AT1 Receptor Expression Research Articles

Low shear stress induces endothelial reactive oxygen species via the AT1R/eNOS/NO pathway.

Reactive oxygen species (ROS) contribute to many aspects of physiological and pathological cardiovascular processes. However, the underlying mechanism of ROS induction by low shear stress (LSS) remains unclear. Accumulating evidence has shown that the angiotensin II type 1 receptor (AT1R) is involved in inflammation, apoptosis, and ROS production. Our aim was to explore the role of AT1R in LSS-mediated ROS induction. We exposed human umbilical vein endothelial cells (HUVECs) to LSS (3 dyn/cm2 ) for different periods of time. Western blotting and immunofluorescence showed that LSS significantly induced AT1R expression in a time-dependent manner. Using immunohistochemistry, we also noted a similar increase in AT1R expression in the inner curvature of the aortic arch compared to the descending aorta in C57BL/6 mice. Additionally, HUVECs were cultured with a fluorescent probe, either DCFH, DHE or DAF, after being subjected to LSS. Cell chemiluminescence and flow cytometry results revealed that LSS stimulated ROS levels and suppressed nitric oxide (NO) generation in a time-dependent manner, which was reversed by the AT1R antagonist Losartan. We also found that Losartan markedly increased endothelial NO synthase (eNOS) phosphorylation at Ser(633,1177) and dephosphorylation at Thr(495), which involved AKT and ERK. Moreover, the ROS level was significantly reduced by endogenous and exogenous NO donors (L-arginine, SNP) and increased by the eNOS inhibitor L-NAME. Overall, we conclude that LSS induces ROS via AT1R/eNOS/NO.

Angiotensins and Huntington's Disease: A Study on Immortalized Progenitor Striatal Cell Lines.

Neurons from mouse models of Huntington’s disease (HD) exhibit altered electrophysiological properties, potentially contributing to neuronal dysfunction and neurodegeneration. The renin–angiotensin system (RAS) is a potential contributor to the pathophysiology of neurodegenerative diseases. However, the role of angiotensin II (Ang II) and angiotensin (1-7) has not been characterized in HD. We investigated the influence of Ang II and angiotensin (1-7) on total potassium current using immortalized progenitor mutant huntingtin-expressing (Q111) and wild-type (Q7) cell lines. Measurements of potassium current were performed using the whole cell configuration of pCLAMP. The results showed that (1) the effect of Ang II administered to the bath caused a negligible effect on potassium current in mutant Q111 cells compared with wild-type Q7 cells and that intracellular administration of Ang II reduced the potassium current in wild type but not in mutant cells; (2) the small effect of Ang II was abolished by losartan; (3) intracellular administration of Ang II performed in mutant huntingtin-expressing Q111 cells revealed a negligible effect of the peptide on potassium current; (4) flow cytometer analysis indicated a low expression of Ang II AT1 receptors in mutant Q111 cells; (5) mutant huntingtin-expressing striatal cells are highly sensitive to Ang (1-7) and that the effect of Ang (1-7) is related to the activation of Mas receptors. In conclusion, mutant huntingtin-expressing cells showed a negligible effect of Ang II on potassium current, a result probably due to the reduced expression of AT1 receptors at the surface cell membrane. In contrast, administration of Ang (1-7) to the bath showed a significant decline of the potassium current in mutant cells, an effect dependent on the activation of Mas receptors. Ang II had an intracrine effect in wild-type cells and Ang (1-7) exerted a significant effect in mutant huntingtin-expressing striatal cells.

Treatment with enalapril and not diltiazem ameliorated progression of chronic kidney disease in rats, and normalized renal AT1 receptor expression as measured with PET imaging.

ACE inhibitors are considered first line of treatment in patients with many forms of chronic kidney disease (CKD). Other antihypertensives such as calcium channel blockers achieve similar therapeutic effectiveness in attenuating hypertension-related renal damage progression. Our objective was to explore the value of positron emission tomography (PET) imaging of renal AT1 receptor (AT1R) to guide therapy in the 5/6 subtotal-nephrectomy (Nx) rat model of CKD. Ten weeks after Nx, Sprague-Dawley rats were administered 10mg/kg/d enalapril (NxE), 30mg/kg/d diltiazem (NxD) or left untreated (Nx) for an additional 8–10 weeks. Kidney AT1R expression was assessed using in vivo [18F]fluoropyridine-losartan PET and in vitro autoradiography. Compared to shams, Nx rats exhibited higher systolic blood pressure that was reduced by both enalapril and diltiazem. At 18–20 weeks, plasma creatinine and albuminuria were significantly increased in Nx, reduced to sham levels in NxE, but enhanced in NxD rats. Enalapril treatment decreased kidney angiotensin II whereas diltiazem induced significant elevations in plasma and kidney levels. Reduced PET renal AT1R levels in Nx were normalized by enalapril but not diltiazem, and results were supported by autoradiography. Reduction of renal blood flow in Nx was restored by enalapril, while no difference was observed in myocardial blood flow amongst groups. Enhanced left ventricle mass in Nx was not reversed by enalapril but was augmented with diltiazem. Stroke volume was diminished in untreated Nx compared to shams and restored with both therapies. [18F]Fluoropyridine-Losartan PET allowed in vivo quantification of kidney AT1R changes associated with progression of CKD and with various pharmacotherapies.

AT1-receptor response to non-saturating Ang-II concentrations is amplified by calcium channel blockers

BackgroundBlockers of angiotensin II type 1 receptor (AT1R) and the voltage gated calcium channel 1.2 (CaV1.2) are commonly used for treatment of hypertension. Yet there is little information about the effect of physiological concentrations of angiotensin II (AngII) on AT1R signaling and whether there is a reciprocal regulation of AT1R signaling by CaV1.2.MethodsTo elucidate these questions, we have studied the Ca2+ signaling response to physiological and pharmacological AngII doses in HEK293a cells, vascular smooth muscle cells and cardiomyocytes using a Ca2+ sensitive dye as the principal sensor. Intra-cellular calcium recordings were performed in presence and absence of CaV1.2 blockers. Semi-quantitative imaging methods were used to assess the plasma membrane expression of AT1R and G-protein activation.ResultsRepeated exposure to pharmacological (100 nM) concentrations of AngII caused, as expected, a down-regulation of the Ca2+ response. In contrast, repeated exposure to physiological (1 nM) AngII concentration resulted in an enhancement of the Ca2+ response. The up-regulation of the Ca2+ response to repeated 1 nM AngII doses and the down-regulation of the Ca2+ response to repeated 100 nM Angll doses were not accompanied by a parallel change of the AT1R plasma membrane expression. The Ca2+ response to 1 nM of AngII was amplified in the presence of therapeutic concentrations of the CaV1.2 blockers, nifedipine and verapamil, in vascular smooth muscle cells, cardiomyocytes and HEK293a cells. Amplification of the AT1R response was also observed following inhibition of the calcium permeable transient receptor potential cation channels, suggesting that the activity of AT1R is sensitive to calcium influx.ConclusionsOur findings have implications for the understanding of hyperactivity of the angiotensin system and for use of Ca2+ channel blockers as mono-therapy in hypertension.

Angiotensin receptors in Dupuytren’s disease: a target for pharmacological treatment?

Background: Attempts at the pharmacological treatment of Dupuytren’s disease have so far been unsuccessful, and the disease is not yet fully understood on a cellular level. The Renin-Angiotensin System has long been understood to play a circulating hormonal role. However, there is much evidence showing Angiotensin II to play a local role in wound healing and fibrosis, with ACE inhibitors being widely used as an anti-fibrotic agent in renal and cardiac disease.Methods: This study was designed to investigate the presence of Angiotensin II receptors 1 (AT1) and 2 (AT2) in Dupuytren’s tissue to form a basis for further study into the pharmacological treatment of this condition. Tissue was harvested from 11 patients undergoing surgery for Dupuytren’s disease. Each specimen was processed into frozen sections and immunostaining was employed to identify AT1 and AT2 receptors.Results: Immunostaining for AT1 receptors was mildly positive in one patient and negative in all the remaining patients. However, all specimens stained extensively for AT2 receptors. This suggests that the expression of AT2 receptors is more prominent than AT1 receptors in Dupuytren’s disease.Conclusion: These findings have opened a new avenue for future research involving ACE inhibitors, AT2 agonists, and AT2 antagonists in Dupuytren’s disease.

Regulation of the angiotensin II-p22phox-reactive oxygen species signaling pathway, apoptosis and 8-oxoguanine-DNA glycosylase 1 retrieval in hyperoxia-induced lung injury and fibrosis in rats.

The present study was designed to explore the impact of hyperoxia on lung injury and fibrosis via the angiotensin II (AngII)-p22phox-reactive oxygen species (ROS) signaling pathway, apoptosis and 8-oxoguanine-DNA glycosylase 1 (OGG1) repair enzyme. Newborn Sprague-Dawley rats were randomly divided in the newborn air group, newborn hyperoxia group and newborn intervention group, the latter of which was administered the chymotrypsin inhibitor, 2-(5-formylamino-6-oxo-2-phenyl-1, 6-dihydropyrimidine-1-yl)-N-[4-dioxo-1-phenyl-7-(2-pyridyloxy)] 2-heptyl-acetamide (NK3201). A group of adult rats also received hyperoxic treatment. Histomorphological changes in lung tissues were dynamically observed. AngII, ROS, angiotensin type 1 receptor (AT1R) and p22phox messenger RNA (mRNA) levels, and OGG1 and peroxisome proliferator-activated receptor-γ (PPARγ) protein levels in the lung tissues were detected at various times after hyperoxia. Hyperoxia led to traumatic changes in the lungs of newborn rats that resulted in decreased viability, increased mortality, morphological changes and the apoptosis of alveolar type II epithelial cells (AT-II), as well as increased expression levels of AngII, AT1R and p22phox, which would ultimately lead to secondary diseases. NK3201 significantly inhibited the hyperoxia-induced increased expression of AngII, AT1R and p22phox and further promoted OGG1 and PPARγ protein expression, thus reducing the intrapulmonary ROS level, the apoptotic index and caspase-3 levels. However, the adult hyperoxia group only exhibited tachypnea and reduced viability. This study suggested that the AngII-p22phox-ROS signaling pathway, PPARγ and OGG1 together contributed to the hyperoxia-induced lung injury and that NK3201 was able to reverse the effects of hyperoxia.

Brain Renin-Angiotensin System and Microglial Polarization: Implications for Aging and Neurodegeneration.

Microglia can transform into proinflammatory/classically activated (M1) or anti-inflammatory/alternatively activated (M2) phenotypes following environmental signals related to physiological conditions or brain lesions. An adequate transition from the M1 (proinflammatory) to M2 (immunoregulatory) phenotype is necessary to counteract brain damage. Several factors involved in microglial polarization have already been identified. However, the effects of the brain renin-angiotensin system (RAS) on microglial polarization are less known. It is well known that there is a “classical” circulating RAS; however, a second RAS (local or tissue RAS) has been observed in many tissues, including brain. The locally formed angiotensin is involved in local pathological changes of these tissues and modulates immune cells, which are equipped with all the components of the RAS. There are also recent data showing that brain RAS plays a major role in microglial polarization. Level of microglial NADPH-oxidase (Nox) activation is a major regulator of the shift between M1/proinflammatory and M2/immunoregulatory microglial phenotypes so that Nox activation promotes the proinflammatory and inhibits the immunoregulatory phenotype. Angiotensin II (Ang II), via its type 1 receptor (AT1), is a major activator of the NADPH-oxidase complex, leading to pro-oxidative and pro-inflammatory effects. However, these effects are counteracted by a RAS opposite arm constituted by Angiotensin II/AT2 receptor signaling and Angiotensin 1–7/Mas receptor (MasR) signaling. In addition, activation of prorenin-renin receptors may contribute to activation of the proinflammatory phenotype. Aged brains showed upregulation of AT1 and downregulation of AT2 receptor expression, which may contribute to a pro-oxidative pro-inflammatory state and the increase in neuron vulnerability. Several recent studies have shown interactions between the brain RAS and different factors involved in microglial polarization, such as estrogens, Rho kinase (ROCK), insulin-like growth factor-1 (IGF-1), tumor necrosis factor α (TNF)-α, iron, peroxisome proliferator-activated receptor gamma, and toll-like receptors (TLRs). Metabolic reprogramming has recently been involved in the regulation of the neuroinflammatory response. Interestingly, we have recently observed a mitochondrial RAS, which is altered in aged brains. In conclusion, dysregulation of brain RAS plays a major role in aging-related changes and neurodegeneration by exacerbation of oxidative stress (OS) and neuroinflammation, which may be attenuated by pharmacological manipulation of RAS components.

Synthetic Receptors Induce Anti Angiogenic and Stress Signaling on Human First Trimester Cytotrophoblast Cells.

The cytotrophoblast (CTB) cells of the human placenta have membrane receptors that bind certain cardiotonic steroids (CTS) found in blood plasma. One of these, marinobufagenin, is a key factor in the etiology of preeclampsia. Herein, we used synthetic receptors (SR) to study their effectiveness on the angiogenic profile of human first trimester CTB cells. The humanextravillous CTB cells (Sw.71) used in this study were derived from first trimester chorionic villus tissue. Culture media of CTB cells treated with ≥1 nM SR level revealed sFlt-1 (Soluble fms-like tyrosine kinase-1) was significantly increased while VEGF (vascular endothelial growth factor) was significantly decreased in the culture media (* p < 0.05 for each) The AT2 receptor (Angiotensin II receptor type 2) expression was significantly upregulated in ≥1 nM SR-treated CTB cells as compared to basal; however, the AT1 (Angiotensin II receptor, type 1) and VEGFR-1 (vascular endothelial growth factor receptor 1) receptor expression was significantly downregulated (* p < 0.05 for each). Our results show that the anti-proliferative and anti-angiogenic effects of SR on CTB cells are similar to the effects of CTS. The observed anti angiogenic activity of SR on CTB cells demonstrates that the functionalized-urea/thiourea molecules may be useful as potent inhibitors to prevent CTS-induced impairment of CTB cells.

Sex differences in molecular mechanisms of vascular aging

Hypertension (HTN) is the most prevalent cardiovascular disease risk factor. Although HTN incidence rises profoundly with age in both sexes, females are relatively protected when they are young and after menopause, females ultimately exceed males in the incidence and adverse outcomes of HTN. In male mice with SMC‐specific deletion of the mineralocorticoid receptor (MR‐KO), we showed protection from the aging‐associated rise in blood pressure (BP). We show that in males, the mechanism involves SMC‐MR transcriptionally repressing vascular miR‐155 expression with age, which then promotes an increase in expression of miR‐155 target genes, Cav1.2, the pore‐forming subunit of the L‐type calcium channel (LTCC) and the angiotensin II type 1 receptor (AT1R). Further studies revealed that restoration of SMC‐specific miR‐155 rescues the aging phenotype observed in male mice. Specifically, raising SMC‐miR‐155 expression resulted in decreased Cav1.2 and AT1R expression and LTCC‐and AT1R‐mediated vasoconstriction. In female mice, however, Cav1.2 and AT1R expression did not change with age. Additionally, vasoconstriction to LTCC agonist BayK‐8644 and Ang II are not increased in aging females compared to young females. These results suggest that mechanism of vascular aging and BP control are distinct between males and females. These data support that sex‐specific therapies may be beneficial in managing vascular aging and BP control.Support or Funding InformationSupported by HL095590‐05, HL119290, and 15POST21300000.

Cardiovascular Protective Effects of AT2R Activation by Peptide Drug NP‐6A4

Introduction and ObjectiveLiterature shows that elevated Angiotensin II Type 2 receptor (AT2R) expression is cardiovascular reparative and mediates protection from hypertension, as well as vascular and renal injury. AT2R is a member of the anti‐inflammatory branch of the renin‐angiotensin system (RAS). Clinically, loss of AT2R expression in men due to the intronic G1675A or A1818T polymorphism is associated with impaired kidney function, pulse pressure and increased arterial stiffness. Given the pathological consequences of the loss of AT2R expression there is need for a drug that can increase AT2R gene (Agtr2) expression to mitigate cardiovascular damage. Currently there are no FDA‐approved drugs that meet these parameters. NP‐6A4 is a peptide agonist for AT2R, recently developed by Novopyxis Inc. We have shown that NP‐6A4 increases the viability of nutrient‐stressed mouse and human cardiovascular cells. Chemotherapeutic agents such as anthracyclins are inducers of cardiotoxicity. Therefore we investigated whether NP‐6A4 can protect cardiac cells from Doxorubicin (Dox; an anthracyclin) induced toxicity. To test the translational potential of a treatment paradigm that utilizes NP‐6A4 to improve cardiac functions and mitigate cardiac structural damage, we investigated the effects of NP‐6A4 treatment in Male Zucker Obese (ZO‐M) rats, a model for heart failure with preserved ejection fraction and cardiomyopathy.MethodsMTS assay (BioVision) was used to determine the effect of Dox treatment (0.5–2 μM) with and without co‐treatment with NP‐6A4 (1μM) on the survival of H9c2 cardiomyoblasts. To evaluate the effect of NP‐6A4 on cardiac structure and function, we treated 11‐week‐old ZO‐M rats that had fully developed cardiovascular disease with NP‐6A4 (1.8mg/kg/day by subcutaneous delivery). Echocardiography was used to assess cardiac function parameters. Cardiac architecture, hypertrophy, capillary density and fibrosis were assessed by staining using hematoxylin and eosin, Helix pomatia agglutinin (HPA), isolectin B4 (IB4) and Picrosirius Red (PSR) respectively. Expression levels of AT2R in the heart were assessed by quantitative RT‐PCR.Results24‐hour Dox treatment of H9c2 cardiomyoblasts suppressed cell viability (78% for 2μM vs untreated) and co‐treatment with NP‐6A4 restored cell survival. NP‐6A4 improved several cardiac parameters including endocardial circumferential strain (p≤0.05), myocardial performance index (MPI) (p≤0.005), and E/E' ratio (p≤0.002). NP‐6A4 reduced cardiomyocyte hypertrophy and fibrosis, and increased capillary density (p≤0.05). Importantly, cardiac Agtr2 (AT2R) mRNA levels were increased by about 9‐fold in NP‐6A4 treated rats vs. saline treated rats. Histopathology staining revealed regions of micro‐infarcts present in the saline group which were largely absent in the NP‐6A4 treated animals.ConclusionNP‐6A4 could mitigate Dox‐induced cardiac cell toxicity in vitro. NP‐6A4 also improved cardiac functions in a murine model of heart failure with preserved ejection fraction and cardiomyopathy, decreased risk of cardiac event (as measured by E/E'), mitigated myocardial structural damage, and improved cardiac vascularization. To our knowledge, NP‐6A4 is the first AT2R agonist that can increase cardiac AT2R expression. Based on these observations, we propose that this therapeutic candidate has the potential to treat chemotherapy‐induced cardiotoxicity as well as heart failure.Support or Funding InformationThis work was supported by NIH grant 1R01HL118376‐01 (LP), a small grant from Novopyxis Inc (LP), and Research Services facilities of HSTMVH.

Inhibitory Effects of Single Component Isolated in Natural Product Complex (No‐ap) on the AT1 Receptor Expression and Oxidative Stress in Angiotensin II‐Stimulated Cells

Inhibitory Effects of Single Component Isolated in Natural Product Complex (No‐ap) on the AT1 Receptor Expression and Oxidative Stress in Angiotensin II‐Stimulated Cells

Treatment with Captopril Enhances the Vascular Relaxation Induced by the Angiotensin II Type 2 Receptor (AT2R) Agonist CGP42112 in Diabetic Rats

The role of the AT2 receptor (AT2R) in physiological and pathophysiological processes is still controversial. Some studies suggest that expression of AT2R increases in certain diseases like diabetes mellitus. This could have beneficial effects as signaling through the AT2 receptor has been shown to counter the effects of angiotensin II mediated through the AT1 receptor, which are believed to contribute to pathological changes; this is supported by the favorable effects of ACE inhibitors and Angiotensin receptor blockers on the course of diabetes induced complications in renal function. We examined the effect of diabetes and of treatment with captopril on the vascular responses to CGP42112 (CGP), an AT2R selective agonist, in Sprague Dawley rats with streptozotocin (85 mg/kg i.v.) induced diabetes. Rats were followed for either 2, 4 or 12 weeks, at the end of which period thoracic aortas were isolated and used for measurement of relaxant responses to CGP42112 after pre‐contraction with phenylephrine. In a subset of the 4‐week study, osmotic pumps were implanted at week 2 to deliver either captopril (50 mg/kg/day) or losartan (5 mg/kg/day) s.c. for 2 weeks. All aortic ring studies were conducted in the presence of losartan (100 μM) to prevent interactions of CGP with the AT1R. CGP induced a mild (10%) relaxation in aortic rings at 2 weeks, 4 weeks and 12 weeks in the control animals. The response to CGP in aortic rings from diabetic rats was not different from controls. Treatment with losartan for 2 weeks did not alter the responses to CGP observed after 4 weeks. However, treatment with captopril for 2 weeks resulted in a markedly exaggerated relaxant response to CGP reaching up to 50% relaxation. These preliminary results suggest that while diabetes, per se, may not alter responses to CGP, treatment of diabetic rats (but not control rats) with captopril produces a marked increase in the sensitivity of the AT2R. Considering the known anti‐proliferative and anti‐ischemic effects of AT2R activation, these results potentially imply that the use of ACE inhibitors together with AT2R agonists could amplify the protective effect of ACE inhibitors against diabetes induced complications. Thus, the pathophysiological and therapeutic implications of this finding require further study.Support or Funding InformationSupported by funds from the Medical Practice Plan, AUB Faculty of Medicine

Angiotensin II promotes the anticoagulant effects of rivaroxaban via angiotensin type 2 receptor signaling in mice

Rivaroxaban is an oral direct factor Xa inhibitor approved for the treatment of stroke and systemic thromboembolism in patients with non-valvular atrial fibrillation. Despite its efficacy, rivaroxaban therapy results in adverse effects and complications, such as bleeding. Angiotensin II (AngII) is implicated in many cardiovascular conditions, such as hypertension and heart failure. In this study, we investigate whether AngII influences anticoagulant effects of rivaroxaban by using an experimental mouse model with type 2 diabetes mellitus and advanced glycation end product (AGE)-exposed human umbilical vein endothelial cells (HUVECs). We found that AngII promoted the anticoagulant effects of rivaroxaban in KKAy mice. The combination of rivaroxaban and AngII enhanced in vivo tissue factor pathway inhibitor (TFPI) activity and induced TFPI expression and activity in AGE-exposed HUVECs. Angiotensin type 2 receptor (AT2R) and Mas antagonists attenuated the AngII-enhanced anticoagulant action of rivaroxaban in vivo, and abolished the increased endothelial TFPI expression and activity. However, angiotensin type 1 receptor (AT1R) antagonist exerted no effects. Additionally, combination of rivaroxaban and AngII induced aortic AT2R and Mas expression. Our data suggest that the anticoagulant effects of rivaroxaban are promoted by AngII via AT2R and Mas signaling. These findings are significant for the clinical administration of rivaroxaban.

Local renin-angiotensin system regulates the differentiation of mesenchymal stem cells into insulin-producing cells through angiotensin type 2 receptor

Differentiation of stem cells into insulin-producing cells (IPCs) suitable for therapeutic transplantation offers a desperately needed approach for the diabetic patients. Elucidation of the molecular mechanisms during the differentiation of mesenchymal stem cells (MSCs) into IPCs assists the successful production of IPCs and provides an important insight into the improvement of the role of MSCs as a therapeutic tool for diabetes mellitus (DM). The present study aimed to investigate the role of local renin-angiotensin system (RAS) on MSCs differentiation into IPCs by measuring the expression of local RAS in MSCs during the differentiation into IPCs and assessing the effect of angiotensin type 1 receptor (AT1R) blocker and angiotensin type 2 receptor (AT2R) blocker on the differentiation process. Our data showed that the differentiation of MSCs into IPCs was associated with an increase in cellular angiotensinogen, angiotensin-converting enzyme (ACE), renin, and AT2R expression and undetectable expression of AT1R. The net effect was an increase in cellular angiotensin II (Ang II) during the differentiation process. AT1R blockade allowed the differentiation of MSCs into IPCs, whereas AT2R blockade alone and blockade of both AT1R and AT2R inhibited the differentiation of MSCs into IPCs. Our data demonstrated an important role of local RAS in the regulation of MSCs differentiation into IPCs and that Ang II mainly orchestrates this role through AT2R activation.

Effects of moderate‑intensity endurance exercise on angiotensin II and angiotensin II type I receptors in the rat heart.

The current study was aimed at examining the effects of moderate-intensity endurance exercise on the expression of angiotensin II (AngII) and AngII type 1 receptor (AT1R) in the rat heart. Male Sprague-Dawley rats were randomly divided into the control group (n=20) and moderate-intensity endurance exercise group (n=20). Cardiac hypertrophy was induced by treadmill endurance training for 8 weeks. The mRNA expression of AngII and AT1R were assessed by reverse transcription-quantitative polymerase chain reaction. The immune response positive area and optical density of AngII and AT1R was measured by immunohistochemistry. AngII was primarily expressed in the cytoplasm and membrane, however infrequently in coronary vascular wall smooth muscle cells. AT1R was primarily expressed in the coronary vessel wall smooth muscle, rarely in cardiac cells. The mRNA expression of cardiac AngII was significantly increased after the 8-week exercise period, while AT1R was significantly decreased. Immunohistochemistry indicated a significant increase in the AngII immune-positive area and optical density after the 8-week exercise. The AT1R immune-positive area and optical density was significantly reduced following the 8-week exercise. In conclusion, subsequent to 8-weeks endurance training, the AngII expression was increased and the AT1R expression was decreased. AT1R may expand the coronary artery, thereby increasing coronary blood flow and ensuring the energy supply of heart during exercise. The expression change in AngII does not reflect the character of cardiac hypertrophy. The exercise-induced change in the expression of AngII and AT1R may be a protective mechanism to avoid cardiac pathological hypertrophy.

Dopamine modulates astroglial and microglial activity via glial renin-angiotensin system in cultures

Dopamine is an immunomodulatory molecule that acts on immune effector cells both in the CNS and peripheral tissues. However, the role of changes in dopamine levels in the neuroinflammatory response is controversial. The local/paracrine renin-angiotensin system (RAS) plays a major role in inflammatory processes in peripheral tissues and brain. In the present study, we investigated the possible role of the brain RAS in the effects of dopamine on the glial inflammatory responses. Astrocytes are the major source of the precursor protein angiotensinogen and angiotensin II (AII) in the brain. Neurotoxins such as MPP+ (1-methyl-4-phenylpyridinium) can act directly on astrocytes to increase levels of angiotensinogen and AII. Conversely, dopamine, via type-2 (D2) receptors, inhibited production of angiotensinogen, decreased expression of angiotensin type-1 (AT1) receptors and increased expression of AT2 receptors. In microglia, dopamine and dopamine agonists also regulated RAS activity. First, indirectly, via downregulation of the astrocyte-derived AII. Second, via dopamine-induced regulation of microglial angiotensin receptors. Dopamine decreased the microglial AT1/AT2 ratio leading to inhibition of the pro-inflammatory AT1/NADPH-oxidase/superoxide axis. D2 receptors were particularly responsible for microglial RAS inhibition in basal culture conditions. However, both D1 and D2 agonists inhibited the AT1/NADPH-oxidase axis in lipopolysaccharide-treated (LPS; i.e. activated) microglia. The results indicate that the decrease in dopamine levels observed in early stages of Parkinson’s disease and aging may promote neuroinflammation and disease progression via glial RAS exacerbation.

Angiotensin II dependent cardiac remodeling in the eel Anguilla anguilla involves the NOS/NO system

Angiotensin II (AngII), the principal effector of the Renin-Angiotensin System (RAS), plays an important role in controlling mammalian cardiac morpho-functional remodelling. In the eel Anguilla anguilla, one month administration of AngII improves cardiac performance and influences the expression and localization of molecules which regulate cell growth.To deeper investigate the morpho-functional chronic influences of AngII on the eel heart and the molecular mechanisms involved, freshwater eels (A. anguilla) were intraperitoneally injected for 2 months with AngII (1 nmol g BW−1). Then the isolated hearts were subjected to morphological and western blotting analyses, and nitrite measurements.If compared to control animals, the ventricle of AngII-treated hearts showed an increase in compacta thickness, vascularization, muscle mass and fibrosis. Structural changes were paralleled by a higher expression of AT2 receptor and a negative modulation of the ERK1-2 pathway, together with a decrease in nitrite concentration, indicative of a reduced Nitric Oxide Synthase (NOS)-dependent NO production. Moreover, immunolocalization revealed, particularly on the endocardial endothelium (EE) of AngII-treated hearts, a significant reduction of phosphorylated NOS detected by peNOS antibody accompanied by an increased expression of the eNOS disabling protein NOSTRIN, and a decreased expression of the positive regulators of NOS activity, pAkt and Hsp90. On the whole, results suggest that, in the eel, AngII modulates cardiac morpho-functional plasticity by influencing the molecular mechanisms that control NOS activity and the ERK1-2 pathway.

Administration of anabolic steroid during adolescence induces long-term cardiac hypertrophy and increases susceptibility to ischemia/reperfusion injury in adult Wistar rats

Chronic administration of anabolic androgenic steroids (AAS) in adult rats results in cardiac hypertrophy and increased susceptibility to myocardial ischemia/reperfusion (IR) injury. Molecular analyses demonstrated that hyperactivation of type 1 angiotensin II (AT1) receptor mediates cardiac hypertrophy induced by AAS and also induces down-regulation of myocardial ATP-sensitive potassium channel (KATP), resulting in loss of exercise-induced cardioprotection. Exposure to AAS during adolescence promoted long-term cardiovascular dysfunctions, such as dysautonomia. We tested the hypothesis that chronic AAS exposure in the pre/pubertal phase increases the susceptibility to myocardial ischemia/reperfusion (IR) injury in adult rats. Male Wistar rats (26day old) were treated with vehicle (Control, n=12) or testosterone propionate (TP) (AAS, 5mgkg−1 n=12) 5 times/week during 5 weeks. At the end of AAS exposure, rats underwent 23days of washout period and were submitted to euthanasia. Langendorff-perfused hearts were submitted to IR injury and evaluated for mechanical dysfunctions and infarct size. Molecular analysis was performed by mRNA levels of α-myosin heavy chain (MHC), βMHC and brain-derived natriuretic peptide (BNP), ryanodine receptor (RyR2) and sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) by quantitative RT-PCR (qRT-PCR). The expression of AT1 receptor and KATP channel subunits (Kir6.1 and SURa) was analyzed by qRT-PCR and Western Blot. NADPH oxidase (Nox)-related reactive oxygen species generation was assessed by spectrofluorimetry. The expression of antioxidant enzymes was measured by qRT-PCR in order to address a potential role of redox unbalance. AAS exposure promoted long-term cardiac hypertrophy characterized by increased expression of βMHC and βMHC/αMHC ratio. Baseline derivative of pressure (dP/dt) was impaired by AAS exposure. Postischemic recovery of mechanical properties was impaired (decreased left ventricle [LV] developed pressure and maximal dP/dt; increased LV end-diastolic pressure and minimal dP/dt) and infarct size was larger in the AAS group. Catalase mRNA expression was significantly decreased in the AAS group. In conclusion, chronic administration of AAS during adolescence promoted long-term pathological cardiac hypertrophy and persistent increase in the susceptibility to myocardial IR injury possible due to disturbances on catalase expression.

Early treatment with losartan effectively ameliorates hypertension and improves vascular remodeling and function in a prehypertensive rat model

AimsPharmacological treatment of prehypertension may ameliorate hypertension and improve vascular structure and function. This study investigated 1) whether early treatment with either losartan or amlodipine at the onset of prehypertension can prevent hypertension and 2) whether losartan and amlodipine equally improve vascular remodeling and function in a rat model of hypertension. Materials and methodsStroke-prone spontaneously hypertensive (SHRSP) rats were administered losartan, amlodipine or saline for 6 or 16weeks at the onset of prehypertension. Wistar-Kyoto rats were used as a control. All groups were observed for 40weeks. Systolic blood pressure was measured using the tail-cuff method. Vascular structure and function were determined by microscopy and vascular ring contractility assays, respectively. Angiotensin II (Ang II) and aldosterone (Aldo) were measured by radioimmunoassays. Angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) expression was measured by western blot. Key findingsLosartan effectively reduced progression from prehypertension to hypertension as well as vascular remodeling and improved vascular contractility in SHRSP rats. Long-term losartan (16weeks) had greater benefits than short-term (6weeks) treatment. Losartan increased Ang II and decreased Aldo levels in the serum and vessel walls of resistance vessels in a time-dependent manner. Losartan significantly decreased AT1R and increased AT2R vascular expression. Amlodipine had no effect on vascular AT1R and AT2R expression. SignificanceLosartan administered at the onset of prehypertension is more effective than amlodipine in ameliorating hypertension and improving vascular remodeling and function, which is likely mediated by the renin–angiotensin–aldosterone system.

Heavy Alcohol Consumption Effects on Blood Pressure and on Kidney Structure Persist After Long-Term Withdrawal

Background/Aims: Heavy ethanol consumption is a risk factor for hypertension and prompts organ damage. There is no information regarding the impact of long-term heavy ethanol consumption on kidney structure and function linking to their hypertensive effects nor the repercussions after withdrawal. Methods: Rats were exposed to ethanol for 24 weeks and, afterwards, a group was assigned to withdrawal for 8 weeks. Blood pressure (BP) was measured and serum biochemical parameters were quantified. Glomerular volume density, areal density of glomerular tuft and renal corpuscles were determined. Angiotensin II type 1 receptor (AT1R) protein expression was evaluated. Results: Twenty-four weeks of ethanol consumption causes atrophy of renal corpuscles and glomeruli and reduces the volume of glomeruli. Glomerular changes induced by ethanol consumption were still evident after withdrawal. Renal AT1R levels were increased in ethanol-treated rats and returned to control levels during withdrawal. Ethanol consumption also induced an increase in BP, uric acid and albumin levels. Upon withdrawal, systolic and mean arterial pressures decreased, but were still higher than in controls rats. Conclusion: Ethanol consumption induces changes in glomerular morphology associated with increased BP and AT1R expression. Long-term withdrawal was inefficient to restore the structural integrity of renal corpuscles and in lowering systolic pressure.