OBJECTIVE

Obesity and diabetes remain among the world's most pervasive health problems. Although the importance of <em>angiotensin</em> II for metabolic regulation is well documented, the role of the <em>angiotensin</em>-(<em>1</em>-<em>7</em>)/Mas axis in this process is poorly understood. The aim of this study was to evaluate the effect of increased <em>angiotensin</em>-(<em>1</em>-<em>7</em>) plasma levels in lipid and glucose metabolism using transgenic rats that express an <em>angiotensin</em>-(<em>1</em>-<em>7</em>)-releasing fusion protein, TGR(A<em>1</em>-<em>7</em>)3292 (TGR).

RESULTS

The increased <em>angiotensin</em>-(<em>1</em>-<em>7</em>) levels in TGR induced enhanced glucose tolerance, insulin sensitivity, and insulin-stimulated glucose uptake. In addition, TGR presented decreased triglycerides and cholesterol levels, as well as a significant decrease in abdominal fat mass, despite normal food intake. These alterations were accompanied by a marked decrease of <em>angiotensin</em>ogen expression and increased Akt in adipose tissue. Furthermore, augmented plasma levels and expression in adipose tissue was observed for adiponectin. Accordingly, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) stimulation increased adiponectin production by primary adipocyte culture, which was blocked by the Mas antagonist A<em>7</em><em>7</em>9. Circulating insulin and muscle glycogen content were not altered in TGR.

CONCLUSIONS

These results show that increased circulating <em>angiotensin</em>-(<em>1</em>-<em>7</em>) levels lead to prominent changes in glucose and lipid metabolism.

Duchenne muscular dystrophy (DMD) is the most common inherited neuromuscular disease, and is characterized by the lack of dystrophin, muscle wasting, increased transforming growth factor (TGF)-β Smad-dependent signalling and fibrosis. Acting via the Mas receptor, <em>angiotensin</em>-<em>1</em>-<em>7</em> [Ang-(<em>1</em>-<em>7</em>)], is part of the renin-<em>angiotensin</em> system, with the opposite effect to that of <em>angiotensin</em> II. We hypothesized that the Ang-(<em>1</em>-<em>7</em>)/Mas receptor axis might protect chronically damaged tissues as in skeletal muscle of the DMD mouse model mdx. Infusion or oral administration of Ang-(<em>1</em>-<em>7</em>) in mdx mice normalized skeletal muscle architecture, decreased local fibrosis and improved muscle function in vitro and in vivo. These positive effects were mediated by the inhibition of TGF-β Smad signalling, which in turn led to reduction of the pro-fibrotic microRNA miR-2<em>1</em> concomitant with a reduction in the number of TCF4 expressing fibroblasts. Mdx mice infused with Mas antagonist (A-<em>7</em><em>7</em>9) and mdx deficient for the Mas receptor showed highly deteriorated muscular architecture, increased fibrosis and TGF-β signalling with diminished muscle strength. These results suggest that this novel compound Ang-(<em>1</em>-<em>7</em>) might be used to improve quality of life and delay death in individuals with DMD and this drug should be investigated in further pre-clinical trials.

We examined the dose-related effects of <em>angiotensin</em>-converting enzyme inhibitors on circulating and tissue levels of <em>angiotensin</em> and bradykinin peptides by administering perindopril or lisinopril to rats in drinking water for <em>7</em> days. A reduction in the ratio of plasma <em>angiotensin</em> II (Ang II) to Ang I was seen for 0.006 mg/kg per day perindopril, with an increase in plasma renin and Ang I at 0.0<em>1</em><em>7</em> mg/kg per day. Plasma Ang II levels did not decrease until <em>1</em>.4 mg/kg per day perindopril, at which dose plasma Ang I levels reached a plateau of an approximate 25-fold increase. The effects of perindopril on Ang II and Ang I levels in heart, lung, aorta, and brown adipose tissue were parallel to those observed for plasma. By contrast, renal Ang I levels did not increase, and renal Ang II levels decreased by 40% at 0.0<em>1</em><em>7</em> mg/kg per day, the same threshold seen for the increase in plasma renin. Perindopril increased circulating bradykinin-(<em>1</em>-9) levels approximately eightfold, with a threshold dose of 0.052 mg/kg per day, and increased bradykinin-(<em>1</em>-9) levels in kidney, heart, and lung in parallel with the changes observed for plasma. By contrast, aortic and brown adipose tissue bradykinin-(<em>1</em>-9) and bradykinin-(<em>1</em>-<em>7</em>) levels increased severalfold for perindopril doses as low as 0.006 mg/kg per day. Lisinopril also increased aortic bradykinin-(<em>1</em>-9) and bradykinin-(<em>1</em>-<em>7</em>) levels at doses below the threshold for the decrease in the ratio of Ang II to Ang I. These data indicate that renal Ang II levels and vascular bradykinin-(<em>1</em>-9) levels respond to low doses of converting enzyme inhibitor and may be important mediators of the effects of these compounds. The parallel increases in bradykinin-(<em>1</em>-9) and bradykinin-(<em>1</em>-<em>7</em>) levels in aorta and brown adipose tissue, at inhibitor doses below the threshold for inhibition of Ang I conversion, may result from a mechanism different from inhibition of "classic" <em>angiotensin</em>-converting enzyme.

The 20<em>1</em>9 novel coronavirus (20<em>1</em>9-nCoV) appeared in December 20<em>1</em>9 and then spread throughout the world rapidly. The virus invades the target cell by binding to <em>angiotensin</em>-converting enzyme (ACE) 2 and modulates the expression of ACE2 in host cells. ACE2, a pivotal component of the renin-<em>angiotensin</em> system, exerts its physiological functions by modulating the levels of <em>angiotensin</em> II (Ang II) and Ang-(<em>1</em>-<em>7</em>). We reviewed the literature that reported the distribution and function of ACE2 in the female reproductive system, hoping to clarify the potential harm of 20<em>1</em>9-nCoV to female fertility. The available evidence suggests that ACE2 is widely expressed in the ovary, uterus, vagina and placenta. Therefore, we believe that apart from droplets and contact transmission, the possibility of mother-to-child and sexual transmission also exists. Ang II, ACE2 and Ang-(<em>1</em>-<em>7</em>) regulate follicle development and ovulation, modulate luteal angiogenesis and degeneration, and also influence the regular changes in endometrial tissue and embryo development. Taking these functions into account, 20<em>1</em>9-nCoV may disturb the female reproductive functions through regulating ACE2.

There is an increasing body of evidence to suggest that the RAS (renin-<em>angiotensin</em> system) contributes to tissue injury and fibrosis in chronic liver disease. A number of studies have shown that components of a local hepatic RAS are up-regulated in fibrotic livers of humans and in experimental animal models. <em>Angiotensin</em> II, the main physiological effector molecule of this system, mediates liver fibrosis by stimulating fibroblast proliferation (myofibroblast and hepatic stellate cells), infiltration of inflammatory cells, and the release of inflammatory cytokines and growth factors such as TGF (transforming growth factor)-beta<em>1</em>, IL (interleukin)-<em>1</em>beta, MCP (monocyte chemoattractant protein)-<em>1</em> and connective tissue growth factor. Furthermore, blockade of the RAS by ACE (<em>angiotensin</em>-converting enzyme) inhibitors and <em>angiotensin</em> type <em>1</em> receptor antagonists significantly attenuate liver fibrosis in experimental models of chronic liver injury. In 2000 ACE2 (<em>angiotensin</em>-converting enzyme 2), a human homologue of ACE, was identified. ACE2 efficiently degrades <em>angiotensin</em> II to <em>angiotensin</em>-(<em>1</em>-<em>7</em>), a peptide which has recently been shown to have both vasodilatory and tissue protective effects. This suggests that ACE2 and its products may be part of an alternate enzymatic pathway in the RAS, which counterbalances the generation and actions of <em>angiotensin</em> II, the ACE2-<em>angiotensin</em>-(<em>1</em>-<em>7</em>)-Mas axis. This review focuses on the potential roles of the RAS, <em>angiotensin</em> II and ACE2 in chronic liver injury and fibrogenesis.

BACKGROUND

Hereditary angioedema caused by mutations in the factor XII gene is a recently described disease entity that occurs mainly in women. It differs from hereditary angioedema caused by C<em>1</em> inhibitor deficiency.

OBJECTIVE

To assess the clinical symptoms, factors triggering acute attacks, and treatments of this disease.

METHODS

Thirty-five female patients with hereditary angioedema and the factor XII mutations p.Thr309Lys and p.Thr309Arg who came from <em>1</em>3 unrelated families were studied. The observation period was 8.4 years on average (range, 2-26 years).

RESULTS

Patients had on average <em>1</em>2.<em>7</em> +/- <em>7</em>.9 angioedema attacks per year. Recurrent facial swellings occurred in all patients; skin swellings other than facial, abdominal pain attacks, tongue swellings, and laryngeal edema occurred less frequently. Some factors that triggered angioedema attacks were trauma, physical pressure, and emotional stress. Clinical symptoms started mainly after intake of oral contraceptives (<em>1</em><em>7</em> women) or pregnancy (3 women). Exacerbation of the symptoms occurred after oral contraceptive use (8 women), pregnancy (<em>7</em> women), hormone replacement therapy (3 women), intake of <em>angiotensin</em>-converting enzyme inhibitors (2 women), and an <em>angiotensin</em> <em>1</em> receptor blocker (<em>1</em> woman). Effective treatments included C<em>1</em> inhibitor concentrate for angioedema attacks (6 women) and, for prophylaxis, progesterone (8 women), danazol (2 women), and tranexamic acid (<em>1</em> woman). No difference between mutation p.Thr309Arg and p.Thr309Lys was found.

CONCLUSIONS

Facial swelling is a cardinal symptom of this condition. Estrogens may have a great influence, but this influence is highly variable. Various treatment options are available.

In contrast to other respiratory viruses, children have less severe symptoms when infected with the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, we discuss proposed hypotheses for the age-related difference in severity of coronavirus disease 20<em>1</em>9 (COVID-<em>1</em>9).Factors proposed to explain the difference in severity of COVID-<em>1</em>9 in children and adults include those that put adults at higher risk and those that protect children. The former include: (<em>1</em>) age-related increase in endothelial damage and changes in clotting function; (2) higher density, increased affinity and different distribution of <em>angiotensin</em> converting enzyme 2 receptors and transmembrane serine protease 2; (3) pre-existing coronavirus antibodies (including antibody-dependent enhancement) and T cells; (4) immunosenescence and inflammaging, including the effects of chronic cytomegalovirus infection; (5) a higher prevalence of comorbidities associated with severe COVID-<em>1</em>9 and (6) lower levels of vitamin D. Factors that might protect children include: (<em>1</em>) differences in innate and adaptive immunity; (2) more frequent recurrent and concurrent infections; (3) pre-existing immunity to coronaviruses; (4) differences in microbiota; (5) higher levels of melatonin; (6) protective off-target effects of live vaccines and (<em>7</em>) lower intensity of exposure to SARS-CoV-2.

Keywords: epidemiology; microbiology; virology.

The ability of skeletal muscles to produce force at a high velocity, which is crucial for success in power and sprint performance, is strongly influenced by genetics and without the appropriate genetic make-up, an individual reduces his/her chances of becoming an exceptional power or sprinter athlete. Several genetic variants (i.e. polymorphisms) have been associated with elite power and sprint performance in the last few years and the current paradigm is that elite performance is a polygenic trait, with minor contributions of each variant to the unique athletic phenotype. The purpose of this review is to summarize the specific knowledge in the field of genetics and elite power performance, and to provide some future directions for research in this field. Of the polymorphisms associated with elite power and sprint performance, the α-actinin-3 R5<em>7</em><em>7</em>X polymorphism provides the most consistent results. ACTN3 is the only gene that shows a genotype and performance association across multiple cohorts of elite power athletes, and this association is strongly supported by mechanistic data from an Actn3 knockout mouse model. The <em>angiotensin</em>-<em>1</em> converting enzyme insertion/deletion polymorphism (ACE I/D, registered single nucleotide polymorphism [rs]4646994), <em>angiotensin</em>ogen (AGT Met235Thr rs699), skeletal adenosine monophosphate deaminase (AMPD<em>1</em>) Gln(Q)<em>1</em>2Ter(X) [also termed C34T, rs<em>1</em><em>7</em>602<em>7</em>29], interleukin-6 (IL-6 -<em>1</em><em>7</em>4 G/C, rs<em>1</em>800<em>7</em>95), endothelial nitric oxide synthase 3 (NOS3 -<em>7</em>86 T/C, rs20<em>7</em>0<em>7</em>44; and Glu298Asp, rs<em>1</em><em>7</em>99983), peroxisome proliferator-activated receptor-α (PPARA Intron <em>7</em> G/C, rs4253<em>7</em><em>7</em>8), and mitochondrial uncoupling protein 2 (UCP2 Ala55Val, rs660339) polymorphisms have also been associated with elite power performance, but the findings are less consistent. In general, research into the genetics of athletic performance is limited by a small sample size in individual studies and the heterogeneity of study samples, often including athletes from multiple-difference sporting disciplines. In the future, large, homogeneous, strictly defined elite power athlete cohorts need to be established though multinational collaboration, so that meaningful genome-wide association studies can be performed. Such an approach would provide unbiased identification of potential genes that influence elite athletic performance.

The anti-inflammatory, antifibrotic, and antiproteinuric properties of vitamin D have been defined in studies using active vitamin D analogs. In this prospective observational study we determined whether nutritional vitamin D repletion can have additional beneficial effects in patients with type 2 diabetic nephropathy already established on renin-<em>angiotensin</em>-aldosterone system inhibition. During a <em>7</em>-month period, 63 patients were enrolled and those with low levels of 25(OH)D were treated with oral cholecalciferol for 4 months. Baseline serum 25(OH)D and <em>1</em>,25(OH)(2)D showed no significant correlation with baseline urinary MCP-<em>1</em>, TGF-β<em>1</em>, or albuminuria measured as the urinary albumin-to-creatinine ratio. Of the 63 patients, 54 had insufficient or deficient levels of serum 25(OH)D and 49 complied with cholecalciferol therapy and follow-up. Both 25(OH)D and <em>1</em>,25(OH)(2)D were significantly increased at 2 and 4 months of treatment. Albuminuria and urinary TGF-β<em>1</em> decreased significantly at both time points compared to their baseline values, while urinary MCP-<em>1</em> did not change. Thus, in the short term, dietary vitamin D repletion with cholecalciferol had a beneficial effect in delaying the progression of diabetic nephropathy above that due to established renin-<em>angiotensin</em>-aldosterone system inhibition.

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [ANG-(<em>1</em>-<em>7</em>)] is a recently described heptapeptide product of the renin-<em>angiotensin</em> system. Because biosynthesis of ANG-(<em>1</em>-<em>7</em>) increases in animals treated with cardioprotective drugs and inactivation of the gene for <em>angiotensin</em> converting enzyme 2 [an enzyme involved in the biosynthesis of ANG-(<em>1</em>-<em>7</em>)] leads to the development of cardiac dysfunction, it has been suggested that ANG-(<em>1</em>-<em>7</em>) has cardioprotective properties. To directly test this possibility, we have generated transgenic rats that chronically overproduce ANG-(<em>1</em>-<em>7</em>) by using a novel fusion protein methodology. TGR(A<em>1</em>-<em>7</em>)3292 rats show testicular-specific expression of a cytomegalovirus promoter-driven transgene, resulting in a doubling of circulating ANG-(<em>1</em>-<em>7</em>) compared with nontransgenic control rats. Radiotelemetry hemodynamic measurements showed that transgenic rats presented a small but significant increase in daily and nocturnal heart rate and a slight but significant increase in daily and nocturnal cardiac contractility estimated by dP/d t measurements. Strikingly, TGR(A<em>1</em>-<em>7</em>)3292 rats were significantly more resistant than control animals to induction of cardiac hypertrophy by isoproterenol. In addition, transgenic rats showed a reduced duration of reperfusion arrhythmias and an improved postischemic function in isolated Langendorff heart preparations. These results support a cardioprotective role for circulating ANG-(<em>1</em>-<em>7</em>) and provide a novel tool for evaluating the functional role of ANG-(<em>1</em>-<em>7</em>).

Cardiovascular disease (CVD) is the most prevalent disease worldwide and there is intense interest in pharmaceutical approaches to reduce the burden of this chronic, aging-related condition. The sirtuin (SIRT) family of NAD(+)-dependent protein deacetylases and ADP-ribosyltransferases have emerged as exciting targets for CVD management that can impact the cardiovascular system both directly and indirectly, the latter by modulating whole body metabolism. SIRT<em>1</em>-4 regulate the activities of a variety of transcription factors, coregulators, and enzymes that improve metabolic control in adipose tissue, liver, skeletal muscle, and pancreas, particularly during obesity and aging. SIRT<em>1</em> and <em>7</em> can control myocardial development and resist stress- and aging-associated myocardial dysfunction through the deacetylation of p53 and forkhead box O<em>1</em> (FoxO<em>1</em>). By modulating the activity of endothelial nitric oxide synthase (eNOS), FoxO<em>1</em>, and p53, and the expression of <em>angiotensin</em> II type <em>1</em> receptor (AT<em>1</em>R), SIRT<em>1</em> also promotes vasodilatory and regenerative functions in endothelial and smooth muscle cells of the vascular wall. Given the array of potentially beneficial effects of SIRT activation on cardiovascular health, interest in developing specific SIRT agonists is well-substantiated. Because SIRT activity depends on cellular NAD+ availability, enzymes involved in NAD+ biosynthesis, including nicotinamide phosphoribosyltransferase (Nampt), may also be valuable pharmaceutical targets for managing CVD. Herein we review the actions of the SIRT proteins on the cardiovascular system and consider the potential of modulating SIRT activity and NAD+ availability to control CVD.

BACKGROUND

Recent clinical trials have suggested that therapy with angiotensin-converting enzyme inhibitors in asymptomatic patients with reduced left ventricular (LV) function can significantly reduce the incidence of congestive heart failure compared with patients receiving placebo. In the present study, we examined the effects of long-term monotherapy with enalapril, metoprolol, and digoxin on the progression of LV systolic dysfunction and LV chamber enlargement in dogs with reduced LV ejection fraction (EF).

RESULTS

LV dysfunction was produced in 28 dogs by multiple sequential intracoronary microembolizations. Embolizations were discontinued when LVEF was 30% to 40%. Three weeks after the last embolization, dogs were randomized to 3 months of oral therapy with enalapril (10 mg twice daily, n = 7), metoprolol (25 mg twice daily, n = 7), digoxin (0.25 mg once daily, n = 7), or no treatment (control, n = 7). As expected, in untreated dogs, LVEF decreased (36 +/- 1% versus 26 +/- 1%, P < .001) and LV end-systolic volume (ESV) and end-diastolic volume (EDV) increased during the 3-month follow-up period (39 +/- 4 versus 57 +/- 6 mL, P < .001, and 61 +/- 6 versus 78 +/- 8 mL, P < .002, respectively). In dogs treated with enalapril or metoprolol, LVEF remained unchanged or increased after therapy compared with before therapy (35 +/- 1% versus 38 +/- 3% and 35 +/- 1% versus 40 +/- 3%, respectively, P < .05), whereas ESV and EDV remained essentially unchanged. In dogs treated with digoxin, EF remained unchanged but ESV and EDV increased significantly.

CONCLUSIONS

In dogs with reduced LVEF, long-term therapy with enalapril or metoprolol prevents the progression of LV systolic dysfunction and LV chamber dilation. Therapy with digoxin maintains LV systolic function but does not prevent progressive LV enlargement.

Recently there has been growing evidence suggesting that beneficial effects of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] in the heart are mediated by its receptor Mas. However, the signaling pathways involved in these effects in cardiomyocytes are unknown. Here, we investigated the involvement of the Ang-(<em>1</em>-<em>7</em>)/Mas axis in NO generation and Ca(2+) handling in adult ventricular myocytes using a combination of molecular biology, intracellular Ca(2+) imaging, and confocal microscopy. Acute Ang-(<em>1</em>-<em>7</em>) treatment (<em>1</em>0 nmol/L) leads to NO production and activates endothelial NO synthase and Akt in cardiomyocytes. Ang-(<em>1</em>-<em>7</em>)-dependent NO raise was abolished by pretreatment with A-<em>7</em><em>7</em>9 (<em>1</em> micromol/L). To confirm that Ang-(<em>1</em>-<em>7</em>) action is mediated by Mas, we used cardiomyocytes isolated from Mas-deficient mice. In Mas-deficient cardiomyocytes, Ang-(<em>1</em>-<em>7</em>) failed to increase NO levels. Moreover, Mas-ablation was accompanied by significant alterations in the proteins involved in the regulation of endothelial NO synthase activity, indicating that endothelial NO synthase and its binding partners are important effectors of the Mas-mediated pathway in cardiomyocytes. We then investigated the role of the Ang-(<em>1</em>-<em>7</em>)/Mas axis on Ca(2+) signaling. Cardiomyocytes treated with <em>1</em>0 nmol/L of Ang-(<em>1</em>-<em>7</em>) did not show changes in Ca(2+)-transient parameters such as peak Ca(2+) transients and kinetics of decay. Nevertheless, cardiomyocytes from Mas-deficient mice presented reduced peak and slower [Ca(2+)](i) transients when compared with wild-type cardiomyocytes. Lower Ca(2+) ATPase of the sarcoplasmic reticulum expression levels accompanied the reduced Ca(2+) transient in Mas-deficient cardiomyocytes. Therefore, chronic Mas-deficiency leads to impaired Ca(2+) handling in cardiomyocytes. Collectively, these observations reveal a key role for the Ang-(<em>1</em>-<em>7</em>)/Mas axis as a modulator of cardiomyocyte function.

BACKGROUND

Cardiomyopathy is a leading cause of death in patients with Duchenne muscular dystrophy and myocardial damage precedes decline in left ventricular systolic function. We tested the efficacy of eplerenone on top of background therapy in patients with Duchenne muscular dystrophy with early myocardial disease.

METHODS

In this randomised, double-blind, placebo-controlled trial, boys from three centres in the USA aged <em>7</em> years or older with Duchenne muscular dystrophy, myocardial damage by late gadolinium enhancement cardiac MRI and preserved ejection fraction received either eplerenone 25 mg or placebo orally, every other day for the first month and once daily thereafter, in addition to background clinician-directed therapy with either <em>angiotensin</em>-converting enzyme inhibitors (ACEI) or <em>angiotensin</em> receptor blockers (ARB). Computer-generated randomisation was done centrally using block sizes of four and six, and only the study statistician and the investigational pharmacy had the preset randomisation assignments. The primary outcome was change in left ventricular circumferential strain (Ecc) at 12 months, a measure of contractile dysfunction. Safety was established through serial serum potassium levels and measurement of cystatin C, a non-creatinine measure of kidney function. This trial is registered with ClinicalTrials.gov, number NCT01521546.

RESULTS

Between Jan 26, 2012, and July 3, 2013, 188 boys were screened and 42 were enrolled. 20 were randomly assigned to receive eplerenone and 22 to receive placebo, of whom 20 in the eplerenone group and 20 in the placebo group completed baseline, 6-month, and 12-month visits. After 12 months, decline in left ventricular circumferential strain was less in those who received eplerenone than in those who received placebo (median ΔEcc 1·0 [IQR 0·3-2·2] vs 2·2 [1·3-3·1]; p=0·020). Cystatin C concentrations remained normal in both groups, and all non-haemolysed blood samples showed normal potassium concentrations. One 23-year-old patient in the placebo group died of fat embolism, and another patient in the placebo group withdrew from the trial to address long-standing digestive issues. All other adverse events were mild: short-lived headaches coincident with seasonal allergies occurred in one patient given eplerenone, flushing occurred in one patient given placebo, and anxiety occurred in another patient given placebo.

CONCLUSIONS

In boys with Duchenne muscular dystrophy and preserved ejection fraction, addition of eplerenone to background ACEI or ARB therapy attenuates the progressive decline in left ventricular systolic function. Early use of available drugs warrants consideration in this population at high risk of cardiac death, but further studies are needed to determine the effect of combination cardioprotective therapy on event-free survival in Duchenne muscular dystrophy.

BACKGROUND

BallouSkies, Parent Project for Muscular Dystrophy, US National Center for Advancing Translational Sciences, and US National Institutes of Health.

The G protein-coupled receptor Mas was recently described as an <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [ANG-(<em>1</em>-<em>7</em>)] receptor. In the present study we evaluated the anatomical localization of Mas using immunofluorescence in the central nervous system of adult male Wistar rats. An abundant labeling was found in the hippocampus, amigdala, anterodorsal thalamic nucleus, cortex, and hypoglossal nucleus. More importantly, a dense ANG-(<em>1</em>-<em>7</em>) receptor Mas immunoreactivity was observed in cardiovascular-related areas of the medulla and forebrain, shown in several previous studies as sites for the action of ANG-(<em>1</em>-<em>7</em>) in the brain. A strong staining was found in the nucleus of the solitary tract, caudal and rostral ventrolateral medulla, inferior olive, parvo and magnocellular portions of the paraventricular hypothalamic nucleus, supraoptic nucleus, and lateral preoptic area. Furthermore, Mas staining was predominantly present in neurons. At the medullary sites, a specific and high-intensity binding for rhodamine-ANG-(<em>1</em>-<em>7</em>) was also shown. The specific ANG-(<em>1</em>-<em>7</em>) binding was completely displaced by the anti-Mas antibody or by the ANG-(<em>1</em>-<em>7</em>) antagonist, A-<em>7</em><em>7</em>9. The data presented provide the first anatomical basis for the physiological role of ANG-(<em>1</em>-<em>7</em>)/Mas axis in the modulation of different cardiovascular functions and give new insights for clarifying the role of ANG-(<em>1</em>-<em>7</em>) in the central nervous system.

BACKGROUND

<em>Angiotensin</em> type <em>1</em> receptor (AT<em>1</em>R) mediates most physiologic and pathophysiologic actions of its endogenous ligand, <em>angiotensin</em> II, with overactivity leading to vascular remodeling and hypertension. Antibodies to AT<em>1</em>R are implicated in several vascular pathologies. The aim of our study was to determine the impact of antibody to AT<em>1</em>R on clinical outcomes including antibody mediated rejection (AMR), with or without C4d deposition, in patients whose sera contained no donor human leukocyte antigen (HLA)-specific antibody (HLA-DSA).

METHODS

Pretransplant sera from 97 recipients and sera obtained at the time of acute rejection (AR) were tested by Luminex-based single-antigen bead assays to determine HLA-DSA and antibodies to major histocompatibility class I chain-related gene A (MICA). The presence of antibody to AT<em>1</em>R was determined by a cell-based ELISA method using a cutoff of <em>1</em>7 units to distinguish high from low binding.

RESULTS

Sera from 63 recipients were determined to have no HLA-DSA and no donor-specific MICA antibodies pretransplant and at the time of AR, and <em>1</em>6 of these recipients were diagnosed with AR including 7 with AMR and 9 with cellular AR (cell-mediated rejection). High-binding AT<em>1</em>R antibodies were identified for six of seven in the AMR+ group and zero of nine in the cell-mediated rejection+ group (P=0.0009).

CONCLUSIONS

A strong association was observed between the presence of high binding to AT<em>1</em>R and AMR in recipients whose sera contained no antibody to donor HLA or MICA. Assessing the AT<em>1</em>R antibody status along with the HLA-DSA provides additional information to determine the immunologic risk for recipients.

In this study, we have utilized electrophysiological single unit recordings to evaluate the effects of nonpeptidergic <em>angiotensin</em> II (ANG II) antagonists on neural responses of hypothalamic paraventricular nucleus (PVN) neurons to either electrical stimulation in subfornical organ (SFO) or direct application of ANG II. Electrical stimulation (200-400 microA; 0.<em>1</em> ms) in the SFO resulted in excitatory responses in 36 of 50 PVN neurons tested. Peristimulus histogram analysis of such excitatory effects demonstrated latencies of < 30 ms and variability of response times of approximately 50 ms in <em>1</em>4 of these 36 neurons. In view of previous anatomic and electrophysiological studies such inputs were therefore considered to be monosynaptically mediated by direct neural inputs from the SFO. The remaining 22 cells excited by such SFO stimulation showed responses of longer latency and duration suggestive of a different underlying synaptic mechanism. Local pressure ejection of ANG II into the PVN resulted in increased neural activity in 50% (9 of <em>1</em>8) of the neurons tested. After systemic (3 mg/kg iv) or local (2 x <em>1</em>0(-2) M; <em>1</em>-25 s; 2-40 psi) microinjection of the nonpeptidergic <em>angiotensin</em> II<em>1</em> (AT<em>1</em>) receptor antagonist losartan, SFO excitations were attenuated in 63.9% (23 of 36) of the PVN neurons tested, such pharmacologically blocked excitatory responses being reduced by 68.3 +/- 5.2% from control stimulation effects (P < 0.00<em>1</em>). Similar losartan-induced attenuations of both short latency (presumed monosynaptic) (50.0%) and longer latency (<em>7</em>2.<em>7</em>%) responses were observed. In addition, losartan also abolished the excitatory effects of local administration of ANG II on <em>7</em><em>7</em>.8% (<em>7</em> of 9) of ANG II-sensitive neurons in PVN tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary arterial hypertension (PAH) is a deadly disease with no cure. Alternate conversion of <em>angiotensin</em> II (AngII) to <em>angiotensin</em>-(<em>1</em>-<em>7</em>) (Ang-(<em>1</em>-<em>7</em>)) by <em>angiotensin</em>-converting enzyme 2 (ACE2) resulting in Mas receptor (Mas<em>1</em>) activation improves rodent models of PAH. Effects of recombinant human (rh) ACE2 in human PAH are unknown. Our objective was to determine the effects of rhACE2 in PAH.We defined the molecular effects of Mas<em>1</em> activation using porcine pulmonary arteries, measured AngII/Ang-(<em>1</em>-<em>7</em>) levels in human PAH and conducted a phase IIa, open-label pilot study of a single infusion of rhACE2 (GSK258688<em>1</em>, 0.2 or 0.4 mg·kg-<em>1</em> intravenously).Superoxide dismutase 2 (SOD2) and inflammatory gene expression were identified as markers of Mas<em>1</em> activation. After confirming reduced plasma ACE2 activity in human PAH, five patients were enrolled in the trial. GSK258688<em>1</em> was well tolerated with significant improvement in cardiac output and pulmonary vascular resistance. GSK258688<em>1</em> infusion was associated with reduced plasma markers of inflammation within 2-4 h and increased SOD2 plasma protein at 2 weeks.PAH is characterised by reduced ACE2 activity. Augmentation of ACE2 in a pilot study was well tolerated, associated with improved pulmonary haemodynamics and reduced markers of oxidant and inflammatory mediators. Targeting this pathway may be beneficial in human PAH.

BACKGROUND

Aortic stiffness is increased in Marfan syndrome contributing to aortic dilatation and rupture, the major cause of premature death in this population. Angiotensin-converting enzyme inhibitors have been shown to reduce arterial stiffness.

OBJECTIVE

To determine whether perindopril therapy reduces aortic stiffness and attenuates aortic dilatation in patients with Marfan syndrome.

METHODS

A randomized, double-blind, placebo-controlled trial of 17 patients with Marfan syndrome (mean [SD], 33 [6] years) taking standard beta-blocker therapy, initiated in January 2004 and completed in September 2006, at Alfred Hospital Marfan Syndrome Clinic, Melbourne, Australia.

METHODS

Patients were administered 8 mg/d of perindopril (n = 10) or placebo (n = 7) for 24 weeks.

METHODS

Indices of arterial stiffness were assessed via systemic arterial compliance, and central and peripheral pulse wave velocities. Aortic root diameters were assessed at 4 sites via transthoracic echocardiography.

RESULTS

Perindopril reduced arterial stiffness as indicated by increased systemic arterial compliance (mean [SEM], 0.33 [0.01] mL/mm Hg at baseline to 0.54 [0.04] mL/mm Hg at 24 weeks in perindopril group vs 0.30 [0.01] mL/mm Hg to 0.29 [0.01] mL/mm Hg in placebo group, P = .004), and reduced central (7.6 [0.4] m/s to 5.9 [0.3] m/s in perindopril group, P < .001 vs placebo) and peripheral (10.9 [0.4] m/s to 8.7 [0.4] m/s in perindopril group, P < .001 vs placebo) pulse wave velocities. In addition, perindopril significantly reduced aortic root diameters relative to placebo in both end-systole and end-diastole (P<.01 to P < .001 for all comparisons between groups). Although perindopril marginally reduced mean arterial pressure (from 81 [2] mm Hg to 80 [1] mm Hg in perindopril group vs 83 [2] mm Hg to 84 [3] mm Hg in placebo group, P = .004), the observed changes in both stiffness and left ventricular outflow tract diameter remained significant when mean arterial pressure was included as a covariate. Transforming growth factor beta (TGF-beta), which contributes to aortic degeneration in Marfan syndrome, was reduced by perindopril compared with placebo in both latent (59 [6] ng/mL to 45 [3] ng/mL in perindopril group, P = .01 vs placebo) and active (46 [2] ng/mL to 42 [1] ng/mL in perindopril group, P = .02 vs placebo) forms.

CONCLUSIONS

Perindopril reduced both aortic stiffness and aortic root diameter in patients with Marfan syndrome taking standard beta-blocker therapy, possibly through attenuation of TGF-beta signaling. Large clinical trials are needed to assess the clinical benefit of angiotensin II blockade in Marfan syndrome.

BACKGROUND

clinicaltrials.gov Identifier: NCT00485368.

TG(mRen2)2<em>7</em> (Ren2) transgenic rats overexpress the mouse renin gene, manifest hypertension, and exhibit increased tissue ANG II levels and oxidative stress. Evidence indicates that elevated tissue ANG II contributes to oxidative stress, increases in glomerular macromolecular permeability, and consequent albuminuria. Furthermore, <em>angiotensin</em> type <em>1</em> receptor (AT<em>1</em>R) blockers reduce albuminuria and slow progression of renal disease. However, it is not known whether improvements in glomerular filtration barrier integrity and albuminuria during treatment are related to reductions in oxidative stress and/or kidney renin-<em>angiotensin</em> system (RAS) activity. To investigate the renal protective effects of AT<em>1</em>R blockade, we treated young (6-<em>7</em> wk old) male Ren2 rats with valsartan (Ren2-V; 30 mg/kg) for 3 wk and measured urine albumin, kidney malondialdehyde (MDA), RAS component mRNAs, and NADPH oxidase subunits (gp9<em>1</em>(phox) and Rac<em>1</em>) compared with age-matched untreated Ren2 and Sprague-Dawley (S-D) rats. Basement membrane thickness, slit pore diameter and number, and foot process base width were measured by transmission electron microscopy (TEM). Results indicate that AT<em>1</em>R blockade lowered systolic blood pressure (30%), albuminuria (9<em>1</em>%), and kidney MDA (80%) in Ren2-V compared with untreated Ren2 rats. Increased slit pore number and diameter and reductions in basement membrane thickness and podocyte foot process base width were strongly associated with albuminuria and significantly improved following AT<em>1</em>R blockade. AT<em>1</em>R blockade was also associated with increased <em>angiotensin</em>-converting enzyme-2 and neprilysin expression, demonstrating a beneficial shift in balance of renal RAS. Thus reductions in blood pressure, albuminuria, and tissue oxidative stress with AT<em>1</em>R blockade were associated with improved indexes of glomerular filtration barrier integrity and renal RAS in Ren2 rats.

The serine protease prolylcarboxypeptidase (PRCP), isolated from human umbilical vein endothelial cells (HUVECs), is a plasma prekallikrein (PK) activator. PRCP cDNA was cloned in pMT/BIP/V5-HIS-C, transfected into Schneider insect (S2) cells, and purified from serum-free media. Full-length recombinant PRCP (rPRCP) activates PK when bound to high-molecular-weight kininogen (HK). Recombinant PRCP is inhibited by leupeptin, <em>angiotensin</em> II, bradykinin, anti-PRCP, diisopropyl-fluorophosphonate (DFP), phenylmethylsulfonyl fluoride (PMSF), and Z-Pro-Proaldehyde-dimethyl acetate, but not by <em>1</em> mM EDTA (ethylenediaminetetraacetic acid), bradykinin <em>1</em>-5, or <em>angiotensin</em> <em>1</em>-<em>7</em>. Corn trypsin inhibitor binds to prekallikrein to prevent rPRCP activation, but it does not directly inhibit the active site of either enzyme. Unlike factor XIIa, the ability of rPRCP to activate PK is blocked by <em>angiotensin</em> II, not by neutralizing antibody to factor XIIa. PRCP antigen is detected on HUVEC membranes using flow cytometry and laser scanning confocal microscopy. PRCP antigen does not colocalize with LAMP<em>1</em> on nonpermeabilized HUVECs, but it partially colocalizes in permeabilized cells. PRCP colocalizes with all the HK receptors, gC<em>1</em>qR, uPAR, and cytokeratin <em>1</em> antigen, on nonpermeabilized HUVECs. PRCP activity and antigen expression on cultured HUVECs are blocked by a morpholino antisense oligonucleotide. These investigations indicate that rPRCP is functionally identical to isolated HUVEC PRCP and is a major HUVEC membrane-expressed, PK-activating enzyme detected in the intravascular compartment.

Previous work from this laboratory demonstrated induction of apoptosis in lung alveolar epithelial cells (AEC) by purified <em>angiotensin</em> II (ANG II) and expression of mRNAs for both ANG II receptor subtypes AT(<em>1</em>) and AT(2) (Wang R, Zagariya A, Ibarra-Sunga O, Gidea C, Ang E, Deshmukh S, Chaudhary G, Baraboutis J, Filippatos G, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 2<em>7</em>6: L885-L889, <em>1</em>999.). The present study was designed to determine the ANG II receptor subtype mediating AEC apoptosis in response to ANG II. Apoptosis was induced with purified ANG II applied to the human lung AEC-derived carcinoma cell line A549 or to primary AEC isolated from Wistar rats. In both cell types, the AT(<em>1</em>)-selective receptor antagonists L-<em>1</em>58809 or losartan inhibited ANG II-induced apoptosis by 90% at concentrations of <em>1</em>0(-8) M and <em>1</em>0(-<em>7</em>) M, respectively. The inhibition was concentration dependent with IC(50) of <em>1</em>0(-<em>1</em>2) M and <em>1</em>0(-<em>1</em><em>1</em>) M on the primary rat AEC. In contrast, the AT(2)-selective antagonists PD-<em>1</em>233<em>1</em>9 or PD-<em>1</em>26055 could not block ANG II-induced apoptosis in either cell type. In primary rat AEC, apoptosis in response to ANG II was blunted in a dose-dependent manner by the protein kinase C inhibitor chelerythrine but not by the tyrosine phosphatase inhibitor sodium orthovanadate. Together, these data indicate that AEC apoptosis in response to ANG II is mediated by receptor subtype AT(<em>1</em>), despite the expression of mRNAs for both AT(<em>1</em>) and AT(2).

Locally formed <em>angiotensin</em> II (Ang II) and mast cells may participate in the development of atherosclerosis. Chymase, which originates from mast cells, is the major Ang II-forming enzyme in the human heart and aorta in vitro. The aim of the present study was to investigate aortic Ang II-forming activity (AIIFA) and the histochemical localization of each Ang II-forming enzyme in the atheromatous human aorta. Specimens of normal (n=9), atherosclerotic (n=8), and aneurysmal (n=6) human aortas were obtained at autopsy or cardiovascular surgery from 23 subjects (<em>1</em>6 men, <em>7</em> women). The total, <em>angiotensin</em>-converting enzyme (ACE)-dependent, and chymase-dependent AIIFAs in aortic specimens were determined. The histologic and cellular localization of chymase and ACE were determined by immunocytochemistry. Total AIIFA was significantly higher in atherosclerotic and aneurysmal lesions than in normal aortas. Most of AIIFA in the human aorta in vitro was chymase-dependent in both normal (82%) and atherosclerotic aortas (90%). Immunocytochemical staining of the corresponding aortic sections with antichymase, antitryptase or anti-ACE antibodies showed that chymase-positive mast cells were located in the tunica adventitia of normal and atheromatous aortas, whereas ACE-positive cells were localized in endothelial cells of normal aorta and in macrophages of atheromatous neointima. The density of chymase- and tryptase-positive mast cells in the atherosclerotic lesions was slightly but not significantly higher than that in the normal aortas, and the number of activated mast cells in the aneurysmal lesions (<em>1</em>8%) was significantly higher than in atherosclerotic (5%) and normal (<em>1</em>%) aortas. Our results suggest that local Ang II formation is increased in atherosclerotic lesions and that chymase is primarily responsible for this increase. The histologic localization and potential roles of chymase in the development of atherosclerotic lesions appear to be different from those of ACE.

The amino terminal <em>angiotensin</em> heptapeptide, Asp-Arg-Val-Tyr-Ile-His-Pro [ANG-(<em>1</em>-<em>7</em>)], is the major product formed during incubation of <em>1</em>25I-labeled ANG I or <em>1</em>25I-labeled ANG II with homogenates obtained from canine dorsomedial medulla oblongata. To determine whether ANG-(<em>1</em>-<em>7</em>) has central-mediated cardiovascular effects, this heptapeptide was microinjected into the dorsal medulla of chloralose-urethan-anesthetized rats. Unilateral injections of ANG-(<em>1</em>-<em>7</em>) into the medial nucleus tractus solitarii caused depressor and bradycardic effects at doses between 0.<em>1</em> and <em>1</em>2.5 ng. Similar hypotensive responses accompanied with bradycardia were produced by injections of ANG-(<em>1</em>-<em>7</em>) into the dorsal motor nucleus of the vagus. In both nuclei, the monophasic depressor responses elicited by ANG-(<em>1</em>-<em>7</em>) were qualitatively similar to those found with injections of ANG II. Biphasic depressor-pressor responses of variable magnitude were produced by the injection of either <em>angiotensin</em> peptide at a high dose (250 ng). Because ANG-(<em>1</em>-<em>7</em>) has no direct vascular or dipsogenic effects, our findings suggest important differences in the receptor requirements for vascular and neural tissue of the dorsal medulla. Moreover, the data support the concept of tissue specific formation and action of <em>angiotensin</em> peptides in the brain.