Administration Of Angiotensin II Research Articles

Angiotensin-II drives changes in microglia–vascular interactions in rats with heart failure

Activation of microglia, the resident immune cells of the central nervous system, leading to the subsequent release of pro-inflammatory cytokines, has been linked to cardiac remodeling, autonomic disbalance, and cognitive deficits in heart failure (HF). While previous studies emphasized the role of hippocampal Angiotensin II (AngII) signaling in HF-induced microglial activation, unanswered mechanistic questions persist. Evidence suggests significant interactions between microglia and local microvasculature, potentially affecting blood–brain barrier integrity and cerebral blood flow regulation. Still, whether the microglial-vascular interface is affected in the brain during HF remains unknown. Using a well-established ischemic HF rat model, we demonstrate the increased abundance of vessel-associated microglia (VAM) in HF rat hippocampi, along with an increased expression of AngII AT1a receptors. Acute AngII administration to sham rats induced microglia recruitment to brain capillaries, along with increased expression of TNFα. Conversely, administering an AT1aR blocker to HF rats prevented the recruitment of microglia to blood vessels, normalizing their levels to those in healthy rats. These results highlight the critical importance of a rather understudied phenomenon (i.e., microglia-vascular interactions in the brain) in the context of the pathophysiology of a highly prevalent cardiovascular disease, and unveil novel potential therapeutic avenues aimed at mitigating neuroinflammation in cardiovascular diseases.

Clinical Response to Third-Line Angiotensin-II vs Epinephrine in Septic Shock: A Propensity-Matched Cohort Study.

The appropriate third-line vasopressor in septic shock patients receiving norepinephrine and vasopressin is unknown. Angiotensin-II (AT-II) offers a unique mechanism of action to traditionally used vasopressors in septic shock. The objective of this study was to compare the clinical efficacy and safety of third-line AT-II to epinephrine in patients with septic shock. A single-center, retrospective cohort study of critically ill patients was performed between April 1, 2019 and July 31, 2022. Propensity-matched (2:1) analysis compared adults with septic shock who received third-line AT-II to controls who received epinephrine following norepinephrine and vasopressin. The primary outcome was clinical response 24 hours after third-line vasopressor initiation. Additional efficacy and safety outcomes were investigated. Twenty-three AT-II patients were compared with 46 epinephrine patients. 47.8% of AT-II patients observed a clinical response at hour 24 compared with 28.3% of epinephrine patients (P = 0.12). In-hospital mortality (65.2% vs 73.9%, P = 0.45), cardiac arrhythmias (26.1% vs 26.1%, P = 0.21), and thromboembolism (4.3% vs 2.2%, P = 0.61) were not observed to be statistically different between groups. Administration of AT-II as a third-line vasopressor agent in septic shock patients was not associated with significantly improved clinical response at hour 24 compared with epinephrine. Although underpowered to detect meaningful differences, the clinical observations of this study warrant consideration and further investigation of AT-II as a third-line vasopressor in septic shock.

Novel preventive effect of isorhamnetin on electrical and structural remodeling in atrial fibrillation.

Isorhamnetin, a natural flavonoid, has strong antioxidant and antifibrotic effects, and a regulatory effect against Ca2+-handling. Atrial remodeling due to fibrosis and abnormal intracellular Ca2+ activities contributes to initiation and persistence of atrial fibrillation (AF). The present study investigated the effect of isorhamnetin on angiotensin II (AngII)-induced AF in mice. Wild-type male mice (C57BL/6J, 8 weeks old) were assigned to three groups: (1) control group, (2) AngII-treated group, and (3) AngII- and isorhamnetin-treated group. AngII (1000 ng/kg/min) and isorhamnetin (5 mg/kg) were administered continuously via an implantable osmotic pump for two weeks and intraperitoneally one week before initiating AngII administration, respectively. AF induction and electrophysiological studies, Ca2+ imaging with isolated atrial myocytes and HL-1 cells, and action potential duration (APD) measurements using atrial tissue and HL-1 cells were performed. AF-related molecule expression was assessed and histopathological examination was performed. Isorhamnetin decreased AF inducibility compared with the AngII group and restored AngII-induced atrial effective refractory period prolongation. Isorhamnetin eliminated abnormal diastolic intracellular Ca2+ activities induced by AngII. Isorhamnetin also abrogated AngII-induced APD prolongation and abnormal Ca2+ loading in HL-1 cells. Furthermore, isorhamnetin strongly attenuated AngII-induced left atrial enlargement and atrial fibrosis. AngII-induced elevated expression of AF-associated molecules, such as ox-CaMKII, p-RyR2, p-JNK, p-ERK, and TRPC3/6, was improved by isorhamnetin treatment. The findings of the present study suggest that isorhamnetin prevents AngII-induced AF vulnerability and arrhythmogenic atrial remodeling, highlighting its therapeutic potential as an anti-arrhythmogenic pharmaceutical or dietary supplement.

Neurons of the median preoptic nucleus contribute to chronic angiotensin II-salt induced hypertension in the rat.

Experiments were designed to test the hypothesis that median preoptic (MnPO) neurons are necessary for the full hypertensive response to chronic angiotensin II (AngII) in rats consuming a high salt diet. The MnPO is implicated in many of the physiologic actions of AngII, primarily acting as a downstream nucleus to AngII binding at circumventricular organs such as the organum vasculosum of the lamina terminalis (OVLT). We have previously shown a prominent effect of lesion of the OVLT on the chronic hypertensive effects of AngII in rats consuming high salt. Additionally, we have shown that lesion of the MnPO attenuated the hypertensive response to chronic intravenous infusion of AngII in rats. However, whether MnPO neurons or fibers of passage contribute to this response is not clear. Male Sprague Dawley rats were randomly assigned to either sham (SHAM; n=8) or ibotenic acid lesion of the MnPO (MnPOx; n=6). In the MnPOx group, 200 nl of ibotenic acid in phosphate buffer saline (5 μg/μl) was injected into each of 3 predetermined coordinates targeted at the entire MnPO. After a week of recovery, rats were instrumented with radiotelemetric pressure transducers, provided 2.0% NaCl diet and distilled water ad libitum and given another week to recover. After 3 days of baseline measurements, osmotic minipumps were implanted subcutaneously in all rats for administration of AngII at a rate of 150 ng/kg/min. Blood pressure measurements were made for 14 days after minipump implantation. By day 7 of AngII treatment, blood pressure responses appeared to plateau in both groups while the hypertensive response was markedly attenuated in MnPOx rats (MnPOx, 122 ± 6 mmHg; SHAM, 143 ± 8 mmHg). These results support the hypothesis that neurons of the MnPO are involved in the central pathway mediating the chronic hypertensive effects of AngII in rats consuming a high salt diet.

Symposium 2-3 - Thrombomodulin as a new therapeutic target for preeclampsia

Thrombomodulin (TM) is a transmembrane glycoprotein abundantly expressed on vascular endothelial cells and have been known as molecules with anticoagulant and anti-inflammatory properties. TM binds to a danger signal, HMGB1, and suppresses the inflammatory response induced by HMGB1. This study aimed to clarify the relationship between HMGB1 and the pathological mechanisms of preeclampsia (PE). Additionally, we explored the therapeutic potential of TM for PE. We used angiotensin II (AngII)-induced PE mouse model, in which continuous administration of Ang II using subcutaneous pump developed PE symptoms in pregnant mice. AngII administration caused placental release of HMGB1, leading to enhanced production of sFlt-1 and the inflammatory cytokines in trophoblast cells. When TM was given to the PE mouse model, improvements in fetal growth, maternal hypertension and urinary protein were observed. Furthermore, in-vitro experiments suggest that antagonism of HMGB1 underlies the therapeutic effect of TM on PE. In the studies using the serum samples and placental tissues obtained from pregnant women, HMGB1 and TM levels in peripheral blood were higher in pregnant women with PE, and there was a negative correlation between the two in pregnant women with PE. Immunostaining of PE placentas showed HMGB1 release from the nucleus and defective staining of membrane-type TM on the surface of syncytiotrophoblast. These findings supported that the increased HMGB1 and TM levels in the peripheral blood of PE women originated from the placenta. In conclusion, HMGB1 released from the dysfunctional placenta is deeply involved in the pathogenesis of PE and that inhibition of HMGB1 action by TM leads to improvement of placental function. Treatment strategy aiming to inhibit HMGB1 action, including TM, is a new therapeutic approach for PE.

PERMISSIVE ROLE OF ANGIOTENSIN II IN MANTENANCE OF ENDOTHELIUM-DEPENDENT VASODILATION IN CEREBRAL MICROCIRCULATION

Objective: A number of data suggest, rather controversial, permissive role of angiotensin II (ANGII) in maintaining vasodilator responses in microcirculation, via its interaction with AT1 receptors (AT1R). This study aimed to determine the role of ANGII and AT1R in the mechanisms of flow-induced dilation (FID) in middle cerebral arteries (MCA) and incidence of oxidative stress at the functional, cellular and molecular level in the cerebral vasculature and in the serum of Sprague-Dawley (SD) rats fed high salt diet (HS). Design and method: Eleven-week old, male SD rats were randomly assigned to control group (CTRL, 0.4% NaCl in rat chow); Losartan group (rats on a standard diet given the AT1R blocker losartan (1 mg/mL) in drinking water); HS group (7 days 4% NaCl in rat chow) or HS+ANGII group (7 days HS with 3 days ANGII administration via osmotic minipumps (100 ng/kg/min on days 4–7)). FID of MCA was determined in absence/presence of the NOS inhibitor L-NAME, the non-selective cyclooxygenases inhibitor indomethacin, and the superoxide dismutase mimetic TEMPOL. Gene and protein expression of antioxidative enzymes and enzymes involved in FID mechanisms were determined by RT-qPCR and western blot. Vascular NO and superoxide/ROS levels were assessed by direct fluorescence. Serum systemic oxidative stress parameters was measured by spectrophotometry. All experimental procedures conformed to the EU Directive 2010/63/EU. Results: HS or AT1R blockade impaired FID and increased oxidative stress. Supplementation of ANGII restored the mechanisms of FID in MCA of rats fed HS diet to the ones present in CTRL rats without affecting arterial blood pressure by decreasing systemic oxidative stress and decreasing superoxide/ROS levels and increased NO bioavailability in the vascular wall of HS treated rats. The AT1R blockade resulted in significantly attenuated endothelium-dependent dilation, changed mechanisms of FID, increased vascular and systemic oxidative stress and decreased expression/ activity of antioxidative enzymes. Conclusions: ANGII is crucial in the preservation of vascular oxidative stress-antioxidant system balance and via AT1R, in maintenance of physiological vasodilation mechanisms of MCA. Suppression of ANGII is a key link between HS intake and development of endothelial dysfunction, and impaired regulation of cerebral blood flow.

High salt induced augmentation of angiotensin II mediated hypertension is associated with differential expression of tumor necrosis factor-alpha receptors in the kidney

Aim: Chronic high salt (HS) intake causes minimal changes in blood pressure (BP) but it induces augmented hypertensive response to angiotensin II (AngII) administration in rodents. The mechanism of this augmentation is not clearly understood. As tumor necrosis factor-alpha (TNF-α) induces natriuresis by activating TNF-α receptor type 1 (TNFR1) but not type 2 (TNFR2), we hypothesize that TNFR1 activity is reduced when HS is given in combination of AngII that leads to enhanced sodium retention and thus, causing augmented hypertension. The aim of this study is to examine the responses to chronic HS intake and AngII administration on the renal tissue protein expressions of TNFR1 and TNFR2 in mice. Methods: Different groups of mice (n = 6–7 in each group) chronically treated with or without AngII (25 ng/min; implanted minipump) for 4 weeks which were fed either normal salt (NS; 0.4% NaCl) or high salt (HS; 4% NaCl) diets. Systemic BP was measured by tail-cuff plethysmography. At the end of treatment period, kidneys were harvested after sacrificing the mice with euthanasia. Immuno-histochemical analysis of TNFR1 and TNFR2 proteins in renal tissues was performed by measuring the staining area and the intensity of receptors’ immunoreactivities using NIS-Elements software. The results were expressed in percent area of positive staining and the relative intensity. Results: HS intake alone did not alter mean BP (HS; 77 ± 1 vs. NS; 76 ± 3 vs. mmHg; tail-cuff plethysmography) but AngII induced increases in BP were augmented in HS group (104 ± 2 vs. 95 ± 2 mmHg; P < 0.05). The area of TNFR1 staining was higher in HS than NS group (6.0 ± 0.9% vs. 3.2 ± 0.7%; P < 0.05) but it was lower in AngII + HS than in AngII + NS group (5.0 ± 0.7% vs. 6.3 ± 0.7%; P = 0.068). TNFR2 immunoreactivity was minimal in NS and HS groups but it was high in AngII + NS and even higher in AngII + HS group. Conclusions: These data suggest that the HS induced increased TNFR1 activity that facilitates enhanced sodium excretion is compromised in elevated AngII condition leading to salt retention and augmented hypertension.

Exogenous central angiotensin fails to stimulate a sodium appetite in diabetes insipidus Brattleboro rats

We investigated the effect of central administration of angiotensin II (AngII) on a specific salt appetite (SSA) in homozygous diabetes insipidus Brattleboro (DI) rats because this stimulus induces such a response in all other rat strains. DI rats have a deficiency in the synthesis of arginine vasopressin (AVP) and a reduced content of pituitary oxytocin (OT). They are characterized also by polyuria, polydipsia, and they seldom ingest high concentrations of NaCl solutions. We also tested if the appetite can be influenced by neurohypophyseal hormones especially oxytocin (OT) because it inhibits SSA in other animals. DI rats and Long Evans (LE) controls were fed ad libitum and given a choice between water, and either 0.9% or 1.8% NaCl.The data showed a significant increase of daily spontaneous water intake in DI compared with LE rats. Both DI and LE ingested similar small spontaneous volumes of the isotonic NaCl solution, but DI rats drank significantly less hypertonic NaCl than the LE controls. I.c.v infusion of AngII induced significant sodium intake in LE rats, but only raised water intake in DI rats. When combined with i.c.v. Ang II, OVT enhanced salt intake in LE animals while AVP attenuated water intake in both groups of rats and blocked NaCl intake completely in LE rats. In conclusion, DI rats did not demonstrate a SSA in response to central administration of AngII, although the drinking of water was enhanced. In combination with i.c.v. AngII, AVP inhibits water drinking in both DI and LE rats. In the LE controls OT attenuates AngII-induced SSA but has no effect in DI rats.

Melatonin alleviates angiotensin-II-induced cardiac hypertrophy via activating MICU1 pathway.

Mitochondrial calcium uptake 1 (MICU1) is a pivotal molecule in maintaining mitochondrial homeostasis under stress conditions. However, it is unclear whether MICU1 attenuates mitochondrial stress in angiotensin II (Ang-II)-induced cardiac hypertrophy or if it has a role in the function of melatonin. Here, small-interfering RNAs against MICU1 or adenovirus-based plasmids encoding MICU1 were delivered into left ventricles of mice or incubated with neonatal murine ventricular myocytes (NMVMs) for 48 h. MICU1 expression was depressed in hypertrophic myocardia and MICU1 knockdown aggravated Ang-II-induced cardiac hypertrophy in vivo and in vitro. In contrast, MICU1 upregulation decreased cardiomyocyte susceptibility to hypertrophic stress. Ang-II administration, particularly in NMVMs with MICU1 knockdown, led to significantly increased reactive oxygen species (ROS) overload, altered mitochondrial morphology, and suppressed mitochondrial function, all of which were reversed by MICU1 supplementation. Moreover, peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α)/MICU1 expression in hypertrophic myocardia increased with melatonin. Melatonin ameliorated excessive ROS generation, promoted mitochondrial function, and attenuated cardiac hypertrophy in control but not MICU1 knockdown NMVMs or mice. Collectively, our results demonstrate that MICU1 attenuates Ang-II-induced cardiac hypertrophy by inhibiting mitochondria-derived oxidative stress. MICU1 activation may be the mechanism underlying melatonin-induced protection against myocardial hypertrophy.

Abstract 15127: Isorhamnetin Reverses Angiotensin Ii-induced Atrial Fibrillation Vulnerability Through Electrophysiological and Structural Reverse Remodeling in Mice

Background: Isorhamnetin (ISO), a natural flavonoid compound, has been shown to have strong antioxidant and antifibrotic effects in vivo and in vitro; however, no report has been shown on the effect of ISO on arrhythmic disorders. This study aimed to investigate the effect of ISO on angiotensin-II (Ag-II) induced atrial fibrillation (AF) in mice. Methods: Wild-type male mice (C57BL/6J, 8 weeks old) were assigned in 3 groups as follows: control group (Cont), Ag-II group treated with Ag-II and Ag-II & ISO group treated with Ag-II and ISO. Ag-II (1000 ng/kg/min) was continuously administered using an implantable osmotic pump for 2 weeks, and ISO (5 mg/kg) was administered intraperitoneally one week before starting Ag-II administration. We performed AF induction and electrophysiologic study via transvenous electrode catheter and Ca 2+ imaging with isolated cardiomyocyte. We also assessed the gene expression levels of AF-related molecules, and histological examination of atrial fibrosis. Results: Compared with Cont, AF inducibility was dramatically increased by Ag-II and significantly decreased by ISO. AF duration was also remarkably prolonged by Ag-II and significantly reduced by ISO. Atrial effective refractory period (A-ERP) (BCL=150msec) was reduced by Ag-II and recovered by ISO. Incidences of diastolic intracellular Ca 2+ abnormal activities (sarcoplasmic reticulum [SR] Ca 2+ leakage) was observed in Ag-II group; however, ISO treatment eliminated these abnormalities. Ag-II induced elevated expression of fibrosis-related molecules (Col1a1, Tgfb1 and Tgfb2) and Ca 2+ -handling related molecules (Camk2 and Ryr2), those were partially normalized by ISO. Histological examination revealed that cell size and the fibrosis ratio were increased by Ag-II, and significantly attenuated by ISO. Conclusion: These results suggest that ISO prevented against Ag-II induced AF vulnerability through both electrophysiological and structural reverse remodeling.

Aldehyde dehydrogenase 2 protects against abdominal aortic aneurysm formation by reducing reactive oxygen species, vascular inflammation, and apoptosis of vascular smooth muscle cells

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is an enzyme that detoxifies aldehydes by converting them to carboxylic acids. ALDH2 deficiency is known to increase oxidative stress. Increased oxidative stress plays a pivotal role in abdominal aortic aneurysm (AAA) pathogenesis. Reactive oxygen species (ROS) promote degradation of the extracellular matrix (ECM) and vascular smooth muscle cell (VSMC) apoptosis. Reducing oxidative stress by an ALDH2 activator could have therapeutic potential for limiting AAA development. We hypothesized that ALDH2 deficiency could increase the risk for AAA by decreasing ROS elimination and that an ALDH2 activator could provide an alternative option for AAA treatment. The National Center for Biotechnology (NCBI) Gene Expression Omnibus (GEO) database was used. Human aortic smooth muscle cells (HASMCs) were used for the in vitro experiments. Gene-targeted ALDH2*2 KI knock-in mice on a C57BL/6J background and apolipoprotein E knockout (ApoE KO) mice were obtained. An animal model of AAA was constructed using osmotic minipumps to deliver 1000ng/kg/min angiotensin II (AngII) for 28days. Patients with AAA had significantly lower ALDH2 expression levels than normal subjects. ALDH2*2 KI mice were susceptible to AngII administration, exhibiting significantly increased AAA incidence rates and increased aortic diameters. Alda-1, an ALDH2 activator, reduced AngII-induced ROS production, NF-kB activation, and apoptosis in HASMCs. Alda-1 attenuated AngII-induced aneurysm formation and decreased aortic expansion in ApoE KO mice. We concluded that ALDH2 deficiency is associated with the development of AAAs in humans and a murine disease model. ALDH2 deficiency increases susceptibility to AngII-induced AAA formation by attenuating anti-ROS effects and increasing VSMC apoptosis and vascular inflammation. Alda-1 was shown to attenuate the progression of experimental AAA in a murine model.

SUN-164 CXCR4-AngII axis plays an important role in glomerular injury through promoting the crosstalk between podocytes and mesangial cells

Glomerular disease has a high incidence worldwide. Podocyte injury and mesangial cell activation play pivotal roles in the progression of proteinuria and glomerulosclerosis. However, the linkage and underlying mechanisms are rarely reported. Herein, we investigated the role of chemokine receptor (CXCR4) and angiotensin II (AngII) in the pathogenesis of podocyte and mesangial cell injury. The association of CXCR4-AngII axis in crosstalk between podocytes and mesangial cells was also testified. 5/6 nephrectomized mice was established. To identify the role of CXCR4, CXCR4 plasmid was injected once a week by tail injection. Some mice were injected with adriamycin (ADR), advanced oxidation protein products (AOPPs) or Losartan. In vitro, MPC5 cells, an immortalized mouse podocyte cell line, were incubated with AOPPs or transfected with CXCR4 expression plasmid or siRNA to CXCR4. RMC cells, an immortalized mesangial cell line, were also adopted. RMC were treated with the conditional medium from AOPPs-treated MPC5 cells to assess the crosstalk between podocytes and mesangial cells. In 5/6NX and ADR mice model, CXCR4 was upregulated predominantly in podocytes. Whereas, SDF-1α, the ligand of CXCR4, was highly expressed in mesangial cells. CXCR4 colocalized with AngII in podocytes. Administration of AOPPs significantly induced the upregulation of CXCR4 and accelerated the progression of proteinuria. AOPPs induced podocyte injury and mesangial cell proliferation. These glomerular lesions were accompanied by upregulation of CXCR4, SDF-1α and activation of ACE, Renin and AT1. In ADR model, ectopic expression of CXCR4 aggravated proteinuria, podocyte injury, mesangial cell proliferation and glomerulosclerosis. Treatment with losartan significantly ameliorated these pathological changes. In cultured MPC5 cells, incubation of AOPPs or overexpression of CXCR4 promoted the activation of ACE, Renin, and AT1. However, blockade of CXCR4 reduced these effects. In RMC cells, administration of AngII stimulated fibrogenesis and cell proliferation. The conditional medium from AOPPs-treated MPC5 cells induced upregulation of fibronectin, PCNA and angiotensin II receptor AT1, but blocked by incubation with losartan. Our studies indicate that CXCR4 could upregulate the expression of RAS system in podocyte. AngII may subsequently bind to its receptor AT1 in mesangial cells to induce matrix deposition. The activation of mesangial cells would lead to the secretion of SDF-1α, the ligand of CXCR4, which further triggers podocyte injury. These studies demonstrate that CXCR4-AngII axis plays a vital role in glomerular injury via mediating pathologic crosstalk between podocytes and mesangial cells. Our findings uncover a new pathogenic mechanism by which CXCR4-AngII axis promotes glomerulosclerosis.

P5443NOD2 knock down worsens diastolic dysfunction in murine angiotensin II-induced heart failure

Abstract Background Heart failure with preserved ejection fraction (HFpEF) is associated with cardiac inflammatory responses, indicating a potential role of the immune system in the pathology of diastolic dysfunction. The cytoplasmatic pattern recognition receptor, nucleotide binding oligomerization domain 2 (NOD2) belongs to the innate immune system and induces among others the NLRP3 inflammasome, known to be involved in myocarditis and coronary heart disease. Purpose The aim of this study was to explore the role of NOD2 in Angiotensin II (AngII)-induced diastolic heart failure. Methods In NOD2−/− knock down and C57Bl6/j-wild type (WT) mice, diastolic dysfunction was induced by subcutaneous administration of 1.4mg/kg*day–1 AngII. Twenty-one days after first AngII administration, left ventricular (LV) function was evaluated by pressure tip catheter. Cardiac fibrosis, inflammation, and the expression of NOD2 and the NLRP3 component Apoptosis-associated speck like protein containing a caspase recruitment domain (ASC) were determined via immunohistochemistry, real-time PCR or Western Blot. Results LV NOD2 mRNA expression was 2.3-fold (p<0.0005) and 1.9-fold (p<0.0005) lower in NOD2−/− control and NOD2−/− AngII mice compared to their respective WT littermates. In parallel, LV protein expression of the downstream NLRP3 component Apoptosis-associated speck like protein containing a caspase recruitment domain (ASC) was 1.5-fold (p<0.05) lower in NOD2−/− AngII mice versus WT AngII mice, whereas LV protein IL-1β levels were unchanged. LV diastolic dysfunction was more pronounced in NOD2−/− AngII mice versus WT AngII mice, as displayed by a 19% (p<0.05) increased LV relaxation time and 24% (p<0.057) impaired dP/dtmin, with no changes in the ejection fraction (EF: NOD2−/− AngII 72.5%±5.4 versus WT AngII 65.6±3.5). In parallel, LV presence of CD68-positive cells was 1.8-fold (p<0.05) higher in NOD2−/− AngII compared to WT AngII mice. Concomitantly, NOD2−/− AngII mice displayed 1.3-fold (p<0.05) and 1.7-fold (p<0.05) higher LV mRNA expression of the chemokine macrophage inflammatory protein (MIP)-2 and monocyte chemotactant protein (MCP)-1 compared to WT AngII mice, respectively. Furthermore, cardiac interstitial fibrosis in NOD2−/− mice with AngII-induced diastolic dysperformance was more pronounced versus the WT AngII group, as indicated by a 2.0-fold (p<0.0005), 2.0-fold, and 1.6-fold (p<0.05) higher LV ColI/ColIII ratio, and TGF-β and TIMP-1 mRNA expression, respectively. Conclusion NOD2−/− deteriorates LV diastolic dysfunction and worsens pathophysiological key mechanisms in mice with AngII-induced diastolic heart failure.

P4476Cardiovascular benefits of GLP-1 (liraglutide) treatment in experimental arterial hypertension are mediated by the endothelial GLP-1 receptor

Abstract Objective The LEADER trial demonstrated that glucagon-like peptide-1 (GLP-1) analogs like Liraglutide (Lira) reduce the risk of cardiovascular events in T2DM, an effect beyond glycemic control. A detailed evaluation of the precise mechanisms underlying the cardiovascular protective effects of GLP-1 has been hampered by the fact that the GLP-1 receptor is expressed on different cell types in the vasculature including platelets, neuronal, endothelial and inflammatory cells. We used endothelial and myeloid cell-specific knockout mice of the GLP-1 receptor (GLP-1r) in an angiotensin-II (ATII)-induced model of hypertension. The aim of the recent study was to investigate the cardioprotective effects of GLP-1 in ATII-induced arterial hypertension and to characterize the cell-specific contribution of GLP-1r signaling. Methods Arterial hypertension was induced by s.c. ATII administration (0.5mg/kg/d; 7 days) in WT (C57/BL6J) as well as endothelial and myeloid cell-specific GLP-1r knockout mice (Cdh5crexGLP-1rfl/fl and LysMcrexGLP-1rfl/fl mice). Animals were treated with Lira (2x30μg/d; 7 days). Blood pressure was measured by tail-cuff. Vascular function was tested by isometric tension recording. Aortic and cardiac tissue was used for Western blotting, qRT-PCR, FACS, IHC and HPLC to determine the extent of inflammation, oxidative stress and fibrosis. ELISA was used to determine GLP-1 and insulin levels in plasma. Results Endogenous GLP-1 (7–36 and 9–36) was reduced in hypertensive animals. Lira ameliorated blood pressure and improved endothelial dysfunction, vascular oxidative stress and inflammation caused by ATII, in both WT and myeloid cell-specific GLP-1r knockout mice. Hypertension led to infiltration of inflammatory monocytes (Ly6G-Ly6Chigh) and neutrophils (Ly6G+Ly6C+) into the vascular wall, which was prevented by Lira. In accordance, Lira suppressed vascular oxidative stress and mRNA expression of iNOS, CD11b and Nox2. Endothelial NO synthase (eNOS) was S-glutathionylated with ATII treatment indicating uncoupled eNOS. Thus, aortic NO levels were reduced, all of which was restored by Lira. Furthermore, vascular fibrosis and cardiac hypertrophy were tremendously reduced by GLP-1. Interestingly, all of these beneficial cardiovascular effects of GLP-1 were abolished in endothelial cell-specific GLP-1r knockout mice. Conclusion We show that Lira reduces blood pressure and improves vascular function, fibrosis and cardiac hypertrophy in experimental arterial hypertension in mice. Mechanistically, Lira prevents the infiltration of inflammatory cells to the vascular wall, leading to reduced oxidative stress and improved NO bioavailability. Beneficial effects of GLP-1 are mediated by the GLP-1r expressed on endothelial and not myeloid cells. With the present study we provide a mechanistic approach to explain the cardioprotective effects of GLP-1 analogs like Lira, for which the endothelial GLP-1 receptor is indispensable. Acknowledgement/Funding Deutsche Forschungsgesellschaft (DFG), Bundesministerium für Bildung und Forschung (BMBF)

Abstract P2026: Involvement of Subfornical Organ Insulin Receptor Signaling in the Development of Angiotensin-II Hypertension

Neurocardiovascular dysfunction due to angiotensin-II (Ang-II) signaling within the brain has emerged as a key mechanism in hypertension development. Interestingly, the hormone insulin is also closely associated with multiple forms of hypertension. Emerging evidence suggests interactions between insulin receptor and Ang-II type 1a receptor (AT 1a R) signaling, although the extent to which this contributes to hypertension development remains unclear. Based on this, we hypothesized that brain insulin receptor signaling is involved in Ang-II hypertension. Male C57Bl/6J mice were instrumented with intracerebroventricular cannula (ICV) and radiotelemeters for conscious cardiovascular measurements. Subsequently, administration of Ang-II (600 ng/kg/min) over 2 wk resulted in the development of hypertension in control animals; daily ICV insulin receptor antagonism with the pharmacological agent S961 (3.5 nM) prevented hypertension development (e.g. day 14 mean arterial pressure 118±6 vs 110±6 mmHg, ICV vehicle vs ICV S961, p=0.05). Given the well-known role for the subfornical organ, a forebrain region that lacks a blood-brain-barrier, in this model of hypertension, we next focused our attention on this nucleus. Insulin receptor immunohistochemistry in AT 1a R eGFP reporter mice (GENSAT NZ44) revealed that insulin receptors were present on 69% of AT 1a R cells (42 of 62, n=3) in the SFO. Acute insulin administration (i.p. 5U) was also used to explore SFO insulin receptor-associated intracellular signaling including phosphorylation of AKT [pAkt; phosphatidylinositol 3-kinase (PI3K) signaling] and ERK1/2 [pERK; mitogen-activated protein kinase (MAPK) signaling]. Analysis of over 4000 SFO cells indicated that insulin resulted in activation of the PI3K pathway as demonstrated by an increase in the percentage of cells with medium to high pAKT expression (5±1 vs 14±1% per section, vehicle vs insulin, n=3-4, 5-10 sections/animal). In contrast, acute insulin administration did not influence SFO pERK. Collectively, these findings indicate that brain insulin receptor signaling is necessary for the development of Ang-II hypertension, and that this response may be mediated by interactions between Ang-II and insulin and/or PI3K signaling in the SFO.

Licochalcone A attenuates abdominal aortic aneurysm induced by angiotensin II via regulating the miR-181b/SIRT1/HO-1 signaling.

Licochalcone A (LA), a chalcone derived from liquorice, exhibits multiple biological activities, including anti-oxidation and anti-inflammation. This study aimed to investigate the role and underlying mechanism of LA in the abdominal aortic aneurysm (AAA). AAA model was established by continuous infusion of 1000 ng/kg/min of angiotensin II (AngII) in ApoE -/- mice for 4 weeks. At 7 days before AngII administration, 5 mg/kg/day or 10 mg/kg/day of LA was intraperitoneally administered to mice and continued for 4 weeks. The characteristics and quantification of AAAs were determined in situ. Real-time PCR or western blot was used to measure mRNA or protein levels of matrix metalloproteinase 2 and matrix metalloproteinase 9; pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6; apoptosis-related proteins Bax, Bcl-2, and active caspase-3; miR-181b; Sirtuin 1 (SIRT1); and heme oxygenase-1 (HO-1). Mouse-aorta-origin vascular smooth muscle (MOVAS) cells were used to confirm the involved pathways in vitro. We found LA administration dose-dependently reduced the incidence of AngII-induced AAA, aneurysm diameter enlargement, elastin degradation, matrix metalloproteinase production, pro-inflammatory cytokines and miR-181b expression, and vascular smooth muscle cell apoptosis. It elevated SIRT1 and HO-1 expression that was suppressed by AngII. AngII enhanced miR-181b but reduced SIRT1 and HO-1 expression in MOVAS cells. In AngII-stimulated MOVAS cells, downregulation of miR-181b significantly upregulated the expression of SIRT1 and HO-1, the effect of which was abrogated by SIRT1 siRNA. Collectively, LA could attenuate AngII-induced AAA by modulating the miR-181b/SIRT1/HO-1 signaling. LA might be a potential medical therapy for small AAA.

Abstract P380: Inverse Circadian Pattern in the Renal Generation of Tumor Necrosis Factor-Alpha (TNFå) and Angiotensinogen (AGT) in Salt-Sensitive Hypertension (SSH) Induced by Angiotensin II (AngII) in Mice

Individuals with SSH show a non-dipping blood pressure (BP) during the inactive night period, a phenomenon which is significantly associated with enhanced cardiovascular morbidity and mortality. However, the pathophysiological mechanism for this phenomenon is not yet clearly understood. Chronic AngII administration in mice (reversed circadian rhythm) induced SSH with an increase in intra-renal AGT formation, a response that was exaggerated in TNFα receptor type 1 (TNFR1) knockout mice, indicating that renal AGT formation is suppressed by TNFR1 activation by TNFα. The present study examined the hypothesis that AngII-induced TNFα formation follows a circadian pattern with increases during the active period compared to the inactive period, that facilitates less AGT formation during the active period. Experiments were performed in mice chronically treated with or without AngII (25 ng/min; osmotic mini-pump) + high salt (4% NaCl) intake for 4 weeks that increased BP from 90±2 to 108±3 mmHg (tail-cuff method). Circadian rhythm in urinary parameters was assessed by 12 hour collections of urine using metabolic cages during the active night period (7PM to 7AM) and the inactive day period (7 AM to 7 PM). In normal mice (n=6), urinary excretion of TNFα (uTNFα) was higher (0.5±0.2 vs 0.13±0.08 pg/hour) but urinary excretion of AGT (uAGT) was lower (0.28±0.18 vs 0.46±0.14 ng/hour) during the active period compared to that during the inactive period. This inverse relationship between uTNFα and uAGT was exaggerated in AngII+HS treated mice (n=6). From the baseline (0 week) to the 2nd and 4 th week period, there were marked incremental changes in uTNFα during active periods (0.68±0.20 to 14.2±5.4 and 44.6±14.6 pg/hour) which were less during inactive periods (0.73±0.48 to 10.3±3.3 and 12.1±4.7 pg/hour). Interestingly, uAGT changed minimally during active periods (0.21±0.14 to 0.05±0.04 and 1.42±0.97 ng/hour) but increased significantly during inactive periods (0.32±0.14 to 1.57±0.71 and 3.98±1.67 ng/hour). These data suggest that an increase in TNFα generation suppresses intra-renal AGT formation during active periods but this phenomenon is inversed during inactive periods. The increase in AGT during the inactive period could lead to a non-dipping BP pattern in SSH.

Cilostazol ameliorates heart failure with preserved ejection fraction and diastolic dysfunction in obese and non-obese hypertensive mice

Cilostazol (Ciloz) a potent Type III phosphodiesterase inhibitor is effective against inflammation, insulin resistance and cardiomyopathy. However, the effect of Ciloz on obesity-associated left ventricular diastolic dysfunction has not been explored yet. Hence, we examined the effect of Ciloz on cardiac remodelling and dysfunction in non-obese and obese-insulin resistant mice infused with AngiotensinII (AngII). Male C57BL/6 J mice were initially subjected to 19 weeks of chow or high fat diet (HFD) regimen and thereafter animals were randomised for AngII (1500 ng/kg/min, s.c) infusion or saline and Ciloz (50 mg/kg, p.o) for another 1 week. Obese and non-obese mice infused with AngII exhibited significant diastolic dysfunction and features of heart failure with preserved ejection fraction (HFpEF) since a decrease in fractional shortening and no change in ejection fraction were observed when compared with respective controls. Administration of AngII and Ciloz in HFD fed mice significantly improved the left ventricular function compared with AngII infused HFD mice as evinced from the echocardiographic data. Further, Ciloz treatment significantly reduced cardiomyocyte area, interstitial and perivascular fibrosis; and collagen deposition. Moreover, Ciloz reduced the inflammatory milieu in the heart as evinced by decreased F4/80+ and CD68+ cells; IL-1β and IL-6 gene transcripts. Quantitative assessment of the expression levels revealed substantial upregulation of MMP-9 (pro- and mature-forms) and α-SMA in the left ventricle of AngII infused HFD-fed mice, which was considerably suppressed by Ciloz regimen. The beneficial effect of Ciloz was associated with the normalization in gene expression of hypertrophic and fibrotic markers. Likewise, Ciloz administration markedly reduced the AngII and HFD induced TGF-β1/SMAD3 and Akt/mTOR signalling. Additionally, AngII administered and HFD-fed mice showed increased glycolytic flux, which was considerably diminished by Ciloz treatment as indicated from suppressed PKM2, HK-2, PDK-2, HIF-1α mRNA and GLUT-1 protein expression. Taken together, Ciloz might be therapeutically exploited against AngII and obesity-associated diastolic dysfunction thereby preventing overt heart failure.

Angiotensin‐II‐Induced Increase in Blood Pressure in Female and Male Mice

The kidney regulates blood pressure by controlling urinary salt and water excretion. Angiotensin II (AngII), a key player in controlling blood pressure, constricts blood vessels, stimulates vasopressin and aldosterone release, stimulates thirst, and increases sodium absorption from the proximal tubule. All effects cause increased blood pressure. Sex differences in the regulation of arterial blood pressure involve events surrounding the renin‐angiotensin‐aldosterone system. Evidence generally supports a protective mechanism for females with AngII‐induced hypertension; although, conflicting evidence exists. The purpose of this study was to investigate sex differences in CD‐1 mice under AngII administration for a period of 10 days. We hypothesized that female mice would be protected from AngII‐induced increase in mean arterial pressure (MAP) compared to male mice and that the renal handling of salt and water contributes to this protection. Four‐week old CD‐1 mice were obtained from Envigo, Inc. (Indianapolis, IN). Two studies, one with females and one with males, were conducted. Mice were placed in metabolic cages and consumed normal diet and water, ad libitum, for 15 days. After the first 5 days, an Alzet™ minipump containing either AngII (n=5) or vehicle (n=5) was implanted subcutaneously in each mouse to ensure continuous AngII infusion (1 mg/kg/min). Daily measurements included MAP via the tail‐cuff method (Kent Scientific, CODA System), urinary output, and urinary sodium excretion. MAP (mmHg) was not different between females and males in the 5‐day control period (85.1 ± 3.7 vs 81.7 ± 4.0, respectively). AngII‐treatment increased MAP in both females and males (110.3 ± 4.4 and 105.8 ± 3.0, p<0.001, compared to respective control period). Interestingly, AngII‐treated females showed higher urine output than AngII‐treated males (9.7 ± 0.8 vs 7.4 ± 0.4 ml/day, p<0.05) and also had higher sodium excretion (242 mEq/day vs 166 mEq/day, p<0.01). Absolute food intake in females and males was not different, however, females consumed more food per body weight. We conclude that female mice are not protected from AngII‐induced increase in MAP compared to male mice for a 10‐day AngII infusion period. However, based on the sodium excretion data, protection may be observed later from increased urine and sodium output in females. Results suggest that the renal handling of salt and water could be an important determinant of sex differences in blood pressure regulation.Support or Funding InformationNational Institute of General Medical Sciences of National Institutes of Health through Grant Number 8P20GM103447This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Determining the Influence of a Novel Americanized Rodent Diet on Renal Health in Wild‐Type Mice Administered Angiotensin II

Diet has an established relationship with morbidity and mortality worldwide. While poor diet is primarily associated with cardiovascular disease, there is evidence that poor diet has a direct negative influence on renal health that can also contribute to deteriorating cardiovascular health. However, due to the lack of a well‐designed disease inducing rodent diet, the physiological link between diet and the cardiovascular and renal systems remain unclear. We have developed a novel Americanized diet (AD) that is based on the nutritional intake of Americans and includes modifications to several nutrients while also accounting for the difference in human and mouse nutritional requirements. We hypothesized that mice consuming the AD will have greater weight gain and exacerbated renal injury, as compared to mice fed standard chow, in response to low‐dose angiotensin II (AngII) administration. All animal experiments were performed following animal protocols approved by the Liberty University IACUC and conforms to the FASEB Statement of Principles for the use of animals in research and education. Weanling (3‐week old) male C57Bl/6J mice were given ad libitum access to the AD (n=5) or standard laboratory chow (n=5) and ddH2O. 24‐hour dietary intake and body weights were recorded weekly. After 5 weeks, a baseline urine sample was collected and a subcutaneous pump delivering AngII (250 ng/kg*min) was implanted in the rear flank. Systolic blood pressure was recorded daily during AngII infusion using a non‐invasive tail‐cuff volume pressure recording method. After 4 weeks of AngII infusion, another urine sample was collected and urinary albumin concentrations were quantified by ELISA. Prior to euthanasia, renal blood flow was estimated using contrast‐enhanced ultrasonography. Body weight and blood pressure data were analyzed using generalized linear models (Two‐Way Repeated Measures), while the remaining parameters were analyzed using a multivariate GLM procedure. All statistical analyses were performed using SPSS. Mice consuming the AD had greater weight gain (P<0.01) and urinary concentration of albumin (P=0.01) prior to AngII administration. Interestingly, the difference in weight was abolished upon initiation of AngII infusion despite a continued difference in estimated daily caloric intake. Albuminuria persisted in the mice consuming the AD after 4 weeks of AngII (38±10 vs 20±5 ug/24 hr, P=0.02), despite a lack of difference in blood pressure between the AD and chow fed mice (P=0.8). Estimated renal blood flow was also greater in mice fed the AD (P=0.02), and likely explains the tendency for higher 24 hour urine output (P=0.2) as compared to mice consuming chow and receiving AngII. In conclusion, mice consuming an AD had greater weight gain and renal damage (albuminuria) despite the absence of a change in systolic blood pressure. These data show that the nutritional quality of a “typical” American diet has direct negative impact on renal health in rodents and further studies are needed to further elucidate the consequences of these findings.Support or Funding InformationThis work was supported by Liberty University, College of Osteopathic Medicine intramural grant #2016‐07.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.