Hypertension alters cerebrovascular regulation and increases the brain's susceptibility to stroke and dementia. We investigated the temporal relationships between the arterial pressure (AP) elevation induced by "slow pressor" <em>angiotensin</em> II (ANG II) infusion, which recapitulates key features of human hypertension, and the resulting cerebrovascular dysfunction. Minipumps delivering saline or ANG II for <em>1</em>4 days were implanted subcutaneously in C5<em>7</em>BL/6 mice (n = 5/group). Cerebral blood flow was assessed by laser-Doppler flowmetry in anesthetized mice equipped with a cranial window. With ANG II (600 ng · kg(-<em>1</em>) · min(-<em>1</em>)), AP started to rise after 9 days (P < 0.05 vs. saline), remained elevated at <em>1</em><em>1</em>-<em>1</em><em>7</em> days, and returned to baseline at 2<em>1</em> days (P>> 0.05). ANG II attenuated the cerebral blood flow increase induced by neural activity (whisker stimulation) or endothelium-dependent vasodilators, an effect observed before the AP elevation (<em>7</em> days), as well as after the hypertension subsided (2<em>1</em> days). Nonpressor doses of ANG II (200 ng · kg(-<em>1</em>) · min(-<em>1</em>)) induced cerebrovascular dysfunction and oxidative stress without elevating AP (P>> 0.05 vs. saline), whereas phenylephrine elevated AP without inducing cerebrovascular effects. ANG II (600 ng · kg(-<em>1</em>) · min(-<em>1</em>)) augmented neocortical reactive oxygen species (ROS) with a time course similar to that of the cerebrovascular dysfunction. Neocortical application of the ROS scavenger manganic(I-II)meso-tetrakis(4-benzoic acid)porphyrin or the NADPH oxidase peptide inhibitor gp9<em>1</em>ds-tat attenuated ROS and cerebrovascular dysfunction. We conclude that the alterations in neurovascular regulation induced by slow pressor ANG II develop before hypertension and persist beyond AP normalization but are not permanent. The findings unveil a striking susceptibility of cerebrovascular function to the deleterious effects of ANG II and raise the possibility that cerebrovascular dysregulation precedes the elevation in AP also in patients with ANG II-dependent hypertension.

Despite evidence that hyperactivity of the vasodeleterious axis (ACE/<em>angiotensin</em> II (Ang II)/AT<em>1</em> receptor) of the renin-<em>angiotensin</em> system (RAS) is associated with the pathogenesis of diabetic retinopathy (DR) use of the inhibitors of this axis has met with limited success in the control of this pathophysiology. We investigated the hypothesis that enhancing the local activity of the recently established protective axis of the RAS, ACE2/Ang-(<em>1</em>-<em>7</em>), using adeno-associated virus (AAV)-mediated gene delivery of ACE2 or Ang-(<em>1</em>-<em>7</em>) would confer protection against diabetes-induced retinopathy. Genes expressing ACE2 and Ang-(<em>1</em>-<em>7</em>) were cloned in AAV vector. The effects of ocular AAV-ACE2/Ang-(<em>1</em>-<em>7</em>) gene transfer on DR in diabetic eNOS(-/-) mice and Sprague-Dawley (SD) rats were examined. Diabetes was associated with approximately tenfold and greater than threefold increases in the ratios of ACE/ACE2 and AT<em>1</em>R/Mas mRNA levels in the retina respectively. Intraocular administration of AAV-ACE2/Ang-(<em>1</em>-<em>7</em>) resulted in significant reduction in diabetes-induced retinal vascular leakage, acellular capillaries, infiltrating inflammatory cells and oxidative damage in both diabetic mice and rats. Our results demonstrate that DR is associated with impaired balance of retinal RAS. Increased expression of ACE2/Ang-(<em>1</em>-<em>7</em>) overcomes this imbalance and confers protection against DR. Thus, strategies enhancing the protective ACE2/Ang-(<em>1</em>-<em>7</em>) axis of RAS in the eye could serve as a novel therapeutic target for DR.

The gut microbiome is essential for physiological host responses and development of immune functions. The impact of gut microbiota on blood pressure and systemic vascular function, processes that are determined by immune cell function, is unknown.

Unchallenged germ-free mice (GF) had a dampened systemic T helper cell type <em>1</em> skewing compared to conventionally raised (CONV-R) mice. Colonization of GF mice with regular gut microbiota induced lymphoid mRNA transcription of T-box expression in T cells and resulted in mild endothelial dysfunction. Compared to CONV-R mice, <em>angiotensin</em> II (AngII; <em>1</em> mg/kg per day for <em>7</em> days) infused GF mice showed reduced reactive oxygen species formation in the vasculature, attenuated vascular mRNA expression of monocyte chemoattractant protein <em>1</em> (MCP-<em>1</em>), inducible nitric oxide synthase (iNOS) and NADPH oxidase subunit Nox2, as well as a reduced upregulation of retinoic-acid receptor-related orphan receptor gamma t (Rorγt), the signature transcription factor for interleukin (IL)-<em>1</em><em>7</em> synthesis. This resulted in an attenuated vascular leukocyte adhesion, less infiltration of Ly6G(+) neutrophils and Ly6C(+) monocytes into the aortic vessel wall, protection from kidney inflammation, as well as endothelial dysfunction and attenuation of blood pressure increase in response to AngII. Importantly, cardiac inflammation, fibrosis and systolic dysfunction were attenuated in GF mice, indicating systemic protection from cardiovascular inflammatory stress induced by AngII.

Gut microbiota facilitate AngII-induced vascular dysfunction and hypertension, at least in part, by supporting an MCP-<em>1</em>/IL-<em>1</em><em>7</em> driven vascular immune cell infiltration and inflammation.

When increased in vascular tissues, <em>angiotensin</em>-converting enzyme 2 (ACE2), a carboxypeptidase that hydrolyzes <em>angiotensin</em> II to <em>angiotensin</em>-(<em>1</em>-<em>7</em>), may augment the growth inhibitory and vasodilatory effects of the heptapeptide. We investigated the regulation of ACE2 and <em>angiotensin</em>-(<em>1</em>-<em>7</em>) expression in aortas and carotid arteries of <em>1</em>2-wk-old male spontaneously hypertensive rats (SHR) by determining the effect of sustained <em>angiotensin</em> type <em>1</em> (AT(<em>1</em>)) receptor blockade with olmesartan (<em>1</em>0 mg.kg(-<em>1</em>).day(-<em>1</em>), n = <em>1</em>3) compared with those that received atenolol (30 mg.kg(-<em>1</em>).day(-<em>1</em>), n = <em>1</em>3), hydralazine (<em>1</em>0 mg.kg(-<em>1</em>).day(-<em>1</em>), n = <em>1</em>3), or vehicle (n = 2<em>1</em>). Systolic blood pressures were approximately 30% lower (P < 0.05) in rats treated for 2 wk with olmesartan compared with vehicle-treated rats. Both atenolol and hydralazine produced similar decreases in systolic blood pressure. ACE2 mRNA in the thoracic aorta of olmesartan-treated rats (n = 8) was fivefold greater (P < 0.05) than that in vehicle-treated rats (n = <em>1</em>6), whereas atenolol (n = 8) or hydralazine (n = 8) had no effect. Immunostaining intensities in rats treated with olmesartan (n = 5) were also associated with increased (P < 0.05) ACE2 and <em>angiotensin</em>-(<em>1</em>-<em>7</em>) in thoracic aorta media compared with vehicle-treated rats. In contrast, immunostaining intensities for both ACE2 and <em>angiotensin</em>-(<em>1</em>-<em>7</em>) were not different from vehicle (n = 5) in carotid arteries of SHR medicated with either atenolol (n = 5) or hydralazine (n = 5). A comparison of vessel wall dimensions showed that olmesartan selectively reduced the thoracic aorta media-to-lumen ratio (P < 0.05) and media thickness (P < 0.05) without an effect on carotid artery morphometry. Compared with vehicle-treated SHR, vascular hypertrophy determined from media and lumen measurements was not changed in SHR given either atenolol or hydralazine. These data represent the first report of ACE2 and <em>angiotensin</em>-(<em>1</em>-<em>7</em>) expression in the aorta and carotid arteries of SHR. Increased ACE2 and <em>angiotensin</em>-(<em>1</em>-<em>7</em>) in association with altered dimensions of the thoracic aorta but not carotid arteries in response to olmesartan treatment provides evidence that this pathway is regulated by AT(<em>1</em>) receptors and may be important in mediating the pressure-independent vascular remodeling effects of <em>angiotensin</em> peptides.

Sex differences exist in the regulation of arterial pressure and renal function by the renin-<em>angiotensin</em> system (RAS). This may in part stem from a differential balance in the pressor and depressor arms of the RAS. In males, the ACE/AngII/AT(<em>1</em>)R pathways are enhanced, whereas, in females, the balance is shifted towards the ACE2/Ang(<em>1</em>-<em>7</em>)/MasR and AT(2)R pathways. Evidence clearly demonstrates that premenopausal women, as compared to aged-matched men, are protected from renal and cardiovascular disease, and this differential balance of the RAS between the sexes likely contributes. With aging, this cardiovascular protection in women is lost and this may be related to loss of estrogen postmenopause but the possible contribution of other sex hormones needs to be further examined. Restoration of these RAS depressor pathways in older women, or up-regulation of these in males, represents a therapeutic target that is worth pursuing.

The <em>angiotensin</em> (Ang) type <em>1</em> receptor (AT(<em>1</em>)R) is highly expressed on renal nuclei and stimulates reactive oxygen species (ROS). It is not known whether other functional components of the Ang system regulate the nuclear Ang II-AT(<em>1</em>)R ROS pathway. Therefore, we examined the expression of Ang receptors in nuclei isolated from the kidneys of young adult (<em>1</em>.5 years) and older adult (3.0 to 5.0 years) sheep. Binding studies in renal nuclei revealed the AT(2)R as the predominant receptor subtype ( approximately 80%) in young sheep, with the Ang-(<em>1</em>-<em>7</em>) (AT(<em>7</em>)R; Mas protein) and AT(<em>1</em>)R antagonists competing for the remaining sites. Conversely, in older sheep, the AT(<em>1</em>)R accounted for approximately 85% of nuclear sites, whereas the Ang type 2 receptor and AT(<em>7</em>)R subtypes comprise approximately 20% of remaining sites. Ang II increased nuclear ROS to a greater extent in older (9<em>7</em>+/-22%; n=6) versus young animals (<em>7</em>+/-2%; P=0.0<em>1</em>; n=4), and this was abolished by an AT(<em>1</em>)R antagonist. The AT(<em>7</em>)R antagonist D-Ala(<em>7</em>)-Ang-(<em>1</em>-<em>7</em>) increased ROS formation to Ang II by approximately 2-fold (<em>1</em><em>7</em>4+/-5% versus 9<em>7</em>+/-22%; P<0.05) in older adults. Immunoblots of renal nuclei revealed protein bands for the AT(<em>7</em>)R and Ang-converting enzyme 2 (ACE2), which metabolizes Ang II to Ang-(<em>1</em>-<em>7</em>). The ACE2 inhibitor MLN4<em>7</em>60 also exacerbated the Ang II-dependent formation of ROS (<em>1</em>56+/-<em>1</em>5%) and abolished the generation of Ang-(<em>1</em>-<em>7</em>) from Ang II. We conclude that an ACE2-Ang-(<em>1</em>-<em>7</em>)-AT(<em>7</em>)R pathway modulates Ang II-dependent ROS formation within the nucleus, providing a unique protective mechanism against oxidative stress and cell damage.

Purpose The primary toxicity of trastuzumab therapy for human epidermal growth factor receptor 2-overexpressing (HER2-positive) breast cancer is dose-independent cardiac dysfunction. <em>Angiotensin</em>-converting enzyme inhibitors and β-blockers are recommended first-line agents for heart failure. We hypothesized that <em>angiotensin</em>-converting enzyme inhibitors and β-blockers could prevent trastuzumab-related cardiotoxicity. Patients and Methods In this double-blinded, placebo-controlled trial, patients with HER2-positive early breast cancer were randomly assigned to receive treatment with perindopril, bisoprolol, or placebo (<em>1</em>:<em>1</em>:<em>1</em>) for the duration of trastuzumab adjuvant therapy. Patients underwent cardiac magnetic resonance imaging at baseline and post-cycle <em>1</em><em>7</em> for the determination of left ventricular volumes and left ventricular ejection fraction (LVEF). Cardiotoxicity was evaluated as the change in indexed left ventricular end diastolic volume and LVEF. Results Thirty-three patients received perindopril, 3<em>1</em> received bisoprolol, and 30 received placebo. Baseline demographic, cancer, and cardiovascular profiles were similar between groups. Study drugs were well tolerated with no serious adverse events. After <em>1</em><em>7</em> cycles of trastuzumab, indexed left ventricular end diastolic volume increased in patients treated with perindopril (+<em>7</em> ± <em>1</em>4 mL/m2), bisoprolol (+8 mL ± 9 mL/m2), and placebo (+4 ± <em>1</em><em>1</em> mL/m2; P = .36). In secondary analyses, trastuzumab-mediated decline in LVEF was attenuated in bisoprolol-treated patients (-<em>1</em> ± 5%) relative to the perindopril (-3 ± 4%) and placebo (-5 ± 5%) groups ( P = .00<em>1</em>). Perindopril and bisoprolol use were independent predictors of maintained LVEF on multivariable analysis. Conclusion Perindopril and bisoprolol were well tolerated in patients with HER2-positive early breast cancer who received trastuzumab and protected against cancer therapy-related declines in LVEF; however, trastuzumab-mediated left ventricular remodeling-the primary outcome-was not prevented by these pharmacotherapies.

Left ventricular dysfunction can be improved with <em>angiotensin</em>-converting-enzyme inhibition started <em>1</em> week after myocardial infarction or later. To see whether earlier intervention may confer greater benefit, a double-blind study was carried out in which <em>1</em>00 patients with Q wave myocardial infarction, but without clinical heart failure, were randomly allocated treatment with captopril 50 mg twice daily or placebo starting 24-48 h after onset of symptoms. Left ventricular volumes were measured regularly during 3 months of treatment and after a 48 h withdrawal period by means of two-dimensional echocardiography. The placebo group showed significant increases in left ventricular end-diastolic (LVEDVI) and end-systolic (LVESVI) volume indices, with the ejection fraction unchanged. By contrast, the captopril group showed a slight but not significant rise in LVEDVI and a significant reduction in LVESVI with ejection fraction increased significantly. At 3 months there was a 4.6% difference in the change in ejection fraction from baseline between the groups (p less than 0.000<em>1</em>). Most of the treatment benefit was evident at <em>1</em> month and there were no changes in left ventricular volumes after 48 h withdrawal of treatment at 3 months. Heart failure requiring treatment with frusemide developed in <em>7</em> patients in each group during the study period; 3 of these (<em>1</em> captopril-treated, 2 placebo-treated) had to be withdrawn from the trial with severe heart failure requiring open treatment. Thus early treatment with captopril is effective in preventing the ventricular dilatation that can occur after Q wave myocardial infarction.

A search was performed for presynaptic, release-modulating receptor systems on the post-ganglionic sympathetic nerves of rabbit pulmonary artery. Strips of the artery were preincubated with (-)-3H-noradrenaline and then superfused and stimulated transmurally. <em>1</em>. Tetrodotoxin, guanethidine, and omission of calcium all suppressed the stimulation-evoked overflow of tritium, thus confirming selective release from noradrenergic neurones. 49% of the stimulation-evoked overflow of total consisted of 3H-noradrenaline, 22% of 3H-3,4-dihydroxyphenyglycol (DOPEG), and 9% of 3H-normetanephrine. Cocaine virtually abolished the evoked overflow of 3H-DOPEG; further addition of corticosterone also abolished that of 3H-normetanephrine. In the presence of cocaine plus corticosterone, unmetabolized 3H-noradrenaline accounted for 86% of the stimulation-evoked overflow of total tritium. The overflow evoked per pulse was 2.2 X <em>1</em>0(-5) of the tritium content of the tissue (<em>1</em> Hz); it increased 2-fold when the frequency was raised to 8 Hz. 2. Presynaptic alpha-adrenoceptors have previously been demonstrated in this tissue (Starke et al., <em>1</em>9<em>7</em>5b). High concentrations of isoprenaline reduced the stimulation-evoked overflow of tritium, presumably by alpha-adrenergic inhibiton. No presynaptic effect of up to <em>1</em>0(-5) M normetanephrine and metanephrine was found. 3. Dopamine slightly diminished the stimulation-evoked overflow of tritium, but only at <em>1</em>00 times the inhibitory threshold concentration of noradrenaline (which is <em>1</em>0(-8) M; Starke et al., <em>1</em>9<em>7</em>5b), probably through activation of presynaptic alpha-adrenoceptors. Apomorphine failed to reduce the evoked overflow whether the superfusion medium contained cocaine and corticosterone or not. 4. Isoprenaline (<em>1</em>0(-9) -<em>1</em>0(-6) M) did not change the evoked overflow whether the medium contained cocaine and corticosterone or not, and whether the frequency was <em>1</em> or 2 Hz. Propranolol also had no effect. 5. <em>Angiotensin</em> II increased the stimulation-evoked overflow both in the absence and in the presence of cocaine and corticosterone. Equieffective concentrations of <em>angiotensin</em> I were <em>1</em>0 times higher. Saralasin had no effect, whereas <em>1</em>-Sar,8-Ile-<em>angiotensin</em> produced a small increase. Both of the latter peptides behaved as presynaptic antagonists of <em>angiotensin</em> II. A presynaptically supramaximal concentration of the alpha-adrenergic agonist oxymetazoline prevented the facilitatory action of yohimbine, but not that of <em>angiotensin</em> II. Separation of 3H-compounds showed that <em>angiotensin</em> II caused a proportionate increase in stimulation-evoked overflow of 3H-noradrenaline, 3H-DOPEG, and 3H-normetanephrine; this finding rules out any inhibition of noradrenaline uptake mechanisms. 6. <em>1</em>0(-4) -<em>1</em>0(-3) M acetylcholine caused hexamethonium-sensitive acceleration of basal tritium outflow. Much lower concentrations (<em>1</em>0(-<em>7</em>) M and higher) reduced the overflow evoked by electrical stimulation. The evoked overflow of 3H-noradrenaline, 3H-DOPEG, and 3H-normetanephrine was proportionately decreased...

We have recently shown that hydrolysis of labeled <em>angiotensin</em> I in canine brainstem homogenate causes a rapid accumulation of the heptapeptide <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)]. Although this <em>angiotensin</em> fragment has no vasopressor activity, its consistent generation in brain homogenate led us to study its potential neurosecretory effects in the rat hypothalamo-neurohypophysial system (HNS) in vitro. Ang-(<em>1</em>-<em>7</em>) or <em>angiotensin</em> II (Ang II) was added to HNS perifusate in concentrations of 0.04, 0.4, and 4 microM, and release of arginine vasopressin (AVP) during each treatment was quantified as a percentage of the AVP release detected in the preceding collection period. Base-line release of AVP averaged 28<em>1</em> +/- 4<em>7</em> pg per <em>1</em>5 min (mean +/- SEM) in HNS explants (five experiments, five explants per chamber) perifused in Krebs solution at 3<em>7</em> degrees C, after a <em>1</em>-hr equilibration period. At 0.04 microM, Ang II or Ang-(<em>1</em>-<em>7</em>) did not stimulate AVP release. Ang II increased AVP release over the control value by <em>1</em><em>7</em>2% +/- 44% and 268% +/- 66% at 0.4 and 4 microM, respectively; the same concentrations of Ang-(<em>1</em>-<em>7</em>) increased AVP release by <em>1</em>34% +/- <em>1</em>2% and 2<em>1</em>6% +/- 45%. The responses to Ang II and Ang-(<em>1</em>-<em>7</em>) at the highest concentration were both significant (P less than 0.05), and comparison by two-way analysis of variance indicated that Ang II and Ang-(<em>1</em>-<em>7</em>) were equipotent in stimulating AVP release over the range of concentrations studied. In the presence of the competitive Ang II antagonist [Sar<em>1</em>,Thr8]Ang II (20 microM), the release of AVP increased approximately equal to 2-fold. Neither Ang II nor Ang-(<em>1</em>-<em>7</em>) (4 microM) caused a further enhancement of AVP release in the presence of [Sar<em>1</em>,Thr8]Ang II. These data suggest that a hydrophobic residue in position 8 of the <em>angiotensin</em> peptide is not essential for activation of <em>angiotensin</em> receptors in the rat HNS. Moreover, the equipotence of Ang II and Ang-(<em>1</em>-<em>7</em>) indicates that Ang-(<em>1</em>-<em>7</em>) may participate in the control of AVP release.

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] is an endogenous seven-amino acid peptide hormone with antiproliferative properties. Our previous studies showed that Ang-(<em>1</em>-<em>7</em>) inhibits the growth of human lung cancer cells in vitro and reduces the size of human lung tumor xenografts in vivo. In the current study, s.c. injection of Ang-(<em>1</em>-<em>7</em>) not only caused a significant reduction in human A549 lung tumor growth but also markedly decreased vessel density, suggesting that the heptapeptide inhibits angiogenesis to reduce tumor size. A decrease in human endothelial cell tubule formation in Matrigel was observed following a <em>1</em>6 h incubation with Ang-(<em>1</em>-<em>7</em>), with a maximal reduction at a <em>1</em>0 nmol/L concentration. Ang-(<em>1</em>-<em>7</em>) had similar antiangiogenic effects in the chick chorioallantoic membrane, causing a >50% decrease in neovascularization. The Ang-(<em>1</em>-<em>7</em>)-induced reduction in both endothelial cell tubule formation and vessel formation in the chick was completely blocked by the specific Ang-(<em>1</em>-<em>7</em>) receptor antagonist [d-proline(<em>7</em>)]-Ang-(<em>1</em>-<em>7</em>), suggesting that these biological actions are mediated by an AT((<em>1</em>-<em>7</em>)) receptor. Ang-(<em>1</em>-<em>7</em>) significantly reduced vascular endothelial growth factor-A protein and mRNA in tumors from mice treated with the heptapeptide compared with saline controls as well as in the parent A549 human lung cancer cells in culture. These results suggest that Ang-(<em>1</em>-<em>7</em>) may attenuate tumor angiogenesis by reducing vascular endothelial growth factor-A, a primary proangiogenic protein. Taken together, this study shows that Ang-(<em>1</em>-<em>7</em>) exhibits significant antiangiogenic activity and may be a novel therapeutic agent for lung cancer treatment targeting a specific AT((<em>1</em>-<em>7</em>)) receptor.

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) (Ang-[<em>1</em>-<em>7</em>]) is a bioactive component of the renin-<em>angiotensin</em> system, which has depressor, vasodilatory, and antihypertensive actions. In normal pregnancy, we questioned whether the known rise in plasma <em>angiotensin</em> II (Ang II) is counterbalanced by an increase in plasma Ang-(<em>1</em>-<em>7</em>) and whether Ang-(<em>1</em>-<em>7</em>) levels are decreased in preeclampsia and may thus be a factor involved in the development of hypertension. Nulliparous preeclamptic subjects, third-trimester normotensive pregnant subjects, and a nonpregnant group were enrolled (n = <em>1</em>5/group). Preeclamptic subjects had no previous history of hypertension or renal, connective-tissue, or metabolic disease, but at the time of delivery had significant hypertension (<em>1</em>59 +/- 3/98 +/- 3 mmHg) and>> or = 3+ proteinuria. Plasma Ang-(<em>1</em>-<em>7</em>) was increased by 5<em>1</em>% in normal pregnancy (p < 0.05). Plasma Ang I, Ang II, and renin activity were also significantly elevated in normal pregnancy. In preeclamptic subjects, Ang-(<em>1</em>-<em>7</em>) was significantly decreased (p < 0.0<em>1</em>) compared with normal pregnant subjects. All other components of the renin-<em>angiotensin</em>-aldosterone system, except serum <em>angiotensin</em>-converting enzyme, were reduced in preeclamptic subjects compared with normal pregnant subjects; only plasma Ang II remained elevated in preeclamptic compared with nonpregnant subjects. These studies demonstrate, for the first time, increased plasma Ang-(<em>1</em>-<em>7</em>) in normal pregnant subjects compared with nonpregnant subjects and decreased Ang-(<em>1</em>-<em>7</em>) in preeclamptic subjects compared with normal pregnant subjects. In preeclampsia the decreased plasma Ang-(<em>1</em>-<em>7</em>) in the presence of elevated Ang II is consistent with the development of hypertension.

The renin-<em>angiotensin</em> system (RAS) is a critical regulator of hypertension, primarily through the actions of the vasoactive peptide Ang II, which is generated by the action of <em>angiotensin</em>-converting enzyme (ACE) mediating an increase in blood pressure. The discovery of ACE2, which primarily metabolises Ang II into the vasodilatory Ang-(<em>1</em>-<em>7</em>), has added a new dimension to the traditional RAS. As a result there has been huge interest in ACE2 over the past decade as a potential therapeutic for lowering blood pressure, especially elevation resulting from excess Ang II. Studies focusing on ACE2 have helped to reveal other actions of Ang-(<em>1</em>-<em>7</em>), outside vasodilation, such as antifibrotic and antiproliferative effects. Moreover, investigations focusing on ACE2 have revealed a variety of roles not just catalytic but also as a viral receptor and amino acid transporter. This paper focuses on what is known about ACE2 and its biological roles, paying particular attention to the regulation of ACE2 expression. In light of the entrance of human recombinant ACE2 into clinical trials, we discuss the potential use of ACE2 as a therapeutic and highlight some pertinent questions that still remain unanswered about ACE2.

Earlier work from this laboratory demonstrated that apoptosis of alveolar epithelial cells (AECs) requires autocrine generation of <em>angiotensin</em> (ANG) II. More recent studies showed that <em>angiotensin</em> converting enzyme-2 (ACE-2), which degrades ANGII to form ANG1-<em>7</em>, is protective but severely downregulated in human and experimental lung fibrosis. Here it was theorized that ACE-2 and its product ANG1-<em>7</em> might therefore regulate AEC apoptosis. To evaluate this hypothesis, the AEC cell line MLE-12 and primary cultures of rat AECs were exposed to the profibrotic apoptosis inducers ANGII or bleomycin (Bleo). Markers of apoptosis (caspase-9 or -3 activation and nuclear fragmentation), steady-state ANGII and ANG1-<em>7</em>, and JNK phosphorylation were measured thereafter. In the absence of Bleo, inhibition of ACE-2 by small interfering RNA or by a competitive inhibitor (DX600 peptide) caused a reciprocal increase in autocrine ANGII and corresponding decrease in ANG1-<em>7</em> in cell culture media (both P < 0.05) and, moreover, induced AEC apoptosis. At baseline (without inhibitor), ANG1-<em>7</em> in culture media was 10-fold higher than ANGII (P < 0.01). Addition of purified ANGII or bleomycin-induced caspase activation, nuclear fragmentation, and JNK phosphorylation in cultured AECs. However, preincubation with ANG1-<em>7</em> (0.1 μM) prevented JNK phosphorylation and apoptosis. Moreover, pretreatment with A<em>7</em><em>7</em>9, a specific blocker of the ANG1-<em>7</em> receptor mas, prevented ANG1-<em>7</em> blockade of JNK phosphorylation, caspase activation, and nuclear fragmentation. These data demonstrate that ACE-2 regulates AEC survival by balancing the proapoptotic ANGII and its antiapoptotic degradation product ANG1-<em>7</em>. They also suggest that ANG1-<em>7</em> inhibits AEC apoptosis through the ANG1-<em>7</em> receptor mas.

In spite of recent advancements in the treatment of pulmonary hypertension, successful control has yet to be accomplished. The abundant presence of <em>angiotensin</em>-converting enzyme 2 (ACE2) in the lungs and its impressive effect in the prevention of acute lung injury led us to test the hypothesis that pulmonary overexpression of this enzyme could produce beneficial outcomes against pulmonary hypertension. Monocrotaline (MCT) treatment of mice for 8 weeks resulted in significant increases in right ventricular systolic pressure, right ventricle:left ventricle plus septal weight ratio, and muscularization of pulmonary vessels. Administration of a lentiviral vector containing ACE2, <em>7</em> days before MCT treatment prevented the increases in right ventricular systolic pressure (control: 25+/-<em>1</em> mm Hg; MCT: 44+/-5 mm Hg; MCT+ACE2: 26+/-<em>1</em> mm Hg; n=6; P<0.05) and right ventricle:left ventricle plus septal weight ratio (control: 0.25+/-0.0<em>1</em>; MCT: 0.3<em>1</em>+/-0.0<em>1</em>; MCT+ACE2: 0.26+/-0.0<em>1</em>; n=8; P<0.05). A significant attenuation in muscularization of pulmonary vessels induced by MCT was also observed in animals overexpressing ACE2. These beneficial effects were associated with an increase in the <em>angiotensin</em> II type 2 receptor:<em>angiotensin</em> II type <em>1</em> receptor mRNA ratio. Also, pulmonary hypertension-induced increases in proinflammatory cytokines were significantly attenuated by lentiviral vector-containing ACE2 treatment. Furthermore, ACE2 gene transfer in mice after 6 weeks of MCT treatment resulted in a significant reversal of right ventricular systolic pressure. These observations demonstrate that ACE2 overexpression prevents and reverses right ventricular systolic pressure and associated pathophysiology in MCT-induced pulmonary hypertension by a mechanism involving a shift from the vasoconstrictive, proliferative, and fibrotic axes to the vasoprotective axis of the renin-<em>angiotensin</em> system and inhibition of proinflammatory cytokines.

OBJECTIVE

In this review we will focus on the recent findings related to <em>angiotensin</em>-(<em>1</em>-<em>7</em>) as an <em>angiotensin</em> II counter-regulatory peptide within the renin-<em>angiotensin</em> system.

RESULTS

The identification of the <em>angiotensin</em>-converting enzyme homologue ACE2 as an <em>angiotensin</em> peptide processing enzyme and of Mas as a receptor for <em>angiotensin</em>-(<em>1</em>-<em>7</em>) has contributed to establishing this heptapeptide as a biologically active member of the renin-<em>angiotensin</em> system cascade.

CONCLUSIONS

The previously unsuspected complexity of the renin-<em>angiotensin</em> system, unmasked by novel findings, has revealed new possibilities for exploring its physiological and pathophysiological roles. In addition, the ACE2-<em>angiotensin</em>-(<em>1</em>-<em>7</em>)-Mas axis may be seriously considered as a putative target for the development of new cardiovascular drugs.

In summary, the prevailing concept is that brain Ang II increases blood pressure by activating AT<em>1</em> receptors, and that these have a neuromodulating effect to increase the activity of autonomic nervous system. Pathways for Ang II stimulating thirst and blood pressure, increased vasopressin release and sympathetic activation have been outlined. Brain RAS synthesis, while incompletely understood, is active in the absence of a peripheral RAS. <em>Angiotensin</em> elicits specific receptor mediated signals in neurons, particularly in the hypothalamus and brainstem. These actions are due to neuronal membrane ionic currents and the regulation of transcription factors. The areas to be explored further are characterization and functional roles of the other AT receptor subtypes, such as AT4, AT(<em>1</em>-<em>7</em>) and nuclear AT-R. Their interactions with other peptides and transmitters, and their signaling pathways need to be investigated. The story that began <em>1</em>00 years ago with renin is certainly not ended and will continue to unfold as further investigations with new techniques progress.

This work was designed to study the expression of the vasodilator peptide <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] and its generating enzyme (ACE2) in the uteroplacental interface. Placentas were obtained from <em>1</em><em>1</em> early pregnancy failures (5 miscarriages and 6 ectopic pregnancies), <em>1</em>5 normotensive, and <em>1</em>0 preeclamptic gestations. In placental villi, the main sites of immunocytochemical expression of Ang-(<em>1</em>-<em>7</em>) and ACE2 were the syncytiotrophoblast, cytotrophoblast, endothelium and vascular smooth muscle of primary and secondary villi. Syncitial Ang-(<em>1</em>-<em>7</em>) expression in samples obtained from miscarriages and ectopic pregnancies was increased compared to normal term pregnancy [2.0 (2.0-2.25 for the 25 and <em>7</em>5% interquartile range) vs <em>1</em>.3 (<em>1</em>.0-<em>1</em>.9), p<0.0<em>1</em>]. In the maternal stroma, Ang-(<em>1</em>-<em>7</em>) and ACE2 were expressed in the invading and intravascular trophoblast and in decidual cells in all 3 groups. Ang-(<em>1</em>-<em>7</em>) and ACE2 staining was also found in arterial and venous endothelium and smooth muscle of the umbilical cord. The expression of Ang-(<em>1</em>-<em>7</em>) and ACE2 was similar in samples obtained from normal term or preeclamptic pregnancies, except for increased expression of ACE2 in umbilical arterial endothelium in preeclampsia [0.5 (0.5-0.8) vs 0.0 (0.0-0.0), p<0.0<em>1</em>]. The uteroplacental location of Ang-(<em>1</em>-<em>7</em>) and ACE2 in pregnancy suggests an autocrine function of Ang-(<em>1</em>-<em>7</em>) in the vasoactive regulation that characterizes placentation and established pregnancy.

BACKGROUND

Cyclic mechanical stretch (CMS) is an important physiological and pathological factor in the heart.

OBJECTIVE

We examined whether CMS can affect localization of gap junctions with regard to the cell axis.

RESULTS

Neonatal rat cardiomyocytes were cultured (<em>7</em> days) on flexible 6-well plates. Thereafter, cells were kept static or stimulated with CMS (<em>1</em> Hz; 0, <em>1</em>0, 20% elongation) for 0, 24, or 48 hours (with or without <em>1</em>0 micromol/L PD98059, 5 micromol/L BIM I (bisindolylmaleimide I), 2 micromol/L H8 [N-(2-methlyamino-ethyl)-5-isoquinoline-sulfonamid], or 0.<em>1</em> micromol/L <em>angiotensin</em> II. Additionally, cells were exposed to 24 hours of CMS followed by 24 hours of static recovery. CMS (24 hour, <em>1</em>0%) induced elongation of the cardiomyocytes and orientation <em>7</em>9+/-8 degrees toward the stretch direction. Moreover, the distribution of connexin (Cx)43 together with N-cadherin changed, so that both proteins were accentuated at the cell poles, whereas in nonstretched cells, they were distributed around the cell without preferential localization. Additional <em>angiotensin</em> II reduced polar Cx43 accentuation. The CMS-induced changes in Cx43 were reversible within 24 hours after end of stretch, and could be completely prevented by the MEK<em>1</em>/2 inhibitor PD98059 but not by BIM I or H8. Moreover, stretch resulted in Cx43 protein and Cx43-mRNA upregulation and in a significant upregulation of the phosphorylated forms of ERK<em>1</em>/2, glycogen synthase kinase 3beta and AKT. Furthermore, CMS resulted in a significant increase of the transcription factors activator protein <em>1</em> and CREB (cAMP response element-binding protein) in the nucleus.

CONCLUSIONS

CMS results in self-organization of cardiomyocytes leading to elongated cells orientated transverse to the stretch axis, enhanced Cx43 expression and Cx43 accentuation at the cell poles. The Cx43-changes seem to depend on the ERK<em>1</em>/2 signaling cascade.

BACKGROUND

Patients with type 2 diabetes and nephropathy have high cardiorenal morbidity and mortality despite optimum treatment including angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs). Residual risk is related to residual albuminuria. We assessed whether CCX140-B, a selective inhibitor of C-C chemokine receptor type 2 (CCR2), could further reduce albuminuria when given in addition to standard care, including ACE inhibitors or ARBs.

METHODS

In this randomised, double-blind, placebo-controlled clinical trial, we recruited patients from 78 research centres in Belgium, Czech Republic, Germany, Hungary, Poland, and the UK. We enrolled patients with type 2 diabetes aged 18-75 years with proteinuria (first morning void urinary albumin to creatinine ratio [UACR] 100-3000 mg/g), estimated glomerular filtration rate of 25 mL/min per 1·73 m(2) or higher, and taking stable antidiabetic treatment and ACE inhibitors or ARBs, for at least 8 weeks before study entry. Patients were stratified based on baseline UACR and renal function (estimated glomerular filtration rate), and then randomly assigned (1:1:1) via an interactive web response system with a minimisation algorithm to oral placebo, 5 mg CCX140-B, or 10 mg CCX140-B once a day. The 12-week dosing period in the initial protocol was extended to 52 weeks by protocol amendment. The primary efficacy measure was change from baseline in UACR during 52 weeks in the modified intention-to-treat population (all patients with uninterrupted dosing, excluding patients who stopped dosing at week 12 either permanently under the original protocol, or temporarily because of delay in approval of the protocol amendment). We did safety analyses on all randomly assigned patients who received at least one dose of study drug. According to a prespecified analysis plan, we analysed the primary endpoint with one-sided statistical testing with calculation of upper 95% confidence limits of the differences between active and control. This trial is registered with ClinicalTrials.gov, number NCT01447147.

RESULTS

The study ran from Dec 7, 2011 (first patient enrolled), until Aug 4, 2014. We enrolled 332 patients: 111 were assigned to receive placebo, 110 to 5 mg CCX140-B, and 111 to 10 mg CCX140-B. Of these, 192 were included in the modified intention-to-treat population. UACR changes from baseline during 52 weeks were -2% for placebo (95% CI -11% to 9%), -18% for 5 mg CCX140-B (-26% to -8%), and -11% for 10 mg CCX140-B (-20% to -1%). We recorded a -16% difference between 5 mg CCX140-B and placebo (one-sided upper 95% confidence limit -5%; p=0·01) and a -10% difference between 10 mg CCX140-B and placebo (upper 95% confidence limit 2%; p=0·08). Adverse events occurred in 81 (73%) of 111 patients in the placebo group versus 71 (65%) of 110 patients in the CCX140-B 5 mg group and 68 (61%) of 111 patients in the CCX140-B 10 mg group; there were no renal events during the study.

CONCLUSIONS

Our data suggest that CCR2 inhibition with CCX140-B has renoprotective effects on top of current standard of care in patients with type 2 diabetes and nephropathy.

BACKGROUND

ChemoCentryx.

BACKGROUND

The renin <em>angiotensin</em> system (RAS) plays an important role in inflammation and fibrosis. The classical axis of the RAS, formed by <em>angiotensin</em> converting en-zyme (ACE), <em>angiotensin</em> II (Ang II) and <em>angiotensin</em> receptor type <em>1</em> (AT<em>1</em>), activates several cell functions and molecular signaling pathways related to tissue injury, inflammation and fibrosis. In sharp contrast, the RAS axis composed by <em>angiotensin</em> converting enzyme 2 (ACE2), <em>angiotensin</em>-(<em>1</em>-<em>7</em>) and Mas receptor exerts opposite effects in relation to inflammatory response and tissue fibrosis.

OBJECTIVE

In this review, we have the aim to summarize recent findings on the anti-inflammatory and anti-fibrogenic role of ACE2/Ang-(<em>1</em>-<em>7</em>)/Mas axis in the context of basic research, experimental human dis-eases and clinical studies.

RESULTS

Several studies showed that ACE2/Angiotensin-(<em>1</em>-<em>7</em>)/Mas axis reduces cytokine release and inhibits signaling pathways of tissue fibrosis in experimental models of human diseases including atherosclerosis, cerebral ischemia, obesity, chronic kidney disease, liver diseases and asthma. On the other hand, very few data was provided by clinical studies.

CONCLUSIONS

Experimental studies clearly support the anti-inflammatory and anti-fibrotic effects of ACE2/ Ang-(<em>1</em>-<em>7</em>)/Mas axis. Clinical studies, especially phase III and IV trials, will be necessary to establish the therapeutic role of ACE2/Ang-(<em>1</em>-<em>7</em>)/Mas axis in controlling inflammation in different human diseases.

BACKGROUND

In the Canagliflozin Cardiovascular Assessment Study (CANVAS) Program, canagliflozin reduced the rates of major adverse cardiovascular events and the results suggested a renal benefit in patients with type 2 diabetes who were at high risk for cardiovascular events, compared with those treated with placebo. Here we report the results of a prespecified exploratory analysis of the long-term effects of canagliflozin on a range of sustained and adjudicated renal outcomes.

METHODS

The CANVAS Program consists of two double-blind, randomised trials that assessed canagliflozin versus placebo in participants with type 2 diabetes who were at high risk of cardiovascular events, done at 66<em>7</em> centres in 30 countries. People with type 2 diabetes and an HbA<em>1</em>c of <em>7</em>·0-<em>1</em>0·5% (53-9<em>1</em> mmol/mol) who were aged at least 30 years and had a history of symptomatic atherosclerotic vascular disease, or who were aged at least 50 years and had at least two cardiovascular risk factors were eligible to participate. Participants in CANVAS were randomly assigned (<em>1</em>:<em>1</em>:<em>1</em>) to receive 300 mg canagliflozin, <em>1</em>00 mg canagliflozin, or matching placebo once daily. Participants in CANVAS-R were randomly assigned (<em>1</em>:<em>1</em>) to receive canagliflozin or matching placebo, at an initial dose of <em>1</em>00 mg daily, with optional uptitration to 300 mg from week <em>1</em>3 or matching placebo. Participants and all study staff were masked to treatment allocations until study completion. Prespecified outcomes reported here include a composite of sustained and adjudicated doubling in serum creatinine, end-stage kidney disease, or death from renal causes; the individual components of this composite outcome; annual reductions in estimated glomerular filtration rate (eGFR); and changes in urinary albumin-to-creatinine ratio (UACR). The trials are registered with ClinicalTrials.gov, numbers NCT0<em>1</em>032629 (CANVAS) and NCT0<em>1</em>989<em>7</em>54 (CANVAS-R).

RESULTS

Between Nov <em>1</em><em>7</em>, 2009, and March <em>7</em>, 20<em>1</em><em>1</em> (CANVAS), and Jan <em>1</em><em>7</em>, 20<em>1</em>4, and May 29, 20<em>1</em>5 (CANVAS-R), <em>1</em>5 494 people were screened, of whom <em>1</em>0 <em>1</em>42 participants (with a baseline mean eGFR <em>7</em>6·5 mL/min per <em>1</em>·<em>7</em>3 m2, median UACR <em>1</em>2·3 mg/g, and 80% of whom were receiving renin-angiotensin system blockade) were randomly allocated to receive either canagliflozin or placebo. The composite outcome of sustained doubling of serum creatinine, end-stage kidney disease, and death from renal causes occurred less frequently in the canagliflozin group compared with the placebo group (<em>1</em>·5 per <em>1</em>000 patient-years in the canagliflozin group vs 2·8 per <em>1</em>000 patient-years in the placebo group; hazard ratio 0·53, 95% CI 0·33-0·84), with consistent findings across prespecified patient subgroups. Annual eGFR decline was slower (slope difference between groups <em>1</em>·2 mL/min per <em>1</em>·<em>7</em>3 m2 per year, 95% CI <em>1</em>·0-<em>1</em>·4) and mean UACR was <em>1</em>8% lower (95% CI <em>1</em>6-20) in participants treated with canagliflozin than in those treated with placebo. Total serious renal-related adverse events were similar between the canagliflozin and placebo groups (2·5 vs 3·3 per <em>1</em>000 patient-years; HR 0·<em>7</em>6, 95% CI 0·49-<em>1</em>·<em>1</em>9).

CONCLUSIONS

In a prespecified exploratory analysis, canagliflozin treatment was associated with a reduced risk of sustained loss of kidney function, attenuated eGFR decline, and a reduction in albuminuria, which supports a possible renoprotective effect of this drug in people with type 2 diabetes.

BACKGROUND

Janssen Research & Development.

OBJECTIVE

To evaluate the effectiveness of long-term <em>angiotensin</em> (Ang) (<em>1</em>-<em>7</em>) treatment to inhibit the progression of atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice.

RESULTS

Ang (<em>1</em>-<em>7</em>) is a heptapeptide fragment that has been proposed to counterregulate the Ang II proatherogenic effects. The effect of long-term 4-week Ang (<em>1</em>-<em>7</em>) treatment on both inhibition of atherosclerotic lesion development and improvement of endothelial function was examined in apolipoprotein E(-/-) mice that had been fed an atherogenic high-fat (2<em>1</em>%) diet for <em>1</em>6 weeks. Chronic Ang (<em>1</em>-<em>7</em>) treatment significantly improved endothelial function, an effect reversed with either <em>angiotensin</em> type 2 (AT(2)) or Mas receptor blockade. In these vessels, Ang (<em>1</em>-<em>7</em>) treatment significantly decreased superoxide production and increased endothelial nitric oxide synthase immunoreactivity when compared with vehicle treatment. These effects were blocked by both AT(2) and Mas receptor antagonists. Lesion development, assessed as both fatty deposits (oil red O) and intima to media ratio, was also significantly decreased with Ang (<em>1</em>-<em>7</em>) treatment compared with respective controls. Cotreatment with either AT(2) or Mas receptor antagonists reversed Ang (<em>1</em>-<em>7</em>)-mediated reduction in lesion development.

CONCLUSIONS

Long-term Ang (<em>1</em>-<em>7</em>) treatment caused both vasoprotection, via improvement in endothelial function, and atheroprotection, with a reduction in lesion progression in a model of atherosclerosis. These effects appear to be mediated by the restoration of nitric oxide bioavailability and involve a complex interaction of both Mas and AT(2) receptors.

BACKGROUND

Albuminuria and hypertension are predictors of poor renal and cardiovascular outcome in diabetic patients. We tested whether dual blockade of the renin-<em>angiotensin</em> system (RAS) with both an <em>angiotensin</em>-converting enzyme (ACE) inhibitor and an <em>angiotensin</em> II receptor blocker (ARB) is superior to maximal recommended dose of ACE inhibitor in type <em>1</em> diabetic patients with diabetic nephropathy (DN).

METHODS

We performed a randomized, double-blind, crossover trial with 8 weeks treatment with placebo and irbesartan 300 mg (once daily), added on top of enalapril 40 mg (once daily). We included 24 type <em>1</em> patients with DN. At the end of each treatment period, albuminuria, 24-hour blood pressure, and glomerular filtration rate (GFR) were measured.

RESULTS

Values on ACE inhibitors + placebo were: albuminuria [mean (95% CI)], 5<em>1</em>9 (342 to 789) mg/24 hours; blood pressure [mean (SEM)], <em>1</em>3<em>1</em> (3)/74 (<em>1</em>) mm Hg, and GFR [mean (SEM)], 65 (5) mL/min/<em>1</em>.73 m2. Dual blockade of the RAS induced a reduction in albuminuria [mean (95% CI)] of 25% (<em>1</em>5, 34) (P < 0.00<em>1</em>), a reduction in systolic blood pressure of 8 mm Hg (4, <em>1</em>2) (P = 0.002), and a reduction of 4 mm Hg (2, 7) (P = 0.003) in diastolic blood pressure. GFR and plasma potassium remained unchanged during both treatment regimes. Dual blockade was safe and well tolerated.

CONCLUSIONS

Dual blockade of the RAS is superior to maximal recommended dose of ACE inhibitors with regard to lowering of albuminuria and blood pressure in type <em>1</em> patients with DN. Long-term trials are needed to further establish the role of dual blockade of the RAS in renal and cardiovascular protection.