AT1 Receptor Antagonist Research Articles

Renal‐derived human sPRR increases blood pressure in female but not male mice

Previous studies showed that soluble prorenin receptor (sPRR) plays an important role in blood pressure regulation. In rodent models, sPRR contributes to AngII production by increasing renin activity and systolic blood pressure (SBP). Moreover, reducing sPRR release into plasma decreased blood pressure and attenuated angiotensin II (AngII) induced hypertension. However, there is a gap of knowledge concerning the functional role of locally produced sPRR from the kidney in blood pressure regulation. Additionally, the role of human sPRR in blood pressure regulation has not been delineated. Therefore, we evaluated the renal derived human sPRR role in SBP control.Human sPRR‐Myc‐tag transgenic mice were bred with mice expressing Hoxb7/Cre to selectively express human sPRR in the collecting duct (RHsPRR). RHsPRR and control (CTL) male and female mice were fed a standard diet for 10 months (n=8‐11/group). Body weight and urine flow rate were examined and SBP measured by radiotelemetry. Western blot analysis depicted the presence of human sPRR‐Myc‐tag (28 KDa) in the cortex and medulla of RHsPRR male mice which validated the humanized mouse model.Male mice have higher body weights than female mice but renal derived human sPRR did not influence body weights (M: 34.1±0.8 and 32.9±0.8g, F: 28.4±0.8 and 29.6±0.5g, P<0.05). Circulating sPRR was unchanged between male and female mice (M: 3995±643 and 4342±500pg/ml, F: 3479±194 and 3948±238pg/ml). SBP increased significantly in female RHsPRR mice compared to CTL (F: 118.7±2 and 127.2±3 mmHg, P<0.05) but not in male mice (M: 123.8±2 and 119.9±6 mmHg). The SBP response to losartan, an AT1R antagonist, was unchanged in females but was impaired in males (M: ‐7.6±1.2 and ‐5.5±0.8 mmHg, F: ‐6.5±1.2 and ‐7.9±1.5 mmHg, P<0.05). Renal derived human sPRR significantly increased AT1R gene expression in females but not in males (M: 1.8±0.6 and 1.2±0.3 2‐ΔΔCT, F: 1.6±0.5 and 3.8±0.9 2‐ΔΔCT, P<0.05). Moreover, renal derived human sPRR decreased the gene expression of renin and angiotensinogen in female mice (Renin: 1.5±0.5 and 0.4±0.1 2‐ΔΔCT, Angiotensinogen: F: 0.9±0.1 and 0.6±0.1 2‐ΔΔCT, P<0.05) suggesting a negative feedback loop involving Angiotensin II‐AT1R pathway. These effects were not observed in males (Renin: 3.3±1.2 and 4.1±1.2 2‐ΔΔCT; Angiotensinogen: M: 1.4±0.4 and 1.4±0.4 2‐ΔΔCT).Overall, our data suggest that renal‐derived human sPRR increases blood pressure in female mice likely via the activation of renal AT1R expression. In contrast, renal‐derived human sPRR expression impaired the blood pressure response to Losartan in male mice. In conclusion, our data support renal derived human sPRR as a promising therapeutic target in hypertension in humans.

Sexually dimorphic role of smooth muscle cell estrogen receptor alpha in aging‐associated arterial stiffness

BackgroundAging is associated with rising arterial stiffness (AS), an independent risk factor for cardiovascular disease. The development of aging‐associated AS follows a sexually dimorphic pattern with women developing AS later in life. Estrogen and estrogen receptor alpha (ERα) are protective in the premenopausal state, potentially due to the regulation of the vasoprotective angiotensin II type 2 receptor (AT2R), however, the role of estrogen/ERα in aging‐associated AS has not been defined and the mechanisms of the sexually‐dimorphic development of aging‐associated AS are unknown.Methods/ResultsAll studies were conducted in accordance with the FASEB Statement of Principles for the use of Animals in Research and Education. We generated novel smooth muscle cell‐specific ERα‐KO mice and measured aortic pulse wave velocity (PWV), an index of AS, in 3, 12, and 18 month old male and female intact and KO mice. Male mice develop increases in AS from 3 to 12 months compared to 3 to 18 months in females (p<0.05). SMC‐ERα‐KO protected females from the aging‐associated increase in AS (p<0.05). Circulating estrogens also declined in 18 month old females vs. 3 mo. (p<0.05), suggesting a role for unliganded SMC‐ERα in aging‐associated AS. SMC‐ERα‐KO females were also protected from the aging‐associated decline in aortic AT2R expression observed in intact females (p<0.05). To further investigate the potential role of unliganded SMC‐ERα in AS, we performed ovariectomy (OVX) or sham surgeries in young female SMC‐ERα‐Intact and KO mice and measured PWV. Results demonstrated an OVX‐driven increase in AS that was prevented by SMC‐ERα‐KO (p<0.05). This was also associated with an OVX‐mediated decrease in aortic expression of the AT2R, which was prevented by SMC‐ERα‐KO. To determine the in vivo role of AT2R in the vascular protection observed in 12 month old female mice, we treated 12 month old male and female mice with AT2R antagonist PD‐123319 (3mg/kg/day) or vehicle via osmotic minipumps for 4 weeks. Chronic inhibition of the AT2R increased AS in 12 month old females (p<0.05), but not males, suggesting that AT2R‐mediated protection contributes to the delay in aging‐associated AS in female mice. In vitro studies also demonstrated that estrogen upregulates AT2R mRNA and protein in female mouse aortic SMC, supporting an estrogen‐mediated mechanism of AT2R driven vascular protection in females.ConclusionThese data indicate sex differences in the time course of the development of aging‐associated arterial stiffness and suggest the presence of unliganded SMC‐ERα contributes specifically to aging‐associated arterial stiffness in females via reductions in vascular AT2R. Further, these results implicate that AT2R agonist therapy may improve cardiovascular outcomes in aging women to retain the beneficial downstream effects of estrogen signaling, without the potential detrimental effects of hormone replacement therapy.

Angiotensin II Type 1 Receptor‐Mediated Hypothalamic Paraventricular Nucleus Neuroinflammation And Blood Brain Barrier Disruption Contribute To Age‐Dependent Hypertension In Male, But Not Female, Sprague Dawley Rats

AimAging is a risk factor for the development of hypertension. Recently, neuroinflammation in cardio‐regulatory centers of the brain, specifically the hypothalamic paraventricular nucleus (PVN), has been identified as a potential driver of hypertension via sympathetically‐mediated mechanisms. We hypothesize neuroinflammatory mechanisms contribute to the progression of age‐dependent hypertension.MethodsIn male (M) Sprague Dawley (SD) rats aged 3, 8, and 16 months old (MO) and female (F) SD rats aged 3 and 16 MO femoral artery cannulation was used to measure mean arterial pressure (MAP; mmHg) and assess sympathetic tone to the vasculature (ΔMAP; mmHg) via depressor response to ganglionic blockade (i.v. hexamethonium 30 mg/kg). Global sympathetic tone was assessed as plasma norepinephrine (nmol/L) via ELISA. Blood brain barrier (BBB) dysfunction in the PVN was assessed via PVN FITC extravasation following co‐infusion of FITC‐Dextran (10 kDa) and rhodamine B isothiocyanate‐Dextran (70 kDa). Immunohistochemistry was performed in male rats only for microglia (CD11b/c), astrocytes (GFAP), interleukin 6 (IL‐6) and tumor necrosis factor‐alpha (TNF‐α) in the PVN. MAP, assessments of sympathetic tone, BBB dysfunction, and microglia and astrocyte activity were also measured in 16 MO M rats treated with subcutaneous (s.c.) losartan, an Angiotensin II Type 1 Receptor (AT1R) antagonist, for 21 days (s.c. 3 mg/kg/day; N=6/group).ResultsM, but not F, SD rats develop age‐dependent hypertension (MAP [mmHg] 3 MO M 124±4, 8 MO M 138±5, 16 MO M 153±5, p<0.05; 3 MO F 132±5, 16 MO F 133±4), increased vascular sympathetic tone (ΔMAP to hexamethonium [mmHg] 3 MO M ‐34±7, 8 MO M ‐68±5, 16 MO M ‐60±9, p<0.05; 3 MO F ‐36±8, 16 MO F ‐34±8) and increased global sympathetic tone (Plasma NE [nmol/L] 3 MO M 39±6, 8 MO M 44±8, 16 MO M 58±9, p<0.05; 3 MO F 44±6, 16 MO F 46±7). In M rats only, BBB dysfunction occurs at 8 MO and 16 MO‐ F rats do not exhibit BBB disruption with age (% FITC extravasation [% area] 3 MO M 1.5±1.0, 8 MO M 9.4±1.3, 16 MO M 9.8±2.6, p<0.05; 3 MO F 1.3±0.6, 16 MO F 2.67±1.37). Further, 16 MO M rats exhibit significant neuroinflammation indicated by increased astrocyte reactivity and microglial activation, and increased IL‐6 and TNF‐α expression in the PVN. Following losartan administration in 16 MO M rats, there is a significant decrease in MAP, vascular and global sympathetic tone. BBB disruption is attenuated and neuroinflammation is ameliorated following losartan treatment as shown by a decrease in microglial activation and astrocytic reactivity.ConclusionsAge‐dependent hypertension develops in a sex‐dependent manner in the SD rat, potentially due to a protective effect of female sex steroids. Amelioration of neuroinflammation and reduction of BBB disruption via AT1R antagonism attenuates hypertension, vascular tone and global sympathetic outflow in an age‐dependent manner in male rats. These results indicate that PVN neuroinflammation modulates sympathetic nervous system activity and age‐related control of blood pressure in male rats potentially by an AT1R‐mediated mechanism.

AT2R activation increases in vitro angiogenesis in pregnant human uterine artery endothelial cells.

Angiogenesis is vital during pregnancy for remodeling and enhancing vasodilation of maternal uterine arteries, and increasing uterine blood flow. Abnormal angiogenesis is associated with decreased uteroplacental blood flow and development of pregnancy disorders such as gestational hypertension, preeclampsia, fetal growth restriction, preterm delivery, stillbirth, and miscarriage. The mechanisms that contribute to normal angiogenesis remain obscure. Our previous studies demonstrated that expression of the angiotensin type 2 receptor (AT2R) is increased while the angiotensin type 1 receptor (AT1R) is unchanged in the endothelium of uterine arteries, and that AT2R-mediated pregnancy adaptation facilitates enhanced vasodilation and uterine arterial blood flow. However, the role of AT2R in regulating angiogenesis during pregnancy has never been studied. This study examines whether or not AT2R activation induces angiogenesis and, if so, what mechanisms are involved. To this end, we used primary human uterine artery endothelial cells (hUAECs) isolated from pregnant and nonpregnant women undergoing hysterectomy. The present study shows that Compound 21, a selective AT2R agonist, induced proliferation of pregnant-hUAECs, but not nonpregnant-hUAECs, in a concentration-dependent manner, and that this C21-induced mitogenic effect was blocked by PD123319, a selective AT2R antagonist. The mitogenic effects induced by C21 were inhibited by blocking JNK—but not ERK, PI3K, and p38—signaling pathways. In addition, C21 concentration dependently increased cell migration and capillary-like tube formation in pregnant-hUAECs. The membrane-based antibody array showed that C21 increased expression of multiple angiogenic proteins, including EGF, bFGF, leptin, PLGF, IGF-1, and angiopoietins. Our qPCR analysis demonstrates that C21-induced increase in expression of these angiogenic proteins correlates with a proportional increase in mRNA expression, indicating that AT2R activates angiogenic proteins at the transcriptional level. In summary, the present study shows that AT2R activation induces angiogenesis of hUAECs in a pregnancy-specific manner through JNK-mediated pathways with associated transcriptional upregulation of multiple proangiogenic proteins.

Effects of Goldblatt hypertension on rats' hippocampal cholinergic system.

BackgroundThe classical renin-angiotensin system (RAS) has an important role in the cardiovascular system and water homeostasis in the body. Recently, the existence of RAS with all of its components has been shown in the mammalian brain. RAS participates in many brain activities, including memory acquisition and consolidation. Since the cholinergic neurotransmission in the hippocampus is crucial for these functions, this study aims to evaluate the hippocampal angiotensin receptors (ATs) and choline acetyltransferase (ChAT) mRNA in the renovascular hypertensive rats in captopril- and losartan-treated hypertensive rats.MethodsThe rats were randomly divided into four groups of eight animals; sham, Goldblatt two kidney one clip (2K1C) hypertensive rats and Goldblatt 2K1C hypertensive rats received 5 mg/kg captopril and Goldblatt 2K1C hypertensive rats received 10 mg/kg losartan. After 8 days of treatment, the rats were sacrificed and angiotensin-converting enzyme (ACE), ChAT, AT1, and AT2 receptor mRNAs in the hippocampus of rats were assessed by real-time PCR. The Morris water maze test was applied to measure the cognitive functioning of the rats.ResultsHypertensive rats showed impaired acquisition and memory function in the Morris water maze test. Treatment with ACE inhibitor (captopril) and AT1 receptor antagonist (losartan) reversed the observed acquisition and memory deficit in hypertensive rats. Overexpression of AChE, AT1, and AT2 and low expression of ChAT were noted in the hippocampus of rats with Goldblatt hypertension compared with that of the sham group. Treatment with captopril significantly reversed these changes, while treatment with losartan slightly reduced the mentioned effects.ConclusionThe memory-enhancing effect of captopril in renovascular hypertensive rats might lead to increased hippocampal ChAT expression.

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

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

Central angiotensinergic mechanisms in female spontaneously hypertensive rats treated with estradiol

Estrogens reduce 0.3 M NaCl intake and palatability in a widely used model of essential hypertension, the spontaneously hypertensive rats (SHRs). Here we investigated whether the inhibitory effects of β-estradiol (E2, 10 μg/kg b.w. subcutaneously for 8 days) on water deprived partially-rehydrated (WD-PR) ovariectomized (OVX) adult female SHRs (fSHRs, n = 4–10/group) are related to interferences on brain angiotensin II AT1 receptors (AT1r). After WD-PR, E2 reduced 0.3 M NaCl intake (1.3 ± 0.6, vs. vehicle: 3.5 ± 1.2 ml/30 min), the number of hedonic responses to intraoral NaCl infusion (57 ± 11, vs. vehicle: 176 ± 32/min), and the relative angiotensin AT1r (Agtr1a) mRNA expression in the hypothalamus. Losartan (AT1r antagonist, 100 μg) intracerebroventricularly in OVX fSHRs treated with vehicle subcutaneously abolished 0.3 M NaCl intake (0.1 ± 0.1 ml/30 min) and only transiently reduced hedonic responses to intraoral NaCl. Losartan combined with E2 decreased the number of hedonic and increased the number of aversive responses to intraoral NaCl and abolished 0.3 M NaCl intake. E2 also reduced the pressor and dipsogenic responses to intracerebroventricular angiotensin II. The results suggest that AT1r activation increases palatability and induces NaCl intake in WD-PR fSHRs. E2 reduced hypothalamic Agtr1a mRNA expression, which may account for the effects of E2 on NaCl intake and palatability and intracerebroventricular angiotensin II-induced pressor and dipsogenic responses in OVX fSHRs. Future studies considering natural fluctuations in estrogen secretion might help to determine the degree of such interference in brain neuronal activity.

Involvement of V1-type vasopressin receptors on NaCl intake by hyperosmotic rats treated with muscimol in the lateral parabrachial nucleus

GABAA receptor activation with agonist muscimol in the lateral parabrachial nucleus (LPBN) induces 0.3 M NaCl intake. In the present study, we investigated water and 0.3 M NaCl intake in male adult rats treated with losartan (angiotensin AT1 receptor antagonist) or MeT-AVP (V1-type vasopressin receptor antagonist) combined with muscimol or methysergide (5-HT2 antagonist) into the LPBN in rats treated with intragastric 2 M NaCl. After 2 M NaCl load and bilateral injections of muscimol (0.5 nmol/0.2 µL) into the LPBN, rats ingested water and 0.3 M NaCl. The pre-treatment of the LPBN with MeT-AVP (1 nmol/0.2 µL) but not losartan (50 µg/0.2 µL) in muscimol treated rats reduced 0.3 M NaCl intake. The pre-treatment of the LPBN with MeT-AVP did not modify the increased 0.3 M NaCl intake in rats treated with methysergide (4 µg/0.2 µL), suggesting that the effect of MeT-AVP was not due to non-specific inhibition of ingestive behavior. The results suggest that endogenous vasopressin in the LPBN facilitates the effects of GABAergic activation driving cell-dehydrated male rats to ingest 0.3 M NaCl.

The Retinal Renin-Angiotensin-Aldosterone System: Implications for Glaucoma.

Aldosterone is one of the main effectors of the renin-angiotensin-aldosterone system (RAAS) along with having roles in hypertension, and cardiovascular and renal diseases. Recent evidence has also shown the presence of an active local RAAS within the human eye. It has been shown that at 12 h after a retinal ischemia-reperfusion injury, there is an upregulation of the protein levels of angiotensin II type 1 receptor (AT1-R) in the retina. Furthermore, at 12 h after reperfusion, there is an increase in reactive oxygen species (ROS) production in the retina that is mediated via an NADPH oxidase pathway. This ischemia-reperfusion injury-induced increase of retinal ROS levels and NADPH oxidase expression can be prevented by the administration of an AT1-R antagonist. This suggests that one of the main retinal ischemic injury pathways is via the local RAAS. It has also been reported that progressive retinal ganglion cell loss and glaucomatous optic nerve degeneration without elevated intraocular pressure occur after administration of local or systemic aldosterone. Elucidation of glaucoma pathogenesis, especially normal-tension glaucoma (NTG) subtype by our current animal model can be used for identifying potential therapeutic targets. Based on these results, we are further evaluating NTG prevalence among primary aldosteronism patients.

Pleiotropic effects of AT-1 receptor antagonists in hypoxia induced by cardiac ischaemia.

The renin-angiotensin system (RAS) plays a crucial role and coordinates multiple body functions through its hormonal mechanism. The RAS is supported in its function by numerous peptides such as angiotensin II (Ang II), Ang IV, Ang III, angiotensin (1-7) and (1-9). The system formed by ACE2/Ang(1-7)/MASr is a regulatory pathway within the RAS system and its functions are different from those of the ACE/Ang II/AT-1r system. Recently, it has been discovered that a key role of the RAS and the ACE2/Ang(1-7)/MASr system is in inflammatory processes such as cardiac hypertrophy and heart failure. Studies are ongoing to better understand and comprehend the function of the RAS in inflammation. Recent evidence associates AT-1r antagonists with a cardioprotective, anti-inflammatory, and anti-hypertrophic role. In this in vitro study, we demonstrate the protective role of treatment (50 and 200μM) of an AT-1r antagonist, irbesartan, on hypoxia and inflammation-induced damage in cardiomyocytes.

Thyroid hormone increases oxygen metabolism causing intrarenal tissue hypoxia; a pathway to kidney disease.

The proposed mechanisms for the development of nephropathy are many, complex and often overlapping. Although recent literature strongly supports a role of kidney hypoxia as an independent pathway to nephropathy, the evidence remains inconclusive since the role of hypoxia is difficult to differentiate from confounding factors such as hyperglycemia, hypertension and oxidative stress. By increasing kidney oxygen consumption using triiodothyronine (T3) and, thus, avoiding these confounding factors, the aim of the present study was to investigate renal hypoxia per se as a causal pathway for the development of nephropathy. Healthy Sprague-Dawley rats were treated with T3 (10 μg/kg/day) and the angiotensin II AT1-receptor antagonist candesartan (1 mg/kg in drinking water) to eliminate effects of T3-induced renin release; and compared to a candesartan treated control group. After 7 weeks of treatment in vivo kidney function, oxygen metabolism and mitochondrial function were evaluated. T3 did not affect glomerular filtration rate or renal blood flow, but increased total kidney oxygen consumption resulting in cortical hypoxia. Nephropathy, demonstrated as albuminuria and tubulointerstitial fibrosis, developed in T3-treated animals. Mitochondria uncoupling mediated by uncoupling protein 2 and the adenosine nucleotide transporter was demonstrated as a mechanism causing the increased kidney oxygen consumption. Importantly, blood glucose levels, mean arterial blood pressure and oxidative stress levels were not affected by T3. In conclusion, the present study provides further evidence for increased kidney oxygen consumption causing intrarenal tissue hypoxia, as a causal pathway for development of nephropathy.

Schizophrenia-like endurable behavioral and neuroadaptive changes induced by ketamine administration involve Angiotensin II AT1 receptor

Schizophrenia is a chronic disease affecting 1% worldwide population, of which 30% are refractory to the available treatments: thus, searching for new pharmacological targets is imperative. The acute and repeated ketamine administration are validated preclinical models that recreate the behavioral and neurochemical features of this pathology, including the parvalbumin-expressing interneurons dysfunction. Angiotensin II, through AT1 receptors (AT1-R), modulates the dopaminergic and GABAergic neurotransmission. We evaluated the AT1-R role in the long-term neuronal activation and behavioral alterations induced by repeated ketamine administration. Adult male Wistar rats received AT1-R antagonist candesartan/vehicle (days 1–10) and ketamine/saline (days 6–10). After 14 days of drug-free, neuronal activation and behavioral analysis were performed. Locomotor activity, social interaction and novel object recognition tests were assessed at basal conditions or after ketamine challenge. Immunostaining for c-Fos, GAD67 and parvalbumin were assessed after ketamine challenge in cingulate, insular, piriform, perirhinal, and entorhinal cortices, striatum, and hippocampus. Additionally, to evaluate the AT1-R involvement in acute ketamine psychotomimetic effects, the same behavioral tests were performed after 6 days of daily-candesartan and a single-ketamine administration. We found that ketamine-induced long-lasting schizophrenia-like behavioral alterations, and regional-dependent neuronal activation changes, involving the GABAergic neurotransmission system and the parvalbumin-expressing interneurons, were AT1-R-dependent. The AT1-R were not involved in the acute ketamine psychotomimetic effects. These results add new evidence to the wide spectrum of action of ketamine and strengthen the AT1-R involvement in endurable alterations induced by psychostimulants administration, previously proposed by our group, as well as their preponderant role in the development of psychiatric pathologies.

Angiotensinogen uptake and stimulation of oxidative stress in human pigment retinal epithelial cells

We previously reported that isolated proximal tubules (PT) internalize the precursor protein angiotensinogen and that the 125Iodine-labeled protein accumulated in the nuclear and mitochondrial fractions of the PT cells; however, whether internalization of angiotensinogen occurs in non-renal epithelial cells is unknown. Therefore, the present study assessed the cellular uptake of 125I-angiotensinogen in human retinal pigment ARPE-19 epithelial cells, a widely utilized cell model for the assessment of retinal injury, inflammation and oxidative stress. ARPE-19 cells, maintained in serum-free media to remove extracellular sources of bovine serum angiotensinogen and renin, were incubated with 125Iodine-angiotensinogen at 37 °C and revealed the time-dependent uptake of angiotensinogen over 24 h. In contrast, incubation with labelled Ang II, Ang-(1−7) or Ang I revealed minimal cellular uptake. Subcellular fractionation following a 4-hour uptake of 125I-angiotensinogen revealed that the majority of the labeled protein localized to the nuclear fraction with lower accumulation in the mitochondrial and cytosolic fractions. Finally, we show that addition of angiotensinogen (2 nM) to the ARPE-19 cells increased oxidative stress as assessed by DCF fluorescence that was blocked by pretreatment of the cells with either the NADPH oxidase 1/4 inhibitor GKT137831, apocynin or atorvastatin, but not the AT1 receptor antagonist losartan. In contrast, treatment of the cells with Angiotensin II at an equivalent dose to angiotensinogen failed to stimulate oxidative stress. We conclude that human retinal pigment cells internalize angiotensinogen to elicit an increase in oxidative stress through a pathway that appears distinct from the Ang II-AT1 receptor axis.

Angiotensin Type-1 Receptor Inhibition Reduces NLRP3 Inflammasome Upregulation Induced by Aging and Neurodegeneration in the Substantia Nigra of Male Rodents and Primary Mesencephalic Cultures.

The tissue renin–angiotensin system (RAS) has been shown to be involved in prooxidative and proinflammatory changes observed in aging and aging-related diseases such as dopaminergic degeneration in Parkinson’s disease (PD). We studied the activation of the NLRP3 inflammasome in the substantia nigra with aging and early stages of dopaminergic degeneration in PD models and, particularly, if the brain RAS, via its prooxidative proinflammatory angiotensin II (AngII) type 1 (AT1) receptors, mediates the inflammasome activation. Nigras from aged rats and mice and 6-hydroxydopamine PD models showed upregulation in transcription of inflammasome-related components (NLRP3, pro-IL1β and pro-IL18) and IL1β and IL18 protein levels, which was inhibited by the AT1 receptor antagonist candesartan. The role of the AngII/AT1 axis in inflammasome activation was further confirmed in rats intraventricularly injected with AngII, and in primary mesencephalic cultures treated with 6-hydroxydopamine, which showed inflammasome activation that was blocked by candesartan. Observations in the nigra of young and aged AT1 and AT2 knockout mice confirmed the major role of AT1 receptors in nigral inflammasome activation. In conclusion, the inflammasome is upregulated by aging and dopaminergic degeneration in the substantia nigra, possibly related with a decrease in dopamine levels, and it is mediated by the AngII/AT1 axis.

Regulation of total LC3 levels by angiotensin II in vascular smooth muscle cells

Hypertension is associated with high circulating angiotensin II (Ang II). We have reported that autophagy regulates Ang II‐induced vascular smooth muscle cell (VSMC) hypertrophy, but the mechanism mediating this effect is still unknown. Therefore, we studied how Ang II regulates LC3 levels in VSMCs and whether Bag3, a co‐chaperone known to regulate LC3 total levels, may be involved in the effects elicited by Ang II. A7r5 cell line or rat aortic smooth muscle cell (RASMC) primary culture were stimulated with Ang II 100 nM for 24 h and LC3 I, LC3 II and Bag3 protein levels were determined by Western blot. MAP1LC3B mRNA levels were assessed by RT‐qPCR. Ang II increased MAP1LC3B mRNA levels and protein levels of LC3 I, LC3 II and total LC3 (LC3 I + LC3 II). Cycloheximide, but not actinomycin D, abolished LC3 II and total LC3 increase elicited by Ang II in RASMCs. In A7r5 cells, cycloheximide prevented the Ang II‐mediated increase of LC3 I and total LC3, but not LC3 II. Moreover, Ang II increased Bag3 levels, but this increase was not observed upon co‐administration with either losartan 1 μM (AT1R antagonist) or Y‐27632 10 μM (ROCK inhibitor). These results suggest that Ang II may regulate total LC3 content through transcriptional and translational mechanisms. Moreover, Bag3 is increased in response to Ang II by a AT1R/ROCK signalling pathway. These data provide preliminary evidence suggesting that Ang II may stimulate autophagy in VSMCs by increasing total LC3 content and LC3 processing.

AMPA and angiotensin type 1 receptors are necessary for hemorrhage-induced vasopressin secretion.

Hypovolemia induced by hemorrhage is a common clinical complication, which stimulates vasopressin (AVP) secretion by the neurohypophysis in order to retain body water and maintain blood pressure. To evaluate the role of brain L-glutamate and angiotensin II on AVP secretion induced by hypovolemia we induced hemorrhage (∼25% of blood volume) after intracerebroventricular (icv) administration of AP5, NBQX, or losartan, which are NMDA, AMPA, and AT1 receptor antagonists, respectively. Hemorrhage significantly increased plasma AVP levels in all groups. The icv injection of AP5 did not change AVP secretion in response to hemorrhage. Conversely, icv administration of both NBQX and losartan significantly decreased plasma AVP levels after hemorrhage. Therefore, the blockade of AMPA and AT1 receptors impaired AVP secretion in response to hemorrhage, suggesting that L-glutamate and angiotensin II acted in these receptors to increase AVP secretion in response to hemorrhage-induced hypovolemia.

Paternal long-term PM2.5 exposure causes hypertension via increased renal AT1R expression and function in male offspring.

Maternal exposure to fine particulate matter (PM2.5) causes hypertension in offspring. However, paternal contribution of PM2.5 exposure to hypertension in offspring remains unknown. In the present study, male Sprague-Dawley rats were treated with PM2.5 suspension (10 mg/ml) for 12 weeks and/or fed with tap water containing an antioxidant tempol (1 mM/L) for 16 weeks. The blood pressure, 24 h-urine volume and sodium excretion were determined in male offspring. The offspring were also administrated with losartan (20 mg/kg/d) for 4 weeks. The expressions of angiotensin II type 1 receptor (AT1R) and G-protein–coupled receptor kinase type 4 (GRK4) were determined by qRT-PCR and immunoblotting. We found that long-term PM2.5 exposure to paternal rats caused hypertension and impaired urine volume and sodium excretion in male offspring. Both the mRNA and protein expression of GRK4 and its downstream target AT1R were increased in offspring of PM2.5-exposed paternal rats, which was reflected in its function because treatment with losartan, an AT1R antagonist, decreased the blood pressure and increased urine volume and sodium excretion. In addition, the oxidative stress level was increased in PM2.5-treated paternal rats. Administration with tempol in paternal rats restored the increased blood pressure and decreased urine volume and sodium excretion in the offspring of PM2.5-exposed paternal rats. Treatment with tempol in paternal rats also reversed the increased expressions of AT1R and GRK4 in the kidney of their offspring. We suggest that paternal PM2.5 exposure causes hypertension in offspring. The mechanism may be involved that paternal PM2.5 exposure-associated oxidative stress induces the elevated renal GRK4 level, leading to the enhanced AT1R expression and its-mediated sodium retention, consequently causes hypertension in male offspring.

Simultaneous Determination of Candesartan and Hydrochlorothiazide in Human Plasma by HPLC-MS/MS

Introduction. Combined drugs have the greatest efficacy and safety in arterial hypertension treatment. The combination of candesartan and hydrochlorothiazide (AT1-receptor antagonist and a thiazide diuretic, respectively) provides high efficiency of antihypertensive combination therapy, therefore it is widely used in medical practice. Developing a method for simultaneous determination of candesartan and hydrochlorithiazide in human blood plasma is necessary for performing the analytical part of pharmacokinetic studies and bioequivalence studies of multicomponent drugs.Aim. The aim of this study is to develop a method for quantitative determination of candesartan and hydrochlorothiazide in human plasma by high-performance liquid chromatography – tandem mass spectrometry (HPLC-MS/MS) for further bioequivalence studies.Materials and methods. Determination of candesartan and hydrochlorothiazide in human plasma by HPLC-MS/MS. The samples were processed by acetonitrile protein precipitation. Internal standard: mixed solution of valsartan and indapamide. Mobile phase: 0.1 % formic acid solution in water (eluent A), 0.1 % formic acid in acetonitrile (eluent B). Column: Phenomenex Luna Phenyl-Hexyl, 50x4.6 mm, 5 μm. Analytical range: 2.00– 300.00 ng/mL for candesartan, 2.00–200.00 ng/mL for hydrochlorothiazide in human plasma. Ionization source: electrospray ionization. Detection conditions: 441.10 → 192.00 m/z, 441.10 → 263.15 m/z (candesartan), 295.85 → 269.00 m/z (hydrochlorothiazide), 436.00 → 207.05 m/z (valsartan), 363.85 → 132.10, 363.85 → 189.00 m/z (indapamide).Results and discussion. This method was validated by selectivity, matrix effect, calibration curve, accuracy, precision, spike recovery, the lower limit of quantification, carry-over effect and stability. The developed method meets the requirements for conducting bioequivalence studies of medicinal products within the framework of the Eurasian Economic Union.Conclusion. The analytical range was 2.00–300.00 ng/mL for candesartan, 2.00–200.00 ng/mL for hydrochlorothiazide in human plasma. The method was applied in BE study of the combination of candesartan and hydrochlorothiazide.

Abstract 14470: Novel Autophagy Mechanisms of NP-6A4 Protection in Heart Disease Induced by Untreated Obesity

Introduction & Hypothesis: Untreated obesity induces insulin resistance and heart disease affecting 42% of the US population. Autophagy, a conserved homeostatic process for cellular quality control, plays a critical role in maintaining health. Heart tissues of obese humans and rodent models show impaired autophagy. Drugs that can regulate autophagy are thus of high clinical significance. We reported recently that NP-6A4, a new peptide agonist of the cardiovascular protective angiotensin receptor AT2R, mitigated obesity-induced cardiac dysfunction and damage in rats with untreated obesity and pre-diabetes. We hypothesized that activation of autophagy by NP-6A4 is involved in NP-6A4-AT2R-induced cardioprotection in untreated obesity. Methods & Results: 7-week old female ZDF rats fed high fat diet (D12468, Research Diet) for 6 weeks were randomized to receive either NP-6A4 (10mg/kg/day), NP6A4+AT2R antagonist PD123319 (PD, 5mg/kg/day) delivered subcutaneously (N=6/group), or vehicle (N=5) for 4 weeks. Echocardiography showed that NP-6A4 reduced abnormal E/A ratio from 2.1 to 1.45 (P<0.006), and isovolumic contraction (P<0.004) and relaxation times (P<0.02), and increased myocardial radial strain (P<0.019) and radial strain rate (P<0.033). PD reversed these effects. Immunohistochemistry (IHC) and Western blot analysis revealed a significant 5.9-fold increase (p<0.0006) in LC3 puncta (Fig. A-B) and 2.2-fold increase (p<0.0008) in lipidated autophagy marker LC3-II (Fig. C) compared to vehicle controls. The autophagy activation by NP-6A4 was not seen with the co-administration of PD (Fig. A-C). Cardiac proteome analysis showed that Sec16a, an important partner of autophagy initiators ULK1/2 is increased in ZDF rat heart by NP-6A4 (2.75 fold; P<0.020), but PD suppressed this effect. Conclusions: We identified NP-6A4’s dual targeting of AT2R and novel autophagy regulation to mitigate cardiac dysfunction induced by untreated obesity in female rats

Abstract 14318: Sex Differences in Treatment Responses to NP-6A4, a Cardiomyopathy Drug With the FDA Designation, in Heart Disease Induced by Untreated Obesity

Introduction: Obesity affects 42% of the US population and exacerbates heart disease. Currently there are no effective treatments for obesity-induced cardiac pathology. We reported that NP-6A4, a peptide agonist of the Angiotensin receptor AT2R mitigates cardiac dysfunction and damage in male rats with untreated obesity. Since cardiac pathology of male and female rats with untreated obesity differ, we hypothesized that there can be differences in their cardiac response to NP-6A4. Methods and Results: Echocardiographic analysis was performed in 15-week old male and female healthy Wistar (N=8) or Zucker lean (N=7) rats treated with saline, and obese and pre-diabetic Zucker rats treated with saline or NP-6A4 (1.8-10mg/kg/day in saline) for four weeks (N=8/group for females, N=7/group for males; subcutaneous delivery). Compared to healthy males, obese males showed significant (P<0.05) reduction in heart rate and increase in anterior wall thickness and left ventricular (LV) filling pressure (E/E’) suggesting concentric hypertrophy. Compared to healthy females, obese females showed significant (P<0.05) reduction in relative wall thickness, heart rate, and ejection fraction (although preserved) and increase in LV internal diameter at both diastole and systole indicating eccentric hypertrophy. NP-6A4 treatment suppressed the pathologic changes in both obese males and females (P<0.05). In another cohort of obese female rats NP-6A4 treatment from 13-to 17-weeks significantly (P<0.05) reduced abnormal E/A ratio and isovolumic contraction and relaxation time, and improved myocardial radial strain. Treatment with AT2R antagonist PD123319 (N=8: 5mg/kg/day delivered subcutaneously) abolished NP-6A4 effects. Cardiac proteome analysis of 18 tissue samples from this cohort using data independent acquisition (DIA) resulted in a total proteome of 4520 proteins and 296 differentially expressed proteins that were significant (P<0.01). Ingenuity pathway analysis predicted that inhibition of NP-6A4-mediated signaling by PD123319 may increase infection of cells (P=<0.001). Conclusion: NP-6A4 attenuated cardiac pathology induced by untreated obesity by altering different cardiac parameters in male and female rats.