Association of Perinatal Cardiovascular Features with Angiotensin System Expressions in Maternal Preeclampsia.

We hypothesized and investigated whether prenatal exposure to preeclampsia (PE) would simultaneously affect perinatal cardiovascular features and angiotensin system expressions. This prospective study was composed of mother-neonate dyads with (n = 49) and without maternal preeclampsia (n = 48) in a single tertiary medical center. The neonates exposed to PE had significantly larger relative sizes for the left and right coronary arteries and a higher cord plasma level of aminopeptidase-N, which positively correlated with the maternal diastolic blood pressures and determined the relative sizes of the left and right coronary arteries, whereas the encoding aminopeptidase-N (ANPEP) mRNA level in the PE cord blood leukocytes was significantly decreased, positively correlated with the neonatal systolic blood pressures (SBPs), and negatively correlated with the cord plasma-induced endothelial vascular cell adhesion molecule-1 mRNA levels. The PE cord plasma significantly induced higher endothelial mRNA levels of angiotensin II type 1 receptor (AT1R) and AT4R, whereas in the umbilical arteries, the protein expressions of AT2R and AT4R were significantly decreased in the PE group. The endothelial AT1R mRNA level positively determined the maternal SBPs, and the AT4R mRNA level positively determined the neonatal chamber size and cardiac output. In conclusion, PE may influence perinatal angiotensin system and cardiovascular manifestations of neonates across placentae. Intriguing correlations between these two warrant further mechanistic investigation.

Bariatric surgery modulates plasma levels of antibodies against angiotensin II type 1 and endothelin 1 type A receptor in severe obesity.

PURPOSE: The contribution of endothelial-targeted autoantibodies against the angiotensin II type 1 receptor (anti-AT1R) and the anti-endothelin 1 type A receptor (anti-ETAR1) has been proposed in the development of cardiovascular diseases. However, no data have been reported yet in obesity. In this observational study we evaluated the relationship between anthropometric and metabolic parameters and anti-AT1R and anti-ETAR1 concentrations in a cohort of patients with severe obesity and associated comorbidities undergoing bariatric surgery. METHODS: Clinical evaluation and metabolic assessment were performed in 36 subjects referring to our Center for the Study and Integrated Treatment of Obesity at the University Hospital of Padova. Circulating inflammatory adipocytokines and the endothelial dysfunction marker asymmetric dimethylarginine (ADMA) were evaluated; plasma levels of anti-AT1R and anti-ETAR1 were also determined. 10 normal-weight subjects were considered as a control group. 29 patients out of 36 were re-evaluated after surgery. RESULTS: With respect to normal-weight controls patients showed significantly higher plasma levels of anti-AT1R (28 ± 20.4 vs 13.5 ± 2.8 U/mL, p < 0.005) and ADMA (0.8 ± 0.1 vs 0.54 ± 0.08 uM/L, p < 0.0001) but not anti-ETAR1 (14.2 ± 1.3 vs 13.3 ± 2 U/mL, p = 0.1). Anti-AT1R concentration showed an increasing trend with the worsening of glycemic status, while the presence of arterial hypertension among the patients did not affect autoantibodies levels. One year after surgery, a significant improvement in body weight and metabolic and inflammatory parameters was observed, along with a significant reduction of anti-AT1R (28.1 ± 20.4 U/mL vs 22.6 ± 16 U/mL, p < 0.05) and anti-ETAR1 (14.2 ± 1.3 U/L vs 13 ± 1.6 U/L, p < 0.01). CONCLUSIONS: Subjects with obesity present higher plasma levels of anti-AT1R which are more related to glycemic profile than blood pressure levels, and are reduced by bariatric surgery. Considering the detrimental effects of these autoantibodies on vascular health, they should be assessed as potential biomarkers in obesity and metabolic diseases.

Adaptive and maladaptive roles of different angiotensin receptors in the development of cardiac hypertrophy and heart failure.

Angiotensin II (Ang II) is formed by the action of angiotensin-converting enzyme (ACE) in the renin-angiotensin system. This hormone is known to induce cardiac hypertrophy and heart failure and its actions are mediated by the interaction of both pro- and antihypertrophic Ang II receptors (AT1R and AT2R). Ang II is also metabolized by ACE 2 to Ang-(1-7), which elicits the activation of Mas receptors (MasR) for inducing antihypertrophic actions. Since heart failure under different pathophysiological situations is preceded by adaptive and maladaptive cardiac hypertrophy, we have reviewed the existing literature to gain some information regarding the roles of AT1R, AT2R, and MasR in both acute and chronic conditions of cardiac hypertrophy. It appears that the activation of AT1R may be involved in the development of adaptive and maladaptive cardiac hypertrophy as well as subsequent heart failure because both ACE inhibitors and AT1R antagonists exert beneficial effects. On the other hand, the activation of both AT2R and MasR may prevent the occurrence of maladaptive cardiac hypertrophy and delay the progression of heart failure, and thus therapy with different activators of these antihypertrophic receptors under chronic pathological stages may prove beneficial. Accordingly, it is suggested that a great deal of effort should be made to develop appropriate activators of both AT2R and MasR for the treatment of heart failure subjects.

The Effect of Treatment With Aminoguanidine, an Advanced Glycation End Product Inhibitor, on Streptozotocin-Induced Diabetic Rats and Its Effects on Physiological and Renal Functions.

BACKGROUND/AIM: Diabetes is a multifactorial syndrome that affects the functioning of the renin-angiotensin system (RAS). The role of advanced glycation end products (AGEs) in diabetes is well known. In the present study, we hypothesized that the prevention of AGE accumulation or abrogation of AGE synthesis using an AGE inhibitor, aminoguanidine (AG), in streptozotocin (STZ)-induced diabetic animal models would affect the progression of diabetes and its related complications. We determined the effects of aminoguanidine (AG), an AGE inhibitor, in STZ-induced diabetic rats by determining various indices of RAS and renal functions. Additionally, we also investigated the effect of the drug, AG, on various hemodynamic and physiological functions in the body of the animals. METHODS: Male Sprague Dawley rats weighing 200-250 g were assigned to four groups (n = 4-6): Vehicle, Vehicle+AG, STZ-induced, and STZ-induced+AG rats. Type 1 diabetes was induced by a single intraperitoneal (IP) injection of streptozotocin (55 mg/kg) dissolved in sodium citrate buffer. The control groups (Vehicle) were injected with buffer. The blood glucose levels were measured after 48 hours, and animals with blood glucose levels > 300 mg/dL were included in the study. Blood glucose levels in the vehicle rats were also determined to ensure non-diabetic conditions. After confirmation, AG was administrated at a dose of 1 g/L in drinking water for two weeks. Urine was collected to measure the glomerular filtration rate (GFR), and the immune reactivity for AT1 and AT2 proteins was analyzed by immunoblotting. Data were expressed as mean ± standard error of the mean (SEM), and a p-value < 0.05 was considered statistically significant. RESULTS: Diabetic rats had a significant drop in body weight, accompanied by increased food and water consumption. The diabetic rats exhibited significantly increased urine flow and GFR. These phenotypes were significantly or considerately reversed by AG treatment in the STZ+AG-treated diabetic rats. Aminoguanidine prevented the increase in blood sugar levels compared to STZ-induced diabetic rats alone (295.9 ± 50.69 versus 462.3 ± 18.6 mg/dL (p < 0.05)). However, it did not affect the glomerular filtration rate (GFR) and glomerular damage, as assessed by the renal histopathological studies. The STZ-induced diabetic rats had an increased sodium excretion (3.24 ± 0.40 mmol) and significantly increased expression of the AT2 receptor and that of the AT1 receptor, which was slightly reversed by the treatment with AG. Treatment with AG decreased sodium excretion (2.12 ± 0.63, as compared to the diabetic rats). These rats also had modestly decreased expression of the AT2 receptor (0.99 ± 0.07 versus 1.12 ± 0.08, as compared to the STZ-induced diabetic rats), while the AT1 receptors showed a slight increase in the STZ+AG-treated rats compared to the STZ-induced diabetic rats (1.1 ± 0.19 versus 1.08 ± 0.12). CONCLUSION: This study highlights the action of the drug AG in not exacerbating any damage in diabetic rats. Employing AG as a pharmacological intervention to prevent an increase in blood sugar adds a new dimension to controlling increased blood sugar and preventing diabetic complications. The employability and pharmacological intervention of the drug AG, in diabetes, therefore, need a renewed and further investigation.

Effect of the renin-angiotensin system on the exacerbation of adrenal glucocorticoid steroidogenesis in diabetic mice: Role of angiotensin-II type 2 receptor.

Prior investigation shows an increase in the activity of both hypothalamus-pituitary-adrenal (HPA) axis and the renin-angiotensin system (RAS) in diabetic patients. Moreover, activation of angiotensin-II type 1 receptor (AT1) has been associated with adrenal steroidogenesis. This study investigates the role of RAS on the overproduction of corticosterone in diabetic mice. Diabetes was induced by intravenous injection of alloxan into fasted Swiss-webster mice. Captopril (angiotensin-converting enzyme inhibitor), Olmesartan (AT1 receptor antagonist), CGP42112A (AT2 receptor agonist) or PD123319 (AT2 receptor antagonist) were administered daily for 14 consecutive days, starting 7 days post-alloxan. Plasma corticosterone was evaluated by ELISA, while adrenal gland expressions of AT1 receptor, AT2 receptor, adrenocorticotropic hormone receptor MC2R, pro-steroidogenic enzymes steroidogenic acute regulatory protein (StAR), and 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) were assessed using immunohistochemistry or western blot. Diabetic mice showed adrenal gland overexpression of AT1 receptor, MC2R, StAR, and 11ßHSD1 without altering AT2 receptor levels, all of which were sensitive to Captopril or Olmesartan treatment. In addition, PD123319 blocked the ability of Olmesartan to reduce plasma corticosterone levels in diabetic mice. Furthermore, CGP42112A significantly decreased circulating corticosterone levels in diabetic mice, without altering the overexpression of MC2R and StAR in the adrenal glands. Our findings revealed that inhibition of both angiotensin synthesis and AT1 receptor activity reduced the high production of corticosterone in diabetic mice via the reduction of MC2R signaling expression in the adrenal gland. Furthermore, the protective effect of Olmesartan on the overproduction of corticosterone by adrenals in diabetic mice depends on both AT1 receptor blockade and AT2 receptor activation.

Histomorphometry Changes and Decreased Reactivity to Angiotensin II in the Ileum and Colon of Streptozotocin-Induced Diabetic Rats.

Diabetes mellitus (DM) is a chronic progressive metabolic disorder associated with several gastrointestinal complications, affecting up to 75% of patients. Knowing that Angiotensin II (AngII) also regulates intestinal contraction, we decided to evaluate changes in ileum and colon histomorphometry and AngII reactivity in a rat model of DM. Streptozotocin (STZ, 55 mg/kg) was administered to induce DM to 24 adult male Wistar rats. Diabetic rats displayed all the characteristic signs of type 1 DM (T1DM) and fecal excretion increased about 4-fold over 14 days, while the excretion of controls remained unaltered. Compared to controls, diabetic ileum and colon presented an increase in both macroscopic (length, perimeter and weight) and microscopic (muscular wall thickness) parameters. Functionally, AngII-induced smooth muscle contraction was lower in diabetic rats, except in the distal colon. These differences in the contractile response to AngII may result from an imbalance between AngII type 1 (antagonized by candesartan, 10 nM) and type 2 receptors activation (antagonized by PD123319, 100 nM). Taken together, these results indicate that an early and refined STZ-induced T1DM rat model already shows structural remodelling of the gut wall and decreased contractile response to AngII, findings that may help to explain diabetic dysmotility.

Increased Arterial Responsiveness to Angiotensin II in Mice Conceived by Assisted Reproductive Technologies.

Since the first report in 1978, the number of individuals conceived by Assisted Reproductive Technologies (ART) has grown incessantly. In parallel, with the recent emergence of possible underlying mechanisms of ART-induced epigenetic changes in the renin-angiotensin system, the cardiovascular repercussions of ART in mice and human offspring (including arterial hypertension, vascular dysfunction, and cardiac remodeling) have become increasingly recognized. Here, we hypothesized that ART may increase arterial responsiveness to angiotensin II (ANG II) by epigenetically modifying the expression of its receptors. To test this hypothesis, we assessed the vasoconstrictor responsiveness to ANG II in isolated aortas from ART and control mice. We also examined ANG II receptor (ATR) type 1 and 2 expression and the promoter methylation of the At1aR, At1bR and At2R genes. We found that the vasoconstrictor response to ANG II was markedly increased in ART mice compared to controls. This exaggerated vasoconstrictor responsiveness in ART mice correlated with a significant increase in the ANG II receptor (ATR) type 1 to ATR type 2 protein expression ratio in the aorta; this was mainly driven by an increase in AT1R expression, and by hypomethylation of two CpG sites located in the At1bR gene promoter leading to increased transcription of the gene. We conclude that in mice, ART increase the vasoconstrictor response to ANG II in the aorta by epigenetically causing an imbalance between the expression of vasoconstrictor (AT1R) and vasodilator (AT2R) ANG II receptors. Unbalanced expression of AT1R and AT2R receptors seems to be a novel mechanism contributing to ART-induced arterial hypertension in mice.

Aging modifies receptor expression but not muscular contractile response to angiotensin II in rat jejunum

The involvement of renin-angiotensin system in the modulation of gut motility and age-related changes in mRNA expression of angiotensin (Ang II) receptors (ATR) are well accepted. We aimed to characterize, in vitro, the contractile responses induced by Ang II, in jejunum from young (3–6 weeks old) and old rats (≥ 1 year old), to evaluate possible functional differences associated to changes in receptor expression. Mechanical responses to Ang II were examined in vitro as changes in isometric tension. ATR expression was assessed by qRT-PCR. Ang II induced a contractile effect, antagonized by losartan, AT1R antagonist, and increased by PD123319, AT2R antagonist, as well by neural blocker ω-conotoxin and by nitric oxide (NO) synthase inhibitor. No difference in the response was observed between young and old groups. AT1 receptor-mediated contractile response was decreased by U-73122, phospholipase C (PLC) inhibitor; or 2-aminoethoxy-diphenylborate (2-APB), inositol triphosphate (IP3) receptor inhibitor; or nifedipine, L-type calcium channel blocker. Age-related changes in the expression of both AT1 receptor subtypes, AT1a and AT1b, and of AT2 receptors were detected. In conclusion, Ang II modulates the spontaneous contractility of rat jejunum via postjunctional AT1 receptors, involving Ca2+ mobilization from intracellular stores, via PLC/IP3 pathway, and Ca2+ influx from extracellular space, via L-type channels. Prejunctional AT2 receptors would counteract AT1 receptor effects, via NO synthesis. The observed age-related differences in the expression of all AT receptor subtypes are not reflected in the muscular contractile response to Ang II. (AU)

Actions of Novel Angiotensin Receptor Blocking Drugs, Bisartans, Relevant for COVID-19 Therapy: Biased Agonism at Angiotensin Receptors and the Beneficial Effects of Neprilysin in the Renin Angiotensin System.

Angiotensin receptor blockers (ARBs) used in the treatment of hypertension and potentially in SARS-CoV-2 infection exhibit inverse agonist effects at angiotensin AR1 receptors, suggesting the receptor may have evolved to accommodate naturally occurring angiotensin 'antipeptides'. Screening of the human genome has identified a peptide (EGVYVHPV) encoded by mRNA, complementary to that encoding ANG II itself, which is an inverse agonist. Thus, opposite strands of DNA encode peptides with opposite effects at AR1 receptors. Agonism and inverse agonism at AR1 receptors can be explained by a receptor 'switching' between an activated state invoking receptor dimerization/G protein coupling and an inverse agonist state mediated by an alternative/second messenger that is slow to reverse. Both receptor states appear to be driven by the formation of the ANG II charge-relay system involving TyrOH-His/imidazole-Carboxylate (analogous to serine proteases). In this system, tyrosinate species formed are essential for activating AT1 and AT2 receptors. ANGII is also known to bind to the zinc-coordinated metalloprotease angiotensin converting enzyme 2 (ACE2) used by the COVID-19 virus to enter cells. Here we report in silico results demonstrating the binding of a new class of anionic biphenyl-tetrazole sartans ('Bisartans') to the active site zinc atom of the endopeptidase Neprilysin (NEP) involved in regulating hypertension, by modulating humoral levels of beneficial vasoactive peptides in the RAS such as vasodilator angiotensin (1-7). In vivo and modeling evidence further suggest Bisartans can inhibit ANG II-induced pulmonary edema and may be useful in combatting SARS-CoV-2 infection by inhibiting ACE2-mediated viral entry to cells.

Estrogen normalizes maternal HFD-induced vascular dysfunction in offspring by regulating ATR.

Previous studies have shown that female offspring are resistant to fetal high-fat diet (HFD)-induced programming of heightened vascular contraction; however, the underlying mechanisms remain unclear. The present study tested the hypothesis that estrogen plays a key role in protecting females from fetal programming of increased vascular contraction induced by maternal HFD exposure. Pregnant rats were fed a normal diet (ND) or HFD (60% kcal from fat). Ovariectomy (OVX) and 17ß-estradiol (E2) replacement were performed on 8-week-old female offspring. Aortas were isolated from adult female offspring. Maternal HFD exposure increased angiotensin II (Ang II)-induced contractions of the aorta in adult OVX offspring, which was abrogated by E2 replacement. The AT1 receptor (AT1R) antagonist losartan (10 µM), but not the AT2 receptor (AT2R) antagonist PD123319 (10 µM), completely blocked Ang II-induced contractions in both ND and HFD offspring. In addition, HFD exposure caused a decrease in endothelium-dependent relaxations induced by acetylcholine (ACh) in adult OVX but not OVX-E2 offspring. However, it had no effect on sodium nitroprusside (SNP)-induced endothelium-independent aorta relaxation in any of the six groups. Maternal HFD feeding increased AT1R, but not AT2R, leading to an increased AT1R/AT2R ratio in HFD-exposed OVX offspring, associated with selective decreases in DNA methylation at the AT1aR promoter, which was ameliorated by E2 replacement. Our results indicated that estrogen play a key role in sex differences of maternal HFD-induced vascular dysfunction and development of hypertensive phenotype in adulthood by differently regulating vascular AT1R and AT2R gene expression through a DNA methylation mechanism.

Aging modifies receptor expression but not muscular contractile response to angiotensin II in rat jejunum.

The involvement of renin-angiotensin system in the modulation of gut motility and age-related changes in mRNA expression of angiotensin (Ang II) receptors (ATR) are well accepted. We aimed to characterize, in vitro, the contractile responses induced by Ang II, in jejunum from young (3-6 weeks old) and old rats (≥ 1 year old), to evaluate possible functional differences associated to changes in receptor expression. Mechanical responses to Ang II were examined in vitro as changes in isometric tension. ATR expression was assessed by qRT-PCR. Ang II induced a contractile effect, antagonized by losartan, AT1R antagonist, and increased by PD123319, AT2R antagonist, as well by neural blocker ω-conotoxin and by nitric oxide (NO) synthase inhibitor. No difference in the response was observed between young and old groups. AT1 receptor-mediated contractile response was decreased by U-73122, phospholipase C (PLC) inhibitor; or 2-aminoethoxy-diphenylborate (2-APB), inositol triphosphate (IP3) receptor inhibitor; or nifedipine, L-type calcium channel blocker. Age-related changes in the expression of both AT1 receptor subtypes, AT1a and AT1b, and of AT2 receptors were detected. In conclusion, Ang II modulates the spontaneous contractility of rat jejunum via postjunctional AT1 receptors, involving Ca2+ mobilization from intracellular stores, via PLC/IP3 pathway, and Ca2+ influx from extracellular space, via L-type channels. Prejunctional AT2 receptors would counteract AT1 receptor effects, via NO synthesis. The observed age-related differences in the expression of all AT receptor subtypes are not reflected in the muscular contractile response to Ang II.

Structural insights into ligand recognition and activation of angiotensin receptors.

G protein-coupled angiotensin II receptors, AT1R and AT2R, are integral components of the renin-angiotensin system (RAS) that regulates blood pressure and fluid balance in humans. While AT1R is a well-established target of angiotensin receptor blockers (ARBs) for managing hypertension and a prime system for studying biased signaling, AT2R has been recognized as a promising target against neuropathic pain and lung fibrosis. In this review, we discuss how recent structural advances illuminate ligand-binding modes and subtype selectivity, shared and distinct features of the receptors, their transducer-coupling patterns, and downstream signaling responses. We also underscore the key ATR aspects that require further studies to fully appreciate the mechanistic framework that fine-tunes their cellular and physiological functions, providing untapped potential for drug discovery.

Estrogen normalizes maternal HFD-induced cardiac hypertrophy in offspring by regulating AT2R.

Our previous study has demonstrated maternal high-fat diet (HFD) caused sex-dependent cardiac hypertrophy in adult male, but not female offspring. The present study tested the hypothesis that estrogen normalizes maternal HFD-induced cardiac hypertrophy by regulating angiotensin II receptor (ATR) expression in adult female offspring. Pregnant rats were divided into the normal diet (ND) and HFD (60% kcal fat) groups. Ovariectomy (OVX) and 17ß-estradiol (E2) replacement were performed on 8-week-old female offspring. Maternal HFD had no effect on left ventricular (LV) wall thickness, cardiac function and molecular markers of cardiac hypertrophy function in sham groups. However, maternal HFD caused cardiac hypertrophy of offspring in OVX groups, which was abrogated by E2 replacement. In addition, maternal HFD had no effect on ERα and ERß in sham groups. In contrast, HFD significantly decreased ERα, but not ERß in OVX groups. In sham groups, there was no difference in the cardiac ATR type 1 (AT1R) and ATR type 2 (AT2R) between ND and HFD offspring. HFD significantly increased AT2R, but not AT1R in OVX groups. Furthermore, maternal HFD resulted in decreased glucocorticoid receptors (GRs) binding to the glucocorticoid response elements at the AT2R promoter, which was due to decreased GRs in hearts from OVX offspring. These HFD-induced changes in OVX groups were abrogated by E2 replacement. These results support a key role of estrogen in the sex difference of maternal HFD-induced cardiac hypertrophy in offspring, and suggest that estrogen protects female offspring from cardiac hypertrophy in adulthood by regulating AT2R.

Morpho-functional remodelling of the adult zebrafish (Danio rerio) heart in response to waterborne angiotensin II exposure.

Angiotensin II (AngII), the principal effector of the Renin-Angiotensin System, is a pluripotent humoral agent whose biological actions include short-term modulations and long-term adaptations. In fish, short-term cardio-tropic effects of AngII are documented, but information on the role of AngII in long-term cardiac remodelling is not fully understood. Here, we describe a direct approach to disclose long-term morpho-functional effects of AngII on the zebrafish heart. Adult fish exposed to waterborne teleost analogue AngII for 8 weeks showed enhanced heart weight and cardio-somatic index, coupled to myocardial structural changes (i.e. augmented compacta thickness and fibrosis), and increased heart rate. These findings were paralleled by an up-regulation of type-1 and type-2 AngII receptors expression, and by changes in the expression of GATA binding protein 4, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 and superoxide dismutase 1 soluble mRNAs, as well as of cytochrome b-245 beta polypeptide protein, indicative of cardiac remodelling. Our results suggest that waterborne AngII can sustain and robustly affect the cardiac morpho-functional remodelling of adult zebrafish.

Analysis of angiotensin II-Induced rat urinary bladder contractions in the presence of angiotensin II receptors blockers.

OBJECTIVE: An application of a specific analysis on recordings obtained from urinary bladder (UB) preparations influenced with Angiotensin II (AngII) and AngII receptor (ATR) blockers was performed. METHODS: UB preparations were divided as follows: group 1 stimulated with AngII only; group 2:PD123319 (ATR type-2 blocker)+AngII; group 3:Losartan (ATR type-1 blocker)+AngII. The averaged time and force parameters of the contractions were processed by a spline interpolation and graphic images of the different patterns of the contractile activity were obtained. RESULTS: The speed of AngII-induced UB contraction, when PD123319 was administered, was significantly higher than those, registered by the application of AngII alone and Losartan + AngII. The presence of Losartan markedly delayed the speed of the overall AngII-induced contraction. CONCLUSION: The study indicates the contribution of both ATR subtypes for the development of AngII-induced UB contraction. Our results showed that probably ATR mediate a reciprocal dynamic response to AngII in the bladder.

Role of angiotensin II type 1 (AT1) and type 2 (AT2) receptors in airway reactivity and inflammation in an allergic mouse model of asthma.

Objective: Angiotensin II (Ang II) exerts its effects through two G-protein coupled receptors: angiotensin II type 1 receptors (AT1) and type 2 receptors (AT2). Both these receptor subtypes are poorly understood in asthma. In this study, we investigated effects of AT1 receptor antagonist losartan, novel AT2 receptor agonist novokinin and AT2 receptor antagonist PD 123319 in a mouse model of asthma. Methods: Mice were divided into control (CON) and allergen sensitized (SEN) groups. SEN was sensitized with ovalbumin (OVA) on days 1 and 6 (30 µg; i.p.), followed by 5% OVA aerosol challenge (days 11-13). Treatments included (a) losartan (SEN + LOS; 20 mg/kg i.p., day 14), (b) novokinin (SEN + NOV; 0.3 mg/kg i.p., day 14), and (c) PD 123319 (SEN + PD; 5 mg/kg i.p., day 14). Experiments for airway responsiveness, bronchoalveolar lavage, and tracheal ring reactivity using isolated organ bath were performed. Results: Airway responsiveness to methacholine (MCh) (48 mg/mL) was significantly higher in SEN (563.71 ± 40% vs. 294.3 ± 123.84 in CON). This response was potentiated in SEN + PD group (757 ± 30%; p < .05 compared to SEN). SEN + LOS (247.61 ± 86.85%) and SEN + NOV (352 ± 11%) had significantly lower response compared to SEN. SEN + LOS (26.22 ± 0.29%) and SEN + NOV (46.20 ± 0.76%) treatment significantly (p < .001) attenuated total cell count and eosinophils compared to SEN group (69.38 ± 1.5%), while SEN + PD (73.04 ± 0.69%) had highest number of eosinophils. Tracheal response to MCh was significantly higher in SEN group compared to controls, and this response was significantly lowered with the losartan and novokinin treatments. Conclusions: These data suggest that AT1 and AT2 receptors have opposite effects in modulating airway hyperresponsiveness and inflammation in asthma.

Mitochondrial angiotensin receptors and cardioprotective pathways.

A growing body of data provides strong evidence that intracellular angiotensin II (ANG II) plays an important role in mammalian cell function and is involved in the pathogenesis of human diseases such as hypertension, diabetes, inflammation, fibrosis, arrhythmias, and kidney disease, among others. Recent studies also suggest that intracellular ANG II exerts protective effects in cells during high extracellular levels of the hormone or during chronic stimulation of the local tissue renin-angiotensin system (RAS). Notably, the intracellular RAS (iRAS) described in neurons, fibroblasts, renal cells, and cardiomyocytes provided new insights into regulatory mechanisms mediated by intracellular ANG II type 1 (AT1Rs) and 2 (AT2Rs) receptors, particularly, in mitochondria and nucleus. For instance, ANG II through nuclear AT1Rs promotes protective mechanisms by stimulating the AT2R signaling cascade, which involves mitochondrial AT2Rs and Mas receptors. The stimulation of nuclear ANG II receptors enhances mitochondrial biogenesis through peroxisome proliferator-activated receptor-γ coactivator-1α and increases sirtuins activity, thus protecting the cell against oxidative stress. Recent studies in ANG II-induced preconditioning suggest that plasma membrane AT2R stimulation exerts protective effects against cardiac ischemia-reperfusion by modulating mitochondrial AT1R and AT2R signaling. These studies indicate that iRAS promotes the protection of cells through nuclear AT1R signaling, which, in turn, promotes AT2R-dependent processes in mitochondria. Thus, despite abundant data on the deleterious effects of intracellular ANG II, a growing body of studies also supports a protective role for iRAS that could be of relevance to developing new therapeutic strategies. This review summarizes and discusses previous studies on the role of iRAS, particularly emphasizing the protective and counterbalancing actions of iRAS, mitochondrial ANG II receptors, and their implications for organ protection.

Clinical Trials on Diabetic Nephropathy: A Cross-Sectional Analysis.

INTRODUCTION: Treatment options and decisions are often based on the results of clinical trials. We have evaluated the public availability of results from completed, registered phase III clinical trials on diabetic nephropathy and current treatment options. METHODS: This was a cross-sectional analysis in which STrengthening the Reporting of OBservational studies in Epidemiology criteria were applied for design and analysis. In June 2017, 34 completed phase III clinical trials on diabetic nephropathy in the ClinicalTrials. gov registry were identified and matched to publications in the ClinicalTrials.gov registry and to those in the PubMed and Google Scholar databases. If no publication was identified, the principal investigator was contacted. The ratio of published and non-published studies was calculated. Various parameters, including study design, drugs, and comparators provided, were analyzed. RESULTS: Drugs/supplements belonged to 26 different categories of medications, with the main ones being angiotensin-converting enzyme inhibitors, angiotensin-II receptors blockers, and dipeptidyl-peptidase-4-inhibitors. Among the trials completed before 2016 (n = 32), 22 (69%) were published, and ten (31%) remained unpublished. Thus, data on 11 different interventions and more than 1000 patients remained undisclosed. Mean time to publication was 26.5 months, which is longer than the time constrictions imposed by the U.S. Food and Drug Administration Amendments Act. Most trials only showed weak effects on micro- and macroalbuminuria, with an absolute risk reduction of 1.0 and 0.3%, respectively, and the number needed to treat varied between 91 and 333, without any relevant effect on end-stage-renal disease by intensive glucose-lowering treatment. Comparison of the results, however, was difficult since study design, interventions, and the renal outcome parameters vary greatly between the studies. CONCLUSION: Despite the financial and human resources involved and the relevance for therapeutic guidelines and clinical decisions, about one-third of phase III clinical trials on diabetic nephropathy remain unpublished. Interventions used in published trials showed a low efficacy on renal outcome. FUNDING: Deutsche Forschungsgemeinschaft (DFG): SFB 1118.

Maternal high-fat diet alters angiotensin II receptors and causes changes in fetal and neonatal rats†.

Maternal high-fat diet (HFD) during pregnancy is linked to cardiovascular diseases in postnatal life. The current study tested the hypothesis that maternal HFD causes myocardial changes through angiotensin II receptor (AGTR) expression modulation in fetal and neonatal rat hearts. The control group of pregnant rats was fed a normal diet and the treatment group of pregnant rats was on a HFD (60% kcal fat). Hearts were isolated from embryonic day 21 fetuses (E21) and postnatal day 7 pups (PD7). Maternal HFD decreased the body weight of the offspring in both E21 and PD7. The ratio of heart weight to body weight was increased in E21, but not PD7, when compared to the control group. Transmission electron microscopy revealed disorganized myofibrils and effacement of mitochondria cristae in the treatment group. Maternal HFD decreased S-phase and increased G1-phase of the cellular cycle for fetal and neonatal cardiac cells. Molecular markers of cardiac hypertrophy, such as Nppa and Myh7, were found to be increased in the treatment group. There was an associated increase in Agtr2 mRNA and protein, whereas Agtr1a mRNA and AGTR1 protein were decreased in HFD fetal and neonatal hearts. Furthermore, maternal HFD decreased glucocorticoid receptors (GRs) binding to glucocorticoid response elements at the Agtr1a and Agtr2 promoter, which correlated with downregulation of GR in fetal and neonatal hearts. These findings suggest that maternal HFD may promote premature termination of fetal and neonatal cardiomyocyte proliferation and compensatory hypertrophy through intrauterine modulation of AGTR1 and AGTR2 expression via GR dependent mechanism.

Reversal of Aortic Enlargement Induced by Increased Biomechanical Forces Requires AT1R Inhibition in Conjunction With AT2R Activation.

Objective- Pharmacological inhibition of the AT1R (angiotensin II type 1 receptor) with losartan can attenuate ascending aortic remodeling induced by transverse aortic constriction (TAC). In this study, we investigated the role of the AT2R (angiotensin II type 2 receptor) and MasR (Mas receptor) in TAC-induced ascending aortic dilation and remodeling. Approach and Results- Wild-type C57BL/6J mice were subjected to sham or TAC surgeries in the presence and absence of various drugs. Aortic diameters were assessed by echocardiography, central blood pressure was measured in the ascending aorta 2 weeks post-operation, and histology and gene expression analyses completed. An angiotensin-converting enzyme inhibitor, captopril, decreased systolic blood pressure to the same level as losartan but did not attenuate aortic dilation, adventitial inflammation, medial collagen deposition, elastin breakage, or Mmp9 (matrix metalloproteinase-9) expression when compared with TAC mice. In contrast, co-administration of captopril with an AT2R agonist, compound 21, attenuated aortic dilation, medial collagen content, elastin breaks, and Mmp9 expression, whereas co-administration of captopril with a MasR agonist (AVE0991) did not reverse aortic dilation and led to aberrant aortic remodeling. An AT2R antagonist, PD123319, reversed the protective effects of losartan in TAC mice. Treatment with compound 21 alone showed no effect on TAC-induced aortic enlargement, blood pressure, elastin breakage, or Mmp9 expression. Conclusions- Our data indicate that when AT1R signaling is blocked, AT2R activation is a key modulator to prevent aortic dilation that occurs with TAC. These data suggest that angiotensin-converting enzyme inhibitor may not be as effective as losartan for slowing aneurysm growth because losartan requires intact AT2R signaling to prevent aortic enlargement.