Blockage of Angiotensin II type 2 receptor prevents thyroxine-mediated cardiac hypertrophy by blocking Akt activation

Although most of effects of Angiotensin II (Ang II) related to cardiac remodelling can be attributed to type 1 Ang II receptor (AT(1)R), the type 2 receptor (AT(2)R) has been shown to be involved in the development of some cardiac hypertrophy models. In the present study, we investigated whether the thyroid hormone (TH) action leading to cardiac hypertrophy is also mediated by increased Ang II levels or by change on AT(1)R and AT(2)R expression, which could contribute to this effect. In addition, we also evaluated the possible contribution of AT(2)R in the activation of Akt and in the development of TH-induced cardiac hypertrophy. To address these questions, Wistar rats were treated with thyroxine (T(4), 0.1 mg/kg BW/day, i.p.), with or without AT(2)R blocker (PD123319), for 14 days. Cardiac hypertrophy was identified based on heart/body weight ratio and confirmed by analysis of atrial natriuretic factor mRNA expression. Cardiomyocyte cultures were used to exclude the influence of TH-related hemodynamic effects. Our results demonstrate that the cardiac Ang II levels were significantly increased (80%, P < 0.001) as well as the AT(2)R expression (50%, P < 0.05) in TH-induced cardiac hypertrophy. The critical involvement of AT(2)R to the development of this cardiac hypertrophy in vivo was evidenced after administration of AT(2) blocker, which was able to prevent in 40% (P < 0.01) the cardiac mass gain and the Akt activation induced by TH. The role of AT(2)R to the TH-induced cardiomyocyte hypertrophy was also confirmed after using PD123319 in the in vitro studies. These findings improve understanding of the cardiac hypertrophy observed in hyperthyroidism and provide new insights into the generation of future therapeutic strategies.

Angiotensin II modulates CD40 expression in vascular smooth muscle cells

The signalling pathway CD40/CD40L (CD40 ligand) plays an important role in atherosclerotic plaque formation and rupture. AngII (angiotensin II), which induces oxidative stress and inflammation, is also implicated in the progression of atherosclerosis. In the present study, we tested the hypothesis that AngII increases CD40/CD40L activity in vascular cells and that ROS (reactive oxygen species) are part of the signalling cascade that controls CD40/CD40L expression. Human CASMCs (coronary artery smooth muscle cells) in culture exposed to IL (interleukin)-1 beta or TNF-alpha (tumour necrosis factor-a) had increased superoxide generation and enhanced CD40 expression, detected by EPR (electron paramagnetic resonance) and immunoblotting respectively. Both phenomena were abolished by previous incubation with membrane-permeant antioxidants or cell transfection with P22(phox) antisense. AngII (50-200 nmol/l) induced an early and sustained increase in CD40 mRNA and protein expression in CASMCs, which was blocked by treatment with antioxidants. Increased CD40 expression led to enhanced activity of the pathway, as AngII-treated cells stimulated with recombinant CD40L released higher amounts of IL-8 and had increased COX-2 (cyclo-oxygenase-2) expression. We conclude that AngII stimulation of vascular cells leads to a ROS-dependent increase in CD40/CD40L signalling pathway activity. This phenomenon may be an important mechanism modulating the arterial injury observed in atherosclerosis-related vasculopathy.

Carboxypeptidases A1 and A2 from the perfusate of rat mesenteric arterial bed differentially process angiotensin peptides

Here we report the isolation of carboxypeptidases A1 and A2 (CPA1 and CPA2) from the rat mesenteric arterial bed perfusate, which were found to be identical with their pancreatic counterparts. Angiotensin (Ang) I, Ang II, Ang-(1-9) and Ang-(1-12) were differentially processed by these enzymes, worthy mentioning the peculiar CPA1-catalyzed conversion of Ang II to Ang-(1-7) and the CPA2-mediated formation of Ang I from Ang-(1-12). We detected gene transcripts for CPA1 and CPA2 in mesentery and other extrapancreatic tissues, indicating that these CPAs might play a role in the renin-angiotensin system in addition to their functions as digestive enzymes. (C) 2011 Elsevier Inc. All rights reserved.

Angiotensin II type 1 receptor blockade partially attenuates hypoxia-induced pulmonary hypertension in newborn piglets: relationship with the nitrergic system

The objective of this study was to observe possible interactions between the renin-angiotensin and nitrergic systems in chronic hypoxia-induced pulmonary hypertension in newborn piglets. Thirteen chronically instrumented newborn piglets (6.3 +/- 0.9 days; 2369 +/- 491 g) were randomly assigned to receive saline (placebo, P) or the AT(1) receptor (AT(1)-R) blocker L-158,809 (L) during 6 days of hypoxia (FiO(2) = 0.12). During hypoxia, pulmonary arterial pressure (Ppa; P < 0.0001), pulmonary vascular resistance (PVR; P < 0.02) and the pulmonary to systemic vascular resistance ratio (PVR/SVR; P < 0.05) were significantly attenuated in the L (N = 7) group compared to the P group (N = 6). Western blot analysis of lung proteins showed a significant decrease of endothelial NOS (eNOS) in both P and L animals, and of AT(1)-R in P animals during hypoxia compared to normoxic animals (C group, N = 5; P < 0.01 for all groups). AT(1)-R tended to decrease in L animals. Inducible NOS (iNOS) did not differ among P, L, and C animals and iNOS immunohistochemical staining in macrophages was significantly more intense in L than in P animals (P < 0.01). The vascular endothelium showed moderate or strong eNOS and AT(1)-R staining. Macrophages and pneumocytes showed moderate or strong iNOS and AT(1)-R staining, but C animals showed weak iNOS and AT(1)-R staining. Macrophages of L and P animals showed moderate and weak AT(2)-R staining, respectively, but the endothelium of all groups only showed weak staining. In conclusion, pulmonary hypertension induced by chronic hypoxia in newborn piglets is partially attenuated by AT(1)-R blockade. We suggest that AT(1)-R blockade might act through AT(2)-R and/or Mas receptors and the nitrergic system in the lungs of hypoxemic newborn piglets.

Angiotensin-(1-7) decreases LPS-induced inflammatory response in macrophages

It has been previously shown that besides its classical role in blood pressure control the reninangiotensin system, mainly by action of angiotensin II on the AT1 receptor, exerts pro-inflammatory effects such as by inducing the production of cytokines. More recently, alternative pathways to this system were described, such as binding of angiotensin-(17) to receptor Mas, which was shown to counteract some of the effects evoked by activation of the angiotensin IIAT1 receptor axis. Here, by means of different molecular approaches we investigated the role of angiotensin-(17) in modulating inflammatory responses triggered in mouse peritoneal macrophages. Our results show that receptor Mas transcripts were up-regulated by eightfold in LPS-induced macrophages. Interestingly, macrophage stimulation with angiotensin-(17), following to LPS exposure, evoked an attenuation in expression of TNF-a and IL-6 pro-inflammatory cytokines; where this event was abolished when the receptor Mas selective antagonist A779 was also included. We then used heterologous expression of the receptor Mas in HEK293T cells to search for the molecular mechanisms underlying the angiotensin-(17)-mediated anti-inflammatory responses by a kinase array; what suggested the involvement of the Src kinase family. In LPS-induced macrophages, this finding was corroborated using the PP2 compound, a specific Src kinase inhibitor; and also by Western blotting when we observed that Ang-(17) attenuated the phosphorylation levels of Lyn, a member of the Src kinase family. Our findings bring evidence for an anti-inflammatory role for angiotensin-(17) at the cellular level, as well as show that its probable mechanism of action includes the modulation of Src kinases activities. J. Cell. Physiol. 227: 21172122, 2012. (C) 2011 Wiley Periodicals, Inc.

Effects of aerobic exercise training on cardiac renin-angiotensin system in an obese Zucker rat strain

Objective: Obesity and renin angiotensin system (RAS) hyperactivity are profoundly involved in cardiovascular diseases, however aerobic exercise training (EXT) can prevent obesity and cardiac RAS activation. The study hypothesis was to investigate whether obesity and its association with EXT alter the systemic and cardiac RAS components in an obese Zucker rat strain. Methods: The rats were divided into the following groups: Lean Zucker rats (LZR); lean Zucker rats plus EXT (LZR+EXT); obese Zucker rats (OZR) and obese Zucker rats plus EXT (OZR+EXT). EXT consisted of 10 weeks of 60-min swimming sessions, 5 days/week. At the end of the training protocol heart rate (HR), systolic blood pressure (SBP), cardiac hypertrophy (CH) and function, local and systemic components of RAS were evaluated. Also, systemic glucose, triglycerides, total cholesterol and its LDL and HDL fractions were measured. Results: The resting HR decreased (, 12%) for both LZR+EXT and OZR+EXT. However, only the LZR+EXT reached significance (p, 0.05), while a tendency was found for OZR versus OZR+EXT (p = 0.07). In addition, exercise reduced (57%) triglycerides and (61%) LDL in the OZR+EXT. The systemic angiotensin I-converting enzyme (ACE) activity did not differ regardless of obesity and EXT, however, the OZR and OZR+EXT showed (66%) and (42%), respectively, less angiotensin II (Ang II) plasma concentration when compared with LZR. Furthermore, the results showed that EXT in the OZR prevented increase in CH, cardiac ACE activity, Ang II and AT2 receptor caused by obesity. In addition, exercise augmented cardiac ACE2 in both training groups. Conclusion: Despite the unchanged ACE and lower systemic Ang II levels in obesity, the cardiac RAS was increased in OZR and EXT in obese Zucker rats reduced some of the cardiac RAS components and prevented obesity-related CH. These results show that EXT prevented the heart RAS hyperactivity and cardiac maladaptive morphological alterations in obese Zucker rats.

Contribution of the genes of the renin-angiotensin system to interindividual differences in blood pressure levels in Caucasians from Rochester, Minnesota

The Renin-Angiotensin system (RAS) regulates blood pressure through its effects on vascular tone, renal hemodynamics, and renal sodium and fluid balance. The genes encoding the four major components of the RAS, angiotensinogen, renin, angiotensin I-converting enzyme (ACE), and angiotensin II receptor type 1 (AT1), have been investigated as candidate genes in the pathogenesis of essential hypertension. However, studies have primarily focused on small samples of diseased individuals, and, therefore, have provided little information about the determinants of interindividual variation in blood pressure (BP) in the general population.^ Using data from a large population-based sample from Rochester, MN, I have evaluated the contribution of variation in the region of the RAS genes to interindividual variation in systolic, diastolic, and mean arterial pressure in the population-at-large. Marker genotype data from four polymorphisms located within or very near these genes were first collected on 3,974 individuals from 583 randomly ascertained three-generation pedigrees. Haseman-Elston regression and variance component methods of linkage analysis were then carried out to estimate the proportion of interindividual variance in BP attributable to the effects of variation at these four measured loci.^ A significant effect of the ACE locus on interindividual variation in mean arterial pressure (MAP) was detected in a sample of siblings belonging to the youngest generation. After allowing for measured covariates, this effect accounted for 15-25% of the interindividual variance in MAP, and was even greater in a subset with a positive family history of hypertension. When gender-specific analyses were carried out, this effect was significant in males but not in females. Extended pedigree analyses also provided evidence for an effect of the ACE locus on interindividual variation in MAP, but no difference between males and females was observed. Circumstantial evidence suggests that the ACE gene itself may be responsible for the observed effects on BP, although the possibility that other genes in the region may be at play cannot be excluded.^ No definitive evidence for an effect of the renin, angiotensinogen, or AT1 loci on interindividual variation in BP was obtained in this study, suggesting that the impact of these genes on BP may not be great in the Caucasian population-at-large. However, this does not preclude a larger effect of these genes in some subsets of individuals, especially among those with clinically manifest hypertension or coronary heart disease, or in other populations. ^

A new strategy to decrease N-methyl-d-aspartate (NMDA) receptor coactivation: Inhibition of d-serine synthesis by converting serine racemase into an eliminase

Serine racemase is a brain-enriched enzyme that synthesizes d-serine, an endogenous modulator of the glycine site of N-methyl-d-aspartate (NMDA) receptors. We now report that serine racemase catalyzes an elimination reaction toward a nonphysiological substrate that provides a powerful tool to study its neurobiological role and will be useful to develop selective enzyme inhibitors. Serine racemase catalyzes robust elimination of l-serine O-sulfate that is 500 times faster than the physiological racemization reaction, generating sulfate, ammonia, and pyruvate. This reaction provides the most simple and sensitive assay to detect the enzyme activity so far. We establish stable cell lines expressing serine racemase and show that serine racemase can also be converted into a powerful eliminase in cultured cells, while the racemization of l-serine is inhibited. Likewise, l-serine O-sulfate inhibits the synthesis of d-serine in primary astrocyte cultures. We conclude that the synthetic compound l-serine O-sulfate is a better substrate than l-serine as well as an inhibitor of d-serine synthesis. Inhibition of serine racemase provides a new strategy to selectively decrease NMDA receptor coactivation and may be useful in conditions in which overstimulation of NMDA receptors plays a pathological role.

Differential suppression by protease inhibitors and cytokines of apoptosis induced by wild-type p53 and cytotoxic agents.

Apoptosis induced in myeloid leukemic cells by wild-type p53 was suppressed by different cleavage-site directed protease inhibitors, which inhibit interleukin-1 beta-converting enzyme-like, granzyme B and cathepsins B and L proteases. Apoptosis was also suppressed by the serine and cysteine protease inhibitor N-tosyl-L-phenylalanine chloromethylketone (TPCK) [corrected], but not by other serine or cysteine protease inhibitors including N alpha-p-tosyl-L-lysine chloromethylketone (TLCK), E64, pepstatin A, or chymostatin. Protease inhibitors suppressed induction of apoptosis by gamma-irradiation and cycloheximide but not by doxorubicin, vincristine, or withdrawal of interleukin 3 from interleukin 3-dependent 32D non-malignant myeloid cells. Induction of apoptosis in normal thymocytes by gamma-irradiation or dexamethasone was also suppressed by the cleavage-site directed protease inhibitors, but in contrast to the myeloid leukemic cells apoptosis in thymocytes was suppressed by TLCK but not by TPCK. The results indicate that (i) inhibitors of interleukin-1 beta-converting enzyme-like proteases and some other protease inhibitors suppressed induction of apoptosis by wild-type p53 and certain p53-independent pathways of apoptosis; (ii) the protease inhibitors together with the cytokines interleukin 6 and interferon-gamma or the antioxidant butylated hydroxyanisole gave a cooperative protection against apoptosis; (iii) these protease inhibitors did not suppress induction of apoptosis by some cytotoxic agents or by viability-factor withdrawal from 32D cells, whereas these pathways of apoptosis were suppressed by cytokines; (iv) there are cell type differences in the proteases involved in apoptosis; and (v) there are multiple pathways leading to apoptosis that can be selectively induced and suppressed by different agents.

Tonic stimulation of renin gene expression by nitric oxide is counteracted by tonic inhibition through angiotensin II.

This study was designed to examine the possible involvement of prostaglandins and nitric oxide (NO) in the renin stimulatory effect of angiotensin II (AngII) antagonists. To this end, plasma renin activities (PRAs) and renal renin mRNA levels were assayed in rats that were treated with the Ang-converting enzyme inhibitor ramipril or with the AngII AT1-receptor antagonist losartan. Ramipril and losartan increased PRA values from 7.5 +/- 1.6 to 86 +/- 6 and 78 +/- 22 ng of AngI per h per ml and renin mRNA levels from 112 +/- 9% to 391 +/- 20% and 317 +/- 10%, respectively. Inhibition of prostaglandin formation with indomethacin did not influence basal or ramipril-affected PRA. Basal renin mRNA levels also were unchanged by indomethacin, while increases in renin mRNA levels after ramipril treatment were slightly reduced by indomethacin. Inhibition of NO synthase by nitro-L-arginine methyl ester (L-NAME) reduced PRA values to 3.2 +/- 0.9, 34 +/- 13, and 12.1 +/- 2.7 ng of AngI per h per ml in control, ramipril-treated, and losartan-treated animals, respectively. Renin mRNA levels were reduced to 77 +/- 14% under basal conditions and ramipril- and losartan-induced increases in renin mRNA levels were completely blunted after addition of L-NAME. The AngII antagonists, furthermore, induced an upstream recruitment of renin-expressing cells in the renal afferent arterioles, which was also blunted by L-NAME. These findings suggest that renin mRNA levels are tonically increased by NO and that the action of NO is counteracted by AngII.

The renin-angiotensin system and male reproduction: new functions for old hormones

The blood-borne renin-angiotensin system (RAS) is known best for its role in the maintenance of blood pressure and electrolyte and fluid homeostasis. However, numerous tissues show intrinsic angiotensin-generating systems that cater for specific local needs through actions that add to, or differ from, the circulating RAS. The male reproductive system has several sites of intrinsic RAS activity. Recent focus on the epididymis, by our laboratories and by others, has contributed important details about the local RAS in this tissue. The RAS components have been localized morphologically and topographically; they have been shown to be responsive to androgens and to hypoxia; and angiotensin has been shown to influence tubular, and consequently, fluid secretion. Components of the RAS have also been found in the testis, vas deferens, prostate and semen. Angiotensin II receptors, type 1 and, to a lesser extent, type 2 are widespread, and angiotensin IV receptors have been localized in the prostate. The roles of the RAS in local processes at these sites are still uncertain and have yet to be fully elucidated, although there is evidence for involvement in tubular contractility, spermatogenesis, sperm maturation, capacitation, acrosomal exocytosis and fertilization. Notwithstanding this evidence for the involvement of the RAS in various important aspects of male reproduction, there has so far been a lack of clinical evidence, demonstrable by changes in fertility, for a crucial role of the RAS in male reproduction. However, it is clear that there are several potential targets for manipulating the activity of the male reproductive system by interfering with the locally generated angiotensin systems.

Evaluation of Antiproliferative, Anti-Type 2 Diabetes, and Antihypertension Potentials of Ellagitannins from Strawberries (Fragaria x ananassa Duch.) Using In Vitro Models

Strawberries represent the main source of ellagic acid derivatives in the Brazilian diet, corresponding to more than 50% of all phenolic compounds found in the fruit. There is a particular interest in the determination of the ellagic acid content in fruits because of possible chemopreventive benefits. In the present study, the potential health benefits of purified ellagitannins from strawberries were evaluated in relation to the antiproliferative activity and in vitro inhibition of alpha-amylase, alpha-glucosidase, and angiotensin I-converting enzyme (ACE) relevant for potential management of hyperglycemia and hypertension. Therefore, a comparison among ellagic acid, purified ellagitannins, and a strawberry extract was done to evaluate the possible synergistic effects of phenolics. In relation to the antiproliferative activity, it was observed that ellagic acid had the highest percentage inhibition of cell proliferation. The strawberry extract had lower efficacy in inhibiting the cell proliferation, indicating that in the case of this fruit there is no synergism. Purified ellagitannins had high alpha-amylase and ACE inhibitory activities. However, these compounds had low alpha-glucosidase inhibitory activity. These results suggested that the ellagitannins and ellagic acid have good potential for the management of hyperglycemia and hypertension linked to type 2 diabetes. However, further studies with animal and human models are needed to advance the in vitro assay-based biochemical rationale from this study.

Evaluation of Indigenous Grains from the Peruvian Andean Region for Antidiabetes and Antihypertension Potential Using In Vitro Methods

The health-relevant functionality of 10 thermally processed Peruvian Andean grains (five cereals, three pseudocereals, and two legumes) was evaluated for potential type 2 diabetes-relevant antihyperglycemia and antihypertension activity using in vitro enzyme assays. Inhibition of enzymes relevant for managing early stages of type 2 diabetes such as hyperglycemia-relevant alpha-glucosidase and alpha-amylase and hypertension-relevant angiotensin I-converting enzyme (ACE) were assayed along with the total phenolic content, phenolic profiles, and antioxidant activity based on the 1,1-diphenyl-2-picrylhydrazyl radical assay. Purple corn (Zea mays L.) (cereal) exhibited high free radical scavenging-linked antioxidant activity (77%) and had the highest total phenolic content (8 +/- 1 mg of gallic acid equivalents/g of sample weight) and alpha-glucosidase inhibitory activity (51% at 5 mg of sample weight). The major phenolic compound in this cereal was protocatechuic acid (287 +/- 15 mu g/g of sample weight). Pseudocereals such as Quinoa (Chenopodium quinoa Willd) and Kaniwa (Chenopodium pallidicaule Aellen) were rich in quercetin derivatives (1,131 +/- 56 and 943 +/- 35 mu g [expressed as quercetin aglycone]/g of sample weight, respectively) and had the highest antioxidant activity (86% and 75%, respectively). Andean legumes (Lupinus mutabilis cultivars SLP-1 and H-6) inhibited significantly the hypertension-relevant ACE (52% at 5 mg of sample weight). No alpha-amylase inhibitory activity was found in any of the evaluated Andean grains. This in vitro study indicates the potential of combination of Andean whole grain cereals, pseudocereals, and legumes to develop effective dietary strategies for managing type 2 diabetes and associated hypertension and provides the rationale for animal and clinical studies.

Antidiabetes and antihypertension potential of commonly consumed carbohydrate sweeteners using in vitro models

Commonly consumed carbohydrate sweeteners derived from sugar cane, palm, and corn (syrups) were investigated to determine their potential to inhibit key enzymes relevant to Type 2 diabetes and hypertension based on the total phenolic content and antioxidant activity using in vitro models. Among sugar cane derivatives, brown sugars showed higher antidiabetes potential than white sugars; nevertheless, no angiotensin I-converting enzyme (ACE) inhibition was detected in both sugar classes. Brown sugar from Peru and Mauritius (dark muscovado) had the highest total phenolic content and 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, which correlated with a moderate inhibition of yeast alpha-glucosidase without showing a significant effect on porcine pancreatic alpha-amylase activity. In addition, chlorogenic acid quantified by high-performance liquid chromatography was detected in these sugars (128 +/- 6 and 144 +/- 2 mu g/g of sample weight, respectively). Date sugar exhibited high alpha-glucosidase, alpha-amylase, and ACE inhibitory activities that correlated with high total phenolic content and antioxidant activity. Neither phenolic compounds or antioxidant activity was detected in corn syrups, indicating that nonphenolic factors may be involved in their significant ability to inhibit alpha-glucosidase, alpha-amylase, and ACE. This study provides a strong biochemical rationale for further in vivo studies and useful information to make better dietary sweetener choices for Type 2 diabetes and hypertension management.

EVALUATION OF RED CURRANTS (RIBES RUBRUM L.), BLACK CURRANTS (RIBES NIGRUM L.), RED AND GREEN GOOSEBERRIES (RIBES UVA-CRISPA) FOR POTENTIAL MANAGEMENT OF TYPE 2 DIABETES AND HYPERTENSION USING IN VITRO MODELS

Red currants (Ribes rubrum L.), black currants (Ribes nigrum L.), red and green gooseberries (Ribes uva-crispa) were evaluated for the total phenolics, antioxidant capacity based on 2, 2-diphenyl-1-picrylhydrazyl radical scavenging assay and functionality such as in vitro inhibition of alpha-amylase, alpha-glucosidase and angiotensin I-converting enzyme (ACE) relevant for potential management of hyperglycemia and hypertension. The total phenolics content ranged from 3.2 (green gooseberries) to 13.5 (black currants) mg/g fruit fresh weight. No correlation was found between total phenolics and antioxidant activity. The major phenolic compounds were quercetin derivatives (black currants and green gooseberries) and chlorogenic acid (red currants and red gooseberries). Red currants had the highest alpha-glucosidase, alpha-amylase and ACE inhibitory activities. Therefore red currants could be good dietary sources with potential antidiabetes and antihypertension functionality to compliment overall dietary management of early stages of type 2 diabetes.