstrates that chymase, as an alternative AngII-generating enzyme, is markedly upregulated in the diabetic kidney and may be associated with the development of diabetic/hypertensive nephropathy. In addition, differential expression of ACE and chymase in the diabetic kidney indicates that both ACE and chymase may be of equal importance for AngII-mediated diabetic nephropathy and vascular disease. Results from this study suggest that blockade of both AngII-generating pathways may provide additional beneficial effect on diabetic nephropathy.