Doses Of Angiotensin II Research Articles

Abstract P3124: Chronic Infusion Of Angiotensin II Promotes Ventricular Fibrosis And Diastolic Dysfunction In Aging Male But Not Female Mouse Hearts

Heart failure prevalence increases with age and affects men and women differently. Chronic activation of the renin-angiotensin system (RAS) in heart failure increases circulating angiotensin II (AII) levels, which elevates blood pressure (BP), promotes fibrosis and leads to adverse remodelling of the heart. Whether maladaptive responses to AII differ between the sexes at older ages is unclear, as most studies use young male animal models. We investigated whether adverse cardiac remodeling induced by chronic exposure to AII differed between the sexes in older C57BL/6 mice. Osmotic minipumps with pressor doses of AII (3 mg/kg/day) or saline were implanted into older mice (≈16 mos) of both sexes for 6 weeks. BP (tail cuff) and ventricular structure/function (echocardiography) were measured at baseline, midpoint, and endpoint. Ventricular fibrosis markers (transforming growth factor-ß (TGF-ß), fibronectin collagen I, collagen III) were quantified with qPCR. Mean arterial pressure increased similarly in both sexes (76.3±4.2 vs. 136.1±4.6 mmHg in males and 73.9±2.9 vs. 116.1±8.7 mmHg in females; p<0.05; n=6-8/group). AII infusion also increased LV mass and reduced ejection fraction in males and females. By contrast, AII increased signs of diastolic dysfunction in males but had little impact on diastolic function in females. For example, isovolumic relaxation time increased over 6 weeks of AII infusion in males (11.7±1.0 vs. 19.0±1.4 ms; p<0.05) but not in females (14.6±0.9 vs. 16.4±1.2 ms). Similar results were seen for E/A ratios. Molecular studies showed a marked increase in mRNA abundance for TGF-ß in male ventricles (1.02±0.13 vs. 1.50±0.16; p<0.05) but not in females (1.01±0.08 vs. 1.25±0.10). Similar male-female differences were seen for collagens I and III (fibronectin was similar in both sexes, and not affected by AII). As TGF-ß signalling increases the production of profibrotic mediators like collagens I and III, these findings suggest that chronic RAS activation increases fibrosis and disrupts diastolic function in aging male hearts. By contrast, older female hearts are resistant to these deleterious effects of RAS activation. These sex-specific differences may modify heart failure expression and responses to treatment in older men and women.

Abstract 273: Smooth Muscle Cell Specific Beclin-1 Deficiency Accelerates Aortic Aneurysms In Male And Female Normolipidemic And Hypercholesterolemic Mice

Background and Objective: Ascending and abdominal aortic aneurysms (AAs) are permanent dilations of the aorta with 80% mortality after rupture. Vascular smooth muscle cell (SMC)-rich aortic media is disrupted in AAs. Autophagy is a process by which cell recycles their cytoplasmic materials for energy purposes under stress. Recent clinical studies highlighted that Beclin-1, an autophagy induction gene, is upregulated in AA tissues from patients. Using Angiotensin II (AngII) infusion model of AAs, we examined the functional role of SMC-derived Beclin-1 during AA development in normolipidemic and hypercholesterolemic mice. Methods and Results: Mice with inducible deletion of Beclin-1 in SMCs were produced by breeding male Acta2-Cre ERT2 hemizygous mice female Beclin-1 floxed mice in normolipidemic and hypercholesterolemic (LDLR-/-) background. At 8 weeks of age, Beclin-1 x Acta2-Cre ERT2 (Cre+) and non-Cre littermates (Cre-) mice were injected with tamoxifen (75 mg/kg, i.p.) for 5 days. After 2 weeks, Western blot analyses showed depletion of Beclin-1 protein in the aortic media from Cre+ mice compared to Cre- littermates. To study the role of SMC-Beclin-1, normolipidemic or hypercholesterolemic male Cre+ and Cre- (N=7 per group) mice were fed either chow or fat-enriched diet (21% fat; 0.15% cholesterol) for 5 weeks. After 1 week of diet feeding, mice were infused with saline or low dose of AngII (500 ng/kg/min) by osmotic mini-pumps for 4 weeks. SMC-Beclin-1 deficiency in either normolipidemic or hypercholesterolemic mice spontaneously but significantly accelerated ascending (Cre-: 1.2 ± 0.07 vs Cre+: 1.8 ± 0.04 mm; P <0.001), arch (Cre-: 1.1 ± 0.04 vs Cre+: 1.8 ± 0.03 mm; P <0.001), descending (Cre-: 1.1 ± 0.04 vs Cre+: 1.5 ± 0.04 mm; P <0.001), and abdominal (Cre-: 1.0 ± 0.02 vs Cre+: 1.5 ± 0.14 mm; P <0.001) aortic expansion. AngII-infusion had no additional influence on SMC-Beclin-1 deficiency accelerated aortic expansion. In addition, SMC-Beclin-1 deficiency significantly increased aortic medial hypertrophy and elastin breaks (P<0.001 Cre- vs Cre+) associated with complete loss of medial alpha-SMC actin positive area. Conclusion: These findings demonstrate that SMC-Beclin-1 plays a critical role in suppressing aortic aneurysmal formation in mice.

Angiotensin II constricts mouse iliac arteries: possible mechanism for aortic aneurysms.

Abdominal aortic aneurysms (AAA) result from maladaptive remodeling of the vascular wall and reduces structural integrity. Angiotensin II (AngII) infusion has become a standard laboratory model for studying AAA initiation and progression. We determined the different vasoactive responses of various mouse arteries to Ang II. Ex vivo isometric tension analysis was conducted on 18-week-old male C57BL/6 mice (n = 4) brachiocephalic arteries (BC), iliac arteries (IL), and abdominal (AA) and thoracic aorta (TA). Arterial rings were mounted between organ hooks, gently stretched and an AngII dose response was performed. Rings were placed in 4% paraformaldehyde for immunohistochemistry analysis to quantify peptide expression of angiotensin type 1 (AT1R) and 2 receptors (AT2R) in the endothelium, media, and adventitia. Results from this study demonstrated vasoconstriction responses in IL were significantly higher at all AngII doses when compared to BC, and TA and AA responses (maximum constriction-IL: 68.64 ± 5.47% vs. BC: 1.96 ± 1.00%; TA: 3.13 ± 0.16% and AA: 2.75 ± 1.77%, p < 0.0001). Expression of AT1R was highest in the endothelium of IL (p < 0.05) and in the media and (p < 0.05) adventitia (p < 0.05) of AA. In contrast, AT2R expression was highest in endothelium (p < 0.05), media (p < 0.01, p < 0.05) and adventitia of TA. These results suggest that mouse arteries display different vasoactive responses to AngII, and the exaggerated response in IL arteries may play a role during AAA development.

Diminazene Aceturate Reduces Angiotensin II Constriction and Interacts with the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus 2.

Diminazene aceturate (DIZE) is a putative angiotensin-converting enzyme 2 (ACE2) activator and angiotensin type 1 receptor antagonist (AT1R). Its simple chemical structure possesses a negatively charged triazene segment that is homologous to the tetrazole of angiotensin receptor blockers (ARB), which explains its AT1R antagonistic activity. Additionally, the activation of ACE2 by DIZE converts the toxic octapeptide angiotensin II (AngII) to the heptapeptides angiotensin 1–7 and alamandine, which promote vasodilation and maintains homeostatic balance. Due to DIZE’s protective cardiovascular and pulmonary effects and its ability to target ACE2 (the predominant receptor utilized by severe acute respiratory syndrome coronavirus 2 to enter host cells), it is a promising treatment for coronavirus 2019 (COVID-19). To determine DIZE’s ability to inhibit AngII constriction, in vitro isometric tension analysis was conducted on rabbit iliac arteries incubated with DIZE or candesartan and constricted with cumulative doses of AngII. In silico docking and ligand interaction studies were performed to investigate potential interactions between DIZE and other ARBs with AT1R and the spike protein/ACE2 complex. DIZE, similar to the other ARBs investigated, was able to abolish vasoconstriction in response to AngII and exhibited a binding affinity for the spike protein/ACE2 complex (PDB 6LZ6). These results support the potential of DIZE as a treatment for COVID-19.

Hypertensive Female Mice Are Protected from Impaired Parenchymal Arteriolar TRPV4‐Mediated Dilation and Reactive Astrogliosis

Hypertension is a leading risk factor for cerebral small vessel disease. Our lab has previously demonstrated that cerebral parenchymal arterioles (PAs) are dependent on transient receptor potential vanilloid 4 (TRPV4) function for endothelium‐dependent dilation, and that this dilation is impaired in angiotensin II (AngII)‐hypertensive male mice. This PA dilation impairment is associated with cognitive deficits and increased inflammation. However, sex differences in hypertension‐associated dementia development are not well understood. To better understand the effect of female sex hormones during hypertension, we tested the hypothesis that young, cycling hypertensive female mice will be protected from impaired TRPV4‐mediated PA dilation and reactive astrogliosis observed in male mice. AngII‐filled osmotic minipumps (800ng/kg/min, 4 weeks) were implanted in 16‐18‐week‐old male C56BL/6 mice. Age‐matched female mice received either an AngII dose that matches the dose used in male mice (800ng/kg/min) or a higher dose (1200ng/kg/day) that produces an elevation in blood pressure similar to male mice. Sham mice served as control. Blood pressure was measured by tail‐cuff plethysmography. Pressure myography was used to assess TRPV4‐mediated dilation in PAs. An anti‐glial fibrillary acidic protein (GFAP) antibody was used in paraformaldehyde‐fixed 40μm coronal brain sections to assess the quantity and morphology of astrocytes. Data are presented as means ± SEM (n=5‐10). Male data were compared by Student’s t‐test and female data were compared by one‐way ANOVA unless otherwise stated. Systolic blood pressure was elevated in AngII‐treated male mice (sham: 133 ± 10, AngII: 181 ± 11mmHg; p=0.005) and in 1200ng AngII‐treated female mice (sham: 146 ± 8, 800ng AngII: 161 ± 13, 1200ng AngII: 179 ± 7mmHg; p=0.0482) vs sham. PA dilation in response to the TRPV4 agonist GSK1016790A (10‐9‐10‐5M) was not impaired in AngII‐treated female mice (sham: 85 ± 5, 800ng AngII: 73 ± 7, 1200ng AngII: 77 ± 8% maximum dilation; p=0.4381), contrasting with what we have previously reported in male mice. There was an increase in astrocytes in the corpus callosum of hypertensive male mice (sham: 65 ± 2; AngII: 77 ± 5; p=0.039). Interestingly, sham female mice had more astrocytes than sham male mice, and AngII infusion reduced the quantity (sham: 81 ± 3, 800ng AngII: 71 ± 3, 1200ng AngII: 69 ± 3; p=0.011). Sholl analysis was used to assess astrocyte morphology. Astrocytes from hypertensive male mice had more arborization further from the soma compared to sham (p=0.0348 at 14‐15μm from the soma, by two‐way ANOVA and Bonferroni correction). Female mice receiving the 1200ng dose AngII, however, had fewer arborizations away from the soma compared to sham (p=0.0263 at 13‐15μm from the soma, by two‐way ANOVA and Bonferroni correction). Female mice are resistant to AngII‐induced hypertension and protected from the associated deleterious cerebrovascular effects, supporting our hypothesis.

The Association Between Angiotensin II and Renin Kinetics in Patients After Cardiac Surgery.

Hyperreninemia after cardiac surgery is associated with cardiovascular instability. Angiotensin II (AT-II) could potentially attenuate hyperreninemia while maintaining target blood pressure. This study assesses the association between AT-II usage and renin levels in cardiac surgery patients with postoperative hyperreninemia and vasoplegia. Between September 2020 and March 2021, we retrospectively identified 40 cardiac surgery patients with high Δ-renin levels (4 hours after cardiopulmonary bypass [CPB] minus preoperative levels) (defined as higher than 3.7 µU/mL) and vasopressor use who received a vasopressor therapy with either AT-II or continued norepinephrine alone. The primary outcome was the renin plasma level at 12 hours after surgery, adjusted by the renin plasma level at 4 hours after surgery. Overall, the median renin plasma concentration increased from a baseline with median of 44.3 µU/mL (Q1-Q3, 14.6-155.5) to 188.6 µU/mL (Q1-Q3, 29.8-379.0) 4 hours after CPB. High Δ-renin (difference between postoperation and preoperation) patients (higher than 3.7 µU/mL) were then treated with norepinephrine alone (median dose of 3.25 mg [Q1-Q3, 1.00-4.75]) or with additional AT-II (norepinephrine dose: 1.33 mg [Q1-Q3, 0.78-2.04]; AT-II dose: 0.34 mg [Q1-Q3, 0.29-0.78]). At 12 hours after surgery, AT-II patients had lower renin levels than standard of care patients (71.7 µU/mL [Q1-Q3, 21.9-211.4] vs 130.6 µU/mL [Q1-Q3, 62.9-317.0]; P = .034 adjusting for the renin plasma level at 4 hours after surgery). In cardiac surgery patients with hypotonia and postoperative high Δ-renin levels, AT-II was associated with reduced renin plasma levels for at 12 hours and significantly decreased norepinephrine use, while norepinephrine alone was associated with increased renin levels. Further studies of AT-II in cardiac surgery appear justified.

Overexpression of NOX2 Exacerbates AngII-Mediated Cardiac Dysfunction and Metabolic Remodelling.

The present study aimed to examine the effects of low doses of angiotensin II (AngII) on cardiac function, myocardial substrate utilization, energetics, and mitochondrial function in C57Bl/6J mice and in a transgenic mouse model with cardiomyocyte specific upregulation of NOX2 (csNOX2 TG). Mice were treated with saline (sham), 50 or 400 ng/kg/min of AngII (AngII50 and AngII400) for two weeks. In vivo blood pressure and cardiac function were measured using plethysmography and echocardiography, respectively. Ex vivo cardiac function, mechanical efficiency, and myocardial substrate utilization were assessed in isolated perfused working hearts, and mitochondrial function was measured in left ventricular homogenates. AngII50 caused reduced mechanical efficiency despite having no effect on cardiac hypertrophy, function, or substrate utilization. AngII400 slightly increased systemic blood pressure and induced cardiac hypertrophy with no effect on cardiac function, efficiency, or substrate utilization. In csNOX2 TG mice, AngII400 induced cardiac hypertrophy and in vivo cardiac dysfunction. This was associated with a switch towards increased myocardial glucose oxidation and impaired mitochondrial oxygen consumption rates. Low doses of AngII may transiently impair cardiac efficiency, preceding the development of hypertrophy induced at higher doses. NOX2 overexpression exacerbates the AngII -induced pathology, with cardiac dysfunction and myocardial metabolic remodelling.

Epigenetic Programming Reverses Cardiometabolic Dysfunctions and Modulates Hypothalamic Genes Involved in Oxidative Stress and Inflammation in Angiotensin II-Treated Male Mice

Cardiometabolic disease is a global health issue that affects millions of people worldwide. Environmental perinatal exposure affects the health outcomes of the offspring and determines their disease susceptibility later in life. Angiotensin-II (Ang-II) is a peptide known to cause vasoconstriction, elevated blood glucose levels and inflammation. Previously, we reported that perinatal exposure to a hypercaloric diet (HD) results in elevated blood pressure (BP), weight gain, fasting hyperglycemia and glucose intolerance only in male mice. In addition, subcutaneous infusion of a sub-pressor dose of Ang-II was associated with a normalization in fasting blood glucose levels and a reversal of glucose intolerance only in programmed male mice. We hypothesize that epigenetic programming blocks the deleterious effects of Ang-II by altering its inflammatory signaling pathway. C57BL6/J dams were fed HD or regular diet (RD) for 1 month before mating with RD-fed males. After weaning, offspring of HD dams (programmed) and of RD dams (controls) were maintained on RD until 3 months of age. Mice then underwent 24 h BP recording (telemetry) and were implanted with Ang-II osmotic pumps (200 ng/kg/min/2 weeks). BP (24 h) was recorded weekly for 2 weeks. Mice were then sacrificed and hypothalami were harvested for mRNA sequencing (Illumina NextSeq). Programmed mice had lower 24 h systolic BP levels compared to control males (area under the curve: 41844 ±263.2 vs. 44522 ±275.6; p<0.0001). For RNAseq analysis, data showed 62 differentially expressed genes (DEG) in programmed males compared to controls. Using iPathway analysis, we found that some of the DEG are correlated to cholinergic synapse pathway (p=0.005) and neuroactive ligand-receptor interaction pathway (p=0.003). Nicotinic acetylcholine alpha-7 receptor (Chrna7) gene, known for its anti-inflammatory and hypoglycemic effects was upregulated in programmed males (p=0.024). On the other hand, genes involved in metabolic pathways and oxidative stress were differentially expressed as well. Phospholipase A2 group 3 (Pla2g3) gene, known to be overexpressed in oxidative stress was downregulated in programmed males (p=0.04). Moreover, Thiosulfate sulfurtransferase (Tst) gene, an antioxidant enzyme and used as a marker for enhanced insulin sensitivity was upregulated (p=0.023) in programmed males. Interestingly, female mice did not show any changes in BP or gene expression between the two groups. In conclusion, perinatal exposure to HD alters the cardiovascular response to Ang-II possibly through the modulation of gene expression of Chrna7 gene and genes involved in oxidative stress. Future experiments will be investigating the signaling pathways used in epigenetic programming to affect inflammation and oxidative stress in male mice.

Sexual Dimorphisms in Hypertension‐Associated Cerebrovascular Damage

Hypertension is a major risk factor for cerebral small vessel disease (CVSD), which increases stroke and dementia risk. Hypertension alters artery structure, impairing blood flow regulation and preventing nutrient delivery to the brain parenchyma. We previously showed angiotensin II (AngII)-hypertension in male mice reduces overall pial blood flow measured by scanning laser Doppler and causes inward hypotrophic remodeling in cerebral parenchymal arterioles (PAs). Epidemiological evidence suggests sex differences exist in CVSD, with large vessel damage occurring more in males and small vessel damage occurring more in females. We hypothesized hypertensive male and female mice would have reductions in pial blood flow measured by laser speckle contrast and inward hypotrophic remodeling in PAs. AngII-filled osmotic minipumps (800ng/kg/min, 4 weeks) were implanted in 16-18-week-old male C56BL/6 mice. Age-matched female mice received either an AngII dose that matches the dose used in male mice (800ng/kg/min) or a higher dose (1200ng/kg/day) that produces an elevation in blood pressure similar to male mice. Sham mice served as control. Blood pressure was measured by tail-cuff plethysmography. Pial artery blood flow was measured in the frontal, parietal, and temporal areas of both brain hemispheres. After euthanasia, PAs were isolated and mounted on a pressure myograph for structure assessment by increasing intraluminal pressure from 3 to 120mmHg in 20mmHg increments. Data are presented as means ± SEM (n=6-9). PA data are reported at an intraluminal pressure of 40mmHg. Systolic blood pressure was elevated in AngII-treated male mice (sham: 133 ± 10, AngII: 181 ± 11mmHg; p<0.05) and in 1200ng AngII-treated female mice (sham: 146 ± 8, 800ng AngII: 161 ± 13, 1200ng AngII: 179* ± 7mmHg; p<0.05) vs sham. There was a sex difference in blood flow alterations during hypertension. Male AngII treated mice had reduced flow in the parietal region (sham: 361 ± 19, AngII: 301 ± 9.3A.U.; p<0.05) but not frontal (sham: 359 ± 19, AngII: 327 ± 11A.U.; p=0.19) or temporal regions (sham: 292 ± 13, AngII: 284 ± 15A.U.; p=0.68) vs sham. AngII treated female mice had reductions in pial flow in the parietal (sham: 403 ± 9, 800ng AngII: 336* ± 19, 1200ng AngII: 314* ± 33 A.U.; p<0.05), frontal (sham: 378 ± 7, 800ng AngII: 314* ± 13, 1200ng AngII: 279* ± 24 A.U.; p<0.05), and temporal (sham: 338 ± 8.4, 800ng AngII: 273* ± 11, 1200ng AngII: 278* ± 25 A.U.; p<0.05) regions vs sham. PAs from male AngII mice had reduced outer diameter (sham: 56 ± 3, AngII: 46 ± 2μm; p<0.05), lumen diameter (sham: 47 ± 1, AngII: 40 ± 2μm; p=0.05), and wall area (sham: 719 ± 115, AngII: 405 ± 24μm2; p<0.05) vs sham, reproducing previous findings. PAs from AngII treated female mice did not exhibit significant reductions in outer diameter (sham: 57 ± 3, 800ng AngII: 50 ± 5, 1200ng AngII: 47 ± 4μm; p=0.17) or lumen diameter (sham: 49 ± 2, 800ng AngII: 43 ± 5, 1200ng AngII: 41 ± 4μm; p=0.22, but showed a modest reduction in wall area (sham: 647 ± 64, 800ng AngII: 534 ± 78, 1200ng AngII: 439 ± 58μm2; p=0.09) vs sham. These data suggest the mechanism of hypertension-induced vascular damage differs between males and females. Understanding sex differences in CVSD is critical for dementia and stroke prevention.

Tumor Necrosis Factor α Receptor Type 1 Activation in the Hypothalamic Paraventricular Nucleus Contributes to Glutamate Signaling and Angiotensin II-Dependent Hypertension.

There are significant neurogenic and inflammatory influences on blood pressure, yet the role played by each of these processes in the development of hypertension is unclear. Tumor necrosis factor α (TNFα) has emerged as a critical modulator of blood pressure and neural plasticity; however, the mechanism by which TNFα signaling contributes to the development of hypertension is uncertain. We present evidence that following angiotensin II (AngII) infusion the TNFα type 1 receptor (TNFR1) plays a key role in heightened glutamate signaling in the hypothalamic paraventricular nucleus (PVN), a key central coordinator of blood pressure control. Fourteen day administration of a slow-pressor dose of AngII in male mice was associated with transcriptional and post-transcriptional (increased plasma membrane affiliation) regulation of TNFR1 in the PVN. Further, TNFR1 was shown to be critical for elevated NMDA-mediated excitatory currents in sympathoexcitatory PVN neurons following AngII infusion. Finally, silencing PVN TNFR1 prevented the increase in systolic blood pressure induced by AngII. These findings indicate that TNFR1 modulates a cellular pathway involving an increase in NMDA-mediated currents in the PVN following AngII infusion, suggesting a mechanism whereby TNFR1 activation contributes to hypertension via heightened hypothalamic glutamate-dependent signaling.SIGNIFICANCE STATEMENT Inflammation is critical for the emergence of hypertension, yet the mechanisms by which inflammatory mediators contribute to this dysfunction are not clearly defined. We show that tumor necrosis factor α receptor 1 (TNFR1) in the paraventricular hypothalamic nucleus (PVN), a critical neuroregulator of cardiovascular function, plays an important role in the development of hypertension in mice. In the PVN, TNFR1 expression and plasma membrane localization are upregulated during hypertension induced by angiotensin II (AngII). Further, TNFR1 activation was essential for NMDA signaling and the heightening NMDA currents during hypertension. Finally, TNFR1 silencing in the PVN inhibits elevated blood pressure induced by AngII. These results point to a critical role for hypothalamic TNFR1 signaling in hypertension.

How predictive is body weight on fluid intake in rats? It depends on sex.

The assumption that body weight is a predictor of fluid intake is often used as rationale for normalizing intake to body weight when examining sex differences in drinking behavior. Nonuniform application of this body weight correction likely contributes to discrepancies in the literature. We, however, previously demonstrated sex differences in the relationship between body weight and angiotensin II (AngII)-stimulated water intake. Only after a pharmacological dose of AngII did water intake correlate with body weight, and only in males. Here we investigated whether body weight correlated with fluid intake stimulated by additional dipsogenic agents in male and female rats. We found that intake stimulated by either water deprivation or furosemide correlated with body weight in male rats. We found no relationship between intake and body weight after water deprivation, furosemide treatment, or isoproterenol treatment in females, nor did we find a relationship between intake and body weight after hypertonic saline treatment in either males or females. Finally, we report that daily water intake correlated with body weight in females. This effect, however, is likely the result of a relationship between body weight and food intake because when food was absent or reduced, the correlation between body weight and intake disappeared. These results demonstrate that multiple factors need to be considered when determining the best way to compare fluid intake between males and females and provides insight to help explain the discrepancies in the literature regarding sex differences in fluid intake.

Editing of the myosin phosphatase regulatory subunit suppresses angiotensin II induced hypertension via sensitization to nitric oxide mediated vasodilation.

Alternative splicing of exon 24 (E24) of the myosin phosphatase regulatory subunit (Mypt1) tunes smooth muscle sensitivity to NO/cGMP-mediated vasorelaxation and thereby controls blood pressure (BP) in otherwise normal mice. This occurs via the toggling in or out of a C-terminal leucine zipper (LZ) motif required for hetero-dimerization with and activation by cGMP-dependent protein kinase cGK1α. Here we tested the hypothesis that editing (deletion) of E24, by shifting to the LZ positive isoform of Mypt1, would suppress the hypertensive response to angiotensin II (AngII). To test this, mice underwent tamoxifen-inducible and smooth muscle-specific deletion of E24 (E24 cKO) at age 6 weeks followed by a chronic slow-pressor dose of AngII (400 ng/kg/min) plus additional stressors. E24 cKO suppressed the hypertensive response to AngII alone or with the addition of a high salt diet. This effect was not a function of altered salt balance as there were no differences in intake or renal excretion of sodium. This effect was NO dependent as L-NAME in the drinking water caused an exaggerated hypertensive response in the E24cKO mice. E24cKO mouse mesenteric arteries were more sensitive to DEA/NO-induced vasorelaxation and less responsive to AngII- and α-adrenergic-induced vasoconstriction at baseline. Only the latter two effects were still present after 2 weeks of chronic AngII treatment. We conclude that editing of Mypt1 E24, by shifting the expression of naturally occurring isoforms and sensitizing to NO-mediated vasodilation, could be a novel approach to the treatment of human hypertension.

Experimental preeclampsia results in post‐partum sensitivity to hypertensive stimuli

Preeclampsia (PE) is a syndrome of new onset high blood pressure (BP) with renal damage during late pregnancy that is associated with increased circulating anti‐angiogenic proteins, including soluble VEGF receptor (sFlt1). Women exposed to PE have increased risk of future cardiovascular disease, including hypertension, with evidence of a “hyper‐responsive” renin‐angiotensin‐aldosterone system (RAAS) persisting post‐partum. Aldosterone (Aldo) and angiotensin II (AngII) can activate vascular smooth muscle cell mineralocorticoid receptor (SMC‐MR), causing changes in gene transcription that contribute to increased BP. Thus, we hypothesized that exposure to increased sFlt1 during pregnancy may induce a state of persistent sensitivity to hypertensive stress by enhanced SMC‐MR activity post‐partum .We established a model that reproduces the hallmarks of PE by viral overexpression of sFlt1 compared to control virus in pregnant C57Bl6 mice. Using telemetry to measure blood pressure, we demonstrate that sFlt1 injection results in increased serum sFlt1, elevated BP and glomerular endotheliosis, the hallmark of PE‐induced renal damage, in late pregnancy that all resolve post‐partum. Two months post‐partum, wire myography studies performed on 3rd order mesenteric resistance vessels ex vivo reveal that sFlt1‐injected females (“prior‐PE”) have a vasoconstrictor sensitivity to AngII (1e‐9 dose: 0.16mN vs. 1.42mN, p=0.059) . To test whether prior PE results in increased sensitivity to hypertensive stimuli, post‐partum mice were exposed to various high salt diet conditions or to a moderate pressor dose of AngII (600mg/kg/day infusion) while BP was monitored via telemetry. Mice with prior PE had a trend toward an increased peak BP in response to 2% sodium (11.71mmHg vs. 3.62mmHg, p=0.09) and a significantly increased BP response to 4% sodium (8.88mmHg vs 5.34mmHg, p&lt;0.05). Prior PE mice also had a significantly enhanced BP response to AngII versus those with normotensive pregnancies (22.83 mmHg vs 4.46 mmHg, p=0.0005). Plasma Aldo measured via radioimmunoassay tended to be higher in mice with prior PE (4.65nM vs 2.09nM, P=0.08). Since SMC‐MR can be activated by AngII, we investigated the extent of AngII‐induced SMC‐MR activation after PE using qPCR to measure known SMC‐MR target genes. In microvessels from mice with prior PE, mRNA expression showed increased AT1R (relative expression 3.69 vs 1.30, p&lt;0.05), Cav1.2 (2.29 vs 1.11, p=0.06) and PlGF (1.92 vs 1.20, p=0.11). Studies are underway to determine whether SMC‐specific deletion of MR or post‐partum pharmacologic MR inhibition can protect against enhanced BP sensitivity to AngII and high salt diet after sFlt1‐induced PE.sFlt1‐induced PE produces a state of enhanced sensitivity to hypertensive stimuli that may be mediated by increased activation of SMC‐MR. If confirmed, these data support the use of spironolactone as a therapy to mitigate the increased risk of cardiovascular disease in women exposed to PE.Support or Funding InformationNIH T32HL007609 (LAB), NIH R25GM066567 (BVC)

Hyperglycemia‐Induced Inhibition of the Angiotensin II Type 1 Receptor Pathway in the Vascular Endothelium

Hyperglycemia, associated with diabetes, is known to damage blood vessels and contribute to negative cardiovascular health outcomes. However, the etiology behind the negative influence of the exposure to prolonged elevated glucose on endothelial cells is not yet fully understood. Our laboratories’ previous study identified changes in endothelial cell surface glycosylation resulting from hyperglycemia. One such modified protein was the Angiotensin II type 1 receptor (AT1R), which was N‐glycosylated adjacent to the active site. Activation of AT1R by angiotensin II (AngII) at subpressor doses is associated with positive endothelial cell growth, proliferation, and tube formation. However, AngII binding to the Angiotensin II type 2 receptor (AT2R), has been shown to elicit opposing effects. The objective of this study was to further investigate the effects of this hyperglycemia‐induced glycosylation of AT1R. A two‐week high glucose treatment (25 mM) of rat cardiac microvascular endothelial cells (RCMVECs) displayed impaired function by tube formation analysis (TFA) in comparison to a normal glucose treatment (4.5 mM). The decrease tube formation was further exacerbated by the addition of a subpressor dose of AngII. To further test the influence of hyperglycemia‐induced AT1R N‐glycosylation on signaling we treated RCMVECs with the PNGaseF enzyme for N‐glycosylation removal at the cell surface in combination with functional assays and AngII treatment under varying glycemic conditions. The efficacy of this treatment was tested through TFA and measurements of ERK1/2 activation through phosphorylation (known contributor to AT1R signaling). We observed a decrease in tube length, tube area, and number of branch points in a high glucose environment plus Ang‐II and were able to recover functionality with the addition of PNGaseF. While normal glucose treated cells had shown ERK1/2 activation, no observable increases in ERK1/2 were observed in response to AngII in the high glucose conditions. In the high glucose plus AngII treatment group we also observed a significant decrease in Vegfr2 gene expression compared to the normal glucose plus AngII treatment group. Further analysis is currently underway to further validate this mechanism of AT1R signaling impairment, but the data we have so far lead us to believe that hyperglycemia‐induced glycosylation blocks the AT1R active site to decrease functionality. It is possible that the hyperglycemia‐induced glycosylation of AT1R inhibits AngII binding, allowing for a higher rate of binding in the AT2R to further impair endothelial cell functionality.Support or Funding InformationSupport for this project has been provided by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (K01‐DK105043 to BRH) and the Department of Biomedical Engineering at the Medical College of Wisconsin and Marquette University.

P4995Neuregulin-1 compensates for endothelial NO synthase deficiency in the heart and kidney

Abstract Background Decreased eNOS activity is the hallmark of endothelial dysfunction and is associated with cardiovascular and renal disorders. Besides NO, endothelial cells produce numerous other small molecules, peptides, and proteins, which modulate the function of adjacent cells. For instance, neuregulin-1 (NRG-1) is an endothelium-derived growth factor, which plays crucial roles in cardiovascular development, has cardioprotective properties, and induces growth and differentiation of cell types in different organs, including the kidney. Purpose Although the cardioprotective effects of endothelium-derived NO and NRG-1 are well established, their interaction is not clear. Therefore, we studied the interaction between the NO/eNOS and NRG-1/ErbB signalling pathways in a transgenic eNOS knock-out mouse model (eNOS−/−) treated with subpressor doses of angiotensin II (AngII). Methods eNOS−/− mice and their wild type (WT) littermates (n=64, 15 weeks old) were randomized for implantation of osmotic minipumps with AngII (400 ng/kg.min) for 28 days or sham surgery. Mice were randomized to receive either daily NRG-1 injections (20 μg/kg, intraperitoneal) or vehicle for 4 weeks (n=8/group). Hypertrophy and fibrosis were measured in left ventricle (LV) and kidneys using echography and immunohistochemical staining. Results Although blood pressure was higher in eNOS−/− mice compared to their WT littermates, it was unaffected by a subpressor dose of AngII. Masson's trichrome staining showed that AngII significantly increased LV (interstitial and perivascular) and renal fibrosis in eNOS−/− mice, but not in WT controls (see figure for LV data). NRG-1 reversed this AngII-induced LV and renal fibrosis caused by eNOS deficiency. There was also significant hypertrophy of LV and kidneys in eNOS−/− mice treated with AngII, which was again normalized by NRG-1 treatment. Moreover, NRG-1 significantly attenuated albuminuria induced by eNOS deficiency under AngII treatment. Conclusions This study demonstrates that the anti-fibrotic and anti-hypertrophic effects of NRG-1 are independent from the NO/eNOS pathway in both heart and kidney. Strikingly, NRG-1 is able to compensate for some of the negative effects of eNOS deficiency, at least in conditions of AngII stimulation. Acknowledgement/Funding supported by university of Antwerp

OR04-5 Stimulation of Protein Disulfide Isomerase Activity by Activation of The Renin-Angiotensin System

Understanding the molecular and cellular mechanisms that are affected by activation of the renin-angiotensin system (RAS) are important for development of novel therapeutic strategies to more specifically treat hypertension and reduce cardiovascular risk. RAS regulates blood pressure (BP) in part via its effector molecule, angiotensin II (AngII), a potent vasopressor that stimulates the production of reactive oxygen species (ROS). Protein disulfide isomerase (PDI) is a multifunctional oxidoreductase that catalyzes thiol/disulfide interchange reactions within the endoplasmic reticulum and regulates ROS production. Also, PDI is present in circulation and at the cell surface of platelets, lymphocytes, erythrocytes and endothelial cells and plays a critical role in initiating thrombus formation. However, the interaction of RAS and PDI remain unclear. We hypothesized that RAS activation would lead to increased PDI levels thus contributing to oxidative stress. First, we studied the in vitro effects of AngII on EA.hy926, a human endothelial cell line, and measured PDI activity. Our results show that AngII dose and time dependently increased extracellular PDI activity (p<0.05, n=6); an event that was reduced to baseline levels by preincubation with 0.5 uM losartan, an AngII type 1 receptor antagonist (ARB). Consistent with these results, AngII stimulated: 1) a 3-fold rise of extracellular PDI levels as determined by ELISA (p<0.05, n=4); 2) a 2.5-fold increase in PDI mRNA levels by RT-PCR (p<0.05, n=4); and 3) increased PDI protein levels by western blot analyses that were blocked by losartan. We also studied the effect of AngII on extracellular PDI activity in differentiated HL-60 cells, a human polymorphonuclear neutrophil cell line, and observed significantly increased extracellular PDI activity following incubation with AngII (p<0.05, n=4). We then characterized the in vivo effects of exogenous AngII infusion on PDI in Sprague-Dawley rats, a model of increased BP, inflammation and target organ damage. We studied three conditions: (1) control; (2) AngII infused (80 ng/min x 28 days); (3) AngII + ARB (10 mg losartan/kg/day x 21 days). AngII infusion led to significant increases in plasma PDI levels that were reduced by 20% following ARB treatment (p<0.05, n=5/group). We then studied a model of naturally increased AngII and hypertension, the Otsuka Long Evans Tokushima Fatty rats (OLETF) and their strain controls that were randomly assigned to the following groups: (1) untreated strain controls; (2) untreated OLETF; (3) OLETF + ARB. OLETF rats had increased BP and significantly greater circulating PDI activity than control rats that was likewise blocked by ARB treatment (p<0.05; n=6/group). Thus, RAS activation represents a novel mechanism for increased PDI levels and activity that may contribute to the deleterious effects of disordered RAS in cardiovascular disease.

Recharacterizing the Murine Chronic Angiotensin II Infusion Model of Cardiovascular and Renal Injury

The chronic angiotensin II (AngII) infusion model is one of the most commonly used to study cardiovascular and kidney injury. However, the dosages of AngII used to induce cardiovascular or renal injury vary significantly in the literature. Therefore, we set out to determine the dosages of angiotensin II needed to produce renal injury and hypertension in mice. All experiments were performed in accordance with protocols approved by the Liberty University IACUC and conform to the FASEB standards for the use of animals in research and education. Male C57Bl/6J mice (~20 grams body weight) were purchased from The Jackson Laboratory and acclimated to our environment for one week. Mice were then assigned to receive 0, 400, 800, or 1200 ng AngII/kg BW*min for four weeks via osmotic minipumps (subcutaneous). Blood pressure (tail‐cuff) was recorded each week of the study. At baseline, 2 and 4 weeks, renal blood flow and renal hemodynamics were estimated using contrast‐enhanced ultrasonography. After 4 weeks, mice were placed in metabolic cages for the quantification of water intake and urinary output. Urine was stored for determination of albumin concentration using a commercially available ELISA. Mice were then euthanized and kidney tissues collected to measure the mRNA expression of several genes involved in the intrarenal renin‐angiotensin system (RAS) (renin, angiotensinogen, angiotensin converting enzyme (ACE), ACE2, angiotensin II‐receptor 1a (AT1a), and AT1b receptor) and tissue inflammation and remodeling [CCL5, α‐smooth muscle actin (αSMA), collagen 1, collagen 3, and tumor growth factor β (TGFβ)]. All data were analyzed using general linear models in SPSS. Mice administered the 800 dose had the greatest and most consistent increase in systolic blood pressure (147 ± 8mmHg) as compared to mice administered the saline (114 ± 9, P=0.004) and the 400 dose (121 ± 11mmHg, P=0.01). Mice administered the 1200 dose tended (P=0.08) to have a greater total renal blood flow throughout the study, with a significant increase (P=0.047) in cortical blood flow as compared to mice administered saline. Despite the modest changes in estimated renal blood flow, mice administered the 800 and 1200 doses of AngII had similar, yet significantly greater (P≤0.001) water intake and urinary output as compared to mice treated with saline or the 400 dose. Similarly, mice with the 800 and 1200 doses had significantly greater evidence of tissue damage evident by increased albuminuria (P≤0.05) and increased renal gene expression of CCL5, TGFβ, collagen 1, and collagen 3. There was also an effect on the intrarenal RAS, with mice administered the 800 and 1200 doses having significant (P&lt;0.05) increase of renin, angiotensinogen, ACE1, ACE2, AT1a, and AT1b expression. In our hands, it appears that a dosage of greater than 400 ng/kg*min is needed to elicit the pressor effect of AngII and its associated effects on kidney structure and function. Our data also suggests that doses greater than 800 do not result in a further increase in blood pressure or several markers of kidney health. Taken together, more studies are needed to better refine this popular animal model and identify potential causes of variability throughout the literature.Support or Funding InformationProject was supported by LUCOM Intramural Grant #2018‐01.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P257: Investigating Angiotensin II Infusion as a Model of Superimposed Preeclampsia in Pregnant Stroke Prone Spontaneously Hypertensive Rats

Background: Hypertensive disorders of pregnancy are a leading cause of maternal mortality worldwide. Chronic hypertension with superimposed preeclampsia is becoming more prevalent in modern society. Animal models are vital for understanding this condition. Our aim was to induce a preeclamptic phenotype in pregnant stroke prone spontaneously hypertensive (SHRSP) rats by angiotensin II (AngII) infusion. Methods: AngII, at a dose of 500ng/kg/min or 1000ng/kg/min, was administered by minipump in pregnant SHRSP rats from gestational day (GD)10. Cardiovascular and renal changes were monitored using radiotelemetry, echocardiography and weekly urine collections. GD18 ex vivo uterine artery function and structure were assessed using myography. Results: Both AngII doses resulted in an immediate and sustained increase in systolic blood pressure compared to vehicle control ( 172.5±5.0mmHg; 500ng/kg/min and 204.1±5.6mmHg; 1000ng/kg/min vs 159.0±0.4mmHg; p&lt;0.001 ). Cardiac output increased over pregnancy in the vehicle group ( 53±6L/min vs 81±5L/min; p&lt;0.01 ), however this trend was not observed in 500ng/kg/min treatment group ( 60±6L/min vs 56±11L/min ) and was reversed in 1000ng/kg/min treatment group ( 60±6L/min vs 36±6L/min; p&lt;0.01 ). GD14 urinary albumin:creatinine ratios were significantly increased in both treatment groups ( 1.3±0.2; 500ng/kg/min, 2.9±0.1; 1000ng/kg/min vs 0.5±0.2; vehicle; p&lt;0.05, p&lt;0.001 ). Uterine arteries in the 1000ng/kg/min treatment group demonstrated significantly reduced cross-sectional area ( 4.8x10 4 ±32μm 2 vs 7.6x10 4 ±16μm 2 ; p&lt;0.01 ). Both treatment groups had a reduced vasorelaxation to 2x10 5 M carbachol ( 93.6±3.9%; 500ng/kg/min, 84.7±4.3%; 1000ng/kg/min vs 56.9±21.1%; vehicle; p&lt;0.05 ) and contractile response to 2x10 5 M noradrenaline demonstrated an increased trend in both treatment groups ( 66.9±16.1KPa; 500ng/kg/min, 61.8±6.2KPa; 1000ng/kg/min vs 45.1±5.7KPa; vehicle ). Conclusion: This study demonstrates that AngII infusion in pregnant SHRSP rats can mimic specific haemodynamic, cardiac and urinary profiles common to preeclamptic women. This rodent model of superimposed preeclampsia will be crucial for detailed investigation of underlying causes and treatment options for this condition.

Sex differences in the drinking response to angiotensin II (AngII): Effect of body weight

Sex differences in fluid intake stimulated by angiotensin II (AngII) have been reported, but the direction of the differences is inconsistent. To resolve these discrepancies, we measured water intake by male and female rats given AngII. Males drank more than females, but when intake was normalized to body weight, the sex difference was reversed. Weight-matched males and females, however, had no difference in intake. Using a linear mixed model analysis, we found that intake was influenced by weight, sex, and AngII dose. We used linear regression to disentangle these effects further. Comparison of regression coefficients revealed sex and weight differences at high doses of AngII. Specifically, after 100ng AngII, weight was a predictor of intake in males, but not in females. Next, we tested for differences in AngII-induced intake in male and females allowed to drink both water and saline. Again, males drank more water than females, but females showed a stronger preference for saline. Drinking microstructure analysis suggested that these differences were mediated by postingestive signals and more bottle switches by the females. Finally, we probed for differences in the expression of components of the renin-angiotensin system in the brains of males and females and found sex differences in several genes in discrete brain regions. These results provide new information to help understand key sex differences in ingestive behaviors, and highlight the need for additional research to understand baseline sex differences, particularly in light of the new NIH initiative to balance sex in biomedical research.

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

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