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Background: Pulmonary arterial hypertension (PAH) is related to inflammation, and the lncRNA H19 is associated with inflammation. However, whether PDGF-BB-H19-let-7b-AT 1 R axis contributes to the pathogenesis of PAH has not been thoroughly elucidated to date. This study investigated the role of H19 in PAH and its related mechanism., Methods: In the present study, SD rats, C57/BL6 mice and H19-/- mice were injected with monocrotaline (MCT) to establish a PAH model. H19 was detected in the cytokine-stimulated pulmonary arterial smooth muscle cells (PASMCs), serum and lungs of rats/mice. H19 overexpression and knockdown experiments were also conducted. A dual luciferase reporter assay was used to explore whether let-7b is a sponge miRNA of H19, and AT 1 R is a novel target of let-7b. A CCK-8 assay and flow cytometry were used to analyse cell proliferation., Results: The results showed that H19 was highly expressed in the serum and lungs of MCT-induced rats/mice, and H19 was upregulated by PDGF-BB in vitro. H19 upregulated AT 1 R expression via sponging miRNA let-7b following PDGF-BB stimulation. AT 1 R is a novel target of let-7b. Moreover, the overexpression of H19 and AT 1 R could facilitate PASMCs proliferation in vitro. H19 knockout protected mice from pulmonary artery remodeling and PAH following MCT treatment., Conclusion: Our study showed that H19 is highly expressed in MCT-induced rodent lungs and upregulated by PDGF-BB. The H19-let-7b-AT 1 R axis contributed to the pathogenesis of PAH by stimulating PASMCs proliferation. The H19 knockout had a protective role in the development of PAH. H19 may be a potential tar-get for the treatment of PAH.

Objective: The current study considers changes of the postnatal brainstem cell number and angiotensin receptors by maternal protein restriction (LP) and LP taurine supplementation (LPT), and its impact on arterial hypertension development in adult life., Methods and Results: The brain tissue studies were performed by immunoblotting, immunohistochemistry, and isotropic fractionator analysis. The current study shows that elevated blood pressure associated with decreased fractional urinary sodium excretion (FENa) in adult LP offspring was reverted by diet taurine supplementation. Also, that 12-day-old LP pups present a reduction of 21% of brainstem neuron counts, and, immunohistochemistry demonstrates a decreased expression of type 1 angiotensin II receptors (AT1R) in the entire medial solitary tract nuclei (nTS) of 16-week-old LP rats compared to age-matched NP and LPT offspring. Conversely, the immunostained type 2 AngII (AT2R) receptors in 16-week-old LP nTS were unchanged., Conclusion: The present investigation shows a decreased FENa that occurs despite unchanged creatinine clearance. It is plausible to hypothesize an association of decreased postnatal nTS cell number, AT1R/AT2R ratio and FENa with the higher blood pressure levels found in taurine-deficient progeny (LP) compared with age-matched NP and LPT offspring., (© The Author(s) 2013.)

Renin-Angiotensin System (RAS) plays an important role in the development of Metabolic Syndrome (MS) and in aging. Angiotensin 1-7 (Ang 1-7) has opposite effects to Ang II. All of the components of RAS are expressed locally in adipose tissue and there is over-activation of adipose RAS in obesity and hypertension. We determined serum and abdominal adipose tissue Ang II and Ang 1-7 in control and MS rats during aging and the expression of AT1, AT2 and Mas in white adipose tissue. MS was induced by sucrose ingestion during 6, 12 and 18 months. During aging, an increase in body weight, abdominal fat and dyslipidemia were found but increases in aging MS rats were higher. Control and MS concentrations of serum Ang II from 6-month old rats were similar. Aging did not modify Ang II seric concentration in control rats but decreased it in MS rats. Ang II levels increased in WAT from both groups of rats. Serum and adipose tissue Ang 1-7 increased during aging in MS rats. Western blot analysis revealed that AT1 expression increased in the control group during aging while AT2 and Mas remained unchanged. In MS rats, AT1 and AT2 expression decreased significantly in aged rats. The high concentration of Ang 1-7 and adiponectin in old MS rats might be associated to an increased expression of PPAR-γ. PPAR-γ was increased in adipose tissue from MS rats. It decreased with aging in control rats and showed no changes during aging in MS rats. Ang 1-7/Mas axis was the predominant pathway in WAT from old MS animals and could represent a potential target for therapeutical strategies in the treatment of MS during aging., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Two G-protein-coupled receptors, angiotensin type 1 receptor (AT1R) and type 2 receptor (AT2R), mediate the majority of angiotensin II (Ang II) effects. It has long been believed that the AT2R is reduced to extremely low levels in adulthood but is abundantly expressed in the fetus. However, recent data from rats and mice do not support this notion. Employing Western blot analysis, we found a gradual increase in AT2R protein expression from fetal life to adulthood in brain, heart, lung, liver, and kidney. The reason for the discrepancy between our observations and the conventional concept is unknown. Evidence supporting a regression of the AT2R during maturation is derived largely from autoradiographic signals that were confined to the outline of fetal rats, implying that the previously reported high AT2R expression in the fetus might be valid only for skin. We therefore hypothesized that the ontogeny of angiotensin receptors in skin is opposite that in other organs. In the current experiment, we employed Western blot analysis to compare AT2R and AT1R protein expressions in skin with that in the brainstem of fetal and adult mice. Fetal brainstem expressed lower AT2R and higher AT1R protein compared to adult brainstem (AT2R/GAPDH: 0.08 ± 0.2 in fetus vs 7.6 ± 0.9 in adult, p < 0.05; AT1R/GAPDH: 5.9 ± 0.8 in fetus vs 0.7 ± 0.3 in adult, p < 0.05). In contrast, fetal skin expresses higher AT2R and lower AT1R protein compared to adult skin (AT2R/GAPDH: 0.95 ± 0.4 in fetus vs 0.04 ± 0.2 in adult, p < 0.05; AT1R/GAPDH: 0.6 ± 0.2 in fetus vs 2.1 ± 0.5 in adult, p < 0.05). This expression profile was also confirmed by immunofluorescence. Using real-time RT-PCR, we could not detect any significant differences in mRNA of these two receptors in brainstem between fetal and adult mice. However, we found a higher AT2R mRNA and lower AT1R mRNA in the fetal skin compared to adult skin. The above results confirm our hypothesis that skin displays an expression profile of angiotensin receptors opposite that of other organs during development, suggesting that the currently held view concerning AT2R maturation is suitable only for skin. In other organs and tissues, adults express higher AT2R protein than does the fetus., (© The Author(s) 2014.)

To investigate the effects of losartan and amlodipine on cell apoptosis in the cerebral cortex of stroke-prone spontaneously hypertensive rats (SHRSP) from the onset of prehypertension or hypertension. SHRSP were randomly divided into five experimental groups that were administered losartan, amlodipine (n=8 in each group; 4 weeks old or 10 weeks old), or vehicle, respectively. Wistar-Kyoto rats were used as control animals. Systolic blood pressure was measured using the tail-cuff method every 2 weeks. At 20 weeks of age, apoptosis was analyzed by TdT-mediated dUTP-biotin nick end labeling, and the level of angiotensin II was measured by radioimmunoassay. Protein expressions of gp91(phox), superoxide dismutase, and angiotensin II type 1 (AT1R) and type 2 (AT2R) receptors in the cerebral cortex were detected by western blot. Losartan and amlodipine effectively delayed the progression of systolic blood pressure elevation, especially from the onset of prehypertension, and they had no obvious effects on the level of angiotensin II. In addition, treatment with losartan or amlodipine significantly decreased cell apoptosis, downregulated the protein expression of gp91(phox), and upregulated the protein expression of superoxide dismutase. The protein expressions of AT1R and AT2R were decreased by the administration of both drugs. No difference was found in the expression of AT1R among the drug treatment groups, whereas the expression of AT2R was increased in rats with increased blood pressure. Amlodipine, especially from the onset of prehypertension, was more effective than losartan in reducing apoptosis in the cerebral cortex in SHRSP. This may be related to the antioxidative stress properties of amlodipine.

Background and Objectives: The renin-angiotensin system (RAS) is considered as a hormonal circulatory system involved in maintaining blood pressure, electrolyte and fluid homeostasis. RAS components can be synthesized in local tissues and are found to play a role in gingival overgrowth. The drug-induced gingival overgrowth (DIGO) is a fibrotic condition, which is associated with multiple factors, including inflammation and adverse drug effects such as cyclosporine A. This study was directed forward to the identification of the angiotensinogen, angiotensin II (Ang II) and its receptors AT₁ /AT₂ expression in DIGO tissues and cyclosporine-treated human gingival fibroblast cells., Material and Methods: Gingival samples were obtained from patients with cyclosporine-induced gingival overgrowth, chronic periodontitis and normal healthy subjects. The total RNA was isolated and reverse transcription-polymerase chain reaction was performed for angiotensinogen, Ang II and AT₁ /AT₂ receptor. Ang II protein was estimated from tissue by enzyme immunoassay. The expression of Ang II and its receptors were also examined in gingival fibroblast cells treated with cyclosporine., Results: Ang II mRNA and protein expression was significantly higher in patients with DIGO than in patients with periodontitis and healthy subjects. The AT₁ mRNA was expressed more than AT₂ in all examined tissues. In gingival fibroblasts, Ang II and AT₁ expressions were increased with cyclosporine incorporation compared to controls., Conclusion: These results suggest that cyclosporine can modulate local expression of RAS components such as angiotensinogen, Ang II and its receptors in gingival tissues and gingival fibroblast cells., (© 2012 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Aim: Living high training low" (LHTL) is an exercise-training protocol that refers living in hypoxia stress and training at normal level of O2. In this study, we investigated whether LHTL caused physiological heart hypertrophy accompanied by changes of biomarkers in renin-angiotensin system in rats., Methods: Adult male SD rats were randomly assigned into 4 groups, and trained on living low-sedentary (LLS, control), living low-training low (LLTL), living high-sedentary (LHS) and living high-training low (LHTL) protocols, respectively, for 4 weeks. Hematological parameters, hemodynamic measurement, heart hypertrophy and plasma angiotensin II (Ang II) level of the rats were measured. The gene and protein expression of angiotensin-converting enzyme (ACE), angiotensinogen (AGT) and angiotensin II receptor I (AT1) in heart tissue was assessed using RT-PCR and immunohistochemistry, respectively., Results: LLTL, LHS and LHTL significantly improved cardiac function, increased hemoglobin concentration and RBC. At the molecular level, LLTL, LHS and LHTL significantly decreased the expression of ACE, AGT and AT1 genes, but increased the expression of ACE and AT1 proteins in heart tissue. Moreover, ACE and AT1 protein expression was significantly increased in the endocardium, but unchanged in the epicardium., Conclusion: LHTL training protocol suppresses ACE, AGT and AT1 gene expression in heart tissue, but increases ACE and AT1 protein expression specifically in the endocardium, suggesting that the physiological heart hypertrophy induced by LHTL is regulated by region-specific expression of renin-angiotensin system components.

Chronic intermittent hypoxia (CIH) raises arterial pressure, impairs vasodilator responsiveness, and increases circulating angiotensin II (Ang II); however, the role of Ang II in CIH-induced vascular dysfunction is unknown. Rats were exposed to CIH or room air (NORM), and a subset of these animals was treated with losartan (Los) during the exposure period. After 28 days, vasodilatory responses to acetylcholine or nitroprusside were measured in isolated gracilis arteries. Superoxide levels and Ang II receptor protein expression were measured in saphenous arteries. After 28 days, arterial pressure was increased and acetylcholine-induced vasodilation was blunted in CIH vs. NORM, and this was prevented by Los. Responses to nitroprusside and superoxide levels did not differ between CIH and NORM. Expression of AT(2)R was decreased and the AT(1)R:AT(2)R ratio was increased in CIH vs. NORM, but this was unaffected by Los. These results indicate that the blood pressure elevation and endothelial dysfunction associated with CIH is dependent, at least in part, on RAS signaling., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Angiotensin-converting enzyme 2 (ACE2) has been suggested to be involved in the central regulation of autonomic function. During chronic heart failure (CHF), elevated central angiotensin II signaling contributes to the sustained increase of sympathetic outflow. This is accompanied by a downregulation of ACE2 in the brain. We hypothesized that central overexpression of ACE2 decreases sympathetic outflow and enhances baroreflex function in CHF. Transgenic mice overexpressing human ACE2 selectively in the brain (SYN-hACE2 [SA]) and wild-type littermates (WT) were used. CHF was induced by permanent coronary artery ligation. Four weeks after coronary artery ligation, both WT and SA mice exhibited a significant decrease in left ventricular ejection fraction (<40%). A slight decrease in mean arterial pressure was found only in SA mice. Compared with WT mice with CHF, brain-selective ACE2 overexpression attenuated left ventricular end-diastolic pressure; decreased urinary norepinephrine excretion; baseline renal sympathetic nerve activity (WT CHF: 71.6±7.6% max versus SA CHF: 49.3±6.1% max); and enhanced baroreflex sensitivity (maximum slope: WT sham: 1.61±0.16%/mm Hg versus SA CHF: 1.51±0.17%/mm Hg). Chronic subcutaneous blockade of mas receptor increased renal sympathetic nerve activity in SA mice with CHF (A779: 67.3±5.8% versus vehicle: 46.4±3.6% of max). An upregulation in angiotensin II type 1 receptor expression was detected in medullary nuclei in WT CHF mice, which was significantly attenuated in SA mice with CHF. These data suggest that central ACE2 overexpression exerts a potential protective effect in CHF through attenuating sympathetic outflow. The mechanism for this effect involves angiotensin (1-7) mas signaling, as well as a decrease in angiotensin II type 1 receptor signaling in the medulla.

Objective: Visfatin is a newly identified proinflammatory adipocytokine whose plasma levels have been reported to be higher in subjects with type 2 diabetes mellitus. Recent studies have shown that visfatin increases the synthesis of profibrotic molecules in mesangial cells (MCs) and thus plays an important role in the pathogenesis of diabetic nephropathy. However, the mechanism by which visfatin induces kidney injury is unknown. The renin-angiotensin system (RAS) plays pivotal roles in renal diseases. Therefore, in this study the effect of visfatin on the regulation of RAS in MCs was examined., Methods: Cultured rat MCs were treated with different doses of visfatin. We used real-time polymerase chain reaction to detect mRNA expression of renin, angiotensinogen (AGT), angiotensin-converting enzyme (ACE), angiotensin II (Ang II) type 1 receptor (AT1), and Ang II type 2 receptor (AT2); western blot analysis for expression of ANG and AT1; and radioimmunoassay to measure Ang II production from MCs in the supernatants of culture media., Results: Visfatin treatments increased renin, angiotensinogen (AGT), AT1 mRNA, and AGT, AT1 protein expression, as well as Ang II levels in a dose-dependent manner but did not affect ACE and AT2 mRNA levels in cultured rat MCs., Conclusions: Our findings suggest that visfatin imparts a detrimental effect on diabetic nephropathy at least partly through the activation of intrarenal RAS.

Hypertension is one of the cardiovascular diseases that might cause cardiovascular remodeling and endothelial dysfunction besides high blood pressure. Angiotensin II (Ang II) receptors are implicated in hypertension. Genetic and epigenetic manipulations of the Ang II receptors play a crucial part in the programming of cardiovascular diseases, and certain variants of the Ang II type 1 and Ang II type 2 receptors are constitutively predisposed to higher cardiovascular risk and hypertension. In this review, we focus on the expression, mode of action of Ang II receptors, and their role in programming the cardiovascular diseases in utero. In addition, we discuss possible therapeutic interventions of Ang II stimulation. Collectively, this information might lead us to new drug designs against cardiovascular diseases., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Excessive sympathetic drive is a hallmark of chronic heart failure (HF). Disease progression can be correlated with plasma norepinephrine concentration. Renal function is also correlated with disease progression and prognosis. Because both the renal nerves and renin-angiotensin II system are activated in chronic HF we hypothesized that excessive renal sympathetic nerve activity decreases renal blood flow in HF and is associated with changes in angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) expression. The present study was carried out in conscious, chronically instrumented rabbits with pacing-induced HF. We found that rabbits with HF showed a decrease in mean renal blood flow (19.8±1.6 in HF vs. 32.0±2.5 ml/min from prepace levels; P<0.05) and an increase in renal vascular resistance (3.26±0.29 in HF vs. 2.21±0.13 mmHg·ml(-1)·min in prepace normal rabbits; P<0.05) while the blood flow and resistance was not changed in HF rabbits with the surgical renal denervation. Renal AT1R expression was increased by ∼67% and AT2R expression was decreased by ∼87% in rabbits with HF; however, kidneys from denervated rabbits with HF showed a near normalization in the expression of these receptors. These results suggest renal sympathetic nerve activity elicits a detrimental effect on renal blood flow and may be associated with alterations in the expression of angiotensin II receptors.

Sepsis causes impaired vascular reactivity, hypotension and acute renal failure. The ability of the Escherichia coli endotoxin (lipopolysaccharide [LPS]) to impair agonist-induced contractility in mesangial cells, which contributes to LPS-induced renal dysfunction, was evaluated. Agonist-induced intracellular calcium ([Ca(2+)]i) mobilization was analyzed using angiotensin II (AngII). The effect of LPS on the levels of the renin-angiotensin system (RAS) components and the roles of vasodilatation-inducing molecules including AT2 receptor (AT2R) and nitric oxide (NO) in the cell reactivity were also evaluated. Confluent human mesangial cells (HMCs) were stimulated with LPS (0111-B4, 100 microg/mL). AngII-induced [Ca(2+)]i mobilization was measured by fluorometric analysis using Fura-2AM in the absence and presence of an AT2R antagonist (PD123319). The mRNA and protein levels for angiotensinogen, renin, angiotensin-converting enzyme, AT1R and AT2R were analyzed by realtime reverse transcriptase-polymerase chain reaction and Western blot, respectively. NO production was measured by the chemiluminescence method in the culture media after 24, 48 and 72 h of LPS incubation. After 24 h, LPS-stimulated HMCs displayed lower basal [Ca(2+)]i and an impaired response to AngII-induced rise in [Ca(2+)]i. LPS significantly increased AT2R levels, but did not cause significant alterations of RAS components. PD123319 restored both basal and AngII-induced [Ca(2+)]i peak, suggesting an involvement of AT2R in these responses. The expected increase in NO production was significant only after 72 h of LPS incubation and it was unaffected by PD123319. Results showed that LPS reduced the reactivity of HMCs to AngII and suggest that the vasodilatation induced by AT2R is a potential mediator of this response through a pathway independent of NO.

The renin angiotensin system (RAS) has profound effects on atherosclerosis development in animal models, which is partially complimented by evidence in the human disease. Although angiotensin II was considered to be the principal effector of the RAS, a broader array of bioactive angiotensin peptides have been identified that have increased the scope of enzymes and receptors in the RAS. Genetic interruption of the synthesis of these peptides has not been extensively performed in experimental or human studies. A few studies demonstrate that interruption of a component of the angiotensin peptide synthesis pathway reduces experimental lesion formation. The evidence in human studies has not been consistent. Conversely, genetic manipulation of the RAS receptors has demonstrated that AT1a receptors are profoundly involved in experimental atherosclerosis. Few studies have reported links of genetic variants of angiotensin II receptors to human atherosclerotic diseases. Further genetic studies are needed to define the role of RAS in atherosclerosis.

We have investigated the effect of benzo[a]pyrene (B[a]P), a carcinogen of tobacco smoke and an agonist for the aryl hydrocarbon receptor (AHR), on hypoxia-induced angiogenesis. Ischemia was induced by femoral artery ligation in wild-type and AHR-null mice, and the animals were subjected to oral administration of B[a]P (125 mg/kg) once a week. Exposure to B[a]P up-regulated the expression of metallothionein in the ischemic hindlimb and markedly inhibited ischemia-induced angiogenesis in wild-type mice. The amounts of interleukin-6 and of vascular endothelial growth factor (VEGF) mRNA in the ischemic hindlimb of wild-type mice were reduced by exposure to B[a]P. These various effects of B[a]P were markedly attenuated in AHR-null mice. Our observations suggest that the loss of the inhibitory effect of B[a]P on ischemia-induced angiogenesis apparent in AHR-null mice may be attributable to maintenance of interleukin-6 expression and consequent promotion of angiogenesis through up-regulation of VEGF expression.

Background: Angiotensin blockade such as with an angiotensin II receptor blocker (ARB) or angiotensinconverting enzyme inhibitor (ACEI) has antifibrotic properties. The aim of this study was to evaluate and compare the antifibrotic effect between ARBs and ACEIs., Methods: Common bile duct-ligated (BDL) adult Sprague-Dawley rats were allocated to five groups (each group, n = 8) as follows: G1, BDL without drug; G2, BDL + captopril 100 mg/kg per day; G3, BDL + ramipril 10 mg/kg per day; G4, BDL + losartan 10 mg/kg per day; G5, BDL + irbesartan 15 mg/kg per day. Four weeks post-BDL, hepatic fibrosis was analyzed histomorphologically using Batts and Ludwig scores. alpha-Smooth muscle actin (alpha-SMA) expression by immunohistochemical staining, hydroxyproline contents of liver tissue by spectrophotometry, and angiotensin receptor, collagen, procollagen, and transforming growth factor beta (TGF-beta) expressions were evaluated by real-time reverse transcriptase-polymerase chain reaction. Angiotensin receptor expression was also determined by Western blotting., Results: Batts and Ludwig scores were 3.8, 2.6, 2.4, 1.8, and 1.6 in G1, G2, G3, G4, and G5, respectively. Histologically, ARB groups (G4, G5) showed significant suppression of hepatic fibrosis compared with ACEI groups or the control. Expressions of alpha-SMA (%) and the content of hydroxyproline (microg liver tissue) were significantly lower in ARB groups (G4, G5) than in ACEI groups (G2, G3) (P < 0.05). Also, ARB reduced the expression of angiotensin receptor, collagen, procollagen, and TGF-beta1 compared with ACEI. Western blot analysis showed that the expression of angiotensin receptor was inhibited in both ARB and ACEI groups., Conclusions: Both ARB and ACEI attenuate hepatic fibrosis through inhibiting hepatic stellate cell activation, and the inhibitory effect of ARBs on hepatic fibrosis is superior to that of ACEIs in the BDL rat model.

Summary The central nervous system (cerebral ganglion) of adult ascidians is linked to the neural gland complex (NGC), which consists of a dorsal tubercle, a ciliated duct and a neural gland. The function of the NGC has been the subject of much debate. The recent publication of the complete genomic sequence of Ciona intestinalis provides new opportunities to examine the presence and distribution of protein families in this basal chordate. We focus here on the ascidian neuropeptide G-protein-coupled receptors (GPCRs), the vertebrate homologues of which are involved in homeostasis. In situ hybridization revealed that five Ciona GPCRs [vasopressin receptor, somatostatin receptor, CRH (corticotropin-releasing hormone) receptor, angiotensin receptor and tachykinin receptor] are expressed in the NGC of adult ascidians. These findings, together with histological and ultrastructural data, provide evidence to support a role for the ascidian NGC in maintaining ionic homeostasis. We further speculate about the potential similarities between the ascidian NGC and the vertebrate choroid plexus, a neural peri-ventricular organ.

To investigate the association between hyperinsulinemia and cardiac hypertrophy, we treated rats with insulin for 7 wk and assessed effects on myocardial growth, vascularization, and fibrosis in relation to the expression of angiotensin II receptors (AT-R). We also characterized insulin signaling pathways believed to promote myocyte growth and interact with proliferative responses mediated by G protein-coupled receptors, and we assessed myocardial insulin receptor substrate-1 (IRS-1) and p110 alpha catalytic and p85 regulatory subunits of phospatidylinositol 3 kinase (PI3K), Akt, MEK, ERK1/2, and S6 kinase-1 (S6K1). Left ventricular (LV) geometry and performance were evaluated echocardiographically. Insulin decreased AT1a-R mRNA expression but increased protein levels and increased AT2-R mRNA and protein levels and phosphorylation of IRS-1 (Ser374/Tyr989), MEK1/2 (Ser218/Ser222), ERK1/2 (Thr202/Tyr204), S6K1 (Thr421/Ser424/Thr389), Akt (Thr308/Thr308), and PI3K p110 alpha but not of p85 (Tyr508). Insulin increased LV mass and relative wall thickness and reduced stroke volume and cardiac output. Histochemical examination demonstrated myocyte hypertrophy and increases in interstitial fibrosis. Metoprolol plus insulin prevented the increase in relative wall thickness, decreased fibrosis, increased LV mass, and improved function seen with insulin alone. Thus our data demonstrate that chronic hyperinsulinemia decreases AT1a-to-AT2 ratio and increases MEK-ERK1/2 and S6K1 pathway activity related to hypertrophy. These changes might be crucial for increased cardiovascular growth and fibrosis and signs of impaired LV function.

Neonatal handling affects the hypothalamus-pituitary-gonadal axis in female rats. Indeed, postnatal handling induces anovulatory estrous cycles and decreases sexual receptiveness. On the other hand, Angiotensin II (Ang II) infused into the medial amygdala (MeA) reduces sexual behavior in male and female rats. Considering this, and that gonadal steroid secretion after copulatory behavior is important for reproductive success, the purpose of the present study was to investigate whether the reduction in sexual receptiveness in neonatally handled female rats is mediated by changes in Ang II receptor density in MeA. Moreover, gonadal steroid secretion after sexual behavior was analyzed. Two groups of female Wistar rats were studied: nonhandled (pups were left undisturbed) and handled (pups were handled for 1 min once a day during the first 10 days of life). Once they were 80-85 days old in the evening of the proestrus day, sexual receptiveness was recorded and after that the animals were killed by decapitation. Trunk blood samples were collected, and plasma estradiol and progesterone were measured by radioimmunoassay. The brains were removed for Ang II receptor autoradiography in MeA. The decreased lordosis quotient in the neonatally handled group was confirmed in the present study. Neonatal handling also reduced the progesterone concentration in the plasma, but did not change the estradiol and the density of Ang II receptors in MeA. The reduced progesterone could be due to the decreased lordosis frequency of handled females. However, this decreased sexual receptiveness is not mediated by changes in Ang II receptors in MeA.

Objectives: The controversy regarding potential long-term side effects of antenatal steroid administration for accelerating fetal lung maturation is still unresolved despite more than 30 years of experience. Studies in animals have demonstrated that administration of glucocorticoids during pregnancy alters renal expression of several key regulatory molecules at different developmental stages followed in most cases with the development of hypertension in the adult. We studied the effects of betamethasone on the expression of (1) NA,K-ATPAse pump; (2) the Na/H exchanger 3 (NAHE3); (3) angiotensin receptor (AT1 and AT2); and (4) the type 1 dopamine receptor (D1R)., Methods: Pregnant sheep were treated with either 0.17 mg/kg betamethasone or vehicle 24 hours apart at 80 and 81 days' gestation. Fetal kidneys were harvested at 81 and 135 days' gestation. Protein and mRNA levels were measured in kidney cortex., Results: Betamethasone had acute and long-term effects on fetal kidney cortex gene expression. Acutely, mRNA abundance for AT2 was significantly lower and that of NHE3 significantly higher than in controls (0.4 +/- 0.02 vs 0.7 +/- 0.05; 1.2 +/- 0.16 vs 0.4 +/- 0.04; P < .05). At 135 days' gestation, AT2 receptor abundance remained lower than control (0.2 +/- 0.02 vs 0.4 +/- 0.02; P < .05), whereas D1R expression was higher (0.8 +/- 0.17 vs 0.5 +/- 0.06; P < .05). No changes in Na,K-ATPase of AT1 receptor at either of the two time points studied were observed. Antenatal steroid administration was not associated with premature labor or a reduction in either body weight or kidney weight., Conclusion: Our findings strongly suggest that antenatal glucocorticoid administration according to National Institutes of Health (NIH) consensus guidelines may alter human fetal renal development. Further studies are needed to establish a direct relationship between alterations in fetal renal gene expression and the development of hypertension in adulthood.

Activation of the renin-angiotensin system has a pivotal role in the pathogenesis of diabetic complications. However, recent evidence suggests that it may also contribute to the development of diabetes itself. In the endocrine pancreas, all the components of an active renin-angiotensin system are present, which modulate a range of activities including local blood flow, hormone release and prostaglandin synthesis. In both types 1 and 2 diabetes, there is an up-regulation of its expression and activity in the endocrine pancreas. Whether these changes have a direct pathogenetic role or reflect a response to local stress or tissue injury remains to be established. Angiotensin-mediated increases in oxidative stress, inflammation and free fatty acids levels potentially contribute to beta-cell dysfunction in diabetes. In addition, activation of the renin-angiotensin system appears to potentiate the action of other pathogenic pathways including glucotoxicity, lipotoxicity and advanced glycation. In experimental models of type 2 diabetes, blockade of the renin-angiotensin system with angiotensin converting enzyme inhibitors or angiotensin receptor antagonists results in the improvement of islet structure and function. Moreover, the incidence of de novo diabetes appears to be significantly reduced by blockade of the renin-angiotensin system in clinical studies. At least two large controlled trials are currently underway to study the role of renin-angiotensin system in the development of diabetes. It is hoped that these studies will demonstrate the true potential of the blockade of the renin-angiotensin system for the prevention of diabetes.

The mechanism of overproduction of aldosterone in primary aldosteronism is unclear. The intraadrenal renin-angiotensin system (RAS) has been suggested to possess the functional role of the synthesizing aldosterone and regulating blood pressure. In order to clarify the pathophysiological roles of adrenal RAS in aldosterone-producing adenoma (APA), we studied the expressions of the messenger RNAs (mRNAs) of renin, angiotensinogen, type 1 (AT1R) and type 2 angiotensin II receptor (AT2R), CYP11B1 (11 beta-hydroxylase gene) and CYP11B2 (aldosterone synthase gene) in 8 patients with angiotensin II-responsive (ATII-R) APA and compared them with the expressions of the same mRNAs in 8 patients with angiotensin II-unresponsive (ATII-U) APA. Quantification of the mRNA of each gene was done using a real-time polymerase chain reaction with specific primers. There were no significant differences between ATII-R APA and ATII-U APA in the mRNA levels of renin, angiotensinogen, AT1 R, CYP11B1 and CYP11B2. The amount of AT2R mRNA was significantly higher in the patients with ATII-R APA than in those with ATII-U APA (p<0.05). These results may suggest that AT2R partially contributes to the overproduction of aldosterone in ATII-R APA.

Background: Although endothelin I (ET-1) is a very potent vasoconstrictor, ET-1 transgenic (ET-1 tg) mice are not hypertensive. This might be due to higher bioavailability of nitric oxide (NO) in ET-1 tg, which counteracts the effect of vasoconstrictors. We hypothesized lower angiotensin II (Ang II) sensitivity of afferent arterioles in ET-1 tg., Methods: Afferent arterioles were manually dissected and microperfused. Changes of the luminal diameter due to application of vasoactive substances were used for assessment of the reactivity of afferent arterioles. We investigated the effect of L-NAME, an unspecific NO synthase inhibitor, on basal tone, and the sensitivity of afferent arterioles to Ang II with and without pre-treatment with L-NAME. The renin-angiotensin-system was characterized by expression analysis of angiotensin-receptors and renin at the mRNA level., Results: L-NAME reduced afferent arterioles diameters similarly in ET-1 tg and wild-types (WT). Ang II sensitivity determined by calculation of EC50 for Ang II was less in ET-1 tg compared with WT (P<0.05). Ang II reduced luminal diameters to a lesser extent in ET-1 tg compared to WT (P<0.05). After pre-treatment with L-NAME, Ang II sensitivity and maximum constriction of afferent arterioles were similar in ET-1 tg and WT. The expression of renin- and Ang II-receptor-mRNA in the kidney did not differ between either group., Conclusion: The loss of differences in the maximum constriction and Ang II sensitivity of afferent arterioles between ET-1 tg and WT in the absence of NO suggests pronounced NO effects in afferent arterioles of ET-1 tg. This might contribute to the maintenance of normal renal arteriolar tone in ET-1 tg mice.

Genetic, biochemical and physiological studies have demonstrated that the renin-angiotensin system (RAS) plays a fundamental role in kidney development. All of the components of the RAS are expressed in the metanephros. Mutations in the genes encoding components of the RAS in mice or pharmacological inhibition of RAS in animals or humans cause diverse congenital abnormalities of the kidney and lower urinary tract. The latter include renal vascular abnormalities, abnormal glomerulogenesis, renal papillary hypoplasia, hydronephrosis, aberrant UB budding, duplicated collecting system, and urinary concentrating defect. Thus, the actions of angiotensin (ANG) II during kidney development are pleiotropic both spatially and temporally. Whereas the role of ANG II in renovascular and glomerular development has received much attention, little is known about the potential role of ANG II and its receptors in the morphogenesis of the collecting system. In this review, we discuss recent genetic and functional evidence gathered from transgenic knockout mice and in vitro organ and cell culture implicating the RAS in the development of the ureteric bud and collecting ducts. A novel conceptual framework has emerged from this body of work which states that stroma-derived ANG II elicits activation of AT(1)/AT(2) receptors expressed on the ureteric bud to stimulate branching morphogenesis as well as collecting duct elongation and papillogenesis.

The influence of the renin-angiotensin system (RAS) is recognized in cardiac and vascular injury. An extrinsic RAS has been known for decades, and an equally important intrinsic RAS has been discovered recently. The latter leads to pathologic tissue alterations in the absence of systemic stimuli and may be the main source of local tissue effects of RAS. A new radiotracer fluorobenzoyl-lisinopril was synthesized by radiolabeling benzoic acid active ester with 18F and reacting that with the epsilon-amino group of lisinopril. The presence of angiotensin-converting enzyme (ACE) activity and angiotensin II receptors was examined in relation to myocardial fibrosis. This tissue-specific radioligand represents the first study of ACE in the human heart. This article presents preliminary data on imaging the RAS in the human cardiac tissue and discusses the potential for clinical application of these imaging techniques to human patients.

The aim of this study was to clarify the role of the renal renin-angiotensin system (RAS) in diabetic nephropathy (DN), which was induced by injection of streptozotocin (STZ). Male CBA/N and CBA/J mice were compared in this study. The former possesses a single renin gene, Ren1, whereas the latter carries two renin genes, Ren1 and Ren2. To examine the molecular dynamics of renal RAS, including renin, angiotensinogen (Agt), angiotensin-converting enzyme (Ace), angiotensin type 1 (Agtr1) and type 2 (Agtr2) receptors in experimental DN, we performed laser-microdissection (LMD) followed by reverse transcriptase nested polymerase chain reaction using each specific primer pairs and immunohistochemistry for renin and angiotensin II. CBA/N mice had a higher response after injection of STZ than CBA/J mice, showing a significant increase of the kidney/body weight ratio, although there was no significant difference between the two strains for the blood glucose level or pancreatic beta-cell response. The onset of renal pathological changes associated with DN was earlier and more severe in CBA/N mice than in CBA/J mice. Distinct immunoreactivities for renin and angiotensin II were newly distributed on the flattened epithelial cells in the dilated distal tubules in the cortex as well as the collecting ducts in the cortex and medulla, and were demonstrated more intensely in CBA/N mice than in CBA/J mice. Microdissectional analysis in both DN models revealed a higher incidence of RAS-related gene expression in CBA/J, Ren 1 Ren 2 mice than in CBA/N, Ren 1 mice. These findings suggest that intrarenal RAS plays an important role in the onset of renal pathological changes associated with DN. Additionally, Ren 1 mice have more severe histopathological nephropathy than Ren1 Ren2 mice, followed by marked production of angiotensin II.

Background: Adequate maternal nutrition during gestation is requisite for fetal nutrition and development. While a large group of epidemiological studies indicate poor fetal nutrition increases heart disease risk and mortality in later life, little work has focused on the effects of impaired maternal nutrition on fetal heart development. We have previously shown that 50% global nutrient restriction from 28-78 days of gestation (early to mid-pregnancy; term = 147 days) in sheep at mid-gestation retards fetal growth while protecting growth of heart and results in hypertensive male offspring at nine months of age. In the present study, we evaluate LV gene transcription using RNA protection assay and real-time reverse transcriptase polymerase chain reaction, and protein expression using western blot, of VEGF and AT1 and AT2 receptors for AngII at mid-gestation in fetuses from pregnant ewes fed either 100% (C) or 50% (NR) diet during early to mid-gestation., Results: No difference between the NR (n = 6) and C (n = 6) groups was found in gene transcription of the AngII receptors. Immunoreactive AT1 (1918.4 +/- 154.2 vs. 3881.2 +/- 494.9; P < 0.01) and AT2 (1729.9 +/- 293.6 vs. 3043.3 +/- 373.2; P < 0.02) was decreased in the LV of NR fetuses compared to C fetuses. The LV of fetuses exposed to NR had greater transcription of mRNA for VEGF (5.42 +/- 0.85 vs. 3.05 +/- 0.19; P < 0.03) than respective C LV, while no change was observed in immunoreactive VEGF., Conclusion: The present study demonstrates that VEGF, AT1 and AT2 message and protein are not tightly coupled, pointing to post-transcriptional control points in the mid gestation NR fetus. The present data also suggest that the role of VEGF and the renin-angiotensin system receptors during conditions inducing protected cardiac growth is distinct from the role these proteins may play in normal fetal cardiac growth. The present findings may help explain epidemiological studies that indicate fetuses with low birth weight carry an increased risk of mortality from coronary and cardiovascular disease, particularly if these individuals have reduced cardiovascular reserve due to an epigenetic decrease in vascularization.

Animal models support human epidemiological studies in demonstrating a relationship between impaired fetal growth and risk of adult hypertension. Undernutrition during pregnancy exerts programming effects on the developing kidney, and modulation of angiotensin receptor (ATR) expression has been observed persisting into adult life. Fetal overexposure to glucocorticoids is thought to be central to the nutritional programming of blood pressure and may act through an interaction with ATR expression. Pregnant female Wistar rats were fed a control (n = 6) or a maternal low-protein diet (MLP; n = 17) throughout pregnancy. The glucocorticoid dependency of MLP effects was tested using metyrapone, an inhibitor of corticosterone synthesis. MLP-fed rats were injected twice daily with metyrapone, metyrapone plus corticosterone, or vehicle over days 1-14 of pregnancy. At delivery, all animals were fed standard laboratory chow. MLP-exposed offspring 4 wk of age exhibited increased systolic blood pressure compared with controls (P < 0.05), which proved to be glucocorticoid dependent in males only. AT(1)R mRNA expression was independent of in utero dietary treatment. AT(2)R mRNA expression was downregulated in MLP-exposed females only (P < 0.05) and in a glucocorticoid-independent manner. Male offspring exhibited glucocorticoid-dependent hypertension with no modulation of renal ATR mRNA expression. In contrast, female offspring exhibited glucocorticoid-independent hypertension associated with reduced expression of renal AT(2)R mRNA. These data do not support the hypothesis that an interaction between glucocorticoid and ATR mRNA expression underlies the nutritional programming of blood pressure but instead suggest two independent mechanisms acting in a sex-specific manner.

Background: Elevated circulating aldosterone level is associated with impaired cardiovascular function. Although the mechanisms are not fully understood, aldosterone antagonists decrease total and cardiovascular mortality in heart failure and myocardial infarction. Aldosterone induces cardiac fibrosis in experimental models, and it is synthesized locally in rat heart. These observations suggest pathological effects of aldosterone in heart that remain unclear., Methods and Results: Transgenic mice (TG) that overexpress the terminal enzyme of aldosterone biosynthesis, aldosterone synthase (AS), in heart have been raised by gene targeting with the alpha-myosin heavy chain promoter. AS mRNA increased 100-fold and aldosterone concentration 1.7-fold in hearts of male TG mice relative to wild-type. No structural or myocardial alterations were evidenced, because ventricle/body weight, AT1 and AT2 receptor binding, and collagen content were unchanged in TG. No alteration in cardiac function was evidenced by echocardiography, isolated perfused heart, or whole-cell patch clamp experiments. In contrast, coronary function was impaired, because basal coronary flow was decreased in isolated perfused heart (-55% of baseline values), and vasodilatation to acetylcholine, bradykinin, and sodium nitroprusside was decreased by 75%, 60%, and 75%, respectively, in TG mice compared with wild-type, showing that the defect was not related to NO production., Conclusions: Increased cardiac aldosterone production in male mice induces a major coronary endothelium-independent dysfunction with no detectable alterations in cardiac structure and function. However, coronary dysfunction may be harmful for coronary adaptation to increased flow demand.

Objectives: We evaluated whether the angiotensin II (Ang II) receptors from perioperation through one-year post-transplantation predict the transplant coronary artery disease (TCAD) progression., Background: The role of Ang II receptors (type 1: AT(1)R; type 2: AT(2)R) in TCAD is uncertain., Methods: We investigated 28 heart donors and the corresponding recipients. The levels of AT(1)R and AT(2)R messenger ribonucleic acid (mRNA) were examined in lymphocytes from the donor spleen and in the donor heart at one-week and one-year posttransplantation to determine their association with the progression of TCAD, measured as changes in maximal intimal thickness (CMIT) and plaque volume (CPV) by intravascular ultrasound (IVUS) examinations., Results: The AT(1)R mRNA in lymphocytes from the donor spleen (CMIT: r = 0.73, p < 0.0001; CPV: r = 0.69, p < 0.0001) and in the donor hearts at one-week (CMIT: r = 0.52, p = 0.005; CPV: r = 0.56, p = 0.002) and at one-year (CMIT: r = 0.63, p < 0.0001; CPV: r = 0.43, p = 0.004) post-transplantation along with AT(2)R mRNA in the donor hearts at one-year post-transplantation (CMIT: r = 0.3, p < 0.0001; CPV: r = 0.53, p = 0.009) were univariate predictors, whereas AT(1)R mRNA in lymphocytes and in the donor hearts at one-year post-transplantation proved to be multivariate predictors of the progression of TCAD., Conclusions: These data suggest a role for Ang II receptors in the pathogenesis of TCAD and support a novel concept that TCAD may have its origin in the donor per se and may be modulated by the recipient's inherent biological factors.

Objective: The normal suppression of vascular sensitivity to angiotensin II (Ang II) in pregnancy is lost in pregnancy-induced hypertension (PIH). To examine the mechanism, we investigated Ang II receptor subtype 1 (AT1R) and 2 (AT2R) expression in human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells (VSMC)., Methods: The HUVEC and VSMC were incubated with serum from normal pregnant women and PIH patients for 0 to 12 h. The AT1R and AT2R mRNA were semiquantified as the ratio to glyceraldehyde-3-phosphate dehydrogenase mRNA, using multiplex reverse transcription-polymerase chain reaction. The AT1R expression was also evaluated by immunocytochemistry., Results: Serum from PIH patients significantly increased AT1R mRNA of HUVEC (1.48 +/- 0.44) after a 12-h incubation compared with that from normal pregnant women (0.25 +/- 0.14). On the other hand, AT2R mRNA of HUVEC incubated with serum from PIH patients (0.14 +/- 0.02) was significantly decreased compared with HUVEC incubated with serum from normal pregnant women (0.31 +/- 0.08). The AT1R mRNA of VSMC was significantly increased by serum from both PIH patients and normal pregnant women. The AT1R-to-AT2R mRNA ratio increased by serum from PIH patients was significantly reduced by anti-tumor necrosis factor-alpha (TNF-alpha) antibody (20 microg/mL). Valsartan (an AT1R antagonist, at 1 to 10 nmol/L) significantly increased AT2R mRNA of HUVEC. Also, immunocytochemistry demonstrated that endothelial AT1R expression was strongly increased by PIH sera and reduced by anti-TNF-alpha antibody., Conclusions: Endothelial AT1R expression is increased and AT2R expression is decreased in PIH. The TNF-alpha is related to the pathogenesis of PIH by reduced AT2R mRNA through an increase of AT1R mRNA.

The transcriptional factor Sp1 is associated with GC-rich promoters and involved in basal promoter activity. A GC-box-related sequence is located within the -58 to -34 base pair region of the angiotensin type 1 receptor gene promoter. We examined whether Sp1 in the hypothalamus was increased in spontaneously hypertensive rats (SHR) and whether inhibition of Sp1 binding sites suppressed angiotensin type 1 receptor expression and thus decreased blood pressure in SHR. Western blot analysis showed that Sp1 protein levels were increased in nuclear extracts of hypothalamus from SHR. Electrophoretic mobility shift assay (EMSA) using oligonucleotides containing Sp1 consensus sequence and -58 to -34 region sequence oligonucleotides showed that DNA-protein complexes were greater in nuclear extracts of hypothalamus from SHR than those of Wistar Kyoto rats (WKY). Sp1 decoy phosphorothioate oligodeoxynucleotides injected into the lateral ventricle produced a decrease in blood pressure in SHR, and decreased angiotensin type 1 receptor mRNA levels and number of angiotensin receptors in the hypothalamus of SHR. Pressor responses to angiotensin II but not to carbachol injected into the lateral ventricle were decreased in the Sp1 decoy-treated SHR. The results of the present study suggest that Sp1 levels in the hypothalamus of SHR are increased, and that inhibition of the binding of Sp1 to its binding sites decreases angiotensin type 1 receptor expression and blood pressure in SHR. The possibility cannot be ruled out that the Sp1 decoy oligodeoxynucleotides (ODN) also suppressed transcriptions of genes other than the angiotensin type 1 receptor gene.

Unlabelled: TIVES: We postulated a change of angiotensin II receptor subtype expression in patients with lone atrial fibrillation (AF) and AF with underlying mitral valve disease (MVD) both compared with sinus rhythm (SR)., Background: Atrial fibrillation is a progressive disease associated with electrical and structural remodeling. Angiotensin II (ANGII) is involved in the process of myocardial remodeling. Actions of ANGII are mediated by ANGII receptor subtypes 1 and 2 (AT(1) and AT(2))., Methods: Left atrial (LA) and right atrial (RA) tissue samples were obtained from patients with AF or SR with or without underlying MVD. The AT(1) and AT(2) protein levels were measured by quantitative Western blotting techniques., Results: The AT(1) protein level in the LA was significantly increased in patients with AF (all forms) compared with SR (p < 0.05), whereas AT(2) expression was not significantly altered. Comparison of the subgroups revealed a similar increase of AT(1) in both paroxysmal AF and chronic AF with or without MVD. Additionally, investigations of ANGII receptor subtypes in the RA did not exhibit any significant changes either in AT(1) or in AT(2) in patients with AF versus SR. Underlying MVD did not significantly affect AT(2) receptor subtype expression in LA., Conclusions: Atrial fibrillation is associated with an up-regulation of AT(1) in LA, but not in RA, and did not appear to influence the AT(2) expression in the atrium. Because we found an enhanced expression of AT(1)in the LA, we conclude that AT(1) might be involved in the pathogenesis of AF in the LA.

Angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists attenuate fibrosis in the kidney, heart, and liver by suppressing transforming growth factor-beta1 mRNA and decreasing production of extracellular matrix proteins. We recently demonstrated that lisinopril, an angiotensin-converting enzyme inhibitor, alleviates pancreatic inflammation and fibrosis in male Wistar Bonn/Kobori rats. The involvement of angiotensin II receptor and its receptor interaction in the pathogenesis of spontaneous chronic pancreatitis was assessed in this model. Candesartan, an angiotensin II receptor antagonist, was administered in drinking water (10.5, 42, or 125 mg/l) to 10-week-old male WBN/Kob rats for 10 weeks and inflammatory parameters, fibrosis, and gene expression of renin-angiotensin system components and transforming growth factor-beta1 were assessed in the pancreas. Immunostaining for alpha-smooth muscle actin was also performed. Candesartan significantly suppressed decrease in pancreatic weight and increases in pancreatic myeloperoxidase activity, hydroxyproline content, ratio of fibrous tissue, histologic scores, and ratio of alpha-smooth muscle actin-positive cells (activated pancreatic stellate cells) at 20 weeks. The high dose enhanced the expression of angiotensinogen and angiotensin II receptor type 2 mRNA and suppressed the overexpression of transforming growth factor-beta1 mRNA. The conclusion is that candesartan alleviates chronic pancreatitis and fibrosis by suppressing the overexpression of transforming growth factor-beta1, resulting in prevention of activation of pancreatic stellate cells in male WBN/Kob rats. We propose that angiotensin II receptor type 1 antagonists may be useful for the treatment of chronic pancreatitis involving angiotensin II interaction with its receptor.

Pericytes are an integral component of blood capillaries, but their involvement in a variety of conditions and diseases, including hypertension and multiple sclerosis, is poorly understood. In order to analyze the mRNA expression of markers related to hypertension and multiple sclerosis in rat brain pericytes, we have established brain capillary pericyte cell lines from temperature-sensitive SV40 large T antigen transgenic rats. The newly established clones showed similar biochemical and morphological properties to primary pericytes. The expression of endothelial cell-related markers Flt-1, Flk-1, Tie-1, and Tie-2 was evaluated by RT-PCR analysis. beta2-Adrenergic receptor (beta2-AR), angiotensin II receptor type1A (AT1A), and klotho were also evaluated as markers related to hypertension and multiple sclerosis. All of the isolated clones expressed beta2-AR, AT1A and klotho genes. They also stably expressed Flt-1 and Tie-2, while Flk-1, Tie-1 and CXCR4 were expressed only at low levels in some of the clones. The expressions of AT1 in TR-PCT1 were determined by Western blotting. Angiotensin II stimulated migration of pericytes. This effect was blocked by an AT1 antagonist. The pericyte cell lines established here are pluripotent, and should be useful for analysis of the reactivity and biological roles of pericytes., (Copyright 2003 Wiley-Liss, Inc.)

Background: Local renin-angiotensin systems (RAS) have been implicated as playing an important role in vascular remodeling. The relationship of this system to the etiology of cerebral aneurysm was investigated., Methods and Results: The aneurysmal wall from patients with a ruptured or unruptured cerebral aneurysm and the cortical cerebral artery in control patients with head trauma or a glioma were taken during surgery for study. Local RAS were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and/or immunohistochemistry. RT-PCR analysis revealed a significantly decreased expression of angiotensin-converting enzyme (ACE), angiotensin type 1 (AT1) receptor, basic fibroblast growth factor, platelet-derived growth factor-AA, and tissue inhibitor of matrix metalloproteinases-1 mRNA in the aneurysmal wall as compared with the control cortical arterial wall. Immunohistochemistry also revealed a decreased expression of ACE, AT1 receptor, and angiotensin II in the aneurysmal wall., Conclusions: Expression of local RAS was decreased in the aneurysmal wall, which may induce aneurysm formation caused by a lack of vascular remodeling that prevents the arterial wall from thickening under increased hemodynamic stress. This is the first report that suggests that a decreased expression of local RAS plays a part in the pathogenesis of any disease.

Despite an increase of circulatory volume and of renin-angiotensin-aldosterone system (RAAS) activity, pregnancy is paradoxically accompanied by a decrease in blood pressure. We have reported that the decrease in blood pressure was maintained in pregnant rats despite overactivation of RAAS following reduction in sodium intake. The purpose of this study was to evaluate the impact of the opposite condition, e.g., decreased activation of RAAS during pregnancy in the rat. To do so, 0.9% or 1.8% NaCl in drinking water was given to nonpregnant and pregnant Sprague-Dawley rats for 7 days (last week of gestation). Increased sodium intakes (between 10- and 20-fold) produced reduction of plasma renin activity and aldosterone in both nonpregnant and pregnant rats. Systolic blood pressure was not affected in nonpregnant rats. However, in pregnant rats, 0.9% sodium supplement prevented the decreased blood pressure. Moreover, an increase of systolic blood pressure was obtained in pregnant rats receiving 1.8% NaCl. The 0.9% sodium supplement did not affect plasma and fetal parameters. However, 1.8% NaCl supplement has larger effects during gestation as shown by increased plasma sodium concentration, hematocrit level, negative water balance, proteinuria, and intrauterine growth restriction. With both sodium supplements, decreased AT1 mRNA levels in the kidney and in the placenta were observed. Our results showed that a high-sodium intake prevents the pregnancy-induced decrease of blood pressure in rats. Nonpregnant rats were able to maintain homeostasis but not the pregnant ones in response to sodium load. Furthermore, pregnant rats on a high-sodium intake (1.8% NaCl) showed some physiological responses that resemble manifestations observed in preeclampsia.

Paraventricular nucleus (PVN) neurons that project to the spinal cord are important in the control of sympathetic outflow. Angiotensin II (Ang II) can stimulate PVN neurons, but its cellular mechanisms are not clear. In this study, we determined the effect of Ang II on the excitatory and inhibitory synaptic inputs to spinally projecting PVN neurons. Whole-cell patch-clamp recordings were performed on PVN neurons labeled by a retrograde fluorescence tracer injected into the thoracic spinal cord of rats. Immunocytochemistry labeling revealed that the immunoreactivity of angiotensin type 1 (AT1) receptors was colocalized with a presynaptic marker, synaptophysin, in the PVN. Application of 0.1-5 microm Ang II significantly decreased the amplitude of evoked GABAergic IPSCs in a concentration-dependent manner. Also, Ang II decreased the frequency of miniature IPSCs from 2.56 +/- 0.45 to 1.05 +/- 0.20 Hz (p < 0.05; n = 12), without affecting the amplitude and the decay time constant. The effect of Ang II on miniature IPSCs was blocked by losartan but not PD123319. However, Ang II had no effect on the evoked glutamatergic EPSCs and did not alter the frequency and amplitude of miniature EPSCs at concentrations that attenuated IPSCs. Furthermore, Ang II increased the firing rate of PVN neurons from 3.75 +/- 0.36 to 7.89 +/- 0.85 Hz (p < 0.05; n = 9), and such an effect was abolished by losartan. In addition, Ang II failed to excite PVN neurons in the presence of bicuculline. Thus, this study provides substantial new evidence that Ang II excites spinally projecting PVN neurons by attenuation of GABAergic synaptic inputs through activation of presynaptic AT1 receptors.

Background: Alcohol abuse increases the risk of acute lung injury in critically ill patients. We have shown that alveolar epithelial cell (AEC) apoptosis in response to inflammatory mediators, including tumor necrosis factor-alpha (TNF-alpha), parallels endotoxin-mediated acute lung injury in ethanol-fed rats. Although angiotensin II mediates TNF-alpha-induced apoptosis of AECs in vitro, its role in ethanol-mediated susceptibility to AEC apoptosis is unknown., Methods: Adult male rats were fed the Lieber-DeCarli diet for 6 weeks. AECs were isolated, and TNF-alpha- and angiotensin II-induced cytotoxicity (by terminal transferase-mediated dUTP nick end labeling staining) was determined with or without the addition of the angiotensin-converting enzyme inhibitor (lisinopril) or a selective blocker of the angiotensin II type 1 receptor (AT(1)) or type 2 receptor (AT(2)). Finally, the relative expression of the AT(1) and AT(2) receptors in AECs was determined by Western blot analysis., Results: TNF-alpha-induced cytotoxicity, but not angiotensin II-induced cytotoxicity, was prevented by lisinopril, indicating that de novo angiotensin II synthesis is required for TNF-alpha-induced apoptosis in these cells. Both TNF-alpha- and angiotensin II-induced cytotoxicity in AECs from control-fed and ethanol-fed rats were inhibited by the selective AT(2) blocker, PD123319, but not by the selective AT(1) blocker, losartan. In parallel, ethanol ingestion doubled AT(2) expression in AECs (by Western blot) but had no significant effect on AT(1) receptor expression., Conclusions: Chronic ethanol ingestion increases AT(2) expression in the alveolar epithelium and enhances TNF-alpha- and angiotensin II-induced cytotoxicity, both of which act via AT(2). Together, these findings suggest that selective AT(2) receptor inhibition could limit the development of acute lung injury in alcoholic patients.

We examined the regulation/expression of angiotensin II (Ang II) receptors in the transgenic (TG) (mRen-2)27 rat compared to the normal Sprague-Dawley (SD) rat. Ang II receptor binding and mRNA expression were determined by quantitative autoradiography and real-time PCR, respectively. Ang II receptors in the rat prostate rat were of the AT(1) receptor subtype and were significantly reduced in the prostate of the TG rat compared to the normal SD rat. However, AT(1) receptor binding was significantly higher in the prostate of the TG rat treated with the ACE inhibitor lisinopril compared to the untreated TG rat and comparable to the control SD rat. In contrast to the protein, AT(1) receptor mRNA expression was not reduced in the prostate of the TG rat compared to the SD rat. However, AT(1) receptor mRNA was markedly reduced in the prostate of the lisinopril-treated TG rat compared to the untreated TG rat or control SD rat. In conclusion, the findings suggest that AT(1) receptors are present in the rat prostate at a protein level and are subject to down-regulation in the TG rat which may be due to receptor internalisation as a consequence of receptor hyper-stimulation by increased local tissue levels of Ang II. Moreover, AT(1) receptor protein and mRNA expression in the prostate may be inversely modulated.

Background: Accumulating evidence suggests that C-reactive protein (CRP), in addition to predicting vascular disease, may actively facilitate lesion formation by inciting endothelial cell activation. Given the central importance of angiotensin type 1 receptor (AT1-R) in the pathogenesis of atherosclerosis, we examined the effects of CRP on AT1-R expression and kinetics in vascular smooth muscle (VSM) cells. In addition, the effects of CRP on VSM migration, proliferation, and reactive oxygen species (ROS) production were evaluated in the presence and absence of the angiotensin receptor blocker, losartan. Lastly, the effects of CRP (and losartan) on neointimal formation were examined in vivo in a rat carotid angioplasty model., Methods and Results: The effects of human recombinant CRP (0 to 100 microg/mL) on AT1-R transcript, mRNA stability, and protein expression were studied in cultured human VSM cells. AT1-R binding was assessed with 125I-labeled angiotensin II (Ang II). VSM migration was assessed with wound cell migration assays, whereas VSM proliferation was determined with [3H]-incorporation and cell number. The effects of CRP (and losartan) on Ang II-induced ROS production were evaluated by 2',7'-dichlorofluorescein fluorescence. Lastly, the effects of CRP (and losartan) on neointimal formation, VSM cell migration, proliferation, and matrix formation were studied in vivo in a rat carotid artery balloon injury model. CRP markedly upregulated AT1-R mRNA and protein expression and increased AT1-R number on VSM cells. CRP promoted VSM migration and proliferation in vitro and increased ROS production. Furthermore, CRP potentiated the effects of Ang II on these processes. In the rat carotid artery angioplasty model, exposure to CRP resulted in an increase in cell migration and proliferation, collagen and elastin content, and AT1-R expression, as well as an increase in neointimal formation; these effects were attenuated by losartan., Conclusions: CRP, at concentrations known to predict cardiovascular events, upregulates AT1-R-mediated atherosclerotic events in vascular smooth muscle in vitro and in vivo. These data lend credence to the notion that CRP functions as a proatherosclerotic factor as well as a powerful risk marker.

Experiments were performed to study angiotensin (Ang) AT1a and AT1b mRNA expression in mice, including, examination of brain distribution and the effect of salt loading. In situ hybridization (ISH) methods showed that the pattern of mRNA expression was identical for AT1a and AT1b, with cellular labeling in rostral forebrain, hypothalamus and brainstem. Receptor mRNAs were concentrated in brain regions involved in the regulation of electrolyte and cardiovascular balance. Immunocytochemistry with AT1 specific antisera showed a pattern that was consistent with the ISH. Reverse transcriptase-polymerase chain reaction (RT-PCR) of hypothalamus and pituitary verified the presence of both AT1a and AT1b mRNA. Using quantitative ISH, we found that AT1a mRNA expression was significantly increased after 5 days of 2% NaCl consumption in anterior third ventricle (AV3V), paraventricular hypothalamus (PVN) and subfornical organ (SFO), but unchanged in anterior pituitary. There were no significant changes in AT1b mRNA. These results document the utility of ISH coupled with quantitative imaging techniques for the study of subtype specific expression. Using ISH and RT-PCR, we verified that AT1a and AT1b receptors are expressed in mouse brain and pituitary and show a similar pattern of distribution. Salt loading produced a specific increase in AT1a mRNA in osmosensitive regions, suggesting that this receptor subtype is regulated by sodium/osmolar input.

Substitutions of Asn111 of the AT(1) angiotensin receptor and mutations of the corresponding amino acids in other G-protein-coupled receptors (GPCRs) cause constitutive receptor activation. Ligand binding and signalling of constitutively active mutant GPCRs are discussed and similarities and differences during the activation of amine and peptide GPCRs are identified. Studies using the AT(1) receptor suggest that conformational selection is not sufficient to explain the mechanism of receptor activation, and that agonist binding to the receptor provides energy to induce activation of the receptor. Because agonist binding also actively facilitates the conformational rearrangements leading to activation of other GPCRs we propose that agonist induction should be considered as a general mechanism of GPCR activation.

We recently reported that overexpression of the angiotensin II type 2 (AT2) receptor downregulates the AT1a receptor through the bradykinin/NO pathway in a ligand-independent manner in vascular smooth muscle cells (VSMCs). In the present study, we investigated the effect of AT2 receptor overexpression on the expression of the AT1a receptor and transforming growth factor-beta (TGF-beta) receptor subtypes in VSMCs from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Transfection of the AT2 receptor gene downregulated expression of the AT1a receptor in VSMCs from WKY, but did not affect expression of the AT1a receptor in VSMCs from SHR. Transfection of the AT2 receptor abolished DNA synthesis in response to angiotensin II in VSMCs from WKY; in VSMCs from SHR, basal DNA synthesis was suppressed, but DNA synthesis in response to Ang II was not altered. The NO substrate L-arginine augmented downregulation of the AT1a receptor in VSMCs from WKY, whereas it did not affect expression of the AT1a receptor in VSMCs from SHR. In response to AT2 receptor transfection, expression of TGF-beta type I receptor mRNA was suppressed significantly in VSMCs from WKY, whereas expression of TGF-beta type I receptor was not altered in VSMCs from SHR. These results suggest that the AT2 receptor downregulates AT1a and TGF-beta type I receptors in normal VSMCs, but not in SHR-derived VSMCs. The lack of downregulation of the AT1a receptor may contribute, in part, to the exaggerated growth of VSMCs from SHR.

Introduction: The circulating renin-angiotensin system (RAS) plays a crucial role in the regulation of blood pressure, electrolytes, and fluid homeostasis. In contrast to the circulating RAS, the presence of an intrinsic RAS has been demonstrated in different tissues/organs, which may affect both local and global functions of a biologic system. Our previous studies provided solid evidence of the existence of a local RAS in rat pancreas. Our further investigation showed that such a pancreatic RAS could be activated by experimental models of chronic hypoxia and chemically induced pancreatitis. These previous findings formed the basis for the current study., Methodology: Adult Sprague-Dawley rats were exposed to isobaric hypoxia (10% O2), and the effects on the circulating and pancreatic RAS were documented., Results: The current study shows that exposure of rats to isobaric hypoxia caused a time-dependent increase in plasma renin activity. The activation of circulating RAS by hypoxia was associated with a parallel upregulation of local RAS components, including the mRNA expression of angiotensinogen and angiotensin II receptor types I and II in the pancreas., Conclusion: The upregulation of local pancreatic RAS, along with its counterpart circulating RAS, may be responsible for both physiologic and pathophysiologic aspects of a biologic system under chronic hypoxic stress.

Background: Surgical therapy for aortic stenosis leads to reverse remodeling, with normalization of left ventricular hypertrophy (LVH). The aim of this study was to examine Renin-Angiotensin system (RAS) gene expression in this setting., Methods: Growing sheep (n=44) underwent supracoronary aortic banding for controlled induction of LVH at the age of 6 to 8 months (A=baseline). Surgical revision to completely release the pressure gradient was performed 8.3+/-1 months later (B). The animals were sacrificed after another 10.1+/-2 months (C). Along with hemodynamic measurements, subtractive hybridization and competitive polymerase chain reaction were applied to quantify mRNA expression for angiotensin-converting enzyme (ACE) and angiotensin receptors 1 and 2 (AT1-R and AT2-R)., Results: Left ventricular mass index was 82+/-21 g (A), 150+/-33 g (B), and 78+/-18 g (C), P<0.01. Left ventricular function and cardiac index remained stable. Myocardial fiber diameter was 11.3+/-0.8 (A), 15.9+/-1.2 (B), and 11.4+/-1 (C) microm, P<0.01. Gene expression was as follows: ACE 0.8+/-0.05 (A), 1.3+/-0.08 (B), and 0.9+/-0.06 (C), P<0.01; AT1-R 0.7+/-0.06 (A), 0.9+/-0.07 (B), and 0.3+/-0.04 (C), P<0.01; AT2-R 0.5+/-0.05 (A), 0.2+/-0.04 (B), and 0.5+/-0.05 (C), P<0.01., Conclusion: LVH in aortic stenosis coincides with significant alterations of the RAS. Surgical therapy leads to reverse remodeling, which is paralleled by regression of RAS gene expression.

Our laboratory demonstrated previously that spontaneously hypertensive rats (SHR) exhibited an elevated basal Fos expression in the nucleus tractus solitarii (NTS), the terminal site for primary baroreceptor afferents, and that Fos protein is required for the re-expression of angiotensin subtype 1 receptor (AT1R) mRNA in the NTS after baroreceptor activation. The present study evaluated the hypothesis that this re-expression of AT1R is augmented in SHR and is promoted by the heightened Fos expression. Reverse transcription-polymerase chain reaction analysis revealed that baroreceptor activation via sustained increase in systemic arterial pressure resulted in a discernible reduction in the expression of AT1R mRNA at the dorsomedial medulla of SHR and normotensive Wistar-Kyoto rats. However, SHR manifested an appreciably larger magnitude of decline, followed by a faster time course of re-expression in AT1R mRNA. Parallel findings were obtained from the pressor response induced by microinjection unilaterally of angiotensin II (40 pmol) into the NTS. Whereas the re-expression of AT1R at both transcriptional and functional expression levels after baroreceptor activation was discernibly blunted by prior bilateral application into the NTS of an antisense c-fos oligonucleotide (50 pmol), the suppression in SHR was again significantly more intense. Control pretreatment with the corresponding sense or scrambled c-fos oligonucleotide was ineffective. We conclude that the heightened Fos expression in SHR is causatively related to the augmented re-expression of AT1R in the NTS at both transcriptional and functional levels.