We examined the impact of fetal programming on the functional responses of renal <em>angiotensin</em> receptors. Fetal sheep were exposed in utero to betamethasone (BMX; 0.<em>1</em><em>7</em> mg/kg) or control (CON) at 80 to 8<em>1</em> days gestation with full-term delivery. Renal nuclear and plasma membrane fractions were isolated from sheep age <em>1</em>.0 to <em>1</em>.5 years for receptor binding and fluorescence detection of reactive oxygen species (ROS) or nitric oxide (NO). Mean arterial blood pressure and blood pressure variability were significantly higher in the BMX-exposed adult offspring versus CON sheep. The proportion of nuclear AT(<em>1</em>) receptors sensitive to losartan was 2-fold higher (6<em>7</em> ± 6% vs 2<em>7</em> ± 9%; P<0.0<em>1</em>) in BMX compared with CON. In contrast, the proportion of AT(2) sites was only one third that of controls (BMX, 25 ± <em>1</em><em>1</em>% vs CON, <em>7</em>8 ± 4%; P<0.0<em>1</em>), with a similar reduction in sites sensitive to the Ang-(<em>1</em>-<em>7</em>) antagonist D-Ala<em>7</em>-Ang-(<em>1</em>-<em>7</em>) with BMX exposure. Functional studies revealed that Ang II stimulated ROS to a greater extent in BMX than in CON sheep (<em>1</em>6 ± 3% vs 6 ± 4%; P<0.05); however, NO production to Ang II was attenuated in BMX (26 ± <em>7</em>% vs 82 ± <em>1</em>4%; P<0.05). BMX exposure was also associated with a reduction in the Ang-(<em>1</em>-<em>7</em>) NO response (<em>7</em>5 ± 8% vs <em>1</em>3<em>1</em> ± 26%; P<0.05). We conclude that altered expression of <em>angiotensin</em> receptor subtypes may be one mechanism whereby functional changes in NO- and ROS-dependent signaling pathways may favor the sustained increase in blood pressure evident in fetal programming.

Mas is the receptor for <em>angiotensin</em>-(<em>1</em>-<em>7</em>) and is involved in cardiovascular and neuronal regulation, in which the heptapeptide also plays a major role. Mas-deficient mice have been generated by us, and their characterization has shown that Mas has important functions in behaviour and cardiovascular regulation. These mice exhibit increased anxiety but, despite an enhanced long-term potentiation in the hippocampus, do not perform better in learning experiments. When Mas-deficient mice are backcrossed to the FVB/N genetic background, a cardiovascular phenotype is uncovered, in that the backcrossed animals become hypertensive. Concordant with our detection by fluorescent in situ hybridization of Mas mRNA in mouse endothelium, this phenotype is caused by endothelial dysfunction based on a dysbalance between nitric oxide and reactive oxygen species in the vessel wall. In agreement with these data, transgenic spontaneously hypertensive stroke-prone rats overexpressing ACE2 in the vessel wall exhibit reduced blood pressure as a result of improved endothelial function. Moreover, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) overexpression in transgenic rats has cardioprotective and haemodynamic effects. In conclusion, the <em>angiotensin</em>-(<em>1</em>-<em>7</em>)-Mas axis has important functional implications for vascular regulation and blood pressure control, particularly in pathophysiological situations.

BACKGROUND

Although certain drugs that target the renin- angiotensin-aldosterone system are linked to an increased risk for angioedema, data on their absolute and comparative risks are limited. We assessed the risk for angioedema associated with the use of angiotensin converting enzyme inhibitors (ACEIs), angiotensin receptor blockers (ARBs), and the direct renin inhibitor aliskiren.

METHODS

We conducted a retrospective, observational, inception cohort study of patients 18 years or older from 17 health plans participating in the Mini-Sentinel program who had initiated the use of an ACEI (n = 1 845 138), an ARB (n = 467 313), aliskiren (n = 4867), or a β-blocker (n = 1 592 278) between January 1, 2001, and December 31, 2010. We calculated the cumulative incidence and incidence rate of angioedema during a maximal 365-day follow-up period. Using β-blockers as a reference and a propensity score approach, we estimated the hazard ratios of angioedema separately for ACEIs, ARBs, and aliskiren, adjusting for age, sex, history of allergic reactions, diabetes mellitus, heart failure, or ischemic heart disease, and the use of prescription nonsteroidal anti-inflammatory drugs.

RESULTS

A total of 4511 angioedema events (3301 for ACEIs, 288 for ARBs, 7 for aliskiren, and 915 for β-blockers) were observed during the follow-up period. The cumulative incidences per 1000 persons were 1.79 (95% CI, 1.73-1.85) cases for ACEIs, 0.62 (95% CI, 0.55-0.69) cases for ARBs, 1.44 (95% CI, 0.58-2.96) cases for aliskiren, and 0.58 (95% CI, 0.54-0.61) cases for β-blockers. The incidence rates per 1000 person-years were 4.38 (95% CI, 4.24-4.54) cases for ACEIs, 1.66 (95% CI, 1.47-1.86) cases for ARBs, 4.67 (95% CI, 1.88-9.63) cases for aliskiren, and 1.67 (95% CI, 1.56-1.78) cases for β-blockers. Compared with the use of β-blockers, the adjusted hazard ratios were 3.04 (95% CI, 2.81-3.27) for ACEIs, 1.16 (95% CI, 1.00-1.34) for ARBs, and 2.85 (95% CI, 1.34-6.04) for aliskiren.

CONCLUSIONS

Compared with β-blockers, ACEIs or aliskiren was associated with an approximately 3-fold higher risk for angioedema, although the number of exposed events for aliskiren was small. The risk for angioedema was lower with ARBs than with ACEIs or aliskiren.

BACKGROUND

Recent studies showed that <em>angiotensin</em> II type <em>1</em> receptor blocker (ARB) slows progression of chronic renal disease in patients with type 2 diabetes, regardless of changes in blood pressure. We showed that the imbalance of nitric oxide (NO) and reactive oxygen species (ROS) due to endothelial NO synthase (eNOS) uncoupling contributed to renal dysfunction in the diabetic nephropathy. The aim of this study was to determine the effects of ARB on uncoupled eNOS in rat diabetic nephropathy.

METHODS

Diabetes was induced in Sprague-Dawley rats with streptozotocin (65 mg/ kg body weight). After 6 weeks, rats were divided into saline (DM; n = <em>1</em><em>1</em>) and ARB, losartan groups (DM+Los; n = <em>1</em><em>1</em>). After 2-week treatment, glomerular ROS production was assessed by 2',7'-dichlorofluorescin diacetate (DCFH-DA)-derived chemiluminescence. Renal NO and ROS production were imaged by confocal laser microscopy after renal perfusion with DCFH-DA and diaminorhodamine-4M acetoxymethyl ester with L-arginine. The dimeric form of eNOS was measured by low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Serum tetrahydrobiopterin (BH4) concentrations were determined by high-performance liquid chromatography. Protein and mRNA expression of GTP cyclohydrolase <em>1</em> (GTPCH<em>1</em>), key enzyme of BH4 synthesis, were examined.

RESULTS

Losartan attenuated glomerular ROS production in DM. Accelerated ROS production and diminished bioavailable NO caused by NOS uncoupling were noted in DM glomeruli. Losartan reversed the decreased GTPCH<em>1</em> and decreased dimeric form of eNOS and glomerular NO production by increased BH4 bioavailability.

CONCLUSIONS

ARB improved the NOS uncoupling in diabetic nephropathy by increasing BH4 bioavailability.

The newly emergent novel coronavirus disease 20<em>1</em>9 (COVID-<em>1</em>9) outbreak, which is caused by SARS-CoV-2 virus, has posed a serious threat to global public health and caused worldwide social and economic breakdown. <em>Angiotensin</em>-converting enzyme 2 (ACE2) is expressed in human vascular endothelium, respiratory epithelium, and other cell types, and is thought to be a primary mechanism of SARS-CoV-2 entry and infection. In physiological condition, ACE2 via its carboxypeptidase activity generates <em>angiotensin</em> fragments (Ang <em>1</em>-9 and Ang <em>1</em>-<em>7</em>), and plays an essential role in the renin-<em>angiotensin</em> system (RAS), which is a critical regulator of cardiovascular homeostasis. SARS-CoV-2 via its surface spike glycoprotein interacts with ACE2 and invades the host cells. Once inside the host cells, SARS-CoV-2 induces acute respiratory distress syndrome (ARDS), stimulates immune response (i.e., cytokine storm) and vascular damage. SARS-CoV-2 induced endothelial cell injury could exacerbate endothelial dysfunction, which is a hallmark of aging, hypertension, and obesity, leading to further complications. The pathophysiology of endothelial dysfunction and injury offers insights into COVID-<em>1</em>9 associated mortality. Here we reviewed the molecular basis of SARS-CoV-2 infection, the roles of ACE2, RAS signaling, and a possible link between the pre-existing endothelial dysfunction and SARS-CoV-2 induced endothelial injury in COVID-<em>1</em>9 associated mortality. We also surveyed the roles of cell adhesion molecules (CAMs), including CD209L/L-SIGN and CD209/DC-SIGN in SARS-CoV-2 infection and other related viruses. Understanding the molecular mechanisms of infection, the vascular damage caused by SARS-CoV-2 and pathways involved in the regulation of endothelial dysfunction could lead to new therapeutic strategies against COVID-<em>1</em>9.

Keywords: ACE2; CD209L; L-SIGN; SARS-CoV-2; endothelial cell injury; endothelial dysfunction.

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] is an endogenous peptide of the renin-<em>angiotensin</em> system with several beneficial effects that are often opposite to those attributed to <em>angiotensin</em> II (Ang II). Since there are no data available so far on the role of Ang-(<em>1</em>-<em>7</em>) after cerebral ischemia/reperfusion, in this paper, we investigated the central administration of Ang-(<em>1</em>-<em>7</em>) modulates in vivo the nitric oxide(NO) release and the endothelial NO synthase (eNOS) expression following focal cerebral ischemia/reperfusion in rats. Cerebral ischemia-reperfusion injury was induced by intraluminal thread occlusion of middle cerebral artery in the adult male rats. The levels of NO in ischemic tissues were measured by NO detection kits. Reverse transcription (RT)-PCR and western blot were used to determine messenger RNA (mRNA) and protein levels of the eNOS in ischemic tissues. The cerebral ischemic lesion resulted in a significant increase of NO release at 3 and 6h compared with sham operation group in our model after reperfusion, whereas both medium and high doses Ang-(<em>1</em>-<em>7</em>) markedly enhanced NO levels at 3-24h, and 3-<em>7</em>2h after reperfusion, respectively. In addition, NO release increased was significantly induced by high-dose Ang-(<em>1</em>-<em>7</em>) compared with medium-dose Ang-(<em>1</em>-<em>7</em>) at 24-<em>7</em>2 h after reperfusion. Medium and high-dose Ang-(<em>1</em>-<em>7</em>) significantly stimulated eNOS activation when compared with artificial cerebrospinal fluid (aCSF) treatment group at 3, 6, <em>1</em>2, 24, and 48h after reperfusion, however, no significant changes in eNOS expression were found between medium and high-dose Ang-(<em>1</em>-<em>7</em>) at different times after the ischemic insult. These findings indicate that medium and high-dose Ang-(<em>1</em>-<em>7</em>) stimulate NO release and upregulate eNOS expression in ischemic tissues following focal cerebral ischemia/reperfusion in rats.

Ang II, the primary effector pleiotropic hormone of the renin-<em>angiotensin</em> system (RAS) cascade, mediates physiological control of blood pressure and electrolyte balance through its action on vascular tone, aldosterone secretion, renal sodium absorption, water intake, sympathetic activity and vasopressin release. It affects the function of most of the organs far beyond blood pressure control including heart, blood vessels, kidney and brain, thus, causing both beneficial and deleterious effects. However, the protective axis of the RAS composed of ACE2, Ang (<em>1</em>-<em>7</em>), alamandine, and Mas and MargD receptors might oppose some harmful effects of Ang II and might promote beneficial cardiovascular effects. Newly identified RAS family peptides, Ang A and angioprotectin, further extend the complexities in understanding the cardiovascular physiopathology of RAS. Most of the diverse actions of Ang II are mediated by AT<em>1</em> receptors, which couple to classical Gq/<em>1</em><em>1</em> protein and activate multiple downstream signals, including PKC, ERK<em>1</em>/2, Raf, tyrosine kinases, receptor tyrosine kinases (EGFR, PDGF, insulin receptor), nuclear factor κB and reactive oxygen species (ROS). Receptor activation via G<em>1</em>2/<em>1</em>3 stimulates Rho-kinase, which causes vascular contraction and hypertrophy. The AT<em>1</em> receptor activation also stimulates G protein-independent signaling pathways such as β-arrestin-mediated MAPK activation and Src-JAK/STAT. AT<em>1</em> receptor-mediated activation of NADPH oxidase releases ROS, resulting in the activation of pro-inflammatory transcription factors and stimulation of small G proteins such as Ras, Rac and RhoA. The components of the RAS and the major Ang II-induced signaling cascades of AT<em>1</em> receptors are reviewed.

It was recently demonstrated that <em>angiotensin</em> II (AngII) concentrations in the renal interstitial fluid (RIF) of anesthetized rats were in the nanomolar range and were not reduced by intra-arterial infusion of an <em>angiotensin</em>-converting enzyme (ACE) inhibitor (enalaprilat). This study was performed to determine changes in RIF AngI and AngII concentrations during interstitial administration of ACE inhibitors (enalaprilat and perindoprilat). Studies were also performed to determine the effects of enalaprilat on the de novo formation of RIF AngII elicited by interstitial infusion of AngI. Microdialysis probes (cut-off point, 30,000 D) were implanted in the renal cortex of anesthetized rats and were perfused at 2 micro l/min. The effluent dialysate concentrations of AngI and AngII were measured by RIA, and reported values were corrected for the equilibrium rates at this perfusion rate. Basal RIF AngI (0.<em>7</em>4 +/- 0.05 nM) and AngII (3.30 +/- 0.<em>1</em><em>7</em> nM) concentrations were much higher than plasma AngI and AngII concentrations (0.<em>1</em>5 +/- 0.0<em>1</em> and 0.<em>1</em>4 +/- 0.0<em>1</em> nM, respectively; n = 2<em>7</em>). Interstitial infusion of enalaprilat through the microdialysis probe (<em>1</em> or <em>1</em>0 mM in the perfusate; n = 5 and 8, respectively) significantly increased RIF AngI concentrations but did not significantly alter AngII concentrations. However, perindoprilat (<em>1</em>0 mM in the perfusate, n = <em>7</em>) significantly decreased RIF AngII concentrations by 22 +/- 4% and increased RIF AngI concentrations. Interstitial infusion of AngI (<em>1</em>00 nM in the perfusate, n = <em>7</em>) significantly increased the RIF AngII concentration to 8.26 +/- 0.<em>7</em>5 nM, whereas plasma AngI and AngII levels were not affected (0.<em>1</em>5 +/- 0.02 and 0.<em>1</em>4 +/- 0.02 nM, respectively). Addition of enalaprilat to the perfusate (<em>1</em>0 mM) prevented the conversion of exogenously added AngI. These results indicate that addition of AngI in the interstitial compartment leads to low but significant conversion to AngII via ACE activity (blocked by enalaprilat). However, the addition of ACE inhibitors directly into the renal interstitium, via the microdialysis probe, either did not reduce RIF AngII levels or reduced levels by a small fraction of the total basal level, suggesting that much of the RIF AngII is formed at sites not readily accessible to ACE inhibition or is formed via non-ACE-dependent pathways.

The sites of action of <em>angiotensin</em> II along the nephron are not well defined and both proximal and distal effects are suggested. Using a microassay that permits measurement of hormone binding in discrete tubule segments, we determined the binding sites of <em>1</em>25I-<em>angiotensin</em> II along the nephron of Sprague-Dawley rats. Specific binding in proximal convoluted tubule (PCT) (at 25 degrees C, pH <em>7</em>.4) was linearly related to tubule length and saturable, with an apparent maximal binding capacity of approximately 300 amol X cm-<em>1</em>. Binding specificity was verified in competition experiments that revealed significant (P less than 0.00<em>1</em>) and comparable competition for radioligand binding by <em>angiotensin</em> II and <em>angiotensin</em> precursor, metabolite, and analogues, whereas unrelated peptides of similar size (bradykinin, ACTH [<em>1</em>-<em>1</em>0]) were without effect. The profile of specific <em>angiotensin</em> II binding along the nephron was: PCT, 2<em>1</em>6 +/- <em>1</em>3; pars recta, 86 +/- <em>1</em>4; medullary thick ascending limb of Henle's loop, 46 +/- 8; cortical thick ascending limb of Henle's loop, <em>7</em><em>7</em> +/- 8; distal convoluted tubule, 49 +/- <em>1</em>0; cortical collecting tubule, <em>1</em>5 +/- <em>1</em>; medullary collecting tubule, 32 +/- <em>7</em> amol X cm-<em>1</em>. These results indicate the presence of specific <em>angiotensin</em> II binding sites in all tubule segments studied, but binding capacity was highest in the proximal convoluted tubule, in agreement with transport studies that localize the effects of the hormone in this segment.

We examined the effect of troglitazone on immunoreactive endothelin-<em>1</em> (ET-<em>1</em>) secretion from cultured bovine vascular endothelial cells (bVECs). Insulin (<em>1</em>0(-9)-<em>1</em>0(-<em>7</em>) M) stimulated ET-<em>1</em> secretion in a dose-dependent fashion without any kinetic change. Troglitazone (<em>1</em>-20 microM) dose-dependently inhibited both spontaneous and insulin-stimulated ET-<em>1</em> secretion. This inhibitory effect of troglitazone was associated with reduced ET-<em>1</em> mRNA levels. Addition of indomethacin (<em>1</em>00 microM) or Nw-nitro-l-arginine methyl ester (<em>1</em> mM) and downregulation of protein kinase C by prolonged pretreatment of the cells with a phorbol ester, <em>1</em>2-O-tetradecanoylphorbol <em>1</em>3-acetate, did not affect the inhibitory effect of troglitazone at concentrations up to <em>1</em>0 microM. Troglitazone did not change the intracellular Ca2+ concentration stimulated by <em>angiotensin</em> II (<em>1</em>0 microM). Other PPARgamma ligands, pioglitazone (<em>1</em>-<em>1</em>0 microM) and <em>1</em>5-deoxy-delta <em>1</em>2, <em>1</em>4-prostaglandin J2 (<em>1</em>-<em>1</em>0 microM), but not a PPARalpha ligand, bezafibrate (<em>1</em>-<em>1</em>0 microM), dose-dependently suppressed spontaneous ET-<em>1</em> secretion from bVECs. These results, taken together, suggest that troglitazone inhibits ET-<em>1</em> mRNA expression and secretion in bVECs possibly through activation of PPARgamma. This inhibition may contribute to the hypotensive effect of troglitazone in insulin-resistant subjects.

The renal <em>angiotensin</em> <em>angiotensin</em> type 2 receptor has been shown to mediate natriuresis, and <em>angiotensin</em> III, not <em>angiotensin</em> II, may be the preferential <em>angiotensin</em> type 2 receptor activator of this response. <em>Angiotensin</em> III is metabolized to <em>angiotensin</em> IV by aminopeptidase N. The present study hypothesizes that inhibition of aminopeptidase N will augment natriuretic responses to intrarenal <em>angiotensin</em> III in angiotension type <em>1</em> receptor-blocked rats. Rats received systemic candesartan for 24 hours before the experiment. After a <em>1</em>-hour control, cumulative renal interstitial infusion of <em>angiotensin</em> III at 3.5, <em>7</em>, <em>1</em>4, and 28 nmol/kg per minute (each dose for 30 minutes) or <em>angiotensin</em> III combined with aminopeptidase N inhibitor PC-<em>1</em>8 was administered into <em>1</em> kidney. The contralateral control kidney received renal interstitial infusion of vehicle. In kidneys infused with <em>angiotensin</em> III alone, renal sodium excretion rate increased from 0.05+/-0.0<em>1</em> micromol/min in stepwise fashion to 0.<em>1</em><em>1</em>+/-0.0<em>1</em> micromol/min at 28 nmol/kg per minute of <em>angiotensin</em> III (overall ANOVA F=3.68; P<0.0<em>1</em>). In <em>angiotensin</em> III combined with PC-<em>1</em>8, the renal sodium excretion rate increased from 0.05+/-0.0<em>1</em> to 0.32+/-0.08 mumol/min at 28 nmol/kg per minute of <em>angiotensin</em> III (overall ANOVA F=6.2; P<0.00<em>1</em>). The addition of intrarenal PD-<em>1</em>233<em>1</em>9, an <em>angiotensin</em> type 2 receptor antagonist, to renal interstitial <em>angiotensin</em> III plus PC-<em>1</em>8 inhibited the natriuretic response. Mean arterial blood pressure and renal sodium excretion rate from control kidneys were unchanged by <em>angiotensin</em> III +/- PC-<em>1</em>8 + PD-<em>1</em>233<em>1</em>9. <em>Angiotensin</em> III plus PC-<em>1</em>8 induced a greater natriuretic response than Ang III alone (overall ANOVA F=<em>1</em>6.9; P=0.000<em>1</em>). Aminopeptidase N inhibition augmented the natriuretic response to <em>angiotensin</em> III, suggesting that <em>angiotensin</em> III is a major agonist of <em>angiotensin</em> type 2 receptor-induced natriuresis.

Many advances have been made in the cardiovascular field in the last several decades. Among them is the progress completed to date on the heptapeptide member of the renin-<em>angiotensin</em> system (RAS), <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)]. The peptide's beneficial actions against pathophysiological processes, such as cardiac arrhythmia, heart failure, hypertension, renal disease, preeclampsia, and even cancer are continuously being uncovered. This review encompasses the pharmacology of Ang-(<em>1</em>-<em>7</em>) and expounds upon the peptide's potential as a therapeutic agent against pathological processes both within and outside the cardiovascular continuum.

Reactive oxygen species have an important pathogenic role in organ damage. We investigated the role of oxidative stress via nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase in the kidney of the Dahl salt-sensitive (DS) rats with heart failure (DSHF). Eleven-week-old DS rats fed an 8%-NaCl diet received either vehicle or imidapril (<em>1</em> mg/kg per day) for <em>7</em> weeks. The renal expression of the NAD(P)H oxidase p4<em>7</em>phox and endothelial NO synthase were evaluated. In DSHF rats, associated with increased renal <em>angiotensin</em> II, mRNA and protein expression of NAD(P)H oxidase p4<em>7</em>phox were enhanced with an increase in renal lipid peroxidation production (0.33+/-0.03 versus 0.22+/-0.0<em>1</em> nmol/mg protein, P<0.05) and urinary excretion of hydrogen peroxide (26.9+/-6.6 versus 9.5+/-2.<em>1</em> U/mg creatinine, P<0.0<em>1</em>) compared with levels in Dahl salt-resistant rats. The endothelial NO synthase expression was decreased in the kidney. Treatment with imidapril reduced renal <em>angiotensin</em> II and NAD(P)H oxidase expression and the oxidative products (kidney lipid peroxidation product: 0.<em>1</em>6+/-0.02, P<0.00<em>1</em>; urinary hydrogen peroxide: 3.<em>1</em>+/-0.2, P<0.0<em>1</em> versus DSHF rats). Imidapril significantly decreased albuminuria and reduced glomerulosclerosis without changes in the blood pressure. In conclusion, DSHF rats showed increased oxidative stress in the kidney via NAD(P)H oxidase. Blockade of local <em>angiotensin</em> II with subpressor dose of imidapril inhibited NAD(P)H oxidase and prevented renal damage.

Recent identification of a counterregulatory axis of the renin-<em>angiotensin</em> system, called <em>angiotensin</em>-converting enzyme 2-<em>angiotensin</em>-(<em>1</em>-<em>7</em>) [ANG-(<em>1</em>-<em>7</em>)]-Mas receptor, may offer new targets for the treatment of renal fibrosis. We hypothesized that therapy with ANG-(<em>1</em>-<em>7</em>) would improve glomerulosclerosis through counteracting ANG II in experimental glomerulonephritis. Disease was induced in rats with the monoclonal anti-Thy-<em>1</em> antibody, OX-<em>7</em>. Based on a three-dose pilot study, 5<em>7</em>6 microg x kg(-<em>1</em>) x day(-<em>1</em>) ANG-(<em>1</em>-<em>7</em>) was continuously infused from day <em>1</em> using osmotic pumps. Measures of glomerulosclerosis include semiquantitative scoring of matrix proteins stained for periodic acid Schiff, collagen I, and fibronectin EDA+ (FN). ANG-(<em>1</em>-<em>7</em>) treatment reduced disease-induced increases in proteinuria by <em>7</em>5%, glomerular periodic acid Schiff staining by 48%, collagen I by 24%, and FN by 25%. The dramatic increases in transforming growth factor-beta<em>1</em>, plasminogen activator inhibitor-<em>1</em>, FN, and collagen I mRNAs seen in disease control animals compared with normal rats were all significantly reduced by ANG-(<em>1</em>-<em>7</em>) administration (P < 0.05). These observations support our hypothesis that ANG-(<em>1</em>-<em>7</em>) has therapeutic potential for reversing glomerulosclerosis. Several results suggest ANG-(<em>1</em>-<em>7</em>) acts by counteracting ANG II effects: <em>1</em>) renin expression in ANG-(<em>1</em>-<em>7</em>)-treated rats was dramatically increased as it is with ANG II blockade therapy; and 2) in vitro data indicate that ANG II-induced increases in mesangial cell proliferation and plasminogen activator inhibitor-<em>1</em> overexpression are inhibited by ANG-(<em>1</em>-<em>7</em>) via its binding to a specific receptor known as Mas.

We provide a new foundation for an alternative interpretation of the biochemical physiology of the brain and other tissue <em>angiotensin</em> systems on the basis of research done in our laboratory. This perspective is prompted by the discovery that <em>angiotensin</em>-(<em>1</em>-<em>7</em>) has cellular functions that differ from those established for <em>angiotensin</em> II. Although <em>angiotensin</em>-(<em>1</em>-<em>7</em>) is not an agonist in terms of activating vasoconstriction, stimulating thirst, or promoting aldosterone release, the heptapeptide caused neuronal excitation and vasopressin release with a potency similar to that found with <em>angiotensin</em> II. Furthermore, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) enhances the production of prostanoids by a receptor-mediated event that causes no associated rise in intracellular Ca2+. These actions of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) provide a new understanding of the heterogeneous functions of <em>angiotensin</em> peptides as modulators of a wide range of regulatory functions in mammals.

Our goal was to determine whether <em>angiotensin</em> II (Ang II) and its metabolic fragments release nitric oxide and the mechanisms by which this occurs in blood vessels from the canine heart. We incubated 20 mg of microvessels or large coronary arteries in phosphate-buffered saline for 20 minutes and measured nitrite release. Nitrite release increased from 2<em>7</em> +/- 2 up to <em>1</em>03 +/- 5, <em>1</em>45 +/- <em>1</em><em>7</em>, 84 +/- 4, <em>1</em>0<em>7</em> +/- <em>1</em>6, and 54 +/- 4 pmol/mg (P < .05) in response to <em>1</em>0(-5) mol/L of Ang I, II, III, IV, and Ang-(<em>1</em>-<em>7</em>), respectively. The effects of all <em>angiotensins</em> were blocked by N omega-nitro-L-arginine methyl ester (<em>1</em>00 mumol/L), indicating that nitrite was a product of nitric oxide metabolism, and by Hoe <em>1</em>40 (<em>1</em>0 mumol/L), a specific bradykinin B2 receptor antagonist, indicating a potential role for local kinin formation. The protease inhibitors aprotinin (<em>1</em>0 mumol/L) and soybean trypsin inhibitor, which block local kinin formation, inhibited nitrite release by all of the <em>angiotensins</em>. <em>Angiotensin</em> nonselective (saralasin), type <em>1</em>-specific (losartan), and type 2-specific (PD <em>1</em>233<em>1</em>9) receptor antagonists abolished the nitrite released in response to all the fragments. <em>Angiotensin</em> type <em>1</em> and type 2 and receptors mediate nitrite release after Ang I, II, III, and Ang-(<em>1</em>-<em>7</em>), whereas only type 2 receptors mediate nitrite release after Ang IV. Similar results were obtained in large coronary arteries. In summary, formation of nitrite from coronary microvessels and large arteries in the normal dog heart in response to <em>angiotensin</em> peptides is due to the activation of local kinin production in the coronary vessel wall.

Spontaneous tone in large arteries may contribute to the pathogenesis of hypertension. Reactive oxygen species and Ca2+ influx have been shown to stimulate the development of spontaneous tone in isolated aortic rings in several models of hypertensive rats. The aim of this study was to investigate the role of the RhoA/Rho-kinase signaling pathway in the development of spontaneous tone in <em>angiotensin</em> II-induced hypertension and to explore the underlying mechanisms of RhoA/Rho-kinase activation. Our results showed that spontaneous tone was greatly enhanced in endothelium-denuded aortic rings from <em>angiotensin</em> II-induced hypertensive rats compared with their normotensive counterparts (<em>7</em>3+/-5 versus <em>7</em>+/-3% of phenylephrine-induced maximal contraction, respectively). The Rho-kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(<em>1</em>-aminoethyl)-cyclohexanecarboxamide (Y-2<em>7</em>632) (0.<em>1</em>-<em>1</em>0 microM) concentration dependently inhibited spontaneous tone in aortic rings from <em>angiotensin</em> II-treated rats. NADPH oxidase inhibitors diphenylene iodonium and apocynin also significantly reduced spontaneous tone. Chronic <em>angiotensin</em> II treatment markedly increased RhoA protein expression (5<em>7</em>%) but had no effect on Rho guanine nucleotide exchange factor mRNA or Rho-kinase protein expression levels. In endothelium-denuded rings from normotensive rats, <em>angiotensin</em> II (<em>1</em>00 nM) increased RhoA membrane translocation and phosphorylation of the myosin light chain phosphatase target subunit, which were both blocked by the NADPH oxidase inhibitor diphenylene iodonium (<em>1</em>0 microM). In conclusion, these data suggest that chronic treatment with <em>angiotensin</em> II leads to up-regulation of the RhoA/Rho-kinase pathway, contributing to spontaneous tone development in rat aorta. Increased NADPH oxidase-dependent reactive oxygen species may be one of the mechanisms mediating the RhoA/Rho-kinase activation.

<em>Angiotensin</em> II induces cardiovascular injury, in part, by activating inflammatory response; however, the initial factors that trigger the inflammatory cascade remain unclear. Microarray analysis of cardiac tissue exposed to systemic <em>angiotensin</em> II infusion revealed that extracellular heterodimeric proteins S<em>1</em>00a8/a9 were highly upregulated. The increase in S<em>1</em>00a8/a9 mRNA of CD<em>1</em><em>1</em>b(+)Gr<em>1</em>(+) neutrophils isolated from both the peripheral blood and heart was highest on day <em>1</em> of <em>angiotensin</em> II infusion and decreased to baseline at day <em>7</em>. Immunostaining showed that S<em>1</em>00a8/a9 was primarily present in infiltrating CD<em>1</em><em>1</em>b(+)Gr<em>1</em>(+) neutrophils in the heart. The receptor for advanced glycation end products, an S<em>1</em>00a8/a9 receptor, was expressed in cardiac fibroblasts (CFs). Microarray analysis and Bio-Plex protein array showed that treatment of CFs with recombinant S<em>1</em>00a8/a9 activated multiple chemokine and cytokines released. Luciferase reporter assay indicated S<em>1</em>00a8/a9-activated nuclear factor-κ B pathway in CFs. Consequently, recombinant S<em>1</em>00a8/a9-treated CFs promoted migration of monocytes and CFs, whereas neutralizing S<em>1</em>00a9 antibody blocked S<em>1</em>00a9 or receptor for advanced glycation end products-suppressed cellular migration. Finally, administration of a neutralizing S<em>1</em>00a9 antibody prevented <em>angiotensin</em> II infusion-induced nuclear factor-κ B activation, inflammatory cell infiltration, cytokine production, subsequent perivascular and interstitial fibrosis, and hypertrophy in heart. Our findings identify neutrophil-produced S<em>1</em>00a8/a9 as an initial proinflammatory factor needed to trigger inflammation and cardiac injury during acute hypertension.

OBJECTIVE

Patients with paroxysmal atrial fibrillation (AF) often present with typical angina pectoris and mildly elevated levels of cardiac troponin (non ST-segment elevation myocardial infarction) during an arrhythmic event. However, in a large proportion of these patients, significant coronary artery disease is excluded by coronary angiography. Here we explored the potential underlying mechanism of these events.

RESULTS

A total of <em>1</em>4 pigs were studied using a closed chest, rapid atrial pacing (RAP) model. In five pigs RAP was performed for <em>7</em> h (600 b.p.m.; n = 5), in five animals RAP was performed in the presence of <em>angiotensin</em>-II type-<em>1</em>-receptor (AT(<em>1</em>)-receptor) inhibitor irbesartan (RAP+Irb), and four pigs were instrumented without intervention (Sham). One-factor analysis of variance was performed to assess differences between and within the three groups. Simultaneous measurements of fractional flow reserve (FFR) and coronary flow reserve (CFR) before, during, and after RAP demonstrated unchanged FFR (P = 0.32<em>7</em>), but decreased CFR during RAP (RAP: 6<em>7</em>.<em>7</em> +/- <em>7</em>.2%, sham: 9<em>7</em>.2 +/- 2.8%, RAP+Irb: 93.2 +/- 3.3; P = 0.00<em>1</em>3) indicating abnormal left ventricular (LV) microcirculation. Alterations in microcirculatory blood flow were accompanied by elevated ventricular expression of NADPH oxidase subunit Nox2 (P = 0.039), lectin-like oxidized low-density lipoprotein receptor-<em>1</em> (LOX-<em>1</em>, P = 0.004), and F(2)-isoprostane levels (P = 0.008) suggesting RAP-related oxidative stress. Plasma concentrations of cardiac troponin-I (cTn-I) increased in RAP (RAP: 6<em>1</em>3.3 +/- <em>1</em>25.8 pmol/L vs. sham: 82.5 +/- <em>1</em>2.5 pmol/L; P = 0.0<em>1</em>3), whereas protein levels of eNOS and LV function remained unchanged. RAP+Irb prevented the increase of Nox2, LOX-<em>1</em>, and F(2)-isoprostanes, and abolished the impairment of microvascular blood flow.

CONCLUSIONS

Rapid atrial pacing induces AT(<em>1</em>)-receptor-mediated oxidative stress in LV myocardium that is accompanied by impaired microvascular blood flow and cTn-I release. These findings provide a plausible mechanism for the frequently observed cTn-I elevation accompanied with typical angina pectoris symptoms in patients with paroxysmal AF and normal (non-stenotic) coronary arteries.

<em>1</em>. The M-like current IK(M,ng) in differentiated NG<em>1</em>08-<em>1</em>5 mouse neuroblastoma x rat glioma hybrid cells has been studied using tight-seal, whole-cell patch-clamp recording. 2. When calculated from steady-state current-voltage curves, the conductance underlying IK(M,ng) showed a Boltzmann dependence on voltage with half-activation voltage Vo = -44 mV (in 3 mM [K+]) and slope factor (a) = 8.<em>1</em> mV/e-fold increase in conductance. In <em>1</em>2 mM [K+] Vo = -38 mV and a = 6.9 mV. The deactivation reciprocal time constant accelerated with hyperpolarization with slope factor <em>1</em><em>7</em> mV/e-fold voltage change. 3. The reversal potential for deactivation tail currents varied with external [K+] as if PNa/PK were 0.005. 4. Steady-state current was increased on removing external Ca2+. In the presence of external Ca2+, reactivation of IK(M, ng) after a hyperpolarizing step was delayed. This delay was preceded by an inward Ca2+ current, and coincided with an increase in intracellular [Ca2+] as measured with Indo-<em>1</em> fluorescence. Elevation of intracellular [Ca2+] with caffeine also reduced IK(M, ng). 5. IK(M, ng) was inhibited by external divalent cations in decreasing order of potency (mM IC50 in parentheses): Zn2+ (0.0<em>1</em><em>1</em>) greater than Cu2+ (0.0<em>1</em>8) greater than Cd2+ (0.0<em>7</em>0) greater than Ni2+ (0.44) greater than Ba2+ (0.4<em>7</em>) greater than Fe2+ (0.69) greater than Mn2+ (0.86) greater than Co2+ (0.92) greater than Ca2+ (5.6) greater than Mg2+ (<em>1</em>6) greater than Sr2+ (33). This was not secondary to inhibition of ICa since: (i) inhibition persisted in Ca(2+)-free solution; (ii) La3+ did not inhibit IK(M, ng) at concentrations which inhibited ICa; and (iii) organic Ca2+ channel blockers were ineffective. Inhibition comprised both depression of the maximum conductance and a positive shift of the activation curve. Addition of Ca2+ (<em>1</em>0 microM free [Ca2+]) or Ba2+ (<em>1</em> mM total [Ba2+]) to the pipette solution did not significantly change IK(M, ng). 6. IK(M, ng) was reduced by 9-amino-<em>1</em>,2,3,4-tetrahydroacridine (IC50 8 microM) and quinine (30 microM) but was insensitive to tetraethylammonium (IC50 greater than 30 mM), 4-aminopyridine (greater than <em>1</em>0 mM), apamin (greater than 3 microM) or dendrotoxin (greater than <em>1</em>00 nM). <em>7</em>. IK(M, ng) was inhibited by bradykinin (<em>1</em>-<em>1</em>0 microM) or <em>angiotensin</em> II (<em>1</em>-<em>1</em>0 microM), but not by the following other receptor agonists: acetylcholine (<em>1</em>0 mM), muscarine (<em>1</em>0 microM), noradrenaline (<em>1</em>00 microM), adrenaline (<em>1</em>00 microM), dopamine (<em>1</em>00 microM), histamine (<em>1</em>00 microM), 5-hydroxytryptamine (<em>1</em>0 microM), Met-enkephalin (<em>1</em> microM), glycine (<em>1</em>00 microM), gamma-aminobutyric acid (<em>1</em>00 microM) or baclofen (500 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

The renin-<em>angiotensin</em> (Ang) system plays a pivotal role in the pathogenesis of cardiovascular disease, with Ang II being the major effector of this system. Multiple lines of evidence have shown that Ang-(<em>1</em>-<em>7</em>) exerts cardioprotective effects in the heart by counterregulating Ang II actions. The questions that remain are how and where Ang-(<em>1</em>-<em>7</em>) exerts its effects. By using a combination of molecular biology, confocal microscopy, and a transgenic rat model with increased levels of circulating Ang-(<em>1</em>-<em>7</em>) (TGR[A<em>1</em>-<em>7</em>]3292), we evaluated the signaling pathways involved in Ang-(<em>1</em>-<em>7</em>) cardioprotection against Ang II-induced pathological remodeling in ventricular cardiomyocytes. Rats were infused with Ang II for 2 weeks. We found that ventricular myocytes from TGR(A<em>1</em>-<em>7</em>)3292 rats are protected from Ang II pathological remodeling characterized by Ca(2+) signaling dysfunction, hypertrophic fetal gene expression, glycogen synthase kinase 3beta inactivation, and nuclear factor of activated T-cells nuclear accumulation. Moreover, cardiomyocytes from TGR(A<em>1</em>-<em>7</em>)3292 rats infused with Ang II presented increased expression levels of neuronal NO synthase. To provide a signaling pathway involved in the beneficial effects of Ang-(<em>1</em>-<em>7</em>), we treated neonatal cardiomyocytes with Ang-(<em>1</em>-<em>7</em>) and Ang II for 36 hours. Treatment of cardiomyocytes with Ang-(<em>1</em>-<em>7</em>) prevented Ang II-induced hypertrophy by modulating calcineurin/nuclear factor of activated T-cell signaling cascade. Importantly, antihypertrophic effects of Ang-(<em>1</em>-<em>7</em>) on Ang II-treated cardiomyocytes were prevented by N(G)-nitro-l-arginine methyl ester and <em>1</em>H-<em>1</em>,2,4oxadiazolo4,2-aquinoxalin-<em>1</em>-one, suggesting that these effects are mediated by NO/cGMP. Taken together, these data reveal a key role for NO/cGMP as a mediator of Ang-(<em>1</em>-<em>7</em>) beneficial effects in cardiac cells.

Alterations in the balance between ANG II/ACE and ANG <em>1</em>-<em>7</em>/ACE2 in ANG II-dependent hypertension could reduce the generation of ANG <em>1</em>-<em>7</em> and contribute further to increased intrarenal ANG II. Upregulation of collecting duct (CD) renin may lead to increased ANG II formation during ANG II-dependent hypertension, thus contributing to this imbalance. We measured ANG I, ANG II, and ANG <em>1</em>-<em>7</em> contents, <em>angiotensin</em>-converting enzyme (ACE) and ACE2 gene expression, and renin activity in the renal cortex and medulla in the clipped kidneys (CK) and nonclipped kidneys (NCK) of 2K<em>1</em>C rats. After 3 wk of unilateral renal clipping, systolic blood pressure and plasma renin activity increased in 2K<em>1</em>C rats (n = <em>1</em><em>1</em>) compared with sham rats (n = 9). Renal medullary <em>angiotensin</em> peptide levels were increased in 2K<em>1</em>C rats [ANG I: (CK = <em>1</em><em>7</em><em>1</em> ± 4; NCK = 25<em>1</em> ± 8 vs. sham = 55 ± 3 pg/g protein; P < 0.05); ANG II: (CK = 558 ± <em>7</em>9; NCK = 328 ± <em>1</em>8 vs. sham = 94 ± <em>7</em> pg/g protein; P < 0.00<em>1</em>)]; and ANG <em>1</em>-<em>7</em> levels decreased (CK = <em>1</em>8 ± 2; NCK = <em>1</em>9 ± 2 pg/g vs. sham = 63 ± <em>1</em>0 pg/g; P < 0.00<em>1</em>). In renal medullas of both kidneys of 2K<em>1</em>C rats, ACE mRNA levels and activity increased but ACE2 decreased. In further studies, we compared renal ACE and ACE2 mRNA levels and their activities from chronic ANG II-infused (n = 6) and sham-operated rats (n = 5). Although the ACE mRNA levels did not differ between ANG II rats and sham rats, the ANG II rats exhibited greater ACE activity and reduced ACE2 mRNA levels and activity. Renal medullary renin activity was similar in the CK and NCK of 2K<em>1</em>C rats but higher compared with sham. Thus, the differential regulation of ACE and ACE2 along with the upregulation of CD renin in both the CK and NCK in 2K<em>1</em>C hypertensive rats indicates that they are independent of perfusion pressure and contribute to the altered content of intrarenal ANG II and ANG <em>1</em>-<em>7</em>.

The renin-<em>angiotensin</em> system (RAS) constitutes one of the most important hormonal systems in the physiological regulation of blood pressure through renal and nonrenal mechanisms. Indeed, dysregulation of the RAS is considered a major factor in the development of cardiovascular pathologies, including kidney injury, and blockade of this system by the inhibition of <em>angiotensin</em> converting enzyme (ACE) or blockade of the <em>angiotensin</em> type <em>1</em> receptor (AT<em>1</em>R) by selective antagonists constitutes an effective therapeutic regimen. It is now apparent with the identification of multiple components of the RAS within the kidney and other tissues that the system is actually composed of different <em>angiotensin</em> peptides with diverse biological actions mediated by distinct receptor subtypes. The classic RAS can be defined as the ACE-Ang II-AT<em>1</em>R axis that promotes vasoconstriction, water intake, sodium retention, and other mechanisms to maintain blood pressure, as well as increase oxidative stress, fibrosis, cellular growth, and inflammation in pathological conditions. In contrast, the nonclassical RAS composed primarily of the AngII/Ang III-AT2R pathway and the ACE2-Ang-(<em>1</em>-<em>7</em>)-AT<em>7</em>R axis generally opposes the actions of a stimulated Ang II-AT<em>1</em>R axis through an increase in nitric oxide and prostaglandins and mediates vasodilation, natriuresis, diuresis, and reduced oxidative stress. Moreover, increasing evidence suggests that these non-classical RAS components contribute to the therapeutic blockade of the classical system to reduce blood pressure and attenuate various indices of renal injury, as well as contribute to normal renal function.

<em>Angiotensin</em> II type <em>1</em> receptor (AT<em>1</em>R) promotes tumor invasion, migration, metastasis and angiogenesis. We explored the potential antitumor effects of AT<em>1</em>R antagonists in breast cancer. We found that <em>angiotensin</em> II promoted cell proliferation and upregulated the expression of vascular endothelial growth factor A (VEGF-A) in MCF-<em>7</em> cells. Losartan downregulated the expression of VEGF-A in MCF-<em>7</em> cells treated with <em>angiotensin</em> II. Candesartan downregulated the expression of VEGF-A in mice bearing MCF-<em>7</em> xenografts and inhibited tumor growth and angiogenesis. AT<em>1</em>R and VEGF-A expression correlated with increased microvascular density in <em>1</em>02 breast cancer patients. Our data suggest that AT<em>1</em>R antagonists might be useful to suppress breast cancer by inhibiting the <em>angiotensin</em> II.