bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 666: Monoclonal Anti-marinobufagenin Antibody Alters Profile Of Expression Of Profibrotic Genes In Aorta In Hypertensive Dahl-S Rats
Authors: Fedorova Olga V , Shilova Victoria , Marshall Courtney A , Zhang Yongqing , Lehrmann Elin , Becker Kevin G , Lakatta Edward G , Bagrov Alexei Y
Published: July 2021

Elevated levels of cardiotonic steroid marinobufagenin (MBG) are implicated in pathogenesis of salt-sensitive hypertension in Dahl-S rats (DS), and induce cardiac fibrosis in rats with experimental uremic cardiomyopathy. We hypothesized that in hypertensive DS immunoneutralization of MBG with a monoclonal antibody (Mab) would affect expression of profibrotic genes and would exhibit anti-remodeling effects. We studied following groups (n=6 each): (1) DS on a low salt (0.3% NaCl) diet (LS); (2) DS on a high salt (8% NaCl) diet for 7 weeks (HS); (3) DS on a high salt diet for 7 weeks, following 3E9 anti-MBG Mab treatment for 5 days (HSAB). Levels of MBG, and levels of mRNA expression in aorta (microarray analysis, Illumina) were assessed. In HS vs. LS, BP increased by 78 mmHg (p<0.01), plasma MBG doubled (p<0.05), renal MBG excretion increased 6-fold (p<0.01), and relative weights of aortae increased (4.44±0.17 vs. 3.01±0.06 mg/mm x kg BW, p<0.01). In HSAB, BP decreased by 35 mmHg (p<0.01), and weights of aortae were markedly reduced (3.72±0.06 mg/mm x kg BW; p<0.01) vs. HS. In hypertensive DS, genes implicated in TGFβ-signaling (Table) were up-regulated, and were down-regulated following immunoneutralization of MBG. Thus, MBG participates in vascular remodeling in DS, and immunoneutralization of MBG produces an anti-remodeling effect associated with down-regulation of genes implicated in TGFβ-induced pro-fibrotic signaling.

Table
. Z-ratio of paired analysis of gene expression in aorta:
Abstract 70: Effect of Mas Receptor Stimulation and loss on Cardiac Pathology in Salt Sensitive Hypertension
Authors: Anene-Maidoh Ogugua T , Greene Andrew S
Published: July 2021

Aim:
We investigated the role of the Mas receptor in the development of cardiac pathology induced by salt loading.

METHODS:
Studies were performed on 8 week old Mas+/+ (n = 30) and Mas-/- (n= 28) rats produced on the genetic background of the SS/MCWi rat using zinc-finger nuclease technology and fed on low salt (LS) or high salt (HS) diets. Mas stimulation was achieved via AVE0991 administered in the drinking water (20nmoles/kg/day). Animals were grouped into Mas+/+ rats on LS, HS, and HS + AVE0991 and Mas-/- rats on LS, HS, (n=10 for all preceding groups) and HS + AVE0991 (n=8). Blood pressure, left ventricular (LV) wall thickness, and LV pressure volume relationships (PVR) were measured daily, weekly and at the end of the 4 week experiment respectively. Hearts were stained to assess LV fibrosis.

Results:
Salt loading led to significant increases in MAP in all HS treated animals compared to LS controls (p<0.05). There was no effect of Mas receptor loss (Mas-/-) or stimulation (AVE0991 treatment) on MAP. Compared to LS controls, HS diet led to significant increases in LV thickness in Mas+/+ (2.1mm vs 2.4mm p<0.05) and Mas-/- (2mm vs 2.6mm p<0.05) rats at 4 weeks following salt loading. AVE0991 stimulation of Mas attenuated salt-induced hypertrophy in Mas+/+ (2.4mm vs 2.2mm p<0.05) but not in Mas-/- (2.6mm vs 2.6mm) rats. HS increased ventricular stiffness (end diastolic PVR) to a greater degree in Mas-/- (2.1 fold) compared to Mas+/+ rats (1.5 fold). Treatment of HS rats with AVE0991 blocked salt induced changes in LV stiffness in Mas+/+ (1.5 vs 1.03 fold p<0.05) but had no effect on Mas-/- rats. Increases in systolic contractility in response to dobutamine infusion was robust in Mas+/+ rats on LS (2.3 fold), HS (1.7 fold) and HS + AVE0991 (1.8 fold) and Mas-/- rats on LS (1.9 fold). There was a blunted response to dobutamine in Mas-/- rats on HS (1.25 fold) and HS +AVE0991 (1.23 fold). Percent LV fibrosis was increased in Mas+/+ and Mas-/- rats on HS compared to LS controls (1.1% vs 2% p<0.05 and 1.2% vs 2.8% p<0.05). AVE0991 treatment abolished the fibrotic response in Mas+/+ rats (2% vs 1.4% p<0.05) but not in Mas-/- rats (2.8% vs. 2.9%)

Conclusion:
The Mas receptor plays a significant role in the modulation of LV hypertrophy, fibrosis and systolic and diastolic dysfunction following salt loading.
Abstract 71: STAT3 Affects Myofibrillar Structure and Its Loss May Contribute to Heart Failure in Hypertension
Authors: Zouein Fouad , Zgheib Carlos , Fuseler John , Hall John E , Kurdi Mazen , Booz George W
Published: July 2021

How hypertension causes heart failure is not known. Since patients with heart failure have reduced cardiac STAT3 and STAT3 KO mice develop heart failure with age, we tested the hypothesis that reduced STAT3 transcriptional activity contributes at an early stage to remodeling that precedes heart failure in hypertension using SA mice with a STAT3 S727A mutation. SA and wild type (WT) mice received angiotensin (A) II (1000 ng/kg/min) or saline (S) for 17 days. Hearts of WT and SA mice had similar levels of STAT3-induced protective proteins Bcl-xL and SOD2, and unlike STAT3 KO mice, cardiac miR-199a levels were not increased in SA mice. AII increased systolic blood pressure measured by telemetry in SA (124 ± 1 to 167 ± 3) and WT (122 ± 3 to 162 ± 3) mice to the same extent. AII increased cardiac levels of cytokines (pg/μg protein) associated with heart failure in both WT and SA mice, but significantly less so (P<0.05) in SA mice; IL-6, 13.6 ± 1.4 vs. 9.1 ± 0.6; TGFβ, 56 ± 4 vs. 38 ± 3 and MCP1 35 ± 2 vs. 22 ± 2. Compared to WT mice, hearts of SA mice showed signs of developing systolic dysfunction with AII as seen by a significant (P<0.05) reduction in ejection fraction (63.7 ± 7.1 to 51.7 ± 6.9) and fractional shortening (34.3 ± 4.9 to 26.4 ± 4.3). AII caused fibrosis in the left ventricle of both WT and SA mice characterized by cardiac myocyte loss and increased % collagen: WT+S, 5.59 ± 0.34; WT+AII, 15.70 ± 1.87; SA+S, 6.70 ± 0.40; SA+AII, 16.50 ± 1.91. In WT+AII mice there was a nonsignificant trend towards a loss of myofibrillar content of cardiac myocytes, but an increase in the mass of the myofibrils (IOD/myofibrillar area). In contrast, cardiac myocytes of SA+AII mice had a significant (P<0.001) % loss in myofibrils (5.71 ± 0.28) compared to SA+S (0.75 ± 0.07), WT+S (0.80 ± 0.06) and WT+AII (1.54 ± 0.10) mice. In addition, the mass of the myofibrils in SA+AII mice (6.01 ± 0.07) was significantly less (P<0.001) than those of SA+S mice (6.46 ± 0.04), although greater than WT+S (4.85 ± 0.06) or WT+AII (5.27 ± 0.08) mice. Our findings reveal that STAT3 transcriptional activity is important for proper morphology of the myofibrils of cardiac myocytes. Loss of STAT3 activity may impair cardiac function in the hypertensive heart due to defective myofibrillar structure and remodeling that may lead to heart failure.
Abstract 77: Abolished Renal Nerve Protection, Aggravated Renal Functional Impairment, and Elevated Blood Pressure in Response to Inhibition of Cyclooxygenase in Mice Fed a Western Diet: Role of Trpv1
Authors: Zhong Beihua , Wang Donna H
Published: July 2021

Salsalate (sodium salicylate, SA), a cyclooxygenase inhibitor, has been shown to increase insulin sensitivity and to suppress inflammation in obesity and type II diabetes in humans and animals. This study tests the hypothesis that SA (120 mg/kg/day x 4 wks) protects renal nerve function in wild type (WT) but not TRPV1-null mutant (TRPV1
-/-
) mice fed a western diet (WD for 4 months), accompanying with deteriorated renal function and elevated blood pressure in TRPV1
-/-
mice fed WD. SA improved glucose tolerance and insulin tolerance in WT-WD-SA and TRPV1-/-WD-SA compared to WT-WD and TRPV1-/-WD groups (P<0.05). Renal pelvis perfusion of capsaicin (CAP, 10
-6
M
), a selective TRPV1 agonist, increased afferent renal nerve activity (ARNA) in WT mice only, with a greater magnitude in WT-WD-SA than in WT-WD mice (P<0.05). Baroreflex (BR) control of renal sympathetic nerve activity (RSNA) in response to IV infusion of sodium nitroprusside (SNP)- or phenylephrine (PE) was improved in WT-WD-SA but impaired in TRPV1-/-WD and TRPV1-/-WD-SA compared to WT-WD mice (P<0.05). SA decreased urinary levels of 6-keto PGF1α (a major metabolite of PGI
2
) and PGE
2
in TRPV1-/-WD-SA and WT-WD-SA compared to TRPV1-/-WD and WT-WD
(P
<0.05). SA decreased the glomerular filtration rate (GFR) and increased plasma urea and creatinine levels in both strains fed WD
(P
<0.05), with greater magnitudes in TRPV1-/-WD-SA than in WT-WD-SA
(P
<0.05). SA also increased urinary albumin levels in TRPV1-/-WD-SA compared to WT-WD, TRPV1-/-WD, and WT-WD-SA groups (WT-WD 15.2± 2.9; TRPV1-/-WD 22.1±3.5; WT-WD-SA18.4 ±5.0; TRPV1-/-WD-SA 35.9 ± 19.8 ug/24hr, P<0.05). Telemetry mean arterial pressure was not altered by SA in WT-WD-SA, but increased at the night time in TRPV1-/-WD and TRPV1-/-WD-SA compared to corresponding WT controls and at the day time in TRPV1-/-WD-SA compared to TRPV1-/-WD (P<0.05). Thus, inhibition of cyclooxygenase with SA in mice fed WD increases ARNA and sensitivity of BR and BR modulation of RSNA. SA-mediated nerve protective effects are abolished when TRPV1 is ablated, accompanying with exacerbated renal functional impairment and elevated blood pressure. These data may have significant clinical implication for obese and diabetic patients with whom impaired TRPV1 occurs.
Abstract 82: In Vivo Memri Reveals Persistent Activation of the Pvn by an Acute Systemic Angiotensin Ii Injection
Authors: Zubcevic Jasenka , Perez Pablo D , Marulanda Carvajal Jessica , Raizada Mohan K , Febo Marcelo
Published: July 2021

Introduction:
An overactive brain renin-angiotensin system is a major factor in the establishment of neurogenic hypertension in the spontaneously hypertensive rat (SHR). However, there is no concrete evidence to indicate that this is associated with enhanced neuronal activity in the brain. The objective here was to use the MRI to establish the effect of ANGII on neuronal activity in the autonomic brain areas. We propose that a single ANGII injection will cause a long-lasting neuronal response in the autonomic brain areas, which will be exaggerated in the SHR.

Methods:
In vivo
basal and ANGII-evoked neuronal activity was measured in the Wistar-Kyoto (WKY) rat and the SHR using manganese-enhanced MRI (MEMRI) at 4.7Tesla. Rats were treated with manganese chloride (MnCl
2
30 mM solution,
i.p
.;16-20 hrs prior to the MRI), which labels active neurons. T
1
-weighted images were obtained 16-20 hrs after a single ANGII injection (0.32μg/kg
i.p.).
Coronal slice scans (caudally from end of the cerebellum towards the hypothalamus) were processed using itkSNAP, and data analyzed for normalized signal intensity.

Results:
Acute ANGII injection caused an immediate pressor response in the WKY (ΔSBP=∼20mmHg), normalizing within 2 hours. Despite this, ANGII evoked a persistent PVN neuronal activation, which was elevated by 22±4% in the WKY, and by 187±45% in the PVN of SHR. As a result, there was a ∼8.5fold increase in the ANGII-dependent neuronal activity in the PVN of SHR compared to WKY. Furthermore, there was a ∼2.5fold decrease in the NTS neuronal activity in the SHR compared to WKY.

Conclusion:
The present study shows for the first time the correlation between ANGII and autonomic neuronal activation. Even a single systemic ANGII injection results in a lasting effect on the brain. This is particularly apparent in the SHR, which exhibited an exaggerated neuronal response to the ANGII stimulus, reflected in the elevated PVN neuronal activation corresponding to the enhanced sympathetic drive, and in the depressed NTS activation corresponding to the dysfunction in the barorereflex processing. Thus, repeated pro-hypertensive stimuli in the autonomic brain areas may lead to pre-sympathetic neuronal plasticity, resulting in heightened sympathetic drive and hypertension.
Abstract 88: Systems Biology Approaches to Exploring Molecular Mechanisms Underlying Blood Pressure Regulation
Authors: Huan Tianxiao , Zhu Jun , Joehanes Roby , Zhang Bin , Wang Zhi , Johnson Andrew D , Munson Peter , Courchesne Paul , O'Donnell Christopher J , Vasan Ramachandran , Derry Jonathan , Friend Stephen , Yang Xia , Levy Daniel
Published: July 2021

Blood pressure (BP) is a complex trait that is influenced by both genetic and environment factors. Recent genome-wide association studies (GWAS) have identified about 30 genetic loci that are associated with systolic and diastolic blood pressure (SBP, DBP) and hypertension. However, the molecular mechanisms underlying the GWAS associations remain unclear. We hypothesize that BP-associated genetic variants with both strong and subtle effects drive shifts in gene subnetworks that in turn affect BP. We surveyed BP-associated molecular interactions in Framingham offspring cohort participants (n=2461; 55% women; age range 40-92 yrs.) by integrating gene expression profiles, expression-associated SNPs (eSNPs), and BP GWAS with network approaches. Peripheral whole blood samples were collected and large-scale transcriptomic microarray analysis was performed on all available participants who attended a clinic visit in 2005-2008. Based on pedigrees the samples were split into a discovery set (n=1421) and a replication set (n=820). The expression levels of 661 genes (FDR < 20%) and the eigengenes of 3 coexpression modules (CoEMs), which were found to be significantly correlated with SBP or DBP in the discovery set, were tested in the replication set. Seventy-two genes were replicated at FDR < 20%. The 72-gene BP-correlated signature and 2 of the three CoEMs were found to be significantly enriched for eSNPs with low p value associations with BP in the International Consortium of Blood Pressure (ICBP) GWAS at p<0.001, suggesting that these gene sets play causal roles in BP regulation. The putative causal BP gene sets were in turn integrated with tissue-specific Bayesian networks and a protein-protein interaction network to identify central network nodes (key drivers or KDs) that drive these BP-related gene sets. One hundred and ten KDs including TSPAN2, ECT2 and KCNK3 were identified. The BP causal gene sets and their KDs are enriched for BP-related biological processes including ion transport, nervous system development, and blood coagulation. Our systems biology analysis not only predicts novel BP risk genes, but also uncovers a network structure that entails the molecular interactions among BP and cardiovascular risk genes in a tissue-specific fashion.
Abstract 11: Improved Prediction of Adverse Pregnancy Outcome With the sFlt-1/PlGF Ratio in Patients Suspected of Preeclampsia?
Authors: Verdonk Koen , van Ingen Manon , Smilde Johanna E , Steegers Eric A , Danser A. H. Jan , van den Meiracker Anton H , Visser Willy
Published: July 2021

The sFlt-1/PlGF ratio has high sensitivity and specificity to diagnose preeclampsia (PE) and to predict pregnancy outcome. Especially in patients with preexisting hypertension and/or proteinuria, diagnosis and management of PE is challenging. We studied the predictive value of the sFlt-1/PlGF ratio for the occurrence of adverse outcome in patients with a high prevalence of preexisting hypertension or proteinuria, clinically suspected of having PE.

A sFlt-1/PlGF ratio >= 85 was considered to be a positive test. Adverse pregnancy outcome was defined as HELPP syndrome, intra-uterine growth restriction, or perinatal death. The predictive value of adverse pregnancy outcome of preeclampsia based on clinical grounds (clinical PE) or of a positive ratio was compared using a logistic regression model corrected for gestational age at testing.

Results:
So far 64 patients with a gestational age (GA) of 29.3 wks (range 20-37 wks) were included. 19 had preexisting hypertension, 5 had preexisting proteinuria and 6 had both conditions. At time of measurement 23 patients had clinical PE (4 with a negative ratio) and 30 patients had a positive sFlt-1/PlGF test (11 without clinical PE at testing of whom 7 developed clinical PE within 2 wks).

27% of patients had an adverse outcome of pregnancy. GA between patients with clinical PE or a positive test did not differ. Patients with clinical PE at the time of testing had an odds ratio of 2.5 (95% CI: 0.75 - 7.8) and patients with a positive test had an odds ratio of 6.8 (95% CI;2.1 - 33.9) for an adverse outcome. Patients with clinical PE had an absolute risk for an adverse outcome of 39% (9/23) compared to 46%(14/30) for patients with a positive sFlt-1/PlGF test (p=.075)

In patients where the diagnosis of PE is challenging because of preexisting hypertension and/or proteinuria a positive sFlt-1/PlGF is a stronger determinant for poor pregnancy outcome than the clinical diagnosis of PE. An explanation could be that a positive ratio can select patients that will develop PE in the near future and because of misclassification of patients with preexisting hypertension and/or proteinuria.
Abstract 153: Angiotensin II-induced Hypertension Alters Endothelial CaMKII
Authors: Charbel Chimene , Toussaint Fanny , Béziau Delphine , Blanchette Alexandre , Comtois Philippe , Ledoux Jonathan
Published: July 2021

The endothelial dysfunction associated with arterial hypertension is characterized by a Ca2+ dyshomeostasis resulting in an alteration of the delicate balance between reactive oxygen species (ROS) and nitric oxide (NO). However, the underlying mechanisms remain controversial.

It has been recently suggested that CaMKII, a Ca2+-calmodulin dependent kinase plays an important role in cardiovascular diseases such as diabetes-associated hypertension. Paradoxically, CaMKII also modulates eNOS expression and activity, and is thereby involved in the regulation of endothelial function. Indeed, acting as a Ca2+ sensor, CaMKII has the unique ability to integrate oscillatory Ca2+ signals into specific outcomes. We recently provided the first evidence that endothelial CaMKII is activated by a local Ca2+ signal, the Ca2+ pulsars, localized within myoendothelial projections (MEP), and results in an increased NO production. The characterization of this novel endothelial CaMKII signalling pathway in pathophysiological conditions will strengthen our understanding of endothelial dysfunction associated with hypertension.

This study aimed at the characterization of the impact of AngII-induced hypertension on the correlation between endothelial Ca2+pulsars and CaMKII in microvasculature.

Real-time confocal imaging showed that acute exposure to AngII (10 μM) increases both ROS intracellular levels (52%) and Ca2+ pulsars frequency (35%). Exposure to lower AngII levels (100 nM) only increased Ca2+ pulsars frequency (49%). However, CaMKIIα and β clustering (and activation) within MEP was observed at both AngII levels. Acute AngII also stimulated NO production (42%), an increase blunted by CaMKII inhibition with KN-93 (17%). Although a significant increase in Ca2+ pulsars frequency was observed (147%) in AngII-hypertensive mice, an AngII-induce increased in endothelial ROS levels was recorded independently of blood pressure levels. Recruitment of CaMKIIα and β at the MEP by chronic AngII was also increased regardless of blood pressure (60% and 43%). Our finding strongly suggests that AngII stimulates CaMKII through both ROS and Ca2+ pulsars . This study consolidates our understanding of CaMKII as a key regulatory component of endothelial function.
Abstract 175: Activation of Nrf2 by Exercise Training and Curcumin Contributes to Sympatho-Inhibition in Heart Failure
Authors: Haack Karla K , Harlow Amanda M , Wang Hanjun , Zucker Irving H
Published: July 2021

Chronic Heart Failure (CHF) is a disease characterized by increased Angiotensin II type 1 receptor (AT1R) signaling, sympathetic outflow, and oxidative stress. In conditions of high oxidant stress, Nuclear factor erythroid 2 related factor 2 (Nrf2) is dissociated from Kelch-like ECH associated protein 1 (Keap1) to activate antioxidant response element (ARE)genes. Nuclear factor kappa B (NF-kB) is a transcription factor downstream of AT1R that inhibits ARE transcription . Exercise training (ExT) decreases sympathetic outflow and oxidative stress, but the mechanism(s) by which ExT is protective remain unclear. The aim of this study was to investigate if ExT indirectly activates Nrf2 and if direct Nrf2 activation by curcumin (Cur) in rats with CHF would decrease sympatho-excitation and normalize AT1R pathway overactivation. Rats were ExT for 4 weeks post-surgery on a treadmill at a final speed of 25 m/min for 60 minutes, 5 days a week for 6 weeks. Western blot analyses of RVLM micropunches are summarized in the Table. Briefly, in CHF, there was a significant upregulation in NF-kB and Keap1 and a decrease in Nrf2 protein compared to sham. ExT significantly increased Nrf2 expression compared to both sham and CHF groups but normalized NF-kB and AT1R expression in CHF animals. Oral Cur (200mg/kg/day) decreased resting HR, urinary norepinephrine excretion, and renal sympathetic nerve activity in CHF animals. Western blot analyses indicated that Cur increased Nrf2, decreased NF-kB and normalized AT1R expression in the RVLM (Table). Taken together, Nrf2 activation may be protective in normalizing AT1R expression in CHF and one of the mechanisms by which ExT exerts its beneficial effects.
Abstract 177: Antenatal Betamethasone Exposure Reduces Ang-(1-7) but Enhances Angiotensin-Converting Enzyme and a Thiol Peptidase to Metabolize Ang-(1-7) in the Cerebrospinal Fluid
Authors: Marshall Allyson C , Shaltout Hossam A , Pirro Nancy T , Rose James C , Diz Debra I , Chappell Mark C
Published: July 2021

Antenatal betamethasone (BM) therapy accelerates lung development in preterm infants but may induce early programming events with long-term cardiovascular consequences. To elucidate these programming events, we developed a model of programming whereby pregnant ewes are administered BM (2 doses of 0.17 mg/kg) or vehicle at the 80th day of gestation and offspring are delivered at term. The BM-exposed (BMX) offspring develop elevated blood pressure, decreased baroreflex sensitivity, and alterations in the circulating, renal, and brain renin-angiotensin systems (RAS) by 6-months of age. While components of the RAS are present in cerebrospinal fluid (CSF), it is not known if BM exposure influences the CSF RAS to the same extent as other tissue compartments. Therefore, we characterized the CSF RAS in 6-month old male sheep, and established the impact of antenatal exposure on the RAS. Consistent with our previous findings of predominant ACE2 activity in the choroid plexus, CSF Ang-(1-7) levels are significantly higher than Ang I (320.8 ± 96.0 vs. 19.3 ± 4.6 pM; p<0.01, N = 12) or Ang II (1.4 ± 0.5 pM; p<0.01, N = 12). Moreover, Ang-(1-7) was 70% lower in the CSF of the BMX group (97.3 ± 31.4 pM; p<0.05) without changes in Ang I or Ang II. We next assessed the metabolism of Ang-(1-7) in the CSF and demonstrate a trend for increased enzyme activity in BMX males. Further analysis revealed two peptidases that contribute to Ang-(1-7) degradation in CSF of both groups. ACE accounted for approximately 15% (85 out of 100) of the overall Ang-(1-7) metabolism that hydrolyzed the peptide to Ang-(1-5) in CSF and ACE was 1.4-fold higher in the BMX group (6.5 ± 0.5 vs. 8.8 ± 0.5 fmol/min/ml; p<0.02, N = 4). We also identified a novel thiol peptidase activity (Ki = 4 μM PCMB) as the predominant activity that metabolized Ang-(1-7) to Ang-(1-4) in CSF; kinetic analysis of this peptidase in pooled CSF revealed Km and Vmax constants of 5.4 μM and 54 nmol/min/mg for control, and 4 μM and 60 nmol/min/mg for BMX. In summary, CSF levels of Ang-(1-7) were markedly lower following antenatal exposure. The reduced expression of central Ang-(1-7) potentially through an enhanced metabolism pathway may contribute to the long-term increase in blood pressure and an attenuated baroreflex in this model of fetal programming.
Abstract 191: Transcriptional Regulation of Renin by Nuclear Receptors Co-regulated With Renin
Authors: Lu Ko-Ting , Weatherford Eric T , Ketsawatsomkron Pimonrat , Grobe Justin L , Sigmund Curt D
Published: July 2021

Expression of the renin gene is required to maintain normal morphological and physiological identity of renal juxtaglomerular (JG) cells, yet the mechanisms regulating renin gene transcription remain elusive. We re-examined data from Brunskill et. al (JASN 22:2213, 2011), investigating genome-wide gene expression in JG and other renal cell types. Based on our previous data implicating nuclear receptors (RAR, RXR, VDR, PPARG, Nr2f2 and Nr2f6) in the regulation of mouse and human renin gene expression, we focused our analysis on the expression of genes encoding the 48 nuclear hormone receptors and their co-regulation with renin. Several nuclear receptors have an expression pattern emulating that of renin, that is, they were similarly enriched in JG cells but not in other cell types. These include Esr1, Nr1h4, Ppara, VDR, Nr1i2, Ppard, Hnf4g, Nr1h3, Thrb, Hnf4a, Esrrg, Nr4a3, Nr3c2, and Ar. We tested the hypothesis that a nuclear receptor that is co-regulated with renin may participate in renin gene regulation. To accomplish this, endogenous renin expression was evaluated in renin-expressing As4.1 cells after siRNA-mediated knock down of selected nuclear receptors. Each experiment included a negative control siRNA duplex (NC) that does not target any known genes. By way of example, siRNA-mediated inhibition of estrogen receptor alpha (Esr1) by 70-80% resulted in a 2-fold decrease in renin mRNA (fold change ± SEM: siEsr1: 0.4±0.2, p<0.001 vs NC). Similar results were obtained with a different siRNA targeting Esr1. Interestingly, loss of Esr1 also caused up-regulation of vitamin D receptor (VDR, 2.8±0.7 fold, p<0.001 vs NC) and Nr2f6 (2.0±0.2 fold, p<0.05 vs NC), both of which are known to be negative regulators of renin. Similarly, both renin (0.1±0.02, p<0.001 vs untreated) and Esr1 (0.3±0.1, p<0.05 vs untreated) mRNA were reduced in the kidney from mice treated with deoxycorticosterone acetate (50mg) and receiving 0.15 M NaCl in drinking water for 21 days (DOCA-salt). These data suggest Esr1 may regulate renin expression. Studies are in progress to assess if Esr1 stimulates renin expression on its own or acts by affecting the level of other nuclear receptors; and to determine if other co-regulated nuclear receptors also regulate expression of the renin gene.
Abstract 203: Angiotensin II Induced ER Stress in the Heart is Attenuated by Exercise
Authors: Masson Gustavo S , R. Nair Anand , White Mary E , Agarwal Deepmala , Michelini Lisete C , Francis Joseph
Published: July 2021

Recent evidence indicates that endoplasmic reticulum (ER) stress plays an important pathophysiological role in a number of chronic diseases including cardiovascular disease. In this study, we demonstrate that ANGII-induced ER stress in the heart is attenuated by exercise.

Methods:
Male rats were implanted with telemetry probes and trained to do exercise. After collecting baseline blood pressure, hypertension was induced by implanting osmotic minipumps containing ANGII 200ng/kg/min for 42 days or saline. Rats were randomized into sedentary (SED) or exercise trained (ExT). BP and cardiac functions were measured by radiotelemetry and echocardiography. At the end of the study, heart tissues were analyzed for ER stress and hypertrophy markers by real time RT-PCR and Western blotting.

Results:
are tabulated. The protein expression pattern for Grp78 and CHOP were similar to mRNA.

Conclusions:
1. ANGII infusion induces ER stress in the heart and contributes to cardiac hypertrophy. 2. EXT attenuates hypertensive response, cardiac hypertrophy and ER stress. 3. ER stress and ROS dependent mechanism contribute to ANGII induced cardiac hypertrophy and contribute to the development of hypertension.
Abstract 208: Augmented Cerebrovascular Responses to Diazoxide, an Opener of MitokAtp Channels, Following Middle Cerebral Artery (MCA) Occlusion in Rats
Authors: Rutkai Ibolya , Dutta Somhrita , Liu Dana , Katakam Prasad V , Busija David W
Published: July 2021

Previous research has not examined the effects of ischemia on cerebral vascular responses to mitochondrial activation in experimental strokes caused by occlusion of the MCA (MCAO). We investigated the role and mechanisms of mitochondrial derived vasoreactivity in the MCA of male SD rats following 90 min ischemia/48 h reperfusion injury. Ischemia was induced ipsilaterally (I) and the contralateral (C) side was non-ischemic. Electron microscopy showed disrupted mitochondrial morphology on the I side. Western blots for expression of mitochondrial proteins (Mean±SEM of immunoband intensity normalized to β-actin and as C vs. I): DRP-1 (1±0.1 vs. 3.1±0.2); VDAC (0.4±0.1 vs. 0.8±0.2); and complex-V (1.3±0.3 vs.2.0±0.3) as well as the non-mitochondrial proteins: phosphorylated (ph) ph-nNOS (0.4±0.1 vs. 0.7±0.1); ph-eNOS (0.2±0.06 vs. 0.7±0.2); COX-1 (0.5±0.1 vs. 0.8±0.1); and COX-2 (0.16±0.04 vs. 0.3±0.03) were elevated on I compared with C. The I mitochondrial membrane potential was greater (165±7 %) compared with C using TMRE fluorescence. Mitochondrial membrane depolarization decreased the TMRE intensity in both groups (100 to 80±3 vs. 165±7 to 70±4). Vascular responses of the MCA were characterized using the isolated, pressurized artery technique. Vasodilation in response to Ach (12±2 vs. 3±1), BK (42±9 vs. 32±6), SNP (75±7 vs. 38±4), and vasoconstriction to serotonin (65±8 vs. 33±7), were significantly decreased in I compared to C MCAs. On the other hand, 50 μM DZ induced vasodilation was enhanced in I arteries compared with C (5±1 vs. 17±2). Diazoxide induced dilation was decreased in the presence of the NOS inhibitor L-NAME (5±1 to 1±2 vs. 17±2 to 3±2) and the non-selective COX inhibitor indomethacin (5±1 to 1±2 vs.17±2 to 6±3). Our results indicate that experimental stoke has an major effect on mitochondria via inducing mitochondrial biogenesis leading to altered morphology, protein expression and function. Furthermore, the nitric-oxide and prostanoid

pathways appear to be involved in enhanced diazoxide mediated vasodilation after MCAO. We speculate that targeting mitochondria may be useful therapy for improving outcome in stroke patients.
Abstract 213: Adenosine Signaling in Hypertensive Chronic Renal Disease
Authors: Zhang Weiru , Zhang Yujin , Wang Wei , Luo Renna , Zhao Cheng , Grenz Almut , Tao Lijian , Blackburn Michael , Eltzschig Holger , Kellems Rodney , Xia Yang
Published: July 2021

Hypertension is the most prevalent life-threatening disease worldwide and is frequently associated with chronic kidney disease (CKD). However, the molecular basis underlying hypertensive CKD is not fully understood. We sought to identify specific factors and signaling pathways that contribute to hypertensive CKD and thereby exacerbate disease progression. Using high throughput quantitative RT-PCR profiling, we discovered that the expression level of 5’-ectonucleotidase (CD73), a key enzyme that produces extracellular adenosine, was significantly increased in the kidneys of angiotensin II (Ang II)-infused mice, an animal model of hypertensive nephropathy. Using both CD73 and A2B adenosine receptor (ADORA2B)-deficient mice coupled with pharmacological studies, we found that elevated CD73-mediated excess renal adenosine preferentially induced the ADORA2B expression and enhanced kidney ADORA2B signaling contributes to Ang II-induced hypertension. Similarly, in humans, we found that both CD73 and ADORA2B levels were significantly elevated in the kidneys of CKD patients compared to normal individuals and further elevated in hypertensive CKD patients. These findings led us to further discover that elevated renal CD73 contributes to excess adenosine signaling via ADORA2B activation that directly stimulates endothelin-1 (ET-1) production in a hypoxia inducible factor-α (HIF-1α)-dependent manner and underlies the pathogenesis of the disease. Lastly, we revealed that HIF-1α is an important factor responsible for Ang II-induced CD73 and ADORA2B expression at the transcriptional level. Overall, our studies reveal that Ang II-induced renal CD73 promotes the production of renal adenosine that is a prominent driver of hypertensive CKD by enhanced ADORA2B signaling-mediated ET-1 induction in a HIF-1α dependent manner. The inhibition of excess adenosine-mediated ADORA2B signaling represents a novel therapeutic target for the disease.
Abstract 240: A Potential Role of Memory T cells in Hypertension
Authors: Itani Hana A , Zhang Feng , Xiao Liang , Chen Wei , Harrison David G
Published: July 2021

Immunological memory is an important component of the adaptive immune response to pathogenic stimuli. Effector memory T cells, which are CD44high CD62Llow, reside in various non-lymphoid tissues and serve as a first line defense against foreign antigens. We have previously shown that T cells are important in hypertension, but the role of memory T cells has not been defined. Thus we hypothesized that CD8+ memory T cells are component of this memory response. To test this hypothesis, mice initially received two weeks of ang II, were allowed to recover for 3 weeks and were then re-infused with low dose of ang II (140 ng/kg/min) that minimally raises blood pressure in naïve mice. This low-dose of ang II increased blood pressure to 137±6 mmHg in mice that had previously received a vehicle infusion, but to 172±12 mmHg in mice that had received ang II. In keeping with a memory T cell response, we found that angiotensin II causes a 5 to 10-fold increase in CD8+ CD62Lhigh/CD44high/CCR7+ central memory cells in the kidney while reducing these effector memory CD8+ T cells in the spleen. CD8+ memory T cells require co-stimulatory signaling between CD70 on macrophages and CD27 on T cells. We found that angiotensin II infusion increased CD70 mRNA in isolated kidney vessels by 5 to 10-fold. Similarly, flow cytometry revealed that angiotensin II increased macrophages expressing CD70 and CD8+ T cells expressing CD27 expression in the kidney. Thus, these studies indicate that repeated exposures to angiotensin II promote an immune memory response of CD8+ T cells and markedly enhance the hypertensive response to this octapeptide. The role of CD27 and CD70 signaling requires additional study but might serve as a therapeutic target.
Abstract 252: 6ß-Hydroxytestosterone, A Cytochrome P450 1B1 Metabolite of Testosterone, Enhances Angiotensin II-Induced Pressor Effect in Male Mice
Authors: Pingili Ajeeth K , Jennings Brett L , Khan Nayaab S , Malik Kafait U
Published: July 2021

Androgens have been implicated in the development of hypertension and castration minimizes the pressor effect of angiotensin (Ang) II. Previously we showed that Ang II-induced hypertension and associated pathophysiological changes are diminished in male cytochrome P450 (CYP) 1B1 gene disrupted mice. Since CYP1B1 metabolizes testosterone to 6β-hydroxytestosterone (6β-OHT); this study was conducted to determine its contribution in modulation of Ang II-induced hypertension. Eight weeks old male Cyp1b1+/+ and Cyp1b1-/- mice were either castrated or injected with 6β-OHT (15 μg/g, i.p. every 3rd day) or vehicle (DMSO, 50 μl), infused with Ang II (700 ng/kg/min) or vehicle for 2 weeks, and systolic blood pressure (SBP) was measured by tail cuff. Castration attenuated Ang II-induced increase in SBP in both Cyp1b1+/+ (184 ± 6 vs. 129 ± 4 mmHg, P < 0.05) and Cyp1b1-/- mice (150 ± 6 vs. 129 ± 4 mmHg, P < 0.05). In Cyp1b1+/+ mice, 6β-OHT did not alter Ang II-induced increase in SBP (184 ± 6 vs. 180 ± 8 mmHg, P < 0.05), but enhanced it in Cyp1b1-/- mice (150 ± 6 vs. 172 ± 8 mmHg, P < 0.05). Castration improved endothelial dysfunction associated with Ang II-induced hypertension in Cyp1b1+/+ mice, as demonstrated by increased relaxation of the aorta to acetylcholine. No endothelial dysfunction was observed in Cyp1b1-/- mice given Ang II with or without castration. In Cyp1b1+/+ mice, 6β-OHT did not alter Ang II-induced endothelial dysfunction, however, in Cyp1b1-/- mice infused with Ang II, 6β-OHT caused endothelial dysfunction. We have shown that Ang II-induced hypertension is associated with increased vascular production of reactive oxygen species (ROS) in Cyp1b1+/+ mice, and this increase is attenuated in Cyp1b1-/- mice, as measured by dihydroethidium fluorescence. In both Cyp1b1+/+ and Cyp1b1-/- mice given Ang II, castration abolished the increased ROS production. In Cyp1b1+/+ mice, 6β-OHT did not alter levels of ROS produced by Ang II, however, 6β-OHT further increased ROS production in Cyp1b1-/- mice given Ang II. These data suggest that 6β-OHT, a CYP1B1 metabolite of testosterone, contributes to the hypertensive effect of Ang II in male mice. Moreover, CYP1B1 could serve as a novel target for the development of agents for the treatment of androgen-mediated hypertension.
Abstract 269: 5-aminoimidazole-4-carboxamide-3-ribonucleoside (AICAR) Decreases Soluble Fms-like Tyrosine Kinase-1 (sFlt-1) and Attenuates Endoplasmic Reticulum Stress in Rat Placental Villi Explants
Authors: Banek Christopher T , Gillham Haley E , Johnson Sarah M , Dreyer Hans C , Gilbert Jeffrey S
Published: July 2021

Preeclampsia, defined by the onset of
de novo
hypertension and proteinuria near the 20th week of gestation, is a major contributor to maternal and fetal morbidity and mortality worldwide. Preeclampsia is often preceded by placental ischemia and an imbalance in circulating angiogenic factors (e.g. VEGF - vascular endothelial growth factor, sFlt-1 - soluble VEGF receptor 1). Recent studies also report increased expression of endoplasmic reticulum (ER) stress products in preeclamptic placentas. Our laboratory recently reported 5-aminoimidazole-4-carboxamide-3-ribonuceloside (AICAR) reduces blood pressure and improves angiogenic balance (increased VEGF, decreased sFlt-1) in rats with placental ischemia-induced hypertension, but the mechanism is unclear. We hypothesized AICAR would decrease sFlt-1, increase AMPK phosphorylation, and decrease ER stress in hypoxic placental villous explants. On day 19 of pregnancy, placentas were isolated from four Sprague-Dawley rats and immediately dissected in ice-cold phosphate-buffered saline. Explants were cultured for 12 hours in physiologic normoxic (8% O2) and hypoxic (1.5% O2) conditions. All experiments were performed in triplicate. VEGF secretion was unaffected by AICAR treatment in both normoxic and hypoxic conditions. AICAR decreased sFlt -1 secretion in hypoxic villi (2147±116 vs. *1411±67, P<0.05). Additionally, AMPK activation ratio was increased with AICAR, and was hypoxic-dependent (8%: 2.9±0.3; 8%+A: 3.3±0.1; 1.5%: 3.5±0.1; 1.5%+A: *4.5±0.01;*P<.05). Moreover, markers of ER stress were increased with hypoxia, and decreased with AICAR treatment (BiP 8%: 1.2±0.2; 8%+A: 1.0±0.0; 1.5%: *8.3±0.7; 1.5%+A: 1.9±0.0.3;*P<.05), (CHOP 8%: 0.5±0.0; 8%+A: 0.3±0.1; 1.5%: *1.7±0.1; 1.5%+A: 0.7±0.1;*P<.05). ATF4 was not changed with hypoxia or AICAR treatment. The present data show that AICAR stimulates AMPK phosphorylation and decreases ER stress response proteins in hypoxic placental villi. Further, the present data support the hypothesis that AICAR restores angiogenic balance by decreasing sFlt-1 rather than increasing VEGF secretion from placental villi. These findings suggest AICAR may improve placental function during pregnancies complicated by placental-ischemia.
Abstract 283: Protective Role of Endogenous 20-HETE in Renal Ischemia-Reperfusion Injury
Authors: Muroya Yoshikazu , Fan Fan , Regner Kevin R , Marthaler Brodie , Falck John R , Garrett Michael R , Juncos Luis , Roman Richard J
Published: July 2021

Previous studies have indicated that inhibitors of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) protect against ischemia reperfusion injury (IRI) in the brain and heart. However, the role of endogenous 20-HETE in renal IRI has not been well characterized. The present study compared the susceptibility to IRI in Dahl salt-sensitive (S) rats in which the renal formation of 20-HETE is impaired versus that seen in normal Sprague-Dawley (SD) rats and in SS.5LEW 4A+ (4A+) congenic and SS.BN5 (BN5) consomic strains in which the CYP4A region of chromosome 5 was transferred from the Lewis or Brown Norway rats into the S genetic background. S, 4A+ and BN5 rats were fed a low salt diet and SD rats were fed a standard diet. IRI was induced by bilateral renal ischemia for 30 minutes followed by reperfusion. The 20-HETE production was measured using liquid chromatography-mass spectrometry (LC/MS). The renal production level of 20-HETE was significantly greater in SD, 4A+ and BN5 rats as compared to S rats and renal 20-HETE levels rose to a greater extent in these strains than in S rats following renal ischemia reperfusion. Plasma creatinine concentration rose to 3.7 ± 0.1 (n=6), 2.0 ± 0.4 (n=6), 1.9 ± 0.3 (n=6) and 2.2 ± 0.3 (n=6) mg/dl 24 hours following renal ischemia in S, 4A+, BN5 and SD rats, respectively. The kidney of S rats following IRI exhibited renal tubular necrosis and protein casts. The % of necrotic tubules in the corticomedullary regions of S rats (42.5 ± 3.5 %) was significantly higher than that seen in 4A+ (10.8 ± 3.8 %) or BN5 (10.2 ± 1.5 %) rats. The number of apoptotic tubular cells in the corticomedullary regions of S rats (125.3 ± 8.2 cells/field) was significantly higher than that seen in BN5 rats (46.8 ± 5.1 cells/field). Administration of the 20-HETE antagonist (6, 15-20-hydroxyeicosadecanoic acid) or the 20-HETE synthesis inhibitor (HET0016) abolished the resistance of 4A+ and BN5 rats to renal IRI and plasma creatinine levels rose to 4.0 ± 0.4 (n=4) and 3.8 ± 0.1 (n=4) mg/dl, respectively. The % of necrotic tubules respectively increased to 33.6 ± 6.9 % and 38.5 ± 5.5 % in 4A+ and BN5 rats and the number of apoptotic tubular cells increased to 119.2 ± 10.7 cells/field in BN5 rats. These data indicate that endogenously released 20-HETE plays a protective role in renal IRI.
Abstract 293: Brain Gai2 Proteins Mediate Acute Neural Blood Pressure Responses to Centrally and Peripherally Administered NaCl
Authors: Carmichael Casey Y , Mahne Sarah , Wainford Richard D
Published: July 2021

Aim:
We have demonstrated hypertension mediated by failure to upregulate PVN Gαi
2
proteins in rats fed a chronic high salt diet, but the role of this mechanism is unknown in acute settings. We examined the effect of central Gαi
2
proteins in the neural control of blood pressure in response to an acute physiological (IV and ICV) and pharmacological challenge.

Methods:
Twenty-four hour (24-h) ICV Gαi
2
or SCR oligodeoxynucleotide (ODN; 25μg/5μl)-pretreated conscious Sprague-Dawley rats were continuously monitored for changes in HR and MAP in response to peripherally-administered NaCl (3M IV bolus; 0.14 ml/100g) or centrally-administered NaCl (1M ICV; 5μL)(N=4/gp). To determine the cardiac baroreflex MAP was slowly raised to ~175 mmHg using phenylephrine and lowered to ~50 mmHg using sodium nitroprusside in separate group of pre-treated rats (N=8/gp).

Results:
In response to IV sodium, peak changes in HR were significantly greater in SCR
vs.
Gαi
2
treated rats (IV 3M NaCl ΔHR [bpm]; SCR -79±15
vs
. Gαi
2
-59±12, P<0.05), but no difference was observed in peak changes in MAP (IV 3M NaCl ΔMAP [mmHg] SCR 147±4 mmHg
vs
. Gαi
2
149±3). In SCR rats, MAP returned to baseline by 100 min whereas Gαi
2
rats remained significantly elevated for 120 min (P<0.05). In response to ICV sodium, we observed no difference between groups in peak HR changes (ICV 1M NaCl ΔHR [bpm] -23±8 bpm
vs
. Gαi
2
-22±8) or peak MAP changes (ICV 1M NaCl ΔMAP [mmHg] 16±3 mmHg
vs
. Gαi
2
9±3). In SCR rats, MAP returned to baseline by 50 min whereas Gαi
2
rats remained elevated for 90 min. The 24h Gαi
2
ODN pretreatment significantly altered the high-, but not low-pressure gain of the baroreflex in response to pharmacological challenge (MAP=180 mmHg, SCR HR=270 bpm, Gαi
2
HR=307 bpm, P<0.05).

Conclusion:
Downregulation of Gαi
2
proteins resulted in altered cardiac baroreflex function by impairing reflex decreases in HR and mediating significantly prolonged elevated MAP in response to peripherally administered sodium. This highlights a previously undiscovered role of brain Gαi
2
proteins in the baroreflex control of HR at elevated blood pressures—a factor that may contribute to elevated MAP in neurogenic models of hypertension.
Abstract 294: Evaluation of the Role of the NAD(P)H Oxidase Subunit p67phox in the Regulation of Glomerular Filtration Rate in Dahl Salt-sensitive Rats
Authors: Evans Louise C , Ryan Robert P , Kurth Terry , Skelton Meredith , Cowley Allen W
Published: July 2021

Studies were performed in male Dahl salt-sensitive (SS) rats with a null mutation in the p67
phox
gene (SSp67
phox
-/-) and wild type littermates (SS-WT) aged 10-12 weeks (n=5/6 /group). In previous studies we have shown that this important subunit of NADPH oxidase is upregulated in the SS rat. A new method for sequential measurement of GFR in conscious rats was applied during the consumption of a high salt diet. Utilizing a miniaturized device, disappearance curves of fluorescein isothiocynate (FITC)-sinistrin were determined by transcutaneous excitation and real time detection of the emitted light through the skin. The rats were surgically prepared with femoral venous catheters for the administration of (FITC)-sinistrin and carotid catheters for MAP measurement by telemetry. Baseline measurement of GFR and MAP were made daily to obtain 2 stable control days on low salt (LS, 0.4% NaCl). High salt (HS, 4.0% NaCl) measurements were made on day 2,5,7,14 and 21. In SS-WT rats, HS resulted in a progressive and significant increase in MAP (from 126±2 mmHg during LS to 158±11 mmHg by d21 HS). GFR decreased significantly from 1.50±0.03 ml/min/100g bwgt during LS to 1.26±0.02 by d21 HS. Notably, a significant increase in blood pressure was observed at d7 HS in the SS-WT preceeding the reduction in GFR which did not occur until d14 HS. In rats with the null mutation of the p67
phox
gene, the pressor response to HS was blunted, MAP averaged 131±5 mmHg by d21, and there was no significant change in GFR (LS values 1.54±0.07 ml/min/100g bwgt comparable to d21 HS values of 1.41±0.04). In summary, when p67
phox
is not functional in the SS rat, the hypertensive response to HS is blunted and GFR is maintained. We conclude that reactive oxygen species production via NADPH oxidase plays a major role in the eventual reduction of GFR in SS rats following the development of hypertension.